Abstract B56: Transfer of Senescent Signals in a Hypoxic Tumor Microenvironment: A Mechanism of Tumor Cell Survival Mediated by Exosomes

Abstract Triple Negative Breast Cancers (TNBCs) are a histological subtype of breast tumors which are innately hypoxic; a tumor microenvironment integral to chemoresistance and aggressive tumor behaviour. Importantly, hypoxia can drive viable cellular fate mechanisms rendering cancer cells capable of by-passing apoptotic cell death when chemotherapeutically challenged. Moreover, exosomes (nanovesicles 40-150nm of endocytic origin) preferentially released in the hypoxic environment can potentially be trafficked into neighbouring cells thereby mediating communication and propagating chemoresistance. The objective of this study was to establish if exosomes released from the TNBC cell line MDA-MB-231 (MSL) grown in a hypoxic environment, can mediate the transfer of viable cellular fate signatures (senescent and autophagic) to non-hypoxic cells rendering them non-apoptotic and chemoresistant. Experimentally, MDA-MB-231 (MSL) TNBC cells were cultured in hypoxia (1%) for 0-96 hours. Senescent, autophagic and cell cycle markers (LC3, p21, HDAC6, TSG101 and MAD2) were analysed by Western Blot. Nanoparticle Tracking Analysis (NTA), performed on the Nanosight LM10 microscope, allowed the enumeration and characterisation of harvested exosomes, while Dil labelled fluorescent imaging confirmed exosomal uptake in vitro. Hypoxic exposure (1%) of the MDA-MB-231 (MSL) confirmed by an increase in HIF1a, demonstrated that cellular senescence (p21), autophagy (LC3II), HDAC6 and the tumor susceptibility gene 101 (TSG101) markers increased in prolonged hypoxic exposure. Prolonged hypoxic exposure was also shown to result in an increase in the number of exosomes released from MDA-MB-231 (MSL) cells. siRNA knockdown of TSG101, a component of the ESCRT pathway involved in exosome release, resulted in a decrease in the amount of exosomes released from MDA-MB-231 (MSL) cells. In summary, it is well established in the literature that TNBCs are more hypoxic and chemoresistant than non-TNBC histological subtypes. The fact that both senescence and autophagy are induced in this environment underlies the ability of cancer cells to maintain viability. Moreover, exosomal release is enhanced in hypoxia thereby facilitating signalling between cancer cells with the potential to propagate chemoresistance. We suggest that targeting TSG101 thereby impeding exosomal release has the potential to cessate communication between cells in hypoxia thereby promoting apoptotic cell death in cells that are chemotherapeutically challenged. Funding: The Mater Surgical Oncology Research Appeal, and Mater Breast Health Research Citation Format: Luke Gubbins, Karolina Weiner-Gorzel, Shiva Sharma, Laura Cooke, Anna Salvati, Jeremy Simpson, Kenneth Dawson, Malcolm Kell, Amanda McCann. Transfer of senescent signals in a hypoxic tumor microenvironment: A mechanism of tumor cell survival mediated by exosomes. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B56. doi:10.1158/1538-7445.CHTME14-B56