ES2695449A1 - Method for designing personalized therapy for an individual suffering from lung cancer and treated with cisplatin - Google Patents

Abstract

Method for designing a personalized therapy for an individual suffering from lung cancer who has been treated with cisplatin. The present invention refers to an in vitro method for designing a personalized therapy for an individual suffering from lung cancer and who is going to be treated with cisplatin, which comprises determining the level of expression of the PGC-1alpha gene before and after treatment with cisplatin. where if the expression level of said gene is higher after than before the treatment, then the therapy comprises the administration of a second treatment based on the combination of cisplatin with a compound aimed at decreasing the OXPHOS function.

Description

DESCRIPTION

Method to design personalized therapy for an individual suffering from lung cancer and treated with cisplatin

The present invention relates to an in vitro method for designing personalized therapy for an individual suffering from lung cancer who is to be treated with cisplatin comprising determining the expression level of the PGC-1alpha gene before and after treatment with cisplatin , where if the level of expression of said gene is greater after than before treatment, then the therapy comprises the administration of a second treatment based on the combination of cisplatin with a compound directed to decrease the OXPHOS function. Therefore, the present invention is encompassed within the field of cancer treatment, more specifically, in the treatment of lung cancer.

BACKGROUND OF THE INVENTION

Lung cancer is the most widespread cancer throughout the world in terms of incidence and is the leading cause of death due to cancer. In the majority of cases, cisplatin-based chemotherapy is the standard treatment. However, it is common for resistance to develop during treatment, limiting the clinical usefulness of this drug. The mechanisms of resistance of cisplatin are complex, because they involve various strategies and metabolic pathways. In recent years, an increase in oxidative metabolism has been suggested as the mechanism of common action for many cancers and resistance to treatment.

Approximately 85% to 90% of diagnoses of lung cancer are non-small cell lung cancer or NSCLC (from their initials in English, Non-Small Cell Lung Cancer). The current classification of NSCLC takes into account molecular alterations that may influence the therapeutic decision. However, up to now, there is no pharmacopoeia adapted to the mutations that show the majority of patients suffering from NSCLC and therefore, platinum-based chemotherapy is still the standard therapy of choice.

Additionally, although lung cancer treatments have progressed from Significantly for some time now, and even more recently with the appearance of immunotherapy, its cost-effectiveness ratio compared to cisplatin-based treatments is still a challenge to overcome. Therefore, cisplatin is one of the main drugs used in the treatment of NSCLC. Unfortunately, it is common to develop chemo resistance during the treatment, or the treatment has to be discontinuous due to the platinum toxicity which limits the clinical utility of this drug.

The mechanisms of resistance to cisplatin are complex. Although an altered energetic metabolism is one of the hallmarks of cancer, little is known about its role in chemo resistance. Mitochondria are organ systems known to be the central energy of the cell, responsible for oxidative phosphorylation or OXPHOS (from its initials in English oxidative phosphorilation). In this process, acetyl CoA from glycolysis or oxidation of fatty acids feeds the cycle of tricarboxylic acids (TCA) that generates reduced cofactors. These cofactors transfer electrons to the OXPHOS system that, through a chain of redox reactions, reduces the oxygen to water. Some of these reactions produce reactive species of oxygen (or ROS, for its acronym in English Reactive Oxigen Species). Simultaneously during this process, the protons are pumped into the intermembrane space generating what is known as the mitochondrial inner membrane potential (MIMP ) which is finally dissipated through the V complex generating ATP. The classic view of tumor metabolism is known as the Warburg effect. According to this theory, the tumor cells increase their aerobic glycolysis by reducing the OXPHOS function with the aim of increasing the provision of metabolic intermediates used in anabolic processes. However, under some circumstances, tumor cells have the ability to adapt their metabolism to different environments and treatments, increasing their adaptability and resistance of the tumor to therapies.

Therefore, there is a need in the state of the art to develop new therapeutic strategies that overcome all these drawbacks, and thus improve! the effectiveness of the treatment administered to the patient.

DESCRIPTION OF THE INVENTION

The inventors have found that, in lung cell lines, the expression levels of the PGC-1alpha gene are increased in response to treatment with cisplatin, resulting in the cell becoming resistant to this drug, but surprisingly, said resistance it is reversed when a compound directed to diminish the OXPHOS function is administered, such as, for example, metformin.

Therefore, this discovery opens a new therapeutic window to the treatment of lung cancer, in particular, lung cancer that is resistant to treatment with cisplatin, because it allows direct treatment to the special characteristics of the individual, avoiding the administration of ineffective therapies. Therefore, based on this discovery, a series of inventive aspects have been developed that will be described below.

Method of the invention

In view of the foregoing, in one aspect the present invention relates to an in vitro method for designing personalized therapy to an individual suffering from lung cancer and to be treated with cisplatin, hereinafter "method of the invention". ", Which comprises

(a) determining the level of expression of the PGC-1alpha gene before and after treatment with cisplatin in an isolated biological sample from said individual, and

(b) comparing the level of expression obtained in step (a) before treatment with the level of expression after treatment,

wherein an expression level of PGC-1alpha after treatment greater than the expression level of PGC-1alpha before treatment is indicative of therapy comprises a second administration of cisplatin in combination with a compound directed to decrease the OXPHOS function.

In the present invention, "therapy" or "treatment" means the administration to an individual of a compound, or combination of compounds, for the purpose of inhibiting a disease or pathological condition, that is, stopping its development; alleviate a disease or pathological condition, that is, cause the regression of the disease or pathological condition; and / or stabilize a disease or pathological condition in an individual. In the present invention, the disease or pathological condition is lung cancer, in particular, non-small cell lung cancer (NSCLC ). In another particular embodiment, lung cancer is metastatic, more in particular, NSCLC in the metastatic state.

In the present invention, "lung cancer" is understood to mean that disease, or group of diseases, resulting from the uncontrolled growth of the cells of the respiratory tract, in particular, of lung tissue In the context of the present invention, the term " Cancer "also includes the concept of" tumors ", I understand these as an aggregate set of cells resulting from the uncontrolled proliferation of a single cell.The great majority of the types of lung cancer are carcinomas, that is, malignant tumors that are born of epithelial cells There are two forms of lung carcinoma, categorized by the size and appearance of the malignant cells seen histopathologically under a microscope: non-small cell and small cell tumors.Asl, examples of lung cancer include, without limitation a, adenocarcinomas, squamous cell carcinomas, large cell carcinomas and small cell carcinomas. bronchioalveolar carcinomas and several mixed forms.

