Hsp70 chaperone controls proteostasis and anti-stress responses in rapidly renewing cancer cells, making it an important target for therapeutic compounds. To date several Hsp70 inhibitors are presented with remarkable anticancer activity, however their clinical application is limited by the high toxicity towards normal cells. This study aimed to develop assays to search for the substances that reduce the chaperone activity of Hsp70 and diminish its protective function in cancer cells. On our mind the resulting compounds alone should be safe and function in combination with drugs widely employed in oncology. We constructed systems for the analysis of substrate-binding and refolding activity of Hsp70 and to validate the assays screened the substances representing most diverse groups of chemicals of InterBioScreen library. One of the inhibitors was AEAC, an N-amino-ethylamino derivative of colchicine, which toxicity was two-orders lower than that of parent compound. In contrast to colchicine, AEAC inhibited substrate-binding and refolding functions of Hsp70 chaperones. The results of a drug affinity responsive target stability assay, microscale thermophoresis and molecular docking show that AEAC binds Hsp70 with nanomolar affinity. AEAC was found to penetrate C6 rat glioblastoma and B16 mouse melanoma cells and reduce there the function of the Hsp70-mediated refolding system. Although the cytotoxic and growth inhibitory activities of AEAC were minimal, the compound was shown to increase the antitumor efficiency of doxorubicin in tumor cells of both types. When the tumors were grown in animals, AEAC administration in combination with doxorubicin exerted maximal therapeutic effect prolonging animal survival by 10–15 days and reducing tumor growth rate by 60%. To our knowledge, this is the first time that this approach to the high-throughput analysis of chaperone inhibitors has been applied, and it can be useful in the search for drug combinations that are effective in the treatment of highly resistant tumors.

The release of Hsp70 chaperone from tumor cells is found to trigger the full-scale anti-cancer immune response. Such release and the proper immune reaction can be induced by the delivery of recombinant Hsp70 to a tumor and we sought to explore how the endogenous Hsp70 can be transported to extracellular space leading to the burst of anti-cancer activity. Hsp70 transport mechanisms were studied by analyzing its intracellular tracks with Rab proteins as well as by using specific inhibitors of membrane domains. To study Hsp70 forms released from cells we employed the assay consisting of two affinity chromatography methods. Hsp70 content in culture medium and extracellular vesicles (EVs) was measured with the aid of ELISA. The properties and composition of EVs were assessed using nanoparticle tracking analysis and immunoblotting. The activity of immune cells was studied using an assay of cytotoxic lymphocytes, and for in vivo studies we employed methods of affinity separation of lymphocyte fractions. Analyzing B16 melanoma cells treated with recombinant Hsp70 we found that the chaperone triggered extracellular transport of its endogenous analog in soluble and enclosed in EVs forms; both species efficiently penetrated adjacent cells and this secondary transport was corroborated with the strong increase of Natural Killer (NK) cell toxicity towards melanoma. When B16 and CT-26 colon cancer cells before their injection in animals were treated with Hsp70-enriched EVs, a powerful anti-cancer effect was observed as shown by a two-fold reduction in tumor growth rate and elevation of life span. We found that the immunomodulatory effect was due to the enhancement of the CD8-positive response and anti-tumor cytokine accumulation; supporting this there was no delay in CT-26 tumor growth when Hsp70-enriched EVs were grafted in nude mice. Importantly, pre-treatment of B16 cells with Hsp70-bearing EVs resulted in a decline of arginase-1-positive macrophages, showing no generation of tumor-associated macrophages. In conclusion, Hsp70-containing EVs generated by specifically treated cancer cells give a full-scale and effective pattern of anti-tumor immune responses.

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Alpha-actinin 4 (ACTN4) is an actin-binding protein of the spectrin superfamily. ACTN4 is found both in the cytoplasm and nucleus of eukaryotic cells. The main function of cytoplasmic ACTN4 is stabilization of actin filaments and their binding to focal contacts. Nuclear ACTN4 takes part in the regulation of gene expression following by activation of certain transcription factors, but the mechanisms of regulation are not completely understood. Our previous studies have demonstrated the interaction of ACTN4 with the RelA/p65 subunit of NF-kappaB factor and the effect on its transcriptional activity in A431 and HEK293T cells. In the present work, we investigated changes in the composition of nuclear ACTN4-interacting proteins in non-small cell lung cancer cells H1299 upon stable RELA overexpression. We showed that ACTN4 was present in the nuclei of H1299 cells, regardless of the RELA expression level. The presence of ectopic RelA/p65 in H1299 cells increased the number of proteins interacting with nuclear ACTN4. Stable expression of RELA in these cells suppressed cell proliferation, which was further affected by simultaneous ACTN4 overexpression. We detected no significant effect on cell cycle but the apoptosis rate was increased in cells with a double RELA/ACTN4 overexpression. Interestingly, when expressed individually ACTN4 promoted proliferation of lung cancer cells. Furthermore, the bioinformatics analysis of gene expression in lung cancer patients suggested that overexpression of ACTN4 correlated with poor survival prognosis. We hypothesize that the effect of RELA on proliferation and apoptosis of H1299 cells can be mediated via affecting the interactome of ACTN4.