Hyang Sook Seol

Epigenetic silencing of microRNA-373 to epithelial-mesenchymal transition in non-small cell lung cancer through IRAK2 and LAMP1 axes

The role of microRNAs (miRNAs) in carcinogenesis as tumor suppressors or oncogenes has been widely reported. Epigenetic change is one of the mechanisms of transcriptional silencing of miRNAs in cancer. To identify lung cancer-related miRNAs that are mediated by histone modification, we conducted microarray analysis in the Calu-6 non-small cell lung cancer (NSCLC) cell line after treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor. The expression level of miR-373 was enhanced by SAHA treatment in this cell line by microarray and the following quantitative RT-PCR analyses. Treatment with another HDAC inhibitor, Trichostatin A, restored the levels of miR-373 expression in A549 and Calu-6 cells, while demethylation drug treatment did not. Importantly, miR-373 was found to be down-regulated in NSCLC tissues and cell lines. Transfection of miR-373 into A549 and Calu-6 cells attenuated cell proliferation, migration, and invasion and reduced the expression of mesenchymal markers. Additional microarray analysis of miR-373-transfected cells and computational predictions identified IRAK2 and LAMP1 as targets of miR-373. Knockdown of these two genes showed similar biological effects to those of miR-373 overexpression. In clinical samples, overexpression of IRAK2 correlated with decreased disease-free survival of patients with non-adenocarcinoma. In conclusion, we found that miR-373 is silenced by histone modification in lung cancer cells and identified its function as a tumor suppressor and negative regulator of the mesenchymal phenotype through downstream IRAK2 and LAMP1 target genes.

Development and characterization of a colon PDX model that reproduces drug responsiveness and the mutation profiles of its original tumor.

Cultures of primary tumors are very useful as a personalized screening system for effective therapeutic options. We here describe an effective method of reproducing human primary colon tumors through primary culture and a mouse xenograft model. A total of 199 primary colon tumor cultures were successfully established under optimized conditions to enrich for tumor cells and to expand it for long-term storage in liquid nitrogen. To examine whether these stored cultures retained original tumor properties, fifty primary cultures were xenografted into NOD-SCID mouse. Histological and tumor marker analysis of four representative tumor xenografts revealed that all of the xenograft retained its primary tumor characteristics. Oncomap analysis further showed no change in the major mutations in the xenografts, confirming that our method faithfully reproduced human colon tumors. A drug sensitivity assay revealed that two of the primary cultures were hypersensitive to oxaliplatin rather than 5-FU, which was used in the patients, suggesting it as an effective therapeutic option. We thus present an effective, reproducible preclinical model for testing various personalized therapeutic options in colon cancer patients.

CD49fhigh Cells Retain Sphere-Forming and Tumor-Initiating Activities in Human Gastric Tumors

Identification of gastric tumor-initiating cells (TICs) is essential to explore new therapies for gastric cancer patients. There are reports that gastric TICs can be identified using the cell surface marker CD44 and that they form floating spheres in culture, but we could not obtain consistent results with our patient-derived tumor xenograft (PDTX) cells. We thus searched for another marker for gastric TICs, and found that CD49fhigh cells from newly-dissected gastric cancers formed tumors with histological features of parental ones while CD49flow cells did not when subcutaneously injected into immunodeficient mice. These results indicate that CD49f, a subunit of laminin receptors, is a promising marker for human gastric TICs. We established a primary culture system for PDTX cells where only CD49fhigh cells could grow on extracellular matrix (ECM) to form ECM-attaching spheres. When injected into immunodeficient mice, these CD49fhigh sphere cells formed tumors with histological features of parental ones, indicating that only TICs could grow in the culture system. Using this system, we found that some sphere-forming TICs were more resistant than gastric tumor cell lines to chemotherapeutic agents, including doxorubicin, 5-fluorouracil and doxifluridine. There was a patient-dependent difference in the tumorigenicity of sphere-forming TICs and their response to anti-tumor drugs. These results suggest that ECM plays an essential role for the growth of TICs, and that this culture system will be useful to find new drugs targeting gastric TICs.

p53-Independent expression of wild-type p53-induced phosphatase 1 (Wip1) in methylmethane sulfonate-treated cancer cell lines and human tumors

