SHP-1 Expression Research Articles

Effects of changes in SHP2 expression on liver fibrosis by influencing the apoptosis of hepatic stellate cells.

Accumulating research has revealed that src-homology domain 2-containing protein tyrosine phosphatase-2 (SHP2), an oncogenic protein tyrosine phosphatase, is associated with liver fibrosis. Currently, it is still unclear whether SHP2 affects liver fibrosis by influencing the apoptosis of hepatic stellate cells (HSC). In present study, we investigate effects of SHP2 expression changes on liver fibrosis, with special emphasis on the apoptosis of HSC. Using adenovirus vector, wild-type SHP2 gene and short hairpin RNA targeting SHP2 were introduced into rats with liver fibrosis and LX-2 cells in vitro. The expressions of type I and III collagen, pathological and functional changes, collagen deposition in rat liver and apoptosis of LX-2 cells were detected by immunohistochemical and HE staining, automated biochemical analyzer, Masson trichrome staining, and TUNEL. This study showed that overexpression of SHP2 exacerbated dysfunction, inflammatory damage, collagen deposition and increased expression of type I and III collagen in rat liver reduced apoptosis of LX-2 cells. On the contrary, low expression of SHP2 alleviated the aforementioned detection indicators of rats and promoted apoptosis of LX-2 cells. In conclusion, the downregulation of SHP2 expression alleviates liver fibrosis by inducing the apoptosis of HSC, while overexpressed SHP2 exacerbates liver fibrosis by inhibiting the apoptosis of HSC.

JAK2V617F-dependent down regulation of SHP-1 expression participates in the selection of myeloproliferative neoplasm cells in the presence of TGF-β.

Myeloproliferative neoplasms (MPNs) are characterized by an increased production of blood cells due to the acquisition of mutations such as JAK2V617F. TGF-β, whose secretion is increased in MPN patients, is known to negatively regulate haematopoietic stem cell (HSC) proliferation. Using an isogenic JAK2V617F or JAK2 wild-type UT-7 cell line we observed that JAK2V617F cells resist to TGF-β antiproliferative activity. Although TGF-β receptors and SMAD2/3 expressions are similar in both cell types, TGF-β-induced phosphorylation of SMAD2/3 is reduced in UT-7 JAK2V617F cells compared with JAK2 WT cells. We confirmed that JAK2V617F mutated cells are resistant to the antiproliferative effect of TGF-β in a competitive assay as we observed a positive selection of JAK2V617F cells when exposed to TGF-β. Using cell lines, CD34-positive cells from MPN patients and bone marrow cells from JAK2V617F knock-in mice we identified a down regulation of the SHP-1 phosphatase, which is required for the regulation of HSC quiescence by TGF-β. The transduction of SHP-1 cDNA (but not a phosphatase inactive cDNA) restores the antiproliferative effect of TGF-β in JAK2V617F mutated cells. Finally, SC-1, a known agonist of SHP-1, antagonized the selection of JAK2V617F mutated cells in the presence of TGF-β. In conclusion, we show a JAK2-dependent down regulation of SHP-1 in MPN patients' cells which is related to their resistance to the antiproliferative effect of TGF-β. This may participate in the clonal selection of cancer cells in MPNs.

MSU crystallization promotes fibroblast proliferation and renal fibrosis in diabetic nephropathy via the ROS/SHP2/TGFβ pathway

