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Figure S1. RHEB mRNA expression in different types of cancer. Figure S2. In vivo growth of MeT-5A mesothelial cells expressing exogenous RHEBL1. Figure S3. RHEBL1 binds to SmgGDS. Figure S4. SmgGDS knockdown suppresses the proliferation of mesothelial cells expressing exogenous RHEBL1. Figure S5. SmgGDS knockdown suppresses the growth of PTEN-deficient MM cells in vitro. Figure S6. Subcellular RHEBL1 localization. Figure S7. SmgGDS knockdown alters RHEBL1 localization in PTEN-deficient MM cells. Figure S8. Amino acid stimulation does not affect RHEBL1 subcellular localization. Figure S9. RHEBL1 farnesylation is essential for SmgGDS binding.

Malignant mesothelioma (MM) is an aggressive tumor that typically develops after a long latency following asbestos exposure. Although mechanistic target of rapamycin complex 1 (mTORC1) activation enhances MM cell growth, the mTORC1 inhibitor everolimus has shown limited efficacy in clinical trials of MM patients. We explored the mechanism underlying mTORC1 activation in MM cells and its effects on cell proliferation and progression. Analysis of the expression profiles of 87 MMs from The Cancer Genome Atlas revealed that 40 samples (46%) displayed altered expression of RPTOR (mTORC1 component) and genes immediately upstream that activate mTORC1. Among them, we focused on RHEB and RHEBL1, which encode direct activators of mTORC1. Exogenous RHEBL1 expression enhanced MM cell growth, indicating that RHEB–mTORC1 signaling acts as a pro-oncogenic cascade. We investigated molecules that directly activate RHEBs, identifying SmgGDS as a novel RHEB-binding protein. SmgGDS knockdown reduced mTORC1 activation and inhibited the proliferation of MM cells with mTORC1 activation. Interestingly, SmgGDS displayed high binding affinity with inactive GDP-bound RHEBL1, and its knockdown reduced cytosolic RHEBL1 without affecting its activation. These findings suggest that SmgGDS retains GDP-bound RHEBs in the cytosol, whereas GTP-bound RHEBs are localized on intracellular membranes to promote mTORC1 activation. We revealed a novel role for SmgGDS in the RHEB–mTORC1 pathway and its potential as a therapeutic target in MM with aberrant mTORC1 activation.Implications:Our data showing that SmgGDS regulates RHEB localization to activate mTORC1 indicate that SmgGDS can be used as a new therapeutic target for MM exhibiting mTORC1 activation.

Supplementary Figure S5 shows role of MFGE8 in MPM.

Supplementary Figure Legends

PDF file, 73K.

Table S6 contains results from the analysis of DNA methylation in SETD2 mutated and BAP1 inactivated samples.

PDF file, 115K, Supplementary Figure S2 E7050 inhibits tumor cell motility induced by HGF overexpression.

Supplementary Table S1 shows the selected genes with statistically significant changes in transcriptome analysis using two different shRNA constructs.

PDF file, 138K, Supplementary Figure S6 Treatment with WZ4002 and/or E7050 does not induce apoptosis in vivo.

Malignant pleural mesothelioma (MPM) is a highly lethal cancer of the lining of the chest cavity. To expand our understanding of MPM, we conducted a comprehensive integrated genomic study, including the most detailed analysis of BAP1 alterations to date. We identified histology-independent molecular prognostic subsets, and defined a novel genomic subtype with TP53 and SETDB1 mutations and extensive loss of heterozygosity. We also report strong expression of the immune-checkpoint gene VISTA in epithelioid MPM, strikingly higher than in other solid cancers, with implications for the immune response to MPM and for its immunotherapy. Our findings highlight new avenues for further investigation of MPM biology and novel therapeutic options.Significance:Through a comprehensive integrated genomic study of 74 MPMs, we provide a deeper understanding of histology-independent determinants of aggressive behavior, define a novel genomic subtype with TP53 and SETDB1 mutations and extensive loss of heterozygosity, and discovered strong expression of the immune-checkpoint gene VISTA in epithelioid MPM.See related commentary by Aggarwal and Albelda, p. 1508.This article is highlighted in the In This Issue feature, p. 1494

PDF file, 181K, Supplementary Figure S3 Sustained inhibition of Akt and Erk phosphorylation by combining WZ4002 and E7050.

PDF file, 423K, Supplementary Figure S4 Cytostatic effects of WZ4002 and E7050 treatment.

PDF file, 315K, Supplementary Figure S5 Relative body weight of mice during treatment with WZ4002 and/or E7050.

