Mitogenic Pathways Research Articles

Abstract 4951: Expression analysis of the LOT1 gene in head and neck cancer

Abstract Head and neck cancer (HNC) which is the sixth most common type of cancer worlwide, comprise a diverse group of tumors including neoplasias of the paranasal sinuses, the oral cavity, the trachea, the pharynx and the larynx. The most well known environmental risk factors for developing HNC are tobacca, alcohol and betel nut use and sexually transmitted viral pathogens such as human papilloma viruses (HPV). In addition to environmental factors, genetic factors may play a key role in the development of HNC. Therofore, genetic abnormalities have been studied extensively. The LOT1 (Lost-on-transformation 1) gene is localized on human chromosome 6q24-25, a chromosomal region that is frequently deleted in many types of human cancer including squamous cell carcinomas of the head and neck. The product of the LOT1 gene is a 463 amino acid protein which contains 7 C2H2-type zinc-finger motifs at the amino terminal, as well as proline and glutamine rich areas at the carboxyl terminal. Functional analysis of LOT1 demonstrated that it may play a significant role as a transcription factor modulating growth suppression through mitogenic signaling pathways. To elucidate the role of the LOT1 gene in tumorigenesis, many studies analyzing alterations of the gene expression were performed. As a result of these studies, the absence or reduced expression of LOT1 gene was reported in many types of tumors including breast, ovarian, pituitary and lung tumors. In this study, we analyzed the role of the LOT1 gene in head and neck carcinogenesis. For this purpose, we investigated the expression level of LOT1 gene in tumor tissue and adjacent non-cancerous tissue samples from 81 patients with HNC by quantitative RT-PCR. We found that LOT1 gene expression was decreased in 64 of 81 tumor tissues (79%) when compared to normal tissue. Statistically, downregulation of the LOT1 gene mRNA expression was significant (p<0.001). No significant correlation was found between LOT1 gene expression and clinicopathological parameters including age, sex, stage, histological grade and tumor location (p>0.05). Our data indicate that loss of the LOT1 gene function may play a role in the etiology of head and neck carcinogenesis. Citation Format: SEDA EKIZOGLU ERATAK, Emin Karaman, NUR A. BUYRU. Expression analysis of the LOT1 gene in head and neck cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4951. doi:10.1158/1538-7445.AM2015-4951

Abstract 704: Scaffold protein KSR1 is negatively regulated by merlin and promotes tumor development in merlin deficient tumors

Abstract Accumulated evidence suggests that the Ras/Raf/MEK/ERK pathway plays a crucial role in the development of Merlin-deficient tumours. However, the mechanism in which Merlin precisely suppresses this mitogenic signalling pathway has remained poorly understood. In addition, drug specificity, side effects and drug resistance are serious problems when treating patients with MEK inhibitors and therefore a more specific therapeutic target needs to be identified. Using our primary human in vitro model and three complementary approaches including lentiviral shRNA, active mutants and use of a clinically approved inhibitor, we report that Ras/Raf/MEK/ERK scaffold protein Kinase Suppressor of Ras 1 (KSR1) plays a vital role in promoting schwannoma proliferation, multipolar morphology and other pathological phenotypes. Proteomic analysis suggests that merlin shares multiple binding partners with KSR1 incl DCAF1 from the E3 Ubiquitin Ligase CRL4DCAF1, which is directly associated with and functionally inhibited by the tumour suppressor Merlin in the nucleus. Functional study suggests that KSR1 and DCAF1 might co-operate and/or complement to regulate schwannoma's proliferation. These findings demonstrate that Merlin disrupts Ras/Raf/MEK/ERK activity at the scaffold protein level and also suggest that KSR1 is a specific therapeutic target for Merlin-deficient tumours. Citation Format: Clemens O. Hanemann, Lu Zhou, Sylwia Ammoun, Edwin Lasonder, Vikram Sharma, Juergen Muller, Emanuela Ercolano. Scaffold protein KSR1 is negatively regulated by merlin and promotes tumor development in merlin deficient tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 704. doi:10.1158/1538-7445.AM2015-704

Abstract 686: Targeting glioblastoma and its stem cells using novel small molecule inhibitors of the mTOR pathway

