E. V. Korobko

Pdcd4 protein and mRNA level alterations do not correlate in human lung tumors

Pdcd4 (programmed cell death 4) gene is tumor suppressor which expression is frequently down-regulated in tumors, which is considered as a diagnostic and prognostic marker as well as promising target for anti-cancer therapy. Pdcd4 protein is a target for post-translational regulation by phosphorylation marking Pdcd4 for degradation. We questioned if Pdcd4 mRNA decline in human lung tumors is accompanied by proportional depletion of Pdcd4 protein. We found that Pdcd4 protein-to-mRNA ratio varies greatly in human lung cancer cell lines. In squamous cell carcinoma samples where Pdcd4 mRNA suppression was found to be a typical event, Pdcd4 protein level frequently remained unchanged or even up-regulated. Our studies demonstrate that at least in squamous cell carcinoma, alterations in Pdcd4 mRNA and protein levels are not directly linked, and this fact should be taken into consideration when developing Pdcd4-based anti-cancer therapeutic approaches.

Immunotherapy with autologous tumor cells engineered to secrete Tag7/PGRP, an innate immunity recognition molecule

Recent studies indicate that the innate component of immune defense plays an important role in the establishment of antigen‐specific immune response. We have previously isolated a novel mouse gene tag7/PGRP that was shown to be involved in the innate component of the immune system, and its insect homologue is an upstream mediator of Toll signaling in Drosophila.

Resistance to tumor necrosis factor induced apoptosis in vitro correlates with high metastatic capacity of cells in vivo.

TNF is one of the cytokines secreted by the cells of the immune system. Our data demonstrate that those cell lines lacking capability to form metastatic tumors in vivo are susceptible to TNF induced apoptosis in vitro. However, cell lines with high metastatic potential are resistant to TNF in vitro. Furthermore, the same cell lines were resistant to cytolytic action of other cytotoxic proteins secreted by LAK cells. Our data showed that TNF resistance in vitro correlates with the increased level of transcription factor NF-kappaB. This finding may provide a tool to improve current protocols of immunotherapy and insights to how tumor cells are or are not killed by LAK cells.

Pdcd4 tumor suppressor: Properties, functions, and possible applications in oncology

The inactivation of tumor suppressors and activation of protooncogenes are critical events in malignant cell transformation and tumor progression. Pdcd4 encodes a protein with tumor suppressor functions, which accounts for the increased interest in Pdcd4 as a potential diagnostic and prognostic marker and a target for anti-neoplastic therapy. The review summarizes the well-known properties and functions of Pdcd4 tumor suppressor and the mechanisms of its regulation in tumor tissues, the role of Pdcd4 in cellular transformation and tumor progression, and its potential practical application in oncology.

Apoptotic cleavage of rabaptin-5-like proteins and a model for rabaptin-5 inactivation in apoptosis.

Intracellular membrane transport from the plasma membrane is one of the processes affected in apoptotic cells. Apoptotic inhibition of endosomal transport occurs due to cleavage of Rabaptin-5, an effector of small GTPase Rab5, which results in inhibition of early endosome fusion. Recently several novel Rabaptin-5-like proteins were identified. We investigated whether Rabaptin-5-like proteins, Rabaptin-5? and Rabaptin-5?, are also cleaved in apoptosis and found that both proteins are cleaved in apoptotic cell extracts by caspase-3-related proteases. This suggests that functional inactivation of these proteins is necessary for apoptotic cell death. We also mapped a novel, N-terminal, putative Rab5 binding site in Rabaptin-5-like proteins, which becomes physically separated from the previously known C-terminal Rab5 binding site after apoptotic cleavage of these proteins. Presence of the second Rab5 binding site provides a new insight into Rabaptin-5 function in early endosome fusion and a mechanistic model for functional inactivation of Rabaptin-5 in apoptosis.

Subcellular localization of MAK-V/Hunk protein kinase expressed in COS-1 cells

MAK‐V/Hunk is a MARK/Par‐1‐related protein kinase, whose function is unknown. We studied the subcellular localization of MAK‐V/Hunk in COS‐1 cells by immunofluorescence. It has a nucleocytoplasmic distribution and is localized to the centrosome, as indicated by co‐localization with γ‐tubulin. A putative kinase‐deficient mutant, with a mutation in the invariant lysine residue in the catalytic domain, was not targeted to the nucleus or centrosome. These results suggest that the nuclear and centrosomal targeting of MAK‐V/Hunk is specific, and is likely to be coupled to its catalytic activity.

