Publications by authors named "Bigang Liu"

34 Publications

Evidence for context-dependent functions of KDM5B in prostate development and prostate cancer.

Oncotarget 2020 Nov 17;11(46):4243-4252. Epub 2020 Nov 17.

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

Prostate cancer (PCa) is one of the leading causes of cancer-related deaths worldwide. Prostate tumorigenesis and PCa progression involve numerous genetic as well as epigenetic perturbations. Histone modification represents a fundamental epigenetic mechanism that regulates diverse cellular processes, and H3K4 methylation, one such histone modification associated with active transcription, can be reversed by dedicated histone demethylase KDM5B (JARID1B). Abnormal expression and functions of KDM5B have been implicated in several cancer types including PCa. Consistently, our bioinformatics analysis reveals that the mRNA levels are upregulated in PCa compared to benign prostate tissues, and correlate with increased tumor grade and poor patient survival, supporting an oncogenic function of KDM5B in PCa. Surprisingly, however, when we generated prostate-specific conditional knockout mice using probasin (Pb) promoter-driven system, we observed that deletion did not affect normal prostate development but instead induced mild hyperplasia. These results suggest that KDM5B may possess context-dependent roles in normal prostate development vs. PCa development and progression.
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http://dx.doi.org/10.18632/oncotarget.27818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679033PMC
November 2020

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

Overactivation of the NF-κB pathway impairs molar enamel formation.

Oral Dis 2020 Oct 9;26(7):1513-1522. Epub 2020 Jul 9.

Division of Oral Anatomy, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan.

Objective: Hypohidrotic ectodermal dysplasia (HED) is a hereditary disorder characterized by abnormal structures and functions of the ectoderm-derived organs, including teeth. HED patients exhibit a variety of dental symptoms, such as hypodontia. Although disruption of the EDA/EDAR/EDARADD/NF-κB pathway is known to be responsible for HED, it remains unclear whether this pathway is involved in the process of enamel formation.

Experimental Subjects And Methods: To address this question, we examined the mice overexpressing Ikkβ (an essential component required for the activation of NF-κB pathway) under the keratin 5 promoter (K5-Ikkβ).

Results: Upregulation of the NF-κB pathway was confirmed in the ameloblasts of K5-Ikkβ mice. Premature abrasion was observed in the molars of K5-Ikkβ mice, which was accompanied by less mineralized enamel. However, no significant changes were observed in the enamel thickness and the pattern of enamel rods in K5-Ikkβ mice. Klk4 expression was significantly upregulated in the ameloblasts of K5-Ikkβ mice at the maturation stage, and the expression of its substrate, amelogenin, was remarkably reduced. This suggests that abnormal enamel observed in K5-Ikkβ mice was likely due to the compromised degradation of enamel protein at the maturation stage.

Conclusion: Therefore, we could conclude that the overactivation of the NF-κB pathway impairs the process of amelogenesis.
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http://dx.doi.org/10.1111/odi.13384DOI Listing
October 2020

The acute myeloid leukemia variant DNMT3A Arg882His is a DNMT3B-like enzyme.

Nucleic Acids Res 2020 04;48(7):3761-3775

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.

We have previously shown that the highly prevalent acute myeloid leukemia (AML) mutation, Arg882His, in DNMT3A disrupts its cooperative mechanism and leads to reduced enzymatic activity, thus explaining the genomic hypomethylation in AML cells. However, the underlying cause of the oncogenic effect of Arg882His in DNMT3A is not fully understood. Here, we discovered that DNMT3A WT enzyme under conditions that favor non-cooperative kinetic mechanism as well as DNMT3A Arg882His variant acquire CpG flanking sequence preference akin to that of DNMT3B, which is non-cooperative. We tested if DNMT3A Arg882His could preferably methylate DNMT3B-specific target sites in vivo. Rescue experiments in Dnmt3a/3b double knockout mouse embryonic stem cells show that the corresponding Arg878His mutation in mouse DNMT3A severely impairs its ability to methylate major satellite DNA, a DNMT3A-preferred target, but has no overt effect on the ability to methylate minor satellite DNA, a DNMT3B-preferred target. We also observed a previously unappreciated CpG flanking sequence bias in major and minor satellite repeats that is consistent with DNMT3A and DNMT3B specificity suggesting that DNA methylation patterns are guided by the sequence preference of these enzymes. We speculate that aberrant methylation of DNMT3B target sites could contribute to the oncogenic potential of DNMT3A AML variant.
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http://dx.doi.org/10.1093/nar/gkaa139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144950PMC
April 2020

The ZBTB24-CDCA7 axis regulates HELLS enrichment at centromeric satellite repeats to facilitate DNA methylation.

