Instruct: Given a scientific claim, retrieve documents that support or refute the claim.
Query: MEK inhibitors are effective treatments in RAS-driven mouse models of cancer.
Somatic mutations that activate phosphoinositide 3-kinase (PI3K) have been identified in the p110-alpha catalytic subunit (encoded by PIK3CA). They are most frequently observed in two hotspots: the helical domain (E545K and E542K) and the kinase domain (H1047R). Although the p110-alpha mutants are transforming in vitro, their oncogenic potential has not been assessed in genetically engineered mouse models. Furthermore, clinical trials with PI3K inhibitors have recently been initiated, and it is unknown if their efficacy will be restricted to specific, genetically defined malignancies. In this study, we engineered a mouse model of lung adenocarcinomas initiated and maintained by expression of p110-alpha H1047R. Treatment of these tumors with NVP-BEZ235, a dual pan-PI3K and mammalian target of rapamycin (mTOR) inhibitor in clinical development, led to marked tumor regression as shown by positron emission tomography-computed tomography, magnetic resonance imaging and microscopic examination. In contrast, mouse lung cancers driven by mutant Kras did not substantially respond to single-agent NVP-BEZ235. However, when NVP-BEZ235 was combined with a mitogen-activated protein kinase kinase (MEK) inhibitor, ARRY-142886, there was marked synergy in shrinking these Kras-mutant cancers. These in vivo studies suggest that inhibitors of the PI3K-mTOR pathway may be active in cancers with PIK3CA mutations and, when combined with MEK inhibitors, may effectively treat KRAS mutated lung cancers.
Germline mutations in the RET tyrosine kinase gene are responsible for the development of multiple endocrine neoplasia 2A and 2B (MEN2A and MEN2B). However, knowledge of the fundamental principles that determine the mutant RET-mediated signaling remains elusive. Here, we report increased expression of mitogen-activated protein kinase phosphatase-2 (MKP-2) in carcinomas developed in transgenic mice carrying RET with the MEN2A mutation (RET-MEN2A). The expression of MKP-2 was not only induced by RET-MEN2A or RET-MEN2B mutant proteins but also by the activation of endogenous RET by its ligand, glial cell line-derived neurotrophic factor (GDNF). MKP-2 expression was also evident in the MKK-f cell line, which was established from a mammary tumor developed in a RET-MEN2A transgenic mouse. Inhibition of MKP-2 attenuated the in vitro and in vivo proliferation of MKK-f cells, which was mediated by the suppression of cyclin B1 expression. Furthermore, we found that MKP-2 is highly expressed in medullary thyroid carcinomas derived from MEN2A patients. These findings suggest that the increased expression of MKP-2 may play a crucial role in oncogenic signaling downstream of mutant RET, leading to deregulation of cell cycle.
The nonreceptor protein tyrosine kinase Src is overexpressed in 70% of pancreatic adenocarcinomas. Here, we describe the effect of molecular and pharmacological down-regulation of Src on incidence, growth, and metastasis of pancreatic tumor cells in an orthotopic model. Src expression in L3.6pl human pancreatic tumor cells was reduced by stable expression of a plasmid encoding small interfering RNA (siRNA) to c-src. In stable siRNA clones, Src expression was reduced >80%, with no change in expression of the related kinases c-Yes and c-Lyn, and proliferation rates were similar in all clones. Phosphorylation of Akt and p44/42 Erk mitogen-activated protein kinase and production of VEGF and IL-8 in culture supernatants were also reduced (P < 0.005). On orthotopic implantation of varying cell numbers into nude mice, tumor incidence was unchanged; however, in the siRNA clones, large tumors failed to develop, and incidence of metastasis was significantly reduced, suggesting that c-Src activity is critical to tumor progression. To examine this possibility further, animals bearing established wild-type tumors were treated with the Src/Abl-selective inhibitor BMS-354825 (dasatinib). Tumor size was decreased, and incidence of metastases was significantly reduced in treated mice compared with controls. These results demonstrate that Src activation contributes to pancreatic tumor progression in this model, offering Src as a candidate for targeted therapy.
Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner.
Phosphatidylinositol-3-kinase (PI3K) pathway deregulation is a common event in human cancer, either through inactivation of the tumor suppressor phosphatase and tensin homologue deleted from chromosome 10 or activating mutations of p110-alpha. These hotspot mutations result in oncogenic activity of the enzyme and contribute to therapeutic resistance to the anti-HER2 antibody trastuzumab. The PI3K pathway is, therefore, an attractive target for cancer therapy. We have studied NVP-BEZ235, a dual inhibitor of the PI3K and the downstream mammalian target of rapamycin (mTOR). NVP-BEZ235 inhibited the activation of the downstream effectors Akt, S6 ribosomal protein, and 4EBP1 in breast cancer cells. The antiproliferative activity of NVP-BEZ235 was superior to the allosteric selective mTOR complex inhibitor everolimus in a panel of 21 cancer cell lines of different origin and mutation status. The described Akt activation due to mTOR inhibition was prevented by higher doses of NVP-BEZ235. NVP-BEZ235 reversed the hyperactivation of the PI3K/mTOR pathway caused by the oncogenic mutations of p110-alpha, E545K, and H1047R, and inhibited the proliferation of HER2-amplified BT474 cells exogenously expressing these mutations that render them resistant to trastuzumab. In trastuzumab-resistant BT474 H1047R breast cancer xenografts, NVP-BEZ235 inhibited PI3K signaling and had potent antitumor activity. In treated animals, there was complete inhibition of PI3K signaling in the skin at pharmacologically active doses, suggesting that skin may serve as surrogate tissue for pharmacodynamic studies. In summary, NVP-BEZ235 inhibits the PI3K/mTOR axis and results in antiproliferative and antitumoral activity in cancer cells with both wild-type and mutated p110-alpha.
Tumour cells, with stem-like properties, are highly aggressive and often show drug resistance. Here, we reveal that integrin αvβ3 serves as a marker of breast, lung and pancreatic carcinomas with stem-like properties that are highly resistant to receptor tyrosine kinase inhibitors such as erlotinib. This was observed in vitro and in mice bearing patient-derived tumour xenografts or in clinical specimens from lung cancer patients who had progressed on erlotinib. Mechanistically, αvβ3, in the unliganded state, recruits KRAS and RalB to the tumour cell plasma membrane, leading to the activation of TBK1 and NF-κB. In fact, αvβ3 expression and the resulting KRAS–RalB–NF-κB pathway were both necessary and sufficient for tumour initiation, anchorage independence, self-renewal and erlotinib resistance. Pharmacological targeting of this pathway with bortezomib reversed both tumour stemness and erlotinib resistance. These findings not only identify αvβ3 as a marker/driver of carcinoma stemness but also reveal a therapeutic strategy to sensitize such tumours to RTK inhibition.
Owing to their integral involvement in cell cycle regulation, the Polo-like kinase (Plk) family, particularly Plk1, has emerged as an attractive therapeutic target in oncology. In recent years, several Plk1 inhibitors have been developed, with some agents showing encouraging results in early-phase clinical trials. This review focuses on volasertib (BI 6727; an investigational agent), a potent and selective Plk inhibitor. Volasertib has shown promising activity in various cancer cell lines and xenograft models of human cancer. Trials performed to date suggest that volasertib has clinical efficacy in a range of malignancies, with the most promising results seen in patients with acute myeloid leukemia (AML). Encouragingly, recent phase II data have demonstrated that volasertib combined with low-dose cytarabine (LDAC) was associated with higher response rates and improved event-free survival than LDAC alone in patients with previously untreated AML. Based on these observations, and its presumably manageable safety profile, volasertib is currently in phase III development as a potential treatment for patients with AML who are ineligible for intensive remission induction therapy. Given that many patients with AML are of an older age and frail, this constitutes an area of major unmet need. In this review, we discuss the biologic rationale for Plk1 inhibitors in cancer, the clinical development of volasertib to date in solid tumors and AML, and the future identification of biomarkers that might predict response to volasertib and help determine the role of this agent in the clinic.
