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.  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.
mammary carcinogenesis; PKB/Akt; PTEN; Wnt-1
MMTV-Wnt1 transgenic mice develop mammary hyperplasia early in development, followed by the appearance of solitary mammary tumors with a high proportion of cells expressing early lineage markers and many myoepithelial cells. The occurrence of tumors is accelerated in experiments that activate FGF proto-oncogenes or remove the tumor suppressor genes Pten or P53, implying that secondary oncogenic events are required for progression from mammary hyperplasia to carcinoma. It is not known, however, which oncogenic pathways contribute to Wnt1-induced tumorigenesis – further experimental manipulation of these mice is needed. Secondary events also appear to be required for mammary tumorigenesis in MMTV-Neu transgenic mice because the transgene in the tumors usually contains an acquired mutation that activates the Neu protein-tyrosine kinase.
cDNA or DNA from the mammary glands and mammary tumors from MMTV-Wnt1, MMTV-Wnt1/p53-/-, MMTV-Neu transgenic mice, and newly generated MMTV-Wnt1/MMTV-Neu bitransgenic mice, was sequenced to seek activating mutations in H-Ras, K-Ras, and N-Ras genes, or in the MMTV-Neu transgene. In addition, tumors from bitransgenic animals were examined to determine the cellular phenotype.
We found activating mutations at codons 12, 13, and 61 of H-Ras in just over half of the mammary tumors in MMTV-Wnt1 transgenic mice, and we confirmed the high frequency of activating mutations of Neu in tumors in MMTV-Neu transgenic mice. Tumors appeared earlier in bitransgenic MMTV-Wnt1/MMTV-Neu mice, but no Ras or MMTV-Neu mutations were found in these tumors, which were phenotypically similar to those arising in MMTV-Wnt1 mice. In addition, no Ras mutations were found in the mammary tumors that arise in MMTV-Wnt1 transgenic mice lacking an intact P53 gene.
Tumorigenic properties of cells undergoing functionally significant secondary mutations in H-Ras or the MMTV-Neu transgene allow selection of those cells in MMTV-Wnt1 and MMTV-Neu transgenic mice, respectively. Alternative sources of oncogenic potential, such as a second transgenic oncogene or deficiency of a tumor suppressor gene, can obviate the selective power of those secondary mutations. These observations are consistent with the notion that somatic evolution of mouse mammary tumors is influenced by the specific nature of the inherited cancer-promoting genotype.
Cancer susceptibility has been attributed to at least one heterozygous genetic alteration in a tumor suppressor gene (TSG)1. It has been hypothesized that subtle variations in TSG expression can promote cancer development2,3. However, this hypothesis has not yet been definitively supported in vivo. PTEN is a TSG frequently lost in human cancer and mutated in inherited cancer-predisposition syndromes4. Here, we analyze Pten hypermorphic mice (Ptenhy/+), expressing 80% normal levels of Pten. Ptenhy/+ mice develop a spectrum of tumors, with breast tumors occurring at the highest penetrance. All breast tumors analyzed here retained two intact copies of Pten and maintained Pten levels above heterozygosis. Notably, subtle downregulation of Pten altered the steady-state biology of the mammary tissues and the expression profiles of genes involved in cancer cell proliferation. We present an alterative working model for cancer development in which subtle reductions in the dose of TSGs predispose to tumorigenesis in a tissue-specific manner.
Loss of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and amplification or elevated expression of ErbB-2 are both involved in human breast cancer. To directly test the importance of these genetic events in mammary tumorigenesis, we have assessed whether mammary-specific disruption of PTEN could cooperate with activation of ErbB-2. Transgenic mice expressing ErbB-2 under the transcriptional control of its endogenous promoter (ErbB-2KI) were interbred with mice carrying conditional PTEN alleles and an MMTV/Cre transgene. Loss of one or both PTEN alleles resulted in a dramatic acceleration of mammary tumor onset and an increased occurrence of lung metastases in the ErbB-2KI strain. Tumor progression in PTEN-deficient/ErbB-2KI strains was associated with elevated ErbB-2 protein levels, which were not due to ErbB-2 amplification or to a dramatic increase in ErbB-2 transcripts. Moreover, the PTEN-deficient/ErbB-2KI–derived mouse mammary tumors display striking morphologic heterogeneity in comparison with the homogeneous pathology of the ErbB-2KI parental strain. Therefore, inactivation of PTEN would not only have a dramatic effect on ErbB-2–induced mammary tumorigenesis but would also lead to the formation of mammary tumors that, in part, display pathologic and molecular features associated with the basal-like subtype of primary human breast cancer.
