ALG3 Is a Potential Biomarker for the Prognosis of Bladder Cancer.

OBJECTIVE: Previous research showed that ALG3 was associated with several cancers, but the function of ALG3 in bladder cancer (BC) was yet unknown. The purpose of this study was to investigate the relative expression of ALG3 in BC tissues and corresponding normal tissues and the relationship between the relative expression of ALG3 and clinical outcome in bladder cancer patients. METHODS: In this study, the expression of ALG3 in bladder cancer was detected by immunochemistry. In order to determine the cell proliferation and migration ability more accurately, we performed colony forming assay, MTT assay and wound healing migration assay. The role of ALG3 on tumor growth and metastasis was explored by animal model in vivo. RESULTS: ALG3 was expressed higher in bladder cancer than that in the normal tissues (P<0.05). At the same time, we found that there was a positive correlation between ALG3 expression and the prognosis (P<0.05). Moreover, we also discovered that the expression of ALG3 was associated with clinical pathological features (P<0.05). The proliferation and migration abilities of bladder cancer cell line T24 and 5637 were inhibited by silencing ALG3. In addition, the growth of bladder cancer cell line T24 cells were inhibited by silencing ALG3 in vivo. CONCLUSION: Silencing ALG3 plays a critical role in bladder cancer development and growth. It inhibits bladder cancer cells growth in vitro and in vivo.

From Fragment to Lead: De Novo Design and Development toward a Selective FGFR2 Inhibitor.

Fibroblast growth factor receptors (FGFRs) are implicated in a range of cancers with several pan-kinase and selective-FGFR inhibitors currently being evaluated in clinical trials. Pan-FGFR inhibitors often cause toxic side effects and few examples of subtype-selective inhibitors exist. Herein, we describe a structure-guided approach toward the development of a selective FGFR2 inhibitor. De novo design was carried out on an existing fragment series to yield compounds predicted to improve potency against the FGFRs. Subsequent iterative rounds of synthesis and biological evaluation led to an inhibitor with nanomolar potency that exhibited moderate selectivity for FGFR2 over FGFR1/3. Subtle changes to the lead inhibitor resulted in a complete loss of selectivity for FGFR2. X-ray crystallographic studies revealed inhibitor-specific morphological differences in the P-loop which were posited to be fundamental to the selectivity of these compounds. Additional docking studies have predicted an FGFR2-selective H-bond which could be utilized to design more selective FGFR2 inhibitors.

Survival of bladder or renal cancer in patients with CHEK2 mutations.

PURPOSE: The purpose of this study was to compare the clinical characteristics and the survival of CHEK2 mutation positive and CHEK2 mutation negative patients diagnosed with bladder or kidney cancer. MATERIALS AND METHODS: 1016 patients with bladder and 402 cases with kidney cancer and 8302 controls were genotyped for four CHEK2 variants: 1100delC, del5395, IVS2+1G>A and I157T. Predictors of survival were determined among CHEK2 pathogenic variant carriers using the Cox proportional hazards model. The median follow-up was 17.5 years. Covariates included age (≤60; >61 years), sex (female; male), clinical characteristics (stage: TNM, grade, histopathological type), smoking status (non-smoking; smoking) and cancer family history (negative; positive). RESULTS: We found no impact of CHEK2 mutations on bladder or kidney cancer survival. However, we observed a possible increased survival in the subgroup of patients with stage T1 bladder cancer with CHEK2 mutations but this did not meet statistical significance (HR = 0.14; 95% CI 0.02-1.04; p = 0.055). Moreover, we observed that the missense mutations were more frequent in the low grade invasive bladder cancer patient group (OR = 7.9; 95% CI 1.50-42.1; p = 0.04) and in patients with bladder cancer with stage Ta (OR = 2.4; 95% CI 1.30-4.55; p = 0.006). The different results where missense mutations occurs less often we observed among patients with high grade invasive bladder cancer (OR = 0.12; 95% CI 0.02-0.66; p = 0.04) and those with stage T1 disease (OR = 0.2; 95% CI 0.07-0.76; p = 0.01). Our investigations revealed that any mutation in CHEK2 occurs more often among patients with stage Ta bladder cancer (OR = 2.0; 95% CI 1.19-3.47; p = 0.01) and less often in patients with stage T1 disease (OR = 0.31; 95% CI 0.12-0.78; p = 0.01). In the kidney cancer patients, truncating mutations were present more often in the group with clear cell carcinoma GII (OR = 8.0; 95% CI 0.95-67.7; p = 0.05). The 10-year survival for all CHEK2 mutation carriers with bladder cancer was 33% and for non-carriers 11% (p = 0.15). The 10-year survival for CHEK2 mutation carriers with kidney cancer 34% and for non-carriers 20% (p = 0.5). CONCLUSION: CHEK2 mutations were not associated with any change in bladder or kidney cancer survival regardless of their age, sex, smoking status and family history. We observed a potentially protective effect of CHEK2 mutations on survival for patients with stage T1 bladder cancer. CHEK2 missense mutations were more common among patients with low grade invasive bladder cancer and in patients with stage Ta diease. The frequencies of the I157T CHEK2 pathogenic variant were less in patients with high grade invasive bladder cancer and those with stage T1 disease. Among patients with bladder cancer with stage Ta disease, the OR for any mutation in CHEK2 was 2.0 but for those with stage T1 disease, the OR was 0.3. We observed truncating CHEK2 mutations were associated with kidney cancer patients with GII clear cell carcinoma.

