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1.  Ubiquitin-Dependent Proteolysis in G1/S Phase Control and Its Relationship with Tumor Susceptibility 
Genes & Cancer  2010;1(7):717-724.
Cell division depends upon the coordinated action of positive and negative regulatory factors that ensure high fidelity replication of the genome and its equivalent separation into daughter cells following cytokinesis. The role of positive factors such as the cyclin-dependent kinases in promoting cell division is firmly established, as is the function of CDK inhibitors and phosphatases that antagonize CDKs. In addition to these, regulated protein destruction is now appreciated as essential for temporal regulation of cell cycle transitions. Protein degradation serves as an irreversible switch that ensures temporally regulated cell cycle transitions. Signal-dependent regulation of protein degradation is best understood with regard to the 26S proteasome. Proteins are directed to this machine subsequent to enzymatic transfer of a highly conserved small polypeptide, ubiquitin. The focus of this review is the regulatory molecules that direct the regulated attachment of ubiquitin, polyubiquitylation, to proteins destined for degradation as cells transition through the G1 phase into S phase. During the past decade, it has become increasingly apparent that these molecules are critical mediators of normal cell proliferation, and as such, they are frequently deregulated in human cancers.
PMCID: PMC2991141  PMID: 21113395
F-box; cullin; cyclin; CDK
2.  Ubiquitin and Cancer 
Genes & Cancer  2010;1(7):679-680.
PMCID: PMC3092239  PMID: 21779465
3.  Monographs Editors 
Genes & Cancer  2010;1(7):677-678.
PMCID: PMC3092243  PMID: 21779464
4.  IGFBP3 promotes esophageal cancer growth by suppressing oxidative stress in hypoxic tumor microenvironment 
Insulin-like growth factor binding protein 3 (IGFBP3), a hypoxia-inducible gene, regulates a variety of cellular processes including cell proliferation, senescence, apoptosis and epithelial-mesenchymal transition (EMT). IGFBP3 has been linked to the pathogenesis of cancers. Most previous studies focus upon proapoptotic tumor suppressor activities of IGFBP3. Nevertheless, IGFBP3 is overexpressed in certain cancers including esophageal squamous cell carcinoma (ESCC), one of the most aggressive forms of squamous cell carcinomas (SCCs). The tumor-promoting activities of IGFBP3 remain poorly understood in part due to a lack of understanding as to how the tumor microenvironment may influence IGFBP3 expression and how IGFBP3 may in turn influence heterogeneous intratumoral cell populations. Here, we show that IGFBP3 overexpression is associated with poor postsurgical prognosis in ESCC patients. In xenograft transplantation models with genetically engineered ESCC cells, IGFBP3 contributes to tumor progression with a concurrent induction of a subset of tumor cells showing high expression of CD44 (CD44H), a major cell surface receptor for hyaluronic acid, implicated in invasion, metastasis and drug resistance. Our gain-of-function and loss-of-function experiments reveal that IGFBP3 mediates the induction of intratumoral CD44H cells. IGFBP3 cooperates with hypoxia to mediate the induction of CD44H cells by suppressing reactive oxygen species (ROS) in an insulin-like growth factor-independent fashion. Thus, our study sheds light on the growth stimulatory functions of IGFPB3 in cancer, gaining a novel mechanistic insight into the functional interplay between the tumor microenvironment and IGFBP3.
PMCID: PMC3902230  PMID: 24482736
CD44; esophageal; squamous cell carcinoma; hypoxia; IGFBP3 and reactive oxygen species
5.  miR-211 is a pro-survival micro-RNA that regulates chop expression in a PERK-dependent manner 
Molecular cell  2012;48(3):353-364.
Micro-RNAs typically function at the level of post-transcriptional gene silencing within the cytoplasm; however increasing evidence suggests that they may also function in nuclear, Argonaut containing complexes, to directly repress target gene transcription. We have investigated the role of micro-RNAs in mediating endoplasmic reticulum (ER) stress responses. ER stress triggers the activation of three signaling molecules: Ire-1α/β, PERK and ATF6 whose function is to facilitate adaption to the ensuing stress. We demonstrate that PERK induces miR-211, which in turn attenuates stress-dependent expression of the pro-apoptotic transcription factor chop/gadd153. MiR-211 directly targets the proximal chop/gadd153 promoter where it increases histone methylation and represses chop expression. Maximal chop accumulation ultimately correlates with miR-211 down regulation. Our data suggests a model where PERK-dependent miR-211 induction prevents premature chop accumulation and thereby provides a window of opportunity for the cell to re-establish homeostasis prior to apoptotic commitment.
