Abstract 2076: HSF1 inhibition accentuates the lethal activity of proteasome inhibitor in human pancreatic cancer cells

Abstract

Abstract Presence of aneuploidy increases the intracellular levels of misfolded proteins and proteotoxic stress phenotype in cancer cells, which is exploited and further accentuated by the therapeutic use of a proteasome inhibitor, e.g., the novel, orally bioavailable carfilzomib (CF). This results in the disruption of the cytosolic complex of the histone deacetylase 6 (HDAC6), the ATPase and segregase p97 and the heat shock factor 1 (HSF1)-a transcription factor involved in the transactivation of heat shock proteins (hsps). In the present studies, we demonstrate that treatment of the pancreatic cancer Hs766T and Panc1 cells with CF increased the levels of misfolded polyubiquitylated proteins, which disrupts the repressive HSF1/hsp90/HDAC6/Vp97 complex. This induces the phosphorylation, nuclear localization and activation of HSF1, which transactivated hsps (e.g., hsp70, hsp40 and hsp27) as an adaptive response to CF-induced proteotoxic stress. Treatment with CF also induced endoplasmic reticulum (ER) stress, represented by upregulation of GRP78 and the pro-apoptotic transcription factor CHOP, which induced the BH3 only pro-apoptotic protein Bim. Treatment with CF also dose-dependently decreased the viability of Hs766T and Panc1 cells. To further elucidate the role of HSF1 in the adaptive, protective response to CF-induced proteotoxic stress, we stably knocked down HSF1 in Hs766T cells. As compared to the control cells, following heat shock, Hs766T cells with knockdown (KD) of HSF1 displayed reduced expression of hsps. In the HSF1-KD versus the control Hs766T cells, treatment with CF resulted in a dose-dependent induction of polyubiquitylated proteins, GRP78, CHOP, and Bim, as well as induced significantly more apoptosis. HSF1-KD versus the control Hs766T cells exhibited markedly lower levels of HDAC6 and, consequently, displayed reduced CF-induced perinuclear protective aggresome formation, as determined by confocal microscopy. Additionally, as compared to the control cells, Hs766T cells with HSF1-KD demonstrated higher levels of p-AMPK and CF (5 to 20 nM) or the pan-HDAC inhibitor panobinostat (PS, 50 nM)-induced autophagy, as demonstrated by the accumulation of LC3-II (by immunoblot analysis) and LC3 puncta by immunofluorescent staining. Importantly, co-treatment with the autophagy inhibitor hydroxy-chloroquine (HCQ) induced more CF-induced cell death of HSF1-KD versus the control Hs766T cells. Using an acid-sensitive mCherry-GFP-LC3 reporter, we determined that the induction of LC3-II in the Hs766T HSF1-KD cells is the result of enhanced autophagic flux and not due to inefficient fusion of autophagosomes with lysosomes. Collectively, these findings indicate that inhibition of the transcriptional activity of HSF1 sensitizes pancreatic cancer cells to CF or PS-induced cells death, which is augmented by co-treatment with an autophagy inhibitor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2076. doi:10.1158/1538-7445.AM2011-2076