Wild-type p53-induced phosphatase 1 (Wip1, PPM1D) is induced by p53 in response to various stressors and dephosphorylates cellular target proteins involved in DNA repair and cell cycle checkpoint pathways. The Wip1 gene is frequently amplified or overexpressed in human cancers, promoting tumor growth by switching off major checkpoint kinases and p53. To explore wild-type p53-independent Wip1 induction, Wip1 promoter activity and its transcript level were evaluated by luciferase assay and real-time PCR, after methylmethane sulfonate (MMS) treatment in breast cancer cell lines and p53-null cell lines. Wip1 promoter activities in response to UV irradiation and various anti-cancer agents were compared between wild-type and a p53-response element (p53RE) mutated construct. Wip1 expression and its effects were examined in primary non-small cell lung cancer (NSCLC) and colon tumor cells by using Wip1-specific siRNA. MMS induced Wip1 promoter activity in Hs578T, MDA-MB-231, and SK-BR-3 cells expressing DNA binding-deficient p53 mutants. A549-E6 and HCT116 (p53(-/-)) cells retained substantial Wip1 induction. Wip1 promoter activity was reduced, but not eliminated, in cells expressing a promoter containing a mutated p53-response element. Wip1 induction was not blocked by SB202190 or SP600125. MMS increased Wip1 expression in primary non-small cell lung cancer cells expressing a p53 R175H mutant. Our data indicate that Wip1 is induced in the absence of functional p53, like p38 MAPK and JNK, as a stress response terminator.

Generation and molecular characterization of pancreatic cancer patient-derived xenografts reveals their heterologous nature

Pancreatic ductal adenocarcinoma (PDAC) is the most challenging type of cancer to treat, with a 5-year survival rate of <10%. Furthermore, because of the large portion of the inoperable cases, it is difficult to obtain specimens to study the biology of the tumors. Therefore, a patient-derived xenograft (PDX) model is an attractive option for preserving and expanding these tumors for translational research. Here we report the generation and characterization of 20 PDX models of PDAC. The success rate of the initial graft was 74% and most tumors were re-transplantable. Histological analysis of the PDXs and primary tumors revealed a conserved expression pattern of p53 and SMAD4; an exome single nucleotide polymorphism (SNP) array and Comprehensive Cancer Panel showed that PDXs retained over 94% of cancer-associated variants. In addition, Polyphen2 and the Sorting Intolerant from Tolerant (SIFT) prediction identified 623 variants among the functional SNPs, highlighting the heterologous nature of pancreatic PDXs; an analysis of 409 tumor suppressor genes and oncogenes in Comprehensive Cancer Panel revealed heterologous cancer gene mutation profiles for each PDX-primary tumor pair. Altogether, we expect these PDX models are a promising platform for screening novel therapeutic agents and diagnostic markers for the detection and eradication of PDAC.

Glutamate release inhibitor, Riluzole, inhibited proliferation of human hepatocellular carcinoma cells by elevated ROS production.

Liver cancer is one of the common malignancies in many countries and an increasing cause of cancer death. Despite of that, there are few therapeutic options available with inconsistent outcome, raising a need for developing alternative therapeutic options. Through a drug repositioning screening, we identified and investigated the action mechanism of the Riluzole, an amyotrophic lateral sclerosis (ALS) drug, on hepatocellular carcinoma (HCC) therapy. Treatment of the Riluzole leads to a suppression of cell proliferation in liver primary cancer cells and cancer cell lines. In addition, Riluzole induced caspase-dependent apoptosis and G2/M cell cycle arrest in SNU449 and Huh7 cell lines. In a line with the known function of glutamate release inhibitor, we found Riluzole-treated cells have increased the level of inner cellular glutamate that in turn decrease the glutathione (GSH) level and finally augment the reactive oxygen species (ROS) production. We confirm this finding in vivo by showing the Riluzole-induced GSH and ROS changes in a Huh7 xenograft cancer model. Altogether, these data suggest the anti-cancer effect of Riluzole on hepatocellular carcinoma and the suppression of glutamate signaling might be a new target pathway for HCC therapy.

The Generation and Application of Patient-Derived Xenograft Model for Cancer Research

Establishing an appropriate preclinical model is crucial for translational cancer research. The most common way that has been adopted by far is grafting cancer cell lines, derived from patients. Although this xenograft model is easy to generate, but has several limitations because this cancer model could not represent the unique features of each cancer patient sufficiently. Moreover, accumulating evidences demonstrate cancer is a highly heterogeneous disease so that a tumor is comprised of cancer cells with diverse characteristics. In attempt to avoid these discrepancies between xenograft model and patients’ tumor, a patient-derived xenograft (PDX) model has been actively generated and applied. The PDX model can be developed by the implantation of cancerous tissue from a patient’s tumor into an immune-deficient mouse directly, thereby it preserves both cell-cell interactions and tumor microenvironment. In addition, the PDX model has shown advantages as a preclinical model in drug screening, biomarker development and co-clinical trial. In this review, we will summarize the methodology and applications of PDX in detail, and cover critical issues for the development of this model for preclinical research.

