WO2019002655A1 - Method for designing a personalised therapy for an individual suffering from cisplatin-resistant lung cancer - Google Patents

Abstract

The present invention relates to an in vitro method for designing personalized therapy for an individual suffering from cisplatin-resistant lung cancer comprising determining the level of expression of the PGC-1alpha gene before and after treatment with cisplatin, wherein if the Expression level of said gene is higher after than before the treatment with cisplatin, then the therapy comprises the administration of a compound directed to decrease the OXPHOS function.

Description

 DESCRIPTION

Method for designing personalized therapy for an individual suffering from cisplatin-resistant lung cancer

The present invention relates to an in vitro method for designing personalized therapy for an individual suffering from cisplatin-resistant lung cancer comprising determining the level of expression of the PGC-1alpha gene before and after treatment with cisplatin, wherein if the level of expression of said gene is higher after than before treatment, then therapy after treatment with cisplatin comprises the administration of a compound directed to decrease 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 in the world in terms of incidence and is the main cause of death due to cancer. In most cases, cisplatin-based chemotherapy is the standard treatment. However, resistance often develops during the treatment, limiting the clinical usefulness of this drug. The mechanisms of resistance of cisplatin are complex, since they involve various strategies and metabolic pathways. In recent years, an increase in oxidative metabolism has been suggested as the common mechanism of 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 its 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, until now, there is no drug 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 significantly for some time, and even more recently with the emergence of immunotherapy, their cost-effectiveness ratio compared with Treatment based on cisplatin 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 treatment, or treatment has to be discontinuous due to platinum toxicity which limits the clinical utility of this drug

The mechanisms of resistance to cisplatin are complex. Although an altered energy metabolism is one of the hallmarks of cancer, little is known about its role in chemo resistance. Mitochondria are organelles known to be the cell's powerhouse, responsible for oxidative phosphorylation or OXPHOS (from their initials in English, oxidative phosphorilation). In this process, acetyl CoA from glycolysis or the oxidation of fatty acids feeds the cycle of tricarboxylic acids (TCA) that generates reduced cofactors. These cofactors transfer electrons to the OXPHOS system, which through a chain of redox reactions reduces oxygen to water. Some of these reactions produce reactive oxygen species (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, tumor cells increase their aerobic glycolysis by reducing the OXPHOS function with the aim of increasing the supply 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 tumor resistance 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 efficacy 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, causing the cell to become resistant to this drug, but, surprisingly, the viability of this cell decreases after the administration of a compound aimed at decreasing OXPHOS function, such as metformin, rotenone or an interfering RNA. PGC-1alpha gene, even reversing said resistance to cisplatin, making the cell sensitive to this drug.

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 to direct the treatment to the special characteristics of the individual, avoiding the administration of ineffective therapies. Thus, 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 above, in one aspect the present invention relates to an in vitro method for designing a personalized therapy to an individual suffering from cisplatin-resistant lung cancer comprising

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

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

wherein a level of PGC-1alpha expression after treatment greater than the level of expression of PGC-1 alpha before treatment is indicative that therapy after treatment with cisplatin comprises the administration of a compound aimed at decreasing 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 the 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 of its acronym in English non-small cell lung cancer). 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 the 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 vast majority of lung cancers are carcinomas, that is, malignant tumors that arise from epithelial cells. There are two forms of lung carcinoma, categorized by the size and appearance of the malignant cells seen histopathologically under a microscope: the tumors of non-small cells and those of small cells. Thus, examples of lung cancer include, without limitation, adenocarcinomas, squamous cell carcinomas, large cell carcinomas and small cell carcinomas. There are also bronchoalveolar carcinomas and several mixed forms.

Also, lung cancer can be metastatic or non-metastatic. In the present invention "metastatic lung cancer" is understood to mean that stage of cancer development in which cancerous respiratory tract cells, in particular, cancerous lung cells, invade the individual's bloodstream, reaching the lymph nodes , and colonizing other tissues than the tissue of origin. The therapy is considered to be "personalized" when the compound (s) to be administered to the individual to treat a disease is specially adapted to the genotypic and phenotypic characteristics of the individual to be treated, avoiding with it the loss of time in ineffective therapies. In the present invention, the characteristic that determines the therapy that is going to be administered to the individual is the expression level of the PGC-1alpha gene. Additionally, as will be explained below, other values such as the mitochondrial inner membrane potential, the levels of the reactive oxygen species and / or the mitochondrial mass can also be measured. In the present invention, it is understood that lung cancer is "resistant to cisplatin" when the number of tumor (or carcinogenic) cells does not decrease in response to the administration of cisplatin as a consequence of the fact that the tumor cells have become progressively resistant to this compound due to the continued administration thereof to the individual suffering from lung cancer. Additionally, in the present invention the criterion followed in clinical practice is also contemplated, where it is considered that an individual presents a lung cancer "resistant to cisplatin" when the individual does not respond to the treatment with cisplatin as established using the response criteria to the treatment of solid tumors (Response Evaluation Criteria In Solid Tumors, RECIST), three-dimensional measurement methods, or both. The RECIST criteria are widely known in the state of the art and are routine practice for the expert in the field.

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-1 alpha gene (PGC-1a) is a gene that, in humans, is located on chromosome 4p15.2. Alternative names that may 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 nucleotide sequence 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-1 alpha 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]. The BLAST programs, for example, BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, are public domain on the website of The National Center for Biotechnology Information (NCBI).

The PGC-1alpha gene encodes the PPAR gamma coactivator-1 protein. In a particular embodiment, the protein encoded by the PGC-1alpha gene comprises an amino acid sequence 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-1 alpha 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 at non-critical positions for the functionality of the protein are evolutionarily neutral mutations that do not affect its overall structure or its functionality , resulting in protein variants. 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-1alpha gene or from the protein encoded by said gene, that is, from the PPAR gamma coactivator-1 protein. Thus, in a particular embodiment of the method of the invention, the determination of the 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 fragment of the PGC-1alpha gene is a fragment of the sequence SEQ ID NO: 1.

Analogously, in the present invention "fragment of the PPAR gamma coactivator-T protein is understood to be the amino acid sequence of the PPAR gamma coactivator-1 protein comprising one or more amino acids absent from its amino terminal or carboxyl terminal end with 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 will be made from the cDNA or mRNA, first it is necessary to extract the nucleic acid from the biological sample isolated from the subject.To this end, the biological sample can be treated to physically or mechanically disintegrate the structure of the tissue or the cell, releasing the intracellular components in an aqueous or organic solution to isolate and prepare the nucleic acids. aen 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 is extracted, the quantification of PGC-gene expression is performed Almost any conventional method can be used within the framework of the invention to detect and quantify the levels of mRNA of the PGC-1alpha gene or of its corresponding cDNA.Finally, non-limiting, mRNA levels 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 amplification product of said MRNA, such as electrophoresis and staining, or alternatively, by means of 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, microarrays, etc.

Similarly, the levels of the cDNA corresponding to said mRNA of the PGC-1alpha gene can also be quantified by the use of conventional techniques; 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 the expression levels is carried out 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-1 alpha 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 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 formed complexes.

