SHP-2 Knockdown Research Articles

Interaction between CD244 and SHP2 regulates inflammation in chronic obstructive pulmonary disease via targeting the MAPK/NF-κB signaling pathway.

This study delved into the interplay between CD244 and Src Homology 2 Domain Containing Phosphatase-2 (SHP2) in chronic obstructive pulmonary disease (COPD) pathogenesis, focusing on apoptosis and inflammation in cigarette smoke extract (CSE)-treated human bronchial epithelial (HBE) cells. Analysis of the GSE100153 dataset identified 290 up-regulated and 344 down-regulated differentially expressed genes (DEGs). Weighted gene co-expression network analysis (WGCNA) highlighted the turquoise module had the highest correlation with COPD samples. Functional enrichment analysis linked these DEGs to critical COPD processes and pathways like neutrophil degranulation, protein kinase B activity, and diabetic cardiomyopathy. Observations on CD244 expression revealed its upregulation with increasing CSE concentrations, suggesting a dose-dependent relationship with inflammatory cytokines (IL-6, IL-8, TNF-α). CD244 knockdown mitigated CSE-induced apoptosis and inflammation, while overexpression exacerbated these responses. Co-immunoprecipitation (Co-IP) confirmed the physical interaction between CD244 and SHP2, emphasizing their regulatory connection. Analysis of Concurrently, the Nuclear Factor-kappa B (NF-κB) and Mitogen-activated protein kinase (MAPK) signaling pathways showed that modulating CD244 expression impacted key pathway components (p-JNK, p-IKKβ, p-ERK, p-P38, p-lkBα, p-P65), an effect reversed upon SHP2 knockdown. These findings underscore the pivotal role of the CD244/SHP2 axis in regulating inflammatory and apoptotic responses in CSE-exposed HBE cells, suggesting its potential as a therapeutic target in COPD treatment strategies.

MEST promotes immune escape in gastric cancer by downregulating MHCI expression via SHP2

BackgroundImmune escape is a major obstacle to T-cell-based immunotherapy for cancers such as gastric cancer (GC). Mesoderm-specific transcript (MEST) is a tumor-promoting factor that regulates multiple oncogenic signaling pathways. However, the role of MEST-mediated immune escape is unclear. MethodsBioinformatics analysis of MEST expression and enrichment pathways were performed Quantitative reverse transcription PCR (qPCR) or western blot was used to detect the expression of MEST, Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2), Major histocompatibility class I (MHCI)-related genes. Cell function was assessed by Cell Counting Kit (CCK)-8, Transwell, Lactate dehydrogenase (LDH) kit, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC). Xenograft nude mice and immune-reconstructed mice were used to test the effects of different treatments on tumor growth and immune escape in vivo. ResultsMEST was upregulated in GC and promoted tumor proliferation, migration, and invasion. Rescue experiments revealed that TNO155 treatment or knockdown of SHP2 promoted the killing ability of CD8+ T cells and the expression of granzyme B (GZMB) and interferon-gamma (IFN-γ), and MEST overexpression reversed the effect. In vivo experiments confirmed that MEST promoted tumor growth, knockdown of MEST inhibited immune escape in GC, and that combination treatment with anti-PD-1 improved anti-tumor activity. ConclusionIn this study, we demonstrated that MEST inhibited IFN-γ secretion from CD8+ T cells by up-regulating SHP2, thereby downregulating MHCI expression in GC cells to promote immune escape and providing a new T cell-based therapeutic potential for GC.

Fragment-Based Discovery of Allosteric Inhibitors of SH2 Domain-Containing Protein Tyrosine Phosphatase-2 (SHP2).

The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signaling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signaling and inhibit the proliferation of RTK-driven cancer cell lines. Here, we describe the first reported fragment-to-lead campaign against SHP2, where X-ray crystallography and biophysical techniques were used to identify fragments binding to multiple sites on SHP2. Structure-guided optimization, including several computational methods, led to the discovery of two structurally distinct series of SHP2 inhibitors binding to the previously reported allosteric tunnel binding site (Tunnel Site). One of these series was advanced to a low-nanomolar lead that inhibited tumor growth when dosed orally to mice bearing HCC827 xenografts. Furthermore, a third series of SHP2 inhibitors was discovered binding to a previously unreported site, lying at the interface of the C-terminal SH2 and catalytic domains.

