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1.  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
2.  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
3.  Increased Notch signaling inhibits anoikis and stimulates proliferation of prostate luminal epithelial cells 
Nature communications  2014;5:4416.
The prostate epithelial lineage hierarchy remains inadequately defined. Recent lineage-tracing studies have implied the existence of prostate luminal epithelial progenitors with extensive regenerative capacity. However, this capacity has not been demonstrated in prostate stem cell activity assays, probably due to the strong susceptibility of luminal progenitors to anoikis. Here we show that constitutive expression of Notch1 intracellular domain impairs secretory function of mouse prostate luminal cells, suppresses anoikis of luminal epithelial cells by augmenting NF-κB activity independent of Hes-1, stimulates luminal cell proliferation by potentiating PI3K-AKT signaling, and rescues the capacities of the putative prostate luminal progenitors for unipotent differentiation in vivo and short-term self-renewal in vitro. Epithelial cell-autonomous AR signaling is dispensable for the Notch-mediated effects. As Notch activity is increased in prostate cancers and anoikis resistance is a hallmark for metastatic cancer cells, this study suggests a pro-metastatic function of Notch signaling during prostate cancer progression.
doi:10.1038/ncomms5416
PMCID: PMC4167399  PMID: 25048699
4.  FGFR1-WNT-TGF-β signaling in prostate cancer mouse models recapitulates human reactive stroma 
Cancer research  2013;74(2):609-620.
The reactive stroma surrounding tumor lesions performs critical roles ranging from supporting tumor cell proliferation to inducing tumorigenesis and metastasis. Therefore, it is critical to understand the cellular components and signaling control mechanisms that underlay the etiology of reactive stroma. Previous studies have individually implicated fibroblast growth factor receptor 1 (FGFR1) and canonical WNT/β-catenin signaling in prostate cancer progression and the initiation and maintenance of a reactive stroma; however, both pathways are frequently found co-activated in cancer tissue. Using autochthonous transgenic mouse models for inducible FGFR1 (JOCK1) and prostate-specific and ubiquitously expressed inducible β-catenin (Pro-Cat and Ubi-Cat, respectively) and bigenic crosses between these lines (Pro-Cat × JOCK1 and Ubi-Cat × JOCK1), we describe WNT-induced synergistic acceleration of FGFR1-driven adenocarcinoma, associated with a pronounced fibroblastic reactive stroma activation surrounding prostatic intraepithelial neoplasia (mPIN) lesions found both in situ and reconstitution assays. Both mouse and human reactive stroma exhibited increased transforming growth factor-beta (TGF-β) signaling adjacent to pathologic lesions likely contributing to invasion. Furthermore, elevated stromal TGF-β signaling was associated with higher Gleason scores in archived human biopsies, mirroring murine patterns. Our findings establish the importance of the FGFR1-WNT-TGF-β signaling axes as driving forces behind reactive stroma in aggressive prostate adenocarcinomas, deepening their relevance as therapeutic targets.
doi:10.1158/0008-5472.CAN-13-1093
PMCID: PMC3905049  PMID: 24305876
Prostate Cancer; Reactive Stroma; FGFR1; WNT; TGF-β
5.  Circulating and disseminated tumor cells from breast cancer patient-derived xenograft-bearing mice as a novel model to study metastasis 
Introduction
Real-time monitoring of biologic changes in tumors may be possible by investigating the transitional cells such as circulating tumor cells (CTCs) and disseminated tumor cells in bone marrow (BM-DTCs). However, the small numbers of CTCs and the limited access to bone marrow aspirates in cancer patients pose major hurdles. The goal of this study was to determine whether breast cancer (BC) patient-derived xenograft (PDX) mice could provide a constant and renewable source of CTCs and BM-DTCs, thereby representing a unique system for the study of metastatic processes.
Methods
CTCs and BM-DTCs, isolated from BC PDX-bearing mice, were identified by immunostaining for human pan-cytokeratin and nuclear counterstaining of red blood cell-lysed blood and bone marrow fractions, respectively. The rate of lung metastases (LM) was previously reported in these lines. Associations between the presence of CTCs, BM-DTCs, and LM were assessed by the Fisher’s Exact and Cochran-Mantel-Haenszel tests. Two separate genetic signatures associated with the presence of CTC clusters and with lung metastatic potential were computed by using the expression arrays of primary tumors from different PDX lines and subsequently overlapped to identify common genes.
Results
In total, 18 BC PDX lines were evaluated. CTCs and BM-DTCs, present as either single cells or clusters, were detected in 83% (15 of 18) and 62.5% (10 to16) of the lines, respectively. A positive association was noted between the presence of CTCs and BM-DTCs within the same mice. LM was previously found in 9 of 18 (50%) lines, of which all nine had detectable CTCs. The presence of LM was strongly associated with the detection of CTC clusters but not with individual cells or detection of BM-DTCs. Overlapping of the two genetic signatures of the primary PDX tumors associated with the presence of CTC clusters and with lung metastatic potential identified four genes (HLA-DP1A, GJA1, PEG3, and XIST). This four-gene profile predicted distant metastases-free survival in publicly available datasets of early BC patients.
