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1.  Combined Histone Deacetylase and Cyclooxygenase Inhibition Achieves Enhanced Antiangiogenic Effects in Lung Cancer Cells 
Molecular carcinogenesis  2011;52(3):218-228.
PGE2 is an important pro-angiogenic and pro-proliferative cytokine and the key enzymes modulating its levels, COX-2 and 15-PGDH play important opposing roles in carcinogenesis. Previously we found loss of 15-PGDH expression in lung cancer and its reactivation leads to strong in vivo tumor-suppressive effect via an antiangiogenic mechanism. Here we find that HDAC inhibitors (HDACI), such as trichostatin A (TSA) and vorinostat could reactivate 15-PGDH expression but overall induce PGE2 generation and this is the result of concomitant induction of COX-1 and 2 leading to functional promotion of endothelial cell proliferation and capillary formation. Direct TSA treatment inhibits endothelial cell proliferation and capillary formation in our study in line with prior reports as HDACIs have been shown to directly inhibit angiogenesis. The elevation of PGE2 levels induced by HDACI is potently neutralized by indomethacin (INN) or celecoxib co-treatment and accordingly, angiogenesis is more effectively inhibited when using conditioned medium of co-treatment than either alone confirming that this effect is mediated via the PGE2 axis. Accordingly, blockage of EP2/4 receptors mitigates the stimulation of angiogenesis by excessive PGE2 generation mediated by TSA. In this study, we identify a potentially adverse effect of HDACIs through induction of both 15-PGDH and COX-2 leading to elevated PGE2 levels and thereby stimulation of angiogenesis. Co-treatment of TSA and INN shows more potent anti-angiogenic effects by inducing 15-PGDH and inhibiting COX-2. Overall, our results suggest that combined HDACI and COX inhibition should be explored clinically to achieve more meaningful benefits from HDACI therapy in lung cancer.
doi:10.1002/mc.21846
PMCID: PMC3980866  PMID: 22121107
2.  Tumor-Associated Mutations in O6-Methylguanine DNA-Methyltransferase (MGMT) Reduce DNA Repair Functionality 
Molecular carcinogenesis  2012;53(3):201-210.
MGMT is the primary vehicle for cellular removal of alkyl lesions from the O-6 position of guanine and the O-4 position of thymine. While key to the maintenance of genomic integrity, MGMT also removes damage induced by alkylating chemotherapies, inhibiting the efficacy of cancer treatment. Germline variants of human MGMT are well-characterized, but somatic variants found in tumors were, prior to this work, uncharacterized. We found that MGMT G132R, from a human esophageal tumor, and MGMT G156C, from a human colorectal cancer cell line, are unable to rescue methyltransferase-deficient Escherichia coli as well as wild type (WT) human MGMT after treatment with a methylating agent. Using pre-steady state kinetics, we biochemically characterized these variants as having a reduced rate constant. G132R binds DNA containing an O6-methylguanine lesion half as tightly as WT MGMT, while G156C has a 40-fold decrease in binding affinity for the same damaged DNA versus WT. Mammalian cells expressing either G132R or G156C are more sensitive to methylating agents than mammalian cells expressing WT MGMT. G132R is slightly resistant to O6-benzylguanine, an inhibitor of MGMT in clinical trials, while G156C is almost completely resistant to this inhibitor. The impared functionality of expressed variants G132R and G156C suggests that the presence of somatic variants of MGMT in a tumor could impact chemotherapeutic outcomes.
doi:10.1002/mc.21964
PMCID: PMC3720794  PMID: 23065697
O(6)-benzyguanine; O(6)-alkylguanine DNA alkyltransferase; MNNG; DNA repair; O-(6)-methylguanine
3.  Polycomb group protein EZH2-mediated E-cadherin repression promotes metastasis of oral tongue squamous cell carcinoma 
Molecular carcinogenesis  2011;52(3):229-236.
Enhancer of zeste homolog 2 (EZH2) is a critical component of the polycomb-repressive complex 2 (PRC2) that regulates many essential biological processes, including embryogenesis and many developmental events. The oncogenic role of EZH2 has recently been implicated in several cancer types. In this study, we first confirmed that the over-expression of EZH2 is a frequent event in oral tongue squamous cell carcinoma (OTSCC). We further demonstrated that EZH2 over-expression is correlated with advanced stages of the disease and is associated with lymph node metastasis. Statistical analysis revealed that EZH2 over-expression was correlated with reduced overall survival. Furthermore, over-expression of EZH2 was correlated with reduced expression of tumor suppressor gene E-cadherin. These observations were confirmed in vitro, in which knockdown of EZH2 induced E-cadherin expression and reduced cell migration and invasion. In contrast, ectopic transfection of EZH2 led to reduced E-cadherin expression and enhanced cell migration and invasion. Furthermore, EZH2 may act on cell migration in part by suppressing the E-cadherin expression. Taken together, these data suggest that EZH2 plays major roles in the progression of OTSCC, and may serve as a biomarker or therapeutic target for patients at risk of metastasis.
doi:10.1002/mc.21848
PMCID: PMC3400713  PMID: 22161744
EZH2; E-cadherin; metastasis; prognosis; squamous cell carcinoma
4.  Effect of Silibinin in Human Colorectal Cancer Cells: Targeting the Activation of NF-κB Signaling 
Molecular carcinogenesis  2011;52(3):195-206.