Inactivation of the Rb-mediated G1 control pathway is a common event found in many types of human tumors. To test how the Rb pathway interacts with other pathways in tumor suppression, we characterized mice with mutations in both the cyclin-dependent kinase (CDK) inhibitor p18Ink4c and the lipid phosphatase Pten, which regulates cell growth. The double mutant mice develop a wider spectrum of tumors, including prostate cancer in the anterior and dorsolateral lobes, with nearly complete penetrance and at an accelerated rate. The remaining wild-type allele of Pten was lost at a high frequency in Pten+/− cells but not in p18+/− Pten+/− or p18−/− Pten+/− prostate tumor cells, nor in other Pten+/− tumor cells, suggesting a tissue- and genetic background-dependent haploinsufficiency of Pten in tumor suppression. p18 deletion, CDK4 overexpression, or oncoviral inactivation of Rb family proteins caused activation of Akt/PKB that was recessive to the reduction of PTEN activity. We suggest that p18 and Pten cooperate in tumor suppression by constraining a positive regulatory loop between cell growth and cell cycle control pathways.
PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. The role of PTEN in carcinogenesis has been validated by knockout mouse models. PTEN heterozygous mice develop neoplasms in multiple organs. Unfortunately, the embryonic lethality of biallelic excision of PTEN has inhibited the study of complete PTEN deletion in the development and progression of cancer. By crossing PTEN conditional knockout mice with transgenic mice expressing a tamoxifen-inducible Cre-ERT under the control of a chicken actin promoter, we have generated a tamoxifen-inducible mouse model that allows temporal control of PTEN deletion. Interestingly, administration of a single dose of tamoxifen resulted in PTEN deletion mainly in epithelial cells, but not in stromal, mesenchymal or hematopoietic cells. Using the mT/mG double-fluorescent Cre reporter mice, we demonstrate that epithelial-specific PTEN excision was caused by differential Cre activity among tissues and cells types. Tamoxifen-induced deletion of PTEN resulted in extremely rapid and consistent formation of endometrial in situ adenocarcinoma, prostate intraepithelial neoplasia and thyroid hyperplasia. We also analyzed the role of PTEN ablation in other epithelial cells, such as the tubular cells of the kidney, hepatocytes, colonic epithelial cells or bronchiolar epithelium, but those tissues did not exhibit neoplastic growth. Finally, to validate this model as a tool to assay the efficacy of anti-tumor drugs in PTEN deficiency, we administered the mTOR inhibitor everolimus to mice with induced PTEN deletion. Everolimus dramatically reduced the progression of endometrial proliferations and significantly reduced thyroid hyperplasia. This model could be a valuable tool to study the cell-autonomous mechanisms involved in PTEN-loss-induced carcinogenesis and provides a good platform to study the effect of anti-neoplastic drugs on PTEN-negative tumors.
Cancer develops through a multistep process in which normal cells progress to malignant tumors via the evolution of their genomes as a result of the acquisition of mutations in cancer driver genes. The number, identity and mode of action of cancer driver genes, and how they contribute to tumor evolution is largely unknown. This study deployed the Mouse Mammary Tumor Virus (MMTV) as an insertional mutagen to find both the driver genes and the networks in which they function. Using deep insertion site sequencing we identified around 31000 retroviral integration sites in 604 MMTV-induced mammary tumors from mice with mammary gland-specific deletion of Trp53, Pten heterozygous knockout mice, or wildtype strains. We identified 18 known common integration sites (CISs) and 12 previously unknown CISs marking new candidate cancer genes. Members of the Wnt, Fgf, Fgfr, Rspo and Pdgfr gene families were commonly mutated in a mutually exclusive fashion. The sequence data we generated yielded also information on the clonality of insertions in individual tumors, allowing us to develop a data-driven model of MMTV-induced tumor development. Insertional mutations near Wnt and Fgf genes mark the earliest “initiating” events in MMTV induced tumorigenesis, whereas Fgfr genes are targeted later during tumor progression. Our data shows that insertional mutagenesis can be used to discover the mutational networks, the timing of mutations, and the genes that initiate and drive tumor evolution.
