Chemoresistance Of Bladder Cancer Cells Research Articles

Abstract 5196: Stepwise molecular mechanisms involved in chemoresistance in bladder cancer cells

Abstract Bladder cancer (BC) is one of the most prevalent genitourinary tumors worldwide, with approximately 573,000 new cases reported. Approximately 80% of BC patients are diagnosed with non-muscle-invasive bladder cancer (NMIBC) with a high five-year survival rate, while the remaining 20% are diagnosed with muscle-invasive bladder cancer (MIBC). Although the surgical operation has been utilized to BC, patients with NMIBC frequently experience disease recurrence, of which about 20% progress to MIBC. However, the response rate of MIBC patients to gemcitabine treatment has been shown to be limited to less than 40% efficacy, and only a small number of patients have potential benefits.Chemotherapy resistance is an obstacle to cancer therapy and is considered a major cause of recurrence. Thus, understanding the mechanisms of chemoresistance is critical to improving the prognosis of patients. Here, we have established a stepwise gemcitabine-resistant T24 bladder cancer cell line to understand the molecular mechanisms of chemoresistance within cancer cells. The characteristics of the stepwise chemoresistance cell line were divided into 4 phases (parental, early, intermediate, and late phases). These four phase cells showed increasingly aggressive phenotypes in vitro and in vivo experiments with increasing phases and revealed the molecular properties of the biological process from parent cells to phased gemcitabine-resistant cell line (GRC). Taken together, through the analysis of gene expression profile data, we have characterized gene set of each phase indicating the response to anticancer drug treatment. Specifically, we identified a multigene signature (23-genes including GATA3, APOBEC3G, NT5E, MYC, STC1, FOXD1, SMAD9) and developed a chemoresistance score consisting of that could predict eventual responsiveness to gemcitabine treatment. We suggest that our findings improve prognosis by predicting to acquiring of chemotherapy resistance in bladder cancer patients. Citation Format: Jae-Jun Kim, Gi-Eun Yang, Tae Hwan Shin, Sun-Hee Leem. Stepwise molecular mechanisms involved in chemoresistance in bladder cancer 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 5196.

Abstract 5214: HYAL4 splice variant induces treatment-resistance in advanced bladder cancer

Abstract Introduction and Objective: Precision-based treatment approaches could improve the prognosis of patients with bladder cancer (BC). We recently discovered a first-in-class human Chondroitinase (Chase) that degrades chondroitin sulfate. This Chase is a splice variant of HYAL4 (V1) and promotes a malignant phenotype and chemoresistance in BC cells. We evaluated V1/Chase expression and functions in BC and inhibitors to overcome V1/Chase-mediated Gemcitabine (Gem) resistance. Methods: Cohort 1: 653 voided urine specimens (BC, 160; non-BC, 493); cohort 2: 40 cystectomy specimens from MIBC patients who received adjuvant Gemcitabine plus cisplatin (G+C) treatment. Gene expression was measured by q-PCR and urine was assayed by the Chase test. V1 was expressed in BC cell lines or silenced and the transfectants were analyzed for sensitivity to Gem and Cisplatin. The mechanism of Gem resistance was evaluated in preclinical in vitro and in vivo models. Results: In cohort 1, Chase levels were 7-10-fold elevated in patients with BC compared to patients with benign genitourinary conditions, history of BC, or other cancers. Chase test detected BC with 94.4% and 87.4% specificity. Chase test had 84% accuracy in correctly diagnosing the cases with atypical cytology inference. Stem cell marker CD44 was the major substrate of Chase in BC cells. V1’s Chase activity induced CD44 cleavage and shedding. Intracellularly, CD44 cleavage induced JAK2/Stat3 pathway and subsequently upregulation of cytidine deaminase. CDA induces Gem metabolism and efflux of dFdU, an inactive Gem metabolite. HPLC analysis showed, V1 transfectants have increased levels of dFdU and higher efflux. STAT3 and CDA inhibitors synergistically re-sensitized V1-expressing cells to Gem. In cohort 2, V1, p-JAK2 and CDA expression predicted failure to G+C treatment (P<0.0001). JAK2, STAT3 and CDA inhibitors synergistically re-sensitized V1-expressing cells to Gem. V1-induced muscle invasive tumors that were metastatic. While V1-expressing tumors were resistant, combination of Gem with a CDA or JAK-2 inhibitor abrogated V1 tumor growth with minimal toxicity. Conclusion: V1/Chase is a potential diagnostic and prognostic biomarker for bladder cancer and drives muscle-invasion, metastasis, and Gem resistance in BC. Inhibitors of the Chase signaling pathway overcome V1-induced Gem resistance, suggesting a precision-based treatment approach to improve treatment response. Support: 1R01CA227277-05A1; 1R01CA283699-01 Citation Format: Karina Aguilar, Sunita Panda, Joshua Van Der Eerden, Luis Lopez, Martha Terris, Pablo Santamaria, Bal Lokeshwar, Vinata B. Lokeshwar. HYAL4 splice variant induces treatment-resistance in advanced 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 5214.

