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author:("mile, Lucio")
1.  Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model 
Combination therapies for melanoma that target immune-regulatory networks are entering clinical practice, and more are under investigation in preclinical or clinical studies. Adenosine plays a key role in regulating melanoma progression. We investigated the effectiveness of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibody (mAb) in combination with either modulators of adenosine receptors (AR) activation or an inhibitor of adenosine production in a murine model of melanoma. We found that treatment with APCP, selective inhibitor of the adenosine-generating nucleotidase CD73, enhanced the activity of anti-CTLA4 mAb, by improving tumor immune response. Blockade of the adenosine A2a receptor (A2aR), which plays a critical role in the regulation of T-cell functions, significantly reduced melanoma growth. Most importantly, combination therapy including an A2aR antagonist with anti-CTLA4 mAb markedly inhibited tumor growth and enhanced anti-tumor immune responses. Targeting A3R and CTLA4 was not as effective in limiting melanoma growth as targeting A2aR. These data suggest that the efficacy of anti-CTLA4 melanoma therapy may be improved by targeting multiple mechanisms of immune suppression within tumor tissue, including CD73 or A2a receptor.
PMCID: PMC3960454  PMID: 24660106
CD73; adenosine receptor; CTLA4; melanoma; immunotherapy
2.  Increased plasma levels of soluble vascular endothelial growth factor (VEGF) receptor 1 (sFlt-1) in women by moderate exercise and increased plasma levels of VEGF in overweight/obese women 
The incidence of breast cancer is increasing worldwide, and this seems to be related to an increase in lifestyle risk factors, including physical inactivity, and overweight/obesity. We previously reported that exercise induced a circulating angiostatic phenotype characterized by increased sFlt-1 and endostatin and decreased unbound-VEGF in men. However, there is no data on women. The present study determines the following: 1) whether moderate exercise increased sFlt-1 and endostatin and decreased unbound-VEGF in the circulation of adult female volunteers; 2) whether overweight/obese women have a higher plasma level of unbound-VEGF than lean women. 72 African American and Caucasian adult women volunteers aged from 18–44 were enrolled into the exercise study. All the participants walked on a treadmill for 30 minutes at a moderate intensity (55–59% heart rate reserve), and oxygen consumption (VO2) was quantified by utilizing a metabolic cart. We had the blood samples before and immediately after exercise from 63 participants. ELISA assays (R&D Systems) showed that plasma levels of sFlt-1 were 67.8±3.7 pg/ml immediately after exercise (30 minutes), significantly higher than basal levels, 54.5±3.3 pg/ml, before exercise (P < 0.01; n=63). There was no significant difference in the % increase of sFlt-1 levels after exercise between African American and Caucasian (P=0.533) or between lean and overweight/obese women (P=0.892). There was no significant difference in plasma levels of unbound VEGF (35.28±5.47 vs. 35.23±4.96 pg/ml; P=0.99) or endostatin (111.12±5.48 vs. 115.45±7.15 ng/ml; P=0.63) before and after exercise. Basal plasma levels of unbound-VEGF in overweight/obese women were 52.26±9.6 pg/ml, significantly higher than basal levels of unbound-VEGF in lean women, 27.34±4.99 pg/ml (P < 0.05). The results support our hypothesis that exercise-induced plasma levels of sFlt-1 could be an important clinical biomarker to explore the mechanisms of exercise training in reducing breast cancer progression and that VEGF is an important biomarker in obesity and obesity-related cancer progression.
doi:10.1097/CEJ.0b013e328353ed81
PMCID: PMC3449013  PMID: 22609636
Exercise; Young adult women; Overweight/obese; sFlt-1; Endostatin; VEGF
3.  Therapeutic targeting of NOTCH signaling ameliorates immune-mediated bone marrow failure of aplastic anemia 
The Journal of Experimental Medicine  2013;210(7):1311-1329.
Notch1 signaling sustains the proinflammatory behavior of Th1 cells, implicated in the development of aplastic anemia in humans and mice.
