Genetic alterations associated with prostate cancer progression

Prostate cancer is the second most commonly diagnosed cancer among men in the United States. In this study, evidence is presented to support the hypothesis that specific chromosomal aberrations (involving one or more chromosomal regions) are associated with prostate cancer progression from organ-confined to locally advanced tumors and that some aberrations seen in high frequency in metastatic tumors may also be present in a subset of primary tumors. To determine the appropriate approach to address this hypothesis, I have established a modified CGH protocol by microdissection and DOP-PCR for use in detecting chromosomal changes in clinical prostate tumor specimens that is more sensitive and accurate than conventional CGH methods. I have successfully performed the improved CGH protocol to screen for genetic changes of 24 organ confined (pT2) and 21 locally advanced (pT3b) clinical prostate cancer specimens without metastases (N0M0). Comparisons of tumors by stage or Gleason scores following contingency table analysis showed that seven regions of the genome differed significantly between pT2 and pT3b tumors or between low and high Gleason tumors suggesting that these regions may be important in local prostate cancer progression. These included losses on 6p21–25, 6q24–27, 8p, 10q25–26, 15q22–26, and 18cen–q12 as well as gain of 3p13–q13. Multivariate analyses showed that loss of 8p (step1) and loss of 6q25–26 (or 6p21–25 or 10q25–26) (step 2) were predictive of pathologic stage or Gleason groups with 80% accuracy. Additional 5–7 steps in the multivariate model increased the predictive value to 91–95%. Comparison of the CGH data from the primary prostate tumors of this study with those obtained from published literature on metastases and recurrent tumors showed that the clinically more aggressive stage pT3b tumors shared more abnormalities in high frequency with metastases and recurrent tumors than less aggressive stage pT2 tumors. Furthermore, loss of 11cen–q22 was shared only between the primary tumors and metastases while gain of Xcen–q13 and loss of 18cen–q12 were in common between primary and recurrent tumors. These analyses suggest that the multistage model of prostate cancer progression is not linear and that some early primary tumors may be predisposed to metastasize or evolve into recurrent tumors due to the presence of specific genetic alterations. ^

Cutaneous malignant melanoma: A study of skin cancer in the United States from 1973--1997

Cutaneous malignant melanoma (CMM) is the cancer of the melanocytes, the cells that produce the pigment melanin, and is an aggressive skin cancer that is most prevalent in the white population. Although most cases of malignant melanoma are white, black and other non-white populations also develop this disease. However, the etiologic factors involved in the development of melanoma in these lower-risk populations are not well known. Generally, survival rates of malignant melanoma have been found to be lower in blacks than for whites with similar stage of disease at diagnosis. ^ This study presents an analysis of the differences in survival between black and white cases with malignant melanoma of the skin as the only or first primary cancer, found in the National Cancer Institute Surveillance, Epidemiology and End Results (SEER) cancer registry from 1973 to 1997. A total of 54,193 cases of CMM were diagnosed in black and white patients between 1973 and 1997. Black patients tended to be older, with a mean age of 64.46 years, compared to 53.14 years for white patients. Eighty-nine percent of patients were diagnosed with CMM as the only cancer. (Abstract shortened by UMI.)^

Role of the transcription factor activator protein-2alpha in prostate carcinogenesis

Activator protein 2α (AP-2) is a transcription factor known to play a crucial role in the progression of malignant melanoma, colorectal carcinoma, and breast cancer. Several AP-2 target genes are known to be deregulated in prostate cancer, therefore, we hypothesize that loss AP-2 expression plays a causal role in prostate carcinogenesis. Immunofluorescent staining for AP-2 of 30 radical prostatectomy specimens demonstrated that while AP-2 was highly expressed in normal prostate epithelium, its expression was lost in most cases of high grade prostatic intraepithelial neoplasia (PIN), and all cases of prostate cancer studied. Additional analyses demonstrated that AP-2 was associated with normal luminal differentiation and it was not expressed in the basal cell layer. In cell lines, AP-2 was strongly expressed in immortalized normal prostate epithelial cells, whereas low expression was observed in the LNCaP, LNCaP-LN3, and PC3M-LN4 prostate cancer cell lines. Transfection of the highly tumorigenic and metastatic cell line PC3M-LN4 with the AP-2 gene significantly decreased tumor growth in the prostate of nude mice (p = 0.032) and inhibited metastases to the lymph nodes. Moreover, transfection of the low tumorigenic, low metastatic cell line LNCaP-LN3 with full length AP-2; resulted in complete inhibition of tumor incidence in the AP-2 transfectants (0/19) vs. neo control (10/16). A potential mechanism for this loss of tumorigenicity was the modulation of gene expression in prostate cancer cells that mimicked the normal phenotype. Analysis of differential expression between neo control- and AP-2-transfected cells in vitro and in tumors demonstrated low VEGF expression in AP-2 transfectants. We further demonstrated that AP-2 acted as a transcriptional repressor of the VEGF promoter by binding to a GC-rich region located between −88 and −66. This region contains an AP-2 consensus element overlapping two Sp1 consensus elements. We found that Sp3 and AP-2 bound to this region in a mutually exclusive manner to promote activation or repression. Increased VEGF expression has been observed in high grade PIN and in prostate cancer. Here we provide evidence that this early molecular change could be a result of loss of AP-2 expression in the prostatic epithelium. ^

