Rosiglitazone and trametinib exhibit potent anti-tumor activity in a mouse model of muscle invasive bladder cancer

Abstract

Muscle invasive bladder cancers (BCs) can be divided into 2 major subgroups-basal/squamous (BASQ) tumors and luminal tumors. Since Pparg has low or undetectable expression in BASQ tumors, we tested the effects of rosiglitazone, Pparg agonist, in a mouse model of BASQ BC. We find that rosiglitazone reduces proliferation while treatment with rosiglitazone plus trametinib, a MEK inhibitor, induces apoptosis and reduces tumor volume by 91% after 1 month. Rosiglitazone and trametinib also induce a shift from BASQ to luminal differentiation in tumors, which our analysis suggests is mediated by retinoid signaling, a pathway known to drive the luminal differentiation program. Our data suggest that rosiglitazone, trametinib, and retinoids, which are all FDA approved, may be clinically active in BASQ tumors in patients.

Fig. 1. Combined Rosi+Tram treatment reduces tumor burden in mice with BBN-induced MIBC.

Wildtype male mice were given BBN (0.05%) in drinking water ad libidum for 5 months. Mice were then treated for one month by daily via oral gavage with vehicle (Control), Rosi alone, Tram alone, or with a combination of Rosi+Tram. Tumor volume was measured prior to euthanasia, and bladders were collected. Ultrasound images and 3D reconstruction of bladders were obtained from mice before and after 1 month treatment. A–H Representative images of ultrasound imaging of a mouse exposed to BBN for 5 months before A and after B administration of vehicle for 1 month. Ultrasound image of a mouse exposed to BBN for 5 months before C and after D administration of Rosi for 1 month. Ultrasound images and 3D reconstruction of a bladder from a mouse treated for 5 months with BBN before E or after F treatment for 1 month with Tram. Ultrasound images and 3D reconstruction of bladders from mice treated for 5 months with BBN before G and after H 1 month of combined Rosi+Tram treatment. I. Box plot showing tumor volume measurements based on 3D reconstruction before and after 1 month vehicle (n = 7), Rosi (n = 6), Tram (n = 6), or Rosi+Tram (n = 13). Box plots display minima, maxima, and interquartile range (IQR). Significance calculated by one-way ANCOVA with Dunnet’s test for pairwise comparisons ***p = 0.0007. J–N Panoramas showing histological analysis of a bladders from BBN exposed mouse after 1 month of vehicle J, after 1 month of Rosi treatment K, after 1 month of Tram L, after 1 month of Rosi+Tram M, and untreated wild type control N. O–S Magnified regions in boxed regions shown in images J–N. Scale bars: 50 µm. Source data are provided as a Source Data file. Cartoons in Fig. 1 created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en).

Fig. 2. Combined Rosi+Tram treatment impairs tumor growth and restores normal urothelial differentiation in mice with BBN-induced BASQ tumors.

A–D Panoramas showing luminal (Upk2, K20) and basal/squamous (K14/K6a) marker expression in mouse exposed to BBN for 5 months then treated for 1 month with vehicle (n = 4) A, B or Rosi+Tram (n = 5) C, D. E Composition of the cell types in the urothelium in animals treated with vehicle, Rosi, Tram, Rosi+Tram, and no BBN controls. F, I, L, O, R. Upk2, K20, and P63 expression in animals exposed to BBN for 5 months then treated with vehicle (n = 4) F, Rosi (n = 5) I, Tram (n = 4) L, Rosi+Tram (n = 5) O, and untreated adult mouse controls (n = 3) R. G, J, M, P, S. Expression of K14, P63, and K6a in the urothelium of mice exposed to BBN for 5 months then treated for one month with vehicle (n = 4) G, Rosi (n = 5) J, Tram (n = 4) M, combined Rosi+Tram (n = 5) P, and untreated controls (n = 3) S. H, K, N, Q, T. Expression of K14, K5, and P63 in the urothelium of mice exposed to BBN for 5 months then treated for one month with vehicle (n = 4) (H), Rosi (n = 5) (K), Tram (n = 4) N, combined Rosi+Tram (n = 5) Q, and untreated controls (n = 3) (T). U–X Pparg/Fabp4 expression in urothelium of BBN-induced tumors after treatment for one month with vehicle U, Rosi V, Tram W and Rosi+Tram X. Y, Z Bar graphs showing changes in the percentage of the basal-most layer of the urothelium expressing Pparg Y and FABP4 Z after 1 month treatment with vehicle (n = 3), Rosi (n = 3), Tram (n = 3), and Rosi+Tram (n = 3). Data given as means ± SD, significance calculated using two-tailed unpaired Welch’s t-test. A′–D′. Expression of K14, Pparg, and FABP4 in basal tumors treated for 7 days with vehicle A′ or Rosi+Tram (B′–D′). E′. Quantification of percentage of lesions expressing FABP4 after 7 days of treatment with vehicle (n = 4) or Rosi+Tram (n = 4). Data given as means ± SD, significance calculated using two-tailed unpaired Welch’s t-test, p = 0.0001. Scale bars: 50 µm. Source data are provided as a Source Data file.

