Preclinical studies have shown that PTEN loss enhances sensitivity to mammalian target of Rapamycin (mTOR) inhibitors because of facilitated PI3K (phosphatidylinositol-3 kinase)/Akt activation and consecutive stimulation of the mTOR pathway. In patients with advanced transitional cell carcinoma (TCC) treated with the mTOR inhibitor everolimus, PTEN loss was, however, associated with resistance to treatment.
Transitional cell carcinoma specimens, human bladder cancer cells and derived mouse xenografts were used to evaluate how the PTEN status influences the activity of mTOR inhibitors.
Transitional cell carcinoma patients with a shorter progression-free survival under everolimus exhibited PTEN deficiency and increased Akt activation. Moreover, PTEN-deficient bladder cancer cells were less sensitive to rapamycin than cells expressing wild-type PTEN, and rapamycin strikingly induced Akt activation in the absence of functional PTEN. Inhibition of Akt activation by the PI3K inhibitor wortmannin interrupted this rapamycin-induced feedback loop, thereby enhancing the antiproliferative effects of the mTOR inhibitor both in vitro and in vivo.
Facilitation of Akt activation upon PTEN loss can have a more prominent role in driving the feedback loop in response to mTOR inhibition than in promoting the mTOR pathway. These data support the use of both PI3K and mTOR inhibitors to treat urothelial carcinoma, in particular in the absence of functional PTEN.
bladder cancer; rapamycin; PTEN; Akt; wortmannin
Urothelial carcinoma of the renal pelvis is a deadly disease with an unclear tumorigenic mechanism. We conducted gene expression profiling on a set of human tumors of this type, and identified a PI3K/AKT activation expression signature in 76.9% (n=13) of our samples. Sequence analysis found both activating mutations of PIK3CA (13.6%, n = 22) and loss of heterozygosity at the PTEN locus (25%, n = 8). In contrast, none of the other subtypes of kidney neoplasms (e.g., clear cell renal cell carcinoma) harbored PIK3CA mutations (n = 87; P < 0.001). Immunohistochemical analysis of urothelial carcinoma samples found loss of PTEN protein expression (36.4%, n = 11) and elevation of phospho-mTOR (63.6%, n = 11). To confirm the role of the PI3K/AKT pathway in urothelial carcinoma, we generated mice containing biallelic inactivation of Pten in the urogenital epithelia. These mice developed typical renal pelvic urothelial carcinomas, with an incidence of 57.1% in mice older than one year. Laser capture microdissection followed by PCR confirmed the deletion of Pten exons 4 and 5 in the animal tumor cells. Immunohistochemical analyses demonstrated increased phospho-mTOR and phospho-S6K levels in the animal tumors. Renal lymph node metastases were found in 15.8% of the animals with urothelial carcinoma. In conclusion, we identified and confirmed an important role for the PI3K/AKT pathway in the development of urothelial carcinoma and suggested that inhibitors of this pathway (e.g. mTOR inhibitor) may serve as effective therapeutic agents.
Phosphatidylinositol-3-kinase; PTEN; urothelial carcinoma; renal pelvis; animal model
The PI3K/PTEN/Akt/mTOR pathway plays critical roles in the regulation of cell growth. The effects of this pathway on drug resistance and cellular senescence of breast cancer cells has been a focus of our laboratory. Introduction of activated Akt or mutant PTEN constructs which lack lipid phosphatase [PTEN(G129E)] or lipid and protein phosphatase [PTEN(C124S)] activity increased the resistance of the cells to the chemotherapeutic drug doxorubicin, and the hormonal drug tamoxifen. Activated Akt and PTEN genes also inhibited the induction of senescence after doxorubicin treatment; a phenomenon associated with unrestrained proliferation and tumorigenesis. Interference with the lipid phosphatase domain of PTEN was sufficient to activate Akt/mTOR/p70S6K as MCF-7 cells transfected with the mutant PTEN gene lacking the lipid phosphatase activity [PTEN(G129E)] displayed elevated levels of activated Akt and p70S6K compared to empty vector transfected cells. Cells transfected with mutant PTEN or Akt constructs were hypersensitive to mTOR inhibitors when compared with the parental or empty vector transfected cells. Akt-transfected cells were cultured for over two months in tamoxifen from which tamoxifen and doxorubicin resistant cells were isolated that were >10-fold more resistant to tamoxifen and doxorubicin than the original Akt-transfected cells. These