RT4 Bladder Cancer Cells Research Articles

Abstract PR008: Modulating the PPARγ pathway to augment NECTIN4-targeting chimeric antigen receptor (CAR) T cell therapy

Abstract Background: We present the development of NECTIN4-CAR T cells targeting bladder cancer and evaluate the ability of thiazolidinedione, a class of anti-diabetic drugs, to specifically upregulate NECTIN4 expression and expand the therapeutic window of NECTIN4-CAR T cells. In addition, we assess efficacy of NECTIN4-CAR T cell therapy against a model of bladder cancer with acquired resistance to enfortumab vedotin (EV). Methods: NECTIN4-targeting chimeric antigen receptor (CAR) constructs were designed and cloned into a second-generation EQ-28ζ CAR backbone construct. Human primary T cells were transduced with lentiviral NECTIN4-CAR constructs and co-cultured with a panel of bladder cancer cell lines with a range of NECTIN4 expression and against NECTIN4 knockout cells to assess specificity. Tumor cell count over time was monitored in vitro using an IncuCyte S3. Bladder cancer cell lines (RT112, UMUC-1, HT1197) were treated with PPARγ agonists (rosiglitazone) and antagonists (T0070907), and total and surface NECTIN4 protein levels were assessed. Anti-tumor effects of the NECTIN4-CAR T were evaluated in vitro and in vivo, either with or without rosiglitazone pre-treatment. Finally, RT112 bladder cancer cells were exposed to “cycles” of escalating doses of EV to generate EV-resistant cell line models, and anti-tumor efficacy against EV-resistant lines was evaluated. Results: NECTIN4-targeting CAR T cells displayed potent and NECTIN4-specific cytotoxic activity against bladder cancer cell lines. The efficacy of NECTIN4-CAR T cells was strongly correlated with NECTIN4 expression. Treatment with PPARγ agonists and antagonists was shown to upregulate and downregulate NECTIN4 expression, respectively. Treatment with the PPARγ agonist rosiglitazone induced NECTIN4 expression in a PPARγ-dependent and dose responsive manner. NECTIN4 expression was increased in bladder cancer xenografts established in immunodeficient NSG mice that were treated with systemic rosiglitazone. Priming with rosiglitazone enhanced sensitivity to NECTIN4-CAR T cells in vitro, and the combination of rosiglitazone pre-treatment and NECTIN4-CAR T cell therapy showed enhanced tumor control in bladder cancer xenograft models. We generated RT112 cells that exhibited resistance to EV in vitro and in vivo, which retained NECTIN4 expression. Interestingly, EV-resistant RT112 cells remained sensitive to NECTIN4-CAR T cell therapy both in vitro and in vivo. Conclusions: We engineered a NECTIN4-targeting CAR T cell therapy that is highly antigen-specific and demonstrates antitumor activity against multiple bladder cancer models, including EV resistant cells. Modulating the PPARγ pathway increases NECTIN4 expression, which we leveraged by repurposing an FDA-approved anti-diabetes drug rosiglitazone to increase targeting and anti-tumor efficacy of NECTIN4-CAR T cells. These preclinical results lay the groundwork for CAR T therapy development in bladder cancer and suggest rational drug combinations that can expand the therapeutic window of NECTIN4-targeting therapies. Citation Format: Kevin Chang, Henry M. Delavan, Jun Zhu, Corynn Kasap, Elizabeth Yip, Roshan Lodha, Sheng-You Liao, Sima Porten, Terence Friedlander, Vadim Koshkin, Felix Feng, Arun Wiita, John Lee, Carissa E. Chu, Jonathan Chou. Modulating the PPARγ pathway to augment NECTIN4-targeting chimeric antigen receptor (CAR) T cell therapy [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR008.

Antibody-dye Conjugates Targeting EGFR and HER2 for the Photoimmunotherapy of Bladder Cancer.

Although there are curative treatment options for non-muscle-invasive bladder cancer, the recurrence of this tumor is high. Therefore, novel targeted therapies are needed for the complete removal of bladder cancer cells in stages of localized disease, in order to avoid local recurrence, to spare bladder cancer patients from stressful and expensive treatment procedures and to increase their quality of life and life expectancy. This study tested a new approach for the photoimmunotherapy (PIT) of bladder cancer. We generated a cysteine modified recombinant version of the antibody cetuximab targeting the epidermal growth factor receptor (EGFR) on the surface of bladder cancer cells. Then, we coupled the novel photoactivatable phthalocyanine dye WB692-CB1 via a maleimide linker to the free cysteines of the antibody. PIT was performed by incubating bladder cancer cells with the antibody dye conjugate followed by irradiation with visible red light. The conjugate was able to induce specific cytotoxicity in EGFR-positive bladder cancer cells in a light dose-dependent manner. Enhanced cytotoxicity in RT112 bladder cancer cells was evoked by addition of a second antibody dye conjugate targeting HER2 or by repeated cycles of PIT. Our new antibody dye conjugate targeting EGFR-expressing bladder cancer cells is a promising candidate for the future PIT of bladder cancer patients.

