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Supplementary Figure 2 from Activation of Hypoxia-Inducible Factor-1α Is Necessary for Lysophosphatidic Acid–Induced Vascular Endothelial Growth Factor Expression

Supplementary Figure 3 from Activation of Hypoxia-Inducible Factor-1α Is Necessary for Lysophosphatidic Acid–Induced Vascular Endothelial Growth Factor Expression

Purpose: Lysophosphatidic acid (LPA) plays an important role in mediating cell proliferation, survival, and tumor invasion and angiogenesis. This bioactive phospholipid at the concentration in ascitic fluid stimulates the growth of malignant ovarian tumors by increasing the expression of vascular endothelial growth factor (VEGF). In the present study, we investigated whether LPA activates hypoxia inducible factor-1 (HIF-1), a key transcriptional complex in tumor progression and metastasis, thereby increasing the expression of VEGF.Experimental Design: Immunoblotting, reverse transcription-PCR, ELISA, immunofluorescence, and chromatin immunoprecipitation assay were used to examine the expression of VEGF and HIF-1α in various cancer cells. Specific HIF-1α small interfering RNA was transfected to various cancer cells to determine the role of HIF-1α in LPA-induced VEGF expression.Results: LPA induced expressions of VEGF and HIF-1α in OVCAR-3, CAOV-3, PC-3, and SK-Hep1 cells but not in SKOV-3 and Hep-3B cells. In OVCAR-3 and PC-3 cells, the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin/p70S6K and p42/p44 mitogen-activated protein kinase pathways were required for LPA-induced HIF-1α and VEGF expressions, whereas only the phosphoinositide 3-kinase/mammalian target of rapamycin/p70S6K pathway was important in SK-Hep1 cells. Immunofluorescence microscopy assay showed translocation of HIF-1α to nucleus by LPA, and chromatin immunoprecipitation assay revealed the binding of HIF-1α to the promoter of VEGF by LPA. Importantly, we found that small interfering RNA–induced reduction of HIF-1α expression significantly attenuated VEGF expression by LPA.Conclusions: Our results show for the first time that LPA induces VEGF via HIF-1α activation and reveal a critical role of HIF-1α in LPA-induced cancer cell proliferation and angiogenesis.

Rab coupling protein (RCP) is upregulated in head and neck squamous cell carcinoma (HNSCC) and is correlated with the progression and survival of patients. However, the role of RCP in one of the aggressive types of HNSCC, oral squamous cell carcinoma (OSCC), remains elusive. In the present study, we identified the important role of Zeb1 in RCP-induced OSCC epithelial-to-mesenchymal transition (EMT) and invasion. RCP induces Zeb1 expression, and silencing Zeb1 expression significantly inhibits RCP-induced OSCC invasion. In addition, Zeb1 upregulates MT1-MMP expression to promote OSCC EMT and invasion. Furthermore, we observed that the β1 integrin/EGFR/β-catenin signaling cascade mediates RCP-induced Zeb1 expression to promote OSCC invasion. Notably, we provide evidence that resveratrol (REV) strongly inhibits RCP-induced Zeb1 expression through blocking β1 integrin endosome recycling and EGFR activation, leading to suppression of RCP-induced OSCC invasion, demonstrating the important role of RCP in OSCC invasion and its reversion by REV. Collectively, the present study provides evidence for the first time that RCP aggravates OSCC invasion through increasing Zeb1 expression and subsequently upregulating MT1-MMP expression and that this process is reversed by REV, providing novel biomarkers and indicating the therapeutic potential of REV in OSCC., Oral cancer: Stopping the spread Resveratrol, a naturally occurring compound present in grape skins, peanuts, and blueberries, reduces the ability of oral squamous cell carcinoma (OSCC) cells to spread to other parts of the body. Over half a million new cases of OSCC are diagnosed each year, and spread beyond the mouth is the most common cause of death. Hoi Young Lee at Konyang University, Daejon, Republic of Korea, and co-workers investigated whether a protein called RCP, which increases the invasiveness of many but not all types of cancer, is implicated in aggressive spread of OSCC. They found that RCP does increase the invasiveness of OSCC. Resveratrol is known to suppress the spread of many cancers, and strongly curtailed the spread of OSCC by blocking RCP activity. These results shed light on OSCC invasiveness, and offer a potential new treatment.

Oxidative stress is one of the principal causes of hypoxia-induced kidney injury. The ceria nanoparticle (CNP) is known to exhibit free radical scavenger and catalytic activities. When zirconia is attached to CNPs (CZNPs), the ceria atom tends to remain in a Ce3+ form and its efficacy as a free radical scavenger thus increases. We determined the effectiveness of CNP and CZNP antioxidant activities against hypoxia-induced acute kidney injury (AKI) and observed that these nanoparticles suppress the apoptosis of hypoxic HK-2 cells by restoring autophagy flux and alleviating mitochondrial damage. In vivo experiments revealed that CZNPs effectively attenuate hypoxia-induced AKI by preserving renal structures and glomerulus function. These nanoparticles can successfully diffuse into HK-2 cells and effectively counteract reactive oxygen species (ROS) to block hypoxia-induced AKI. This suggests that these particles represent a novel approach to controlling this condition.

Rab coupling protein (RCP) has been known to induce cancer invasion and metastasis, and STAT3 is one of major oncogenic factors. In the present study, we identify the critical role of STAT3 in RCP-induced cancer cell invasion. Immunohistochemical data of ovarian cancer tissues presented that levels of RCP expression are closely correlated with those of phospho-STAT3 (p-STAT3). In addition, ovarian cancer patients with high expression of both RCP and p-STAT3 had significantly lower progress-free and overall survival rates compared to those with low either RCP or p-STAT3 expression. Mechanistically, RCP induced STAT3 phosphorylation in both ovarian and breast cancer cells. Silencing or pharmacological inhibition of STAT3 significantly inhibited RCP-induced cancer cell invasion. In addition, we provide evidence that the β1 integrin/EGFR axis is important for RCP-induced STAT3 phosphorylation. Furthermore, STAT3 activated NF-κB for Slug expression that in turn upregulated MT1-MMP expression for cancer cell invasion. Collectively, our present data demonstrate that STAT3 is located downstream of the β1 integrin/EGFR axis and induces Slug and MT1-MMP expression for cancer cell invasion.

