© 1999 by Oxford University Press
Journal of the National Cancer Institute, Vol. 91, No. 22, 1922-1932,
November 17, 1999
© 1999 Oxford University Press
REVIEW |
Mutational Spectra of PTEN/MMAC1 Gene: a Tumor Suppressor With Lipid Phosphatase Activity
Affiliations of authors: I. U. Ali, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD; L. M. Schriml, M. Dean, Laboratory of Genomic Diversity, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD.
Correspondence to: Iqbal U. Ali, Ph.D., National Institutes of Health, Executive Plaza North, Rm. 201, Bethesda, MD 20892-7332 (e-mail: ia1t{at}nih.gov).
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ABSTRACT |
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 Top Abstract IntroductionI. Germline PTEN/MMAC1 Mutations...II. Inactivation of PTEN/MMAC1...III. PTEN/MMAC1 as Tumor...NotesReferences |
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PTEN/MMAC1 (phosphatase, tensin homologue/mutated in multiple advanced cancers) is a tumor suppressor protein that has sequence homology with dual-specificity phosphatases, which are capable of dephosphorylating both tyrosine phosphate and serine/threonine phosphate residues on proteins. The in vivo function of PTEN/MMAC1 appears to be dephosphorylation of phosphotidylinositol 3,4,5-triphosphate. The PTEN/MMAC1 gene is mutated in the germline of patients with rare autosomal dominant cancer syndromes and in subsets of specific cancers. Here we review the mutational spectra of the PTEN/MMAC1 gene in tumors from various tissues, especially endometrium, brain, prostate, and ovary, in which the gene is inactivated very frequently. Germline and somatic mutations in the PTEN/MMAC1 gene occur mostly in the protein coding region and involve the phosphatase domain and poly(A)6 stretches. Compared with germline alterations found in the PTEN/MMAC1 gene, there is a substantially increased frequency of frameshift mutations in tumors. Glioblastomas and endometrial carcinomas appear to have distinct mutational spectra, probably reflecting differences in the underlying mechanisms of inactivation of the PTEN/MMAC1 gene in the two tissue types. Also, depending on the tissue type, the gene appears to be involved in the initiation or the progression of cancers. Further understanding of PTEN/MMAC1 gene mutations in different tumors and the physiologic consequences of these mutations is likely to open up new therapeutic opportunities for targeting this critical gene.
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INTRODUCTION |
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TopAbstractIntroductionI. Germline PTEN/MMAC1 Mutations...II. Inactivation of PTEN/MMAC1...III. PTEN/MMAC1 as Tumor...NotesReferences |
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Genetic alterations in a variety of critical genes underlie the formation and progression of human cancers. Some of these genes, such as tumor suppressors p53 (also known as TP53), Rb (retinoblastoma protein), and p16, are involved in central processes, such as transcriptional modulation and cell cycle regulation, and are frequently mutated in human cancers. Inactivation of another tumor suppressor gene PTEN/MMAC1 (phosphatase, tensin homologue/mutated in multiple advanced cancers) has been reported in several types of human tumors, including those from brain, breast, endometrium, kidney, and prostate (1-3). The name PTEN reflects the presence of the protein tyrosine phosphatase domain as well as the domain with homology to tensin. MMAC1 conveys the fact that the gene is mutated in multiple advanced cancers. Considerable information has accumulated within the last year on the mutations in the PTEN/MMAC1 gene in a wide variety of human cancers. In this review, we present the mutational spectra of the PTEN/MMAC1 gene in the germline as well as in tumors of various tissue types. The information was searched in the English language literature by use of the MEDLINE® database. We briefly discuss the enzymatic function of PTEN/MMAC1 with the purpose of understanding possible associations between distinct mutations and functional changes in specific malignancies.
PTEN/MMAC1 is located on chromosome 10q23.3 and encodes a predicted protein product of 403 amino acids containing a protein tyrosine phosphatase domain with a critical motif, IHCKAGKGRTG, found in dual specificity phosphatases (1-3), which dephosphorylate tyrosine as well as serine and threonine residues. The protein also has extensive identity to two cytoskeletal proteins—tensin, which appears to interact with actin filaments in focal adhesions, and auxilin, which is involved in synaptic vesicle transport (1-3). Mapping of homozygous deletions on chromosome 10q23 followed by representational difference analysis and positional cloning led to the isolation of the gene independently by two groups (1,2). Another approach utilized the conserved sequence motifs of phosphatases to isolate a novel protein tyrosine phosphatase, which turned out to be identical with PTEN/MMAC1 and whose transcription was modulated by transforming growth factor-ß (TGF-ß), giving the protein another name, TEP1 (TGF-ß-regulated and epithelial cell-enriched phosphatase) (3).