cells had a decreased induction of both activated p53 and total p21Cip1 upon doxorubicin treatment. Furthermore, these cells had an increased inactivation of GSK-3β and decreased expression of the estrogen receptor-α. In these drug resistant cells, there was an increased activation of ERK which is associated with proliferation. These drug resistant cells were hypersensitive to mTOR inhibitors and also sensitive to MEK inhibitors, indicating that the enhanced p70S6K and ERK expression was relevant to their drug and hormonal resistance. Given that Akt is overexpressed in greater than 50% of breast cancers, our results point to potential therapeutic targets, mTOR and MEK. These studies indicate that activation of the Akt kinase or disruption of the normal activity of the PTEN phosphatase can have dramatic effects on activity of p70S6K and other downstream substrates and thereby altering the therapeutic sensitivity of breast cancer cells. The effects of doxorubicin and tamoxifen on induction of the Raf/MEK/ERK and PI3K/Akt survival pathways were examined in unmodified MCF-7 breast cells. Doxorubicin was a potent inducer of activated ERK and to a lesser extent Akt. Tamoxifen also induced ERK. Thus a consequence of doxorubicin and tamoxifen therapy of breast cancer is the induction of a pro-survival pathway which may contribute to the development of drug resistance. Unmodified MCF-7 cells were also sensitive to MEK and mTOR inhibitors which synergized with both tamoxifen and doxorubicin to induce death. In summary, our results point to the key interactions between the PI3K/PTEN/Akt/mTOR and Raf/MEK/ERK pathways in regulating chemotherapeutic drug resistance/sensitivity in breast cancer and indicate that targeting these pathways may prevent drug and hormonal resistance.
Conventional therapy; targeted therapy; signal transduction inhibitors; breast cancer
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
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
Plasmacytoid urothelial carcinoma is a rare but aggressive variant of bladder cancer with no clear therapeutic guidelines. Dysregulation of the mammalian target of rapamycin (mTOR) pathway has been linked to oncogenesis in conventional bladder cancer. Several antineoplastic agents targeting mTOR pathway are currently available. This study assesses mTOR pathway status as well as c-myc and p27 expression. We retrieved 19 archival cases of plasmacytoid urothelial carcinoma from two institutions. Whole tissue sections were evaluated for immunoexpression of phosphatase and tensin homolog (PTEN), phosphorylated mTOR, phosphorylated protein kinase B (AKT), phosphorylated S6, c-myc, and p27. We evaluated intensity (0 to 3+) and extent (0%–100%) of expression for all markers. An H score was calculated as the sum of products of intensity and extent for each marker and used during analysis. In addition, PTEN loss was defined as absence of expression in >10% of tumor cells. We encountered PTEN loss in 28%. Higher H score for nuclear phosphorylatedAKT and a lowerHscore for phosphorylated S6 was encountered in muscle invasive tumors compared to non-muscle invasive tumors (P = .007 and P = .009, respectively). Although a trend for negative prognostic impact on overall survival for higher phosphorylated mTOR expression was noted (P = .051), markers expression levels failed to predict survival in our cohort. We found dysregulation of mTOR pathway members in urinary bladder plasmacytoid urothelial carcinoma, suggesting that the use of mTOR pathway inhibitors might be beneficial for patients with this aggressive tumor.
Mammalian target of rapamycin; PTEN; Plasmacytoid urothelial carcinoma; Bladder
Glycogen Synthase Kinase-3 (GSK-3) α and β are two serine-threonine kinases controlling insulin, Wnt/β-catenin, NF-κB signaling and other cancer-associated transduction pathways. Recent evidence suggests that GSK-3 could function as growth-promoting kinases, especially in malignant cells. In this study, we have investigated GSK-3α and GSK-3β function in multiple myeloma (MM).
GSK-3 α and β expression and cellular localization were investigated by Western blot (WB) and immunofluorescence analysis in a panel of MM cell lines and in freshly isolated plasma cells from patients. MM cell growth, viability and sensitivity to bortezomib was assessed upon treatment with GSK-3 specific inhibitors or transfection with siRNAs against GSK-3 α and β isoforms. Survival signaling pathways were studied with WB analysis.
