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1.  Ex Vivo 4D Lung Cancer Model Mimics Metastasis 
The Annals of thoracic surgery  2015;99(4):1149-1156.
Background
We have developed a 4D lung cancer model that forms perfusable tumor nodules. We determined if the model could be modified to mimic metastasis.
Methods
We modified the 4D lung cancer model by seeding H1299, A549, or H460 cells via the trachea only to the left lobes of the acellular lung matrix. The model was modified so that the tumor cells can reach the right lobes of the acellular lung matrix only through the pulmonary artery as circulating tumor cells (CTC). We determined the gene expressions of the primary tumor, CTCs, and metastatic lesions using the Human OneArray chip.
Results
All cell lines formed a primary tumor in the left lobe of the ex vivo 4D lung cancer model. The CTCs were identified in the media and increased over time. All cell lines formed metastatic lesions with H460 forming significantly more metastatic lesions than H1299 and A549 cells. The CTC gene signature predicted poor survival in lung cancer patients. Unique genes were significantly expressed in CTC compared to the primary tumor and metastatic lesion.
Conclusions
The 4D lung cancer model can isolate tumor cells in three phases of tumor progression. This 4D lung cancer model may mimic the biology of lung cancer metastasis and may be used to determine its mechanism and potential therapy in the future.
doi:10.1016/j.athoracsur.2014.08.085
PMCID: PMC4387006  PMID: 25701100
4D model; lung cancer; Metastasis
2.  Fatty Acid Oxidation-Driven Src Links Mitochondrial Energy Reprogramming and Regulation of Oncogenic Properties in Triple Negative Breast Cancer 
Cell reports  2016;14(9):2154-2165.
Summary
Transmitochondrial cybrids and multiple OMICs approaches were used to understand mitochondrial reprogramming and mitochondria-regulated cancer pathways in triple negative breast cancer (TNBC). Analysis of cybrids and established breast cancer (BC) cell lines showed that metastatic TNBC maintains high levels of ATP through fatty acid β-oxidation (FAO) and activates Src oncoprotein through autophosphorylation at Y419. Manipulation of FAO including the knocking down of carnitine palmitoyltransferase-1 (CPT1) and 2 (CPT2), the rate-limiting proteins of FAO, and analysis of patient-derived xenograft models, confirmed the role of mitochondrial FAO in Src activation and metastasis. Analysis of TCGA and other independent BC clinical data further reaffirmed the role of mitochondrial FAO and CPT genes in Src regulation and their significance in BC metastasis.
doi:10.1016/j.celrep.2016.02.004
PMCID: PMC4809061  PMID: 26923594
3.  Multilevel Genomics-based Taxonomy of Renal Cell Carcinoma 
Cell reports  2016;14(10):2476-2489.
Summary
On the basis of multidimensional and comprehensive molecular characterization (including DNA methylation and copy number, and RNA and protein expression), we classified 894 renal cell carcinomas (RCCs) of various histologic types into nine major genomic subtypes. Site of origin within the nephron was one major determinant in the classification, reflecting differences between clear cell, chromophobe, and papillary RCC. Widespread molecular changes associated with chromatin modifier genes or TFE3 gene fusion were present within specific subtypes as well as spanning multiple subtypes. Differences in patient survival and in alteration of specific pathways—including hypoxia, metabolism, MAP kinase, NRF2-ARE, Hippo, immune checkpoint, and PI3K/AKT/mTOR—could further distinguish the subtypes. Immune checkpoint markers and molecular signatures of T cell infiltrates were both highest in the subtype associated with aggressive clear cell RCC. Differences between the genomic subtypes suggest that therapeutic strategies could be tailored to each RCC disease subset.
Graphical Abstract
doi:10.1016/j.celrep.2016.02.024
PMCID: PMC4794376  PMID: 26947078
4.  Increased COUP-TFII Expression in Adult Hearts Induces Mitochondrial Dysfunction Resulting in Heart Failure 
Nature communications  2015;6:8245.
Mitochondrial dysfunction and metabolic remodeling are pivotal in the development of cardiomyopathy. Here, we show that myocardial COUP-TFII overexpression causes heart failure in mice, suggesting a causal effect of elevated COUP-TFII levels on development of dilated cardiomyopathy. COUP-TFII represses genes critical for mitochondrial electron transport chain enzyme activity, oxidative stress detoxification and mitochondrial dynamics, resulting in increased levels of reactive oxygen and lower rates of oxygen consumption in mitochondria. COUP-TFII also suppresses the metabolic regulator PGC-1 network and decreases expression of key glucose and lipid utilization genes, leading to a reduction in both glucose and oleate oxidation in hearts. These data suggest that COUP-TFII affects mitochondrial function, impairs metabolic remodeling and plays a key role in dilated cardiomyopathy. Lastly, COUP-TFII haploinsufficiency attenuates the progression of cardiac dilation and improves survival in a calcineurin transgenic mouse model, indicating that COUP-TFII may serve as a therapeutic target for treatment of dilated cardiomyopathy.
