WO2012110425A1 - Methods for monitoring the response to treatment and for treating colorectal cancer - Google Patents

Abstract

The present invention relates to a calpain inhibitor for use for delaying or inhibiting the secondary resistance to an anti-cancer agent in a patient suffering from colorectal cancer. The invention also refers to a method for monitoring the response to a treatment of a patient suffering from a cancer chosen from colorectal cancer, gastric cancer, esophageal cancer, non- small-cell and small-cell lung cancers, leukemia, lymphomas, central nervous system maligant gliomas and ovarian carcinomas, and preferably colorectal cancer, comprising: a. treating said patient with an anti-cancer agent for a time period of at least 5 months; then b. measuring the level of expression of at least one gene selected from the group consisting of calpain, annexin A2 and S100A10 genes, in cancer cells of said patient.

Description

Methods for monitoring the response to treatment and for treating colorectal cancer

FIELD OF THE INVENTION

The present invention relates to methods for monitoring the response to treatment and for treating colorectal cancer.

BACKGROUND OF THE INVENTION

Colorectal cancer (CRC) is a highly prevalent disease that is associated with high mortality and morbidity rates, with > 1,000,000 new cases and 500,000 death worldwide every year (1). The poor therapeutic outcome is largely due to the development of resistance to conventional drugs. The standard treatment for metastatic patients for the last 40 years was 5-fluorouracil (5-FU) and leucovorin-based therapy. Major progress has been made by the introduction of regimens containing cytotoxic drugs such as CPT-l l/SN-38 (also called camptothecin- 11 or irinotecan). It may be sold under the tradename Camptosar (2). However, these combinations remain inactive in about half of the patients (innate or primary resistance), and in addition, resistance to treatment appears in almost all patients who initially responded (acquired or secondary resistance) (3). Thus, identification of new targets that are responsible for resistance and/or of new biomarkers that can predict resistance to CPT-l l/SN-38 represents a great challenge for the treatment and eradication of colorectal cancer. CPT-l l/SN-38 is a semisynthetic analog of camptothecin. It interacts with cellular topoisomerase I-DNA complexes (Topo I) which results in induction of a S -phase- specific cytotoxicity (5). CPT-11 is a pro-drug which is converted into the active molecule SN-38 by carboxylesterases and a clear relationship between carboxylesterase level and the chemosensitivity of human lung cancer cells has been demonstrated in vitro. The mechanisms of CPT-l l/SN-38 resistance have not been fully understood [reviewed in (6)]. It has been reported that the efflux of CPT-11 or SN-38 is due to the overexpression of the ATP-binding cassette (ABC) transmembrane transporters such as P-glycoprotein (P-gp) (7) and the breast cancer protein (BCRP) (8). In addition, tumor cells may escape irinotecan cytotoxic effects by reducing the level of Topo I expression (9). Interestingly, CPT-l l/SN-38 has been shown to activate the nuclear factor kappa B (NF-KB) which may account for an important mechanism for resistance in several tumor models (10-13). NF-κΒ is an ubiquitous transcription factor. In the absence of stimulation, NF-κΒ is sequestered in the cytoplasm of most cells, by binding to ΙκΒ inhibitory subunits. Upon stimulation, ΙκΒ molecules are phosphorylated by the specific kinases IKK (ΙκΒ kinase) l/α and IKK2/B which, together with NEMO (NF-κΒ Essential Modulator)/IKKy, form the IKK complex that integrates signals for NF-κΒ activation. Serine phosphorylation is followed by polyubiquitination and subsequent degradation of IKB via the 26S proteasome pathway (14). NF-κΒ then translocates into the nucleus where it controls the transcription of a wide variety of genes especially involved in cell survival, proliferation and chemoresistance (15). Nevertheless, the mechanism responsible for CPT-l l/SN-38-induced activation of NF-κΒ remains largely unknown. It has been documented that there is an interplay between death and survival complexes in the development of CPT-l l/SN-38 resistance (16, 17).

Therefore, identifying the molecules which influence this balance would be of great interest. Particularly, said molecules would help in delaying or preventing the onset of secondary resistance in patients suffering from colorectal cancers.

There is also a need for monitoring the onset of secondary resistance in patients suffering from colorectal cancer. Said method would allow a much better efficient treatment of colorectal cancer. The inventors surprisingly discovered that a resistance to CPT-l l/SN-38 was acquired concomitantly with an increase in NF-κΒ activation, and that said NF-κΒ activation results from a calpain 2-dependent ΙκΒ-α degradation. They also showed that the expression of a specific set of genes was increased in a resistant cell line established from a resistant tumor (HT-29R) compared to the HT-29 sensitive parental cell line. SUMMARY OF THE INVENTION

The invention relates to a method for delaying or inhibiting the secondary resistance to an anti-cancer agent, particularly CPT-l l/SN-38, in a patient suffering from colorectal cancer, comprising the administration of a calpain inhibitor to said patient. The present invention also relates to a calpain inhibitor for use for delaying or inhibiting the secondary resistance to an anti-cancer agent, particularly CPT-l l/SN-38, in a patient suffering from colorectal cancer. Also provided is a product containing an anti-cancer agent, particularly CPT-l l/SN-38, and a calpain inhibitor as a combined preparation for simultaneous, separate or sequential use in colorectal cancer therapy.

The present invention also relates to a method for monitoring the response to a treatment of a patient suffering from a cancer chosen from colorectal cancer, gastric cancer, esophageal cancer, non-small-cell and small-cell lung cancers, leukemia, lymphomas, central nervous system maligant gliomas and ovarian carcinomas, comprising:

a. treating said patient with an anti-cancer agent, particularly CPT-l l/SN- 38, for a time period of at least 5 months ; then

b. measuring the level of expression of at least one gene selected from the group consisting of calpain, annexin A2 and S 100A10, in cancer cells of said patient.

Preferably, said patient is suffering from colorectal cancer.

DETAILED DESCRIPTION OF THE INVENTION Definitions As used herein, the term "calpain inhibitor" refers to compounds which inhibit signalling through calpain 1 (calpain 1 inhibitors) or calpain 2 (calpain 2 inhibitors), as well as compounds which inhibit the expression of the calpain 1 or calpain 2 genes. Compounds which inhibit signalling through calpain 1 or calpain 2 are called calpain antagonists. They include compounds which inhibit the activity of calpain 1 or calpain 2, by binding to said calpain, or by inhibiting calpain 1 or calpain 2 signalling by other mechanisms.

