Phosphatase Homolog Research Articles

Hepatic HuR protects against the pathogenesis of non-alcoholic fatty liver disease by targeting PTEN

The liver plays an important role in lipid and glucose metabolism. Here, we show the role of human antigen R (HuR), an RNA regulator protein, in hepatocyte steatosis and glucose metabolism. We investigated the level of HuR in the liver of mice fed a normal chow diet (NCD) and a high-fat diet (HFD). HuR was downregulated in the livers of HFD-fed mice. Liver-specific HuR knockout (HuRLKO) mice showed exacerbated HFD-induced hepatic steatosis along with enhanced glucose tolerance as compared with control mice. Mechanistically, HuR could bind to the adenylate uridylate-rich elements of phosphatase and tensin homolog deleted on the chromosome 10 (PTEN) mRNA 3′ untranslated region, resulting in the increased stability of Pten mRNA; genetic knockdown of HuR decreased the expression of PTEN. Finally, lentiviral overexpression of PTEN alleviated the development of hepatic steatosis in HuRLKO mice in vivo. Overall, HuR regulates lipid and glucose metabolism by targeting PTEN.

Overall survival of patients with metastatic castrate-resistant prostate cancer (mCRPC) who have PTEN tumor suppressor gene loss of function.

58 Background: It is estimated that more than 40% of patients with mCRPC have functional loss of phosphatase and tensin homolog (PTEN) tumor suppressor gene, which is associated with unfavorable prognosis and reduced response to androgen receptor-targeting therapy. We describe patient characteristics and survival outcomes by PTEN LOF status among patients with mCRPC in real-world clinical practice. Methods: We conducted a retrospective cohort study using data from the nationwide Flatiron Health-Foundation Medicine mCRPC Clinico-Genomic database (FH-FMI CGDB), a de-identified database linking data derived from electronic health records with genomic data derived from FMI comprehensive genomic profiling (CGP) tests. The study included patients ≥18 years old, with a primary diagnosis of mCRPC between 1/1/2013 and 6/30/2019 who underwent FMI CGP testing and who had a valid PTEN LOF status. Patients were included if their PTEN report date and mCRPC diagnosis date occurred before death or censoring. PTEN LOF status was identified via FMI’s CGP testing. Kaplan-Meier (KM) methods assessed overall survival (OS) by PTEN LOF status from the date of mCRPC diagnosis (later of metastasis and castration resistance) until death or end of study follow-up. A stratified Cox regression model was used to estimate the hazard of death. The Cox model was adjusted for age, race and sequence of metastasis/CRPC diagnoses, and was stratified by the year of mCRPC diagnosis. Adjustments to account for left-truncation and survivorship bias were made in the KM analysis and the Cox regression model. Results: Among the 458 patients who met the eligibility criteria, 174 (38%) had PTEN LOF. The majority of the study sample (76%) was diagnosed with castration-resistance after metastasis. The PTEN LOF group had a higher percentage of white patients (80% vs. 68%; p= 0.01) compared to the PTEN non-LOF group. The mean age of the study sample was 68 years, and there was no difference in mean age at diagnosis by PTEN LOF status ( p= 0.17). Based on the KM estimates adjusted for left-truncation, the median OS was 14.3 months (95% confidence interval [CI]: 11.1-19.7) in the PTEN LOF group compared to 18.3 months (95%CI: 15.5-21.5) in the PTEN non-LOF group (log-rank p= 0.049). In the multivariable Cox model, the PTEN LOF group had numerically 30% higher risk of death compared to the PTEN non-LOF group (hazard ratio = 1.30; 95% CI: 0.99-1.71; p= 0.057). Conclusions: Among real-world patients with mCRPC in the CGDB, PTEN LOF could be associated with poorer survival outcomes, potentially highlighting the unmet need among these patients. Additional studies with larger cohorts are needed to better evaluate the survival outcomes of patients with PTEN LOF. Therapeutic agents acting on the PTEN/PI3K/AKT/mTOR pathway are being tested in clinical trials, and could potentially improve outcomes in this subgroup of patients with mCRPC.