doi:10.1038/ncomms9245
PMCID: PMC4568566  PMID: 26356605
5.  In-silico QTL mapping of postpubertal mammary ductal development in the mouse uncovers potential human breast cancer risk loci 
Genetic background plays a dominant role in mammary gland development and breast cancer (BrCa). Despite this, the role of genetic diversity in mammary gland development is only partially understood. This study used strain-dependent variation in an inbred mouse mapping panel, to identify quantitative trait loci (QTL) underlying structural variation in mammary ductal development, and determined if these QTL correlated with genomic intervals conferring breast cancer susceptibility in humans. For about half of the traits, the observed variation among the complete set of strains in this study was greater (P<0.05) than that observed with previously studied strains or with strains that are in current common use for mammary gland biology. Correlations were also detected with previously reported variation in mammary tumor latency and metastasis. In silico genome-wide association (GWAS) identified 20 mammary development QTL (Mdq). Of these, 5 were syntenic with previously reported human BrCa loci. The most highly significant (P=1×10−11) association of the study was on MMU6 and contained the genes Plxna4, Plxna4os1, and Chchd3. On MMU5, a QTL was detected (p=8×10−7) that was syntenic to a human BrCa locus on h12q24.5 containing the genes Tbx3 and Tbx5. Intersection of high-association SNP (r2 >0.8) with genomic and epigenomic features, and intersection of candidate genes with gene expression and survival data from human BrCa highlighted several for further study. These results support the conclusion that genetic variation in mammary ductal development is greater than previously appreciated. They also suggest that mammary tumor latency and metastatic index may be influenced by variations in the same factors that control normal mammary ductal development and that further studies of genetically diverse mice can improve our understanding of the connection between breast development and breast cancer in humans by identifying novel susceptibility genes.
doi:10.1007/s00335-014-9551-x
PMCID: PMC4548818  PMID: 25552398
6.  Functional annotation of rare gene aberration drivers of pancreatic cancer 
Nature Communications  2016;7:10500.
As we enter the era of precision medicine, characterization of cancer genomes will directly influence therapeutic decisions in the clinic. Here we describe a platform enabling functionalization of rare gene mutations through their high-throughput construction, molecular barcoding and delivery to cancer models for in vivo tumour driver screens. We apply these technologies to identify oncogenic drivers of pancreatic ductal adenocarcinoma (PDAC). This approach reveals oncogenic activity for rare gene aberrations in genes including NAD Kinase (NADK), which regulates NADP(H) homeostasis and cellular redox state. We further validate mutant NADK, whose expression provides gain-of-function enzymatic activity leading to a reduction in cellular reactive oxygen species and tumorigenesis, and show that depletion of wild-type NADK in PDAC cell lines attenuates cancer cell growth in vitro and in vivo. These data indicate that annotating rare aberrations can reveal important cancer signalling pathways representing additional therapeutic targets.
Next generation sequencing allows the identification of oncogenic driver genes in pancreatic cancer. Here, in an effort to identify additional causal genes, the authors develop a high throughput in vivo screen and identify genes that whilst infrequently mutated in pancreatic cancer contribute to tumour formation.
doi:10.1038/ncomms10500
PMCID: PMC4737758  PMID: 26806015
7.  KAP1 promotes proliferation and metastatic progression of breast cancer cells 
Cancer research  2014;75(2):344-355.
KAP1 (TRIM28) is a transcriptional regulator in embryonic development that controls stem cell self-renewal, chromatin organization and the DNA damage response, acting as an essential co-repressor for KRAB family zinc finger proteins (KRAB-ZNF). To gain insight into the function of this large gene family, we developed an antibody that recognizes the conserved zinc fingers linker region (ZnFL) in multiple KRAB-ZNF. Here we report that the expression of many KRAB-ZNF along with active SUMOlyated KAP1 is elevated widely in human breast cancers. KAP1 silencing in breast cancer cells reduced proliferation and inhibited the growth and metastasis of tumor xenografts. Conversely, KAP1 overexpression stimulated cell proliferation and tumor growth. In cells where KAP1 was silenced, we identified multiple downregulated genes linked to tumor progression and metastasis, including EREG/epiregulin, PTGS2/COX2, MMP1, MMP2 and CD44, along with downregulation of multiple KRAB-ZNF proteins. KAP1-dependent stabilization of KRAB-ZNF required direct interactions with KAP1. Together, our results show that KAP1-mediated stimulation of multiple KRAB-ZNF contributes to the growth and metastasis of breast cancer.
doi:10.1158/0008-5472.CAN-14-1561
PMCID: PMC4297582  PMID: 25421577
breast cancer; KAP1; KRAB-ZNF; degradation
8.  The microRNA-200/Zeb1 axis regulates ECM-dependent β1-integrin/FAK signaling, cancer cell invasion and metastasis through CRKL 
Scientific Reports  2016;6:18652.
