Phosphatase Homolog Research Articles

Prevalence of APC and PTEN Alterations in Urachal Cancer

Urachal carcinoma (UrC) is a rare tumor with remarkable histological and molecular similarities to colorectal cancer (CRC). Adenomatous polyposis coli (APC) is the most frequently affected gene in CRC, but the prevalence and significance of its alterations in UrC is poorly understood. In addition, loss of phosphatase and tensin homologue (PTEN) was shown to be associated with therapy resistance in CRC. Our primary aim was to assess specific genetic alterations including APC and PTEN in a large series of UrC samples in order to identify clinically significant genomic alterations. We analyzed a total of 40 UrC cases. Targeted 5-gene (APC, PTEN, DICER1, PRKAR1A, TSHR, WRN) panel sequencing was performed on the Illumina MiSeq platform (n = 34). In addition, ß-catenin (n = 38) and PTEN (n = 30) expressions were assessed by immunohistochemistry. APC and PTEN genes were affected in 15% (5/34) and 6% (2/34) of cases. Two of five APC alterations (p.Y1075*, p.K1199*) were truncating pathogenic mutations. One of the two PTEN variants was a pathogenic frameshift insertion (p.C211fs). In 29% (11/38) of samples, at least some weak nuclear ß-catenin immunostaining was detected and PTEN loss was observed in 20% (6/30) of samples. The low prevalence of APC mutations in UrC represents a characteristic difference to CRC. Based on APC and ß-catenin results, the Wnt pathway seems to be rarely affected in UrC. Considering the formerly described involvement of PTEN protein loss in anti-EGFR therapy-resistance its immunohistochemical testing may have therapeutic relevance.

Histone H2A-Reactive B Cells Are Functionally Anergic in Healthy Mice With Potential to Provide Humoral Protection Against HIV-1.

Peripheral tolerance is essential for silencing weakly autoreactive B cells that have escaped central tolerance, but it is unclear why these potentially pathogenic B cells are retained rather than being eliminated entirely. Release from peripheral tolerance restraint can occur under certain circumstances (i.e., strong TLR stimulus), that are present during infection. In this regard, we hypothesized that autoreactive B cells could function as a reserve population that can be activated to contribute to the humoral immune response, particularly with pathogens, such as HIV-1, that exploit immune tolerance to avoid host defense. In this study, we identify a population of autoreactive B cells with the potential to neutralize HIV-1 and experimentally release them from the functional restrictions of peripheral tolerance. We have previously identified murine monoclonal antibodies that displayed autoreactivity against histone H2A and neutralized HIV-1 in vitro. Here, we identify additional H2A-reactive IgM monoclonal antibodies and demonstrate that they are both autoreactive and polyreactive with self and foreign antigens and are able to neutralize multiple clades of tier 2 HIV-1. Flow cytometric analysis of H2A-reactive B cells in naïve wildtype mice revealed that these B cells are present in peripheral B cell populations and we further document that murine H2A-reactive B cells are restrained by peripheral tolerance mechanisms. Specifically, we show endogenous H2A-reactive B cells display increased expression of the inhibitory mediators CD5 and phosphatase and tensin homolog (PTEN) phosphatase and fail to mobilize calcium upon immunoreceptor stimulation; all characterized markers of anergy. Moreover, we show that toll-like receptor stimulation or provision of CD4 T cell help induces the in vitro production of H2A-reactive antibodies, breaking tolerance. Thus, we have identified a novel poly/autoreactive B cell population that has the potential to neutralize HIV-1 but is silenced by immune tolerance.