Plasma-derived exosomal protein SHP2 deficiency induces neutrophil hyperactivation in Behcet's uveitis

To investigate the effect of plasma-derived exosomal proteins on neutrophil hyperactivation in Behcet's uveitis (BU), we treated neutrophils from healthy controls with plasma-derived exosomes from active BU patients, and determined the level of neutrophil activation by real-time quantitative PCR (RT-qPCR) and cytokine detection assay. The results revealed that exosomes from active BU patients could activate neutrophils as shown by increasing the expression levels of pro-inflammatory cytokines (IL-17 and IL-6), chemokines (IL-8 and MCP-1), and NETs (MPO and ELANE). Label-free quantitative proteomic analysis of plasma-derived exosomes from patients and healthy controls found a remarkably distinct protein profile and identified differentially expressed proteins (DEPs) between the two groups. The results of GO, KEGG, and GSEA enrichment analysis showed that DEPs were enriched in innate immune-mediated and neutrophil hyperactivation-related signaling pathways. The protein-protein interaction (PPI) analysis determined that SHP2 was a downregulated key hub protein in the exosomes of active BU patients. Knockdown of SHP2 in human neutrophil cell lines (NB4 cells) was shown to promote the secretion of pro-inflammatory cytokines, chemokines, and NETs. The converse effects were observed following SHP2 overexpression. In conclusion, we highlighted a pathogenic role of plasma-derived exosomal SHP2 deficiency in facilitating neutrophil activation and suggested that SHP2 might be an immunoprotective factor in BU pathologic process.

Abstract C141: Targeting SHP2-dependent adaptive resistance to BRAF and MEK inhibition in gliomas

Abstract Introduction: BRAF-mutant gliomas, common in children, are sensitive to MEK and/or BRAF inhibitors. However, responses are incomplete and can be short-lived, due to poorly understood mechanisms of resistance that are likely to involve kinome re-programming, treatment-emergent resistance mutations, and adaptive resistance, by which upstream signaling components become activated due to release of negative feedback. Based on the critical role of SHP2 upstream of RAS pathway, we postulated that the combination of SHP2i and BRAFi/MEKi would maintain superior ERK pathway inhibition in gliomas. Here, we demonstrate that combined SHP2i+BRAFi can be efficacious in BRAFV600E mutant gliomas, potentially without the toxicity posed by SHP2i+MEKi in human participants. Methods: Using a panel of BRAFV600E mutant and wild-type glioma cell lines, we examined cell growth with Incucyte live-cell imaging, RAS activity with pull-down assay, and immunoblots of ERK signaling activity in response to MEKi (Trametinib), BRAFi (Dabrafenib), and/or SHP2i (TNO155). NSG immune-deficient mice were implanted subcutaneously with human-derived glioma lines and patient-derived xenografts (PDXs), then treated via oral gavage for ≤28 days while recording tumor volume and weight. Results: In BRAFV600E glioma cells, we observed ERK reactivation and increased RAS activity in response to BRAFi or MEKi. SHP2 knockdown using PTPN11 pooled siRNA partially inhibited cell growth, while the addition of BRAFi/MEKi enhanced growth inhibition compared with either intervention alone. BRAFV600E cells showed insensitivity to small molecule SHP2i monotherapy, but augmented growth inhibition upon the combination with either BRAFi or MEKi due to prolonged ERK pathway suppression. BRAF wt glioma cell lines exhibit sensitivity to TNO155 monotherapy, and combination with MEKi demonstrates sustained pERK suppression in NF1-mut and NF1 wt cell lines. Additionally, we evaluated the effect of combined BRAFi+SHP2i in heterotopic xenografts using human-derived glioma lines and PDXs. In both models, tumor growth was significantly slower in the combined treatment group than either monotherapy. Moreover, mice tolerated the combination well with no weight loss or lethargy. Conclusion. Combined MEKi and SHP2i attenuate RAS reactivation in response to MEKi and demonstrates additive effects in glioma cell lines that are resistant to MEKi monotherapy. Moreover, SHP2i in combination with BRAFi in BRAFV600E mutant glioma cells synergistically attenuated tumor growth in vitro and shows superior preclinical activity to BRAFi or SHP2i alone in BRAFV600E models of gliomas. Citation Format: Abiola Abdulrahman Ayanlaja, Kriti Lalwani, Maria Ioannou, Christine Pratilas, Karisa Schreck. Targeting SHP2-dependent adaptive resistance to BRAF and MEK inhibition in gliomas [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C141.

