PTEN Knockout Mice Research Articles

Non-Targeted Metabolomics Reveal Apomorphine’s Therapeutic Effects and Lysophospholipid Alterations in Steatohepatitis

Metabolic dysfunction-associated steatohepatitis (MASH), characterized by progressive inflammation and fibrosis, evolves from metabolic dysfunction-associated steatotic liver disease and significantly heightens the risk of cirrhosis and hepatocellular carcinoma. Understanding metabolic pathways that influence MASH progression is crucial for developing targeted therapies. Non-targeted metabolomics offer a comprehensive view of metabolic alterations, enabling identification of novel biomarkers and pathways without preconceived ideas. Conversely, targeted metabolomics deliver precise and reproducible measurements, focusing on predefined metabolites to accurately quantify established pathways. This study utilized hepatocyte-specific PTEN knockout mice as a model to explore metabolic shifts associated with MASH. By integrating non-targeted metabolomics and targeted metabolomics, we analyzed liver samples from three groups: normal, pathological (MASH-affected), and MASH-affected, but treated with apomorphine, an antioxidant and recently reported ferroptosis inhibitor with potential therapeutic effects. Metabolic profiling identified lysophospholipids (LPLs) as significantly altered metabolites, with elevated levels in the MASH model and a notable reduction after apomorphine treatment. This suggests that LPLs are central to the etiology of MASH and may serve as targets for therapeutic intervention. Our findings underscore the effectiveness of apomorphine in modulating disease-specific metabolic disruptions, offering insights into its potential as a treatment for human MASH.

A cross-species analysis of fecal microbiomes in humans and mice reveals similarities and dissimilarities associated with prostate cancer risk.

Prostate cancer is a complex disease that develops over time and is influenced by several lifestyle factors that also impact gut microbes. Gut dysbiosis is intricately linked to prostate carcinogenesis, but the precise mechanisms remain poorly understood. Mice are crucial for studying the relationships between gut microbes and prostate cancer, but discovering similarities between humans and mice may aid in elucidating new mechanisms. We used 16s rRNA sequencing data from stool samples of tumor-bearing prostate-specific conditional Pten-knockout mice, disease-free wildtype mice, and a human cohort suspected of having prostate cancer to conduct taxonomic and metagenomic profiling. Features were associated with prostate cancer status and low risk (a negative biopsy of Gleason grade <2) or high risk (Gleason grade ≥2) in humans. In both humans and mice, community composition differed between individuals with and without prostate cancer. Odoribacter spp. and Desulfovibrio spp. were taxa associated with prostate cancer in mice and humans. Metabolic pathways associated with cofactor and vitamin synthesis were common in mouse and human prostate cancer, including bacterial synthesis of folate (vitamin B9), ubiquinone (CoQ10), phylloquinone (vitamin K1), menaquinone (vitamin K2), and tocopherol (vitamin E). Our study provides valuable data that can help bridge the gap between human and mouse microbiomes. Our findings provide evidence to support the notion that certain bacterial-derived metabolites may promote prostate cancer, as well as a preclinical model that can be used to characterize biological mechanisms and develop preventive interventions.

Regulation of YAP translocation by myeloid Pten deficiency alleviates acute lung injury via inhibition of oxidative stress and inflammation

BackgroundPhosphatase and tensin homolog deleted on chromosome 10 (PTEN) is intricately involved in modulating the inflammatory response in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Nevertheless, the myeloid PTEN governing Hippo-YAP pathway mediated oxidative stress and inflammation in lipopolysaccharide (LPS)-induced ALI remains to be elucidate. MethodsThe floxed Pten (PtenFL/FL) and myeloid-specific Pten knockout (PtenM−KO) mice were intratracheal instill LPS (5 mg/kg) to establish ALI, then Yap siRNA mix with the mannose-conjugated polymers was used to knockdown endogenous macrophage YAP in some PtenM−KO mice before LPS challenged. The bone marrow-derived macrophages (BMMs) from PtenFL/FL and PtenM−KO mice were obtained, and BMMs were transfected with CRISPR/Cas9-mediated glycogen synthase kinase 3 Beta (GSK3β) knockout (KO) or Yes-associated protein (YAP) KO vector subjected to LPS (100 ng/ml) challenged or then cocultured with MLE12 cells. ResultsHere, our findings demonstrate that myeloid-specific PTEN deficiency exerts a protective against LPS-induced oxidative stress and inflammation dysregulated in ALI model. Moreover, ablation of the PTEN-YAP axis in macrophages results in reduced nuclear factor-E2-related factor-2 (NRF2) expression, a decrease in antioxidant gene expression, augmented levels of free radicals, lipid and protein peroxidation, heightened generation of pro-inflammatory cytokines, ultimately leading to increased apoptosis in MLE12 cells. Mechanistically, it is noteworthy that the deletion of myeloid PTEN promotes YAP translocation and regulates NRF2 expression, alleviating LPS-induced ALI via the inhibition of GSK3β and MST1 binding. ConclusionsOur study underscores the crucial role of the myeloid PTEN-YAP-NRF2 axis in governing oxidative stress and inflammation dysregulated in ALI, indicating its potential as a therapeutic target for ALI.