Severe aplastic anemia (AA) is a bone marrow (BM) failure (BMF) disease frequently caused by aberrant immune destruction of blood progenitors. Although a Th1-mediated pathology is well described for AA, molecular mechanisms driving disease progression remain ill defined. The NOTCH signaling pathway mediates Th1 cell differentiation in the presence of polarizing cytokines, an action requiring enzymatic processing of NOTCH receptors by γ-secretase. Using a mouse model of AA, we demonstrate that expression of both intracellular NOTCH1IC and T-BET, a key transcription factor regulating Th1 cell differentiation, was increased in spleen and BM-infiltrating T cells during active disease. Conditionally deleting Notch1 or administering γ-secretase inhibitors (GSIs) in vivo attenuated disease and rescued mice from lethal BMF. In peripheral T cells from patients with untreated AA, NOTCH1IC was significantly elevated and bound to the TBX21 promoter, showing NOTCH1 directly regulates the gene encoding T-BET. Treating patient cells with GSIs in vitro lowered NOTCH1IC levels, decreased NOTCH1 detectable at the TBX21 promoter, and decreased T-BET expression, indicating that NOTCH1 signaling is responsive to GSIs during active disease. Collectively, these results identify NOTCH signaling as a primary driver of Th1-mediated pathogenesis in AA and may represent a novel target for therapeutic intervention.
doi:10.1084/jem.20112615
PMCID: PMC3698520  PMID: 23733784
4.  Non-Canonical Notch Signaling Drives Activation and Differentiation of Peripheral CD4+ T Cells 
Cleavage of the Notch receptor via a γ-secretase, results in the release of the active intra-cellular domain of Notch that migrates to the nucleus and interacts with RBP-Jκ, resulting in the activation of downstream target genes. This canonical Notch signaling pathway has been documented to influence T cell development and function. However, the mechanistic details underlying this process remain obscure. In addition to RBP-Jκ, the intra-cellular domain of Notch also interacts with other proteins in the cytoplasm and nucleus, giving rise to the possibility of an alternate, RBP-Jκ independent Notch pathway. However, the contribution of such RBP-Jκ independent, “non-canonical” Notch signaling in regulating peripheral T cell responses is unknown. In this report, we specifically demonstrate the requirement of Notch1 for regulating signal strength and signaling events distal to the T cell receptor in peripheral CD4+ T cells. By using mice with a conditional deletion in Notch1 or RBP-Jκ, we show that Notch1 regulates activation and proliferation of CD4+ T cells independently of RBP-Jκ. Furthermore, differentiation to TH1 and iTreg lineages although Notch dependent, is RBP-Jκ independent. Our striking observations demonstrate that many of the cell-intrinsic functions of Notch occur independently of RBP-Jκ. Such non-canonical regulation of these processes likely occurs through NF-κ B. This reveals a previously unknown, novel role of non-canonical Notch signaling in regulating peripheral T cell responses.
doi:10.3389/fimmu.2014.00054
PMCID: PMC3921607  PMID: 24611064
Notch1; CD4+ T cell; non-canonical; activation; differentiation
5.  Blockade of A2b Adenosine Receptor Reduces Tumor Growth and Immune Suppression Mediated by Myeloid-Derived Suppressor Cells in a Mouse Model of Melanoma12 
Neoplasia (New York, N.Y.)  2013;15(12):1400-1409.
The A2b receptor (A2bR) belongs to the adenosine receptor family. Emerging evidence suggest that A2bR is implicated in tumor progression in some murine tumor models, but the therapeutic potential of targeting A2bR in melanoma has not been examined. This study first shows that melanoma-bearing mice treated with Bay 60-6583, a selective A2bR agonist, had increased melanoma growth. This effect was associated with higher levels of immune regulatory mediators interleukin-10 (IL-10) and monocyte chemoattractant protein 1 (MCP-1) and accumulation of tumor-associated CD11b positive Gr1 positive cells (CD11b+Gr1+) myeloid-derived suppressor cells (MDSCs). Depletion of CD11b+Gr1+ cells completely reversed the protumor activity of Bay 60-6583. Conversely, pharmacological blockade of A2bR with PSB1115 reversed immune suppression in the tumor microenvironment, leading to a significant melanoma growth delay. PSB1115 treatment reduced both levels of IL-10 and MCP-1 and CD11b+Gr1+ cell number in melanoma lesions. These effects were associated with higher frequency of tumor-infiltrating CD8 positive (CD8+) T cells and natural killer T (NKT) cells and increased levels of T helper 1 (Th1)-like cytokines. Adoptive transfer of CD11b+Gr1+ cells abrogated the antitumor activity of PSB1115. These data suggest that the antitumor activity of PSB1115 relies on its ability to lower accumulation of tumor-infiltrating MDSCs and restore an efficient antitumor T cell response. The antitumor effect of PSB1115 was not observed in melanoma-bearing nude mice. Furthermore, PSB1115 enhanced the antitumor efficacy of dacarbazine. These data indicate that A2bR antagonists such as PSB1115 should be investigated as adjuvants in the treatment of melanoma.
