WO2016097175A1 - New method for treating pancreatic cancer - Google Patents

Abstract

The present invention relates to a compound which is an antagonist of LDLR or an inhibitor of the LDLR expression for use in the treatment of pancreatic cancer.

Description

NEW METHOD FOR TREATING PANCREATIC CANCER

FIELD OF THE INVENTION:

The inventions relates to a compound which is an antagonist of LDLR or an inhibitor of the LDLR expression for use in the treatment of pancreatic cancer.

BACKGROUND OF THE INVENTION:

Pancreatic ductal adenocarcinoma (PDAC) is one of the top cancer killers as it ranks to the 4th leading cause of cancer-related death in United States and Europe, with a 5-year survival rate of about 4% and a median survival less than 6 months [Malvezzi M et al, 2013 and Bosetti C et al, 2012]. In the absence of early symptoms, 80% of patients are diagnosed with locally advanced or metastatic pancreatic cancer. For these patients, the palliative chemotherapy with gemcitabine (GEM) remains the most delivered treatment although its survival benefit is limited (5.6 months survival). Recently, two alternative therapies, Nab- paclitaxel plus GEM or Folfirinox (folinic acid, 5-fluorouracil, irinotecan and oxaliplatin) shown an efficacy superior to GEM alone and prolong patient survival by a few months [Assaf E et al, 2011 and Von Hoff DD et al, 2013].

This poor chemotherapeutic response results, in part, to the abnormal development of the stromal compartment during carcinogenesis leading to vascular distortion, oxygen and nutrients deprivation and impairment of drug delivery to tumoral cells. To survive and growth in this hostile microenvironment, cancer cells develop an adaptive metabolic response to meet their excessive demand in energy and biomass. Most of the researches in metabolism focus on the two major blood nutrients, glucose and glutamine, used by pancreatic cancer cells to produce energy and building molecules. However, cell growth can not only rely on these two nutrients sources. The metabolic reprogramming is complex and depends on the type, stage, environmental and genetic contexts of the tumor, all orienting the nature of nutrients up-taken by cancer cells and the metabolic routes used to sustain tumor growth over time. Hence, establishing the metabolic signature of PDAC may highlight key metabolic actors that may constitute interesting therapeutic targets. - -

The inventors defined the metabolic fingerprint of advanced PDAC, induced by both pancreas-specific K-Ras^G12D mutation and Ink4a/Arf deletion [Aguirre AJ et al, 2003], which demonstrates a strong enrichment of dysregulated genes involved in carbohydrate, amino acid and lipid pathways. The lipid enriched-pathways are the most abundant in tumors and those related to cholesterol synthesis and lipoprotein catabolism are among the most activated and enriched pathways in PDAC compared to non-malignant pancreas. These results highlight that pancreatic cancer cells are highly dependent on cholesterol.

Tumor cells have elevated cholesterol requirements that need to be finely regulated. They can increase their cholesterol pools either through activation of endogenous synthesis (i.e. mevalonate pathway), hydrolysis of cholesterol ester (CE) stores or through receptor- mediated endocytosis of plasma cholesterol-rich low density lipoproteins (LDL) via the LDL receptor (LDLR). Cholesterol is highly represented in membrane, especially in micro- domains, named lipid rafts, wherein reside key cell signaling molecules associated to malignant progression [Staubach S et al., 2011]. In cancer cells, changes in cholesterol content of lipid rafts modulate growth factor receptors signaling, such as PI3K/Akt- and EGFR-dependent survival pathway. To prevent the toxic effects of free cholesterol (FC) loading of subcellular organelles, cells esterify and retain excessive cholesterol into CE droplets. This stored cholesterol can then be mobilized by tumor cells in case of and increased demand. Excessive FC is also converted into oxysterols and steroid hormones derivatives. Therefore, the proportion of FC and CE and their distribution within and among organelles and plasma membrane need to be finely regulated at the transcriptional and post-translational levels.

Evidence from pre-clinical studies shows that inhibition of cholesterol synthesis by statins or zoledronic acid, limits tumor growth and formation of metastasis [Sumi S et al., 1992; Kusama T et al, 2002; Gbelcova H et al, 2008], though clinically no significant benefit have been observed for advanced PDAC-patients [Hong JY et al, Cancer Chemother Pharmacol, 2014].

SUMMARY OF THE INVENTION:

Here, the inventors proposed a novel strategy, based on the blockade of LDLR, the main selective route of cholesterol rich- lipoproteins entrance into cancer cells, to limit cholesterol supply to pancreatic tumors. They firstly evaluated whether shRNA- silencing of - -

LDLR, suppresses the tumorigenic properties of pancreatic cancer cells and then elucidated the signaling pathways involved. Secondly, they examined if a reduction in cholesterol uptake affects in vitro PDAC cell sensitivity to standard drugs, and the PDAC syngeneic grafts regression in GEM-treated mice.

Thus, the inventions relates to a compound which is an antagonist of LDLR or an inhibitor of the LDLR expression for use in the treatment of pancreatic cancer.

DETAILED DESCRIPTION OF THE INVENTION: Therapeutic method

The invention relates to a compound which is an antagonist of LDLR or an inhibitor of the LDLR expression for use in the treatment of pancreatic cancer. In another embodiment, the compound according to the invention is administrated in combination with a chemotherapeutic agent.

Thus, the invention also relates to i) a compound according to the invention, and ii) a chemotherapeutic agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of pancreatic cancer.

According to the invention chemotherapeutic agents may be Gemcitabine, Paclitaxel,

Nab-Paclitaxel, Folfirinox, (folinic acid, 5-fluorouracil, irinotecan, oxaliplatin, Erlotinib.

In another particular embodiment, the compound according to the invention is administrated in combination with Gemcitabine.

Thus, the invention also relates to i) compound according to the invention, and ii) the Gemcitabine, as a combined preparation for simultaneous, separate or sequential use in the treatment of pancreatic cancer.

In another embodiment, the invention also relates to i) compound according to the invention, ii) a chemotherapeutic agent and iii) a radiotherapy, as a combined preparation for simultaneous, separate or sequential use in the treatment of pancreatic cancer.

In other word, the invention also relates to i) compound according to the invention, ii) a chemotherapeutic agent and iii) a radio therapeutic agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of pancreatic cancer. - -

In another embodiment, the invention also relates to i) compound according to the invention, ii) the Gemcitabine and iii) a radiotherapy, as a combined preparation for simultaneous, separate or sequential use in the treatment of pancreatic cancer.

