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Cox proportional hazard regression analysis on a comparison between Cluster 1 and other clusters as selected by cluster analysis of the methylation status of mucin genes MUC1, MUC2, and MUC4 in non-neoplastic regions (A) and neoplastic regions (B). Red solid line: Cluster 1, black dashed line: other clusters.

Cox proportional hazard regression analysis on a comparison between Cluster 1 and Cluster 2 selected by cluster analysis of mRNA expression levels of MUC1, MUC2, and MUC4 in non-neoplastic regions (A) and neoplastic regions (B). Red solid line: Cluster 1, black dashed line: Cluster 2.

Purpose:Pancreatic cancer remains a disease of high mortality despite advanced diagnostic techniques. Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in pancreatic cancers. MUC1 and MUC4 expression are related to the aggressive behavior of human neoplasms and a poor patient outcome. In contrast, MUC2 is a tumor suppressor, and we have previously reported that MUC2 is a favorable prognostic factor in pancreatic neoplasia. This study investigates whether the methylation status of three mucin genes from postoperative tissue specimens from patients with pancreatic neoplasms could serve as a predictive biomarker for outcome after surgery.Experimental Design:We evaluated the methylation status of MUC1, MUC2, and MUC4 promoter regions in pancreatic tissue samples from 191 patients with various pancreatic lesions using methylation-specific electrophoresis. Then, integrating these results and clinicopathologic features, we used support vector machine-, neural network-, and multinomial-based methods to develop a prognostic classifier.Results:Significant differences were identified between the positive- and negative-prediction classifiers of patients in 5-year overall survival (OS) in the cross-validation test. Multivariate analysis revealed that these prognostic classifiers were independent prognostic factors analyzed by not only neoplastic tissues but also nonneoplastic tissues. These classifiers had higher predictive accuracy for OS than tumor size, lymph node metastasis, distant metastasis, and age and can complement the prognostic value of the TNM staging system.Conclusions:Analysis of epigenetic changes in mucin genes may be of diagnostic utility and one of the prognostic predictors for patients with pancreatic ductal adenocarcinoma.

Difference in the expression level of mucins between Cluster 1 and other clusters selected by cluster analysis of the methylation status of mucin genes MUC1, MUC2, and MUC4. Expression levels show relative quantification (log10).

Univariate and multivariate analyses for overall survival (Cox proportional hazard model).

Prognosis prediction by machine learning classifier in the k-fold CV test. Cox proportional hazard regression analysis for the comparison between the positive and negative groups selected by each classifier. Red solid line: predicted high-risk group (positive), blue line: other groups (negative). (A) 3-fold cross-validation test. (B) 4-fold cross-validation test. The insets show p-value (p) and hazard ratio (HR) of each test.

Comparison of feature between training and test cohort.

Comparison of SVM predict ability among these kernels in LOOCV

Overview ideogram of CGH data of all chordoma cell lines. Every cell line corresponds to one color. Lines on the left side are losses, and on the right side are gains of chromosomal material. Arrows mark the CDK6 region on 7q and p16 region on 9p.

Detailed CGH karyograms of the chordoma cell lines as shown in the ideogram in Supplementary Figure 1 (according to ISCN: International System for Human Cytogentic Nomenclature).

Results of the single tandem repeats analysis of the cell lines U-CH1, U-CH2, UCH3, UCH6, UCH7, U-CH10, U-CH11 and UCH12. The results of the sex typing marker AMEL and the detected alleles for the markers D3S1358, D1S1656, D2S441, D10S1248, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA, D22S1045 are given.

Expression data of mRNA as shown by box plot graphs for brachyury (A), collagen 2A1 (B) and CD24 (C).

Unsupervised cluster analysis of mRNA expression data of all chordoma cell lines. The T-gene (i.e., brachyury) is overexpressed (red). Cluster Analysis of chordoma cell lines with chondrocyte, liposarcoma and sarcoma expression data from GEO and own data from a cell line with cartilaginous differentiation and from tissue of a vertebral disc.

Effect of Abemaciclib (LY2835219) on the growth of chordoma lines U-CH1, U-CH6, CAL51 (negative control), and MCF7 (positive control). Cells were grown in 96-well-plates were incubated for 3 days at 37{degree sign}C with increasing concentrations of Abemaciclib (3, 6, 10, 30, 60, 100, 300, 600, 1000 nM) in the medium or left untreated for control. The amount of viable cells was determined by using the MTS cell viability test (GraphPad Prism software, Version 6).

Western blot analyses of 8 chordoma cell lines showing expression of brachyury at 47 kDa.