PMCID: PMC3496065  PMID: 23022383
miR-211; histone methylation; PERK; CHOP
6.  Regulation of autophagy during ECM detachment is linked to a selective inhibition of mTORC1 by PERK 
Oncogene  2012;32(41):4932-4940.
Adhesion to the extracellular matrix (ECM) is critical for epithelial tissue homeostasis and function. ECM detachment induces metabolic stress and programmed cell death via anoikis. ECM-detached mammary epithelial cells are able to rapidly activate autophagy allowing for survival and an opportunity for re-attachment. However, the mechanisms controlling detachment-induced autophagy remain unclear. Here we uncover that the kinase PERK rapidly promotes autophagy in ECM-detached cells by activating AMP-activated protein kinase (AMPK), resulting in downstream inhibition of mTORC1-p70S6K signaling. LKB1 and TSC2, but not TSC1, are required for PERK-mediated inhibition of mammalian target of rapamycinin MCF10A cells and mouse embryo fibroblast cells. Importantly, this pathway shows fast kinetics, is transcription-independent and is exclusively activated during ECM detachment, but not by canonical endoplasmic reticulum stressors. Moreover, enforced PERK or AMPK activation upregulates autophagy and causes luminal filling during acinar morphogenesis by perpetuating a population of surviving autophagic luminal cells that resist anoikis. Hence, we identify a novel pathway in which suspension-activated PERK promotes the activation of LKB1, AMPK and TSC2, leading to the rapid induction of detachment-induced autophagy. We propose that increased autophagy, secondary to persistent PERK and LKB1-AMPK signaling, can robustly protect cells from anoikis and promote luminal filling during early carcinoma progression.
PMCID: PMC3600386  PMID: 23160380
anoikis; breast cancer; unfolded protein response
7.  Disruption of cyclin D1 nuclear export and proteolysis accelerates mammary carcinogenesis 
Oncogene  2007;27(9):1231-1242.
Cyclin D1 levels are maintained at steady state by phosphorylation-dependent nuclear export and polyubiquitination by the SCFFBX4-αB crystallin E3 ligase. Inhibition of cyclin D1 proteolysis has been implicated as a causative factor leading to its overexpression in carcinomas of the breast and esophagus; however evaluation of the contribution of stable cyclin D1 to the genesis of such carcinomas has not been performed. We therefore generated transgenic mice wherein expression of either wild-type or a stable cyclin D1 allele (D1T286A) is regulated by MMTV-LTR. MMTV-D1T286A mice developed mammary adenocarcinomas at an increased rate relative to MMTV-D1 mice. Similar to human cancers that overexpress cyclin D1, D1T286A tumors were estrogen receptor positive and exhibited estrogen-dependent growth. MMTV-D1T286A tumors specifically overexpressed genes involved in DNA replication and DNA damage checkpoints suggesting that stabilization and nuclear accumulation of cyclin D1-dependent kinase contributes to genomic instability through perturbations in DNA replication. Collectively, these results suggest that temporal control of cyclin D1 subcellular localization and proteolysis is critical for the maintenance of homeostasis within the mammary epithelium.
PMCID: PMC3733559  PMID: 17724472
αB crystallin; CDK4; cyclin D1; FBX4; mammary gland
8.  DNA Damage-dependent cyclin D1 proteolysis: GSK3β holds the smoking gun 
Cell cycle (Georgetown, Tex.)  2009;8(6):824-827.