In other word, the invention also relates to i) compound according to the invention, ii) the Gemcitabine and iii) a radio therapeutic agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of pancreatic cancer.

As used herein, "radiotherapy" may consist of gamma-radiation, X-ray radiation, electrons or photons, external radiotherapy or curitherapy.

As used herein, the term "radiotherapeutic agent", is intended to refer to any radio therapeutic agent known to one of skill in the art to be effective to treat or ameliorate cancer, without limitation. For instance, the radiotherapeutic agent can be an agent such as those administered in brachytherapy or radionuclide therapy. Such methods can optionally further comprise the administration of one or more additional cancer therapies, such as, but not limited to, chemotherapies, and/or another radiotherapy.

As used herein, the term "LDLR" for "Low-Density Lipoprotein Receptor" denotes a mosaic mature protein of 839 amino acids (after removal of 21 -amino acid signal sequence) that mediates the endocytosis of cholesterol-rich LDL. It is a cell-surface receptor that recognizes the apo lipoprotein B100, which is embedded in the outer phospholipid layer of LDL particles. The receptor also recognizes the apolipoprotein E found in Very Low Density Lipoprotein (VLDL) and Intermediate-Density Lipoprotein (IDL), the precursors of LDL.

In a particular embodiment, the pancreatic cancer is a pancreatic ductal adenocarcinoma (PDAC) or an acinar tumor.

In one embodiment, the antagonist according to the invention may be a low molecular weight antagonist, e. g. a small organic molecule (natural or not).

The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Particular small organic molecules range in size up to about 10000 Da, more particularly up to 5000 Da, more particularly up to 2000 Da and most particularly up to about 1000 Da. - -

In one embodiment, the antagonist may bind to LDLR and block the binding of other compound like the apo lipoprotein B100 or E on LDLR.

In another embodiment, antagonist of LDLR of the invention may be an anti-LDLR antibody which neutralizes LDLR or an anti-LDLR fragment thereof which neutralizes LDLR.

Antibodies directed against LDLR 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. Antibodies useful in practicing the invention can be polyclonal or monoclonal antibodies. Monoclonal antibodies against LDLR 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-LDLR single chain antibodies. LDLR antagonists useful in practicing the present invention also include anti-LDLR antibody 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 LDLR.

Humanized anti-LDLR antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized - - antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397).

Then, for this invention, neutralizing antibodies of LDLR are selected. In a particular embodiment, the antibody anti-LDLR according to the invention may be an antibody as explained in the patent application WO 2001068710.

In a particular embodiment, the antibody anti-LDLR according to the invention may be an antibody as explained in the patent application WO2007014992A2

In still another embodiment, LDLR antagonists may be selected from aptamers. 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. and Gold L., 1990. 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 consists of a conformationally constrained antibody variable region 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).

Then, for this invention, neutralizing aptamers of LDLR are selected.

In another embodiment, LDLR antagonists may be selected from peptides or peptides mimetic. Such peptides or peptides mimetic can be identified thank to their ability to bind the LDLR to inhibit the binding the apo lipoprotein B100 and E on the LDLR and thus blocking the LDL entry in cells. - -

In a particular embodiment, the compound according to the invention is an inhibitor of the LDLR expression.

Small inhibitory R As (siRNAs) or short hairpin RNA (shRNAs) can also function as inhibitors of LDLR gene expression for use in the present invention. LDLR gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that LDLR gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as inhibitors of LDLR gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of LDLR mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both siRNAs, shRNAs (antisense oligonucleotides) and ribozymes useful as inhibitors of LDLR gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the - -

T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides, siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA or ribozyme nucleic acid to the cells and particularly cells expressing LDLR. Particularly, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, the siRNA or ribozyme nucleic acid sequences. Viral vectors are a particular type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno- associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Particular viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991). - -

Particular viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno- associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter may be specific for Muller glial cells, microglia cells, - - endothelial cells, pericyte cells and astrocytes. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.

In one embodiment, nucleases, endonucleases or meganucleases which target the gene which codes for the LDLR can be used as compound according to the invention.

The term "nuclease" or "endonuclease" means synthetic nucleases consisting of a DNA binding site, a linker, and a cleavage module derived from a restriction endonuclease which is used for gene targeting efforts. The synthetic nucleases according to the invention exhibit increased preference and specificity to bipartite or tripartite DNA target sites comprising DNA binding (i.e. TALE recognition site(s)) and restriction endonuclease target site while cleaving at off-target sites comprising only the restriction endonuclease target site is prevented.

Example of nucleases which may be used in the present invention are disclosed in WO 2010/079430, WO2011072246, WO2013045480, Mussolino C, et al (Curr Opin Biotechnol. 2012 Oct;23(5):644-50) and Papaioannou I. et al (Expert Opinion on Biological Therapy, March 2012, Vol. 12, No. 3 : 329-342) all of which are herein incorporated by reference.

A further object of the invention relates to a method for treating pancreatic cancer comprising administering to a subject in need thereof a therapeutically effective amount of a compound which is an antagonist of LDLR or an inhibitor of the LDLR expression.

In another embodiment, the invention relates to a method for treating pancreatic cancer comprising administering to a subject in need thereof a therapeutically effective amount of i) compound according to the invention, and ii) a chemotherapeutic agent, as a combined preparation for simultaneous, separate or sequential.

Compounds of the invention may be administered in the form of a pharmaceutical composition, as defined below.

In one embodiment, said compound is an antagonist of LDLR.

By a "therapeutically effective amount" is meant a sufficient amount of compound to treat pancreatic cancer.

It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of - - the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific antagonist employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Particularly, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, particularly from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The present invention also provides a pharmaceutical composition comprising an effective dose of an antagonist of LDLR and/or compound according to the invention.

Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.

Particularly, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in - - particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

Pharmaceutical compositions of the present invention may comprise a further therapeutic active agent. The present invention also relates to a kit comprising a compound according to the invention and a further therapeutic active agent.

In one embodiment, the pharmaceutical composition is administrated in combination with radiotherapy and/or chimiotherapy.

In one embodiment said therapeutic active agent is an anticancer agent. For example, said anticancer agents include but are not limited to fludarabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbazine, 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, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin A. In one embodiment, additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, anthracyclines, MDR inhibitors and Ca2+ ATPase inhibitors. - -

Additional anticancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.

Additional anticancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.

In the present methods for treating cancer the further therapeutic active agent can be an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiefhylperazine, thioproperazine and tropisetron. In a particular embodiment, the antiemetic agent is granisetron or ondansetron.

