Min-Goo Lee

Promoter CpG hypermethylation and downregulation of XAF1 expression in human urogenital malignancies: implication for attenuated p53 response to apoptotic stresses.

XIAP-associated factor 1 (XAF1) is a new candidate tumor suppressor, which has been known to exert proapoptotic effects by interfering with the caspase-inhibiting activity of XIAP. To explore the XAF1s candidacy for a suppressor in urogenital tumorigenesis, we investigated the XAF1 status in a series of cancer cell lines and primary tumors derived from the bladder, kidney and prostate. Expression of XAF1 transcript was undetectable or extremely low in 60% (3/5) of bladder, 66% (10/15) of kidney, and 100% (3/3) prostate cancer cell lines. Abnormal reduction of XAF1 was also found in 33% (18/55) of primary bladder and 40% (8/20) of primary kidney tumors, and showed a correlation with advanced stage and high grade of bladder tumor. Hypermethylation at 14 CpG sites in the 5′ proximal region of the XAF1 promoter was highly prevalent in cancers versus adjacent normal or benign tissues and tightly associated with reduced gene expression. XAF1 expression enhanced the apoptotic response of tumor cells to chemotherapeutic agents, such as etoposide or 5-FU. While XAF1 expression did not influence the subcellular distribution or expression of XIAP, it elevated the protein stability of p53 and its target gene expression. Moreover, the apoptosis-sensitizing and growth suppression function of XAF1 was markedly impeded by blockade of p53 function. Collectively, our study demonstrates that epigenetic alteration of XAF1 is frequent in human urogenital cancers and may contribute to the malignant progression of tumors by rendering tumor cells a survival advantage partially through the attenuated p53 response to apoptotic stresses.

Opposite functions of HIF-α isoforms in VEGF induction by TGF-β1 under non-hypoxic conditions

Transforming growth factor (TGF)-β1 has biphasic functions in prostate tumorigenesis, having a growth-inhibitory effect in the early stages, but in the late stages promoting tumor angiogenesis and metastasis. We demonstrate here that tumor-producing TGF-β1 induces vascular endothelial growth factor (VEGF) in prostate cancer cells, and hypoxia-inducible factor (HIF)-1α and HIF-2α has opposite functions in TGF-β1 regulation of VEGF expression under non-hypoxic conditions. The promoter response of VEGF to TGF-β1 was upregulated by the transfection of HIF-2α or siHIF-1α but downregulated by HIF-1α and siHIF-2α. Both HIF-1α and HIF-2α were induced by TGF-β1 at mRNA and protein levels, however, their nuclear translocation was differentially regulated by TGF-β1, suggesting its association with their opposite effects. VEGF induction by TGF-β1 occurred in a Smad3-dependent manner, and the Smad-binding element 2 (SBE2, −992 to −986) and hypoxia response element (−975 to −968) in the VEGF promoter were required for the promoter response to TGF-β1. Smad3 cooperated with HIF-2α in TGF-β1 activation of VEGF transcription and Smad3 binding to the SBE2 site was greatly impaired by knockdown of HIF-2α expression. Moreover, the VEGF promoter response to TGF-β1 was synergistically elevated by co-transfection of Smad3 and HIF-2α but attenuated by HIF-1α in a dose-dependent manner. Additionally, TGF-β1 was found to increase the stability of VEGF transcript by facilitating the cytoplasmic translocation of a RNA-stabilizing factor HuR. Collectively, our data show that tumor-producing TGF-β1 induces VEGF at the both transcription and post-transcriptional levels through multiple routes including Smad3, HIF-2α and HuR. This study thus suggests that autocrine TGF-β1 production may contribute to tumor angiogenesis via HIF-2α signaling under non-hypoxic conditions, providing a selective growth advantage for prostate tumor cells.

