Aberrant activation and mutation status of proteins in the phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and the mitogen activated protein kinase (MAPK) signaling pathways have been linked to tumorigenesis in various tumors including urothelial carcinoma (UC). However, anti-tumor therapy with small molecule inhibitors against mTOR turned out to be less successful than expected. We characterized the molecular mechanism of this pathway in urothelial carcinoma by interfering with different molecular components using small chemical inhibitors and siRNA technology and analyzed effects on the molecular activation status, cell growth, proliferation and apoptosis. In a majority of tested cell lines constitutive activation of the PI3K was observed. Manipulation of mTOR or Akt expression or activity only regulated phosphorylation of S6K1 but not 4E-BP1. Instead, we provide evidence for an alternative mTOR independent but PI3K dependent regulation of 4E-BP1. Only the simultaneous inhibition of both S6K1 and 4E-BP1 suppressed cell growth efficiently. Crosstalk between PI3K and the MAPK signaling pathway is mediated via PI3K and indirect by S6K1 activity. Inhibition of MEK1/2 results in activation of Akt but not mTOR/S6K1 or 4E-BP1. Our data suggest that 4E-BP1 is a potential new target molecule and stratification marker for anti cancer therapy in UC and support the consideration of a multi-targeting approach against PI3K, mTORC1/2 and MAPK.
The current standard of care for metastatic urothelial carcinoma is cisplatin-based chemotherapy but treatment is generally not curative. Mechanisms of resistance to conventional cytotoxic regimens include tumor cell drug efflux pumps, intracellular anti-oxidants, and enhanced anti-apoptotic signaling. Blockade of signaling pathways with small molecule tyrosine kinase inhibitors has produced dramatic responses in subsets of other cancers. Multiple potential signaling pathway targets are altered in Urothelial carcinoma (UC). Blockade of the PI3K/Akt/mTOR pathway may prove efficacious because 21% have activating PI3K mutations and another 30% have PTEN inactivation (which leads to activation of this pathway). The fibroblast growth factor receptor 3 protein may be overactive in 50–60% and agents which block this pathway are under development. Blockade of multiple other pathways including HER2 and aurora kinase also have potential efficacy. Anti-angiogenic and immunotherapy strategies are also under development in UC and are discussed in this review. Novel therapeutic approaches are needed in UC. We review the various strategies under investigation and discuss how best to evaluate and optimize their efficacy.
urothelial cancer; bladder cancer; oncogenes; chemotherapy; resistance mechanisms
PTEN, a tumor suppressor whose function is frequently lost in human cancers, possesses a lipid phosphatase activity that represses phosphatidylinositol 3-kinase (PI3K) signaling, controlling cell growth, proliferation, and survival. The potential for PTEN to regulate the synthesis of RNA polymerase (Pol) III transcription products, including tRNAs and 5S rRNAs, was evaluated. The expression of PTEN in PTEN-deficient cells repressed RNA Pol III transcription, whereas decreased PTEN expression enhanced transcription. Transcription repression by PTEN was uncoupled from PTEN-mediated effects on the cell cycle and was independent of p53. PTEN acts through its lipid phosphatase activity, inhibiting the PI3K/Akt/mTOR/S6K pathway to decrease transcription. PTEN, through the inactivation of mTOR, targets the TFIIIB complex, disrupting the association between TATA-binding protein and Brf1. Kinetic analysis revealed that PTEN initially induces a decrease in the serine phosphorylation of Brf1, leading to a selective reduction in the occupancy of all TFIIIB subunits on tRNALeu genes, whereas prolonged PTEN expression results in the enhanced serine phosphorylation of Bdp1. Together, these results demonstrate a new class of genes regulated by PTEN through its ability to repress the activation of PI3K/Akt/mTOR/S6K signaling.
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.