Abstract B41: Modeling marginal zone lymphomagenesis

Abstract Introduction: Indolent B-cell non-Hodgkin’s lymphomas (NHL) represent a heterogeneous group of lymphoproliferative malignancies that remain largely incurable. Marginal zone lymphomas (MZL) are the second most common subtype of indolent NHL and lack a unique cytogenetic identifying abnormality. The B-cell receptor signaling pathway is activated in B-cell malignancy and mediates its activity mainly through the phosphoinositide 3-kinase (PI3K) pathway. Furthermore, novel PI3K inhibitors, such as copanlisib and parsaclisib, have shown impressive clinical activity in several indolent lymphomas, including MZL. This further supports the important role of the PI3K pathway in the pathogenesis of this tumor. Therefore, we hypothesized that the PI3K-mTOR pathway is sufficient for driving the pathogenesis of MZL. Methods: In order to test our hypothesis, we generated a genetically engineered mouse model carrying heterozygous global knockout alleles of both the tumor suppressor genes Phosphatase and Tensin Homolog (PTEN) and Liver Kinase B1 (LKB1). This led to overactivation of the PI3K-mTOR pathway in all mouse tissues. We closely monitored these mice for tumor formation via weekly physical examinations for several months. Upon tumor detection, the mouse was sacrificed and tumors were sectioned for histologic characterization. In order to generate a more specific model of B cells, and more accurately mimic the underlying human disease, we used the Cre-LoxP system to create the CD19-Cre-PTENfl/fl-LKB1fl/fl. Results: Thirty mice of global KO PTEN+/- LKB1 +/- died or were sacrificed due to disease progression, defined as either lymph node enlargement and/or splenomegaly. All mice showed either abnormal lymphadenopathy or splenomegaly. By Kaplan-Meier analysis, we saw a steady decrease in both tumor-free and overall survival after 3 months of age. Utilizing the product limit method, the median survival time was 6 months (95% CI: 6, 8). A total of 51 lymph nodes were sent for immunohistochemistry and pathologic characterization. Of the 51 nodes, 61.5% (N=32) showed indolent non-Hodgkin’s lymphoma, 25% (N=13) were atypical, and 11.5% (N=6) were reactive. All lymph nodes with indolent NHL were of MZL subtype. Compared to wild-type (n=3), the new CD19-Cre-PTENfl/fl-LKB1fl/fl (n=3) showed an overall increase in spleen mass (120 vs. 196 mg, p=0.0564), % B1 cells (4% vs. 59%, p= 0.0075), % MZ cells (5% vs. 30%, p=0.0547), % plasma cells (1% vs. 12%, p=0.0729), and a decrease in % FO cells (80% vs. 12%, p=0.0003) by flow cytometry. Further characterization of the new model is currently under way. Conclusion: Marginal zone lymphoma remains an incurable lymphoma that lacks reliable preclinical models. Our data provide, for the first time, a proof of concept on the role of the PI3K-mTOR pathway in the pathogenesis of marginal zone lymphoma and pave the way for future studies understanding the biology of this disease and developing rational therapies for this incurable malignancy. Citation Format: Victor Yazbeck, Ian McConnell, Emily Harris, Joseph Lownick, Ariel Sindel, Roy Sabo, Alden Chesney, Guanhua Lai, Adolfo Mauro, Chad Cain, Fadi Salloum, Steven Grant, Jamal Zweit, Jolene Windle, Steven Grossman. Modeling marginal zone lymphomagenesis [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B41.

Circ100284 promotes invasion and migration of osteosarcoma cells by down-regulating PTEN and EMP1.

To investigate the role of circ100284 in osteosarcoma (OS) and the underlying mechanism. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to detect the level of circ100284 in OS tissues and cells, and to examine the association between its level and clinicopathological features such as tumor size, tumor stage, and survival time. In addition, circ100284 was knocked out in MG63 and U2OS cells to observe the effect of circ100284 on cell viability, migration, cycle, and apoptosis by Cell Counting Kit-8 (CCK-8), transwell assay, and flow cytometry assay. Correlations of circ100284 with lysine-specific histone demethylase 1A (LSD1) and enhancer of zeste homolog 2 (EZH2) target proteins were analyzed by RNA co-precipitation experiments. Furthermore, the chromatin immunoprecipitation assay (ChIP)-qPCR assay was performed to analyze the relationship between circ100284 and its target protein and target gene. Circ100284 had a high level in OS tissues. The high expression of circ100284 was positively correlated with tumor size, pathological stage, and lung metastasis, and negatively correlated with patient survival time. Knocking down circ100284 in OS cells damaged the cell viability and invasiveness, blocked cell cycle, and promoted cell apoptosis. Further experiments showed that circ100284 could epigenetically inhibit cell proliferation by negatively regulating Phosphatase and tensin homolog (PTEN) and epithelial membrane protein 1 (EMP1) in OS. Circ100284 promotes the progression of OS cells by downregulating PTEN and EMP1.

LncRNA TP73-AS1 Regulates miR-21/PTEN Axis to Affect Cell Proliferation in Acute Myeloid Leukemia

Objective: TP73-AS1 has been reported as an overexpressed oncogenic lncRNA in several types of cancer. However, these analyses of The Cancer Genome Atlas data set revealed downregulation of TP73-AS1 in acute myeloid leukemia (AML). In this study, we aimed to study the molecular mechanism between TP73-AS1 and cell proliferation in AML. Methods: Bone marrow (BM) samples were obtained from 50 AML patients and 50 healthy controls. Cell transient transfections were performed to analyze gene interactions. Dual-luciferase reporter assay, quantitative polymerase chain reaction and Western blot were used to study the gene expressions. Cell proliferation was analyzed by CCK-8 method. Results: TP73-AS1 was confirmed to be downregulated in AML. TP73-AS1 was predicted to interact with miR-21, while overexpression of TP73-AS1 and miR-21 did not affect the expression of each other. Instead, overexpression of TP73-AS1 led to the upregulation of phosphatase and tensin homologue (PTEN), a downstream target of miR-21. Cell proliferation analysis showed that overexpression of TP73-AS1 and PTEN led to a decreased proliferation rate of AML cells. Overexpression of miR-21 played an opposite role and reduced the effects of overexpressing TP73-AS1 and PTEN. Conclusion: TP73-AS1 may regulate the miR-21/PTEN axis to affect cell proliferation in AML.

