Pten
Pten/Mmac1+/- heterozygous mice exhibit neoplasms in multiple organs including the endometrium, liver, prostate, gastrointestinal tract, thyroid, and thymus. Loss
of the wild-type allele is detected in neoplasms of the thymus and liver. Surprisingly, tumors of the gastrointestinal epithelium develop in association with gut
lymphoid tissue. Tumors of the endometrium, thyroid, prostate, and liver are not associated with lymphoid tissue and appeared to be highly mitotic. In addition,
these mice have nonneoplastic hyperplasia of lymph nodes that is caused by an inherited defect in apoptosis detected in B cells and macrophages. Examination of
peripheral lymphoid tissue including lymphoid aggregates associated with polyps reveal that the normal organization of B and T cells is disrupted in heterozygous
animals. Taken together, these data suggest that PTEN is a regulator of apoptosis and proliferation that behaves as a 'landscaper' tumor suppressor in the gut and a 'gatekeeper' tumor suppressor in other organs (Podsypanina, 1999).
PTEN phosphatase acts as a tumor suppressor by negatively regulating the phosphoinositide 3-kinase (PI3K) signaling pathway. It is
unclear which downstream components of this pathway are necessary for oncogenic transformation. Transformed cells of PTEN+/- mice have elevated levels of phosphorylated Akt and activated p70/S6 kinase associated with an
increase in proliferation. Pharmacological inactivation of mTOR/RAFT/FRAP reduces neoplastic proliferation, tumor size, and p70/S6 kinase activity, but does not affect the status of Akt. These data suggest that p70/S6K and possibly other targets of mTOR contribute significantly to tumor development and that inhibition of these proteins may be therapeutic for cancer patients with deranged PI3K signaling (Podsypanina, 2001).
A functional test was carried out for PTEN candidacy as a growth suppressor in glioma cells. A combination of Northern blot analysis, protein
truncation assays, and sequence analysis was used to determine the types and frequency of PTEN mutations in glioma cell lines so that appropriate recipients could be defined to assess the growth suppressive function of PTEN by gene transfer. Introduction of wild-type PTEN into glioma cells containing endogenous mutant alleles causes growth suppression, but was without effect in cells containing endogenous wild-type PTEN. The ectopic expression of PTEN alleles that carry mutations found
in primary tumors and have been shown or are expected to inactivate its phosphatase activity causes little growth suppression. These data strongly suggest that PTEN is a protein phosphatase that exhibits functional and specific growth-suppressing activity (Furnari, 1997).
Recombinant P-TEN dephosphorylates protein and peptide substrates phosphorylated on serine, threonine, and tyrosine residues, indicating that P-TEN is a dual-specificity phosphatase. In addition, P-TEN exhibits a high degree of substrate specificity, showing selectivity for extremely acidic substrates in vitro. Furthermore, mutations in P-TEN, identified from primary tumors, tumor cells lines, and a patient with Bannayan-Zonana syndrome, result in the ablation of phosphatase activity, demonstrating that enzymatic activity of P-TEN is
necessary for its ability to function as a tumor suppressor (Myers, 1997).
Biochemical and functional evidence is provided
that PTEN/MMAC1 acts a negative regulator of the phosphoinositide 3-kinase (PI3-kinase)/Akt pathway. PTEN/MMAC1 impairs activation of endogenous Akt in
cells and inhibits phosphorylation of 4E-BP1, a downstream target of the PI3-kinase/Akt pathway involved in protein translation, whereas a catalytically inactive,
dominant negative PTEN/MMAC1 mutant enhances 4E-BP1 phosphorylation. In addition, PTEN/MMAC1 represses gene expression in a manner that is rescued
by Akt but not PI3-kinase. Finally, higher levels of Akt activation are observed in human prostate cancer cell lines and xenografts lacking PTEN/MMAC1
expression when compared with PTEN/MMAC1-positive prostate tumors or normal prostate tissue. Because constitutive activation of either PI3-kinase or Akt is
known to induce cellular transformation, an increase in the activation of this pathway caused by mutations in PTEN/MMAC1 provides a potential mechanism for its
tumor suppressor function (Wu, 1998).
Mutated in multiple advanced cancers 1/phosphatase and tensin homologue (MMAC1/PTEN) is a novel tumor suppressor gene candidate located on chromosome
10 that is commonly mutated in human glioblastoma multiforme and several other cancer types. To evaluate the function of this gene as a tumor suppressor, a replication-defective adenovirus (MMCB) was constructed for efficient, transient transduction of MMAC1 into tumor cells. Infection of MMAC1-mutated U87MG
glioblastoma cells with MMCB results in dose-dependent exogenous MMAC1 protein expression as detected by Western blotting of cell lysates. In vitro
proliferation of U87MG cells is inhibited by MMCB in comparison to several control adenoviruses at equal viral doses, implying a specific effect of MMAC1
expression. Anchorage-independent growth in soft agar is also inhibited by MMCB compared to control adenovirus. Tumorigenicity in nude mice of transiently
transduced mass cell cultures was then assessed. MMCB-infected U87MG cells are almost completely nontumorigenic compared to untreated and several control
adenovirus-treated cells at equal viral doses. These data support an in vivo tumor suppression activity of MMAC1/PTEN and suggest that in vivo gene transfer with
this recombinant adenoviral vector has a potential use in cancer gene therapy (Cheney, 1998.