NAT10 drives cisplatin chemoresistance by enhancing ac4C-associated DNA repair in bladder cancer.

Epitranscriptomic RNA modifications constitute a critical gene regulatory component that can affect cancer progression. Among these, the RNA N4-acetylcytidine (ac4C) modification, which is mediated by the ac4C writer N-acetyltransferase 10 (NAT10), regulates the stabilization of mRNA. Here, we identified that the ac4C modification is induced upon cisplatin treatment and correlates with chemoresistance in bladder cancer (BCa). Both in vitro and in vivo, NAT10 promoted cisplatin chemoresistance in BCa cells by enhancing DNA damage repair. Mechanistically, NAT10 bound and stabilized AHNAK mRNA by protecting it from exonucleases, and AHNAK-mediated DNA damage repair was required for NAT10-induced cisplatin resistance. Clinically, NAT10 overexpression was associated with chemoresistance, recurrence, and worse clinical outcome in BCa patients. Cisplatin-induced NFκB signaling activation was required for the upregulation of NAT10 expression, and NFκB p65 directly bound to the NAT10 promoter to activate transcription. Moreover, pharmacological inhibition of NAT10 with Remodelin sensitized BCa organoids and mouse xenografts to cisplatin. Overall, the present study uncovered a mechanism of NAT10-mediated mRNA stabilization in BCa, laying the foundation for NAT10 as a therapeutic target to overcome cisplatin resistance in BCa.

Inhibition of SND1 overcomes chemoresistance inbladder cancer cells by promoting ferroptosis.

Chemotherapy remains one of the most important adjuvant treatments for bladder cancer (BC). However, similar to other malignancies, BC is prone to chemotherapy resistance and only approximately half of muscle‑invasive patients with BC respond to chemotherapy. The present study aimed to reveal the mechanisms underlying chemoresistance in BC cells. Cell viabilities were assessed by CCK‑8 assay. The differentiated expression of genes in chemoresistant and their parental BC cells were examined by RNA sequencing. Cell death was determined by flow cytometry. Different cell death inhibitors were used to determine the types of cell death. Levels of reactive oxygen species, iron, glutathione and malondialdehyde were assessed using the corresponding commercial kits. ChIP and dual luciferase activity assays were performed to investigate the interaction between staphylococcal nuclease and tumour domain containing1 (SND1) and glutathione peroxidase4 (GPX4) mRNA. RNAi was used to knockdown SND1 or GPX4. The results revealed that SND1 in BC cells were insensitive to cisplatin, and inhibition of SND1 overcame this resistance. Silencing of SND1 enhanced cell death induced by cisplatin by promoting ferroptosis in BC cells. Mechanistically, SND1 was revealed to bind to the 3'UTR region of GPX4 mRNA and stabilise it. Knockdown of GPX4 could also overcome chemoresistance, and overexpressing GPX4 reversed the effects of silencing of GPX4 on the chemosensitivity of BC cells. Thus, targeting the SND1‑GPX4 axis may be a potential strategy to overcome chemoresistance in BC cells.

Stepwise molecular mechanisms responsible for chemoresistance in bladder cancer cells

Chemotherapy resistance is an obstacle to cancer therapy and is considered a major cause of recurrence. Thus, understanding the mechanisms of chemoresistance is critical to improving the prognosis of patients. Here, we have established a stepwise gemcitabine-resistant T24 bladder cancer cell line to understand the molecular mechanisms of chemoresistance within cancer cells. The characteristics of the stepwise chemoresistance cell line were divided into 4 phases (parental, early, intermediate, and late phases). These four phase cells showed increasingly aggressive phenotypes in vitro and in vivo experiments with increasing phases and revealed the molecular properties of the biological process from parent cells to phased gemcitabine-resistant cell line (GRC). Taken together, through the analysis of gene expression profile data, we have characterized gene set of each phase indicating the response to anticancer drug treatment. Specifically, we identified a multigene signature (23 genes including GATA3, APOBEC3G, NT5E, MYC, STC1, FOXD1, SMAD9) and developed a chemoresistance score consisting of that could predict eventual responsiveness to gemcitabine treatment. Our data will contribute to predicting chemoresistance and improving the prognosis of bladder cancer patients.

