Homology-2 Domain-containing Research Articles

Capsaicin Enhances Temozolomide-Resistant Glioblastoma Cells’ Chemosensitivity and Ferroptosis through FHOD1/IRF2 Downregulation

Resistance of tumor cells to chemotherapy, particularly in the case of glioblastoma (GBM), a common brain tumor, presents a substantial challenge in oncology. In this study, we investigated the potential of capsaicin to overcome drug resistance in temozolomide (TMZ)-resistant U87 and U251 cells by targeting ferroptosis-mediated interferon regulatory factor (IRF)-2 and formin homology-2 domain-containing protein-1 (FHOD1) signal pathways. First, we induced TMZ resistance in these cells by treating them with TMZ for three weeks. Subsequently, we assessed the impacts of capsaicin on various aspects, including cell viability, proliferation, ferroptosis markers, levels of IRF2 and FHOD1, intracellular iron concentrations, and cell migration in these cells. Our results indicate that capsaicin treatment resulted in a significant decrease in both cell viability and proliferation in TMZ-resistant U87-R and U251-R cells. In addition, it effectively suppressed cell migration rates in these cells, targeting TMZ resistance. Furthermore, capsaicin demonstrated its ability to downregulate FHOD1, IRF2, glutathione, and glutathione peroxidase 4 levels in TMZ-resistant cells. This was accompanied by an increase in intracellular iron, total oxidant status, and increased malondialdehyde levels. Significantly, the treatment with capsaicin led to a notable decrease in the expressions of FHOD1 and IRF2 at both the mRNA and protein levels in U87-R and U251-R cells. In summary, our results emphasize the substantial potential of capsaicin in enhancing the sensitivity of TMZ-resistant GBM cells to chemotherapy. This effect is achieved through its modulation of ferroptosis-related pathways, involving the regulation of FHOD1 and IRF2 expressions.

From bench to bedside: current development and emerging trend of KRAS-targeted therapy.

Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) is the most frequently mutated oncogene in human cancers with mutations predominantly occurring in codon 12. These mutations disrupt the normal function of KRAS by interfering with GTP hydrolysis and nucleotide exchange activity, making it prone to the GTP-bound active state, thus leading to sustained activation of downstream pathways. Despite decades of research, there has been no progress in the KRAS drug discovery until the groundbreaking discovery of covalently targeting the KRASG12C mutation in 2013, which led to revolutionary changes in KRAS-targeted therapy. So far, two small molecule inhibitors sotorasib and adagrasib targeting KRASG12C have received accelerated approval for the treatment of non-small cell lung cancer (NSCLC) harboring KRASG12C mutations. In recent years, rapid progress hasbeen achieved in the KRAS-targeted therapy field, especially the exploration of KRASG12C covalent inhibitors in other KRASG12C-positive malignancies, novel KRAS inhibitors beyond KRASG12C mutation or pan-KRAS inhibitors, and approaches to indirectly targeting KRAS. In this review, we provide a comprehensive overview of the molecular and mutational characteristics of KRAS and summarize the development and current status of covalent inhibitors targeting the KRASG12C mutation. We also discuss emerging promising KRAS-targeted therapeutic strategies, with a focus on mutation-specific and direct pan-KRAS inhibitors and indirect KRAS inhibitors through targeting the RAS activation-associated proteins Src homology-2 domain-containing phosphatase 2 (SHP2) and son of sevenless homolog 1 (SOS1), and shed light on current challenges and opportunities for drug discovery in this field.

From Stem to Sternum: The Role of Shp2 in the Skeleton.

Src homology-2 domain-containing phosphatase 2 (SHP2) is a ubiquitously expressed phosphatase that is vital for skeletal development and maintenance of chondrocytes, osteoblasts, and osteoclasts. Study of SHP2 function in small animal models has led to insights in phenotypes observed in SHP2-mutant human disease, such as Noonan syndrome. In recent years, allosteric SHP2 inhibitors have been developed to specifically target the protein in neoplastic processes. These inhibitors are highly specific and have great potential for disease modulation in cancer and other pathologies, including bone disorders. In this review, we discuss the importance of SHP2 and related signaling pathways (e.g., Ras/MEK/ERK, JAK/STAT, PI3K/Akt) in skeletal development. We review rodent models of pathologic processes caused by germline mutations that activate SHP2 enzymatic activity, with a focus on the skeletal phenotype seen in these patients. Finally, we discuss SHP2 inhibitors in development and their potential for disease modulation in these genetic diseases, particularly as it relates to the skeleton.

