Biallelic inactivating mutations and an occult germline mutation of PTEN in primary cervical carcinomas

A tumor suppressor gene on chromosome sub-band 10q23.3, PTEN, is frequently mutated or deleted in a variety of human cancers. Germline mutations in PTEN, that encodes a dual-specificity phosphatase, have been implicated in two hamartoma-tumor syndromes that exhibit some clinical overlap, Cowden syndrome and Bannayan-Zonana syndrome. Although cervical cancer is not a known component of these two syndromes, loss of heterozygosity (LOH) of markers on chromosome arm 10q is frequently observed in cervical cancers. To determine the potential role that PTEN mutation may play in cervical tumorigenesis, we screened 20 primary cervical cancers for LOH of polymorphic markers within and flanking the PTEN gene, and for intragenic mutations in the entire coding region and exon-intron boundaries of the PTEN gene. LOH was observed in 7 of 19 (36.8%) cases. Further, one sample may have homozygous deletion. Three (15%) intragenic mutations were found: two were somatic missense mutations in exon 5, that encodes the phosphatase motif, and an occult germline intronic sequence variant in intron 7, that we show to be associated with aberrant splicing. All three samples with the mutations also had LOH of the wild-type allele. These data indicate that disruption of PTEN by allelic loss or mutation may contribute to tumorigenesis in cervical cancers. In cervical cancer, unlike some other human primary carcinomas, e.g., those of the breast and thyroid, biallelic structural PTEN defects seem necessary for carcinogenesis. Further, one in 20 unselected cervical carcinomas was found to have a germline PTEN mutation; it is unclear whether the patient with this mutation had Cowden disease or a related syndrome.     

A comprehensive functional analysis of PTEN mutations: implications in tumor- and autism-related syndromes

The PTEN (phosphatase and tensin homolog) phosphatase is unique in mammals in terms of its tumor suppressor activity, exerted by dephosphorylation of the lipid second messenger PIP(3) (phosphatidylinositol 3,4,5-trisphosphate), which activates the phosphoinositide 3-kinase/Akt/mTOR (mammalian target of rapamycin) oncogenic pathway. Loss-of-function mutations in the PTEN gene are frequent in human cancer and in the germline of patients with PTEN hamartoma tumor-related syndromes (PHTSs). In addition, PTEN is mutated in patients with autism spectrum disorders (ASDs), although no functional information on these mutations is available. Here, we report a comprehensive in vivo functional analysis of human PTEN using a heterologous yeast reconstitution system. Ala-scanning mutagenesis at the catalytic loops of PTEN outlined the critical role of residues within the P-catalytic loop for PIP(3) phosphatase activity in vivo. PTEN mutations that mimic the P-catalytic loop of mammalian PTEN-like proteins (TPTE, TPIP, tensins and auxilins) affected PTEN function variably, whereas tumor- or PHTS-associated mutations targeting the PTEN P-loop produced complete loss of function. Conversely, Ala-substitutions, as well as tumor-related mutations at the WPD- and TI-catalytic loops, displayed partial activity in many cases. Interestingly, a tumor-related D92N mutation was partially active, supporting the notion that the PTEN Asp92 residue might not function as the catalytic general acid. The analysis of a panel of ASD-associated hereditary PTEN mutations revealed that most of them did not substantially abrogate PTEN activity in vivo, whereas most of PHTS-associated mutations did. Our findings reveal distinctive functional patterns among PTEN mutations found in tumors and in the germline of PHTS and ASD patients, which could be relevant for therapy.     

PTEN mutations do not cause nuclear beta-catenin accumulation in endometrial carcinomas