Tumor cell metastasis is a complex process that has been mechanistically linked to the epithelial-mesenchymal transition (EMT). The double-negative feedback loop between the microRNA-200 family and the Zeb1 transcriptional repressor is a master EMT regulator, but there is incomplete understanding of how miR-200 suppresses invasion. Our recent efforts have focused on the tumor cell-matrix interactions essential to tumor cell activation. Herein we utilized both our Kras/p53 mutant mouse model and human lung cancer cell lines to demonstrate that upon miR-200 loss integrin β1-collagen I interactions drive 3D in vitro migration/invasion and in vivo metastases. Zeb1-dependent EMT enhances tumor cell responsiveness to the ECM composition and activates FAK/Src pathway signaling by de-repression of the direct miR-200 target, CRKL. We demonstrate that CRKL serves as an adaptor molecule to facilitate focal adhesion formation, mediates outside-in signaling through Itgβ1 to drive cell invasion, and inside-out signaling that maintains tumor cell-matrix contacts required for cell invasion. Importantly, CRKL levels in pan-cancer TCGA analyses were predictive of survival and CRKL knockdown suppressed experimental metastases in vivo without affecting primary tumor growth. Our findings highlight the critical ECM-tumor cell interactions regulated by miR-200/Zeb1-dependent EMT that activate intracellular signaling pathways responsible for tumor cell invasion and metastasis.
doi:10.1038/srep18652
PMCID: PMC4700473  PMID: 26728244
9.  Increased COUP-TFII expression in adult hearts induces mitochondrial dysfunction resulting in heart failure 
Nature Communications  2015;6:8245.
Mitochondrial dysfunction and metabolic remodelling are pivotal in the development of cardiomyopathy. Here, we show that myocardial COUP-TFII overexpression causes heart failure in mice, suggesting a causal effect of elevated COUP-TFII levels on development of dilated cardiomyopathy. COUP-TFII represses genes critical for mitochondrial electron transport chain enzyme activity, oxidative stress detoxification and mitochondrial dynamics, resulting in increased levels of reactive oxygen species and lower rates of oxygen consumption in mitochondria. COUP-TFII also suppresses the metabolic regulator PGC-1 network and decreases the expression of key glucose and lipid utilization genes, leading to a reduction in both glucose and oleate oxidation in the hearts. These data suggest that COUP-TFII affects mitochondrial function, impairs metabolic remodelling and has a key role in dilated cardiomyopathy. Last, COUP-TFII haploinsufficiency attenuates the progression of cardiac dilation and improves survival in a calcineurin transgenic mouse model, indicating that COUP-TFII may serve as a therapeutic target for the treatment of dilated cardiomyopathy.
Transcription factor COUP-TFII is elevated in the hearts of non-ischaemic cardiomyopathy patients, but the nature of this correlation is unknown. Here the authors show that forced cardiac expression of COUP-TFII in mice causes dilated cardiomyopathy because of altered mitochondrial function and impaired metabolic remodelling.
doi:10.1038/ncomms9245
PMCID: PMC4568566  PMID: 26356605
10.  DRUG-REPOSITIONING SCREENING IDENTIFIED PIPERLONGUMINE AS A DIRECT STAT3 INHIBITOR WITH POTENT ACTIVITY AGAINST BREAST CANCER 
Oncogene  2014;34(11):1341-1353.
Signal transducer and activator of transcription (STAT) 3 regulates many cardinal features of cancer including cancer cell growth, apoptosis resistance, DNA damage response, metastasis, immune escape, tumor angiogenesis, the Warburg effect, and oncogene addiction and has been validated as a drug target for cancer therapy. Several strategies have been employed to identify agents that target Stat3 in breast cancer but none has yet entered into clinical use. We used a high-throughput fluorescence microscopy search strategy to identify compounds in a drug-repositioning library (Prestwick library) that block ligand-induced nuclear translocation of Stat3 and identified piperlongumine (PL), a natural product isolated from the fruit of the pepper Piper longum. Piperlongumine inhibited Stat3 nuclear translocation, inhibited ligand-induced and constitutive Stat3 phosphorylation, and modulated expression of multiple Stat3-regulated genes. Surface plasmon resonance assay revealed that piperlongumine directly inhibited binding of Stat3 to its phosphotyrosyl (pY) peptide ligand. Phosphoprotein antibody array analysis revealed that PL does not modulate kinases known to activate Stat3 such as JAKs, Src kinase family members, or RTKs. PL inhibited anchorage-independent and anchorage-dependent growth of multiple breast cancer cell lines having increased pStat3 or total Stat3, and induced apoptosis. PL also inhibited mammosphere formation by tumor cells from patient derived xenografts (PDX). PL’s anti-tumorigenic function was causally linked to its Stat3-inhibitory effect. PL was non-toxic in mice up to a dose of 30 mg/Kg/day for 14 days and caused regression of breast cancer cell line xenografts in nude mice. Thus, PL represents a promising new agent for rapid entry into the clinic for use in treating breast cancer, as well as other cancers in which Stat3 plays a role.