Fig. 3. Rosi/Tram treatment induces apoptosis and reduces proliferation in BBN-induced BASQ tumors after 7 days.

A, B Expression of activated Caspase-3 in tumors of mice exposed to BBN for 5 months then treated with vehicle for 7 days, image shown with A and without B Ecad. C, D Expression of activated Caspase-3 in 5 M BBN tumors treated with Rosi+Tram for 7 days, image shown with C and without D Ecad. E Bar graph showing the percentage of cells expressing activated caspase-3 in BBN-induced tumors treated for 7 days with vehicle (n = 3) or Rosi+Tram (n = 3). Data given as means ± SD, significance calculated using two-tailed Mann-Whitney test, ***p = 0.0001. F Bar graph showing the percentage of cells expressing Ki67 in BBN-treated mice after vehicle (n = 3) or Rosi+Tram (n = 3) for 7 days. Data given as means ± SD, significance calculated using two-tailed Mann-Whitney test, ****p < 0.0001. G Apoptosis assayed by AnnexinV-eFluor450/7-AAD double staining in BBN963 cells treated with DMSO, Rosi, Tram, or combined Rosi+Tram for 72 h. Cells undergoing early apoptosis were AnnexinV+/7-AAD: late apoptosis: AnnexinV+/7-AAD+. H Heatmap showing normalized expression of proapoptotic genes, caspases, and pro-survival genes in Rosi+Tram treated BBN963 cells compared to controls based on RNAseq analysis. I–L Decreased proliferation determined by Ki67 staining in BBN963 cells after Rosi (n = 4) J, Tram (n = 4) K, or combined Rosi+Tram (n = 4) L compared to controls (n = 4) I. M–P Down-regulation of Ccnd1 in BBN963 cells after treatment with Rosi N, Tram O, or Rosi+Tram P compared to controls M. Scale bars: 50 µm. Q Heatmap of normalized gene expression showing down-regulation of Ccnd1 and genes that positively regulate cell cycle progression and up-regulation of genes that inhibit progression in BBN963 cells treated with Rosi+Tram for 72 h compared to DMSO treated controls. R Bar graph showing number of Ccnd1-positive BBN963 cells after 72-h treatment with DMSO (n = 4), Rosi (n = 4), Tram (n = 4), and combined Rosi+Tram (n = 4). Data given as means ± SD, significance calculated using two-tailed unpaired Welch’s t-test, ***p = 0.0006, ****p < 0.0001. Source data are provided as a Source Data file. Cartoons in Fig. 3 created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en).

Fig. 4. RosiTram treatment of BBN-induced tumors increases Kdm6a and retinoid signaling and decreases AP-1.

