WO2020161614A1 - Methods for treating triple negative breast cancer using a human erbb-2 mutant protein - Google Patents

Abstract

A method of treating Triple Negative Breast Cancer in a subject, which comprises delivering a nucleic acid sequence encoding a mutant Erb B-2 polypeptide in an amount effective to inhibit cancer cell proliferation, wherein the mutant Erb B-2 lacks a functional nuclear localization signal, cannot translocate to the nucleus of the cell in which it is present, and functions as a dominant-negative inhibitor of endogenous Erb B-2 by inhibiting nuclear translocation of endogenous Erb B-2 in the cell in which the mutant is present, wherein the mutant Erb B-2 polypeptide retains intrinsic tyrosine kinase activity and does not inhibit endogenous Erb B-2 tyrosine kinase activity.

Description

METHODS FOR TREATING TRIPLE NEGATIVE BREAST CANCER USING A

HUMAN ERBB-2 MUTANT PROTEIN

FIELD OF THE INVENTION

The present invention concerns a method of treating Triple Negative Breast Cancer (TNBC) by inhibiting TNBC cell proliferation, particularly a method of treating TNBC by inhibiting nuclear localization of ErbB-2 receptor by means of hErbB-2ANLS, a human ErbB-2 nuclear localization domain mutant unable to translocate to the nucleus, which functions as a dominant negative (DN) inhibitor of endogenous ErbB-2 nuclear migration.

BACKGROUND OF THE INVENTION

The ErbB/HER family of receptor tyrosine kinases, includes major factors in breast cancer.

The ErbBs family of membrane receptor tyrosine kinases is composed of four members: epidermal growth factor receptor (EGF-R/ErbB-1), ErbB-2 (also known as HER-2/neu), ErbB-3, and ErbB-4. ErbBs ligands include all isoforms of heregulins (HRG), which bind to ErbB-3 and ErbB-4 and recognize EGF-R and ErbB-2 as co-receptors, and the epidermal growth factor (EGF) which binds to EGF-R. Upon ligand binding, ErbBs dimerize and their intrinsic tyrosine kinase activity is stimulated, which leads to the activation of signal transduction pathways that mediate ErbBs proliferative effects. Although ErbB-2 is an orphan receptor, it participates in an extensive network of ligand-induced formation of ErbBs dimers. Overexpression of ErbB-2 at the cytoplasmic membrane or ERBB-2 gene amplification is associated with increased metastatic potential, poor prognosis, and therapeutic resistance in breast cancer.

ErbB-2 has also been shown to migrate to the nuclear compartment where it binds DNA at specific sequences, HER-2 associated sequences (HAS) (Wang, S. C., Lien, H. C., Xia, W., Chen, I. F., Lo, H. W., Wang, Z., Ali-Seyed, M., Lee, D. F., Bartholomeusz, G., Ou-Yang, F., et al. (2004). Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2. Cancer Cell 6, 251-261). Through this function as a transcription factor (TF), ErbB-2 modulates the expression of the cyclooxigenase-2 (COX-2) gene (Wang et.al., 2004, op.cit .). Association of ErbB-2 with the COX-2 promoter was detected in breast cancer cell lines overexpressing ErbB-2, as well as in human primary breast tumors from the BC subtype that overexpresses ErbB-2 at the cytoplasmic membrane or shows ERBB-2 gene amplification, called ErbB-2-enriched (ErbB-2E) (Wang et al, 2004, op.cit.). Triple negative breast cancer (TNBC) refers to the subgroup of breast cancers (BCs) (15- 20%) with poor prognosis which do not express clinically significant levels of the steroid hormone receptors for estrogen (ER) and progesterone (PR), and lack membrane ErbB-2 overexpression or ERBB2 gene amplification. There are neither established biomarkers to predict outcome nor treatment for women bearing TNBCs, besides standard chemotherapy, highlighting that only deep understanding of the mechanisms involved in TNBC growth will enable the design of targeted therapies for this disease. Although in the molecular classification of breast cancer most TNBCs (70%) fall into the basal-like subtype (BLBC), the rest show a variety of molecular signatures, different from the gene expression (GE) profile that defines BLBC, indicating that TNBC is indeed a heterogeneous group. TNBCs are typically high grade tumors which most frequently develop in young women. Also, TNBC incidence is higher in certain populations such as in African Americans, Asians and Hispanics. Four different GE profile clusters were identified in TNBC: basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal (M), and luminal androgen receptor (LAR). At present, it is clear that new treatments against TNBC are urgently required.

The inventors’ novel findings through clinical studies in TNBC cohorts revealed that around 40% of TNBC express ErbB-2 at the nuclear compartment (hereinafter, nuclear ErbB-2 will be referred to as N ErbB-2). Their findings revealed a direct correlation between N ErbB-2 expression and local recurrence and distant metastasis during follow-up. Furthermore, their Kaplan-Meier analysis revealed that TNBCs patients bearing NErbB-2-positive tumors showed significantly shorter overall survival (OS) and disease-free survival (DFS) probabilities, as compared to patients whose tumors lacked N ErbB-2. Also, local relapse- free survival (LRFS) and distant metastasis-free survival (DMFS) probabilities are much lower in NErbB-2-positive tumors than in NErbB-2-negative ones.

These findings place ErbB-2 in a new and unanticipated scenario, which is the nucleus of TNBC.

Based on these novel clinical results, the inventors explored the biological significance of NErbB-2 expression in TNBC. Their results, using in vitro and in vivo models that mimic the different TNBC subtypes, disclosed that NErbB-2 drives proliferation of all four TNBC subtypes. In the frame of these discoveries, the inventors designed a novel treatment, which involves blocking nuclear ErbB-2 presence in TNBC. The treatment generally involves inhibiting nuclear ErbB-2 presence in TNBC cells by transfection with hErbB-2ANLS, a human ErbB-2 nuclear localization domain mutant, unable to translocate to the nucleus, which functions as a dominant negative (DN) inhibitor of endogenous ErbB-2 nuclear migration.

US patent N° 9,427,458 B2 (also by the applicant of the present invention) discloses a mutant ErbB-2 polypeptide in an amount effective to inhibit cancer cell proliferation, wherein the mutant lacks a functional nuclear localization signal, cannot translocate to the nucleus of the cell in which it is present, and functions as a dominant-negative inhibitor of endogenous ErbB-2 by inhibiting nuclear translocation of endogenous ErbB-2 in the cell in which the mutant is present, wherein the cancer overexpresses ErbB-2, and the mutant ErbB-2 polypeptide retains intrinsic tyrosine kinase activity and does not inhibit endogenous ErbB-2 tyrosine kinase activity. This patent is incorporated herein by reference in its entirety.

The present invention addresses previous shortcomings in the art by providing a method of treating TNBC by inhibiting cancer cell proliferation, particularly a method of treating TNBC by inhibiting nuclear localization of ErbB-2 receptor by means of hErbB-2ANLS, a human ErbB-2 nuclear localization domain mutant (having an amino acid sequence as set forth in SEQ ID NO:4), unable to translocate to the nucleus, which functions as a dominant negative (DN) inhibitor of endogenous ErbB-2 nuclear migration.

SUM MARY OF THE INVENTION

A first aspect of the invention is a method of treating TNBC in a subject, comprising delivering to a subject in need of such treatment a nucleic acid sequence that encodes and expresses a mutant of ErbB-2 in an amount effective to inhibit cancer cell proliferation, wherein the mutant cannot translocate to a nucleus of a cell in which it is present and functions as a dominant-negative inhibitor of endogenous ErbB-2.

According to a preferred embodiment of the present invention, the nucleic acid sequence used in the method of the invention is as set forth in SEQ ID NO: 13.

The ErbB-2 mutant according to the present invention has an amino acid sequence as set forth in SEQ ID NO:4.

A second aspect of the invention is the use of a mutant of ErbB-2 for carrying out a method of the present invention.

A further aspect of the invention is the use of a mutant of ErbB-2 for the preparation of a medicament for carrying out a method of the present invention. The foregoing and other aspects of the present invention will now be described in more detail with respect to other embodiments described herein. It should be appreciated that the invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1C. NErbB-2 expression in TNBC patients. (1A) NErbB-2 levels were evaluated by immunofluorescence (IF) and confocal microscopy using the C-18 ErbB-2 polyclonal antibody, raised against the human ErbB-2 carboxy (C) terminal region (amino acids 1242 to 1255). NErbB-2 was scored as previously reported (Schillaci, R., Guzman, P., Cayrol, F., Beguelin, W., Diaz Flaque, M. C., Proietti, C. J., Pineda, V., Palazzi, J., Frahm, I., Charreau, E. H., et al. (2012). Clinical relevance of ErbB-2/HER2 nuclear expression in breast cancer. BMCCancer 12, 74), considering both the percentage of positive cells and the staining intensity. A score of 0 represents faint or no staining in less than 10% of cells, 1+ weak nuclear staining in 10-25%, 2+ moderate staining in 26-50%, and 3+ strong staining in > 50% of cells. Scores of 2+ and 3+ were considered positive for NErbB-2. Left and middle panels (Cases #1 to #4): Shown are representative samples of tumors displaying 0 to 3+ NErbB-2 staining. Low membrane ErbB-2 (MErbB-2) levels (1+ score) are not considered as membrane ErbB-2 overexpression in the clinical setting. Right panels (control cases #1 and #2): As control of specificity, NErbB-2-negative BC samples were used either lacking MErbB-2 or displaying 3+ MErbB-2 score. Thick arrows indicate MErbB-2 and slim arrows, NErbB-2. Nuclei were stained with DAPI. (1 B) NErbB-2 IF staining using the C-18 ErbB-2 polyclonal antibody (upper panel) or the ErbB-2 A-2 monoclonal antibody raised against amino acids 1180-1197 at the ErbB-2 C-terminus (lower panel). One representative tumor displaying 3+ NErbB-2 score is shown. Nuclei were stained with DAPI. (1C) NErbB-2 expression was detected with the C-18 antibody by IF, as described in Fig. 1A, or by immunohistochemistry (IHC). Shown are representative images of tumors displaying 3+ NErbB-2 score. Boxed areas in the IHC staining are shown in detail in the inset (400X).

Figures 2A-2D. NErbB-2 shows clinical relevance in TNBC. Relationship between NErbB-2 positivity and survival in terms of overall survival (2A), disease-free survival (2B), local relapse-free survival (2C) and distant metastasis-free survival (2D) probabilities (%), as assessed by a Kaplan-Meier analysis and log-rank test. The TNBC clinical study, is based on the monitoring of 99 patients, 38 therefrom being NErbB-2-positive (full line) and 61 NErbB-2-negative (dotted line).

Figures 3A-3I. ErbB-2 isoforms expression and activation in BC cells. (3A) Representative Western Blot (WB) of ErbB-2 expression in BC cells (50 pg of protein). Total cell lysates were analyzed with the ErbB-2 C-18 antibody. MDA-MB-453 cells (LAR subtype) expressed full-length ErbB-2 (185 kDa, p185ErbB-2, ErbB-2 wild-type) comparable to the canonical ErbB-2 present in BT-474 cells, used as a control. MDA-MB-468 (BL1 subtype) showed an ErbB-2 variant of lower MW (-165 kDa, p165ErbB-2). HCC-70 and MDA-MB-231 cells (BL2 and M subtype, respectively) presented both wild-type (WT) and p165ErbB-2. Additionally, the ErbB-2 truncated isoforms p95ErbB-2/CTFs (-90-115 kDa) produced by proteolytic cleavage and alternative initiation of translation were observed. (3B) Signal intensities of p185ErbB-2 (black bars) and p165ErbB-2 (grey bars) in four independent WBs performed as in Fig. 3A, were analyzed by densitometry and normalized to b-tubulin protein bands. Densitometry was performed at different exposures to assure quantification within the linear detection range, preventing signal saturation. Data are presented as mean ± S.D. One-way ANOVA test with Dunnett’s multiple comparisons was applied to determine significant differences between BT-474 and TNBC cells. For b vs a: P < 0.001. (3C) Signal intensities of p95ErbB-2/CTFs (white bars) were quantified and plotted as in Fig. 3B. For b vs a: P < 0.001. (3D) Proportion of each ErbB-2 isoform (p185ErbB-2, black bars; p165ErbB-2, grey bars; and p95ErbB-2/CTFs, white bars) among BC cell lines. Mean values obtained in Figs. 3B-3C were represented as percentage relative to total ErbB-2. (3E) Representative WB analysis, performed as in Fig. 3A, in a panel of BC lines. (3F) Unsupervised hierarchical clustering of BC cells carried out on quantification profiles of p185, p165 and p95ErbB- 2/CTFs protein bands from three independent WBs performed as in Fig. 3E. Color scale of heatmap represents the logarithm of mean protein intensities normalized to b-tubulin. (3G) WB analysis of ErbB-2 isoforms expression using the monoclonal antibody A-2 raised against amino acids 1180-1197 located at the C-terminus of p185ErbB-2. (3H) WB analysis of ErbB-2 isoforms expression using the monoclonal antibody C-3 against amino acids 251- 450 located at the N-terminus of p185ErbB-2. (3I) Total cell lysates were analyzed by WB using the anti-phospho-ErbB-2 Tyr877 antibody or with the total ErbB-2 C-18 antibody. BT- 474 cells were used as a control of ErbB-2 expression and activation. The experiments shown in Figs. 3G to 3I are representative of three independent ones with similar results. Figures 4A-4D. ErbB-2 subcellular localization in BC cells. (4A) ErbB-2 localization was studied by IF and confocal microscopy using the ErbB-2 C-18 antibody followed by incubation with an IgG-Alexa Fluor 488 secondary antibody. Thick arrows indicate the presence of MErbB-2 and slim ones show NErbB-2 presence in TNBC cell lines and in the control BT-474 cells upon heregulin (HRG) stimulation. Nuclei were stained with DAPI. (4B) Percentage of nuclear ErbB-2 presence in confocal images from Fig. 4A. Integrated density (mean fluorescence intensity per unit area) of subcellular compartments was quantified in 50 cells from each cell line and was analyzed as percentages (mean ± SD), relative to the total content (integrated density) of ErbB-2 in each cell, which was set to 100%. One-way ANOVA with Dunnett’s multiple comparisons test was applied to determined significant differences between control and HRG-bI -treated cells. For b vs a, and c vs a: P < 0.001. (4C) Nuclear ErbB-2 expression was analyzed in BT-474 and MDA-MB-468 cells by IF and confocal microscopy using the ErbB-2 A-2 and C-3 antibodies, followed by incubation with an IgG-Alexa Fluor 488 secondary antibody. Nuclei were stained with propidium iodide (PI). (4D) p185 and p165ErbB-2 are located in the nucleus of TNBC cells. Cytosolic (C), Nuclear (N) and Membrane (M) protein lysates were analyzed by WB with the ErbB-2 C-18 antibody. Total (T) protein lysates were blotted in parallel. Histone H3 and b-tubulin were used to control cellular fractionation efficiency. The experiments shown in Figs. 4A to 4D are representative of three independent ones.

