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Fam20C Dual Regulation of Bone Resorption and Breast Cancer Bone Metastasis through Osteopontin and BMP4

ABSTRACT: Samples: Conditioned medium from cultured wild-type or Fam20C-VavCre bone marrow cells. Six 10-cm plates of bone marrow cells from each genotype (Wild-type or Fam20C-VavCre) of 2-month-old female littermate mice at a density of 2.8*107 cells/plate were cultured in the presence of 40 ng/mL of mouse M-CSF for 3 days. Cells were washed with PBS for four times and with serum-free medium twice. Then cells were cultured in serum-free medium (MEMalpha without phenol red) for 48 hours. Conditioned medium was collected and spin down at 1,000 g for 5 minutes. The supernatant was spin down at 10,000 g for 20 minutes. The supernatant was added with 1 mM Na3VO4, 1 mM PMSF and 1x protease inhibitor cocktail (Sigma-Aldrich), and concentrated with Amicon Ultracel-3K units (EMD Millipore). Protein concentrations were measured with Pierce BCA protein assay (Fisher Scientific), and 600 ug of protein each sample were subjected to following procedures

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Yihong Wan

PROVIDER: MSV000086418 | MassIVE | Fri Nov 06 12:18:00 GMT 2020

REPOSITORIES: MassIVE

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Project description:Disseminated tumor cells (DTCs) in the bone marrow can be detected in patients with solid tumors early on in disease progression. Via interaction with mesenchymal stromal cells (MSCs) these tumor cells may interfere with hematopoiesis. Using appropriate co-culture models, we investigated whether DTCs can change the bone marrow microenvironment by modulating MSC function with a special emphasis on their chemoattractive activity towards hematopoietic stem and progenitor cells (HSPCs). Human bone marrow derived MSCs as well as an immortalised MSC line (SCP-1) were co-cultured with MCF-7, MDA-MB231 breast carcinoma or MCF-10A non-malignant breast epithelial cells or their conditioned medium. Gene expression analysis of SCP-1 cells cultured with MCF-7 conditioned medium revealed SDF-1/CXCL12 as one of the significantly downregulated genes. Both tumor cell lines caused an inhibited SDF-1 promoter activity in SCP-1 cells, whereby MCF-7 medium decreased it to 77% and MDA-MB231 to 47%. Moreover, the SDF-1 mRNA and protein levels were significantly reduced. As a functional consequence of lower SDF-1 levels, we detected a decreased trans-well migration potential of CD34+ HSPC to MSC/tumor cell co-cultures or conditioned medium. The specificity of this chemokine mediated effect was confirmed by blocking studies with the CXCR4 antagonist AMD3100. Downregulation of SP1 transcription factor and increased miR23a levels in MSCs after contact with tumor cell medium as well as an enhanced TGFb1 expression were identified as potential molecular regulators of SDF-1 activity in MSCs. We propose an additional mechanism by which tumor cells affect the niche environment of HSPCs and therefore negatively impact hematopoiesis. Gene expression of human immortalized mesenchymal stromal cells (SCP-1) was investigated after incubation with conditioned medium of the breast carcinoma cell line MCF-7 for 24, 48, and 72 hours. Three independent experiments were performed at each time.

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Fam20C Dual Regulation of Bone Resorption and Breast Cancer Bone Metastasis through Osteopontin and BMP4

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