GSK-3α and GSK-3β were differently expressed and phosphorylated in MM cells. Inhibition of GSK-3 with the ATP-competitive, small chemical compounds SB216763 and SB415286 caused MM cell growth arrest and apoptosis through the activation of the intrinsic pathway. Importantly, the two inhibitors augmented the bortezomib-induced MM cell cytotoxicity. RNA interference experiments showed that the two GSK-3 isoforms have distinct roles: GSK-3β knock down decreased MM cell viability, while GSK-3α knock down was associated with a higher rate of bortezomib-induced cytotoxicity. GSK-3 inhibition caused accumulation of β-catenin and nuclear phospho-ERK1, 2. Moreover, GSK-3 inhibition and GSK-3α knockdown enhanced bortezomib-induced AKT and MCL-1 protein degradation. Interestingly, bortezomib caused a reduction of GSK-3 serine phosphorylation and its nuclear accumulation with a mechanism that resulted partly dependent on GSK-3 itself.
These data suggest that in MM cells GSK-3α and β i) play distinct roles in cell survival and ii) modulate the sensitivity to proteasome inhibitors.
To investigate whether atorvastatin can promote formation of neurites in cultured cortical neurons and the signaling mechanisms responsible for this effect.
Cultured rat cerebral cortical neurons were incubated with atorvastatin (0.05–10 μmol/L) for various lengths of time. For pharmacological experiments, inhibitors were added 30 min prior to addition of atorvastatin. Control cultures received a similar amount of DMSO. Following the treatment period, phase-contrast digital images were taken. Digital images of neurons were analyzed for total neurite branch length (TNBL), neurite number, terminal branch number, and soma area by SPOT Advanced Imaging software. After incubation with atorvastatin for 48 h, the levels of phosphorylated 3-phosphoinoside-dependent protein kinase-1 (PDK1), phospho-Akt, phosphorylated mammalian target of rapamycin (mTOR), phosphorylated 4E-binding protein 1 (4E-BP1), p70S6 kinase (p70S6K), and glycogen synthase kinase-3β (GSK-3β) in the cortical neurons were evaluated using Western blotting analyses.
Atorvastatin (0.05–10 μmol/L) resulted in dose-dependent increase in neurite number and length in these neurons. Pretreatment of the cortical neurons with phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 (30 μmol/L) and wortmannin (5 μmol/L), Akt inhibitor tricribine (1 μmol/L) or mTOR inhibitor rapamycin (100 nmol/L) blocked the atorvastatin-induced increase in neurite outgrowth, suggesting that atorvastatin promoted neurite outgrowth via activating the PI3K/Akt/mTOR signaling pathway. Atorvastatin (10 μmol/L) significantly increased the levels of phosphorylated PDK1, Akt and mTOR in the cortical neurons, which were prevented by LY294002 (30 μmol/L). Moreover, atorvastatin (10 μmol/L) stimulated the phosphorylation of 4E-BP1 and p70S6K, the substrates of mTOR, in the cortical neurons. In addition, atorvastatin (10 μmol/L) significantly increased the phosphorylated GSK-3β level in the cortical neurons, which was prevented by both LY294002 and tricribine.
These results suggest that activation of both the PI3K/Akt/mTOR and Akt/GSK-3β signaling pathways is responsible for the atorvastatin-induced neurite outgrowth in cultured cortical neurons.
statin; atorvastatin; cortical neurons; neuritogenesis; PI3-kinase; GSK-3β
Prostaglandin F2α (PGF2α) increases reactive oxygen species (ROS) and induces vascular smooth muscle cell (VSMC) hypertrophy by largely unknown mechanism(s). To investigate the signaling events governing PGF2α –induced VSMC hypertrophy we examined the ability of the PGF2α analog, fluprostenol to elicit phosphorylation of Akt, the mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6k), glycogen synthase kinase-3β (GSK-3β), phosphatase and tensin homolog (PTEN), extracellular signal-regulated kinase 1/2 (ERK1/2) and Jun N-terminal kinase (JNK) in growth arrested A7r5 VSMC. Fluprostenol-induced hypertrophy was associated with increased ROS, mTOR translocation from the nucleus to the cytoplasm, along with Akt, mTOR, GSK-3β, PTEN and ERK1/2 but not JNK phosphorylation. Whereas inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002 blocked fluprostenol-induced changes in total protein content, pretreatment with rapamycin or with the ERK1/2-MAPK inhibitor UO126 did not. Taken together, these findings suggest that fluprostenol-induced changes in A7R5 hypertrophy involve mTOR translocation and occur through PI3K-dependent mechanisms.
A7R5; VSMC; PGF-2α; hypertrophy; p70S6k; PTEN
Basal-like carcinomas (BLCs) and human epidermal growth factor receptor 2 overexpressing (HER2+) carcinomas are the subgroups of breast cancers that have the most aggressive clinical behaviour. In contrast to HER2+ carcinomas, no targeted therapy is currently available for the treatment of patients with BLCs. In order to discover potential therapeutic targets, we aimed to discover deregulated signalling pathways in human BLCs.
