Hepatocyte growth factor is an invasion/migration factor of rat urothelial carcinoma cells in vitro.

Hepatocyte growth factor (HGF) plays an important role in the growth, progression and angiogenesis of various tumors. It is reported that patients with urinary bladder cancer have elevated levels of HGF in urine and that bladder cancer tissue contains an increased amount of HGF. Thus, the data suggest a functional role of HGF in urinary bladder cancer. We evaluated the mechanistic role of HGF in urinary bladder carcinoma in vitro using the rat urothelial cell lines MYP3 (anchorage-dependent and non-tumorigenic in athymic nude mice), LMC19, MYU3L, T6 and AS-HTB1 (anchorage-independent and tumorigenic). The HGF receptor c-met was expressed by all of the cell lines, as determined by northern blot. In MYP3 cells, HGF strongly stimulated anchorage-dependent growth, but not migration, invasion or secretion of matrix metalloproteinases (MMPs). In LMC19, T6 and AS-HTB1 cells, HGF stimulated migration, invasion and secretion of MMPs. Anchorage-dependent growth stimulation was limited to AS-HTB1 cells. MYU3L cells were refractory to HGF in both growth and invasion assays. However, a neutralizing antibody and an anti-sense oligonucleotide to HGF partially inhibited the growth only of MYU3L cells, the finding being indicative of an autocrine stimulatory mechanism. HGF mRNA expression and protein synthesis were induced in bladder stromal cells by the conditioned medium of carcinoma cell lines, and IL-1beta and basic fibroblast growth factor were identified as cancer cell-derived HGF-releasing factors. These results suggest that HGF acts as a mitogen in a non-tumorigenic cell line, whereas in tumorigenic cell lines it acts as an invasion and migration factor by either a paracrine or an autocrine mechanism.     

Proepithelin promotes migration and invasion of 5637 bladder cancer cells through the activation of ERK1/2 and the formation of a paxillin/FAK/ERK complex

The growth factor proepithelin (also known as progranulin, acrogranin, PC-derived growth factor, or granulin-epithelin precursor) is a secreted glycoprotein that functions as an important regulator of cell growth, migration, and transformation. Proepithelin is overexpressed in a great variety of cancer cell lines and clinical specimens of breast, ovarian, and renal cancer as well as glioblastomas. In this study, we have investigated the effects of proepithelin on bladder cancer cells using human recombinant proepithelin purified to homogeneity from 293-EBNA cells. Although proepithelin did not appreciably affect cell growth, it did promote migration of 5637 bladder cancer cells and stimulate in vitro wound closure and invasion. These effects required the activation of the mitogen-activated protein kinase pathway and paxillin, which upon proepithelin stimulation formed a complex with focal adhesion kinase and active extracellular signal-regulated kinase. Our results provide the first evidence for a role of proepithelin in stimulating migration and invasion of bladder cancer cells, and support the hypothesis that this growth factor may play a critical role in the establishment of the invasive phenotype.     

CXCR4 expression reflects tumor progression and regulates motility of bladder cancer cells

Transitional cell carcinoma of the urinary bladder remains life threatening due to the high occurrence of metastases. Emerging evidence suggests that chemokines and their receptors play a critical role in tumor metastases. In our study, we performed a systematic analysis of the mRNA and protein expression levels of all 18 chemokine receptors in normal urothelium and bladder cancer. CXCR4 was the only chemokine receptor whose mRNA expression level was upregulated in bladder cancer cell lines as well as in invasive and locally advanced bladder cancer tissue samples (pT2-pT4). In contrast, superficial bladder tumors (pTa and pT1) displayed low CXCR4 expression levels and normal urothelial cells were negative for CXCR4. Immunohistochemistry of a bladder cancer tissue microarray (TMA) confirmed that a subgroup of invasive bladder cancers revealed a high CXCR4 protein expression, while superficial bladder tumors showed low immunoreactivity. To investigate the functional significance of CXCR4 expression, we performed migration and invasion assays. Exposure of CXCR4-positive bladder cancer cells to CXCL12 in a Boyden chamber type assay provoked a significant increase in migration as well as invasion across a Matrigel barrier. Enhanced migration and invasion were inhibited by a CXCR4-specific blocking antibody. In contrast, normal urothelial cells did not respond to CXCL12 and lacked chemotactic migration. In conclusion, bladder cancer cells express CXCR4 progressively with advanced tumorigenesis and this receptor interacts with CXCL12 to mediate tumor chemotaxis and invasion through connective tissue. These properties identify CXCR4 as a potential target for the attenuation of bladder cancer metastases.     