Argon inhibits reactive oxygen species oxidative stress via the miR-21-mediated PDCD4/PTEN pathway to prevent myocardial ischemia/reperfusion injury

ABSTRACT The objective of this study was to explore the effect of argon preconditioning on myocardial ischemia reperfusion (MI/R) injury and its mechanism. Cardiomyocytes H2C9 were pre-treated with 50% argon, and a cell model of oxygen-glucose deprivation (OGD) was established. CCK-8 and cytotoxicity detection kits were used to detect cell viability and lactate dehydrogenase (LDH) release. The miR-21 expression was detected using quantitative real-time polymerase chain reaction. Western blot analysis was performed to detect the expression of programmed cell death protein 4 (PDCD4) and homologous phosphatase and tensin homolog (PTEN) proteins. The levels of inflammatory factors (IL-1β, IL-6, and IL-8) and oxidative stress factors (reactive oxygen species ROS], malondialdehyde [MDA], and superoxide dismutase [SOD]) were measured using an enzyme-linked immunosorbent assay. The effect of argon on cell apoptosis was detected using flow cytometry. Argon increased the proliferation of cardiomyocytes induced by OGD, decreased the release of LDH in cell culture medium, increased miR-21 expression in cells, decreased the expression of miR-21 target proteins PDCD4 and PTEN, decreased the levels of inflammatory factors (interleukin-1β [IL-1β], interleukin-6 [IL-6], and interleukin-8 [IL-8]) and oxidative stress factors (ROS and MDA), increased the SOD content, and decreased the cell apoptosis rate. Our results suggest that argon preconditioning inhibited the PDCD4/PTEN pathway via miR-21, thereby inhibiting ROS oxidative stress and preventing MI/R injury.

Age-Related Increased Onset and Progression of Prostate Cancer Is Revealed in Novel Pten-Null Mouse Models

Prostate cancer (PCa) is associated with advanced age. To better understand how age impacts PCa, it is critical to use PCa animal models generated at different ages (aged vs. non-aged). The PB-Cre4 driven phosphatase and tensin homolog (Pten) conditional knockout mouse model, which closely imitates human PCa initiation and progression. However, the Pten deletion is triggered in a 2-week-old prostate, when comparing the extent of PCa between aged and non-aged mice, it is difficult to distinguish the extent to which the onset and progression of PCa are due to the acceleration of the normal aging process or due to the manifestation of PCa pathologies over time. We present here a protocol to inject Cre-expressing adenovirus with luciferin tag intraductally into the prostate anterior lobes of Pten floxed mice, thus, the Pten-loss will be triggered at different ages post-Cre expression. The in vivo imaging of luciferin signals following viral infection was conducted to confirm the Cre expression and activity. Immunohistochemical staining was performed to confirm the Cre expression, Pten loss, and p-Akt and p-S6 activation. Prostate weight and histopathology were compared between aged and non-aged mice. The results showed that the virus infection was limited in the prostate glands and aged mice had significantly increased PCa onset and progression compared to young mice. Although technical skill is required to carry out this procedure and the success rate of viral infection is about 80%, this model of PCa is of great use to all investigators in the aging and cancer research field.

Phosphatase and Tensin Homolog Mutation in Immune Cell Infiltration and Clinicopathological Features of Low-Grade Gliomas.

The mutation of phosphatase and tensin homolog (PTEN) genes frequently occur in low-grade gliomas (LGGs) and are deeply associated with a poor prognosis and survival rate. In order to identify the crucial signaling pathways and genes associated with the PTEN mutation, we performed bioinformatics analysis on the RNA sequencing results, which were obtained from The Cancer Genome Atlas database. A total of 352 genes were identified as differentially expressed genes (DEGs). The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that the DEGs were significantly enriched in categories associated with cell division and multiple metabolic progressions. The histological stage was significantly associated with PTEN expression levels. In addition, the PTEN mutation was associated with an abundance of B cells, neutrophils, macrophages, dendritic cells, and CD8+ T cells during tumor infiltration. The results showed that patients with LGGs harboring the PTEN mutation had a poor prognosis and more serious immune cell infiltration occurred depending on the mRNA expression level. These results demonstrated that multiple genes and signaling pathways play a key role in LGG from low grade to high grade, and are associated with PTEN mutations. In this study, we outlined an approach to assess the influence of PTEN mutations on prognosis, overall survival, and messenger RNA (mRNA) expression. Our results provided alternative strategies for the personalized treatment of patients with LGGs harboring the PTEN mutation.