Abstract Mammalian target of rapamycin (mTOR), which functions in two distinct multiprotein complexes, termed mTORC1 and mTORC2, is a downstream signaling kinase in the PI3K/AKT pathway. mTOR is often deregulated in glioblastoma (GBM), the most aggressive primary brain tumor in adults, due to the frequent loss of tumor suppressor, PTEN. mTORC1 and mTORC2 control cell growth, proliferation, migration, survival, and stem cell regulation in response to nutrients and growth factors. The presence of stem cells within the tumor mass and the surrounding areas precludes the possibility for elimination of GBM. Furthermore, the substrate of activated AKT, the proline-rich AKT substrate of 40 kDa (PRAS40), is a surrogate marker of activated AKT in response to mTOR inhibition therapy in GBM patients. PRAS40 regulates mTORC1 and has recently been considered as a target for cancer therapy. Rapamycin and its analogs failed in clinical trials for GBM patients due to their incomplete inhibition of mTORC1 as well as the activation of mitogenic pathways via negative feedback loops. The aim of this study is to provide evidence that mTORC1 and mTORC2 inhibitors [Torin 1 (tricyclic benzonaphthyridinone inhibitor), Torin 2, and XL 388 ([7-(6-Amino-3-pyridinyl)-2,3-dihydro-1,4-benzoxazepin-4(5H)-yl][3-fluoro-2-methyl-4-(methylsulfonyl)phenyl]-methanone)] effectively target mTORC1/2, suppressing cell growth, dissemination, and altering self-renewal properties of GBM stem cells. Results demonstrated that: 1) A significant number of tumors showed an increased expression of pAKTSer473 and pmTORSer2448 using immunohistochemistry, suggesting an overactivation of mTOR pathway; 2) Co-expression of mTOR and stem cell marker, nestin was evident in GBM tumors as shown by immunofluorescence analysis, implying its role in stem cell regulation; 3) Torin 2, but not Torin 1 or XL 388, was able to suppress mTORC1 activity completely as shown by the reduced expression of its downstream substrate pS6KSer235/236 in a dose-dependent manner; 4) Torin 1, Torin 2, and XL 388 inhibited the phosphorylation of 4E-BP1 and PRAS40; 5) Torin 1, Torin 2, and XL 388 suppressed the mTORC2 activity as reported by complete dephosphorylation of pAKTSer473; 6) Functional analysis revealed that Torin 1, Torin 2, and XL 388 effectively suppressed proliferation as shown by MTT analysis and S-phase entry as evidenced by EdU incorporation; 7) Both Torin 1 and Torin 2 inhibited GBM cell migration, however XL 388 was ineffective; 8) Torin 1 and Torin 2 suppressed the self-renewal of GBM stem cells and inhibited their proliferation, XL 388 remained ineffective. These results suggested that Torin 1 and Torin 2, but not XL 388 are useful in suppressing mTORC1 and mTORC2, thereby inhibiting GBM cell proliferation and dissemination. Moreover, Torin 1 and Torin 2 were effective in inhibiting stem cell self-renewal, underscoring their potential use for the treatment of GBM. Citation Format: John L. Gillick, Zachary E. Thwing, Sudeepta Sridhara, Raj Murali, Meena Jhanwar-Uniyal. Targeting glioblastoma and its stem cells using novel small molecule inhibitors of the mTOR pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 686. doi:10.1158/1538-7445.AM2015-686