PTEN: and beta-catenin mutations constitute the predominant genetic alterations in endometrioid carcinomas of the endometrium. PTEN encodes a dual-specificity phosphatase with lipid phosphatase and protein tyrosine phosphatase activities that regulate both apoptosis and interactions with the extracellular matrix. Recent studies have associated PTEN mutations with tumorigenesis of prostate carcinoma via the Wnt signaling pathway, leading to nuclear beta-catenin accumulation. To elucidate the potential interaction of PTEN and beta-catenin in endometrial cancer, we performed mutation analyses of the entire PTEN gene and of exon 3 of the beta-catenin gene that is most frequently targeted by mutations. A total of 82 endometrial carcinomas comprising 62 type I endometrioid carcinomas and 20 type II high-grade carcinomas were investigated. In addition in a subset of 22 carcinomas, the intracellular beta-catenin distribution was analyzed by immunohistochemistry. Overall, 20 (24.4%) of 82 tumors revealed mutations in the PTEN gene, and 16 (19.5%) of 82, in the beta-catenin gene. Six tumors (7.3%) showed mutations in both the PTEN and beta-catenin gene. Mutations were mainly detected in endometrioid carcinomas of the endometrium. As expected, a striking nuclear accumulation of beta-catenin could be shown in tumors with beta-catenin mutations. In the vast majority of tumors with PTEN mutations, a regular staining pattern of the cytoplasmic and membranous compartments was found. We therefore conclude that, in contrast to prostate cancer, mutations in the PTEN gene seem not to affect cellular distribution of the beta-catenin protein in endometrial carcinomas.     

Germline SDHx variants modify breast and thyroid cancer risks in Cowden and Cowden-like syndrome via FAD/NAD-dependant destabilization of p53

Cowden syndrome (CS), a Mendelian autosomal-dominant disorder, predisposes to breast, thyroid and other cancers. Germline mutations in phosphatase and tensin homolog (PTEN) have been recently reported in 23% of a large series of classic CS. Here, we validated our small (n = 10) pilot study in a large patient series that germline variations in succinate dehydrogenase genes (SDHx) occur in 8% (49/608) of PTEN mutation-negative CS and CS-like (CSL) individuals (SDH(var+)). None of these SDHx variants was found in 700 population controls (P < 0.0001). We then found that SDHx variants also occur in 6% (26/444) of PTEN mutation-positive (PTEN(mut+)) CS/CSL individuals (PTEN(mut+)/SDH(var+)). Of 22 PTEN(mut+)/SDH(var+) females, 17 had breast cancers compared with 34/105 PTEN(mut+) (P < 0.001) or 27/47 SDH(var+) patients (P = 0.06). Notably, individuals with SDH(var+) alone had the highest thyroid cancer prevalence (24/47) compared with PTEN(mut+) patients (27/105, P = 0.002) or PTEN(mut+)/SDH(var+) carriers (6/22, P = 0.038). Patient-derived SDH(var+) lymphoblastoid cells had elevated cellular reactive oxygen species, highest in PTEN(mut+)/SDH(var+) cells, correlating with apoptosis resistance. SDH(var+) cells showed stabilized and hyperactivated hypoxia inducible factor (HIF)1α signaling. Most interestingly, we also observed the loss of steady-state p53 in the majority of SDH(var+) cells. This loss of p53 was regulated by MDM2-independent NADH quinone oxidoreductase 1-mediated protein degradation, likely due to the imbalance of flavin adenine dinucleotide/nicotinamide adenine dinucleotide in SDH(var+) cells. Our data suggest the potential regulation of HIF1α, p53 and PTEN signaling by mitochondrial metabolism in CS/CSL tumorigenesis. Together, our findings suggest the importance of considering SDHx as candidate predisposing and modifier genes for CS/CSL-related malignancy risks, and a mechanism which suggests ways of therapeutic reversal or prevention.     

Germline and germline mosaic PTEN mutations associated with a Proteus-like syndrome of hemihypertrophy, lower limb asymmetry, arteriovenous malformations and lipomatosis

