Patent Application
20230063059
The Sequence Listing for this application is labeled “Seq-List-replace.txt” which was created on May 4, 2022 and is 10484 bytes. The entire content of the sequence listing is incorporated herein by reference in its entirety.
The present invention refers to substances for use in the treatment of cancers, to a method for screening candidate molecules for treating cancers, and to a process for the diagnosis of cancer.
Therefore, the present invention has utility in pharmaceutical field.
In the description below, the references into brackets ([ ]) refer to the listing of references situated at the end of the text.
Pancreatic ductal adenocarcinoma (PDAC) accounts for 90% of all diagnosed pancreatic cancers; and is one of the leading causes of cancer deaths in men and women. In recent years, there has been very little improvement in PDAC patient survival rates. The factors that contribute to the very poor prognosis for patients with PDAC therefore include its rapid progression and invasion, the absence of specific symptoms, and the little impact of the available chemotherapy. The outcome of this cancer is unfortunately almost always fatal despite the available treatments, with a current five-year survival rate of about 6%. Indeed, most patients die within a year after the detection of cancer, especially because about 50% of these patients are diagnosed at invasive metastatic stages of the disease not accessible to surgery.
Despite some progress over the past decade, systemic chemotherapy to treat PDAC has globally limited efficacy and significant toxicity, and PDAC are notoriously resistant to agents molecularly targeted and to immunotherapy. Surgical resection is the reference treatment that is to remove the tumor when still possible; it remains to date the only reliable curative approach. Surgical operation is only considered if the size, location and general condition of the patient allows it. This is the case in 10 to 20% of exocrine pancreatic cancers. When the tumor is unoperable or that it could not be completely removed, different types of chemotherapy, often in combination (gemcitabine, 5-FU, irinotecan, oxaliplatin and nab-paclitaxel), are being considered, and radiotherapy is proposed in case of not operable tumor without distant metastasis.
The favourable therapeutic results remain low in the treatment of PDAC, just like the results of the evaluation of targeted therapies in the treatment of this cancer have been disappointing. This is partly related to the absence of validated biomarkers for the selection of patients eligible for treatment and therefore for the optimal clinical decision. In fact, a major obstacle to effective PDAC treatment is heterogeneity of the disease, which is reflected in various patterns of clinical response to therapy and very heterogeneous clinical evolutions of the disease. It is therefore necessary to identify biomarkers that provide a measure of disease activity and response to treatment for identifying subpopulations of patients likely to benefit to a specific and optimal therapy.
Pancreatic cancer (PC) is thus characterized by extremely high mortality and poor prognosis that are largely attributed to limited effective therapies (in 85% of pancreatic cancer cases), difficulties of early diagnosis and low number of reliable prognostic markers and therapeutic stratification of patients, making this cancer a major public health problem. The PDAC should become the second leading cause of cancer deaths in the United States and Europe by 2030, highlighting the need urging the development of more effective strategies for the control and the detection of pancreatic cancer.
Thus, a need exists of alternative tools for the diagnostic, the stratification, the prognostic and the treatment of pancreatic cancer.
Significant work has led the Applicant to develop a new therapeutic target for cancers, especially pancreatic cancer. The Applicant has surprisingly succeeded in demonstrating that STIM1 (Stromal Interacting Molecule 1) protein located at the plasma membrane, and in particular the membrane fraction of the STIM1 protein orientated with its C terminal domain (Cter) located in the extracellular medium, is a specific therapeutic target of cancer, especially pancreatic cancer.
The Applicant identifies for the first time, in the context of the present invention, a calcium influx pathway—the constitutive entry of calcium—regulated by a newly characterized protein, namely the protein STIM1 located at the plasma membrane (mSTIM1). Indeed, the Applicant characterized in pancreatic epithelial cells a new pathway for Ca2+ entry that is independent of the release of intracellular calcium stores and is regulated by the STIM1 pool located at the plasma membrane (mSTIM1) of these cells. The Applicant demonstrates the amount of STIM1 expression is correlated to constitutive calcium entry in cancer cell lines.
This Ca2+ entry pathway supports the survival of pancreatic epithelial cells, and is involved in their migration.
The Applicant has been able first to demonstrate that the expression of the STIM1 protein in the tumor tissues is correlated with the prognosis of the patients, and that a high expression of STIM1 in these tissues is linked to a decrease in the patient's survival. The measurement of STIM1 expression in cancerous tissues is therefore a new prognostic biomarker. In addition, mSTIM1 protein and the signalling pathways that it regulates have never been proposed as a therapeutic target.
Also, the Applicant has surprisingly demonstrated for the first time that mSTIM1 presents a double topology with an extracellular localization of the N terminal domain of the protein (called N ter out) but also with a C terminal domain orientation (called C ter) of mSTIM1 to the extracellular medium.
Surprisingly, the Applicant demonstrated that mSTIM1 oriented with its extracellular C-terminal domain is present in different cancer cell lines. This dual topology is observed at least in pancreatic cancer cell lines, Burkitt lymphoma cell lines, colon adenocarcinoma cell lines, breast cancer adenocarcinoma cell lines and cell from patients suffering from Chronic Lymphocytic Leukemia.
So, the Applicant proposes mSTIM1 oriented with its extracellular C-terminal domain as a new therapeutic target for pancreatic cancer and the use of substances specifically targeting this part of mSTIM1, especially anti-STIM1 antibodies against C terminal domain of STIM1, as new therapeutic tools. The Applicant demonstrated that treating pancreatic cells with an anti-mSTIM1 antibody targeting the C terminus of STIM1 modulates the constitutive entry of Ca2+.Treating the cells with an anti-mSTIM1 antibody targeting the C terminus of STIM1 do not affect store operated calcium entry (SOCEE) in pancreatic cancer cell lines.