Discovery of Novel Src Homology-2 Domain-Containing Phosphatase 2 and Histone Deacetylase Dual Inhibitors with Potent Antitumor Efficacy and Enhanced Antitumor Immunity.

Both Src homology-2 domain-containing phosphatase 2 (SHP2) and histone deacetylase (HDAC) are important oncoproteins and potential immunomodulators. In this study, we first observed a synergistic antiproliferation effect of an allosteric SHP2 inhibitor (SHP099) and HDAC inhibitor (SAHA) in MV4-11 cells. Inspired by this result, a series of SHP2/HDAC dual inhibitors were designed based on the pharmacophore fusion strategy. Among these inhibitors, the most potent compound 8t showed excellent inhibitory activities against SHP2 (IC50 = 20.4 nM) and HDAC1 (IC50 = 25.3 nM). In particular, compound 8t exhibited improved antitumor activities compared with those of SHP099 and SAHA in vitro and in vivo. Our study also indicated that treatment with 8t could trigger efficient antitumor immunity by activating T cells, enhancing the antigen presentation function and promoting cytokine secretion. To our knowledge, we report the first small molecular SHP2/HDAC dual inhibitor and demonstrate a new strategy for cancer immunotherapy.

Discovery of a Novel Src Homology-2 Domain Containing Protein Tyrosine Phosphatase-2 (SHP2) and Cyclin-Dependent Kinase 4 (CDK4) Dual Inhibitor for the Treatment of Triple-Negative Breast Cancer.

The treatment of triple-negative breast cancer (TNBC) remains a huge clinical challenge and dual-targeted small-molecule drugs might provide new therapeutic options for this type of breast cancer. In this work, we discovered a series of SHP2 and CDK4 dual inhibitors through a fused pharmacophore strategy and structural optimization. Notably, lead compound 10 with excellent SHP2 (IC50 = 4.3 nM) and CDK4 (IC50 = 18.2 nM) inhibitory activities effectively induced G0/G1 arrest to prevent the proliferation of TNBC cell lines. Furthermore, compound 10 showed great in vivo pharmacokinetic properties (F = 45.8%) and exerted significant antitumor efficacy in the EMT6 syngeneic mouse model. Western blotting and immunohistochemical analysis confirmed that 10 effectively targeted on both SHP2 and CDK4 and activated the immune response in tumors. These results indicate that lead compound 10, as the first SHP2 and CDK4 dual inhibitor, merits further development for treating TNBC.

TGF-beta-mediated lung fibroblast-to-myofibroblast transition exhibits IFN-γ resistance via SHP2/SOCS3 activation

Abstract Idiopathic pulmonary fibrosis is a lethal disease of unknown etiology. Abnormal activation of TGF-β is a common cause in developing fibrotic lesions, where Src homology-2 domain-containing phosphatase 2 (SHP2) plays a critical regulator in fibroblast activation and fibrosis. We found that a marked expression of phospho-SHP2, phospho-STAT3 and the suppressor of cytokine signaling 3 (SOCS3) were detected in bleomycin-induced rat lung fibrosis, which was highly association with pulmonary parenchymal lesions and collagen deposition. Human pulmonary fibroblasts differentiated into myofibroblasts via TGF-β1 treatment were characterized with the increased bundles of F-actin, enhanced expression of α-smooth muscle actin (α-SMA), fibronectin and collagen, which also exhibited the activation of SHP2, SOCS1/3, PIAS1, STAT3, IL-6 and IL-10. An AKT-regulated glycogen synthase kinase (GSK)-3β/SHP2 axis was induced in ROS-dependent regulation during myofibroblast differentiation. The significant retardation of interferon (IFN)-γ signaling in myofibroblasts was revealed by the decrease expression of phospho-STAT1 and IFN-γ-associated genes along with IFN-γ-inducible protein 10 (IP-10) reduction. Treatment of nintedanib (bibf1120) in myofibroblasts effectively inhibits both SHP2 and SOCS3 activation accompanying by a suppression of fibroblast-to-myofibroblast transition (FMT). Moreover, synergistic treatment of nintedanib and IFN-γ markedly ameliorated TGF-β1-mediated FMT. Accordingly, we speculate that the activation of both ROS/AKT/GSK-3β/SHP2 and IL-6/STAT3/SOCS3 axis signaling causes myofibroblasts hyposensitivity to IFN-γ whereby escaping from IFN-γ immune-surveillance during FMT.