Clinical data of chordoma patients. Every sample was positive for brachyury. Asterisks (*) mark the parental chordomas of the established chordoma cell lines.

Proliferation assay using Alamar Blue assay for U-CH2, 3, 6 and 10. Increasing concentrations of Palbociclib lead to an increase of growth inhibition of chordoma cells.

Inversion microscopy views of the published chordoma cell lines U-CH1, U-CH2, U-CH 3, 6, 7, 10, 11, and 12. The cells showing the typical physaliphorous cytology; bars=100µm.

Supplementary Figure Legend

Immunocytology of U-CH1 control vs inhibited cells with 1000 nM Palbociclib for 3 days. Ki-67 decreases from 20% down to

Data Supplement from Preclinical Characterization of Novel Chordoma Cell Systems and Their Targeting by Pharmocological Inhibitors of the CDK4/6 Cell-Cycle Pathway

Expression data of mRNA as shown by box plot graphs for brachyury (A), collagen 2A1 (B) and CD24 (C).

List of up and down regulated genes on expression level with a p-value of 10-4 a FC of 10.

FISH results regarding the CDKN2A status in chordoma cell lines and the corresponding parental tumor.

Western blot analysis for cleaved PARP and cleaved caspase 3 of all chordoma cell lines as compared to HEK-293 cells (+/- stimulation with TNFα and Bortezomib for 18h). No corresponding bands are apparent in the cell lines after inhibition with 1000 nM Palbociclib for 3 days. U-CH 1-6 (A) and U-CH 7-12 (A).

Introduction: Pancreatic cancer cells are exposed to multiple stressors with both endogenous (proliferation, metabolism and nutrient deprivation) and exogenous (medical treatment) origins. The c-Jun N-terminal protein kinases (JNKs) JNK1 and JNK2 are important stress-activated kinases, responsible for the cellular response to these stressors. Both were demonstrated to exert both tumor-suppressing and tumor-promoting effects in human cancers Most previous studies rely on nonspecific pharmacological JNK inhibition. In this study, we planned to determine the isoform-specific roles in PDAC. Methods: Specific JNK1- and JNK2-knockdown (KD) clones were established in cultured human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 by using specific shRNA plasmids. Anchorage dependent and independent growth, as well as migration and invasion, were analyzed in JNK-1/2 KD clones and compared to pharmacological JNK inhibition using SP600125. Xenograft growth was assessed using an orthotopic mouse model. Mechanistically, we assessed changes in EMT markers both in vitro and in vivo. Results: JNKs were expressed in both cultured pancreatic cancer cell lines as well as in resected human pancreatic cancer samples. Specific downregulation of JNK2 resulted in an overall increase in cell proliferation, invasion and alterations in cytoskeleton structure, while JNK1-KD revealed opposite effects. Nonspecific JNK-inhibition using SP600125 inhibited cell growth of all cell lines except PANC-1. Conclusion: JNK1 and JNK2 play opposing roles in human pancreatic cancer. Specifically targeting JNK1 with the retained function of JNK2 may provide a potential, individualized approach for targeting pancreatic cancer progression. Citation Format: Jingwei Shi, Xiaodong Tian, Marko Kornmann, Benno Traub. JNK2 suppresses the growth and invasion of pancreatic cancer and is opposed by JNK1 [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-093.

Chordomas are tumors that arise at vertebral bodies and the base of the skull. Although rare in incidence, they are deadly owing to slow growth and a lack of effective therapeutic options. In this study, we addressed the need for chordoma cell systems that can be used to identify therapeutic targets and empower testing of candidate pharmacologic drugs. Eight human chordoma cell lines that we established exhibited cytology, genomics, mRNA, and protein profiles that were characteristic of primary chordomas. Candidate responder profiles were identified through an immunohistochemical analysis of a chordoma tissue bank of 43 patients. Genomic, mRNA, and protein expression analyses confirmed that the new cell systems were highly representative of chordoma tissues. Notably, all cells exhibited a loss of CDKN2A and p16, resulting in universal activation of the CDK4/6 and Rb pathways. Therefore, we investigated the CDK4/6 pathway and responses to the CDK4/6–specific inhibitor palbociclib. In the newly validated system, palbociclib treatment efficiently inhibited tumor cell growth in vitro and a drug responder versus nonresponder molecular signature was defined on the basis of immunohistochemical expression of CDK4/6/pRb (S780). Overall, our work offers a valuable new tool for chordoma studies including the development of novel biomarkers and molecular targeting strategies. Cancer Res; 75(18); 3823–31. ©2015 AACR.