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The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (PtencKO) retinas. Wnt signaling molecules are also enriched in PtencKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.

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.

Objective To investigate the effect and mechanism of targeted regulation of phosphatase and tensin homolog (PTEN) and phosphatidylinositol 3-kinase (PI3K) on the proliferation and apoptosis of nephroblastoma cells. Methods Posto-perative tumor tissue and normal paracancerous tissue were collected from 15 children with nephroblastoma, and Western blotting and quantitative real-time PCR were used to measure the protein and mRNA expression levels of PTEN and PI3K in tissue. Nephroblastoma SK-NEP-1 cells were divided into control group (group A, transfected with NC siRNA and Flag empty vector), PTEN overexpression group (group B, transfected with NC siRNA and Flag-PTEN expression vector), PI3K knockdown group (group C, transfected siPI3K and Flag empty vector), a nd combined targeting group (group D, transfected siPI3K and FLAG-PTEN). Western blotting was used to measure the expression levels of PI3K, protein kinase A (AKT), and phosphorylated AKT (p-AKT) in SK-NEP-1 cells at 24 h after transfection; CCK-8 assay was used to observe the proliferation of SK-NEP-1 cells at 24, 48, and 72 h of intervention; flow cytometry was used to observe apoptosis rate and cell cycle at 24 h after transfection. Results Compared with normal paracancerous tissue, nephroblastoma tumor tissue showed significant increases in the protein and mRNA expression levels of PI3K (t=22.862,7.098,P

This paper explores the potential of rAAV2-retro to deliver gene modifying cargoes to the cells of origin of multiple pathways that are interrupted by spinal cord injury (SCI), summarizing data from previous studies and new data from additional experiments. rAAV-retro exhibits uniquely robust and reliable long-distance retrograde transport from pre-terminal axons and synapses back to neuronal bodies. Previous studies have documented that various AAV-based genetic modifications can enable axon regeneration after SCI, but these have targeted the cells of origin of one pathway at a time. In contrast, rAAV-retro can simultaneously transduce large numbers of neurons of origin of multiple spinal pathways with single injections into the spinal cord. Our initial studies use RosatdTomato and double transgenic PTENf/f; RosatdTomato mice in which transfection with rAAV-retro/Cre deletes PTEN and activates tdT expression in the same neurons. Injections of rAAV-retro/Cre into the cervical, thoracic and lumbar spinal cord led to topographically specific retrograde transduction in cortical motoneurons and neurons in subcortical regions that give rise to different spinal pathways. Our results confirm and extend previous studies indicating selective transduction of neurons that terminate at the level of the injection with minimal retrograde transduction of axons in transit to lower levels. We document feasibility of using rAAV-retro expressing shRNA against PTEN along with a GFP reporter (rAAV-retro-shPTEN/GFP) to effectively knock down PTEN in multiple populations of neurons, which can be used in any species. Some limitations and caveats of currently available rAAV-retros are discussed. Together, our results support the potential applications of rAAV-retro for AAV-based gene-modifications for SCI.

M2 macrophages in the tumor microenvironment are important drivers of cancer metastasis. Exosomes play a critical role in the crosstalk between different cells by delivering microRNAs or other cargos. Whether exosomes derived from pro-tumorigenic M2 macrophages (M2-Exos) could modulate the metastatic behavior of renal cell carcinoma (RCC) is unclear. This study found that M2-Exos promotes migration and invasion in RCC cells. Inhibiting miR-21-5p in M2-Exos significantly reversed their pro-metastatic effects on RCC cells in vitro and in the avian embryo chorioallantoic membrane in vivo tumor model. We further found that the pro-metastatic mechanism of miR-21-5p in M2-Exos is by targeting PTEN-3'UTR to regulate PTEN/Akt signaling. Taken together, our results demonstrate that M2-Exos carries miR-21-5p promote metastatic features of RCC cells through PTEN/Akt signaling. Reversing this could serve as a novel approach to control RCC metastasis.

About 15-20% of breast cancer (BCa) is triple-negative BCa (TNBC), a devastating disease with limited therapeutic options. Aberrations in the PI3K/PTEN signaling pathway are common in TNBC. However, the therapeutic impact of PI3K inhibitors in TNBC has been limited and the mechanism(s) underlying this lack of efficacy remain elusive. Here, we demonstrate that a large subset of TNBC expresses significant levels of MAPK4, and this expression is critical for driving AKT activation independent of PI3K and promoting TNBC cell and xenograft growth. The ability of MAPK4 to bypass PI3K for AKT activation potentially provides a direct mechanism regulating tumor sensitivity to PI3K inhibition. Accordingly, repressing MAPK4 greatly sensitizes TNBC cells and xenografts to PI3K blockade. Altogether, we conclude that high MAPK4 expression defines a large subset or subtype of TNBC responsive to MAPK4 blockage. Targeting MAPK4 in this subset/subtype of TNBC both represses growth and sensitizes tumors to PI3K blockade.

BackgroundPTEN is a well-established risk gene for autism spectrum disorder (ASD). Yet, little is known about how PTEN mutations and associated molecular processes influence neurobehavioral function in mutation carriers with (PTEN-ASD) and without ASD (PTEN no-ASD). The primary aim of the present study was to examine group differences in peripheral blood-derived PTEN pathway protein levels between PTEN-ASD, PTEN no-ASD, and idiopathic macrocephalic ASD patients (macro-ASD). Secondarily, associations between protein levels and neurobehavioral functions were examined in the full cohort.MethodsPatients were recruited at four tertiary medical centers. Peripheral blood-derived protein levels from canonical PTEN pathways (PI3K/AKT and MAPK/ERK) were analyzed using Western blot analyses blinded to genotype and ASD status. Neurobehavioral measures included standardized assessments of global cognitive ability and multiple neurobehavioral domains. Analysis of variance models examined group differences in demographic, neurobehavioral, and protein measures. Bivariate correlations, structural models, and statistical learning procedures estimated associations between molecular and neurobehavioral variables. To complement patient data, Western blots for downstream proteins were generated to evaluate canonical PTEN pathways in the PTEN-m3m4 mouse model.ResultsParticipants included 61 patients (25 PTEN-ASD, 16 PTEN no-ASD, and 20 macro-ASD). Decreased PTEN and S6 were observed in both PTEN mutation groups. Reductions in MnSOD and increases in P-S6 were observed in ASD groups. Elevated neural P-AKT/AKT and P-S6/S6 from PTEN murine models parallel our patient observations. Patient PTEN and AKT levels were independently associated with global cognitive ability, and p27 expression was associated with frontal sub-cortical functions. As a group, molecular measures added significant predictive value to several neurobehavioral domains over and above PTEN mutation status.LimitationsSample sizes were small, precluding within-group analyses. Protein and neurobehavioral data were limited to a single evaluation. A small number of patients were excluded with invalid protein data, and cognitively impaired patients had missing data on some assessments.ConclusionsSeveral canonical PTEN pathway molecules appear to influence the presence of ASD and modify neurobehavioral function in PTEN mutation patients. Protein assays of the PTEN pathway may be useful for predicting neurobehavioral outcomes in PTEN patients. Future longitudinal analyses are needed to replicate these findings and evaluate within-group relationships between protein and neurobehavioral measures.Trial registrationClinicalTrials.gov Identifier NCT02461446.

Neoadjuvant PD-1 blockade may be efficacious in some individuals with high-risk, resectable oral cavity head and neck cancer. To explore correlates of response patterns to neoadjuvant nivolumab treatment and post-surgical recurrences, we analyzed longitudinal tumor and blood samples in a cohort of 12 individuals displaying 33% responsiveness. Pretreatment tumor-based detection of FLT4 mutations and PTEN signature enrichment favors response, and high tumor mutational burden improves recurrence-free survival. In contrast, preexisting and/or acquired mutations (in CDKN2A, YAP1, or JAK2) correlate with innate resistance and/or tumor recurrence. Immunologically, tumor response after therapy entails T cell receptor repertoire diversification in peripheral blood and intratumoral expansion of preexisting T cell clones. A high ratio of regulatory T to T helper 17 cells in pretreatment blood predicts low T cell receptor repertoire diversity in pretreatment blood, a low cytolytic T cell signature in pretreatment tumors, and innate resistance. Our study provides a molecular framework to advance neoadjuvant anti-PD-1 therapy for individuals with resectable head and neck cancer.