Conclusion
This study suggests that CTCs and BM-DTCs detected in BC PDX-bearing mice may represent a valuable and unique preclinical model for investigating the role of these rare cells in tumor metastases.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0508-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0508-5
PMCID: PMC4318479  PMID: 25572662
6.  Amplification and over-expression of MAP3K3 gene in human breast cancer promotes formation and survival of breast cancer cells 
The Journal of pathology  2014;232(1):75-86.
Gene amplifications in the 17q chromosomal region are observed frequently in breast cancers. An integrative bioinformatics analysis of this region nominated the MAP3K 3 gene as a potential therapeutic target in breast cancer. This gene encodes mitogen-activated protein kinase kinase kinase 3 (MAP3K3/MEKK3), which has not yet been reported to be associated with cancer-causing genetic aberrations. We found that MAP3K3 was amplified in approximately 8–20% of breast cancers. Knockdown of MAP3K3 expression significantly inhibited cell proliferation and colony formation in MAP3K3-amplified breast cancer cell lines MCF-7 and MDA-MB-361 but not in MAP3K3 non-amplified breast cancer cells. Knockdown of MAP3K3 expression in MAP3K3-amplified breast cancer cells sensitized breast cancer cells to apoptotic induction by TNFα and TRAIL, as well as doxorubicin, VP-16 and fluorouracil, three commonly used chemotherapeutic drugs for treating breast cancer. In addition, ectopic expression of MAP3K3, in collaboration with Ras, induced colony formation in both primary mouse embryonic fibroblasts and immortalized human breast epithelial cells (MCF-10A). Combined, these results suggest that MAP3K3 contributes to breast carcinogenesis and may endow resistance of breast cancer cells to cytotoxic chemotherapy. Therefore, MAP3K3 may be a valuable therapeutic target in patients with MAP3K3-amplified breast cancers, and blocking MAP3K3 kinase activity with a small molecule inhibitor may sensitize MAP3K3-amplified breast cancer cells to chemotherapy.
doi:10.1002/path.4283
PMCID: PMC3966110  PMID: 24122835
MAP3K3; oncogene; breast cancer; chemo-resistance
7.  A dosage-dependent pleiotropic role of Dicer in prostate cancer growth and metastasis 
Oncogene  2013;33(24):3099-3108.
Dicer is as an RNase III enzyme essential for the maturation of the majority of microRNAs. Recent studies have revealed down-regulation or hemizygous loss of Dicer in many tumor models and demonstrated that suppressing Dicer activity enhances tumorigenic activities of lung and breast cancer cells, which support Dicer as a haploinsufficient tumor suppressor in these cancer models. Surprisingly, we found that knocking down Dicer expression suppresses the growth and tumorigenic capacity of human prostate cancer cell lines, but enhances migratory capacities of some prostate cancer cell lines. Dicer is up-regulated in human prostate cancer specimens, but lower Dicer expression portends a shorter time to recurrence. Complete ablation of Dicer activity in a Pten null mouse model for prostate cancer significantly halts tumor growth and progression, demonstrating that microRNAs play a critical role in maintaining cancer cell fitness. In comparison, hemizygous loss of Dicer in the same model also reduces primary tumor burden, but induces a more locally invasive phenotype and causes seminal vesicle obstruction at high penetrance. Disrupting Dicer activity leads to an increase in apoptosis and senescence in these models, presumably through up-regulation of P16/INK4a and P27/Kip1. Collectively, these results highlight a pleotropic role of Dicer in tumorigenesis that is not only dosage-dependent but also tissue context-dependent.
doi:10.1038/onc.2013.281
PMCID: PMC3916938  PMID: 23851498
Prostate cancer; Dicer; Pten; Senescence; Apoptosis; Invasiveness
8.  Stromal TGF-β signaling induces AR activation in prostate cancer 
Oncotarget  2014;5(21):10854-10869.
AR signaling is essential for the growth and survival of prostate cancer (PCa), including most of the lethal castration-resistant PCa (CRPC). We previously reported that TGF-β signaling in prostate stroma promotes prostate tumor angiogenesis and growth. By using a PCa/stroma co-culture model, here we show that stromal TGF-β signaling induces comprehensive morphology changes of PCa LNCaP cells. Furthermore, it induces AR activation in LNCaP cells in the absence of significant levels of androgen, as evidenced by induction of several AR target genes including PSA, TMPRSS2, and KLK4. SD-208, a TGF-β receptor 1 specific inhibitor, blocks this TGF-β induced biology. Importantly, stromal TGF-β signaling together with DHT induce robust activation of AR. MDV3100 effectively blocks DHT-induced, but not stromal TGF-β signaling induced AR activation in LNCaP cells, indicating that stromal TGF-β signaling induces both ligand-dependent and ligand-independent AR activation in PCa. TGF-β induces the expression of several growth factors and cytokines in prostate stromal cells, including IL-6, and BMP-6. Interestingly, BMP-6 and IL-6 together induces robust AR activation in these co-cultures, and neutralizing antibodies against BMP-6 and IL-6 attenuate this action. Altogether, our study strongly suggests tumor stromal microenvironment induced AR activation as a direct mechanism of CRPC.