The pattern of deletion, mutation,
and expression of MMAC1/PTEN has been studied in 35 unrelated melanoma cell lines. Nine (26%) of the cell lines showed partial or complete homozygous deletion of the
MMAC1/PTEN gene, and another six (17%) harbored a mutation in combination with loss of the second allele. Mutations could also be demonstrated in uncultured
tumor specimens from which the cell lines had been established, and cell lines derived from two different metastases from one individual carried the same missense
mutation. Collectively, these findings suggest that disruption of MMAC1/PTEN by allelic loss or mutation may contribute to the pathogenesis or neoplastic evolution
in a large proportion of malignant melanomas (Guldberg, 1997).
In preliminary screens, mutations of PTEN are detected in 31% (13/42) of glioblastoma cell lines and xenografts,
100% (4/4) of prostate cancer cell lines, 6% (4/65) of breast cancer cell lines and xenografts, and 17% (3/18) of primary glioblastomas. The predicted PTEN
product has a protein tyrosine phosphatase domain and extensive homology to tensin, a protein that interacts with actin filaments at focal adhesions. These
homologies suggest that PTEN may suppress tumor cell growth by antagonizing protein tyrosine kinases and may regulate tumor cell invasion and metastasis through
interactions at focal adhesions (J. Li, 1997).
Cowden disease (CD) is an autosomal dominant cancer predisposition syndrome associated with an elevated risk for tumours of the breast, thyroid and skin.
Lhermitte-Duclos disease (LDD) cosegregates with a subset of CD families and is associated with macrocephaly, ataxia and dysplastic cerebellar
gangliocytomatosis. The common feature of these diseases is a predisposition to hamartomas, benign tumours containing differentiated but disorganized cells
indigenous to the tissue of origin. Linkage analysis has determined that a single locus within chromosome 10q23 is likely to be responsible for both of these diseases.
A candidate tumour suppressor gene (PTEN) within this region is mutated in sporadic brain, breast and prostate cancer. Another group has independently isolated
the same gene, termed MMAC1, and also found somatic mutations throughout the gene in advanced sporadic cancers. Mutational analysis of PTEN in CD kindreds
has identified germline mutations in four of five families. Nonsense and missense mutations were found that are predicted to disrupt the protein tyrosine/dual-specificity
phosphatase domain of this gene. Thus, PTEN appears to behave as a tumour suppressor gene in the germline. These data also imply that PTEN may play a role in
organizing the relationship of different cell types within an organ during development (Liaw, 1997).
Loss of heterozygosity (LOH) from chromosome 10 is a hallmark of glioblastoma, the most malignant (grade IV) form of glioma. A candidate tumor suppressor
gene, PTEN/MMAC1, that may be targeted for deletion in association with chromosome 10 LOH has recently been identified. 63
glioblastomas have been investigated for PTEN/MMAC1 alterations and DNA sequence changes that would affect the encoded protein have been identified in 17 (27%) tumors. Microsatellite
analyses of normal-tumor DNA pairs were performed on 14 of these cases and revealed LOH at locations flanking and/or near PTEN/MMAC1 in all but 1
instance, suggesting that deletion of the remaining wild-type allele had occurred in the large majority of tumors with PTEN/MMAC1 mutations. Competitive PCR
assays were developed to address the possible occurrence of PTEN/MMAC1 homozygous deletions in glioblastomas, and this analysis identified three samples
having loss of both PTEN/MMAC1 alleles. EGFR amplification was determined to occur at similar frequencies among cases with or without PTEN/MMAC1
homozygous deletions or mutations, suggesting that a growth-promoting effect resulting from amplification-associated increases in epidermal growth factor receptor
signaling is not necessarily dependent on the inactivation of PTEN/MMAC1 (Liu, 1997).
In the present study,
123 brain tumors, including various grades and histological types of gliomas occurring in children and adults, were analyzed for PTEN mutations by SSCP assay and
sequencing. Mutations in the PTEN gene were found in 13 of 42 adult glioblastomas and 3 of 13 adult anaplastic astrocytomas, whereas none of the 21 low-grade
adult gliomas or the 22 childhood gliomas of all grades showed mutations. The single medulloblastoma with a mutation was a recurrent tumor that also possessed a
p53 mutation. High-grade adult gliomas with PTEN mutations included cases that also contained gene amplification or p53 gene mutations, as well as cases that did
not contain either of these abnormalities. There was no obvious relationship between presence of PTEN mutation and survival; however, there was a tendency for
PTEN mutations to occur in older age group patients. This analysis suggest that PTEN gene mutations are restricted to high-grade adult gliomas and that this
abnormality is independent of the presence or absence of gene amplification or p53 gene mutation in these tumors (Rasheed, 1997).
Endometrial carcinomas represent the most common gynecological cancer in the United States, yet the molecular genetic events that underlie the development of
these tumors remain obscure. Because PTEN is included in the region of LOH in many endometrial carcinomas, 70
endometrial carcinomas were examined for alterations in PTEN/MMAC1. Somatic mutations were detected in 24 cases (34%) including 21 cases that resulted in premature
truncation of the protein, 2 tumors with missense alterations in the conserved phosphatase domain, and 1 tumor with a large insertion. These data indicate that
PTEN/MMAC1 is more commonly mutated than any other known gene in endometrial cancers (Risinger, 1997).
Deletions involving regions of chromosome 10 occur in the vast majority of human glioblastoma multiformes. A region at chromosome 10q23-24 was
implicated to contain a tumour suppressor gene and the identification of homozygous deletions in four glioma cell lines further refined the location. A gene, designated MMAC1, has been identified that spans these deletions and encodes a widely expressed 5.5-kb mRNA. The predicted MMAC1 protein contains sequence motifs
with significant homology to the catalytic domain of protein phosphatases and to the cytoskeletal proteins, tensin and auxilin. MMAC1 coding-region mutations were
observed in a number of glioma, prostate, kidney and breast carcinoma cell lines or tumour specimens. These results identify a strong candidate tumour suppressor
gene at chromosome 10q23.3, whose loss of function appears to be associated with the oncogenesis of multiple human cancers (Steck, 1997).