Novel Insights of Lymphomagenesis of Helicobacter pylori-Dependent Gastric Mucosa-Associated Lymphoid Tissue Lymphoma

Gastric mucosa-associated lymphoid tissue (MALT) lymphoma is the most common subtype of gastric lymphoma. Most gastric MALT lymphomas are characterized by their association with the Helicobacter pylori (HP) infection and are cured by first-line HP eradication therapy (HPE). Several studies have been conducted to investigate why most gastric MALT lymphomas remain localized, are dependent on HP infection, and show HP-specific intratumoral T-cells (e.g., CD40-mediated signaling, T-helper-2 (Th2)-type cytokines, chemokines, costimulatory molecules, and FOXP3+ regulatory T-cells) and their communication with B-cells. Furthermore, the reason why the antigen stimuli of these intratumoral T-cells with tonic B-cell receptor signaling promote lymphomagenesis of gastric MALT lymphoma has also been investigated. In addition to the aforementioned mechanisms, it has been demonstrated that the translocated HP cytotoxin-associated gene A (CagA) can promote B-cell proliferation through the activation of Src homology-2 domain-containing phosphatase (SHP-2) phosphorylation-dependent signaling, extracellular-signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), B-cell lymphoma (Bcl)-2, and Bcl-xL. Furthermore, the expression of CagA and these CagA-signaling molecules is closely associated with the HP-dependence of gastric MALT lymphomas (completely respond to first-line HPE). In this article, we summarize evidence of the classical theory of HP-reactive T-cells and the new paradigm of direct interaction between HP and B-cells that contributes to the HP-dependent lymphomagenesis of gastric MALT lymphomas. Although the role of first-line HPE in the treatment of HP-negative gastric MALT lymphoma remains uncertain, several case series suggest that a proportion of HP-negative gastric MALT lymphomas remains antibiotic-responsive and is cured by HPE. Considering the complicated interaction between microbiomes and the genome/epigenome, further studies on the precise mechanisms of HP- and other bacteria-directed lymphomagenesis in antibiotic-responsive gastric MALT lymphomas are warranted.

Expressions of the CagA protein and CagA-signaling molecules predict Helicobacter pylori dependence of early-stage gastric DLBCL

AbstractWe previously reported that early-stage gastric diffuse large B-cell lymphomas (DLBCLs), including DLBCLs with mucosa-associated lymphoid tissue (DLBCL[MALT]) and without (“pure” DLBCL) the features of MALT lymphomas, can achieve long-term complete remission after frontline Helicobacter pylori (HP) eradication (HPE). We recently reported that expression of cytotoxin-associated gene A (CagA) and CagA-signaling molecules (phospho–Src homology-2 domain-containing phosphatase [p-SHP2] and phospho–extracellular signal-regulated kinase [p-ERK]) is associated with HP dependence of gastric MALT lymphoma. However, the significance of CagA and CagA-signaling molecules in gastric DLBCL remains unexplored. The association between expression of CagA, p-SHP-2, and p-ERK in malignant B cells and tumor response to HPE was evaluated in 63 patients with stage IE/IIE1 HP-positive gastric DLBCL who received HPE as frontline treatment. We detected CagA expression in 20 of 42 DLBCL (MALT) cases (47.6%) and in 13 of 21 “pure” DLBCL cases (61.9%). CagA expression was higher in HP-dependent tumors than in HP-independent tumors (74.3% [26 of 35] vs 25.0% [7 of 28]). Patients with CagA expression responded to HPE quicker than those without expression (median time to complete remission, 4.0 months vs 5.0 months). The expression of CagA was closely associated with p-SHP-2 and p-ERK expression. Combined CagA, p-SHP-2, and p-ERK expression showed an increased positive predictive value (81.8% vs 75.9%) and an increased specificity (84.0% vs 75.0%) for HP dependence compared with CagA expression alone. Our results indicated that CagA and its signaling molecules can be detected in the malignant B cells of gastric DLBCL, and the expression of these molecules is clinically and biologically associated with HP dependence.