Abstract B028: Epithelial-specific loss of PIK3R1 in a murine model result in endometrial hyperplasia

Abstract Somatic mutations in the phosphatidylinositol 3-kinase (PI3K) pathway occur in 84% of endometrial carcinomas (EC) and up to 68% of endometrial hyperplasia (EH). Regulation of PI3K/Akt/mTOR signaling is integral for normal endometrial function and is governed by tumor suppressor genes PTEN and PIK3R1 and proto-oncogene PIK3CA. While the actions of PTEN and PIK3CA are in direct opposition as a phosphatase and kinase, respectively, PIK3R1 regulates PI3K/Akt/mTOR signaling in two distinct ways. As the regulatory subunit of the PI3K enzyme, PIK3R1 controls the catalytic activity of PIK3CA and augments PTEN activity. Mutations or alterations in PIK3R1 are present in 33% of EC and 9% of EH, the majority of which lead to loss of PIK3R1 protein (p85a). Both loss of PTEN and PIK3R1 lead to increased proliferation through PI3K/Akt/mTOR signaling, but the structural, functional, and mechanistic implications of PIK3R1 loss in the endometrial epithelium (EE) in vivo are largely unexplored. To further interrogate the changes in the endometrium resulting from PIK3R1 loss, we have created a murine model of endometrial epithelial-specific PIK3R1 loss driven by Pax8-Cre. Mice were estrus cycle staged and sacrificed in the diestrus phase to reduce confounding variables when assessing phenotypes between genotypes. PIK3R1 loss was confirmed by immunohistochemistry (IHC). Histology and immunostaining of control and PIK3R1 knockout (KO) mice were assessed by IHC and immunofluorescence. We found that PIK3R1 loss in the EE leads to EH distinct from what is observed with PTEN loss. In contrast to the EH with atypia seen In PTEN KO mice, the EH resulting from PIKR1 loss more closely resembles EH without atypia. The endometrium of PIK3R1 KO mice is characterized by thickened luminal epithelia with jagged edges and irregularities, mild glandular crowding, and occasional nuclear atypia. PIK3R1 KO mice also showed an increase in proliferation (Ki-67) and downstream PI3K signaling (phospho-S6) in the EE. Gene expression changes occurring within the EE from PIK3R1 KO and Cre-negative control mice were also assessed by RNA-seq. In vivo genomic analyses were compared to in vitro knockdown of PIK3R1 and publicly available patient data. We found that PIK3R1 loss leads to an increase in signaling through the PI3K/Akt/mTOR and MAPK pathways. Our data show that the effect of PIK3R1 loss is sufficient to cause EH and may have shared and distinct actions from PTEN. Citation Format: Shannon K. Harkins, Hilary J. Skalski, Amelia Arendt, Laura Pavliscak, Mike R. Wilson, Ronald L. Chandler. Epithelial-specific loss of PIK3R1 in a murine model result in endometrial hyperplasia [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B028.

Enhanced Chemoprevention of Prostate Cancer by Combining Arctigenin with Green Tea and Quercetin in Prostate-Specific Phosphatase and Tensin Homolog Knockout Mice.

The low bioavailability of most phytochemicals limits their anticancer effects in humans. The present study was designed to test whether combining arctigenin (Arc), a lignan mainly from the seed of Arctium lappa, with green tea (GT) and quercetin (Q) enhances the chemopreventive effect on prostate cancer. We performed in vitro proliferation studies on different cell lines. We observed a strong synergistic anti-proliferative effect of GT+Q+Arc in exposing androgen-sensitive human prostate cancer LNCaP cells. The pre-malignant WPE1-NA22 cell line was more sensitive to this combination. No cytotoxicity was observed in normal prostate epithelial PrEC cells. For an in vivo study, 3-week-old, prostate-specific PTEN (phosphatase and tensin homolog) knockout mice were treated with GT+Q, Arc, GT+Q+Arc, or the control daily until 16 weeks of age. In vivo imaging using prostate-specific membrane antigen (PSMA) probes demonstrated that the prostate tumorigenesis was significantly inhibited by 40% (GT+Q), 60% (Arc at 30 mg/kg bw), and 90% (GT+Q+Arc) compared to the control. A pathological examination showed that all control mice developed invasive prostate adenocarcinoma. In contrast, the primary lesion in the GT+Q and Arc alone groups was high-grade prostatic intraepithelial neoplasia (PIN), with low-grade PIN in the GT+Q+Arc group. The combined effect of GT+Q+Arc was associated with an increased inhibition of the androgen receptor, the PI3K/Akt pathway, Ki67 expression, and angiogenesis. This study demonstrates that combining Arc with GT and Q was highly effective in prostate cancer chemoprevention. These results warrant clinical trials to confirm the efficacy of this combination in humans.