Chronic inflammation is one of the primary causes of colorectal cancer (CRC), and major inflammatory pathways implicated in CRC are COX2 and iNOS; both regulated by NF-κB suggesting that inhibitors of these pathways could be ideal against CRC. Silibinin has shown promising efficacy against various malignancies including CRC, and therefore here we assessed whether silibinin targets NF-κB activation and associated signaling as a mechanism of its anti-inflammatory and anti-cancer effects in CRC. Our results indicated that silibinin treatment (50–200 μM) of human CRC SW40, LoVo and HT29 cells strongly inhibits TNFα-induced NF-κB activation together with decreased nuclear levels of both p65 and p50 sub-units. Silibinin also significantly increased IκBα level with a concomitant decrease in phospho-IκBα, without any effect on TNFR1, TRADD and RIP2, indicating its inhibitory effect on IKKα kinase activity. Next we assessed the effect of oral silibinin feeding on NF-κB pathway in SW480 (COX-2 negative) and LoVo (COX-2 positive) tumor xenografts in nude mice. Together with its inhibitory efficacy on tumor growth and progression, silibinin inhibited NF-κB activation in both xenografts. The protein levels of various NF-κB-regulated molecules such as Bcl-2, COX2, iNOS, VEGF and MMPs were also decreased by silibinin in both cell culture studies and xenograft analyses, suggesting its potential to alter NF-κB transcriptional activity. Together, these findings are highly significant in establishing for the first time that silibinin suppresses CRC growth and progression possibly through its anti-inflammatory activity by interfering with NF-κB activation and thus has potential against human CRC.
doi:10.1002/mc.21843
PMCID: PMC3563833  PMID: 22086675
NF-κB signaling; colorectal cancer; chemoprevention; silibinin
5.  Isosilybin A Induces Apoptosis in Human Prostate Cancer Cells via Targeting Akt, NF-κB and Androgen Receptor Signaling 
Molecular carcinogenesis  2010;49(10):902-912.
Prostate cancer (PCA) is second most malignancy in American men. Advanced stage PCA cells possess unlimited replication potential as well as resistance to apoptosis. Therefore, targeting survival mechanisms and activating apoptotic machinery in PCA cells using non-toxic phytochemicals is suggested as an attractive strategy against this deadly malignancy. In the present study, we assessed the effect of one such botanical agent, namely isosilybin A, on apoptotic machinery and key members of cell survival signaling [Akt, NF- κB and androgen receptor (AR)] in different PCA cells. Results showed that isosilybin A (90–180 μM) treatment significantly induces apoptotic death by activating both extrinsic (increased level of DR5 and cleaved caspase 8) and intrinsic pathways (caspase 9 and caspase 3 activation) of apoptosis in three different human PCA cell lines namely 22Rv1, LAPC4 and LNCaP. Further, isosilybin A treatment decreased the levels of phospho-Akt (Serine-473), total Akt and the nuclear levels of NF-κB constituents (p50 and p65). Isosilybin A treatment also decreased the AR and PSA level in 22Rv1, LAPC4 and LNCaP cells. Employing pan-caspase inhibitor (Z-VAD.fmk), we confirmed that isosilybin A-mediated decreased AR is independent of caspases activation. Temporal kinetics analysis showed that the primary effect of isosilybin A is on AR, as decrease in AR was evident much earlier (4h) relative to caspase activation and apoptosis induction (12h). Overall, our results demonstrated that isosilybin A activates apoptotic machinery in PCA cells via targeting Akt-NF-κB-AR axis; thereby, indicating a promising role for this phytochemical in the management of clinical PCA.
doi:10.1002/mc.20670
PMCID: PMC3924888  PMID: 20721970
Prostate cancer; isosilybin A; chemoprevention; apoptosis; androgen receptor
6.  Overexpression of Evi-1 Oncoprotein Represses TGF-β Signaling in Colorectal Cancer 
Molecular carcinogenesis  2011;52(4):255-264.
Human colorectal cancer (CRC) cells are resistant to the anti-proliferative effect of transforming growth factor-β (TGF-β), suggesting that disruption of TGF-β signaling plays an important role in colorectal carcinogenesis. Ecotropic virus integration site-1 (Evi-1) oncoprotein represses TGF-β signaling by interacting with Smads, but its role in CRC has not been established. The purpose of this study is to determine whether Evi-1 plays role(s) in CRCs and to characterize Evi-1 transcript(s) in CRCs. Evi-1 was overexpressed in 53% of human CRC samples, 100% of colon adenoma samples, and 100% of human colon cancer cell lines tested. Using 5′ RACE, we cloned a novel Evi-1 transcript (Evi-1e) from a human CRC tissue and found that this novel transcript was expressed at a higher level in CRC tissues than in normal tissues and was the major Evi-1 transcript in CRCs. Transient Evi-1 transfection inhibited TGF-β-induced transcriptional activity and reversed the growth inhibitory effect of TGF-β in MC-26 mouse colon cancer cells. In conclusion, we have identified overexpression of Evi-1 oncoprotein as a novel mechanism by which a subset of human CRCs may escape TGF-β regulation. We have also identified a novel Evi-1 transcript, Evi-1e, as the major Evi-1 transcript expressed in human CRCs.
doi:10.1002/mc.21852
PMCID: PMC3922648  PMID: 22161860
colorectal cancer; ecotropic virus integration site-1; transforming growth factor-β; Smad proteins; rapid amplification of cDNA ends; growth inhibition
7.  Quercetin-3-methyl ether inhibits lapatinib-sensitive and -resistant breast cancer cell growth by inducing G2/M arrest and apoptosis 
Molecular carcinogenesis  2011;52(2):134-143.
