PTEN expression is reduced in a subset of sporadic thyroid carcinomas: evidence that PTEN-growth suppressing activity in thyroid cancer cells mediated by p27kip1

The dual-specificity phosphatase PTEN/MMAC1/TEP1 has recently been identified as the tumor suppressor gene most frequently mutated and/or deleted in human tumors. Germline mutations of PTEN give rise to Cowden Disease (CD), an autosomal dominantly-inherited cancer syndrome which predisposes to increased risk of developing breast and thyroid tumors. However, PTEN mutations have rarely been detected in sporadic thyroid carcinomas. In this study, we confirm that PTEN mutations in sporadic thyroid cancer are infrequent as we found one point mutation and one heterozygous deletion of PTEN gene in 26 tumors and eight cell lines screened. However, we report that PTEN expression is reduced both at the mRNA and at the protein level - in five out of eight tumor-derived cell lines and in 24 out of 61 primary tumors. In most cases, decreased PTEN expression is correlated with increased phosphorylation of the PTEN-regulated protein kinase Akt/PKB. Moreover, we demonstrate that PTEN may act as a suppressor of thyroid cancerogenesis as the constitutive re-expression of PTEN into two different thyroid tumor cell lines markedly inhibits cell growth. PTEN-dependent inhibition of BrdU incorporation is accompanied by enhanced expression of the cyclin-dependent kinase inhibitor p27kip1 and can be overcome by simultaneous co-transfection of an excess p27kip1 antisense plasmid. Accordingly, in a subset of thyroid primary carcinomas and tumor-derived cell lines, a striking correlation between PTEN expression and the level of p27kip1 protein was observed. In conclusion, our findings demonstrate that inactivation of PTEN may play a role in the development of sporadic thyroid carcinomas and that one key target of PTEN suppressor activity is represented by the cyclin-dependent kinase inhibitor p27kip1.     

No evidence for germline PTEN mutations in families with breast and brain tumours.

Germline mutations of the PTEN gene are involved in Cowden disease, a genetic condition associated with an increased risk of breast cancer. Further somatic PTEN mutations have been found in glioblastomas and to a lesser extent in meningiomas. Therefore, PTEN germline mutations were searched for in a series of 20 unrelated women with breast cancer who also had a personal or familial breast-brain tumour history. Inclusion criteria were 1. family history of breast cancer; 2. absence of germline BRCA1 and p53 mutation; and 3. at least one case of brain tumour (glioblastoma, meningioma, or medulloblastoma) in either the index case or one of their first or second degree relatives. Any stigmata of Cowden disease was an exclusion criteria. Screening of the PTEN gene for point mutations or small rearrangements were performed using the denaturing gradient gel electrophoresis method on the 9 coding exons. No disease-associated mutation of the PTEN gene has been detected in our series. It is, thus, unlikely that PTEN is a significant BRCA predisposing locus. However, one might ask whether breast cancer cases resulting from germline PTEN mutation could occur without any mammary histological feature of Cowden disease.     

Germline PTEN mutations are rare and highly penetrant

Cowden syndrome (multiple hamartoma syndrome, MIM 158350) is an early onset syndrome characterized by multiple hamartomas in the skin, mucous membranes, breast, thyroid and endometrium. Patients with Cowden syndrome have increased risk of breast cancer, thyroid cancer and endometrial cancer. In 1997 germline mutations in PTEN were demonstrated to cause Cowden syndrome. We report the results of diagnostic and predictive testing in all families with Cowden syndrome or suspected Cowden syndrome registered at the Norwegian cancer family clinics. PTEN mutations were found in all six families meeting the clinical criteria for Cowden syndrome, in none of the two families assumed to have Cowden syndrome but not fulfilling the criteria, and in none of the eight families selected in our computerized medical files to have a combination of breast and thyroid cancers. Age-related penetrances for the various neoplasms are given. All families but one were small and de novo mutations were found.     

Absence of PTEN germ-line mutations in men with a potential inherited predisposition to prostate cancer.