Said calpain antagonists may be chosen from specific calpain 1 antagonists and specific calpain 2 antagonists. By "specific" or "selective" it is meant that the affinity of the antagonist for said calpain (i.e. calpain 1 or calpain 2) is at least 10-fold, preferably 25- fold, more preferably 100-fold, still preferably 500-fold higher than the affinity for the other calpains. Said calpain antagonists may also be chosen from specific calpain 1/2 antagonists. By "specific calpain 1/2 antagonists", it is meant that the affinity of the antagonist for said calpains (i.e. calpain 1 and calpain 2) is at least 10-fold, preferably 25-fold, more preferably 100-fold, still preferably 500-fold higher than the affinity for the other calpains.

Typically, calpain antagonists are synthetic compounds such as ALLN (calpain inhibitor I) and ALLM (calpain inhibitor II) peptides developed by Peptides International. ALLN is Acetyl-L-Leucyl-L-Leucyl-L-Norleucinal (N-Ac-Leu-Leu-Nle-CHO (aldehyde)), and inhibits preferentially calpain 1, but also calpain 2 to a less extent. It is also a proteasome and cathepsin inhibitor.

ALLM is Acetyl-L-Leucyl-L-Leucyl-L-Methioninal (N-Ac-Leu-Leu-Met-CHO (aldehyde)), and inhibits preferentially calpain 1, but also calpain 2 to a less extent. It is also a cathepsin inhibitor.

Calpain antagonists also include:

calpastatin, which is encoded by the CAST gene and which is the natural antagonist of calpains 1 and 2 (Paquet-Durand F et al., Journal of neurochemistry, 115:930-940, 2010);

calpeptin, also known as benzyloxycarbonyldipeptidyl aldehyde;

- SNJ- 1945. MDL28170 and SJA6017 and A-705253 (N-( l -ben/y!-2-carbamoyl- 2-oxoethyl )-2-(H-2-(4-cliethylaminomethylp enyl )ethen- 1 -v! )ben/amicle). SNJ- 1945 is also known as 2-(2-methoxyethoxy)ethyl N-[(1S)-1-[[(2S)-1- (cyclopropylcarbamoyl)-l-oxo-3-phenyl-propan-2-yl]carbamoyl]-3-methyl- butyl] carbamate, which is an orally available calpain inhibitor. SJA6017 is also known as N-(4-Fluorophenylsulfonyl)-L-Valyl-L-Leucinal ; AK295, also known as Cbz-Leu-aminobutyrate-CONH(CH₂)3 morpholine; and tetracyclines, like chlortetracycline (CTC) and demeclocycline (DMC).

Calpain inhibitors also include compounds which inhibit the expression of the calpain 1 or calpain 2 genes; said compounds are called inhibitors of calpain gene expression. An "inhibitor of gene expression" refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of said gene. Consequently an inhibitor of calpain 1 or calpain 2 gene expression refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of the calpain 1 or calpain 2 genes respectively.

The inhibitors of calpain gene expression include, but are not limited to, antisense oligonucleotides, siRNAs, shRNAs, ribozymes and DNAzymes.

Preferably, said inhibitor of calpain gene expression is chosen from siRNAs. As used herein, the term "colorectal cancer" refers to the pathological condition in mammals that is typically characterized by unregulated cell growth in the colon, rectum and appendix. It is also called colon cancer or large bowel cancer.

As used herein, the term "patient" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a patient according to the invention is a human.

In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.

As used herein, the expression "anti-cancer agent" or "chemotherapeutic agent" refers to compounds which are used in the treatment of colorectal cancer.

Anti-cancer agents include but are not limited to fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5- fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan (CPT-11), SN-38, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, monoclonal antibodies against EGF receptor or VEGF, such as bevacizumab, cetuximab and panitumumab, imatimb mesylate, hexamethyhnelamine, topotecan, genistein, erbstatin, lavendustin and also bortezomib (also called PS341, and sold by Millenium Pharmaceuticals under the name Velcade).

Preferably, the anti-cancer agent is irinotecan or SN-38.

As used herein, the expression "secondary resistance to an anti-cancer agent", or "acquired resistance to an anti-cancer agent", refers to the resistance to both apoptosis and antiproliferative effect that occurs during the treatment, and that is induced by said anti-cancer agent in a patient suffering from colorectal cancer, said patient being an initial responder (sensitive) to said treatment. The secondary resistance appears in almost all patients who initially responded to the first line chemotherapy with said anticancer agent, particularly with CPT-11 or SN-38; the colorectal cancerous cells of the patient are then less or no more sensitive to the anti-cancer agent.

Secondary resistance to an anti-cancer agent has to be distinguished from primary resistance (or innate resistance), which is the resistance to both apoptosis and antiproliferative effect that occurs in a patient suffering from colorectal cancer at the beginning of the treatment. In the case of primary resistance, said patient is an initial non responder (not sensitive) to said treatment.

Therapeutic method

The present invention relates to a calpain inhibitor for use for delaying or inhibiting the secondary resistance to an anti-cancer agent in a patient suffering from colorectal cancer. The invention also relates to a method for delaying or inhibiting the secondary resistance to an anti-cancer agent in a patient suffering from colorectal cancer, comprising the administration of a calpain inhibitor to said patient.

In other terms, the invention relates to the use of a calpain inhibitor for the manufacture of a medicament for delaying or inhibiting the secondary resistance to an anti-cancer agent in a patient suffering from colorectal cancer.

Preferably, the calpain inhibitor is used for delaying or inhibiting the secondary resistance to irinotecan or SN-38 in a patient suffering from colorectal cancer.

Said calpain inhibitor is preferably a calpain 2 inhibitor.

By "delaying the secondary resistance to an anti-cancer agent", it is meant postponing the onset of secondary resistance to said anti-cancer agent in a patient.

By "inhibiting the secondary resistance to an anti-cancer agent", it is meant preventing the onset of secondary resistance to said anti-cancer agent in a patient.

