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PDF file - 75KB, Supplemental Figure S1. Inactivation of NR4A1 induces ER stress and autophagy in vitro and in vivo.

PDF file - 36KB, Supplemental Table S2. Proteins showing differential expression in Panc1 cells by transfection of siNR4A1.

PDF file - 33KB, Supplemental Figure S2. Inactivation of NR4A1 induces DNA fragmentation and ER Ca2+ release in Panc-1 cells.

PDF file - 50KB, Supplemental Figure S5. Effect of siNR4A2 on induction of apoptosis and ER stress in pancreatic cancer cells.

PDF file - 20KB, Supplemental Table S1. List of real-time PCR primer sequences.

PDF file - 53KB, Supplemental Figure S4. DIM-C-pPhOH induces ER stress through inactivation of nuclear NR4A1.

Hepatocellular carcinoma (HCC) is a heterogeneous disease. Mouse models are commonly used as preclinical models to study hepatocarcinogenesis, but how well these models recapitulate molecular subtypes of human HCC is unclear. Here, integration of genomic signatures from molecularly and clinically defined human HCC (n = 11) and mouse models of HCC (n = 9) identified the mouse models that best resembled subtypes of human HCC and determined the clinical relevance of each model. Mst1/2 knockout (KO), Sav1 KO, and SV40 T antigen mouse models effectively recapitulated subtypes of human HCC with a poor prognosis, whereas the Myc transgenic model best resembled human HCCs with a more favorable prognosis. The Myc model was also associated with activation of β-catenin. E2f1, E2f1/Myc, E2f1/Tgfa, and diethylnitrosamine (DEN)-induced models were heterogeneous and were unequally split into poor and favorable prognoses. Mst1/2 KO and Sav1 KO models best resemble human HCC with hepatic stem cell characteristics. Applying a genomic predictor for immunotherapy, the six-gene IFNγ score, the Mst1/2 KO, Sav1 KO, SV40, and DEN models were predicted to be the least responsive to immunotherapy. Further analysis showed that elevated expression of immune-inhibitory genes (Cd276 and Nectin2/Pvrl2) in Mst1/2 KO, Sav1 KO, and SV40 models and decreased expression of immune stimulatory gene (Cd86) in the DEN model might be accountable for the lack of predictive response to immunotherapy.Implication: The current genomic approach identified the most relevant mouse models to human liver cancer and suggests immunotherapeutic potential for the treatment of specific subtypes. Mol Cancer Res; 16(11); 1713–23. ©2018 AACR.

PDF file - 125KB, Supplemental Table S3. mRNAs showing differential expression in Panc1 cells by transfection of siNR4A1.

PDF file - 37KB, Supplemental Figure S3. Top 10 significant enriched canonical pathways altered by transfection of Panc-1 cells with siNR4A1.

NR4A1 (Nur77, TR3) is an orphan nuclear receptor that is overexpressed in pancreatic cancer and exhibits pro-oncogenic activity. RNA interference of NR4A1 expression in Panc-1 cells induced apoptosis and subsequent proteomic analysis revealed the induction of several markers of endoplasmic reticulum stress, including glucose-related protein 78 (GRP78), CCAAT/enhancer-binding protein-homologous protein (CHOP), and activating transcription factor-4 (ATF-4). Treatment of pancreatic cancer cells with the NR4A1 antagonist 1,1-bis(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) gave similar results. Moreover, both NR4A1 knockdown and DIM-C-pPhOH induced reactive oxygen species (ROS), and induction of ROS and endoplasmic reticulum stress by these agents was attenuated after cotreatment with antioxidants. Manipulation of NR4A1 expression coupled with gene expression profiling identified a number of ROS metabolism transcripts regulated by NR4A1. Knockdown of one of these transcripts, thioredoxin domain containing 5 (TXNDC5), recapitulated the elevated ROS and endoplasmic reticulum stress; thus, demonstrating that NR4A1 regulates levels of endoplasmic reticulum stress and ROS in pancreatic cancer cells to facilitate cell proliferation and survival. Finally, inactivation of NR4A1 by knockdown or DIM-C-pPhOH decreased TXNDC5, resulting in activation of the ROS/endoplasmic reticulum stress and proapoptotic pathways.Implications: The NR4A1 receptor is pro-oncogenic, regulates the ROS/endoplasmic reticulum stress pathways, and inactivation of the receptor represents a novel pathway for inducing cell death in pancreatic cancer. Mol Cancer Res; 12(4); 527–38. ©2014 AACR.

