TCF21 Promotes Luminal-Like Differentiation and Suppresses Metastasis in Bladder Cancer Running Title: TCF21 in the Suppression of Metastasis in Bladder Cancer

Author Manuscript Published OnlineFirst on March 2, 2020; DOI: 10.1158/1541-7786.MCR-19-0766 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

TCF21 promotes luminal-like differentiation and suppresses metastasis in bladder cancer Running title: TCF21 in the suppression of metastasis in bladder cancer

Sharada Mokkapati1, Sima P. Porten2, Vikram M. Narayan1, Amy H. Lim1, Isuru S. Jayaratna3, Beat Roth4,10, Tiewei Cheng1, Neema Navai1, Matthew Wszolek5, Jonathan Melquist6, Ganiraju Manyam7, Woonyoung Choi8, Bradley Broom7, Shanna Pretzsch1, Bogdan Czerniak9, David J. McConkey8, and Colin P. N. Dinney1

1Department of Urology, University of Texas MD Anderson Cancer Center, Houston, TX, USA 2Department of Urology, University of California San Francisco, San Francisco, CA, USA 3Department of Urology, Icahn School of Medicine at Mount Sinai, New York, NY, USA 4Department of Urology, University Hospital of Bern, University of Bern, Bern, Switzerland 5Department of Urology, Massachusetts General Hospital, Boston, MA, USA 6Department of Urology, Baptist MD Anderson Cancer Center, Jacksonville, FL, USA 7Department of Bioinformatics and Computational Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA 8Greenberg Bladder Cancer Institute, Johns Hopkins University, Baltimore, MD, USA 9Department of Pathology, University of Texas MD Anderson Cancer Center, Houston, TX, USA 10Department of Urology, University Hospital of Lausanne (CHUV), University of Lausanne, Lausanne, Switzerland

Address correspondence to:

Colin P.N. Dinney, M.D. Department of Urology, Unit 1373 University of Texas MD Anderson Cancer Center Houston, TX 77030 USA Tel (713) 563-7465 E-mail: [email protected]

Conflicts of Interest Statement: CPND participates in research for FKD Therapies Oy, the NCI, and the University of Finland, Faculty of Health Sciences. The remaining authors declare no potential conflicts of interest.

Downloaded from mcr.aacrjournals.org on September 28, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 2, 2020; DOI: 10.1158/1541-7786.MCR-19-0766 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Little is known regarding the subclone evolution process in advanced bladder cancer (BLCA), particularly with respect to the genomic alterations that lead to the development of metastatic lesions. In this project, we identify gene expression signatures associated with metastatic BLCA through mRNA expression profiling of RNA isolated from 33 primary BLCA and corresponding lymph node (LN) metastasis samples. Gene expression profiling (GEP) was performed on RNA isolated using the Illumina DASL platform. We identified the developmental transcription factor TCF21 as being significantly higher in primary BLCA compared to LN metastasis samples. To elucidate its function in BLCA, loss- and gain-of- function experiments were conducted in BLCA cell lines with high and low expression of TCF21, respectively. We also performed GEP in BLCA cell lines following TCF21 overexpression. We identified 2390 genes differentially expressed in primary BLCA and corresponding LN metastasis pairs at an FDR cutoff of 0.1 and a fold change of 1. Among those significantly altered, expression of TCF21 was higher in the primary tumor compared to LN metastasis. We validated this finding with Q-PCR and IHC on patient samples. Moreover, TCF21 expression was higher in luminal cell lines and knockdown of TCF21 increased invasion, tumor cell dissemination and metastasis. In contrast, overexpression of TCF21 in highly metastatic basal BLCA cell lines decreased their invasive and metastatic potential.

Implications statement: TCF21 is differentially overexpressed in primary BLCA compared to matched LN metastasis, with in vitro and in vivo studies demonstrating a metastasis suppressor function of this transcription factor.