An increase in galectin-3 causes cellular unresponsiveness to IFN-γ-induced signal transduction and growth inhibition in gastric cancer cells.

Glycogen synthase kinase (GSK)-3β facilitates interferon (IFN)-γ signaling by inhibiting Src homology-2 domain-containing phosphatase (SHP) 2. Mutated phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN) cause AKT activation and GSK-3β inactivation to induce SHP2-activated cellular unresponsiveness to IFN-γ in human gastric cancer AGS cells. This study investigated the potential role of galectin-3, which acts upstream of AKT/GSK-3β/SHP2, in gastric cancer cells. Increasing or decreasing galectin-3 altered IFN-γ signaling. Following cisplatin-induced galectin-3 upregulation, surviving cells showed cellular unresponsiveness to IFN-γ. Galectin-3 induced IFN-γ resistance independent of its extracellular β-galactoside-binding activity. Galectin-3 expression was not regulated by PI3K activation or by a decrease in PTEN. Increased galectin-3 may cause GSK-3β inactivation and SHP2 activation by promoting PDK1-induced AKT phosphorylation at a threonine residue. Overexpression of AKT, inactive GSK-3βR96A, SHP2, or active SHP2D61A caused cellular unresponsiveness to IFN-γ in IFN-γ-sensitive MKN45 cells. IFN-γ-induced growth inhibition and apoptosis in AGS cells were observed until galectin-3 expression was downregulated. These results demonstrate that an increase in galectin-3 facilitates AKT/GSK-3β/SHP2 signaling, causing cellular unresponsiveness to IFN-γ.

Synthesis of Aromatic Amide Derivatives and Their Biological Evaluation against Protein Tyrosine Phosphatase 1B and Scr Homology-2 Domain Containing Protein Tyrosine Phosphatase-2

To explore the chemical space of protein tyrosine phosphatase 1B (PTP1B) inhibitors by changing bis-aromatic amide moiety into aromatic amide moiety, a series of aromatic amide derivatives were designed, synthesized and their biological activities were evaluated against PTP1B and Scr homology-2 domain containing protein tyrosine phosphatase-2 (SHP2). Among them, compound 3c displayed moderate inhibitory activity with IC50 of (5.13±0.21) μmol/L against PTP1B and showed two times selectivity for other related PTPs. Interestingly, compound 12 [IC50=(7.47±1.26) μmol/L] showed moderate inhibitory activity against SHP2 and 2-fold selectivity against PTP1B, T-cell protein tyrosine phosphatase (TCPTP) or Src homology-2 domain containing protein tyrosine phosphatase-1 (SHP1) respectively, and offered a novel scaffold to develop new SHP2 inhibitors.

SHP-2 Mediates Cryptosporidium parvum Infectivity in Human Intestinal Epithelial Cells.

The parasite, Cryptosporidium parvum, induces human gastroenteritis through infection of host epithelial cells in the small intestine. During the initial stage of infection, C. parvum is reported to engage host mechanisms at the host cell-parasite interface to form a parasitophorous vacuole. We determined that upon infection, the larger molecular weight proteins in human small intestinal epithelial host cells (FHs 74 Int) appeared to globally undergo tyrosine dephosphorylation. In parallel, expression of the cytoplasmic protein tyrosine phosphatase Src homology-2 domain-containing phosphatase 2 (SHP-2) increased in a time-dependent manner. SHP-2 co-localized with the C. parvum sporozoite and this interaction increased the rate of C. parvum infectivity through SH2-mediated SHP-2 activity. Furthermore, we show that one potential target that SHP-2 acts upon is the focal adhesion protein, paxillin, which undergoes moderate dephosphorylation following infection, with inhibition of SHP-2 rescuing paxillin phosphorylation. Importantly, treatment with an inhibitor to SHP-2 and with an inhibitor to paxillin and Src family kinases, effectively decreased the multiplicity of C. parvum infection in a dose-dependent manner. Thus, our study reveals an important role for SHP-2 in the pathogenesis of C. parvum. Furthermore, while host proteins can be recruited to participate in the development of the electron dense band at the host cell-parasite interface, our study implies for the first time that SHP-2 appears to be recruited by the C. parvum sporozoite to regulate infectivity. Taken together, these findings suggest that SHP-2 and its down-stream target paxillin could serve as targets for intervention.

