AACR abstracts out: here are couple..will keep digging
Abstract Number: 712
Presentation Title: RNA-dependent protein kinase (PKR) indirectly regulates AKT in human lung cancer cells
Presentation Start/End Time: Sunday, Apr 02, 2006, 1:00 PM - 5:00 PM
Location: Exhibit Hall, Washington Convention Center
Poster Section: 1
Poster Board Number: 6
Author Block: Abujiang Pataer, Urs V. Holzen, Dora Bocangel, Sunil Chada, Jack A. Roth, Kelly K. Hunt, Stephen G. Swisher. MD Anderson Cancer Center, Houston, TX, Introgen Therapeutics Inc, Houston, TX
We have previously demonstrated that adenoviral mediated overexpression of mda-7 (Ad-mda7) leads to a rapid induction and activation of PKR, which correlates with apoptosis in A549 and H1299 human lung cancer cells. To our surprise, we found that Ad-mda7 induced activation of not only PKR, but also AKT in these cells. Western analysis revealed that Ad-mda7-mediated activation of AKT is induced by phosphorylation at the S437 site, in A549 and H1299 human lung cancer cells in a time- and dose-dependent manner. Inhibition of phospho-AKT by geldanamycin (GA) enhances Ad-mda7-mediated cell killing in these cells. Additionally, PKR siRNA 48 h treatment of these cells induces a dramatic down-regulation of PKR and phospho-AKT protein levels. To determine whether PKR is required for activation of AKT, we used Western blot analysis to evaluate the effects of PKR, phospho-PKR, AKT, and phospho-AKT in mouse embryo fibroblasts (MEFs) derived from PKR-deficient mice. Although similar AKT protein expression levels were detected in both PKR null (-/-) and wild-type MEFs, only PKR wild-type MEFs demonstrated phosphorylation of AKT at the S473 site; we did not detect phosphorylated AKT in PKR null (-/-) cells. We then performed a coimmunoprecipitation assay for PKR and AKT, and detected an indirect interaction between the endogenous proteins in PKR MEFs and A549 and H1299 human lung cancer cells. We did not detect AKT, PDK1, PDK2, ILK, ATM, or STAT3 proteins from anti-PKR immunoprecipitated samples or PKR protein from anti-AKT immunoprecipitated samples. Our results clearly show that although PKR proteins do not directly interact with AKT proteins in these cells, phosphorylation of AKT in these cells clearly depends on induction and/or activation of PKR.
Abstract Number: 1460
Presentation Title: Interaction of the tumor suppressor FUS1 with PDGFRß inhibits PDGFR-mediated proliferation of human lung cancer cells
Presentation Start/End Time: Sunday, Apr 02, 2006, 3:25 PM - 3:40 PM
Location: Room 150, Washington Convention Center
Author Block: Guanglin Wu, Wuguo Deng, Gitanjali Jayachandran, John D. Minna, Jack A. Roth, Lin Ji. U.T.M.D.Anderson Cancer Center, Houston, TX, UT Southwestern Medical Center, Dallas, TX
Platelet-derived growth factors (PDGFs) play crucial roles in diverse biological processes like cell migration, proliferation, apoptosis, and survival. PDGFs activate their protein tyrosine kinase (PTK) receptors, PDGFRa and PDGFRß, which subsequently stimulate the downstream targets MEK and ERK to activate PI3-K/Akt signaling, thus promoting cell survival and anti-apoptotic processes in many cell systems. Blocking constitutive oncogenic PDGFRs with selective PTK inhibitors has promoted anti-angiogenesis and suppressed tumor proliferation in vitro and in animal models of human cancers. FUS1 is a novel tumor suppressor gene identified in human chromosome region 3p21.3, where genomic and genetic abnormalities are common and occur early in the development of many types of cancer. We previously found that FUS1 protein expression was deficient in many human primary lung cancer specimens and cell lines and that reactivating FUS1 in 3p21.3-deficient lung cancer cells inhibited their growth and induced apoptosis, in part by inhibiting the activity of oncogenic PTKs such as c-Abl, c-Kit, and PDGFR. Here we examined PDGFR expression in various non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines and found high expression of PDGFRß but not PDGFRa in almost all cell lines tested. Immunoprecipitation and