Young-Ah Suh

ROS1 Receptor Tyrosine Kinase, a Druggable Target, is Frequently Overexpressed in Non-Small Cell Lung Carcinomas Via Genetic and Epigenetic Mechanisms

BackgroundMicroarray analyses have revealed significantly elevated expression of the proto-oncogene ROS1 receptor tyrosine kinase in 20–30xa0% of non-small cell lung carcinomas (NSCLC). Selective and potent ROS1 kinase inhibitors have recently been developed and oncogenic rearrangement of ROS1 in NSCLC identified.MethodsWe performed immunohistochemical evaluation of expression of ROS1 kinase and its downstream molecules in 399 NSCLC cases. ROS1 expression in primary and recurring lesions of 92 recurrent NSCLC cases was additionally analyzed. To elucidate mechanism of expression, two ROS1-nonexpressing NSCLC cell lines (Calu6 and H358) and fresh frozen tissues from 28 consecutive NSCLC patients were examined for ROS1 promoter methylation status and ROS1 expression.ResultsOverall expression rate of ROS1 was 22xa0% (19xa0% for adenocarcinomas and 25xa0% for nonadenocarcinomas) in NSCLC. ROS1 expression was a worse prognostic factor for overall survival in adenocarcinomas of stage I NSCLC. In recurred NSCLC, ROS1 expression was significantly higher in recurring tumors (38xa0%) than primary tumors (19xa0%). Two NSCLC cell lines showed increased ROS1 expression after treatment with 5-aza-2′deoxycytidine and/or trichostatin A. Among the 14 adenocarcinomas examined, two (14xa0%) showed more than twice the level of ROS1 expression in tumor tissue than was observed in matched normal tissue and statistically significant differences in the ROS1 promoter methylation level.ConclusionsA subset of NSCLC revealed overexpression of ROS1 receptor tyrosine kinase, possibly in relation to epigenetic changes. ROS1 expression was an independent prognostic factor for overall survival in adenocarcinomas of stage I NSCLC. Further studies are needed to validate our results.

KIF3A binds to β-arrestin for suppressing Wnt/β-catenin signalling independently of primary cilia in lung cancer.

Aberrant Wnt/β-catenin signalling is implicated in the progression of several human cancers, including non-small cell lung cancer (NSCLC). However, mutations in Wnt/β-catenin pathway components are uncommon in NSCLC, and their epigenetic control remains unclear. Here, we show that KIF3A, a member of the kinesin-2 family, plays a role in suppressing Wnt/β-catenin signalling in NSCLC cells. KIF3A knockdown increases both β-catenin levels and transcriptional activity with concomitant promotion of malignant potential, such as increased proliferation and migration and upregulation of stemness markers. Because KIF3A binds β-arrestin, KIF3A depletion allows β-arrestin to form a complex with DVL2 and axin, stabilizing β-catenin. Although primary cilia, whose biogenesis requires KIF3A, are thought to restrain the Wnt response, pharmacological inhibition of ciliogenesis failed to increase β-catenin activity in NSCLC cells. A correlation between KIF3A loss and a poorer NSCLC prognosis as well as β-catenin and cyclin D1 upregulation further suggests that KIF3A suppresses Wnt/β-catenin signalling and tumourigenesis in NSCLC.

A patient-derived xenograft mouse model generated from primary cultured cells recapitulates patient tumors phenotypically and genetically

BackgroundPreclinical trials of cancer therapeutics require both in vitro and in vivo evaluations. Recently, a patient-derived xenograft model in immunodeficient mice has been reported as a valuable in vivo evaluation system. In our current study, we aimed to establish a more efficient and accurate system for preclinical trials by generating primary cancer cells from patients and performing xenograft transfers of these cells into mice.MethodsHuman lung cancer specimens (nxa0=xa04) obtained from chemo-naive patients were cultured in bronchiolar epithelial basal medium supplemented with growth factors, followed by inoculation into non-obese diabetic/severe combined immunodeficient mice. The generated tumors in the mice were validated phenotypically and genetically using the original specimen and primary cancer cells.ResultsImmunohistochemical analysis of marker proteins, including cytokeratin 7, cytokeratin 20, epidermal growth factor receptor, thyroid transcription factor-1, CD56, chromogranin, and synaptophysin, demonstrated that the xenograft tumors were originated from the patient tumors. Moreover, mutation profiling using the OncoMap System, which analyzes mutations at 440 sites in 41 tumor-related genes, showed the same patterns in both the patient and xenograft tumors.ConclusionsThese results indicate that our animal system is suitable for the amplification of patient tumors and will therefore be beneficial for both in vivo and in vitro assessments and preclinical trials of chemotherapeutics. This has the potential to provide a very effective tool for future personalized therapy and for conducting translational lung cancer research.