Figures 5A-5B. hErbB-2ANLS acts as dominant negative inhibitor of NErbB-2 localization in TNBC cells. (5A) TNBC cell lines were transiently transfected with hErbB-2ANLS vector. Green fluorescent protein (GFP) from hErbB-2ANLS vector was visualized by direct fluorescence imaging, and total ErbB-2 was localized by IF and confocal microscopy using the ErbB-2 C-18 antibody followed by incubation with an IgG-Alexa Fluor 546 secondary antibody. Solid arrows: hErbB-2ANLS- transfected cells, dashed arrows: wild-type cells that did not uptake the vector. Nuclei were stained with DAPI. Data are representative of three independent experiments with similar results. (5B) Inhibition of in vitro proliferation of TNBC cells by hErbB-2ANLS. TNBC cell lines were transiently transfected with hErbB-2ANLS or pEGFP-N1 empty vector. Cell proliferation was evaluated by a [³H]-thymidine incorporation assay. Data are presented as mean ± S.D of three independent experiments. Differences between groups were analyzed by unpaired two-tailed Student’s t test. For b vs a: P < 0.001. Figures 6A-6D. Preclinical models of in vivo blockade of ErbB-2 nuclear localization. Female NIH(S)-nude mice were inoculated in the mammary fat pad with MDA-MB-468 (6A) or with luciferase-expressing MDA-MB-231 cells (MDA-MB-231-luc) (6B). Once tumors were established (average tumor volume of 50-70 mm³), mice received pEGFP-N1 (empty vector) or hErbB-2ANLS intratumoral injections (0.55 mg/kg) once a week. Each point represents the mean volume ± S.D. (n=6). (6C) In vivo bioluminescence imaging (BLI) analysis of MDA-MB-231-luc tumors at the end of the experiment. Shown are representative images of the inhibitory effect of hErbB-2ANLS on tumor growth. (6D) Tumor burden was measured based on BLI signal quantified as total photon flux (photons/second), followed by anatomical confirmation of tumor sites post mortem. Data was analyzed by unpaired two- tailed Student’s t-test. For b vs a: P < 0.05.

Figures 7A-7H. Histopathological analysis. Representative hematoxylin and eosin (H&E) staining of histological sections from MDA-MB-468 (7A) and MDA-MB-231-luc (7B) tumors excised at the end of the experiment. Mitotic figures are indicated with black arrows. Tumor proliferation was quantified by mitotic figures count per high power field (HPF) in MDA-MB- 468 (7C) and MDA-MB-231-luc (7D) tumor samples. Data are presented as mean ± S.D. For b vs a: P < 0.05, unpaired two-tailed Student’s t test. Representative H&E staining of histological sections from MDA-MB-468 (7E) and MDA-MB-231-luc (7F) tumors showing areas with extensive necrosis (pale eosinophilic areas) after hErbB-2ANLS treatment. Percentage of necrosis was measured at lower magnification in MDA-MB-468 (7G) and MDA-MB-231-luc (7H) tumors. Data are presented as mean ± S.D. For b vs a: P < 0.05, unpaired two-tailed Student’s t test.

Figures 8A-8B. NErbB-2 was not detected in tumors injected with hErbB-2ANLS. IF and confocal microscopy of histological sections from MDA-MB-468 (8A) and MDA-MB-231-luc (8B) tumors excised at the end of the experiment. hErbB-2ANLS vector was visualized with an anti-GFP antibody, followed by incubation with an IgG-Alexa Fluor 488 secondary antibody. Total ErbB-2 was detected using the ErbB-2 C-18 antibody, followed by incubation with an IgG-Alexa Fluor 546 secondary antibody. Nuclei were stained with DAPI. Representative images are shown.

Figures 9A-9G. NErbB-2 function as TF induces Erk5 expression in TNBC to promote growth. (9A) WB analysis of Erk5 levels in BC cells. Fold change was calculated by normalizing the absolute levels of Erk5 to those of b-tubulin, setting the value of BT-474 cells to 1. Representative image of three independent experiments is shown. (9B) Schematic diagram showing the location of a HAS site on Erk5 gene. Primers (SEQ ID NO:9 and 10) flanking said region (black arrows), were used for ChIP-qPCR.. (9C) Recruitment of ErbB-2 to a HAS site on Erk5 gene was analyzed by chromatin immunoprecipitation (ChIP) in TNBC cells. Immunoprecipitated DNA was amplified by qPCR using primers (SEQ ID NO:9 and 10) flanking the HAS site at position +1321 , relative to Erk5 transcription start site (TSS). The arbitrary qPCR number obtained for each sample was normalized to the input, setting the value of the IgG samples as 1. (9D) H4 acetylation levels (H4Ac) at the HAS site of Erk5 were analyzed by ChIP. (9E) MDA-MB-468 and MDA- MB-231 cells were transfected with hErbB-2ANLS or pEGFP-N1 vectors. Recruitment of ErbB-2 at the HAS site of Erk5 was analyzed by ChIP-qPCR as in Fig. 9C. Data in Figs. 9C to 9E are presented as mean ± S.D. of three independent experiments. For b vs a: P < 0.001. (9F) TNBC cells were transfected as in Fig. 9E and Erk5 levels were assessed by WB. Fold change was calculated as in Fig. 9A, setting the value of pEGFP-N1 -transfected cells to 1. Shown is a representative experiment of a total of three. (9G) MDA-MB-468 and MDA-MB-453 cells were transfected with Erk5 siRNA of SEQ ID NO: 11 (ON-TARGETplus Set of 4, Human MAPK7 siRNA #2 cat #LQ-003513-00-0020, Dharmacon, Lafayette, CO, USA) or Control siRNA of SEQ ID NO:12 (siGENOME Non-Targeting siRNA #5 cat #D- 001210-05-20, Dharmacon, Lafayette, CO, USA) (100 nM) and proliferation was evaluated by [³H]-thymidine uptake. Data are presented as mean ± S.D of three independent experiments. For b vs a: P < 0.001 , by unpaired two-tailed Student’s t test.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in detail. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present invention.

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms“a”, “an” and“the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

Also as used herein,“and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed.

As used herein, the transitional phrase“consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps“and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Thus, the term“consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

The term“about,” as used herein when referring to a measurable value such as an amount or concentration (e.g., the amount of overexpression of ErbB-2) and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1 % of the specified amount. i. Definitions

“ErbB-2” as used herein refers to the tyrosine kinase receptor ErbB-2 that belongs to the epidermal growth factor receptor family. ErbB-2 can be natural or synthetic (e.g., derived from PCR and/or recombinant DNA techniques). ErbB-2 can be from a mammal, such as a human. Reference nucleic acid sequences and/or amino acid sequences can be obtained through publicly available databases, such as the National Center for Biotechnology Information (NCBI) database or commercially available databases, such as from Celera Genomics, Inc. (Rockville, Md.). Sequence information for human ErbB-2 can be found at NCBI Gene ID: 2064.

An exemplary wild-type ErbB-2 nucleic acid sequence is NCBI GenBank Accession No. NG_007503.1 (SEQ ID NO: 1), as shown in the attached Sequence Listing.

“Mutant” as used herein refers to a protein, such as ErbB-2, which comprises, consists of, or consists essentially of at least one amino acid substitution, insertion, deletion, and/or any combination thereof, i.e. , the mutant may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 50, 100, or more amino acid substitutions, insertions, deletions, and/or any combination thereof. These substitutions, insertions, deletions, and/or any combination thereof may or may not be confined to one location of the protein sequence and may be at multiple locations of the protein amino acid sequence. The mutation, i.e., the substitution, insertion, deletion, and/or any combination thereof, can be made to a wild-type protein, i.e., a protein existing naturally in an organism or subject, a protein substantially identical to a wild-type protein, or to a protein already comprising a mutation.

Mutants of the present invention can be produced by any suitable method known in the art. Such methods include conventional techniques in molecular biology, microbiology, and recombinant DNA. The mutant can be prepared by the construction of nucleotide sequences encoding the respective mutant and expressing the amino acid sequence in a suitable transfected host. The mutant can also be produced by chemical synthesis or by a combination of chemical synthesis and recombinant DNA technology. The mutant can be produced by obtaining the desired nucleotide sequence from a vector harboring the desired sequence or synthesized completely or in part using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques where appropriate.

“Substantially identical” or“substantially similar” as used herein refers to a reference amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical or similar, respectively, to the reference amino acid sequence. In some embodiments the reference amino acid sequence is the wild-type protein amino acid sequence.

“Dominant-negative inhibitor” and grammatical variations thereof as used herein refer to a mutant resulting from a dominant negative mutation. A dominant negative mutation occurs when a mutant affects one or more of the activities and/or functions of the normal, wild-type protein within the same cell in which it is present. A dominant negative mutation usually occurs if the product of the mutation (i.e., the dominant-negative inhibitor) can still interact with the same elements as the wild-type protein, but blocks or inhibits some aspect of the wild-type protein's activity and/or function. Such dominant-negative inhibitors can act in a variety of manners. “Dominant-negative inhibitor” as used herein is not intended to be limited in the manner in which the dominant-negative inhibitor acts as they can act in a variety of manners. In some cases, the dominant-negative inhibitor includes a binding domain and is capable of interacting with the wild-type protein to induce an inactive conformational change or the dominant-negative inhibitor may prevent an activating conformational change. In other cases, the dominant-negative inhibitor competitively binds to a substrate; thus, preventing binding of the substrate to the wild-type protein. Additionally, it is not intended to be limited in the manner in which the dominant-negative inhibitor is made as the dominant-negative inhibitors of the present invention may be made by any method known in the art. Some embodiments contemplate that it is produced synthetically. “Dominant-negative inhibitor” as used herein is also intended to include a mutant that provides partial inhibition or alteration of activity and/or function. It is not intended to require total inhibition or alteration, but in some embodiments the dominant-negative inhibitor may totally or substantially inhibit one or more functions of the wild-type protein. Exemplary dominant-negative inhibitors of the present invention include, but are not limited to, mutants of ErbB-2, which inhibit one or more activities and/or functions of endogenous (i.e. , wild- type) ErbB-2 in a cell in which they are present. In some embodiments the ErbB-2 mutant inhibits cancer cell proliferation. In other embodiments the ErbB-2 mutant inhibits nuclear translocation of endogenous ErbB-2. In certain embodiments the ErbB-2 mutant inhibits cancer cell proliferation and inhibits nuclear translocation of endogenous ErbB-2.

“Subject” as used herein is generally a human subject and includes, but is not limited to, a cancer patient. The subject may be male or female and may be of any race or ethnicity, including, but not limited to, Caucasian, African-American, African, Asian, Hispanic, Indian, etc. The subject may be of any age, including newborn, neonate, infant, child, adolescent, adult, and geriatric. Subjects may also include animal subjects, particularly mammalian subjects such as canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g. rats and mice), lagomorphs, primates (including non-human primates), etc., treated or screened for veterinary medicine or pharmaceutical drug development purposes.