Lapatinib, an oral, small-molecule, reversible inhibitor of both EGFR and HER2, is highly active in HER2 positive breast cancer as a single agent and in combination with other therapeutics. However, resistance against lapatinib is an unresolved problem in clinical oncology. Recently, interest in the use of natural compounds to prevent or treat cancers has gained increasing interest because of presumed low toxicity. Quercetin-3-methyl ether, a naturally occurring compound present in various plants, has potent anticancer activity. Here, we found that quercetin-3-methyl ether caused in a significant growth inhibition of lapatinib-sensitive and -resistant breast cancer cells. Western blot data showed that quercetin-3-methyl ether had no effect on Akt or ERKs signaling in resistant cells. However, quercetin-3-methyl ether caused a pronounced G2/M block mainly through the Chk1-Cdc25c-cyclin B1/Cdk1 pathway in lapatinib-sensitive and -resistant cells. In contrast, lapatinib produced an accumulation of cells in the G1 phase mediated through cyclin D1, but only in lapatinib-sensitive cells. Moreover, quercetin-3-methyl ether induced significant apoptosis, accompanied with increased levels of cleaved caspase 3, caspase 7 and poly (ADP-ribose) polymerase (PARP) in both cell lines. Overall, these results suggested that quercetin-3-methyl ether might be a novel and promising therapeutic agent in lapatinib-sensitive or -resistant breast cancer patients.
doi:10.1002/mc.21839
PMCID: PMC3340451  PMID: 22086611
Breast cancer; HER2; lapatinib; natural product; quercetin-3-methyl ether; apoptosis
8.  JAK/STAT/SOCS-signaling pathway and colon and rectal cancer 
Molecular carcinogenesis  2011;52(2):155-166.
The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway is involved in immune function and cell growth. We evaluated the association between genetic variation in JAK1 (10 SNPs), JAK2 (9 SNPs), TYK2 (5 SNPs), SOCS1 (2 SNPs), SOCS2 (2 SNPs), STAT1 (16 SNPs), STAT2 (2 SNPs), STAT3 (6 SNPs), STAT4 (21 SNPs), STAT5A (2 SNPs), STAT5B (3 SNPs), STAT6 (4 SNPs) with risk of colorectal cancer. We used data from population-based case-control studies (colon cancer n=1555 cases, 1956 controls; rectal cancer n=754 cases, 959 controls). JAK2, SOCS2, STAT1, STAT3, STAT5A, STAT5B, and STAT6 were associated with colon cancer; STAT3, STAT4, STAT6, and TYK2 were associated with rectal cancer. Given the biological role of the JAK/STAT-signaling pathway and cytokines, we evaluated interaction with IFNG, TNF, and IL6; numerous statistically significant associations after adjustment for multiple comparisons were observed. The following statistically significant interactions were observed: TYK2 with aspirin/NSAID use; STAT1, STAT4, and TYK2 with estrogen status; and JAK2, STAT2, STAT4, STAT5A, STAT5B, and STAT6 with smoking status and colon cancer risk; JAK2, STAT6, and TYK2 with aspirin/NSAID use; JAK1 with estrogen status; STAT2 with cigarette smoking and rectal cancer. JAK2, SOCS1, STAT3, STAT5, and TYK2 were associated with colon cancer survival (HRR of 3.3 95% CI 2.01, 5.42 for high mutational load). JAK2, SOCS1, STAT1, STAT4, and TYK2 were associated with rectal cancer survival (HRR 2.80 95 %CI 1.63, 4.80). These data support the importance of the JAK/STAT-signaling pathway in colorectal cancer and suggest targets for intervention.
doi:10.1002/mc.21841
PMCID: PMC3430812  PMID: 22121102
JAK; STAT; SOCS; colon cancer; rectal cancer; estrogen; NSAIDs; cigarette smoking
9.  SMAD4-Dependent Polysome RNA Recruitment in Human Pancreatic Cancer Cells 
Molecular carcinogenesis  2011;51(10):10.1002/mc.20845.
Pancreatic cancer is the fourth leading cause of cancer death in the United States because most patients are diagnosed too late in the course of the disease to be treated effectively. Thus, there is a pressing need to more clearly understand how gene expression is regulated in cancer cells and to identify new biomarkers and therapeutic targets. Translational regulation is thought to occur primarily through non-SMAD directed signaling pathways. We tested the hypothesis that SMAD4-dependent signaling does play a role in the regulation of mRNA entry into polysomes and that novel candidate genes in pancreatic cancer could be identified using polysome RNA from the human pancreatic cancer cell line BxPC3 with or without a functional SMAD4 gene. We found that (i) differentially expressed whole cell and cytoplasm RNA levels are both poor predictors of polysome RNA levels; (ii) for a majority of RNAs, differential RNA levels are regulated independently in the nucleus, cytoplasm, and polysomes; (iii) for most of the remaining polysome RNA, levels are regulated via a “tagging” of the RNAs in the nucleus for rapid entry into the polysomes; (iv) a SMAD4-dependent pathway appears to indeed play a role in regulating mRNA entry into polysomes; and (v) a gene list derived from differentially expressed polysome RNA in BxPC3 cells generated new candidate genes and cell pathways potentially related to pancreatic cancer.
doi:10.1002/mc.20845
PMCID: PMC3859243  PMID: 22965423
polysomes; differential gene expression; pancreatic cancer; BxPC3; SMAD4
10.  Transgenic Insulin-like Growth Factor-1 stimulates activation of COX-2 signaling in mammary glands 
Molecular carcinogenesis  2011;51(12):973-983.
