Olive Mill Wastewater Inhibits Growth and Proliferation of Cisplatin- and Gemcitabine-Resistant Bladder Cancer Cells In Vitro by Down-Regulating the Akt/mTOR-Signaling Pathway

Bladder cancer patients whose tumors develop resistance to cisplatin-based chemotherapy often turn to natural, plant-derived products. Beneficial effects have been particularly ascribed to polyphenols, although their therapeutic relevance when resistance has developed is not clear. The present study evaluated the anti-tumor potential of polyphenol-rich olive mill wastewater (OMWW) on chemo-sensitive and cisplatin- and gemcitabine-resistant T24, RT112, and TCCSUP bladder cancer cells in vitro. The cells were treated with different dilutions of OMWW, and tumor growth and clone formation were evaluated. Possible mechanisms of action were investigated by evaluating cell cycle phases and cell cycle-regulating proteins. OMWW profoundly inhibited the growth and proliferation of chemo-sensitive as well as gemcitabine- and cisplatin-resistant bladder cancer cells. Depending on the cell line and on gemcitabine- or cisplatin-resistance, OMWW induced cell cycle arrest at different phases. These differing phase arrests were accompanied by differing alterations in the CDK-cyclin axis. Considerable suppression of the Akt-mTOR pathway by OMWW was observed in all three cell lines. Since OMWW blocks the cell cycle through the manipulation of the cyclin-CDK axis and the deactivation of Akt-mTOR signaling, OMWW could become relevant in supporting bladder cancer therapy.

Oncogenic E3 ubiquitin ligase NEDD4 binds to KLF8 and regulates the microRNA-132/NRF2 axis in bladder cancer

The neuronally expressed developmentally downregulated 4 (NEDD4) gene encodes a ubiquitin ligase that targets the epithelial sodium channel for degradation and has been implicated in tumor growth in various cancers. Hence, in this study, we intended to characterize the functional relevance of the NEDD4-mediated Kruppel-like factor 8/microRNA-132/nuclear factor E2-related factor 2 (KLF8/miR-132/NRF2) axis in the development of bladder cancer. NEDD4 and KLF8 were overexpressed in bladder cancer tissues and were associated with poorer patient survival rates. In bladder cancer cells, NEDD4 intensified the stability and transcriptional activity of KLF8 through ubiquitination to augment cell viability and migratory ability. Our investigations revealed that NEDD4 promotes the binding of KLF8 to the miR-132 promoter region and inhibits the expression of miR-132. KLF8 inhibited the expression of miR-132 to augment the viability and migratory ability of bladder cancer cells. Furthermore, miR-132 downregulated the expression of NRF2 to restrict the viability and migratory ability of bladder cancer cells. In addition, in vivo findings verified that NEDD4 regulates the KLF8/miR-132/NRF2 axis by accelerating tumor growth and lung metastasis. In conclusion, this study highlights NEDD4 as a potential therapeutic target against tumor recurrence and metastasis in bladder cancer.

EGF-induced nuclear translocation of SHCBP1 promotes bladder cancer progression through inhibiting RACGAP1-mediated RAC1 inactivation

Bladder cancer is a highly heterogeneous and aggressive malignancy with a poor prognosis. EGF/EGFR activation causes the detachment of SHC-binding protein 1 (SHCBP1) from SHC adapter protein 1 (SHC1), which subsequently translocates into the nucleus and promotes cancer development via multiple signaling pathways. However, the role of the EGF-SHCBP1 axis in bladder cancer progression remains unexplored. Herein, we report that SHCBP1 is upregulated in bladder cancer tissues and cells, with cytoplasmic or nuclear localization. Released SHCBP1 responds to EGF stimulation by translocating into the nucleus following Ser273 phosphorylation. Depletion of SHCBP1 reduces EGF-induced cell migration and invasiveness of bladder cancer cells. Mechanistically, SHCBP1 binds to RACGAP1 via its N-terminal domain of amino acids 1 ~ 428, and this interaction is enhanced following EGF treatment. Furthermore, SHCBP1 facilitates cell migration by inhibiting RACGAP-mediated GTP-RAC1 inactivation, whose activity is indispensable for cell movement. Collectively, we demonstrate that the EGF-SHCBP1-RACGAP1-RAC1 axis acts as a novel regulatory mechanism of bladder cancer progression, which offers a new clinical therapeutic strategy to combat bladder cancer.

Wnt/β-Catenin Signaling Contributes to Paclitaxel Resistance in Bladder Cancer Cells with Cancer Stem Cell-Like Properties

The Wnt/β-catenin pathway plays an important role in tumor progression and chemotherapy resistance and seems to be essential for the maintenance of cancer stem cells (CSC) in several tumor types. However, the interplay of these factors has not been fully addressed in bladder cancer. Here, our goal was to analyze the role of the Wnt/β-catenin pathway in paclitaxel resistance and to study the therapeutic efficacy of its inhibition in bladder cancer cells, as well as to determine its influence in the maintenance of the CSC-like phenotype in bladder cancer. Our results show that paclitaxel-resistant HT1197 cells have hyperactivation of the Wnt/β-catenin pathway and increased CSC-like properties compared with paclitaxel-sensitive 5637 cells. Paclitaxel sensitivity diminishes in 5637 cells after β-catenin overexpression or when they are grown as tumorspheres, enriched for the CSC-like phenotype. Additionally, downregulation of β-catenin or inhibition with XAV939 sensitizes HT1197 cells to paclitaxel. Moreover, a subset of muscle-invasive bladder carcinomas shows aberrant expression of β-catenin that associates with positive expression of the CSC marker ALDH1A1. In conclusion, we demonstrate that Wnt/β-catenin signaling contributes to paclitaxel resistance in bladder cancer cells with CSC-like properties.

