ATDC mediates a TP63-regulated basal cancer invasive program

Abstract

Basal subtype cancers are deadly malignancies but the molecular events driving tumor lethality are not completely understood. Ataxia-telangiectasia group D complementing gene (ATDC, also known as TRIM29), is highly expressed and drives tumor formation and invasion in human bladder cancers but the factor(s) regulating its expression in bladder cancer are unknown. Molecular subtyping of bladder cancer has identified an aggressive basal subtype, which shares molecular features of basal/squamous tumors arising in other organs and is defined by activation of a TP63-driven gene program. Here, we demonstrate that ATDC is linked with expression of TP63 and highly expressed in basal bladder cancers. We find that TP63 binds to transcriptional regulatory regions of ATDC and KRT14 directly, increasing their expression, and that ATDC and KRT14 execute a TP63-driven invasive program. In vivo, ATDC is required for TP63-induced bladder tumor invasion and metastasis. These results link TP63 and the basal gene expression program to ATDC and to aggressive tumor behavior. Defining ATDC as a molecular determinant of aggressive, basal cancers may lead to improved biomarkers and therapeutic approaches.

Fig. 1.

ATDC is correlated with TP63 and basal gene expression and is enriched in basal subtype bladder cancers. (a) ATDC is correlated with TP63, KRT14 and KRT5 mRNA levels in bladder, breast, prostate and esophageal cancers based on analysis of TCGA data. Pearson and Spearman correlations with ATDC: Bladder (n=413): TP63 (0.46, 0.54), KRT14 (0.34, 0.42), KRT5 (0.5, 0.52). Breast (n=1105): TP63 (0.43, 0.56), KRT14 (0.49, 0.74), KRT5 (0.5, 0.79). Prostate (n=499): TP63 (0.88, 0.94), KRT14 (0.4, 0.69), KRT5 (0.93, 0.97). Esophageal (n=185): TP63 (0.41, 0.77), KRT14 (0.47,0.79), KRT5 (0.72, 0.85). p-values for all Pearson correlations are < 0.00001. (b) Hierarchical clustering analysis of basal and luminal gene expression in TCGA human bladder cancers demonstrates that ATDC and TP63 expression is most closely associated with basal subtype bladder cancer. Heatmap was generated using z-scores calculated per-gene across all patients. (c-d) Scatter plots representing TP63 and ATDC expression in the luminal or basal tumors identified in Fig 1b. Line represents mean log2 RSEM expression. Expression differences between luminal and basal subtypes were highly statistically significant: ATDC p = 3.59 × 10⁻²² and TP63 p = 2.6 × 10⁻²¹. (e) Immunohistochemical staining for ATDC, KRT14, KRT20 and TP63 in human muscle invasive bladder cancer samples demonstrates ATDC co-expression in representative tumors with basal marker expression (top 2 panels), but not in luminal tumors expressing the marker KRT20 (bottom 2 panels). (f) Bar graph showing ATDC protein expression as measured by IHC in basal (KRT14+, n =15) and luminal (KRT20+, n = 30) human bladder tumors. Expression of ATDC was scored 0 (no staining) to 3 (intense staining) by experience observer blinded to experimental conditions. Student t test p value = 0.02. Error bars = SEM.

Fig. 2.

dNp63α is the most commonly expressed isoform of TP63 in bladder cancer and drives ATDC and basal gene expression. (a) Schematic diagram of TP63 isoforms. (b) Analysis of TCGA RNA sequencing data from bladder cancer samples (BLCA) classified as basal or luminal subtype demonstrates that dNp63α is predominate isoform expressed in human bladder cancer followed by dNp63β and dNp63γ respectively. dNp63α, β and γ were all more highly expressed in basal vs luminal subtypes (5.65 fold increase, p =8 × 10⁻²²; 6.06 fold increase, p = 2.3 × 10⁻¹⁷; 4.1 fold increase, p = 1.7 × 10⁻²⁴ respectively). (c) Similar TP63 isoform expression patterns were observed in a panel of 20 human bladder cancer cell lines. Line indicates median and box indicates first-third interquartile range for (b-c). (d) ATDC expression is significantly correlated with expression of dNp63α (Pearson 0.659), dNp63β (Pearson 0.662) and dNp63γ (Pearson 0.600) as well as total TP63 (Pearson 0.655) in the bladder cancer TCGA data set (all p values < 0.0001). (e) Overexpression of dNp63α and TAp63α drives upregulation of ATDC expression in UC10 and UC14 bladder cancer cell lines as measured by western blotting. (f) Overexpression of dNp63α in UC14 and UC10 human bladder cancer cell lines promoted expression of KRT14 and KRT6A genes as well as ATDC as measured by quantitative RT-PCR. n = 3 for each condition. Error bars = S.D. * indicates p < 0.05 compared to controls.

