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F. Cognetti



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    MO20 - Preclinical Therapeutic Models II (ID 93)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO20.06 - Histone deacetylase inhibition downregulates thymidylate synthase (TS) expression and enhances pemetrexed-induced cytotoxicity in NSCLC models (ID 2010)

      10:30 - 12:00  |  Author(s): F. Cognetti

      • Abstract
      • Presentation
      • Slides

      Background
      Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Pemetrexed (PEM), a multi-target folate antagonist, has demonstrated targeted efficacy in NSCLC histological subtypes characterized by low thymidylate synthase (TS, one of PEM’s molecular targets) expression. Recently, TS expression has been found to be regulated by histone acetylation status, thus raising the interesting hypothesis that histone deacetylase inhibitors (HDACi) may sensitize NSCLC cells to PEM cytotoxicity.

      Methods
      Molecular and functional effects of single and combined HDAC inhibition and PEM exposure were assessed in NSCLC cell lines (A549, H1299, H1650, Calu-1) and patient-derived lung cancer stem cells (L-CSC). Pharmacologic interactions were assessed by conservative isobologram analysis using the Chou-Talalay method and the Calcusyn software. TS expression was studied by WB analysis and real-time PCR. Apoposis induction was assessed by flow cytometry and WB. Autophagy was assessed by analysis of autophagosome formation in EGFP-LC3B expressing cells, detection of acidic vesicle organelles (AVO) formation and WB. In vivo experiments were conducted in xenograft models established by i.m. injection of NSCLC cells into 6-8 week-old male athymic mice (nu/nu).

      Results
      In NSCLC cell lines and L-CSC, the HDACi ITF2357 dose-dependently inhibited cell growth (IC~50~: <1-20 mM), induced histone H3 acetylation, and downregulated TS expression at the mRNA and protein levels. Combined HDAC inhibition and PEM exposure was then tested using three different administration schedules: simultaneous exposure to both drugs, ITF2357 followed by PEM, and the reverse sequence. Simultaneous PEM/ITF2357 treatment resulted in antagonistic growth inhibitory interactions (combination index – CI >1) in all cell lines tested, while ITF2357 followed by PEM had additive effects in A549 cells and slightly synergistic effects in H1299 and Calu-1 cells; conversely, PEM followed by ITF2357 had strikingly synergistic effects (CI <<1) in all NSCLC cell lines, as well as in the L-CSC143. Most notably, only the ITF2357 followed by PEM sequence synergistically induced apoptosis, resulting in approximately 50% Annexin V-positive cells; apoptosis was only partially rescued by caspase inhibition by z-VAD-fmk, which led us to investigate autophagy as an alternative mechanism of combination-induced cell death. Indeed, ITF2357, and to a significantly greater extent PEM followed by ITF2357, induced autophagy as evidenced by AVO formation, LC3BII processing, p62 downregulation, and Beclin1 induction. Most importantly, autophagy induction was instrumental to the cytotoxic interaction between PEM and ITF2357, as Beclin1 silencing by shRNA completely reversed their growth inhibitory synergism and prevented both autophagy and apoptosis induction. The synergistic cytotoxic interaction between PEM and ITF2357 was at least partly due to ITF2357 ability to prevent PEM-induced TS upregulation, as TS silencing by siRNA further enhanced apoptosis induction by single and combined PEM/ITF2357 exposure. Finally, both H1650 and H1299 xenografts had a robust response to sequential PEM/ITF2357 administration in vivo, resulting in an approximately doubled mice survival in the H1650 model.

      Conclusion
      Overall, our data indicate that HDAC inhibition by ITF2357 downregulates TS expression and synergistically potentiates apoptosis and autophagy induction following PEM exposure, supporting the clinical investigation of sequential PEM/ITF2357 schedules for the treatment of advanced NSCLC.

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    P1.06 - Poster Session 1 - Prognostic and Predictive Biomarkers (ID 161)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.06-041 - Prognostic impact of cytoskeleton regulatory protein human Mena (hMena) isoforms in resected, node-negative, non-small-cell lung cancer: validation of a clinic-molecular prognostic model. (ID 2609)

      09:30 - 16:30  |  Author(s): F. Cognetti

      • Abstract

      Background
      Human Mena and the isoform hMena[+11a] are cytoskeleton regulatory proteins involved in adhesion, motility, regulated in the epithelio-mesenchimal transition. Here, we investigated their potential prognostic value in node-negative non-small-cell lung cancer (NSCLC) patients.

      Methods
      Pan-hMena, hMena[+11a], E-cadherin, vimentin, ER-beta, EGFR, HER-2, pAKT, detected immunohystochemically on duplicate TMA and clinical factors (sex, age, histology, grading, T-size, number of resected nodes, RN) were correlated to 3-yr disease-free (DFS), cancer-specific (CSS), and overall survival (OS) using a Cox model. ROC analysis provided optimal cut-off values and model validation. A logistic equation including regression analysis coefficients was constructed to estimate individual patients’ probability (IPP) of relapse. Internal cross-validation (100 simulations with 80% of the dataset) and external validation was accomplished.

      Results
      In a training set of 248 patients (median follow-up: 36 months, range 1-96), Pan-hMmena and hMena+11a were the only biological variables displaying significant correlation with outcome(s), confirmed by the cross-validation (replication rate: 78%, 83%), with a prognostic model accuracy of 61% (standard error 0.04, p=0.0001). Patients with high pan-hMENA expression had a non-significant trend towards a worse outcome, while patients with high hMena+11a expression had a significant and borderline significant advantage in DFS (p=0.03) and OS (p=0.056), respectively, and a non-significant trend towards a better CSS. Univariate and multivariate 3-yr median individual patient probabilities of recurrence were 70.9 (range 40.3-94.4) and 41.2 (range 13.6-86.5), respectively (data not shown). The subgroup of patients with High Pan-hMena/Low hMena11a relative expression fared significantly better than any of the other 3 groups (p≤0.002 for all outcomes). On the basis of the combination between this molecular hybrid variable and T-size and RN, a 3-class risk stratification model was generated; the derived 3-risk class survival model strikingly discriminated between patients at different risk of relapse, cancer-related death, and death for any cause, with a prognostic accuracy of 61% (standard error 0.03, p=0.01), according to ROC analysis. The 3-risk class survival model was externally validated in an independent dataset of 133 patients, and significantly discriminated between patients at Intermediate- and High-Risk of relapse and cancer-related death.

      Conclusion
      The expression of the hMena and its isoform may represent a powerful prognostic factor in early NSCLC and usefully complements clinical parameters to accurately predict individual patient risk..