Growth Factor Receptor Signaling Cascade Research Articles

Linker Engineering of Ligand-Decorated DNA Origami Nanostructures Affects Biological Activity.

News from an old acquaintance: The streptavidin (STV)-biotin binding system is frequently used for the decoration of DNA origami nanostructures (DON) to study biological systems. Here, a surprisingly high dynamic of the STV/DON interaction is reported, which is affected by the structure of the DNA linker system. Analysis of different mono- or bi-dentate linker architectures on DON with a novel high-speed atomic force microscope (HS-AFM) enabling acquisition times as short as 50ms per frame gave detailed insights into the dynamics of the DON/STV interaction, revealing dwell times in the sub-100millisecond range. The linker systems are also used to present biotinylated epidermal growth factor on DON to study the activation of the epidermal growth factor receptor signaling cascade in HeLa cells. The studies confirm that cellular activation correlated with the binding properties of linker-specific STV/DON interactions observed by HS-AFM. This work sheds more light on the commonly used STV/DON system and will help to further standardize the use of DNA nanostructures for the study of biological processes.

N‐cadherin‐mediated Regulation of Metabolism in Oral Precancer

Cadherins are transmembrane proteins traditionally known to have a structural role in cellular adhesion. These proteins display tissue-specific changes in expression that have been correlated with poor prognosis in a variety of cancers. N-cadherin expression specifically has been associated with poor oral cancer prognosis. An estimated 80% of oral cancers exhibit aberrant N-cadherin expression. We have previously shown that cadherins can alter growth factor receptor signaling cascades, which have been shown in other studies to direct metabolic changes essential to oral cancer metastasis. The goal of the current study was to characterize the mechanism(s) by which abnormal expression of N-cadherin may alter metabolic activity in oral epithelia. We have utilized a dysplastic oral keratinocyte (DOK) line to develop retrovirally-transduced cells which express control levels or reduced levels of N-cadherin protein. Using these lines, we evaluated the effect of N-cadherin expression on EGF-dependent EGFR phosphorylation as well as downstream effectors within the mammalian target of rapamycin (mTOR) signaling pathway. Western blot analysis of our EGF-treated DOK lines suggested that N-cadherin expression suppressed phosphorylation of EGFR at Tyr 1068, which plays a role in EGFR degradation, and Tyr 1045, which can mediate cell survival through activation of signal transducer and activator of transcription 3 (STAT3). We also found minimal changes in phosphorylation of Tyr 992, a phosphosite associated with mitogenic signaling. EGFR activation has been shown to direct metabolism through phosphatidyl inositol-3 kinase (PI3K)/Akt/mTOR signaling. Our data indicated that N-cadherin expression is necessary for maximal EGF-directed phosphorylation of both Akt at Ser 473 and of mTORC1 at Ser 2448. mTOR has been shown to increase synthesis of the transcription factor hypoxia inducible factor-1 alpha (HIF-1 alpha) to redirect glycolytic intermediates from oxidative metabolism to anaerobic glycolysis by way of lactate dehydrogenase (LDH), a hallmark of the Warburg effect. We found that N-cadherin expression suppressed HIF-1 alpha synthesis but did not appear to alter lactate dehydrogenase expression. It is important to note that reduced expression of N-cadherin did not alter levels of either E- or P-cadherin expression. Taken together, these findings suggest a role for N-cadherin in the regulation of mTOR-related metabolic signaling, and in modulation of the Warburg effect in oral precancer. Given the high incidence of N-cadherin expression in oral cancer as well as its correlation with poor prognosis, elucidating the mechanisms by which N-cadherin regulates metabolism could reveal novel and promising targets for future therapeutic approaches.

Quercetin‐mediated regulation of signal transduction cascades and microRNAs: Natural weapon against cancer

Recent technological and analytical breakthroughs in genomics and proteomics have deepened our understanding related to the multifaceted nature of cancer. Because of therapeutically challenging nature of cancer, there has been a renewed interest in phytochemistry, and much attention is currently being given to the identification of signaling pathway inhibitors. Data obtained through high-throughput technologies has provided a broader landscape of wiring maps of complex oncogenic signaling networks, thus revealing novel therapeutic opportunities. Increasingly, it is being realized that although our knowledge related to physiological and pathophysiological roles of signal transduction cascades has evolved rapidly, the clinical development of signaling pathway inhibitors has been challenging. Quercetin has attracted considerable attention because of its amazingly high pharmacological value. Research over decades has sequentially shown that quercetin effectively inhibited cancer development and progression. In this review, we have attempted to set the spotlight on the regulation of different cell signaling pathways by quercetin. We partition this multicomponent review into how quercetin effectively regulates the Wnt/β-catenin pathway, Janus kinase-signal transducer and activator of transcription pathway, and vascular endothelial growth factor/vascular endothelial growth factor receptor signaling cascade in different types of cancers. We also provide an overview of the regulation of NOTCH and SHH pathways by quercetin. MicroRNAs (miRNAs) have also emerged as versatile regulators of cancer, and contemporary studies have shed light on the ability of quercetin to control different miRNAs in various cancers. We have scattered information related to NOTCH and SHH pathways, and future studies must converge on the investigation of these pathways to see how quercetin modulates the signaling machinery of these pathways.