Polypyrimidine tract-binding protein 1-mediated down-regulation of ATG10 facilitates metastasis of colorectal cancer cells.

Autophagy plays complex roles in tumor initiation and development, and the expression of autophagy-related genes (ATGs) is differentially regulated in various cancer cells, depending on their environment. In this study, we analyzed the expressional relationship between polypyrimidine tract-binding protein 1 (PTBP1) and ATG10 in metastatic colorectal cancer. PTBP1 is associated with tumor metastasis in primary colorectal tumors and colorectal cancer liver metastasis (CLM) tissues. In addition, PTPB1 directly interacts with mRNA of ATG10, and regulates ATG10 expression level in colorectal cancer cells. Ectopic expression of PTBP1 decreased ATG10 expression, whereas down-regulation of PTBP1 increased ATG10 level. In contrast to PTBP1, expression of ATG10 was decreased in CLM tissues. Knock down of ATG10 promoted cell migration and invasion of colorectal cancer cells. Moreover, depletion of ATG10 modulated epithelial-mesenchymal transition-associated proteins in colorectal cancer cells: N-cadherin, TCF-8/ZEB1, and CD44 were up-regulated, whereas E-cadherin was down-regulated. Taken together, our findings suggest that expression of ATG10 negatively regulated by PTBP1 is associated with metastasis of colorectal cancer cells.

Targeting FGFR Pathway in Human Hepatocellular Carcinoma: Expressing pFGFR and pMET for Antitumor Activity

The MET receptor tyrosine kinase, the receptor for hepatocyte growth factor (HGF), has been implicated in cancer growth, invasion, migration, angiogenesis, and metastasis in a broad variety of human cancers, including human hepatocellular carcinoma (HCC). Recently, MET was suggested to be a potential target for the personalized treatment of HCC with an active HGF–MET signaling pathway. However, the mechanisms of resistance to MET inhibitors need to be elucidated to provide effective treatment. Here, we show that HCC cells exhibit different sensitivities to the MET inhibitor PHA665752, depending on the phosphorylation status of FGFR. Treatment of cells expressing both phospho-FGFR and phospho-MET with the inhibitor PHA665752 did not cause growth inhibition and cell death, whereas treatment with AZD4547, a pan-FGFR inhibitor, resulted in decreased colony formation and cleavage of caspase-3. Moreover, silencing of endogenous FGFR1 and FGFR2 by RNAi of HCC cells expressing phospho-FGFR, phospho-FGFR2, and phospho-MET overcame the resistance to PHA665752 treatment. Treatment of primary cancer cells from patients with HCC expressing both phospho-FGFR and phospho-MET with PHA665752 did not induce cell death, whereas AZD4547 treatment induced cell death through the cleavage of caspase-3. In addition, treatment of cells resistant to PHA665752 with AZD4547 abrogated the activation of downstream effectors of cell growth, proliferation, and survival. On the basis of these results, we conclude that the FGFR pathway is critical for HCC survival, and that targeting this pathway with AZD4547 may be beneficial for the treatment of patients with HCC-expressing phospho-FGFR and phospho-MET. Mol Cancer Ther; 14(11); 2613–22. ©2015 AACR.