Many cancers, including breast cancer, harbor loss of function mutations in the catalytic domain of the PTEN phosphatase or have reduced PTEN expression through loss of heterozygosity (LOH) and/or epigenetic silencing mechanisms. However, specific phenotypic effects of PTEN inactivation in human cancer cells remain poorly defined without a direct causal connection between the loss of PTEN function and the development or progression of cancer. To evaluate the biological and clinical relevance of reduced or deleted PTEN expression, a novel in vitro model system was generated using human somatic cell knock-out technologies. Targeted homologous recombination allowed for a single and double allelic deletion which resulted in reduced and deleted PTEN expression, respectively. We determined that heterozygous loss of PTEN in the non-tumorigenic human mammary epithelial cell line, MCF-10A, was sufficient for activation of the PI3K/Akt and MAPK pathways, while the homozygous absence of PTEN expression led to a further increased activation of both pathways. The deletion of PTEN was able to confer growth factor-independent proliferation which was confirmed by the resistance of the PTEN−/− MCF-10A cells to small molecule inhibitors of the EGF receptor. However, neither heterozygous nor homozygous loss of PTEN expression was sufficient to promote anchorage-independent growth, but the loss of PTEN did confer apoptotic resistance to cell rounding and matrix detatchment. Finally, MCF-10A cells with the reduction or loss of PTEN showed increased susceptibility to the chemotherapeutic drug doxorubicin, but not paclitaxel.
PTEN; breast cancer; anoikis; tumor dormancy; doxorubicin
The phosphatase PTEN regulates growth, adhesion, and apoptosis, among many other cell processes. To investigate its role during mouse mammary gland development, we generated MK-PTEN, a transgenic mouse model in which human PTEN is overexpressed in ductal and alveolar mammary epithelium during puberty, pregnancy, lactation, and involution. No obvious phenotype was observed in mammary tissue of pubescent virgin mice. However, MK-PTEN females could not lactate normally, and ∼30% of pups died, with survivors exhibiting growth retardation. Transgenic offspring nursed by wild-type foster mothers, conversely, developed normally. This phenotype is consistent with a reduced number of alveolar epithelial cells due to a decrease in cell proliferation and an increase in apoptosis. Using mammary-enriched cDNA microarrays, we identified several genes that were preferentially expressed in MK-PTEN mammary tissue, including the IGF-binding protein-5 (Igfbp5) gene, and others whose expression was reduced, including the genes for c-Jun amino-terminal kinase. Secretory epithelial cell differentiation was impaired, as measured by the expression of specific milk protein genes. MK-PTEN mice also exhibited a 50% decrease in the phosphorylation state of Akt. Taken together, these results suggest that PTEN controls mammary gland development and, consequently, lactation.
cDNA microarray-derived expression profiles of MMTV-Wnt-1 and MMTV-Neu transgenic mice reveal several hundred genes to be differentially expressed at each stage of breast tumor development.
In human breast cancer normal mammary cells typically develop into hyperplasia, ductal carcinoma in situ, invasive cancer, and metastasis. The changes in gene expression associated with this stepwise progression are unclear. Mice transgenic for mouse mammary tumor virus (MMTV)-Wnt-1 exhibit discrete steps of mammary tumorigenesis, including hyperplasia, invasive ductal carcinoma, and distant metastasis. These mice might therefore be useful models for discovering changes in gene expression during cancer development.
We used cDNA microarrays to determine the expression profiles of five normal mammary glands, seven hyperplastic mammary glands and 23 mammary tumors from MMTV-Wnt-1 transgenic mice, and 12 mammary tumors from MMTV-Neu transgenic mice. Adipose tissues were used to control for fat cells in the vicinity of the mammary glands. In these analyses, we found that the progression of normal virgin mammary glands to hyperplastic tissues and to mammary tumors is accompanied by differences in the expression of several hundred genes at each step. Some of these differences appear to be unique to the effects of Wnt signaling; others seem to be common to tumors induced by both Neu and Wnt-1 oncogenes.
