REVIEW
Mutational Spectra of PTEN/MMAC1 Gene: a Tumor
Suppressor With Lipid Phosphatase Activity
Iqbal Unnisa Ali, Lynn M. Schriml, Michael Dean
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. [J Natl Cancer Inst 1999;91:
1922–32]
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
1922 REVIEW
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-␤ (TGF-␤), giving the protein another name, TEP1 (TGF-␤-regulated and epithelial cellenriched 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).
I. GERMLINE PTEN/MMAC1 MUTATIONS IN
PATIENTS WITH HAMARTOMA SYNDROMES AND
PREDISPOSITION TO CANCERS
The hamartomatous polyposis syndromes, Cowden’s disease
(Mendelian Inheritance in Man [MIM] database No. 158350),
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: [email protected]).
See “Notes” following “References.”
Journal of the National Cancer Institute, Vol. 91, No. 22, November 17, 1999
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).
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
Fig. 1. Germline PTEN/MMAC1 mutations in patients with Cowden’s disease and
Bannayan–Zonana syndrome. Red ⳱ frameshift mutations; green ⳱ missense
mutations; blue ⳱ nonsense mutations; black arrows below the bar ⳱ splice-site
variants generated by point mutations; arrows marked with B ⳱ mutations in
patients with Bannayan–Zonnana syndrome; and unmarked arrows ⳱ mutations
in patients with Cowden’s disease. Structural and functional domains of the PTEN/
MMAC1 gene are shown. Black vertical lines inside the bar ⳱ intron/exon
boundaries. Exon numbers are shown under the bar. The strongest homology
region to tensin/auxilin is in blue, with the hatched region showing the phosphatase domain and the solid red showing the phosphatase core motif. Poly(A)6
stretches between codons 265–267 (in exon 7) and codons 321–323 (in exon 8) and
the palindromic sequence preceding the second poly(A)6 stretch encompassing
codons 317–320 are shown in orange. Tyrosine phosphorylation sites with tyrosine residues at codons 240 (in exon 7), 315 (in exon 8), and 336 (in exon 8) are
shown in purple, and serine phosphorylation sites with serine residues at codons
338 (in exon 8) and 355 (in exon 9) are shown in green.
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).
II. INACTIVATION OF PTEN/MMAC1 GENE
MULTIPLE HUMAN TUMORS
IN
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
Journal of the National Cancer Institute, Vol. 91, No. 22, November 17, 1999
REVIEW 1923
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.
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 instabilitynegative 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 instabilitypositive 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.
Table 1. Mutations of the PTEN/MMAC1 gene in tumors of various tissue types*
Mutation type
No. of tumors
analyzed
HD
FS
MS
NS
SS
No. of
mutations
Frequency of
mutations, %
Reference
No.
Endometrium
Carcinoma
Hyperplasia
286
80
2
ND
74
11
25
5
20
2
7
0
128†
18†
45
22.5
(39–45)
(45,47)
Brain
GBM/AA
Others‡
674
427
16
ND
49
1
57
4
19
2
18
0
159
7
24
2
(1,2,51–63)
(52,55,56,59–61,63,90)
Prostate
271
16
5
2
2
1
26
10
(70–74)
38
1
3
3
3
0
10
26
(76,77)
116
1
4
1
0
0
6
5
(2,53,78,79)
Thyroid
95
0
1
0
0
0
1
>1
(81)
Head and neck
67
5
0
1
1
0
7
10
(82,83)
8
0
1
0
0
0
1
12.5
(53)
Lung
99
2
1
0
1
0
4
4
(53,84–86)
Melanoma
27
1
1
1
0
0
3
11
(53,87)
Gastric
29
0
1
0
0
0
1
3.4
Lymphoma
68
ND
0
2
0
1
3
4
2285
44
152
101
50
27
374
16
Tumor type
Ovary (endometrioid)
Breast
Kidney
All tumors
(41)
(88,89)
*AA ⳱ anaplastic astrocytoma; FS ⳱ frameshift mutation; GBM ⳱ glioblastoma multiforme; HD ⳱ homozygous deletions; MS ⳱ missense mutations; ND
⳱ not done/not detected; NS ⳱ nonsense mutations; SS ⳱ splice-site mutations. In-frame deletions and insertions, although they do not generate frameshifts, are
also included in FS mutations.