"Antibody" means a glycoprotein of the gamma globulin type that forms part of the humoral immune system that binds specifically to an antigen. The term "antibody", as used herein, includes monoclonal antibodies, polyclonal antibodies, recombinant antibody fragments, combibodies, Fab fragments and scFv antibodies, as well as the ligand binding domains. Antibodies specific for 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. Also, the methods for producing antibodies are widely known in the state of the art.

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 trademark 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, and techniques based on 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 carried out 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 that is 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 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 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, 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, brain extracts and the like), and that can 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 has been separated or extracted from the rest of the components that accompany it naturally. Techniques for obtaining biological samples from an individual are well known in the state of the art, and any of them can 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 levels of expression obtained between them. If the level of expression of PGC-1alpha after treating the individual with cisplatin is greater than the level of expression of PGC-1 alpha before treatment with cisplatin, then the therapy to be administered to the individual comprises the administration of a compound directed to decrease the OXPHOS function. Otherwise, the administration of a compound aimed at decreasing the OXPHOS function 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 the administration of a compound directed to diminish the function OXPHOS.

In the present invention, "compound aimed at decreasing OXPHOS function" is understood as the compound that decreases the ability to generate energy in the form of ATP by the OXPHOS system (the oxidative phosphorylation system that through a series of protein complexes). that act in chain finishes producing molecules of ATP), or through to diminish the contribution of reduced cofactors, the transport of electrons, the activity or quantity of some of their complexes or to dissipate the proton gradient generated. In a particular embodiment, the compound directed to decrease the OXPHOS function is an inhibitor of the expression of the PGC-1alpha gene, or of the protein encoded by said gene, or is a compound that inhibits the complex I of the mitochondrial electron transport chain , preferably, a biguanide or rotenone. Both the biguanides and the rotetone are compounds that inhibit the complex I or NADH dehydrogenase or NADH: ubiquinone oxidoreductase of the mitochondrial electron transport chain.

In the present invention, "inhibitor of PGC-1 alpha gene expression" is understood as that compound capable of reducing, blocking or preventing the transcription and / or translation of the PGC-1 alpha gene. Analogously, "inhibitor of the protein encoded by the PGC-1 alpha gene" is understood to mean that compound that is able to reduce, block or prevent the activity or function of the protein encoded by said gene, that is, the protein PPAR gamma coactivator-1. As previously explained, the function of PGC-1alpha is to act as a transcriptional cofactor that favors the transcription of various genes related to the antioxidant response and the OXPHOS function. Examples of compounds capable of inhibiting the protein encoded by the PGC-1alpha gene include, but are not limited to, anti-PPAR gamma coactivator-1 antibodies as described above. An assay to determine whether a given compound is an inhibitor of the activity or function of the protein encoded by the PGC-1 alpha gene is, for example, and without excluding others, the measurement of expression levels of genes regulated by PGC -1alpha and involved in the antioxidant response and OXPHOS function, such as the genes SOD2, UCP-2, Prx5, TFAM, TOM20, and any of the 13 mitochondria, such as MT-C01, MT-C02, MT-C03, MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MT-ATP6, MT-ATP8 and MT-CYTB, among others. Methods for measuring the expression levels of a gene have been explained in the present description. In a particular embodiment, the inhibitor of PGC-1alpha gene expression is an interfering RNA. In another still more particular embodiment, said interference RNA comprises the sequence CCUGUUUGAUGACAGCGAAtt (SEQ ID NO: 3).

In the present invention, "compound that inhibits complex I of the mitochondrial electron transport chain" is understood to mean that compound that is capable of blocking, decreasing, preventing or stopping mitochondrial electron transport at the level of complex I or NADH dehydrogenase, so that the cell is impaired or hindered its ability to produce ATP. Examples of assays useful for determining whether a given compound is capable of inhibiting complex I of the electron transport chain include, without limitation, the measurement of mitochondrial membrane potential or the production of ATP. In the present invention, "biguanide" is understood to mean that compound classified within category A10B of the ATC code (Anatomical Classification System, Therapeutics, Chemistry). In another still more particular embodiment, the biguanide is selected from the group consisting of phenformin, metformin and buformin. In another still more particular embodiment, the compound directed to decrease the OXPHOS function is metformin.

In the present invention, rotenone is understood to mean that compound with insecticidal activity, isolated from plants of the genus Lonchocarpus, which inhibits mitochondrial electron transport at the level of complex I or NADH dehydrogenase. The administration of 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, cutaneous, parenteral, nasal and intraperitoneal. Also within the present invention is the possibility that the compound directed to decrease the OXPHOS function is carried out in combination with a new administration of cisplatin. Therefore, in another particular embodiment of the method of the invention, the administration of the compound directed to decrease the OXPHOS function is carried out in combination with a new administration of cisplatin. The administration of both compounds (the compound aimed at decreasing the function OXPHOS and cisplatin) can be carried out at the same time or separately. Thus, in a particular embodiment of the method of the invention, the administration of the compound directed to decrease the OXPHOS function is carried out sequentially, simultaneously or separately with the new administration of cisplatin.

In the present invention it is understood that the administration is "sequential" when the cisplatin and the compound directed to decrease the OXPHOS function are administered to the individual at different moments in time, so that first one is administered and subsequently another is administered. The order in which cisplatin and the compound aimed at decreasing 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, ie 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, the "separate" administration can be the same "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 to be treated with cisplatin. Among these parameters are the internal mitochondrial membrane potential (MIMP), ROS levels and / or mitochondrial mass. Thus, in a particular embodiment, the method of the invention further comprises measuring the MIMP before and after the 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. is indicative that therapy after treatment with cisplatin involves the administration of a compound aimed at decreasing OXPHOS function. The MIMP can be measured, for example, by the use of DiOC6 (3) dye by flow cytometry. In another particular embodiment, the method of the invention further comprises measuring ROS levels before and after treatment with cisplatin in a biological sample isolated from said individual, wherein ROS levels after treatment are greater than the level of ROS before treatment is indicative that therapy after treatment with cisplatin involves the administration of a compound aimed at decreasing OXPHOS function. ROS levels can be measured, for example, by determining catalase activity or quantifying malondialdehyde, among others.

In another particular embodiment, the method of the invention further comprises measuring the mitochondrial mass before and after treatment with cisplatin in a biological sample isolated from said individual, wherein a mitochondrial mass after the treatment is greater than the mitochondrial mass before the Treatment is indicative that therapy after treatment with cisplatin involves the administration of a compound aimed at decreasing OXPHOS function.

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

In view of the above, within the present invention the use of expression levels of the PGC-1alpha gene is also contemplated to design personalized therapy for an individual suffering from a cisplatin-resistant lung cancer.

