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1.  Abrogation of p53 function leads to metastatic transcriptome networks that typify tumor progression in human breast cancer xenografts 
International journal of oncology  2010;37(5):1167-1176.
Development of chromosomal instability (CIN) and consequent phenotypic heterogeneity represent common events during breast cancer progression. Breast carcinomas harboring extensive chromosomal aberrations display a more aggressive behavior characterized by chemoresistance and the propensity to give rise to distant metastases. The tumor suppressor p53 plays a key role in the maintenance of chromosomal stability and tissue homeostasis through activation of cell cycle checkpoints following DNA damage and control of centrosome duplication that ensures equal chromosome segregation during cell division. Furthermore, p53 suppresses CD44 expression and the acquisition of stem cell-like properties responsible for epithelial to mesenchymal transition (EMT) and metastasis. In this study we employed MCF-7 breast cancer cells with endogenous wild-type p53, an engineered MCF-7 variant (vMCF-7DNP53) overexpressing a dominant negative p53val135 mutant, and cells re-cultured from vMCF-7DNP53 tumor xenografts. We carried out an integrative transcriptome and cytogenetic analysis to characterize the mechanistic linkage between loss of p53 function, EMT and consequent establishment of invasive gene signatures during breast cancer progression. We demonstrate that abrogation of p53 function drives the early transcriptome changes responsible for cell proliferation, EMT and survival, while further transcriptome changes that occur during in vivo tumor progression are mechanistically linked to the development of CIN leading to a more invasive and metastatic breast cancer phenotype. Here we identified distinct novel non-canonical transcriptome networks involved in cell proliferation, EMT, chemoresistance and invasion that arise following abrogation of p53 function in vitro and development of CIN in vivo. These studies also have important translational implications since some of the nodal genes identified here are ‘druggable’ making them appropriate molecular targets for the treatment of breast carcinomas displaying mutant p53, EMT, CIN and high metastatic potential.
PMCID: PMC4312700  PMID: 20878064
breast cancer; centrosome amplification; chromosomal instability; epithelial mysenchymal transition; invasive gene networks; tumor cell heterogeneity; tumor progression
2.  Raf-1 oncogenic signaling is linked to activation of mesenchymal to epithelial transition pathway in metastatic breast cancer cells 
International journal of oncology  2012;40(6):1858-1864.
Aberrant activation of the Raf/MEK/MAPK pathway plays a key role in breast cancer development and progression. Dysregulation of Raf/MEK/MAPK oncogenic signaling often results from overexpression of the HER-2/Neu tyrosine kinase receptor leading to chemoendocrine resistance, development of distant metastases and ultimately poor prognosis in breast cancer patients. HER-2/Neu overexpression is also linked to activation of the epithelial to mesenchymal transition (EMT) pathway, loss of adhesion molecules and metastasis. Recently, it has been demonstrated that cancer cells that undergo EMT acquire a CD44+/CD24−/low basal cancer stem cell-like phenotype and are characterized by activation of HER-2/Neu and TGFβ oncogenic signaling pathways with increased capacity of self-renewal, drug resistance, invasion and distant metastases. Following meta-static dissemination, cancer cells re-activate certain epithelial properties through mesenchymal to epithelial transition (MET) to establish neoplastic lesions at secondary sites, although the molecular mechanisms regulating MET remain elusive. In this study we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces HER-2/Neu overexpression leading to the development of distant metastases in ERα+ MCF-7 breast cancer xenografts. Importantly, development of distant metastases in xenograft models was linked to activation of the MET pathway characterized by reduced expression of EMT inducer genes (TGFB2, TWIST1 and FOXC1) and overexpression of BMB7, CXCR7 and EGR family of transcription factors. In summary, our results demonstrate for the first time that amplification of Raf/MEK/MAPK oncogenic signaling during tumor growth promotes the genesis of metastatic lesions from primary tumors by activating the mesenchymal epithelial transition.
PMCID: PMC4058770  PMID: 22447278
Raf-1; breast cancer; Raf/MEK/MAPK pathway; HER-2/Neu; tyrosine kinase receptor
3.  Personalized therapy for metastatic melanoma: could timing be everything? 
Future oncology (London, England)  2012;8(11):1401-1406.