Monosodium urate (MSU) crystallisation deposited in local tissues and organs induce inflammatory reactions, resulting in diseases such as gout. MSU has been recognized as a common and prevalent pathology in various clinical conditions. In this study, we investigated the role of MSU in the pathogenesis of diabetic kidney disease (DKD). We induced renal injury in diabetic kidney disease mice using streptozotocin (STZ) and assessed renal histopathological damage using Masson's trichrome staining and Collagen III immunofluorescence staining. We measured the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and uric acid (UA) using ELISA. Protein expression levels of NLRP3, p-NF-κB, SHP2, p-STAT3, and p-ERK1/2 were analyzed by Western blot. To further investigate the role of MSU in diabetic kidney disease, we conducted in vitro experiments. In our in vivo experiments, we found that compared to the Model group, there was a significant increase in interstitial fibrosis in the kidneys of mice after treatment with MSU, accompanied by elevated levels of MDA, SOD, and UA. Furthermore, the protein expression of NLRP3, p-NF-NB, SHP2, p-STAT3, and p-ERK1/2 was upregulated. In our subsequent studies on mouse fibroblasts (L929 cells), we discovered that high glucose, MSU, and TGF-β could promote the expression of P22, GP91, NLRP3, NF-κB, p-NF-κB, p-SHP2, p-EGFR, p-STAT3, and Collagen-III proteins. Additionally, we found that SHP2 could counteract the upregulation trend induced by MSU on the expression of p-SHP2, p-EGFR, p-STAT3, and Collagen-III proteins, and inhibitors YQ128, NAC, and Cetuximab exhibited similar effects. Furthermore, immunofluorescence results indicated that SHP2 could inhibit the expression of the fibrosis marker α-SMA in L929 cells. These findings suggest that MSU can promote renal fibroblast SHP2 expression, induce oxidative stress, activate the NLRP3/NF-κB pathway, and enhance diabetic kidney disease fibroblast proliferation through the TGFβ/STAT3/ERK1/2 signaling pathway, leading to renal fibrosis.

Treatment of tumor-associated macrophages with PD-1 monoclonal antibodies affects vascular generation in cervical cancer via the PD-1/IRE1α/SHP2/HIF1α signaling pathway.

To investigate the effect of PD-1 monoclonal antibodies in tumor-associated macrophages on angiogenesis in cervical cancer and its mechanism of action. The effect of PD-1 monoclonal antibodies on the progression of cervical cancer was assessed using the nude mouse xenograft model and HE staining; the impact of PD-1 monoclonal antibodies on cervical cancer cell migration was evaluated using wound healing assay and Transwell assay; the effect on vascular formation in cervical cancer cells was examined using an angiogenesis assay; the impact on the expression of related proteins was tested using Western blotting. PD-1 monoclonal antibodies in tumor-associated macrophages can regulate and thus inhibit the progression of cervical cancer while promoting the expression of SHP2. Additionally, Sindilizumab inhibited the expression of tissue-type fibrinogen activator K and HIF1α through the PD-1/IRE1α/SHP2 signaling pathway, which inhibited the migration and neovascularization of cervical cancer cells. This study discovered that PD-1 monoclonal antibodies in tumor-associated macrophages inhibit vascular generation inside cervical cancer by affecting the PD-1/IRE1α/SHP2/HIF1α signaling pathway, providing a new therapeutic target for the treatment of cervical cancer.

WWP1 inhibition increases SHP2 inhibitor efficacy in colorectal cancer

Protein tyrosine phosphatase SHP2 activates RAS signaling, which is a novel target for colorectal cancer (CRC) therapy. However, SHP2 inhibitor monotherapy is ineffective for metastatic CRC and a combination therapy is required. In this study, we aimed to improve the antitumor efficacy of SHP2 inhibition and try to explore the resistance mechanism of SHP2 inhibitor. Results showed that WWP1 promoted the proliferation of CRC cells. Genetic or pharmacological inhibition of WWP1 enhanced the effect of SHP2 inhibitor in suppressing tumor growth in vitro and in vivo. WWP1 may mediate feedback reactivation of AKT signaling following SHP2 inhibition. Furthermore, nomogram models constructed with IHC expression of WWP1 and SHP2 greatly improved the accuracy of prognosis prediction for patients with CRC. Our findings indicate that WWP1 inhibitor I3C can synergize with SHP2 inhibitor and is expected to be a new strategy for clinical trials in treating advanced CRC patients.

RGD-modified ZIF-8 nanoparticles as a drug carrier for MR imaging and targeted drug delivery in myocardial infarction.

Aim: A multifunctional nanoplatform has been developed to enhance the targeting capability and biosafety of drug/siRNA for better diagnosis and treatment of myocardial infarction (MI).Materials & methods: The nanoplatform's chemical properties, biodistribution, cardiac magnetic resonance imaging (MRI) capabilities, therapeutic effects and biocompatibility were investigated.Results: The nanoplatform exhibited MI-targeting properties and pH-sensitivity, allowing for effective cardiac MRI and delivery of drugs to the infarcted myocardium. The GCD/Qt@ZIF-RGD demonstrated potential as a reliable MRI probe for MI diagnosis. Moreover, the GCD/si-SHP1/Qt@ZIF-RGD effectively suppressed SHP-1 expression, increased pro-angiogenesis gene expression and reduced cell apoptosis in HUVECs exposed to hypoxia/reoxygenation.Conclusion: Our newly developed multifunctional drug delivery system shows promise as a nanoplatform for both the diagnosis and treatment of MI.