Lysophosphatidic acid (LPA) has been known to induce epithelial-mesenchymal transition (EMT) to stimulate cancer cell invasion, and resveratrol (3,5,4'-trans-trihydroxystilbene; REV) suppresses the invasion and metastasis of various cancers. The current study aimed to identify the underlying mechanism by which LPA aggravates breast cancer cell invasion and the reversal of this phenomenon. Immunoblotting and quantitative RT-PCR analysis revealed that LPA induces amphiregulin (AREG) expression. Silencing of Y-box binding protein 1 (YB-1) or enhancer of zeste homolog 2 (EZH2) expression efficiently inhibited LPA-induced AREG expression. In addition, transfection of the cells with YB-1 siRNA abrogated LPA-induced EZH2 and AREG expression, leading to attenuation of breast cancer cell invasion. Furthermore, we observed that both REV and 5-fluorouracil (5-Fu) significantly reduce LPA-induced YB-1 phosphorylation and subsequent breast cancer invasion. Importantly, combined treatment of REV with 5-Fu showed more significant inhibition of LPA-induced breast cancer invasion compared to single treatment. Therefore, our data demonstrate that the YB-1/EZH2 signaling axis mediates LPA-induced AREG expression and breast cancer cell invasion and its inhibition by REV and 5-Fu, providing potential therapeutic targets and inhibition of breast cancer.

Lysophosphatidic acid (LPA), a bioactive lipid produced extracellularly by autotaxin (ATX), has been known to induce various pathophysiological events, including cancer cell invasion and metastasis. Discoidin domain receptor 2 (DDR2) expression is upregulated in ovarian cancer tissues, and is closely associated with poor clinical outcomes in ovarian cancer patients. In the present study, we determined a critical role and signaling cascade for the expression of DDR2 in LPA-induced ovarian cancer cell invasion. We also found ectopic expression of ATX or stimulation of ovarian cancer cells with LPA-induced DDR2 expression. However, the silencing of DDR2 expression significantly inhibited ATX- and LPA-induced ovarian cancer cell invasion. In addition, treatment of the cells with pharmacological inhibitors of phosphoinositide 3-kinase (PI3K), Akt, and mTOR abrogated LPA-induced DDR2 expression. Moreover, we observed that HIF-1α, located downstream of the mTOR, is implicated in LPA-induced DDR2 expression and ovarian cancer cell invasion. Finally, we provide evidence that LPA-induced HIF-1α expression mediates Twist1 expression to upregulate DDR2 expression. Collectively, the present study demonstrates that ATX, and thereby LPA, induces DDR2 expression through the activation of the PI3K/Akt/mTOR/HIF-1α/Twist1 signaling axes, aggravating ovarian cancer cell invasion.

Rab coupling protein (RCP) aggravates cancer cell metastasis and has been implicated in various cancer patient outcomes. Recently, we showed that RCP induces Slug expression and cancer cell invasion by stabilizing the β1 integrin protein. In the present study, we demonstrated that FAK is implicated in RCP-induced EGFR phosphorylation and ovarian cancer cell invasion with inhibition by curcumin. Ectopic expression of RCP induced FAK phosphorylation, which links β1 integrin with EGFR and participates in a positive regulation loop with EGFR. Interestingly, we observed for the first time that curcumin attenuates RCP-induced ovarian cancer cell invasion by blocking stabilization of β1 integrin and consequently inhibiting FAK and EGFR activation, providing potential biomarkers for ovarian cancer and therapeutic approaches for this deadly disease., Ovarian cancer: Natural compound suppresses cancer cell invasiveness Rab coupling protein (RCP)-induced tumor cell migration has been implicated in tumor pathophysiology and patient outcomes. Hoi Young Lee and colleagues at Konyang University in Daejeon, South Korea, have previously shown that RCP promotes ovarian cancer cell invasiveness by stabilizing cell adhesion receptors. In their latest study they find that RCP also increases the levels of two of its protein-binding partners and activates an important mediator of growth factor signaling, Focal Adhesion Kinase (FAK). Interestingly, treating ovarian cancer cells with curcumin, a natural compound extracted from the spice turmeric, not only blocked the effects of RCP on cell adhesion and FAK activation, it also potentiated the inhibitory effects of the chemotherapeutic agent doxorubicin on cell invasiveness. Further research will determine whether curcumin could be used to halt ovarian cancer progression.

Background Colistin (polymyxin E) is an important constituent of the polymyxin class of cationic polypeptide antibiotics. Intrarenal oxidative stress can contribute to colistin-induced nephrotoxicity. Nicotinamide adenine dinucleotide 3-phosphate oxidases (Noxs) are important sources of reactive oxygen species. Among the various types of Noxs, Nox4 is predominantly expressed in the kidney. Objectives We investigated the role of Nox4 and benefit of Nox4 inhibition in colistin-induced acute kidney injury using in vivo and in vitro models. Methods Human proximal tubular epithelial (HK-2) cells were treated with colistin with or without NOX4 knockdown, or GKT137831 (most specific Nox1/4 inhibitor). Effects of Nox4 inhibition on colistin-induced acute kidney injury model in Sprague-Dawley rats were examined. Results Nox4 expression in HK-2 cells significantly increased following colistin exposure. SB4315432 (transforming growth factor-β1 receptor I inhibitor) significantly inhibited Nox4 expression in HK-2 cells. Knockdown of NOX4 transcription reduced reactive oxygen species production, lowered the levels of pro-inflammatory markers (notably mitogen-activated protein kinases) implicated in colistin-induced nephrotoxicity and attenuated apoptosis by altering Bax and caspase 3/7 activity. Pretreatment with GKT137831 replicated these effects mediated by downregulation of mitogen-activated protein kinase activities. In a rat colistin-induced acute kidney injury model, administration of GKT137831 resulted in attenuated colistin-induced acute kidney injury as indicated by attenuated impairment of glomerulus function, preserved renal structures, reduced expression of 8-hydroxyguanosine and fewer apoptotic cells. Conclusions Collectively, these findings identify Nox4 as a key source of reactive oxygen species responsible for kidney injury in colistin-induced nephrotoxicity and highlight a novel potential way to treat drug-related nephrotoxicity.