There is ample evidence that immune-related processes in humans are under temporal regulation. The circadian variation of humoral and cellular immunity is well documented and appears to be hormonally modulated via the hypothalamic–pituitary–adrenal axis. In advanced melanoma, it has recently been demonstrated that systemic immunity is repolarized toward a global state of chronic inflammation (Th2 dominance) and appears to be governed by infradian biorhythms of cytokines and immune cell subsets, which extend beyond the 24-h circadian variability reported in healthy volunteers. It is suggested that synchronizing administration of lymphodepleting chemotherapy (temozolomide) with these endogenous (individualized) immune dynamics (biorhythms) in patients with advanced/metastatic melanoma improves clinical outcomes compared with temozolomide used in a conventional ‘random delivery’ fashion.
PMCID: PMC4012533  PMID: 23148614
biorhythms; cancer; chemotherapy; cytokine; immunity; infradian; melanoma
4.  Impaired tissue regeneration corresponds with altered expression of developmental genes that persists in the metabolic memory state of diabetic zebrafish 
As previously reported by our lab streptozocin-induced Diabetes mellitus (DM) in adult zebrafish results in an impairment of tissue regeneration as monitored by caudal fin regeneration. Following streptozocin withdrawal, a recovery phase occurs to re-establish euglycemia, via pancreatic beta-cell regeneration. However, DM-associated impaired fin regeneration continues indefinitely in the metabolic memory state (MM) allowing for subsequent molecular analysis of the underlying mechanisms of MM. This study focuses on elucidating the molecular basis that explains the DM-associated impaired fin regeneration and why it persists into the MM state with the aim of better understanding MM. Using a combination of microarray analysis and bioinformatics approaches our study found that of the 14,900 transcripts analyzed, aberrant expression of 71 genes relating to tissue developmental and regeneration processes were identified in DM fish and the altered expression of these 71 genes persisted in MM fish. Key regulatory genes of major development, and signal transduction pathways were identified among this group of 71. The aberrant expression of key regulatory genes in the DM state that persist into the MM state provides a plausible explanation for how hyperglycemia induced impaired fin regeneration in the adult zebrafish DM/MM model.
PMCID: PMC3594430  PMID: 23438205
diabetes; metabolic memory; regeneration; zebrafish; epigenetics
5.  Autophagy Control by the VEGF-C/NRP-2 axis in Cancer and its Implication for Treatment Resistance 
Cancer research  2012;73(1):160-171.
A major contributor to cancer mortality is recurrence and subsequent metastatic transformation following therapeutic intervention. Therefore, in order to develop new treatment modalities and improve the efficacy of current ones, it is important to understand the molecular mechanisms that promote resistance to therapy in cancer cells. One pathway contributing to therapy resistance is autophagy, a self-digestive process that can eliminate unnecessary or damaged organelles to protect cancer cells from death. We have found that the VEGF-C/NRP-2 axis is involved in the activation of autophagy, which helps cancer cell survival following treatment. Inhibition of mTOR complex 1 activity by this axis is the underlying mechanism for the activation of autophagy. Furthermore, we identified two VEGF-C/NRP-2-regulated genes, LAMP-2 and WDFY-1 that have previously been suggested to participate in autophagy and vesicular trafficking. Up-regulation of WDFY-1 following VEGF-C or NRP-2 depletion contributes to cytotoxic drug-mediated cell death. Together, these data suggest a link between the VEGF-C axis and cancer cell survival despite the presence of chemotherapy-induced stress. Effective targeting of this pathway may lead to the development of new cancer therapies.
PMCID: PMC3805049  PMID: 23149913
VEGF-C; NRP-2; autophagy; therapy resistance
6.  The immunomodulatory effects of bevacizumab on systemic immunity in patients with metastatic melanoma 
Oncoimmunology  2013;2(5):e24436.