Alterations of the PTEN gene occur in glioblastoma multiforme. To determine the frequency of PTEN alteration, 34 consecutive glioblastomas were studied in detail.
Sequencing each of the nine exons amplified from tumor DNA revealed 11 mutations. Analysis of polymorphic markers within and surrounding the PTEN gene
identified an additional four homozygous deletion mutations. Loss of heterozygosity (LOH) was observed in 25 of 34 (74%) cases. All mutations occurred in the
presence of LOH. PTEN was mutated in 44% (15 of 34) of all glioblastomas studied and 60% (15 of 25) of tumors with LOH on 10q. Thus, PTEN appears to be
the major target of inactivation on chromosome 10q in glioblastoma multiforme (Wang, 1997).
To determine if
PTEN is a target of 10q loss of heterozygosity in carcinomas of the endometrium, 32 primary endometrial carcinomas were examined for mutations in PTEN. The
tumors included the two major histopathological types of endometrial carcinoma: endometrioid (n = 26; 14 microsatellite instability (MI)-positive and 12
MI-negative) and serous (n = 6). Overall, mutations were detected in 50% of the endometrial carcinomasanalyzed. Mutations were present in 12 of 14 (86%)
MI-positive and 4 of 12 (33%) MI-negative endometrioid tumors. Furthermore, mutations were found in all three histological grades of MI-positive endometrioid
carcinoma. All six serous endometrial carcinomas lacked detectable mutations. To evaluate the role of PTEN in other common malignancies of the female genital
tract, 12 serous ovarian carcinomas and 10 squamous cervical carcinomas were analyzed and were negative for mutations. These results support PTEN as a tumor
suppressor gene and suggest that mutations in PTEN play a significant role in the pathogenesis of the endometrioid type of endometrial carcinoma (Tashiro, 1997).
Constitutive DNA from 37 Cowden disease (CD) families and seven Bannayan-Zonana Syndrome (BZS) families was screened for germline PTEN mutations. PTEN mutations were identified in 30 of 37
(81%) CD families, including missense and nonsense point mutations, deletions, insertions, a deletion/insertion and splice site mutations. These mutations were
scattered over the entire length of PTEN , with the exception of the first, fourth and last exons. A 'hot spot' for PTEN mutation in CD was identified in exon 5 that
contains the PTPase core motif, with 13 of 30 (43%) CD mutations identified in this exon. Seven of 30 (23%) were within the core motif, the majority (five of seven)
of which were missense mutations, possibly pointing to the functional significance of this region. Germline PTEN mutations were identified in four of seven (57%)
BZS families studied. Interestingly, none of these mutations was observed in the PTPase core motif. It is also worthy of note that a single nonsense point mutation,
R233X, was observed in the germline DNA from two unrelated CD families and one BZS family. Genotype-phenotype studies were not performed on this small
group of BZS families. However, genotype-phenotype analysis in the group of CD families revealed two possible associations worthy of follow-up in independent
analyses. The first was an association noted in the group of CD families with breast disease. A correlation was observed between the presence/absence of a PTEN
mutation and the type of breast involvement (unaffected versus benign versus malignant). Specifically and more directly, an association was also observed between
the presence of a PTEN mutation and malignant breast disease. Secondly, there appeared to be an interdependent association between mutations upstream and
within the PTPase core motif, the core motif containing the majority of missense mutations, and the involvement of all major organ systems (central nervous system,
thyroid, breast, skin and gastrointestinal tract). However, these observations would need to be confirmed by studying a larger number of CD families (Marsh, 1998).
PTEN is a tumor suppressor gene located on chromosome 10q23 that encodes a protein and phospholipid phosphatase. Somatic mutations of PTEN are found in a number of human malignancies, and loss of expression, or mutational inactivation of PTEN, leads to the constitutive activation of protein kinase B (PKB)/Akt via enhanced phosphorylation of Thr-308 and Ser-473. The integrin-linked kinase (ILK) can phosphorylate PKB/Akt on Ser-473 in a phosphoinositide phospholipid-dependent manner. The activity of ILK is constitutively elevated in a serum- and anchorage-independent manner in PTEN-mutant cells, and transfection of wild-type (WT) PTEN into these cells inhibits ILK activity. Transfection of a kinase-deficient, dominant-negative form of ILK or exposure to a small molecule ILK inhibitor suppresses the constitutive phosphorylation of PKB/Akt on Ser-473, but not on Thr-308, in the PTEN-mutant prostate carcinoma cell lines PC-3 and LNCaP. Transfection of dominant-negative ILK and WT PTEN into these cells also results in the inhibition of PKB/Akt kinase activity. Furthermore, dominant-negative ILK or WT PTEN induces G(1) phase cycle arrest and enhanced apoptosis. Together, these data demonstrate a critical role for ILK in PTEN-dependent cell cycle regulation and survival and indicate that inhibition of ILK may be of significant value in PTEN-mutant tumor therapy (Persad, 2000).