Fig. 3.

TP63 binds to ATDC enhancer and KRT14 promoter elements. (a) TP63 chromatin immunoprecipitation (ChIP) sequencing data from human keratinocytes (GEO accession number GSE32061) identifies 3 putative TP63 binding sites within ATDC enhancer. Putative TP63 binding sites correspond to transcriptionally active regions marked by H3K27 acetylation. P1, P2 and P3 refer to location of PCR products used in (b-d). (b) ChIP of TP63 (p63α IP) in the UC14 bladder cancer cell line transduced with control or dNp63α overexpression (dNp63α OE) vectors indicates enrichment of ATDC P1, P2 and P3 sequence compared to IgG controls. dNp63α overexpression increased TP63 occupancy of binding sites in UC14. CDKN1A is a positive control target of TP63 binding. Satellite and RPL30 are negative controls. (c) TP63 ChIP in UC5 bladder cancer cell line confirms TP63 occupancy on ATDC P1, 2 and 3 binding sites. (d) TP63 ChIP in UC10 bladder cancer cells (low endogenous TP63 and ATDC) with and without ectopic dNP63α expression demonstrates TP63 occupancy at ATDC promoter sites only in cells with ectopic dNp63α expression. (e and f) TP63 ChIP in UC10 and UC14 cells with ectopic dNp63α demonstrates enrichment of KRT14 promoter element. All samples are done in triplicate. Error Bars = S.D. * denotes a statistically significant difference (p < 0.05, student t test) compared to satellite negative control for all panels. # denotes a statistically significant difference (p < 0.05, student t test) compared to both satellite negative control and vector control conditions for all panels.

Fig. 4.

TP63 Drives Expression of ATDC and KRT14 in Bladder Cancer. (a) siRNA-mediated knockdown of TP63 reduced ATDC expression in UC5 and UC14 bladder cancer cell lines, but siRNA-mediated knockdown of ATDC had no effect on TP63 expression as measured by western blotting. IRF6 serves as a known TP63 target control. (b and c) siRNA-mediated knockdown of TP63 reduces TP63 and ATDC mRNA expression as measured by qRT-PCR in UC14. Error Bar = S.D. * p < 0.0001 compared to siCtl (d-e) Overexpression of dNp63α (dNp63α-OE) in UC10 (d) and UC14 (e) promotes increased expression of TP63 mRNA (qRT-PCR) compared to empty vector control (Vector) which was not effected by knockdown of ATDC with siRNA. Error Bar = S.D. N = 3. * p < 0.0001, ** p = 0.0099 compared to Vector siCtl for (d). * p = 0.0005, ** p = 0.05, each compared to Vector siCtl for (e). (f-g) dNp63α-OE upregulated expression of ATDC mRNA (dNp63α-OE siCtl compared to Vector siCtl) in UC10 (f) and UC14 (g). Error Bar = S.D. N = 3. * p < 0.0001 compared to Vector siCtl, ** p = 0.0007 compared to Vector siCtl, *** p < 0.0001 compared to dNp63α-OE siCtl for (f). * p < 0.0001 compared to Vector siCtl, ** p < 0.0001 compared to Vector siCtl, *** p = 0.0001 each compared to dNp63α-OE siCtl for (g). (h-i) dNp63α-OE upregulated expression of KRT14 mRNA (dNp63α-OE siCtl compared to Vector siCtl) but this upregulation was reduced by ATDC siRNA mediated KD (dNp63α-OE siCtl compared to dNp63α-OE siATDC) in UC10 (h) and UC14 (i). Error Bar = S.D. N = 3. * p = 0.06 compared to Vector siCtl, ** p = 0.0012 compared to Vector siCtl, *** p = 0.03 compared to dNp63α-OE siCtl for (h). * p = 0.007 compared to Vector siCtl, ** p = 0.0313 compared to dNp63α-OE siCtl for (i). (j) CRISPR-Cas9 mediated genomic ablation of ATDC in UC14 (ATDC Knockout #1 and 2) results in loss of ATDC mRNA. Error Bars = S.D. n = 3. * p < 0.0001 compared to ATDC wild type. (k) ATDC Knockout results in significant decrease in KRT14 mRNA expression. Error Bars = S.D. n = 3. * < 0.001 compared to wild type. (l) UC14 ATDC knockout (KO) cells have reduced ATDC and KRT14 protein expression compared to ATDC wild type (Wt). dNp63α-OE increased ATDC and KRT14 expression.