Supplementary materials and methods, as well as supplementary figures S1-S7.

PDF file, 40K, Supplementary Table S1 IC50 values of each compound in MTT assay.

Although the EGF receptor tyrosine kinase inhibitors (EGFR-TKI) erlotinib and gefitinib have shown dramatic effects against EGFR mutant lung cancer, patients become resistant by various mechanisms, including gatekeeper EGFR-T790M mutation, Met amplification, and HGF overexpression, thereafter relapsing. Thus, it is urgent to develop novel agents to overcome EGFR-TKI resistance. We have tested the effects of the mutant-selective EGFR-TKI WZ4002 and the mutant-selective Met-TKI E7050 on 3 EGFR mutant lung cancer cell lines resistant to erlotinib by different mechanisms: PC-9/HGF cells with an exon 19 deletion, H1975 with an L858R mutation, and HCC827ER with an exon 19 deletion, with acquired resistance to erlotinib because of HGF gene transfection, gatekeeper T790M mutation, and Met amplification, respectively. WZ4002 inhibited the growth of H1975 cells with a gatekeeper T790M mutation, but did not inhibit the growth of HCC827ER and PC-9/HGF cells. HGF triggered the resistance of H1975 cells to WZ4002, whereas E7050 sensitized HCC827ER, PC-9/HGF, and HGF-treated H1975 cells to WZ4002, inhibiting EGFR and Met phosphorylation and their downstream molecules. Combined treatment potently inhibited the growth of tumors induced in severe-combined immunodeficient mice by H1975, HCC827ER, and PC-9/HGF cells, without any marked adverse events. These therapeutic effects were associated with the inhibition of EGFR and Met phosphorylation in vivo. The combination of a mutant-selective EGFR-TKI and a Met-TKI was effective in suppressing the growth of erlotinib-resistant tumors caused by gatekeeper T790M mutation, Met amplification, and HGF overexpression. Further evaluations in clinical trials are warranted. Mol Cancer Ther; 11(10); 2149–57. ©2012 AACR.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that has a low overall survival; however, no significant treatment advances have been made in the past 15 years. Large-scale molecular studies have identified a poor prognostic subset of MPM linked to the epithelial–mesenchymal transition (EMT) that may contribute toward resistance to chemotherapy, suggesting that EMT could be targeted to treat patients with MPM. Previously, we reported that histone modifiers regulating EMT could be therapeutic targets; therefore, in this study, we investigated whether targeting lysine-specific demethylase 1 (LSD1/KDM1), a histone-modifying enzyme responsible for demethylating histone H3 lysine 4 and lysine 9, could represent a novel therapeutic strategy for MPM. We suppressed LSD1 and investigated the EMT phenotype using EMT marker expression and wound-healing assay; and chemosensitivity using apoptosis assay. We found that suppressing LSD1 induces an epithelial phenotype in sarcomatoid MPM cells, while attenuating the mesenchymal phenotype sensitized MPM cells to cisplatin-induced apoptosis. Subsequent genome-wide identification, comprehensive microarray analysis, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to assess genome-wide changes in chromatin accessibility suggested that LSD1 directly regulates milk fat globulin protein E8 (MFGE8), an integrin ligand that is involved in the FAK pathway. Furthermore, we found that LSD1 regulates the mesenchymal phenotype and apoptosis by activating the FAK–AKT–GSK3β pathway via a positive feedback loop involving MFGE8 and Snail expression, thereby leading to cisplatin resistance.Implications:This study suggests that LSD1 regulates the mesenchymal phenotype and apoptosis, and that LSD1 inhibitors could be combined with the cisplatin as a novel therapy for patients with MPM.

Malignant pleural mesothelioma (MPM) is a malignant neoplasm of the pleura caused by asbestos exposure. For diagnosis of MPM, immunohistochemistry using multiple markers is recommended to rule out differential diagnoses, such as pulmonary adenocarcinoma. However, the specificity of currently used markers is not fully satisfactory. We previously developed a monoclonal antibody named S1, which recognizes 6-sulfo sialyl Lewis x, an L-selectin ligand expressed on high endothelial venules. During the screening process, we discovered that this antibody stained normal pleural mesothelium. This finding prompted us to hypothesize that the epitope recognized by S1 might serve as a new diagnostic marker for MPM.To test this hypothesis, we immunostained human MPM (n = 22) and lung adenocarcinoma (n = 25) tissues using S1 antibody.77.3% of MPM were S1 positive, and if limited to epithelioid type, the positivity rate was 100%, while that of lung adenocarcinoma was only 36.0%. Statistical analysis revealed a significant difference in the S1 positivity rate between each disease. Furthermore, immunohistochemistry using a series of anti-carbohydrate antibodies combined with glycosidase digestion revealed the structure of sulfated glycans expressed in MPM to be 6-sulfo sialyl N-acetyllactosamine attached to core 2-branched O-glycans.We propose that the S1 glycoepitope could serve as a new diagnostic marker for MPM.

