Slavica Brnjic

Inhibition of proteasome deubiquitinating activity as a new cancer therapy

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.

Induction of Tumor Cell Apoptosis by a Proteasome Deubiquitinase Inhibitor Is Associated with Oxidative Stress

AIMS b-AP15 is a recently described inhibitor of the USP14/UCHL5 deubiquitinases (DUBs) of the 19S proteasome. Exposure to b-AP15 results in blocking of proteasome function and accumulation of polyubiquitinated protein substrates in cells. This novel mechanism of proteasome inhibition may potentially be exploited for cancer therapy, in particular for treatment of malignancies resistant to currently used proteasome inhibitors. The aim of the present study was to characterize the cellular response to b-AP15-mediated proteasome DUB inhibition. RESULTS We report that b-AP15 elicits a similar, but yet distinct, cellular response as the clinically used proteasome inhibitor bortezomib. b-AP15 induces a rapid apoptotic response, associated with enhanced induction of oxidative stress and rapid activation of Jun-N-terminal kinase 1/2 (JNK)/activating protein-1 signaling. Scavenging of reactive oxygen species and pharmacological inhibition of JNK reduced b-AP15-induced apoptosis. We further report that endoplasmic reticulum (ER) stress is induced by b-AP15 and is involved in apoptosis induction. In contrast to bortezomib, ER stress is associated with induction of α-subunit of eukaryotic initiation factor 2 phosphorylation. INNOVATION The findings establish that different modes of proteasome inhibition result in distinct cellular responses, a finding of potential therapeutic importance. CONCLUSION 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.

Identification of a Novel Topoisomerase Inhibitor Effective in Cells Overexpressing Drug Efflux Transporters

Background Natural product structures have high chemical diversity and are attractive as lead structures for discovery of new drugs. One of the disease areas where natural products are most frequently used as therapeutics is oncology. Method and Findings A library of natural products (NCI Natural Product set) was screened for compounds that induce apoptosis of HCT116 colon carcinoma cells using an assay that measures an endogenous caspase-cleavage product. One of the apoptosis-inducing compounds identified in the screen was thaspine (taspine), an alkaloid from the South American tree Croton lechleri. The cortex of this tree is used for medicinal purposes by tribes in the Amazonas basin. Thaspine was found to induce conformational activation of the pro-apoptotic proteins Bak and Bax, mitochondrial cytochrome c release and mitochondrial membrane permeabilization in HCT116 cells. Analysis of the gene expression signature of thaspine-treated cells suggested that thaspine is a topoisomerase inhibitor. Inhibition of both topoisomerase I and II was observed using in vitro assays, and thaspine was found to have a reduced cytotoxic effect on a cell line with a mutated topoisomerase II enzyme. Interestingly, in contrast to the topoisomerase II inhibitors doxorubicin, etoposide and mitoxantrone, thaspine was cytotoxic to cell lines overexpressing the PgP or MRP drug efflux transporters. We finally show that thaspine induces wide-spread apoptosis in colon carcinoma multicellular spheroids and that apoptosis is induced in two xenograft mouse models in vivo. Conclusions The alkaloid thaspine from the cortex of Croton lechleri is a dual topoisomerase inhibitor effective in cells overexpressing drug efflux transporters and induces wide-spread apoptosis in multicellular spheroids.

Restriction of cisplatin induction of acute apoptosis to a subpopulation of cells in a three-dimensional carcinoma culture model

Cisplatin is a clinically important chemotherapeutical agent used to treat epithelial malignancies. High concentrations (20–100 μM) of cisplatin have been used in numerous studies to induce apoptosis of carcinoma cells grown in monolayer culture over 24–48 hr. These conditions may not be relevant to 3‐D tumor tissue in vivo and the importance of apoptosis for tumor response is controversial. We here studied the effects of cisplatin on a 3‐D colon carcinoma in vitro model (multicellular spheroids). Cisplatin at a dose of 40 μM induced active caspase‐3 preferentially in the peripheral 30 μm cell layer of spheroids, mainly during late stages (72–96 hr). The p53 response to cisplatin was also largely confined to peripheral cell layers. Despite the use of a high cisplatin concentration, a significant fraction of the cells in the spheroids survived treatment. A high proportion of surviving cells stained positive for β‐galactosidase, a marker of premature senescence. Cells growth‐arrested by cisplatin treatment showed a higher spontaneous cell death rate than untreated proliferating cells. We propose that acute apoptosis is of minor significance for the overall response of carcinoma cells to cisplatin treatment.

Specific demonstration of drug-induced tumour cell apoptosis in human xenografts models using a plasma biomarker

Pharmacodynamic (PD) assays should be used before advancing new drugs to clinical trials. Most PD assays measure the response to drugs in tissue, a procedure which requires tissue biopsies. The M30-Apoptosense ELISA is a PD biomarker assay for the quantitative determination of caspase-cleaved cytokeratin 18 (CK18) released from apoptotic carcinoma cells into blood. We here demonstrate that whereas the M30-Apoptosense ELISA assay detects human caspase-cleaved CK18, the mouse and rat CK18 caspase cleavage products are detected with low affinity. The M30-Apoptosense ELISA therefore facilitates the determination of drug-induced apoptosis in human tumour xenografts in rodents using plasma samples, largely independently from host toxicity. Increases of caspase-cleaved CK18 were observed in plasma from different carcinoma xenograft models in response to anticancer drugs. The appearance caspase-cleaved CK18 in plasma was found to reflect formation of the caspase-cleaved epitope in FaDu head-neck carcinomas and in cultured cells. The M30-Apoptosense assay allows determination of tumour response in blood from xenograft models and from patients, providing a powerful tool for translational studies of anticancer drugs.

