The EBNA3 family of Epstein-Barr virus nuclear proteins associates with the USP46/USP12 deubiquitination complexes to regulate lymphoblastoid cell line growth.

The Epstein-Barr virus (EBV) nuclear proteins EBNA3A, EBNA3B, and EBNA3C interact with the cell DNA binding protein RBPJ and regulate cell and viral genes. Repression of the CDKN2A tumor suppressor gene products p16(INK4A) and p14(ARF) by EBNA3A and EBNA3C is critical for EBV mediated transformation of resting B lymphocytes into immortalized lymphoblastoid cell lines (LCLs). To define the composition of endogenous EBNA3 protein complexes, we generated lymphoblastoid cell lines (LCLs) expressing flag-HA tagged EBNA3A, EBNA3B, or EBNA3C and used tandem affinity purification to isolate each EBNA3 complex. Our results demonstrated that each EBNA3 protein forms a distinct complex with RBPJ. Mass-spectrometry revealed that the EBNA3A and EBNA3B complexes also contained the deubquitylation complex consisting of WDR48, WDR20, and USP46 (or its paralog USP12) and that EBNA3C complexes contained WDR48. Immunoprecipitation confirmed that EBNA3A, EBNA3B, and EBNA3C association with the USP46 complex. Using chromatin immunoprecipitation, we demonstrate that WDR48 and USP46 are recruited to the p14(ARF) promoter in an EBNA3C dependent manner. Mapping studies were consistent with WDR48 being the primary mediator of EBNA3 association with the DUB complex. By ChIP assay, WDR48 was recruited to the p14(ARF) promoter in an EBNA3C dependent manner. Importantly, WDR48 associated with EBNA3A and EBNA3C domains that are critical for LCL growth, suggesting a role for USP46/USP12 in EBV induced growth transformation.

CD133-targeted niche-dependent therapy in cancer: a multipronged approach.

Cancer treatment continues to be challenged by the development of therapeutic resistances and relapses in the clinical setting, which are largely attributed to tumor heterogeneity, particularly the existence of cancer stem cells (CSCs). Thus, targeting the CSC subpopulation may represent an effective therapeutic strategy. However, despite advances in identifying and characterizing CD133(+) CSCs in various human cancers, efforts to translate these experimental findings to clinical modalities have been slow in the making, especially in light of the growing awareness of CSC plasticity and the foreseeable pitfall of therapeutically targeting CSC base sorely on a surface marker. We, and others, have demonstrated that the CD133(+) CSCs reside in complex vascular niches, where reciprocal signaling between the CD133(+) CSCs and their microenvironment may govern niche morphogenesis and homeostasis. Herein, we discuss the multifaceted functional role of the CD133(+) cells in the context of their niche, and the potential of targeting CD133 as a niche-dependent approach in effective therapy.

CD133+ melanoma subpopulations contribute to perivascular niche morphogenesis and tumorigenicity through vasculogenic mimicry.

Tumor cell subpopulations that express cancer stem cell markers such as CD133 (prominin1) or ABCB5 are thought to be crucial for tumor initiation and heterogeneity, but their biological significance in melanoma has been controversial. Here, we report that CD133(+) and ABCB5(+) subpopulations are colocalized in melanomas in perivascular niches that contain CD144 (VE-cadherin)(+) melanoma cells forming vessel-like channels, a phenomenon termed vasculogenic mimicry (VM). RNAi-mediated attenuation of CD133 established its critical function in morphogenesis of these perivascular niches as well as in melanoma tumorigenicity. Niche-associated genes CD144 and ABCB5 were downregulated in tumors derived from CD133 knockdown (KD) melanoma cells compared with controls. CD133KD cells also lacked the ability to form CD144(+) VM-like channels in a manner that was associated with a depletion of the ABCB5(+) cell subpopulation. Finally, CD133 KD cells exhibited poorer tumor growth in vivo. Taken together, our findings corroborate models in which CD133(+)/ABCB5(+) melanoma cells reside in a complex anastomosing microvascular niche that encompasses CD144(+) VM channels as well as authentic endothelial cell-lined blood vessels. Further, they indicate that CD133(+) cells act as stem-like cells, which drive tumor growth by promoting VM and the morphogenesis of a specialized perivascular niche in melanoma.