Introduction

Bladder cancer (BLCA) accounts for nearly 200,000 deaths worldwide and is a source of significant morbidity, with over 500,000 new cases diagnosed each year. Among all malignancies in the United States, BLCA is the fifth most common overall.[1] It is estimated that up to one-third of patients present with muscle-invasive bladder cancer (MIBC), which can result in mortality within two years of diagnosis in 85% of cases when left untreated.[2,3] The majority of patients diagnosed with BLCA are diagnosed with urothelial carcinoma, a heterogenous epithelial malignancy with a high somatic mutation rate.[4,5]

Large-scale mRNA expression profiling efforts of MIBC have led to the development of several genomic taxonomies for BLCA, classifying the disease into molecular subtypes to better elucidate disease pathology and potentially guide treatment decisions.[4,6,7] Patients with metastatic BLCA have a poor prognosis, and LN positive disease is an independent predictor of worse survival.[8,9] To date, little is known regarding the subclone evolution process in advanced BLCA, particularly with respect to the genomic alterations that lead to the development of metastatic lesions. Previous reports have suggested that genomic assessments originating from the primary tumor alone may underestimate the true mutational burden that exists within heterogenous tumors.[10] Phylogenetic reconstructions have demonstrated that tumors can exhibit an evolutionary pattern of growth, with metastatic lesions demonstrating greater intratumoral heterogeneity than the primary tumor. [11,12] This heterogeneity can confer challenges in personalizing therapy, and additional investigation into the genomic drivers of metastatic lesions in BLCA is required.

In this study, we describe the results of our efforts to use gene expression profiling to identify differences between primary BLCA tissue and their corresponding metastatic lesions, based on mRNA expression profiling on matched LN tissue from human specimens. Metastatic xenograft models were then used to confirm the role of novel candidate genes in the development of tumor metastasis, and to identify potential targets for biomarkers and therapy.

Materials and Methods Human subjects

Thirty-three well annotated clinically homogeneous patients were identified. Patients treated with neoadjuvant chemotherapy were excluded from the study. Tumor tissues from the same histologic component of both the primary tumor and metastases were macrodissected by an experienced genitourinary pathologist from formalin-fixed paraffin embedded (FFPE) sections and RNA was isolated. A schematic representation of our experimental design is shown (Figure 1A). RNA was extracted from FFPE matched primary and lymph node metastasis using an AllPrep DNA/RNA FFPE kit (Qiagen GmBH, Germany).

Gene expression profiling After RNA isolation, RNA purity and integrity were measured by a NanoDrop ND1000 and an Agilent Bioanalyzer, respectively. High quality RNA was then used for the synthesis of biotin-labeled cRNA using the Illumina RNA amplification kit (Ambion, USA). Briefly, 500 ng of total RNA was converted to cRNA by in vitro transcription, purified, and 1.5 μg cRNA was hybridized to Illumina HT12 v4 chips (Illumina, USA). The slides from Direct Hybridization (Illumina, USA) were washed, scanned with a Bead Station 500 (Illumina, USA), and the signal intensities from the scanner were quantified using Genome Studio (Illumina, USA). Quantile normalization in linear models was used to normalize the data, which were processed by established techniques. Unsupervised analysis was performed to identify outliers and assess overall similarity/differences amongst the samples. Differential expression analysis was performed using t-tests and false discovery rate (FDR) was estimated using the BUM method.[13]

Quantitative Real Time PCR For Q-PCR, 20 ng of RNA was used along with the AgPAth-ID One-Step RT-PCR kit (ThermoFisher, USA) with Taqman probes to detect TCF21 expression. The comparative CT method was used to determine relative gene expression.[14]

Tumor tissue staining (H&E) and immunohistochemistry The histology of primary tumor and metastases in human tumors was confirmed by H&E staining. Antigen retrieval was accomplished by a combination of heat-mediated retrieval and enzymatic retrieval (trypsin). In the histopathological studies, endogenous peroxidase was blocked with 3% H2O2 in PBS before blocking with 5% horse and 1% goat serum. Rabbit anti-TCF21 (Sigma, USA) was used as a primary antibody. HRP-conjugated goat anti-rabbit antibody (1:100; BioRad, USA) was used as a secondary antibody. After

DAB incubation, slides were counterstained with hematoxylin. Images were captured using a Nikon Microphot FXA (Nikon, Japan).

Tumor cell lines and culture conditions The cell lines used were obtained from the U.T. MD Anderson Cancer Center Bladder

SPORE Tissue Bank. Their identities were validated by DNA fingerprinting kits

(AmpFlSTR® Identifiler® Amplification or AmpFlSTR® Profiler® PCR Amplification,

Applied Biosystems, USA). The cell lines were cultured as monolayers in modified MEM supplemented with 10% FBS, 1% vitamin solution (Mediatech), and 0.5% each of sodium pyruvate, L-glutamine (Life Technologies, USA), penicillin/streptomycin solution, and non- essential amino acids (BioWhittaker, USA) at 37°C in a 5% CO2 incubator. Cells were tested every month for mycoplasma contamination by fixing in Carnoy’s fixative and staining with

Hoechst 33258 (Sigma B2261, 0.5 micrograms/ml in HBSS). Extra-nuclear fluorescence was considered positive mycoplasma contamination.

