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Definitions

• Dr. Stephen Paget proposed the "seed and soil" hypothesis to describe the supporting role of the tumor microenvironment (the soil) in promoting the growth and survival of metastatic cancer cells (the seeds).
• the seeds the supporting role of the tumor microenvironment
• metastatic cancer cells the seeds
• Cav-1 -deficient cancer stroma revealed an elevation of autophagy and glycolysis pathways [1 1]. Also, previous studies show that the tumor stroma provides metabolic support directly to cancer cells [12]. Tumor initiation begins when cancer cells recruit the surrounding stromal cells by producing reactive-oxygen species (ROS), and inducing oxidative stress. Consequently, cancer-associated fibroblasts (CAFs) undergo DNA damage, which initiates several catabolic pathways, such as autophagy and mitophagy (mitochondrial degradation) [13]. Lacking functional mitochondria, CAFs then shift their metabolism towards glycolysis, producing energy-rich molecules, such as L-lactate, ketone bodies, and glutamine.
• ROS reactive-oxygen species
• CAFs cancer-associated fibroblasts
• CAFs Lacking functional mitochondria, CAFs then shift their metabolism towards glycolysis, producing energy-rich molecules, such as L-lactate, ketone bodies, and glutamine.
• Breast cancer type 1 susceptibility protein (BRCAl) is a tumor suppressor that is mutated in 45% of hereditary breast cancers and down-regulated in sporadic breast cancers [15-17].
• BRCAl is involved in several signaling pathways and cellular processes, such as the DNA damage response, the regulation cell cycle progression, apoptosis, and ubiquitination [15].
• BRCAl has been described recently as an autophagy inhibitor [22, 23]. Esteve et al. observed that starved cells down-regulate BRCAl expression and display an elevation of ROS and autophagy. Conversely, BRCAl was found to induce several anti-oxidant genes that are responsible for ROS inhibition [22-24]. Unfortunately, all previous BRCAl studies focused only on the role of BRCAl in epithelial cancer cells, and not the tumor stroma.
• This invention provides a method for treating a subject afflicted with breast cancer, comprising concurrently administering to the subject (i) a PARP inhibitor and (ii) an autophagy inhibitor, wherein the amounts of the PARP inhibitor and autophagy inhibitor, when concurrently administered, are therapeutically effective.
• This invention also provides a method for inducing the death of a breast cancer cell, comprising concurrently contacting the cell with (i) a PARP inhibitor and (ii) an autophagy inhibitor, wherein the amounts of the PARP inhibitor and autophagy inhibitor, when concurrently contacted with the cell, are effective to induce the cell's death.
• this invention provides a composition comprising (i) a PARP inhibitor, (ii) an autophagy inhibitor, and (iii) a pharmaceutically acceptable carrier.
• FIG. 1 Generating BRCAl knock-down fibroblasts. Using shRNA targeted against BRCAl , a BRCAl knock-down was attempted in hTERT-immortalized human fibroblasts using four different constructs. Construct number 2 displayed the most significant inhibition of BRCAl expression. Therefore, all subsequent experiments used only this fibroblast cell line.
• FIG. 2 Increased proliferation in shB RCA 1 fibroblasts.
• FIG. 3 BRCAl knock-down induces autophagy and mitophagy.
• A Immuno-blotting shows an increase in the expression of markers for autophagy (LC3) and mitophagy (BNIP3) in shBRCAl fibroblasts.
• B Increased autophagy in shBRCAl fibroblasts when co-cultured with breast cancer cells. Immuno-fluorescence shows that shBRCAl fibroblasts demonstrate higher expression of the mitophagy marker (BNIP3, red color, top panel) when co-cultured with triple negative breast cancer cell line MDA-MB-231 -GFP (green color). Also, shBRCAl fibroblasts displayed an elevation of the autophagy marker (LAMP1, red color, low panel). Scale bar, 20 ⁇ .
• FIG. 4 BRCAl knock-down induces HIF1 and mitochondrial dysfunction.
• A Knock-down of BRCAl induces HIF- ⁇ expression.
• shBRCAl fibroblasts were incubated with 10 ⁇ of MG132 (proteasome inhibitor) plus lmM of DMOG (HIF-1 a stabilizer) for 5 hours.
