PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (33)
 

Clipboard (0)
None

Select a Filter Below

Journals
more »
Year of Publication
1.  Lung Cancer Lymph Node Micrometastasis Detection Using RT-PCR – Correlation with Vascular Endothelial Growth Factor (VEGF) expression 
Objectives
Lymph node (LN) staging provides critical information in non-small cell lung cancer (NSCLC) patients. Lymphangiogenesis may be an important contributor to the pathophysiology of lymphatic metastases. We hypothesized that the presence of lymph node micrometastases positively correlates with VEGF-A/C/D and VEGF-receptor-3 (lymphangiogenic factors) expression in lymph nodes.
Methods
Forty NSCLC patients had pre-operative PET-CT and mediastinoscopy. RT-PCR assays for mRNA expression of epithelial markers (CK-7, CEACAM-5 and PLUNC) were performed in selected fluorodeoxyglucose (FDG)-avid lymph nodes. VEGF-A/C/D and VEGF-receptor-3 expression levels were measured in primary tumors and lymph nodes. Wilcoxon rank sum test was run for the association between the RT-PCR epithelial marker levels and VEGF expression levels in the LNs.
Results
RT-PCR for CK-7, CEACAM5 or PLUNC indicated lymph node micrometastatic disease in 19 of 35 patients (54%). There was a high correlation between detection of micrometastases and VEGF-A/C/D or VEGF-receptor-3 expression levels in lymph nodes. Median follow-up was 12.6 months.
Conclusions
RT-PCR analysis of FDG-avid lymph nodes results in up-staging of patients. Micrometastases correlate with the expression of VEGF in lymph nodes in NSCLC patients. This may reflect the role of lymphangiogenesis in promoting metastases.
doi:10.1016/j.jtcvs.2012.12.023
PMCID: PMC3584695  PMID: 23414988
2.  Androgen Receptor Status Is a Prognostic Marker in Non-Basal Triple Negative Breast Cancers and Determines Novel Therapeutic Options 
PLoS ONE  2014;9(2):e88525.
Triple negative breast cancers are a heterogeneous group of tumors characterized by poor patient survival and lack of targeted therapeutics. Androgen receptor has been associated with triple negative breast cancer pathogenesis, but its role in the different subtypes has not been clearly defined. We examined androgen receptor protein expression by immunohistochemical analysis in 678 breast cancers, including 396 triple negative cancers. Fifty matched lymph node metastases were also examined. Association of expression status with clinical (race, survival) and pathological (basal, non-basal subtype, stage, grade) features was also evaluated. In 160 triple negative breast cancers, mRNA microarray expression profiling was performed, and differences according to androgen receptor status were analyzed. In triple negative cancers the percentage of androgen receptor positive cases was lower (24.8% vs 81.6% of non-triple negative cases), especially in African American women (16.7% vs 25.5% of cancers of white women). No significant difference in androgen receptor expression was observed in primary tumors vs matched metastatic lesions. Positive androgen receptor immunoreactivity was inversely correlated with tumor grade (p<0.01) and associated with better overall patient survival (p = 0.032) in the non-basal triple negative cancer group. In the microarray study, expression of three genes (HER4, TNFSF10, CDK6) showed significant deregulation in association with androgen receptor status; eg CDK6, a novel therapeutic target in triple negative cancers, showed significantly higher expression level in androgen receptor negative cases (p<0.01). These findings confirm the prognostic impact of androgen receptor expression in non-basal triple negative breast cancers, and suggest targeting of new androgen receptor-related molecular pathways in patients with these cancers.
doi:10.1371/journal.pone.0088525
PMCID: PMC3914993  PMID: 24505496
3.  Pharmacological blockade of FAK autophosphorylation decreases human glioblastoma tumor growth and synergizes with temozolomide 
Molecular cancer therapeutics  2012;12(2):162-172.
Malignant gliomas are characterized by aggressive tumor growth with a mean survival of 15–18 months and frequently developed resistance to temozolomide. Therefore, strategies that sensitize glioma cells to temozolomide have a high translational impact. We have studied focal adhesion kinase (FAK), a tyrosine kinase and emerging therapeutic target that is known to be highly expressed and activated in glioma. In this report we tested the FAK autophosphorylation inhibitor, Y15 in DBTRG and U87 glioblastoma cells. Y15 significantly decreased viability and clonogenicity in a dose-dependent manner, increased detachment in a dose and time-dependent manner, caused apoptosis and inhibited cell invasion in both cell lines. In addition, Y15 treatment decreased autophosphorylation of FAK in a dose-dependent manner and changed cell morphology by causing cell rounding in DBTRG and U87 cells. Administration of Y15 significantly decreased subcutaneous DBTRG tumor growth with decreased Y397-FAK autophosphorylation, activated caspase-3 and PARP. Y15 was administered in an orthotopic glioma model, leading to an increase in mouse survival. The combination of Y15 with temozolomide was more effective than either agent alone in decreasing viability and activating caspase-8 in DBTRG and U87 cells in vitro. In addition, the combination of Y15 and temozolomide synergistically blocked U87 brain tumor growth in vivo. Thus, pharmacologic blockade of FAK autophosphorylation with the oral administration of a small molecule inhibitor Y15 has a potential to be an effective therapy approach for glioblastoma either alone or in combination with chemotherapy agents such as temozolomide.
doi:10.1158/1535-7163.MCT-12-0701
PMCID: PMC3570595  PMID: 23243059
Focal Adhesion Kinase; Y397 site; autophosphorylation; brain; glioblastoma
4.  Ovarian Cancer Spheroid Cells with Stem Cell-Like Properties Contribute to Tumor Generation, Metastasis and Chemotherapy Resistance through Hypoxia-Resistant Metabolism 
PLoS ONE  2014;9(1):e84941.
