Prognostic Significance of Lineage Diversity in Bladder Cancer Revealed by Single-Cell Sequencing.

Bladder cancer is the most common malignant tumor of the urinary system. We investigated the clinical implications of cell lineages in bladder cancer by integrating single-cell and bulk transcriptome data. By investigating the single-cell transcriptional profiles of 12,424 cells from normal bladder, eleven cell types and five types of epithelial sub-population were identified. Based on the signature of cell types identified in single-cell profiles, deconvolution analysis was employed to estimate cell types and epithelial lineages in the bulk RNA sequencing bladder cancer cohort. Cancer subtypes with clinical implications were further identified based on the heterogeneity of the epithelial lineage across patients. This study suggests that the EMT-like subtype is robustly correlated with poor prognosis and the umbrella subtype is a positive factor for the patient survival. Our research has a high potential for accurate prognostic and therapeutic stratification of bladder cancer.

Identification and Development of Inflammatory Response-Related Genes Signature Associated With Prognosis Evaluation and Immune Status of Bladder Cancer.

Background: Bladder urothelial carcinoma (BLCA) is one of the most common malignant tumors with high morbidity and recurrence rate. The study aims to establish a prediction model to elaborate the relation between inflammatory response and prognosis of BLCA and thus to evaluate the potential prognostic value of inflammatory response-related genes (IRGs) in therapeutic choices. Methods: The study utilized the gene expression profiles from the The Cancer Genome Atlas and Gene Expression Omnibus (GSE32894) datasets. Differentially expressed IRGs between normal and tumor tissues were identified, and 10 of them were correlated with overall survival (OS) (p < 0.05). Then, the LASSO-Cox regression analysis was applied to optimize the signature. RNA sequencing data of patients with BLCA from GSE32894 were applied as a validation set. Cox regression analyses of the seven-gene signature were performed to examine the efficiency of signature in predicting prognosis. Receiver operating characteristic curve analysis was applied to measure the predictive performance of the risk score for OS. Analysis of independent prognostic factors, downstream functional enrichment, drug sensitivity, and immune features were included in this study. Results: The IRG signature (LDLR, ROS1, MMP14, TNFAIP6, MYC, PTGER4, and RIPK2) was used to divide patients into high- and low-risk groups. Cox regression analyses revealed that the risk score was an independent predictive factor. Functional enrichment analysis revealed that genes were enriched in prognosis-related molecular functions and immune-related biological processes. Drug sensitivity and tumor microenvironment correlation analysis indicated that the signature was related to immunotherapy effect. Conclusion: The study defined a new prognostic signature consisting of seven IRGs, which could effectively predict the prognosis of patients with BLCA and reveal relationship of immune features in BLCA with different risk scores. The study also provided a possible indicator for targeted therapy.

Down-regulation of Polo-like kinase 4 (PLK4) induces G1 arrest via activation of the p38/p53/p21 signaling pathway in bladder cancer.

Polo-like kinase 4 (PLK4) has been reported to contribute to tumor growth, invasion, and metastasis. However, the role of PLK4 in human bladder cancer (BC) remains unclear. Here, we demonstrate the regulatory function of PLK4 in human BC progression. PLK4 is overexpressed in BC cell lines and tissues, and its overexpression correlated with poor prognosis. Our transcriptome analysis combined with subsequent functional assays indicated that PLK4 inhibition can suppress BC cell growth and induce cell cycle arrest at G1 phase via activation of the p38/p53/p21 pathway in vitro and in vivo. Overall, our data suggest that PLK4 is a critical regulator of BC cell proliferation, and thus, it may have potential as a novel molecular target for BC treatment.

HSP47 contributes to angiogenesis by induction of CCL2 in bladder cancer.

Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone and is involved in tumor progression by promoting angiogenesis. However, the regulatory network of HSP47 in angiogenesis remains elusive. In this study, we report a novel mechanism of HSP47-induced angiogenesis in bladder cancer (BC). We find that HSP47 is abnormally overexpressed in BC and is correlated with poor prognosis. HSP47 down-regulation suppresses angiogenesis in BC cells. Mechanistically, activation of the ERK pathway and induction of C-C Motif Chemokine Ligand 2 (CCL2) are responsible for HSP47-induced angiogenesis. The correlation between HSP47 with CCL2 and angiogenesis is further confirmed in BC clinical samples. Taken together, our findings suggest that HSP47 contributes to BC angiogenesis by induction of CCL2 and provide a potential anti-angiogenesis target for BC therapy.

CDK7 blockade suppresses super-enhancer-associated oncogenes in bladder cancer.

