Function Of PTEN Tumor Suppressor Research Articles

Notch Signaling Suppresses Melanoma Tumor Development in BRAF/Pten Mice.

Both oncogenic and tumor suppressor roles have been assigned to Notch signaling in melanoma. In clinical trials, Notch inhibitors proved to be ineffective for melanoma treatment. Notch signaling has also been implicated in melanoma transdifferentiation, a prognostic feature in primary melanoma. In this study, we investigated the role of Notch signaling in melanoma tumor development and growth using the genetic model of mouse melanoma by crossing BRAFCA/+/Pten+/+/Tyr-CreER+ (B) and BRAFCA/+/Pten-/-/Tyr-CreER + (BP) mice with Notch1 or Notch2 floxed allele mice. The topical application of tamoxifen induced tumors in BP mice but not in B mice with or without the deletion of either Notch1 or Notch2. These data show that the loss of either Notch1 nor Notch2 can substitute the tumor suppressor function of Pten in BRAFV600E-induced melanomagenesis. However, in Pten-null background, the loss of either Notch1 or Notch2 appeared to accelerate BRAFV600E-induced tumor development, suggesting a tumor suppressor role for Notch1 and Notch2 in BRAFV600E/Pten-null driven melanomagenesis. Quantitative immunochemical analysis of a human cutaneous melanoma tissue microarray that consists of >100 primary tumors with complete clinical history showed a weak to moderate correlation between NOTCH protein levels and clinical and pathological parameters. Our data show that Notch signaling is involved during melanomagenesis and suggest that the identification of genes and signaling pathways downstream of Notch could help devise strategies for melanoma prevention.

Identification of the PTEN-ARID4B-PI3K pathway reveals the dependency on ARID4B by PTEN-deficient prostate cancer

PTEN is frequently mutated in prostate cancer. The tumor suppressor function of PTEN is attributed to its lipid phosphatase activity that counters PI3K action. Here, we report a PTEN-ARID4B-PI3K axis in which PTEN inhibits expression of ARID4B, while ARID4B is a transcriptional activator of the PI3K subunit genes PIK3CA and PIK3R2 that are crucial for activation of the PI3K/AKT pathway. Reciprocal binding of ARID4B and histone H1 to the PIK3CA and PIK3R2 promoters modulates chromatin condensation, suggesting a mechanism by which ARID4B activates these promoters. Functional analyses reveals that ARID4B is required for prostate tumorigenesis when PTEN is deficient. The biological significance is further substantiated by the existence of a PTEN/ARID4B/PIK3CA three-gene signature that improves the predictive power for prostate cancer recurrence in patients. In summary, we identify ARID4B as a master regulator in the PTEN-PI3K pathway, thus providing a potential therapeutic target for prostate cancer carrying PTEN mutations.

Clinical Significance of PTEN Deletion, Mutation, and Loss of PTEN Expression in De Novo Diffuse Large B-Cell Lymphoma

PTEN loss has been associated with poorer prognosis in many solid tumors. However, such investigation in lymphomas is limited. In this study, PTEN cytoplasmic and nuclear expression, PTEN gene deletion, and PTEN mutations were evaluated in two independent cohorts of diffuse large B-cell lymphoma (DLBCL). Cytoplasmic PTEN expression was found in approximately 67% of total 747 DLBCL cases, more frequently in the activated B-cell–like subtype. Nuclear PTEN expression was less frequent and at lower levels, which significantly correlated with higher PTEN mRNA expression. Remarkably, loss of PTEN protein expression was associated with poorer survival only in DLBCL with AKT hyperactivation. In contrast, high PTEN expression was associated with Myc expression and poorer survival in cases without abnormal AKT activation. Genetic and epigenetic mechanisms for loss of PTEN expression were investigated. PTEN deletions (mostly heterozygous) were detected in 11.3% of DLBCL, and showed opposite prognostic effects in patients with AKT hyperactivation and in MYC rearranged DLBCL patients. PTEN mutations, detected in 10.6% of patients, were associated with upregulation of genes involved in central nervous system function, metabolism, and AKT/mTOR signaling regulation. Loss of PTEN cytoplasmic expression was also associated with TP53 mutations, higher PTEN-targeting microRNA expression, and lower PD-L1 expression. Remarkably, low PTEN mRNA expression was associated with down-regulation of a group of genes involved in immune responses and B-cell development/differentiation, and poorer survival in DLBCL independent of AKT activation. Collectively, multi-levels of PTEN abnormalities and dysregulation may play important roles in PTEN expression and loss, and that loss of PTEN tumor-suppressor function contributes to the poor survival of DLBCL patients with AKT hyperactivation.

