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
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.
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.
Many components of Wnt/β-catenin signaling pathway also play critical roles in mammary tumor development, yet the role of the tumor suppressor gene APC (adenomatous polyposis coli) in breast oncongenesis is unclear. To better understand the role of Apc in mammary tumorigenesis, we introduced conditional Apc mutations specifically into two different mammary epithelial populations using K14-cre and WAP-cre transgenic mice that express Cre-recombinase in mammary progenitor cells and lactating luminal cells, respectively. Only the K14-cre–mediated Apc heterozygosity developed mammary adenocarcinomas demonstrating histological heterogeneity, suggesting the multilineage progenitor cell origin of these tumors. These tumors harbored truncation mutation in a defined region in the remaining wild-type allele of Apc that would retain some down-regulating activity of β-catenin signaling. Activating mutations at codons 12 and 61 of either H-Ras or K-Ras were also found in a subset of these tumors. Expression profiles of acinar-type mammary tumors from K14-cre; ApcCKO/+ mice showed luminal epithelial gene expression pattern, and clustering analysis demonstrated more correlation to MMTV-neu model than to MMTV-Wnt1. In contrast, neither WAP-cre–induced Apc heterozygous nor homozygous mutations resulted in predisposition to mammary tumorigenesis, although WAP-cre–mediated Apc deficiency resulted in severe squamous metaplasia of mammary glands. Collectively, our results suggest that not only the epithelial origin but also a certain Apc mutations are selected to achieve a specific level of β-catenin signaling optimal for mammary tumor development and explain partially the colon- but not mammary-specific tumor development in patients that carry germline mutations in APC.
Breast cancer is one of the most common malignanices in women in Western countries. Many components of Wnt/β-catenin signaling pathway are known to play critical roles in mammary tumor development, yet the role of the tumor suppressor gene APC (adenomatous polyposis coli) in breast oncongenesis is unclear. To study the role of Apc in mammary tumorigenesis, we introduced conditional Apc mutations specifically into two different mammary epithelial populations using K14 (Keratin 14)-cre and WAP (Whey Acidic Protein)-cre transgenic mice that express Cre recombinase in mammary progenitor cells and lactating luminal cells, respectively. In this study, we show that a specific type of Apc somatic mutations in mammary progenitor/stem cell population in mice induces mammary carcinomas with histological and molecular heterogeneity, but a complete deletion leads to squamous metaplasia. Our data show that mutations in a multilineage progenitor cell are important in certain mammary tumors. We also show that certain Apc mutations are selected to achieve a specific level of β-catenin signaling optimal for mammary tumor development and explain partially the reason why breast cancers do not develop as frequently as colorectal tumors in patients that carry germline mutations in APC.
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.
In human somatic tumorigenesis, mutations are thought to arise sporadically in individual cells surrounded by unaffected cells. This contrasts with most current transgenic models where mutations are induced synchronously in entire cell populations. Here we have modeled sporadic oncogene activation using a transgenic mouse in which c-MYC is focally activated in prostate luminal epithelial cells. Focal c-MYC expression resulted in mild pathology, but prostate-specific deletion of a single allele of the Pten tumor suppressor gene cooperated with c-MYC to induce high grade prostatic intraepithelial neoplasia (HGPIN)/cancer lesions. These lesions were in all cases associated with loss of Pten protein expression from the wild type allele. In the prostates of mice with concurrent homozygous deletion of Pten and focal c-MYC activation, double mutant (i.e. c-MYC+;Pten-null) cells were of higher grade and proliferated faster than single mutant (Pten-null) cells within the same glands. Consequently, double mutant cells outcompeted single mutant cells despite the presence of increased rates of apoptosis in the former. The p53 pathway was activated in Pten-deficient prostate cells and tissues, but c-MYC expression shifted the p53 response from senescence to apoptosis by repressing the p53 target gene p21Cip1. We conclude that c-MYC overexpression and Pten deficiency cooperate to promote prostate tumorigenesis, but a p53-dependent apoptotic response may present a barrier to further progression. Our results highlight the utility of inducing mutations focally to model the competitive interactions between cell populations with distinct genetic alterations during tumorigenesis.