doi:10.1038/onc.2014.72
PMCID: PMC4182178  PMID: 24681959
Stat3; Piperlongumine; Breast Cancer; Drug repositioning; High throughput
11.  The somatic genomic landscape of chromophobe renal cell carcinoma 
Cancer cell  2014;26(3):319-330.
Summary
We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) based on multidimensional and comprehensive characterization, including mitochondrial DNA (mtDNA) and whole genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared to other kidney cancers with more proximal origins. Combined mtDNA and gene expression analysis implicates changes in mitochondrial function as a component of the disease biology, while suggesting alternative roles for mtDNA mutations in cancers relying on oxidative phosphorylation. Genomic rearrangements lead to recurrent structural breakpoints within TERT promoter region, which correlates with highly elevated TERT expression and manifestation of kataegis, representing a mechanism of TERT up-regulation in cancer distinct from previously-observed amplifications and point mutations.
doi:10.1016/j.ccr.2014.07.014
PMCID: PMC4160352  PMID: 25155756
12.  Loss of TRIM62 Expression Is an Independent Adverse Prognostic Factor in Acute Myeloid Leukemia 
Clinical lymphoma, myeloma & leukemia  2014;15(2):115-127.e15.
TRIM62 is a putative tumor suppressor gene. We investigated the levels of expression of TRIM62 protein in 511 patients with acute myeloid leukemia (AML) by reverse-phase protein array technology. Low TRIM62 levels were associated with markedly poorer outcomes and improved the prognostic impact of NPM1 and FLT3 mutations. Low TRIM62 levels, therefore, is an independent adverse prognostic factor in AML.
Background
Tripartite motif (TRIM)-62 is a putative tumor suppressor gene whose role in leukemia is unknown.
Materials and Methods
We evaluated the effect of TRIM62 protein expression in patients with acute myeloid leukemia (AML). We used reverse-phase protein array methodology to determine TRIM62 levels in leukemia-enriched protein samples from 511 patients newly diagnosed with AML.
Results
TRIM62 levels in AML cells were significantly lower than in normal CD34-positive cells, suggesting that TRIM62 loss might be involved in leukemogenesis, but was not associated with specific karyotypic abnormalities or Nucleophosmin (NPM1), Fms-like Tyrosine Kinase-3 (FLT3), or rat sarcoma viral oncogene (RAS) mutational status. Low TRIM62 levels were associated with shorter complete remission duration and significantly shorter event-free and overall survival rates, particularly among patients with intermediate-risk cytogenetics. In that AML subgroup, age and TRIM62 levels were the most powerful independent prognostic factors for survival. TRIM62 protein levels further refined the risk associated with NPM1 and FLT3 mutational status. TRIM62 loss was associated with altered expression of proteins involved in leukemia stem cell homeostasis (β-catenin and Notch), cell motility, and adhesion (integrin-β3, ras-related C3 botulinum toxin substrate [RAC], and fibronectin), hypoxia (Hypoxia-inducible factor 1-alpha [HIF1α], egl-9 family hypoxia-inducible factor 1 [Egln1], and glucose-regulated protein, 78kDa [GRP78]), and apoptosis (B-cell lymphoma-extra large (BclXL) and caspase 9).
Conclusion
Low TRIM62 levels, consistent with a tumor suppressor role, represent an independent adverse prognostic factor in AML.
doi:10.1016/j.clml.2014.07.011
PMCID: PMC4560255  PMID: 25248926
AML; Proteomics; Reverse phase protein array; RPPA; TRIM62
13.  Ror2 regulates branching, differentiation, and actin-cytoskeletal dynamics within the mammary epithelium 
The Journal of Cell Biology  2015;208(3):351-366.
Intricate cross-talk between classical and alternative Wnt signaling pathways includes an essential role for Ror2 in mammary epithelial development and differentiation.