In this study, we focused on the oncogenic phosphatidylinositol 3-kinase (PI3K) pathway in 13 BLCs, and compared it with a control series of 11 hormonal receptor negative- and grade III-matched HER2+ carcinomas. The two tumour populations were first characterised by immunohistochemistry and gene expression. The PI3K pathway was then investigated by gene copy-number analysis, gene expression profiling and at a proteomic level using reverse-phase protein array technology and tissue microarray. The effects of the PI3K inhibition pathway on proliferation and apoptosis was further analysed in three human basal-like cell lines.
The PI3K pathway was found to be activated in BLCs and up-regulated compared with HER2+ tumours as shown by a significantly increased activation of the downstream targets Akt and mTOR (mammalian target of rapamycin). BLCs expressed significantly lower levels of the tumour suppressor PTEN and PTEN levels were significantly negatively correlated with Akt activity within that population. PTEN protein expression correlated significantly with PTEN DNA copy number and more importantly, reduced PTEN DNA copy numbers were observed specifically in BLCs. Similar to human samples, basal-like cell lines exhibited an activation of PI3K/Akt pathway and low/lack PTEN expression. Both PI3K and mTOR inhibitors led to basal-like cell growth arrest. However, apoptosis was specifically observed after PI3K inhibition.
These data provide insight into the molecular pathogenesis of BLCs and implicate the PTEN-dependent activated Akt signalling pathway as a potential therapeutic target for the management of patients with poor prognosis BLCs.
Dendritic cells (DCs), key players of immunity, are regulated by glycogen synthase kinase GSK3. GSK3 activity is suppressed by PKB/Akt and SGK isoforms, which are in turn stimulated by the PI3K pathway. Exposure to bacterial lipopolysaccharides increases cytosolic Ca2+-concentration ([Ca2+]i), an effect augmented in DCs isolated from mutant mice expressing PKB/SGK-resistant GSK3α,β (gsk3KI). Factors affecting [Ca2+]i include Ca2+-release from intracellular stores (CRIS), store-operated Ca2+-entry (SOCE) through STIM1/STIM2-regulated Orai1, K+-dependent Na+/Ca2+-exchangers (NCKX), K+-independent Na+/Ca2+-exchangers (NCX) and calbindin-D28k. The present study explored whether PKB/SGK-dependent GSK3α, β-activity impacts on CRIS, SOCE, NCKX, NCX or calbindin. DCs were isolated from gsk3KI mice and respective wild-type mice (gsk3WT), [Ca2+]i estimated from Fura2 fluorescence, Orai1, STIM1, STIM2 as well as calbindin-D28k protein abundance determined by Western blotting and mRNA levels quantified by real time PCR. As a result, thapsigargin-induced CRIS and SOCE were significantly blunted by GSK3-inhibitors SB216763 (1–10 µM, 30 min) or GSK-XIII (10 µM, 30 min) but were significantly lower in gsk3WT than in gsk3KIDCs. Orai1, STIM1 and STIM2 protein abundance was significantly lower and calbindin-D28k abundance significantly higher in gsk3KI than in gsk3WTDCs. Activity of NCKX and NCX was significantly higher in gsk3KI than in gsk3WTDCs and was significantly increased by SB216763 (1 µM, 30 min) or GSK-XIII (10 µM, 30 min). Treatment of gsk3WT DCs with SB216763 (1 µM, 4–24 h) or GSK-XIII (10 µM, 4–24 h) did not significantly modify the protein abundance of Orai1, STIM1 and STIM2. The present observations point to a dual role of GSK3 in the regulation of Ca2+ in DCs. Acute inhibition of GSK3 blunted the increase of [Ca2+]i following CRIS and SOCE and stimulated NCKX/NCX activity. However, expression of PKB/SGK-resistant GSK3α, β downregulated the increase of [Ca2+]i following CRIS and SOCE, an effect at least partially due to downregulation of Orai1, STIM1 and STIM2 expression as well as upregulation of Na+/Ca2+-exchanger activity and calbindin D28k expression.