Compartmentalization of signals generated by receptor tyrosine kinase (RTK) endocytosis has emerged as a major determinant of various cell functions. Here, using tumour-associated Met-activating mutations, we demonstrate a direct link between endocytosis and tumorigenicity. Met mutants exhibit increased endocytosis/recycling activity and decreased levels of degradation, leading to accumulation on endosomes, activation of the GTPase Rac1, loss of actin stress fibres and increased levels of cell migration. Blocking endocytosis inhibited mutants’ anchorage-independent growth, in vivo tumorigenesis and metastasis while maintaining their activation. One mutant resistant to inhibition by a Met-specific tyrosine kinase inhibitor was sensitive to endocytosis inhibition. Thus, oncogenicity of Met mutants results not only from activation but also from their altered endocytic trafficking, indicating that endosomal signalling may be a crucial mechanism regulating RTK-dependent tumorigenesis.
Angiogenesis, the formation of new blood vessels from an existing vasculature, is requisite for tumor growth. It entails intercellular coordination of endothelial and tumor cells through angiogenic growth factor signaling. Interruption of these events has implications in the suppression of tumor growth. PD166285, a broad-spectrum receptor tyrosine kinase (RTK) inhibitor, and PD173074, a selective FGFR1TK inhibitor, were evaluated for their anti-angiogenic activity and anti-tumor efficacy in combination with photodynamic therapy (PDT). To evaluate the anti-angiogenic and anti-tumor activities of these compounds, RTK assays, in vitro tumor cell growth and microcapillary formation assays, in vivo murine angiogenesis and anti-tumor efficacy studies utilizing RTK inhibitors in combination with photodynamic therapy were performed. PD166285 inhibited PDGFR-β-, EGFR-, and FGFR1TKs and c-src TK by 50% (IC50) at concentrations between 7−85nM. PD173074 displayed selective inhibitory activity towards FGFR1TK at 26nM. PD173074 demonstrated (>100 fold) selective growth inhibitory action towards human umbilical vein endothelial cells compared with a panel of tumor cell lines. Both PD166285 and PD173074 (at 10nM) inhibited the formation of microcapillaries on Matrigel-coated plastic. In vivo anti-angiogenesis studies in mice revealed that oral administration (p.o.) of either PD166285 (1−25 mg/kg) or PD173074 (25−100 mg/kg) generated dose dependent inhibition of angiogenesis. Against a murine mammary 16c tumor, significantly prolonged tumor regressions were achieved with daily p.o. doses of PD166285 (5−10 mg/kg) or PD173074 (30−60 mg/kg) following PDT compared with PDT alone (p<0.001). Many long-term survivors were also noted in combination treatment groups. PD166285 and PD173074 displayed potent anti-angiogenic and anti-tumor activity and prolonged the duration of anti-tumor response to PDT. Interference in membrane signal transduction by inhibitors of specific RTKs (e.g. FGFR1TK) should result in new chemotherapeutic agents having the ability to limit tumor angiogenesis and regrowth following cytoreductive treatments such as PDT.