Thyroid cancer surveillance (TCS) with ultrasound (US) is advised for PTEN hamartoma tumour syndrome (PHTS) patients due to increased thyroid cancer (TC) risk. However, data supporting TCS guidelines are scarce. We aimed to assess the detection and yield of annual TCS with US in adult PHTS patients without a TC history and to evaluate the impact of a reduced US interval on the TCS yield. A retrospective cohort study was conducted, including adult PHTS patients and medical record data between 2005 and 2021. The yield from annual TCS was compared with hypothetical biennial and triennial TCS after two initial US with annual interval by counting delayed detection of nodular growth, thyroid adenoma, and TC. During 279 follow-up years, 84 patients (median age 40 years) underwent 349 US. Thyroidectomy was performed in 6/84 (7%) patients, revealing a minimally invasive follicular TC in one patient aged 22 and a thyroid adenoma in two patients aged 21 and 53. Multiple thyroid nodules were diagnosed in 73/84 (87%) patients (median age 36 years). Nodular growth was detected in 9/56 (16%) patients, and its detection would have been delayed in 4–7% US rounds with biennial TCS, and in 2–6% US rounds with triennial TCS. US-based thyroiditis and indeterminate non-malignant lymph nodes were found in 8/74 (11%) and 7/72 (10%) patients, respectively. Following our findings combined with the literature, we propose starting TCS before age 18 and reducing the follow-up frequency after the initial two US from annual to biennial if no suspicious findings are detected. [ABSTRACT FROM AUTHOR]

Glioblastoma IDH-wildtype presents with a wide histological spectrum. Some features are so distinctive that they are considered as separate histological variants or patterns for the purpose of classification. However, these usually lack defined (epi-)genetic alterations or profiles correlating with this histology. Here, we describe a molecular subtype with overlap to the unique histological pattern of glioblastoma with primitive neuronal component. Our cohort consists of 63 IDH-wildtype glioblastomas that harbor a characteristic DNA methylation profile. Median age at diagnosis was 59.5 years. Copy-number variations and genetic sequencing revealed frequent alterations in TP53, RB1 and PTEN, with fewer gains of chromosome 7 and homozygous CDKN2A/B deletions than usually described for IDH-wildtype glioblastoma. Gains of chromosome 1 were detected in more than half of the cases. A poorly differentiated phenotype with frequent absence of GFAP expression, high proliferation index and strong staining for p53 and TTF1 often caused misleading histological classification as carcinoma metastasis or primitive neuroectodermal tumor. Clinically, many patients presented with leptomeningeal dissemination and spinal metastasis. Outcome was poor with a median overall survival of only 12 months. Overall, we describe a new molecular subtype of IDH-wildtype glioblastoma with a distinct histological appearance and genetic signature.

SOX9 (Sex-determining region Y Box 9) is a well-characterized transcription factor that is a marker for progenitor cells in various tissues. In the liver, cells delineated by SOX9 are responsible for regenerating liver parenchyma when cell proliferation is impaired following chronic injury. However, whether these SOX9+ cells play a role in liver carcinogenesis has not been fully understood, although high SOX9 expression has been linked to poor survival outcome in liver cancer patients. To address this question, we developed a liver cancer mouse model (PtenloxP/loxP; Sox9-CreERT+; R26RYFP) where tumor suppressor Pten (phosphatase and tensin homolog deleted on chromosome ten) is deleted in SOX9+ cells following tamoxifen injection. In this paper, we employ lineage-tracing to demonstrate the tumorigenicity potential of the Pten-, SOX9+ cells. We show that these cells are capable of giving rise to mixed-lineage tumors that manifest features of both hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CCA). Our results suggest that PTEN loss induces the transformation of SOX9+ cells. We further show that to activate these transformed SOX9+ cells, the presence of liver injury is crucial. Liver injury, induced by hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or high-fat diet (HFD), substantially increases tumor incidence and accelerates liver carcinogenesis from SOX9+ cells in Pten null mice but not in control mice. We further examine the mechanisms underlying tumor formation in this model to show that concurrent with the induction of niche signal (i.e., Wnt signaling), liver injury significantly stimulates the expansion of tumor-initiating cells (TICs). Together, these data show that (1) SOX9+ cells have the potential to become TICs following the primary transformation (i.e. Pten deletion) and that (2) liver injury is necessary for promoting the activation and proliferation of transformed SOX9+ cells, resulting in the genesis of mixed-lineage liver tumors.

Abstract Background There is pressing needs to find the biomarker in the selection of neoadjuvant therapy in postmenopausal luminal breast cancer patients. We examined the hypothesis that PIK3CA mutations and low phosphatase and tensin homolog (PTEN) expression affect the response to neoadjuvant therapy and prognosis in postmenopausal luminal breast cancer patients. Methods Postmenopausal patients with estrogen receptor-positive, human epidermal growth factor receptor 2-negative breast cancer, up to stage II, who underwent neoadjuvant chemotherapy (NAC; n = 60) or neoadjuvant endocrine therapy (NAE; n = 55) were selected. PIK3CA exon 9 and exon 20 mutations were screened by high resolution melting analysis and confirmed by Sanger sequence. PTEN expression was evaluated by immunohistochemistry. The relationships among PIK3CA mutations, PTEN expression, clinicopathological features, the pathological effect of neoadjuvant therapy, recurrence-free survival (RFS) and overall survival were analyzed. Results Among 115 patients, PIK3CA mutations and low PTEN expression before treatment were detected in 35 patients (30.4%) and in 28 patients (24.3%), respectively. In the NAC group, tumor with PIK3CA mutations showed significantly poorer response than tumor with PIK3CA wild-type (p = 0.03). On the other hand, in the NAE group, there was no significant difference in pathological therapeutic effect between tumor with PIK3CA mutations and tumor with PIK3CA wild-type (p = 0.54). In the NAC group, the log-rank test showed no difference in RFS between patients with PIK3CA mutations and PIK3CA wild-type (p = 0.43), but patients with low PTEN expression showed significantly worse RFS compared to patients with high PTEN expression (5 year RFS 0.64 vs. 0.87, p = 0.01). In the Cox proportional hazards model for RFS, PTEN expression, progesterone receptor, and pathological therapeutic effect were predictive factors for time to recurrence (All p

Background and aimsEpitranscriptomic modification of RNA has emerged as the most prevalent form of regulation of gene expression that affects development, differentiation, metabolism, viral infections, and most notably cancer. We have previously shown that hepatitis B virus (HBV) transcripts are modified by N6 methyladenosine (m6 A) addition. HBV also affects m6 A modification of several host RNAs, including phosphatase and tensin homolog (PTEN), a well-known tumor suppressor. PTEN plays a critical role in antiviral innate immunity and the development of hepatocellular carcinoma (HCC). Reports have shown that PTEN controlled interferon regulatory factor 3 (IRF-3) nuclear localization by negative phosphorylation of IRF-3 at Ser97, and PTEN reduced carcinogenesis by inhibiting the phosphatidylinositol-3-kinase (PI3K)/AKT pathway.Approach and resultsHere, we show that HBV significantly increases the m6 A modification of PTEN RNA, which contributes to its instability with a corresponding decrease in PTEN protein levels. This is reversed in cells in which the expression of m6 A methyltransferases is silenced. PTEN expression directly increases activated IRF-3 nuclear import and subsequent interferon synthesis. In the absence of PTEN, IRF-3 dephosphorylation at the Ser97 site is decreased and interferon synthesis is crippled. In chronic HBV patient biopsy samples, m6 A-modified PTEN mRNA levels were uniformly up-regulated with a concomitant decrease of PTEN mRNA levels. HBV gene expression also activated the PI3K/AKT pathway by regulating PTEN mRNA stability in HCC cell lines.ConclusionsThe m6 A epitranscriptomic regulation of PTEN by HBV affects innate immunity by inhibiting IRF-3 nuclear import and the development of HCC by activating the PI3K/AKT pathway. Our studies collectively provide new insights into the mechanisms of HBV-directed immune evasion and HBV-associated hepatocarcinogenesis through m6 A modification of the host PTEN mRNAs.

Chronic hepatitis B virus (HBV) infections are one of the leading causes of cirrhosis and hepatocellular carcinoma. N6-methyladenosine (m6A) modification of cellular and viral RNAs is the most prevalent internal modification that occurs cotranscriptionally. Previously, we reported the dual functional role of m6A modification of HBV transcripts in the viral life cycle. Here, we show that viral HBV X (HBx) protein is responsible for the m6A modifications of viral transcripts. HBV genomes defective in HBx failed to induce m6A modifications of HBV RNAs during infection/transfection, while ectopic expression of HBx restores m6A modifications of the viral RNAs but not the mutant HBx carrying the nuclear export signal. Using chromatin immunoprecipitation assays, we provide evidence that HBx and m6A methyltransferase complexes are localized on the HBV minichromosome to achieve cotranscriptional m6A modification of viral RNAs. HBx interacts with METTL3 and 14 to carry out methylation activity and also modestly stimulates their nuclear import. This role of HBx in mediating m6A modification also extends to host phosphatase and tensin homolog (PTEN) mRNA. This study provides insight into how a viral protein recruits RNA methylation machinery to m6A-modify RNAs.

