Tumorigenesis facilitated by Pten deficiency in the skin: evidence of p53-Pten complex formation on the initiation phase.
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Activation of synovial fibroblasts in rheumatoid arthritis: lack of Expression of the tumour suppressor PTEN at sites of invasive growth and destruction.
AIMS: PTEN is a novel tumour suppressor which exhibits tyrosine phosphatase activity as well as homology to the cytoskeletal proteins tensin and auxilin. Mutations of PTEN have been described in several human cancers and associated their invasiveness and metastatic properties. Although not malignant, rheumatoid arthritis synovial fibroblasts (RA-SF) exhibit certain tumour-like features such as attachment to cartilage and invasive growth. In the present study, we analyzed whether mutant transcripts of PTEN were present in RA-SF. In addition, we used in situ hybridization to study the expression of PTEN messenger (m)RNA in tissue samples of RA and normal individuals as well as in cultured RA-SF and in the severe combined immunodeficiency (SCID) mouse model of RA. Synovial tissue specimens were obtained from seven patients with RA and from two nonarthritic individuals. Total RNA was isolated from synovial fibroblasts and after first strand complementary (c)DNA synthesis, polymerase chain reaction (PCR) was performed to amplify a 1063 base pair PTEN fragment that encompassed the coding sequence of PTEN including the phosphatase domain and all mutation sites described so far. The PCR products were subcloned in Escherichia coli, and up to four clones were picked from each plate for automated sequencing. For in situ hybridization, digoxigenin-labelled PTEN-specific RNA probes were generated by in vitro transcription. For control in situ hybridization, a matrix metalloproteinase (MMP)-2-specific probe was prepared. To investigate the expression of PTEN in the absence of human macrophage or lymphocyte derived factors, we implanted RA-SF from three patients together with normal human cartilage under the renal capsule of SCID mice. After 60 days, mice were sacrificed, the implants removed and embedded into paraffin. RESULTS: PCR revealed the presence of the expected 1063 base pair PTEN fragment in all (9/9) cell cultures (Fig.1). No additional bands that could account for mutant PTEN variants were detected. Sequence analysis revealed 100% homology of all RA-derived PTEN fragments to those from normal SF as well as to the published GenBank sequence (accession number U93051). However, in situ hybridization demonstrated considerable differences in the expression of PTEN mRNA within the lining and the sublining layers of RA synovial membranes. As shown in Figure 2a, no staining was observed within the lining layer which has been demonstrated to mediate degradation of cartilage and bone in RA. In contrast, abundant expression of PTEN mRNA was found in the sublining of all RA synovial tissues (Figs 2a and b). Normal synovial specimens showed homogeneous staining fo PTEN within the thin synovial membrane (Fig. 2c). In situ hybridization using the sense probe gave no specific staining (Fig. 2d). We also performed in situ hybridization on four of the seven cultured RA-SF and followed one cell line from the first to the sixth passage. Interestingly, only 40% of cultured RA-SF expressed PTEN mRNA (Fig. 3A), and the proportion of PTEN expressing cells did not change throughout the passages. In contrast, control experiments using a specific RNA probe fo MMP-2 revealed mRNA expression by nearly all cultured cells (Fig. 3B). As seen before, implantation of RA-SF into the SCID mice showed considerable cartilage degradation. Interestingly, only negligible PTEN expression was found in those RA-SF aggressively invading the cartilage (Fig. 3c). In situ hybridization for MMP-2 showed abundant staining in these cells (Fig. 3d). DISCUSSION: Although this study found no evidence for mutations of PTEN in RA synovium, the observation that PTEN expression is lacking in the lining layer of RA synovium as well as in more that half of cultured RA-SF is of interest. It suggests that loss of PTEN function may not exclusively be caused by genetic alterations, yet at the same time links the low expression of PTEN to a phenotype of cells that have been shown to invade cartilage aggressively. It has been proposed that the tyrosine phosphatase activity of counteracting th actions o protein tyrosine kinases. As some studies have demonstrated an upregulation of tyrosine kinase activity in RA synovial cells, it might be speculated that the lack of PTEN expression in aggressive RA-SF contributes to the imbalance of tyrosine kinases and phosphatases in this disease. However, the extensive amino-terminal homology of the predicted protein to the cytoskeletal proteins tensin and auxilin suggests a complex regulatory function involving cellular adhesion molecules and phosphatase-mediated signalling. The tyrosine phosphatase TEP1 has been shown to be identical to the protein encoded by PTEN, and gene transcription of TEP1 has been demonstrated to be downregulated by transforming growth factor (TGF)-beta. Therefore, it could be hypothesized that TGF-beta might be responsible for the downregulation of PTEN. Low expression of PTEN may belong to the features that distinguish between the activated phenotype of RA-SF and the sublining, proliferating but nondestructive cells.
