Chronic Binge Alcohol Exposure During Pregnancy Alters mTOR System in Rat Fetal Hippocampus.

BACKGROUND: Gestational alcohol exposure can contribute to fetal alcohol spectrum disorders (FASD), an array of cognitive, behavioral, and physical developmental impairments. Mammalian target of rapamycin (mTOR) plays a key role in regulating protein synthesis in response to neuronal activity, thereby modulating synaptic plasticity and long-term memory formation in the brain. Based on our previous quantitative mass spectrometry proteomic studies, we hypothesized that gestational chronic binge alcohol exposure alters mTOR signaling and downstream pathways in the fetal hippocampus. METHODS: Pregnant Sprague-Dawley rats were assigned to either a pair-fed control (PF-Cont) or a binge alcohol (Alcohol) treatment group. Alcohol dams were acclimatized via a once-daily orogastric gavage of 4.5 g/kg alcohol (peak BAC, 216 mg/dl) from GD 5-10 and progressed to 6 g/kg alcohol (peak BAC, 289 mg/dl) from GD 11-21. Pair-fed dams similarly received isocaloric maltose dextrin. RESULTS: In the Alcohol group, following this exposure paradigm, fetal body weight and crown-rump length were decreased. The phosphorylation level of mTOR (P-mTOR) in the fetal hippocampus was decreased in the Alcohol group compared with controls. Alcohol exposure resulted in dysregulation of fetal hippocampal mTORC1 signaling, as evidenced by an increase in total 4E-BP1 expression. Phosphorylation levels of 4E-BP1 and p70 S6K were also increased following alcohol exposure. P-mTOR and P-4E-BP1 were exclusively detected in the dentate gyrus and oriens layer of the fetal hippocampus, respectively. DEPTOR and RICTOR expression levels in the fetal hippocampus were increased; however, RAPTOR was not altered by chronic binge alcohol exposure. CONCLUSION: We conclude that chronic binge alcohol exposure during pregnancy alters mTORC1 signaling pathway in the fetal hippocampus. We conjecture that this dysregulation of mTOR protein expression, its activity, and downstream proteins may play a critical role in FASD neurobiological phenotypes.

Targeting RICTOR Sensitizes SMAD4-Negative Colon Cancer to Irinotecan.

Deciphering molecular targets to enhance sensitivity to chemotherapy is becoming a priority for effectively treating cancers. Loss of function mutations of SMAD4 in colon cancer are associated with metastatic progression and resistance to 5-fluorouracil (5-FU), the most extensively used drug of almost all chemotherapy combinations used in the treatment of metastatic colon cancer. Here, we report that SMAD4 deficiency also confers resistance to irinotecan, another common chemotherapeutic frequently used alone or in combination with 5-FU against colon cancer. Mechanistically, we find that SMAD4 interacts with and inhibits RICTOR, a component of the mTORC2 complex, resulting in suppression of downstream effector phosphorylation of AKT at Serine 473. In silico meta-analysis of publicly available gene expression datasets derived from tumors indicates that lower levels of SMAD4 or higher levels of RICTOR/AKT, irrespective of the SMAD4 status, correlate with poor survival, suggesting them as strong prognostic biomarkers and targets for therapeutic intervention. Moreover, we find that overexpression of SMAD4 or depletion of RICTOR suppresses AKT signaling and increases sensitivity to irinotecan in SMAD4-deficient colon cancer cells. Consistent with these observations, pharmacologic inhibition of AKT sensitizes SMAD4-negative colon cancer cells to irinotecan in vitro and in vivo. Overall, our study suggests that hyperactivation of the mTORC2 pathway is a therapeutic vulnerability that could be exploited to sensitize SMAD4-negative colon cancer to irinotecan. IMPLICATIONS: Hyperactivation of the mTORC2 pathway in SMAD4-negative colon cancer provides a mechanistic rationale for targeted inhibition of mTORC2 or AKT as a distinctive combinatorial therapeutic opportunity with chemotherapy for colon cancer.

[MicroRNA-152 and microRNA-448 inhibit proliferation of colorectal cancer cells in vitro by targeting Rictor].

OBJECTIVE: To screen the microRNAs (miRNAs) targeting Rictor and investigate their effects in regulating the biological behaviors of colorectal cancer (CRC). METHODS: Human colorectal cancer cell line KM12SM was transfected with the miRNAs targeting Rictor identified by prediction software to test inhibitory effects of these miRNAs on Rictor expression using qRT-PCR and Western blotting. Dual luciferase reporter assay was used to further confirm the binding of these miRNAs to the 3'UTR of Rictor mRNA. Cell survival and colony formation assays were used to investigate the effects of these miRNAs on survival and colony formation in KM12SM cells. RESULTS: miR-152 and miR-448 were identified as the Rictor-targeting miRNAs, which significantly inhibited the expression of Rictor in KM12SM cells (P < 0.05). The two miRNAs were confirmed to bind to the 3'UTR of Rictor mRNA and significantly inhibited luciferase activity in KM12SM cells (P < 0.01, P < 0.05); they also showed activities of posttranscriptional modulation of Rictor. Overexpression of miR-152 and miR-448 both significantly inhibited the growth and colony formation of KM12SM cells. CONCLUSIONS: miR-152 and miR-448 can down-regulate the protein expression of Rictor by targeting Rictor mRNA to negatively regulate the growth and colony formation of colorectal cancer cells.