PMCID: PMC3884531  PMID: 24403862
6.  Chemoresistance to gemcitabine in hepatoma cells induces epithelial-mesenchymal transition and involves activation of PDGF-D pathway 
Oncotarget  2013;4(11):1999-2009.
Hepatocellular carcinoma (HCC) is one of the common malignances in the world and has high mortality in part due to development of acquired drug resistance. Therefore, it is urgent to investigate the molecular mechanism of drug resistance in HCC. To explore the underlying mechanism of drug resistance in HCC, we developed gemcitabine-resistant (GR) HCC cells. We used multiple methods to achieve our goal including RT-PCR, Western blotting analysis, transfection, Wound-healing assay, migration and invasion assay. We observed that gemcitabine-resistant cells acquired epithelial-mesenchymal transition (EMT) phenotype. Moreover, we found that PDGF-D is highly expressed in GR cells. Furthermore, down-regulation of PDGF-D in GR cells led to partial reversal of the EMT phenotype. Our findings demonstrated that targeting PDGF-D could be a novel strategy to overcome gemcitabine resistance in HCC.
PMCID: PMC3875765  PMID: 24158561
Hepatocellular carcinoma; chemoresistance; PDGF-D; EMT; gemcitabine
7.  Down-Regulation of Notch-1 Is Associated With Akt and FoxM1 in Inducing Cell Growth Inhibition and Apoptosis in Prostate Cancer Cells 
Journal of cellular biochemistry  2011;112(1):78-88.
Although many studies have been done to uncover the mechanisms by which down-regulation of Notch-1 exerts its anti-tumor activity against a variety of human malignancies, the precise molecular mechanisms remain unclear. In the present study, we investigated the cellular consequence of Notch-1 down-regulation and also assessed the molecular consequence of Notch-1-mediated alterations of its downstream targets on cell viability and apoptosis in prostate cancer (PCa) cells. We found that the down-regulation of Notch-1 led to the inhibition of cell growth and induction of apoptosis, which was mechanistically linked with down-regulation of Akt and FoxM1, suggesting for the first time that Akt and FoxM1 are downstream targets of Notch-1 signaling. Moreover, we found that a “natural agent” (genistein) originally discovered from soybean could cause significant reduction in cell viability and induced apoptosis of PCa cells, which was consistent with down-regulation of Notch-1, Akt, and FoxM1. These results suggest that down-regulation of Notch-1 by novel agents could become a newer approach for the prevention of tumor progression and/or treatment, which is likely to be mediated via inactivation of Akt and FoxM1 signaling pathways in PCa.
doi:10.1002/jcb.22770
PMCID: PMC3792569  PMID: 20658545
NOTCH-1; PROSTATE CANCER; CELL GROWTH; APOPTOSIS; Akt; FoxM1
8.  Notch Signaling Pathway in Pancreatic Cancer Progression 
Pancreatic disorders & therapy  2013;3(114):1000114.
PMCID: PMC3767173  PMID: 24027656
Notch; Pancreatic cancer; EMT; Stem cell; Microrna; Therapy
9.  Notch signaling: targeting cancer stem cells and epithelial-to-mesenchymal transition 
OncoTargets and therapy  2013;6:1249-1259.
Notch signaling is an evolutionarily conserved pathway involved in cell fate control during development, stem cell self-renewal, and postnatal tissue differentiation. Roles for Notch in carcinogenesis, the biology of cancer stem cells, tumor angiogenesis, and epithelial-to-mesenchymal transition (EMT) have been reported. This review describes the role of Notch in the “stemness” program in cancer cells and in metastases, together with a brief update on the Notch inhibitors currently under investigation in oncology. These agents may be useful in targeting cancer stem cells and to reverse the EMT process.
doi:10.2147/OTT.S36162
PMCID: PMC3772757  PMID: 24043949
Notch signaling; EMT; cancer stem cells; mesenchymal stem cells; metastases; Notch inhibitors
10.  Comprehensive Assessment and Network Analysis of the Emerging Genetic Susceptibility Landscape of Prostate Cancer 
Cancer Informatics  2013;12:175-191.
Background
Recent advances in high-throughput genotyping have made possible identification of genetic variants associated with increased risk of developing prostate cancer using genome-wide associations studies (GWAS). However, the broader context in which the identified genetic variants operate is poorly understood. Here we present a comprehensive assessment, network, and pathway analysis of the emerging genetic susceptibility landscape of prostate cancer.