Leptin activates multiple signaling pathways in cells, including the
phosphatidylinositol 3-kinase pathway, indicating a degree of cross-talk with
insulin signaling. The exact mechanisms by which leptin alters this signaling
pathway and how it relates to functional outputs are unclear at present. A
previous study has established that leptin inhibits the activity of the
phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10), an
important tumor suppressor and modifier of phosphoinositide signaling. In this
study we demonstrate that leptin phosphorylates multiple sites on the
C-terminal tail of PTEN in hypothalamic and pancreatic β-cells, an action
not replicated by insulin. Inhibitors of the protein kinases CK2 and glycogen
synthase kinase 3 (GSK3) block leptin-mediated PTEN phosphorylation. PTEN
phosphorylation mutants reveal the critical role these sites play in
transmission of the leptin signal to F-actin depolymerization. CK2 and GSK3
inhibitors also prevent leptin-mediated F-actin depolymerization and
consequent ATP-sensitive K+ channel opening. GSK3 kinase activity
is inhibited by insulin but not leptin in hypothalamic cells. Both hormones
increase N-terminal GSK3 serine phosphorylation, but in hypothalamic cells
this action of leptin is transient. Leptin, not insulin, increases GSK3
tyrosine phosphorylation in both cell types. These results demonstrate a
significant role for PTEN in leptin signal transmission and identify GSK3 as a
potential important signaling node contributing to divergent outputs for these
The tumor suppressor gene PTEN (phosphatase and tensin homologue deleted on chromosome 10) is frequently mutated or deleted in various human cancers. PTEN localizes predominantly to the cytoplasm and functions as a lipid phosphatase, thereby negatively regulating the phosphatidylinositol 3-kinase-AKT signaling pathway. PTEN can also localize to the nucleus, where it binds and regulates p53 protein level and transcription activity. However, the precise function of nuclear PTEN and the factors that control PTEN nuclear localization are still largely unknown. In this study, we identified oxidative stress as one of the physiological stimuli that regulate the accumulation of nuclear PTEN. Specifically, oxidative stress inhibits PTEN nuclear export, a process depending on phosphorylation of its amino acid residue Ser-380. Nuclear PTEN, independent of its phosphatase activity, leads to p53-mediated G1 growth arrest, cell death, and reduction of reactive oxygen species production. Using xenografts propagated from human prostate cancer cell lines, we reveal that nuclear PTEN is sufficient to reduce tumor progression in vivo in a p53-dependent manner. The data outlined in this study suggest a unique role of nuclear PTEN to arrest and protect cells upon oxidative damage and to regulate tumorigenesis. Since tumor cells are constantly exposed to oxidative stress, our study elucidates the cooperative roles of nuclear PTEN with p53 in tumor suppression.
The tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10) and the androgen receptor (AR) play important roles in tumor development and progression in prostate carcinogenesis. Among many functions, PTEN negatively regulates the cytoplasmic phosphatidylinositol-3-kinase/AKT anti-apoptotic pathway; and nuclear PTEN affects the cell cycle by also negatively regulating the MAPK pathway via cyclin D. Decreased PTEN expression is correlated with prostate cancer progression. Over-expression of AR and upregulation of AR transcriptional activity are often observed in the later stages of prostate cancer. Recent studies indicate that PTEN regulates AR activity and stability. However, the mechanism of how AR regulates PTEN has never been studied. Furthermore, resveratrol, a phytoalexin enriched in red grapes, strawberries and peanuts, has been shown to inhibit AR transcriptional activity in prostate cancer cells. In this study, we use prostate cancer cell lines to test the hypothesis that resveratrol inhibits cellular proliferation in both AR-dependent and -independent mechanisms. We show that resveratrol inhibits AR transcriptional activity in both androgen-dependent and -independent prostate cancer cells. Additionally, resveratrol stimulates PTEN expression through AR inhibition. In contrast, resveratrol directly binds epidermal growth factor receptor (EGFR) rapidly inhibiting EGFR phosphorylation, resulting in decreased AKT phosphorylation, in an AR-independent manner. Thus, resveratrol may act as potential adjunctive treatment for late-stage hormone refractory prostate cancer. More importantly, for the first time, our study demonstrates the mechanism by which AR regulates PTEN expression at the transcription level, indicating the direct link between a nuclear receptor and the PI3K/AKT pathway.
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.