Wnt signaling encompasses β-catenin–dependent and –independent networks. How receptor context provides Wnt specificity in vivo to assimilate multiple concurrent Wnt inputs throughout development remains unclear. Here, we identified a refined expression pattern of Wnt/receptor combinations associated with the Wnt/β-catenin–independent pathway in mammary epithelial subpopulations. Moreover, we elucidated the function of the alternative Wnt receptor Ror2 in mammary development and provided evidence for coordination of this pathway with Wnt/β-catenin–dependent signaling in the mammary epithelium. Lentiviral short hairpin RNA (shRNA)-mediated depletion of Ror2 in vivo increased branching and altered the differentiation of the mammary epithelium. Microarray analyses identified distinct gene level alterations within the epithelial compartments in the absence of Ror2, with marked changes observed in genes associated with the actin cytoskeleton. Modeling of branching morphogenesis in vitro defined specific defects in cytoskeletal dynamics accompanied by Rho pathway alterations downstream of Ror2 loss. The current study presents a model of Wnt signaling coordination in vivo and assigns an important role for Ror2 in mammary development.
doi:10.1083/jcb.201408058
PMCID: PMC4315251  PMID: 25624393
14.  Wild type N-Ras, overexpressed in basal-like breast cancer, promotes tumor formation by inducing IL8 secretion via JAK2 activation 
Cell reports  2015;12(3):511-524.
SUMMARY
Basal-like breast cancers (BLBCs) are aggressive, and their drivers are unclear. We have found that wild-type N-RAS is overexpressed in BLBCs, but not in other breast cancer subtypes. Repressing N-RAS inhibits transformation and tumor growth, while overexpression enhances these processes even in preinvasive BLBC cells. We identified N-Ras-responsive genes, most of which encode chemokines, e.g., IL8. Expression levels of these chemokines and N-RAS in tumors correlate with outcome. N-Ras, but not K-Ras, induces IL8 by binding and activating the cytoplasmic pool of JAK2; IL8 then acts on both the cancer cells and stromal fibroblasts. Thus BLBC progression is promoted by increasing activities of wild-type N-Ras, which mediates autocrine/paracrine signaling that can influence both cancer and stroma cells.
Graphical Abstract
doi:10.1016/j.celrep.2015.06.044
PMCID: PMC4512851  PMID: 26166574
15.  FGF23 promotes prostate cancer progression 
Oncotarget  2015;6(19):17291-17301.
Prostate cancer is the most common cancer in US men and the second leading cause of cancer deaths. Fibroblast growth factor 23 (FGF23) is an endocrine FGF, normally expressed by osteocytes, which plays a critical role in phosphate homeostasis via a feedback loop involving the kidney and vitamin D. We now show that FGF23 is expressed as an autocrine growth factor in all prostate cancer cell lines tested and is present at increased levels in prostate cancer tissues. Exogenous FGF23 enhances proliferation, invasion and anchorage independent growth in vitro while FGF23 knockdown in prostate cancer cell lines decreases these phenotypes. FGF23 knockdown also decreases tumor growth in vivo. Given that classical FGFs and FGF19 are also increased in prostate cancer, we analyzed expression microarrays hybridized with RNAs from of LNCaP cells stimulated with FGF2, FGF19 or FGF23. The different FGF ligands induce overlapping as well as unique patterns of gene expression changes and thus are not redundant. We identified multiple genes whose expression is altered by FGF23 that are associated with prostate cancer initiation and progression. Thus FGF23 can potentially also act as an autocrine, paracrine and/or endocrine growth factor in prostate cancer that can promote prostate cancer progression.
PMCID: PMC4627308  PMID: 26019137
prostate cancer; FGF23; signal transduction; fibroblast growth factors; endocrine fibroblast growth factors
16.  Reverse phase protein arrays in signaling pathways: a data integration perspective 
The reverse phase protein array (RPPA) data platform provides expression data for a prespecified set of proteins, across a set of tissue or cell line samples. Being able to measure either total proteins or posttranslationally modified proteins, even ones present at lower abundances, RPPA represents an excellent way to capture the state of key signaling transduction pathways in normal or diseased cells. RPPA data can be combined with those of other molecular profiling platforms, in order to obtain a more complete molecular picture of the cell. This review offers perspective on the use of RPPA as a component of integrative molecular analysis, using recent case examples from The Cancer Genome Altas consortium, showing how RPPA may provide additional insight into cancer besides what other data platforms may provide. There also exists a clear need for effective visualization approaches to RPPA-based proteomic results; this was highlighted by the recent challenge, put forth by the HPN-DREAM consortium, to develop visualization methods for a highly complex RPPA dataset involving many cancer cell lines, stimuli, and inhibitors applied over time course. In this review, we put forth a number of general guidelines for effective visualization of complex molecular datasets, namely, showing the data, ordering data elements deliberately, enabling generalization, focusing on relevant specifics, and putting things into context. We give examples of how these principles can be utilized in visualizing the intrinsic subtypes of breast cancer and in meaningfully displaying the entire HPN-DREAM RPPA dataset within a single page.