Abstract A39: PI3K inhibition in preclinical models of HNSCC

Abstract Head and neck squamous cell carcinoma (HNSCC) is characterized by alteration of the PI3K pathway; especially mutation and/or amplification of the oncogene PIK3CA, and these alterations are enriched in HNSCC associated with human papilloma virus infection (HPV(+)HNSCC). We sought to test the hypothesis that HNSCC cells/tumors with mutant or amplified PIK3CA are more sensitive to anti-tumor effects of PI3K pathway inhibitors, including BKM-120 and BYL-719, and to assess the effect of HPV status on the efficacy of these agents. Proliferation assays and immunobloting were employed to examine the effects on growth and signaling of BKM-120 and BYL-719 administration to HNSCC cell lines with endogenous PIK3CA mutation, gene amplification, or WT, unamplified PIK3CA. The cytotoxic effects of BKM-120 and BYL-719 were comparable in a panel of 8 HNSCC cell lines, with IC50s in the micromolar range. BYL-719 demonstrated greater cytotoxic activity in cell lines with endogenous PIK3CA alterations compared with cells harboring endogenous WT, unamplified PIK3CA. Treatment with BKM-120 decreased the levels of phosphorylated AKT in a dose-dependent fashion to a greater degree in PIK3CA altered cells than in PIK3CA WT cells. In contrast, BYL-719 treatment inhibited phosphorylation of AKT in both PIK3CA altered and PIK3CA WT HNSCC cells, with comparable efficacy. Phosphorylation of S6 and MTOR however, was inhibited by BYL-719 to a greater degree in PIK3CA altered cell lines vs. PIK3CA WT cell lines. More than half of all HPV(+)HNSCC harbor some form of PI3K alteration; generally PIK3CA mutation or amplification. However, there is a paucity of HPV(+)HNSCC cell lines, and no cell lines to date derived from HPV(+)HNSCC tumors contain PIK3CA mutations. Although we included two HPV(+)HNSCC cell lines in our studies, neither are known to harbor endogenous PIK3CA mutations, and they did not appear to be more sensitive to PI3K pathway targeting with BKM-120 or BYL-719. Our in vivo treatment experiments, utilizing patient derived xenografts (PDX) of varying HPV and PIK3CA status, demonstrate that the growth of HPV(+)HNSCC tumors can be inhibited by BYL-719 treatment to a greater degree than the growth of HPV(-)HNSCC tumors. These cumulative findings suggest that PI3K inhibition may represent an effective therapeutic strategy in the subset of HNSCC patients whose tumors harbor PI3K alteration. As the PI3K pathway is the most commonly altered mitogenic pathway in HNSCC; the completion of this work, and its extension into clinical trials, will likely have translational significance. Citation Format: Matthew Louis Hedberg, Hua Li, Yan Zeng, Jennifer Rubin Grandis. PI3K inhibition in preclinical models of HNSCC. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A39.

Comprehensive assessment of expression of insulin signaling pathway components in subcutaneous adipose tissue of women with and without polycystic ovary syndrome

ObjectiveInsulin resistance is a common feature of polycystic ovary syndrome (PCOS). The insulin signaling pathway consists of two major pathways, the metabolic and the mitogenic cascades. The many components of these pathways have not been comprehensively analyzed for differential expression in insulin-responsive tissues in PCOS. The goal of this study was to determine whether the core elements of the insulin signal transduction cascade were differentially expressed in subcutaneous adipose tissue (SAT) between PCOS and controls. Materials/methodsQuantitative real-time PCR for 36 insulin signaling pathway genes was performed in subcutaneous adipose tissue from 22 white PCOS and 13 healthy controls. ResultsGenes in the insulin signaling pathway were not differentially expressed in subcutaneous adipose tissue between PCOS and controls (P > 0.05 for all). Components mainly of the mitogenic pathway were correlated with both androgens and metabolic phenotypes. Expression levels of five genes (MKNK1, HRAS, NRAS, KRAS, and GSK3A) were positively correlated with total testosterone level (ρ > 0, P < 0.05). Inverse correlation was found between expression of six genes (HRAS, MAP2K2, NRAS, MAPK3, GRB2, and SHC1) and metabolic traits (body mass index, fasting glucose, fasting insulin, and HOMA-IR) (ρ < 0, P < 0.05). ConclusionsDifferential expression of core insulin signaling pathway components in subcutaneous adipose tissue is not a major contributor to the pathogenesis of PCOS. Correlation between clinical phenotypes and expression of several genes in the mitogenic limb of the insulin signaling pathway suggests mitogenic signaling by insulin may regulate steroidogenesis and glucose homeostasis.

Ligularia fischeri inhibits endothelial cell proliferation, invasion and tube formation through the inactivation of mitogenic signaling pathways and regulation of vascular endothelial cadherin distribution and matrix metalloproteinase expression.