Methods
We created a comprehensive catalog of genetic variants and associated genes by mining published reports and accompanying websites hosting supplementary data on GWAS. We then performed network and pathway analysis using single nucleotide polymorphism (SNP)-containing genes to identify gene regulatory networks and pathways enriched for genetic variants.
Results
We identified multiple gene networks and pathways enriched for genetic variants including IGF-1, androgen biosynthesis and androgen signaling pathways, and the molecular mechanisms of cancer. The results provide putative functional bridges between GWAS findings and gene regulatory networks and biological pathways.
doi:10.4137/CIN.S12128
PMCID: PMC3769142  PMID: 24031161
prostate cancer GWAS network pathway analysis
11.  Analysis of Patterns of Gene Expression Variation within and between Ethnic Populations in Pediatric B-ALL 
Cancer Informatics  2013;12:155-173.
B-Precursor acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. Although 80% of B-ALL patients are able to be cured, significant challenges persist. Significant disparities in clinical outcomes and mortality rates exist between racial/ethnic populations. The objective of this study was to determine whether gene expression levels significantly differ between ethnic populations. We compared gene expression levels between four ethnic populations (Whites, Blacks, Hispanics, and Asians) in the United States. Additionally, we performed network and pathway analysis to identify gene networks and pathways. Gene expression data involved 198 samples distributed as follows: 126 Whites, 51 Hispanics, 13 Blacks, and 8 Asians. We identified 300 highly significantly (P < 0.001) differentially expressed genes between the four ethnic populations. Among the identified genes included the genes PHF6, BRD3, CRLF2, and RNF135 which have been implicated in pediatric B-ALL. We identified key pathways implicated in B-ALL including the PDGF, PI3/AKT, ERBB2-ERBB3, and IL-15 signaling pathways.
doi:10.4137/CIN.S11831
PMCID: PMC3762614  PMID: 24023509
leukemia gene expression variation pediatric B-ALL
12.  17β-Estradiol Enhances Signalling Mediated by VEGF-A-Delta-Like Ligand 4-Notch1 Axis in Human Endothelial Cells 
PLoS ONE  2013;8(8):e71440.
Estrogens play a protective role in coronary artery disease. The mechanisms of action are still poorly understood, although a role for estrogens in stimulation of angiogenesis has been suggested. In several cell types, estrogens modulate the Notch pathway, which is involved in controlling angiogenesis downstream of vascular endothelial growth factor A (VEGF-A). The goal of our study was to establish whether estrogens modulate Notch activity in endothelial cells and the possible consequences on angiogenesis. Human umbilical vein endothelial cells (HUVECs) were treated with 17β-estradiol (E2) and the effects on Notch signalling were evaluated. E2 increased Notch1 processing as indicated by i) decreased levels of Notch1 transmembrane subunit ii) increased amount of Notch1 in nuclei iii) unaffected level of mRNA. Similarly, E2 increased the levels of the active form of Notch4 without altering Notch4 mRNA. Conversely, protein and mRNA levels of Notch2 were both reduced suggesting transcriptional repression of Notch2 by E2. Under conditions where Notch was activated by upregulation of Delta-like ligand 4 (Dll4) following VEGF-A treatment, E2 caused a further increase of the active form of Notch1, of the number of cells with nuclear Notch1 and of Hey2 mRNA. Estrogen receptor antagonist ICI 182.780 antagonized these effects suggesting that E2 modulation of Notch1 is mediated by estrogen receptors. E2 treatment abolished the increase in endothelial cells sprouting caused by Notch inhibition in a tube formation assay on 3D Matrigel and in mouse aortic ring explants. In conclusion, E2 affects several Notch pathway components in HUVECs, leading to an activation of the VEGF-A-Dll4-Notch1 axis and to a modulation of vascular branching when Notch signalling is inhibited. These results contribute to our understanding of the molecular mechanisms of cardiovascular protection exerted by estrogens by uncovering a novel role of E2 in the Notch signalling-mediated modulation of angiogenesis.
doi:10.1371/journal.pone.0071440
PMCID: PMC3742772  PMID: 23967210
13.  Mitochondrial Dysfunction Promotes Breast Cancer Cell Migration and Invasion through HIF1α Accumulation via Increased Production of Reactive Oxygen Species 
PLoS ONE  2013;8(7):e69485.