Mechanistic study of inhibitory peptides with SHP-1 in hypertonic environment for infection model

Cutaneous Leishmaniasis, an infectious disease is globally the most prevalent form of leishmaniasis accounting for approximately 1 million cases every year as per world health organization. Infected individuals develop skin lesion which has been reported to be infiltrated by immune cells and parasite with high sodium accumulation creating hypertonic environment. In our work, we tried to mimic the hypertonic environment in virtual environment to study dynamicity of SHP-1 and NFAT5 along with their interactions through molecular dynamics simulation. We validated the SHP-1 and NFAT5 dynamics in infection and HSD conditions to study the impact of hypertonicity derived NFAT5 mediated response to L.major infection. We also evaluated our therapeutic peptides for their binding to SHP-1 and to form stable complex. Membrane stability with the peptides was analyzed to understand their ability to sustain mammalian membrane. We identified PepA to be a potential candidate to interact with SHP-1. Inhibition of SHP-1 through PepA to discern IL-10 and IL-12 reciprocity may be assessed in future and furnish us with a potential therapeutic molecule. HSD mice exhibited high pro-inflammatory response to L.major infection which resulted in reduced lesion size. Contrary to observations in HSD mice, infection model exhibited low pro-inflammatory response and increased lesion size with high parasite load. Thus, increase in NFAT5 expression and reduced SHP-1 expression may result in disease resolving effect which can be further studied through incorporation of synthetic circuit using PepA to modulate IL-10 and IL-12 reciprocity.

Inhibiting SHP2 reduces glycolysis, promotes microglial M1 polarization, and alleviates secondary inflammation following spinal cord injury in a mouse model.

JOURNAL/nrgr/04.03/01300535-202503000-00030/figure1/v/2024-06-17T092413Z/r/image-tiff Reducing the secondary inflammatory response, which is partly mediated by microglia, is a key focus in the treatment of spinal cord injury. Src homology 2-containing protein tyrosine phosphatase 2 (SHP2), encoded by PTPN11, is widely expressed in the human body and plays a role in inflammation through various mechanisms. Therefore, SHP2 is considered a potential target for the treatment of inflammation-related diseases. However, its role in secondary inflammation after spinal cord injury remains unclear. In this study, SHP2 was found to be abundantly expressed in microglia at the site of spinal cord injury. Inhibition of SHP2 expression using siRNA and SHP2 inhibitors attenuated the microglial inflammatory response in an in vitro lipopolysaccharide-induced model of inflammation. Notably, after treatment with SHP2 inhibitors, mice with spinal cord injury exhibited significantly improved hind limb locomotor function and reduced residual urine volume in the bladder. Subsequent in vitro experiments showed that, in microglia stimulated with lipopolysaccharide, inhibiting SHP2 expression promoted M2 polarization and inhibited M1 polarization. Finally, a co-culture experiment was conducted to assess the effect of microglia treated with SHP2 inhibitors on neuronal cells. The results demonstrated that inflammatory factors produced by microglia promoted neuronal apoptosis, while inhibiting SHP2 expression mitigated these effects. Collectively, our findings suggest that SHP2 enhances secondary inflammation and neuronal damage subsequent to spinal cord injury by modulating microglial phenotype. Therefore, inhibiting SHP2 alleviates the inflammatory response in mice with spinal cord injury and promotes functional recovery postinjury.

Kaempferide triggers apoptosis and paraptosis in pancreatic tumor cells by modulating the ROS production, SHP-1 expression, and the STAT3 pathway.

