The crosstalk between enteric nervous system and immune system in intestinal development, homeostasis and diseases.

The gut is the largest digestive and absorptive organ, which is essential for induction of mucosal and systemic immune responses, and maintenance of metabolic-immune homeostasis. The intestinal components contain the epithelium, stromal cells, immune cells, and enteric nervous system (ENS), as well as the outers, such as gut microbiota, metabolites, and nutrients. The dyshomeostasis of intestinal microenvironment induces abnormal intestinal development and functions, even colon diseases including dysplasia, inflammation and tumor. Several recent studies have identified that ENS plays a crucial role in maintaining the immune homeostasis of gastrointestinal (GI) microenvironment. The crosstalk between ENS and immune cells, mainly macrophages, T cells, and innate lymphoid cells (ILCs), has been found to exert important regulatory roles in intestinal tissue programming, homeostasis, function, and inflammation. In this review, we mainly summarize the critical roles of the interactions between ENS and immune cells in intestinal homeostasis during intestinal development and diseases progression, to provide theoretical bases and ideas for the exploration of immunotherapy for gastrointestinal diseases with the ENS as potential novel targets.

A Treg-specific long noncoding RNA maintains immune-metabolic homeostasis in aging liver.

Regulatory T (Treg) cells modulate several aging-related liver diseases. However, the molecular mechanisms regulating Treg function in this context are unknown. Here we identified a long noncoding RNA, Altre (aging liver Treg-expressed non-protein-coding RNA), which was specifically expressed in the nucleus of Treg cells and increased with aging. Treg-specific deletion of Altre did not affect Treg homeostasis and function in young mice but caused Treg metabolic dysfunction, inflammatory liver microenvironment, liver fibrosis and liver cancer in aged mice. Depletion of Altre reduced Treg mitochondrial integrity and respiratory capacity, and induced reactive oxygen species accumulation, thus increasing intrahepatic Treg apoptosis in aged mice. Moreover, lipidomic analysis identified a specific lipid species driving Treg aging and apoptosis in the aging liver microenvironment. Mechanistically, Altre interacts with Yin Yang 1 to orchestrate its occupation on chromatin, thereby regulating the expression of a group of mitochondrial genes, and maintaining optimal mitochondrial function and Treg fitness in the liver of aged mice. In conclusion, the Treg-specific nuclear long noncoding RNA Altre maintains the immune-metabolic homeostasis of the aged liver through Yin Yang 1-regulated optimal mitochondrial function and the Treg-sustained liver immune microenvironment. Thus, Altre is a potential therapeutic target for the treatment of liver diseases affecting older adults.

N6 - Methyladenosine defines a new checkpoint in γδ T cell development.

T cells, which are derived from hematopoietic stem cells (HSCs), are the most important components of adaptive immune system. Based on the expression of αß and γδ receptors, T cells are mainly divided into αß and γδ T cells. In the thymus, they share common progenitor cells, while undergoing a series of well-characterized and different developmental processes. N6 -Methyladenosine (m6 A), one of the most abundant modifications in mRNAs, plays critical roles in cell development and maintenance of function. Recently, we have demonstrated that the depletion of m6 A demethylase ALKBH5 in lymphocytes specifically induces an expansion of γδ T cells through the regulation of Jag1/Notch2 signaling, but not αß T cells, indicating a checkpoint role of ALKBH5 and m6 A modification in the early development of γδ T cells. Based on previous studies, many key pathway molecules, which exert dominant roles in γδ T cell fate determination, have been identified as the targets regulated by m6 A modification. In this review, we mainly summarize the potential regulation between m6 A modification and these key signaling molecules in the γδ T cell lineage commitment, to provide new perspectives in the checkpoint of γδ T cell development.

RNA m6A demethylase ALKBH5 regulates the development of γδ T cells.