Ubiquitin mediated degradation of cyclin D1 following the G1/S transition counters its mitogen-dependent accumulation during G1 phase of the cell cycle. Although the cellular machinery responsible for this process has been identified, how this regulatory pathway interfaces to cellular stress responses, often referred to as checkpoints, remains to be established. One intensely investigated checkpoint is the cellular response to DNA damage. When DNA damage is sensed, the corresponding DNA damage checkpoint triggers the inhibition of CDK-dependent cell cycle progression, with arrest coordinated by induction of CDK inhibitors and rapid degradation of specific cyclins, such as cyclin D1. In recent work, we identified a phosphorylation- and Fbx4-dependent cyclin D1 degradation mechanism in response to genotoxic stress.18 This work revealed that loss of cyclin D1 regulation compromises the intra-Sphase response to DNA damage, promoting genomic instability and sensitization of cells to S-phase chemotherapy, highlighting a potential therapeutic strategy for cancers exhibiting cyclin D1 accumulation.
PMCID: PMC3703625  PMID: 19221502
Cyclin D1; phosphorylation; ATM; CDK4; DNA damage; intra-S-phase checkpoint; SCFFbx4-αBCrystallin; GSK3β
9.  PERK Is Required in the Adult Pancreas and Is Essential for Maintenance of Glucose Homeostasis 
Molecular and Cellular Biology  2012;32(24):5129-5139.
Germ line PERK mutations are associated with diabetes mellitus and growth retardation in both rodents and humans. In contrast, late embryonic excision of PERK permits islet development and was found to prevent onset of diabetes, suggesting that PERK may be dispensable in the adult pancreas. To definitively establish the functional role of PERK in adult pancreata, we generated mice harboring a conditional PERK allele in which excision is regulated by tamoxifen administration. Deletion of PERK in either young adult or mature adult mice resulted in hyperglycemia associated with loss of islet and β cell architecture. PERK excision triggered intracellular accumulation of proinsulin and Glut2, massive endoplasmic reticulum (ER) expansion, and compensatory activation of the remaining unfolded-protein response (UPR) signaling pathways specifically in pancreatic tissue. Although PERK excision increased β cell death, this was not a result of decreased proliferation as previously reported. In contrast, a significant and specific increase in β cell proliferation was observed, a result reflecting increased cyclin D1 accumulation. This work demonstrates that contrary to expectations, PERK is required for secretory homeostasis and β cell survival in adult mice.
PMCID: PMC3510549  PMID: 23071091
10.  Nuclear cyclin D1: An oncogenic driver in human cancer 
Journal of cellular physiology  2009;220(2):292-296.
Perturbations in the regulation of the core cell cycle machinery are frequently observed in human cancers. Cyclin D1 which functions as a mitogenic sensor and allosteric activator of CDK4/6, is one of the more frequently altered cell cycle regulators in cancers. Cyclin D1 is frequently overexpressed in cancers and its overexpression can be attributed to many factors including increased transcription, translation, and protein stability. Although cyclin D1 overexpression is clearly implicated in the affected cancers, overexpression of cyclin D1 is not sufficient to drive oncogenic transformation. Rather, emerging evidence suggests that nuclear retention of cyclin D1 resulting from altered nuclear trafficking and proteolysis is critical for the manifestation of its oncogenicity. This review provides a brief overview of current data documenting various mechanisms underlying aberrant cyclin D1 regulation in human cancers and their impact on neoplastic transformation.
PMCID: PMC2874239  PMID: 19415697
11.  Lysine 269 is essential for cyclin D1 ubiquitylation by the SCFFbx4/αB-crystallin ligase and subsequent proteasome-dependent degradation 
Oncogene  2009;28(49):4317-4325.
Protein ubiquitylation is a complex enzymatic process that results in the covalent attachment of ubiquitin, via Gly-76 of ubiquitin, to an ε-NH2-group of an internal lysine residue in a given substrate. While E3 ligases frequently utilize lysines adjacent to the degron within the substrate, many substrates can be targeted to the proteasome via polyubiquitylation of any lysine. We have assessed the role of lysine residues proximal to the cyclin D1 phosphodegron for ubiquitylation by the SCFFbx4/αB-crystallin ubiquitin ligase and subsequent proteasome-dependent degradation of cyclin D1. The work described herein reveals a requisite role for Lys-269 (K269) for the rapid, poly-ubiquitin mediated degradation of cyclin D1. Mutation of lysine 269, which is proximal to the phosphodegron sequence surrounding Thr-286 in cyclin D1, not only stabilizes cyclin D1, but also triggers cyclin D1 accumulation within the nucleus thereby promoting cell transformation. In addition, D1-K269R is resistant to genotoxic stress induced degradation, similar to non-phosphorylatable D1-T286A, supporting the critical role for the post-translational regulation of cyclin D1 in the response to DNA damaging agents. Strikingly, while mutation of lysine 269 to arginine inhibits cyclin D1 degradation, it does not inhibit cyclin D1 ubiquitylation in vivo demonstrating that ubiquitylation of a specific lysine can influence substrate targeting to the 26S proteasome.