In a preferred embodiment, the calpain inhibitor is used in combination with chemotherapy, i.e. in combination with an anti-cancer agent. In a preferred embodiment, the anti-cancer agent is selected from 5-fluorouracil, bevacizumab, irinotecan, SN38, bortezomib, oxaliplatin, cetuximab, panitumumab, leucovorine and capecitabine. In a most preferred embodiment, the anti-cancer agent is selected from irinotecan and SN38.

The present invention thus also relates to a product containing an anti-cancer agent and a calpain inhibitor as a combined preparation for simultaneous, separate or sequential use in colorectal cancer therapy.

Without wishing to be bound by theory, the inventors have discovered that said calpain inhibitors prevent the degradation of the ΙκΒ-α subunit, thus inhibiting NF-κΒ activation and subsequent development of secondary resistance to anti-cancers agents, particularly by CPT-l l/SN-38. Without said development of secondary resistance, the anti-cancers agents are still efficient in treating colorectal cancer. The present invention relates to a method for treating colorectal cancer in a secondary resistant patient comprising administering a therapeutically effective amount of a calpain inhibitor and a therapeutically effective amount of an anti-cancer agent. Typically medicaments according to the invention comprise a calpain inhibitor, and optionally an anti-cancer agent, together with a pharmaceutically-acceptable carrier. A person skilled in the art will be aware of suitable carriers. Suitable formulations for administration by any desired route may be prepared by standard methods, for example by reference to well-known text such as Remington; The Science and Practice of Pharmacy.

Method for monitoring the response to treatment

The inventors surprisingly discovered that the expression of the genes of calpain 2, annexin A2 and S 100A10, was increased in a resistant cell line established from a resistant tumor (HT-29R) compared to the HT-29 sensitive parental cell line. Thus, high levels of expression of said genes in the colon cancer cells of a patient are indicative of a secondary resistance to treatment with an anti-cancer agent, particularly CPT-l l/SN- 38, of said patient compared to patients who do not express or express low levels of said genes in said colon cancer cells. In such a case, the treatment has to be re-evaluated, and the addition of a calpain inhibitor has to be considered.

Moreover, cancers like gastric cancer, esophageal cancer, non- small-cell and small-cell lung cancers, leukemia, lymphomas, central nervous system maligant gliomas and ovarian carcinomas, may be treated with CPT-11. This compound has indeed been shown as clinically active against these cancers (Rothenberg ML et al., 2001; 6(1), 66- 80; and (4)).

The invention thus provides a method for monitoring the response to a treatment of a patient suffering from a cancer chosen from colorectal cancer, gastric cancer, esophageal cancer, non- small-cell and small-cell lung cancers, leukemia, lymphomas, central nervous system maligant gliomas and ovarian carcinomas, and preferably colorectal cancer, comprising:

a. treating said patient with an anti-cancer agent for a time period of at least 5 months; then

b. measuring the level of expression of at least one gene selected from the group consisting of calpain, annexin A2 and S 100A10 genes, in cancer cells of said patient.

Typically, in a patient suffering from colorectal cancer, high levels of expression of calpain, annexin A2 and S 100A10 genes, particularly of at least calpain 2 gene, in colon cancer cells is indicative of secondary resistance to treatment with an anti-cancer agent. Indeed, the inventors have demonstrated in the example that calpain 2, S 100A10 and annexin A2 are overexpressed in resistant colon cancer cells, and that calpain 2 overexpression is involved in secondary resistance to CPT-l l/SN-38 treatment.

Thus preferably, if at least S 100A10 and annexin A2 genes, and optionally calpain 2 gene, are overexpressed in colon cancer cells of a patient treated with an anti-cancer agent like CPT-l l/SN-38, further treatment with a calpain inhibitor besides said anticancer agent has to be seriously considered. Preferably, the anti-cancer agent is as defined above. Most preferably, the anti-cancer agent is selected from CPT-11 and SN-38.

Step a. comprises preferably the treatment of said patient with the anti-cancer agent during at least 5 months, preferably at least 6 months.

Preferably, step b. of the method according to the invention comprises measuring the level of expression of at least the calpain 2 gene in cancer cells of said patient.

Preferably, step b. of the method according to the invention comprises measuring the level of expression of at least S100A10 and annexin A2 genes in cancer cells of said patient. Preferably, step b. of the method according to the invention comprises measuring the levels of expression of all the calpain, annexin A2 and S IOOAIO genes, in cancer cells of said patient.

Preferably, step b. of the method according to the invention comprises measuring the levels of expression of all the calpain 2, annexin A2 and S IOOAIO genes, in cancer cells of said patient.

Calpain genes refer to calpain 1 gene and calpain 2 gene.

Annexin A2, also known as annexin II, is a protein that in humans in encoded by the ANXA2 gene. It is a member of the annexin family, and it has interactions with some ligands, including S IOOAIO.

S IOOAIO, also known as pi 1, is a protein that is encoded by the S IOOAIO gene in humans, and the S IOOAIO gene in other species. It is a member of the SlOO family of proteins containing two EF-hand calcium binding motifs, and it is not calcium- dependent.

As used herein, the term "gene expression level" or "level of expression of a gene" refers to an amount or a concentration of a transcription product, for instance mRNA, or of a translation product, for instance a protein or polypeptide. Typically, a level of mRNA expression can be expressed in units such as transcripts per cell or nanograms per microgram of tissue. A level of a polypeptide can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example. Alternatively, relative units can be employed to describe a gene expression level.

As used herein, the expression of "measuring the level of expression of a gene" encompasses the step of measuring the quantity of a transcription product, preferably mRNA obtained through transcription of said gene, and/or the step of measuring the quantity of translation product, preferably the protein obtained through translation of said gene. Preferably, the step of measuring the expression of a gene refers to the step of measuring the quantity of mRNA obtained through transcription of said gene.

In one embodiment of the invention, step b. of measuring the gene expression level is performed by the following method: a) obtaining a biological sample comprising cancer cells, preferably colon cancer cells, from said patient,

b) measuring the level of expression of said gene(s) in said cancer cells in said biological sample.

According to the invention, in case of monitoring the response to a treatment of a patient suffering from colorectal cancer, a biological sample may be a sample of the colorectal tumor tissue or colon cancer cells obtained from the patient according to methods known in the art. Said biological sample is for example a biopsy.

