Conversion of Osteoclasts into Bone-Protective, Tumor-Suppressing Cells

Osteoclasts are a driver of a vicious bone-destructive cycle with breast cancer cells. Here, we examined whether this vicious cycle can be altered into a beneficial one by activating Wnt signaling with its activating agent, BML284. The conditioned medium, derived from Wnt-activated RAW264.7 pre-osteoclast cells (BM CM), reduced the proliferation, migration, and invasion of EO771 mammary tumor cells. The same inhibitory effect was obtained with BML284-treated primary human macrophages. In a mouse model, BM CM reduced the progression of mammary tumors and tumor-induced osteolysis and suppressed the tumor invasion to the lung. It also inhibited the differentiation of RANKL-stimulated osteoclasts and enhanced osteoblast differentiation. BM CM was enriched with atypical tumor-suppressing proteins such as Hsp90ab1 and enolase 1 (Eno1). Immunoprecipitation revealed that extracellular Hsp90ab1 interacted with latent TGFβ (LAP-TGFβ) as an inhibitor of TGFβ activation, while Hsp90ab1 and Eno1 interacted and suppressed tumor progression via CD44, a cell-adhesion receptor and a cancer stem cell marker. This study demonstrated that osteoclast-derived CM can be converted into a bone-protective, tumor-suppressing agent by activating Wnt signaling. The results shed a novel insight on the unexplored function of osteoclasts as a potential bone protector that may develop an unconventional strategy to combat bone metastasis.

Treatment of Triple Negative Cell Lines with Olaparib to Block DNA Repair

Background: The most aggressive breast cancer is the triple negative histological type and the gold standard for its treatment is platinum salts, such as carboplatin. Due to high recurrence, there is a need to test new drugs, such as PARP inhibitors (PARPi) that induce lethality in cells with DNA damage. Olaparib is a PARPi, already used in some tumors, but not tested in canine species. Thus, the aim of this study was demonstrating the efficacy of olaparib in inhibiting DNA repair and controlling disease progression by decreasing the migration capacity of mammary tumor cells. Methods: The cell lines, CF41.Mg and MDA-MB-468, were cultured and was performed the MTT to define the best dose of carboplatin. Next, the cells were treated with 10 µM carboplatin, olaparib and with combination of both for 24 hours. PARP-1 protein and gene expression was evaluated by immunofluorescence, western blotting and qRT-PCR, respectively. The analysis of cell migration was performed in transwell chambers. Results: For CF41.Mg and MDA-MB-468 cell lines, there was decrease in PARP-1 protein and gene expression after treatment with carboplatin, olaparib and both in combination compared to the group without treatment (control) (p<0.05). Moreover, in both lines, reduction in invasion rate was observed after treatment with carboplatin, olaparib and when combined, compared to the control group (p<0.05). Conclusion: Our data suggests that carboplatin and olaparib were able to block DNA repair and control the cancer invasion, especially when used in combination. The results with olaparib in the canine line are unpublished. The olaparib should be a possible agent against human breast cancer and canine mammary tumors.

Parsing β-catenin’s cell adhesion and Wnt signaling functions in malignant mammary tumor progression

During malignant progression, epithelial cancer cells dissolve their cell–cell adhesion and gain invasive features. By virtue of its dual function, β-catenin contributes to cadherin-mediated cell–cell adhesion, and it determines the transcriptional output of Wnt signaling: via its N terminus, it recruits the signaling coactivators Bcl9 and Pygopus, and via the C terminus, it interacts with the general transcriptional machinery. This duality confounds the simple loss-of-function analysis of Wnt signaling in cancer progression. In many cancer types including breast cancer, the functional contribution of β-catenin’s transcriptional activities, as compared to its adhesion functions, to tumor progression has remained elusive. Employing the mouse mammary tumor virus (MMTV)–PyMT mouse model of metastatic breast cancer, we compared the complete elimination of β-catenin with the specific ablation of its signaling outputs in mammary tumor cells. Notably, the complete lack of β-catenin resulted in massive apoptosis of mammary tumor cells. In contrast, the loss of β-catenin’s transcriptional activity resulted in a reduction of primary tumor growth, tumor invasion, and metastasis formation in vivo. These phenotypic changes were reflected by stalled cell cycle progression and diminished epithelial–mesenchymal transition (EMT) and cell migration of breast cancer cells in vitro. Transcriptome analysis revealed subsets of genes which were specifically regulated by β-catenin’s transcriptional activities upon stimulation with Wnt3a or during TGF-β–induced EMT. Our results uncouple the signaling from the adhesion function of β-catenin and underline the importance of Wnt/β-catenin–dependent transcription in malignant tumor progression of breast cancer.

