Reflux conditions induce E-cadherin cleavage and EMT via APE1 redox function in oesophageal adenocarcinoma.

OBJECTIVE: Chronic gastro-oesophageal reflux disease, where acidic bile salts (ABS) reflux into the oesophagus, is the leading risk factor for oesophageal adenocarcinoma (EAC). We investigated the role of ABS in promoting epithelial-mesenchymal transition (EMT) in EAC. DESIGN: RNA sequencing data and public databases were analysed for the EMT pathway enrichment and patients' relapse-free survival. Cell models, pL2-IL1ß transgenic mice, deidentified EAC patients' derived xenografts (PDXs) and tissues were used to investigate EMT in EAC. RESULTS: Analysis of public databases and RNA-sequencing data demonstrated significant enrichment and activation of EMT signalling in EAC. ABS induced multiple characteristics of the EMT process, such as downregulation of E-cadherin, upregulation of vimentin and activation of ß-catenin signalling and EMT-transcription factors. These were associated with morphological changes and enhancement of cell migration and invasion capabilities. Mechanistically, ABS induced E-cadherin cleavage via an MMP14-dependent proteolytic cascade. Apurinic/apyrimidinic endonuclease (APE1), also known as redox factor 1, is an essential multifunctional protein. APE1 silencing, or its redox-specific inhibitor (E3330), downregulated MMP14 and abrogated the ABS-induced EMT. APE1 and MMP14 coexpression levels were inversely correlated with E-cadherin expression in human EAC tissues and the squamocolumnar junctions of the L2-IL1ß transgenic mouse model of EAC. EAC patients with APE1high and EMThigh signatures had worse relapse-free survival than those with low levels. In addition, treatment of PDXs with E3330 restrained EMT characteristics and suppressed tumour invasion. CONCLUSION: Reflux conditions promote EMT via APE1 redox-dependent E-cadherin cleavage. APE1-redox function inhibitors can have a therapeutic role in EAC.

Helicobacter pylori-induced cell death is counteracted by NF-κB-mediated transcription of DARPP-32.

OBJECTIVE: DARPP-32 is a frequently amplified and overexpressed gene that promotes several oncogenic functions in gastric cancer. Herein, we investigated the relationship between Helicobacter pylori infection, proinflammatory NF-κB activation and regulation of DARPP-32. DESIGN: The study used in vivo and in vitro experiments. Luciferase reporter, quantitative real-time PCR, immunoblot, chromatin immunoprecipitation (ChIP), cell viability, H. pylori infection, tissue microarrays and immunohistochemical assays were used. RESULTS: Our results indicated that H. pylori infection increased the DARPP-32 mRNA and protein levels in gastric cancer cell lines and gastric mucosa of mice. H. pylori infection increased the activity of NF-κB reporter and p-NF-κB (S536) protein level in vitro and in vivo. To investigate the transcriptional regulation of DARPP-32, we cloned a 3019 bp of the DARPP-32 promoter into the luciferase reporter (pGL3-Luc). Both H. pylori infection and tumour necrosis factor-α treatment induced DARPP-32 reporter activity (p<0.01). Using deletion constructs of DARPP-32 promoter and ChIP assay, we demonstrated that the sequence -996 to -1008 bp containing putative NF-κB-binding sites is the most active region. The induction of DARPP-32 expression by H. pylori infection counteracted H. pylori-induced cell death through activation of serine/threonine-specific protein kinase (AKT), as determined by ATP-Glo and clonogenic survival assays. Immunohistochemistry analysis demonstrated a significant positive correlation between NF-κB and DARPP-32 expression levels in gastric cancer tissues (r2=0.43, p<0.01). CONCLUSIONS: Given the high frequency of DARPP-32 overexpression and its prosurvival oncogenic functions, the induction of DARPP-32 expression following H. pylori infection and activation of NF-κB provides a link between infection, inflammation and gastric tumourigenesis.

Gastric tumour-derived ANGPT2 regulation by DARPP-32 promotes angiogenesis.