Cells with sphere forming capacity, spheroid cells, are present in the malignant ascites of patients with epithelial ovarian cancer (EOC) and represent a significant impediment to efficacious treatment due to their putative role in progression, metastasis and chemotherapy resistance. The exact mechanisms that underlie EOC metastasis and drug resistance are not clear. Understanding the biology of sphere forming cells may contribute to the identification of novel therapeutic opportunities for metastatic EOC. Here we generated spheroid cells from human ovarian cancer cell lines and primary ovarian cancer. Xenoengraftment of as few as 2000 dissociated spheroid cells into immune-deficient mice allowed full recapitulation of the original tumor, whereas >105 parent tumor cells remained non-tumorigenic. The spheroid cells were found to be enriched for cells with cancer stem cell-like characteristics such as upregulation of stem cell genes, self-renewal, high proliferative and differentiation potential, and high aldehyde dehydrogenase (ALDH) activity. Furthermore, spheroid cells were more aggressive in growth, migration, invasion, scratch recovery, clonogenic survival, anchorage-independent growth, and more resistant to chemotherapy in vitro. 13C-glucose metabolic studies revealed that spheroid cells route glucose predominantly to anaerobic glycolysis and pentose cycle to the detriment of re-routing glucose for anabolic purposes. These metabolic properties of sphere forming cells appear to confer increased resistance to apoptosis and contribute to more aggressive tumor growth. Collectively, we demonstrated that spheroid cells with cancer stem cell-like characteristics contributed to tumor generation, progression and chemotherapy resistance. This study provides insight into the relationship between tumor dissemination and metabolic attributes of human cancer stem cells and has clinical implications for cancer therapy.
doi:10.1371/journal.pone.0084941
PMCID: PMC3883678  PMID: 24409314
5.  Genome-wide methylation patterns provide insight into differences in breast tumor biology between American women of African and European ancestry 
Oncotarget  2013;5(1):237-248.
American women of African ancestry (AA) are more likely than European-Americans (EA) to be diagnosed with aggressive, estrogen receptor (ER) negative breast tumors; mechanisms underlying these disparities are poorly understood. We conducted a genome wide (450K loci) methylation analysis to determine if there were differences in DNA methylation patterns between tumors from AA and EA women and if these differences were similar for both ER positive and ER negative breast cancer. Methylation levels at CpG loci within CpG islands (CGI)s and CGI-shores were significantly higher in tumors (n=138) than in reduction mammoplasty samples (n=124). In hierarchical cluster analysis, there was separation between tumor and normal samples, and in tumors, there was delineation by ER status, but not by ancestry. However, differential methylation analysis identified 157 CpG loci with a mean β value difference of at least 0.17 between races, with almost twice as many differences in ER-negative tumors compared to ER-positive cancers. This first genome-wide methylation study to address disparities indicates that there are likely differing etiologic pathways for the development of ER negative breast cancer between AA and EA women. Further investigation of the genes most differentially methylated by race in ER negative tumors can guide new approaches for cancer prevention and targeted therapies, and elucidate the biologic basis of breast cancer disparities.
PMCID: PMC3960204  PMID: 24368439
DNA methylation; breast cancer; disparities; estrogen receptor; African-American; genome-wide
6.  Computational methods for detecting copy number variations in cancer genome using next generation sequencing: principles and challenges 
Oncotarget  2013;4(11):1868-1881.
Accurate detection of somatic copy number variations (CNVs) is an essential part of cancer genome analysis, and plays an important role in oncotarget identifications. Next generation sequencing (NGS) holds the promise to revolutionize somatic CNV detection. In this review, we provide an overview of current analytic tools used for CNV detection in NGS-based cancer studies. We summarize the NGS data types used for CNV detection, decipher the principles for data preprocessing, segmentation, and interpretation, and discuss the challenges in somatic CNV detection. This review aims to provide a guide to the analytic tools used in NGS-based cancer CNV studies, and to discuss the important factors that researchers need to consider when analyzing NGS data for somatic CNV detections.
PMCID: PMC3875755  PMID: 24240121
copy number variation; next generation sequencing; cancer genome analysis; somatic mutations
7.  6p22.3 amplification as a biomarker and potential therapeutic target of advanced stage bladder cancer 
Oncotarget  2013;4(11):2124-2134.