Also, lung cancer can be metastasic or non-metastatic. In the present invention, "metastatic lung cancer" is understood to mean that stage of cancer development in which cells of the cancer-causing respiratory tract, in particular, cancer-causing lung cells, invade the blood stream of the individual, reaching the lymph nodes. , and colonizing other tissues than the tissue of origin.

It is considered that the therapy is "personalized" when the compound (s) to be administered to the individual to treat a disease that is specially adapted to the genotypic and phenotypic characteristics of the individual to be treated, avoiding Thus, the loss of time in non-effective therapies In the present invention, the characteristic that determines the therapy that is going to be administered to the individual is the level of expression of the PGC-1alpha gene, as will be explained later, also other values can be measured, such as the mitochondrial inner membrane potential and / or the levels of the reactive oxygen species.

In the present invention the term "individual" is equivalent to the term "subject", so both terms can be used interchangeably throughout the present description. It is understood by "individual", any animal belonging to any species. However, in a particular embodiment, the subject is a mammal, preferably a primate, more preferably, a human being of any race, sex or age. The individual subject of the present invention suffers from lung cancer and will be treated with cisplatin as a first treatment.

The method of the invention comprises in a first step, step a), determining the level of expression of the PGC-1alpha gene before and after treatment with cisplatin in an isolated biological sample from said individual.

The PGC-1alpha gene is a gene that, in humans, is located on chromosome 4p15.2. Alternative names that can be used in the scientific literature to refer to the PGC-1alpha gene include, but are not limited to, " peroxisome proliferative activated receptor, gamma, coactivator 1", " peroxisome proliferative activated receptor, gamma, coactivator 1, alpha", "peroxisome proliferator-activated gamma receptor, coactivator 1 alpha", "PPARG coactivator 1 alpha ", PPARGC1A, LEM6, PGC-1v, PGC1, PGC1A and PPARGC1. In a particular embodiment, the PGC-1alpha gene comprises a sequence of nucleotides with a sequence identity of at least 80, 85, 90, 95, 96, 97, 98, 99% with the nucleotide sequence SEQ ID NO: 1 (access number to GenBank AF106698 version AF106698.1). In another particular embodiment, the PGC-1alpha gene comprises a nucleotide sequence with a sequence identity of 100% with the nucleotide sequence SEQ ID NO: 1.

In the present invention, "sequence identity" is understood to be the degree of similarity between two nucleotide (or amino acid) sequences obtained by aligning the two sequences. Depending on the number of common residues between the aligned sequences, a degree of identity expressed as a percentage will be obtained. The degree of identity between two nucleotide (or amino acid) sequences can be determined by conventional methods, for example, by standard algorithms of sequence alignment known in the state of the art, such as BLAST [Altschul SF et al. Basic local alignment search tool. J Mol Biol.

1990 Oct 5; 215 (3): 403-10]. BLAST programs, for example, BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, are public domain on the National Center for Biotechnology Information (NCBI) website.

The PGC-1alpha gene encodes the PPAR gamma coactivator-1 protein. In a particular embodiment, the protein encoded by the PGC-1alpha gene comprises a sequence of amino acids with a sequence identity of at least 80, 85, 90, 95, 96, 97, 98, 99% with the sequence SEQ ID NO: 2 (access number to GenBank NP_037393 version NP_037393.1). In another particular embodiment, the protein encoded by the PGC-1alpha gene comprises an amino acid sequence with a sequence identity of 100% with the sequence SEQ ID NO: 2. The term "sequence identity" has been previously defined.

The person skilled in the art understands that mutations in the nucleotide sequence of the genes that give rise to conservative amino acid substitutions in non-critical positions for the functionality of the protein are evolutionarily neutral mutations that do not affect its overall structure or functionality , giving rise to variants of the protelna. Said variants fall within the scope of the present invention, that is, those variants of the protein encoded by the PGC-1alpha gene that present insertions, deletions or modifications of one or more amino acids with respect to the sequence SEQ ID NO: 2.

In the present description, the terms "expression" and "gene expression" include the transcription and / or translation of the nucleic acid, therefore, the quantification of the expression of the PGC-1alpha gene can be determined from the nucleic acid of the PGC gene. -1alpha or of the protein encoded by said gene, that is, of the PPAR gamma coactivator-1 protein.

Thus, in a particular embodiment of the method of the invention, the determination of expression levels of the PGC-1alpha gene comprises measuring the level of cDNA, or a fragment thereof, the level of mRNA, or a fragment thereof, and / or the level of the protein encoded by said gene, or a fragment of said protein.

In the present invention, "mRNA fragment" or "cDNA fragment" is understood to mean the nucleotide sequence of the PGC-1alpha gene comprising one or more nucleotides absent from the 3 'and / or 5' ends with respect to the sequence of complete nucleotides of the PGC-1alpha gene. In a particular embodiment, the PGC-1alpha gene fragment is a fragment of the sequence SEQ ID NO: 1.

Analogously, in the present invention "fragment of the PPAR gamma coactivator-1 protein" is understood to mean the amino acid sequence of the PPAR gamma coactivator-1 protein comprising one or more amino acids absent from its amino terminal or terminal carboxyl end. with respect to the complete amino acid sequence of the PPAR gamma coactivator-1 protein. In a particular embodiment, the fragment of the PPAR gamma coactivator-1 protein is a fragment of the sequence SEQ ID NO: 2.

If the quantification of the expression of the PGC-1alpha gene is to be performed from the cDNA or the mRNA, first the extraction of the nucleic acid from the biological sample isolated from the subject is necessary. For this purpose, the biological sample can be treated to physically or mechanically disintegrate the structure of the tissue or cell, releasing the intracellular components in an aqueous or organic solution to isolate and prepare the nucleic acids. The nucleic acids are extracted by methods known to the person skilled in the art and commercially available (Sambroock, J., et al., 2012, " Molecular cloning: a Laboratory Manual", 4th ed., Cold Spring Harbor Laboratory Press, NY, Vol. 1-3.).