In another embodiment, the further therapeutic active agent can be an hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.

In still another embodiment, the other therapeutic active agent can be an opioid or non-opioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.

In yet another embodiment, the further therapeutic active agent can be an anxiolytic agent. Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam. - -

Prognostic method

The invention also relates to an in vitro method for the prognosis of the survival time of a patient suffering from a pancreatic cancer comprising the steps consisting of i) determining the expression level of LDLR in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good outcome prognosis when the expression level is lower than the predetermined reference value and a poor outcome prognosis when the expression level is higher than the predetermined reference value.

Typically, the sample according to the invention may be a blood, plasma, serum sample or a cancer biopsy. In a particular embodiment, said sample is a pancreatic cancer biopsy. In another embodiment, the invention relates to a method for predicting the overall survival (OS) of a patient suffering from a pancreatic cancer comprising the steps consisting of i) determining the expression level of LDLR in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good outcome prognosis when the expression level is lower than the predetermined reference value and a poor outcome prognosis when the expression level is higher than the predetermined reference value.

In another embodiment, the invention relates to a method for predicting the disease- free survival (EFS) of a patient suffering from a pancreatic cancer comprising the steps consisting of i) determining the expression level of LDLR in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good outcome prognosis when the expression level is lower than the predetermined reference value and a poor outcome prognosis when the expression level is higher than the predetermined reference value.

In other words, the invention relates to a method for predicting the recurrence of a patient which has suffered of a pancreatic cancer, to have a tumor relapse comprising the steps consisting of i) determining the expression level of LDLR in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good outcome prognosis when the expression level is lower than the predetermined reference - - value and a poor outcome prognosis when the expression level is higher than the predetermined reference value.

As used herein, the term "Overall survival (OS)" denotes the percentage of people in a study or treatment group who are still alive for a certain period of time after they were diagnosed with or started treatment for a disease, such as pancreatic cancer (according to the invention). The overall survival rate is often stated as a twelve months survival rate, which is the percentage of people in a study or treatment group who are alive twelve months after their diagnosis or the start of treatment.

As used herein, the term "Disease-Free Survival (DFS)" denotes the length of time after primary treatment for a cancer ends that the patient remains free of certain complications or events that the treatment was intended to prevent or delay. These events may include the return of the cancer or the onset of certain symptoms, such as hepatic or pulmonary metastases.

As used herein, the term "Good Prognosis" denotes a patient with more than 50% chance of survival for the next 2 years after the treatment after surgery.

The term "detecting" as used above includes qualitative and/or quantitative detection (measuring levels) with or without reference to a control. Typically LDLR expression may be measured for example by enzyme-labeled and mediated immunoassays (such as ELISA), flow cytometry assessment or qRT-PCR performed on the sample.

The "reference value" may be a healthy subject, i.e. a subject who does not suffer from any cancer and particularly pancreatic cancer. Particularly, said control is a healthy subject.

Detection of LDLR expression in the sample may be performed by measuring the level of LDLR protein or the Ldlr gene.

In the case of the detection of LDLR protein, the methods may comprise contacting a sample with a binding partner capable of selectively interacting with LDLR protein present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, particularly monoclonal.

The presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; - - biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.

The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. 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.

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is 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 labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.

Various immunoenzymatic staining methods are known in the art for detecting a protein of interest. For example, immunoenzymatic interactions can be visualized using different enzymes such as peroxidase, alkaline phosphatase, or different chromogens such as DAB, AEC, or Fast Red; or fluorescent labels such as FITC, Cy3, Cy5, Cy7, Alexafluors, etc. Counterstains may include H&E, DAPI, Hoechst, so long as such stains are compatable with other detection reagents and the visualization strategy used. As known in the art, amplification reagents may be used to intensify staining signal. For example, tyramide reagents may be used. The staining methods of the present invention may be accomplished using any suitable method or system as would be apparent to one of skill in the art, including automated, semi-automated or manual systems.

The method of the invention may comprise a further step consisting of comparing

LDLR expression with a control reference.

In the case of detection of the Ldlr gene, the term "expression level of LDLR " refers to an amount or a concentration of a transcription product, for instance mRNA coding for Ldlr - - gene. 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 protein 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 an expression level. Methods to detect a level of mRNA are well known in the state of art.

Typically, a "threshold value", "threshold level", "reference value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. Particularly, the person skilled in the art may compare the expression levels of LDLR obtained according to the method of the invention with a defined threshold value.

Particularly, said threshold value is the mean expression level of LDLR of a population of healthy individuals. As used herein, the term "healthy individual" denotes a human which is known to be healthy, i.e. which does not suffer from a cancer and in particular from a pancreatic cancer and does not need any medical care.

Typically, the skilled person in the art may determine the expression level of LDLR in a biological sample, particularly a biopsy of a pancreatic cancer, of 100 individuals known to be healthy. The mean value of the obtained expression levels is then determined, according to well known statistical analysis, so as to obtain the mean expression level of LDLR. Said value is then considered as being normal and thus constitutes a threshold value. By comparing the expression levels of LDLR to this threshold value, the physician is then able to classify and prognostic the cancer.

Accordingly, the physician would be able to adapt and optimize appropriate medical care of a subject in a critical and life-threatening condition suffering from cancer. The determination of said prognosis is highly appropriate for follow-up care and clinical decision making.

The present invention also relates to kits useful for the methods of the invention, comprising means for detecting LDLR expression.

According to the invention, the kits of the invention may comprise an anti- LDLR protein antibody; and another molecule coupled with a signalling system which binds to said LDLR protein antibody or any molecule which bind to the mRNA of Ldlr gene like a probe. - -

Typically, the antibodies or combination of antibodies are in the form of solutions ready for use. In one embodiment, the kit comprises containers with the solutions ready for use. Any other forms are encompassed by the present invention and the man skilled in the art can routinely adapt the form to the use in immunohistochemistry.

In another embodiment, the invention relates to an in vitro method for monitoring a patient's response cancer treatment which comprises a step of measuring the level of LDLR expression, in a sample from a patient.

Thus, the present invention relates to the use of LDLR as a biomarker for the monitoring of anti pancreatic cancer therapies.

According to the invention, the expression level of LDLR may be determined to monitor a patient's response to pancreatic cancer treatment.

Another aspect of the invention relates to a compound which is an antagonist of LDLR or an inhibitor of the LDLR expression for use in the treatment of patient suffering of a pancreatic cancer with a high expression level of LDLR.

