BCa Cells Research Articles

Abstract 506: TRIP13 is a promising therapeutic target for bladder cancer

Abstract Bladder cancer (BCa) is the second most common genitourinary cancer. High recurrence rate, progression to MIBC and poor prognosis in metastatic disease are major challenges for this disease management. Therapeutic options for advanced stage BCa are very limited, hence developing agents to treat this disease progression are highly needed. Thyroid receptor-interacting protein 13 (TRIP13), a member of the AAA-ATPase family, regulates various cellular processes such as DNA repair and spindle assembly. TCGA data analysis showed significant overexpression of TRIP13 that correlates with disease stage and mortality. Recent studies have shown that TRIP13 amplification occurs in several types of human cancers, including BCa, and it regulates cell proliferation, tumor growth, and drug resistance. However, the role of TRIP13 in BCa and its potential as a therapeutic target remain elusive. Therefore, in the present study we performed a preclinical investigation of the TRIP13 role in BCa progression, underlying molecular mechanisms, and develop promising therapeutics against BCa. Initially we profiled TRIP13 expression in 10 different bladder cancer cell lines from a panel of human and rodent origin and found elevated protein expression in multiple BCa cell lines. TRIP13 levels were also significantly elevated in the BBN induced rat- and SV40T driven mouse- bladder tumor tissues compared to normal bladder tissues. Pharmacological inhibition of TRIP13 with a potent drug DCZ0415 significantly suppressed cell proliferation (IC50 ~7μM) in J82, NBT-II and MB49 cell lines and induced cell death. DCZ0415 treatment led to cell cycle arrest at G1 phase (P<0.001) in BCa cells. In mechanistic investigations on its effect on cell cycle progression by DCZ0415 treatment decreased the expression of CDK4, CDK6, Cyclin D1, and PCNA protein expression. TRIP13 inhibition also suppressed the migration and invasion potential of BCa cells. Annexin V and γ-H2AX staining suggested that DCZ0415 induced apoptosis in BCa cells and increased DNA damage. These findings were also complemented by an increase in cleaved caspase-3 and PARP proteins. We also observed modulation of cGAS-STING signaling pathway in BCa cells with DCZ0415 treatment, indicating potential for its combination with cGAS-STING stimulators. Importantly, we found that TRIP13 inhibition with DCZ0415 significantly suppressed the growth of 3D BCa models as well BBN-induced rat BCa tumoroids and spheroids. Overall, these results suggest that TRIP13 could be a potential therapeutic target for prevention of bladder cancer and warrants further validation in in vivo models (Funding supported by P30CA225520 and ACS-IRG grant). Citation Format: Surya Pratap Singh, Krishnendu Goswami, Adam S. Asch, Chinthalapally V. Rao, Venkateshwar Madka. TRIP13 is a promising therapeutic target for bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 506.

Abstract 261: Akt1 and Cyclin D1 govern polarity of self-renewing divisions in breast cancer stem cells

Abstract Background. Stem-like cells contribute to the development and progression of human cancers. Genes that control symmetric cell divisions have an evolutionarily conserved role in the regulation of cell polarity and in tumorigenesis. Two cells generated by asymmetric divisions have reduced proliferative potential compared with symmetric divisions. The cell survival oncoprotein Akt (Protein Kinase B) is encoded by three isoforms that are altered in human cancers. The Akt isoform-specific impact on breast cancer stem cell function and polarity is controversial, with Akt1 either reducing (1) or promoting (2) cell polarity and migration in tissue culture. Deletion of Akt1 reduced mammary tumor progression and metastasis in transgenic mice (2,3). Methods. Analysis of Akt1, cyclin D1 and Notch signaling was conducted from public databases, and human as well as murine breast cancer samples. Cells derived from Akt1 gene deletion MMTV-ErbB2 breast oncomice (MMTV-ErbB2/Akt1+/+ and MMTV-ErbB2/Akt1−/−) and from the human MCF-7 breast cancer cell line with regulated expression of cyclin D1 and p53 were analyzed for the symmetry of cell division, stem cell function, and signaling pathways. Results. In breast tumors derived from MMTV-ErbB2 transgenics, associated with reduced metastasis, Akt1 genetic deletion reduced the frequency of symmetric cell divisions and the production of mammospheres. Akt1 deletion increased p53 and reduced cyclin D1. Pharmacological reactivation of p53 with Nutlin correlated with the reinduction of asymmetric cellular divisions and reduction in mammospheres in murine and human BCa cells. Akt1 deletion abrogated Nutlin-mediated induction of asymmetric cell divisions. Cyclin D1 induced symmetrical cell divisions, mammosphere formation and reduced p53 and Numb. Conclusions. Akt1 promoted symmetrical cell division at least in part via cyclin D1 suppression of Numb, and induction of γ secretase activity and thereby Notch signaling. Reference. (1) Irie HY, Pearline RV, Grueneberg D, Hsia M, Ravichandran P, Kothari N, et al. Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-mesenchymal transition. J Cell Biol 2005;171:1023-34. (2) Ju X, Katiyar S, Wang C, Liu M, Jiao X, Li S, et al. Akt1 governs breast cancer progression in vivo. Proc Natl Acad Sci U S A 2007;104:7438-43. (3) Chen X, Ariss MM, Ramakrishnan G, Nogueira V, Blaha C, Putzbach W, et al. Cell-Autonomous versus Systemic Akt Isoform Deletions Uncovered New Roles for Akt1 and Akt2 in Breast Cancer. Mol Cell 2020;80:87-101 e5 Citation Format: Xuanmao Jiao, Xin Sun, Zhiping Li, Xiaoming Ju, Danni Li, Ritika Harish, Gordon Robertson, Anthony W. Ashton, Richard G. Pestell. Akt1 and Cyclin D1 govern polarity of self-renewing divisions in breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 261.