Long noncoding RNA TUG1 decreases bladder cancer chemo-sensitivity toward doxorubicin through elevating KPNA2 expression and activating the PI3K/AKT pathway via adsorbing miR-582-5p.

Long noncoding RNA taurine-upregulated gene1 (TUG1) has been reported to be implicated in the chemo-resistance of bladder cancer. Hence, this study aimed to survey regulatory mechanism by which TUG1 regulates the chemo-resistance of bladder cancer cells to doxorubicin (DOX). Relative expression of TUG1, miR-582-5p, and karyopherin alpha 2 (KPNA2) was detected by qRT-PCR. The viability and proliferation of DOX-resistant bladder cancer cells were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Protein levels were measured by western blot analysis. The apoptosis, migration, and invasion of DOX-resistant bladder cancer cells were determined by flow cytometry or transwell assays. The relationship between TUG1 or KPNA2 and miR-582-5p was verified by dual-luciferase reporter assay. TUG1 and KPNA2 were upregulated while miR-582-5p was downregulated in resistant bladder cancer tissues and cells. TUG1 inhibition elevated cell chemo-sensitivity, facilitated cell apoptosis, and curbed proliferation, migration, invasion, and autophagy of DOX-resistant bladder cancer cells. Also, TUG1 acted as a sponge for miR-582-5p, and miR-582-5p inhibitor reversed TUG1 knockdown-mediated influence on DOX chemo-sensitivity and malignant behaviors in DOX-resistant bladder cancer cells. Furthermore, miR-582-5p targeted KPNA2, and KPNA2 overexpression counteracted the inhibitory impact of miR-582-5p mimic on DOX chemo-resistance and malignant behaviors in DOX-resistant bladder cancer cells. Additionally, TUG1 silencing inactivated the PI3K/AKT pathway through sponging miR-582-5p. TUG1 sponged miR-582-5p to increase KPNA2 expression and activated the KPNA2/PI3K/AKT pathway, thereby elevating DOX chemo-resistance and malignant behaviors in bladder cancer cells.