“Triple Negative Breast Cancer” or“TNBC” as used herein refers to the subgroup of breast cancers (BCs) (15-20%) with poor prognosis which do not express clinically significant levels of the steroid hormone receptors for estrogen (ER) and progesterone (PR), and lack membrane ErbB-2 (MErbB-2) overexpression or ERBB2 gene amplification. Although in the molecular classification of breast cancer most TNBCs (70%) fall into the basal-like subtype (BLBC), the rest show a variety of molecular signatures, different from the gene expression (GE) profile that defines BLBC, indicating that TNBC is indeed a heterogeneous group. TNBCs are typically high grade tumors which most frequently develop in young women. Also, TNBC incidence is higher in certain populations such as in African Americans, Asians and Hispanics. Four different GE profile clusters were identified in TNBC: basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal (M), and luminal androgen receptor (LAR).

In some embodiments of the present invention, the TNBC may be resistant to one or more cancer therapies. The term“resistant,”“resistance,” and grammatical variations thereof as used herein refers to the response of a cell when contacted with an agent or therapy. A cancer cell is said to be resistant to a therapy or agent when the therapy or agent inhibits the cell growth or proliferation of the cancer cell to a lesser degree than is expected compared to an appropriate control, such as an average of other cancer cells that have been matched by suitable criteria, including but not limited to, tissue type, doubling rate or metastatic potential. In some embodiments, lesser degree refers to about 10%, 15%, 20%, 25%, 50%, or 100% less than the control cell. Exemplary cancer therapies that a cancer may become resistant to include, but are not limited to, ErbB-2 targeting therapies such as trastuzumab, lapatinib, and pertuzumab; hormonal therapies, such as tamoxifen and anastrozole; docetaxel; dacarbazine; paclitaxel; carboplatin; cisplatin; and gemcitabine.

“Proliferation” and“proliferating” as used herein refer to cells undergoing mitosis. Thus, “cancer cell proliferation” refers to cell division and a resulting increase in the number of cancer cells.

“Inhibit” as used herein refers to the prevention or slowing of a certain activity or function and includes a partial reduction in the activity. The term“inhibit” as used herein does not require complete blockage or elimination of the activity, but complete blockage or elimination of the activity may be seen in some embodiments of the present invention.

“Inhibition of proliferation” and grammatical variations thereof as used herein refer to a decrease in the rate of proliferation (e.g., a decrease or slowing in the rate of cellular division), cessation of proliferation (e.g., entry into GO phase or senescence), or death of a cell, including necrotic cell death or apoptosis.

“Treat,”“treating” or“treatment” as used herein refer to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the disease, reduction in the severity of the disorder or the symptoms of the disorder, the disorder is partially or entirely eliminated, as compared to that which would occur in the absence of treatment, etc. Treatment does not require the achievement of a complete cure of the disorder and can refer to stabilization of disease.

“Effective amount” or“amount effective” as used herein refer to the amount of a therapeutic active agent that when administered or delivered to a subject by an appropriate dose and regimen produces the desired result.

“Pharmaceutically acceptable” as used herein means that the active agent is suitable for administration or delivery to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

Active agents of the present invention may optionally be administered in conjunction with other compounds useful in the treatment of cancer. The other compounds may optionally be administered concurrently. As used herein, the word“concurrently” means sufficiently close in time to produce a combined effect (that is, concurrently may be simultaneously, or it may be two or more events occurring within a short time period before or after each other, e.g., sequentially). Simultaneous concurrent administration may be carried out by mixing the compounds prior to administration or delivery, or by administering or delivering the compounds at the same point in time but at different anatomic sites and/or by using different routes of administration. ii. Active Agents and their Methods of Use

The present invention refers to a method for treating TNBC by using active agents or compounds that comprise, consist of, or consist essentially of mutants of ErbB-2. The mutants of ErbB-2 of the present invention cannot translocate to the nucleus of the cell in which they are present or are not as effective at translocating to the nucleus of the cell in which they are present compared to wild-type ErbB-2. The effectiveness of the ErbB-2 mutant in translocating to the nucleus of the cell in which it is present can be reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% compared to wild-type ErbB-2. The inability or reduced effectiveness or ability of the ErbB-2 mutant to translocate to the nucleus of the cell may be due to many factors, such as, but not limited to, a mutation in a necessary binding domain or signaling sequence. In some embodiments of the present invention the ErbB-2 mutant (SEQ ID NO:4) lacks a functional nuclear localization signal. A“functional nuclear localization signal” as used herein refers to a nuclear localization signal having the characteristics of the wild-type protein. In certain embodiments the ErbB-2 mutant's nuclear localization signal does not allow for the mutant to be translocated to the nucleus or is not as effective as the nuclear localization signal of the wild-type ErbB-2 in translocating to the nucleus. The nuclear localization signal sequence of the ErbB-2 mutant may be mutated in any manner to result in a non-functional nuclear localization signal. A“non-functional nuclear localization signal” as used herein refers to a nuclear localization signal that inhibits translocation of the ErbB-2 mutant to the nucleus of the cell in which it is present. The inhibition provided by the non-functional nuclear localization signal can be a partial inhibition, i.e., result in a reduced effectiveness or ability of the mutant to translocate to the nucleus, or it can be a total inhibition of translocation to the nucleus. A non-functional nuclear localization signal includes where part or the entire nuclear localization signal sequence has been deleted in the ErbB-2 mutant.

The nuclear localization signal sequence of wild-type ErbB-2 comprises the amino acid sequence of KRRQQKIRKYTMRR (SEQ ID NO:3). In some embodiments of the present invention the nuclear localization signal sequence, e.g., SEQ ID NO:3, in the ErbB-2 mutant (SEQ ID NO:4) is deleted. In other embodiments amino acids at positions 676 to 689 of SEQ ID NO:2 are deleted and in certain embodiments amino acids at positions 676 to 692 of SEQ ID NO:2 are deleted. Deletion of the nuclear localization signal sequence may comprise removing or deleting a portion or segment of the nuclear localization signal sequence or removing or deleting the entire nuclear localization signal sequence. Deletion of the nuclear localization signal sequence does not foreclose the possibility that more of the ErbB-2 amino acid sequence than just the nuclear localization signal sequence is mutated. In some embodiments more of the ErbB-2 sequence is mutated than the amino acids of SEQ ID NO:3. The ErbB-2 mutants of the present invention may be mutated in more than one location. In other embodiments only a portion of the nuclear localization signal sequence or SEQ ID NO:3 is mutated. In some embodiments the mutant of ErbB-2 may be shortened by the number of amino acids in the nuclear localization signal sequence, i.e. the entire nuclear localization signal sequence is deleted. In other embodiments the nuclear localization signal sequence may be replaced or substituted with one or more amino acids.

In certain embodiments the ErbB-2 mutant (SEQ ID NO:4) is generated by deleting the nuclear localization signal sequence KRRQQKIRKYTMRR (SEQ ID NO:3) at amino acids 676 to 689 to result in the amino acid sequence of KLM at the deletion junction. For this ErbB-2 mutant N-terminal (aa 1 to 675) and C-terminal (aa 690 to 1234) portions of ErbB-2 can be PCR amplified using a high-fidelity PCR kit (Roche) and two sets of primers, 5 - ATCGCTAGCATGGAGCTGGCGGCCTTG-3' (SEQ ID NO:5) with 5'- AT CAAGCTT GAT GAGGATCCCAAAGAC-3' (SEQ ID NO:6) and 5'- ATCAAGCTTATGCTGCTGCAGGAAACGGAG-3' (SEQ ID NO:7) with 5'- AT CACCGGT AACACT GGCACGTCCAGACC-3' (SEQ ID N0:8), respectively. The amplified N-terminal portion that contains Nhel (5' end) and Hindi 11 (3' end) and the C-terminal portion that contains Hindi II (5' end) and Agel (3' end) can be digested and sequentially cloned into the pEGFP-NI vector (BD Biosciences) (Giri, D. K., Ali-Seyed, M., Li, L. Y., Lee, D. F., Ling, P., Bartholomeusz, G., Wang, S. C., and Hung, M. C. (2005). Endosomal transport of ErbB- 2: mechanism for nuclear entry of the cell surface receptor. MolCell Biol 25, 1 1005-11018)

In some embodiments of the present invention the mutants of ErbB-2 function as dominant negative inhibitors of endogenous ErbB-2 (i.e. , wild-type ErbB-2). Thus, the ErbB-2 mutant inhibits one or more functions and/or activities of endogenous ErbB-2 in a cell in which it is present. In some embodiments of the present invention the ErbB-2 mutant inhibits nuclear translocation of endogenous ErbB-2. The ErbB-2 mutant may inhibit nuclear translocation of endogenous ErbB-2 by about 10%, 15%, 20%, 25%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or more compared to a control cell or cancerous cell in which the ErbB-2 mutant is not present. ErbB-2 is a transmembrane protein that upon inducement or activation translocates or migrates to the nucleus of a cell. In some embodiments the ErbB-2 mutant prevents inducement or activation of endogenous ErbB-2 and in other embodiments it blocks or inhibits activated ErbB-2 from translocating to the nucleus.

Resistance to cancer therapies may occur with TNBC. In some embodiments of the present invention the ErbB-2 mutant overcomes or lessens resistance to one or more cancer therapies. Resistance to a cancer therapy may be decreased by the ErbB-2 mutant by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or more. Exemplary cancer therapies that a TNBC may become resistant to include, but are not limited to, well- established therapeutic options with standard anthracycline- and/or taxane-based chemotherapy. Additional therapies to which TNBC may be resistant to, when used alone and/or with anthracycline and/or taxane chemotherapeutic regimens, include: a) chemotherapy treatments with carboplatin, capecitabine and cyclophosphamide ; b) anti androgen receptor (AR) therapies using bicalutamide or enzalutamide; c) treatment with the anti-PD-11 antibodies nivolumab, pembrolizumab, atezolizumab, avelumab and durvalumab; d) endocrine therapy for estrogen receptor-beta-positive TNBC, using toremifene or anastrozole; e) immunotherapy with the PVX-410 multi-peptide vaccine; treatment with: f) the anti-EGF-R antibody cetuximab; g) the Hedgehog signaling inhibitor vismodegib; h) the anti-vascular endothelial growth factor receptor (VEGF-R) monoclonal antibody bevacizumab; i) the poly (ADP Ribose) polymerase inhibitors olaparib, talazoparib and veliparib; j) phosphoinositide 3-kinase (PI3K), AKT and mammalian target of rapamycin (mTOR) inhibitors, such as the pan-PI3K inhibitor buparlisib (BKM120), the mTOR inhibitor everolimus, and the three AKT isoforms inhibitor Ipatasertib; k) MEK Inhibitors such as cobimetinib.

In certain embodiments the ErbB-2 mutant sensitizes the TNBC to one or more cancer therapies or makes the TNBC more susceptible to one or more cancer therapies. A TNBC cell is more susceptible or sensitive to a cancer therapy or agent when the therapy inhibits the cell growth or proliferation of the TNBC cell to a greater degree than is expected for an appropriate control, such as an average of other TNBC cells that have been matched by suitable criteria, including but not limited to, tissue type, doubling rate or metastatic potential. In some embodiments, the TNBC is more susceptible or sensitive to a cancer therapy by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or more compared to a control cell or the response of cancer cells prior to treatment with the ErbB-2 mutant. Exemplary cancer therapies that a TNBC may become resistant to include, but are not limited to, well-established therapeutic options with standard anthracycline- and/or taxane-based chemotherapy. Additional therapies to which TNBC may be resistant to, when used alone and/or with anthracycline and/or taxane chemotherapeutic regimens, include: a) chemotherapy treatments with carboplatin, capecitabine, and cyclophosphamide; b) anti-androgen receptor (AR) therapies using bicalutamide or enzalutamide; c) treatment with the anti-PD-11 antibodies nivolumab, pembrolizumab, atezolizumab, avelumab and durvalumab; d) endocrine therapy for estrogen receptor-beta-positive TNBC, using toremifene or anastrozole; e) immunotherapy with the PVX-410 multi-peptide vaccine; treatment with: f) the anti-EGF-R antibody cetuximab; g) the Hedgehog signaling inhibitor vismodegib; h) the anti-vascular endothelial growth factor receptor (VEGF-R) monoclonal antibody bevacizumab; i) the poly (ADP Ribose) polymerase inhibitors olaparib, talazoparib and veliparib ) phosphoinositide 3-kinase (PI3K), AKT and mammalian target of rapamycin (mTOR) inhibitors, such as the pan-PI3K inhibitor buparlisib (BKM120), the mTOR inhibitor everolimus, and the three AKT isoforms inhibitor Ipatasertib; k) MEK Inhibitors such as cobimetinib.

In some embodiments of the present invention methods for treating TNBC are provided. In certain embodiments a method of treating TNBC in a subject is provided comprising delivering to a subject in need of such treatment a mutant of ErbB-2 in an amount effective to inhibit TNBC cell proliferation, wherein the mutant cannot translocate to a nucleus of a cell in which it is present and functions as a dominant-negative inhibitor of endogenous ErbB-2. In other embodiments a method for slowing the growth of a TNBC tumor are provided comprising delivering to a subject in need of such treatment a mutant of ErbB-2 in an amount effective to inhibit TNBC cell proliferation, wherein the mutant cannot translocate to a nucleus of a cell in which it is present and functions as a dominant-negative inhibitor of endogenous ErbB-2.

