Jennifer D. Tibodeau

The anticancer effects of chaetocin are independent of programmed cell death and hypoxia, and are associated with inhibition of endothelial cell proliferation

Background:We previously reported that chaetocin has potent and selective anti-myeloma activity attributable to reactive oxygen species (ROS) induction imposed by inhibition of the redox enzyme thioredoxin reductase; we now detail its effects in solid tumours.Methods:Cellular assays, transcriptional profiling and the NCI60 screen were used to assess the effects of chaetocin in solid tumour and endothelial cells.Results:NCI-60 screening demonstrated chaetocin to even more potently inhibit proliferation in solid tumour than in haematological cell lines; transcriptional profiling revealed a signature consistent with induction of inflammatory response and cell death pathways. Chaetocin induced ROS, oxidative damage to cellular proteins and apoptosis, with 2–10 nM IC50s (24 h exposures) in all tested solid tumour cell lines. The pan-caspase inhibitor zVAD-fmk did not block chaetocin-induced cell death despite inhibiting mitochondrial membrane depolarisation and apoptosis. Further, Molt-4 rho0 cells lacking metabolically functional mitochondria were readily killed by chaetocin; in addition chaetocin-induced cytotoxicity was unaffected by autophagy inhibitors or hypoxia and consequent HIF-1α upregulation. Moreover, chaetocin inhibited SKOV3 ovarian cancer xenografts producing less vascular tumours, and inhibited human umbilical vein endothelial cell proliferation.Conclusion:Chaetocin has intriguing and wide-ranging in vitro and in vivo anticancer effects, and is an attractive candidate for further preclinical and clinical development.

Drug efflux by breast cancer resistance protein is a mechanism of resistance to the benzimidazole insulin-like growth factor receptor/insulin receptor inhibitor, BMS-536924

Preclinical investigations have identified insulin-like growth factor (IGF) signaling as a key mechanism for cancer growth and resistance to clinically useful therapies in multiple tumor types including breast cancer. Thus, agents targeting and blocking IGF signaling have promise in the treatment of solid tumors. To identify possible mechanisms of resistance to blocking the IGF pathway, we generated a cell line that was resistant to the IGF-1R/InsR benzimidazole inhibitors, BMS-554417 and BMS-536924, and compared expression profiles of the parental and resistant cells lines using Affymetrix GeneChip Human Genome U133 arrays. Compared with MCF-7 cells, breast cancer resistance protein (BCRP) expression was increased 9-fold in MCF-7R4, which was confirmed by immunoblotting and was highly statistically significant (P = 7.13E-09). BCRP was also upregulated in an independently derived resistant cell line, MCF-7 924R. MCF-7R4 cells had significantly lower intracellular accumulation of BMS-536924 compared with MCF-7 cells. Expression of BCRP in MCF-7 cells was sufficient to reduce sensitivity to BMS-536924. Furthermore, knockdown of BCRP in MCF-7R4 cells resensitized cells to BMS-536924. Four cell lines selected for resistance to the pyrrolotriazine IGF-1R/InsR inhibitor, BMS-754807, did not have upregulation of BCRP. These data suggest that benzimidazole IGF-1R/InsR inhibitors may select for upregulation and be effluxed by the ATP-binding cassette transporter, BCRP, contributing to resistance. However, pyrrolotriazine IGF-1R/InsR inhibitors do not appear to be affected by this resistance mechanism. Mol Cancer Ther; 10(1); 117–25. ©2011 AACR.

Drug efflux by breast cancer resistance protein (BCRP) is a mechanism of resistance to the insulin-like growth factor receptor/insulin receptor inhibitor, BMS-536924.

Abstract #2149 Background: Inhibitors of the insulin-like growth factor 1 receptor (IGF-1R) are currently undergoing clinical testing. Preclinical investigations have identified IGF-1 signaling as a key mechanism for breast cancer growth and resistance to clinically useful therapies, including tamoxifen and trastuzumab. Thus, agents targeting IGF-1R have promise in the treatment of breast cancer. Determining mechanisms that can confer resistance to these agents may aid their clinical development.
 Methods: To understand factors may be important in predicting sensitivity to targeting the IGF-1 signaling pathway, we developed a cell line (MCF-7R4) that is resistant to BMS-554417, a small molecule, dual-kinase inhibitor of IGF-1R and insulin receptor (InsR). Compared with the parental MCF-7 cells, MCF-7R4 cells are 40- to 50-fold resistant to BMS-554417 and cross-resistant to the similar compound BMS-536924. The expression profiles of MCF-7R4 and that of MCF-7 were compared using Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. Intracellular concentrations of BMS-536924 were examined by reverse phase high performance liquid chromatography. BMS-536924 cellular accumulation in vitro was visualized by fluorescence microscopy using a DAPI filter set. MCF-7 cells stably transfected with either the empty mammalian expression vector pcDNA (MCF-EV) or full length BCRP (MCF-BCRP) were examined for sensitivity to BMS 536924 by MTS assays.
 Results: Compared to MCF-7 cells, BCRP expression was increased 9-fold in MCF-7R4, which was highly statistically significant by t-test (p= 7.13E-09). Little change was observed in other ABC transporter proteins, including ABCB1. No change was observed in IGF-1R or InsR expression. BCRP overexpression in MCF-7R4 cells was confirmed by western blotting. MCF-7R4 cells had significantly lower intracellular accumulation of BMS-536924 compared to MCF-7 cells. Confirming these results, MCF-BCRP cells were significantly less sensitive to the cytoxic effects of BMS-536924 cells than MCF-EV cells.
 Conclusions: BCRP expression was stimulated by prolonged exposure of MCF-7 cells to BMS-554417. Upregulation of BCRP is one of the most significant changes observed in MCF-7R4 cells in comparison to parental cells. BCRP expression decreased cellular exposure to BMS-536924 and was sufficient to confer resistance. These data suggest that BSM-536924 is a substrate for BCRP-mediated efflux. Expression of BCRP may be important in de novo and acquired resistance to benzimidazole –based inhibitors of IGF-1R/InsR. Supported in part by the Mayo Clinic Breast SPORE (CA116201-03), NIH K12 (CA090628-05) and the Mayo Clinic Cancer Center (CA15083). Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2149.

