The Role of TKS5 in Chromosome Stability and Bladder Cancer Progression

TKS5 promotes invasion and migration through the formation of invadopodia in some tumour cells, and it also has an important physiological function in cell migration through podosome formation in various nontumour cells. To date, the role of TKS5 in urothelial cells, and its potential role in BC initiation and progression, has not yet been addressed. Moreover, the contribution of TKS5 to ploidy control and chromosome stability has not been reported in previous studies. Therefore, in the present study, we wished to address the following questions: (i) Is TKS5 involved in the ploidy control of urothelial cells? (ii) What is the mechanism that leads to aneuploidy in response to TKS5 knockdown? (iii) Is TKS5 an oncogene or tumour-suppressor gene in the context of BC? (iv) Does TKS5 affect the proliferation, migration and invasion of BC cells? We assessed the gene and protein expressions via qPCR and Western blot analyses in a set of nontumour cell strains (Y235T, HBLAK and UROtsa) and a set of BC cell lines (RT4, T24, UMUC3 and J82). Following the shRNA knockdown in the TKS5-proficient cells and the ectopic TKS5 expression in the cell lines with low/absent TKS5 expression, we performed functional experiments, such as metaphase, invadopodia and gelatine degradation assays. Moreover, we determined the invasion and migration abilities of these genetically modified cells by using the Boyden chamber and wound-healing assays. The TKS5 expression was lower in the bladder cancer cell lines with higher invasive capacities (T24, UMUC3 and J82) compared to the nontumour cell lines from human ureter (Y235T, HBLAK and UROtsa) and the noninvasive BC cell line RT4. The reduced TKS5 expression in the Y235T cells resulted in augmented aneuploidy and impaired cell division. According to the Boyden chamber and wound-healing assays, TKS5 promotes the invasion and migration of bladder cancer cells. According to the present study, TKS5 regulates the migration and invasion processes of bladder cancer (BC) cell lines and plays an important role in genome stability.     

Enhancement of Farnesoid X Receptor Inhibits Migration,Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

(1) Background: Bladder cancer is a malignant tumor mainly caused by exposure to environmental chemicals, with a high recurrence rate. NR1H4, also known as Farnesoid X Receptor (FXR), acts as a nuclear receptor that can be activated by binding with bile acids, and FXR is highly correlated with the progression of cancers. The aim of this study was to verify the role of FXR in bladder cancer cells. (2) Methods: A FXR overexpressed system was established to investigate the effect of cell viability, migration, adhesion, and angiogenesis in low-grade TSGH8301 and high-grade T24 cells. (3) Results: After FXR overexpression, the ability of migration, adhesion, invasion and angiogenesis of bladder cancer cells declined significantly. Focal adhesive complex, MMP2, MMP9, and angiogenic-related proteins were decreased, while FXR was overexpressed in bladder cancer cells. Moreover, FXR overexpression reduced vascular endothelial growth factor mRNA and protein expression and secretion in bladder cancer cells. After treatment with the proteosome inhibitor MG132, the migration, adhesion and angiogenesis caused by FXR overexpression were all reversed in bladder cancer cells. (4) Conclusions: These results may provide evidence on the role of FXR in bladder cancer, and thus may improve the therapeutic efficacy of urothelial carcinoma in the future.     

缺氧诱导因子-1α与缺氧状态下胃癌细胞侵袭的相关性

目的探讨缺氧状态下及缺氧诱导因子-1α(Hypoxia-inducible factor-1α,HIF-1α)RNA干扰后,HIF-1α的表达及其与胃癌细胞黏附、游走、侵袭能力的相关性。方法将HIF-1α shRNA真核表达质粒pGPU6/GFP/Neo-HIF-1α-2484转染胃癌细胞SGC-7901,构建靶向干扰HIF-1α表达的SGC-7901稳定转染细胞株;采用Western blot法检测缺氧及HIF-1αRNA干扰对SGC-7901细胞HIF-1α表达的影响;MTT法检测缺氧及HIF-1αRNA干扰对SGC-7901细胞黏附能力的影响;采用Boyden Chamber膜侵袭系统,观察缺氧及HIF-1αRNA干扰对SGC-7901细胞游走及侵袭能力的影响。结果 SGC-7901细胞在缺氧培养4、8和16h时,HIF-1α均呈阳性表达,并随时间的延长而显著增加;细胞粘贴率、游走和侵袭穿膜细胞数分别随时间延长而显著增加,与HIF-1α表达呈显著正相关。HIF-1αRNA干扰后,缺氧SGC-7901细胞HIF-1α表达显著下降,细胞粘贴率、游走和侵袭穿膜细胞数均显著下降。结论 HIF-1α的过度表达导致胃癌细胞黏附、游走及侵袭能力增强,可能是胃癌局部侵袭和远处转移的重要原因之一。     