AIMS AND BACKGROUND The PI3 kinase signalling pathway is now accepted as being at least as important as the ras-MAP kinase pathway in cell survival and proliferation, and hence its potential role in cancer is of great interest. The purpose of this review is briefly to examine evidence for an involvement of PI3K in human cancers, discuss the mechanisms by which its activation promotes tumor progression, and consider its utility as a novel target for anticancer therapy. METHODS AND STUDY DESIGN A Medline review of recent literature concerning the role of PI3 kinase in tumor progression--mechanisms of action and clinical implications. RESULTS Evidence is presented that misregulation of the PI3 kinase pathway is a feature of many common cancers, either by loss of the suppressor protein PTEN, or by constitutive activation of PI3 kinase isoforms or downstream elements such as AKT and mTOR. This activation potentiates not only cell survival and proliferation, but also cytoskeletal deformability and motility; key elements in tumor invasion. In addition the PI3K pathway is implicated in many aspects of angiogenesis, including upregulation of angiogenic cytokines due to tumor hypoxia or oncogene activation and endothelial cell responses to them. These cytokines signal though receptors such as VEGF-R, FGF-R and Tie-2 and potentiate processes essential for neoangiogenesis including cell proliferation, migration, differentiation into tubules and "invasion" of these capillary sprouts into extracellular matrix (ECM). CONCLUSIONS A more complete understanding of the role of the PI3 kinase pathway in cancer will lead the way to the development of more potent and selective inhibitors which should be a useful adjunct to conventional therapies, potentially interfering with tumor progression at several pivotal points; in particular cell survival, invasion and angiogenesis.
MAP kinase phosphatase-2 (MKP-2) is a member of the family of dual specificity phosphatases that functions to inactivate the ERK and JNK MAP kinase signalling pathways. Here, we identify a novel human MKP-2 variant (MKP-2-S) lacking the MAP kinase binding site but retaining the phosphatase catalytic domain. Endogenous MKP-2-S transcripts and proteins were found in PC3 prostate and MDA-MB-231 breast cancer cells and also human prostate biopsies. Cellular transfection of MKP-2-S gave rise to a nuclear protein of 33kDa which displayed phosphatase activity comparable to the formerly described long form of MKP-2 (MKP-2-L). Due to its lack of a kinase interacting motif (KIM), MKP-2-S did not bind to JNK or ERK; MKP-2-L bound ERK and to a lesser extent JNK. Protein turnover of adenoviral expressed MKP-2-S was accelerated relative to MKP-2-L, with a greater susceptibility to proteosomal-mediated degradation. MKP-2-S retained its ability to deactivate JNK in a similar manner as MKP-2-L and was an effective inhibitor of LPS-stimulated COX-2 induction. However, unlike MKP-2-L, MKP-2-S was unable to reverse serum-induced ERK activation or significantly inhibit endothelial cell proliferation. These findings reveal the occurrence of a novel splice variant of MKP-2 which is unable to bind ERK and may be significant in the dysregulation of MAP kinase activity in certain disease states, particularly in breast and prostate cancers.
Activating mutations in the EGF receptor (EGFR) are associated with clinical responsiveness to EGFR tyrosine kinase inhibitors (TKI), such as erlotinib and gefitinib. However, resistance eventually arises, often due to a second EGFR mutation, most commonly T790M. Through a genome-wide siRNA screen in a human lung cancer cell line and analyses of murine mutant EGFR-driven lung adenocarcinomas, we found that erlotinib resistance was associated with reduced expression of neurofibromin, the RAS GTPase-activating protein encoded by the NF1 gene. Erlotinib failed to fully inhibit RAS-ERK signaling when neurofibromin levels were reduced. Treatment of neurofibromin-deficient lung cancers with a MAP-ERK kinase (MEK) inhibitor restored sensitivity to erlotinib. Low levels of NF1 expression were associated with primary and acquired resistance of lung adenocarcinomas to EGFR TKIs in patients. These findings identify a subgroup of patients with EGFR-mutant lung adenocarcinoma who might benefit from combination therapy with EGFR and MEK inhibitors.