Targeting CD20 in melanoma patients at high risk of disease recurrence.

Melanomas contain distinct cell subpopulations. Several of these subpopulations, including one expressing CD20, may harbor stem cell-like or tumor-initiating characteristics. We hypothesized that patients at high risk of disease recurrence could benefit from an adjuvant anti-CD20 therapy. Therefore, we initiated a small pilot trial to study the effect of the anti-CD20 antibody rituximab in a group of melanoma patients with stage IV metastatic disease who had been rendered without evident disease by way of surgery, chemotherapy and/or radiation therapy. The major objective was safety, while secondary objectives were description of recurrence-free intervals (RFI) and overall survival (OS). Nine patients received rituximab at 375 mg/m(2) qw for 4 weeks followed by a maintenance therapy every 8 weeks. Treatment was discontinued after 2 years or with disease recurrence. Treatment was well tolerated. After a median observation of 42 months, the median neither of RFI nor of OS has been reached. Despite therapy that ended after 2 years, six out of nine patients are still alive and five of them are recurrence-free. Though the patient number is too small for definitive conclusions, our data may represent a first example of the potential therapeutic value of targeting CD20(+) cell populations-at least for a subset of patients.

Expression of the embryonic stem cell transcription factor SOX2 in human skin: relevance to melanocyte and merkel cell biology.

SOX2 is a gene located on chromosome 3q26.33 that encodes a transcription factor important to maintenance of embryonic neural crest stem cell pluripotency. We have identified rare SOX2-immunoreactive cells in normal human skin at or near the established stem cell niches. Three subsets of SOX2-positive cells were defined in these regions: those expressing only SOX2 and those that co-expressed SOX2 and either CK20 or microphthalmia-associated transcription factor, which are consistent with dichotomous differentiation of SOX2-expressing precursors along neuroendocrine (Merkel cell) or melanocytic lines, respectively. Examination of Merkel cell carcinomas confirmed nuclear SOX2 expression in this tumor type. In human patient melanoma, strong nuclear expression of SOX2 was noted in a subset of tumors, and the ability to detect SOX2 in lesional cells significantly correlated with primary tumor thickness in a survey cohort. To assess the potential role of SOX2 in melanoma growth, an in vivo tumorigenesis assay was used. Whereas SOX2 knockdown failed to influence proliferation of cultured melanoma cells in vitro, tumor xenografts generated with the SOX2-knockdown cell line showed significant decrease in mean tumor volume as compared with controls. In aggregate, these findings suggest that SOX2 is a novel biomarker for subpopulations of normal skin cells that reside in established stem cell niches and that might relate to Merkel cell and melanocyte ontogeny and tumorigenesis.

Epstein-Barr virus nuclear protein 3C domains necessary for lymphoblastoid cell growth: interaction with RBP-Jkappa regulates TCL1.

