Identification and characterisation of calcitonin receptor isoforms expressed in glioblastoma derived glioma stem and U-87 MG cells.

Abstract

Glioblastoma (GBM) is a highly lethal brain cancer in which the calcitonin receptor (CT Receptor), encoded by the CALCR gene, is expressed in 78-88% of patient biopsies. Here, we investigate whether the CT Receptor plays a role in cancer cell survival. In cancer cell lines, knockdown of CALCR disrupts the cell cycle and induces apoptosis, supporting an essential pro-survival role. The CALCR gene produces three main transcripts in humans, of which Transcript 1 encodes CALCR_b mRNA including exon 10 and is translated into the CT_b Receptor isoform, and Transcripts 2 and 3 which are translated into the CT_a Receptor. CALCR_b expression is conserved across a diverse range of mammalian species. We examined the expression of all CT Receptor isoforms (CALCR_total) and CALCR_b expression in four high-grade glioma stem-like cell lines and in U-87 MG glioblastoma cells. Using qPCR, we observed stable levels of both CALCR_total and CALCR_b expression under conditions of autophagy or apoptosis, consistent with a requirement for CALCR_b in cell survival. As alternative splicing (AS) of key genes in cancers confers tumour resilience, we investigated AS of CALCR transcript 2 using long-read nanopore sequencing. Unexpectedly, we discovered a novel AS event causing inclusion of exon 10 within Transcript 2 in all glioblastoma cell lines investigated. This finding, together with stable CALCR_b expression under cellular stress and the finding by other groups confirming that knockdown of CT Receptor compromises cell survival, implicates the CT_b Receptor as a potential oncoprotein.