A Pathways changed in ATAC-seq/RNA-seq analysis after 1 M and 5 days of RosiTram vs vehicle. Bubble color/size corresponds to the p-value (p < 0.05) and number of genes enriched in pathways (GeneRatio), respectively. B Scatter plot showing differentially expressed/accessible genes after 5 days RosiTram vs vehicle. Plotted as Fold Change ATACseq RPM [Sample/Control] and Fold Change RNAseq RPM [Sample/Control], each dot is one gene. C IGV tracks showing gene expression and chromatin accessibility of retinoid signaling pathway genes in tumors treated for 5 days with RosiTram or vehicle. D Heatmap showing normalized gene expression of RA targets and RA synthesizing enzymes after 5 days of RosiTram treatment versus vehicle controls (from RNA-seq). E–L Expression of Aldh1a1 and Aldh1a2 in 5 M BBN tumors treated with vehicle (n = 3) E, I Rosi (n = 3) F, J, Tram (n = 4) G, K, and combined Rosi+Tram (n = 3) H, L. Scale bars: 50 µm M. Volcano plot showing upregulated (red) and down-regulated (blue) differentially expressed genes (DEGs) from RNAseq analysis of BBN963 cells after treatment with RosiTram (n = 5) vs DMSO controls (n = 5). Each dot is one DEG, colored points have a p-value ≤ 0.05. N–S Expression of Krt14, Kdm6a, and Fabp4 in BBN-induced tumors after treatment with vehicle (n = 3) N, Rosi (n = 3) O, Tram (n = 3) Q, or Rosi+Tram (n = 3) R, patient MIBC BASQ tumor (n = 2) P, and patient luminal papillary tumor (n = 3) S. Scale bars: 50 µm. T. HOMER motif analysis after ATAC-seq showing enrichment of the Junb regulatory network in less accessible peaks after Rosi+Tram for 5 days compared to vehicle. Heatmap showing normalized expression of AP-1 pathway genes after 5-day treatment with RosiTram compared to vehicle (bulk RNA-seq). U–X. Expression of Fosb U, V or JunB W, X after 7-day RosiTram or vehicle treatment of BBN-induced tumors. Scale bars: 50 µm. Y, Z Bar graphs quantitating the percentage of FosB Y and JunB Z-positive tumor cells after 7-day treatment with vehicle (n ≥ 3) or Rosi+Tram (n = 3). Data given as means ± SD, significance calculated using two-tailed unpaired Welch’s t-test, ****p < 0.0001. Source data are provided as a Source Data file.

Fig. 5. Retinoic acid induces BBN 963 cells to undergo a Basal-to-luminal shift in differentiation and lowers proliferation.

A, B Ki67 and Foxa1 staining in an orthotopic tumors from BBN963 cells engrafted into bladders of C57Bl/6 mice after treated with vehicle for 7 days. C, D Ki67 and Foxa1 staining in an orthotopic tumors from BBN963 cells engrafted into bladders of C57Bl/6 mice after treatment with RA for 7 days. Scale bars: 50 µm E. Heatmap showing normalized gene expression changes in BASQ and luminal markers after RA treatment of BBN963 cells compared to controls from RNAseq analysis. F Bar graph showing decreased proliferation (via percentage of Ki67+ cells) in BBN963 orthotopic tumors after treatment with RA (n = 4) versus vehicle (n = 4) for 7 days. Data given as means ± SD, significance calculated using two-tailed unpaired Welch’s t-test. G Volcano plot showing upregulated (red) and down-regulated (blue) differentially expressed genes (DEGs) from RNAseq analysis of BBN963 cells treated either with vehicle (n = 4) or RA (n = 4). Each dot is one DEG, colored points have a p-value ≤ 0.05. Source data are provided as a Source Data file. Cartoons in Fig. 5 created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en).

Fig. 6. Combined and single activities of Rosi+Tram regulate tumor survival and subtype.

Rosi or Tram alone decrease tumors size and growth, but combined Rosi + Tram treatment induces apoptosis of BASQ tumors 7 days after treatment. Tram helps maintain Pparg activity by preventing its phosphorylation, which can lead to degradation. Rosi induces Pparg activation, which leads to up-regulation of RA-synthesizing enzymes that activate the retinoid signaling pathway. Retinoids and Kdm6a cooperate to promote luminal differentiation, suppressing AP-1, a pathway that promotes BASQ differentiation. Source data are provided as a Source Data file. Cartoons in Fig. 6 created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). Cartoons in Fig. 6 created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en).