TCF21 Promotes Luminal-Like Differentiation and Suppresses Metastasis in Bladder Cancer Sharada Mokkapati1, Sima P

Published OnlineFirst March 2, 2020; DOI: 10.1158/1541-7786.MCR-19-0766

MOLECULAR CANCER RESEARCH | CANCER GENES AND NETWORKS

TCF21 Promotes Luminal-Like Differentiation and Suppresses Metastasis in Bladder Cancer Sharada Mokkapati1, Sima P. Porten2, Vikram M. Narayan1, Amy H. Lim1, Isuru S. Jayaratna3, Beat Roth4,5, Tiewei Cheng1, Neema Navai1, Matthew Wszolek6, Jonathan Melquist7, Ganiraju Manyam8, Woonyoung Choi9, Bradley Broom8, Shanna Pretzsch1, Bogdan Czerniak10, David J. McConkey9,and Colin P. N. Dinney1

ABSTRACT ◥ Little is known regarding the subclone evolution process in differentially expressed in primary bladder cancer and correspond- advanced bladder cancer, particularly with respect to the genomic ing LN metastasis pairs at an FDR cutoff of 0.1 and a fold change of alterations that lead to the development of metastatic lesions. In this 1. Among those significantly altered, expression of TCF21 was project, we identify gene expression signatures associated with higher in the primary tumor compared with LN metastasis. We metastatic bladder cancer through mRNA expression profiling of validated this finding with qPCR and IHC on patient samples. RNA isolated from 33 primary bladder cancer and corresponding Moreover, TCF21 expression was higher in luminal cell lines and lymph node (LN) metastasis samples. Gene expression profiling knockdown of TCF21 increased invasion, tumor cell dissemination, (GEP) was performed on RNA isolated using the Illumina DASL and metastasis. In contrast, overexpression of TCF21 in highly platform. We identified the developmental transcription factor metastatic basal bladder cancer cell lines decreased their invasive TCF21 as being significantly higher in primary bladder cancer and metastatic potential. compared with LN metastasis samples. To elucidate its function in bladder cancer, loss- and gain-of-function experiments were Implications: TCF21 is differentially overexpressed in primary conducted in bladder cancer cell lines with high and low expression bladder cancer compared with matched LN metastasis, with of TCF21, respectively. We also performed GEP in bladder cancer in vitro and in vivo studies demonstrating a metastasis suppressor cell lines following TCF21 overexpression. We identified 2,390 genes function of this transcription factor.

Introduction of patients diagnosed with bladder cancer are diagnosed with urothelial carcinoma, a heterogenous epithelial malignancy with Bladder cancer accounts for nearly 200,000 deaths worldwide and a high somatic mutation rate (4, 5). is a source of significant morbidity, with over 500,000 new cases Large-scale mRNA expression profiling efforts of MIBC have diagnosed each year. Among all malignancies in the United States, led to the development of several genomic taxonomies for bladder cancer is the fifth most common overall (1). It is estimated bladder cancer, classifying the disease into molecular subtypes that up to one-third of patients present with muscle-invasive to better elucidate disease pathology and potentially guide treat- bladder cancer (MIBC), which can result in mortality within 2 years ment decisions (4, 6, 7). Patients with metastatic bladder cancer of diagnosis in 85% of cases when left untreated (2, 3). The majority have a poor prognosis, and lymph node (LN) positive disease is an independent predictor of worse survival (8, 9). To date, little is known regarding the subclone evolution process in advanced 1Department of Urology, University of Texas MD Anderson Cancer Center, bladder cancer, particularly with respect to the genomic altera- Houston, Texas. 2Department of Urology, University of California San Francisco, tions that lead to the development of metastatic lesions. Previous San Francisco, California. 3Department of Urology, Icahn School of Medicine at 4 reports have suggested that genomic assessments originating Mount Sinai, New York, New York. Department of Urology, University Hospital from the primary tumor alone may underestimate the true of Bern, University of Bern, Bern, Switzerland. 5Department of Urology, University Hospital of Lausanne (CHUV), University of Lausanne, Lausanne, mutational burden that exists within heterogenous tumors (10). Switzerland. 6Department of Urology, Massachusetts General Hospital, Boston, Phylogenetic reconstructions have demonstrated that tumors can Massachusetts. 7Department of Urology, Baptist MD Anderson Cancer Center, exhibit an evolutionary pattern of growth, with metastatic lesions Jacksonville, Florida. 8Department of Bioinformatics and Computational demonstrating greater intratumoral heterogeneity than the pri- Biology, University of Texas MD Anderson Cancer Center, Houston, Texas. 9 mary tumor (11, 12). This heterogeneity can confer challenges Greenberg Bladder Cancer Institute, Johns Hopkins University, Baltimore, in personalizing therapy, and additional investigation into Maryland. 10Department of Pathology, University of Texas MD Anderson Cancer Center, Houston, Texas. the genomic drivers of metastatic lesions in bladder cancer is required. Note: Supplementary data for this article are available at Molecular Cancer In this study, we describe the results of our efforts to use gene Research Online (http://mcr.aacrjournals.org/). expression profiling to identify differences between primary blad- Corresponding Author: Colin P.N. Dinney, University of Texas MD Anderson der cancer tissue and their corresponding metastatic lesions, Cancer Center, 1515 Holcombe Blvd, Unit 1373, Houston, TX 77030. Phone: 713- fi 792-3250; Fax: 713-794-4824; E-mail: [email protected] based on mRNA expression pro ling on matched LN tissue from human specimens. Metastatic xenograft models were then used to – Mol Cancer Res 2020;XX:XX XX confirm the role of novel candidate genes in the development of doi: 10.1158/1541-7786.MCR-19-0766 tumor metastasis, and to identify potential targets for biomarkers 2020 American Association for Cancer Research. and therapy.

