Amin Momin

Immunoproteasome deficiency is a feature of non-small cell lung cancer with a mesenchymal phenotype and is associated with a poor outcome

Significance The success rate of therapeutic trials that target tumor antigens is quite limited. We demonstrate for the first time to our knowledge that lung cancer cells that have undergone epithelial-to-mesenchymal transition lose immunoproteasome expression, resulting in markedly reduced antigen presentation. Reduced expression of the immunoproteasome was associated with and can predict poor outcome in non-small cell lung carcinoma (NSCLC) patients. Induction of the immunoproteasome with IFNγ or 5-aza-2′-deoxycytidine (5-aza-dC) treatment can overcome this immune escape mechanism of mesenchymal cells by restoring functional HLA class I-bound peptides. These findings have substantial relevance for development of effective strategies to target tumor cells with inherent resistance to T cell-mediated immunotherapy. The immunoproteasome plays a key role in generation of HLA peptides for T cell-mediated immunity. Integrative genomic and proteomic analysis of non-small cell lung carcinoma (NSCLC) cell lines revealed significantly reduced expression of immunoproteasome components and their regulators associated with epithelial to mesenchymal transition. Low expression of immunoproteasome subunits in early stage NSCLC patients was associated with recurrence and metastasis. Depleted repertoire of HLA class I-bound peptides in mesenchymal cells deficient in immunoproteasome components was restored with either IFNγ or 5-aza-2′-deoxycytidine (5-aza-dC) treatment. Our findings point to a mechanism of immune evasion of cells with a mesenchymal phenotype and suggest a strategy to overcome immune evasion through induction of the immunoproteasome to increase the cellular repertoire of HLA class I-bound peptides.

ETS2 Mediated Tumor Suppressive Function and MET Oncogene Inhibition in Human Non–Small Cell Lung Cancer

Purpose: The ETS2 transcription factor is an evolutionarily conserved gene that is deregulated in cancer. We analyzed the transcriptome of lung adenocarcinomas and normal lung tissue by expression profiling and found that ETS2 was significantly downregulated in adenocarcinomas. In this study, we probed the yet unknown functional role of ETS2 in lung cancer pathogenesis. Experimental Design: Lung adenocarcinomas (n = 80) and normal lung tissues (n = 30) were profiled using the Affymetrix Human Gene 1.0 ST platform. Immunohistochemical (IHC) analysis was conducted to determine ETS2 protein expression in non–small cell lung cancer (NSCLC) histologic tissue specimens (n = 201). Patient clinical outcome, based on ETS2 IHC expression, was statistically assessed using the log-rank and Kaplan–Meier tests. RNA interference and overexpression strategies were used to assess the effects of ETS2 expression on the transcriptome and on various malignant phenotypes. Results: ETS2 expression was significantly reduced in lung adenocarcinomas compared with normal lung (P < 0.001). Low ETS2 IHC expression was a significant predictor of shorter time to recurrence in NSCLC (P = 0.009, HR = 1.89) and adenocarcinoma (P = 0.03, HR = 1.86). Moreover, ETS2 was found to significantly inhibit lung cancer cell growth, migration, and invasion (P < 0.05), and microarray and pathways analysis revealed significant (P < 0.001) activation of the HGF pathway following ETS2 knockdown. In addition, ETS2 was found to suppress MET phosphorylation and knockdown of MET expression significantly attenuated (P < 0.05) cell invasion mediated by ETS2-specific siRNA. Furthermore, knockdown of ETS2 augmented HGF-induced MET phosphorylation, cell migration, and invasion. Conclusion(s): Our findings point to a tumor suppressor role for ETS2 in human NSCLC pathogenesis through inhibition of the MET proto-oncogene. Clin Cancer Res; 19(13); 3383–95. ©2013 AACR.

Estrogen-Regulated Genes in Rat Testes and Their Relationship to Recovery of Spermatogenesis after Irradiation

