We report a novel mechanism of action of ONC201 as a mitochondria-targeting drug in cancer cells. ONC201 was originally identified as a small molecule that induces transcription of TNF-related apoptosis-inducing ligand (TRAIL) and subsequently kills cancer cells by activating TRAIL death receptors. In this study, we examined ONC201 toxicity on multiple human breast and endometrial cancer cell lines. ONC201 attenuated cell viability in all cancer cell lines tested. Unexpectedly, ONC201 toxicity was not dependent on either TRAIL receptors nor caspases. Time-lapse live cell imaging revealed that ONC201 induces cell membrane ballooning followed by rupture, distinct from the morphology of cells undergoing apoptosis. Further investigation found that ONC201 induces phosphorylation of AMP-dependent kinase and ATP loss. Cytotoxicity and ATP depletion were significantly enhanced in the absence of glucose, suggesting that ONC201 targets mitochondrial respiration. Further analysis indicated that ONC201 indirectly inhibits mitochondrial respiration. Confocal and electron microscopic analysis demonstrated that ONC201 triggers mitochondrial structural damage and functional impairment. Moreover, ONC201 decreased mitochondrial DNA (mtDNA). RNAseq analysis revealed that ONC201 suppresses expression of multiple mtDNA-encoded genes and nuclear-encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions. Importantly, fumarate hydratase deficient cancer cells and multiple cancer cell lines with reduced amounts of mtDNA were resistant to ONC201. These results indicate that cells not dependent on mitochondrial respiration are ONC201-resistant. Our data demonstrate that ONC201 kills cancer cells by disrupting mitochondrial function and further suggests that cancer cells that are dependent on glycolysis will be resistant to ONC201.

The tumor microenvironment has an important role in cancer progression. Here we show that miR-148a is downregulated in 15 out of 16 samples (94%) of cancer-associated fibroblasts (CAFs) compared with matched normal tissue fibroblasts (NFs) established from patients with endometrial cancer. Laser-capture microdissection of stromal cells from normal tissue and endometrial cancer confirmed this observation. Treatment of cells with 5-aza-deoxycytidine stimulated the expression of miR-148a in the majority of CAFs implicating DNA methylation in the regulation of miR-148a expression. Investigation of miR-148a function in fibroblasts demonstrated that conditioned media (CM) from CAFs overexpressing miR-148a significantly impaired the migration of five endometrial cancer cell lines without affecting their growth rates in co-culture experiments. Among predicted miR-148a target genes are two WNT family members, WNT1 and WNT10B. Activation of the WNT/β-catenin pathway in CAFs was confirmed by microarray analysis of gene expression and increased activity of the SuperTOPFIash luciferase reporter. We found elevated levels of WNT10B protein in CAFs and its level decreased when miR-148a was re-introduced by lentiviral infection. The 3′-UTR of WNT10B, cloned downstream of luciferase cDNA, suppressed luciferase activity when co-expressed with miR-148a indicating that WNT10B is a direct target of miR-148a. In contrast to the effect of miR-148a, WNT10B stimulated migration of endometrial cancer cell lines. Our findings have defined a molecular mechanism in the tumor microenvironment that is a novel target for cancer therapy.

Centrosomal localization of kinase-active CK1δ is required for neurite outgrowth in response to Wnt-3a.

Background: Wnt signaling causes phosphorylation of Dishevelled, but its functional significance is unclear. Results: Sites of Wnt-induced phosphorylation were mapped in Dvl2 and mutated to permit functional testing. Conclusion: Three CK1 phosphorylation sites in the C-terminal of Dvl2 account for the Wnt-induced mobility shift and modulate signaling. Significance: Wnt-induced phosphorylation of Dvl differentially regulates canonical and noncanonical Wnt signaling.

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/β-catenin signaling pathway has been previously described. In fact, Cripto-1 is a downstream target gene of the canonical Wnt/β-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/β-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 β-catenin and elevated β-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/β-catenin signaling co-operate in regulating motility and in vitro transformation of mammary epithelial cells.