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extracted from FFPE matched primary and lymph node metastasis using Materials and Methods an AllPrep DNA/RNA FFPE Kit (Qiagen GmBH). Human subjects Thirty-three well annotated clinically homogeneous patients were Gene expression profiling identified. Patients treated with neoadjuvant chemotherapywere exclud- After RNA isolation, RNA purity and integrity were measured by ed from the study. Tumor tissues from the same histologic component of a NanoDrop ND1000 and an Agilent Bioanalyzer, respectively. High- both the primary tumor and metastases were macrodissected by an quality RNA was then used for the synthesis of biotin-labeled cRNA experienced genitourinary pathologist from formalin-fixed paraffin using the Illumina RNA Amplification Kit (Ambion). Briefly, 500 ng of embedded (FFPE) sections and RNA was isolated. A schematic repre- total RNA was converted to cRNA by in vitro transcription, purified, sentation of our experimental design is shown (Fig. 1A). RNA was and 1.5 mg cRNA was hybridized to Illumina HT12 v4 chips (Illumina).

Figure 1. Transcriptome analysis of primary bladder cancer (BLCA) and LN metastases. A, Schematic representation of experimental study design to perform differential expression analysis between matched primary bladder cancer and LN metastasis samples. B, PCA showing that the primary tumors and metastases form two distinct groups. C, Heatmap of hierarchical clustering showing gene expression of 64 of 2,390 genes that are differentially expressed between the primary and LN metastases at FDR cutoff of 0.1 and with a log fold change of 1. Red: increased relative expression; green: decreased relative expression.