Abstract 5640: Unlocking the therapeutic potential of SF3B1 inhibitors for bladder cancer treatment

Abstract RNA splicing dysregulation is a hallmark of cancer, occurring in multiple solid and hematological malignancies. Splicing factor 3 subunit B1 (SF3B1), a key component of the U2 small nuclear ribonucleoprotein (snRNP) complex that regulates RNA splicing, is frequently altered in a variety of cancer types, including non-muscle-invasive and muscle-invasive bladder cancer, where the E902K mutation is common. In this study, we investigated the therapeutic potential of the SF3B1 inhibitor, pladienolide B, in bladder cancer using in vitro models. We used a recombinant plasmid containing the SF3B1E902K mutation and established an UMUC3E902K mutant urothelial cell line. Both UMUC3WT and UMUC3E902K cells were treated with pladienolide B alone or in combination with cisplatin to evaluate differences in cell viability. Flow cytometry using annexin V staining and propidium iodide exclusion was utilized to assess the cell death mechanism. RNAseq was performed after RNA extraction from UMUC3WT, UMUC3E902K, RT4, and HT1376 bladder cancer cell lines were treated with pladienolide alone or combined with cisplatin. We determined that the IC50 for pladienolide B is 5nM for UMUC3 cells observed 72 hours post-treatment. Treatment with pladienolide B halted the cell cycle and caused cell death by apoptosis for both UMUC3WT and UMUC3E902K cell lines. We also observed that UMUC3E902K cells were significantly more sensitive to pladienolide B compared to UMUC3WT cells (p≤0.05). Furthermore, there was a synergistic effect observed after co-treatment with low-dose pladienolide B (2 nM) and cisplatin (3 µM) for both UMUC3WT and UMUC3E902K cell lines, with UMUC3E902K cells showing greater sensitivity to combination treatment. Our results provide insights into the potential use of splicing inhibitors, alone or in combination with chemotherapy, for treating bladder cancer, specifically for tumors that bear driver mutations in splicing factors. Currently, we are evaluating the mechanism by which SF3B1 mutations make cells sensitive to pladienolide B in vitro and in vivo, with the goal of identifying specific mRNA splicing vulnerabilities that can be targeted by anti-sense or splice-switching oligonucleotides to achieve anti-tumor outcomes. Additional differential splicing patterns and preliminary in vivo results will be presented. Citation Format: Rod Carlo Columbres, Arup Chakraborty, Andrea Apolo, Rouf Banday. Unlocking the therapeutic potential of SF3B1 inhibitors for bladder cancer treatment [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 5640.

Abstract 4366: Loss of EP300 triggers IL-1α signaling and subsequent activation of the IL-6/JAK/STAT3 axis to drive oncogenesis in bladder cancer

Abstract Bladder Cancer (BLCA) is the sixth most common cancer in the United States with 81,180 new cases and 17,100 deaths in 2022. Next Generation Sequencing of BLCA has revealed an enrichment of alterations in genes classified as chromatin modifiers compared to other solid tumors. Among these is EP300, the gene encoding the p300 histone acetyltransferase, which is mutated in ~12% of BLCA with the majority putative loss of function frameshift and nonsense mutations. To investigate the biological role of EP300 loss-of-function in BLCA pathogenesis, we generated isogenic EP300 null RT112 bladder cancer cells using a 2-step CRISPR knockout (KO) and knockin strategy. EP300 KO cells demonstrated increased proliferation, anchorage independent cell growth, tumor formation in mice, and enhanced invasive potential. To investigate the mechanism(s) whereby EP300 loss-of-function enhances oncogenicity, we performed an unbiased Tandem Tag Mass Spectrometry (TMT MS) screen to identify pathways dysregulated in EP300 KO versus parental cells. EP300 loss-of-function was found to induce IL-6/JAK/STAT3 signaling via upregulated expression of IL-1α, a canonical pro-inflammatory cytokine. Knock down of IL1A gene expression was sufficient to downregulate STAT3 activation in EP300 KO cells, whereas ectopic stimulation using IL-1α increased STAT3 activation in parental RT112 cells, suggesting that IL-1α was the predominant upstream activator of JAK/STAT3 signaling in our model. Pharmacological inhibition of downstream targets of IL-1α signaling such as IRAK1/4 were also able to inhibit STAT3 signaling in EP300 KO cells in a dose and time dependent manner. Further investigations indicated that activated IL-1α signaling not only drove transcriptional upregulation of IL-6 but also the production of soluble IL-6R, activating IL-6 trans signaling. Finally, to elucidate how IL-1α upregulation is triggered by EP300 loss-of-function, we performed immunoprecipitation and immunofluorescence assays, which revealed that IL-1α binds directly to p300. Additionally, ectopic p300 expression in EP300 KO clones dramatically reduced IL-1α protein expression, indicating that p300 acts as a negative regulator of IL-1α in a bladder cancer context. In summary, EP300 loss-of-function relieves p300’s transcriptional and/or physical-tethering inhibition on IL-1α signaling, subsequently activating the IL-6/JAK/STAT3 pathway to drive oncogenesis in BLCA. Citation Format: James A. Rodrigues, Jiaqian Luo, Amanda R. Sabel, Ziyu Chen, Xinran Tang, Fengshen Kuo, Hikmat Al-Ahmadie, Kwanghee Kim, Eugene Pietzak, Gopa Iyer, David B. Solit, Sizhi P. Gao. Loss of EP300 triggers IL-1α signaling and subsequent activation of the IL-6/JAK/STAT3 axis to drive oncogenesis in 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 4366.