†Some of the endometrial tumors had two mutations each, probably one on each allele.
‡See text for details of tumor types.
1924 REVIEW
Journal of the National Cancer Institute, Vol. 91, No. 22, November 17, 1999
Fig. 2. Mutational spectra of PTEN/MMAC1 in human cancers. Mutations reported in various tumors are compiled (see Table 1 for references). Red ⳱
frameshift mutations; green ⳱ missense mutations; and blue ⳱ nonsense mutations. a) Mutation spectrum in endometrial carcinoma. b) Mutation spectrum
in brain tumors. c) Mutations in various tumors are as indicated: B ⳱ breast; G
⳱ gastric; HN ⳱ head and neck squamous cell carcinoma; K ⳱ kidney; Lu ⳱
lung; Ly ⳱ lymphoma; M ⳱ melanoma; O ⳱ ovarian cancer; P ⳱ prostate; and
T ⳱ thyroid. d) Spectrum of missense mutations. e) Spectrum of nonsense
mutations. f) Spectrum of frameshift mutations. Mutations in d–f are pooled
from a–c to present an overall representation of missense, nonsense, and frameshift mutations in the PTEN/MMAC1 gene. Black and red arrows under the
bar ⳱ splice-site variants generated by point mutations and frameshift mutations, respectively. Structural and functional domains of the PTEN/MMAC1
gene are described in the legend for Fig. 1.
Journal of the National Cancer Institute, Vol. 91, No. 22, November 17, 1999
REVIEW 1925
Prostate Carcinoma
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.
1926 REVIEW
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
Journal of the National Cancer Institute, Vol. 91, No. 22, November 17, 1999
(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 lowgrade 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).
Table 2. Summary of germline and somatic mutations in the
PTEN/MMAC1 gene*
Mutations
Total
No. of
mutations
Mutation type
FS†
Germline
95
Somatic
330‡
25 (26%)
Endometrium§
144‡
85 + 2 (60%)
Brain§
150‡
50 + 6 (37%)
MS NS SS
PM†
24
35
11
59 + 11 (74%)
153 + 10 (49%) 101
50
26
151 + 16 (51%)
30
22
7
52 + 5 (40%)
61
21
18
82 + 12 (63%)
*FS ⳱ frameshift; MS ⳱ missense; NS ⳱ nonsense; SS ⳱ splice site; PM
⳱ point mutations (missense, nonsense, and splice-site mutations generated by
single-base substitutions).
†Second number in FS and PM columns represents splice-site mutations generated by FS and PM, respectively.
‡Numbers do not include homozygous deletions.
§Mutations from the tumors of the endometrium and the brain are included in
this summary because they constitute 90% of total mutations reported.
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
Journal of the National Cancer Institute, Vol. 91, No. 22, November 17, 1999
REVIEW 1927
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.
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
Table 3. Frequently mutated codons in the PTEN/MMAC1 gene
Features
Total
germline
mutations
(n ⳱ 75)
Total
somatic
mutations
(n ⳱ 273)
Phosphatase core motif
Phosphatase core motif; CpG
Tertiary structure
CpG; conserved in tensin and auxilin
CpG; Tyr-P site
Poly(A)6; repeat motif; Tyr-P site
335 CpG; Tyr-P and Ser-P sites
6 (8%)
11 (14.7%)
4 (5.3%)
0
6 (8%)
1 (1.3%)
5 (7%)
3 (1%)
20 (7.3%)
0
12 (4%)
8 (2.9%)
24 (9%)
11 (4.0%)
Codon
No.
129
130
157
173
233
316–323
332–338
1928 REVIEW
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.
III. PTEN/MMAC1 AS TUMOR SUPPRESSOR WITH
DEFINED ENZYMATIC FUNCTION
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, DAF18, 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).
Journal of the National Cancer Institute, Vol. 91, No. 22, November 17, 1999
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 growthsuppressive 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 in-
verse 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 actincontaining 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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NOTES
We thank Dr. Barbara Dunn for her critical comments on the manuscript.
Manuscript received February 23, 1999; revised August 13, 1999; accepted
September 21, 1999.
Journal of the National Cancer Institute, Vol. 91, No. 22, November 17, 1999