Once the nucleic acid has been extracted, the expression of the PGC-1alpha gene is quantified. Practically any conventional method can be used within the framework of the invention to detect and quantify the mRNA levels of the PGC-1alpha gene or its corresponding cDNA. By way of illustration, not limitation, the levels of mRNA of said gene can be quantified by the use of conventional methods, for example, methods comprising the amplification of the mRNA and the quantification of the product of the amplification of said mRNA, such as electrophoresis and staining, or alternatively, by Southern blot and use of appropriate probes, northern blot and use of specific probes of the mRNA of the gene of interest ( PTGDR gene ) or of its corresponding cDNA, mapping with nuclease S1, RT-PCR, hybridization, microarray , etc.

Analogously, the cDNA levels corresponding to said mRNA of the PGC-1alpha gene can also be quantified by employing techniques conventional in this case, the method of the invention includes a step of synthesizing the corresponding cDNA by reverse transcription (RT) of the corresponding mRNA followed by amplification and quantification of the amplification product of said cDNA. Conventional methods of quantifying expression levels can be found, for example, in Sambrook et al. cited ad supra. In a particular embodiment, the quantification of expression levels is performed by means of a quantitative polymerase chain reaction (PCR), an array of DNA or RNA, or RNA-Seq or Mass Sequencing applied to the study of RNA. In another still more particular embodiment, the level of expression of the mRNA of the PGC-1alpha gene is determined by qRT-PCR using the Taqman® gene expression assay (Taqman® gene expression assay) with the probe Hs01016719_m1.

If the quantification of PGC-1alpha gene expression is to be performed from the protein encoded by the PGC-1alpha gene, ie the PPAR gamma coactivator-1 protein, then the biological sample isolated from the subject has to be treated to extract the proteins. Methods for extracting or isolating proteins are known to the person skilled in the art and are commercially available (Sambroock, J., et al.

2012, cited ad supra).

The levels of the PPAR gamma coactivator-1 protein can be quantified by any conventional method that allows to detect and quantify said protein in a sample of a subject. By way of illustration, not limitation, the levels of said protein can be quantified, for example, by the use of antibodies capable of binding to the PPAR gamma coactivator-1 protein and the subsequent quantification of the complexes formed.

"Antibody" means a glycoprotein of the gamma globulin type that is part of the humoral immune system that binds specifically to an antigen. The term "antibody", as used herein, includes monoclonal antibodies, polyclonal antibodies, recombinant fragments of antibodies, combibodies, Fab fragments and scFv antibodies, so! as ligand binding domains. Specific antibodies of the protein encoded by the PGC-1alpha gene are commercially available. Examples of such antibodies include, but are not limited to, antibodies 3G6 # 2178 from Cell Signaling Company, ab54481 from Abcam and H-300 sc-13067 from Santa Cruz Biotechnology.

The antibodies used in these assays may or may not be labeled. The terms "label" or "label" refer to a composition capable of producing a detectable signal indicative of the presence of the labeled molecule. Illustrative examples of suitable labels include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a brand is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. There is a wide variety of known assays that can be used in the present invention, which utilize unlabeled antibodies (primary antibody) and labeled antibodies (secondary antibody); these techniques include Western-blot or Western blot, ELISA (RIA enzyme-linked immunosorbent assay (radioimmunoassay), competitive EIA (competitive enzyme-linked immunosorbent assay), DAS-ELISA (double-antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, employment-based techniques of biochips or protein microarrays that include specific antibodies or assays based on colloidal precipitation in formats such as dipsticks Other ways to detect and quantify the PPAR gamma coactivator-1 protein include affinity chromatography techniques, ligand binding assays, spectrometry However, in a particular embodiment, the quantification of protein levels is done by means of Western blot, ELISA, an array of proteins or a binding study.When an immunological method is used, any antibody or antibody can be used. reagent known to bind to the PPAR gamma coactivator-1 protein with high affinity to detect the amount of it. Examples of antibodies or reagents capable of binding to said PPAR gamma coactivator-1 protein include, but are not limited to, polyclonal sera, supernatants of hybridomas or monoclonal antibodies, antibody fragments, Fv, Fab, Fab 'and F (ab') 2, scFv, diabodies, triabodies, tetrabodies and humanized antibodies. In the market, there are commercial antibodies against the PPAR gamma coactivator-1 protein that can be used in the context of the present invention, such as those cited above.

As it has been shown in previous paragraphs, in order to carry out the first stage of the method of the invention, it is necessary to have a biological sample isolated from the individual under study. In the context of the present invention, the term "biological sample" refers to any biological material that can be obtained from the individual, such as a biopsy, a tissue, a cell or a fluid (serum, saliva, semen, sputum, tears, mucus, sweat, milk, extracts of brain and the like), and which may hold information about the characteristic genetic endowment of a person.In a particular embodiment, the biological sample is blood, serum or tissue from a biopsy.The term "isolated" implies that the biological sample it has been separated or removed from the rest of the components that accompany it naturally. Techniques for obtaining biological samples from an individual are widely known in the state of the art, and any of them may be employed in the practice of the present invention.

Once step (a) has been carried out, that is, the expression levels of the PGC-1alpha gene have been quantified before and after the treatment with cisplatin, the method of the invention comprises a step (b) comprising compare the expression levels obtained between them. If the expression level of PGC-1alpha after treating the individual with cisplatin is greater than the level of expression of PGC-1alpha before treatment with cisplatin, then the therapy to be administered to the individual comprises a second administration of cisplatin in combination with a compound aimed at decreasing the OXPHOS function. Otherwise, the administration of a compound aimed at decreasing the OXPHOS function to continue with the cisplatin-based treatment is not necessary.

In the present invention it is understood that one level of expression is "greater" than another when one value of the level of expression is higher than another.In particular, it is understood that an expression level is "higher" when the expression levels of the PGC-1alpha gene after treatment with cisplatin are at least 1.1 times, 1.5 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or even more with respect to the expression levels of the PGC-1alpha gene before treatment with cisplatin. Depending on the result of said comparison, it can be concluded whether the therapy to be administered to the individual comprises a second administration of cisplatin in combination with a compound directed to decrease the OXPHOS function.

In the present invention, "compound directed to diminish OXPHOS function" is understood as the compound that decreases the capacity to generate energy in the form of ATP by the OXPHOS system, either by decreasing the contribution of reduced cofactors, the transport of electrons, the activity or quantity of some of its complexes or dissipating the generated proton gradient. In a particular embodiment, the compound directed to decrease the OXPHOS function is a biguanide. In the present invention, "biguanide" is understood to mean that compound classified within category A10B of the code ATC (Anatomical, Therapeutic, Qulmica Classification System.) In another still more particular embodiment, biguanide is selected from the group consisting of phenformin , metformin and buformin In another even more particular embodiment, the compound aimed at decreasing the OXPHOS function is metformin.