In most human cancers, mutations are thought to arise in a single cell or few cells surrounded by their unaffected neighbors. Expansion of mutant cells can then allow the accumulation of additional mutations. The cell–cell interactions that may occur between mutant and unaffected cells or between cells with distinct mutations during tumorigenesis have not been well studied due to the lack of suitable in vivo models. To help fill this gap, we generated and characterized transgenic mice in which the oncogene c-MYC is activated focally in prostate epithelial cells. We have also analyzed mice in which prostate epithelial cells with two mutations (c-MYC overexpression and loss of Pten tumor suppressor) are found next to cells with a single mutation (loss of Pten). Although loss of Pten in the prostate is tumorigenic, it also activates a cellular senescence response which restrains further tumor progression. We found that concurrent c-MYC expression suppressed the senescence response in Pten-null cells in favor of apoptosis. c-MYC+;Pten-null cells proliferated faster than Pten-null cells in the same glands, with the net result that c-MYC+;Pten-null cells outcompete Pten-null cells. Our results demonstrate the utility of accurate models to mimic the heterogeneous and incremental nature of human prostate carcinogenesis.
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
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.
Retinoic acid signaling pathways are disabled in human breast cancer suggesting a controlling role in normal mammary growth that might be lost in tumorigenesis. We tested a single receptor isotype, RARα1 (retinoic acid receptor isotype alpha, isoform 1), for its role in mouse mammary gland morphogenesis and mouse mammary tumor virus (MMTV)-wingless-related MMTV integration site 1 (wnt1)-induced oncogenesis.
The role of RARα1 in mammary morphogenesis was tested in RARα1-knockout (KO) mice and in mammary tumorigenesis in bi-genic (RARα1/KO crossed with MMTV-wnt1) mice. We used whole mounts analysis, stem cells/progenitor quantification, mammary gland repopulation, quantitative polymerase chain reaction (Q-PCR), test of tumor-free survival, tumor fragments and cell transplantation.
In two genetic backgrounds (129/Bl-6 and FVB) the neo-natal RARα1/KO-mammary epithelial tree was two-fold larger and the pubertal tree had two-fold more branch points and five-fold more mature end buds, a phenotype that was predominantly epithelial cell autonomous. The stem/progenitor compartment of the RARα1/KO mammary, defined as CD24low/ALDHhigh activity was increased by a median 1.7-fold, but the mammary stem cell (MaSC)-containing compartment, (CD24low/CD29high), was larger (approximately 1.5-fold) in the wild type (wt)-glands, and the mammary repopulating ability of the wt-gland epithelium was approximately two-fold greater. In MMTV-wnt1 transgenic glands the progenitor (CD24low/ALDHhigh activity) content was 2.6-fold greater than in the wt and was further increased in the RARα1/KO-wnt1 glands. The tumor-free survival of RARα1/KO-wnt1 mice was significantly (P = 0.0002, Kaplan Meier) longer, the in vivo growth of RARα1/KO-wnt1 transplanted tumor fragments was significantly (P = 0.01) slower and RARα1/KO-wnt1 tumors cell suspension produced tumors after much longer latency.
In vitamin A-replete mice, RARα1 is required to maintain normal mammary morphogenesis, but paradoxically, also efficient tumorigenesis. While its loss increases the density of the mammary epithelial tree and the content of luminal mammary progenitors, it appears to reduce the size of the MaSC-containing compartment, the mammary repopulating activity, and to delay significantly the MMTV-wnt1-mammary tumorigenesis. Whether the delay in tumorigenesis is solely due to a reduction in wnt1 target cells or due to additional mechanisms remains to be determined. These results reveal the intricate nature of the retinoid signaling pathways in mammary development and carcinogenesis and suggest that a better understanding will be needed before retinoids can join the armament of effective anti-breast cancer therapies.