Treating pancreatic cancer with anti-mSTIM1 antibody targeting the C terminus of STIM1 induces a decrease in the survival and migration of treated pancreatic cancer cells. Advantageously, the use of such an anti-mSTIM1 antibody according to the invention allows to decrease the survival of targeted cancer cells, in particular by inducing their entry into apoptosis and to reduce their proliferation. Similar observations on cancer cell survival were done for colon and breast cancer cells with effects correlated to the presence of STIM1 Cter out in the plasma membranes of the cells.
Thus, the invention is the first to propose to modulate the activity of mSTIM1 by using a substance targeting the extracellular C ter domain of mSTIM1, for example an antibody directed against the extracellular C ter domain of mSTIM1, in order to modulate cellular responses implied in pancreatic cancer (migration, survival, cell proliferation) and thus bring a new therapeutic solution in the treatment of pancreatic cancer. The substances of the invention, and especially the antibodies, are the first pharmacological modulators specific for the membrane fraction of STIM1 protein with its C terminal end out. It is an alternative to existing treatments, which can advantageously improve their effectiveness while reducing their side effects. The applicant demonstrated an improvement of gemcitabine efficiency for killing pancreatic cancer cells when cells are treated with low dose of gemcitabine along with a low dose of anti-mSTIM1 antibody targeting the C terminus of STIM1.
The use of an anti-STIM1 monoclonal antibody directed against the Cter fraction of STIM1 thus constitutes a novel immunotherapy targeting an alternative signalling pathway to currently targeted signalling pathways in the treatment of cancers, and more particularly pancreatic cancer, Burkitt lymphoma, colon adenocarcinoma, breast cancer adenocarcinoma, Chronic Lymphocytic Leukemia. The development of immunotherapy based on the use of an anti-STIM1 antibody is also a “first in class” therapy to fight against this cancer, and a fortiori against the other types of cancer in which this therapeutic target is present.
Advantageously, such an anti-STIM1 antibody may be used, according to the present invention, alone or in combination with existing chemotherapies, for example gemcitabine. Advantageously, it may potentiate effects of existing chemotherapies. Advantageously, it may potentiate effects of low doses gemcitabine administered to a patient in need thereof. Also, that immunotherapy can be advantageously proposed in the first line of treatment.
In addition, the Applicant has also demonstrated the interest of STIM1, in the prediction of the evolution of pancreatic cancer.
Accordingly, in a first aspect, the present invention provides a substance interacting with C terminal fragment of the STIM1 fraction localized to the plasma membrane of the cells, for its use in the treatment of cancers.
“STIM1” refers, according to the present invention, to the Stromal Interaction Molecule 1, also referenced in the literature as “GOK”. STIM1 is a protein that is encoded in humans by the STIM1 gene. STIM1 is a multidomain transmembrane protein. The human STIM1 molecule is a protein with sequence ID NO: 1 corresponding to the Uniprot sequence: Q13586 or NCBI: NP_003147.2. This protein is encoded by the sequence corresponding to the NCBI sequence: NM_003156.3 (mRNA transcript). The STIM1 N-terminal region is located in the ER lumen and contains a SAM domain (sterile a motif domain, a protein-protein interaction module) and an EF-hand motif (calcium-binding motif). In the middle of the protein, there is a transmembrane domain, which is followed by a C-terminal region, including a coiled coil, an ERM domain (ezrin-radixin-moesin) and a basic/serine/proline region. The peptide sequence of the C terminal fragment of the STIM1 protein is given in SEQ ID NO: 2 corresponding to amino acids 234 a 685 of the STIM1 protein:
STIM1 is mostly localized to the endoplasmic reticulum, and to a much lesser extent to the plasma membrane.
“Fraction of the STIM1 protein localized to the plasma membrane of the cells” or “Fraction of the STIM1 protein localized to the plasma membrane of the cells with its C terminal end out” means, in the sense of the present invention, the fraction of the STIM1 protein localized to the plasma membrane of the cells. Unless otherwise indicated, “STIM1” refers to the total cell fraction, i.e. comprising STIM1 localized in the endoplasmic reticulum membrane and to the plasma membrane, although “mSTIM1” refers to the fraction of STIM1 only located to the plasma membrane.
“C terminal fragment of the STIM1 fraction localized to the plasma membrane of the cells” refers, in the sense of the present invention, to the region between the amino acids 234 and 685 of the STIM1 amino acid sequence SEQ ID NO: 1, i.e. the peptide of sequence SEQ ID NO: 2. The presence of STIM1 at the plasma membrane presenting its C-Terminal end exposed to the extracellular environment is independent of STIM1 glycosylation. The presence of mSTIM1 with its C ter out is regulated by the TGFβ pathway. This pathway is up-regulated in pancreatic cancer and stimulation of pancreatic cancer cells with TGFβ induces an increase in the amount of mSTIM1 with the C ter our orientation.
“Substance” means, in the sense of the present invention, any molecule displaying interaction with C terminal fragment of the STIM1 protein localized to the plasma membrane (mSTIM1), advantageously for inhibiting the constitutive entry of calcium or modulating the presence of mSTIM1 at the plasma membrane in targeted cells to correct all or part of the defects in the cellular functions in which it is involved. The inhibition of the constitutive entry of calcium in targeted cells may be measured or controlled by any means known by the skilled person, for example by studying calcium flows or measuring the variations in intracellular calcium concentration by fluorescence.
The presence of mSTIM1 at the plasma membrane in targeted cells may be measured or controlled by any means known by the skilled person, for example by flow cytometry (Fluorescence activated cell sorting), or Elisa.
The substance may be for example any compound that binds, or specifically binds to the C terminal fragment of the mSTIM1 protein. In this case, the term “interact” means “bind” or “specifically bind to”.
The substance may be of natural or synthetic origin. It may be a protein produced chemically or by any method of bioengineering, such as purification. The substance may notably be identified by applying the method of screening as defined below. Advantageously, the substance is not able to cross the plasma membrane and interacts specifically with the fraction of the STIM1 protein localized to the plasma membrane of the cells without penetrating the cells.
