sphingosine-kinase and Urinary-Bladder-Neoplasms

Hypoxia facilitates an aggressive phenotype and immune evasion in solid tumors including bladder cancer (BC). Sphingosine kinase 1 (SphK1) is aberrantly expressed and correlated with poor prognosis in BC patients. However, its roles in hypoxia-evoked malignancies and immune evasion in BC remain elusive.. The expression of SphK1 in BC tissues was analysed using a bioinformatics database. BC cells were transfected with si-SphK1 or recombinant HIF-1α plasmids under hypoxic conditions. The mRNA level, activity and protein expression of SphK1 were determined. Transwell assay was performed to evaluate cell invasion. After co-culture with natural killer (NK) cells, NK cell cytotoxicity to BC cells was assessed. The involvement of sphingosine-1-phosphate (S1P)/HIF-1α signaling was analysed by ELISA, qRT-PCR and western blot.. UALCAN and GEPIA database confirmed high expression of SphK1 in BC tissues. Moreover, hypoxia increased the expression and activity of SphK1. Loss of SphK1 inhibited hypoxia-induced cell invasion. IL-2 induced NK cell activation by secreting TNF-α and IFN-γ. Hypoxia antagonized NK cell activation-evoked cytotoxicity to BC cells. Intriguingly, SphK1 knockdown reversed hypoxia-induced cell resistance to NK cell killing. Mechanically, SphK1 loss inhibited hypoxia-activated the S1P/HIF-1α signaling. However, S1P addition reversed the inhibitory effects of SphK1 down-regulation on hypoxia-activated S1P/HIF-1α signaling. Notably, reactivating HIF-1α overturned the suppressive roles of SphK1 loss in decreasing hypoxia-induced cell invasion and resistance to NK cell cytotoxicity.. Targeting SphK1 may inhibit hypoxia-evoked invasion and immune evasion via the S1P/HIF-1α signaling, indicating a promising therapeutic target for BC.

Topics: Carcinoma; Cell Death; Humans; Hypoxia; Interleukin-2; Killer Cells, Natural; Oncogenes; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; Tumor Necrosis Factor-alpha; Urinary Bladder; Urinary Bladder Neoplasms

Sphingosine‑1‑phosphate (S1P) serves an important role in various physiological and pathophysiological processes, including the regulation of cell apoptosis, proliferation and survival. Sphingosine kinase 1 (SPHK1) is a lipid kinase that phosphorylates sphingosine to generate S1P. S1P has been proven to be positively correlated with chemotherapy resistance in breast cancer, colorectal carcinoma and non‑small cell lung cancer. However, whether SPHK1 is involved in the development of cisplatin resistance remains to be elucidated. The present study aimed to identify the association between SPHK1 and chemoresistance in bladder cancer cells and to explore the therapeutic implications in patients with bladder cancer. Bladder cancer cell proliferation and apoptosis were determined using Cell Counting Kit‑8 assays and flow cytometry, respectively. Apoptosis‑related proteins were detected via western blotting. The results revealed that SPHK1 was positively correlated with cisplatin resistance in bladder cancer cells, exhibiting an antiapoptotic effect that was reflected by the downregulation of apoptosis‑related proteins (Bax and cleaved caspase‑3) and the upregulation of an antiapoptotic protein (Bcl‑2) in SPHK1‑overexpression cell lines. Suppression of SPHK1 by small interfering RNA or FTY‑720 significantly reversed the antiapoptotic effect. A potential mechanism underlying SPHK1‑induced cisplatin resistance and apoptosis inhibition may be activation of STAT3 via binding non‑POU domain containing octamer binding. In conclusion, the present study suggested that SPHK1 displayed significant antiapoptotic effects in cisplatin‑based treatment, thus may serve as a potential novel therapeutic target for the treatment for bladder cancer.