Pancreatic cancer is one of the deadliest diseases with a poor prognosis and a five-survival rate. The STAT3 pathway is hyperactivated which contributes to the sustained proliferative signals in pancreatic cancer cells. We have isolated kaempferide (KF), an O-methylated flavonol, from the green propolis of Mimosa tenuiflora and examined its effect on two forms of cell death namely, apoptosis and paraptosis. KF significantly increased the cleavage of caspase-3 and PARP. It also downmodulated the expression of Alix (an intracellular inhibitor of paraptosis) and increased the expression of CHOP and ATF4 (transcription factors that promote paraptosis) indicating that KF promotes apoptosis as well as paraptosis. KF also increased intracellular reactive oxygen species (ROS) suggesting the perturbance of the redox state. N-acetylcysteine reverted the apoptosis- and paraptosis-inducing effects of KF. Some ROS inducers are known to suppress the STAT3 pathway and investigation revealed that KF downmodulates STAT3 and its upstream kinases (JAK1, JAK2, and Src). Additionally, KF also elevated the expression of SHP-1, a tyrosine phosphatase which is involved in the negative modulation of the STAT3 pathway. Knockdown of SHP-1 prevented KF-driven STAT3 inhibition. Altogether, KF has been identified as a promoter of apoptosis and paraptosis in pancreatic cancer cells through the elevation of ROS generation and SHP-1 expression.

Mechanism study of tyrosine phosphatase shp-1 in inhibiting hepatocellular carcinoma progression by regulating the SHP2/GM-CSF pathway in TAMs

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Macrophage-mediated innate immune responses play a crucial role in tumor development. This study revealed the mechanism of SHP-1 in regulating HCC progression. SHP-1 inhibits tumour development in vivo. Increasing SHP-1 expression in macrophages promotes the expression of p-SHP-1, SHP2, and p-SHP-2. In macrophages GM-CSF recruits SHP-2 to the GM-CSF receptor GM-CSFR induces p-SHP-2 dephosphorylation. GM-CSF recruits p-SHP-2 for dephosphorylation by up-regulating HoxA10HOXA10 activates the transcription of TGFβ2 by interacting with tandem cis-elements in the promoter thereby regulating the proliferation and migration of liver cancer cells. GM-CSF inhibits SHP-1 regulation of p-SHP-1, SHP2, and p-SHP-2 in macrophages. Detailed studies have shown that SHP-1 regulates SHP2 expression, and SHP-1 and SHP2 are involved in macrophage M2 polarisation. SHP-1 inhibits HOXA10 and TGFβ2 which in turn regulates the expression of the migration-associated proteins, MMP2/9, and the migration of hepatocellular carcinoma cells. Overexpression of SHP-1 inhibits macrophage M2 polarisation via the p-STAT3/6 signalling pathway Classical markers arginase-1, CD206, CD163 and regulate the expression of M2 polarisation cytokines IL-4 and IL-10. In addition, hypoxia-induced ROS inhibited SHP-1 regulation by suppressing the expression of p-SHP-1. The combined effect of GM-CSF and ROS significantly increased p-HOXA10/TGFβ2 and macrophage M2 polarisation, and the regulatory effect of ROS was significantly suppressed by GM-CSF knockdown. These findings suggest that increasing the expression of tyrosine phosphatase SHP-1 can inhibit hepatocellular carcinoma progression by modulating the SHP2/GM-CSF pathway in TAM and thus inhibit the progression of hepatocellular carcinoma.

Effect of SHP-1 knockout in airway epithelial cells on emphysema phenotype in chronic obstructive pulmonary disease in mice

Objective: To construct and characterize conditional Src homology region 2 protein tyrosine phosphatase 1 (SHP-1) knockout mice in airway epithelial cells and to observe the effect of defective SHP-1 expression in airway epithelial cells on the emphysema phenotype in chronic obstructive pulmonary disease (COPD). Methods: To detect the expression of SHP-1 in the airway epithelium of COPD patients. CRISPR/Cas9 technology was used to construct SHP-1flox/flox transgenic mice, which were mated with airway epithelial Clara protein 10-cyclase recombinase and estrogen receptor fusion transgenic mice (CC10-CreER+/+), and after intraperitoneal injection of tamoxifen, airway epithelial SHP-1 knockout mice were obtained (SHP-1flox/floxCC10-CreER+/-, SHP-1Δ/Δ). Mouse tail and lung tissue DNA was extracted and PCR amplified to discriminate the genotype of the mice; the knockout effect of SHP-1 gene in airway epithelial cells was verified by qRT-PCR, Western blotting, and immunofluorescence. In addition, an emphysema mouse model was constructed using elastase to assess the severity of emphysema in each group of mice. Results: Airway epithelial SHP-1 was significantly downregulated in COPD patients. Genotyping confirmed that SHP-1Δ/Δ mice expressed CC10-CreER and SHP-1-flox. After tamoxifen induction, we demonstrated the absence of SHP-1 protein expression in airway epithelial cells of SHP-1Δ/Δ mice at the DNA, RNA, and protein levels, indicating that airway epithelial cell-specific SHP-1 knockout mice had been successfully constructed. In the emphysema animal model, SHP-1Δ/Δ mice had a more severe emphysema phenotype compared with the control group, which was manifested by disorganization of alveolar structure in lung tissue and rupture and fusion of alveolar walls to form pulmonary alveoli. Conclusions: The present study successfully established and characterized the SHP-1 knockout mouse model of airway epithelial cells, which provides a new experimental tool for the in-depth elucidation of the role of SHP-1 in the emphysema process of COPD and its mechanism.