Pluronic F-127-coated ZnO nanoparticles as superior photosensitizers for effective bladder cancer photodynamic therapy: In-vitro evaluation

Bladder cancer is the most prevalent malignancy of the urinary system and ranks among the top ten most common cancers in humans. Photodynamic therapy (PDT) demonstrates significant promise in cancer treatment due to its high selectivity and minimal side effects. Zinc oxide nanoparticles (ZnO NPs) exhibit substantial potential in various biomedical applications, including cancer therapy. This study systematically investigates the potential of bare and Pluronic F-127-coated ZnO NPs as photosensitizers (PSs) for effective photodynamic therapy against the RT4 bladder cancer cell line, marking the first such exploration. Transmission electron microscopy (TEM) micrographs reveal the formation of a Pluronic F-127 coating layer with thickness ranging from approximately 3 nm–7 nm on the particle surface. The Pluronic coating process significantly enhances the cytotoxicity of ZnO NPs under dark conditions and during photodynamic therapy on the RT4 bladder cancer cell line. Additionally, the impact of PDT using bare and coated ZnO NPs is further evaluated through a 3D spheroid assay, which better replicates the real tumor environment for photodynamic therapy. Hoechst and rhodamine assays confirm the induction of apoptosis, characterized by nuclear condensation and disruption of mitochondrial membrane potential in the cells. This study advances the potential of ZnO NPs in bladder cancer photodynamic therapy and highlights the importance of Pluronic F-127 coatings, paving the way for more effective and targeted cancer treatments.

Sulforaphane Inhibits Adhesion and Migration of Cisplatin- and Gemcitabine-Resistant Bladder Cancer Cells In Vitro.

Only 20% of patients with muscle-invasive bladder carcinoma respond to cisplatin-based chemotherapy. Since the natural phytochemical sulforaphane (SFN) exhibits antitumor properties, its influence on the adhesive and migratory properties of cisplatin- and gemcitabine-sensitive and cisplatin- and gemcitabine-resistant RT4, RT112, T24, and TCCSUP bladder cancer cells was evaluated. Mechanisms behind the SFN influence were explored by assessing levels of the integrin adhesion receptors β1 (total and activated) and β4 and their functional relevance. To evaluate cell differentiation processes, E- and N-cadherin, vimentin and cytokeratin (CK) 8/18 expression were examined. SFN down-regulated bladder cancer cell adhesion with cell line and resistance-specific differences. Different responses to SFN were reflected in integrin expression that depended on the cell line and presence of resistance. Chemotactic movement of RT112, T24, and TCCSUP (RT4 did not migrate) was markedly blocked by SFN in both chemo-sensitive and chemo-resistant cells. Integrin-blocking studies indicated β1 and β4 as chemotaxis regulators. N-cadherin was diminished by SFN, particularly in sensitive and resistant T24 and RT112 cells, whereas E-cadherin was increased in RT112 cells (not detectable in RT4 and TCCSup cells). Alterations in vimentin and CK8/18 were also apparent, though not the same in all cell lines. SFN exposure resulted in translocation of E-cadherin (RT112), N-cadherin (RT112, T24), and vimentin (T24). SFN down-regulated adhesion and migration in chemo-sensitive and chemo-resistant bladder cancer cells by acting on integrin β1 and β4 expression and inducing the mesenchymal-epithelial translocation of cadherins and vimentin. SFN does, therefore, possess potential to improve bladder cancer therapy.

Validation of hyperthermia as an enhancer of chemotherapeutic efficacy: insights from a bladder cancer organoid model

Objective This study aimed to evaluate the combined efficacy of hyperthermia and chemotherapy using a bladder cancer organoid model and to explore hyperthermia-related molecular pathways. Method Tumor organoids were generated by embedding RT4 bladder cancer cells into Matrigel. The resulting organoids were treated with pirarubicin or gemcitabine at 37 °C or 42 °C. Proliferation was determined by Ki67 immunofluorescence staining, and apoptosis was assessed using a TdT-mediated dUTP nick end labeling (TUNEL) assay. RNA sequencing was used to identify the differentially expressed genes. Results Bladder cancer organoids were successfully established and exhibited robust proliferative abilities. Treatment with gemcitabine or pirarubicin under hyperthermic conditions caused pronounced structural damage to the organoids and increased cell death compared to that in the normothermically treated group. Furthermore, Ki67 labeling and TUNEL assays showed that the hyperthermia chemotherapy group showed a significantly reduced proliferation rate and high level of apoptosis. Finally, RNA sequencing revealed the IFN-γ signaling pathway to be associated with hyperthermia. Conclusion Overall, hyperthermia combined with chemotherapy exerted better therapeutic effects than those of normothermic chemotherapy in grade 1-2 non-muscle-invasive bladder cancer, potentially through activation of the IFN-γ-JAK-STAT pathway.