Frequent epigenetic inactivation of hSRBC in gastric cancer and its implication in attenuated p53 response to stresses.

hSRBC is a putative tumor suppressor located at 11p15.4, at which frequent genomic loss has been observed in several human malignancies. To explore the candidacy of hSRBC as a suppressor of gastric tumorigenesis, we analyzed the expression and mutation status of hSRBC in gastric tissues and cell lines. hSRBC transcript was expressed in all normal and benign tumor tissues examined, but undetectable or very low in 73% (11/15) cancer cell lines and 41% (46/111) primary tumors. Loss or reduction of hSRBC expression was tumor‐specific and correlated with stage and grade of tumors. While allelic loss or somatic mutations of the gene were infrequent, its expression was restored in tumor cells by 5‐aza‐2′‐deoxycytidine treatment and aberrant hypermethylation of 23 CpG sites in the promoter region showed a tight association with altered expression. Transient or stable expression of hSRBC led to a G1 cell cycle arrest and apoptosis of tumor cells, and strongly suppresses colony forming ability and xenograft tumor growth. In addition, hSRBC elevated apoptotic sensitivity of tumor cells to genotoxic agents, such as 5‐FU, etoposide and ultraviolet. Interestingly, hSRBC increased the protein stability of p53 and expression of p53 target genes, such as p21Waf1, PUMA and NOXA, while hSRBC‐mediated cell cycle arrest and apoptosis were abolished by blockade of p53 function. Our findings suggest that hSRBC is a novel tumor suppressor whose epigenetic inactivation contributes to the malignant progression of gastric tumors, in part, through attenuated p53 response to stresses.

Microarray data mining using landmark gene-guided clustering

BackgroundClustering is a popular data exploration technique widely used in microarray data analysis. Most conventional clustering algorithms, however, generate only one set of clusters independent of the biological context of the analysis. This is often inadequate to explore data from different biological perspectives and gain new insights. We propose a new clustering model that can generate multiple versions of different clusters from a single dataset, each of which highlights a different aspect of the given dataset.ResultsBy applying our SigCalc algorithm to three yeast Saccharomyces cerevisiae datasets we show two results. First, we show that different sets of clusters can be generated from the same dataset using different sets of landmark genes. Each set of clusters groups genes differently and reveals new biological associations between genes that were not apparent from clustering the original microarray expression data. Second, we show that many of these new found biological associations are common across datasets. These results also provide strong evidence of a link between the choice of landmark genes and the new biological associations found in gene clusters.ConclusionWe have used the SigCalc algorithm to project the microarray data onto a completely new subspace whose co-ordinates are genes (called landmark genes), known to belong to a Biological Process. The projected space is not a true vector space in mathematical terms. However, we use the term subspace to refer to one of virtually infinite numbers of projected spaces that our proposed method can produce. By changing the biological process and thus the landmark genes, we can change this subspace. We have shown how clustering on this subspace reveals new, biologically meaningful clusters which were not evident in the clusters generated by conventional methods. The R scripts (source code) are freely available under the GPL license. The source code is available [see Additional File 1] as additional material, and the latest version can be obtained at http://www4.ncsu.edu/~pchopra/landmarks.html. The code is under active development to incorporate new clustering methods and analysis.

ZNF313 is a novel cell cycle activator with an E3 ligase activity inhibiting cellular senescence by destabilizing p21WAF1

ZNF313 encoding a zinc-binding protein is located at chromosome 20q13.13, which exhibits a frequent genomic amplification in multiple human cancers. However, the biological function of ZNF313 remains largely undefined. Here we report that ZNF313 is an ubiquitin E3 ligase that has a critical role in the regulation of cell cycle progression, differentiation and senescence. In this study, ZNF313 is initially identified as a XIAP-associated factor 1 (XAF1)-interacting protein, which upregulates the stability and proapoptotic effect of XAF1. Intriguingly, we found that ZNF313 activates cell cycle progression and suppresses cellular senescence through the RING domain-mediated degradation of p21WAF1. ZNF313 ubiquitinates p21WAF1 and also destabilizes p27KIP1 and p57KIP2, three members of the CDK-interacting protein (CIP)/kinase inhibitor protein (KIP) family of cyclin-dependent kinase inhibitors, whereas it does not affect the stability of the inhibitor of CDK (INK4) family members, such as p16INK4A and p15INK4B. ZNF313 expression is tightly controlled during the cell cycle and its elevation at the late G1 phase is crucial for the G1-to-S phase transition. ZNF313 is induced by mitogenic growth factors and its blockade profoundly delays cell cycle progression and accelerates p21WAF1-mediated senescence. Both replicative and stress-induced senescence are accompanied with ZNF313 reduction. ZNF313 is downregulated during cellular differentiation process in vitro and in vivo, while it is commonly upregulated in many types of cancer cells. ZNF313 shows both the nuclear and cytoplasmic localization in epithelial cells of normal tissues, but exhibits an intense cytoplasmic distribution in carcinoma cells of tumor tissues. Collectively, ZNF313 is a novel E3 ligase for p21WAF1, whose alteration might be implicated in the pathogenesis of several human diseases, including cancers.