PMCID: PMC4279415  PMID: 25333263
TGF-β; AR; tumor microenvironment; prostate stroma; prostate cancer; co-culture
9.  EZH2–miR-30d–KPNB1 pathway regulates malignant peripheral nerve sheath tumour cell survival and tumourigenesis 
The Journal of pathology  2014;232(3):308-318.
Malignant peripheral nerve sheath tumours (MPNSTs), which develop sporadically or from neurofibromatosis, recur frequently with high metastatic potential and poor outcome. The polycomb group protein enhancer of zeste homologue 2 (EZH2) is an important regulator for various human malignancies. However, the function of EZH2 in MPNSTs is unknown. Here we report that the EZH2–miR-30d–KPNB1 signalling pathway is critical for MPNST tumour cell survival in vitro and tumourigenicity in vivo. Up-regulated EZH2 in MPNST inhibits miR-30d transcription via promoter binding activity, leading to enhanced expression of the nuclear transport receptor KPNB1 that is inhibited by miR-30d targeting of KPNB1 3′ UTR region. Furthermore, inhibition of EZH2 or KPNB1, or miR-30d over-expression, induces MPNST cell apoptosis in vitro and suppresses tumourigenesis in vivo. More importantly, forced over-expression of KPNB1 rescues MPNST cell apoptosis induced by EZH2 knockdown. Immunohistochemical analyses show that EZH2 and KPNB1 over-expression is observed in human MPNST specimens and is negatively associated with miR-30d expression. Our findings identify a novel signalling pathway involved in MPNST tumourigenesis, and also suggest that EZH2–miR-30d–KPNB1 signalling represents multiple potential therapeutic targetable nodes for MPNST.
doi:10.1002/path.4294
PMCID: PMC4166508  PMID: 24132643
EZH2; miR-30d; KPNB1; MPNST; tumourigenesis
10.  Overcoming endocrine resistance due to reduced PTEN levels in estrogen receptor-positive breast cancer by co-targeting mammalian target of rapamycin, protein kinase B, or mitogen-activated protein kinase kinase 
Introduction
Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in estrogen receptor α (ER)-positive breast cancer is associated with reduced ER expression and activity, luminal B subtype, and poor outcome. Phosphatase and tensin homolog (PTEN), a negative regulator of this pathway, is typically lost in ER-negative breast cancer. We set out to clarify the role of reduced PTEN levels in endocrine resistance, and to explore the combination of newly developed PI3K downstream kinase inhibitors to overcome this resistance.
Methods
Altered cellular signaling, gene expression, and endocrine sensitivity were determined in inducible PTEN-knockdown ER-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer cell and/or xenograft models. Single or two-agent combinations of kinase inhibitors were examined to improve endocrine therapy.
Results
Moderate PTEN reduction was sufficient to enhance PI3K signaling, generate a gene signature associated with the luminal B subtype of breast cancer, and cause endocrine resistance in vitro and in vivo. The mammalian target of rapamycin (mTOR), protein kinase B (AKT), or mitogen-activated protein kinase kinase (MEK) inhibitors, alone or in combination, improved endocrine therapy, but the efficacy varied by PTEN levels, type of endocrine therapy, and the specific inhibitor(s). A single-agent AKT inhibitor combined with fulvestrant conferred superior efficacy in overcoming resistance, inducing apoptosis and tumor regression.
Conclusions
Moderate reduction in PTEN, without complete loss, can activate the PI3K pathway to cause endocrine resistance in ER-positive breast cancer, which can be overcome by combining endocrine therapy with inhibitors of the PI3K pathway. Our data suggests that the ER degrader fulvestrant, to block both ligand-dependent and -independent ER signaling, combined with an AKT inhibitor is an effective strategy to test in patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0430-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0430-x
PMCID: PMC4303114  PMID: 25212826
11.  HOXA1 drives melanoma tumor growth and metastasis and elicits an invasion gene expression signature that prognosticates clinical outcome 
Oncogene  2013;33(8):1017-1026.