PMCID: PMC2794935  PMID: 19767775
12.  Expression of Constitutively Nuclear Cyclin D1 in Murine Lymphocytes Induces B-Cell Lymphoma 
Oncogene  2006;25(7):998-1007.
Mantle cell lymphoma (MCL) is a B-cell lymphoma characterized by overexpression of cyclin D1 due to the t(11;14) chromosomal translocation. While expression of cyclin D1 is correlates with MCL development, expression of wild type cyclin D1 transgene in murine lymphocytes is unable to drive B-cell lymphoma. Because cyclin D1 mutants that are refractory to nuclear export display heighten oncogenicity in vitro compared with wild type D1, we generated mice expressing FLAG-D1/T286A, a constitutively nuclear mutant, under the control of the immunoglobulin enhancer, Eµ. D1/T286A transgenic mice universally develop a mature B-cell lymphoma. Expression of D1/T286A in B lymphocytes results in promiscuous S-phase entry and increased apoptosis in spleens of young pre-malignant mice. Lymphoma onset correlates with loss of p53 suggesting that inactivation of the p53 signaling axis precedes lymphoma development. Our results describe a cyclin D1-driven model of B-cell lymphomagenesis and provide evidence that nuclear-retention of cyclin D1 is oncogenic in vivo.
PMCID: PMC2832762  PMID: 16247460
CDK4; cyclin D1; mantle cell lymphoma; apoptosis
13.  PERK-dependent regulation of IAP translation during ER stress 
Oncogene  2008;28(6):910-920.
Exposure of cells to Endoplasmic Reticulum (ER) stress leads to activation of phosphatidylinositol 3-kinase (PI3K)–Akt signaling pathway and transcriptional induction of the inhibitor of apoptosis family of proteins. One of the proximal effectors of the ER stress response, the PKR-like ER kinase (PERK), leads to cellular adaptation to stress by multiple mechanisms, including attenuation of protein synthesis, and transcriptional induction of pro-survival genes. While PERK activity leads to cellular adaptation to ER stress, we now demonstrate that PERK activity also inhibits the ER stress-induced apoptotic program through induction of cellular inhibitor of apoptosis (cIAP1 and cIAP2) proteins. This induction of IAPs occurs through both transcriptional and translational responses that are PERK-dependent. Reintroduction of cIAP1 or cIAP2 expression into PERK−/− MEFs during ER stress delays the early onset of ER stress-induced caspase activation and apoptosis observed in these cells. Furthermore, we demonstrate that activation of the PI3K-Akt pathway by ER stress is dependent on PERK, suggesting additional ways in which PERK activity protects cells from ER stress-induced apoptosis.
PMCID: PMC2642534  PMID: 19029953
PERK; ER Stress; Apoptosis; IAP
14.  PERK Utilizes Intrinsic Lipid Kinase Activity To Generate Phosphatidic Acid, Mediate Akt Activation, and Promote Adipocyte Differentiation 
Molecular and Cellular Biology  2012;32(12):2268-2278.
The endoplasmic reticulum (ER) resident PKR-like kinase (PERK) is necessary for Akt activation in response to ER stress. We demonstrate that PERK harbors intrinsic lipid kinase, favoring diacylglycerol (DAG) as a substrate and generating phosphatidic acid (PA). This activity of PERK correlates with activation of mTOR and phosphorylation of Akt on Ser473. PERK lipid kinase activity is regulated in a phosphatidylinositol 3-kinase (PI3K) p85α-dependent manner. Moreover, PERK activity is essential during adipocyte differentiation. Because PA and Akt regulate many cellular functions, including cellular survival, proliferation, migratory responses, and metabolic adaptation, our findings suggest that PERK has a more extensive role in insulin signaling, insulin resistance, obesity, and tumorigenesis than previously thought.