S1. Correlation of prognostic probability and risk scores for mouse HCCs. S2. Glycolytic gene expression signature in mouse liver. S3. Glycolytic expression signature in mouse HCC tumors from 9 models. S4. Correlation of hepatic stem cell probability with SOH probability. S5. Expression of hepatic stem cell markers in 9 mouse models. S6. Expression of Krt-7 in Sav1 KO liver issues. S7. Expression of downstream target genes in mouse tumors. S8. Expression of Pdcd1 and Cd274 and their correlation with IFNG6 score in mouse HCCs in nine models. S9. Regulation of Cd276 by Yap1.

Purpose: The Hippo pathway is a tumor suppressor in the liver. However, the clinical significance of Hippo pathway inactivation in HCC is not clearly defined. We analyzed genomic data from human and mouse tissues to determine clinical relevance of Hippo pathway inactivation in HCC.Experimental Design: We analyzed gene expression data from Mst1/2−/− and Sav1−/− mice and identified a 610-gene expression signature reflecting Hippo pathway inactivation in the liver [silence of Hippo (SOH) signature]. By integrating gene expression data from mouse models with those from human HCC tissues, we developed a prediction model that could identify HCC patients with an inactivated Hippo pathway and used it to test its significance in HCC patients, via univariate and multivariate Cox analyses.Results: HCC patients (National Cancer Institute cohort, n = 113) with the SOH signature had a significantly poorer prognosis than those without the SOH signature [P < 0.001 for overall survival (OS)]. The significant association of the signature with poor prognosis was further validated in the Korean (n = 100, P = 0.006 for OS) and Fudan University cohorts (n = 242, P = 0.001 for OS). On multivariate analysis, the signature was an independent predictor of recurrence-free survival (HR, 1.6; 95% confidence interval, 1.12–2.28: P = 0.008). We also demonstrated significant concordance between the SOH HCC subtype and the hepatic stem cell HCC subtype that had been identified in a previous study (P < 0.001).Conclusions: Inactivation of the Hippo pathway in HCC is significantly associated with poor prognosis. Clin Cancer Res; 22(5); 1256–64. ©2015 AACR.

Recurrence risk score and its clinical significance.

Purpose: Despite continual efforts to develop a prognostic model of gastric cancer by using clinical and pathologic parameters, a clinical test that can discriminate patients with good outcomes from those with poor outcomes after gastric cancer surgery has not been established. We aim to develop practical biomarker-based risk score that can predict relapse of gastric cancer after surgical treatment.Experimental Design: Microarray technologies were used to generate and analyze gene expression profiling data from 65 gastric cancer patients to identify biomarker genes associated with relapse. The association of expression patterns of identified genes with relapse and overall survival was validated in independent gastric cancer patients.Results: We uncovered two subgroups of gastric cancer that were strongly associated with the prognosis. For the easy translation of our findings into practice, we developed a scoring system based on the expression of six genes that predicted the likelihood of relapse after curative resection. In multivariate analysis, the risk score was an independent predictor of relapse in a cohort of 96 patients. We were able to validate the robustness of the six-gene signature in an additional independent cohort.Conclusions: The risk score derived from the six-gene set successfully prognosticated the relapse of gastric cancer patients after gastrectomy. Clin Cancer Res; 17(7); 1850–7. ©2011 AACR.

Distribution of candidate genes in biological process categories of the Gene Ontology database.