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The slides from Direct Hybridization (Illumina) were washed, scanned from The Jackson Laboratory and used for xenograft experiments. with a Bead Station 500 (Illumina), and the signal intensities from the Between 19 and 20 mice were used in each group. The mice were scanner were quantified using Genome Studio (Illumina). Quantile housed under specific pathogen-free conditions and all surgeries were normalization in linear models was used to normalize the data, which conducted under anesthesia by approved animal protocols. were processed by established techniques. Unsupervised analysis was Labeled (luciferase and red fluorescent protein; luc-RFP) and performed to identify outliers and assess overall similarity/differences orthotopically recycled human bladder cancer cells UM-UC-3, UM- among the samples. Differential expression analysis was performed UC13, and UM-UC-14 were used. Recycling was performed to enrich using t tests and FDR was estimated using the BUM method (13). the spontaneous metastatic phenotype as described previously (15). Cell lines were transfected with the lentiviral TCF21 shRNAi (UM- qPCR UC14) or the TCF21 overexpression pLOC construct (UM-UC3, UM- For qPCR, 20 ng of RNA was used along with the AgPAth-ID One- UC13) were injected at a concentration of 105/50 mL using HBSS fi Step RT-PCR Kit (Thermo Fisher Scienti c) with Taqman probes to (Gibco) by intramural injection into bladder wall. For shRNAi therapy detect TCF21 expression. The comparative CT method was used to experiments, UM-UC14 tumors were allowed to establish for 5 days determine relative gene expression (14). before randomization and induction of knockdown using doxycycline (25 mg/kg/day). Tail vein blood (50–100 mL) was taken weekly to Tumor tissue staining (hematoxylin and eosin) and IHC calculate the number of circulating tumor cells (CTC) as described The histology of primary tumor and metastases in human tumors previously (15). At the end of the study, whole blood was drawn under fi was con rmed by hematoxylin and eosin (H&E) staining. Antigen anesthesia by heart puncture (terminal procedure) before the mice retrieval was accomplished by a combination of heat-mediated retriev- were euthanized. Tumors were then excised, and samples were either al and enzymatic retrieval (trypsin). In the histopathologic studies, formalin fixed and embedded in paraffin, embedded in OCT (Miles, endogenous peroxidase was blocked with 3% H2O2 in PBS before Inc.), or frozen rapidly in liquid nitrogen and stored at 80 C for RNA blocking with 5% horse and 1% goat serum. Rabbit anti-TCF21 and protein extraction. Investigators were blinded to treatment groups (Sigma) was used as a primary antibody. HRP-conjugated goat when performing tumor measurements to minimize the risk of bias. anti-rabbit antibody (1:100; Bio-Rad) was used as a secondary anti- RNA was isolated from cell line with UM-UC3 TCF21-pLOC and body. After DAB incubation, slides were counterstained with hema- UM-UC3 pLOC cells grown in vitro. For UC13, RNA was isolated toxylin. Images were captured using a Nikon Microphot FXA (Nikon). from cells of LN and distant metastasis. Gene expression profiling was then performed. Differential gene expression analysis was Tumor cell lines and culture conditions performed and FDR was estimated using the BUM method (13). The cell lines used were obtained from the U.T. MD Anderson Significant genes were definedbyusingaFDRcut-offof0.1andlog Cancer Center Bladder SPORE Tissue Bank. Their identities were fold change of 1. validated by DNA fingerprinting kits (AmpFlSTR Identifiler Ampli- fi fi fi cation or AmpFlSTR Pro ler PCR Ampli cation; Applied Biosys- In vivo bioluminescence imaging fi tems). The cell lines were cultured as monolayers in modi ed MEM Bioluminescence imaging was conducted on an IVIS 100 imaging supplemented with 10% FBS, 1% vitamin solution (Mediatech), and system with Living Image software (Xenogen; ref. 15). In brief, animals 0.5% each of sodium pyruvate, L-glutamine (Life Technologies), were anesthetized before imaging in a chamber containing a 2.5% penicillin/streptomycin solution, and non-essential amino acids (Bio- fl iso urane/O2 mixture and injected subcutaneously with 15 mg/mL of Whittaker) at 37 C in a 5% CO2 incubator. Cells were tested every luciferin potassium salt in PBS at a dose of 150 mg/kg body weight. A fi fi month for mycoplasma contamination by xing in Carnoy's xative digital gray-scale animal image was then overlaid with a pseudo- and staining with Hoechst 33258 (Sigma B2261, 0.5 micrograms/mL in colored image representing the spatial distribution of detected photons fl HBSS). Extra-nuclear uorescence was considered positive mycoplas- emerging from active luciferase. Signal intensity was quantified as the ma contamination. sum of all detected photons within the region of interest. Each primary tumor and each metastatic site were calculated separately. Silencing with inducible short hairpin RNA; overexpression with precision LentiORFs (pLOC) Statistical analysis For short hairpin RNA (shRNA)mir-based knockdown, recycled Statistical analyses were performed using GraphPad Prism Software UM-UC14 cells (sixth cycle) were plated in six-well plates (105 cells/ (GraphPad). Tumor growth curves in xenografts were analyzed using well) and transfected 24 hours later with a TCF21 lentiviral vector two-way ANOVA with Bonferroni multiple comparisons. Statistical (TRIPZ shRNAmir, Tet-On). Polybrene (Santa Cruz Biotechnology) significance was set at P < 0.05. was used to increase the efficiency of infection. Cells were continuously cultured and selected with puromycin (5 mg/mL). For pLOC stable TCF21 overexpression experiments, recycled UM-UC3 and UM- Results UC13 cells (eight cycle) were transfected 24 hours after plating with Identification of genes critical for LN metastasis in bladder the construct. Cells were continuously cultured and selected with cancer blasticidin. Total RNA and protein lysates were collected to confirm Clinical characteristics of the 33 patients with matched primary efficacy of overexpression. bladder cancer and LN metastasis are shown in Table 1. Importantly, all patients were clinically understaged, and 91% were found to have Tumor xenograft studies lymphovascular invasion (LVI) on final pathology. All mouse experiments were conducted in accordance with the Unsupervised clustering was performed using principal component Institutional Animal Care and Use Committee at MD Anderson Cancer analysis (PCA) using log normalized expression data. The first two Center (IACUC protocol no. 110012735). Female athymic nude mice components of PCA were used to illustrate the variation among the (NCr-nu) that were 5 to 8 weeks old and 20 g in weight were purchased samples. Our analysis demonstrated that the primary tumors and LN