Prediction of the potency of active compounds in pasak bumi root as the treatment for prostate cancer through phosphatase and tensin homolog (PTEN) pathway

Prediction of the potency of active compounds in pasak bumi root as the treatment for prostate cancer through phosphatase and tensin homolog (PTEN) pathway

Bone marrow mesenchymal stem cells derived miRNA-130b enhances epithelial sodium channel by targeting PTEN

AimsAcute lung injury (ALI) is a clinical syndrome with high morbidity and mortality, and severe pulmonary edema is one of the characteristics. Epithelial sodium channel (ENaC) located on the apical side of alveolar type 2 epithelial (AT2) cells is the primary rate limiting segment in alveolar fluid clearance. Many preclinical studies have revealed that mesenchymal stem cells (MSCs) based therapy has great therapeutic potential for ALI, while the role of ENaC in this process is rarely known.MethodsWe studied the effects of bone marrow-derived MSCs (BMSCs) on the protein/mRNA expression and activity of ENaC in primary mouse AT2 and human H441 cells by co-culture with them, respectively. Moreover, the changes of miRNA-130b in AT2 cells were detected by qRT-PCR, and we studied the involvement of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and the downstream PI3K/AKT pathway in the miRNA-130b regulation of ENaC.ResultsOur results demonstrated that BMSCs could increase ENaC protein expression and function, as well as the expression level of miRNA-130b. The dual luciferase target gene assay verified that PTEN was one of the target genes of miR-130b, which showed adverse effects on the protein expression of α/γ-ENaC and PTEN in AT2 cells. Upregulating miR-130b and/or knocking down PTEN resulted in the increase of α/γ-ENaC protein level, and the protein expression of p-AKT/AKT was enhanced by miR-130b. Both α and γ-ENaC protein expressions were increased after AT2 cells were transfected with siPTEN, which could be reversed by the co-administration of PI3K/AKT inhibitor LY294002.ConclusionIn summary, miRNA-130b in BMSCs can enhance ENaC at least partially by targeting PTEN and activating PI3K/AKT pathway, which may provide a promising new direction for therapeutic strategy in ALI.

Polyphenols Extracted from Chinese Hickory (Carya cathayensis) Promote Apoptosis and Inhibit Proliferation through the p53-Dependent Intrinsic and HIF-1α-VEGF Pathways in Ovarian Cancer Cells.

Ovarian cancer is the second most common gynecologic cancer with an estimated 13,940 mortalities across the United States in 2020. Natural polyphenols have been shown to double the survival time of some cancer patients due to their anticancer properties. Therefore, the effect of polyphenols extracted from Chinese hickory seed skin Carya cathayensis (CHSP) on ovarian cancer was investigated in the present study. Cell viability results showed that CHSP is more effective in inhibiting ovarian cancer cells than normal ovarian cells, with the IC50 value for inhibition of cell proliferation of Ovarian cancer cells (OVCAR-3) being 10.33 ± 0.166 μg/mL for a 24 h treatment. Flow cytometry results showed that the apoptosis rate was significantly increased to 44.21% after 24 h treatment with 20 μg/mL of CHSP. Western blot analysis showed that CHSP induced apoptosis of ovarian cancer cells through a p53-dependent intrinsic pathway. Compared with control values, levels of VEGF excreted by OVCAR-3 cancer cells were reduced to 7.87% with a 40 μg/mL CHSP treatment. Consistent with our previous reports, CHSP inhibits vascular endothelial growth factor (VEGF) secretion by regulating the HIF-1α-VEGF pathway. In addition, we also found that the inhibitory effect of CHSP on ovarian cancer is related to the up-regulation of Phosphatase and tension homolog (PTEN) and down-regulation of nuclear factor kappa-B (NF-kappa B). These findings provide some evidence of the anti-ovarian cancer properties of CHSP and support the polyphenols as potential candidates for ovarian cancer adjuvant therapy.

Abstract PO-054: A stromal PTEN/STAT3 signaling axis promotes immune evasion and tumor progression in pancreatic cancer