Loss of activator protein-2alpha results in overexpression of the thrombin receptor and correlates with the malignant phenotype of human melanoma

Increasing evidence demonstrates that the thrombin receptor (protease activated receptor-1, PAR-1) plays a major role in tumor invasion and contributes to the metastatic phenotype of human melanoma. We demonstrate that the metastatic potential of human melanoma cells correlates with overexpression of PAR-1. The promoter of the PAR-1 gene contains multiple putative AP-2 and Sp1 consensus elements. We provide evidence that an inverse correlation exists between the expression of AP-2 and the expression of PAR-1 in human melanoma cells. Re-expression of AP-2 in WM266-4 melanoma cells (AP-2 negative) resulted in decreased mRNA and protein expression of PAR-1 and significantly reduced the tumor potential in nude mice. ChIP analysis of the PAR-1 promoter regions bp −365 to −329 (complex 1) and bp −206 to −180 (complex 2) demonstrates that in metastatic cells Sp1 is predominantly binding to the PAR-1 promoter, while in nonmetastatic cells AP-2 is bound. In vitro analysis of complex 1 demonstrates that AP-2 and Sp1 bind to this region in a mutually exclusive manner. Transfection experiments with full-length and progressive deletions of the PAR-1 promoter luciferase constructs demonstrated that metastatic cells had increased promoter activity compared to low and nonmetastatic melanoma cells. Our data shows that exogenous AP-2 expression decreased promoter activity, while transient expression of Sp1 further activated expression of the reporter gene. Mutational analysis of complex 1 within PAR-1 luciferase constructs further demonstrates that the regulation of PAR-1 is mediated through interactions with AP-2 and Sp1. Moreover, loss of AP-2 in metastatic cells alters the AP-2 to Sp1 ratio and DNA-binding activity resulting in overexpression of PAR-1. In addition, we evaluated the expression of AP-2 and PAR-1 utilizing a tissue microarray of 93 melanocytic lesions spanning from benign nevi to melanoma metastasis. We report loss of AP-2 expression in malignant tumors compared to benign tissue while PAR-1 was expressed more often in metastatic melanoma cells than in benign melanocytes. We propose that loss of AP-2 results in increased expression of PAR-1, which in turn results in upregulation of gene products that contribute to the metastatic phenotype of melanoma. ^

Clinical, microbiological, and immunological markers of gingival and periodontal disease induction and progression

The potential impact of periodontal disease, a suspected risk factor for systemic diseases, presents challenges for health promotion and disease prevention strategies. This study examined clinical, microbiological, and immunological factors in a disease model to identify potential biomarkers that may be useful in predicting the onset and severity of both inflammatory and destructive periodontal disease. This project used an historical cohort design based on data obtained from 47 adult, female nonhuman primates followed over a 6-year period for 5 unique projects where the ligature-induced model of periodontitis was utilized. Standardization of protocols for sample collection allowed for comparison over time. Bleeding and pocket depth measures were selected as the dependent variables of relevance to humans based upon the literature and historical observations. Exposure variables included supragingival plaque, attachment level, total bacteria, black-pigmented bacteria, Gram-negative and Gram-positive bacteria, total IgG and IgA in crevicular fluid, specific IgG antibody in both crevicular fluid and serum, and IgG antibody to four select pathogenic microorganisms. Three approaches were used to analyze the data from this study. The first approach tested for differences in the means of the response variables within the group and among longitudinal observations within the group at each time point. The second approach examined the relationship among the clinical, microbiological, and immunological variables using correlation coefficients and stratified analyses. Multivariable models using GEE for repeated measures were produced as a predictive description of the induction and progression of gingivitis and periodontal disease. The multivariable models for bleeding (gingivitis) include supragingival plaque, total bacteria and total IgG while the second also contains supragingival plaque, Gram-positive bacteria, and total IgG. Two multivariable models emerged for periodontal disease. One multivariable model contains plaque, total bacteria, total IgG and attachment level. The second model includes black-pigmented bacteria, total bacteria, antibody to Campylobacter rectus, and attachment level. Utilization of the nonhuman primate model to prospectively examine causal hypotheses can provide a focus for human research on the mechanisms of progression from health to gingivitis to periodontitis. Ultimately, causal theories can guide strategies to prevent disease initiation and reduce disease severity. ^