The phosphatidylinositol 3-kinase (PI3K) signaling pathway modulates growth, proliferation and cell survival in diverse tissue types and plays specialized roles in the nervous system including influences on neuronal polarity, dendritic branching and synaptic plasticity. The tumor-suppressor phosphatase with tensin homology (PTEN) is the central negative regulator of the PI3K pathway. Germline PTEN mutations result in cancer predisposition, macrocephaly and benign hamartomas in many tissues, including Lhermitte-Duclos disease, a cerebellar growth disorder. Neurological abnormalities including autism, seizures and ataxia have been observed in association with inherited PTEN mutation with variable penetrance. It remains unclear how loss of PTEN activity contributes to neurological dysfunction. To explore the effects of Pten deficiency on neuronal structure and function, we analyzed several ultra-structural features of Pten-deficient neurons in Pten conditional knockout mice. Using Golgi stain to visualize full neuronal morphology, we observed that increased size of nuclei and somata in Pten-deficient neurons was accompanied by enlarged caliber of neuronal projections and increased dendritic spine density. Electron microscopic evaluation revealed enlarged abnormal synaptic structures in the cerebral cortex and cerebellum. Severe myelination defects included thickening and unraveling of the myelin sheath surrounding hypertrophic axons in the corpus callosum. Defects in myelination of axons of normal caliber were observed in the cerebellum, suggesting intrinsic abnormalities in Pten-deficient oligodendrocytes. We did not observe these abnormalities in wild-type or conditional Pten heterozygous mice. Moreover, conditional deletion of Pten drastically weakened synaptic transmission and synaptic plasticity at excitatory synapses between CA3 and CA1 pyramidal neurons in the hippocampus. These data suggest that Pten is involved in mechanisms that control development of neuronal and synaptic structures and subsequently synaptic function.
PTEN; PI3K; brain; neurons; myelin; hypertrophy; synaptic plasticity
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
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
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.
Adiponectin is an adipokine possessing beneficial effects on obesity-related medical complications. A negative association of adiponectin levels with breast cancer development has been demonstrated. However, the precise role of adiponectin deficiency in mammary carcinogenesis remains elusive.
In the present study, MMTV-polyomavirus middle T antigen (MMTV-PyVT) transgenic mice with reduced adiponectin expressions were established and the stromal effects of adiponectin haploinsufficiency on mammary tumor development evaluated. In mice from both FVB/N and C57BL/6J backgrounds, insufficient adiponectin production promoted mammary tumor onset and development. A distinctive basal-like subtype of tumors, with a more aggressive phenotype, was derived from adiponectin haplodeficient MMTV-PyVT mice. Comparing with those from control MMTV-PyVT mice, the isolated mammary tumor cells showed enhanced tumor progression in re-implanted nude mice, accelerated proliferation in primary cultures, and hyperactivated phosphatidylinositol-3-kinase (PI3K)/Akt/beta-catenin signaling, which at least partly attributed to the decreased phosphatase and tensin homolog (PTEN) activities. Further analysis revealed that PTEN was inactivated by a redox-regulated mechanism. Increased association of PTEN-thioredoxin complexes was detected in tumors derived from mice with reduced adiponectin levels. The activities of thioredoxin (Trx1) and thioredoxin reductase (TrxR1) were significantly elevated, whereas treatment with either curcumin, an irreversible inhibitor of TrxR1, or adiponectin largely attenuated their activities and resulted in the re-activation of PTEN in these tumor cells. Moreover, adiponectin could inhibit TrxR1 promoter-mediated transcription and restore the mRNA expressions of TrxR1.
Adiponectin haploinsufficiency facilitated mammary tumorigenesis by down-regulation of PTEN activity and activation of PI3K/Akt signalling pathway through a mechanism involving Trx1/TrxR1 redox regulations.