Genetic and epigenetic alterations have been identified as to contribute directly or indirectly to the generation of transitional cell carcinoma of the urinary bladder (TCC-UB). In a comparative fashion much less is known about copy number alterations in TCC-UB, but it appears that amplification of chromosome 6p22 is one of the most frequent changes. Using fluorescence in situ hybridization (FISH) analyses, we evaluated chromosomal 6p22 amplification in a large cohort of bladder cancer patients with complete surgical staging and outcome data. We have also used shRNA knockdown candidate oncogenes in the cell based study. We found that amplification of chromosome 6p22.3 is significantly associated with the muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) (22%) in contrast to superficial TCC-UB (9%) (p=7.2-04). The rate of 6p22.3 amplification in pN>1 patients (32%) is more than twice that in pN0 (16%) patients (p=0.05). Interestingly, we found that 6p22.3 amplification is as twice as high (p=0.0201) in African American (AA) than European American (EA) TCC-UB patients. Moreover, we showed that the expression of some candidate genes (E2F3, CDKAL1 and Sox4) in the 6p22.3 region is highly correlated with the chromosomal amplification. In particular, knockdown of E2F3 inhibits cell proliferation in a 6p22.3-dependent manner, whereas knockdown of CDKAL1 and Sox4 has no effect on cell proliferation. Using gene expression profiling, we further identified some common as well as distinctive subset targets of the E2F3 family members. In summary, our data indicate that E2F3 is a key regulator of cell proliferation in a subset of bladder cancer and the 6p22.3 amplicon is a biomarker of aggressive phenotype in this tumor type.
PMCID: PMC3875774  PMID: 24231253
bladder cancer; chromosome 6p22; FISH; outcome; survival
8.  Aldehyde dehydrogenase and ATP binding cassette transporter G2 (ABCG2) functional assays isolate different populations of prostate stem cells where ABCG2 function selects for cells with increased stem cell activity 
Introduction
High expression of aldehyde dehydrogenase1A1 (ALDH1A1) is observed in many organs and tumors and may identify benign and cancer stem cell populations.
Methods
In the current study, the stem cell characteristics were determined in cells isolated from human prostate cell lines and clinical prostate specimens based upon the ALDEFLUOR™ assay. Cells isolated based on the ALDEFLUOR™ assay were compared to cells isolated based on ATP binding cassette transporter G2 (ABCG2) activity using the side population assay. To test for stem cell characteristics of self-renewal and multipotency, cells with high and low ALDH1A1 activity, based on the ALDEFLUOR™ assay (ALDHHi and ALDHLow), were isolated from prostate clinical specimens and were recombined with rat urogenital sinus mesenchyme to induce prostate gland formation.
Results
The percentage of ALDHHi cells in prostate cell lines (RWPE-1, RWPE-2, CWR-R1, and DU-145) was 0.5 to 6%, similarly in non-tumor and tumor clinical specimens the percentage of ALDHHi cells was 0.6 to 4%. Recombinants using ALDHHi cells serially generated prostate tissue up to three generations with as few as 250 starting cells. Immunohistochemical analysis of the recombinants using ALDHHi cells contained prostatic glands frequently expressing androgen receptor (AR), p63, chromogranin A, ALDH1A1, ABCG2, and prostate specific antigen (PSA), compared to their ALDHLow counterparts. Inhibition of ALDH resulted in the reduction of sphere formation capabilities in the CWR-R1, but not in the RWPE-2 and DU-145, prostate cell lines. ABCG2 inhibition resulted in a more robust decrease of sphere formation in androgen sensitive cell lines, CWR-R1 and RWPE-2, but not androgen insensitive DU-145. ALDH1A1 expression was enriched in ALDHHi cells and non-side population cells. ABCG2 expression was only enriched in side population cells.
Conclusions
The percentage of ALDHHi cells in prostate cell lines and prostate tissue was consistently higher compared to cells with high ABCG2 activity, identified with the side population assay. The expression of the stem and differentiation markers indicates the ALDHHi recombinants contained cells with self-renewal and multipotency activity. When the two assays were directly compared, cells with the side population phenotype demonstrated more stem cell potential in the tissue recombination assay compared to ALDHHi cells. The increased stem cell potential of side population cells in the tissue recombination assay and the decrease in sphere formation when ABCG2 is inhibited indicates that the side population enriches for prostate stem cells.
doi:10.1186/scrt343
PMCID: PMC3854760  PMID: 24405526
9.  Isolation and genome-wide expression and methylation characterization of CD31+ cells from normal and malignant human prostate tissue 
Oncotarget  2013;4(9):1472-1483.
Endothelial cells (ECs) are an important component involved in the angiogenesis. Little is known about the global gene expression and epigenetic regulation in tumor endothelial cells. The identification of gene expression and epigenetic difference between human prostate tumor-derived endothelial cells (TdECs) and those in normal tissues may uncover unique biological features of TdEC and facilitate the discovery of new anti-angiogenic targets. We established a method for isolation of CD31+ endothelial cells from malignant and normal prostate tissues obtained at prostatectomy. TdECs and normal-derived ECs (NdECs) showed >90% enrichment in primary culture and demonstrated microvascular endothelial cell characteristics such as cobblestone morphology in monolayer culture, diI-acetyl-LDL uptake and capillary-tube like formation in Matrigel®. In vitro primary cultures of ECs maintained expression of endothelial markers such as CD31, von Willebrand factor, intercellular adhesion molecule, vascular endothelial growth factor receptor 1, and vascular endothelial growth factor receptor 2. We then conducted a pilot study of transcriptome and methylome analysis of TdECs and matched NdECs from patients with prostate cancer. We observed a wide spectrum of differences in gene expression and methylation patterns in endothelial cells, between malignant and normal prostate tissues. Array-based expression and methylation data were validated by qRT-PCR and bisulfite DNA pyrosequencing. Further analysis of transcriptome and methylome data revealed a number of differentially expressed genes with loci whose methylation change is accompanied by an inverse change in gene expression. Our study demonstrates the feasibility of isolation of ECs from histologically normal prostate and prostate cancer via CD31+ selection. The data, although preliminary, indicates that there exist widespread differences in methylation and transcription between TdECs and NdECs. Interestingly, only a small proportion of perturbed genes were overlapped between American (AA) and Caucasian American (CA) patients with prostate cancer. Our study indicates that identifying gene expression and/or epigenetic differences between TdECs and NdECs may provide us with new anti-angiogenic targets. Future studies will be required to further characterize the isolated ECs and determine the biological features that can be exploited in the prognosis and therapy of prostate cancer.