The substance may be for example chosen among an antibody or an isolated binding fragment thereof, a peptide, a protein, a chemical compound and an aptamer. Preferably, it is an isolated antibody or an isolated binding fragment thereof, for example a monoclonal antibody.
The binding of the substance of the invention may be measured by any known techniques, for example by FACS (Fluorescence activated cell sorting), ELISA, or Biacore™ assay.
The phrase “specifically (or selectively) binds to” refers to a binding reaction that is determinative of the presence of the antigen, i.e. the region between amino acid residues 234 and 685 of the STIM1 amino acid sequence SEQ ID NO: 1, in a heterogeneous population of proteins and other biologics. Specific binding between two entities means a binding with an equilibrium constant (KA) (kon/koff) between 102M−1 and 5×1015M−1.
In addition to the equilibrium constant (KA) noted above, the compound of the invention may advantageously also have a dissociation rate constant (KD) (koff/kon) of 5×10−2M or lower, and binds to the antigen as defined above with an affinity that is at least twofold greater than its affinity for binding to a non-specific antigen.
In one embodiment, the compound of the invention may have dissociation constant (Kd) of less than 3000 pM as assessed using a method described herein or known to one of skill in the art, e.g., a BIAcore™ assay (Biacore™ International AB, Uppsala, Sweden), ELISA, surface plasmon resonance or FACS.
The term “antibody” as used herein refers to whole antibodies that interact with, e.g., by binding, steric hindrance, stabilizing/destabilizing or spatial distribution, of the C terminal fragment of the STIM1 fraction localized to the plasma membrane of the cells, or to an “antibody fragment”, which refers to one or more portions of an antibody that retain(s) the ability to specifically interact with the C terminal fragment of the STIM1 fraction localized to the plasma membrane of the cells. The antibody of the invention may be a naturally occurring antibody, which is a glycoprotein comprising at least two heavy (abbreviated herein as H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system as effector cells and the first component (Clq) of the classical complement system.
The term “antibody” or “antibody fragment” include for example, monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), Fab fragments, F(ab′) fragments, and anti-idiotypic (anti-Id) antibodies including, e.g., anti-Id antibodies to antibodies of the invention), a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, F(ab2)′, F(ab)2′, scFv, VHH (sdAb fragment, which consists of a VH domain), a Fd fragment consisting of the VH and CHI domains; and an isolated complementarity determining region (CDR). The antibody fragments may be obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
The framework and/or constant region of the antibody, in the case of an entire antibody, may be from mammals or non-mammals such as human, rodent, camel, canine, feline, shark.
Preferably, the antibody may be a chimeric, a human or a humanized antibody, so that its immunogenicity is reduced in humans, and/or its effectiveness is improved upon therapeutic administration to humans.
The antibodies can be of any isotype for example IgG, IgE, IgM, IgD, IgA and IgY, of any class, for example IgG1, IgG2, for example IgG2b, IgG3, IgG4, IgA1 and IgA2, or of any subclass.
According to a preferred embodiment of the invention, the constant region of each of the light chains of the antibody according to the invention is of K type. Any allotype is suitable for the implementation of the invention, e.g. Km(1), Km(1, 2), Km(1, 2, 3) or Km(3), but the preferred allotype is Km(3). Preferably, the antibodies of the invention are IgG2b/kappa, especially murine IgG2b/kappa.
The phrase “isolated antibody” as used herein refers to an antibody that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
For example, the substance may be the anti-STIM1 antibodies the targeting the terminal C (Cter) such as the clone CDN3H4, sc-66173 (Santacruz), the clone HPA012123 (Sigma Aldrich), or the clone HPA011088 (Sigma Aldrich).
The antibody may be manufactured by any method known by the skilled person, for example by culturing a host cell expressing the antibody under conditions that result in production of the antibody, and isolating the antibody of the invention from the host cell or culture medium of the host cell.
The host cell refers to a cell expressing the antibody, by transfection with a nucleic acid molecule or infection with phagemid or a bacteriophage, and the progeny or potential progeny of such a cell. It may be any cell known in the prior art, for example SP2/0, YB2/0, IR983F, Namalwa human myeloma, PERC6, CHO cell, as CHO-DG44, CHO-DUK-B11, CHO-K-1, CHO-Lec10, CHO-Lec1, CHO-Lec13, CHO Pro-5 or CHO/DHFR-, Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, BHK, K6H6, NS0, SP2/0-Ag 14 or P3X63Ag8.653.
For expression of the nucleic acid, the expression vector(s) may be transfected into the host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, such as electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
Cell culture production, purification and characterization of antibodies can be realized by well-known methods of the prior art. For example, cell can be allow to grow and die (4 to 5 days) before supernatant collection, clarification by low-speed centrifugation and volume reduction by ultra-filtration, for example on Pellicon XL Filter (Millipore). The concentrated culture supernatants can be injected into a HiTrap protein A FF column (GE Healthcare), bound antibodies can be eluted with sodium citrate buffer, and fractions can be neutralized using Tris. Fractions containing the antibodies can be pooled and dialyzed into PBS, and the samples can be sterile-filtered and stored at 4° C. The purified antibodies can be characterized by SDS-PAGE under non-reducing and reducing conditions.
In another embodiment of the invention, the substance may be an aptamer, i.e. nucleic acid molecules having specific binding affinity to non-nucleic acid or nucleic acid molecules through interactions other than classic Watson-Crick base pairing Aptamers may be for example RNA or DNA aptamers. Aptamers are known to be able to substitute for monoclonal antibodies in various applications.
“Cancer” means, in the sense of the present invention, any cancer having a STIM1 fraction localized to the plasma membrane of the cells with at least a part of its C ter end exposed to the external medium. For example, it may be a pancreatic cancer, and more specifically pancreatic ductal adenocarcinoma. It may also be colon cancer (adenocarcinoma), breast cancer (adenocarcinoma), Burkitt lymphoma or Chronic Lymphocytic Leukemia.