doi:10.2147/DDDT.S38375
PMCID: PMC4500628  PMID: 26185419
RPPA; proteomics; molecular profiling; integrative analysis; breast cancer; TCGA
17.  PKA turnover by the REGγ-proteasome modulates FoxO1 cellular activity and VEGF-induced angiogenesis 
The REGγ-proteasome serves as a short-cut for the destruction of certain intact mammalian proteins in the absence of ubiquitin-and ATP. The biological roles of the proteasome activator REGγ are not completely understood. Here we demonstrate that REGγ controls degradation of protein kinase A catalytic subunit-α (PKAca) both in primary human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblast cells (MEFs). Accumulation of PKAca in REGγ-deficient HUVECs or MEFs results in phosphorylation and nuclear exclusion of the transcription factor FoxO1, indicating that REGγ is involved in preserving FoxO1 transcriptional activity. Consequently, VEGF-induced expression of the FoxO1 responsive genes, VCAM-1 and E-Selectin, was tightly controlled by REGγ in a PKA dependent manner. Functionally, REGγ is crucial for the migration of HUVECs. REGγ−/− mice display compromised VEGF-instigated neovascularization in cornea and aortic ring models. Implanted matrigel plugs containing VEGF in REGγ−/− mice induced fewer capillaries than in REGγ+/+ littermates. Taken together, our study identifies REGγ as a novel angiogenic factor that plays an important role in VEGF-induced expression of VCAM-1 and E-Selectin by antagonizing PKA signaling. Identification of the REGγ–PKA–FoxO1 pathway in endothelial cells (ECs) provides another potential target for therapeutic intervention in vascular diseases.
doi:10.1016/j.yjmcc.2014.02.007
PMCID: PMC4237316  PMID: 24560667
Angiogenesis; E-Selectin; FoxO1; PKAca; REGγ; VCAM-1
18.  Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma 
The Journal of Clinical Investigation  2015;125(3):1147-1162.
Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde–derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde–derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma.
doi:10.1172/JCI74725
PMCID: PMC4362236  PMID: 25664850
19.  A Murine Uterine Transcriptome, Responsive to Steroid Receptor Coactivator-2, Reveals Transcription Factor 23 as Essential for Decidualization of Human Endometrial Stromal Cells1 
Biology of Reproduction  2014;90(4):75.
ABSTRACT
Recent data from human and mouse studies strongly support an indispensable role for steroid receptor coactivator-2 (SRC-2)—a member of the p160/SRC family of coregulators—in progesterone-dependent endometrial stromal cell decidualization, an essential cellular transformation process that regulates invasion of the developing embryo into the maternal compartment. To identify the key progesterone-induced transcriptional changes that are dependent on SRC-2 and required for endometrial decidualization, we performed comparative genome-wide transcriptional profiling of endometrial tissue RNA from ovariectomized SRC-2flox/flox (SRC-2f/f [control]) and PRcre/+/SRC-2flox/flox (SRC-2d/d [SRC-2-depleted]) mice, acutely treated with vehicle or progesterone. Although data mining revealed that only a small subset of the total progesterone-dependent transcriptional changes is dependent on SRC-2 (∼13%), key genes previously reported to mediate progesterone-driven endometrial stromal cell decidualization are present within this subset. Along with providing a more detailed molecular portrait of the decidual transcriptional program governed by SRC-2, the degree of functional diversity of these progesterone mediators underscores the pleiotropic regulatory role of SRC-2 in this tissue. To showcase the utility of this powerful informational resource to uncover novel signaling paradigms, we stratified the total SRC-2-dependent subset of progesterone-induced transcriptional changes in terms of novel gene expression and identified transcription factor 23 (Tcf23), a basic-helix-loop-helix transcription factor, as a new progesterone-induced target gene that requires SRC-2 for full induction. Importantly, using primary human endometrial stromal cells in culture, we demonstrate that TCF23 function is essential for progesterone-dependent decidualization, providing crucial translational support for this transcription factor as a new decidual mediator of progesterone action.
Induction of transcription factor 23 by progesterone requires steroid receptor coactivator-2 and is essential for endometrial decidualization.
doi:10.1095/biolreprod.114.117531
PMCID: PMC4076383  PMID: 24571987
decidualization; endometrium; human; microarray; mouse; progesterone; steroid receptor coactivator-2; transcription factor 23
20.  Function of phosphorylation of NF-kB p65 ser536 in prostate cancer oncogenesis 
Oncotarget  2015;6(8):6281-6294.