The invention will be further illustrated by the following figures and examples.

However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Up-regulation of cholesterol uptake correlates to cholesterol overload in PDAC. A. Mean LDLR and HMGCR protein levels in control pancreas and PDAC (from n=5 mice) are normalized to total loaded-protein. B. Free (FC) and esterified cholesterol (CE) quantities in control pancreas (n=5) and PDAC (n=6) normalized to respective total lipid content. For the figure, data are mean ± SEM, *P < 0.05, Student's t-test or Mann- Whitney U- test.

Figure 2. LDLR silencing, by modifying cholesterol distribution, potentiates the gemcitabine (GEM)-induced PDAC regression. Tumor regression and weight measured in - -

GEM treated-mice implanted with Ldlr3 and Ctrl shR A and cells (n=10 per group). Data are mean ± SEM and are expressed as the fold-decrease in tumor growth or weight measured in GEM-treated mice relative to untreated mice. P value is relative to growth curve obtained from GEM treated-mice implanted with Ctrl shRNA cells.

Figure 3. Cutpoints, relative to disease-free survival (DFS) and overall survival (OS), determined using maximally selected rank statistics which allow partition of the patient cohort into two groups (low and high Ldlr expression). Figure 4. A high expression of Ldlr in human PDAC is associated with an increased risk of recurrence. Kaplan-Meier disease-free survival (A) and overall survival (B) curves for 23 PDAC patients, divided into high- and low Ldlr expression groups. Differences between group survival distributions have been estimated with log rank test.

EXAMPLE: Material & Methods Human PDAC samples. Tumor specimens from 23 patients (54-79 years old) were taken, with patient consent, during surgery (Tissue collection: DC-2013-1857). PDAC stages were classified according to the American Joint Committee on Cancer staging system. A postoperative follow-up including clinical, biochemical and radiological assessment was performed for all patients.

Mouse strains and tissue collection. We used male Pdxl-Cre; Ink4a/Arffl/fl; LSL- KrasG12D mice, developing spontaneous PDAC, between 8 to 12 weeks of age, and their mating control littermates, as previously described (1). After sacrifice, pieces of tumor or control pancreas were fixed in 4% formaldehyde or frozen in cold isopentane for further analysis, or directly homogenized in 4M guanidinium isothiocyanate lysis buffer for efficient pancreatic RNA extraction, according to Chirgwin's procedure (2). All animal care and experimental procedures were performed in accordance with French Guidelines for animal handling. - -

Microarray analysis. Expression array data (Mouse Transcriptome Assay 1.0, Affymetrix,) were normalized with standard RMA (Bioconductor, Package Affy) and filtered from a pre-compiled GO terms list, annotated with the term "metabolic" (NCBI GO-gene association file downloaded the 1st of April, 2013), to restrain analysis to metabolic transcripts. Significant PDAC dysregulated transcripts and enriched pathways were determined by the SAM method (MeV software) and a Bonferroni-corrected hypergeometric distribution, respectively. Results can be accessed from the GEO database (http://www.ncbi.nlm.nih.gov/geo/, accession number: GSE61412). Human tumor collection and mRNA extraction. A portion of each human tumor, collected during surgery, was fixed in 10% formalin for histology analysis, and another portion was used for RNA isolation using the AllPrep DNA/RNA mini kit (Qiagen, France). RNA quality was verified on RNA Nano chips (Agilent, Santa Clara, CA, USA). Establishment of stable shRNA PK4A cell lines. 293T cells were cotransfected with lentiviral vectors expressing shRNA targeting LDLR (Ldlrl, clone ID: NM O 10700.1 - 1457slcl; Ldlr2, clone ID: NM_010700.1-2457slcl; Ldlr3, clone ID: M_010700.2- 3407s21cl, Ldlr4, clone ID: NM_010700.1-1304slcl; Ldlr5, clone ID: NM 010700.1- 1864slcl, Ldlr6, clone ID: NM_010700.2-1124s21cl and clone ID: M_010700.2- 642s21cl; Sigma- Aldrich) or a non-mammalian control shRNA (SHC202V, Sigma- Aldrich), together with pCMV-delta helper and pCMV-VsVg plasmids, using lipofectamine 2000 (Invitrogen). PK4A cells (1.3.106), were infected twice at 24h intervals with shRNA- expressing lentiviruses. Puromycin selected cells were grown in glutamax high glucose DMEM medium (Life Technologies), supplemented with 10% fetal bovine serum GOLD (PAA Laboratories), 1% antibiotic-antimycotic solution (Life Technologies) and puromycin (0.2 μg/ml).

PDAC syngenic graft models. Under isoflurane anesthesia (induction: 4% and maintenance: 1.5%), five week-old male NMRI nude mice (Harlan, France) were subcutaneously implanted, between the shoulders, with 2.106 control or Ldlr3 shRNA PK4A cells. Two times each week, GEM (125mg/kg, Lilly France) was intraperitoneally administered and tumor size was determined with calipers. Tumor volume was established with the following formula: (L x W2)/2 and tumoral weight was determined at the experimental endpoint. - -

Cell viability assays. 1.105 Ldlr shRNA and control shRNA PK4A cells were plated in 12-well plates for 24h before replacement of the standard medium with DMEM supplemented with 5% FBS and 1% antibiotic- antimycotic solution. Cells were trypsinized after either 24 or 48 or 72 hours and live cells were determined by trypan blue exclusion using the automated cell countess apparatus (Life Technologies) or cell viability analyzer (Vi-Cell, Beckman Coulter). Diameter of live cells was determined with Vi-Cell counter. Cell proliferation was monitored every hour for 80 hours using the iCELLigence Sytem (ACEA Biosciences). Cell electrode impedance was expressed as an arbitrary unit, called cell index corrected for background impedance of the media alone.

Dil-LDL uptake. Control and Ldlr3 shRNA PK4A cells (1.106) were seeded in 6-well culture plates and cultured in standard medium overnight. After 1 hour of serum starvation, cells were incubated with Dil-LDL (5μg/ml, Molecular Probes) for 30min. Trypsinized cells were then resuspended in IX PBS and Dil-LDL uptake was analyzed in 1.104 cells using a MACSQuant VYB instrument (Miltenyi Biotec). Untreated cells were used as negative controls for background fluorescence.

Clonogenic assays. 8.102 control and Ldlr3 shRNA PK4A cells were seeded into a 12-well plate and cultured in high glucose DMEM media with 10% FBS and 1% antibiotic- antimycotic solution. Five days later, colonies were fixed in 10% formalin, stained with 0.5% crystal violet, imaged and quantified by Image J software (NIH, MD, USA).