Animals↗
[Prokaryotic expression of functional PTEN in Escherichia coli and preparation of polyclonal antibody].
PTEN, a dual-specificity phosphatase, exerts its tumor-suppressive effects through the inhibition of cell cycle progression and cell immigration, therefore could be an important candidate for tumor-suppression. As study on prokaryotic expression of PTEN and its anti-tumor functions has not been reported, the present study aims at an efficient expression of functional PTEN in Escherichia coli and the investigation of its tumor-suppression activity. PTEN cDNA cloned in our lab previously was recombined into prokaryotic expression vector pET-44a(+) to construct pET-PTEN (pEP) and pET-Nus-PTEN (pENP). PTEN was fused with 6 x His tag in pEP, and with Nus in pENP, which could be useful for a stable and soluble expression. The recombinant vectors were transformed into both BL21 (DE3) (BL) and Rosetta-gami (DE3) pLysS (RG). The former is a normal expression host while the latter is optimized for expression of eukaryotic genes and folding of target proteins. On the induction of 0.5mmol/L IPTG, 55kD and 118kD specific protein bands were observed, corresponding to His-PTEN and Nus-PTEN fusion proteins, respectively. Western blot analysis showed the recombinant fusion proteins could react with PTEN polyclonal antibody. The recombinant HTEN was expressed both in soluble fraction and inclusion body. Higher expression levels of recombinant PTEN were obtained in BL (His-PTEN: 10.3%; NusA-PTEN: 18.7%), whereas the higher percentages of soluble recombinant proteins were observed in RG (His-PTEN: 4.7%; Nus-PTEN: 6.6%). The obtained recombinant fusion proteins were purified by affinity chromatography and were showed to be homogeneous in SDS-PAGE. In tumor-suppression experiments, His-PTEN was proved to have significant inhibition on growth of mice solid tumor with an inhibitory ratio of 58.76%, and on the proliferation of DU-145 tumor cells with an inhibitory ratio of 46.16%. The cell cycle progression of DU-145 tumor cells was also arrested from G0/G1 to S phase. His-PTEN from RG was proved to have significantly higher tumor-suppression activity than that from BL, indicating that there may be some advantages for eukaryotic genes to be expressed in the former host. This is the report of functional recombinant PTEN expressed in Escherichia coli. Purified His-PTEN was used for immunizing Kunming mice, and ascitic polyclonal antibodies raised against His-PTEN were generated using sarcoma 180 cells. At 1:2000 dilution, the antibodies could interact with PTEN by western blot. The present study has laid a foundation for application of PTEN in cancer therapy.
Animals↗
PTEN methylation and expression in glioblastomas.
The tumor suppressor gene PTEN on chromosome 10q23.3 regulates the Akt signaling pathway and modulates cell growth and apoptosis. The PTEN gene is mutated in 20-40% of glioblastomas. In this study, we assessed whether loss of PTEN expression is also caused epigenetically. Methylation-specific PCR revealed that CpG islands of the PTEN promoter were methylated in 27 of 77 (35%) glioblastomas and in 4 of 11 (36%) glioblastoma cell lines. Only two glioblastomas showed loss of PTEN immunoreactivity in the entire biopsy; both had a missense PTEN mutation and LOH at the PTEN locus, but lacked PTEN methylation. In biopsy specimens with focal loss of PTEN expression, DNA samples extracted from microdissected foci showed PTEN methylation only in areas with loss of PTEN expression. These results suggest that PTEN methylation occurs frequently in glioblastomas and may be associated with focal loss of PTEN expression. However, the correlation between PTEN methylation, PTEN mutations, LOH at the PTEN locus, and loss of PTEN protein expression was inconsistent. Possible reasons for discrepancies between gene status and protein expression include differences in the biological effect of specific PTEN mutations and the possibility that the processed PTEN pseudogene on 9p21 is expressed in glioblastomas and co-reacts with the PTEN antibody.
Adult↗
Mutation and expression analyses reveal differential subcellular compartmentalization of PTEN in endocrine pancreatic tumors compared to normal islet cells.
