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Oncogenic ras represses transforming growth factor-beta /Smad signaling by degrading tumor suppressor Smad4
D Saha1, P K Datta, R D Beauchamp
1Department of Surgery, Vanderbilt University Medical Center, Vanderbilt-Ingram Cancer Center, Nashville, Tennessee 37232, USA.
Abstract:
The loss of growth-inhibitory responses to transforming growth factor-beta (TGF-beta) is a frequent consequence of malignant transformation. Smad2, Smad3, and Smad4 proteins are important mediators of the antiproliferative responses to TGF-beta and may become inactivated in some human cancers. Epithelial cells harboring oncogenic Ras mutations often exhibit a loss of TGF-beta antiproliferative responses. To further investigate the effect of oncogenic Ras in TGF-beta signaling, we used an isopropyl-1-thio-beta-d-galactopyranoside-inducible expression system to express Ha-Ras(Val-12) in intestinal epithelial cells. Induction of Ha-Ras(Val-12) caused a decrease in the level of Smad4 expression, inhibited TGF-beta-induced complex formation between Smad2/Smad3 and Smad4, blocked Smad4 nuclear translocation, inhibited the TGF-beta-mediated decrease in [(3)H]thymidine incorporation, and repressed TGF-beta-activated transcriptional responses. The withdrawal of isopropyl-1-thio-beta-d-galactopyranoside or the addition of an inhibitor of the ubiquitin-proteasome pathway restored the Smad4 level and TGF-beta-induced Smad complex formation. Forced expression of Smad4 resulted in partial recovery of the TGF-beta-mediated growth inhibition and transcriptional responses in the presence of oncogenic Ras. Further, PD98059, a specific inhibitor of the MEK/ERK/mitogen-activated protein kinase pathway prevented the Ras-induced decrease in Smad4 expression and complex formation. Our results suggest a novel mechanism by which oncogenic Ras represses TGF-beta signaling by mitogen-activated protein kinase-dependent down-regulation of Smad4, thereby subverting the tumor suppressor function of TGF-beta.
Insights
Oncogenic Ras disrupts transforming growth factor-beta (TGF-beta) signaling by reducing Smad4 protein levels via the MAPK pathway. This interference impairs TGF-beta
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Signaling
Background:
- Loss of transforming growth factor-beta (TGF-beta) growth inhibition is common in cancer.
- Smad proteins (Smad2, Smad3, Smad4) mediate TGF-beta's antiproliferative effects and can be inactivated in cancers.
- Oncogenic Ras mutations in epithelial cells often correlate with diminished TGF-beta responses.
Purpose of the Study:
- To investigate how oncogenic Ras influences TGF-beta signaling pathways.
- To elucidate the molecular mechanisms by which Ras may subvert TGF-beta's tumor suppressor functions.
Main Methods:
- Utilized an inducible expression system for Ha-Ras(Val-12) in intestinal epithelial cells.
- Assessed Smad4 protein levels, Smad complex formation, nuclear translocation, and TGF-beta-induced gene transcription.
- Employed ubiquitin-proteasome pathway inhibitors and MEK/ERK pathway inhibitors (PD98059).
Main Results:
- Induction of Ha-Ras(Val-12) decreased Smad4 expression and inhibited TGF-beta-induced Smad complex formation and nuclear translocation.
- Oncogenic Ras blocked TGF-beta's inhibition of DNA synthesis and repressed TGF-beta-activated transcription.
- Smad4 levels and TGF-beta signaling were restored by inhibiting the ubiquitin-proteasome pathway or by forced Smad4 expression.
- MEK/ERK pathway inhibition prevented Ras-induced Smad4 down-regulation and complex disruption.
Conclusions:
- Oncogenic Ras represses TGF-beta signaling through a mitogen-activated protein kinase-dependent mechanism.
- This repression involves the down-regulation of Smad4 protein.
- Ras-induced disruption of TGF-beta signaling contributes to tumor development by inactivating a key tumor suppressor pathway.