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PubMed2003

PTEN immunohistochemical expression is suppressed in G1 endometrioid adenocarcinoma of the uterine corpus.

PURPOSE: PTEN is a tumor suppressor gene that inhibits cell proliferation by regulating intracellular signaling pathways, and this activity can be abolished by mutations of the PTEN gene. This study was designed to examine the correlation of PTEN expression with the expression of cell cycle regulators and with clinicopathological parameters in endometrioid adenocarcinoma of the uterine corpus. METHODS: Tissue samples of 117 endometrioid adenocarcinomas in addition to those of 19 normal endometria and 20 endometrial hyperplasias were used for the study. Immunohistochemical staining for PTEN protein was performed with the labeled streptavidin-biotin method on formalin-fixed and paraffin-embedded tissue samples. PTEN expression was represented as the staining score. RESULTS: Immunohistochemistry showed that the nuclei of cells were positive for PTEN. The PTEN staining score of normal endometrium was significantly higher in the proliferative phase than in the secretory phase. The scores of various endometrial hyperplasias were not significantly different from each other, regardless of the type of hyperplasia. The PTEN staining scores of endometrioid adenocarcinomas were 7.6+/-5.2 in G1, 9.6+/-5.2 in G2, and 11.9+/-3.7 in G3, and increased significantly as the histological grade increased. PTEN staining score was not significantly correlated with clinicopathological parameters such as FIGO stage, myometrial invasion, lymph-vascular space invasion (LVSI), lymph node metastasis or group, but was significantly correlated with labeling indices (LIs) of cell cycle regulators such as Ki-67, cdk2, cyclin A, cyclin D1, cyclin E, p27, and p53. The PTEN staining score of p53-wild cases was significantly lower than that of p53-mutant ones, but there was no significant difference of the score in cases with different PTEN gene status. PTEN expression was significantly lower in cases with both high levels of estrogen receptor and progesterone receptor. CONCLUSION: PTEN protein expression was decreased in well-differentiated and less growth-aggressive endometrial carcinoma with wild-type p53 gene and high levels of ER and PR. This suggests that disturbed PTEN expression occurs in an early phase of the tumorigenesis of well-differentiated endometrial carcinoma.

Adenocarcinoma↗
PubMed2004

Functional definition of relevant epitopes on the tumor suppressor PTEN protein.

The binding of PTEN to PDZ-domain-containing proteins appears to play an important role in the control of cell growth, motility and apoptosis. In turn, this binding can be abrogated by cleavage of the PTEN C-terminal region by caspase-3. We have generated and characterized monoclonal antibodies (mAb) directed against distinct epitopes at the C-terminal region of PTEN, and used them to define protein-binding epitopes on PTEN and to study its cleavage by caspase-3. mAb directed against epitopes at the far C-terminus of PTEN blocked binding to PTEN cognate PDZ domains and did not recognize the caspase-3 cleaved PTEN fragments. On the other hand, mAb that recognized an epitope within the C2 domain of PTEN did not prevent binding to PDZ domains, but could detect the caspase-3 cleaved PTEN fragments. The analysis of PTEN cleavage by caspase-3 revealed that the lipid phosphatase activity of PTEN controls its own degradation by interfering with the PI3-K anti-apoptotic activity. Our results define protein-binding sites on the PTEN tumor suppressor at the immunochemical level, and suggest a regulatory link between PTEN phosphatase activity, caspase-3 sensitivity and PTEN-protein interactions.

Binding Sites↗
PubMed2002

PTEN mutational spectra, expression levels, and subcellular localization in microsatellite stable and unstable colorectal cancers.