Abstract Pancreatic adenocarcinoma (PDAC) is among the most lethal cancer types, with more than 90% of patients succumbing to the disease within five years of diagnosis. The most notable hallmark of PDAC tumors is an inordinately dense stromal reaction comprised of extracellular matrix proteins, immune infiltrates, endothelial cells, and activated fibroblasts. The stroma experiences a dramatic expansion in parallel with the progression of the tumor, suggesting it is an active participant in PDAC development. However, key studies targeting stromal development through Hedgehog signaling revealed tumor-suppressive functions of the stroma, further complicating the strategy to target the stroma as a single component. Thus, our understanding of the complex relationship between the tumor and its microenvironment and how to effectively target the stroma itself remains incomplete. Previous work by our lab demonstrates that phosphatase and tensin homolog (PTEN) expression is lost in approximately 25% of patients in a PDAC cohort, and lower PTEN expression correlates with worsened patient outcome. Additionally, inhibition of Hedgehog signaling results in loss of PTEN expression and subsequent activation of Akt signaling pathways, and increased tumor growth. The purpose of the current study is to determine the effect of PTEN loss in PDAC cancer-associated fibroblasts (CAFs) on tumor progression and dissemination, and immune evasion. Using a combination of in vitro and in vivo experiments, we have found robust activation of Signal Transducer and Activator of Transcription 3 (STAT3) in PTEN-null pancreatic fibroblasts. We also see selective alterations in the secretome of pancreatic fibroblasts, including a marked increase in IL-6, CXCL1, and CXCL2. Our group has developed a genetically engineered mouse model of PDAC using a dual recombinase system in which the fibroblast-specific protein-1 (FspCre) transgene is used to conditionally delete genes of interest in the fibroblasts of Pdx1Flpki/+;FSF-KrasG12D/+;p53frt/+ (KPF) mice while driving tumorigenesis in the pancreatic epithelium. Conditional deletion of STAT3 in stromal fibroblasts of these mice results in increased overall survival, reduced tumor progression, and alterations within the immune landscape of the tumor microenvironment. Based on our current findings, we believe that a PTEN/STAT3/CXCL1/2 signaling axis within stromal CAFs produces an immunosuppressive tumor microenvironment that favors PDAC progression. Future experiments are needed to validate and confirm this conclusion, including defining the exact mechanisms by which PTEN loss leads to STAT3 activation and elevated secretion of CXCL1/2. This project further highlights the nuanced and complex relationship between PDAC tumor and stroma, and may serve to advance our understanding of how the stroma can be more precisely and effectively targeted to reduce tumor burden and increase the efficacy of immune therapy. Citation Format: Catherine B. MarElia-Bennett, Julia E. Lefler, Michael C. Ostrowski. A stromal PTEN/STAT3 signaling axis promotes immune evasion and tumor progression in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-054.

Prediction of PIK3CA mutations from cancer gene expression data.

Breast cancers with PIK3CA mutations can be treated with PIK3CA inhibitors in hormone receptor-positive HER2 negative subtypes. We applied a supervised elastic net penalized logistic regression model to predict PIK3CA mutations from gene expression data. This regression approach was applied to predict modeling using the TCGA pan-cancer dataset. Approximately 10,000 cases were available for PIK3CA mutation and mRNA expression data. In 10-fold cross-validation, the model with λ = 0.01 and α = 1.0 (ridge regression) showed the best performance, in terms of area under the receiver operating characteristic (AUROC). The final model was developed with selected hyper-parameters using the entire training set. The training set AUROC was 0.93, and the test set AUROC was 0.84. The area under the precision-recall (AUPR) of the training set was 0.66, and the test set AUPR was 0.39. Cancer types were the most important predictors. Both insulin like growth factor 1 receptor (IGF1R) and the phosphatase and tensin homolog (PTEN) were the most significant genes in gene expression predictors. Our study suggests that predicting genomic alterations using gene expression data is possible, with good outcomes.

Abstract PO-24: 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 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, Jamal Zweit, Steven Grant, Jolene Windle, Steven Grossman. Modeling marginal zone lymphomagenesis [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-24.

SET8 suppression mediates high glucose-induced vascular endothelial inflammation via the upregulation of PTEN

Hyperglycemia-mediated endothelial inflammation participates in the pathogenesis of cardiovascular complications in subjects with diabetes. Previous studies reported that phosphatase and tensin homolog deleted on chromosome ten (PTEN) and SET8 participate in high glucose-mediated endothelial inflammation. In this study, we hypothesize that SET8 regulates PTEN expression, thus contributing to high glucose-mediated vascular endothelial inflammation. Our data indicated that plasma soluble intercellular adhesion molecule-1 (sICAM-1) and endothelial selectin (e-selectin) were increased in patients with diabetes and diabetic rats. PTEN expression was augmented in the peripheral blood mononuclear cells of patients with diabetes and in the aortic tissues of diabetic rats. Our in vitro study indicated that high glucose increased monocyte/endothelial adhesion, endothelial adhesion molecule expression and p65 phosphorylation in human umbilical vein endothelial cells (HUVECs). Moreover, high glucose led to endothelial inflammation via upregulation of PTEN. Furthermore, high glucose inhibited SET8 expression and histone H4 lysine 20 methylation (H4K20me1), a downstream target of SET8. SET8 overexpression reversed the effects of high-glucose treatment. shSET8-mediated endothelial inflammation was counteracted by siPTEN. Furthermore, SET8 was found to interact with FOXO1. siFOXO1 attenuated high glucose-mediated endothelial inflammation. FOXO1 overexpression-mediated endothelial inflammation was counteracted by siPTEN. H4K20me1 and FOXO1 were enriched in the PTEN promoter region. shSET8 increased PTEN promoter activity and augmented the positive effect of FOXO1 overexpression on PTEN promoter activity. Our in vivo study indicated that SET8 was downregulated and FOXO1 was upregulated in the peripheral blood mononuclear cells of patients with diabetes and the aortic tissues of diabetic rats. In conclusion, SET8 interacted with FOXO1 to modulate PTEN expression in vascular endothelial cells, thus contributing to hyperglycemia-mediated endothelial inflammation.