PURPOSE Therapies to target prostate cancer bone metastases have only limited effects. New treatments are focused on the interaction between cancer cells, bone marrow cells and the bone matrix. Osteoclasts play an important role in the development of bone tumors caused by prostate cancer. Since Src kinase has been shown to be necessary for osteoclast function, we hypothesized that dasatinib, a Src family kinase inhibitor, would reduce osteoclast activity and prostate cancer (PC-3) cell-induced osteoclast formation. RESULTS Dasatinib inhibited RANKL-induced osteoclast differentiation of bone marrow-derived monocytes with an EC(50) of 7.5 nM. PC-3 cells, a human prostate cancer cell line, were able to differentiate RAW 264.7 cells, a murine monocytic cell line, into osteoclasts, and dasatinib inhibited this differentiation. In addition, conditioned medium from PC-3 cell cultures was able to differentiate RAW 264.7 cells into osteoclasts and this too, was inhibited by dasatinib. Even the lowest concentration of dasatinib, 1.25 nmol, inhibited osteoclast differentiation by 29%. Moreover, dasatinib inhibited osteoclast activity by 58% as measured by collagen 1 release. EXPERIMENTAL DESIGN We performed in vitro experiments utilizing the Src family kinase inhibitor dasatinib to target osteoclast activation as a means of inhibiting prostate cancer bone metastases. CONCLUSION Dasatinib inhibits osteoclast differentiation of mouse primary bone marrow-derived monocytes and PC-3 cell-induced osteoclast differentiation. Dasatinib also inhibits osteoclast degradation activity. Inhibiting osteoclast differentiation and activity may be an effective targeted therapy in patients with prostate cancer bone metastases.
We examined the effects of an inhibitor of PI3K, XL147, against human breast cancer cell lines with constitutive PI3K activation. Treatment with XL147 resulted in dose-dependent inhibition of cell growth and levels of pAKT and pS6, signal transducers in the PI3K/AKT/TOR pathway. In HER2-overexpressing cells, inhibition of PI3K was followed by up-regulation of expression and phosphorylation of multiple receptor tyrosine kinases, including HER3. Knockdown of FoxO1 and FoxO3a transcription factors suppressed the induction of HER3, InsR, IGF1R, and FGFR2 mRNAs upon inhibition of PI3K. In HER2(+) cells, knockdown of HER3 with siRNA or cotreatment with the HER2 inhibitors trastuzumab or lapatinib enhanced XL147-induced cell death and inhibition of pAKT and pS6. Trastuzumab and lapatinib each synergized with XL147 for inhibition of pAKT and growth of established BT474 xenografts. These data suggest that PI3K antagonists will inhibit AKT and relieve suppression of receptor tyrosine kinase expression and their activity. Relief of this feedback limits the sustained inhibition of the PI3K/AKT pathway and attenuates the response to these agents. As a result, PI3K pathway inhibitors may have limited clinical activity overall if used as single agents. In patients with HER2-overexpressing breast cancer, PI3K inhibitors should be used in combination with HER2/HER3 antagonists.
S-trans,trans-Farnesylthiosalicylic acid (FTS) is a novel farnesylated rigid carboxylic acid derivative. In cell-free systems, it acts as a potent competitive inhibitor (Ki = 2.6 microM) of the enzyme prenylated protein methyltransferase (PPMTase), which methylates the carboxyl-terminal S-prenylcysteine in a large number of prenylated proteins including Ras. In such systems, FTS inhibits Ras methylation but not Ras farnesylation. Inhibition of the PPMTase by FTS in homogenates or membranes of a variety of tissues and cell lines is inferred from a block in the methylation of exogenously added substrates such as N-acetyl-S-trans,trans-farnesyl-L-cysteine and of endogenous substrates including small GTP-binding proteins. FTS can also inhibit methylation of these proteins in intact cells (e.g. in Rat-1 fibroblasts, Ras-transformed Rat-1, and B16 melanoma cells). Unlike in cell-free systems, however, relatively high concentrations of FTS (50-100 microM) are required for partial blocking (10-40%) of protein methylation in the intact cells. Thus, FTS is a weak inhibitor of methylation in intact cells. Because methylation is the last step in the processing of Ras and related proteins, FTS is not likely to affect steps that precede it, e.g. protein prenylation. This may explain why the growth and gross morphology of a variety of cultured cell types (including Chinese hamster ovary, NIH3T3, Rat1, B16 melanoma, and PC12) is not affected by up to 25 microM FTS and is consistent with the observed lack of FTS-induced cytotoxicity. Nevertheless, FTS reduces the levels of Ras in cell membranes and can inhibit Ras-dependent cell growth in vitro, independently of methylation. It inhibits the growth of human Ha-ras-transformed cells (EJ cells) and reverses their transformed morphology in a dose-dependent manner (0.1-10 microM). The drug does not interfere with the growth of cells transformed by v-Raf or T-antigen but inhibits the growth of ErbB2-transformed cells and blocks the mitogenic effects of epidermal and basic fibroblast growth factors, thus implying its selectivity toward Ras growth signaling, possibly via modulation of Ras-Raf communication. Taken together, the results raise the possibility that FTS may specifically interfere with the interaction of Ras with a farnesylcysteine recognition domain in the cell membrane.(ABSTRACT TRUNCATED AT 400 WORDS)