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 tumour 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 human umbilical vein endothelial cells 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 signal transducer and activator of transcription 3 (STAT3), known regulators of angiogenesis. The results ruled out NF-κB and showed induction of STAT3 phosphorylation, activation and nuclear localisation. Inhibition or knockdown of STAT3 significantly attenuated the induction of ANGPT2 by DARPP-32 proteins. In vivo xenograft models demonstrated that overexpression of DARPP-32 promotes angiogenesis and tumour growth. Analyses of human gastric cancer tissues showed a strong correlation between DARPP-32 and ANGPT2. CONCLUSIONS: Our novel findings establish the role of DARPP-32-STAT3 axis in regulating ANGPT2 in cancer cells to promote angiogenesis and tumorigenesis.

Activation of ß-catenin signalling by TFF1 loss promotes cell proliferation and gastric tumorigenesis.

OBJECTIVE: In this study, we investigated the role of Trefoil factor 1 (TFF1) in regulating cell proliferation and tumour development through ß-catenin signalling using in vivo and in vitro models of gastric tumorigenesis. DESIGN: Tff1-knockout (Tff1-KO) mice, immunohistochemistry, luciferase reporter, qRT-PCR, immunoblot, and phosphatase assays were used to examine the role of TFF1 on ß-catenin signalling pathway. RESULTS: Nuclear localisation of ß-catenin with transcriptional upregulation of its target genes, c-Myc and Ccnd1, was detected in hyperplastic tissue at an early age of 4-6 weeks and maintained during all stages of gastric tumorigenesis in the Tff1-KO mice. The reconstitution of TFF1 or TFF1 conditioned media significantly inhibited the ß-catenin/T-cell factor (TCF) transcription activity in MKN28 gastric cancer cells. In agreement with these results, we detected a reduction in the levels of nuclear ß-catenin with downregulation of c-MYC and CCND1 mRNA. Analysis of signalling molecules upstream of ß-catenin revealed a decrease in phosphorylated glycogen synthase kinase 3ß (p-GSK3ß) (Ser9) and p-AKT (Ser473) protein levels following the reconstitution of TFF1 expression; this was consistent with the increase of p-ß-catenin (Ser33/37/Thr41) and decrease of p-ß-catenin (Ser552). This TFF1-induced reduction in phosphorylation of GSK3ß, and AKT was dependent on protein phosphatase 2A (PP2A) activity. The treatment with okadaic acid or knockdown of PP2A abrogated these effects. Consistent with the mouse data, we observed loss of TFF1 and an increase in nuclear localisation of ß-catenin in stages of human gastric tumorigenesis. CONCLUSIONS: Our data indicate that loss of TFF1 promotes ß-catenin activation and gastric tumorigenesis through regulation of PP2A, a major regulator of AKT-GSK3ß signalling.

Aurora kinase A in gastrointestinal cancers: time to target.

Gastrointestinal (GI) cancers are a major cause of cancer-related deaths. During the last two decades, several studies have shown amplification and overexpression of Aurora kinase A (AURKA) in several GI malignancies. These studies demonstrated that AURKA not only plays a role in regulating cell cycle and mitosis, but also regulates a number of key oncogenic signaling pathways. Although AURKA inhibitors have moved to phase III clinical trials in lymphomas, there has been slower progress in GI cancers and solid tumors. Ongoing clinical trials testing AURKA inhibitors as a single agent or in combination with conventional chemotherapies are expected to provide important clinical information for targeting AURKA in GI cancers. It is, therefore, imperative to consider investigations of molecular determinants of response and resistance to this class of inhibitors. This will improve evaluation of the efficacy of these drugs and establish biomarker based strategies for enrollment into clinical trials, which hold the future direction for personalized cancer therapy. In this review, we will discuss the available data on AURKA in GI cancers. We will also summarize the major AURKA inhibitors that have been developed and tested in pre-clinical and clinical settings.

Trefoil factor 1 expression suppresses Helicobacter pylori-induced inflammation in gastric carcinogenesis.