The pathogenesis of sporadic endocrine pancreatic tumors (EPTs) is still primarily unknown. Comparative genomic hybridization studies revealed loss of 10q in a significant number (nine of 31) of EPTs. The tumor suppressor gene PTEN lies on 10q23, and so, is a candidate to play some role in EPT pathogenesis. Germline PTEN mutations are found in Cowden and Bannayan-Riley-Ruvalcaba syndromes, whereas somatic mutations and deletions are found in a variety of sporadic cancers. The mutation and expression status of PTEN in EPTs has not yet been examined. Mutation analysis of the entire coding region of PTEN including splice sites was performed in 33 tumors, revealing one tumor with somatic L182F (exon 6). Loss of heterozygosity of the 10q23 region was detected in eight of 15 informative malignant (53%) and in none of seven benign EPTs. PTEN expression was assessed in 24 available EPTs by immunohistochemistry using a monoclonal anti-PTEN antibody. Of these 24, 23 tumors showed strong immunoreactivity for PTEN. Only the EPTs with PTEN mutation lacked PTEN protein expression. Although normal islet cells always exhibited predominantly nuclear PTEN immunostaining, 19 of 23 EPTs had a predominantly cytoplasmic PTEN expression pattern. Exocrine pancreatic tissue was PTEN-negative throughout. PTEN mutation is a rare event in malignant EPTs and PTEN protein is expressed in most (23 of 24) EPTs. Thus, intragenic mutation or another means of physical loss of PTEN is rarely involved in the pathogenesis of EPTs. Instead, either an impaired transport system of PTEN to the nucleus or some other means of differential compartmentalization could account for impaired PTEN function. Loss of heterozygosity of the 10q23 region is a frequent event in malignant EPTs and might suggest several hypotheses: a different tumor suppressor gene in the vicinity of PTEN might be principally involved in EPT formation; alternatively, 10q loss, including PTEN, seems to be associated with malignant transformation, but the first step toward neoplasia might involve altered subcellular localization of PTEN.
Adult↗
Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels, but not associated with p27 and cyclin D1 expression, in primary epithelial ovarian carcinomas.
PTEN (MMAC1/TEP1), a tumor suppressor gene on chromosome subband 10q23.3, is variably mutated and/or deleted in a variety of human cancers. Germline mutations in PTEN, which encode a dual-specificity phosphatase, have been implicated in at least two hamartoma tumor syndromes that exhibit some clinical overlap, Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome. Among several series of ovarian cancers, the frequency of loss of heterozygosity (LOH) of markers flanking and within PTEN, is approximately 30 to 50%, and the somatic intragenic PTEN mutation frequency is <10%. In this study, we screened primary adenocarcinomas of the ovary for LOH of polymorphic markers within and flanking the PTEN gene and for intragenic mutations of the PTEN gene and compared them to PTEN expression using immunohistochemistry. Furthermore, we sought to detect the expression of the presumed downstream targets of PTEN, such as P-Akt, p27, and cyclin D1 by immunohistochemistry. LOH at 10q23 was observed in 29 of 64 (45%) cases. Of the 117 samples, 6 somatic intragenic PTEN mutations, 1 germline mutation, and 1 novel polymorphism were found in 7 (6%) patients. Immunostaining of 49 ovarian cancer samples revealed that 13 (27%) were PTEN immunostain-negative, 25 (51%) had reduced staining, and the rest (22%) were PTEN expression-positive. Among the 44 informative tumors assessed for 10q23 LOH and PTEN immunostaining, there was an association between 10q23 LOH and decreased or absent staining (P = 0.0317). Of note, there were five (11%) tumors with neither mutation nor deletion that exhibited no PTEN expression and 10 (25%) others without mutation or deletion but had decreased PTEN expression. Among the 49 tumors available for immunohistochemistry, 28 (57%) showed P-Akt-positive staining, 24 (49%) had decreased p27 staining, and cyclin D1 was overexpressed in 35 (79%) cases. In general, P-Akt expression was inversely correlated with PTEN expression (P = 0.0083). These data suggest that disruption of PTEN by several mechanisms, allelic loss, intragenic mutation, or epigenetic silencing, all contribute to epithelial ovarian carcinogenesis, and that epigenetic silencing is a significant mechanism. The Akt pathway is prominently involved, but clearly not in all cases. Surprisingly, despite in vitro demonstration that p27 and cyclin D1 lies downstream of PTEN and Akt, there was no correlation between p27 and cyclin D1 expression and PTEN or P-Akt status. Thus, in vivo, although PTEN and Akt play a prominent role in ovarian carcinogenesis, p27 and cyclin D1 might not be the primary downstream targets. Alternatively, these observations could also suggest that pathways involving other than Akt, p27 and cyclin D1 that lie downstream of PTEN play roles in ovarian carcinogenesis.
Carcinoma↗
Alteration of PTEN expression in endometrial carcinoma is associated with down-regulation of cyclin-dependent kinase inhibitor, p27.