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Table 1. Clinical characteristics of patients in the study. and LN metastasis sample pairs (Fig. 2A). IHC on patient primary/met pairs showed that out of six samples tested, three clearly had a Clinicopathologic characteristics (N ¼ 33) reduction in TCF21 staining in LN metastasis when compared with Mean age (years) SD 64.3 12.2 the primary tumors. A representative image from one of the primary- Gender met pairs is shown (Fig. 2Ba–d). Expression of TCF21 was nuclear and Female 3 (9%) was detectable in epithelial cells in primary bladder cancer (Fig. 2Ba,c) Male 30 (91%) but was reduced or undetectable in LN tumor cells (Fig. 2Bb,d). H&E Race staining of the sections are shown as insets. We also assessed the Caucasian 27 (82%) African American 2 (6%) expression of TCF21 in the normal mouse urothelium and found that Hispanic 3 (9%) TCF21 was moderately expressed in urothelial cells (Supplementary Asian 1 (3%) Fig. S2A). Moderate nuclear expression has also been reported in Clinical stage human bladder urothelium (18). TisN0M0 1 (3%) T1N0M0 4 (12%) TCF21high expression is associated with improved survival in T2N0M0 22 (67%) patients with bladder cancer and correlates with the luminal T3N0M0 6 (18%) subtype of bladder cancer Pathologic stage To identify whether TCF21 expression correlated with survival, we pT2bNþM0 4 (12%) pT3aNþM0 6 (18%) performed a survival analysis on this cohort. At a mean cutoff of 8.2, high n ¼ fi pT3bNþM0 18 (55%) TCF21 tumor samples ( 24) had a statistically signi cant low pT3bNþM1 1 (3%) improvement in survival when compared with TCF21 tumors pT4aNþM0 4 (12%) (n ¼ 22; Fig. 3A). We validated this finding in an independent MD Concomitant CIS 28 (85%) Anderson patient cohort (n ¼ 73) that has been published previously (6). LVI 30 (91%) Samples with low expression (TCF21low, quartile 1) and samples with a Variant histology 13 (40%) high expression (TCF21high, quartile 4) were compared. All three probes in the dataset demonstrated that TCF21high patients had improved a Variant histology defined if features of squamous differentiation, glandular survival when compared with TCF21low patients (Fig. 3B). Probes differentiation, micropapillary architecture, or sarcomatoid transformation were ILMN_1719351 and ILMN_1728570 target the same transcript. Two observed. of the three probes showed statistically significant differences in overall survival (Fig. 3B). Using the UALCAN database (19), we compared metastases formed distinct groups (Fig. 1B). We also performed a expression of TCF21 in bladder cancer tumor and normal bladder tissue. differential expression analysis of the contrast of interest using t tests. TCF21 expression was significantly lower in bladder cancer tissue With an FDR cutoff of 0.1 and log fold change of 1, we identified compared with normal urothelium; significantly lower expression was 2,390 genes that were differentially expressed. A heatmap with the top identified at all tumor stages (Supplementary Figs. S2B and S2C) (20). differentially expressed genes is shown (Fig. 1C). Top 10 candidate up- bladder cancer is heterogenous and tumors can be classified into distinct and downregulated genes in the LN metastases are listed in Supple- subtypes based on expression profiles (5, 21). We also considered the mentary Table S1. This included TCF21, which was found to be relative expression of TCF21 in primary bladder cancer tumor subtypes downregulated in metastatic tissue in two different probes. Upregu- using tumor samples in The Cancer Genome Atlas (TCGA) database lated genes included MIR142 and FCER2, among others (Supplemen- (n ¼ 402). With the super-subtype classification that divides bladder tary Table S1). Several pathways were differentially enriched in the cancer into three subtypes (basal, luminal, and neuroendocrine), we primary and LN metastasis samples; the top-ranked among these (FDR found that TCF21 was higher in the luminal subtype (Fig. 3C,allP < q value < 0.12) are shown in Supplementary Tables S2 and S3. GSEA 0.001). In the five-subtype TCGA classification that includes basal- plots for significant Hallmark pathways (FDR q value < 0.01) for LN squamous, luminal, luminal-infiltrated, luminal-papillary, and neuro- metastasis and primary bladder cancer are shown in Supplementary endocrine subtypes, TCF21 was higher in the luminal and luminal- Figs. S1A and S1B. infiltrated subtype (Fig. 3C,allP < 0.001). Hallmark pathways associated with the primary bladder cancer tissues included epithelial–mesenchymal transition (EMT), myogen- Modulation of TCF21 expression has a major impact on esis, angiogenesis, TNFa, TGFb, and Notch signaling, which have all metastases in orthotopic human bladder cancer xenograft been implicated in bladder cancer pathogenesis (16). Interestingly, models several metabolic pathways such as adipogenesis, glycolysis, and fatty All orthotopic and recycled xenograft models showed much higher acid metabolism were also upregulated in the primary bladder cancer. TCF21 expression than the originally injected 2D cells in vitro (Sup- Metastases, in contrast, were enriched in IFNa and IFNg response plementary Fig. S3A). Strikingly, however, the TCF21 RNA expression pathways and the IL6/JAK/STAT3 or IL2/JAK/STAT5 signaling path- was higher in the “epithelial” xenograft models (UM-UC6, UM-UC9, ways. Importantly, E2F targets which are known to enhance metastasis UM-UC14) when compared with the metastatic “mesenchymal” in other epithelial cancers such as breast cancer were enriched in the xenograft models (UM-UC3, UM-UC13) (Supplementary Fig. S3A) metastasis samples (17). and primary tumors in these metastatic models showed much higher expression of TCF21 than their matched CTCs, LN metastases, and TCF21 expression in tumor tissues distant metastases (Supplementary Fig. S3B). We used the established, Because TCF21 was found to be downregulated using two separate recycled “epithelial” UM-UC14 model, which is highly tumorigenic, probes and is a transcription factor that can control other genes, we but infrequently metastatic (15) to inducibly knockdown TCF21. A sought to further understand its role in bladder cancer metastasis. We schematic of the experimental setup is shown in Supplementary confirmed the sequencing data by performing qPCR on primary tumor Fig. S3C. We confirmed reduced expression of TCF21 in the

