Jikhyon Han

NF-κB Activates Transcription of the RNA-Binding Factor HuR, via PI3K-AKT Signaling, to Promote Gastric Tumorigenesis

BACKGROUND & AIMS HuR is a RNA-binding factor whose expression is commonly upregulated in some human tumor types. We explored the molecular mechanism underlying HuR elevation and its role in gastric cancer tumorigenesis. METHODS HuR expression and subcellular localization were determined by polymerase chain reaction, immunoblot, and immunohistochemical analyses. Its effect on tumor growth was characterized using flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and soft agar analyses. Luciferase reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays were used to measure transcriptional activation by nuclear factor kappaB (NF-kappaB) signaling. RESULTS Compared with normal gastric tissues, HuR was expressed at higher levels in gastric tumors, particularly in advanced versus early tumors; this increase was associated with enhanced cytoplasmic translocation of HuR. HuR overexpression increased proliferation of tumor cells, activating the G(1) to S transition of the cell cycle, DNA synthesis, and anchorage-independent growth. Small interfering RNA-mediated knockdown of HuR expression reduced tumor cell proliferation and response to apoptotic stimuli. No genetic or epigenetic alterations of HuR were observed in gastric tumor cell lines or primary tumors; overexpression depended on phosphatidylinositol 3-kinase/AKT signaling and NF-kappaB activity. AKT activation increased p65/RelA binding to a putative NF-kappaB binding site in the HuR promoter, the stability of HuR target transcripts, and the cytoplasmic import of HuR. CONCLUSIONS HuR is a direct transcription target of NF-kappaB; its activation in gastric cancer cell lines depends on phosphatidylinositol 3-kinase/AKT signaling. HuR activation by this pathway has proliferative and antiapoptotic effects on gastric cancer cells.

Caveolin-1 Increases Aerobic Glycolysis in Colorectal Cancers by Stimulating HMGA1-Mediated GLUT3 Transcription

Caveolin-1 (CAV1) acts as a growth suppressor in various human malignancies, but its expression is elevated in many advanced cancers, suggesting the oncogenic switch of its role during tumor progression. To understand the molecular basis for the growth-promoting function of CAV1, we characterized its expression status, differential roles for tumor growth, and effect on glucose metabolism in colorectal cancers. Abnormal elevation of CAV1 was detected in a substantial fraction of primary tumors and cell lines and tightly correlated with promoter CpG sites hypomethylation. Depletion of elevated CAV1 led to AMPK activation followed by a p53-dependent G1 cell-cycle arrest and autophagy, suggesting that elevated CAV1 may contribute to ATP generation. Furthermore, CAV1 depletion downregulated glucose uptake, lactate accumulation, and intracellular ATP level, supporting that aerobic glycolysis is enhanced by CAV1. Consistently, CAV1 was shown to stimulate GLUT3 transcription via an HMGA1-binding site within the GLUT3 promoter. HMGA1 was found to interact with and activate the GLUT3 promoter and CAV1 increased the HMGA1 activity by enhancing its nuclear localization. Ectopic expression of HMGA1 increased glucose uptake, whereas its knockdown caused AMPK activation. In addition, GLUT3 expression was strongly induced by cotransfection of CAV1 and HMGA1, and its overexpression was observed predominantly in tumors harboring high levels of CAV1 and HMGA1. Together, these data show that elevated CAV1 upregulates glucose uptake and ATP production through HMGA1-mediated GLUT3 transcription, suggesting that CAV1 may render tumor cells growth advantages by enhancing aerobic glycolysis.

Epigenetic alteration of PRKCDBP in colorectal cancers and its implication in tumor cell resistance to TNFα-induced apoptosis

Purpose: PRKCDBP is a putative tumor suppressor in which alteration has been observed in several human cancers. We investigated expression and function of PRKCDBP in colorectal cells and tissues to explore its candidacy as a suppressor in colorectal tumorigenesis. Experimental Design: Expression and methylation status of PRKCDBP and its effect on tumor growth were evaluated. Transcriptional regulation by NF-κB signaling was defined by luciferase reporter and chromatin immunoprecipitation assays. Results: PRKCDBP expression was hardly detectable in 29 of 80 (36%) primary tumors and 11 of 19 (58%) cell lines, and its alteration correlated with tumor stage and grade. Promoter hypermethylation was commonly found in cancers. PRKCDBP expression induced the G1 cell-cycle arrest and increased cellular sensitivity to various apoptotic stresses. PRKCDBP was induced by TNFα, and its level correlated with tumor cell sensitivity to TNFα-induced apoptosis. PRKCDBP induction by TNFα was disrupted by blocking NF-κB signaling while it was enhanced by RelA transfection. The PRKCDBP promoter activity was increased in response to TNFα, and this response was abolished by disruption of a κB site in the promoter. PRKCDBP delayed the formation and growth of xenograft tumors and improved tumor response to TNFα-induced apoptosis. Conclusions: PRKCDBP is a proapoptotic tumor suppressor which is commonly altered in colorectal cancer by promoter hypermethylation, and its gene transcription is directly activated by NF-κB in response to TNFα. This suggests that PRKCDBP inactivation may contribute to tumor progression by reducing cellular sensitivity to TNFα and other stresses, particularly under chronic inflammatory microenvironment. Clin Cancer Res; 17(24); 7551–62. ©2011 AACR.