Rab coupling protein (RCP)-induced tumor cell migration has been implicated in tumor pathophysiology and patient outcomes. In the present study, we demonstrate that RCP stabilizes β1 integrin leading to increased β1 integrin levels and activation of a signaling cascade culminating in Slug induction, epithelial-to-mesenchymal transition and increased invasion. Ectopic expression of RCP induced Slug expression. Silencing β1 integrin efficiently inhibited RCP-induced Slug expression and subsequent cancer cell invasion. Conversely, ectopic expression of β1 integrin was sufficient to induce Slug expression. Pharmacological inhibition of integrin linked kinase (ILK), EGFR and NF-κB, as well as transfection of a dominant-negative mutant of Ras (RasN17), significantly inhibited RCP-induced Slug expression and cancer cell invasion. Strikingly, ectopic expression of RCP was sufficient to enhance metastasis of ovarian cancer cells to the lung. Collectively, we demonstrate a mechanism by which RCP promotes cancer cell aggressiveness through sequential β1 integrin stabilization, activation of an ILK/EGFR/Ras/NF-κB signaling cascade and subsequent Slug expression.

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The small GTP-binding protein Rab25 is associated with tumor formation and progression. However, recent studies have shown discordant effects of Rab25 on cancer cell progression depending on cell lineage. In the present study, we elucidate the underlying mechanisms by which Rab25 induces cellular invasion. We demonstrate that Rab25 increases β1 integrin levels and subsequent activation of EGFR and upregulation of VEGF-A expression, leading to increased Snail expression, epithelial-to-mesenchymal transition and cancer cell invasiveness. Strikingly, we identify that Snail mediates Rab25-induced cancer cell invasiveness through fascin expression and that ectopic expression of Rab25 aggravates metastasis of ovarian cancer cells to the lung. We thus demonstrate a novel role of a β1 integrin/EGFR/VEGF-A/Snail signaling cascade in Rab25-induced cancer cell aggressiveness through induction of fascin expression, thus providing novel biomarkers and potential therapeutic targets for Rab25-expressing cancer cells.

MicroRNAs (miRNAs) are recognized as crucial posttranscriptional regulators of gene expression, and play critical roles as oncogenes or tumor suppressors in various cancers. Here, we show that miR-196b is upregulated in mesenchymal-like-state non-small cell lung cancer (NSCLC) cells and lung cancer tissues. Moreover, miR-196b upregulation stimulates cell invasion and a change in cell morphology to a spindle shape via loss of cell-to-cell contacts. We identified homeobox A9 (HOXA9) as a target gene of miR-196b by using public databases such as TargetScan, miRDB, and microRNA.org. HOXA9 expression is inversely correlated with miR-196b levels in clinical NSCLC samples as compared to that in corresponding control samples, and with the migration and invasion of NSCLC cells. Ectopic expression of HOXA9 resulted in a suppression of miR-196b-induced cell invasion, and HOXA9 reexpression increased E-cadherin expression. Furthermore, HOXA9 potently attenuated the expression of snail family zinc finger 2 (SNAI2/SLUG) and matrix metallopeptidase 9 (MMP9) by controlling the binding of nuclear factor-kappa B to the promoter of SLUG and MMP9 genes, respectively. Therefore, we suggest that HOXA9 plays a central role in controlling the aggressive behavior of lung cancer cells and that miR-196b can serve as a potential target for developing anticancer agents. © 2015 Wiley Periodicals, Inc.

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Background: Rab coupling protein (RCP), also called family interacting protein 1 (Rab11fip1) is a protein encoded by the RAB11FIP1 gene and has been known to augment cancer tumorigenesis, invasion, and metastasis. Recently, RCP was reported to aggravate cancer cell invasion and metastasis by stabilizing beta1 integrin and inducing epithelial-mesenchymal transition (EMT) in breast and ovarian cancer. Further, a high RCP expression was significantly correlated with shorter overall survival and disease-free survival of HNSCC patients. However, the molecule mechanisms by which RCP increases HNSCC invasion and metastasis are unclear. In the present study, we demonstrate the role of RCP in oral cancer cell invasiveness and its underlying mechanism. Methods: Oral cancer YD-10B and YD-38 cells were transiently transfected with RCP constructs. siRNAs were used to analyze the role of specific proteins for Slug and Zeb1 expression. Immunoblotting and immunofluorescence were used to analyze protein expression. Matrigel-coated in vitro insert and wound healing assay were used to analyze cancer cell invasion and migration, respectively. Results: We observed that ectopic expression of RCP induces invasion of oral cancer cells. Conversely, silencing of RCP expression increased E-cadherin expression and reduced oral cancer cell invasion. In addition, RCP upregulated Zeb1 and Slug expression that is important for RCP-induced oral cancer cell EMT and invasion. Furthermore, we identified that silencing of beta1 integrin efficiently attenuates RCP-induced Zeb1 and Slug expression. In addition, we observed that a selective inhibitor of EGFR kinase activity, Gefitinib, significantly reduces RCP-induced Zeb1 and Slug expression and that RasN17 recovers RCP-induced Zeb1 expression. Finally, RCP increased the viability of oral cancer cells against a chemotherapeutic agent cisplatin. Conclusion: Collectively, we demonstrate that RCP augments oral cancer cell EMT and invasiveness through a beta1 integrin/EGFR/Ras/Slug and Zeb1 signaling axis, providing potential biomarkers for oral cancer patients therapy. Citation Format: Jin Young Kim, Kyung Hwa Cho, Bo Young Jeong, Zi Hae Song, Chang Gyo Park, Hoi Young Lee. RCP augments oral cancer cell invasion through Zeb1 and Slug expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2860.

BACKGROUND Epidermal growth factor (EGF) has been known to induce epithelial–mesenchymal transition (EMT) and prostate cancer cell progression. However, a detailed underlying mechanism by which EGF induces EMT and prostate cancer cell progression remained to be answered. Hypoxia-inducible factor (HIF)-1α and TWIST1 are transcription factors implicated in EMT and cancer metastasis. The purpose of this study is to determine the underlying mechanism of EGF-induced TWIST1 expression and prostate cancer invasion. METHODS siRNAs were used to silence genes. Immunoblotting, quantitative RT-PCR and immunofluorescence analysis were used to examine protein or mRNA expression. Modified Boyden chamber and invasion assay kit with Matrigel-coated inserts were used to determine prostate cancer cell migration and invasion, respectively. RESULTS We observed that EGF induced HIF-1α expression and morphological change of prostate cancer epithelial cells to mesenchymal cells. Silencing HIF-1α expression dramatically reduced EGF-induced TWIST1 expression and prostate cancer cell EMT. Conversely, transfection of the cells with HIF-1α siRNA reversed the reduced E-cadherin expression by EGF. Pretreatment of the cells with pharmacological inhibitors of reactive oxygen species [ROS, N-acetylcysteine (NAC)] and STAT3 (WP1066) but not p38 MAPK (SB203580) significantly reduced EGF-induced HIF-1α mRNA and protein expression. Further, pretreatment of the cells with NAC attenuated EGF-induced STAT3 phosphorylation. In addition, we showed that TWIST1 mediated EGF-induced N-cadherin expression, leading to prostate cancer invasion. CONCLUSIONS We demonstrate a mechanism by which EGF promotes prostate cancer cell progression through a ROS/STAT3/HIF-1α/TWIST1/N-cadherin signaling cascade, providing novel biomarkers and promising therapeutic targets for prostate cancer cell progression. Prostate 74:528–536, 2014. © 2014 Wiley Periodicals, Inc.