PTEN on 10q23.3 encodes a dual-specificity phosphatase that negatively regulates the phosphoinositol-3-kinase/Akt pathway and mediates cell-cycle arrest and apoptosis. Germline PTEN mutations cause Cowden syndrome and a range of several different hamartoma-tumor syndromes. Hereditary nonpolyposis colon cancer (HNPCC) syndrome is characterized by germline mutations in the mismatch repair (MMR) genes and by microsatellite instability (MSI) in component tumors. Although both colorectal carcinoma and endometrial carcinoma are the most frequent component cancers in HNPCC, only endometrial cancer has been shown to be a minor component of Cowden syndrome. We have demonstrated that somatic inactivation of PTEN is involved in both sporadic endometrial cancers and HNPCC-related endometrial cancers but with different mutational spectra and different relationships to MSI. In the current study, we sought to determine the relationship of PTEN mutation, 10q23 loss of heterozygosity, PTEN expression, and MSI status in colorectal cancers (CRCs). Among 11 HNPCC CRCs, 32 MSI+ sporadic cancers, and 39 MSI- tumors, loss of heterozygosity at 10q23.3 was found in 0%, 8%, and 19%, respectively. Somatic mutations were found in 18% (2 of 11) of the HNPCC CRCs and 13% (4 of 32) of the MSI+ sporadic tumors, but not in MSI- cancers (P = 0.015). All somatic mutations occurred in the two 6(A) coding mononucleotide tracts in PTEN, suggestive of the etiological role of the deficient MMR. Immunohistochemical analysis revealed 31% (14 of 45) of the HNPCC CRCs and 41% (9 of 22) of the MSI+ sporadic tumors with absent or depressed PTEN expression. Approximately 17% (4 of 23) of the MSI- CRCs had decreased PTEN expression, and no MSI- tumor had complete loss of PTEN expression. Among the five HNPCC or MSI+ sporadic CRCs carrying frameshift somatic mutations with immunohistochemistry data, three had lost all PTEN expression, one showed weak PTEN expression levels, and one had mixed tumor cell populations with weak and moderate expression levels. These results suggest that PTEN frameshift mutations in HNPCC and sporadic MSI+ tumors are a consequence of mismatch repair deficiency. Further, hemizygous deletions in MSI- CRCs lead to loss or reduction of PTEN protein levels and contribute to tumor progression. Finally, our data also suggest that epigenetic inactivation of PTEN, including differential subcellular compartmentalization, occurs in CRCs.

Base Pair Mismatch↗
PubMed1999

Regulation of PTEN expression in neuronal apoptosis.

PTEN phosphatase is a tumor suppressor gene that dephosphorylates phosphatidylinositol phosphates. PTEN restrains the function of a major antiapoptotic and survival pathway involving phosphoinositide 3-kinase and Akt kinase. Our purpose was to find out whether apoptotic inducers affect the expression of PTEN in cerebellar granule neurons and neuroblastoma 2a cells (Neuro-2a). PTEN mRNA expression showed a major 5.5-kb and a lower abundance 2.5-kb transcripts. In Neuro-2a cells, serum withdrawal induced a prominent, continuous decrease both in 5.5- and 2.5-kb transcripts of PTEN mRNA. Simultaneously, the expression level of 56-kDa PTEN protein decreased in Neuro-2a cells. The decrease in PTEN expression precedes apoptotic changes observed after serum withdrawal. On the contrary, okadaic acid and etoposide only slightly affected the expression of PTEN although they induce a prominent apoptosis in Neuro-2a cells. In cerebellar granule neurons, okadaic acid treatment induced a prominent increase in PTEN mRNA expression after 6-h treatment, both at the 5.5- and 2.5-kb transcripts. The early response in PTEN mRNA expression disappeared in 5.5-kb transcripts already at 12 h and in the case of 2.5-kb transcripts it lasted up to 24 h. Potassium deprivation, known to induce apoptosis in cerebellar granule cells, did not affect PTEN mRNA expression but together with serum deprivation induced a clear decrease in the 5. 5-kb PTEN transcripts. It seems that the changes in PTEN expression level and neuronal apoptosis are not related to each other in general but the expression of PTEN phosphatase seems to regulate certain apoptotic signals affecting phosphoinositide 3-kinase function.

Animals↗
PubMed2000

Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases.