Loss of PTEN causes SHP2 activation, making lung cancer cells unresponsive to IFN-γ

Src homology-2 domain-containing phosphatase (SHP) 2, an oncogenic phosphatase, inhibits type II immune interferon (IFN)-γ signaling by subverting signal transducers and activators of transcription 1 tyrosine phosphorylation and activation. For cancer immunoediting, this study aimed to investigate the decrease of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein, leading to cellular impairment of IFN-γ signaling. In comparison with human lung adenocarcinoma A549 cells, the natural PTEN loss in another human lung adenocarcinoma line, PC14PE6/AS2 cells, presents reduced responsiveness in IFN-γ-induced IFN regulatory factor 1 activation and CD54 expression. Artificially silencing PTEN expression in A549 cells also caused cells to be unresponsive to IFN-γ without affecting IFN-γ receptor expression. IFN-γ-induced inhibition of cell proliferation and cytotoxicity were demonstrated in A549 cells but were defective in PC14PE6/AS2 cells and in PTEN-deficient A549 cells. Aberrant activation of SHP2 by ROS was specifically shown in PC14PE6/AS2 cells and PTEN-deficient A549 cells. Inhibiting ROS and SHP2 rescued cellular responses to IFN-γ-induced cytotoxicity and inhibition of cell proliferation in PC14PE6/AS2 cells. These results demonstrate that a decrease in PTEN facilitates ROS/SHP2 signaling, causing lung cancer cells to become unresponsive to IFN-γ.

Atg7 enhances host defense against infection via downregulation of superoxide but upregulation of nitric oxide.

Pseudomonas aeruginosa is an opportunistic bacterium that can cause serious infection in immunocompromised individuals. Although autophagy may augment immune responses against P. aeruginosa infection in macrophages, the critical components and their role of autophagy in host defense are largely unknown. In this study, we show that P. aeruginosa infection-induced autophagy activates JAK2/STAT1α and increases NO production. Knocking down Atg7 resulted in increased IFN-γ release, excessive reactive oxygen species, and increased Src homology-2 domain-containing phosphatase 2 activity, which led to lowered phosphorylation of JAK2/STAT1α and subdued expression of NO synthase 2 (NOS2). In addition, we demonstrated the physiological relevance of dysregulated NO under Atg7 deficiency as atg7(-/-) mice were more susceptible to P. aeruginosa infection with increased mortality and severe lung injury than wild-type mice. Furthermore, P. aeruginosa-infected atg7(-/-) mice exhibited increased oxidation but decreased bacterial clearance in the lung and other organs compared with wild-type mice. Mechanistically, atg7 deficiency suppressed NOS2 activity by downmodulating JAK2/STAT1α, leading to decreased NO both in vitro and in vivo. Taken together, these findings revealed that the JAK2/STAT1α/NOS2 dysfunction leads to dysregulated immune responses and worsened disease phenotypes.

PD-1/SHP-2 negatively regulate Tc1/Th1 phenotypic responses and activation of T cells in the tumor microenvironment

