We performed a genome-wide association study of esophageal squamous cell carcinoma (ESCC) by genotyping 1,077 individuals with ESCC and 1,733 control subjects of Chinese Han descent. We selected 18 promising SNPs for replication in an additional 7,673 cases of ESCC and 11,013 control subjects of Chinese Han descent and 303 cases of ESCC and 537 control subjects of Chinese Uygur-Kazakh descent. We identified two previously unknown susceptibility loci for ESCC: PLCE1 at 10q23 (P(Han combined for ESCC) = 7.46 x 10(-56), odds ratio (OR) = 1.43; P(Uygur-Kazakh for ESCC) = 5.70 x 10(-4), OR = 1.53) and C20orf54 at 20p13 (P(Han combined for ESCC) = 1.21 x 10(-11), OR = 0.86; P(Uygur-Kazakh for ESCC) = 7.88 x 10(-3), OR = 0.66). We also confirmed association in 2,766 cases of gastric cardia adenocarcinoma cases and the same 11,013 control subjects (PLCE1, P(Han for GCA) = 1.74 x 10(-39), OR = 1.55 and C20orf54, P(Han for GCA) = 3.02 x 10(-3), OR = 0.91). PLCE1 and C20orf54 have important biological implications for both ESCC and GCA. PLCE1 might regulate cell growth, differentiation, apoptosis and angiogenesis. C20orf54 is responsible for transporting riboflavin, and deficiency of riboflavin has been documented as a risk factor for ESCC and GCA.

ABSTRACT:Nicotine, a major constituent of tobacco, plays a critical role in smoking addiction. In humans, nicotine is primarily metabolized to cotinine, which is further metabolized to trans-3-hydroxycotinine. Recently, we have demonstrated that heterologously expressed human CYP2A13 is highly active in the metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a nicotine-derived carcinogen. In the present study, CYP2A13-catalyzed NNK metabolism was found to be inhibited competitively by nicotine and Nnitrosonornicotine (NNN), suggesting that both nicotine and NNN are also substrates of CYP2A13. We have further demonstrated that human CYP2A13 is indeed an efficient enzyme in catalyzing C-oxidation of nicotine to form cotinine, with the apparent K m and V max values of 20.2 M and 8.7 pmol/min/pmol, respectively. CYP2A13 also catalyzes the 3-hydroxylation of cotinine to form trans-3-hydroxycotinine, with the apparent K m and V max values of 45.2 M and 0.7 pmol/min/pmol, respectively. The importance of CYP2A13-catalyzed nicotine and cotinine metabolism in vivo remains to be determined.Nicotine is a major constituent of tobacco, playing a critical role in establishing and maintaining tobacco dependence (Henningfield et al., 1985). The major pathway of nicotine metabolism in humans is the C-oxidation to form cotinine, which is a two-step process. The first step is catalyzed by the cytochrome P450 (P450) system to produce the intermediate nicotine-⌬ Ϫ1Ј(5Ј) -iminium ion, which is further oxidized to cotinine by cytosolic aldehyde oxidase (Gorrod and Hibberd, 1982;Peterson et al., 1987;Williams et al., 1990). Other major metabolites of nicotine identified in human urine so far include trans-3Ј-hydroxycotinine and nicotine NЈ-oxide (Jacob et al., 1988). The conversion of cotinine to 3Ј-hydroxycotinine is highly stereospecific in vivo, with the trans isomer as a predominating product (Jacob et al., 1990). trans-3Ј-Hydroxycotinine is excreted in the urine to a much greater extent than cotinine itself. Cotinine may also be metabolized to 5Ј-hydroxycotinine, norcotinine, and cotinine N-oxide (Murphy et al., 1999).In humans, liver is the primary site of nicotine metabolism. Nicotine is also metabolized to some extent in the lung and kidney (Benowitz et al., 1987). Previous studies demonstrated that human CYP2A6 is a key enzyme for the metabolism of nicotine to cotinine, and cotinine to trans-3Ј-hydroxycotinine (Flammang et al., 1992;McCracken et al., 1992; Nakajima et al., 1996a,b;Shimada et al., 1996;Messina et al., 1997). CYP2A6 is the best characterized member in the CYP2A subfamily (Pelkonen and Raunio, 1995; FernandezSalguero and Gonzalez, 1995;Pelkonen et al., 2000). The other two members are CYP2A7 and CYP2A13, which share a high degree of sequence identity with CYP2A6 (Fernandez-Salguero and Gonzalez, 1995). Although the transcripts of these three genes were all found in the liver, CYP2A6 was more abundant than CYP2A7 and CYP2A13 (Koskela et al., 1999). The highest level of CYP2A13 mRNA was found in huma...

ABSTRACT:Human cytochrome P450 2A13 (CYP2A13) is highly efficient in the metabolic activation of a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and another potent carcinogen, aflatoxin B1 (AFB1). Although previous studies demonstrated that CYP2A13 mRNA is predominantly expressed in human respiratory tissues, expression of CYP2A13 protein in these tissues and the involved cell types have not been determined because of the lack of CYP2A13-specific antibodies. To explore the toxicological and physiological function of CYP2A13, it is important to understand the tissue/cellular distribution of CYP2A13 protein.In this study, we generated a peptide-specific antibody against human CYP2A13 and demonstrated by immunoblot analysis that this antibody does not cross-react with heterologously expressed human CYP2A6 and mouse CYP2A5 proteins, both sharing a high degree of amino acid sequence similarity with CYP2A13. Nor does the antibody cross-react with heterologously expressed human CYP3A4, CYP2S1, or any of the cytochrome P450 enzymes present in the human liver microsomes. Using this highly specific antibody for immunohistochemical staining, we detected a high level of CYP2A13 protein expression in the epithelial cells of human bronchus and trachea, but a rare distribution in the alveolar cells. There was little expression of CYP2A13 protein in different types of lung cancers. In consideration of the high efficiency of CYP2A13 in NNK metabolic activation, our result is consistent with the reported observations that most smoking-related human lung cancers are bronchogenic and supports that CYP2A13-catalyzed in situ activation may play a critical role in human lung carcinogenesis related to NNK and AFB1 exposure.