Although mitochondrial dysfunction has been observed in various types of human cancer cells, the molecular mechanism underlying mitochondrial dysfunction mediated tumorigenesis remains largely elusive. To further explore the function of mitochondria and their involvement in the pathogenic mechanisms of cancer development, mitochondrial dysfunction clones of breast cancer cells were generated by rotenone treatment, a specific inhibitor of mitochondrial electron transport complex I. These clones were verified by mitochondrial respiratory defect measurement. Moreover, those clones exhibited increased reactive oxygen species (ROS), and showed higher migration and invasive behaviors compared with their parental cells. Furthermore, antioxidant N-acetyl cysteine, PEG-catalase, and mito-TEMPO effectively inhibited cell migration and invasion in these clones. Notably, ROS regulated malignant cellular behavior was in part mediated through upregulation of hypoxia-inducible factor-1 α and vascular endothelial growth factor. Our results suggest that mitochondrial dysfunction promotes cancer cell motility partly through HIF1α accumulation mediated via increased production of reactive oxygen species.
doi:10.1371/journal.pone.0069485
PMCID: PMC3726697  PMID: 23922721
14.  Novel Integrative Genomics Approach for Associating GWAS Information with Intrinsic Subtypes of Breast Cancer 
Cancer Informatics  2013;12:125-142.
Genome-wide association studies (GWAS) have achieved great success in identifying common variants associated with increased risk of developing breast cancer. However, GWAS do not typically provide information about the broader context in which genetic variants operate in different subtypes of breast cancer. The objective of this study was to determine whether genes containing single nucleotide polymorphisms (SNPs, herein called genetic variants) are associated with different subtypes of breast cancer. Additionally, we sought to identify gene regulator networks and biological pathways enriched for these genetic variants. Using supervised analysis, we identified 201 genes that were significantly associated with the six intrinsic subtypes of breast cancer. The results demonstrate that integrative genomics analysis is a powerful approach for linking GWAS information to distinct disease states and provide insights about the broader context in which genetic variants operate in different subtypes of breast cancer.
doi:10.4137/CIN.S11452
PMCID: PMC3663490  PMID: 23761956
GWAS subtypes breast cancer
15.  EGCG, a major green tea catechin suppresses breast tumor angiogenesis and growth via inhibiting the activation of HIF-1α and NFκB, and VEGF expression 
Vascular Cell  2013;5:9.
The role of EGCG, a major green tea catechin in breast cancer therapy is poorly understood. The present study tests the hypothesis that EGCG can inhibit the activation of HIF-1α and NFκB, and VEGF expression, thereby suppressing tumor angiogenesis and breast cancer progression. Sixteen eight-wk-old female mice (C57BL/6 J) were inoculated with 10^6 E0771 (mouse breast cancer) cells in the left fourth mammary gland fat pad. Eight mice received EGCG at 50–100 mg/kg/d in drinking water for 4 weeks. 8 control mice received drinking water only. Tumor size was monitored using dial calipers. At the end of the experiment, blood samples, tumors, heart and limb muscles were collected for measuring VEGF expression using ELISA and capillary density (CD) using CD31 immunohistochemistry. EGCG treatment significantly reduced tumor weight over the control (0.37 ± 0.15 vs. 1.16 ± 0.30 g; P < 0.01), tumor CD (109 ± 20 vs. 156 ± 12 capillary #/mm^2; P < 0.01), tumor VEGF expression (45.72 ± 1.4 vs. 59.03 ± 3.8 pg/mg; P < 0.01), respectively. But, it has no effects on the body weight, heart weight, angiogenesis and VEGF expression in the heart and skeletal muscle of mice. EGCG at 50 μg/ml significantly inhibited the activation of HIF-1α and NFκB as well as VEGF expression in cultured E0771 cells, compared to the control, respectively. These findings support the hypothesis that EGCG, a major green tea catechin, directly targets both tumor cells and tumor vasculature, thereby inhibiting tumor growth, proliferation, migration, and angiogenesis of breast cancer, which is mediated by the inhibition of HIF-1α and NFκB activation as well as VEGF expression.
