Activation Of Ribosomal Protein S6 Research Articles

Upregulated DNMT3a coupling with inhibiting p62-dependent autophagy contributes to NNK tumorigenicity in human bronchial epithelial cells

NNK, formally known as 4-(methyl nitrosamine)-1-(3-pyridyl)-1-butanoe, is a potent chemical carcinogen prevalent in cigarette smoke and is a key contributor to the development of human lung adenocarcinomas. On the other hand, autophagy plays a complex role in cancer development, acting as a "double-edged sword" whose impact varies depending on the cancer type and stage. Despite this, the relationship between autophagy and NNK-induced lung carcinogenesis remains largely unexplored. Our current study uncovers a marked reduction in p62 protein expression in both lung adenocarcinomas and lung tissues of mice exposed to cigarette smoke. Interestingly, this reduction appears to be contingent upon the activity of extrahepatic cytochrome P450 (CYP450), revealing that NNK metabolic activation by CYP450 enzyme escalates its potential to induce p62 downregulation. Further mechanistic investigations reveal that NNK suppresses autophagy by accelerating the degradation of p62 mRNA, thereby promoting the malignant transformation of human bronchial epithelial cells. This degradation process is facilitated by the hypermethylation of the Human antigen R (HuR) promoter, resulting in the transcriptional repression of HuR - a key regulator responsible for stabilizing p62 mRNA through direct binding. This hypermethylation is triggered by the activation of ribosomal protein S6, which is influenced by NNK exposure and subsequently amplifies the translation of DNA methyltransferase 3 alpha (DNMT3a). These findings provide crucial insights into the nature of p62 in both the development and potential treatment of tobacco-related lung cancer.

Reducing Fatty Acid Oxidation Improves Cancer-free Survival in a Mouse Model of Li-Fraumeni Syndrome.

Germline mutations of TP53, which cause the cancer predisposition disorder Li-Fraumeni syndrome (LFS), can increase mitochondrial activity as well as fatty acid β-oxidation (FAO) in mice. Increased fatty acid metabolism can promote cancer malignancy, but its specific contribution to tumorigenesis in LFS remains unclear. To investigate this, we crossed LFS mice carrying the p53 R172H knock-in mutation (p53172H/H , homolog of the human TP53 R175H LFS mutation) with myoglobin-knockout (MB-/- ) mice known to have decreased FAO. MB-/- p53172H/H double-mutant mice also showed mildly reduced FAO in thymus, a common site of T lymphoma development in LFS mice, in association with an approximately 40% improvement in cancer-free survival time. RNA sequencing profiling revealed that the p53 R172H mutation promotes mitochondrial metabolism and ribosome biogenesis, both of which are suppressed by the disruption of MB. The activation of ribosomal protein S6, involved in protein translation and implicated in cancer promotion, was also inhibited in the absence of MB. To further confirm the role of FAO in lymphomagenesis, mitochondrial FAO enzyme, carnitine palmitoyltransferase 2 (CPT2), was specifically disrupted in T cells of p53172H/H mice using a Cre-loxP-mediated strategy. The heterozygous knockout of CPT2 resulted in thymus FAO haploinsufficiency and an approximately 30% improvement in survival time, paralleling the antiproliferative signaling observed with MB disruption. Thus, this study demonstrates that moderating FAO in LFS can suppress tumorigenesis and improve cancer-free survival with potential implications for cancer prevention. PREVENTION RELEVANCE: Mildly inhibiting the increased fatty acid oxidation observed in a mouse model of Li-Fraumeni syndrome, a cancer predisposition disorder caused by inherited mutations of TP53, dampens aberrant pro-tumorigenic cell signaling and improves the survival time of these mice, thereby revealing a potential strategy for cancer prevention in patients.