ß-Catenin is a protein that plays a role in intercellular adhesion as well as in the regulation of gene expression. The latter role of ß-catenin
is associated with its oncogenic properties due to the loss of expression or inactivation of the tumor suppressor adenomatous polyposis
coli (APC) or mutations in ß-catenin itself. Another tumor suppressor, PTEN, is also involved in the regulation
of nuclear ß-catenin accumulation and T cell factor (TCF) transcriptional activation in an APC-independent manner. Nuclear ß-catenin expression is constitutively elevated in PTEN null cells and this elevated expression is reduced upon reexpression of PTEN. TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF
transcriptional activity. Furthermore, the constitutively elevated expression of cyclin D1, a ß-catenin/TCF-regulated gene, is also suppressed upon reexpression of
PTEN. Mechanistically, PTEN increases the phosphorylation of ß-catenin and enhances its rate of degradation. A pathway is defined that involves mainly
integrin-linked kinase and glycogen synthase kinase 3 in the PTEN-dependent regulation of ß-catenin stability, nuclear ß-catenin expression, and transcriptional activity. These data indicate that ß-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression (Persad, 2001).
Mechanistically, these results indicate that PTEN induces an increase in the phosphorylation of ß-catenin, thereby increasing its relative rate of degradation in PTEN-transfected PC3 cells compared with the control cells. It is likely that this increased phosphorylation is a direct result of the observed increase in GSK-3 activity induced by PTEN. It is well known that ß-catenin stability is regulated by phosphorylation of the protein at Ser 33/37/45 and Thr 41 by GSK-3 at its NH2 terminus, followed by ubiquitination proteasome-mediated degradation. This increased degradation of ß-catenin may effectively lower cellular concentration of the protein and prevent its further accumulation in the nucleus, leading to decreased nuclear ß-catenin. It is also possible that PTEN may regulate the translocation of ß-catenin from the nucleus and subsequently induce its degradation in the cytosol. However, further studies are required to explore this latter hypothesis (Persad, 2001).
PTEN-transfected PC3 cells also appear to exhibit a more prominent membranal localization of ß-catenin. This may be related to the fact that PTEN appears to induce the transcription of E-cadherin in PC3 cells, which do not normally express E-cadherin. Also, it is possible that the reexpression of
E-cadherin in PC3 cells may contribute to the observed decrease in nuclear ß-catenin. ß-Catenin is known to interact with the cytoplasmic domains of E-cadherin, linking it to the actin cytoskeleton. Thus, the more prominent localization of ß-catenin to the cell surface may be related
to the reappearance of its cell membrane anchor, E-cadherin, in the PTEN-reexpressing cells. However, no E-cadherin-ß-catenin
complexes could be detected in PTEN-transfected PC3 cells. Also, the lack of interaction between ß-catenin and E-cadherin demonstrates that stimulation of E-cadherin expression is unlikely to play a significant role in the observed decreased expression of nuclear ß-catenin. However, it should be noted that PC3 cells do express N-cadherin and this expression is unaffected by reexpression of PTEN. Loss of E-cadherin expression has been linked to the
acquisition of an invasive and/or metastatic phenotype and it is this antiinvasive and/or antimetastatic property of
E-cadherin that may be of significance in relation to the tumor suppressor PTEN. In support of this, reexpression of E-cadherin by transfection has been shown to
suppress the invasive phenotype in E-cadherin-negative prostate tumor cell clones. Overexpression or constitutive activation of integrin-linked kinase (ILK) has been shown to result in an invasive phenotype concomitant with downregulation of E-cadherin expression, translocation of ß-catenin to the nucleus, and formation of the
LEF-1-ß-catenin bipartite complex. Also, ILK has been shown to be regulated by the tumor suppressor PTEN (Persad, 2001).
Cyclin D1 is known to be one of the oncogenic targets of ß-catenin. In PC3 cells, the expression level of cyclin D1 is upregulated in a constitutive, serum-independent manner. More
importantly, replacement of PTEN or inhibition of ILK results in dramatic suppression of the expression levels of cyclin D1. Furthermore, transient overexpression of
GSK-3-WT also suppresses cyclin D1 expression. These results are supported by the observation that overexpression of ILK stimulates cyclin D1
expression. Also, ILK has been shown recently to regulate cyclin D1 transcription via a pathway involving GSK-3 and the cyclic AMP
response element binding protein transcription factor. Cyclin D1 expression in PC3 cells changes in a parallel manner to
ß-catenin expression in response to reexpression of PTEN or expression of ILK-KD and GSK-3. Therefore, it is proposed that the alterations in cyclin D1 expression
very likely represent the physiological end result of the regulation of ß-catenin by PTEN and ILK via GSK-3. It should be pointed out that although nuclear ß-catenin
and cyclin D1 expressions undergo parallel alterations due to reexpression of PTEN or inhibition of ILK, the expression of the CDK inhibitors p27Kip and p21Cip
remain unchanged. This illustrates the specificity of the alterations induced upon ß-catenin and cyclin D1. PTEN, ILK-KD, and GSK-3-WT all dramatically reduce cyclin D1 promoter activity. Furthermore, Northern blot analysis demonstrates that ILK-KD, PTEN-WT, and
GSK-3 induce dramatic inhibitory effects upon cyclin D1 transcriptional expression. This supports the working hypothesis that PTEN and ILK can regulate nuclear ß-catenin through GSK-3. This is in agreement with the fact that ß-catenin is known to be regulated by GSK-3 and recent studies
have identified GSK-3 as a critical regulatory component for the transcriptional activity and binding of the TCF-LEF-1-ß-catenin complex transcription factors (Persad, 2001).
