Advances in Targeting Signal Transduction Pathways

Over the past few years, significant advances have occurred in both our understanding of the complexity of signal transduction pathways as well as the isolation of specific inhibitors which target key components in those pathways. Furthermore critical information is being accrued regarding how genetic mutations can affect the sensitivity of various types of patients to targeted therapy. Finally, genetic mechanisms responsible for the development of resistance after targeted therapy are being discovered which may allow the creation of alternative therapies to overcome resistance. This review will discuss some of the highlights over the past few years on the roles of key signaling pathways in various diseases, the targeting of signal transduction pathways and the genetic mechanisms governing sensitivity and resistance to targeted therapies

Apocynin attenuates motility and induces transition from sustained to transient EGF-dependent Akt activation in MCF-7 cells that overexpress adaptor protein Ruk/CIN85

Aim. To study a possible involvement of NADPH oxidases in the control of cell motility and Akt signaling in the human breast adenocarcinoma MCF-7 cells that stably overexpress the full-length form of adaptor protein Ruk/CIN85. Methods. Cell motility was studied by a transwell migration assay. The dynamics of EGF-induced Akt activation was investigated by Western blot analysis. Results. It has been shown that apocynin, an inhibitor of the assembly of plasma membrane NADPH oxidases, substantially attenuates the motility of Ruk/CIN85 overexpressing MCF-7 cells (subclone G10) in comparison with control cells. In addition, apocynin induced the transition from sustained to transient EGF-dependent Akt activation in G10 cells and did not influence transient Akt activation in control cells. Conclusions. The data obtained can suggest that ROS produced by NADPH oxidases are signaling components, upstream to Akt kinase, that mediate the increased migratory potential of Ruk/CIN85 overexpressing MCF-7 cells

Adaptor protein Ruk/CIN85 modulates resistance to doxorubicin of murine 4T1 breast cancer cells

The acquisition of chemoresistance in the course of tumor progression includes activation of membrane ABC transporters, detoxification enzymes, cell cycle deceleration and activation of specific signaling pathways such as Akt/mTOR, MAPK, NF-κB. Adaptor proteins play an essential role in the assembly of supramolecular signaling complexes, maintaining and directing the intracellular signaling. One of such proteins, called Ruk/CIN85, is strongly associated with malignant transformation and metastasis. In present study we investigated the Ruk/CIN85 effect of up/down-regulation on the transforming potential and doxorubicin resistance of highly aggressive mouse breast adenocarcinoma 4T1 cells. It was demonstrated that 4T1 cells overexpressing Ruk/CIN85 possessed increased resistance to doxorubicin (in the range of concentrations 0.1–10.0 µM) while knockdown cells were the most sensitive. Also, high levels of Ruk/CIN85 in 4T1 cells positively correlated with their ability to form colonies in semi-solid agar. Ruk/CIN85-overexpressing cells formed four times more colonies in comparison with Ruk/CIN85 nockdown cells, the growth of which revealed higher resistance to doxorubicin action

Apocynin attenuates motility and induces transition from sustained to transient EGF-dependent Akt activation in MCF-7 cells that overexpress adaptor protein Ruk/CIN85

Aim. To study a possible involvement of NADPH oxidases in the control of cell motility and Akt signaling in the human breast adenocarcinoma MCF-7 cells that stably overexpress the full-length form of adaptor protein Ruk/CIN85. Methods. Cell motility was studied by a transwell migration assay. The dynamics of EGF-induced Akt activation was investigated by Western blot analysis. Results. It has been shown that apocynin, an inhibitor of the assembly of plasma membrane NADPH oxidases, substantially attenuates the motility of Ruk/CIN85 overexpressing MCF-7 cells (subclone G10) in comparison with control cells. In addition, apocynin induced the transition from sustained to transient EGF-dependent Akt activation in G10 cells and did not influence transient Akt activation in control cells. Conclusions. The data obtained can suggest that ROS produced by NADPH oxidases are signaling components, upstream to Akt kinase, that mediate the increased migratory potential of Ruk/CIN85 overexpressing MCF-7 cells

Transcriptional regulation of NOX genes expression in human breast adenocarcinoma MCF-7 cells is modulated by adaptor protein Ruk/CIN85

