Investigation of <Emphasis Type="Italic">MMAC/PTEN</Emphasis> Gene Mutations and Protein Expression in Low Grade Gliomas

The MMAC/PTEN tumor suppressor gene has an essential biological role in the formation of glioblastomas. It is known that there are variations in genetic alterations in tumors that develop in patients with different ethnic backgrounds; thus, we aimed to evaluate the incidence of MMAC/PTEN mutations and protein expression among various low grade gliomas of Turkish patients. We investigated 28 low grade gliomas for mutations of the MMAC/PTEN gene using single strand conformational polymorphism method followed by DNA sequencing. Additionally, the level of MMAC/PTEN protein expression in the tissues of 26 tumors was assessed by immunohistochemistry. In our investigation, MMAC/PTEN mutations were detected in 2 of 28 tumors (7.14%). One novel sequence variant G → A transition at codon 159 was identified. This missense variation was a result of an alteration from AGG (Arginine) to AAG (Lysine). Moreover, it was observed that MMAC/PTEN protein expression was reduced to 73.08% of tumors. In conclusion, reduced MMAC/PTEN expression by genetic and/or epigenetic mechanisms in low grade gliomas might be associated with glioma tumorigenesis.     

mTOR inhibitors radiosensitize PTEN‐deficient non‐small‐cell lung cancer cells harboring an EGFR activating mutation by inducing autophagy

Clinical resistance to gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), in patients with lung cancer has been linked to acquisition of the T790M resistance mutation in activated EGFR or amplification of MET. Phosphatase and tensin homolog (PTEN) loss has been recently reported as a gefitinib resistance mechanism in lung cancer. The aim of this study was to evaluate the efficacy of radiotherapy in non‐small‐cell lung cancer (NSCLC) with acquired gefitinib resistance caused by PTEN deficiency to suggest radiotherapy as an alternative to EGFR TKIs. PTEN deficient‐mediated gefitinib resistance was generated in HCC827 cells, an EGFR TKI sensitive NSCLC cell line, by PTEN knockdown with a lentiviral vector expressing short hairpin RNA‐targeting PTEN. The impact of PTEN knockdown on sensitivity to radiation in the presence or absence of PTEN downstream signaling inhibitors was investigated. PTEN knockdown conferred acquired resistance not only to gefitinib but also to radiation on HCC827 cells. mTOR inhibitors alone failed to reduce HCC827 cell viability, regardless of PTEN expression, but ameliorated PTEN knockdown‐induced radioresistance. PTEN knockdown‐mediated radioresistance was accompanied by repression of radiation‐induced cytotoxic autophagy, and treatment with mTOR inhibitors released the repression of cytotoxic autophagy to overcome PTEN knockdown‐induced radioresistance in HCC827 cells. These results suggest that inhibiting mTOR signaling could be an effective strategy to radiosensitize NSCLC harboring the EGFR activating mutation that acquires resistance to both TKIs and radiotherapy due to PTEN loss or inactivation mutations. J. Cell. Biochem. 114: 1248–1256, 2013. © 2012 Wiley Periodicals, Inc.     

The gene dosage of class Ia PI3K dictates the development of PTEN hamartoma tumor syndrome

The PTEN hamartoma tumor syndrome (PHTS) is a complex disorder caused by germline inactivating mutations of the tumor suppressor gene PTEN. Loss of PTEN function leads to unimpeded phosphatidylinositol-3′-kinase (PI3K) activity and PI3K-driven cell division. Individuals with PHTS develop benign hamartomas in various tissues and have an increased risk of developing malignant diseases. Notably, no effective therapy currently exists for this disorder. Using both genetic mouse models and pharmacological approaches, we recently demonstrated that PI3K p110α and p110β isoforms play spatially distinct but concerted roles in the skin that are required for the development and maintenance of PHTS. We also show that treatment with a pan-PI3K inhibitor prevents the development of skin PHTS and reverses advanced-stage skin hamartomas in vivo. Here, we report that genetic ablation of only 3 out of 4 p110 alleles is sufficient to block the development of skin hamartomas resulting from the complete loss of Pten in mice. Similar to our findings in skin, we now also show that mammary gland neoplastic lesions can be prevented or reversed upon PI3K inhibition in our PHTS mouse model. Our data suggest a possible route to chemoprevention using reduced doses of PI3K inhibitors for PTEN-deficient carrier patients.     