The method for treating TNBC, in some embodiments, may comprise identifying a subject having a TNBC tumor that is characterized by expression of nuclear ErbB-2; and delivering to the subject a mutant of ErbB-2 in an amount effective to inhibit TNBC cell proliferation, wherein the mutant cannot translocate to a nucleus of a cell in which it is present and functions as a dominant-negative inhibitor of endogenous ErbB-2. In other embodiments, a method of inhibiting the proliferation of a TNBC cell is provided comprising delivering to a TNBC cell a mutant of ErbB-2 in an amount effective to inhibit TNBC cell proliferation, wherein the mutant cannot translocate to the nucleus of the cell and functions as a dominant-negative inhibitor of endogenous ErbB-2.

In some embodiments of the present invention, other therapies, including but not limited to cancer therapies, can be used in combination with the methods of the present invention. Exemplary therapies include, but are not limited to, radiotherapeutic agents and factors; surgery; antibiotics such as doxorubicin, daunorubicin, mitomycin, actinomycin D, and bleomycin; chemotherapeutic agents such as cisplatin, VP16, adriamycin, verapamil, and podophyllotoxin; tumor necrosis factor; plant alkaloids such as taxol, vincristine, and vinblastine; and alkylating agents such as carmustine, melphalan, cyclophosphamide, chlorambucil, busulfan, and lomustine. Additional exemplary cancer therapies include, but are not limited to docetaxel; dacarbazine; paclitaxel; carboplatin; and gemcitabine. In some embodiments the mutant of ErbB-2 is delivered in combination with at least one additional cancer therapy. In certain embodiments the cancer therapy is a TNBC therapy selected from the following therapeutic options 1) anti-androgen receptor (AR) therapies using bicalutamide or enzalutamide; 2) treatment with the anti-PD-11 antibodies nivolumab, pembrolizumab, atezolizumab, avelumab and durvalumab; 3) endocrine therapy for estrogen receptor-beta-positive TNBC, using toremifene or anastrozole; 4) immunotherapy with the PVX-410 multi-peptide vaccine; treatment with: 5) the anti-EGF-R antibody cetuximab; 6) the Hedgehog signaling inhibitor vismodegib; h) the anti-vascular endothelial growth factor receptor (VEGF-R) monoclonal antibody bevacizumab; 7) the poly (ADP Ribose) polymerase inhibitors olaparib, talazoparib and veliparib; ,8) phosphoinositide 3- kinase (PI3K), AKT and mammalian target of rapamycin (mTOR) inhibitors, such as the pan-PI3K inhibitor buparlisib (BKM120), the mTOR inhibitor everolimus, and the three AKT isoforms inhibitor Ipatasertib; 9) MEK Inhibitors such as cobimetinib. In other embodiments of the present invention, the ErbB-2 mutant is delivered as a single agent therapy to treat the TNBC. A“single-agent therapy,” as used herein, is one in which no other agent or therapy is utilized to treat the TNBC or to sensitize the cancer cell to the ErbB-2 mutant, i.e., the ErbB-2 mutant is administered or delivered as a single therapeutic or agent to treat the TNBC. In some embodiments the ErbB-2 mutant is delivered as a single-agent therapy in the first-line therapeutic approach. The “first-line therapeutic approach,”“first-line therapy,” and grammatical variations thereof, as used herein, refer to a therapeutic utilized in the initial treatment of a disease or disorder. The first-line therapeutic approach as used herein is not limited to single-agent therapies, but may also apply to combination therapies. Thus, in some embodiments the ErbB-2 mutant is utilized as a first- line therapy for the initial treatment of cancer, wherein the ErbB-2 mutant is delivered as a single-agent therapy or as a combination therapy. In other embodiments the ErbB-2 mutant is utilized as a therapeutic in the second-line therapeutic approach or in any subsequent therapeutic approach. The second-line therapeutic approach and any subsequent therapeutic approaches refer to therapeutic approaches after the initial therapeutic approach, i.e., the first-line therapeutic approach. These approaches may be the same as or different than the first-line therapeutic approach and may comprise a single-agent therapy or a combination therapy. iii. Pharmaceutical Formulations and Methods of Delivery

The active agents and/or compositions thereof described herein may be formulated for administration or delivery in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (9^th Ed. 1995). In the manufacture of a pharmaceutical formulation according to the invention, the active compound(s) (including the physiologically acceptable salts thereof) is typically admixed with, inter alia, an acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient. The carrier may be a solid or a liquid, or both, and is preferably formulated with the compound(s) as a unit-dose formulation.

Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the active compound, which preparations are preferably isotonic with the blood of the intended recipient. These preparations may contain anti-oxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents. The formulations may be presented in unit\dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. For example, in one aspect of the present invention, there is provided an injectable, stable, sterile composition comprising an active compound or composition in a unit dosage form in a sealed container. The compound or composition is provided in the form of a lyophilizate that is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject. The unit dosage form typically comprises from about 10 mg to about 10 grams of the compound or composition. When the compound or composition is substantially water-insoluble, a sufficient amount of emulsifying agent that is physiologically acceptable may be employed in sufficient quantity to emulsify the compound or composition in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.

Further, the present invention provides liposomal formulations of the compounds disclosed herein and compositions thereof. The technology for forming liposomal suspensions is well known in the art. When the compound or composition thereof is an aqueous-soluble composition, using conventional liposome technology, the same may be incorporated into lipid vesicles. In such an instance, due to the water solubility of the compound or composition, the compound or composition will be substantially entrained within the hydrophilic center or core of the liposomes. The lipid layer employed may be of any conventional composition and may either contain cholesterol or may be cholesterol-free. When the compound or composition of interest is water-insoluble, again employing conventional liposome formation technology, the composition may be substantially entrained within the hydrophobic lipid bilayer that forms the structure of the liposome. In either instance, the liposomes that are produced may be reduced in size, as through the use of standard sonication and homogenization techniques.

Liposomal formulations containing the compounds disclosed herein or compositions thereof (e.g., ErbB-2 mutants), may be lyophilized to produce a lyophilizate, which may be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension. Examples of liposomal formulations that can be used include the neutral lipid 1 ,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DPOC)

Other pharmaceutical compositions may be prepared from the water-insoluble compounds disclosed herein, or compositions thereof, such as aqueous base emulsions. In such an instance, the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of the compound or composition thereof. Particularly useful emulsifying agents include phosphatidyl cholines, and lecithin.

In addition to active compounds, the pharmaceutical compositions may contain other additives, such as pH-adjusting additives. In particular, useful pH-adjusting agents include acids, such as hydrochloric acid, bases or buffers, such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate. Further, the compositions may contain microbial preservatives. Useful microbial preservatives include methylparaben, propylparaben, and benzyl alcohol. The microbial preservative is typically employed when the formulation is placed in a vial designed for multidose use. Of course, as indicated, the pharmaceutical compositions of the present invention may be lyophilized using techniques well-known in the art.

The therapeutically effective dosage of any one active agent, the use of which is in the scope of present invention, will vary somewhat from compound to compound, and patient to patient, and will depend upon factors such as the age and condition of the patient and the route of delivery. Such dosages can be determined in accordance with routine pharmacological procedures.

As a general proposition, the initial pharmaceutically effective amount of the active compound or composition administered parenterally will be in the range of about 0.1 to 50 mg/kg of patient body weight per day. The desired dosage can be delivered by a single bolus administration, by multiple bolus administrations, or by continuous infusion administration of active compound, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve.

The active compound(s) is administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of active compound(s) is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 0.1 , 0.5, 1 , 10 or 100 pg/kg up to 100, 200 or 500 mg/kg, or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. A more particular dosage of the active compound will be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g., such that the patient receives from about two to about twenty, e.g. about six doses of the ErbB-2 mutant). An initial higher loading dose, followed by one or more lower doses may be administered. An exemplary dosing regimen comprises administering an initial loading dose of about 0.5 to 10 mg/kg, followed by a weekly maintenance dose of about 0.5 to 10 mg/kg of the active compound. However, other dosage regimens may be useful. The progress of this therapy can be monitored by conventional techniques and assays.

Subjects treated by the methods of the present invention can also be administered one or more additional therapeutic agents. Chemotherapeutic agents may be administered for example systemically, by direct injection into the cancer, or by localization at the site of the cancer by associating the desired chemotherapeutic agent with an appropriate slow release material or intra-arterial perfusing of the tumor. The preferred dose may be chosen by the practitioner based on the nature of the cancer to be treated, and other factors routinely considered in administering.

Subjects may also be treated by radiation therapy, including, but not limited to, external beam radiotherapy, which may be at any suitable dose (e.g., 20 to 70 Gy or more per tumor, typically delivered over a fractionated schedule).

The ErbB-2 mutants of the present invention can be delivered or administered to a cell (e.g., a cancer cell) in vivo, ex vivo, or in vitro. In some embodiments the ErbB-2 mutant is delivered as a nucleic acid sequence that encodes and expresses the ErbB-2 mutant. According to the present invention, said nucleic acid sequence is as set forth in SEQ ID NO:13. In certain embodiments the ErbB-2 mutant is delivered to a subject as a nucleic acid sequence (SEQ ID NO: 13) that encodes the mutant and expresses the mutant in the subject. The nucleic acid sequence may comprise deoxyribonucleic acids and/or ribonucleic acids.

Delivery of the nucleic acids of the present invention to an organelle, cell, tissue, and/or organism may be accomplished by delivering or introducing genetic material into a cell by transfection or transduction procedures. Transfection refers to the acquisition by a cell of new genetic material by incorporation of added nucleic acid molecules. Transfection can occur by physical or chemical methods. Transduction refers to the process of transferring nucleic acid into a cell using a DNA or RNA virus. Such methods for delivering nucleic acids to an organelle, cell, tissue, and/or organism include, but are not limited to, direct delivery of DNA such as by ex vivo transfection, by injection, including microinjection; by electroporation; by calcium phosphate precipitation; by using DEAE-dextran followed by polyethylene glycol; by direct sonic loading; by liposome mediated transfection and receptor-mediated transfection; by microprojectile bombardment; by agitation with silicon carbide fibers; by Agrobacterium-mediated transformation; by PEG-mediated transformation of protoplasts; by desiccation/inhibition-mediated DNA uptake, naked plasmid adsorption, and any combination of such methods. Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed.

A vector may be utilized in some embodiments as a carrier for the nucleic acid sequence. A “vector” as used herein refers to a carrier nucleic acid molecule into which a nucleic acid sequence encoding the ErbB-2 mutant can be inserted for introduction into a cell where it can be replicated. The vector may comprise deoxyribonucleic acids (DNA) and/or ribonucleic acids (RNA). When the vector is a DNA molecule it is capable of being transcribed and subsequently translated into the ErbB-2 mutant. When the vector is a RNA molecule it is capable of being translated into the ErbB-2 mutant. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), and may be constructed through standard recombinant techniques. Non-limiting examples of vectors include plasmid vectors such as E. coir, phage vectors; and viral vectors such as adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, vaccinia viruses, and Semliki Forest virus vectors.

Treatment of cells, or contacting cells, with recombinant nucleic acid molecules can take place in vitro, in vivo, or ex vivo. For ex vivo treatment, cells are isolated from an animal (e.g., a human), transformed (i.e., transduced or transfected in vitro) with a delivery vehicle containing a nucleic acid molecule encoding an ErbB-2 mutant, and then administered to a recipient. Procedures for removing cells from mammals are well known in the art. In addition to cells, tissue or the whole or parts of organs may be removed, treated ex vivo and then returned to the patient. Thus, cells, tissue or organs may be cultured, bathed, perfused and the like under conditions for introducing the recombinant nucleic acid molecules of the invention into the desired cells.

For in vivo treatment, cells of a subject are transformed in vivo with a recombinant nucleic acid molecule of the invention. The in vivo treatment may involve, but is not limited to, systemic intravenous treatment with a recombinant nucleic acid molecule, local internal treatment with a recombinant nucleic acid molecule, such as by localized perfusion or topical treatment, and the like.

In certain embodiments of the present invention, a nucleic acid sequence encoding an ErbB-2 mutant is delivered to a cell or subject and is expressed in the cell or subject. In some embodiments the nucleic acid sequence encoding the ErbB-2 mutant is delivered to the cell or subject by injection. According to the invention, said nucleic acid sequence encoding the mutant of ErbB-2 is as set forth in SEQ ID NO:13. The injection (e.g., needle injection) may comprise one or more injections and can be, for example, subcutaneous, intradermal, intramuscular, intervenous, intraperitoneal, intrathecal, and/or intratumor. Methods of injection may include injection of a composition comprising a saline solution. Further embodiments of the present invention include the introduction of a nucleic acid by direct microinjection.