MP03-03 TARGETING V1/CHASE TO OVERCOME GEMCITABINE RESISTANCE

You have accessJournal of UrologyCME1 May 2022MP03-03 TARGETING V1/CHASE TO OVERCOME GEMCITABINE RESISTANCE Semih Sarcan, Daley S. Morera, Marie C. Hupe, Karina Aguilar, Anuj Sharma, Sung Alexander, Joseph McDaniels, Huabin Zhu, Martin Hennig, Martha Terris, Axel S. Merseburger, and Vinata B. Lokeshwar Semih SarcanSemih Sarcan More articles by this author , Daley S. MoreraDaley S. Morera More articles by this author , Marie C. HupeMarie C. Hupe More articles by this author , Karina AguilarKarina Aguilar More articles by this author , Anuj SharmaAnuj Sharma More articles by this author , Sung AlexanderSung Alexander More articles by this author , Joseph McDanielsJoseph McDaniels More articles by this author , Huabin ZhuHuabin Zhu More articles by this author , Martin HennigMartin Hennig More articles by this author , Martha TerrisMartha Terris More articles by this author , Axel S. MerseburgerAxel S. Merseburger More articles by this author , and Vinata B. LokeshwarVinata B. Lokeshwar More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002515.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Precision-based treatment approaches could improve the prognosis of patients with bladder cancer (BC). We recently discovered a first-in-class human Chondroitinase (Chase) that degrades chondroitin sulfate. This Chase is a splice variant of HYAL4 (V1) and promotes a malignant phenotype and chemoresistance in BC cells. We evaluated V1/Chase expression and functions in BC and inhibitors to overcome V1/Chase-mediated Gemcitabine (Gem) resistance. METHODS: Cohort 1: 86 bladder tissues (normal (NBL)=34; tumor (TBL)=52); cohort 2: 40 cystectomy specimens from MIBC patients who received adjuvant Gemcitabine plus cisplatin (G+C) treatment. Cohort 3: 144 urine specimens (BCA=42, non-BCa=102). Gene expression was measured by q-PCR in all cohorts and urine was assayed for Chase activity. V1/Chase was stably expressed or silenced in normal urothelial and three BC cell lines. Transfectants were analyzed for sensitivity to Gemcitabine and Cisplatin. The mechanism of Gem resistance was evaluated in preclinical in vitro and in vivo models. RESULTS: In cohort 1, V1 mRNA levels were an independent predictor of metastasis and death due to BC (P=0.0008). In cohort 2, high V1 levels significantly predicted with G+C treatment failure (P <0.0001). In cohort 3, Chase levels were 13 to 75-fold elevated in low and high-grade BC patients’ urine compared to non-BC cases (92.7% sensitivity; 88.2% specificity). V1-expressing urothelial and BC cells were resistant to Gem but not to Cisplatin. V1 expression increased Gem metabolism and subsequent efflux of an inactive metabolite dFdU. V1 increased CD44 shedding the conditioned media. This triggered intracellular activation of the JAK2/STAT3 pathway, resulting in the upregulation of cytidine deaminase (CDA). CDA induces Gem metabolism and dFdU efflux. JAK2, STAT3 and CDA inhibitors, (some FDA-approved), synergistically re- sensitized V1-expressing cells to Gem. V1-induced muscle invasive tumors that were metastatic. While Gem inhibited BC xenograft growth, V1-expressing tumors were resistant. Combination of Gem with a CDA inhibitor (tetrahydrouridine) abrogated V1 tumor growth with minimal toxicity. CONCLUSIONS: V1 is a functional biomarker that drives muscle-invasion, metastasis and Gem resistance in BC. Inhibitors of the JAK2/STAT3/CDA pathway overcome V1- mediated Gem resistance, suggesting a precision-based treatment approach to improve treatment response. Source of Funding: 1R01CA227277-01A1 (VBL); Department of Defence-PRCRP (W81XWH1810277 (CA170470; PRCRP) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e19 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Semih Sarcan More articles by this author Daley S. Morera More articles by this author Marie C. Hupe More articles by this author Karina Aguilar More articles by this author Anuj Sharma More articles by this author Sung Alexander More articles by this author Joseph McDaniels More articles by this author Huabin Zhu More articles by this author Martin Hennig More articles by this author Martha Terris More articles by this author Axel S. Merseburger More articles by this author Vinata B. Lokeshwar More articles by this author Expand All Advertisement PDF DownloadLoading ...

SPHK1 contributes to cisplatin resistance in bladder cancer cells via the NONO/STAT3 axis

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

Downregulated exosomal microRNA-148b-3p in cancer associated fibroblasts enhance chemosensitivity of bladder cancer cells by downregulating the Wnt/\u03b2-catenin pathway and upregulating PTEN

ObjectiveExosomes derived from cancer-associated fibroblasts (CAFs) are known as important drivers of tumor progression. Previously, microRNA (miR)-148b-3p has been found to be upregulated in bladder cancers as well as in body fluids (blood, urine) of bladder cancer patients. Here, we aimed to explore the role of CAF-derived exosome miR-148b-3p in bladder cancer progression and chemosensitivity.MethodsTranswell, MTT, flow cytometry and colony formation assays were applied to assess the effects of CAF-derived exosomes on bladder cancer cell metastasis, epithelial-mesenchymal transition (EMT) and chemosensitivity. A dual luciferase reporter assay was employed to evaluate the targeting relationship between miR-148b-3p and PTEN. Gain- and loss- of function assays were conducted to explore the roles of miR-148b-3p and PTEN in the behavior of bladder cancer cells. The role of PTEN in the metastasis, EMT and chemosensitivity of bladder cancer cells was assessed both in vivo and in vitro.ResultsWe found that CAF-derived exosomes promoted the metastasis, EMT and drug resistance of bladder cancer cells. We also found that CAF-derived exosomes could directly transport miR-148b-3p into bladder cancer cells. In a xenograft mouse model we found that CAF-derived exosomes increased miR-148b-3p expression levels and promoted tumor proliferation, metastasis and drug resistance. PTEN was validated as a target of miR-148b-3p. Concordantly, we found that PTEN overexpression inhibited EMT, metastasis and chemoresistance in bladder cancer cells, reversing the tumor promoting effects of miR-148b-3p via the Wnt/β-catenin pathway.ConclusionsOur results suggest that miR-148b-3p downregulation in CAF-derived exosomes, thereby inhibiting the Wnt/β-catenin pathway and promoting PTEN expression, may offer potential opportunities for bladder cancer treatment.