TTYH3 Modulates Bladder Cancer Proliferation and Metastasis via FGFR1/H-Ras/A-Raf/MEK/ERK Pathway

Tweety family member 3 (TTYH3) is a calcium-activated chloride channel with a non-pore-forming structure that controls cell volume and signal transduction. We investigated the role of TTYH3 as a cancer-promoting factor in bladder cancer. The mRNA expression of TTYH3 in bladder cancer patients was investigated using various bioinformatics databases. The results demonstrated that the increasingly greater expression of TTYH3 increasingly worsened the prognosis of patients with bladder cancer. TTYH3 knockdown bladder cancer cell lines were constructed by their various cancer properties measured. TTYH3 knockdown significantly reduced cell proliferation and sphere formation. Cell migration and invasion were also significantly reduced in knockdown bladder cancer cells, compared to normal bladder cancer cells. The knockdown of TTYH3 led to the downregulation of H-Ras/A-Raf/MEK/ERK signaling by inhibiting fibroblast growth factor receptor 1 (FGFR1) phosphorylation. This signaling pathway also attenuated the expression of c-Jun and c-Fos. The findings implicate TTYH3 as a potential factor regulating the properties of bladder cancer and as a therapeutic target.     

BMAL1 Knockdown Leans Epithelial–Mesenchymal Balance toward Epithelial Properties and Decreases the Chemoresistance of Colon Carcinoma Cells

The circadian clock coordinates biological and physiological functions to day/night cycles. The perturbation of the circadian clock increases cancer risk and affects cancer progression. Here, we studied how BMAL1 knockdown (BMAL1-KD) by shRNA affects the epithelial–mesenchymal transition (EMT), a critical early event in the invasion and metastasis of colorectal carcinoma (CRC). In corresponding to a gene set enrichment analysis, which showed a significant enrichment of EMT and invasive signatures in BMAL1_high CRC patients as compared to BMAL1_low CRC patients, our results revealed that BMAL1 is implicated in keeping the epithelial–mesenchymal equilibrium of CRC cells and influences their capacity of adhesion, migration, invasion, and chemoresistance. Firstly, BMAL1-KD increased the expression of epithelial markers (E-cadherin, CK-20, and EpCAM) but decreased the expression of Twist and mesenchymal markers (N-cadherin and vimentin) in CRC cell lines. Finally, the molecular alterations after BMAL1-KD promoted mesenchymal-to-epithelial transition-like changes mostly appeared in two primary CRC cell lines (i.e., HCT116 and SW480) compared to the metastatic cell line SW620. As a consequence, migration/invasion and drug resistance capacities decreased in HCT116 and SW480 BMAL1-KD cells. Together, BMAL1-KD alerts the delicate equilibrium between epithelial and mesenchymal properties of CRC cell lines, which revealed the crucial role of BMAL1 in EMT-related CRC metastasis and chemoresistance.     

Pharmacological Inhibition of miR-130 Family Suppresses Bladder Tumor Growth by Targeting Various Oncogenic Pathways via PTPN1

Previously, we have revealed that the miR-130 family (miR-130b, miR-301a, and miR-301b) functions as an oncomiR in bladder cancer. The pharmacological inhibition of the miR-130 family molecules by the seed-targeting strategy with an 8-mer tiny locked nucleic acid (LNA) inhibits the growth, migration, and invasion of bladder cancer cells by repressing stress fiber formation. Here, we searched for a functionally advanced target sequence with LNA for the miR-130 family with low cytotoxicity and found LNA #9 (A(L)^i^i^A(L)^T(L)^T(L)^G(L)^5(L)^A(L)^5(L)^T(L)^G) as a candidate LNA. LNA #9 inhibited cell growth in vitro and in an in vivo orthotopic bladder cancer model. Proteome-wide tyrosine phosphorylation analysis suggested that the miR-130 family upregulates a wide range of receptor tyrosine kinases (RTKs) signaling via the expression of phosphorylated Src (pSrc^Tyr416). SILAC-based proteome analysis and a luciferase assay identified protein tyrosine phosphatase non-receptor type 1 (PTPN1), which is implicated as a negative regulator of multiple signaling pathways downstream of RTKs as a target gene of the miR-130 family. The miR-130-targeted LNA increased and decreased PTPN1 and pSrc^Tyr416 expressions, respectively. PTPN1 knockdown led to increased tumor properties (cell growth, invasion, and migration) and increased pSrc^Tyr416 expression in bladder cancer cells, suggesting that the miR-130 family upregulates multiple RTK signaling by targeting PTPN1 and subsequent Src activation in bladder cancer. Thus, our newly designed miR-130 family targeting LNA could be a promising nucleic acid therapeutic agent for bladder cancer.     

