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Ph. D. Project : Restoring Chemotherapy Sensitivity in TNBC “claudin 1 low cells” by expressing claudin 1: a promising perspective?
Dates : 2018/10/01 - 2021/09/30
Manager(s) CRAN: Isabelle GRILLIER-VUISSOZ , Sandra KUNTZ
Full reference: Three major groups of tumors can be identified using the presence or the lack of three biomarkers: luminal tumors that express estrogen receptor alpha and progesterone receptor, HER2 “enriched” tumors that overexpress human EGF receptor 2, and triple negative breast cancer (TNBC) that do not express ERα, PR and HER2. These markers allowed the development of hormone therapies against estrogen sensitive tumors and targeted therapies against HER2 overexpressing tumors. Unfortunately, no specific therapy is available for TNBC. The latter accounts for 15 to 20% of breast cancers and is more common in young women. TNBC can be particularly aggressive and has a higher risk of recurrence as compared to other subtypes of breast cancer. These clinical data highlight the urgent need for the characterization of predictive marker to chemotherapy response. Such markers could lead to the development of new personalized treatment strategy. In 40% of TNBC, the expression of claudin 1, a major constituent of tight junctions, has been shown to be absent or strongly diminished. Most studies reported that a loss of claudin 1 expression correlates with increased malignant and metastasis potential associated with recurrence of disease in invasive breast carcinoma patients and specifically those with TNBC. Tight junctions play crucial roles in the maintenance of cell adhesion and polarity. Although the role of claudin 1 in breast cancer is not clearly defined, several in vitro studies suggested that claudin 1 functions as a tumor suppressor. Indeed, downregulation of claudin 1 was associated with increased motility of non-invasive T-47D breast cancer cells and with neoplastic transformation of mammary epithelial MCF-12A cells. We and others showed that the re-expression of claudin 1 was shown to be sufficient to induce apoptosis in some TNBC cells type. Recently, it was reported in MCF-7 (ER+) cancer cells that claudin 1 increased the sensitivity to Tamoxifen, Etoposide and Cisplatin. Taken together, these data led us to hypothesize that claudin 1 re-expression could constitute a new therapeutic option in “claudin 1-low” TNBC. Our preliminary data, showed that a demethylating agent, the 5 aza-cytidine, stimulates claudin 1 expression in MDA-MB-231 and Hs578T (TNBC “claudin 1 low” cells) accordingly to previous results. In MDA-MB-231 cells, claudin 1 expression is stimulated by Doxorubicine and Paclitaxel, two classical chemotherapy agents. The claudin 1 expression level is correlated to the apoptotic response. Moreover, 5-FU could not induce claudin 1 expression, nor an apoptotic response. Interestingly, claudin 1 re-expression by pre-treatment with 5 aza-cytidine restore the sensitivity of resistant TNBC “claudin 1-low” to 5FU.
The aim of the Ph.D is to answer the following biological questions:
1) Could claudine 1 re-expression in resistant TNBC “claudin 1 low” cells, re-sensitize them to chemotherapeutic agent? 2) What are the key factor that stimulated claudin 1 expression? 3) Could Claudine 1 be a predictive marker for the response to chemotherapy?
Experiment design
1.The Ph.D. student will analyze if claudin 1 expression could re-sensitize the TNBC “claudin 1 low” cells MDA-MB-231 and Hs578T to a combination of various chemotherapy agents standardly use in clinic. The apoptotic response will be measured by PARP and caspase 7 cleavage using western blot analysis and by TUNEL staining. The involvement of claudin 1 will be determine using si RNA strategy. Ph.D. student will complete this study by a three-dimensional (3D) cell culture study with both cell lines. First he will determine the experimental condition to re-expresse claudin 1 by the 5-azacitidin. More over in the laboratory we have MDA-MB-231 clones stably overexpressing claudin 1 at various level. He will developed the same models for Hs578T cells. The mammosphere re-expressing claudin 1 protein either by chemical stimulation or by stable transfection will be treated by the most efficient therapy on bi- dimensional (2D) culture. The apoptotic death will be analyze as described below. The role of claudin 1 will be confirm in vivo using Zebrafish model in collaboration with Pr. Marc Diederich, LBMCC Luxembourg. The mammosphere will be treated as previously, and then injected in the yolk sac of the zebrafish. After 3 days, the tumor size will be analyze and correlated to claudin 1 expression.
2. The second part of the project will aim to identify new regulators and/or co-markers which could prognostic the biological sensitivity to a claudin 1 re-expression. The Ph.D. student will determine the minimal promoter region involved in claudin 1 regulation using both experimental biological and bioinformatics approaches. The region responsible for the sensitivity will be determined by increasing deletion of the promoter. The potential site will be confirm by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay
3. The third part will be focused on aspect that is more clinical and will be developed in collaboration with the “Institut de Cancérologie de Lorraine”. Its purpose is to evaluate if the absence of claudin 1 protein could be a predictive marker for tumor resistance. The Ph.D fellow will analyze by immunohistochemistry samples from tumors with various profile of sensitivity to chemotherapy and characterize the expression of claudin 1 and eventually its key regulators (identified in the previous part of the project). In fine the candidate should evaluated if a correlation could be establish between claudin 1 and or other co-regulators of its expression and the chemo-sensitivity or resistance to the treatment .
In conclusion, this work will determine 1) if claudin 1 re-expression could be a promising path to restore response to chemotherapy agent by appropriated co-treatments and 2) if the status of claudin 1 and its transcriptional regulators could provide guidance for the treatments.

This project is part of the SBS department and integrates the two axes from the sticmo project’s. The role of claudin 1 in breast cancer is a recent topic and very few teams investigated if a modulation of claudin 1 could modify the response to chemotherapy. Recently Zhou et al showed on the luminal (ER+) MCF-7 cells that claudin 1 overexpression increased the sensitivity to tamoxifen, etoposide and cisplatin.
Keywords: Breast cancer, claudin 1, chemotherapy agents, tight junction, apoptosis
Conditions: , UMR CNRS 7039 SBS départent
Université de Lorraine, Campus science, boulevard des aiguillettes, entrée 1B,9eme étage BP 70 239 54 506 Vandoeuvre-les -Nancy France
1680 euros per month.
The candidate should have a Master degree in cell biology from an internationally recognized university. High level of competency in cell and molecular biology is require. The laureate should have obtained his diploma with honor (M1 and M2) and his academic cursus should be very good in order to be able to postulate to a Ph.D. fellowship.
Department(s):
Health - Biology - Signal
Financial aspects: doctoral felowship
Publications: hal-00746870v1,hal-01094210v1,hal-01094216v1,hal-00917939v1,hal-01589665v1.    + CRAN - Publications