BACKGROUND: Infection with Helicobacter pylori, a high-risk factor for gastric cancer, is frequently associated with chronic inflammation through activation of nuclear factor κB (NF-κB). Trefoil factor 1 (TFF1) is a constitutively expressed protein in the stomach that has tumor-suppressor functions and plays a critical role in maintaining mucosal integrity. This study investigated the role of TFF1 in regulating the proinflammatory response to H. pylori infections. METHODS: For in vitro studies, immunofluorescence, luciferase reporter assays, Western blots, and quantitative real-time polymerase chain reaction were performed to investigate the activation of NF-κB and its target genes in response to infections with H. pylori strains J166 and 7.13. In addition, Tff1-knockout (KO) and Tff1-wild-type mice were used for infections with the H. pylori strain called premouse Sydney strain 1. RESULTS: The reconstitution of TFF1 expression in gastric cancer cells significantly suppressed H. pylori-mediated increases in NF-κB-p65 nuclear staining, transcriptional activity, and expression of proinflammatory cytokine genes (tumor necrosis factor α, interleukin 1ß, chemokine [C-X-C motif] ligand 5, and interleukin 4 receptor) that were associated with reductions in the expression and phosphorylation of NF-κB-p65 and IκB kinase α/ß proteins. The in vivo studies using the Tff1-KO mouse model of gastric neoplasia confirmed the in vitro findings. Furthermore, they demonstrated increases in chronic inflammation scores and in the frequency of invasive gastric adenocarcinoma in the Tff1-KO mice infected with H. pylori versus the uninfected Tff1-KO mice. CONCLUSIONS: These findings underscore an important protective role of TFF1 in abrogating H. pylori-mediated inflammation, a crucial hallmark of gastric tumorigenesis. Therefore, loss of TFF1 expression could be an important step in H. pylori-mediated gastric carcinogenesis.

Glutathione peroxidase 7 has potential tumour suppressor functions that are silenced by location-specific methylation in oesophageal adenocarcinoma.

OBJECTIVE: To investigate the potential tumour suppressor functions of glutathione peroxidase 7 (GPX7) and examine the interplay between epigenetic and genetic events in regulating its expression in oesophageal adenocarcinomas (OAC). DESIGN: In vitro and in vivo cell models were developed to investigate the biological and molecular functions of GPX7 in OAC. RESULTS: Reconstitution of GPX7 in OAC cell lines, OE33 and FLO-1, significantly suppressed growth as shown by the growth curve, colony formation and EdU proliferation assays. Meanwhile, GPX7-expressing cells displayed significant impairment in G1/S progression and an increase in cell senescence. Concordant with the above functions, Western blot analysis displayed higher levels of p73, p27, p21 and p16 with a decrease in phosphorylated retinoblastoma protein (RB), indicating its increased tumour suppressor activities. On the contrary, knockdown of GPX7 in HET1A cells (an immortalised normal oesophageal cell line) rendered the cells growth advantage as indicated with a higher EdU rate, lower levels of p73, p27, p21 and p16 and an increase in phosphorylated RB. We confirmed the tumour suppressor function in vivo using GPX7-expressing OE33 cells in a mouse xenograft model. Pyrosequencing of the GPX7 promoter region (-162 to +138) demonstrated location-specific hypermethylation between +13 and +64 in OAC (69%, 54/78). This was significantly associated with the downregulation of GPX7 (p<0.01). Neither mutations in the coding exons of GPX7 nor DNA copy number losses were frequently present in the OAC examined (<5%). CONCLUSIONS: Our data suggest that GPX7 possesses tumour suppressor functions in OAC and is silenced by location-specific promoter DNA methylation.

Loss of glutathione peroxidase 7 promotes TNF-α-induced NF-κB activation in Barrett's carcinogenesis.

Esophageal adenocarcinoma (EAC) is a classic example of inflammation-associated cancer, which develops through GERD (gastroesophageal reflux disease)-Barrett's esophagus (BE)-dysplasia-adenocarcinoma sequence. The incidence of EAC has been rising rapidly in the USA and Western countries during the last few decades. The functions of glutathione peroxidase 7 (GPX7), an antioxidant enzyme frequently silenced during Barrett's tumorigenesis, remain largely uncharacterized. In this study, we investigated the potential role of GPX7 in regulating nuclear factor-kappaB (NF-κB) activity in esophageal cells. Western blot analysis, immunofluorescence and luciferase reporter assay data indicated that reconstitution of GPX7 expression in CP-A (non-dysplastic BE cells) and FLO-1 (EAC cells) abrogated tumor necrosis factor-α (TNF-α)-induced NF-κB transcriptional activity (P < 0.01) and nuclear translocation of NF-κB-p65 (P = 0.01). In addition, we detected a marked reduction in phosphorylation levels of components of NF-κB signaling pathway, p-p65 (S536), p-IκB-α (S32) and p-IKKα/ß (S176/180), as well as significant suppression in induction of NF-κB target genes [TNF-α, interleukin (IL)-6, IL-8, IL-1ß, CXCL-1 and CXCL-2] following treatment with TNF-α in GPX7-expressing FLO-1 cells as compared with control cells. We validated these effects by knockdown of GPX7 expression in HET1A (normal esophageal squamous cells). We found that GPX7-mediated suppression of NF-κB is independent of reactive oxygen species level and GPX7 antioxidant function. Further mechanistic investigations demonstrated that GPX7 promotes protein degradation of TNF-receptor 1 (TNFR1) and TNF receptor-associated factor 2 (TRAF2), suggesting that GPX7 modulates critical upstream regulators of NF-κB. We concluded that the loss of GPX7 expression is a critical step in promoting the TNF-α-induced activation of proinflammatory NF-κB signaling, a major player in GERD-associated Barrett's carcinogenesis.