Epidemiological studies have demonstrated that men with a family history of prostate cancer are at an increased risk for this disease. This important observation has led a number of research teams, including our own, to collect DNA samples and clinical data from prostate cancer families, with the goal of localizing and characterizing prostate cancer susceptibility genes. The candidate tumor suppressor gene PTEN (also called MMAC1) has recently been shown to be somatically altered in several common malignancies, including cancers of the brain, kidney, skin, thyroid, endometrium, breast, and prostate. Germ-line mutations in this gene, which maps to chromosome 10q23, have been associated with Cowden disease, an autosomal dominant cancer predisposition syndrome that is characterized by multiple hamartomas. Although prostate cancer is not typically associated with Cowden disease, previous studies of sporadic prostate cancers demonstrate loss of heterozygosity at 10q23 loci in approximately 25% of cases. We, therefore, hypothesized that germ-line mutations in the PTEN gene may predispose to prostate cancer in a subset of families, particularly those in which cancers of the breast, kidney, and/or thyroid also segregate. To test this hypothesis, DNA was isolated from whole blood of 11 prostate cancer patients from 10 unrelated families. Four of the 10 families met the previously established clinical criteria for hereditary prostate cancer. Eight of the II men had at least one second primary malignancy, including cases of neuroendocrine cancer, glioblastoma multiforme, melanoma, kidney, and thyroid cancer. Although we identified some common as well as some unique polymorphisms, no nonsense or missense mutations were identified in any of the 11 samples. To further examine the possibility that PTEN mutations contribute to prostate cancer predisposition, we also studied the probands from each of 10 families with early-onset and/or multiple individuals with prostate cancer. Sequence analysis of the PTEN gene in these 10 men also revealed no mutations or novel polymorphisms. We conclude that germ-line mutations in the PTEN are unlikely to contribute in a significant way to the inherited predisposition to prostate cancer.     

PTEN loss in the continuum of common cancers, rare syndromes and mouse models

PTEN is among the most frequently inactivated tumour suppressor genes in sporadic cancer. PTEN has dual protein and lipid phosphatase activity, and its tumour suppressor activity is dependent on its lipid phosphatase activity, which negatively regulates the PI3K-AKT-mTOR pathway. Germline mutations in PTEN have been described in a variety of rare syndromes that are collectively known as the PTEN hamartoma tumour syndromes (PHTS). Cowden syndrome is the best-described syndrome within PHTS, with approximately 80% of patients having germline PTEN mutations. Patients with Cowden syndrome have an increased incidence of cancers of the breast, thyroid and endometrium, which correspond to sporadic tumour types that commonly exhibit somatic PTEN inactivation. Pten deletion in mice leads to Cowden syndrome-like phenotypes, and tissue-specific Pten deletion has provided clues to the role of PTEN mutation and loss in specific tumour types. Studying PTEN in the continuum of rare syndromes, common cancers and mouse models provides insight into the role of PTEN in tumorigenesis and will inform targeted drug development.     

Cowden syndrome

Cowden syndrome (CS), due to germline mutations of the PTEN tumor-suppressor gene, is an often overlooked cancer predisposition syndrome associated with an increased risk of breast, thyroid, and endometrial cancers, as well as benign manifestations. Germline PTEN mutations also are associated with syndromes that have not been historically connected to an increase in risk for malignancy. These disorders include Bannayan-Riley-Ruvalcaba syndrome (BRRS), Proteus syndrome (PS), and Proteus-like syndrome (PSL). These syndromes can be described under the umbrella of PTEN hamartoma tumor syndrome (PHTS). As one would expect in allelic disorders, there is broad phenotypic overlap in the PHTS; however, the syndromes are clinically distinct. As additional information is discovered about new syndromes of cancer predisposition and their concordant genes, oncologists and allied healthcare providers must maintain vigilance to appropriately identify, and screen, individuals at an increased risk. Although CS is the only PHTS with a clearly documented predisposition to malignancies, pending further data, for precautionary reasons all individuals with a germline PTEN mutation are recommended to follow the cancer surveillance recommendations for CS.     