Abstract B07: TAK228 enhances antitumor activity of eribulin in triple-negative breast cancer

Abstract Background: Phosphatase and tensin homologue deleted from chromosome 10 (PTEN) negatively regulates the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway. Triple-negative breast cancers (TNBC) are often PTEN-deficient, making mTOR a compelling target. We evaluated the efficacy of catalytic mTOR inhibitor TAK228 alone and in combination with eribulin in TNBC. Methods: We tested TAK228 in combination with eribulin in a panel of TNBC cell lines with cell proliferation assays. Western blot analysis was performed to assess PTEN status and PI3K pathway inhibition. In vivo, antitumor efficacy of TAK228 as a single agent as well as in combination with eribulin was evaluated. Results: Five of eight triple-negative breast cell lines were sensitive to TAK228, independent of PIK3CA/PTEN status. Western blotting demonstrated inhibition of mTORC1/2 signaling as demonstrated by decreased phospho-AKT, phospho-S6 and phospho-4EBP1. In vitro, TAK228 was synergistic with eribulin in all eight TNBC cell lines. The combination of TAK228 and eribulin did not enhance apoptosis but increased G2/M growth arrest (p<0.001). In vivo, TAK228 led to modest growth inhibition in TNBC derived xenografts with no tumor regression observed. In two TNBC PDXs with PTEN loss, one with intrinsic eribulin sensitivity, another eribulin resistance, TAK228 in combination with eribulin did not enhance in vivo efficacy. In a third PTEN-negative TNBC model, eribulin alone achieved disease stabilization, but the combination of TAK228 and eribulin led to significantly smaller tumor volumes compared to eribulin alone (p<0.0001). Conclusion: TAK228 enhanced the antitumor efficacy of eribulin in TNBC models in vitro and enhanced in vivo activity in selected models. Further study is needed to determine the potential of this combination and optimal patient selection strategies. Citation Format: Nicci Owusu-Brackett, Kurt W. Evans, Argun Akcakanat, Erkan Yuca, Ecaterina Ileana Dumbrava, Filip Janku, Funda Meric-Bernstam. TAK228 enhances antitumor activity of eribulin in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B07.

Clinical Significance of miR-575 in Ovarian Cancer and Its Effect on Ovarian Cancer Cells

Ovarian cancer (OC) is a common form of gynecological cancer that increases in incidence annually, seriously threatening the health and safety of patients. The critical role of miR-575 in multiple neoplastic diseases has already been well established. We suspected that miRNA-575 may be strongly associated with the occurrence of OC and may be the key to OC diagnosis and treatment in the future. To test this hypothesis, we selected 68 OC patients (research group, or RG) who were admitted to our hospital and 60 concurrent healthy controls (control group, or CG) as the study participants. Human OC cell lines A2780, OVCAR-3, and SK-OV-3, and human normal ovarian epithelial cells IOSE80 were used in this study. Nanoparticle-assisted polymerase chain reaction (Nano-PCR), was used to identify miRNA-575 expression and its diagnostic implications in OC. miRNA-575 expression in OC cells and its influence on OC cell biological behaviors were investigated, and the correlation between miR-575 and the phosphatase and tensin homolog (PTEN) was discussed. The results revealed a highly expressed miRNA-575 in OC. Detection of biological behaviors was conducted after transfection of miRNA-575 into OC cells. Cell proliferation and invasion in the mimics-miRNA-575 group was statistically enhanced compared to the negative control (NC) and ininhibition-miRNA-575 groups, and the apoptosis and apoptosis protein count significantly declined (P < 0.05). Subsequently, PTEN was observed to present a lower expression in OC cells. Online target gene analysis website (http://www.targetscan.org/vert_72/) identified the related sites that could bind miR-575 and PTEN. PTEN protein detection in the mimics-miR-575, ininhibition-miR-575, and NC groups showed that the PTEN protein expression in the mimics-miR-575 group was reduced in comparison with that of the other groups, while that for ininhibition-miR-575 was elevated compared to that of the NC group (P < 0.05). Therefore, we came to the conclusion that a high expression of miR-575 can bolster OC cell proliferation and invasion and inhibit apoptosis, while the impact of miR-575 on OC cells may be achieved via targeting the PTEN protein. miR-575 presents important clinical applications in OC diagnosis as an underlying therapeutic target of OC and is a major breakthrough toward future OC research.