B lymphocytes converted into lymphoblastoid cell lines (LCLs) by an Epstein-Barr virus that expresses a conditional EBNA3C require complementation with EBNA3C for growth under nonpermissive conditions. Complementation with relatively large EBNA3C deletion mutants identified amino acids (aa) 1 to 506 (which includes the RBP-Jkappa/CSL [RBP-Jkappa] binding domain) and 733 to 909 to be essential for LCL growth, aa 728 to 732 and 910 to 992 to be important for full wild-type (wt) growth, and only aa 507 to 727 to be unimportant (S. Maruo, Y. Wu, T. Ito, T. Kanda, E. D. Kieff, and K. Takada, Proc. Natl. Acad. Sci. USA 106:4419-4424, 2009). When mutants with smaller deletions were used, only aa 51 to 400 and 851 to 900 were essential for LCL growth; aa 447 to 544, 701 to 750, 801 to 850, and 901 to 992 were important for full wt growth; and aa 4 to 50, 401 to 450, 550 to 707, and 751 to 800 were unimportant. These data reduce the EBNA3C essential residues from 68% to 40% of the open reading frame. Point mutations confirmed RBP-Jkappa binding to be essential for wt growth and indicated that SUMO and CtBP binding interactions were important only for full wt growth. EBNA3C aa 51 to 150, 249 to 311, and 851 to 900 were necessary for maintaining LCL growth, but not RBP-Jkappa interaction, and likely mediate interactions with other key cell proteins. Moreover, all mutants null for LCL growth had fewer S+G(2)/M-phase cells at 14 days, consistent with EBNA3C interaction with RBP-Jkappa as well as aa 51 to 150, 249 to 311, and 851 to 900 being required to suppress p16(INK4A) (S. Maruo, Y. Wu, S. Ishikawa, T. Kanda, D. Iwakiri, and K. Takada, Proc. Natl. Acad. Sci. USA 103:19500-19505, 2006). We have confirmed that EBNA3C upregulates TCL1 and discovered that EBNA3C upregulates TCL1 through RBP-Jkappa, indicating a central role for EBNA3C interaction with RBP-Jkappa in mediating cell gene transcription.

Aggressive melanoma cells escape from BMP7-mediated autocrine growth inhibition through coordinated Noggin upregulation.

Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily responsible for mediating a diverse array of cellular functions both during embryogenesis and in adult life. Previously, we reported that upregulation of BMP7 in human melanoma correlates with tumor progression. However, melanoma cells are either inhibited by or become resistant to BMP7 as a function of tumor progression, with normal melanocytes being most susceptible. Herein, real-time quantitative reverse transcriptase-polymerase chain reactions and western blotting revealed that the expression of BMP antagonist, Noggin, correlates with resistance to BMP7 in advanced melanoma cells. To test the hypothesis that coordinated upregulation of Noggin protects advanced melanoma cells from autocrine inhibition by BMP7, functional expression of Noggin in susceptible melanoma cells was achieved by adenoviral gene transfer. The Noggin-overexpressing cells exhibited a growth advantage in response to subsequent BMP7 transduction in vitro under anchorage-dependent and -independent conditions, in three-dimensional skin reconstructs, as well as in vivo in severe combined immunodeficient mice. In concordance, Noggin knockdown by lentiviral shRNA confers sensitivity to BMP7-induced growth inhibition in advanced melanoma cells. Our findings suggest that, like TGF-beta, BMP7 acts as an autocrine growth inhibitor in melanocytic cells, and that advanced melanoma cells may escape from BMP7-induced inhibition through concomitant aberrant expression of Noggin.

Hsp72 up-regulates Epstein-Barr virus EBNALP coactivation with EBNA2.

The Epstein-Barr virus (EBV) transcriptional coactivator EBNALP specifically associates and colocalizes with Hsp72 in lymphoblastoid cell lines. We now find that overexpression of Hsp72 more than doubled EBNALP coactivation with EBNA2 of a transfected EBV LMP1 promoter in B lymphoblasts, did not affect EBNA2 or EBNALP protein levels, and strongly up-regulated EBNA2 and EBNALP coactivation of LMP1 protein expression from the endogenous EBV genome in latency I infected Akata cells. The Hsp72 ATP, protein binding, and the C-terminal regulatory domains were required for full activity. An EBNALP deletion mutant, EBNALPd45, which does not associate with Hsp72, coactivated with EBNA2, but was not affected by Hsp72 overexpression, despite Hsp72 up-regulation of wild-type EBNALP coactivation with EBNA2 at all levels of EBNALP expression, indicating the importance of Hsp72 association with EBNALP for Hsp72 up-regulation of coactivation. Of importance, a 90% RNAi knockdown of Hsp72 reduced EBNALP coactivation with EBNA2 of transfected EBV LMP1 and Cp promoters by approximately 50%. Overexpression of the Hsp72 C-terminal interacting and regulatory protein, CHIP, strongly down-regulated EBNALP coactivation, independently of CHIP ubiquitin ligase activity. CHIP effects were Hsp72 dependent, indicating a background downmodulating role for CHIP in Hsp72 augmentation of EBNA2 and EBNALP coactivation. Based on these and other cited data, we favor a model in which Hsp72 chaperones EBNALP shuttling of repressors from EBNA2-enhanced promoters.

