Background: Rheb activates mTORC1 to stimulate mRNA translation. Results: Rheb binds to CAD and activates CPSase activity. Conclusion: Rheb affects intracellular pyrimidine nucleotide pools. Significance: Our results provide a new mechanism that TSC/Rheb signaling regulates cell growth.

Malignant mesothelioma (MM) is an aggressive tumor that typically develops after a long latency following asbestos exposure. Although mechanistic target of rapamycin complex 1 (mTORC1) activation enhances MM cell growth, the mTORC1 inhibitor everolimus has shown limited efficacy in clinical trials of MM patients. We explored the mechanism underlying mTORC1 activation in MM cells and its effects on cell proliferation and progression. Analysis of the expression profiles of 87 MMs from The Cancer Genome Atlas revealed that 40 samples (46%) displayed altered expression of RPTOR (mTORC1 component) and genes immediately upstream that activate mTORC1. Among them, we focused on RHEB and RHEBL1, which encode direct activators of mTORC1. Exogenous RHEBL1 expression enhanced MM cell growth, indicating that RHEB–mTORC1 signaling acts as a pro-oncogenic cascade. We investigated molecules that directly activate RHEBs, identifying SmgGDS as a novel RHEB-binding protein. SmgGDS knockdown reduced mTORC1 activation and inhibited the proliferation of MM cells with mTORC1 activation. Interestingly, SmgGDS displayed high binding affinity with inactive GDP-bound RHEBL1, and its knockdown reduced cytosolic RHEBL1 without affecting its activation. These findings suggest that SmgGDS retains GDP-bound RHEBs in the cytosol, whereas GTP-bound RHEBs are localized on intracellular membranes to promote mTORC1 activation. We revealed a novel role for SmgGDS in the RHEB–mTORC1 pathway and its potential as a therapeutic target in MM with aberrant mTORC1 activation. Implications: Our data showing that SmgGDS regulates RHEB localization to activate mTORC1 indicate that SmgGDS can be used as a new therapeutic target for MM exhibiting mTORC1 activation.

<p>Figure S1. RHEB mRNA expression in different types of cancer. Figure S2. In vivo growth of MeT-5A mesothelial cells expressing exogenous RHEBL1. Figure S3. RHEBL1 binds to SmgGDS. Figure S4. SmgGDS knockdown suppresses the proliferation of mesothelial cells expressing exogenous RHEBL1. Figure S5. SmgGDS knockdown suppresses the growth of PTEN-deficient MM cells in vitro. Figure S6. Subcellular RHEBL1 localization. Figure S7. SmgGDS knockdown alters RHEBL1 localization in PTEN-deficient MM cells. Figure S8. Amino acid stimulation does not affect RHEBL1 subcellular localization. Figure S9. RHEBL1 farnesylation is essential for SmgGDS binding.</p>

<p>Figure S1. RHEB mRNA expression in different types of cancer. Figure S2. In vivo growth of MeT-5A mesothelial cells expressing exogenous RHEBL1. Figure S3. RHEBL1 binds to SmgGDS. Figure S4. SmgGDS knockdown suppresses the proliferation of mesothelial cells expressing exogenous RHEBL1. Figure S5. SmgGDS knockdown suppresses the growth of PTEN-deficient MM cells in vitro. Figure S6. Subcellular RHEBL1 localization. Figure S7. SmgGDS knockdown alters RHEBL1 localization in PTEN-deficient MM cells. Figure S8. Amino acid stimulation does not affect RHEBL1 subcellular localization. Figure S9. RHEBL1 farnesylation is essential for SmgGDS binding.</p>

<div>Abstract<p>Malignant mesothelioma (MM) is an aggressive tumor that typically develops after a long latency following asbestos exposure. Although mechanistic target of rapamycin complex 1 (mTORC1) activation enhances MM cell growth, the mTORC1 inhibitor everolimus has shown limited efficacy in clinical trials of MM patients. We explored the mechanism underlying mTORC1 activation in MM cells and its effects on cell proliferation and progression. Analysis of the expression profiles of 87 MMs from The Cancer Genome Atlas revealed that 40 samples (46%) displayed altered expression of <i>RPTOR</i> (mTORC1 component) and genes immediately upstream that activate mTORC1. Among them, we focused on <i>RHEB</i> and <i>RHEBL1</i>, which encode direct activators of mTORC1. Exogenous <i>RHEBL1</i> expression enhanced MM cell growth, indicating that RHEB–mTORC1 signaling acts as a pro-oncogenic cascade. We investigated molecules that directly activate RHEBs, identifying SmgGDS as a novel RHEB-binding protein. SmgGDS knockdown reduced mTORC1 activation and inhibited the proliferation of MM cells with mTORC1 activation. Interestingly, SmgGDS displayed high binding affinity with inactive GDP-bound RHEBL1, and its knockdown reduced cytosolic RHEBL1 without affecting its activation. These findings suggest that SmgGDS retains GDP-bound RHEBs in the cytosol, whereas GTP-bound RHEBs are localized on intracellular membranes to promote mTORC1 activation. We revealed a novel role for SmgGDS in the RHEB–mTORC1 pathway and its potential as a therapeutic target in MM with aberrant mTORC1 activation.</p>Implications:<p>Our data showing that SmgGDS regulates RHEB localization to activate mTORC1 indicate that SmgGDS can be used as a new therapeutic target for MM exhibiting mTORC1 activation.</p></div>