Ubiquitin-tagged substrates are degraded by the 26S proteasome, which is a multisubunit complex comprising a proteolytic 20S core particle capped by 19S regulatory particles. The approval of bortezomib for the treatment of multiple myeloma validated the 20S core particle as an anticancer drug target. Here we describe the small molecule b-AP15 as a previously unidentified class of proteasome inhibitor that abrogates the deubiquitinating activity of the 19S regulatory particle. b-AP15 inhibited the activity of two 19S regulatory-particle-associated deubiquitinases, ubiquitin C-terminal hydrolase 5 (UCHL5) and ubiquitin-specific peptidase 14 (USP14), resulting in accumulation of polyubiquitin. b-AP15 induced tumor cell apoptosis that was insensitive to TP53 status and overexpression of the apoptosis inhibitor BCL2. We show that treatment with b-AP15 inhibited tumor progression in four different in vivo solid tumor models and inhibited organ infiltration in an acute myeloid leukemia model. Our results show that the deubiquitinating activity of the 19S regulatory particle is a new anticancer drug target.

Background: Human melanoma cells are able to sustain low pH conditions characterizing many solid tumors. Results: Acidic stress in melanoma cells induces reduced nutrients uptake, inhibition of mammalian target of rapamycin, and activation of autophagy. Conclusion: Melanoma cells activate autophagy as a protective and adaptive response to acidic stress. Significance: Adaptation to acidosis via autophagy confirms the feasibility of anticancer therapy targeting autophagy.

Our data show that enhanced oxidative stress and ER stress are major determinants of the strong apoptotic response elicited by the 19S DUB inhibitor b-AP15.

Background: Intracellular free calcium ([Ca 2+ ] i ) is a key element in apoptotic signaling and a number of calcium-dependent apoptosis pathways have been described. We here used a chemical biology strategy to elucidate the relative importance of such different pathways.

The mechanism of multicellular drug resistance, defined as the reduced efficacy of chemotherapeutic drugs in solid tumors is incompletely understood. Here we report that colon carcinoma cells cultured as 3D microtissues (spheroids) display dramatic increases in the expression of a subset of type I interferon-(IFN)-stimulated genes (ISGs). A similar gene signature was associated previously with resistance to radiation and chemotherapy, prompting us to examine the underlying biological mechanisms. Analysis of spheroids formed by different tumor cell lines and studies using knock-down of gene expression showed that cell crowding leads to the induction of IFN regulatory factor-9 (IRF9) which together with STAT2 and independently of IFNs, is necessary for ISG upregulation. Increased expression of IRF9 alone was sufficient to induce the ISG subset in monolayer cells and to confer increased resistance to clinically used cytotoxic drugs. Our data reveal a novel mechanism of regulation of a subset of ISGs, leading to drug resistance in solid tumors.Therapy resistance represents a major hurdle in the management of cancer patients. Several resistance mechanisms have been described (e.g., overexpression of efflux pumps, mutations in the DNA repair system, activation of the antiapoptotic machinery). Multicellular resistance, suggested to be important for the resistance of advanced solid tumors, 1 is due both to limited penetration of drugs into tumor parenchyma 2 and to the presence of quiescent cell populations in hypoxic regions. These populations are insensitive to clinically used drugs and are believed to repopulate tumors between therapy cycles ("regrowth resistance"). 3 Most experimental studies of resistance mechanisms and anticancer drug testing are performed with monolayer cell cultures, which is not an optimal experimental system and has limitations. Three-dimensional (3D) cell cultures, as the well-established multicellular spheroid (MCS) system, reflect the in vivo structure of solid tumors and more adequately reproduce the metabolic microenvironment of tumor tissue. The formation of a 3D structure by tumor cells is accompanied by changes in cells and their microenvironment: the cells are more tightly packed than in 2D and have differences in nutrient supply, oxygen tension, pH, proliferation, resistance to apoptosis. 4,5 MCS are more resistant to chemotherapy than cells grown in 2D. 6,7 To examine the mechanisms of drug resistance, we performed a gene expression profiling of a colon cancer cell line MCS. Among highly upregulated genes in MCS we found a subset of type I interferonstimulated genes (ISGs). A similar signature designated the interferon-related DNA damage signature (IRDS) was previously associated with resistance to radiotherapy. 8,9 The full set of ISGs is activated by the transcription factor complex ISGF3, consisting of tyrosine-phosphorylated STAT1 and STAT2, and the DNA-binding protein IRF9. However, ISGF3 lacking tyrosine phosphorylation of STAT1 and STAT2 (unphosphorylated ISGF3, U-ISGF3) can ...