Lee Goodglick

Global Levels of Histone Modifications Predict Prognosis in Different Cancers

Cancer cells exhibit alterations in histone modification patterns at individual genes and globally at the level of single nuclei in individual cells. We demonstrated previously that lower global/cellular levels of histone H3 lysine 4 dimethylation (H3K4me2) and H3K18 acetylation (ac) predict a higher risk of prostate cancer recurrence. Here we show that the cellular levels of both H3K4me2 and H3K18ac also predict clinical outcome in both lung and kidney cancer patients, with lower levels predicting significantly poorer survival probabilities in both cancer groups. We also show that lower cellular levels of H3K9me2, a modification associated with both gene activity and repression, is also prognostic of poorer outcome for individuals with either prostate or kidney cancers. The predictive power of these histone modifications was independent of tissue-specific clinicopathological variables, the proliferation marker Ki-67, or a p53 tumor suppressor mutation. Chromatin immunoprecipitation experiments indicated that the lower cellular levels of histone modifications in more aggressive cancer cell lines correlated with lower levels of modifications at DNA repetitive elements but not with gene promoters across the genome. Our results suggest that lower global levels of histone modifications are predictive of a more aggressive cancer phenotype, revealing a surprising commonality in prognostic epigenetic patterns of adenocarcinomas of different tissue origins.

Cyclooxygenase-2–Dependent Regulation of E-Cadherin: Prostaglandin E2 Induces Transcriptional Repressors ZEB1 and Snail in Non–Small Cell Lung Cancer

Elevated tumor cyclooxygenase-2 (COX-2) expression is associated with tumor invasion, metastasis, and poor prognosis in non-small cell lung cancer (NSCLC). Here, we report that COX-2-dependent pathways contribute to the modulation of E-cadherin expression in NSCLC. First, whereas genetically modified COX-2-sense (COX-2-S) NSCLC cells expressed low E-cadherin and showed diminished capacity for cellular aggregation, genetic or pharmacologic inhibition of tumor COX-2 led to increased E-cadherin expression and resulted in augmented homotypic cellular aggregation among NSCLC cells in vitro. An inverse relationship between COX-2 and E-cadherin was shown in situ by double immunohistochemical staining of human lung adenocarcinoma tissue sections. Second, treatment of NSCLC cells with exogenous prostaglandin E(2) (PGE(2)) significantly decreased the expression of E-cadherin, whereas treatment of COX-2-S cells with celecoxib (1 mumol/L) led to increased E-cadherin expression. Third, the transcriptional suppressors of E-cadherin, ZEB1 and Snail, were up-regulated in COX-2-S cells or PGE(2)-treated NSCLC cells but decreased in COX-2-antisense cells. PGE(2) exposure led to enhanced ZEB1 and Snail binding at the chromatin level as determined by chromatin immunoprecipitation assays. Small interfering RNA-mediated knockdown of ZEB1 or Snail interrupted the capacity of PGE(2) to down-regulate E-cadherin. Fourth, an inverse relationship between E-cadherin and ZEB1 and a direct relationship between COX-2 and ZEB1 were shown by immunohistochemical staining of human lung adenocarcinoma tissue sections. These findings indicate that PGE(2), in autocrine or paracrine fashion, modulates transcriptional repressors of E-cadherin and thereby regulates COX-2-dependent E-cadherin expression in NSCLC. Thus, blocking PGE(2) production or activity may contribute to both prevention and treatment of NSCLC.

Inhibition of the Raf–MEK1/2–ERK1/2 Signaling Pathway, Bcl-xL Down-Regulation, and Chemosensitization of Non-Hodgkin’s Lymphoma B Cells by Rituximab