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Figure 2. A 71 Primary tumor TCF21 expression in primary tumor and 61 Lymph node metastases metastasis in patients with bladder 51 cancer (BLCA). A, TCF21 expression in 41 primary tumors and metastasis pairs 31 using qPCR from RNA isolated from 21 FFPE sections of 7 patients with blad- 11 der cancer shows higher expression in 1 1.0 the primary tumor when compared with its corresponding lymph node metastasis. B, Loss/reduced expres- Relative expression sion was conﬁrmed by IHC in matched 0.5 primary bladder cancer (b, f, j) and LN metastasis (c, g, k) and comparable in some samples (n, r, v, o, r, s). H&E staining of corresponding primary 0.0 ABCDEFG bladder cancer (a, e, i, m, q, u) and LN Patients metastasis (d, h, l, p, t, x) is also shown. TCF21 Scale bar: 200 mm. Insets show higher B TCF21 magniﬁcation of TCF21 staining. bc H&E H&E a d

fg e h

j k I l

no m p

rs q t

vw u x

Primary BLCA LN metastasis

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Figure 3. High expression levels of TCF21 correlates with better survival outcomes and is correlated with luminal and luminal-infiltrated subtypes of bladder cancer (BLCA). A, Survival analysis of patients with high and low TCF21 expression in the study cohort at mean cutoff of 8.2 showing significant difference in disease-specific (DSS) and overall survival (OS). B, In the MDA cohort, high TCF21 expression was associated better survival outcome in all the three probes tested and was statistically significant in two of three probes. P values are indicated in the graph. C, Comparative analysis of TCF21 expression in the bladder cancer subtypes using the super- subtype classifier and the five-subtype classifier in TCGA dataset (n ¼ 402) shows significant difference in expression among the subtypes.

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A UC14 primary BLCA UC14 LN metastases Tumor weight

1.5 × 1011 3.0 × 1009 Doxycycline Doxycycline Control Control 300 Control P = n.s. Doxycycline P < 0.01 1.0 × 1011 2.0 × 1009 200 mg

Photon count 5.0 × 1010 Photon count 1.0 × 1009 100

840−4 24201612 28 840−4 2824201612 Treatment day Treatment day P = 0.468

B Day 14 Day 28 No. of LN No. of distant 600 200,000 metastases metastases 150,000 100,000 40,000 4 2.5

L blood µ L blood µ L 30,000 400 20,000 10,000 3 2.0 200 1.5 150 2 200 1.0 100 1 50

Number of LN mets 0.5 Number of CTCs per 100 Number of CTCs per 100

0 0 Number of distant mets 0 0.0 Control Doxycycline Control Doxycycline Control Doxycycline Control Doxycycline

P = 0.052 P < 0.001 P < 0.001 P = 0.084 C UC3 Primary BLCA UC3 LN metastases Tumor weight

5.0 × 1010 8.0 × 1009 TCF21-pLOC TCF21-pLOC pLOC pLOC 800 4.0 × 1010 6.0 × 1009 TCF21 pLOC pLOC 3.0 × 1010 600 4.0 × 1009 2.0 × 1010 400 mg Photon count Photon count

2.0 × 1009 1.0 × 1010 200

0 840−4 2824201612 840−4 2824201612 Treatment day Treatment day P = 0.832 *P = n.s. according bonferroni multiple comparisons *P < 0.001 according bonferroni multiple comparisons

D 3 weeks 4 weeks No. of LN No. of distant metastases metastases 400,000 800,000

300,000 600,000 200,000 400,000 8 2.5 100,000

L blood µ L blood µ L 200,000 8,000 2.0 6,000 6 4,000 8,000 2,000 6,000 4,000 1.5 50 2,000 4 40 20 1.0 30 15 2 20 10 0.5 Number of LN met

10 5 No. of distant mets Number of CTCs per 100 Number of CTCs per 100 0 0 0 pLOC TCF21-pLOC pLOC TCF21-pLOC pLOC TCF21 pLOC pLOC TCF21 pLOC P < 0.001 P < 0.0001 P < 0.001 P = 0.066