Abstract 1897: Determining the role of PRCP/PREP in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) accounts for 15-20% of all breast cancer cases. Currently, the only effective treatment for TNBC is chemotherapy agents. A well-established characteristic of TNBC is the ability of these cells to become metastatic following chemotherapy treatment resulting in increased mortality. New forms of treatment which target TNBC are urgently needed. Insulin receptor substrate 1 (IRS1) has been extensively studied in the regulation of the insulin and insulin-like growth factor receptor signaling cascades, specifically in the induction of the intracellular PI3K/AKT/mTORC1 and MAP kinase pathways. The roles of such pathways in cancer are currently being studied and considered for therapeutic targeting. We previously showed that the stability and levels of IRS1 are maintained by prolylcarboxypeptidase proteins PRCP/PREP in pancreatic cancer cells. Our hypothesis is that PRCP/PREP are potential therapeutic targets in TNBC. Specifically, we hypothesize that blocking the expression or reducing the activity of PRCP/PREP will inhibit the PI3K/AKT/mTORC1 pathway in TNBC cells and, in turn, reduce growth and survival in these cells. To test this, we will be treating a number of TNBC cell lines with a PRCP/PREP inhibitor and determining IRS1/2 protein levels, AKT/mTORC1 signaling, and growth and survival. TNBC cells were screened for sensitivity against the PRCP/PREP inhibitor, Y-ox. Sensitive cells treated with Y-ox showed earlier reduction in the IRS1/AKT/mTORC1 signaling, as well as earlier loss of cell viability compared to those cells with relative resistance to Y-ox. Similarly, cells treated with the IRS1/2 inhibitor NT-157 and the AKT inhibitor, MK-2206 showed loss of cell viability similarly seen with the PRCP/PREP inhibitor, Y-ox. In conclusion, the results shown in this study show a possible new therapeutic target against triple negative breast cancer in the inhibition of PRCP/PREP leading to loss of cell proliferation and viability. Citation Format: Ricardo E. Perez, Lei Duan, Carl G. Maki. Determining the role of PRCP/PREP in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1897.

Abstract A170: Analysis of marine sponge Cribrochalina vasculum compounds demonstrate selective antitumor properties by activation of intrinsic apoptotic signaling and impaired growth factor receptor signaling cascades.

Abstract Introduction and Aim of Study: Marine derived compounds have been explored and considered as possible antitumor agents. Some natural products from marine organisms are currently in clinical trials, such as: Eribulin, an analogue of halichondrin B, isolated from the marine sponge Halichondria okadai, which is approved by FDA for the treatment of metastatic breast cancer; and Trabectedin, from Ecteinascidia turbinate, approved for treatment of soft tissue sarcomas and ovarian cancer also in Europe. In this study we analyzed extracts of the sponge Cribrochalina vasculum for the inhibition of tumor growth and hence hold potential to be used as novel antitumor agents. Material and Methods: Extracts were obtained from the Caribbean sponge Cribrochalina vasculum. The tumor specific cytotoxicity of the extracts was determined by MTT assay using non-small cell lung carcinoma (NSCLC) cells and normal lung fibroblasts WI-38. The active crude extract showing tumor specific toxicity was fractionated by HPLC to obtain pure compounds and their structures elucidated by physiochemical methods. Various concentrations of the pure compounds were rescreened on several non-small and small lung carcinoma cell lines (NSCLC and SCLC), on ovarian carcinoma cells as well as on normal lung fibroblasts WI-38. Induction of apoptosis was assessed as caspase-3, caspase-9 and PARP cleavage on western blot. To detect and quantify apoptotic morphology, cells treated were stained with DAPI and examined using a fluorescence microscope. Conformational change in proapoptotic protein Bak in response to treatment for 16 h was examined in NSCLC U-1810 using flow cytometric analyses. Cell cycle distribution was examined in NSCLC and diploid fibroblast by propidium iodide (PI) staining, using a flow cytometer. Results: Screening of crude extracts from C. vasculum identified certain extracts that showed tumor specific toxicity while having no effect on diploid fibroblasts. From these extracts purified compounds were identified which showed antitumor activity in both NSCLC, SCLC as well as in ovarian carcinoma cells. The purified compounds were found to cause a time dependent increase in activation of apoptotic signaling in NSCLC cells, but not in normal fibroblasts. Thus exposure of NSCLC cells to these compounds caused cleavage of caspase-9, caspase-3 and PARP, as well as induction of apoptotic cell morphology and Bak conformational change. Cell cycle analyses revealed that these compounds caused a pronounced block in the G2/M phase of the cell cycle of NSCLC cells whereas normal fibroblasts were not influenced. In vivo analyses of the compounds in mice tumor models is ongoing. Conclusion: We have identified, by screening extracts from the sponge C. vasculum, compounds that hold promise as antitumor agents in NSCLC and SCLC as well as in ovarian carcinoma. Further work aims to identify putative cellular targets of these compounds within tumor cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A170. Citation Format: Ana Zovko, Kristina Viktorsson, Petra Hååg, Micha Ilan, Dimitry Kovalerchick, Shmuel Carmeli, Andrea Alimonti, Rolf Lewensohn. Analysis of marine sponge Cribrochalina vasculum compounds demonstrate selective antitumor properties by activation of intrinsic apoptotic signaling and impaired growth factor receptor signaling cascades. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A170.