Metastatic melanoma is a highly lethal disease notorious for its aggressive clinical course and eventual resistance to existing therapies. Currently we possess a limited understanding of the genetic events driving melanoma progression, and much effort is focused on identifying pro-metastatic aberrations or perturbed signaling networks that constitute new therapeutic targets. In this study, we validate and assess the mechanism by which homeobox transcription factor A1 (HOXA1), a pro-invasion oncogene previously identified in a metastasis screen by our group, contributes to melanoma progression. Transcriptome and pathway profiling analyses of cells expressing HOXA1 reveals up-regulation of factors involved in diverse cytokine pathways that include the TGFβ signaling axis, which we further demonstrate to be required for HOXA1-mediated cell invasion in melanoma cells. Transcriptome profiling also shows HOXA1’s ability to potently down-regulate expression of microphthalmia-associated transcription factor (MITF) and other genes required for melanocyte differentiation, suggesting a mechanism by which HOXA1 expression de-differentiates cells into a pro-invasive cell state concomitant with TGFβ activation. Our analysis of publicly available datasets indicate that the HOXA1-induced gene signature successfully categorizes melanoma specimens based on their metastatic potential and, importantly, is capable of stratifying melanoma patient risk for metastasis based on expression in primary tumors. Together, these validation data and mechanistic insights suggest that patients whose primary tumors express HOXA1 are among a high-risk metastasis subgroup that should be considered for anti-TGFβ therapy in adjuvant settings. Moreover, further analysis of HOXA1 target genes in melanoma may reveal new pathways or targets amenable to therapeutic intervention.
doi:10.1038/onc.2013.30
PMCID: PMC3982326  PMID: 23435427
HOXA1; melanoma; metastasis; MITF; TGFβ
12.  Novel MicroRNAs Regulating Proliferation and Apoptosis in Uterine Papillary Serous Carcinomas 
Cancer letters  2013;335(2):314-322.
MicroRNAs (miRNAs) are endogenous, non-coding RNA transcripts that regulate gene expression. Here, we report 175 putative novel miRNAs identified in uterine cancers profiled by Next Generation Sequencing. Our data indicate that one of these putative miRNAs (BCM-173) is conserved across multiple species and is expressed at levels similar to known human miRNAs. Functionally, this miRNA promotes the growth and migration of uterine cancer cell lines by targeting vinculin and altering the distribution of focal adhesions. These results expand our insight into the repertoire of human miRNAs and identify novel pathways by which dysregulated miRNA expression promotes uterine cancer growth.
doi:10.1016/j.canlet.2013.02.043
PMCID: PMC3669647  PMID: 23454583
MicroRNA; Uterine Cancer; Vinculin; Focal adhesions; Migration
13.  Pathway-Centric Integrative Analysis Identifies RRM2 as a Prognostic Marker in Breast Cancer Associated with Poor Survival and Tamoxifen Resistance123 
Neoplasia (New York, N.Y.)  2014;16(5):390-402.
Breast cancer (BCa) molecular subtypes include luminal A, luminal B, normal-like, HER-2–enriched, and basal-like tumors, among which luminal B and basal-like cancers are highly aggressive. Biochemical pathways associated with patient survival or treatment response in these more aggressive subtypes are not well understood. With the limited availability of pathologically verified clinical specimens, cell line models are routinely used for pathway-centric studies. We measured the metabolome of luminal and basal-like BCa cell lines using mass spectrometry, linked metabolites to biochemical pathways using Gene Set Analysis, and developed a novel rank-based method to select pathways on the basis of their enrichment in patient-derived omics data sets and prognostic relevance. Key mediators of the pathway were then characterized for their role in disease progression. Pyrimidine metabolism was altered in luminal versus basal BCa, whereas the combined expression of its associated genes or expression of one key gene, ribonucleotide reductase subunit M2 (RRM2) alone, associated significantly with decreased survival across all BCa subtypes, as well as in luminal patients resistant to tamoxifen. Increased RRM2 expression in tamoxifen-resistant patients was verified using tissue microarrays, whereas the metabolic products of RRM2 were higher in tamoxifen-resistant cells and in xenograft tumors. Both genetic and pharmacological inhibition of this key enzyme in tamoxifen-resistant cells significantly decreased proliferation, reduced expression of cell cycle genes, and sensitized the cells to tamoxifen treatment. Our study suggests for evaluating RRM2-associated metabolites as noninvasive markers for tamoxifen resistance and its pharmacological inhibition as a novel approach to overcome tamoxifen resistance in BCa.
doi:10.1016/j.neo.2014.05.007
PMCID: PMC4198742  PMID: 25016594
14.  Uterine Leiomyosarcoma Management, Outcome, and Associated Molecular Biomarkers: A Single Institution’s Experience 
Annals of surgical oncology  2013;20(7):2364-2372.
Background
Uterine leiomyosarcoma (ULMS) is an aggressive, rapidly progressive tumor lacking clinical and molecular predictors of outcome.
