Loss of the urothelial differentiation marker FOXA1 is associated with high grade, late stage bladder cancer and increased tumor proliferation

Abstract

Approximately 50% of patients with muscle-invasive bladder cancer (MIBC) develop metastatic disease, which is almost invariably lethal. However, our understanding of pathways that drive aggressive behavior of MIBC is incomplete. Members of the FOXA subfamily of transcription factors are implicated in normal urogenital development and urologic malignancies. FOXA proteins are implicated in normal urothelial differentiation, but their role in bladder cancer is unknown. We examined FOXA expression in commonly used in vitro models of bladder cancer and in human bladder cancer specimens, and used a novel in vivo tissue recombination system to determine the functional significance of FOXA1 expression in bladder cancer. Logistic regression analysis showed decreased FOXA1 expression is associated with increasing tumor stage (p<0.001), and loss of FOXA1 is associated with high histologic grade (p<0.001). Also, we found that bladder urothelium that has undergone keratinizing squamous metaplasia, a precursor to the development of squamous cell carcinoma (SCC) exhibited loss of FOXA1 expression. Furthermore, 81% of cases of SCC of the bladder were negative for FOXA1 staining compared to only 40% of urothelial cell carcinomas. In addition, we showed that a subpopulation of FOXA1 negative urothelial tumor cells are highly proliferative. Knockdown of FOXA1 in RT4 bladder cancer cells resulted in increased expression of UPK1B, UPK2, UPK3A, and UPK3B, decreased E-cadherin expression and significantly increased cell proliferation, while overexpression of FOXA1 in T24 cells increased E-cadherin expression and significantly decreased cell growth and invasion. In vivo recombination of bladder cancer cells engineered to exhibit reduced FOXA1 expression with embryonic rat bladder mesenchyme and subsequent renal capsule engraftment resulted in enhanced tumor proliferation. These findings provide the first evidence linking loss of FOXA1 expression with histological subtypes of MIBC and urothelial cell proliferation, and suggest an important role for FOXA1 in the malignant phenotype of MIBC.

Figure 1. Analysis of FOXA1 and uroplakin expression in vitro and in human bladder tissue.

A: A panel of commonly used urothelial cell lines was screened via traditional RT-PCR for the presence of FOXA1, FOXA2, and FOXA3 transcripts, as well as members of the uroplakin (UPK) family. HepG2 cells, which express each member of the FOXA subfamily, were used as positive controls (data not shown). RT4 cells exhibited robust expression of FOXA1 and as previously reported, UPK family members . FOXA2 expression was detected in T24 cells, but was not correlated with UPK family member expression. FOXA3 was not detected in any tested cell line (data not shown). B:Q-RT-PCR analysis shows FOXA1 expression in the SCaBER cell line derived from a primary SCC and in T24 is significantly lower compared to RT4. C∶Decreased FOXA1 expression is associated with decreased UPK expression in human tissue. Archival normal adjacent tissue (top panel) and muscle invasive bladder tumor was immunostained with a pan-UPK antibody, AUM as well as an antibody directed against FOXA1. Out of 10 samples with FOXA1 positive NAT, and FOXA1 negative associated tumor, 4 tumors exhibited negative AUM staining.

Figure 2. Silencing of FOXA1 in RT4 cells results in increased expression of UPK family members.

A: Stable knockdown of FOXA1 in human RT4 bladder cancer cells (See Figure 6 for FOXA1 protein levels following knockdown in RT4). B: No significant changes in UPK1A were detected following FOXA1 KD. (C-F): mRNA levels of UPK1B, UPK2, UPK3A, and UPK3B were significantly increased following FOXA1 knockdown.

Figure 3. FOXA1 expression is lost in most in high grade, advanced stage muscle-invasive bladder cancers.

AJCC stage Ta, T1, T2, T3 and T4 bladder tumors were immunostained for FOXA1 and FOXA2. Representative cases are illustrated in top panels. Tis area is depicted in a patient diagnosed with AJCC T1 stage bladder tumor. Association between loss of FOXA1 expression and increasing stage was confirmed by logistic regression (bottom panel, p<0.001). A small subset of invasive tumors exhibited nuclear expression of FOXA2.

Figure 4. FOXA1 expression is absent in keratinizing squamous metaplasia and squamous cell carcinoma (SCC) of the urinary bladder.

(A) H&E and immunostaining of non-keratinizing (left panel) and keratinizing (right panel) squamous metaplasia for FOXA1, the squamous cell marker cytokeratin 10, and the basal cell marker keratin 14. Inset on left panel shows positive FOXA1 staining at high magnification. (B) H&E (left panel) of FOXA1-positive (top) and FOXA1-negative (bottom) samples of human SCC of the urinary bladder are depicted. Most cases (81%) of bladder SCC showed loss of FOXA1 expression. (C) FOXA1 expression is lost in lymph node metastases of some patients with SCC: H&E (left panel) of FOXA1-positive UCC (top) and FOXA1-negative SCC (middle and bottom) metastatic lymph node samples isolated from bladder cancer patients are depicted.

Figure 5. FOXA1 negative tumor cells are proliferative.

Dual immunofluorescence of radical cystectomy patient samples depicting co-localization of FOXA1 (green) and proliferation marker Ki67 (red). Left panel is normal adjacent tissue (NAT, top left) and matched tumor (bottom left) from a patient with UCC, while right panels depict normal adjacent tissue (NAT, top right) and squamous cell carcinoma (bottom right). Note that while NAT from UCC depicted in left panel expresses FOXA1 and is Ki67 negative, NAT from patient with SCC displays a subpopulation of FOXA1-negative, Ki67-positive cells. Both tumors (bottom panel) show a subpopulation of cells exhibiting mutually exclusive expression of FOXA1 and Ki67.

Figure 6. Alterations in FOXA1 expression in RT4 and T24 cells results in changes in E-cadherin expression and cell behavior.

(A) Generation of RT4-Scrambled and RT4-FOXA1 KD cells, as well as T24-pLPCX (empty vector) and T24-FOXA1 overexpressing cells: FOXA1 knockdown in RT4 cells resulted in decreased E-cadherin expression (A) and significantly increased cell proliferation at day 4 (B). Overexpression of FOXA1 in T24 cells resulted in increased E-cadherin expression (A), and significantly decreased cell proliferation (C). While manipulation of FOXA1 had no impact on in vitro invasion of RT4 cells (D), overexpression of FOXA1 in T24 cells significantly decreased cell invasion (D). RT4 cells stably expressing scrambled construct (RT4-Scrambled) or FOXA1-specific shRNA (RT4-FOXA1 KD) were recombined with embryonic bladder mesenchyme (eBLM) isolated from embryonic-16 day old rats and inserted under the kidney capsule of immunocompromised mice. After three weeks, host mice were injected with BrdU and sacrificed. (E and F) Tumor volume was significantly increased in FOXA1-KD RT4 cells. (G) H&E (top panel) and BRDU staining (bottom panel) of RT4-Scrambled and RT4-FOXA1 KD cells. H&E staining shows presence of fibromuscular stroma following recombination with both cell lines. RT4-FOXA1 KD cells showed increased incorporation of BrdU (G), indicating FOXA1 knock down results in increased bladder cancer cell proliferation.