Signal transduction via cannabinoid receptors.

The endocannabinoids anandamide and 2-arachidonoylglycerol are lipid mediators that signal via CB(1) and CB(2) cannabinoid receptors and Gi/o-proteins to inhibit adenylyl cyclase and stimulate mitogen-activated protein kinase. In the brain, CB(1) receptors interact with opioid receptors in close proximity, and these receptors may share G-proteins and effector systems. In the striatum, CB(1) receptors function in coordination with D(1) and D(2) dopamine receptors, and combined stimulation of CB(1)-D(2) receptor heteromeric complexes promotes a unique interaction to stimulate cAMP production. CB(1) receptors also trigger growth factor receptor signaling cascades in cells by engaging in cross-talk or interreceptor signal transmission with the receptor tyrosine kinase (RTK) family. Mechanisms for CB(1) receptor-RTK transactivation can include stimulation of signal transduction pathways regulated by second messengers such as phospholipase C, metalloprotease cleavage of membrane-bound precursor proteins such as epidermal growth factor which activate RTKs, RTK autophosphorylation, and recruitment of non-receptor tyrosine kinases. CB(1) and CB(2) receptors are expressed in peripheral tissues including liver and adipose tissue, and are induced in pathological conditions. Novel signal transduction resulting from endocannabinoid regulation of AMP-regulated kinase and peroxisome proliferator-activated receptors have been discovered from studies of hepatocytes and adipocytes. It can be predicted that drug discovery of the future will be based upon these novel signal transduction mechanisms for endocannabinoid mediators.

EGFR point mutation in non‐small cell lung cancer is occasionally accompanied by a second mutation or amplification

Activating mutations of EGFR are found frequently in a subgroup of patients with non-small cell lung cancer (NSCLC) and are highly correlated with the response to gefitinib and erlotinib. In the present study, we searched for mutations of EGFR, HER2 and KRAS in 264 resected primary NSCLC from Japanese patients and determined whether there is a correlation between genetic alterations of these genes and clinicopathological factors, together with 85 tumors that we reported previously. EGFR mutations were found in 102 of the total 349 tumors, and seven tumors had two missense mutations. Reverse transcription-polymerase chain reaction of EGFR and subsequent subcloning analyses identified that the double mutations occurred in the same allele. Furthermore, in 202 NSCLC analyzed by Southern blotting, we identified 11 tumors with gene amplification of EGFR, with eight tumors containing a mutation in EGFR. Sequence analysis detected only weak or no signals of the wild-type allele in the eight tumors, strongly suggesting that the mutated allele was amplified selectively. These findings indicate that a dual genetic change of EGFR can occur in the same allele either with a possible second-hit mutation or with amplification, which may imply a more selective growth advantage in a cancer cell. Meanwhile, HER2 mutations and amplifications were found in six of 349 tumors and three of 202 tumors, respectively, and KRAS mutations in 21 of 349 tumors. Mutations of the EGFR and HER2 genes were more frequently found in female never or light-smoking patients with adenocarcinoma, and there were no tumors that had two or more mutations simultaneously among EGFR, HER2 and KRAS. The current study further demonstrates that a double genetic event in EGFR can occasionally occur in lung cancer, thus providing new clues for understanding the involvement of epidermal growth factor receptor signaling cascades in the pathogenesis of NSCLC.