Chronic elevated free fatty (FFA) levels are linked to metabolic disorders and tumorigenesis. However, the molecular mechanism by which FFAs induce cancer remains poorly understood. Here, we show that the tumor suppressor PTEN protein levels were decreased in high fat diet (HFD) fed mice. As palmitic acid (PA, C16:0) showed a significant increase in the HFD fed mice, we further investigated its role in PTEN down regulation. Our studies revealed that exposure of cells to high doses of PA induced mTOR/S6K-mediated phosphorylation of PTEN at T366. The phosphorylation subsequently enhanced the interaction of PTEN with the E3 ubiquitin ligase WW domain-containing protein 2 (WWP2), which promoted polyubiquitination of PTEN and protein degradation. Consistent with PTEN degradation, exposure of cells to increased concentrations of PA also promoted PTEN-mediated AKT activation and cell proliferation. Significantly, a higher level of S6K activation, PTEN T366 phosphorylation, and AKT activation were also observed in the livers of the HFD fed mice. These results provide a molecular mechanism by which a HFD and elevated PA regulate cell proliferation through inactivation of tumor suppressor PTEN.

Hepatocellular carcinoma (HCC) is the sixth most common primary cancer with an unsatisfactory long-term survival. Gain of function mutations of PIK3CA occur in a subset of human HCC. Alpelisib, a selective PIK3CA inhibitor, has been approved by the FDA to treat PIK3CA mutant breast cancers. In this manuscript, we evaluated the therapeutic efficacy of alpelisib, either alone or in combination, for the treatment of HCC. We tested alpelisib in mouse HCC induced by hydrodynamic injection of c-Met/PIK3CA(H1047R) (c-Met/H1047R), c-Met/PIK3CA(E545K) (c-Met/E545K), and c-Met/sgPten gene combinations. Alpelisib slowed down the growth of c-Met/H1047R and c-Met/E545K HCC but was ineffective in c-Met/sgPten HCC. Mechanistically, alpelisib inhibited p-ERK and p-AKT in c-Met/H1047R and c-Met/E545K HCC progression but did not affect the mTOR pathway or genes involved in cell proliferation. In human HCC cell lines transfected with PIK3CA(H1047R), alpelisib synergized with the mTOR inhibitor MLN0128 or the CDK4/6 inhibitor palbociclib to suppress HCC cell growth. In c-Met/H1047R mice, alpelisib/MLN0128 or alpelisib/palbociclib combination therapy caused tumor regression. Our study demonstrates that alpelisib is effective for treating PIK3CA-mutated HCC by inhibiting MAPK and AKT cascades. Furthermore, combining alpelisib with mTOR or CDK4/6 inhibitors has a synergistic efficacy against PIK3CA-mutated HCC, providing novel opportunities for precision medicine against HCC.

BackgroundPTEN loss is a putative driver in histotypes of ovarian cancer (high-grade serous (HGSOC), endometrioid (ENOC), clear cell (CCOC), mucinous (MOC), low-grade serous (LGSOC)). We aimed to characterise PTEN expression as a biomarker in epithelial ovarian cancer in a large population-based study.MethodsTumours from 5400 patients from a multicentre observational, prospective cohort study of the Ovarian Tumour Tissue Analysis Consortium were used to evaluate associations between immunohistochemical PTEN patterns and overall survival time, age, stage, grade, residual tumour, CD8+ tumour-infiltrating lymphocytes (TIL) counts, expression of oestrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR) by means of Cox proportional hazard models and generalised Cochran-Mantel-Haenszel tests.ResultsDownregulation of cytoplasmic PTEN expression was most frequent in ENOC (most frequently in younger patients; p value = 0.0001) and CCOC and was associated with longer overall survival in HGSOC (hazard ratio: 0.78, 95% CI: 0.65-0.94, p value = 0.022). PTEN expression was associated with ER, PR and AR expression (p values: 0.0008, 0.062 and 0.0002, respectively) in HGSOC and with lower CD8 counts in CCOC (p value

Accumulating evidence has documented that microRNAs (miRNAs or miRs) function as important post‑transcriptional regulators of the differentiation of mesenchymal stem cells (MSCs), including human adipose‑derived mesenchymal stem cells (hADSCs); however, their roles in hADSC osteogenic differentiation require further investigation. The present study aimed to investigate the role of miRNAs in the osteogenic differentiation of hADSCs and to elucidate the underlying molecular mechanisms. Using an miRNA microarray, it was found that 24 miRNAs were upregulated and 14 miRNAs were downregulated compared with the undifferentiated cells, and miR‑29b‑3p (miR‑29b) was selected for further experiments. Functional experiments revealed that the upregulation of miR‑29b by agomir‑29b significantly enhanced alkaline phosphatase (ALP) activity and the mineralization of extracellular matrix (ECM), and led to an increase in the mRNA and protein levels of osteogenic marker genes, including runt‑related transcription factor 2 (Runx2), osteopontin (OPN), osteocalcin (OCN) and bone sialoprotein (BSP), whereas the knockdown of miR‑29b suppressed these processes. In addition, phosphatase and tensin homolog (PTEN), a negative regulator of the AKT/β‑catenin pathway, was identified as a direct target of miR‑29b in the hADSCs. Moreover, it was observed that the overexpression of miR‑29b activated the AKT/β‑catenin signaling pathway by inhibiting PTEN expression in the hADSCs. Most importantly, it was also found that the overexpression of PTEN reversed the promoting effects of miR‑29b on osteogenic differentiation. On the whole, these findings suggest that miR‑29b promotes the osteogenic differentiation of hADSCs by modulating the PTEN/AKT/β‑catenin signaling pathway. Thus, this miRNA may be a promising target for the active modulation of hADSC‑derived osteogenesis.

Cetuximab, an mAb targeting EGFR, is a standard of care for the treatment for locally advanced or metastatic head and neck squamous cell carcinoma (HNSCC). However, despite overexpression of EGFR in more than 90% of HNSCC lesions, most patients with HNSCC fail to respond to cetuximab treatment. In addition, there are no available biomarkers to predict sensitivity or resistance to cetuximab in the clinic. Here, we sought to advance precision medicine approaches for HNSCC by identifying PI3K/mTOR signaling network-specific cetuximab resistance mechanisms. We first analyzed the frequency of genomic alterations in genes involved in the PI3K/mTOR signaling circuitry in the HNSCC TCGA dataset. Experimentally, we took advantage of CRISPR/Cas9 genome editing approaches to systematically explore the contribution of genomic alterations in each tumor suppressor gene (TSG) controlling the PI3K-mTOR pathway to cetuximab resistance in HNSCC cases that do not exhibit PIK3CA mutations. Remarkably, we found that many HNSCC cases exhibit pathway-specific gene copy number loss of multiple TSGs that normally restrain PI3K/mTOR signaling. Among them, we found that both engineered and endogenous PTEN gene deletions can mediate resistance to cetuximab. Our findings suggest that PTEN gene copy number loss, which is highly prevalent in HNSCC, may result in sustained PI3K/mTOR signaling independent of EGFR, thereby representing a promising mechanistic biomarker predictive of cetuximab resistance in this cancer type. Further prospective studies are needed to investigate the impact of PTEN loss on cetuximab efficacy in the clinic.

Metastasis is responsible for 90% of human cancer mortality, yet it remains a challenge to model human cancer metastasis in vivo. Here we describe mouse models of high-grade serous ovarian cancer, also known as high-grade serous carcinoma (HGSC), the most common and deadliest human ovarian cancer type. Mice genetically engineered to harbor Dicer1 and Pten inactivation and mutant p53 robustly replicate the peritoneal metastases of human HGSC with complete penetrance. Arising from the fallopian tube, tumors spread to the ovary and metastasize throughout the pelvic and peritoneal cavities, invariably inducing hemorrhagic ascites. Widespread and abundant peritoneal metastases ultimately cause mouse deaths (100%). Besides the phenotypic and histopathological similarities, mouse HGSCs also display marked chromosomal instability, impaired DNA repair, and chemosensitivity. Faithfully recapitulating the clinical metastases as well as molecular and genomic features of human HGSC, this murine model will be valuable for elucidating the mechanisms underlying the development and progression of metastatic ovarian cancer and also for evaluating potential therapies.

