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E. Bracco



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    P1.05 - Poster Session 1 - Preclinical Models of Therapeutics/Imaging (ID 156)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.05-015 - Assessment of the activity of Pemetrexed and Dasatinib as single agents and in combination in three malignant pleural mesothelioma cell lines (ID 2339)

      09:30 - 09:30  |  Author(s): E. Bracco

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM), an asbestos exposure related disease, is a highly aggressive tumor. Pemetrexed is a third-generation multitargeted antifolate approved as single agent or in combination with cisplatin as standard of care in first/second line treatment of unresectable MPM. Thymidylate synthase (TS), a key enzyme in the de novo synthesis of thymidine, is the main target of Pemetrexed and its overexpression has been related to Pemetrexed-resistance. Experimental data suggest that c-SRC tyrosine kinase hyperactivation has a key role in MPM. The effect of c-SRC pharmacological inhibition in correlation with TS expression levels and Pemetrexed resistance, has been investigated in three MPM cell lines.

      Methods
      Cell growth inhibitory effects of both Pemetrexed and Dasatinib (10nM-100μM) were evaluated by MTS proliferation assay in epithelial (MPP89, REN) and biphasic (MSTO-211) MPM cell lines. Apoptosis was detected by AnnexinV-propidium iodide method using a FACScan, while drug-mediated changes in invasive ability were tested using “wound healing” scratch assay. Real-Time PCR and Western blot were assessed to identify drugs-associated genes and/or proteins modulation.

      Results
      The cell lines assayed displayed different sensitivity to both Pemetrexed and Dasatinib treatments. Among the three cell lines, MSTO-211 was the most sensitive to Pemetrexed (IC~50 ~0.5 μM); on the contrary REN was the most resistant (IC~50 ~5 μM). A similar trend was observed upon Dasatinib treatment with IC50 values ranging from 1 to 5 μM. The synergistic effect of Dasatinib and Pemetrexed was also evaluated, being significantly relevant in MPP89 and REN after 72h treatment while, in MSTO-211, was already detectable after 48h. Early and late apoptosis assessment confirmed, for both drugs, the ability to induce apoptosis, being MPP89 the most Dasatinib-sensitive and MSTO-211 the most Pemetrexed-sensitive cell line. In MPP89 and REN cells co-administration of Pemetrexed/Dasatinib significantly increased the apoptotic rate of 16 folds and this behaviour was enhanced in MSTO-211 (up to 27 folds). Both TS, gene and protein levels were higher in REN compared to MPP89 and MSTO-211 cells. Pemetrexed administration increased TS levels over time, in those cells most sensitive to the drug. Interestingly, in REN cells Pemetrexed treatment did not affect the high baseline TS levels but Dasatinib administration suppressed TS protein and, to a lesser extent, mRNA expression, thus increasing sensitivity to Pemetrexed. In addition, in REN cells the pretreatment with Dasatinib (5 μM) enhanced Pemetrexed sensitivity leading to a strong cell viability reduction. In all 3 cell lines, SRC was expressed (mRNA and protein), decreasing its levels from MSTO and MPP89 to REN, and activated. Dasatinib impaired also cell migration, as observed by wound-healing assay.

      Conclusion
      In vitro data suggest that inhibition of both TS and SRC might represent a potential therapeutic strategy in MPM. The evidence indicates that Dasatinib plays a role by inhibiting cell motility and, more surprisingly, by down-regulating TS. Dasatinib-mediated TS expression impairment suggests a cross-talk between SRC and TS pathways thus leading to hypothesize a therapeutic use of Dasatinib to sensitize those Pemetrexed-resistant MPM patients’ cohort.

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    P3.02 - Poster Session 3 - Novel Cancer Genes and Pathways (ID 149)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.02-013 - ATF2 contributes to Platinum resistance in Non Small Cell Lung Cancer and cJUN/ATF2 Celastrol mediated modulation restores Platinum sensitization. (ID 2275)

      09:30 - 09:30  |  Author(s): E. Bracco

      • Abstract

      Background
      ATF2 is a member of the basic helix-loop-helix (b-ZIP) transcription factor family playing important roles in Stress and DNA damage. Several studies reported correlation between ATF2/JNK-mediated activation and resistance to damaging agents. Celastrol is an ATF2 inhibitor extracted from a Chinese plant known as “Tunder of God Vine”. Celastrol is used in traditional Chinese medicine for anti-inflammatory properties. In addition, Celastrol is a triterpene with promising anticancer activity in several cancer models both in vivo and in vitro. The purpose of the present study was to investigate whether ATF2 might play a role in inducing drug resistance in Non-Small Cell Lung Cancer (NSCLC).

      Methods
      In NSCLC cell lines ATF2 expression levels were evaluated by quantitative PCR (qPCR) and Western Blot (WB) and correlated to cisplatin (CDDP) resistance. Celastrol mediated ATF2/cJUN activity was assessed by Luminometry, qPCR and western blotting (WB). Furthermore, matched tumors and corresponding normal tissues of 88 surgically resected NSCLC specimens, were collected and both qPCR and immunohistochemical analyses for ATF2 were performed.

      Results
      NSCLC cell lines CDDP-resistant (H522 and H1395) expressed high levels of ATF2 protein. Moreover, CDDP treatment increased ATF2 phosphorylation levels leading to an enrichment within the nuclear cell compartment. In our study, Celastrol reduced ATF2 activity by decreasing the production of ATF2 mRNA and blocking the CDDP-mediated phosphorylation/mRNA expression of cJUN, a main ATF2 partner. Furthermore, ATF2/cJUN functional inhibition mediated by Celastrol restored the response to CDDP in resistant lung cell Lines. ATF2, at both protein and mRNA level, was significantly up-modulated in NSCLC tumor samples compared to the paired normal lung tissue (mRNA: p<<0.01, mean Log2(FC)=+4.7). Moreover, high expression of ATF2 mRNA was correlated with the smoking status of the patients. Relevantly, smoker or former smoker patients expressed significantly high ATF2 mRNA levels compared to non-smokers (p=0.02 and p=0.04, respectively).

      Conclusion
      This study shows that in NSCLC cell lines a correlation between ATF2 protein expression and CDDP resistance occurs. Furthermore, our results suggest a potential increase of CDDP sensitivity, following the Celastrol-mediated ATF2/cJUN inhibition. For the first time it has been shown in NSCLC an up-regulation of ATF2 mRNA/protein levels compared to normal tissues and consistent with that detected in CDDP resistant cell lines. This data suggest a possible involvement of ATF2 in NSCLC CDDP-resistance.