PTEN Regulates Glucose Transporter Recycling by Impairing SNX27 Retromer Assembly

SummaryThe tumor suppressor PTEN executes cellular functions predominantly through its phosphatase activity. Here we identified a phosphatase-independent role for PTEN during vesicular trafficking of the glucose transporter GLUT1. PTEN physically interacts with SNX27, a component of the retromer complex that recycles transmembrane receptors such as GLUT1 from endosomes to the plasma membrane. PTEN binding with SNX27 prevents GLUT1 accumulation at the plasma membrane because of defective recycling and thus reduces cellular glucose uptake. Mechanistically, PTEN blocks the association of SNX27 with VPS26 and thereby hinders assembly of a functional retromer complex during the receptor recycling process. Importantly, we found a PTEN somatic mutation (T401I) that is defective in disrupting the association between SNX27 and VPS26, suggesting a critical role for PTEN in controlling optimal GLUT1 levels at the membrane to prevent tumor progression. Together, our results reveal a fundamental role of PTEN in the regulation of the SNX27 retromer pathway, which governs glucose transport and might contribute to PTEN tumor suppressor function.

Editorial: Lipid Signaling in T Cell Development and Function.

Editorial: Lipid Signaling in T Cell Development and Function.

Mutant PTEN in Cancer: Worse Than Nothing

Tumor suppressors block the development of cancer and are often lost during tumor development. Papa etal. show that partial loss of normal PTEN tumor suppressor function can be compounded by additional disruption caused by the expression of inactive mutant PTEN protein. This has significant implications for patients with PTEN gene mutations.

How Lipids Mediate Pten Tumor Suppressor Function

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is an important regulator of the PI 3-kinase signaling pathway. Mutation or deletion of one copy of this protein results in a tumorigenic state and PTEN has been identified as the second most important human tumor suppressor, rivaled only by p53. Not surprisingly, PTEN function is tightly regulated at the post-translational level as well as through interactions with lipids and other proteins. We have shown that PTEN binds synergistically to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylserine (PS), which leads to membrane association and allosteric activation. In this talk we will present molecular details about the interaction of PI(4,5)P2 with PTEN's N-terminal end as well as the interaction of PS with PTEN's C2 domain. In addition, we will discuss how PTEN function is affected by the lateral distribution of phosphoinositide lipids.

Abstract 5539: Low levels of estrogen sensitize PTEN-deficient endometrial tumors to poly-ADP ribose polymerase (PARP) inhibition in vivo.