Hippo/YAP signaling is implicated in tumorigenesis and progression of various cancers. By inhibiting a plethora signaling cascades, resveratrol has strong anti-tumorigenic and anti-metastatic activity. In the present study, we demonstrate that resveratrol decreases the expression of YAP target genes. In addition, our data showed that resveratrol attenuates breast cancer cell invasion through the activation of Lats1 and consequent inactivation of YAP. Strikingly, we also demonstrate that resveratrol inactivates RhoA, leading to the activation of Lats1 and induction of YAP phosphorylation. Further, resveratrol in combination with other agents that inactivate RhoA or YAP showed more marked suppression of breast cancer cell invasion compared with single treatment. Collectively, these findings indicate the beneficial effects of resveratrol on breast cancer patients by suppressing the RhoA/Lats1/YAP signaling axis and subsequently inhibiting breast cancer cell invasion.

Lysophosphatidic acid (LPA) is a biolipid that has diverse biological activities implicated in ovarian cancer initiation and progression. Previous studies have shown the critical role of the Rho/Rho-associated kinase (ROCK) pathway in LPA-induced ovarian cancer progression. However, detailed underlying mechanism by which the Rho/ROCK pathway induces ovarian cancer cell invasion is still incompletely understood. In the present study, we observed that the Rho/ROCK pathway is implicated in the production of proteolytic enzymes, leading to LPA-induced ovarian cancer cell invasion. LPA induced matrix metalloproteinase (MMP)-9 expression in CAOV-3 and PA-1 cells and urokinase-type plasminogen activator (uPA) expression in SKOV-3 cells. LPA-induced proteolytic enzyme expression was required for the invasion of ovarian cancer cells expressing corresponding enzymes. Pretreatment of cells with a pharmacological inhibitor of Rho/ROCK (Y-27632) or overexpression of a dominant-negative mutant of Rho (Rho N19) profoundly inhibited LPA-induced proteolytic enzyme expression as well as the invasive potential of ovarian cancer cells. In addition, transfection with dominant-negative Ras (Ras N17) significantly inhibited LPA-induced Rho activation as well as MMP-9 and uPA expression. Consistently, Y-27632 reduced LPA-induced nuclear factor (NF)-κB activation that is critical for proteolytic enzyme expression and cellular invasion. Collectively, we demonstrate a mechanism by which LPA promotes ovarian cancer progression through coordinate activation of a Ras/Rho/ROCK/NF-κB signaling pathway and the proteolytic enzyme secretion, providing novel biomarkers and promising therapeutic targets for ovarian cancer cell progression.

Early detection of lung cancer provides the highest potential for saving lives. To date, no routine screening method enabling early detection is available, which is a key factor in the disease’s high mortality rate. Copy number changes and DNA methylation alterations are good indicators of carcinogenesis and cancer prognosis. In this study, we attempted to combine profiles of DNA copy number and methylation patterns in 20 paired cancerous and noncancerous tissue samples from non-small cell lung cancer (NSCLC) patients, and we detected several clinically important genes with genetic and epigenetic relationships. Using array comparative genomic hybridization (aCGH), statistically significant differences were observed across the histological subtypes for gains at 1p31.1, 3q26.1, and 3q26.31–3q29 as well as for losses at 1p21.1, 2q33.3, 2q37.3, 3p12.3, 4q35.2, and 13q34 in squamous cell carcinoma (SQ) patients, and losses at 12q24.33 were measured in adenocarcinoma (AD) patients (p < 0.05). In an analysis of DNA methylation at 1505 autosomal CpG loci that are associated with 807 cancer-related genes, we identified six and nine loci with higher and lower DNA methylation levels, respectively, in tumor tissue compared to non-tumor lung tissues from AD patients. In addition, three loci with higher and seven loci with lower DNA methylation levels were identified in tumor tissue from SQ patients compared to non-tumor lung tissue. Subsequently, we searched for regions exhibiting concomitant hypermethylation and genomic loss in both ADs and SQs. One clone representing 7p15.2 (which includes candidate genes such as HOXA9 and HOXA11) and one target ID representing HOXA9_E252_R were detected. Quantitative real-time PCR identified the potential candidate gene HOXA9 as being down-regulated in the majority of NSCLC patients. Moreover, following HOXA9 over-expression, the invasion of representative cell lines, A549 and HCC95, were significantly inhibited. Taken together, our results show that the combined profiling analysis technique is a useful tool for identifying biomarkers in lung cancer and that HOXA9 might be a potential candidate gene for the pathogenesis and diagnosis of NSCLC patients.

Dieckol, a polyphenol compound extracted from Brown algae, Ecklonia cava, is known to have anti-inflammatory and anti-tumorigenic activities. However, the underlying molecular mechanism by which dieckol regulates cancer cell growth and proliferation remains elusive. Here we report that dieckol extracted from Ecklonia cava inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced human hepatocellular carcinoma SK-Hep1 cell motility, and that dieckol decreased extracellular signal-regulated kinases 1/2 and c-Jun N-terminal kinases (JNKs) activities, but not p38 Mitogen-Activated Protein Kinase (MAPK), in dose-dependent manner. Dieckol reduced TPA-induced activator protein-1 transcriptional activity. TPA-induced matrix metalloproteinase-9 (MMP-9) activity was greatly reduced in SK-Hep1 cells by treatment of dieckol. These findings suggest that dieckol functions as a potent inhibitor for tumor promoter-mediated MAPK signaling pathways leading to Activator Protein 1 (AP-1) and MMP-9 activation thereby regulates cancer cell motility.

