Jiansong Wang

miR-9 promotes cell proliferation and inhibits apoptosis by targeting LASS2 in bladder cancer.

MicroRNA-9 upregulation was reported in several tumors. However, its function and mechanism in human bladder cancer remains obscure. The present study aims to identify the expression pattern, biological roles and potential mechanism of miR-9 in human bladder cancers. We found that expression level of miR-9 in bladder cancer tissues was higher than normal tissues. miR-9 mimic transfection was performed in T24 and 5637 cells with low miR-9 expression, and miR-9 inhibitor was employed in BIU-87 cell line with high endogenous expression. miR-9 increased cell proliferation, cell cycle progression, invasion and chemoresistance, with upregulation of cyclin D1, MMP9, Bcl-2, and survivin and downregulation of E-cadherin. Using luciferase reporter assay, we confirmed that LASS2 was a direct target of miR-9 in bladder cancer cells. Transfection of miR-9 mimic downregulated LASS2 expression. LASS2 transfection downregulated Bcl-2 and survivin expression, which were induced by miR-9 mimic in both cell lines. In conclusion, these results indicate that miR-9 upregulation might be associated with malignant phenotype of bladder cancer. miR-9 promotes chemoresistance of bladder cancer cells by target LASS2.

GALNT1-Mediated Glycosylation and Activation of Sonic Hedgehog Signaling Maintains the Self-Renewal and Tumor-Initiating Capacity of Bladder Cancer Stem Cells

The existence of bladder cancer stem cells (BCSC) has been suggested to underlie bladder tumor initiation and recurrence. Sonic Hedgehog (SHH) signaling has been implicated in promoting cancer stem cell (CSC) self-renewal and is activated in bladder cancer, but its impact on BCSC maintenance is unclear. In this study, we generated a mAb (BCMab1) against CD44(+) human bladder cancer cells that recognizes aberrantly glycosylated integrin α3β1. The combination of BCMab1 with an anti-CD44 antibody identified a BCMab1(+)CD44(+) cell subpopulation as BCSCs with stem cell-like properties. Gene expression analysis revealed that the hedgehog pathway was activated in the BCMab1(+)CD44(+) subpopulation and was required for BCSC self-renewal. Furthermore, the glycotransferase GALNT1 was highly expressed in BCMab1(+)CD44(+) cells and correlated with clinicopathologic features of bladder cancers. Mechanistically, GALNT1 mediated O-linked glycosylation of SHH to promote its activation, which was essential for the self-renewal maintenance of BCSCs and bladder tumorigenesis. Finally, intravesical instillation of GALNT1 siRNA and the SHH inhibitor cyclopamine exerted potent antitumor activity against bladder tumor growth. Taken together, our findings identify a BCSC subpopulation in human bladder tumors that appears to be responsive to the inhibition of GALNT1 and SHH signaling, and thus highlight a potential strategy for preventing the rapid recurrence typical in patients with bladder cancer.

Expression of a tumor-associated gene, LASS2, in the human bladder carcinoma cell lines BIU-87, T24, EJ and EJ-M3

Homo sapiens longevity assurance homolog 2 of yeast LAG1 (LASS2), a metastasis suppressor gene of human cancer, is the most abundantly expressed member of the ceramide synthase gene family. Expression of LASS2 has been reported in carcinomas of the prostate, liver and breast. However, there has been no report on the expression of LASS2 in human bladder cancer cell lines. In order to investigate the expression and potential role of this new tumor metastasis supressor gene in human bladder cancer, we compared the proliferation, metastasis and invasion among the BIU-87, T24, EJ and EJ-M3 human bladder cancer cell lines. The mRNA expression levels of the LASS2 gene were examined using real-time quantitative PCR (qPCR). The expression levels of LASS1 and LASS3 mRNA were used as references. The protein expression level of the LASS2 gene was detected using western blotting. The most aggressive of these four human cancer cell lines was observed to be EJ-M3. The expression of LASS2 mRNA was significantly correlated with diverse proliferation, metastasis and invasion. The expression levels of LASS1 and LASS3 mRNA were not correlated with these parameters. At the protein level, we observed that the more aggressive the cancer cell line, the lower the LASS2 protein expression level. Therefore, LASS2 expression may be correlated with the development and progression of human bladder cancer and may be a prognostic indicator for this cancer.

Apoptin induces apoptosis in nude mice allograft model of human bladder cancer by altering multiple bladder tumor-associated gene expression profiles.

