The tumour-suppressive role of GPR15L and its interaction with GPR15 in colorectal cancer

Abstract

Colorectal cancer (CRC) remains one global concern due to its prevalence and unfavourable outcomes, while modulation of the tumour microenvironment is crucial in driving its aggressivity. Hence, searching for novel therapeutic targets remains an important field of CRC study. Increasing investigations have identified the role of G protein-coupled receptor 15 ligand (GPR15L) beyond immune modulation, such as enhancing the proliferation of keratinocytes through inhibiting cell cycle arrest in dermatological disorders. However, this becomes a dilemma when one study in colorectal cancer suggested that the GPR15L may induce G1 arrest. Alternatively, activation of its receptor, G protein-coupled receptor 15 (GPR15), showed repressive outcomes on pancreatic ductal adenocarcinoma, yet its activation in endothelial cells promotes angiogenesis. Given that GPR15 and GPR15L are dysregulated in CRC, this study attempted to delineate the controversial characteristics of GPR15L and GPR15, investigate their interactions, and identify their clinicopathological significance in CRC.

First, the downregulation of GPR15L in CRC tissues was identified. A comparison of GPR15L mRNA expression in 141 paired CRC and normal adjacent tissues (NATs) revealed the correlation between reduced GPR15L in tumours and poor prognosis, including later TNM staging, increased tumour size, presence of metastasis and reduced disease-free survival. Reduced GPR15L in NATs was also associated with adverse development of tumours. Epigenetically, hypermethylation was found in 17/28 CpGs in proposed enhancers, promoters, and gene body of GPR15L when compared CRC with NATs, while early-stage cases showed a reduced number of hypermethylated CpGs. Treatment of cell lines with demethylating agent promoted GPR15L expression. In vitro assays suggested the correlation of GPR15L with increased chemosensitivity, especially to 5-fluorouracil, reduced migration and colony formation, whereas in vivo study revealed a significant reduction in tumour volume and increased chemosensitivity. Western blotting also identified the inversed correlation of GPR15L and oncoproteins PI3K p110β, p-Akt, Bcl-2, Bcl-xL and cyclin D1, suggesting its tumour-suppressive characteristics.

Serving as a secreted protein, treatment of cell lines with secreted GPR15L resulted in comparable outcomes, functionally and molecularly, to GPR15L overexpression in vitro, while knockdown of GPR15 diminished the tumour-suppressive effect of secreted GPR15L. Accordantly, such treatment induced the chemosensitivity of 5FU-resistant CRC cells. Supplement of secreted GPR15L also reduced cell surface GPR15 expression. Comparing the transcriptional changes in GPR15L-overexpressing and secreted GPR15L-treated cells, a concordant downregulation of 23 proto-oncogenes was identified by RNA sequencing.

Finally, upregulation of GPR15 protein was detected in CRC tissues, while online databases suggested the association of GPR15 mRNA overexpression with poor survival. Functionally, in vitro assays revealed its oncogenic effect in elevating proliferation, migration and colony formation, but also promoting chemosensitivity, whereas knockdown of GPR15 resulted in reversed outcomes. Positive correlations were identified between GPR15 expression and PI3K/Akt signalling, Wnt signalling, and anti-apoptotic proteins. Based on the opposite functional and molecular effects of GPR15L and GPR15, it was concluded that GPR15L may serve as both a prognostic marker and therapeutic strategy to suppress oncogenic characteristics partially by inducing endocytosis and degradation of GPR15, in addition to its GPR15-independent signalling, resulted in tumour suppression.