The Inhibitory Effect of Sulforaphane on Bladder Cancer Cell Depends on GSH Depletion-Induced by Nrf2 Translocation.

Sulforaphane (SFN), an isothiocyanate (ITCs) derived from glucosinolate that is found in cruciferous vegetables, has been reported to exert a promising anticancer effect in a substantial amount of scientific research. However, epidemical studies showed inconsistencies between cruciferous vegetable intake and bladder cancer risk. In this study, human bladder cancer T24 cells were used as in vitro model for revealing the inhibitory effect and its potential mechanism of SFN on cell growth. Here, a low dose of SFN (2.5 µM) was shown to promote cell proliferation (5.18-11.84%) and migration in T24 cells, whilst high doses of SFN (>10 µM) inhibited cell growth significantly. The induction effect of SFN on nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression at both low (2.5 µM) and high dose (10 µM) was characterized by a bell-shaped curve. Nrf2 and glutathione (GSH) might be the underlying mechanism in the effect of SFN on T24 cell growth since Nrf2 siRNA and GSH-depleting agent L-Buthionine-sulfoximine abolished the effect of SFN on cell proliferation. In summary, the inhibitory effect of SFN on bladder cancer cell growth and migration is highly dependent on Nrf2-mediated GSH depletion and following production. These findings suggested that a higher dose of SFN is required for the prevention and treatment of bladder cancer.

ALPK2 acts as tumor promotor in development of bladder cancer through targeting DEPDC1A.

Bladder cancer is one of the most common malignant tumors in the urinary system. The development and improvement of treatment efficiency require the deepening of the understanding of its molecular mechanism. This study investigated the role of ALPK2, which is rarely studied in malignant tumors, in the development of bladder cancer. Our results showed the upregulation of ALPK2 in bladder cancer, and data mining of TCGA database showed the association between ALPK2 and pathological parameters of patients with bladder cancer. In vitro and in vivo experiments demonstrated that knockdown of ALPK2 could inhibit bladder cancer development through regulating cell proliferation, cell apoptosis, and cell migration. Additionally, DEPDC1A is identified as a potential downstream of ALPK2 with direct interaction, whose overexpression/downregulation can inhibit/promote the malignant behavioral of bladder cancer cells. Moreover, the overexpression of DEPDC1A can rescue the inhibitory effects of ALPK2 knockdown on bladder cancer. In conclusion, ALPK2 exerts a cancer-promoting role in the development of bladder cancer by regulating DEPDC1A, which may become a promising target to improve the treatment strategy of bladder cancer.

The role of matrix metalloproteinases in pathogenesis of human bladder cancer.

