Impact of loss of PRLHR on aneuploidy and initiation of bladder cancer.

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854 Background: The question still remains for the presence of yet unknown genetic modifiers in the initiation and/or progression of bladder cancer (BC). It remains challenging to identify the key factors, the mechanism and potential target behind. A genome-wide CRISPR library colony formation assay was performed using UROtsa cells, an immortalized ureter epithelium cell line, in order to identify novel genes that promote the transformation of normal urothelial cells. As one of the top candidate genes, the Prolactin Releasing Hormone Receptor (PRLHR) was identified. To date, only a few studies addressed the impact and the correlation of PRLHR in cancer and reports in the context of BC are missing altogether. Methods: We assessed the gene and protein expressions via public database, qPCR and Western blot analyses in a set of nontumour cell strains (Y235T, UROtsa) and a set of BC cell lines (RT4, T24). Following the shRNA knockdown in the PRLHR-proficient cells(Y235T) and the ectopic PRLHR expression in the cell lines with low/absent PRLHR expression(T24), we performed functional experiments, such as soft agar colony formation assay, MTT, invadopodia and gelatine degradation assays. We determined the invasion and migration abilities of these genetically modified cells by using the and wound-healing, Boyden chamber and porcine bladder ex vivo organ culture invasion assay. Metaphase spread assay and Immunofluorescence was performed in modified Y235T cells for chromosome counting and microscopy observation (centrsomes, chromosomal misalignments and lagging chromosomes). Results: The available in silico expression data and expression analyses in BC cell lines and matched normal/tumour patient samples revealed the repression of PRLHR early in BC tumorigenesis. Soft agar experiments confirmed that ectopic PRLHR impairs colony formation of T24 cells, providing indirect evidence that the CRISPR-Cas9 mediated deficiency of PRLHR promoted UROtsa cells transformation in the screening assay. Functional experiments using the MTT and the wound healing assays showed that ectopic PRLHR improved the viability and migration abilities of BC cells. The Boyden chamber assay and the porcine bladder organ culture model revealed that PRLHR impairs BC cell invasion, likely by the inhibition of the AKT/PKB pathway. Moreover, we observed an abnormal number of centrosomes, an increasing number of lagging chromosomes in the anaphase and telophase of the PRLHR-knockdown cells during mitosis. Conclusions: The screening procedure identified PRHLR as a new candidate tumour suppressor factor, and that loss/down-regulation of this protein contributes to the progression of bladder cancer. Downregulation of PRLHR leads to aneuploidy and genome instability in urothelial cells. PRLHR might serve as a novel biomarker or potential target for BC.

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