CpG island hypermethylation of BRCA1 and loss of pRb as co-occurring events in basal/triple-negative breast cancer

Triple-negative breast cancer (TNBC) occurs in approximately 15% of all breast cancer patients, and the incidence of TNBC is greatly increased in BRCA1 mutation carriers. This study aimed to assess the impact of BRCA1 promoter methylation with respect to breast cancer subtypes in sporadic disease. Tissue microarrays (TMAs) were constructed representing tumors from 303 patients previously screened for BRCA1 germline mutations, of which a subset of 111 sporadic tumors had previously been analyzed with respect to BRCA1 methylation. Additionally, a set of eight tumors from BRCA1 mutation carriers were included on the TMAs. Expression analysis was performed on TMAs by immunohistochemistry (IHC) for BRCA1, pRb, p16, p53, PTEN, ER, PR, HER2, CK5/6, EGFR, MUC1 and Ki-67. Data on BRCA1 aberrations and IHC expression was examined with respect to breast cancer-specific survival. The results demonstrate that CpG island hypermethylation of BRCA1 significantly associates with the basal/triple-negative subtype. Low expression of pRb, and high/intense p16, were associated with BRCA1 promoter hypermethylation, and the same effects were seen in BRCA1 mutated tumors. The expression patterns of BRCA1, pRb, p16 and PTEN were highly correlated, and define a subgroup of TNBCs characterized by BRCA1 aberrations, high Ki-67 (≥40%) and favorable disease outcome. In conclusion, our findings demonstrate that epigenetic inactivation of the BRCA1 gene associates with RB/p16 dysfunction in promoting TNBCs. The clinical implications relate to the potential use of targeted treatment based on PARP inhibitors in sporadic TNBCs, wherein CpG island hypermethylation of BRCA1 represents a potential marker of therapeutic response.     

Loss of protein expression and recurrent DNA hypermethylation of the GNG7 gene in squamous cell carcinoma of the head and neck

Although down-regulation of GNG7 in cancer was reported before, its role in carcinogenesis is poorly understood. It belongs to a family of large G-proteins that may be involved in cell-contact-induced growth arrest and function in tumor suppression. In the present study, we stained immunohistochemically 188 tumors derived from larynx or floor of the mouth for GNG7 protein and confronted it with clinicopathologic data. Moreover, we performed bisulfite pyrosequencing to analyze GNG7 promoter methylation. We identified recurrent loss of GNG7 protein expression in 68/188 (36%) cases and promoter hypermethylation in (42/98; 43%) primary tumors, predominantly in young patients (p?相似文献   

Germline PIK3CA and AKT1 Mutations in Cowden and Cowden-like Syndromes

Cowden syndrome (CS) is a difficult-to-recognize multiple hamartoma syndrome with high risks of breast, thyroid, and other cancers. Germline mutations in PTEN on 10q23 were found to cause 85% of CS when accrued from tertiary academic centers, but prospective accrual from the community over the last 12 years has revealed a 25% PTEN mutation frequency. PTEN is the phosphatase that has been implicated in a heritable cancer syndrome and subsequently in multiple sporadic cancers and developmental processes. PTEN antagonizes the AKT1/PI3K signaling pathway and has roles in cell cycle, migration, cell polarity, and apoptosis. We report that 8 of 91 (8.8%) unrelated CS individuals without germline PTEN mutations carried 10 germline PIK3CA mutations (7 missense, 1 nonsense, and 2 indels) and 2 (2.2%) AKT1 mutations. These mutations result in significantly increased P-Thr308-AKT and increased cellular PIP3. Our observations suggest that PIK3CA and AKT1 are CS susceptibility genes.     