We described gene-expression patterns associated with breast-cancer development in mice, and identified genes that may be significant targets for oncogenic events. The expression data developed provide a resource for illuminating the molecular mechanisms involved in breast cancer development, especially through the identification of genes that are critical in cancer initiation and progression.
The phosphatase and tensin homolog located on chromosome ten, PTEN, is one of the most commonly mutated tumor suppressor genes (TSGs) in human cancer [1–3]. PTEN catalyzes the conversion of the membrane lipid second messenger PIP3 to PIP2 and is therefore a key mediator of the AKT/PKB pathway [4,5]. Although inherited PTEN mutations predispose to the development of Cowden syndrome, which is also a breast cancer susceptibility syndrome, the role of PTEN in breast tumorigenesis has been considered minor when compared to that of other TSGs such as BRCA1 or p53 . There is no current evidence that mutations in PTEN account for a substantial proportion of familial breast cancer in the absence of Cowden syndrome . Moreover, PTEN mutations or deletions are not common in sporadic breast tumors, especially when compared with other tumor types (<5%) such as prostate cancer [7, 8].
Despite this evidence, recent studies have demonstrated that PTEN protein down-regulation is frequently observed (more than 50%) in sporadic breast tumors, highlighting the relevance of the dose of this TSG for the pathogenesis of breast cancer [7–9]. Our paper, in the last month’s issue of Nature Genetics provides additional evidence of the role of PTEN dose in breast cancer susceptibility, braking current dogmas regarding the development of cancer and opening to novel clinical and therapeutic implications .
Basal-like breast cancer (BBC) is a subtype of breast cancer with poor prognosis1–3. Inherited mutations of BRCA1, a cancer susceptibility gene involved in double-strand DNA break (DSB) repair, lead to breast cancers that are nearly always of the BBC subtype3–5; however, the precise molecular lesions and oncogenic consequences of BRCA1 dysfunction are poorly understood. Here we show that heterozygous inactivation of the tumor suppressor gene Pten leads to the formation of basal-like mammary tumors in mice, and that loss of PTEN expression is significantly associated with the BBC subtype in human sporadic and BRCA1-associated hereditary breast cancers. In addition, we identify frequent gross PTEN mutations, involving intragenic chromosome breaks, inversions, deletions and micro copy number aberrations, specifically in BRCA1-deficient tumors. These data provide an example of a specific and recurrent oncogenic consequence of BRCA1-dependent dysfunction in DNA repair and provide insight into the pathogenesis of BBC with therapeutic implications. These findings also argue that obtaining an accurate census of genes mutated in cancer will require a systematic examination for gross gene rearrangements, particularly in tumors with deficient DSB repair.
Skin cancer is the most common cancer in the United States. UV radiation in sunlight is the major environmental factor causing skin cancer development. PTEN (phosphatase and tensin homolog deleted on chromosome 10), a recently discovered tumor suppressor gene, is frequently mutated, deleted, or epigenetically silenced in various human cancers. PTEN negatively regulates the oncogenic phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways. PTEN is clearly a critical tumor suppressor for skin cancer in humans and in mice. This review summarizes the recent progress in the function of PTEN in the development of skin cancer, including basal-cell carcinoma, squamous-cell carcinoma, and melanoma. The regulation of PTEN by UV radiation is also discussed in association with skin carcinogenesis. Understanding the fundamental mechanisms that lead to the reduction of PTEN function in skin carcinogenesis and the essential association with UV radiation opens up new opportunities for molecular chemoprevention and therapy of skin cancer by targeting PTEN pathways.
Expression of PTEN tumor suppressor is frequently lost in breast cancer in the absence of mutation or promoter methylation through as yet undetermined mechanisms. In this study, we demonstrated that the Rak tyrosine kinase physically interacts with PTEN and phosphorylates PTEN on Tyr 336. The knockdown of Rak enhanced the binding of PTEN to its E3 ligase, NEDD4-1, and promoted PTEN polyubiquitination, leading to PTEN protein degradation. Notably, ectopic expression of Rak effectively suppressed breast cancer cell proliferation, invasion, and colony formation in vitro and tumor growth in vivo. Furthermore, Rak knockdown was sufficient to transform normal mammary epithelial cells. Therefore, Rak acts as a bona fide tumor suppressor gene through the mechanism of regulating PTEN protein stability and function.