Cell cytotoxicity assay. 8.104 control and Ldlr3 shRNA PK4A cells (8.104), seeded in 96-well plates, were treated with gemcitabine (Gemzar®, Lilly), oxaliplatin (Hospira), (1/4 dilution scheme: 1 to 1000 nM) or with SN38 (Sigma-Aldrich, H-0165) (1/4 dilution scheme: 0.1 to ΙΟΟηΜ) for 48h. Cell viability was determined with PrestoBlue reagent (Life Technologies) and fluorescence intensity was measured detected with a microplate reader (TriStar, Berthold Technologies). The half inhibitory concentrations (IC50) were calculated using a four parameter logistic non- linear regression with BioDataFit 1.02 software.

Cell cycle analysis. Control and Ldlr3 shRNA PK4A cells (1.106) were fixed in 70% ethanol. After RNA removal by RNase digestion (100 μg/ml), cells were stained with propidium iodide (50μg/ml, Sigma-Aldrich) and analyzed by flow cytometry using a - -

MACSQuant VYB instrument (Miltenyi Biotec). The cell number in each phase was determined using the Watson Pragmatic model with FlowJo software (Tree Star Inc., Ashland, OR, USA). Cholesterol assays. Total lipids from control or tumoral pancreas, control and Ldlr3 shRNA -implanted tumors or control and Ldlr3 shRNA PK4A cells were extracted according to the Bligh and Dyer method (3). Free and esterified cholesterol were separated by High Performance Thin Layer Chromatography (Camag, Switzerland) and quantified by scanning densitometry with a TLC Visualizer (Camag, Switzerland).

Glucose and Lactate assays. Control and Ldlr3 shRNA PK4A cells (1.105) were grown in High glucose DMEM supplemented with 5% FBS and 1% antibiotic- antimycotic solution for 24, 48 or 72 hours. The concentrations of L-lactate and D-glucose (mmol/1) from cultured-cell supernatants were determined electro-enzymatically using an YSI 2950 Biochemistry Analyzer (Yellow Springs Instruments, USA). Each metabolite concentration was normalized to viable cells number determined using the Vi-Cell cell counter (Beckman Coulter).

Doxorubicin uptake. Control and Ldlr3 shRNA PK4A cells (1.106) were seeded in 6- well culture plates and cultured in standard medium overnight. Cells were then treated with doxorubicin (DOX, lmg/ml) for 15, 30, 60, 120 or 240 min. Trypsinized cells were then resuspended in IX PBS and DOX uptake was analyzed in 1.104 cells using a MACSQuant VYB instrument (Miltenyi Biotec). Untreated cells were used as negative controls for background fluorescence.

Immunohistochemistry. Formalin-fixed, paraffin-embedded mouse PDAC sections (5μιη) were deparaffmized in xylene and rehydrated through a serie of graded ethanol concentrations. Antigen retrieval was performed at 95°C in target retrieval solution (pH6, Dako), before quenching endogenous peroxidase activity (3% H202). Tissue sections were then incubated with goat anti-LDLR antibody (R&D systems, AF2255) and immunoreactivity was visualized using the Vectastain ABC kit (PK-4001 , Vector Laboratories) according to the manufacturer's protocol. Peroxidase activity was revealed using the liquid-DAB+ substrate chromogen system (Dako). Counterstaining with Mayer hematoxylin was followed by a - - bluing step in 0.1% sodium bicarbonate buffer, before final dehydration, clearance and mounting of the sections.

Immunohisto chemistry with an anti-human LDLR antibody (LifeSpan Biosciences, CI 93443) was performed on formalin- fixed and paraffin-embedded human PDAC sections using the Ventana Discovery XT automated stainer (Ventana Medical Systems, Tucson, USA) in the Pathology Department (Hopital Nord, Marseille). Antigen retrieval was performed with CCl buffer (Cell Conditioning 1; citrate buffer pH 6.0, Ventana Medical Systems). Immunofluorescence. Cryostat tumor and control pancreas sections (8μιη) were fixed in cold acetone and pre incubated in blocking solution (3% BSA, 10% donkey serum). Tissue- sections were then incubated with primary antibody(ies), followed by incubation with alexa fluor 568 and/or alexa fluor 488-conjugated secondary antibodies (Molecular probes, 1 :500). Stained tissue sections were mounted using Prolong Gold Antifade reagent with DAPI (Life Technologies).

Filipin staining. Frozen tissue sections (tumor or control pancreas) or Ldlr3 shR A PK4A cells were fixed in 4% formaldehyde and incubated with 1.5 mg/ml glycine. A heat induced-antigen retrieval step, in 10 mM Sodium citrate, 0.05%> Tween 20, pH6, was performed for tissue section.

Tissue sections were incubated overnight at 4°C with filipin (200 μg/ml, Sigma- Aldrich, F4767) and then with anti-Lamin A/C antibody (1 : 100, Cell Signalling Technology) for 1 hour at room temperature. Lamin A/C staining was revealed with alexa fluor 568- conjugated secondary antibody (Molecular probes, 1 :500). Tissue sections were mounted using Prolong Gold Antifade reagent without DAPI (Life Technologies).

Cells were stained 30 minutes with filipin (50 μg/ml) and then incubated in Syto Red fluorescent nucleic acid stains (1/20000, Molecular Probes) for 10 minutes before to be mounted as described above. Filipin staining was viewed by fluorescence microscopy using a DAPI filter and green pseudocolor was assigned to it to improve reader visibility.

Western-blot. Whole cell proteins (50-65 μg/lane) were resolved by SDS-PAGE using a 10% acrylamide gel and were transferred onto nitrocellulose membranes which were then blocked in 5% milk in PBS, before being incubated overnight with primary antibodies raised against LDLR (R&D systems, AF2255), HMGCR, P-ERK1/2, ERK1/2 or D-actin. ECL - - protein detection (Milipore) was performed with a Fusion Fx7 chemiluminescent imager and the band intensities of the protein(s) of interest were determined by densitometry using Image J software (NIH, MD, USA). The band intensities of the proteins of interest were normalized to respective β-actin band intensities for cell protein extracts, or to the total protein loading and stained by amido black for tissue protein extracts.