Figure 4. Role of TCF21 in metastasis in mouse models by overexpression and knockdown experiments. A, In the doxycycline-inducible UC14 TCF21 KD, primary tumor burden as assessed by IVIS Spectrum imaging showed comparable growth patterns in the control and TCF21 KD cells (P ¼ n.s.) whereas LN metastases were more readily detected in the TCF21 KD group (P < 0.01). Final bladder weights were also comparable (P ¼ 0.468). B, CTCs were collected at day 14 and day 28; CTCs were significantly higher in the TCF21 KD group on day 28 (P < 0.001). The final number of LN mets detected in the KD group was statistically significant (P < 0.001); distant mets were higher in the KD group but not significant (P ¼ 0.084). C, TCF21 overexpression in UM-UC3 did not alter the primary tumor growth but detection of LN metastases was lower in the TCF21-OE group when compared with pLOC group (P < 0.001). Primary tumor weights were comparable. D, TCF21 OE significantly reduced the number of CTCs in TCF21 OE group 3 and 4 weeks after injection (P < 0.001 and 0.0001, respectively). The final number of LN metastases was significantly lower in the TCF21 OE group (P < 0.001). The number of distant metastases was higher in pLOC group, although it was not statistically significant between the groups (P ¼ 0.066).

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doxycycline treated group by qPCR (Supplementary Fig. S4A). Inac- performed qPCR probing for the luminal marker CD24 and basal tivation of TCF21 resulted in a significant increase in CTCs and marker CD44 in the UC3 cell line and confirmed an increase in the metastases (both LN and distant metastases) compared with vehicle luminal marker and decrease in the basal marker in TCF21-over- controls (Fig. 4A and B). We compared the primary tumor growth by expressed cells (Fig. 5F). luciferase imaging and by measuring final tumor weights. Primary tumor growth was comparable between the two groups (Fig. 4A). We then transduced the highly metastatic, UM-UC3 cells (recycled eight Discussion times) with an overexpression construct (UM-UC3 TCF21-pLOC). Current therapy for metastatic bladder cancer provides effective We confirmed overexpression by qPCR, Western blotting and by IHC palliation but only a modest prolongation of life. A principal barrier (Supplementary Figs. S4B, S4C, and S4D). In vitro invasion was to the effective eradication of metastasis is our lack of insight into significantly reduced by TCF21 overexpression as assessed by depth- the cellular and molecular properties that mediate the development code assay in both UC3 and UC13 cells (Supplementary Figs. S4E and of metastatic disease. In this study, we establish that primary S4F). In mice, the number of CTCs and the number of metastases bladder cancer tissues express distinct gene signatures compared decreased dramatically in the TCF21 overexpressing group relative to with their corresponding LN metastases, in a population of che- empty vector controls (UM-UC3 pLOC), whereas no change in motherapy-na€ve patients with occult LN metastases found at the primary tumor growth was observed (Fig. 4C and D). Together, these time of cystectomy. In particular, we observed that the expression of data indicate that TCF21 is a strong regulator of tumor cell dissem- TCF21 was lost in LN metastasis. TCF21 is a transcription factor ination and metastasis in our xenograft models. expressed in embryonic tissues and is important for the normal development of organs such as the heart and kidney (22–24). It has TCF21 overexpression promotes luminal-like differentiation a broad expression pattern in adult tissues and is expressed by and suppresses EMT specialized epithelial cells such as podocytes of kidneys and in To identify mechanisms underlying TCF21-mediated metastasis epicardial progenitors of the heart (25, 26). TCF21 is also expressed suppression, we performed gene expression profiling using RNA in normal bladder urothelium (18), where it supports the normal extracted from UM-UC3 and UM-UC13 TCF21 overexpression sys- growth of the bladder during embryogenesis (M.E.R. Pramod; tems. For UC13, cells were isolated from two sources, LN and distant personal communication). TCF21 is implicated in multiple biolog- metastasis (MET, in figure). Heatmaps showing hierarchical clustering ical processes such as EMT, invasion, metastasis, cell cycle, and of significant genes for the three cell lines are shown (Fig. 5A–C). Six autophagy (27). TCF21 has been reported to suppress the progres- genes were significant in the UC3 group, which included TCF21, sion of various cancers including breast, lung, squamous cell CD24, TMEM158, NLRP3, DKFZp761P0423, and ADRB2 (Supple- carcinoma, and renal cell carcinoma (28–32). Tumor cells acquire mentary Table S4). Ninety significant probes representing 84 genes metastatic transformation by either activating genes that contribute were identified in the UC13 LN group whereas 22 significant probes to tumor cell dissemination and invasion and/or by inactivating representing 20 genes were detected in the UC13 MET group (Sup- genes that suppress the process (33). Our clinical observation that plementary Tables S5 and S6). Along with TCF21, we identified CD24 TCF21 is differentially expressed by primary bladder cancer com- as a gene that is commonly upregulated in all three data sets with pared with its corresponding metastasis suggested a role for TCF21 TCF21 overexpression. Boxplots showing increased expression of in the development of bladder cancer metastasis. CD24 in TCF21 overexpression with the two probe sets on the array There is a growing body of knowledge supporting the concept that for all three cell lines is shown in Supplementary Fig. S5A. We also LN metastases act as the gateway for metastatic tumor cell dissemi- checked for expression of basal marker CD44 in these three cell lines. nation (34, 35). We observed that several hallmark pathways including CD44 expression was significantly downregulated in all three probe those related to EMT, myogenesis, angiogenesis, TNFa, TGFb, and sets in the in UC13 LN cells whereas reduction of CD44 was detected in Notch signaling, as well as metabolic pathways involving adipogenesis, two of three probe sets for UC13 MET and UC3 cells (Supplementary glycolysis, and fatty acid metabolism were principally expressed by the Fig. S5B). bladder primary. In contrast, LN metastases were characterized by an GSEA analysis revealed several key signaling pathways enriched in enrichment of IFNa and IFNg response as well as STAT signaling the basal metastatic cell lines UC3 and UC13. EMT was enriched in the pathways. E2F and MYC pathways were also enriched in the LN pLOC group in both the cell lines suggesting that TCF21 overexpres- metastases (17, 36). Thus, the observation that the EMT pathway was sion suppressed EMT and thereby metastasis (Fig. 5D). Several other enriched in primary bladder cancer is of particular interest as activa- key pathways such as glycolysis, inflammatory response, and protein tion of EMT is a critical step in the development of metastasis (37). In secretion were also enriched in the control pLOC cells when compared bladder cancer, the TGFb and Notch pathways have been shown to with TCF21 pLOC cells (Supplementary Fig. S6). promote EMT (38, 39), and the classic mesenchymal gene SNAIL, has Using luminal markers that we have described previously (6), we been shown to promote metastasis (15, 40). generated a luminal score and compared control pLOC to TCF21 In the highly metastatic UC3 and UC13 cell lines, overexpression of overexpression in UC13 cell lines. The UC13 cell line showed a TCF21 significantly inhibited invasion in vitro, and reduced the significantly higher number of altered genes following TCF21 over- number of CTCs and metastases following the orthotopic implanta- expression when compared with UC3. TCF21 overexpression tion of UC3, with no change in the primary tumor burden. Similarly, in increased the luminal score in both the UC13 LN and UC13 MET the poorly metastatic UM-UC14 cell line, TCF21 knockdown pro- lines (Fig. 5E), suggesting that TCF21 promotes more luminal differ- moted metastasis, also without change in the primary tumor burden. entiation in bladder cancer cells. Hierarchical clustering and heatmaps Interestingly, profiling of TCF21 overexpressing UM-UC3 and UM- showing differential expression between UC13 LN and UC13 MET UC13 cells revealed changes in several common pathways, EMT being cells is shown (Supplementary Figs. S7A and S7B). TCF21 clearly one among them. TCF21 has previously been shown to act to suppress upregulated expression of luminal markers KRT7, CD24, and PPARg EMT by downregulating EMT markers such as SNAI1 and vimentin in both cell lines (Supplementary Figs. S7A and S7B). We also while upregulating epithelial markers such as WNT4 and CDH1 in