Supplementary Tables 1-4 from Integrative Analysis of Proteomic Signatures, Mutations, and Drug Responsiveness in the NCI 60 Cancer Cell Line Set

Methods

Purpose: This study was aimed at developing and validating a quantitative multigene assay for predicting tumor recurrence after gastric cancer surgery.Experimental Design: Gene expression data were generated from tumor tissues of patients who underwent surgery for gastric cancer (n = 267, training cohort). Genes whose expression was significantly associated with activation of YAP1 (a frequently activated oncogene in gastrointestinal cancer), 5-year recurrence-free survival, and 5-year overall survival were first identified as candidates for prognostic genes (156 genes, P < 0.001). We developed the recurrence risk score (RRS) by using quantitative RT-PCR to identify genes whose expression levels were significantly associated with YAP1 activation and patient survival in the training cohort.Results: We based the RRS assay on 6 genes, IGFBP4, SFRP4, SPOCK1, SULF1, THBS, and GADD45B, whose expression levels were significantly associated with YAP1 activation and prognosis in the training cohort. The RRS assay was further validated in an independent cohort of 317 patients. In multivariate analysis, the RRS was an independent predictor of recurrence [HR, 1.6; 95% confidence interval (CI), 1.02–2.4; P = 0.03]. In patients with stage II disease, the RRS had an HR of 2.9 (95% CI, 1.1–7.9; P = 0.03) and was the only significant independent predictor of recurrence.Conclusions: The RRS assay was a valid predictor of recurrence in the two cohorts of patients with gastric cancer. Independent prospective studies to assess the clinical utility of this assay are warranted. Clin Cancer Res; 22(24); 6228–35. ©2016 AACR.

Aberrations in oncogenes and tumor suppressors frequently affect the activity of critical signal transduction pathways. To analyze systematically the relationship between the activation status of protein networks and other characteristics of cancer cells, we did reverse phase protein array (RPPA) profiling of the NCI60 cell lines for total protein expression and activation-specific markers of critical signaling pathways. To extend the scope of the study, we merged those data with previously published RPPA results for the NCI60. Integrative analysis of the expanded RPPA data set revealed five major clusters of cell lines and five principal proteomic signatures. Comparison of mutations in the NCI60 cell lines with patterns of protein expression showed significant associations for PTEN, PIK3CA, BRAF, and APC mutations with proteomic clusters. PIK3CA and PTEN mutation enrichment were not cell lineage-specific but were associated with dominant yet distinct groups of proteins. The five RPPA-defined clusters were strongly associated with sensitivity to standard anticancer agents. RPPA analysis identified 27 protein features significantly associated with sensitivity to paclitaxel. The functional status of those proteins was interrogated in a paclitaxel whole genome small interfering RNA (siRNA) library synthetic lethality screen and confirmed the predicted associations with drug sensitivity. These studies expand our understanding of the activation status of protein networks in the NCI60 cancer cell lines, demonstrate the importance of the direct study of protein expression and activation, and provide a basis for further studies integrating the information with other molecular and pharmacological characteristics of cancer. Mol Cancer Ther; 9(2); 257–67

Figure S1: Significant concordance between silence of Hippo signature and YAP1 expression in human hepatocellular carcinoma (HCC); Figure S2: Venn diagram of gene lists from two different gene expression signatures; Table S1: Genes in the SOH signature; Table S2: Functional categories of genes in the SOH signature; Table S3: Canonical Pathways enriched in the SOH signature; Table S4: Significant association between the SOH subtype and YAP1 protein expression in HCC; Table S5: Univariate and multivariate Cox regression analyses of recurrence-free survival with continuous SOH probability; Table S6: Univariate and multivariate Cox regression analyses of overall survival; Table S7: Univariate and multivariate Cox regression analyses of overall survival with continuous SOH probability; Table S8: Drop in concordance index in multivariable analysis; Table S9: Concordance of silence of Hippo signature with other prognostic HCC gene expression signatures in the NCI cohort; Table S10: Characteristics of HCC patients in the MSH cohort, stratified by SOH signature.

Supplementary Figures S1-S6; Supplementary Tables S1-S2.