Matrix metalloproteinases (MMPs) play an important role in many physiological and pathological processes, including neoplastic processes. They belong to a group of enzymes called endopeptidases and have the ability to hydrolyze all proteins in the extracellular matrix (ECM). They are produced in most connective tissue cells, macrophages, leukocytes, endothelial cells, microglial cells and in cancer cells. Neoplastic diseases are one of the main causes of death in Poland and in the world, therefore learning about the process of carcinogenesis seems to be particularly important. The process of carcinogenesis is currently widely studied and MMPs play one of the key roles in the development of cancer. They do this by regulating local tumor growth, stromal invasion, stimulating angiogenesis and metastasis formation. Bladder cancer is the 7th most common cancer in the male population and the 11th most common cancer in the world. In bladder cancer, most studies have been devoted to MMP-2 and MMP-9, that are enzymes responsible for the degradation of type IV collagen in the first place, which through the destruction of basement membranes and ECM, play an essential role in the tumor invasion process. Since bladder cancer is characterized by the ability to relapse, from the point of view of clinical practice it seems particularly important to develop a marker of early bladder tumor recurrence. MMPs detected in the urine and serum of patients with bladder cancer are potential factors that could play such a role.

Circular RNA circSETD3 hampers cell growth, migration, and stem cell properties in bladder cancer through sponging miR-641 to upregulate PTEN.

Bladder cancer (BLCA) is a common malignant urothelial cancer in the world. Although circular RNAs (circRNAs) involve in regulating BLCA progression, the role of a novel circular RNA circSETD3 in regulating BLCA pathogenesis has not been studied. The expression of circSETD3, miR-641, PTEN mRNA in BLCA tissues and cell lines were measured using RT-qPCR. The gain-of-function experiments were performed in vitro and in vivo to detect the effects of circSETD3 on cell proliferation, migration, EMT, and stemness maintenance. Besides, rescue experiments were performed to demonstrate the regulatory mechanism of circSETD3/miR-641/PTEN in BLCA cell malignant phenotypes in vitro. CircSETD3 was remarkably downregulated in the cancerous clinical tissues and cell lines, in contrast with their normal counterparts, and circSETD3 tended to be deficient in BLCA patients with larger tumor size, advanced clinical stages, positive lymph metastasis and worse prognosis. In addition, circular isoforms of circSETD3 were more resistant to RNase R+ and actinomycetes D treatment compared to their linear isoforms, and circSETD3 mainly distributed in the cytoplasm of the BLCA cells. Further gain-of-function experiments showed that circSETD3 acted as a tumor suppressor to suppress BLCA cell proliferation, migration, EMT and stemness, and the underlying mechanisms had also been elucidated. Mechanistically, circSETD3 sponged miR-641 to upregulate PTEN, resulting in the blockage of BLCA progression. Our findings indicated that circSETD3 acted as a vital tumor suppressor in BLCA via regulating the miR-641/PTEN axis.

Elastolytic activity is associated with inflammation in bladder cancer.

Cancer development and progression is often associated with inflammation. Late diagnosis of inflammation that directly leads to the development of neoplasm-cancer is associated with a reduction in the chance of successful treatment or is associated with therapeutic difficulties. A panel of chromogenic substrates was used for the qualitative determination of the specific activity of enzymes in urine of patients with confirmed inflammatory reaction and/or epithelial neoplasms in particular tumours at various stages of development. Urine of people with excluded inflammation was used as a control group. Proteolytic activity was determined in urine samples collected from patients with epithelial neoplasms and/or inflammation. What is more, we determine human neutrophil elastase activity-related inflammation based on the examination of urine samples. We suspect that the proteolytical activity of urine samples is due to neutrophil response to inflammation, which is directly related to cancer. This is the first study to determine elastolytic activity in bladder cancer urine samples. It supports wider use of urine for inflammation screening.

TDO2 overexpression correlates with poor prognosis, cancer stemness, and resistance to cetuximab in bladder cancer.