PTEN is the most frequently mutated gene in uterine endometriod carcinoma (UEC) and its precursor complex atypical hyperplasia (CAH). Because the mutation frequency is similar in CAH and UEC, PTEN mutations are thought to occur relatively early in endometrial tumorigenesis. Previous work from our laboratory using the Pten+/− mouse model has demonstrated somatic inactivaton of the wild type allele of Pten in both CAH and UEC. In the present study, we injected adenoviruses expressing Cre into the uterine lumen of adult Pten floxed mice in an attempt to somatically delete both alleles of Pten specifically in the endometrium. Our results demonstrate that biallelic inactivation of Pten results in an increased incidence of carcinoma as compared to the Pten+/− mouse model. In addition, the carcinomas were more aggressive with extension beyond the uterus into adjacent tissues and were associated with decreased expression of nuclear ERα as compared to associated CAH. Primary cultures of epithelial and stromal cells were prepared from uteri of Pten floxed mice and Pten was deleted in vitro using Cre expressing adenovirus. Pten deletion was evident in both the epithelial and stromal cells and the treatment of the primary cultures with estrogen had different effects on Akt activation as well as Cyclin D3 expression in the two purified components. This study demonstrates that somatic biallelic inactivation of Pten in endometrial epithelium in vivo results in an increased incidence and aggressiveness of endometrial carcinoma compared to mice carrying a germline deletion of one allele and provides an important in vivo and in vitro model system for understanding the genetic underpinnings of endometrial carcinoma.
Pten; endometrial carcinoma; biallelic inactivation
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.
The PTEN/Akt/β-catenin pathway is important for maintaining stem or progenitor cells in normal and cancerous breast tissue and may be a promising target for effective, long-lasting cancer treatment.
Recent evidence suggests that many malignancies, including breast cancer, are driven by a cellular subcomponent that displays stem cell-like properties. The protein phosphatase and tensin homolog (PTEN) is inactivated in a wide range of human cancers, an alteration that is associated with a poor prognosis. Because PTEN has been reported to play a role in the maintenance of embryonic and tissue-specific stem cells, we investigated the role of the PTEN/Akt pathway in the regulation of normal and malignant mammary stem/progenitor cell populations. We demonstrate that activation of this pathway, via PTEN knockdown, enriches for normal and malignant human mammary stem/progenitor cells in vitro and in vivo. Knockdown of PTEN in normal human mammary epithelial cells enriches for the stem/progenitor cell compartment, generating atypical hyperplastic lesions in humanized NOD/SCID mice. Akt-driven stem/progenitor cell enrichment is mediated by activation of the Wnt/β-catenin pathway through the phosphorylation of GSK3-β. In contrast to chemotherapy, the Akt inhibitor perifosine is able to target the tumorigenic cell population in breast tumor xenografts. These studies demonstrate an important role for the PTEN/PI3-K/Akt/β-catenin pathway in the regulation of normal and malignant stem/progenitor cell populations and suggest that agents that inhibit this pathway are able to effectively target tumorigenic breast cancer cells.
Healthy adult tissues are maintained through the regulated proliferation of a subset of cells known as tissue stem and progenitor cells. Many cancers, including breast cancer, also are thought to arise from and be maintained by a small population of cells that display stem cell-like properties. These so-called “cancer stem cells” may also contribute to tumor spread (metastasis), resistance to treatment, and disease relapse. Effective, long-lasting cancer treatments likely will need to target and eliminate these cancer stem cells specifically. Regulatory pathways responsible for maintaining cancer stem cells therefore may be promising targets for treatment. Breast cancers in humans frequently display abnormalities in the PTEN/PI3K/Akt pathway. We demonstrate using cell culture and a mouse model of breast cancer that stem or progenitor cells in both normal breast tissue and breast tumors are dependent for their continued growth on this pathway and on the Wnt/β-catenin pathway. We further show that the drug perifosine, which inhibits the kinase Akt, is able specifically to reduce the population of breast cancer stem or progenitor cells growing in mice. Our findings support the idea that drugs that selectively target breast cancer stem cells through the PTEN/PI3K/Akt pathway may reduce tumor growth and metastasis and result in improved patient outcomes.
An early age at Breast Cancer (BC) onset may be a hallmark of inherited predisposition, but BRCA1/2 mutations are only found in a minority of younger BC patients. Among the others, a fraction may carry mutations in rarer BC genes, such as TP53, STK11, CDH1 and PTEN. As the identification of women harboring such mutations allows for targeted risk-management, the knowledge of associated manifestations and an accurate clinical and family history evaluation are warranted.