Abstract B074: Sapanisertib in combination with trametinib in glioblastoma models enhances target inhibition and antitumor activity

Abstract Objective: To determine the biological and signaling effects of dual-pathway inhibition with mTORC1/2 and MEK inhibitors in glioblastoma neurosphere cell lines. Background: The PI3K/mTOR signaling pathway is frequently dysregulated in glioblastoma (GBM), making it the focus of active scientific and drug development efforts in brain tumors. Unfortunately, small molecule targeted therapies against EGFR and its downstream effector pathways, while promising in the pre-clinical setting, have been disappointing in glioma clinical trials. The mTOR pathway target ribosomal protein S6 (RPS6) can also be activated by the ERK pathway target p90-RSK. We therefore tested the hypothesis that small molecule inhibition of both ERK and mTOR signaling pathways would result in enhanced anti-tumor activity. Methods: Seven glioblastoma neurosphere lines were tested for sensitivity to the ATP-competitive mTORC1/2 inhibitor sapanisertib (MLN0128) alone or in combination with the allosteric MEK1/2 inhibitor trametinib (GSK1120212) using cell growth assays and immunoblots as indicated. Results: The dual mTORC1/2 inhibitor sapanisertib inhibits mTOR signaling and cell growth in all GBM neurosphere lines, but only partially inhibits phosphorylation of the downstream target ribosomal protein S6 (phospho-RPS6) in these models. The MEK inhibitor, trametinib, inhibited ERK phosphorylation in all neurosphere lines, though phosphorylation of downstream targets and cell growth were inhibited to varying degrees. Inhibition of RPS6 activation was dose-dependent and correlated with cell line sensitivity when treated with trametinib but not with sapanisertib. In combination, sapanisertib plus trametinib showed more profound inhibition of phospho-RPS6, as well as enhanced growth inhibition and induction of apoptosis in sensitive neurosphere lines. Conclusions: The combination of sapanisertib and trametinib may have significant antitumor activity in glioblastoma neurospheres by enhanced inhibition of S6, which serves as the convergence point between these effector pathways. Citation Format: Karisa C Schreck, Amy Allen, Jiawan Wang, Christine A Pratilas. Sapanisertib in combination with trametinib in glioblastoma models enhances target inhibition and antitumor activity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B074. doi:10.1158/1535-7163.TARG-19-B074

Axl-mediated Akt phosphorylation leads to mTORC1 activation via NFk-B in renal mesangial cells

Abstract Proliferative glomerulonephritis (GN) is a critical condition in many forms of kidney diseases and characterized by the proliferation of mesangial cells with influx of inflammatory cells. Receptor tyrosine kinase Axl belongs to the TAM family, along with Mer and Tyro-3. Receptors from this family contribute to clearance of apoptotic cells and suppression of inflammation. Previously, we reported an important role of Axl-mediated mesangial proliferation in the anti-GBM induced mouse model of lupus nephritis. In this study, we investigated the signaling cascade induced by Axl activation in renal mesangial cells. We found that Axl activation led to the phosphorylation of mTORC1 complex, proceeded with the ribosomal protein S6 activation. Inhibition of NFκ-B activity diminished mTOR phosphorylation in mesangial cells, indicating that NFκ-B is a critical regulator for mTORC1 activation in the Axl/Akt pathway. Axl expression also participated in mesangial cell efferocytosis. Interestingly, mesangial efferocytosis is not mediated by Mer receptor tyrosine kinase, the canonical TAM family receptor facilitating apoptotic cell engulfment in macrophages. Taken together, data suggest that Axl mediated mTOR activation and apoptotic cell engulfment is renal mesangial cell specific, as those functions were not observed in similarly treated peritoneal macrophages.

Calpain-1 deletion impairs mGluR-dependent LTD and fear memory extinction

Recent studies indicate that calpain-1 is required for the induction of long-term potentiation (LTP) elicited by theta-burst stimulation in field CA1 of hippocampus. Here we determined the contribution of calpain-1 in another type of synaptic plasticity, the long-term depression (LTD) elicited by activation of type-I metabotropic glutamate receptors (mGluR-LTD). mGluR-LTD was associated with calpain-1 activation following T-type calcium channel opening, and resulted in the truncation of a regulatory subunit of PP2A, B56α. This signaling pathway was required for both the early and late phase of Arc translation during mGluR-LTD, through a mechanism involving mTOR and ribosomal protein S6 activation. In contrast, in hippocampal slices from calpain-1 knock-out (KO) mice, application of the mGluR agonist, DHPG, did not result in B56α truncation, increased Arc synthesis and reduced levels of membrane GluA1-containing AMPA receptors. Consistently, mGluR-LTD was impaired in calpain-1 KO mice, and the impairment could be rescued by phosphatase inhibitors, which also restored Arc translation in response to DHPG. Furthermore, calpain-1 KO mice exhibited impairment in fear memory extinction to tone presentation. These results indicate that calpain-1 plays a critical role in mGluR-LTD and is involved in many forms of synaptic plasticity and learning and memory.