Targeting FGFRs by pemigatinib induces G1 phase cell cycle arrest, cellular stress and upregulation of tumor suppressor microRNAs

BackgroundFibroblast growth factor receptor (FGFR) gene family alterations are found in several cancers, indicating their importance as potential therapeutic targets. The FGFR-tyrosine kinase inhibitor (TKI) pemigatinib has been introduced in the treatment of advanced cholangiocarcinoma and more recently for relapsed or refractory myeloid/lymphoid neoplasms with FGFR2 and FGFR1 rearrangements, respectively. Several clinical trials are currently investigating the possible combination of pemigatinib with immunotherapy.In this study, we analyzed the biological and molecular effects of pemigatinib on different cancer cell models (lung, bladder, and gastric), which are currently objective of clinical trial investigations.MethodsNCI-H1581 lung, KATO III gastric and RT-112 bladder cancer cell lines were evaluated for FGFR expression by qRT-PCR and Western blot. Cell lines were treated with Pem and then characterized for cell proliferation, apoptosis, production of intracellular reactive oxygen species (ROS), and induction of senescence. The expression of microRNAs with tumor suppressor functions was analyzed by qRT-PCR, while modulation of the proteins coded by their target genes was evaluated by Western blot and mRNA. Descriptive statistics was used to analyze the various data and student’s t test to compare the analysis of two groups.ResultsPemigatinib exposure triggered distinct signaling pathways and reduced the proliferative ability of all cancer cells, inducing G1 phase cell cycle arrest and strong intracellular stress resulting in ROS production, senescence and apoptosis. Pemigatinib treatment also caused the upregulation of microRNAs (miR-133b, miR-139, miR-186, miR-195) with tumor suppressor functions, along with the downregulation of validated protein targets with oncogenic roles (c-Myc, c-MET, CDK6, EGFR).ConclusionsThese results contribute to clarifying the biological effects and molecular mechanisms mediated by the anti-FGFR TKI pemigatinib in distinct tumor settings and support its exploitation for combined therapies.

Deep convolutional neural network accurately classifies different types of bladder cancer cells based on their pH fingerprints and morphology

Non-invasive identification of different populations of cells at the single-cell level carries significant biomedical implications. We recently developed a novel fast and cost-effective method that, combining pH imaging and machine learning, enabled us to classify normal and cancer cells based on their different intracellular acidity. Here, we sought to capture and utilize intracellular structural features, known to undergo changes during cancer progression, in addition to intracellular pH patterns in order to make robust predictions. Leveraging both the biophysical and biochemical markers acquired via pH imaging with deep learning allowed us to classify cancer cells, at single-cell resolution, with very high accuracy. Specifically, the deep Convolutional Neural Network (CNN)-based strategy classified individual cells from the RT4 and J82 bladder cancer cell lines with an accuracy of 99.9%, compared to 94% achieved with our previously reported pH-based method.

Abstract 6135: Dynamics of ligand-induced epidermal growth factor receptor internalization in cancer cell lines

Abstract Antibody-drug conjugates (ADCs), comprised of cancer cell-specific antibodies linked to cytotoxic drugs, represent an innovative and promising class of anticancer agents. Due to its amplified expression in various tumor types, the epidermal growth factor receptor (EGFR) presents an attractive target for ADC therapy, and three anti-EGFR ADCs are currently studied in clinical trials. As ADCs depend on receptor internalization for intracellular release of drugs in the targeted cancer cells, studying the dynamics of receptor internalization can aid in their development and optimization. We studied the internalization of EGFR induced by its ligand EGF, as a mimic of ADCs, in cell lines with different surface receptor densities. BxPC-3 (pancreas) and RT-112 (bladder) cancer cells were seeded in 6-well plates and allowed to adhere for 24 hours. Cells were serum-starved overnight, followed by stimulation with 150 ng/mL EGF for 2, 4, 6, 24 and 48 hours. Internalization was studied by quantification of the number of surface-expressed receptors using flow cytometry with Quantibrite PE beads and EGFR-PE antibody. Moreover, incorporation of the ligand-receptor complex into acidic endo-lysosomes was studied using EGF conjugated to the fluorescent pH sensor pHrodo™ Green. Total EGFR and EGFR phosphorylation levels were assessed by western blot analysis. RNA was isolated and converted into cDNA to perform TaqMan qPCR. Unstimulated BxPC-3 cells showed higher numbers of EGFR per cell compared to unstimulated RT-112 cells. Since high receptor expression is key for some ADCs to induce effective endocytosis, this may indicate BxPC-3 cells as a more attractive model to study EGFR internalization. Stimulation with EGF induced a strong decrease in surface receptor density for both BxPC-3 and RT-112 cells during the first two hours, suggesting receptor internalization. Accordingly, treatment of BxPC-3 cells with pHrodo™ Green EGF demonstrated the incorporation of ligand-receptor complexes into endo-lysosomes. During following hours, the cell surface expression of EGFR remained stable, whereas it partially recovered after 24 to 48 hours. In contrast, the lysosomal incorporation of EGF-EGFR complexes was not decreased at 24 or 48 hours, suggesting that the increase in surface EGFR may be due to de novo protein synthesis rather than recycling. No increase of EGFR mRNA was, however, detected. Finally, increased phosphorylation of EGFR was observed after two to six hours of stimulation, whereas total EGFR levels were highest in unstimulated and 24- or 48-hour-stimulated cells, which is in concordance with the flow cytometry data. In conclusion, both BxPC-3 and RT-112 cells show dynamic changes in EGFR internalization upon EGF stimulation, despite differences in EGFR expression levels. These cell lines can therefore successfully be used for internalization studies, which may contribute to future ADC development. Citation Format: Mandy W. Smeets, Eef F. Smits, Janneke J. Melis, Dimitri Pappaioannou, Guido J. Zaman. Dynamics of ligand-induced epidermal growth factor receptor internalization in cancer cell lines. [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 6135.