SHP2 potentiates anti-PD-1 effectiveness through intervening cell pyroptosis resistance in triple-negative breast cancer

Triple negative breast cancer (TNBC) presents a formidable challenge due to the lack of effective treatment modalities. Immunotherapy stands as a promising therapeutic approach; however, the emergence of drug resistance mechanisms within tumor cells, particularly those targeting apoptosis and pyroptosis, has hampered its clinical efficacy. SHP2 is intricately involved in diverse physiological processes, including immune cell proliferation, infiltration, and tumor progression. Nevertheless, the precise contribution of SHP2 to tumor cell pyroptosis resistance remains inadequately understood. Herein, we demonstrate that SHP2 inhibition hampers the proliferative, migratory, and invasive capabilities of TNBC, accompanied by noticeable alterations in cellular membrane architecture. Mechanistically, we provide evidence that SHP2 depletion triggers the activation of Caspase-1 and GSDMD, resulting in GSDMD-dependent release of LDH, IL-1β, and IL-18. Furthermore, computational analyses and co-localization investigations substantiate the hypothesis that SHP2 may hinder pyroptosis through direct binding to JNK, thereby impeding JNK phosphorylation. Our cellular experiments further corroborate these findings by demonstrating that JNK inhibition rescues pyroptosis induced by SHP2 knockdown. Strikingly, in vivo experiments validate the suppressive impact of SHP2 knockdown on tumor progression via enhanced JNK phosphorylation. Additionally, SHP2 knockdown augments tumor sensitivity to anti-PD-1 therapy, thus reinforcing the pro-pyroptotic effects and inhibiting tumor growth. In summary, our findings elucidate the mechanism by which SHP2 governs TNBC pyroptosis, underscoring the potential of SHP2 inhibition to suppress cell pyroptosis resistance and its utility as an adjunctive agent for tumor immunotherapy.

Knockdown of SHP2 attenuated LPS-induced ferroptosis via downregulating ACSL4 expression in acute lung injury.

Acute lung injury (ALI) is a complex disease with a high mortality. Src homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) is a protein tyrosine phosphatase that participates in pathogenesis of multiple diseases. Nevertheless, the role of SHP2 in ALI remains unknown. The in vivo and in vitro lipopolysaccharide (LPS)-induced ALI models were successfully established. The histopathological changes were evaluated by hematoxylin and eosin staining. The vascular permeability of lungs was assessed by Evans blue assay. The expression of ACSL4 and SHP2 was detected by western blot and qRT-PCR assay. The lactate dehydrogenase (LDH) activity, malondialdehyde (MDA), iron, and glutathione (GSH) levels were measured by commercial kits. The SHP2 was upregulated in LPS-induced ALI mice and LPS-stimulated MLE-12 cells. In loss-of function experiment, the knockdown of SHP2 attenuated LPS-induced lung injury, microvessels damage, pulmonary edema, and increase of lung vascular permeability in vivo. Mechanically, shSHP2-rescued LPS induced increase in LDH activity, MDA, and iron levels, and decrease in GSH levels, as well as the accumulation of reactive oxygen species in vivo and in vitro, leading to an alleviation of LPS-induced ferroptosis. Notably, shSHP2 reduced the expression of Acyl-CoA synthetase long-chain 4 (ACSL4). In the rescued experiments, overexpression of ACSL4 abolished the shSHP2-induced reduction of LDH activity, MDA, and iron levels, and increase in GSH levels, thereby aggravating the LPS-induced ferroptosis. These findings concluded that the knockdown of SHP2 attenuated LPS-induced ferroptosis via downregulation of ACSL4 expression in ALI, providing a novel sight for ALI treatment.

Early Combined SHP2 Targeting Reverses the Therapeutic Resistance of Vemurafenib in Thyroid Cancer.

The BRAFV600E mutation is the most common oncogenic mutation in thyroid cancer, suggesting an aggressive subtype of thyroid cancer and poor prognosis. Vemurafenib, a selective inhibitor of BRAFV600E, may provide therapeutic benefit in various cancers including thyroid cancer. However, the prevalence of drug resistance remains a challenge because of the feedback activation of the MAPK/ERK and PI3K/AKT pathways. In treating thyroid cancer cells with vemurafenib, we have detected reactivation of the MAPK/ERK signaling pathway as a result of the release of multiple receptor tyrosine kinases (RTKs) from the negative feedback of ERK phosphorylation. SHP2 is an important target protein downstream of the RTK signaling pathway. Decreasing it through SHP2 knockdown or the use of an inhibitor of SHP2 (SHP099) was found to significantly increase the early sensitivity and reverse the late resistance to vemurafenib in BRAFV600E mutant thyroid cancer cells. Overall, our findings suggest that blocking SHP2 reverses the reactivation of the MAPK/ERK signaling pathway caused by the activation of RTKs and improves the sensitivity of thyroid cancer to vemurafenib, which has potential implications for mechanism-based early combination strategies to treat thyroid cancer.