Silencing with inducible short hairpin RNA (shRNAi); overexpression with Precision LentiORFs (pLOC) For shRNAmir-based knockdown, recycled UM-UC14 cells (6th cycle) were plated in 6-well plates (105 cells/well) and transfected 24 hours later with a TCF21 lentiviral vector (TRIPZ shRNAmir, Tet-On®). Polybrene (Santa Cruz, USA) was used to increase the efficiency of infection. Cells were continuously cultured and selected with puromycin (5μg/ml). For pLOC stable TCF21 overexpression experiments, recycled UM-UC3 and UM- UC13 cells (8th cycle) were transfected 24 hours after plating with the construct. Cells were continuously cultured and selected with blasticidin. Total RNA and protein lysates were collected to confirm efficacy of overexpression.

Tumor xenograft studies All mouse experiments were conducted in accordance with the Institutional Animal Care and Use Committee at MD Anderson Cancer Center (IACUC protocol #110012735). Female athymic nude mice (NCr-nu) that were 5 to 8 weeks old and 20g in weight were

purchased from The Jackson Laboratory (USA) and used for xenograft experiments. Between 19 to 20 mice were used in each group. The mice were housed under specific pathogen-free conditions and all surgeries were conducted under anesthesia by approved animal protocols. Labeled (luciferase and red fluorescent protein; luc-RFP) and orthotopically recycled human BLCA cells UM-UC-3, UM-UC13 and UM-UC-14 were used. Recycling was performed to enrich the spontaneous metastatic phenotype as previously described.[15] Cell lines were transfected with the lentiviral TCF21 shRNAi (UM-UC14) or the TCF21 overexpression pLOC construct (UM-UC3, UM-UC13) were injected at a concentration of 105/50μL using HBSS (GIBCO®, USA) by intramural injection into bladder wall. For shRNAi therapy experiments, UM-UC14 tumors were allowed to establish for 5 days before randomization and induction of knockdown using doxycycline (25mg/kg per day). Tail vein blood (50-100μl) was taken weekly in order to calculate the number of circulating tumor cells (CTCs) as previously described.[15] At the end of the study, whole blood was drawn under anesthesia by heart puncture (terminal procedure) before the mice were euthanized. Tumors were then excised, and samples were either formalin fixed and embedded in paraffin, embedded in OCT (Miles, Inc, USA), or frozen rapidly in liquid nitrogen and stored at -80°C for RNA and protein extraction. Investigators were blinded to treatment groups when performing tumor measurements to minimize the risk of bias. RNA was isolated from cell line with UM-UC3 TCF21-pLOC and UM-UC3 pLOC cells grown in vitro. For UC13, RNA was isolated from cells of LN and distant metastasis. Gene expression profiling was then performed. Differential gene expression analysis was performed and false discovery rate (FDR) was estimated using the BUM method.[13] Significant genes were defined by using a FDR cut-off of 0.1 and log fold change of 1.

In vivo bioluminescence imaging Bioluminescence imaging was conducted on an IVIS 100 imaging system with Living Image software (Xenogen, USA).[15] In brief, animals were anesthetized before imaging in a chamber containing a 2.5% isoflurane/O2 mixture and injected subcutaneously with 15 mg/mL of luciferin potassium salt in PBS at a dose of 150 mg/kg body weight. A digital gray- scale animal image was then overlaid with a pseudo-colored image representing the spatial distribution of detected photons emerging from active luciferase. Signal intensity was quantified as the sum of all detected photons within the region of interest. Each primary tumor and each metastatic site were calculated separately.

Statistics Statistical analyses were performed using GraphPad Prism Software (GraphPad, USA). Tumor growth curves in xenografts were analyzed using two-way ANOVA with Bonferroni multiple comparisons. Statistical significance was set at p<0.05.

