Publications by authors named "Collene Jeter"

26 Publications

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Author Correction: LRIG1 is a pleiotropic androgen receptor-regulated feedback tumor suppressor in prostate cancer.

Nat Commun 2020 06 4;11(1):2895. Epub 2020 Jun 4.

Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, 14263, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-16615-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272421PMC
June 2020

LRIG1 is a pleiotropic androgen receptor-regulated feedback tumor suppressor in prostate cancer.

Nat Commun 2019 12 2;10(1):5494. Epub 2019 Dec 2.

Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, 14263, USA.

LRIG1 has been reported to be a tumor suppressor in gastrointestinal tract and epidermis. However, little is known about the expression, regulation and biological functions of LRIG1 in prostate cancer (PCa). We find that LRIG1 is overexpressed in PCa, but its expression correlates with better patient survival. Functional studies reveal strong tumor-suppressive functions of LRIG1 in both AR and AR xenograft models, and transgenic expression of LRIG1 inhibits tumor development in Hi-Myc and TRAMP models. LRIG1 also inhibits castration-resistant PCa and exhibits therapeutic efficacy in pre-established tumors. We further show that 1) AR directly transactivates LRIG1 through binding to several AR-binding sites in LRIG1 locus, and 2) LRIG1 dampens ERBB expression in a cell type-dependent manner and inhibits ERBB2-driven tumor growth. Collectively, our study indicates that LRIG1 represents a pleiotropic AR-regulated feedback tumor suppressor that functions to restrict oncogenic signaling from AR, Myc, ERBBs, and, likely, other oncogenic drivers.
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http://dx.doi.org/10.1038/s41467-019-13532-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889295PMC
December 2019

USP22 controls multiple signaling pathways that are essential for vasculature formation in the mouse placenta.

Development 2019 02 22;146(4). Epub 2019 Feb 22.

Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA

USP22, a component of the SAGA complex, is overexpressed in highly aggressive cancers, but the normal functions of this deubiquitinase are not well defined. We determined that loss of USP22 in mice results in embryonic lethality due to defects in extra-embryonic placental tissues and failure to establish proper vascular interactions with the maternal circulatory system. These phenotypes arise from abnormal gene expression patterns that reflect defective kinase signaling, including TGFβ and several receptor tyrosine kinase pathways. USP22 deletion in endothelial cells and pericytes that are induced from embryonic stem cells also hinders these signaling cascades, with detrimental effects on cell survival and differentiation as well as on the ability to form vessels. Our findings provide new insights into the functions of USP22 during development that may offer clues to its role in disease states.
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http://dx.doi.org/10.1242/dev.174037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398448PMC
February 2019

Linking prostate cancer cell AR heterogeneity to distinct castration and enzalutamide responses.

Nat Commun 2018 09 6;9(1):3600. Epub 2018 Sep 6.

Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, 14263, USA.

Expression of androgen receptor (AR) in prostate cancer (PCa) is heterogeneous but the functional significance of AR heterogeneity remains unclear. Screening ~200 castration-resistant PCa (CRPC) cores and whole-mount sections (from 89 patients) reveals 3 AR expression patterns: nuclear (nuc-AR), mixed nuclear/cytoplasmic (nuc/cyto-AR), and low/no expression (AR). Xenograft modeling demonstrates that AR CRPC is enzalutamide-sensitive but AR CRPC is resistant. Genome editing-derived AR and AR-knockout LNCaP cell clones exhibit distinct biological and tumorigenic properties and contrasting responses to enzalutamide. RNA-Seq and biochemical analyses, coupled with experimental combinatorial therapy, identify BCL-2 as a critical therapeutic target and provide proof-of-concept therapeutic regimens for both AR and AR CRPC. Our study links AR expression heterogeneity to distinct castration/enzalutamide responses and has important implications in understanding the cellular basis of prostate tumor responses to AR-targeting therapies and in facilitating development of novel therapeutics to target AR PCa cells/clones.
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http://dx.doi.org/10.1038/s41467-018-06067-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6127155PMC
September 2018

Histone 2B-GFP Label-Retaining Prostate Luminal Cells Possess Progenitor Cell Properties and Are Intrinsically Resistant to Castration.

Stem Cell Reports 2018 01 21;10(1):228-242. Epub 2017 Dec 21.

Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY 14263, USA; Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA; Cancer Stem Cell Institute, Research Center for Translational Medicine, East Hospital, Tongji University, Shanghai 200120, PR China. Electronic address:

The existence of slow-cycling luminal cells in the prostate has been suggested, but their identity and functional properties remain unknown. Using a bigenic mouse model to earmark, isolate, and characterize the quiescent stem-like cells, we identify a label-retaining cell (LRC) population in the luminal cell layer as luminal progenitors. Molecular and biological characterizations show that these luminal LRCs are significantly enriched in the mouse proximal prostate, exhibit relative dormancy, display bipotency in both in vitro and in vivo assays, and express a stem/progenitor gene signature with resemblance to aggressive prostate cancer. Importantly, these LRCs, compared with bulk luminal cells, maintain a lower level of androgen receptor (AR) expression and are less androgen dependent and also castration resistant in vivo. Finally, analysis of phenotypic markers reveals heterogeneity within the luminal progenitor cell pool. Our study establishes luminal LRCs as progenitors that may serve as a cellular origin for castration-resistant prostate cancer.
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http://dx.doi.org/10.1016/j.stemcr.2017.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768933PMC
January 2018

GCN5 Regulates FGF Signaling and Activates Selective MYC Target Genes during Early Embryoid Body Differentiation.