Fig. 5.

TP63 promotes invasion and requires ATDC and KRT14. Overexpression of dNp63α or ATDC drives increased invasion as measured by transwell assays for 253J (a), UC10 (b) or UC14 (c). n = 5. Error Bars = S.D. * p < 0.05 compared to control. (d-e) siRNA-mediated KD of ATDC and KRT14 blocked dNp63α induced invasion in transwell assays in UC10 (d) and UC14 (e). n = 4. Error Bars = S.D. * p < 0.05 compared to control. (f and g) dNp63α overexpression (OE) in UC5 (f) and UC14 (g) bladder cancer cells promoted invasion which was blocked by ATDC knockout. n = 3. Error Bars = S.D. * p < 0.05 compared to vector control. ** and *** p < 0.05 compared to dNp63α control. (h-k) dNp63a overexpression promoted invasion in 3D tumor spheroid invasion assays using UC14 (h) and UC5 (j) and this effect was blocked by ATDC knockout. Circles indicate extent of tumor prior to invasion. (i and k) Quantification of linear invasion distance of tumor spheroids shown in (h and j). Graphs shows mean of 4 independent measurements of farthest invaded distance. Error Bars = S.D.

Fig. 6.

TP63 drives in vivo tumor growth and invasion and requires ATDC. TP63 overexpression in UC14 (a) and UC10 (b) increased tumor growth in bladder orthotopic models. UC14 control vs dNp63α OE p value = 0.0053. UC10 control vs dNp63α OE p value = 0.21. n = 8–14 animals per group. Error bars = S.E.M. (c-d) H&E images of orthotopic UC14 (c) and UC10 (d) bladder tumors demonstrate increased invasion in tumors with dNp63α-OE. Arrows indicate areas of tumor invasion into the muscularis propria. Bar = 50μM. (e) Knockout of ATDC blocks TP63 induced tumor growth and invasion in an orthotopic bladder tumor model (UC14 cells) at 4 weeks. Four representative tumor bearing mouse bladders are shown. Upper panels show 2× cross-sectional H&E image of bladder plus tumor (Bar = 500μm). Black arrows indicate tumor. Inset image of corresponding bladder from necropsy. Lower panels show 20× H&E image of bladder tumor (Bar = 50μm). Yellow arrows and dashed line indicate muscle invasion. (f) Quantitation of tumor volumes from (e). Wt = ATDC wildtype. KO = ATDC knockout. Ctl = empty control overexpression vector. OE = dNp63α overexpression. * p = 0.0025 compared to ATDC Wt, TP63 Vector Ctl. ** p = 0.0173 compared to ATDC Wt, TP63 OE. Error bars = S.E.M. (g) Quantification of luciferase activity of bioluminescent primary bladder tumors demonstrated that knock down of ATDC (shATDC) blocked TP63-induced tumor growth of UC10 orthotopic tumors. Error bars = S.E.M. * p = 0.03 compared to UC10 dNp63αOE. (h) Knock down of ATDC (shATDC) blocked TP63-induced metastases from UC10 orthotopic tumors (Control n = 10, dNp63αOE n = 8 and dNp63aOE+shATDC n = 10). Bioluminescent imaging of 3 representative mice from each group are shown 1, 3 and 5 weeks after inoculation of bladder. Numbers and arrows indicate liver metastases confirmed by serial bioluminescent imaging, necropsy and H&E. Numbered H&E images of liver metastases shown on the right match numbered bioluminescent images. Bar = 50μM.