Supplementary Data from Hepatocyte Growth Factor Reduces Susceptibility to an Irreversible Epidermal Growth Factor Receptor Inhibitor in EGFR-T790M Mutant Lung Cancer

CCR Translation for This Article from Reciprocal and Complementary Role of MET Amplification and EGFR T790M Mutation in Acquired Resistance to Kinase Inhibitors in Lung Cancer

Purpose: In epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy for lung cancer patients, acquired resistance develops almost inevitably and this limits the improvement in patient outcomes. EGFR T790M mutation and MET amplification are the two main mechanisms underlying this resistance, but the relationship between these two mechanisms is unclear. In this study, we explored their relationship using in vitro models and autopsy specimens.Experimental Design: Erlotinib-resistant HCC827 (HCC827ER) cells were developed by chronic exposure to erlotinib at increasing concentrations. HCC827EPR cells were also developed by chronic exposure to erlotinib in the presence of PHA-665,752 (a MET TKI). The erlotinib-resistant mechanisms of these cells were analyzed. In addition, 33 autopsy tumor samples from 6 lung adenocarcinoma patients harboring multiple gefitinib-refractory tumors were analyzed.Results: HCC827ER developed MET amplification, and clinically relevant resistance occurred at ≥4-fold MET gene copy number gain (CNG). By contrast, HCC827EPR developed T790M without MET CNG. Of six patients harboring gefitinib-refractory tumors, three exhibited T790M only, one exhibited MET amplification only, and the other two exhibited T790M and/or MET amplification depending on the lesion sites. In these gefitinib-refractory tumors, T790M developed in 93% (14 of 15) of tumors without MET gene CNGs, in 80% (4 of 5) of tumors with moderate MET gene CNGs (MET amplification (≥4-fold).Conclusions: These results indicate a reciprocal and complementary relationship between T790M and MET amplification and the necessity of concurrent inhibition of both for further improving patient outcomes. Clin Cancer Res; 16(22); 5489–98. ©2010 AACR.

Supplementary Table 2 from LATS2 Is a Tumor Suppressor Gene of Malignant Mesothelioma

Supplementary Figure 2 from LATS2 Is a Tumor Suppressor Gene of Malignant Mesothelioma

Supplementary Figure 3 from LATS2 Is a Tumor Suppressor Gene of Malignant Mesothelioma

Emerging evidence, although currently very sparse, suggests the presence of “lineage-specific dependency” in the survival mechanisms of certain cancers. TTF-1 has a decisive role as a master regulatory transcription factor in lung development and in the maintenance of the functions of terminal respiratory unit (TRU) cells. We show that a subset of lung adenocarcinoma cell lines expressing TTF-1, which presumably represent those derived from the TRU lineage, exhibit marked dependence on the persistent expression of TTF-1. The inhibition of TTF-1 by RNA interference (RNAi) significantly and specifically induced growth inhibition and apoptosis in these adenocarcinoma cell lines. Furthermore, a fraction of TTF-1–expressing tumors and cell lines displayed an increase in the gene dosage of TTF-1 in the analysis of 214 patients with non–small-cell lung cancer, including 174 adenocarcinomas, showing a tendency of higher frequency of increased gene copies at metastatic sites than at primary sites (P = 0.07, by two-sided Fisher's exact test). These findings strongly suggest that in addition to the development and maintenance of TRU lineages in normal lung, sustained TTF-1 expression may be crucial for the survival of a subset of adenocarcinomas that express TTF-1, providing credence for the lineage-specific dependency model. [Cancer Res 2007;67(13):6007–11]

Supplementary Figure from A Polycistronic MicroRNA Cluster, miR-17-92, Is Overexpressed in Human Lung Cancers and Enhances Cell Proliferation

Supplementary Table 2 from Roles of Achaete-Scute Homologue 1 in DKK1 and E-cadherin Repression and Neuroendocrine Differentiation in Lung Cancer

Supplementary Methods and Figures 1-2 from Roles of Achaete-Scute Homologue 1 in DKK1 and E-cadherin Repression and Neuroendocrine Differentiation in Lung Cancer