Abstract 5678: Hypoxia promotes cell attachment and proliferation due to extracellular matrix compositional and structural changes

Abstract Background: Hypoxia is a common feature of cancer that promotes hallmarks such as cell migration and sustained proliferation. Hypoxia also has a strong link with extracellular matrix (ECM) remodeling, affecting both composition and structure. ECM changes also affect cancer hallmarks through integrin signaling. However, effects of long-term chronic hypoxia stress on ECM have not been studied in bladder cancer. We aimed to investigate the changes induced by hypoxia in the ECM and how they affected cell attachment and proliferation. Methods: UMUC3, J82, RT4 and T24 bladder cancer cell lines were cultured for 7 days in 21% (normoxic) or 0.2% (hypoxic) O2. For obtaining the cell-derived ECM (CDM), cell cultures were decellularized with NH4OH. CDM samples were in-gel trypsin digested and tandem mass spectrometry (MS) performed. IF was performed for FN and COL5A1 protein. Hypoxic ECM effects on cancer cell behavior were studied using attachment and proliferation assays. Attachment assays involved incubating cells at 37°C for 20min (T24, J82, RT4) or 2h (UMUC3) on hypoxic or normoxic CDM. Cells were fixed, stained with crystal violet and absorbance measured at 540nm. Proliferation was measured after seeding cells on hypoxic or normoxic CDM, analyzing cell growth up to 7 days using an IncuCyte S3 system. Results: MS detected 186 ECM proteins, with 66 differentially expressed due to hypoxia in ≥1 cell line (p<0.05). MS data clustering showed variation in CDM composition due to both hypoxia and cell line. Gene Ontology analysis showed hypoxia-induced enrichment (p<0.001) of pathways associated with ECM structure, cell adhesion and integrin adhesion. IF confirmed morphological changes in FN and COL5A1 protein fibres (p<0.05) for 3 of 4 cell lines. All cell lines showed a significant increase in attachment on hypoxic vs normoxic CDM (p<0.05). Initial data showed a significant increase in proliferation of T24 cells grown on hypoxic vs normoxic matrix (p<0.05). Conclusion: Hypoxia induces ECM remodeling in vitro, significantly affecting the level of one third of the proteins detected by MS. In silico analysis of the MS results correlated compositional changes with structural changes correlated compositional changes with structural changes. Those changes were confirmed morphologically for FN and COL5A1 fibers. Cell and integrin adhesion changes predicted in silico were also observed when performing attachment assays, where hypoxic ECM significantly increased cell attachment. Finally, pilot proliferation data showed that hypoxic ECM promotes T24 cell proliferation. To our knowledge, this is the first comprehensive study to report compositional and structural changes in bladder cancer ECM due to hypoxia stress. Further studies should focus on the role of integrin, whose interaction with hypoxic ECM may be key for the induction of cell proliferation and attachment reported in this study. Citation Format: Conrado Guerrero Quiles, Julia Gonzalez Abalos, Jonathan Humphries, Martin Humphries, Ananya CHOUDHURY, Catharine West. Hypoxia promotes cell attachment and proliferation due to extracellular matrix compositional and structural changes [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 5678.

Dual targeting of FGFR3 and ERBB3 enhances the efficacy of FGFR inhibitors in FGFR3 fusion-driven bladder cancer

BackgroundMutations and fusions in Fibroblast Growth Factor Receptor 3 (FGFR3) occur in 10–20% of metastatic urothelial carcinomas and confer sensitivity to FGFR inhibitors. However, responses to these agents are often short-lived due to the development of acquired resistance. The objective of this study was to identify mechanisms of resistance to FGFR inhibitors in two previously uncharacterised bladder cancer cell lines harbouring FGFR3 fusions and assess rational combination therapies to enhance sensitivity to these agents.MethodsAcquired resistance to FGFR inhibitors was generated in two FGFR3 fusion harbouring cell lines, SW780 (FGFR3-BAIAP2L1 fusion) and RT4 (FGFR3-TACC3 fusion), by long-term exposure to the FGFR inhibitor BGJ398. Changes in levels of receptor tyrosine kinases were assessed by phospho-RTK arrays and immunoblotting. Changes in cell viability and proliferation were assessed by the Cell-Titre Glo assay and by propidium iodide staining and FACS analysis.ResultsLong term treatment of FGFR3-fusion harbouring SW780 and RT4 bladder cancer cell lines with the FGFR inhibitor BGJ398 resulted in the establishment of resistant clones. These clones were cross-resistant to the clinically approved FGFR inhibitor erdafitinib and the covalently binding irreversible FGFR inhibitor TAS-120, but remained sensitive to the MEK inhibitor trametinib, indicating resistance is mediated by alternate activation of MAPK signalling. The FGFR inhibitor-resistant SW780 and RT4 lines displayed increased expression of pERBB3, and strikingly, combination treatment with an FGFR inhibitor and the ATP-competitive pan-ERBB inhibitor AZD8931 overcame this resistance. Notably, rapid induction of pERBB3 and reactivation of pERK also occurred in parental FGFR3 fusion-driven lines within 24 h of FGFR inhibitor treatment, and combination treatment with an FGFR inhibitor and AZD8931 delayed the reactivation of pERBB3 and pERK and synergistically inhibited cell proliferation.ConclusionsWe demonstrate that increased expression of pERBB3 is a key mechanism of adaptive resistance to FGFR inhibitors in FGFR3-fusion driven bladder cancers, and that this also occurs rapidly following FGFR inhibitor treatment. Our findings demonstrate that resistance can be overcome by combination treatment with a pan-ERBB inhibitor and suggest that upfront combination treatment with FGFR and pan-ERBB inhibitors warrants further investigation for FGFR3-fusion harbouring bladder cancers.