Results

Identification of genes critical for LN metastasis in BLCA Clinical characteristics of the 33 patients with matched primary BLCA and LN metastasis are shown in Table 1. Importantly, all patients were clinically understaged, and 91% were found to have lymphovascular invasion (LVI) on final pathology. Unsupervised clustering was performed using Principal Component Analysis (PCA) using log normalized expression data. The first two components of PCA were used to illustrate the variation among the samples. Our analysis demonstrated that the primary tumors and LN metastases formed distinct groups (Figure 1B). We also performed a differential expression analysis of the contrast of interest using t-tests. With an FDR cut off of 0.1 and log fold change of 1, we identified 2390 genes that were differentially expressed. A heatmap with the top differentially expressed genes is shown (Figure 1C). Top 10 candidate up- and downregulated genes in the LN metastases are listed in Supplemental Table 1. This included TCF21, which was found to be downregulated in metastatic tissue in two different probes. Upregulated genes included MIR142 and FCER2, among others (Supplemental Table 1). Several pathways were differentially enriched in the primary and LN metastasis samples; the top-ranked among these (FDR q value<0.12) are shown in Supplemental Table 2 & 3. GSEA plots for significant Hallmark pathways (FDR q value <0.01) for LN metastasis and primary BLCA are shown in Supplemental Figure 1 A,B. Hallmark pathways associated with the primary BLCA tissues included epithelial- mesenchymal transition (EMT), myogenesis, angiogenesis, TNFα, TGFβ and Notch signaling which have all been implicated in BLCA pathogenesis.[16] Interestingly, several metabolic pathways such as adipogenesis, glycolysis, and fatty acid metabolism were also upregulated in the primary BLCA. Metastases, in contrast, were enriched in interferon-α and interferon-γ response pathways and the IL6/JAK/STAT3 or IL2/JAK/STAT5 signaling pathways. Importantly, E2F targets which are known to enhance metastasis in other epithelial cancers such as breast cancer were enriched in the metastasis samples.[17]

TCF21 expression in tumor tissues

Because TCF21 was found to be downregulated using two separate probes and is a transcription factor that can control other genes, we sought to further understand its role in BLCA metastasis. We confirmed the sequencing data by performing Q-PCR on primary tumor and LN metastasis sample pairs (Figure 2A). Immunohistochemistry (IHC) on patient primary/met pairs showed that out of 6 samples tested, 3 clearly had a reduction in TCF21 staining in LN metastasis when compared to the primary tumors. A representative image from one of the primary-met pairs is shown (Figure 2B a-d). Expression of TCF21 was nuclear and was detectable in epithelial cells in primary BLCA (Figure 2B a,c) but was reduced or undetectable in LN tumor cells (Figure 2B b,d). H&E staining of the sections are shown as insets (Figure 2B a’-d’). We also assessed the expression of TCF21 in the normal mouse urothelium and found that TCF21 was moderately expressed in urothelial cells (Supplemental Figure 2A). Moderate nuclear expression has also been reported in human bladder urothelium.[18]

TCF21high expression is associated with improved survival in BLCA cancer patients and correlates with the luminal subtype of BLCA

To identify whether TCF21 expression correlated with survival, we performed a survival analysis on this cohort. At a mean cut off of 8.2, TCF21high tumor samples (n=24) had a statistically significant improvement in survival when compared to TCF21low tumors (n=22) (Figure 3A). We validated this finding in an independent MD Anderson patient cohort (n=73) that has been previously published.[6] Samples with low expression (TCF21low, quartile 1) and samples with high expression (TCF21high, quartile 4) were compared. All three probes in the dataset demonstrated that TCF21high patients had improved survival when compared to TCF21low patients (Figure 3B). Probes ILMN_1719351 and ILMN_1728570 target the same transcript. Two of the three probes showed statistically significant differences in overall survival (Figure 3B). Using the UALCAN database,[19] we compared expression of TCF21 in BLCA tumor and normal bladder tissue. TCF21 expression was significantly lower in BLCA tissue compared to normal urothelium; significantly lower expression was identified at all tumor stages (Supplemental 2B,C).[20] Bladder cancer is heterogenous and tumors can be classified into distinct subtypes based on expression profiles.[5,21] We also considered the relative expression of TCF21 in primary BLCA tumor subtypes using tumor samples in The Cancer Genome Atlas (TCGA) database (n=402). With the super-subtype classification that divides BLCA into three subtypes (basal, luminal and neuroendocrine), we found that TCF21 was higher in the luminal subtype (Figure 3C, all p<0.001). In the 5-

subtype TCGA classification that includes basal-squamous, luminal, luminal-infiltrated, luminal-papillary and neuroendocrine subtypes, TCF21 was higher in the luminal and luminal-infiltrated subtype (Figure 3C, all p<0.001).

