High grade bladder cancer is an extremely aggressive malignancy associated with high rates of morbidity and mortality. Understanding how exosomes may affect bladder cancer progression could reveal novel therapeutic targets. Exosomes derived from human bladder cancer cell lines and the urine of patients with high grade bladder cancer were assessed for the ability to promote cancer progression in standard assays. Exosomes purified from the high grade bladder cancer cell line TCC-SUP and the nonmalignant urothelial cell line SV-HUC were submitted for mass spectrometry analysis. EDIL-3 was identified and selected for further analysis. Western blot was done to determine EDIL-3 levels in urinary exosomes from patients with high grade bladder cancer. shRNA gene knockdown and recombinant EDIL-3 were applied to study EDIL-3 function. Exosomes isolated from high grade bladder cancer cells and the urine of patients with high grade bladder cancer promoted angiogenesis and migration of bladder cancer cells and endothelial cells. We silenced EDIL-3 expression and found that shEDIL-3 exosomes did not facilitate angiogenesis, and urothelial and endothelial cell migration. Moreover, exosomes purified from the urine of patients with high grade bladder cancer contained significantly higher EDIL-3 levels than exosomes from the urine of healthy controls. EDIL-3 activated epidermal growth factor receptor signaling while blockade of epidermal growth factor receptor signaling abrogated this EDIL-3 induced bladder cell migration. Exosomes derived from the urine of patients with bladder cancer contains bioactive molecules such as EDIL-3. Identifying these components and their associated oncogenic pathways could lead to novel therapeutic targets and treatment strategies.

Angiogenesis is an essential step in initial tumor development and metastasis. Consequently, compounds that inhibit angiogenesis would be useful in treating cancer. A variety of antitumor effects mediated by 1α, 25-dihydroxyvitamin D₃ (1,25-VD) have been reported, one of which is anti-angiogenesis; however, detailed mechanisms remain unclear. We have demonstrated that 1,25-VD inhibits prostate cancer (PCa) cell-induced human umbilical vein endothelial cell migration and tube formation, two critical steps involved in the angiogenesis. An angiogenesis factor, interleukin-8 (IL-8), secreted from PCa cell was suppressed by 1,25-VD at both mRNA and protein levels. Mechanistic dissection found that 1,25-VD inhibits NF-κB signal, one of the most important IL-8 upstream regulators. The 1,25-VD-mediated NF-κB signal reduction was shown to result from the blocking of nuclear translocation of p65, a subunit of the NF-κB complex, and was followed by attenuation of the NF-κB complex binding to DNA. The role of IL-8 in PCa progression was further examined by PCa tissue microarray analyses. We found that IL-8 expression was elevated during PCa progression, which suggests that IL-8 may play a role in tumor progression mediated through its stimulation on angiogenesis. These findings indicate that 1,25-VD could prevent PCa progression by interrupting IL-8 signaling, which is required in tumor angiogenesis, and thus applying vitamin D in PCa treatment may be beneficial for controlling disease progression.

Overproduction of reactive oxygen species (ROS), through either endogenous or exogenous sources, could induce DNA damage, and accumulation of DNA damage might lead to multistep carcinogenesis. The antioxidative effects of vitamin D have been suggested by epidemiological and many in vitro and in vivo laboratory studies. While exploring the antioxidative effects of vitamin D in prostate cells, we found that the active form of vitamin D, 1α, 25‐dihydroxyvitamin D₃ (1,25‐VD), can protect nonmalignant human prostate epithelial cell lines, BPH‐1 and RWPE‐1, but not malignant human prostate epithelial cells, CWR22R and DU 145, from oxidative stress‐induced cell death. Glucose‐6‐phosphate dehydrogenase (G6PD), a key antioxidant enzyme, was dose‐ and time‐dependently induced by 1,25‐VD. Mechanistic studies using chromatin immunoprecipitation (ChIP) assay revealed that a direct repeat‐3 (DR3) vitamin D response element located in the first intron of the G6PD genome can be bound by liganded vitamin D receptor, thereby regulating G6PD gene expression. Increasing G6PD activity and glutathione level by 1,25‐VD can scavenge cellular ROS. Moreover, the protective effects of 1,25‐VD were abolished by dehydroepiandrosterone, a noncompetitive inhibitor of G6PD activity. Together, our results showed that 1,25‐VD can protect nonmalignant prostate cells from oxidative stress‐induced cell death by elimination of ROS‐induced cellular injuries through transcriptional activation of G6PD activity. The antioxidative effect of vitamin D strengthens its roles in cancer chemoprevention and adds to a growing list of beneficial effects of vitamin D against cancer.