Germline PTEN mutations cause Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome (BRR), two hamartoma-tumour syndromes, and somatic PTEN alterations have been shown to participate, to a greater or lesser extent, in a wide variety of sporadic neoplasia. PTEN is a tumour suppressor and dual-specificity phosphatase which affects apoptosis via its lipid phosphatase activity in the phosphoinositol-3-kinase and AKT pathway as well as inhibiting cell spreading via the focal adhesion kinase pathway. CS and BRR share some features, such as hamartomas and lipomatosis. To determine whether other syndromes characterized by overgrowth and lipomas are part of the PTEN syndrome spectrum, we ascertained six individuals with overgrowth and lipomas but who did not meet the diagnostic criteria for CS or BRR. Five had Proteus syndrome and one, a Proteus-like syndrome. When germline DNA and DNA from at least one involved tissue per case were examined for PTEN mutations, only the Proteus-like patient was found to harbour a germline R335X mutation. Interestingly, a lipomatous mass, an epidermoid naevus and arteriovenous malformation tissue, all of which were sampled from physically distinct sites, were all found to carry a second hit R130X mutation on the allele opposite the germline R335X. Both mutations have been described in CS and BRR. We postulate that the second hit, R130X, occurred early in embryonic development and may even represent germline mosaicism. Thus, PTEN may be involved in Proteus-like syndrome with its implications for cancer development in the future.     

p53 alterations and their relationship to SDHD mutations in parasympathetic paragangliomas.

Experimental and observational evidence suggests that chronic hypoxic stimulation can induce parasympathetic paraganglioma. This is emphasized by the identification of germline mutations in genes of the mitochondrial succinate dehydrogenase enzyme complex II in hereditary paraganglioma. Because of inactivating mutations in the succinate dehydrogenase subunit B (SDHB), C (SDHC), or D (SDHD) gene, the paraganglia undergo a chronic hypoxic stimulus leading to proliferation of the paraganglionic cells. Hypoxia is a known inducer of p53 up-regulation, which triggers cell cycle arrest and apoptosis. Inactivation of the p53 pathway, by gene mutation or by MDM2 overexpression, would enable cells to escape from cell cycle arrest and apoptosis and could contribute to tumorigenesis. To determine whether p53 inactivation plays a role in paraganglioma tumorigenesis, we investigated a series of 43 paragangliomas from 41 patients (of whom 24 patients harbored a germline SDHD mutation) for mutations in p53 exons 5-8 by PCR-SSCP. In addition, these tumors were investigated for p53 and MDM2 protein expression by immunohistochemistry, and the results were compared with clinical data and the presence of SDHD mutations. No aberrations in p53 exons 5-8 were found. The immunohistochemical experiments showed nuclear p53 expression in 15 tumors. Three tumors were positive for MDM2 that were also positive for p53. There was no correlation between p53 and MDM2 expression and clinical data or SDHD status. Given the fact that hypoxia induces p53 expression and regarding the absence of p53 mutations, these results suggest that p53 inactivation does not play a major role in the tumorigenesis of hereditary and sporadic paragangliomas.     

Novel PTEN mutations in neurodevelopmental disorders and macrocephaly

Somatic mutations of the phosphatase and tensin ( PTEN ) gene have been frequently detected in many types of human cancer. However, germline mutations can determine multiple hamartoma syndromes and, as more recently ascertained, syndromes clinically characterized by autism associated with macrocephaly. To determine whether germline mutations of PTEN may lead to different phenotypes, we screened all the nine exons of the PTEN gene in 40 patients with neurodevelopmental disorders, with or without features of autism spectrum disorder, associated with macrocephaly. Three novel de novo missense mutations were found (p.H118P, p.Y176C, p.N276S) in two severely mentally retarded patients with autism and in a subject with neurodevelopmental disorders without autistic features. Our results provide evidence that PTEN germline mutations may sustain a more wide phenotypical spectrum than previously suggested.     

Detection of new PTEN/MMAC1 mutations in head and neck squamous cell carcinomas with loss of chromosome 10.

Alterations of the candidate tumor suppressor gene PTEN/MMAC1 and the cell cycle control gene p16((CDKN2/MTS-1/INK4a)) have been detected in many types of human cancer. Here, we wanted to study the role of PTEN/MMAC1 in head and neck squamous cell carcinomas (HNSCC) in correlation to mutation and methylation of p16 and to previous in situ hybridization results concerning loss of chromosomes 9 and 10. We screened for alterations of PTEN/MMAC1 and p16 in 52 HNSCC of different sites. Mutations of PTEN/MMAC1 were found in 23% of tumor samples (missense mutations in 7 carcinomas, 13%). A loss of chromosome 10 was detected in five carcinomas with missense PTEN/MMAC1 mutations (71%). The missense mutations of PTEN/MMAC1 occurred in exons 5 (five different mutations in the neighborhood of the protein tyrosine phosphatase domain), 6, 7, and 8. Only one of these mutations had been described before. In addition, in three laryngeal carcinomas (6%), missense mutations of p16 (in exon 2) were detected and 14% of carcinomas showed a methylation of p16. Our results focus on the essential but not solitary role of PTEN/MMAC1 in the tumorigenesis or progression of a subset of HNSCC.     