Aurora kinase A promotes inflammation and tumorigenesis in mice and human gastric neoplasia.

BACKGROUND & AIMS: Chronic inflammation contributes to the pathogenesis of gastric tumorigenesis. The aurora kinase A (AURKA) gene is frequently amplified and overexpressed in gastrointestinal cancers. We investigated the roles of AURKA in inflammation and gastric tumorigenesis. METHODS: We used quantitative real-time reverse transcription polymerase chain reaction, immunofluorescence, immunohistochemistry, luciferase reporter, immunoblot, co-immunoprecipitation, and in vitro kinase assays to analyze AGS and MKN28 gastric cancer cells. We also analyzed Tff1(-/-) mice, growth of tumor xenografts, and human tissues. RESULTS: We correlated increased expression of AURKA with increased levels of tumor necrosis factor-α and inflammation in the gastric mucosa of Tff1(-/-) mice (r = 0.62; P = .0001). MLN8237, an investigational small-molecule selective inhibitor of AURKA, reduced nuclear staining of nuclear factor-κB (NF-κB) p65 in human gastric cancer samples and mouse epithelial cells, suppressed NF-κB reporter activity, and reduced expression of NF-κB target genes that regulate inflammation and cell survival. Inhibition of AURKA also reduced growth of xenograft tumors from human gastric cancer cells in mice and reversed the development of gastric tumors in Tff1(-/-) mice. AURKA was found to regulate NF-κB activity by binding directly and phosphorylating IκBα in cells. Premalignant and malignant lesions from the gastric mucosa of patients had increased levels of AURKA protein and nuclear NF-κB, compared with healthy gastric tissue. CONCLUSIONS: In analyses of gastric cancer cell lines, human tissue samples, and mouse models, we found AURKA to be up-regulated during chronic inflammation to promote activation of NF-κB and tumorigenesis. AURKA inhibitors might be developed as therapeutic agents for gastric cancer.

The combination of alisertib, an investigational Aurora kinase A inhibitor, and docetaxel promotes cell death and reduces tumor growth in preclinical cell models of upper gastrointestinal adenocarcinomas.

BACKGROUND: Upper gastrointestinal adenocarcinomas (UGCs) respond poorly to current chemotherapeutic regimes. The authors and others have previously reported frequent Aurora kinase A (AURKA) gene amplification and mRNA and protein overexpression in UGCs. The objective of the current study was to determine the therapeutic potential of alisertib (MLN8237) alone and in combination with docetaxel in UGCs. METHODS: After treatment with alisertib and/or docetaxel, clonogenic cell survival, cell cycle analyses, Western blot analyses, and tumor xenograft growth assays were carried out to measure cell survival, cell cycle progression, apoptotic protein expression, and tumor xenograft volumes, respectively. RESULTS: By using the AGS, FLO-1, and OE33 UGC cell lines, which have constitutive AURKA overexpression and variable tumor protein 53 (p53) status, significantly enhanced inhibition of cancer cell survival was observed with alisertib and docetaxel treatment in combination (P < .001), compared with single-agent treatments. Cell cycle analyses, after 48 hours of treatment with alisertib, produced a significant increase in the percentage of polyploidy in UGC cells (P < .01) that was further enhanced by docetaxel (P < .001). In addition, an increase in the percentage of cells in sub-G1-phase observed with alisertib (P < .01) was significantly enhanced with the combination treatment (P < .001). Western blot analysis demonstrated higher induction of cleaved caspase 3 protein expression with the combined treatment compared with single-agent treatments. In addition, FLO-1 and OE33 cell xenograft models demonstrated enhanced antitumor activity for the alisertib and docetaxel combination compared with single-agent treatments (P < .001). CONCLUSIONS: The current study demonstrated that alisertib combined with docetaxel can mediate a better therapeutic outcome in UGC cell lines.