Inhibition of invasion and metastasis has become a new approach for treatment of advanced prostate cancer in which secondary hormone therapy has failed. Accumulating evidence indicates that 1α,25-dihydroxyvitamin D ₃ (1,25-VD) suppresses prostate cancer progression by inhibition of tumor growth and metastasis. However, the detailed mechanisms underlying these effects remain to be determined. Here, we used the in vitro cell invasion assay to demonstrate that 1,25-VD inhibits the invasive ability of human prostate cancer cell lines, LNCaP, PC-3 and DU 145. Three major groups of proteases, the matrix metalloproteinases (MMPs), the plasminogen activators (PAs) and the cathepsins (CPs), that are involved in tumor invasion were then examined for changes in activity and expression after 1,25-VD treatment. We found that 1,25-VD decreased MMP-9 and CPs, but not PAs activities, while it increased the activity of their counterparts, tissue inhibitors of metalloproteinase-1 (TIMP-1) and cathepsin inhibitors. Mechanistic studies showed that 1,25-VD did not suppress MMP-9 expression at the transcriptional level, but reduced its mRNA stability. In addition, 1,25-VD increased AP-1 complexes binding to TIMP-1 promoter, which contributed to the enhancement of TIMP-1 activity, and thus resulted in inhibition of MMP activity and tumor invasion. These findings support the idea that vitamin D-based therapies might be beneficial in the management of advanced prostate cancer, especially among patients who have higher MMP-9 and CPs activities.

The anti-tumor effect of vitamin D has been well recognized but its translational application is hindered by side effects induced by supra-physiological concentration of vitamin D required for cancer treatment. Thus, exploring the vitamin D tumor suppressive functional mechanism can facilitate improvement of its clinical application. We screened miRNA profiles in response to vitamin D and found that a tumor suppressive miRNA, miR-98, is transcriptionally induced by 1α,25-dihydroxyvitamin D₃ (1,25-VD) in LNCaP. Mechanistic dissection revealed that 1,25-VD-induced miR-98 is mediated through both a direct mechanism, enhancing the VDR binding response element in the promoter region of miR-98, and an indirect mechanism, down-regulating LIN-28 expression. Knockdown of miR-98 led to a reduction of 1,25-VD anti-growth effect and overexpression of miR-98 suppressed the LNCaP cells growth via inducing G2/M arrest. And CCNJ, a protein controlling cell mitosis, is down-regulated by miR-98 via targeting 3′-untranslated region of CCNJ. Interestingly, miR-98 levels in blood are increased upon 1,25-VD treatment in mice suggesting the biomarker potential of miR-98 in predicting 1,25-VD response. Together, the finding that growth inhibitive miR-98 is induced by 1,25-VD provides a potential therapeutic target for prostate cancer and a potential biomarker for 1,25-VD anti-tumor action.

The field cancerization effect has been proposed to explain bladder cancer’s multifocal and recurrent nature, yet the mechanisms of this effect remain unknown. In this work, using cell biology, flow cytometry, and qPCR analyses, along with a xenograft mouse tumor model, we show that chronic exposure to tumor-derived extracellular vesicles (TEVs) results in the neoplastic transformation of non-malignant human SV-HUC urothelial cells. Inhibition of EV uptake prevented this transformation. Transformed cells not only possessed several oncogenic properties, such as increased genome instability, loss of cell–cell contact inhibition, and invasiveness, but also displayed altered morphology and cell structures, such as an enlarged cytoplasm with disrupted endoplasmic reticulum (ER) alignment and the accumulation of smaller mitochondria. Exposure of SV-HUC cells to TEVs provoked the unfolded protein response in the endoplasmic reticulum (UPR^_ER). Prolonged induction of UPR^_ER signaling activated the survival branch of the UPR^_ER pathway, in which cells had elevated expression of inositol-requiring enzyme 1 (IRE1), NF-κB, and the inflammatory cytokine leptin, and incurred loss of the pro-apoptotic protein C/EBP homologous protein (CHOP). More importantly, inhibition of ER stress by docosahexaenoic acid prevented TEV-induced transformation. We propose that TEVs promote malignant transformation of predisposed cells by inhibiting pro-apoptotic signals and activating tumor-promoting ER stress–induced unfolded protein response and inflammation. This study provides detailed insight into the mechanisms underlying the bladder cancer field effect and tumor recurrence.