Multi-omics analyses reveal ClpP activators disrupt essential mitochondrial pathways in triple-negative breast cancer.

ClpP activators ONC201 and related small molecules (TR compounds, Madera Therapeutics), have demonstrated significant anti-cancer potential in vitro and in vivo studies, including clinical trials for refractory solid tumors. Though progress has been made in identifying specific phenotypic outcomes following ClpP activation, the exact mechanism by which ClpP activation leads to broad anti-cancer activity has yet to be fully elucidated. In this study, we utilized a multi-omics approach to identify the ClpP-dependent proteomic, transcriptomic, and metabolomic changes resulting from ONC201 or the TR compound TR-57 in triple-negative breast cancer cells. Applying mass spectrometry-based methods of proteomics and metabolomics, we identified ∼8,000 proteins and 588 metabolites, respectively. From proteomics data, 113 (ONC201) and 191 (TR-57) proteins significantly increased and 572 (ONC201) and 686 (TR-57) proteins significantly decreased in this study. Gene ontological (GO) analysis revealed strong similarities between proteins up- or downregulated by ONC201 or TR-57 treatment. Notably, this included the downregulation of many mitochondrial processes and proteins, including mitochondrial translation and mitochondrial matrix proteins. We performed a large-scale transcriptomic analysis of WT SUM159 cells, identifying ∼7,700 transcripts (746 and 1,100 significantly increasing, 795 and 1,013 significantly decreasing in ONC201 and TR-57 treated cells, respectively). Less than 21% of these genes were affected by these compounds in ClpP null cells. GO analysis of these data demonstrated additional similarity of response to ONC201 and TR-57, including a decrease in transcripts related to the mitochondrial inner membrane and matrix, cell cycle, and nucleus, and increases in other nuclear transcripts and transcripts related to metal-ion binding. Comparison of response between both compounds demonstrated a highly similar response in all -omics datasets. Analysis of metabolites also revealed significant similarities between ONC201 and TR-57 with increases in α-ketoglutarate and 2-hydroxyglutaric acid and decreased ureidosuccinic acid, L-ascorbic acid, L-serine, and cytidine observed following ClpP activation in TNBC cells. Further analysis identified multiple pathways that were specifically impacted by ClpP activation, including ATF4 activation, heme biosynthesis, and the citrulline/urea cycle. In summary the results of our studies demonstrate that ONC201 and TR-57 induce highly similar and broad effects against multiple mitochondrial processes required for cell proliferation.

The TRAIL receptor agonist drozitumab targets basal B triple-negative breast cancer cells that express vimentin and Axl.

Previously, we found that GST-tagged tumor necrosis factor-related apoptosis inducing ligand preferentially killed triple-negative breast cancer (TNBC) cells with a mesenchymal phenotype by activating death receptor 5 (DR5). The purpose of this study was to explore the sensitivity of breast cancer cell lines to drozitumab, a clinically tested DR5-specific agonist; identify potential biomarkers of drozitumab-sensitive breast cancer cells; and determine if those biomarkers were present in tumors from patients with TNBC. We evaluated viability, caspase activity, and sub-G1 DNA content in drozitumab-treated breast cancer cell lines and we characterized expression of potential biomarkers by immunoblot. Expression levels of vimentin and Axl were then explored in 177 TNBC samples from a publically available cDNA microarray dataset and by immunohistochemistry (IHC) in tumor tissue samples obtained from 53 African-American women with TNBC. Drozitumab-induced apoptosis in mesenchymal TNBC cell lines but not in cell lines from other breast cancer subtypes. The drozitumab-sensitive TNBC cell lines expressed the mesenchymal markers vimentin and Axl. Vimentin and Axl mRNA and protein were expressed in a subset of human TNBC tumors. By IHC, ~15 % of TNBC tumors had vimentin and Axl expression in the top quartile for both. These findings indicate that drozitumab-sensitive mesenchymal TNBC cells express vimentin and Axl, which can be identified in a subset of human TNBC tumors. Thus, vimentin and Axl may be useful to identify TNBC patients who would be most likely to benefit from a DR5 agonist.

Cripto-1 enhances the canonical Wnt/ß-catenin signaling pathway by binding to LRP5 and LRP6 co-receptors.