Modulation of TCF21 expression has a major impact on metastases in orthotopic human BLCA xenograft models.

All orthotopic and recycled xenograft models showed much higher TCF21 expression than the originally injected 2D cells in vitro (Supplemental Figure 3A). Strikingly, however, the TCF21 RNA expression was higher in the “epithelial” xenograft models (UM-UC6, UM- UC9, UM-UC14) when compared to the metastatic “mesenchymal” xenograft models (UM- UC3, UM-UC13) (Supplemental Figure 3A) and primary tumors in these metastatic models showed much higher expression of TCF21 than their matched CTCs, LN metastases and distant metastases (Supplemental Figure 3B). We used the established, recycled “epithelial” UM-UC14 model which is highly tumorigenic, but infrequently metastatic[15] to inducibly knockdown TCF21. A schematic of the experimental setup is shown in Supplemental Figure 3C. We confirmed reduced expression of TCF21 in the doxycycline treated group by Q-PCR (Supplemental Figure 4A). Inactivation of TCF21 resulted in a significant increase in CTCs and metastases (both LN and distant metastases) compared to vehicle controls (Figure 4A,B). We compared the primary tumor growth by luciferase imaging and by measuring final tumor weights. Primary tumor growth was comparable between the two groups (Figure 4A). We then transduced the highly metastatic, UM-UC3 cells (recycled 8-times) with an overexpression construct (UM-UC3 TCF21-pLOC). We confirmed overexpression by Q- PCR, western blotting and by IHC (Supplemental Figure 4B,C,D). In vitro invasion was significantly reduced by TCF21 overexpression as assessed by depthcode assay in both UC3 and UC13 cells (Supplemental Figure 4E,F). In mice, the number of CTCs and the number of metastases decreased dramatically in the TCF21 overexpressing group relative to empty vector controls (UM-UC3 pLOC), while no change in primary tumor growth was observed (Figure 4C and 4D). Together, these data indicate that TCF21 is a strong regulator of tumor cell dissemination and metastasis in our xenograft models.

TCF21 overexpression promotes luminal-like differentiation and suppresses EMT

In order to identify mechanisms underlying TCF21-mediated metastasis suppression, we performed gene expression profiling using RNA extracted from UM-UC3 and UM-UC13 TCF21 overexpression systems. For UC13, cells were isolated from two sources, LN and

distant metastasis (MET, in figure). Heatmaps showing hierarchical clustering of significant genes for the three cell lines are shown (Figure 5A-C). Six genes were significant in the UC3 group, which included TCF21, CD24, TMEM158, NLRP3, DKFZp761P0423 and ADRB2 (Supplemental Table 4). Ninety significant probes representing 84 genes were identified in the UC13 LN group whereas 22 significant probes representing 20 genes were detected in the UC13 MET group (Supplemental Table 5 & 6). Along with TCF21, we identified CD24 as a gene that is commonly upregulated in all three data sets with TCF21 overexpression. Boxplots showing increased expression of CD24 in TCF21 overexpression with the two probe sets on the array for all three cell lines is shown in Supplemental Figure 5A. We also checked for expression of basal marker CD44 in these 3 cell lines. CD44 expression was significantly downregulated in all three probe sets in the in UC13 LN cells while reduction of CD44 was detected in 2 of 3 probe sets for UC13 MET and UC3 cells (Supplemental Figure 5B).

GSEA analysis revealed several key signaling pathways enriched in the basal metastatic cell lines UC3 and UC13. EMT was enriched in the pLOC group in both the cell lines suggesting that TCF21 overexpression suppressed EMT and thereby metastasis (Figure 5D). Several other key pathways such as glycolysis, inflammatory response and protein secretion were also enriched in the control pLOC cells when compared to TCF21 pLOC cells (Supplemental Figure 6).