Three lines of evidence have established PTEN/MMAC1 as a tumor suppressor gene. First, germline mutations in PTEN/MMAC1 are associated with autosomal dominant hamartomatous and often cancer-predisposing syndromes, Cowden's disease and Bannayan-Zonana syndrome (see Section I). Second, the PTEN/MMAC1 gene is homozygously inactivated in a variety of sporadic human cancers (see Section II). Third, it is a highly conserved protein, with phosphatase activity capable of dephosphorylating tyrosine and serine/threonine residues of proteins as well as position 3 on the inositol ring of the lipid phosphatidylinositol 3,4,5-triphosphate (PIP3). Introduction of the PTEN/MMAC1 gene into cancer cell lines leads to growth suppression, and the heterozygous or homozygous inactivation of the gene in the mouse germline results in increased tumor susceptibility or embryonic lethality, respectively (see Section III).
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I. GERMLINE PTEN/MMAC1 MUTATIONS IN PATIENTS WITH HAMARTOMA SYNDROMES AND PREDISPOSITION TO CANCERS |
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TopAbstractIntroductionI. Germline PTEN/MMAC1 Mutations...II. Inactivation of PTEN/MMAC1...III. PTEN/MMAC1 as Tumor...NotesReferences |
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The hamartomatous polyposis syndromes, Cowden's disease (Mendelian Inheritance in Man [MIM] database No. 158350), Bannayan-Zonana syndrome (MIM No. 153480), and juvenile polyposis coli (MIM No. 174900), are rare autosomal dominant disorders with the common feature of the development of hamartomas, which are hyperplastic, disorganized, and nonmalignant growths. All three syndromes appear to have distinct clinical features as well as partial overlap in their clinical phenotype. The predominant phenotype of Cowden's disease is hamartomas of the skin; other organ sites developing hamartomas include breast, thyroid, endometrium, gastrointestinal tract, and central nervous system, with breast cancers developing in 25%-50% of affected women and thyroid cancer in 3%-10% of affected individuals (4,5). Lhermitte-Duclos disease, which is characterized by an unusual central nervous system tumor, cerebellar dysplastic gangliocytoma, is also associated with Cowden's disease (6). Juvenile polyposis coli patients develop hamartomatous polyps of the digestive tract and are reported to be predisposed to gastrointestinal cancer and possibly to pancreatic cancer (7), whereas in the case of Bannayan-Zonana syndrome, no definite documentation exists regarding an increased risk of malignancy (8).
Germline mutations in the PTEN/MMAC1 gene have been detected in Cowden's disease and in Bannayan-Zonana syndrome families (9-25). In juvenile polyposis coli syndrome, however, the presence of PTEN/MMAC1 germline mutations has been controversial (26,27) for multiple reasons, including overlap of phenotypic features with Cowden's disease and Bannayan-Zonana syndrome, age-related penetrance of the disease, and genetic heterogeneity (28,29). Recently, linkage for juvenile polyposis coli was established on the chromosomal region 18q21.1 (30), and germline mutations in the SMAD4 tumor suppressor gene were detected in a subset of juvenile polyposis coli families (31).
The frequency and types of mutations reported in multiple studies in Cowden's disease
and in Bannayan-Zonana syndrome families are shown in Fig. 1.
Of the 95
germline mutations reported, 25 are frameshift mutations and the rest are point mutations
comprising 35 nonsense mutations, 24 missense mutations, and 11 base substitutions generating
splice variants. Altogether, 75% of the germline mutations corresponding to nonsense,
frameshift, and splice-site mutations result in the generation of a truncated protein. A very high
proportion of the missense mutations, 13 (54%) of 24, cluster in the phosphatase core
motif beween residues 122 and 132 in exon 5, altering primarily glycine 129 and arginine 130. A
similar proportion, 19 (54%) of 35 nonsense mutations, target three codons, 130 and 157
in exon 5 and 233 in exon 7 of the PTEN/MMAC1 gene. Residue 233 is altered by nonsense
mutations in one Bannayan-Zonana syndrome and in five Cowden's disease families (9,11,14,15).
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A relatively low frequency or even absence of PTEN/MMAC1 mutations in some Cowden's disease and Bannayan-Zonana syndrome (10,22,32) patients has been reported, suggesting genetic heterogeneity. Nevertheless, the germline mutations reported so far argue that, in most Cowden's disease families and in a subset of Bannayan-Zonana syndrome families, PTEN/MMAC1 is the susceptibility gene and that these hamartomatous polyposis syndromes are allelic to each other. Furthermore, deletion of the wild-type allele of the PTEN/MMAC1 gene detected in the DNAs from hamartomas of various organs analyzed in Cowden's disease patients and from normal tissues adjacent to the tumors indicates that loss of its expression is an early event leading to increased proliferation, tissue disorganization, and susceptibility to tumor development (11,14). This contention is strongly supported by the recent observation that mice with germline heterozygous mutations in PTEN/MMAC1 develop increased susceptibility to tumors of various tissues (33-35).