Protein Cell 2020 03;11(3):214-218

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

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http://dx.doi.org/10.1007/s13238-019-00682-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026229PMC
March 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

DNMT3L facilitates DNA methylation partly by maintaining DNMT3A stability in mouse embryonic stem cells.

Nucleic Acids Res 2019 01;47(1):152-167

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

DNMT3L (DNMT3-like), a member of the DNMT3 family, has no DNA methyltransferase activity but regulates de novo DNA methylation. While biochemical studies show that DNMT3L is capable of interacting with both DNMT3A and DNMT3B and stimulating their enzymatic activities, genetic evidence suggests that DNMT3L is essential for DNMT3A-mediated de novo methylation in germ cells but is dispensable for de novo methylation during embryogenesis, which is mainly mediated by DNMT3B. How DNMT3L regulates DNA methylation and what determines its functional specificity are not well understood. Here we show that DNMT3L-deficient mouse embryonic stem cells (mESCs) exhibit downregulation of DNMT3A, especially DNMT3A2, the predominant DNMT3A isoform in mESCs. DNA methylation analysis of DNMT3L-deficient mESCs reveals hypomethylation at many DNMT3A target regions. These results confirm that DNMT3L is a positive regulator of DNA methylation, contrary to a previous report that, in mESCs, DNMT3L regulates DNA methylation positively or negatively, depending on genomic regions. Mechanistically, DNMT3L forms a complex with DNMT3A2 and prevents DNMT3A2 from being degraded. Restoring the DNMT3A protein level in DNMT3L-deficient mESCs partially recovers DNA methylation. Thus, our work uncovers a role for DNMT3L in maintaining DNMT3A stability, which contributes to the effect of DNMT3L on DNMT3A-dependent DNA methylation.
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http://dx.doi.org/10.1093/nar/gky947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326784PMC
January 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

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

MicroRNA-141 suppresses prostate cancer stem cells and metastasis by targeting a cohort of pro-metastasis genes.

Nat Commun 2017 01 23;8:14270. Epub 2017 Jan 23.

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

MicroRNAs play important roles in regulating tumour development, progression and metastasis. Here we show that one of the miR-200 family members, miR-141, is under-expressed in several prostate cancer (PCa) stem/progenitor cell populations in both xenograft and primary patient tumours. Enforced expression of miR-141 in CD44 and bulk PCa cells inhibits cancer stem cell properties including holoclone and sphere formation, as well as invasion, and suppresses tumour regeneration and metastasis. Moreover, miR-141 expression enforces a strong epithelial phenotype with a partial loss of mesenchymal phenotype. Whole-genome RNA sequencing uncovers novel miR-141-regulated molecular targets in PCa cells including the Rho GTPase family members (for example, CDC42, CDC42EP3, RAC1 and ARPC5) and stem cell molecules CD44 and EZH2, all of which are validated as direct and functionally relevant targets of miR-141. Our results suggest that miR-141 employs multiple mechanisms to obstruct tumour growth and metastasis.
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http://dx.doi.org/10.1038/ncomms14270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264244PMC
January 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

Regional regulation of Filiform tongue papillae development by Ikkα/Irf6.

Dev Dyn 2016 09 13;245(9):937-46. Epub 2016 Jul 13.

Division of Oral Anatomy, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.

Background: Non-gustatory filiform papillae play critical roles in helping to grip food, drawing food to the esophagus, cleaning the mouth, and spreading saliva. The molecular mechanisms of filiform tongue papillae development however are not fully understood.

Results: We found Ikkα and Irf6 expression in developing tongue epithelium, and describe here specific tongue abnormalities in mice with mutation of these genes, indicating a role for Ikkα and Irf6 in filiform papillae development. Ikkα and Irf6 mutant tongues showed ectopic vertical epithelium at the midline, while lateral sides of mutant tongues adhered to the oral mucosa. Both the ectopic median vertical epithelium and adhered epithelium exhibited the presence of filiform tongue papillae, whereas epithelium between the median vertical epithelium and adhered tongue showed a loss of filiform tongue papillae. Timing of filiform papillae development was found to be slightly different between the midline and lateral regions of the wild-type tongue.