Rituximab (Rituxan, IDEC-C2B8) has been shown to sensitize non-Hodgkin’s lymphoma (NHL) cell lines to chemotherapeutic drug-induced apoptosis. Rituximab treatment of Bcl-2–deficient Ramos cells and Bcl-2–expressing Daudi cells selectively decreases Bcl-xL expression and sensitizes the cells to paclitaxel-induced apoptosis. This study delineates the signaling pathway involved in rituximab-mediated Bcl-xL down-regulation in Ramos and Daudi NHL B cells. We hypothesized that rituximab may interfere with the extracellular signal-regulated kinase (ERK) 1/2 pathway, leading to decreased Bcl-xL expression. Rituximab (20 μg/mL) inhibited the kinase activity of mitogen-activated protein kinase kinase (MEK) 1/2 and reduced the phosphorylation of the components of the ERK1/2 pathway (Raf-1, MEK1/2, and ERK1/2) and decreased activator protein-1 DNA binding activity and Bcl-xL gene expression. These events occurred with similar kinetics and were observed 3 to 6 hours after rituximab treatment. Rituximab-mediated effects were corroborated by using specific inhibitors of the ERK1/2 pathway, which also reduced Bcl-xL levels and sensitized the NHL B cells to paclitaxel-induced apoptosis. Previous findings implicated a negative regulatory role of the Raf-1 kinase inhibitor protein (RKIP) on the ERK1/2 pathway. Rituximab treatment of NHL B cells significantly up-regulated RKIP expression, thus interrupting the ERK1/2 signaling pathway through the physical association between Raf-1 and RKIP, which was concomitant with Bcl-xL down-regulation. These novel findings reveal a signaling pathway triggered by rituximab, whereby rituximab-mediated up-regulation of RKIP adversely regulates the activity of the ERK1/2 pathway, Bcl-xL expression, and subsequent chemosensitization of drug-refractory NHL B cells. The significance of these findings is discussed.

Estrogen receptor signaling pathways in human non-small cell lung cancer

Lung cancer is the most common cause of cancer mortality in male and female patients in the US. The etiology of non-small cell lung cancer (NSCLC) is not fully defined, but new data suggest that estrogens and growth factors promote tumor progression. In this work, we confirm that estrogen receptors (ER), both ERalpha and ERbeta, occur in significant proportions of archival NSCLC specimens from the clinic, with receptor expression in tumor cell nuclei and in extranuclear sites. Further, ERalpha in tumor nuclei was present in activated forms as assessed by detection of ER phosphorylation at serines-118 and -167, residues commonly modulated by growth factor receptor as well as steroid signaling. In experiments using small interfering RNA (siRNA) constructs, we find that suppressing expression of either ERalpha or ERbeta elicits a significant reduction in NSCLC cell proliferation in vitro. Estrogen signaling in NSCLC cells may also include steroid receptor coactivators (SRC), as SRC-3 and MNAR/PELP1 are both expressed in several lung cell lines, and both EGF and estradiol elicit serine phosphorylation of SRC-3 in vitro. EGFR and ER also cooperate in promoting early activation of p42/p44 MAP kinase in NSCLC cells. To assess new strategies to block NSCLC growth, we used Faslodex alone and with erlotinib, an EGFR kinase inhibitor. The drug tandem elicited enhanced blockade of the growth of NSCLC xenografts in vivo, and antitumor activity exceeded that of either agent given alone. The potential for use of antiestrogens alone and with growth factor receptor antagonists is now being pursued further in clinical trials.

Raf-1 Kinase Inhibitor Protein: Structure, Function, Regulation of Cell Signaling, and Pivotal Role in Apoptosis

