While bone is a frequent target of breast cancer-associated metastasis, little is known about the effects of tumor-bone interactions on the efficacy of tumor-suppressing agents. Here we examined the effect of two FDA-approved dopamine modulators, fluphenazine and trifluoperazine, on mammary tumor cells, osteoclasts, osteoblasts, and osteocytes. These agents suppressed proliferation and migration of mammary tumor cells chiefly by antagonizing dopamine receptor D2 and reduced bone resorption by downregulating nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1). Three-dimensional spheroid formation assays revealed that tumor cells have high affinity to osteocytes and type I collagen, and interactions with osteocytes as well as administration of fluphenazine and trifluoperazine downregulated Snail and suppressed migratory behaviors. Unlike the inhibitory action of fluphenazine and trifluoperazine on tumor growth, tumor-osteocyte interactions stimulated tumor proliferation by upregulating NFκB and Akt. In the bone microenvironment, osteocytes downregulated Snail and acted as an attractant as well as a stimulant to mammary tumor cells. These results demonstrate that tumor-osteocyte interactions strengthen dopamine receptor-mediated suppression of tumor migration but weaken its inhibition of tumor proliferation in the osteocyte-rich bone microenvironment. These findings provide novel insight into the cellular cross-talk in the bone microevironment and the effects of dopamine modulators on mammary tumor cells and osteocytes. .

Background: Advanced breast cancer metastasizes to many organs including bone, but few effective treatments are available. Here we report that induced tumor-suppressing (iTS) MSCs protected bone from metastases while un-induced MSCs did not. Methods: iTS MSCs were generated by overexpressing Lrp5, β-catenin, Snail, or Akt. Their tumor-suppressing capability was tested using a mouse model of mammary tumors and bone metastasis, human breast cancer tissues and cancer cell lines. Results: In a mouse model, the induced MSC-derived conditioned medium (MSC CM) reduced mammary tumors and suppressed tumor-induced osteolysis. Tumor-promoting genes such as CXCL2 and LIF, as well as PDL1, a blocker of T-cell-based immune responses were downregulated. Proteomics analysis revealed that heat shock protein 90 (Hsp90ab1), calreticulin (Calr) and peptidylprolyl isomerase B (Ppib), which are highly expressed intracellular proteins in many cancers, were enriched in MSC CM as atypical tumor suppressors. Thus, overexpressing selected genes that were otherwise tumorigenic rendered MSCs the tumor-suppressing capability through the atypical suppressors, as well as p53 and Trail. Notably, the inhibitory effect of Lrp5- and Akt-overexpressing MSC CMs, Hsp90ab1 and Calr presented selective inhibition to tumor cells than non-tumor cells. The development of bone-resorbing osteoclasts was also suppressed by MSC CMs. Conclusion: Collectively, the results showed an anti-tumor effect of iTS MSCs and suggested novel therapeutic approaches to suppress the progression of tumors into the bone.

Breast cancer is the most common cancer detected in women and current screening methods for the disease are not sensitive. Volatile organic compounds (VOCs) include endogenous metabolites that provide information about health and disease which might be useful to develop a better screening method for breast cancer. The goal of this study was to classify mice with and without tumors and compare tumors localized to the mammary pad and tumor cells injected into the iliac artery by differences in VOCs in urine. After 4T1.2 tumor cells were injected into BALB/c mice either in the mammary pad or into the iliac artery, urine was collected, VOCs from urine headspace were concentrated by solid phase microextraction and results were analyzed by gas chromatography-mass spectrometry quadrupole time-of-flight. Multivariate and univariate statistical analyses were employed to find potential biomarkers for breast cancer and metastatic breast cancer in mice models. A set of six VOCs classified mice with and without tumors with an area under the receiver operator characteristic (ROC AUC) of 0.98 (95% confidence interval [0.85, 1.00]) via five-fold cross validation. Classification of mice with tumors in the mammary pad and iliac artery was executed utilizing a different set of six VOCs, with a ROC AUC of 0.96 (95% confidence interval [0.75, 1.00]).