PURPOSE: Transcriptional addiction plays a pivotal role in maintaining the hallmarks of cancer cells. Thus, targeting super-enhancers (SEs), which modulate the transcriptional activity of oncogenes, has become an attractive strategy for cancer therapy. As yet, however, the molecular mechanisms of this process in bladder cancer (BC) remain to be elucidated. Here, we aimed to provide detailed information regarding the SE landscape in BC and to investigate new potential pharmaceutical targets for BC therapy. METHODS: We employed THZ1 as a potent and specific CDK7 inhibitor. In vitro and in vivo studies were carried out to investigate the anticancer and apoptosis-inducing effects of THZ1 on BC cells. Whole-transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) were performed to investigate the mechanism and function of SE-linked oncogenic transcription in BC cells. RESULTS: We found that THZ1 serves as an effective and potent inhibitor with suppressive activity against BC cells. An integrative analysis of THZ1-sensitive and SE-associated oncogenes yielded potential new pharmaceutical targets, including DDIT4, B4GALT5, PSRC1 and MED22. Combination treatment with THZ1 and the DDIT4 inhibitor rapamycin effectively suppressed BC cell growth. In addition, we found that THZ1 and rapamycin sensitized BC cells to conventional chemotherapy. CONCLUSIONS: Our data indicate that exploring BC gene regulatory mechanisms associated with SEs through integrating RNA-seq and ChIP-seq data improves our understanding of BC biology and provides a basis for innovative therapies.

Targeting TIGIT Inhibits Bladder Cancer Metastasis Through Suppressing IL-32.

Bladder cancer is a highly metastatic tumor and one of the most common malignancies originating in the urinary tract. Despite the efficacy of immune checkpoints, including programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the effect of immunotherapy for bladder cancer remains unsatisfactory. Therefore, it is urgent to develop new targets to expand immunotherapeutic options. In this study, we utilized single-cell sequencing to explore the cell composition of tumors and detected a subset of Treg cells with high expression of T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) and interleukin (IL)-32. The antitumor immune response was suppressed by this subset of Treg cells, while IL-32 promoted bladder cancer metastasis. Nevertheless, targeting TIGIT not only reversed immunosuppression by restoring the antitumor immune response mediated by T cells but also suppressed the secretion of IL-32 and inhibited the metastasis of bladder cancer cells. Thus, our study provided novel insights into immunosuppression in bladder cancer and highlighted TIGIT as a novel target for immunotherapy of bladder cancer. We also illustrated the mechanism of the dual effect of targeting TIGIT and revealed the metastasis-promoting effect of IL-32 in bladder cancer. Collectively, these findings raise the possibility of utilizing TIGIT as a target against bladder cancer from the bench to the bedside.

Multi-Omics Characterization of Tumor Microenvironment Heterogeneity and Immunotherapy Resistance Through Cell States-Based Subtyping in Bladder Cancer.

Due to the strong heterogeneity of bladder cancer (BC), there is often substantial variation in the prognosis and efficiency of immunotherapy among BC patients. For the precision treatment and assessment of prognosis, the subtyping of BC plays a critical role. Despite various subtyping methods proposed previously, most of them are based on a limited number of molecules, and none of them is developed on the basis of cell states. In this study, we construct a single-cell atlas by integrating single cell RNA-seq, RNA microarray, and bulk RNA-seq data to identify the absolute proportion of 22 different cell states in BC, including immune and nonimmune cell states derived from tumor tissues. To explore the heterogeneity of BC, BC was identified into four different subtypes in multiple cohorts using an improved consensus clustering algorithm based on cell states. Among the four subtypes, C1 had median prognosis and best overall response rate (ORR), which characterized an immunosuppressive tumor microenvironment. C2 was enriched in epithelial-mesenchymal transition/invasion, angiogenesis, immunosuppression, and immune exhaustion. Surely, C2 performed the worst in prognosis and ORR. C3 with worse ORR than C2 was enriched in angiogenesis and almost nonimmune exhaustion. Displaying an immune effective environment, C4 performed the best in prognosis and ORR. We found that patients with just an immunosuppressive environment are suitable for immunotherapy, but patients with an immunosuppressive environment accompanied by immune exhaustion or angiogenesis may resist immunotherapy. Furthermore, we conducted exploration into the heterogeneity of the transcriptome, mutational profiles, and somatic copy-number alterations in four subtypes, which could explain the significant differences related to cell states in prognosis and ORR. We also found that PD-1 in immune and tumor cells could both influence ORR in BC. The level of TGFß in a cell state can be opposite to the overall level in the tissues, and the level in a specific cell state could predict ORR more accurately. Thus, our work furthers the understanding of heterogeneity and immunotherapy resistance in BC, which is expected to assist clinical practice and serve as a supplement to the current subtyping method from a novel perspective of cell states.