Hypoxia-mediated tumor progression, metastasis, and drug resistance are major clinical challenges in ovarian cancer. Exosomes released in the hypoxic tumor microenvironment may contribute to these challenges by transferring signaling proteins between cancer cells and normal cells. We observed that ovarian cancer cells exposed to hypoxia significantly increased their exosome release by upregulating Rab27a, downregulating Rab7, LAMP1/2, NEU-1, and also by promoting a more secretory lysosomal phenotype. STAT3 knockdown in ovarian cancer cells reduced exosome release by altering the Rab family proteins Rab7 and Rab27a under hypoxic conditions. We also found that exosomes from patient-derived ascites ovarian cancer cell lines cultured under hypoxic conditions carried more potent oncogenic proteins—STAT3 and FAS that are capable of significantly increasing cell migration/invasion and chemo-resistance in vitro and tumor progression/metastasis in vivo. Hypoxic ovarian cancer cells derived exosomes (HEx) are proficient in re-programming the immortalized fallopian tube secretory epithelial cells (FT) to become pro-tumorigenic in mouse fallopian tubes. In addition, cisplatin efflux via exosomes was significantly increased in ovarian cancer cells under hypoxic conditions. Co-culture of HEx with tumor cells led to significantly decreased dsDNA damage and increased cell survival in response to cisplatin treatment. Blocking exosome release by known inhibitor Amiloride or STAT3 inhibitor and treating with cisplatin resulted in a significant increase in apoptosis, decreased colony formation, and proliferation. Our results demonstrate that HEx are more potent in augmenting metastasis/chemotherapy resistance in ovarian cancer and may serve as a novel mechanism for tumor metastasis, chemo-resistance, and a point of intervention for improving clinical outcomes.
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We thank Dr. Zhang Liwen, PhD for proteomic analysis using Fusion Orbitrap instrument at OSU supported by NIH Award Number Grant S10 OD018056; Dr. Xiaokui Mo, Ph.D for analysis of Bioinformatics data; and Dr. Min Gao, Ph.D, Kent University for TEM analysis. Confocal images presented in this manuscript were generated using the services of the Campus Microscopy and Imaging Facility at OSU supported in part by grant P30 CA016058, National Cancer Institute, Bethesda, MD. We also thank Dr. John Olesik, Trace Element Research laboratory at OSU who helped us to quantify the Cisplatin concentration in exosomes by ICP-Mass Spectrometry; Dr. John Hays for his willingness to pre-review this manuscript and provide valuable comments and suggestions; Dr. Matthew Ringel, MD and his laboratory member Dr. Moto Saji for their invaluable help in exosome isolation protocols; and Brentley Smith, GYN/ONC fellow at OSU, Graduate students Dongju Park and Christopher Koivisto, Medical student Rashmi Madhukar, and Undergraduate student Maria Riley, for the IHC, cell culture, and basic assay help. This work was funded by Ovarian Cancer Research Fund (OCRF), NCI RO1-CA176078 grant (to KS and DEC) and KOH ovarian cancer foundation grant to KDPD.
KS, KDPD, and DEC designed all experiments. KDPD and RW performed most of the in vitro and in vivo studies, exosome isolation, STAT3 knockdown experiments, patient ascites cell characterization, cisplatin measurement, and analyzed the data collected. JJW and RZ collected patient ascites and performed WB. AAS performed the tumor histopathology. US performed the transfection work. KDPD, JJW, DEC, and KS wrote, edited, and proofread the manuscript.
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The role of hypoxic signalling in metastasis: towards translating knowledge of basic biology into novel anti-tumour strategies
Clinical & Experimental Metastasis (2018)