Knockdown of ADAM8 inhibits the proliferation, migration, invasion, and tumorigenesis of renal clear cell carcinoma cells to enhance the immunotherapy efficacy.

The current study performed bioinformatics and in vitro and in vivo experiments to explore the effects of ADAM8 on the malignant behaviors and immunotherapeutic efficacy of renal clear cell carcinoma (ccRCC) Cells. The modular genes most associated with immune cells were screened. Then, prognostic risk models were constructed by univariate COX analysis, LASSO regression analysis and multivariate COX analysis, and their diagnostic value was determined. The correlation between tumor mutation load (TMB) scores and the prognosis of ccRCC patients was clarified. Finally, six key genes (ABI3, ADAM8, APOL3, MX2, CCDC69, and STAC3) were analyzed for immunotherapy efficacy. Human and mouse ccRCC cell lines and human proximal tubular epithelial cell lines were used for in vitro cell experiments. The effect of ADAM8 overexpression or knockdown on tumor formation and survival in ccRCC cells was examined by constructing subcutaneous transplanted tumor model. Totally, 636 Black module genes were screened as being most associated with immune cell infiltration. Six genes were subsequently confirmed for the construction of prognostic risk models, of which ABI3, APOL3 and CCDC69 were low-risk factors, while ADAM8, MX2 and STAC3 were high-risk factors. The constructed risk model based on the identified six genes could accurately predict the prognosis of ccRCC patients. Besides, TMB was significantly associated with the prognosis of ccRCC patients. Furthermore, ABI3, ADAM8, APOL3, MX2, CCDC69 and STAC3 might play important roles in treatment concerning CTLA4 inhibitors or PD-1 inhibitors or combined inhibitors. Finally, we confirmed that ADAM8 could promote the proliferation, migration and invasion of ccRCC cells through in vitro experiments, and further found that in in vivo experiments, ADAM8 knockdown could inhibit tumor formation in ccRCC cells, improve the therapeutic effect of anti-PD1, and prolong the survival of mice. Our study highlighted the alleviative role of silencing ADAM8 in ccRCC patients.

Histone deacetylase 2 regulates STAT1-dependent upregulation of atypical chemokine receptor 3 to induce M2 macrophage migration and immune escape in hepatocellular carcinoma.

Atypical chemokine receptor 3 (ACKR3) has been linked to the tumor microenvironment. This work investigates the effects of ACKR3 and its regulatory molecules on the chemotactic migration of tumor-associated macrophages (TAMs) in hepatocellular carcinoma (HCC). RT-qPCR and western blot assays identified elevated ACKR3 and HDAC2 levels in HCC tissues and cells. Knockdown or overexpression of ACKR3 was induced in HCC cells through vectors of lentivirus plasmids, and then the conditioned medium of the HCC cells was collected to stimulate TAMs. The stimulated TAMs were co-cultured with CD3+ T cells. ACKR3 knockdown in HCC cells inhibited migration of TAMs and promoted M1 polarization, which restored the activity of T cells. Histone deacetylase 2 (HDAC2) recruited signal transducer and activator of transcription 1 (STAT1) to the ACKR3 promoter to activate ACKR3 transcription. HDAC2 silencing suppressed nuclear translocation of STAT1 and decreased ACKR3 expression. HDAC2 knockdown in HCC cells similarly suppressed TAM migration, promoted M1 polarization, and restored T cell function, but these changes were inversed by ACKR3 upregulation. HDAC2 or ACKR3 silencing weakened tumor growth and immune escape in mice. In conclusion, this study demonstrates that HDAC2 upregulates ACKR3 via STAT1 to induce migration of M2 macrophages and immune escape in HCC.

METTL3 promotes m6A hypermethylation of RBM14 via YTHDF1 leading to the progression of hepatocellular carcinoma.