Pten loss in the mouse thyroid causes goiter and follicular adenomas: insights into thyroid function and Cowden disease pathogenesis

Inactivation and silencing of the tumor suppressor PTEN are found in many different epithelial tumors, including thyroid neoplasia. Cowden Disease patients, who harbor germ-line PTEN mutations, often display thyroid abnormalities, including multinodular goiter and follicular adenomas, and are at increased risk of thyroid cancer. To gain insights into the role PTEN plays in thyroid function and disease, we have generated a mouse strain, in which Cre-mediated recombination is used to specifically delete Pten in the thyrocytes. We found that Pten mutant mice develop diffuse goiter characterized by extremely enlarged follicles, in the presence of normal thyroid-stimulating hormone and T4 hormone levels. Loss of Pten resulted in a significant increase in the thyrocyte proliferative index, which was more prominent in the female mice, and in increased cell density in the female thyroid glands. Surprisingly, goitrogen treatment did not cause a substantial increase of the mutant thyroid size and increased only to some extent the proliferation index of the female thyrocytes, suggesting that a relevant part of the thyroid-stimulating hormone-induced proliferation signals are funneled through the phosphatidylinositol-3-kinase (PI3K)/Akt cascade. Although complete loss of Pten was not sufficient to cause invasive tumors, over two thirds of the mutant females developed follicular adenomas by 10 months of age, showing that loss of Pten renders the thyroid highly susceptible to neoplastic transformation through mechanisms that include increased thyrocyte proliferation. Our findings show that constitutive activation of the PI3K/Akt cascade is sufficient to stimulate continuous autonomous growth and provide novel clues to the pathogenesis of Cowden Disease and sporadic nontoxic goiter.     

Nuclear PTEN expression and clinicopathologic features in a population-based series of primary cutaneous melanoma

Germline mutations of the PTEN tumor-suppressor gene, on 10q23, cause Cowden syndrome, an inherited hamartoma syndrome with a high risk of breast, thyroid and endometrial carcinomas and, some suggest, melanoma. To date, most studies which strongly implicate PTEN in the etiology of sporadic melanomas have depended on cell lines, short-term tumor cultures and noncultured metastatic melanomas. The only study which reports PTEN protein expression in melanoma focuses on cytoplasmic expression, mainly in metastatic samples. To determine how PTEN contributes to the etiology or the progression of primary cutaneous melanoma, we examined cytoplasmic and nuclear PTEN expression against clinical and pathologic features in a population-based sample of 150 individuals with incident primary cutaneous melanoma. Among 92 evaluable samples, 30 had no or decreased cytoplasmic PTEN protein expression and the remaining 62 had normal PTEN expression. In contrast, 84 tumors had no or decreased nuclear expression and 8 had normal nuclear PTEN expression. None of the clinical features studied, such as Clark's level and Breslow thickness or sun exposure, were associated with cytoplasmic PTEN expressional levels. An association with loss of nuclear PTEN expression was indicated for anatomical site (p = 0.06) and mitotic index (p = 0.02). There was also an association for melanomas to either not express nuclear PTEN or to express p53 alone, rather than both simultaneously (p = 0.02). In contrast with metastatic melanoma, where we have shown previously that almost two-thirds of tumors have some PTEN inactivation, only one-third of primary melanomas had PTEN silencing. This suggests that PTEN inactivation is a late event likely related to melanoma progression rather than initiation. Taken together with our previous observations in thyroid and islet cell tumors, our data suggest that nuclear-cytoplasmic partitioning of PTEN might also play a role in melanoma progression.     