It results from the above that the invention also includes a method for treating cancers, comprising administering a medicament comprising a substance interacting with C terminal fragment of the STIM1 fraction localized to the plasma membrane of the cells, to a patient in need thereof.
It also results from the above that the invention additionally includes the use of a substance interacting with C terminal fragment of the STIM1 fraction localized to the plasma membrane of the cells, for the manufacture of a medicament for the treatment of cancers.
A method of treating a cancer, comprising administering a substance interacting with C terminal fragment of the STIM1 fraction localized to the plasma membrane of the cells in a subject in need thereof, is also described.
In a second aspect, the present invention provides a pharmaceutical composition comprising at least one substance interacting with C terminal fragment of the STIM1 fraction localized to the plasma membrane of the cells, and at least one pharmaceutically acceptable excipient.
Such a composition may comprise any excipients and/or additives that facilitate the formulation of the substance in preparations that may be used pharmaceutically. The expression “pharmaceutically acceptable” encompasses any vehicle that does not interfere negatively with the efficacy of the substance for treating cancer, and that is not toxic to the host to whom or to which it is administered. In particular, suitable pharmaceutically acceptable vehicles for a composition according to the invention are vehicles that are suitable in particular for systemic application. Suitable pharmaceutically acceptable vehicles are well known in the prior art and are described for example in Remington Pharmaceutical Sciences (Mack Publishing Company, Easton, USA, 1985 ([2])), a standard reference text in this field. It may be for example one or more components selected from sodium citrate, polysorbate 80, sodium chloride, sodium hydroxide, hydrochloric acid, and water for injection.
Advantageously, the composition according to the invention may find application as a medicinal product. Particularly advantageously, the composition of the invention may find application as a medicinal product in the treatment and/or the diagnosis, stratification and prognostic of cancer as mentioned above.
In the pharmaceutical composition of the invention, the above-mentioned substance may be the only active principal for treatment or diagnosis of cancer. Alternatively, the pharmaceutical composition of the invention may comprise in addition any other active ingredient, for example any other active ingredient that potentiates the effect of the substance or is potentiated by the substance as defined above. It may be for example an association of anti-STIM1 targeting the C terminal of STIM1 and gemcitabine, which decreases pancreatic cell survival compared to the use of active ingredient alone, as demonstrated by the applicant in the Examples.
In a third aspect, the present invention provides the use of isolated C terminal fragment of the STIM1 fraction localized to the plasma membrane of the cells in a method for screening candidate molecules for treating cancers, especially pancreatic cancers.
“Method of screening” means, in the sense of the present invention, any method allowing identification of a substance interacting with the membrane fraction of the STIM1 protein, especially the C-terminal fragment, or modulating its membrane expression. Preferably, the fraction is located on the plasma membrane of intact cells, which means that the plasma membrane is non broken and/or non permeabilized, and advantageously does not allow non-permeant molecules to penetrate the cells. It may be any method known by a person skilled in the art, for example biological screening, for example a technique selected from the group comprising immunofluorescence, FRET, BRET, other luminescence or fluorescence complementation assays, Proximity ligation Assays, Western blot, immunoprecipitation, surface plasmon resonance (SPR), flow cytometry, video microscopy, study of calcium flows, enzyme-linked immunosorbent assay (ELISA), and confocal microscopy, or biophysical screening, for example by measuring the variations in intracellular calcium concentration by fluorescence. Advantageously, the method of screening allows identifying substances that interact selectively with the fraction of the STIM1 protein localized with its C terminal out at the plasma membrane of the cells, without penetrating the cell. In other words, the method of screening allows identifying non-penetrating substances that interact selectively with the fraction of the STIM1 protein localized at the plasma membrane of the cells with its C terminal out. The method of screening may be realized in vitro, for example on a sample containing intact cells expressing on their plasma membrane the mSTIM1 protein.
“Candidate molecule” means, in the sense of the present invention, any molecule that interacts with the C terminal fragment of the fraction of the STIM1 protein localized to the plasma membrane of the cells. The interaction may be as defined above in relation with the substance. For example, the interaction may be a modulation of the activity or expression of this protein. Modulation of the activity of the protein may be reflected in a change of the calcium flows such as a change in the constitutive entry of intracellular calcium. The modulation of expression may be an increase or a decrease in expression of the STIM1 protein localized to the plasma membrane relative to a level measured on the same cell or a comparable cell before application of the candidate molecule. The modulation of expression of the STIM1 protein may for example be linked to transcriptional modifications, epigenetic modifications or changes in mechanisms regulating STIM1 protein incorporation or stability at the plasma membrane, such as modification of STIM1 phosphorylation, ubiquitinylation or modulation of the glycosylation process that is necessary for membrane addressing of the STIM1 protein. Advantageously, the selected candidate molecules interact specifically with the fraction of the STIM1 protein localized to the plasma membrane of the cells. As the selected candidate molecules do not cross the plasma membrane, they do not interact with the STIM1 protein localized to the endoplasmic reticulum in the method of screening of the invention.
“Cells” means, in the sense of the present invention, any cell expressing STIM1 at the level of the plasma membrane and with the C terminus end of STIM1 exposed to the extracellular environment. Advantageously, in the screening method of the invention, cells are intact and/or non broken and/or not permeabilized, in order to strictly screen for molecules that modulate the membrane expression of STIM1 and/or that modulate its functionality such a modification of the constitutive entry of extracellular Ca2+. Advantageously, screening method of the invention allows selecting molecules that do not penetrate into the cells and that stay at the plasma membrane, due to their specific interaction with the fraction of the STIM1 protein localized to the plasma membrane. In other words, the method of screening allows selecting non permeant molecules, i.e. molecules that do not cross the plasma membrane. The cells may be isolated cells, and may be provided for the method of screening of the invention in the form of a sample. Advantageously, the cells may be cancer cells.