Therefore, in another aspect, the present invention relates to the in vitro use of the expression level of the PGC-1 alpha gene to design personalized therapy for an individual suffering from a lung cancer resistant to cisplatin. Hereinafter, this inventive aspect will be referred to as "use of the invention". How to use the level of expression of the PGC-1alpha gene to design such personalized therapy has been explained in previous paragraphs. Thus, in a particular embodiment, when the level of expression of PGC-1alpha after treatment with cisplatin is greater than the level of expression of PGC-1alpha before said treatment, then therapy after treatment with cisplatin comprises administration of a compound aimed at decreasing the OXPHOS function. The terms used in the present inventive aspect, such as level of expression, PGC-1 alpha gene, personalized therapy, etc. they have been explained in previous paragraphs and are applicable to the present inventive aspect. Also, 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, ROS levels, and / or mitochondrial mass to design personalized therapy for an individual suffering from cisplatin-resistant lung cancer. Thus, in another particular embodiment, a MIMP, ROS levels and / or a mitochondrial mass after treatment with cisplatin greater than the MIMP, ROS levels and / or the mitochondrial mass before said treatment, are indicative that the Therapy after treatment with cisplatin comprises a compound aimed at decreasing OXPHOS function. All these terms and expressions have been explained in the above aspect of the invention.

In a particular embodiment, the compound directed to decrease the OXPHOS function is an inhibitor of the expression of the PGC-1alpha gene, or of the protein encoded by said gene, or is a compound that inhibits the complex I of the mitochondrial electron transport chain , preferably, a biguanide or rotenone. In another more particular embodiment, the inhibitor of PGC-1alpha gel expression is an interfering RNA, which in another still more particular embodiment, comprises the sequence SEQ ID NO: 3. In another more particular embodiment, the biguanide is selected from group consisting of phenformin, metformin and buformin.

In a particular embodiment of the use of the invention, the administration of the compound directed to decrease the OXPHOS function is carried out in combination with a new administration of cisplatin. As previously explained, this new administration of cisplatin can be carried out sequentially, simultaneously or separately.

Thus, in another particular embodiment, the new administration of cisplatin in combination with the compound aimed at decreasing the OXPHOS function is carried out sequentially, simultaneously or separately. The terms "sequential", "simultaneous" or "separate" have been previously defined in the present description. In the context of the present invention, the expression levels of the PGC-1 alpha gene can be used to design a therapy for an individual suffering from any type of cisplatin-resistant lung cancer. However, in a particular embodiment, lung cancer is metastatic. In another particular embodiment, lung cancer is NSCLC. In another still more particular embodiment of 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-1 alpha 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 the present description. Thus, in a particular embodiment the use of the invention, the level of expression of the PGC-1 alpha 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 a compound directed to decrease OXPHOS function in the preparation of a drug for the treatment of cisplatin-resistant lung cancer in an individual is contemplated.

Thus, in another aspect, the present invention relates to the use of a compound directed to decrease OXPHOS function in the manufacture of a drug for the treatment of cisplatin-resistant lung cancer in an individual, wherein said individual comprises a level of expression of the PGC-1alpha gene after treatment with cisplatin greater than the level of expression of the PGC-1alpha gene before treatment.

Similarly, the present invention also relates to a compound directed to decrease OXPHOS function for use in the treatment of cisplatin-resistant lung cancer in an individual, wherein said individual comprises an expression level of the PGC-1 alpha gene after the treatment with cisplatin greater than the level of expression of the PGC-1alpha gene before treatment. Likewise, a method for the treatment of cisplatin-resistant lung cancer in an individual comprising the administration to said individual of a compound directed to decrease the OXPHOS function, wherein said individual comprises a level of expression of the PGC-1alpha gene after treatment with cisplatin greater than the level of expression of the PGC-1alpha gene before treatment.

Particular embodiments of the present inventive aspect comprise:

 - the compound aimed at decreasing the OXPHOS function is used in combination with cisplatin in the preparation of the drug.

 - the individual also comprises a MIMP after treatment with cisplatin greater than MIMP before treatment, ROS levels after treatment with cisplatin greater than ROS levels before treatment, and / or a mitochondrial mass after treatment with cisplatin greater than the mitochondrial mass before treatment with cisplatin;

 - 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 an inhibitor of the expression of the PGC-1alpha gene, or of the protein encoded by said gene, or is a compound that inhibits the complex I of the mitochondrial electron transport chain, preferably a biguanide or rotenone. In another particular embodiment, the inhibitor of PGC-1 alpha gene expression is an interfering RNA. 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. To implement the present invention it is necessary to have a kit comprising the reagents necessary to determine the expression levels of the gene PGC-1alfa. Therefore, in another aspect, the present invention relates to a kit for designing a personalized therapy to an individual suffering from lung cancer and 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 that contains 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 term "reagent 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 protein level. Therefore, reagents of the first type include nucleic acids, e.g. primers and probes, capable of specifically hybridizing with the mRNA encoded by the gene involved. The reagents of the second type are compounds that specifically bind 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 specifically 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.

Example of a nucleic acid that hybridizes specifically with the PGC-1 alpha gene is, for example, a probe. In the present invention, "probe" is understood to mean the nucleic acid molecule whose nucleotide sequence hybridizes specifically with the nucleotide sequence of a target gene. In the present invention, the target gene is the PGC-1alpha gene. The term "specifically-form hybrid", as used herein, refers to conditions that allow the hybridization of two polynucleotides in highly or moderately rigorous conditionals. High stringency conditions involve, in general: (1) low ionic strength and high temperature for washing, for example sodium chloride 0.015M / sodium citrate 0.0015M / 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), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 times Denhardt's solution, sonicated salmon sperm DNA (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 the 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 case the expression levels of the PGC-1 alpha gene are determined by measuring the levels of protein encoded by said gene, the kit of the invention comprises reagents that are capable of binding specifically to said protein, such as antibodies Examples of antibodies that can specifically recognize the protein encoded by the PGC-1 alpha gene and bind to it have been described in previous paragraphs. In another particular embodiment, the PGC-1alpha gene comprises a nucleotide sequence 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 nucleotide sequence with a sequence identity of 100% with the sequence SEQ ID NO: 1.

Additionally, the kit of the invention may comprise reagents aimed at determining other parameters useful for designing personalized therapy for an individual suffering from cisplatin-resistant lung cancer. As indicated above, among these parameters are MIMP, ROS levels and / or mitochondrial mass. Thus, in a particular embodiment, the kit further comprises reagents for measuring MIMP, ROS levels and / or mitochondrial mass. 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 cisplatin-resistant lung cancer, wherein said individual comprises a level of expression of the PGC-1 alpha gene after treatment with cisplatin greater than the level of PGC-1alpha gene expression before treatment.

As indicated above, when designing therapy personalized to the individual, other types of parameters can be taken into account. Thus, in a particular embodiment, the individual further comprises a MIMP after treatment with cisplatin greater than MIMP before treatment, ROS levels after treatment with cisplatin greater than ROS levels before treatment, and / or a mitochondrial mass after treatment with cisplatin greater than the mitochondrial mass before treatment with cisplatin.

In another particular embodiment, lung cancer is metastatic and / or lung cancer is non-small cell lung cancer.