In other embodiments the nucleic acid sequence (SEQ ID N 0 : 13) encoding the ErbB-2 mutant is delivered to the cell or subject by liposome-mediated transfection. When the nucleic acid sequence encoding the ErbB-2 mutant is delivered to the cell or subject by liposome-mediated transfection the nucleic acid is entrapped in a lipid complex such as, for example, a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. Also contemplated is a nucleic acid complexed with Lipofectamine™ (Gibco BRL) or Superfect (Qiagen). In certain embodiments of the invention, a liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome- encapsulated DNA. In other embodiments, a liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1). In yet further embodiments, a liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In other embodiments, a delivery vehicle may comprise a ligand and a liposome.

In other embodiments TNBC cancer will be removed from the patient, tumors will be disgregated and primary cultures of the tumors cells will be either transfected with the ErbB- 2 mutant or the nuclear localization signal of ErbB-2 will be knocked out by CRISPR Cas9 in the laboratory. The tumor cells will be selected ex vivo, expanded, irradiated, and infused back into patients (10⁶ cells) in one cycle. Cyclophosphamide at 20 mg/kg single dose will be administered 3 days i.v. before cell infusion.

EXAMPLES

The present invention is explained in greater detail in the following non-limiting Examples.

Example 1 - NErbB-2 correlates with poor prognosis in TNBC

The role of NErbB-2 in TNBC remains completely unknown. The present inventors explored NErbB-2 presence and clinical relevance in a cohort of 99 TNBC samples (see Table 1 below). Samples were stained by immunofluorescence (IF) with the C-18 polyclonal antibody, raised against the human ErbB-2 C-terminal region. This protocol shows significantly higher sensitivity for detection of NErbB-2 than IHC procedures, while its specificity and sensitivity to detect MErbB-2 are comparable to those of IHC (Schillaci et al. , 2012, op.cit). High NErbB-2 expression (2+ and 3+ according to the scoring system described by the present inventors (Schillaci et al., 2012, op.cit.)) was found in 38.4% of the tumors (FIG. 1A and Table 3). Either absence (85.9% of the samples) or low MErbB-2 levels (1+ score, 14.1% of the samples), which would not be considered as membrane overexpression in the clinical setting, were found in the TN tumors (FIG. 1A and Table 1). Similar scores of NErbB-2 were found when selected TN samples were stained with an ErbB-2 monoclonal antibody, ErbB-2 A-2, raised against the C-terminal domain of human ErbB-2 (FIG. 1 B). The present inventors also explored NErbB-2 by IHC using the same antibody as in IF and found substantial to excellent overall concordance regarding NErbB-2 positivity detected by both techniques (91.4%, K=0.82, Table 2 below) (FIG. 1C).

Kaplan-Meier analysis revealed that TNBC patients bearing NErbB-2-positive tumors showed significantly shorter overall survival (OS) and disease-free survival (DFS) compared to patients whose tumors lacked NErbB-2 (FIG. 2A and FIG. 2B). Local relapse-free survival (LRFS) and distant metastasis-free survival (DMFS) were also lower in NErbB-2-positive tumors than in NErbB- 2-negative ones (FIG. 2C and FIG. 2D). Univariate analysis revealed that NErbB-2 and higher clinical stage were associated with lower OS, DFS and DMFS (see Table 4 below). Higher lymph node status was also associated with shorter OS and DMFS. In addition, multivariate analysis adjusted for the clinical stage identified NErbB-2 as an independent predictor of shorter OS (HR, 2.54; 95% Cl, 1.22-5.28; P = 0.013), DFS (HR, 2.91 ; 95% Cl, 1.44-5.87; P = 0.003), and DMFS (HR, 2.59; 95% Cl, 1.20-5.60; P = 0.015) in TNBC. TABLE 1. Clinicopathological characteristics of TNBC patients. NErbB-2 presence was explored in a cohort of 99 TNBC samples by IF and confocal microscopy:

Characteristic N° patients %

99

Total number of patients

Age (years)

Mean 53.51

SD 1 1 .93

Range 28-81

Length of follow up (months)

Median 58.50

Range 2.4-140.90

Menopausal status

Premenopausal 41 41 .41

Postmenopausal 58 58.59

Tumor size

T1 16 16.16

T2 45 45.46

T3 26 26.26

T4 9 9.09

Not documented 3 3.03

Lymph node status

NO 55 55.56

N1 19 19.19

N2 17 17.17

N3 8 8.08

Distant metastasis at diagnosis

MO 99 100.00

M1 0 0.00

Clinical Stage

8 8.08

50 50.51

41 41 .41

0 0.00

Tumor grade

1 2 2.02

2 24 24.24

3 70 70.71

Not documented 3 3.03

Membrane ErbB-2 Status (IHC)^a

0 85 85.86

1 + 14 14.14

2+ 0 0.00

3+ 0 0.00

Operation

Breast conserving surgery 44 44.44

Mastectomy 54 54.55

Not documented 1 1 .01

Chemotherapy (Anthracycline-containing regimen)

No 0 0

Yes 99 100

Radiotherapy

No 19 19.19

Yes 79 79.80

Not documented 1 1 .01

Events during follow up

No 66.00 66.67

Yes 33.00 33.33

Events description

Local recurrence plus metastasis 3.00 9.09

Only local recurrence 4.00 12.12 Only metastasis 24.00 72.73

_ Not documented 2Ό0 _ 6.06

a IHC: immunohistochemistry.

TABLE 2. Concordance between detection of NErbB-2 expression by immunofluorescence and immunohistochemistry. NErbB-2 expression determined by immunohistochemistry (IHC) using the C18 antibody shows substantial to excellent overall concordance regarding NErbB-2 positivity detected by both techniques (91.4%, K=0.82):

Overall

NErbB-2 (IF^a C-18), n (%) Total N^c K

concordance

- (%) statistics⁶

(%)

Negative Positive

NErbB-2 (IHC^b C-18),

n (%)

Negative 12 (34.3)^d 1 (2.9) 13 (37.1) 91 .4 0.82

Positive 2 (5.7) 20 (57.1) 22 (62.9)

a IF: Immunofluorescence.

b IHC: Immunohistochemistry.

c Concordance between detection of NErbB-2 expression by IF and IHC was evaluated in 35 patients from our cohort.

d Percentage of the total number of patients analyzed by both IF and IHC.

⁶ K statistics (with a value of 1 .0 indicating perfect agreement and a value of -1 .0 indicating perfect disagreement) revealed almost perfect levels of concordance between detection of NErbB-2 positivity by IF and IHC with C-18 antibody.

TABLE 3. Univariate analysis of clinicopathological characteristics of 99 TNBC patients in relation to NErbB-2 expression positivity using Odds ratio model.

a Chi-Square Test

b Fisher’s exact test

cWell to moderately differentiated: tumor grade 1 + 2, poorly differentiated: tumor grade 3 TABLE 4. Univariate and multivariate analyses of overall survival (OS), disease-free survival (DFS) and distant metastasis-free survival (DMFS) in TNBC patients.

Multivariate analysis adjusted for the clinical stage identified NErbB-2 positivity as a significant and independent predictor of shorter OS (HR, 2.54; 95% Cl, 1.22-5.28; P = 0.013), DFS (HR, 2.91 ; 95% Cl, 1.44-5.87; P = 0.003), and DMFS (HR, 2.59; 95% Cl, 1.20- 5.60; P = 0.015):

HR: Hazard ratio

b Cl: Confidence interval

c Well to moderately differentiated: tumor grade 1 + 2, poorly differentiated: tumor grade 3

Example 2 - ErbB-2 protein variants expression and activation in TNBC cells

The present inventors explored the presence and activation state of ErbB-2 in TNBC cell lines from all four TNBC molecular subtypes (TNBC-4type) (Lehmann, B. D., Bauer, J. A., Chen, X., Sanders, M. E., Chakravarthy, A. B., Shyr, Y., and Pietenpol, J. A. (2011). Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. JCIinlnvest 121 , 2750-2767; Lehmann, B. D., Jovanovic, B., Chen, X., Estrada, M. V., Johnson, K. N., Shyr, Y., Moses, H. L., Sanders, M. E., and Pietenpol, J. A. (2016). Refinement of Triple-Negative Breast Cancer Molecular Subtypes: Implications for Neoadjuvant Chemotherapy Selection. PloS one 11 , e0157368; Prat et al. , 2013a). While ErbB-2 expression was detected in cell lysates from all lines, using the C-18 antibody, ErbB-2 variants of different molecular weight (MW) where found among subtypes. Comparable to control BT-474 cells, from the ErbB-2-enriched (ErbB-2E) intrinsic BC subtype, MDA-MB-453 cells (LAR subtype) express wild-type (WT) ErbB-2 (MW of 185 kDa, p185ErbB-2) (FIG. 3A). MDA-MB-468 cells (BL1 subtype) express only an isoform with a SDS-PAGE MW of 165 kDa (p165ErbB-2) (FIG. 3A). HCC-70 (BL2 subtype) and MDA-MB- 231 (M subtype) display both p185ErbB-2 and p165ErbB-2 (FIG. 3A). Consistent with the inventors’ finding on the expression of p165ErbB-2, ErbB-2 bands between 130-170 kDa were previously detected in clinically ErbB-2-negative BC samples (Gautrey, H., Jackson, C., Dittrich, A. L, Browell, D., Lennard, T., and Tyson-Capper, A. (2015). SRSF3 and hnRNP H1 regulate a splicing hotspot of HER2 in breast cancer cells. RNABiol 12, 1139- 1151).

As reported (Brand, T. M., lida, M., Dunn, E. F., Luthar, N., Kostopoulos, K. T., Corrigan, K. L, Wleklinski, M. J., Yang, D., Wisinski, K. B., Salgia, R., and Wheeler, D. L. (2014). Nuclear epidermal growth factor receptor is a functional molecular target in triple-negative breast cancer. Molecular cancer therapeutics 13, 1356-1368), TNBC lines express significantly lower p185ErbB-2 levels than those in BT-474 cells (FIG. 3A and FIG. 3B). Levels of p165ErbB-2 isoform in MDA-MB-468, HCC-70 and MDA-MB-231 cells were also lower than those of p185ErbB-2 in BT-474 cells (FIG. 3A and FIG. 3B). Proteolytic cleavage and alternative initiation of translation of ErbB-2 result in carboxy-terminal fragments from 90 to 115 kDa in BC cells and tumors, collectively referred to as p95ErbB-2/CTFs variants (Anido, J., Scaltriti, M., Bech Serra, J. J., Santiago, J. B., Todo, F. R., Baselga, J., and Arribas, J. (2006). Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation. EMBO J 25, 3234-3244; Lin, Y. Z., and Clinton, G. M. (1991). A soluble protein related to the HER-2 proto-oncogene product is released from human breast carcinoma cells. Oncogene 6, 639-643; Pupa, S. M., Menard, S., Morelli, D., Pozzi, B., De Palo, G., and Colnaghi, M. I. (1993). The extracellular domain of the c-erbB-2 oncoprotein is released from tumor cells by proteolytic cleavage. Oncogene 8, 2917-2923), which are associated with nodal metastasis, resistance to anti-MErbB-2 and to endocrine therapies (Anido, J., Scaltriti, M., Bech Serra, J. J., Santiago, J. B., Todo, F. R., Baselga, J., and Arribas, J. (2006). Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation. EMBO J 25, 3234-3244; Scaltriti, M., Rojo, F., Ocana, A., Anido, J., Guzman, M., Cortes, J., Di Cosimo, S., Matias-Guiu, X., Cajal, S., Arribas, J., and Baselga, J. (2007). Expression of p95HER2, a truncated form of the HER2 receptor, and response to anti-HER2 therapies in breast cancer. JNatICancer Inst 99, 628-638; Warri, A. M., Isola, J. J., and Harkonen, P. L. (1996). Anti-oestrogen stimulation of ERBB2 ectodomain shedding from BT-474 human breast cancer cells with ERBB2 gene amplification. EurJCancer 32A, 134-140). Upon longer exposition of the blots, p95ErbB-2 variants were detected in all TNBC cells, again at levels significantly lower than those in BT-474 cells (FIG. 3A and FIG. 3C). Proportional representation of ErbB-2 isoforms highlights that in TNBC cells which express p165ErbB-2, this is the highest represented variant (FIG. 3D). Similar results on ErbB-2 isoforms and levels of expression were found when TNBC lines were compared with additional ErbB-2E cells (SK-BR-3 and HCC-1419) (FIG. 3E). Also, it was found that in T47D cells, from the luminal B (LumB) intrinsic BC subtype, p185ErbB-2 levels were comparable to or even lower than those in TNBC lines (FIG. 3E). T47D cells do not express of p165ErbB-2 (FIG. 3E). Unsupervised hierarchical clustering based on differential expression of ErbB-2 isoforms showed that ErbB-2E lines clustered together (FIG. 3F). T47D and MDA-MB-453 were placed together in a cluster which forms another major one with the ErbB-2E lines (FIG. 3F). MDA-MB-468, MDA-MB-231 and HCC-70 segregated in another major cluster (FIG. 3F). The present inventors found similar results when expression of ErbB-2 isoforms was investigated using the monoclonal antibodies A-2 and C- 3 raised against ErbB-2 C- and N-terminus, respectively (FIG. 3G and FIG. 3H). These antibodies showed lower sensitivity to detect p165ErbB-2 than C-18, likely because polyclonal antibodies, such as C-18, recognize a broad range of epitopes, amplifying the signal from proteins with low expression level. The present inventors previously reported that in ErbB-2E BC cells, N ErbB-2 is phosphorylated at tyrosine (Tyr) 877 (Beguelin, W., Diaz Flaque, M. C., Proietti, C. J., Cayrol, F., Rivas, M. A., Tkach, M., Rosemblit, C., Tocci, J. M., Charreau, E. H., Schillaci, R., and Elizalde, P. V. (2010). Progesterone receptor induces ErbB-2 nuclear translocation to promote breast cancer growth via a novel transcriptional effect: ErbB-2 function as a coactivator of Stat3. MolCell Biol 30, 5456-5472; Cordo Russo, R. I., Beguelin, W., Diaz Flaque, M. C., Proietti, C., Venturutti, L., Galigniana, N. M., Tkach, M., Guzman, P., Roa, J. C., O'Brien, N., et al. (2015). Targeting ErbB-2 nuclear localization and function inhibits breast cancer growth and overcomes trastuzumab resistance. Oncogene 34, 3413-3428; Diaz Flaque, M. C., Galigniana, N. M., Beguelin, W., Vicario, R., Proietti, C. J., Russo, R. C., Rivas, M. A., Tkach, M., Guzman, P., Roa, J. C., et al. (2013a). Progesterone receptor assembly of a transcriptional complex along with activator protein 1, signal transducer and activator of transcription 3 and ErbB-2 governs breast cancer growth and predicts response to endocrine therapy. Breast Cancer Res 15, R118; Diaz Flaque, M. C., Vicario, R., Proietti, C. J., Izzo, F., Schillaci, R., and Elizalde, P. V. (2013b). Progestin drives breast cancer growth by inducing p21(CIP1) expression through the assembly of a transcriptional complex among Stat3, progesterone receptor and ErbB-2. Steroids 78, 559-567; Venturutti, L., Romero, L. V., Urtreger, A. J., Chervo, M. F., Cordo Russo, R. I., Mercogliano, M. F., Inurrigarro, G., Pereyra, M. G., Proietti, C. J., Izzo, F., et al. (2015). Stat3 regulates ErbB-2 expression and co-opts ErbB-2 nuclear function to induce miR-21 expression, PDCD4 downregulation and breast cancer metastasis. Oncogene), a site different from the autophosphorylation ones, located at the kinase domain (Guo W, Pylayeva Y, Pepe A, Yoshioka T, Muller WJ, Inghirami G & Giancotti FG 2006 Beta 4 Integrin Amplifies ErbB2 Signaling to Promote Mammary Tumorigenesis. Cell 126 489-502; Xu W, Yuan X, Beebe K, Xiang Z & Neckers L 2007 Loss of Hsp90 Association Up-Regulates Src-Dependent ErbB2 Activity. Mol Cell Biol 27 220-228). This phosphorylation appears to be mandatory for ErbB-2 nuclear migration in said cells (Beguelin et al., 2010, op.cit. Cordo Russo et al., 2015, op.cit.] Diaz Flaque et al., 2013a, op.cit:, Diaz Flaque et al., 2013b, op.cit.] Venturutti et al., 2015, op.cit). As shown herein, ErbB-2 Tyr877 phosphorylation was found in all TNBC cells at both p165 and p185 ErbB-2 (FIG. 3I).