Gender Differences in a Mouse Model of Hepatocellular Carcinoma Revealed Using Multi-Modal Imaging.

The worldwide incidence of hepatocellular carcinoma (HCC) continues to rise, in part due to poor diet, limited exercise, and alcohol abuse. Numerous studies have suggested that the loss or mutation of PTEN plays a critical role in HCC tumorigenesis through the activation of the PI3K/Akt signaling axis. The homozygous knockout of PTEN in the livers of mice results in the accumulation of fat (steatosis), inflammation, fibrosis, and eventually progression to HCC. This phenotype bears a striking similarity to non-alcoholic steatohepatitis (NASH) which is thought to occupy an intermediate stage between non-alcoholic fatty liver disease (NAFLD), fibrosis, and HCC. The molecular and physiological phenotypes that manifest during the transition to HCC suggest that molecular imaging could provide a non-invasive screening platform to identify the hallmarks of HCC initiation prior to the presentation of clinical disease. We have carried out longitudinal imaging studies on the liver-specific PTEN knockout mouse model using CT, MRI, and multi-tracer PET to interrogate liver size, steatosis, inflammation, and apoptosis. In male PTEN knockout mice, significant steatosis was observed as early as 3 months using both magnetic resonance spectroscopy (MRS) and computed tomography (CT). Enhanced uptake of the apoptosis tracer 18F-TBD was also observed in the livers of male PTEN homozygous knockout mice between 3 and 4 months of age relative to heterozygous knockout controls. Liver uptake of the inflammation tracer [18F]4FN remained relatively low and constant over 7 months in male PTEN homozygous knockout mice, suggesting the suppression of high-energy ROS/RNS with PTEN deletion relative to heterozygous males where the [18F]4FN liver uptake was elevated at early and late time points. All male PTEN homozygous mice developed HCC lesions by month 10. In contrast to the male cohort, only 20% (2 out of 10) of female PTEN homozygous knockout mice developed HCC lesions by month 10. Steatosis was significantly less pronounced in the female PTEN homozygous knockout mice relative to males and could not accurately predict the eventual occurrence of HCC. As with the males, the [18F]4FN uptake in female PTEN homozygous knockout mice was low and constant throughout the time course. The liver uptake of 18F-TBD at 3 and 4.5 months was higher in the two female PTEN knockout mice that would eventually develop HCC and was the most predictive imaging biomarker for HCC in the female cohort. These studies demonstrate the diagnostic and prognostic role of multi-modal imaging in HCC mouse models and provide compelling evidence that disease progression in the PTEN knockout model is highly dependent on gender.

Rapamycin improves social and stereotypic behavior abnormalities induced by pre-mitotic neuronal subset specific Pten deletion.

The mechanistic target of rapamycin (mTOR) pathway is a signaling system integral to neural growth and migration. In both patients and rodent models, mutations to the phosphatase and tensin homolog gene (PTEN) on chromosome 10 results in hyperactivation of the mTOR pathway, as well as seizures, intellectual disabilities and autistic behaviors. Rapamycin, an inhibitor of mTOR, can reverse the epileptic phenotype of neural subset specific Pten knockout (NS-Pten KO) mice, but its impact on behavior is not known. To determine the behavioral effects of rapamycin, male and female NS-Pten KO and wildtype (WT) mice were assigned as controls or administered 10 mg/kg of rapamycin for 2 weeks followed by behavioral testing. Rapamycin improved social behavior in both genotypes and stereotypic behaviors in NS-Pten KO mice. Rapamycin treatment resulted in a reduction of several measures of activity in the open field test in both genotypes. Rapamycin did not reverse the reduced anxiety behavior in KO mice. These data show the potential clinical use of mTOR inhibitors by showing its administration can reduce the production of autistic-like behaviors in NS-Pten KO mice.

Inducible TgfbR1 and Pten deletion in a model of tongue carcinogenesis and chemoprevention.