Primary antibodies. Wide-spectrum KRT (Abeam, ab9377), E-Cadherin (Invitrogen, 13-1900), N-Cadherin (Hypromatrix, HM1049), HMGCR (Santa Cruz Biotechnology, sc- 33827), P-ERK1/2 (Sigma-Aldrich, M8159), ERK1/2 (Sigma-Aldrich, M5670), Lamin A/C (Cell Signalling Technology, 2032) and β-actin (Sigma-Aldrich, A5316).

PathScan signaling antibody array analysis. An antibody array for simultaneous detection of 18 well characterized intracellular signaling molecules was used (Cell Signaling Technology, 7323). Control and Ldlr3 shRNA PK4A cells were washed with ice-cold IX PBS and lysed in the IX Cell Lysis buffer provided. Total protein extracts were then hybridized, to the slide containing pre-spotted target-specific antibodies, overnight at 4°C. A cocktail of biotinylated detection antibodies was then added to each well and incubated for lh at room temperature and Horseradish peroxidase (HRP)-conjugated streptavidin was then added for 30 min. The slide was covered with the LumiGLO/Peroxide reagent provided and chemiluminescent signals were detected with a Fusion Fx7 imager.

Reverse transcript PCR and qPCR. Before reverse transcription, RNA quality was verified with the RNA Nano Chip kit (Agilent) on an Agilent Bioanalyzer and treatment with DNase was systematically performed using the RNase-free DNase set (Qiagen). Then, 5μg of total RNA from each sample was used to synthesize the first-strand cDNA by using the PrimeScript RT reagent kit (Promega) and the provided oligo-dT primers, according to manufacturer's instructions. Quantitative PCR reactions were performed with specific primers (Table S7) and the GoTaq qPCR master mix kit (Promega) using the Mx3000P Stratagene system. Differential expressions of transcripts of interest were calculated in relation to the RplpO housekeeping transcript.

Statistical analysis. Significant differences between two experimental groups were determined using the Mann- Whitney U test or T-Test. One-way analysis of variance (ANOVA) was used to compare more than two experimental groups followed by Tukey's HSD post hoc tests (Real Statistics). The patients cohort was separated in two groups, based - - on the levels of Ldlr expression in tumors (high and low Ldlr expression groups), to maximize the p-value of the Kaplan-Meier model. Overall- and disease free-survival curves, estimated using the Kaplan-Meier method, were compared by the log-rank test, and a P-value of 0.05 was considered statistically significant. All data are expressed as mean ± SEM or s.e. and statistical significance was defined as *, P <0.05.

Results Up-regulation of cholesterol and lipoprotein metabolic pathways in PDAC

Here, we used DNA microarray technology to identify transcripts involved in metabolic processes, which were differentially expressed between invasive PDAC and control pancreas. We used control mice (Ink4a/Arffl/fl; LSL-KrasG12D) and mice bearing spontaneous PDAC (Pdxl-Cre; Ink4a/Arffl/fl; LSL-KrasG12D) with histological and clinical features similar to those reported in humans.

We filtered the entire murine genome, to select only metabolic transcripts, which constitute 12% of the mouse genome (i.e. 2177 transcripts), and encodes for known enzymes or transporters. Using the Significance Analysis of Microarrays (SAM) method, we showed that 427 transcripts were significantly up-regulated and 320 were down-regulated in PDAC, when compared to control pancreas.

Subsequent Bonferroni-corrected hypergeometric distribution analysis, showed that the PDAC metabolic signature was highly enriched in up- and down-regulated pathways associated with amino acid, carbohydrate and lipid metabolism. Of these, the lipid class contained the greatest number of disrupted pathways in PDAC. In particular, pathways associated with lipoprotein catabolism and its negative regulation, and those involved in regulating cholesterol homeostasis were the most highly enriched in PDAC (enrichment of 100%). Biosynthetic lipoprotein and retinoic acid pathways and glycosphingo lipid metabolism were also over-represented in PDAC (enrichment ranging from 57 to 75%). In contrast, enriched- fatty acid, acylglycerol and triglyceride metabolic processes were highly depleted in PDAC when compared to control pancreas. Heat map representations of the differential expression of cholesterol and lipoprotein associated transcripts in PDAC and control pancreas, revealed that LDLR facilitating circulating lipoprotein uptake, the apolipoprotein B100 and E (ApoB, ApoE) which form lipoproteins, and the ATP binding cassette transporter Al (ABCAl), mediating cholesterol efflux, were drastically up-regulated - - in the tumor. Moreover, up-regulation of transcripts involved in the cholesterol synthesis pathway and in the synthesis of its derivatives (oxysterols, steroid hormones), was also observed in PDAC when compared to control pancreas (data not shown). Finally, the master transcriptional activator of cholesterol synthesis and uptake, the sterol response element- binding factor 2 (Srebf2)(10), was also over-expressed in PDAC.

So, the abundance and large diversity of transcripts encoding key enzymes, transporters and apo lipoproteins involved in cholesterol-related metabolic pathways, strongly indicate that pancreatic cancer cells have a high dependency on cholesterol. Cholesterol uptake contributes to the increase of cholesterol content in PDAC

We next validated the over-expression of transcripts involved in cholesterol synthesis and storage, oxysterol and steroid synthesis and cholesterol uptake in four additional PDAC. The expression of Hmgcr, (3-Hydroxy-3-methylglutaryl coenzyme A reductase), which encodes for the rate-limiting enzyme of cholesterol synthesis, was 23 times higher in PDAC than in control pancreas, though its protein level was not so abundant, probably due to an increase in its degradation rate (10). Other cholesterologenic transcripts and those involved in cholesterol storage, Lipase A (Lipa) and, cholesterol acyltransferase 1 (Acatl) displayed high expression levels in the tumor when compared to control pancreas. These data indicate that cholesterol synthesis and processes preventing cytotoxic FC loading, such as cholesterol esterification by AC ATI , or promoting LIPA induced hydrolysis of CE, contained in droplets or lipoproteins, co-exist in the tumor. Interestingly, in PDAC, the most up-regulated transcript in the steroid hormone synthesis pathway encodes for SRD5A1, which promotes the conversion of testosterone into its active metabolite, the dihydrotestosterone. Finally, concerning lipoprotein-dependent cholesterol uptake, Ldlr and ApoE are 8.2 and 6.2 times higher in PDAC when compared to control pancreas, suggesting that, along with cholesterol synthesis, cholesterol uptake is also strongly stimulated in the tumor. Importantly, whereas HMGCR levels are enhanced slightly in PDAC, the Ldlr increase is associated with a 7.7 fold- increase in its protein level (Figure 1 A). This latter result demonstrates the pivotal role of the key cholesterol uptake facilitator, LDLR, and to a much lesser extent HMGCR, in cholesterol supply to PDAC cells. In the entire tumor, we effectively demonstrated a 3.5 fold- increase in the total cholesterol (TC) content which is furthermore composed of 50% stored- CE (Figure IB). In PDAC, filipin labeled-FC is detected in epithelial cell membranes and the cytoplasm while it is mainly present in islets of Langerhans in control pancreas. - -

These results demonstrate a high avidity of pancreatic tumors for cholesterol, which appears mostly satisfied by cholesterol uptake. Since, targeting of cholesterol synthesis has proven ineffective for PDAC treatment, therefore, blocking cholesterol uptake with LDLR silencing, in order to alter the content and distribution of cholesterol in the tumor may help to define the impact of such a blockade on the tumorigenic properties of pancreatic cancer cells.