The present inventors also explored N ErbB-2 presence in TNBC cells by immunofluorescence (IF) and confocal microscopy with the C-18 antibody. NErbB-2 presence was found in all lines, at levels comparable to those in BT-474 cells treated with heregulin (HRG), an ErbBs^' ligand which induces ErbB-2 nuclear migration (Cordo Russo et al., 2015, op.cit.) (FIG. 4A and FIG. 4B). TNBC cells also display very low to moderate levels of MErbB-2 staining (FIG. 4A and FIG. 4B). The inventors found comparable results using the anti-ErbB-2 A-2 and C-3 antibodies (FIG. 4C, shows results in MDA-MB-468 and BT-474 cells). Subcellular fractionation and immunoblotting studies, using C-18 antibody showed that the major ErbB-2 nuclear isoform reflects its abundance in each cell type (FIG. 4D).

Example 3 - Blockade of all nuclear ErbB-2 isoforms as therapeutic strategy to inhibit TNBC

As discussed in the previous example, the present inventors demonstrated that 70-90% of ErbB-2 is located in the nucleus of TNBC cells.

Since previous findings by the present inventors showed that transfection with a human ErbB-2 nuclear localization domain mutant named hErbB~2ANLS, unable to translocate to the nucleus and which acts as a dominant negative inhibitor of endogenous ErbB-2 nuclear migration, blocks ErbB-2 nuclear localization in ErbB-2E BC (Beguelin et a!., 2010, op.cit .; Cordo Russo et al., 2015, op.cit .; Girl et al. , 2005, op.cit.), in the present invention it was explored whether this strategy, based on the heferodimerization of endogenous ErbB-2 and hErbB-2ANLS while both are in the cytoplasm, works in TNBC where ErbB-2 is constitutively in the nucleus. Indeed, hErbB-2ANLS transfection inhibited ErbB-2 nuclear localization and abrogated in vitro growth in TNBC cells (FIG. 5A and FIG. SB).

Next, tumor xenografts were established in nude mice using MDA-MB-468 and MDA-MB- 231 cells stably expressing luciferase (MDA-MB-231-luc). Once tumors reached 50 mm³, animals received intratumoral injections of hErbB-2ANLS or of the empty vector once a week. Volumes and growth rates of tumors injected with hErbB-2ANLS were significantly lower than those of tumors injected with the empty vector (FIG. 6A and FIG. 6B).

Growth rates were calculated as the slopes of growth curves. Volumes and percentages of growth inhibition were calculated at the end of the experiment and analyzed by unpaired two-tailed Student’s t test. # vs *: P < 0.001. b: with respect to pEGFP-N1 vector, P < 0.001. (see Tables 5 and 6 below).

TABLE 5 - Tumor growth analysis of MDA-MB-468 xenografts

3

MPD MR.4R» Mean tumor volume Mean growth rate % Growth

(mm⁵) ± S.D. (mm³ /day) ± S.D, inhibition

pEGFP-NI 145.5 ± 18.33* 1.887 + 0.1429*

hErbB-2ANLS 31.43 ± 10.40^# 0.3256 ± 0.1531^# 78.398^b

TABLE 6 - Tumor growth analysis of MDA-MB-231 -luc xenografts

a

Mean tumor volume Mean growth rate % Growth

MDA-MB-231-luc

(mm³) ± S.D. (mm³ /day) ± S.D, inhibition

pEGFP-NI 255.5 ± 26.33* 5.061 ± 0.4703*

HErbB-2ANLS 51.97 ± 23.86^# 1.063 ± 0.5728^#

Also, MDA-MB-231-luc tumor burden, measured by bioluminescence imaging (BLI) at the end of the experiment, showed that hErbB-2ANLS injection results in a significant inhibition of tumor growth (FIG. 6C and FIG. 6D). Histopathological analysis of MDA-MB-468 and MDA-MB-231-luc tumors revealed that tumors receiving hErbB-2ANLS showed significantly lower mitotic figures count per HPF than those receiving the empty vector (FIG. 7A to FIG. 7D). Larger percentages of tumor mass from hErbB-2ANLS-treated mice were necrotic as compared to empty vector-treated mice (FIG. 7E to FIG. 7H). Notably, NErbB-2 was not detected in neither preclinical models injected with hErbB-2ANLS (FIG. 8A and FIG. 8B). No weight loss or signs of overt toxicity were found in mice from hErbB-2ANLS or control groups in both tumor models. In addition, histological examination of liver, lung, heart and spleen did not reveal any pathological changes (data not shown). Example 4 - NErbB-2 function as transcription factor induces Erk5 expression in TNBC to promote growth

In order to explore whether NErbB-2 growth- pro oting effect is due to its functions as a TF, the present inventors performed a bioinformatics analysis searching for ErbB-2 binding sites (HAS) (Wang, S. C., H. C. Lien, W. Xia, I. F. Chen, H. W. Lo, Z. Wang, M. Ali-Seyed, D. F. Lee, G. Bartholomeusz, F. Ou-Yang, D. K. Giri, and M. C. Hung. 2004. Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2. Cancer Cell 6:251-261) in genes involved in TNBC growth. The inventors focused on kinases due to their key role in BC and identified Erk5 (extracellular signal-regulated kinase 5), a member of the mitogen-activated protein kinases (MAPKs) family, which has been reported to be overexpressed in TNBC (Al Ejeh, F., Miranda, M., Shi, W., Simpson, P. T., Song, S., Vargas, A. C., Saunus, J. M., Smart, C. E., Mariasegaram, M., Wiegmans, A. P., et al. (2014). Kinome profiling reveals breast cancer heterogeneity and identifies targeted therapeutic opportunities for triple negative breast cancer. Oncotarget 5, 3145-3158; Hsu, Y. H., Yao, J., Chan, L. C., Wu, T. J., Hsu, J. L, Fang, Y. F., Wei, Y., Wu, Y., Huang, W. C., Liu, C. L., et al. (2014). Definition of PKC-alpha, CDK6, and MET as therapeutic targets in triple-negative breast cancer. Cancer research 74, 4822-4835). Limited studies associate Erk5 expression with TNBC growth (Al Ejeh et al., 2014, op.cit .; Ortiz-Ruiz, M. J., Alvarez- Fernandez, S., Parrott, T., Zaknoen, S., Burrows, F. J., Ocana, A., Pandiella, A., and Esparis-Ogando, A. (2014). Therapeutic potential of ERK5 targeting in triple negative breast cancer. Oncotarget 5, 11308-11318) and the mechanisms involved in the transcriptional regulation of Erk5 in TNBC remain unknown. The present inventors found higher Erk5 levels in TNBC cells compared to BT-474 (FIG. 9A), as reported (Al Ejeh et al., 2014, op.cit .; Ortiz- Ruiz et al., 2014, op.cit.). The inventors identified a HAS site at position +1321 , relative to Erk5 transcription start site (TSS) (FIG. 9B). Chromatin immunoprecipitation (ChIP) assays using primers flanking this region (Forward primer: 5’-CACACCGCTGCCTCTGTAG-3’ (SEQ ID NO:9) and Reverse primer: 5’- T GCCT ATGGT CT CGAT GATCT -3’) (SEQ ID NO: 10), showed constitutive ErbB-2 loading to this site in TNBC cells. Levels of ErbB-2 recruitment were higher in cells expressing p165ErbB-2 (MDA-MB-468) or both p165ErbB-2 and p185ErbB-2 (MDA-MB-231) than in cells expressing only p185ErbB-2 (MDA-MB-453) (FIG. 9C). The inventors also found high levels of histone H4 acetylation at this HAS site, a marker of active gene transcription (FIG. 9D). Consistent with its ability to block ErbB-2 nuclear presence, transfection with the hErbB-2ANLS vector evicted ErbB-2 from the HAS site at Erk5 in TNBC cells expressing either p165ErbB-2 or p165ErbB-2 plus p185ErbB-2 (FIG. 9E). These inventors’ findings discovered a molecular mechanism underlying Erk5 expression in TNBC. As expected, the present inventors found that Erk5 protein levels decreased by hErbB-2ANLS transfection in all three TNBC models (FIG. 9F). The present inventors’ findings reveal that Erk5 is a downstream mediator of NErbB-2 in vivo induction of TNBC growth. As previously reported for M subtype (Ortiz-Ruiz et al., 2014), silencing of Erk5 inhibits in vitro proliferation in BL and LAR subtypes (FIG. 9G), confirming Erk5 critical role in TNBC.