PMCID: PMC3824530  PMID: 23978847
Endothelial cells; Gene expression; DNA methylation; prostate cancer
10.  TAZ induces growth factor-independent proliferation through activation of EGFR ligand amphiregulin 
Cell Cycle  2012;11(15):2922-2930.
The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. We previously showed that the pivotal effector of this pathway, YAP, is amplified in tumors and promotes epithelial-to-mesenchymal transition (EMT) and malignant transformation. Here, we report that overexpression of TAZ, a paralog of YAP, in human mammary epithelial cells promotes EMT and, in particular, some invasive structures in 3D cultures. TAZ also leads to cell migration and anchorage-independent growth in soft agar. Furthermore, we identified amphiregulin (AREG), an epidermal growth factor receptor (EGFR) ligand, as a target of TAZ. We show that AREG functions in a non-cell-autonomous manner to mediate EGF-independent growth and malignant behavior of mammary epithelial cells. In addition, ablation of TEAD binding completely abolishes the TAZ-induced phenotype. Last, analysis of breast cancer patient samples reveals a positive correlation between TAZ and AREG in vivo. In summary, TAZ-dependent secretion of AREG indicates that activation of the EGFR signaling is an important non-cell-autonomous effector of the Hippo pathway, and TAZ as well as its targets may play significant roles in breast tumorigenesis and metastasis.
doi:10.4161/cc.21386
PMCID: PMC3419062  PMID: 22825057
amphiregulin (AREG); epithelial-to-mesenchymal transition (EMT); Hippo pathway; TAZ; tumor metastasis
11.  Disruption of focal adhesion kinase and p53 interaction with small molecule compound R2 reactivated p53 and blocked tumor growth 
BMC Cancer  2013;13:342.
Background
Focal Adhesion Kinase (FAK) is a 125 kDa non-receptor kinase that plays a major role in cancer cell survival and metastasis.
Methods
We performed computer modeling of the p53 peptide containing the site of interaction with FAK, predicted the peptide structure and docked it into the three-dimensional structure of the N-terminal domain of FAK involved in the complex with p53. We screened small molecule compounds that targeted the site of the FAK-p53 interaction and identified compounds (called Roslins, or R compounds) docked in silico to this site.
Results
By different assays in isogenic HCT116p53+/+ and HCT116 p53-/- cells we identified a small molecule compound called Roslin 2 (R2) that bound FAK, disrupted the binding of FAK and p53 and decreased cancer cell viability and clonogenicity in a p53-dependent manner. In addition, dual-luciferase assays demonstrated that the R2 compound increased p53 transcriptional activity that was inhibited by FAK using p21, Mdm-2, and Bax-promoter targets. R2 also caused increased expression of p53 targets: p21, Mdm-2 and Bax proteins. Furthermore, R2 significantly decreased tumor growth, disrupted the complex of FAK and p53, and up-regulated p21 in HCT116 p53+/+ but not in HCT116 p53-/- xenografts in vivo. In addition, R2 sensitized HCT116p53+/+ cells to doxorubicin and 5-fluorouracil.
Conclusions
Thus, disruption of the FAK and p53 interaction with a novel small molecule reactivated p53 in cancer cells in vitro and in vivo and can be effectively used for development of FAK-p53 targeted cancer therapy approaches.
doi:10.1186/1471-2407-13-342
PMCID: PMC3712010  PMID: 23841915
Focal adhesion kinase; p53Cancer; Small molecule; p21; Tumor; Apoptosis
12.  Identification of somatic mutations in non-small cell lung carcinomas using whole-exome sequencing 
Carcinogenesis  2012;33(7):1270-1276.