Topics: Aged; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cisplatin; DNA-Binding Proteins; Drug Resistance, Neoplasm; Female; Gene Knockdown Techniques; Humans; Male; Middle Aged; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; RNA-Binding Proteins; Signal Transduction; STAT3 Transcription Factor; Urinary Bladder Neoplasms

MAFG-AS1 is an oncogenic lncRNA in multiple types of cancer. However, its role in bladder cancer (BC) remains unclear. The present study aimed to investigate the function of MAFG-AS1 in BC. BC and paired non-tumor tissues were collected. Two BC cell lines HT01197 and HT-1376 were used. Dual luciferase activity assay, RT-qPCR, western blot, CCK-8, transwell invasion assay, and wound healing assay were performed. We found that MAFG-AS1 was significantly up-regulated in BC tissues and predicted a poor survival rate. MAFG-AS1 interacted with miR-125b-5p. However, the expression levels of MAFG‑AS1 and miR-125b-5p were not obviously correlated in BC tissues, and MAFG‑AS1 and miR-125b-5p did not regulate the expression of each other. Interestingly, we found that SphK1, a downstream target of miR-125b-5p, was negatively correlated with miR-125b-5p, while it was positively correlated with MAFG-AS1 across BC tissues. In addition, overexpression of MAFG‑AS1 upregulated the expression of SphK1 in BC cells, and attenuated the inhibitory effects of miR-125b-5p on the expression of SphK1. Functional assays showed that overexpression of MAFG‑AS1 promoted BC cell proliferation, migration, and invasion, while its effects were attenuated by overexpression of miR-125b-5p. Moreover, overexpression of miR-125b-5p inhibited BC cell proliferation, migration, and invasion, while its effects were alleviated by overexpression of SphK1. Taken together, our findings demonstrated that MAFG-AS1 has an oncogenic role in BC by regulating the miR-125b-5p/SphK1 axis. MAFG-AS1 might serve as a good diagnostic marker and a potential therapeutic target of BC.

Topics: Adult; Aged; Biomarkers, Tumor; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; MafG Transcription Factor; Male; MicroRNAs; Middle Aged; Molecular Targeted Therapy; Neoplasm Invasiveness; Phosphotransferases (Alcohol Group Acceptor); RNA, Long Noncoding; Urinary Bladder Neoplasms

Upregulation of sphingosine kinase 1 (SPHK1) protein has been reported to be associated with a poor prognosis in a variety of malignant tumors. However, the role of SPHK1 in bladder cancer (BC) has not been thoroughly elucidated. The purpose of this study was to assess SPHK1 expression and to explore its contribution to BC. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was conducted to detect SPHK1 mRNA expression in 37 pairs of fresh-frozen BC tissues and corresponding noncancerous tissues. Results showed that SPHK1 mRNA expression level in BC tissues was significantly higher than that in corresponding noncancerous tissues. To investigate the association between SPHK1 protein expression and clinicopathological characteristics of BC, immunohistochemistry (IHC) was performed in 153 archived paraffin-embedded BC samples. Interestingly, high SPHK1 expression was significantly associated with histologic grade (P = 0.045) and tumor stage (P < 0.001) of patients with BC. The Kaplan-Meier survival curve showed that patients with high SPHK1 expression had significantly reduced overall 5-year survival rates (P < 0.001). Multivariate Cox regression analysis further suggested that the increased expression of SPHK1 was an independent poor prognostic factor for this disease. In conclusion, our data offer the convincing evidence for the first time that the increased expression of SPHK1 may be involved in the pathogenesis and progression of BC. SPHK1 might be a potential marker to predict the prognosis in BC.

Topics: Aged; Biomarkers, Tumor; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Middle Aged; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Proportional Hazards Models; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Urinary Bladder Neoplasms

Mechanisms by which cancer cells communicate with the host organism to regulate lung colonization/metastasis are unclear. We show that this communication occurs via sphingosine 1-phosphate (S1P) generated systemically by sphingosine kinase 1 (SK1), rather than via tumour-derived S1P. Modulation of systemic, but not tumour SK1, prevented S1P elevation, and inhibited TRAMP-induced prostate cancer growth in TRAMP(+/+) SK1(-/-) mice, or lung metastasis of multiple cancer cells in SK1(-/-) animals. Genetic loss of SK1 activated a master metastasis suppressor, Brms1 (breast carcinoma metastasis suppressor 1), via modulation of S1P receptor 2 (S1PR2) in cancer cells. Alterations of S1PR2 using pharmacologic and genetic tools enhanced Brms1. Moreover, Brms1 in S1PR2(-/-) MEFs was modulated by serum S1P alterations. Accordingly, ectopic Brms1 in MB49 bladder cancer cells suppressed lung metastasis, and stable knockdown of Brms1 prevented this process. Importantly, inhibition of systemic S1P signalling using a novel anti-S1P monoclonal antibody (mAb), Sphingomab, attenuated lung metastasis, which was prevented by Brms1 knockdown in MB49 cells. Thus, these data suggest that systemic SK1/S1P regulates metastatic potential via regulation of tumour S1PR2/Brms1 axis.