Liver is a well-known immunological organ with unique microenvironment. In normal conditions, the rich immune-infiltrating cells cooperate with non-parenchymal cells, such as Kupffer cells (KCs). The presence of liver immunosuppressive microenvironment underlines the importance to dissect this interaction to understand how this cross-talk promotes tumor growth in hepatocellular carcinoma (HCC). Therefore, the aim of the study here was to probe the role of methyltransferase-like 3 (METTL3) in the HCC progression and its effect on the polarization of KCs. KCs showed M2 polarization, and METTL3 was overexpressed in our collected HCC tissues relative to adjacent tissues. METTL3 depletion inhibited the M2 polarization of KCs, thereby reverting the malignant phenotype of HCC cells in vitro and growth and metastasis in vivo. Mechanistically, YTH domain-containing family protein 1 (YTHDF1) bound to RNA-binding protein 14 (RBM14), whereas METTL3 knockdown in KCs cells suppressed RBM14 expression by decreasing N-methyladenosine (m6A) methylation. Overexpression of RBM14 mitigated the anti-tumor effects of sh-METTL3 in vitro and in vivo. It is suggested that the mechanism of sh-METTL3 suppressing the polarization of KCs and the progression of HCC is to regulate the RBM14 expression via YTHDF1-dependent m6A modification.

ALDH2 Hampers Immune Escape in Liver Hepatocellular Carcinoma through ROS/Nrf2-mediated Autophagy.

Aldehyde dehydrogenase 2 (ALDH2) has been implicated in the progression of liver hepatocellular carcinoma (LIHC). The most important feature of LIHC is the immune escape process. This study sets to study the role of ALDH2 in regulating immune escape in LIHC. Bioinformatics analysis was applied to examine the expression of ALDH2 in LIHC and its impact on patients' survival. The effect of ALDH2 expression on malignant phenotype of LIHC cells was assessed by gain-of-function assays. RT-qPCR and Western blot were conducted to examine the expression of related factors, thus investigating the downstream mechanisms of ALDH2. ELISA assay was carried out to measure the level of oxidative stress in cells, and crystal violet staining was conducted to observe the killing effect of T cells on tumor cells. Finally, xenograft assay was carried out to verify the role of ALDH2 in vivo.ALDH2 was poorly expressed in LIHC, which predicted dismal prognoses for patients. ALDH2 inhibited the malignant aggressiveness of LIHC cells. ALDH2 blocked the activation of Nrf2 by suppressing reactive oxygen species (ROS) in LIHC, and Nrf2 significantly reversed the tumor-suppressing properties of ALDH2. Nrf2 hindered autophagy and led to immune escape of LIHC cells. Moreover, ALDH2 considerably suppressed the growth of xenografts, increased autophagy and promoted the accumulation of T cells in tumors. In contrast, Nrf2 drastically reversed the repressive effect of ALDH2 on tumor growth.ALDH2 impaired the ROS/Nrf2 axis to promote autophagy, thereby repressing immune escape in LIHC.

Discovery of novel sulphonamide hybrids that inhibit LSD1 against bladder cancer cells.

Aim:A series of sulphonamide hybrids were designed, synthesised, and identified as potential lysine-specific demethylase 1 (LSD1) inhibitors.Materials and methods: Bladder cancer cell lines were cultured to evaluate the antiproliferative activity. Inhibitory evaluation of sulphonamide hybrids against LSD1 were performed.Conclusion: sulphonamide derivative L8 exhibited the antiproliferative activity against HTB5, HTB3, HT1376, and HTB1 cells with IC50 values of 1.87, 0.18, 0.09, and 0.93 µM, respectively. Compound L8 as a selective and reversible LSD1 inhibitor could inhibit LSD1 with the IC50 value of 60 nM. It effectively inhibited LSD1 by increasing the expression levels of H3K4me1, H3K4me2, and H3K9me2 in HT1376 cells. To the best of our knowledge, this was the first report which showed that sulphonamide-quinoline-dithiocarbamate hybrids potently inhibited LSD1 in bladder cancer cells. Our studies give the potential application of the sulphonamide-based scaffold for developing LSD1 inhibitors to treat bladder cancer.

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.

LINC00511/miRNA-143-3p Modulates Apoptosis and Malignant Phenotype of Bladder Carcinoma Cells via PCMT1.