MiR-223 Promotes Smooth Muscle Cell Proliferation and Atherosclerotic Plaque Formation by Inhibiting Phosphatase and Tensin Homolog (PTEN)/ Phosphatidylinositol 3-Kinase (PI3K) and Protein Kinase B (AKT) Signaling Pathway

Abnormal vascular smooth muscle cells (VSMCs) proliferation is the pathological basis of atherosclerosis (AS) pathogenesis. miR-223 is abnormally expressed in AS plaques and affects the proliferation of VSMCs, but the mechanism of miR-223 affecting the proliferation of VSMCs is unclear. Our study intends to investigate the mechanism of miR-223 in VSMCs proliferation and AS formation. Healthy SD rats and miR-223 knockout SD rats took high-fat diet to induce AS model. Oil red O staining was done to observe AS formation. miR-223 mimics/NC was transferred to VSMCs followed by analysis of miR-214 expression by real-time PCR, cell proliferation by CCK8 assay, phosphatase and tensin homolog gene (PTEN) level by Western blot detection. Compared with control group, after knocking out miR-223, the AS level was significantly decreased and PTEN expression was significantly elevated (P < 0 05). After transfection of miR-223 mimics into VSMCs, PTEN expression protein was significantly decreased and the number of cells was increased (P < 0 05). In addition, the luciferase signal of miR-223 mimics and pmirGLO-PTEN-3 UTR-wt co-transfection group was significantly reduced (P < 0 05). miR-223 promotes VSMCs proliferation and AS plaque formation by targeting PTEN/PI3K/Akt signaling pathway.

MiR-141 Regulates Proliferation and Apoptosis of Renal Clear Cell Carcinoma by Targeting Phosphatase and Tensin Homolog (PTEN)

Objective: PTEN inhibits the activity of PI3K/AKT pathway. Abnormal miR-141 expression is associated with kidney cancer. Bioinformatics analysis revealed a relationship of miR-141 with PTEN. This study assessed miR-141's role in renal cancer cells. Methods: The dual luciferase reporter gene assay validated the relationship of miR-141 with PTEN. The tumor tissues and adjacent tissues of patients with renal cell carcinoma were collected to measure miR-141 and PTEN level. A498 cells were divided into miR-NC group and miR-141 inhibitor group followed by analysis of the expressions of miR-141, PTEN and p-AKT, cell apoptosis and proliferation by flow cytometry. Results : There is a relationship of miR-141 with PTEN. Compared with those in adjacent tissues, miR-141 was upregulated and PTEN mRNA was downregulated in tumor tissues. There was a negative correlation between miR-141 and PTEN mRNA (r = – 0 646,P < 0 001). Compared with that in HK-2 cells, miR-141 expression was increased in RCC4 and A498 cells, with decreased PTEN expression. Transfection of miR-141 inhibitor significantly up-regulated PTEN in A498 cells, reduced PI3K/AKT signaling activity, decreased cell proliferation and colony formation, as well as promoted cell apoptosis. Conclusion: miR-141 participates in reducing PTEN expression and promoting the pathogenesis of renal cancer. Inhibiting miR-141 expression up-regulates PTEN, inhibits PI3K/AKT signaling, and attenuates proliferation and promotes apoptosis of renal cancer cells.

Effects of miR-32 targeting PTEN on proliferation and apoptosis of myeloma cells.

To explore the effects of micro ribonucleic acid (miR)-32 on the proliferation and apoptosis of myeloma cells, and to verify whether it exerts its function by targeting phosphatase and tensin homolog deleted on chromosome ten (PTEN). The differentially expressed miRNAs were screened in healthy people and myeloma patients. The myeloma U266 cells transfected with negative control (NC) were used as control group, those transfected with miR-32 inhibitor as transfection group, and those transfected with miR-32 inhibitor and treated with PTEN inhibitor SF1670 as the transfection + inhibitor group. Then, the cell proliferation and apoptosis in each group were detected using the 5-Ethynyl-2'-deoxyuridine (EdU) kit and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, respectively. Finally, the expressions of apoptosis-related proteins B-cell lymphoma-2 (Bcl-2), Bcl-2 homologous antagonist/killer (Bak), caspase-9, and survivin were detected. The expressions of some miRNAs and genes in myeloma patients were significantly different from those in healthy people. In myeloma patients, miR-32, miR-126, miR-123, and miR-183 were significantly highly expressed, while miR-5, miR-76, and miR-50 were remarkably lowly expressed. After myeloma U266 cells were transfected with the miR-32 inhibitor, the expression of miR-32 markedly declined. In addition, the mRNA expression of PTEN in myeloma cells rose after transfection with the miR-32 inhibitor, and declined after addition of the PTEN inhibitor SF1670, which were consistent with the results of Western blotting. Besides, the proliferation ability of myeloma cells was evidently weakened after transfection with the miR-32 inhibitor, while it was restored to a certain extent after addition of the PTEN inhibitor SF1670. Moreover, the number of apoptotic myeloma cells was remarkably larger after transfection with the miR-32 inhibitor, while it was remarkably smaller after addition of the PTEN inhibitor SF1670. The expressions of pro-apoptotic proteins Bak and caspase-9 in myeloma cells were significantly increased after transfection with the miR-32 inhibitor (p<0.05), and significantly decreased after addition of the PTEN inhibitor SF1670, while the expressions of anti-apoptotic proteins Bcl-2 and survivin were opposite to those of Bak and caspase-9. MiR-32 targeting PTEN will have certain effects on the proliferation and apoptosis of myeloma cells.