Metastatic melanoma is near-to-invariably a fatal disease. As novel therapeutic strategies against metastatic melanoma are urgently needed, we have tested a combinatorial regimen consisting of conventional chemotherapy coupled to bevacizumab, a monoclonal antibody that inhibit angiogenesis, demonstrating some clinical benefit. A preliminary assessment of one of our clinical trials points to a previously unrecognized immunomodulatory effect of bevacizumab. Herein, we evaluate the immunomodulatory effect of bevacizumab when administered together with conventional chemotherapy to patients with metastatic melanoma. To this aim, we measured the abundance of various lymphocyte subsets among peripheral blood mononuclear cells (PBMCs) as well as the circulating levels of 42 cytokines, chemokines and growth factors in patients with metastatic melanoma who received albumin-bound paclitaxel plus carboplatin, either as a standalone intervention (AC, 55 subjects) or combined with bevacizumab (ACB, 39 individuals), in the context of clinical trials N057e and N0775, respectively. Relative shifts in PBMC subsets and cytokine levels were calculated (relative to baseline levels) when patients underwent restaging evaluation after two cycles of therapy. The Mann–Whitney U test was used to compare responses between the groups. Bevacizumab failed to affect the TH1/TH2 cell ratio in this patient cohort. However, we observed a significant increase in CD8+ lymphocytes in patients who received ACB (+38%) but not in subjects treated with AC only (−10%) (p = 0.03). Moreover, circulating interleuikin-6 (IL-6) levels were reduced in patients treated with ACB (−42%) but not in individuals receiving AC only (28%) (p = 0.0018). Thus, the addition of bevacizumab to chemotherapy for the treatment of metastatic melanoma exerts immunomodulatory effects.
PMCID: PMC3667915  PMID: 23762809
bevacizumab; cytotoxic T cells; IL-6; immunity; metastatic melanoma
7.  Inhibition of Cdk2 activity decreases Aurora-A kinase centrosomal localization and prevents centrosome amplification in breast cancer cells 
Oncology Reports  2013;29(5):1785-1788.
Centrosome amplification plays a key role in the origin of chromosomal instability (CIN) during cancer development and progression. In this study, MCF-7 breast cancer cell lines harboring abrogated p53 function (vMCF-7DNp53) were employed to investigate the relationship between induction of genotoxic stress, activation of cyclin-A/Cdk2 and Aurora-A oncogenic signalings and development of centrosome amplification. Introduction of genotoxic stress in the vMCF-7DNp53 cell line by treatment with hydroxyurea (HU) induced centrosome amplification that was mechanistically linked to Aurora-A kinase activity. In cells carrying defective p53, the development of centrosome amplification also occurred following treatment with another DNA damaging agent, methotrexate. Importantly, we demonstrated that Aurora-A kinase-induced centrosome amplification was mediated by Cdk2 kinase since molecular inhibition of Cdk2 activity by SU9516 suppressed Aurora-A centrosomal localization and consequent centrosome amplification. In addition, we employed vMCF-7DRaf-1 cells that display high levels of endogenous cyclin-A and demonstrated that molecular targeting of Aurora-A by Alisertib reduces cyclin-A expression. Taken together, these findings demonstrate a novel positive feed-back loop between cyclin-A/Cdk2 and Aurora-A pathways in the development of centrosome amplification in breast cancer cells. They also provide the translational rationale for targeting ‘druggable cell cycle regulators’ as an innovative therapeutic strategy to inhibit centrosome amplification and CIN in breast tumors resistant to conventional chemotherapeutic drugs.
PMCID: PMC3658847  PMID: 23446853
breast cancer; centrosome amplification; Aurora-A; Cdk2; genotoxic stress
8.  Heritable Transmission of Diabetic Metabolic Memory in Zebrafish Correlates With DNA Hypomethylation and Aberrant Gene Expression 
Diabetes  2012;61(2):485-491.
Metabolic memory (MM) is the phenomenon whereby diabetes complications persist and progress after glycemic recovery is achieved. Here, we present data showing that MM is heritable and that the transmission correlates with hyperglycemia-induced DNA hypomethylation and aberrant gene expression. Streptozocin was used to induce hyperglycemia in adult zebrafish, and then, following streptozocin withdrawal, a recovery phase was allowed to reestablish a euglycemic state. Blood glucose and serum insulin returned to physiological levels during the first 2 weeks of the recovery phase as a result of pancreatic β-cell regeneration. In contrast, caudal fin regeneration and skin wound healing remained impaired to the same extent as in diabetic fish, and this impairment was transmissible to daughter cell tissue. Daughter tissue that was never exposed to hyperglycemia, but was derived from tissue that was, did not accumulate AGEs or exhibit increased levels of oxidative stress. However, CpG island methylation and genome-wide microarray expression analyses revealed the persistence of hyperglycemia-induced global DNA hypomethylation that correlated with aberrant gene expression for a subset of loci in this daughter tissue. Collectively, the data presented here implicate the epigenetic mechanism of DNA methylation as a potential contributor to the MM phenomenon.