Abstract 292: Cell non-autonomous downregulation of the tumor suppressor DAB2IP in the cancer microenvironment: Molecular mechanism and biological implications

Abstract Background and Purpose: The tumor suppressor DAB2IP is a cytoplasmic Ras-GAP and an adaptor protein that restrains oncogenic signaling by extracellular inputs, contributing to modulate the dynamic crosstalk established between tumor cells and their stromal compartment. In human malignancies, DAB2IP is frequently disabled by epigenetic and post-transcriptional means. DAB2IP loss-of-function in cancer cells promotes their aberrant proliferation, dissemination, and tumor-promoting inflammation, potentially favor progression of tumors. Intriguingly, endothelial-specific DAB2IP depletion in mouse models fosters the formation of a pro-metastatic niche, implicating that also the loss-of-function of DAB2IP in stromal cells might dramatically impact cancer progression. We previously found that the secretome of prostate cancer cells reduced DAB2IP levels in non-transformed prostate epithelial cell lines, and in primary endothelial cells, thereby fostering their proliferation, migration and expression of angiogenesis markers. Accordingly, cell non-autonomous DAB2IP inactivation in the stromal compartment may reprogram the response to paracrine and autocrine signals, thus supporting a tumor-promoting behavior; however, molecular basis and clinical implication of this phenomenon await clarification. Materials and Methods: We used conditioned medium (CM) from prostate (PCa) and breast (BCa) cancer cell lines, to study its effects in mediating DAB2IP downregulation and in inducing tumor-associated phenotypes in endothelial cell and fibroblasts. Through multiple approaches, we isolated extracellular vesicles from PCa and BCa conditioned media, to test their involvement in mediating DAB2IP downregulation in recipient cells. Finally, we used cancer cells’CM or specific siRNAs to study the consequence of DAB2IP loss in fibroblasts. Results: The treatment with CM from PCa and BCa cells induces DAB2IP downregulation in immortalized and in primary fibroblasts, and in endothelial cells. Analysis of DAB2IP levels in primary fibroblasts from PCa and BCa surgical samples confirmed differential expression of DAB2IP protein in normal vs. tumor-associated fibroblasts. Exploring the biochemical nature of extracellular signals involved in DAB2IP downregulation by cancer cells’ CM, we found that DAB2IP inhibition in recipient cells is partially mediated by miR-149-3p contained in extracellular vesicles. Finally, we found that DAB2IP reduction potentiates fibroblasts proliferation and invasion, facilitates gel contraction and extracellular matrix remodeling, and fosters expression of markers of activated fibroblasts. Conclusions: Together, these data support the hypothesis that cancer cells can modulate DAB2IP levels in nearby stromal cells, pushing them to acquire a metastasis-supporting phenotypes. Citation Format: Arianna Bellazzo, Rossella De Florian Fania, Chiara Agostinis, Roberta Bulla, Ilenia Segatto, Barbara Belletti, Vito G. D’Agostino, Ian M. Bonapace, Licio Collavin. Cell non-autonomous downregulation of the tumor suppressor DAB2IP in the cancer microenvironment: Molecular mechanism and biological implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 292.

Circular STAG2 RNA Modulates Bladder Cancer Progression via miR-145-5p/TAGLN2 and Is Considered as a Biomarker for Recurrence.

The current study aimed to elucidate the regulatory mechanisms of the circRNA hsa_circ_0139697 (circSTAG2(16-25)) in BCa and to consider the opportunity of using circSTAG2(16-25) isolated from BCa patient urine as a marker for disease development prediction. The selection of this circRNA was determined by the special role of its parental gene STAG2 in BCa biology. The circRNA hsa_circ_0139697 was chosen from 25 STAG2 circRNAs due to its differential expression in the urine of BCa patients and healthy volunteers. Higher levels of circSTAG2(16-25) were detected in urine samples obtained from patients with recurrent tumors. A higher expression of circSTAG2(16-25) was also detected in more tumorigenic BCa cell lines. The overexpression of circSTAG2(16-25) in BCa cells induced the elevation of proliferation, motility, and invasion. To study the mechanisms of circSTAG2(16-25) activity, we confirmed that circSTAG2(16-25) can bind miR-145-5p in vitro as was predicted by bioinformatic search. miR-145-5p was shown to suppress some genes that promoted BCa progression. One of these genes, TAGLN2, encodes the protein Transgelin 2, which plays a role in BCa cell motility and invasion. Therefore, the possible mechanism of action of circSTAG2(16-25) could be sponging the tumor suppressor miR-145-5p, which results in activation of TAGLN2. In addition, circSTAG2(16-25) might be considered as a potential biomarker for recurrence prediction.