The suppression of cell motility through the reduction of FAK activity and expression of cell adhesion proteins by hAMSCs secretome in MDA-MB-231 breast cancer cells.

Breast cancer is a leading cause of death in women worldwide. Cancer therapy based on stem cells is considered as a novel and promising platform. In the present study, we explore the therapeutic effects of human amniotic mesenchymal stromal cells (hAMSCs) through the reduction of focal adhesion kinase (FAK) activity, SHP-2, and cell adhesion proteins such as Paxillin, Vinculin, Fibronectin, Talin, and integrin αvβ3 expression in MDA-MB-231 breast cancer cells. For this purpose, we employed a co-culture system using 6-well plate transwell. After 72h, hAMSCs-treated MDA-MB-231 breast cancer cells, the activity of focal adhesion kinase (FAK) and the expression of SHP-2 and cell adhesion proteins such as Paxillin, Vinculin, Fibronectin, Talin, and integrin αvβ3 expression were analyzed using western blot. The shape and migration of cells were also analyzed. Based on our results, a significant reduction in tumor cell motility through downregulation of the tyrosine phosphorylation level of FAK (at Y397 and Y576/577 sites) and cell adhesion expression in MDA-MB-231 breast cancer cells was demonstrated. Our findings indicate that hAMSCS secretome has therapeutic effects on cancer cell migration through downregulation of FAK activity and expression of cell adhesion proteins.

Expression of several deubiquitinase and tyrosine phosphatase genes involved in inflammatory response in lymphoma

Lymphoma represents a heterogeneous group of cancers affecting the lymphatic system, encompassing non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL). The majority of NHL cases originate from B cells. HL is characterized by the presence of rare Hodgkin and Reed-Sternberg cells. Within the context of the inflammatory response, two SH2 domain-containing protein tyrosine phosphatases (SHPs), namely SHP-1 and SHP-2, serve as negative regulators. Deubiquitinating enzymes (DUBs) play a crucial role in inhibiting NF-κB activation in response to various stimuli. This study focuses on the DUBs OTUB1, OTUB2, and Cezanne. Blood samples were collected from 50 NHL patients, 26 HL patients, and 50 healthy individuals. Quantitative RT-PCR was employed to assess the mRNA expression of OTUB1, OTUB2, Cezanne, SHP-1,and SHP-2, while ELISA was used to determine IL-6 and CA125 concentrations. The results revealed that the mRNA level of OTUB1 was significantly downregulated in NHL patients but not in HL patients. Notably, Cezanne expression was downregulated in lymphoma patients, with significantly lower levels observed in HL compared to NHL patients. Furthermore, SHP-1 mRNA expression was significantly lower in the NHL group compared to the HL group or healthy individuals. Conversely, SHP-2 gene expression was upregulated in NHL patients but remained unchanged in HL patients. In conclusion, these findings highlight significant differences in the expressions of DUB and SHP genes and the inflammatory response in lymphoma patients. This study provides a foundation for further investigation into the roles of DUBs and tyrosine phosphatases in regulating the functional activation of lymphoma cells.

Allicin treats myocardial infarction in I/R through the promotion of the SHP2 axis to inhibit p-PERK-mediated oxidative stress.