Head and neck squamous cell carcinoma (HNSCC) is a significant public health problem, with a need for novel approaches to chemoprevention and treatment. Preclinical models that recapitulate molecular alterations that occur in clinical HNSCC patients are needed to better understand molecular and immune mechanisms of HNSCC carcinogenesis, chemoprevention, and efficacy of treatment. We optimized a mouse model of tongue carcinogenesis with discrete quantifiable tumors via conditional deletion of Tgfβr1 and Pten by intralingual injection of tamoxifen. We characterized the localized immune tumor microenvironment, metastasis, systemic immune responses, associated with tongue tumor development. We further determined the efficacy of tongue cancer chemoprevention using dietary administration of black raspberries (BRB). Three Intralingual injections of 500 µg tamoxifen to transgenic K14 Cre, floxed Tgfbr1, Pten (2cKO) knockout mice resulted in tongue tumors with histological and molecular profiles, and lymph node metastasis similar to clinical HNSCC tumors. Bcl2, Bcl-xl, Egfr, Ki-67, and Mmp9, were significantly upregulated in tongue tumors compared to surrounding epithelial tissue. CD4+ and CD8 + T cells in tumor-draining lymph nodes and tumors displayed increased surface CTLA-4 expression, suggestive of impaired T-cell activation and enhanced regulatory T-cell activity. BRB administration resulted in reduced tumor growth, enhanced T-cell infiltration to the tongue tumor microenvironment and robust antitumoral CD8+ cytotoxic T-cell activity characterized by greater granzyme B and perforin expression. Our results demonstrate that intralingual injection of tamoxifen in Tgfβr1/Pten 2cKO mice results in discrete quantifiable tumors suitable for chemoprevention and therapy of experimental HNSCC.

Neuropathological signatures revealed by transcriptomic and proteomic analysis in Pten-deficient mouse models

PTEN hamartoma tumour syndrome is characterised by mutations in the human PTEN gene. We performed transcriptomic and proteomic analyses of neural tissues and primary cultures from heterozygous and homozygous Pten-knockout mice. The somatosensory cortex of heterozygous Pten-knockout mice was enriched in immune response and oligodendrocyte development Gene Ontology (GO) terms. Parallel proteomic analysis revealed differentially expressed proteins (DEPs) related to dendritic spine development, keratinisation and hamartoma signatures. However, primary astrocytes (ASTs) from heterozygous Pten-knockout mice were enriched in the extracellular matrix GO term, while primary cortical neurons (PCNs) were enriched in immediate-early genes. In ASTs from homozygous Pten-knockout mice, cilium-related activity was enriched, while PCNs exhibited downregulation of forebrain neuron generation and differentiation, implying an altered excitatory/inhibitory balance. By integrating DEPs with pre-filtered differentially expressed genes, we identified the enrichment of traits of intelligence, cognitive function and schizophrenia, while DEPs in ASTs were significantly associated with intelligence and depression.

Targeting pleckstrin-2/Akt signaling reduces proliferation in myeloproliferative neoplasm models.

Myeloproliferative neoplasms (MPNs) are characterized by the activated JAK2/STAT pathway. Pleckstrin-2 (Plek2) is a downstream target of the JAK2/STAT5 pathway and is overexpressed in patients with MPNs. We previously revealed that Plek2 plays critical roles in the pathogenesis of JAK2-mutated MPNs. The nonessential roles of Plek2 under physiologic conditions make it an ideal target for MPN therapy. Here, we identified first-in-class Plek2 inhibitors through an in silico high-throughput screening approach and cell-based assays, followed by the synthesis of analogs. Plek2-specific small-molecule inhibitors showed potent inhibitory effects on cell proliferation. Mechanistically, Plek2 interacts with and enhances the activity of Akt through the recruitment of downstream effector proteins. The Plek2-signaling complex also includes Hsp72, which protects Akt from degradation. These functions were blocked by Plek2 inhibitors via their direct binding to the Plek2 dishevelled, Egl-10 and pleckstrin (DEP) domain. The role of Plek2 in activating Akt signaling was further confirmed in vivo using a hematopoietic-specific Pten-knockout mouse model. We next tested Plek2 inhibitors alone or in combination with an Akt inhibitor in various MPN mouse models, which showed significant therapeutic efficacies similar to that seen with the genetic depletion of Plek2. The Plek2 inhibitor was also effective in reducing proliferation of CD34-positive cells from MPN patients. Our studies reveal a Plek2/Akt complex that drives cell proliferation and can be targeted by a class of antiproliferative compounds for MPN therapy.