The use of cells, for example isolated intact, expressing on their plasma membrane the STIM1 protein with C terminus out, in a method for in vitro screening candidate molecules useful for treating cancers, especially pancreatic cancer, is included in the invention.
The method of screening may comprise the steps of:
In another aspect, the present invention provides a process for predicting the progression and/or monitoring the progression of a cancer, comprising the in vitro detection of the expression of STIM1 in a biological sample from the subject. Advantageously, the measurements of STIM1 are made in the total cell fraction STIM1.
The diagnosis of a cancer, for example a pancreatic cancer in a subject, may be realised by measuring STIM1 expression in pancreatic cells. A STIM1 expression in these cells that is reduced compared to STIM1 level in control pancreatic non-cancerous cells means that these cells are cancerous, and thereby allows diagnosing pancreatic cancer in the subject.
By “predicting the progression and/or monitoring the progression” it is meant in the invention that the method allows to predict the likely outcome of a cancer. More particularly, the prognosis method can evaluate the survival rate, said survival rate indicating the percentage of people, in a study, who are alive for a given period of time, usually 5 years, after diagnosis of a cancer. This information allows the practitioner to determine if a medication is appropriated, and in the affirmative, what type of medication is more appropriate for the patient.
The prediction of progression and/or the monitoring of the progression of a cancer, for example a pancreatic cancer in a subject, may be realised by measuring STIM1 expression in pancreatic cancer cells. An increase of STIM1 expression in tumor tissues over time in the subject is associated with a decreased overall survival in pancreatic cancer, i.e. a bad prognosis of the cancer.
The change in expression may be an increased expression of STIM1 within the tumor, thereby allowing a bad prognosis or progression of cancer. “Increased expression” means, in the sense of the invention, an expression with a grade of more than 2 using our IHC expression scale.
Generally, a good prognosis, in the sense of the invention, is associated with median survival higher than 3 years, whereas a bad prognosis is associated with a median survival lower than 3 years.
For the measurement of STIM1 expression, STIM1 detection may be realized with any compound able to recognize specifically any part of STIM1, or preferably the C terminal fragment of the STIM1. It may be for example an antibody or a binding-fragment thereof, a protein, a peptide, a chemical compound or an aptamer. Preferably, detection is made with an antibody directed against the C terminal fragment of the STIM1 fraction located at the cell plasma membrane, for example anti-STIM1 antibodies (STIM1: clone CDN3H4, sc-66173, Santacruz) targeting the terminal C fragment of mSTIM1.
This invention is further illustrated by the following examples with regard to the annexed drawings that should not be construed as limiting.
We have demonstrated that the expression of STIM1 protein, in cancer cells of patients with pancreatic adenocarcinoma (Pancreatic Ductal AdenoCarcinoma: PDAC) constitutes a pathology evolution marker (see
The expression of STIM1 (average grade) in healthy pancreatic tissues and pancreatic tumor tissues (PDAC) (n=48) is shown in Table 1.
The expression of STIM1 in healthy pancreatic tissues and cancer pancreatic tissues defined by the percentage of low and high grades (n=48) is shown in Table 2.
The expression of STIM1 protein is decreased in cancer cells compared to normal peri-tumoral cells. However the increase in STIM1 expression in cancer cells is correlated with poor prognosis.
STIM1 expression was determined using classical immunohistochemistry protocols. A panel of two different validated antibodies directed against STIM1 was used. Briefly, for IHC analysis of STIM1 expression, formalin-fixed, paraffin-embedded (FFPE) tissue blocks containing both tumor and normal pancreatic tissue were obtained from patients at different pancreatic cancer stages and who had surgery. IHC staining was performed using automated IHC staining system (Roche Diagnostics Ventana Benchmark, N750-BMKU-FS) in accordance with the manufacturer's recommendations. The level of expression of our proteins is scored (1 to 3) corresponding for the score 0 to a weak or nonexistent expression of STIM1 and 3 corresponding to a strong expression of our proteins of interests. Statistical analysis was done using Wilcoxon paired tests.
The level of expression of STIM1 modulates the constitutive entry of calcium: (see
Panc cells were transfected with 5 μg of an expression vector containing the STIM1 gene (pSTIM1 vector) or an empty vector using the transfecting agent lipo293 following commercial recommendations. Cells were transfected with 5 μM of an SiRNA targeting STIM1 (SEQ ID NO: 3: ugagggaagaccucaauua) or a SiCtrl using the transfecting agent lipoRNAimax following commercial recommendations.
For constitutive Ca2+ entry (CCE) measurements, Panc cells were loaded with 2 μM of the Fura-2/AM fluorescent dye in the presence of 2 μM Pluronic acid for 60 min at 37° C. in a medium containing: 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 10 mM Glucose with an 7.4-adjusted pH supplemented with 5 mM CaCl2). Cells were then washed and displayed on coverslips for single cell fluorescence imaging. Fura-2 was excited alternatively at 340 and 380 nm using a monochromator, and fluorescence emission was recorded at 510 nm using a fluorescence microscope equipped with a dichroic mirror and a 14-bit CCD camera. After the stabilization of basal fluorescence, the extracellular medium was replaced by Buffer A supplemented with 0.5 mM CaCl2) for 100 s and again with the original 5 mM CaCl2)—containing Buffer A after curve stabilization. Values of the ratio of fluorescence measured at 340 and 380 nm are collected over time and normalized.
The amount of mSTIM1 is evaluated by flow cytometry using 5×106 cells per condition. Cells were taken off culture plates using an Enzyme free solution for 5 min at 37° C. Cells were then centrifuged for 5 min at 1500 rpm and then incubated with 1 μl of an anti-STIM1 antibody coupled with PE (GOK-PE, BD Biosciences; 20 μg/ml) or with 0.5 μl of an isotype control coupled with PE in 50 μl of PBS on cells for 30 min on ice. After 3 washes, cells were read in PBS using a Flow cytometer (Navios, Beckman coulter).