Pten (Phosphatase and tensin homolog deleted on chromosome 10) is a recently identified tumor suppressor gene which is deleted or mutated in a variety of primary human cancers and in three cancer predisposition syndromes [1]. Pten regulates apoptosis and cell cycle progression through its phosphatase activity on phosphatidylinositol (PI) 3,4,5-trisphosphate (PI(3,4,5)P(3)), a product of PI 3-kinase [2-5]. Pten has also been implicated in controlling cell migration [6], but the exact mechanism is not very clear. Using the isogenic Pten(+/+) and Pten(-/-) mouse fibroblast lines, here we show that Pten deficiency led to increased cell motility. Reintroducing the wild-type Pten, but not the catalytically inactive Pten C124S or lipid-phosphatase-deficient Pten G129E mutant, reduced the enhanced cell motility of Pten-deficient cells. Moreover, phosphorylation of the focal adhesion kinase p125(FAK) was not changed in Pten(-/-) cells. Instead, significant increases in the endogenous activities of Rac1 and Cdc42, two small GTPases involved in regulating the actin cytoskeleton [7], were observed in Pten(-/-) cells. Overexpression of dominant-negative mutant forms of Rac1 and Cdc42 reversed the cell migration phenotype of Pten(-/-) cells. Thus, our studies suggest that Pten negatively controls cell motility through its lipid phosphatase activity by down-regulating Rac1 and Cdc42.

Animals↗
PubMed2004

PTEN mutations are common in sporadic microsatellite stable colorectal cancer.

The tumour suppressor gene PTEN, located at chromosome sub-band 10q23.3, encodes a dual-specificity phosphatase that negatively regulates the phosphatidylinositol 3'-kinase (PI3 K)/Akt-dependent cellular survival pathway. PTEN is frequently inactivated in many tumour types including glioblastoma, prostate and endometrial cancers. While initial studies reported that PTEN gene mutations were rare in colorectal cancer, more recent reports have shown an approximate 18% incidence of somatic PTEN mutations in colorectal tumours exhibiting microsatellite instability (MSI+). To verify the role of this gene in colorectal tumorigenesis, we analysed paired normal and tumour DNA from 41 unselected primary sporadic colorectal cancers for PTEN inactivation by mutation and/or allelic loss. We now report PTEN gene mutations in 19.5% (8/41) of tumours and allele loss, including all or part of the PTEN gene, in a further 17% (7/41) of the cases. Both PTEN alleles were affected in over half (9/15) of these cases showing PTEN genetic abnormalities. Using immunohistochemistry, we have further shown that all tumours harbouring PTEN alterations have either reduced or absent PTEN expression and this correlated strongly with later clinical stage of tumour at presentation (P=0.02). In contrast to previous reports, all but one of the tumours with PTEN gene mutations were microsatellite stable (MSI-), suggesting that PTEN is involved in a distinct pathway of colorectal tumorigenesis that is separate from the pathway of mismatch repair deficiency. This work therefore establishes the importance of PTEN in primary sporadic colorectal cancer.

Alleles↗
PubMed2004

PTEN can inhibit in vitro organotypic and in vivo orthotopic invasion of human bladder cancer cells even in the absence of its lipid phosphatase activity.