Studies show that elevated IGF-1 levels are associated with an increased risk of breast cancer; however, mechanisms through which IGF-1 promotes mammary tumorigenesis in vivo have not been fully elucidated. To assess the possible involvement of COX-2 signaling in the protumorigenic effects of IGF-1 in mammary glands, we used the unique BK5.IGF-1 mouse model in which transgenic (Tg) mice have significantly increased incidence of spontaneous and DMBA–induced mammary cancer compared to wild type (WT) littermates. Studies revealed that COX-2 expression was significantly increased in Tg mammary glands and tumors, compared to age-matched WTs. Consistent with this, PGE2 levels were also increased in Tg mammary glands. Analysis of expression of the EP receptors that mediate the effects of PGE2 showed that among the four G-protein-coupled receptors, EP3 expression was elevated in Tg glands. Up-regulation of the COX-2/PGE2/EP3 pathway was accompanied by increased expression of VEGF and a striking enhancement of angiogenesis in IGF-1 Tg mammary glands. Treatment with celecoxib, a selective COX-2 inhibitor, caused a 45% reduction in mammary PGE2 levels, attenuated the influx of mast cells and reduced vascularization in Tg glands. These findings indicate that the COX-2/PGE2/EP3 signaling pathway is involved in IGF-1–stimulated mammary tumorigenesis and that COX-2–selective inhibitors may be useful in the prevention or treatment of breast cancer associated with elevated IGF-1 levels in humans.
doi:10.1002/mc.20868
PMCID: PMC3790642  PMID: 22006370
mammary cancer; IGF-1; COX-2; stroma; transgenic mouse
11.  AMPK-Mediated Phosphorylation of Murine p27 at T197 Promotes Binding of 14-3-3 Proteins and Increases p27 Stability 
Molecular carcinogenesis  2010;49(5):10.1002/mc.20613.
The Tuberous Sclerosis Complex 2 (TSC2) gene product, tuberin, acts as a negative regulator of mTOR signaling, and loss of tuberin function leads to tumors of the brain, skin, kidney, heart and lungs. Previous studies have shown that loss of tuberin function affects the stability and subcellular localization of the cyclin-dependent kinase inhibitor (CKI) p27, although the mechanism(s) by which tuberin modulates p27 stability have not been elucidated. Previous studies have also shown that AMPK, which functions in an energy-sensing pathway in the cell, becomes activated in the absence of tuberin. Here we show that in Tsc2-null tumors and cell lines, AMPK activation correlates with an increase in p27 levels, and inhibition of AMPK signaling decreases p27 levels in these cells. In addition, activation of AMPK led to phosphorylation of p27 at the conserved terminal threonine residue of murine p27 (T197) in both in vitro kinase assays and in cells. Phosphorylation of p27 at T197 led to increased interaction between p27 and 14-3-3 proteins and increased the protein stability of p27. Furthermore, activation of AMPK signaling promoted the interaction between p27 and 14-3-3 proteins and increased the stability of the p27 protein in a manner that was dependent on T197. These data identify a conserved mechanism for regulation of p27 stability via phosphorylation at the terminal threonine (mT197/hT198), which when AMPK is activated, results in stabilization of the p27 protein.
doi:10.1002/mc.20613
PMCID: PMC3818129  PMID: 20146253
Tuberous sclerosis complex; TSC2; energy sensing
12.  PTBP1-dependent Regulation of USP5 Alternative RNA Splicing Plays a Role in Glioblastoma Tumorigenesis 
Molecular carcinogenesis  2011;51(11):895-906.
Aberrant RNA splicing is thought to play a key role in tumorigenesis. The assessment of its specific contributions is limited by the complexity of information derived from genome-wide array-based approaches. We describe how performing splicing factor-specific comparisons using both tumor and cell line datasets may more readily identify physiologically relevant tumor-specific splicing events. Affymetrix exon array data derived from glioblastoma (GBM) tumor samples with defined PTBP1 levels were compared with data from U251 GBM cells with and without PTBP1 knockdown. This comparison yielded overlapping gene sets that comprised only a minor fraction of each dataset. The identification of a novel GBM-specific splicing event involving the USP5 gene led us to further examine its role in tumorigenesis. In GBM, USP5 generates a shorter isoform 2 through recognition of a 5′ splice site within exon 15. Production of the USP5 isoform 2 was strongly correlated with PTBP1 expression in GBM tumor samples and cell lines. Splicing regulation was consistent with the presence of an intronic PTBP1 binding site and could be modulated through antisense targeting of the isoform 2 splice site to force expression of isoform 1 in GBM cells. The forced expression of USP5 isoform 1 in two GBM cell lines inhibited cell growth and migration, implying an important role for USP5 splicing in gliomagenesis. These results support a role for aberrant RNA splicing in tumorigenesis and suggest that changes in relatively few genes may be sufficient to drive the process.
doi:10.1002/mc.20859
PMCID: PMC3319169  PMID: 21976412
Exon Array; USP5; isopeptidase-T; GBM
13.  Genetic Variation in the PNPLA3 Gene and Hepatocellular Carcinoma in USA: Risk and Prognosis Prediction 
Molecular carcinogenesis  2013;52(0):10.1002/mc.22057.
Nonalcoholic fatty liver disease (NAFLD) is an emerging epidemic with high prevalence in Western countries. Genome-wide association studies had reported that a variation in the patatin-like phospholipase domain containing 3 (PNPLA3) gene is associated with high susceptibility to NAFLD. However, the relationship between this variation and hepatocellular carcinoma (HCC) has not been well established. We investigated the impact of PNPLA3 genetic variation (rs738409: C>G) on HCC risk and prognosis in the United States by conducting a case–control study that included 257 newly diagnosed and pathologically confirmed Caucasian patients with HCC (cases) and 494 healthy controls. Multivariate logistics and Cox regression models were used to control for the confounding effects of HCC risk and prognostic factors. We observed higher risk of HCC for subjects with a homozygous GG genotype than for those with CC or CG genotypes, the adjusted odds ratio (OR) was 3.21 (95% confidence interval [CI], 1.68–6.41). We observed risk modification among individuals with diabetes mellitus (OR = 19.11; 95% CI, 5.13–71.20). The PNPLA3 GG genotype was significantly associated with underlying cirrhosis in HCC patients (OR = 2.48; 95% CI, 1.05–5.87). Moreover, GG allele represents an independent risk factor for death. The adjusted hazard ratio of the GG genotype was 2.11 (95% CI, 1.26–3.52) compared with CC and CG genotypes. PNPLA3 genetic variation (rs738409: C>G) may determine individual susceptibility to HCC development and poor prognosis. Further experimental investigations are necessary for thorough assessment of the hepatocarcinogenic role of PNPLA3.
doi:10.1002/mc.22057
PMCID: PMC3808509  PMID: 23776098
molecular epidemiology; genetic susceptibility; case–control; single nucleotide polymorphism
14.  MicroRNA target site polymorphisms in the VHL-HIF1α pathway predict renal cell carcinoma risk 
Molecular carcinogenesis  2012;10.1002/mc.21917.