The administration of cisplatin in combination with a compound directed to diminish the OXPHOS function can be carried out by any of the forms of administration that exist in the state of the art. Examples of routes of administration include, but are not limited to, oral, skin, parenteral, nasal and intraperitoneal. Also, the administration of both compounds can be carried out at the same time or separately. Thus, in a particular embodiment, the administration of cisplatin in combination with the compound aimed at decreasing the OXPHOS function is carried out sequentially, simultaneously or separately.

In the present invention it is understood that the administration is "sequential" when the cisplatin and the compound intended to decrease the OXPHOS function are administered to the individual at different times in time, so that one is administered first and another is subsequently administered. order in which the cisplatin and the compound directed to decrease the OXPHOS function are administered is indifferent.

In the present invention it is understood that the administration is "simultaneous" when the cisplatin and the compound directed to decrease the OXPHOS function are part of the same composition and are administered together, that is, at the same time to the individual.

In the present invention it is understood that the administration is "separate" when the cisplatin and the compound directed to decrease the OXPHOS function are part of different compositions, each of them being administered individually.

As the person skilled in the art understands, "separate" administration can be "simultaneous" if both compositions are administered at the same time.

Additionally, the method of the invention may comprise additional steps aimed at determining other parameters useful for designing personalized therapy for an individual suffering from lung cancer and who is going to be treated with cisplatin. Among these parameters are the internal mitochondrial membrane potential (MIMP) and ROS levels.

Thus, in a particular embodiment, the method of the invention further comprises measuring the MIMP before and after treatment with cisplatin in an isolated biological sample from said individual, wherein a MIMP after the treatment is greater than the MIMP before the treatment. it is indicative that the therapy comprises a second administration of cisplatin in combination with a compound directed to decrease the OXPHOS function.

In another particular embodiment, the method of the invention further comprises measuring ROS levels before and after treatment with cisplatin in an isolated biological sample from said individual, where ROS levels after treatment are greater than the level of ROS before treatment is indicative that the therapy comprises a second administration of cisplatin in combination with a compound directed to decrease the OXPHOS function.

The reagents, conditions and methods for measuring MIMP and ROS levels are widely known in the state of the art.

In view of what has been explained above, the use of expression levels of the PGC-1alpha gene to design personalized therapy for an individual suffering from lung cancer who is going to be treated with cisplatin is also contemplated within the present invention. .

Therefore, in another aspect, the present invention relates to the in vitro use of the expression level of the PGC-1alpha gene to design personalized therapy to an individual suffering from lung cancer and to be treated with cisplatin. From here on, this inventive aspect will be called "use of the invention".

How to use the expression level of the PGC-1alpha gene to design such personalized therapy has been explained in previous paragraphs. Thus, in a particular embodiment, when the expression level of PGC-1alpha after treatment with cisplatin is greater than the expression level of PGC-1alpha before said treatment, then the therapy comprises a second administration of cisplatin in combination with a compound aimed at decreasing the OXPHOS function.

The terms used in the present inventive aspect, such as level of expression, PGC-1alpha gene, personalized therapy, etc. they have been explained in previous paragraphs and are applicable to the present inventive aspect.

Also, the particular embodiments that have been described for the method of the invention are also applicable to the use of the invention.

In a particular embodiment, the use of the invention further comprises the use of MIMP and / or ROS levels to design personalized therapy for an individual suffering from lung cancer who will be treated with cisplatin. Thus, in another particular embodiment, a MIMP and / or ROS levels after treatment with cisplatin greater than the MIMP and / or ROS levels prior to said treatment, are indicative that the therapy comprises a second administration of cisplatin in combination with a compound aimed at decreasing the OXPHOS function.

In a particular embodiment, the compound directed to decrease the OXPHOS function is a biguanide. In another more particular embodiment, the biguanide is selected from the group consisting of phenformin, metformin and buformin.

In another particular embodiment, the administration of cisplatin in combination with the compound directed to decrease the OXPHOS function is carried out sequentially, simultaneously or separately. The terms "sequential", "simultaneous" or "separate" have been previously defined.

In the context of the present invention, the expression levels of the PGC-1alpha gene can be used to design a therapy for an individual suffering from any type of lung cancer and who will be treated with cisplatin. However, in a particular embodiment, lung cancer is metastatic. In another embodiment In particular, lung cancer is NSCLC. In another still more particular realization the use of the invention, lung cancer is metastatic NSCLC.

In the present invention, any of the molecules representative of the expression levels of the PGC-1alpha gene, such as the cDNA, the mRNA and / or the protein encoded by said gene can be used. All these molecules, as well as explanations on the use of them to determine the expression level of the PGC-1alpha gene, have been described in previous paragraphs of this description. Thus, in a particular embodiment the use of the invention, the level of expression of the PGC-1alpha gene comprises determining the level of cDNA, mRNA and / or protein encoded by said gene.

Additionally, within the inventive aspects included within the present invention, the use of cisplatin combined with a compound directed to diminish the OXPHOS function in the elaboration of a drug for the treatment of lung cancer in an individual that is going to be treated with cisplatin.

Thus, in another aspect, the present invention relates to the use of cisplatin combined in a sequential, simultaneous or separate manner with a compound intended to decrease the OXPHOS function in the manufacture of a medicament for the treatment of lung cancer in an individual who goes to be treated with cisplatin, wherein said individual comprises an expression level of PGC-1alpha after treatment with cisplatin greater than the expression level of PGC-1alpha before treatment.

Particular embodiments of the present inventive aspect comprise:

- the individual also understands MIMP after treatment greater than MIMP before treatment and / or ROS levels after treatment greater than ROS levels before treatment;

- Lung cancer is NSCLC and / or metastatic;

- The level of expression of the PGC-1alpha gene corresponds to the level of cDNA, mRNA and / or the protein encoded by said gene;

- The compound aimed at decreasing the OXPHOS function is a biguanide. In another more particular embodiment, the biguanide is selected from the group consisting of phenformin, metformin and buformin.