Rejection of tumor cells by a robust cellular immune response relies on production of type 1 cytokines (such as IFN-γ) and cytolytic activity of T cells. Programmed Death 1 (PD-1), a co-inhibitory receptor proposed to represent T cell dysfunction, is highly expressed on tumor infiltrating lymphocytes (TIL) [1], and may reflect T cell exhaustion marked by decreased proliferation, production of type 1 cytokines and poor cytolytic activity [2]. T-bet, a T-box transcription factor which can be activated by phosphorylated signal transducers and activators of transcription 1 (p-STAT1), plays an important role in Tc1/Th1 skewing. Although anti-PD-1 antibodies enhance IFN-γ secretion after TCR stimulation [3], the mechanistic link between PD-1 and Tc1/Th1 skewing remains unclear. In prospectively collected cancer tissues, TIL manifested dampened Tc1/Th1 skewing and activation compared to PBL (Figure ​(Figure11 and ​and2).2). In addition, PD-1 triggering using PD-L1 coated beads further suppressed TCR-stimulated upregulation of p-STAT1, T-bet and p-S6 as well as Th1 cytokines, while PD-1 blockade reversed suppressive effects of PD-1: PD-L1 ligation (Figure ​(Figure3).3). We also found that Src homology-2 domain-containing phosphatase (SHP-2) is higher in TIL than in PBL, tightly correlates with PD-1 expression (Figure ​(Figure4),4), and negatively regulates STAT1 and T-bet activation (Figure ​(Figure5).5). Thus, the PD-1/SHP-2/p-STAT1/T-bet axis provides an important mechanism for PD-1 suppression of type 1 immunity at tumor sites. PD-1 blocking Abs, which are clinically effective in several solid cancers, should improve T cell-based cancer immunotherapy by restoring robust type 1 immunity and T cell activation to reverse immunosuppression in the tumor microenvironment. SHP-2 inhibitory strategies may also be useful to improve type 1-biased TIL. Figure 1 CD8+ TIL have dampened Tc1 phenotypic responses and activation compared to PBL. p-STAT1, T-bet, p-STAT4 and p-S6 levels in CD8+ PBL and TIL from HNC patients were analyzed by intracellular flow cytometry. Representative figures (A) and summary data (B) ... Figure 2 CD4+ TIL show abortive Th1 differentiation and low activation compared with PBL. p-STAT1, T-bet, p-STAT4 and p-S6 levels in CD4+ PBL and TIL from HNC patients were analyzed by intracellular flow cytometry. Representative figures (A) and summary data (B) ... Figure 3 PD-1 ligation with bead-coated PD-L1 suppressed TCR-stimulated up-regulation of p-STAT1, T-bet and production of Th1 cytokines, while anti-PD-1 blockade could reverse the suppressive effects of PD-1. Total TIL were stimulated with anti-CD3/-CD28/hIgG1 ... Figure 4 SHP-2 activation by fusaruside suppresses p-STAT1/T-bet and production of Th1 cytokines upon TCR stimulation. Total TIL were stimulated with anti-CD3/-CD28/hIgG1 beads (bead: cell = 10:1) or anti-CD3/-CD28/PD-L1 beads plus 100 ug/mL anti-PD-1 blockade ... Figure 5 SHP-2 activation by fusaruside suppresses p-STAT1/T-bet and production of Th1 cytokines upon TCR stimulation. Total TIL were stimulated with anti-CD3/-CD28/hIgG1 beads (bead: cell = 10:1) or anti-CD3/-CD28/PD-L1 beads plus 100 ug/mL anti-PD-1 blockade ...

Abstract 5281: Role of SHP-2 tyrosine phosphatase in colorectal carcinogenesis

Abstract SHP-2 (Src homology-2 domain-containing phosphatase 2) is a tyrosine phosphatase ubiquitously expressed. Previous work done in SHP-2-/- fibroblasts demonstrated its implication in proliferation and migration as well as in the regulation of many signaling pathways including the Ras-Raf/MEK/ERK, the PI3K/AKT and the Jak/STAT pathways(1). Importantly, gain-of-function mutations were discovered in some colorectal cancers (CRCs)(2). However, the role of SHP-2 in intestinal tumorigenesis has never been investigated. Methods: SHP-2 expression and catalytic activity were analyzed by Western blot, qPCR and immunohistochemistry in human CRC cells and in human tumours from different stages. SHP-2 expression was also analyzed in polyps (adenomas) from mice bearing a mutation in Apc gene (APCMin/+ mice). To elucidate the role of SHP-2, RNA interference was used to specifically downregulate its expression in intestinal epithelial cells transformed or not by the oncogenic form of KRASG12V (IEC/KRas). Proliferation (cell counting), invasion (through Matrigel in Boyden chamber), anchorage-independent growth (soft agar) and tumor formation in nude mice (xenograft assays) were analyzed. Results: 1- SHP-2 mRNA and protein levels were significantly increased in human CRC cells and tumors, especially in adenomas, in comparison to healthy adjacent tissues. 2- Interestingly, polyps from APCMin/+ mice also exhibited marked increase in SHP-2 protein expression in comparison to normal adjacent intestinal epithelium. 3- SHP-2 silencing in IEC/KRAS cells markedly reduced their proliferation rate, their growth in soft agar, their capacity to migrate and invade Matrigel and finally, their capacity to form tumor in vivo, in nude mice. 4- Of note, phosphorylated and activated levels of MEK and ERK kinases were significantly decreased following SHP-2 silencing in IECs particularly in those expressing the mutated form of KRAS. Conclusion: Our data suggest that SHP-2 promotes malignancy of intestinal epithelial cells by sustaining the oncogenic activation of MEK/ERK signalling during intestinal tumorigenesis. (1)Shi ZQ, Lu W, Feng GS., J Biol Chem. 1998 Feb 27;273(9):4904-8. (2)Bentires-Alj M. et al., Cancer Res. 2004 Dec 15;64(24):8816-20. Citation Format: Jessica Gagne Sansfacon, Geneviève Coulombe, Nadia Bourdages, Nathalie Rivard. Role of SHP-2 tyrosine phosphatase in colorectal carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5281. doi:10.1158/1538-7445.AM2014-5281