Bladder cancer has easy recurrence characteristics, but its occurrence and development mechanism are still unclear. Non-coding RNA is a kind of RNA that exists widely and cannot be translated into proteins, which has played a key role in the regulation of biological functions of tumor cells. However, the regulation mechanism of non-coding RNA on bladder tumors is not fully understood. By microarray analysis and database analysis, we found that LINC00511 was significantly highly expressed in bladder cancer. The expressions of LINC00511, miR-143-3p, and PCMT in bladder cancer tissues and cells were detected by quantitative reverse transcription-polymerase chain reaction. The relationship between the expressions of miR-143-3p and PCMT1 and the clinicopathological parameters of the tumor was analyzed. The proliferation and invasion of bladder cancer cells were detected by MTT assay and Transwell assay. The expression levels of E-cadherin and vimentin in bladder cancer cells were detected by Western blot. Cell apoptosis was detected by flow cytometry. In vivo, TCCSUP or SW780 cells were inoculated into BALB/c nude mice to detect tumor volume and weight. Bioinformatics and dual luciferase reporter gene were used to analyze the relationship between LINC00511 and miR-143-3p and its downstream target gene PCMT1. The results showed that LINC00511 could target miR-143-3p/PCMT1 to regulate the proliferation, migration, and apoptosis of bladder cancer TCCSUP or SW780 cells and promote the occurrence and development of bladder cancer.

OCT3/4 enhances tumor immune response by upregulating the TET1-dependent NRF2/MDM2 axis in bladder cancer.

This study aimed to investigate the function of OCT3/4 on tumor immune escape in bladder cancer. Initially, the expression of OCT3/4, TET1, NRF2 and MDM2 was quantified in tumor tissues and cells, followed by gain- or loss-of-function studies to define their roles in cell migration, invasion and apoptosis and tumorigenicity in nude mice. Bladder cancer presented with abundant expression levels of OCT3/4, TET1, NRF2 and MDM2. We found that OCT3/4 promoted TET1 expression via binding to its promoter and that TET1 recruited MLL protein to NRF2 promoter and upregulated its expression, while NRF2 enhanced MDM2 expression. Upregulated MDM2 accelerated tumor immune escape in bladder cancer in mice. OCT3/4 knockdown suppressed the cell migration and invasion while inducing apoptosis, and consequently prevented tumor growth and immune escape in mice. Collectively, OCT3/4 may promote the progression of tumor immune escape in bladder cancer through acting as a promoter of the TET1/NRF2/MDM2 axis.

Enoxacin Exerts Anti-Tumor Effects Against Prostate Cancer Through Inducing Apoptosis.

BACKGROUND: Prostate cancer is the most commonly diagnosed cancer and second leading cause of cancer death in men. Enoxacin, a third-generation fluoroquinolone antibiotic, was found with anti-proliferative effects against many cancer types. This study was to further investigate its effects against prostate cancer and explore the underlying molecular mechanisms. METHODS: PC-3 cells were treated with Enoxacin at different concentrations. Tumor model was established by subcutaneously injecting PC-3 cells into nude mice. MTT assay was used to detect cell viability. ELISA assay, Annexin V/PI staining and TUNEL assay were used to detect apoptosis. RT-qPCR and western blot were used to detect the gene and protein expression, respectively. RESULTS: Our data showed that Enoxacin inhibited PC-3 cell proliferation and induced the apoptosis through up-regulating the expression of pro-apoptotic proteins, while down-regulating expression levels of anti-apoptotic proteins. Moreover, Enoxacin increased the gene and protein expression of the autophagy and endoplasmic reticulum stress markers. Treating tumor-bearing mice with Enoxacin significantly inhibited tumor growth in xenograft tumor model. CONCLUSION: Our results suggested that Enoxacin could be developed as a potential anti-tumor agent against prostate carcinoma.

Exosomes-mediated transfer of long noncoding RNA LINC01133 represses bladder cancer progression via regulating the Wnt signaling pathway.