Conclusions: Filiform papillae thus develop through distinct molecular mechanisms between the regions of tongue dorsum in the medio-lateral axis, with some filiform papillae developing under the control of Ikkα and Irf6. Developmental Dynamics 245:937-946, 2016. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/dvdy.24427DOI Listing
September 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

Longitudinal tracking of subpopulation dynamics and molecular changes during LNCaP cell castration and identification of inhibitors that could target the PSA-/lo castration-resistant cells.

Oncotarget 2016 Mar;7(12):14220-40

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

We have recently demonstrated that the undifferentiated PSA-/lo prostate cancer (PCa) cell population harbors self-renewing long-term tumor-propagating cells that are refractory to castration, thus representing a therapeutic target. Our goals here are, by using the same lineage-tracing reporter system, to track the dynamic changes of PSA-/lo and PSA+ cells upon castration in vitro, investigate the molecular changes accompanying persistent castration, and develop large numbers of PSA-/lo PCa cells for drug screening. To these ends, we treated LNCaP cells infected with the PSAP-GFP reporter with three regimens of castration, i.e., CDSS, CDSS plus bicalutamide, and MDV3100 continuously for up to ~21 months. We observed that in the first ~7 months, castration led to time-dependent increases in PSA-/lo cells, loss of AR and PSA expression, increased expression of cancer stem cell markers, and many other molecular changes. Meanwhile, castrated LNCaP cells became resistant to high concentrations of MDV3100, chemotherapeutic drugs, and other agents. However, targeted and medium-throughput library screening identified several kinase (e.g., IGF-1R, AKT, PI3K/mTOR, Syk, GSK3) inhibitors as well as the BCL2 inhibitor that could effectively sensitize the LNCaP-CRPC cells to killing. Of interest, LNCaP cells castrated for >7 months showed evidence of cyclic changes in AR and the mTOR/AKT signaling pathways potentially involving epigenetic mechanisms. These observations indicate that castration elicits numerous molecular changes and leads to enrichment of PSA-/lo PCa cells. The ability to generate large numbers of PSA-/lo PCa cells should allow future high-throughput screening to identify novel therapeutics that specifically target this population.
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http://dx.doi.org/10.18632/oncotarget.7303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924710PMC
March 2016

Epithelial-intrinsic IKKα expression regulates group 3 innate lymphoid cell responses and antibacterial immunity.

J Exp Med 2015 Sep 14;212(10):1513-28. Epub 2015 Sep 14.

Jill Roberts Institute for Research in Inflammatory Bowel Disease, Weill Cornell Medical College, Cornell University, New York, NY 10021

Innate lymphoid cells (ILCs) are critical for maintaining epithelial barrier integrity at mucosal surfaces; however, the tissue-specific factors that regulate ILC responses remain poorly characterized. Using mice with intestinal epithelial cell (IEC)-specific deletions in either inhibitor of κB kinase (IKK)α or IKKβ, two critical regulators of NFκB activation, we demonstrate that IEC-intrinsic IKKα expression selectively regulates group 3 ILC (ILC3)-dependent antibacterial immunity in the intestine. Although IKKβ(ΔIEC) mice efficiently controlled Citrobacter rodentium infection, IKKα(ΔIEC) mice exhibited severe intestinal inflammation, increased bacterial dissemination to peripheral organs, and increased host mortality. Consistent with weakened innate immunity to C. rodentium, IKKα(ΔIEC) mice displayed impaired IL-22 production by RORγt(+) ILC3s, and therapeutic delivery of rIL-22 or transfer of sort-purified IL-22-competent ILCs from control mice could protect IKKα(ΔIEC) mice from C. rodentium-induced morbidity. Defective ILC3 responses in IKKα(ΔIEC) mice were associated with overproduction of thymic stromal lymphopoietin (TSLP) by IECs, which negatively regulated IL-22 production by ILC3s and impaired innate immunity to C. rodentium. IEC-intrinsic IKKα expression was similarly critical for regulation of intestinal inflammation after chemically induced intestinal damage and colitis. Collectively, these data identify a previously unrecognized role for epithelial cell-intrinsic IKKα expression and TSLP in regulating ILC3 responses required to maintain intestinal barrier immunity.
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http://dx.doi.org/10.1084/jem.20141831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4577836PMC
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

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

An IKKα-nucleophosmin axis utilizes inflammatory signaling to promote genome integrity.