Olive Mill Wastewater Inhibits Growth and Proliferation of Cisplatin- and Gemcitabine-Resistant Bladder Cancer Cells In Vitro by Down-Regulating the Akt/mTOR-Signaling Pathway.

Bladder cancer patients whose tumors develop resistance to cisplatin-based chemotherapy often turn to natural, plant-derived products. Beneficial effects have been particularly ascribed to polyphenols, although their therapeutic relevance when resistance has developed is not clear. The present study evaluated the anti-tumor potential of polyphenol-rich olive mill wastewater (OMWW) on chemo-sensitive and cisplatin- and gemcitabine-resistant T24, RT112, and TCCSUP bladder cancer cells in vitro. The cells were treated with different dilutions of OMWW, and tumor growth and clone formation were evaluated. Possible mechanisms of action were investigated by evaluating cell cycle phases and cell cycle-regulating proteins. OMWW profoundly inhibited the growth and proliferation of chemo-sensitive as well as gemcitabine- and cisplatin-resistant bladder cancer cells. Depending on the cell line and on gemcitabine- or cisplatin-resistance, OMWW induced cell cycle arrest at different phases. These differing phase arrests were accompanied by differing alterations in the CDK-cyclin axis. Considerable suppression of the Akt-mTOR pathway by OMWW was observed in all three cell lines. Since OMWW blocks the cell cycle through the manipulation of the cyclin-CDK axis and the deactivation of Akt-mTOR signaling, OMWW could become relevant in supporting bladder cancer therapy.

STAG2 loss-of-function affects short-range genomic contacts and modulates the basal-luminal transcriptional program of bladder cancer cells.

Cohesin exists in two variants containing STAG1 or STAG2. STAG2 is one of the most mutated genes in cancer and a major bladder tumor suppressor. Little is known about how its inactivation contributes to tumorigenesis. Here, we analyze the genomic distribution of STAG1 and STAG2 and perform STAG2 loss-of-function experiments using RT112 bladder cancer cells; we then analyze the genomic effects by integrating gene expression and chromatin interaction data. Functional compartmentalization exists between the cohesin complexes: cohesin-STAG2 displays a distinctive genomic distribution and mediates short and mid-ranged interactions that engage genes at higher frequency than those established by cohesin-STAG1. STAG2 knockdown results in down-regulation of the luminal urothelial signature and up-regulation of the basal transcriptional program, mirroring differences between STAG2-high and STAG2-low human bladder tumors. This is accompanied by rewiring of DNA contacts within topological domains, while compartments and domain boundaries remain refractive. Contacts lost upon depletion of STAG2 are assortative, preferentially occur within silent chromatin domains, and are associated with de-repression of lineage-specifying genes. Our findings indicate that STAG2 participates in the DNA looping that keeps the basal transcriptional program silent and thus sustains the luminal program. This mechanism may contribute to the tumor suppressor function of STAG2 in the urothelium.

PD37-02 FOXF1 IS A NOVEL REGULATOR OF NODAL METASTASIS IN BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (PD37)1 Sep 2021PD37-02 FOXF1 IS A NOVEL REGULATOR OF NODAL METASTASIS IN BLADDER CANCER Anirban P. Mitra, Andrea Kokorovic, Tanner S. Miest, Vikram M. Narayan, Debasish Sundi, Amy Lim, Sharada Mokkapati, and Colin P. N. Dinney Anirban P. MitraAnirban P. Mitra More articles by this author , Andrea KokorovicAndrea Kokorovic More articles by this author , Tanner S. MiestTanner S. Miest More articles by this author , Vikram M. NarayanVikram M. Narayan More articles by this author , Debasish SundiDebasish Sundi More articles by this author , Amy LimAmy Lim More articles by this author , Sharada MokkapatiSharada Mokkapati More articles by this author , and Colin P. N. DinneyColin P. N. Dinney More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002047.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Members of the forkhead transcription factor (FOX) family mediate embryonic development and are altered in many cancers. The functional role of FOXF1 in bladder cancer progression is unclear thus far. We investigated the clinical implications of differential FOXF1 expression in bladder cancer and potential mechanisms by which its alteration can lead to metastasis. METHODS: Whole genome expression profiling performed on paired primary tumors and nodal metastases from a radical cystectomy discovery cohort using Illumina HT12 v3-4 BeadChip arrays identified FOXF1 as a top differentially expressed gene. Prognostic role of differential FOXF1 expression was validated in two independent cystectomy cohorts. Differential FOXF1 expression was also evaluated in murine orthotopic xenografts. Small interfering RNA was used to knock down FOXF1 in RT112 and UC6 bladder cancer cell lines to develop an in vitro model for assessment of metastatic potential. Next-generation sequencing and hierarchical clustering analysis were used to identify differentially altered genes secondary to FOXF1 knockdown. 186 biologically curated pathways were interrogated with internal validation to elucidate downstream biologic mechanisms of metastasis. RESULTS: FOXF1 was a top differentially expressed gene in the discovery cohort with 3.6-fold lower expression in nodal metastases than paired primary tumors (n=33, p<0.001). Multivariable analyses in two validation cohorts (total n=128) indicated that FOXF1 underexpression was associated with worse cancer-specific (p=0.046) and overall survival (p=0.006). Murine orthotopic xenografts (n=13) established from human bladder cancer cell lines (UC3, UC6, UC14) showed FOXF1 underexpression in metastatic deposits compared with primary tumors (p=0.004). Hierarchical clustering identified 40 differentially expressed genes between FOXF1-knockdown bladder cancer cell lines and their corresponding controls. Biological pathway interrogation showed differential enrichment for genes associated with mitogen-activated protein kinase signaling, focal adhesion and other carcinogenic pathways in FOXF1-knockdown cells compared with controls (normalized enrichment score ≥1.3). CONCLUSIONS: We identify and characterize FOXF1 as a novel regulatory molecule that drives bladder cancer metastasis. This may be modulated through alterations in intracellular signaling and cellular adhesion. FOXF1 may serve as a prognostic biomarker that can identify patients at impending risk for metastasis who may benefit from more aggressive management. Source of Funding: The University of Texas MD Anderson SPORE in Genitourinary Cancer P50CA091846, NIH/NCI under P30CA016672 © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e655-e655 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Anirban P. Mitra More articles by this author Andrea Kokorovic More articles by this author Tanner S. Miest More articles by this author Vikram M. Narayan More articles by this author Debasish Sundi More articles by this author Amy Lim More articles by this author Sharada Mokkapati More articles by this author Colin P. N. Dinney More articles by this author Expand All Advertisement Loading ...