Macrophage polarization by MSC-derived CXCL12 determines tumor growth

Background Phenotypic and functional heterogeneity of macrophages is known to be the main reason for their ability to regulate inflammation and promote tumorigenesis. Mesenchymal stem cells (MSCs) are one of the principal cells commonly found in the tumor stromal niche, with capability of macrophage phenotypic switching. The objective of this study was to evaluate the role of C-X-C motif chemokine ligand 12 (CXCL12) produced by marrow-derived MSCs in the phenotypic and functional pattern of bone marrow-derived macrophages (BMDMs). Methods First, the CRISPR/Cas9 system was used for the CXCL12 gene knock-out in MSCs. Then, coculture systems were used to investigate the role of MSCsCXCL12−/− and MSCsCXCL12+/+ in determination of macrophage phenotype. To further analyze the role of the MSC-derived CXCL12 niche, cocultures of 4T1 mammary tumor cells and macrophages primed with MSCsCXCL12−/− or MSCsCXCL12+/+ as well as in-vivo limiting dilution assays were performed. Results Our results revealed that the expression of IL-4, IL-10, TGF-β and CD206 as M2 markers was significantly increased in macrophages co-cultured with MSCsCXCL12+/+ , whereas the expression of IL-6, TNF-α and iNOS was conversely decreased. The number and size of multicellular tumor spheroids were remarkably higher when 4T1 cells were cocultured with MSCCXCL12+/+-induced M2 macrophages. We also found that the occurrence of tumors was significantly higher in coinjection of 4T1 cells with MSCCXCL12+/+-primed macrophages. Tumor initiating cells were significantly decreased after coinjection of 4T1 cells with macrophages pretreated with MSCsCXCL12−/−. Conclusions In conclusion, our findings shed new light on the role of MSC-derived CXCL12 in macrophage phenotypic switching to M2, affecting their function in tumorigenesis.

Fucoxanthin Exerts Anti-Tumor Activity on Canine Mammary Tumor Cells via Tumor Cell Apoptosis Induction and Angiogenesis Inhibition

Fucoxanthin is a carotenoid derived from brown algae. It is known to exhibit anticancer activity, including the promotion of apoptosis and cell cycle arrest in several tumors. However, it remains unclear whether fucoxanthin exhibits anticancer activity against mammary gland tumors. In this study, we evaluated fucoxanthin activity against canine mammary tumor cells (CMT-U27) and human umbilical vein endothelial cells (HUVECs) to investigate its effect on cell viability, migration, tube formation, and angiopoietin 2 (Ang2) expression. Our results showed that fucoxanthin induced apoptosis via caspase activation in CMT-U27 cells. In rat aortic ring assay, fucoxanthin suppressed endothelial cell sprouting. Furthermore, fucoxanthin inhibited tube formation and migration in HUVECs. The number of migrated cells was assessed using CMT-U27 cells. The results demonstrated that fucoxanthin exerted anti-angiogenic activity on HUVECs and CMT-U27 cells by promoting Ang2 expression. In conclusion, our results demonstrated that fucoxanthin induced tumor cell death and inhibited angiogenesis, suggesting that fucoxanthin could be considered as a promising therapeutic agent for canine mammary gland tumors.

Immune Milieu Established by Postpartum Liver Involution Promotes Breast Cancer Liver Metastasis

In rodents, we identified a physiologic process within the normal liver that creates a pre-metastatic niche. This physiology is weaning-induced liver involution, characterized by hepatocyte cell death, immune influx, and extracellular matrix remodeling. Here, using weaning-induced liver involution as a model of a physiologically regulated pro-metastatic niche, we investigate how liver involution supports breast cancer metastasis. Liver metastases were induced in BALB/c immune competent hosts by portal vein injection of D2OR (low metastatic) or D2A1 (high metastatic) mouse mammary tumor cells. Tumor incidence and multiplicity increased in involution hosts with no evidence of a proliferation advantage. D2OR tumor cell extravasation, seeding, and early survival were not enhanced in the involuting group compared to the nulliparous group. Rather, the involution metastatic advantage was observed at 14 days post tumor cell injection. This metastatic advantage associated with induction of immune tolerance in the involution host liver, reproductive state dependent intra-tumoral immune composition, and CD8-dependent suppression of metastases in nulliparous hosts. Our findings suggest that the normal postpartum liver is in an immune suppressed state, which can provide a pro-metastatic advantage to circulating breast cancer cells. Potential relevance to women is suggested as a postpartum diagnosis of breast cancer is an independent predictor of liver metastasis.

Targeting the Aryl Hydrocarbon Receptor Signaling Pathway in Breast Cancer Development

Activation of the aryl hydrocarbon receptor (AhR) through environmental exposure to known human carcinogens including dioxins can lead to the promotion of breast cancer. While the repressor protein of the AhR (AhRR) blocks the canonical AhR pathway, the function of AhRR in the development of breast cancer is not well-known. In the current study we examined the impact of suppressing AhR activity using its dedicated repressor protein AhRR. AhRR is a putative tumor suppressor and is silenced in several cancer types, including breast, where its loss correlates with shorter patient survival. Using the AhRR transgenic mouse, we demonstrate that AhRR overexpression opposes AhR-driven and inflammation-induced growth of mammary tumors in two different murine models of breast cancer. These include a syngeneic model using E0771 mammary tumor cells as well as the Polyoma Middle T antigen (PyMT) transgenic model. Further AhRR overexpression or knockout of AhR in human breast cancer cells enhanced apoptosis induced by chemotherapeutics and inhibited the growth of mouse mammary tumor cells. This study provides the first in vivo evidence that AhRR suppresses mammary tumor development and suggests that strategies which lead to its functional restoration and expression may have therapeutic benefit.