Family history is among the strongest known risk factors for prostate cancer (PCa). Emerging data suggest molecular subtypes of PCa, including two somatic genetic aberrations: fusions of androgen-regulated promoters with ERG and, separately, phosphatase and tensin homolog (PTEN) loss. We examined associations between family history and incidence of these subtypes in 44,126 men from the prospective Health Professionals Follow-up Study. ERG and PTEN status were assessed by immunohistochemistry. Multivariable competing risks models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for associations between self-reported family history of PCa and molecular subtypes of disease. Thirteen percent of men had a positive family history of PCa at baseline. During a median follow-up of 18.5 years, 5,511 PCa cases were diagnosed. Among them, 888 were assayed for ERG status (47% ERG-positive) and 715 were assayed for PTEN loss (14% PTEN null). Family history was more strongly associated with risk of ERG-negative (HR: 2.15; 95% CI: 1.71-2.70) than ERG-positive (HR: 1.49; 95% CI: 1.13-1.95) disease (pheterogeneity : 0.04). The strongest difference was among men with an affected father (HRERG-negative : 2.09; 95% CI: 1.64-2.66; HRERG-positive : 1.30; 95% CI: 0.96-1.76; pheterogeneity : 0.01). Family history of PCa was positively associated with both PTEN null (HR: 2.10; 95% CI: 1.26-3.49) and PTEN intact (HR: 1.72; 95% CI: 1.39-2.13) PCa (pheterogeneity : 0.47). Our results indicate that PCa family history may be positively associated with PCa in all ERG and PTEN subtypes, suggesting a role of genetic susceptibility in their development. It is possible that ERG-negative disease could be especially associated with positive family history.

Autism is characterized by repetitive behaviors, impaired social interactions, and communication deficits. It is a prevalent neurodevelopmental disorder, and available treatments offer little benefit. Here, we show that genetically reducing the protein tau prevents behavioral signs of autism in two mouse models simulating distinct causes of this condition. Similar to a proportion of people with autism, both models have epilepsy, abnormally enlarged brains, and overactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B)/ mammalian target of rapamycin (mTOR) signaling pathway. All of these abnormalities were prevented or markedly diminished by partial or complete genetic removal of tau. We identify disinhibition of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a negative PI3K regulator that tau controls, as a plausible mechanism and demonstrate that tau interacts with PTEN via tau's proline-rich domain. Our findings suggest an enabling role of tau in the pathogenesis of autism and identify tau reduction as a potential therapeutic strategy for some of the disorders that cause this condition.

Genetic deletion or knockdown of PTEN enables regeneration of CNS axons, enhances sprouting of intact axons after injury, and induces de novo growth of uninjured adult neurons. It is unknown, however how PTEN deletion in mature neurons alters neuronal physiology. As a first step to address this question, we used immunocytochemistry for activity-dependent markers to assess consequences of PTEN knockdown in cortical neurons and granule cells of the dentate gyrus. In adult rats that received unilateral intra-cortical injections of AAV expressing shRNA against PTEN, immunostaining for c-fos under resting conditions (home cage, HC) and after 1 h of exploration of a novel enriched environment (EE) revealed no hot spots of c-fos expression that would suggest abnormal activity. Counts revealed similar numbers of c-fos positive neurons in the area of PTEN deletion vs. homologous areas in the contralateral cortex in the HC and similar induction of c-fos with EE. However, IEG induction in response to high frequency stimulation (HFS) of the cortex was attenuated in areas of PTEN deletion. In rats with AAVshRNA-mediated PTEN deletion in the dentate gyrus, induction of the IEGs c-fos and Arc with HFS of the perforant path was abrogated in areas of PTEN deletion. Immunostaining using phosphospecific antibodies for phospho-S6 (a downstream marker for mTOR activation) and phospho-ERK1/2 revealed abrogation of S6 phosphorylation in PTEN-deleted areas but preserved activation of phosphorylation of ERK1/2. SIGNIFICANCE STATEMENT: Deletion or knockdown of the tumor suppressor gene PTEN enables regenerative growth of adult CNS axons after injury, which is accompanied by enhanced recovery of function. Consequently, PTEN represents a potential target for therapeutic interventions to enhance recovery after CNS injury. Here we show that activity-dependent IEG induction is attenuated in PTEN-depleted neurons. These findings raise the intriguing possibility that functional recovery due to regenerative growth may be limited by the disruption of plasticity-related signaling pathways, and that recovery might be enhanced by restoring PTEN expression after regenerative growth has been achieved.

Manipulations that enhance GABAergic inhibition have been associated with improved behavioral phenotypes in autism models, suggesting that autism may be treated by correcting underlying deficits of inhibition. Interneuron transplantation is a method for increasing recipient synaptic inhibition, and it has been considered a prospective therapy for conditions marked by deficient inhibition, including neuropsychiatric disorders. It is unknown, however, whether interneuron transplantation may be therapeutically effective only for conditions marked by reduced inhibition, and it is also unclear whether transplantation improves behavioral phenotypes solely by normalizing underlying circuit defects. To address these questions, we studied the effects of interneuron transplantation in male and female mice lacking the autism-associated gene, Pten, in GABAergic interneurons. Pten mutant mice exhibit social behavior deficits, elevated synaptic inhibition in prefrontal cortex, abnormal baseline and social interaction-evoked electroencephalogram (EEG) signals, and an altered composition of cortical interneuron subtypes. Transplantation of wild-type embryonic interneurons from the medial ganglionic eminence into the prefrontal cortex of neonatal Pten mutants rescued social behavior despite exacerbating excessive levels of synaptic inhibition. Furthermore, transplantation did not normalize recipient EEG signals measured during baseline states. Interneuron transplantation can thus correct behavioral deficits even when those deficits are associated with elevated synaptic inhibition. Moreover, transplantation does not exert therapeutic effects solely by restoring wild-type circuit states. Our findings indicate that interneuron transplantation could offer a novel cell-based approach to autism treatment while challenging assumptions that effective therapies must reverse underlying circuit defects.SIGNIFICANCE STATEMENT Imbalances between neural excitation and inhibition are hypothesized to contribute to the pathophysiology of autism. Interneuron transplantation is a method for altering recipient inhibition, and it has been considered a prospective therapy for neuropsychiatric disorders, including autism. Here we examined the behavioral and physiological effects of interneuron transplantation in a mouse genetic model of autism. They demonstrate that transplantation rescues recipient social interaction deficits without correcting a common measure of recipient inhibition, or circuit-level physiological measures. These findings demonstrate that interneuron transplantation can exert therapeutic behavioral effects without necessarily restoring wild-type circuit states, while highlighting the potential of interneuron transplantation as an autism therapy.

Many cellular models aimed at elucidating cancer biology do not recapitulate pathobiology including tumor heterogeneity, an inherent feature of cancer that underlies treatment resistance. Here we introduce a cancer modeling paradigm using genetically engineered human pluripotent stem cells (hiPSCs) that captures authentic cancer pathobiology. Orthotopic engraftment of the neural progenitor cells derived from hiPSCs that have been genome-edited to contain tumor-associated genetic driver mutations revealed by The Cancer Genome Atlas project for glioblastoma (GBM) results in formation of high-grade gliomas. Similar to patient-derived GBM, these models harbor inter-tumor heterogeneity resembling different GBM molecular subtypes, intra-tumor heterogeneity, and extrachromosomal DNA amplification. Re-engraftment of these primary tumor neurospheres generates secondary tumors with features characteristic of patient samples and present mutation-dependent patterns of tumor evolution. These cancer avatar models provide a platform for comprehensive longitudinal assessment of human tumor development as governed by molecular subtype mutations and lineage-restricted differentiation.

ObjectiveIncreased de novo fatty acid (FA) synthesis and cholesterol biosynthesis have been independently described in many tumour types, including hepatocellular carcinoma (HCC).DesignWe investigated the functional contribution of fatty acid synthase (Fasn)-mediated de novo FA synthesis in a murine HCC model induced by loss of Pten and overexpression of c-Met (sgPten/c-Met) using liver-specific Fasn knockout mice. Expression arrays and lipidomic analysis were performed to characterise the global gene expression and lipid profiles, respectively, of sgPten/c-Met HCC from wild-type and Fasn knockout mice. Human HCC cell lines were used for in vitro studies.ResultsAblation of Fasn significantly delayed sgPten/c-Met-driven hepatocarcinogenesis in mice. However, eventually, HCC emerged in Fasn knockout mice. Comparative genomic and lipidomic analyses revealed the upregulation of genes involved in cholesterol biosynthesis, as well as decreased triglyceride levels and increased cholesterol esters, in HCC from these mice. Mechanistically, loss of Fasn promoted nuclear localisation and activation of sterol regulatory element binding protein 2 (Srebp2), which triggered cholesterogenesis. Blocking cholesterol synthesis via the dominant negative form of Srebp2 (dnSrebp2) completely prevented sgPten/c-Met-driven hepatocarcinogenesis in Fasn knockout mice. Similarly, silencing of FASN resulted in increased SREBP2 activation and hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase (HMGCR) expression in human HCC cell lines. Concomitant inhibition of FASN-mediated FA synthesis and HMGCR-driven cholesterol production was highly detrimental for HCC cell growth in culture.ConclusionOur study uncovers a novel functional crosstalk between aberrant lipogenesis and cholesterol biosynthesis pathways in hepatocarcinogenesis, whose concomitant inhibition might represent a therapeutic option for HCC.