Majority of prostate cancer (PCa) patients carry TMPRSS2/ERG (T/E) fusion genes and there has been tremendous interest in understanding how the T/E fusion may promote progression of PCa. We showed that T/E fusion can activate NF-kB pathway by increasing phosphorylation of NF-kB p65 Ser536 (p536), but the function of p536 has never been studied in PCa. We report here that active p536 can significantly increase cell motility and transform PNT1a cells (an immortalized normal cell line), suggesting p536 plays a critical role in promoting PCa tumorigenesis. We have discovered a set of p536 regulated genes, among which we validated the regulation of CCL2 by p536. Based on all evidence, we favor that T/E fusion, NF-kB p536 and CCL2 form a signaling chain. Finally, PNT1a cells (not tumorigenic) can form tumors in SCID mice when overexpressing of either wild type or active p65 in the presence of activated AKT, demonstrating synergistic activities of NF-kB and AKT signals in promoting PCa tumorigenesis. These findings indicate that combination therapies targeting T/E fusion, NF-kB, CCL2 and/or AKT pathways may have efficacy in T/E fusion gene expressing PCa. If successful, such targeted therapy will benefit more than half of PCa patients who carry T/E fusions.
PMCID: PMC4467437  PMID: 25749044
Prostate cancer; TMPRSS2/ERG; NF-κB; AKT; CCL2
21.  Effects of TP53 Mutational Status on Gene Expression Patterns Across Ten Human Cancer Types 
The Journal of pathology  2014;232(5):522-533.
Mutations in the TP53 tumor suppressor gene occur in half of all human cancers, indicating its critical importance in inhibiting cancer development. Despite extensive studies, the mechanisms by which mutant p53 enhances tumor progression remain only partially understood. Here, using data from The Cancer Genome Atlas (TCGA), genomic and transcriptomic analyses were performed on 2256 tumors from ten human cancer types. We show that tumors with TP53 mutations have altered gene expression profiles compared to tumors retaining two wildtype TP53 alleles. Among 113 known p53 upregulated target genes identified from cell culture assays, ten were consistently upregulated in at least 8 of 10 cancer types that retain both copies of wildtype TP53. RPS27L, CDKN1A (p21CIP1), and ZMAT3 were significantly upregulated in all ten cancer types retaining wildtype TP53. Using this p53-based expression analysis as a discovery tool, we used cell-based assays to identify five novel p53 target genes from genes consistently upregulated in wildtype p53 cancers. Global gene expression analyses revealed that cell cycle regulatory genes and transcription factors E2F1, MYBL2, and FOXM1 were disproportionately upregulated in many TP53 mutant cancer types. Finally, over 93% of tumors with a TP53 mutation exhibited greatly reduced wildtype p53 messenger expression due to loss of heterozygosity or copy neutral loss of heterozygosity, supporting the concept of p53 as a recessive tumor suppressor. The data indicate that tumors with wildtype TP53 retain some aspects of p53-mediated growth inhibitory signaling through activation of p53 target genes and suppression of cell cycle regulatory genes.
doi:10.1002/path.4321
PMCID: PMC4362779  PMID: 24374933
p53; TP53; TCGA; nonsense-mediated mRNA decay; MYBL2; FOXM1; E2F1; RPS27L; CDKN1A; ZMAT3
22.  Genomics of chromophobe renal cell carcinoma: implications from a rare tumor for pan-cancer studies 
Oncoscience  2015;2(2):81-90.
Chromophobe Renal Cell Carcinoma (ChRCC) is a rare subtype of the renal cell carcinomas, a heterogenous group of cancers arising from the nephron. Recently, The Cancer Genome Atlas (TCGA) profiled this understudied disease using multiple data platforms, including whole exome sequencing, whole genome sequencing (WGS), and mitochondrial DNA (mtDNA) sequencing. The insights gained from this study would have implications for other types of kidney cancer as well as for cancer biology in general. Global molecular patterns in ChRCC provided clues as to this cancer's cell of origin, which is distinct from that of the other renal cell carcinomas, illustrating an approach that might be applied towards elucidating the cell of origin of other cancer types. MtDNA sequencing revealed loss-of-function mutations in NADH dehydrogenase subunits, highlighting the role of deregulated metabolism in this and other cancers. Analysis of WGS data led to the discovery of recurrent genomic rearrangements involving TERT promoter region, which were associated with very high expression levels of TERT, pointing to a potential mechanism for TERT deregulation that might be found in other cancers. WGS data, generated by large scale efforts such as TCGA and the International Cancer Genomics Consortium (ICGC), could be more extensively mined across various cancer types, to uncover structural variants, mtDNA mutations, themes of tumor metabolic properties, as well as noncoding point mutations. TCGA's data on ChRCC should continue to serve as a resource for future pan-cancer as well as kidney cancer studies, and highlight the value of investigations into rare tumor types to globally inform principals of cancer biology.