A novel mechanism for the pyruvate protection against zinc-induced cytotoxicity: mediation by the chelating effect of citrate and isocitrate

Intracellular accumulation of free zinc contributes to neuronal death in brain injuries such as ischemia and epilepsy. Pyruvate, a glucose metabolite, has been shown to block zinc neurotoxicity. However, it is largely unknown how pyruvate shows such a selective and remarkable protective effect. In this study, we sought to find a plausible mechanism of pyruvate protection against zinc toxicity. Pyruvate almost completely blocked cortical neuronal death induced by zinc, yet showed no protective effects against death induced by calcium (ionomycin, NMDA) or ferrous iron. Of the TCA cycle intermediates, citrate, isocitrate, and to a lesser extent oxaloacetate, protected against zinc toxicity. We then noted with LC–MS/MS assay that exposure to pyruvate, and to a lesser degree oxaloacetate, increased levels of citrate and isocitrate, which are known zinc chelators. While pyruvate added only during zinc exposure did not reduce zinc toxicity, citrate and isocitrate added only during zinc exposure, as did extracellular zinc chelator CaEDTA, completely blocked it. Furthermore, addition of pyruvate after zinc exposure substantially reduced intracellular zinc levels. Our results suggest that the remarkable protective effect of pyruvate against zinc cytotoxicity may be mediated indirectly by the accumulation of intracellular citrate and isocitrate, which act as intracellular zinc chelators.

Abstract A22: Cell growth regulation of gain-of-function mutant p53 via miRNAs on metabolic inhibition

There is growing evidence that tumor cells depend on unique metabolism for their continued growth and survival, and that cancer cells are peculiarly addicted to the rapacious uptake of glucose and glutamine. Due to the gain-of-function, mutant p53 protein contributes to tumor metastasis and shortening the latency of tumor progression, and thereby activated K-ras/mutant p53 mouse is preferentially used as lung adenocarcinoma preclinical model. Epigenetic regulation through histone modification or miRNA also plays a pivotal role on gene regulation, as regarded as global epigenetic markers, especially in tumor related genes. Hence, chemical approaches targeting histone-modifying enzymes have emerged onto the main stage of anticancer drug discovery. Here, we have investigated the involvement of miRNAs in cell growth regulation upon the treatment of metabolic inhibitors. We have generated different genotypes of mouse tumor cells derived from activated mutant p53 and/or K-ras mutant mice. We have tested whether inhibitors regulating metabolism induce adverse effect on cell growth and changes of miRNA expression through microarray analysis, with the hint of enormous demands for nutrients in fast growing tumor cells. We then analyzed affected genes and miRNAs via quantitative RT-PCR to confirm the involvement of miRNAs on cell growth when tumor metabolic environment were damaged. Citation Format: Se-Young Jo, Hae-Min Moon, ChuHee Lee, Se Jin Jang, Young-Ah Suh. Cell growth regulation of gain-of-function mutant p53 via miRNAs on metabolic inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A22.

Abstract 2295: AMP-activated protein kinase (AMPK) signaling in the context of gain-of-function mutant p53 in vivo

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnThere is growing evidence that tumor cells depend on unique metabolism for their continued growth and survival, and that cancer cells are peculiarly addicted to the rapacious uptake of glucose and glutamine. AMP-activated protein kinase (AMPK) is one of the central regulators of cellular and organismal metabolism in eukaryotes, playing critical roles in regulating cell growth and reprogramming metabolism. Several of the downstream effects of AMPK on metabolic adaptation can be attributed to the AMPK-dependent activation of the p53 tumor suppressor, resulting in its transcriptional activation and initiating a metabolic cell cycle checkpoint. However, in the absence of p53, treatment with the blood glucose-lowering drug metformin delays tumor progression, suggesting that the AMPK-p53 signaling axis responds in diverse manner depending on genetic alteration. Due to the gain-of-function, mutant p53 protein contributes to tumor metastasis and shortening the latency of tumor progression. Thereby activated K-ras/mutant p53 mouse is preferentially used as lung adenocarcinoma preclinical model. Here, we have established mouse tumor cells derived from activated K-ras/mutant p53 mouse to generate syngeneic lung cancer mouse model, allowing in vivo imaging. We asked whether the gain-of-function metastatic phenotype observed in the mouse is responsible for unique metabolic conditions resulting in the formation of lung tumors. We have evaluated tumor metabolic environment of this model and examined AMPK signaling pathways.nnCitation Format: Se-Young Jo, Hye-Min Moon, ChuHee Lee, Se Jin Jang, Young-Ah Suh. AMP-activated protein kinase (AMPK) signaling in the context of gain-of-function mutant p53 in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2295. doi:10.1158/1538-7445.AM2015-2295