Using luminal markers that we have previously described,[6] we generated a luminal score and compared control pLOC to TCF21 overexpression in UC13 cell lines. The UC13 cell line showed a significantly higher number of altered genes following TCF21 overexpression when compared to UC3. TCF21 overexpression increased the luminal score in both the UC13 LN and UC13 MET lines (Figure 5E), suggesting that TCF21 promotes more luminal differentiation in BLCA cells. Hierarchical clustering and heatmaps showing differential expression between UC13 LN and UC13 MET cells is shown (Supplemental Figure 7 A, B). TCF21 clearly upregulated expression of luminal markers KRT7, CD24 and PPAR in both cell lines (Supplemental Figure 7 A, B). We also performed Q-PCR probing for the luminal marker CD24 and basal marker CD44 in the UC3 cell line and confirmed an increase in the luminal marker and decrease in the basal marker in TCF21-overexpressed cells (Figure 5F).

Discussion

Current therapy for metastatic BLCA provides effective palliation but only a modest prolongation of life. A principal barrier to the effective eradication of metastasis is our lack of insight into the cellular and molecular properties that mediate the development of metastatic disease. In this study, we establish that primary BLCA tissues express distinct gene signatures compared to their corresponding LN metastases, in a population of chemotherapy-naïve patients with occult LN metastases found at the time of cystectomy. In particular, we observed that the expression of TCF21 was lost in LN metastasis. TCF21 is a transcription factor expressed in embryonic tissues and is important for the normal development of organs such as the heart and kidney.[22-24] It has a broad expression pattern in adult tissues and is expressed by specialized epithelial cells such as podocytes of kidneys and in epicardial progenitors of the heart.[25,26] TCF21 is also expressed in normal bladder urothelium,[18] where it supports the normal growth of the bladder during embryogenesis.[27] TCF21 is implicated in multiple biological processes such as EMT, invasion, metastasis, cell cycle and autophagy.[28] TCF21 has been reported to suppress the progression of various cancers including breast, lung, squamous cell carcinoma, and renal cell carcinoma.[29-33] Tumor cells acquire metastatic transformation by either activating genes that contribute to tumor cell dissemination and invasion and/or by inactivating genes that suppress the process.[34] Our clinical observation that TCF21 is differentially expressed by primary BLCA compared to its corresponding metastasis suggested a role for TCF21 in the development of BLCA metastasis. There is a growing body of knowledge supporting the concept that LN metastases act as the gateway for metastatic tumor cell dissemination.[35,36] We observed that several hallmark pathways including those related to EMT, myogenesis, angiogenesis, TNFα, TGFβ and Notch signaling, as well as metabolic pathways involving adipogenesis, glycolysis, and fatty acid metabolism were principally expressed by the bladder primary. In contrast, LN metastases were characterized by an enrichment of interferon-α and interferon-γ response as well as STAT signaling pathways. E2F and MYC pathways were also enriched in the LN metastases.[17,37] Thus, the observation that the EMT pathway was enriched in primary BLCA is of particular interest as activation of EMT is a critical step in the development of metastasis.[38] In BLCA, the TGFβ and Notch pathways have been shown to promote EMT,[39,40] and the classic mesenchymal gene SNAIL, has been shown to promote metastasis.[15,41] In the highly metastatic UC3 and UC13 cell lines, overexpression of TCF21 significantly inhibited invasion in vitro, and reduced the number of CTCs and metastases following the orthotopic implantation of UC3, with no change in the primary tumor burden.