Platelet derived growth factor receptor (PDGFR) activity is deregulated in human GBM due to amplification and rearrangement of the PDGFR-alpha gene locus or overexpression of the PDGF ligand, resulting in the activation of downstream kinases such as phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). Aberrant PDGFR signaling is observed in approximately 25-30% of human GBMs, which are frequently molecularly classified as the proneural subclass. It would be valuable to understand how PDGFR driven GBMs respond to Akt and mTOR inhibition.
Using genetically engineered PTEN-intact and PTEN-deficient PDGF-driven mouse models of GBM that closely mimic the histology and genetics of the human PDGF subgroup, we investigated the effect of inhibiting Akt and mTOR alone or in combination in vitro and in vivo. We used perifosine and CCI-779 to inhibit Akt and mTOR, respectively. Here, we show in vitro data demonstrating that the most effective inhibition of Akt and mTOR activity in both PTEN-intact and PTEN-null primary glioma cell cultures is obtained when using both inhibitors in combination. We next investigated if the effects we observed in culture could be duplicated in vivo by treating mice with gliomas for 5 days. The in vivo treatments with the combination of CCI-779 and perifosine resulted in decreased Akt and mTOR signaling, which correlated to decreased proliferation and increased cell death independent of PTEN status, as monitored by immunoblot analysis, histology and MRI.
These findings underline the importance of simultaneously targeting Akt and mTOR to achieve significant down-regulation of the PI3K pathway and support the rationale for testing the perifosine and CCI-779 combination in the human PDGF-subgroup of GBM.
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
Although formation of urothelial carcinoma of the bladder (UCB) requires multiple steps and proceeds along divergent pathways, the underlying genetic and molecular determinants for each step and pathway remain undefined. By developing transgenic mice expressing single or combinatorial genetic alterations in urothelium, we demonstrated here that overcoming oncogene-induced compensatory tumor barriers was critical for urothelial tumor initiation. Constitutively active Ha-ras (Ras*) elicited urothelial hyperplasia that was persistent and did not progress to tumors over a 10 months period. This resistance to tumorigenesis coincided with increased expression of p53 and all pRb family proteins. Expression of a Simian virus 40 T antigen (SV40T), which disables p53 and pRb family proteins, in urothelial cells expressing Ras* triggered early-onset, rapidly-growing and high-grade papillary UCB that strongly resembled the human counterpart (pTaG3). Urothelial cells expressing both Ras* and SV40T had defective G1/S checkpoint, elevated Ras-GTPase and hyperactivated AKT-mTOR signaling. Inhibition of the AKT-mTOR pathway with rapamycin significantly reduced the size of high-grade papillary UCB but hyperactivated mitogen-activated protein kinase (MAPK). Inhibition of AKT-mTOR, MAPK and STAT3 altogether resulted in much greater tumor reduction and longer survival than did inhibition of AKT-mTOR pathway alone. Our studies provide the first experimental evidence delineating the combinatorial genetic events required for initiating high-grade papillary UCB, a poorly defined and highly challenging clinical entity. Furthermore, they suggest that targeted therapy using a single agent such as rapamycin may not be highly effective in controlling high-grade UCB and that combination therapy employing inhibitors against multiple targets are more likely to achieve desirable therapeutic outcomes.
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.
Hyperactivation of the phosphatidylinositol 3-kinase/Akt signaling through disruption of PTEN function is common in glioblastoma multiforme, and these genetic changes are predicted to enhance sensitivity to mammalian target of rapamycin (mTOR) inhibitors such as RAD001 (everolimus).
To test whether PTEN loss could be used as a predictive marker for mTOR inhibitor sensitivity, the response of 17 serially transplantable glioblastoma multiforme xenografts was evaluated in an orthotopic therapy evaluation model. Of these 17 xenograft lines, 7 have either genomic deletion or mutation of PTEN.
Consistent with activation of Akt signaling, there was a good correlation between loss of PTEN function and elevated levels of Akt phosphorylation. However, of the 7 lines with disrupted PTEN function, only 1 tumor line (GBM10) was significantly sensitive to RAD001 therapy (25% prolongation in median survival), whereas1 of 10 xenograft lines with wild-type PTEN was significantly sensitive to RAD001 (GS22; 34% prolongation in survival). Relative to placebo, 5 days of RAD001 treatment was associated with a marked 66% reduction in the MIB1 proliferation index in the sensitive GBM10 line (deleted PTEN) compared with a 25% and 7% reduction in MIB1 labeling index in the insensitive GBM14 (mutant PTEN) and GBM15 (wild-type PTEN) lines, respectively. Consistent with a cytostatic antitumor effect, bioluminescent imaging of luciferase-transduced intracranial GBM10 xenografts showed slowed tumor growth without significant tumor regression during RAD001 therapy.