Despite numerous observations of the effects of estrogens on spermatogenesis, identification of estrogen-regulated genes in the testis is limited. Using rats in which irradiation had completely blocked spermatogonial differentiation, we previously showed that testosterone suppression with gonadotropin-releasing hormone-antagonist acyline and the antiandrogen flutamide stimulated spermatogenic recovery and that addition of estradiol (E2) to this regimen accelerated this recovery. We report here the global changes in testicular cell gene expression induced by the E2 treatment. By minimizing the changes in other hormones and using concurrent data on regulation of the genes by these hormones, we were able to dissect the effects of estrogen on gene expression, independent of gonadotropin or testosterone changes. Expression of 20 genes, largely in somatic cells, was up- or downregulated between 2- and 5-fold by E2. The unexpected and striking enrichment of transcripts not corresponding to known genes among the E2-downregulated probes suggested that these might represent noncoding mRNAs; indeed, we have identified several as miRNAs and their potential target genes in this system. We propose that genes for which expression levels are altered in one direction by irradiation and in the opposite direction by both testosterone suppression and E2 treatment are candidates for controlling the block in differentiation. Several genes, including insulin-like 3 (Insl3), satisfied those criteria. If they are indeed involved in the inhibition of spermatogonial differentiation, they may be candidate targets for treatments to enhance recovery of spermatogenesis following gonadotoxic exposures, such as those resulting from cancer therapy.

Lysyl Hydroxylase 2 Is Secreted By Tumor Cells and Can Modify Collagen in the Extracellular Space

Lysyl hydroxylase 2 (LH2) catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens, which leads to the formation of stable collagen cross-links. Recently we reported that LH2 enhances the metastatic propensity of lung cancer by increasing the amount of stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), which generate a stiffer tumor stroma (Chen, Y., et al. (2015) J. Clin. Invest. 125, 125, 1147–1162). It is generally accepted that LH2 modifies procollagen α chains on the endoplasmic reticulum before the formation of triple helical procollagen molecules. Herein, we report that LH2 is also secreted and modifies collagen in the extracellular space. Analyses of lung cancer cell lines demonstrated that LH2 is present in the cell lysates and the conditioned media in a dimeric, active form in both compartments. LH2 co-localized with collagen fibrils in the extracellular space in human lung cancer specimens and in orthotopic lung tumors generated by injection of a LH2-expressing human lung cancer cell line into nude mice. LH2 depletion in MC3T3 osteoblastic cells impaired the formation of HLCCs, resulting in an increase in the unmodified lysine aldehyde-derived collagen cross-link (LCC), and the addition of recombinant LH2 to the media of LH2-deficient MC3T3 cells was sufficient to rescue HLCC formation in the extracellular matrix. The finding that LH2 modifies collagen in the extracellular space challenges the current view that LH2 functions solely on the endoplasmic reticulum and could also have important implications for cancer biology.

Latent feature decompositions for integrative analysis of diverse high-throughput genomic data

A general method for regressing a continuous response upon large groups of diverse genetic covariates via dimension reduction is developed and exemplified. It is shown that allowing latent features derived from different covariate groups to interact aids in prediction when interactions subsist among the original covariates. A means of selecting a subset of relevant covariates from the original set is proposed, and a simulation study is performed to demonstrate the effectiveness of the procedure for prediction and variable selection. The procedure is applied to a high-dimensional lung cancer data set to model the effects of gene expression, copy number variation, and methylation on a drug response.

Expression Profiling Stratifies Mesothelioma Tumors and Signifies Deregulation of Spindle Checkpoint Pathway and Microtubule Network with Therapeutic Implications

BACKGROUND Malignant pleural mesothelioma (MPM) is a lethal neoplasm exhibiting resistance to most treatment regimens and requires effective therapeutic options. Though an effective strategy in many cancer, targeted therapy is relatively unexplored in MPM because the therapeutically important oncogenic pathways and networks in MPM are largely unknown. MATERIALS AND METHODS We carried out gene expression microarray profiling of 53 surgically resected MPMs tumors along with paired normal tissue. We also carried out whole transcriptomic sequence (RNA-seq) analysis on eight tumor specimens. Taqman-based quantitative Reverse-transcription polymerase chain reaction (qRT-PCR), western analysis and immunohistochemistry (IHC) analysis of mitotic arrest deficient-like 1 (MAD2L1) was carried out on tissue specimens. Cell viability assays of MPM cell lines were carried out to assess sensitivity to specific small molecule inhibitors. RESULTS Bioinformatics analysis of the microarray data followed by pathway analysis revealed that the mitotic spindle assembly checkpoint (MSAC) pathway was most significantly altered in MPM tumors with upregulation of 18 component genes, including MAD2L1 gene. We validated the microarray data for MAD2L1 expression using quantitative qRT-PCR and western blot analysis on tissue lysates. Additionally, we analyzed expression of the MAD2L1 protein by IHC using an independent tissue microarray set of 80 MPM tissue samples. Robust clustering of gene expression data revealed three novel subgroups of tumors, with unique expression profiles, and showed differential expression of MSAC pathway genes. Network analysis of the microarray data showed the cytoskeleton/spindle microtubules network was the second-most significantly affected network. We also demonstrate that a nontaxane small molecule inhibitor, epothilone B, targeting the microtubules have great efficacy in decreasing viability of 14 MPM cell lines. CONCLUSIONS Overall, our findings show that MPM tumors have significant deregulation of the MSAC pathway and the microtubule network, it can be classified into three novel molecular subgroups of potential therapeutic importance and epothilone B is a promising therapeutic agent for MPM.