PMCID: PMC3372262  PMID: 22493067
15.  A NOTCH3-mediated squamous cell differentiation program limits expansion of EMT competent cells that express the ZEB transcription factors 
Cancer research  2011;71(21):6836-6847.
Zinc finger E-box binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in transforming growth factor (TGF)-β-mediated senescence, epithelial to mesenchymal transition (EMT) and cancer stem cell function. ZEBs are negatively regulated by members of the miR-200 microRNA family, but precisely how tumor cells expressing ZEBs emerge during invasive growth remains unknown. Here we report that NOTCH3-mediated signaling prevents expansion of a unique subset of ZEB-expressing cells. ZEB expression was associated with the lack of cellular capability of undergoing NOTCH3-mediated squamous differentiation in human esophageal cells. Genetic inhibition of the Notch-mediated transcriptional activity by dominant-negative Mastermind-like1 (DNMAML1) prevented squamous differentiation and induction of Notch target genes including NOTCH3. Moreover, DNMAML1 enriched EMT competent cells exhibited robust upregulation of ZEBs, downregulation of the miR-200 family, and enhanced anchorage independent growth and tumor formation in nude mice. RNA interference (RNAi) experiments suggested the involvement of ZEBs in anchorage independent colony formation, invasion and TGF-β-mediated EMT. Invasive growth and impaired squamous differentiation was recapitulated upon Notch inhibition by DNMAML1 in organotypic 3D culture, a form of human tissue engineering. Together, our findings indicate that NOTCH3 is a key factor limiting the expansion of ZEB-expressing cells, providing novel mechanistic insights into the role of Notch signaling in the cell fate regulation and disease progression of squamous esophageal cancers.
PMCID: PMC3206139  PMID: 21890822
Notch; EMT; squamous cell differentiation; ZEB1; miR-200
16.  PERK and GCN2 Contribute to eIF2α Phosphorylation and Cell Cycle Arrest after Activation of the Unfolded Protein Response Pathway 
Molecular Biology of the Cell  2005;16(12):5493-5501.
Exposure of cells to endoplasmic reticulum (ER) stress leads to activation of PKR-like ER kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, repression of cyclin D1 translation, and subsequent cell cycle arrest in G1 phase. However, whether PERK is solely responsible for regulating cyclin D1 accumulation after unfolded protein response pathway (UPR) activation has not been assessed. Herein, we demonstrate that repression of cyclin D1 translation after UPR activation occurs independently of PERK, but it remains dependent on eIF2α phosphorylation. Although phosphorylation of eIF2α in PERK–/– fibroblasts is attenuated in comparison with wild-type fibroblasts, it is not eliminated. The residual eIF2α phosphorylation correlates with the kinetics of cyclin D1 loss, suggesting that another eIF2α kinase functions in the absence of PERK. In cells harboring targeted deletion of both PERK and GCN2, cyclin D1 loss is attenuated, suggesting GCN2 functions as the redundant kinase. Consistent with these results, cyclin D1 translation is also stabilized in cells expressing a nonphosphorylatable allele of eIF2α; in contrast, repression of global protein translation still occurs in these cells, highlighting a high degree of specificity in transcripts targeted for translation inhibition by phosphorylated eIF2α. Our results demonstrate that PERK and GCN2 function to cooperatively regulate eIF2α phosphorylation and cyclin D1 translation after UPR activation.
PMCID: PMC1289396  PMID: 16176978
17.  The Cell Biology of the Unfolded Protein Response 
Gastroenterology  2011;141(1):38-41.e2.
The Unfolded Protein Response (UPR) is an ensemble of signal transduction pathways that respond to perturbations in the oxidative, pro-folding environment of the endoplasmic reticulum. During the past decade, ongoing research implicated these pathways in maintaining homeostasis of cells and organisms exposed to various stresses. Herein, we highlight recent findings regarding the functional role of the UPR in both normal and pathophysiological processes.