IL-3 Maintains Activation of the p90S6K/RPS6 Pathway and Increases Translation in Human Eosinophils.

IL-5 is a major therapeutic target to reduce eosinophilia. However, all of the eosinophil-activating cytokines, such as IL-5, IL-3, and GM-CSF, are typically present in atopic diseases, including allergic asthma. As a result of the functional redundancy of these three cytokines on eosinophils and the loss of IL-5R on airway eosinophils, it is important to take IL-3 and GM-CSF into account to efficiently reduce tissue eosinophil functions. Moreover, these three cytokines signal through a common β-chain receptor but yet differentially affect protein production in eosinophils. Notably, the increased ability of IL-3 to induce the production of proteins, such as semaphorin-7A, without affecting mRNA levels suggests a unique influence of IL-3 on translation. The purpose of this study was to identify the mechanisms by which IL-3 distinctively affects eosinophil function compared with IL-5 and GM-CSF, with a focus on protein translation. Peripheral blood eosinophils were used to study intracellular signaling and protein translation in cells activated with IL-3, GM-CSF, or IL-5. We establish that, unlike GM-CSF or IL-5, IL-3 triggers prolonged signaling through activation of ribosomal protein S6 (RPS6) and the upstream kinase 90-kDa ribosomal S6 kinase (p90S6K). Blockade of p90S6K activation inhibited phosphorylation of RPS6 and IL-3-enhanced semaphorin-7A translation. Furthermore, in an allergen-challenged environment, in vivo phosphorylation of RPS6 and p90S6K was enhanced in human airway compared with circulating eosinophils. Our findings provide new insights into the mechanisms underlying differential activation of eosinophils by IL-3, GM-CSF, and IL-5. These observations identify IL-3 and its downstream intracellular signals as novel targets that should be considered to modulate eosinophil functions.

Prenylation inhibition-induced cell death in melanoma: reduced sensitivity in BRAF mutant/PTEN wild-type melanoma cells.

While targeted therapy brought a new era in the treatment of BRAF mutant melanoma, therapeutic options for non-BRAF mutant cases are still limited. In order to explore the antitumor activity of prenylation inhibition we investigated the response to zoledronic acid treatment in thirteen human melanoma cell lines with known BRAF, NRAS and PTEN mutational status. Effect of zoledronic acid on proliferation, clonogenic potential, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were investigated in vitro with SRB, TUNEL and PARP cleavage assays and videomicroscopy and immunoblot measurements, respectively. Subcutaneous and spleen-to-liver colonization xenograft mouse models were used to evaluate the influence of zoledronic acid treatment on primary and disseminated tumor growth of melanoma cells in vivo. Zoledronic acid more efficiently decreased short-term in vitro viability in NRAS mutant cells when compared to BRAF mutant and BRAF/NRAS wild-type cells. In line with this finding, following treatment decreased activation of ribosomal protein S6 was found in NRAS mutant cells. Zoledronic acid demonstrated no significant synergism in cell viability inhibition or apoptosis induction with cisplatin or DTIC treatment in vitro. Importantly, zoledronic acid could inhibit clonogenic growth in the majority of melanoma cell lines except in the three BRAF mutant but PTEN wild-type melanoma lines. A similar pattern was observed in apoptosis induction experiments. In vivo zoledronic acid did not inhibit the subcutaneous growth or spleen-to-liver colonization of melanoma cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. Nevertheless, we also demonstrated that therapeutic sensitivity might be influenced by the PTEN status of BRAF mutant melanoma cells. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target prenylation in melanoma cells.

Abstract B37: Prenylation inhibition induces cell death in NRAS mutant melanoma cells

