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.

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.

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.

The Cyp2c44 epoxygenase regulates epithelial sodium channel activity and the blood pressure responses to increased dietary salt.

Hypertension is a major risk factor for cerebral, cardiovascular, and renal disease, and its prevalence and devastating consequences raises a need for new strategies for its early diagnosis and treatment. We show here that lack of a Cyp2c44 epoxygenase causes dietary salt-sensitive hypertension, a common form of the human disease. Cyp2c44(-/-) mice on normal salt diets are normotensive but become hypertensive when fed high salt. Hypertensive Cyp2c44(-/-) mice show a hyperactive kidney epithelial sodium channel (ENaC) and reductions in ERK1/2 and ENaC subunit phosphorylation. The demonstration that amiloride, an ENaC inhibitor, lowers the blood pressure of hypertensive Cyp2c44(-/-) mice identifies a role for the channel in the hypertensive phenotype of the animals. These studies: (a) identify an antihypertensive role for the kidney Cyp2c44 epoxygenase and for its epoxyeicosatrienoic acid (EET) metabolites in the in vivo control of ENaC activity and the activation of mitogenic kinase pathways; (b) provide evidence for a Cyp2c44 epoxygenase, EET-mediated mechanism of ENaC regulation involving an ERK1/2-catalyzed threonine phosphorylation of the channel γ subunit: and (c) characterize a common scientific platform that could explain the seemingly unrelated biological activities attributed to the epoxygenase metabolites in cell proliferation, angiogenesis, channel activity, and blood pressure control. It is expected that these results will serve as a basis for the development of novel strategies for the early diagnosis and treatment of hypertension and of pathophysiologies associated with dysfunctional mitogenic signaling.

Epoxyeicosatrienoic acids (EETs) regulate epithelial sodium channel activity by extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated phosphorylation.

The epithelial sodium channel (ENaC) participates in the regulation of plasma sodium and volume, and gain of function mutations in the human channel cause salt-sensitive hypertension. Roles for the arachidonic acid epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs), in ENaC activity have been identified; however, their mechanisms of action remain unknown. In polarized M1 cells, 14,15-EET inhibited amiloride-sensitive apical to basolateral sodium transport as effectively as epidermal growth factor (EGF). The EET effects were associated with increased threonine phosphorylation of the ENaC ß and γ subunits and abolished by inhibitors of (a) mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal regulated kinases 1 and 2 (MEK/ERK1/2) and (b) EGF receptor signaling. CYP2C44 epoxygenase knockdown blunted the sodium transport effects of EGF, and its 14,15-EET metabolite rescued the knockdown phenotype. The relevance of these findings is indicated by (a) the hypertension that results in mice administered cetuximab, an inhibitor of EGF receptor binding, and (b) immunological data showing an association between the pressure effects of cetuximab and reductions in ENaCγ phosphorylation. These studies (a) identify an ERK1/2-dependent mechanism for ENaC inhibition by 14,15-EET, (b) point to ENaC as a proximal target for EET-activated ERK1/2 mitogenic kinases, (c) characterize a mechanistic commonality between EGF and epoxygenase metabolites as ENaC inhibitors, and (d) suggest a CYP2C epoxygenase-mediated pathway for the regulation of distal sodium transport.

Oxidized phospholipids induce type VIII collagen expression and vascular smooth muscle cell migration.