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II. INACTIVATION OF PTEN/MMAC1 GENE IN MULTIPLE HUMAN TUMORS |
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TopAbstractIntroductionI. Germline PTEN/MMAC1 Mutations...II. Inactivation of PTEN/MMAC1...III. PTEN/MMAC1 as Tumor...NotesReferences |
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Besides the germline mutations in inherited hamartomatous syndromes, somatic mutations in the PTEN/MMAC1 gene are frequently found in a variety of sporadic human tumors in which the wild-type allele of the gene is inactivated mostly by deletions, thus conforming to the classical paradigm of tumor suppressor genes. The region on chromosome 10q that harbors the PTEN/MMAC1 gene and very likely other tumor suppressor gene(s) is hemizygously deleted in many human cancers, with a frequency reaching 60%-80% in the case of prostate cancer, endometrial carcinoma, and advanced glial tumors. Studies carried out within the last year have established PTEN/MMAC1 as the likely target gene of these deletions in many human tumors. The gene is inactivated by multiple mechanisms, including homozygous genomic deletions. In tumors with hemizygous deletions at chromosome 10q23, frameshift or nonsense mutations or mutations resulting in splice variants prematurely terminate the open reading frame of the remaining copy of the gene, thus producing a truncated and nonfunctional protein. Alternatively, the remaining copy of the gene is altered by missense mutations that are predicted to severely impair the function of its protein product. A detailed analysis of the nature and frequency of PTEN/MMAC1 gene mutations in different cancers may provide insights into the underlying genetic mechanisms of tumorigenesis in specific tissue types.
Endometrial Cancer
Endometrial carcinoma is the second most common noncolonic malignancy in hereditary nonpolyposis colon cancer (HNPCC) families, whose members carry germline mutations in mismatch repair genes resulting in replication errors and microsatellite instability (36). Among sporadic endometrial cancers, microsatellite instability is present only at a frequency of 20% (37). The two histologic variants of endometrial carcinoma, the endometrioid and serous types, appear to carry distinct genetic lesions. The less common but more aggressive serous carcinoma harbors mutations in the p53 gene with about 90% frequency, whereas K-ras mutations are restricted to the endometrioid type and are present in 20% of the tumors (38,39).
Several reports (39-45) show that PTEN/MMAC1 is the most
frequently mutated gene identified yet in endometrial cancers. Inactivation of the PTEN/MMAC1
gene appears to be confined to tumors of endometrioid histology. In six different studies analyzing
a total of 286 tumors (39-45), PTEN/MMAC1 mutations were detected
in 33%-55% of the endometrial cancers, the highest frequency among the different
tumor types examined so far (Table 1). Furthermore, PTEN/MMAC1
mutations occur in early well-differentiated lesions (stage I/grade 1) as well as in very advanced
and invasive tumors (stage IV/grade 3), suggesting their involvement in the initiation of
endometrial tumorigenesis. That the inactivation of the PTEN/MMAC1 gene is an early event in
endometrial carcinogenesis is also supported by data from two independent studies (46,47) showing a 22% frequency of PTEN/MMAC1 mutations in premalignant
lesions of endometrial hyperplasia both with and without atypia.
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Other striking features of the mutational profile of PTEN/MMAC1 in endometrial cancer are the substantially high frequency of mutations in microsatellite instability-positive tumors (78%-86%) compared with the microsatellite instability-negative tumors (
30%) as well as the relative abundance of frameshift mutations (Fig. 2, A). It is not clear if the elevated frequency of frameshift mutations in endometrial
carcinoma is a manifestation of the tumor's microsatellite instability-positive phenotype. A
causal relationship is believed to exist between defective mismatch repair systems and alterations
in microsatellite sequences in HNPCC (36). DNA polymerase slippage
events at microsatellite repeat sequences lead to misalignment of DNA strands, which, if not
repaired, give rise to deletions or insertions, resulting in frameshift mutations (48). However, the mutations in the PTEN/MMAC1 gene in endometrial carcinoma do
not appear to be a consequence of a generalized replication error phenotype, as suggested by the
following observations: 1) The frequency of mutations in the known DNA repair genes in
endometrial carcinomas appears to be very low, 2) the repeat sequence in the transforming
growth factor receptor BII gene, which is almost always mutated in microsatellite
instability-positive colon cancers, is rarely mutated in endometrial cancers (49), and 3) the mutational spectrum of the PTEN/MMAC1 gene in microsatellite
instability-negative endometrioid tumors does not differ from that in microsatellite
instability-positive tumors (40,41). These observations argue that the
PTEN/MMAC1 gene may be specifically targeted for mutations in endometrial carcinoma and is
necessary for tumorigenesis.