Cell Rep 2013 Dec 27;5(5):1243-55. Epub 2013 Nov 27.

Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21701, USA. Electronic address:

The inflammatory microenvironment promotes skin tumorigenesis. However, the mechanisms by which cells protect themselves from inflammatory signals are unknown. Downregulation of IKKα promotes skin tumor progression from papillomas to squamous cell carcinomas, which is frequently accompanied by genomic instability, including aneuploid chromosomes and extra centrosomes. In this study, we found that IKKα promoted oligomerization of nucleophosmin (NPM), a negative centrosome duplication regulator, which further enhanced NPM and centrosome association, inhibited centrosome amplification, and maintained genome integrity. Levels of NPM hexamers and IKKα were conversely associated with skin tumor progression. Importantly, proinflammatory cytokine-induced IKKα activation promoted the formation of NPM oligomers and reduced centrosome numbers in mouse and human cells, whereas kinase-dead IKKα blocked this connection. Therefore, our findings suggest a mechanism in which an IKKα-NPM axis may use inflammatory signals to suppress centrosome amplification, promote genomic integrity, and prevent tumor progression.
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http://dx.doi.org/10.1016/j.celrep.2013.10.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4159076PMC
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

Dissociated primary human prostate cancer cells coinjected with the immortalized Hs5 bone marrow stromal cells generate undifferentiated tumors in NOD/SCID-γ mice.

PLoS One 2013 22;8(2):e56903. Epub 2013 Feb 22.

Department of Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Science Park, Smithville, Texas, United States of America.

Reconstitution of tumor development in immunodeficient mice from disaggregated primary human tumor cells is always challenging. The main goal of the present study is to establish a reliable assay system that would allow us to reproducibly reconstitute human prostate tumor regeneration in mice using patient tumor-derived single cells. Using many of the 114 untreated primary human prostate cancer (HPCa) samples we have worked on, here we show that: 1) the subcutaneum represents the most sensitive site that allows the grafting of the implanted HPCa pieces; 2) primary HPCa cells by themselves fail to regenerate tumors in immunodeficient hosts; 3) when coinjected in Matrigel with rUGM (rat urogenital sinus mesenchyme), CAF (carcinoma-associated fibroblasts), or Hs5 (immortalized bone marrow derived stromal) cells, primary HPCa cells fail to initiate serially transplantable tumors in NOD/SCID mice; and 4) however, HPCa cells coinjected with the Hs5 cells into more immunodeficient NOD/SCID-IL2Rγ(-/-) (NSG) mice readily regenerate serially transplantable tumors. The HPCa/Hs5 reconstituted 'prostate' tumors present an overall epithelial morphology, are of the human origin, and contain cells positive for AR, CK8, and racemase. Cytogenetic analysis provides further evidence for the presence of karyotypically abnormal HPCa cells in the HPCa/Hs5 tumors. Of importance, HPCa/Hs5 xenograft tumors contain EpCAM(+) cells that are both clonogenic and tumorigenic. Surprisingly, all HPCa/Hs5 reconstituted tumors are undifferentiated, even for HPCa cells derived from Gleason 7 tumors. Our results indicate that primary HPCa cells coinjected with the immortalized Hs5 stromal cells generate undifferentiated tumors in NSG mice and we provide evidence that undifferentiated HPCa cells might be the cells that possessed tumorigenic potential and regenerated HPCa/Hs5 xenograft tumors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056903PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3579939PMC
September 2013

The role of Irf6 in tooth epithelial invagination.

Dev Biol 2012 May 14;365(1):61-70. Epub 2012 Feb 14.

Department of Craniofacial Development and Comprehensive Biomedical Research Centre, Dental Institute, King's College London, UK.

Thickening and the subsequent invagination of the epithelium are an important initial step in ectodermal organ development. Ikkα has been shown to play a critical role in controlling epithelial growth, since Ikkα mutant mice show protrusions (evaginations) of incisor tooth, whisker and hair follicle epithelium rather than invagination. We show here that mutation of the Interferon regulatory factor (Irf) family, Irf6 also results in evagination of incisor epithelium. In common with Ikkα mutants, Irf6 mutant evagination occurs in a NF-κB-independent manner and shows the same molecular changes as those in Ikkα mutants. Irf6 thus also plays a critical role in regulating epithelial invagination. In addition, we also found that canonical Wnt signaling is upregulated in evaginated incisor epithelium of both Ikkα and Irf6 mutant embryos.
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http://dx.doi.org/10.1016/j.ydbio.2012.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7462630PMC
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

Role of IKKα in skin squamous cell carcinomas.