In conclusion, a novel pathway involving PI-3 kinase/PTEN, ILK, and GSK-3 has been demonstrated that maintains tight control over the levels and localization of ß-catenin. In prostate cancer cells, as well in other malignancies where PTEN is either lost or inactive, this control may be eliminated, resulting in elevated ß-catenin levels, its accumulation in the nucleus, and increased transcription of its oncogenic targets. Cyclin D1 is a known target of ß-catenin and it is also the first
participant of the chain of cyclins and CDKs that control progression through the G1 and S phase of the cell cycle. Therefore, it is conceivable that PTEN and ILK,
by virtue of their capacity to regulate ß-catenin and subsequently cyclin D1, may ultimately regulate the progression of cells through the cell cycle. By virtue of their ability to regulate the expression of E-cadherin, PTEN/PI-3 kinase and ILK may also control the metastatic potential of cancer cells. Therefore, the inhibition of a potent regulator such as ILK may present a feasible alternative means of treating the numerous forms of tumors where the PI-3 kinase-dependent signal transduction pathway is dysregulated due to mutations of the tumor suppressor PTEN (Persad, 2001).
Recent evidence places the FRAP/mTOR kinase downstream of the phosphatidyl inositol 3-kinase/Akt-signaling pathway, which is up-regulated
in multiple cancers because of loss of the PTEN tumor suppressor gene.
TOR (target of rapamycin) is the protein kinase FRAP/mTOR, an evolutionarily
conserved member of the phosphoinositide kinase-related kinase family that includes DNA-PK, ATM, and ATR. Biological and biochemical studies were performed to determine whether PTEN-deficient cancer cells are sensitive to pharmacologic inhibition of FRAP/mTOR by using the rapamycin derivative CCI-779. In vitro and in vivo studies of isogenic PTEN+/+ and PTEN-/- mouse cells as well as human cancer cells with defined PTEN status show that the growth of PTEN null cells is blocked preferentially by pharmacologic FRAP/mTOR inhibition. Enhanced tumor growth caused by constitutive activation of Akt in PTEN+/+ cells also is reversed by CCI-779 treatment, indicating that FRAP/mTOR functions downstream of Akt in tumorigenesis. Loss of PTEN correlates with increased S6 kinase activity and phosphorylation of ribosomal S6 protein, providing evidence for activation of the FRAP/mTOR pathway in these cells. Differential sensitivity to CCI-779 is not explained by differences in biochemical blockade of the FRAP/mTOR pathway, because S6 phosphorylation is inhibited in sensitive and resistant cell lines. It is concluded that PTEN-deficient cells are sensitive to growth inhibition
caused by pharmacologic mTOR blockade. These results provide rationale for testing FRAP/mTOR inhibitors in PTEN null human cancers (Neshat, 2001).
PTEN tumor suppressor is frequently mutated in human cancers, including breast cancers. Female patients with inherited PTEN mutations suffer from virginal hypertrophy of the breast with high risk of
malignant transformation. However, the exact mechanisms of PTEN in controlling mammary gland development and tumorigenesis are unclear. In this study, mice were generated with a mammary-specific deletion of the Pten gene. Mutant mammary tissue displays precocious lobulo-alveolar development, excessive ductal branching, delayed involution and severely reduced apoptosis. Pten null mammary epithelial cells are disregulated and hyperproliferative. Mutant females develop mammary tumors early in life. Similar phenotypes were observed in Pten-null mammary epithelia that had been transplanted into wild-type stroma, suggesting that PTEN plays an essential and cell-autonomous role in controlling the proliferation, differentiation and apoptosis of mammary epithelial cells (Li, 2002).
Germline mutations in LKB1 (Drosophila homolog: lkb1), TSC2, or PTEN tumor suppressor genes result in hamartomatous syndromes with shared tumor biological features. The recent observations of LKB1-mediated activation of AMP-activated protein kinase (AMPK) and AMPK inhibition of mTOR through TSC2 prompted an examination of the biochemical and biological relationship between LKB1 and mTOR regulation. LKB1 is required for repression of mTOR under low ATP conditions in cultured cells in an AMPK- and TSC2-dependent manner, and Lkb1 null MEFs and the hamartomatous gastrointestinal polyps from Lkb1 mutant mice show elevated signaling downstream of mTOR. These findings position aberrant mTOR activation at the nexus of these germline neoplastic conditions and suggest the use of mTOR inhibitors in the treatment of Peutz-Jeghers syndrome (Shaw, 2004).
Pten heterozygous (Pten+/-) mice develop increased papilloma numbers and show decreased carcinoma latency time in comparison with controls after skin treatment with dimethyl benzanthracene (DMBA) and tetradecanoyl-phorbol acetate (TPA). H-ras mutation is normally a hallmark of DMBA-TPA-induced skin tumors, but 70% of carcinomas from Pten+/- mice do not exhibit this mutation, and in all cases have lost the wild-type Pten allele. Tumors that retain the Pten wild-type allele also have H-ras mutations, indicating that activation of H-ras and complete loss of Pten are mutually exclusive events in skin carcinomas. Mitogen-activated protein kinase (MAPK) is consistently activated in the tumors with H-ras mutations, but is strongly down-regulated in Pten-/- tumors, suggesting that this pathway is dispensable for skin carcinoma formation. These data have important implications in designing individual therapeutic strategies for the treatment of cancer (Mao, 2004).