Abstract Prior in vitro work suggests that PARP inhibition can destroy tumor cells with defects in homologous recombination resulting from loss of PTEN. Clinical trials have tested the efficacy of this strategy in BRCA mutant ovarian tumors. This approach has not been reported in endometrial carcinomas in vivo. Loss of PTEN tumor suppressor function is the most common genetic alteration associated with endometrial cancer that is the leading gynecologic cancer in the U.S. We hypothesized that oral administration of the PARP inhibitor AZD2281 (Olaparib) would be an effective therapeutic strategy for treating endometrial carcinomas resulting from PTEN loss. This hypothesis was tested in a mouse model for endometrial cancer established by our group. Uterine epithelia were harvested from PTENloxP/loxP mice, dissociated and infected with a Cre lentivirus resulting in PTEN deletion in uterine epithelia. These cells were combined with wild type stroma and placed subrenally in SCID mice resulting in formation of endometrial tumors that accurately recapitulate human disease. After tumor establishment (8 weeks), mice were divided into two cohorts: Group 1 was subjected to a continuous supply of estrogen and Group 2 was deprived of estrogen. Ten SCID mice were treated with a PARP inhibitor, AZD2281, administered orally for 3 weeks while the other 10 SCID mice were treated with vehicle in both hormonal conditions. At completion of therapy, tumors were harvested and analyzed. Forty mice were included in this preclinical trial. AZD2281 was successful in achieving a significant reduction in tumor size in a low estrogenic hormonal milieu. In striking contrast, no response to AZD2281 was seen in tumors exposed to high levels of estrogen. Hormonal deprivation resulted in a 20-fold increase in circulating levels of AZD2281 concomitant with increased inhibition of PARP enzyme activity at the tumor bed. Low levels of estrogen sensitize PTEN deficient tumors to PARP inhibition via two potential mechanisms: 1) down-regulation of key components of the homologous recombination pathway in low compared to high levels of estrogen and 2) a significant increase in the serum concentration of AZD2281 coupled with increased efficacy of PARP inhibition at the tumor bed. We suspect that withdrawal of estrogen results in a decrease in homologous recombination proteins and an increase in circulating levels of AZD2281 by decreasing its metabolism. The net result is greater circulating levels and effectiveness of PARP inhibitors in estrogen deprived treated mice. The results of this preclinical trial suggest that orally administered PARP inhibitors in a low estrogenic hormonal milieu can be effective in targeting PTEN-null endometrial tumors. Extension of this work to a clinical trial can result in personalizing therapies for women afflicted with endometrial cancer using well tolerated orally administered therapeutic agents. Citation Format: Daniel Y. Paik, Miguel Rosales, Deanna Janzen, Brian Yep, Donghui Cheng, Owen Witte, Huseyin Kayadibi, Kym Faull, Sanaz Memarzadeh. Low levels of estrogen sensitize PTEN-deficient endometrial tumors to poly-ADP ribose polymerase (PARP) inhibition in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5539. doi:10.1158/1538-7445.AM2013-5539

Abstract 4608: Autophagy is required for prostate cancer progression.

Abstract Autophagy (also referred to as macroautophagy) is a self-catabolic process that maintains intracellular homeostasis and prolongs cell survival by the autophagosomic-lysosomal degradation of cytoplasmic contents during metabolic stress or nutrient starvation. Inactivation of PTEN tumor suppressor function is a common characteristic of human prostate cancer that leads to the upregulation of the PI3K/Akt/mTOR signaling pathway, and has emerged as promising candidate for targeted therapies. mTOR is one of the upstream regulators of autophagy, therefore, inhibiting the PI3K/Akt/mTOR axis putatively stimulates autophagy. However, the role of autophagy in cancer is complex, acting both as a tumor suppressor and promoter, and its role in prostate tumorigenesis remains to be defined. Here we demonstrate that prostate cancer is dependent on autophagy for its growth and progression through the use genetically modified mouse models of prostate cancer. We had previously reported a conditional knockout mouse model in which homozygous deletion of PTEN lead to the stage-specific development of invasive adenocarcinoma. Characterization of tumors from the mice showed that although autophagy was suppressed early in tumor development, tumors eventually restored autophagic function despite increased PI3K/Akt/mTOR signaling. To determine the effect of defective autophagy on prostate cancer progression, we generated prostate-specific conditional double knockout mice harboring both inactivated PTEN and ATG7, a key regulator for the formation of autophagosomes. Prostate-specific inactivation of ATG7 function alone did not result in changes to the development or function of the prostate gland. Prostate-specific inactivation of both PTEN and ATG7 led to the early development of prostate adenocarcinoma similar to homozygous PTEN-mutant mice. However, prostate tumors failed to progress and showed regression. Our data suggests that impaired autophagy can be induced independent of mTOR and defective autophagy contributes to suppress tumor progression. Thus, our findings support the role of autophagy as a therapeutic target for advance prostate cancer. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Emiko Fukushima, Yuji Hatanaka, Yutaka Yamamoto, Nobutaka Shimizu, Kazuhiro Yoshimura, Masahiro Nozawa, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Autophagy is required for prostate cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4608. doi:10.1158/1538-7445.AM2013-4608