Abstract A017: Epigenetic activation of SOX11 is associated with recurrence and progression of DCIS to invasive breast cancer

Abstract Background: Screening mammography has dramatically increased incidental detection of Ductal Carcinoma in situ (DCIS), a non-obligate precursor of breast cancer. Approximately 10-50% of DCIS progress to the invasive disease and the risk of developing recurrence or progression after surgery is still not well defined based on clinical and pathological characteristics. Our study investigated the genetic and epigenetic mechanisms associated with recurrence and progression of DCIS to invasive cancer. Methods: To identify genes associated with recurrence and progression of DCIS to invasive cancer, we performed Nanostring nCounter analysis (MDACC, n = 40) and RNA-sequencing (Sloane study, n = 183) in two independent prospective primary DCIS cohorts. Mammary epithelial-specific Pten knockout mouse model developed in our lab was used to interrogate the genetic pathway deregulated during development of mammary intraepithelial neoplasia to adenocarcinoma. ATAC-seq and ChIP-qPCR were performed to assess chromatin accessibility in the genome and validate binding of histone-modifying enzymes on target promoters. Histopathological analyses were performed to determine DCIS grades, subtypes, and protein expression profiles. Results: RNA expression profiles of normal mammary and primary DCIS tissues (n = 40) identified 10 gene transcripts that were significantly and differentially expressed between normal and high-grade DCIS. Only SOX11 expression was significantly and progressively correlated with KI67 and the DCIS recurrence score as described by Solin (JNCI 2013). Mammary intraepithelial neoplasia in Pten knockout mice and 21T DCIS to IDC progression cell line model also revealed SOX11 upregulation compared to controls. Since PTEN/PI3K/AKT pathway is known to regulate global active histone marks and open accessible chromatin, we investigated the role of AKT on SOX11 expression. Results demonstrated that inhibition of AKT altered enrichment of histone-modifying enzymes KDM5A and EZH2 on the promoter accompanying downregulated SOX11 expression in DCIS cell lines. SOX11 and EZH2 expression profiles revealed significant correlation in DCIS tissue samples. In two independent DCIS cohorts, SOX11 was found significantly upregulated in HER2+ and basal-like DCIS subtypes (P &amp;lt; 0.001). RNA sequencing in 183 longitudinal DCIS cases of the Sloane study (median follow-up of 96 months) revealed significantly shorter recurrence-free survival in SOX11+ than SOX11- patients (P &amp;lt; 0.02 in all subtypes; P = 0.002 in HER2+ subtype). Based on COX proportional hazards model, SOX11 alone was significantly associated with the risk of developing recurrence and invasive breast cancer (HR = 1.7, 95% CI = 1.1 to 2.6; P &amp;lt; 0.02). Conclusions: Epigenetic activation of SOX11 is associated with recurrence and progression of DCIS to invasive breast cancer. SOX11 and its epigenetic regulators can be developed as prognostic markers for DCIS patients with recurrence risk independent of traditional clinical and pathologic characteristics. Citation Format: Warapen Treekitkarnmongkol, Vandna Shah, Kazuharu Kai, Hiroshi Katayama, Justin W. Wong, Farah A Ladha, Brian Menegaz, Sarah E. Pinder, Wei Lu, Fei Yang, Ximing Tang, Savitri Krishnamurthy, Ignacio I. Wistuba, Elinor J. Sawyer, Alastair M. Thompson, Subrata Sen. Epigenetic activation of SOX11 is associated with recurrence and progression of DCIS to invasive breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr A017.

Loss of Pten in Renal Tubular Cells Leads to Water Retention by Upregulating AQP2

Introduction: Phosphatase and tensin (PTEN) is a multifunctional gene associated with the normal development and physiological function of various tissues including the kidney. However, its role in renal tubular reabsorption function has not been well elucidated. Methods: We generated a renal tubule-specific Pten knockout mouse model by crossing Pten^fl/fl mice with Ksp-Cre transgenic mice, evaluated the effect of Pten loss on renal tubular function, and investigated the underlying mechanisms. Results: Pten loss resulted in abnormal renal structure and function and water retention in multiple organs. Our results also demonstrated that aquaporin-2 (AQP2), an important water channel protein, was upregulated and concentrated on the apical plasma membrane of collecting duct cells, which could be responsible for the impaired water balance in Pten loss mice. The regulation of Pten loss on AQP2 was mediated by protein kinase B (AKT) activation. Conclusions: Our results reveal a connection between PTEN gene inactivation and water retention, suggesting the importance of PTEN in normal kidney development and function.

Deletion of monoamine oxidase A in a prostate cancer model enhances anti-tumor immunity through reduced immune suppression

We have previously shown that monoamine oxidase A (MAO A) mediates prostate cancer growth and metastasis. Further, MAO A/Pten double knockout (DKO) mice were generated and demonstrated that the deletion of MAO A delayed prostate tumor development in the Pten knockout mouse model of prostate adenocarcinoma. Here, we investigated its effect on immune cells in the tumor microenvironment in MAO A/Pten DKO mouse model. Our results shows that Paraffin embedded prostate tissues from MAO A/Pten DKO mice had elevated markers of immune stimulation (CD8+ cytotoxic T cells, granzyme B, and IFNγ) and decreased expression of markers of immune suppression (FoxP3, CD11b, HIF-1-alpha, and arginase 1) compared to parental Pten knockouts (MAO A wildtype). CD11b+ myeloid derived suppressor cells (MDSC) were the primary immunosuppressive cell types in these tumors. The data suggest that deletion of MAO A reduces immune suppression in prostate tumors to enhance antitumor immunity in prostate cancer. Thus, MAO A inhibitor may alleviate immune suppression, increase the antitumor immune response and be used for cancer immunotherapy.