Promoter hypermethylation using 24-gene array in early head and neck cancer

Silencing of tumor suppressor genes (TSGs) by DNA promoter hypermethylation is an early event in carcinogenesis and a potential target for personalized cancer treatment. In head and neck cancer, little is known about the role of promoter hypermethylation in survival. Using methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) we investigated the role of promoter hypermethylation of 24 well-described genes (some of which are classic TSGs), which are frequently methylated in different cancer types, in 166 HPV-negative early oral squamous cell carcinomas (OSCC), and 51 HPV-negative early oropharyngeal squamous cell carcinomas (OPSCC) in relation to clinicopathological features and survival. Early OSCC showed frequent promoter hypermethylation in RARB (31% of cases), CHFR (20%), CDH13 (13%), DAPK1 (12%), and APC (10%). More hypermethylation (≥ 2 genes) independently correlated with improved disease specific survival (hazard ratio 0.17, P = 0.014) in early OSCC and could therefore be used as prognostic biomarker. Early OPSCCs showed more hypermethylation of CDH13 (58%), TP73 (14%), and total hypermethylated genes. Hypermethylation of two or more genes has a significantly different effect on survival in OPSCC compared with OSCC, with a trend toward worse instead of better survival. This could have a biological explanation, which deserves further investigation and could possibly lead to more stratified treatment in the future.     

Mutation-specific non-canonical pathway of PTEN as a distinct therapeutic target for glioblastoma

PTEN is one of the most frequently altered tumor suppressor genes in malignant tumors. The dominant-negative effect of PTEN alteration suggests that the aberrant function of PTEN mutation might be more disastrous than deletion, the most frequent genomic event in glioblastoma (GBM). This study aimed to understand the functional properties of various PTEN missense mutations and to investigate their clinical relevance. The genomic landscape of PTEN alteration was analyzed using the Samsung Medical Center GBM cohort and validated via The Cancer Genome Atlas dataset. Several hotspot mutations were identified, and their subcellular distributions and phenotypes were evaluated. We established a library of cancer cell lines that overexpress these mutant proteins using the U87MG and patient-derived cell models lacking functional PTEN. PTEN mutations were categorized into two major subsets: missense mutations in the phosphatase domain and truncal mutations in the C2 domain. We determined the subcellular compartmentalization of four mutant proteins (H93Y, C124S, R130Q, and R173C) from the former group and found that they had distinct localizations; those associated with invasive phenotypes (‘edge mutations’) localized to the cell periphery, while the R173C mutant localized to the nucleus. Invasive phenotypes derived from edge substitutions were unaffected by an anti-PI3K/Akt agent but were disrupted by microtubule inhibitors. PTEN mutations exhibit distinct functional properties regarding their subcellular localization. Further, some missense mutations (‘edge mutations’) in the phosphatase domain caused enhanced invasiveness associated with dysfunctional cytoskeletal assembly, thus suggesting it to be a potent therapeutic target.Subject terms: Cancer, Oncogenes     

Germline mutations and variants in the succinate dehydrogenase genes in Cowden and Cowden-like syndromes