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Malignant Glial Tumors
Evolution of glioblastomas, the highest malignancy grade glial tumors of the brain, is believed to occur via multiple genetic pathways. Notably, two pathways for which the molecular details are well characterized (50) are readily discernible in the formation of malignant gliomas. One subset of glioblastomas evolves from low-grade gliomas, which commonly harbor p53 mutations, via a progression pathway accumulating multiple genetic lesions that often include loss of chromosome 10 sequences. Alternatively, glioblastomas can develop in a de novo fashion following epidermal growth factor receptor amplification and gene losses on chromosome 10. Thus, loss of heterozygosity on chromosome 10 is a characteristic feature of high-malignancy-grade gliomas arising via both genetic pathways and occurs in 60%-80% of the tumors (50). PTEN/MMAC1 appears to be one of the target genes on chromosome 10 in glioblastomas and is found to be mutated in combination with either p53 mutations or epidermal growth factor receptor amplification (51). However, in many glioblastomas, the PTEN/MMAC1 gene is inactivated without accompanying alterations in either p53 or epidermal growth factor receptor genes. Thus, amplification of epidermal growth factor receptor, which harbors a receptor tyrosine kinase activity, and mutations in PTEN/MMAC1, with an associated phosphatase activity, appear to be independent of each other (51,52).
A total of 674 malignant glial tumors (anaplastic astrocytomas and glioblastomas multiforme) were analyzed, and PTEN/MMAC1 mutations were reported in 159 of these tumors, i.e., at a frequency of 24% (1,2,51-63). That PTEN/MMAC1 is not the only target for inactivation on chromosome 10 in gliomas is suggested by the fact that a considerable fraction of tumors with allelic losses on chromosome 10 retain a normal copy of the gene. Deletion mapping as well as functional studies have provided evidence for the presence of multiple tumor suppressor genes on both arms of chromosome 10 that may be involved in the development of glial neoplasms of different malignancy grades (64). Another putative tumor suppressor gene, DMBT1 (deleted in malignant brain tumors) with homology to the scavenger receptor cysteine-rich superfamily, has been identified on chromosome 10q25.3-26.1, which appears to be homozygously deleted in a subset of glioblastomas (65).
The spectrum of somatic mutations in the PTEN/MMAC1 gene in glioblastomas encompasses
insertions, deletions, and point mutations, with the latter constituting about 60% of total
mutations (Fig. 2,
B). It is not clear if this mutational profile is
attributable to alterations in the mismatch repair genes. Glioblastomas have been reported to be
weakly microsatellite instability positive (66), and an association appears
to exist between reduced levels of expression of multiple mismatch repair genes and tumor
progression (67). It has been reported that MSH6, along with MSH2,
functions in repairing single-base mismatches and smaller insertions/deletions and that cells
derived from MSH6-deficient mice have a single nucleotide mismatch repair defect (68). Molecular profiling of the mismatch repair genes, in particular MSH6, in
glioblastomas would be helpful in understanding the mutational spectrum of PTEN/MMAC1 with
a relative abundance of base substitutions, a feature characteristic so far of the p53 gene, as
opposed to frameshift mutations.
Prostate Carcinoma
Deletion mapping analysis of chromosome 10 in prostate carcinoma
identified a complex pattern of allelic losses that includes 10q23.3,
the region where the PTEN/MMAC1 gene resides (69). Analysis of
200 primary prostate carcinomas and 71 metastases from multiple sites,
including those from pelvic lymph nodes, revealed 16 homozygous
deletions and 10 mutations (70-74) (Table 1; Fig.
2, C). Of
these, 13 (18%) tumors with homozygous deletions or point mutations
derived from 71 metastases. The rather low frequency of mutations
(10%) in primary and metastatic prostate carcinoma, which included
both homozygous deletions and intragenic mutations, could be due to
several factors. First, loss of the PTEN/MMAC1 gene may be particularly
relevant to the progression and metastasis of advanced prostate
carcinomas, a conclusion suggested by two studies (70,71).
Second, tumors were not always screened for homozygous deletions and
there may be a higher frequency of homozygous deletions than reported.
Finally, the possibility exists that inactivation of the PTEN/MMAC1
gene occurs by mechanisms other than deletions and mutations. In this
context, loss of expression or reduced expression of PTEN/MMAC1, both
at the messenger RNA and protein levels, was recently demonstrated in
xenografted prostate tumors (75). Restoration of PTEN/MMAC1
expression by demethylation in cells derived from the xenografts
suggests loss of expression by promoter methylation (75).