Germline and somatic PTEN mutations are found in Cowden syndrome (CS) and multiple sporadic malignancies, respectively. PTEN function appears to be modulated by subcellular compartmentalization, and mislocalization may affect function. We have shown that cellular ATP levels affect nuclear PTEN levels. Here, we examined the ATP-binding capabilities of PTEN and functional consequences, relevant to cancer-associated mutations. PTEN mutation analysis of CS patients and sporadic colorectal carcinomas and comparative aminoacid analysis were utilized to identify mutations in ATP-binding motifs. The ability of wild-type (WT) or mutant PTEN to bind ATP was assessed by ATP–agarose-binding assays. Subcellular fractionation, western blotting, confocal microscopy and growth assays were used to determine relative nuclear-cytoplasmic localization and function. Somatic colorectal carcinoma-derived PTEN missense mutations were associated with nuclear mislocalization. These mutations altered cellular proliferation, apoptosis and anchorage-dependent growth. Examination of PTEN's amino acid sequence revealed these mutations resided in previously undescribed ATP-binding motifs (c.60–73; c.122–136). In contrast to WT PTEN, both cancer-associated somatic and germline-derived PTEN missense mutations, which lie within the ATP-binding motifs, result in mutant PTEN that does not bind ATP efficiently. We also show that CS patients with germline ATP-binding motif-mutations had nuclear PTEN mislocalization. Of four unrelated patients with functional germline ATP-binding domain mutations, all three female patients had breast cancers. Germline and somatic mutations within PTEN's ATP-binding domain play important pathogenic roles in both heritable and sporadic carcinogenesis by PTEN nuclear mislocalization resulting in altered signaling and growth. Manipulation of ATP may represent novel therapies in tumors with such PTEN alterations.
Membrane type 1 (MT1)-matrix metalloproteinase (MT1-MMP) is a membrane-tethered MMP that has been shown to play a key role in promoting cancer cell invasion. MT1-MMP is highly expressed in bone metastasis of prostate cancer (PC) patients and promotes intraosseous tumor growth of PC cells in mice. The majority of metastatic prostate cancers harbor loss-of-function mutations or deletions of the tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten). However, the role of PTEN inactivation in MT1-MMP expression in PC cells has not been examined. In this study, prostate epithelial cell lines derived from mice that are either heterozygous (PTEN+/-) or homozygous (PTEN-/-) for PTEN deletion or harboring a wild type PTEN (PTEN+/+) were used to investigate the expression of MT1-MMP. We found that biallelic loss of PTEN is associated with posttranslational regulation of MT1-MMP protein in mouse PC cells. PTEN-/- PC cells display higher levels of MT1-MMP at the cell surface when compared to PTEN+/+ and PTEN+/- cells and consequently exhibited enhanced migratory and collagen-invasive activities. MT1-MMP displayed by PTEN-/- cells is differentially O-glycosylated and exhibits a slow rate of turnover. MT1-MMP expression in PTEN-/- cells is under control of the PI3K/AKT signaling pathway, as determined using pharmacological inhibitors. Interestingly, rapamycin, an mTOR inhibitor, up-regulates MT1-MMP expression in PTEN+/+ cells via PI3K activity. Collectively, these data in a mouse prostate cell system uncover for the first time a novel and complex relationship between PTEN loss-mediated PI3K/AKT activation and posttranslational regulation of MT1-MMP, which may play a role in PC progression.
matrix metalloproteinases; prostate cancer; PTEN; glycosylation; posttranslational modification
Tumor suppressor gene PTEN is important in the initiation and progression of human prostate carcinoma, whereas the role of TP53 remains controversial. Since Pten/Trp53 double conditional knockout mice show earlier onset and fast progression of prostate cancer when compared to Pten knockout mice, we asked whether heterozygosity of these two tumor suppressor genes was sufficient to accelerate prostatic tumorigenesis. To answer this question we examined prostatic lesion progression of Pten/Trp53 double heterozygous mice and a series of controls such as Pten heterozygous, Pten conditional knockout, Trp53 heterozygous and Trp53 knockout mice. Tissue recombination of adult prostatic epithelium coupled with embryonic rat seminal vesicle mesenchyme was used as a tool to stimulate prostatic epithelial proliferation. In our study, high-grade prostatic intraepithelial neoplasia (PIN) was found with high frequency at 8 weeks post-tissue recombination transplantation. PIN lesions in Pten/Trp53 double heterozygous mice were more severe than those seen in Pten heterozygous alone. Furthermore, morphologic features attributable to Pten or Trp53 loss appeared to be enhanced in double heterozygous tissues. LOH analysis of Pten and Trp53 in genomic DNA collected from high-grade PIN lesions in Pten heterozygous and Pten/Trp53 double heterozygous mice showed an intact wild-type allele for both genes in all samples examined. In conclusion, simultaneous heterozygosity of Pten and Trp53 accelerates prostatic tumorigenesis in this mouse model of prostate cancer independently of loss of heterozygosity of either gene.
