Abstract 4524: The effect of acute intraoperative hypoxia in breast cancer

Abstract

Abstract Background: Hypoxia is defined as oxygen levels in tumor microenvironment of less than that in blood (90-100 mm Hg) and influences many aspects of tumour biology. During surgery tumour vasculature is cut off gradually leading to induction of acute hypoxia.The present study aims to experimentally test the genotypic and phenotypic effects of surgically induced acute hypoxia in breast cancer tumor samples and cell lines. Methodology: Core biopsy samples were collected from breast tumors (N=8 patients) at three time points during their curative surgery: prior (pre), mid-way (intra) and at the end (post). The samples were subjected to RNA-Seq and a list of differentially expressed genes (DEG) was prepared. A set of 26 DEG (‘pre’ Vs ‘intra’ Vs ‘post’) obtained from RNA-Seq analysis and additional 17 genes involved in inflammation, EMT and hypoxia pathways were chosen for validation in tumor samples. These genes were validated using a customized qPCR Array (Qiagen). A gene was considered validated if it was significantly deregulated in at least 4 out of 8 patients. In another experiment, MCF-7 cells were exposed to varying levels of oxygen concentrations (0.1-20%) for varying time periods ranging from 30 minutes to 72 hours, to study time and dose dependent effects of hypoxia on following functional characteristics: proliferation, invasion and cell cycle changes. Results: Concordant, statistically significant up-regulation of FOS, DUSP1, JUNB, FOSB, ZFP36, RGS1, S100A4, CXCL8 and CCL2 were observed in RNA-Seq and qPCR experiments while MMP13, HIF1A and VEGFA were up-regulated only in qPCR. However, 7 protein markers of inflammation, EMT and hypoxia did not show any significant change between pre, intra and post-operative samples. In MCF-7 cells, a dose and time dependent decrease in cell viability was observed with increasing severity of hypoxia as well as decrease in invasiveness, but there was no significant impact on cell cycle phases. When hypoxic cells were re-incubated under normoxic conditions an increase in cell proliferation and accumulation of cells in S phase (with a reduction in G2-M fraction) were observed, compared to cells grown under only normoxic conditions. Conclusion: Acute intra-operative hypoxia up-regulates expression of genes related to cell survival, chemoresistance, invasiveness, inflammation and angiogenesis in breast tumors. Breast cancer cells exposed to acute severe hypoxia followed by normoxia show increased proliferation. These effects may have implications for tumor cells that disseminate during surgery. Citation Format: Dimple R. Bhatia, Shalaka Joshi, Rohan Chaubal, Poonam Gera, Prajakta Kalkar, Farheen Naeem, Nisanth Mathew Raju, Nilesh Gardi, Nita Nair, Vaibhav Vanmali, Rohini W. Hawaldar, Amit Dutt, Rajendra A. Badwe, Sudeep Gupta. The effect of acute intraoperative hypoxia in breast 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 4524. doi:10.1158/1538-7445.AM2017-4524