Unique biochemical properties of the protein tyrosine phosphatase activity of PTEN—Demonstration of different active site structural requirements for phosphopeptide and phospholipid phosphatase activities of PTEN

Missense PTEN mutations of the active site residues Asp-92, Cys-124 and Gly-129 contribute to Cowden syndrome. How their mutations affect phospholipid phosphatase activity and tumor suppressor function of PTEN has been defined. In this study, we investigated how their mutations affect the kinetics and catalytic mechanism of PTEN phosphoprotein phosphatase activity. Our data suggest that PTEN catalysis of phosphoprotein dephosphorylation follows a two-step mechanism with Cys-124 transiently phosphorylated to form the phosphoenzyme intermediate. In spite of this, we were unable to trap the genuine phosphoenzyme intermediate; instead, we unexpectedly discovered a novel phosphotransfer reaction in which the phosphate group is transferred from a tyrosyl phosphopeptide to PTEN to form a unique phosphorylated protein. Even though the finding is novel, the phosphotransfer reaction is likely an in vitro non-enzymatic reaction. Kinetic analysis revealed that mutation of Asp-92 has negligible impacts on phosphopeptide phosphatase activity of PTEN, suggesting that Asp-92 does not participate in the phosphopeptide dephosphorylation reaction. The results also imply that allosteric regulators facilitating the recruitment of Asp-92 to participate in catalysis will increase the activity of PTEN in dephosphorylating phosphoprotein and phosphopeptide substrates. Furthermore, kinetic analysis revealed that the G129E mutation has different effects on phospholipid and phosphoprotein phosphatase activities. Taken together, the data show that while the two phosphatase activities of PTEN follow a similar catalytic mechanism, they have notable differences in the requirements of the active site structure.

The Protein Phosphatase Activity of PTEN Regulates Src Family Kinases and Controls Glioma Migration

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is mutated or lost in 60% to 70% of advanced gliomas and is associated with malignant phenotypic changes such as migration, which contribute to the morbidity and mortality of this disease. Most of the tumor suppressor function of PTEN has been attributed to its ability to dephosphorylate the second messenger, phosphatidylinositol 3,4,5-triphosphate, resulting in the biological control of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. Despite recent work suggesting that the protein phosphatase activity of PTEN controls glioma cell migration, the mechanisms by which this occurs are unclear. Herein, we show using glioma cell lines (U87MG and U373MG) stably transfected with wild-type PTEN or catalytically altered mutants of PTEN that PTEN controls integrin-directed migration in a lipid phosphatase, PI3K/AKT-independent manner. Confirming this observation, we show that the stable overexpression of COOH-terminal Src kinase, the physiologic negative regulator of SRC family kinases (SFK), or treatment with the SFK inhibitor PP1 abrogates glioma migration. The results provide direct evidence that the downstream effect of the protein phosphatase activity of PTEN is to suppress SFK and FYN, and to regulate RAC-GTPase activity after alpha(v) integrin stimulation. Furthermore, studying vitronectin-directed migration using (a) Fyn small interfering RNA and (b) astrocytes from Fyn heterozygous (+/-) mice, Pten heterozygous (+/-) mice, Pten and Fyn double heterozygous (+/-) mice, or Fyn knockout (-/-) mice confirmed a role of FYN in alpha(v) integrin-mediated haptotaxis in glial cells. Our combined results provide direct biochemical and genetic evidence that PTEN's protein phosphatase activity controls FYN kinase function in glioma cells and regulates migration in a PI3K/AKT-independent manner.