The study attempted to explore how allicin reduces oxidative stress levels by promoting SHP2 expression to inhibit p-PERK in I/R mice. The GEO database and RNA sequencing were used to predict downstream gene. TTC staining was used to visualize the myocardial infarction area. Masson staining was used to assess the level of fibrosis. IF was used to examine the expression of SHP2, CTGF, ROS. RT-PCR analysis was used to quantify the expression of SHP2 mRNA. Western blot was used to detect the protein expression levels of SHP2, p-PERK, MFN1, NLRP3, NOX2, and NOX3. GEO and transcriptomic data revealed low expression of SHP2 in the heart tissues I/R mice. In the I/R mouse model, TTC staining result showed that allicin can reduce the area of myocardial infarction; Masson staining results indicated that allicin can reduce fibrosis; Macrophage transcriptome sequencing found SHP2 is a target gene of allicin; Immunofluorescence showed allicin can increase SHP2; qPCR results showed allicin can raise SHP2 mRNA level; Immunofluorescence indicated that allicin can inhibit ROS in myocardial infarction tissue, but the specific SHP2-KD eliminates changes in ROS. Western blot analysis demonstrated allicin can increase SHP2 protein and reduce the expression of p-PERK, MFN1, NLRP3, NOX2, and NOX3; SHP2-KD eliminates the expression differences in p-PERK, MFN1, NLRP3, NOX2, and NOX3. Allicin can modulate p-PERK activation by enhancing the expression of SHP2, thereby inhibiting myocardial ischemia-reperfusion-induced oxidative stress in mice.

The low expression of miR-155 promotes the expression of SHP2 by inhibiting the activation of the ERK1/2 pathway and improves cell pyroptosis induced by I/R in mice.

This study aims to explore the specific mechanism by which miR-155 regulates SHP2 expression in mouse ischemia-reperfusion (I/R) induced necroptosis. Various methods including cardiac ultrasound, TTC staining, Masson staining, TUNEL staining, and Western blotting were used to examine changes in the morphology and function of the rat left ventricle, myocardial fibrosis, as well as the expression of proteins related to tissue and cardiomyocyte necroptosis pathways. In vivo results showed that knockdown (KD) of miR-155 significantly improved cardiac ultrasound parameters (EF, FS, LVAW;d, and LVAW;s), reduced the myocardial infarction area, myocardial fibrosis, and cell apoptosis in I/R mice, upregulated cardiac SHP2 protein expression, and other proteins including p-ERK1/2, NLRP3, GSDMD, caspase-3, caspase-4, and caspase-11 were also significantly decreased. In vitro experiments showed that compared with the SHP2 WT miR-155 KD group, SHP2 protein expression was significantly increased in the SHP2 WT miR-155 KD group, while the expression of other proteins was significantly reduced, consistent with in vivo results. MiR-155 can regulate ERK1/2 and NLRP3 through SHP2. After adding the ERK1/2 inhibitor U0126 to cardiomyocytes from SHP2 KO mice, it was found that the expression of proteins other than SHP2 significantly decreased compared to SHP2 KO cells without the inhibitor. In summary, low expression of miR-155 promoted the expression of SHP2 and improved mouse I/R-induced necroptosis by inhibiting the activation of the ERK1/2 pathway.

1-Pyrroline-5-carboxylate inhibit T cell glycolysis in prostate cancer microenvironment by SHP1/PKM2/LDHB axis

BackgroundOur previous studies demonstrated that 1-Pyrroline-5-carboxylate (P5C) released by prostate cancer cells inhibits T cell proliferation and function by increasing SHP1 expression. We designed this study to further explore the influence of P5C on T cell metabolism, and produced an antibody for targeting P5C to restore the functions of T cells.MethodWe co-immunoprecipated SHP1 from T cells and analyzed the proteins that were bound to it using liquid chromatography mass spectrometry (LC/MS-MS). The influence of P5C on T cells metabolism was also detected by LC/MS-MS. Seahorse XF96 analyzer was further used to identify the effect of P5C on T cells glycolysis. We subsequently designed and produced an antibody for targeting P5C by monoclonal technique and verified its effectiveness to restore the function of T cells in vitro and in vivo.ResultPKM2 and LDHB bind SHP1 in T cells, and P5C could increase the levels of p-PKM2 while having no effect on the levels of PKM2 and LDHB. We further found that P5C influences T cell energy metabolism and carbohydrate metabolism. P5C also inhibits the activity of PKM2 and decreases the content of intracellular lactic acid while increasing the activity of LDH. Using seahorse XF96 analyzer, we confirmed that P5C remarkably inhibits glycolysis in T cells. We produced an antibody for targeting P5C by monoclonal technique and verified that the antibody could oppose the influence of P5C to restore the process of glycolysis and function in T cells. Meanwhile, the antibody also inhibits the growth of prostate tumors in an animal model.ConclusionOur study revealed that P5C inhibits the process of glycolysis in T cells by targeting SHP1/PKM2/LDHB complexes. Moreover, it is important that the antibody for targeting P5C could restore the function of T cells and inhibit the growth of prostate tumors.