Carbosilane Dendrimers Loaded with siRNA Targeting Nrf2 as a Tool to Overcome Cisplatin Chemoresistance in Bladder Cancer Cells.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is considered as the master regulator of antioxidant and cytoprotective gene expressions. Moreover, it plays a pivotal role in cancer progression. Nrf2 mediates the adaptive response which contributes to the resistance to chemotherapeutic pro-oxidant drugs, such as cisplatin (CDDP), in various tumors, including bladder cancers. For this reason, Nrf2 could be a promising target to overcome chemoresistance. There are several known Nrf2 pharmacological inhibitors; however, most of them are not specific. The use of a specific small interfering RNA (siRNA) targeting the Nrf2 gene (siNrf2) loaded into nanovehicles is an attractive alternative, since it can increase specificity. This study aimed to evaluate the biological activity of siNrf2 loaded on guanidine-terminated carbosilane dendrimers (GCDs) in overcoming CDDP resistance in bladder cancer cells with a high level of Nrf2. Parameters such as viability, proliferation, apoptosis, migration, and oxidative stress level were taken into account. Results demonstrated that siNrf2-GCD treatment sensitized CDDP-resistant cells to CDDP treatment. Moreover, data obtained by treating the non-cancerous human kidney HK-2 cell line strongly suggest a good safety profile of the carbosilane dendrimers loaded with siNrf2. In conclusion, we suggest that siNrf2-GCD is a promising drug delivery system for gene therapy to be used in vivo; and it may represent an important tool in the therapy of CDDP-resistant cancer.

&lt;p&gt;Circular RNA circRNA_103809 Accelerates Bladder Cancer Progression and Enhances Chemo-Resistance by Activation of miR-516a-5p/FBXL18 Axis&lt;/p&gt;

BackgroundNumerous researches have suggested that circular RNAs (circRNAs) play critical functions in bladder cancer (BC) progression. This study aims to investigate the potential roles of circRNA_103809 in regulating BC development.MethodsqRT-PCR was used to analyze gene expression. CCK8 and colony formation were used to analyze cell proliferation. Transwell was utilized to examine cell migration and invasion. Gemcitabine was used to analyze the effect of circRNA_103809 on the chemo-resistance of BC cells. Luciferase reporter assay was performed to detect the RNA interactions.ResultscircRNA_103809 was highly expressed in BC tissues and cell lines. CircRNA_103809 high expression was associated with a poor progression in BC patients. CircRNA_103809 knockdown impaired the growth and metastasis of BC cells. Furthermore, circRNA_103809 silencing increased the sensitivity of BC cells to Gemcitabine treatment. CircRNA_103809 was the sponge for miR-516a-5p and promoted FBXL18 expression via restraining miR-516a-5p activity.ConclusioncircRNA_103809 promotes proliferation, migration, invasion and chemo-resistance of BC cells through regulating miR-516a-5p/FBXL18 axis.

MKP-1 overexpression is associated with chemoresistance in bladder cancer via the MAPK pathway.

Mitogen activated protein kinase phosphatase-1 (MKP-1) has been revealed to be overexpressed in bladder cancer, particularly in non-muscle invasive bladder cancer. MKP-1 may also be associated with chemotherapy resistance. However, the underlying mechanism is yet to be elucidated. The current study investigated the expression of MKP-1 by performing immunohistochemistry in surgically resected specimens obtained from primary and recurrent patients with bladder cancer. The results revealed that MKP-1 expression increased in recurrent patients. Additionally, a 3D model of the human bladder cancer cell line, RT112, was established to determine the role of MKP-1 in drug resistance. The results demonstrated that MKP-1 overexpression protected bladder cancer cells against cell death. Contrarily, MKP-1 knockdown was revealed to sensitize cells to death. In addition, the application of MAPK inhibitors effectively increased RT112 cell sensitivity to pirarubicin. In conclusion, the results of the current study indicated that MKP-1 treatment resulted in bladder cancer cell chemoresistance via JNK, ERK and p38 pathways. MKP-1 may also serve as a potential therapeutic target for chemoresistance in patients with bladder cancer.

Forkhead Box R2 Knockdown Decreases Chemoresistance to Cisplatin via MYC Pathway in Bladder Cancer.