Abstract 89: Development of preclinical lung adenocarcinoma model

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnLung cancer is a leading cause of cancer death in worldwide. 85% of lung cancers are classified as non-small cell lung cancers (NSCLC), and adenocarcinoma (ADC) type is the most prevalent cancer in NSCLC. Approximately 50% of non-small cell lung cancer (NSCLC) patients have mutations in some oncogenes. The K-ras activation due to the mutant is notorious in many kinds of cancer types, including pancreatic cancer and lung adenocarcinoma, as is generally accepted as driving mutation for lung malignancy of lung adenocarcinoma. Another predominantly alternated gene in lung cancer is the EGFR that would hit the similar signaling with K-ras activation, to which small molecules and antibodies target to halt the tumor progression. The tumor suppressor p53 is also often altered in almost all kinds of cancers and lung cancer. In this reason, K-ras activated mouse is utilized as lung cancer mouse model. K-ras activated mouse eventually develops lung adenocarcinoma, which can be accelerated with mutated p53. Due to the gain-of-function, mutant p53 protein contributes to tumor metastasis and shortening the latency of tumor progression. Thereby activated K-ras/mutant p53 mouse is preferentially used as lung adenocarcinoma preclinical model. Here, we have used mouse tumor cells derived from activated K-ras/mutant p53 mouse to establish syngeneic lung cancer mouse model, allowing in vivo imaging. We asked whether the gain-of-function metastatic phenotype observed in the mouse is responsible for the formation of lung tumors when inoculated intravenously. Tumor cells from the mice could efficiently develop tumors in four weeks at the same genetic background. Tumors from the mice were histologically identical with original lung adenocarcinoma, suggesting that these cells are the efficient tools to generate preclinical lung tumor mouse model. We have examined this model for evaluating the efficiency of chemotherapeutics and signaling pathways involved in tumor growth regulation.nnCitation Format: Young-Ah Suh, Minsuh Kim, Sun-Hye Lee, Chae Lim Jung, Hye-Min Mun, Ju-Hee Oh, Eun Kyung Choi, Se Jin Jang. Development of preclinical lung adenocarcinoma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 89. doi:10.1158/1538-7445.AM2014-89

Abstract 338: Regulation of mutant p53 retarded cell growth established from mouse lung cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnThe genetic defects in lung cancer patient are the most important factor for malignancies, and it has been reported that approximately 50% of non-small cell lung cancer (NSCLC) patients have mutations in certain tumor-related genes. Therefore, exhaustive efforts have been devoted to uncovering the genetic alterations of lung cancer, although its driving genes and genetic determinants that attribute to the development of lung cancer so far remain elusive.nnThe K-ras activation due to the mutant is notorious in many kinds of cancer types, including pancreatic cancer and lung adenocarcinoma, as is generally accepted as driving mutation for lung malignancy of lung adenocarcinoma. Another predominantly alternated gene in lung cancer is the EGFR that hits the similar signaling with K-ras activation, to which small molecules and antibodies target to halt the tumor progression. The tumor suppressor p53 is also often altered in almost all kinds of cancers and lung cancer. In this reason, K-ras activated mouse is utilized as lung cancer mouse model. K-ras activated mouse eventually develops lung adenocarcinoma, which can be accelerated with mutated p53.nnDue to the gain-of-function, mutant p53 protein contributes to tumor metastasis and shortening of the latency of tumor progression. Thereby activated K-ras/mutant p53 mouse is preferentially used as lung adenocarcinoma preclinical model. Here, we have regulated mutant p53 protein expression in mouse embryo fibroblast (MEF) or tumor cells derived from activated K-ras/mutant p53 mouse with small molecules to inhibit tumor growth. The cell growth was inhibited when the mutant p53 was destabilized. We assessed the mechanism that is responsible for the regulation of gain-of-function metastatic phenotype observed in the p53 mutant mousennCitation Format: Young-Ah Suh, Chae Lim Jung, Ji-Ho Park, Se Jin Jang. Regulation of mutant p53 retarded cell growth established from mouse lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 338. doi:10.1158/1538-7445.AM2013-338