Future Oncol 2011 Jan;7(1):123-34

Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.

Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are two major types of skin cancer derived from keratinocytes. SCC is a more aggressive type of cancer than BCC in humans. One significant difference between SCC and BCC is that SCC development is generally associated with cell dedifferentiation and morphological changes. When SCC is converted to spindle cell carcinoma, the latest stage of cancer, the tumor cells change to a fibroblastic cell morphology (epithelial-to-mesenchymal transition) and lose their differentiation markers. Recently, several laboratories have reported altered IκB kinase α (IKKα) protein localization, downregulated IKKα, and IKKα gene deletions and mutations in human SCCs of the skin, lung, esophagus, and neck and head. In addition, IKKα reduction promotes chemical carcinogen- and ultraviolet B-induced skin carcinogenesis, and IKKα deletion in keratinocytes causes spontaneous skin SCCs, but not BCCs, in mice. Thus, IKKα emerges as a bona fide skin tumor suppressor. In this article, we will discuss the role of IKKα in skin SCC development.
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http://dx.doi.org/10.2217/fon.10.166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365019PMC
January 2011

Reduction of IKKalpha expression promotes chronic ultraviolet B exposure-induced skin inflammation and carcinogenesis.

Am J Pathol 2010 May 19;176(5):2500-8. Epub 2010 Mar 19.

Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, USA.

Ultraviolet B light (UVB) is a common cause of human skin cancer. UVB irradiation induces mutations in the tumor suppressor p53 gene as well as chronic inflammation, which are both essential for UVB carcinogenesis. Inhibitor of nuclear factor kappaB kinase-alpha (IKKalpha) plays an important role in maintaining skin homeostasis, and expression of IKKalpha was found to be down-regulated in human and murine skin squamous cell carcinomas. However, the role of IKKalpha in UVB skin carcinogenesis has not been investigated. Thus, here we performed UVB carcinogenesis experiments on Ikkalpha(+/+) and Ikkalpha(+/-) mice. Ikkalpha(+/-) mice were found to develop a twofold greater number of skin tumors than Ikkalpha(+/+) mice after chronic UVB irradiation. In addition, tumor latency was significantly shorter and tumors were bigger in Ikkalpha(+/-) than in Ikkalpha(+/+) mice. At an early stage of carcinogenesis, an increase in UVB-induced p53 mutations as well as macrophage recruitment and mitogenic activity, and a decrease in UVB-induced apoptosis, were detected in Ikkalpha(+/-) compared with those in Ikkalpha(+/+) skin. Also, reduction of IKKalpha levels in keratinocytes up-regulated the expression of monocyte chemoattractant protein-1 (MCP-1/CCL2), TNFalpha, IL-1, and IL-6, and elevated macrophage migration, which might promote macrophage recruitment and inflammation. Therefore, these findings suggest that reduction of IKKalpha expression orchestrates UVB carcinogen, accelerating tumorigenesis.
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http://dx.doi.org/10.2353/ajpath.2010.091041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2861114PMC
May 2010

A tale of terminal differentiation: IKKalpha, the master keratinocyte regulator.

Cell Cycle 2009 Feb 10;8(4):527-31. Epub 2009 Feb 10.

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

Keratinocyte differentiation is the process of cellular maturation from a mitotic state to a terminally differentiated state during which skin builds up a tough yet soft skin barrier to protect the body. Its irreversibility also allows the shedding of excessive keratinocytes, thereby maintaining skin homeostasis and preventing skin diseases. Although the entire journey of keratinocyte differentiation is intricate and not well understood, it is known that Ras is able to block keratinocyte terminal differentiation and instead induce keratinocyte proliferation and transformation. It appears that uncontrolled proliferation actually interrupts differentiation. However, it has been unclear whether there are any innate surveillants that would be able to induce terminal differentiation by antagonizing excessive mitotic activities. Inhibitor of nuclear factor kappaB kinase-alpha (IKKalpha, previously known as Chuk) emerges as a master regulator in the coordinative control of keratinocyte differentiation and proliferation and as a major tumor suppressor in human and mouse skin squamous cell carcinomas. IKKalpha does so largely by integrating into the epidermal growth factor receptor (EGFR)/Ras/extracellular signal-regulated kinase (Erk)/EGFR ligand pathways during mitosis and differentiation. We discuss these findings herein to extend our understanding of how IKKalpha-mediated terminal differentiation serves as an innate surveillant in skin.
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http://dx.doi.org/10.4161/cc.8.4.7598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7243803PMC
February 2009

Critical role of IkappaB kinase alpha in embryonic skin development and skin carcinogenesis.