MATERIALS AND METHODS

i. Patients

The Review Boards on Human Research from Universidad de La Frontera-Hospital de Temuco (Chile) and Hospital General de Agudos“Juan A. Fernandez” (Argentina) reviewed and approved the collection of tumor specimens, the survey data, and all clinical and pathological information, as well as the retrospective biomarker analysis on anonymized specimens from their corresponding archival cohorts. Paraffin-embedded tissue sections from 99 consecutively archived triple negative invasive breast carcinomas were selected from the files of the Histopathology Department of Hospital Temuco (from 2001 to 2008) and of Hospital Fernandez (from 2003 to 2015). The median follow-up time was 58.5 months (range 2.4-140.9 months). Pre-established patient inclusion criteria: women aged 18-85 (alive or deceased), diagnosed with stage l-lll TNBC as primary tumor, treated with surgery, who received radiotherapy and/or standard chemotherapy with anthracyclins, taxanes and/or platinum compounds in the adjuvant setting. This study was conducted according to the provisions of the Declaration of Helsinki and informed written consents were obtained from all patients before inclusion. The primary endpoints were disease-free survival (DFS), distant metastasis-free survival (DMFS), and local relapse-free survival (LRFS). DFS was defined as the time from BC diagnosis to the first recording of a recurrence or death, whichever came first. DMFS and LRFS were defined as the time from BC diagnosis to the first recording of a distant metastasis or a local recurrence, respectively. Local relapse was defined as recurrences of BC occurring in the ipsilateral breast, regional lymph nodes, and skin from the breast. Distant relapse was defined as recurrences of BC occurring beyond the confines of the ipsilateral breast, chest wall, or regional lymph nodes. Sites of distant relapse included: brain (and central nervous system), liver, lung, bone, pleural/peritoneal, and supraclavicular nodes. The secondary endpoint was the overall survival (OS). Pre treatment patient staging was classified according to the American Joint Committee on Cancer (AJCC) system (Singletary, S. E., Allred, C., Ashley, P., Bassett, L. W., Berry, D., Bland, K. I., Borgen, P. I., Clark, G., Edge, S. B., Hayes, D. F., et al. (2002). Revision of the American Joint Committee on Cancer staging system for breast cancer. JCIinOncol 20, 3628-3636) through the Elston and Ellis histological grading system (Page, D. L, Ellis, I. O., and Elston, C. W. (1995). Histologic grading of breast cancer. Let's do it. AmJCIinPathol 103, 123-124). Clinicopathological data of the cohort is shown in Table 1. ii. Cell lines and treatments

MDA-MB-468, HCC-70, MDA-MB-231 , BT-474, SK-BR-3 and T47D cells were obtained from American Type Culture Collection (Manassas, VA, USA). MDA-MB-453 and HCC-1419 were a gift from DJ Slamon (University of California, Los Angeles, CA, USA). Luciferase- expressing MDA-MB-231 cells (MDA-MB-231-luc) were kindly provided by MC Hung (University of Texas, M. D. Anderson Cancer Center, Houston, TX, USA). BT-474, SK-BR-3 and T47D cells were cultured as described previously (Beguelin et al. , 2010, op.cit .; Cordo Russo et al., 2015, op.cit.] Diaz Flaque et al., 2013a, op.cit .; Proietti, C. J., Rosemblit, C., Beguelin, W., Rivas, M. A., Diaz Flaque, M. C., Charreau, E. H., Schillaci, R., and Elizalde, P. V. (2009). Activation of Stat3 by heregulin/ErbB-2 through the co-option of progesterone receptor signaling drives breast cancer growth. MolCell Biol 29, 1249-1265; Rivas, M. A., Tkach, M., Beguelin, W., Proietti, C. J., Rosemblit, C., Charreau, E. H., Elizalde, P. V., and Schillaci, R. (2010). Transactivation of ErbB-2 induced by tumor necrosis factor alpha promotes NF-kappaB activation and breast cancer cell proliferation. Breast Cancer ResTreat 122, 1 11-124). MDA-MB-453 and HCC-1419 cells were cultured as described by DJ Slamon O'Brien, N. A., Browne, B. C., Chow, L., Wang, Y., Ginther, C., Arboleda, J., Duffy, M. J., Crown, J., O'Donovan, N., and Slamon, D. J. (2010). Activated phosphoinositide 3-kinase/AKT signaling confers resistance to trastuzumab but not lapatinib. MolCancer Ther 9, 1489-1502). MDA-MB-468, HCC-70 and MDA-MB-231 were maintained according to the supplier’s instructions. MDA-MB-231-luc cells were cultured as described by MC Hung (Xie, X., Li, L., Xiao, X., Guo, J., Kong, Y., Wu, M., Liu, W., Gao, G., Hsu, J. L., Wei, W., et al. (2012). Targeted expression of BikDD eliminates breast cancer with virtually no toxicity in noninvasive imaging models. Molecular cancer therapeutics 1 1 , 1915-1924). All cell lines were authenticated by short tandem repeat DNA profiling and were routinely tested for mycoplasma infection. All experiments were performed in complete media. In experiments assessing the effects of heregulin b1 (HRG b1) on ErbB-2 nuclear migration, BT-474 cells were starved in 0.1 % charcoalized fetal calf serum for 48 h before stimulation with HRG b1 (40 ng/ml) (R&D Systems Inc.). iii. Antibodies

The following antibodies were used for Western Blot: rabbit polyclonal anti-ErbB-2 clone C-18 (sc-284, raised against the C-terminus), mouse monoclonal anti-ErbB-2 clone A-2 (sc- 393712, raised against the C-terminus), mouse monoclonal anti-ErbB-2 clone C-3 (sc- 377344, raised against the N-terminus), and rabbit polyclonal anti-Histone H3 clone C-16 (sc- 8654-R), all from Santa Cruz Biotechnology; rabbit polyclonal anti-pErbB-2 Tyr877 (2241) from Cell Signaling Technology; mouse monoclonal anti^-Tubulin clone T0198 (T0198) from Sigma-Aldrich; rabbit monoclonal anti-Erk5 clone EP791Y (ab40809) from Abeam; and HRP-conjugated secondary antibodies from Vector Laboratories.

The antibodies used for immunofluorescence (IF) and confocal microscopy were anti-ErbB-2 C-18, anti-ErbB-2 A-2, anti-ErbB-2 C-3, and mouse monoclonal anti-green fluorescence protein (GFP) clone B-2 (SC-9996), all from Santa Cruz Biotechnology, and Alexa Fluor- conjugated secondary antibodies from Thermo Fisher Scientific. The antibodies used for immunohistochemistry (IHC) were anti-ErbB-2 C-18 and anti-ErbB-2 A-2, from Santa Cruz Biotechnology.

The antibodies used for chromatin immunoprecipitation (ChIP) assays were anti-ErbB-2 C-18 from Santa Cruz Biotechnology, and rabbit polyclonal anti-acetyl- Histone H4 (06-866) from Millipore. Rabbit IgG polyclonal antibody (PP64) from Millipore was used as negative control. iv. Western Blot and SDS-PAGE molecular weight (MW) calculation

Total lysates were obtained from cells growing in complete media or subjected to the different treatments. 25-50 pg of lysates were separated on a 6-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane and blotted as described (Beguelin et al., 2010, op.cit.) with the antibodies detailed in each experiment. Signal intensities of phospho-ErbB-2 bands were analyzed by densitometry using Image J software (National Institutes of Health) and normalized to total ErbB-2 protein bands. Experiments assessing total protein content were also repeated three to five times and signal intensities were normalized to b-tubulin bands, used as loading control. Subcellular protein fractionation kit for cultured cells (78840, Thermo Fisher Scientific Inc., Waltham, MA, USA) was performed in order to obtain cytoplasmic, membrane, and nuclear protein extracts, according to manufacturer^'s instructions. Molecular weight (MW) determination was performed as described (Guan Y, Zhu Q, Huang D, Zhao S, Jan Lo L, Peng J. An equation to estimate the difference between theoretically predicted and SDS PAGE-dispiayed molecular weights for an acidic peptide. Sci Rep. 2015;5: 13370). Briefly, for each western blot (WB), a MW standard protein ladder (Amersham ECL Rainbow Marker Full range, GE Healthcare) was loaded along with different protein samples. Then, an Rf value, defined as the migration distance of a protein through the gel divided by the migration distance of the dye front, was calculated for each standard protein. The obtained Rf values were plotted against the log(MW) of its corresponding standard protein to get the linear formula log(MW) = aRf + b (where a is the slope and b is the y-intercept). Finally, the Rf value for each protein sample was then obtained and used for the calculation of the SDS-PAGE-displayed MW of the ErbB-2 protein band. v. Hierarchical clustering

Unsupervised hierarchical clustering of BC cells was carried out on the quantification profiles of p185-, p165- and p95-ErbB-2 from WBs. Normalized signal intensities of the different ErbB-2 isoforms were log2 transformed and clustering analysis was performed using the R's heatmap.2 function. The heatmap and associated dendrogram were generated with the Euclidean distance and complete linkage clustering method without additional normalization. Color scale reflects the standardized expression of each isoform with red indicating highest expression and green indicating lowest expression. vi. Plasmids and transient transfections

The green fluorescence protein (GFP)-tagged human ErbB-2 mutant, which lacks the putative nuclear localization signal (NLS) (aa 676-KRRQQKIRKYTMRR-689), resulting in the sequence of KLM at the deletion junction (hErbB-2ANLS) (Giri et al., 2005), and which blocks the nuclear migration of the endogenous ErbB-2 (Beguelin et al., 2010, op.cit .; Cordo Russo et al., 2015, op.cit.), was generously provided by Dr MC Hung (University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA). The empty vector pEGFP-N1 was obtained from BD Biosciences-Clontech (Palo Alto, CA, USA). Cells were transfected for 72 h with 2 pg of expression vectors using X-tremeGENE HP (Roche) as described (Beguelin et al., 2010, op.cit. Cordo Russo et al., 2015, op.cit.] Venturutti et al., 2015). Transfection efficiencies, evaluated using the pEGFP-N1 vector and determined by the percentage of cells that exhibited GFP 96 h after transfection, varied between 60-70%. vii. Cell proliferation

Cell proliferation was evaluated by the incorporation of 1 pCi [³H]-thymidine (PerkinElmer; specific activity 6.7 Ci/mmol) during the last 16 hours of the corresponding treatments as previously described (Beguelin et al., 2010, op.cit.] Rivas, M. A., Venturutti, L, Huang, Y. W., Schillaci, R., Huang, T. H., and Elizalde, P. V. (2012). Downregulation of the tumor- suppressor miR-16 via progestin-mediated oncogenic signaling contributes to breast cancer development. Breast Cancer Res 14, R77). viii. Preclinical models of in vivo inhibition of ErbB-2 nuclear localization

Two-month-old female NIH(S)-nude mice (La Plata National University, Argentina) were inoculated into their inguinal mammary fat pad with MDA-MB-468 or MDA-MB-231-luc cells (5x10⁶/mouse) resuspended in 1 :1 v/v DMEM F12:Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA). Once tumors were established (volume of 50-70 mm³), mice were randomly assigned into either a treatment group or a control group (n=6). hErbB-2ANLS or pEGFP-N1 vectors (0.55 mg/kg) were administered by intratumoral injections once a week. For each injection, hErbB-2ANLS or pEGFP-N1 vector was appropriately diluted in serum free medium (DMEM-F12) to a final concentration of 11 pg plasmid DNA/25 pi medium (0.44 pg/pl), and complexed with 25 mI of X-tremeGENE HP (Roche). Tumor growth was measured routinely with a Vernier caliper and volume was calculated as previously described (Cordo Russo et al., 2015, op.cit). Mice were sacrificed 7 days after the last treatment and tumors from each group were fixed in 10% formalin for downstream studies. At necropsy, whole organ specimens were also fixed and processed similar to tumor pieces for histopathological examination. All animal studies were conducted in accordance with the highest standards of animal care as outlined by the NIH Guide for the Care and Use of Laboratory Animals and were approved by the IBYME Animal Research Committee. ix. Bioluminescence imaging (BLI)

The in vivo growth of MDA-MB-231-luc tumors was imaged at the end of the experiment. Before imaging, mice were anaesthetized with a mixture of oxygen and isoflurane and were injected i.p. with 150 mg/kg D-Luciferin (PerkinElmer) in sterile PBS. Fifteen minutes later, MDA-MB-231-luc tumor bearing mice were subjected to in vivo bioluminescence imaging (BLI) using an I VIS Lumina Bioluminometer (Xenogen) (exposure time: 10 s; field of view: D; subject height: 1.5 cm; luminescent binning: 8; luminescent f-stop: 1). Data were analyzed with I VIS Living Image 3.0 software (Xenogen) by drawing regions of interest (ROIs) around tumor masses and BLI signal was quantified as total photon flux, which is the radiance in each pixel summed or integrated over the ROI area (ah2)c4p giving units of photons/s. x. Histopathological analysis

Hematoxylin and eosin (H&E) staining was performed on 5 pm slide sections of MDA-MB- 468 and MDA-MB~231-!uc tumors, and used for histopathological examination. Mitotic figure counts were performed in 10 consecutive high power fields (HPF, 400 X magnification) using a Leica DM500 light microscope (0.45 mm diameter of the HPF). The identification of well-defined mitotic figures was performed as previously described (van Diest PJ, Baak JP, Matze-Cok P, Wisse-Brekelmans EC, van Galen CM, Kurver PH, et al. Reproducibility of mitosis counting in 2,469 breast cancer specimens: results from the Multicenter Morphometric Mammary Carcinoma Project. Hum Pathol. . 1992;23:603-7; Baak JP, van Diest PJ, Voorhorst FJ, van der Wall E, Beex LV, Vermorken JB, et al. Prospective multicenter validation of the independent prognostic value of the mitotic activity index in lymph node-negative breast cancer patients younger than 55 years. J Clin Oncol. 2005;23:5993-6001). in brief, the most poorly differentiated peripheral area of the tumor was used for counting mitoses. Necrotic, heavily inflamed, or benign areas were avoided. This area, called the measurement area, was minimally 1 x 1 mm and maximally 5 x 5 mm. In the measurement area, at 400 X magnification (objective 40, field diameter 450 pm at the specimen level), mitoses were counted in 10 consecutive neighboring fields of vision in the most cellular area. Only certain mitoses were counted, doubtful structures and apoptotic bodies were ignored. Percentage of tumor necrosis was evaluated at 40 X magnification using a Leica DM500 light microscope as previously described (Elmore SA, Dixon D, Hailey JR, Harada T, Herbert RA, Maronpot RR, et al. Recommendations from the INHAND Apoptosis/Necrosis Working Group. Toxicol Pathol. 2016;44:173-88). Necrotic areas were characterized by cell and nuclear swelling, pale eosinophilic cytoplasm, nuclear dissolution (karyolysis), nuclear fragments (karyorrhexis) and loss of cellular detail with shadows of tumor ceils visible to variable extent. Some degree of nuclear condensation (pyknosis) may be present. Adjacent cellular debris and inflammation (neutrophils, macrophages, etc.) may also be present if ceil membrane leakage or rupture has occurred. xi. ChIP assays and real-time quantitative PCR