PMCID: PMC4381700  PMID: 25859550
ChRCC; chromophobe; kidney cancer; genomics; TERT; TCGA; mitochondria
23.  T lymphocytes redirected against the chondroitin sulfate proteoglycan-4 control the growth of multiple solid tumors both in vitro and in vivo 
Purpose
Because of its high expression on various types of tumors and its restricted distribution in normal tissues, chondroitin sulfate proteoglycan-4 (CSPG4) represents an attractive target for the antibody-based therapy of several solid tumors. We tested whether T cells transduced with a CSPG4-specific chimeric antigen receptor (CAR) inhibited the growth of CSPG4-expressing tumor cells both in vitro and in vivo.
Experimental Design
We first independently validated by immunohistochemistry (IHC) the expression of CSPG4 in an extensive panel of tumor arrays and normal tissues as well as queried public gene expression profiling datasets of human tumors. We constructed a second generation CSPG4-specific CAR also encoding the CD28 costimulatory endodomain (CAR.CSPG4). We then evaluated human T lymphocytes expressing this CAR for their ex vivo and in vivo anti-tumor activity against a broad panel of solid tumors.
Results
IHC showed that CSPG4 is highly expressed in melanoma, breast cancer, head and neck squamous cell carcinoma (HNSCC) and mesothelioma. In addition, in silico analysis of microarray expression data identified other important potential tumors expressing this target, including glioblastoma, clear cell renal carcinoma and sarcomas. T lymphocytes genetically modified with a CSPG4-CAR controlled tumor growth in vitro and in vivo in NSG mice engrafted with human melanoma, HNSCC and breast carcinoma cell lines.
Conclusions
CAR.CSPG4-redirected T cells should provide an effective treatment modality for a variety of solid tumors.
doi:10.1158/1078-0432.CCR-13-2218
PMCID: PMC3944408  PMID: 24334762
24.  Gene Expression Profile of A549 Cells from Tissue of 4D Model Predicts Poor Prognosis in Lung Cancer Patients 
The tumor microenvironment plays an important role in regulating cell growth and metastasis. Recently, we developed an ex vivo lung cancer model (4D) that forms perfusable tumor nodules on a lung matrix that mimics human lung cancer histopathology and protease secretion pattern. We compared the gene expression profile (Human OneArray v5 chip) of A549 cells, a human lung cancer cell line, grown in a petri dish (2D), and of the same cells grown in the matrix of our ex vivo model (4D). Furthermore, we obtained gene expression data of A549 cells grown in a petri dish (2D) and matrigel (3D) from a previous study and compared the 3D expression profile with that of 4D. Expression array analysis showed 2,954 genes differentially expressed between 2D and 4D. Gene ontology (GO) analysis showed upregulation of several genes associated with extracellular matrix, polarity, and cell fate and development. Moreover, expression array analysis of 2D versus 3D showed 1006 genes that were most differentially expressed, with only 36 genes (4%) having similar expression patterns as observed between 2D and 4D. Finally, the differential gene expression signature of 4D cells (versus 2D) correlated significantly with poor survival in patients with lung cancer (n = 1,492), while the expression signature of 3D versus 2D correlated with better survival in lung cancer patients with lung cancer. Since patients with larger tumors have a worse rate of survival, the ex vivo 4D model may be a good mimic of natural progression of tumor growth in lung cancer patients.
doi:10.1002/ijc.28428
PMCID: PMC4070715  PMID: 23934967
Lung cancer; matrigel; ex vivo 4D model; gene expression profile; survival
25.  Androgen deprivation–induced NCoA2 promotes metastatic and castration-resistant prostate cancer 
The Journal of Clinical Investigation  2014;124(11):5013-5026.
A major clinical hurdle for the management of advanced prostate cancer (PCa) in patients is the resistance of tumors to androgen deprivation therapy (ADT) and their subsequent development into castration-resistant prostate cancer (CRPC). While recent studies have identified potential pathways involved in CRPC development, the drivers of CRPC remain largely undefined. Here we determined that nuclear receptor coactivator 2 (NCoA2, also known as SRC-2), which is frequently amplified or overexpressed in patients with metastatic PCa, mediates development of CRPC. In a murine model, overexpression of NCoA2 in the prostate epithelium resulted in neoplasia and, in combination with Pten deletion, promoted the development of metastasis-prone cancer. Moreover, depletion of NCoA2 in PTEN-deficient mice prevented the development of CRPC. In human androgen-sensitive prostate cancer cells, androgen signaling suppressed NCoA2 expression, and NCoA2 overexpression in murine prostate tumors resulted in hyperactivation of PI3K/AKT and MAPK signaling, promoting tumor malignance. Analysis of PCa patient samples revealed a strong correlation among NCoA2-mediated signaling, disease progression, and PCa recurrence. Taken together, our findings indicate that androgen deprivation induces NCoA2, which in turn mediates activation of PI3K signaling and promotes PCa metastasis and CRPC development. Moreover, these results suggest that the inhibition of NCoA2 has potential for PCa therapy.
doi:10.1172/JCI76412
PMCID: PMC4347241  PMID: 25295534

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