• Pseudohypoxic shBRCAl fibroblasts demonstrate an elevation of HIF-la expression.
• Immuno- blotting for LC3 shows an increase in activated LC3 (LC3 II) in pseudo-hypoxic shBRCAl fibroblasts. Beta-actin is shown as a loading control.
• B Down-regulation of SDHB in BRCA1- deficient fibroblasts. Immuno-blot analysis displays a decrease of SDHB expression in shBRCAl fibroblasts. SDHB down-regulation will abrogate mitochondrial oxidative
• FIG. 1 Knock-down of BRCAl induces Akt-activation.
• shBRCAl fibroblasts were incubated with 10 ⁇ of MG132 (proteasome inhibitor) plus 1 mM of D OG (HIF-1 a stabilizer) for 5 hrs.
• shBRCAl fibroblasts demonstrate an increased activation of the Akt pathway, as judged by the elevated phosphorylation of Akt and mTOR.
• ⁇ -actin is shown as a loading control.
• FIG. 6 Ketone body production and metabolic reprogramming of cancer cells towards oxidative mitochondrial metabolism.
• A Knock-down of BRCAl induces elevated ketone body production. To evaluate if BRCAl knock-down induces mitochondrial dysfunction, ketone body generation was measured in the condition media of control and shBRCAl fibroblasts. It is worth noting that ketone body generation is increased up to 5.5-fold in shBRCAl fibroblasts, relative to control cells.
• Figure 7 shBRCAl fibroblasts induce tumor growth when co-injected with cancer cells.
• Cancer cells release ROS (such as H 2 0 2 ) into the tumor micro-environment, thereby affecting neighboring stromal cells. Oxidative stress and starvation induces DNA damage in stromal cells. Consequently, an activated tumor stroma initiates autophagy and mitophagy related signaling pathways, which will switch fibroblast metabolism towards glycolysis. Moreover, oxidative stress stabilizes HIF-1 a expression, which will up-regulate glycolytic molecules and induce mitophagy. High-energy mitochondrial fuels (such as ketones) are secreted into the tumor micro-environment and are then transferred by MCT1 (monocarboxylate transporter) into cancer cells and are consumed by cancer cell mitochondria, via OXPHOS.
• MCT1 monocarboxylate transporter
• PARP inhibitors alone can induce autophagy in tumor stroma, which can be detrimental for breast cancer patients. Thus, by combining autophagy inhibitors with PARP inhibitors, cancer cells can be deprived from utilizing stromal autophagy as a
• breast cancer can be any type of breast cancer, such as noninvasive (in situ) breast cancer and invasive breast cancer.
• breast cancer includes a breast tumor that is Luminal A, Luminal B, triple negative/basal-like or HER2-type, as well as ductal carcinoma in situ (DCIS).
• Breast cancer also includes, for example, neoplasms having a subtype of ER(+), PR(+), HER2(+), triple negative (ER(-)/PR(-)/HER2(-)), ER(-), PR(-), all tumor and nodal stages, and all tumor grades.
• a first and second agent are concurrently administered to a subject if, beginning on the first day of treatment, the first agent is administered once per week for 10 weeks, and the second agent is administered daily for the first, third, fifth and seventh weeks.
• a first and a second agent are concurrently administered to a subject if they are administered in the form of a single composition containing both agents.
• PARP inhibitor i.e., an inhibitor of poly ADP ribose polymerase
• PARP inhibitor shall mean an agent that inhibits PARP more than it inhibits any other polymerase.
• the PARP inhibitor inhibits PARP at least two-fold more than it inhibits any other polymerase.
• the PARP inhibitor inhibits PARP at least 10-fold more than it inhibits any other polymerase.
• the PARP inhibitor inhibits PARP more than it inhibits any other enzyme.
• a pharmaceutical composition typically comprises an active agent and a "pharmaceutically acceptable carrier.”
• Pharmaceutical compositions can be in the form of a liquid (e.g., an injectable), a solid (e.g., a tablet), or an emulsion, for example.
• Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.01-0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions.
• non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
• Aqueous carriers include water, alcoholic/aqueous solutions, emulsions and suspensions, including saline and buffered media.
• Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
• Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as Ringer's dextrose, those based on Ringer's dextrose, and the like. Fluids used commonly for i.v.
• Tablets typically contain excipients such as magnesium stearate, binders such as gelatin or sorbitol, HPMC,
• subject shall mean any animal, such as a human, non-human primate, mouse, rat, guinea pig or rabbit.
• treating a subject afflicted with breast cancer shall mean slowing, stopping or reversing the cancer's progression.
• treating a subject afflicted with breast cancer can mean increasing the subject's progression-free survival ("PFS") by a factor of 1.1 , 1.2, 1.5, 2, or greater than 2.
• PFS progression-free survival
• treating a subject afflicted with breast cancer means reversing the cancer's progression, ideally to the point of eliminating the cancer.
• Therapeutic agents can be administered by any known means including, for example, orally, intravenously, intramuscularly, topically (such as by injection into, or other direct contact with, the tumor) and subcutaneously.
• the two agents administered, i.e., the PARP inhibitor and autophagy inhibitor, can be administered by the same route or different routes.
• This invention provides a method for treating a subject, preferably human, afflicted with breast cancer, comprising concurrently administering to the subject (i) a PARP inhibitor and (ii) an autophagy inhibitor, wherein the amounts of the PARP inhibitor and autophagy inhibitor, when concurrently administered, are therapeutically effective.
• the amount of PARP inhibitor administered would be therapeutically effective even if it were administered without the autophagy inhibitor. In another embodiment, the amount of autophagy inhibitor administered would be therapeutically effective even if it were administered without the PARP inhibitor. In a further embodiment, the amount of PARP inhibitor administered would not be therapeutically effective if it were administered without the autophagy inhibitor. In yet a further embodiment, the amount of autophagy inhibitor administered would not be therapeutically effective if it were administered without the PARP inhibitor.
• the PARP inhibitor can be any agent that inhibits PARP.
• the PARP inhibitor is olaparib, rucaparib, veliparib, CEP 9722, MK 4827, BMN-673 or 3- aminobenzamide.
• the PARP inhibitor can be administered, for example, according to any dosing regimen, known or otherwise, that would be appropriate for breast cancer treatment in conjunction with an autophagy inhibitor.
• the PARP inhibitor is administered once or twice weekly, for one or more cycles of three weeks or more.
• the PARP inhibitor is administered once or twice daily, for one or more cycles of three weeks or more.
• the autophagy inhibitor can be any agent that inhibits autophagy.
• the autophagy inhibitor is chloroquine, N-acetyl-L-cysteine, L-asparagine, bafilomycin Al , catalase, DBeQ, E-64d protease inhibitor, or GMX1778.
• the autophagy inhibitor is chloroquine, which is preferably administered orally.
• the autophagy inhibitor can be administered, for example, according to any dosing regimen, known or otherwise, that would be appropriate for breast cancer treatment in conjunction with a PARP inhibitor.
• chloroquine is administered in tablet form at the dosing regimen prescribed for malarial suppression or for treating an acute malarial attack.
• the chloroquine is administered according to one of the following dosing regimens: (i) 500mg chloroquine phosphate once per week; (ii) 5mg chloroquine phosphate per kg body weight per week; and (iii) 5mg chloroquine phosphate per kg body weight every 12 hours for a cycle of at least two days, three days, four days, five days, six days, seven days, or more.
• the inhibitors can be administered in the form of one or more pharmaceutical compositions. In one embodiment, the inhibitors are administered in the form of one pharmaceutical composition. In another embodiment, the inhibitors are administered in the form of two pharmaceutical compositions, each with its own dosing regimen.
• This invention also provides a method for inducing the death of a breast cancer cell (preferably a human cell), comprising concurrently contacting the cell with (i) a PARP inhibitor and (ii) an autophagy inhibitor, wherein the amounts of the PARP inhibitor and autophagy inhibitor, when concurrently contacted with the cell, are effective to induce the cell's death.
• a breast cancer cell preferably a human cell
• the PARP and autophagy inhibitors can be any agents that inhibit PARP or autophagy, respectively.
