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International audience; The p53 protein is a key tumor suppressor in mammals. In response to various forms of genotoxic stress p53 stimulates expression of genes whose products induce cell cycle arrest and/or apoptosis. An E3-ubiquitin ligase, Mdm2 (mouse-double-minute 2) and its human ortholog Hdm2, physically interact with the amino-terminus of p53 to mediate its ubiquitin-mediated degradation via the proteasome. Thus, pharmacological inhibition of the p53-Mdm2 interaction leads to overall stabilization of p53 and stimulation of its anti-tumorigenic activity. In this study we characterize the biological effects of a novel class of non-genotoxic isatin Schiff and Mannich base derivatives (ISMBDs) that stabilize p53 on the protein level. The likely mechanism behind their positive effect on p53 is mediated via the competitive interaction with Mdm2. Importantly, unlike Nutlin, these compounds selectively promoted p53-mediated cell death. These novel pharmacological activators of p53 can serve as valuable molecular tools for probing p53-positive tumors and set up the stage for development of new anti-cancer drugs.

The proteasome is the key player in targeted degradation of cellular proteins and serves as a therapeutic target for treating several blood malignancies. Although in general, degradation of proteins via the proteasome requires their ubiquitination, a subset of proteins can be degraded independently of their ubiquitination by direct interaction with subunits of the 20S proteasome core. Thus, investigation of the proteasome-associated proteins may help identify novel targets of proteasome degradation and provide important insights into the mechanisms of malignant cell proteostasis. Here, using biochemical purification of proteasomes from multiple myeloma (MM) cells followed by mass-spectrometry we have uncovered 77 proteins in total that specifically interacted with the 20S proteasome via its PSMA3 subunit. Our GST pull-down assays followed by western blots validated the interactions identified by mass-spectrometry. Eleven proteins were confirmed to bind PSMA3 only upon apoptotic conditions induced by a combined treatment with the proteasome inhibitor, bortezomib, and genotoxic drug, doxorubicin. Nine of these eleven proteins contained bioinformatically predicted intrinsically disordered regions thus making them susceptible to ubiquitin-independent degradation. Importantly, among those proteins five interacted with the ubiquitin binding affinity matrix suggesting that these proteins may also be ubiquitinylated and hence degraded via the ubiquitin-dependent pathway. Collectively, these PSMA3-interacting proteins represent novel potential substrates for 20S proteasomes upon apoptosis. Furthermore, these data may shed light on the molecular mechanisms of cellular response to chemotherapy. Abbreviations: BD: bortezomib/doxorubicin treatment; CDK: cyclin-dependent kinases; CHCA: α-cyanohydroxycinnamic acid; IDP: intrinsically disordered proteins; IDR: intrinsically disordered regions; IPG: immobilized pI gradient; MALDI TOF/TOF: matrix-assisted laser desorption/ionization time-of-flight tandem mass-spectrometry; MM: multiple myeloma; ODC: ornithine decarboxylase; PI: proteasomal inhibitors; PSMA: alpha-type 20S proteasome subunits; PTMs: post-translational modifications; SDS-PAGE: sodium dodecylsulphate polyacrylamide gel electrophoresis; UIP: ubiquitin-independent proteasomal proteolysis.

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.

The main feature of tumor cells is their ability to divide unrestrictedly. Cellular division critically depends on successful mitosis. Thus, perhaps unsurprisingly, among the most powerful anticancer drugs are the ones that target mitosis. Proper segregation of chromosomes during mitosis is ensured by a highly coordinated and dynamic process of polymerization/depolymerization of mitotic spindle microtubules. Hence, natural microtubule-poisoning drugs, such as Paclitaxel (PTX or Taxol) and vinblastine are commonly used as anti-neoplastic agents for the treatment of solid tumors, including breast, ovarian, lung, and head and neck malignancies.1 The consequence of treatment with these drugs is mitotic arrest, which, in turn, activates the spindle assembly checkpoint (SAC). Tumor cells employ several mechanisms to skip SAC, including ubiquitin-dependent degradation/inactivation of the cyclin B/Cdk1 complex by the anaphase-promoting complex/cyclosome (APC/C). The latter is an E3 ubiquitin ligase that operates via 26S proteasome2 and is positively regulated by cyclin B/Cdk1-mediated phosphorylation. Inactivation of Cdk1 results in attenuation of the APC/C activity, which, in turn, is required for degradation of securin and the release of sister chromatids, thus promoting the metaphase-to-anaphase transition. Another mechanism of slippage from SAC is attenuation of mitotic kinase Aurora A, which is necessary for the proper separation of centrosomes after the mitotic spindle has been formed.3 However, for escaping SAC, cancer cells pay their toll, i.e., they die because of mitotic catastrophe.4 Unfortunately, during the prolonged anti-mitotic therapy, tumor cells acquire resistance to PTX by overexpressing mutated versions of tubulin proteins inert to PTX, or by modulating the expression of micro-RNAs that target the Bcl-2 family of proteins. Therefore, the problem of overcoming PTX resistance urgently requires its solution and warrants extensive studies in this area. The idea of combining physiological (39.5–45 °C) hyperthermia (HT) with cancer therapy is currently being actively explored in clinical studies. For example, several phase III trials comparing radiotherapy alone or with hyperthermia have shown a beneficial effect of hyperthermia in terms of local control of recurrent breast cancer and malignant melanoma and survival (e.g., head and neck lymph-node metastases, glioblastoma, cervical carcinoma, etc.).5 In this respect, the study by Giovinazzi et al. published in the present issue of Cell Cycle is timely and interesting. The authors showed that hyperthermia (HT) and APC/C inhibition have opposite effects on the fate of tumor cells arrested in mitosis; HT provoked mitotic slippage of breast tumor cells, resulting in mitotic catastrophe, whereas the inhibitor of APC/C, proTAME, on the contrary, prolonged the time of mitosis and led to apoptosis. Importantly, these effects were independent of the means by which cells were arrested in mitosis, i.e., either by treatment with Aurora A inhibitor MLN8054 or with Paclitaxel (PTX). On the therapeutic side, the authors showed that HT enhanced the cytotoxic effect of PTX on breast cancer cells irrespective of their status toward sensitivity to PTX. How HT promotes the escape of PTX-arrested cells is yet not clear. In general, HT was reported to disrupt the microtubule network. However, in the case of prior PTX treatment, the microtubule bundles remained intact.6 The most apparent effect of HT was observed at the level of centrosomes. In this respect, members of the heat shock proteins family (HSPs) play the pivotal role in assisting repair of centrosomes after heat shock. Microinjection of Hsp73 antibodies retarded recovery of the interphase centrosome structure and microtubule re-assembly after HT, whereas injection of purified Hsp73 before heat shock enhanced these processes.7 Collectively, these findings suggest an intriguing possibility that the combination of chemical inhibitors of HSPs (17-AAG and KNK437, or quercetin)8 with PTX and HT will facilitate the slippage of cancer cells from mitosis and, hence, augment the antitumor effect of combination therapy, especially in the case of PTX-resistant malignancies.