Lung cancer is the leading cause of cancer-related death, with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Most lung cancer is caused by the accumulation of genomic alterations due to tobacco exposure. To uncover its mutational landscape, we performed whole-exome sequencing in 31 NSCLCs and their matched normal tissue samples. We identified both common and unique mutation spectra and pathway activation in lung adenocarcinomas and squamous cell carcinomas, two major histologies in NSCLC. In addition to identifying previously known lung cancer genes (TP53, KRAS, EGFR, CDKN2A and RB1), the analysis revealed many genes not previously implicated in this malignancy. Notably, a novel gene CSMD3 was identified as the second most frequently mutated gene (next to TP53) in lung cancer. We further demonstrated that loss of CSMD3 results in increased proliferation of airway epithelial cells. The study provides unprecedented insights into mutational processes, cellular pathways and gene networks associated with lung cancer. Of potential immediate clinical relevance, several highly mutated genes identified in our study are promising druggable targets in cancer therapy including ALK, CTNNA3, DCC, MLL3, PCDHIIX, PIK3C2B, PIK3CG and ROCK2.
doi:10.1093/carcin/bgs148
PMCID: PMC3499051  PMID: 22510280
13.  Aberrant expression of DNA damage response proteins is associated with breast cancer subtype and clinical features 
Landmark studies of the status of DNA damage checkpoints and associated repair functions in preneoplastic and neoplastic cells has focused attention on importance of these pathways in cancer development, and inhibitors of repair pathways are in clinical trials for treatment of triple negative breast cancer. Cancer heterogeneity suggests that specific cancer subtypes will have distinct mechanisms of DNA damage survival, dependent on biological context. In this study, status of DNA damage response (DDR)-associated proteins was examined in breast cancer subtypes in association with clinical features; 479 breast cancers were examined for expression of DDR proteins γH2AX, BRCA1, pChk2, and p53, DNA damage-sensitive tumor suppressors Fhit and Wwox, and Wwox-interacting proteins Ap2α, Ap2γ, ErbB4, and correlations among proteins, tumor subtypes, and clinical features were assessed. In a multivariable model, triple negative cancers showed significantly reduced Fhit and Wwox, increased p53 and Ap2γ protein expression, and were significantly more likely than other subtype tumors to exhibit aberrant expression of two or more DDR-associated proteins. Disease-free survival was associated with subtype, Fhit and membrane ErbB4 expression level and aberrant expression of multiple DDR-associated proteins. These results suggest that definition of specific DNA repair and checkpoint defects in subgroups of triple negative cancer might identify new treatment targets. Expression of Wwox and its interactor, ErbB4, was highly significantly reduced in metastatic tissues vs. matched primary tissues, suggesting that Wwox signal pathway loss contributes to lymph node metastasis, perhaps by allowing survival of tumor cells that have detached from basement membranes, as proposed for the role of Wwox in ovarian cancer spread.
doi:10.1007/s10549-010-1248-6
PMCID: PMC3677189  PMID: 21069451
Triple negative breast cancer; DNA damage response proteins; Lymph node metastases; Fhit; Wwox; Tissue microarrays
14.  Human Prostate Side Population Cells Demonstrate Stem Cell Properties in Recombination with Urogenital Sinus Mesenchyme 
PLoS ONE  2013;8(1):e55062.
Stem cell enrichment provides a tool to examine prostate stem cells obtained from benign and malignant tissue. Functional assays can enrich stem cells based on common stem cell phenotypes, such as high ATP binding cassette (ABC) transporter mediated efflux of Hoechst substrates (side population assay). This functional assay is based upon mechanisms that protect cells from environmental insult thus contributing to the survival and protection of the stem cell population. We have isolated and analyzed cells digested from twelve clinical prostate specimens based on the side population assay. Prostate stem cell properties of the isolated cells were tested by serial recombination with rat urogenital mesenchyme. Recombinants with side population cells demonstrate an increase in the frequency of human ductal growth and the number of glands per recombinant when compared to recombinants with non-side population cells. Isolated cells were capable of prostatic growth for up to three generations in the recombination assay with as little as 125 sorted prostate cells. The ability to reproducibly use cells isolated by fluorescence activated cell sorting from human prostate tissue is an essential step to a better understanding of human prostate stem cell biology. ABC transporter G2 (ABCG2) was expressed in recombinants from side population cells indicating the side population cells have self-renewal properties. Epithelial cell differentiation of recombinants was determined by immunohistochemical analysis for expression of the basal, luminal, and neuroendocrine markers, p63, androgen receptor, prostate specific antigen, and chromogranin A, respectively. Thus, the ABCG2 expressing side population demonstrates multipotency and self-renewal properties indicating stem cells are within this population.
doi:10.1371/journal.pone.0055062
PMCID: PMC3561453  PMID: 23383057
15.  Carcinogen-induced squamous papillomas and oncogenic progression in the absence of the SSeCKS/AKAP12 metastasis suppressor correlates with FAK upregulation 
The ability of SSeCKS/Gravin/AKAP12 (SSeCKS) to negatively regulate cell cycle progression is thought to relate to its spatiotemporal scaffolding activity for key signaling molecules such as protein kinase A and C, calmodulin, and cyclins. SSeCKS is downregulated upon progression to malignancy in many cancer types, including melanoma and non-melanoma skin cancer. The forced re-expression of SSeCKS is especially potent in suppressing metastasis through the inhibition of VEGF-mediated neovascularization. We have previously shown that SSeCKS-null (KO) mice exhibit hyperplasia and focal dysplasia in the prostate marked by activated Akt. To address whether KO-mice exhibit increased skin carcinogenesis, WT and KO C57BL/6 mice were treated topically with 12-O-tetradecanoylphorbol-13-acetate and 7,12-dimethylbenzanthracene. Compared to WT mice, KO mice developed squamous papillomas more rapidly and in greater numbers, and also exhibited significantly increased progression to squamous cell carcinoma. Untreated KO epidermal layers were thicker than those in age-matched WT mice, and exhibited significantly increased levels of FAK and phospho-ERK1/2, known mediators of carcinogen-induced squamous papilloma progression to carcinoma. Compared to protein levels in WT mouse embryo fibroblasts (MEF), SSeCKS levels were increased in FAK-null cells whereas FAK levels were increased in SSeCKS-null cells. RNAi studies in WT MEF cells suggest that SSeCKS and FAK attenuate each other’s expression. Our study implicates a role for SSeCKS in preventing of skin cancer progression possibly through negatively regulating FAK expression.
