Woo-Young Kim

Implication of the Insulin-like Growth Factor-IR Pathway in the Resistance of Non–small Cell Lung Cancer Cells to Treatment with Gefitinib

Purpose: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors have been found to be effective against lung cancer in vitro, but clinical resistance to these agents has developed as their usage has increased. In this study, we determined whether the insulin-like growth factor I (IGF-I) signaling pathway induces resistance of non–small cell lung cancer (NSCLC) cells to the EGFR tyrosine kinase inhibitor gefitinib. Experimental Design: The effects of gefitinib and cetuximab on NSCLC cells, alone or with an IGF-I receptor (IGF-IR) inhibitor, were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the flow cytometry–based terminal nucleotidyl transferase–mediated nick end labeling assay, coimmunoprecipitation, and Western blot analysis. EGFR and IGFR expression in NSCLC tissues were examined by Western blot analysis. Results: Gefitinib inhibited NSCLC cell proliferation by inducing apoptosis when IGF-IR signaling was suppressed. Treatment with gefitinib, but not cetuximab, induced EGFR:IGF-IR heterodimerization and activation of IGF-IR and its downstream signaling mediators, resulting in increased survivin expression in NSCLC cell lines with high levels of IGF-IR expression. Inhibition of IGF-IR activation and knockdown of survivin expression led to increased apoptosis. In contrast, overexpression of survivin protected cells with low IGF-IR expression from gefitinib-induced apoptosis. Most NSCLC tissues with EGFR overexpression had associated high levels of IGF-IR expression. Conclusions: IGF-IR expression may be useful as a predictive marker for gefitinib treatment of NSCLC. Suppression of IGF-IR signaling pathways may prevent or delay development of gefitinib resistance in patients with NSCLC.

Targeting Heat Shock Protein 90 Overrides the Resistance of Lung Cancer Cells by Blocking Radiation-Induced Stabilization of Hypoxia-Inducible Factor-1α

Hypoxia-inducible factor-1 (HIF-1) has been suggested to play a major role in tumor radioresistance. However, the mechanisms through which irradiation regulates HIF-1alpha expression remain unclear. The purpose of this study was to investigate the mechanisms that mediate HIF-1 activation and thus radioresistance. Here, we show that irradiation induces survival and angiogenic activity in a subset of radioresistant lung cancer cell lines by elevating HIF-1alpha protein expression. Radiation induced HIF-1alpha protein expression mainly through two distinct pathways, including an increase in de novo protein synthesis via activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and stabilization of HIF-1alpha protein via augmenting the interaction between heat shock protein 90 (Hsp90) and HIF-1alpha protein. Whereas the PI3K/Akt/mTOR pathway was activated by irradiation in all the lung cancer cells examined, the Hsp90-HIF-1alpha interaction was enhanced in the resistant cells only. Inhibition of Hsp90 function by 17-allylamino-17-demethoxygeldanamycin or deguelin, a novel natural inhibitor of Hsp90, suppressed increases in HIF-1alpha/Hsp90 interaction and HIF-1alpha expression in radioresistant cells. Furthermore, combined treatment of radiation with deguelin significantly decreased the survival and angiogenic potential of radioresistant lung cancer cells in vitro. We finally determined in vivo that systemic administration of deguelin resulted in profound inhibition of tumor growth and angiogenesis when combined with radiation. These results provide a strong rationale to target Hsp90 as a means to block radiation-induced HIF-1alpha and thus to circumvent radioresistance in lung cancer cells.

Presenilins are required for maintenance of neural stem cells in the developing brain.

