Conditional ablation of TGF-β signaling inhibits tumor progression and invasion in an induced mouse bladder cancer model

The role of transforming growth factor-β (TGF-β) signaling in cancer progression is still under debate. To determine the function of TGF-β signaling in bladder cancer progression, we conditionally knocked out the Tgfbr2 in mouse model after a N-butyl-N-4-hydroxybutyl Nitrosamine induced bladder carcinogenesis. We found the ablation of TGF-β signaling could inhibit the cancer cell proliferation, cancer stem cell population and EMT, hence suppressed the invasive cancer progression, which is similar with the result of TGF-β receptor I inhibitor treatment. These findings recognize the roles and mechanisms of TGF-β signaling in bladder cancer progression in vivo for the first time.


BBN-induced bladder carcinogenesis.
To investigate the role of TGF-β signaling in bladder cancer progression in vivo, we made use of a BBN-induced bladder carcinogenesis mouse model that shares molecular similarities to human disease 15 . Only male mice with correct genotype were selected for the study since the female mice are more resistant to BBN treatment 16 . Bladders of the BBN treated mice developed progressively from dysplasia and carcinoma in situ to invasive carcinoma (Fig. 1A). Compared with the vehicle treated group, all mice (8/8) exposed to BBN for 26 weeks were observed to develop muscle-invasive bladder cancers, which were confirmed by H&E staining in histologic sections showing invasion into muscle layers (Fig. 1A). Previously studies have demonstrated that keratin5 (K5)-expressing basal cells are main progenitors of carcinoma in situ and invasive carcinoma in bladder by lineage-tracing experiments 17 . Immunohistochemistry results showed that a large portion of the invasive carcinoma induced by BBN were keratin14 (K14) and K5 positive (Fig. 1B). In addition, indicated by Ki-67 staining, the invasive tumor cells are highly proliferating, while most normal urothelial cells are postmitotic (Fig. 1B).

TGF-β signaling is required for BBN-induced invasive bladder cancer progression. TGF-β signa-
ling is well-known for its involvement in cancer invasion 11 , to examine the role of TGF-β pathway in bladder cancer invasion in vivo, we crossed Tgfbr2 flox mice with K5 Cre-ER mice, and tamoxifen was applied to tumor-bearing mice after 26 week of BBN treatment. Four weeks after Tamoxifen administration, tumors from both control and conditional Tgfbr2 knockout mice were collected for further analysis ( Fig. 2A). All control mice developed invasive bladder cancers, while only 37.5% of conditional knockout mice developed invasive bladder cancer, as confirmed by H&E staining (Fig. 2B,D). As a function of cancer progression, relative gross bladder/body weight ratio was calculated to estimate the tumor growth in mice of bladder cancer model. The average ratio of the knockout group was 2.23% ± 1.00%, which was significantly lower than that of the control group(4.65% ± 1.31%) (P = 0.022). Immunohistochemistry analysis revealed strong phosphorylation of Smad2 of Ser465/467, which is an activation marker of TGF signaling 18 , in control invasive cancer cells. In contrast, Tamoxifen administration to K5Cre-ER; Tgfbr2 flox/flox mice led to significant decrease of Smad2 phosphorylation in tumors (Fig. 2D,E). Blockage of TGF-β signaling led to decrease of cancer stem cell population. K14-expressing tumor cells have been previously identified as cancer stem cells population in bladder cancer 19 . We reasoned that deletion of Tgfbr2 may cause a decrease of K14 + cancer stem cells population, which subsequently impaired the invasive capability of tumor. Our data displayed a noticeable decrease of K14 expression in conditional Tgfbr2 knockout tumors relative to the normal control (Fig. 3A,B). In addition, K5 showed similar expression pattern with K14 (data not shown). Consistently, we detected a substantially increase of Keratin18 (K18), a differentiated tumor cell marker, in Tgfbr2 conditional knockout tumors (Fig. 3A,B). In invasive bladder tumors from wild-type mice, there were 65.4% K14 + tumor cells and 34.6% K18 + differentiated cells. However, in the tumors from Tgfbr2 conditional knockout mice, these cell population ratio were considerably altered to 16.3%(K14 + ) and 83.7%(K18 + ), respectively (Fig. 2C). The frequency of these cellular compartments confirmed a functional role of TGF-β signaling in regulating cancer stem cell population.

TGF-β signaling affects BBN-induced tumor proliferation and apoptosis. To examine whether
TGF-β signaling can influence bladder cancer proliferation and apoptosis in vivo, we assessed cell proliferation and apoptotic cell death by immunostaining for Ki67, active-caspase3 and TUNEL assay, respectively, in the BBN-induced bladder tumors harvested from mice mentioned above. The proliferation index, defined as the percentages of Ki67 + in tumors, was statistically different between control and Tgfbr2 conditional knockout groups (Fig. 4A,B) (proliferation index: 31.5 ± 6.2 versus 17.1 ± 4.2, respectively). We also detected significant increase of apoptotic cells percentage in Tgfbr2 conditional knockout tumors compared with control tumors (apoptotic index: determined by AcCap3: 0.5 ± 0.1 versus 3.7 ± 1.8, and by TUNEL: 1.3 ± 0.3 versus 5.6 ± 0.9, respectively) ( Fig. 4A,B). Thus, these findings indicated that the TGF-β signaling contributes to bladder cancer cell proliferation and apoptosis in BBN-induced bladder cancer and support the hypothesis that TGF-β signaling promotes bladder cancer progression.