AIMS: PTEN is a recently identified tumour suppressor inactivated in a wide variety of human cancers, including endometrial cancers. Mutation of the PTEN tumour suppressor gene has been reported in approximately 50-83% of endometrial adenocarcinoma. Despite this fact, study of the expression of PTEN protein in human tumours is limited. PTEN protein functions as a tumour suppressor by regulating the cell cycle and survival through signal transduction pathway. PTEN protein was considered to have a dual-specificity phosphatase activity, but it is now known that its principal physiological activity is mainly derived from its lipid phosphatase activity. The cyclin-dependent kinase inhibitor, p27, has been suggested as a downstream target of cell cycle arrest of PTEN in various in vitro studies. In this study, we evaluated the alteration of PTEN protein expression in endometrial carcinoma and assessed its relationship to the expression of p27, the presumed downstream target of PTEN. METHODS AND RESULTS: Immunohistochemical staining was performed on 66 cases of endometrial carcinoma including 61 endometrioid type and five serous type, using antibodies to PTEN and p27. Loss or decrease of PTEN expression was observed in 66% (40/61 cases) of uterine endometrioid carcinoma, whereas most uterine serous carcinoma (4/5 cases) showed intense PTEN expression. Four (30%) of 13 endometrial hyperplasia synchronous with endometrioid carcinoma demonstrated complete loss of PTEN expression. All endometrioid carcinoma synchronous with PTEN-negative endometrial hyperplasia showed loss of PTEN expression. Alteration of PTEN expression was not correlated with histological grade or stage. Decreased immunoreactivity of p27 was found in 48 cases (79%) of 61 endometrioid carcinoma, and 76% (36 cases) of them also showed loss or decrease of PTEN expression. Four of five uterine serous carcinoma revealed strong p27 immunoreactivity, all of which showed intense PTEN expression. A positive correlation between PTEN and p27 expression was statistically significant (Mantel-Haenszel chi2 test, P=0.001). Immunoreactivity of p27 was not related to histological grade and clinical stage. CONCLUSION: These results show that PTEN and p27 are differentially expressed in endometrioid type carcinoma compared with those of the serous type, and suggest that the cyclin-dependent kinase inhibitor, p27, is a downstream target of PTEN-dependent cell cycle arrest in endometrial carcinoma.
Carcinoma, Endometrioid↗
[Research on the mechanisms of PTEN gene inactivation in ovarian cancer].
OBJECTIVE: To investigate the mechanisms of PTEN gene inactivation starting from DNA, mRNA and protein levels in ovarian cancers. METHODS: Tumor tissue samples were obtained from 48 patients with epithelial ovarian cancers. Using four polymorphic markers (D10s541, D10s583, D10s1687 and D10s2491) within and flanking the PTEN gene located in chromosome 10q 23.3, polymerase chain reaction (PCR) and loss of heterozygosity (LOH) were introduced to examine LOH of PTEN gene; PCR-single strand conformation polymorphism (PCR-SSCP) was introduced to examine mutations of the fifth, sixth, seventh, and eighth exons of PTEN. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (SP method) were applied to detect PTEN mRNA and PTEN protein expressions, respectively. RESULTS: LOH of PTEN gene was observed in 19 of 48 (39.6%) ovarian cancers. PTEN mutations were found only in 2 (4.2%) of the cases. Absence of PTEN mRNA expression was 18.8% (9 of 48). Immunostaining of 48 cancer samples revealed that 13 (27.1%) were PTEN immunostain negative. Of these 13 samples, only 2 (15.4%) had structural, biallelic inactivation by intragenic PTEN mutations and loss of the remaining wild-type allele; 7 (53.8%) showed evidence of LOH, 5 of these 7 samples showed deletion of PTEN mRNA expression, another 2 samples showed positive expression of PTEN mRNA; 4 (30.8%) tumors had neither PTEN gene mutation nor LOH but exhibited no PTEN protein expression, 2 of these 4 cases showed deletion of PTEN mRNA expression, another 2 showed positive expression of PTEN mRNA. For the cases of PTEN protein absent staining, the rate of LOH was 69.2% (9 of 13), higher than 28.6% (10 of 35) for the positive staining (P < 0.05). CONCLUSIONS: PTEN gene inactivation may contribute to epithelial ovarian carcinogenesis. There may be several mechanisms of PTEN gene inactivation in ovarian cancers. Protein expression deletions may be a significant mechanism.
Adult↗
Growth and activation of PI-3K/PKB and Akt by stromal cell-derived factor 1alpha in endometrial carcinoma cells with expression of suppressor endoprotein PTEN.