Tyrosine kinase inhibitors (TKIs) exhibit impressive activity against advanced renal cell carcinoma. However, recent clinical studies have demonstrated an equivocal response to sunitinib in patients with castration-resistant prostate cancer. The tumor suppressor phosphatase and tensin homolog (PTEN) acts as a gatekeeper of the PI3K/Akt/mTOR cell-survival pathway. Our experiments demonstrate that PTEN expression inversely correlates with sunitinib resistance in renal and prostate cancer cells. Restoration of PTEN expression markedly increases sensitivity of tumor cells to sunitinib both in vitro and in vivo. In addition, pharmacologic manipulation of PI3K/Akt/mTOR signaling with PI3K/mTOR inhibitor, GDC-0980, mTOR inhibitor, temsirolimus, or pan-Akt inhibitor, GSK690693, was able to overcome sunitinib resistance in cancer cells. Our findings underscore the importance of PTEN expression in relation to sunitinib resistance and imply a direct cytotoxic effect by sunitinib on tumor cells in addition to its anti-angiogenic actions.
PTEN; mTOR; sunitinib; temsirolimus; cancer
Loss of the PTEN tumor suppressor is a common occurrence in human prostate cancer, particularly in advanced disease. In keeping with its role as a pivotal upstream regulator of the phosphatidylinositol 3-kinase signaling pathway, experimentally-induced deletion of Pten in the murine prostate invariably results in neoplasia. However, and unlike humans where prostate tumorigenesis likely evolves over decades, disease progression in the constitutively Pten deficient mouse prostate is relatively rapid, culminating in invasive cancer within several weeks post-puberty. Given that the prostate undergoes rapid androgen-dependent growth at puberty, and that Pten excisions during this time might be especially tumorigenic, we hypothesized that delaying prostate-specific Pten deletions until immediately after puberty might alter the pace of tumorigenesis. To this end we generated mice with a tamoxifen-inducible Cre recombinase transgene enabling temporal control over prostate-specific gene alterations. This line was then interbred with mice carrying floxed Pten alleles. Despite evidence of increased Akt/mTOR/S6K axis activity at early time points in Pten-deficient epithelial cells, excisions induced in the post-pubertal (6 wk-old) prostate yielded gradual acquisition of a range of lesions. These progressed from pre-malignant changes (nuclear atypia, focal hyperplasia) and low grade prostatic intraepithelial neoplasia (PIN) at 16–20 wks post-tamoxifen exposure, to overtly malignant lesions by ∼1 yr of age, characterized by high-grade PIN and microinvasive carcinoma. In contrast, when Pten excisions were triggered in the pre-pubertal (2 week-old) prostate, neoplasia evolved over a more abbreviated time-frame, with a spectrum of premalignant lesions, as well as overt PIN and microinvasive carcinoma by 10–12 wks post-tamoxifen exposure. These results indicate that the developmental stage at which Pten deletions are induced dictates the pace of PIN development.
DJ-1 and HSP90α play a significant role in the progression of various types of cancer and are known to be associated with phosphatase and tensin homolog deleted on chromosome 10 (PTEN), PI3K-p110α and pAkt, the signaling molecule proteins from the phosphatidylinositol 3-kinase (PI3K) pathway. However, the expression of these proteins and their clinical significance are not well characterized in urothelial carcinoma (UC). Immunohistochemical analysis of DJ-1, HSP90α, PTEN, pAkt and PI3K-p110α expression was performed on tumor samples from 102 patients with UC to assess the relationship between the expression of each protein and the pathological parameters. The expression of DJ-1 and HSP90α was positively correlated with the pathological stage of UC, whereas PTEN expression negatively correlated with, not only the pathological stage, but also the growth pattern and histological grade of UC. Although PI3K-p110α expression was significantly correlated with DJ-1 as well as PTEN expression in UC, PI3K-p110α expression itself failed to reveal any significant correlation with the clinicopathological parameters. In conclusion, the overexpression of DJ-1 and HSP90α, and a loss of PTEN are associated with invasive UC, and PI3K-p110α expression is correlated with DJ-1 and PTEN expression in UC.