Individuals with PTEN mutations have Cowden syndrome (CS), associated with breast, thyroid, and endometrial neoplasias. Many more patients with features of CS, not meeting diagnostic criteria (termed CS-like), are evaluated by clinicians for CS-related cancer risk. Germline mutations in succinate dehydrogenase subunits SDHB-D cause pheochromocytoma-paraganglioma syndrome. One to five percent of SDHB/SDHD mutation carriers have renal cell or papillary thyroid carcinomas, which are also CS-related features. SDHB-D may be candidate susceptibility genes for some PTEN mutation-negative individuals with CS-like cancers. To address this hypothesis, germline SDHB-D mutation analysis in 375 PTEN mutation-negative CS/CS-like individuals was performed, followed by functional analysis of identified SDH mutations/variants. Of 375 PTEN mutation-negative CS/CS-like individuals, 74 (20%) had increased manganese superoxide dismutase (MnSOD) expression, a manifestation of mitochondrial dysfunction. Among these, 10 (13.5%) had germline mutations/variants in SDHB (n = 3) or SDHD (7), not found in 700 controls (p < 0.001). Compared to PTEN mutation-positive CS/CS-like individuals, those with SDH mutations/variants were enriched for carcinomas of the female breast (6/9 SDH versus 30/107 PTEN, p < 0.001), thyroid (5/10 versus 15/106, p < 0.001), and kidney (2/10 versus 4/230, p = 0.026). In the absence of PTEN alteration, CS/CS-like-related SDH mutations/variants show increased phosphorylation of AKT and/or MAPK, downstream manifestations of PTEN dysfunction. Germline SDH mutations/variants occur in a subset of PTEN mutation-negative CS/CS-like individuals and are associated with increased frequencies of breast, thyroid, and renal cancers beyond those conferred by germline PTEN mutations. SDH testing should be considered for germline PTEN mutation-negative CS/CS-like individuals, especially in the setting of breast, thyroid, and/or renal cancers.     

Promoter Hypermethylation of ARID1A Gene Is Responsible for Its Low mRNA Expression in Many Invasive Breast Cancers

ARID1A (AT-rich interactive domain 1A) has recently been identified as a tumor suppressor gene. Its mRNA expression is significantly low in many breast cancers; this is often associated with more aggressive phenotypes. However, the underlying molecular mechanism for its low expression has not been fully understood. This study was undertaken to evaluate the contribution of gene copy number variation, mutations, promoter methylation and histone modification to ARID1A’s low expression. 38 pairs of breast invasive ductal carcinomas and their normal breast tissue counterparts from the same patients were randomly selected for gene expression and copy number variation detection. Promoter methylation and histone modification levels were evaluated by MeDIP-qPCR and ChIP-qPCR, respectively. PCR product Sanger sequencing was carried out to detect the exon mutation rate. Twenty-two out of 38 invasive ductal carcinomas in the study (57.9%) revealed ARID1A mRNA low expression by realtime RT-PCR. The relative promoter methylation level was, significantly higher in ARID1A mRNA low expression group compared with its high expression group (p<0.001). In the low expression group, nineteen out of 22 invasive ductal carcinomas (86.4%) exhibited ARID1A promoter hypermthylation. In addition, the promoter hypermethylation was accompanied with repressive histone modification (H3K27Me3). Although five out of 38 invasive ductal carcinomas (13.2%) exhibited loss of ARID1A gene copy number by realtime PCR and nine exon novel mutations are seen from eight out of 33 invasive ductal carcinomas (24.2%), there was no statistically significant difference in both ARID1A mRNA low and high expression groups (p = 0.25,and p = 0.68, respectively). We demonstrate that promoter hypermethylation was the main culprit for ARID1A mRNA low expression in invasive ductal carcinomas. The influence of mutation and copy number variation on the expression were statistically insignificant at mRNA level, and were, therefore, not considered the main causes for ARID1A mRNA low expression in invasive breast cancer.     

Dynamics and structural stability effects of germline PTEN mutations associated with cancer versus autism phenotypes

Individuals with germline mutations in the tumor suppressor gene phosphatase and tensin homolog (PTEN), irrespective of clinical presentation, are diagnosed with PTEN hamartoma tumor syndrome (PHTS). PHTS confers a high risk of breast, thyroid, and other cancers or autism spectrum disorder (ASD) with macrocephaly. It remains unclear why mutations in one gene can lead to seemingly disparate phenotypes. Thus, we sought to identify differences in ASD vs. cancer-associated germline PTEN missense mutations by investigating putative structural effects induced by each mutation. We utilized a theoretical computational approach combining in silico structural analysis and molecular dynamics (MD) to interrogate 17 selected mutations from our patient population: six mutations were observed in patients with ASD (only), six mutations in patients with PHTS-associated cancer (only), four mutations shared across both phenotypes, and one mutation with both ASD and cancer. We demonstrate structural stability changes where all six cancer-associated mutations showed a global decrease in structural stability and increased dynamics across the domain interface with a proclivity to unfold, mediating a closed (inactive) active site. In contrast, five of the six ASD-associated mutations showed localized destabilization that contribute to the partial opening of the active site. Our results lend insight into distinctive structural effects of germline PTEN mutations associated with PTEN-ASD vs. those associated with PTEN-cancer, potentially aiding in identification of the shared and separate molecular features that contribute to autism or cancer, thus, providing a deeper understanding of genotype–phenotype relationships for germline PTEN mutations.     

