NSCLC Growth Research Articles

Abstract 4608: RRBP1 overexpression is frequently found In lung cancer patients and alleviates intracellular stress induced apoptosis through positive regulation of GRP78 in lung cancer

Abstract How tumors evade apoptotic signals and survive during cancer progression has not yet been resolved. To unravel the molecular mechanisms involved in lung carcinogenesis will greatly improve therapy. Endoplasmic reticulum is a cellular organelle in which secretory and proteins are synthesized and modified. These processes are always enhanced in cancer cells. Ribosome binding protein1(RRBP1) was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum. In this report, we showed that RRBP1 was highly expressed in the early stage of lung cancer compared to their adjunct normal tissue samples by utilizing lung cancer tissue array(n=87). Depletion of RRBP1 by short-hairpin RNAs significantly reduced cell growth and induced apoptosis in A549 and CL1-5 cells, but not in normal lung cells(BEAS2B). Furthermore, RRBP1-depleted NSCLC cells could activate CD95/caspase signaling pathways. Treatment with caspase8 inhibitor (ZVAD-FMK) or with shRNA against caspase8 was sufficient to suppress apoptosis in RRBP1-depleted cells. This effect is associated with significantly reduction in the expression of GRP78. GRP78, an antiapoptotic protein widely upregulated in cancer, plays a critical role in chemotherapy resistance in some cancers. According to our data, ectopic expression of RRBP1 alleviated tunicamycin induced apoptosis by upregulation GRP78 protein level in lung cancer cells. A strong correlation was observed between the expression of RRBP1 and GRP78 in tumor biopsies from NSCLC patients, supporting the concept that GRP78 expression levels are controlled by RRBP1. Taken together, this study not only showed that GRP78 is regulated by RRBP1 but also report that RRBP1 and GRP78 expression is involved in NSCLC growth and progression. suggest that RRBP1 is required for lung cancer growth and development, which may be a useful target in lung cancer treatment and expected to provide the basis for a rational approach for the development of new molecular targeted therapies Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4608. doi:1538-7445.AM2012-4608

Abstract A26: GATA2 is necessary for oncogene-mutant non-small cell lung tumorigenesis

Abstract Non-small cell lung cancer (NSCLC) is the most common histological cancer type in terms of both prevalence and mortality worldwide. In NSCLC, KRAS and EGFR are the most frequently mutated oncogenes, constituting half of all patients. Thus, there is a profound need to develop therapies targeting these lesions. To address this need, we previously performed an RNA interference screen for factors required in KRAS mutant cells. Through this screen, we found KRAS mutant cells depend on the transcription factor GATA2. Subsequent analysis of a broad panel of NSCLC lines revealed GATA2 depletion suppressed mutant cell viability. We then explored the role of GATA2 in NSCLC cells with a variety of oncogene mutations. We observed lethality with GATA2 loss in mutant NSCLC cells, with no effect in wild type NSCLC cells. Moreover, transplanted mutant NSCLC cells depleted for GATA2 exhibited abrogated tumor growth, while wild type tumors were unaffected, suggesting GATA2 is needed in oncogene-mutant NSCLC. To elucidate the mechanism of GATA2 function in NSCLC gene expression, we performed gene expression analysis of mutant cells with GATA2 suppression. With GATA2 loss, we observed global downregulation of the proteasome and subsequent inhibition of proteasome activity. This inhibited proteasome function is biologically salient, as restoration of proteasome activity rescued growth in response to GATA2 depletion. We further explored the role of GATA2 in NSCLC by integrating the above gene expression data with global genome occupancy with chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-seq). Through these approaches, GATA2 loss suppressed expression of the IL-1 signaling pathway in mutant cells, down-regulating pathway flux. Consequently, restored IL-1 signaling reestablished growth in response to GATA2 depletion. Moreover, GATA2 occupancy was enriched in Rho signaling pathway components, functionally inactivating Rho signaling. Accordingly, activated Rho signaling rescued viability in response to GATA2 loss. Analyzing the DNA motifs from our GATA2 ChIP of mutant NSCLC cells, we observed enrichment of the STAT5 response element at Rho target genes. We subsequently isolated a GATA2-STAT5 complex in mutant NSCLC cells. Overall, our integrative genomic analyses have revealed a transcriptional network governed by GATA2 in oncogene-driven NSCLC. To examine the effect of GATA2 loss in genetically engineered mouse models (GEMMs) of NSCLC, we combined a conditional allele of Gata2 with Cre-inducible oncogenic Kras, where Gata2 deletion suppressed tumor growth compared to wild type animals. We then examined the role of whole-body loss of GATA2 in established lung tumors via systemic deletion of Gata2. In this system, GATA2 depletion caused complete regression of established lesions. We then explored whether inhibition of GATA2-regulated pathways with clinically approved compounds would recapitulate the effects of GATA2 loss in NSCLC GEMMs. To do so, we combined bortezomib, an inhibitor of the proteasome, and fasudil, an inhibitor of Rho signaling, in the Kras lung tumor model. Treatment of the Kras-mutant GEMM with these compounds caused a near-complete clearance of tumors. These combined genetic and therapeutic approaches reveal GATA2 is required for autocthonous oncogene-driven tumorigenesis and that combined inhibition of GATA2-controlled pathways with licensed agents suppresses NSCLC growth. In sum, these results demonstrate GATA2 is necessary for oncogene-mutant NSCLC cell survival. Most strikingly, these findings suggest that the functional pleiotropy of GATA2, not a traditional druggable target, represents a network of druggable pathways for therapeutic exploitation.

