Abstract LB-97: Identification of a novel splice variant of Hyaluronan Synthase-2 (HAS2) in ovarian cancer stem cells

We have identified a novel cellular structure consisting of a chain of 4–72 cells with a pericellular sugar coat (glycocalyx) in metastatic human ovarian cancer (OvCa) ascites samples. We have termed these “catenae” (Latin for “chains”). Catenae develop clonally and are exclusively composed of cancer stem cells (CSC) since a single catena cell forms intraperitoneal tumor (Abs #1376, AACR 2011). The glycocalyx is necessary for the maintenance of cancer stem cell state of catena and confers resistance to small molecule, antibody and immune cell-mediated killing. Microarray gene expression analysis identified hyaluronan synthase-2 (HAS2) as the most upregulated gene in catena compared to differentiated OvCa monolayer cells. HAS2 enzyme catalyzes the production of hyaluronan, which is an essential component of the catena glycocalyx. 454 deep sequencing analysis of the HAS2 gene in catenae showed that the read coverage was specific to the exon 1 and exon 4. Limited coverage was detected for exon 2 and exon 3, which suggested the possibility of an alternative splice variant. RT-PCR analysis of HAS2 identified a band of 1115 bp as a new splice variant of the HAS2 gene lacking exon 2, which we have termed the “Greenwich variant”. The Greenwich variant is in the same sequence frame as the wild-type HAS2 and represents a novel form of HAS2 protein starting from amino acid 215 to the C-terminus. The variant still has half the cytoplasmic enzymatic domain and it retains three extracellular domains. The Greenwich variant was expressed in catena cells with high CSC (50–100%) frequencies but not in differentiated OvCa monolayer cells with low CSC (1–5%) frequencies. The variant was present in 14 out of 16 (88%) ascites samples from patients with advanced stage metastatic serous ovarian adenocarcinoma and in 15 out of 25 (60%) metastatic serosal cancer samples of other histologies including breast, lung, gastric, colon and pancreatic cancer. Deep sequencing analysis identified 2 intronic point mutations at the splicing acceptor site of exon 4 of the HAS2 gene in catena cells. 5 out of 220 OvCa patients in The Cancer Genome Atlas database had HAS2 mutations. 4 out of 5 of these mutations were located at the exon 2-exon 3 junction. These mutations could result in alternative splicing of HAS2 and generation of the Greenwich variant. We also analyzed the expression of miRNAs in catena cells by Agilent arrays. hsa-miR-23b was the most downregulated miRNA in catena compared to differentiated OvCa cells. hsa-miR-23b is a negative regulator of HAS2 gene, and our results suggest that downregulation of hsa-miR-23b is associated with upregulation of wild-type HAS2 and the Greenwich variant. The Greenwich variant is a novel CSC-specific marker and its presence could be linked to catena formation, increase in cancer stem cell frequency, drug resistance, metastasis to serosal cavities (ascites, pleural and pericardial effusions) and poor prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-97. doi:10.1158/1538-7445.AM2011-LB-97