Ligularia fischeri (LF) has been used as an edible herb and traditional medicine for the treatment of inflammatory and infectious diseases. In the present study, we report the effects and molecular mechanism of the ethanolic extract of LF on cell proliferation, invasion and tube formation in human umbilical vein endothelial cells (HUVECs). LF-mediated inhibition of cell proliferation was accompanied by reduced expression of cell cycle-related proteins such as cyclin-dependent kinases (Cdks) and cyclins, leading to pRb hypophosphorylation and G1 phase cell cycle arrest. We also show that LF treatment inhibited cell invasion and tube formation in HUVECs. These anti-angiogenic activities of LF were associated with the inactivation of mitogenic signaling pathways, induction of vascular endothelial (VE)-cadherin distribution at cell-cell contacts and inhibition of matrix metalloproteinase (MMP) expression. Collectively, our findings demonstrate the pharmacological functions and molecular mechanisms of LF in regulating endothelial cell fates, and support further development as a potential therapeutic agent for the treatment and prevention of angiogenesis-related disorders including cancer.

Inflammation in Colorectal Cancer Inflammatory Bowel Disease, and Diabetes Mellitus: The Link

IntroductionVarious chronic diseases like Inflammatory bowel disease (IBD), colorectal cancer (CRC) and Type 2 diabetes mellitus (T2DM) are triggered by infection and or unregulated chronic inflammation. Where is the link?InflammationInflammation is basically a beneficial complex response which may involve a panel of bioactive pro‐and anti‐inflammatory molecules. However, a persistent or an inadequately resolved chronic inflammation may increase the risk of several pathologies such as IBD, CRC and T2DM, through toxic factors and bioactive molecules promoting epithelial‐mesenchymal transition (EMT) and facilitating carcinogenesis through the activation of JAK/STAT signaling pathway (IL‐6, TNF‐α), the differential phosphorylation of SMAD's which induce EMT (TGFB's), or mechanisms involving reduced oxygen species (ROS) and reactive nitrogen species (RNS).CRC and IBD: It is widely accepted that IBD patients have a higher risk of CRC. The IBD's are multifactorial pathologies involving alterations in the microbiota which can induce chronic inflammation leading to a potential role in carcinogenesis through mediators relevant in EMT induction like TGFB, TNF‐α , IL‐6, ROS and others.T2DM: Multiple inflammatory mechanisms provoke the pathogenesis of T2DM through mediators like (TNF‐α, IL‐6, IL‐8, and TGFB, which may alter the normal structure of the β‐cells by inducing pancreatic islet's apoptosis, thus the leading to the release of insulin‐like growth factor ‐1 (IGF‐1) which stimulates EMT, and carcinogenesis. In T2DM, Hyperglycemia contributes to the onset of inflammation, the increase of ROS, and triggering of inflammatory cytokines while hyperinsulinemia activates IGF‐1 over production which induces EMT and activates two mitogenic pathways that favor cancer growth: P13K/AKK and β‐catenin pathwaysConclusionIBD, CRC and T2DM are commonly interrelated clinical problems. They share a common basis influenced by disease‐related inflammation enhanced by changes in the endocrine milieu.

A Potential Role of H. Pylori PPIase in the Transformation of Gastric Epithelial Cell and the Development of Gastric Cancer

Helicobacter pylori (H. pylori) is the causative agent of chronic gastritis and gastric cancer, the second leading cause of cancer mortality worldwide. H. pylori colonizes the stomach in more than half of the world's population and has been classified as a class I carcinogen. H. pylori produces several virulence factors, including CagA, VacA and urease. Because the clinical outcomes of H. pylori infection do not necessarily correlate with the presence of these proteins, there is a need to identify new H. pylori factors that may modulate the clinical course of H. pylori‐associated diseases. Recently, we identified several gastric cancer‐associated H. pylori genes, including FKBP‐type peptidyl‐prolyl cis‐trans isomerase (PPIase‐FKBP). In this study, we show that co‐culture of gastric epithelial cells (AGS) with PPIase‐FKBP deletion H. pylori strain prevented H. pylori‐induced proliferation of AGS cells. Consistent with this observation, ectopic expression of H. pylori PPIase‐FKBP promoted AGS cell proliferation and anchorage‐independent growth. To gain insight into the biochemical mechanism of PPIase‐FKBP‐induced effect, early signal events involved in mitogenic signal pathways were evaluated. Expression of PPIase‐FKBP induced increases in basal as well as EGF‐stimulated phosphorylation of ERK and EGF receptor at Tyr1086. Pretreatment with MEK inhibitor completely blocked PPIase‐FKBP‐induced cell proliferation, suggesting that the action of PPIase‐FKBP is mediated by activation of ERK1/2 mitogenic signal pathway. H. pylori PPIase‐FKBP may represent a novel target for therapeutic management of gastric cancer patients.