Many primary tumours have low levels of molecular oxygen (hypoxia), and hypoxic tumours respond poorly to therapy. Pan-cancer molecular hallmarks of tumour hypoxia remain poorly understood, with limited comprehension of its associations with specific mutational processes, non-coding driver genes and evolutionary features. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancers across 38 tumour types, we quantify hypoxia in 1188 tumours spanning 27 cancer types. Elevated hypoxia associates with increased mutational load across cancer types, irrespective of underlying mutational class. The proportion of mutations attributed to several mutational signatures of unknown aetiology directly associates with the level of hypoxia, suggesting underlying mutational processes for these signatures. At the gene level, driver mutations in TP53, MYC and PTEN are enriched in hypoxic tumours, and mutations in PTEN interact with hypoxia to direct tumour evolutionary trajectories. Overall, hypoxia plays a critical role in shaping the genomic and evolutionary landscapes of cancer.

BackgroundHepatocellular carcinoma (HCC) is a leading cause of cancer related deaths worldwide. The PI3K cascade is one of the major signaling pathways underlying HCC development and progression. Activating mutations of PI3K catalytic subunit alpha (PIK3CA) and/or loss of Pten often occur in human HCCs. Serum and glucocorticoid kinase 3 (SGK3) belongs to the SGK family of AGK kinases and functions in parallel to AKT downstream of PI3K. Previous studies have shown that SGK3 may be the major kinase responsible for the oncogenic potential of PIK3CA helical domain mutants, such as PIK3CA(E545K), but not kinase domain mutants, such as PIK3CA(H1047R).MethodsWe investigated the functional contribution of SGK3 in mediating activated PIK3CA mutant or loss of Pten induced HCC development using Sgk3 knockout mice.ResultsWe found that ablation of Sgk3 does not affect PIK3CA(H1047R) or PIK3CA(E545K) induced lipogenesis in the liver. Using PIK3CA(H1047R)/c-Met, PIK3CA(E545K)/c-Met, and sgPten/c-Met murine HCC models, we also demonstrated that deletion of Sgk3 moderately delays PIK3CA(E545K)/c-Met driven HCC, while not affecting PIK3CA(H1047R)/c-Met or sgPten/c-Met HCC formation in mice. Similarly, in human HCC cell lines, silencing of SGK3 reduced PIK3CA(E545K) -but not PIK3CA(H1047R)- induced accelerated tumor cell proliferation.ConclusionAltogether, our data suggest that SGK3 plays a role in transducing helical domain mutant PIK3CA signaling during liver tumor development.

Background: There is pressing needs to find the biomarker in the selection of neoadjuvant therapy in postmenopausal luminal breast cancer patients. We examined the hypothesis that PIK3CA mutations and low phosphatase and tensin homolog (PTEN) expression affect the response to neoadjuvant therapy and prognosis in postmenopausal luminal breast cancer patients. Methods: Postmenopausal patients with estrogen receptor-positive, human epidermal growth factor receptor 2-negative breast cancer, up to stage II, who underwent neoadjuvant chemotherapy (NAC; n = 60) or neoadjuvant endocrine therapy (NAE; n = 55) were selected. PIK3CA exon 9 and exon 20 mutations were screened by high resolution melting analysis and confirmed by Sanger sequence. PTEN expression was evaluated by immunohistochemistry. The relationships among PIK3CA mutations, PTEN expression, clinicopathological features, the pathological effect of neoadjuvant therapy, recurrence-free survival (RFS) and overall survival were analyzed. Results: Among 115 patients, PIK3CA mutations and low PTEN expression before treatment were detected in 35 patients (30.4%) and in 28 patients (24.3%), respectively. In the NAC group, tumor with PIK3CA mutations showed significantly poorer response than tumor with PIK3CA wild-type (p = 0.03). On the other hand, in the NAE group, there was no significant difference in pathological therapeutic effect between tumor with PIK3CA mutations and tumor with PIK3CA wild-type (p = 0.54). In the NAC group, the log-rank test showed no difference in RFS between patients with PIK3CA mutations and PIK3CA wild-type (p = 0.43), but patients with low PTEN expression showed significantly worse RFS compared to patients with high PTEN expression (5 year RFS 0.64 vs. 0.87, p = 0.01). In the Cox proportional hazards model for RFS, PTEN expression, progesterone receptor, and pathological therapeutic effect were predictive factors for time to recurrence (All p < 0.05). Conclusions: PIK3CA mutations are associated with resistance to NAC but do not affect the response to NAE. Low PTEN expression does not affect response to either NAC or NAE but correlates with shorter RFS in patients who received NAC. These biomarkers will be further evaluated for clinical use to treat postmenopausal luminal breast cancer patients. [ABSTRACT FROM AUTHOR]

E-cadherin complexes with the actin cytoskeleton via cytoplasmic catenins and maintains the functional characteristics and integrity of the epithelia in normal epithelial tissues. Lost expression of E-cadherin disrupts this complex resulting in loss of cell polarity, epithelial denudation and increased epithelial permeability in a variety of tissues. Decreased expression of E-cadherin has also been observed in invasive and metastatic human tumors. In this study, we investigated the effect of E-cadherin loss in prostatic epithelium using newly developed genetically engineered mouse models. Deletion of E-cadherin in prostatic luminal epithelial cells with modified probasin promoter driven Cre (PB-Cre4) induced the development of mouse prostatic intraepithelial neoplasia (PIN). An increase in levels of cytoplasmic and nuclear β-catenin appeared in E-cadherin deleted atypical cells within PIN lesions. Using various experimental approaches, we further demonstrated that the knockdown of E-cadherin expression elevated free cytoplasmic and nuclear β-catenin and enhanced androgen-induced transcription and cell growth. Intriguingly, pathological changes representing prostatic epithelial cell denudation and increased apoptosis accompanied the above PIN lesions. The essential role of E-cadherin in maintaining prostatic epithelial integrity and organization was further demonstrated using organoid culture approaches. To directly assess the role of loss of E-cadherin in prostate tumor progression, we generated a new mouse model with bigenic Cdh1 and Pten deletion in prostate epithelium. Early onset, aggressive tumor phenotypes presented in the compound mice. Strikingly, goblet cell metaplasia was observed, intermixed within prostatic tumor lesions of the compound mice. This study provides multiple lines of novel evidence demonstrating a comprehensive role of E-cadherin in maintaining epithelial integrity during the course of prostate oncogenic transformation, tumor initiation and progression.

PIP3-dependent Rac exchanger 1 (P-Rex1) is activated downstream of G protein-coupled receptors to promote neutrophil migration and metastasis. The structure of more than half of the enzyme and its regulatory G protein binding site are unknown. Our 3.2 Å cryo-EM structure of the P-Rex1-Gβγ complex reveals that the carboxyl-terminal half of P-Rex1 adopts a complex fold most similar to those of Legionella phosphoinositide phosphatases. Although catalytically inert, the domain coalesces with a DEP domain and two PDZ domains to form an extensive docking site for Gβγ. Hydrogen-deuterium exchange mass spectrometry suggests that Gβγ binding induces allosteric changes in P-Rex1, but functional assays indicate that membrane localization is also required for full activation. Thus, a multidomain assembly is key to the regulation of P-Rex1 by Gβγ and the formation of a membrane-localized scaffold optimized for recruitment of other signaling proteins such as PKA and PTEN.

Neurogenin-3 (NEUROG3) is a helix-loop-helix (HLH) transcription factor involved in the production of endocrine cells in the intestine and pancreas of humans and mice. However, the human NEUROG3 loss-of-function phenotype differs subtly from that in mice, but the reason for this difference remains poorly understood. Because NEUROG3 expression precedes exit of the cell cycle and the expression of endocrine cell markers during differentiation, we investigated the effect of lentivirus-mediated overexpression of the human NEUROG3 gene on the cell cycle of BON4 cells and various human nonendocrine cell lines. NEUROG3 overexpression induced a reversible cell cycle exit, whereas expression of a neuronal lineage homolog, NEUROG1, had no such effect. In endocrine lineage cells, the cellular quiescence induced by short-term NEUROG3 expression required cyclin-dependent kinase inhibitor 1A (CDKN1A)/p21CIP1 expression. Expression of endocrine differentiation markers required sustained NEUROG3 expression in the quiescent, but not in the senescent, state. Inhibition of the phosphatase and tensin homolog (PTEN) pathway reversed quiescence by inducing cyclin-dependent kinase 2 (CDK2) and reducing p21CIP1 and NEUROG3 protein levels in BON4 cells and human enteroids. We discovered that NEUROG3 expression stimulates expression of CDKN2a/p16INK4a and BMI1 proto-oncogene polycomb ring finger (BMI1), with the latter limiting expression of the former, delaying the onset of CDKN2a/p16INK4a -driven cellular senescence. Furthermore, NEUROG3 bound to the promoters of both CDKN1a/p21CIP1 and BMI1 genes, and BMI1 attenuated NEUROG3 binding to the CDKN1a/p21CIP1 promoter. Our findings reveal how human NEUROG3 integrates inputs from multiple signaling pathways and thereby mediates cell cycle exit at the onset of differentiation.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor and bi-functional lipid and protein phosphatase. We report that the metabolic regulator pyruvate dehydrogenase kinase1 (PDHK1) is a synthetic-essential gene in PTEN-deficient cancer and normal cells. The PTEN protein phosphatase dephosphorylates nuclear factor κB (NF-κB)-activating protein (NKAP) and limits NFκB activation to suppress expression of PDHK1, a NF-κB target gene. Loss of the PTEN protein phosphatase upregulates PDHK1 to induce aerobic glycolysis and PDHK1 cellular dependence. PTEN-deficient human tumors harbor increased PDHK1, a biomarker of decreased patient survival. This study uncovers a PTEN-regulated signaling pathway and reveals PDHK1 as a potential target in PTEN-deficient cancers.