Stem Cell Reports 2018 01 14;10(1):287-299. Epub 2017 Dec 14.

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA; Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA; Program in Epigenetics and Molecular Carcinogenesis, The Graduate School of Biomedical Sciences (GSBS) of the University of Texas Health Science Center at Houston, Houston, TX 77030, USA; Program in Genes and Development, The Graduate School of Biomedical Sciences (GSBS) of the University of Texas Health Science Center at Houston, Houston, TX 77030, USA. Electronic address:

Precise control of gene expression during development is orchestrated by transcription factors and co-regulators including chromatin modifiers. How particular chromatin-modifying enzymes affect specific developmental processes is not well defined. Here, we report that GCN5, a histone acetyltransferase essential for embryonic development, is required for proper expression of multiple genes encoding components of the fibroblast growth factor (FGF) signaling pathway in early embryoid bodies (EBs). Gcn5 EBs display deficient activation of ERK and p38, mislocalization of cytoskeletal components, and compromised capacity to differentiate toward mesodermal lineage. Genomic analyses identified seven genes as putative direct targets of GCN5 during early differentiation, four of which are cMYC targets. These findings established a link between GCN5 and the FGF signaling pathway and highlighted specific GCN5-MYC partnerships in gene regulation during early differentiation.
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http://dx.doi.org/10.1016/j.stemcr.2017.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768892PMC
January 2018

Transgenic overexpression of NanogP8 in the mouse prostate is insufficient to initiate tumorigenesis but weakly promotes tumor development in the Hi-Myc mouse model.

Oncotarget 2017 Aug 18;8(32):52746-52760. Epub 2017 Apr 18.

Department of Molecular Carcinogenesis, University of Texas M.D Anderson Cancer Center, Science Park, Smithville, TX 78957, USA.

This project was undertaken to address a critical cancer biology question: Is overexpression of the pluripotency molecule Nanog sufficient to initiate tumor development in a somatic tissue? Nanog1 is critical for the self-renewal and pluripotency of ES cells, and its retrotransposed homolog, NanogP8 is preferentially expressed in somatic cancer cells. Our work has shown that shRNA-mediated knockdown of NanogP8 in prostate, breast, and colon cancer cells inhibits tumor regeneration whereas inducible overexpression of NanogP8 promotes cancer stem cell phenotypes and properties. To address the key unanswered question whether tissue-specific overexpression of NanogP8 is sufficient to promote tumor development , we generated a NanogP8 transgenic mouse model, in which the ARRPB promoter was used to drive NanogP8 cDNA. Surprisingly, the ARRPB-NanogP8 transgenic mice were viable, developed normally, and did not form spontaneous tumors in >2 years. Also, both wild type and ARRPB-NanogP8 transgenic mice responded similarly to castration and regeneration and castrated ARRPB-NanogP8 transgenic mice also did not develop tumors. By crossing the ARRPB-NanogP8 transgenic mice with ARRPB-Myc (i.e., Hi-Myc) mice, we found that the double transgenic (i.e., ARRPB-NanogP8; Hi-Myc) mice showed similar tumor incidence and histology to the Hi-Myc mice. Interestingly, however, we observed white dots in the ventral lobes of the double transgenic prostates, which were characterized as overgrown ductules/buds featured by crowded atypical Nanog-expressing luminal cells. Taken together, our present work demonstrates that transgenic overexpression of NanogP8 in the mouse prostate is insufficient to initiate tumorigenesis but weakly promotes tumor development in the Hi-Myc mouse model.
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http://dx.doi.org/10.18632/oncotarget.17186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5581066PMC
August 2017

NANOG reprograms prostate cancer cells to castration resistance via dynamically repressing and engaging the AR/FOXA1 signaling axis.

Cell Discov 2016 15;2:16041. Epub 2016 Nov 15.

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, USA; Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY, USA; Cancer Stem Cell Institute, Research Center for Translational Medicine, East Hospital, Tongji University School of Medicine, Shanghai, China; Centers for Cancer Epigenetics, Stem Cell and Developmental Biology, RNA Interference and Non-coding RNAs and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Houston, TX, USA.