BACKGROUND: Mutation or deletion in the phosphatase and tensin homologue deleted on chromosome ten (PTEN) gene has been identified as an important cause of endometrial carcinoma; stromal cell derived factor-1alpha (SDF-1alpha) exerts growth-promoting effects on endometrial cancer cells through activation of the PI-3 kinase/Akt pathway and downstream effectors such as extracellular-responsive kinase (ERK). In this study, a plasmid containing the PTEN gene was transfected into Ishikawa cells to investigate the difference in growth and signal transduction between Ishikawa-PTEN and Ishikawa cells after SDF-1alpha stimulation, and to study mechanisms of the involvement of PTEN protein in endometrial carcinoma development. METHODS: Ishikawa cells were transfected with a plasmid (pLXSN-PTEN) containing the PTEN gene and a plasmid (pLXSN-EGFP) with enhanced green fluorescent protein (EGFP). Cells were then screened to obtain Ishikawa-PTEN cells and Ishikawa-neo cells that can both stably express PTEN protein and EGFP. Expression of PTEN protein, phosphorylation levels of AKT and ERK (pAKT and pERK) and growth differences in Ishikawa-PTEN, Ishikawa-neo and Ishikawa cells before and after SDF-1alpha stimulation were then determined by Western blots and MTT assays. RESULTS: Western blot analysis showed that Ishikawa cells produced PTEN after transfection with the PTEN gene. At 15 minutes after SDF-1alpha stimulation, the pAKT level of Ishikawa-PTEN cells was lower than that of Ishikawa-neo cells and Ishikawa cells. There was no significant difference in pERK levels among the three cell lines. The positive effect of SDF-1alpha on Ishikawa-PTEN cells growth was markedly less than the effect on Ishikawa-neo and Ishikawa cells. However, in the absence of SDF-1alpha stimulation (baseline), the pAKT level in Ishikawa-PTEN cells was less than that in Ishikawa cells. There was a significant difference in growth between the Ishikawa-PTEN cells and the Ishikawa-neo cells. CONCLUSIONS: PTEN gene transfection can regulate the level of pAKT but not pERK in Ishikawa-PTEN cells. PTEN protein may suppress the growth-promoting effect of SDF-1alpha on endometrial carcinoma by inhibiting the PI-3K/AKT signal transduction pathway.
Cell Line, Tumor↗
Nuclear-cytoplasmic partitioning of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) differentially regulates the cell cycle and apoptosis.
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase that dephosphorylates both protein and lipid substrates, is found to be mutated in both heritable and sporadic breast cancer. Cellular PTEN has been shown to regulate Akt phosphorylation, mitogen-activated protein kinase (MAPK) phosphorylation, p27(kip1), and cyclin D1 protein levels. Additionally, we and others have shown that PTEN can regulate not only the cell cycle but also cellular apoptosis. Until recently, the functions of PTEN have been thought to occur through cytoplasmic PTEN. However, we have shown that PTEN localizes to the nucleus and that this localization coincides with the G0-G1 phases of the cell cycle. Furthermore, we have shown that PTEN has bipartite nuclear localization sequence (NLS)-like sequences that are required for major vault protein-mediated nuclear import. These findings suggest that subcellular localization of PTEN may regulate its function and that nuclear-localized PTEN may regulate unique cellular functions that have been attributed to cytoplasmic PTEN. To examine this possibility, we analyzed downstream PTEN readouts using MCF-7 Tet-Off breast cancer cell lines stably transfected with two different NLS mutant PTEN constructs, which do not localize to the nucleus, and compared these with cells transfected with wild-type PTEN and empty vector control cells. We found that cytoplasmic PTEN down-regulates phosphorylation of Akt and up-regulates p27(kip1), whereas nuclear PTEN down-regulates cyclin D1 and prevents the phosphorylation of MAPK. Additionally, whereas we observe that nuclear PTEN is required for cell cycle arrest, we found that cytoplasmic PTEN is required for apoptosis. Our observations show that nuclear-cytoplasmic partitioning differentially regulates the cell cycle and apoptosis and, in this manner, provide further evidence that nuclear import of PTEN should play a role in carcinogenesis.
Apoptosis↗
Lack of PTEN expression in non-small cell lung cancer could be related to promoter methylation.
PURPOSE: The PTEN gene at chromosome 10q23.3 is a tumor-suppressor genethat is inactivated in several types of human tumors. Althoughmutation and homozygous deletion are the most commonmechanisms of PTEN inactivation, promoter methylation and translational modification can also account for PTEN silencing. The aim of this study was to investigate the expression of PTEN protein in primary non-small cell lung cancer (NSCLC) samples and to investigate the promoter methylation status of the gene in a panel of NSCLC cell lines as well as primary tumors. EXPERIMENTAL DESIGN: We analyzed PTEN expression by immunohistochemistry in tissue samples from 125 patients with early-stage NSCLC. We also evaluated PTEN promoter methylation status by methylation-specific PCR in 20 microdissected PTEN-negative primary tumors from among the last specimens as well as in a panel of 16 NSCLC cell lines. Western and Northern blotting were performed in the same panel of NSCLC cell lines. RESULTS: Thirty (24%) of the 125 specimens showed a lack of staining for PTEN. PTEN methylation was detected in 7 (35%) of the 20 PTEN-negative NSCLC samples and in none of the 10 PTEN-positive NSCLC samples that were microdissected. Furthermore, PTEN methylation was observed in 11 (69%) of the 16 NSCLC cell lines tested. PTEN mRNA expression was increased in the NCI-H1299 cell line by in vitro treatment with the demethylating agent 5-aza-2'-deoxycytidine. PTEN methylation was well correlated with PTEN expression in NSCLC cell lines by Western and Northern blot (P = 0.025). CONCLUSIONS: Although genetic alterations of the PTEN gene are rare in NSCLC, loss of PTEN protein is not an uncommon event in early-stage NSCLC. Lack of PTEN expression may be partially explained by promoter methylation.