PTEN is an important tumor suppressor gene. Hereditary mutation of PTEN causes tumor-susceptibility diseases such as Cowden disease. The Cre-loxP system was used to generate an endothelial cell-specific mutation of Pten (Tie2CrePten) in mice. Tie2CrePtenflox/+ mice display enhanced tumorigenesis due to an increase in angiogenesis driven by vascular growth factors. This effect is partially dependent on the PI3K subunits p85alpha and p110gamma. In vitro, Tie2CrePtenflox/+ endothelial cells show enhanced proliferation/migration. Tie2CrePtenflox/flox mice die before embryonic day 11.5 (E11.5) due to bleeding and cardiac failure caused by impaired recruitment of pericytes and vascular smooth muscle cells to blood vessels, and of cardiomyocytes to the endocardium. These phenotypes depend strongly on p110gamma rather than on p85alpha and are associated with decreased expression of Ang-1, VCAM-1, connexin 40, and ephrinB2 but increased expression of Ang-2, VEGF-A, VEGFR1, and VEGFR2. Pten is thus indispensable for normal cardiovascular morphogenesis and post-natal angiogenesis, including tumor angiogenesis (Hamada, 2005).
RT-PCR analyses of gene expression in whole yolk sacs from E8.5 Tie2CrePten+/+ and Tie2CrePtenflox/flox embryos show that a lack of Pten significantly reduces expression of connexin-40, Ang-1, ephrinB2, and VCAM-1, but increased expression of Ang-2, VEGF-A, VEGFR1, VEGFR2, TGF-ß, and PAI-1 . These differences were confirmed by RT-PCR analyses of VEGFR2+ cells from E9.5 Tie2CrePten+/+ and Tie2CrePtenflox/flox embryos and protein analyses of human umbilical vein endothelial cells (HUVECs) in which PTEN expression was reduced by siRNA. These results suggest that Pten deficiency leads directly to an altered VGF profile that may be responsible for the cardiovascular defects of Tie2CrePtenflox/flox mice (Hamada, 2005).
In Tie2CrePten+/+ endothelial cells, expression levels of VEGF-A and its receptors VEGFR1 and VEGFR2 is significantly increased after stimulation with VEGF-A, as expected. However, this expression is further increased in Tie2CrePtenflox/+ endothelial cells. This enhanced expression of VEGF-A and its receptors may thus partly contribute to the enhanced angiogenesis observed in Tie2CrePtenflox/+ mice (Hamada, 2005).
The precise functions of PI3K isoforms in endothelial cells have been difficult to ascertain. Because most VGF receptors have tyrosine kinase activity, class IA PI3Ks likely play major roles in cardiovasculogenesis and tumor angiogenesis. Indeed, endothelial cell growth/survival and angiogenesis are enhanced following ectopic expression of constitutively active p110alpha, the catalytic subunit of class IA PI3Ks. Consistent with this finding, the enhanced angiogenesis and accelerated tumor growth observed in Tie2CrePtenflox/+ mice, and the impaired cardiovascular morphogenesis observed in Tie2CrePtenflox/flox mice are partially resolved by loss of p85alpha, the major regulatory subunit of class IA PI3Ks (Hamada, 2005).
This study sheds light on the potential roles of the class IB PI3K in cardiovascular morphogenesis and post-natal angiogenesis. Double mutant mice lacking both Pten and class IB PI3K functions were generated, and it was demonstrated that the post-natal angiogenic responses of Tie2CrePtenflox/+ mice are rescued to the same degree by loss of p110gamma, the catalytic subunit of PI3Kgamma, as by loss of p85alpha. Furthermore, compared with p85alpha deficiency, p110gamma deficiency dramatically resolves the defective cardiovasculogenesis observed in Tie2CrePtenflox/flox mice. In the p85alpha-deficient mice used in this study, only the p85alpha isoform was deleted, not its alternative splicing isoforms p55alpha and p50alpha. Moreover, p85ß and p55gamma, the alternative regulatory subunits of class IA PI3Ks, still exist in these mice. However, since p85alpha is the major regulatory subunit of class IA PI3Ks, it is believed that the class IB PI3K may have a more important function in cardiovasculogenesis than do the class IA PI3Ks (Hamada, 2005).
It was not expected that the enhanced angiogenesis induced in Tie2CrePtenflox/+ mice by RTK agonists (e.g., VEGF and Ang-1) would be partially rescued by p110gamma deficiency. Up until now, p110gamma has been postulated to be activated downstream of GPCR but not downstream of RTK. Indeed, the activation of PKB/Akt and MAPK induced by VEGF and Ang-1 is not suppressed by p110gamma deficiency in vitro. It is thus unlikely that an RTK type VGF receptor directly couples with p110gamma, an interaction noted for PDGF receptors and erythropoietin receptors. It may be that, in vivo, an unknown VGF (possibly a GPCR ligand) activates p110gamma and influences RTK signaling that is initiated by VEGF or Ang-1 and leads to angiogenesis. In endothelial cells, identified GPCR ligands include sphingosine-1-phosphate (S1P), angiotensin II, CXCL-16, and shear stress. One of the candidate GPCR ligands that may activate p110gamma in endothelial cells is sphingosine-1-phosphate (S1P). S1P induces endothelial cell proliferation, migration, and morphogenesis in vitro and in vivo. Moreover, EDG1, a GPCR-type S1P receptor, is essential for vascular maturation. This study shows that the p110gamma deficiency partially blocks enhanced angiogenesis driven not only by Ang-1 or VEGF-A but also by S1P. However, S1P-induced activation of PKB/Akt and MAPK is not suppressed by p110gamma deficiency, indicating that the major downstream target of S1P may not be p110gamma (Hamada, 2005).
Although PTEN has dual lipid and protein phosphatase activities, the results clearly demonstrate that a primary function of PTEN is to fine-tune the intracellular level of PIP3 produced by PI3Ks and thereby regulate vascular remodeling and tumor angiogenesis. The data also suggest the functional overlapping of class IA and IB PI3Ks in angiogenesis. This hypothesis is supported by the lack of an endothelial cell phenotype in mice lacking p85alpha, p85ß, p110ß, or p110gamma or in p110delta 'kinase dead' knock-in mice. Furthermore, the results demonstrate that, among the multiple PIP3 phosphatases, PTEN has an exclusive role in down-regulating PIP3 in endothelial cells. Various PI3Kgamma-specific inhibitors that are under investigation as anti-inflammatory drugs may therefore also be useful as cancer therapies targeting tumor angiogenesis (Hamada, 2005).