DJ-1; HSP90α; PTEN; PI3K-p110a; pAkt; urothelial carcinoma
The widespread distribution of the tumor suppressor PTEN in the nervous system suggests a role in a broad range of brain functions. PTEN negatively regulates the signaling pathways initiated by protein kinase B (Akt) thereby regulating signals for growth, proliferation and cell survival. Pten deletion in the mouse brain has revealed its role in controlling cell size and number. In this study, we used Cre-loxP technology to specifically inactivate Pten in dopamine (DA) neurons (Pten KO mice). The resulting mutant mice showed neuronal hypertrophy, and an increased number of dopaminergic neurons and fibers in the ventral mesencephalon. Interestingly, quantitative microdialysis studies in Pten KO mice revealed no alterations in basal DA extracellular levels or evoked DA release in the dorsal striatum, despite a significant increase in total DA tissue levels. Striatal dopamine receptor D1 (DRD1) and prodynorphin (PDyn) mRNA levels were significantly elevated in KO animals, suggesting an enhancement in neuronal activity associated with the striatonigral projection pathway, while dopamine receptor D2 (DRD2) and preproenkephalin (PPE) mRNA levels remained unchanged. In addition, PTEN inactivation protected DA neurons and significantly enhanced DA-dependent behavioral functions in KO mice after a progressive 6OHDA lesion. These results provide further evidence about the role of PTEN in the brain and suggest that manipulation of the PTEN/Akt signaling pathway during development may alter the basal state of dopaminergic neurotransmission and could provide a therapeutic strategy for the treatment of Parkinson's disease, and other neurodegenerative disorders.
To investigate whether mammalian target of rapamycin (mTOR) inhibition by rapamycin is therapeutically efficacious in combination with cisplatin for bladder cancer.
MATERIALS AND METHODS
Using a panel of human urothelial carcinoma cell lines, we determined the effect of rapamycin on cell viability, cell-cycle progression, signalling and apoptosis.
The effect of mTOR inhibition on chemosensitivity was investigated by treating cells with rapamycin, alone, or with cisplatin.
The effect of rapamycin or cisplatin treatment was assessed in xenograft mice inoculated with urothelial carcinoma cells.
Expression of p-mTOR in human bladder cancer specimens was assessed using a tissue microarray.
Treatment with rapamycin significantly decreased cell viability in UMUC3 (P=0.004) and 253J (P<0.001) cells. It induced arrest in the G0–G1 phase and decreased activation of p-mTOR and its downstream effector, p-S6K, in both cell lines.
Treatment with rapamycin increased the ability of cisplatin to inhibit cell viability in UMUC3 (P=0.002) and 253J (P=0.03) cells. No evidence for apoptosis induction was noted after treatment with rapamycin alone.
Mouse xenografts of UMUC3 cells revealed that rapamycin significantly prolonged survival and enhanced the therapeutic efficacy of cisplatin.
In patient urothelial carcinoma specimens, p-mTOR expression was increased in cancer vs. non-tumour bladder tissue in 65/203 (32.0%) tumours.
mTOR blockade inhibits urothelial carcinoma cell proliferation and enhances the effectiveness of cisplatin.
Suppression of the mTOR pathway has the potential to be a therapeutic target in bladder cancer for selected patients.
bladder cancer; mTOR; chemotherapy; rapamycin; radical cystectomy
In 1997, the PTEN gene (phosphatase and tensin homolog deleted on chromosome 10) was identified as a tumor suppressor gene on the long arm of chromosome 10. Since then, important progress has been made with respect to the understanding of the role of the Pten protein in the normal development of the brain as well as in the molecular pathogenesis of human gliomas. This review summarizes the current state of the art concerning the involvement of aberrant Pten function in the development of different biologic features of malignant gliomas, such as loss of cell-cycle control and uncontrolled cell proliferation, escape from apoptosis, brain invasion, and aberrant neoangiogenesis. Most of the tumor-suppressive properties of Pten are dependent on its lipid phosphatase activity, which inhibits the phosphatidylinositol-3'-kinase (PI3K)/Akt signaling pathway through dephosphorylation of phosphatidylinositol-(3,4,5)-triphosphate. The additional function of Pten as a dual-specificity protein phosphatase may also play a role in glioma pathogenesis. Besides the wealth of data elucidating the functional roles of Pten, recent studies suggest a diagnostic significance of PTEN gene alterations as a molecular marker for poor prognosis in anaplastic astrocytomas and anaplastic oligodendrogliomas. Furthermore, the possibility of selective targeting of PTEN mutant tumor cells by specific pharmacologic inhibitors of members of the Pten/PI3K/Akt pathway opens up new perspectives for a targeted molecular therapy of malignant gliomas.