Discovery of 1H-pyrazolo[3,4-b]pyrazine derivatives as selective allosteric inhibitor of protein tyrosine phosphatase SHP2 for the treatment of KRASG12C-mutant non-small cell lung cancer

The high expression or mutation of SHP2 can induce cancer, so targeting SHP2 has become a new strategy for cancer treatment. In this study, we used the previously reported SHP2 allosteric inhibitor IACS-13909 as a lead drug for structural derivation and modification, and synthesized three SHP2 inhibitors. Among them, 1H-pyrazolo[3,4-b]pyrazine derivative 4b was a highly selective SHP2 allosteric inhibitor, with an IC50 value of 3.2 nM, and its inhibitory activity was 17.75 times than that of the positive control IACS-13909. The cell proliferation experiment detected that compound 4b would markedly inhibit the proliferation of various cancer cells. Interestingly, compound 4b was highly sensitive to KRASG12C-mutant non-small cell lung cancer NCI-H358 cells, with an IC50 value of 0.58 μM and its antiproliferative activity was 4.79 times than that of IACS-13909. Furthermore, the combination therapy of compound 4b and KRASG12C inhibitor sotorasib would play a strong synergistic effect against NCI-H358 cells. The western blot experiment detected that compound 4b markedly downregulated the phosphorylation levels of ERK and AKT in NCI-H358 cells. Molecular docking study predicted that compound 4b bound to the allosteric site of SHP2 and formed H-bond interactions with key residues Thr108, Glu110, Arg111, and Phe113. In summary, this study aims to provide new ideas for the development of SHP2 allosteric inhibitors for the treatment of KRASG12C mutant non-small cell lung cancer. Communicated by Ramaswamy H. Sarma

Abstract B073: Understanding treatment response in pancreatic cancer: NetraAI provides genetic differentiation in FOLFIRINOX and gemcitabine response

Abstract Pancreatic cancer is known as a “silent killer” due to vague symptoms, late diagnosis, rapid progression, and poor response to therapy. Due to the highly malignant nature of this disease, prolonging progression-free survival (PFS) and overall survival (OS) for patients is critical. Standard of care involves combination chemotherapy using FOLFIRINOX (FFX) or Gemcitabine with nab-paclitaxel (GnP). The COMPASS trial performed whole genome sequencing (WGS) and RNA sequencing (RNASeq) on pancreatic ductal adenocarcinoma (PDAC) patients prior to FFX or GnP first-line combination therapy, finding molecular heterogeneity between patients and differing response to chemotherapy. Using the COMPASS trial dataset consisting of only 208 patients and NetraAI, a novel machine learning (ML) platform using a novel causal feature set discovery methodology, we explored generated hypotheses to differentiate causal factors that affect patient response to FFX and GnP. Differentiating FFX and GnP treatment response in stable disease patients revealed 3 distinct subpopulations with genetic differentiation related to neuron death and regulation, neurotrophin signaling, and neuron apoptotic processes. NetraAI identified genes driving patient subpopulations, such as LRRC8E, PTPRH, and UTG1A13p. Further investigation revealed that LRRC29 and CLEC19A distinguished a group of patients that responded well to FFX. LRRC29 relates to HOOK1, which negatively regulates epithelial-to-mesenchymal transition and has been reported to be decreased in PDAC. Low HOOK1 and SHP2 expression in non-small cell lung cancer is associated with a good response to GnP, suggesting that chemotherapy response depends on the interaction of the chemotherapeutic agent and genetic factors involved in disease progression as opposed to the tumor type. These results suggest that LRRC29 expression can act as a biomarker to choose between GnP and FFX treatment. In this way, NetraAI's ability to generate explainable hypotheses around patient subpopulations can help oncologists personalize treatments and better understand clinical trial patient populations. By identifying distinct genetic signatures within patient datasets. By identifying unique molecular heterogeneity that explains differing responses to therapy, NetraAI is a powerful tool that can extract insights from multi-dimensional and highly heterogenous patient population datasets that can help to inform clinical trial enrichment decisions to improve the success of pivotal trials. This work has a significant impact on precision medicine and clinical trial de-risking, while identifying specific genes and pathways involved in patient response to therapy. Citation Format: Bessi Qorri, Mike Tsay, Josh Spiegel, Douglas Cook, Joseph Geraci. Understanding treatment response in pancreatic cancer: NetraAI provides genetic differentiation in FOLFIRINOX and gemcitabine response [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B073.