γδ T cells are an abundant T cell population at the mucosa and are important in providing immune surveillance as well as maintaining tissue homeostasis. However, despite γδ T cells' origin in the thymus, detailed mechanisms regulating γδ T cell development remain poorly understood. N6-methyladenosine (m6A) represents one of the most common posttranscriptional modifications of messenger RNA (mRNA) in mammalian cells, but whether it plays a role in γδ T cell biology is still unclear. Here, we show that depletion of the m6A demethylase ALKBH5 in lymphocytes specifically induces an expansion of γδ T cells, which confers enhanced protection against gastrointestinal Salmonella typhimurium infection. Mechanistically, loss of ALKBH5 favors the development of γδ T cell precursors by increasing the abundance of m6A RNA modification in thymocytes, which further reduces the expression of several target genes including Notch signaling components Jagged1 and Notch2. As a result, impairment of Jagged1/Notch2 signaling contributes to enhanced proliferation and differentiation of γδ T cell precursors, leading to an expanded mature γδ T cell repertoire. Taken together, our results indicate a checkpoint role of ALKBH5 and m6A modification in the regulation of γδ T cell early development.

m6A mRNA modification maintains colonic epithelial cell homeostasis via NF-κB-mediated antiapoptotic pathway.

Colonic mucosal barrier dysfunction is one of the major causes of inflammatory bowel disease (IBD). However, the mechanisms underlying mucosal barrier dysfunction are poorly understood. N6-methyladenosine (m6A) mRNA modification is an important modulator of epitranscriptional regulation of gene expression, participating in multiple physiological and pathological processes. However, the function of m6A modification in colonic epithelial cells and stem cells is unknown. Here, we show that m6A modification is essential for maintaining the homeostatic self-renewal in colonic stem cells. Specific deletion of the methyltransferase 14 (Mettl14) gene in mouse colon resulted in colonic stem cell apoptosis, causing mucosal barrier dysfunction and severe colitis. Mechanistically, we revealed that Mettl14 restricted colonic epithelial cell death by regulating the stability of Nfkbia mRNA and modulating the NF-κB pathway. Our results identified a previously unidentified role for m6A modification in colonic epithelial cells and stem cells, suggesting that m6A modification may be a potential therapeutic target for IBD.

m6A demethylase ALKBH5 controls CD4+ T cell pathogenicity and promotes autoimmunity.

N6-methyladenosine (m6A) modification is dynamically regulated by "writer" and "eraser" enzymes. m6A "writers" have been shown to ensure the homeostasis of CD4+ T cells, but the "erasers" functioning in T cells is poorly understood. Here, we reported that m6A eraser AlkB homolog 5 (ALKBH5), but not FTO, maintains the ability of naïve CD4+ T cells to induce adoptive transfer colitis. In addition, T cell-specific ablation of ALKBH5 confers protection against experimental autoimmune encephalomyelitis. During the induced neuroinflammation, ALKBH5 deficiency increased m6A modification on interferon-γ and C-X-C motif chemokine ligand 2 messenger RNA (mRNA), thus decreasing their mRNA stability and protein expression in CD4+ T cells. These modifications resulted in attenuated CD4+ T cell responses and diminished recruitment of neutrophils into the central nervous system. Our findings reveal an unexpected specific role of ALKBH5 as an m6A eraser in controlling the pathogenicity of CD4+ T cells during autoimmunity.

lncRNA PVT1 Promotes Tumorigenesis of Colorectal Cancer by Stabilizing miR-16-5p and Interacting with the VEGFA/VEGFR1/AKT Axis.

Recently, the long noncoding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) was reported to be involved in the pathogenesis of several cancers, including human colorectal cancer (CRC). However, the molecular basis for cancer initiation, development, and progression remains unclear. In this study, we observe that upregulated PVT1 is associated with poor prognosis and bad clinicopathological features of CRC patients. In vitro means of PVT1 loss in a CRC cell line inhibit cell proliferation, migration, and invasion. Furthermore, dual-luciferase reporter and RNA pull-down assays indicated that PVT1 binds to miR-16-5p, which has been shown to play strong tumor suppressive roles in CRC. Targeted loss of miR-16-5p partially rescues the suppressive effect induced by PVT1 knockdown. Vascular endothelial growth factor A (VEGFA), a direct downstream target of miR-16-5p, was suppressed by PVT1 knockdown in CRC cells. Overexpression of VEGFA is known to modulate the AKT signaling cascade by activating vascular endothelial growth factor receptor 1 (VEGFR1). We, therefore, show that PVT1 loss combined with miR-16-5p overexpression reduces tumor volume maximally when propagated within a mouse xenograft model. We conclude that the PVT1-miR-16-5p/VEGFA/VEGFR1/AKT axis directly coordinates the response in CRC pathogenesis and suggest PVT1 as a novel target for potential CRC therapy.

