br Involvement of Notch receptors in tumors
Involvement of Notch receptors in tumors has been previously described in several types of cancer, where Notch receptors serve as oncogenes or tumor suppressors . Notch3, in particular, is overexpressed by breast cancer Doxorubicinol  whereby down-regulation of Notch3 leads to reduced prolif-eration . Moreover, in vitro and in vivo studies with breast cancer cell lines revealed constitutive activa-tion of Notch3 especially in the basal-like subtype, promoting tumor cell growth . Overexpression of activated Notch3 in vivo induced transformation and tumor growth in mammary tissues, revealing the oncogenic properties of Notch3 signaling [11,12].
Cartilage Oligomeric Matrix Protein (COMP) is a secreted, pentameric protein comprising a polymeriza-tion region located at the N-terminus, followed by 4 epidermal growth factor domains (EGF), 8 thrombos-pondin domains (TSP), and a globular C-terminus. COMP binds to integrins on the cell surface through the RGD domain providing a structural scaffold for the simultaneous binding of different ligands . COMP is a multifunctional protein important for various cellular functions and expressed in diverse cell types and tissues . COMP is secreted by connective tissue, where it interacts with several components of the ECM  and plays a major role in the organization of cartilage [16–18]. Mutations in COMP are associated with skeletal diseases such as pseudoachondroplasia [19,20] and COMP is highly expressed under fibrotic conditions , especially in scleroderma . The role of COMP is also evident in the reorganization of the vascular wall  and thrombin regulation .
Importantly, COMP is expressed by breast cancer epithelial cells, in which high levels of COMP expres-sion are correlated with poor survival, metastases,
enhanced invasiveness, and deregulation of cancer related pathways, revealed by a tumor mRNA expres-sion microarray analysis using a xenograft mouse model . Furthermore, COMP is expressed in prostate cancer, and correlates with recurrence. Additionally, prostate cancer cells expressing COMP were protected against apoptosis and exhibited an enhanced Warburg metabolic effect, due to COMP-induced changes in intracellular calcium homeostasis .
In this study, we found that plasma membrane-bound COMP acts as a cancer stem cell regulator by mediating an enhanced interaction between Notch3 and Jagged1, leading to activation of Notch3. Expres-sion of COMP increased the population of cancer stem cells in vivo and in vitro. COMP knock-out in the MMTV-PyMT mouse model of breast cancer led to reduced tumor growth compared to wild type animals. Thus, the pro-oncogenic effect of COMP is related to its ability to facilitate the interaction between Notch3 and Jagged1, leading to specific activation of the Notch3 pathway, which orchestrates the initiation of cancer stem cells.
Materials and methods
Cell lines & tumorspheres formation
BT-20 and MDA-MB-231 breast cancer cell lines were grown in DMEM High Glucose medium contain-ing 10% FBS, 1% penicillin and streptomycin (Thermo Fisher Scientific). COMP expressing cells were gener-ated previously  and maintained with 0.8 μg/ml G418 (Thermo Fisher Scientific). Both cell lines were originally purchased from American Type Culture Collection (ATCC) and cells were frozen immediately after re-cultivation of the original aliquot and all the experiments were performed on cultures originating from these secondary aliquots within no N5 passages. The cells were mycoplasma free and tested monthly with the VenorGEM Classic kit (Minerva Biolabs). Cells were transiently transfected with 2.5 μg of plasmid, 7.5 μl of Lipofectamine 3000, and 5 μl of Reagent 3000 per sample (Thermo Fisher Scientific) for 48 h. DAPT (Sigma), a γ-secretase inhibitor, was used at 10 μM final concentration. Tumorsphere formation assay was performed in 6-well ultra-low attachment plates (Corning) with Mammocult Medium (STEM-CELL Technologies) supplemented with 4 μg/ml hep-arin (STEMCELL Technologies), 0.48 μg/ml hydrocortisone (STEMCELL Technologies) following manufacturer instructions. In brief, 4 × 104 single suspension free of serum cells seeded in 2 ml of complete Mammocult Medium and incubated at 37 °C with 5% CO2 for 7 days. At least 10 images were captured per well with EVOS XL Core Cell Imaging System (Thermo Fisher Scientific) and 10 tumorsphere lengths were measured per well utilizing ImageJ image analysis software.
COMP leads to activation of Notch3 109
qPCR array and real time PCR
RNA was purified from MDA-MB-231 COMP-expressing and control cells seeded in 6-well plate using RNeasy Plus Mini kit (Qiagene). In total 4 RNA samples were collected from each group. The PAHS-059Y RT2 Profiler PCR Array Human Notch Signaling Pathway Plus (Qiagene) process was performed and analyzed following manufacturer instructions and online tools. For real time PCR, RNA was reverse transcribed to DNA with SuperScript III Reverse Transcriptase and gene expression was quantified with TaqMan Gene Expression Assays, CD133 Hs01009259_m1, Notch1 Hs01062014_m1, Notch2 Hs01050702_m1, Notch3 Hs01128537_m1 (Thermo Fisher Scientific).