Prostate cancer; Tumor suppressor genes; Pten; Trp53; Mouse models; Pathology; AKT; Tissue recombinants; Embryonic mesenchyme
Cowden Syndrome (CS) patients with germ line point mutations in the PTEN gene are at high risk for developing breast cancer. It is believed that cells harboring these mutant PTEN 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.
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 PTEN 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 Pten-/- mouse embryo fibroblasts (MEFS). Histidine (His)-tagged PTEN fusion protein was generated in Sf9 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.
We found a mutation in the PTEN 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 Pten-/- MEFS.
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.
Indole-3-carbinol (I3C) is a phytochemical (derived from broccoli, cabbage, and other cruciferous vegetables) with proven anticancer efficacy including the reduction of cervical intraepithelial neoplasia (CIN) and its progression to cervical cancer. In a breast cancer cell line, I3C inhibited cell adhesion, spreading, and invasion associated with an upregulation of the tumor suppressor gene PTEN, suggesting that PTEN is important in inhibition of late stages in the development of cancer. The goal of this study was to determine the expression of PTEN during the development of cervical cancer and whether I3C affected expression of PTEN in vivo. We show diminished PTEN expression during the progression from low-grade to high-grade cervical dysplasia in humans and in a mouse model for cervical cancer, the K14HPV16 transgenic mice promoted with estrogen. The implication is that loss of PTEN function is required for this transition. Additionally, dietary I3C increased PTEN expression in the cervical epithelium of the transgenic mouse, an observation that suggests PTEN upregulation by I3C is one mechanism by which I3C inhibits development of cervical cancer.
Somatic and germline mutations in PTEN (phosphatase and tensin homolog deleted on chromosome 10) are found in sporadic cancers and Cowden syndrome patients, respectively. Recent identification of naturally occurring cancer and germline mutations within the ATP-binding motifs of PTEN (heretofore referred to as PTEN ATP-binding mutations) has revealed that these mutations disrupted the subcellular localization and tumor-suppressor activity of PTEN. However, very little is known about the underlying mechanisms of PTEN ATP-binding mutations in tumorigenesis. Here we show that these mutations impair PTEN's function both qualitatively and quantitatively. On the one hand, PTEN ATP-binding mutants lose their phosphatase activity and the effect of downregulation of cyclin D1. On the other, the mislocalized mutant PTEN results in a significantly decreased nuclear p53 protein level and transcriptional activity, enhanced production of reactive oxygen species, induction of Cu/Zn superoxide dismutase as well as dramatically increased DNA double-strand breaks (DSBs). When compared with wild-type PTEN, the ATP-binding mutant PTEN has reduced half-life in vitro and decreased protein expression levels in vivo. Our data, thus, reveal a novel mechanism of tumorigenesis in patients with germline or somatic mutations affecting PTEN ATP-binding motifs, i.e. qualitative and quantitative impairment of PTEN due to the loss of its phosphatase activity, and nuclear mislocalization, resulting in rapid PTEN protein degradation, suppression of p53-mediated transcriptional activity, loss of protection against oxidative stress as well as accumulation of spontaneous DNA DSBs.
The gene encoding Na+/H+ exchanger regulatory factor 1 (NHERF1) is a putative tumor suppressor gene that harbors frequent loss of heterozygosity (LOH) and intragenic mutations in breast carcinoma. The exact biologic activity of NHERF1 in mammary glands, however, remains unclear. It was recently proposed that NHERF1 forms a ternary complex with platelet-derived growth factor receptor (PDGFR) and phosphatase and tensin homolog (PTEN), linking NHERF1 suppressor activity to PDGF-initiated phosphoinositide-3 kinase (PI3K)/PTEN signaling.