BACKGROUND: Bladder cancer (BC) is the 10th most common cancer in the world. BC with muscle invasion results in a poor prognosis and is usually fatal. Cancer cell metabolism has an essential role in the development and progression of tumors. Expression of tryptophan 2,3-dioxygenase (TDO2) is associated with tumor progression and worse survival in some other cancers. However, no studies have been performed to uncover the biofunctional roles of TDO2 in BC. AIM: This study aim to investigate the clinicopathologic significance of TDO2 in BC. METHODS AND RESULTS: TDO2 expression was evaluated by qRT-PCR and immunohistochemistry in an integrated analysis with the Cancer Genome Atlas (TCGA) and other published datasets. TDO2 overexpression was significantly associated with T classification, N classification, and M classification, tumor stage, recurrence, and basal type, and with the expression of CD44 and aldehyde dehydrogenase 1 (ALDH1) in BC. High TDO2 expression correlated with poor outcome of BC patients. Using BC cell lines with knockdown and forced expression of TDO2, we found that TDO2 was involved in the growth, migration, and invasiveness of BC cells. Moreover, TDO2 was found to be crucial for spheroid formation in BC cells. Importantly, TDO2 promoted BC cells resistance to cetuximab through integration of the EGFR pathway. CONCLUSION: Our results indicate that TDO2 might take an essential part in BC progression and could be a potential marker for targeted therapy in BC.

Pegylated Recombinant Human Arginase 1 Induces Autophagy and Apoptosis via the ROS-Activated AKT/mTOR Pathway in Bladder Cancer Cells.

Bladder cancer is one of the most commonly diagnosed cancers worldwide, especially in males. Current therapeutic interventions, including surgery, radiation therapy, chemotherapy, and immunotherapy, have not been able to improve the clinical outcome of bladder cancer patients with satisfaction. Recombinant human arginase (rhArg, BCT-100) is a novel agent with great anticancer effects on arginine-auxotrophic tumors. However, the effects of BCT-100 on bladder cancer remain unclear. In this study, the in vitro anticancer effects of BCT-100 were assessed using four bladder cancer cell lines (J82, SCaBER, T24, and 5637), while the in vivo effects were evaluated by establishing T24 nude mice xenograft models. Intracellular arginine level was observed to be sharply decreased followed by the onset of apoptotic events. Furthermore, BCT-100 was found to induce H2O2 production and mitochondrial membrane depolarization, leading to the release of mitochondrial cytochrome c and Smac to the cytosol. Treatment with BCT was observed to upregulate the expression of LC3B and Becllin-1, but downregulate the expression of p62 in a time-dependent manner. Autophagic flux was also observed upon BCT-100 treatment. Besides, the phosphorylation of the AKT/mTOR pathway was suppressed in a time-dependent fashion in BCT-100-treated T24 cells. While N-acetyl-L-cysteine was shown to alleviate BCT-100-induced apoptosis and autophagy, chloroquine, MK-2206, and rapamycin were found to potentiate BCT-100-triggered apoptosis. Finally, BCT-100 was demonstrated to induce autophagy and apoptosis via the ROS-mediated AKT/mTOR signaling pathway in bladder cancer cells.

Anti-Cancer Effects and Tumor Marker Role of Glutathione S-Transferase Mu 5 in Human Bladder Cancer.

Our previous study demonstrated that the glutathione S-transferase Mu 5 (GSTM5) gene is highly CpG-methylated in bladder cancer cells and that demethylation by 5-aza-dC activates GSTM5 gene expression. The aim of the present study was to investigate the role of GSTM5 in bladder cancer. The levels of GSTM5 gene expression and DNA methylation were analyzed in patients with bladder cancer, and functional studies of GSTM5 were conducted using GSTM5 overexpression in cultured bladder cancer cells. Clinical analysis revealed that the GSTM5 mRNA expression was lower in bladder cancer tissues than in normal tissues and that the level of GSTM5 DNA methylation was higher in bladder cancer tissues than in normal urine pellets. Overexpression of GSTM5 decreased cell proliferation, migration and colony formation capacity. Glutathione (GSH) assay results indicated that cellular GSH concentration was decreased by GSTM5 expression and that GSH supplementation reversed the decrease in proliferation and migration of cells overexpressing GSTM5. By contrast, a GSH synthesis inhibitor significantly decreased 5637 cell GSH levels, survival and migration. Furthermore, GSTM5 overexpression inhibited the adhesion of cells to the extracellular matrix protein fibronectin. To elucidate the effect of GSTM5 on anticancer drugs used to treat bladder cancer, cellular viability was compared between cells with or without GSTM5 overexpression. GSTM5-overexpressed cells showed no significant change in the cytotoxicity of cisplatin or mitomycin C in 5637, RT4 and BFTC 905 cells. Though a degree of resistance to doxorubicin was noted in 5637 cells overexpressing GSTM5, no such resistance was observed in RT4 and BFTC 905 cells. In summary, GSTM5 plays a tumor suppressor role in bladder cancer cells without significantly affecting chemoresistance to cisplatin and mitomycin C, and the cellular GSH levels highlight a key mechanism underlying the cancer inhibition effect of GSTM5. These findings suggest that low gene expression and high DNA methylation levels of GSTM5 may act as tumor markers for bladder cancer.