The DSP Domain of Pseudophosphatase MK‐STYX Decreases Stress Granules

MK‐STYX [MAPK (mitogen activated protein kinase) phosphoserine/threonine/tyrosine binding protein] is a pseudophosphatase. Due to point mutations in the HC(X5)R catalytic active site signature motif, MK‐STYX (FSTQGISR) can only bind, but cannot dephosphorylate phosphorylated residues. This interaction of MK‐STYX allows it to play important roles in various cellular pathways including the stress response pathway. MK‐STYX decreases stress granules (SG) independently of serine 149 phosphorylation of G3BP‐1 (stress granule nucleator). Stress granules are cytoplasmic aggregates of untranslated mRNA and proteins formed in response to stressors. The aim of this study is to determine which domain of MK‐STYX decreases stress granules. MK‐STYX has two domains: a dual specificity phosphatase domain (DSP) and a rhodanese/cdc 25 phosphatase homology domain. In our study, HEK‐293 cells are cotransfected with G3BP‐GFP and mCherry‐DSP, mCherry‐CH2, or mChrerry. Cells expressing G3BP‐GFP and mCherry‐DSP exhibit fewer SG than the control (G3BP‐GFP and mCherry). Whereas, cells cotransfected with G3BP‐GFP and mCherry‐CH2 have similar levels of SG compared to the control, indicating that the DSP domain of MK‐STYX is responsible for decreasing the number of stress granules. Since the “active site” lies within the DSP domain, the study suggests that MK‐STYX decreases SG by binding, and perhaps blocking dephosphorylation of certain proteins. Future studies will focus on characterizing size and exact number of these SGs, as well as the interactors of MK‐STYX‐DSP.

PTEN activation contributes to neuronal and synaptic engulfment by microglia in tauopathy

Phosphatase and tensin homolog (PTEN) regulates synaptic density in development; however, whether PTEN also regulates synapse loss in a neurodegenerative disorder such as frontotemporal lobar degeneration with Tau deposition (FTLD-Tau) has not been explored. Here, we found that pathological Tau promotes early activation of PTEN, which precedes apoptotic caspase-3 cleavage in the rTg4510 mouse model of FTLD-Tau. We further demonstrate increased synaptic and neuronal exposure of the apoptotic signal phosphatidylserine that tags neuronal structures for microglial uptake, thereby linking PTEN activation to synaptic and neuronal structure elimination. By applying pharmacological inhibition of PTEN's protein phosphatase activity, we observed that microglial uptake can be decreased in Tau transgenic mice. Finally, we reveal a dichotomous relationship between PTEN activation and age in FTLD-Tau patients and healthy controls. Together, our findings suggest that in tauopathy, PTEN has a role in the synaptotoxicity of pathological Tau and promotes microglial removal of affected neuronal structures.

Correlation between the deletion of phosphatase and tensin homolog gene on chromosome 10, syncytin-1 and the damage of maternal endothelium and trophoblast in preeclampsia placenta