• the PARP inhibitor is olaparib, rucaparib, veliparib, CEP 9722, MK 4827, BMN-673, or 3-aminobenzamide
• the autophagy inhibitor is chloroquine, N-acetyl-L-cysteine, L-asparagine, bafilomycin Al , catalase, DBeQ, E-64d protease inhibitor, or GMX1778.
• the autophagy inhibitor is chloroquine.
• the inhibitors can be contacted with the breast cancer cell in the form of one or more
• the inhibitors are contacted with the breast cancer cell in the form of one pharmaceutical composition. In another embodiment, the inhibitors are contacted with the breast cancer cell in the form of two pharmaceutical compositions.
• this invention provides a composition comprising (i) a PARP inhibitor, (ii) an autophagy inhibitor, and (iii) a pharmaceutically acceptable carrier.
• the PARP and autophagy inhibitors can be any agents that inhibit PARP or autophagy, respectively.
• the PARP inhibitor is olaparib, rucaparib, veliparib, CEP 9722, MK 4827, BMN-673, or 3-aminobenzamide
• the autophagy inhibitor is chloroquine, N-acetyl-L-cysteine, L-asparagine, bafilomycin Al, catalase, DBeQ, E-64d protease inhibitor, or GMX1778.
• the autophagy inhibitor is chloroquine.
• PARP inhibitors olaparib (AZD-2281), rucaparib (AG014699, PF-01367338), veliparib (ABT-888), CEP 9722, MK 4827, BMN-673, and 3-aminobenzamide (ABZ).
• Autophagy inhibitors chloroquine, N-acetyl-L-cysteine (NAC), L-asparagine (L-Asp), bafilomycin Al (Baf), catalase, DBeQ, E-64d protease inhibitor (E-64d), and GMX1778.
• iniparib is envisioned for use in lieu of olaparib, mutatis mutandis, in each of the subject methods and compositions.
• shBRCAl fibroblasts showed a significant increase in cell proliferation and an elevation of both autophagy and mitophagy markers.
• shBRCAl fibroblasts expressed elevated levels of HIF- ⁇ . This was accompanied by a decrease in succinate dehydrogenase B (SDHB) expression levels.
• SDHB succinate dehydrogenase B
• hTERT-immortalized human fibroblasts hTERT-BJl cells
• shBRCAl short hairpin RNA's targeting BRCAl
• FIG 1 shows that only one shBRCAl construct (designated as #2) effectively knocked-down BRCAl expression.
• BRCA1 knock-down accelerates fibroblasts proliferation.
• shBRCAl fibroblasts were plated in a 12-well dish. For 10 days, cells were trypsinized and counted using a hemocytometer chamber. At day 10, shBRCAl fibroblasts demonstrated a 5-fold increase in cell number, as compared to control fibroblasts ( Figure 2).
• shBRCAl fibroblasts display higher levels of autophagy and mitophagy markers.
• knocking-down BRCA1 induces autophagy [22-24].
• shBRCAl fibroblasts displayed an increase in the autophagy marker (LC3) and the mitophagy marker (BNIP3) ( Figure 3A).
• shBRCAl fibroblasts overexpressed BNIP3 and LAMP 1 (autophagy marker), when co- cultured with a triple negative breast cancer cell line (namely, MDA-MB-231 cells) (Figure 3B).
• shBRCAl fibroblasts express HIF-la, under chemical pseudo-hypoxia.
• BRCA1 functions mainly as a DNA guardian, it is also important in maintaining the integrity of mitochondria [22, 29].
• BRCA1 is mainly present in the cell nucleus
• phosphorylated BRCA1 has also been found to be localized in the mitochondrial matrix [30]
• succinate dehydrogenase B (SDHB, mitochondrial complex II subunit) was found to be a hot spot gene that frequently showed loss of heterozygosity and allelic instability in cancer stroma [31].
• shBRCAl fibroblast lysates showed a down-regulation of SDHB expression (Figure 4B).
• ketone body generation was measured in the conditioned media of control and shBRCAl fibroblasts.
• Figure 6A shows that ketone body generation is increased up to -5.5 -fold in shBRCAl fibroblasts, relative to control cells.
• shBRCAl fibroblasts markedly increased tumor growth by ⁇ 2.2-fold, as compared with control fibroblasts ( Figure 7). This study highlights the potential lethality of a BRCA1- deficient tumor stroma.