Inhibiting H2AX Can Ameliorate Myocardial Ischemia/Reperfusion Injury by Regulating P53/JNK Signaling Pathway.

Myocardial ischemia-reperfusion (I/R) injury is a significant area of focus in cardiovascular disease research. I/R injury can increase intracellular oxidative stress, leading to DNA damage. H2AX plays a crucial role in DNA repair. This study utilized mouse and cell models of myocardial I/R to investigate the impact of H2AX on cardiomyocytes during I/R. This study initially assessed the expression of H2AX in MI/R mice compared to a sham surgery group. Subsequently, cardiac function, infarct area, and mitochondrial damage were evaluated after inhibiting H2AX in MI/R mice and a negative control group. Furthermore, the study delved into the molecular mechanisms by analyzing the expression of H2AX, P53, p-JNK, SHP2, p-SHP2, p-RAS, parkin, Drp1, Cyt-C, Caspase-3, and Caspase-8 in cardiomyocytes following the addition of JNK or P53 agonists. The results from western blotting in vivo indicated significantly higher H2AX expression in the MI/R group compared to the sham group. Inhibiting H2AX improved cardiac function, reduced myocardial infarct area, and mitigated mitochondrial damage in the MI/R group. In vitro experiments demonstrated that inhibiting H2AX could attenuate mitochondrial damage and apoptosis in myocardial cells by modulating the P53 and JNK signaling pathways. These findings suggested that inhibiting H2AX may alleviate myocardial I/R injury through the regulation of the P53/JNK pathway, highlighting H2AX as a potential target for the treatment of myocardial ischemia/reperfusion injury.

M-CSF secreted by gastric cancer cells exacerbates the progression of gastric cancer by increasing the expression of SHP2 in tumor-associated macrophages.

To investigate the effect of Src homology 2 domain-containing tyrosine phosphatase-2 (SHP2) in tumor-associated macrophages (TAMs), which is mediated by macrophage colony-stimulating factor (M-CSF) secreted by gastric cancer cells, on the development of gastric cancer and its molecular mechanism. The progression of gastric cancer was detected by nude mouse tumor-bearing experiments. Colony formation assay and cell counting kit-8 (CCK8) assay were used to detect the proliferation capacity of gastric cancer cells. The migration capacity of gastric cancer cells was examined by wound healing assay. Transwell migration and invasion assays were performed on gastric cancer cells. Detection of relevant protein expression using western blotting. Overexpression of SHP2 could promote the progression of gastric cancer in nude mice. The results of colony formation assay and CCK8 assay showed that overexpression of SHP2 could enhance the proliferation of gastric cancer cells. It was found by wound healing assay and Transwell assay that overexpression of SHP2 could facilitate the migration and invasion of gastric cancer cells. The results of Western blotting revealed that overexpression of SHP2 could increase the expressions of p-STAT3, s-PD-1, p-Src, p-Lyn, p-PI3K, p-AKT, Arginase-1, MMP1 and MMP3 but decrease the expressions of TBK1 and SOCS1 in TAMs, and also increase the expressions of CD9, TSG101 and s-PD-1 in exosomes. M-CSF secreted by gastric cancer cells can promote the proliferation, invasion and migration of gastric cancer cells by increasing the expression of SHP2 in TAMs.