BackgroundBladder cancer is a very common urological cancer globally, and cisplatin- or gemcitabine-based chemotherapy is essential for advanced bladder cancer patients. Many patients with bladder cancer have a relatively poor response to chemotherapy, leading to failure of clinical treatment. We mined the GSE77883 GEO dataset, identifying FoxR2 as being a significantly upregulated gene in T24 chemoresistant cells. Herein, we assessed how FoxR2 functions in bladder cancer cell chemoresistance.Material/MethodsCisplatin-resistant T24 (T24/DDP) cells were constructed by administering increasing concentrations of cisplatin, and differences in expression of FoxR2 were examined in T24/DDP and T24 cells. FoxR2 loss- and gain-of-function cells models were established in T24/DDP and T24 cells, respectively. Cell survival, clone formation, cell cycle, and cell apoptosis were assessed, and the MYC pathway was verified.ResultsFoxR2 was significantly upregulated in T24/DDP cells compared to T24 cells. Knockdown of FoxR2 in T24/DDP cells, survival rate, and clone formation were decreased, G1/S phase transition was suppressed, and cell apoptosis was promoted. These results were reversed by restoration of FoxR2 levels in T24 cells. We found that FoxR2 knockdown enhanced sensitivity to cisplatin, whereas MYC overexpression antagonized chemosensitivity in T24/DDP cells.ConclusionsFoxR2 knockdown decreases chemoresistance to cisplatin via the MYC pathway in bladder cancer cells, and this may be a target for overcoming chemoresistance in bladder cancer.

FGFR3S249C mutation promotes chemoresistance by activating Akt signaling in bladder cancer cells.

Fibroblast growth factor receptor 3 (FGFR3) is a high frequency mutant gene in bladder cancer (BCa) and has become a promising therapeutic target due to its involvement in cell proliferation and migration. However, whether and how FGFR3 mutations affects BCa cell chemosensitivity is unknown. The current study aimed to elucidate the role of the FGFR3S249C mutation in the development of chemoresistance in BCa cells. The results revealed that 97-7 (FGFR3S249C) cells had decreased sensitivity to cisplatin compared with 5637 (FGFR3WT) and T24 (FGFR3WT) cells. The ratio of phosphorylated-Akt/total-Akt was higher in 97-7 (FGFR3S249C) cells, which was reversed by knockdown of FGFR3. Furthermore, inhibition of Akt signaling by GDC0068 or LY294002 increased the cisplatin sensitivity of 97-7 (FGFR3S249C) cells. GDC0068 or LY294002 was also revealed to augment the effects of cisplatin on 97-7 (FGFR3S249C) cell proliferation and apoptosis. The results of the present study demonstrated that the FGFR3S249C mutation promotes chemoresistance in BCa cells by activating the Akt signaling pathway. The FGFR3S249C mutation may therefore be used as a predictor of chemosensitivity in patients with BCa.

A Novel Long Non-Coding RNA KMU15 Promotes Growth and Chemoresistance of Bladder Cancer.

Chemotherapy constitutes one of the most important adjuvant treatments for bladder cancer. However, many patients usually develop chemoresistance during chemotherapy. At present, lncRNA has been confirmed not only to be involved in tumorigenesis and progression, but also in tumor chemoresistance. However, the relationship between lncRNAs and chemoresistance of bladder cancer have been rarely reported. The novel lncRNA-KMU15 was screened by lncRNAs microarray and determination of IC50 in bladder cancer. The expression of KMU15 was evaluated by qRT-PCR. The correlation between KMU15 and clinicopathological parameters was analyzed from clinical cases. The effects of KMU15 on the biological behavior and chemoresistance were investigated by [3H]-TdR incorporation assay and other experiments. The effects of KMU15 on the growth of xenograft tumors and the survival of nude mice under cisplatin were examined in a xenograft mouse model. We confirmed that KMU15 was expressed higher in bladder cancer tissues than paired control tissues. Moreover, the expression of KMU15 was significantly positively correlated with the grade, stage, metastasis, and recurrence of bladder cancer and was significantly negatively correlated with the prognosis. In addition, KMU15 knockdown could significantly inhibit bladder cell proliferation, adhesion, migration, and chemoresistance and promoted apoptosis. Knockdown of KMU15 inhibited the growth of xenografts in nude mice and significantly prolonged the survival of tumor-bearing mice under cisplatin. The novel lncRNA KMU15, which is highly expressed in bladder cancer tissues, could promote the proliferation and progression and was closely related to the malignant degree of bladder cancer. It could also significantly enhance the chemoresistance of bladder cancer cells. Therefore, it was expected to be a new therapy target for bladder cancer and a potential prognosis biomarker for chemotherapy.

Comprehensive analysis of microRNA-messenger RNA regulatory network in gemcitabine-resistant bladder cancer cells.