Glutathione peroxidase 7 protects against oxidative DNA damage in oesophageal cells.

OBJECTIVE: Exposure of the oesophageal mucosa to gastric acid and bile acids leads to the accumulation of reactive oxygen species (ROS), a known risk factor for Barrett's oesophagus and progression to oesophageal adenocarcinoma (OAC). This study investigated the functions of glutathione peroxidase 7 (GPX7), frequently silenced in OAC, and its capacity in regulating ROS and its associated oxidative DNA damage. DESIGN: Using in-vitro cell models, experiments were performed that included glutathione peroxidase (GPX) activity, Amplex UltraRed, CM-H(2)DCFDA, Annexin V, 8-oxoguanine, phospho-H2A.X, quantitative real-time PCR and western blot assays. RESULTS: Enzymatic assays demonstrated limited GPX activity of the recombinant GPX7 protein. GPX7 exhibited a strong capacity to neutralise hydrogen peroxide (H(2)O(2)) independent of glutathione. Reconstitution of GPX7 expression in immortalised Barrett's oesophagus cells, BAR-T and CP-A led to resistance to H(2)O(2)-induced oxidative stress. Following exposure to acidic bile acids cocktail (pH4), these GPX7-expressing cells demonstrated lower levels of H(2)O(2), intracellular ROS, oxidative DNA damage and double-strand breaks, compared with controls (p<0.01). In addition, these cells demonstrated lower levels of ROS signalling, indicated by reduced phospho-JNK (Thr183/Tyr185) and phospho-p38 (Thr180/Tyr182), and demonstrated lower levels of apoptosis following the exposure to acidic bile acids or H(2)O(2)-induced oxidative stress. The knockdown of endogenous GPX7 in immortalised oesophageal squamous epithelial cells (HET1A) confirmed the protective functions of GPX7 against pH4 bile acids by showing an increase in the levels of H(2)O(2), intracellular ROS, oxidative DNA damage, double-strand breaks, apoptosis, and ROS-dependent signalling (p<0.01). CONCLUSION: The dysfunction of GPX7 in oesophageal cells increases the levels of ROS and oxidative DNA damage, which are common risk factors for Barrett's oesophagus and OAC.

Altered expression of AXL receptor tyrosine kinase in gastrointestinal cancers: a promising therapeutic target.

Gastrointestinal (GI) cancers that include all cancers of the digestive tract organs are generally associated with obesity, lack of exercising, smoking, poor diet, and heavy alcohol consumption. Treatment of GI cancers typically involves surgery followed by chemotherapy and/or radiation. Unfortunately, intrinsic or acquired resistance to these therapies underscore the need for more effective targeted therapies that have been proven in other malignancies. The aggressive features of GI cancers share distinct signaling pathways that are connected to each other by the overexpression and activation of AXL receptor tyrosine kinase. Several preclinical and clinical studies involving anti-AXL antibodies and small molecule AXL kinase inhibitors to test their efficacy in solid tumors, including GI cancers, have been recently carried out. Therefore, AXL may be a promising therapeutic target for overcoming the shortcomings of standard therapies in GI cancers.

DARPP-32 increases interactions between epidermal growth factor receptor and ERBB3 to promote tumor resistance to gefitinib.