The pluripotency transcription factor NANOG has been implicated in tumor development, and NANOG-expressing cancer cells manifest stem cell properties that sustain tumor homeostasis, mediate therapy resistance and fuel tumor progression. However, how NANOG converges on somatic circuitry to trigger oncogenic reprogramming remains obscure. We previously reported that inducible NANOG expression propels the emergence of aggressive castration-resistant prostate cancer phenotypes. Here we first show that endogenous NANOG is required for the growth of castration-resistant prostate cancer xenografts. Genome-wide chromatin immunoprecipitation sequencing coupled with biochemical assays unexpectedly reveals that NANOG co-occupies a distinctive proportion of androgen receptor/Forkhead box A1 genomic loci and physically interacts with androgen receptor and Forkhead box A1. Integrative analysis of chromatin immunoprecipitation sequencing and time-resolved RNA sequencing demonstrates that NANOG dynamically alters androgen receptor/Forkhead box A1 signaling leading to both repression of androgen receptor-regulated pro-differentiation genes and induction of genes associated with cell cycle, stem cells, cell motility and castration resistance. Our studies reveal global molecular mechanisms whereby NANOG reprograms prostate cancer cells to a clinically relevant castration-resistant stem cell-like state driven by distinct NANOG-regulated gene clusters that correlate with patient survival. Thus, reprogramming factors such as NANOG may converge on and alter lineage-specific master transcription factors broadly in somatic cancers, thereby facilitating malignant disease progression and providing a novel route for therapeutic resistance.
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http://dx.doi.org/10.1038/celldisc.2016.41DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5109294PMC
November 2016

miR-199a-3p targets stemness-related and mitogenic signaling pathways to suppress the expansion and tumorigenic capabilities of prostate cancer stem cells.

Oncotarget 2016 Aug;7(35):56628-56642

Cancer Stem Cell Institute, Research Center for Translational Medicine, East Hospital, Tongji University School of Medicine, Shanghai 200120, China.

Human cancers exhibit significant cellular heterogeneity featuring tumorigenic cancer stem cells (CSCs) in addition to more differentiated progeny with limited tumor-initiating capabilities. Recent studies suggest that microRNAs (miRNAs) regulate CSCs and tumor development. A previous library screening for differential miRNA expression in CD44+ (and other) prostate CSC vs. non-CSC populations identified miR-199a-3p to be among the most highly under-expressed miRNAs in CSCs. In this study, we characterized the biological functions of miR-199a-3p in CD44+ prostate cancer (PCa) cells and in tumor regeneration. Overexpression of miR-199a-3p in purified CD44+ or bulk PCa cells, including primary PCa, inhibited proliferation and clonal expansion without inducing apoptosis. miR-199a-3p overexpression also diminished tumor-initiating capacities of CD44+ PCa cells as well as tumor regeneration from bulk PCa cells. Importantly, inducible miR-199a-3p expression in pre-established prostate tumors in NOD/SCID mice inhibited tumor growth. Using target prediction program and luciferase assays, we show mechanistically that CD44 is a direct functional target of miR-199a-3p in PCa cells. Moreover, miR-199a-3p also directly or indirectly targeted several additional mitogenic molecules, including c-MYC, cyclin D1 (CCND1) and EGFR. Taken together, our results demonstrate how the aberrant loss of a miRNA-mediated mechanism can lead to the expansion and tumorigenic activity of prostate CSCs, further supporting the development and implementation of miRNA mimics for cancer treatment.
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http://dx.doi.org/10.18632/oncotarget.10652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302940PMC
August 2016

Defining a Population of Stem-like Human Prostate Cancer Cells That Can Generate and Propagate Castration-Resistant Prostate Cancer.

Clin Cancer Res 2016 Sep 8;22(17):4505-16. Epub 2016 Apr 8.

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, Texas. Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York. Cancer Stem Cell Institute, Research Center for Translational Medicine, East Hospital, Tongji University School of Medicine, Shanghai, China. Centers for Cancer Epigenetics, Stem Cell and Developmental Biology, RNA Interference and Non-coding RNAs, and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, Texas.

Purpose: We have shown that the phenotypically undifferentiated (PSA(-/lo)) prostate cancer cell population harbors long-term self-renewing cancer stem cells (CSC) that resist castration, and a subset of the cells within the PSA(-/lo) population bearing the ALDH(hi)CD44(+)α2β1(+) phenotype (Triple Marker(+)/TM(+)) is capable of robustly initiating xenograft tumors in castrated mice. The goal of the current project is to further characterize the biologic properties of TM(+) prostate cancer cell population, particularly in the context of initiating and propagating castration-resistant prostate cancer (CRPC).

Experimental Design: The in vivo CSC activities were measured by limiting-dilution serial tumor transplantation assays in both androgen-dependent and androgen-independent prostate cancer xenograft models. In vitro clonal, clonogenic, and sphere-formation assays were conducted in cells purified from xenograft and patient tumors. qPCR, Western blot, lentiviral-mediated gene knockdown, and human microRNA arrays were performed for mechanistic studies.

Results: By focusing on the LAPC9 model, we show that the TM(+) cells are CSCs with both tumor-initiating and tumor-propagating abilities for CRPC. Moreover, primary patient samples have TM(+) cells, which possess CSC activities in "castrated" culture conditions. Mechanistically, we find that (i) the phenotypic markers are causally involved in CRPC development; (ii) the TM(+) cells preferentially express castration resistance and stem cell-associated molecules that regulate their CSC characteristics; and (iii) the TM(+) cells possess distinct microRNA expression profiles and miR-499-5p functions as an oncomir.

Conclusions: Our results define the TM(+) prostate cancer cells as a population of preexistent stem-like cancer cells that can both mediate and propagate CRPC and highlight the TM(+) cell population as a therapeutic target. Clin Cancer Res; 22(17); 4505-16. ©2016 AACR.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-2956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5010458PMC
September 2016

Systematic dissection of phenotypic, functional, and tumorigenic heterogeneity of human prostate cancer cells.