Effect of SHP2 knockdown on the proliferation and osteogenic differentiation of human periodontal ligament stem cells under inflammatory environment

Effect of SHP2 knockdown on the proliferation and osteogenic differentiation of human periodontal ligament stem cells under inflammatory environment

Abstract A020: Combined inhibition of SHP2 and CDK4/6 is active in preclinical models of NF1-associated malignant peripheral nerve sheath tumor

Abstract Background: NF1 is an essential negative regulator of RAS activity and its function is lost in nearly 90% of malignant peripheral nerve sheath tumors (MPNST). Additional recurrent molecular changes include loss of function alterations in CDKN2A, TP53, EED and SUZ12, but molecular targeting of these genomic events represents a unique challenge. We previously reported that the efficacy of MEK inhibitor is limited by adaptive activation of receptor tyrosine kinases and the adaptor protein SHP2, and that combined inhibition of MEK and SHP2 is effective in MPNST. However, the clinical potential of combination MEK and SHP2 inhibitors may be limited by the overlapping toxicity induced by ERK pathway inhibition. Loss of CDKN2A, inactivation of RB1, and hyperactivation of cyclin dependent kinases (CDK) in MPNST also suggest that small-molecule CDK4/6 inhibitors (CDK4/6i) may be a potential therapeutic strategy, but monotherapy with CDK4/6i also demonstrates limited activity. We hypothesize that the anti-tumor response of SHP2i may be potentiated by agents targeting the cell cycle in combination. Methods: The effects of shRNA-mediated inducible SHP2 knockdown on RAS signaling, short-term and long-term cell growth, and response to CDK4/6i were examined using immunoblotting, high throughput proliferation assays, and colony formation assays. Combined effects of SHP2i plus CDK4/6i on signaling, cell cycle, apoptosis, cell and in vivo tumor growth were assessed. Pharmacodynamic (PD) assays were performed on tumors extracted following drug treatment in patient-derived xenograft (PDX) models of MPNST. Results: Despite a modest effect of SHP2 knockdown on ERK signaling, shPTPN11 reduced MPNST cell growth. SHP2 knockdown or SHP2i treatment alleviated activation of ERK signaling and cyclin D1 expression induced by CDK4/6i, and enhanced the sensitivity to CDK4/6i. Combination benefit was observed in in vitro cell growth and in vivo PDX. Although some PDX models demonstrated similar responses to SHP2i alone or SHP2i + CDK4/6i during the initial four weeks on treatment, we found sustained tumor growth inhibition exerted by the combination on longer therapy. PD studies demonstrated a decrease in p-ERK levels in tumors treated with either SHP2i alone or the SHP2i/CDK4/6i combination, as well as a synergistic suppression of cell cycle regulators by the combination. Conclusions: Our preliminary data demonstrate that the combined inhibition of SHP2 and CDK4/6 is active and produces deep and durable response in models of NF1-associated MPNST. This combination strategy may represent a novel treatment approach for patients with MPNST. Citation Format: Jiawan Wang, Ana Calizo, Kai Pollard, Lindy Zhang, John M. Gross, Nicolas Llosa, Angela C. Hirbe, Christine A. Pratilas. Combined inhibition of SHP2 and CDK4/6 is active in preclinical models of NF1-associated malignant peripheral nerve sheath tumor [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A020.

Deficiency of the Src homology phosphatase 2 in podocytes is associated with renoprotective effects in mice under hyperglycemia.