Lysophosphatidic acid (LPA), produced extracellularly by autotaxin (ATX), has diverse biological activities implicated in tumor initiation and progression, including increasing cell survival, angiogenesis, invasion and metastasis. ATX, LPA and the matrix metalloproteinase (MMP)-9 have all been implicated in hepatocellular carcinoma (HCC) invasion and metastasis. We, thus sought to determine whether ATX with subsequent LPA production and action, including induction of MMP-9 could provide a unifying mechanism. ATX transcripts and LPA receptor type 1 (LPA1) protein are elevated in HCC compared with normal tissues. Silencing or pharmacological inhibition of LPA1 significantly attenuated LPA-induced MMP-9 expression and HCC cell invasion. Further, reducing MMP-9 activity or expression significantly inhibits LPA-induced HCC cell invasion, demonstrating that MMP-9 is downstream of LPA1. Inhibition of phosphoinositide-3 kinase (PI3K) signaling or dominant-negative mutants of protein kinase Cδ and p38 mitogen-activated protein kinase (MAPK) abrogated LPA-induced MMP-9 expression and subsequent invasion. We thus demonstrate a mechanistic cascade of ATX-producing LPA with LPA activating LPA1 and inducing MMP-9 through coordinate activation of the PI3K and the p38 MPAK signaling cascades, providing novel biomarkers and potential therapeutic targets for HCC.

A growing number of studies have demonstrated that physiological factors can influence the progression of several cancers via cellular immune function, angiogenesis and metastasis. Recently, stress-induced catecholamines have been shown to increase the expression of various cancer progressive factors, including vascular endothelial growth factor (VEGF), matrix metalloproteinases and interleukins. However, a detailed mechanism remains to be identified. In this study, we investigated the role of adrenergic receptors and hypoxia-inducible factor (HIF)-1α protein in catecholamine-induced VEGF expression and angiogenesis. Treatment of the cells with norepinephrine (NE) or isoproterenol induced VEGF expression and HIF-1α protein amount in a dose-dependent manner. Induction of VEGF expression by NE was abrogated when the cells were transfected with HIF-1α-specific siRNA. Similarly, adenylate cyclase activator forskolin and cyclic AMP-dependent protein kinase A inhibitor H-89 enhanced and decreased HIF-1α protein amount, respectively. More importantly, conditioned medium of NE-stimulated cancer cells induced angiogenesis in a HIF-1α protein-dependent manner. In addition, pretreatment of cells with propranolol, a β-adrenergic receptor (AR) blocker, completely abolished induction of VEGF expression and HIF-1α protein amount by NE in all of the tested cancer cells. However, treatment with the α1-AR blocker prazosin inhibited NE-induced HIF-1α protein amount and angiogenesis in SK-Hep1 and PC-3 but not MDA-MB-231 cells. Collectively, our results suggest that ARs and HIF-1α protein have critical roles in NE-induced VEGF expression in cancer cells, leading to stimulation of angiogenesis. These findings will help to understand the mechanism of cancer progression by stress-induced catecholamines and design therapeutic strategies for cancer angiogenesis.

Sphingosine kinase (SK) is an enzyme that converts sphingosine to sphingosine 1-phosphate, a lysophospholipid with various cellular functions. Here, we report cloning and characterization of a novel isoform of rat SK1 (rSK1) with an N-terminal extension (long-rSK1). Long-rSK1 is 458 amino acid-long and has a calculated molecular weight of 49.8 kDa. Deduced amino acid sequences of rat and human long-SK1 have high homology (71.3% identity and 78.9% similarity). Long-rSK1 mRNA is widely expressed throughout the tissues including brain, heart, lung, liver, kidney, and spleen, whereas mRNA encoding rSK1 has been shown not to be expressed in heart or liver. When the long-rSK1 was transfected in COS-7 cells, SK activity was 53-fold increased. Substrate specificity and dependency on divalent cations of long-rSK1 were similar to those of rSK1. Taken together, the fact that long-rSK1 with similar enzymatic characteristics to rSK1 displays differential tissue distribution may suggest an additional role of long-rSK1.

Background Cancer metastasis is a multi-step event including epithelial-to-mesenchymal transition (EMT). Breast cancer metastasis suppressor 1 (BRMS1) is a novel metastasis suppressor protein without anti-proliferating activity. However, a detailed underlying mechanism by which BRMS1 attenuates cancer cell EMT and invasion remained to be answered. In the present study, we report an additional mechanism by which BRMS1 attenuates Transforming growth factor-beta1 (TGF-β1)-induced breast cancer cell EMT and invasion. Methods Experimental analysis involving chromosome immunoprecipitation (ChIP) and luciferase reporter assays were used to validate hypoxia inducible factor-1alpha (HIF-1α) as a transcriptional regulator of TWIST1 and Snail. Quantitative RT-PCR was used to analyze transcript expression. Immunoblotting and immunofluorescence were used to analyze protein expression. Matrigel-coated in vitro invasion insert was used to analyze cancer cell invasion. Results BRMS1 strongly inhibited TGF-β1-induced breast cancer cell EMT and invasion. Unexpectedly, we observed that BRMS1 downregulates not only TWIST1 but also Snail expression, thereby inhibiting breast cancer cell invasion. In addition, we provide evidence that HIF-1α is required for Snail and TWIST1 expression. Further, BRMS1 reduced TGF-β1-induced HIF-1α transcript expression through inactivation of nuclear factor kappaB (NF-κB). Conclusion Collectively, the present study demonstrates a mechanical cascade of BRMS1 suppressing cancer cell invasion through downregulating HIF-1α transcript and consequently reducing Snail and TWIST1 expression.

Stress hormone norepinephrine (NE) has been associated with acquisition of cancer progression, and naturally occurring phytoalexin resveratrol (REV) has been known to suppress cancer growth and progression. In the present study, we determine the effect of REV on NE-induced ovarian cancer invasiveness. Pretreatment of REV significantly inhibited NE-induced ovarian cancer cell epithelial-to-mesenchymal transition with concomitant recovery of E-cadherin expression. In addition, our data showed that REV downregulates NE-induced human telomerase reverse transcriptase (hTERT) expression through inhibiting Src phosphorylation and HIF-1α expression. Further, REV reduced NE-induced Slug expression and subsequent ovarian cancer invasion. More importantly, combined treatment of REV with a pharmacological inhibitor of beta adrenergic receptor significantly attenuated NE-induced ovarian cancer invasion compared to single treatment. Therefore, we demonstrate interference of a Src and HIF-1α/hTERT/Slug signaling cascade by REV, providing potential therapeutic targets and inhibition of ovarian cancer.