The effects of CTX damage or inhibition of bone marrow hematopoiesis and GM-CSF stimulation of bone marrow hematopoiesis on the peripheral blood TCRß CDR3 repertoire of BALB/c mice.

Objective: This paper aims to investigate the dynamic changes of the T-cell receptor (TCR) ß complementarity-determining region 3 (CDR3) repertoire during cyclophosphamide or Cytoxan (CTX) damage or inhibition of bone marrow hematopoiesis caused by a reduction of peripheral blood white blood cells (WBCs) in BALB/c mice.Methods: We analyze TCR CDR3 repertoire of BALB/c mice including (1) NS control group (2) CTX damage group (3) CTX damage + GM-CSF recovery group (4) CTX damage + auto-recovery group.Results: The number of WBCs in the CTX group is significantly lower than that in the NS group and after GM-CSF injection, the GM-CSF group is higher than that in the NS group. The diversity of the CTX damage group is the highest and there is a significant difference in high-frequency clonal proliferation between the CTX damage group and CTX damage + GM-CSF recovery group compared with the NS control group. In addition, the numbers of unique productive CDR3 overlapping numbers in the four experimental groups are similar.Conclusions: These data reveal that CTX significantly reduced the number of WBCs and ratio of high-frequency TCR CDR3 sequences, and indirectly increased the diversity of the TCR CDR3 repertoire. GM-CSF quickly restored the number of WBCs, and partially restored changes in the TCR CDR3 repertoire induced by CTX. Results from monitoring the dynamic changes of the TCR CDR3 repertoire can be used to assess the effects of CTX and GM-CSF on the function of peripheral blood T cells and to explore the possible underlying mechanisms.

INPP4B exerts a dual function in the stemness of colorectal cancer stem-like cells through regulating Sox2 and Nanog expression.

Inositol polyphosphate 4-phosphatase type II (INPP4B), a lipid phosphatase, was identified as a negative regulator of phosphatidylinositol 3-kinase (PI3K)/Akt signaling in several cancers. The expression and biological function of INPP4B in human colorectal cancer (CRC) are controversial, while the role and molecular mechanism of INPP4B in colorectal cancer stem-like cells (CR-CSLCs) remains unclear. Here, we observed that INPP4B expression was markedly decreased in primary non-metastatic CR-CSLCs and increased in highly metastatic CR-CSLCs compared with corresponding control non-CSLCs. INPP4B overexpression inhibited self-renewal, and chemoresistance of primary non-metastatic CR-CSLCs, but exerted the opposite roles in highly metastatic CR-CSLCs in vitro. Similarly, INPP4B knockdown had dual functions in the self-renewal and chemoresistance of different CR-CSLCs. In addition, we demonstrated that INPP4B overexpression suppressed the tumorigenicity of primary non-metastatic CR-CSLCs while induced the tumorigenicity of highly metastatic CR-CSLCs in nude mice. Furthermore, INPP4B was found to modulate the stemness of CR-CSLCs by regulating Sox2 and Nanog expression, which was dependent on PI3K/PTEN/Akt signaling. In conclusion, our results highlight an important role of INPP4B in the stemness of CR-CSLCs for the first time and emphasize INPP4B as a dual therapeutic target for suppressing primary cancer cell proliferation and for preventing metastasis in CRC patients.

INPP4B inhibits cell proliferation, invasion and chemoresistance in human hepatocellular carcinoma.

Background: Inositol polyphosphate 4-phosphatase type II (INPP4B) has been identified as a negative regulator of phosphatidyl inositol 3-kinase (PI3K)/Akt signaling in human several cancers. However, the expression, clinical significance and biological function of INPP4B in human hepatocellular carcinoma (HCC) clinical tissues and cell lines are little known. Materials and methods: We evaluated the expression of INPP4B in 86 cases of paired human HCC samples by immunohistochemistry, and the clinical significance of INPP4B expression was analyzed. The expression of INPP4B in five HCC cell lines was detected through using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses. The role of INPP4B gene on HCC cell proliferation, apoptosis, migration, invasion as well as epithelial-to-mesenchymal transition (EMT) and chemoresistance was examined via INPP4B mammalian expression vector and small interfering RNA (siRNA) transfection in vitro. Western blot analysis was used to explore the downstream molecules modulated by INPP4B. Results: Immunohistochemistry analysis revealed that INPP4B was significantly downregulated in HCC tissues compared with the corresponding normal tissues. The rate of INPP4B-positive staining was markedly lower in metastatic samples than in those of non-metastatic samples. Univariate analysis showed that INPP4B expression was indicated to have a marked association with histological grades, tumor size and tumor metastasis. Moreover, INPP4B overexpression suppressed cell proliferation, migration, invasion and EMT, but induced cell apoptosis and chemosensitivity in human HCC cell lines. In contrast, INPP4B knockdown had the opposite effects on the biological behaviors of HCC cells. Furthermore, INPP4B was found to inhibit the activation of PI3K/Akt signaling in HCC cells. Conclusion: Our findings suggest that INPP4B is a tumor suppressing gene in human HCC, and might act as a novel therapeutic target for HCC patients.