Renal cell carcinoma (RCC) accounts for ~4% of all human malignancies and is the 9th leading cause of male cancer death in the United States. The purpose of this study was to determine the effect of variation within microRNA (miRNA) binding sites of genes in the VHL-HIF1α pathway on RCC risk. We identified 429 miRNA binding site single nucleotide polymorphisms (SNPs) in 102 pathway genes and assessed 53 tagging-SNPs for 31 of these genes for risk in a case-control study consisting of 894 RCC cases and 1,516 controls. Results showed that five SNPs were significantly associated with RCC risk. The most significant finding was rs743409 in MAPK1. Under the additive model, the variant was associated with a 10% risk reduction (OR: 0.90, 95% CI, 0.77-0.98). Other significant findings were for SNPs in CDCP1, TFRC, and DEC1. Cumulative effects analysis showed that subjects carrying four or five unfavorable genotypes had a 2.14-fold increase in risk (95% CI, 1.03-4.43, P = 0.04) than those with no unfavorable genotypes. Potential higher-order gene-gene interactions were identified and categorized subjects into different risk groups. The OR of the high-risk group defined by two SNPs: CDCP1:rs6773576 (GG) and DEC1:rs10982724 (GG) was 4.46-times higher than that of low-risk reference group (95% CI, 1.31-15.08). Overall, our study provides the first evidence supporting a connection between miRNA binding site SNPs within the VHL-HIF1α pathway and RCC risk. These novel genetic risk factors might help identify individuals at high risk to enable detection of tumors at an early, curable stage.
doi:10.1002/mc.21917
PMCID: PMC3424388  PMID: 22517515
VHL-HIF1α pathway; microRNA; renal cell carcinoma
15.  Genetic variability in DNA repair and cell cycle control pathway genes and risk of smoking-related lung cancer 
Molecular carcinogenesis  2011;51(Suppl 1):E11-E20.
DNA repair and cell cycle control play an important role in the repair of DNA damage caused by cigarette smoking. Given this role, functionally relevant single nucleotide polymorphisms (SNPs) in genes in these pathways may well affect the risk of smoking-related lung cancer. We examined the relationship between 240 SNPs in DNA repair and cell cycle control pathway genes and lung cancer risk in a case-control study of white current and ex-cigarette smokers (722 cases and 929 controls). Additive, dominant and recessive genetic models were evaluated for each SNP. A genetic risk summary score was also constructed. Odds ratios (OR) for lung cancer risk and 95% confidence intervals (95% CI) were estimated using logistic regression models. Thirty-eight SNPs were associated with lung cancer risk in our study population at P<0.05. The strongest associations were observed for rs2074508 in GTF2H4 (Padditive=0.003), rs10500298 in LIG1 (Precessive=2.7×10−4), rs747658 and rs3219073 in PARP1 (rs747658: Padditive=5.8×10−5; rs3219073: Padditive=4.6×10−5), and rs1799782 and rs3213255 in XRCC1 (rs1799782: Pdominant=0.006; rs3213255: Precessive=0.004). Compared to individuals with first quartile (lowest) risk summary scores, individuals with third and fourth quartile summary score results were at increased risk for lung cancer (OR: 2.21, 95% CI: 1.66–2.95 and OR: 3.44, 95% CI: 2.58–4.59, respectively; Ptrend<0.0001). Our data suggests that variation in DNA repair and cell cycle control pathway genes is associated with smoking-related lung cancer risk. Additionally, combining genotype information for SNPs in these pathways may assist in classifying current and ex-cigarette smokers according to lung cancer risk.
doi:10.1002/mc.20858
PMCID: PMC3289753  PMID: 21976407
SNP; case-control; lung cancer
16.  Genetic variants of a BH3-only pro-apoptotic gene, PUMA, and risk of HPV16-associated squamous cell carcinoma of the head and neck 
Molecular carcinogenesis  2011;51(Suppl 1):E54-E64.
P53 up-regulated modulator of apoptosis (PUMA) is a critical factor in the intrinsic apoptotic pathway. Through PUMA-dependent mechanisms, human papillomavirus 16 (HPV16) oncoprotein may affect apoptosis by E6-mediated p53 degradation. To examine whether the PUMA variants modify the association between HPV16 serology and risk of squamous cell carcinoma of the head and neck (SCCHN), we genotyped two polymorphisms in the PUMA promoter (rs3810294 and rs2032809) in 380 cases and 335 cancer-free controls of non-Hispanic whites, who were frequency-matched by age (± 5 years), sex, smoking and drinking status. We found that each individual polymorphism had only a modest impact on risk of SCCHN, particularly in oropharyngeal cancer for rs3810294 and non-oropharyngeal cancer for rs2032809. After we stratified the individuals by HPV16 serology, and used those with the corresponding common homozygous genotype and HPV16 seronegativity as the reference group, for each polymorphism we found that the risk of SCCHN associated with HPV16 seropositivity was higher among those with variant genotypes than those with the corresponding common homozygous genotype. Notably, this effect modification was particularly pronounced in several subgroups including never smokers, never drinkers, younger patients, and patients with oropharyngeal cancer. Furthermore, we also characterized the functional relevance of the two polymorphisms to explore the genotype-phenotype correlation. Our results suggested that the PUMA promoter polymorphisms may be a biomarker for risk of HPV16-associated SCCHN, particularly in never smokers, never drinkers, younger patients, and patients with oropharyngeal cancer. Larger studies are needed to validate our findings.
doi:10.1002/mc.21838
PMCID: PMC3326219  PMID: 22086558
PUMA polymorphisms; HPV16; genetic susceptibility; molecular epidemiology; squamous cell carcinoma of the head and neck
17.  Combined effects of E2F1 and E2F2 polymorphisms on risk and early onset of squamous cell carcinoma of the head and neck 
Molecular carcinogenesis  2012;51(Suppl 1):E132-E141.