BackgroundThe phosphatidylinositol 3-kinase (PI3K) pathway is frequently altered in cancer. This report describes the landscape of PI3K alterations in solid tumors as well as co-alterations serving as potential resistance/attenuation mechanisms.MethodsConsecutive samples were analyzed in a commercial Clinical Laboratory Improvement Amendment-certified laboratory using comprehensive genomic profiling performed by next-generation sequencing (315 genes). The co-alterations evaluated included the Erb-B2 receptor tyrosine kinase 2 (ERBB2), ERBB3, ERBB4, RAS, MET proto-oncogene tyrosine kinase (MET), and mitogen-activated protein kinase kinase (MAP2K) genes as well as tumor protein 53 (TP53), estrogen receptor 1 (ESR1), and androgen receptor (AR).ResultsAlterations in any of 18 PI3K-pathway associated genes were identified in 44% of 60,991 tumors. Although single base and insertions/deletions (indels) were the most frequent alterations, copy number changes and rearrangements were identified in 11% and 0.9% of patients, respectively. Overall, the most frequently altered genes were PIK3 catalytic subunit α (PIK3CA) (13%), phosphatase and tensin homolog (PTEN) (9%), and serine/threonine kinase 11 (STK11) (5%). Tumor types that frequently harbored at least 1 PI3K alteration were uterine (77%), cervical (62%), anal (59%), and breast (58%) cancers. Alterations also were discerned frequently in tumors with carcinosarcoma (89%) and squamous cell carcinoma (62%) histologies. Tumors with a greater likelihood of co-occurring PI3K pathway and MAPK pathway alterations included colorectal cancers (odds ratio [OR], 1.64; P

Cigarette smoke (CS)-induced accumulation of mitochondrial damage has been widely implicated in chronic obstructive pulmonary disease (COPD) pathogenesis. Mitophagy plays a crucial role in eliminating damaged mitochondria, and is governed by the PINK1 (PTEN induced putative protein kinase 1)-PRKN (parkin RBR E3 ubiquitin protein ligase) pathway. Although both increased PINK1 and reduced PRKN have been implicated in COPD pathogenesis in association with mitophagy, there are conflicting reports for the role of mitophagy in COPD progression. To clarify the involvement of PRKN-regulated mitophagy in COPD pathogenesis, prkn knockout (KO) mouse models were used. To illuminate how PINK1 and PRKN regulate mitophagy in relation to CS-induced mitochondrial damage and cellular senescence, overexpression and knockdown experiments were performed in airway epithelial cells (AEC). In comparison to wild-type mice, prkn KO mice demonstrated enhanced airway wall thickening with emphysematous changes following CS exposure. AEC in CS-exposed prkn KO mice showed accumulation of damaged mitochondria and increased oxidative modifications accompanied by accelerated cellular senescence. In vitro experiments showed PRKN overexpression was sufficient to induce mitophagy during CSE exposure even in the setting of reduced PINK1 protein levels, resulting in attenuation of mitochondrial ROS production and cellular senescence. Conversely PINK1 overexpression failed to recover impaired mitophagy caused by PRKN knockdown, indicating that PRKN protein levels can be the rate-limiting factor in PINK1-PRKN-mediated mitophagy during CSE exposure. These results suggest that PRKN levels may play a pivotal role in COPD pathogenesis by regulating mitophagy, suggesting that PRKN induction could mitigate the progression of COPD. Abbreviations: AD: Alzheimer disease; AEC: airway epithelial cells; BALF: bronchoalveolar lavage fluid; AKT: AKT serine/threonine kinase; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; COPD: chronic obstructive pulmonary disease; CS: cigarette smoke; CSE: CS extract; CXCL1: C-X-C motif chemokine ligand 1; CXCL8: C-X-C motif chemokine ligand 8; HBEC: human bronchial epithelial cells; 4-HNE: 4-hydroxynonenal; IL: interleukin; KO: knockout; LF: lung fibroblasts; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; 8-OHdG: 8-hydroxy-2'-deoxyguanosine; OPTN: optineurin; PRKN: parkin RBR E3 ubiquitin protein ligase; PCD: programmed cell death; PFD: pirfenidone; PIK3C: phosphatidylinositol-4:5-bisphosphate 3-kinase catalytic subunit; PINK1: PTEN induced putative kinase 1; PTEN: phosphatase and tensin homolog; RA: rheumatoid arthritis; ROS: reactive oxygen species; SA-GLB1/β-Gal: senescence-associated-galactosidase, beta 1; SASP: senescence-associated secretory phenotype; SNP: single nucleotide polymorphism; TNF: tumor necrosis factor.

Cardiovascular disease (CVD) remains the leading cause of death globally, and heart failure is a major component of CVD-related morbidity and mortality. The development of cardiac hypertrophy in response to hemodynamic overload is initially considered to be beneficial; however, this adaptive response is limited and, in the presence of prolonged stress, will transition to heart failure. Yes-associated protein (YAP), the central downstream effector of the Hippo signaling pathway, regulates proliferation and survival in mammalian cells. Our previous work demonstrated that cardiac-specific loss of YAP leads to increased cardiomyocyte (CM) apoptosis and impaired CM hypertrophy during chronic myocardial infarction (MI) in the mouse heart. Because of its documented cardioprotective effects, we sought to determine the importance of YAP in response to acute pressure overload (PO). Our results indicate that endogenous YAP is activated in the heart during acute PO. YAP activation that depended upon RhoA was also observed in CMs subjected to cyclic stretch. To examine the function of endogenous YAP during acute PO, Yap+/flox;Creα-MHC (YAP-CHKO) and Yap+/flox mice were subjected to transverse aortic constriction (TAC). We found that YAP-CHKO mice had attenuated cardiac hypertrophy and significant increases in CM apoptosis and fibrosis that correlated with worsened cardiac function after 1 week of TAC. Loss of CM YAP also impaired activation of the cardioprotective kinase Akt, which may underlie the YAP-CHKO phenotype. Together, these data indicate a prohypertrophic, prosurvival function of endogenous YAP and suggest a critical role for CM YAP in the adaptive response to acute PO.

Ionizing radiation (IR) and chemotherapy are standard-of-care treatments for glioblastoma (GBM) patients and both result in DNA damage, however, the clinical efficacy is limited due to therapeutic resistance. We identified a mechanism of such resistance mediated by phosphorylation of PTEN on tyrosine 240 (pY240-PTEN) by FGFR2. pY240-PTEN is rapidly elevated and bound to chromatin through interaction with Ki-67 in response to IR treatment and facilitates the recruitment of RAD51 to promote DNA repair. Blocking Y240 phosphorylation confers radiation sensitivity to tumors and extends survival in GBM preclinical models. Y240F-Pten knockin mice showed radiation sensitivity. These results suggest that FGFR-mediated pY240-PTEN is a key mechanism of radiation resistance and is an actionable target for improving radiotherapy efficacy.

Mucosal melanoma is a rare and poorly characterized subtype of human melanoma. Here we perform a cross-species analysis by sequencing tumor-germline pairs from 46 primary human muscosal, 65 primary canine oral and 28 primary equine melanoma cases from mucosal sites. Analysis of these data reveals recurrently mutated driver genes shared between species such as NRAS, FAT4, PTPRJ, TP53 and PTEN, and pathogenic germline alleles of BRCA1, BRCA2 and TP53. We identify a UV mutation signature in a small number of samples, including human cases from the lip and nasal mucosa. A cross-species comparative analysis of recurrent copy number alterations identifies several candidate drivers including MDM2, B2M, KNSTRN and BUB1B. Comparison of somatic mutations in recurrences and metastases to those in the primary tumor suggests pervasive intra-tumor heterogeneity. Collectively, these studies suggest a convergence of some genetic changes in mucosal melanomas between species but also distinctly different paths to tumorigenesis.

