Characterization of a novel PTEN mutation in MDA-MB-453 breast carcinoma cell line

<p>Abstract</p> <p>Background</p> <p>Cowden Syndrome (CS) patients with germ line point mutations in the <it>PTEN </it>gene are at high risk for developing breast cancer. It is believed that cells harboring these mutant <it>PTEN </it>alleles are predisposed to malignant conversion. This article will characterize the biochemical and biological properties of a mutant PTEN protein found in a commonly used metastatic breast cancer cell line.</p> <p>Methods</p> <p>The expression of PTEN in human breast carcinoma cell lines was evaluated by Western blotting analysis. Cell line MDA-MB-453 was selected for further analysis. Mutation analysis of the <it>PTEN </it>gene was carried out using DNA isolated from MDA-MB-453. Site-directed mutagenesis was used to generate a PTEN E307K mutant cDNA and ectopic expressed in PC3, U87MG, MCF7 and <it>Pten</it>^-/-mouse embryo fibroblasts (MEFS). Histidine (His)-tagged PTEN fusion protein was generated in <it>Sf9 </it>baculovirus expression system. Lipid phosphatase and ubiquitination assays were carried out to characterize the biochemical properties of PTEN E307K mutant. The intracellular localization of PTEN E307K was determined by subcellular fractionation experiments. The ability of PTEN E307K to alter cell growth, migration and apoptosis was analyzed in multiple PTEN-null cell lines.</p> <p>Results</p> <p>We found a mutation in the <it>PTEN </it>gene at codon 307 in MDA-MB-453 cell line. The glutamate (E) to lysine (K) substitution rendered the mutant protein to migrate with a faster mobility on SDS-PAGE gels. Biochemically, the PTEN E307K mutant displayed similar lipid phosphatase and growth suppressing activities when compared to wild-type (WT) protein. However, the PTEN E307K mutant was present at higher levels in the membrane fraction and suppressed Akt activation to a greater extent than the WT protein. Additionally, the PTEN E307K mutant was polyubiquitinated to a greater extent by NEDD4-1 and displayed reduced nuclear localization. Finally, the PTEN E307K mutant failed to confer chemosensitivity to cisplatinum when re-expressed in <it>Pten</it>^-/-MEFS.</p> <p>Conclusions</p> <p>Mutation at codon 307 in PTEN C2 loop alters its subcellular distribution with greater membrane localization while being excluded from the cell nucleus. This mutation may predispose breast epithelial cells to malignant transformation. Also, tumor cells harboring this mutation may be less susceptible to the cytotoxic effects of chemotherapeutics.</p

Reduced PTEN expression in the pancreas overexpressing transforming growth factor-beta 1

PTEN is a candidate tumour suppressor gene and frequently mutated in multiple cancers, however, not in pancreatic cancer. Recently, it has been demonstrated that PTEN expression is regulated by TGF-β1. Using TGF-β1 transgenic mice (n=7) and wildtype littermates (n=6), as well as pancreatic tissues obtained from organ donors (n=10) and patients with pancreatic cancer (n=10), we assessed the expression of PTEN by means of immunohistochemistry and semiquantitative PCR analysis. In addition, PANC-1 cells were treated with TGF-β1 in vitro and the levels of PTEN mRNA were determined in these cells. In human pancreatic cancers PTEN mRNA levels were significantly decreased (P<0.05). In addition, in the pancreas of TGF-β1 transgenic mice the expression of PTEN was significantly reduced (P<0.01), as compared to wildtype littermates and incubation of PANC-1 cells with TGF-β1 decreased PTEN mRNA levels after 24 h. Inasmuch as TGF-β1 decreases PTEN expression in human pancreatic cancer cells and human pancreatic cancers overexpress TGF-β1, the reduced expression of PTEN in pancreatic cancer may be mediated by TGF-β1 overexpression. Thus, although PTEN is not mutated in pancreatic cancers, the reduction of its expression may give pancreatic cancer cells an additional growth advantage

Tumours and tremors: how PTEN regulation underlies both

Mutations of the tumour suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) are seen in many human cancers. However, dysregulation of PTEN may be involved in other disease states such as Parkinson's disease. This minireview describes recent work examining PTEN regulation and its implications for the development of both cancer and neurodegenerative disease

Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells

Recent advances have highlighted extensive phenotypic and functional similarities between normal stem cells and cancer stem cells. This raises the question of whether disease therapies can be developed that eliminate cancer stem cells without eliminating normal stem cells. Here we address this issue by conditionally deleting the Pten tumour suppressor gene in adult haematopoietic cells. This led to myeloproliferative disease within days and transplantable leukaemias within weeks. Pten deletion also promoted haematopoietic stem cell (HSC) proliferation. However, this led to HSC depletion via a cell-autonomous mechanism, preventing these cells from stably reconstituting irradiated mice. In contrast to leukaemia-initiating cells, HSCs were therefore unable to maintain themselves without Pten. These effects were mostly mediated by mTOR as they were inhibited by rapamycin. Rapamycin not only depleted leukaemia-initiating cells but also restored normal HSC function. Mechanistic differences between normal stem cells and cancer stem cells can thus be targeted to deplete cancer stem cells without damaging normal stem cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62514/1/nature04703.pd

