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 7

TGF-β is a driver of Wnt7b expression in KRC cells.

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TGF-β is a driver of Wnt7b expression in KRC cells. (A) KRC cells (white...
(A) KRC cells (white bars) expressed high Wnt7b mRNA levels compared with KC (control, black bars; *P < 0.04) and KSC (gray bars; *P < 0.02) cells. (B and C) TGF-β1 (0.5 nM) increased Wnt7b mRNA (B, white bars; *P = 0.032) and protein (C) levels in KRC cells. (C) Representative blot from two independent experiments. ERK2 was used to confirm equivalent lane loading. (D and E) SB505124 (D, 2 μM, white bars) and anti–TGF-β2 (E, 5 mg/ml, white bars) reduced Wnt7b mRNA levels in KRC cells (**P < 0.001) compared with their respective controls (black bars). (F) TGF-β1 (0.5 nM, white bars) did not alter Wnt7b mRNA levels in KSC cells. (G) TGF-β1 (0.5 nM) increased wild-type Wnt7b promoter activity (left panel) in KRC cells (*P = 0.002), whereas KSC cells had low basal activity that was not increased by TGF-β1. TGF-β1 failed to increase the activity of an SBE-mutated Wnt7b reporter in KRC cells (right panel). (H) ChIP assays revealed that Smad2-4 (Smad) bound the Wnt7b promoter in TGF-β1–stimulated (0.5 nM) KRC cells. Left panel: quantification; **P < 0.001. Right panel: representative gel image from three independent experiments. (I) hPDAC PCCs with strong cytoplasmic Wnt7b immunoreactivity showed nuclear Smad4 (top panels), whereas hPDACs that lack Wnt7b did not demonstrate Smad4 immunoreactivity (bottom panels). (I) Representative images from two PDACs. Scale bars: 50 μm. (A, B, and D–H) Data represent the means ± SEM from three independent experiments.