CREB3L1 has been recently proposed as a novel metastasis suppressor gene in breast cancer. Our current study highlights CREB3L1 expression, regulation, and function in bladder cancer. We demonstrate a significant downregulation of CREB3L1 mRNA expression (n = 64) in primary bladder cancer tissues caused by tumor-specific CREB3L1 promoter hypermethylation (n = 51). Based on pyrosequencing CREB3L1 methylation was shown to be potentially associated with a more aggressive phenotype of bladder cancer. These findings were verified by an independent public data set containing data from 184 bladder tumors. In addition, immunohistochemical evaluation showed that CREB3L1 protein expression is decreased in bladder cancer tissues as well. Interestingly, protein loss is predominately observed in the nuclei of aggressive tumor cells. Based on in vitro models we clearly show that CREB3L1 re-expression mediates suppression of tumor cell migration and colony growth of high grade and invasive bladder cancer cells. The candidate tumor suppressor and TGF-β signaling inhibitor HTRA3 was furthermore identified as putative target gene of CREB3L1 in both invasive J82 bladder cells and primary bladder tumors. Hence, our data provide for the first time evidence that the transcription factor CREB3L1 may have an important role as a putative tumor suppressor in bladder cancer.
Keywords: ATCC, American Type Culture Collection; BMP-2, bone morphogenetic protein 2; CA, California; CIS, Carcinoma in situ; CREB3L1, element binding protein 3-like 1; DAB, 3-3′ diaminobenzidine; DAC, 5-aza-2′-deoxycytidine; DNA, desoxyribonucleic acid; EK, ethics committee; ER, endoplasmic reticulum; FC, fold change; FFPE, formalin fixed paraffin embedded; G1, well differentiated; G2, moderately differentiated; G3, poorly differentiated; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HCV, Hepatitis C virus; HPV, human papilloma virus; HTRA (1-4), high-temperature requirement factor A (1-4); HTRA3; IQR, interquartile range; IRS, immunoreactive score; LMU, Ludwig-Maximilians-University; M, methylated; MIBC, muscle invasive bladder cancer; MSP, methylation specific PCR; NMIBC, non-muscle invasive bladder cancer; NU, normal urothelium; OASIS / CREB3L1; OASIS, old astrocyte specifically-induced substance; PCR, polymerase chain reaction; RIP, regulated intramembrane proteolysis; RWTH, Rheinisch Westfälisch Technische Hochschule; SP1, site 1 protease; SP2, site 2 protease; TCGA, The Cancer Genome Atlas; TGF-β, transforming growth factor beta; TSA, trichostatin A; TSS, transcription start site; U, unmethylated; UC, urothelial cell cancer; UPR, unfold protein response; USA, United States of America; WHO, World Health Organization; WI, Wisconsin; bladder cancer; cDNA, copy number desoxyribonucleic acid; mRNA, messenger ribo nucleic acid; n, number; ns, not significant; pTa, papillary non-invasive tumors; promoter methylation; s.e.m., standard error of the margin; tumor cell migration; tumor suppressor gene.