In adipose tissue, insulin controls glucose and lipid metabolism through the intracellular mediators phosphatidylinositol 3-kinase and serine-threonine kinase AKT. Phosphatase and a tensin homolog deleted from chromosome 10 (PTEN), a negative regulator of the phosphatidylinositol 3-kinase/AKT pathway, is hypothesized to inhibit the metabolic effects of insulin. Here we report the generation of mice lacking PTEN in adipose tissue. Loss of Pten results in improved systemic glucose tolerance and insulin sensitivity, associated with decreased fasting insulin levels, increased recruitment of the glucose transporter isoform 4 to the cell surface in adipose tissue, and decreased serum resistin levels. Mutant animals also exhibit increased insulin signaling and AMP kinase activity in the liver. Pten mutant mice are resistant to developing streptozotocin-induced diabetes. Adipose-specific Pten deletion, however, does not alter adiposity or plasma fatty acids. Our results demonstrate that in vivo PTEN is a potent negative regulator of insulin signaling and insulin sensitivity in adipose tissue. Furthermore, PTEN may be a promising target for nutritional and/or pharmacological interventions aimed at reversing insulin resistance.
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
Inactivation and silencing of PTEN have been observed in multiple cancers, including follicular thyroid carcinoma. PTEN (phosphatase and tensin homologue deleted from chromosome 10) functions as a tumour suppressor by opposing the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signalling pathway. Despite correlative data, how deregulated PTEN signalling leads to thyroid carcinogenesis is not known. Mice harbouring a dominant-negative mutant thyroid hormone receptor β (TRβPV/PV mice) spontaneously develop follicular thyroid carcinoma and distant metastases similar to human cancer. To elucidate the role of PTEN in thyroid carcinogenesis, we generated TRβPV/PV mice haploinsufficient for Pten (TRβPV/PVPten+/− mouse). PTEN deficiency accelerated the progression of thyroid tumour and increased the occurrence of metastasis spread to the lung in TRβPV/PVPten+/− mice, thereby significantly reducing their survival as compared with TRβPV/PVPten+/+ mice. AKT activation was further increased by two-fold in TRβPV/PVPten+/− mice thyroids, leading to increased activity of the downstream mammalian target of rapamycin (mTOR)–p70S6K signalling and decreased activity of the forkhead family member FOXO3a. Consistently, cyclin D1 expression was increased. Apoptosis was decreased as indicated by increased expression of nuclear factor-κB (NF-κB) and decreased caspase-3 activity in the thyroids of TRβPV/PVPten+/− mice. Our results indicate that PTEN deficiency resulted in increased cell proliferation and survival in the thyroids of TRβPV/PVPten+/− mice. Altogether, our study provides direct evidence to indicate that in vivo, PTEN is a critical regulator in the follicular thyroid cancer progression and invasiveness.
thyroid cancer; Pten; carcinogenesis; mouse model; mutations
The phosphoinositide-3 kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway is a cellular pathway involved in cell growth, tumorigenesis and cell invasion which is frequently activated in various types of cancer. The downstream effector of the pathway is mTOR which is important in cellular growth and homeostasis and aberrant activation of mTOR has been reported in several types of cancer. The tumor suppressor gene phosphatase and tensin homolog (PTEN) is essential in this pathway for inhibiting tumor invasion and metastasis. However, the involvement of mTOR and PTEN in the progression of human gastric cancer remains to be identified. Immunohistochemical staining was performed to detect the expression of mTOR and PTEN in paraffin-embedded gastric tissue sections obtained from 33 patients with gastric cancer and 30 normal controls. The expressed mTOR was mainly distributed in the cytoplasm, while PTEN was mainly localized to the nucleus. By considering negative mTOR expression with positive PTEN expression as one group and negative PTEN expression with positive mTOR expression as the other, significant statistical differences were observed in various categories, including histological types and metastatic and clinical pathology stages, between the 2 groups (P<0.01 or 0.05). The results indicated that the expression levels of mTOR and PTEN were negatively correlated in the PI3K-AKT-mTOR signaling pathway. Combined detection of mTOR and PTEN expression may be used to evaluate the degree of malignancy in gastric cancer and may be a useful marker for the early diagnosis of gastric cancer.