Probiotics suppress nonalcoholic steatohepatitis and carcinogenesis progression in hepatocyte-specific PTEN knockout mice

Nonalcoholic fatty liver disease (NAFLD), a hepatic characteristic of metabolic syndrome, received significant attention in clinical settings. The multiple-hit theory is one of the proposed mechanisms of NAFLD, and gut dysbiosis is considered a hit. Thus, controlling gut microbiota is a potential target in the management of NAFLD, and probiotics can be used as a treatment agent for NAFLD. The current study aimed to investigate the efficacy of probiotics against nonalcoholic steatohepatitis in a hepatocyte-specific PTEN knockout mouse model that mimics the characteristics of human NAFLD. Probiotics were administered to male knockout mice for 8 or 40 weeks. Next, we assessed hepatic inflammation, fibrosis, carcinogenesis, and oxidative stress. Probiotics were found to reduce serum transaminase levels, NAFLD activity score, and the gene expression of pro-inflammatory cytokines. In addition, they decreased liver fibrosis grade, which was examined via Sirius red staining, gene expression of fibrotic markers, and hydroxyproline. Furthermore, probiotics suppressed the number of liver tumors, particular in HCC. Probiotics reduced oxidative stresses, including glutathione levels, and anti-oxidative stress marker, which may be an underlying mechanism for their beneficial effects. In conclusion, probiotics treatment had beneficial effects against NAFLD and carcinogenesis in hepatocyte-specific PTEN knockout mice.

The role of mitochondria in the recovery of neurons after injury.

The role of mitochondria in the recovery of neurons after injury.

Targeting HIC1/TGF-β axis-shaped prostate cancer microenvironment restrains its progression

Prostate cancer (PCa) is a malignant tumor that seriously threatens men’s health worldwide. Recently, stromal cells in the tumor microenvironment (TME) have been reported to contribute to the progression of PCa. However, the role and mechanism of how PCa cells interact with stromal cells to reshape the TME remain largely unknown. Here, using a spontaneous prostate adenocarcinoma (PRAD) model driven by the loss of Pten and Hic1, we found that M2 macrophages markedly infiltrated the stroma of Pten and Hic1 double conditional knockout (dCKO) mice compared with those in control (Ctrl) mice due to higher TGF-β levels secreted by HIC1-deleted PCa cells. Mechanistically, TGF-β in TME promoted the polarization of macrophages into “M2” status by activating the STAT3 pathway and modulating c-Myc to upregulate CXCR4 expression. Meanwhile, TGF-β activated the fibroblasts to form cancer-associated fibroblasts (CAFs) that secrete higher CXCL12 levels, which bound to its cognate receptor CXCR4 on M2 macrophages. Upon interaction with CAFs, M2 macrophages secreted more CXCL5, which promoted the epithelial-mesenchymal transition (EMT) of PCa via CXCR2. Moreover, using the TGF-β receptor I antagonist, galunisertib, significantly inhibited the tumor growth and progression of the TRAMP-C1 cell line-derived subcutaneous tumor model. Finally, we confirmed that the stromal microenvironment was shaped by TGF-β in HIC1-deficient PCa and was associated with the progression of PCa.

Abstract 5647: PTEN loss is associated with elevation of cell cycle signature in human glioblastoma transcriptome