Nanoantibodies for detection and blocking of bioactivity of human vascular endothelial growth factor A(165).

Nanoantibodies (single-domain antibodies, nanobodies) derived from noncanonical single-chain immunoglobulins provide an attractive tool for in vitro and in vivo diagnostics as well as for development of targeted drugs for clinical use. Nanoantibodies against several clinically important targets have been developed and are actively investigated. However, no development of nanoantibodies against vascular endothelial growth factor VEGF-A165 has been reported. We describe here the generation of nanoantibodies derived from single-chain Bactrian camel immunoglobulins directed against VEGF-A165. We demonstrate that these nanoantibodies are suitable for enzyme-linked immunoassay to quantify human VEGF-A165 as well as for blocking its activity. Our results provide a basis for diagnostic kit development for quantification of VEGF-A165, which emerges as a biomarker useful in various pathological conditions. In addition, the nanoantibodies might be used for development of therapeutic molecules targeting VEGF-A165-dependent pathological neoangiogenesis.

Pro-survival activity of the MAK-V protein kinase in PC12 cells

MAK-V/HUNK is an AMPK-like protein kinase which role remains largely enigmatic. MAK-V is preferentially expressed in specific cell populations in brain, kidney, mammary gland and in embryonic development.1–4 Besides that, mak-v cDNA has been isolated in a result of comparative studies of mouse tumor kinomes2,5 with later study demonstrated its frequent overexpression in human breast tumors.6 Together the data obtained strongly suggest that MAK-V might play some role in developmental processes and in nervous system and potentially its expression might determine some specific properties of tumor cells. However these suggestions are largely based on the mak-v expression pattern and just a few experimental evidences supporting them became currently available.3,7 In an attempt to reveal effects of MAK-V on cell behavior, we have used PC12 cells with doxycycline (DOX)-inducible expression of the C-terminally FLAG-tagged MAK-V protein (referred as WT cells). To ultimately address a question of specificity for the effects observed upon MAK-V expression, we generated PC12 cells with inducible expression of the kinase-dead MAK-V mutant with catalytic lysine substituted for arginine thus rendering enzyme inactive (KR cells, Sup. Fig. 1). Based on the expression pattern, MAK-V is expected to play a role in nervous system which has been recently confirmed by its involvement in Xenopus brain pattering and morphogenesis.7 We have used WT cells to directly assess if MAK-V expression affects differentiation along the neural pathway induced by nerve growth factor (NGF). NGF treatment resulted in appearance of larger number of WT cells with neurites when MAK-V expression has been induced but percentages of differentiated cells were the same irrespectively of MAK-V expression. Instead, total cell number at the end of NGF treatment was higher when cells were treated with DOX thus indicating their improved viability (Sup. Fig. 2). Indeed, when grown on collagen IV-treated surface, the conditions used in differentiation experiments, WT but not parental PC12TetOn cells showed reduced viability when not treated with DOX to induce MAK-V expression (Fig. 1A and Sup. Fig. 3). This was accompanied by the increase in lactate dehydrogenase (LDH) release into cell culture medium, higher caspase-3/7 activation and elevated Annexin V-FITC immunoreactivity (Fig. 1B–D). This indicates that collagen IV induces apoptotic cell death in WT cells and MAK-V expression allows cells to escape from it. Similar to WT cells, KR cells showed suppressed viability when plated on collagen IV (Sup. Fig. 3). However induction of catalytically inactive MAK-V mutant expression failed to improve their viability that was concomitant with no change in LDH release (Fig. 1A and B). Therefore improved viability of WT cells upon contact with collagen IV can be entirely attributed to the expression of catalytically active MAK-V protein kinase. WT and KR cells showed decreased viability upon contacting with collagen IV but this effect was not so pronounced when cells were grown on laminin-coated surface (Sup. Fig. 4A). At the same time, only when plated on collagen IV but not on laminin WT cells acquired spreaded morphology (Sup. Fig. 4B) indicating that formation of contacts with collagen IV but not with laminin induces rearrangements in actin cytoskeleton. Thus it is feasible that collagen IV-induced actin rearrangement can result in activation of apoptotic machinery by some unknown mechanism which is alleviated by MAK-V protein kinase activity. Interestingly, recent study linked MAK-V with actin cytoskeleton rearrangement through regulation of cofilin phoshorylation.8 However induction of MAK-V expression in WT cells had no effect on cofilin phosphorylation (Sup. Fig. 4C). This observation further supports the emerging suggestion that MAK-V activity depends on the specific cellular context and might significantly differ in different types of cells8–11 and rules out a possibility that MAK-V in WT PC12 clonal derivative directly affects actin cytoskeleton rearrangement induced by collagen IV through cofilin-1. Integrin signaling activates numerous signal transduction events including those affecting cell viability and survival, among them ERK and Akt pathways. Moreover, MAK-V was identified as one of the candidate protein kinases that might modulate EGF signaling.12 However MAK-V expression in WT cells did not alter activation of ERK signaling. Similarly, no change in Akt signaling pathway activation was evident in MAK-V expressing WT cells (Sup. Fig. 5). Therefore MAK-V protein kinase affects distinct from the assayed molecular pathway in WT cells to inhibit their apoptotic death induced by collagen IV binding. Summarizing, we showed that MAK-V expression can improve cell viability as exemplified by WT cells grown on collagen IV. This is the first study to demonstrate pro-survival activity of the MAK-V protein kinase molecular mechanisms underlying which are yet to be identified. Our findings ultimately prove previously suggested role of MAK-V in cell viability regulation derived from the results of two high-throughput RNAi screens.10,11 Importantly, MAK-V expression in tumor cells might benefit their survival in otherwise restrictive conditions, and this conclusion is supported by the reported predominant MAK-V expression in aggressive subsets of tumors.9 Although functional consequences of MAK-V expression might vary depending on the cellular context,8–11 the revealed pro-survival and anti-apoptotic activity of MAK-V signifies its role in tumor cells and prompt future research aiming to validate MAK-V as a novel target to treat cancer. In addition, MAK-V is characterized by highly patterned expression in embryonic development assuming its involvement in developmental processes. Our findings suggest a novel role of MAK-V in developmental processes through regulating cell survival and directed migration in response to contacts with specific components of extracellular matrix.