CagA+ H. pylori filtrate induces cytokine IL-8 secretion by esophageal squamous carcinoma EC 109 cells via a p38 pathway.

The relationship between Helicobacter pylori (Hp) infection and the risk of esophageal squamous cell carcinoma is unclear. The purpose was to investigate the effects of CagA+ Hp on esophageal squamous carcinoma Ec 109 cells in vitro and explore the molecular mechanisms underlying these effects. Ec 109 cells were treated with CagA+ Hp filtrate at a concentration of 1.0 mg/mL or 50 μg/mL in vitro, proliferation and apoptosis of Ec 109 cells were assayed, the secretion of IL-8 was measured by ELISA, and the levels of Src homology-2 domain-containing phosphatase (SHP-2) mRNAs was assayed by RT-PCR.. Furthermore, after pretreatment of Ec109 cells with the specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, the p38 pathway was detected. CagA+ Hp filtrate enhanced both the proliferation and apoptosis of Ec 109 cells. In addition, cytokine IL-8 release was significantly increased, and the expression of SHP-2 mRNA declined sharply in the CagA+ Hp group. Furthermore, after pretreatment of Ec109 cells with the specific p38 MAPK inhibitor SB203580, Ec109 cells proliferation and IL-8 secretion were inhibited. Our results suggest that CagA+ Hp filtrates could induce proliferation and the secretion of IL-8 by Ec109 cells in vitro. IL-8 secretion was induced through the activation of the p38 MAPK signal pathway.

Research progress of protein tyrosine phosphatase SHP-2

The Src homology-2 domain-containing phosphatase SHP-2 encoded by PTPN11 is an essential component in several signaling pathways.Different types of mutation in SHP-2 have been confirmed in several types of leukemia and solid tumors. Elucidation of the events underlying Shp2-evoked transformation may provide new insights into the novel targets for anti-cancer therapy.

Regulation of SHP2 by PTEN/AKT/GSK-3β signaling facilitates IFN-γ resistance in hyperproliferating gastric cancer (162.8)

Abstract In addition to oncogenic activation, and tumor suppressor gene inactivation escape from immune surveillance, such as IFN-γ resistance, is critical for cancer cell growth and survival. In this study, we investigated the crosstalk signaling between IFN-γ resistance and signaling of hyperproliferation in gastric cancer cells. IFN-γ inhibited the cell growth of MKN45 cells but not hyperproliferating AGS cells. AGS cells did not respond to IFN-γ because of a decrease in STAT1 but not due to dysfunctional IFN-γ receptors. Signaling of PI3K/AKT, as well as MEK/ERK, was required for the hyperproliferation; notably, PI3K/AKT alone mediated the IFN-γ resistance. Aberrant Src homology-2 domain-containing phosphatase (SHP) 2 determined IFN-γ resistance but unexpectedly had no effects on hyperproliferation or ERK activation. In the IFN-γ resistant cells, inactivation of glycogen synthase kinase (GSK)-3β by PI3K/AKT was important for SHP2 activation but not for hyperproliferation. An imbalance of AKT/GSK-3β/SHP2 caused by a reduction of PTEN was important for the crosstalk between IFN-γ resistance and hyperproliferation. PI3K is constitutively expressed in AGS cells and immunohistochemical staining showed a correlation between hyperproliferation and expression of SHP2 and STAT1 in gastric tumors. These results demonstrate the effects of PTEN/AKT/GSK-3β/SHP2 signaling on IFN-γ resistance in hyperproliferating gastric cancer cells.