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TCF21 in the Suppression of Metastasis in Bladder Cancer

Figure 5. Mechanisms underlying TCF21-mediated metastasis suppression in bladder cancer (BLCA). Heat map of hierarchical clustering showing differentially expressed genes at FDR cutoff of 0.1 with a fold change of 1 in A. UM-UC3 TCF21 pLOC (six probes). B, UM-UC13 TCF21 LN (90 probes) C, UM-UC13 TCF21 Met (22 probes) when compared with controls. D, GSEA analysis showing enrichment of EMT in control UC3 pLOC and UC13 pLOC samples when compared with UC3 TCF21 OE and UC13 TCF21 OE samples. E, Luminal score comparison between the pLOC control and TCF21 OE samples in cells from UC13 LN and UC13 distant MET samples showing increased luminal score after TCF21 overexpression. F, qPCR analysis conﬁrming increased expression of luminal marker CD24 and decreased expression of basal markers CD44 in UM-UC3 TCF21 pLOC cells.

several cancers (28, 41–43). In bladder cancer, TCF21 also appears to Molecular profiling of bladder cancer has identified distinct geno- suppress EMT. Taken together with our orthotopic mouse model data, mic subtypes, broadly classified as basal or luminal, with differing this convincingly demonstrates a metastasis suppressor function of biology and responses to conventional therapies (5, 21, 44). We found TCF21 in both early and late metastasis events. TCF21 expression to be enriched within the luminal subtype, which is