Abstract While targeted therapeutic approaches are now available for the treatment of BRAF mutant melanomas, non-BRAF mutant melanoma still lacks this modality. In order to explore the potential of prenylation inhibition we investigated the response of NRAS or BRAF mutant and double wild-type melanoma to zoledronic acid treatment. Effect of zoledronic acid on proliferation, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were characterized in vitro. In vivo subcutaneous xenograft and spleen to liver colonization models were used to evaluate the influence of zoledronic acid treatment on primary tumor growth and metastatic potential of melanoma cells. Zoledronic acid significantly decreased proliferation in NRAS mutant cells as compared to BRAF mutant and double wild-type cells, in vitro. Similarly, TUNEL assay revealed apoptosis induction in an oncogenic mutation specific manner. In line with this finding, activation of ribosomal protein S6 was decreased. Unexpected activation of the downstream effector Erk1/2 and increase in migration was found in BRAF mutant cells. Zoledronic acid failed to show significant synergism with cytotoxic cisplatin or DTIC treatment in vitro. In vivo zoledronic acid did not inhibit the subcutaneous growth of melanoma cells irrespective of oncogenic mutations. In contrast, the lower dose of zoledronic acid resulted in significantly higher liver metastasis in double wild-type cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target melanoma cells. Citation Format: Tamas Garay, Istvan Kenessey, Viktoria Laszlo, Jozsef Tovari, Walter Berger, Laszlo Kovacs, Jozsef Timar, Balazs Hegedus. Prenylation inhibition induces cell death in NRAS mutant melanoma cells. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B37. doi: 10.1158/1557-3125.RASONC14-B37

Resistance to Selumetinib (AZD6244) in Colorectal Cancer Cell Lines is Mediated by p70S6K and RPS6 Activation

Selumetinib (AZD6244, ARRY-142886) is a MEK1/2 inhibitor that has gained interest as an anti-tumour agent. We have determined the degree of sensitivity/resistance to Selumetinib in a panel of colorectal cancer cell lines using cell proliferation and soft agar assays. Sensitive cell lines underwent G1 arrest, whereas Selumetinib had no effect on the cell cycle of resistant cells. Some of the resistant cell lines showed high levels of ERK1/2 phosphorylation in the absence of serum. Selumetinib inhibited phosphorylation of ERK1/2 and RSK and had no effect on AKT phosphorylation in both sensitive and resistant cells. Furthermore, mutations in KRAS, BRAF, or PIK3CA were not clearly associated with Selumetinib resistance. Surprisingly, Selumetinib was able to inhibit phosphorylation of p70 S6 kinase (p70S6K) and its downstream target ribosomal protein S6 (RPS6) in sensitive cell lines. However, p70S6K and RPS6 phosphorylation remained unaffected or even increased in resistant cells. Moreover, in some of the resistant cell lines p70S6K and RPS6 were phosphorylated in the absence of serum. Interestingly, colorectal primary cultures derived from tumours excised to patients exhibited the same behaviour than established cell lines. Pharmacological inhibition of p70S6K using the PI3K/mTOR inhibitor NVP-BEZ235, the specific mTOR inhibitor Rapamycin and the specific p70S6K inhibitor PF-4708671 potentiated Selumetinib effects in resistant cells. In addition, biological inhibition of p70S6K using siRNA rendered responsiveness to Selumetinib in resistant cell lines. Furthermore, combination of p70S6K silencing and PF-47086714 was even more effective. We can conclude that p70S6K and its downstream target RPS6 are potential biomarkers of resistance to Selumetinib in colorectal cancer.

Synthetic Lethality Screen Identifies RPS6KA2 as Modifier of Epidermal Growth Factor Receptor Activity in Pancreatic Cancer

Pancreatic cancer is characterized by a high degree of resistance to chemotherapy. Epidermal growth factor receptor (EGFR) inhibition using the small-molecule inhibitor erlotinib was shown to provide a small survival benefit in a subgroup of patients. To identify kinases whose inhibition acts synergistically with erlotinib, we employed a kinome-wide small-interfering RNA (siRNA)-based loss-of-function screen in the presence of erlotinib. Of 779 tested kinases, we identified several targets whose inhibition acted synergistically lethal with EGFR inhibition by erlotinib, among them the S6 kinase ribosomal protein S6 kinase 2 (RPS6KA2)/ribosomal S6 kinase 3. Activated RPS6KA2 was expressed in approximately 40% of 123 human pancreatic cancer tissues. RPS6KA2 was shown to act downstream of EGFR/RAS/mitogen-activated protein kinase kinase (MEK)/extracellular-signal regulated kinase (ERK) signaling and was activated by EGF independently of the presence of KRAS mutations. Knockdown of RPS6KA2 by siRNA led to increased apoptosis only in the presence of erlotinib, whereas RPS6KA2 activation or overexpression rescued from erlotinib- and gemcitabine-induced apoptosis. This effect was at least in part mediated by downstream activation of ribosomal protein S6. Genetic as well as pharmacological inhibition of RPS6KA2 by the inhibitor BI-D1870 acted synergistically with erlotinib. By applying this synergistic lethality screen using a kinome-wide RNA interference-library approach, we identified RPS6KA2 as potential drug target whose inhibition synergistically enhanced the effect of erlotinib on tumor cell survival. This kinase therefore represents a promising drug candidate suitable for the development of novel inhibitors for pancreatic cancer therapy.

