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Supplementary Figure S1-S10 from Targeting RICTOR Sensitizes SMAD4-Negative Colon Cancer to Irinotecan

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posted on 2023-04-03, 17:05 authored by Chen Khuan Wong, Arthur W. Lambert, Sait Ozturk, Panagiotis Papageorgis, Delia Lopez, Ning Shen, Zaina Sen, Hamid M. Abdolmaleky, Balázs Győrffy, Hui Feng, Sam Thiagalingam

S1: SMAD4+/+ and SMAD4-/- colon cancer cells were serum-starved and seeded on transwells and allowed to cross the membrane for 24 hours (transwell migration; RFU, relative fluorescence unit; mean {plus minus} SD, n = 3 biological replicates; **P < 0.01). S2: RICTOR depletion in SMAD4-negative colon cancer cells using shRNA#2 was measured by RT-qPCR (RT-qPCR; mean {plus minus} SD, n = 3 technical replicates; ***P < 0.001). B, Western blotting shows the relative levels of RICTOR, p-AKTS473, and cleaved-caspase 3 upon knockdown of RICTOR and in response to irinotecan. C, The indicated cell lines were treated with irinotecan (10μM) for 72 hours. S3: Cells depleted of RICTOR were serum starved and seeded on transwells and allowed to cross the membrane for 24 hours (transwell migration; RFU, relative fluorescence unit; mean {plus minus} SD, n = 6 biological replicates; ***P < 0.001). S4: A, Western blot shows the relative levels of RICTOR and p-AKTS47 in SW403 cells upon transfection with the indicated siRNAs. B, siRNA-transfected SW403 cells were treated with irinotecan (10μM) for 120 hours. Viability of cells was normalized to DMSO-treated controls (cell viability assay; mean {plus minus} SD, n = 3 biological replicates; **P < 0.01). S5: High RICTOR/AKT1 expression correlates with worse survival in colon cancer. S6: Targeting AKT with MK2206 sensitizes SMAD4-negative SW403 colon cancer cells to irinotecan. S7: Targeting AKT with either AKTi-1/2 or MK2206 sensitizes SMAD4-negative colon cancer cells to irinotecan. S8: No toxicity observed in mice exposed to MK2206 and irinotecan. S9: Targeting AKT with MK2206 sensitizes SMAD4-negative ASPC1 and CFPAC1 pancreatic cancer cells to irinotecan. S10: In silico Kaplan-Meier analyses showing the correlation between SMAD4, RICTOR, or AKT1 expression and overall survival (OS) in pancreatic cancer patients.

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ARTICLE ABSTRACT

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. 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.

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    Molecular Cancer Research

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    BiomarkersPrognostic biomarkersChemotherapyCombination chemotherapyDrug ResistanceGastrointestinal CancersColorectal cancerOncogenes & Tumor Suppressors

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