In another aspect, the present invention relates to the use of a kit comprising a compound directed to decrease OXPHOS function in the treatment of cisplatin-resistant lung cancer in an individual, wherein said individual comprises expression levels of the PGC gene. -1 alpha after treatment with cisplatin greater than PGC-1alpha gene expression levels before treatment. In a particular embodiment of the kit, the compound directed to decrease the OXPHOS function is an inhibitor of the expression of the PGC-1alpha gene, or of the protein encoded by said gene, or is a compound that inhibits the complex I of the transport chain of mitochondrial electrons, preferably, a biguanide or rotenone. In another more particular embodiment, the biguanide is selected from the group consisting of phenformin, metformin and buformin. In another particular embodiment, the inhibitor of PGC-1alpha gene expression is an interfering RNA. Other parameters that the individual may have increased after treatment with cisplatin compared to said parameters before treatment with cisplatin are MIMP, ROS levels and / or mitochondrial mass. On the other hand, within the present invention also the following inventive aspects are contemplated along with their particular embodiments: In one aspect, the invention relates to an in vitro method for designing personalized therapy for an individual suffering from lung cancer and to be treated with cisplatin comprising

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

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

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

In another particular embodiment, the method further comprises measuring the levels of oxygen free radicals (ROS) before and after the 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 aimed at decreasing OXPHOS function.

In another particular embodiment, the lung cancer of the method is metastatic and / or non-small cell lung cancer (NSCLC). Method according to any one of claims 1 to 3, wherein the lung cancer is metastatic.

In another particular embodiment, the measurement or quantification of the expression levels of the PGC-1alpha gene comprises measuring the level of cDNA, mRNA and / or the protein encoded by said gene. In another particular embodiment, the biological sample is blood, serum or tissue from a biopsy.

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.

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

In the context of the present inventive aspect, combinations of the particular embodiments described herein are also included, as well as the simultaneous presence of all particular embodiments in the method described.

In another aspect, the invention relates to the in vitro use of the expression level of the PGC-1alpha gene to design personalized therapy for an individual suffering from lung cancer and to be treated with cisplatin. In a particular embodiment, in addition to the expression levels of the PGC-1alpha gene, the use of MIMP and / or ROS levels is also contemplated in order to design personalized therapy for an individual suffering from lung cancer and going to be treated with cisplatin. In another particular embodiment, an expression level of PGC-1alpha after treatment with cisplatin greater than the level of expression 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.

In another particular embodiment, a MIMP and / or ROS levels after treatment with cisplatin greater than 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. In another particular embodiment, the compound directed to decrease the OXPHOS function is a biguanide, preferably, 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.

In another particular embodiment, the lung cancer is metastatic and / or non-small cell lung cancer.

In another particular embodiment, the level of expression of the PGC-1alpha gene comprises determining the level of cDNA, mRNA and / or protein encoded by said gene. In the context of the present inventive aspect, combinations of the particular embodiments described herein are also included, as well as the simultaneous presence of all the particular embodiments.

In another aspect, the invention relates to the use of cisplatin combined in a sequential, simultaneous or separate manner with a compound aimed at decreasing 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 level of PGC-1alpha expression after treatment greater than the level of expression of PGC-1 alpha before treatment.

In a particular embodiment, the individual further comprises a MIMP after the treatment greater than the MIMP before treatment and / or ROS levels after treatment, greater than the ROS levels before treatment. In another particular embodiment, the lung cancer is metastatic and / or non-small cell lung cancer.

In another particular embodiment, the expression level of the PGC-1alpha gene corresponds to the level of cDNA, mRNA and / or protein encoded by said gene. In another particular embodiment, the compound directed to decrease the OXPHOS function is a biguanide, preferably, the biguanide is selected from the group consisting of phenformin, metformin and buformin. In the context of the present inventive aspect, combinations of the particular embodiments described herein are also included, as well as the simultaneous presence of all the particular embodiments.

In another aspect, the invention relates to a kit for designing a personalized therapy to an individual suffering from lung cancer and to be treated with cisplatin comprising the reagents necessary to determine the expression levels of the PGC-1alpha gene.

In a particular embodiment, the reagents of the kit comprise

- a nucleic acid that specifically hybridizes the PGC-1 alpha gene, and / or

 - antibodies that specifically recognize the protein encoded by the PGC-1alpha gene.

In another particular embodiment, the PGC-1alpha gene comprises a nucleotide sequence 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 kit further comprises reagents for measuring MIMP and / or ROS levels.

In the context of the present inventive aspect, combinations of the particular embodiments described herein are also included, as well as the simultaneous presence of all the particular embodiments. In another aspect, the present invention relates to the in vitro use of a kit as described in previous paragraphs, to design a personalized therapy to an individual suffering from lung cancer and to be treated with cisplatin. 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 aspects. Throughout the description and the claims the word "comprises" and its variants do not intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly 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 μΜ CDDP, resistant versions were obtained. The graphs represent the percentage of viable cells (Y-axis) after 48 hours of different treatments with CDDP (X-axis). Characterization of the cell cycle of the parental lines and resistant to CDDP (D, E and F). No significant differences were observed between the cell lines in the percentage of cells in the G1, S and G2 phases of the cell cycle. The data are the average of at least three different experiments and are represented as a percentage relative to 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. (A) Glucose consumption of cell lines. (B) Relationship between the growth rate in galactose and glucose of the parental cell lines and resistant to CDDP, an increase in this ratio is indicative of an increase in OXPHOS function. The mitochondrial inner membrane potential (C) and the total ROS levels (D) were measured by flow cytometry with the fluorescent probes TMRE and DCFHDA respectively. (E) OXPHOS Supercomplexes in lines H1299 and H460. (F and G) Mitochondrial mass measured by Mitotracker Green (MTG) and TOM20 immunofluorescence. (H) Relative levels of mRNA for the PGC-1a gene compared to the parental lines. The error bars indicate the standard deviation. The data are means of at least three different experiments and are represented by relative to your parental line. 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: Increase of PGC-1a and mitochondrial mass in PDX models after treatment with CDDP. (A) PGC-1 a mRNA levels relative to untreated controls for tumors obtained from the PDX6 and PDX8 models. (B) Evaluation of mitochondrial mass by IHC for TOM20 and quantification of fluorescence in controls and samples treated with CDDP for the same PDX.

Figure 4: Resistance to Cisplatin increases the sensitivity to PGC-1 to siRNA, Metformin and Rotenone. (A) Levels of PGC-1 a after gene silencing with a non-specific (control) siRNA or specific to the sequence of the PGC-1a gene. (B) The interference of PGC-1 a decreases the mitochondrial mass measured with MTG. (C) Sensitivity to interference from PGC-1 a in A549 and H1299. (D) Treatment with metformin reduces MIMP in both the parental and resistant lines. (E and F) Sensitivity to metformin and rotenone for the parental and resistant lines A549 and H1299. 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 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: Changes in the early response to CDDP treatment. The CDDP-resistant and parental cell lines were treated with 5 μΜ of CDDP for 24 hours. Changes in ROS (A), levels of PGC1-a (B), mitochondrial mass (C) and MIMP (D) were evaluated. Simultaneous treatment with 10 mM NAC is able to avoid the increase in ROS and MIMP induced by CDDP treatment at 24 hours (E). Cell cycle analysis and MIMP during treatment with CDDP (F).