Taraxasterol stimulates apoptosis of BCa cells via restraining PI3K/AKT and epidermal growth factor receptor (EGFR)

Taraxasterol stimulates apoptosis of BCa cells via restraining PI3K/AKT and epidermal growth factor receptor (EGFR)

Bioevaluation of spiro N-propargylic β-enaminones as anti-breast cancer agents: Cell cycle and molecular docking studies.

The study aimed to investigate the in vitro inhibitory activities of spiro N-propargylic β-enaminones (SPEs) (1-31) against breast cancer cells (BCa). Cytotoxic activity was investigated via MTT assay. DNA fragmentation was evaluated via ELISA assay. Cell cycle distributions were investigated by flow cytometry. Expression levels of Bcl-2, Bax, p21 and Cyclin D1 were measured by qRT-PCR and western blot analysis. Autodock/vina software was used to understand the interaction between the SPEs and the ERα, PR, EGFR, and Her2. ADMETlab 2.0 tool was used to determine the drug-likeness properties. Notably, SPEs 1, 22, and 28 displayed selective cytotoxicity and apoptosis against BCa cells. They arrested the cell cycle at the S phase, inducing p21 and Cyclin D1. SPE1, SPE22, and SPE28 exhibited strong affinity for BCa receptors and drug-like properties. Pending in vivo validation with safety assurance for human health, these compounds hold potential as chemotherapy candidates.

Circ_0000235 targets MCT4 to promote glycolysis and progression of bladder cancer by sponging miR-330-5p

Warburg effect plays a crucial role in bladder cancer (Bca) development. However, the mechanism by which glycolysis is involved in Bca remains poorly understood. CircRNAs commonly play a regulatory role in tumor progression. Our study discovered and identified a novel circRNA, hsa_circ_0000235 (circ235), and investigated its role in the glycolytic process, which further results in the progression of Bca. We applied qRT-PCR to assess its clinicopathological relevance and evaluated its proliferation, migration, and glycolytic capacity. We investigated its mechanism using RNA immunoprecipitation, dual-luciferase reporters, and fluorescence in situ hybridization. The findings demonstrated that circ235 was dramatically increased in Bca tissues and was related to a worse prognosis. In vitro studies revealed that circ235 accelerated the rate of extracellular acidification and promoted glucose uptake and lactate manufacture in Bca cells. Additionally, it strengthened the proliferative and migratory capacities. Experiments on animals revealed that downregulating circ235 dramatically reduced carcinogenesis and tumor growth. Circ235 activates monocarboxylate transporter 4 (MCT4) by sponging miR-330-5p, which promotes glycolysis and tumor growth. In conclusion, these findings suggest that circ235 may be a viable molecular marker and therapeutic target for Bca.

Recent Progress on Urinary Exosomal miRNAs in Bladder Cancer

Bladder cancer (Bca) is one of the most common tumors in the urinary system. Cystoscopy biopsy is a gold standard for diagnosis of Bca in the clinic. However, cystoscopy biopsy is invasive, costly and invasive process causing pain in the patients. Because the Bca cells are closely in contact with urine, and the exosomes of the Bca can pass through the base membrane and transport the microRNA (miRNAs) to the urine, so the detection of the exosome-derived miRNAs in the urine is expected to be a new non-invasive method for diagnostics of Bca. Recently, liquid biopsy for miRNAs in urine is the recent research hotspot. In this review, we mainly introduce the feasibility of the application of the miRNAs from exosome to Bca, and its feasibility to detect Bca.

Abstract 1802: Personalization of oncolytic virus therapy: Heterogenous anti-tumor responses in preclinical prostate and bladder cancer models