Chemotherapy is still a standard treatment of unresectable bladder cancer or distant metastases. The chemotherapy resistance always occurs after a period of treatment indicating poor prognosis. The current study aimed to explore the molecular mechanism of chemoresistance in bladder cancer cells. The gene expression profiles of GSE77883, including three untreated T24 cells samples and three gemcitabine-resistant T24 cells samples, was downloaded from Gene Expression Omnibus database. The screening of differentially expressed genes (DEGs), gene function analysis, and interaction prediction between microRNAs (miRNAs) and DEGs were performed by R software. The protein-protein interaction (PPI) and miRNA-DEGs networks were constructed and visualized by Cytoscape software. Then, the small molecules, with potential synergistic or antagonistic effects to gemcitabine resistance, were identified using the Connectivity Mapdatabase. Finally, gemcitabine-resistant T24 cell line was established and key genes were validated by quantitative real-time polymerase chain reaction (qRT-PCR). In total, 536 upregulated and 513 downregulated genes were screened and mainly enriched in oxidative stress response and signaling pathways related to extracellular matrix-receptor interaction and focal adhesion. PPI network showed interleukin 6, tumor necrosis factor, kinesin family member 11, and BUB1 mitotic checkpoint serine/threonine kinase B were key genes. The miRNA-DEGs regulatory networks included 18 miRNAs and 185 DEGs, including miR-182-5p, miR-590-3p, miR-320a and serum- and glucocorticoid-regulated kinase 1 (SGK1). Then, the related key genes and miRNAs were confirmed by qRT-PCR. Furthermore, 81 small molecules with antagonistic or synergistic effect to GEM were screened. We have investigated the molecular mechanisms driving GEM-resistance in bladder cancer cells that would contribute to the development of chemotherapy for advanced bladder cancer.

MiR‑214 reduces cisplatin resistance by targeting netrin‑1 in bladder cancer cells.

miR‑214 has been reported to be downregulated in several cancer types, such as bladder cancer. However, its involvement in apoptosis and chemoresistance has not been investigated. The present study aimed to clarify the biological function of miR‑214 and potential mechanisms in chemoresistance of bladder cancer cells. Reverse transcription‑quantitative polymerase chain reaction demonstrated that miR‑214 was downregulated in bladder cancer tissues compared with the level in normal tissues. miR‑214 was downregulated in bladder cancer cell lines compared with the level in the normal cell line SV‑HUC‑1. miR‑214 mimics were transfected into T24 and J82 cell lines to restore its expression. The results indicated that miR‑214 mimic inhibited proliferation and invasion in these cell lines. In addition, miR‑214 mimic reduced cisplatin resistance in T24 and J82 cells, indicated by the inhibition of cell viability and upregulation of cell apoptosis. Western blotting demonstrated that miR‑214 mimic was able to upregulate cleaved caspase‑3 and cleaved poly (ADP‑ribose) polymerase (PARP), while downregulate caspase‑3 and PARP expression, and AKT phosphorylation. Using prediction software, it was revealed that the netrin‑1 oncoprotein is on the target list of miR‑214. miR‑214 also downregulated netrin‑1 protein and mRNA expression levels in the T24 and J82 cell lines. Luciferase reporter assays demonstrated that netrin‑1 acted as a direct target of miR‑214. A negative correlation between netrin‑1 and miR‑214 expression in bladder cancer tissues was also observed. In addition, cisplatin treatment could induce netrin‑1 protein expression in bladder cancer cells and miR‑214 mimic partly blocked this phenomenon. Netrin‑1 plasmid transfection inhibited cisplatin‑induced apoptosis, upregulated AKT phosphorylation, and downregulated caspase‑3 and PARP cleavage. Netrin‑1 was restored in cells transfected with miR‑214 mimic using plasmid transfection. Netrin‑1 transfection restored AKT phosphorylation and blocked caspase/PARP cleavage in the T24 and J82 cell lines. In conclusion, the present study demonstrated that miR‑214 is downregulated in bladder cancer tissues and cell lines. miR‑214 reduces chemoresistance by targeting netrin‑1 in bladder cancer cell lines.

LASS2 regulates invasion and chemoresistance via ERK/Drp1 modulated mitochondrial dynamics in bladder cancer cells.