Factors influencing time to HIV disease progression in Romanian children

The purpose of this research was two-fold; to investigate the effect of institutionalization on death and CD4 decline in a cohort of 325 HIV-infected Romanian children, and to investigate the effect of disclosure of the child's own HIV status in this cohort. All children were treated with Kaletra-based highly active antiretroviral therapy, and were followed from November, 2001 through October, 2004. The mean age of the children included in the cohort is 13. The study found that children in biological families were more likely to experience disease progression through either death or CD4 decline than children in institutions (p=0.04). The family home-style institution may prove to be a replicable model for the safe and appropriate care of HIV-infected orphaned and abandoned children and teens. The study also found that children who do not know their own HIV infection status were more likely to experience disease progression through either death or CD4 decline than children who know their HIV diagnosis (p=0.03). This evidence suggests that, in the context of highly active anti retroviral therapy, knowledge of one's own HIV infection status is associated with delayed HIV disease progression. ^

Ligand-directed targeting of malignant gliomas

The dramatic poor survival of patients diagnosed with glioblastoma multiforme (GBM) is a reflection of the struggles that accompany traditional treatments. Thus, the development of molecular-based targeted therapies represents new windows for intervention. In this study, we hypothesized that we could select peptide-ligands that selectively target GBM based on the idea that the glioma microenvironment may induce or modify the expression of cell surface receptors that could be accessed by circulating peptides. To select the peptides we employed two distinct in vivo screenings. First, a random phage-displayed peptide library was injected into mice bearing intracranial tumors. Phage that bound to tumor were recovered and sequenced. We found that the tumor-derived phage CLSYKGRC, CNKVSTKC and CQSSREKC were recovered with the highest frequencies and used for subsequent targeting experiments. Second, the phage peptide library was injected into mice without tumors and phage were recovered from brain and sequenced. A phage-displayed peptide (CRTIGPSVC) with homology to transferrin (Tf) was selected and injected into brain tumor-bearing mice. Results showed that after 6 hours of circulation, the CLSYKGRC, CNKVSTKC and CQSSREKC-phage selectively targeted GBM vasculature. In contrast, Tf-like phage accumulated outside the tumor blood vessels in the cytoplasm of cells located within GBM, suggesting it was internalized in vivo. However, after short periods of circulation this phage was restricted to the tumor vasculature. Importantly, none of the selected phage targeted normal brain cells in animals bearing intracranial tumors. An affinity column coupled to the CNKVSTKC zpeptide was used to identify receptors from GBM. Using mass-spectrometry Vimentin, a marker of glial malignancy, was identified as a potential receptor. Other studies showed that the Tf-like phage bound selectively to Apo-Tf (iron free), with no binding to Holo-Tf (iron loaded) or to Tf receptor (TfR). However, the binding of Tf-like phage to glioma cells that express TfR increased in the presence of Apo-Tf. Thus, the Tf-like phage could indirectly target TfR using the endogenous Tf pathway. We propose that the novel peptides identified in this study could be conjugated to therapeutic or imaging agents for use GBM. ^

BLyS/BAFF-R signaling pathway in human aggressive non Hodgkin's lymphoma B cell and normal peripheral blood B lymphocytes