doi:10.1002/ijc.25828
PMCID: PMC3107938  PMID: 21128249
SSeCKS; TPA; DMBA; FAK; AKAP12; Gravin; papilloma; squamous cell carcinoma; skin cancer
16.  Epigenetic deregulation of TCF21 inhibits metastasis suppressor KISS1 in metastatic melanoma 
Carcinogenesis  2011;32(10):1467-1473.
Metastatic melanoma is a fatal disease due to the lack of successful therapies and biomarkers for early detection and its incidence has been increasing. Genetic studies have defined recurrent chromosomal aberrations, suggesting the location of either tumor suppressor genes or oncogenes. Transcription factor 21 (TCF21) belongs to the class A of the basic helix-loop-helix family with reported functions in early lung and kidney development as well as tumor suppressor function in the malignancies of the lung and head and neck. In this study, we combined quantitative DNA methylation analysis in patient biopsies and in their derived cell lines to demonstrate that TCF21 expression is downregulated in metastatic melanoma by promoter hypermethylation and TCF21 promoter DNA methylation is correlated with decreased survival in metastatic skin melanoma patients. In addition, the chromosomal location of TCF21 on 6q23–q24 coincides with the location of a postulated metastasis suppressor in melanoma. Functionally, TCF21 binds the promoter of the melanoma metastasis-suppressing gene, KiSS1, and enhances its gene expression through interaction with E12, a TCF3 isoform and with TCF12. Loss of TCF21 expression results in loss of KISS1 expression through loss of direct interaction of TCF21 at the KISS1 promoter. Finally, overexpression of TCF21 inhibits motility of C8161 melanoma cells. These data suggest that epigenetic downregulation of TCF21 is functionally involved in melanoma progression and that it may serve as a biomarker for aggressive tumor behavior.
doi:10.1093/carcin/bgr138
PMCID: PMC3179423  PMID: 21771727
17.  PP2A-B56α Controls Oncogene-Induced Senescence in Normal and Tumor Human Melanocytic Cells 
Oncogene  2011;31(12):1484-1492.
Oncoprotein C-MYC is overexpressed in human metastatic melanomas and melanoma-derived cells where it is required for suppression of oncogene-induced senescence (OIS). The genetic events that maintain high levels of C-MYC in melanoma cells and their role in OIS are unknown. Here, we report that C-MYC in cells from several randomly chosen melanoma lines was up-regulated at the protein level, and largely due to the increased protein stability. Of all known regulators of C-MYC stability, levels of B56α subunit of the PP2A tumor suppressor complex were substantially suppressed in all human melanoma cells compared to normal melanocytes. Accordingly, immuno-histochemical analysis revealed that the lowest and the highest amounts of PP2A-B56α were predominantly detected in metastatic melanoma tissues and in primary melanomas from patients with good clinical outcome, respectively. Importantly, PP2A-B56α overexpression suppressed C-MYC in melanoma cells and induced OIS, whereas depletion of PP2A-B56α in normal human melanocytes up-regulated C-MYC protein levels and suppressed BRAFV600E- and, less efficiently, NRASQ61R-induced senescence. Our data reveal a mechanism of C-MYC overexpression in melanoma cells and identify a functional role for PP2A-B56α in OIS of melanocytic cells.
doi:10.1038/onc.2011.339
PMCID: PMC3213274  PMID: 21822300
PP2A-B56α; C-MYC; melanoma; senescence
18.  LOH of chromosome 12p correlates with Kras2 mutation in non-small cell lung cancer 
Oncogene  2003;22(8):1243-1246.
Previous observation has shown that the wild-type Kras2 allele is a suppressor of lung cancer in mice. Here we report that loss of heterozygosity (LOH) of chromosome 12p was detected in ~50% of human lung adenocarcinomas and large cell carcinomas, and Kras2 mutations were detected at codon 12 in ~40% of adenocarcinomas and large cell carcinomas. Interestingly, all of the lung adenocarcinomas and large cell carcinomas containing a Kras2 mutation exhibited allelic loss of the wild-type Kras2 allele when a correlation between LOH of the region on chromosome 12p and Kras2 mutation was made. These results from human lung cancer tissues provide a strong evidence in support of our previous observation in mouse models that the wild-type Kras2 is a tumor suppressor of lung cancer.
doi:10.1038/sj.onc.1206192
PMCID: PMC3438910  PMID: 12606951
wild-type Kras2; mutations: lung cancer; loss of heterozygosity; tumor suppressor
20.  TAK1-TAB2 signaling contributes to bone destruction by breast carcinoma cells 
Molecular cancer research : MCR  2011;9(8):1042-1053.