Histol Histopathol 2009 02;24(2):265-71

Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Smithville, Texas, USA.

IkappaB kinase alpha (IKKalpha), IKKbeta, and IKKgamma/NEMO form the IKK complex, which is essential for NF-kappaB activation. However, genetic studies have shown that the role of IKKalpha is distinct from that of IKKbeta or IKKgamma in the development of the mouse embryonic skin. Loss of IKKalpha has been shown to cause epidermal hyperplasia, prevent keratinocyte terminal differentiation, and impair the formation of the skin, resulting in the deaths of IKKalpha-deficient (Ikkalpha-/-) mice soon after birth. Recent experimental data from several laboratories have revealed that IKKalpha functions as a tumor suppressor in human squamous cell carcinomas (SCCs) of skin, lungs, and head and neck. Chemical carcinogenesis studies using mice have shown that reduction in IKKalpha expression increases the number and size of Ras-initiated skin tumors and promotes their progression, indicating that reduced IKKalpha expression provides a selective growth advantage that cooperates with Ras activity to promote skin carcinogenesis. In this review, we will summarize these findings from our and other studies on the role that IKKalpha plays in development of the mouse embryonic skin and skin carcinogenesis.
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http://dx.doi.org/10.14670/HH-24.265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7243875PMC
February 2009

IKKalpha is required to maintain skin homeostasis and prevent skin cancer.

Cancer Cell 2008 Sep;14(3):212-25

Department of Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA.

It has long been known that excessive mitotic activity due to H-Ras can block keratinocyte differentiation and cause skin cancer. It is not clear whether there are any innate surveillants that are able to ensure that keratinocytes undergo terminal differentiation, preventing the disease. IKKalpha induces keratinocyte terminal differentiation, and its downregulation promotes skin tumor development. However, its intrinsic function in skin cancer is unknown. Here, we found that mice with IKKalpha deletion in keratinocytes develop a thickened epidermis and spontaneous squamous cell-like carcinomas. Inactivation of epidermal growth factor receptor (EGFR) or reintroduction of IKKalpha inhibits excessive mitosis, induces terminal differentiation, and prevents skin cancer through repressing an EGFR-driven autocrine loop. Thus, IKKalpha serves as an innate surveillant.
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http://dx.doi.org/10.1016/j.ccr.2008.07.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263012PMC
September 2008

ALG-2 interacting protein AIP1: a novel link between D1 and D3 signalling.

Eur J Neurosci 2008 Apr;27(7):1626-33

Academic Neurology Unit, The Henry Wellcome Laboratories for Medical Research, Division of Genomic Medicine, School of Medicine and Biomedical Sciences, University of Sheffield, Beech Hill Road, Sheffield S10 2RX, UK.

Dopamine signalling is a critically important process in the human brain that controls mood, cognition and motor activity. In order to gain detailed insight into this signalling pathway at the molecular level, we carried out yeast two-hybrid screens with D1-like (D1, D5) and D2-like (D2, D3, D4) dopamine receptors and identified 11 dopamine receptor interacting proteins (DRIPs). Using the C-terminal domain of D1 receptor as bait, we identified AIP1 (ALG-2 interacting protein 1), a known modulator of caspase-dependent and caspase-independent cell death, including neuronal cell death, that is also part of the endosomal transport system. In a separate yeast two-hybrid screen, using the third intracellular cytoplasmic loop of D3 as bait, we again identified AIP1. The interaction of AIP1 with both D1 and D3 was confirmed in vitro and in vivo using a variety of methods, including glutathione S-transferase (GST) pull-down, blot overlay and coimmunoprecipitation from mouse brain lysates. We have also observed colocalization of D1 and D3 with AIP1 in mouse brain tissue. In addition, coexpression of AIP1 with D1 resulted in > 50% reduction in binding capacity of D1 to its antagonist. Finally, AIP1 up-regulates D1 and D3 expression and appears to be important for their stability and trafficking.
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http://dx.doi.org/10.1111/j.1460-9568.2008.06135.xDOI Listing
April 2008