Isolation and characterization of beta-ketoacyl-acyl carrier protein reductase (fabG) mutants of Escherichia coli and Salmonella enterica serovar Typhimurium.

FabG, beta-ketoacyl-acyl carrier protein (ACP) reductase, performs the NADPH-dependent reduction of beta-ketoacyl-ACP substrates to beta-hydroxyacyl-ACP products, the first reductive step in the elongation cycle of fatty acid biosynthesis. We report the first documented fabG mutants and their characterization. By chemical mutagenesis followed by a tritium suicide procedure, we obtained three conditionally lethal temperature-sensitive fabG mutants. The Escherichia coli [fabG (Ts)] mutant contains two point mutations: A154T and E233K. The beta-ketoacyl-ACP reductase activity of this mutant was extremely thermolabile, and the rate of fatty acid synthesis measured in vivo was inhibited upon shift to the nonpermissive temperature. Moreover, synthesis of the acyl-ACP intermediates of the pathway was inhibited upon shift of mutant cultures to the nonpermissive temperature, indicating blockage of the synthetic cycle. Similar results were observed for in vitro fatty acid synthesis. Complementation analysis revealed that only the E233K mutation was required to give the temperature-sensitive growth phenotype. In the two Salmonella enterica serovar Typhimurium fabG(Ts) mutants one strain had a single point mutation, S224F, whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the twofold axes of symmetry at the dimer interfaces in this homotetrameric protein, suggesting that the quaternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature.

Beta-ketoacyl-acyl carrier protein synthase III (FabH) is essential for bacterial fatty acid synthesis.

beta-Ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, also called acetoacetyl-ACP synthase) encoded by the fabH gene is thought to catalyze the first elongation reaction (Claisen condensation) of type II fatty acid synthesis in bacteria and plant plastids. However, direct in vivo evidence that KAS III catalyzes an essential reaction is lacking, because no mutant organism deficient in this activity has been isolated. We report the first bacterial strain lacking KAS III, a fabH mutant constructed in the Gram-positive bacterium Lactococcus lactis subspecies lactis IL1403. The mutant strain carries an in-frame deletion of the KAS III active site region and was isolated by gene replacement using a medium supplemented with a source of saturated and unsaturated long-chain fatty acids. The mutant strain is devoid of KAS III activity and fails to grow in the absence of supplementation with exogenous long-chain fatty acids demonstrating that KAS III plays an essential role in cellular metabolism. However, the L. lactis fabH deletion mutant requires only long-chain unsaturated fatty acids for growth, a source of long-chain saturated fatty acids is not required. Because both saturated and unsaturated fatty acids are required for growth when fatty acid synthesis is blocked by biotin starvation (which prevents the synthesis of malonyl-CoA), another pathway for saturated fatty acid synthesis must remain in the fabH deletion strain. Indeed, incorporation of [1-14C]acetate into fatty acids in vivo showed that the fabH mutant retained about 10% of the fatty acid synthetic ability of the wild-type strain and that this residual synthetic capacity was preferentially diverted to the saturated branch of the pathway. Moreover, mass spectrometry showed that the fabH mutant retained low levels of palmitic acid upon fatty acid starvation. Derivatives of the fabH deletion mutant strain were isolated that were octanoic acid auxotrophs consistent with biochemical studies indicating that the major role of FabH is production of short-chain fatty acid primers. We also confirmed the essentiality of FabH in Escherichia coli by use of a plasmid-based gene insertion/deletion system. Together these results provide the first genetic evidence demonstrating that FabH conducts the major condensation reaction in the initiation of type II fatty acid biosynthesis in both Gram-positive and Gram-negative bacteria.