PDF file - 416KB, Supplementary methods, figures and tables. Fig. S1. Gene expression profiling of patient-matched brain and extracranial metastases. Fig. S2. PTEN gene expression in BM_03 (homozygous copy loss) and EM_03 (one copy loss). Gene expression was measured by microarray. Fig. S3. Quantitative RT-PCR measurement of SGK3, SGSM2, and ELOVL2 expression in five pairs of brain metastases (BM) and extracranial metastases (EM). Fig. S4. Log2 expression levels of RB_pS807_S811 in seven pairs of matched metastases. Fig. S5 Examples of PTEN-absent tumors. PTEN IHC of BM_03 and EM_03 were photographed at 00X magnification. Fig. S6 GSK3/_pS21/S9 IHC staining in 26 pairs of matched brain metastases (BM) and extracranial metastases (EM). Fig. S7. Results of RB_pS807_S811 IHC in 25 pairs of matched brain metastases (BM) and extracranial metastases (EM). Fig. S8. Kaplan-Meier survival analysis of nude mice with BRAF-mutant melanoma brain metastases treated with BKM120 and/or LGX818. Fig. S9. AKT_pS473 level in subcutaneously (sc) and intracranially (ic) implanted melanomas. Table S1. List of clinical samples analyzed. Table S2. Clinical features and genetic aberrations of matched samples analyzed by high-throughput techniques. Table S3. Hotspot mutations analyzed by Sequenom. Table S4. Antibodies used in RPPA analysis. Table S5. Genes with copy number differences (P

Supplementary Figure Legends 1-4 from Integrative Analysis of Proteomic Signatures, Mutations, and Drug Responsiveness in the NCI 60 Cancer Cell Line Set

Supplementary Figures 1-4 from Integrative Analysis of Proteomic Signatures, Mutations, and Drug Responsiveness in the NCI 60 Cancer Cell Line Set

Supplementary Figures S1-S5. Supplementary Figure S1. Expression of YAP1 in NCI-H716 CRC cells. Supplementary Figure S2. YAP1-specific gene expression signature used in our study of YAP1 activation in CRC patients. Supplementary Figure S3. Gene set enrichment analysis (GSEA) of gene expression data from two cancer cells with activated YAP1. Supplementary Figure S4. Prognostic impact of the YAP1 signature for CRC according to disease stages. Supplementary Figure S5. PFS according to KRAS mutation status (Cohort 5)

Purpose: Activation of YAP1, a novel oncogene in the Hippo pathway, has been observed in many cancers, including colorectal cancer. We investigated whether activation of YAP1 is significantly associated with prognosis or treatment outcomes in colorectal cancer.Experimental Design: A gene expression signature reflecting YAP1 activation was identified in colorectal cancer cells, and patients with colorectal cancer were stratified into two groups according to this signature: activated YAP1 colorectal cancer (AYCC) or inactivated YAP1 colorectal cancer (IYCC). Stratified patients in five test cohorts were evaluated to determine the effect of the signature on colorectal cancer prognosis and response to cetuximab treatment.Results: The activated YAP1 signature was associated with poor prognosis for colorectal cancer in four independent patient cohorts with stage I–III disease (total n = 1,028). In a multivariate analysis, the impact of the YAP1 signature on disease-free survival was independent of other clinical variables [hazard ratio (HR), 1.63; 95% confidence interval (CI), 1.25–2.13; P < 0.001]. In patients with stage IV colorectal cancer and wild-type KRAS, IYCC patients had a better disease control rate and progression-free survival (PFS) after cetuximab monotherapy than did AYCC patients; however, in patients with KRAS mutations, PFS duration after cetuximab monotherapy was not different between IYCC and AYCC patients. In multivariate analysis, the effect of YAP1 activation on PFS was independent of KRAS mutation status and other clinical variables (HR, 1.82; 95% CI, 1.05–3.16; P = 0.03).Conclusions: Activation of YAP1 is highly associated with poor prognosis for colorectal cancer and may be useful in identifying patients with metastatic colorectal cancer resistant to cetuximab. Clin Cancer Res; 21(2); 357–64. ©2014 AACR.