Amphiregulin activates human hepatic stellate cells and is upregulated in non alcoholic steatohepatitis.

Amphiregulin (AR) involvement in liver fibrogenesis and hepatic stellate cells (HSC) regulation is under study. Non-alcoholic fatty liver disease (NAFLD) and its more severe form non-alcoholic steatohepatitis (NASH) may progress to cirrhosis and hepatocellular cancer (HCC). Our aim was to investigate ex vivo the effect of AR on human primary HSC (hHSC) and verify in vivo the relevance of AR in NAFLD fibrogenesis. hHSC isolated from healthy liver segments were analyzed for expression of AR and its activator, TNF-α converting enzyme (TACE). AR induction of hHSC proliferation and matrix production was estimated in the presence of antagonists. AR involvement in fibrogenesis was also assessed in a mouse model of NASH and in humans with NASH. hHSC time dependently expressed AR and TACE. AR increased hHSC proliferation through several mitogenic signaling pathways such as EGFR, PI3K and p38. AR also induced marked upregulation of hHSC fibrogenic markers and reduced hHSC death. AR expression was enhanced in the HSC of a murine model of NASH and of severe human NASH. In conclusion, AR induces hHSC fibrogenic activity via multiple mitogenic signaling pathways, and is upregulated in murine and human NASH, suggesting that AR antagonists may be clinically useful anti-fibrotics in NAFLD.

TNFα Signaling Exposes Latent Estrogen Receptor Binding Sites to Alter the Breast Cancer Cell Transcriptome

The interplay between mitogenic and proinflammatory signaling pathways plays key roles in determining the phenotypes and clinical outcomes of breast cancers. Using GRO-seq in MCF-7 cells, we defined the immediate transcriptional effects of crosstalk between estradiol (E2) and TNFα, identifying a large set of target genes whose expression is rapidly altered with combined E2+ TNFα treatment, but not with either agent alone. The pleiotropic effects on gene transcription in response to E2+ TNFα are orchestrated by extensive remodeling of the ERα enhancer landscape in an NF-κB- and FoxA1-dependent manner. In addition, expression of the de novo and synergistically regulated genes is strongly associated with clinical outcomes in breast cancers. Together, our genomic and molecular analyses indicate that TNFα signaling, acting in pathways culminating in the redistribution of NF-κB and FoxA1 binding sites across the genome, creates latent ERα binding sites that underlie altered patterns of gene expression and clinically relevant cellular responses.

Sorafenib induces paradoxical phosphorylation of the extracellular signal-regulated kinase pathway in acute myeloid leukemia cells lacking FLT3-ITD mutation

Gain-of-function mutations in the RAS and FLT3 genes are frequently found in cells of acute myeloid leukemia (AML), leading to constitutive activation of signaling pathways that regulate fundamental cellular processes, and are therefore attractive targets for AML therapy. The multi-targeted kinase inhibitor sorafenib is efficacious in AML with FLT3-internal tandem duplication (ITD), but resistance to therapy is an important clinical problem. It is unclear whether AML lacking FLT3-ITD responds to sorafenib. Using AML cell lines, we have shown that a low concentration of sorafenib induces opposing effects depending on the oncogenic background. In FLT3-ITD positive cells sorafenib blocks Erk activity and cell proliferation, and triggers apoptosis. However, in cells lacking FLT3-ITD, sorafenib paradoxically activates Erk2, and stimulates cellular proliferation and metabolic activity. Thus, depending on the genetic context, sorafenib is a beneficial inhibitor or paradoxical activator of mitogenic signaling pathways in AML. These results harbor important consequences in planning clinical trials in AML.

