CISPLATINUM SENSITIVITY OF BRCA1-MUTATED HCC1937 BREAST CANCER CELLS IS LINKED TO IMPAIRMENT OF NOTCH SIGNALING AND IS INCREASED BY GAMMA-SECRETASE INHIBITORS

Obietti vo: BRCA1 plays a criti cal role in DNA-damage repair mechanisms elicited by cell exposure to anti -tumor agents. HCC1937 is a BRCA1-defecti ve breast cancer cell line which discloses higher sensiti vity to cisplati num (CDDP) as compared to its derivati ve clone, HCC1937/wtBRCA1, generated in our laboratory by full-lenght BRCA1 cDNA transfecti on. To identi fy the molecular bases of BRCA1-related diff erenti al sensiti vity to CDDP, we analyzed the whole gene expression profi le of HCC1937 and HCC1937/wtBRCA1 cells following in vitro exposure to CDDP;with this experimental approach, we identi fi ed CDDPinduced trascripti onal changes involving the Notch pathway. Preclinical evidence suggests that combinati on strategies with plati num compounds and Notch inhibitors may exert anti -tumor eff ects. At this aim, we evaluated in vitro anti - proliferati ve eff ect of a pan-Notch inhibitor, ?-secretase inhibitor XII(GSI-XII) in HCC1937 and HCC1937/wtBRCA1, alone or in combinati on with CDDP, to assess its anti tumor acti vity in this specifi c setti ng. Materiali e metodi: RNA was isolated from HCC1937 and HCC1937/wtBRCA1 cell lines exposed to CDDP at IC50 doses of 30 and 70?M respecti vely, for 3,12 and 24 hours. Gene expression profi ling was performed by Array 1. 0ST (Aff ymetrix). Array data were analyzed using Gene Expression Console and Ingenuity®Pathway Analysis(IPA). Western Blotti ng was performed in both cell lines aft er exposure to CDDP using Notch3 M20 sc-7424 goat polyclonal IgG. Cell proliferati on was analyzed by MTT assay on both cell lines in presence of increasing concentrati on of CDDP, GSI-XII and combinati on of both compounds. Risultati : By cDNA microarray whole gene expression profi le and IPA, we demonstrated a diff erenti al modulati on of Notch signaling aft er CDDP exposure in HCC1937 cell line as compared to HCC1937/wtBRCA1,with strong down-regulati on of Notch1, 2 and 3 expression in HCC1937 together with other genes involved in the Notch pathway. We demonstrated a signifi cant reducti on of Notch3 protein's expression following CDDP exposure at the IC50 dose for 12,18 and 24 hours in HCC1937 as compared to HCC1937/wtBRCA1 consistently with gene expression results. Finally, we observed a ti me- and dose-dependent decrease of cell growth of HCC1937 following GSI-XII exposure as compared to HCC1937/ wtBRCA1, with an IC50 between 20 and 25 ?M. Conversely, HCC1937/wtBRCA1 where highly resistant to the drug. The combinati on of 10 ?M of GSI-XII plus CDDP at diff erent concentrati ons produced a signifi cant sinergisti c eff ect at 48 hours in HCC1937, which did not occurred in HCC1937/wtBRCA1. Conclusioni: Our fi ndings suggest that the high sensiti vity of BRCA1-defecti ve cells to CDDP exposure may be related not only to depression of the DNAdamage repair machinery but also to down-modulati on of the Notch survival pathway. GSIs deserve investi gati on as a novel therapeuti c tool in the specifi c setti ng of BRCA1 defecti ve tumors (Supported by Italian Ministry of Educati on).