The PEAK1-PPP1R12B axis inhibits tumor growth and metastasis by regulating Grb2/PI3K/Akt signalling in colorectal cancer.

Pseudopodium enriched atypical kinase 1 (PEAK1), a novel non-receptor tyrosine kinase, was recently implicated in cancer pathogenesis. However, its functional role in colorectal cancer (CRC) is not well known. Herein, we demonstrated that PEAK1 was frequently downregulated in CRC and significantly associated with tumor size, differentiation status, metastasis, and clinical stage. PEAK1 overexpression suppressed CRC cell growth, invasion, and metastasis in vitro and in vivo, whereas knockout had the opposite effects. Further evaluation revealed that PEAK1 expression was positively correlated with protein phosphatase 1 regulatory subunit 12B (PPP1R12B) in CRC cell lines and clinical tissues, and this protein was found to suppress activation of the Grb2/PI3K/Akt pathway. Moreover, PPP1R12B knockdown markedly abrogated PEAK1-mediated tumor suppressive effects, whereas its upregulation recapitulated the effects of PEAK1 knockout on cell behaviours and the activation of signalling. Mechanistically, PI3K and Akt inhibitors reversed impaired the effect of PEAK1 function on cell proliferation, migration, and invasion. Our results provide compelling evidence that the PEAK1-PPP1R12B axis inhibits colorectal tumorigenesis and metastasis through deactivation of the Grb2/PI3K/Akt pathway, which might provide a novel therapeutic strategy for CRC treatment.

Intestinal microbiota: a novel perspective in colorectal cancer biotherapeutics.

It is believed that genetic factors, immune system dysfunction, chronic inflammation, and intestinal microbiota (IM) dysbiosis contribute to the pathogenesis of colorectal cancer (CRC). The beneficial role played by the direct regulation of IM in inflammatory bowel disease treatment is identified by the decreased growth of harmful bacteria and the increased production of anti-inflammatory factors. Interestingly, gut microbiota has been proven to inhibit tumor formation and progression in inflammation/carcinogen-induced CRC mouse models. Recently, evidence has indicated that IM is involved in the negative regulation of tumor immune response in tumor microenvironment, which then abolishes or accelerates anticancer immunotherapy in several tumor animals. In clinical trials, a benefit of IM-based CRC therapies in improving the intestinal immunity balance, epithelial barrier function, and quality of life has been reported. Meanwhile, specific microbiota signature can modulate host's sensitivity to chemo-/radiotherapy and the prognosis of CRC patients. In this review, we aim to 1) summarize the potential methods of IM-based therapeutics according to the recent results; 2) explore its roles and underlying mechanisms in combination with other therapies, especially in biotherapeutics; 3) discuss its safety, deficiency, and future perspectives.

Overexpression of PEAK1 contributes to epithelial-mesenchymal transition and tumor metastasis in lung cancer through modulating ERK1/2 and JAK2 signaling.