PMCID: PMC3129462  PMID: 21620842
18.  Notch receptor inhibition reveals the importance of cyclin D1 and Wnt signaling in invasive esophageal squamous cell carcinoma 
Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of squamous cell carcinomas. Common genetic lesions in ESCC include p53 mutations and EGFR overexpression, both of which have been implicated in negative regulation of Notch signaling. In addition, cyclin D1 is overexpressed in ESCC and can be activated via EGFR, Notch and Wnt signaling. To elucidate how these genetic lesions may interact during the development and progression of ESCC, we tested a panel of genetically engineered human esophageal cells (keratinocytes) in organotypic 3D culture (OTC), a form of human tissue engineering. Notch signaling was suppressed in culture and mice by dominant negative Mastermind-like1 (DNMAML1), a genetic pan-Notch inhibitor. DNMAML1 mice were subjected to 4-Nitroquinoline 1-oxide-induced oral-esophageal carcinogenesis. Highly invasive characteristics of primary human ESCC were recapitulated in OTC as well as DNMAML1 mice. In OTC, cyclin D1 overexpression induced squamous hyperplasia. Concurrent EGFR overexpression and mutant p53 resulted in transformation and invasive growth. Interestingly, cell proliferation appeared to be regulated differentially between those committed to squamous-cell differentiation and those invading into the stroma. Invasive cells exhibited Notch-independent activation of cyclin D1 and Wnt signaling. Within the oral-esophageal squamous epithelia, Notch signaling regulated squamous-cell differentiation to maintain epithelial integrity, and thus may act as a tumor suppressor by preventing the development of a tumor-promoting inflammatory microenvironment.
PMCID: PMC3410579  PMID: 22860235
Esophageal squamous cell carcinoma; organotypic 3D culture; EGFR; P53; cyclin D1; Wnt; Notch; squamous-cell differentiation; invasion; 4-Nitroquinoline 1-oxide
19.  Detecting and targeting mesenchymal-like subpopulations within squamous cell carcinomas 
Cell Cycle  2011;10(12):2008-2016.
Curative eradication of all cells within carcinomas is seldom achievable with chemotherapy alone. This limitation may be partially attributable to tumor cell subpopulations with intrinsic resistance to current drugs. Within squamous cell carcinoma (SCC) cell lines, we previously characterized a subpopulation of mesenchymal-like cells displaying phenotypic plasticity and increased resistance to both cytotoxic and targeted agents. These mesenchymal-like (Ecad-lo) cells are separable from epithelial-like (Ecad-hi) cells based on loss of surface E-cadherin and expression of vimentin. Despite their long-term plasticity, both Ecad-lo and Ecad-hi subsets in short-term culture maintained nearly uniform phenotypes after purification. This stability allowed testing of segregated subpopulations for relative sensitivity to the cytotoxic agent cisplatin in comparison to salinomycin, a compound with reported activity against CD44+CD24− stem-like cells in breast carcinomas. Salinomycin showed comparable efficacy against both Ecad-hi and Ecad-lo cells in contrast to cisplatin, which selectively depleted Ecad-hi cells. An in vivo correlate of these mesenchymal-like Ecad-lo cells was identified by immunohistochemical detection of vimentin-positive malignant subsets across a part of direct tumor xenografts (DTXs) of advanced stage SCC patient samples. Cisplatin treatment of mice with established DTXs caused enrichment of vimentin-positive malignant cells in residual tumors, but salinomycin depleted the same subpopulation. These results demonstrate that mesenchymal-like SCC cells, which resist current chemotherapies, respond to a treatment strategy developed against a stem-like subset in breast carcinoma. Further, they provide evidence of mesenchymal-like subsets being well-represented across advanced stage SCCs, suggesting that intrinsic drug resistance in this subpopulation has high clinical relevance.
PMCID: PMC3154419  PMID: 21558812
EMT; squamous cell carcinoma; head and neck cancer; esophageal cancer; chemotherapy resistance; salinomycin; tumor heterogeneity
20.  The Fbx4 Tumor Suppressor Regulates Cyclin D1 Accumulation and Prevents Neoplastic Transformation ▿  
Molecular and Cellular Biology  2011;31(22):4513-4523.