LDLR is expressed in the epithelial compartment of PDAC

Using histological analysis, we determined the spatial LDLR expression in stromal and epithelial PDAC compartments. We showed that LDLR was restricted to PDAC, since no specific staining was observable in controls. In tumors, LDLR was chiefly expressed in the epithelial compartment and more specifically in well differentiated cells, which were organized into glands and stained with the pan-cytokeratin (pan-KRT) epithelial marker. Moreover, LDLR was also present in undifferentiated cancer cells, which were disseminated into the stroma. Although these LDLR-undifferentiated cells were stained with pan-KRT marker, they had lost the E-Cadherin epithelial marker, and acquired the N-Cadherin mesenchymal marker.

These results illustrate that LDLR is present in the epithelial compartment of the tumor, both in differentiated and aggressive cells which exhibit an epithelial to mesenchymal transition phenotype.

LDLR silencing, by modifying cholesterol distribution, inhibits ERK survival pathway and reduces the proliferative and clonogenic potential of pancreatic cancer cells

PK4A cells, established from Pdxl-Cre; LSL-Kras^G12D; Ink4a/Arf^a/fl tumors, were used to investigate the impact of LDLR inhibition on tumorigenic properties of PDAC cells. PK4A cells, expressing shRNA which targeted different Ldlr sequences, were established and validated for LDLR knock-down (data not shown). Successful LDLR knockdown was achieved with Ldlr3 shRNA (i.e. a 70% decrease in LDLR levels when compared to control shRNA cells), which significantly correlated to a 50% reduction in the uptake of labeled-LDL (Ι,ΐ' -dioctadecyl-l-3,3,3,3-tetramethyl-indocarbocyanine perchlorate-LDL, Dil-LDL). Interestingly, LDLR silencing did not disturb TC content, but modified its distribution, as the CE content was reduced by 46%, while the FC fraction was 1.9-fold increased and detected in membrane and cytoplasm of PDAC cells. However, the increase in FC was not associated with an over-activation of cholesterol synthesis, since HMGCR protein levels were not altered - - by LDLR silencing, nor the uptake of glucose from which the cholesterol is synthesized (data not shown). Moreover, the same quantities of glucose were shifted away from the tricarboxylic acid (TCA) cycle, a prerequisite for cholesterol synthesis, toward lactate formation in the two shRNA PK4A cells. Thus, these data show that LDLR-depleted cells do not use compensatory mechanisms, as activation of cholesterol synthesis, to counteract the inefficient cholesterol uptake.

To investigate the role of LDLR in supporting the growth and survival of PDAC cells, we performed real-time impedimetric cell proliferation monitoring along with colony formation assays. We showed that LDLR silencing significantly decreased the proliferation rate as well as the number of colonies formed, by 50%. Importantly, the morphology and size of PDAC cells were not impacted by LDLR silencing. We then investigated which survival signaling pathways are disturbed in LDLR-depleted cells. By using a PathScan Intracellular Signaling array, we directly detected a reduction in expression of phosphorylated ER l/2 (pERKl/2) and an increase of pGSK-3P in Ldlr3 shRNA PK4A cells when compared to control shRNA cells, among other unchanged pro- and anti-proliferative signaling pathways. The ERKl/2 survival pathway is particularly interesting since it is constitutively activated by the K-Ras^G12D oncogene in PDAC cells, and here we reported that LDLR depletion drastically reduced pERKl/2 levels over time (24, 48 and 72 hours) when compared to control shRNA cells. Thus, silencing LDLR may be an alternative strategy to prevent the constitutive K- Ras^G12D ERK activation.

LDLR silencing enhances both the cytotoxic effects of chemotherapy drugs on PDAC cells and GEM-induced PDAC regression

Gemcitabine and FOLFIRINOX, despite limited benefits, remain the drugs commonly used for PDAC treatment. Efforts are currently being made to increase the therapeutic efficacy of these drugs by combination with other antitumoral agents. Here, we investigated whether cholesterol distribution disorder, induced by inhibition of cholesterol uptake, altered the drug sensitivity of PDAC cells. We showed that LDLR depleted-cells were 2- and 3 -times more sensitive to GEM and SN38 (an active metabolite of irinotecan) than control cells. Importantly, the half-inhibitory concentration (IC50) of each of these drugs shifted towards the inhibitory concentrations 1.9 and 3 times lower in LDLR depleted-cells when compared to control shRNA cells. In contrast, LDLR depletion did not affect the oxaliplatin dose-response. These results indicate that cholesterol-associated metabolic disruption strengthens the cytotoxic effects of several drugs on PDAC cells. - -

Using pancreatic syngeneic tumor graft mice, we then evaluated whether LDLR inhibition potentiates GEM-dependent tumor regression. One week after tumor establishment, half of the Ldlr3 and control shRNA PK4A implanted-mice were treated, twice weekly, with GEM. We noted that, tumor growth was reduced by 50% in GEM treated Ldlr3 shRNA mice when compared to GEM treated control shRNA mice (Figure 2A), along with a 2.3-fold reduction in tumor weight in GEM-treated Ldlr3 shRNA mice when compared to GEM treated control shRNA mice (Figure 2B). Importantly, at this time, LDLR protein levels remain efficiently reduced in Ldlr3 shRNA implanted-tumors when compared to control shRNA tumors, and HMGCR expression was not disturb by LDLR silencing, as previously shown in vitro. Moreover, LDLR silencing, associated or not with GEM, did not modify TC content when compared to control shRNA tumors. Interestingly, when combined to LDLR silencing, GEM altered the cholesterol distribution (i.e. the FC:CE ratio). Indeed, the FC was increased by 80% and CE stores were almost depleted in GEM treated Ldlr3 shRNA tumors compared to untreated counterpart (FC:CE ratio of 0.5:0.5 in untreated vs 0.9:0.1 in GEM treated Ldlr3 shRNA tumors).