Recent studies have found a higher frequency of the PTEN tumor-suppressor gene alterations in invasive bladder carcinoma than in superficial disease, suggesting that PTEN is important in this process. A role of PTEN in bladder cancer invasion is further suggested by the fact that PTEN is a regulator of cell motility, a necessary component of tumor invasion. However, it is unknown whether PTEN is mechanistically involved in 'in vivo' tumor invasion or merely an epiphenomenon and, if the former is true, whether this process is dependent on its protein or lipid phosphatase activities. To address these issues, we stably transfected several commonly used human bladder cancer cell lines with known invasive phenotypes with either wild-type PTEN constructs or those deficient in the lipid phosphatase (G129E) or both protein and lipid phosphatase (G129R) activities. Here we show that chemotaxis was inhibited by both the wild-type and G129E mutant of PTEN but not by G129R-transfected cells. Using a novel organotypic in vitro invasion assay, we evaluated the impact of wild-type and mutant PTEN transgene expression on the invasive ability of T24T, a human bladder cancer cell line with a functionally impaired PTEN. Results indicate that the G129E mutant blocks invasion as efficiently as wild-type PTEN transfection. In contrast to the wild-type gene, this mutant has no effect on cell clonogenicity in agar. To further establish the role of PTEN in tumor invasion, we evaluated vector- and PTEN-transfected T24T cells in an orthotopic in vivo assay that faithfully reproduces human disease. Microscopic examination of murine bladders at the completion of this experiment parallels the results obtained with the organotypic assay. Our results are the first demonstration: (1) that the inhibitory effects of PTEN on cell motility translate into suppression of in vivo invasion; (2) that PTEN can inhibit tumor invasion even in the absence of its lipid phosphatase activity; (3) how organotypic in vitro approaches can be used as surrogates of in vivo invasion allowing rapid dissection of molecular processes leading to this phenotype while reducing the number of animals used in research.

Amino Acid Substitution↗
PubMed2005

PTEN as an effector in the signaling of antimigratory G protein-coupled receptor.

PTEN, a tumor suppressor phosphatase, is important in the regulation of cell migration and invasion. Physiological regulation of PTEN (phosphatase and tensin homolog deleted on chromosome 10) by cell surface receptors has not been described. Here, we show that the bioactive lipid sphingosine 1-phosphate (S1P), which acts through the S1P2 receptor (S1P2R) G protein-coupled receptor (GPCR) to inhibit cell migration, utilizes PTEN as a signaling intermediate. S1P2R inhibition of cell migration is abrogated by dominant-negative PTEN expression. S1P was unable to efficiently inhibit the migration of Pten(DeltaloxP/DeltaloxP) mouse embryonic fibroblasts; however, the antimigratory effect was restored upon the expression of PTEN. S1P2R activation of Rho GTPase is not affected in Pten(DeltaloxP/DeltaloxP) cells, and dominant-negative Rho GTPase reversed S1P inhibition of cell migration in WT cells but not in Pten(DeltaloxP/DeltaloxP) cells, suggesting that PTEN acts downstream of the Rho GTPase. Ligand activation of the S1P2R receptor stimulated the coimmunoprecipitation of S1P2R and PTEN. Interestingly, S1P2R signaling increased PTEN phosphatase activity in membrane fractions. Furthermore, tyrosine phosphorylation of PTEN was stimulated by S1P2R signaling. These data suggest that the S1P2R receptor actively regulates the PTEN phosphatase by a Rho GTPase-dependent pathway to inhibit cell migration. GPCR regulation of PTEN maybe a general mechanism in signaling events of cell migration and invasion.

Animals↗
PubMed1999

PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway.

The tumor suppressor PTEN is a phosphatase with sequence homology to tensin. PTEN dephosphorylates phosphatidylinositol 3,4, 5-trisphosphate (PIP3) and focal adhesion kinase (FAK), and it can inhibit cell growth, invasion, migration, and focal adhesions. We investigated molecular interactions of PTEN and FAK in glioblastoma and breast cancer cells lacking PTEN. The PTEN trapping mutant D92A bound wild-type FAK, requiring FAK autophosphorylation site Tyr397. In PTEN-mutated cancer cells, FAK phosphorylation was retained even in suspension after detachment from extracellular matrix, accompanied by enhanced PI 3-K association with FAK and sustained PI 3-K activity, PIP3 levels, and Akt phosphorylation; expression of exogenous PTEN suppressed all five properties. PTEN-mutated cells were resistant to apoptosis in suspension, but most of the cells entered apoptosis after expression of exogenous PTEN or wortmannin treatment. Moreover, overexpression of FAK in PTEN-transfected cells reversed the decreased FAK phosphorylation and PI 3-K activity, and it partially rescued PIP3 levels, Akt phosphorylation, and PTEN-induced apoptosis. Our results show that FAK Tyr397 is important in PTEN interactions with FAK, that PTEN regulates FAK phosphorylation and molecular associations after detachment from matrix, and that PTEN negatively regulates the extracellular matrix-dependent PI 3-K/Akt cell survival pathway in a process that can include FAK.