Ribosomal Protein S6 Phosphorylation is Associated with Epithelial Dysplasia and Squamous Cell Carcinoma of the Oral Cavity

Ribosomal protein S6 (RPS6), a downstream effector of the mammalian target of rapamycin pathway (mTOR), is activated in many cancers including oral squamous cell carcinoma (OSCC). However, the role of RPS6 in the progression of potentially malignant disorders (or premalignant lesions) to OSCC is unknown. The purpose of this study was to examine the expression of RPS6 in epithelial dysplasia and OSCC to determine the association of RPS6 in tumor progression. In our study, an immunohistochemical analysis of RPS6 was performed on tissue microarrays containing 30 control samples, 15 epithelial dysplasia cases, and 53 OSCC cases. Correlations between the clinicopathologic features of OSCC and RPS6 expression were analyzed using the Chi-square test. We found RPS6 phosphorylation (p-RPS6) in 15/30 (50%) control normal oral mucosa samples, 15/15 (100%) epithelial dysplasia cases, and 47/53 (88.68%) OSCC cases. The frequency of p-RPS6 in epithelial dysplasia or OSCC showed a statistically significant difference compared to control (P < 0.001). However, there were no significant correlations between p-RPS6 and the clinicopathologic features of OSCC. Our findings suggest that RPS6 activation is associated with the early events of tumor progression, suggesting p-RPS6 as a potential marker for early detection of oral cancer.

Synthetic Lethality Screens Reveal RPS6 and MST1R as Modifiers of Insulin-like Growth Factor-1 Receptor Inhibitor Activity in Childhood Sarcomas

The insulin-like growth factor-1 receptor (IGF1R) is emerging as a promising therapeutic target in human cancers. In the high-risk childhood sarcomas Ewing family tumor and rhabdomyosarcoma, IGF1R-blocking antibodies show impressive antitumor activity in some but not all patients, and acquired resistance is observed. Because tumor IGF1R mutations are not described, the basis of IGF1R inhibitor resistance remains unknown. We hypothesized that compensatory signaling cascades bypassing targeted IGF1R inhibition might be involved. To test this systematically, we performed small interfering RNA (siRNA) screens in sarcoma cell lines to identify IGF1R pathway components or related protein tyrosine kinase (PTK) networks that modulate the antitumor efficacy of the BMS-536924 IGF1R kinase inhibitor. This strategy revealed (a) that sarcoma cells are exquisitely sensitive to loss of distal rather than proximal IGF1R signaling components, such as ribosomal protein S6 (RPS6); (b) that BMS-536924 fails to block RPS6 activation in resistant sarcoma cell lines; and (c) that siRNA knockdown of the macrophage-stimulating 1 receptor tyrosine kinase (MST1R; also known as RON) restores BMS-536924 efficacy, even in highly drug-resistant cell lines. We confirmed MST1R expression across a broad panel of childhood sarcomas, and found that loss of MST1R by RNA interference blocks downstream RPS6 activation when combined with BMS-536924 in vitro. These findings underscore the importance of fully understanding PTK networks for successful clinical implementation of kinase inhibitor strategies.

Abstract 5084: Treadmill exercise modulates AMP-activated protein kinase and downstream signaling in mammary gland of p53+/−, but not p53+/+ mice