MicroRNAs (miRNAs) are recognized as crucial posttranscriptional regulators of gene expression, and play critical roles as oncogenes or tumor suppressors in various cancers. Here, we show that miR-196b is upregulated in mesenchymal-like-state non-small cell lung cancer (NSCLC) cells and lung cancer tissues. Moreover, miR-196b upregulation stimulates cell invasion and a change in cell morphology to a spindle shape via loss of cell-to-cell contacts. We identified homeobox A9 (HOXA9) as a target gene of miR-196b by using public databases such as TargetScan, miRDB, and microRNA.org. HOXA9 expression is inversely correlated with miR-196b levels in clinical NSCLC samples as compared to that in corresponding control samples, and with the migration and invasion of NSCLC cells. Ectopic expression of HOXA9 resulted in a suppression of miR-196b-induced cell invasion, and HOXA9 reexpression increased E-cadherin expression. Furthermore, HOXA9 potently attenuated the expression of snail family zinc finger 2 (SNAI2/SLUG) and matrix metallopeptidase 9 (MMP9) by controlling the binding of nuclear factor-kappa B to the promoter of SLUG and MMP9 genes, respectively. Therefore, we suggest that HOXA9 plays a central role in controlling the aggressive behavior of lung cancer cells and that miR-196b can serve as a potential target for developing anticancer agents. © 2015 Wiley Periodicals, Inc.

Lysophosphatidic acid (LPA) mediates diverse biological responses, including cell migration, through the activation of G-protein-coupled receptors. Recently, we have shown that LPA stimulates p21-activated kinase (PAK) that is critical for focal adhesion kinase (FAK) phosphorylation and cell motility. Here, we provide the direct evidence that p85 beta-PIX is required for cell motility of NIH-3T3 cells by LPA through FAK and p38 MAP kinase phosphorylations. LPA induced p85 beta-PIX binding to FAK in NIH-3T3 cells that was inhibited by pretreatment of the cells with phosphoinositide 3-kinase inhibitor, LY294002. Furthermore, the similar inhibition of the complex formation was also observed, when the cells were transfected with either p85 beta-PIX mutant that cannot bind GIT or dominant negative mutants of Rac1 (N17Rac1) and PAK (PAK-PID). Transfection of the cells with specific p85 beta-PIX siRNA led to drastic inhibition of LPA-induced FAK phosphorylation, peripheral redistribution of p85 beta-PIX with FAK and GIT1, and cell motility. p85 beta-PIX was also required for p38 MAP kinase phosphorylation induced by LPA. Finally, dominant negative mutant of Rho (N19Rho)-transfected cells did not affect PAK activation, while the cells stably transfected with p85 beta-PIX siRNA or N17Rac1 showed the reduction of LPA-induced PAK activation. Taken together, the present data suggest that p85 beta-PIX, located downstream of Rac1, is a key regulator for the activations of FAK or p38 MAP kinase and plays a pivotal role in focal complex formation and cell motility induced by LPA.

The small GTP-binding protein Rab25 is associated with tumor formation and progression. However, recent studies have shown discordant effects of Rab25 on cancer cell progression depending on cell lineage. In the present study, we elucidate the underlying mechanisms by which Rab25 induces cellular invasion. We demonstrate that Rab25 increases β1 integrin levels and subsequent activation of EGFR and upregulation of VEGF-A expression, leading to increased Snail expression, epithelial-to-mesenchymal transition and cancer cell invasiveness. Strikingly, we identify that Snail mediates Rab25-induced cancer cell invasiveness through fascin expression and that ectopic expression of Rab25 aggravates metastasis of ovarian cancer cells to the lung. We thus demonstrate a novel role of a β1 integrin/EGFR/VEGF-A/Snail signaling cascade in Rab25-induced cancer cell aggressiveness through induction of fascin expression, thus providing novel biomarkers and potential therapeutic targets for Rab25-expressing cancer cells.

The antimetabolite 5-fluorouracil (5-FU) is one of the more prominent clinical antitumor agents available for the treatment of stomach and colorectal cancers. In the present study, we characterized the effects of 5-FU on nitric oxide (NO) production by cells from the stomach cancer cell line NCI-N87. A cytokine mixture [interleukin (IL)-1beta/interferon (IFN)-gamma] increased the production of NO by stomach cancer cells in a concentration- and time-dependent manner. Pretreatment with 5-FU inhibited the production of NO that was stimulated by the cytokine mixture and reduced the expression of iNOS. The cytokine mixture activated nuclear factor kappaB (NF-kappaB) in a concentration- and time-dependent manner, which was blocked by 5-FU pretreatment. The pretreatment with 5-FU stabilized IkappaBalpha and inactivated IkappaB kinase. Collectively, these data suggest that the efficacy of 5-FU may include the inactivation of IkappaB kinase and the inhibition of NO production.

Doxorubicin (DOX) is an active and broad spectrum chemotherapeutic agent. Increased inducible nitric oxide synthase (NOS) expression and/or activity have been reported in several human tumors. While the relationship between DOX treatment and the enzymatic activity of endothelial NOS has been well characterized, little is known about the effects of DOX on the expression of iNOS in human cancer cells. In the present study, we characterized the effects of DOX on the nitric oxide (NO) production by colorectal cancer cells, DLD-1. IFN-gamma/IL-1beta (CM) increased the production of NO, whereas pretreatment of DOX inhibited the production of NO in response to CM in a dose dependent manner. The increased expressions of iNOS mRNA and protein by CM were completely blocked by DOX without affecting the iNOS mRNA stability. However, DOX activated nuclear factor-kappaB (NF-kappaB) in response to CM. Furthermore, the expression of inhibitor kappaB alpha was reduced by DOX in a dose dependent manner. Collectively, DOX inhibited the production of NO by DLD-1 cells, which is not linked to well known transcription factor, NF-kappaB. Therefore, further studies on the possible mechanisms of inhibitory effects of NO production by DOX would be worth pursuing.

Autotaxin (ATX), originally isolated from human melanoma cells, is a novel metastasis-enhancing motogen and angiogenesis factor. In the present study, we compared the expression level of ATX mRNA between normal and breast cancer tissues and found that the expression of ATX mRNA was closely linked to invasiveness of cancer cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis showed higher cellular ATX mRNA expression in the cancer than normal breast tissues. MDA-MB-435S breast cancer cells, expressing higher amount of ATX mRNA, showed greater relative invasiveness to fibroblast-conditioned medium (FCM) than MCF7, MDA-MB-231, and HBL-100 breast cancer cells. Furthermore, ATX-transfected MCF7 cells showed increased motility and invasiveness than vector-transfected MCF7 cells. Collectively, our results suggest that the expression of ATX is closely linked to the invasiveness of breast cancer cells.