Cowden disease and the PTEN gene: a successfully clinical and biological combined approach]

Cowden disease is an autosomal dominant inherited cancer syndrome characterized by the occurrence of multiple hamartomas, tumors or hyperplastic lesions that may develop in any organ. The disease is related to germline mutation of the PTEN gene, a recently cloned tumor suppressor gene involved in the pathogenesis of sporadic glioblastoma and endometrial carcinoma. It has been shown that the PTEN gene product was a phosphatase able for dephosphorylating a lipid substrate: the phosphatidylinositol (3,4,5)-triphosphate (PIP3). So PTEN appears to negatively control the PI3K-AKT signaling pathway implicated in regulation of cell growth and survival.     

Cutaneous manifestations of breast cancer

Breast cancer may present with cutaneous symptoms. The skin manifestations of breast cancer are varied. Some of the more common clinical presentations of metastatic cutaneous lesions from breast cancer will be described. Paraneoplastic cutaneous dermatoses have been reported as markers of breast malignancy and include erythema gyratum repens, acquired ichthyosis, dermatomyositis, multicentric reticulohistiocytosis, and hypertrichosis lanuginosa acquisita. Mammary Paget’s disease, often associated with an underlying breast cancer, and Cowden syndrome, which has an increased risk of breast malignancy, each have specific dermatologic findings. Recognition of these distinct cutaneous signs is important in the investigation of either newly diagnosed or recurrent breast cancer.     

PTEN suppresses breast cancer cell growth by phosphatase activity-dependent G1 arrest followed by cell death

PTEN/MMAC1/TEP1, a tumor suppressor gene, is frequently mutated in a variety of human cancers. Germ-line mutations of phosphatase and tensin homolog, deleted on chromosome ten (PTEN) are found in two inherited hamartoma tumor syndromes: Cowden syndrome, which has a high risk of breast, thyroid, and other cancers; and Bannayan-Zonana syndrome, a related disorder. PTEN encodes a phosphatase that recognizes both protein substrates and phosphatidylinositol-3,4,5-triphosphate. The lipid phosphatase activity of PTEN seems to be important for growth suppression through inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. We established clones with stable PTEN expression controlled by a tetracycline-inducible system to examine the consequences of increased levels of wild-type and mutant PTEN expression in a well-characterized breast cancer line, MCF-7. When we overexpressed PTEN in MCF-7, growth suppression was observed, but only if PTEN phosphatase activity is preserved. The initial growth suppression was attributable to G1 cell cycle arrest, whereas subsequent growth suppression was attributable to a combination of G1 arrest and cell death. Of note, the decrease in Akt phosphorylation preceded the onset-of suppression of cell growth. Treatment of MCF-7 cells with wortmannin, a PI3K inhibitor, caused cell growth inhibition in a way similar to the effects of overexpression of PTEN in this cell. In general, the inverse correlation between PTEN protein level and Akt phosphorylation was found in a panel of breast cancer cell lines. Therefore, PTEN appears to suppress breast cancer growth through down-regulating PI3K signaling, which leads to the blockage of cell cycle progression and the induction of cell death, in a sequential manner.     

Exclusion of a major role for the PTEN tumour-suppressor gene in breast carcinomas

PTEN is a novel tumour-suppressor gene located on chromosomal band 10q23.3. This region displays frequent loss of heterozygosity (LOH) in a variety of human neoplasms including breast carcinomas. The detection of PTEN mutations in Cowden disease and in breast carcinoma cell lines suggests that PTEN may be involved in mammary carcinogenesis. We here report a mutational analysis of tumour specimens from 103 primary breast carcinomas and constitutive DNA from 25 breast cancer families. The entire coding region of PTEN was screened by single-strand conformation polymorphism (SSCP) analysis and direct sequencing using intron-based primers. No germline mutations could be identified in the breast cancer families and only one sporadic carcinoma carried a PTEN mutation at one allele. In addition, all sporadic tumours were analysed for homozygous deletions by differential polymerase chain reaction (PCR) and for allelic loss using the microsatellite markers D10S215, D10S564 and D10S573. No homozygous deletions were detected and only 10 out of 94 informative tumours showed allelic loss in the PTEN region. These results suggest that PTEN does not play a major role in breast cancer formation.     