Abstract Epidemiological data show that exercise is generally protective against breast cancer. Inhibition of mammalian target of rapamyocin (mTOR) through activation of AMP-activated protein kinase (AMPK) has been shown to be responsive to exercise. Decreased activation of ribosomal protein S6, a marker for mTOR inhibition, is associated with decreased growth and proliferation. Recently, this energy-sensing pathway has been shown to be inhibited by increased production of reactive oxygen species (ROS) leading to AMPK activation. ROS levels are maintained by p53-inducible anti-oxidant enzymes. Therefore, we hypothesized that energy sensing pathways that mediate the protective effects of exercise in breast cancer are modulated by p53 gene dosage. To test this hypothesis, we fed ovariectomized mice a high-fat diet (60% kcal) ad libitum for 10 weeks to model post-menopausal obesity. Mice were then randomized to a sedentary or exercise regimen (n=15/group). Exercised mice ran on a variable speed treadmill (5% incline, 20 m/min, 40 min/day, 5 days/week for 22 weeks). Changes in percent body fat were measured every 2 weeks using quantitative magnetic resonance. Immunoblot analysis was performed to measure phosphorylation of AMPK (Thr 172) and ribosomal protein S6 (Ser 240/244) relative to total levels in the mammary fat pad (MFP). Also, qRT-PCR was performed to measure mRNA expression of the p53-inducible anti-oxidant enzyme sestrin 1. At the end of the diet-induced obesity regimen, there was no effect of p53 genotype on percent body fat (54.4 ± 0.7 in p53+/+ versus 55.9 ± 1.1 in p53+/−). After 22 weeks of exercise, the EX mice displayed signficantly less percent body fat than sedentary controls, with no effect of genotype (44.4 ± 0.6 versus 47.8 ± 0.8 % respectively, p&amp;lt;0.05). Immunoblot analysis revealed that in p53+/− mice, but not p53+/+ mice, exercise increased AMPK phosphorylation. This increased AMPK phosphorylation was associated with decreased phosphorylation of ribosomal protein S6, a marker of mTOR pathway activation (n=3/group). The observed increase in AMPK phosphorylation in p53+/− exercised mice relative to sedentary p53+/− was associated with decreased mRNA levels of sestrin 1 (n=4-6/group), indicating that the p53+/− mice may have higher levels of ROS in the MFP. In conclusion, exercise may increase AMPK activation in MFP of p53+/− mice relative to sedentary p53+/− mice through an ROS-mediated mechanism. Current analysis is focused on understanding how exercise affects AMPK activation in the mammary fat pad of MMTV-Wnt-1 transgenic mice heterozygous for p53, and how this impacts mammary tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5084.

Abstract 3422: Synthetic lethal siRNA screening reveals novel modifiers of insulin-like growth factor-1 receptor (IGF1R) inhibitor activity in childhood sarcomas

Abstract The insulin-like growth factor-1 receptor (IGF1R) is emerging as a therapeutic target in many cancers including Ewing family tumors and other childhood sarcomas, where IGF1R blocking antibodies show promising anti-tumor activity in some but not all patients. A key question remains as to what underlies IGF1R inhibitor sensitivity versus resistance. IGF1R mutations have to date not been found in these tumors. Instead, we hypothesize that compensatory signaling circuits by-passing targeted IGF1R inhibition are involved. We performed synthetic lethal siRNA screens in Ewing family tumor cell lines to study the IGF1R signaling axis and related protein tyrosine kinase networks in the presence and absence of sub-lethal doses of an IGF1R kinase inhibitor, with the goal of identifying siRNAs that restored full inhibitor activity. This strategy revealed (1) that Ewing sarcoma and rhabdomyosarcoma cells are exquisitely sensitive to loss of distal IGF1R signaling components such as the ribosomal protein S6 (RPS6); (2) IGF1R inhibitors fail to block RPS6 activation in resistant cell lines; and (3) that siRNA knock-down of certain protein tyrosine kinases such as the MET family receptor MST1R restores inhibitor efficacy, even in highly drug resistant cell lines. Moreover, we confirmed MST1R expression in childhood sarcomas and found that loss of MST1R blocks downstream RPS6 activation when combined with IGF1R inhibition in vitro. Taken together, we present an approach in principle applicable to any promising drug of interest. We demonstrate that key signaling effectors of the IGF1R axis in childhood sarcomas represent distal pathway proteins such as RPS6, and we identify the MST1R receptor tyrosine kinase as a potential therapeutic target whose knock-down markedly enhances sensitivity to IGF1R inhibition in high-risk sarcoma cell types, including resistant lines. These data underscore the importance of fully understanding protein tyrosine kinase networks for successful implementation of kinase inhibitor strategies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3422.