We analyzed vascular responses (endothelial function, oxidant stress) to postprandial hypertriglyceridemia (PHTG) in patients with coronary artery disease (CAD) to reveal potential therapeutical effects of angiotensin converting enzyme inhibition (ACE-I) and of lipid lowering (fibrate). The study population (n=39, mean age: 60 years) consisted of four groups, all of which had angiographically documented CAD. A high fat group (n=9) consumed a high fat meal, a low fat group (n=9) a low fat meal, and ACE-I (n=10) or fibrate (n=11) groups consumed a high fat meal plus lisinopril or fenofibrate. Serum triglycerides (TG) increased significantly 2 h after eating a test meal in all groups with the exception of the low fat group. In the high and low fat groups changes of serum TG were positively correlated (r=0.664, P0.005) with changes of phorbol ester-activated leukocyte superoxide anion radical (O(2-.)) formation and were negatively correlated (r=-0.488, P0.05) with flow-mediated brachial artery dilation (FMD). There was a negative correlation (r=-0.419, P=0.094) between FMD and changes of O(2-.) formation in the high and low fat groups. In the ACE-I and fibrate groups, O(2-.) formation decreased 2 h after eating a test meal (from 5.34+/-1.01 to 3.81+/-1.15 nmol/10(6)cells per min, P0.01, and from 4.66+/-0.91 to 4.26+/-0.97 nmol/10(6)cells per min, P=0.374, respectively). However, endothelial function did not show any significant changes 2 h after eating a test meal in all groups. PHTG increases oxidant stress and further deteriorates endothelial function, even in patients with CAD. Both ACE-I and fibrates have an antioxidant effect but no acute beneficial effects in terms of endothelial function under conditions of PHTG in CAD patients.

Epidermal growth factor (EGF) has been known to induce epithelial-mesenchymal transition (EMT) and prostate cancer cell progression. However, a detailed underlying mechanism by which EGF induces EMT and prostate cancer cell progression remained to be answered. Hypoxia-inducible factor (HIF)-1α and TWIST1 are transcription factors implicated in EMT and cancer metastasis. The purpose of this study is to determine the underlying mechanism of EGF-induced TWIST1 expression and prostate cancer invasion.siRNAs were used to silence genes. Immunoblotting, quantitative RT-PCR and immunofluorescence analysis were used to examine protein or mRNA expression. Modified Boyden chamber and invasion assay kit with Matrigel-coated inserts were used to determine prostate cancer cell migration and invasion, respectively.We observed that EGF induced HIF-1α expression and morphological change of prostate cancer epithelial cells to mesenchymal cells. Silencing HIF-1α expression dramatically reduced EGF-induced TWIST1 expression and prostate cancer cell EMT. Conversely, transfection of the cells with HIF-1α siRNA reversed the reduced E-cadherin expression by EGF. Pretreatment of the cells with pharmacological inhibitors of reactive oxygen species [ROS, N-acetylcysteine (NAC)] and STAT3 (WP1066) but not p38 MAPK (SB203580) significantly reduced EGF-induced HIF-1α mRNA and protein expression. Further, pretreatment of the cells with NAC attenuated EGF-induced STAT3 phosphorylation. In addition, we showed that TWIST1 mediated EGF-induced N-cadherin expression, leading to prostate cancer invasion.We demonstrate a mechanism by which EGF promotes prostate cancer cell progression through a ROS/STAT3/HIF-1α/TWIST1/N-cadherin signaling cascade, providing novel biomarkers and promising therapeutic targets for prostate cancer cell progression.

Histone deacetylases (HDACs) have been shown to play important roles in the regulation of chromatin remodeling by histone deacetylation, and their expression is induced in several types of cancer. In addition, they are known to be associated with resistance to anticancer drugs. However, the relevance of HDAC4 in chemoresistance remains unclear. Therefore, we investigated the interaction between HDAC4 expression and chemoresistance in breast cancer cells. We found that increased HDAC4 expression in MDA-MB-231 cells was associated with resistance to the anticancer drug 5-fluorouracil (5-FU). To verify these results, a cell line stably overexpressing HDAC4 was generated using MCF-7 cells (HDAC4OE). This cell line displayed increased 5-FU resistance, and HDAC4 knockdown in HDAC4OE cells restored 5-FU sensitivity. Consequently, we concluded that HDAC4 is a critical gene associated with 5‑FU chemoresistance. Further investigation using a microarray approach revealed that 355 genes were differentially expressed following HDAC4 overexpression. Based on functional annotation of the array results, HDAC4 overexpression was found to downregulate genes related to the transforming growth factor (TGF) β signaling pathway, including SMAD4, SMAD6, bone morphogenetic protein 6, inhibitor of DNA binding 1 and TGFβ2. We also found that HDAC4 expression regulates SMAD4 expression by inducing deacetylation of histone H3 in the SMAD4 promoter region. In addition, SMAD4 knockdown in MCF‑7 cells increased 5-FU resistance. In summary, our data suggest that HDAC4‑mediated deacetylation of the SMAD4 promoter may lead to 5-FU resistance in breast cancer cells.

PURPOSE Corneal neovascularization (CNV) is associated with Chlamydia trachomatis. The minimal components of bacterial cell walls are recognized by nucleotide-binding oligomerization domain-containing protein (Nod), which is important for host defense--a mechanism manifested in human corneal cells. We aimed to examine whether Nod stimulation is associated with CNV. METHODS Three groups of mice with alkali-induced CNV were topically treated with tripeptide L-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP, a Nod1 agonist), muramyl dipeptide (a Nod2 agonist), or phosphate-buffered saline twice daily for 8 days. The time course responses were quantified using biomicroscopic examinations and immunohistochemistry. Angiogenic factor expression was evaluated by quantitative real-time reverse transcription-polymerase chain reaction. To confirm the involvement of Nod1 signaling in CNV, RICK (an essential molecule in Nod signaling)-knockout mice treated with Tri-DAP were examined biomicroscopically and immunohistochemically 8 days after injury. RESULTS According to the biomicroscopic camera images and histology, Tri-DAP and muramyl dipeptide promoted CNV. Significantly, Tri-DAP increased the number and size of the neovascularized areas. The messenger RNA expression level of vascular endothelial growth factor was elevated in the Tri-DAP-treated mice after alkali injury. Compared with wild-type mice, CNV was attenuated in RICK-deficient mice treated with Tri-DAP. CONCLUSIONS These data suggest that Nod1 stimulation is an important inducer of CNV and that Nod1 might be useful in the development of CNV therapies.