Diabetic nephropathy (DN) is a significant complication of diabetes and the leading cause of end-stage renal disease. Hyperglycemia-induced dysfunction of the glomerular podocytes is a major contributor to the deterioration of renal function in DN. Previously, we demonstrated that podocyte-specific disruption of the Src homology phosphatase 2 (Shp2) ameliorated lipopolysaccharide-induced renal injury. This study aims to evaluate the contribution of Shp2 to podocyte function under hyperglycemia and explore the molecular underpinnings. We report elevated Shp2 in the E11 podocyte cell line under high glucose and the kidney under streptozotocin- and high-fat diet-induced hyperglycemia. Consistently, Shp2 disruption in podocytes was associated with partial renoprotective effects under hyperglycemia, as evidenced by the preserved renal function. At the molecular level, Shp2 deficiency was associated with altered renal insulin signaling and diminished hyperglycemia-induced renal endoplasmic reticulum stress, inflammation, and fibrosis. Additionally, Shp2 knockdown in E11 podocytes mimicked the in vivo deficiency of this phosphatase and ameliorated the deleterious impact of high glucose, whereas Shp2 reconstitution reversed these effects. Moreover, Shp2 deficiency attenuated high glucose-induced E11 podocyte migration. Further, we identified the protein tyrosine kinase FYN as a putative mediator of Shp2 signaling in podocytes under high glucose. Collectively, these findings suggest that Shp2 inactivation may afford protection to podocytes under hyperglycemia and highlight this phosphatase as a potential target to ameliorate glomerular dysfunction in DN.

Elaiophylin triggers paraptosis and preferentially kills ovarian cancer drug-resistant cells by inducing MAPK hyperactivation

Finely tuned mitogen-activated protein kinase (MAPK) signaling is important for cancer cell survival. Perturbations that push cells out of the MAPK fitness zone result in cell death. Previously, in a screen of the North China Pharmaceutical Group Corporation’s pure compound library of microbial origin, we identified elaiophylin as an autophagy inhibitor. Here, we demonstrated a new role for elaiophylin in inducing excessive endoplasmic reticulum (ER) stress, ER-derived cytoplasmic vacuolization, and consequent paraptosis by hyperactivating the MAPK pathway in multiple cancer cells. Genome-wide CRISPR/Cas9 knockout library screening identified SHP2, an upstream intermediary of the MAPK pathway, as a critical target in elaiophylin-induced paraptosis. The cellular thermal shift assay (CETSA) and surface plasmon resonance (SPR) assay further confirmed the direct binding between the SHP2 and elaiophylin. Inhibition of the SHP2/SOS1/MAPK pathway through SHP2 knockdown or pharmacological inhibitors distinctly attenuated elaiophylin-induced paraptosis and autophagy inhibition. Interestingly, elaiophylin markedly increased the already-elevated MAPK levels and preferentially killed drug-resistant cells with enhanced basal MAPK levels. Elaiophylin overcame drug resistance by triggering paraptosis in multiple tumor-bearing mouse models resistant to platinum, taxane, or PARPi, suggesting that elaiophylin might offer a reasonable therapeutic strategy for refractory ovarian cancer.

Sciatic nerve block downregulates the BDNF pathway to alleviate the neonatal incision-induced exaggeration of incisional pain via decreasing microglial activation

Sciatic nerve block is under investigation as a possible therapeutic strategy for neonatal injury-induced exaggeration of pain responses to reinjury. Spinal microglial priming, brain-derived neurotrophic factor (BDNF) and Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) participate in exaggerated incisional pain induced by neonatal incision. However, effects of sciatic nerve block on exacerbated incisional pain and underlying mechanisms remain unclear. Here, we demonstrated that sciatic nerve block alleviates pain hypersensitivity and microglial activation in rats subjected to neonatal incision and adult incision (nIN-IN). Chemogenetic activation or inhibition of spinal microglia attenuates or mimics effects of sciatic nerve block on pain hypersensitivity, respectively. Moreover, α-amino-3-hydroxy- 5-methy- 4-isoxazole propionate (AMPA) receptor subunit GluA1 contributes to the exaggeration of incisional pain. The inhibition of BDNF or SHP2 blocks upregulations of downstream molecules in nIN-IN rats. Knockdown of SHP2 attenuates the increase of GluA1 induced by injection of BDNF in adult rats with only neonatal incision. The inhibition of microglia or ablation of microglial BDNF attenuates upregulations of SHP2 and GluA1. Additionally, sciatic nerve block downregulates the expression of these three molecules. Upregulation of BDNF, SHP2 or AMPA receptor attenuates sciatic nerve block-induced reductions of downstream molecules and pain hypersensitivity. Microglial activation abrogates reductions of these three molecules induced by sciatic nerve block. These results suggest that decreased activation of spinal microglia contributes to beneficial effects of sciatic nerve block on the neonatal incision-induced exaggeration of incisional pain via downregulating BDNF/SHP2/GluA1-containing AMPA receptor signaling. Thus, sciatic nerve block may be a promising therapy.