We demonstrated by cytometry and Elisa approaches using anti-STIM1 antibodies targeting the C terminal end (clone CDN3H4, sc-66173, Santacruz) and the N terminal end (clone GOK, BD transduction Laboratory) of the STIM1 protein that the fraction of this protein STIM1 localized to the plasma membrane (mSTIM1) has a double topology with an orientation in which the N-terminal domain of the protein is extracellular (called N ter out), as previously established, but also an orientation in which the terminal C domain of mSTIM1 is extracellular (Cter out orientation) (see
Panc cells were transfected with 5 μg of an expression vector containing the STIM1 gene (pSTIM1 vector) or an empty vector using the transfecting agent lipo293 following commercial recommendations. Cells were transfected with 5 μM of an SiRNA targeting STIM1 (sequence: UGAGGGAAGACCUCAAUUA, SEQ ID NO: 3) or a SiCtrl using the transfecting agent lipoRNAimax following commercial recommendations.
The amount of STIM1 was evaluated using flow cytometry or Elisa approaches.
For the ELISA measurements, 106 cells were loaded on 96 wells plates in DMEM medium. Cells were fixed using PFA 2% for 10 min at room temperature (RT). Cells were then washed with Phosphate Buffer Solution (PBS) and next incubated with PBS supplemented with 5% of fat milk for 30 minutes. Cells were next incubated with the anti-STIM1 antibody directed against the N terminus (0.1 μl GOK, BD Biosciences; 1 μg/ml) or the C terminus (10 μl CDN3H4 sc-66173, Santacruz; 1 μg/ml) for 1 h30 at RT. After 3 washes with PBS, cells were incubated with in PBS +5% of fat milk containing the peroxidase conjugated secondary antibody for 30 min at RT. After 3 washes, the substrate for peroxidase conjugated secondary antibody (SIGMAFAST™ OPD tablets, Sigma-Aldrich) was added for 20 min at 37° C. and the reaction was stopped using H2504 solution. ELISA plate was read at 392 nm in absorbance and the optical density normalized using absorbance emitted at 495 nm by cells following incubation with Janus green.
For determination of the amount of STIM1 using flow cytometry, 5×106 cells were used per condition. Pancreatic cells were taken off culture plates using Enzyme free solution for 5 min at 37° C. Cells were then centrifuged for 5 min at 1500 rpm and incubated with 50 μL of PBS containing anti-STIM1 antibody directed against the N terminus (1 μl GOK-PE, BD Biosciences; 20 μg/ml) or the C terminus (5 μl CDN3H4 sc-66173, Santacruz; 10 μg/ml) or 0.5 μL of an isotype control for 30 min on ice. In case of incubation with the uncoupled anti-STIM1, cells were washed and then incubate with a PE conjugated secondary antibody for 20 min on ice. After 3 washes, cells were read in PBS using a Flow cytometer (Navios, Beckman Coulter Life Sciences).
The dual orientation of mSTIM1 has been observed in various pancreatic cell lines (see
For the ELISA measurements, 106 cells were loaded on 96 wells plates in DMEM medium. Cells were fixed using PFA 2% for 10 min at room temperature (RT). Cells were then washed with Phosphate Buffer Solution (PBS) and next incubated with PBS supplemented with 5% of fat milk for 30 minutes. Cells were next incubated with the anti-STIM1 antibody directed against the N terminus (0.1 μl GOK, BD Biosciences; 1 μg/ml) or the C terminus (10 μl CDN3H4 sc-66173, Santacruz; 1 μg/ml) for 1 h30 at RT. After 3 wash with PBS, cells were incubated in PBS +5% of fat milk containing the peroxidase conjugated secondary antibody for 30 min at RT. After 3 washes, the substrate for peroxidase conjugated secondary antibody (SIGMAFAST™ OPD tablets, Sigma-Aldrich) was added for 20 min at 37° C. and the reaction was stopped using H2504 solution. ELISA plate was read at 392 nm in absorbance and the optical density normalized using absorbance emitted at 495 nm by cells following incubation with Janus green.
For determination of the amount of STIM1 using flow cytometry, 5×106 cells were used per condition. Pancreatic cells were taken off culture plates using Enzyme free solution for 5 min at 37° C. Cells were then centrifuged for 5 min at 1500 rpm and incubated with 50 uL of PBS containing anti-STIM1 antibody directed against the N terminus (1 μl GOK-PE, BD Biosciences; 20 μg/ml) or the C terminus (5 μl CDN3H4 sc-66173, Santacruz; 10 μg/ml) or 0.5 μL of an isotype control for 30 min on ice. In case of incubation with the uncoupled anti-STIM1, cells were washed and then incubate with a PE conjugated secondary antibody for 20 min on ice. After 3 washes, cells were read in PBS using a Flow cytometer (Navios, Beckman Coulter Life Sciences).
By western blot approaches (see
Protein extraction was performed by scratching 107 Panc 1-wt cells on ice with a lysis buffer containing: 20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X100, 2.5 mM Na+ pyrosodium tetraphosphate, 1 mM glycerophosphate, 1 mM Na+ orthovanadate, 1 μg/ml leupeptin and a protease inhibitor cocktail. Protein extracts were sonicated and centrifuged for 12 min at 16,000 g. Protein concentration of cell lysates were determined using the Folin method. 75 μg of proteins were run on SDS-PAGE 7.5% polyacrylamide gels in denaturing conditions, and then transferred onto PVDF (PolyVinyliDene Fluoride) membrane sheets. Unspecific blocking was done by incubation with 5% fat milk in PBS, 0.1% tween 20 for 1 hour. Blots were incubated overnight with 5% fat milk in PBS, 0.1% tween 20, containing mouse monoclonal anti-STIM1 (CDN3H4 clone Santacruz; 1:1,000 dilution) or mouse monoclonal anti-GAPDH antibody (6C5 clone Abcam; 1:10,000 dilution). Blots were incubated with Horseradish Peroxydase (HRP)-conjugated goat anti-mouse after washing with PBS, 0.1% tween 20 and revealed with the Luminata Forte reagent. All results were normalized upon GAPDH quantification.