Objective To study the correlation between phosphatase and tensin homolog deleted on chromosome ten(PTEN), Syncytin-1 in preeclamptic placenta and maternal endothelial injury, trophoblast injury. Methods From February 2015 to December 2017, 46 patients diagnosed as preeclampsia and delivered in Xiaolan Hospital Affiliated to Southern Medical University and 80 pregnant women with normal delivery in the same period were selected.Before delivery, maternal peripheral blood was collected and the content of endothelial injury markers was determined; after delivery, the placenta was retained and the expression of PTEN, Syncytin-1, apoptosis gene and the contents of oxidative stress markers were measured. Results The mRNA expression of PTEN in placental tissue of preeclampsia was significantly higher than that of normal placental tissue(t=32.797, P<0.000), and the mRNA expression of Syncytin-1 was significantly lower than that of normal placental tissue(t=28.506, P<0.001); the content of ET-1 and sFlt-1 in the blood circulation of the maternal body of preeclampsia was significantly higher than thsoe of normal pregnant women(t=39.884, 40.243; all P<0.000) and positively correlated with the mRNA expression of PTEN in placenta(correlation coefficient=0.674, 0.596; P=0.004, 0.007), negatively correlated with the mRNA expression of Syncytin-1 in placenta(correlation coefficient=-0.615, -0.637; P<0.009, 0.008); the content of NO, PGI2, VEGF and PLGF were significantly lower than thse of normal pregnant women(t=30.944, 27.404, 32.827, 26.644; all P<0.001) and negatively correlated with the mRNA expression of PTEN in placenta(correlation coefficient=-0.648, -0.592, -0.537, -0.613; P=0.006, 0.009, 0.013, 0.007), positively correlated with the mRNA expression of Syncytin-1 in placenta(correlation coefficient=0.632, 0.572, 0.537, 0.654; P<0.010, 0.013, 0.015, 0.009); The contents of NOX4, MDA, 8-OHdG and the mRNA expression of Fas, FasL, Bax, GRP78, CHOP, Caspase-3 in placental tissue of preeclampsia was significantly higher than that of normal placental tissue(t=27.714, 27.704, 26.786, 27.964, 22.622, 25.496, 25.893, 30.837, 28.975; all P<0.001) and positively correlated with the mRNA expression of PTEN in placenta(correlation coefficient=0.703, 0.651, 0.593, 0.628, 0.449, 0.552, 0.612, 0.569, 0.502; P<0.003, 0.007, 0.004, 0.006, 0.016, 0.010, 0.006, 0.012, 0.018), negatively correlated with the mRNA expression of Syncytin-1 in placenta(correlation coefficient=-0.615, -0.714, -0.577, -0.548, -0.512, -0.585, -0.619, -0.495, -0.662; P<0.009, 0.001, 0.013, 0.015, 0.018, 0.011, 0.009, 0.023, 0.003); The contents of PrxII, PrxIII, SOD and mRNA expression of Bcl-2 in placental tissue of preeclampsia was significantly lower than that of normal placental tissue(t=26.115, 23.502, 17.194; all P<0.000) and negatively correlated with the mRNA expression of PTEN in placenta(correlation coefficient=-0.713, -0.657, -0.591, -0.642; P<0.001, 0.006, 0.009, 0.007), positively correlated with the mRNA expression of Syncytin-1 in placenta(correlation coefficient=0.562, 0.711, 0.682, 0.608, P<0.013, <0.001, 0.002, 0.008). Conclusion The high expression of PTEN and the low expression of syncytin-1 in placenta of preeclampsia can aggravate the damage of endothelium and trophoblast. Key words: Preeclampsia; Phosphatase and tensin homologue-deleted chromosome ten gene; Syncytin-1; Endothelial injury; Trophoblast injury

MiR-630 Regulates Proliferation and Apoptosis of Clear Cell Renal Cell Carcinoma Through Targeted Binding to Phosphatase and Tensin Homolog

Clear cell renal cell carcinoma (ccRCC) occurs as tumor cells in the kidney quickly accumulate and forms a lump (mass). Previous evidence showed that miR-630 overexpression is associated with gastric cancer progression. In this study, the differentially expressed miRNAs were screened in ccRCC patients using the gene expression profile chip. The possible targets for miR-630 were predicted by Target Scan and assessed via the luciferase reporter gene assay. The cell proliferation and apoptosis were detected via CCK-8 assay and flow cytometry. The Bcl-2, Bax, PTEN as well as downstream PI3K/AKT/mTOR signaling were detected via Western blotting. The result revealed that in silico analysis using the Target Scan software predicted that PTEN might be a potential target for miR-630, which was further verified via the luciferase reporter gene assay. The increase of miR-630 expression significantly raised the proliferation rate (P &lt; 0.05) and reduced cellapoptosis (P &lt; 0.05). In addition, the results of Western blotting also revealed that miR-630 mimic could activate PI3K/AKT/mTOR signaling (P &lt; 0.05). In conclusion, miR-630 cantargeted reduce the PTEN expression, and then activate the PI3K/AKT/mTOR signaling, ultimately promoting cell proliferation and inhibiting apoptosis of ccRCC.

MiR-19a suppress apoptosis of myocardial cells in rats with myocardial ischemia/reperfusion through PTEN/Akt/P-Akt signaling pathway.