Abstract Glioblastoma is the most lethal type of brain cancer with universally poor prognosis. Despite advanced treatment, the cancer recurs frequently. Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene whose mutations have been found to be one of the most frequent events in GBM. PTEN acts as a negative regulator of the phosphoinositide 3-kinase (PI3K) pathway. GBM transcriptome data from The Cancer Genome Atlas (TCGA) revealed a positive enrichment of cell cycle and PI3K pathway. Moreover, weighted gene co-expression network analysis (WGCNA) on TCGA GBMLGG cohort revealed a cell cycle-related module (‘cell-cycle’ module), which was shown to be positively correlated with PTEN mutation status. This ‘cell-cycle’ module also showed a positive correlation with glioma aggressiveness.We then utilized a nestin-cre;Pten-fl/fl mouse model to investigate the consequences of PTEN loss that may be involved in GBM. Based on our preliminary data, transcriptomic profiling of primary astrocytes derived from Pten knockout mice revealed an overlapping enrichment of cell cycle related pathways with human GBM. Four cell cycle genes, RCC1, RCC2, NEK2, and CHK2, were validated to be significantly upregulated in Pten-null astrocytes by qRT-PCR. Regulator of chromatin condensation 1 (RCC1) and Regulator of chromatin condensation 2 (RCC2) both belong to the RCC1 family. Accumulating evidence demonstrated that both RCC1 and RCC2 are involved in tumorigenesis of multiple cancers and cell cycle progression. By siRNA inhibition, we demonstrated that silencing of either RCC1 or RCC2 in glioblastoma cell lines suppresses GBM cell growth. Moreover, we found that RCC2 is essential for both G1-S and G2-M transition. Additionally, RCC2 silencing and a PI3K inhibitor LY294002 inhibit glioblastoma cell viability in a synergistic manner. In summary, PTEN deficiency leads to the alteration of cell cycle progression, which may promote tumorigenesis of glioblastoma. RCC1 and RCC2 have been demonstrated to be potential targets regulated by PTEN and affects cell viability. RCC2 was shown to be a key regulator of cell cycle progression. To further explore the RCC1 and RCC2 roles, we will find the potential downstream target of RCC1 and RCC2 and its potential therapeutic effect in vivo. Funding: This work is supported by a General Research Fund grant (#14113519) from the University Grants Committee of Hong Kong. Citation Format: Tian Liu, Penelope Mei-Yu Or, Chi-Wai Wong, Stanley Kwok-Kuen Cheung, Yubing Wang, Yiwei Wang, Wayne Lut-Heng Ho, Andrew M. Chan. PTEN loss is associated with elevation of cell cycle signature in human glioblastoma transcriptome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5647.

Abstract 816: Cyclin c suppresses pancreatic intraepithelial neoplasm progression and neuroendocrine neoplasm development in a murine Kras model

Abstract Cyclin c is a component of the Cdk8 kinase module that plays both a positive and negative role in transcription. In addition, cellular damage induces cyclin c re-localization to the mitochondria where it stimulates fission and recruits Bax in preparation for apoptotic initiation. Previous studies revealed that cyclin c suppresses thyroid hyperplasia in a Pten knockout mouse model. In the present study, a role for cyclin c in suppressing pancreatic cancer progression was investigated in the Pdx1-Cre LSL-KrasG12D murine pancreatic cancer model. Within eight weeks, a 6-8 fold increase in the appearance of pancreatic intraepithelial neoplasia (PanIN) and acinar ductal metaplasia (ADM) lesions was observed in the KrasG12D+Ccnc-/- animals compared to KrasG12D alone. Surprisingly, high-grade neuroendocrine neoplasms (NEN) were also observed. These aggressive NEN showed mixed morphology with areas of acinar-like differentiation. High grade NEN exhibited pronounced proliferative index (Ki67), high EZH2 expression, and elevated Notch1, suggesting a connection between aberrant Notch signaling and NEN formation. Our previous studies identified a positive role for cyclin c in autophagy gene transcription. Similarly, cell lines derived from a KrasG12D+Ccnc-/- pancreas exhibited a reduction in autophagy compared to KrasG12D+Ccnc+/+ cells. Autophagic deficiency induces pancreatic cell stress and is associated with apoptosis. Consistent with this model, high caspase 3 expression was observed in early KrasG12D+Ccnc-/- NEN but not in KrasG12D+Ccnc+/+ tissues. Taken together, these results demonstrate a role for cyclin c in suppressing both progression of early PanIN and the development of high-grade NEN and suggest a model that cyclin c loss suppresses autophagy and increases apoptosis. Acknowledgements: Support was provided by NCI CCSG grant CA06927 for the FCCC Histopathology and Cell Culture Facilities, the PA Department of Health CURE fund (KSC), the Boye Foundation (RS), and the New Jersey Health Foundation (RS). Citation Format: Kathy Q. Cai, Sara E. Hanley, Andres Klein-Szanto, Kerry S. Campbell, Randy Strich. Cyclin c suppresses pancreatic intraepithelial neoplasm progression and neuroendocrine neoplasm development in a murine Kras model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 816.