Typically, step b. of measuring the gene expression level may be performed according to the routine techniques, well known of the person skilled in the art.

More preferably, the measurement comprises contacting the cancer cells of the biological sample with selective reagents such as probes, primers, ligands or antibodies, and thereby detecting the presence of nucleic acids or proteins of interest originally in the sample.

In a preferred embodiment, the expression may be measured by measuring the level of mRNA.

Methods for measuring the level of mRNA are well known in the art. For example the nucleic acid contained in the samples (e.g., isolated cancer cells prepared from the patient, like those included in biopsies) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid- binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT- PCR). In a preferred embodiment, the expression of the calpain, annexin A2 or S 100A10 is measured by RT-PCR, preferably quantitative or semi-quantitative RT- PCR, even more preferably real-time quantitative or semi-quantitative RT-PCR.

Other methods of amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).

Probes typically comprise single- stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).

The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.

In a particular embodiment, the methods of the invention comprise contacting the cancer cells of the biological sample with a binding partner capable of selectively interacting with the calpain, annexin A2 or S 100A10 proteins present in the biological sample. The binding partner may be an antibody that may be polyclonal or monoclonal, preferably monoclonal. In another embodiment, the binding partner may be an aptamer. Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.

Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV- hybridoma technique (Cole et al. 1985).

Alternatively, techniques described for the production of single chain antibodies (see e.g. U.S. Pat. No. 4,946,778) can be adapted to produce anti-calpain or anti-annexin A2 or anti-S lOOAlO single chain antibodies. Antibodies useful in practicing the present invention also include anti-calpain fragments and anti-annexin A2 fragments and anti- S 100A10 fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to calpain, annexin A2 or S 100A10. For example, phage display of antibodies may be used. In such a method, single-chain Fv (scFv) or Fab fragments are expressed on the surface of a suitable bacteriophage, e. g., M13. Briefly, spleen cells of a suitable host, e. g., mouse, that has been immunized with a protein are removed. The coding regions of the VL and VH chains are obtained from those cells that are producing the desired antibody against the protein. These coding regions are then fused to a terminus of a phage sequence. Once the phage is inserted into a suitable carrier, e. g., bacteria, the phage displays the antibody fragment. Phage display of antibodies may also be provided by combinatorial methods known to those skilled in the art. Antibody fragments displayed by a phage may then be used as part of an immunoassay.

In another embodiment, the binding partner may be an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. 1997. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by a platform protein, such as E. coli Thioredoxin A, that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

The binding partners of the invention, such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.

As used herein, the term "labelled", with regard to the antibody, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Inl l l, Rel86, Rel88.

The aforementioned assays generally involve the binding of the binding partner (ie. antibody or aptamer) to a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like. The gene expression level of the calpain, annexin A2 or SIOOAIO protein in cancer cells may be measured by using standard immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. In such embodiments, cancers cells are purified from the isolated biological sample. Such assays include, but are not limited to, agglutination tests; enzyme-labelled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis ; immunoprecipitation.

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the target (ie calpain, annexin A2 or S IOOAIO). The cancer cells of the biological sample that are suspected of containing calpain, annexin A2 or S IOOAIO, are then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

FIGURE LEGENDS

Figure 1

In vivo effect of NF-κΒ inhibition on primary and secondary CPT-11 resistance

Evolution of the volume of s.c. HT-29 xenografts. Nude mice received daily oral injections of AS602868, 5 days a week (black squares), and (grey circles)/or (grey triangles) CPT-11 i.p. injections twice a week, or vehicle buffer (black lozenges). Data are the mean + SD of tumor measurements using 10 mice/group and are representative of 2 experiments. Statistically significant differences between control and AS602868 treated groups on the 7^th week and between CPT-11 and CPT-11 + AS602868 treated groups on the 10^th week (first course of treatment), are indicated on the figure (n.s. : not significant). Figure 2

In vitro and in vivo caracterization of HT-29R cells

A-B: Study of HT-29 and HT-29R viability. HT-29 and HT-9R cells were incubated for 5 days with AS602868, SN-38, both compounds simultaneously (A) or several concentrations of 5-FU or etoposide (B). Cytotoxicity was evaluated using the MTT assay. Data are expressed as mean + SD of quadruplicates of one representative experiment out of 5 (A) or 3 (B). Statistically significant differences between HT-29 and HT-29R cells are indicated on the figure (***p<0.001).

C: Clivage of pro-caspase 3 in HT-29 and HT-29R cells stimulated for 3 days with AS602868, SN-38 or both compounds, was studied by Western blotting.

D: Quantification of AS602868 + SN-38 effect on HT-29 and HT-29R cell proliferation by ELISA based on BrdU incorporation in DNA. Statistically significant differences between HT-29 and HT-29R cells are indicated on the figure (*p<0.05, **p<0.01, ***p<0.001).

E: Evolution of HT-29 and HT-29R tumor volumes. HT-29 and HT-29R tumor-bearing mice received respectively CPT-11 i.p. injections (20 mg/ml) twice a week daily with (black squares) (black circles) or without (grey lozenges) (grey triangles) oral injections of AS602868 (20 mg/ml), 5 days a week. Data are the mean + SD of tumor measurements using 5 mice/group and are representative of 2 experiments. Statistically significant differences between CPT-11 treated HT-29 and HT-29R tumor-bearing mice and CPT-11 + AS602868 treated HT-29 and HT-29R tumor-bearing mice on the 10^th week are indicated on the figure.

Figure 3

Study of the NF-κΒ pathway in HT-29 resistant cells and tumors

A-B: NF-κΒ activation was visualized by EMS A. (A) HT-29 and HT29R cells were treated with AS602868 30 min before stimulation with SN-38 for lh. (B) Sensitive tumors (ST) were harvested from mice at the end of the first course of bitherapy (i.e. 10^th week). Resistant tumors (RT) were harvested at the end of the second course of treatment (i.e. 22^nd week). These results correspond to one representative experiment from 3. C: NF-KB activation was measured by reporter gene assay. Luciferase (RLU), Bgal activities and the protein concentration were measured in cell extracts. The luciferase activity was normalized and adjusted to 1 μg protein.

D: Total ΙκΒ- and ΙκΒ- phosphorylation levels were studied by Western blotting in HT-29 and HT-29R cells stimulated for 3 days with AS602868, SN-38 or both compounds. HSP60 was used as loading control.

