The therapeutic efficacy of two bis(thiosemicarbazonato) copper complexes, glyoxalbis[N4-methylthiosemicarbazonato]Cu(II) [Cu(II)(gtsm)] and diacetylbis[N4-methylthiosemicarbazonato]Cu(II) [Cu(II)(atsm)], for the treatment of prostate cancer was assessed in cell culture and animal models. Distinctively, copper dissociates intracellularly from Cu(II)(gtsm) but is retained by Cu(II)(atsm). We further demonstrated that intracellular H2gtsm [reduced Cu(II)(gtsm)] continues to redistribute copper into a bioavailable (exchangeable) pool. Both Cu(II)(gtsm) and Cu(II)(atsm) selectively kill transformed (hyperplastic and carcinoma) prostate cell lines but, importantly, do not affect the viability of primary prostate epithelial cells. Increasing extracellular copper concentrations enhanced the therapeutic capacity of both Cu(II)(gtsm) and Cu(II)(atsm), and their ligands (H2gtsm and H2atsm) were toxic only toward cancerous prostate cells when combined with copper. Treatment of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model with Cu(II)(gtsm) (2.5 mg/kg) significantly reduced prostate cancer burden (∼70%) and severity (grade), while treatment with Cu(II)(atsm) (30 mg/kg) was ineffective at the given dose. However, Cu(II)(gtsm) caused mild kidney toxicity in the mice, associated primarily with interstitial nephritis and luminal distention. Mechanistically, we demonstrated that Cu(II)(gtsm) inhibits proteasomal chymotrypsin-like activity, a feature further established as being common to copper-ionophores that increase intracellular bioavailable copper. We have demonstrated that increasing intracellular bioavailable copper can selectively kill cancerous prostate cells in vitro and in vivo and have revealed the potential for bis(thiosemicarbazone) copper complexes to be developed as therapeutics for prostate cancer.

p53 is regarded as a central player in tumour suppression, as it controls programmed cell death (apoptosis) as well as cellular senescence. While apoptosis eliminates cells at high risk for oncogenic transformation, senescence acts as a barrier to tumourigenesis by imposing irreversible cell cycle arrest. p53 can act directly or indirectly at multiple levels of the tumour suppression network by invoking a myriad of mechanisms. p53 induces the extrinsic and intrinsic apoptotic pathways at multiple steps to ensure an efficient death response. This response involves transcriptional activation or repression of target genes, as well as the recently identified microRNAs, and transcription-independent functions. Importantly, p53 loss of function is required for tumour maintenance. Therefore, therapeutic strategies aimed at reactivation of p53 in tumours emerge as a promising approach for the treatment of cancer patients.

IntroductionA link between proteasomal degradation and cancer development has been established and a general proteasome inhibitor, Velcade (bortezomib), is in clinical use for the treatment of multiple myeloma and mantle cell lymphoma. 1,2 Deregulation of E3 ubiquitin ligases can be sufficient to suppress the expression and function of key tumor suppressors. For example, the inhibition of p53 as a consequence of Mdm2 amplification is frequently observed in human sarcomas and retinoblastoma. [3][4][5] Interestingly, in human papilloma virus (HPV)-infected cells the suppression of p53 is not achieved by Mdm2, but rather by the cellular E6AP (E6-associated protein) ubiquitin ligase, which is recruited to p53 by the HPV-E6 protein. [6][7][8] E6AP is encoded by the UBE3A locus, which is mutated in Angelman syndrome (AS), a human neuro-developmental disorder. 9 E6AP was the first mammalian ubiquitin E3 ligase to be identified. It is the prototype of the subfamily of E3 ligases that covalently bind ubiquitin and are characterized by a C-terminal HECT (homologous to the E6AP C terminus) domain. 10 We recently demonstrated that E6AP regulates the stability of the promyelocytic leukemia (PML) protein and the formation of PML nuclear bodies (PML-NBs). 11 PML is a tumor suppressor that was identified as a consequence of the chromosomal translocation of its gene in acute promyelocytic leukemia (APL). 12 Consistent with the role of PML as a tumor suppressor, PML deficient mice showed abnormally increased susceptibility to carcinogen 13,14 and oncogene-induced tumorigenesis. 15 Importantly, PML expression was found to be down-regulated or lost in a variety of human cancer types, including prostate, breast, and colon adenocarcinomas. 16,17 PML protein and the PML-NBs were found to play critical roles in cellular stress responses, including those that elicit apoptosis or cellular senescence. 18-21 Cellular senescence is emerging as an important mechanism for tumor suppression. 22,23 It represents a profound arrest of cellular proliferation, accompanied by a distinct set of alterations in the cellular phenotype, such as the formation of senescence-associated heterochromatin foci (SAHF, eg, H3K9me3) and up-regulation of certain inhibitors of cell growth, such as p21, PAI-1, and p16. 24 In this study, we explored the role of the E6AP-PML axis in HPV-independent cancer development. We chose pre-B/B-cell lymphomagenesis as a model For personal use only. on May 7, 2018. by guest www.bloodjournal.org From because of the high frequency of PML down-regulation in nonHodgkin lymphomas (NHLs). 16 For this purpose we used the well established E-myc transgenic mice, a mouse model for Burkitt lymphoma and other NHLs. 25 We found that the loss of one allele of E6AP significantly delayed Myc-driven B-cell lymphomagenesis and this was accompanied by elevated PML expression and the induction of cellular senescence. Importantly, E6AP expression was observed to be elevated in human Burkitt lymphoma and cell lines derived from these tumors. Our findi...

A role for the RUNX genes in cancer failsafe processes has been suggested by their induction of senescence-like growth arrest in primary murine fibroblasts and the failure of RAS-induced senescence in Runx2 deficient cells. We now show that RUNX1 induces senescence in human primary fibroblasts. High affinity DNA binding is necessary but not sufficient, as shown by the functional attenuation of the truncated RUNX1/AML1a isoform and the TEL-RUNX1 fusion oncoprotein. However, a similar phenotype was potently induced by the RUNX1-ETO (AML1-ETO) oncoprotein, despite its dominant negative potential. Detailed comparison of H-RASV12, RUNX1 and RUNX1-ETO senescent phenotypes showed that the RUNX effectors induce earlier growth stasis with only low levels of DNA damage signalling and a lack of chromatin condensation, a marker of irreversible growth arrest. In human fibroblasts, all effectors induced p53 in the absence of detectable p14ARF, while only RUNX1-ETO induced senescence in p16INK4a null cells. Correlation was noted between induction of p53, reactive oxygen species and phospho-p38, while p38MAPK inhibition rescued cell growth markedly. These findings reveal a role for replication-independent pathways in RUNX and RUNX1-ETO senescence, and show that the context-specific oncogenic activity of RUNX1 fusion proteins are mirrored in their distinctive interactions with failsafe responses.