This study is the first report of the functional analysis of Pten and PI3Kgamma in murine endothelial cells in vivo. The normal function of the PI3K-PKB/Akt-Pten pathway in endothelial cells is required for cardiovascular development, and loss of Pten-mediated control of this pathway enhances tumor angiogenesis. Deficiency in Pten function thus contributes both to susceptibility to new tumorigenic mutations and to accelerated tumor growth. Inhibition of the PI3K pathway, including PI3Kgamma, is thus an attractive therapeutic target for the treatment of various malignancies (Hamada, 2005).
The role of tumor suppressor haploinsufficiency in oncogenesis is
still poorly understood. The PTEN and TSC2 tumor suppressors function
to antagonize mTOR (mammalian target of rapamycin) activation by Akt;
hence, compound heterozygous inactivation of Pten and
Tsc2 in the mouse may in principle exacerbate the tumor
phenotypes observed in the single mutants in a reciprocal manner. In
contrast, it was found that while Tsc2 heterozygosity unmasks
Pten haploinsufficiency in growth and tumor suppression,
tumorigenesis in Tsc2+/- mutants is surprisingly not
accelerated by Pten heterozygosity, even though mTOR
activation is cooperatively enhanced by compound Pten/Tsc2
heterozygosity. The wild-type alleles of both
Pten and Tsc2 are retained in prostate tumors from both
Pten+/- and Pten+/-Tsc2+/- mice,
whereas TSC-related tumor lesions are invariably associated with
Tsc2 loss of heterozygosity (LOH) in both
Tsc2+/- and Pten+/-Tsc2+/-
mice. These findings demonstrate that inactivation of TSC2 is
epistatic to PTEN in the control of tumor initiation and
progression and, importantly, that both Pten and Tsc2
are haploinsufficient for suppression of tumorigenesis initiated by
Pten heterozygosity, while neither Pten nor Tsc2
is haploinsufficient for repression of carcinogenesis arising from
Tsc2 heterozygosity, providing a rationale for the
differential cancer susceptibility of the two human conditions
associated with PTEN or TSC2 heterozygous
mutations (Ma, 2005).
The PTEN and TSC2 tumor suppressors inhibit mammalian target of rapamycin (mTOR) signaling and are defective in distinct hamartoma syndromes. Using mouse genetics, it has been found that Pten and Tsc2 act synergistically to suppress the severity of a subset of tumors specific to loss of each of these genes. Interestingly, the slow-growing tumors specific to Tsc2+/- mice exhibit defects in signaling downstream of Akt. However, Pten haploinsufficiency restores Akt signaling in these tumors and dramatically enhances their severity. This study demonstrates that attenuation of the PI3K-Akt pathway in tumors lacking TSC2 contributes to their benign nature (Manning, 2005).
The enzyme mTOR (mammalian target of rapamycin) is a major target for therapeutic intervention to treat many human diseases, including cancer, but very little is known about the processes that control levels of mTOR protein. This study shows that mTOR is targeted for ubiquitination and consequent degradation by binding to the tumor suppressor protein FBXW7. Human breast cancer cell lines and primary tumors showed a reciprocal relation between loss of FBXW7 and deletion or mutation of PTEN (phosphatase and tensin homolog), which also activates mTOR. Tumor cell lines harboring deletions or mutations in FBXW7 are particularly sensitive to rapamycin treatment, which suggests that loss of FBXW7 may be a biomarker for human cancers susceptible to treatment with inhibitors of the mTOR pathway (Mao, 2008).
The tumour stroma is believed to contribute to some of the most malignant characteristics of epithelial tumours. However, signalling between stromal and tumour cells is complex and remains poorly understood. This study shows that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumours. This was associated with the massive remodelling of the extracellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumours ameliorated disruption of the tumour microenvironment and was sufficient to decrease tumour growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumour stroma of patients with breast cancer. These findings identify the Pten-Ets2 axis as a critical stroma-specific signalling pathway that suppresses mammary epithelial tumours (Trimboli, 2009).
Genetic S6K1 (see Drosophila S6k) inactivation can induce apoptosis in PTEN-deficient cells (see Drosophila Pten). This study analyzed the therapeutic potential of S6K1 inhibitors in PTEN-deficient T cell leukemia and glioblastoma. Results revealed that the S6K1 inhibitor LY-2779964 was relatively ineffective as a single agent, while S6K1-targeting AD80 induced cytotoxicity selectively in PTEN-deficient cells. In vivo, AD80 rescued 50% of mice transplanted with PTEN-deficient leukemia cells. Cells surviving LY-2779964 treatment exhibited inhibitor-induced S6K1 phosphorylation due to increased mTOR-S6K1 (see Drosophila Tor) co-association, which primed the rapid recovery of S6K1 signaling. In contrast, AD80 avoided S6K1 phosphorylation and mTOR co-association, resulting in durable suppression of S6K1-induced signaling and protein synthesis. Kinome analysis revealed that AD80 coordinately inhibits S6K1 together with the TAM family tyrosine kinase AXL. TAM suppression by BMS-777607 or genetic knockdown potentiated cytotoxic responses to LY-2779964 in PTEN-deficient glioblastoma cells. These results reveal that combination targeting of S6K1 and TAMs is a potential strategy for treatment of PTEN-deficient malignancy (Liu, 2017).