Synergistic Cytotoxicity Effect of 5-Fluorouracil and SHP2 Inhibitor Demethylincisterol A3 on Cervical Cancer Cell.

Demethylincisterol A3 (DTA3) has been identified as an SHP2 inhibitor and suppresses the growth of many cancer cells. 5-Fluorouracil (5-FU) is widely used for the clinical treatment of various cancers. However, the combination effects of 5-FU and DTA3 on cervical cancer cells remain unknown. This study evaluates the mechanism of the combination effects of 5-FU and DTA3 in cervical cancer cells. The synergistic cytotoxic effects of 5-FU and DTA3 in cervical cancer cells were calculated. Apoptosis was analysed by flow cytometry. Western blot analyses were used to examine the related signalling pathways. DTA3 and 5-FU synergized to induce apoptosis and repress proliferation of cervical cancer cells by downregulating the activation of PI3K/AKT and NF-κB signalling pathways. We provided evidence that the upregulation of SHP2 expression by transfection significantly inhibited the cytotoxicity of 5-FU and DTA3. SHP2 knockdown enhanced the anti-proliferation activity of 5-FU, indicating targeting SHP2 sensitized cervical cancer cells to 5-FU. Our study demonstrates that SHP2 inhibitor DTA3 and 5-FU have a synergistic cytotoxic effect on cervical cancer cells. The synergistic combination of SHP2 inhibitor and 5-FU may present a promising strategy for the treatment of cervical cancer.

Abstract P125: Combined inhibition of SHP2 and CDK4/6 is active in NF1-associated malignant peripheral nerve sheath tumor

Abstract Background: NF1 is an essential negative regulator of RAS activity and is altered in nearly 90% of malignant peripheral nerve sheath tumors (MPNST). Additional recurrent molecular changes include loss of function alterations in CDKN2A, TP53, EED and SUZ12, but molecular targeting of these genomic events represents a unique challenge. We previously reported that the efficacy of MEK inhibitor is limited by adaptive activation of receptor tyrosine kinases and the adaptor protein SHP2, and that combined inhibition of MEK and SHP2 is effective in MPNST. Loss of CDKN2A, inactivation of RB1, and hyperactivation of cyclin dependent kinases (CDK) in MPNST also suggest that small-molecule CDK4/6 inhibitors (CDK4/6i) may be a potential therapeutic strategy, but monotherapy with CDK4/6i also demonstrates limited activity. Given the dependency of D-cyclins on RAS signaling, we hypothesize that the anti-tumor effects of CDK4/6i may be potentiated by agents (SHP2i) targeting the upstream activators of RAS. Methods: The effects of shRNA-mediated inducible SHP2 knockdown on RAS signaling, short-term and long-term cell growth, and response to CDK4/6i were examined using immunoblotting, high throughput proliferation assays, and colony formation assays. Combined effects of SHP2i plus CDK4/6i on signaling, cell and tumor growth were assessed. Pharmacodynamic (PD) assays were performed on tumors extracted following drug treatment in patient-derived xenograft (PDX) models. Results: Despite a modest effect of SHP2 knockdown on ERK signaling, shSHP2 reduced MPNST cell growth. SHP2 knockdown or SHP2i treatment alleviated activation of ERK signaling and cyclin D1 expression induced by CDK4/6i, and enhanced the sensitivity to CDK4/6i. Combination benefit was observed in in vitro cell growth and in vivo PDX. Although some PDX models demonstrated similar responses to SHP2i alone or SHP2i + CDK4/6i during the initial 4 weeks on treatment, we found more sustained growth inhibition exerted by the combination. PD studies demonstrated a decrease in p-ERK levels in tumors treated with either SHP2i alone or the SHP2i/CDK4/6i combination. Conclusions: Our preliminary data demonstrate that the combined inhibition of SHP2 and CDK4/6 is active and produces durable response in models of NF1-associated MPNST. This combination strategy may represent a novel treatment strategy for patients with MPNST. Citation Format: Jiawan Wang, Ana Calizo, Kai Pollard, Angela C. Hirbe, Christine A. Pratilas. Combined inhibition of SHP2 and CDK4/6 is active in NF1-associated malignant peripheral nerve sheath tumor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P125.

SHP2 as a Potential Therapeutic Target in Diffuse-Type Gastric Carcinoma Addicted to Receptor Tyrosine Kinase Signaling.