To detect differentially expressed micro ribonucleic acids (miRNAs) in rats with myocardial ischemia/reperfusion (MIR), and to explore the influence of miR-19a on MIR rats and its mechanism. Firstly, the Sprague-Dawley (SD) rats were used to prepare MIR models, RNAs were extracted, and miRNA sequencing analysis was carried out to determine differentially expressed miRNAs related to MIR. Secondly, the predicted target genes of miR-19a were collected, and WebGestalt was applied to analyze gene ontology (GO) and pathway enrichment. Thirdly, the expression of the related proteins and the apoptosis of myocardial cells in MIR rats were detected via Western blotting. Fourthly, the interaction between miR-19a and the target gene phosphatase and tensin homolog (PTEN) was examined through Luciferase reporter assay. Compared with that in the Sham operation (Sham) group, the miR-19a expression in rat myocardial tissues in the MIR group was significantly increased (p<0.05). Compared with those in the miR-negative control (miR-NC) group, the messenger RNA (mRNA) and protein expressions of PTEN in the miR-19a group were notably decreased (p<0.05). In comparison with the miR-NC group, miR-19a group had elevated expression of phosphorylated protein kinase B (p-Akt) (p<0.05). The Luciferase reporter gene assay manifested the direct binding of miR-19a to PTEN mRNA. MiR-19a inhibits the PTEN expression by directly binding to the 3'-UTR of PTEN mRNA, thus activating the Akt/p-Akt signaling pathway to suppress the apoptosis of myocardial cells in MIR injury.

A specific amino acid context in EGFR and HER2 phosphorylation sites enables selective binding to the active site of Src homology phosphatase 2 (SHP2)

The Src homology phosphatase 2 (SHP2) is a cytoplasmic enzyme that mediates signaling induced by multiple receptor tyrosine kinases, including signaling by the epidermal growth factor receptor (EGFR) family (EGFR1-4 or the human homologs HER1-4). In EGFR (HER1) and EGFR2 (HER2) signaling, SHP2 increases the half-life of activated Ras by blocking recruitment of Ras GTPase-activating protein (RasGAP) to the plasma membrane through dephosphorylation of docking sites on the receptors. However, it is unclear how SHP2 selectively recognizes RasGAP-binding sites on EGFR and HER2. In this report, we show that SHP2-targeted pTyr residues exist in a specific amino acid context that allows selective binding. More specifically, we show that acidic residues N-terminal to the substrate pTyr in EGFR and HER2 mediate specific binding by the SHP2 active site, leading to blockade of RasGAP binding and optimal signaling by the two receptors. Molecular modeling studies revealed that a peptide derived from the region of pTyr992-EGFR packs well and makes stronger interactions with the SHP2 active site than with the SHP1 active site, suggesting a built-in mechanism that enables selective substrate recognition by SHP2. A phosphorylated form of this peptide inhibits SHP2 activity in vitro and EGFR and HER2 signaling in cells, suggesting inhibition of SHP2 protein tyrosine phosphatase activity by this peptide. Although we do not expect this peptide to be a strong inhibitor by itself, we foresee that the insights into SHP2 selectivity described here will be useful in future development of active-site small molecule-based inhibitors.

The effect of miRNA-34a antisense oligonucleotide on non-small cell lung cancer cell line HCC827

Objective To investigate the effect of antisense oligonucleotides of miRNA-34a on non-small cell lung cancer (NSCLC) and its molecular mechanism. Methods The expression of miRNA-34a in human non-small cell lung cancer cell line HCC827 and human normal lung cell MRC-5 was detected by real time fluorescence quantitative polymerase chain reaction (qRT-PCR). HCC827 cells were divided into three groups: blank control group, negative control group, anti-sense oligonucleotide group(liposome 2000 transfected anti-sense oligonucleotide miRNA-34a); cell counting kit-8 (CCK-8) method was used to detect cell proliferation, Jimsa staining was used to detect cell cloning ability, Transwell test was used to detect cell migration and invasion ability; RT-PCR and Western blot were used to detect phosphatase and tensin homolog (PTEN), phosphorylation-protein kinase B (p-Akt), phosphatidylinositol-3-kinase (PI3K) mRNA and protein expression. Results The relative expression of miRNA34a in HCC827 cells was significantly higher than that in human normal lung cells (P 0.05). At 48 h, 72 h and 96 h, the proliferation level of HCC827 cells in anti-sense oligonucleotide miRNA-34a group was significantly lower than that in negative control group and blank control group (P<0.05). The cell cloning rate of antisense oligonucleotide miRNA-34a group was significantly lower than that of negative control group and blank control group (P<0.01). The number of migration and invasion of HCC827 cells in antisense oligonucleotide RNA-34a group was significantly lower than that in negative control group and blank control group (P<0.01). The relative expression of PTEN mRNA and protein in antisense oligonucleotide miRNA-34a group was significantly higher than that in negative control group and blank control group (P<0.05); the relative expression of p-Akt, PI3K mRNA and protein in antisense oligonucleotide miRNA-34a group were significantly lower than that in negative control group and blank control group (P<0.05). Conclusions The expression level of miRNA-34a in human non-small cell lung cancer cells is significantly higher than that in human normal lung cells. Antisense oligonucleotides of miRNA-34a can inhibit the proliferation, cloning, migration and invasion of human non-small cell lung cancer cells. The mechanism may be related to the negative regulation of PTEN/p-Akt/PI3K signaling pathway. Key words: Oligonucleotides, antisense; miRNA-34a; Carcinoma, non-small-cell lung; Cell line, tumor