We describe the case of a woman who developed an infiltrating ductal carcinoma of the right breast at the age of 32, a contralateral BC at age 36 and another BC of the right breast at 40. When she was 39 years-old, during a dermatological examination, mucocutaneous features suggestive of Cowden Syndrome, a disorder associated to germ-line PTEN mutations, were noticed. PTEN genetic testing revealed the novel c.71A > T (p.Asp24Val) mutation, whose deleterious effect, suggested by conservation data and in silico tools, was definitely demonstrated by the incapacity of mutant PTEN to inhibit Akt phosphorylation when used to complement PTEN-null cells. In BC tissue, despite the absence of LOH or somatic mutations of PTEN, Akt phosphorylation was markedly increased in comparison to normal tissue, thus implying additional somatic events into the deregulation of the PI3K/Akt/mTOR pathway and, presumably, into carcinogenesis. Hence, known oncogenic mutations in PIK3CA (exons 10 and 21) and AKT1 (exon 2) were screened in tumor DNA with negative results, which suggests that the responsible somatic event(s) is a different, uncommon one.
This case stresses the importance of clinical/genetic assessment of early-onset BC patients in order to identify mutation carriers, who are at high risk of new events, so requiring tailored management. Moreover, it revealed a novel PTEN mutation with pathogenic effect, pointing out, however, the need for further efforts to elucidate the molecular steps of PTEN-associated carcinogenesis.
Hereditary breast cancer; PTEN; Cowden syndrome; PI3K/Akt/mTOR pathway
Breast cancer is genetically and clinically a heterogeneous disease. However, the exact contribution of different cell types and oncogenic mutations to this heterogeneity are not well understood. Recently, we discovered an interaction between Wnt and integrin-linked kinase (ILK) within the signaling cascade that regulates cell growth and survival. Interestingly, mammary-specific expression of either one of these proteins has been shown to promote mammary tumorigenesis. In light of our recent findings and to investigate the potential interaction between Wnt and ILK proteins during mammary tumor formation and progression, we established a transgenic mouse model that expresses both Wnt and ILK in mammary epithelial cells.
A novel transgenic mouse model with mammary-specific expression of both Wnt1 and ILK was generated by crossing the two previously characterized mouse models, MMTV-Wnt1 and MMTV-ILK. The resulting MMTV-Wnt/ILK mice were closely monitored for tumor development and growth, as well as for the tumor onset. The molecular phenotypes of both tumors and premalignant mammary glands were investigated by using biochemical and global gene-expression analysis approaches.
A significant acceleration in mammary tumor incidence and growth was observed in the MMTV-Wnt/ILK mice. Pre-neoplastic mammary glands also display lobuloalveolar hyperplasia and an increase in ductal epithelium proliferation. Apart from elevated expression of Wnt/ILK targets, such as β-catenin and cyclin D1, gene-expression profiling identified the surprising activation of the FOXA1 transcription factor. Upregulation of FOXA1, which is also known as the molecular marker of differentiated mammary luminal cells, was consistent with the expansion of the enriched luminal progenitor population or CD29loCD24hiCD61+ cells in MMTV-Wnt/ILK tumors.
These results show cooperation between Wnt1 and ILK transgenes during mammary carcinogenesis, leading to changes in a transcriptional network, which could dictate a specific breast cancer phenotype with enhanced growth dynamics. The MMTV-Wnt/ILK can be used as a model to identify further the genes downstream of the estrogen receptor-β/FOXA1 and to investigate the mechanisms targeting the expansion of the luminal progenitor cells leading to hyperplasia and tumorigenesis.
Inflammation and genetic instability are enabling characteristics of prostate carcinoma (PCa). Inactivation of the tumour suppressor gene phosphatase and tensin homolog (PTEN) is prevalent in early PCa. The relationship of PTEN deficiency to inflammatory signalling remains to be characterised.
To determine how loss of PTEN functionality modulates expression and efficacy of clinically relevant, proinflammatory chemokines in PCa.
Design, setting, and participants:
Experiments were performed in established cell-based PCa models, supported by pathologic analysis of chemokine expression in prostate tissue harvested from PTEN heterozygous (Pten+/−) mice harbouring inactivation of one PTEN allele.
Small interfering RNA (siRNA)–or small hairpin RNA (shRNA)–directed strategies were used to repress PTEN expression and resultant interleukin-8 (CXCL8) signalling, determined under normal and hypoxic culture conditions.