Complement proteins C7 and CFH control the stemness of liver cancer cells via LSF-1

Tumor-initiating cells are important for the formation and maintenance of tumor bulks in various tumors. To identify surface markers of liver tumor-initiating cells, we performed primary tumorsphere culture and analyzed the expression of cluster of differentiation (CD) antigen genes using NanoString. Interestingly, we found significant upregulation of the complement proteins (p = 1.60 × 10(-18)), including C7 and CFH. Further studies revealed that C7 and CFH are required to maintain stemness in liver cancer cells. Knockdown of C7 and CFH expression abrogated tumorsphere formation and induced differentiation, whereas overexpression stimulated stemness factor expression as well as in vivo cell growth. Mechanistically, by studying C7 and CFH-dependent LSF-1 expression and its direct role on stemness factor transcription, we found that LSF-1 is involved in this regulation. Taken together, our data demonstrate the unprecedented role of complement proteins on the maintenance of stemness in liver tumor-initiating cells.

Development of Cell-Defined Lentivirus-Based Microarray for Mammalian Cells

Although reverse transfection cell microarray (RTCM) is a powerful tool for mammalian cell studies, the technique is not appropriate for cells that are difficult to transfect. The lentivirus-infected cell microarray (LICM) technique was designed to improve overall efficiency. However, LICM presents new challenges because individual lentiviral particles can spread through the cell population, leading to cross-contamination. Therefore, we designed a cell-defined lentivirus microarray (CDLM) technique using cell-friendly biomaterials that are controlled by cell attachment timing. We selected poly-l-lysine (PLL) with Matrigel as the best combination of biomaterials for cell-defined culture. We used 2 µL PLL to determine by titration the optimum concentration required (0.04% stock, 0.005% final concentration). We also determined the optimum concentration of 10 µL of lentivirus particles for maximum reverse infection efficiency (1 × 108 infectious units [IFU]/mL stock, 62.5% final concentration) and established the best combination of components for the lentivirus mixture (10 µL of lentivirus particles and 2 µL each of siGLO Red dye, Matrigel, and 0.04% PLL). Finally, we validated both the effect of reverse infection in various cell lines and lentivirus spot activity in CDLM by storage period. This method provides an effective lentivirus-infected cell microarray for large-scale gene function studies.

Abstract 2987: Oncogenic Wip1 is activated and negatively regulates DNA-damage induced apoptosis in p53-null cancer cells

Wip1 (wild-type p53-induced phosphatase, or PPM1D) belongs to the protein phosphatase type 2C family and its expression is induced by p53 in response to various stresses. Wip1 is known to dephosphorylate cellular target proteins such as p38 MAPK, p53, and uracil DNA glycosylase that are involved in DNA-repair and cell-cycle-checkpoint pathways. Here, we show that after methylmethane sulfonate (MMS) treatment Wip1 is induced in various breast-cancer cell lines in the absence of wild-type p53 and p38 MAPK activity. Wip1 expression in response to MMS was not blocked by the p38 MAPK inhibitor SB202190 or in p38-silenced MCF-7 cells using p38 MAPK small-interfering RNA (siRNA). The Wip1 promoter displayed great induction in breast cancer cell lines, which have DNA-binding deficient p53 mutants, and p53-null cancer cells such as Saos-2, A549-E6 and HCT116(p53−/−), whereas a control promoter, p53-Luc, showed the least promoter activity after MMS treatment. The mutation of p53 response element in the Wip1 promoter decreased its activity in both unstressed and stressed cells, but the overall level of fold-induction was similar. In addition, cell death was greatly increased in Wip1-siRNA and MMS treated cells in comparison with scrambled siRNA and MMS treated cells in the both cell lines of HCT116 (p53 +/+) and HCT116 (p53−/−). Our data indicate that Wip1 induction in response to various stimuli is not solely controlled by p53, and other damage response signaling appears to compensate for p53. Furthermore, Wip1 negatively regulates DNA-damage induced cell death regardless of the presence wild-type p53. 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 2987.