Abstract B15: Targeting PBX1 signaling to sensitize carboplatin cytotoxicity in ovarian cancer

Abstract

Introduction: Resistance to chemotherapy represents a major obstacle to achieving long-term remission in ovarian cancer patients. Thereby, there is an unmet need to understand the pathogenesis of chemoresistance and to develop effective strategy to overcome drug resistance. To better understand the molecular etiology of drug resistance, we previously studied ovarian high-grade serous carcinoma (HGSC) to identify the genes and pathways they controlled in the development of chemoresistance. Toward this goal, we have demonstrated Notch3 pathway as one of the molecular contributors to carboplatin resistance in ovarian cancer cells. We further identified that PBX1, a TALE homeodomain transcription factor and proto-oncogene, mediates the functions of Notch3 signaling. This finding together with the fact that PBX1 is involved in several tumor-promoting functions raises the possibility that PBX1 may play an important role in chemoresistance. Thus, in this study, we aim to determine the roles of PBX1 in the development of carboplatin resistance in ovarian HGSC. Experimental procedures: Western blot and immunohistochemistry were performed to determine the expression levels of PBX1 in primary and recurrent/chemoresistant ovarian HGSC tissues and cell lines. To assess the functional roles of PBX1, PBX1 siRNA or cDNA was transfected into ovarian cancer cell lines. Stem cell-like characteristics were measured using an ALDH1 staining kit and flow cytometry to identify verapamil-sensitive side populations. A small molecule inhibitor targeting the binding between PBX1 and DNA was employed in mouse ovarian cancer xenograft models. Results: Immunohistochemistry demonstrated that PBX1 protein was upregulated in recurrent post-chemotherapy HGSCs as compared to the matched primary tumors. Western blot analysis showed that carboplatin-resistant (CR) ovarian cancer cell lines expressed higher protein levels of PBX1 than both parental cells and paclitaxel resistant cells. Gene knockdown of PBX1 using siRNA significantly suppressed growth in CR cell lines, indicating the dependence of CR cells on PBX1 expression. Since ALDH1, a stem cell marker, was also upregulated in CR cells as compared to the parental cells, we determined whether ectopic expression of PBX1 in PBX1-low ovarian cancer cells enhanced stem-cell phenotypes. Indeed, the fraction of verapamil-sensitive side population and the number of ALDH1 positive cells both increased in ovarian cells transfected with the PBX1-expressing plasmids, as were the stem cell associated markers including ABCB1 and GPR160. Moreover, we designed a novel small molecule inhibitor that disrupted the Pbx1/DNA complex and this PBX1 inhibitor potently interrupted the binding of PBX1 to the consensus DNA motif responsible for PBX1 binding. We observed an anti-proliferative effect of this inhibitor in CR ovarian cancer lines and an anti-tumorigenic effect in ovarian cancer mouse xenografts. Conclusion: This study demonstrates that PBX1 is a marker for recurrent post-chemotherapy HGSCs. Inactivating PBX1 using gene silencing and small molecule inhibitors enhances cytotoxicity of CR-resistant ovarian cancer cells. The above findings should have significant implications in cancer biology, and promise a novel strategy to combat ovarian cancer patients suffering from chemoresistance. Citation Format: Jin G. Jung, Joon T. Park, Alexader Stoeck, Tasmeen Hussain, Ren-Chin Wu, Ie-Ming Shih, Tian-Li Wang. Targeting PBX1 signaling to sensitize carboplatin cytotoxicity in ovarian cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B15.

Alternative splicing links histone modifications to stem cell fate decision

“…We also exemplified our hypothesized mechanism by an H3K36me3-associated isoforms switch of PBX1. In addition to its well-known functions in lymphoblastic leukemia (Kamps and Baltimore 1993;Foa et al 2000;Tesanovic et al 2014;Melendez et al 2015) and a number of cancers (Berge et al 2006;Qiu et al 2007;Park et al 2008;Magnani et al 2011;Thiaville et al 2012;Li et al 2014;Feng et al 2015;Jung et al 2015;Magnani et al 2015;Jung et al 2016), PBX1 was also found to promote hESC self-renewal by corporately binding to the regulatory elements of NANOG with KLF4 (Chan et al 2009). We found that the transcriptions of two isoforms of PBX1, PBX1a and PBX1b, are putatively regulated by H3K36me3 during hESC differentiation.…”

“…PBX1 was one of the genes that their ASs are positively associated with H3K36me3 ( Figures 5A, B). Its protein, PBX1, is a member of the TALE (three-amino acid loop extension) family homeodomain transcription factors (Chang et al 1997;Merabet and Mann 2016) and well known for its functions in lymphoblastic leukemia (Kamps and Baltimore 1993;Foa et al 2000;Tesanovic et al 2014;Melendez et al 2015) and several cancers (Berge et al 2006;Qiu et al 2007;Park et al 2008;Magnani et al 2011;Thiaville et al 2012;Li et al 2014;Feng et al 2015;Jung et al 2015;Magnani et al 2015;Jung et al 2016). PBX1 also plays roles in regulating developmental gene expression (Kim et al 2002) and maintaining stemness and self-renewal (Ficara et al 2008;Jung et al 2016), and in cell-cycle progression by repressing the cell-cycle inhibitor CDKN2B to promote transition to S phase (Koss et al 2012).…”