Subunits of the SWI/SNF chromatin remodeling complex are mutated in a significant proportion of human cancers. Malignant rhabdoid tumors (MRTs) are lethal pediatric cancers characterized by a deficiency in the SWI/SNF subunit SMARCB1. Here, we employ an integrated molecular profiling and chemical biology approach to demonstrate that the receptor tyrosine kinases (RTKs) PDGFRα and FGFR1 are coactivated in MRT cells and that dual blockade of these receptors has synergistic efficacy. Inhibitor combinations targeting both receptors and the dual inhibitor ponatinib suppress the AKT and ERK1/2 pathways leading to apoptosis. MRT cells that have acquired resistance to the PDGFRα inhibitor pazopanib are susceptible to FGFR inhibitors. We show that PDGFRα levels are regulated by SMARCB1 expression, and assessment of clinical specimens documents the expression of both PDGFRα and FGFR1 in rhabdoid tumor patients. Our findings support a therapeutic approach in cancers with SWI/SNF deficiencies by exploiting RTK coactivation dependencies.
Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop acute myelogenous leukemia (AML). The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is loss of chromosomal material (genomic instability). Using our two-step mouse model for myeloid leukemic disease progression involving overexpression of human mutant NRAS and BCL2 genes, we show that there is a stepwise increase in the frequency of DNA damage leading to an increased frequency of error-prone repair of double-strand breaks (DSB) by nonhomologous end-joining. There is a concomitant increase in reactive oxygen species (ROS) in these transgenic mice with disease progression. Importantly, RAC1, an essential component of the ROS-producing NADPH oxidase, is downstream of RAS, and we show that ROS production in NRAS/BCL2 mice is in part dependent on RAC1 activity. DNA damage and error-prone repair can be decreased or reversed in vivo by N-acetyl cysteine antioxidant treatment. Our data link gene abnormalities to constitutive DNA damage and increased DSB repair errors in vivo and provide a mechanism for an increase in the error rate of DNA repair with MDS disease progression. These data suggest treatment strategies that target RAS/RAC pathways and ROS production in human MDS/AML.
PURPOSE Peroxisome proliferator-activated receptors (PPAR) regulate lipid and glucose metabolism but their anticancer properties have been recently studied as well. We previously reported the antimetastatic activity of the PPARalpha ligand, fenofibrate, against melanoma tumors in vivo. Here we investigated possible molecular mechanisms of fenofibrate anti metastatic action. EXPERIMENTAL DESIGN Monolayer cultures of mouse (B16F10) and human (SkMell88) melanoma cell lines, soft agar assay, and cell migration assay were used in this study. In addition, we analyzed PPARalpha expression and its transcriptional activity in response to fenotibrate by using Western blots and liciferase-based reporter system. RESULTS Fenofibrate inhibited migration of B16F10 and SkMel188 cells in Transwell chambers and colony formation in soft agar. These effects were reversed by PPAR inhibitor, GW9662. Western blot analysis revealed time-dependent down-regulation of Akt and extracellular signal-regulated kinase l/2 phosphorylation in fenofibrate-treated cells. A B16F10 cell line stably expressing constitutively active Akt mutant was resistant to fenofibrate. In contrast, Akt gene silencing with siRNA mimicked the fenofibrate action and reduced the migratory ability of B16F1O cells. In addition, fenofibrate strongly sensitized BI6FIO cells to the proapoptotic drug staurosporine, further supporting the possibility that fenofibrate-induced down-regulation of Akt function contributes to fenofibrate-mediated inhibition of metastatic potential in this experimental model. CONCLUSIONS Our results show that the PPAR-dependent antimetastatic activity of fenofibrate involves down-regulation of Akt phosphorylation and suggest that supplementation with this drug may improve the effectiveness of melanoma chemotherapy.