Regulation of PTEN Activity by Its Carboxyl-terminal Autoinhibitory Domain

The regulation of PTEN intrinsic biochemical properties has not been fully elucidated. In this report, we investigated the role of the PTEN carboxyl-terminal tail domain in regulating its membrane targeting and catalytic functions. Characterization of a panel of PTEN phosphorylation site mutants revealed that mutating Ser-385 to alanine (S385A) promoted membrane localization in vivo and phosphatase activity in vitro. Furthermore, S385A mutation was associated with a substantial reduction in the phosphorylation of the Ser-380/Thr-382/Thr-383 cluster. Therefore, Ser-385 could prime additional dephosphorylation events to regulate PTEN catalytic activity. Moreover, substituting Ser-380/Thr-382/Thr-383 to phosphomimic residues reversed the phosphatase activity of the S385A mutation. Next, we further defined the underlying mechanisms responsible for the COOH-terminal tail region in modulating PTEN biological activity. We have identified an interaction between the 71-amino acid carboxyl-terminal tail region and the CBRIII motif of the C2 domain, which has been implicated in membrane binding. In addition, a synthetic phosphomimic peptide encompassing the phosphorylation site cluster between amino acids 368 and 390 within the tail region mediated the suppression of PTEN catalytic activity in vitro. This same peptide when expressed in cultured cells also impeded PTEN membrane localization and enhanced phospho-Akt levels. Thus, our data suggest that the COOH-terminal tail can act as an autoinhibitory domain to control both PTEN membrane recruitment and phosphatase activity.

A novel model to identify interaction partners of the PTEN tumor suppressor gene in human bladder cancer

Phosphatase and tensin homolog (PTEN), deleted on chromosome 10, is a potent tumor suppressor. PTEN expression is reduced in advanced bladder cancer and reduction correlates with disease stage. To gain insights into the function of PTEN in human bladder cancer by identifying its binding partners, we developed a novel IPTG inducible PTEN expression system and evaluated this system in the PTEN null UMUC-3 human bladder cancer xenograft model. In this model, induction of PTEN in vivo resulted in reduced tumor growth. We used mass spectrometry to identify PTEN interaction partners in these cells, which identified known interaction partners major vault protein (MVP) and paxillin as well as a novel interaction partner, TRK fused gene (TFG). In conclusion, using a biologically relevant model system to dissect PTEN tumor suppressor function in human bladder cancer, we identified three molecules important for many cellular functions in complex with PTEN.

PTEN is recruited to specific microdomains of the plasma membrane during lactacystin-induced neuronal apoptosis

The tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10) is a novel phosphatase displaying both protein and lipid phosphatase activities. In the central nervous system, PTEN plays an important role in the regulation of cell growth, differentiation and death. The tumor suppressor function of PTEN is attributed to its phospholipid phosphatase activity that dephosphorylates the plasma membrane phosphatidylinositol-(3,4,5)-triphosphate (PtdIns(3,4,5)P 3). Since PTEN is normally localized in the cytosol, it needs to be targeted to the plasma membrane to dephosphorylate PtdIns(3,4,5)P 3. We previously demonstrated that lactacystin-induced apoptosis of culture cortical neuron is associated with: (i) cleavage of PTEN (55 kDa) to a 50 kDa truncated form and (ii) accumulation of PTEN and all the truncated PTEN in a detergent-insoluble membrane fraction of the neuronal cells. Herein we demonstrate that a caspase-3 inhibitor suppresses cleavage of PTEN to the 50 kDa truncated form in culture cortical neurons in response to lactacystin treatment. Using immunogold transmission electron microscopy, we examined the distribution of PTEN in plasma membrane sheets stripped from cultured cortical neurons with and without treatment of lactacystin. Our results demonstrate that lactacystin treatment induces accumulation of PTEN to the inner surface of the plasma membrane sheets of the neuronal cells. Taken together, our data suggest that caspase-3-like proteases are involved in the conversion of PTEN to a 50-kDa truncated form and that PTEN and its truncated form accumulate at specific microdomains of the plasma membrane in neuronal cells undergoing apoptosis.