Germline mutations in PTEN, the gene that encodes phosphatase and tensin homolog, have been identified in up to 20% of children with autism spectrum disorder (ASD) and macrocephaly and are associated with marked abnormalities in the white matter of the brain. This study sought to characterize the neurobehavioral phenotype of PTEN-ASD. Comprehensive neurobehavioral evaluations were conducted in 36 participants (ages 3-21 years) with PTEN-ASD and compared to two groups of controls: non-syndromic ASD with macrocephaly (Macro-ASD, n = 25) and those with PTEN mutations without ASD (PTEN-no ASD, n = 23). Linear regression analysis or Kruskal-Wallis tests were used to examine group differences on neurobehavioral measures (cognitive, behavioral, sensory, and adaptive functioning) and, for select measures, one-sample t-tests were used to compare group performance to healthy control norms. These analyses revealed a distinct neuropsychological profile associated with mutations in PTEN suggesting primary disruption of frontal lobe systems (i.e., attention, impulsivity, reaction time, processing speed, and motor coordination). Cognitive deficits in PTEN-ASD are more severe than those in PTEN-no ASD and extend to other areas of neurobehavioral function, specifically, adaptive behavior and sensory deficits. While core ASD symptoms are similar in PTEN-ASD and Macro-ASD, PTEN-ASD had lower clinical ratings of autism severity and showed more sensory abnormalities suggestive of less sensory responsiveness. Together, these results suggest that PTEN-ASD has a distinct neurobehavioral phenotype compared to idiopathic ASD that is likely to warrant special consideration for overall assessment and treatment.

Sepsis-induced acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) remains the leading complication for mortality caused by bacterial infection. The regulatory T (Treg) cells appear to be an important modulator in resolving lung injury. Despite the extensive studies, little is known about the role of macrophage HMGB1/PTEN/β-catenin signaling in Treg development during ALI. Objectives: This study was designed to determine the roles and molecular mechanisms of HMGB1/PTEN/β-catenin signaling in mediating CD4+CD25+Foxp3+ Treg development in sepsis-induced lung injury in mice. Setting: University laboratory research of First Affiliated Hospital of Anhui Medical University. Subjects: PTEN/β-catenin Loxp and myeloid-specific knockout mice. Interventions: Groups of PTENloxp/β-cateninloxp and myeloid-specific PTEN/β-catenin knockout (PTENM-KO/β-cateninM-KO) mice were treated with LPS or recombinant HMGB1 (rHMGB1) to induce ALI. The effects of HMGB1-PTEN axis were further analyzed by in vitro co-cultures. Measures and Main Results: In a mouse model of ALI, blocking HMGB1 or myeloid-specific PTEN knockout (PTENM-KO) increased animal survival/body weight, reduced lung damage, increased TGF-β production, inhibited the expression of RORγt and IL-17, while promoting β-catenin signaling and increasing CD4+CD25+Foxp3+ Tregs in LPS- or rHMGB-induced lung injury. Notably, myeloid-specific β-catenin ablation (β-cateninM-KO) resulted in reduced animal survival and increased lung injury, accompanied by reduced CD4+CD25+Foxp3+ Tregs in rHMGB-induced ALI. Furthermore, disruption of macrophage HMGB1/PTEN or activation of β-catenin significantly increased CD4+CD25+Foxp3+ Tregs in vitro. Conclusions: HMGB1/PTEN/β-catenin signaling is a novel pathway that regulates Treg development and provides a potential therapeutic target in sepsis-induced lung injury.

In this study, we aimed to identify mutations of key genes associated with docetaxel resistance in nine endometrial cancer cell lines. Endometrial cancers are associated with several critical gene mutations, including PIK3A, PTEN, and KRAS. Different gene mutations in endometrial cancer cells have varied responses to anticancer drugs and cancer therapies. The most frequently altered gene in endometrioid endometrial carcinoma tumors is PTEN. PTEN protein has lipid phosphatase and protein phosphatase activity, as well as other functions in the nucleus. Although the tumor-suppressive function of PTEN has mainly been attributed to its lipid phosphatase activity, a role for PTEN protein phosphatase activity in cell cycle regulation has also been suggested. Various tumor type-specific PTEN mutations are well documented. Here, nine endometrioid endometrial cancer cell lines with PIK3A, PTEN, and KRAS gene mutations were treated with docetaxel and radiation. One mutation with a docetaxel drug-resistant effect was a truncated form of PTEN. Among PTEN mutations in endometrial cancer cells, the Y68 frame shift mutation of PTEN constitutes a major mechanism of resistance to docetaxel treatment. The molecular mechanism involves truncation of the 403 amino acid PTEN protein at amino acid 68 by the Y68 frame shift, leading to the loss of PTEN protein phosphatase and lipid phosphatase activities.

Sequencing data have been instrumental in identifying oncogenic drivers in prostatic carcinoma and highlighting biomarkers that define aggressive disease. A review of a series of 30 primary and metastatic prostatic carcinomas clinically sequenced at our cancer genomics laboratory utilizing a targeted gene panel identified recurrent structural variants in the TP53 gene. These structural variants were found in 27% of all sequenced cases and represented 36% of the cases with metastatic disease. TP53 structural rearrangements have been previously reported in a significant subset of osteosarcomas, where they result in loss of p53 protein expression by immunohistochemistry. Similarly, in our prostate cases with TP53 structural rearrangements for which tissue was available for testing, we find loss of p53 protein expression by immunohistochemistry. In the eight TP53-rearranged cases, concurrent PTEN loss was identified in 4 cases, TMPRSS2-ERG fusion in 5 cases, and AR and FOXA1 amplification in 1 case each. Our results from this small case series suggest that TP53 rearrangements with loss of expression represent a frequent alternative mechanism of inactivation of this key tumor suppressor gene with potential utility as a marker of aggressive disease. Recognition of this TP53 rearrangement pathway is essential to accurately identify prostatic carcinomas with loss of TP53 function.

Malignant melanoma is an aggressive tumor of the skin and still lacks effective preventive and therapeutic treatments. In melanoma, both the BRAF/MEK/ERK and PI3-K/AKT signaling pathways are constitutively activated through multiple mechanisms, which result in cell-cycle progression and prevention of apoptosis. Therefore, the development of novel strategies for targeting BRAF and PI3K are of utmost importance. In this study, we found that Ashitaba (Angelica keiskei) chalcones, 4-hydroxyderricin (4HD) and xanthoangelol (XAG), suppressed melanoma development by directly targeting both BRAFV600E and PI3K, which blocked the activation of downstream signaling. This led to the induction of G1 phase cell-cycle arrest and apoptosis in melanoma cells. Importantly, 4HD or XAG dramatically attenuated tumor incidence and volume in the BRAF-activated Pten-deficient melanoma mouse model. Our findings suggest that 4HD and XAG are promising chemopreventive or potential therapeutic agents against melanomagenesis that act by targeting both BRAF and PI3K, providing hope for rapid clinical translation. Cancer Prev Res; 11(10); 607-20. ©2018 AACR.

Emerging evidence suggests that neuronal signaling is important for oligodendrocyte myelination; however, the necessity of this signaling during development is unclear. By eliminating dynamic neuronal signaling along the developing optic nerve, we find that oligodendrocyte differentiation is not dependent on neuronal signaling and that the initiation of myelination is dependent on a permissive substrate, namely supra-threshold axon caliber. Furthermore, we show that loss of dynamic neuronal signaling results in hypermyelination of axons. We propose that oligodendrocyte differentiation is regulated by non-neuronal factors during optic nerve development, whereas myelination is sensitive to the biophysical properties of axonal diameter.

Salivary gland tumor (SGT) is a rare tumor type, which exhibits broad-spectrum phenotypic, biological, and clinical heterogeneity. Currently, the molecular mechanisms that cause SGT pathogenesis remain poorly understood. A lack of animal models that faithfully recapitulate the naturally occurring process of human SGTs has hampered research progress on this field. In this report, we developed an inducible keratin 5-driven conditional knockout mouse model to delete gene(s) of interest in murine salivary gland upon local RU486 delivery. We have deleted two major tumor suppressors, Pten, a negative regulator of the PI3K pathway, and Smad4, the central signaling mediator of TGFβ pathway, in the murine salivary gland. Our results have shown that deletion of either Pten or Smad4 in murine salivary gland resulted in pleomorphic adenomas, the most common tumor in human SGT patients. Deletion of both Pten and Smad4 in murine salivary gland developed several malignancies, with salivary adenoid cystic carcinoma (SACC) being the most frequently seen. Molecular characterization showed that SACC exhibited mTOR activation and TGFβ1 overexpression. Examination of human SGT clinical samples revealed that loss of Pten and Smad4 is common in human SACC samples, particularly in the most aggressive solid form, and is correlated with survival of SACC patients, highlighting the human relevance of the murine models. In summary, our results offer significant insight into synergistic role of Pten and Smad4 in SGT, providing a rationale for targeting mTOR and/or TGFβ signaling to control SGT formation and progression.