These data suggest that loss of PTEN function is insufficient to adequately predict responsiveness to mTOR inhibitors in glioblastoma multiforme.
Sphingadienes (SDs) derived from soy and other natural sphingolipids are cytotoxic to colon cancer cells via an Akt-dependent mechanism and reduce adenoma formation in Apc
Min/+ mice. Wnt signaling is fundamental to colon carcinogenesis and is the basis for spontaneous tumorigenesis in Apc
Min/+ mice and patients with familial adenomatous polyposis. In the present study, we investigated the impact of SDs on Wnt signaling. Oral SD administration reduced levels of active β-catenin and Wnt targets c-Myc and cyclin D1 in Apc
Min/+ mouse intestinal tissues. Colon cancer cells treated with SDs exhibited reduced Wnt transcriptional activity, as well as reduced nuclear β-catenin localization and subsequent reduction in active-β-catenin levels. Further, we observed a decrease in phosphorylated (inactive) GSK3β in SD-treated mice and colon cancer cells. Expression of constitutively active myristoylated-Akt or inactivation of GSK3β using LiCl attenuated SD-mediated inhibition of Wnt transcriptional activity and active-β-catenin levels. SDs exhibited additive effects with inhibitors of the phosphatidylinositol-3-kinase/Akt/mTOR pathway to induce cytotoxicity. Further, a combination regime of SDs and low-dose rapamycin decreased visible polyps in Apc
Min/+ mice and reduced the levels of Wnt target gene expression and mTOR target activation. SD-mediated inhibition of Akt and Wnt pathways and cytotoxicity in colon cancer cells was dependent upon the activity of protein phosphatase 2A, as shown by reversal of these effects by pretreatment with okadaic acid or calyculin A. Our cumulative findings indicate that SDs inhibit Wnt signaling through a protein phosphatase 2A/Akt/GSK3β-dependent mechanism that may contribute to their chemopreventive effects in intestinal tumorigenesis.
Abbreviations:4EBP-1eIF4E-binding protein1APCadenomatous polyposis coliGSK3βGlycogen synthase Kinase 3βLCMlaser capture microscopyLEFlymphoid enhancer factormTORmammalian target of RapamycinPI3KPhosphatidylinositol-3-kinasePKCprotein kinase CPP2Aprotein phosphatase 2ATCFT-cell factorSDSphingadiene
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
Survival rate for patients presenting muscle invasive bladder cancer is very low, and useful therapeutic target has not been identified yet. In the present study, new COX2 downstream signals involved in urothelial carcinoma cell survival were investigated in vitro and in vivo.
COX2 gene was silenced by siRNA transfection. Orthotopic implantation animal model and transurethral instillation of siRNA with atelocollagen was constructed to examine the effects of COX2 knockdown in vivo. Cell cycle was examined by flowcytoketry. Surgical specimens derived from patients with urinary bladder cancer (all were initially diagnosed cases) were used for immunohistochemical analysis of the indicated protein expression in urothelial carcinoma cells.
Treatment with the COX2 inhibitor or knockdown of COX2 reduced expression of casein kinase (CK) 2 α, a phophorylated Akt and urokinase type plasminogen activator (uPA), resulting in p27 induction, cell cycle arrest at G1 phase and cell growth suppression in human urothelial carcinoma cell lines expressing COX2. Silencing of CK2α exhibited the similar effects. Even in UMUC3 cells lacking the COX2 gene, COX2 inhibition also inhibited cell growth through down-regulation of the CK2α-Akt/uPA axis. The mouse orthotropic bladder cancer model demonstrated that the COX2 inhibitor, meloxicam significantly reduced CK2α, phosphorylated Akt and uPA expression, whereas induced p27 by which growth and invasiveness of bladder cancer cells were strongly inhibited. Immunohistochemically, high expression of COX2, CK2α and phosphorylated form of Akt was found in high-grade, invasive carcinomas as well as carcinoma in situ, but not in low-grade and noninvasive phenotypes.
COX2-dependent and independent activation of CK2α-Akt/uPA signal is mainly involved in urothelial carcinoma cell survival, moreover, not only COX2 but also CK2α could be direct targets of COX2 inhibitors.
cyclooxygenase 2; urothelial carcinoma; casein kinase 2α; Akt
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
Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in estrogen receptor α (ER)-positive breast cancer is associated with reduced ER expression and activity, luminal B subtype, and poor outcome. Phosphatase and tensin homolog (PTEN), a negative regulator of this pathway, is typically lost in ER-negative breast cancer. We set out to clarify the role of reduced PTEN levels in endocrine resistance, and to explore the combination of newly developed PI3K downstream kinase inhibitors to overcome this resistance.
