Humam Kadara

Ursolic Acid Inhibits STAT3 Activation Pathway Leading to Suppression of Proliferation and Chemosensitization of Human Multiple Myeloma Cells

The activation of signal transducers and activators of transcription 3 (STAT3) has been linked with the proliferation of a variety of human cancer cells, including multiple myeloma. Agents that can suppress STAT3 activation have potential for prevention and treatment of cancer. In the present report, we tested an agent, ursolic acid, found in basil, apples, prunes, and cranberries, for its ability to suppress STAT3 activation. We found that ursolic acid, a pentacyclic triterpenoid, inhibited both constitutive and interleukin-6–inducible STAT3 activation in a dose- and time-dependent manner in multiple myeloma cells. The suppression was mediated through the inhibition of activation of upstream kinases c-Src, Janus-activated kinase 1, Janus-activated kinase 2, and extracellular signal–regulated kinase 1/2. Vanadate treatment reversed the ursolic acid–induced down-regulation of STAT3, suggesting the involvement of a tyrosine phosphatase. Indeed, we found that ursolic acid induced the expression of tyrosine phosphatase SHP-1 protein and mRNA. Moreover, knockdown of SHP-1 by small interfering RNA suppressed the induction of SHP-1 and reversed the inhibition of STAT3 activation, thereby indicating the critical role of SHP-1 in the action of this triterpene. Ursolic acid down-regulated the expression of STAT3-regulated gene products such as cyclin D1, Bcl-2, Bcl-xL, survivin, Mcl-1, and vascular endothelial growth factor. Finally, ursolic acid inhibited proliferation and induced apoptosis and the accumulation of cells in G1-G0 phase of cell cycle. This triterpenoid also significantly potentiated the apoptotic effects of thalidomide and bortezomib in multiple myeloma cells. Overall, these results suggest that ursolic acid is a novel blocker of STAT3 activation that may have a potential in prevention and treatment of multiple myeloma and other cancers. (Mol Cancer Res 2007;5(9):943–55)

Co-occurring genomic alterations define major subsets of KRAS - mutant lung adenocarcinoma with distinct biology, immune profiles, and therapeutic vulnerabilities

UNLABELLED The molecular underpinnings that drive the heterogeneity of KRAS-mutant lung adenocarcinoma are poorly characterized. We performed an integrative analysis of genomic, transcriptomic, and proteomic data from early-stage and chemorefractory lung adenocarcinoma and identified three robust subsets of KRAS-mutant lung adenocarcinoma dominated, respectively, by co-occurring genetic events in STK11/LKB1 (the KL subgroup), TP53 (KP), and CDKN2A/B inactivation coupled with low expression of the NKX2-1 (TTF1) transcription factor (KC). We further revealed biologically and therapeutically relevant differences between the subgroups. KC tumors frequently exhibited mucinous histology and suppressed mTORC1 signaling. KL tumors had high rates of KEAP1 mutational inactivation and expressed lower levels of immune markers, including PD-L1. KP tumors demonstrated higher levels of somatic mutations, inflammatory markers, immune checkpoint effector molecules, and improved relapse-free survival. Differences in drug sensitivity patterns were also observed; notably, KL cells showed increased vulnerability to HSP90-inhibitor therapy. This work provides evidence that co-occurring genomic alterations identify subgroups of KRAS-mutant lung adenocarcinoma with distinct biology and therapeutic vulnerabilities. SIGNIFICANCE Co-occurring genetic alterations in STK11/LKB1, TP53, and CDKN2A/B-the latter coupled with low TTF1 expression-define three major subgroups of KRAS-mutant lung adenocarcinoma with distinct biology, patterns of immune-system engagement, and therapeutic vulnerabilities.