Jolkinolide B targets thioredoxin and glutathione systems to induce ROS-mediated paraptosis and apoptosis in bladder cancer cells.

Bladder cancer is a clinically heterogeneous disease with a poor prognosis. In the current study, anti-proliferation assay of a Euphorbiaceae diterpenoid library led to the identification of an anti-bladder cancer agent Jolkinolide B (JB). JB showed significant cytotoxicity against a panel of bladder cancer cell lines and suppressed the growth of cisplatin (CDDP)-resistant bladder cancer xenografts in single or combination treatments. Mechanistic study revealed that, besides inducing mitogen-activated protein kinase (MAPK)-related apoptosis, JB could trigger the paraptosis via activation of reactive oxygen species (ROS)-mediated endoplasmic reticulum (ER) stress and extracellular signal-regulated kinase (ERK) pathway. The excessive production of ROS could be induced by JB via inhibition of thioredoxin reductase 1 (TrxR1) and depletion of glutathione (GSH). Collectively, JB that targets thioredoxin and GSH systems to induce two distinct cell death modes may serve as a promising candidate in future anti-bladder cancer drug development.

CDK7 inhibition by THZ1 suppresses cancer stemness in both chemonaïve and chemoresistant urothelial carcinoma via the hedgehog signaling pathway.

Urothelial carcinoma (UC) is the most common type of bladder cancer, with a 5-year survival rate of only 4.6% in metastatic UC. Despite the advances related to immune-checkpoint inhibitor therapy, chemotherapy remains the standard of care for metastatic diseases, with a 50% response rate. The covalent cyclin-dependent kinase 7 (CDK7) inhibitor THZ1 interferes with transcription machinery and is reported to be effective in cancers without targetable mutations. Therefore, we investigated the therapeutic effect of THZ1 on UC and examined possible mechanisms underlying its effects in both chemonaïve and chemosensitive cancers. CDK7 expression is increased in bladder cancer tissues, especially in patients with chemoresistance. THZ1 induced apoptosis and decreased viability in RT4, BFTC905, HT1376, T24, and T24/R UC cell lines. RNA-sequencing, immunoblotting, and sphere-formation assays confirmed that THZ1 suppressed cancer stemness. In the mouse xenograft model, THZ1 suppressed both chemonaïve and chemoresistant tumors. These results indicate that CDK7 inhibition-related cancer stemness suppression is a potential therapeutic strategy for both chemonaïve and chemoresistant UC.

E3 ubiquitin ligase RNF126 affects bladder cancer progression through regulation of PTEN stability.

E3 ubiquitin ligase RNF126 (ring finger protein 126) is highly expressed in various cancers and strongly associated with tumorigenesis. However, its specific function in bladder cancer (BCa) is still debatable. Here, we found that RNF126 was significantly upregulated in BCa tissue by TCGA database, and our studies indicated that downregulation of RNF126 significantly inhibited cell proliferation and metastasis through the EGFR/PI3K/AKT signaling pathway in BCa cells. Furthermore, we identified PTEN, an inhibitor of the PI3K/AKT signaling pathway, as a novel substrate for RNF126. By co-immunoprecipitation assays, we proved that RNF126 directly interacts with PTEN. Predominantly, PTEN binds to the C-terminal containing the RING domain of RNF126. The in vivo ubiquitination assay showed that RNF126 specifically regulates PTEN stability through poly-ubiquitination. Furthermore, PTEN knockdown restored cell proliferation, metastasis, and tumor formation of BCa cells inhibited by RNF126 silencing in vitro and in vivo. In conclusion, these results identified RNF126 as an oncogene that functions through ubiquitination and degradation of PTEN in BCa.

Profiling of CYP4Z1 and CYP1B1 expression in bladder cancers.