Figure 4

Role of the increase in NF-κΒ activation in CPT-ll/SN-38 secondary resistance A : NF-κΒ activation of HT-29, RAS, RSN and RAS+SN cells was visualized by EMSA.

B : The cytotoxic effect of SN-38 on HT-29, RAS, RSN and RAS+SN cells was evaluated using the MTT assay after 5 days of incubation. Data are extressed as mean + SD of quadruplicates of one representative experiment out of 4. Statistically significant differences between HT-29 and other cell lines are indicated on the figure (* p<0.05; ***p<0.001).

C : HT-29R and RSN cells were stably transfected with a plasmid coding for the ΙκΒ super-repressor. NF-κΒ activation was visualized by EMSA. The cytotoxic effect of SN-38 on these cell lines was evaluated using the MTT assay after 5 days of incubation. Data are expressed as mean + SD of quadruplicates of one representative experiment out of 3. Statistically significant differences between the parental and the ΙκΒ- super- repressor transfected cell line are indicated on the figure (*p<0.05, **p<0.01, ***p<0.001). Figure 5

Identification of overexpressed and down-regulated genes in HT-29R cells

A : Diagram showing the number of genes that are diferentially expressed between sensitive and resistant HT-29 cells and their expected involvement in the regulation of various biological processes through Ingenuity pathway program analysis.

B : HT-29 and HT-29R (R) cells were analyzed for their RNA level of S 100A10, annexin A2 and calpain 2 by RT-PCR. B actin served as an invariant loading control. C : HT-29, HT-29R, RAS, RSN and RAS+SN cells were analyzed for S 100A10, annexin A2 and calpain 2 expression by western blotting. HSP60 served as an invariant loading control.

D : Sensitive and resistant tumors were analyzed for S100A10, annexin A2 and calpain 2 expression by western blotting. HSP60 served as an invariant loading control.

Figure 6

Study of S100A10, annexin A2 and Calpain 2 involvement in NF-κΒ induction and CPT-ll/SN-38 secondary resistance

A : S 100A10, annexin A2 and calpain 2 expression was analyzed by western blotting after transient transfection of respective siRNA in HT-29R cells. Protein expression was observed after 3, 5 or 7 days of siRNA tranfection. HT-29R cells transfected with non relevant siRNA served as negative control. HSP60 served as an invariant loading control.

B : NF-κΒ activation of HT-29R cells transfected with S100A10, annexin A2 or calpain 2 siRNAs and NF-κΒ activation of RSN cells transfected with Calpain 2 siRNA was visualized by EMSA. HT-29R and RSN cells transfected with non relevant siRNA served as negative control.

C : Sensitive (HT-29) and resistant (HT-29R and RSN) cell lines transfected or not with calpain 2 siRNA or non relevant siRNA (c-), were analyzed for ΙκΒ- expression by western blotting as well as sensitive (ST1 and ST2) and resistant (RT1 and RT2) tumors. HSP60 served as an invariant loading control.

D : The cytotoxic effect of SN-38 on HT-29,HT-29R or RSN cells transfected with calpain 2 siRNA or non relevant siRNA was evaluated using the MTT assay after 5 days of incubation. Data are expressed as mean + SD of quadruplicates of one representative experiment out of 3.

Statistically significant differences between the siRNA control and siRNA calpain 2 transfected cell lines are indicated on the figure (*p<0.05, **p<0.01, ***p<0.001). Supplemental Figure SI

Effect of increasing concentrations of AS602868 or CPT-ll/SN-38 on HT-29 versus HT-29R cell.

Study of HT-29 and HT-29R viability. HT-29 and HT-9R cells were incubated for 5 days with growing concentrations of AS602868 or SN-38. Cytotoxicity was evaluated using the MTT assay. Data are expressed as mean + SD of quadruplicates of one representative experiment out of 5.

Supplemental Figure S2

Comparison of the expression of the proteins from the IKK complex in HT-29 versus HT-29R cells

IKK1, IKK2 and NEMO expression was analyzed by western blotting. HSP60 served as an invariant loading control. Supplemental Figure S3

Comparison of the expression of ABC transpoters and Topol in HT-29 versus HT- 29R cells

P-gp, BCRP and Topoisomerase 1 expression was analyzed by western blotting. HSP60 served as an invariant loading control.

Supplemental Figure S5

Effect of the calpain inhibitor I ALLN on NF-κΒ activation and ΙκΒ-α expression in HT-29R and RSN cells

Left pannel : NF-κΒ activation was visualized by EMSA. HT-29R and RSN cells were treated with 10 μΜ ALLN lh before harvesting.

Right pannel :ΙκΒ-α expression was analyzed by western blotting. HSP60 served as an invariant loading control.

Supplemental Figure S6

Effect of siRNA silencing of SIOOAIO, ANNEXIN A2 or both on the sensitivity of HT-29R cells to CPT-ll/SN-38 S 100A10 and/or Annexin A2 were down regulated by siRNA transfection in HT-29R cells and the sensitivity of these transfected cells to SN-38 was studied by the MTT assay. Data are expressed as mean + SD of quadruplicates of one representative experiment out of 2.

EXAMPLE 1 Materials and Methods Drugs and antibodies

AS602868 is an anilino-pyrimidine derivative and ATP competitor selected for its inhibitory effect in vitro on IKKee, a constitutively active version of IKK2. Detailed descriptions of the AS602868 have been previously published (10, 11). AS602868 in sterile cyclodextrin solution as well as CPT-11 were supplied by Merck-Serono S.A. (Geneva, Switzerland). SN-38 was a kind gift from Dr. J.L. Fischel from Antoine Lacassagne oncology center (Nice, France). Etoposide and 5-fluorouracil were respectively obtained from Sigma Aldrich (Saint Quentin Fallavier, France) and MERCK generiques (Lyon, France). Anti-procaspase 3 was purchased from Medical & Biological laboratories (Woburn, MA); anti-phospho-ΙκΒα from Cell Signaling (Beverly, MA); anti-HSP 60, anti-ΙκΒα, anti-Annexin A2 and anti-Calpain 2 from Santa Cruz Biotechnology (Santa Cruz, CA); anti S 100A10 from BD Biosciences (San Jose, CA).