Loss of Drosophila pseudouridine synthase triggers apoptosis-induced proliferation and promotes cell-nonautonomous EMT.

Many developing tissues display regenerative capability that allows them to compensate cell loss and preserve tissue homeostasis. Because of their remarkable regenerative capability, Drosophila wing discs are extensively used for the study of regenerative phenomena. We thus used the developing wing to investigate the role played in tissue homeostasis by the evolutionarily conserved eukaryotic H/ACA small nucleolar ribonucleoprotein pseudouridine synthase. Here we show that localized depletion of this enzyme can act as an endogenous stimulus capable of triggering apoptosis-induced proliferation, and that context-dependent effects are elicited in different sub-populations of the silenced cells. In fact, some cells undergo apoptosis, whereas those surrounding the apoptotic foci, although identically depleted, overproliferate. This overproliferation correlates with ectopic induction of the Wg and JAK-STAT (Janus kinase-signal transducer and activator of transcription) mitogenic pathways. Expression of a p35 transgene, which blocks the complete execution of the death program and generates the so-called ‘undead cells', amplifies the proliferative response. Pseudouridine synthase depletion also causes loss of apicobasal polarity, disruption of adherens cell junctions and ectopic induction of JNK (c-Jun N-terminal kinase) and Mmp1 (matrix metalloproteinase-1) activity, leading to a significant epithelial reorganization. Unexpectedly, cell-nonautonomous effects, such as epithelial mesenchymal transition in the contiguous unsilenced squamous epithelium, are also promoted. Collectively, these data point out that cell–cell communication and long-range signaling can take a relevant role in the response to pseudouridine synthase decline. Considering that all the affected pathways are highly conserved throughout evolution, it is plausible that the response to pseudouridine synthase depletion has been widely preserved. On this account, our results can add new light on the still unexplained tumor predisposition that characterizes X-linked dyskeratosis, the human disease caused by reduced pseudouridine synthase activity.

Systematic analysis of BRAF(V600E) melanomas reveals a role for JNK/c-Jun pathway in adaptive resistance to drug-induced apoptosis.

Drugs that inhibit RAF/MEK signaling, such as vemurafenib, elicit profound but often temporary anti-tumor responses in patients with BRAFV600E melanoma. Adaptive responses to RAF/MEK inhibition occur on a timescale of hours to days, involve homeostatic responses that reactivate MAP kinase signaling and compensatory mitogenic pathways, and attenuate the anti-tumor effects of RAF/MEK inhibitors. We profile adaptive responses across a panel of melanoma cell lines using multiplex biochemical measurement, single-cell assays, and statistical modeling and show that adaptation involves at least six signaling cascades that act to reduce drug potency (IC50) and maximal effect (i.e., Emax ≪ 1). Among these cascades, we identify a role for JNK/c-Jun signaling in vemurafenib adaptation and show that RAF and JNK inhibitors synergize in cell killing. This arises because JNK inhibition prevents a subset of cells in a cycling population from becoming quiescent upon vemurafenib treatment, thereby reducing drug Emax. Our findings demonstrate the breadth and diversity of adaptive responses to RAF/MEK inhibition and a means to identify which steps in a signaling cascade are most predictive of phenotypic response.

Platelet-derived growth factor-BB enhances MSC-mediated cardioprotection via suppression of miR-320 expression.