The early embryonic lethality of mutant mice bearing germ-line deletions of both presenilin genes precluded the study of their functions in neural development. We therefore employed the Cre-loxP technology to generate presenilin conditional double knockout (PS cDKO) mice, in which expression of both presenilins is inactivated in neural progenitor cells (NPC) or neural stem cells and their derivative neurons and glia beginning at embryonic day 11 (E11). In PS cDKO mice, dividing NPCs labeled by BrdU are decreased in number beginning at E13.5. By E15.5, fewer than 20% of NPCs remain in PS cDKO mice. The depletion of NPCs is accompanied by severe morphological defects and hemorrhages in the PS cDKO embryonic brain. Interkinetic nuclear migration of NPCs is also disrupted in PS cDKO embryos, as evidenced by displacement of S-phase and M-phase nuclei in the ventricular zone of the telencephalon. Furthermore, the depletion of neural progenitor cells in PS cDKO embryos is due to NPCs exiting cell cycle and differentiating into neurons rather than reentering cell cycle between E13.5 and E14.5 following PS inactivation in most NPCs. The length of cell cycle, however, is unchanged in PS cDKO embryos. Expression of Notch target genes, Hes1 and Hes5, is significantly decreased in PS cDKO brains, whereas Dll1 expression is up-regulated, indicating that Notch signaling is effectively blocked by PS inactivation. These findings demonstrate that presenilins are essential for neural progenitor cells to re-enter cell cycle and thus ensure proper expansion of neural progenitor pool during embryonic neural development.

Elevated Epithelial Insulin-like Growth Factor Expression Is a Risk Factor for Lung Cancer Development

Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling has been implicated in several human neoplasms. However, the role of serum levels of IGFs in lung cancer risk is controversial. We assessed the role of tissue-derived IGFs in lung carcinogenesis. We found that IGF-I and IGF-II levels in bronchial tissue specimens containing high-grade dysplasia were significantly higher than in those containing normal epithelium, hyperplasia, and squamous metaplasia. Derivatives of human bronchial epithelial cell lines with activation mutation in KRAS(V12) or loss of p53 overexpressed IGF-I and IGF-II. The transformed characteristics of these cells were significantly suppressed by inactivation of IGF-IR or inhibition of IGF-I or IGF-II expression but enhanced by overexpression of IGF-IR or exposure to the tobacco carcinogens (TC) 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone and benzo(a)pyrene. We further determined the role of IGF-IR signaling in lung tumorigenesis by determining the antitumor activities of the selective IGF-IR tyrosine kinase inhibitor cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo [1,5-a]pyrazin-8-ylamine using an in vitro progressive cell system and an in vivo mouse model with a lung-specific IGF-I transgene after exposure to TCs, including 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone plus benzo(a)pyrene. Our results show that airway epithelial cells produce IGFs in an autocrine or paracrine manner, and these IGFs act jointly with TCs to enhance lung carcinogenesis. Furthermore, the use of selective IGF-IR inhibitors may be a rational approach to controlling lung cancer.

Brain angiogenesis in developmental and pathological processes: mechanism and therapeutic intervention in brain tumors

Formation of new blood vessels is required for the growth and metastasis of all solid tumors. New blood vessels are established in tumors mainly through angiogenesis. Brain tumors in particular are highly angiogenic. Therefore, interventions designed to prevent angiogenesis may be effective at controlling brain tumors. Indeed, many recent findings from preclinical and clinical studies of antiangiogenic therapy for brain tumors have shown that it is a promising approach to managing this deadly disease, especially when combined with other cytotoxic treatments. In this minireview, we summarize the basic characteristics of brain tumor angiogenesis and the role of known angiogenic factors in regulating this angiogenesis, which may be targets of antiangiogenic therapy. We also discuss the current status of antiangiogenic therapy for brain tumors, the suggested mechanisms of this therapy and the limitations of this strategy.