doi:10.1186/2045-824X-5-9
PMCID: PMC3649947  PMID: 23638734
16.  Elevated Jagged-1 and Notch-1 expression in high grade and metastatic prostate cancers 
Background: Emerging evidence has suggested that Notch signaling pathway may be involved in the development, progression and metastasis of prostate cancer (PCa). In the present study, we investigated the expression levels of Jagged-1 and Notch-1 in human prostate tumors and their associations with PCa progression and metastasis. Methods: Immunohistochemistry (IHC) for Jagged-1 and Notch-1 was performed on tissue microarray (TMA) slides containing 286 formalin-fixed and paraffin-embedded (FFPE) tissue specimens with various prostatic pathologies, including benign changes, high grade prostatic intraepithelial neoplasia (HGPIN), low- and high-grade PCas as well as metastatic PCa. Results: Cytoplasmic and membranous IHC scores for Jagged-1 in both metastatic PCa and high grade PCa were significantly higher than those in low grade PCa and in benign prostatic tissues. Similarly, cytoplasmic IHC scores of Notch-1 in both metastatic PCa and high grade PCa were significantly elevated compared with those observed in low grade PCa and in benign prostatic tissues. A statistically significant correlation was identified between the expression of Jagged-1 and Notch-1 in human prostatic tissues. Furthermore, significantly more highly expressed Jagged-1 in membrane was observed in Caucasian patients with high-grade or metastatic PCa (vs. African Americans) and in PCa patients with positive surgical margins (vs. negative surgical margins). Conclusion: Our results provide strong evidence that up-regulation of Jagged1-Notch1 signaling plays a role in PCa progression and metastasis and suggest that Jagged-1 and Notch-1 may be useful markers in distinguishing indolent and aggressive PCas.
PMCID: PMC3633979  PMID: 23634247
Prostate cancer (PCa); cancer metastasis; Jagged-1; Notch-1; tissue microarray (TMA); immunohistochemistry (IHC)
17.  Correlation of Notch1, pAKT and nuclear NF-κB expression in triple negative breast cancer 
Gene expression profiling reveals elevated Notch1 mRNA expression in triple negative breast cancers (TNBC), both basaloid and claudin-low subtypes. Notch ligands, Jagged1 and Jagged2, have been correlated with poor prognosis in TNBC. AKT, an oncogenic protein kinase family that is activated downstream of Notch in breast cancer cell lines, is frequently activated in breast cancer. Recent publications suggest that inhibition of cell growth, migration, invasion, and induction of apoptosis caused by Notch1 or Jagged1 inhibition may be attributed in part to inactivation of the AKT signaling pathway. There is significant evidence that Notch1 activates NF-κB in several models, and that AKT can mediate NF-κB activation. In this study, we evaluated Notch1 protein expression by immunohistochemistry (IHC) and correlated this with expression of pAKT and nuclear NF-κB p65 (RelA) in TNBC. A tissue microarray (TMA) containing 32 formalin-fixed, paraffin-embedded (FFPE) TNBC tumor specimens was constructed from the archival tissue database of the Department of Pathology at UMMC and IHC for Notch1 protein, pAKT 1/2/3 (Ser473), and NF-κB, p65 subunit was performed on the TMA with appropriate positive and negative controls. Of the 32 TNBC in our cohort, 100% expressed Notch1 protein by IHC: 24 (75%) showed cytoplasmic expression, 25 (78%) showed membranous expression, and 17 (53%) showed both cytoplasmic and membranous expression. Overall, 29 (91%) expressed pAKT by IHC: 28 (97%) showed cytoplasmic expression, 14 (48%) showed nuclear expression and 13 (45%) showed both cytoplasmic and nuclear expression. Nuclear staining for NF-κB p65 was detected in all 32 TNBC specimens with variable intensities. On bivariate analysis, cytoplasmic Notch1 was significantly correlated with cytoplasmic pAKT (r = 0.373, P = 0.035) and nuclear NF-κB (r = 0.483, P = 0.005); both cytoplasmic and nuclear pAKT significantly correlated with nuclear NF-κB (r = 0.391, P = 0.027; r = 0.525, P = 0.002, respectively). These results suggest that 1) the cross-talk between Notch1, AKT and NF-κB identified in preclinical models may operate in a significant fraction of human TNBC, and 2) combination therapy with agents targeting these pathways warrants further investigation.
PMCID: PMC3623841  PMID: 23593544
Triple negative breast cancers (TNBC); Notch1; AKT; NF-κB; immunohistochemistry (IHC); tissue microarray (TMA)
18.  Identifying Biomarkers and Drug Targets Using Systems Biology Approaches for Pancreatic Cancer 
Pancreatic disorders & therapy  2012;2(4):1000e128.
PMCID: PMC3559026  PMID: 23378937
Biomarkers; Drug target; Microarray; MicroRNA; Pancreatic cancer; Systems biology
19.  An Integrative Genomics Approach for Associating GWAS Information with Triple-Negative Breast Cancer 
Cancer Informatics  2013;12:1-20.