Esophageal adenocarcinoma (EAC) is characterized by resistance to chemotherapy and poor outcome. Although Cisplatin (CDDP) has been used as a first-line therapy in patients with EAC, resistance remains a major clinical problem. The AXL receptor tyrosine kinase, originally isolated as a transforming gene from leukemia, is overexpressed in several solid tumors. Herein, we assessed AXL protein expression in human EACs and examined its role in CDDP resistance in human EAC cells. AXL overexpression was detected in >50% of tumors examined. Elevating AXL in non-overexpressing cells doubled the sensitivity to CDDP cytotoxicity and increased cell survival 3-fold, whereas attenuating AXL in ovexpressing cells reduced survival 2-fold. The effects of AXL modulation on cell survival associated with changes in cellular and molecular markers of apoptosis. Mechanistic investigations revealed that AXL blocked CDDP-induced activation of endogenous p73β (TP73), reducing its protein half-life, and inhibited CDDP-induced levels of p-c-ABL(Y412) and p-p73β(Y99). These changes were associated with a disruption of c-ABL/p73β protein interactions due to association with c-ABL in the cytoplasm, thereby blocking nuclear accumulation of c-ABL and phosphorylation of p73β in response to DNA damage. Together, our results establish that AXL promotes CDDP resistance in esophageal adenocarconima and argue that therapeutic targeting of AXL may sensitize these cancers to DNA damaging drugs.

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Esophageal adenocarcinoma (EAC) is an aggressive malignancy with a poor outcome. Although targeting ERBB2 with trastuzumab is evaluated in clinical trials, the molecular mechanisms of trastuzumab resistance remain uncharacterized in EAC. The dopamine and cyclic AMP-regulated phosphoprotein of Mr 32,000 (DARPP-32), also known as PPP1R1B, is located together with ERBB2 at the 17q12-q21 amplicon. We evaluated the expression of a transcript variant of DARPP-32 (t-DARPP) and ERBB2 in 141 primary tumors and investigated the role of t-DARPP in trastuzumab resistance using OE19 and OE33 EAC cell models. Overexpression of t-DARPP mRNA was detected in two-thirds of tumors with a correlation between ERBB2 and t-DARPP overexpression levels (r=0.58, p=0.003). Cell viability and clonogenic survival assays showed that t-DARPP increased survival by 40% in response to trastuzumab (p<0.01). The Annexin-V staining and Western blot analysis indicated that t-DARPP effectively abrogated trastuzumab-induced apoptosis, inhibited cleavage of caspase-3 and blocked trastuzumab-induced dephosphorylation of ERBB2 and AKT proteins. The knockdown of endogenous t-DARPP reversed these effects and sensitized cells to trastuzumab (p<0.01). The cycloheximide-based protein degradation analysis indicated that t-DARPP extended the half-life of ERBB2 explaining the increase in the basal levels of ERBB2, p-ERBB2(Y1248), and p-AKT(S473). Coimmunoprecipitation and Western blot analysis demonstrated that t-DARPP associated with ERBB2 in a protein complex, and interfered with trastuzumab binding to the ERBB2 receptor. Using EAC-xenografted mouse model, t-DARPP enhanced tumor growth and rendered tumors unresponsive to trastuzumab. This study establishes t-DARPP as a mediator of trastuzumab resistance and underscores its potential importance in clinical trials of EAC.

Objective Overexpression of dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32), and its truncated isoform (t-DARPP) are associated with gastric tumorigenesis. Herein, we investigated the role of DARPP-32 proteins in regulating angiopoietin 2 (ANGPT2) and promoting tumor angiogenesis. Design Quantitative real-time RT-PCR, immunoblotting, luciferase reporter, immunofluorescence, immunohistochemistry, and angiogenesis assays were applied to investigate the regulation of angiogenesis by DARPP-32 proteins. Results Overexpression of DARPP-32 significantly increased the mRNA and protein levels of ANGPT2 in gastric cancer cells. The overexpression of DARPP-32 T34A mutant or the N-terminal truncated isoform, t-DARPP, led to similar effects ruling out the T34-dependent regulation of protein phosphatase 1 activity in regulating ANGPT2. DARPP-32 proteins induced a secreted form of ANGPT2, which was detectable in the media, functionally active, and able to induce angiogenesis, measured by the HUVEC tube formation assay. Antibody blocking of the secreted ANGPT2 abrogated its function. To identify the mechanism by which DARPP-32 regulates ANGPT2, we examined the activities of NF-κB and STAT3, known regulators of angiogenesis. The results ruled out NF-κB and showed induction of STAT3 phosphorylation, activation, and nuclear localization. Inhibition or knockdown of STAT3 significantly attenuated the induction of ANGPT2 by DARPP-32 proteins. In vivo xenografts models demonstrated that overexpression of DARPP-32 promotes angiogenesis and tumor growth. Analyses of human gastric cancer tissues showed a strong correlation between DARPP-32 and ANGPT2. Conclusion Our novel findings establish the role of DARPP-32-STAT3 axis in regulating ANGPT2 in cancer cells to promote angiogenesis and tumorigenesis.