Reduced Intestinal Absorption of Dipeptides via PepT1 in Mice with Diet-induced Obesity Is Associated with Leptin Receptor Down-regulation

Leptin is a major determinant of energy homeostasis, acting both centrally and in the gastrointestinal tract. We previously reported that acute leptin treatment enhances the absorption of di- and tripeptides via the proton-dependent PepT1 transporter. In this study, we investigated the long term effect of leptin on PepT1 levels and activity in Caco2 cell monolayers in vitro. We then assessed the significance of the regulation of PepT1 in vivo in a model of diet-induced obesity. We demonstrated that 1) leptin regulated PepT1 at the transcriptional level, via the MAPK pathway, and at the translational level, via ribosomal protein S6 activation, in Caco2 cells and 2) this activation was systematically followed by a time- and concentration-dependent loss of leptin action reflecting desensitization. Deciphering this desensitization, we demonstrated that leptin induced a down-regulation of its own receptor protein and mRNA expression. More importantly, we showed, in mice with diet-induced obesity, that a 4-week hypercaloric diet resulted in a 46% decrease in PepT1-specific transport, because of a 30% decrease in PepT1 protein and a 50% decrease in PepT1 mRNA levels. As shown in Caco2 cells, these changes in PepT1 were supported by a parallel 2-fold decrease in leptin receptor expression in mice. Taken together, these results indicate that during induction of obesity, leptin resistance may also occur peripherally in the gastrointestinal tract, disrupting the absorption of oligopeptides and peptidomimetic drugs.

The Effects of Adiponectin and Metformin on Prostate and Colon Neoplasia Involve Activation of AMP-Activated Protein Kinase

Population studies provide evidence that obesity and insulin resistance are associated not only with elevated serum insulin levels and reduced serum adiponectin levels but also with increased risk of aggressive prostate and colon cancer. We show here that adiponectin activates AMP-activated protein kinase (AMPK) in colon (HT-29) and prostate (PC-3) cancer cells. These results are consistent with prior observations in myocytes, but we show that in epithelial cancer cells AMPK activation is associated with reduction in mammalian target of rapamycin activation as estimated by Ser(2448) phosphorylation, with reduction in p70S6 kinase activation as estimated by Thr(389) phosphorylation, with ribosomal protein S6 activation as estimated by Ser(235/236) phosphorylation, with reduction in protein translation as estimated by [(35)S]methionine incorporation, and with growth inhibition. Adiponectin-induced growth inhibition is significantly attenuated when AMPK level is reduced using small interfering RNA, indicating that AMPK is involved in mediating the antiproliferative action of this adipokine. Thus, adiponectin has the characteristics of a AMPK-dependent growth inhibitor that is deficient in obesity, and this may contribute to the adverse effects of obesity on neoplastic disease. Furthermore, metformin was observed to activate AMPK and to have growth inhibitory actions on prostate and colon cancer cells, suggesting that this compound may be of particular value in attenuating the adverse effects of obesity on neoplasia.

Inducible Nitric Oxide Synthase Deficiency Protects the Heart From Systolic Overload–Induced Ventricular Hypertrophy and Congestive Heart Failure

Inducible nitric oxide synthase (iNOS) protein is expressed in cardiac myocytes of patients and experimental animals with congestive heart failure (CHF). Here we show that iNOS expression plays a role in pressure overload-induced myocardial chamber dilation and hypertrophy. In wild-type mice, chronic transverse aortic constriction (TAC) resulted in myocardial iNOS expression, cardiac hypertrophy, ventricular dilation and dysfunction, and fibrosis, whereas iNOS-deficient mice displayed much less hypertrophy, dilation, fibrosis, and dysfunction. Consistent with these findings, TAC resulted in marked increases of myocardial atrial natriuretic peptide 4-hydroxy-2-nonenal (a marker of lipid peroxidation) and nitrotyrosine (a marker for peroxynitrite) in wild-type mice but not in iNOS-deficient mice. In response to TAC, myocardial endothelial NO synthase and iNOS was expressed as both monomer and dimer in wild-type mice, and this was associated with increased reactive oxygen species production, suggesting that iNOS monomer was a source for the increased oxidative stress. Moreover, systolic overload-induced Akt, mammalian target of rapamycin, and ribosomal protein S6 activation was significantly attenuated in iNOS-deficient mice. Furthermore, selective iNOS inhibition with 1400W (6 mg/kg per hour) significantly attenuated TAC induced myocardial hypertrophy and pulmonary congestion. These data implicate iNOS in the maladaptative response to systolic overload and suggest that selective iNOS inhibition or attenuation of iNOS monomer content might be effective for treatment of systolic overload-induced cardiac dysfunction.