Adult↗
Genetic and epigenetic alterations of the PTEN gene in soft tissue sarcomas.
The PTEN/MMAC1 ( PTEN ) gene was identified as a tumor suppressor gene encoding a cytoplasmic protein that controls cellular processes. To investigate the potential role and the alteration of the PTEN gene in soft tissue sarcomas (STSs), we searched for homozygous deletion and promoter hypermethylation in a series of 48 STSs that was composed of malignant fibrous histiocytoma, leiomyosarcoma, malignant peripheral nerve sheath tumor, including 2 cases with a mutation that we previously reported; differential polymerase chain reaction and methylation-specific polymerase chain reaction, respectively, were used for the analyses. Furthermore, to determine whether PTEN gene alterations are involved in the down-regulation of PTEN expression, we examined the expression of PTEN protein in 38 cases in which paraffin-embedded tissues were available for immunohistochemical analysis. In addition to our previous results showing that 2 (4%) of 51 cases had a PTEN mutation, promoter methylation was recognized in 6 (13%) of 48 cases, and homozygous deletion was detected in 1 (2%) of 48 cases in the current study. Of 6 cases with promoter methylation of PTEN gene, 5 were malignant peripheral nerve sheath tumor. Decreased expression of PTEN protein was recognized in 11 (29%) of 38 STS cases. Of 9 cases with PTEN alterations (6 cases with promoter methylation, 2 with mutation, and 1 with homozygous deletion), 3 (33%) showed decreased expression of PTEN protein. Furthermore, decreased expression of the PTEN gene showed a statistically significant correlation with high MIB-1 labeling index in 38 STS cases examined ( P = .0441). In conclusion, promoter methylation and homozygous deletion of the PTEN gene were found to be relatively rare events in cases of STS, as is mutation of the gene. Of 9 cases with a PTEN alteration, 3 (33%) showed a decrease in PTEN expression, indicating that PTEN gene alterations seem to play a minor role in the inactivation of PTEN in these tumors. Furthermore, although a further detailed analysis of a larger number of cases is still necessary, the present results suggest that PTEN expression may be a useful indicator of cell proliferation in patients with STS.
Adolescent↗
Src family protein-tyrosine kinases alter the function of PTEN to regulate phosphatidylinositol 3-kinase/AKT cascades.
Src family protein-tyrosine kinases, which play an important role in signal integration, have been implicated in tumorigenesis in multiple lineages, including breast cancer. We demonstrate, herein, that Src kinases regulate the phosphatidylinositol 3-kinase (PI3K) signaling cascade via altering the function of the PTEN tumor suppressor. Overexpression of activated Src protein-tyrosine kinases in PTEN-deficient breast cancer cells does not alter AKT phosphorylation, an indicator of signal transduction through the PI3K pathway. However, in the presence of functional PTEN, Src reverses the activity of PTEN, resulting in an increase in AKT phosphorylation. Activated Src reduces the ability of PTEN to dephosphorylate phosphatidylinositols in micelles and promotes AKT translocation to cellular plasma membranes but does not alter PTEN activity toward water-soluble phosphatidylinositols. Thus, Src may alter the capacity of the PTEN C2 domain to bind cellular membranes rather than directly interfering with PTEN enzymatic activity. Tyrosine phosphorylation of PTEN is increased in breast cancer cells treated with pervanadate, suggesting that PTEN contains sites for tyrosine phosphorylation. Src kinase inhibitors markedly decreased pervanadate-mediated tyrosine phosphorylation of PTEN. Further, expression of activated Src results in marked tyrosine phosphorylation of PTEN. SHP-1, a SH2 domain-containing protein-tyrosine phosphatase, selectively binds and dephosphorylates PTEN in Src transfected cells. Both Src inhibitors and SHP-1 overexpression reverse Src-induced loss of PTEN function. Coexpression of PTEN with activated Src reduces the stability of PTEN. Taken together, the data indicate that activated Src inhibits PTEN function leading to alterations in signaling through the PI3K/AKT pathway.
Animals↗
[Mutations of tumor suppressor gene PTEN mutations in hepatocellular carcinoma and its implications in tumor proliferation and apoptosis].