Oncotarget 2015 Sep;6(27):23959-86

Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Science Park, Smithville, TX 78957, USA.

Human cancers are heterogeneous containing stem-like cancer cells operationally defined as cancer stem cells (CSCs) that possess great tumor-initiating and long-term tumor-propagating properties. In this study, we systematically dissect the phenotypic, functional and tumorigenic heterogeneity in human prostate cancer (PCa) using xenograft models and >70 patient tumor samples. In the first part, we further investigate the PSA-/lo PCa cell population, which we have recently shown to harbor self-renewing long-term tumor-propagating cells and present several novel findings. We show that discordant AR and PSA expression in both untreated and castration-resistant PCa (CRPC) results in AR+PSA+, AR+PSA-, AR-PSA-, and AR-PSA+ subtypes of PCa cells that manifest differential sensitivities to therapeutics. We further demonstrate that castration leads to a great enrichment of PSA-/lo PCa cells in both xenograft tumors and CRPC samples and systemic androgen levels dynamically regulate the relative abundance of PSA+ versus PSA-/lo PCa cells that impacts the kinetics of tumor growth. We also present evidence that the PSA-/lo PCa cells possess distinct epigenetic profiles. As the PSA-/lo PCa cell population is heterogeneous, in the second part, we employ two PSA- (Du145 and PC3) and two PSA+ (LAPC9 and LAPC4) PCa models as well as patient tumor cells to further dissect the clonogenic and tumorigenic subsets. We report that different PCa models possess distinct tumorigenic subpopulations that both commonly and uniquely express important signaling pathways that could represent therapeutic targets. Our results have important implications in understanding PCa cell heterogeneity, response to clinical therapeutics, and cellular mechanisms underlying CRPC.
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http://dx.doi.org/10.18632/oncotarget.4260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4695164PMC
September 2015

Regulation of NANOG in cancer cells.

Mol Carcinog 2015 Sep 27;54(9):679-87. Epub 2015 May 27.

Department of Epigenetics and Molecular Carcinogenesis, University of Texas M.D Anderson Cancer Center, city, Smithville, Texas.

As one of the key pluripotency transcription factors, NANOG plays a critical role in maintaining the self-renewal and pluripotency in normal embryonic stem cells. Recent data indicate that NANOG is expressed in a variety of cancers and its expression correlates with poor survival in cancer patients. Of interest, many studies suggest that NANOG enhances the defined characteristics of cancer stem cells and may thus function as an oncogene to promote carcinogenesis. Therefore, NANOG expression determines the cell fate not only in pluripotent cells but also in cancer cells. Although the regulation of NANOG in normal embryonic stem cells is reasonably well understood, the regulation of NANOG in cancer cells has only emerged recently. The current review provides a most updated summary on how NANOG expression is regulated during tumor development and progression.
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http://dx.doi.org/10.1002/mc.22340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536084PMC
September 2015

Concise Review: NANOG in Cancer Stem Cells and Tumor Development: An Update and Outstanding Questions.

Stem Cells 2015 Aug 13;33(8):2381-90. Epub 2015 May 13.

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Science Park, Smithville, Texas, USA.

The homeobox domain transcription factor NANOG, a key regulator of embryonic development and cellular reprogramming, has been reported to be broadly expressed in human cancers. Functional studies have provided strong evidence that NANOG possesses protumorigenic attributes. In addition to promoting self-renewal and long-term proliferative potential of stem-like cancer cells, NANOG-mediated oncogenic reprogramming may underlie clinical manifestations of malignant disease. In this review, we examine the molecular origin, expression, biological activities, and mechanisms of action of NANOG in various malignancies. We also consider clinical implications such as correlations between NANOG expression and cancer prognosis and/or response to therapy. We surmise that NANOG potentiates the molecular circuitry of tumorigenesis, and thus may represent a novel therapeutic target or biomarker for the diagnosis, prognosis, and treatment outcome of cancer. Finally, we present critical pending questions relating NANOG to cancer stem cells and tumor development.
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http://dx.doi.org/10.1002/stem.2007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4509798PMC
August 2015

miRNA-128 suppresses prostate cancer by inhibiting BMI-1 to inhibit tumor-initiating cells.

Cancer Res 2014 Aug 5;74(15):4183-95. Epub 2014 Jun 5.

Department of Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Science Park, Smithville; Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China

microRNA-128 (miR128) is reduced in prostate cancer relative to normal/benign prostate tissues, but causal roles are obscure. Here we show that exogenously introduced miR128 suppresses tumor regeneration in multiple prostate cancer xenograft models. Cancer stem-like cell (CSC)-associated properties were blocked, including holoclone and sphere formation as well as clonogenic survival. Using a miR128 sensor to distinguish cells on the basis of miR128 expression, we found that miR128-lo cells possessed higher clonal, clonogenic, and tumorigenic activities than miR128-hi cells. miR128 targets the stem cell regulatory factors BMI-1, NANOG, and TGFBR1, the expression of which we found to vary inversely with miR128 expression in prostate cancer stem/progenitor cell populations. In particular, we defined BMI-1 as a direct and functionally relevant target of miR128 in prostate cancer cells, where these genes were reciprocally expressed and exhibited opposing biological functions. Our results define a tumor suppressor function for miR128 in prostate cancer by limiting CSC properties mediated by BMI-1 and other central stem cell regulators, with potential implications for prostate cancer gene therapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-0404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4174451PMC
August 2014

Nanog1 in NTERA-2 and recombinant NanogP8 from somatic cancer cells adopt multiple protein conformations and migrate at multiple M.W species.