Abstract 4712: Evidence of clinical efficacy of the combination of flavopiridol (Alvocidib) and cisplatin in platin-resistant ovarian and primary peritoneal carcinoma: Phase 2 trial MC0261

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Based upon preclinical synergy and prior phase 1 study results, the clinical efficacy of flavopiridol combined with cisplatin was assessed in patients with recurrent ovarian and primary peritoneal cancers. Methods: A two cohort phase 2 trial of cisplatin (60 mg/m2 IV) followed by flavopiridol (100 mg/m2 IV, 24 h continuous infusion; 21 day cycles) was undertaken in patients with recurrent platin-sensitive or platin-resistant ovarian/primary peritoneal cancers (defined by disease progression > vs. 2X the post-treatment nadir – was required, as was ECOG performance <2 and exposure to only one prior treatment regimen. Results: Forty-five patients were enrolled between April 20, 2004 and March 4, 2010 – 40 platin-resistant patients (Group 1), and 5 platin-sensitive patients (Group 2). In Group 1, the median number of treatment cycles was 3 (range 2-12); 39 of the 40 eligible patients have now discontinued treatment. While only 10% of all patients incurred grade 4 toxicities, grade 3 toxicities were seen in the majority (65%). The most frequent grade 3 and 4 toxicities were neutropenia (all grade 3, 17.5%); nausea (12.5%); vomiting, fatigue, thrombosis, anemia (10% each). Sensory neuropathy, grade 1 or 2, was observed in 75% of all patients – with grade 3 and 4 neuropathy not observed primarily due to pre-specified aggressive dose reductions. Six patients (15%) in Group 1 achieved a confirmed response (1 CR, 5 PR), with a median response duration of 119 days (range 84-212). Ten additional Group 1 patients (32.5%) experienced maintained stable disease. Median Group 1 overall time to progression was 3.7 months; overall survival was 17.2 months. Pilot assessment of attained ascites flavopiridol level and sensitivity of patient ascitic tumor cells to flavopiridol confirmed that patient flavopiridol levels were consistent with observed clinical antitumor efficacy. In Group 2, although 2 of 5 patients also responded (40%; 2 PR), the cohort was closed due to poor accrual. Conclusions: The combination of flavopiridol and cisplatin has promising clinical activity in both platin-sensitive and platin-resistant ovarian and primary peritoneal cancers. Supported in part by NCI [CA097129][1], CA15083 and CM62205; clinicaltrials.gov identifier [NCT00083122][2] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4712. doi:10.1158/1538-7445.AM2011-4712 [1]: /lookup/external-ref?link_type=GEN&access_num=CA097129&atom=%2Fcanres%2F71%2F8_Supplement%2F4712.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00083122&atom=%2Fcanres%2F71%2F8_Supplement%2F4712.atom

Abstract 3571: The Thioredoxin Reductase Inhibitor Chaetocin has Potent Antineoplastic Effects in Solid Tumors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: We had previously reported that the natural product chaetocin has potent and selective in vitro, ex vivo and in vivo anti-myeloma activity attributable to the imposition of cellular oxidative stress (ROS) in part mediated via competitive inhibition of the redox enzyme thioredoxin reductase. Having also observed chaetocin-induced cytotoxicity in solid tumor cell lines, we now extend prior work to characterize the effects of chaetocin in solid tumor cell lines and in human umbilical vein endothelial cells (HUVECs). Methods: The effects of chaetocin in solid tumor cell lines were assessed using colony forming assays, trypan blue exclusion assays, apoptosis and autophagy assays, electron microscopy, transcriptional profiling, and the National Cancer Institute 60 cell line screen. Results: Chaetocin demonstrated potent anti-cancer activity in all assessed solid tumor cell lines with IC50 values between 2-10 nM (24 h exposures, colony forming assays). While apoptosis was induced in a cell line-dependant fashion, it was not required for chaetocin-induced cytotoxicity, as ZVAD-fmk prevented apoptosis but not cell death. Markers of autophagy were not altered by chaetocin treatment. Interestingly, results form the NCI 60 cell line screen showed that hematological cell lines were generally more resistant to chaetocin than solid tumor lines despite our prior report indicating the activity of chaetocin in myeloma. Transcriptional profiling results were consistent with those anticipated from an agent producing cell death via imposition of cellular ROS, with heme oxidase-1 prominently induced along with other transcripts in pathways related to inflammatory response and cell death. Results from OxyBlot protein oxidation kit analyses (Millipore, Billerica, MA) confirmed a generalized increase in the carbonyl modification of proteins, a hallmark of cellular oxidative damage, in response to chaetocin treatment. Experiments using Rho mitochondrial inactive cells indicated that cellular ROS is induced by chaetocin independent of respiratory functional mitochondria. Chaetocin was also shown to block the interleukin-, fibroblast growth factor- or EGM2 media-induced proliferation of HUVEC cells at low-nanomolar concentrations. Conclusions: Chaetocin has wide-ranging antineoplastic activity across not only hematological, but also solid tumor, cell lines and displays evidence of antiangiogentic activity in HUVEC proliferation assays. Overall, chaetocin appears to be an attractive agent for further development as a candidate anti-cancer therapeutic in a variety of neoplasms. Supported in part by CA125750. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3571.