The effect of NHERF1 on the kinetics of PDGF-induced Akt activation was determined in cells with varied NHERF1 background. Levels of active Akt in mammary gland of NHERF1 knockout and wild-type mice were compared. We also examined how NHERF1 expression status affects cell sensitivity to PDGFR inhibitor. A plausible connection between NHERF1 and PTEN pathway was explored at the genetic level.
We showed that NHERF1, through its PDZ-I domain, interacts directly with the carboxyl-terminal tail of PTEN. Knocking down NHERF1 expression in Zr75.1 cells markedly delayed the turnover of PDGF-induced phospho-Akt. Conversely, NHERF1 over-expression in MCF10A cells led to accelerated phospho-Akt degradation. The slowed decay of phospho-Akt that resulted from NHERF1 loss was evident in mouse embryonic fibroblasts isolated from NHERF1 knockout mice. In agreement with this, mammary gland tissues from these mice exhibited markedly elevated phospho-Akt. The responses of breast cancer cells to PDGFR inhibition were also altered by changes in NHERF1 expression level. Zr75.1 cells with NHERF1 knockdown were more resistant to STI-571-induced apoptosis than parental cells. Similarly, over-expression of NHERF1 rendered MCF10A cells more sensitive to STI-571. NHERF1-induced apoptotic response relies on an intact PTEN pathway; over-expression of NHERF1 in MCF10A cells with PTEN knockdown did not affect STI-571 sensitivity. It was found that NHERF1 LOH-positive breast cancer cells had reduced NHERF1 expression. Interestingly, these cells more frequently had wild-type PTEN or PI3KCA gene than the LOH-negative lines.
Our data indicate that the interaction of NHERF1 with PTEN counterbalances PI3K/Akt oncogenic signaling and may affect how cells respond to PDGFR inhibition in breast cancer. The dependence of NHERF1 responses on PTEN and genetic segregation of NHERF1 and PTEN (or PI3KCA) alterations suggest that NHERF1 is an active component of the PTEN pathway. Collectively, our study indicates that the biologic activity of NHERF1 in mammary gland is related to PTEN signaling.
The tumor suppressor gene, PTEN, has previously been demonstrated to be involved in breast tumorigenesis and tumor progression. The aim of the present study was to investigate the expression and significance of PTEN in breast carcinomas, to detect the mutation frequency of PTEN in sporadic breast carcinoma tissues and to determine the association between PTEN promoter methylation and gene expression. Immunohistochemical methods were used to analyze the expression of the PTEN gene in 146 cases of breast carcinoma and 10 cases of normal breast tissue closely adjacent to the carcinoma. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis was used to analyze conformation polymorphisms in 45 breast carcinoma and 10 normal breast tissues. Point mutations of abnormal single stranded conformation were detected by DNA sequencing. The methylation of the PTEN promoter was analyzed by methylation-specific PCR. Expression of PTEN was detected in 57.5% (84/146) of patients with breast carcinoma. By contrast, PTEN expression was detected in 100% of normal samples. Expression of PTEN was found to negatively correlate with the tumor size, the pathological stage and the expression of the estrogen receptor (ER) and the progesterone receptor (PR) in breast cancer. The 2-year disease-free survival of patients with a high expression of PTEN was higher compared with those with low PTEN expression (P<0.05). Missense mutations in exon 2 of PTEN were identified in 1/45 breast cancer cases. PTEN promoter methylation was detected in 31.1% (14/45) of breast carcinomas, of which 64.3% (9/14) were associated with a loss of PTEN expression. The tumor suppressor gene, PTEN, was abnormally expressed in the breast carcinomas. The number of PTEN mutations were low (1/45) in the sporadic breast cancer cases analyzed in the present study and PTEN promoter methylation may have been the main mechanism leading to the decreased expression of PTEN. These results indicate that PTEN is important for the tumorigenesis, development and prognosis of breast cancer.