gastric carcinoma; mTOR; PTEN; immunohistochemistry
Phosphatase and tensin homolog (PTEN), deleted on chromosome 10, is a potent tumor suppressor. PTEN expression is reduced in advanced bladder cancer and reduction correlates with disease stage. To gain insights into the function of PTEN in human bladder cancer by identifying its binding partners, we developed a novel IPTG inducible PTEN expression system and evaluated this system in the PTEN null UMUC-3 human bladder cancer xenograft model. In this model, induction of PTEN in vivo resulted in reduced tumor growth. We used mass spectrometry to identify PTEN interaction partners in these cells, which identified known interaction partners Major Vault Protein (MVP) and Paxillin as well as a novel interaction partner, TRK Fused Gene (TFG). In conclusion, using a biologically relevant model system to dissect PTEN tumor suppressor function in human bladder cancer, we identified three molecules important for many cellular functions in complex with PTEN.
PTEN; Major Vault Protein; AKT; EGFR
Phosphatidylinositol 3-kinase (PI3K) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling pathway play an important role in multiple cellular functions such as cell metabolism, proliferation, cell-cycle progression, and survival. PI3K is activated by growth factors and angiogenesis inducers such as vascular endothelial growth factor (VEGF) and angiopoietins. The amplification and mutations of PI3K and the loss of the tumor suppressor PTEN are common in various kinds of human solid tumors. The genetic alterations of upstream and downstream of PI3K signaling molecules such as receptor tyrosine kinases and AKT, respectively, are also frequently altered in human cancer. PI3K signaling regulates tumor growth and angiogenesis by activating AKT and other targets, and by inducing HIF-1 and VEGF expression. Angiogenesis is required for tumor growth and metastasis. In this review, we highlight the recent studies on the roles and mechanisms of PI3K and PTEN in regulating tumorigenesis and angiogenesis, and the roles of the downstream targets of PI3K for transmitting the signals. We also discuss the crosstalk of these signaling molecules and cellular events during tumor growth, metastasis, and tumor angiogenesis. Finally, we summarize the potential applications of PI3K, AKT, and mTOR inhibitors and their outcome in clinical trials for cancer treatment.
Xp11.2 translocation renal cell carcinomas (TRCCs) are a rare family of tumors newly recognized by the World Health Organization (WHO) in 2004. These tumors result in the fusion of partner genes to the TFE3 gene located on Xp11.2. They are most common in the pediatric population, but have been recently implicated in adult renal cell carcinoma (RCC) presenting at an early age. TFE3-mediated direct transcriptional upregulation of the Met tyrosine kinase receptor triggers dramatic activation of downstream signaling pathways including the protein kinase B (Akt)/phosphatidylinositol-3 kinase (PI3K) and mammalian target of rapamycin (mTOR) pathways. Temsirolimus is an inhibitor of mammalian target of rapamycin (mTOR) kinase, a component of intracellular signaling pathways involved in the growth and proliferation of malignant cells. Here we present a case of a 22-year old female who has been treated with temsirolimus for her Xp11.2/TFE3 gene fusion RCC.
renal cell carcinoma; Xp11.2 translocation; temsirolimus.
The phosphatase and tensin homologue on chromosome 10 (PTEN) suppresses the activity of the phosphoinositide-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, a signaling cascade critically involved in the regulation of cell proliferation and growth. Human patients carrying germline PTEN mutations have an increased predisposition to tumors, and also display a variety of neurological symptoms and increased risk of epilepsy and autism, implicating PTEN in neuronal development and function. Consistently, loss of Pten in mouse neural cells results in ataxia, seizures, cognitive abnormalities, increased soma size and synaptic abnormalities. To better understand how Pten regulates the excitability of principal forebrain neurons, a factor that is likely to be altered in cognitive disorders, epilepsy and autism, we generated a novel conditional knockout mouse line (NEX-Pten) in which Cre, under the control of the NEX promoter, drives the deletion of Pten specifically in early postmitotic, excitatory neurons of the developing forebrain. Homozygous mutant mice exhibited a massive enlargement of the forebrain, and died shortly after birth due to excessive mTOR activation. Analysis of the neonatal cerebral cortex further identified molecular defects resulting from Pten deletion that likely affect several aspects of neuronal development and excitability.