COWDEN SYNDROME DIAGNOSIS FROM ORAL MANIFESTATIONS: A CASE REPORT

Cowden syndrome (CS) is a rare condition, inherited with autosomal dominant trait, resulting from mutation in the Phosphatase and Tensin homolog gene on chromosome 10 and characterized mainly by multiple hamartomas and increased risk of malignant development. The report describes a case in a 43-year-old white male patient, complaining of a nodule on the tongue. In the anamnesis, a previous history of malignant neoplasms in the thyroid and testicles treated 20 years ago was been reported. Physical examination showed multiple small papules on the facial skin around eyes and mouth, multiple asymptomatic hamartomas in the labial mucosa, buccal mucosa, dorsal tongue and gingivae, and small superficial keratotic plaques in the dorsal surface of the hands. Three biopsies were performed in different oral sites with histopathological diagnosis of fibrous hyperplasia. Diagnosis of CS was confirmed by medical history, clinical, and histopathological characteristics. The patient is followed up in medical and dental care.

Cowden syndrome is a risk factor for multiple neoplasm: a case report

BackgroundCowden’s syndrome is an autosomal dominant disease with variable penetrance, involving the tumor suppressor phosphatase and tension homolog gene, located on chromosome 10q22-23, responsible for cell proliferation, migration, and cellular apoptosis. Its clinical presentation encompasses mucocutaneous lesions, which are present around 99% of the time; macrocephaly; and cognitive impairment, and it precedes the appearance of neoplasms such as thyroid carcinoma, breast cancer, among others.In addition to these malformations, arteriovenous malformations of the brain and spine, endocrine abnormalities, skeletal defects, and cardiopulmonary lesions may also be found.The relevance of the case is due to the fact that, through a certain phenotype, the patient’s genotype can be inferred and thus followed up closely.Case representationThe clinical case concerns a 28-year-old Caucasian and Portuguese woman with palmar pits, macrocephaly, and cognitive impairment. She was diagnosed with papillary thyroid carcinoma at 22 years of age and proposed total thyroidectomy.At age 27, a pregnancy was diagnosed with a Breast Imaging-Reporting and Data System 2-rated breast lump. After the histological verification, it was concluded that it was a high metastatic breast sarcoma, opting for palliative mastectomy. A genetic evaluation confirmed alteration in the phosphatase and tension homolog gene, confirming Cowden’s syndrome. The patient died at age 29 due to neoplastic pathology.ConclusionThis report aims to alert to the clinical signs of this entity and the clinical supervision and follow-up of these patients. In order to prevent premature deaths and to improve patient’s quality of life, genetic diseases with cancer impact should be diagnosed as early as possible.

Abstract 1434: Loss of PTEN confers resistance to neddylation inhibition through TOP2A in glioblastoma