Purpose: An improved understanding of the molecular pathogenesis of brain metastases, one of the most common and devastating complications of advanced melanoma, may identify and prioritize rational therapeutic approaches for this disease. In particular, the identification of molecular differences between brain and extracranial metastases would support the need for the development of organ-specific therapeutic approaches.Experimental Design: Hotspot mutations, copy number variations (CNV), global mRNA expression patterns, and quantitative analysis of protein expression and activation by reverse-phase protein array (RPPA) analysis were evaluated in pairs of melanoma brain metastases and extracranial metastases from patients who had undergone surgical resection for both types of tumors.Results: The status of 154 previously reported hotspot mutations, including driver mutations in BRAF and NRAS, were concordant in all evaluable patient-matched pairs of tumors. Overall patterns of CNV, mRNA expression, and protein expression were largely similar between the paired samples for individual patients. However, brain metastases demonstrated increased expression of several activation-specific protein markers in the PI3K/AKT pathway compared with the extracranial metastases.Conclusions: These results add to the understanding of the molecular characteristics of melanoma brain metastases and support the rationale for additional testing of the PI3K/AKT pathway as a therapeutic target in these highly aggressive tumors. Clin Cancer Res; 20(21); 5537–46. ©2014 AACR.

Supplementary materials and methods

Supplementary Figures 1-8. Supplementary Fig. 1. CD11b+Gr1+ populations were isolated by FACS from spleens of tumor-bearing p120-/- and control mice. Supplementary Fig. 2. CD38 is expressed by the CD11b+Gr1+ population in a murine model of Barrett's Esophagus. Supplementary Fig. 3. The HNM007 tumors induce CD38 expression on MDSC population similarly to the autochthonous p120-/- tumors. Supplementary Fig. 4. Immunohistochemical analysis of HNM007 tumors co-injected with CD38High or CD38Low MDSCs. Supplementary Fig. 5. CD38high CD11b+Gr1+ cells produce higher levels of iNOS and pNFκB. Supplementary Fig.6. Network analysis of differentially regulated genes in CD38high and CD38low MDSCs shows upregulation of RB1 pathway. Supplementary Fig.7. Distribution of G-MDSC, M-MDSC and mature monocytes is perturbed in tumor-bearing Cd38-/- mice. Supplementary Fig. 8. CD38 is expressed on human MDSC-like cell population that is expanded in the peripheral blood of advanced-stage cancer patients.

Supplementary Tables 1 and 2. Supplementary Table 1. List of the genes differentially expressed in CD11b+Gr1+ splenocytes from tumor-bearing, compared to control mice. Supplementary Table 2. List of the genes differentially expressed in CD38high, compared to CD38low, CD11b+Gr1+ splenocytes from tumor-bearing mice.

Human squamous cell cancers are the most common epithelially derived malignancies. One example is esophageal squamous cell carcinoma (ESCC), which is associated with a high mortality rate that is related to a propensity for invasion and metastasis. Here, we report that periostin, a highly expressed cell adhesion molecule, is a key component of a novel tumor-invasive signature obtained from an organotypic culture model of engineered ESCC. This tumor-invasive signature classifies with human ESCC microarrays, underscoring its utility in human cancer. Genetic modulation of periostin promotes tumor cell migration and invasion as revealed in gain-of-loss and loss-of-function experiments. Inhibition of epidermal growth factor receptor signaling and restoration of wild-type p53 function were each found to attenuate periostin, suggesting the interdependence of two common genetic alterations with periostin function. Collectively, our studies reveal periostin as an important mediator of ESCC tumor invasion and they indicate that organotypic (three-dimensional) culture can offer an important tool to discover novel biological effectors in cancer. Cancer Res; 70(13); 5281–92. ©2010 AACR.

