Marine Cyanobacteria Compounds with Anticancer Properties: Implication of Apoptosis

Marine cyanobacteria have been proved to be an important source of potential anticancer drugs. Although several compounds were found to be cytotoxic to cancer cells in culture, the pathways by which cells are affected are still poorly elucidated. For some compounds, cancer cell death was attributed to an implication of apoptosis through morphological apoptotic features, implication of caspases and proteins of the Bcl-2 family, and other mechanisms such as interference with microtubules dynamics, cell cycle arrest and inhibition of proteases other than caspases

Hypoxia and TGF-β Drive Breast Cancer Bone Metastases through Parallel Signaling Pathways in Tumor Cells and the Bone Microenvironment

BACKGROUND: Most patients with advanced breast cancer develop bone metastases, which cause pain, hypercalcemia, fractures, nerve compression and paralysis. Chemotherapy causes further bone loss, and bone-specific treatments are only palliative. Multiple tumor-secreted factors act on the bone microenvironment to drive a feed-forward cycle of tumor growth. Effective treatment requires inhibiting upstream regulators of groups of prometastatic factors. Two central regulators are hypoxia and transforming growth factor (TGF)- beta. We asked whether hypoxia (via HIF-1alpha) and TGF-beta signaling promote bone metastases independently or synergistically, and we tested molecular versus pharmacological inhibition strategies in an animal model. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed interactions between HIF-1alpha and TGF-beta pathways in MDA-MB-231 breast cancer cells. Only vascular endothelial growth factor (VEGF) and the CXC chemokine receptor 4 (CXCR4), of 16 genes tested, were additively increased by both TGF-beta and hypoxia, with effects on the proximal promoters. We inhibited HIF-1alpha and TGF-beta pathways in tumor cells by shRNA and dominant negative receptor approaches. Inhibition of either pathway decreased bone metastasis, with no further effect of double blockade. We tested pharmacologic inhibitors of the pathways, which target both the tumor and the bone microenvironment. Unlike molecular blockade, combined drug treatment decreased bone metastases more than either alone, with effects on bone to decrease osteoclastic bone resorption and increase osteoblast activity, in addition to actions on tumor cells. CONCLUSIONS/SIGNIFICANCE: Hypoxia and TGF-beta signaling in parallel drive tumor bone metastases and regulate a common set of tumor genes. In contrast, small molecule inhibitors, by acting on both tumor cells and the bone microenvironment, additively decrease tumor burden, while improving skeletal quality. Our studies suggest that inhibitors of HIF-1alpha and TGF-beta may improve treatment of bone metastases and increase survival

Characterization of cell death induced by vinflunine, the most recent Vinca alkaloid in clinical development

Vinflunine, the most recent Vinca alkaloid in clinical development, demonstrated superior antitumour activity to other Vincas in preclinical tumour models. This study aimed to define its molecular mechanisms of cell killing in both parental sensitive and vinflunine-resistant P388 leukaemia cells. Vinflunine treatment of these cells resulted in apoptosis characterized by DNA fragmentation and proteolytic cleavage of poly-(ADP-ribose) polymerase. Apoptosis-inducing concentrations of vinflunine caused c-Jun N-terminal kinase 1 stimulation, as well as caspases-3/7 activation. This activation of caspases and the induction of apoptosis could be inhibited by the caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde. Interestingly, the apoptosis signal triggered by vinflunine in these P388 cells was not mediated through Bcl-2 phosphorylation. In addition, when vinflunine resistance was developed in P388 cells, it was associated with resistance to vinflunine-induced apoptosis, as reflected by a loss of capacity to induce DNA fragmentation and PARP degradation, and characterized by increased levels of Bcl-2 and Bfl-1/A1. Therefore, these data indirectly implicate Bcl-2 and Bfl-1/A1 in vinflunine-induced cell death mechanisms

Abstract P5-04-04: Eribulin differentially disrupts TGF-β signaling pathway in BT-549 and HCC1937 breast cancer cell lines