Pten; mTOR; glutamate; autism; seizure; NMDA receptor; epilepsy
Although deregulation of the Wnt signalling pathway has been implicated in urothelial cell carcinoma (UCC), the functional significance is unknown. To test its importance, we have targeted expression of an activated form of β-catenin to the urothelium of transgenic mice using Cre-Lox technology (UroIICRE+ β-cateninexon3/+). Expression of this activated form of β-catenin led to the formation of localised hyperproliferative lesions by 3 months, which did not progress to malignancy. These lesions were characterised by a marked increase of the PTEN tumour suppressor protein. This appears to be a direct consequence of activating Wnt signalling in the bladder as conditional deletion of the Apc (Adenomatous Polyposis coli) gene within the adult bladder led rapidly to coincident β-catenin and PTEN expression. This PTEN expression blocked proliferation. Next, we combined PTEN deficiency with β-catenin activation and found this caused papillary UCC. These tumours had increased pAKT signalling and were dependent on mTOR. Importantly in human UCC, there was a significant correlation between high levels of β-catenin and pAKT (and low levels of PTEN). Taken together these data definitively show that deregulated Wnt signalling plays a critical role in driving UCC, and suggests that human UCC which have high levels of Wnt and PI3 kinase signalling may be responsive to mTOR inhibition.
β-catenin; PTEN; Urothelial Cell Carcinoma; Bladder Cancer
Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene, and loss of function mutations are common and appear to be important in the pathogenesis of endometrial carcinomas. Loss of PTEN causes deregulated phosphatidylinositol-3 kinase/serine-threonine kinase/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling which may provide neoplastic cells with a selective survival advantage by enhancing angiogenesis, protein translation, and cell cycle progression. Temsirolimus, an ester derivative of rapamycin that inhibits mTOR, was evaluated in this setting.
Patients and Methods
Sequential phase II studies evaluated single-agent activity of temsirolimus in women with recurrent or metastatic chemotherapy-naive or chemotherapy-treated endometrial cancer. Temsirolimus 25 mg intravenously was administered weekly in 4-week cycles.
In the chemotherapy-naive group, 33 patients received a median of four cycles (range, one to 23 cycles). Of the 29 patients evaluable for response, four (14%) had an independently confirmed partial response and 20 (69%) had stable disease as best response, with a median duration of 5.1 months (range, 3.7 to 18.4 months) and 9.7 months (range, 2.1 to 14.6 months). Only five patients (18%) had progressive disease. In the chemotherapy-treated group, 27 patients received a median of three cycles (range, one to six cycles). Of the 25 patients evaluable for response, one (4%) had an independently confirmed partial response, and 12 patients (48%) had stable disease, with a median duration of 4.3 months (range, 3.6 to 4.9 months) and 3.7 months (range, 2.4 to 23.2 months). PTEN loss (immunohistochemistry and mutational analysis) and molecular markers of PI3K/Akt/mTOR pathway did not correlate with the clinical outcome.
mTOR inhibition with temsirolimus has encouraging single-agent activity in endometrial cancer which is higher in chemotherapy-naive patients than in chemotherapy-treated patients and is independent of PTEN status. The difference in activity according to prior therapy should be factored into future clinical trial designs.
PTEN is a tumor suppressor that antagonizes phosphatidylinositol-3 kinase (PI3K) by dephosphorylating the D3 position of phosphatidylinositol (3,4,5)-triphosphate (PtdIns-3,4,5-P3). Given the importance of PTEN in regulating PtdIns-3,4,5-P3 levels, we used Affymetrix GeneChip arrays to identify genes regulated by PTEN. PTEN expression rapidly reduced the activity of Akt, which was followed by a G1 arrest and eventually apoptosis. The gene encoding insulin receptor substrate 2 (IRS-2), a mediator of insulin signaling, was found to be the most induced gene at all time points. A PI3K-specific inhibitor, LY294002, also upregulated IRS-2, providing evidence that it was the suppression of the PI3K pathway that was responsible for the message upregulation. In addition, PTEN, LY294002, and rapamycin, an inhibitor of mammalian target of rapamycin, caused a reduction in the molecular weight of IRS-2 and an increase in the association of IRS-2 with PI3K. Apparently, PTEN inhibits a negative regulator of IRS-2 to upregulate the IRS-2–PI3K interaction. These studies suggest that PtdIns-3,4,5-P3 levels regulate the specific activity and amount of IRS-2 available for insulin signaling.