TSHZ3在膀胱癌组织中的表达及其对膀胱癌细胞生物学功能的影响

目的:采用生物信息学分析TSHZ3基因在膀胱癌组织中的表达水平,并通过体外实验探讨TSHZ3基因对膀胱癌细胞增殖、迁移和侵袭的影响,以及对上皮-间质转化相关蛋白的调控作用。方法:运用Oncomine和GEPIA数据库分析TSHZ3基因在膀胱癌组织中的表达水平,利用HPA数据库分析TSHZ3蛋白在正常膀胱和膀胱癌组织中的表达情况,并收集膀胱癌标本验证TSHZ3蛋白的表达。在膀胱癌T24细胞中转染TSHZ3质粒,CCK8法和克隆形成实验检测过表达TSHZ3基因对T24细胞增殖的影响,Transwell实验检测过表达TSHZ3基因对T24细胞迁移和侵袭的影响,Western blot检测过表达TSHZ3对上皮-间质转化相关蛋白的影响。结果:Oncomine和GEPIA数据库显示TSHZ3基因在膀胱癌组织中的表达水平降低。HPA数据库表明TSHZ3蛋白在正常膀胱尿路上皮组织中呈中等强度染色,而在膀胱尿路上皮癌组织中呈弱-中等强度染色。免疫组化结果显示TSHZ3在癌旁组织中呈现高表达,在膀胱癌组织中呈现低表达。过表达TSHZ3显著抑制T24细胞的增殖、迁移和侵袭能力。过表达TSHZ3增加E-cadherin蛋白的表达,降低N-cadherin和Vimentin蛋白的表达。结论:TSHZ3基因在膀胱癌中低表达,可作为抑癌基因抑制膀胱癌的发展。     

Heregulin regulation of autocrine motility factor expression in human tumor cells

The exposure of cells to growth factors has been shown to induce cytoskeleton reorganization, leading to stimulation of cell motility and invasion. Heregulin beta1 (HRG), a combinatorial ligand for human epidermal growth factor receptor 3 and human epidermal growth factor receptor 4 receptors, is a regulatory secretory polypeptide with a distinctive function in promoting motility and invasiveness of breast cancer cells. In addition to HRG, motility and invasiveness of tumor cells may also involve up-regulation of expression and function of the autocrine motility factor (AMF). Here we explored the possible involvement of AMF in the motility-promoting action of HRG in the MCF-7 breast cancer cell model system. We report that HRG increases the expression of AMF mRNA by 3-8-fold in an actinomycin D-sensitive manner and does not require de novo protein synthesis. The HRG-induced stimulation of AMF expression was inhibited by specific inhibitors of p42/44MAPK and p38MAPK kinases, but not by an inhibitor of the phosphatidylinositol 3'-kinase pathway. Other HRG-responsive human cell lines demonstrated that HRG does indeed significantly up-regulate AMF expression. Furthermore, HRG-stimulated increased motility was partially suppressed by inclusion of an anti-AMF antibody to breast cancer cells, suggesting that a HRG-mediated increase in cell motility may be mediated, at least in part, via induction of AMF. The present study is the first demonstration of AMF regulation by a growth factor and suggests a potential role for AMF in HRG regulation of breast cancer cell motility and a novel function of HRG as a regulator of motility factor expression.     

Heat-shock protein 70-2 (HSP70-2) expression in bladder urothelial carcinoma is associated with tumour progression and promotes migration and invasion

PurposeTestis specific heat-shock protein 70-2 (HSP70-2), a member of HSP70 chaperone family, is essential for the growth of spermatocytes and cancer cells. We investigated the association of HSP70-2 expression with clinical behaviour and progression of urothelial carcinoma of bladder.Experimental designWe assessed the HSP70-2 expression by RT-PCR and HSP70-2 protein expression by immunofluorescence, flow cytometry, immunohistochemistry and Western blotting in urothelial carcinoma patient specimens and HTB-1, UMUC-3, HTB-9, HTB-2 and normal human urothelial cell lines. Further, to investigate the role of HSP70-2 in bladder tumour development, HSP70-2 was silenced in the high-grade invasive HTB-1 and UMUC-3 cells. The malignant properties of urothelial carcinoma cells were examined using colony formation, migration assay, invasion assay in vitro and tumour growth in vivo.ResultsOur RT-PCR analysis and immunohistochemistry analysis revealed that HSP70-2 was expressed in both moderate to well-differentiated and high-grade invasive urothelial carcinoma cell lines studied and not in normal human urothelial cells. In consistence with these results, HSP70-2 expression was also observed in superficially invasive (70%) and muscle-invasive (90%) patient’s tumours. Furthermore, HSP70-2 knockdown significantly suppressed cellular motility and invasion ability. An in vivo xenograft study showed that inhibition of HSP70-2 significantly suppressed tumour growth.ConclusionsIn conclusion, our data suggest that the HSP70-2 expression is associated with early spread and progression of urothelial carcinoma of bladder cancer and that HSP70-2 can be the potential therapeutic target for bladder urothelial carcinoma.     