Bladder cancer is the tenth most common cancer worldwide, where its burden remains a challenge and needs new novel therapies. Several reports indicate expression of CYP4Z1 and CYP1B1 in many tumours. Their expressions are associated with a poor prognosis, and therefore proposed as promising biomarkers or targets for anticancer therapy. By using immunohistochemistry, expression of CYP4Z1 and CYP1B1 was evaluated in a panel of different types of bladder cancer, and the enzymes' relation to histopathological features were assessed. Results showed an increased expression of CYP4Z1 (54.3%) and CYP1B1 (76.9%) in the majority of bladder cancers compared to weak or lack of expression of both enzymes in normal tissues. CYP4Z1expression was significantly associated with tumour grade and stage where the expression was markedly increased in a high grade and advanced stage of the disease (p < 0.05). Additionally, CYP1B1 expression was also associated with TNM staging (p < 0.05) and its expression was increased in patients with lymph node metastasis. The expression profiles of CYP4Z1 and CYP1B1 suggest that both enzymes have the potential to be biomarkers or targets for novel anticancer therapy for bladder cancer. Nevertheless, further studies are needed to better delineate whether these enzymes are druggable targets.

Targeting natural splicing plasticity of APOBEC3B restricts its expression and mutagenic activity.

APOBEC3A (A3A) and APOBEC3B (A3B) enzymes drive APOBEC-mediated mutagenesis. Identification of factors affecting the activity of these enzymes could help modulate mutagenesis and associated clinical outcomes. Here, we show that canonical and alternatively spliced A3A and A3B isoforms produce corresponding mutagenic and non-mutagenic enzymes. Increased expression of the mutagenic A3B isoform predicted shorter progression-free survival in bladder cancer. We demonstrate that the production of mutagenic vs. non-mutagenic A3B protein isoforms was considerably affected by inclusion/skipping of exon 5 in A3B. Furthermore, exon 5 skipping, resulting in lower levels of mutagenic A3B enzyme, could be increased in vitro. Specifically, we showed the effects of treatment with an SF3B1 inhibitor affecting spliceosome interaction with a branch point site in intron 4, or with splice-switching oligonucleotides targeting exon 5 of A3B. Our results underscore the clinical role of A3B and implicate alternative splicing of A3B as a mechanism that could be targeted to restrict APOBEC-mediated mutagenesis.

Rac Family Small GTPase 3 Correlates with Progression and Poor Prognosis in Bladder Cancer.

Bladder cancer (BC) is a common genitourinary malignancy worldwide. However, the molecular pathogenesis of BC remains unclear. The current study conducted bioinformatic analyses to discover key genes involved in BC progression. A total of 375 differentially expressed genes (DEGs) were screened in the GEO database and The Cancer Genome Atlas (TCGA) database, which were further evaluated by the core level in the protein-protein interaction network. RAC3 (Rac family small GTPase 3), one of the top hub genes, was focused on for its gene expression and prognostic value in BC. Immunohistochemical assays indicated elevated RAC3 levels in BC tissues compared with normal tissues. Overexpression of RAC3 expression was closely associated with poor differentiation (p = 0.035), advanced TNM stage (p = 0.014), lymph metastasis (p = 0.033), and recurrence (p < 0.001). Kaplan-Meier and Cox proportional hazards analyses demonstrated that high RAC3 expression indicated poor survival of BC patients, which could serve as an independent prognostic factor for overall survival (HR = 3.159, p = 0.023) and disease-free survival (HR = 4.633, p = 0.002). Moreover, bioinformatic analyses indicated that RAC3 might be correlated with malignant phenotypes and immune infiltration of BC. Taken together, RAC3 could be a novel prognostic biomarker for BC.

SPRTN protease-cleaved MRE11 decreases DNA repair and radiosensitises cancer cells.