• BRCA1 expression was successfully knocked-down using a lentiviral shRNA approach in hTERT-immortalized fibroblasts.
• shBRCAl fibroblasts displayed increased proliferation.
• shBRCAl fibroblasts display an increase in markers of autophagy (LAMP1 and LC3) and mitophagy (BNIP3).
• shBRCAl fibroblasts show an increase in HIF- ⁇ expression, when treated with chemical proteasome inhibitors to inhibit HIF-1 a degradation.
• SDHB succinate dehydrogenase subunit B
• PLD prolyl-4-hydroxylase
• this current study demonstrates the potential lethal effects of a BRCA1- deficient tumor stroma on breast cancer tumor growth.
• BRCAl is a homologous recombination (HR) protein that is involved in the repair of DNA double strand breaks (DSB's).
• HR homologous recombination
• NHEJ non-homologous end joining
• PARP poly (ADP-ribose) polymerase
• the PARP enzyme is responsible for base excision repair (BER) in single strand breaks (SSB's).
• BRCAl -mutation carriers have heterozygous BRCAl mutations in normal tissue, while tumor cells display homozygous BRCAl mutations after loss of heterozygosity (LOH) [31].
• LHO heterozygosity
• Antibodies used in this paper were purchased from commercial sources: anti-BRCAl (Santa Cruz, C-20, SC-642), anti-BNIP3 (abl0433, Abeam), anti-LC3A/B (Ab58610, Abeam), anti- HIF-1 a (NB 100- 123, Novus), anti-beta actin (A5441 , Sigma), anti-SDHB (NBP-54154, Novus), and anti-LAMPl (Santa Cruz, SC-17768).
• Other reagents were purchased as follows; Hoechst- 33258 nuclear stain was from Sigma, Anti-fade reagent (S2828) was from Invitrogen, MG-132 purchased from Calbiochem and DMOG was from Cayman Chemical.
• MDA-MB-231 cells a human triple-negative breast cancer cell line
• hTERT-immortalized human fibroblasts hTERT-BJl
• DMEM fetal bovine serum
• hTERT-immortalized fibroblasts were transduced by lentivirus particles in the presence of 5 ⁇ g/ml of Polybrene (Santa Cruz Biotech). Transduced cells were then selected with 1.5 ⁇ g/ml of puromycin.
• fibroblasts were plated in 10 cm dishes. The next day, the media was discarded and replaced with complete DMEM, supplemented with 10 ⁇ MG-132 (a proteasome inhibitor) plus ImM dimethyloxallyl glycine (DMOG, a stabilizer of HIF-1 a) for 5 hours. Then, the cells were lysed by scraping into lysis-buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100 and 60 mM n-octylglucoside), containing protease inhibitors (Boehringer Mannheim, Indianapolis, IN).
• lysis-buffer 10 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100 and 60 mM n-octylglucoside
• MDA-MB-231 cells and hTERT-fibroblasts were plated on coverslips in 12-well plates for 48 hours in DMEM supplemented with 10% Nu serum. Then, the cells were rinsed with PBS with 0.1 mM CaC12 and ImM MgC12 (PBS/CM), and fixed with 2% paraformaldehyde in PBS/CM for 30 minutes. After fixation, cells were washed three times with PBS/CM, and permeablized with IF buffer (PBS/CM with 0.1 % Triton-Xl OO and 0.2% bovine serum albumin) for 10 minutes.
• IF buffer PBS/CM with 0.1 % Triton-Xl OO and 0.2% bovine serum albumin
• hTERT-BJl fibroblasts (3 x 10 5 cells) with tumor cells (MDA-MB-231 ; 1 x 10 6 cells) in 100 ⁇ of sterile PBS were co-injected into the flanks of athymic NCr nude mice (NCRNU; Taconic Farms; 6-8 weeks of age). Mice were then sacrificed at 31 days post- injection; tumors were excised to determine their weight.
• MCT4 is a marker of oxidative stress in cancer-associated fibroblasts. Cell Cycle; 10: 1772-83.
• poly(ADPribose) polymerase enhances cell death and improves tumor growth delay in irradiated lung cancer models. Clin Cancer Res 2007; 13 :3033-42.

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