Pseudopodium-enriched atypical kinase 1 (PEAK1), a novel non-receptor tyrosine kinase, has been demonstrated to act as an oncogenic regulator in breast and pancreatic cancers. However, the role of PEAK1 in the progression and metastasis of lung cancer is still unknown. Here, we observed that ectopic PEAK1 expression promoted lung cancer cell migration and invasion, while PEAK1 knockout resulted in suppressed cell migration and invasion. Interestingly, cell proliferation did not significantly increase or decrease in either the PEAK1 overexpression or knockout groups compared with the corresponding control cells. In addition, PEAK1 overexpression could induce epithelial-to-mesenchymal transition (EMT) and the expression of matrix metalloproteinase-2 (MMP2) and MMP9 both in vitro and in vivo, whereas PEAK1 knockout had the opposite effects. Then, we had confirmed that PEAK1 was significantly upregulated in lung cancer tissues, and correlated with a higher tumor node metastasis stage. Moreover, PEAK1 upregulation markedly enhanced the activation of extracellular signal-regulated kinase-1/2 (ERK1/2) and Janus kinase-2 (JAK2) signaling in lung cancer cells. Further work demonstrated that the combination of PD98059 with AZD1480 could reverse the effects of PEAK1-induced EMT, cell migration and invasion. Our findings highlight a newer mechanism for PEAK1 in regulating EMT and metastasis in lung cancer, which might serve as a therapeutic target for lung cancer patients.

ERCC1_202 Is A Prognostic Biomarker in Advanced Stage Non-Small Cell Lung Cancer Patients Treated with Platinum-Based Chemotherapy.

Purpose: To develop a qPCR method to examine the 202 isoform of excision repair cross-complementation group 1 (ERCC1_202) and to evaluate its clinical utility as a predictive biomarker for platinum-based chemotherapy in non-small cell lung cancer (NSCLC). Methods: The relative complementary DNA (cDNA) quantification for ERCC1_202 was conducted using a fluorescence-based, real-time detection method and ß-actin was used as a reference gene. Results: A strong correlation was observed between ERCC1_202 mRNA and ERCC1 mRNA levels in NSCLC cells (P < 0.001). 28 patients completed this research. Our results implied that as ERCC1_202 levels increased, the risk of progression (HR = 4.296, P = 0.011) and death (HR = 6.503, P = 0.001) increased. At multivariate analysis, high expression of ERCC1_202 was shown to be an independent predictive factor for time to progression (P = 0.047), and progression-free survival (P = 0.014). However, the high expression of ERCC1_202 was not an independent predictive factor for response (P = 0.324). Conclusions: This study suggests that the efficacy of platinum-based chemotherapy can be improved when customized according to the expression of ERCC1_202.

Elevated Gab2 induces tumor growth and angiogenesis in colorectal cancer through upregulating VEGF levels.

BACKGROUND: Grb2-associated binder 2 (Gab2) is a scaffolding protein that serves as a critical signaling amplifier downstream of tyrosine kinase receptors. Our previous study has shown that Gab2 induces epithelial-to-mesenchymal transition (EMT) and promotes metastasis in colorectal cancer (CRC). However, the role of Gab2 in CRC growth and angiogenesis remains unclear. METHODS: The expression of vascular endothelial growth factor (VEGF) in different colorectal tissues was detected by immunohistochemistry and qRT-PCR to evaluate its correlation with Gab2. Lentiviral vectors bearing Gab2 gene and its small interfering RNAs were constructed and transfected into CRC cell lines. The effects of Gab2 on the cell proliferation in vitro and tumorigenesis in vivo, were examined via CCK­8 assay, colony formation assay as well as tumorigenicity assay respectively. Moreover, to assess its potential role in tumor growth and angiogenesis, the expression of Ki67, CD34 and vascular endothelial growth factor receptor-2 (VEGFR2) were detected by immunohistochemistry in CRC cells tumors. Finally, we evaluated the impact of Gab2 on the expression of c-Myc and VEGF, and the probable effect of mechanistic targeted extracellular signal-regulated kinase (ERK) pathway in suppressing tumor growth and angiogenesis. RESULTS: Up-regulation of Gab2 expression was found to be positively correlated with VEGF in CRC tissues. Exogenous expression of Gab2 obviously promoted, whereas silencing of Gab2 inhibited, proliferation and clone formation of human CRC cells in vitro. Of note, Gab2 enhanced tumorigenesis and tumor growth in mouse xenografts with high Ki67 expression, and led to an increased vessel density with strong CD34 and VEGFR2 activity. In addition, elevated Gab2 expression obviously up-regulated the expression of VEGF, and stimulated the activation of its downstream genes, ERK1/2 and c-Myc in CRC cells. Instead, down-regulated Gab2 expression significantly reduced the levels of VEGF, and inhibited the transduction of ERK/c-Myc pathway. Finally, we revealed that mechanistic target of mitogen-activated protein kinase (MEK) could attenuate Gab2-induced tumor growth and angiogenesis via altering VEGF and c-Myc levels. CONCLUSIONS: The results from our study suggest that Gab2 promotes intestinal tumor growth and angiogenesis through upregulation of VEGF expression mediated by the MEK/ERK/c-Myc pathway.

