The Effects Of Cyclooxygenase-2Signal On Regulating The Proliferative And Anti-apoptotic Capabilities Of Canine Mammary Tumor Initiating Cells

Introduction:Cyclooxygense-2and prostaglanding (COX-2/PGE2) play multiple biological functions on CTMs progression. To elucidate the underlying mechanisms of COX-2/PGE2signal in regulating the biological properites of TICs, we carried out investigations in histological and cytological level:Methods:1. COX-2, CD44, Oct-3/4, EGFR and Ki-67expression were detected in normal and neoplasic canine mammary tissues repectively, and the associations between these markers with clinicopathological characteristics and their relationship among primary mammary tumors and tumorspheres were also analized.2. Gene expression analysis of COX-2/PGE2, Wnt/β-catenin signal pathways and TIC associated transcription factors in tumorsphere cells established from two canine mammary tumor cell lines, CMT1211and CMT1016, versus their adherent counterpart were performed by RT-PCR. The CD44+/CD24-, ALDH+phenotypes of spheroid cells, and their abilities regarding proliferation, invasion and tumor formation were tested.3. COX-2experssoin in tumor cells was knockdowned via siRNA interference or drug stimulation (Celecoxib) and changes of cell phenotypes, tumorspheres formation efficiency and effect on Wnt signaling were observed. Besides that, the suppressive effect of celecoxib combined with cisplatin in NOD/SCID mouse tumor models was verified in terms of targeting TICs.Results:1.Significant differential COX-2expression was observed among histotypes of CMTs and its elevated expression indicated unfavorable factors. Heterogeneous COX-2expression was strongly associated with CD44and EGFR, and its expression was stronger in TSs derived from more malignant tumors along with similar staining intensity with CD44and Oct-3/4in tumorspheres.2. In TICs enriched tumorspheres, COX-2, EP2, EP4, β-catenin, Cyclin-D1, Oct-3/4and ALDH1A3mRNA upregulated robustly and15-PDGH mRNA downregulated. FACS analysis showed that spheroid cells contained higher number of CD44+/CD24-and ALDH+subpopupation than corresponding parental cells, and obtained aggressive phenotype and tumor formation capacity.3. Knockdown of COX-2expression could effectively decreased CD44+/CD24-, ALDH+cells and suppressed malignant behaviors of tumor cells, while these effcts could be conteracted by exogenenous PGE2. COX-2inhibition would downregulate β-catenin, Cyclin-D1and upregulate Phospho-β-catenin (Ser33/37Thr41), and decreased tumorsphere formation efficiency and sensitized TICs to cisplatin treatment. Daily of20mg/kg celecoxib (Oral administration, daily) combined with6mg/kg cisplatin (peritoneal injection of, three times a week) could suppress xenograft growth and promote tumor remission.Conclusions:COX-2/PGE2plays potential role in canine mammary tumor initiation and biological perperites of TICs which was possibly mediated by Wnt/catenin pathway. Targeting COX-2could be a promising neoadjvant chemotherapy approach for eliminating TICs in CMTs.