Germline mutations but not somatic changes at the MYH locus contribute to the pathogenesis of unselected colorectal cancers

MYH-associated polyposis is a recently described, autosomal recessive condition comprising multiple colorectal adenomas and cancer. This disease is caused by germline mutations in the base excision repair (BER) gene MYH. Genes involved in the BER pathway are thus good candidates for involvement in the pathogenesis of sporadic tumors of the large bowel. We have screened a set of 75 sporadic colorectal cancers for mutations in MYH, MTH1, and OGG1. Allelic loss at MYH was also assessed. Selected samples were screened for mutations and allele loss at APC and mutations in p53, K-ras, and beta-catenin. A panel of 35 colorectal cancer cell lines was screened for MYH mRNA and protein expression. One of 75 cancers had bi-allelic germline mutations in MYH and on retrospective analysis of medical records this patient was found to have synchronous multiple small adenomas in addition to carcinoma. No somatic MYH mutations were found and mRNA and protein were expressed in all of our cell lines. There were no clearly pathogenic mutations in MTH1 or OGG1 in any tumor. Bi-allelic germline MYH mutations cause approximately 1 to 3% of unselected colorectal cancers, but appear always to be associated with multiple adenomas. Somatic inactivation of the DNA glycosylases involved in the BER pathway however does not appear to be involved in colorectal tumorigenesis.     

Mutations of the human PTEN gene

PTEN (phosphatase and tensin homolog deleted on chromosome ten), a recently discovered tumor suppressor gene, appears to negatively control the phosphoinositide 3-kinase signaling pathway for regulation of cell proliferation and cell survival by dephosphorylating the phosphatidylinositol 3,4,5-triphosphate. To date, 110 germline PTEN mutations have been reported in patients affected with two tumor predisposing syndromes, each having overlapping clinical features: Cowden disease and Bannayan-Riley-Ruvalcaba syndrome. These germline mutations are scattered along the length of the gene, with the exception of exon 9 (no mutation reported) and exon 1 (only two mutations reported). A mutational hot spot is found in exon 5, which encodes the phosphatase catalytic core motif, and recurrent mutations are also found at CpG dinucleotides suggesting deamination-induced mutations. PTEN has also been found to be defective in a large number of sporadic human tumors. In this article, 332 somatic point mutations of PTEN, occurring in primary tumors or metastasis, have been reviewed. Somatic PTEN mutations are more particularly involved in two types of human cancers: endometrial carcinomas and glioblastomas. In most cases, these somatic mutations result in protein inactivation and, as with germline mutations, recurrent somatic mutations are found in CpG dinucleotides. A mutagenesis by insertion-deletion in repetitive elements is however specifically observed in endometrial carcinomas.     

Ellagic acid,extracted from Sanguisorba officinalis,induces G1 arrest by modulating PTEN activity in B16F10 melanoma cells

In Chinese medicine, herbal medicine is commonly used to treat individuals suffering from many types of diseases. We thus expected that some herbal medicines would contain promising compounds for cancer chemotherapy. Indeed, we found that Sanguisorba officinalis extracts strongly inhibit the growth of B16F10 melanoma cells, and we identified ellagic acid (EA) as the responsible ingredient. B16F10 cells treated with EA exhibited strong G1 arrest accompanied by accumulation of p53, followed by inactivation of AKT. Addition of a PTEN inhibitor, but not a p53 inhibitor, abrogated the EA‐induced AKT inactivation and G1 arrest. The PTEN inhibitor also diminished EA‐induced p53 accumulation. Furthermore, EA apparently increased the protein phosphatase activity of PTEN, as demonstrated by the reduced phosphorylation level of FAK, a protein substrate of PTEN. Furthermore, an in vitro PTEN phosphatase assay on PIP3 showed the direct modulation of PTEN activity by EA. These results suggest that EA functions as an allosteric modulator of PTEN, enhancing its protein phosphatase activity while inhibiting its lipid phosphatase activity. It is notable that a combination of EA and cisplatin, a widely used chemotherapy agent, dramatically enhanced cell death in B16F10 cells, suggesting a promising strategy in chemotherapy.     