Phenotypic switching of vascular smooth muscle cells (VSMCs) is known to play a critical role in the development of atherosclerosis. However, the factors present within lesions that mediate VSMC phenotypic switching are unclear. Oxidized phospholipids (OxPLs), including 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC), are active components of minimally modified low density lipoprotein and have been previously shown to induce multiple proatherogenic events in endothelial cells and macrophages, but their effects on VSMCs have been largely unexplored until recently. We previously showed that OxPLs induced phenotypic switching of VSMCs, including suppression of SMC differentiation marker genes. The goal of the present studies was to test the hypothesis that OxPLs alter extracellular matrix production and VSMC migration. Results showed that POVPC activated expression of several extracellular matrix proteins in VSMC. POVPC increased expression of type VIII collagen alpha1 chain (Col8a1) mRNA in cultured VSMCs and in vivo in rat carotid arteries by 9-fold and 4-fold, respectively. POVPC-induced activation of Col8a1 gene expression was reduced by small interfering RNA-mediated suppression of Krüppel-like factor 4 (Klf4) and Sp1, and was abolished in Klf4-knockout VSMCs. POVPC increased Klf4 binding to the Col8a1 gene promoter both in vivo in rat carotid arteries and in cultured VSMCs based on chromatin immunoprecipitation assays. Moreover, POVPC-induced VSMC migration was markedly reduced in Klf4- or type VIII collagen-knockout VSMCs. Given evidence that OxPLs are present within atherosclerotic lesions, it is interesting to suggest that OxPL-induced changes in VSMC phenotype may contribute to the pathogenesis of atherosclerosis at least in part through changes in extracellular matrix composition.

Oxidized phospholipids induce phenotypic switching of vascular smooth muscle cells in vivo and in vitro.

Atherosclerosis is a vascular disease characterized by lipid deposition and inflammation within the arterial wall. Oxidized phospholipids (oxPLs), such as 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) and its constituents 1-palmytoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) are concentrated within atherosclerotic lesions and are known to be potent proinflammatory mediators. Phenotypic switching of smooth muscle cells (SMCs) plays a critical role in the development, progression, and end-stage clinical consequences of atherosclerosis, yet little is known regarding the effects of specific oxPLs on SMC phenotype. The present studies were focused on determining whether oxPLs regulate expression of SMC differentiation marker genes and the molecular mechanisms involved. Results showed that POVPC and PGPC induced profound suppression of smooth muscle (SM) alpha-actin and SM myosin heavy chain expression while simultaneously increasing expression of MCP-1, MCP-3, and cytolysin. OxPLs also induced nuclear translocation of Krüppel-like transcription factor 4 (KLF4), a known repressor of SMC marker genes. siRNA targeting of KLF4 nearly blocked POVPC-induced suppression of SMC marker genes, and myocardin. POVPC-induced repression of SMC marker genes was also significantly attenuated in KLF4 knockout SMCs. Taken together, these results suggest a novel role for oxPLs in phenotypic modulation of SMCs and indicate that these effects are dependent on the transcription factor, KLF4. These results may have important novel implications for the mechanisms by which oxPLs contribute to the pathogenesis of atherosclerosis.

Darpp-32: a novel antiapoptotic gene in upper gastrointestinal carcinomas.

We show the molecular mechanisms involved in Darpp-32 overexpression and its biological role in upper gastrointestinal adenocarcinomas (UGC). A tumor tissue array of 377 samples was developed and used to detect DARPP-32 DNA amplification and protein overexpression, which occurred in 32% and 60% of UGCs, respectively. Concomitant overexpression of mRNA for Darpp-32 and its truncated isoform t-Darpp was observed in 68% of tumors (P < 0.001). When Darpp-32 and t-Darpp were overexpressed in AGS and RKO gastrointestinal cells, up to a 4-fold reduction in the apoptosis rate was observed (terminal deoxynucleotidyl transferase-mediated nick-end labeling and Annexin V assays) in response to camptothecin, sodium butyrate, and ceramide. However, the introduction of mutations in phosphorylation sites abrogated this effect. Expression of Darpp-32 and t-Darpp preserved the mitochondrial transmembrane potential and was associated with increased levels of Bcl2 protein. A reversal of Bcl2 protein level was obtained using small interfering RNAs for Darpp-32 and t-Darpp. Luciferase assays using the p53 and p21 reporter plasmids and probing of immunoblots with antibodies specific for p53 transcriptional targets, such as Hdm2 and p21, indicated that neither Darpp-32 nor t-Darpp interfere with p53 function. Altogether, we show more frequent mRNA and protein overexpression of Darpp-32 than DNA amplification, suggesting that, in addition to amplification, transcriptional or posttranscriptional mechanisms may play an important role. The expression of Darpp-32 and t-Darpp is associated with a potent antiapoptotic advantage for cancer cells through a p53-independent mechanism that involves preservation of mitochondrial potential and increased Bcl2 levels.