Similarly, in the poorly metastatic UM-UC14 cell line, TCF21 knockdown promoted metastasis, also without change in the primary tumor burden. Interestingly, profiling of TCF21 overexpressing UM-UC3 and UM-UC13 cells revealed changes in several common pathways, EMT being one among them. TCF21 has previously been shown to act to suppress EMT by downregulating EMT markers such as SNAI1 and vimentin while upregulating epithelial markers such as WNT4 and CDH1 in several cancers.[29, 42-44] In BLCA, TCF21 also appears to suppress EMT. Taken together with our orthotopic mouse model data, this convincingly demonstrates a metastasis suppressor function of TCF21 in both early and late metastasis events. Molecular profiling of BLCA has identified distinct genomic subtypes, broadly classified as basal or luminal, with differing biology and responses to conventional therapies.[5,21,45] We found TCF21 expression to be enriched within the luminal subtype, which is less metastatic than the basal subtype of BLCA. Using the 5-subtype TCGA classification, TCF21 was enriched in the luminal and luminal-infiltrated subtypes. The latter shares gene expression profiles with the p53-like subtype described by our group.[21] The p53-like subtype is characterized by a wild-type p53 gene signature, high tumor infiltration, and resistance to cisplatin-based chemotherapy. Recent studies have identified TCF21 as a direct transcriptional target of the p53 pathway in endometrial carcinoma.[46] The basal cell lines UC3 and UC13 selectively express the canonical basal marker CD44 with a corresponding loss of expression of the luminal marker CD24. TCF21 overexpression altered the expression pattern of both by UM-UC3 and UC13 cells, suggesting that TCF21 overexpression directs the basal UC3 and UC13 cells to adopt a luminal cell type with increased expression of CD24 and decreased expression of CD44. Thus, TCF21 appears to inhibit tumor cell dissemination and metastasis by at least two distinct mechanisms: (1) by suppressing EMT, and (2) by altering biomarker expression of other metastases related genes, which together contribute to the suppression of metastasis. The identification of distinct gene expression patterns that characterize the primary bladder tumor and their corresponding metastases has important therapeutic implications. For instance, gene expression profiling of the primary BLCA is commonly used to identify distinct molecular subtypes with differing sensitivity to neoadjuvant chemotherapy.[47] Our data suggests that it may not be sufficient to simply prescribe therapy based solely on the genomic assessment of the primary tumor, as this does not fully capture the molecular heterogeneity of the corresponding metastasis. This is especially important in light of the current debate regarding whether a pT0 state is an appropriate surrogate endpoint for cancer

outcomes, given that NAC is delivered to treat micrometastasis in patients with clinically undetectable metastatic disease.

Limitations of this study include the fact that matched primary tumors and LN samples were analyzed as distinct groups, rather than as independent samples. Although this allowed us to identify major patterns of differential gene expression, it may have masked more granular differences in metastasis development pathways that can only be discerned on a per- patient level. Only one area of the primary tumor was sequenced (as opposed to sampling multiple different sections). Additionally, our study did not directly assess the effect of TCF21 on CD24 and CD44 on a transcriptional level, and this remains an area of ongoing study. Murine studies included only a water control, instead of an shRNA/doxycycline control, which could introduce confounding interactions into our findings. Some of the gene expression findings may also reflect the presence of contaminating immune cells within the lymph node metastasis tissue, although care was taken to histologically identify and dissect out specifically the tumor (and not stromal tissue) present within each tissue block.

In summary, TCF21 is a metastasis suppressor for BLCA and appears to play an important role in determining the metastatic behavior of BLCA cells. At higher expression levels, TCF21 conferred a more epithelial phenotype and reduced LN metastasis without affecting primary tumor growth in mouse models. Interestingly, TCF21 was enriched in the p53-like subtype of BLCA that is inherently resistant to cisplatin-based chemotherapy. Defining the direct transcriptional targets and studying its effect on chemotherapeutic responses will further enable us to increase our current understanding of TCF21 in BLCA.

Acknowledgements

Funding support: C.D.: University of Texas MD Anderson Cancer Center SPORE in Genitourinary Cancer (Grant P50CA091846).

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Clinicopathologic Characteristics (N=33) Mean Age (y) ± SD 64.3 ± 12.2 Gender Female 3 (9%) Male 30 (91%)

Race Caucasian 27 (82%) African American 2 (6%) Hispanic 3 (9%)

Asian 1 (3%) Clinical Stage TisN0M0 1 (3%)

T1N0M0 4 (12%)

T2N0M0 22 (67%) T3N0M0 6 (18%) Pathologic Stage

pT2bN+M0 4 (12%) pT3aN+M0 6 (18%) pT3bN+M0 18 (55%) pT3bN+M1 1 (3%)

pT4aN+M0 4 (12%) Concomitant CIS 28 (85%) Lymphovascular Invasion 30 (91%)

Variant Histology* 13 (40%)

Table I. Clinical characteristics of patients in the study.

*Variant histology defined if features of squamous differentiation, glandular differentiation, micropapillary architecture, or sarcomatoid transformation were observed.