FC022: IPI3K/AKT/MTOR Pathway Regulates Renal Expression of Klotho

Abstract BACKGROUND AND AIMS Chronic kidney disease (CKD) represents an important and growing burden on health worldwide, mainly driven by the increase in the incidence of associated risk factors like diabetes. As kidney function declines, changes in mineral metabolism occur, which contribute to the increased risk of cardiovascular disease in CKD patients. In this respect, Klotho was described as an anti-ageing protein, mainly expressed in the kidney, where it acts as an obligatory co-receptor of the Fibroblast Growth Factor (FGF23), which is involved in urinary phosphate excretion. The earliest mineral metabolism change in CKD is the fall in renal and plasma Klotho levels, followed by an increase in FGF23. Klotho loss is related to inflammatory and fibrotic processes contributing not only to the progression of CKD but also to the increase in cardiovascular risk. Clinically, there is a growing interest in maintaining renal Klotho expression in patients, but the mechanisms in Klotho decline remain unknown. PTEN is the main negative regulator of PI3K-AKT-mTOR pathway. PTEN degrades PIP3 to PIP2 decreasing the activation of the canonical insulin pathway. The involvement of PTEN in cancer has been broadly investigated. Furthermore, the PI3K/PTEN signalling pathways are involved in a wide variety of diseases including cardiac hypertrophy, heart failure, hypertension and acute kidney injury. However, its potential role in CKD has not been properly assessed. The objective of this study was to investigate the role of PTEN and the PI3K/AKT/mTOR pathways in kidney Klotho levels and the modulation of phosphorus metabolism. METHOD We generated a proximal tubule conditional PTEN knockout mouse model (PT-PTEN-cKO), in which a high phosphorus diet (HPD) and rapamycin treatment were administered in order to analyse how PI3K/AKT/mTOR pathway participates in phosphorus metabolism and kidney Klotho levels. We used human proximal tubular cell (PTC) line HK-2 to knock down PTEN by means of transfection with shPTEN lentiviral vector, to study the direct effect of PTEN elimination in Klotho levels in vitro. RESULTS PT-PTEN-cKO mice present a specific activation of the PI3K/AKT/mTOR pathway in the proximal tubule. PTEN mRNA decrease correlates with a drop in kidney Klotho levels, accompanied by an increment of inflammatory and fibrotic markers. In addition, our model showed an increase in both, plasma phosphate and FGF23, with a decrease in the fractional excretion of phosphate (% FEPi). This fact can be explained by the inability of kidney cells to respond to FGF23, because of Klotho loss. Moreover, parathyroid hormone (PTH) and bone resorption were increased. We observe that PT-PTEN-KO mice in an HPD needed higher FGF23 levels in order to excrete phosphate than control mice. Rapamycin administration restored Klotho levels. In vitro, PTEN silencing in PTC, leads to an increase in PI3K/AKT/mTOR activity and also to a reduction in Klotho expression. Finally, the inhibition of mTOR phosphorylation by rapamycin reestablished Klotho expression in vitro. CONCLUSION The overactivation of PI3K/AKT/mTOR pathway, in PTC, decreases Klotho kidney levels. Our findings constitute an important breakthrough in the research of new therapeutic targets in order to maintain renal Klotho levels and it may be useful in the treatment of kidney disease patients.

Cyclin C Suppresses Pancreatic Intraepithelial Neoplasia and Neural Endocrine Neoplasia Progression in a Murine Kras Model

Cyclin C is a component of the Cdk8 kinase module that plays both a positive and negative role in transcription. In addition, cellular damage induces cyclin C re‐localization to the mitochondria where it stimulates fission and recruits Bax in preparation for apoptotic initiation. Previous studies revealed that cyclin C suppresses thyroid hyperplasia in a Pten knockout mouse model. In the present study, a role for cyclin C in suppressing pancreatic cancer progression was investigated in the LSL‐KrasG12Dmurine cancer model. Within eight weeks, a 6‐8 fold increase in the appearance of pancreatic Intraepithelial neoplasia (PanIN) and acinar ductal metaplasia (ADM) lesions were observed in the KrasG12D+; Ccnc‐/‐ animals compared to KrasG12D alone. Surprisingly, high‐grade, poorly differentiated pancreatic neuroendocrine tumors/carcinomas (PNET/PNEC) were also observed. These lesions coexisted in the pancreas with some PanIN regions possessing intermixed cells expressing PNEC markers. Our previous studies identified a positive role for cyclin C in autophagy gene transcription. Similarly, cell lines derived from a KrasG12D+; Ccnc‐/‐ pancreas exhibited a reduction in autophagy compared to KrasG12D+; Ccnc+/+ cells. Autophagic deficiency induces pancreatic cell stress and is associated with elevated apoptosis. Consistent with this model, caspase 3 activation was observed in early KrasG12D+; Ccnc‐/‐PNETs but not in KrasG12D+ tissues. In addition, KrasG12D+; Ccnc‐/‐ cells showed decreased proteasomal activity and a 30% increase in cell death compared to KrasG12D+; Ccnc+/+ cells following treatment with the proteasome inhibitor, Bortezomib. Taken together, these results demonstrate a role for cyclin C in suppressing both PDAC and PNEN cancer progression and suggest a model that its loss promotes pre‐cancerous lesions by stimulating pancreatic cell injury.