We demonstrate that constitutive Ca2+ entry of pancreatic epithelial cells is inhibited by the use of the anti-STIM1 antibody clone CDN3H4 (clone CDN3H4, sc-66173, Santacruz) (see
For SOCE measurement 106 cells were seeded in 96 wells. Cells are loaded with Fura-2 acetoxymethyl ester (Fura-2 QBT™, Molecular Probes) fluorochrome according to the manufacturer's protocol. The Fura-2 QBT™ was aspirated and replaced by an equal volume of free Ca2+ Hepes-buffered solution containing (in mM): 135 NaCl, 5 KCl, 1 MgCl2, 1 EGTA, 10 Hepes, 10 glucose, pH adjusted at 7.45 with NaOH. Intracellular calcium level variations were monitored by using the FlexStation 3™ (Molecular Devices, Berkshire, UK), Dual excitation wavelength capability permits ratiometric measurements of Fura-2AM peak emissions (510 nm) after excitations at 340 nm (bound to Ca2+) and 380 nm (unbound to Ca2+). Modifications in the 340/380 ratio reflect changes in intracellular-free Ca2+ concentrations. The SOCE was elicited by releasing the Ca2+ stores from the endoplasmic reticulum with thapsigargin (2 μM) solution under Ca2+-free conditions to determine the magnitude of intracellular Ca2+ release (Hepes-buffered solution). Next, cells were returned to a Ca2+-containing Hepes-buffered solution to measure SOCE. The magnitude and speed of SOCE were estimated.
For constitutive Ca2+ entry (CCE) measurements, Panc-1 cells were loaded with 2 μM of the Fura-2/AM fluorescent dye in the presence of 2 μM Pluronic acid for 60 min at 37° C. in a medium containing: 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 10 mM Glucose with an 7.4-adjusted pH supplemented with 5 mM CaCl2). Cells were then washed and displayed on coverslips for single cell fluorescence imaging. Fura-2 was excited alternatively at 340 and 380 nm using a monochromator, and fluorescence emission was recorded at 510 nm using a fluorescence microscope equipped with a dichroic mirror and a 14-bit CCD camera. After the stabilization of basal fluorescence, the extracellular medium was replaced by Buffer A supplemented with 0.5 mM CaCl2) for 100 s and again with the original 5 mM CaCl2)—containing Buffer A after curve stabilization. Values of the ratio of fluorescence measured at 340 and 380 nm are collected over time and normalized.
The presence at the plasma membrane of mSTIM1 having a Cter out orientation is independent of glycosylation of STIM1 (glycosylation at position AA N131 and N171) in contrast to its Nter out orientation (see
The amount of mSTIM1 having a Cter out orientation is regulated by different cell signalling pathways and especially by the way TGFβ, an over-activated pathway in pancreatic cancer. TGFβ pathway stimulation induces an increased amount of mSTIM1 with Cter orientation in pancreatic cancer cells (see
Panc and Miapaca cells were overexpressed with 5 μg of the expression vector pSTIM1 vector containing wild type STIM1 (pSTIM1) or mutated STIM1 (N131Q N171Q STIM1) or an empty vector using the transfecting reagent lipo293 following commercial recommendations. To evaluate the influence of TGFβ signaling cells were treated with 25 ng/ml TGFβ1 for 5 min, 15 min, 30 min, 24 h and 48 h in DMEM medium at 37° C.
Amount of STIM1 at the plasma membrane was evaluated by ELISA. 106 cells were loaded on 96 wells plates in DMEM medium. Cells were fixed using PFA 2% for 10 min at room temperature (RT). Cells were then washed with Phosphate Buffer Solution (PBS) and next incubated with PBS supplemented with 5% of fat milk for 30 minutes. Cells were next incubated with the anti-STIM1 antibody directed against the N terminus (1 μl GOK, BD Biosciences; 1 μg/ml) or the C terminus (5 μl CDN3H4 sc-66173, Santacruz; 10 μg/ml) for 1 h30 at RT. After 3 washes with PBS, cells were incubated with in PBS +5% of fat milk containing the peroxidase conjugated secondary antibody for 30 min at RT. After 3 washes, the substrate for peroxidase conjugated secondary antibody (SIGMAFAST™ OPD tablets, Sigma-Aldrich) was added for 20 min at 37° C. and the reaction was stopped using H2504 solution. ELISA plate was read at 392 nm in absorbance and the optical density normalized using absorbance emitted at 495 nm by cells following incubation with Janus green.
Anti-STIM1 antibody targeting the C terminal fraction of STIM1 (clone CDN3H4, Santa Cruz) reduces in vitro migration of Panc-1-Wt cells evaluated by transwell migration (Boyden chambers) or by wound healing technique (see
For migration assays based on migration through filters, 5×106 cells were loaded on the top of a Boyden chamber (transwell Corning 8 μM pores) and incubated with or without anti-STIM1 targeting the C terminus of STIM1 (CDN3H4 clone 10 μg/ml) for 48 hours at 37° C. in DMEM media without SVF. The bottom plate was loaded with 10% SVF DMEM medium. After removing cells from the upper face of the filter, five fields of cells were manually counted after fixation of migrated cells and Dapi nucleus coloration.