Overexpression and overactivation of Akt in thyroid carcinoma

Enhanced activation of Akt occurs in Cowden's disease, an inherited syndrome of follicular thyroid, breast, colon, and skin tumors, via inactivation of its regulatory protein, PTEN. Whereas PTEN inactivation is uncommon in sporadic thyroid cancer, activation of growth factor pathways that signal through Akt is frequently identified. We hypothesized that Akt overactivation could be a common finding in sporadic thyroid cancer and might be important in thyroid cancer biology. We examined thyroid cancer cells lines and benign and malignant thyroid tissue for total Akt activation and isoform-specific Akt expression. In thyroid cancer cells, Akt 1, 2, and 3 proteins were expressed, total Akt was activated by insulin phosphatidylinositol 3'-kinase, and inhibition of phosphatidylinositol 3'-kinase reduced cell viability. In human thyroid tissue, increased levels of phosphorylated total Akt were identified in follicular but not papillary cancers compared with normal tissue. Levels of Akt 1 and 2 proteins and Akt 2 RNA were elevated only in the follicular cancers. In paired samples, Akt 1, 2, 3, and phospho-Akt levels were higher in five of six cancers, including three of three follicular cancers. These data suggest that Akt activation may play a role in the pathogenesis or progression of sporadic thyroid cancer.     

Perinucleolar compartment prevalence is a phenotypic pancancer marker of malignancy

BACKGROUND: The perinucleolar compartment (PNC) is a subnuclear structure localized at the nucleolar periphery. Previous studies using breast cancer as a model system demonstrated that PNC prevalence (the percentage of cells with 1 or more PNC) increased with disease progression and was associated with poor patient outcomes. METHODS: To evaluate the validity of developing PNC prevalence as a novel pancancer prognostic marker, the authors investigated whether PNC prevalence was correlated with malignancy in a spectrum of tissue types and evaluated its selective association with malignancy under various experimental conditions. RESULTS: PNC prevalence was low in primary and immortalized cells and in cell lines derived from hematologic malignancies, but it was heterogeneous in cell lines derived from solid tumors, including those of epithelial and nonepithelial origins. Studies using human myometrial tissue and thyroid cancer cell lines with various levels of malignancy demonstrated a correlation between high PNC prevalence and malignant potential. Furthermore, PNC prevalence corresponded directly to metastatic capacities in a series of well characterized cell lines of the same origin that were selected for various levels of metastatic capacity in a mouse model. Conversely, PNC prevalence was reduced experimentally by over expressing an antimetastatic protein in breast cancer cells. However, PNC prevalence was not associated with traits that were shared by both cancer and normal cells, including proliferation, glycolysis, and differentiation. CONCLUSIONS: Together, these observations helped to verify that PNC prevalence selectively represents malignancy in a broad spectrum of solid tissue tumors, demonstrating its potential to be developed as a pancancer prognostic marker of malignancy.     

Common genetic variation at PTEN and risk of sporadic breast and prostate cancer.

PTEN frequently shows loss of heterozygosity in breast and prostate cancers, and mutations in this gene are responsible for Cowden disease, a rare Mendelian syndrome that includes breast cancer as part of its phenotype. Thus, PTEN serves as a candidate susceptibility gene for both breast and prostate cancer risk. Whether common inherited variation (either coding or noncoding) at the PTEN locus contribute to nonfamilial, sporadic breast and prostate cancer risk is not known. In this study, we employed a linkage disequilibrium-based approach to test for association between common genetic variation at the PTEN locus and breast and prostate cancer risk in African-American, Native Hawaiian, Japanese, Latina, and White men and women in the Multiethnic Cohort Study. We genotyped 17 common single nucleotide polymorphisms (SNP; >/=5% frequency in at least one ethnic group) spanning the PTEN gene to define the common alleles in these populations. These SNPs were in strong linkage disequilibrium, indicating that our survey captured most of the common sequence variation across this locus. Eight tagging SNPs were selected to predict the common PTEN haplotypes (>/=0.05 frequency) in these populations (two additional tagging SNPs were required for African Americans). These SNPs were evaluated in a breast cancer case-control study (cases, n = 1,615; controls, n = 1,962) and prostate cancer case-control study (cases, n = 2,320; controls, n = 2,290) nested within the Multiethnic Cohort Study. Multiple testing was explicitly accounted for by applying a permutation-based framework. We found no strong association with any common haplotype in relation to breast or prostate cancer risk. In summary, our results show that common variants in PTEN do not substantially influence risk of these two common cancers.     