Cell Adhesion Molecules↗
PubMed2006

Distinct expression profiles for PTEN transcript and its splice variants in Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome.

Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome (BRRS) are autosomal dominant hamartoma syndromes. Germline PTEN mutations have been associated with 85% of CS cases and 65% of BRRS cases and also with other disorders, which are collectively referred to as the "PTEN hamartoma tumor syndrome." The human PTEN gene has been previously found to express two naturally occurring splice variants (SVs). Recently, we identified eight novel naturally occurring PTEN SVs that result in different downstream signaling effects: SV3a, SV3b, SV3c (inclusion of various lengths of intron 3 3' of exon 3), SV5a, SV5b, SV5c, SV5d (inclusion of various lengths of intron 5 3' of exon 5), and SV Delta Ex6 (deletion of exon 6). We therefore sought to characterize the relative expression of 5', middle, and 3' full-length PTEN mRNA (FL-PTEN) and also of these eight PTEN SVs in 85 (65 female and 20 male) patients with CS/BRRS (with or without PTEN mutations) compared with 27 controls, using a SYBR green quantitative polymerase chain reaction method. Significantly reduced FL-PTEN levels were found in the probands, compared with those of controls (P < .01). Apart from FL-PTEN, SV3a is the most consistently relatively underexpressed in patients compared with controls. The patients showed relative underexpression of SV3a and SV3b and overexpression of SV5b (P = .005, P = .02, and P = .04, respectively). Indeed, there appears to be an SV expressional genotype-phenotype correlation in which the SV expressional profiles are distinct among CS, CS-like, and BRRS. The reduced FL-PTEN transcript expression, associated with differential expression of PTEN SVs, regardless of PTEN mutation status, supports the concept that modulation of PTEN inactivation may also occur at the transcription level influencing the specific phenotypes seen in these syndromes.

Base Sequence↗
PubMed1999

PTEN is inversely correlated with the cell survival factor Akt/PKB and is inactivated via multiple mechanismsin haematological malignancies.

PTEN is a novel tumour suppressor gene that encodes a dual-specificity phosphatase with homology to adhesion molecules tensin and auxillin. It recently has been suggested that PTEN dephosphorylates phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3, 4,5)P3], which mediates growth factor-induced activation of intracellular signalling, in particular through the serine-threonine kinase Akt, a known cell survival-promoting factor. PTEN has been mapped to 10q23.3, a region disrupted in several human tumours including haematological malignancies. We have analysed PTEN in a series of primary acute leukaemias and non-Hodgkin's lymphomas (NHLs) as well as in cell lines. We have also examined whether a correlation could be found between PTEN and Akt levels in these samples. We show here that the majority of cell lines studied carries PTEN abnormalities. At the structural level, we found mutations and hemizygous deletions in 40% of these cell lines, while a smaller number of primary haematological malignancies, in particular NHLs, carries PTEN mutations. Moreover, one-third of the cell lines had low PTEN transcript levels, and 60% of these samples had low or absent PTEN protein, which could not be attributed to gene silencing by hypermethylation. In addition, we found that PTEN and phosphorylated Akt levels are inversely correlated in the large majority of the examined samples. These findings suggest that PTEN plays a role in the pathogenesis of haematological malignancies and that it might be inactivated through a wider range of mechanisms than initially considered. The finding that PTEN levels inversely correlate with phosphorylated Akt supports the hypothesis that PTEN regulates PtdIns(3,4,5)P3and suggests a role for PTEN in apoptosis.