Latent feature decompositions for integrative analysis of multi-platform genomic data

Increased availability of multi-platform genomics data on matched samples has sparked research efforts to discover how diverse molecular features interact both within and between platforms. In addition, simultaneous measurements of genetic and epigenetic characteristics illuminate the roles their complex relationships play in disease progression and outcomes. However, integrative methods for diverse genomics data are faced with the challenges of ultra-high dimensionality and the existence of complex interactions both within and between platforms. We propose a novel modeling framework for integrative analysis based on decompositions of the large number of platform-specific features into a smaller number of latent features. Subsequently we build a predictive model for clinical outcomes accounting for both within- and between-platform interactions based on Bayesian model averaging procedures. Principal components, partial least squares and non-negative matrix factorization as well as sparse counterparts of each are used to define the latent features, and the performance of these decompositions is compared both on real and simulated data. The latent feature interactions are shown to preserve interactions between the original features and not only aid prediction but also allow explicit selection of outcome-related features. The methods are motivated by and applied to a glioblastoma multiforme data set from The Cancer Genome Atlas to predict patient survival times integrating gene expression, microRNA, copy number and methylation data. For the glioblastoma data, we find a high concordance between our selected prognostic genes and genes with known associations with glioblastoma. In addition, our model discovers several relevant cross-platform interactions such as copy number variation associated gene dosing and epigenetic regulation through promoter methylation. On simulated data, we show that our proposed method successfully incorporates interactions within and between genomic platforms to aid accurate prediction and variable selection. Our methods perform best when principal components are used to define the latent features.

Abstract A060: Immunoproteasome deficiency is a feature of NSCLC with a mesenchymal phenotype and is associated with restricted antigen presentation and poor outcome in patients

Proteasomes are multi-subunit complexes that degrade intracellular proteins through the ubiquitin-proteasome pathway. The immunoproteasome generates peptides that are particularly suitable for binding onto HLA I molecules, thus facilitating antigen presentation leading to CD8+ T cell responses. Lack of expression or down regulation of the immunoproteasome may contribute to immune evasion through antigen loss. The expression of the immunoproteasome and its impact on antigen presentation in tumors of epithelial origin is not well established. Here, we have investigated the constitutive and induced expression patterns of immunoproteasome subunits in lung cancer and their consequence on antigen presentation. We have also assessed the impact of immunoproteasome expression on prognosis for non-small cell lung carcinoma (NSCLC) patients. Proteomic profiling of the immunoproteasome in 42 NSCLC cell lines revealed significantly reduced expression of immunoproteasome components and their regulators associated with epithelial to mesenchymal transition. We observed highly variable immunoproteasome expression among NSCLC cell lines and tumor tissues. Immunohistochemistry data revealed loss of immunoproteasome subunit is significantly correlated with loss of E-cadherin and expression of N-cadherin in NSCLC tumors. Loss of immunoproteasome subunits was also significantly associated with advanced stage (p = 0.014), recurrence (p = 0.02) and metastasis (p Citation Format: Satyendra C. Tripathi, Haley L. Peters, Edwin J. Ostrin, Ayumu Taguchi, Hiroyuki Katayama, Hong Wang, Amin Momin, Mohit K. Jolly, Muge Celiktas, Jaime Rodriguez, Carmen Behrens, Ignacio I. Wistuba, Eshel Ben Jacob, Herbert Levine, Jeffrey J. Molldrem, Samir M. Hanash. Immunoproteasome deficiency is a feature of NSCLC with a mesenchymal phenotype and is associated with restricted antigen presentation and poor outcome in patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A060.