Genome-wide association studies (GWAS) have identified genetic variants associated with an increased risk of developing breast cancer. However, the association of genetic variants and their associated genes with the most aggressive subset of breast cancer, the triple-negative breast cancer (TNBC), remains a central puzzle in molecular epidemiology. The objective of this study was to determine whether genes containing single nucleotide polymorphisms (SNPs) associated with an increased risk of developing breast cancer are connected to and could stratify different subtypes of TNBC. Additionally, we sought to identify molecular pathways and networks involved in TNBC. We performed integrative genomics analysis, combining information from GWAS studies involving over 400,000 cases and over 400,000 controls, with gene expression data derived from 124 breast cancer patients classified as TNBC (at the time of diagnosis) and 142 cancer-free controls. Analysis of GWAS reports produced 500 SNPs mapped to 188 genes. We identified a signature of 159 functionally related SNP-containing genes which were significantly (P <10−5) associated with and stratified TNBC. Additionally, we identified 97 genes which were functionally related to, and had similar patterns of expression profiles, SNP-containing genes. Network modeling and pathway prediction revealed multi-gene pathways including p53, NFkB, BRCA, apoptosis, DNA repair, DNA mismatch, and excision repair pathways enriched for SNPs mapped to genes significantly associated with TNBC. The results provide convincing evidence that integrating GWAS information with gene expression data provides a unified and powerful approach for biomarker discovery in TNBC.
doi:10.4137/CIN.S10413
PMCID: PMC3565545  PMID: 23423317
triple negative breast cancer GWAS gene expression
20.  Notch signaling in pediatric soft tissue sarcomas 
BMC Medicine  2012;10:141.
Pediatric soft tissue sarcomas are rare tumors of childhood, frequently characterized by specific chromosome translocations. Despite improvements in treatment, their clinical management is often challenging due to the low responsiveness of metastatic forms and aggressive variants to conventional therapeutic approaches, which leads to poor overall survival. It is widely thought that soft tissue sarcomas derive from mesenchymal progenitor cells that, during embryonic life, have developed chromosomal aberrations with de-regulation of the main pathways governing tissue morphogenesis. The Notch signaling pathway is one of the most important molecular networks involved in differentiation processes. Emerging evidence highlights the role of Notch signaling de-regulation in the biology of these pediatric sarcomas. In this review, we present an outline of recently gathered evidence on the role of Notch signaling in soft tissue sarcomas, highlighting its importance in tumor cell biology. The potential challenges and opportunities of targeting Notch signaling in the treatment of pediatric soft tissue sarcomas are also discussed.
doi:10.1186/1741-7015-10-141
PMCID: PMC3520771  PMID: 23158439
soft tissue sarcoma; Notch; mesenchymal cells; γ-secretase; Synovial sarcoma; Ewing sarcoma; Rhabdomyosarcoma
21.  The Role of EMT in Pancreatic Cancer Progression 
doi:10.4172/2165-7092.1000e121
PMCID: PMC3491903  PMID: 23145368
Pancreatic cancer; EMT; Stem cell; MicroRNA; Natural compounds
22.  Dicer-Mediated Upregulation of BCRP Confers Tamoxifen Resistance in Human Breast Cancer Cells 
Clinical Cancer Research  2011;17(20):6510-6521.
Purpose
Tamoxifen (Tam) is the most prescribed hormonal agent for treatment of estrogen receptor α-positive breast cancer patients. Using microarray analysis, we observed that metastatic breast tumors resistant to Tam therapy had elevated levels of Dicer.
Experimental Design
We overexpressed Dicer in ERα-positive MCF-7 human breast cancer cells, and observed a concomitant increase in expression of the breast cancer resistance protein BCRP. We thus hypothesized that Tam resistance associated with Dicer overexpression in ERα-positive breast cancer cells may involve BCRP. We analyzed BCRP function in Dicer-overexpressing cells using growth in soft agar and mammosphere formation, and evaluated intracellular Tam efflux.
Results
In the presence of Tam, Dicer-overexpressing cells formed resistant colonies in soft agar, and treatment with BCRP inhibitors restored Tam sensitivity. Tumor xenograft studies confirmed that Dicer-overexpressing cells were resistant to Tam in vivo. Tumors and distant metastases could be initiated with as few as 5 mammosphere cells from both vector and Dicer-overexpressing cells, indicating that the mammosphere assay selected for cells with enhanced tumor initiating and metastatic capacity. Dicer-overexpressing cells with elevated levels of BCRP, effluxed Tam more efficiently than control cells, and BCRP inhibitors were able to inhibit efflux.