Bladder cancer (BC) is one of the most common human malignancies that account for major death in the world. Apoptin that is derived from chicken anemia virus (CAV) has displayed tumor-specific cytotoxic activity in a variety of human carcinomas. However, the magical function of apoptin in bladder carcinoma cell lines has not been identified yet. In our study, we delivered apoptin into bladder-originating T24, EJ, and HCV29 cell lines by adenovirus system. The selective cytotoxic effect of apoptin was determined by cell viability assay, active caspase-3 measurement, and annexin V/PI double staining. Importantly, we have examined the differential expression patterns of tumor-associated genes including Ki67, C-erbB-2, Rb, and nm23 by flow cytometry and western blot in vitro. In an animal study, apoptin was infused into animal models by AAV system, and immunohistochemistry and quantitative real-time PCR (qRT-PCR) were employed to validate results in vivo. The results indicated that apoptin could selectively induce apoptosis in bladder tumorigenic cells coupled with tumor-specific nucleus accumulation in vitro. Interestingly, apoptin could downregulate expression levels of Ki67 and C-erbB-2 and upregulate the expression of Rb both in vitro and in vivo. Moreover, the animal models treated with AAV-apoptin have shown smaller tumor volumes and displayed better prognosis than controls. In conclusion, apoptin could selectively induce apoptosis in bladder tumor cells through altering expression profiles of tumor-associated genes.

Impact of invasive bladder cancer and orthotopic urinary diversion on general health-related quality of life: An SF-36 survey

Bladder cancer is a common type of genitourinary cancer, and radical cystectomy with urinary diversion is considered to be the most effective local treatment for invasive bladder cancer. In order to assess the functional results and health-related quality of life (QOL) in bladder cancer patients with an orthotopic neobladder, and to provide a reasonable basis for the evaluation of urinary diversion in situ, we conducted a study on 96 neobladder patients. In December, 2011, questionnaires were mailed to 96 patients that had undergone urinary diversion surgery between January, 2007 and December, 2009. The questionnaire included the validated health-related QOL questionnaire and the MOS 36-item Short-Form Health Survey (SF-36). We compared the functional results between patients with an orthotopic neobladder and those with other types of urinary diversion at 6, 12 and 24 months after surgery. Data from 82 patients (54 with orthotopic and 28 with non-orthotopic urinary diversion) were included in the analysis. The SF-36 patient scores following orthotopic urinary diversion were significantly higher than those following non-orthotopic urinary diversion. The majority of patients with orthotopic urinary diversion considered themselves as healthy and their scores of total health were higher compared to those of patients with non-orthotopic urinary diversion. However, there were no differences in the scores of physical functioning between patients with orthotopic and those with non-orthotopic urinary diversion. Our findings regarding health-related QOL and the frequency of complications in the orthotopic and the non-orthotopic urinary diversion groups were similar. However, the mental health of patients with orthotopic urinary diversion was more easily restored compared to that of patients with non-orthotopic urinary diversion, which reduced their overall recovery time.

MicroRNA-92 promotes invasion and chemoresistance by targeting GSK3β and activating Wnt signaling in bladder cancer cells

AbstractmiR-92 has been reported to be upregulated in several human cancers. Until now, its expression pattern and biological roles in human bladder cancer still remains unexplored. The present study aims to clarify its expression, function, and potential molecular mechanisms in bladder cancer. Using real-time PCR, we found that miR-92 was upregulated in bladder cancer tissues compared with normal bladder tissues. We transfected miR-92 mimic and inhibitor in T24 and 5637 bladder cancer cells separately. We found that miR-92 mimic promoted T24 proliferation and invasion, with increased expression of cyclin D1, c-myc, and MMP7 at both mRNA and protein levels. Further investigation found that miR-92 could also promote epithelial-mesenchymal transition by downregulating E-cadherin protein and upregulating vimentin. In addition, miR-92 mimic also promoted activation of Wnt signaling. Meanwhile, miR-92 inhibitor displayed the opposite effects in 5637 cell line. By use of bioinformatic prediction software and luciferase reporter assay, we discovered that GSK3β acted as a direct target of miR-92. Additionally, GSK3β siRNA abrogated the effects of miR-92 mimic on cyclin D1 and MMP7. Moreover, we observed a negative correlation between GSK3β and miR-92 in bladder cancer tissues. In conclusion, our study demonstrated that upregulation of miR-92 is closely related with malignant progression of bladder cancer and miR-92 promotes proliferation, invasion, and Wnt/c-myc/MMP7 signaling by targeting GSK3β.