Expression of transcription factor SALL4 in bladder urothelial carcinoma and its relationship with epithelial mesenchymal transformation

Purpose To evaluate the expression levels of spalt-like transcription factor 4 (SALL4) in bladder urothelial carcinoma, and determine its role and underlying mechanism of action in mediating the proliferation, migration and invasion of bladder cancer cells. Methods SALL4 expression was examined in 170 bladder patient urothelial carcinoma tissue samples by immunohistochemistry. Using a SALL4 overexpression plasmid and siRNA-SALL4, the effects of overexpressing and silencing SALL4 on the proliferation, migration and invasion of T24 and 5637 bladder cancer cells was examined using CCK8, migration and invasion assays. Western blot analysis was performed to detect epithelial mesenchymal transition (EMT)-related protein expression. Results The expression rate of SALL4 in low-grade and high-grade urothelial carcinomas was found to be 10% and 49.12%, respectively (P < 0.001), while SALL4 expression was not observed in the normal urothelium. SALL4 protein expression was positively correlated with histological grade, depth of invasion, lymphatic metastasis and vascular invasion of bladder urothelial carcinoma (P < 0.05). In addition, a shorter overall survival time and poor prognosis was observed in the SALL4 protein expression group. Overexpression of SALL4 led to significantly increased cell proliferation, migration and invasion, while knockdown of SALL4 had the opposite effect. In the SALL4 overexpression group, N-cadherin, vimentin, Snail and β-catenin expression were significantly increased, while E-cadherin expression was significantly decreased (P < 0.05). Promotion of EMT was also observed in SALL4-overexpressing cells. In contrast, in the SALL4-siRNA-treated group, EMT was reversed and β-catenin expression was reduced. Conclusions Our data show that the SALL4 gene is associated with the proliferation, invasion and poor prognosis of bladder urothelial carcinoma, and may mediate its effects via the Wnt/β-catenin signaling pathway, which regulates the EMT pathway. Thus, down-regulation of SALL4 may provide a novel therapeutic strategy for the treatment of bladder urothelial carcinoma.

Hypofractionated Radiation Induced the Immunogenic Death of Bladder Cancer Cells Leading to the Immune Sensitization of Dendritic Cells

Purpose Radiotherapy is a commonly used method in the treatment of bladder cancer (BC). Radiation induced immunogenic death (ID) and antitumor immune response are related to the prognosis of radiotherapy. As the most powerful antigen-presenting cell in the body, the role of dendritic cells (DCs) is not very clear. Methods Apoptosis level, cell cycle analysis and expression levels of high mobility group protein 1 (HMGB1), calreticulin (CRT) and heat shock protein 70 (HSP70) were performed for bladder cancer cells after hypofractionated radiotherapy. The effects of the conditioned media on DCs for antitumor immune response activation were studied as well. Results The significantly increased apoptosis level, G2/M phase cell cycle arrest and significantly increased HMGB1, CRT and HSP70 expressions, and increased secretion of CCL5 and CCL21 in the supernatant of bladder cancer cells after hypofractionated radiotherapy. The expression of CD80, CD86, CCR5 and CCR7 on DCs was upregulated in the conditioned media of bladder cancer cells after hypofractionated radiotherapy. Conclusion Hypofractionated radiation blocked the cell cycle of BC cells in the G2/M phase and induced ID occurrence, resulting in DCs immune sensitization, which is of great clinical significance in understanding the radiotherapy of BC and the immunoregulation function of DCs.

An HGF-dependent positive feedback loop between bladder cancer cells and fibroblasts mediates lymphangiogenesis and lymphatic metastasis

Cancer-associated fibroblasts (CAFs) are essential etiologic actors in promoting tumor progression via extensive reciprocal interactions with cancer cells. Yet, the biological role and regulatory mechanism of CAFs phenotype underlying lymph node (LN) metastasis of bladder cancer (BCa) remain unclear. Here, we report that BCa cell-secreted extracellular vesicles (EVs) played an important role in the CAF-enriched microenvironment, which correlated with BCa lymphangiogenesis and LN metastasis. RNA sequencing identified an EV-associated long noncoding RNA, LINC00665, which acted as a crucial mediator of CAF infiltration in BCa. LINC00665 mediated EV release from BCa cells to endow fibroblasts with the CAF phenotype, which reciprocally induced LINC00665 upregulation to form a RAB27B-HGF-c-Myc positive feedback loop, facilitating BCa lymphangiogenesis and LN metastasis. Importantly, we demonstrate that Cabozantinib significantly suppressed LINC00665-mediated BCa LN metastasis in an orthotopic xenograft model. Our study highlights a molecular mechanism by which LINC00665 induces a RAB27B-HGF-c-Myc positive feedback loop between cancer cells and fibroblasts to sustain BCa LN metastasis, and represents LINC00665 as a potential therapeutic target in BCa LN metastasis.