B-lymphocyte stimulator (BLyS also called BAFF), is a potent cell survival factor expressed in many hematopoietic cells. BLyS levels are elevated in the serum of non-Hodgkin lymphoma (NHL) patients, and have been reported to be associated with disease progression, and prognosis. To understand the mechanisms involved in BLyS gene expression and regulation, we examined expression, function, and regulation of the BLyS gene in B cell non-Hodgkin's lymphoma (NHL-B) cells. BLyS is constitutively expressed in aggressive NHL-B cells including large B cell lymphoma (LBCL) and mantle cell lymphoma (MCL) contributing to survival and proliferation of malignant B cells. Two important transcription factors, NF-κB and NFAT, were found to be involved in regulating BLyS expression through at least one NF-κB and two NFAT binding sites in the BLyS promoter. Further study indicates that the constitutive activation of NF-κB and BLyS in NHL-B cells forms a positive feedback loop contributing to cell survival and proliferation. In order to further investigate BLyS signaling pathway, we studied the function of BAFF-R, a major BLyS receptor, on B cells survival and proliferation. Initial study revealed that BAFF-R was also found in the nucleus, in addition to its presence on plasma membrane of B cells. Nuclear presentation of BAFF-R can be increased by anti-IgM and soluble BLyS treatment in normal peripheral B lymphocytes. Inhibition of BLyS expression decreases nuclear BAFF-R level in LBCL cells. Furthermore, we showed that BAFF-R translocated to nucleus through the classic karyopherin pathway. A candidate nuclear localization sequence (NLS) was identified in the BAFF-R protein sequence and mutation of this putative NLS can block BAFF-R entering nucleus and LBCL cell proliferation. Further study showed that BAFF-R co-localized with NF-κB family member, c-rel in the nucleus. We also found BAFF-R mediated transcriptional activity, which could be increased by c-rel. We also found that nuclear BAFF-R could bind to the NF-κB binding site on the promoters of NF-κB target genes such as BLyS, CD154, Bcl-xL, Bfl-1/A1 and IL-8. These findings indicate that BAFF-R may also promote survival and proliferation of normal B cells and NHL-B cells by directly functioning as a transcriptional co-factor with NF-κB family member. ^

A coalescent analysis for modeling the mutation process in colorectal cancer

Colorectal cancer is the forth most common diagnosed cancer in the United States. Every year about a hundred forty-seven thousand people will be diagnosed with colorectal cancer and fifty-six thousand people lose their lives due to this disease. Most of the hereditary nonpolyposis colorectal cancer (HNPCC) and 12% of the sporadic colorectal cancer show microsatellite instability. Colorectal cancer is a multistep progressive disease. It starts from a mutation in a normal colorectal cell and grows into a clone of cells that further accumulates mutations and finally develops into a malignant tumor. In terms of molecular evolution, the process of colorectal tumor progression represents the acquisition of sequential mutations. ^ Clinical studies use biomarkers such as microsatellite or single nucleotide polymorphisms (SNPs) to study mutation frequencies in colorectal cancer. Microsatellite data obtained from single genome equivalent PCR or small pool PCR can be used to infer tumor progression. Since tumor progression is similar to population evolution, we used an approach known as coalescent, which is well established in population genetics, to analyze this type of data. Coalescent theory has been known to infer the sample's evolutionary path through the analysis of microsatellite data. ^ The simulation results indicate that the constant population size pattern and the rapid tumor growth pattern have different genetic polymorphic patterns. The simulation results were compared with experimental data collected from HNPCC patients. The preliminary result shows the mutation rate in 6 HNPCC patients range from 0.001 to 0.01. The patients' polymorphic patterns are similar to the constant population size pattern which implies the tumor progression is through multilineage persistence instead of clonal sequential evolution. The results should be further verified using a larger dataset. ^