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less metastatic than the basal subtype of bladder cancer. Using the five- out specifically the tumor (and not stromal tissue) present within subtype TCGA classification, TCF21 was enriched in the luminal and each tissue block. luminal-infiltrated subtypes. The latter shares gene expression profiles In summary, TCF21 is a metastasis suppressor for bladder cancer with the p53-like subtype described by our group (21). The p53-like and appears to play an important role in determining the metastatic subtype is characterized by a wild-type p53 gene signature, high tumor behavior of bladder cancer cells. At higher expression levels, TCF21 infiltration, and resistance to cisplatin-based chemotherapy. Recent conferred a more epithelial phenotype and reduced LN metastasis studies have identified TCF21 as a direct transcriptional target of the without affecting primary tumor growth in mouse models. Interest- p53 pathway in endometrial carcinoma (45). The basal cell lines UC3 ingly, TCF21 was enriched in the p53-like subtype of bladder cancer and UC13 selectively express the canonical basal marker CD44 with a that is inherently resistant to cisplatin-based chemotherapy. Defining corresponding loss of expression of the luminal marker CD24. TCF21 the direct transcriptional targets and studying its effect on chemo- overexpression altered the expression pattern of both by UM-UC3 and therapeutic responses will further enable us to increase our current UC13 cells, suggesting that TCF21 overexpression directs the basal understanding of TCF21 in bladder cancer. UC3 and UC13 cells to adopt a luminal cell type with increased expression of CD24 and decreased expression of CD44. Thus, TCF21 Disclosure of Potential Conflicts of Interest appears to inhibit tumor cell dissemination and metastasis by at least S.P. Porten has received speakers bureau honoraria from Bristol Myers Squibb two distinct mechanisms: (i) by suppressing EMT, and (ii) by altering andPhotocure.D.J.McConkeyisaConsultant at Janssen, H3 Biomedicine, biomarker expression of other metastases related genes, which togeth- Ranier Pharmaceuticals; also reports of receiving commercial research grant from Astra-Zeneca and Ranier Pharmaceuticals. No potential conflicts of interest were er contribute to the suppression of metastasis. disclosed by the other authors. The identification of distinct gene expression patterns that char- acterize the primary bladder tumor and their corresponding metas- Authors’ Contributions tases has important therapeutic implications. For instance, gene Conception and design: S.P. Porten, B. Roth, M. Wszolek, D.J. McConkey, expression profiling of the primary bladder cancer is commonly used C.P.N. Dinney to identify distinct molecular subtypes with differing sensitivity to Development of methodology: S. Mokkapati, V.M. Narayan, B. Roth, T. Cheng, neoadjuvant chemotherapy (46). Our data suggest that it may not be J. Melquist, D.J. McConkey, C.P.N. Dinney sufficient to simply prescribe therapy based solely on the genomic Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Mokkapati, S.P. Porten, V.M. Narayan, I.S. Jayaratna, B. Roth, assessment of the primary tumor, as this does not fully capture the N. Navai, M. Wszolek, J. Melquist, G. Manyam, W. Choi, B. Czerniak, D.J. McConkey, molecular heterogeneity of the corresponding metastasis. This is C.P.N. Dinney especially important in light of the current debate regarding whether Analysis and interpretation of data (e.g., statistical analysis, biostatistics, a pT0 state is an appropriate surrogate endpoint for cancer outcomes, computational analysis): S. Mokkapati, S.P. Porten, V.M. Narayan, I.S. Jayaratna, given that NAC is delivered to treat micrometastasis in patients with B. Roth, T. Cheng, J. Melquist, G. Manyam, W. Choi, B. Broom, D.J. McConkey, clinically undetectable metastatic disease. C.P.N. Dinney Writing, review, and/or revision of the manuscript: S. Mokkapati, S.P. Porten, Limitations of this study include the fact that matched primary V.M. Narayan, A.H. Lim, I.S. Jayaratna, T. Cheng, N. Navai, G. Manyam, tumors and LN samples were analyzed as distinct groups, rather D.J. McConkey, C.P.N. Dinney than as independent samples. Althoughthisallowedustoidentify Administrative, technical, or material support (i.e., reporting or organizing data, major patterns of differential gene expression, it may have masked constructing databases): V.M. Narayan, S. Pretzsch more granular differences in metastasis development pathways that Study supervision: C.P.N. Dinney can only be discerned on a per-patient level. Only one area of the primary tumor was sequenced (as opposed to sampling multiple Acknowledgments different sections). In addition, our study did not directly assess the University of Texas MD Anderson Cancer Center SPORE in Genitourinary Cancer (Grant No. P50CA091846, to C.P.N. Dinney). effect of TCF21 on CD24 and CD44 on a transcriptional level, and this remains an area of ongoing study. Murine studies included only The costs of publication of this article were defrayed in part by the payment of page a water control, instead of an shRNA/doxycycline control, which charges. This article must therefore be hereby marked advertisement in accordance could introduce confounding interactions into our findings. Some with 18 U.S.C. Section 1734 solely to indicate this fact. of the gene expression findings may also reflect the presence of contaminating immune cells within the lymph node metastasis Received July 30, 2019; revised December 10, 2019; accepted February 26, 2020; tissue, although care was taken to histologically identify and dissect published first March 2, 2020.

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TCF21 Promotes Luminal-Like Differentiation and Suppresses Metastasis in Bladder Cancer

Sharada Mokkapati, Sima P. Porten, Vikram M. Narayan, et al.

Mol Cancer Res Published OnlineFirst March 2, 2020.

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