Skp1-Cul1-F-box (SCF) E3 ubiquitin ligase complexes modulate the accumulation of key cell cycle regulatory proteins. Following the G1/S transition, SCFFbx4 targets cyclin D1 for proteasomal degradation, a critical event necessary for DNA replication fidelity. Deregulated cyclin D1 drives tumorigenesis, and inactivating mutations in Fbx4 have been identified in human cancer, suggesting that Fbx4 may function as a tumor suppressor. Fbx4+/− and Fbx4−/− mice succumb to multiple tumor phenotypes, including lymphomas, histiocytic sarcomas and, less frequently, mammary and hepatocellular carcinomas. Tumors and premalignant tissue from Fbx4+/− and Fbx4−/− mice exhibit elevated cyclin D1, an observation consistent with cyclin D1 as a target of Fbx4. Molecular dissection of the Fbx4 regulatory network in murine embryonic fibroblasts (MEFs) revealed that loss of Fbx4 results in cyclin D1 stabilization and nuclear accumulation throughout cell division. Increased proliferation in early passage primary MEFs is antagonized by DNA damage checkpoint activation, consistent with nuclear cyclin D1-driven genomic instability. Furthermore, Fbx4−/− MEFs exhibited increased susceptibility to Ras-dependent transformation in vitro, analogous to tumorigenesis observed in mice. Collectively, these data reveal a requisite role for the SCFFbx4 E3 ubiquitin ligase in regulating cyclin D1 accumulation, consistent with tumor suppressive function in vivo.
PMCID: PMC3209253  PMID: 21911473
21.  Deletion of p120-catenin results in a tumor microenvironment with inflammation and cancer that establishes it as a tumor suppressor gene 
Cancer cell  2011;19(4):470-483.
P120ctn interacts with E-cadherin, but no formal proof that p120ctn functions as a bone fide tumor suppressor gene has emerged. We report herein that p120ctn loss leads to tumor development in mice. We have generated a conditional knockout model of p120ctn whereby mice develop pre-neoplastic and neoplastic lesions in the oral cavity, esophagus and squamous forestomach. Tumor derived cells secrete granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α (TNFα). The tumors contain significant desmoplasia and immune cell infiltration. Immature myeloid cells comprise a significant percentage of the immune cells present, and likely participate in fostering a favorable tumor microenvironment, including the activation of fibroblasts.
PMCID: PMC3077713  PMID: 21481789
22.  PERK Integrates Autophagy and Oxidative Stress Responses To Promote Survival during Extracellular Matrix Detachment▿  
Molecular and Cellular Biology  2011;31(17):3616-3629.
Mammary epithelial cells (MECs) detached from the extracellular matrix (ECM) produce deleterious reactive oxygen species (ROS) and induce autophagy to survive. The coordination of such opposing responses likely dictates whether epithelial cells survive ECM detachment or undergo anoikis. Here, we demonstrate that the endoplasmic reticulum kinase PERK facilitates survival of ECM-detached cells by concomitantly promoting autophagy, ATP production, and an antioxidant response. Loss-of-function studies show that ECM detachment activates a canonical PERK-eukaryotic translation initiation factor 2α (eIF2α)-ATF4-CHOP pathway that coordinately induces the autophagy regulators ATG6 and ATG8, sustains ATP levels, and reduces ROS levels to delay anoikis. Inducible activation of an Fv2E-ΔNPERK chimera by persistent activation of autophagy and reduction of ROS results in lumen-filled mammary epithelial acini. Finally, luminal P-PERK and LC3 levels are reduced in PERK-deficient mammary glands, whereas they are increased in human breast ductal carcinoma in situ (DCIS) versus normal breast tissues. We propose that the normal proautophagic and antioxidant PERK functions may be hijacked to promote the survival of ECM-detached tumor cells in DCIS lesions.
PMCID: PMC3165554  PMID: 21709020
23.  Periostin, a cell adhesion molecule, facilitates invasion in the tumor microenvironment and annotates a novel tumor invasive signature in esophageal cancer 
Cancer Research  2010;70(13):5281-5292.
Human squamous cell cancers are the most common epithelially derived malignancies. One example is esophageal squamous cell carcinoma (ESCC), which is associated with a high mortality rate (1) that is related to a propensity for invasion and metastasis (2). Here we report that periostin, a highly expressed cell adhesion molecule, is a key component of a novel tumor invasive signature obtained from an organotypic culture model of engineered ESCC. This tumor invasive signature classifies with human ESCC microarrays, underscoring its utility in human cancer. Genetic modulation of periostin promotes tumor cell migration and invasion as revealed in gain of and loss of function experiments. Inhibition of EGFR signaling and restoration of wild-type p53 function were each found to attenuate periostin, suggesting interdependence of two common genetic alterations with periostin function. Collectively, our studies reveal periostin as an important mediator of ESCC tumor invasion and they indicate that organotypic (3D) culture can offer an important tool to discover novel biologic effectors in cancer.