Abstract A18: Syndecan-1 is required for oncogenic Kras-driven PDAC tumorigenesis and maintenance

Although the universal presence of KRAS mutations and their critical role in human pancreatic ductal adenocarcinoma (PDAC) designates it as an ideal therapeutic target, oncogenic KRAS (KRAS*) is still regarded as ‘undruggable’ to date. Therefore, to identify therapeutic points of intervention, it is critical to understand the impact of KRAS*-mediated pathways on in vivo tumor pathogenesis. We have recently generated an inducible Kras G12D -driven (iKRAS*) mouse PDAC model and established a critical role for sustained KRAS* activity in tumor maintenance, providing a model to characterize pathways required for KRAS*-dependent tumorigenicity. Cell surface proteins are relatively accessible and can provide candidates for biomarker discovery, as well as be utilized as therapeutic targets. To characterize the KRAS*-specific organization of cell surface proteins toward the development of possible diagnostic or therapeutic tools, we conducted an unbiased surfaceome analysis of tumor cells in the presence and absence of KRAS* signaling using the iKRAS* mouse PDAC model. Syndecans (SDC), a family of heparin sulfate proteoglycans, were identified as candidates whose membrane expression is correlated with KRAS* activity. Specifically, Syndecan-1 (SDC1) expression was upregulated in response to KRAS* induction in acinar-ductal metaplasia (ADM) and early pancreatic intraepithelial neoplasia (PanIN), but not in cerulein-induced chronic pancreatitis. Moreover, SDC1 membrane expression was abolished upon KRAS* extinction, and our data indicated that the MAPK pathway, but not the PI3K pathway, drives KRAS*-mediated SDC1 surface localization. Supportive of our findings, it has been shown that SDC1 expression is increased in premalignant and malignant pancreatic lesions of primary human PDAC, as well as in various other KRAS*-driven pancreatic cancer mouse models. To further study the role of SDC1 in KRAS*-driven pancreatic cancers, we generated an SDC1-knockout mouse model and crossed with LSL-Kras G12D -PDAC models. Our preliminary data indicate that genetic ablation of SDC1 expression effectively suppressed KRAS*-driven PDAC initiation and progression. The mechanisms of KRAS*-mediated SDC1 membrane localization and its impact on PDAC initiation and progression will be further explored. Citation Format: Wantong Yao, Wei Wang, Ayumu Taguchi, Avnish Kapoor, Amin Momin, Hong Jiang, Johnathon Rose, Muge Celiktas, Angela Deem, Samir Hanash, Ronald DePinho, Haoqiang Ying, Giulio Draetta. Syndecan-1 is required for oncogenic Kras-driven PDAC tumorigenesis and maintenance. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A18.

Abstract PR03: Global quantitative proteomic and phosphoproteomic analysis of oncogenic Kras-driven mouse pancreatic cancer

Background: The most common genetic abnormality in pancreatic cancer is oncogenic mutation of KRAS, which is an initial key event in pancreatic carcinogenesis. Oncogenic KRAS activates downstream signaling pathways, such as PI3K and MAPK pathways, indicating the essential role of phosphorylation in pancreatic cancer. However, the effect of KRAS on the global phosphoproteome in pancreatic cancer has not been fully elucidated. We investigated proteomic and phosphoproteomic alterations induced by oncogenic Kras using mouse pancreatic cancer cell lines established from doxycycline-inducible KrasG12D pancreatic cancer mouse model. Materials and Methods: Three mouse pancreatic cancer cell lines were established from triple transgenic inducible KrasG12D pancreatic cancer mouse model (tetO_LSL- KrasG12D; ROSA26A-LSL-rtTA-IRES-GFP; p48-Cre). To identify direct downstream pathways of Kras, we performed two different experiments for each cell line: (1) 20 hours Kras-off followed by 5 hours Kras-on (designated “Re-on”) vs. 25 hours Kras-off, and (2) Continuous Kras-on (designated “On”) vs. 24 hours Kras-off. Whole cell lysates were fractionated and subjected to analysis with using mass spectrometry. Results and Conclusion: Overall 4,133 phosphorylation sites were identified. Phosphorylation levels of 2,670 sites were quantified in at least one sample, including 2,347 (87.9%) at serine, 293 (11.0%) threonine, and 30 (1.1%) tyrosine residues. Phosphorylation levels were more than 1.25 fold higher at 386 sites (139 proteins) and 558 sites (256 proteins) in “Re-on” and “On” samples, respectively. 190 sites were common between “Re-on” and “On”, 196 sites were “Re-on” unique, and 368 sites were “On” unique. mTOR and MAPK pathways were activated in both “Re-on” and “On”, while Rho kinase, Phospholipase C, and Rac signaling pathways were predominantly associated with “On”, suggestive of differential downstream pathway activation at different phases of Kras signaling. This abstract is also presented as Poster A21. Citation Format: Ayumu Taguchi, Avnish Kapoor, Amin Momin, Hiroyuki Katayama, Wantong Yao, Hong Wang, Haoqiang Ying, Ronald DePinho, Samir Hanash. Global quantitative proteomic and phosphoproteomic analysis of oncogenic Kras-driven mouse pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr PR03. doi: 10.1158/1557-3125.RASONC14-PR03