Abstract As a disease that has not seen lasting advances in its standard of care for decades, glioblastoma (GBM) calls for novel therapeutic approaches, and given GBM's heterogeneous and aggressive nature, precision medicine stands out as an appealing treatment modality for this tragic disease. Neddylation is a post-translational modification process that regulates protein function and turnover. MLN4924, or Pevonedistat, inhibits the E1 ligase Nedd8 activating enzyme (NAE) and, in doing so, the E1, E2, and E3 neddylation cascade. In its most researched role, NEDD-8 is conjugated to the cullin subunit of cullin-RING ligases (CRLs), a family of E3 ligases that facilitate the ubiquitination of different substrates. Thus, inhibition of neddylation decreases ubiquitination and degradation of particular substrates: e.g., p21, p27, wee-1, and other cell cycle regulatory and pro-apoptotic proteins. We and others have shown that neddylation stands out a promising therapeutic approach to cancer and GBM, given the vulnerability to MLN4924 in subset of different in vitro and in vivo models. In this work, we establish the selective vulnerability to MLN4924 in GBM and explore the biological mechanisms that underlie this differential response. Using the Cancer Cell Line Encyclopedia (CCLE) and the Genomics of Drug Sensitivity in Cancer (GDSC) databases, we found that mutations and deletions in phosphatase and tensin homolog (PTEN) are associated with resistance to MLN4924 in both low-grade gliomas and GBM. Our established models validated these findings, as our sensitive models – GB1 (IC50= 0.28 μM) and LN18 (IC50 = 0.19 μM), – expressed PTEN, while our resistant models – M059K (IC50 = 5.5 μM) and SNU1105 (IC50 = 20.9 μM) – did not. Knocking down PTEN with siRNA in our sensitive models increased their IC50 values three- to four-fold. This relationship was further validated in multiple isogenic systems. We sought to uncover whether this PTEN-status dependent resistance was contingent on its well-studied lipid phosphatase activity and PI3K/AKT signaling. While the resistant models had higher baseline levels of phosphorylated AKT (p-AKT), we found that MLN4924 treatment did not consistently alter p-AKT levels in our sensitive and/or resistant models. We also used a PTEN null isogenic glioma stem cell system (GSC23), in which wild-type PTEN and lipid phosphatase dead (G129A) PTEN was re-expressed, and found that both PTEN models sensitized GSC23 to MLN4924 treatment equally. While independent of the PI3K/AKT axis, we did find that there were significant increases in topoisomerase II alpha (TOP2A) levels in the post-treatment lysate of our resistant (PTEN-null) models. Targeting this increase in TOP2A expression, we found strong synergy with two different TOP2 poisons – etoposide and doxorubicin – and MLN4924 in a combination DDR. For the first time, we show that PTEN status not only serves as a novel biomarker for MLN4924 response but also reveals a vulnerability to TOP2A inhibitors when used in combination with MLN4924. Citation Format: Brett Taylor, Shayesteh Ferdosi, Nanyun Tang, Rita Bybee, George Reid, Lauren Hartman, Darren Finlay, Kristiina Vuori, Matthew Lee, Sen Peng, Frank Furnari, Harshil Dhruv, Michael Berens. Loss of PTEN confers resistance to neddylation inhibition through TOP2A in glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1434.

Abstract 5314: A novel dopamine receptor D2 antagonist (ONC206) potentiates the effects of olaparib in endometrial cancer cells through inhibition of cell proliferation and modulation of the mTOR pathway

Abstract Objectives: Poly ADP-ribose polymerase (PARP) inhibitors are effective therapies for patients with homologous recombination (HR) deficient tumors. While HR mutations are rare in endometrial cancers (ECs), emerging data suggests that loss of function of the tumor suppressor gene Phosphatase and Tensin homolog (PTEN), which is mutated in 80% of endometrioid ECs, may also sensitize tumors to PARP inhibition. The imipridone ONC206, a chemical derivative of ONC201, is an orally bioavailable dopamine receptor D2 (DRD2) antagonist that has anti-tumorigenic effects in EC via induction of apoptosis and activation of the integrated stress response. ONC206 exhibits enhanced non-competitive DRD2 antagonism, potency, and bioavailability relative to ONC201. Both olaparib and imipridones (i.e. ONC201) are being evaluated in EC clinical trials, but have yet to be explored in combination. Thus, we sought to examine the effects of olaparib in combination with ONC206 in human endometrioid EC cell lines. Methods: The ECC-1 and HEC-1 EC cell lines were used. Cell proliferation was assessed after exposure to varying doses of olaparib (MedChemExpress) and ONC206 (Oncoceutics) via the MTT assay. Synergy between olaparib and ONC206 was assessed by the combination index (CI) method of Chou-Talalay. Cellular stress was evaluated using the DCFH-DA assay. Apoptosis was assessed using the cleaved caspase-3 assay. Western immunoblotting was performed to determine effects of olaparib and ONC206 on apoptosis and the mTOR pathway. Results: Both drugs inhibited cell proliferation in a dose-dependent manner (IC50 for olaparib of 15 uM for ECC-1 and 25 uM for HEC-1; IC50 for ONC206 of 3.3 uM for ECC-1 and 0.9 uM for HEC-1). Simultaneous exposure of OC cells to ONC206 and olaparib at low doses resulted in synergistic anti-proliferative effects (CI<1). Combination treatment with ONC206 and olaparib at low doses increased cellular stress and apoptosis in both cell lines (p=0.05-0.001), compared to either drug alone. Dual therapy with olaparib and ONC206 decreased expression of the anti-apoptotic protein Bcl-2 and decreased phosphorylation of S6, a downstream target of the mTOR pathway, with greater effects compared to either drug alone. Conclusions: Olaparib and ONC206 had synergistic anti-proliferative effects in human endometrioid EC cell lines, suggesting that this novel dual therapy may be worthy of further exploration in clinical trials in EC. Citation Format: Sarah E. Paraghamian, Gabrielle M. Hawkins, Wenchuan Sun, Yali Fan, Xin Zhang, Hongyan Suo, Varun Vijay Prabhu, Joshua E. Allen, Chunxiao Zhou, Victoria Bae-Jump. A novel dopamine receptor D2 antagonist (ONC206) potentiates the effects of olaparib in endometrial cancer cells through inhibition of cell proliferation and modulation of the mTOR pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5314.

