TGFβRII mutation does not abrogate autocrine TGFβ signaling in mismatch repair deficient colorectal cancers

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The majority of microsatellite instability high (MSI-H) colorectal cancers (CRCs) possess truncating mutations in the type II receptor for transforming growth factor β (TGFβRII) and are thus expected to be insensitive to TGFβ. However, research using dominant negative TGFβRII has supported the phenomenon of receptor bypass and the present study was undertaken in order to determine the extent of TGFβ insensitivity in a series of MSI-H CRC cell lines. Specifically, the sensitivity of cells to autocrine TGFβ was investigated using SB 431542, a selective inhibitor of the type I TGFβ receptor (TGFβRI). The mutation status of TGFβRII in each cell line was first confirmed by sequencing of the polyA tract. Time dependent alterations in signaling mediators downstream of the receptor were investigated using Western blotting for the activated forms of Smad2, Smad3, Erk, p38 and Akt. Transcriptional responses to various TGFβ regulated genes were examined using quantitative real time PCR. Sensitivity to the growth modulating and pro-migratory properties of TGFβ were investigated using MTT and Matrigel invasion chamber assays, respectively. The ability of each cell line to bind TGFβ was confirmed using a flow cytometric fluorokine assay which demonstrated positive binding regardless of TGFβRII expression status. Results demonstrated that all MSI-H CRC cell lines retained some sensitivity to the autocrine effects of TGFβ, though to varying degrees. In particular, the Erk pathway was found to be activated in all cell lines within 2 hours of incubation with the TGFβRI inhibitor. Additionally, levels of activated Smad proteins fell below baseline levels within an hour of receptor inhibition. Downstream effects of these signaling changes were evident upon examination of mRNA expression since transcript levels of PAI, Fibronectin, Smad7 and TGFβ itself were altered in all MSI-H cell lines upon TGFβRI inhibition. Interestingly, while autocrine TGFβ promoted the proliferation of both non-MSI-H cell lines studied, none of the MSI-H CRCs retained sensitivity to the growth modulating properties of TGFβ. In contrast, inhibition of TGFβRI decreased the migratory capacity of several MSI-H cell lines, particularly in those showing the strongest signaling and transcriptional responsiveness. Taken together, these findings support the phenomenon of TGFβRII bypass in MSI-H CRCs and demonstrate retained sensitivity to autocrine TGFβ pathway activation and transcriptional regulation in cells harboring naturally occurring mutations in this receptor. The TGFβRI-mediated responsiveness was found to be biologically significant in terms of cell migration, implying that effective anti-TGFβ therapies may need to target both TGFβ receptors in order to reach maximal potency.

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