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The ALKBH family of proteins are highly expressed in various types of human cancer where they are involved in tumor growth and progression. However, multiple isoforms of ALKBH exist and the effect of individual isoforms on the development of urinary bladder cancer is unknown, particularly the molecular mechanisms involved in the progression from a noninvasive to invasive phenotype. We examined the role and function of ALKBH2 in human bladder cancer development in vitro and provide the first report that suppression of ALKBH2 in a human urothelial carcinoma cell line, KU7, reduces the expression of the transmembrane mucin protein, MUC1, and induces G1 cell cycle arrest. Moreover, reduction of ALKBH2 suppressed epithelial to mesenchymal transition (EMT) via increasing E-cadherin and decreasing vimentin expression. Transfection of MUC1 siRNA inhibited cell proliferation and EMT to the same extent as ALKBH2 gene silencing in vitro. ALKBH2 knockdown significantly suppressed MUC1 expression and tumor volume of bladder cancers in vivo as assessed in an orthotopic mouse model using ALKBH2 shRNA transfected KU7 cells. Immunohistochemical examination showed high expression levels of ALKBH2 in human urothelial carcinoma samples, especially in high-grade, superficially and deeply invasive carcinomas (pT(1) and >pT(2)), and in carcinoma in situ but not in normal urothelium. This study demonstrates that ALKBH2 is an upstream molecule of the oncoprotein, MUC1, and regulates cell cycle and EMT, resulting in progression of urothelial carcinomas.
Urinary bladder cancer is a common cancer of the urogenital system. In 2006, it was estimated that 16 510 new patients were found with bladder cancer, with 6804 projected deaths from the disease in Japan. Urinary bladder cancer is characterized by frequent recurrence and progression that invades the muscularis propria. The role of alterations in molecular pathways that control normal cellular homeostasis in the oncogenesis of bladder cancer is well established. The most common histological type of bladder cancer is urothelial carcinoma. Approximately 80% of patients with urothelial carcinoma present with nonmuscular invasive urothelial carcinoma (from noninvasive papillary to superficially invasive phenotype). Superficially-invasive non-muscular-invasive urothelial carcinoma is notorious for its tendency to invade the muscular layer when it is left untreated, but rarely progress to muscular invasive life-threatening cancer when patients undergo medical treatment. In contrast, muscular invasive urothelial carcinoma and high-grade carcinoma in situ exhibit aggressive behavior with a tendency to develop distal metastatic disease and seem highly resistant to immunotherapy as an initial treatment for bladder cancer as well as surgical intervention and chemoradiation therapy for advanced phenotypes. These two different types of cancer harbor different genetic and molecular alterations.[2, 3] Low-grade noninvasive urothelial carcinomas exhibit constitutively activated fibroblast growth factor receptor 3 and receptor tyrosine kinase-RAS pathways. In contrast, tumors that demonstrate high-grade or invasive phenotype show structural and functional defects in the p53 and retinoblastoma (RB) proteins. Loss of heterozygosity of chromosome 9 is frequently found in both types of urothelial carcinomas.
In Escherichia coli, the AlkB protein is involved in the repair of methylation-induced DNA after damage by oxidative demethylation.[4, 5] To date, eight mammalian homologues, human AlkB homologues 1–8, have been identified by bioinformatic analysis.[5, 6] Only two of the corresponding human proteins, hABH2 and hABH3, have been shown to possess a similar repair activity as AlkB from E. coli. Accumulating molecular evidence suggests that ABH2 is the primary dioxygenase to repair methyl and εADNA damage. We previously found that ALKBH8 contributes to human urothelial carcinoma development via NADPH oxidase 1-dependent generation of reactive oxygen species. Furthermore, it has been demonstrated that overexpression of ALKBH3 is involved in the genesis and progression of prostate cancer, non-small cell lung cancer, pancreatic cancer and urinary bladder cancer.[9, 10, 12] Thus, ALKBH family members may be involved in development of various types of human malignancies.
The membrane-bound oncoprotein MUC1 is expressed at the apical plasma membranes of normal secretory epithelial cells. MUC1 is aberrantly overexpressed in diverse human malignancies and inhibits stress-induced apoptosis,[14-16] enhances invasion and metastasis, and promotes epithelial to mesenchymal transition (EMT) via the upregulation of the expressions of transcription factors Snail and Slug, together with downregulation of E-cadherin expression.(16)
In the present study, we found that ALKBH2 is overexpressed in bladder cancer and that MUC1 functions as an important downstream signal. In addition, our results demonstrate that ALKBH2/MUC1 signals strongly affect proliferation in urothelial carcinogenesis.