Xenograft growth assay

Animal experiments were performed in accordance with the regulations of our institution's ethics commission. Fifty NMRI female nude mice (6-8 weeks of age) were inoculated s.c. with 1 x 10⁶ tumor cells. Mice were then dispached into 3 groups of 10 and one group of 20. Treatment schedules and tumors measures have been performed as previously described (10). At the end of the treatments, tumors were removed, minced, put into liquid nitrogen or RNA later (Ambion, Huntingdon, UK) and stored at -80°C. Cell lines and cell drug treatments

The human colon cancer cell line HT-29 was obtained from the ATCC (Bethesda, MD). The HT-29R cell line was established in our laboratory from a HT-29 xenograft resistant to CPT-11 plus AS602868 treatment. RAS, RSN and RAS+SN cell lines were obtained by adding increasing concentrations of either AS602868 or SN-38 or both in the medium for 6 months. At the end, RAS cells were grown in the presence of 3 μΜ AS602868, RSN cells with 10 nM SN-38 and RAS+SN-38 cells with both.

Cytotoxicity and cell proliferation assays

Cytotoxic studies were carried out using a MTT assay (18), representing the percentage of viability inhibition induced by treatments. Five hundred or one thousand HT-29 or HT-29R or RSN cells were respectively plated per well in 96-well plates with medium and various concentrations of AS602868 + SN-38 for 5 days.

Cell proliferation was measured using the ELISA BrdU kit from Roche Diagnostics (Meylan, France). Assays were performed in triplicate following manufacturer's instructions.

EMSA

Nucleic extracts of cells and tumors were prepared according to the method described by Dignam et al. (19). Nucleic extracts and EMSA were performed as described previously (10, 11). The density of bands was quantified using the ImageJ software (NIH, USA).

Luciferase assays

HT-29 and HT-29R cells were transfected using FuGENE (Roche) and 2 μg of a luciferase reporter gene controlled by a minimal thymidine kinase promoter with three reiterated κΒ sites (κΒχ3 thymidine kinase luc). Detailed protocol has been previously published (11) Generation of stably tranfected cell lines

HT-29, HT-29R or RSN cells were transfected with 1 μg of mutated ΙκΒ-α cDNA (pcDNA3-Myc-huS3236AI-KB-a) a construct from our group, or empty vectors and 0.1 μg of the pBABE-puro plasmid (Addgene N°1764) which confers puromycin resistance using FuGENE (Roche) in 6-well plates. Cells were then grown in DMEM medium containing 0.5 μg/ml puromycin permanently. Western blot

Western blot analysis has been described elsewhere (10, 11). The density of bands was quantified using the ImageJ software (NIH, USA).

Reverse Transcriptase-Polymerase Chain Reaction

Total RNA from cells or tumors was prepared in 2-4 ml of Trizol reagent (Invitrogen, Amsterdam, The Netherlands) according to Chomczynski and Sacchi (20). A total of 1 μg RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) following manufacturer's instructions and resuspended in 12 μΐ final volume. Two μΐ of the reverse-transcribed material were amplified by polymerase chain reaction (PCR) in 20 μΐ reactions containing 0.5 μΐ sense and antisense primers (Eurogentec, Angers, France) ; 0.6 μΐ dNTP (20 mM) ; 2μ1 of Taq polymerase (New England Biolabs, Herts, UK) at 5000 u/μΐ of commercial buffer for a total of 22 or 28 cycles consisting of 94°C for 40s, 57°C for 40s and 73°C for 60s for actin, S 100A10 and calpain 2. The Hybridization temperature for annexin A2 was of 64°C. Ten microliters amplification products were analyzed by electrophoresis in ethidium bromide- stained agarose gels. Primer sequences are available upon request.

Knockdown by siRNA

HT-29R cells were forward transfected with control siRNA or a pool of 3 siRNAs (Invitrogen, stealth RNAi) directed against either S 100A10 (HSS 143791,143792, 143793), annexin A2 (HSS 179172, 179173, 179174) or calpain 2 (HSS 101347, 101348, 188705) using the lipofectamine RNAimax (Invitrogen) protocol.

Microarray Experiments

RNAs were extracted using the Rneasy kit Mini (Qiagen) and quantified by nanodrop spectrophotometry. RNA quality was evaluated using the Agilent Bioanalyser 2100 and Lab-on -Chip Nano 6000 chip (ratio of the 28S / 18S RNA > 1.5). Microarrays dedicated for study of cancer (2728 genes linked to proliferation, cell death, cell signalling, invasion, migration and inflammation) were printed using a selection of oligonucleotides from the human Reseau National des Genopoles/Medical Research Council oligonucleotides collection (21). The list of the 2728 probes spotted on the microarray is available on http://www. microarray. fr:8080/merge/index (follow the link: Mediante, Informations, Download files, human local set). RNA were labeled and hybridized as previously described (22). Experimental data and associated microarray designs have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under series GSE23433 and platform record GPL4719.

Statistical analysis

Statistical significance of in vivo drug treatment effectiveness on tumor growth was calculated using ANOVA and the protective least significant difference Fisher test. A probability of less than 0.05 was considered as significant. Statistical significance of drug sensitivity was determined using a nonparametric Mann-Withney U test. Statistical tests were two-sided. Microarray data were normalized by the print tip lowess method (within-array normalization method) and by quantile (between array normalization) using the LimmaGUI package in the software package R Bioconductor (23).

Mean of ratios from all comparisons were calculated for each gene, and B test analysis was done using the Limma package available from Bioconductor. We voluntarily restricted our analysis to genes that exhibited > 1.5-fold modulation and a "p-value" < 0.001. All normalized data sets were registered in the GEO database under the accession number GSE23433.

Biological Theme Analysis

Ontologies attached to each modulated gene were then used to classified theme according to main biological themes. To this end, we used Ingenuity software (http://www.ingeniiity.com/). The data were analyzed for themes using the gene ontology cellular compartment, molecular function, and biological process provided by Ingenuity. Ingenuity software and Mediante (24), an information system containing diverse information about the probes set and the data sets (http://www.microarray.fr), were also used to find gene links and build biological networks.