However, this mode of inactivation requires further exploration, since
promoter methylation of the PTEN/MMAC1 gene could not be detected in
prostate tumors with allelic loss of the gene and wild-type sequence of
the remaining copy (70).
Ovarian Tumors
Epithelial ovarian tumors exist as four major histologic types
(endometrioid, serous, mucinous, and clear cell), which probably evolve
via distinct molecular pathways. Analysis of all four histologic types
of ovarian tumors for the loss of heterozygosity on chromosome 10 and
mutations in the PTEN/MMAC1 gene indicated that the gene is mutated
predominantly, if not exclusively, in ovarian tumors of endometrioid
origin (76,77). Although allelic losses on chromosome 10 were
common in both endometrioid (43%) and serous (28%) tumors, PTEN/MMAC1
mutations were detected only in endometrioid ovarian tumors with a 26%
frequency (Table 1) and in one of the 10 mucinous tumors examined that
displayed a mixed mucinous and endometrioid histology. Thus, an
important observation is that PTEN/MMAC1 mutations are common in
endometrial as well as ovarian tumors of endometrioid histology and
that tumors of both types containing such mutations are well or
moderately differentiated, suggesting the involvement of PTEN/MMAC1
tumor suppressor function in disease initiation.
Other Cancers
Approximately 25%-50% of women with Cowden's disease develop
breast cancers (5,6). It was, therefore, of interest to
determine if PTEN/MMAC1 is involved in the pathogenesis of sporadic
breast tumors. Screening of the PTEN/MMAC1 gene in 116 primary breast
tumors, most of which included stage I and II tumors, showed an
extremely low frequency of mutations (5%) (53,78,79) (Table
1). It is conceivable that PTEN/MMAC1 is inactivated mainly in advanced
and metastatic breast tumors (79), as is the case in glial and
prostatic tumors. It is, however, also possible that different subsets
of genes are involved in sporadic versus inherited breast cancers, as
is suggested by the absence of mutations in sporadic tumors of BRCA1
and BRCA2 genes responsible for inherited breast cancer in a number of
pedigrees (80).
Thyroid cancer is also among the clinical manifestations of Cowden's disease. In an initial screen of 95 sporadic thyroid tumors encompassing many histologic types, one frameshift mutation in a papillary thyroid carcinoma was detected (81). It is possible that inactivation of the gene is associated with a particular histologic type. Alternatively, the gene may be inactivated in some tumor types by mechanisms other than mutations in the coding region.
Genetic alterations in the PTEN/MMAC1 gene were reported in seven of 67 head and neck
squamous cell carcinomas (53,82,83), in one of eight kidney cancers (53), in four of 99 lung carcinomas (53,84-86), in
three of 27 melanomas (53,87), in one of 29 gastric carcinomas (41), and in three of 68 lymphomas (88,89)
examined (Table 1). Although the PTEN/MMAC1 gene is very frequently
inactivated in high-malignancy-grade gliomas, mutations in the gene were detected at a much
lower frequency in many other types of brain tumors (52,55,56,59-61,63,90). Of the 427 brain tumors of various types examined, including low-grade
astrocytomas, pilocytic astrocytomas, medulloblastomas, oligodendrogliomas, oligoastrocytomas,
and meningiomas, PTEN/MMAC1 alterations were detected only in seven tumors. Similarly,
PTEN/MMAC1 appears to play a role in the development of endometrial and ovarian tumors of
endometrioid histology, but mutations were not detected in other tumors of the female genital
tract, such as the serous type of ovarian and endometrial tumors and squamous cervical carcinoma
(40,76,77,91). Thus, the data obtained so far suggest that inactivation of
the PTEN/MMAC1 gene is relevant in the formation of tumors of specific histology and tissue
type.
Distribution of Mutations in PTEN/MMAC1 Gene
We have compiled PTEN/MMAC1 mutations in a variety of cancers
reported through May 1999. In a total of about 2300 tumors analyzed and
sequenced, 374 inactivating mutations, which include 44 homozygous
deletions, have been reported (Table 1). Many tumor types were not
examined specifically for homozygous deletions of the PTEN/MMAC1 gene
or for mutations in the regulatory sequences; therefore, the overall
mutation frequency of 16% may be an underestimation. Although the
mutations are scattered over the entire gene, clustering of missense
mutations in exons 5 and 6 (Fig. 2, D), of nonsense mutations in exon 7
(Fig. 2, E), and of frameshift mutations in and around the
poly(A)6 stretches and phosphorylation sites in exon 7 and
especially in exon 8 (Fig. 2, F) is obvious. The phosphatase domain
located in exon 5 is the target of missense mutations. Most striking is
the observation that the phosphatase core motif that constitutes less
than 3% of the gene is the site of about 31% and 23% of point
mutations reported so far in germline and sporadic tumors,
respectively. A vast majority of germline mutations (74%) are point
mutations compared with 51% of the somatic mutations detected in
various tumors (two-sided P<.00003; Fisher's exact test );
in particular, nonsense mutations are substantially elevated in the
germline. In contrast, 49% of the inactivating mutations in the
PTEN/MMAC1 gene in tumors were frameshift mutations versus 26% in the
germline (Table 2).