PLoS One 2014 5;9(3):e90615. Epub 2014 Mar 5.

Department of Molecular Carcinogenesis, University of Texas M.D Anderson Cancer Center, Science Park, Smithville, Texas, United States of America; Cancer Stem Cell Institute, Research Center for Translational Medicine, East Hospital, Tongji University School of Medicine, Shanghai, China.

Human Nanog1 is a 305-amino acid (aa) homeodomain-containing transcription factor critical for the pluripotency of embryonic stem (ES) and embryonal carcinoma (EC) cells. Somatic cancer cells predominantly express a retrogene homolog of Nanog1 called NanogP8, which is ~99% similar to Nanog at the aa level. Although the predicted M.W of Nanog1/NanogP8 is ∼35 kD, both have been reported to migrate, on Western blotting (WB), at apparent molecular masses of 29-80 kD. Whether all these reported protein bands represent authentic Nanog proteins is unclear. Furthermore, detailed biochemical studies on Nanog1/NanogpP8 have been lacking. By combining WB using 8 anti-Nanog1 antibodies, immunoprecipitation, mass spectrometry, and studies using recombinant proteins, here we provide direct evidence that the Nanog1 protein in NTERA-2 EC cells exists as multiple M.W species from ~22 kD to 100 kD with a major 42 kD band detectable on WB. We then demonstrate that recombinant NanogP8 (rNanogP8) proteins made in bacteria using cDNAs from multiple cancer cells also migrate, on denaturing SDS-PAGE, at ~28 kD to 180 kD. Interestingly, different anti-Nanog1 antibodies exhibit differential reactivity towards rNanogP8 proteins, which can spontaneously form high M.W protein species. Finally, we show that most long-term cultured cancer cell lines seem to express very low levels of or different endogenous NanogP8 protein that cannot be readily detected by immunoprecipitation. Altogether, the current study reveals unique biochemical properties of Nanog1 in EC cells and NanogP8 in somatic cancer cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0090615PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3944193PMC
January 2015

The cancer gene WWOX behaves as an inhibitor of SMAD3 transcriptional activity via direct binding.

BMC Cancer 2013 Dec 11;13:593. Epub 2013 Dec 11.

Department of Molecular Carcinogenesis, Science Park, The University of Texas M,D, Anderson Cancer Center, Smithville, TX 78957, USA.

Background: The WW domain containing protein WWOX has been postulated to behave as a tumor suppressor in breast and other cancers. Expression of this protein is lost in over 70% of ER negative tumors. This prompted us to investigate the phenotypic and gene expression effects of loss of WWOX expression in breast cells.

Methods: Gene expression microarrays and standard in vitro assays were performed on stably silenced WWOX (shRNA) normal breast cells. Bioinformatic analyses were used to identify gene networks and transcriptional regulators affected by WWOX silencing. Co-immunoprecipitations and GST-pulldowns were used to demonstrate a direct interaction between WWOX and SMAD3. Reporter assays, ChIP, confocal microscopy and in silico analyses were employed to determine the effect of WWOX silencing on TGFβ-signaling.

Results: WWOX silencing affected cell proliferation, motility, attachment and deregulated expression of genes involved in cell cycle, motility and DNA damage. Interestingly, we detected an enrichment of targets activated by the SMAD3 transcription factor, including significant upregulation of ANGPTL4, FST, PTHLH and SERPINE1 transcripts. Importantly, we demonstrate that the WWOX protein physically interacts with SMAD3 via WW domain 1. Furthermore, WWOX expression dramatically decreases SMAD3 occupancy at the ANGPTL4 and SERPINE1 promoters and significantly quenches activation of a TGFβ responsive reporter. Additionally, WWOX expression leads to redistribution of SMAD3 from the nuclear to the cytoplasmic compartment. Since the TGFβ target ANGPTL4 plays a key role in lung metastasis development, we performed a meta-analysis of ANGPTL4 expression relative to WWOX in microarray datasets from breast carcinomas. We observed a significant inverse correlation between WWOX and ANGPTL4. Furthermore, the WWOX(lo)/ANGPTL4(hi) cluster of breast tumors is enriched in triple-negative and basal-like sub-types. Tumors with this gene expression signature could represent candidates for anti-TGFβ targeted therapies.

Conclusions: We show for the first time that WWOX modulates SMAD3 signaling in breast cells via direct WW-domain mediated binding and potential cytoplasmic sequestration of SMAD3 protein. Since loss of WWOX expression increases with breast cancer progression and it behaves as an inhibitor of SMAD3 transcriptional activity these observations may help explain, at least in part, the paradoxical pro-tumorigenic effects of TGFβ signaling in advanced breast cancer.
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http://dx.doi.org/10.1186/1471-2407-13-593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3871008PMC
December 2013

In vivo functional studies of tumor-specific retrogene NanogP8 in transgenic animals.

