Pancreatic cancer–associated retinoblastoma 1 dysfunction enables TGF-β to promote proliferation
A. Jesse Gore, Samantha L. Deitz, Lakshmi Reddy Palam, Kelly E. Craven, Murray Korc
A. Jesse Gore, Samantha L. Deitz, Lakshmi Reddy Palam, Kelly E. Craven, Murray Korc
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Research Article

Pancreatic cancer–associated retinoblastoma 1 dysfunction enables TGF-β to promote proliferation

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is often associated with overexpression of TGF-β. Given its tumor suppressor functions, it is unclear whether TGF-β is a valid therapeutic target for PDAC. Here, we found that proliferating pancreatic cancer cells (PCCs) from human PDAC patients and multiple murine models of PDAC (mPDAC) often exhibit abundant levels of phosphorylated retinoblastoma 1 (RB) and Smad2. TGF-β1 treatment enhanced proliferation of PCCs isolated from KrasG12D-driven mPDAC that lacked RB (KRC cells). This mitogenic effect was abrogated by pharmacological inhibition of type I TGF-β receptor kinase, combined inhibition of MEK/Src or MEK/PI3K, and restoration of RB expression. TGF-β1 promoted epithelial-to-mesenchymal transition (EMT), invasion, Smad2/3 phosphorylation, Src activation, Wnt reporter activity, and Smad-dependent upregulation of Wnt7b in KRC cells. Importantly, TGF-β1–induced mitogenesis was markedly attenuated by inhibition of Wnt secretion. In an in vivo syngeneic orthotopic model, inhibition of TGF-β signaling suppressed KRC cell proliferation, tumor growth, stroma formation, EMT, metastasis, ascites formation, and Wnt7b expression, and markedly prolonged survival. Together, these data indicate that RB dysfunction converts TGF-β to a mitogen that activates known oncogenic signaling pathways and upregulates Wnt7b, which synergize to promote PCC invasion, survival, and mitogenesis. Furthermore, this study suggests that concomitantly targeting TGF-β and Wnt7b signaling in PDAC may disrupt these aberrant pathways, which warrants further evaluation in preclinical models.

Authors

A. Jesse Gore, Samantha L. Deitz, Lakshmi Reddy Palam, Kelly E. Craven, Murray Korc

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Figure 9

TGF-β1 induces EMT and invasion in KRC cells, whereas TβRI inhibition attenuates tumor growth and prolongs survival.

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TGF-β1 induces EMT and invasion in KRC cells, whereas TβRI inhibition at...
(A) TGF-β1 (0.5 nM) altered the epithelial morphology of KRC cells (phase contrast). Immunofluorescence for E-cadherin (red) and N-cadherin (green) shows that TGF-β1 altered the localization of E-cadherin (insets) and changed cell morphology. Scale bars: 50 μm. (B) TGF-β1 decreased E-cadherin and upregulated N-cadherin, ZEB1, and SNAIL. Shown are representative blots from three independent experiments. ERK2 was used to confirm equivalent lane loading. (C) TGF-β1 (white bars) enhanced invasion (left panel), and using KRC1022-4 cells, Wnt7b siRNA blocked this effect. *P < 0.032. Data represent the means ± SEM from three independent experiments. (D) High-resolution ultrasound images show that vehicle-treated mice harbored large tumors (T, arrows) and had formed ascites (A), as evidenced by color doppler (blue), whereas SB505124-treated tumors were smaller and ascites were not detectable. Shown are representative images from day 17. (E) Quantitation shows that SB505124 (SB, white bars) significantly attenuated tumor volumes. *P < 0.031. (F) Kaplan-Meier survival curves reveal that SB-505124 significantly prolonged the survival of mice bearing KRC1017- (green versus blue line, P = 0.026) and KRC1022-4–derived (red versus orange line, P = 0.007) tumors. Horizontal line indicates 50% survival. (G) Compared with vehicle (V, black bars), SB505124 (SB, white bars) significantly reduced the percentage of CK19-positive PCCs with Ki67. **P < 0.001. (E and G) Data represent the means ± SEM.