Role of Periostin Expression in Non-Small Cell Lung Cancer: Periostin Silencing Inhibits the Migration and Invasion of Lung Cancer Cells via Regulation of MMP-2 Expression

The involvement of periostin (POSTN) in non-small-cell lung cancer (NSCLC) migration, invasion, and its underlying mechanisms has not been well established. The present study aims to determine epithelial POSTN expression in NSCLC and to assess associations with clinicopathological factors and prognosis as well as to explore the effects of POSTN knockdown on tumor microenvironment and the migration and invasion of lung cancer cells. Immunohistochemistry was used to evaluate epithelial POSTN expression in NSCLC. POSTN mRNA expression in the dissected lung cancer cells was confirmed by laser capture microdissection and real-time PCR. A549 cells were used for transfecting shRNA-POSTN lentiviral particles. Wound healing and Transwell invasion assays were used to assess the migratory and invasive abilities of A549 cells transfected with POSTN-specific short hairpin (sh)RNA. The results demonstrated significantly higher cytoplasmic POSTN expression in the whole NSCLC group compared to non-malignant lung tissue (NMLT). POSTN expression in cancer cells may be considered to be an independent prognostic factor for survival in NSCLC. POSTN knockdown significantly inhibited A549 cell migration and invasion capabilities in vitro. The activity and the expression level of matrix metalloproteinase-2 (MMP-2) were significantly decreased in A549.shRNA compared to control cells. In summary, POSTN may regulate lung cancer cell invasiveness by modulating the expression of MMP-2 and may represent a potential target for novel therapeutic intervention for NSCLC.     

联氨基姜黄素脂质体纳米颗粒对小鼠乳腺癌4T1细胞生物学行为的影响

目的探讨联氨基姜黄素(Hydrazinocurcumin,HC)脂质体纳米颗粒(Liposome nanoparticles,NPs)对小鼠乳腺癌4T1细胞增殖、凋亡、侵袭及迁移的影响。方法采用薄膜分散-超声法制备HC-NPs,透射电镜检测纳米颗粒的粒径大小。将4T1细胞分为PBS组、NPs组和HC-NPs组,分别加入10%(v/v)PBS、10%(v/v)NP、10%(v/v)HC-NPs,培养24 h后,MTT法检测细胞的增殖活力;流式细胞术检测细胞周期和凋亡率的变化;刘氏染色法检测细胞的形态;Transwell试验检测4T1细胞侵袭和迁移能力;Western blot检测转导和转录激活因子STAT3及细胞增殖、凋亡、侵袭、迁移相关蛋白的表达水平。结果透射电镜下可见HC-NPs为150 nm左右的脂质体颗粒。HC-NPs对4T1细胞的增殖抑制率明显高于NPs组(P<0.01);与PBS组和NPs组相比,HC-NPs可使细胞形态不规则,诱导细胞周期发生G2/M期阻滞,细胞凋亡率明显增加(P<0.01),明显抑制细胞侵袭和迁移(P<0.01)及STAT3的激活,下调CyclinD1、c-Myc、Bcl-2、Survivin、MMP-9的表达,同时上调Bax的表达(P<0.05)。结论 HC-NPs可能通过抑制STAT3的激活,抑制4T1细胞增殖、侵袭和迁移能力,促进细胞凋亡。     

Interleukin-24 Immunobiology and Its Roles in Inflammatory Diseases

Interleukin (IL)-24 belongs to the IL-10 family and signals through two receptor complexes, i.e., IL-20RA/IL-20RB and IL-20RB/IL22RA1. It is a multifunctional cytokine that can regulate immune response, tissue homeostasis, host defense, and oncogenesis. Elevation of IL-24 is associated with chronic inflammation and autoimmune diseases, such as psoriasis, rheumatoid arthritis (RA), and inflammatory bowel disease (IBD). Its pathogenicity has been confirmed by inducing inflammation and immune cell infiltration for tissue damage. However, recent studies also revealed their suppressive functions in regulating immune cells, including T cells, B cells, natural killer (NK) cells, and macrophages. The tolerogenic properties of IL-24 were reported in various animal models of autoimmune diseases, suggesting the complex functions of IL-24 in regulating autoimmunity. In this review, we discuss the immunoregulatory functions of IL-24 and its roles in autoimmune diseases.     