Cripto-1 is implicated in multiple cellular events, including cell proliferation, motility and angiogenesis, through the activation of an intricate network of signaling pathways. A crosstalk between Cripto-1 and the canonical Wnt/ß-catenin signaling pathway has been previously described. In fact, Cripto-1 is a downstream target gene of the canonical Wnt/ß-catenin signaling pathway in the embryo and in colon cancer cells and T-cell factor (Tcf)/lymphoid enhancer factor binding sites have been identified in the promoter and the first intronic region of the mouse and human Cripto-1 genes. We now demonstrate that Cripto-1 modulates signaling through the canonical Wnt/ß-catenin/Tcf pathway by binding to the Wnt co-receptors low-density lipoprotein receptor-related protein (LRP) 5 and LRP6, which facilitates Wnt3a binding to LRP5 and LRP6. Cripto-1 functionally enhances Wnt3a signaling through cytoplasmic stabilization of ß-catenin and elevated ß-catenin/Tcf transcriptional activation. Conversely, Wnt3a further increases Cripto-1 stimulation of migration, invasion and colony formation in soft agar of HC11 mouse mammary epithelial cells, indicating that Cripto-1 and the canonical Wnt/ß-catenin signaling co-operate in regulating motility and in vitro transformation of mammary epithelial cells.

Retention mechanism of hypoxia selective nuclear imaging/radiotherapeutic agent cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) in tumor cells.

The retention mechanism of the novel imaging/radiotherapeutic agent, Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) in tumor cells was clarified in comparison with that in normal tissue in vitro. With Cu-ATSM and reversed phase HPLC analysis, the reductive metabolism of Cu-ATSM in subcellular fractions obtained from Ehrlich ascites tumor cells was examined. As a reference, mouse brain was used. To determine the contribution of enzymes in the retention mechanisms, and specific inhibitor studies were performed. In subcellular fractions of tumor cells, Cu-ATSM was reduced mainly in the microsome/cytosol fraction rather than in the mitochondria. This finding was completely different from that found in normal brain cells. The reduction process in the microsome/cytosol was heat-sensitive and enhanced by adding exogenous NAD(P)H, an indication of enzymatic reduction of Cu-ATSM in tumor cells. Among the known bioreductive enzymes, NADH-cytochrome b5 reductase and NADPH-cytochrome P450 reductase in microsome played a major role in the reductive retention of Cu-ATSM in tumors. This enzymatic reduction was enhanced by the induction of hypoxia. Radiocopper labeled Cu-ATSM provides useful information for the detection of hypoxia as well as the microsomal bioreductive enzyme expression in tumor.

Cytosolic/microsomal redox pathway: a reductive retention mechanism of a PET-oncology tracer, cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (cu-PTSM).

OBJECTIVE: To clarify the retention mechanism of a PET imaging agent Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (Cu-62-PTSM) in tumor cells, reductive metabolism of non-radioactive Cu-PTSM in five cultured tumor cell lines, a tumor specimen and non-tumor tissues in vitro was evaluated by electron spin resonance spectrometry (ESR). RESULTS: In the brain, mitochondrial electron transport enzyme reduced Cu-PTSM specifically. On the other hand, Cu-PTSM was not reduced in tumor mitochondria. The mitochondrial electron transport enzyme in tumor cells was not damaged, but NADH was considered to be depleted. In compensation for that, the tumor cells acquired complementary reduction activity in the microsome/cytosol. The reduction was enzymatic and NADH-dependent, possibly similar to the activation mechanism of bioreductive anticancer drugs. CONCLUSION: Cu-PTSM and its derivatives are considered to be used as a marker for microsome/cytosol redox ability in PET oncology, although the physiological role of the redox enzyme system in tumor cells has not been clarified. The change in electron (NADH) flow in tumor cells might be a mechanism supporting aerobic glycolysis in tumor cells.

A novel 111In-labeled antisense DNA probe with multi-chelating sites (MCS-probe) showing high specific radioactivity and labeling efficiency.

A multi-aminolinked oligodeoxynucleotide (ODN) was synthesized by substitution of dT with aminolinked dU in the sequence, following conjugation with isothiocyanobenzyl-EDTA (IBE) for 111In labeling. As a model target gene, the c-erbB-2 protooncogene was used. The probability of the number of aminolinked dU in the 20mer ODN was 5, but there were actually 3 and 4 in the selected antisense and sense ODNs, respectively. The IBE/ODN conjugation levels of probes with multi-chelating sites (MCS-probe) were 1.6 (antisense) and 2.4 (sense), more than 50 times higher than those of our previous studies using 5'-end aminolinked ODNs (IBE/ODN = 0.03). Labeling studies using the MCS-probe and 111In indicated that specific radioactivity as high as 48 MBq/nmol could be obtained with a labeling efficiency of over 90%. The 111In-antisense-MCS-probe could bound to sense ODN under physiological conditions, but the 111In-sense-MCA-probe could not. Thus, side-chain modification of ODN for metal labeling is considered to be useful for antisense techniques.