BackgroundPhosphatase and tensin homolog (Pten) is capable of mediating microbe-induced immune responses in the gut. Thus, Pten deficiency in the intestine accelerates colitis development in Il10-/- mice. As some ambient pollutants inhibit Pten function and exposure to ambient pollutants may increase inflammatory bowel disease (IBD) incidence, it is of interest to examine how Pten inhibition could affect colitis development in genetically susceptible hosts.MethodsWith human colonic mucosa biopsies from pediatric ulcerative colitis and non-IBD control subjects, we assessed the mRNA levels of the PTEN gene and the gene involved in IL10 responses. The data from the human tissues were corroborated by treating Il10-/-, Il10rb-/-, and wild-type C57BL/6 mice with Pten-specific inhibitor VO-OHpic. We evaluated the severity of mouse colitis by investigating the tissue histology and cytokine production. The gut microbiome was investigated by analyzing the 16S ribosomal RNA gene sequence with mouse fecal samples.ResultsPTEN and IL10RB mRNA levels were reduced in the human colonic mucosa of pediatric ulcerative colitis compared with non-IBD subjects. Intracolonic treatment of the Pten inhibitor induced colitis in Il10-/- mice, characterized by reduced body weight, marked colonic damage, and increased production of inflammatory cytokines, whereas Il10rb-/- and wild-type C57BL/6 mice treated with the inhibitor did not develop colitis. Pten inhibitor treatment changed the fecal microbiome, with increased abundance of colitogenic bacteria Bacteroides and Akkermansia in Il10-/- mice.ConclusionsLoss of Pten function increases the levels of colitogenic bacteria in the gut, thereby inducing deleterious colitis in an Il10-deficient condition.

We report that PTEN-deficient prostate cancer cells use macropinocytosis to survive and proliferate under nutrient stress. PTEN loss increased macropinocytosis only in the context of AMPK activation, revealing a general requirement for AMPK in macropinocytosis and a novel mechanism by which AMPK promotes survival under stress. In prostate cancer cells, albumin uptake did not require macropinocytosis, but necrotic cell debris proved a specific macropinocytic cargo. Isotopic labeling confirmed that macropinocytosed necrotic cell proteins fueled new protein synthesis in prostate cancer cells. Supplementation with necrotic debris, but not albumin, also maintained lipid stores, suggesting that macropinocytosis can supply nutrients other than amino acids. Nontransformed prostatic epithelial cells were not macropinocytic, but patient-derived prostate cancer organoids and xenografts and autochthonous prostate tumors all exhibited constitutive macropinocytosis, and blocking macropinocytosis limited prostate tumor growth. Macropinocytosis of extracellular material by prostate cancer cells is a previously unappreciated tumor-microenvironment interaction that could be targeted therapeutically.Significance: As PTEN-deficient prostate cancer cells proliferate in low-nutrient environments by scavenging necrotic debris and extracellular protein via macropinocytosis, blocking macropinocytosis by inhibiting AMPK, RAC1, or PI3K may have therapeutic value, particularly in necrotic tumors and in combination with therapies that cause nutrient stress. Cancer Discov; 8(7); 866-83. ©2018 AACR.See related commentary by Commisso and Debnath, p. 800This article is highlighted in the In This Issue feature, p. 781.

Axons within the peripheral nervous system are capable of regeneration, but full functional recovery is rare. Recent work has shown that conditional deletion of two key signaling inhibitors of the PI3K and Jak/Stat pathways-phosphatase and tensin homolog (PTEN) and suppressor of cytokine signaling-3 (SOCS3), respectively-promotes regeneration of normally non-regenerative central nervous system axons. Moreover, in studies of optic nerve regeneration, co-deletion of both PTEN and SOCS3 has an even greater effect. Here, we test the hypotheses (1) that PTEN deletion enhances axon regeneration following sciatic nerve crush and (2) that PTEN/SOCS3 co-deletion further promotes regeneration. PTENfl/fl and PTEN/SOCS3fl/fl mice received direct injections of AAV-Cre into the fourth and fifth lumbar dorsal root ganglia (DRG) two weeks prior to sciatic nerve crush. Western blot analysis of whole cell lysates from DRG using phospho-specific antibodies revealed that PTEN deletion did not enhance or prolong PI3K signaling following sciatic nerve crush. However, PTEN/SOCS3 co-deletion activated PI3K for at least 7 days post-injury in contrast to controls, where activation peaked at 3 days. Quantification of SCG10-expressing regenerating sensory axons in the sciatic nerve after crush injury revealed longer distance regeneration at 3 days post-injury with both PTEN and PTEN/SOCS3 co-deletion. Additionally, analysis of noxious thermosensation and mechanosensation with PTEN/SOCS3 co-deletion revealed enhanced sensation at 14 and 21 days after crush, respectively, after which all treatment groups reached the same functional plateau. These findings indicate that co-deletion of PTEN and SOCS3 results in modest but measureable enhancement of early regeneration of DRG axons following crush injury.

Deletion of the phosphatase and tensin (PTEN) gene in neonatal mice leads to enlargement of the cell bodies of cortical motoneurons (CMNs) in adulthood (Gutilla et al., 2016). Here, we assessed whether PTEN deletion in adult mice would trigger growth of mature neurons. PTEN was deleted by injecting AAV-Cre into the sensorimotor cortex of adult transgenic mice with a lox-P flanked exon 5 of the PTEN gene and Cre-dependent reporter gene tdTomato. PTEN-deleted CMN's identified by tdT expression and retrograde labeling with fluorogold (FG) were significantly enlarged four months following PTEN deletion, and continued to increase in size through the latest time intervals examined (12-15 months post-deletion). Sholl analyses of tdT-positive pyramidal neurons revealed increases in dendritic branches at 6 months following adult PTEN deletion, and greater increases at 12 months. 12 months after adult PTEN deletion, axons in the medullary pyramids were significantly larger and G-ratios were higher. Mice with PTEN deletion exhibited no overt neurological symptoms and no seizures. Assessment of motor function on the rotarod and cylinder test revealed slight impairment of coordination with unilateral deletion; however, mice with bilateral PTEN deletion in the motor cortex performed better than controls on the rotarod at 8 and 10 months post-deletion. Our findings demonstrate that robust neuronal growth can be induced in fully mature cortical neurons long after the developmental period has ended and that this continuous growth occurs without obvious functional impairments.

BACKGROUND & AIMS:Intraductal papillary mucinous neoplasias (IPMNs) are precancerous cystic lesions that can develop into pancreatic ductal adenocarcinomas (PDACs). These large macroscopic lesions are frequently detected during medical imaging, but it is unclear how they form or progress to PDAC. We aimed to identify cells that form IPMNs and mutations that promote IPMN development and progression. METHODS:We generated mice with disruption of Pten specifically in ductal cells (Sox9CreERT2;Ptenflox/flox;R26RYFP or PtenΔDuct/ΔDuct mice) and used PtenΔDuct/+ and Pten+/+ mice as controls. We also generated KrasG12D;PtenΔDuct/ΔDuct and KrasG12D;PtenΔDuct/+ mice. Pancreata were collected when mice were 28 weeks to 14.5 months old and analyzed by histology, immunohistochemistry, and electron microscopy. We performed multiplexed droplet digital polymerase chain reaction to detect spontaneous Kras mutations in PtenΔDuct/ΔDuct mice and study the effects of Ras pathway activation on initiation and progression of IPMNs. We obtained 2 pancreatic sections from a patient with an invasive pancreatobiliary IPMN and analyzed the regions with and without the invasive IPMN (control tissue) by immunohistochemistry. RESULTS:Mice with ductal cell-specific disruption of Pten but not control mice developed sporadic, macroscopic, intraductal papillary lesions with histologic and molecular features of human IPMNs. PtenΔDuct/ΔDuct mice developed IPMNs of several subtypes. In PtenΔDuct/ΔDuct mice, 31.5% of IPMNs became invasive; invasion was associated with spontaneous mutations in Kras. KrasG12D;PtenΔDuct/ΔDuct mice all developed invasive IPMNs within 1 month. In KrasG12D;PtenΔDuct/+ mice, 70% developed IPMN, predominately of the pancreatobiliary subtype, and 63.3% developed PDAC. In all models, IPMNs and PDAC expressed the duct-specific lineage tracing marker yellow fluorescent protein. In immunohistochemical analyses, we found that the invasive human pancreatobiliary IPMN tissue had lower levels of PTEN and increased levels of phosphorylated (activated) ERK compared with healthy pancreatic tissue. CONCLUSIONS:In analyses of mice with ductal cell-specific disruption of Pten, with or without activated Kras, we found evidence for a ductal cell origin of IPMNs. We also showed that PTEN loss and activated Kras have synergistic effects in promoting development of IPMN and progression to PDAC.