RESULTS

Inhibition of NF-κΒ using AS602868 does not prevent the appearance of secondary resistance to CPT-ll/SN-38

Fifty mice were inoculated s.c. with HT-29 human colon tumor cells. Treatment started when the mean tumor volume was 150 + 38 mm³ (Figure 1). After 7 weeks, no significant differences in tumor size were observed between control mice and mice treated with AS602868. These mice had to be euthanized for ethical reasons. After 10 weeks of treatment (first course), CPT-11 delayed tumor development (p< 0.0058) and as previously shown (10), the addition of AS602868 significantly improved CPT-11 antitumor effect (p<0.0356). At that time, 10 out of 20 mice of the group which was treated with the bitherapy were euthanized, their tumors harvested, frozen and named "sensitive tumors". For the 10 remaining mice, the treatment was interrupted for 2 weeks. Then, the mice received a second course of bitherapy for 10 weeks. From week 11 till 15, the tumors grew very quickly. From week 15 till 19, the bitherapy was efficient and tumor growth decreased. However, from week 19 till 22 (end of the second course of bitherapy), tumors escaped the treatment (Figure 1). At the 22^nd week, the 10 mice were killed, their tumors removed and named "resistant tumors". From one of those tumors, a cell line was established, cultured and named HT-29R for resistant HT- 29 cells. The carcino-embryonic antigen (CEA) was similarly present on HT-29R and the HT-29 parental cell line showing the absence of contaminating cells (not shown).

In vitro and in vivo characterization of HT-29R cells

Figure 2A shows that in vitro HT-29R cells were not resistant to 3 μΜ of the NF-KB inhibitor AS602868 but rather appeared slightly more sensitive. By contrast, HT-29R cells were significantly (p<0.001) less sensitive to the cytotoxic effect of 10 nM CPT- l l/SN-38 associated or not to AS602868. These results have been confirmed in dose- response experiments using concentrations of AS602868 ranging from 0.3 to 10 μΜ and from 3 to 100 nM for SN-38 (supplemental figure SI). The resistance of HT-29R cells to CPT-1 l/SN-38 appeared rather specific since HT-29R cells were not resistant to the cytotoxic effect of etoposide or 5-Fluorouracil (Figure 2B). Previously, it has been demonstrated that CPT-l l/SN-38 induced apoptosis of HT-29 cells and inhibited their proliferation (10). Western blotting experiments performed on HT-29 and HT-29R cells (Figure 2C) showed that SN-38 (10 nM) and to a lower extend AS602868 (3 μΜ) mediated the cleavage of pro-caspase 3 in HT-29 cells. The addition of AS602868 to SN-38 potentiated SN-38 effect on pro-caspase 3 cleavage. On HT-29R cells, no cleavage of pro-caspase 3 was observed regardless to the treatments. BrdU incorporation (Figure 2D) showed that AS602868 (3 μΜ) weakly inhibited HT-29 and HT-29R cell proliferation. The suboptimal concentration of SN-38 (10 nM) had an antiproliferative effect on HT-29 cells (23%) which reached 68% in the presence of 3 μΜ of AS602868. On HT-29R cells the antiproliferative effect of SN-38 with or without AS602868 was significantly lower than on HT-29 cells. Therefore, HT-29R cells were resistant to the apoptotic and the antiproliferative effects of 10 nM SN-38 in vitro. In vivo, the antitumor effect of CPT-11 and its potentiation by inhibition of the NF-KB pathway was confirmed on HT-29 tumors but these treatments showed a highly reduced efficiency on HT-29R tumors (Figure 2E). NF-KB activation is increased in HT-29 resistant cells and tumors

Unstimulated HT-29 cells displayed a weak constitutive activation of NF-κΒ (Figure 3A, lane 1) that could be decreased by AS602868 (3 μΜ, lane 2). By contrast, SN-38 (10 nM) induced a strong NF-κΒ activation (1.8 fold, lane 3) that was affected by AS602868 (lane 4). Similar results were observed in HT-29R cells except that the cells displayed a higher (1.8 x fold) constitutive NF-κΒ activation (lane compared to lane 1) than in HT-29 cells. A very high level of activated NF-κΒ was also observed ex-vivo (Figure 3B) in 2 HT-29 resistant tumors compared to 2 HT-29 sensitive tumors. This increase in NF-κΒ activation in the resistant cell line was further confirmed in a luciferase reporter gene (Figure 3C) and Western blotting experiments (Figure 3D). The level of phosphorylated ΙκΒ-α in HT-29R cells was globally 1.5 higher than in HT-29 cells when the level of total ΙκΒα was twice lower. Increase of NFKB activation is involved in secondary resistance to CPT-ll/SN-38

To determine if the increase of NFKB activation in HT-29R cells was due to CPT-11, AS602868 or the bitherapy, drug-resistant derivatives of HT-29 cells were obtained after chronic in vitro treatment with either AS602868, SN-38 or both and respectively named RAS, RSN and RAS+SN cells. Figure 4A indicates that NFKB activation was high in RSN cells, moderate in RAS+SN cells and completely decreased in RAS cells compared to the parental HT-29 cell line. Concurrently, MTT assays (Figure 4B) showed that there was a good correlation between the level of NFKB activation and the resistance of these cell lines to SN-38. To evaluate whether the increase of NFKB activation was involved in CPT-l l/SN-38 secondary resistance, HT-29R and RSN cells were stably transfected with a plasmid coding a super-repressor form of ΙκΒ-α which resulted in the down regulation of NF-κΒ activation (Figure 4C). Interestingly, inhibition of NF-κΒ abrogated nearly completely the resistance of HT-29R cells and partially that of RSN cells to SN-38.