The acquisition of resistance to conventional therapies such as radiation and chemotherapeutic drugs remains the major obstacle in the successful treatment of cancer patients. Tumor cells acquire resistance to apoptotic stimuli and it has been demonstrated that conventional therapies exert their cytotoxic activities primarily by inducing apoptosis in the cells. Resistance to radiation and chemotherapeutic drugs has led to the development of immunotherapy and gene therapy approaches with the intent of overcoming resistance to drugs and radiation as well as enhancing the specificity to eliminate tumor cells. However, cytotoxic lymphocytes primarily kill by apoptosis and, therefore, drug-resistant tumor cells may also be cross-resistant to immunotherapy. To evade apoptosis, tumor cells have adopted various mechanisms that interfere with the apoptotic signaling pathways and promote constitutive activation of cellular proliferation and survival pathways. Thus, modifications of the antiapoptotic genes in cancer cells are warranted for the effectiveness of conventional therapies as well as novel immunotherapeutic approaches. Such modifications will avert the resistant phenotype of the tumor cells and will render them susceptible to apoptosis. Current studies, both in vitro and preclinically in vivo, have been aimed at the modification and regulation of expression of apoptosis-related gene products and their activities. A novel protein designated Raf-1 kinase inhibitor protein (RKIP) has been partially characterized. RKIP is a member of the phosphatidylethanolamine-binding protein family. RKIP has been shown to disrupt the Raf-1-MEK1/2 [mitogen-activated protein kinase-ERK (extracellular signal-regulated kinase) kinase-1/2]-ERK1/2 and NF-kappaB signaling pathways, via physical interaction with Raf-1-MEK1/2 and NF-kappaB-inducing kinase or transforming growth factor beta-activated kinase-1, respectively, thereby abrogating the survival and antiapoptotic properties of these signaling pathways. In addition, RKIP has been shown to act as a signal modifier that enhances receptor signaling by inhibiting G protein-coupled receptor kinase-2. By regulating cell signaling, growth, and survival through its expression and activity, RKIP is considered to play a pivotal role in cancer, regulating apoptosis induced by drugs or immune-mediated stimuli. Overexpression of RKIP sensitizes tumor cells to chemotherapeutic drug-induced apoptosis. Also, induction of RKIP by drugs or anti-receptor antibodies sensitizes cancer cells to drug-induced apoptosis. In this review, we discuss the discovery, structure, function, and significance of RKIP in cancer.

COX-2-dependent stabilization of survivin in non-small cell lung cancer

Elevated tumor cyclooxygenase 2 (COX‐2) expression is associated with increased angiogenesis, tumor invasion and promotion of tumor cell resistance to apoptosis. The mechanism(s) by which COX‐2 exerts its cytoprotective effects are not completely understood but may be due to an imbalance of pro‐ and anti‐apoptotic gene expression. To analyze COX‐2‐dependent gene expression and apoptosis, we created cell lines constitutively expressing COX‐2 cDNA in sense and antisense orientations. Whereas COX‐2 sense cells have significantly heightened resistance to radiation and drug‐induced apoptosis, COX‐2 antisense cells are highly sensitive to apoptosis induction. We found that the expression of the anti‐apoptotic protein survivin correlated positively with COX‐2 expression. A COX‐2‐dependent modulation of survivin ubiquitination led to its stabilization in COX‐2 overexpressing cells, and this effect was replicated by exogenous PGE2 treatment of parental tumor cells. In contrast to previous studies in other cell types, in nonsmall cell lung cancer cells survivin was expressed in a cell cycle‐independent manner. When established in SCID mice in vivo, COX‐2 antisense‐derived tumors had significantly decreased survivin levels while COX‐2 sense‐derived tumors demonstrated elevated levels compared with controls. In accord with these findings, survivin and COX‐2 were frequently upregulated and co‐expressed in human lung cancers in situ.

Aromatase inhibitors in human lung cancer therapy.

Lung cancer is the most common cancer in the world. It is a highly lethal disease in women and men, and new treatments are urgently needed. Previous studies implicated a role of estrogens and estrogen receptors in lung cancer progression, and this steroidal growth-stimulatory pathway may be promoted by tumor expression and activity of aromatase, an estrogen synthase. We found expression of aromatase transcripts and protein in human non-small cell lung cancer (NSCLC) cells using reverse transcription-PCR and Western immunoblots, respectively. Aromatase staining by immunohistochemistry was detected in 86% of archival NSCLC tumor specimens from the clinic. Further, biological activity of aromatase was determined in NSCLC tumors using radiolabeled substrate assays as well as measure of estradiol product using ELISA. Significant activity of aromatase occurred in human NSCLC tumors, with enhanced levels in tumor cells compared with that in nearby normal cells. Lung tumor aromatase activity was inhibited by anastrozole, an aromatase inhibitor, and treatment of tumor cells in vitro with anastrozole led to significant suppression of tumor cell growth. Similarly, among ovariectomized nude mice with A549 lung tumor xenografts, administration of anastrozole by p.o. gavage for 21 days elicited pronounced inhibition of tumor growth in vivo. These findings show that aromatase is present and biologically active in human NSCLCs and that tumor growth can be down-regulated by specific inhibition of aromatase. This work may lead to development of new treatment options for patients afflicted with NSCLC.

