Effect of adenovirus-mediated hepaCAM gene expression on cell cycle of bladder cancer cell lines

253 Background: Human epidermal receptors (HER) are overexpressed and HER signaling is biologically relevant in bladder cancer and may mediate chemotherapy resistance. Dacomitinib is a novel, potent, irreversible pan-HER inhibitor with activity against several solid tumors, currently in a phase III clinical trial in NSCLC. We hypothesized that dacomitinib has antitumor activity in bladder cancer models. Methods: Expression level of EGFR and HER2 protein was measured semi-quantitatively in 8 bladder cancer cell lines. We treated UM-UC-3, UM-UC-6, UM-UC-9 cell lines with dacomitinib (1nM-10uM) for 24-72 hours, and measured cell viability, proliferation, apoptosis, and cell cycle effects. Correlations between dose and cell viability were measured by two-way ANOVA (GraphPad Prism 5.0). We injected age-matched male NOD/SCID mice SC with 1x106 UM-UC-6 and UM-UC-9 cells, respectively, generating xenografts. Mice were randomized and treated with dacomitinib, 6mg/kg p.o. daily, starting 1 day or 1 week after cell injection; controls were treated with vehicle. Mice were monitored daily, weighed weekly, sacrificed at 4 weeks and tumors weighed. Results: In vitro, significant cytostatic effect was noted with as low as 50nM in UM-UC6 cells and 100nM in UM-UC9 cells. UM-UC3 cells did not exhibit cytostatic effect even with 1000nM, corresponding to differential target protein (HER) expression. Dacomitinib (2uM) induced apoptosis (UM-UC-6), and G1 cell cycle arrest in both cell lines. These effects corresponded to dacomitinib-mediated inhibition of EGFR, ERK, AKT phosphorylation. In vivo, xenograft weights in both cell lines were significantly lower in dacomitinib-treated mice vs control (p<0.001), corresponding to pharmacodynamic effects (decreased E-cadherin, p-EGFR, p-ERK, mitotic count). Dacomitinib 6mg/kg p.o. daily resulted in significantly lower tumor weights vs lapatinib 50 mg/kg p.o. daily in UM-UC-9 xenograft model (p=0.0052). Conclusions: Dacomitinib demonstrated single-agent activity in bladder cancer cell lines and xenografts. Induction of apoptosis and G1 phase arrest are the suggested mechanisms for anti-tumor activity. Further investigation of this inhibitor in bladder cancer models is being pursued.

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Heterogeneity in Nectin-4 expression across molecular subtypes of urothelial cancer mediates sensitivity to enfortumab vedotin.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.6_suppl.463 ◽

2021 ◽

Vol 39 (6_suppl) ◽

pp. 463-463

Author(s):

Carissa Chu ◽

Martin Sjöström ◽

Emily A Egusa ◽

Ewan Gibb ◽

Michelle L Badura ◽

...

Keyword(s):

Gene Expression ◽

Bladder Cancer ◽

Cell Lines ◽

Cancer Cell ◽

Urothelial Cancer ◽

Molecular Subtypes ◽

Cancer Cell Lines ◽

Bladder Cancer Cell ◽

Antibody Drug Conjugate ◽

Metastatic Urothelial Cancer

463 Background: Enfortumab vedotin (EV) is an antibody-drug conjugate (ADC) targeting Nectin-4 (encoded by the PVRL4/NECTIN4 gene ) approved for treatment-refractory metastatic urothelial cancer. Factors that mediate sensitivity or resistance to EV are unknown. In the present study, we sought to 1) examine heterogeneity of NECTIN4 gene expression across molecular subtypes of bladder cancer and 2) determine if Nectin-4 expression mediates EV sensitivity or resistance. Methods: NECTIN4 expression data from seven muscle-invasive bladder cancer clinical cohorts (n = 1912 total patients) were used to compare relative NECTIN4 expression across molecular subtypes. The outcome of the gene expression analysis was relative NECTIN4 expression in the consensus molecular subtypes of bladder cancer. Expression of NECTIN4 was validated in multiple bladder cancer cell lines. NECTIN4 was stably over-expressed or knocked down in basal (TCCSUP and UMUC-3) and luminal (HT-1376, HT-1197 and UMUC-9) bladder cancer cell lines, respectively, and EV dose-response assays were performed, as measured by cell proliferation and clonogenic assays. Results: NECTIN4 expression is heterogenous across molecular subtypes of bladder cancer and significantly enriched in luminal subtypes (p < 0.001). NECTIN4 expression is positively correlated with the luminal markers GATA3, FOXA1, and PPARG across cohorts (Spearman’s rank correlation r = 0.57, p < 0.0001 for GATA3, r = 0.37, p < 0.0001 for FOXA1, and r = 0.56, p < 0.0001 for PPARG). NECTIN4 expression is both necessary and sufficient for EV sensitivity in luminal and basal subtypes of urothelial bladder cancer cells. Downregulation of NECTIN4 led to EV resistance, and EV-resistant cell lines expressed decreased levels of Nectin-4. Conclusions: Results of this pre-clinical study suggest that sensitivity to EV is mediated by expression of NECTIN4, which is significantly enriched in luminal subtypes of bladder cancer. Downregulation of NECTIN4 leads to resistance to EV. These findings have implications for biomarker development, patient selection and the inclusion of molecular subtyping in ongoing and future EV clinical trials. Further investigation into Nectin-4 loss as a mechanism of resistance in patients treated on EV is warranted.

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