Abstract P5-12-04: Osteogenic cocktail induces calcifications in human breast cancer cell line via placental alkaline phosphatase expression
Authors: Fushimi Atsushi , Takeyama Hiroshi , Tachibana Toshiaki , Manome Yoshinobu 
Published: March 2022

Breast cancer is frequently characterized by calcifications in mammography. Although breast calcifications were previously thought to be produced passively as the final stage of cell regression, recent studies have shown active production. In another context, vascular smooth muscle cells have been found to produce vascular calcifications by acquiring osteoblast-like characteristics. The same mechanism has been suggested to account for breast calcifications. A report showed that breast calcifications are clearly generated by the mouse breast cancer cell line 4T1 through osteoblast-like characteristics. However, the details underlying such a mechanism of mammary calcification is unknown. Understanding this mechanism will improve diagnostic accuracy for breast cancer. We induced calcifications using osteogenic cocktail on the human breast cancer cell line MDA-MB-231. Our hypothesis was that breast cancer calcifications are generated in a process similar to that of osteoblast differentiation. By a comprehensive analysis of transcription compared with osteoblastic cell line MC3T3-E1 in a microarray, we showed that placental alkaline phosphatase (PLAP) is involved in producing calcifications through PI3K-Akt signaling pathways. Herein, we demonstrated that calcifications occur and that alkaline phosphatase enzyme activity increases in MDA-MB-231 cells cultured using an osteogenic cocktail-containing medium. Microarray transcript analysis showed that the PI3K-Akt signaling pathway was significantly involved, with recruitment of placental alkaline phosphatase. Calcifications and alkaline phosphatase enzyme activity were suppressed by silencing placental alkaline phosphatase using a small interfering RNA. Inhibition of the PI3K-Akt signaling pathway suppressed phospho-c-Jun and placental alkaline phosphatase and resulted in absence of calcifications. These findings reveal that breast cancer cells acquire alkaline phosphatase enzyme activity via placental alkaline phosphatase expression and suggest that breast calcification formation is closely associated with the PI3K-Akt signaling pathway. Our results suggest that PLAP produces ALP enzyme activity required for calcification in MDA-MB-231 cells. PLAP is clinically expressed in some breast cancers. A report showed that dexamethasone induces PLAP enzyme activity and mRNA in the human breast cancer BC-M1 cell line. Therefore, dexamethasone in osteogenic cocktail may be associated with PLAP expression in the MDA-MB-231 cells. On the other hand, c-Jun binds to the promoter region of ALPP in MDA-MB-231, and we showed that c-Jun phosphorylation appears in MDA-MB-231 cells cultured using osteogenic cocktail. We suggest that PLAP transcript would be activated when phospho-c-Jun enters the nucleus and binds to the promoter region of PLAP. Our proposed mechanism in breast calcifications is that osteogenic cocktail induces c-Jun phosphorylation via PI3K-Akt signaling pathway and that PLAP transcription would be activated when phospho-c-Jun enters the nucleus and binds to the promoter region of PLAP. Future research might assess whether increased PLAP levels are the main driver of intracellular calcifications in breast cancer, as well as whether the PI3K-Akt signaling pathway has a similar effect in vivo that we demonstrated to cause calcifications in vitro.

Citation Format: Atsushi Fushimi, Hiroshi Takeyama, Toshiaki Tachibana, Yoshinobu Manome. Osteogenic cocktail induces calcifications in human breast cancer cell line via placental alkaline phosphatase expression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-04.