Bladder cancer (BC), as one of the most common malignant cancers of the urinary system, has a high incidence and mortality rates. Recently, increasing studies have indicated that exosomes can mediate cellular communication in assorted cancers, including BC. Long noncoding RNAs (lncRNAs) have also been confirmed to take part in the regulation of many cancers. Long intergenic non-protein coding RNA 1133 (LINC01133) is an lncRNA and its roles in several cancers have been revealed. However, the functions of exosomes and LINC01133 in BC are still not elucidated. In our research, functional assays were conducted to evaluate the function of LINC01133, as well as the influence of exosomes and LINC01133 on BC cells. Western blot assay, immunofluorescence assay, electron microscope, and nanoparticle tracking analysis were applied for detecting the characteristics of exosomes. Bioinformatics tools and quantitative reverse-transcription polymerase chain reaction were performed to test the expression of LINC01133 in BC cells and exosomes of the immortalized human uroepithelial cell line (SV-HUC-1). Luciferase reporter assay was performed to measure the activity of the Wnt pathway. We discovered that LINC01133 expression was high in exosomes of SV-HUC-1 and low in that of BC cells. Additionally, exosomes restrained cell viability, proliferation, migration, and invasion. Similarly, LINC01133 exerted the same function on BC cells. In addition, the Wnt signaling pathway could be inactivated by LINC01133. Finally, in vivo experiments demonstrated that cell growth could be suppressed by overexpressed LINC01133. In short, exosomes-mediated transfer of lncRNA LINC01133 repressed BC progression via regulating the Wnt signaling pathway.

Anticancer evaluation of a novel dithiocarbamate hybrid as the tubulin polymerization inhibitor.

Novel quinoline-dithiocarbamate hybrids were synthesized and designed by the molecular hybridization strategy. All these derivatives were evaluated for their antiproliferative activity against three selected cancer cell lines (MGC-803, HepG-2 and PC-3). Among them, compound 10c displayed the best antiproliferative activity against PC-3 cells with an IC50 value of 0.43 µM. Celluar mechanisms investigated that compound 10c could inhibit the migration against PC-3 cells by regulation the expression levels of E-cadherin and N-cadherin. Compound 10c induced morphological changes of PC-3 cells and regulated apoptosis-related proteins (Bcl-2, Bax and Cleaved-Parp). In addition, compound 10c inhibited tubulin polymerization in vitro with an IC50 value of 4.02 µM. Importantly, compound 10c inhibited the growth of PC-3 cells in vivo with the low toxicity toward mice. These results suggested that compound 10c might be an antitumor agent with potential for treating prostate cancer.

Additive antitumor effect of arsenic trioxide combined with intravesical bacillus Calmette-Guerin immunotherapy against bladder cancer through blockade of the IER3/Nrf2 pathway.

BACKGROUND: As an inorganic compound used to treat various cancers and other diseases, arsenic trioxide (As2O3) has been reported to induce cellular apoptosis in certain kinds of cancers including bladder cancer. The aim of the present study was to elucidate the crucial cooperative role of As2O3 and intravesical bacillus Calmette-Guerin (BCG) immunotherapy and its ability to protect against bladder cancer by targeting the IER3/Nrf2 pathway. METHOD: Initially, an orthotopic bladder cancer model was established in mice by means of intravesical instillation of the human bladder cancer cell line 5637. The expression of IL-6/IL-8 in dendritic cells (DCs) and the proportion of CD4+ cells and ratio of CD4+/CD8+ T cells were subsequently determined. RT-qPCR and Western blot assay methods were employed to determine the expressions of IER3, Nrf2, NQO1, IL-6 and IL-8. Finally, tumor cell apoptosis and the volume and weight of the in vivo tumors were evaluated in an attempt to determine the contributory role of As2O3 in combination with BCG immunotherapy in treating bladder cancer. RESULTS: The additive effect of As2O3 and BCG was demonstrated to promote the expressions of IL-6/IL-8 among DCs. Additionally, the proportion of CD4+ cells, ratio of CD4+/CD8+ T cells and rate of tumor cell apoptosis were all elevated, while decreased in vivo tumor volume and weight were detected. Of importance, we determined the role that ad-shNrf2 (adenoviral vectors expressing shRNA against Nrf2) played in inhibiting the effects of As2O3 on bladder cancer. CONCLUSION: Taken together, the key findings of the present study provide evidence defining the effect of As2O3 on inducing the inhibitory effect of BCG on the development of bladder cancer via the IER3/Nrf2 pathway, highlighting the potential of As2O3 as a treatment option for bladder cancer through its enhancement of intravesical BCG.