Activation of insulin-like growth factor receptor signaling mediates resistance to histone deacetylase inhibitors

Histone deacetylases (HDACs) are considered promising targets in the treatment of hematologic malignancies and several types of solid tumors, including non-small cell lung cancer (NSCLC). However, the efficacy of HDAC inhibitors in solid tumors is marginal, and the mechanisms underlying resistance to HDAC inhibitors are largely unknown. Here, we demonstrate the involvement of type 1 insulin-like growth factor receptor (IGF-1R) signaling in resistance to HDAC inhibitors in NSCLC. Using MTT and soft-agar colony formation assays, we selected NSCLC cell lines that exhibited intrinsic resistance to vorinostat. Treatment with vorinostat activated IGF-1R signaling in vorinostat-resistant but not vorinostat-sensitive NSCLC cells. Other HDAC inhibitors, including trichostatin A, sodium butyrate, and depsipeptide, also activated IGF-1R signaling in vorinostat-resistant NSCLC cells. Blockade of IGF-1R signaling via IGF-1R monoclonal antibodies (mAbs) or through knockdown of IGF-1R via RNA interference sensitized vorinostat-resistant cells to HDAC inhibition. Finally, IGF-1R mAbs sensitized xenograft tumors of vorinostat-resistant cells to vorinostat treatment in vivo. These findings suggest that IGF-1R activation is generally involved in resistance to HDAC inhibitors and that targeting IGF-1R is an effective strategy for overcoming resistance to HDAC inhibitors in NSCLC.

Brain angiogenesis: Mechanism and Therapeutic Intervention in Brain Tumors

Formation of new blood vessels is required for the growth and metastasis of all solid tumors. New blood vessels are established in tumors mainly through angiogenesis. Brain tumors in particular are highly angiogenic. Therefore, interventions designed to prevent angiogenesis may be effective at controlling brain tumors. Indeed, many recent findings from preclinical and clinical studies of antiangiogenic therapy for brain tumors have shown that it is a promising approach to managing this deadly disease, especially when combined with other cytotoxic treatments. In this minireview, we summarize the basic characteristics of brain tumor angiogenesis and the role of known angiogenic factors in regulating this angiogenesis, which may be targets of antiangiogenic therapy. We also discuss the current status of antiangiogenic therapy for brain tumors, the suggested mechanisms of this therapy and the limitations of this strategy.

Abstract LB-41: Histone deacetylase (HDAC) inhibition induces a resistance mechanism via the insulin-like growth factor-1 receptor (IGF-1R) signaling pathway: Rational basis for cotargeting of IGF-1R and HDAC in non-small cell lung cancer