Background: Rab25 is a member of the Rab11 subfamily and exclusively expressed in epithelial cells. As a small GTP-binding protein, Rab25 has been known to mediate recycling of proteins from the endosome to the plasma membrane. Cancer metastasis is multi-step events including epithelial-to-mesenchymal transition (EMT). Various transcription factors mediate EMT through downregulation of E-cadherin. In the present study, we investigate whether Rab25 induces EMT transcription factors, thereby increasing cancer invasiveness. Methods: Immunoblotting, quantitative RT-PCR and immunofluorescence assay were used to examine the expression of Rab25 and EMT factors. siRNAs of Rab25 and Snail were transfected to determine their role in Rab25-induced cancer cell EMT and invasiveness. Transwell invasion assay were used to determine cancer cell invasiveness. Results: Ectopic expression of Rab25 marked reduced E-cadherin expression, while Snail expression was upregulated. In addition, Rab25 significantly aggravated cancer cell invasiveness. However, silencing Snail expression by utilizing specific siRNA significantly attenuated cancer cell invasion. In addition, Rab25 induced phosphorylation of Akt and GSK-3beta. However, pharmacological inhibition of Akt and GSK-3beta marked attenuated Rab25-induced Snail expression. Further, we observed that a beta1integrin located upstream of an Akt/GSK-3beta signaling is important for Rab25-induced Snail expression and cancer cell invasion. Conclusion: Our results show for the first time that Snail mediates Rab25-induced cancer cell EMT and invasiveness through a beta1 integrin /Akt/GSK-3beta signaling cascade, providing novel biomarkers, and potential therapeutic targets for deadly cancers. Citation Format: Bo Young Jeong, Kyung Hwa Cho, Seung Hwa Kim, Yu Na Kim, Chang Gyo Park, Hoi Young Lee. Rab25 aggravates cancer cell invasion through Snail expression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B36.

TGF-β1 induces epithelial-mesenchymal transition (EMT) to stimulate cancer cell progression, and TWIST1 is a critical regulator of EMT. In the present study, we determined the underlying mechanisms of TGF-β1-induced TWIST1 expression and its effect on prostate cancer cell invasion. TGF-β1 stimulated STAT3 phosphorylation and HIF-1α expression. Silencing either STAT3 or HIF-1α efficiently attenuated TGF-β1-induced TWIST1 expression. Further ectopic expression of a dominant negative mutant of STAT3 reduced TGF-β1-induced TWIST1 expression. In addition, STAT3 and HIF-1α up-regulated TWIST1 expression by direct binding to a TWIST1 promoter. Strikingly, STAT3 also enhanced TGF-β1-induced TWIST1 expression through HIF-1α stabilization. Collectively, we demonstrate a mechanistic cascade of TGF-β1 up-regulating STAT3 activation and HIF-1α stabilization and subsequent TWIST1 expression, leading to prostate cancer invasion.

Lysophosphatidic acid (LPA) augments proliferation and metastasis of various cancer cells. We recently identified a critical role of the Rho/ROCK pathway for LPA-induced proteolytic enzyme expression and cancer cell progression. In the present study, we elucidate the underlying mechanisms by which LPA induces Rho activation and subsequent cellular invasion, and the reversal of these effects by resveratrol. We observed that both Gi and G13 contribute to LPA-induced EGFR activation. The activated EGFR in turn initiates a Ras/Rho/ROCK signaling cascade, leading to proteolytic enzyme secretion. Further we provide evidence that resveratrol inhibits EGFR phosphorylation and subsequent activation of a Ras/Rho/ROCK signaling. Therefore, we demonstrate a mechanistic cascade of LPA activating EGFR through Gi and G13 thus inducing a Ras/Rho/ROCK signaling for proteolytic enzyme expression and ovarian cancer cell invasion, as well as interference of the cascade by resveratrol through blocking EGFR phosphorylation.

Histone deacetylases (HDACs) are associated with the development and progression of cancer, but it is not known which of the HDAC isoforms play important roles in breast cancer metastasis. This study identified the specific HDAC isoforms that are necessary for invasion and/or migration in human breast cancer cell lines. MDA-MB-231 cells were significantly more invasive and expressed higher levels of matrix metalloproteinase-9 (MMP-9) compared to MCF-7 cells. We compared the expression of HDAC isoforms between MCF-7 and MDA-MB-231 cells and found greater expression of HDAC4, 6 and 8 in MDA-MB-231 cells by RT-PCR and Western blot analyses. In addition, apicidin, a histone deacetylase inhibitor, was shown to attenuate the invasion, migration and MMP-9 expression in MDA-MB-231 cells. Using specific siRNAs directed against HDAC1, 4, 6 and 8, we show that inhibition of HDAC1, 6 and 8, but not HDAC4, are responsible for invasion and MMP-9 expression in MDA-MB-231 cells. We analyzed the invasiveness of MCF-7 cells overexpressing HDAC1, 4, 6 or 8 and found that overexpression of HDAC1, 6 or 8 increased invasion and MMP-9 expression. By developing HDAC isoforms as potential biomarkers for breast cancer metastasis, the present study can be extended to developing therapies for breast cancer invasion.

Lung cancer is the most common cause of cancer death worldwide. Smoking is known as the strongest single factor in the development of lung cancer. However, there are inherited genetic factors that cause different responses to cigarette smoking exposure among individuals. We tried to identify these differences in heavy smokers by examining copy number variations (CNVs) between lung cancer patients and healthy controls. Analysis by array comparative genomic hybridization which was tested with 20-person training set (10 lung cancer patients, 10 healthy controls) showed 26 significant (adjusted P < 0.05) clones with either copy number gains or losses. Three genes, KCTD11, FGF11, and PTPRH on chromosomal regions 17p13.1 (KCTD11 and FGF11) and 19q13.42 (PTPRH), were selected (adjusted P < 0.001) and tested by real-time quantitative PCR with 34 healthy controls and 54 lung cancer patients. KCTD11 on the chromosomal region 17p13.1 showed significant high odds ratio (OR = 16.0) in heavy smokers, implying that this is a susceptibility region for lung cancer in this group. Therefore, CNVs of 17p13.1 is a promising candidate to identify individuals with a high genetic risk for the development of lung cancer.