Advanced-stage breast cancers frequently metastasize to the bones and cause bone destruction, but the underlying mechanism is not fully understood. This study presents evidence that TGF-β-activated protein kinase 1 (TAK1) signaling in tumor cells promotes bone destruction by metastatic breast carcinoma cells, controlling expression of pro-metastatic factors, including MMP-9 and COX2. Suppression of TAK1 signaling by dominant-negative (dn) TAK1 in breast carcinoma MDA-MB-231 cells impairs bone colonization by carcinoma cells and bone osteolysis in the intra-cardiac injection model. Mechanistic studies showed that inhibition of TAK1 by dn-TAK1 or siRNA blocked expression of factors implicated in bone metastasis, such as MMP-9, COX2/PTGS2, PTHrP, and IL8, but did not affect activation of p38MAPK by TGF-β. TAK1 signaling is mediated by TAK1-binding partners TAB1, TAB2 and TAB3. Carcinoma cells express elevated mRNA levels of TAB2 and TAB3, whereas the TAB1 expression is noticeably low. Accordingly, depletion of TAB2 by siRNA reduced expression of MMP-9 and COX2. Together, these studies demonstrate that the TAK1-TAB2/TAB3 signaling axis is critical for carcinoma-induced bone lesions, mediating expression of pro-invasive and osteolytic factors. These findings identify the TAK1-TAB2 axis as a potential therapeutic target in bone metastasis.
doi:10.1158/1541-7786.MCR-10-0196
PMCID: PMC3157546  PMID: 21700681
Invasion; metastasis; bone metastasis; TAK1; TGF-beta
21.  Differential vitamin D 24-hydroxylase/CYP24A1 gene promoter methylation in endothelium from benign and malignant human prostate 
Epigenetics  2011;6(8):994-1000.
Epigenetic alterations occur in tumor-associated vessels in the tumor microenvironment. Methylation of the CYP24A1 gene promoter differs in endothelial cells isolated from tumors and non-tumor microenvironments in mice. The epigenetic makeup of endothelial cells of human tumor-associated vasculature is unknown due to difficulty of isolating endothelial cells populations from a heterogeneous tissue microenvironment. To ascertain CYP24A1 promoter methylation in tumor-associated endothelium, we utilized laser microdissection guided by CD31 immunohistochemistry to procure endothelial cells from human prostate tumor specimens. Prostate tissues were obtained following robotic radical prostatectomy from men with clinically localized prostate cancer. Adjacent histologically benign prostate tissues were used to compare endothelium from benign versus tumor microenvironments. Sodium bisulfite sequencing of CYP24A1 promoter region showed that the average CYP24A1 promoter methylation in the endothelium was 20% from the tumor microenvironment compared with 8.2% in the benign microenvironment (p < 0.05). A 2-fold to 17-fold increase in CYP24A1 promoter methylation was observed in the prostate tumor endothelium compared with the matched benign prostate endothelium in four patient samples, while CYP24A1 promoter methylation remained unchanged in two patient samples. In addition, there is no correlation of the level of CYP24A1 promoter methylation in prostate tumor-associated endothelium with that of epithelium/stroma. This study demonstrates that the CYP24A1 promoter is methylated in tumor-associated endothelium, indicating that epigenetic alterations in CYP24A1 may play a role in determining the phenotype of tumor-associated vasculature in the prostate tumor microenvironment.
doi:10.4161/epi.6.8.16536
PMCID: PMC3219083  PMID: 21725204
CYP24A1; DNA methylation; human prostate cancer; tumor endothelium; laser microdissection
22.  NY-ESO-1 Cancer Testis Antigen Demonstrates High Immunogenicity in Triple Negative Breast Cancer 
PLoS ONE  2012;7(6):e38783.
Purpose
NY-ESO-1 cancer testis (CT) antigen is an attractive candidate for immunotherapy as a result of its high immunogenicity. The aim of this study was to explore the potential for NY-ESO-1 antigen directed immunotherapy in triple negative breast cancer (TNBC) by determining the frequency of expression by immunohistochemistry (IHC) and the degree of inherent immunogenicity to NY-ESO-1.
Experimental Design
168 TNBC and 47 ER+/HER2- primary breast cancer specimens were used to determine NY-ESO-1 frequency by IHC. As previous studies have shown that patients with a robust innate humoral immune response to CT antigens are more likely to develop CD8 T-cell responses to NY-ESO-1 peptides, we evaluated the degree to which patients with NY-ESO-1 expression had inherent immunogenicity by measuring antibodies. The relationship between NY-ESO-1 expression and CD8+ T lymphocytes was also examined.
Results
The frequency of NY-ESO-1 expression in the TNBC cohort was 16% versus 2% in ER+/HER2- patients. A higher NY-ESO-1 score was associated with a younger age at diagnosis in the TNBC patients with NY-ESO-1 expression (p = 0.026). No differences in OS (p = 0.278) or PFS (p = 0.238) by NY-ESO-1 expression status were detected. Antibody responses to NY-ESO-1 were found in 73% of TNBC patients whose tumors were NY-ESO-1 positive. NY-ESO-1 positive patients had higher CD8 counts than negative patients (p = 0.018).