Simple SummaryDiffuse-type gastric carcinoma (DGC) is characterized by rapid infiltrative growth associated with massive stroma and frequent peritoneal dissemination, which leads to poor patient outcomes. In this study, we found that the oncogenic tyrosine phosphatase SHP2 is tyrosine-phosphorylated downstream of the amplified receptor tyrosine kinases (RTKs) Met and fibroblast growth factor receptor 2 (FGFR2) in DGC cell lines. SHP2 knockdown or pharmacological inhibition selectively suppressed the growth of DGC addicted to amplified Met and FGFR2. Moreover, targeting SHP2 abrogated malignant phenotypes, including peritoneal dissemination, of Met-addicted DGC and could overcome acquired resistance to Met inhibitors. Our findings suggest that SHP2 is a potential target for the treatment of DGC addicted to amplified RTK signaling.Diffuse-type gastric carcinoma (DGC) exhibits aggressive progression associated with rapid infiltrative growth, massive fibrosis, and peritoneal dissemination. Gene amplification of Met and fibroblast growth factor receptor 2 (FGFR2) receptor tyrosine kinases (RTKs) has been observed in DGC. However, the signaling pathways that promote DGC progression downstream of these RTKs remain to be fully elucidated. We previously identified an oncogenic tyrosine phosphatase, SHP2, using phospho-proteomic analysis of DGC cells with Met gene amplification. In this study, we characterized SHP2 in the progression of DGC and assessed the therapeutic potential of targeting SHP2. Although SHP2 was expressed in all gastric carcinoma cell lines examined, its tyrosine phosphorylation preferentially occurred in several DGC cell lines with Met or FGFR2 gene amplification. Met or FGFR inhibitor treatment or knockdown markedly reduced SHP2 tyrosine phosphorylation. Knockdown or pharmacological inhibition of SHP2 selectively suppressed the growth of DGC cells addicted to Met or FGFR2, even when they acquired resistance to Met inhibitors. Moreover, SHP2 knockdown or pharmacological inhibition blocked the migration and invasion of Met-addicted DGC cells in vitro and their peritoneal dissemination in a mouse xenograft model. These results indicate that SHP2 is a critical regulator of the malignant progression of RTK-addicted DGC and may be a therapeutic target.

Shp2 regulates PM2.5-induced airway epithelial barrier dysfunction by modulating ERK1/2 signaling pathway

The impact of fine particulate matter (PM2.5) on public health has received increasing attention. Through various biochemical mechanisms, PM2.5 alters the normal structure and function of the airway epithelium, causing epithelial barrier dysfunction. Src homology domain 2-containing protein tyrosine phosphatase 2 (Shp2) has been implicated in various respiratory diseases; however, its role in PM2.5-induced epithelial barrier dysfunction remains unclear. Herein, we assessed the regulatory effects of Shp2 on PM2.5-mediated epithelial barrier function and tight junction (TJ) protein expression in both mice and human pulmonary epithelial (16HBE) cells. We observed that Shp2 levels were upregulated and claudin-4 levels were downregulated after PM2.5 stimulation in vivo and in vitro. Mice were exposed to PM2.5 to induce acute lung injury, and disrupted epithelial barrier function, with decreased transepithelial electrical resistance (TER) and increased paracellular flux that was observed in 16HBE cells. In contrast, the selective inhibition or knockdown of Shp2 retained airway epithelial barrier function and reversed claudin-4 downregulation that triggered by PM2.5, and these effects may occur through the ERK1/2 MAPK signaling pathway. These data highlight an important role of Shp2 in PM2.5-induced airway epithelial barrier dysfunction and suggest a possible new course of therapy for PM2.5-induced respiratory diseases.

Abstract 1817: Epidermal growth factor receptor alters major histocompatibility complex class I expression on pancreatic cancer cells