Simultaneous inhibition of EGFR and c-Met pathways in a panel of NSCLC cell lines

22223 Background: The EGFR inhibitor, erlotinib, prolongs survival in chemotherapy-resistant NSCLC patients, although < 50% respond. Reportedly, simultaneous inhibition of EGFR (by gefitinib) and c-Met (by PHA-665752) was antiproliferative in a line selected for resistance to gefitinib alone. We hypothesized that in lines not previously exposed to either agent, simultaneous inhibition of EGFR and c-Met pathways would slow NSCLC growth more than targeting either single pathway. Methods: NSCLC lines were cultured in 96-well plates at 5000 cells/well. After 24 h, erlotinib (OSI) and the c-Met inhibitor SU11274 (Sigma) were added. Cells were recovered 3–5 d later by trypsinization and counted by hemacytometer. Receptor activation and signaling were studied in A549 by Western blotting following serum deprivation, incubation with inhibitors (3.5 h) and EGF (15 m). Results: In A549, E2 suppressed EGFR phosphorylation (pY1068), and S3 suppressed c-Met pY1230/1234/1235, confirming pathway targeting. Both drugs suppressed phosphorylation of Akt at S473 and Erk1/2 at T202/Y204, consistent with growth inhibition. Conclusions: For all lines except Calu3, S5 was more antiproliferative than E10, demonstrating that c-Met is a target of interest for NSCLC. By the Bliss criterion, the effect of drugs acting independently should equal the product of the effects of each drug alone. Antiproliferation was independent or nearly so (60% to 167% of prediction) for 14/18 combinations with one or both drugs at their lesser concentrations (bold values). Except in H1437, the combination S5E10 was more potent than either single agent, but weaker than predicted by independence. We speculate that EGFR and c-Met signaling pathways overlap in NSCLC, or E10 and S5 share off-target effects. Lack of pronounced synergism in drug-naive cells distinguishes de novo from acquired resistance to EGFR inhibition. We conclude that combined use of EGFR and c-Met inhibitors is rational even in front-line NSCLC therapy, if dose-limiting toxicities of the drugs differ. Relative proliferation of NSCLC lines incubated with erlotinib (E) and/or SU11274 (S). Cell Line S1 S5 E2 E10 S1E2 S5E2 S1E10 S5E10 A549 0.74 0.04 0.72 0.26 0.52 0.13 0.29 0.08 Calu3 0.88 0.23 0.16 0.15 0.36 0.22 0.18 0.10 H1299 0.81 0.18 0.66 0.24 0.63 0.20 0.25 0.14 H1437 0.97 0.10 0.90 0.37 0.58 0.20 0.46 0.12 H1975 0.65 0.10 0.65 0.23 0.61 0.04 0.23 0.06 H2122 0.64 0.07 0.66 0.26 0.44 0.06 0.13 0.04 Proliferation P = # cells drug/# cells DMSO. E2 = 2 uM erlotinib, etc. 1 SD = 4–11% of P. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration OSI Oncology

TGFα-PE40 inhibits non-small cell lung cancer growth

The ability of a chimeric toxin containing transforming growth factor α (TGFα) and truncated Pseudomonas exotoxin A to inhibit NSCLC growth was investigated. TGFα-PE40 inhibited binding of 125I-EGF to NSCLC cell lines with an IC 50 value of 0.5–3 μg/ml. Similarly, other forms of the fusion protein, TGFα-PE38 and TGFα-PE40 Asp553, which have active TGFα binding domains, inhibited specific 125I-EGF binding to NSCLC cells with IC 50 values of 0.1–2 and 0.05–0.5 μg/ml respectively. TGFα-PE40 inhibited 35S-methionine uptake by NSCLC cells with an ED 50 value of 1–30 ng/ml. TGFα-PE38, which has one of the two disulfide pairs of PE40, inhibited amino acid uptake with ED 50 values of 3–50 ng/ml whereas TGFα-PE40 Asp553, which lacks ADP ribosylation activity, had an ED 50 > 100 ng/ml. TGFα-PE40 inhibited colony formation of NSCLC cells with an LD 50 value of 0.008–0.1 ng/ml. Similarly, TGFα-PE38 inhibited NSCLC colony formation with LD 50 values of 0.002–0.1 ng/ml whereas TGFα-PE40 Asp553 had an LD 50 > 10 ng/ml. Also, TGFα-PE40 and TGFα-PE38 inhibited NSCLC xenograft formation in nude mice whereas TGFα-PE40 Asp553 was inactive. These data suggest that TGFα-PE40 and TGFα-PE38 may be useful agents to inactive NSCLC cells.