All of these particular embodiments as well as the expressions and terms employed have been described in previous paragraphs, and are applicable to the present inventive aspect.

Techniques and materials for preparing medicines (excipients, vehicles, galenic forms, formulations, etc.) are widely known in the state of the art and their application is routine practice for the expert in the field. The terms "medicament" and "pharmaceutical composition" are equivalent and may be used interchangeably in the context of the present invention.

In order to implement the present invention it is necessary to have a kit comprising the reagents necessary to determine the expression levels of the PGC-1alpha gene. Therefore, in another aspect, the present invention relates to a kit for designing personalized therapy for an individual suffering from lung cancer who is going to be treated with cisplatin, hereinafter "kit of the invention", which It comprises the reagents necessary to determine the expression levels of the PGC-1alpha gene.

In the context of the present invention, kit is understood as the product containing the reagents necessary to carry out the method of the invention adapted to allow its transport and storage. Suitable materials for packaging the kit components include, but are not limited to, polyethylene, polypropylene, polycarbonate and the like, glass, plastic, etc. The kit can also include bottles, jars, paper, envelopes, etc.

The expression "reactive that allows determining the level of expression of a gene" means a compound or a group of compounds that allows determining the level of expression of a gene, both by means of the determination of the level of cDNA or mRNA and by means of the protelna level. Therefore, reagents of the first type include nucleic acids, eg primers and probes, capable of hybridizing specifically with the mRNA encoded by the gene involved. The reagents of second type are compounds that bind specifically to the protein encoded by the gene and, preferably, the antibodies are included although they may be specific aptamers.

Thus, in a particular embodiment, the reagents necessary to determine the expression levels of the PGC-1alpha gene comprise

- a nucleic acid that hybridizes in a specific manner with the PGC-1alpha gene, or with a fragment thereof, and / or

- antibodies that specifically recognize the protein encoded by the PGC-1alpha gene, or a fragment thereof.

An example of a nucleic acid that hybridizes in a specific manner with the PGC-1alpha gene is, for example, a probe. In the present invention, "probe" is understood to mean the nucleic acid molecule whose nucleotide sequence hybridizes in a specific manner with the nucleotide sequence of a target gene. In the present invention, the target gene is the PGC-1alpha gene. The expression "specifically hybridized", as used herein, refers to conditions that allow the hybridization of two polynucleotides in highly or moderately stringent conditions. High stringency conditions generally involve: (1) low ionic strength and high temperature for washing, for example 0.015M sodium chloride / 0.0015M sodium citrate / 0.1% sodium dodecyl sulfate at 50 ° C , (2) use during the hybridization of a denaturing agent, such as formamide, for example, 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1 % polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 with sodium chloride at 750 mM, 75 mM sodium citrate at 42 ° C, or (3) use of 50% formamide, 5xSSC (0.75 M) NaCl, 0.075 M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 times Denhardt's solution, salmon sperm DNA sonicate (50 mg / mL), 0.1 % SDS, and 10% dextran sulfate at 42 ° C, washed at 42 ° C in 0.2xSSC (sodium chloride / sodium citrate) and 50% formamide, followed by a high-level wash consisting of in 0.1xSSC containing EDTA at 55 ° C. "Moderately stringent conditions" include the use of wash solution and less stringent hybridization conditions than those described above. How to carry out the hybridization of two nucleotide sequences can be found in Sambroock, J., et al. 2012, cited ad supra.

In the case that the expression levels of the PGC-1alpha gene are determined by measuring the levels of protein encoded by said gene, the kit of the invention comprises reagents that are capable of specifically binding said protein, such as antibodies. . Examples of antibodies that can specifically recognize the protein encoded by the PGC-1alpha gene and bind to it have been described in previous paragraphs.

In another particular embodiment, the PGC-1alpha gene comprises a sequence of nucleotides with a sequence identity of at least 80, 85, 90, 95, 96, 97, 98 or 99% with the sequence SEQ ID NO: 1. In another particular embodiment, the PGC-1alpha gene comprises a sequence of nucleotides with a sequence identity of 100% with the sequence SEQ ID NO: 1.

Additionally, the kit of the invention may comprise reagents directed to determine other parameters useful for designing personalized therapy to an individual suffering from lung cancer and to be treated with cisplatin. As indicated above, among these parameters are the MIMP and ROS levels. Thus, in a particular embodiment, the kit further comprises reagents for measuring MIMP and / or ROS levels.

Finally, in another aspect, the invention is directed to the in vitro use of the kit of the invention to design personalized therapy for an individual suffering from lung cancer who is to be treated with cisplatin.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be apparent in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Generation of lines resistant to CDDP (cisplatin). After the continued treatment of cell lines A549 (A), H1299 (B) and H460 (C) with 5 pM CDDP, they got resistant versions. The graphs represent the percentage of viable cells (Y-axis) after 48 hours of different treatments with CDDP (X-axis). The data are the average of at least three different experiments and are represented as a percentage relative to the untreated cells. The error bars indicate the standard deviation. The p-value of the Student t-test was considered statistically significant when it was less than 0.05 (* = P <0.05, ** = P <0.01, *** = p <0.001).

Figure 2 : Metabolic reprogramming in lines resistant to CDDP. The mitochondrial inner membrane potential (A) and the total ROS levels (B) were measured by flow cytometry with the fluorescent probes TMRE and DCFHDA respectively. The data are means of at least three different experiments and are represented as percentages relative to their parental line. The error bars indicate standard deviation. (C) The bar graph represents the relative levels of mRNA for the PGC-1alpha gene compared to the parental lines. The error bars indicate the standard deviation. The p-value of the Student t test was considered statistically significant when it was less than 0.05 (* = P <0.05, ** = P <0.01, *** = P <0.001).

Figure 3 : Changes in MIMP and ROS in early response to treatment with CDDP. The parental lines resistant to CDDP were treated with 5 pM CDDP for 24 hours and their MIMP (A) and ROS (B) levels were evaluated by flow cytometry with the fluorescent probes TMRE and DCFHDA respectively. The results represent the means of at least three independent experiments and are represented as a relative percentage on the untreated parental cells. The error bars indicate the standard deviation. The p-value of the Student t test was considered statistically significant when it was less than 0.05 (* = P <0.05, ** = P <0.01, *** = P <0.001).