Identification of two novel CT antigens and their capacity to elicit antibody response in hepatocellular carcinoma patients

FATE and TPTE genes were originally reported to be specifically expressed in the adult testis. We searched for the databases of Unigene and serial analysis of gene expression ( SAGE) implying that these two gene transcripts might also be expressed in tumours. Herein, we demonstrated that FATE and TPTE mRNA transcripts were expressed in different histological types of tumours and normal testis. Both are cancer-testis (CT) antigens and renamed as FATE/BJ-HCC-2 and TPTE/BJ-HCC-5, respectively. Comparison at nucleotide sequence, the FATE/BJ-HCC-2 cDNA, was identical to that of FATE, whereas the TPTE/BJ-HCC-5 was found to have two isoforms in both cancers and testis: one was identical in cDNA sequence to TPTE, encoding a protein of 551 amino acids, and the other variant lacked an exon of 54 bp, encoding a protein of 533 amino acids. The mRNA expression was analysed by RT-PCR and real-time PCR. FATE/BJ-HCC-2 mRNA was detected in 66% ( 41 out of 62) in hepatocellular carcinoma (HCC) samples and 21% ( three out of 14) in colon cancer samples, whereas the TPTE/BJ-HCC-5 mRNA was detected in 39% ( 24 out of 62) and 36% ( five out of 14) in HCC and non-small lung cancer samples, respectively. The recombinant proteins were prepared and the reactivity of allogenic sera to these two antigens was screened. The frequency of antibody response against FATE/BJ-HCC-2 and TPTE/BJ-HCC-5 proteins was 7.3% ( three out of 41) and 25.0% ( six out of 24), respectively, in HCC patients bearing respective gene transcripts. Therefore, FATE/BJ-HCC-2 and TPTE/BJ-HCC-5 are the novel CT antigens capable of eliciting antibody response in cancer patients.OncologySCI(E)PubMed22ARTICLE2291-2978

A novel germline mutation of PTEN associated with brain tumours of multiple lineages

We have identified a novel germline mutation in the PTEN tumour suppressor gene. The mutation was identified in a patient with a glioma, and turned out to be a heterozygous germline mutation of PTEN (Arg234Gln), without loss of heterozygosity in tumour DNA. The biological consequences of this germline mutation were investigated by means of transfection studies of the mutant PTEN molecule compared to wild-type PTEN. In contrast to the wild-type molecule, the mutant PTEN protein is not capable of inducing apoptosis, induces increased cell proliferation and leads to high constitutive PKB/Akt activation, which cannot be increased anymore by stimulation with insulin. The reported patient, in addition to glioma, had suffered from benign meningioma in the past but did not show any clinical signs of Cowden disease or other hereditary diseases typically associated with PTEN germline mutations. The functional consequences of the mutation in transfection studies are consistent with high proliferative activity. Together, these findings suggest that the Arg234Gln missense mutation in PTEN has oncogenic properties and predisposes to brain tumours of multiple lineages

HD-PTP Is a Catalytically Inactive Tyrosine Phosphatase Due to a Conserved Divergence in Its Phosphatase Domain

The HD-PTP protein has been described as a tumor suppressor candidate and based on its amino acid sequence, categorized as a classical non-transmembrane protein tyrosine phosphatase (PTP). To date, no HD-PTP phosphorylated substrate has been identified and controversial results concerning its catalytic activity have been recently reported

JNK3 Maintains Expression of the Insulin Receptor Substrate 2 (IRS2) in Insulin-Secreting Cells: Functional Consequences for Insulin Signaling

We have recently shown that silencing of the brain/islet specific c-Jun N-terminal Kinase3 (JNK3) isoform enhances both basal and cytokine-induced beta-cell apoptosis, whereas silencing of JNK1 or JNK2 has opposite effects. While it is known that JNK1 or JNK2 may promote apoptosis by inhibiting the activity of the pro-survival Akt pathway, the effect of JNK3 on Akt has not been documented. This study aims to determine the involvement of individual JNKs and specifically JNK3 in the regulation of the Akt signaling pathway in insulin-secreting cells. JNK3 silencing strongly decreases Insulin Receptor Substrate 2 (IRS2) protein expression, and blocks Akt2 but not Akt1 activation by insulin, while the silencing of JNK1 or JNK2 activates both Akt1 and Akt2. Concomitantly, the silencing of JNK1 or JNK2, but not of JNK3, potently phosphorylates the glycogen synthase kinase3 (GSK3β). JNK3 silencing also decreases the activity of the transcription factor Forkhead BoxO3A (FoxO3A) that is known to control IRS2 expression, in addition to increasing c-Jun levels that are known to inhibit insulin gene expression. In conclusion, we propose that JNK1/2 on one hand and JNK3 on the other hand, have opposite effects on insulin-signaling in insulin-secreting cells; JNK3 protects beta-cells from apoptosis and dysfunction mainly through maintenance of a normal IRS2 to Akt2 signaling pathway. It seems that JNK3 mediates its effects mainly at the transcriptional level, while JNK1 or JNK2 appear to mediate their pro-apoptotic effect in the cytoplasm