The contribution of transforming growth factor- and epidermal growth factor signalling to airway remodelling in chronic asthma

Asthma is increasing in prevalence in the developing world, affecting approximately 10% of the world's population. It is characterised by chronic lung inflammation and airway remodelling associated with wheezing, shortness of breath, acute bronchial hyperresponsiveness to a variety of innocuous stimuli and a more rapid decline in lung function over time. Airway remodelling, involving proliferation and differentiation of mesenchymal cells, particularly myofibroblasts and smooth muscle cells, is generally refractory to corticosteroids and makes a major contribution to disease chronicity. Transforming growth factor-beta is a potent profibrogenic factor whose expression is increased in the asthmatic airways and is a prime candidate for the initiation and persistence of airway remodelling in asthma. This review highlights the role of transforming growth factor-beta in the asthmatic lung, incorporating biosynthesis, signalling pathways and functional outcome. In vivo, however, it is the balance between transforming growth factor-beta and other growth factors, such as epidermal growth factor, which will determine the extent of fibrosis in the airways. A fuller comprehension of the actions of transforming growth factor-beta, and its interaction with other signalling pathways, such as the epidermal growth factor receptor signalling cascade, may enable development of therapies that control airway remodelling where there is an unmet clinical need.

A dimeric version of the short N-cadherin binding motif HAVDI promotes neuronal cell survival by activating an N-cadherin/fibroblast growth factor receptor signalling cascade

The HAVDI and INPISGQ sequences have been identified as functional binding motifs in extracellular domain 1 (ECD1) of N-cadherin. Cyclic peptides containing a tandem repeat of the individual motifs function as N-cadherin agonists and stimulate neurite outgrowth. We now show that the cyclic peptide N-Ac- CHAVDINGHAVDIC-NH 2 (SW4) containing the HAVDI sequence in tandem is efficacious also in promoting the in vitro survival of several populations of central nervous system neurons in paradigms where fibroblast growth factor-2 (FGF-2) is active. SW4 supported the survival of rat postnatal cerebellar granule neurons plated in serum-free medium and limited the death of differentiated granule neurons induced to die by switch to low K + medium. In addition, SW4 rescued embryonic hippocampal and cortical neurons from injury caused by glutamic acid excitotoxicity. The neuroprotective effects of SW4 displayed a concentration dependence similar to those inducing neuritogenesis, were inhibited by a monomeric version of the same motif and by a specific FGF receptor antagonist (PD173074), and were not mimicked by the linear peptide. Inhibitors of the phosphatidylinositol 3-kinase (PI 3-kinase), MAP kinase, and p38 kinase signalling pathways did not interfere with SW4 function. These data suggest that SW4 functions by binding to and clustering N-cadherin in neurons and thereby activating and N-cadherin/FGF receptor signalling cascade, and propose that such agonists may represent a starting point for the development of therapeutic agents promoting neuronal cell survival and regeneration.

Epidermal growth factor receptor signaling cascade as target for tyrphostin (RG 50864) in epithelial cells: Paradoxical effects on mitogen-activated protein kinase kinase and mitogen-activated protein kinase activities

Tyrphostins are synthetic compounds that have been described as in vitro inhibitors of epidermal growth factor receptor (EGF-R) tyrosine kinase activity. The inhibitory effect of tyrphostins in intact cells has been shown only after prolonged treatment. However, these compounds appear to be readily incorporated, which suggests that tyrphostin acts indirectly on EGF-R. We studied the effects of a tyrphostin derivative, RG 50864, without preincubation in intact epithelial cells. We selected two human cell lines differing in degree of expression of the p185 erbB2 protein, which is closely related to EGF-R. We showed that tyrphostin (RG 50864) had no effect on EGF-dependent EGF-R tyrosine phosphorylation in the parental cell line. On the contrary, it prolonged the EGF-dependent EGF-R and p185 erbB2(V-E) tyrosine phosphorylation in p185 erbB2(V-E)-expressing cells. Because tyrphostin has been shown to be an inhibitor of p185 erbB2 and EGF-R in vitro, this finding indicates that the tyrphostin effect on p185 erbB2(V-E) and EGF-R was the result of an indirect mechanism in transfected cells. Tyrphostin treatment alone led to the activation of mitogen-activated protein (MAP) kinase kinase or MAP kinase or extracellular signal-regulated kinase kinase (MEK), suggesting that one of the tyrphostin targets was upstream of MEK1. MAP kinase, however, was not activated after tyrphostin treatment. This finding indicates that tyrphostin had another target in intact cells because MEK1 activation by tyrphostin alone did not correlate with MAP kinase activation. In the two cell lines, tyrphostin modified the time course of EGF-dependent MEK and MAP kinase activation. We conclude that whereas tyrphostins were designed to inhibit EGF-R tyrosine kinase activity, under our conditions EGF-R is not a physiological target for tyrphostin, nor is one of its related protein tyrosine kinases, p185 erbB2(V-E). On the contrary, our results show that tyrphostin targets are multiple, leading to complex effects on receptor signaling in these epithelial cells.