Methods
ULMS patients (n = 349) were classified by disease status at presentation to MDACC as having intra-abdominal (n = 157) or distant metastatic disease (n = 192). Patient, tumor, treatment, and outcome variables were retrospectively retrieved. Formalin-fixed, paraffin-embedded tumor and control tissues from these patients (n = 109) were assembled in a tissue microarray and evaluated for hormone receptors and markers of angiogenesis, cell-cycle progression and survival. Patient, tumor, and treatment variables were correlatively analyzed.
Results
The 5- and 10-year disease-specific survival (DSS) for the cohort was 42 and 27 %, respectively. Patients with primary intra-abdominal tumors had better outcomes than those with recurrent intraperitoneal tumors. Whites had a more favorable prognosis. In patients with intra-abdominal tumors, only mitotic count >10M/10HPF portended poorer prognosis. Patients with pulmonary metastasis had improved outcomes with “curative” metastasectomy. ULMS samples exhibited loss of ER and PR expression, overexpressed Ki-67, and altered p53, Rb, p16, cytoplasmic β-catenin, EGFR, PDGFR-α, PDGFR-β, and AXL levels. Metastatic tumors had increased VEGF, Ki-67, and survivin expression versus localized disease. Survivin and β-catenin expression were associated with intraperitoneal recurrence; high bcl-2 expression predicted longer DSS.
Conclusions
Analysis of both clinicopathologic factors and immunohistochemical biomarkers in ULMS identified several prognostic clinical and molecular factors, suggesting that further study may lead to improved ULMS understanding and treatment.
doi:10.1245/s10434-012-2834-0
PMCID: PMC3913478  PMID: 23334251
15.  An epigenomic approach to therapy for tamoxifen-resistant breast cancer 
Cell Research  2014;24(7):809-819.
Tamoxifen has been a frontline treatment for estrogen receptor alpha (ERα)-positive breast tumors in premenopausal women. However, resistance to tamoxifen occurs in many patients. ER still plays a critical role in the growth of breast cancer cells with acquired tamoxifen resistance, suggesting that ERα remains a valid target for treatment of tamoxifen-resistant (Tam-R) breast cancer. In an effort to identify novel regulators of ERα signaling, through a small-scale siRNA screen against histone methyl modifiers, we found WHSC1, a histone H3K36 methyltransferase, as a positive regulator of ERα signaling in breast cancer cells. We demonstrated that WHSC1 is recruited to the ERα gene by the BET protein BRD3/4, and facilitates ERα gene expression. The small-molecule BET protein inhibitor JQ1 potently suppressed the classic ERα signaling pathway and the growth of Tam-R breast cancer cells in culture. Using a Tam-R breast cancer xenograft mouse model, we demonstrated in vivo anti-breast cancer activity by JQ1 and a strong long-lasting effect of combination therapy with JQ1 and the ER degrader fulvestrant. Taken together, we provide evidence that the epigenomic proteins BRD3/4 and WHSC1 are essential regulators of estrogen receptor signaling and are novel therapeutic targets for treatment of Tam-R breast cancer.
doi:10.1038/cr.2014.71
PMCID: PMC4085766  PMID: 24874954
epigenomic; tamoxifen; breast cancer
16.  miR-1 and miR-133b Are Differentially Expressed in Patients with Recurrent Prostate Cancer 
PLoS ONE  2014;9(6):e98675.
Prostate cancer (PCa) is currently the most frequently diagnosed malignancy in the western countries. It is more prevalent in older men with 75% of the incident cases above 65 years old. After radical prostatectomy, approximately 30% of men develop clinical recurrence with elevated serum prostate-specific antigen levels. Therefore, it is important to unravel the molecular mechanisms underlying PCa progression to develop novel diagnostic/therapeutic approaches. In this study, it is aimed to compare the microRNA (miRNA) profile of recurrent and non-recurrent prostate tumor tissues to explore the possible involvement of miRNAs in PCa progression. Total RNA from 41 recurrent and 41 non-recurrent PCa tissue samples were used to investigate the miRNA signature in PCa specimens. First of all, 20 recurrent and 20 non-recurrent PCa samples were profiled using miRNA microarray chips. Of the differentially expressed miRNAs, miR-1, miR-133b and miR-145* were selected for further validation with qRT-PCR in a different set of 21 recurrent and 21 non-recurrent PCa samples. Data were statistically analyzed using two-sided Student's t-test, Pearson Correlation test, Receiver operating characteristic analysis. Our results demonstrated that miR-1 and mir-133b have been significantly downregulated in recurrent PCa specimens in comparison to non-recurrent PCa samples and have sufficient power to distinguish recurrent specimens from non-recurrent ones on their own. Here, we report that the relative expression of miR-1 and mir-133b have been significantly reduced in recurrent PCa specimens in comparison to non-recurrent PCa samples, which can serve as novel biomarkers for prediction of PCa progression.
doi:10.1371/journal.pone.0098675
PMCID: PMC4072786  PMID: 24967583
17.  BONE MARROW STROMA SECRETED CYTOKINES PROTECT JAK2V617F-MUTATED CELLS FROM THE EFFECTS OF A JAK2 INHIBITOR 
Cancer research  2011;71(11):3831-3840.