Background: Histone deacetylase (HDAC), which modulate chromatin structure and gene expression, has attracted attention as a promising therapeutic target for hematologic and solid tumors including non-small cell lung cancer (NSCLC). However, the mechanisms resulting in the resistance to HDAC inhibitors are not yet well-known. In the current study, we investigated the involvement of insulin-like growth factor 1 receptor (IGF-1R) signaling in resistance to HDAC inhibitor in NSCLC.] Material and Methods: We assessed response of a panel of 14 NSCLC cells to vorinostat-based treatment by MTT assay. Because cells growing in monolayer on standard tissue culture plates could have different response to drug treatment compared with cells growing in vivo in three-dimensional environment, we also tested the effects of vorinostat on the cells cultured as spheroids on poly-HEMA coated plate or in soft agar. The antitumor effects of vorinostat, either alone or in combination with a novel anti-IGF-1R antibody, MK0646 (Merck) were evaluated in vitro using representative vorinostat-resistant NSCLC cell lines. The antitumor potency of the combination was evaluated in vivo using xenograft tumor models established in nude mice. The mice were treated twice a week with vorinostat (50 mg/kg), MK0646 (15 mg/kg) or their combination by intraperitoneal (IP) injection. Western blot, real time quantitative PCR, and immunohistochemical analyses were performed to assess biochemical changes, especially those related with IGF-1R signaling components. Results: Treatment with vorinostat at doses ranging from 0.5 to 50 μ M exhibited a range of sensitivities after 3-day treatment. IGF-1R expression remained unchanged or increased by vorinostat treatment in relatively resistant cells while relatively sensitive cells showed decreased expression of IGF-1R after the drug treatment. Addition of MK0646 enhanced cytotoxic activities of vorinostat in relatively resistant cells. In xenograft model, combined treatment of vorinostat and MK0646 significantly delayed the growth of H1299 and H226Br xenograft tumors compared with vehicle or single agent treatment. Conclusions: Our results suggested that (a) resistance to vorinostat is related to induction expression and subsequent activation of IGF-1R and (b) integration of IGF-IR-targeted agents is needed to the treatment regimens with vorinostat for patients with lung cancer. Results from this study provide a proof-of principle for clinical trials of MK0646 and vorinostat combination in patients with vorinostat-resistant NSCLC. Background: Histone deacetylase (HDAC), which modulate chromatin structure and gene expression, has attracted attention as a promising therapeutic target for hematologic and solid tumors including non-small cell lung cancer (NSCLC). However, the mechanisms resulting in the resistance to HDAC inhibitors are not yet well-known. In the current study, we investigated the involvement of insulin-like growth factor 1 receptor (IGF-1R) signaling in resistance to HDAC inhibitor in NSCLC. Materials and Methods: We assessed response of a panel of 14 NSCLC cells to vorinostat-based treatment by MTT assay. Because cells growing in monolayer on standard tissue culture plates could have different response to drug treatment compared with cells growing in vivo in three-dimensional environment, we also tested the effects of vorinostat on the cells cultured as spheroids on poly-HEMA coated plate or in soft agar. The antitumor effects of vorinostat, either alone or in combination with a novel anti-IGF-1R antibody, MK0646 (Merck) were evaluated in vitro using representative vorinostat-resistant NSCLC cell lines. The antitumor potency of the combination was evaluated in vivo using xenograft tumor models established in nude mice. The mice were treated twice a week with vorinostat (50 mg/kg), MK0646 (15 mg/kg) or their combination by intraperitoneal (IP) injection. Western blot, real time quantitative PCR, and immunohistochemical analyses were performed to assess biochemical changes, especially those related with IGF-1R signaling components. Results: Treatment with vorinostat at doses ranging from 0.5 to 50 μ M exhibited a range of sensitivities after 3-day treatment. IGF-1R expression remained unchanged or increased by vorinostat treatment in relatively resistant cells while relatively sensitive cells showed decreased expression of IGF-1R after the drug treatment. Addition of MK0646 enhanced cytotoxic activities of vorinostat in relatively resistant cells. In xenograft model, combined treatment of vorinostat and MK0646 significantly delayed the growth of H1299 and H226Br xenograft tumors compared with vehicle or single agent treatment. Conclusions: Our results suggested that (a) resistance to vorinostat is related to induction expression and subsequent activation of IGF-1R and (b) integration of IGF-IR-targeted agents is needed to the treatment regimens with vorinostat for patients with lung cancer. Results from this study provide a proof-of principle for clinical trials of MK0646 and vorinostat combination in patients with vorinostat-resistant NSCLC. Supported by the National Institutes of Health Grants R01 CA109520 (to H-Y. Lee) and CA100816 (to H-Y. Lee) and a Study Agreement with Merck and Company. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-41.

Brain angiogenesis in developmental and pathological processes: mechanism and therapeutic intervention in brain tumors: Brain tumor angiogenesis

Formation of new blood vessels is required for the growth and metastasis of all solid tumors. New blood vessels are established in tumors mainly through angiogenesis. Brain tumors in particular are highly angiogenic. Therefore, interventions designed to prevent angiogenesis may be effective at controlling brain tumors. Indeed, many recent findings from preclinical and clinical studies of antiangiogenic therapy for brain tumors have shown that it is a promising approach to managing this deadly disease, especially when combined with other cytotoxic treatments. In this minireview, we summarize the basic characteristics of brain tumor angiogenesis and the role of known angiogenic factors in regulating this angiogenesis, which may be targets of antiangiogenic therapy. We also discuss the current status of antiangiogenic therapy for brain tumors, the suggested mechanisms of this therapy and the limitations of this strategy.