Frequent allelic loss at 10q23 but low incidence of PTEN mutations in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare, highly metastatic skin tumor of neuroectodermal origin. The disease shares clinical and histopathological features with small cell lung carcinoma (SCLC). The genetic mechanisms underlying the development and tumor progression of MCC are poorly understood. We recently showed by comparative genomic hybridization (CGH) that the pattern of chromosomal abnormalities in MCC resembles that of SCLC. One of the most frequently observed losses involved the entire chromosome 10 or partial loss of the chromosome 10 long arm (33% of examined MCC cases). The PTEN tumor-suppressor gene has been mapped to 10q23.3 and was shown to be mutated in a variety of human cancers including SCLC. Germline PTEN mutations have been observed in familial predisposing cancer syndromes including Cowden disease. Interestingly, an association between Cowden syndrome and Merkel cell carcinoma has been reported. To study the possible role of PTEN in MCC oncogenesis, loss of heterozygosity (LOH) analysis for the 10q23 region was performed on 26 MCC tumor samples from 23 MCC patients. The PTEN locus was deleted in 9 of 21 (43%) informative MCC tumor samples [7 of 18 (39%) MCC patients]. Despite this high frequency of LOH at 10q23, mutation and homozygous deletion screening of the PTEN gene revealed only one tumor with a nonsense mutation and a second with a homozygous deletion of exon 9. These data suggest that either alternative mechanisms lead to inactivation of the PTEN gene or that other tumor-suppressor genes at chromosome 10 are implicated in the development of MCC.     

PTEN deficiency: a role in mammary carcinogenesis

The PTEN gene is often mutated in primary human tumors and cell lines, but the low rate of somatic PTEN mutation in human breast cancer has led to debate over the role of this tumor suppressor in this disease. The involvement of PTEN in human mammary oncogenesis has been implicated from studies showing that germline PTEN mutation in Cowden disease predisposes to breast cancer, the frequent loss of heterozygosity at the PTEN locus, and reduced PTEN protein levels in sporadic breast cancers. To assay the potential contribution of PTEN loss in breast tumor promotion, Li et al. [1] crossed Pten heterozygous mice with mouse mammary tumor virus-Wnt-1 transgenic (Wnt-1 TG, Pten+/-) mice. Mammary ductal carcinoma developed earlier in Wnt-1 TG, Pten+/- mice than in mice bearing either genetic change alone, and showed frequent loss of the remaining wild-type PTEN allele. These data indicate a role for PTEN in breast tumorigenesis in an in vivo model.     

Breast carcinoma and basal cell epithelioma after x-ray therapy for hirsutism.

We report a 60-year-old woman with a history of x-ray therapy for generalized hirsutism at 20 years of age who at the age of 37 years developed the first of numerous basal cell epitheliomas on her trunk, including chest, on a background of radiation damaged skin. At the age of 51 years one of the basal cell epitheliomas was biopsied and an incidental histologic finding was a breast carcinoma. The basal cell epithelioma is clearly linked with x-ray exposure; breast cancer is less so although there is impressive epidemiologic evidence supporting an association between human breast cancer and radiation exposure. In view of an association between thyroid cancer and dermatologic x-ray therapy, further investigation of such an association with breast cancer should be considered. It may be wise to evaluate patients who received dermatologic x-ray exposure to their breasts for possible breast cancer and to consider radiation induced skin damage on or near the skin overlying the thyroid or breasts as a cutaneous marker of internal malignancy or potential internal malignancy.