TGF-β signaling affects epithelial-mesenchymal transition (EMT) of tumor cells. TGF-β recep-
tor signaling plays an essential role in promoting EMT and modulating the migration and invasion of cancer cells 20 . Here we collected tumor samples from control and Tgfbr2 conditional knockout mice and analyzed the expression of a number of EMT marker genes. Following Tamoxifen treatment, the mRNA expression levels of the mesenchymal markers, including Vimentin, Slug, Snai1, Twist, Zeb1 were downregulated in conditional knockout tumors (Fig. 5A). Conversely, the mRNA expression of the epithelial marker E-cadherin was upregulated (Fig. 5A). Consistently, Western blot results confirmed that protein levels of Vimentin, Slug, Snai1, Twist, Zeb1 were reduced in conditional knockout tumors (Fig. 5B). While augment expression of E-cadherin protein was detected in conditional knockout tumors as compared with control tumors (Fig. 5B). Our data revealed that TGF-β receptor signaling regulated genes involved in EMT.

Small molecular inhibitor of TGF-β signaling inhibited BBN-induced tumor invasion. To test
whether inhibition of endogenous TGF-β signaling by specific small molecule inhibitors can restrain the invasion and progression of bladder cancer, 16 male Balb/c mice with 26 weeks of BBN treatment were injected intraperitoneally with LY364947 21 (1 mg/kg) 3 times a week for 4 weeks or PBS, respectively (8 mice per group, Fig. 6A). As expected, bladder cancers were sensitive to LY364947, which significantly inhibited tumor formation and growth induced by BBN in mice (Fig. 6B). In tissue sections from LY364947-treated bladder cancer, considerably more activated caspase-3 positive nuclei were demonstrated than in sections from vehicle treated cancers (Fig. 6C,D). On the other hand, the number of proliferating (Ki-67 + ) cells was dramatically diminished in the tumors treated with LY364947 (Fig. 6C,D). Thus, TGF-β signal inhibitors like LY364947 may present an effective therapeutic avenue for bladder cancer.

Discussion
Genetic alterations, as well as epigenetic gene regulation, in various signaling pathways are involved cancer development [22][23][24][25] . The TGF-β signaling pathway is involved in various aspects of psychological and physiological processes, including genesis and progression of urinary bladder cancer 26,27 . It has been shown that TGF-β 1 secretion levels correlates with more aggressive phenotype of bladder cancer cell lines 28 . Moreover, knockdown of TGFβ RI by siRNA significantly reduced invasiveness of bladder cancer cells 29 . In the present study, we set off to define the role of TGF-β signaling pathway in bladder cancer invasion in vivo. We showed that depletion of Tgfbr2 reduced the invasiveness of bladder cancer induced by BBN. Moreover, chemical inhibitor of TGF-β signaling also diminished bladder cancer progression. Therefore, TGF-β signaling pathway components can serve as promising candidates for the development of novel therapeutic strategies for anti-bladder cancer treatment. A series clinical trials of TGF-β inhibitors on different types of cancer have been launched so far, which mainly aim to influences tumor microenvironment by inhibiting fibrosis, angiogenesis, metastasis, and activating immune-related host response 30 . Among which a TGFbR/ALK1 inhibitor PF-03446962 has been approved for phase II trial of bladder cancer treatment 31 .
TGF-β signaling exerts its pro-proliferating and anti-apoptotic effects through activated either Smad proteins or the extracellular signal regulated kinases 1 and 2 (ERK1/ERK2) 32 . In Tgfbr2 conditional knockout mice, we observed downregulation of p-Smad2 in tumor cells, which correlated with increase of tumor cell apoptosis and increase of proliferation activity. In addition, we made the novel observation that LY364947, a Tgfbr1 inhibitor, could also inhibit cell survival and proliferation in BBN-induced carcinogenesis. Hence, TGF-β signaling plays a key role in cell survival for supporting the invasive and metastatic process of bladder cancer.
Long-lived cancer stem cells are the roots of cancer, which are able to self-renew and differentiate, initiate and propagate cancers 33 . Study has shown that TGF-β -activated cells are the cancer stem cell population in skin squamous cell carcinoma. These cells display slow-cycle and chemoresistant properties 34 . Although whether TGF-β -activated are also cancer stem cells in bladder cancer remains closer investigation, our results revealed that ablation of TGF-β signaling led to decrease of K14 + cancer stem cells population, which might be responsible for the decrease of tumor progression and invasion.
TGF-β can also promote tumor invasion and metastasis by inducing an EMT. Epithelial tumor cells undergoing EMT become more invasive, resulting in dissemination of cancer cells to metastatic location from the carcinoma in situ. Essentially, expression of all known EMT transcription factors, including Snail, ZEB and bHLH families, is activated by TGF-β , either through a Smad-dependent mechanism or indirectly through activation of other transcription factors or relief of repression [35][36][37] . How TGF-β regulates EMT in bladder cancer is not completely understood. In conclusion, this study demonstrated that genetic deletion of Tgfbr2 or treatment of Tgfbr1 small molecule inhibitor LY364947 led to decrease of bladder tumor invasion and progression. Therefore, the results obtained in this study utilizing TGF-β signaling antagonists provide a rationale for further pre-clinical studies in order to dissect the TGF-β signaling pathway to obtain full benefit from this pathway targeted therapy as a single agent or in combination against bladder cancer as well as other solid tumors dependent upon TGF-β signaling.