PMCID: PMC3274349  PMID: 20516120
tumor microenvironment; periostin; EGFR; p53
24.  NOTCH1 and NOTCH3 coordinate esophageal squamous differentiation through a CSL-dependent transcriptional network 
Gastroenterology  2010;139(6):2113-2123.
Background & Aims
The Notch receptor family regulates cell fate through cell-cell communication. CSL (CBF-1/RBP-jκ, Su(H), Lag-1) drives canonical Notch-mediated gene transcription during cell lineage specification, differentiation and proliferation in the hematopoietic system, the intestine, the pancreas and the skin. However, the functional roles of Notch in esophageal squamous epithelial biology remain unknown.
Normal esophageal keratinocytes were stimulated with calcium chloride to induce terminal differentiation. The squamous epithelia were reconstituted in organotypic three-dimensional culture, a form of human tissue engineering. Notch was inhibited in culture with a γ-secretase inhibitor or dominant negative mastermind-like1 (DNMAML1). The roles of Notch receptors were evaluated by in vitro gain-of-function and loss-of-function experiments. Additionally, DNMAML1 was targeted to the mouse esophagus by cytokeratin K14 promoter-driven Cre (K14Cre) recombination of Lox-STOP-Lox-DNMAML1. Notch-regulated gene expression was determined by reporter transfection, chromatin immunoprecipitation (ChIP) assays, quantitative reverse-transcription polymerase chain reactions (RT-PCR), Western blotting, immunofluorescence and immunohistochemistry.
NOTCH1 (N1) was activated at the onset of squamous differentiation in the esophagus. Intracellular domain of N1 (ICN1) directly activated NOTCH3 (N3) transcription, inducing HES5 and early differentiation markers such as involucrin (IVL) and cytokeratin CK13 in a CSL-dependent fashion. N3 enhanced ICN1 activity and was required for squamous differentiation. Loss of Notch signaling in K14Cre;DNMAML1 mice perturbed esophageal squamous differentiation and resulted in N3 loss and basal cell hyperplasia.
Notch signaling is important for esophageal epithelial homeostasis. In particular, the crosstalk of N3 with N1 during differentiation provides novel, mechanistic insights into Notch signaling and squamous epithelial biology.
PMCID: PMC2997138  PMID: 20801121
NOTCH1; NOTCH3; esophageal epithelium; squamous differentiation
25.  Oncogenic stress sensitizes murine cancers to hypomorphic suppression of ATR 
Oncogenic Ras and p53 loss-of-function mutations are common in many advanced sporadic malignancies and together predict a limited responsiveness to conventional chemotherapy. Notably, studies in cultured cells have indicated that each of these genetic alterations creates a selective sensitivity to ataxia telangiectasia and Rad3-related (ATR) pathway inhibition. Here, we describe a genetic system to conditionally reduce ATR expression to 10% of normal levels in adult mice to compare the impact of this suppression on normal tissues and cancers in vivo. Hypomorphic suppression of ATR minimally affected normal bone marrow and intestinal homeostasis, indicating that this level of ATR expression was sufficient for highly proliferative adult tissues. In contrast, hypomorphic ATR reduction potently inhibited the growth of both p53-deficient fibrosarcomas expressing H-rasG12V and acute myeloid leukemias (AMLs) driven by MLL-ENL and N-rasG12D. Notably, DNA damage increased in a greater-than-additive fashion upon combining ATR suppression with oncogenic stress (H-rasG12V, K-rasG12D, or c-Myc overexpression), indicating that this cooperative genome-destabilizing interaction may contribute to tumor selectivity in vivo. This toxic interaction between ATR suppression and oncogenic stress occurred without regard to p53 status. These studies define a level of ATR pathway inhibition in which the growth of malignancies harboring oncogenic mutations can be suppressed with minimal impact on normal tissue homeostasis, highlighting ATR inhibition as a promising therapeutic strategy.
PMCID: PMC3248295  PMID: 22133876

Results 1-25 (61)