Epigenetic inactivation of the NORE1 gene correlates with malignant progression of colorectal tumors

BackgroundNORE1 (RASSF5) is a newly described member of the RASSF family with Ras effector function. NORE1 expression is frequently inactivated by aberrant promoter hypermethylation in many human cancers, suggesting that NORE1 might be a putative tumor suppressor. However, expression and mutation status of NORE1 and its implication in colorectal tumorigenesis has not been evaluated.MethodsExpression, mutation, and methylation status of NORE1A and NORE1B in 10 cancer cell lines and 80 primary tumors were characterized by quantitative PCR, SSCP, and bisulfite DNA sequencing analyses. Effect of NORE1A and NORE1B expression on tumor cell growth was evaluated using cell number counting, flow cytometry, and colony formation assays.ResultsExpression of NORE1A and NORE1B transcript was easily detectable in all normal colonic epithelial tissues, but substantially decreased in 7 (70%) and 4 (40%) of 10 cancer cell lines and 31 (38.8%) and 25 (31.3%) of 80 primary carcinoma tissues, respectively. Moreover, 46 (57.6%) and 38 (47.5%) of 80 matched tissue sets exhibited tumor-specific reduction of NORE1A and NORE1B, respectively. Abnormal reduction of NORE1 was more commonly observed in advanced stage and high grade tumors compared to early and low grade tumors. While somatic mutations of the gene were not identified, its expression was re-activated in all low expressor cells after treatment with the demethylating agent 5-aza-dC. Bisulfite DNA sequencing analysis of 31 CpG sites within the promoter region demonstrated that abnormal reduction of NORE1A is tightly associated with promoter CpG sites hypermethylation. Moreover, transient expression and siRNA-mediated knockdown assays revealed that both NORE1A and NORE1B decrease cellular growth and colony forming ability of tumor cells and enhance tumor cell response to apoptotic stress.ConclusionOur data indicate that epigenetic inactivation of NORE1 due to aberrant promoter hypermethylation is a frequent event in colorectal tumorigenesis and might be implicated in the malignant progression of colorectal tumors.

Real-time phase-contrast imaging of photothermal treatment of head and neck squamous cell carcinoma: an in vitro study of macrophages as a vector for the delivery of gold nanoshells

Abstract. Photothermal treatment (PTT) using nanoparticles has gained attention as a promising alternative therapy for malignant tumors. One strategy for increasing the selectivity of PTT is the use of macrophages as a cellular vector for delivering nanoparticles. The aim of the present study is to examine the use of macrophages as a cellular vector for efficient PTT and determine the appropriate irradiation power and time of a near-infrared (NIR) laser using real-time phase-contrast imaging. Thermally induced injury and death of cancer cells were found to begin at 44°C to 45°C, which was achieved using the PTT effect with gold nanoshells (NS) and irradiation with a NIR laser at a power of 2 W for 5 min. The peritoneal macrophage efficiently functioned as a cellular vector for the NS, and the cancer cells surrounding the NS-loaded macrophages selectively lost their cellular viability after being irradiated with the NIR laser.

Identification of XAF1–MT2A mutual antagonism as a molecular switch in cell-fate decisions under stressful conditions