Targeting PI3K in alveolar rhabdomyosarcoma (aRMS)

10025 Background: aRMS is a pediatric solid tumor with a poor prognosis despite aggressive therapy. HGF/c-Met & PTEN-PI3K-AKT pathways are aberrant, leading to aRMS growth & metastasis. We hypothesize that recapitulating the tumor suppressor (TS) function of PTEN with small molecular inhibitors (SMIs) may control aRMS. SMIs of HGF/c-Met & PI3K pathways were examined for their control of aRMS growth & migration. Design/Methods: Pax3/fkhr-associated aRMS cells, RH30, were infected with MIEG3/PTEN (RH30MP1), MIEG3 retroviral vector (RH30MV1) or CMV-driven luciferase (RH30Luc). Migration was assessed by a transwell migration chamber; proliferation with a MTT assay; biochemical analysis by Western blot. Tumor growth and spontaneous lung metastasis in IACUC-approved animal experiments, in which cell lines were injected s.c. in nude mice, were analysed by bioluminescent imaging (BLI) or by direct measurements. Students t-test (p<.05) determined significance. Results: Western analysis revealed RH30 cells have low PTEN, high levels of c-Met & p-AKT. HGF stimulation of RH30 cells results in increased p-Met, p-Akt & p-Erk1/2 levels, correlating with enhanced migration and proliferation (<.001). SMI of PI3K (LY294002) controls RH30 proliferation (p<.01), not migration; c-Met (PHA665752) & MAPK (PD98059) SMIs stop HGF-directed migration through control of p-Erk levels (p<.01). PTEN induction in RH30 cells via PPAR-gamma agonist, rosiglitazone, results in decreased basal p-Akt levels. RH30 cells with stable, retroviral-infected PTEN have significantly reduced migration& proliferation as compared to cells with vector alone (p<.001). In nude mice xenograft models, PTEN & novel PI3K SMI, SF1126 at 50mg/kg, control RH30 tumoriginesis. SF1126 also controls spontaneous RH30Luc metastasis to the lungs. Conclusions: High c-Met-expressing RH30 cells are sensitive to HGF-stimulated migration through MAPK & proliferation via PI3K. PTEN’s control of aRMS migration, proliferation & tumorigenesis suggests that PTEN controls both PI3K & MAPK. Novel PI3K inhibitor, SF1126, controls tumor growth in vivo & prevents spontaneous lung metastasis. Recapitulating PTEN TS function with SF1126 alone or in combination with SMIs of the HGF/c-Met or MAPK may benefit patients with aRMS & deserves further pre-clinical study. [Table: see text]

Tumor suppressor PTEN acts through dynamic interaction with the plasma membrane

The tumor suppressor function of PTEN is strongly linked to its ability to dephosphorylate phosphatidylinositol-3,4,5 trisphosphate and, thereby, control cell growth, survival, and migration. However, the mechanism of action of PTEN in living cells is largely unexplored. Here we use single-molecule TIRF microscopy in living cells to reveal that the enzyme binds to the membrane for a few hundred milliseconds, sufficient to degrade several phosphatidylinositol-3,4,5 trisphosphate molecules. Deletion of an N-terminal lipid-binding motif completely abrogates membrane interaction and in vivo function. Several mechanisms, including C-terminal tail phosphorylations, appear to hold PTEN in a constrained conformation that limits its rate of association with the membrane. The steady-state level of bound PTEN is highest at sites of retracting membrane, including the rear of highly polarized cells. The dynamic membrane association could be modulated temporally or spatially to alter PTEN activity in specific physiological situations and could have important implications for tumor suppressor function.

Cooperation between FGF8b Overexpression and PTEN Deficiency in Prostate Tumorigenesis