BACKGROUND & AIMS: Dopamine and adenosine 3',5'-cyclic monophosphate-regulated phosphoprotein, Mr 32000 (DARPP-32), is overexpressed during gastric carcinogenesis. Gastric tumors can become resistant to gefitinib, an inhibitor of the epidermal growth factor receptor (EGFR). We investigated the role of DARPP-32 in gastric tumor resistance to gefitinib. METHODS: Cell survival was determined by clonogenic survival and ATP-Glo Viability Assays. Apoptosis was assessed by Annexin-V and immunoblot analyses. The association between DARPP-32 and EGFR was evaluated by immunofluorescence and co-immunoprecipitation assays. Findings were validated in mice with gastric xenograft tumors. DARPP-32 expression was reduced using small hairpin RNAs in the human gastric cancer cell lines SNU-16 and MKN-45 cells. RESULTS: Overexpression of DARPP-32 in MKN-28 cells, which do not normally express DARPP-32, blocked gefitinib-induced apoptosis and increased the drug's IC(50) 10-fold, compared to that of control cells (P < .01). Reduced expression of DARPP-32 in SNU-16 cells increased the sensitivity to gefitinib (P < .01). DARPP-32 activated phosphatidylinositol-3-kinase-AKT signaling, increased stability of the EGFR, and suppressed EGF- or gefitinib-induced degradation of the EGFR. DARPP-32 colocalized with EGFR on the cell membrane in a complex with EGFR and the EGF receptor ERBB3. DARPP-32-mediated resistance to gefitinib resulted from increased phosphorylation of and interaction between EGFR and ERBB3, which led to phosphorylation of AKT (at serine 473). Knockdown of DARPP-32 in gastric cancer cells reduced the mean size of tumors in mice and increased their response to gefitinib. CONCLUSIONS: DARPP-32 promotes resistance of gastric cancer cells to gefitinib by promoting interaction between EGFR and ERBB3 and activating phosphatidylinositol-3-kinase-AKT signaling.

AXL Promotes Metformin-Induced Apoptosis Through Mediation of Autophagy by Activating ROS-AMPK-ULK1 Signaling in Human Esophageal Adenocarcinoma.

AXL receptor tyrosine kinase promotes an invasive phenotype and chemotherapy resistance in esophageal adenocarcinoma (EAC). AXL has been implicated in the regulation of autophagy, but the underlying molecular mechanism remains poorly understood. Herein, we investigate the mechanistic role of AXL in autophagy as well as metformin-induced effects on the growth and survival of EAC. We demonstrate that AXL mediates autophagic flux through activation of AMPK-ULK1 signaling in a reactive oxygen species (ROS)-dependent mechanism by glucose starvation. AXL positively regulates basal cellular ROS levels without significantly affecting mitochondrial ROS production in EAC cells. Pharmacological inhibition of cellular ROS using Trolox abrogates glucose starvation-induced AMPK signaling and autophagy. We demonstrate that AXL expression is required for metformin-induced apoptosis in EAC cells in vitro. The apoptosis induction by metformin is markedly attenuated by inhibition of autophagy through genetic silencing of Beclin1 or ATG7 autophagy mediators, thereby confirming the requirement of intact autophagy for enhancing metformin-induced apoptosis in EAC cells. Our data indicate that metformin-induced autophagy displays a pro-apoptotic function in EAC cells. We show that the metformin-induced suppression of tumor growth in vivo is highly dependent on AXL expression in a tumor xenograft mouse model of EAC. We demonstrate that AXL promotes metformin-induced apoptosis through activation of autophagy in EAC. AXL may be a valuable biomarker to identify tumors that are sensitive to metformin. Therefore, AXL expression could inform the selection of patients for future clinical trials to evaluate the therapeutic efficacy of metformin in EAC.

Regulation of ß-catenin by t-DARPP in upper gastrointestinal cancer cells.

BACKGROUND: Truncated dopamine and cyclic-AMP-regulated phosphoprotein (t-DARPP) is frequently overexpressed in gastrointestinal malignancies. In this study, we examined the role of t-DARPP in regulating ß-catenin. RESULTS: The pTopFlash construct that contains multiple TCF/LEF-binding sites was used as a measure of ß-catenin/TCF transcription activity. Gastric (AGS, MKN28) and esophageal (FLO-1) adenocarcinoma cancer cell lines that lack t-DARPP expression were utilized to establish stable and transient in vitro expression models of t-DARPP. The expression of t-DARPP led to a significant induction of the pTOP reporter activity, indicative of activation of ß-catenin/TCF nuclear signaling. Immunofluorescence assays supported this finding and showed accumulation and nuclear translocation of ß-catenin in cells expressing t-DARPP. These cells had a significant increase in their proliferative capacity and demonstrated up-regulation of two transcription targets of ß-catenin/TCF: Cyclin D1 and c-MYC. Because phosphorylated GSK-3ß is inactive and loses its ability to phosphorylate ß-catenin and target it towards degradation by the proteasome, we next examined the levels of phospho-GSK-3ß. These results demonstrated an increase in phospho-GSK-3ß and phospho-AKT. The knockdown of endogenous t-DARPP in MKN45 cancer cells demonstrated a reversal of the signaling events. To examine whether t-DARPP mediated GSK-3ß phosphorylation in an AKT-dependent manner, we used a pharmacologic inhibitor of PI3K/AKT, LY294002, in cancer cells expressing t-DARPP. This treatment abolished the phosphorylation of AKT and GSK-3ß leading to a reduction in ß-catenin, Cyclin D1, and c-MYC protein levels. CONCLUSIONS: Our findings demonstrate, for the first time, that t-DARPP regulates ß-catenin/TCF activity, thereby implicating a novel oncogenic signaling in upper gastrointestinal cancers.