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Among the missense mutations, transitions and transversions occur with about equal frequency. Altogether, 50 base substitutions in tumors result in a termination codon, and 22 of these (i.e., 44%) occur in three codons containing the dinucleotide CpG. There are nine codons containing CpG dinucleotides in the PTEN/MMAC1 open reading frame, three of which occur as CGA at codons 130, 233, and 335. The cytosine residue in these three codons is frequently mutated to thymine, thus terminating the open reading frame at the stop codon. Ten tumors had mutations at codon 130, which is located in the phosphatase core motif, and eight and four tumors each were mutated at codons 233 and 335, respectively. Another CpG dinucleotide present in codon 173 as CGC is mutated in 12 tumors; in eight of those tumors, the cytosine is replaced by thymine, resulting in the substitution of arginine for cysteine. This is consistent with the observation that frequent methylation of cytosine residues in CpG dinucleotides renders them prone to spontaneous deamination and a consequent C to T transition that occurs at a much higher rate than in unmethylated bases (92). The spectrum of point mutations in human tumors as well as in the germline also indicates that about one third of all base substitutions occur at a CpG dinucleotide.
Most of the PTEN/MMAC1 mutations were detected in glioblastomas and endometrial,
ovarian, and prostate carcinomas. The mutational spectrum appears to have characteristic features
for the tumors of each tissue type. In endometrial carcinoma, 60% of the mutations are
frameshift versus 37% in glioblastomas (two-sided P<.0004; Fisher's
exact test) (Table 2). Most of these frameshift mutations are 1-4 base
pairs (bp) in length and occur predominantly in the N-terminal region of the gene or in exons 7
and 8, which contain potential tyrosine and/or serine phosphorylation sites (Fig. 2, A). Most notably, 15 single-base frameshift mutations (seven insertions and eight
deletions of A) are clustered within the poly(A)6 stretch in codons 321-323 and nine
tumors sustained a loss of 4 bp at the palindromic repeat sequence (TACTTACTTTA) at codons
317-320 (Fig. 2, A). Similarly, at another poly(A)6 stretch
between codons 265-267, there was a loss of a single A in six tumors. One of the mechanisms for
the generation of short deletions and insertions involves mispairing of the DNA strands at repeat
sequences. Mononucleotide runs are believed to be the site of slipped misalignment of the
template DNA strand, resulting in deletion or insertion if the nucleotide(s) excluded from pairing
is on the template or primer strand, respectively. There is a clustering of missense mutations (23
[77%] of 30 missense mutations) in exon 5 and nonsense mutations (10
[45%] of 22 nonsense mutations) in exon 7 (Fig. 2, A).
The mutational spectrum of PTEN/MMAC1 in glioblastomas is distinct from that in
endometrial carcinomas. In comparison with endometrial tumors, glioblastomas exhibit
substantially fewer frameshift alterations but more missense mutations (Table 2; Fig. 2, B). This may reflect a different role for
PTEN/MMAC1 in these two tumor types and/or differences in the mechanisms of mutation. Of
the 150 mutations reported, 94 are single-base substitutions (i.e., 63% versus 40%
in endometrial carcinoma), generating 61 missense, 21 nonsense, and 12 splice site variants (Table
2). Conspicuously, 17 mutations are clustered between codons 170 and
codon 173 in exon 6, 10 of which (one frameshift and nine missense mutations) target codon 173,
which contains one of the nine CpG sites of the gene and encodes for a highly conserved residue.
Mutations reported in various other cancers are shown in Fig. 2, C.
Although the numbers are small, about 72% of the mutations (26 of 36) result in truncation
of the protein. Five of the nine mutations reported in ovarian carcinoma are in the phosphatase
core motif, four of them targeting codon 130.
The codons most frequently and differentially targeted by both germline as well as somatic
mutations are listed in Table 3. Codons 129 and 130, which are located in
the phosphatase core motif, are mutated at much higher frequencies in the germline than in
various tumors. Codon 157, which is probably important for stabilizing the tertiary structure of
the protein, is mutated in four Cowden's disease families but is not altered in any of the
tumors. Of the four codons containing CpG dinucleotides that are frequently mutated in the
germline and/or tumors, codon 130 is in the phosphatase core motif; 173 is conserved in tensin,
auxilin, and bacterial phosphatase (2,3); and codons 233 and 335 are
within or near tyrosine and/or serine phosphorylation sites. One striking feature is that codon 173
and its neighboring residues are predominantly mutated in glioblastomas with nine of the 12 point
mutations occurring in glial tumors. Analysis of the enzymatic as well as tumor suppressor
functions of the PTEN/MMAC1 protein mutated at codon 173 and elucidation of its signaling
pathways in glial cells would be helpful in understanding the significance of mutational targeting
of Arg 173 in glioblastomas.