Significance Epigenetic inactivation of XAF1 tumor suppressor is frequently observed in multiple human cancers. However, the mechanisms underlying its function remain largely undefined. Here, we present evidence that XAF1 plays a critical role in cell-fate decisions under heavy-metal–induced stress conditions through the mutual antagonism with MT2A. XAF1 is activated as a transcription target of MTF-1 and destabilizes MT2A through the interaction-directed lysosomal degradation, whereas it is destabilized by MT2A under cytostatic stress conditions. XAF1-mediated MT2A inactivation leads to elevation of free intracellular zinc level and up- and down-regulates p53 and XIAP, respectively, to promote apoptosis. XAF1–MT2A antagonism thus represents one critical regulator of cell-fate decisions, suggesting that it could be an attractive target for the therapeutic intervention of tumor progression. XIAP-associated factor 1 (XAF1) is a tumor suppressor that is commonly inactivated in multiple human neoplasms. However, the molecular mechanism underlying its proapoptotic function remains largely undefined. Here, we report that XAF1 induction by heavy metals triggers an apoptotic switch of stress response by destabilizing metallothionein 2A (MT2A). XAF1 directly interacts with MT2A and facilitates its lysosomal degradation, resulting in the elevation of the free intercellular zinc level and subsequent activation of p53 and inactivation of XIAP. Intriguingly, XAF1 is activated as a unique transcription target of metal-regulatory transcription factor-1 (MTF-1) in signaling apoptosis, and its protein is destabilized via the lysosomal pathway by MTF-1–induced MT2A under cytostatic stress conditions, indicating the presence of mutual antagonism between XAF1 and MT2A. The antagonistic interplay between XAF1 and MT2A acts as a key molecular switch in MTF-1–mediated cell-fate decisions and also plays an important role in cell response to various apoptotic and survival factors. Wild-type (WT) XAF1 but not MT2A binding-deficient mutant XAF1 increases the free intracellular zinc level and accelerates WT folding of p53 and degradation of XIAP. Consistently, XAF1 evokes a more drastic apoptotic effect in p53+/+ versus isogenic p53−/− cells. Clinically, expression levels of XAF1 and MT2A are inversely correlated in primary colon tumors and multiple cancer cell lines. XAF1-depleted xenograft tumors display an increased growth rate and a decreased apoptotic response to cytotoxic heavy metals with strong MT2A expression. Collectively, this study uncovers an important role for XAF1–MT2A antagonism as a linchpin to govern cell fate under various stressful conditions including heavy metal exposure.

RASSF1A suppresses colorectal tumor cell growth through p53-dependent p21WAF1 activation: RASSF1A suppresses colorectal tumor cell growth through p53-dependent p21WAF1 activation

Despite the frequent loss of Ras association domain family 1 isoform A (RASSF1A) expression in various cancers, the precise mechanism underlying its tumor‐suppressive effect is not fully understood. To elucidate the growth‐inhibitory role for RASSF1A in colorectal tumorigenesis, this study investigated the RASSF1A regulation of the p53‐p21WAF1 pathway.

Abstract 5068: Identification of RASSF1A as a novel RhoA antagonist: direct interaction with and Smurf1-mediated ubiquitination of RhoA

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnRASSF1A is a tumor suppressor which plays a critical role in the regulation of various aspects of cellular functions, including cell proliferation, apoptosis, and motility. RASSF1A is frequently inactivated in various types of human cancers by transcriptional silencing of the gene due to aberrant promoter hypermethylation. However, molecular mechanisms underlying its tumor suppressive functions were largely undefined. We characterized RASSF1A regulation of a small GTPase RhoA and its implication in tumor cell migration and invasion. Effect of RASSF1A expression on RhoA activity was examined by immunoblot, immunopricipitation, and ubiquitinylation assays, and RASSF1A modulation of RhoA-mediated cell migration and invasion was determined using wound healing and in vitro invasion assay. We identified that RASSF1A inhibits serum- or LPA-induced RhoA activation in both cancer cells and noncancerous cells. SiRNA-mediated knockdown of RASSF1A increases RhoA activity and RhoA-mediated tumor cell migration. RASSF1A promotes the protesomal degradation of GTP-bound RhoA via Smurf1-mediated ubiquitinylation, but does not affect the activity and stability of Rac1 and CDC42. Immunofluoroscence and immunopricipitation assays revealed that RASSF1A colocalizes and interacts with RhoA and Smurf1. The N-terminal region of RASSF1A interacts with Smurf1, and its C-terminal residues (amino acids 266-272) are critical for the interaction with RhoA. The mutant RASSF1A (delNC-RASSF1A) having a sequence alteration in the Smurf1 and RhoA binding region fails to inhibit the RhoA-mediated tumor cell migration and invasion. Consistent with theses results, loss or abnormal reduction of RASSF1A expression exhibits a tight correlation with RhoA activation in both cancer cell lines and primary tumors of various tissues origins. Our study demonstrates first that RASSF1A regulates cell motility through the proteosomal degradation of activated RhoA through Smurf1-mediated ubiquitinylation, indicating that genetic and epigenetic inactivation of RASSF1A during tumorigenic process increases the invasive and metastatic potential of tumor cells by deregulation of RhoA.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5068.