Topics: Animals; Disease Models, Animal; Humans; Lung Neoplasms; Lysophospholipids; Male; Mice; Mice, Knockout; Neoplasm Metastasis; Phosphotransferases (Alcohol Group Acceptor); Prostatic Neoplasms; Receptors, Lysosphingolipid; Repressor Proteins; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Urinary Bladder Neoplasms

Overexpression of vascular endothelial growth factor receptors (VEGFRs) has been reported in a variety of tumor types. Here we find that 11 out of the 14 bladder tumor cell lines examined express one or more VEGF receptors. Analysis of the T24 bladder tumor cell line reveals a functional autocrine loop involving VEGF and the Flk-1 receptor. Blocking VEGF expression in T24 cells results in a decrease in DNA synthesis. The Flk-1 receptor in T24 cells is phosphorylated in response to VEGF-121 or VEGF-165, and an Flk-1 inhibitor blocks VEGF to ERK signaling. We report that VEGF stimulation of T24 cells results in activation of H- and N-Ras and this is dependent on cellular sphingosine kinase 1 (SPK1) activity. Previously, we found VEGF-induced activation of Ras appears to be independent of a Ras-guanine nucleotide exchange factors (GEFs). Here we report that sphingosine can stimulate Ras-GTPase activating protein (GAP) activity in vitro, and sphingosine-1-phosphate (SPP) can block the stimulatory effects of sphingosine. We present a model where the balance between sphingosine and SPP regulates Ras-GAP activity such that stimulation of SPK1 favors downregulation of Ras-GAP and thereby the activation of Ras proteins. These data highlight a VEGF pathway that may be involved in the survival and proliferation of bladder tumor cells as well as other tumor cell types.

Topics: Angiogenesis Inducing Agents; DNA; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; rac GTP-Binding Proteins; ras GTPase-Activating Proteins; ras Proteins; Receptors, Vascular Endothelial Growth Factor; Signal Transduction; Sphingolipids; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A

Vascular endothelial growth factor (VEGF) signaling is critical to the processes of angiogenesis and tumor growth. Here, evidence is presented for VEGF stimulation of sphingosine kinase (SPK) that affects not only endothelial cell signaling but also tumor cells expressing VEGF receptors. VEGF or phorbol 12-myristate 13-acetate treatment of the T24 bladder tumor cell line resulted in a time- and dose-dependent stimulation of SPK activity. In T24 cells, VEGF treatment reduced cellular sphingosine levels while raising that of sphingosine-1-phosphate. VEGF stimulation of T24 cells caused a slow and sustained accumulation of Ras-GTP and phosphorylated extracellular signal-regulated kinase (phospho-ERK) compared with that after EGF treatment. Small interfering RNA (siRNA) that targets SPK1, but not SPK2, blocks VEGF-induced accumulation of Ras-GTP and phospho-ERK in T24 cells. In contrast to EGF stimulation, VEGF stimulation of ERK1/2 phosphorylation was unaffected by dominant-negative Ras-N17. Raf kinase inhibition blocked both VEGF- and EGF-stimulated accumulation of phospho-ERK1/2. Inhibition of SPK by pharmacological inhibitors, a dominant-negative SPK mutant, or siRNA that targets SPK blocked VEGF, but not EGF, induction of phospho-ERK1/2. We conclude that VEGF induces DNA synthesis in a pathway which sequentially involves protein kinase C (PKC), SPK, Ras, Raf, and ERK1/2. These data highlight a novel mechanism by which SPK mediates signaling from PKC to Ras in a manner independent of Ras-guanine nucleotide exchange factor.

Topics: Carcinoma; Endothelial Growth Factors; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Humans; Intercellular Signaling Peptides and Proteins; Lymphokines; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; ras GTPase-Activating Proteins; ras Guanine Nucleotide Exchange Factors; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors