Activation Of Major Signaling Pathways Research Articles

Lung Cancer Stem Cell Markers as Therapeutic Targets: An Update on Signaling Pathways and Therapies.

Cancer stem cells, a relatively small group of self-renewing cancer cells, were first isolated from acute myeloid leukemia. These cells can play a crucial role in tumor metastasis, relapse, and therapy resistance. The cancer stem cell theory may be applied to lung cancer and explain the inefficiency of traditional treatments and eventual recurrence. However, because of the unclear accuracy and illusive biological function of cancer stem cells, some researchers remain cautious about this theory. Despite the ongoing controversy, cancer stem cells are still being investigated, and their biomarkers are being discovered for application in cancer diagnosis, targeted therapy, and prognosis prediction. Potential lung cancer stem cell markers mainly include surface biomarkers such as CD44, CD133, epithelial cell adhesion molecule, and ATP-binding cassette subfamily G member 2, along with intracellular biomarkers such as aldehyde dehydrogenase, sex-determining region Y-box 2, NANOG, and octamer-binding transcription factor 4. These markers have different structures and functions but are closely associated with the stem potential and uncontrollable proliferation of tumor cells. The aberrant activation of major signaling pathways, such as Notch, Hedgehog, and Wnt, may be associated with the expression and regulation of certain lung cancer stem cell markers, thus leading to lung cancer stem cell maintenance, chemotherapy resistance, and cancer promotion. Treatments targeting lung cancer stem cell markers, including antibody drugs, nanoparticle drugs, chimeric antigen receptor T-cell therapy, and other natural or synthetic specific inhibitors, may provide new hope for patients who are resistant to conventional lung cancer therapies. This review provides comprehensive and updated data on lung cancer stem cell markers with regard to their structures, functions, signaling pathways, and promising therapeutic target approaches, aiming to elucidate potential new therapies for lung cancer.

How to win the ovarian cancer stem cell battle: destroying the roots.

Ovarian cancer has the highest mortality rate among gynecologic malignancies. The combination of cytoreductive surgery and chemotherapy is the standard regimen for the treatment of ovarian cancer. The initial treatment is usually effective, but many patients with ovarian cancer experience recurrence, and treatment options for recurrent disease remain challenging. Cancer stem cells (CSCs) are suggested to play an essential role in cancer recurrence after initial chemotherapy. Furthermore, they are of great interest as CSCs may also be involved in chemotherapy susceptibility. Thus, understanding the characteristics and mechanisms by which CSCs display resistance to therapeutic agents is important to design effective cancer treatments. In this review, we describe and discuss current therapeutic regimens for ovarian cancer, as well as the various CSC markers, association between CSCs and disease progression, correlation of CSCs with poor prognosis, enrichment of CSCs in tumor tissues following repeated chemotherapy cycles, activation of major signaling pathways following chemotherapy, and potential inhibitors that suppress these signaling cascades. In addition, clinical trials evaluating novel targeted therapies to overcome chemotherapy resistance will be reviewed. The combination of traditional chemotherapy and CSC-targeted therapy could be an effective and promising anticancer treatment for ovarian cancer. Understanding the biological properties of CSCs and the mechanism of chemotherapy resistance are critical to design and develop new therapeutic strategies to overcome CSC-associated chemotherapy resistance.

A Dynamic Role of Mastermind-Like 1: A Journey Through the Main (Path)ways Between Development and Cancer.

Major signaling pathways, such as Notch, Hedgehog (Hh), Wnt/β-catenin and Hippo, are targeted by a plethora of physiological and pathological stimuli, ultimately resulting in the modulation of genes that act coordinately to establish specific biological processes. Many biological programs are strictly controlled by the assembly of multiprotein complexes into the nucleus, where a regulated recruitment of specific transcription factors and coactivators on gene promoter region leads to different transcriptional outcomes. MAML1 results to be a versatile coactivator, able to set up synergistic interlinking with pivotal signaling cascades and able to coordinate the network of cross-talking pathways. Accordingly, despite its original identification as a component of the Notch signaling pathway, several recent reports suggest a more articulated role for MAML1 protein, showing that it is able to sustain/empower Wnt/β-catenin, Hh and Hippo pathways, in a Notch-independent manner. For this reason, MAML1 may be associated to a molecular “switch”, with the function to control the activation of major signaling pathways, triggering in this way critical biological processes during embryonic and post-natal life. In this review, we summarize the current knowledge about the pleiotropic role played by MAML proteins, in particular MAML1, and we recapitulate how it takes part actively in physiological and pathological signaling networks. On this point, we also discuss the contribution of MAML proteins to malignant transformation. Accordingly, genetic alterations or impaired expression of MAML proteins may lead to a deregulated crosstalk among the pathways, culminating in a series of pathological disorders, including cancer development. Given their central role, a better knowledge of the molecular mechanisms that regulate the interplay of MAML proteins with several signaling pathways involved in tumorigenesis may open up novel opportunities for an attractive molecular targeted anticancer therapy.

Systematic analysis of the IL-17 receptor signalosome reveals a robust regulatory feedback loop.

IL‐17 mediates immune protection from fungi and bacteria, as well as it promotes autoimmune pathologies. However, the regulation of the signal transduction from the IL‐17 receptor (IL‐17R) remained elusive. We developed a novel mass spectrometry‐based approach to identify components of the IL‐17R complex followed by analysis of their roles using reverse genetics. Besides the identification of linear ubiquitin chain assembly complex (LUBAC) as an important signal transducing component of IL‐17R, we established that IL‐17 signaling is regulated by a robust negative feedback loop mediated by TBK1 and IKKε. These kinases terminate IL‐17 signaling by phosphorylating the adaptor ACT1 leading to the release of the essential ubiquitin ligase TRAF6 from the complex. NEMO recruits both kinases to the IL‐17R complex, documenting that NEMO has an unprecedented negative function in IL‐17 signaling, distinct from its role in NF‐κB activation. Our study provides a comprehensive view of the molecular events of the IL‐17 signal transduction and its regulation.

Impact of ischemia on sample quality of human pancreatic tissues

BackgroundSuccessful clinical evaluation of human tumors relies on proper handling of tissue samples to maximally preserve the cellular and metabolic states in vivo. Pancreatic samples are particularly sensitive to sample mishandling due to the abundance of digestive enzymes. We study how the duration of ischemia, in vivo and ex vivo, both of which are unavoidable lagging periods following surgical dissection, significantly impact the utility of pancreatic samples. MethodsWe systematically characterize a wide range of tissue integrity features, including histological patterns, cellular structures, DNA/RNA quality and activity of major signaling pathways in normal pancreases and pancreatic ductal adenocarcinoma (PDAC) tumor tissues from 41 patients with different ischemia. ResultsWe reveal that tissues experiencing longer periods of ischemia exhibit significant deterioration and could potentially mislead disease diagnosis and preclinical research. Based on these analyses, we propose an optimal procedure that balances better clinical practice and high tissue sample quality. ConclusionsOur work provides a guideline for pancreatic sample handling and could have wide implications in clinical diagnosis and translational research.

Small-Molecule Screening in Zebrafish Embryos Identifies Signaling Pathways Regulating Early Thyroid Development.