Figure 4 : Resistance to CDDP increases sensitivity to metformin. Treatment with metformin reduces MIMP in the cell lines A549 (A) and H1299 (B), independently of their resistance to CDDP. The bars show the means of at least three independent experiments and represent the fluorescence of TMRE. The error bars indicate standard deviation. (C and D) The sensitivity to metformin was evaluated for the parental lines A549 and H1299 as! as for its CDDP resistant versions. The graphs represent the percentage of viable cells (Y-axis) after 48 hours of different treatments with CDDP (X-Axis). The data are the average of at least three different experiments and are represented as a percentage relative to the untreated cells. The error bars indicate the standard deviation. The p-value of the Student t test was considered statistically significant when it was less than 0.05 (* = P <0.05, ** = P <0.01, *** = P <0.001).

Figure 5 : Concomitant treatment of metformin and CDDP in the parental cell lines and resistant to CDDP. Treatment with metformin reduces the increase in MIMP produced by treatment with CDDP in the cell lines A549 (A) and H1299 (B). The sensitivity to different concentrations of metformin in the presence or absence of 5 pM CDDP was evaluated for the parental lines A549 and H1299 (C and D) as! as for their respective versions resistant to CDDP (E and F). Metformin eliminates the differences to CDDP treatment between the parental lines and CDDP-resistant in both cases A549 and H1299 (G and H). The graphs represent the percentage of viable cells (Y-axis) after 48 hours of different treatments with CDDP (X axis). The data are the average of at least three different experiments and are represented as a percentage relative to the untreated cells. The error bars indicate the standard deviation. The p-value of the Student t-test was considered statistically significant when it was less than 0.05 (* = P <0.05, ** = P <0.01, *** = p <0.001).

EXAMPLES

The invention will be illustrated below by means of tests carried out by the inventors, which highlights the effectiveness of the product of the invention.

I. MATERIAL AND METHODS

1.1 Cell lines and reagents.

The cell lines A549 (ATCC® CCL-185 ™), H1299 (ATCC® CRL-5803 ™), and H460 (ATCC® HTB-177 ™) were obtained from the ATCC.

Line A549 comes from a primary tumor of non-small cell lung cancer (NSCLC or NSCLC), while lines H1299 and H460 come from metastasis of NSCLC, more specifically, H1299 cells are derived from lymph node and H460 from a pleural effusion.

All cells were cultured routinely in DM EM ( Dulbecco's Modified Eagle's medium) supplemented with 10% fetal bovine serum, penicillin and streptomycin. Cisplatin was obtained from therapeutic surpluses of the Oncology Medical Service of the Puerta de Hierro Majadahonda Hospital. Metformin was obtained from sigma.

1.2 Cell viability assays.

3x103 cells were seeded in a 96-well multi-well plate and cultured in DMEM until the following day. The following morning the medium was replaced by DMEM containing the different treatments according to the legends of the figures. Cell viability was determined by the commercial kit Cell counting Kit-8 (CCK-8) (Dojindo EU GmBH, Munich, Germany) following the manufacturer's instructions.

1.3 Flow Cytometry

The cytoplasmic ROS were determined using 2 ', 7'-Dichlorofluorescein diacetate (H2DCFHDA, Invitrogen). The internal mitochondrial potential MIMP was determined using Tetramethylrhodamine ethyl ester (TMRE, Invitrogen)

For these tests, 1x105 cells were cultured. After the addition of the fluorophores, (30 pM DCFHDA and 100 nM TMRE) and their incubation for 30 minutes at 37 ° C in the dark, the cells were recovered in DMEM and analyzed immediately on a Cytomic FC500 MPL cytometer (BeckmanCoulter). The size and complexity were used to determine the viable population of the cells, and the average intensity of the fluorescence was determined with the MXP software (BeckmanCoulter). The experiments were performed in duplicate in at least three independent passes.

1.4 Analysis of expression.

RNA from the cells was extracted using the kit "RNeasy mini Kit with DNAse" (Qiagen) and the cDNA was synthesized using the "NZY First-StrandcDNASynthesis Kit" (NZYtech) kit.

The expression levels of PGC-1alpha were assessed by qRT-PCR using the Taqman® expression assay (Hs01016719_m1). TBP levels (Hs00427621_m1) were used as endogenous control (Soes, S., et al (2013), LungCancer 81, 180-186).

1.5 Statistics.

The results were presented as means ± the standard deviation. The Student t test was used for the statistical analysis between the groups and this significant difference was considered when the p-value was less than 0.05 (* = P <0.05, ** = P <0.01, ** * = P <0.001).

II. RESULTS

2.1 Generation of lines resistant to cisplatin.

As a first step in the study of metabolic changes associated with cisplatin resistance in NSCLC, resistant cell lines were obtained.

To do this, starting with three of the most common CPCNP cell lines (A549 primary tumor, H1299 lymph node and H460 pleural effusion), resistant lines were generated by continuous treatment with 5 pM CDDP for three months.

After this selection process, the cells were evaluated for their sensitivity to treatment with cisplatin (Figure 1). As expected, similar to what occurs in patients, the results showed that the continued treatment with CDDP on cell lines led to the generation of an increase in resistance to this treatment for the three cell lines.

Although the curves appear similar, significant differences were observed for the 3 cell lines in the concentration range ranging from 3 to 12 pM CDDP. In addition, these results were obtained in a short period of time, 48 hours, so that at higher exposures, an increase in the differences between the parental and resistant lines would be expected. The following experiments were performed with 5 pM cisplatin, since it is a low concentration, but it allowed to show significant differences for the 3 lines of the study.

2.2 Cell lines resistant to cisplatin show an increase in MIMP and the ROS levels.

Once the three cell models of CDDP resistance were obtained, the metabolic changes associated with this stable increase in resistance were characterized (Figure 2). The mitochondrial internal potential (MIMP) was measured since it is a good reflection of the mitochondrial function and can be easily evaluated by flow cytometry with the fluorescent probe TMRE. The results showed a significant increase of the MIMP for the resistant lines H1299 and H460 compared with their respective parent lines, but not for line A549 (Figure 2A).

On the other hand, the electron transport chain (ETC) is one of the main sources of reactive oxygen species (ROS), therefore the levels of total ROS in our cell lines were evaluated. For this, the fluorescent probe DCFHDA was used. The results show a significant increase close to 20-30% for the three cell lines resistant to CDDP (Figure 2B).