Outcome measurements and statistical analysis:
Changes in chemokine expression in PCa cells and tissue were analysed by real-time polymerase chain reaction (PCR), immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry; effects of chemokine signalling on cell function were assessed by cell cycle analysis, apoptosis, and survival assays.
Results and limitations:
Transient (siRNA) or prolonged (shRNA) PTEN repression increased expression of CXCL8 and its receptors, chemokine (C-X-C motif) receptor (CXCR) 1 and CXCR2, in PCa cells. Hypoxia-induced increases in CXCL8, CXCR1, and CXCR2 expression were greater in magnitude and duration in PTEN-depleted cells. Autocrine CXCL8 signalling was more efficacious in PTEN-depleted cells, inducing hypoxia-inducible factor-1 (HIF-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription and regulating genes involved in survival and angiogenesis. Increased expression of the orthologous chemokine KC was observed in regions displaying atypical cytologic features in Pten+/− murine prostate tissue relative to normal epithelium in wild-type PTEN (PtenWT) glands. Attenuation of CXCL8 signalling decreased viability of PCa cells harbouring partial or complete PTEN loss through promotion of G1 cell cycle arrest and apoptosis. The current absence of clinical validation is a limitation of the study.
PTEN loss induces a selective upregulation of CXCL8 signalling that sustains the growth and survival of PTEN-deficient prostate epithelium.
Inflammation; Apoptosis; CXCL8; CXCR2; Hypoxia; Prostate cancer; PTEN
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.
Insulin controls glucose metabolism via multiple signalling pathways, including the phosphatidylinositol 3-kinase (PI3K) pathway in muscle and adipose tissue. The protein/lipid phosphatase Pten (phosphatase and tensin homologue deleted on chromosome 10) attenuates PI3K signalling by dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate generated by PI3K. The current study was aimed at investigating the effect of haploinsufficiency for Pten on insulin-stimulated glucose uptake.
Materials and methods
Insulin sensitivity in Pten heterozygous (Pten+/−) mice was investigated in i.p. insulin challenge and glucose tolerance tests. Glucose uptake was monitored in vitro in primary cultures of myocytes from Pten+/− mice, and in vivo by positron emission tomography. The phosphorylation status of protein kinase B (PKB/Akt), a downstream signalling protein in the PI3K pathway, and glycogen synthase kinase 3β (GSK3β), a substrate of PKB/Akt, was determined by western immunoblotting.
Following i.p. insulin challenge, blood glucose levels in Pten+/− mice remained depressed for up to 120 min, whereas glucose levels in wild-type mice began to recover after approximately 30 min. After glucose challenge, blood glucose returned to normal about twice as rapidly in Pten+/− mice. Enhanced glucose uptake was observed both in Pten+/− myocytes and in skeletal muscle of Pten+/− mice by PET. PKB and GSK3β phosphorylation was enhanced and prolonged in Pten+/− myocytes.
Pten is a key negative regulator of insulin-stimulated glucose uptake in vitro and in vivo. The partial reduction of Pten due to Pten haploinsufficiency is enough to elicit enhanced insulin sensitivity and glucose tolerance in Pten+/− mice.
Glucose uptake; Insulin hypersensitivity; Insulin sensitivity; Pten haploinsufficiency
Vulval development in Caenorhabditis elegans serves as an excellent model to examine the crosstalk between different conserved signaling pathways that are deregulated in human cancer. The concerted action of the RAS/MAPK, NOTCH, and WNT pathways determines an invariant pattern of cell fates in three vulval precursor cells. We have discovered a novel form of crosstalk between components of the Insulin and the RAS/MAPK pathways. The insulin receptor DAF-2 stimulates, while DAF-18 PTEN inhibits, RAS/MAPK signaling in the vulval precursor cells. Surprisingly, the inhibitory activity of DAF-18 PTEN on the RAS/MAPK pathway is partially independent of its PIP3 lipid phosphatase activity and does not involve further downstream components of the insulin pathway, such as AKT and DAF-16 FOXO. Genetic and biochemical analyses indicate that DAF-18 negatively regulates vulval induction by inhibiting MAPK activation. Thus, mutations in the PTEN tumor suppressor gene may result in the simultaneous hyper-activation of two oncogenic signaling pathways.