MicroRNAs (miRNAs) are small noncoding RNAs, thought to be involved in physiologic and developmental processes by negatively regulating expression of target genes. We have previously reported frequent down-regulation of the let-7 miRNA family in lung cancers and, in the present study, assessed alteration in a panel of 19 lung cancer cell lines. As a result, we found for the first time that the miR-17-92 cluster, which comprises seven miRNAs and resides in intron 3 of the C13orf25 gene at 13q31.3, is markedly overexpressed in lung cancers, especially with small-cell lung cancer histology. Southern blot analysis revealed the presence of increased gene copy numbers of the miRNA cluster in a fraction of lung cancer cell lines with overexpression. In addition, we were able to show predominant localization of C13orf25 transcripts within the nucleus and introduction of the expression construct of the miR-17-92 cluster, but not the putative open reading frame of C13orf25, enhancing lung cancer cell growth. These findings clearly suggest that marked overexpression of the miR-17-92 cluster with occasional gene amplification may play a role in the development of lung cancers, especially in their most aggressive form, small-cell lung cancer, and that the C13orf25 gene may well be serving as a vehicle in this regard.

Supplementary Figure 1 from Roles of Achaete-Scute Homologue 1 in DKK1 and E-cadherin Repression and Neuroendocrine Differentiation in Lung Cancer

Supplementary Figure 1 from LATS2 Is a Tumor Suppressor Gene of Malignant Mesothelioma

Supplementary Materials and Methods from LATS2 Is a Tumor Suppressor Gene of Malignant Mesothelioma

Supplementary Table 1 from LATS2 Is a Tumor Suppressor Gene of Malignant Mesothelioma

Supplementary Figure 1 from Lineage-Specific Dependency of Lung Adenocarcinomas on the Lung Development Regulator TTF-1

Supplementary Table 3 from Roles of Achaete-Scute Homologue 1 in DKK1 and E-cadherin Repression and Neuroendocrine Differentiation in Lung Cancer

PDF file - 785K, Supplementary Figure 1. Expression of total BIM and ratio of BIM isoforms in NSCLC cell lines. Supplementary Figure 2. Flow cytometric analysis showing that EGFR mutated cell lines carrying the BIM polymorphism have low susceptibility to gefitinib-induced apoptosis. Supplementary Figure 3. Vorinostat-induced up-regulation of BIM enhances apoptosis in the HCC2279 cell line with the BIM polymorphism.Supplementary Figure 4. Knockdown or overexpression of BIM affects activation of caspase-3/7 induced by gefitinib and vorinostat in EGFR mutant cells with BIM polymorphism. Supplementary Figure 5. Detection of BIM polymorphism in human peripheral blood mononuclear cells (PBMC) and serum. Supplementary Table 1. Status of EGFR mutation and BIM polymorphism in NSCLC cell lines.

Supplementary Information from Lineage-Specific Dependency of Lung Adenocarcinomas on the Lung Development Regulator TTF-1

Supplementary Figure Legends 1-3 from LATS2 Is a Tumor Suppressor Gene of Malignant Mesothelioma

PDF file - 89K

PDF - 157KB, Figure S1. Characterization of GSCs. Figure S2. Significance of EZH2/PRC2 in GBMs and GSCs. Figure S3. ChIP-chip analysis and MCAM analysis in GSCs. Figure S4. Effects of Wnt1-KD and BMP5-overexpression in GSCs. Figure S5. Downregulation of EZH2 abrogates tumorigenicity. Supplementary Table S1. Primer sequence data Supplementary Table S2. Tumorigenicity of GSCs and S-BTCs in the brain of NOD-SCID mice Supplementary Table S3. Validation of microarray analyses Supplementary Table S4. Targets of H3K27me3 in 1228-GSC, 1228-S-BTC, 0316-GSC, and 0316-S-BTC Supplementary Table S5. List of genes with commonly altered expression in 1228- and 0316-GSCs after serum exposure.