Figure 6: Treatment with ZLN005 induces an increase in mitochondrial mass and reduces sensitivity to CDDP in cell lines. (A) Mitochondrial mass measured by MTG and TOM20 IHC in cells treated with ZLN005 for 48 hours. The bars are means of at least three different experiments and are represented as a percentage relative to cells without treatment. (B) ZLN005 reduces sensitivity to CDDP decreasing apoptosis. Left panel, percentage of viable cells after 48 h of treatments. Right panel, the bars of the graph represent the average percentage of annexin-positive 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 7: Metabolic inhibition in concomitance with CDDP in parental cells resistant to CDDP. The interference of PGC-1a reduces the increase in MTG produced by CDDP (A). Effect of the combined treatment of siRNA against PGC-1a and CDDP on cell viability (B). Treatment with metformin reduces the increase in MIMP produced by treatment with CDDP (C). Metformin and rotenone eliminate differences between parental cell lines and resistant to cisplatin treatment for A549 and H1299 cells (D and E). The graphs represent the percentage of viable cells (Y axis) after 48 hours of different treatments with Metformin or Rotenone (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 now be illustrated 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.

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 NSCLC metastasis, more specifically, H1299 cells are derived from lymph node and H460 of a pleural effusion. All cells were cultured routinely in DMEM (Dulbecco's Modified Eagle's medium) supplemented with 10% fetal bovine serum, penicillin and streptomycin. Cisplatin was obtained from therapeutic surpluses of the Medical Oncology service of the Hospital Puerta de Hierro Majadahonda. Metformin was obtained from sigma. 1.2 Patients:

 All the experiments carried out in this study complied with the current laws of Spain and the European Union and the principles of the Declaration of Helsinki. The study and the experimental protocols were approved by the Ethics Committee of the Puerta de Hierro University Hospital and written informed consent was obtained for all the patients recruited.

1.3 Cell viability assays.

3x10 ³ cells were seeded in a 96 well multi-well plate and cultured in DMEM until the next day. The next morning the medium was replaced by DMEM that contained 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.

1.4 Growth curves in glucose and galactose:

Cell growth was evaluated after plating 25,000 cells / well and culturing the cells for 4 days in DMEM containing 4.5 g / L glucose or 0.9 g / L galactose. The cells were collected and counted every 24 hours. Duplication times were calculated using the software Doubling Time v1.0.10 (http://www.doubling-time.com). The assays were performed in duplicate in at least 3 independent experiments.

1.5 Flow Cytometry.

The cytoplasmic ROS were determined using 2 ', 7'-Dichlorofluorescein diacetate (H2DCFHDA). The internal mitochondrial MIMP potential was determined using Tetramethylrhodamine ethyl ester (TMRE). The cell cycle was determined by the use of Vybrant ™ DyeCycle ™ Violet Stain. The mitochondrial mass was determined using Mitotracker Green FM. All fluorescent probes were purchased from Thermo Scientific. For these tests, 1x10 ⁵ cells were cultured. After the addition of the fluorophores, (5μΜ Vybrant, 30μΜ DCFHDA, 100nM TMRE, 100nM MTG) and their incubation during 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.

1.6 Glucose consumption:

The glucose consumption was determined by measuring the concentration present in the cell medium over time. To do this, 3x10 ⁵ cells were seeded in a 6-well plate. The next day, the medium was removed and 500μΙ_ of fresh DMEM was added. After 6 hours of incubation, the concentration present in the medium was determined. To measure the concentration of glucose in the medium, an Accu-Check Performa (Roche) glucometer was calibrated and used. Both the samples and the standard curve (500-0.15 g / L) were diluted ½ with distilled water before the measurement. The results are shown as milligrams of glucose consumed at 6 hours. At least three independent measurements were obtained to generate the mean.

1.7 Oxygen consumption:

Oxygen consumption of the intact cells was measured in DMEM at 37 ° C using a Clark type oxygen electrode (Hansatech Instruments). Basal respiration was calculated as the rate of oxygen consumption minus non-mitochondrial respiration after the addition of 1 mM KCN. At least three independent measurements were obtained to generate the mean.

1.8 OXPHOS Blue Native PAGE:

Mitochondria were purified from cell cultures as described in Pello et al., 2008 (Hum Mol Genet 17: 4001-401 1). Protein concentrations were determined using the microBCA protein kit (Thermo Scientific). To obtain native mitochondrial proteins, the pellets were solubilized with 4 grams of digitonin (Sigma) per gram of protein in 1.5M aminocaproic acid, 50 mM Bis-Tris, pH 7.0. Native electrophoresis was performed as described in Wittig et al., 2006. NDUFA9 (Abcam, ab14713) was used for the immunodetection of supercomplexes I + II I2 + IV. SDHA (Abcam, ab14715) was used for normalization. The quantification of 4 different membranes from 2 different extracts was performed with ImageJ. 1.9 Immunofluorescence:

The cells were fixed with 4% paraformaldehyde, incubated overnight with TOM20 antibody (Santa Cruz Biotechnology, sc-17764, 1/50). Alexa Fluor 488 anti-mouse (Invitrogen Life Technologies, 1/500) was used as a secondary antibody. The nuclei were stained with Topro-3 (Invitrogen Life Technologies, 1/1000). Five images of each sample were collected with a TCS SP5 confocal microscope (Leica Microsystems) equipped with 20 ^χ HCX PL APO (0.7 numerical aperture). Two channels were acquired sequentially with the following excitation and emission parameters: (488 nm, 500-540 nm) for the TOM20 signal, and (633 nm, 645-750 nm) for Topro-3. Data processing and quantification of the fluorescence signal were performed with LASF SOFTWARE VERSION 2.6.07266.

1. 10 Analysis of expression.

 RNA from the cells was extracted using the "RNeasy mini Kit with DNAse" kit (Qiagen) and the cDNA was synthesized using the "NZY First-StrandcDNASynthesis Kit" (NZYtech) kit. The expression levels of PGC-1 a were assessed by qRT-PCR using the Taqman® expression assay (Hs01016719_m1). The levels of TBP (Hs00427621_m1) were used as endogenous control. 1. 11 Models of xenograft derived from CPCNP patients (PDX):

 Four different models of PDX were generated from four patients with NSCLC. All treatments were carried out in passage 2 mice, with palpable bilateral tumors. At least two mice per group were injected intraperitoneally with 2.5 mg / kg CDDP or vehicle twice a week on non-consecutive days. After the treatments, the animals were sacrificed and the tumors were collected for further analysis.

1. 12 Silencing of PGC-1ct by siRNA:

 Cells were transfected 24 hours before functional experiments by Silencer Select ™ siRNA directed to PGC-1a (s21394, 5 '-> 3' sequence: CCUGUUUGAUGACAGCGAAtt (SEQ ID NO: 3)) or using a non-specific control (Silencer ™ Select Negative Control No. 1) using Lipofectamine RNAiMAX following the manufacturer's instructions (Thermo Scientific). 1.13 Measurement of apoptosis

The cells were seeded in a 24-well plate in DMEM. The next day, the media were changed to DMEM or DMEM containing the different treatments (5μΜ CDDP and / or 20μΜ ZLN005). After 48 hours of treatment, the cells were harvested, washed twice in PBS and resuspended in 50μl of annexin V binding solution diluted 10 times. 1 μΙ_ of annexin V-FITC conjugate was added to the cells and incubated for 15 minutes at room temperature. Finally, the cells were further diluted with 150μΙ_ of annexin V binding solution diluted 10 times and analyzed immediately by flow cytometry. Frontal and lateral scattering were used to select the cell population. The bars on the graph represent the average percentage of annexin-positive cells from 4 different assays.