Blotting, Northern↗
PubMed2006

PTEN autoregulates its expression by stabilization of p53 in a phosphatase-independent manner.

PTEN (phosphatase and tensin homologue, deleted on chromosome 10) is a tumor suppressor with dual phosphatase activity and mutations of its gene, PTEN, have been associated with many sporadic cancers and heritable neoplasia syndromes, including Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome. However, accumulating evidence now shows that PTEN may have novel functions other than as a phosphatase. In the present study, we show that PTEN is able to autoregulate its expression through the stabilization of another tumor suppressor p53. We further show that PTEN enhances p53 transactivation, a relationship that requires the interaction between PTEN and p53 and is PTEN phosphatase independent. We show that cell lines from Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome patients with germ line PTEN promoter mutations in the vicinity of the p53-binding motifs have altered p53 regulation. This seems to be due to reduced PTEN stability and decreased PTEN-p53 interactions. Our data provide clues to better understand the regulation of PTEN expression and the possible mechanisms of the pathogenesis of the subset of Cowden syndrome individuals with germ line promoter variation and who lack mutations in the PTEN coding region and splice sites. Importantly, this mechanism also holds for those sporadic tumors that lack intragenic mutations but have hemizygous deletion of PTEN, which includes the promoter region as manifested by loss-of-heterozygosity of 10q markers. The importance of our observations is underlined by the broad spectrum of neoplasias that harbor somatic PTEN or p53 alterations, or both.

Gene Expression Regulation↗
PubMed2005

Reduced expression of PTEN protein and its prognostic implications in invasive ductal carcinoma of the breast.

OBJECTIVE: The PTEN tumor suppressor gene has been demonstrated to be inactivated in a variety of human tumors. In breast cancer, the PTEN gene mutation is not commonly found whereas loss of heterozygosity affecting the PTEN locus is frequently found. The aim of this study was to analyze PTEN protein expression in breast cancer and to evaluate the prognostic significance of PTEN protein expression. METHODS: Paraffin-embedded sections ofinvasive ductal carcinoma of the breast were immunohistochemically stained for PTEN protein expression in 236 breast cancers. The immunohistochemical expression of breast cancer cells was judged to be either normal or reduced compared with the PTEN protein expression of the normal mammary gland. RESULTS: The expression of PTEN protein was found to have decreased in 67 (28%) of 236 breast cancers. The reduced expression correlated with lymph node metastasis (p = 0.0371), but not with tumor size, nuclear grade, MIB-1 counts or p53 protein expression. Univariate analysis indicated that patients with a reduced PTEN expression had a shorter disease-free survival (DFS) than those with a normal PTEN expression (p = 0.0174). Univariate analyses also determined tumor size, lymph node metastases, nuclear grade, MIB-1 counts, p53 protein as well as PTEN protein expression to be significant factors for DFS, while multivariate analysis determined lymph node metastases and the MIB-1 counts to be independent significant factors for DFS. CONCLUSIONS: The inactivation of PTEN, demonstrated by a reduced expression of PTEN protein by immunohistochemistry, was found in about one third of all breast cancers. The reduced expression of PTEN protein correlated with lymph node metastases and a worse prognosis in the patients with breast cancer.

Adult↗
PubMed2000

Profiling the downstream genes of tumor suppressor PTEN in lung cancer cells by complementary DNA microarray.