Altered cellular signaling, gene expression, and endocrine sensitivity were determined in inducible PTEN-knockdown ER-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer cell and/or xenograft models. Single or two-agent combinations of kinase inhibitors were examined to improve endocrine therapy.
Moderate PTEN reduction was sufficient to enhance PI3K signaling, generate a gene signature associated with the luminal B subtype of breast cancer, and cause endocrine resistance in vitro and in vivo. The mammalian target of rapamycin (mTOR), protein kinase B (AKT), or mitogen-activated protein kinase kinase (MEK) inhibitors, alone or in combination, improved endocrine therapy, but the efficacy varied by PTEN levels, type of endocrine therapy, and the specific inhibitor(s). A single-agent AKT inhibitor combined with fulvestrant conferred superior efficacy in overcoming resistance, inducing apoptosis and tumor regression.
Moderate reduction in PTEN, without complete loss, can activate the PI3K pathway to cause endocrine resistance in ER-positive breast cancer, which can be overcome by combining endocrine therapy with inhibitors of the PI3K pathway. Our data suggests that the ER degrader fulvestrant, to block both ligand-dependent and -independent ER signaling, combined with an AKT inhibitor is an effective strategy to test in patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0430-x) contains supplementary material, which is available to authorized users.
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
Despite treatment with agents that enhance β-cell function and insulin action, reduction in β-cell mass is relentless in patients with insulin resistance and type 2 diabetes mellitus. Insulin resistance is characterized by impaired signaling through the insulin/insulin receptor/insulin receptor substrate/PI-3K/Akt pathway, leading to elevation of negatively regulated substrates such as glycogen synthase kinase-3β (Gsk-3β). When elevated, this enzyme has antiproliferative and proapoptotic properties. In these studies, we designed experiments to determine the contribution of Gsk-3β to regulation of β-cell mass in two mouse models of insulin resistance. Mice lacking one allele of the insulin receptor (Ir+/−) exhibit insulin resistance and a doubling of β-cell mass. Crossing these mice with those having haploinsufficiency for Gsk-3β (Gsk-3β+/−) reduced insulin resistance by augmenting whole-body glucose disposal, and significantly reduced β-cell mass. In the second model, mice missing two alleles of the insulin receptor substrate 2 (Irs2−/−), like the Ir+/− mice, are insulin resistant, but develop profound β-cell loss, resulting in early diabetes. We found that islets from these mice had a 4-fold elevation of Gsk-3β activity associated with a marked reduction of β-cell proliferation and increased apoptosis. Irs2−/− mice crossed with Gsk-3β+/− mice preserved β-cell mass by reversing the negative effects on proliferation and apoptosis, preventing onset of diabetes. Previous studies had shown that islets of Irs2−/− mice had increased cyclin-dependent kinase inhibitor p27kip1 that was limiting for β-cell replication, and reduced Pdx1 levels associated with increased cell death. Preservation of β-cell mass in Gsk-3β+/−Irs2−/− mice was accompanied by suppressed p27kip1 levels and increased Pdx1 levels. To separate peripheral versus β-cell–specific effects of reduction of Gsk3β activity on preservation of β-cell mass, mice homozygous for a floxed Gsk-3β allele (Gsk-3F/F) were then crossed with rat insulin promoter-Cre (RIP-Cre) mice to produce β-cell–specific knockout of Gsk-3β (βGsk-3β−/−). Like Gsk-3β+/− mice, βGsk-3β−/− mice also prevented the diabetes of the Irs2−/− mice. The results of these studies now define a new, negatively regulated substrate of the insulin signaling pathway specifically within β-cells that when elevated, can impair replication and increase apoptosis, resulting in loss of β-cells and diabetes. These results thus form the rationale for developing agents to inhibit this enzyme in obese insulin-resistant individuals to preserve β-cells and prevent diabetes onset.