A Transition Zone Showing Highly Discontinuous or Alternating Levels of Stem Cell and Proliferation Markers Characterizes the Development of PTEN-Haploinsufficient Colorectal Cancer

Background

Stepwise acquisition of oncogene mutations and deletion/inactivation of tumor suppressor genes characterize the development of colorectal cancer (CRC). These genetic events interact with discrete morphologic transitions from hyperplastic mucosa to adenomatous areas, followed by in situ malignant transformation and finally invasive carcinoma. The goal of this study was to identify tissue markers of the adenoma-carcinoma morphogenetic transitions in CRC.

Methods and Findings

We analyzed the patterns of expression of growth regulatory and stem cell markers across these distinct morphologic transition zones in 735 primary CRC tumors. In 202 cases with preserved adenoma-adenocarcinoma transition, we identified, in 37.1% of cases, a zone of adenomatous epithelium, located immediately adjacent to the invasive component, that showed rapidly alternating intraglandular stretches of PTEN+ and PTEN- epithelium. This zone exactly overlapped with similar alternating expression of Ki-67 and inversely with the transforming growth factor-beta (TGF-β) growth regulator SMAD4. These zones also show parallel alternating levels and/or subcellular localization of multiple cancer stem/progenitor cell (CSC) markers, including β-catenin/CTNNB1, ALDH1, and CD44. PTEN was always re-expressed in the invasive tumor in these cases, unlike those with complete loss of PTEN expression. Genomic microarray analysis of CRC with prominent CSC-like expansions demonstrated a high frequency of PTEN genomic deletion/haploinsufficiency in tumors with CSC-like transition zones (62.5%) but not in tumors with downregulated but non-alternating PTEN expression (14.3%). There were no significant differences in the levels of KRAS mutation or CTNNB1 mutation in CSC-like tumors as compared to unselected CRC cases.

Conclusions

In conclusion, we have identified a distinctive CSC-like pre-invasive transition zone in PTEN-haploinsufficient CRC that shows convergent on-off regulation of the PTEN/AKT, TGF-β/SMAD and Wnt/β-catenin pathways. This bottleneck-like zone is usually followed by the emergence of invasive tumors with intact PTEN expression but dysregulated TP53 and uniformly high proliferation rates.     

PTEN is involved in the signal transduction pathway of contact inhibition in endometrial cells

PTEN is involved in the regulation of normal cellular functions in addition to its well–known role as a tumor suppressor. In the present study, we have shown that stable transfection of the PTEN gene into PTEN–mutated endometrial carcinoma cells leads to contact inhibition accompanied by a decreased level of phosphorylated–Akt (p–Akt) expression, an increase in p27^Kip1, and a decrease in β–catenin. PTEN–induced cells with contact inhibition exhibit G0–G1 cell-cycle arrest, and the Ki–67 labeling index is reduced. These changes are canceled by transfection of a double–stranded short–interfering RNA against the PTEN gene. Normal endometrial stromal cells increase their PTEN expression when reaching confluence; this is followed by changes in the expression of Akt–related proteins in the same way as in tumor cells. These results indicate that PTEN, p–Akt, p27, and β–catenin are involved in the signal transduction of contact inhibition and suggest that PTEN may, in part, control the proliferation of endometrial carcinoma cells through the induction of contact inhibition.     