I.14 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 cisplatin resistant lines.

 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 from 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 5μΜ CDDP for three months.

After this selection process, the cells were evaluated for their sensitivity to treatment with cisplatin (Figure 1A-C). As would be expected in a similar way 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 seem similar, significant differences were observed for the 3 cell lines in the concentration range ranging from 3 to 12 μΜ 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 5μΜ cisplatin, since it is a low concentration, but it allowed to show significant differences for the 3 lines of the study.

Given that CDDP affects the process of DNA replication, to exclude that continuous CDDP treatment had selected cells with a more quiescent phenotype and metabolism, the cell cycle of CDDP-resistant and parental cells was studied. The results showed no differences in any of the phases of the cell cycle (G1, S, G2) between the parental cell lines and their CDDP-resistant versions (Figure 1 E-F).

2.2 Cell lines resistant to cisplatin show an increase in oxidative metabolism and mitochondrial biogenesis.

Once the three cell models of CDDP resistance were obtained, the metabolic changes associated with this stable increase in resistance were characterized (Figure 2). Glucose consumption was measured, and a decrease was observed for the CDDP-resistant H1299 and H460 lines (Figure 2A). In addition, the relationship between the cell growth rate in galactose and glucose, indicative of the OXHPOS capacity of the cells, was calculated. The results show an increase in this parameter for the lines H1299 and H460 resistant to CDDP compared to their parental versions (Figure 2B). The mitochondrial internal potential (MIMP) was measured since it is a good reflection of 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 parental lines, but not for the A549 line (Figure 2C). In addition, to confirm the change in oxidative metabolism, the oxygen consumption of the cells was evaluated. The results show an increase in oxygen consumption for the lines H1299 and H460 resistant to CDDP (Figure 2D).

To explain the mechanism behind the increase in oxygen consumption and MIMP, the levels of OXPHOS complexes were studied by BN-PAGE in H1299 and H460 cell lines in which there was a net increase in OXPHOS function (Figure 2E). Surprisingly, the results showed no significant differences in the amount of mitochondrial supercomplexes I + II I2 + IV. An explanation for this apparent contradiction (increase in oxygen consumption and MIMP but not changes in the amount of complexes) could be an increase in mitochondrial biogenesis. In this way, the mitochondrial network would harbor the same density of OXPHOS complexes but having more mitochondrial mass, the total amount of OXPHOS complexes per cell would be higher.

 To test this hypothesis, mitochondrial mass levels were measured using the Mitotracker Green molecular probe (MTG) (Figure 2F), as well as through immunofluorescence with the TOM20 antibody (Figure 2G). The results show an increase in mitochondrial mass for cell lines H1299 and H460 resistant to CDDP. On the other hand, no changes were observed for the cell line A549 resistant to CDDP. Interestingly, this cell line also showed no increase in OXPHOS function.

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

2.3 Xenografts derived from patients (PDX) treated with cisplatin show an increase in PGC-1a and mitochondrial mass.

Four PDX models of 4 patients with CPCNP were generated. The mice were randomized into two groups, controls and treated with CDDP. Tumors of two of four PDX showed an increase in the levels of PGC-1a (PDX6 and PDX8) after treatment with CDDP (Figure 3A), no differences were observed for PDX9 and PDX4 (data not shown). Additionally, for PDX6 and PDX8 the mitochondrial mass was evaluated by IHC of TOM20 (Figure 3B). Consistent with their increased levels of PGC-1a, there was a significant increase in mitochondrial mass after treatment with CDDP.

2.4 OXPHOS inhibition in parental CPCNP lines resistant to CDDP. In view of the results, it seems clear that a mechanism associated with treatment with 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 or by gene silencing of PGC-1a by siRNAs. For these studies we worked with the H1299 cell line since its CDDP-resistant version showed the highest MIMP increase. In addition, the A549 line was used as a negative control since in this line this phenomenon was not observed.

First, it was verified if the silencing of PGC-1a was able to reduce the mitochondrial mass. Using a specific siRNA against PGC-1a, approximately 70% reduction of PGC-1a expression was achieved compared to the controls (Figure 4A). Under these conditions, the interference of PGC-1a reduced the mitochondrial mass measured with MTG in the cell lines (Figure 4B). Interestingly, differences in mitochondrial mass between the parental H1299 and CDDP resistant cells are lost when PGC-1 a is interfered with.

Next, we measured the sensitivity of the cells to gene silencing of PGC-1a (Figure 4C). The interference of PGC-1a decreases the number of viable cells for all the lines, however, this effect was greater for the cell line resistant to CDDP H1229 compared to its parental version. On the other hand, no differences were found in the sensitivity to the interference of PGC-1 between the parental A549 and CDDP-resistant cell lines. In fact, the siRNA sensitivity of PGC-1a seems to correlate with the OXPHOS function measured. Next, it was checked whether the drug metformin was able to reduce the MIMP of the cell lines. Treatment with metformin to lines A549 and H1299 decreased its MIMP independently of its resistance to CDDP (Figure 4D). In addition, the effect of different concentrations of metformin on the cell viability of the parental lines and resistant to CDDP was evaluated (Figure 4E). The results show a decrease in cell viability with an increase in the concentration of metformin. Notably, this effect is much greater in the case of the CD12-resistant H1299 line compared to its parental line. On the other hand, there were no differences between the CDDP-resistant line A549 and its parental line.

Finally, we evaluated the sensitivity of cell lines to rotenone, an inhibitor Specific to mitochondrial Complex I (Figure 4F). Similar to metformin, there is no significant difference in rotenone sensitivity between parental and CDDP-resistant A549 cells. However, CD12 resistant H1299 cells are significantly more sensitive to rotenone than their parental version.

2.5 Treatment with cisplatin induces a metabolic reprogramming in cell lines of ROS-mediated NSCLC independent of the cell cycle.

The results obtained with the resistant lines led to the question whether these changes could occur as an early response to the drug. For this purpose, the levels of ROS, PGC-1a, mitochondrial mass and MIMP in the cell lines after 24 hours of treatment were analyzed (Figure 5).

Since PGC-1a is induced by ROS, we first studied whether cisplatin was able to increase ROS levels in cell lines. For this purpose, ROS levels were analyzed 16h after treatment with CDDP in the different cell lines (Figure 5A). The results showed an increase in ROS when the parental cells are treated with CDDP 5μΜ for 16 h. Similarly, this early change of ROS in response to treatment also occurs in resistant lines, which further increase their increased parameters. Therefore, this phenomenon seems to be common to the parental and resistant cell lines.

Then it was verified that there was a process of metabolic reprogramming induced by CDDP treatment in the short term. We evaluated the levels of PGC-1a (Figure 5B), mitochondrial mass (Figure 5C) and MIMP (Figure 5D) after 24h exposure to 5μΜ CDDP. The results showed an increase in all the parameters. Interestingly, this increase occurs in the 3 cell lines, although the line resistant to CDDP A549 does not have a stable increase in any of them. Also, this early change of MIMP and ROS in response to treatment also occurred in resistant lines, which further increased their parameters.