Abstract Clinical studies revealed that single-agent immunotherapies yield limited efficacy in the management of prostate and bladder cancer (PCa, BCa). PCa and BCa often induce immunosuppressive and immune evasion mechanisms that restrict anti-tumor responses. Oncolytic viruses (OVs) offer promising and safe therapeutic options in the treatment of cancer as OVs target and or preferentially replicate in tumor cells, potentially evoking systemic anti-tumor immune responses. Combination of immunotherapy with OVs may, therefore, unleash the full potential of immunotherapy for these cancers. To achieve beneficial anti-tumor responses a more personalized OV therapy approach seems crucial. Strikingly, only few studies describe head-to-head comparison of the effects of different OVs. Together with our partners in the oncolytic viro-immunotherapy (OVIT) consortium we are developing and evaluating multiple OVs for their abilities to induce oncolysis and subsequent immunomodulation. Furthermore, we are developing tools to predict oncolytic virus effectiveness and design strategies to personalize oncolytic virus therapy. The OVs that were generated by our consortium encompass the spontaneous Orthoreovirus mutant (jin-3), a non-human primate Adenovirus (AdV-lumc007dRbD), and a Newcastle Disease Virus variant (NDV-F0).In this study we determined the anti-tumor and immunomodulatory effects of selected OVs in multiple ‘near-patient and patient-derived human PCa and BCa models; 1) tumor cell line cultures, 2) cocultures of tumor cell lines and monocyte-derived dendritic cells (mo-DCs), and 3) ex-vivo cultured tumor tissue slices and explants from patient material or patient-derived xenograft (PDX) models. The selected OVs infected a panel of PCa and BCa cell lines and displayed heterogenous effects on tumor cell viability and expression of immunogenic cell death markers (HSP90, Calreticulin, and HMGB1). For instance, the jin-3 virus showed enhanced tumor tropism in most PCa cells, while AdV-lumc007dRbD was more efficient in BCa cells. Virus infection of tumor cells resulted in maturation and activation of moDCs. Furthermore, OVs were able to infect and replicate in ex-vivo cultured tumor tissue slices and viability of ex-vivo tumor tissue explants was reduced following viral inoculation. Transcriptomic analysis of infected patient tissues revealed heterogenous responses to the tested OVs.Taken together, we established and compared the oncolytic properties of various OVs in multiple preclinical models of PCa and BCa. The data indicate heterogeneity of response for the tested OVs in urological cancers. In order for OV therapy to reach robust clinical efficacy, better appreciation of underlying molecular and cellular mechanisms is crucial for the personalization of oncolytic viro-immunotherapy. Citation Format: Thijs J. Janssen, Geertje van der Horst, Arjanneke F. van der Merbel, Maaike van der Mark, Martine L. Lamfers, Bernadette G. van den Hoogen, Sjoerd S. van der Burg, Dana A. Mustafa, Niven Mehra, Tilly Aalders, Nadine van Montfoort, Jack Schalken, Rob C. Hoeben, Gabri van der Pluijm. Personalization of oncolytic virus therapy: Heterogenous anti-tumor responses in preclinical prostate and bladder cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1802.

Abstract 547: Role of KDM4A in bladder cancer

Abstract Bladder cancer (BCa) is the most common malignancy of the urinary system in the United States. Most patients present with non-muscle invasive bladder cancer (NMIBC) that is treated with bladder preserving approaches. However, recurrence is high with a 50% chance to progression into muscle invasive bladder cancer (MIBC). Patients with MI disease are treated with aggressive surgery and/or systemic chemo-radiation, and more recently immune- and targeted therapies. Patients with BCa require life-long surveillance with expensive imaging, and repeated treatment courses, making BCa one of the most expensive malignancies throughout the patients’ lifespan. BCa therapies have been stagnant for almost four decades until 2016 when new immune and targeted therapies were approved for patients with locally advanced, recurrent, or metastatic disease, with an overall response of 15-29%. Thus, better understanding of the pathobiology of BCa is needed to identify actionable targets and develop novel therapeutics. In this respect, we have identified histone lysine demethylase 4A (KDM4A), also known as Jumanji D2A (JMJD2A) as a BCa onco-protein. We found that the expression of nuclear KDM4A significantly correlated with disease stage in patients’ tumors and in tobacco carcinogen-induced murine bladder tumors. KDM4A transcript expression significantly increased in bladder tumors compared to normal in TCGA data and three independent publicly available datasets. Knockdown of KDM4A in BCa cell lines significantly inhibited their proliferation and invasiveness. Interestingly, KDM4A transcripts exhibited significant inverse correlation with BCa tumor suppressor SPARC in five independent datasets. In addition, using phenotypic screening and computational modeling, we found that KDM4A is a direct target of verteporfin, a potent inducer of SPARC expression in BCa cell lines. Mechanistically, knockdown of KDM4A in BCa cell lines increased SPARC expression, whereas its overexpression inhibited basal and verteporfin-induced SPARC expression. Chromatin immune-precipitation (ChIP) assays indicated that verteporfin significantly inhibited the association of KDM4A with SPARC promoter. Together, our data underscore the oncogenic role of KDM4A in BCa and its potential as an actionable target. Citation Format: Bailey O. Warren, Ammar Y. Abdelfattah, Alia Ghoneum, Freddie Salsbury, Neveen A. Said. Role of KDM4A in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 547.