Delivery of bone marrow-derived mesenchymal stem cells (MSCs) to myocardium protects ischemic tissue through the paracrine release of beneficial angiogenic and cytoprotective factors. Platelet-derived growth factor (PDGF)-BB, a potent mitogen of MSCs, is involved in the pathophysiology of ischemic heart disease. However, the role(s) of PDGF in MSC-mediated cardioprotection remains unknown. Here, we found that PDGF treatment of MSCs resulted in rapid activation of both Akt and ERK (central intracellular signal mediators), upregulated VEGF, and induced phosphorylation of the activator protein-1 (AP-1) transcription factor c-Jun. Examination of several microRNA genes having predicted promoter c-Jun-binding sites showed that PDGF treatment resulted in upregulation of miR-16-2 and downregulation of miRs-23b, -27b, and -320b. To examine possible PDGF augmentation of therapeutic potential, we evaluated the effects of PDGF using an ex vivo isolated mouse heart ischemia-reperfusion model. Human MSCs, with or without PDGF preconditioning, were infused into the coronary circulation of isolated mouse hearts. The hearts that received PDGF-treated MSCs exhibited a greater functional recovery compared with naïve MSC-infused hearts, following ischemia-reperfusion injury. This enhanced functional recovery was abolished by overexpression of miR-320, a microRNA we found downregulated by PDGF-activated c-Jun. Overexpression of miR-320 also resulted in upregulation of insulin-like growth factor binding protein (IGFBP) family members, suggesting PDGF "cross talk" with the mitogenic IGF signaling pathway. Collectively, we conclude that PDGF enhances MSC-mediated cardioprotection via a c-Jun/miR-320 signaling mechanism and PDGF MSC preconditioning may be an effective therapeutic strategy for cardiac ischemia.

Helicobacter pylori FKBP-type PPIase promotes gastric epithelial cell proliferation and anchorage-independent growth through activation of ERK-mediated mitogenic signaling pathway.

Though Helicobacter pylori (H. pylori) has been classified as class I carcinogen, key virulence factor(s) generated by H. pylori that causes gastric cancer remains to be fully determined. Here, we show that deletion of peptidyl-prolyl cis-trans isomerase (PPIase) prevented H. pylori from stimulating human gastric epithelial cell (AGS) proliferation. Consistent with this observation, ectopic expression of H. pylori PPIase promoted AGS cell proliferation and anchorage-independent growth. To gain insight into the biochemical mechanism of PPIase-induced effect, early signal events involved in mitogenic signaling pathways were evaluated. Expression of H. pylori PPIase caused an increase in basal as well as EGF-stimulated phosphorylation of ERK and EGF receptor at Tyr1086. Treatment with MEK inhibitor completely blocked PPIase-induced cell proliferation. Our results suggest that H. pylori PPIase has the potential to activate mitogenic signaling pathway and to promote transformation of gastric epithelial cells. H. pylori PPIase may represent a novel target for therapeutic management of gastric cancer patients.

Allosteric Effects of the Oncogenic RasQ61L Mutant on Raf-RBD

The Ras/Raf/MEK/ERK signal transduction pathway is a major regulator of cell proliferation activated by Ras-guanosine triphosphate (GTP). The oncogenic mutant RasQ61L is not able to hydrolyze GTP in the presence of Raf and thus is a constitutive activator of this mitogenic pathway. The Ras/Raf interaction is essential for the activation of the Raf kinase domain through a currently unknown mechanism. We present the crystal structures of the Ras-GppNHp/Raf-RBD and RasQ61L-GppNHp/Raf-RBD complexes, which, in combination with MD simulations, reveal differences in allosteric interactions leading from the Ras/Raf interface to the Ras calcium-binding site and to the remote Raf-RBD loop L4. In the presence of Raf, the RasQ61L mutant has a rigid switch II relative to the wild-type and increased flexibility at the interface with switch I, which propagates across Raf-RBD. We show that in addition to local perturbations on Ras, RasQ61L has substantial long-range effects on the Ras allosteric lobe and on Raf-RBD.

Ras and p53: An unsuspected liaison

The precise mechanism by which Ras proteins mediate mitogenic signaling remains obscure. We have identified the p53/p21Cip1 axis as an essential component of the Ras mitogenic pathway. Moreover, cells carrying an inactive p53/p21Cip1/Rb pathway sustain cell proliferation by inducing retroactivation of the MAPK signaling cascade in a Ras-independent manner.

Identification of novel focal adhesion kinase substrates: role for FAK in NFκB signaling.