Regulation of SHP2 by PTEN/AKT/GSK-3β signaling facilitates IFN-γ resistance in hyperproliferating gastric cancer

Oncogenic activation accompanied by escape from immune surveillance, such as IFN-γ resistance, is critical for cancer cell growth and survival. In this study, we investigated the crosstalk signaling between IFN-γ resistance and signaling of hyperproliferation in gastric cancer cells. IFN-γ inhibited the cell growth of MKN45 cells but not hyperproliferating AGS cells. AGS cells did not respond to IFN-γ because of a decrease in STAT1 but not due to dysfunctional IFN-γ receptors. Signaling of PI3K/AKT, as well as MEK/ERK, was required for the hyperproliferation; notably, PI3K/AKT alone mediated the IFN-γ resistance. Aberrant Src homology-2 domain-containing phosphatase (SHP) 2 determined IFN-γ resistance but unexpectedly had no effects on hyperproliferation or ERK activation. In the IFN-γ resistant cells, inactivation of glycogen synthase kinase (GSK)-3β by PI3K/AKT was important for SHP2 activation but not for hyperproliferation. An imbalance of AKT/GSK-3β/SHP2 caused by a reduction of PTEN was important for the crosstalk between IFN-γ resistance and hyperproliferation. PI3K is constitutively expressed in AGS cells and immunohistochemical staining showed a correlation between hyperproliferation and expression of SHP2 and STAT1 in gastric tumors. These results demonstrate the effects of PTEN/AKT/GSK-3β/SHP2 signaling on IFN-γ resistance in hyperproliferating gastric cancer cells.

Glycogen synthase kinase‐3β indirectly facilitates interferon‐γ‐induced nuclear factor‐κB activation and nitric oxide biosynthesis

Either glycogen synthase kinase (GSK)-3β or nuclear factor (NF)-κB regulates interferon (IFN)-γ-induced nitric oxide (NO) biosynthesis; however, the inter-regulation between GSK-3β and NF-κB is unknown. We have previously shown that IFN-γ-activated GSK-3β negatively regulates Src homology-2 domain-containing phosphatase (SHP) 2 to facilitate Janus kinase (Jak) 2-signal transducer and activator of transcription (STAT) 1 activation. Because Jaks-IFN-inducible dsRNA-activated serine-threonine protein kinase (PKR) axis signaling is essential for IFN-γ-activation of NF-κB, in this study we investigate the potential mechanism for GSK-3β-facilitated NF-κB activation in IFN-γ-stimulated RAW264.7 murine macrophages. Pharmacological inhibitors of GSK-3β or NF-κB signaling, such as the inhibitor of κB (IκB) kinase β (IKKβ) and IκBα, inhibited IFN-γ-induced inducible NO synthase (iNOS) and thus NO biosynthesis. Inhibiting GSK-3β decreased IFN-γ-induced NF-κB phosphorylation (Ser536) and activation. The upstream regulators for GSK-3β activation, including okadaic acid-sensitive protein phosphatase and proline-rich tyrosine kinase 2, were also important for IFN-γ-induced IκBα phosphorylation (Ser32) and degradation. Under IFN-γ stimulation, Jak2-PKR axis signaling induced IκBα inactivation as well as iNOS/NO biosynthesis. It is notable that inhibiting GSK-3β caused SHP2-mediated dephosphorylation of PKR (Thr446), IKKβ (Ser180), and NF-κB (Ser536). Taken together, we provide the first evidence to demonstrate that GSK-3β indirectly facilitates IFN-γ-induced NF-κB activation by inhibiting SHP2, in turn activating the PKR-IKKβ-IκBα axis signaling pathway.