The Role of Customer Satisfaction and Service Quality on Customer Loyalty: Case Study of M-Banking Services

The cause of this look at is to investigate the direct impact of provider first-class on customer satisfaction of mobile banking customers, to analyze the direct effect of carrier exceptional on customer loyalty of cellular banking users, to investigate the direct effect of patron pleasure on purchaser loyalty of mobile banking customers and to analyze the indirect effect of carrier exceptional of client loyalty through consumer pleasure of bank BCA cell banking users. The population in this study is 1,050 customers using mobile banking. The sample in this study was taken purposively. The number of samples is 91 people. The studies device used on this study became a questionnaire or questionnaire that turned into made by means of the researcher himself. The studies device is a tool used to degree an observed natural or social phenomenon. The primary data used in this study is data obtained based on answers to questionnaires distributed to customers. The conclusion from the study is that there is a significant influence between service quality variables on customer satisfaction for customers who use mobile banking, so that improving the quality of online services will have an impact on customer satisfaction to use mobile banking. There is a significant influence between the variables of customer satisfaction on customer loyalty, the better the online service quality, the greater the loyalty level of mobile banking application users. There may be a sizeable have an impact on between the variables of customer delight on client loyalty, the higher the level of customer delight, the higher the level of loyalty of mobile banking application users

PHF6 recruits BPTF to promote HIF-dependent pathway and progression in YAP-high breast cancer

BackgroundAberrant epigenetic remodeling events contribute to progression and metastasis of breast cancer (Bca). The specific mechanims that epigenetic factors rely on to mediate tumor aggressiveness remain unclear. We aimed to elucidate the roles of epigenetic protein PHF6 in breast tumorigenesis.MethodsPublished datasets and tissue samples with PHF6 staining were used to investigate the clinical relevance of PHF6 in Bca. CCK-8, clony formation assays were used to assess cell growth capacity. Cell migration and invasion abilities were measured by Transwell assay. The gene mRNA and protein levels were measured by quantitative real-time PCR and western blot. Chromatin immunoprecipitation (ChIP)-qPCR assays were used to investigate transcriptional relationships among genes. The Co-immunoprecipitation (Co-IP) assay was used to validate interactions between proteins. The CRISPR/Cas9 editing technology was used to construct double HIF knockout (HIF-DKO) cells. The subcutaneous xenograft model and orthotopic implantation tumor model were used to asess in vivo tumor growth.ResultsIn this study, we utilized MTT assay to screen that PHF6 is required for Bca growth. PHF6 promotes Bca proliferation and migration. By analyzing The Cancer Genome Atlas breast cancer (TCGA-Bca) cohort, we found that PHF6 was significantly higher in tumor versus normal tissues. Mechanistically, PHF6 physically interacts with HIF-1α and HIF-2α to potentiate HIF-driven transcriptional events to initiate breast tumorigenesis. HIF-DKO abolished PHF6-mediated breast tumor growth, and PHF6 deficiency in turn impaired HIF transcriptional effects. Besides, hypoxia could also rely on YAP activation, but not HIF, to sustain PHF6 expressions in Bca cells. In addition, PHF6 recuits BPTF to mediate epigenetic remodeling to augment HIF transcriptional activity. Targeting PHF6 or BPTF inhibitor (AU1) is effective in mice models. Lastly, PHF6 correlated with HIF target gene expression in human breast tumors, which is an independent prognostic regulator.ConclusionsCollectively, this study identified PHF6 as a prognostic epigenetic regulator for Bca, functioning as a HIF coactivator. The fundamental mechanisms underlying YAP/PHF6/HIF axis in breast tumors endowed novel epigenegtic targets for Bca treatment.

Knockdown of pyruvate kinase M2 suppresses bladder cancer progression

Abstract Objectives Bladder cancer (BCa) is one of the most frequently diagnosed cancers of the urinary tract and has a high mortality. The M2 splice isoform of pyruvate kinase (PKM2) is a key regulator of the Warburg effect in cancer cells. This study aimed to evaluate metabolic alterations and biological behaviours after knocking down PKM2. Methods In this study, 36 pairs of BCa tissues and adjacent normal tissues were collected to analyse the expression level of PKM2 and to explore the relationship between PKM2 level and tumour and patient status. After PKM2 knockdown in T24 cells, cell survival, migration, invasion, glucose uptake, lactate production, and apoptosis were detected. The tumour-forming ability of PKM2-reducing T24 cells was examined in vivo. Results The results showed that PKM2 expression correlates with BCa stage and grade. PKM2 knockdown decreases glucose consumption and lactate production and suppresses cell proliferation, migration, and invasion while increasing reactive oxygen species levels and apoptosis in T24 BCa cells in vitro. In nude mouse models, PKM2 knockdown reduced xenograft and orthotopic tumour size. Moreover, PKM2 knockdown decreased vimentin and fibronectin expression and increased E-cadherin expression. Analysis of high-throughput sequencing data revealed that PKM2 may also be associated with biological processes and diseases. Conclusions Overall, these results indicate that PKM2 may be a therapeutic target for BCa patients.