Identification of Five Candidate Lung Cancer Biomarkers by Proteomics Analysis of Conditioned Media of Four Lung Cancer Cell Lines

Detection of lung cancer at an early stage is necessary for successful therapy and improved survival rates. We performed a bottom-up proteomics analysis using a two-dimensional LC-MS/MS strategy on the conditioned media of four lung cancer cell lines of different histological backgrounds (non-small cell lung cancer: H23 (adenocarcinoma), H520 (squamous cell carcinoma), and H460 (large cell carcinoma); small cell lung cancer: H1688) to identify secreted or membrane-bound proteins that could be useful as novel lung cancer biomarkers. Proteomics analysis of the four conditioned media allowed identification of 1,830 different proteins (965, 871, 726, and 847 from H1688, H23, H460, and H520, respectively). All proteins were assigned a subcellular localization, and 38% were classified as extracellular or membrane-bound. We successfully identified the internal control proteins (also detected by ELISA), kallikrein-related peptidases 14 and 11, and IGFBP2. We also identified known or putative lung cancer tumor markers such as squamous cell carcinoma antigen, carcinoembryonic antigen, chromogranin A, creatine kinase BB, progastrin-releasing peptide, neural cell adhesion molecule, and tumor M2-PK. To select the most promising candidates for validation, we performed tissue specificity assays, functional classifications, literature searches for association to cancer, and a comparison of our proteome with the proteome of lung-related diseases and serum. Five novel lung cancer candidates, ADAM-17, osteoprotegerin, pentraxin 3, follistatin, and tumor necrosis factor receptor superfamily member 1A were preliminarily validated in the serum of patients with lung cancer and healthy controls. Our results demonstrate the utility of this cell culture proteomics approach to identify secreted and shed proteins that are potentially useful as serological markers for lung cancer.

Aromatase Expression Predicts Survival in Women with Early-Stage Non-Small Cell Lung Cancer

Estrogen signaling is critical in the progression of tumors that bear estrogen receptors. In most patients with breast cancer, inhibitors that block interactions of estrogen with its receptors or suppress the production of endogenous estrogens are important interventions in the clinic. Recent evidence now suggests that estrogen also contributes to the pathogenesis of non-small cell lung cancer (NSCLC). We used a human lung cancer xenograph model system to analyze the effect of aromatase or estradiol on tumor growth. We further examined the level of protein expression of aromatase in 422 patients with NSCLC using a high-density tissue microarray. Results were confirmed and validated on an independent patient cohort (n = 337). Lower levels of aromatase predicted a greater chance of survival in women 65 years and older. Within this population, the prognostic value of aromatase was greatest in earlier stage lung cancer (stage I/II). In addition, for women with no history of smoking, lower aromatase levels were a strong predictor of survival. Our findings implicate aromatase as an early-stage predictor of survival in some women with NSCLC. We predict that women whose lung cancers have higher levels of aromatase might be good candidates for targeted treatment with aromatase inhibitors.

Higher Levels of GATA3 Predict Better Survival in Women with Breast Cancer

The GATA family members are zinc finger transcription factors involved in cell differentiation and proliferation. GATA3 in particular is necessary for mammary gland maturation, and its loss has been implicated in breast cancer development. Our goal was to validate the ability of GATA3 expression to predict survival in breast cancer patients. Protein expression of GATA3 was analyzed on a high-density tissue microarray consisting of 242 cases of breast cancer. We associated GATA3 expression with patient outcomes and clinicopathologic variables. Expression of GATA3 was significantly increased in breast cancer, in situ lesions, and hyperplastic tissue compared with normal breast tissue. GATA3 expression decreased with increasing tumor grade. Low GATA3 expression was a significant predictor of disease-related death in all patients, as well as in subgroups of estrogen receptor-positive or low-grade patients. In addition, low GATA3 expression correlated with increased tumor size and estrogen and progesterone receptor negativity. GATA3 is an important predictor of disease outcome in breast cancer patients. This finding has been validated in a diverse set of populations. Thus, GATA3 expression has utility as a prognostic indicator in breast cancer.