The phosphatase and tensin homology deleted on chromosome 10 (PTEN) is a tumor suppressor gene with sequence homology to tyrosine phosphatases and the cytoskeletal proteins tensin and auxilin. PTEN has recently been shown to inhibit cell migration and the spreading and formation of focal adhesions. This study investigated the role of PTEN in carcinoma invasion in a lung-cancer cell line and examined the downstream genes regulated by PTEN. We have previously established a cell-line model in human lung adenocarcinoma with different invasive abilities and metastatic potentials. Examining PTEN gene expression in these cell lines, we found that a homozygous deletion in exon 5 is associated with high invasive ability. We then constructed stable constitutive and inducible wild-type PTEN-overexpressed transfectants in the highly invasive cell line CL(1-5). We found that an overexpression of PTEN can inhibit invasion in lung cancer cells. To further explore the downstream genes regulated by PTEN, a high-density complementary DNA (cDNA) microarray technique was used to profile gene changes after PTEN overexpression. Our results indicate a panel of genes that can be modulated by PTEN. PTEN overexpression downregulated genes, including integrin alpha(6), laminin beta(3), heparin-binding epidermal growth factor-like growth factor, urokinase-type plasminogen activator, myb protein B, Akt2, and some expressed sequence tag (EST) clones. In contrast, PTEN overexpression upregulated protein phosphatase 2A1B, ubiquitin protease (unph), secreted phosphoprotein 1, leukocyte elastase inhibitor, nuclear factor-kappaB, cyclic adenosine monophosphate response element binding protein, DNA ligase 1, heat shock protein 90, and some EST genes. Northern hybridization and flow cytometry analysis also confirmed that PTEN overexpression results in the reduced expression of the integrin alpha(6) subunit. The results of this study indicate that PTEN overexpression may inhibit lung cancer invasion by downregulation of a panel of genes including integrin alpha(6). The cDNA microarray technique may be an effective tool to study the downstream function of a tumor suppressor gene.

Adenocarcinoma↗
PubMed2006

Atheroma development in apolipoprotein E-null mice is not affected by partial inactivation of PTEN.

PTEN is a dual-specificity phosphatase that has been shown to inhibit vascular smooth muscle cell (VSMC) proliferation and migration, two key events in the ethiopathogenesis of atherosclerosis. Adenovirus-mediated PTEN overexpression inhibited the formation of vascular obstructive lesions induced by mechanical injury of the vessel wall. In this study, we investigated whether PTEN protects against atheroma formation in apolipoprotein E-null mice (apoE-/-), a widely used animal model characterized by the development of hypercholesterolemia and atherosclerosis. We examined atheroma development in the aorta of apoE-/- mice with an intact Pten gene and apoE-/- mice lacking one allele of Pten (Pten(+/-)apoE-/-) that were challenged for six weeks with an atherogenic diet. Compared with apoE-/- controls, Western blot analysis of arterial cell lysates from Pten(+/-)apoE-/- mice revealed a decrease in PTEN expression. This correlated with increased phosphorylation of AKT, thus demonstrating that Pten inactivation in Pten(+/-)apoE-/- mice has functional consequences. However, the extent of atherosclerosis was undistinguishable in both groups of fat-fed mice. Likewise, the atheroma of Pten(+/-)apoE-/- and apoE-/- mice displayed similar VSMC content, cellularity and rates of proliferation and apoptosis. Thus, in spite of the cytostatic and antimigratory activities of PTEN, and in contrast to previous studies demonstrating that Pten is haplo-insufficient for tumor suppression, our results demonstrate that atherosclerosis in hypercholesterolemic mice is not aggravated by partial inactivation of Pten.

Animals↗
PubMed2000

Functional evaluation of PTEN missense mutations using in vitro phosphoinositide phosphatase assay.

The tumor suppressor gene PTEN is frequently mutated in diverse human cancers and in autosomal dominant cancer predisposition disorders. Recent studies have shown that the lipid phosphatase activity of PTEN is critical for its tumor suppressor function and that PTEN negatively regulates the phosphatidylinositol 3'-kinase-protein kinase B pathway. Although more than half of PTEN mutations result in protein truncation, a significant fraction of PTEN mutations are missense mutations. To examine whether tumor-derived and germ-line-derived missense mutations inactivate PTEN lipid phosphatase function, we constructed 42 distinct types of PTEN missense mutations and expressed them in Escherichia coli. The purified (His)6-tagged PTEN proteins were tested for their ability to dephosphorylate inositol 1,3,4,5-tetrakisphosphate and phosphatidylinositol 3,4,5-triphosphate. In addition, we examined the effect of mutant PTENs on the ability of PTEN to bind to the phospholipid membrane. The results revealed that the majority of PTEN missense mutations [38 of 42 (90%)] eliminated or reduced phosphatase activity and that all of the mutations examined had no effect on the membrane binding activity of PTEN. Our study indicated that phosphoinositide phosphatase activity is important for the tumor suppressor function of PTEN and that there may be other mechanisms of PTEN inactivation that are not monitored by in vitro phosphatase assay and in vitro membrane binding assay.