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The most obvious difference between the germline and somatic mutations is a very high density of somatic frameshift mutations at and around the two poly(A)6 stretches encompassing codons 265-267 and especially between codons 321 and 323, which is directly preceded by a palindromic sequence and a tyrosine phosphorylation site at codon 315. While there are differences between the germline and somatic mutations in terms of both the types and the frequency of mutations at specific codons/sites, the net effect in both cases is the generation of a truncated and probably nonfunctional or severely impaired protein.
Comparison of Mutational Spectra of Various Tumor Suppressor Genes With PTEN/MMAC1
Germline mutations in the PTEN/MMAC1 gene detected in the familial
Cowden's disease and Bannayan-Zonana syndrome as well as somatic
mutations in various tumors are distributed over the entire gene, with
a clustering in three regions, i.e., phosphorylation sites in exons 7
and 8 and particularly in the phosphatase core motif in exon 5 (Figs. 1
and 2). A great majority of these mutations, 62%-83%, are
made up of
frameshift, nonsense, and splice-site mutations, resulting in a
truncated protein, a feature in common with the mutational spectra of
other tumor suppressor genes. The germline mutations in BRCA1, BRCA2
(80), and patched (PTC) (93) genes are also
scattered
throughout these genes, and most are predicted to result in the
truncation of the proteins. In particular, in the case of BRCA2,
frameshift mutations constitute about 80% of the total mutations
(94,95). A high density of the somatic adenomatous polyposis
coli (APC) gene mutations occurs in the mutation cluster region,
whereas the germline mutations are scattered throughout the 5' half of
the gene (94). The mutational spectrum of the p53 gene is
unique in the sense that a vast majority of p53 mutations are missense
alterations (74%), with 22% of all mutations occurring in three hot
spots on amino acids 175, 248, and 273 (94,95). A
preponderance of missense mutations in the PTEN/MMAC1 gene (61 [41%]
of a total of 150 mutations) is observed only in glioblastomas. The
mutational spectrum of the PTEN/MMAC1 gene thus shares some common
features with other tumor suppressor genes, such as distribution of
mutations over the entire gene, clustering of mutations in three
important functional domains, i.e., the phosphatase core motif in exon
5 and phosphorylation sites in exons 7 and 8, and a large number of
missense mutations in some tumor types.
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III. PTEN/MMAC1 AS TUMOR SUPPRESSOR WITH DEFINED ENZYMATIC FUNCTION |
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TopAbstractIntroductionI. Germline PTEN/MMAC1 Mutations...II. Inactivation of PTEN/MMAC1...III. PTEN/MMAC1 as Tumor...NotesReferences |
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Structural and functional conservation is a hallmark of tumor suppressor genes. PTEN/MMAC1 exhibits remarkable structural similarity across species. The human gene is highly similar to yeast, mouse, and dog sequences, with a virtual sequence identity at the amino acid level between human and mouse genes (1-3). The protein product of the gene appears to have lipid phosphatase activity dephosphorylating the second messenger PIP3 (101-103). In the nematode Caenorhabditis elegans, DAF-18, a homologue of PTEN/MMAC1 functions in the insulin receptor-like signaling pathway. The role of DAF-18 is probably to decrease second messenger signaling by PIP3, thereby regulating metabolism, development, and lifespan of the worm (96,97). The conservation of signaling pathways between the nematodes and mammals raises the possibility that human PTEN/MMAC1, by virtue of its ability to modulate the phosphorylation of the second messenger-PIP3, may play a role not only in cancer development but also in aging and metabolic diseases (96,97).
