Hyaluronan Synthase 3 (HAS3) is a New Target of Sorafenib in Renal Cell Carcinoma

Abstract

Currently, kidney cancer is a common cancer among men and women. More than 85% of kidney tumors are renal cell carcinoma (RCC). Characterized as a hypervascularized tumor type, RCC is resistant to both radiotherapy and chemotherapy. One-third of RCC patients have metastasis at the time of diagnosis, and another one-third develop metastasis after the initial treatment. Therefore, the five-year survival of patients with metastatic RCC is about 12%. Despite the considerable improvement in RCC treatments, including the newly approved immunotherapies, the mortality associated with RCC is significant; 14,830 deaths in 2020. Strategies to develop new therapeutic options and to identify reliable biomarkers still remain as urgent clinical needs to improve outcome. The clinical data used in this study, either collected from The Cancer Genome Atlas (TCGA) or from an institutional cohort, were assessed to evaluate the possibility of Hyaluronan Synthase 3 (HAS3) as a biomarker. Two classic RCC cell lines (786-O cell and Caki-1) were stably transfected using a full-length HAS3 cDNA construct. Empty vector (EV) transfected cells were used as a control. Knock-down was performed using a HAS3 short hairpin RNA (shRNA). Both HAS3 overexpression and knock-down transfectants were validated by immunoblott analysis. Transfectants were subsequently assessed for proliferation, clonogenicity, motility & invasion, cell cycle, apoptosis, and hyaluronic acid (HA) production. The underlying signaling pathways were detected by immunoblott analysis. Tumor growth and metastasis were evaluated in a Sorafenib (SF) resistant (Caki-1) model using subcutaneous and orthotopic implants of Caki-1 EV and HAS3 transfectants. Analysis based on the clinical cohort and TCGA dataset showed that HAS3 expression was elevated in RCC and was a potential prognosticator of metastasis and overall survival (OS). In vitro, low doses of Sorafenib (SF: 5 µM) and 7-Hydroxy-4-Methylcoumerin (MU: 0.1 mM or 0.2 mM) synergistically inhibited cell proliferation, motility, invasion, and caused cell cycle arrest and apoptosis. Contrarily, HAS3 transfectants were resistant to the above combination treatment. Two different sources of endothelial cells: HMEC-1 (from dermal microvascular) and HULEC-5a (from lung microvascular endothelium), were resistant to SF+MU treatment in the presence of HA. Both subcutaneous and orthotopic mouse models showed that SF+MU inhibited tumor angiogenesis, growth and metastasis with minimal toxicity; HAS3-overexpressing tumors were resistant to SF+MU treatment. This study sheds new light on targeting specific mechanisms that cause drug resistance, which is a principal limiting factor for improving the outcome of patients with RCC. HAS3 is a promising potential candidate for developing a biomarker-based targeted therapy RCC.