In addition, this metabolic reprogramming induced by CDDP treatment was avoided by concomitant treatment with 10 mM N-Acetyl Cysteine (NAC), indicating that this process is mediated by ROS (Figure 5E). On the other hand, given that CDDP is an intercalating agent that hinders phase S, it was analyzed whether this increase in MIMP in short times could be a consequence of alterations in the cell cycle (Figure 5F). For this, measuring the cell cycle, it was observed that, in fact, the treatment with CDDP retains the cells in the S-G2 phase, decreasing the proportion of cells in G1. However, this effect is less in the resistant lines. As for the mitochondrial function, the cells in S-G2 phase showed more MIMP, therefore, part of the increase we saw is due to the fact that the cells stop in this phase. In any case, if we compare the cells within the same phase of the cell cycle, there is an increase of the MIMP independent of the phase of the cell cycle induced during the treatment with CDDP. 2.6 Mitochondrial mass levels elevated by treatment with ZLN005 reduce CDDP-induced apoptosis in cells.

 To support that the increase of the mitochondrial mass is a mechanism of resistance to CDDP in CPCNP, it was decided to increase the levels of mitochondrial mass by another approach to check if this reduced the sensitivity to CDDP. Compound ZLN005 after 48 hours of incubation increased mitochondrial mass levels measured by MTG or immunofluorescence of TOM20 in the three parental lines A549, H1299 and H460. (Figure 6A). Under these conditions, with an increased mitochondrial mass, sensitivity to CDDP was reduced in the parental lines (Figure 6B). Similarly, the percentage of apoptotic cells, determined by positive labeling of Annexin V, was significantly reduced in lines A549 and H1299 treated with ZLN005 (Figure 6C).

2.7 Concomitant treatment based on inhibition of OXPHOS and CDDP in parental cell lines resistant to CDDP.

Since an increase in mitochondrial mass and MIMP was observed as an early response to treatment with CDDP, it was verified whether joint treatment with metformin, rotenone or PGC-1a siRNA could increase the effect of cisplatin.

We proceeded in a manner similar to section 2.4, first analyzing whether the siRNA was able to reduce the early increase in mitochondrial mass induced by CDDP (Figure 7A). The results show that PGC-1a siRNA is able to control the increase in mitochondrial mass induced by cisplatin in the two lines A549 and H1299, both for parental and CDDP-resistant versions. Subsequently, we measured cell viability using PGC-1a siRNA in the presence of 5μΜ concomitant CDDP (Figure 7B). The silencing of PGC-1 a increases the effect of CDDP in all cases. However, the differences in CDDP sensitivity between The parental A549 and its CDDP-resistant version are maintained. On the other hand, probably due to an increased sensitivity to PGC-1a silencing in CDDP-resistant H1299 cells, differences in CDDP sensitivity between the parental 1299 H and CDDP-resistant cells are lost.

Second, we tested whether treatment with metformin was able to reverse the increase in MIMP produced by exposure to CDDP (Figure 7C). Treatment with metformin restricts the increase in MIMP induced by CDDP for all cases. In addition, treatment with metformin or rotenone eliminates differences between parental cell lines and resistant to cisplatin treatment for both cell lines (Figure 7 D-E). The differences between the parental and resistant cells are no longer significant from a concentration of 0.1 mM metformin or 1 nM rotenone for A549 cells, and 1 mM metformin or 1 nM rotenone for H1299 cells.

III. DISCUSSION

Resistant versions of three different cell lines were generated after a continuous treatment with cisplatin for three months. Interestingly, there were no changes in the phases of the cell cycle. However, two of three cell lines showed stable changes towards an increase in OXPHOS function (increase in MIMP and mitochondrial mass), although interestingly, all three responded similarly with an increase in these parameters as an early response to CDDP treatment.

The results shown here show an increase in PGC-1a (activator of the OXPHOS function), precisely in the two cell lines (H 1299 and H460) that showed an increase in MIMP, mitochondrial mass and oxygen consumption. Supporting the importance of PGC-1a 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-1a. These results reinforce a specific stable change in the OXPHOS function for CDDP-resistant cell lines of H1299 and H460. Similarly, changes were observed after treatment with CDDP on PDX models, which reinforces that this phenomenon occurs in the tumors of patients with physiological form.

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 CDDP-resistant cell lines and also as an early response to 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 tumor cells preferentially use the aerobic glycolysis strategy despite their lower energy efficiency (compared to the more efficient use of the OXPHOS system) since this "Warburg metabolism" is adapted to support exponential growth. However, as the results indicate, 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 the resistance and treatment of this drug in this type of tumors. This mechanism of resistance associated with CDDP is reinforced by the fact that a pharmacological treatment with compound ZLN005 that induces an increase in mitochondrial mass produces a parallel increase in resistance to CDDP, reducing the entry into apoptosis of cells.

We investigated the possibility of eliminating this group of resistant cells with an enhanced OXPHOS function by inhibiting mitochondrial function, by different treatments (Metformin, Rotenone and PGC-1a siRNA) and under different approaches (alone or in concomitance with CDDP).

In the present study it was demonstrated that both the silencing of PGC-1a, as well as the treatment with the mitochondrial inhibitors Metformin or Rotenone, reduced the viability of the cell lines. This reduction was greater in the H1299 CDDP resistant cell line in which an increase in OXPHOS function was observed compared to its parental version. However, the line A549 CDDP resistant for which no increase was observed in any parameter OXPHOS maintained the same profile of sensitivity to different treatments. The similar results between the effect of rotenone and metformin reinforce that the greater sensitivity to metformin of the HDPC cells resistant to CDDP was due to the ability of metformin to inhibit the OXPHOS system and not to other non-dependent effects of OXPHOS. to metformin. These results also support that the detrimental effect of the inhibition of PGC-1a on the resistant H1299 line is due to its role as responsible for maintaining an elevated OXPHOS function. On the other hand, these data confirm that there is a stable metabolic change towards greater dependence on the OXPHOS system in the cell line H1299 resistant to CDDP.

On the other hand, the concomitant treatment of CDDP with metformin, rotenone or with the silencing of PGC-1a, increases the effect of CDDP, in all cases. In fact, the combined therapy of metformin or rotenone with CDDP eliminates the sensitivity differences between parent and resistant for both cell lines A549 and H1299. Likewise, the combined therapy of PGC-1 a and CDDP eliminates the sensitivity differences between parent and resistant for line H1299 but not for line A549.

To conclude, it can be said that an increase in the mitochondrial mass and therefore of the OXPHOS function is a mechanism of resistance to treatment with cisplatin in NSCLC, and that this characteristic of cisplatin-resistant cells can be used therapeutically by treatment with metformin , rotenone or interference of the PGC-1 gene a.

