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Hypoxia-induced oxidative stress promotes MUC4 degradation via autophagy to enhance pancreatic cancer cells survival

Oncogene volume 35, pages 5882–5892 (2016)Cite this article

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Abstract

Pancreatic cancer (PC) and associated pre-neoplastic lesions have been reported to be hypoxic, primarily due to hypovascular nature of PC. Though the presence of hypoxia under cancerous condition has been associated with the overexpression of oncogenic proteins (MUC1), multiple emerging reports have also indicated the growth inhibitory effects of hypoxia. In spite of being recognized as the top-most differentially expressed and established oncogenic protein in PC, MUC4 regulation in terms of micro-environmental stress has not been determined. Herein, for the first time, we are reporting that MUC4 protein stability is drastically affected in PC, under hypoxic condition in a hypoxia inducible factor 1α (HIF-1α)-independent manner. Mechanistically, we have demonstrated that hypoxia-mediated induction of reactive oxygen species (ROS) promotes autophagy by inhibiting pAkt/mTORC1 pathway, one of the central regulators of autophagy. Immunohistofluorescence analyses revealed significant negative correlation (P-value=0.017) between 8-hydroxy guanosine (8-OHG) and MUC4 in primary pancreatic tumors (n=25). Moreover, we found pronounced colocalization between MUC4 and LAMP1/LC3 (microtubule-associated protein 1A/1B-light chain 3) in PC tissues and also observed their negative relationship in their expression pattern, suggesting that areas with high autophagy rate had less MUC4 expression. We also found that hypoxia and ROS have negative impact on overall cell growth and viability, which was partially, though significantly (P<0.05), rescued in the presence of MUC4. Altogether, hypoxia-mediated oxidative stress induces autophagy in PC, leading to the MUC4 degradation to enhance survival, possibly by offering required metabolites to stressed cells.

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Abbreviations

8-OHG:

8-hydroxyguanosine

CHX:

cycloheximide

CoCl2:

cobalt chloride

DCFDA:

dihydrodichlorofluorescein diacetate

DMEM:

Dulbecco’s modified Eagle’s medium

H2O2:

hydrogen peroxide

HIF-1α:

hypoxia inducible factor-1 alpha

kd:

knocked down

LC3:

microtubule-associated protein 1A/1B-light chain 3

MDC:

monodansylcadaverine

MFI:

mean fluorescence intensity

MG132:

carbobenzoxy-l-leucyl-l-leucyl-l-leucinal

MTT:

3-[4,5-dimethylthiozol-2-yl]-2,5diphenyltetrazoliumbromide

NAC:

N-acetyl-cysteine

ns:

non-significant

qRT–PCR:

quantitative real-time PCR

RAP:

rapamycin

ROS:

reactive oxygen species

VB:

vinblastine

VNTR:

variable number of tandem repeats

WB:

western blot

α-TS:

alpha-tocopherol succinate.

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Acknowledgements

We thank Dr Jerred Garrison, Dr Pankaj K Singh and Dr Michael Hollingsworth for providing us hypoxia chamber, HIF-1α Sh RNA constructs and human MUC1 antibody, respectively. We would also like to thank Kavita Mallya, James Talaska, Janice Taylor, Dr Nilesh Wagh and Dr Phillip Hexley for technical support. Finally, we would like to thank RAPID autopsy program and related personnel and UNMC graduate studies fellowship. The work was supported, in parts, by grants from the NIH (UO1 CA111294, P50 CA127297, U54 CA163120 and RO1 CA183459).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA

    S Joshi, S Kumar, M P Ponnusamy & S K Batra

  2. Buffett Cancer Center, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA

    S K Batra

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Joshi, S., Kumar, S., Ponnusamy, M. et al. Hypoxia-induced oxidative stress promotes MUC4 degradation via autophagy to enhance pancreatic cancer cells survival. Oncogene 35, 5882–5892 (2016). https://doi.org/10.1038/onc.2016.119

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