Background: Defects in embryonic development of the thyroid gland are a major cause for congenital hypothyroidism in human newborns, but the underlying molecular mechanisms are still poorly understood. Organ development relies on a tightly regulated interplay between extrinsic signaling cues and cell intrinsic factors. At present, however, there is limited knowledge about the specific extrinsic signaling cues that regulate foregut endoderm patterning, thyroid cell specification, and subsequent morphogenetic processes in thyroid development. Methods: To begin to address this problem in a systematic way, we used zebrafish embryos to perform a series of in vivo phenotype-driven chemical genetic screens to identify signaling cues regulating early thyroid development. For this purpose, we treated zebrafish embryos during different developmental periods with a panel of small-molecule compounds known to manipulate the activity of major signaling pathways and scored phenotypic deviations in thyroid, endoderm, and cardiovascular development using whole-mount in situ hybridization and transgenic fluorescent reporter models. Results: Systematic assessment of drugged embryos recovered a range of thyroid phenotypes including expansion, reduction or lack of the early thyroid anlage, defective thyroid budding, as well as hypoplastic, enlarged, or overtly disorganized presentation of the thyroid primordium after budding. Our pharmacological screening identified bone morphogenetic protein and fibroblast growth factor signaling as key factors for thyroid specification and early thyroid organogenesis, highlighted the importance of low Wnt activities during early development for thyroid specification, and implicated drug-induced cardiac and vascular anomalies as likely indirect mechanisms causing various forms of thyroid dysgenesis. Conclusions: By integrating the outcome of our screening efforts with previously available information from other model organisms including Xenopus, chicken, and mouse, we conclude that signaling cues regulating thyroid development appear broadly conserved across vertebrates. We therefore expect that observations made in zebrafish can inform mammalian models of thyroid organogenesis to further our understanding of the molecular mechanisms of congenital thyroid diseases.

Inhibitory Effect of (2R)-1-(1-Benzofuran-2-yl)-N-propylpentan-2-amine on Lung Adenocarcinoma

BPAP is a potent enhancer substance with catecholaminergic and serotoninergic activity in the brain. It was discovered that it is also effective against certain types of experimental cancers, showing the most promising results in case of lung cancer. That is why we tested its efficacy in two different doses in a newly developed EGFR wild type mouse lung adenocarcinoma xenograft model. Experiments were conducted on FVB/N and SCID mouse strains treated with low and high dose of BPAP. Body weight, survival, and tumor volumes were recorded. Furthermore, the activity of major signaling pathways of NSCLC such as MAPK and Akt/mTOR as well as cell cycle regulation were determined. Significant inhibition of tumor growth was exerted by both doses, but the mechanism of action was different. High dose directly inhibited, whereas low dose activated the main signaling pathways. Exposure to low dose BPAP resulted in elevated activity of the mTOR pathway together with p16INK-induced cell cycle arrest, a typical feature of geroconversion, a senescent state characterized by loss of cell proliferation. Finally the events culminated in cell cycle inhibition point in case of both doses mirrored by the decrease of cyclin D1, CDK4 and PCNA. In addition, BPAP treatment had a beneficial effect on bodyweight suggesting that the compound at least in part is able to compensate the cancer-related wasting. In view of the low toxicity and confirmed antitumor effect of BPAP against experimental lung adenocarcinoma, this novel compound deserves further attention.

SOSTDC1 inhibits follicular thyroid cancer cell proliferation, migration, and EMT via suppressing PI3K/Akt and MAPK/Erk signaling pathways.

The sclerostin domain containing protein 1 (SOSTDC1) is a cell signaling regulator involved in cell physiology and pathology. SOSTDC1 is known to have a suppressive effect on certain kinds of cancer. However, the role of SOSTDC1 in follicular thyroid cancer (FTC) remains unknown. We aimed to investigate if the expression of SOSTDC1 plays any roles in carcinogenesis and metastasis of FTC. We found a significantly down-regulated SOSTDC1 expression in follicular thyroid cancer samples. In addition, our data showed that ectopic expression of SOSTDC1 dramatically inhibited thyroid cancer cell proliferation in vitro and in nude mice. SOSTDC1 also compromised the migratory, invasive property, and epithelial-mesenchymal transition (EMT) activity of FTC cell. Mechanically, SOSTDC1 exerted its tumor suppressor function by inhibiting the activity of major signaling pathways including the phosphatidylinositol-3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/Erk pathways. Altogether, our findings provide insight into the role of SOSTDC1 as a novel functional tumor suppressor in follicular thyroid cancer through modulating the activities of PI3K/Akt and MAPK/Erk signaling pathways.

Abstract 612: High Dimensional Single-Cell Intracellular Signaling Identifies CCL5 as a Potential Key Driver of Myeloid Cell Reprogramming in Atherosclerotic Patients

Atherosclerosis is a disease characterized by chronic inflammation. However, identifying new molecularly targeted interventions in human atherosclerosis has proven difficult. In this study, we used phosphoCyTOF to comprehensively characterize functional responses of peripheral immune populations in human atherosclerosis at the single-cell level. Healthy PBMCs were exposed to plasma of patients with atherosclerosis (n=20) or healthy donors (n=10) and the activation of major signaling pathways across all major immune subsets using 18 surface markers and 10 intracellular phospho-proteins. Using viSNE we visualized and defined 10 major immune cell populations: B cells, Basophils, CD1c DCs, CD4 T cells, CD8 T cells, CD14 and CD16 monocytes, NK cells, NKT cells, and pDCs. Next, we evaluated the relative expression of 10 phospho-proteins (IkBa, pCREB, pERK1/2, pMAPKAP2, pp38, pPLCg2, pS6, pSTAT1, pSTAT3, and pSTAT5) across this immunological map and visualized functional pathways that were differentially induced in response to plasma of atherosclerotic patients vs. healthy donors. Monocytes exhibited the greatest immune activation, showing increased phosphorylation of p38, MAPKAP2, ERK1/2, CREB and S6 in response to atherosclerotic plasma. To identify plasma cytokines responsible for this effect, we measured a panel of 41 cytokines and identified CCL5, CXCL10 (IP-10), CXCL1 (GRO), PDGF-AA and PDGF-BB as the most differentially expressed in atherosclerotic vs. healthy plasma. PhosphoCyTOF analysis showed that only CCL5 reproduced the phosphorylation pattern induced by atherosclerotic plasma in monocytes, an effect that was completely inhibited by a CCL5 blocking antibody (Fig. 1). These results suggest that CCL5 in plasma of patients with atherosclerotic disease drives specific innate immune cell signaling functions that mark the inflammatory response in atherosclerotic disease.

PAX3 is a novel tumor suppressor by regulating the activities of major signaling pathways and transcription factor FOXO3a in thyroid cancer.

Paired box 3 (PAX3) is expressed early during embryonic development in spatially restricted domains in the nervous system and in some mesodermally-derived structure. In recent years, it is found to be overexpressed in different types of cancer tissues and cell lines including glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, Ewing sarcomas and gastric cancers, suggesting that it may function as an oncogene in these cancers. However, its role in thyroid cancer remains totally unclear. The aim of this study was to explore the functions and related molecular mechanism of PAX3 in thyroid tumorigenesis. Using quantitative RT-PCR (qRT-PCR) and Methylation-specific PCR (MSP) assays, we demonstrated that PAX3 was frequently down-regulated by promoter methylation in both primary thyroid cancer tissues and thyroid cancer cell lines. In addition, our data showed that ectopic expression of PAX3 dramatically inhibited thyroid cancer cell proliferation, colony formation, migration and invasion, induced cell cycle arrest and apoptosis and retarded tumorigenic potential in nude mice. Mechanically, PAX3 exerted its tumor suppressor function by inhibiting the activity of major signaling pathways including the phosphatidylinositol-3-kinase (PI3K)/Akt and MAPK/Erk pathways, and enhancing expression and activity of transcription factor FOXO3a. Altogether, our findings provided insight into the role of PAX3 as a novel functional tumor suppressor in thyroid cancer through modulating the activities of PI3K/Akt and MAPK signaling pathways and transcription factor FOXO3a, and demonstrated that epigenetic alterations such as promoter methylation should be a major mechanism of PAX3 inactivation in this cancer.

EGF stimulates the activation of EGF receptors and the selective activation of major signaling pathways during mitosis

Mitosis and epidermal growth factor (EGF) receptor (EGFR) are both targets for cancer therapy. The role of EGFR signaling in mitosis has been rarely studied and poorly understood. The limited studies indicate that the activation of EGFR and downstream signaling pathways is mostly inhibited during mitosis. However, we recently showed that EGFR is phosphorylated in response to EGF stimulation in mitosis. Here we studied EGF-induced EGFR activation and the activation of major signaling pathways downstream of EGFR during mitosis. We showed that EGFR was strongly activated by EGF during mitosis as all the five major tyrosine residues including Y992, Y1045, Y1068, Y1086, and Y1173 were phosphorylated to a level similar to that in the interphase. We further showed that the activated EGFR is able to selectively activate some downstream signaling pathways while avoiding others. Activated EGFR is able to activate PI3K and AKT2, but not AKT1, which may be responsible for the observed effects of EGF against nocodazole-induced cell death. Activated EGFR is also able to activate c-Src, c-Cbl and PLC-γ1 during mitosis. However, activated EGFR is unable to activate ERK1/2 and their downstream substrates RSK and Elk-1. While it activated Ras, EGFR failed to fully activate Raf-1 in mitosis due to the lack of phosphorylation at Y341 and the lack of dephosphorylation at pS259. We conclude that contrary to the dogma, EGFR is activated by EGF during mitosis. Moreover, EGFR-mediated cell signaling is regulated differently from the interphase to specifically serve the needs of the cell in mitosis.

Nanolipolee-007, a novel nanoparticle-based drug containing leelamine for the treatment of melanoma.

Malignant melanoma is a difficult cancer to treat due to the rapid development of resistance to drugs targeting single proteins. One response to this observation is to identify single pharmacologic agents that, due to a unique mechanism of action, simultaneously target multiple key pathways involved in melanoma development. Leelamine has been identified as functioning in this manner but has poor bioavailability in animals and causes lethality when administered intravenously. Therefore, a nanoliposomal-based delivery system has been developed, called Nanolipolee-007, which stably loads 60% of the compound. The nanoparticle was as effective at killing melanoma cells as leelamine dissolved in DMSO and was more effective at killing cultured melanoma compared with normal cells. Mechanistically, Nanolipolee-007 inhibited PI3K/Akt, STAT3, and MAPK signaling mediated through inhibition of cholesterol transport. Nanolipolee-007 inhibited the growth of preexisting xenografted melanoma tumors by an average of 64% by decreasing cellular proliferation, reducing tumor vascularization, and increasing cellular apoptosis, with negligible toxicity. Thus, a unique clinically viable nanoparticle-based drug has been developed containing leelamine for the treatment of melanoma that acts by inhibiting the activity of major signaling pathways regulating the development of this disease.

The Wnt Antagonist Dkk1 Regulates Intestinal Epithelial Homeostasis and Wound Repair

Dkk1 is a secreted antagonist of the Wnt/β-catenin signaling pathway. It is induced by inflammatory cytokines during colitis and exacerbates tissue damage by promoting apoptosis of epithelial cells. However, little is known about the physiologic role of Dkk1 in normal intestinal homeostasis and during wound repair following mucosal injury. We investigated whether inhibition of Dkk1 affects the morphology and function of the adult intestine. We used doubleridge mice (Dkk1d/d), which have reduced expression of Dkk1, and an inhibitory Dkk1 antibody to modulate Wnt/β-catenin signaling in the intestine. Intestinal inflammation was induced with dextran sulfate sodium (DSS), followed by a recovery period in which mice were given regular drinking water. Animals were killed before, during, or after DSS administration; epithelial homeostasis and the activity of major signaling pathways were investigated by morphometric analysis, bromo-2'-deoxyuridine incorporation, and immunostaining. Reduced expression of Dkk1 increased proliferation of epithelial cells and lengthened crypts in the large intestine, which was associated with increased transcriptional activity of β-catenin. Crypt extension was particularly striking when Dkk1 was inhibited during acute colitis. Dkk1d/d mice recovered significantly faster from intestinal inflammation but exhibited crypt architectural irregularities and epithelial hyperproliferation compared with wild-type mice. Survival signaling pathways were concurrently up-regulated in Dkk1d/d mice, including the AKT/β-catenin, ERK/Elk-1, and c-Jun pathways. Dkk1, an antagonist of Wnt/β-catenin signaling, regulates intestinal epithelial homeostasis under physiologic conditions and during inflammation. Depletion of Dkk1 induces a strong proliferative response that promotes wound repair after colitis.