The anti-STIM1 antibody targeting the C terminal fraction of STIM1 (clone CDN3H4, Santa Cruz) reduces in vitro the survival of pancreatic cancer cells (see
For cell proliferation, cytotoxicity, and cell survival measurements, 106 cells were seeded in 96 wells plates in 100 μL of DMEM medium. Cells were incubated for 48H hours at 37° C. with an anti-STIM1 antibody targeting the C terminal of STIM1 (CDN3H4, Santa Cruz; HPA011088 or HPA012123 clones, Sigma). Cell proliferation was evaluated using the Cell Titer kit proliferation assay (Promega). Cytotoxicity was evaluated using the Cell Tox green cytotoxicity assay (Promega) and cell survival measured using the CCK8 kit from Sigma. All these kits were used following manufacturer recommendations and using a plate reader
For the apoptosis measurements, 5×106 cells were seeded in 12 wells plates. Cells were incubated for 48 hours with anti-STIM1 CDN3H4 antibody (10 ug/ml) at 37° C. After wash, cells were taken off plates using an enzyme free solution. Cells were centrifuged for 5 min at 1500 rmp and suspended in 50 μL of PBS containing 1 μL of AnnexinV-FiTC and 1 μL of propidium iodure (PI) for 15 min at RT in the dark. Evaluation of Anexin and PI staining was realized using a flow cytometer (Navios, Beckman Counter) to evaluate cell apoptosis and necrosis.
A low dose (5 μg/ml) of the anti-STIM1 antibody targeting the C terminal fraction of STIM1 (clone CDN3H4, Santa Cruz) or a low dose of Gemcitabine (15 nM) slightly but significantly reduce the survival and proliferation of pancreatic cancer cells (see
For cell proliferation, cytotoxicity, and cell survival measurements, 106 cells were seeded in 96 wells plates in 100 μL of DMEM medium. Cells were incubated for 48H hours at 37° C. with 5 μg/ml of the anti-STIM1 antibody targeting the C terminal of STIM1 (CDN3H4, Santa Cruz) alone, with 15 nM Gemcitabine alone or with both 5 ug/ml of the anti-STIM1 clone CDN3H4 and 15 nM Gemcitabine. Relevant controls (isotype or/and DMSO treatments) were also realized. Cell proliferation was evaluated using the Cell Titer kit proliferation assay (Promega). Cytotoxicity was evaluated using the Cell Tox green cytotoxicity assay (Promega) and cell survival measured using the CCK8 kit from Sigma. All these kits were used following manufacturer recommendations and using a plate reader
Screening of the molecules modulating the STIM1 fraction localized to the plasma membrane (mSTIM1) with its C terminus out is carried out on cells expressing sufficient amount of mSTIM1 with its C terminus out such as Panc-1 pancreatic cancer cells. Two types of Panc-1 cells are used: Panc-1 cells stably transfected with STIM1 or Panc-1 with endogenous amount of STIM1. These cells display a measurable constitutive calcium entry. Screening consists in a first approach of measuring the effects of the molecules targeting the fraction of STIM1 localized to the plasma membrane of the cells on the constitutive entry of extracellular calcium. The effects of these molecules on calcium entry dependent on the release of reserves SOCE (Store Operated Calcium Entry) are also evaluated in order to determine the effect of the molecules on the influx SOCE of the molecules acting on constitutive calcium entry. The amplitude of these two calcium flows is measured by monitoring the variations in intracellular calcium concentration using a fluorescent probe (Fura-2 QBT, Molecular Devices).
For constitutive Ca2+ entry (CCE) measurements, Panc cells were loaded with 2 μM of the Fura-2 QBT fluorescent dye for 60 min at 37° C. in a medium containing: 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 10 mM Glucose with an 7.4-adjusted pH supplemented with 1.8 mM CaCl2). Cells were then washed and displayed on coverslips for single cell fluorescence imaging. Fura-2 was excited alternatively at 340 and 380 nm using a monochromator, and fluorescence emission was recorded at 510 nm using a fluorescence microscope equipped with a dichroic mirror and a 14-bit CCD camera. After the stabilization of basal fluorescence, the extracellular medium was replaced by Buffer A supplemented with 0.5 mM CaCl2) for 100 s and again with the original 5 mM CaCl2-containing Buffer A after curve stabilization. Values of the ratio of fluorescence measured at 340 and 380 nm are collected over time and normalized.
For SOCE and Constitutive Ca2+ entry measurements, 106 cells were seeded in 96 wells. Cells are loaded with Fura-2 acetoxymethyl ester (Fura-2 QBT™, Molecular Probes) fluorochrome according to the manufacturer's protocol. The Fura-2 QBT™ was aspirated and replaced by an equal volume of free Ca2+ Hepes-buffered solution containing (in mM): 135 NaCl, 5 KCl, 1 MgCl2, 1 EGTA, 10 Hepes, 10 glucose, pH adjusted at 7.45 with NaOH. Changes in fluorescence are monitored by using the FlexStation 3™ (Molecular Devices, Berkshire, UK). Dual excitation wavelength capability permits ratiometric measurements of Fura-2AM peak emissions (510 nm) after excitations at 340 nm (bound to Ca2+) and 380 nm (unbound to Ca2+). Modifications in the 340/380 ratio reflect changes in intracellular-free Ca2+ concentrations. The SOCE was elicited by releasing the Ca2+ stores from the endoplasmic reticulum with thapsigargin (2 μM) solution under Ca2+—free conditions to determine the magnitude of intracellular Ca2+ release (Hepes-buffered solution). Next, cells were returned to a Ca2+-containing Hepes-buffered solution to measure SOCE. The magnitude and speed of SOCE were estimated. Constitutive entry is measured in the absence of any stimulation by following the quench of Fura 2 fluorescence recorded at 360 nm wavelength excitation when manganese (Mn2+) is added to the extracellular medium. The rate of fluorescence quenching represents a good approximation of Ca2+ entry.
The cells are put in contact with the test compound at the moment of loading the cells with the fluorescent probe and throughout measurement of the variations in intracellular calcium concentration.
The effects of screened compounds on STIM1 location at the plasma membrane is realized for lead candidates. Screening is also done by following the effects of promising molecules on cell migration and cell survival.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19305374.1 | Mar 2019 | EP | regional |
This application is the U.S. national stage application of International Patent Application No. PCT/EP2020/057916, filed Mar. 23, 2020.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/057916 | 3/23/2020 | WO |