Focal adhesion kinase (FAK) is a major signaling molecule which functions downstream of integrins or in conjunction with mitogenic signaling pathways. FAK is overexpressed and/or activated in many types of human tumors, in which it promotes cell adhesion, survival, migration and invasion. In addition to FAK's ability to regulate signaling through its scaffolding activities, FAK encodes an intrinsic kinase activity. Although some FAK substrates have been identified, a more comprehensive analysis of substrates is lacking. In this study, we use a protein microarray to screen the human proteome for FAK substrates. We confirm that several of the proteins identified are bona fide in vitro FAK substrates, including several factors which are known to regulate the NFκB pathway. Finally, we identify a role for FAK's kinase activity in both canonical and non-canonical NFκB signaling. Our screen therefore represents the first high throughput screen for FAK substrates and provides the basis for future in-depth analysis of the role of FAK's kinase activity in the processes of tumorigenesis.

A Breast Tissue Protein Expression Profile Contributing to Early Parity-Induced Protection Against Breast Cancer

Background/Aims: Early parity reduces breast cancer risk, whereas, late parity and nulliparity increase breast cancer risk. Despite substantial efforts to understand the protective effects of early parity, the precise molecular circuitry responsible for these changes is not yet fully defined. Methods: Here, we have conducted the first study assessing protein expression profiles in normal breast tissue of healthy early parous, late parous, and nulliparous women. Breast tissue biopsies were obtained from 132 healthy parous and nulliparous volunteers. These samples were subjected to global protein expression profiling and immunohistochemistry. GeneSpring and MetaCore bioinformatics analysis software were used to identify protein expression profiles associated with early parity (low risk) versus late/nulliparity (high risk). Results: Early parity reduces expression of key proteins involved in mitogenic signaling pathways in breast tissue through down regulation of EGFR1/3, ESR1, AKT1, ATF, Fos, and SRC. Early parity is also characterized by greater genomic stability and reduced tissue inflammation based on differential expression of aurora kinases, p53, RAD52, BRCA1, MAPKAPK-2, ATF-1, ICAM1, and NF-kappaB compared to late and nulli parity. Conclusions: Early parity reduces basal cell proliferation in breast tissue, which translates to enhanced genomic stability, reduced cellular stress/inflammation, and thus reduced breast cancer risk.

Integrative analysis of high-throughput RNAi screen data identifies the FER and CRKL tyrosine kinases as new regulators of the mitogenic ERK-dependent pathways in transformed cells.

BackgroundCell proliferation is a hallmark of cancer and depends on complex signaling networks that are chiefly supported by protein kinase activities. Therapeutic strategies have been used to target specific kinases but new methods are required to identify combined targets and improve treatment. Here, we propose a small interfering RNA genetic screen and an integrative approach to identify kinase networks involved in the proliferation of cancer cells.ResultsThe functional siRNA screen of 714 kinases in HeLa cells identified 91 kinases implicated in the regulation of cell growth, most of them never being reported in previous whole-genome siRNA screens. Based on gene ontology annotations, we have further discriminated between two classes of kinases that, when suppressed, result in alterations of the mitotic index and provoke cell-cycle arrest. Extinguished kinases that lead to a low mitotic index mostly include kinases implicated in cytosolic signaling. In contrast, extinguished kinases that result in a high mitotic index mostly include kinases implicated in cell division. By mapping hit kinases in the PhosphPOINT phosphoprotein database, we generated scale-free networks consisting of 449 and 661 protein-protein interactions for kinases from low MI and high MI groups, respectively. Further analyses of the kinase interactomes revealed specific modules such as FER- and CRKL-containing modules that connect three members of the epidermal growth factor receptor (EGFR) family, suggesting a tight control of the mitogenic EGF-dependent pathway. Based on experimental studies, we confirm the involvement of these two kinases in the regulation of tumor cell growth.ConclusionBased on a combined approach of large kinome-wide siRNA screens and ontology annotations, our study identifies for the first time two kinase groups differentially implicated in the control of cell proliferation. We further demonstrate that integrative analysis of the kinase interactome provides key information which can be used to facilitate or optimize target design for new therapeutic strategies. The complete list of protein-protein interactions from the two functional kinase groups will provide a useful database for future investigations.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1169) contains supplementary material, which is available to authorized users.