Muge Celiktas

Stability of the hybrid epithelial/mesenchymal phenotype

Epithelial-to-Mesenchymal Transition (EMT) and its reverse – Mesenchymal to Epithelial Transition (MET) – are hallmarks of cellular plasticity during embryonic development and cancer metastasis. During EMT, epithelial cells lose cell-cell adhesion and gain migratory and invasive traits either partially or completely, leading to a hybrid epithelial/mesenchymal (hybrid E/M) or a mesenchymal phenotype respectively. Mesenchymal cells move individually, but hybrid E/M cells migrate collectively as observed during gastrulation, wound healing, and the formation of tumor clusters detected as Circulating Tumor Cells (CTCs). Typically, the hybrid E/M phenotype has largely been tacitly assumed to be transient and ‘metastable’. Here, we identify certain ‘phenotypic stability factors’ (PSFs) such as GRHL2 that couple to the core EMT decision-making circuit (miR-200/ZEB) and stabilize hybrid E/M phenotype. Further, we show that H1975 lung cancer cells can display a stable hybrid E/M phenotype and migrate collectively, a behavior that is impaired by knockdown of GRHL2 and another previously identified PSF - OVOL. In addition, our computational model predicts that GRHL2 can also associate hybrid E/M phenotype with high tumor-initiating potential, a prediction strengthened by the observation that the higher levels of these PSFs may be predictive of poor patient outcome. Finally, based on these specific examples, we deduce certain network motifs that can stabilize the hybrid E/M phenotype. Our results suggest that partial EMT, i.e. a hybrid E/M phenotype, need not be ‘metastable’, and strengthen the emerging notion that partial EMT, but not necessarily a complete EMT, is associated with aggressive tumor progression.

Molecular Portraits of Epithelial, Mesenchymal, and Hybrid States in Lung Adenocarcinoma and Their Relevance to Survival

Epithelial-to-mesenchymal transition (EMT) is a key process associated with tumor progression and metastasis. To define molecular features associated with EMT states, we undertook an integrative approach combining mRNA, miRNA, DNA methylation, and proteomic profiles of 38 cell populations representative of the genomic heterogeneity in lung adenocarcinoma. The resulting data were integrated with functional profiles consisting of cell invasiveness, adhesion, and motility. A subset of cell lines that were readily defined as epithelial or mesenchymal based on their morphology and E-cadherin and vimentin expression elicited distinctive molecular signatures. Other cell populations displayed intermediate/hybrid states of EMT, with mixed epithelial and mesenchymal characteristics. A dominant proteomic feature of aggressive hybrid cell lines was upregulation of cytoskeletal and actin-binding proteins, a signature shared with mesenchymal cell lines. Cytoskeletal reorganization preceded loss of E-cadherin in epithelial cells in which EMT was induced by TGFβ. A set of transcripts corresponding to the mesenchymal protein signature enriched in cytoskeletal proteins was found to be predictive of survival in independent datasets of lung adenocarcinomas. Our findings point to an association between cytoskeletal and actin-binding proteins, a mesenchymal or hybrid EMT phenotype and invasive properties of lung adenocarcinomas.

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.

A Search for Novel Cancer/Testis Antigens in Lung Cancer Identifies VCX/Y Genes, Expanding the Repertoire of Potential Immunotherapeutic Targets

Cancer/testis (CT) antigens are potential immunotherapeutic targets in cancer. However, the expression of particular antigens is limited to a subset of tumors of a given type. Thus, there is a need to identify antigens with complementary expression patterns for effective therapeutic intervention. In this study, we searched for genes that were distinctly expressed at a higher level in lung tumor tissue and the testes compared with other nontumor tissues and identified members of the VCX/Y gene family as novel CT antigens. VCX3A, a member of the VCX/Y gene family, was expressed at the protein level in approximately 20% of lung adenocarcinomas and 35% of squamous cell carcinomas, but not expressed in normal lung tissues. Among CT antigens with concordant mRNA and protein expression levels, four CT antigens, XAGE1, VCX, IL13RA2, and SYCE1, were expressed, alone or in combination, in about 80% of lung adenocarcinoma tumors. The CT antigen VCX/Y gene family broadens the spectrum of CT antigens expressed in lung adenocarcinomas for clinical applications.