Epigenetic regulator KDM4A activates Notch1-NICD-dependent signaling to drive tumorigenesis and metastasis in breast cancer

BackgroundAltered epigenetic reprogramming and events contribute to breast cancer (Bca) progression and metastasis. How the epigenetic histone demethylases modulate breast cancer progression remains poorly defined. We aimed to elucidate the biological roles of KDM4A in driving Notch1 activation and Bca progression. MethodsThe KDM4A expression in Bca specimens was analyzed using quantitative PCR and immunohistochemical assays. The biological roles of KDM4A were evaluated using wound-healing assays and an in vivo metastasis model. The Chromatin Immunoprecipitation (ChIP)-qPCR assay was used to determine the role of KDM4A in Notch1 regulation. ResultsHere, we screened that targeting KDM4A could induce notable cell growth suppression. KDM4A is required for the growth and progression of Bca cells. High KDM4A enhances tumor migration abilities and in vivo lung metastasis. Bioinformatic analysis suggested that KDM4A was highly expressed in tumors and high KDM4A correlates with poor survival outcomes. KDM4A activates Notch1 expressions via directly binding to the promoters and demethylating H3K9me3 modifications. KDM4A inhibition reduces expressions of a list of Notch1 downstream targets, and ectopic expressions of ICN1 could restore the corresponding levels. KDM4A relies on Notch1 signaling to maintain cell growth, migration and self-renewal capacities. Lastly, we divided a panel of cell lines into KDM4Ahigh and KDM4Alow groups. Targeting Notch1 using specific LY3039478 could efficiently suppress cell growth and colony formation abilities of KDM4Ahigh Bca. ConclusionTaken together, KDM4A could drive Bca progression via triggering the activation of Notch1 pathway by decreasing H3K9me3 levels, highlighting a promising therapeutic target for Bca.

PSMD2 promotes the progression of bladder cancer and is correlated with immune infiltration.

PSMD2 plays an oncogenic role in multiple human malignancies, while it is still unclear that the potential roles and underlying mechanisms of PSMD2 in BCa. The RNA-seq from TCGA and GTEx database was utilized to preliminarily analyze the expression of PSMD2 in BCa tissues, qRT-PCR was adopted to verify the PSMD2 expression in BCa cell lines. Cox regression analyses were applied to assess the prognostic values of PSMD2 in BCa. GSEA analysis was used to explore the underlying mechanisms of PSMD2. In vitro assays such as wound healing and colony formation assays were applied to determine the carcinogenesis of PSMD2 in BCa. xCell and ssGSEA algorithms were applied to analyze the associations of PSMD2 with TIME. The results revealed that in comparison with normal bladder tissues and cell line, PSMD2 was found to be significantly elevated in BCa tissues and cell lines. Elevated expression of PSMD2 can independently predict unfavorable OS for BCa patients. The PSMD2 expression and other clinicopathologic factors were combined to develop a nomogram, which can help to predict OS for BCa patients. GSEA analyses revealed that PSMD2 is correlated with the cell cycle, antigen processing and presentation, JAK-STAT signaling pathway, Toll like receptor signaling pathway, P53 and MAPK signaling pathway. Knockdown of PSMD2 could remarkably inhibit the wound healing and colony formation efficiency of BCa cells. xCell analysis revealed that overexpressed PSMD2 is positively related to the Th2 cells infiltrates and expression levels of immune escape markers, and negatively associated with the infiltrating levels of NK T cell and CD8+ T cell. In conclusion, overexpressed PSMD2 is tightly linked to the immune infiltrates and promotes the progression of BCa.

HOXA1 promotes proliferation and metastasis of bladder cancer by enhancing SMAD3 transcription.

Recent studies showed that HOXA1 can promote or suppress the transcription of target genes via binding to their promoter region, therefore regulating the development and progression of various cancers. However, the biological function of HOXA1 in bladder cancer (Bca) remains unknown. qRT-PCR and Western blot assay was performed to measure the mRNA protein level of HOXA1 in Bca cells. CCK-8 and cell colony formation assay were carried out to detect cell proliferation ability. Wound healing assay was applied to detect cell migration ability, while transwell assay was applied to detect cell invasion ability. Chromatin Immunoprecipitation (ChIP) and dual-luciferase reporter assay were used to investigate the molecular mechanisms underlying HOXA1. In this study, we discovered that HOXA1 mRNA and protein was dramatically increased in Bca tissues and cells compared to matched normal tissues and normal bladder epithelial cell. Enhanced HOXA1 expression was positively correlated with bigger tumor size and lymphatic metastasis, causing shorter overall survival to Bca patients. Knockdown of HOXA1 obviously impaired cell proliferation and metastasis ability. Further experiments proved that HOXA1 could strength the transcription of SMAD3 via binding to the promoter region of SMAD3. In conclusion, our study suggested that HOXA1 contributed to the growth and metastasis of Bca and it might serve as a tumor biomarker for Bca treatment and prognosis monitoring.

TFAP2C Knockdown Sensitizes Bladder Cancer Cells to Cisplatin Treatment via Regulation of EGFR and NF-κB.

