Introduction: Ovarian cancer is the most lethal gynecological malignancy. Despite its clinical significance, the factors that regulate the development and tumor progression and metastasis are among the least understood of all major human malignancies. In this study we show that the oncogenic proteins which are specifically packed in the cargo of the exosomes released from the ovarian cancer cells play a role in tumor progression, metastasis and chemo-resistance.
Methods: The exosomes were isolated using Exo-quick solution from different primary ovarian cancer cells (POCC), HGSOC cell lines, normal ovarian epithelial cells (OSE) and patient ascites. Further quantified by NTA and morphologically characterized by TEM, their purity was validated by the presence of exosome specific markers and absence of golgi matrix protein. Protein profiling in exosomes was done using Capillary LCMS/MS analysis. Proteomic data sets were analyzed by (http://www.qiagen.com/ingenuity) Ingenuity Pathway Analysis (IPA) to gain biological insight in disease mechanisms, associated to the observed expression changes. Cell proliferation assay and cell migration assay were carried out in normal and ovarian cancer cell lines with and without exosome co-culture. STAT3 and Hepatocyte growth factor (HGF) expression in exosomes and Matrix metalloproteinases (MMPs) in different cell lines were analyzed using Western Blotting. Also in-vivo studies were carried out using orthotopic tumor model in mice. In addition cisplatin accumulation studies were done using ICP-MS.
Results: The exosome concentration from POCC and HGSOC cells was much higher (2-10 fold times) than from the OSE cells. Expression of CD9, CD63, TSG101 and EpCAM confirmed the exosomes. HGF, Complement factor H (CFH) and Lysyl oxidase- 2 (LOXL2) were selected from the top 25 proteins identified by IPA based on their expression ratio and disease relevance. IPA scored STAT3 as the top regulator effect network and furthermore HGF was activated in our data set. The expression of activated STAT3 and HGF were confirmed in the exosomes from HGSOC cells and patient ascites. Cell proliferation and migration was significantly increased with concurrent increased expression of MMP-2 and -9 in- vitro favoring the role of exosomal oncogenic proteins in tumor progression and metastasis as evidenced in in-vivo orthotopic tumor mice. The decrease in cisplatin accumulation in OVCAR8 cells observed, when co-cultured with exosomes from Cisplatin resistant cell line suggests its role in chemo-resistance as well. In addition, blocking exosome function inhibits cell proliferation and increases the cytotoxicity to cisplatin.
Conclusion: The results conclude that tumor exosomes carry a unique set of highly elevated oncogenic proteins that play a very important role in mediating tumor progression and metastasis while inducing chemo-resistance as well and are evident from our in-vitro and in-vitro studies.
Citation Format: Kalpana Deepa Priya Dorayappan, Ross A. Wanner, Roman A. Zingarelli, Uksha Saini, Selvendiran Karuppaiyah, David E. Cohn. Exosomal oncogenic proteins promote tumor progression, metastasis and chemoresistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5798. doi:10.1158/1538-7445.AM2017-5798