Study on the mechanism of inhibiting invasion of human laryngeal squamous cell carcinoma Hep-2 and TU212 cells after the downregulation of miRNA-106b

Objective To investigate the effect of miRNA-106b (miR-106b) on human laryngeal squamous cell carcinoma Hep-2 and TU212 cells and its mechanism. Methods Hep-2 and TU212 cells were divided into miR-106b inhibitory sequence transfected group (the experimental group), miR-106b competitive negative sequence transfected group (the negative control group) and non-intervention group (the blank group). The inhibitory effect of miR-106b inhibitory sequence on the expression of miR-106b was verified by using reverse transcription quantitative polymerase chain reaction (qRT-PCR). Whether phosphatase and tensin homolog (PTEN) was the target gene of miR-106b was analyzed by using bioinformatics and luciferase report vector. PTEN small interfering RNA (siRNA) was used to inhibit the expression of PTEN in Hep-2 and TU212 cells. Transwell method and Western blot were used to detect the change of invasion ability of Hep-2 and TU212 cells after miR-106b silencing or the PTEN intervening, and the expression change of PTEN, epithelial cadherin and vimentin. Results The relative expression levels of miR-106b in Hep-2 and TU212 cells in the experimental group were 0.110 ± 0.037 and 0.074 ± 0.009, respectively, which were lower than those in the negative control group (1.013±0.059 and 1.035±0.062, respectively; all P < 0.05). In Transwell experiments, the number of invasive cells in each field of Hep-2 and TU212 cells in the experimental group was less than that in the negative control group [(37.09±4.02) vs. (95.65±4.77), (29.16±2.49) vs. (103.19±6.08), all P < 0.05]. The bioinformatics analysis results showed that 3'-UTR region of PTEN mRNA was complementary to 3'-UTR region of miR-106b. Dual-luciferase reporter system analysis showed that the luciferase reporter activity of wild-type PTEN gene transfected with miR-106b was decreased to (22.84±2.68)%, and that of mutant PTEN gene transfected with miR-106b was almost unchanged [(92.08±3.44)%], and the difference was statistically significant (P < 0.001). The expression level of PTEN protein of Hep-2 and TU212 cells in the experimental group was higher than that in the negative control group. Transwell method showed that the number of invasive cells in each field of Hep-2 and TU212 cells in the experimental group with the inhibition of PTEN expression was more than that in the experimental group without the inhibition of PTEN expression [(65.08±3.57) vs. (26.72±2.58), (57.38±4.96) vs. (31.81±2.97), all P < 0.05]. Western blot showed that the expression level of epithelial-cadherin was up-regulated and vimentin was down-regulated of Hep-2 and TU212 cells in the experimental group with the inhibition of PTEN expression. Conclusions The human laryngeal squamous cell carcinoma Hep-2 and TU212 cell miR-106b can influence the downstream invasion-related protein of PTEN and change the cell invasion ability through the targeted regulation of PTEN expression. Key words: Laryngeal neoplasms; Carcinoma, squamous cell; Neoplasm invasiveness; RNA interference; miRNA-106b; Phosphatase and tensin homolog

Abstract P1-09-08: Development and characterization of PTEN IHC assay for testing breast cancer patients specimens

Abstract Background: Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene that is a major negative regulator of the Phosphatidylinositol 3-kinase (PI3K) pathway. Loss of PTEN protein expression has been mechanistically linked to tumor progression. Blocking the PI3K pathway might inhibit the growth and proliferation of cells that have deletions in PTEN. Thus characterization of PTEN expression in patient tumor samples may assist prediction of potential response to PI3K inhibitor therapies. Methods: We developed and validated an immunohistochemical assay on Ventana BenchMark XT to detect PTEN in formalin fixed and paraffin-embedded (FFPE) tissue by utilizing a rabbit monoclonal antibody (clone 138G6) from Cell Signaling Technologies that recognizes the carboxy-terminal domain of PTEN. PTEN immunohistochemical staining was performed on 1577 breast tumor specimens to determine PTEN protein loss. A subset of these cases (n=663) was also assessed for PTEN mutation. Results: Cellular localization of PTEN expression was observed in both the cytoplasm and the nucleus. Both cellular compartments were scored and used in the staining intensity determination. We observed that PTEN staining is sensitive to variation in tissue handling, fixation and antigen retrieval. PTEN staining was affected significantly by antigen retrieval method. Optimal staining conditions were determined to be 1:60 antibody dilution using Citrate pH 6.0 as antigen retrieval buffer. In a cohort of 1577 hormone receptor positive HER2-negative locally advanced or metastatic breast cancer patients, loss of PTEN protein was observed in 8.6% (135/1577) of patients. There was a positive correlation between PTEN mutation rate (17 out of 66 PTEN IHC positive cases vs 14 out of 587 negative cases) and loss of PTEN protein expression. PTEN loss was observed to correlate with better clinical response to PI3K inhibitor. Conclusions: Pre-analytical handling of samples is important for PTEN IHC staining. PTEN mutations and insertions/deletions contribute to PTEN protein loss. This study validates a simple method to interrogate PTEN status in clinical specimens and supports the utility of this test in selecting patients who are likely to respond to PI3K inhibitor treatment. Citation Format: Hua Gong, Emmanuel Pacia, Sharmila Manjeshwar, Beiru Chen, Xun Li, Bashar Dabbas, Jelveh Lameh, Naveen Babbar, Shabnam Tangri. Development and characterization of PTEN IHC assay for testing breast cancer patients specimens [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-09-08.