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.

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.

XAF1 directs apoptotic switch of p53 signaling through activation of HIPK2 and ZNF313

Significance Epigenetic inactivation of X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is frequently observed in multiple human malignancies. However, the mechanisms underlying its tumor-suppression function remain largely undefined. Here, we identify XAF1 as a positive feedback regulator of p53, which directs the apoptotic switch of p53 signaling. As a unique transcription target of p53 in signaling apoptosis, XAF1 acts as a competitor of E3 ubiquitin ligase MDM2 in binding to p53 and thus disrupts the p53–MDM2 regulatory loop. Moreover, XAF1 appears to promote homeodomain-interacting protein kinase 2 (HIPK2)-mediated p53 phosphorylation by interrupting HIPK2-targeting function of E3 ubiquitin ligase Siah2 and promotes zinc finger protein 313 (ZNF313)-induced p21WAF1 ubiquitination. XAF1 thus represents one critical regulator of p53’s cell-fate decision function, suggesting that restoring the p53–XAF1 feedback loop could be an attractive avenue for the therapeutic intervention of malignant tumor progression. X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a tumor suppressor that is frequently inactivated in many human cancers. However, the molecular mechanism underlying its growth-inhibitory function remains largely unknown. Here, we report that XAF1 forms a positive feedback loop with p53 and acts as a molecular switch in p53-mediated cell-fate decisions favoring apoptosis over cell-cycle arrest. XAF1 binds directly to the N-terminal proline-rich domain of p53 and thus interferes with E3 ubiquitin ligase MDM2 binding and ubiquitination of p53. XAF1 stimulates homeodomain-interacting protein kinase 2 (HIPK2)-mediated Ser-46 phosphorylation of p53 by blocking E3 ubiquitin ligase Siah2 interaction with and ubiquitination of HIPK2. XAF1 also steps up the termination of p53-mediated cell-cycle arrest by activating zinc finger protein 313 (ZNF313), a p21WAF1-targeting ubiquitin E3 ligase. XAF1 interacts with p53, Siah2, and ZNF313 through the zinc finger domains 5, 6, and 7, respectively, and truncated XAF1 isoforms preferentially expressed in cancer cells fail to form a feedback loop with p53. Together, this study uncovers a novel role for XAF1 in p53 stress response, adding a new layer of complexity to the mechanisms by which p53 determines cell-fate decisions.

PPARδ promotes oncogenic redirection of TGF-β1 signaling through the activation of the ABCA1-Cav1 pathway.

TGF-β1 plays biphasic functions in prostate tumorigenesis, inhibiting cell growth at early stages but promoting malignant progression at later stages. However, the molecular basis for the oncogenic conversion of TGF-β1 function remains largely undefined. Here, we demonstrate that PPARδ is a direct transcription target of TGF-β1 and plays a critical role in oncogenic redirection of TGF-β1 signaling. Blockade of PPARδ induction enhances tumor cell response to TGF-β1-mediated growth inhibition, while its activation promotes TGF-β1-induced tumor growth, migration and invasion. PPARδ-mediated switch of TGF-β1 function is associated with down- and upregulation of Smad and ERK signaling, respectively, and tightly linked to its function to activate ABCA1 cholesterol transporter followed by caveolin-1 (Cav1) induction. Intriguingly, TGF-β1 activation of the PPARδ-ABCA1-Cav1 pathway facilitates degradation of TGF-β receptors (TβRs) and attenuates Smad but enhances ERK response to TGF-β1. Expression of PPARδ and Cav1 is tightly correlated in both prostate tissues and cell lines and significantly higher in cancer vs. normal tissues. Collectively, our study shows that PPARδ is a transcription target of TGF-β1 and contributes to the oncogenic conversion of TGF-β1 function through activation of the ABCA1-Cav1-TβR signaling axis.

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.

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, DC RASSF1A 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. Citation 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.

Gene expression and regulation of wax moth transferrin by PAMPs and heavy metals

Abstract A complete mRNA sequence of transferrin from the wax moth, Galleria mellonella, was obtained, and compared with those of other species. We previously reported that the sequence was most similar to those of Manduca sexta and Bombyx mori. As in other moths, G. mellonella transferrin had only one iron‐binding site at its N‐terminal region. Semi‐qRT PCR was conducted to investigate tissue‐specific distribution and transcriptional regulation of the wax moth transferrin mRNA. Larval muscle and fat body contained larger quantity of mRNA than other tested tissues. In this study, it was observed that iron and cadmium regulated transferrin transcription, and this regulation pattern was tissue specific. Iron up‐regulated transferrin mRNA level in fat body, while suppressed it in the Malpighian tubules and silk glands. Cadmium decreased the mRNA level in fat body, muscle, and Malpighian tubules, but significantly increased the mRNA level in silk glands. In addition, the mRNA expression was induced by all tested pathogen‐associated molecular patterns (PAMPs) including LPS, lipoteichoic acid (LTA), glucan, and even chitin.