These data show that inhibition of LDLR combined with GEM treatment, by inducing cholesterol distribution damage, impedes tumor growth more efficiently than GEM alone.

Increased Ldlr gene expression is an indicator of poor prognosis in human pancreatic cancer

To investigate LDLR inhibition as a potential therapy for PDAC resected-patients, we used immunohistochemistry to clinically validate the LDLR expression in 10 human PDAC samples. All tumors expressed LDLR, predominantly in the membrane and cytoplasm compartments of epithelial cancer cells, as shown previously in mice. The analysis of Ldlr transcript profiles in 23 other tumors showed that although expression varied between samples, this differential expression was not correlated with tumor stage. Then, two patient cohorts (low and high Ldlr expression groups) were defined on either side of a cut-off point determined using receiver operating characteristic curve analysis (Figure 3 A and B). We then determined the impact of such variation in Ldlr expression on overall- and disease-free survival (OS and DFS, respectively) by Kaplan-Meier method. We showed that high Ldlr expression was significantly associated with an increased risk of primitive tumor or metastasis recurrence (showed by a reduction of DFS, p=0.008) (Figure 4A), while the OS was not significantly impacted by Ldlr expression levels (p=0.201) (Figure 4B). - -

Thus, in human PDAC, enhanced Ldlr expression, regardless of tumor stage, indicates a poor patient prognosis. Therefore, using a therapy designed to block the activity of this receptor, independently, or in combination with chemotherapy, must be considered as a promising therapeutic strategy for these patients.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Aguirre AJ, Bardeesy N, Sinha M, Lopez L, Tuveson DA, Horner J, et al. Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma. Genes Dev 2003;17(24):3112-26.

Assaf E, Verlinde-Carvalho M, Delbaldo C, Grenier J, Sellam Z, Pouessel D, et al. 5- fluorouracil/leucovorin combined with irinotecan and oxaliplatin (FOLFIRINOX) as second- line chemotherapy in patients with metastatic pancreatic adenocarcinoma. Oncology 2011;80(5-6):301-6.

Bovenschen Niels, Koen Mertens, Lihui Hu, Louis M. Havekes, and Bart J. M. van

Vlijmen. LDL receptor cooperates with LDL receptor-related protein in regulating plasma levels of coagulation factor VIII in vivo. Blood, 1 august 2005.

Bosetti C, Bertuccio P, Negri E, La Vecchia C, Zeegers MP, Boffetta P. Pancreatic cancer: overview of descriptive epidemiology. Mol Carcinog 2012;51(1):3-13.

Gbelcova H, Lenicek M, Zelenka J, Knejzlik Z, Dvorakova G, Zadinova M, et al.

Differences in antitumor effects of various statins on human pancreatic cancer. Int J Cancer

2008;122(6): 1214-21.

Hong JY, Nam EM, Lee J, Park JO, Lee SC, Song SY, Choi SH, Heo JS, Park SH,

Lim HY, Kang WK, Park YS. Randomized double-blinded, placebo-controlled phase II trial of simvastatin and gemcitabine in advanced pancreatic cancer patients. Cancer Chemother

Pharmacol. 2014 Jan;73(l): 125-30. doi: 10.1007/s00280-013-2328-l. Epub 2013 Oct 27.

Kang Qiaohua and Anping Chen. Curcumin suppresses expression of low-density lipoprotein (LDL) receptor, leading to the inhibition of LDL-induced activation of hepatic stellate cells. British Journal of Pharmacology (2009), 157, 1354-1367. - -

Kusama T, Mukai M, Iwasaki T, Tatsuta M, Matsumoto Y, Akedo H, et al. 3-hydroxy- 3-methylglutaryl-coenzyme a reductase inhibitors reduce human pancreatic cancer cell invasion and metastasis. Gastroenterology 2002;122(2):308-17.

Malvezzi M, Bertuccio P, Levi F, La Vecchia C, Negri E. European cancer mortality predictions for the year 2013. Ann Oncol 2013;24(3):792-800.

Sumi S, Beauchamp RD, Townsend CM, Jr., Uchida T, Murakami M, Rajaraman S, et al. Inhibition of pancreatic adenocarcinoma cell growth by lovastatin. Gastroenterology 1992;103(3):982-9.

Staubach S, Hanisch FG. Lipid rafts: signaling and sorting platforms of cells and their roles in cancer. Expert Rev Proteomics 2011;8(2):263-77.

Von Hoff DD, Ervin T, Arena FP, Chiorean EG, Infante J, Moore M, et al. Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine. N Engl J Med 2013;369(18): 1691-703.

Claims

CLAIMS:

1. A compound which is an antagonist of LDLR or an inhibitor of the LDLR expression for use in the treatment of pancreatic cancer.

2. A i) compound according to claim 1 and ii) a chemo therapeutic agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of pancreatic cancer.

3. A combined preparation for use according to claim 2 wherein the chemotherapeutic agent is the Gemcitabine.

4. A compound for use according to claim 1 or a combined preparation for use according to claims 2 or 3 wherein the pancreatic cancer is a pancreatic ductal adenocarcinoma (PDAC).

5. A compound according to claims 1 to 4 wherein the inhibitor of the LDLR expression is a shRNA.

6. A pharmaceutical composition comprising an effective dose of a compound according to claim 1 and a pharmaceutically acceptable excipient.

7. An in vitro method for the prognosis of the survival time of a patient suffering from a pancreatic cancer comprising the steps consisting of i) determining the expression level of LDLR in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good outcome prognosis when the expression level is lower than the predetermined reference value and a poor outcome prognosis when the expression level is higher than the predetermined reference value.

8. An in vitro method for predicting the disease-free survival (EFS) of a patient suffering from a pancreatic cancer comprising the steps consisting of i) determining the expression level of LDLR in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good outcome prognosis when the expression level is lower than the predetermined reference value and a poor outcome prognosis when the expression level is higher than the predetermined reference value.

9. A method for treating pancreatic cancer comprising administering to a subject in need thereof a therapeutically effective amount of a compound which is an antagonist of LDLR or an inhibitor of the LDLR expression.

10. The method for treating pancreatic cancer of claim 9 which further comprises administering to a subject in need thereof a therapeutically effective amount of a chemotherapeutic agent, as a combined preparation for simultaneous, separate or sequential.