Figure Legends Figure 1. Transcriptome analysis of primary BLCA and LN metastases. A. Schematic representation of experimental study design to perform differential expression analysis between matched primary BLCA and LN metastasis samples. B. Principal Component Analysis (PCA) showing that the primary tumors and metastases form two distinct groups. C. Heatmap of hierarchical clustering showing gene expression of 64 of 2390 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. Figure 2. TCF21 expression in primary tumor and metastasis in BLCA patients. A. TCF21 expression in primary tumors and metastasis pairs using Q-PCR from RNA isolated from FFPE sections of 7 BLCA patients shows higher expression in the primary tumor when compared to its corresponding lymph node metastasis. B. Loss/reduced expression was confirmed by IHC in matched primary BLCA (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 BLCA (a, e, i, m, q, u) and LN metastasis (d, h, l, p, t, x) is also shown Scale bar: 200 μm. Insets show higher magnification of TCF21 staining. Figure 3. High expression levels of TCF21 correlates with better survival outcomes and is correlated with luminal and luminal-infiltrated subtypes of 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 BLCA subtypes using the super-subtype classifier and the 5-subtype classifier in TCGA dataset (n=402) shows significant difference in expression among the subtypes. 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=n.s) while LN metastases were more readily detected in the TCF21 KD group (p<0.01). Final bladder weights were also comparable (p=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=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 to 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=0.066). Figure 5. Mechanisms underlying TCF21-mediated metastasis suppression in BLCA. Heat map of hierarchical clustering showing differentially expressed genes at FDR cut off of 0.1 with a fold change of 1 in A. UM-UC3 TCF21 pLOC (6 probes). B. UM-UC13 TCF21

LN (90 probes) C. UM-UC13 TCF21 Met (22 probes) when compared to controls. D. GSEA analysis showing enrichment of EMT in control UC3 pLOC and UC13 pLOC samples when compared to 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. Q-PCR analysis confirming increased expression of luminal marker CD24 and decreased expression of basal markers CD44 in UM-UC3 TCF21 pLOC cells.

Figure 1. matched A LN mets Primary BLCA B . BLCA (n=33) . (n=33)

Gene expression profiling using Illumina DASL platform

Differential gene expression analysis FDR cut off 0.1 & log fold change of 1

2390 differentially expressed genes

GSEA analysis

C .

Downloaded from mcr.aacrjournals.orgPrimary BLCA on September LN28, Metastasis2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 2, 2020; DOI: 10.1158/1541-7786.MCR-19-0766 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 2. A

. 71 Primary tumor 61 Lymph node metastases 51 41

n o

i 21

s s

e 11

r p

x 1 e

1.0

e R

0.5

0.0 A B C D E F G Patients

Figure 2. B TCF21 TCF21 b c . H&E H&E a d

f g e h

j k I l

n o m p

r s q t

v w u x

PrimaryDownloaded BLCA from mcr.aacrjournals.org on September 28, 2021.LN Metastasis © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 2, 2020; DOI: 10.1158/1541-7786.MCR-19-0766 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 3. A. DSS OS

S u rv iv a l p ro p o r tio n s : T C F 2 1 (M e a n C u to ff, 8 .2 ) S u rv iv a l p ro p o r tio n s : T C F 2 1 (M e a n C u to ff, 8 .2 )

p = 0 .0 3 1 4

1 0 0 p = 0 .0 4 4 6 1 0 0

5 0 t 5 0

P P

0 0 0 5 0 1 0 0 1 5 0 2 0 0 0 5 0 1 0 0 1 5 0 2 0 0 S u rv iv a l (m o ) S u rv iv a l (m o ) T C F 2 1 1 (lo w ) T C F 2 1 1 (h ig h ) T C F 2 1 1 (lo w ) T C F 2 1 1 (h ig h ) B.

Figure 4. A. UC14 Primary BLCA UC14 LN Metastases Tumor Weight

p=n.s p<0.01

B. Day 14 Day 28 No. of LN No. of Distant Metastases Metastases

C. UC3 Primary BLCA UC3 LN Metastases Tumor Weight

D. 3 weeks 4 weeks No. of LN No. of Distant Metastases Metastases

Figure 5. A. B. C.

UC3 PLOC UC3 TCF21

UC13 LN PLOC UC13 LN TCF21 UC13 MET PLOC UC13 MET TCF21 D.

FDR 0.0013 FDR 0.046 FDR 0.00

UC3 PLOC UC3 TCF21 UC13 LN PLOC UC13 LN TCF21 UC13 MET PLOC UC13 MET TCF21

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C E O C E O O O L 1 L 1 P 2 P 2 F F C C Downloaded from mcr.aacrjournals.org on SeptemberT 28, 2021. © 2020T American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 2, 2020; DOI: 10.1158/1541-7786.MCR-19-0766 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-19-0766

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