immunoblot analyses showed that FUS1 protein interacted directly with PDGFRß but not with the PDGF ligands. Forced expression of wt-FUS1 by nanoparticle-mediated gene transfer in the PDGFRß-expressing SCLC H128 and NSCLC H358 cell lines inactivated PDGFR, as evidenced by significant reductions in phospho-PDGFRß relative to untransfected or LacZ-transfected controls. Levels of phospho-PI3-K and phospho-AKT proteins, downstream targets of the PDGF/PDGFR signaling pathway, were markedly reduced as well. Finally, combined treatment with FUS1-nanoparticles and the PTK inhibitor imatinib (Gleevec) synergistically inhibited growth and induced apoptosis in SCLC and NSCLC cell lines that contained highly activated phospho-PDGFRß. Our results suggest that the interaction of the FUS1 protein with PDGFRß can interrupt PDGF/PDGFR oncogenic signaling, block PDGFR-mediated tumor cell survival, and inhibit proliferation by promoting apoptosis in lung cancer cells. These findings also indicate that combination treatments with a pro-apoptotic tumor suppressor FUS1 and selective PTK inhibitors may be a useful therapeutic strategy for human lung cancer. This work was supported by grants from the NCI (P50 CA70907) and Department of Defense (TARGET, DAMD17002-1-0706).
Presentation Title: Overcoming gefitinib resistance in NSCLC via inactivation of the PI3K/AKT signaling pathway by a combination of FUS1 nanoparticles and EGFR inhibitors
Presentation Start/End Time: Wednesday, Apr 05, 2006, 8:00 AM -12:00 PM
Location: Exhibit Hall, Washington Convention Center
Poster Section: 14
Poster Board Number: 20
Author Block: Hiroyuki Kawashima, Gitanjali Jayachandran, Wuguo Deng, Kai Xu, John D. Minna, Jack A. Roth, Lin Ji. UT M.D. Anderson Cancer Center, Houston, TX, UT Southwestern Medical Center, Dallas, TX
Gefitinib (Iressa) is an orally active epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has been shown to be clinically effective in a subpopulation of non small cell lung cancer (NSCLC) patients. Despite initial responses to gefitinib in some NSCLC patients, cancer eventually progresses by unknown mechanisms of acquired resistance. We explored the use of the novel tumor suppressor FUS1 to enhance the chemotherapeutic potency of gefitinib and overcome gefitinib resistance in NSCLC. We found that reactivation of wild-type FUS1 by FUS1 nanoparticle-mediated gene transfer into FUS1-deficient and gefitinib-resistant NSCLC H1299, H322, H358, and H460 cells significantly (P < 0.001) sensitized their response to gefitinib treatment and synergistically induced apoptosis in vitro and in an H322 orthotopic lung cancer mouse model. To understand the mechanism of gefitinib-induced resistance, we established a gefitinib-resistant HCC827GR NSCLC cell line (IC50 = 16 µM) by selecting against gefitinib from the parental HCC827 cells that contain an activating deletion mutation of the EGFR gene and are extremely sensitive to gefitinib treatment (IC50 = 0.016 µM). We found no secondary mutations in the EGFR gene in the HCC827GR cells, but these cells registered a significantly elevated level of phosphorylated AKT protein. Combination treatment with FUS1 nanoparticles and gefitinib at a dose level of IC10 significantly re-sensitized the cells to gefitinib, as demonstrated by synergistically enhanced growth inhibition and apoptosis. FUS1 nanoparticle treatment alone or with gefitinib markedly inactivated EGFR and AKT, as demonstrated by decreased phosphorylation levels of both proteins on Western blots, compared with either agent alone. Cleavage of caspase-3, caspase-9, and PARP was also significantly induced by the combination of FUS1 and gefitinib in HCC872GR and other gefitinib-resistant NSCLC cells. Our results suggest that a combination treatment of FUS1 nanoparticles and gefitinib could overcome drug-induced resistance by simultaneously inactivating EGFR and the AKT signaling pathway and by facilitating apoptosis. This abstract is supported by grants from NCI (SPORE P50CA70907) and DOD (TARGET, DAMD17002-1-0706).
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