HER2 Ile655Val and PTEN IVS4 polymorphisms in patients with breast cancer

Although HER2/PTEN pathway is commonly disrupted in cancer, association of HER2 and PTEN polymorphisms with breast cancer (BC) remains controversial. We investigated the HER2 Ile655 Val and PTEN IVS4 polymorphisms in patients with BC in Turkish population. HER2 Ile655Val (rs 1136201) and PTEN IVS4 (rs 3830675) polymorphisms were determined using polymerase chain reaction-based restriction fragment length polymorphism (PCR–RFLP) in blood samples of 118 BC patients and 118 age-matched healthy controls. We found that the frequency of the Ile/Val genotype of HER2 Ile655Val gene was significantly higher in BC patients (p < 0.009; OR: 1,983 95 % CI: 1.181—3.328). The presence of ATCTT insertion (+/+) genotype at downstream of exon 4 in intron 4 of PTEN IVS4 gene was also associated with 1.83 fold decreased risk of BC development (p < 0.033; OR: 1.83, 95 % CI: 1.11—3.03). Analysis on clinico-pathological parameters showed neither HER2 Ile655Val nor PTEN IVS4 genotypes were not associated with any of the variables (p > 0.05).In conclusion, our findings suggest that the Ile/Val genotype of HER2 and ATCTT insertion (+/+) genotype of PTEN IVS4 gene may play an important role as genetic markers for breast cancer risk, but both genes genotypes may not be useful for predicting tumor prognosis in Turkish population.     

CpG island hypermethylation of BRCA1 and loss of pRb as co-occurring events in basal/triple-negative breast cancer

Triple-negative breast cancer (TNBC) occurs in approximately 15% of all breast cancer patients, and the incidence of TNBC is greatly increased in BRCA1 mutation carriers. This study aimed to assess the impact of BRCA1 promoter methylation with respect to breast cancer subtypes in sporadic disease. Tissue microarrays (TMAs) were constructed representing tumors from 303 patients previously screened for BRCA1 germline mutations, of which a subset of 111 sporadic tumors had previously been analyzed with respect to BRCA1 methylation. Additionally, a set of eight tumors from BRCA1 mutation carriers were included on the TMAs. Expression analysis was performed on TMAs by immunohistochemistry (IHC) for BRCA1, pRb, p16, p53, PTEN, ER, PR, HER2, CK5/6, CK8, CK18, EGFR, MUC1, and Ki-67. Data on BRCA1 aberrations and IHC expression was examined with respect to breast cancer-specific survival. The results demonstrate that CpG island hypermethylation of BRCA1 significantly associates with the basal/triple-negative subtype. Low expression of pRb, and high/intense p16, were associated with BRCA1 promoter hypermethylation, and the same effects were seen in BRCA1 mutated tumors. The expression patterns of BRCA1, pRb, p16 and PTEN were highly correlated, and define a subgroup of TNBCs characterized by BRCA1 aberrations, high Ki-67 (≥ 40%) and favorable disease outcome. In conclusion, our findings demonstrate that epigenetic inactivation of the BRCA1 gene associates with RB/p16 dysfunction in promoting TNBCs. The clinical implications relate to the potential use of targeted treatment based on PARP inhibitors in sporadic TNBCs, wherein CpG island hypermethylation of BRCA1 represents a potential marker of therapeutic response.Key words: BRCA1, methylation, epigenetics, triple negative, breast cancer, retionblastoma tumor suppressor gene, pRb, p16     

Combined image and genomic analysis of high-grade serous ovarian cancer reveals PTEN loss as a common driver event and prognostic classifier

Background

TP53 and BRCA1/2 mutations are the main drivers in high-grade serous ovarian carcinoma (HGSOC). We hypothesise that combining tissue phenotypes from image analysis of tumour sections with genomic profiles could reveal other significant driver events.