Mitochondria coordinated a lot of vital cellular processes of energy production and distribution. Change of mitochondrial functions has been implicated in cancer progression. The present study aims to investigate the involvement of mitochondria dynamics in LASS2 induced invasion and chemoresistance of bladder cancer cells. J82 and BIU87 cell lines were used for LASS2 plasmid transfection while siRNA knockdown was carried out in 5637 cell line. Matrigel invasion assay and Annexin V/PI staining demonstrated that LASS2 negatively regulated cancer cell invasion and chemoresistance. JC-1 staining suggested that LASS2 overexpression downregulated mitochondrial membrane potential. Mitotracker staining showed that LASS2 induced mitochondrial fusion and inhibited mitochondrial fission. In addition, LASS2 overexpression downregulated expression of mitochondrial fission protein p-Drp1 Drp1 and Fis1. While depletion of LASS2 exhibited the opposite effects. Drp1 inhibitor Mdivi abolished invasion and chemoresistance induced by LASS2 siRNA. Furthermore, we found that LASS2 overexpression could inhibit phosphorylation of ERK, which act upstream of Drp1. ERK inhibitor PD98059 suppressed Drp1 phosphorylation and abrogated the effects of LASS2 depletion. In conclusion, the present study demonstrated that LASS2 inhibits bladder cancer invasion and chemoresistance through regulation of ERK-Drp1 induced mitochondrial dynamics.

MicroRNA-34a Attenuates Metastasis and Chemoresistance of Bladder Cancer Cells by Targeting the TCF1/LEF1 Axis

Background/Aims: Chemoresistance is largely responsible for relapses of bladder cancer during clinical therapy. However, the molecular mechanisms involved in the chemoresistance of bladder cancer are unclear. Growing evidence supports the theory that microRNAs (miRNAs) play an important role in chemotherapeutic drug resistance because they are downregulated in many malignancies that have been implicated in the regulation of diverse processes in cancer cells. More specifically, the extent and precise mechanism of the involvement of miR-34as in chemoresistance to epirubicin (EPI) in the treatment of bladder cancer remains unclear. Methods: In this study, real-time quantitative polymerase chain reaction (PCR) was used to analyze the expression of miR-34a in bladder cancer cell line BIU87 and its EPI chemoresistant cell line BIU87/ADR. The miR-34a profiles in bladder cancer tissues were obtained from The Cancer Genome Atlas database. The effect of miR-34a on chemosensitivity was evaluated by cell viability assays, colony formation assays, and in vivo experimentation. Apoptosis and the cell cycle were examined by flow cytometry. A luciferase reporter assay was used to assess the target genes of miR-34a. Western blot and qPCR were used to analyze the expression of target proteins and downstream molecules. Results: The downregulation of miR-34a in bladder cancer serves as an independent predictor of reduced patient survival. The CCK-8 assay showed that miR-34a overexpression resulted in increased sensitivity to EPI, while miR-34a downregulation resulted in chemoresistance to EPI in vitro. Moreover, it was found that miR-34a increased the sensitivity of BIU87/ADR cells to chemotherapy in vivo. The luciferase reporter assay ascertained that TCF1 and LEF1 are direct target genes of miR-34a. It was found that miR-34a increased chemosensitivity in BIU87/ADR cells by inhibiting the TCF1/LEF1 axis. Conclusions: The results of this study indicate that miR-34a contributes to the chemosensitivity of BIU87/ADR by inhibiting the TCF1/LEF1 axis. Consequently, miR-34a is a determinant of BIU87 chemosensitivity and may therefore serve as a potential therapeutic target in bladder cancer treatment.

Heterogeneous nuclear ribonucleoprotein K is associated with poor prognosis and regulates proliferation and apoptosis in bladder cancer.

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an essential RNA‐ and DNA‐binding protein that regulates diverse biological events, especially DNA transcription. hnRNPK overexpression is related to tumorigenesis in several cancers. However, both the expression patterns and biological mechanisms of hnRNPK in bladder cancer are unclear. We investigated hnRNPK expression by immunohistochemistry in 188 patients with bladder cancer, and found that hnRNPK expression levels were significantly increased in bladder cancer tissues and that high‐hnRNPK expression was closely correlated with poor prognosis. Loss‐ and gain‐of‐function assays demonstrated that hnRNPK promoted proliferation, anti‐apoptosis, and chemoresistance in bladder cancer cells in vitro, and hnRNPK knockdown suppressed tumorigenicity in vivo. Mechanistically, hnRNPK regulated various functions in bladder cancer by directly mediating cyclin D1, G0/G1 switch 2 (G0S2), XIAP‐associated factor 1, and ERCC excision repair 4, endonuclease catalytic subunit (ERCC4) transcription. In conclusion, we discovered that hnRNPK plays an important role in bladder cancer, suggesting that it is a potential prognostic marker and a promising target for treating bladder cancer.