Diabetes is often characterized by a failure of insulin production by pancreatic β-cells to properly regulate glucose homeostasis. Insulin resistance can lead to β-cell failure, and our studies have focused on elucidating the mechanisms involved in this postnatal failure. In this study, we evaluated a new, negatively regulated enzyme of the insulin signaling pathway, glycogen synthase kinase 3 (Gsk-3), specifically within insulin-producing pancreatic β-cells. When this enzyme is elevated, it can impair replication and increase cell death, resulting in loss of insulin-producing cells and diabetes. Gsk-3 is also known to regulate cell death and proliferation in neurons. We assessed the role of Gsk-3 on glucose homeostasis in two different mouse models of insulin resistance. We demonstrated that genetically reducing the levels of Gsk-3β in the insulin-resistant mouse improved glucose homeostasis. In another model in which severe insulin resistance is associated with destruction of β-cells, reducing Gsk-3β not only preserved β-cells by increasing proliferation and reducing cell death, but it also corrected diabetes. Controlling activity of Gsk-3 could lead to new hopes for maintaining or improving β-cell number and prevention of diabetes.
The insulin signaling pathway negatively regulates glycogen synthase kinase 3. The current studies demonstrate that this enzyme is rate limiting for pancreatic β-cell replication and survival.
The Akt - GSK3 signaling pathway has been recently implicated in psychostimulant-induced behavioral and cellular effects. Here, the ability of cocaine to regulate the activity of Akt and GSK3 was investigated by measuring the phosphorylation states of the two kinases. The anatomical specificity of the response was determined, as was the contributions of dopamine and NMDA receptors to the actions of cocaine. As GSK3 activity was found to be increased by cocaine, subsequent experiments investigated the importance of GSK3 activation in cocaine conditioned reward. Adult male CD-1 mice were injected with cocaine or saline, and levels of phosphorylated Akt and GSK3α/β were measured 30 minutes later. Acute administration of cocaine significantly decreased the phosphorylation of Akt-Thr308 (pAkt-Thr308) and GSK3β in the caudate putamen and nucleus accumbens core, without altering pAkt-Ser473 and pGSK3α. To investigate the role of dopamine and NMDA receptors in the regulation of Akt and GSK3 by cocaine, specific receptor antagonists were administered prior to cocaine. Blockade of dopamine D2 receptors with eticlopride prevented the reduction of pAkt-Thr308 produced by cocaine, whereas antagonists at dopamine D1, dopamine D2 or glutamatergic NMDA receptors each blocked cocaine-induced reductions in pGSK3β. The potential importance of GSK3 activity in the rewarding actions of cocaine was determined using a cocaine conditioned place preference procedure. Administration of the selective GSK3 inhibitor, SB 216763, prior to cocaine conditioning sessions blocked the development of cocaine place preference. In contrast, SB 216763 did not alter the acquisition of a contextual fear conditioning response, demonstrating that SB 216763 did not globally inhibit contextual learning processes. The results of this study indicate that phosphorylation of GSK3β is reduced, hence GSK3β activity is increased following acute cocaine, an effect that is contingent upon both dopaminergic and glutamatergic receptors. Further, GSK3 activity is required for the development of cocaine conditioned reward.
Amplification of the gene encoding the epidermal growth factor (EGF) receptor (EGFR) occurs commonly in glioblastoma, leading to activation of downstream kinases including phosphatidylinositol 3′-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). Here, we show that phosphorylation of mTOR and its downstream substrate rpS6 (ribosomal protein S6) are robust biomarkers for the antiproliferative effect of EGFR inhibitors. Inhibition of EGFR signaling correlated with decreased abundance of phosphorylated mTOR (p-mTOR) and rpS6 (p-rpS6) in cells wild type for the gene encoding PTEN (phosphatase and tensin homolog on chromosome 10), a negative regulator of PI3K. In contrast, inhibition of EGFR signaling failed to affect p-mTOR or p-rpS6 in cells mutant for PTEN, which are resistant to EGFR inhibitors. Although the abundance of phosphorylated Akt (p-Akt) decreased in response to inhibition of EGFR signaling, Akt was dispensable for signaling between EGFR and mTOR. We identified an Akt-independent pathway linking EGFR to mTOR that was critically dependent on protein kinase C (PKC). Consistent with these observations, the abundance of EGFR generally correlated with phosphorylation of rpS6 and PKC in primary human glioblastoma tumors, and correlated poorly with phosphorylation of Akt. Inhibition of PKC led to decreased viability of glioma cells regardless of PTEN or EGFR status, suggesting that PKC inhibitors should be tested in glioma. These findings underline the importance of signaling between EGFR and mTOR in glioma, identify PKCα as essential to this network, and question the necessity of Akt as a critical intermediate coupling EGFR and mTOR in glioma.