Simple SummaryBladder cancer (BCa) is considered one of the most common neoplasms of the urology system. Cisplatin-based chemotherapy has been the primary treatment for patients with advanced or metastatic BCa. Nevertheless, cisplatin resistance often limits the treatment of bladder cancer. We expect to find approaches to improve the therapeutic efficacy of cisplatin in bladder cancer. In recent years, many studies have shown that transcription factor AP-2 gamma (TFAP2C) acts as a key player in cancer development and and its expression level is closely related to the sensitivity of tumors to cisplatin. Our study investigated whether TFAP2C affects the sensitivity of BCa cells to cisplatin and the possible mechanisms. We found that TFAP2C expression was significantly upregulated in most BCa tissues compared to adjacent normal tissues. The present study confirmed that TFAP2C knockdown enhanced the anti-tumor effects of cisplatin by decreasing cisplatin-induced activation levels of epidermal growth factor receptor (EGFR) and nuclear factor kappaB (NF-κB). Specifically, this study provides a novel approach to improve the efficacy of cisplatin.Cisplatin is the first-line chemotherapy for advanced or metastatic bladder cancer. Nevertheless, approximately half of patients with BCa are insensitive to cisplatin therapy or develop cisplatin resistance during the treatment process. Therefore, it is especially crucial to investigate ways to enhance the sensitivity of tumor cells to cisplatin. Transcription factor AP-2 gamma (TFAP2C) is involved in cancer development and chemotherapy sensitivity. However, its relationship with chemotherapy has not been studied in BCa. In this study, we aimed to investigate the therapeutic potential of TFAP2C in human BCa. Results based on TCGA (The Cancer Genome Atlas), GTEx (The Genotype-Tissue Expression) and GEO (Gene Expression Omnibus) data showed that TFAP2C expression was upregulated in BCa tissues and that its high expression was associated with poor prognosis. Meanwhile, we demonstrated the overexpression of TFAP2C in BCa clinical specimens. Subsequently, in vitro, we knocked down TFAP2C in BCa cells and found that TFAP2C knockdown further increased cell cycle arrest and apoptosis caused by cisplatin. In addition, the inhibitory effect of cisplatin on BCa cell migration and invasion was enhanced by TFAP2C knockdown. Our data indicated that cisplatin increased epidermal growth factor receptor (EGFR) and nuclear factor-kappaB (NF-κB) activation levels, but TFAP2C knockdown suppressed this effect. Finally, in vivo data further validated these findings. Our study showed that TFAP2C knockdown affected the activation levels of EGFR and NF-κB and enhanced the anti-tumor effects of cisplatin in vivo and in vitro. This provides a new direction to improve the efficacy of traditional cisplatin chemotherapy.

SMAR1 inhibits proliferation, EMT and Warburg effect of bladder cancer cells by suppressing the activity of the Wnt/β-catenin signaling pathway

ABSTRACT This study aimed to investigate the effects of scaffold matrix attachment region binding protein 1 (SMAR1) on the development of bladder cancer (BCa). SMAR1 expression in paired tumor and corresponding adjacent normal tissues from 55 BCa patients was detected by quantitative reverse transcription-polymerase chain reaction. BCa cells were transfected to regulate SMAR1 expression. BCa cells were treated with XAV-939, LiCl and 2-deoxyglucose. The effect of SMAR1 on the viability, proliferation, migration, invasion and Warburg effect of BCa cells was researched by counting kit-8, colony formation assay, Transwell and aerobic glycolysis assays. Western blot was performed to detect protein expression. BCa cell growth in vivo was recorded in nude mice. Immunohistochemical staining was performed for clinical and xenografted tumor tissue specimens. SMAR1 expression was down-regulated in BCa patients, associating with worse prognoses. SMAR1 knockdown enhanced the viability, proliferation, migration, invasion, EMT and Warburg effect of BCa cells. The opposite effect was found in the SMAR1 overexpression BCa cells. XAV-939 treatment reversed the elevation of β-catenin, c-Myc and Cyclin D1 proteins expression and Warburg effect in Bca cells post-SMAR1 knockdown. LiCl treatment abrogated the inhibition of β-catenin, c-Myc and Cyclin D1 proteins expression and Warburg effect proteins due to SMAR1 overexpression in BCa cells. SMAR1 overexpression inhibited the growth of BCa cells in vivo. SMAR1 might suppress the Wnt/β-catenin signaling pathway activity to inhibit the progression of BCa. It might be an effective treatment target for BCa.