Cell Cycle 2013 Aug 26;12(15):2395-408. Epub 2013 Jun 26.

Department of Molecular Carcinogenesis; University of Texas MD Anderson Cancer Center; Smithville, TX USA; Program in Molecular Carcinogenesis; University of Texas Graduate School of Biomedical Sciences (GSBS); Houston, TX USA.

The current study was undertaken to investigate potential oncogenic functions of NanogP8, a tumor-specific retrogene homolog of Nanog (expressed in pluripotent cells), in transgenic animal models. To this end, human primary prostate tumor-derived NanogP8 was targeted to the cytokeratin 14 (K14) cellular compartment, and two lines of K14-NanogP8 mice were derived. The line 1 animals, expressing high levels of NanogP8, experienced perinatal lethality and developmental abnormalities in multiple organs, including the skin, tongue, eye, and thymus in surviving animals. On postnatal day 5 transgenic skin, for example, there was increased c-Myc expression and Ki-67(+) cells accompanied by profound abnormalities in skin development such as thickened interfollicular epidermis and dermis and lack of hypodermis and sebaceous glands. The line 3 mice, expressing low levels of NanogP8, were grossly normal except cataract development by 4-6 mo of age. Surprisingly, both lines of mice do not develop spontaneous tumors related to transgene expression. Even more unexpectedly, high levels of NanogP8 expression in L1 mice actually inhibited tumor development in a two-stage chemical carcinogenesis model. Mechanistic studies revealed that constitutive NanogP8 overexpression in adult L1 mice reduced CD34(+)α6(+) and Lrig-1(+) bulge stem cells, impaired keratinocyte migration, and repressed the expression of many stem cell-associated genes, including Bmp5, Fgfr2, Jmjd1a, and Jun. Our study, for the first time, indicates that transgenically expressed human NanogP8 is biologically functional, but suggests that high levels of NanogP8 may disrupt normal developmental programs and inhibit tumor development by depleting stem cells.
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http://dx.doi.org/10.4161/cc.25402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841319PMC
August 2013

The PSA(-/lo) prostate cancer cell population harbors self-renewing long-term tumor-propagating cells that resist castration.

Cell Stem Cell 2012 May;10(5):556-69

Department of Molecular Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Science Park, Smithville, TX 78957, USA.

Prostate cancer (PCa) is heterogeneous and contains both differentiated and undifferentiated tumor cells, but the relative functional contribution of these two cell populations remains unclear. Here we report distinct molecular, cellular, and tumor-propagating properties of PCa cells that express high (PSA(+)) and low (PSA(-/lo)) levels of the differentiation marker PSA. PSA(-/lo) PCa cells are quiescent and refractory to stresses including androgen deprivation, exhibit high clonogenic potential, and possess long-term tumor-propagating capacity. They preferentially express stem cell genes and can undergo asymmetric cell division to generate PSA(+) cells. Importantly, PSA(-/lo) PCa cells can initiate robust tumor development and resist androgen ablation in castrated hosts, and they harbor highly tumorigenic castration-resistant PCa cells that can be prospectively enriched using ALDH(+)CD44(+)α2β1(+) phenotype. In contrast, PSA(+) PCa cells possess more limited tumor-propagating capacity, undergo symmetric division, and are sensitive to castration. Altogether, our study suggests that PSA(-/lo) cells may represent a critical source of castration-resistant PCa cells.
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http://dx.doi.org/10.1016/j.stem.2012.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3348510PMC
May 2012

The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44.

Nat Med 2011 Feb 16;17(2):211-5. Epub 2011 Jan 16.

Department of Molecular Carcinogenesis, the University of Texas M.D. Anderson Cancer Center, Science Park, Smithville, Texas, USA.

Cancer stem cells (CSCs), or tumor-initiating cells, are involved in tumor progression and metastasis. MicroRNAs (miRNAs) regulate both normal stem cells and CSCs, and dysregulation of miRNAs has been implicated in tumorigenesis. CSCs in many tumors--including cancers of the breast, pancreas, head and neck, colon, small intestine, liver, stomach, bladder and ovary--have been identified using the adhesion molecule CD44, either individually or in combination with other marker(s). Prostate CSCs with enhanced clonogenic and tumor-initiating and metastatic capacities are enriched in the CD44(+) cell population, but whether miRNAs regulate CD44(+) prostate cancer cells and prostate cancer metastasis remains unclear. Here we show, through expression analysis, that miR-34a, a p53 target, was underexpressed in CD44(+) prostate cancer cells purified from xenograft and primary tumors. Enforced expression of miR-34a in bulk or purified CD44(+) prostate cancer cells inhibited clonogenic expansion, tumor regeneration, and metastasis. In contrast, expression of miR-34a antagomirs in CD44(-) prostate cancer cells promoted tumor development and metastasis. Systemically delivered miR-34a inhibited prostate cancer metastasis and extended survival of tumor-bearing mice. We identified and validated CD44 as a direct and functional target of miR-34a and found that CD44 knockdown phenocopied miR-34a overexpression in inhibiting prostate cancer regeneration and metastasis. Our study shows that miR-34a is a key negative regulator of CD44(+) prostate cancer cells and establishes a strong rationale for developing miR-34a as a novel therapeutic agent against prostate CSCs.
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http://dx.doi.org/10.1038/nm.2284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3076220PMC
February 2011

Methodologies in assaying prostate cancer stem cells.