Proteomic signatures associated with p53 mutational status in lung adenocarcinoma

p53 is commonly mutated in lung adenocarcinoma. Mutant p53 loses wild‐type function and some missense mutations further acquire oncogenic functions, while p53 wild‐type may also induce pro‐survival signaling. Therefore identification of signatures based on p53 mutational status has relevance to our understanding of p53 signaling pathways in cancer and identification of new therapeutic targets. To this end, we compared proteomic profiles of three cellular compartments (whole‐cell extract, cell surface, and media) from 28 human lung adenocarcinoma cell lines that differ based on p53 mutational status. In total, 11 598, 11 569, and 9090 protein forms were identified in whole‐cell extract, cell surface, and media, respectively. Bioinformatic analysis revealed that representative pathways associated with epithelial adhesion, immune and stromal cells, and mitochondrial function were highly significant in p53 missense mutations, p53 loss and wild‐type p53 cell lines, respectively. Of note, mRNA levels of peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC1‐α), a transcription coactivator that promotes mitochondrial oxidative phosphorylation and mitochondrial biogenesis, was substantially higher in p53 wild‐type cell lines compared to either cell lines with p53 loss or with missense mutation. Small interfering RNA targeting PGC1‐α inhibited cell proliferation in p53 wild‐type cell lines, indicative of PGC1‐α and its downstream molecules as potential therapeutic targets in p53 wild‐type lung adenocarcinoma.

Distinguishing mechanisms underlying EMT tristability

BackgroundThe Epithelial-Mesenchymal Transition (EMT) endows epithelial-looking cells with enhanced migratory ability during embryonic development and tissue repair. EMT can also be co-opted by cancer cells to acquire metastatic potential and drug-resistance. Recent research has argued that epithelial (E) cells can undergo either a partial EMT to attain a hybrid epithelial/mesenchymal (E/M) phenotype that typically displays collective migration, or a complete EMT to adopt a mesenchymal (M) phenotype that shows individual migration. The core EMT regulatory network - miR-34/SNAIL/miR-200/ZEB1 - has been identified by various studies, but how this network regulates the transitions among the E, E/M, and M phenotypes remains controversial. Two major mathematical models – ternary chimera switch (TCS) and cascading bistable switches (CBS) - that both focus on the miR-34/SNAIL/miR-200/ZEB1 network, have been proposed to elucidate the EMT dynamics, but a detailed analysis of how well either or both of these two models can capture recent experimental observations about EMT dynamics remains to be done.ResultsHere, via an integrated experimental and theoretical approach, we first show that both these two models can be used to understand the two-step transition of EMT - E→E/M→M, the different responses of SNAIL and ZEB1 to exogenous TGF-β and the irreversibility of complete EMT. Next, we present new experimental results that tend to discriminate between these two models. We show that ZEB1 is present at intermediate levels in the hybrid E/M H1975 cells, and that in HMLE cells, overexpression of SNAIL is not sufficient to initiate EMT in the absence of ZEB1 and FOXC2.ConclusionsThese experimental results argue in favor of the TCS model proposing that miR-200/ZEB1 behaves as a three-way decision-making switch enabling transitions among the E, hybrid E/M and M phenotypes.

MCAM mediates chemoresistance in small-cell lung cancer via the PI3K/AKT/ SOX2 signaling pathway

Despite favorable responses to initial therapy, small-cell lung cancer (SCLC) relapse occurs within a year and exhibits resistance to multiple drugs. Because of limited accessibility of patient tissues for research purposes, SCLC patient-derived xenografts (PDX) have provided the best opportunity to address this limitation. Here, we sought to identify novel mechanisms involved in SCLC chemoresistance. Through in-depth proteomic profiling, we identified MCAM as a markedly upregulated surface receptor in chemoresistant SCLC cell lines and in chemoresistant PDX compared with matched treatment-naïve tumors. MCAM depletion in chemoresistant cells reduced cell proliferation and reduced the IC50 inhibitory concentration of chemotherapeutic drugs in vitro This MCAM-mediated sensitization to chemotherapy occurred via SOX2-dependent upregulation of mitochondrial 37S ribosomal protein 1/ATP-binding cassette subfamily C member 1 (MRP1/ABCC1) and the PI3/AKT pathway. Metabolomic profiling revealed that MCAM modulated lactate production in chemoresistant cells that exhibit a distinct metabolic phenotype characterized by low oxidative phosphorylation. Our results suggest that MCAM may serve as a novel therapeutic target to overcome chemoresistance in SCLC. Cancer Res; 77(16); 4414-25. ©2017 AACR.

Role of CPS1 in cell growth, metabolism, and prognosis in LKB1-inactivated lung adenocarcinoma