Claims

1. In vitro method for designing personalized therapy for an individual suffering from cisplatin-resistant lung cancer comprising  (a) determining the level of expression of the PGC-1alpha gene before and after treatment with cisplatin in a biological sample isolated from said individual, and  (b) comparing the level of expression obtained in step (a) before treatment with the level of expression after treatment, wherein a level of PGC-1alpha expression after treatment greater than the level of expression of PGC-1 alpha before treatment is indicative that therapy after treatment with cisplatin comprises the administration of a compound aimed at decreasing OXPHOS function . The method according to claim 1, further comprising measuring the internal mitochondrial membrane potential (MIMP) before and after treatment with cisplatin in a biological sample isolated from said individual, wherein a MIMP after the treatment is greater than the MIMP before of the treatment is indicative that the therapy after treatment with cisplatin comprises the administration of a compound directed to decrease the OXPHOS function. The method according to claim 1 or 2, further comprising measuring the levels of oxygen free radicals (ROS) before and after treatment with cisplatin in a biological sample isolated from said individual, wherein ROS levels after treatment greater than the level of ROS before treatment is indicative that therapy after treatment with cisplatin involves the administration of a compound aimed at decreasing OXPHOS function. Method according to any one of claims 1 to 3, further comprising measuring the mitochondrial mass before and after treatment with cisplatin in a biological sample isolated from said individual, wherein a mitochondrial mass after treatment is greater than the mitochondrial mass before treatment is indicative that therapy after treatment with cisplatin involves the administration of a compound aimed at decreasing OXPHOS function. The method according to any one of claims 1 to 4, wherein the administration of the compound directed to decrease the OXPHOS function is carried out in combination with a new administration of cisplatin. 6. Method according to any one of claims 1 to 5, wherein the lung cancer is metastatic. The method according to any one of claims 1 to 6, wherein the lung cancer is non-small cell lung cancer. The method according to any one of claims 1 to 7, 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. 9. Method according to any one of claims 1 to 8, wherein the biological sample is blood, serum or tissue from a biopsy. The method according to any one of claims 5 to 9, wherein the administration of the compound directed to decrease the OXPHOS function is carried out sequentially, simultaneously or separately with the new administration of cisplatin. The method according to any of claims 1 to 10, wherein the compound directed to decrease the OXPHOS function is an inhibitor of the expression of the PGC-1alpha gene, or of the protein encoded by said gene, or is a compound that inhibits complex I of the mitochondrial electron transport chain, preferably a biguanide or rotenone. 12. The method of claim 1, wherein the inhibitor of PGC-1alpha gene expression is an interfering RNA. The method according to claim 1, wherein the biguanide is selected from the group consisting of phenformin, metformin and buformin. 14. In vitro use of the expression level of the PGC-1 alpha gene to design personalized therapy for an individual suffering from cisplatin-resistant lung cancer. 15. Use according to claim 14, further comprising the use of MIMP, ROS levels and / or mitochondrial mass. 16. Use according to claim 14 or 15, wherein a level of expression of PGC-1 alpha after treatment with cisplatin greater than the level of expression of PGC-1alpha before said treatment, is indicative that therapy after treatment with cisplatin comprises the administration of a compound directed to decrease OXPHOS function. 17. Use according to claim 15 or 16, wherein a MIMP, ROS levels and / or a mitochondrial mass after treatment with cisplatin greater than the MIMP, the levels of ROS and / or the mitochondrial mass before said treatment, is indicative that therapy after treatment with cisplatin involves the administration of a compound aimed at decreasing OXPHOS function. 18. Use according to claim 16 or 17, wherein the administration of the compound directed to decrease the OXPHOS function is carried out in combination with a new administration of cisplatin. 19. Use according to any one of claim 16 to 18, wherein the compound directed to decrease the OXPHOS function is an inhibitor of the expression of the PGC-lalfa gene, or of the protein encoded by said gene, or is a compound that inhibits the complex I of the mitochondrial electron transport chain, preferably a biguanide or rotenone. 20. Use according to claim 19, wherein the inhibitor of PGC-alpha gene expression is an interfering RNA. 21. Use according to claim 19, wherein the biguanide is selected from the group consisting of phenformin, metformin and buformin. 22. Use according to any one of claims 18 to 21, wherein the new administration of cisplatin in combination with the compound directed to decrease the OXPHOS function is carried out sequentially, simultaneously or separately. 23. Use according to any one of claims 14 to 22, wherein the lung cancer is metastatic. 24. Use according to any one of claims 14 to 23, wherein the lung cancer is non-small cell lung cancer. 25. Use according to any one of claims 14 to 24, wherein the level of expression of the PGC-1alpha gene comprises determining the level of cDNA, mRNA and / or protein encoded by said gene. 26. Use of a compound directed to decrease OXPHOS function in the manufacture of a drug for the treatment of cisplatin-resistant lung cancer in an individual, wherein said individual comprises a level of expression of the PGC-1alpha gene after treatment with cisplatin greater than the level of PGC-1 alpha gene expression before treatment. 27. Use according to claim 26, wherein the individual further comprises a MIMP after treatment with cisplatin greater than the MIMP before treatment, ROS levels after treatment with cisplatin greater than ROS levels before treatment, and / or a mitochondrial mass after treatment with cisplatin greater than a mitochondrial mass before treatment. 28. Use according to claim 26 or 27, wherein the lung cancer is metastatic. 29. Use according to any one of claims 26 to 28, wherein the lung cancer is non-small cell lung cancer. 30. Use according to any one of claims 26 to 29, wherein the level of expression of the PGC-1alpha gene comprises determining the level of cDNA, mRNA and / or protein encoded by said gene. 31. Use according to any one of claims 26 to 30, wherein the compound directed to decrease the OXPHOS function is an inhibitor of the expression of the PGC-1alpha gene, or of the protein encoded by said gene, or is a compound that inhibits the complex I of the mitochondrial electron transport chain, preferably a biguanide or rotenone. 32. Use according to claim 31, wherein the inhibitor of PGC-1alpha gene expression is an interfering RNA. 33. Use according to claim 31, wherein the biguanide is selected from the group consisting of phenformin, metformin and buformin. 34. A kit for designing personalized therapy for an individual suffering from cisplatin-resistant lung cancer, comprising the reagents necessary to determine the expression levels of the PGC-1alpha gene. 35. Kit according to claim 34, wherein the reagents comprise - a nucleic acid that specifically hybridizes the PGC-1 alpha gene,  - antibodies that specifically recognize the protein encoded by the PGC-1alpha gene. 36. Kit according to claim 34 or 35, wherein the PGC-1alpha gene comprises a nucleotide sequence with a sequence identity of at least 80, 85, 90, 95, 96, 97, 98 or 99% with the sequence SEQ ID NO: 1. 37. Kit according to any one of claims 34 to 36, further comprising reagents for measuring MIMP, ROS levels and / or mitochondrial mass. 38. In vitro use of a kit according to any one of claims 34 to 37 for designing personalized therapy for an individual suffering from cisplatin-resistant lung cancer, wherein said individual comprises an expression level of the PGC-1 alpha gene after of the treatment with cisplatin greater than the level of expression of the PGC-1alpha gene before treatment. 39. Use according to claim 38, wherein the lung cancer is metastatic. 40. Use according to claim 38 or 39, wherein the lung cancer is non-small cell lung cancer.