Deregulated expression of most members of the E2F family has been detected in many human cancers. We examined the association of common single nucleotide polymorphisms (SNPs) of E2F1 and E2F2 with risk of squamous cell carcinoma of the head and neck (SCCHN) in 1,096 SCCHN patients and 1,090 cancer-free controls. We genotyped ten selected SNPs in E2F1 and E2F2, including those at the near 5′ UTR, miRNA binding sites at the near 3′ UTR and tagSNPs according to bioinfotmatics analysis. Although none of the selected SNPs alone was significantly associated with risk of SCCHN, there was a statistically significantly increased risk of SCCHN associated with the combined risk genotypes (i.e. rs3213182 AA, rs3213183 GG, rs3213180 GG, rs321318121 GG, rs2742976 GT+TT, rs6667575 GA+AA, rs3218203 CC, rs3218148 AA, rs3218211 CC, rs3218123 GT+TT). Compared with those with 0–4 risk genotypes, an increased risk was observed for those who carried 5–8 risk genotypes (adjusted OR = 1.04; 95% CI = 0.86–1.26) and 9–10 risk genotypes (adjusted OR = 1.62; 95% CI = 1.14–2.30) in a dose-response manner (P = 0.045). Furthermore, the joint effect was more pronounced among patients with oropharyngeal cancer, younger adults (≤57 years old), men, non-smokers, non-drinkers, and individuals with family history of cancer first-degree relatives. Additionally, we also observed that those with 5–10 risk genotypes had an earlier SCCHN onset than those with 0–4 risk genotypes, particularly for non-smokers and/or non-drinkers. We concluded that E2F1 and E2F2 genetic variants may jointly play important roles in head and neck carcinogenesis.
doi:10.1002/mc.21882
PMCID: PMC3370129  PMID: 22344756
E2F1; E2F2; head and neck cancer; polymorphisms; age at onset
18.  GSTM1 and GSTT1 copy numbers and mRNA expression in lung cancer 
Molecular carcinogenesis  2012;51(Suppl 1):E142-E150.
Large fractions of the human population do not express GSTM1 and GSTT1 (GSTM1/T1) enzymes because of deletions in these genes. These variations affect xenobiotic metabolism and have been evaluated in relation to lung cancer risk, mostly based on null/present gene models. We measured GSTM1/T1 heterozygous deletions, not tested in genome-wide association studies, in 2120 controls and 2100 cases from the Environment And Genetics in Lung cancer Etiology (EAGLE) study. We evaluated their effect on mRNA expression on lung tissue and peripheral blood samples and their association with lung cancer risk overall and by histology types. We tested the null/present, dominant and additive models using logistic regression. Cigarette smoking and gender were studied as possible modifiers. Gene expression from blood and lung tissue cells was strongly down-regulated in subjects carrying GSTM1/T1 deletions by both trend and dominant models (p<0.001). In contrast to the null/present model, analyses distinguishing subjects with 0, 1 or 2 GSTM1/T1 deletions revealed several associations. There was a decreased lung cancer risk in never-smokers (OR=0.44;95%CI=0.23–0.82; p=0.01) and women (OR=0.50;95%CI=0.28–0.90; p=0.02) carrying 1 or 2 GSTM1 deletions. Analogously, male smokers had an increased risk (OR=1.13;95%CI=1.0–1.28; p=0.05) and women a decreased risk (OR=0.78;95%CI=0.63–0.97; p=0.02) for increasing GSTT1 deletions. The corresponding gene-smoking and gene-gender interactions were significant (p<0.05). Our results suggest that decreased activity of GSTM1/T1 enzymes elevates lung cancer risk in male smokers, likely due to impaired carcinogens’ detoxification. A protective effect of the same mutations may be operative in never-smokers and women, possibly because of reduced activity of other genotoxic chemicals.
doi:10.1002/mc.21890
PMCID: PMC3376678  PMID: 22392686
GST; copy numbers; gene expression; lung cancer; smoking and gender differences
19.  Functional polymorphisms in the insulin-like binding protein-3 gene may modulate susceptibility to differentiated thyroid carcinoma in Caucasian Americans 
Molecular carcinogenesis  2012;51(Suppl 1):E158-E167.
Purpose
The insulin-like growth factor (IGF) pathway is believed to play a pivotal role in thyroid carcinogenesis. Polymorphisms of IGF-1 and IGF binding protein-3 (IGFBP-3) have been associated with modulation of risk for the emergence of assorted common malignancies, but studies of the influence of such polymorphisms on risk of differentiated thyroid carcinoma (DTC) are lacking.
Methods
In a case-control study of 173 DTC patients, 101 patients with benign thyroid disease, and 401 controls, an unconditional logistical regression model adjusted for age and sex was applied to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for the associations between polymorphisms of IGF-1 and IGFBP-3 and DTC risk.