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We demonstrate for the first time that the human alkB homologue, ALKBH2, contributes to urothelial carcinoma development by regulating MUC1-dependent EMT and cell growth. Reduction in ALKBH2 levels dramatically upregulated the E-cadherin expression while downregulating vimentin expression (a reversal of what is observed at the EMT) and induced cell cycle arrest at G1 phase. MUC1, a transmembrane mucin glycoprotein, is a key modulator of several signaling pathways that affect oncogenesis, motility and metastasis.[22-25] In addition, its expression is closely associated with poor prognosis in patients with several types of malignancies.[22, 26, 27] Recent investigations have found that MUC1 regulates growth factors such as transforming growth factor and platelet-derived growth factor, both of which are well documented to accelerate cell cycle progression, the cadherin switch, stress kinase-mediated snail induction and extracellular matrix remodeling in cancer cells.[16, 28, 29] MUC1 is a transmembrane mucin expressed at the apical surface and affects cell polarity by cooperation with β-catenin at the apical–lateral membrane and epidermal growth factor receptor at the basolateral membrane. Therefore, upregulation of MUC1 can lead to loss of E-cadherin/β-catenin complexes at the adherens junctions, and nuclear shuffling of β-catenin/MUC1-CT (cytoplasmic tail) complexes, resulting ultimately in the upregulation and nuclear localization of the Wnt nuclear effector, Lef-1.[30, 31] The importance of signaling pathways in urinary bladder cancer development has yet to be clarified, but they may target ALKBH2-mediated cell growth and EMT.
The present study provides evidence that ALKBH2 tightly controls MUC1-dependent cell proliferation and EMT in urothelial carcinoma cells. The direct regulation of EMT by a DNA repair molecule seems biologically reasonable in cancer cells. Growing data demonstrates that cells with an EMT phenotype contain an abundant source of cancer stem cells that harbor cytotoxic damage-induced repair signals, suggesting a biological link between EMT and the DNA repair system: EMT will provide the most appropriate microenvironment for DNA repair molecules to perform their functions.
To date, the biological function of MUC1 on tumor progression has been well studied, particularly in ductal adenocarcinoma of the pancreas. Such studies have led to increasing recognition of MUC1 as a potential diagnostic marker and therapeutic target in pancreatic carcinomas. However, immune neutralization against MUC1 alone is not sufficient for clinical application. The present results suggest a more successful therapeutic strategy by targeting MUC1 and its upstream molecule, ALKBH2, in urinary bladder cancer.
It is of great interest that ALKBH2 is overexpressed not only in invasive but also in noninvasive papillary urothelial carcinoma cells and that the expression is much higher in invasive phenotypes. Immunohistochemical analysis of MUC1 indicated the similar correlation with tumor grade in agreement with in vitro/in vivo data (data not shown). No histological evidence of EMT was found, including loss of E-cadherin or increased expression of vimentin in non-invasive urothelial carcinoma cells. We postulate that when the expression of MUC1, tightly parallel to that of ALKBH2, increases above a certain level, EMT is activated, resulting in acquisition of an invasive phenotype. Moreover, low-grade, noninvasive urothelial carcinomas predominantly showed apical and superficial MUC1 expression, whereas basal cells with cytoplasmic and/or circumferential membrane positivity were frequently observed in high-grade invasive phenotypes. ALKBH2 might affect the localization of MUC1 at the apical membrane, basolateral membrane or cytoplasm, thereby modulating the E-cadherin–β-catenin interaction, resulting in the gain of EMT and advanced phenotype in urothelial carcinoma cells. This issue should be further evaluated to address whether any other unidentified molecules affect MUC1-dependent EMT and cell cycle progression after ALKBH2 activation.
It is well known that CIS frequently progresses to invasive urothelial carcinoma; however, the key mechanisms directing this process remain unclear. The present study showed that ALKBH2 was overexpressed in CIS; therefore, the positive regulation of MUC1-dependent enhancement of EMT by ALKBH2 provides a molecular mechanism to explain why CIS cancer cells are more likely to progress to invasion.
In summary, a member of the AlkB family, ALKBH2 plays an important role in cell survival and cancer development by regulating MUC1-dependent signaling in human urothelial carcinoma. Amplification of ALKBH2/MUC1 expression is closely associated with transformation from a noninvasive to an invasive phenotype. Molecular therapy focusing on ALKBH2 may represent an attractive tool to achieve successful therapy in the treatment of human bladder carcinoma.