Supplementary Figure 2 from Lineage-Specific Dependency of Lung Adenocarcinomas on the Lung Development Regulator TTF-1

The proneural basic-helix-loop-helix protein achaete-scute homologue 1 (ASH1) is expressed in a very limited spectrum of normal and cancerous cells in a lineage-specific manner, including normal pulmonary neuroendocrine cells and lung cancer cells with neuroendocrine features. Our previous results indicated that ASH1 may play a crucial role in the growth and survival of lung cancers with neuroendocrine features, which prompted us to investigate the molecular function of ASH1 in relation to its involvement in carcinogenic processes. Herein, we report for the first time that ASH1 functions as a dual transcription factor by activating neuroendocrine differentiation markers and also repressing putative tumor suppressors. This protein was found to inactivate DKK1 and DKK3, negative regulators of Wnt/β-catenin signaling, E-cadherin, and integrin β1 through ASH1-mediated deacetylation and repressive trimethylation of lysine 27 (H3K27me3) of histone H3 in the promoter regions of DKK1 and E-cadherin. In addition, ASH1-transduced A549 adenocarcinoma cells exhibited markedly altered morphology characteristics compared with lung cancer cells with neuroendocrine features both in vitro and in vivo and also grew faster in vivo. Our results provide important clues for a better understanding of the molecular and cellular biological roles of ASH1 in the process of carcinogenesis of lung cancers with neuroendocrine features and warrant future investigations to shed light on the lineage-specific dependency of this transcription factor with dual functions. [Cancer Res 2008;68(6):1647–55]

Malignant pleural mesothelioma (MPM) is a fatal thoracic malignancy, the epigenetics of which are poorly defined. We performed high-throughput methylation analysis covering 6,157 CpG islands in 20 MPMs and 20 lung adenocarcinomas. Newly identified genes were further analyzed in 50 MPMs and 56 adenocarcinomas via quantitative methylation-specific PCR. Targets of histone H3 lysine 27 trimethylation (H3K27me3) and genetic alterations were also assessed in MPM cells by chromatin immunoprecipitation arrays and comparative genomic hybridization arrays. An average of 387 genes (6.3%) and 544 genes (8.8%) were hypermethylated in MPM and adenocarcinoma, respectively. Hierarchical cluster analysis showed that the two malignancies have characteristic DNA methylation patterns, likely a result of different pathologic processes. In MPM, a separate subset of genes was silenced by H3K27me3 and could be reactivated by treatment with a histone deacetylase inhibitor alone. Integrated analysis of these epigenetic and genetic alterations revealed that only 11% of heterozygously deleted genes were affected by DNA methylation and/or H3K27me3 in MPMs. Among the DNA hypermethylated genes, three (TMEM30B, KAZALD1, and MAPK13) were specifically methylated only in MPM and could serve as potential diagnostic markers. Interestingly, a subset of MPM cases (4 cases, 20%) had very low levels of DNA methylation and substantially longer survival, suggesting that the epigenetic alterations are one mechanism affecting progression of this disease. Our findings show a characteristic epigenetic profile of MPM and uncover multiple distinct epigenetic abnormalities that lead to the silencing of tumor suppressor genes in MPM and could serve as diagnostic or prognostic targets. [Cancer Res 2009;69(23):9073–82]

BIM (BCL2L11) is a BH3-only proapoptotic member of the Bcl-2 protein family. BIM upregulation is required for apoptosis induction by EGF receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKI) in EGFR-mutant forms of non–small cell lung cancer (NSCLC). Notably, a BIM deletion polymorphism occurs naturally in 12.9% of East Asian individuals, impairing the generation of the proapoptotic isoform required for the EGFR-TKIs gefitinib and erlotinib and therefore conferring an inherent drug-resistant phenotype. Indeed, patients with NSCLC, who harbored this host BIM polymorphism, exhibited significantly inferior responses to EGFR-TKI treatment than individuals lacking this polymorphism. In an attempt to correct this response defect in the resistant group, we investigated whether the histone deacetylase (HDAC) inhibitor vorinostat could circumvent EGFR-TKI resistance in EGFR-mutant NSCLC cell lines that also harbored the BIM polymorphism. Consistent with our clinical observations, we found that such cells were much less sensitive to gefitinib-induced apoptosis than EGFR-mutant cells, which did not harbor the polymorphism. Notably, vorinostat increased expression in a dose-dependent manner of the proapoptotic BH3 domain-containing isoform of BIM, which was sufficient to restore gefitinib death sensitivity in the EGFR mutant, EGFR-TKI–resistant cells. In xenograft models, while gefitinib induced marked regression via apoptosis of tumors without the BIM polymorphism, its combination with vorinostat was needed to induce marked regression of tumors with the BIM polymorphism in the same manner. Together, our results show how HDAC inhibition can epigenetically restore BIM function and death sensitivity of EGFR-TKI in cases of EGFR-mutant NSCLC where resistance to EGFR-TKI is associated with a common BIM polymorphism. Cancer Res; 73(8); 2428–34. ©2013 AACR.