Repression of the miR-93-enhanced sensitivity of bladder carcinoma to chemotherapy involves the regulation of LASS2

The aberrant expression of miRNA has an important function in bladder cancer (BC). Previous studies indicate that LASS2 is involved in the development of sensitivity to chemotherapy in cancer cells. In the present study, the miRNAs related to LASS2 were selected by using miRNA profiling to distinguish chemo-resistant and chemo-sensitive tumor specimens from patients. Higher levels of miR-93 were observed in the cisplatin-resistant BC cell line RT4, compared to the cell line T24. The role of miR-93 in chemo-sensitivity was demonstrated both in cell culture and mouse tumor xenograft models. We found that inhibiting miR-93 promoted cisplatin-induced apoptosis due to the accumulation of DNA damage. A reporter gene assay was performed, and the results showed miR-93 was not a target of the 3′ untranslated region of LASS2, but had an altered protein expression level. Inhibitors of miR-93 could also enhance the chemo-sensitivity of tumor cells transfected with si-LASS2, but the effect was very slight. These findings suggest that miR-93 plays an important role in the chemo-sensitivity of BC, and may be involved in regulating the LASS2 gene.

BCMab1-Ra, a novel immunotoxin that BCMab1 antibody coupled to Ricin A chain, can eliminate bladder tumor

Bladder cancer is one of the most common malignancies. However, there is no ideal therapy to cure bladder cancer so far, especially invasive carcinoma. Here, we developed a new antibody-based drug BCMab1-Ra, which was generated by conjugation of BCMab1 (a new monoclonal antibody that specifically recognized the aberrantly glycosylated Integrin a3b1 in bladder cancer) with the ricin A chain (Ra). A patient with multiple bladder cancer received intravescical administration of BCMab1-Ra treatment as a volunteer. After 30 weeks of treatment, no tumor was observed by cystoscope examination. We did not observe any local or systemic side effects. Human anti-mouse antibody (HAMA) was not detectable in the circulation. Results follow-up showed no tumor had been found in every half year review in 3 years.

LASS2 inhibits growth and invasion of bladder cancer by regulating ATPase activity

Homo sapiens longevity assurance homolog 2 of yeast LAG1 (LASS2) is a novel suppressor of human cancer metastasis, and downregulation of LASS2 has been associated with a poor prognosis in patients with bladder cancer (BC). However, the molecular mechanism underlying LASS2-mediated inhibition of tumor invasion and metastasis in BC remains unclear. LASS2 has been reported to directly bind to subunit C of vacuolar H+-ATPase (V-ATPase) in various types of cancer, suggesting that LASS2 may inhibit cancer invasion and metastasis by regulating the function of V-ATPase. The present study investigated the effect of LASS2-specific small interfering (si)RNA on the invasion and metastasis of the RT4 human BC cell line, which has a low metastatic potential, and its functional interaction with V-ATPase. Silencing of LASS2 in RT4 cells was able to increase V-ATPase activity, the extracellular hydrogen ion concentration and, in turn, the activation of secreted matrix metalloproteinase (MMP)-2 and MMP-9, which occurred simultaneously with enhanced cell proliferation, cell survival and cell invasion in vitro, as well as acceleration of BC growth in vivo. In this process, it was found that siRNA-LASS2 treatment was able to suppress cell apoptosis induced by doxorubicin. These findings suggest that silencing of LASS2 may enhance the growth, invasion and metastasis of BC by regulating ATPase activity.

LASS2 regulates invasion and chemoresistance via ERK/Drp1 modulated mitochondrial dynamics in bladder cancer cells

Mitochondria coordinated a lot of vital cellular processes of energy production and distribution. Change of mitochondrial functions has been implicated in cancer progression. The present study aims to investigate the involvement of mitochondria dynamics in LASS2 induced invasion and chemoresistance of bladder cancer cells. J82 and BIU87 cell lines were used for LASS2 plasmid transfection while siRNA knockdown was carried out in 5637 cell line. Matrigel invasion assay and Annexin V/PI staining demonstrated that LASS2 negatively regulated cancer cell invasion and chemoresistance. JC-1 staining suggested that LASS2 overexpression downregulated mitochondrial membrane potential. Mitotracker staining showed that LASS2 induced mitochondrial fusion and inhibited mitochondrial fission. In addition, LASS2 overexpression downregulated expression of mitochondrial fission protein p-Drp1 Drp1 and Fis1. While depletion of LASS2 exhibited the opposite effects. Drp1 inhibitor Mdivi abolished invasion and chemoresistance induced by LASS2 siRNA. Furthermore, we found that LASS2 overexpression could inhibit phosphorylation of ERK, which act upstream of Drp1. ERK inhibitor PD98059 suppressed Drp1 phosphorylation and abrogated the effects of LASS2 depletion. In conclusion, the present study demonstrated that LASS2 inhibits bladder cancer invasion and chemoresistance through regulation of ERK-Drp1 induced mitochondrial dynamics.