Signals emanating from the bone marrow microenvironment, including stromal cells, are thought to support the survival and proliferation of the malignant cells in patients with myeloproliferative neoplasms (MPN). To examine this hypothesis we established a co-culture platform (cells co-cultured directly [cell-on-cell] or indirectly [separated by micropore membrane]) designed to interrogate the interplay between JAK2V617F–positive cells and the stromal cells. Treatment with atiprimod, a potent JAK2 inhibitor, caused marked growth inhibition and apoptosis of human (SET2) and mouse (FDCP-EpoR) JAK2V617F–positive cells, and of primary blood or bone marrow mononuclear cells from patients with polycythemia vera, but these effects were attenuated when any of these cell types were co-cultured (cell-on-cell) with human marrow stromal cell lines (HS5, NK.tert, or TM-R1). Co-culture with stromal cells hampered the ability of atiprimod to inhibit the phosphorylation of JAK2 and the downstream signal transducer and activators of transcription (STAT) 3, and STAT5. This protective effect was maintained in non-contact co-culture assays (JAK2V617F–positive cells separated by 0.4 μm micropore membranes from stromal cells), suggesting a paracrine effect. Cytokine profiling of supernatants from non-contact co-culture assays detected distinctly high levels of IL-6, FGF, and CXCL10/IP10. Anti-IL-6, -FGF, or -CXCL10/IP10 neutralizing antibodies ablated the protective effect of stromal cells and restored atiprimod-induced apoptosis of JAK2V617F–positive cells. Thus, our results suggest that humoral factors secreted by stromal cells protect MPN clones from JAK2 inhibitor therapy, underscoring the importance of targeting the marrow niche in MPN for therapeutic purposes.
doi:10.1158/0008-5472.CAN-10-4002
PMCID: PMC4067142  PMID: 21512135
myeloproliferative neoplasms; myelofibrosis; polycythemia vera; stroma; cytokines; JAK2
18.  The Epidermal Growth Factor Receptor Critically Regulates Endometrial Function during Early Pregnancy 
PLoS Genetics  2014;10(6):e1004451.
Infertility and adverse gynecological outcomes such as preeclampsia and miscarriage represent significant female reproductive health concerns. The spatiotemporal expression of growth factors indicates that they play an important role in pregnancy. The goal of this study is to define the role of the ERBB family of growth factor receptors in endometrial function. Using conditional ablation in mice and siRNA in primary human endometrial stromal cells, we identified the epidermal growth factor receptor (Egfr) to be critical for endometrial function during early pregnancy. While ablation of Her2 or Erbb3 led to only a modest reduction in litter size, mice lacking Egfr expression are severely subfertile. Pregnancy demise occurred shortly after blastocyst implantation due to defects in decidualization including decreased proliferation, cell survival, differentiation and target gene expression. To place Egfr in a genetic regulatory hierarchy, transcriptome analyses was used to compare the gene signatures from mice with conditional ablation of Egfr, wingless-related MMTV integration site 4 (Wnt4) or boneless morphogenic protein 2 (Bmp2); revealing that not only are Bmp2 and Wnt4 key downstream effectors of Egfr, but they also regulate distinct physiological functions. In primary human endometrial stromal cells, marker gene expression, a novel high content image-based approach and phosphokinase array analysis were used to demonstrate that EGFR is a critical regulator of human decidualization. Furthermore, inhibition of EGFR signaling intermediaries WNK1 and AKT1S1, members identified in the kinase array and previously unreported to play a role in the endometrium, also attenuate decidualization. These results demonstrate that EGFR plays an integral role in establishing the cellular context necessary for successful pregnancy via the activation of intricate signaling and transcriptional networks, thereby providing valuable insight into potential therapeutic targets.
Author Summary
Approximately 10% of reproductive aged women are considered infertile. While great strides have been made in assisted reproductive technologies, overall success rates, especially considering the cost, remain low. Studies indicate that due to its sequential nature, nearly 75% of pregnancy failures are due to defects that occur very early in gestation. Therefore, understanding the physiological changes that occur in the endometrium during this period and how those changes are regulated is of paramount importance if we are to improve our ability to address female reproductive health concerns. We investigated a family of growth factor receptors and identified one that critically regulates the growth and survival of the endometrium in response to the implanting embryo. Furthermore, we used unbiased approaches to identify which signaling pathways and genetic networks are activated downstream of this receptor to execute each of the processes necessary for a successful pregnancy. Understanding the mechanisms and genetic networks with which pregnancy is regulated is a prerequisite to the development of effective pharmaceutical therapeutics.
doi:10.1371/journal.pgen.1004451
PMCID: PMC4063709  PMID: 24945252
19.  A sumoylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis 
Science (New York, N.Y.)  2011;335(6066):348-353.