The human MRE11/RAD50/NBS1 (MRN) complex plays a crucial role in sensing and repairing DNA DSB. MRE11 possesses dual 3'-5' exonuclease and endonuclease activity and forms the core of the multifunctional MRN complex. We previously identified a C-terminally truncated form of MRE11 (TR-MRE11) associated with post-translational MRE11 degradation. Here we identified SPRTN as the essential protease for the formation of TR-MRE11 and characterised the role of this MRE11 form in its DNA damage response (DDR). Using tandem mass spectrometry and site-directed mutagenesis, the SPRTN-dependent cleavage site for MRE11 was identified between 559 and 580 amino acids. Despite the intact interaction of TR-MRE11 with its constitutive core complex proteins RAD50 and NBS1, both nuclease activities of truncated MRE11 were dramatically reduced due to its deficient binding to DNA. Furthermore, lack of the MRE11 C-terminal decreased HR repair efficiency, very likely due to abolished recruitment of TR-MRE11 to the sites of DNA damage, which consequently led to increased cellular radiosensitivity. The presence of this DNA repair-defective TR-MRE11 could explain our previous finding that the high MRE11 protein expression by immunohistochemistry correlates with improved survival following radical radiotherapy in bladder cancer patients.

Acetylation of putative arylamine and alkylaniline carcinogens in immortalized human fibroblasts transfected with rapid and slow acetylator N-acetyltransferase 2 haplotypes.

Exposure to alkylanilines found in tobacco smoke and indoor air is associated with risk of bladder cancer. Genetic factors significantly influence the metabolism of arylamine carcinogens and the toxicological outcomes that result from exposure. We utilized nucleotide excision repair (NER)-deficient immortalized human fibroblasts to examine the effects of human N-acetyltransferase 1 (NAT1), CYP1A2, and common rapid (NAT2*4) and slow (NAT2*5B or NAT2*7B) acetylator human N-acetyltransferase 2 (NAT2) haplotypes on environmental arylamine and alkylaniline metabolism. We constructed SV40-transformed human fibroblast cells that stably express human NAT2 alleles (NAT2*4, NAT2*5B, or NAT2*7B) and human CYP1A2. Human NAT1 and NAT2 apparent kinetic constants were determined following recombinant expression of human NAT1 and NAT2 in yeast for the arylamines benzidine, 4-aminobiphenyl (ABP), and 2-aminofluorene (2-AF), and the alkylanilines 2,5-dimethylaniline (DMA), 3,4-DMA, 3,5-DMA, 2-6-DMA, and 3-ethylaniline (EA) compared with those of the prototype NAT1-selective substrate p-aminobenzoic acid and NAT2-selective substrate sulfamethazine. Benzidine, 3,4-DMA, and 2-AF were preferential human NAT1 substrates, while 3,5-DMA, 2,5-DMA, 3-EA, and ABP were preferential human NAT2 substrates. Neither recombinant human NAT1 or NAT2 catalyzed the N-acetylation of 2,6-DMA. Among the alkylanilines, N-acetylation of 3,5-DMA was substantially higher in human fibroblasts stably expressing NAT2*4 versus NAT2*5B and NAT2*7B. The results provide important insight into the role of the NAT2 acetylator polymorphism (in the presence of competing NAT1 and CYP1A2-catalyzed N-acetylation and N-hydroxylation) on the metabolism of putative alkyaniline carcinogens. The N-acetylation of two alkylanilines associated with urinary bladder cancer (3-EA and 3,5-DMA) was modified by NAT2 acetylator polymorphism.

Krüppel-like factor 12 suppresses bladder cancer growth through transcriptionally inhibition of enolase 2.

Krüppel-like factors (KLFs) are transcription factors and play important roles in bladder cancer (BC). Clarifying the function of KLFs will provide new strategies for clinical treatment of BC. In this study, we found that Krüppel-like factor 12 (KLF12) was decreased in BC tissues and cells. Knockdown of KLF12 by siRNA dramatically elevated the proliferation and colony formation of BC cells. By contrast, overexpressing KLF12 suppressed the cell viability and the number of clones. Overexpression of KLF12 also regulated cell cycle progression, apoptosis and migration of BC cells. Furthermore, KLF12 bound to the promoter of enolase 2 (ENO2) and transcriptionally inhibited the expression of ENO2, which was highly expressed in BC tissues. KLF12 suppressed, while ENO2 promoted glycolysis. Lastly, ENO2 overexpression and knockdown promoted and suppressed the proliferation and migration of BC cells, respectively. These results suggest that KLF12 acts as a tumor suppressor by negatively regulated ENO2. Targeting ENO2 is a promising treatment strategy for this malignancy.