Sorafenib inhibits tumor growth and vascularization of rhabdomyosarcoma cells by blocking IGF-1R-mediated signaling

The growth of many soft tissue sarcomas is dependent on aberrant growth factor signaling, which promotes their proliferation and motility. With this in mind, we evaluated the effect of sorafenib, a receptor tyrosine kinase inhibitor, on cell growth and apoptosis in sarcoma cell lines of various histological subtypes. We found that sorafenib effectively inhibited cell proliferation in rhabdomyosarcoma, synovial sarcoma and Ewing’s sarcoma with IC50 values <5 μM. Sorafenib effectively induced growth arrest in rhabdomyosarcoma cells, which was concurrent with inhibition of Akt and Erk signaling. Studies of ligand-induced phosphorylation of Erk and Akt in rhabdomyosarcoma cells showed that insulin-like growth factor-1 is a potent activator, which can be blocked by treatment with sorafenib. In vivo sorafenib treatment of rhabdomyosarcoma xenografts had a significant inhibitory effect on tumor growth, which was associated with inhibited vascularization and enhanced necrosis in the adjacent tumor stroma. Our results demonstrate that in vitro and in vivo growth of rhabdomyosarcoma can be suppressed by treatment with sorafenib, and suggests the possibilities of using sorafenib as a potential adjuvant therapy for the treatment of rhabdomyosarcoma.

Cell crowding induces interferon regulatory factor 9, which confers resistance to chemotherapeutic drugs.

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.

Phenotype-based drug screening in primary ovarian carcinoma cultures identifies intracellular iron depletion as a promising strategy for cancer treatment

Primary cultures of patient tumor cells (PCPTC) have been used for prediction of diagnosis-specific activity and individual patient response to anticancer drugs, but have not been utilized as a model for identification of novel drugs in high throughput screening. In the present study, ovarian carcinoma cells from three patients were tested in response to a library of 3000 chemically diverse compounds. Eight hits were retrieved after counter screening using normal epithelial cells, and one of the two structurally related hit compounds was selected for further preclinical evaluation. This compound, designated VLX 50, demonstrated a broad spectrum of activity when tested in a panel of PCPTCs representing different forms of leukemia and solid tumors and displayed a high tumor to normal cell activity. VLX 50 induced delayed cell death with some features of classical apoptosis. Significant in vivo activity was confirmed on primary cultures of human ovarian carcinoma cells in mice using the hollow fiber model. Mechanistic exploration was performed using gene expression analysis of drug exposed tumor cells to generate a drug-specific signature. This query signature was analyzed using the Gene Set Enrichment Analysis and the Connectivity Map database. Strong connections to hypoxia inducible factor 1 and iron chelators were retrieved. The mechanistic hypothesis of intracellular iron depletion leading to hypoxia signaling was confirmed by a series of experiments. The results indicate the feasibility of using PCPTC for cancer drug screening and that intracellular iron depletion could be a potentially important strategy for cancer therapy.

The phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 is effective in inhibiting regrowth of tumour cells after cytotoxic therapy

PURPOSE Regrowth of tumour cells between cycles of chemotherapy is a significant clinical problem. Treatment strategies where antiproliferative agents are used to inhibit tumour regrowth between chemotherapy cycles are attractive, but such strategies are difficult to test using conventional monolayer culture systems. METHODS We used the in vitro tumour spheroid model to study regrowth of 3-D colon carcinoma tissue after cytotoxic therapy. Colon carcinoma cells with wild-type or mutant phosphatidyl inositol 3-kinase catalytic subunit (PI3KCA) or KRAS alleles were allowed to form multicellular spheroids and the effects of different pharmacological compounds were studied after sectioning and staining for relevant markers of cell proliferation and apoptosis. RESULTS Studies using colon cancer cells with gene disruptions suggested that the phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway was essential for proliferation in 3-D culture. The dual PI3K-mTOR inhibitor NVP-BEZ235, currently in clinical trials, was found to inhibit phosphorylation of the mTOR target 4EBP1 in 3-D cultured cells. The ability of NVP-BEZ235 to inhibit tumour cell proliferation and to induce apoptosis was markedly more pronounced in 3-D cultures compared to monolayer cultures. It was subsequently found that NVP-BEZ235 was effective in inhibiting regrowth of 3-D cultured cells after treatment with two cytotoxic inhibitors of the ubiquitin-proteasome system (UPS), methyl-13-hydroxy-15-oxokaurenoate (MHOK) and bortezomib (Velcade®). CONCLUSIONS The dual PI3K-mTOR inhibitor NVP-BEZ235 was found to reduce cell proliferation and to induce apoptosis in 3-D cultured colon carcinoma cells, NVP-BEZ235 is a promising candidate for use in sequential treatment modalities together with cytotoxic drugs to reduce the cell mass of solid tumours.

Abstract 1736: The anticancer activity of the DUB inhibitor b-AP15 is associated with accumulation of proteasome bound ubiquitin and oxidative stress

The anticancer activity of the DUB inhibitor b-AP15 is associated with accumulation of proteasome bound ubiquitin and oxidative stress