OBJECTIVE: To study mutations of tumor suppressor gene PTEN in human hepatocellular carcinomas and its effects on the proliferation and apoptosis of hepatocellular carcinoma cell line HHCC. METHODS: (1) PCR-SSCP and sequence analysis were used to detect the mutations of the 5th and 8th exon of PTEN in 42 cases of human primary hepatocellular carcinoma. (2) Eukaryotic expression vectors of the wild-type (pEGFP-wt-PTEN) and the mutant type (pEGFP-PTEN, G129R) of PTEN were constructed. Lipofectamine 2000 mediated gene transfection was used to transfect hepatocellular carcinoma cell line HHCC, in which the PTEN protein is not expressed. Culture medium containing G418 was used to select stable transfectants. MTT colorimetry was used to analyze the proliferation ability of selected cell lines. Naive HHCC cells and HHCC cells transfected with empty vector (pEGFP-C1) served as controls. (3) TNF-alpha was used to induce apoptosis of selected cell clones. RESULTS: (1) Point mutation involving the 5th exon of PTEN was detected in 4 of 42 primary hepatocellular carcinomas. (2) Compared with the control groups, the proliferation of hepatocellular carcinoma cells was significantly inhibited by the transfection of wild-type PTEN gene, while the transfection with mutant PTEN construct did not significantly change the proliferation. (3) The apoptosis indices of cells transfected with the wild-type and the mutant PTEN genes were 13.8% and 8.1% respectively. Compared with the control, the apoptosis index of HHCC cell transfected by the wild type PTEN was significantly lower (P < 0.05). There were no significant differences between HHCC cells transfected with mutated PTEN gene and the control (P > 0.05). The expression of internal 473-phosphorylated Akt of HHCC was weak, but was enhanced when the cells treated with TNF-alpha. However, it was down regulated by the wild type PTEN. CONCLUSIONS: (1) First time report that PTEN mutations can be found in 9.5% human primary hepatocellular carcinomas. (2) The expression of the wild-type PTEN can suppress the proliferation of HHCC cells, and such suppression was lost when PTEN gene was mutated. (3) PTEN inhibition of the proliferation and the enhancement of apoptosis of hepatocellular carcinoma cells is likely related to a down-regulation of the TNF-alpha induced activation of protein kinase Akt pathway.
Apoptosis↗
The role of PTEN and its signalling pathways, including AKT, in breast cancer; an assessment of relationships with other prognostic factors and with outcome.
PTEN is a novel tumour suppressor gene located on chromosome 10. PTEN mutations are believed to exert their effects through the putative PI3K-AKT-mTOR signalling pathway. Specifically, loss of PTEN leads to activation of AKT, which in turn promotes anti-apoptotic and pro-cell cycle entry pathways believed to be essential in tumourigenesis. Whilst PTEN mutations are frequent in a variety of sporadic cancers and inherited cancer syndromes, it is not clear how frequently PTEN mutations and immunohistochemical loss of PTEN expression occur in sporadic breast cancer. This study used tissue microarrays (TMAs) to assess wild-type PTEN and pAKT immunohistochemical staining in 670 and 691 cases, respectively, of primary operable breast cancer. Scores of 0, 1, and 2 were given for negative, weakly positive, and strongly positive degrees of immunoreactivity, respectively. In addition, immunohistochemical assessment of epidermal growth factor receptor (EGFR), Her2, and proliferation by MIB1 expression was performed on the same TMAs and the scores were compared with those of PTEN and pAKT. Eight per cent of cases did not express wild-type PTEN. No correlation was observed between patient, tumour and outcome variables and PTEN. pAKT expression correlated inversely with adverse tumour variables such as tumour grade (p< 0.001) and correlated positively with ER status (p< 0.001). No correlation was seen between either PTEN or AKT and EGFR, Her2 or MIB1. No association of PTEN or pAKT was seen in Kaplan-Meier or multivariate analysis for overall survival. The results indicate that loss of PTEN expression is infrequent in breast cancer. PTEN and AKT do not appear to be prognostic markers. The study argues against the current model of a simple linear tumourigenic PTEN-PI3K-AKT-mTOR pathway in breast cancer. It also suggests that, in this group of breast cancers, the most common upstream regulator of AKT may be ER rather than PTEN, EGFR or Her2.
Adult↗
The alteration of PTEN tumor suppressor expression and its association with the histopathological features of human primary hepatocellular carcinoma.
PURPOSE: Although deletions or inactivating mutations of the tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10) are involved in the development of a variety of tumors including glioblastoma, melanoma, prostate cancer, breast cancer, endometrial cancers etc., the role of PTEN expression in human primary hepatocellular carcinoma (HCC) has not yet been clarified. The aim of this study is to investigate the involvement of PTEN mRNA and protein expression in HCC. METHODS: The level of PTEN mRNA expression in HCC specimens was analyzed by Northern blot. PTEN poly-clonal antibody was raised by immunizing New Zealand white rabbit with (His)(6)-tagged PTEN fusion protein and characterized by Western blot. The level of PTEN protein expression was determined by immunohistochemistry. The significance of PTEN in HCC was analyzed by comparing its expression level with the clinicopathological parameters of HCC patients. RESULTS: Four transcripts of PTEN mRNA at 5.5 kb, 4.4 kb, 2.4 kb, and 1.8 kb were detected in most para-carcinoma liver tissues, and the expression level of PTEN mRNA in carcinoma liver tissues was found to decrease significantly. The poly-clonal antibody raised against histidine-tagged fusion PTEN protein showed specific immuno-reactivity to PTEN protein. Using the specific poly-clonal antibody prepared and characterized by ourselves, we found that PTEN protein was significantly down-regulated in HCC tissues compared with paired para-carcinoma tissues. The protein expression of PTEN is negatively associated with the pathological grading and presence of cancer thrombus of HCC. CONCLUSIONS: Down-regulation of PTEN expression may play an important role in the development of HCC and the level of PTEN expression may be a potential adjuvant parameter in forecasting the progression and prognosis of HCC patients.