Upregulated stromal EGFR and vascular remodeling in mouse xenograft models of angiogenesis inhibitor–resistant human lung adenocarcinoma

Angiogenesis is critical for tumor growth and metastasis, and several inhibitors of angiogenesis are currently in clinical use for the treatment of cancer. However, not all patients benefit from antiangiogenic therapy, and those tumors that initially respond to treatment ultimately become resistant. The mechanisms underlying this, and the relative contributions of tumor cells and stroma to resistance, are not completely understood. Here, using species-specific profiling of mouse xenograft models of human lung adenocarcinoma, we have shown that gene expression changes associated with acquired resistance to the VEGF inhibitor bevacizumab occurred predominantly in stromal and not tumor cells. In particular, components of the EGFR and FGFR pathways were upregulated in stroma, but not in tumor cells. Increased activated EGFR was detected on pericytes of xenografts that acquired resistance and on endothelium of tumors with relative primary resistance. Acquired resistance was associated with a pattern of pericyte-covered, normalized revascularization, whereas tortuous, uncovered vessels were observed in relative primary resistance. Importantly, dual targeting of the VEGF and EGFR pathways reduced pericyte coverage and increased progression-free survival. These findings demonstrated that alterations in tumor stromal pathways, including the EGFR and FGFR pathways, are associated with, and may contribute to, resistance to VEGF inhibitors and that targeting these pathways may improve therapeutic efficacy. Understanding stromal signaling may be critical for developing biomarkers for angiogenesis inhibitors and improving combination regimens.

Sex determining region Y-box 2 (SOX2) is a potential cell-lineage gene highly expressed in the pathogenesis of squamous cell carcinomas of the lung

Background Non-small cell lung cancer (NSCLC) represents the majority (85%) of lung cancers and is comprised mainly of adenocarcinomas and squamous cell carcinomas (SCCs). The sequential pathogenesis of lung adenocarcinomas and SCCs occurs through dissimilar phases as the former tumors typically arise in the lung periphery whereas the latter normally arise near the central airway. Methodology/Principal Findings We assessed the expression of SOX2, an embryonic stem cell transcriptional factor that also plays important roles in the proliferation of basal tracheal cells and whose expression is restricted to the main and central airways and bronchioles of the developing and adult mouse lung, in NSCLC by various methodologies. Here, we found that SOX2 mRNA levels, from various published datasets, were significantly elevated in lung SCCs compared to adenocarcinomas (all p<0.001). Moreover, a previously characterized OCT4/SOX2/NANOG signature effectively separated lung SCCs from adenocarcinomas in two independent publicly available datasets which correlated with increased SOX2 mRNA in SCCs. Immunohistochemical analysis of various histological lung tissue specimens demonstrated marked nuclear SOX2 protein expression in all normal bronchial epithelia, alveolar bronchiolization structures and premalignant lesions in SCC development (hyperplasia, dysplasia and carcinoma in situ) and absence of expression in all normal alveoli and atypical adenomatous hyperplasias. Moreover, SOX2 protein expression was greatly higher in lung SCCs compared to adenocarcinomas following analyses in two independent large TMA sets (TMA set I, n = 287; TMA set II, n = 511 both p<0.001). Furthermore, amplification of SOX2 DNA was detected in 20% of lung SCCs tested (n = 40) and in none of the adenocarcinomas (n = 17). Conclusions/Significance Our findings highlight a cell-lineage gene expression pattern for the stem cell transcriptional factor SOX2 in the pathogenesis of lung SCCs and suggest a differential activation of stem cell-related pathways between squamous cell carcinomas and adenocarcinomas of the lung.

High Expression of Folate Receptor Alpha in Lung Cancer Correlates with Adenocarcinoma Histology and Mutation

Introduction: Folate receptor alpha (FR&agr;) and reduced folate carrier-1 (RFC1) regulate uptake of folate molecules inside the cell. FR&agr; is a potential biomarker of tumors response to antifolate chemotherapy, and a target for therapies using humanized monocloncal antibody. Information on the protein expression of these receptors in non–small-cell lung carcinoma (NSCLC) is limited. Material and Methods: Expressions of FR&agr; and RFC1 were examined by immunohistochemistry (IHC) in 320 surgically resected NSCLC (202 adenocarcinomas and 118 squamous cell carcinomas) tissue specimens and correlated with patients’ clinico-pathologic characteristics. Folate receptor &agr; gene (FOLR1) mRNA expression was examined using publicly available microarray datasets. FR&agr; expression was correlated with thymidylate synthase and p53 expression in NSCLCs, and with epidermal growth factor receptor (EGFR) and V-Ki-ras2 Kirsten rat sarcoma viral (KRAS) gene mutations in adenocarcinomas. Results: NSCLC overexpressed FR&agr; and RFC1. In a multivariate analysis, lung adenocarcinomas were more likely to express FR&agr; in the cytoplasm (OR = 4.39; p < 0.0001) and membrane (OR = 5.34; p < 0.0001) of malignant cells than squamous cell carcinomas. Tumors from never-smokers were more likely to express cytoplasmic (OR = 3.35; p<0.03) and membrane (OR = 3.60; p=0.0005) FR&agr; than those from smokers. In adenocarcinoma, EGFR mutations correlated with higher expression of membrane FR&agr; and FOLR1 gene expressions. High levels of FR&agr; expression was detected in 42 NSCLC advanced metastatic tumor tissues. Conclusions: FR&agr; and RFC1 proteins are overexpressed in NSCLC tumor tissues. The high levels of FR&agr; in lung adenocarcinomas may be associated to these tumors’ better responses to antifolate chemotherapy and represents a potential novel target for this tumor type.