Abstract Microtubule targeting agents (MTAs) continue to be valuable in treating breast cancer. While these drugs were classically identified as only antimitotic agents, recent evidence demonstrates that the ability of MTAs to disrupt key signaling components in interphase cells likely contributes to their anticancer actions. Yoshida and colleagues demonstrated that eribulin reverses TGF-β-induced epithelial-to-mesenchymal transition (EMT) in preclinical models of triple negative breast cancer (TNBC) within 7days. To gain a deeper understanding of the interphase effects of eribulin in comparison with other MTAs on the TGF-β signaling pathway, we tested the hypothesis that a short-term, 2 h treatment of TNBC cells with MTAs would disrupt TGF- β signaling. The effects of the microtubule destabilizers eribulin and vinorelbine and the microtubule stabilizers paclitaxel and ixabepilone on downstream targets of the TGF-β pathway were evaluated in two TNBC cell lines. In the mesenchymal type BT-549 cells, eribulin and vinorelbine significantly inhibited TGF-β induced expression of Snail and Slug, while the microtubule stabilizers had no effect on their expression. In the basal-like 1 type HCC1937 cells, the microtubule destabilizers inhibited the expression of Snail when compared to vehicle and stabilizer-treated cells, but caused increased expression of Slug. These data suggest that MTAs have different effects on the TGF-β signaling pathway in distinct molecular contexts. Our efforts are directed towards deciphering the effects of eribulin and other MTAs on the TGF- β pathway and how this contributes to differential sensitivity. Preliminary data suggest that eribulin initiated inhibition of Snail and Slug in BT-549 cells is mediated by the scaffold protein NEDD9 and we are testing whether the differential levels of expression of NEDD9 help explain the differences in the effects of eribulin in BT-549 and HCC1937 cells. These results will begin to decipher differences among MTAs in different molecular contexts and might help identify biomarkers for optimal therapies. Funding for this work was provided by Eisai Inc. Citation Format: Kaul R, Risinger AL, Mooberry SL. Eribulin differentially disrupts TGF-β signaling pathway in BT-549 and HCC1937 breast cancer cell lines [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-04-04.</jats:p

Abstract P4-04-04: Eribulin impairs TGF-β type I receptor localization and signaling in BT-549 cells

Abstract Microtubule targeting agents (MTAs) continue to be some of the most valuable drugs used to treat breast cancer. While decades of research have shown that these drugs cause mitotic arrest in cells by suppressing the dynamic instability of microtubules, recent evidence demonstrates that the ability of MTAs to disrupt microtubule-dependent transport of key signaling components, including proteins and microRNAs, in interphase cells likely contributes to their anticancer actions. TGF-β signaling is a driver of oncogenesis and epithelial-to-mesenchymal transition (EMT) that involves multiple protein trafficking and intracellular signaling events. Ligand mediated activation of the cell surface TGF-β receptors leads to downstream signaling in the canonical and the non-canonical pathways that collectively lead to the expression of proteins implicated in EMT, including Snail and Slug. Additionally, TGF-β type 1 receptor (TGF-βR1) undergoes constant cycling from the plasma membrane to the cytosol, a microtubule-dependent process. We tested the hypothesis that a short-term treatment of breast cancer cells with eribulin or 3 other clinically relevant MTAs would differentially disrupt interphase microtubules and alter the transport and downstream signaling of TGF-βR1. BT-549 cells were treated for 2 h with concentrations of MTAs that cause comparable disruption of the interphase microtubule network; 100 nM was used for the destabilizers, eribulin or vinorelbine and 1 µm was used for the stabilizers, paclitaxel or ixabepilone. The results show that TGF-βR1 was extensively localized along the stabilizer-induced microtubule bundles, a phenomenon not observed with destabilizer-treated cells. The downstream consequences were further assessed. The expression of Snail and Slug were evaluated in cells pretreated with MTAs followed by TGF-β stimulation. Eribulin and vinorelbine significantly inhibited the TGF-β-induced expression of Snail and Slug while stabilizers did not alter their expression levels. We hypothesize that eribulin and vinorelbine are inhibiting the non-canonical TGF-β signaling pathway since they had no effect on the localization and expression of Smad2/3, proteins involved in the canonical pathway. Eribulin induced inhibition of TGF-β signaling is consistent with previous studies that show that a 7 day treatment reversed TGF-β mediated EMT in breast cancer cells1. Data from patients shows that eribulin decreases the plasma concentration of TGF-β within 7 days of treatment initiation2. Our data suggest that eribulin rapidly inhibits non-canonical TGF-β signaling indicating that this is a potential mechanism for the eribulin-mediated EMT reversal. This information, together with previously published reports, suggest that eribulin has multiple effects leading to inhibition of TGF-β signaling. These studies begin to shed light into the diverse mechanisms of action of MTAs. 1. Yoshida T, et al., Brit J Cancer 110(6): 1497-505, 2014 2. Ueda S, et al., Brit J Cancer, 2016 This work is funded by Eisai Inc. Citation Format: Kaul R, Risinger AL, Mooberry SL. Eribulin impairs TGF-β type I receptor localization and signaling in BT-549 cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-04-04.</jats:p