PTEN gene; breast cancer; gene mutation; promoter methylation
Although Wnt signaling activation is frequently observed in human breast cancer, mutations in the genes encoding intracellular components of the Wnt signaling pathway are rare. We found that expression of Wnt signaling co-receptor LRP6 is up-regulated in a subset of human breast cancer tissues and cell lines. To examine whether overexpression of LRP6 in mammary epithelial cells is sufficient to activate Wnt signaling and promote cell proliferation, we generated transgenic mice overexpressing LRP6 in mammary epithelial cells driven by the mouse mammary tumor virus (MMTV) promoter. We found that mammary glands from MMTV-LRP6 mice exhibit significant Wnt activation evidenced by the translocation of β-catenin from membrane to cytoplasmic/nuclear fractions. Expression of several Wnt-target genes including Axin2, Cyclin D1 and c-Myc was also increased in MMTV-LRP6 mice. More importantly, mammary glands from virgin MMTV-LRP6 mice exhibit significant hyperplasia, a precursor to breast cancer, when compared to wild-type littermate controls. Several matrix metalloproteinases are up-regulated in MMTV-LRP6 mice that could contribute to the hyperplasia phenotype. Our results suggest that Wnt signaling activation at the cell surface receptor level can contribute to breast cancer tumorigenesis.
LRP6; Wnt signaling; mammary gland; breast cancer
The PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) tumor suppressor gene is frequently mutated or deleted in a wide variety of solid tumors, and these cancers are generally more aggressive and difficult to treat than those possessing wild type PTEN. While PTEN lies upstream of the phosphoinositide-3 kinase signaling pathway, the mechanisms that mediate its effects on tumor survival remain incompletely understood. Renal cell carcinoma (RCC) is associated with frequent treatment failures (~90% in metastatic cases), and these tumors frequently contain PTEN abnormalities.
Using the ACHN cell line containing wild type PTEN, we generated a stable PTEN knockdown RCC cell line using RNA interference. We then used this PTEN knockdown cell line to show that PTEN attenuation increases resistance to cisplatin-induced apoptosis, a finding associated with increased levels of the cyclin kinase inhibitor p21. Elevated levels of p21 result from stabilization of the protein, and they are dependent on the activities of phosphoinositide-3 kinase and Akt. More specifically, the accumulation of p21 occurs preferentially in the cytosolic compartment, which likely contributes to both cell cycle progression and resistance to apoptosis.
Since p21 regulates a decision point between repair and apoptosis after DNA damage, our data suggest that p21 plays a key role in mechanisms used by PTEN-deficient tumors to escape chemotherapy. This in turn raises the possibility to use p21 attenuators as chemotherapy sensitizers, an area under active continuing investigation in our laboratories.
Correlative data suggest that thyroid hormone receptor-β (TRβ) mutations could increase the risk of mammary tumor development, but unequivocal evidence is still lacking. To explore the role of TRβ mutants in vivo in breast tumor development and progression, we took advantage of a knock-in mouse model harboring a mutation in the Thrb gene encoding TRβ (ThrbPV mouse). Although in adult nulliparous females, a single ThrbPV allele did not contribute to mammary gland abnormalities, the presence of two ThrbPV alleles led to mammary hyperplasia in ~36% ThrbPV/PV mice. The ThrbPV mutation further markedly augmented the risk of mammary hyperplasia in a mouse model with high susceptibility to mammary tumors (Pten+/− mouse), as demonstrated by the occurrence of mammary hyperplasia in ~60% of Thrbpv/+Pten+/− and ~77% of ThrbPV/PV Pten+/− mice versus ~33% of Thrb+/+Pten+/− mice. The ThrbPV mutation increased the activity of signal transducer and activator of transcription (STAT5) to increase cell proliferation and the expression of the STAT5 target gene encoding β-casein in the mammary gland. We next sought to understand the molecular mechanism underlying STAT5 overactivation by TRβPV. Cell-based studies with a breast cancer cell line (T47D cells) showed that thyroid hormone (T3) repressed STAT5 signaling in TRβ-expressing cells through decreasing STAT5-mediated transcription activity and target gene expression, whereas sustained STAT5 signaling was observed in TRβPV-expressing cells. Collectively, these findings show for the first time that a TRβ mutation promotes the development of mammary hyperplasia via aberrant activation of STAT5, thereby conferring a fertile genetic ground for tumorigenesis.
thyroid hormone receptor; breast cancer; mammary tumors; Pten; mouse models