Background Liver kinase B1 ( LKB1 ) is a tumor suppressor in lung adenocarcinoma (LADC). We investigated the proteomic profiles of 45 LADC cell lines with and without LKB1 inactivation. Carbamoyl phosphate synthetase 1 (CPS1), the first rate-limiting mitochondrial enzyme in the urea cycle, was distinctively overexpressed in LKB1-inactivated LADC cell lines. We therefore assessed the role of CPS1 and its clinical relevance in LKB1-inactivated LADC. Methods Mass spectrometric profiling of proteome and metabolome and function of CPS1 were analyzed in LADC cell lines. CPS1 and LKB1 expression in tumors from 305 LADC and 160 lung squamous cell carcinoma patients was evaluated by immunohistochemistry. Kaplan-Meier and Cox regression analyses were applied to assess the association between overall survival and CPS1 and LKB1 expression. All statistical tests were two-sided. Results CPS1 knockdown reduced cell growth, decreased metabolite levels associated with nucleic acid biosynthesis pathway, and contributed an additive effect when combined with gemcitabine, pemetrexed, or CHK1 inhibitor AZD7762. Tissue microarray analysis revealed that CPS1 was expressed in 65.7% of LKB1-negative LADC, and only 5.0% of LKB1-positive LADC. CPS1 expression showed statistically significant association with poor overall survival in LADC (hazard ratio = 3.03, 95% confidence interval = 1.74 to 5.25, P < .001). Conclusions Our findings suggest functional relevance of CPS1 in LKB1-inactivated LADC and association with worse outcome of LADC. CPS1 is a promising therapeutic target in combination with other chemotherapy agents, as well as a prognostic biomarker, enabling a personalized approach to treatment of LADC.BACKGROUND Liver kinase B1 (LKB1) is a tumor suppressor in lung adenocarcinoma (LADC). We investigated the proteomic profiles of 45 LADC cell lines with and without LKB1 inactivation. Carbamoyl phosphate synthetase 1 (CPS1), the first rate-limiting mitochondrial enzyme in the urea cycle, was distinctively overexpressed in LKB1-inactivated LADC cell lines. We therefore assessed the role of CPS1 and its clinical relevance in LKB1-inactivated LADC. METHODS Mass spectrometric profiling of proteome and metabolome and function of CPS1 were analyzed in LADC cell lines. CPS1 and LKB1 expression in tumors from 305 LADC and 160 lung squamous cell carcinoma patients was evaluated by immunohistochemistry. Kaplan-Meier and Cox regression analyses were applied to assess the association between overall survival and CPS1 and LKB1 expression. All statistical tests were two-sided. RESULTS CPS1 knockdown reduced cell growth, decreased metabolite levels associated with nucleic acid biosynthesis pathway, and contributed an additive effect when combined with gemcitabine, pemetrexed, or CHK1 inhibitor AZD7762. Tissue microarray analysis revealed that CPS1 was expressed in 65.7% of LKB1-negative LADC, and only 5.0% of LKB1-positive LADC. CPS1 expression showed statistically significant association with poor overall survival in LADC (hazard ratio = 3.03, 95% confidence interval = 1.74 to 5.25, P < .001). CONCLUSIONS Our findings suggest functional relevance of CPS1 in LKB1-inactivated LADC and association with worse outcome of LADC. CPS1 is a promising therapeutic target in combination with other chemotherapy agents, as well as a prognostic biomarker, enabling a personalized approach to treatment of LADC.

Abstract 2775: CPS1 as a therapeutic target and prognostic indicator in LKB1-inactivated lung adenocarcinoma

Liver Kinase B1 (LKB1), encoded by STK11, is a tumor suppressor and somatically mutated in approximately 20% of lung adenocarcinoma. Aside from the effects of LKB1 inactivation on tumor initiation, LKB1-mutant cancers are biologically distinct from cancer with functional LKB1, and the loss of LKB1 uniquely confers invasive and metastatic properties in genetically engineered mouse models of cancer. While various pathways, including energy metabolism, cell polarity, and cell growth, are regulated by LKB1 and can play a pleiotropic role in cancer initiation and progression, no therapies are currently available for clinical use that specifically target LKB1 inactivation. Therefore, elucidation of the functional mechanisms associated with LKB1 inactivation has translational relevance. We analyzed proteomic profiles of 45 lung adenocarcinoma cell lines with and without LKB1 inactivation to identify molecular features associated with LKB1 inactivation. Carbamoyl phosphate synthase 1 (CPS1) was identified as a markedly overexpressed protein in LKB1-inactivated lung adenocarcinoma cell lines. CPS1 is the first rate-limiting mitochondrial enzyme in the urea cycle, and plays an intricate role in arginine and pyrimidine metabolism. CPS1 knockdown reduced cell growth, decreased levels of metabolites associated with nucleic acid biosynthesis pathway, and contributed an additive effect when combined with conventional chemotherapy agents including gemcitabine, pemetrexed, or CHK1 inhibitor AZD7762. Tissue microarray analysis using 305 lung adenocarcinoma tumors revealed that CPS1 was expressed in 65.7% of LKB1-negative and only 5.0% of LKB1-positive lung adenocarcinomas. In addition, CPS1 expression was significantly and independently associated with poor overall survival of lung adenocarcinoma patients (P Citation Format: Muge Celiktas, Ichidai Tanaka, Satyendra Chandra Tripathi, Johannes F. Fahrmann, Clemente Aguilar-Bonavides, Pamela Villalobos, Oliver Delgado, Dilsher Dhillon, Jennifer B. Dennison, Edwin J. Ostrin, Hong Wang, Carmen Behrens, Kim-Anh Do, Adi F. Gazdar, Samir M. Hanash, Ayumu Taguchi. CPS1 as a therapeutic target and prognostic indicator in LKB1-inactivated lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2775. doi:10.1158/1538-7445.AM2017-2775

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.