Hunk/Mak-v is a negative regulator of intestinal cell proliferation

BackgroundConditional deletion of the tumour suppressor gene Apc within the murine intestine results in acute Wnt signalling activation. The associated over-expression of a myriad of Wnt signalling target genes yields phenotypic alterations that encompass many of the hallmarks of neoplasia. Previous transcriptomic analysis aimed at identifying genes that potentially play an important role in this process, inferred the Hormonally upregulated Neu-associated kinase (HUNK/Mak-v/Bstk1) gene as a possible candidate. Hunk is a SNF1 (sucrose non fermenting 1)-related serine/threonine kinase with a proposed association with many different tumour types, including colorectal cancer.MethodsHere we describe the generation of a novel Hunk kinase deficient mouse which has been used to investigate the involvement of Hunk-kinase activity in intestinal homeostasis and tumourigenesis.ResultsWe show that in the morphologically normal intestine, Hunk-kinase negatively regulates epithelial cell proliferation. However, the increase in cell proliferation observed in the Hunk kinase deficient intestine is counteracted by increased cell migration, thereby maintaining intestinal homeostasis. Using qRT-PCR, we further demonstrate that Hunk is significantly over-expressed in Apc deficient / Wnt-signalling activated intestinal tissue. Using the classical intestinal tumourigenesis ApcMin mouse model we show that loss of Hunk-kinase activity significantly reduced tumour initiation rates in the small intestine. However, an accompanying increase in the size of the tumours counteracts the impact this has on overall tumour burden or subsequently survival.ConclusionsIn the intestinal setting we demonstrate that Hunk has a role in normal intestinal proliferation and homeostasis and, although it does not alter overall survival rates, activity of this kinase does impact on tumour initiation rates during the early stages in tumourigenesis in the small intestine.

Identification of Nedd4 E3 ubiquitin ligase as a binding partner and regulator of MAK-V protein kinase.

MAK-V/Hunk is a scantily characterized AMPK-like protein kinase. Recent findings identified MAK-V as a pro-survival and anti-apoptotic protein and revealed its role in embryonic development as well as in tumorigenesis and metastasis. However molecular mechanisms of MAK-V action and regulation of its activity remain largely unknown. We identified Nedd4 as an interaction partner for MAK-V protein kinase. However, this HECT-type E3 ubiquitin ligase is not involved in the control of MAK-V degradation by the ubiquitin-proteasome system that regulates MAK-V abundance in cells. However, Nedd4 in an ubiquitin ligase-independent manner rescued developmental defects in Xenopus embryos induced by MAK-V overexpression, suggesting physiological relevance of interaction between MAK-V and Nedd4. This identifies Nedd4 as the first known regulator of MAK-V function.