A broad spectrum of mutations in PTEN, encoding a lipid phosphatase that inactivates the P13-K/AKT pathway, is found associated with primary tumors. Some of these mutations occur outside the phosphatase domain, suggesting that additional activities of PTEN function in tumor suppression. This study reports a nuclear function for PTEN in controlling chromosomal integrity. Disruption of Pten leads to extensive centromere breakage and chromosomal translocations. PTEN was found localized at centromeres and physically associated with CENP-C, an integral component of the kinetochore. C-terminal PTEN mutants disrupt the association of PTEN with centromeres and cause centromeric instability. Furthermore, Pten null cells exhibit spontaneous DNA double-strand breaks (DSBs). PTEN acts on chromatin and regulates expression of Rad51, which reduces the incidence of spontaneous DSBs. These results demonstrate that PTEN plays a fundamental role in the maintenance of chromosomal stability through the physical interaction with centromeres and control of DNA repair. It is proposed that PTEN acts as a guardian of genome integrity (Shen, 2007).
Scribble (SCRIB) localizes to cell-cell junctions and regulates establishment of epithelial cell polarity. Loss of expression of SCRIB functions as a tumor suppressor in Drosophila and mammals, conversely, overexpression of SCRIB promotes epithelial differentiation in mammals. This study reports that SCRIB is frequently amplified, mRNA over-expressed and protein is mislocalized from cell-cell junctions in human breast cancers. High levels of SCRIB mRNA are associated with poor clinical prognosis identifying an unexpected role for SCRIB in breast cancer. Transgenic mice expressing a SCRIB mutant (Pro 305 to Leu (P305L)) that fails to localize to cell-cell junctions, under the control of the mouse mammary tumor virus long terminal repeat promoter, develop multifocal hyperplasia that progresses to highly pleomorphic and poorly differentiated tumors with basal characteristics. SCRIB interacts with PTEN and the expression of P305L, but not wild-type SCRIB, promotes an increase in PTEN levels in the cytosol. Overexpression of P305L, but not wild type SCRIB, activates the Akt/mTOR/S6K signaling pathway. Human breast tumors overexpressing SCRIB have high levels of S6K but do not harbor mutations in PTEN or PIK3CA, identifying SCRIB amplification as a mechanism of activating PI3K signaling in tumors without mutations in PIK3CA or PTEN. Thus, this study has demonstrated that high levels of mislocalized SCRIB functions as a neomorph to promote mammary tumorigenesis by affecting subcellular localization of PTEN and activating an Akt/mTOR/S6kinase signaling pathway (Feigin, 2014).
The phosphatidylinositol 3-kinase signaling pathway has inherent
oncogenic potential. It is up-regulated in diverse human cancers by either a
gain of function in PI3K itself or in its downstream target Akt, or by a loss of
function in the negative regulator PTEN. However, the complete consequences of
this up-regulation are not known. Insulin and epidermal growth
factor or an inactivating mutation in the tumor suppressor PTEN specifically
increase the protein levels of hypoxia-inducible factor (HIF) 1alpha but not of
HIF-1beta in human cancer cell lines. This specific elevation of HIF-1alpha
protein expression requires PI3K signaling. In the prostate carcinoma-derived
cell lines PC-3 and DU145, insulin- and epidermal growth factor-induced
expression of HIF-1alpha is inhibited by the PI3K-specific inhibitors LY294002
and wortmannin in a dose-dependent manner. HIF-1beta expression is not affected
by these inhibitors. Introduction of wild-type PTEN into the PTEN-negative PC-3
cell line specifically inhibits the expression of an HIF-1alpha but not that of
HIF-1beta. In contrast to the HIF-1alpha protein, the level of HIF-1alpha mRNA
is not significantly affected by PI3K signaling. Vascular endothelial growth
factor reporter gene activity is induced by insulin in PC-3 cells and is
inhibited by the PI3K inhibitor LY294002 and by the coexpression of a HIF-1
dominant negative construct. Vascular endothelial growth factor reporter gene
activity is also inhibited by expression of a dominant negative PI3K construct
and by the tumor suppressor PTEN (Jiang, 2001).
Chondrocytes within the growth plates acclimatize themselves to a variety of stresses that might otherwise disturb cell fate. The tumor suppressor PTEN has been implicated in the maintenance of cell homeostasis. However, the functions of PTEN in regulating chondrocytic adaptation to stresses remain largely unknown. This study created chondrocyte-specific Pten knockout mice (Ptenco/co;Col2a1-Cre) using the Cre-loxP system. Following AKT activation, Pten mutant mice exhibit dyschondroplasia resembling human enchondroma. Cartilaginous nodules originate from Pten mutant resting chondrocytes that suffer from impaired proliferation and differentiation, and this is coupled with enhanced endoplasmic reticulum (ER) stress. It was further found that ER stress in Pten mutant chondrocytes only occurs under hypoxic stress, characterized by an upregulation of unfolded protein response-related genes as well as an engorged and fragmented ER in which collagens are trapped. An upregulation of hypoxia-inducible factor 1alpha (HIF1alpha) and downstream targets followed by ER stress induction was also observed in Pten mutant growth plates and in cultured chondrocytes, suggesting that PI3K/AKT signaling modulates chondrocytic adaptation to hypoxic stress via regulation of the HIF1alpha pathway. These data demonstrate that PTEN function in chondrocytes is essential for their adaptation to stresses and for the inhibition of dyschondroplasia (Yang, 2008).
Pten Evolutionary homologs part 1/3 | part 2/3 | part 3/3|
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