Melatonin has been shown to have oncostatic effects on malignant melanoma in vitro and in vivo. We studied the growth suppressive effects of melatonin over a wide range of concentrations in four melanoma cell lines (SBCE2, WM-98, WM-164 and SKMEL-188) representative for different growth stages and phenotype. Melanoma cells were incubated with melatonin 10(-12)-10(-3) M, and proliferation and clonogenicity was assessed at 12 h and 14 days, respectively. We also determined the expression of cytosolic quinone oxidoreductases NQO1, NQO2 (known as MT3 receptor) and nuclear receptor RORalpha by RT-PCR. Melatonin at pharmacological concentrations (10(-3)-10(-7) M) suppressed proliferation in all melanoma cell lines. In SKMEL-188 cells cultured in serum-free media, melatonin at low concentrations (10(-12)-10(-10) M) also slightly attenuated the proliferation. The effects of pharmacological doses of melatonin were confirmed in the clonogenic assay. Expression of NQO1 was detected in all cell lines, whereas NQO2 and nuclear receptor RORalpha including its isoform RORalpha4 were present only in SBCE2, WM-164 and WM-98. Thus, melatonin differentially suppressed proliferation in melanoma cell lines of different behaviour. The intensity of the oncostatic response to melatonin could be related to the cell-line specific pattern of melatonin cellular receptors and cytosolic binding protein expression.
The target of rapamycin (TOR) is a conserved Ser/Thr kinase that regulates cell growth and metabolism in response to environmental cues. Here, highlighting contributions from studies in model organisms, we review mammalian TOR complexes and the signaling branches they mediate. TOR is part of two distinct multiprotein complexes, TOR complex 1 (TORC1), which is sensitive to rapamycin, and TORC2, which is not. The physiological consequences of mammalian TORC1 dysregulation suggest that inhibitors of mammalian TOR may be useful in the treatment of cancer, cardiovascular disease, autoimmunity, and metabolic disorders.
BACKGROUND/AIMS The small GTPase of Ras and Rho families are widely involved in human tumorgenesis and metastasis. It has recently been reported that pamidronate inhibits the mevalonate pathway, which is required for the prenylation of the small GTPase. We demonstrated a possible beneficial use of pamidronate in the treatment of hepatocellular carcinoma (HCC). METHODS The effect of pamidronate on cell proliferation was analyzed with five hepatoma cell lines using MTT assay. Apoptosis was evaluated by staining with DAPI and a histon ELISA assay. A cell migration assay was performed using the Modified Boyden Chamber. To analyze anti-proliferation effect of pamidronate in vivo, tumor volumes were monitored with the intraperitoneal injection of pamidronate after subcutaneous inoculation of PLC/PRF/5 cells into nude mice. RESULTS Pamidronate inhibited cell growth for all hepatoma cell lines. The amount of membrane associated Ras and phosphorylated extracellular signal-regulated kinase 2 (ERK 2) were reduced after pamidronate treatment. Pamidronate increased apoptosis and cleavage of Caspase-3, and -9. Pamidronate suppressed membrane associated RhoA and cell motility. In vivo, tumor volumes were significantly suppressed by pamidronate at three weeks (P<0.03). CONCLUSIONS We conclude that pamidronate has therapeutic potential in inducing anti-proliferative, apoptotic, and anti-migratory effects in HCC.