The human tumor suppressor PTEN is mutated in many different types of cancer. Using the roundworm C. elegans as a model to study how cells communicate during animal development, we discovered a new mechanism by which PTEN inhibits the activity of the oncogenic RAS/MAPK signaling pathway. Focusing on the development of the vulva, the egg-laying organ of the hermaphrodite, as a model to investigate the regulation of RAS/MAPK signaling, we could distinguish between two distinct inhibitory activities of PTEN on the RAS/MAPK signaling pathway. On the one hand, PTEN acts as a lipid phosphatase that inhibits the production of PIP3, which in turn stimulates RAS/MAPK signaling. On the other hand, PTEN acts as a protein phosphatase that negatively regulates RAS/MAPK signaling by inhibiting signal transduction at the level of the MAPK, which is a key component in the pathway. Understanding the detailed molecular mechanism by which the PTEN tumor suppressor homolog regulates signal transduction in C. elegans can help predict the consequences of mutations in human PTEN for cancer development in humans.
PPARγ is a nuclear receptor that regulates gene transcription associated with intermediary metabolism, adipocyte differentiation, as well as tumor suppression and proliferation. To understand the role of PPARγ in tumorigenesis, transgenic mice were generated with mammary gland-directed expression of the dominant-negative transgene, Pax8PPARγ. Transgenic mice were phenotypically indistinguishable from wild-type mice, but mammary epithelial cells expressed a greater a high percentage of CD29hi/CD24neg, CK5+ and double positive CK14/CK18 cells. These changes correlated with reduced PTEN and increased Ras, ERK and AKT activation. Although spontaneous tumorigenesis did not occur, transgenic animals were highly susceptible to progestin/DMBA-induced mammary carcinogenesis, which in contrast to wild-type mice, resulted in a high tumor multiplicity and most importantly, in the appearance of predominantly estrogen receptorα-positive (ER+) ductal adenocarcinomas. Tumors expressed a similar PTENlo/pERKhi/pAKThi phenotype as mammary epithelium, and exhibited high activation of ERE-dependent reporter gene activity. Tumorigenesis in MMTV-Pax8PPARγ mice was insensitive to the chemopreventive effect of a PPARγ agonist, but was profoundly inhibited by the ER antagonist fulvestrant. These results reveal important new insights into the previously unrecognized role of PPARγ in the specification of mammary lineage and the development of ER+ tumors.
PPARγ; ERK; ER; mammary carcinogenesis; stem cells
BACKGROUND: Melanoma is an aggressive tumor with a propensity to rapidly metastasize. The PTEN gene encodes a phosphatase with an unusual dual specificity for proteins and lipids. Mutations of PTEN have been found in various human cancers, including glioblastoma, prostate, breast, lung, and melanoma. Here we investigate in vitro the effects of blocking PI3K signaling using adenoviral-delivered PTEN (Ad-PTEN) in cell lines derived from both early- and late-stage melanoma. MATERIALS AND METHODS: Ad-PTEN transduced melanoma cell lines or normal cells were assayed for cell death, apoptosis, gene expression, invasion and migration, and regulation of angiogenesis. RESULTS: The PTEN locus from RGP and metastatic melanoma cell lines was sequenced; no coding region mutations were found. Adenoviral transfer of PTEN into melanoma cells containing wild-type PTEN alleles led to tumor-specific apoptosis and growth inhibition, with coordinate inhibition of AKT phosphorylation. Ad-PTEN suppressed cell migration by metastatic melanoma cells with concomitant increase in the level of cell surface E-cadherin. Immunohistochemical and confocal analyses localized PTEN to the cytoplasm and demonstrated enrichment at the cell membrane. Ad-PTEN inhibited angiogenesis as demonstrated by the tube formation assay using human vascular endothelial cells. CONCLUSIONS: These studies indicate that Ad-PTEN can inhibit tumor cells via multiple mechanisms and has pro-apoptotic, anti-metastatic, and anti-angiogenic properties. Thus, PI3K blockade via Ad-PTEN may be a promising approach for the treatment of early- and late-stage melanoma, even in tumors that do not harbor PTEN mutations.