NADPH oxidases are key components of redox-dependent signaling networks involved in the control of cancer cell proliferation, survival and invasion. The data have been accumulated that demonstrate specific expression patterns and levels of NADPH oxidase homologues (NOXs) and accessory genes in human cancer cell lines and primary tumors as well as modulation of these parameters by extracellular cues. Our previous studies revealed that ROS production by human colorectal adenocarcinoma HT-29 cells is positively correla­ted with adaptor protein Ruk/CIN85 expression while increased levels of Ruk/CIN85 in weakly invasive human breast adenocarcinoma MCF-7 cells contribute to their malignant phenotype through the constitutive activation of Src/Akt pathway. In this study, to investigate whether overexpression of Ruk/CIN85 in MCF-7 cells can influence transcriptional regulation of NOXs genes, the subclones of MCF-7 cells with different levels­ of Ruk/CIN85 were screened for NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2 as well as for regulatory subunit p22Phox mRNA contents by quantitative RT-PCR (qPCR). Systemic multidirectional changes in mRNA levels for NOX1, NOX2, NOX5, DUOX2 and p22Phox were revealed in Ruk/CIN85 overexpressing cells in comparison to control WT cells. Knocking down of Ruk/CIN85 using technology of RNA-interference resulted in the reversion of these changes. Further studies are necessary to elucidate, by which molecular mechanisms Ruk/CIN85 could affect transcriptional regulation of NOXs genes

Multiple molecular forms of adaptor protein Ruk/CIN85 specifically associate with different subcellular compartments in human breast adenocarcinoma MCF-7 cells

Ruk/CIN85 is a receptor-proximal ‘signalling’ adaptor that possesses three SH3 domains, Pro- and Ser-rich regions and C-terminal coiled-coil domain. It employs distinct domains and motifs to act as a transducer platform in intracellular signalling. Based on cDNA analysis, various isoforms of Ruk/CIN85 with different combination of protein-protein interaction domains as well as additional Ruk/CIN85 forms that are the products of post-translational modifications have been demonstrated. Nevertheless, there is no precise information regarding both the subcellular distribution and the role of Ruk/CIN85 multiple molecular forms in cellular responses. Using MCF-7 human breast adenocarcinoma cells and cell fractionation technique, specific association of Ruk/CIN85 molecular forms with different subcellular compartments was demonstrated. Induction of apoptosis of MCF-7 cells by doxorubicin treatment or by serum deprivation resulted in the system changes of Ruk/CIN85 molecular forms intracellular localization as well as their ratio. The data obtained provide a new insight into potential physiological significance of Ruk/CIN85 molecular forms in the regulation of various cellular functions

Ruk is ubiquitinated but not degraded by the proteasome

The regulator of ubiquitous kinase (Ruk) protein, also known as CIN85 or SETA, is an adaptor-type protein belonging to the CD2AP/CMS family. It was found in complexes with many signaling proteins, including phosphoinositol (PtdIns) 3-kinase (EC 2.7.1.137), Cbl, GRB2, p130Cas and Crk. Functional analysis of these interactions, implicated Ruk in the regulation of apoptosis, receptor endocytosis and cytoskeletal rearrangements. We have recently demonstrated that overexpression of Ruk induces apoptotic death in neurons, which could be reversed by activated forms of PtdIns 3-kinase and PKB/Akt. Furthermore, Ruk was shown to be a negative regulator of PtdIns 3-kinase activity through binding to its P85 regulatory subunit [Gout, I., Middleton, G., Adu, J., Ninkina, N. N., Drobot, L. B., Filonenko, V., Matsuka, G., Davies, A.M., Waterfield, M. & Buchman, V. L. (2000) Embo J.19, 4015–4025]. Here, we report for the first time, that all three isoforms of Ruk (L, M and S) are ubiquitinated. Specific interaction between the E3 ubiquitin ligase Cbl and all three Ruk isoforms was demonstrated by coexpression studies in Hek293 cells. The interaction of Ruk M and S isoforms with Cbl was found to be mediated via heterodimerization with Ruk L. The use of proteosomal and lysosomal inhibitors clearly indicated that ubiquitination of Ruk L does not lead to its degradation. Based on this study, we propose a possible mechanism for the regulation of Ruk function by ubiquitination