High-throughput screening for natural compound-based autophagy modulators reveals novel chemotherapeutic mode of action for arzanol

Autophagy is an intracellular recycling pathway with implications for intracellular homeostasis and cell survival. Its pharmacological modulation can aid chemotherapy by sensitizing cancer cells toward approved drugs and overcoming chemoresistance. Recent translational data on autophagy modulators show promising results in reducing tumor growth and metastasis, but also reveal a need for more specific compounds and novel lead structures. Here, we searched for such autophagy-modulating compounds in a flow cytometry-based high-throughput screening of an in-house natural compound library. We successfully identified novel inducers and inhibitors of the autophagic pathway. Among these, we identified arzanol as an autophagy-modulating drug that causes the accumulation of ATG16L1-positive structures, while it also induces the accumulation of lipidated LC3. Surprisingly, we observed a reduction of the size of autophagosomes compared to the bafilomycin control and a pronounced accumulation of p62/SQSTM1 in response to arzanol treatment in HeLa cells. We, therefore, speculate that arzanol acts both as an inducer of early autophagosome biogenesis and as an inhibitor of later autophagy events. We further show that arzanol is able to sensitize RT-112 bladder cancer cells towards cisplatin (CDDP). Its anticancer activity was confirmed in monotherapy against both CDDP-sensitive and -resistant bladder cancer cells. We classified arzanol as a novel mitotoxin that induces the fragmentation of mitochondria, and we identified a series of targets for arzanol that involve proteins of the class of mitochondria-associated quinone-binding oxidoreductases. Collectively, our results suggest arzanol as a valuable tool for autophagy research and as a lead compound for drug development in cancer therapy.

Evaluation of Loading Strategies to Improve Tumor Uptake of Gemcitabine in a Murine Orthotopic Bladder Cancer Model Using Ultrasound and Microbubbles

In this study we compared three different microbubble-based approaches to the delivery of a widely used chemotherapy drug, gemcitabine: (i) co-administration of gemcitabine and microbubbles (Gem+MB); (ii) conjugates of microbubbles and gemcitabine-loaded liposomes (GemlipoMB); and (iii) microbubbles with gemcitabine directly bound to their surfaces (GembioMB). Both in vitro and in vivo investigations were carried out, respectively, in the RT112 bladder cancer cell line and in a murine orthotopic muscle-invasive bladder cancer model. The in vitro (in vivo) ultrasound exposure conditions were a 1 (1.1) MHz centre frequency, 0.07 (1.0) MPa peak negative pressure, 3000 (20,000) cycles and 100 (0.5) Hz pulse repetition frequency. Ultrasound exposure produced no significant increase in drug uptake either in vitro or in vivo compared with the drug-only control for co-administered gemcitabine and microbubbles. In vivo, GemlipoMB prolonged the plasma circulation time of gemcitabine, but only GembioMB produced a statistically significant increase in cleaved caspase 3 expression in the tumor, indicative of gemcitabine-induced apoptosis.

Characterization of FOXF1 as a novel regulator of nodal metastasis in bladder cancer.