Histologic Patterns and Molecular Characteristics of Lung Adenocarcinoma Associated With Clinical Outcome

Lung adenocarcinoma is histologically heterogeneous and has 5 distinct histologic growth patterns: lepidic, acinar, papillary, micropapillary, and solid. To date, there is no consensus regarding the clinical utility of these patterns.

EZH2 Protein Expression Associates with the Early Pathogenesis, Tumor Progression, and Prognosis of Non–Small Cell Lung Carcinoma

Purpose: Enhancer of zeste homolog 2 (EZH2) promotes carcinogenesis by epigenetically silencing tumor suppressor genes. We studied EZH2 expression by immunohistochemistry in a large series of non–small cell lung carcinomas (NSCLC) in association with tumor characteristics and patient outcomes. Experimental Design: EZH2 immunohistochemistry expression was analyzed in 265 normal and premalignant bronchial epithelia, 541 primary NSCLCs [221 squamous cell carcinomas (SCC) and 320 adenocarcinomas] and 36 NSCLCs with paired brain metastases. An independent set of 91 adenocarcinomas was also examined. EZH2 expression was statistically correlated with clinico-pathological information, and EGFR/KRAS mutation status. Results: EZH2 expression was significantly (P < 0.0001) higher in SCCs compared with adenocarcinomas and in brain metastasis relative to matched primary tumors (P = 0.0013). EZH2 expression was significantly (P < 0.0001) elevated in bronchial preneoplastic lesions with increasing severity. In adenocarcinomas, higher EZH2 expression significantly correlated with younger age, cigarette smoking, and higher TNM stage (P = 0.02 to P < 0.0001). Higher EZH2 expression in adenocarcinoma was associated with worse recurrence-free survival (RFS; P = 0.025; HR = 1.54) and overall survival (OS; P = 0.0002; HR = 1.96). Furthermore, lung adenocarcinomas with low EZH2 levels and high expression of the lineage-specific transcription factor, TTF-1, exhibited significantly improved RFS (P = 0.009; HR = 0.51) and OS (P = 0.0011; HR = 0.45), which was confirmed in the independent set of 91 adenocarcinomas. Conclusion: In lung, EZH2 expression is involved in early pathogenesis of SCC and correlates with a more aggressive tumor behavior of adenocarcinoma. When EZH2 and TTF-1 expressions are considered together, they serve as a prognostic marker in patients with surgically resected lung adenocarcinomas. Clin Cancer Res; 19(23); 6556–65. ©2013 AACR.

A five-gene and corresponding-protein signature for stage-I lung adenocarcinoma prognosis