MicroRNA-92a: The Administrator of Certain Diseases

MicroRNA-92a (miR-92a) is an evolutionarily conserved noncoding small RNA that can regulate gene expression after transcription. Previous studies have found that miR-92a is overexpressed in many tumors and can regulate numerous tumor suppressor genes negatively, with relevant effects on the development of different tumors, by regulating the DUSP10/c-Jun N-terminal kinase (JNK), phosphatase and tensin homologs (PTEN)/AKT, Wnt and EP4/Notch1 signaling axes. MiR-92a also promotes the proliferation and migration of vascular smooth muscle cells (VSMCs) through the Rho-associated coiled-coil-forming kinase/myosin light chain kinase signaling pathway and inhibits VSMC apoptosis through the MKK4/JNK signaling pathway. Moreover, miR-92a affects endothelial functions; mediates endothelial dysfunction in chronic kidney diseases; mediates THBS1 inhibition; promotes the migration, proliferation and angiogenesis of neighboring endothelial cells (ECs); mediates the Nrf2/KEAP1/ARE signaling pathway to regulate vascular endothelial aging; and is involved in immune responses to activate ECs. This review summarizes the potential role and pathogenic mechanism of the miR-92a gene in certain diseases to provide possible new treatment options.

Mesenchymal stem cells promote human melanocytes proliferation and resistance to apoptosis through PTEN pathway in vitiligo

BackgroundVitiligo is an acquired chronic and recurrent skin disease that causes a depigmentation disorder, resulting in selective destruction of melanocytes (MC). However, the mechanism that leads to melanocyte dysfunction and death remains unclear.MethodsWe performed RNA sequencing, immunohistochemistry, and immunoblotting to characterize the patterns of phosphatase and tensin homolog (PTEN)/phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway activation in vitiligo. We also cocultured primary melanocytes with mesenchymal stem cells (MSCs) in a Transwell system to explore how MSCs inhibit the PTEN/PI3K/AKT pathway in melanocytes.ResultsWe identified that vitiligo normal-lesional junction skin presented with high expression of PTEN, which led to the inhibition of AKT phosphorylation (p-AKT) at S-473. Furthermore, PTEN overexpression led to oxidative stress-induced apoptosis in melanocytes. Coculturing with MSCs enhanced the cell proliferation of human melanocytes and repressed PTEN expression, which inhibited oxidative stress-induced apoptosis.ConclusionWe report that vitiligo patients present with high PTEN expression, which may play a role in the impairment of melanocytes. Furthermore, our study provides evidence that MSCs target the PTEN/PI3K/AKT pathway to regulate cell proliferation and apoptosis in human melanocytes, indicating that MSCs may serve as a promising therapy for vitiligo.

MiR-203 Regulates DJ-1/Phosphatase and Tensin Homologue Deleted on Chromosome Ten/Phosphatidylinositol-3 Kinase/Protein Kinase B Pathway and Affects Proliferation, Apoptosis and Cisplatin Resistance of Ovarian Cancer Cells

DJ-1 negatively regulates PTEN and plays a role in tumorigenesis and progression. Abnormal miR-203 expression is associated with ovarian cancer. miR-203 was predicted to have a relationship with DJ-1 3-UTR. Our study assessed miR-203’s role in ovarian cancer cell cisplatin (CDDP) resistance. The CDDP-resistant cell line SKOV3/CDDP was established and compared with the expression of miR-203 and DJ-1 in the parental SKOV3 cells. SKOV3/CDDP cells were separated into miR-NC group and miR-203 mimic group followed by analysis of DJ-1, PTEN and p-AKT expression, cell apoptosis and proliferation. There was a targeted relationship between miR-203 and DJ-1 mRNA. miR-203 and PTEN protein expression in SKOV3/CDDP cells was significantly reduced compared to SKOV3 cells with significantly upregulated DJ-1. miR-203 mimic significantly upregulated miR-203, decreased DJ-1 and p-AKT protein expression and elevated PTEN expression along with increased cell apoptosis and reduced cell proliferative ability. Decreased miR-203 expression and increased DJ-1 expression participate in drug resistance in ovarian cancer cells. miR-203 inhibits DJ-1 expression, affects PTEN-PI3K/AKT signaling, inhibits ovarian cancer cell proliferation and promotes apoptosis, and reduces CDDP resistance.