Abstract Pancreatic cancer is one of the deadliest neoplasms, with a poor 5-year survival rate of just 10%. The ability of pancreatic cancer cells to evade the immune system hinders an anti-tumor response and contributes to the poor survival rate. In some cases, down regulation of major histocompatibility complex (MHC) class I cell-surface expression aids in immune evasion by preventing endogenous tumor antigens from being presented to cytotoxic T cells. The goal of this study is to evaluate the role epidermal growth factor receptor (EGFR) plays in the regulation of MHC class I expression by pancreatic cancer cells. Pancreatic cancer cell lines were treated with epidermal growth factor (EGF) or transforming growth factor alpha (TGF-alpha) to activate EGFR or with the small molecule drug erlotinib to inhibit EGFR. Immunoprecipitation, western blot analysis, and flow cytometry were used to analyze cells' MHC class I expression post treatment. Human pancreatic cancer cell lines (BxPC-3, T3M4, S2-013, Panc-1) were found to decrease MHC class I expression after EGFR activation by EGF. However, activation of EGFR via TGF-alpha led to differential effects on MHC class I expression in a cell line-dependent manner. EGFR inhibition by erlotinib led to increased MHC class I expression by greater than 2.5-fold on both human and mouse pancreatic cancer cells. Mechanistic studies were done to evaluate the role of src homology 2 phosphatase (SHP2) in EGFR's regulation of MHC class I in pancreatic cancer cells, since in head and neck cancer cells SHP2 can be phosphorylated by activated EGFR and subsequently dephosphorylate STAT1 and reduce MHC class I expression. Following SHP2 knockdown by siRNA transfection, a slight increase in MHC class I expression was observed on pancreatic cancer cells, suggesting SHP2 has a minor down-regulatory role in MHC class I expression in these cells. EGFR was found to co-immunoprecipitate with MHC class I, indicating that EGFR might directly affect MHC class I expression by facilitating its endocytosis. However, after EGFR inhibition, no change in the MHC class I cell surface turnover rate was observed. Other potential mechanisms for EGFR regulation of MHC class I expression are currently being evaluated. Our discoveries defining a role for activated EGFR in reducing MHC class I expression and revealing that EGFR inhibitors can boost MHC class I levels on cancer cells could facilitate the adoption of new, more effective therapeutic tactics that combine the use of EGFR inhibitors with immunotherapies to augment patients' immune systems for effective targeting and killing of cancer cells. Citation Format: Shelby M. Knoche, Joyce C. Solheim. Epidermal growth factor receptor alters major histocompatibility complex class I expression on pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1817.

Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Promotes Inflammation and Accelerates Osteoarthritis by Activating β-Catenin

Osteoarthritis (OA) is a chronic articular disease characterized by cartilage degradation, subchondral bone remodeling and osteophyte formation. Src homology 2 domain-containing protein tyrosine phosphatase (SHP2) has not been fully investigated in the pathogenesis of OA. In this study, we found that SHP2 expression was significantly increased after interleukin-1β (IL-1β) treatment in primary mouse chondrocytes. Inhibition of SHP2 using siRNA reduced MMP3, MMP13 levels, but increased AGGRECAN, COL2A1, SOX9 expression in vitro. On the contrary, overexpression of SHP2 exerted the opposite results and promoted cartilage degradation. Mechanistically, SHP2 activated Wnt/β-catenin signaling possibly through directly binding to β-catenin. SHP2 also induced inflammation through activating Mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways. Our in vivo studies showed that SHP2 knockdown effectively delayed cartilage destruction and reduced osteophyte formation in the mouse model of OA induced by destabilization of the medial meniscus (DMM). Altogether, our study identifies that SHP2 is a novel and potential therapeutic target of OA.

SHP-2-Induced Activation of c-Myc Is Involved in PDGF-B-Regulated Cell Proliferation and Angiogenesis in RMECs.

Background: Aberrant neovascularization resulting from inappropriate angiogenic signaling is closely related to many diseases, such as cancer, cardiovascular disease, and proliferative retinopathy. Although some factors involved in regulating pathogenic angiogenesis have been identified, the molecular mechanisms of proliferative retinopathy remain largely unknown. In the present study, we determined the role of platelet-derived growth factor-B (PDGF-B), one of the HIF-1-responsive gene products, in cell proliferation and angiogenesis in retinal microvascular endothelial cells (RMECs) and explored its regulatory mechanism.Methods: Cell counting kit-8 (CCK-8), bromodeoxyuridine (BrdU) incorporation, tube formation, cell migration, and Western blot assays were used in our study.Results: Our results showed that PDGF-B promoted cell proliferation and angiogenesis by increasing the activity of Src homology 2 domain-containing tyrosine phosphatase 2 (SHP-2) in RMECs, which was attenuated by the inhibition of PDGF receptor (PDGFR) or SHP-2 knockdown. Moreover, activation of c-Myc was involved in the processes of PDGF-B/SHP-2-driven cell proliferation in RMECs. The promoting effects of PDGF-B/SHP-2 on c-Myc expression were mediated by the Erk pathway.Conclusion: These results indicate that PDGF-B facilitates cell proliferation and angiogenesis, at least in part, via the SHP-2/Erk/c-Myc pathway in RMECs, implying new potential treatment candidates for retinal microangiopathy.