SLC12A5 interacts and enhances SOX18 activity to promote bladder urothelial carcinoma progression via upregulating MMP7

Solute carrier family 12 member 5 (SLC12A5) has an oncogenic role in bladder urothelial carcinoma. The present study aimed to characterize the molecular mechanisms of SLC12A5 in bladder urothelial carcinoma pathogenesis. Functional assays identified that in bladder urothelial carcinoma SLC12A5 interacts with and stabilizes SOX18, and then upregulates matrix metalloproteinase 7 (MMP7). In vivo and in vitro assays were performed to confirm the effect of SLC12A5’s interaction with SOX18 on MMP7‐mediated bladder urothelial carcinoma progression. SLC12A5 was upregulated in human bladder tumors, and correlated with the poor survival of patients with bladder urothelial carcinoma tumor invasion and metastasis, promoted by SLC12A5 overexpression. We demonstrated that SLC12A5 interacted with SOX18, and then upregulated MMP7, thus enhancing tumor progression. Importantly, SLC12A5 expression correlated positively with SOX18 and MMP7 expression in bladder urothelial carcinoma. Furthermore, SLC12A5 expression was suppressed by miR‐133a‐3p. Ectopic expression of SLC12A5 partly abolished miR‐133a‐3p‐mediated suppression of cell migration. SLC12A5‐SOX18 complex‐mediated upregulation on MMP7 was important in bladder urothelial carcinoma progression. The miR‐133a‐3p/SLC12A5/SOX18/MMP7 signaling axis was critical for progression, and provided an effective therapeutic approach against bladder urothelial carcinoma.     

HDAC6 and SIRT2 promote bladder cancer cell migration and invasion by targeting cortactin

Histone deacetylase 6 (HDAC6) promotes cell motility and contributes to the metastasis of cancers. The purpose of this study was to investigate the role of HDAC6 in human bladder cancer for the first time. The results showed that HDAC6 promotes the migration and invasion of bladder cancer cells by targeting the cytoskeletal protein cortactin. Furthermore, a colony formation assay as well as in?vitro migration and invasion assays demonstrated that this migration and invasion was suppressed by the HDAC6-specific inhibitor tubacin. In addition, cortactin is the substrate of SIRT2, which also belongs to the family of histone deacetylases. We demonstrated that by using SIRT2-specific siRNA combined with tubacin treatment, the cell migratory and invasive abilities were dramatically suppressed. Taken together, we conclude that HDAC6 and SIRT2 work synergistically to promote cell migration and invasion in bladder cancer, and the HDAC6-specific inhibitor tubacin may be regarded as a novel therapeutic agent for bladder cancer.     

miR-144-3p靶向调控E2F3抑制膀胱癌T24细胞的增殖与侵袭

目的:探讨miR144-3p在膀胱癌组织及细胞中的表达及其对T24细胞增殖与侵袭的影响.方法:选用2018年2月至2018年12月空军军医大学唐都医院手术切除的36例膀胱癌组织及10例正常膀胱上皮组织标本,以及人膀胱癌细胞株T24和正常尿路上皮细胞株SV-HUC-1,用qPCR法检测膀胱癌组织和细胞中miR144-3p...     

Adenovirus-mediated PEDF expression inhibits prostate cancer cell growth and results in augmented expression of PAI-2

PEDF is one of the most potent inhibitor of angiogenesis. Loss of PEDF was found in human prostate tumors and associated with the progression toward a metastatic phenotype. To test the therapeutic potential of PEDF, we constructed a replication-defective adenoviral vector capable of efficient transduction and expression of PEDF (Ad-PEDF) in PC-3 prostate carcinoma cells. As controls, we used adenoviruses expressing beta-galactosidase (Ad-LacZ). We showed that overexpression of PEDF inhibited proliferation of cells and augmented apoptosis in serum-starved cells, in comparison with Ad-LacZ. Furthermore, Ad-PEDF suppresses anchorage-independent growth cells in soft agar and tumor formation in athymic nude mice associated with decreased microvessel density. Microarray analysis showed that 56 out of 8464 genes were found to be upregulated and downregulated in the PC-3 infected with Ad-PEDF as compared with Ad-LacZ. The differentially expressed genes cover a broad range of functional activities including catalytic activity, protein binding, signal transduction activity and cell invasion. Among them, PAI-2 and DRHC were confirmed to be upregulated with real-time PCR and Western blot, suggesting a possible association with PEDF-induced signaling for cell motility and metastasis. These results can contribute to our understanding of the molecular mechanisms of treatment strategies of PEDF for prostate cancer.