Methods Mol Biol 2009 ;568:85-138

Department of Carcinogenesis, University of Texas MD Anderson Cancer Center, Smithville, TX, USA.

The cancer stem cell (CSC) theory posits that only a small population of tumor cells within the tumor has the ability to reinitiate tumor development and is responsible for tumor homeostasis and progression. Tumor initiation is a defining property of putative CSCs, which have been reported in both blood malignancies and solid tumors. In order to test whether any given human tumor cell population has CSC properties, the relatively enriched single cells have to be put into a foreign microenvironment in a recipient animal to test their tumorigenic potential. Furthermore, various in vitro assays need be performed to demonstrate that the presumed CSCs have certain biological properties normally associated with the stem cells (SCs). Herein, we present a comprehensive review of the experimental methodologies that our lab has been using in assaying putative prostate cancer (PCa) SCs in culture, xenograft tumors, and primary tumor samples.
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http://dx.doi.org/10.1007/978-1-59745-280-9_7DOI Listing
November 2009

Functional evidence that the self-renewal gene NANOG regulates human tumor development.

Stem Cells 2009 May;27(5):993-1005

Department of Carcinogenesis, University of Texas MD Anderson Cancer Center, Science Park-Research Division, Smithville, TX 78957, USA.

Tumor development has long been known to resemble abnormal embryogenesis. The embryonic stem cell (ESC) self-renewal gene NANOG is purportedly expressed by some epithelial cancer cells but a causal role in tumor development has remained unclear. Here, we provide compelling evidence that cultured cancer cells, as well as xenograft- and human primary prostate cancer cells express a functional variant of NANOG. NANOG mRNA in cancer cells is derived predominantly from a retrogene locus termed NANOGP8. NANOG protein is detectable in the nucleus of cancer cells and is expressed higher in patient prostate tumors than matched benign tissues. NANOGP8 mRNA and/or NANOG protein levels are enriched in putative cancer stem/progenitor cell populations. Importantly, extensive loss-of-function analysis reveals that RNA interference-mediated NANOG knockdown inhibits tumor development, establishing a functional significance for NANOG expression in cancer cells. Nanog short hairpin RNA transduced cancer cells exhibit decreased long-term clonal and clonogenic growth, reduced proliferation and, in some cases, altered differentiation. Thus, our results demonstrate that NANOG, a cell-fate regulatory molecule known to be important for ESC self-renewal, also plays a novel role in tumor development.
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http://dx.doi.org/10.1002/stem.29DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3327393PMC
May 2009

Abnormal differentiation, hyperplasia and embryonic/perinatal lethality in BK5-T/t transgenic mice.

Differentiation 2009 Mar 21;77(3):324-34. Epub 2008 Nov 21.

Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, 1808 Park Road 1-C, P.O. Box 389, Smithville, TX 78957, USA.

The cell-of-origin has a great impact on the types of tumors that develop and the stem/progenitor cells have long been considered main targets of malignant transformation. The SV40 (SV40-Simian Virus 40) large T and small t antigens (T/t), have been targeted to multiple-differentiated cellular compartments in transgenic mice. In most of these studies, transgenic animals develop tumors without apparent defects in animal development. In this study, we used the bovine keratin 5 (BK5) promoter to target the T/t antigens to stem/progenitor cell-containing cytokeratin 5 (CK5) cellular compartment. A transgene construct, BK5-T/t, was made and microinjected into the male pronucleus of FVB/N mouse oocytes. After implanting approximately 1700 embryos, only 7 transgenics were obtained, including 4 embryos (E9.5, E13, E15, and E20) and 3 postnatal animals, which died at P1, P2, and P18, respectively. Immunohistological analysis revealed aberrant differentiation and prominent hyperplasia in several transgenic CK5 tissues, especially the upper digestive organs (tongue, oral mucosa, esophagus, and forestomach) and epidermis, the latter of which also showed focal dysplasia. Altogether, these results indicate that constitutive expression of the T/t antigens in CK5 cellular compartment results in abnormal epithelial differentiation and leads to embryonic/perinatal animal lethality.
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http://dx.doi.org/10.1016/j.diff.2008.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2680132PMC
March 2009

Critical and distinct roles of p16 and telomerase in regulating the proliferative life span of normal human prostate epithelial progenitor cells.

J Biol Chem 2008 Oct 28;283(41):27957-27972. Epub 2008 Jul 28.

Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957; Program in Molecular Carcinogenesis, Graduate School of Biomedical Sciences, Houston, Texas 77030. Electronic address:

Normal human prostate (NHP) epithelial cells undergo senescence in vitro and in vivo, but the underlying molecular mechanisms remain obscure. Here we show that the senescence of primary NHP cells, which are immunophenotyped as intermediate basal-like cells expressing progenitor cell markers CD44, alpha2beta1, p63, hTERT, and CK5/CK18, involves loss of telomerase expression, up-regulation of p16, and activation of p53. Using genetically defined manipulations of these three signaling pathways, we show that p16 is the primary determinant of the NHP cell proliferative capacity and that hTERT is required for unlimited proliferative life span. Hence, suppression of p16 significantly extends NHP cell life span, but both p16 inhibition and hTERT are required to immortalize NHP cells. Importantly, immortalized NHP cells retain expression of most progenitor markers, demonstrate gene expression profiles characteristic of proliferating progenitor cells, and possess multilineage differentiation potential generating functional prostatic glands. Our studies shed important light on the molecular mechanisms regulating the proliferative life span of NHP progenitor cells.
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http://dx.doi.org/10.1074/jbc.M803467200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2562067PMC
October 2008

Plant responses to extracellular nucleotides: Cellular processes and biological effects.

Purinergic Signal 2006 Sep 11;2(3):443-9. Epub 2006 Aug 11.

Science Park-Research Division, Department of Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, 78957, USA.

Higher plants exhibit cellular responsiveness to the exogenous application of purine nucleotides in a manner consistent with a cell-cell signaling function for these molecules. Like animals, plants respond to extracellular ATP, ADP, and stable analogues (e.g., ATPgammaS and ADPbetaS) by increasing the cytoplasmic concentration of calcium. Agonist substrate specificity and concentration dependency suggest receptor mediation of these events, and, although the identity of the plant receptor is currently unknown, pharmacological analysis points to the involvement of a plasma membrane-localized calcium channel. Extracellular ATP can also induce the production of reactive oxygen species and stimulate an increase in the mRNA levels of a number of stress- and calcium-regulated genes, suggesting a role for nucleotide-based signaling in plant wound and defense responses. Furthermore, the growth and development of plants can also be altered by the application of external ATP. Recent studies are only beginning to uncover the complexities of plant signaling networks activated in response to extracellular ATP and how these might interact to affect plant physiological processes.
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http://dx.doi.org/10.1007/s11302-005-3981-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2254475PMC
September 2006

Prostate cancer stem/progenitor cells: identification, characterization, and implications.

Mol Carcinog 2007 Jan;46(1):1-14

Department of Carcinogenesis, The University of Texas MD Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957, USA.

Several solid tumors have now been shown to contain stem cell-like cells called cancer stem cells (CSC). These cells, although generally rare, appear to be highly tumorigenic and may be the cells that drive tumor formation, maintain tumor homeostasis, and mediate tumor metastasis. In this Perspective, we first provide our insight on how a CSC should be defined. We then summarize our current knowledge of stem/progenitor cells in the normal human prostate (NHP), an organ highly susceptible to hyperproliferative diseases such as benign prostate hyperplasia (BPH) and prostate cancer (PCa). We further review the evidence that cultured PCa cells, xenograft prostate tumors, and patient tumors may contain stem/progenitor cells. Along with our discussion, we present several methodologies that can be potentially used to identify putative tumor-reinitiating CSC. Finally, we present a hypothetical model for the hierarchical organization of human PCa cells and discuss the implications of this model in helping understand prostate carcinogenesis and design novel diagnostic, prognostic, and therapeutic approaches.
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http://dx.doi.org/10.1002/mc.20255DOI Listing
January 2007

Evidence of a novel cell signaling role for extracellular adenosine triphosphates and diphosphates in Arabidopsis.

Plant Cell 2004 Oct 14;16(10):2652-64. Epub 2004 Sep 14.

Section of Molecular Cell and Developmental Biology, University of Texas, Austin, Texas 78712, USA.

Extracellular ATP is a known receptor agonist in animals and was previously shown to alter plant growth, and so we investigated whether ATP derivatives could function outside plant cells as signaling agents. Signaling responses induced by exogenous nucleotides in animal cells typically include increases in free cytoplasmic calcium concentration ([Ca(2+)](cyt)). We have evaluated the ability of exogenously applied adenosine 5'-[gamma-thio]triphosphate (ATPgammaS), adenosine 5'-[beta-thio]diphosphate (ADPbetaS), and adenosine 5'-O-thiomonophosphate to alter [Ca(2+)](cyt) in intact apoaequorin transgenic Arabidopsis thaliana seedlings. ATPgammaS and ADPbetaS increase [Ca(2+)](cyt), and this increase is enhanced further when the nucleotides are added with the elicitor oligogalacturonic acid. Exogenous treatment with ATP also increases the level of transcripts encoding mitogen-activated protein kinases and proteins involved in ethylene biosynthesis and signal transduction. The increase in [Ca(2+)](cyt) induced by nucleotide derivatives can be ablated by Ca(2+)-channel blocking agents and by the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and the changes in gene expression can be partially blocked by these agents. These observations suggest that extracellular ATP can activate calcium-mediated cell-signaling pathways in plants, potentially playing a physiological role in transducing stress and wound responses.
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http://dx.doi.org/10.1105/tpc.104.023945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC520962PMC
October 2004