Myc is an oncogenic transcription factor frequently dysregulated in human cancer. To identify pathways supporting the Myc oncogenic program, we employed a genome-wide RNAi screen for Myc-synthetic-lethal genes and uncovered a role for the SUMO-activating-enzyme (SAE1/2). Loss of SAE1/2 enzymatic activity drives synthetic lethality with Myc. Inactivation of SAE2 leads to mitotic catastrophe and cell death selectively upon Myc hyper-activation. Mechanistically, SAE2 inhibition switches a transcriptional subprogram of Myc from activated to repressed. A subset of these SUMOylation-dependent-Myc-switchers (SMS genes) is required for mitotic spindle function and to support the Myc oncogenic program. SAE2 is required for Myc-dependent tumor growth, and patient survival significantly correlates with SAE1/SAE2 levels in Myc-high tumors. These studies reveal a mitotic vulnerability of Myc-driven cancers, demonstrate that inhibiting sumoylation impairs Myc-dependent tumorigenesis, and suggest inhibiting SUMOylation may have therapeutic benefits for patients with Myc-driven cancer.
doi:10.1126/science.1212728
PMCID: PMC4059214  PMID: 22157079
20.  COUP-TFII inhibits TGF-β-induced growth barrier to promote prostate tumorigenesis 
Nature  2012;493(7431):236-240.
Mutations in phosphatase and tensin homologue (PTEN) or genomic alterations in the phosphatidylinositol-3-OH kinase-signalling pathway are the most common genetic alterations reported in human prostate cancer1–4. However, the precise mechanism underlying how indolent tumours with PTEN alterations acquire meta-static potential remaisns poorly understood. Recent studies suggest that upregulation of transforming growth factor (TGF)-β signalling triggered by PTEN loss will form a growth barrier as a defence mechanism to constrain prostate cancer progression5, underscoring that TGF-β signalling might represent a pre-invasive checkpoint to prevent PTEN-mediated prostate tumorigenesis. Here we show that COUP transcription factor II (COUP-TFII, also known as NR2F2)6–9, a member of the nuclear receptor superfamily, serves as a key regulator to inhibit SMAD4-dependent transcription, and consequently overrides the TGF-β-dependent checkpoint for PTEN-null indolent tumours. Overexpression of COUP-TFII in the mouse prostate epithelium cooperates with PTEN deletion to augment malignant progression and produce an aggressive metastasis-prone tumour. The functional counteraction between COUP-TFII and SMAD4 is reinforced by genetically engineered mouse models in which conditional loss of SMAD4 diminishes the inhibitory effects elicited by COUP-TFII ablation. The biological significance of COUP-TFII in prostate carcinogenesis is substantiated by patient sample analysis, in which COUP-TFII expression or activity is tightly correlated with tumour recurrence and disease progression, whereas it is inversely associated with TGF-β signalling. These findings reveal that the destruction of the TGF-β-dependent barrier by COUP-TFII is crucial for the progression of PTEN-mutant prostate cancer into a life-threatening disease, and supports COUPTFII as a potential drug target for the intervention of metastatic human prostate cancer.
doi:10.1038/nature11674
PMCID: PMC4022346  PMID: 23201680
21.  ZEB1 sensitizes lung adenocarcinoma to metastasis suppression by PI3K antagonism 
The Journal of Clinical Investigation  2014;124(6):2696-2708.
Epithelial tumor cells that have undergone epithelial-to-mesenchymal transition (EMT) are typically prone to metastasis and drug resistance and contribute to a poor clinical outcome. The transcription factor ZEB1 is a known driver of EMT, and mediators of ZEB1 represent potential therapeutic targets for metastasis suppression. Here, we have shown that phosphatidylinositol 3-kinase–targeted (PI3K-targeted) therapy suppresses metastasis in a mouse model of Kras/Tp53-mutant lung adenocarcinoma that develops metastatic disease due to high expression of ZEB1. In lung adenocarcinoma cells from Kras/Tp53-mutant animals and human lung cancer cell lines, ZEB1 activated PI3K by derepressing miR-200 targets, including amphiregulin (AREG), betacellulin (BTC), and the transcription factor GATA6, which stimulated an EGFR/ERBB2 autocrine loop. Additionally, ZEB1-dependent derepression of the miR-200 and miR-183 target friend of GATA 2 (FOG2) enhanced GATA3-induced expression of the p110α catalytic subunit of PI3K. Knockdown of FOG2, p110α, and RHEB ameliorated invasive and metastatic propensities of tumor cells. Surprisingly, FOG2 was not required for mesenchymal differentiation, suggesting that mesenchymal differentiation and invasion are distinct and separable processes. Together, these results indicate that ZEB1 sensitizes lung adenocarcinoma cells to metastasis suppression by PI3K-targeted therapy and suggest that treatments to selectively modify the metastatic behavior of mesenchymal tumor cells are feasible and may be of clinical value.
doi:10.1172/JCI72171
PMCID: PMC4038569  PMID: 24762440
22.  Endocrine fibroblast growth factor FGF19 promotes prostate cancer progression 
Cancer research  2013;73(8):2551-2562.