ChIP was performed as described (Beguelin et al., 2010, op.cit.). Briefly, chromatin was sonicated to an average of about 500 bp. Sonicated chromatin was then immunoprecipitated by using 4 pg of the indicated antibodies and IgG as a control. The immunoprecipitate was collected by using either protein A or G beads (MilliporeSigma), which were washed repeatedly to remove nonspecific DNA binding. The chromatin was eluted from the beads, and cross-links were removed overnight at 65°C. DNA was then purified using the QIAquick PCR purification kit (QIAGEN) according to the manufacturer’s instructions. ChIP DNA was amplified by real-time quantitative PCR (qPCR) using FastStart Universal SYBR Green Master mix (Roche, MilliporeSigma), and performed in a StepOne Real Time PCR System (Applied Biosystems) under the following conditions: 40 cycles with 15 s of denaturing at 95°C and annealing and extension at 60°C for 1 min. Primers used were designed to amplify a region of the human Erk5/MAPK7 gene containing one HAS site (position +1321). xii. Immunofluorescence (IF) and confocal microscopy in cell cultures

ErbB-2 was localized using either a rabbit polyclonal (C-18) or a mouse monoclonal (C-3 or A-2) ErbB-2 antibody (all from Santa Cruz Biotechnology). Secondary antibodies for ErbB-2 C-18 were goat anti-rabbit IgG-Alexa Fluor 488 or donkey anti-rabbit IgG-Alexa Fluor 546. For ErbB-2 C-3 or A-2 a goat anti-mouse IgG-Alexa 488 was used, all from Invitrogen. Negative controls were carried out using PBS instead of primary antibodies. When cells were transfected with hErbB-2ANLS, green fluorescent protein (GFP) from this expression vector was visualized by direct fluorescence imaging. Cells were analyzed using a Nikon Eclipse E800 confocal laser microscopy system. A quantitative analysis of confocal images with Image J software was performed to evaluate the percentages of ErbB-2 localized at the nucleus and the cytoplasmic membrane. Segmentation of the whole cell was performed using ErbB-2 images. The cytoplasmic membrane compartment was defined as the difference between the image of the cell and a binary erosion (iterations: 5-25), and the nuclear compartment was defined according to the nuclear stain (DAPI (4’,6-diamidino-2- phenylindole) or propidium iodide). An integrated density value (mean fluorescence intensity per unit area) was obtained for total ErbB-2 (tErbB-2), for membrane ErbB-2 (mErbB-2), and for nuclear ErbB-2 (nErbB-2). To compute ErbB-2 cellular distribution, the ratio of integrated density of nErbB-2/tErbB-2 (NErbB-2) and mErbB-2/tErbB-2 (MErbB-2) was calculated for at least 50 cells from each group and an average value was obtained. Quantitative analysis of ErbB-2 subcellular localization is expressed as the percentage of MErbB-2 or NErbB-2. xiii. Immunofluorescence (IF) and immunohistochemistry (IHC) analysis of TNBC patient tissues

IF analysis was performed as described (Schillaci et al., 2012, op.cit .). Briefly, antigen retrieval was performed by immersing the sections in 10 mM sodium citrate buffer pH 6 and microwaving at high power for 4 min. Slides were blocked in Modified Hank’s Buffer (MHB) with 5% bovine serum albumin for 30 min and were incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal ErbB-2 C-18 antibody and mouse monoclonal ErbB-2 A-2 antibody, both from Santa Cruz Biotechnology. Slides were then incubated with anti-rabbit or anti-mouse IgG-Alexa 488 antibody (Invitrogen). Reduction of the autofluorescent background was performed by incubation with Sudan Black B 0.1 % (MilliporeSigma). Nuclei were stained with DAPI. Slides were analyzed by a Nikon Eclipse E800 confocal laser microscopy system. Negative controls were carried out with MHB instead of primary antibodies. C4HD tumors from the model of mammary tumors induced by progestins were also used as controls (Beguelin et al., 2010, op.cit.). Slides were independently scored by three pathologists. Score discrepancies were re-evaluated and reconciled on a two-headed microscope. Membrane ErbB-2 (MErbB-2) expression levels detected by IF were semiquantified using the same scores as those used in IHC staining (see below). Nuclear ErbB-2 levels detected by IF were scored considering both the percentage of positive cells and staining intensity. A score of 0 represents faint or no staining in less than 10% of cells, 1+ weak nuclear staining in 10-25%, 2+ moderate staining in 26-50%, and 3+ strong staining in > 50% of cells. Scores of 2+ and 3+ were considered positive for NErbB-2 presence (Schillaci et al., 2012, op.cit). NErbB-2 was also evaluated by IHC with the ErbB-2 C-18 antibody as follows: heat-induced antigen retrieval was performed in 10 mM Tris, 1 mM EDTA pH 9 for 30 min. Slides were incubated with the ErbB-2 C-18 antibody (dilution 1 :200) overnight at 4°C. Sections were subsequently incubated with the anti-rabbit EnVision+ System-HRP Labelled Polymer (K4003, Dako, Agilent) and developed using 3,3'-diaminobenzidine chromogen solution (Cell Marque, MilliporeSigma) according to manufacturer's protocol. The score was performed as detailed for IF.

MErbB-2 expression was evaluated by IHC with c-erb-B2 clone A0485 (Dako), as we already described (Schillaci et al. , 2012, op.cit .). MErbB-2 was scored according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines (Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J. Clin. Oncol. 2013;31 :3997-4013). Scores 2+ in which FISH confirmed ErbB-2 amplification and 3+ were considered positive for MErbB-2 overexpression. Estrogen (ER) and progesterone receptor (PR) were evaluated by IHC with clone 6F11 (Novocastra Laboratories) and clone hPRa2+hPRa3 (NeoMarkers), respectively and scored as described (Schillaci et al., 2012, op.cit.). xiv. Statistics

Analyses were performed using GraphPad Prism 5 (GraphPad) and STATA version 15 (Stata Corp. LLC). Information on biological replicates and statistical significance are reported in the respective figure legends. When two groups were compared, the unpaired two-tailed Student’s t-test was used. When three or more groups were compared, the one-way ANOVA with Tukey’s or Dunnett’s multiple comparisons test was used. Homoscedasticity of the variances was analyzed in every case. No statistical methods were used a priori to pre determine sample sizes, but sizes were in line with those indicated in previous reports (Cordo Russo et al., 2015, op.cit .; Rivas et al., 2012, op.cit.). Two way ANOVA with repeated measures plus Bonferroni post-test was applied to assess statistical significance of differences in tumor growth kinetics among groups as previously described (Proietti CJ, Izzo F, Diaz Flaque MC, Cordo RR, Venturutti L, Mercogliano MF, et al. Heregulin Co-opts PR Transcriptional Action Via Stat3 Role As a Coregulator to Drive Cancer Growth. Mol. Endocrinol. 2015;29:1468-1485). Comparison of tumor volumes and percentage of growth inhibition among different groups was performed at the end of each experiment and analyzed by unpaired two-tailed Student’s t-test test. Growth rates were calculated as the slopes of growth curves. Briefly, linear regression analysis was performed on tumor growth curves and the slopes were compared by using unpaired two-tailed Student’s t-test test to evaluate statistical differences. Correlations between categorical variables were performed using x2-test or Fisher’s exact test when the number of observations obtained for analysis was small. Specifically, Fisher’s exact test was selected when the number of expected values was under five, because it uses the exact hypergeometric distribution to compute the P value (Upton GJG. Fisher's exact test. Journal of the Royal Statistical Society. Series A (Statistics in Society). 1992; 155:395-402). Cumulative survival probabilities were calculated according to the Kaplan-Meier method, and statistical significance was analyzed by log-rank test. Multivariate analysis was performed using the Cox multiple hazards model. Adjustment for significant confounders was done to avoid increased bias and variability, unreliable confidence interval coverage, and problems with the model associated to the small size of our sample (Vittinghoff E, McCulloch CE. Relaxing the rule of ten events per variable in logistic and Cox regression. Am. J. Epidemiol. 2007; 165:710-718). Only variables that were statistically significant in a univariate model were included in the multivariate analysis. Guidelines for Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) were followed in this work (McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM. Reporting recommendations for tumor marker prognostic studies. J. Clin. Oncol. 2005;23:9067-9072). All tests of statistical significance were two-sided. P values < 0.05 were regarded as statistically significant.

Claims

1. A method of treating Triple Negative Breast Cancer in a subject in need thereof, the method comprising delivering to said subject a nucleic acid sequence encoding the mutant ErbB-2 polypeptide in an amount effective to inhibit cancer cell proliferation, wherein said nucleic acid sequence is as set forth in SEQ ID NO: 13, said mutant of ErbB-2 comprises the sequence of SEQ ID NO:4, and wherein said mutant lacks a functional nuclear localization signal, cannot translocate to the nucleus of the cell in which it is present, and functions as a dominant-negative inhibitor of endogenous ErbB-2 by inhibiting nuclear translocation of endogenous ErbB-2 in the cell in which the mutant is present, wherein the mutant ErbB-2 polypeptide retains intrinsic tyrosine kinase activity and does not inhibit endogenous ErbB-2 tyrosine kinase activity.

2. The method of claim 1 , wherein the nuclear localization signal comprises the sequence of SEQ ID NO:3.

3. The method of claim 1 , wherein the cancer is resistant to cancer therapies selected from the group consisting of anthracycline- and/or taxane-based chemotherapy; chemotherapy treatments with carboplatin, capecitabine, and cyclophosphamide; anti-androgen receptor (AR) therapies using bicalutamide or enzalutamide; the anti-PD-11 antibodies nivolumab, pembrolizumab, atezolizumab, avelumab and durvalumab; endocrine therapy for estrogen receptor-beta-positive TNBC using toremifene or anastrozole; immunotherapy with the PVX-410 multi peptide vaccine; the anti-EGF-R antibody cetuximab; the Hedgehog signaling inhibitor vismodegib; the anti-vascular endothelial growth factor receptor (VEGF-R) monoclonal antibody bevacizumab; the poly (ADP Ribose) polymerase inhibitors olaparib, talazoparib and veliparib; the phosphoinositide 3-kinase (PI3K), AKT and mammalian target of rapamycin (mTOR) inhibitors, the pan-PI3K inhibitor buparlisib (BKM120), the mTOR inhibitor everolimus, and the three AKT isoforms of inhibitor Ipatasertib; and the MEK Inhibitor cobimetinib.

4. The method of claim 1 , wherein the nucleic acid sequence encoding the mutant of ErbB-2 is delivered as a single-agent therapy.

5. The method of claim 1 , wherein the nucleic acid sequence encoding the mutant of ErbB-2 is delivered in combination with at least one additional cancer therapy.

6. The method of claim 5, wherein the at least one additional cancer therapy is selected from the group consisting of anthracycline- and/or taxane-based chemotherapy; chemotherapy treatments with carboplatin, capecitabine, and cyclophosphamide; anti-androgen receptor (AR) therapies using bicalutamide or enzalutamide; the anti-PD-11 antibodies nivolumab, pembrolizumab, atezolizumab, avelumab and durvalumab; endocrine therapy for estrogen receptor-beta-positive TNBC using toremifene or anastrozole; immunotherapy with the PVX-410 multi peptide vaccine; the anti-EGF-R antibody cetuximab; the Hedgehog signaling inhibitor vismodegib; the anti-vascular endothelial growth factor receptor (VEGF-R) monoclonal antibody bevacizumab; the poly (ADP Ribose) polymerase inhibitors olaparib, talazoparib and veliparib; the phosphoinositide 3-kinase (PI3K), AKT and mammalian target of rapamycin (mTOR) inhibitors, the pan-PI3K inhibitor buparlisib (BKM120), the mTOR inhibitor everolimus, and the three AKT isoforms of inhibitor Ipatasertib; and the MEK Inhibitor cobimetinib.

7. The method of claim 1 , wherein the nucleic acid sequence encoding the mutant of ErbB-2 is delivered to the subject by injection.

8. The method of claim 1 , wherein the nuclear localization signal of ErbB- 2 is knocked out by CRISPR Cas9.

9. The method of claim 1 , wherein the nucleic acid sequence encoding the mutant of ErbB-2 is delivered to the subject by liposome-mediated transfection.

10. The method of claim 1 , wherein the nucleic acid sequence encoding the mutant of ErbB-2 is delivered to the subject by viral vector-mediated transfection.

11. The method of claim 1 , wherein the cell retains endogenous ErbB-2 expression.