14-3-3 zeta overexpression in early stage breast disease and tumor progression

Recent progress in diagnostic tools allows many breast cancers to be detected at an early pre-invasive stage. Thus, a better understanding of the molecular basis of early breast cancer progression is essential. 14-3-3 is a family of highly conserved and ubiquitously expressed proteins that are expressed in all eukaryotic organisms. In mammals there are seven isoforms, which bind to phosphor-serine/threonine residues regulating essential cellular processes such as signal transduction, cell cycle progression, and apoptosis. Our laboratory has discovered that a particular 14-3-3 family member, Zeta, is overexpressed in over 40% of breast tumor tissues. Furthermore, I examined the stage of breast disease in which 14-3-3ζ overexpression occurs and found that increased expression of 14-3-3ζ begins at the stage of atypical ductal hyperplasia, a very early stage of breast disease that confers increased risk for progress toward breast cancer. To determine whether 14-3-3ζ overexpression is a decisive early event in breast cancer, I overexpressed 14-3-3ζ in MCF10A cells, a non-transformed mammary epithelial cell (MEC) line and examined its impact on acini formation in a three dimensional (3D) culture model which simulates a basic unit of structure in the mammary gland. I discovered that 14-3-3ζ overexpression severely disrupted the acini architecture resulting in the disruption of polarity and luminal filling. Both are critical morphological events in the pre-neoplastic breast disease. This thesis focuses on the molecular mechanism of luminal filling. Proper lumen formation is a result of anoikis, a specific type apoptosis of cells not attached to the basement membrane. I found that 14-3-3ζ overexpression conferred a resistance to anoikis. Additionally, 14-3-3ζ overexpression in MCF10A cells and in MECs from 14-3-3ζ transgenic mice reduced expression of p53, which is known to mediate anoikis. Mechanistically, 14-3-3ζ induced hyperactivation of the PI3K/Akt pathway which led to phosphorylation and translocation of the MDM2 to the nucleus resulting in increased p53 degradation. Ectopic expression of p53 restored luminal apoptosis in 14-3-3ζ overexpressing MCF10A acini in 3D cultures. These data suggest that 14-3-3ζ overexpression is a critical event in early breast disease and down-regulation of p53 is one of the mechanisms by which 14-3-3ζ alters MEC acini structure and may increase the risk of progression to breast cancer. ^

Insulin-like growth factor binding protein 2 (IGFBP2) promotes glioma development and progression

Overexpression of insulin-like growth factor binding protein 2 (IGFBP2) is associated with progression and poor survival in many types of human cancer (such as prostate, ovarian, adrenocortical, breast, colorectal carcinomas, leukemia, and high-grade gliomas). We therefore hypothesize that IGFBP2 is a key regulator of tumor progression. We tested our hypothesis in gliomas using the somatic gene transfer RCAS-tva mouse model system, which permits the introduction of specific genes into specific, cell lineages, in this case glial cells (RCAS: Replication competent avian sarcomavirus, tv-a: avian RCAS virus receptor). Mice are transgenic and harbor the tv-a receptor under the control of a glial-specific promoter and study genes are cloned into the RCAS vector for post-natal intracranial delivery. For these experiments, the study genes were IGFBP2, platelet-derived growth factor B (PDGFB), K-Ras, Akt, and IIp45 (invasion inhibitory protein 45 kDa; known to bind and block IGFBP2 activity), which were delivered separately and in combination. Our results show that PDGFB signaling leads exclusively to the formation of low-grade (WHO grade II) oligodendrogliomas. PDGFB delivered in combination with IGFBP2 results in the formation of anaplastic oligodendrogliomas (WHO grade III), which are characterized by increased cellularity, vascular proliferation, small regions of necrosis, increased mitotic activity, and increased activation of the Akt pathway. IIp45 injected in combination with PDGFB and IGFBP2 ablates IGFBP2-induced tumor progression, which results in formation of low-grade oligodendrogliomas, and an overall reduction in tumor incidence. K-Ras expression was required to form astrocytomas with either IGFBP2 or Akt, indicating the activation of two separate pathways is necessary for gliomagenesis. In ex vivo experiments, blockade of Akt by an inhibitor led to decreased viability of cells co-expressing IGFBP2 versus PDGFB expression alone. This study provides definitive evidence, for the first time, that: (1) IGFBP2 plays a role in activation of the Akt pathway, (2) IGFBP2 collaborates with K-Ras or PDGFB in the development and progression of two major types of glioma, and (3) IGFBP2-induced tumor progression can be ablated by IIp45 or by specific inhibition of the Akt pathway. ^

Micro RNA expression alterations in patients with glioblastoma

Glioblastoma, also known as glioblastoma multiform or GBM, is the most common and most malignant primary brain tumor. The clinical history of patients with glioblastoma is short, usually less than 3 months in more than 50% of cases after diagnosis. Currently, the methods of glioblastoma treatment are chemotherapy, radiotherapy and surgery. Even with the more effective treatment options, patients with glioblastoma most likely have a median survival time of 10 to 12 months. It is necessary to seek other treatment methods, including gene-targeted treatment. The success of gene-targeted treatment depends critically on the knowledge of genes that may be the cause of, or contribute to disease. To establish a correlate between glioblastoma survival timeline and micro RNA expression alteration, a study of 91 glioblastoma patients was conducted at the University of Texas M. D. Anderson Cancer Center. These 91 glioblastoma patients were newly diagnosed from 2002 to 2007. Statistical analysis was conducted to test the association of miRNA expression alteration between long-term survival and short-term survival glioblastoma. The completion of this proposed study will provide a better understanding of the regulatory role of miRNA in glioblastoma progression.^