These results were completed with the analysis of expression levels of PGC-lalfa, known to be the main regulator of mitochondrial biogenesis (Figure 2C). The expression levels of PGC-1alpha were significantly increased in the lines resistant to CDDP H1299 and H460. However, no changes were seen in the line A549 resistant to CDDP, similar to what was observed for the MIMP.

2.3 Treatment with cisplatin induces a metabolic reprogramming in NSCLC cell lines

The results obtained with resistant lines led to the question whether these changes could occur as an early response to the drug. For this purpose, the levels of MIMP and ROS were analyzed after 24 hours of treatment with CDDP in the different cell lines (Figure 3).

The results show an increase in MIMP and ROS when the parental lines are treated for 24 hours with 5 pM CDDP. Interestingly for the MIMP, this increase occurs in the three cell lines, although line A549 does not show a stable increase in MIMP.

Similarly, this early change of MIMP and ROS in response to treatment also occurred in the resistant lines, which further increased their parameters.

Therefore, this early adaptation to treatment seems to be common to the parental and resistant lines. However, the relative increase compared to untreated cells is similar for both parental cell lines and CDDP-resistant (although the levels of MIMP and ROS are significantly higher for resistant cell lines).

2.4 Treatment with metformin in lines of CPCNP parental and resistant to CDDP.

In view of the results, it seems clear that a mechanism of resistance to cisplatin would be an increase in MIMP and mitochondrial biogenesis. Deepening this hypothesis, it was investigated whether this group of resistant cells with an increased MIMP could be attacked with a drug that inhibits mitochondrial function.

For these studies we worked with the H1299 cell line since its CDDP-resistant version was the one that showed a greater increase of the MIMP. In addition line A549 was used as a negative control since in this line this phenomenon was not observed.

First it was verified if the drug metformin was able to reduce the MIMP of the cell lines. Treatment with metformin at lines A549 and H1299 decreased its MIMP independently of its resistance to CDDP (Figure 4 A-B).

Secondly, the effect of different concentrations of metformin on the cell viability of the parental lines and resistant to CDDP was evaluated (Figure 4 C-D).

The results show a decrease in cell viability with an increase in metformin concentration. Notably, this effect is much greater in the case of the line H1299 resistant to CDDP compared to its parental line. On the other hand, no differences were observed between line A549 resistant to CDDP and its parental line.

3.5 Concomitant treatment of metformin and CDDP in parental cell lines resistant to CDDP.

Since an increase in MIMP was observed as an early response to treatment With CDDP, it was checked whether joint treatment with metformin could increase the effect of cisplatin. We proceeded in a similar way to the previous section, first we analyzed if metformin was able to block the early increase of MIMP produced by the CDDP (Figure 5 AB).

The results show that metformin is capable of reversing the increase in MIMP induced by cisplatin in the two cell lines A549 and H1299, for the parental and cisplatin resistant versions.

Second, cell viability was measured for all cell lines with different concentrations (0, 0.1, 1, 5, and 10 mM) in the presence or absence of 5 pM CDDP (Figure 5 C-F).

The results showed a decrease in the percentage of viable cells for all cell lines treated with metformin. Notably, this effect is maintained when the cells are treated with the combination metformin plus CDDP, although in some cases a slight protective effect that does not become significant is observed for metformin concentrations lower than 1 mM.

Finally, treatment with metformin eliminates the differences between the parental lines and resistant to CDDP treatment for both cell lines (Figure 5 G-H). The differences between the parental line and the resistant lines are no longer significant from a metformin concentration of 0.1 mM for line A549 and from 1 mM for line H1299.

III. DISCUSSION

Resistant versions of three different cell lines were generated after a continuous treatment with cisplatin for three months. Two out of three cell lines showed stable changes towards an increase in MIMP, although interestingly, all three responded in a similar way to the increase in this parameter as an early response to CDDP treatment.

The results shown here show an increase of PGC-1alpha (activator of the OXPHOS function), precisely in the two cell lines (H1299 and H460) that showed an increase in MIMP.

Supporting the importance of PGC-1alpha in the metabolic change, the cell line A549 resistant to CDDP, without changes observed in the MIMP, did not show an increase in the expression of PGC-1alpha. These results reinforce a stable specific change in the OXPHOS function for CDDP-resistant cell lines of H1299 and H460.

An increase in OXPHOS function can lead to an increase in the levels of superoxide and hydrogen peroxide. The ROS levels measured with DCFHDA were significantly increased. Surprisingly, this increase occurs both as a stable modification in the CDDP-resistant cell lines and also as an early response to the exposure to cisplatin in all cell lines studied.

All these results indicate a process of metabolic reprogramming towards a greater OXPHOS function against the Warburg Effect. Most of the tumor cells use the aerobic glycolysis strategy in spite of their lower energy efficiency (in comparison with the use of the more efficient OXPHOS system) since this "Warburg metabolism" is adapted to sustain an exponential growth. However, as indicated by the results, there is an increase in the function of OXPHOS both in cells treated with CDDP and in cells resistant to CDDP in a stable manner. Thus, this study demonstrates a metabolic reprogramming associated with CDDP resistance in this type of tumors.

The possibility of attacking this group of resistant cells with an enhanced OXPHOS function was investigated by inhibiting mitochondrial function.

In the present study it was demonstrated that the addition of metformin to the cell lines A549 and H1299 decreases its MIMP, most likely by inhibiting the function of complex I independently of its resistance to CDDP (Figure 4A-B) or the presence of concomitant cisplatin (Figure 5A-B). In addition, metformin decreased, in a dose-dependent manner, the viability of the cells proportionally to their OXPHOS requirements. As expected, no differences in viability were observed between the parental and CDDP-resistant A549 cells, since no there were changes in the OXPHOS function for its CDDP-resistant version.

However, there were notable differences for the H1299 line, whose CDDP-resistant version was much more sensitive to metformin. These results again support a specific metabolic reprogramming towards a more oxidative metabolism in H1299 resistant cells, since this line is much more sensitive than its parent to a mitochondrial inhibitor such as metformin.

Metformin caused a reduction in the percentage of viable cells for all cell lines, even in the presence of concomitant cisplatin. However, treatment with metformin eliminated the differences between parental cell lines resistant to cisplatin treatment for cells A549 and H1299. The CDDP-resistant A549 cells, which survived the treatment with cisplatin than the parental cell line, lose these differences when treated with a concentration as low as 0.1 mM metformin. As of this moment, the behavior of both cell lines with metformin treatment was similar.