Carcinoma, Hepatocellular↗
Immunohistochemical evidence of loss of PTEN expression in primary ductal adenocarcinomas of the breast.
Germline mutations in PTEN, encoding a dual-specificity phosphatase on 10q23.3, cause Cowden syndrome (CS), which is characterized by a high risk of breast and thyroid cancers. Loss of heterozygosity of 10q22-24 markers and somatic PTEN mutations have been found to a greater or lesser extent in a variety of sporadic component and noncomponent cancers of CS. Among several series of sporadic breast carcinomas, the frequency of loss of flanking markers around PTEN is approximately 30 to 40%, and the somatic intragenic PTEN mutation frequency is <5%. In this study, we analyzed PTEN expression in 33 sporadic primary breast carcinoma samples using immunohistochemistry and correlated this to structural studies at the molecular level. Normal mammary tissue had a distinctive pattern of expression: myoepithelial cells uniformly showed strong PTEN expression. The PTEN protein level in mammary epithelial cells was variable. Ductal hyperplasia with and without atypia exhibited higher PTEN protein levels than normal mammary epithelial cells. Among the 33 carcinoma samples, 5 (15%) were immunohistochemically PTEN-negative; 6 (18%) had reduced staining, and the rest were PTEN-positive. In the PTEN-positive tumors as well as in normal epithelium, the protein was localized in the cytoplasm and in the nucleus (or nuclear membrane). Among the immunostain negative group, all had hemizygous PTEN deletion but no structural alteration of the remaining allele. Thus, in these cases, an epigenetic phenomenon such as hypermethylation, -ecreased protein synthesis or increased protein degradation may be involved. In the cases with reduced staining, 5 of 6 had hemizygous PTEN deletion and 1 did not have any structural abnormality. Finally, clinicopathological features were analyzed against PTEN protein expression. Three of the 5 PTEN immunostain-negative carcinomas were also both estrogen and progesterone receptor-negative, whereas only 5 of 22 of the PTEN-positive group were double receptor-negative. The significance of this last observation requires further study.
Antibodies, Monoclonal↗
PTEN gene transfer in human malignant glioma: sensitization to irradiation and CD95L-induced apoptosis.
The tumor suppressor gene PTEN (MMAC1, TEP1) encodes a dual-specificity phosphatase and is considered a progression-associated target of genetic alterations in human gliomas. Recently, it has been reported that the introduction of wild type PTEN into glioma cells containing endogenous mutant PTEN alleles (U87MG, LN-308), but not in those which retain wild-type PTEN (LN-18, LN-229), causes growth suppression and inhibits cellular migration, spreading and focal adhesion. Here, we show that PTEN gene transfer has no effect on the chemosensitivity of the four cell lines. Further, a correlational analysis of the endogenous PTEN status of 12 human glioma cell lines with their sensitivity to seven different cancer chemotherapy drugs reveals no link between PTEN and chemosensitivity. In contrast, ectopic expression of wild type PTEN, but not the PTEN(G129R) mutant, in PTEN-mutant gliomas markedly sensitizes these cells to irradiation and to CD95-ligand (CD95L)-induced apoptosis. PTEN-mediated facilitation of CD95L-induced apoptosis is associated with enhanced CD95L-evoked caspase 3 activity. Protein kinase B (PKB/Akt), previously shown to inhibit CD95L-induced apoptosis in nonglial COS7 cells, is inactivated by dephosphorylation. Interestingly, both PTEN-mutant U87MG and PTEN-wild-type LN-229 cells contain phosphorylated PKB constitutively. Wild-type PTEN gene transfer promotes dephosphorylation of PKB specifically in U87MG cells but not in LN-229 cells. Sensitization of U87MG cells to CD95L-apoptosis by wild-type PTEN is blocked by insulin-like growth factor-1 (IGF-1). The protection by IGF-1 is inhibited by the phosphoinositide 3-OH (PI 3) kinase inhibitor, wortmannin. Although PKB is a down-stream target of PI 3 kinase, the protection by IGF-1 was not associated with the reconstitution of PKB phosphorylation. Thus, PTEN may sensitize human malignant glioma cells to CD95L-induced apoptosis in a PI 3 kinase-dependent manner that may not require PKB phosphorylation.
Antineoplastic Agents↗