Abstract 1256: MUC4 is negatively regulated by hypoxia in ROS-dependent manner in pancreatic cancer
Authors: Joshi Suhasini , Kumar Sushil , Ponnusamy Moorthy P. , Batra Surinder K. 
Published: August 2015

Pancreatic cancer (PC) stands as the fourth leading cause of cancer-related death in the United States, with the 5-year survival rate of less than 6%. Hypoxia is one of the most prominent features of the PC microenvironment, which is also characterized by the presence of extremely high desmoplasia. Emerging evidence demonstrates that HIF-1α regulates MUC1 expression under hypoxia in PC. It enhances hypoxia driven angiogenesis and tumor cells survival by regulating the metabolic programming of PC cells. Our group has demonstrated that MUC4, which remains undetectable in the normal pancreas, is aberrantly overexpressed in the precursor lesions and progressively increase with the severity of PC. It is one of the most differentially expressed transmembrane proteins and has strongly been implicated in the progression, metastasis and chemoresistance of PC.

Given the substantial increase in hypoxia and MUC4 in PC, we hypothesized that hypoxia positively regulates MUC4 expression. To investigate the role of hypoxia on MUC4 expression, we treated PC cell lines (CD18/HPAF, T3M4 and CAPAN1) with 1% hypoxia. On contrary to our anticipation, we observed significant (p&lt;0.05) downregulation (two-fold) of MUC4 at the protein level in all tested PC cell lines. To explore whether hypoxia mediated downregulation of MUC4 is HIF-1α dependent, HIF-1α was selectively inhibited by ShRNA and YC1 inhibitor in PC cells, and interestingly, significant downregulation of MUC4 was observed which further goes down under hypoxic condition, suggesting that MUC4 is positively regulated by HIF-1α and the observed negative effect of hypoxia on MUC4 expression is HIF-1α independent. Our clinical data also revealed positive correlation between MUC4 and HIF-1α expression. As hypoxia also induces ROS production, our next objective was to observe the effect of exogenous hydrogen peroxide (H2O2; 50 μM) and hypoxia in combination with free radical scavenger N-acetyl cysteine (NAC; 5 mM) compound on MUC4 expression, and noticed significant reduction in MUC4 expression, which was attenuated in the presence of NAC. In order to relate our findings with the PC phenotype, we performed MTT assay and growth kinetics for 48 hours for cells treated with H2O2 and hypoxia, and observed significant (p&lt;0.05) cell death (by 20%-30%), which was abolished by NAC treatment as it was able to neutralize hypoxia induced ROS, as determined by DCFDA florescence measurement. This hypoxia mediated inhibitory effect on PC cell survival and proliferation was attributed to inhibition on Akt pathway.

In spite of commonly perceived cancer promoting role of hypoxia, growing bodies of evidence start pointing towards the inhibitory effects of hypoxia on cancer cell survival, which is further strengthened by our findings. This study demonstrates the novel role of ROS on MUC4 regulation in PC and emphasizes the need to optimize antioxidant therapies against PC.

Citation Format: Suhasini Joshi, Sushil Kumar, Moorthy P. Ponnusamy, Surinder K. Batra. MUC4 is negatively regulated by hypoxia in ROS-dependent manner in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1256. doi:10.1158/1538-7445.AM2015-1256