Supplementary Figure 4 from Periostin, a Cell Adhesion Molecule, Facilitates Invasion in the Tumor Microenvironment and Annotates a Novel Tumor-Invasive Signature in Esophageal Cancer

Supplementary Table 2 from Periostin, a Cell Adhesion Molecule, Facilitates Invasion in the Tumor Microenvironment and Annotates a Novel Tumor-Invasive Signature in Esophageal Cancer

Supplementary Table 1 from Periostin, a Cell Adhesion Molecule, Facilitates Invasion in the Tumor Microenvironment and Annotates a Novel Tumor-Invasive Signature in Esophageal Cancer

Supplementary Figure 2 from Periostin, a Cell Adhesion Molecule, Facilitates Invasion in the Tumor Microenvironment and Annotates a Novel Tumor-Invasive Signature in Esophageal Cancer

Supplementary Figure 1 A-C from Periostin, a Cell Adhesion Molecule, Facilitates Invasion in the Tumor Microenvironment and Annotates a Novel Tumor-Invasive Signature in Esophageal Cancer

Supplementary Figure 3 from Periostin, a Cell Adhesion Molecule, Facilitates Invasion in the Tumor Microenvironment and Annotates a Novel Tumor-Invasive Signature in Esophageal Cancer

We studied the influence of GaAs top‐layer thickness on the small‐signal modulation response and 56 Gb/s four‐level pulse‐amplitude modulation eye quality of 850 nm vertical‐cavity surface‐emitting lasers (VCSELs). We considered the proportionality of the gain‐saturation coefficient to the photon lifetime. The simulation results that employed the transfer‐matrix method and laser rate equations led to the conclusion that the proportionality should be considered for proper explanation of the experimental results. From the obtained optical eyes, we could determine an optimum thickness of the GaAs top layer that rendered the best eye quality of VCSEL. We also compared two results: one result with a fixed gain‐saturation coefficient and the other that considered the proportionality. The former result with the constant gain‐saturation coefficient demonstrated a better eye quality and a wider optimum range of the GaAs top‐layer thickness because the resultant higher damping reduced the relaxation oscillation.

We have investigated the transverse-mode behavior of 850 nm VCSELs using time-average lasing spectra and eye patterns. Measurements were done at varying DC bias currents, bitrates, and modulation amplitudes. Bitrates range from 0.5 to 10 Gb/s and pseudo-random bit sequence of 215–1 was applied to the VCSEL with ∼6 major modes. DC bias currents were 4, 6, and 8 mA. The amplitude of modulation current is adjusted so that the off-state current is higher than 2 times the threshold current. Correlation coefficients and normalized differences were calculated at each bias current for quantitative analysis using 2.5 Gb/s DC spectrum as a reference. Measurement results show that lasing spectra are determined by DC bias current and almost independent of bitrates and modulation amplitudes. The correlation coefficients are nearly unity and the normalized differences are small. Also, the correlation coefficients and optical-eye shapes remain almost unchanged even with multimode-fiber-VCSEL misalignment. The results mean that transverse modes respond to the modulation current in a synchronized manner when the off-state current is much higher than threshold. This behavior together with the DC-bias determined lasing spectra is called the quasi-static mode behavior. This quasi-static behavior leads to linear relationship between the optical modulation and the modulation current amplitudes, and to optical-eye shapes invariant with fiber-VCSEL misalignment. The quasi-static behavior is weakened as the bitrate is decreased below ∼2 Gb/s and modulation current amplitude is increased so that the off-state current decreases near to the threshold current.

We investigated the effects of metal leachates from hydrothermal ore particulates on the physiological and molecular responses of the marine copepod Tigriopus japonicus. We evaluated its life cycle parameters including survival, development, and fecundity and the transcriptional profiles of its genes regulating detoxification (cytochrome P450 [CYP]), antioxidation (glutathione S-transferase [GST]), and development (vitellogenin [Vtg]) in response to hydrothermal ore and beneficiation leachates. There were preponderances of copper, iron, and zinc in the hydrothermal ore and the leachates derived from it. There were no significant changes in copepod developmental time in response to leachate exposure. However, copepod survival and fecundity significantly changed in response to the leachates. Moreover, the transcriptional profiles of the detoxification, antioxidation, and development-related genes were significantly modulated following leachate treatment. Collectively, our results suggest that metal leachates from hydrothermal ore particulates can promote the transcriptional expression of detoxification, antioxidation, and development-related genes, and altered survival and fecundity in T. japonicus.