Escherichia coli↗
PubMed2000

High incidence of breast and endometrial neoplasia resembling human Cowden syndrome in pten+/- mice.

PTEN is one of the most commonly mutated tumor suppressor genes in human cancer. PTEN mutations have been implicated in the development of a variety of human neoplasia, including high-grade glioblastoma, prostate, breast, endometrial, and thyroid carcinoma. Germ-line mutations of PTEN cause Cowden's syndrome (CS), a multiple hamartoma condition resulting in increased susceptibility for the development of cancer. When more than 6 months old, pten+/- mice develop a range of tumors, partially resembling the spectrum of neoplasia observed in CS patients. One-half (32 of 65) of pten+/- females developed breast tumors, whereas all (65 of 65) of the females had endometrial hyperplasia, and there was a high incidence (14 of 65) of endometrial cancer. Hamartoamous tumors of the gastrointestinal tract, as well as prostate and adrenal neoplasia, were also frequently observed. Significantly, the spectrum of neoplasia observed in pten+/- mice partially overlaps with the types of tumors frequently detected in CS patients. The majority of tumors in pten+/- mice exhibit loss of heterozygosity at the pten locus, which indicates the importance for loss of PTEN function in tumor formation. Consistent with the role of PTEN in negative regulation of PKB/Akt phosphorylation and activity, pten loss of heterozygosity is accompanied by hyperphosphorylation of PKB/Akt in tumors. Taken together, our results establish pten+/- mice as an excellent animal model system for the investigation of PTEN-related hamartoma syndromes, as well as the role of PTEN in breast and endometrial carcinogenesis.

Aging↗
PubMed2002

PI3 kinase blockade by Ad-PTEN inhibits invasion and induces apoptosis in RGP and metastatic melanoma cells.

BACKGROUND: Melanoma is an aggressive tumor with a propensity to rapidly metastasize. The PTEN gene encodes a phosphatase with an unusual dual specificity for proteins and lipids. Mutations of PTEN have been found in various human cancers, including glioblastoma, prostate, breast, lung, and melanoma. Here we investigate in vitro the effects of blocking PI3K signaling using adenoviral-delivered PTEN (Ad-PTEN) in cell lines derived from both early- and late-stage melanoma. MATERIALS AND METHODS: Ad-PTEN transduced melanoma cell lines or normal cells were assayed for cell death, apoptosis, gene expression, invasion and migration, and regulation of angiogenesis. RESULTS: The PTEN locus from RGP and metastatic melanoma cell lines was sequenced; no coding region mutations were found. Adenoviral transfer of PTEN into melanoma cells containing wild-type PTEN alleles led to tumor-specific apoptosis and growth inhibition, with coordinate inhibition of AKT phosphorylation. Ad-PTEN suppressed cell migration by metastatic melanoma cells with concomitant increase in the level of cell surface E-cadherin. Immunohistochemical and confocal analyses localized PTEN to the cytoplasm and demonstrated enrichment at the cell membrane. Ad-PTEN inhibited angiogenesis as demonstrated by the tube formation assay using human vascular endothelial cells. CONCLUSIONS: These studies indicate that Ad-PTEN can inhibit tumor cells via multiple mechanisms and has pro-apoptotic, anti-metastatic, and anti-angiogenic properties. Thus, PI3K blockade via Ad-PTEN may be a promising approach for the treatment of early- and late-stage melanoma, even in tumors that do not harbor PTEN mutations.

Adenoviridae↗