Epigenetic PTEN silencing in malignant melanomas without PTEN mutation

A tumor suppressor gene at 10q 23.3, designated PTEN, encoding a dual specificity phosphatase with lipid and protein phosphatase activity, has been shown to play an important role in the pathogenesis of a variety of human cancers. Germline mutations in PTEN cause Cowden syndrome (CS), which is characterized by multiple hamartomas and a high risk of breast and thyroid cancers. Frequent loss of heterozygosity at 10q is found in both early and advanced-stage sporadic melanomas; however, mutations or deletions in PTEN are detected mainly in melanoma cell lines. In this study, we examined PTEN expression in 34 unselected sporadic melanomas (4 primary melanomas, 30 metastases) using immunohistochemistry and correlated this with the results of structural studies of this gene. Immunostaining of 34 melanoma samples revealed no PTEN expression in 5 (15%) and low PTEN expression in 17 (50%), whereas the rest of the tumors (35%) had high levels of expression. Hemizygous deletion was found in 32% of the tumors but neither intragenic PTEN mutation nor biallelic deletion was found in any of the samples. Of the 5 melanomas showing no PTEN expression, 4 had no mutation or deletion of PTEN. Of the 13 tumors having weak PTEN immunoreactivity and informative loss of heterozygosity results, 6 had evidence of hemizygous allelic loss of PTEN while the remaining 7 had intact PTEN. These results strongly support PTEN as a major tumor suppressor on 10q involved in melanoma tumorigenesis and suggest an epigenetic mechanism of biallelic functional inactivation not previously observed in other cancers where PTEN might be involved.     

Generation of loss of heterozygosity and its dependency on p53 status in human lymphoblastoid cells

Loss of heterozygosity (LOH) is a critical event in the development of human cancers. LOH is thought to result from either a large deletion or recombination between homologous alleles during repair of DNA double-strand breaks (DSBs). These types of genetic alterations produce mutations in the TK gene mutation assay, which detects a wide mutational spectrum, ranging from point mutations to LOH-type mutations. TK6, a human lymphoblastoid cell line, is heterozygous for the thymidine kinase (TK) gene and has a wild-type p53 gene. The related cell lines, TK6-E6 and WTK-1, which are p53-deficient and p53-mutant (Ile237), respectively, are also heterozygous for the TK gene and LOH-type mutation can be detected in these cells. Therefore, comparative studies of TK mutation frequency and spectrum with these cell lines are useful for elucidating the role of p53 in generating LOH and maintaining genomic stability in human cells. We demonstrate here that LOH and its associated genomic instability strongly depend on the p53 status in these cells. TK6-E6 and WTK-1 are defective in the G1/S checkpoint and in apoptosis. Unrepaired DSBs that escape from the checkpoint can potentially initiate genomic instability after DNA replication, resulting in LOH and a variety of chromosome changes. Moreover, genomic instability is enhanced in WTK-1 cells. It is likely that the mutant p53 protein in WTK-1 cells increases LOH in a dominant-negative manner due to its abnormal recombination capacity. We discuss the mutator phenotype and genomic instability associated with p53 inactivation with the goal of elucidating the mechanisms of mutation and DNA repair in untargeted mutagenesis.     