Peroxiredoxin 1 - an antioxidant enzyme in cancer.

Peroxiredoxins (PRDXs), a ubiquitous family of redox-regulating proteins, are reported of potential to eliminate various reactive oxygen species (ROS). As a major member of the antioxidant enzymes, PRDX1 can become easily over-oxidized on its catalytically active cysteine induced by a variety of stimuli in vitro and in vivo. In nucleus, oligomeric PRDX1 directly associates with p53 or transcription factors such as c-Myc, NF-κB and AR, and thus affects their bioactivities upon gene regulation, which in turn induces or suppresses cell death. Additionally, PRDX1 in cytoplasm has anti-apoptotic potential through direct or indirect interactions with several ROS-dependent (redox regulation) effectors, including ASK1, p66Shc , GSTpi/JNK and c-Abl kinase. PRDX1 is proven to be a versatile molecule regulating cell growth, differentiation and apoptosis. Recent studies have found that PRDX1 and/or PRDX1-regulated ROS-dependent signalling pathways play an important role in the progression and metastasis of human tumours, particularly in breast, oesophageal and lung cancers. In this paper, we review the structure, effector functions of PRDX1, its role in cancer and the pivotal role of ROS in anticancer treatment.

Combined application of anti-VEGF and anti-EGFR attenuates the growth and angiogenesis of colorectal cancer mainly through suppressing AKT and ERK signaling in mice model.

BACKGROUND: Angiogenesis is generally involved during the cancer development and hematogenous metastasis. Vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) are considered to have an important role in tumor-associated angiogenesis. However, the effects of simultaneously targeting on VEGF and EGFR on the growth and angiogenesis of colorectal cancer (CRC), and its underlying mechanisms remain unknown. METHODS: Immunohistochemical staining was used to detect the VEGF and EGFR expression in different CRC tissue specimens, and the correlation between VEGF/EGFR expression with the clinicopathologic features was analyzed. Cell counting kit­8 (CCK-8) and transwell assays were used to assess the cellular proliferation and invasion of CRC cells after treated with anti-VEGF antibody and/or anti-EGFR antibody in vitro, respectively. Moreover, in vivo tumor formation was performed on nude mice model, and the tumor microvessel density (MVD) was determined by anti-CD34 staining in different groups. Finally, we evaluated the impact of anti-VEGF antibody and/or anti-EGFR antibody on the activation of downstream signaling effectors using western blot. RESULTS: VEGF and EGFR were upregulated in CRC tissues, and their expression levels were correlated with hepatic metastasis. Blockage on VEGF or EGFR alone could inhibit the cellular proliferation and metastasis while their combination could reach a good synergism in vitro. In addition, in vivo xenograft mice model demonstrated that the tumor formation and angiogenesis were strongly suppressed by combination treatment of anti-VEGF and anti-EGFR antibodies. Besides, the combination treatment significantly reduced the activation of AKT and ERK1/2, but barely affected the activation of c-Myc, NF-κB/p65 and IκBα in CRC cells tumors. Interestingly, anti-VEGF antibody or anti-EGFR antibody alone could attenuate the phosphorylation of STAT3 as compared with negative control group, whereas the combined application not further suppressed but at least partially restored the activation of STAT3 in vivo. CONCLUSIONS: Simultaneous targeting on VEGF and EGFR does show significant inhibition on CRC tumor growth and angiogenesis in mice model, and these effects are mainly attributed to suppression of the AKT and ERK signaling pathways.

Gab2 facilitates epithelial-to-mesenchymal transition via the MEK/ERK/MMP signaling in colorectal cancer.