Background TNF-related apoptosis-inducing ligand (TRAIL) receptor agonists are attractive anti-tumor agents because of their capability to induce apoptosis in cancer cells by activating death receptors (DR) 4 and 5 with little toxicity against normal cells. Despite an attractive mechanism of action, previous clinical efforts to use TRAIL receptor agonists have been unsuccessful. In this study, we examined MEDI3039, a highly potent multivalent DR5 agonist, in breast cancer cell lines and in vivo models. Methods As in vitro model systems, we used 19 breast cancer cell lines that are categorized into four subtypes: ER+, HER2 amplified, basal A (triple-negative breast cancer) TNBC, and basal B TNBC. Cell viability was analyzed by MTS and RealTime live/dead assays. As in vivo model systems, MDA-MB231T orthotopic primary tumor growth in the mammary fat pad (MFP) and two experimental lung metastasis models were used. The effect of MEDI3039 on MFP tumors was assessed with immunohistochemical analysis. Lung metastases were analyzed with Bouin’s and H&E staining. Results MEDI3039 killed multiple breast cancer cell lines, but the sensitivity varied among different subtypes. Sensitivity was basal B TNBC >> basal A TNBC > HER2 amplified > ER+ (average IC 50 = 1.4, 203, 314, 403 pM, respectively). While the pattern of relative sensitivity was similar to GST-TRAIL in most cell lines, MEDI3039 was at least two orders of magnitude more potent compared with GST-TRAIL. In the MFP model, weekly treatment with 0.1 or 0.3 mg/kg MEDI3039 for 5 weeks inhibited tumor growth by 99.05% or 100% (median), respectively, compared with the control group, and extended animal survival ( p = 0.08 or p = 0.0032 at 0.1 or 0.3 mg/kg, respectively). MEDI3039-induced caspase activation was confirmed in tumors grown in MFP ( p < 0.05). In an experimental pulmonary metastasis model, MEDI3039 significantly suppressed outgrowth of surface ( p < 0.0001) and microscopic metastases ( p < 0.05). In an established lung metastasis model, MEDI3039 significantly inhibited growth of metastases ( p < 0.01 in surface [> 4 mm], p < 0.01 in tumor percentage) and extended animal survival ( p < 0.0001). Conclusion MEDI3039 is a potent DR5 agonist in breast cancer cells in vitro and in vivo and has potential as a cancer drug in breast cancer patients, especially those with basal B TNBC. Electronic supplementary material The online version of this article (10.1186/s13058-019-1116-1) contains supplementary material, which is available to authorized users.

Inhibition of casein kinase 1 delta (CK1δ) blocks primary ciliogenesis in human telomerase reverse transcriptase immortalized retinal pigmented epithelial and mouse inner medullary collecting duct cells-3. Mouse embryonic fibroblasts (MEFs) and retinal cells from Csnk1d (CK1δ)-null mice also exhibit ciliogenesis defects. CK1δ catalytic activity and centrosomal localization signal (CLS) are required to rescue cilia formation in MEFs . Furthermore, expression of a truncated derivative containing the CLS displaces full-length CK1δ from the centrosome and decreases ciliary length in control MEFs, suggesting that centrosomal CK1δ has a role in ciliogenesis. CK1δ inhibition also alters pericentrosomal or ciliary distribution of several proteins involved in ciliary transport, including Ras-like in rat brain-11A, Ras-like in rat brain-8A, centrosomal protein of 290 kDa, pericentriolar material protein 1, and polycystin-2, as well as the Golgi distribution of its binding partner, A-kinase anchor protein 450 (AKAP450). As reported for AKAP450, CK1δ was required for microtubule nucleation at the Golgi and maintenance of Golgi integrity. Overexpression of an AKAP450 fragment containing the CK1δ-binding site inhibits Golgi-derived microtubule nucleation, Golgi distribution of intraflagellar transport protein 20 homologue, and ciliogenesis. Our results suggest that CK1δ mediates primary ciliogenesis by multiple mechanisms, one involving its centrosomal function and another dependent on its interaction with AKAP450 at the Golgi, where it is important for maintaining Golgi organization and polarized trafficking of multiple factors that mediate ciliary transport.

Rspo2 was identified as a novel common integration site (CIS) for the mouse mammary tumor virus (MMTV) in viral induced mouse mammary tumors. Here we show that Rspo2 modulates Wnt signaling in mouse mammary epithelial cells. Co-expression of both genes resulted in an intermediate growth phenotype on plastic and had minor effects on the growth-promoting properties of Wnt1 in soft agar. However, individual Rspo2 and Wnt1 HC11 transfectants as well as the double transfectant were tumorigenic in athymic nude mice, with tumors from each line having distinctive histological characteristics. Rspo2 and Rspo2/Wnt1 tumors contained many spindle cells, consistent with an epithelial-mesenchymal transformation (EMT) phenotype. When Rspo2 and Rspo2/Wnt1 tumor cells were transferred into naïve mice, they exhibited greater metastatic activity than cells derived from Wnt1 tumors. For comparison, C57MG/Wnt1/Rspo2 co-transfectants exhibited invasive properties in three-dimensional (3D) Matrigel cultures that were not seen with cells transfected only with Wnt1 or Rspo2. Use of Dickkopf-1, a specific antagonist of the Wnt/β-catenin pathway, or short hairpin RNA targeting β-catenin expression demonstrated that the invasive activity was not mediated by β-catenin. Our results indicate that Rspo2 and Wnt1 have mutually distinct effects on mammary epithelial cell growth and these effects are context-dependent. While Rspo2 and Wnt1 act synergistically in the β-catenin pathway, other mechanisms are responsible for the invasive properties of stable double transfectants observed in 3D Matrigel cultures.