Purpose: Identification of effective markers for outcome is expected to improve the clinical management of non–small cell lung cancer (NSCLC). Here, we assessed in NSCLC the prognostic efficacy of genes, which we had previously found to be differentially expressed in an in vitro model of human lung carcinogenesis. Experimental Design: Prediction algorithms and risk-score models were applied to the expression of the genes in publicly available NSCLC expression data sets. The prognostic capacity of the immunohistochemical expression of proteins encoded by these genes was also tested using formalin-fixed paraffin-embedded (FFPE) tissue specimens from 156 lung adenocarcinomas and 79 squamous cell carcinomas (SCCs). Results: The survival of all-stages (P < 0.001, HR = 2.0) or stage-I (P < 0.001, HR = 2.84) adenocarcinoma patients that expressed the five-gene in vitro lung carcinogenesis model (FILM) signature was significantly poorer than that of patients who did not. No survival differences were observed between SCCs predicted to express or lack FILM signature. Moreover, all stages (P < 0.001, HR = 1.95) or stage-I (P = 0.001, HR = 2.6) adenocarcinoma patients predicted to be at high risk by FILM transcript exhibited significantly worse survival than patients at low risk. Furthermore, the corresponding protein signature was associated with poor survival (all stages, P < 0.001, HR = 3.6; stage-I, P < 0.001, HR = 3.5; stage-IB, P < 0.001, HR = 4.6) and mortality risk (all stages, P = 0.001, HR = 4.0; stage-I, P = 0.01, HR = 3.4; stage-IB, P < 0.001, HR = 7.2) in lung adenocarcinoma patients. Conclusions: Our findings highlight a gene and corresponding protein signature with effective capacity for identification of stage-I lung adenocarcinoma patients with poor prognosis that are likely to benefit from adjuvant therapy. Clin Cancer Res; 17(6); 1490–501. ©2010 AACR.

Knockout of the Tumor Suppressor Gene Gprc5a in Mice Leads to NF-κB Activation in Airway Epithelium and Promotes Lung Inflammation and Tumorigenesis

Mouse models can be useful for increasing the understanding of lung tumorigenesis and assessing the potential of chemopreventive agents. We explored the role of inflammation in lung tumor development in mice with knockout of the tumor suppressor Gprc5a. Examination of normal lung tissue and tumors from 51 Gprc5a+/+ (adenoma incidence, 9.8%; adenocarcinoma, 0%) and 38 Gprc5a−/− mice (adenoma, 63%; adenocarcinoma, 21%) revealed macrophage infiltration into lungs of 45% of the Gprc5a−/− mice and 8% of Gprc5a+/+ mice and the direct association of macrophages with 42% of adenomas and 88% of adenocarcinomas in the knockout mice. Gprc5a−/− mouse lungs contained higher constitutive levels of proinflammatory cytokines and chemokines and were more sensitive than lungs of Gprc5a+/+ mice to stimulation of NF-κB activation by lipopolysaccharide in vivo. Studies with epithelial cells cultured from tracheas of Gprc5a−/− and Gprc5a+/+ mice revealed that Gprc5a loss is associated with increased cell proliferation, resistance to cell death in suspension, and increased basal, tumor necrosis factor α–induced, and lipopolysaccharide-induced NF-κB activation, which were reversed partially in Gprc5a−/− adenocarcinoma cells by reexpression of Gprc5a. Compared with Gprc5a+/+ cells, the Gprc5a−/− cells produced higher levels of chemokines and cytokines and their conditioned medium induced more extensive macrophage migration. Silencing Gprc5a and the p65 subunit of NF-κB in Gprc5a+/+ and Gprc5a−/− cells, respectively, reversed these effects. Thus, Gprc5a loss enhances NF-κB activation in lung epithelial cells, leading to increased autocrine and paracrine interactions, cell autonomy, and enhanced inflammation, which may synergize in the creation of a tumor-promoting microenvironment. Cancer Prev Res; 3(4); 424–37. ©2010 AACR.

Pulmonary adenocarcinoma: a renewed entity in 2011.

Lung cancer, of which non‐small‐cell lung cancer comprises the majority, is the leading cause of cancer‐related deaths in the United States and worldwide. Lung adenocarcinomas are a major subtype of non‐small‐cell lung cancers, are increasing in incidence globally in both males and females and in smokers and non‐smokers, and are the cause for almost 50% of deaths attributable to lung cancer. Lung adenocarcinoma is a tumour with complex biology that we have recently started to understand with the advent of various histological, transcriptomic, genomic and proteomic technologies. However, the histological and molecular pathogenesis of this malignancy is still largely unknown. This review will describe advances in the molecular pathology of lung adenocarcinoma with emphasis on genomics and DNA alterations of this disease. Moreover, the review will discuss recognized lung adenocarcinoma preneoplastic lesions and current concepts of the early pathogenesis and progression of the disease. We will also portray the field cancerization phenomenon and lineage‐specific oncogene expression pattern in lung cancer and how both remerging concepts can be exploited to increase our understanding of lung adenocarcinoma pathogenesis for subsequent development of biomarkers for early detection of adenocarcinomas and possibly personalized prevention.