Conclusion
Dicer-overexpressing breast cancer cells enriched for cells with enhanced BCRP function. We hypothesize that it is this population which may be involved in the emergence of Tam-resistant growth. BCRP may be a novel clinical target to restore Tam sensitivity.
doi:10.1158/1078-0432.CCR-11-1403
PMCID: PMC3281508  PMID: 21878538
Tamoxifen; Breast Cancer; Breast Cancer Resistance Protein; Dicer; Hormone Resistance
24.  Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells 
Journal of cellular biochemistry  2011;112(9):2296-2306.
FoxM1 is known to play important role in the development and progression of many malignancies including pancreatic cancer. Studies have shown that the acquisition of Epithelial-to-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotypes are highly inter-related, and contributes to drug resistance, tumor recurrence and metastasis. The molecular mechanism(s) by which FoxM1 contributes to the acquisition of EMT phenotype and induction of CSC self-renewal capacity is poorly understood. Therefore, we established FoxM1 over-expressing pancreatic cancer (AsPC-1) cells, which showed increased cell growth, clonogenicity and cell migration. Moreover, over-expression of FoxM1 led to the acquisition of EMT phenotype by activation of mesenchymal cell markers, ZEB1, ZEB2, Snail2, E-cadherin, and vimentin, which is consistent with increased sphere-forming (pancreatospheres) capacity and expression of CSC surface markers (CD44 and EpCAM). We also found that over-expression of FoxM1 led to decreased expression of miRNAs (let-7a, let-7b, let-7c, miR-200b and miR-200c); however, re-expression of miR-200b inhibited the expression of ZEB1, ZEB2, vimentin as well as FoxM1, and induced the expression of E-cadherin, leading to the reversal of EMT phenotype. Finally, we found that genistein, a natural chemo-preventive agent, inhibited cell growth, clonogenicity, cell migration and invasion, EMT phenotype, and formation of pancreatospheres consistent with reduced expression of CD44 and EpCAM. These results suggest, for the first time, that FoxM1 over-expression is responsible for the acquisition of EMT and CSC phenotype, which is in part mediated through the regulation of miR-200b and these processes, could be easily attenuated by genistein.
doi:10.1002/jcb.23150
PMCID: PMC3155646  PMID: 21503965
FoxM1; EMT phenotype; miRNAs; CSC-self renewal; genistein
25.  Notch-1 induces Epithelial-mesenchymal transition consistent with cancer stem cell phenotype in pancreatic cancer cells 
Cancer letters  2011;307(1):26-36.
Activation of Notch-1 is known to be associated with the development and progression of human malignancies including pancreatic cancer. Emerging evidence suggest that the acquisition of epithelial-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotype are interrelated and contributes to tumor recurrence and drug resistance. The molecular mechanism(s) by which Notch-1 contributes to the acquisition of EMT phenotype and CSC self-renewal capacity has not been fully elucidated. Here we show that forced over-expression of Notch-1 leads to increased cell growth, clonogenicity, migration and invasion of AsPC-1 cells. Moreover, over-expression of Notch-1 led to the induction of EMT phenotype by activation of mesenchymal cell markers such as ZEB1, CD44, EpCAM, and Hes 1. Here we also report, for the first time, that over-expression of Notch-1 leads to increased expression of miR-21, and decreased expression of miR-200b, miR-200c, let-7a, let-7b, and let-7c. Re-expression of miR-200b led to decreased expression of ZEB1, and vimentin, and increased expression of E-cadherin. Over-expression of Notch-1 also increased the formation of pancreatospheres consistent with expression of CSC surface markers CD44 and EpCAM. Finally, we found that genistein, a known natural anti-tumor agent inhibited cell growth, clonogenicity, migration, invasion, EMT phenotype, formation of pancreatospheres and expression of CD44 and EpCAM. These results suggest that the activation of Notch-1 signaling contributes to the acquisition of EMT phenotype, which is in part mediated through the regulation of miR-200b and CSC self-renewal capacity, and these processes could be attenuated by genistein treatment.
doi:10.1016/j.canlet.2011.03.012
PMCID: PMC3104092  PMID: 21463919
Notch-1; EMT phenotype; miRNAs; CSC-self renewal; genistein

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