BACKGROUND: Grb2-associated binder 2 (Gab2), a scaffolding adaptor protein, has recently been implicated in cancer progression. However, the role of Gab2 in the progression and metastasis of colorectal cancer (CRC) remains unclear. METHODS: Gab2 expression was assessed in CRC patient specimens as well as in CRC cell lines. Recombinant lentivirus vector containing Gab2 gene and its small interfering RNAs were constructed and introduced into CRC cells. Cell migration and invasion ability were evaluated by transwell assays in vitro, and in vivo metastasis was performed on nude mice model. Moreover, the expression of Gab2 and epithelial-to-mesenchymal transition (EMT)-associated proteins (E-cadherin and vimentin) were assessed by western blot and qRT-PCR in CRC cells to evaluate the correlation between Gab2 and EMT. Finally, we evaluated the impact of Gab2 on the activation of its downstream signaling effectors, and furthermore the effects of these pathways on Gab2 induced-EMT were also detected. RESULTS: We confirmed that increased Gab2 expression correlated with higher tumor node metastasis stage and highly invasive CRC cell lines. Ectopic expression of Gab2 promoted metastasis of CRC cells, whereas silencing of Gab2 resulted in inhibited metastasis both in vitro and in vivo. Overexpression of Gab2 in CRC cells induced EMT, whereas knockdown of Gab2 had the opposite effect. Furthermore, upregulation of Gab2 expression obviously stimulated the activation of extracellular signal-regulated kinase-1/2 (ERK1/2), and increased the expression of matrix metalloproteinase-7 (MMP7) and matrix metalloproteinase-9 (MMP9) in CRC cells. Conversely, downregulation of Gab2 expression significantly decreased the activation of ERK1/2, and inhibited MMP7 and MMP9 expression. U0126, an inhibitor of mitogen-activated protein kinase (MEK), can reverse the effects of Gab2 on EMT. CONCLUSIONS: Our work highlights that Gab2 induces EMT through the MEK/ERK/MMP pathway, which in turn promotes intestinal tumor metastasis.

Gab2 is a novel prognostic factor for colorectal cancer patients.

Gab2 (Grb2-associated binder 2), a member of the DOS/Gab family of scaffolding adapters, serves as a critical signal amplifier downstream of various growth factor receptors. Recent studies have identified that Gab2 is overexpressed in several cancer types and that increased Gab2 expression promotes cell proliferation, cell transformation, and tumor progression. Here, we show for the first time that Gab2 protein is overexpressed in clinical colorectal cancer (CRC) specimens. Elevated mRNA (P=0.014) expression and protein (P=0.003) expression of Gab2 were found in most CRC tissues compared with the matched adjacent non-tumor tissues using real-time quantitative reverse transcription PCR (qRT-PCR) and western blotting, respectively. Immunohistochemical analyses showed that Gab2 protein was upregulated in CRC tissues relative to adjacent normal tissues (P<0.001), and this overexpression was significantly correlated with lymph node metastasis (P=0.007), distant metastasis (P<0.001) and TNM stage (P=0.002). According to Kaplan-Meier model, CRC patients with Gab2-positive had a significantly poorer prognosis compared to those with Gab2-negative (P=0.007). Multivariate analysis suggested that the positive expression of Gab2 protein was an independent prognostic factor for CRC patients. In conclusion, our data demonstrated that Gab2 expression may play an important role in the progression of CRC, and underscored that Gab2 has the potential value as a prognostic predictor for CRC patients.

Structure and function of Gab2 and its role in cancer (Review).

The docking proteins of the Grb-associated binder (Gab) family transduce cellular signals between receptors and intracellular downstream effectors, and provide a platform for protein­protein interactions. Gab2, a key member of the Gab family of proteins, is involved in the amplification and integration of signal transduction, evoked by a variety of extracellular stimuli, including growth factors, cytokines and antigen receptors. Gab2 protein lacks intrinsic catalytic activity; however, when phosphorylated by protein­tyrosine kinases (PTKs), Gab2 recruits several Src homology­2 (SH2) domain­containing proteins, including the SH2­containing protein tyrosine phosphatase 2 (SHP2), the p85 subunit of phosphoinositide­3 kinase (PI3K), phospholipase C­Î³ (PLCγ)1, Crk, and GC­GAP. Through these interactions, the Gab2 protein triggers various downstream signal effectors, including SHP2/rat sarcoma viral oncogene/RAF/mitogen­activated protein kinase kinase/extracellular signal­regulated kinase and PI3K/AKT, involved in cell growth, differentiation, migration and apoptosis. It has been previously reported that aberrant Gab2 and/or Gab2 signaling is closely associated with human tumorigenesis, particularly in breast cancer, leukemia and melanoma. The present review aimed to focus on the structure and effector function of Gab2, its role in cancer and its potential for use as an effective therapeutic target.