In the H1299 cell line, its parental version is cisplatin resistant and metformin resistant. On the other hand, the resistant version was sensitive to metformin and less sensitive to cisplatin. Therefore, as the concentration of metformin increases, the graphs come together because CDDP-resistant cells die from the effect of metformin but not by cisplatin (Figure 5F). In contrast, metformin had practically no effect on the parental cell line (as seen in Figure 5D), with the reduction in cell viability being mainly produced by cisplatin.

To conclude, it can be said that an increase in the function of OXPHOS is a mechanism of resistance to treatment with cisplatin in NSCLC, and that this characteristic of cells resistant to cisplatin can be exploited therapeutically by treatment with metformin.

Claims (32)

1. In vitro method for designing personalized therapy for an individual suffering from lung cancer who will be treated with cisplatin comprising (a) determining the level of expression of the PGC-1alpha gene before and after treatment with cisplatin in an isolated biological sample from said individual, and (b) comparing the level of expression obtained in step (a) before treatment with the level of expression after treatment, wherein an expression level of PGC-1alpha after treatment greater than the expression level of PGC-1alpha before treatment is indicative that the therapy comprises a second administration of cisplatin in combination with a compound directed to decrease the OXPHOS function. 2. Method according to claim 1, further comprising measuring the internal mitochondrial membrane potential (MIMP) before and after treatment with cisplatin in an isolated biological sample from said individual, wherein a MIMP after the treatment is greater than the MIMP before of the treatment is indicative that the therapy comprises a second administration of cisplatin in combination with a compound directed to decrease the OXPHOS function. 3. Method according to claim 1 or 2, which further comprises measuring the levels of free oxygen radicals (ROS) before and after treatment with cisplatin in an isolated biological sample from said individual, where ROS levels after treatment greater than the level of ROS before treatment is indicative that the therapy comprises a second administration of cisplatin in combination with a compound directed to decrease the OXPHOS function. Method according to any one of claims 1 to 3, wherein the lung cancer is metastatic. Method according to any one of claims 1 to 4, wherein the lung cancer is lung cancer of non-small cells. Method according to any one of claims 1 to 5, wherein determining the level of expression of the PGC-1alpha gene comprises measuring the level of cDNA, mRNA and / or the protein encoded by said gene. Method according to any one of claims 1 to 6, wherein the biological sample is blood, serum or tissue from a biopsy. The method according to any one of claims 1 to 7, wherein the administration of cisplatin in combination with the compound directed to decrease the OXPHOS function is carried out sequentially, simultaneously or separately. 9. Method according to any of claims 1 to 8, wherein the compound directed to decrease the OXPHOS function is a biguanide. The method according to claim 9, wherein the biguanide is selected from the group consisting of phenformin, metformin and buformin. 11. In vitro use of the expression level of the PGC-1alpha gene to design personalized therapy for an individual suffering from lung cancer who will be treated with cisplatin. 12. Use according to claim 11, which further comprises the use of MIMP and / or ROS levels. 13. Use according to claim 11 or 12, wherein an expression level of PGC-1alpha after treatment with cisplatin greater than the expression level of PGC-1alpha before said treatment is indicative that the therapy comprises a second administration. of cisplatin in combination with a compound aimed at decreasing the OXPHOS function. 14. Use according to claim 12 or 13, wherein a MIMP and / or ROS levels after treatment with cisplatin greater than the MIMP and / or ROS levels prior to said treatment, is indicative that the therapy comprises a second administration of cisplatin in combination with a compound aimed at decreasing the OXPHOS function. 15. Use according to any one of claim 13 or 14, wherein the compound directed to decrease the OXPHOS function is a biguanide. 16. Use according to claim 15, wherein the biguanide is selected from the group consisting of phenformin, metformin and buformin. 17. Use according to any one of claims 13 to 16, wherein the administration of cisplatin in combination with the compound directed to decrease the OXPHOS function is carried out sequentially, simultaneously or separately. 18. Use according to any one of claims 11 to 17, wherein the lung cancer is metastatic. 19. Use according to any one of claims 11 to 18, wherein the lung cancer is lung cancer of non-small cells. 20. Use according to any one of claims 11 to 19, wherein the level of expression of the PGC-1alpha gene comprises determining the level of cDNA, mRNA and / or protein encoded by said gene. 21. Use of cisplatin combined in a sequential, simultaneous or separate manner with a compound intended to decrease the OXPHOS function in the preparation of a drug for the treatment of lung cancer in an individual to be treated with cisplatin, wherein said individual comprises a PGC-1alpha expression level after treatment greater than the level of PGC-1alpha expression before treatment. 22. Use according to claim 21, wherein the individual further comprises a MIMP after treatment greater than the MIMP before treatment and / or ROS levels after treatment, greater than ROS levels before treatment. 23. Use according to claim 21 or 22, wherein the lung cancer is metastatic. 24. Use according to any one of claims 21 to 23, wherein the lung cancer is lung cancer of non-small cells. 25. Use according to any one of claims 21 to 24, wherein the level of expression of the PGC-1alpha gene corresponds to the level of cDNA, mRNA and / or protein encoded by said gene. 26. Use according to any one of claims 21 to 25, wherein the compound directed to decrease the OXPHOS function is a biguanide. 27. Use according to claim 26, wherein the biguanide is selected from the group consisting of phenformin, metformin and buformin. 28. A kit for designing personalized therapy for an individual suffering from lung cancer who will be treated with cisplatin comprising the reagents necessary to determine the expression levels of the PGC-1alpha gene. 29. Kit according to claim 28, wherein the reagents comprise - a nucleic acid that hybridizes in a specific way the PGC-1alpha gene, and / or - antibodies that specifically recognize the protein encoded by the PGC-1alpha gene. 30. Kit according to claim 28 or 29, wherein the PGC-1alpha gene comprises a sequence of nucleotides with a sequence identity of at least 80, 85, 90, 95, 96, 97, 98 or 99% with the sequence SEQ ID NO: 1. 31. Kit according to any one of claims 28 to 30, further comprising reagents for measuring MIMP and / or ROS levels. 32. In vitro use of a kit according to any one of claims 28 to 31 for designing personalized therapy for an individual suffering from lung cancer who will be treated with cisplatin.