Abstract 5675: Pathological crosstalk between osteocytes and breast cancer cells in bone metastasis

Abstract Bone provides an ideal niche for breast cancer cells (BCa) to reside and proliferate and is a frequent site of BCa metastasis. Osteocytes are the most abundant skeletal cells and permanent residents of bone. However, the role of osteocytes in BCa bone metastasis remains understudied. Here, we investigated the crosstalk between metastatic BCa cells and osteocytes. In vitro cultures revealed that conditioned media (CM) derived from metastatic E0771 BCa cells decreased by 50% the number of alive MLO-A5 and MLO-Y4 osteocyte-like cell lines. BCa cells modestly increased osteocyte apoptosis (2% vs. 6%, alone vs. exposed to BCa, respectively). Treatment with DEVD, a caspase 3 inhibitor, blocked osteocyte apoptosis but did not prevent the decrease in osteocytes induced by BCa. Thus, we studied osteocyte senescence, a process that causes irreversible cell cycle arrest and profound changes in gene expression. We found that BCa-CM upregulated the expression of markers of senescence markers (p16, p21) and senescence-associated secretory phenotype (Mmp13, Mmp9, Vcam-1) in osteocyte-like cell lines. Similarly, BCa-CM increased the expression of senescent markers in ex vivo bone organ cultures containing primary osteocytes. Osteocytes increased BCa cell proliferation by increasing cyclin D1 expression Through both cell-to-cell interactions and secretion of soluble factors., osteocytes increased BCa cell proliferation by increasing cyclin D1 expression. Moreover, osteocyte-derived CM stimulated BCa cells migration and invasion capabilities and upregulated the expression of genes involved in migration (Vcam-1 and N-cadherin). Co-culture with stromal cells also stimulated BCa cell proliferation, invasion, and migration. However, the increases induced by stromal cells were four-fold lower than those seen with osteocytes. Overall, our results suggest that BCa cells reprogram osteocytes, which acquire a senescent phenotype, whereas osteocytes produce factors that stimulate tumor growth, migration, and invasion. Together, these findings support the existence of a crosstalk between osteocytes and BCa cells in the metastatic niche that favors the progression of breast cancer in bone. Future studies are warranted to examine the contribution of osteocyte senescence and osteocytes-derived factors in animal models of breast cancer metastasis. Citation Format: Manish Adhikari, Hayley Sabol, Aric anloague, Sharmin Khan, Jesus Delgado-Calle. Pathological crosstalk between osteocytes and breast cancer cells in bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5675.

Abstract 3991: Ts65Dn Down syndrome mice reveal a potential therapeutic vulnerability in a BBN bladder cancer model and subset of bladder cancer cell lines

Abstract Trisomy 21, Down Syndrome, results in a constellation of symptoms including increased risk for leukemia. In contrast, mounting evidence indicates that people with Down Syndrome have lower rates of numerous solid tumors, particularly carcinogen driven cancers, even when adjusted for age. The mechanisms that dictate why this occurs are not well understood but could potentially yield actionable targets for treating or preventing cancer formation. We hypothesized Ts65Dn mice, a murine model of Down Syndrome, would be protected against BCa formation and could be evaluated as a genetic basis for the discovery of novel therapeutic targets in bladder cancer. Using the well characterized N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) chemical carcinogen bladder cancer model we initiated tumors in Ts65Dn and WT mice. After 16 weeks BBN exposure, TS65Dn mice were found to have smaller bladder tumors by weight with a trend toward lower stages as compared to WT animals. RNA-sequencing was used to assess broad genomic changes in the tumors. Reactome analysis of the RNA-sequencing data indicated pathways associated with mitosis and the cell cycle were most affected in Ts65Dn mice, including downregulation of pathways associated with Aurora Kinase B (AURKB). AURKB is a pro-tumorigenic kinase known to enhance cell survival and proliferation. Bioinformatics analysis indicates that while AURKB is broadly expressed in most tissues, it is potently upregulated in bladder cancers, offering a potential therapeutic target. Using the AURKB specific inhibitor barasertib-HQPA we evaluated drug sensitivity in a set of BCa cell lines. Cell lines associated with a basal subtype gene signature (5637, HT-1376) were largely susceptible to barasertib with IC50 values in nM concentrations, whereas other BCa cell lines (HTB-5, T24) had IC50 values nearly 1,000X higher. We next assessed histone H3 phosphorylation, a known substrate of AURKB, to validate drug efficacy. Interestingly, inhibition of histone H3 phosphorylation was noted in all cell lines at nM concentrations, indicating that while barasertib is functionally active in both sets of cells, the anti-proliferative activity is not dependent upon phosphorylation of histone H3, but rather other factors that we have yet to determine. In conclusion, we have found Ts65Dn mice have reduced tumor formation when exposed to the BCa carcinogen BBN and furthermore, have identified AURKB as a potential therapeutic target. Use of Ts65Dn mice may be a relevant model for identifying potentially undefined tumorigenic agents in multiple cancers including bladder. Citation Format: Binh Vo, Erika Abbott, Carolyn J. Vivian, Xena Moore, Ganeshkumar Rajendran, Katie Dennis, Benjamin L. Woolbright, Scott J. Weir, John A. Taylor. Ts65Dn Down syndrome mice reveal a potential therapeutic vulnerability in a BBN bladder cancer model and subset of bladder cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3991.