The diagnostic value of a 12-lead electrocardiogram in patients with cardiac tamponade due to a malignant pericardial effusion

Background. Cardiac tamponade can occur when a large amount of fluid, gas, singly or in combination, accumulating within the pericardium, compresses the heart causing circulatory compromise. Although previous investigators have found the 12-lead ECG to have a poor predictive value in diagnosing cardiac tamponade, very few studies have evaluated it as a follow up tool for ruling in or ruling out tamponade in patients with previously diagnosed malignant pericardial effusions. ^ Methods. 127 patients with malignant pericardial effusions at the MD Anderson Cancer Center were included in this retrospective study. While 83 of these patients had a cardiac tamponade diagnosed by echocardiographic criteria (Gold standard), 44 did not. We computed the sensitivity (Se), specificity (Sp), positive (PPV) and negative predictive values (NPV) for individual and combinations of ECG abnormalities. Individual ECG abnormalities were also entered singly into a univariate logistic regression model to predict tamponade. ^ Results. For patients with effusions of all sizes, electrical alternans had a Se, Sp, PPV and NPV of 22.61%, 97.61%, 95% and 39.25% respectively. These parameters for low voltage complexes were 55.95%, 74.44%, 81.03%, 46.37% respectively. The presence of all three ECG abnormalities had a Se = 8.33%, Sp = 100%, PPV = 100% and NPV = 35.83% while the presence of at least one of the three ECG abnormalities had a Se = 89.28%, Sp = 46.51%, PPV = 76.53%, NPV = 68.96%. For patients with effusions of all sizes electrical alternans had an OR of 12.28 (1.58–95.17, p = 0.016), while the presence of at least one ECG abnormality had an OR of 7.25 (2.9–18.1, p = 0.000) in predicting tamponade. ^ Conclusions. Although individual ECG abnormalities had low sensitivities, specificities, NPVs and PPVs with the exception of electrical alternans, the presence of at least one of the three ECG abnormalities had a high sensitivity in diagnosing cardiac tamponade. This could point to its potential use as a screening test with a correspondingly high NPV to rule out a diagnosis of tamponade in patients with malignant pericardial effusions. This could save expensive echocardiographic assessments in patients with previously diagnosed pericardial effusions. ^

EphA2 expression is regulated by EGFR and KRAS and promotes non-small cell lung cancer progression

Lung cancer is the leading cause of cancer deaths worldwide. The development of improved systemic therapy is needed for the most common form of the disease, non-small cell lung cancer (NSCLC). This will depend on the identification of valid molecular targets. Recent studies point to the receptor tyrosine kinase EphA2 as a novel therapeutic target. Overexpression of EphA2 has been demonstrated in a number of epithelial cancers, and its expression has been associated with more severe disease. Regulation of EphA2 in cancer is poorly understood. Recently, regulation of EphA2 by EGFR and KRAS has been reported in a number of in vitro models, but no examination of this relationship has been undertaken in patient tumors. Because of the established importance of EGFR and KRAS in NSCLC, we have investigated the relationship between these mutations and EphA2 in NSCLC patient tissues and cell lines. The significance of Epha2 expression was further examined by testing for correlation with survival, metastases, histology, and smoking status in patient tissues, and tumor cell proliferation and migration in vitro. EphA2 expression was analyzed in by immunohistochemistry in tissue microarray (TMA) format utilizing surgically resected lung cancer specimens. EGFR and KRAS mutation status was determined for the majority of specimens. EphA2 expression was detected in >90% of NSCLC tumors. High EphA2 expression was associated with decreased time to recurrence and metastases, and predicted poorer progression free and overall survival. Expression of EphA2 was positively correlated with activated EGFR and with KRAS mutation. Expression of EphA2 was also positively correlated with a history of smoking. There was no association between gender or histology and EphA2 expression. In H322 cells, activation of EGFR or KRAS resulted in an increase in EphA2 protein expression. Downregulation of EphA2 resulted in decreased proliferation in a clonal growth assay, and inhibited migration in a wound healing assay, in a panel of cell lines. The decrease in proliferation correlated with a transient decrease in the levels of phospho-ERK, a downstream effector of EGFR and KRAS. Based on these data, the potential of EphA2 as a therapeutic target for NSCLC should be further investigated. ^