HT-29 resistant cells and tumors overexpress the potential NF-κΒ inducers S100A10, Annexin A2 and Calpain 2

The increase of NF-κΒ activation in resistant cells and tumors was neither due to a higher expression of IKK1, IKK2, NEMO (Supplemental figure S2) nor to that of P-gp, BCRP or topol (supplemental figure S3). To gain insight into the resistance mechanisms, gene expression profiles of parental HT-29 and HT-29R resistant cells were performed using local microarrays spotted with 2728 genes dedicated to NF-KB, inflammation, apoptosis... These experiments were done on two independent cell cultures for each cell lines and gave rise to the diagram presented Figure 5A. We found that 36 and 11 genes were respectively significantly overexpressed or downregulated in HT-29R cells (data not shown). The biological theme analysis revealed that these themes were essentially linked to "cancer", "cell movement and morphology" and "cell signaling". As the cancer group of genes was the most important and was organized around NF-κΒ (data not shown), we searched down-regulated NF-κΒ repressor and/or overexpressed NF-κΒ inducers in this gene network. We did not find any repressor gene. However, we found that 2 potential NF-κΒ inducer genes, S 100A10/pl l (25) and calpain 2 (26), were overexpressed. Furthermore, the annexin A2 gene which codes for S IOOAIO main ligand (27) was also overexpressed. The overexpression of these 3 genes in HT-29R cells was then confirmed at the RNA (figure 5B) and protein levels (Figure 5C). Their expression was correlated with the resistance of the cell lines to CPT-l l/SN- 38: weak expression in HT-29 and RAS cell lines and high expression in HT-29R, RSN and RAS+SN cell lines. The expression of these 3 proteins was also significantly increased in 6 resistant tumors compared to 6 sensitive-one (Figure 5D). The expression of S IOOAIO and Annexin A2 was twice and that of calpain 2 was 1.6 times higher in resistant tumors. Calpain 2 overexpression is involved in secondary resistance to CPT-ll/SN-38

Then, we investigated the effect of SIOOAIO, annexin A2 and calpain 2 in the resistance to CPT-l l/SN-38 by siRNA silencing. The expression of each protein was studied 3, 5 and 7 days after transfection with control or specific siRNA (Figure 6A). A complete knockdown of the 3 proteins was evidence 3 days following the addition of the corresponding siRNA whereas a control siRNA had no effect. On the 5^th and 7^th day, the expression of calpain 2 stayed nearly undetectable, that of and annexin A2 was still very low at 5^th but was twice less expressed than in control cells at 7^th day. The expression of S IOOAIO nearly returned to its basal level after 7 days of transfection. Figure 6B showed that the knockdown of SIOOAIO or annexin A2 by siRNA transfection for 3 days, had no effect on NF-κΒ activation nor had that of S IOOAIO and annexin A2 (not shown). By contrast, knockdown of calpain 2 resulted in a sharp decrease in NF-KB activation in HT-29R and RSN resistant cells. As calpain 2 has been demonstrated to induce NF-κΒ activation via the degradation of ΙκΒ-α (26, 28), Western blotting experiments studying ΙκΒ-α expression were performed. As shown in figure 6C, the expression of ΙκΒ-α was lower in the HT-29R (x 2) as well as in RSN cells (x 1.25) and tumors (x 2.6) than in the sensitive HT-29 cells or sensitive tumors, while the transfection of siCalpain 2 in HT-29R or RSN cells resulted in an increased expression of ΙκΒ-α in the 2 resistant cell lines (respectively x 1.7 and x 1.25). Control siRNA had no effect. Similar results were obtained with ΙΟμΜ ALLN, the calpain inhibitor 1 (Supplemental figure S5). HT-29R cells were then incubated or not with control or siCalpain 2 and the effect of increasing concentrations of CPT-l l/SN-38 on their viability was measured (Figure 6D). Calpain 2 knockown was able to significantly resensitize HT-29R cells to CPT-l l/SN-38 at all concentrations tested though calpain 2 knockdown could not completely restore the sensitivity of HT-29R to the level of the HT-29 parental cell line. Similar results were obtained in the resistant RSN cell line. However, silencing S IOOAIO and/or Annexin A2 using siS lOOAlO and/or siAnnexin A2 had no effect on the sensitivity of HT-29R to CPT-l l/SN-38 (supplemental figure S6).

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Claims

1. Calpain inhibitor for use for delaying or inhibiting the secondary resistance to an anticancer agent in a patient suffering from colorectal cancer.

2. Product containing an anti-cancer agent and a calpain inhibitor as a combined preparation for simultaneous, separate or sequential use in colorectal cancer therapy.

3. Calpain inhibitor according to claim 1 or product according to claim 2, wherein said calpain inhibitor is a calpain 1 or a calpain 2 inhibitor.

4. Calpain inhibitor according to claim 1 or product according to claim 2, wherein said calpain inhibitor is a calpain antagonist.

5. Calpain inhibitor or product according to claim 4, wherein said calpain inhibitor is chosen from ALLN, ALLM, calpastatin, calpeptin, SNJ-1945, MDL28170, SJA6017, A- 705253, AK295, and tetracyclines, like chlortetracycline and demeclocycline.

6. Calpain inhibitor according to claim 1 or product according to claim 2, wherein said calpain inhibitor is an inhibitor of calpain gene expression.

7. Calpain inhibitor or product according to claim 6, wherein said calpain inhibitor is chosen from antisense oligonucleotides, siRNAs, shRNAs, ribozymes and DNAzymes.

8. Calpain inhibitor or product according to claim 7, wherein said calpain inhibitor is chosen from siRNAs.

9. A method for monitoring the response to a treatment of a patient suffering from a cancer chosen from colorectal cancer, gastric cancer, esophageal cancer, non-small- cell and small-cell lung cancers, leukemia, lymphomas, central nervous system maligant gliomas and ovarian carcinomas, and preferably colorectal cancer, comprising: a. treating said patient with an anti-cancer agent for a time period of at least 5 months ; then

b. measuring the level of expression of at least one gene selected from the group consisting of calpain, annexin A2 and S 100A10 genes, in cancer cells of said patient.

10. Calpain inhibitor according to any one of claims 1 or 3 to 8, or product according to any one of claims 2 to 8, or method according to claim 9, wherein said anti-cancer agent is selected from 5-fluorouracil, bevacizumab, irinotecan, SN38, oxaliplatin, cetuximab, panitumumab, leucovorine, bortezomib and capecitabine.

11. Calpain inhibitor or product or method, according to claim 10, wherein said anticancer agent is selected from irinotecan and SN38.

12. The method according to any one of claims 9 to 11, wherein step b. comprises the step of measuring the levels of expression of the calpain, annexin A2 and S 100A10 genes, in cancer cells of said patient.

13. The method according to any one of claims 9 to 12, wherein step b. comprises the step of measuring the levels of expression of the calpain 2, annexin A2 and S 100A10 genes, in cancer cells of said patient.