Abstract 1902: Enrichment of putative cancer stem cells during anti-angiogenic therapies promotes relapse formation in hepatocellular carcinoma
Authors: Castven Darko , Czauderna Carolin , Becker Diana , Wörns Marcus A. , Thorgeirsson Snorri S. , Grimminger Peter , Lang Hauke , Galle Peter R. , Marquardt Jens U. 
Published: July 2017

Abstract
Background and Aims: Activation of neo-angiogenic processes in hepatocellular carcinoma (HCC) during disease progression is frequently associated with poor clinical outcome. Consequently, inhibition of neo-angiogenesis is an effective treatment strategy for advanced HCC. However, development of chemoresistance is observed in the majority of patients. Compelling evidence suggest that cancer stem cells (CSCs) may contribute to the acquisition of resistant properties in many solid tumors, but their exact role in this process for HCC remains to be defined. Here, we evaluate the importance of CSCs in the development of resistance and relapse formation after exposure to different anti-angiogenic therapies in HCC and define the concomitant adaptive molecular changes.
Methods: Five HCC cell lines and one primary HCC isolate were exposed to sorafenib and sunitinib for a total of 14 days. The treatment effects on CSCs were estimated by sphere forming capacity in vitro as well as the side-population (SP) approach. Expression levels of key oncogenic and CSC markers, such as EpCAM, CD133 and ABCG2 transporter, were assessed by qRT-PCR and flow cytometry. Furthermore, whole transcriptome analyses were performed at different time points.
Results: Both treatment regimens effectively reduced oncogenic properties in all investigated HCC cells. However, sustained anti-proliferative effect after treatments was observed in only one cell line. In three other hepatoma lines an initial treatment effect was subsequently followed by rapid re-growth thereby mimicking the responses observed in patients. Interestingly, two cell lines showed differential response to applied drugs, showing anti-proliferative effects to sorafenib, while relapse formation occurred after sunitinib treatments. While anti-oncogenic effects in sensitive cell lines were associated with significant reduction in sphere forming capacity, CSC marker EpCAM as well as SP cells, resistant cell line showed a transient increased in CSC properties. Importantly, acquired resistance to both drugs uniformly developed in the cell lines suggesting that common molecular mechanisms might be operative. These adaptive molecular changes involved signaling pathways known to be associated to cell survival and proliferation (RAS, AKT, MYC), as well as angiogenesis (VEGFR, PDGFR, HIF1a). Furthermore, the resistant cell lines showed compensatory upregulation of key oncogenic molecules such as EGFR as well as multidrug resistance ABC transporters.
Conclusions: Our in vitro model recapitulates features of drug resistance observed in human HCC patients. Resistance to anti-angiogenic therapies might be fueled by transient expansion of CSCs. Therefore, specific targeting of CSCs as well as pro-oncogenic compensatory signaling pathways might be an effective therapeutic strategy to overcome resistance in HCC.
Citation Format: Darko Castven, Carolin Czauderna, Diana Becker, Marcus A. Wörns, Snorri S. Thorgeirsson, Peter Grimminger, Hauke Lang, Peter R. Galle, Jens U. Marquardt. Enrichment of putative cancer stem cells during anti-angiogenic therapies promotes relapse formation in hepatocellular carcinoma [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 1902. doi:10.1158/1538-7445.AM2017-1902