Results
IGFBP-3 rs2132572 GA/AA genotypes were associated with a decreased risk of DTC (adjusted OR=0.6, 95% CI: 0.4–0.9), particularly multifocal DTC (adjusted OR=0.3, 95% CI: 0.1–0.7). The association with DTC was more evident in subjects with a first-degree family history of cancer (adjusted OR=0.4, 95% CI: 0.2–0.7, Pinteraction=0.013) and nondrinkers (adjusted OR=0.4, 95% CI: 0.2–0.7, Pinteraction=0.028). A 4 SNP haplotype of IGFBP-3 was associated with a decreased risk of DTC (adjusted OR=0.7, 95% CI: 0.5–1.0, P=0.030).
Conclusions
Our study suggests that polymorphic IGFBP-3 may be involved in susceptibility to DTC.
doi:10.1002/mc.21900
PMCID: PMC3473095  PMID: 22415807
IGF-1; multifocal; genetic susceptibility; papillary thyroid carcinoma; case-control study
20.  A Single Nucleotide Polymorphism in Tumor Suppressor Gene SEL1L as a Predictive and Prognostic Marker for Pancreatic Ductal Adenocarcinoma in Caucasians 
Molecular carcinogenesis  2011;51(5):433-438.
SEL1L is a putative tumor suppressor gene that is frequently down-regulated in pancreatic ductal adenocarcinoma (PDA). A single nucleotide polymorphism (SNP) rs12435998 in intron3 of SEL1L has previously been reported to be associated with susceptibility to Alzheimer’s disease. We hypothesized that this SNP may influence clinical outcomes of patients with PDA. We analyzed DNA samples from 497 Caucasian patients with pathologically confirmed primary PDA. Of these, 98 had been enrolled in a clinical trial of neoadjuvant chemo-radiotherapy and 77 of the 98 had subsequently undergone pancreaticoduodenectomy (PD). We performed Kaplan–Meier analysis to evaluate the correlation between different SNP genotypes and age at diagnosis, survival time after diagnosis, and survival time after PD. In nonsmokers, we found a significant difference in median age at diagnosis between variant genotypes (AG/GG) carriers and wild-type genotype (AA) carriers (58 versus 62 years; log-rank test, P=0.017). Patients with variant genotypes also showed an increased hazard ratio (HR) of 1.45 (95% confidence interval [CI], 1.07–1.97) relative to wild-type genotype. Among the patients in the clinical trial, the variant genotypes carriers had a median post-PD survival time that was 34.7 months shorter than wild-type genotype carriers (log-rank test, P=0.019; HR, 1.91; 95% CI, 1.09–3.34). Our results suggest that the rs12435998 SNP in SEL1L gene plays a role in modifying age at diagnosis of PDA in Caucasian nonsmokers. In addition, this SNP may serve as a prognostic marker in PDA patients who undergo the same or similar treatment as the clinical trials.
doi:10.1002/mc.20808
PMCID: PMC3780592  PMID: 21656579
pancreatic cancer; SNP; biologic marker; cigarette smoking; treatment
21.  Imiquimod attenuates the growth of UVB-induced SCC in mice through Th1/Th17 cells 
Molecular Carcinogenesis  2012;10.1002/mc.21901.
Imiquimod (IMQ), a Toll-like receptor (TLR) 7/8 agonist, has been used to treat various skin neoplasms, including genital warts, actinic keratoses and superficial basal cell carcinomas. Although IMQ has been recognized to activate both innate and adaptive immunity, the underlying mechanism(s) by which IMQ exerts its anti-tumor activity in vivo remains largely unknown. In this study, we took advantage of skin cancer-prone mice to characterize the effects of IMQ on ultraviolet irradiation (UV)-induced de novo carcinogenesis. Transgenic mice with keratinocytes expressing constitutively activated Stat3 (K5.Stat3C mice) developed squamous cell carcinomas (SCC in situ) as early as after 14 weeks of UVB irradiation, while wild-type mice required much higher doses of UVB with more than 25 weeks of UVB irradiation to produce SCC. Topical treatment of K5.Stat3C mice with IMQ attenuated UVB-induced epidermal dysplasia (SCC in situ). In addition, SCC growth due to increased total irradiation doses was significantly attenuated by IMQ treatment. Topical IMQ treatment induced T cell and plasmacytoid dendritic cell infiltrates at the tumor sites, where levels of IL-12/23p40, IL-12p35, IL-23p19, IL-17A and IFN-γ mRNAs were up-regulated. Immunohistochemistry revealed T cell infiltrates consisting of T1, Th17 and CD8+ T cells. We speculate that topical IMQ treatment attenuates the de novo growth of UVB-induced SCC through activation of Th17/Th1 cells and cytotoxic T lymphocytes.
doi:10.1002/mc.21901
PMCID: PMC3397281  PMID: 22431065
TLR agonist; UVB-induced skin cancer; Th1/Th17
22.  A keratin 15 containing stem cell population from the hair follicle contributes to squamous papilloma development in the mouse 
Molecular carcinogenesis  2012;10.1002/mc.21896.
The multistage model of non-melanoma skin carcinogenesis has contributed significantly to our understanding of epithelial cancer in general. We used the Krt1-15CrePR1;R26R transgenic mouse to determine the contribution of keratin 15+ cells from the hair follicle to skin tumor development by following the labeled progeny of the keratin 15 expressing cells into papillomas. We present three novel observations. First, we found that keratin 15 expressing cells contribute to most of the papillomas by 20 weeks of promotion. Second, in contrast to the transient behavior of labeled keratin 15-derived progeny in skin wound healing, keratin 15 progeny persist in papillomas and some malignancies for many months following transient induction of the reporter gene. Third, papillomas have surprising heterogeneity not only in their cellular composition, but also in their expression of the codon 61 signature Ha-ras mutation with approximately 30 percent of keratin 15-derived regions expressing the mutation. Together, these results demonstrate that keratin 15 expressing cells of the hair follicle contribute to cutaneous papillomas with long term persistence and a subset of which express the Ha-ras signature mutation characteristic of initiated cells.
doi:10.1002/mc.21896
PMCID: PMC3424369  PMID: 22431489
skin carcinogenesis; stem cells; hair follicles; papillomas
23.  Loss of Trp53 Promotes Medulloblastoma Development but not Skin Tumorigenesis in Sufu Heterozygous Mutant Mice 
Molecular Carcinogenesis  2011;51(9):754-760.
