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We identified a novel pathway responsible for attenuation of p53 activity in human cancers. We demonstrate that AGR2, which is overexpressed in a variety of human cancers and provides a poor prognosis, up-regulates DUSP10 which subsequently inhibits p38 MAPK and prevents p53 activation by phosphorylation. Analysis of human breast cancers reveals that AGR2 specifically provides a poor prognosis in ERþ breast cancers with wildtype p53 but not ER-or mutant p53 breast cancers, and analysis of independent data sets show that DUSP10 levels also have prognostic significance in this specific sub-group of patients. These data not only reveal a novel pro-oncogenic signaling pathway mediating resistance to DNA damaging agents in human tumors, but also has implications for designing alternative strategies for modulation of wild-type p53 activity in cancer therapy.

BackgroundCisplatin and its derivatives are commonly used anti-cancer drugs. However, cisplatin has clinical limitations including serious side effects and frequent emergence of intrinsic or acquired resistance. Thus, the novel platinum(IV) complex LA-12 represents a promising treatment modality, which shows increased intracellular penetration resulting in improved cytotoxicity in various cancer cell lines, including cisplatin resistant cells.ResultsLA-12 disrupts cellular proliferation regardless of the p53 status in the cells, however the potency of the drug is greatly enhanced by the presence of a functional p53, indicating several mechanisms of action. Similarly to cisplatin, an interaction of LA-12 with molecular chaperone Hsp90 was proposed. Binding of LA-12 to Hsp90 was demonstrated by Hsp90 immunoprecipitation followed by platinum measurement using atomic absorption spectrometry (AAS). An inhibitory effect of LA-12 on Hsp90 chaperoning function was shown by decrease of Hsp90-assisted wild-type p53 binding to p21WAF1 promoter sequence in vitro and by accelerated ubiqutination and degradation of primarily unfolded mutant p53 proteins in cells exposed to LA-12.ConclusionsTo generalize our findings, LA-12 induced degradation of other Hsp90 client proteins such as Cyclin D1 and estrogen receptor was shown and proved as more efficient in comparison with cisplatin. This newly characterised molecular mechanism of action opens opportunities to design new cancer treatment strategy profitable from unique LA-12 properties, which combine DNA damaging and Hsp90 inhibitory effects.

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Regionální centrum aplikované molekulární onkologie, Masarykův onkologický ústav, Brno Souhrn Studium protein-proteinových interakcí in vivo se v současné době dostává do popředí zájmuumožňuje prokázat nebo upřesnit již známé protein-proteinové interakce a odhalit jejich inhibitory, zachytit konformační změny proteinů, objasnit nebo upřesnit signální kaskády v živé buňce s minimálním ovlivněním jejího buněčného prostředí. Jedním z možných přístupů umožňujících tuto charakteristiku jsou metody využívající rezonančního přenosu energiefl uorescenční (FRET) a jeho pozdější modifi kace bio luminiscenční (BRET). Tyto metody jsou založeny na zviditelnění proteinových interakcí pomocí excitace fl uorescenčních proteinů, ať už světelně nebo enzymaticky. Tyto přístupy umožňují nejen lokalizovat proteiny v buňce nebo jejich organelách (případně i v malých živočiších), ale i kvantifi kovat intenzitu fl uorescenčního nebo luminiscenčního signálu a odhalit pevnost vazby mezi interakčními partnery. V tomto příspěvku je objasněn princip metod FRET a BRET, jejich konkrétní aplikace při studiu protein-proteinových interakcí a jsou popsány dosavadní poznatky získané s využitím těchto metod a upřesňující ně kte ré molekulární a buněčné mechanizmy a signalizace související s nádorovou bio logií. Klíčová slova FRET-BRET-zobrazovací metody-protein-proteinové interakce in vivo Summary Nowadays, in vivo protein-protein interaction studies have become preferable detecting methods that enable to show or specify (already known) protein interactions and discover their inhibitors. They also facilitate detection of protein conformational changes and discovery or specifi cation of signaling pathways in living cells. One group of in vivo methods enabling these fi ndings is based on fl uorescent resonance energy transfer (FRET) and its bio luminescent modifi cation (BRET). They are based on visualization of protein-protein interactions via light or enzymatic excitation of fl uorescent or bio luminescent proteins. These methods allow not only protein localization within the cell or its organelles (or small animals) but they also allow us to quantify fl uorescent signals and to discover weak or strong interaction partners. In this review, we explain the principles of FRET and BRET, their applications in the characterization of protein-protein interactions and we describe several fi ndings using these two methods that clarify molecular and cellular mechanisms and signals related to cancer bio logy.