Previous studies reported that miR-146a was involved in small intestine ischemia-reperfusion (I/R) injury, but the mechanism is largely vague. Here, we aimed to identify the change of miR-146a in patients with mesenteric ischemia and explore the potential regulatory mechanism of miR-146a in intestine epithelial cells survival under ischemia and I/R injury. The plasma of 20 patients with mesenteric ischemia and 25 controls was collected to examine the miR-146a expression by qPCR. Rat intestinal epithelial cells (IEC-6) and 24 male Sprague-Dawley rats were included to build ischemia and I/R model in vitro and in vivo. The qPCR results showed that miR-146a decreased both in the plasma of patients with mesenteric ischemia and in IEC-6 cells and rat small intestine tissues in ischemia and I/R model compared to controls. Both the in vitro and in vivo results showed that I/R resulted in more severe apoptotic injury than ischemia. Cleaved-caspase 3, TLR4, TRAF6, and nuclear NF-κB p65 were up-regulated accompanying reduced XIAP and SOCS3 expression in intestinal ischemia and I/R injury. After up-regulation of miR-146a in IEC-6 cells, increased cell survival and decreased cell apoptosis were observed, concomitant with decreased cleaved-caspase 3 and down-regulated TLR4/TRAF6/NF-κB pathway. What is more, this protective effect was blocked by TRAF6 overexpression and increased nuclear NF-κB p65 nuclear. Taken together, this study revealed that miR-146a expression was decreased in small intestine ischemia and I/R injury. And miR-146a improves intestine epithelial cells survival under ischemia and I/R injury through inhibition TLR4, TRAF6, and p-IκBα, subsequently leading to decreased NF-κB p65 nuclear translocation.

Dopaminergic signaling plays a critical role in the nervous system, but little is known about its potential role in breast cancer and bone metabolism. A screening of ~1,000 biologically active compounds revealed that a selective agonist of dopamine receptor D1 (DRD1), A77636, inhibited proliferation of 4T1.2 mammary tumor cells as well as MDA-MB-231 breast cancer cells. Herein, we examined the effect of A77636 on bone quality using a mouse model of bone metastasis from mammary tumor. A77636 inhibited migration of cancer cells in a DRD1-dependent fashion and suppressed development of bone-resorbing osteoclasts by downregulating NFATc1 through the elevation of phosphorylation of eIF2α. In the mouse model of bone metastasis, A77636 reduced osteolytic lesions and prevented mechanical weakening of the femur and tibia. Collectively, we expect that dopaminergic signaling might provide a novel therapeutic target for breast cancer and bone metastasis.

Osteocytes are mechanosensitive bone cells, but little is known about their effects on tumor cells in response to mechanical stimulation. We treated breast cancer cells with osteocyte-derived conditioned medium (CM) and fluid flow-treated conditioned medium (FFCM) with 0.25 Pa and 1 Pa shear stress. Notably, CM and FFCM at 0.25 Pa induced the mesenchymal-to-epithelial transition (MET), but FFCM at 1 Pa induced the epithelial-to-mesenchymal transition (EMT). This suggested that the effects of fluid flow on conditioned media depend on flow intensity. Fluorescence resonance energy transfer (FRET)-based evaluation of Src activity and vinculin molecular force showed that osteopontin was involved in EMT and MET switching. A mouse model of tumorinduced osteolysis was tested using dynamic tibia loadings of 1, 2, and 5 N. The low 1 N loading suppressed tumor-induced osteolysis, but this beneficial effect was lost and reversed with loads at 2 and 5 N, respectively. Changing the loading intensities in vivo also led to changes in serum TGFβ levels and the composition of tumor-associated volatile organic compounds in the urine. Collectively, this study demonstrated the critical role of intensity-dependent mechanotransduction and osteopontin in tumorosteocyte communication, indicating that a biophysical factor can tangibly alter the behaviors of tumor cells in the bone microenvironment.