Prostate cancer (PCa) is the most common visceral malignancy and the second leading cause of cancer deaths in US men. There is broad evidence that FGF receptors are important in PCa initiation and progression, but the contribution of particular FGFs in this disease is not fully understood. The FGF family members FGF19, FGF21 and FGF23 comprise a distinct subfamily that circulate in serum and act in an endocrine manner. These endocrine FGFs require α-Klotho (KL) and/or β-Klotho (KLB), two related single-pass transmembrane proteins restricted in their tissue distribution, to act as co-receptors along with classic FGFRs to mediate potent biological activity. Here we show that FGF19 is expressed in primary and metastatic PCa tissues where it functions as an autocrine growth factor. Exogenous FGF19 promoted the growth, invasion, adhesion and colony formation of PCa cells at low ligand concentrations. FGF19 silencing in PCa cells expressing autocrine FGF19 decreased invasion and proliferation in vitro and tumor growth in vivo. Consistent with these observations, KL and/or KLB were expressed in PCa cells in vitro and in vivo, raising the possibility that additional endocrine FGFs may also exert biological effects in PCa. Our findings support the concept that therapies targeting FGFR signaling these therapies may have efficacy in PCa and they highlight FGF19 as a relevant endocrine FGF in this setting.
doi:10.1158/0008-5472.CAN-12-4108
PMCID: PMC3630260  PMID: 23440425
prostate cancer; FGF19; signal transduction; fibroblast growth factors; endocrine; fibroblast growth factors; Klotho
23.  Integrative radiogenomic profiling of squamous cell lung cancer 
Cancer research  2013;73(20):10.1158/0008-5472.CAN-13-1616.
Radiation therapy is one of the mainstays of anti-cancer treatment, but the relationship between the radiosensitivity of cancer cells and their genomic characteristics is still not well-defined. Here we report the development of a high-throughput platform for measuring radiation survival in vitro and its validation by comparison to conventional clonogenic radiation survival analysis. We combined results from this high-throughput assay with genomic parameters in cell lines from squamous cell lung carcinoma, which is standardly treated by radiation therapy, to identify parameters that predict radiation sensitivity. We showed that activation of NFE2L2, a frequent event in lung squamous cancers, confers radiation resistance. An expression-based, in silico screen nominated inhibitors of PI3K as NFE2L2 antagonists. We showed that the selective PI3K inhibitor, NVP-BKM120, both decreased NRF2 protein levels and sensitized NFE2L2 or KEAP1 mutant cells to radiation. We then combined results from this high-throughput assay with single-sample gene set enrichment analysis (ssGSEA) of gene expression data. The resulting analysis identified pathways implicated in cell survival, genotoxic stress, detoxification, and innate and adaptive immunity as key correlates of radiation sensitivity. The integrative, high-throughput methods shown here for large-scale profiling of radiation survival and genomic features of solid-tumor derived cell lines should facilitate tumor radiogenomics and the discovery of genotype-selective radiation sensitizers and protective agents.
doi:10.1158/0008-5472.CAN-13-1616
PMCID: PMC3856255  PMID: 23980093
TP53; cmap; radiomodifier
25.  CXCR4/CXCL12 mediate autocrine cell cycle progression in NF1-associated malignant peripheral nerve sheath tumors 
Cell  2013;152(5):1077-1090.
Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are soft tissue sarcomas that arise in connective tissue surrounding peripheral nerves. They occur sporadically in a subset of patients with Neurofibromatosis type-1 (NF1). MPNSTs are highly aggressive, therapeutically resistant, and typically fatal. Using comparative transcriptome analysis, we identified CXCR4, a G protein-coupled receptor, as highly expressed in mouse models of NF1-deficient MPNSTs, but not in non-transformed precursor cells. The chemokine receptor CXCR4 and its ligand, CXCL12, promote MPNST growth by stimulating cyclin D1 expression and cell cycle progression through PI3-Kinase (PI3K) and β-catenin signaling. Suppression of CXCR4 activity, either by shRNA or pharmacological inhibition decreases MPNST cell growth in culture and inhibits tumorigenesis in allografts and in spontaneous genetic mouse models of MPNST. We further demonstrate conservation of these activated molecular pathways in human MPNSTs. Our findings indicate a role for CXCR4 in NF1-associated MPNST development, and identify a novel therapeutic target.
doi:10.1016/j.cell.2013.01.053
PMCID: PMC3594500  PMID: 23434321

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