Phosphorylation and dephosphorylation are key regulatory mechanisms in a variety of fundamental cellular processes, including cellular proliferation and differentiation. A critical balance among these processes is maintained by protein kinases, which are widely implicated in carcinogenesis, and protein phosphatases, which are the natural antagonists of the action of normal as well as oncogenic protein kinases and have been associated with cell cycle regulation and inhibition of cell proliferation (98,99). PTEN/MMAC1 is the first tumor suppressor gene identified in the phosphatase family (100), and the principal function of its gene product appears to be dephosphorylation of the second-messenger PIP3 (101-103). The growth-inhibitory function of PTEN/MMAC1 is evident when its wild-type complementary DNA is transfected into a variety of cancer cell lines, including glioblastoma, melanoma, and renal, breast, and prostate carcinomas (104-111). Mutant forms of PTEN/MMAC1 that retain protein phosphatase but lose lipid phosphatase activity fail to suppress the growth of glioma cell lines (107,110). The same mutations have been detected in Cowden's disease patients as well as in various sporadic tumors, providing strong evidence that lipid phosphatase activity is essential for its role in normal development and its tumor suppressor function. The growth-suppressive activity of PTEN/MMAC1 may be cell type specific and mediated by multiple mechanisms. In some cell lines, growth is suppressed by the ability of PTEN/MMAC1 to block cell cycle progression at the G1 phase by causing an increased synthesis of cyclin-dependent kinase inhibitor p27Kip1 (110,112,113), whereas, in other tumor cell lines, PTEN/MMAC1 has been shown to directly induce apoptosis (programmed cell death) (107,111,114-116).
The results in knockout mutant mice also demonstrate that the PTEN/MMAC1 plays an important role in both embryonic development and tumor suppression. Three independent knockout studies using different deletions in the PTEN/MMAC1 gene and mice with different genetic backgrounds showed differences in the details, such as timing of embryonic lethality, in the phenotype and in the spectrum of the lesions (34-36). One study (34) reported hyperplastic/dysplastic changes in the prostate, skin, and colon of PTEN/MMAC1 heterozygotes, resembling the features characteristic of Cowden's disease and Bannayan-Zonana syndrome. Neoplasms in multiple organs, including prostate, thyroid, gastrointestinal tract, and endometrium, were reported in another study (36), with multifocal endometrial complex atypical hyperplasia developing in all heterozygous females. This situation closely resembles human endometrial cancer, where PTEN/MMAC1 is the most commonly mutated gene and is involved in the early events of endometrial tumorigenesis with more than 20% of complex atypical hyperplasia containing mutations in the gene (46,47). Although leukemia is not a feature of Cowden's disease, a high incidence of T-cell lymphoma/leukemia was reported in two knockout studies (35,36).
Cells derived from PTEN/MMAC1-deficient mouse embryos contain elevated levels of intracellular PIP3 supporting a role of PTEN/MMAC1 in this signaling pathway (116). The intracellular levels of PIP3 directly control the phosphorylation of protein kinase B/Akt protooncogene, which is one of the key regulatory molecules of cell survival in response to multiple apoptotic stimuli (117). In PTEN/MMAC1-deficient mouse embryo fibroblasts, constitutively elevated activity of PKB/Akt together with decreased sensitivity to apoptosis suggests a role for PTEN/MMAC1 as a negative regulator of cell survival (116). An inverse association between the PTEN/MMAC1 protein and AKT/PKB was also detected in cell lines derived from hematologic malignancies (118).
Besides regulating the intracellular PIP3 levels, the phosphatase activity of the PTEN/MMAC1 protein might impact other cellular substrates/functions. In addition to modulating signal transduction and cell cycle progression, phosphorylation affects other cellular processes, such as cell-cell and cell-matrix adhesion, migration, and angiogenesis, processes that are important not only for normal cellular physiology but also impact tumor growth, invasion, and metastasis. The cytoplasmic location of the PTEN/MMAC1 protein (3) and its homology with tensin, which binds to actin filaments at focal adhesions and is implicated in the assembly of the signaling complexes, suggest that its physiologic substrates may reside in the cytoplasm. That PTEN/MMAC1 may regulate cellular signals originating from the cytoskeleton and/or focal adhesions is suggested by its inhibitory effects on integrin-mediated cell spreading, formation of actin-containing microfilaments and focal adhesions, and cell migration (119,120). These biologic activities of PTEN/MMAC1, at least in vitro, appear to be arbitrated by tyrosine dephosphorylation of the focal adhesion kinase, and the phosphatase domain of the gene is required for these functions (119,120). Inactivation of the PTEN/MMAC1 in human cancers may, therefore, contribute not only to their proliferation but also to their invasive potential.
Although the PTEN/MMAC1 is inactivated in multiple advanced cancers, it does show selectivity for specific target organs, as suggested by the absence or relative infrequency of mutations in various tumors. The pathway involving PTEN/MMAC1, PI3 kinase, and PKB/Akt appears to be critical in regulating cell survival in various tissues. A detailed characterization of this pathway and identification of other physiologic substrate(s)/mechanism(s) of action of PTEN/MMAC1 will be helpful in understanding the signaling pathways that are subverted not only during the progression of advanced cancers like glioblastomas and prostate carcinomas but also during the initiation of a large subset of endometrial and ovarian carcinomas.
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We thank Dr. Barbara Dunn for her critical comments on the manuscript.
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