Conclusion
NY-ESO-1 is expressed in a substantial subset of TNBC patients and leads to a high humoral immune response in a large proportion of these individuals. Given these observations, patients with TNBC may benefit from targeted therapies directed against NY-ESO-1.
doi:10.1371/journal.pone.0038783
PMCID: PMC3386262  PMID: 22761704
23.  Expression of FACT in mammalian tissues suggests its role in maintaining of undifferentiated state of cells 
Oncotarget  2011;2(10):783-796.
The Facilitates Chromatin Transcription (FACT) chromatin remodeling complex, comprised of two subunits, SSRP1 and SPT16, is involved in transcription, replication and DNA repair. We recently showed that curaxins, small molecules with anti-cancer activity, target FACT and kill tumor cells in a FACT-dependent manner. We also found that FACT is overexpressed in human and mouse tumors and that tumor cells are sensitive to FACT downregulation. To clarify the clinical potential of FACT inhibition, we were interested in physiological role(s) of FACT in multicellular organisms. We analyzed SSRP1 and SPT16 expression in different cells, tissues and conditions using Immunohistochemical (IHC) staining of mouse and human tissues and analysis of publically available high-content gene expression datasets. Both approaches demonstrated coordinated expression of the two FACT subunits, which was primarily associated with the stage of cellular differentiation. Most cells of adult tissues do not have detectable protein level of FACT. High FACT expression was associated with stem or less-differentiated cells, while low FACT levels were seen in more differentiated cells. Experimental manipulation of cell differentiation and proliferation in vitro, as well as tissue staining for the Ki67 proliferation marker, showed that FACT expression is related more to differentiation than to proliferation. Thus, FACT may be part of a stem cell-like gene expression signature and play a role in maintaining cells in an undifferentiated state, which is consistent with its potential role as an anti-cancer target.
PMCID: PMC3248156  PMID: 21998152
chromatin remodeling; transcription; SPT16; SSRP1; cancer; differentiation; proliferation; oncotarget
24.  Epigenetic Regulation of Vitamin D 24-Hydroxylase/CYP24A1 in Human Prostate Cancer 
Cancer research  2010;70(14):5953-5962.
Calcitriol, a regulator of calcium homeostasis with antitumor properties, is degraded by the product of the CYP24A1 gene which is downregulated in human prostate cancer by unknown mechanisms. We found that CYP24A1 expression is inversely correlated with promoter DNA methylation in prostate cancer cell lines. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) activates CYP24A1 expression in prostate cancer cells. In vitro methylation of the CYP24A1 promoter represses its promoter activity. Furthermore, inhibition of histone deacetylases by trichostatin A (TSA) enhances the expression of CYP24A1 in prostate cancer cells. ChIP-qPCR reveals that specific histone modifications are associated with the CYP24A1 promoter region. Treatment with TSA increases H3K9ac and H3K4me2 and simultaneously decreases H3K9me2 at the CYP24A1 promoter. ChIP-qPCR assay reveals that treatment with DAC and TSA increases the recruitment of VDR to the CYP24A1 promoter. RT-PCR analysis of paired human prostate samples reveals that CYP24A1 expression is down-regulated in prostate malignant lesions compared to adjacent histologically benign lesions. Bisulfite pyrosequencing shows that CYP24A1 gene is hypermethylated in malignant lesions compared to matched benign lesions. Our findings indicate that repression of CYP24A1 gene expression in human prostate cancer cells is mediated in part by promoter DNA methylation and repressive histone modifications.
doi:10.1158/0008-5472.CAN-10-0617
PMCID: PMC2928678  PMID: 20587525
CYP24A1 expression; DNA methylation; histone modification; prostate cancer
25.  Copy number gain and oncogenic activity of YWHAZ/14-3-3ζ in head and neck squamous cell carcinoma 
Gene amplification, a common mechanism for oncogene activation in cancers, has been used in the discovery of novel oncogenes. . Low level copy number gains are frequently observed in head and neck squamous cell carcinomas (HNSCCs) where numerous amplification events and potential oncogenes have already been reported. Recently, we applied restriction landmark genome scanning (RLGS) to study gene amplifications in HNSCC and located novel and uncharacterized regions in primary tumor samples. Gain on chromosome 8q22.3, the location of YWHAZ (14-3-3ζ), is found in 30-40% HNSCC cases. Data obtained from fluorescence in situ hybridization (FISH) and immunohistochemistry on HNSCC tissue microarrays confirmed frequent low-level YWHAZ copy number gain and protein overexpression. YWHAZ mRNA was frequently upregulated in patients’ tumor tissues. Furthermore, YWHAZ RNAi significantly suppressed the growth rate of HNSCC cell lines, and overexpression of YWHAZ in HaCaT immortalized human skin keratinocytes promotes overgrowth, as well as morphological changes. Reduced YWHAZ levels increased the G1/G0-phase proportion, decreased the S-phase proportion and the rate of DNA synthesis. Based on this evidence, we suggest that YWHAZ is a candidate proto-oncogene and deserves further investigation into its role in HNSCC carcinogenesis.
doi:10.1002/ijc.24346
PMCID: PMC2756013  PMID: 19405126
HNSCC; amplification; oncogene; YWHAZ/14-3-3ζ

Results 1-25 (33)