Extrahepatic cytochrome P450 epoxygenases: pathophysiology and clinical significance in human gastrointestinal cancers.

Cytochrome P450 (CYP) epoxygenases, a multi-gene superfamily of heme-containing enzymes, are commonly known to metabolize endogenous arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs). The role of CYPs is mostly studied in liver drugs metabolism, cardiac pathophysiology, and hypertension fields. Particularly, the biological functions of these enzymes have increasingly attracted a growing interest in cancer biology. Most published studies on CYPs in cancer have been limited to their role as drug metabolizing systems. The activity of these enzymes may affect drug pharmacokinetics and bioavailability as well as exogenous compounds turnover. Some CYP isoforms are selectively highly expressed in tumors, suggesting a potential mechanistic role in promoting resistance to chemotherapy. Majority of drugs elicit their effects in extrahepatic tissues whereby their metabolism can significantly determine treatment outcome. Nonetheless, the role of extrahepatic CYPs is not fully understood and targeting these enzymes as effective anti-cancer therapies are yet to be developed. This review article summarizes an up-to-date body of information from published studies on CYP enzymes expression levels and pathophysiological functions in human normal and malignant gastrointestinal (GI) tract tissues. Specifically, we reviewed and discussed the current research initiatives by emphasizing on the clinical significance and the pathological implication of CYPs in GI malignancies of esophagus, stomach, and colon.

t-DARPP regulates phosphatidylinositol-3-kinase-dependent cell growth in breast cancer.

BACKGROUND: Recent reports have shown that t-DARPP (truncated isoform of DARPP-32) can mediate trastuzumab resistance in breast cancer cell models. In this study, we evaluated expression of t-DARPP in human primary breast tumors, and investigated the role of t-DARPP in regulating growth and proliferation in breast cancer cells. RESULTS: Quantitative real time RT-PCR analysis using primers specific for t-DARPP demonstrated overexpression of t-DARPP in 36% of breast cancers (13/36) as opposed to absent to very low t-DARPP expression in normal breast tissue (p < 0.05). The mRNA overexpression of t-DARPP was overwhelmingly observed in ductal carcinomas, including invasive ductal carcinomas and intraductal carcinomas, rather than other types of breast cancers. The immunohistochemistry analysis of DARPP-32/t-DARPP protein(s) expression in breast cancer tissue microarray that contained 59 tumors and matched normal tissues when available indicated overexpression in 35.5% of primary breast tumors that were more frequent in invasive ductal carcinomas (43.7%; 21/48). In vitro studies showed that stable overexpression of t-DARPP in MCF-7 cells positively regulated proliferation and anchorage-dependent and -independent growth. Furthermore, this effect was concomitant with induction of phosphorylation of AKT(ser473) and its downstream target phospho(ser9) GSK3ß, and increased Cyclin D1 and C-Myc protein levels. The knockdown of endogenous t-DARPP in HCC1569 cells led to a marked decrease in phosphorylation of AKTs(ser473) and GSK3ß(ser9). The use of PI3K inhibitor LY294002 or Akt siRNA abrogated the t-DARPP-mediated phosphorylation of AKT(ser473) and led to a significant reduction in cell growth. CONCLUSIONS: Our findings underscore the potential role of t-DARPP in regulating cell growth and proliferation through PI3 kinase-dependent mechanism.

Co-overexpression of AXL and c-ABL predicts a poor prognosis in esophageal adenocarcinoma and promotes cancer cell survival.