Proteomic Analysis of Ischemia-Reperfusion Injury upon Human Liver Transplantation Reveals the Protective Role of IQGAP1

Ischemia-reperfusion injury (IRI) represents a major determinant of liver transplantation. IRI-induced graft dysfunction is related to biliary damage, partly due to a loss of bile canaliculi (BC) integrity associated with a dramatic remodeling of actin cytoskeleton. However, the molecular mechanisms associated with these events remain poorly characterized. Using liver biopsies collected during the early phases of organ procurement (ischemia) and transplantation (reperfusion), we characterized the global patterns of expression and phosphorylation of cytoskeleton-related proteins during hepatic IRI. This targeted functional proteomic approach, which combined protein expression pattern profiling and phosphoprotein enrichment followed by mass spectrometry analysis, allowed us to identify IQGAP1, a Cdc42/Rac1 effector, as a potential regulator of actin cytoskeleton remodeling and maintenance of BC integrity. Cell fractionation and immunohistochemistry revealed that IQGAP1 expression and localization were affected upon IRI and related to actin reorganization. Furthermore using an IRI model in human hepatoma cells, we demonstrated that IQGAP1 silencing decreased the basal level of actin polymerization at BC periphery, reflecting a defect in BC structure coincident with reduced cellular resistance to IRI. In summary, this study uncovered new mechanistic insights into the global regulation of IRI-induced cytoskeleton remodeling and led to the identification of IQGAP1 as a regulator of BC structure. IQGAP1 therefore represents a potential target for the design of new organ preservation strategies to improve transplantation outcome.

Protein Kinase C-dependent Protein Kinase D Activation Modulates ERK Signal Pathway and Endothelial Cell Proliferation by Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is essential for many angiogenic processes both in normal conditions and in pathological conditions. However, the signaling pathways involved in VEGF-induced angiogenesis are not well defined. Protein kinase D (PKD), a newly described serine/threonine protein kinase, has been implicated in many signal transduction pathways and in cell proliferation. We hypothesized that PKD would mediate VEGF signaling and function in endothelial cells. Here we found that VEGF rapidly and strongly stimulated PKD phosphorylation and activation in endothelial cells via VEGF receptor 2 (VEGFR2). The pharmacological inhibitors for phospholipase Cgamma (PLCgamma) and protein kinase C (PKC) significantly inhibited VEGF-induced PKD activation, suggesting the involvement of the PLCgamma/PKC pathway. In particular, PKCalpha was critical for VEGF-induced PKD activation since both overexpression of adenovirus PKCalpha dominant negative mutant and reduction of PKCalpha expression by small interfering RNA markedly inhibited VEGF-induced PKD activation. Importantly, we found that small interfering RNA knockdown of PKD and PKCalpha expression significantly attenuated ERK activation and DNA synthesis in endothelial cells by VEGF. Taken together, our results demonstrated for the first time that VEGF activates PKD via the VEGFR2/PLCgamma/PKCalpha pathway and revealed a critical role of PKD in VEGF-induced ERK signaling and endothelial cell proliferation.