Basal cell carcinoma of the skin typically carries genetic alterations in components of the hedgehog (HH) signaling pathway. Previously, we generated a knockout mouse with a loss-of-function mutation in suppressor of fused (Sufu), an essential repressor of the pathway downstream of Hh ligand cell surface reception. Mice heterozygous for the mutated Sufu allele develop a skin phenotype that includes lesions similar to basaloid follicular hamartomas. The purpose of the current study was to test the possibility that the simultaneous loss of the tumor suppressor gene, Trp53, would aggravate the Sufu skin phenotype since Trp53 loss is known to enhance the growth of other Hh-driven tumors. Consistent with previous reports, medulloblastomas and rhabdomyosarcomas developed in Sufu+/−;Trp53−/− mice. However, the characteristic Sufu+/− skin phenotype was not altered in the absence of Trp53, and showed no changes in latency, multiplicity, cellular phenotype or proliferative capacity of the basaloid lesions. This finding was both novel and intriguing and demonstrated a differential, tissue-specific sensitivity to Sufu and Trp53 tumor suppressor gene loss, which may be linked to developmental stage and the degree of proliferative activity in specific cell types.
doi:10.1002/mc.20852
PMCID: PMC3235237  PMID: 21882258
Hedgehog; suppressor of fused; basal cell carcinoma; p53
24.  Inhibition of Constitutively Activated Phosphoinositide 3-Kinase/AKT Pathway Enhances Antitumor Activity of Chemotherapeutic Agents in Breast Cancer Susceptibility Gene 1-Defective Breast Cancer Cells 
Molecular carcinogenesis  2012;52(9):667-675.
Loss or decrease of wild type BRCA1 function, by either mutation or reduced expression, has a role in hereditary and sporadic human breast and ovarian cancers. We report here that the PI3K/AKT pathway is constitutively active in BRCA1-defective human breast cancer cells. Levels of phospho-AKT are sustained even after serum starvation in breast cancer cells carrying deleterious BRCA1 mutations. Knockdown of BRCA1 in MCF7 cells increases the amount of phospho-AKT and sensitizes cells to small molecule protein kinase inhibitors (PKIs) targeting the PI3K/AKT pathway. Restoration of wild type BRCA1 inhibits the activated PI3K/AKT pathway and de-sensitizes cells to PKIs targeting this pathway in BRCA1 mutant breast cancer cells, regardless of PTEN mutations. In addition, clinical PI3K/mTOR inhibitors, PI-103 and BEZ235, showed anti-proliferative effects on BRCA1 mutant breast cancer cell lines and synergism in combination with chemotherapeutic drugs, cisplatin, doxorubicin, topotecan, and gemcitabine. BEZ235 synergizes with the anti-proliferative effects of gemcitabine by enhancing caspase-3/7 activity. Our results suggest that the PI3K/AKT pathway can be an important signaling pathway for the survival of BRCA1-defective breast cancer cells and pharmacological inhibition of this pathway is a plausible treatment for a subset of breast cancers.
doi:10.1002/mc.21905
PMCID: PMC3586771  PMID: 22488590
constitutive activation; PI3K/AKT pathway; BRCA1-defective breast cancer; kinase inhibitor; chemotherapeutic agents; synergism
25.  Differential Effects of Black Raspberry and Strawberry Extracts on BaPDE-Induced Activation of Transcription Factors and Their Target Genes 
Molecular carcinogenesis  2008;47(4):286-294.
The chemopreventive properties of edible berries have been demonstrated both in vitro and in vivo, however, the specific molecular mechanisms underlying their anti-cancer effects are largely unknown. Our previous studies have shown that a methanol extract fraction of freeze-dried black raspberries inhibits benzoapyrene (BaP)-induced transformation of Syrian hamster embryo cells. This fraction also blocks activation of activator protein-1 (AP-1) and nuclear factor κB (NF-κB) induced by benzoapyrene diol-epoxide (BaPDE) in mouse epidermal JB6 Cl 41 cells. To determine if different berry types exhibit specific mechanisms for their anti-cancer effects, we compared the effects of extract fractions from both black raspberries and strawberries on BaPDE-induced activation of various signaling pathways in Cl 41 cells. Black raspberry fractions inhibited the activation of AP-1, NF-κB, and nuclear factor of activated T cells (NFAT) by BaPDE as well as their upstream PI-3K/Akt-p70S6K and mitogen-activated protein kinase pathways. In contrast, strawberry fractions inhibited NFAT activation, but did not inhibit the activation of AP-1, NF-κB or the PI-3K/Akt-p70S6K and mitogen-activated protein kinase pathways. Consistent with the effects on NFAT activation, tumor necrosis factor-α (TNF-α) induction by BaPDE was blocked by extract fractions of both black raspberries and strawberries, whereas vascular endothelial growth factor (VEGF) expression, which depends on AP-1 activation, was suppressed by black raspberry fractions but not strawberry fractions. These results suggest that black raspberry and strawberry components may target different signaling pathways in exerting their anti-carcinogenic effects.
doi:10.1002/mc.20377
PMCID: PMC3758121  PMID: 18085529
transcription factors; BaPDE; berry; gene expression; chemoprevention

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