BubR1 Acts as a Promoter in Cellular Motility of Human Oral Squamous Cancer Cells through Regulating MMP-2 and MMP-9

BubR1 is a critical component of spindle assembly checkpoint, ensuring proper chromatin segregation during mitosis. Recent studies showed that BubR1 was overexpressed in many cancer cells, including oral squamous cell carcinomas (OSCC). However, the effect of BubR1 on metastasis of OSCC remains unclear. This study aimed to unravel the role of BubR1 in the progression of OSCC and confirm the expression of BubR1 in a panel of malignant OSCC cell lines with different invasive abilities. The results of quantitative real-time PCR showed that the mRNA level of BubR1 was markedly increased in four OSCC cell lines, Ca9-22, HSC3, SCC9 and Cal-27 cells, compared to two normal cells, normal human oral keratinocytes (HOK) and human gingival fibroblasts (HGF). Moreover, the expression of BubR1 in these four OSCC cell lines was positively correlated with their motility. Immunofluorescence revealed that BubR1 was mostly localized in the cytosol of human gingival carcinoma Ca9-22 cells. BubR1 knockdown significantly decreased cellular invasion but slightly affect cellular proliferation on both Ca9-22 and Cal-27 cells. Consistently, the activities of metastasis-associated metalloproteinases MMP-2 and MMP-9 were attenuated in BubR1 knockdown Ca9-22 cells, suggesting the role of BubR1 in promotion of OSCC migration. Our present study defines an alternative pathway in promoting metastasis of OSCC cells, and the expression of BubR1 could be a prognostic index in OSCC patients.     

Wnt/β-Catenin Signalling and Its Cofactor BCL9L Have an Oncogenic Effect in Bladder Cancer Cells

Bladder cancer (BC) is characterised by a high recurrence and progression rate. However, the molecular mechanisms of BC progression remain poorly understood. BCL9L, a coactivator of β-catenin was mutated in the 5′ and 3′ untranslated regions (UTRs). We assessed the influence of UTRs mutations on BCL9L, and the role of BCL9L and Wnt/β-catenin signalling in BC cells. UTR mutations were analysed by a luciferase reporter. BCL9L protein was assessed by immunohistochemistry in BC tissues. Cell proliferation was examined by crystal violet staining and by the spheroid model. Moreover, migration and invasion were analysed in real-time using the xCelligence RTCA system. The A > T mutation at 3′ UTR of BCL9L reduces the luciferase reporter mRNA expression and activity. BCL9L is predominantly increased in dysplastic urothelial cells and muscle-invasive BC. Knockdown of BCL9L and inhibition of Wnt/β-catenin signalling significantly repress the proliferation, migration and invasion of Cal29 and T24. In addition, BCL9L knockdown reduces mRNA level of Wnt/β-catenin target genes in Cal29 but not in T24 cells. BCL9L and Wnt/β-catenin signalling play an oncogenic role in bladder cancer cells and seems to be associated with BC progression. Nevertheless, the involvement of BCL9L in Wnt/β-catenin signalling is cell-line specific.     

Inhibition of Autophagy Potentiates Atorvastatin-Induced Apoptotic Cell Death in Human Bladder Cancer Cells in Vitro

Statins are cholesterol reduction agents that exhibit anti-cancer activity in several human cancers. Because autophagy is a crucial survival mechanism for cancer cells under stress conditions, cooperative inhibition of autophagy acts synergistically with other anti-cancer drugs. Thus, this study investigates whether combined treatment of atorvastatin and autophagy inhibitors results in enhancing the cytotoxic effects of atorvastatin, upon human bladder cancer cells, T24 and J82, in vitro. To measure cell viability, we performed the EZ-Cytox cell viability assay. We examined apoptosis by flow cytometry using annexin-V/propidium iodide (PI and western blot using procaspase-3 and poly (ADP-ribose) polymerase (PARP) antibodies. To examine autophagy activation, we evaluated the co-localization of LC3 and LysoTracker by immunocytochemistry, as well as the expression of LC3 and p62/sequestosome-1 (SQSTM1) by western blot. In addition, we assessed the survival and proliferation of T24 and J82 cells by a clonogenic assay. We found that atorvastatin reduced the cell viability of T24 and J82 cells via apoptotic cell death and induced autophagy activation, shown by the co-localization of LC3 and LysoTracker. Moreover, pharmacologic inhibition of autophagy significantly enhanced atorvastatin-induced apoptosis in T24 and J82 cells. In sum, inhibition of autophagy potentiates atorvastatin-induced apoptotic cell death in human bladder cancer cells in vitro, providing a potential therapeutic approach to treat bladder cancer.     