480 Background: Members of the forkhead transcription factor (FOX) family are important mediators of embryonic development and are known to be altered in a variety of cancers. The functional role of FOXF1 in bladder tumorigenesis and progression has not been clearly characterized thus far. This study investigated the clinical implications of differential FOXF1 expression in bladder cancer, and potential mechanisms by which its alteration can lead to tumor metastasis. Methods: Whole genome expression profiling was performed on paired primary tumors and nodal metastases from a radical cystectomy discovery cohort using Illumina HT12 v3-4 BeadChip arrays to identify FOXF1 as a top differentially expressed gene. Prognostic role of differential FOXF1 expression was validated on two independent cystectomy cohorts. Differential FOXF1 expression was also evaluated in murine orthotopic xenografts. Small interfering RNA was used to knock down FOXF1 in RT112 and UC6 bladder cancer cell lines to develop an in vitro model for assessment of metastatic potential. Next-generation sequencing and hierarchical clustering analysis were used to identify differentially altered genes secondary to FOXF1 knockdown. 186 biologically curated pathways were interrogated with internal validation to elucidate the downstream biologic mechanisms of metastasis. Results: In the discovery cohort, FOXF1 was a top differentially expressed gene with 3.6-fold lower expression in nodal metastases than paired primary tumors (n = 33, p &lt; 0.001). Multivariable analyses in two validation cohorts (total n = 128) indicated that FOXF1 underexpression was associated with worse cancer-specific (p = 0.046) and overall survival (p = 0.006). Murine orthotopic xenografts (n = 13) established from human bladder cancer cell lines (UC3, UC6, UC14) showed FOXF1 underexpression in metastatic deposits compared with primary tumors (p = 0.004). Hierarchical clustering identified 40 differentially expressed genes between FOXF1-knockdown bladder cancer cell lines and their corresponding controls. Biological pathway interrogation showed differential enrichment for genes associated with mitogen-activated protein kinase signaling, focal adhesion and other carcinogenic pathways in FOXF1-knockdown cells compared with controls (normalized enrichment score ≥ 1.3). Conclusions: We identify and characterize FOXF1 as a novel regulatory molecule that potentially drives bladder cancer metastasis. This may be modulated through alterations in intracellular signaling and cellular adhesion. FOXF1 may serve as a prognostic biomarker that can identify patients at impending risk for metastasis who may benefit from more aggressive management.

Radiosensitisation of bladder cancer cells via short-chain fatty acids and/or other metabolites produced by the gut microbiota

New non-toxic radiosensitisers are needed in the treatment of muscle-invasive bladder cancer because elderly patients are very vulnerable to chemotherapy-related toxicity of currently available radiosensitisers. Our previous study showed that high fibre diets sensitised RT112 xenografts to irradiation by modifying the gut microbiome and this phenotype was positively correlated with B. acidifaciens abundance. Short chain fatty acids (SCFAs) are major products of fibre fermentation by the gut microbiota. Therefore, we hypothesise that B. acidifaciens may radiosensitise tumours via secretion of SCFAs and/or other metabolites. We treated the RT112 human bladder cancer cell line with SCFAs to determine histone acetylation levels by Western Blot and radiosensitivity by clonogenic assay. We also used a cell viability assay to validate the cytotoxic effects of SCFAs and bacterial supernatants on cancer cells. We showed that all three SCFAs increased histone acetylation (10 mM acetate p=0.014, 10 mM propionate p=0.004, 10 mM butyrate p<0.001) of RT112 bladder cancer cells, in a dose-dependent manner. All SCFAs tended to increase radiosensitivity in the clonogenic assay, with butyrate having a statistically significant effect (p=0.002 for butyrate at 8Gy). In a cell viability assay, we saw that the combination of SCFAs in a physiological ratio conferred a stronger phenotype than single SCFAs, in a time-dependent manner. To validate the anti-tumoural effects of B. acidifaciens, we treated the bladder tumour cells with bacterial supernatants of B. acidifaciens and its cross-feeding with F. prausnitzii, and compared the effects to Bifidobacterium (acetate-producer) and F. prausnitzii (butyrate-producer). Bacterial supernatants of B. acidifaciens and its cross-feeding with F. prausnitzii significantly increased the cytotoxic response of bladder tumour cells compared to the other supernatants. In conclusion, our in vitro experiments in RT112 human bladder cancer cells suggest a role for short-chain fatty acids and/or other metabolites generated by the microbiota in radiosensitisation of tumour cells.

Exploring the Mechanism of Flaccidoxide-13-Acetate in Suppressing Cell Metastasis of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common liver or hepatic cancer, accounting for 80% of all cases. The majority of this cancer mortality is due to metastases, rather than orthotopic tumors. Therefore, the inhibition of tumor metastasis is widely recognized as the key strategy for successful intervention. A cembrane-type diterpene, flaccidoxide-13-acetate, isolated from marine soft coral Sinularia gibberosa, has been reported to have inhibitory effects against RT4 and T24 human bladder cancer invasion and cell migration. In this study, we investigated its suppression effects on tumor growth and metastasis of human HCC, conducting Boyden chamber and Transwell assays using HA22T and HepG2 human HCC cell lines to evaluate invasion and cell migration. We utilized gelatin zymography to determine the enzyme activities of matrix metalloproteinases MMP-2 and MMP-9. We also analyzed the expression levels of MMP-2 and MMP-9. Additionally, assays of tissue inhibitors of metalloproteinase-1/2 (TIMP-1/2), the focal adhesion kinase (FAK)/phosphatidylinositide-3 kinases (PI3K)/Akt/mammalian target of the rapamycin (mTOR) signaling pathway, and the epithelial-mesenchymal transition (EMT) process were performed. We observed that flaccidoxide-13-acetate could potentially inhibit HCC cell migration and invasion. We postulated that, by inhibiting the FAK/PI3K/Akt/mTOR signaling pathway, MMP-2 and MMP-9 expressions were suppressed, resulting in HCC cell metastasis. Flaccidoxide-13-acetate was found to inhibit EMT in HA22T and HepG2 HCC cells. Our study results suggested the potential of flaccidoxide-13-acetate as a chemotherapeutic candidate; however, its clinical application for the management of HCC in humans requires further research.