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

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 CALCRb mRNA including exon 10 and is translated into the CTb Receptor isoform, and Transcripts 2 and 3 which are translated into the CTa Receptor. CALCRb expression is conserved across a diverse range of mammalian species. We examined the expression of all CT Receptor isoforms (CALCRtotal) and CALCRb 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 CALCRtotal and CALCRb expression under conditions of autophagy or apoptosis, consistent with a requirement for CALCRb 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 CALCRb expression under cellular stress and the finding by other groups confirming that knockdown of CT Receptor compromises cell survival, implicates the CTb Receptor as a potential oncoprotein.