Abstract 97: Analysis of Pten-null mouse astrocytes revealed overlapping transcribed genes with human glioblastoma transcriptome

Abstract Glioblastoma is an aggressive form of brain cancer with universally poor prognosis. Even with optimal therapy, the cancer recurs frequently. Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene whose mutations are often found in multiple human cancers. PTEN encodes a lipid phosphatase and serves as a negative regulator of the phosphoinositide 3-kinase (PI3K) pathway. Cumulative evidence suggests that PTEN mutations play significant roles in tumorigenesis of glioblastoma as 60% of glioblastoma patients acquired mutations in this gene. Based on our preliminary data, transcriptomic profiling of primary astrocytes derived from Pten knockout mice revealed an overlapping enrichment of mitotic regulators and cell cycle related pathways with human GBM. 4 genes (RCC1, RCC2, NEK2, and CHK2) were validated to be significantly upregulated in PTEN KO astrocytes by qRT-PCR. Regulator of chromatin condensation 2 (RCC2) has been reported to be guanine-nucleotide exchange factor (GEF) for Rac1 and RalA GTPase and act as a regulator of cell cycle progression. Accumulating evidence revealed a pro-tumorigenic role of RCC2 in multiple cancers such as tumor growth and tumor metastasis. Via siRNA inhibition of RCC2, we demonstrated that knocking down of RCC2 in glioblastoma cell lines suppresses cell growth and RCC2 is essential for both G1-S and G2-M progression. Additionally, RCC2 knocking down and a PI3K inhibitor LY294002 inhibit glioblastoma cell viability in a synergistic manner. In summary, PTEN deficiency leads to the upregulation of RCC2, which may promote tumorigenesis of glioblastoma by dysregulating cell cycle progression.To further explore the RCC2 roles, we will find the potential downstream target of RCC2 in dysregulating cell cycle and its potential therapeutic effect in vivo. Funding: General Research Fund Grant #14113519 from The Research Grants Council of Hong Kong Citation Format: Tian Liu, Penelope Mei-Yu Or, Chi-Wai Wong, Stanley Kwok-Kuen Cheung, Yubing Wang, Yiwei Wang, Andrew M. Chan. Analysis of Pten-null mouse astrocytes revealed overlapping transcribed genes with human glioblastoma transcriptome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 97.

Schwann cell-specific Pten inactivation reveals essential role of the sympathetic nervous system activity in adipose tissue development

There is increasing evidence that the sympathetic nervous system (SNS) plays an important role in adipose tissue development. However, the underlying molecular mechanism(s) associated with this remains unclear. SNS innervation of white adipose tissue (WAT) is believed to be necessary and sufficient to elicit WAT lipolysis. In this current study, mice with Schwann cell (SC)-specific inactivation of phosphatase and tensin homolog (Pten) displayed enlarged inguinal white adipose tissue (iWAT). This serendipitous observation implicates the role of SCs in mediating SNS activity associated with mouse adipose tissue development. Mice with SC-specific Pten inactivation displayed enlarged iWAT. Interestingly, the SNS activity in iWAT of SC-specific Pten-deficient mice was reduced as demonstrated by decreased tyrosine hydroxylase (TH) expression level and neurotransmitters, such as norepinephrine (NE) and histamine (H). The lipolysis related protein, phosphorylated hormone sensitive lipase (pHSL), was also decreased. As expected, AKT-associated signaling pathway was hyperactivated and hypothesized to induce enlarged iWAT in SC-specific Pten-deficient mice. Moreover, preliminary experiments using AKT inhibitor AZD5363 treatment ameliorated the enlarged iWAT condition in SC-specific Pten-deficient mice. Taken together, SCs play an essential role in the regulation of SNS activity in iWAT development via the AKT signaling pathway. This novel role of SCs in SNS function allows for better understanding into the genetic mechanisms of peripheral neuropathy associated obesity.