Results

Automatic estimates of stromal content combined with genomic analysis of TCGA HGSOC tumours show that stroma strongly biases estimates of PTEN expression. Tumour-specific PTEN expression was tested in two independent cohorts using tissue microarrays containing 521 cases of HGSOC. PTEN loss or downregulation occurred in 77% of the first cohort by immunofluorescence and 52% of the validation group by immunohistochemistry, and is associated with worse survival in a multivariate Cox-regression model adjusted for study site, age, stage and grade. Reanalysis of TCGA data shows that hemizygous loss of PTEN is common (36%) and expression of PTEN and expression of androgen receptor are positively associated. Low androgen receptor expression was associated with reduced survival in data from TCGA and immunohistochemical analysis of the first cohort.

Conclusion

PTEN loss is a common event in HGSOC and defines a subgroup with significantly worse prognosis, suggesting the rational use of drugs to target PI3K and androgen receptor pathways for HGSOC. This work shows that integrative approaches combining tissue phenotypes from images with genomic analysis can resolve confounding effects of tissue heterogeneity and should be used to identify new drivers in other cancers.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0526-8) contains supplementary material, which is available to authorized users.     

A reinvestigation of somatic hypermethylation at the PTEN CpG island in cancer cell lines

Background

PTEN is an important tumour suppressor gene that is mutated in Cowden syndrome as well as various sporadic cancers. CpG island hypermethylation is another route to tumour suppressor gene inactivation, however, the literature regarding PTEN hypermethylation in cancer is controversial. Furthermore, investigation of the methylation status of the PTEN CpG island is challenging due to sequence homology with the PTEN pseudogene, PTENP1. PTEN shares a CpG island promoter with another gene known as KLLN. Here we present a thorough reinvestigation of the methylation status of the PTEN CpG island in DNA from colorectal, breast, ovarian, glioma, lung and haematological cancer cell lines.

Results

Using a range of bisulphite-based PCR assays we investigated 6 regions across the PTEN CpG island. We found that regions 1-4 were not methylated in cancer cell lines (0/36). By allelic bisulphite sequencing and pyrosequencing methylation was detected in regions 5 and 6 in colorectal, breast and haematological cancer cell lines. However, methylation detected in this region was associated with the PTENP1 promoter and not the PTEN CpG island.

Conclusions

We show that methylation of the PTEN CpG island is a rare event in cancer cell lines and that apparent methylation most likely originates from homologous regions of the PTENP1 pseudogene promoter. Future studies should utilize assays that reliably discriminate between PTEN and PTENP1 to avoid data misinterpretation.     

Estrogen receptor beta (ERβ) produces autophagy and necroptosis in human seminoma cell line through the binding of the Sp1 on the phosphatase and tensin homolog deleted from chromosome 10 (PTEN) promoter gene

Testicular germ cell tumors are the most common tumor in male and the least studied. We focused on human seminoma using the TCAM2 cell line. Through ERβ, 10 nM estradiol (E2) was able to induce PTEN gene expression and promoter transactivation. Transient transfections, ChIP and EMSA assays evidenced the 5′-flanking region of PTEN gene promoter E2-responsive. The ERβ binding to the Sp1 on PTEN promoter decreased cell survival. The presence of ERβ or PTEN is necessary to induce the loss of cell survival upon E2, addressing their cooperation in this action. pAKT and AKT expression decreased under E2 and DPN, while known apoptotic markers appeared to be unchanged. The PI3K/AKT pathway inhibition also leads to autophagy: E2 and DPN enhanced the expression of autophagy-related markers such as PI3III, Beclin 1, AMBRA and UVRAG. MDC and TEM assays confirmed E2-induced autophagy. The absence of DNA fragmentation, caspase 9 and PARP1 cleavages suggested that necroptosis and/or parthanatos may occur. FACS analysis, LDH assay and RIP1 expression attested this hypothesis. Our study reveals a unique mechanism through which ERβ/PTEN signaling induces cell death in TCAM2 by autophagy and necroptosis. These data, supporting estrogen-dependency of human seminoma, propose ERβ ligands for therapeutic use in the treatment of this pathological condition.