PMCID: PMC3266410  PMID: 22228713
9.  Biosignatures for Parkinson’s Disease and Atypical Parkinsonian Disorders Patients 
PLoS ONE  2012;7(8):e43595.
Diagnosis of Parkinson’ disease (PD) carries a high misdiagnosis rate due to failure to recognize atypical parkinsonian disorders (APD). Usually by the time of diagnosis greater than 60% of the neurons in the substantia nigra are dead. Therefore, early detection would be beneficial so that therapeutic intervention may be initiated early in the disease process. We used splice variant-specific microarrays to identify mRNAs whose expression is altered in peripheral blood of early-stage PD patients compared to healthy and neurodegenerative disease controls. Quantitative polymerase chain reaction assays were used to validate splice variant transcripts in independent sample sets. Here we report a PD signature used to classify blinded samples with 90% sensitivity and 94% specificity and an APD signature that resulted in a diagnosis with 95% sensitivity and 94% specificity. This study provides the first discriminant functions with coherent diagnostic signatures for PD and APD. Analysis of the PD biomarkers identified a regulatory network with nodes centered on the transcription factors HNF4A and TNF, which have been implicated in insulin regulation.
PMCID: PMC3428307  PMID: 22952715
10.  Deep Sequence Analysis of Non-Small Cell Lung Cancer: Integrated Analysis of Gene Expression, Alternative Splicing, and Single Nucleotide Variations in Lung Adenocarcinomas with and without Oncogenic KRAS Mutations 
KRAS mutations are highly prevalent in non-small cell lung cancer (NSCLC), and tumors harboring these mutations tend to be aggressive and resistant to chemotherapy. We used next-generation sequencing technology to identify pathways that are specifically altered in lung tumors harboring a KRAS mutation. Paired-end RNA-sequencing of 15 primary lung adenocarcinoma tumors (8 harboring mutant KRAS and 7 with wild-type KRAS) were performed. Sequences were mapped to the human genome, and genomic features, including differentially expressed genes, alternate splicing isoforms and single nucleotide variants, were determined for tumors with and without KRAS mutation using a variety of computational methods. Network analysis was carried out on genes showing differential expression (374 genes), alternate splicing (259 genes), and SNV-related changes (65 genes) in NSCLC tumors harboring a KRAS mutation. Genes exhibiting two or more connections from the lung adenocarcinoma network were used to carry out integrated pathway analysis. The most significant signaling pathways identified through this analysis were the NFκB, ERK1/2, and AKT pathways. A 27 gene mutant KRAS-specific sub network was extracted based on gene–gene connections from the integrated network, and interrogated for druggable targets. Our results confirm previous evidence that mutant KRAS tumors exhibit activated NFκB, ERK1/2, and AKT pathways and may be preferentially sensitive to target therapeutics toward these pathways. In addition, our analysis indicates novel, previously unappreciated links between mutant KRAS and the TNFR and PPARγ signaling pathways, suggesting that targeted PPARγ antagonists and TNFR inhibitors may be useful therapeutic strategies for treatment of mutant KRAS lung tumors. Our study is the first to integrate genomic features from RNA-Seq data from NSCLC and to define a first draft genomic landscape model that is unique to tumors with oncogenic KRAS mutations.
PMCID: PMC3356053  PMID: 22655260
transcriptome sequencing; RNA-Seq; KRAS mutation; NSCLC; bioinformatics; network analysis; data integration and computational methods
11.  MicroRNA Expression in Ileal Carcinoid Tumors: Down-regulation of MicroRNA-133a with Tumor Progression 
MicroRNAs are involved in cell proliferation, differentiation, and apoptosis and can function as tumor suppressor genes or oncogenes. The role of microRNAs in neuroendocrine tumors such as ileal carcinoids is largely unknown. We examined the differential expression of 95 microRNAs by RT-PCR using the QuantiMir System in eight matching primary and metastatic carcinoid tumors from the ileum. All microRNAs chosen for the QuantiMir System Array were based on their potential functions related to cancer biology, cell development and apoptosis. The expression of microRNAs for the samples was normalized to microRNA-197, and the matching primary and metastatic tumors were compared. There was down-regulation of microRNA-133a, 145, 146, 222 and 10b in all samples between the primary and matching metastatic tumors and up-regulation of microRNA-183, 488 and 19a + b in six of eight metastatic carcinoids compared to the primary tumors. MicroRNA-133a was further analyzed by TaqMan Real Time RT-PCR and Northern hybridization using six additional matching primary and metastatic samples which supported the PCR Array findings. There were significant differences in microRNA-133a expression with down-regulation in the metastasis compared to the primary in the eight original cases (p<0.009) and in the six additional cases used for validation (p<0.014). Laser capture microdissection and Real Time RT-PCR analysis using normal ileum found microRNA-133a expression in normal enterochromaffin cells. In situ hybridization in normal ileum showed that some of the mucosal endocrine cells expressed microRNA-133a. Both primary and metastatic ileal carcinoid tumors expressed microRNA-133a by in situ hybridization. These results provide information about novel marker microRNAs that may be used as biomarkers and/or therapeutic targets in intestinal carcinoid tumors.