Background: Esophageal adenocarcinoma (EAC) is highly aggressive and characterized by poor prognosis. AXL expression has been linked to Barrett's tumorigenesis and resistance to chemotherapy, which is associated with c-ABL intracellular localization. However, the molecular and functional relationship between AXL and c-ABL and the clinical significance of the co-expression of these proteins in EAC remain unclear. Methods: We used immunohistochemical analysis (IHC) on tissue microarrays containing human EAC samples (n=53) and normal esophageal tissues (n=11) in combination with corresponding deidentified clinicopathological information to evaluate the expression and the prognostic significance of AXL and c-ABL in EAC. The data were statistically analyzed using Kruskal-Wallis, the chi-square, the Fisher's exact, and Pearson tests. The Kaplan-Meier method and Cox proportional hazards regression model were used to evaluate cancer patient survival. We used a serum deprivation EAC cell model to investigate the pro-survival function of AXL and c-ABL using cell viability, apoptosis, and lactate dehydrogenase activity assays. We performed in vitro assays, including Western blotting, quantitative real-time PCR, and translational chromatin immunoprecipitation (TrIP-Chip) to study the molecular relationship between AXL and c-ABL in EAC cells. Results: IHC analysis revealed that AXL and c-ABL were overexpressed in 55% and 66% of EAC samples, respectively, as compared to normal tissues. Co-overexpression of the two proteins was observed in 49% of EAC samples. The chi-square test indicated a significant association between AXL and c-ABL expression in the EAC samples (χ2 = 6.873, p = 0.032), and the expression of these proteins was significantly associated with EAC patient age (p < 0.001), tumor stage (p < 0.01), and lymph node status (p < 0.001). AXL and c-ABL protein expression data analysis exhibited an identical clinicopathological association profile. Additionally, we found a significant association between expression of AXL (χ2 = 16.7, p = 0.002) or c-ABL (χ2 = 13.4, p = 0.001) and survival of EAC patients. The Cox proportional hazards model and log rank test predicted a significant increase in mortality of patients with high expression of AXL [hazard ratio (HR): 2.86, 95% confidence interval (CI): 1.53 - 5.34, p = 0.003] or c-ABL [HR: 3.29, 95% CI: 1.35 - 8.03, p = 0.001] as compared to those patients with low expression of AXL or c-ABL proteins. Molecular investigations indicated that AXL positively regulates c-ABL protein expression through increased cap-dependent protein translation involving phosphorylation of EIF4E in EAC cells. Next, we investigated the functional relationship between AXL and c-ABL in EAC cells. We demonstrated that the pro-survival activity of AXL requires c-ABL expression in response to serum deprivation. Conclusion: This study highlights the importance of the co-overexpression of AXL and c-ABL proteins as a valuable prognostic biomarker and targeting these proteins could be an effective therapeutic approach in EAC or other solid tumors expressing high levels of AXL and c-ABL proteins.

Expression of t-DARPP mediates trastuzumab resistance in breast cancer cells.

PURPOSE: We have investigated the role of t-DARPP in trastuzumab resistance in ERBB2-amplified and overexpressed breast cancer cell lines. EXPERIMENTAL DESIGN: We have used the HR-5 and HR-6 trastuzumab-resistant cells that were established from tumors that recurred in the presence of trastuzumab therapy following xenografts of BT-474 cells in nude mice. In addition, SKBR-3 cells, engineered for stable expression of t-DARPP, and HCC-1569 cells, which have constitutive expression of t-DARPP and are de novo resistant to trastuzumab, were used. RESULTS: We reported > or =15-fold up-regulation of mRNA and protein levels of t-DARPP in HR-5 and HR-6 cells compared with their progenitor BT-474 trastuzumab-sensitive cells. The t-DARPP expression was not regulated by changes in its promoter DNA methylation levels. The SKBR-3 cells stably expressing t-DARPP developed resistance to trastuzumab compared with their parental cells and empty vector controls (P < 0.01). The trastuzumab-resistant cell lines showed a significant increase in pAKT (Ser(473)) and BCL2 protein levels. The small interfering RNA knockdown of t-DARPP in all trastuzumab-resistant cells led to a significant reduction in ERBB2, pAKT (Ser(473)), and BCL2 protein levels with a significant decrease in cell viability (P < or = 0.001) and an increase in cleaved caspase-3 levels, indicating the progression of these cells toward apoptosis. The t-DARPP protein was associated with both heat shock protein 90 and ERBB2 forming a potential protein complex. This association may play a role in regulating ERBB2 protein in trastuzumab-resistant cells. CONCLUSION: We conclude that t-DARPP is a novel molecular target that can mediate the therapeutic resistance to trastuzumab in breast cancer cells.