Two commonly occurring genetic aberrations of human prostate cancer [i.e., overexpression of a mitogenic polypeptide (fibroblast growth factor 8, isoform b or FGF8b) and loss of function of PTEN tumor suppressor] were recapitulated into a new combinatorial mouse model. This model harboring the Fgf8b transgene and haploinsufficiency in Pten, both in a prostate epithelium-specific manner, yielded prostatic adenocarcinoma with readily detectable lymph node metastases, whereas single models with each of the defects were shown earlier to progress generally only up to prostatic intraepithelial neoplasia (PIN). In addition to late age-related development of typical adenocarcinoma, the model also displayed a low incidence of mucinous adenocarcinoma, a rare variant type of human prostatic adenocarcinoma. The cooperation between FGF8b activation and PTEN deficiency must be linked to acquisition of additional genetic alterations for the progression of the lesions to primary adenocarcinoma. Here, we identified loss of heterozygosity at the Pten gene leading to bialleic loss, as a necessary secondary event, indicating that a complete loss of PTEN function is required in the development of invasive cancer in the model. Analyses of expression of downstream mediators phospho-AKT (p-AKT) and p27(KIP1), in various types of lesions, however, revealed a complex picture. Although PIN lesions displayed relatively strong expression of p-AKT and p27(KIP1), there was a notable heterogeneity with variable decrease in their immunostaining in adenocarcinomas. Together, the results further underscore the notion that besides activation of AKT by loss of PTEN function, other PTEN-regulated pathways must be operative for progression of lesions from PIN to adenocarcinoma.

Lactacystin-induced apoptosis of cultured mouse cortical neurons is associated with accumulation of PTEN in the detergent-resistant membrane fraction.

The tumor suppressor function of PTEN is attributed to its phospholipid phosphatase activity that dephosphorylates the plasma membrane phosphatidylinositol-(3,4,5)-triphosphate [PtdIns(3,4,5)P3]. Implicit in this notion is that PTEN needs to be targeted to the plasma membrane to dephosphorylate PtdIns(3,4,5)P3. However, the recruitment of PTEN to the plasma membrane is not fully understood. Here, we demonstrate PTEN accumulation in the detergent-insoluble fraction of neuronal cells in response to treatment by the proteasome inhibitor lactacystin. First, lactacystin induces apoptosis and the activation of caspase-3 in cultured cortical neurons. Second, PTEN undergoes proteolysis to form a truncated 50-kDa form that lacks parts of its C-terminal tail. Third, the truncated PTEN is stably associated with the detergent-insoluble fraction in which the plasma membrane marker protein flotillin-1 resides. Taken together, our results suggest that truncation and accumulation of PTEN to the detergent-insoluble membrane fraction are two events associated with the apoptotic signals of the proteasome inhibitor in cortical neurons.

Frequent inactivation of PTEN by promoter hypermethylation in microsatellite instability-high sporadic colorectal cancers.

Loss of PTEN tumor suppressor function is observed in tumors of breast, prostate, thyroid, and endometrial origin. Allelic losses in the proximity of the PTEN locus (10q23) also occur in sporadic colorectal cancers (CRCs), but biallelic inactivation of this site has not been frequently demonstrated. We hypothesized that alternative mechanisms of PTEN allelic inactivation, such as promoter hypermethylation, might be operative in CRC and that PTEN inactivation may be related to recognized forms of genomic instability. We characterized a cohort of 273 sporadic CRCs by determining their microsatellite instability (MSI) status. Of these, 146 cancers were examined for PTEN promoter methylation by methylation-specific PCR. Mutations at the poly(A)6 repeat sequences in PTEN exons 7 and 8 and deletions at the 10q23 locus were also identified using microsatellite analysis. The presence of PTEN protein was determined by immunostaining, and the results were correlated with the promoter methylation status. We observed that PTEN promoter hypermethylation was a frequent occurrence in MSI-high (MSI-H) tumors (19.1% of MSI-H versus 2.2% of MSI-low/microsatellite stable tumors; P = 0.002). A PTEN mutation or a deletion event was present in 60% of the tumors with promoter region hypermethylation. Hypermethylation of the PTEN promoter correlated significantly with either decreased or complete loss of PTEN protein expression (P = 0.004). This is the first demonstration of PTEN inactivation as a result of promoter hypermethylation in MSI-H sporadic CRCs. These data suggest that this silencing mechanism plays a major role in PTEN inactivation and, in colon cancer, may be more important than either allelic losses or inactivating mutations. The significant correlation of PTEN hypermethylation with MSI-H tumors further suggests that PTEN is an additional important "target" of methylation along with the hMLH1 gene in the evolution of MSI-H CRCs and also confers the "second hit" in the biallelic inactivation mechanism for some proportion of tumors.