PMCID: PMC2886953  PMID: 20037573
PCR array; carcinoids; enterochromaffin cells; RT-PCR; in situ hybridization
12.  Evidence of systemic Th2 driven chronic inflammation in patients with metastatic melanoma 
Immunotherapeutic modalities are commonly used for treatment of patients with melanoma. The therapeutic success in pre-clinical models has not yielded the expected clinical results. To understand this discrepancy, we attempted to define immune homeostasis of 209 patients with melanoma across stages of disease relative to normal controls.
Patients and Methods
PBMC and plasma were collected from patients and healthy donors. PBMC were analyzed for frequencies of natural killer, dendritic, and T-cells, and their functional status. Matched plasma samples were analyzed for the concentrations of 27 cytokines, chemokines, and growth factors. RNA was isolated from 24 metastatic melanoma tumor biopsies and profiled by microarray analysis.
The frequency of NK, T-cells, and DC in patients does not significantly change across stages of melanoma. However, plasma concentrations of Th2 cytokines (IL-4, IL-5, IL-10 and IL-13) in tumor bearing patients were significantly higher than those with resected melanoma. Expression array analysis of metastatic melanoma revealed that the malignant melanocytes were not the source of the Th2 cytokines, but did highly up-regulate VEGF transcripts, consistent with plasma VEGF concentrations. In vitro VEGF exposure of normal PBMC lead to re-polarization from Th1 to Th2 emulating the state of metastatic melanoma.
Patients with metastatic melanoma exist in a state of Th2 mediated “chronic inflammation” as a result of at least VEGF overproduction by malignant tumors. These data support prior observations regarding the impact of VEGF on immune cell function and suggests consideration of VEGF inhibitors in future cancer immunotherapy clinical studies in metastatic melanoma.
PMCID: PMC2757059  PMID: 19240164
Melanoma; Inflammation; Cytokines
The signs and symptoms of Graves’ ophthalmopathy (GO) result from inflammation and increased volume of the orbital adipose tissues and extraocular muscles.
To identify differentially regulated genes that may be involved in stimulating the orbital adipose tissue expansion seen in GO.
Gene expression profiling was used to compare genes expressed in orbital adipose tissues from GO patients and normal individuals.
Private practice tertiary referral center.
Orbital adipose tissues were collected at transantral orbital decompression surgery from 20 euthyroid patients undergoing this procedure for severe GO and at early autopsy from 8 normal individuals having no evidence of thyroid or ocular disease.
Of the 12,686 genes analyzed, 25 known genes were increased in expression (> 4 fold) in GO orbital tissues, while 11 genes were decreased (> 4 fold). Upregulated genes, confirmed by quantitative RT-PCR, included secreted frizzled related protein-1 (sFRP-1; 18.5 fold) and several adipocyte-related genes, including peroxisome proliferator activated receptor-γ (44.1 fold) and adiponectin (25 fold). Treatment in vitro of GO orbital preadipocytes with recombinant sFRP-1 (100 nM) significantly increased adiponectin (2.0 fold; p<.05), leptin (7 fold; p<.002), and thyrotropin receptor mRNA (13 fold; p<.003) levels, and enhanced Oil red-O staining in the cultures.
These results support the concept that orbital adipogenesis is enhanced in GO, and suggest that elevated levels of sFRP-1 in the GO orbit may be involved in stimulating this pathogenic process.
PMCID: PMC1236982  PMID: 15886250
sFRP-1; Graves’ disease; Graves’ ophthalmopathy; adipogenesis

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