S1P Lyase siRNA Dampens Malignancy of DLD-1 Colorectal Cancer Cells

Sphingosine-1-phosphate lyase 1 (S1P lyase or SGPL1) is an essential sphingosine-1-phosphate-degrading enzyme. Its manipulation favors onset and progression of colorectal cancer and others in vivo. Thus, SGPL1 is an important modulator of cancer initiation. However, in established cancer, the impact of retrospective SGPL1 modulation is elusive. Herein, we analyzed how SGPL1 siRNA affects malignancy of the human colorectal cancer cells DLD-1 and found that in parallel to the reduction of SGPL1 expression levels, migration, invasion, and differentiation status changed. Diminished SGPL1 expression was accompanied with reduced cell migration and cell invasion in scratch assays and transwell assays, whereas metabolic activity and proliferation was not altered. Decreased migration was attended by increased cell–cell-adhesion through upregulation of E-cadherin and formation of cadherin-actin complexes. Spreading cell islets showed lower vimentin abundance in border cells. Furthermore, SGPL1 siRNA treatment induced expression of epithelial cell differentiation markers, such as intestinal alkaline phosphatase and cytokeratin 20. Hence, interference with SGPL1 expression augmented a partial redifferentiation of colorectal cancer cells toward normal colon epithelial cells. Our investigation showed that SGPL1 siRNA influenced tumorigenic activity of established colorectal cancer cells. We therefore suggest SGPL1 as a target for lowering malignant potential of already existing cancer.     

Establishment of Three-Dimensional Bioprinted Bladder Cancer-on-a-Chip with a Microfluidic System Using Bacillus Calmette–Guérin

Immunotherapy of bladder cancer is known to have favorable effects, although it is difficult to determine which patients will show a good response because of the different tumor microenvironments (TME). Here, we developed a bladder cancer-on-a-chip (BCOC) to mimic the TME using three-dimensional (3D) bioprinting and microfluidic technology. We fabricated a T24 and a 5637-cell line-based BCOC that also incorporated MRC-5, HUVEC, and THP-1 cells. We evaluated the effects of TME and assessed the immunologic reactions in response to different concentrations of Bacillus Calmette–Guérin (BCG) via live/dead assay and THP-1 monocytic migration, and concentrations of growth factors and cytokines. The results show that cell viability was maintained at 15% filling density in circle-shaped cell constructs at 20 μL/min microfluidic flow rate. A 3D co-culture increased the proliferation of BCOCs. We found that the appropriate time to evaluate the viability of BCOC, concentration of cytokines, and migration of monocytes was 6 h, 24 h, and three days after BGC treatment. Lastly, the immunotherapeutic effects of BCOC increased according to BCG dosage. To predict effects of immunotherapeutic agent in bladder cancer, we constructed a 3D bioprinted BCOC model. The BCOC was validated with BCG, which has been proven to be effective in the immunotherapy of bladder cancer.     

Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK,SRC, and JNK Pathways

Bladder cancer is a common global cancer with a high percentage of metastases and high mortality rate. Thus, it is necessary to identify new biomarkers that can be helpful in diagnosis. Pyruvate dehydrogenase kinase 4 (PDK4) belongs to the PDK family and plays an important role in glucose utilization in living organisms. In the present study, we evaluated the role of PDK4 in bladder cancer and its related protein changes. First, we observed elevated PDK4 expression in high-grade bladder cancers. To screen for changes in PDK4-related proteins in bladder cancer, we performed a comparative proteomic analysis using PDK4 knockdown cells. In bladder cancer cell lines, PDK4 silencing resulted in a lower rate of cell migration and invasion. In addition, a PDK4 knockdown xenograft model showed reduced bladder cancer growth in nude mice. Based on our results, PDK4 plays a critical role in the metastasis and growth of bladder cancer cells through changes in ERK, SRC, and JNK.