Abstract Phosphoinositide 3-kinases (PI3Ks) are central to the control of cell growth, proliferation and survival, and drive the progression of tumours by activating phosphoinositide-dependent kinase, protein kinase B and the target of rapamycin. Other downstream effectors link PI3K to cell motility and the control of cardiovascular parameters. Current knowledge indicates that PI3Ks might qualify as drug targets for the treatment of cancer, chronic inflammation, allergy and cardiovascular failure. However, PI3Ks also modulate vital processes such as metabolic control and nutrient uptake. Here, mechanistic data and mouse phenotypic analyses are summarised, and the possible success of therapeutic inhibition of distinct PI3K isoforms is discussed.
The mammalian CIP/KIP family of cyclin-dependent kinase (CDK) inhibitors (CKIs) comprises three proteins--p21(Cip1/WAF1), p27(Kip1), and p57(Kip2)--that bind and inhibit cyclin-CDK complexes, which are key regulators of the cell cycle. CIP/KIP CKIs have additional independent functions in regulating transcription, apoptosis and actin cytoskeletal dynamics. These divergent functions are performed in distinct cellular compartments and contribute to the seemingly contradictory observation that the CKIs can both suppress and promote cancer. Multiple ubiquitin ligases (E3s) direct the proteasome-mediated degradation of p21, p27 and p57. This review analyzes recent data highlighting our current understanding of how distinct E3 pathways regulate subpopulations of the CKIs to control their diverse functions.
The mechanisms by which melanoma and other cancer cells evade anti-tumor immunity remain incompletely understood. Here, we show that the growth of tumors formed by mutant Braf(V600E) mouse melanoma cells in an immunocompetent host requires their production of prostaglandin E2, which suppresses immunity and fuels tumor-promoting inflammation. Genetic ablation of cyclooxygenases (COX) or prostaglandin E synthases in Braf(V600E) mouse melanoma cells, as well as in Nras(G12D) melanoma or in breast or colorectal cancer cells, renders them susceptible to immune control and provokes a shift in the tumor inflammatory profile toward classic anti-cancer immune pathways. This mouse COX-dependent inflammatory signature is remarkably conserved in human cutaneous melanoma biopsies, arguing for COX activity as a driver of immune suppression across species. Pre-clinical data demonstrate that inhibition of COX synergizes with anti-PD-1 blockade in inducing eradication of tumors, implying that COX inhibitors could be useful adjuvants for immune-based therapies in cancer patients.
Cytokinesis is the final stage in cell division. Although integrins can regulate cytokinesis, the mechanisms involved are not fully understood. In this study, we demonstrate that integrin-regulated ERK (extracellular signal-related kinase) and RSK (p90 ribosomal S6 kinase) signaling promotes successful cytokinesis. Inhibiting the activation of ERK and RSK in CHO cells by a mutation in the integrin β1 cytoplasmic tail or with pharmacological inhibitors results in the accumulation of cells with midbodies and the formation of binucleated cells. Activation of ERK and RSK signaling by the expression of constitutively active RAF1 suppresses the mutant phenotype in a RSK-dependent manner. Constitutively active RSK2 also restores cytokinesis inhibited by the mutant integrin. Importantly, the regulatory role of the RSK pathway is not specific to CHO cells. MCF-10A human mammary epithelial cells and HPNE human pancreatic ductal epithelial cells exhibit a similar dependence on RSK for successful cytokinesis. In addition, depriving mitotic MCF10A cells of integrin-mediated adhesion by incubating them in suspension suppressed ERK and RSK activation and resulted in a failure of cytokinesis. Furthermore, inhibition of RSK or integrins within the 3D context of a developing salivary gland organ explant also leads to an accumulation of epithelial cells with midbodies, suggesting a similar defect in cytokinesis. Interestingly, neither ERK nor RSK regulates cytokinesis in human fibroblasts, suggesting cell-type specificity. Taken together, our results identify the integrin-RSK signaling axis as an important regulator of cytokinesis in epithelial cells. We propose that the proper interaction of cells with their microenvironment through integrins contributes to the maintenance of genomic stability by promoting the successful completion of cytokinesis.