Rationale : The progression of cancer cells depends on the soil and building an inhibitory soil might be a therapeutic option. We previously created tumor-suppressive secretomes by activating Wnt signaling in MSCs. Here, we examined whether the anti-tumor secretomes can be produced from tumor cells. Methods: Wnt signaling was activated in tumor cells by overexpressing β-catenin or administering BML284, a Wnt activator. Their conditioned medium (CM) was applied to cancer cells or tissues, and the effects of CM were evaluated. Tumor growth in the mammary fat pad and tibia in C57BL/6 female mice was also evaluated through μCT imaging and histology. Whole-genome proteomics analysis was conducted to determine and characterize novel tumor-suppressing proteins, which were enriched in CM. Results: The overexpression of β-catenin or the administration of BML284 generated tumor-suppressive secretomes from breast, prostate and pancreatic cancer cells. In the mouse model, β-catenin-overexpressing CM reduced tumor growth and tumor-driven bone destruction. This inhibition was also observed with BML284-treated CM. Besides p53 and Trail, proteomics analysis revealed that CM was enriched with enolase 1 (Eno1) and ubiquitin C (Ubc) that presented notable tumor-suppressing actions. Importantly, Eno1 immunoprecipitated CD44, a cell-surface adhesion receptor, and its silencing suppressed Eno1-driven tumor inhibition. A pan-cancer survival analysis revealed that the downregulation of MMP9, Runx2 and Snail by CM had a significant impact on survival outcomes ( p < 0.00001). CM presented a selective inhibition of tumor cells compared to non-tumor cells, and it downregulated PD-L1, an immune escape modulator. Conclusions: The tumor-suppressive secretome can be generated from tumor cells, in which β-catenin presented two opposing roles, as an intracellular tumor promoter in tumor cells and a generator of extracellular tumor suppressor in CM. Eno1 was enriched in CM and its interaction with CD44 was involved in Eno1's anti-tumor action. Besides presenting a potential option for treating primary cancers and metastases, the result indicates that aggressive tumors may inhibit the growth of less aggressive tumors via tumor-suppressive secretomes.

Background: Advanced breast cancer frequently metastasizes to bone, but inhibiting tumor progression in chemotherapy may occasionally enhance tumorigenesis. Here, we employed a counterintuitive approach of overexpressing Yamanaka factors (Oct4, c-Myc, Sox2, and Klf4) and examined a conditioned medium (CM)-based treatment option with induced tumor-suppressing cells (iTSCs). Methods: In vitro proliferation and migration assays were conducted using tumor cell lines derived from breast cancer, as well as prostate and pancreatic cancers, and osteosarcoma. The tumor-suppressing capability of iTSC-derived CM was evaluated using freshly isolated breast cancer tissues and a mouse model of mammary tumors and tumor-induced osteolysis. The regulatory mechanism was evaluated using Western blotting, immunoprecipitation, pull-down, gene overexpression, and RNA interference based on mass spectrometry-based proteomics data. Results: The overexpression of Oct4 and c-Myc in tumor cells and MSCs, but not Sox2 or Klf4, generated anti-tumor CM, which suppressed the progression of mammary tumors and tumor-induced bone loss. Notably, CM downregulated histone demethylase, and PDL-1, a blocker of T-cell-based immune responses. Whole-genome proteomics predicted enolase 1 (Eno1), Hsp90ab1, Eef2, and vinculin as extracellular tumor suppressors. Specifically, CD44 was co-immunoprecipitated with Eno1 and the silencing of CD44 suppressed Eno1's anti-tumor action. The overexpression of Oct4 and c-Myc also generated secretomes that inhibited the development of bone-resorbing osteoclasts. Conclusions: In analogous to cell competition in which Myc-overexpressing cells in Drosophila and mouse embryos remove neighboring cells with a lower level of Myc, this study presented the possibility of eliminating tumor cells by the secretory proteomes derived from Myc/Oc4-overexpressing iTSCs.