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

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

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

Subcellular localization of adapter protein Ruk, in HEK293 cells

Subcellular localization of adapter protein Ruk1 has been investigated in human embryonic kidney HEK293 cells transfected with pRc/CMV2/Ruk1-Glu-tag. Using immunofluorescence microscopy, it was shown that the recombinant protein was distributed diffusely in both cytoplasm and nucleus, but with punctated structures in the nucleus, which correspond in all probability to the nucleolus. The Ruk1 localization in the nucleus was confirmed by Western blotting of nucleic extracts prepared from transfected HEK293 cells using monoclonal anti-Glu-tag antibodies, as well as from nontransfected HEK293 and U937 cells using polyclonal anti-Ruk antibodies. The ability of affine purified Ruk1 Glu-tagged preparation to bind to DNA from calf thymus, but not to Escherichia coli DNA, was revealed by the immunodot-blot analysis. The nuclear localization of Ruk1 suggests that this adapter protein is involved in some new, yet unrecognized functions, in the eukaryotic cell nucleus

Comparative study of expression of adaptor proteins Ruk/CIN85 and CD2AP/CMS in normal andtumor human uterus tissues

Adaptor proteins play an important role in facilitating protein-protein interactions and subsequent formation of signalling networks. These proteins recruit binding partners to a specific location inside the cell, and also regulate their activity. Adaptor protein Ruk/CIN85 and its structural and functional homologue CD2AP/CMS are important components of different regulatory pathways involved in control of cell proliferation, adhesion, invasion and survival, and, thus, can play a role in uterine carcinogenesis. In this work, we set out a comparative study of expression of Ruk/CIN85 and CD2AP/CMS at the level of mRNA and protein in intact uterine tissues, as well as in benign and malignant uterine tumors of different histological types. In most cases, an increase of expression levels of Ruk/CIN85 full-length form mRNA and protein, as well as CD2AP/CMS protein, were observed in uterine tumors, comparing with surrounding normal uterine tissues. Characteristic feature of conditionally normal uterine tissues, as well as benign uterine lesions, was an elevated content of high-molecular mass Ruk/CIN85 forms of 140 and 130 kDa, while an increased expression level of low-molecular mass 40 and 30 kDa Ruk/CIN85 forms was observed in the malignant tissue samples. Our findings suggest that an abnormal expression patterns of adaptor proteins Ruk/CIN85 and CD2AP/CMS in uterine tumors, and, thus, the corresponding changes in the activity of downstream signalling pathways, might be involved in maintenance of the malignant phenotype