In vitro functional studies of naturally occurring pathogenic PRKAR1A mutations that are not subject to nonsense mRNA decay

Patients presenting with primary pigmented nodular adrenocortical disease (PPNAD), Carney complex (CNC), or sporadic tumors were previously found to carry germline mutations in the human type Ialpha regulatory subunit (RIalpha) of adenosine 3',5'-cyclic monophosphate (cyclic AMP [cAMP])-dependent protein kinase (PKA; PRKAR1A). Although about 90% of disease-causing PRKAR1A mutations lead to premature stop codon generation and subsequent degradation of the mutant message by nonsense-mediated mRNA decay (NMD), here we describe seven PRKAR1A mutations whose mRNAs do not seem to undergo NMD and instead result in an expressed mutant RIalpha protein. The expressed mutations (p.Ser9Asn, p.Glu60_Lys116del [Delta-exon 3], p.Arg74Cys, p.Arg146Ser, p.Asp183Tyr, p.Ala213Asp, and p.Gly289Trp) were spread over all the functional RIalpha domains, and all of them exhibited increased PKA activity, which we attribute to decreased binding to cAMP and/or the catalytic subunit. Our data further corroborate the previous finding that altered PRKAR1A function, not only haploinsufficiency, is enough to elevate PKA activity which is apparently associated with tumorigenesis in tissues affected by CNC. In some cases, as with the Delta-exon 3 mutation, we may even conclude that the presence of a mutant PRKAR1A protein may be more harmful than allelic loss.     

Absence of germline mutations in exons 5–9 of the p53 gene in patients with Li-Fraumeni-like (SBLA) and familial adenomatous polyposis heritable cancer syndromes

Although acquired mutations in the human p53 gene occur in many tumor types, germline mutations are rare. An exception is the occurrence of germline p53 mutations in a fraction of families afflicted with the Li-Fraumeni syndrome (LFS). Previous studies from our laboratory demonstrated increased levels of wild type p53 protein in skin fibroblasts (SF) of patients from heritable cancer syndrome, including familial adenomatous polyposis (FAP), neurofibromatosis type 1 (NF1), and bilateral retinoblastoma (bRB) (Kopelovich and DeLeo, 1984,1986). Here, we further address the association between germline p53 alterations and genetic predisposition to cancer in the SBLA syndrome and in FAP. DNA sequencing and single-stranded conformational polymorphism analysis (SSCP) were utilized to screen for the presence of mutations within exons 5–9 of the p53 gene in SF and in benign tumors. Thus we observed no germline mutations in exons 5–9 of the p53 gene in SF from SBLA or FAP patients, including the Gardner variant. In addition, we observed no acquired mutations in exons 5–9 of the p53 gene in benign tumors from FAP patients. In conclusion, we found no association between germline p53 mutations and SBLA or FAP. How mechanisms that involve nonmutational activation of the p53 protein might affect genetic predisposition to cancer remains to be established.     

PTEN coordinates G(1) arrest by down-regulating cyclin D1 via its protein phosphatase activity and up-regulating p27 via its lipid phosphatase activity in a breast cancer model

The tumour suppressor gene PTEN/MMAC1/TEP1 encodes a dual-specificity phosphatase that recognizes phosphatidylinositol-3,4,5-triphosphate and protein substrates. We have shown previously that over-expression of PTEN in a tetracycline-controlled inducible system blocks cell cycle progression and induces apoptosis in MCF-7 breast cancer cells. Here, we demonstrate that over-expression of wild-type PTEN leads to the suppression of cell growth through the blockade of cell cycle progression, an increase in the abundance of p27, a decrease in the protein levels of cyclin D1 and the inhibition of Akt phosphorylation. In contrast, expression of the phosphatase-dead mutant, C124S, promotes cell growth and has the opposite effect on the abundance of p27, cyclin D1 levels and the phosphorylation of Akt. The G129E mutant, which does not have lipid phosphatase activity but retains protein phosphatase activity, behaves like C124S except that the former causes decreases in cyclin D1 levels similar to wild-type PTEN. Therefore, PTEN exerts its growth suppression through lipid phosphatase-dependent and independent activities and most likely, via the coordinate effect of both protein phosphatase and lipid phosphatase activities. Addition of either estrogen or insulin abrogates PTEN-mediated up-regulation of p27 and partially blocks PTEN-mediated growth suppression, whereas the combination of estrogen and insulin eliminates the alterations of p27 and cyclin D1 and completely blocks PTEN-mediated growth suppression. Our findings demonstrate that PTEN blocks cell cycle progression differentially through down-regulating the positive cell cycle regulator, cyclin D1, by its protein phosphatase activity, and up-regulating the negative cell cycle regulator, p27, by its lipid phosphatase activity.