Fig 2: Downregulation of PDL-1 and Kdm3a in 4T1.2 mammary tumor cells by Hsp90ab1, Eno1, Eef2, and VCL, and the suppression of the development of osteoclasts. CN = control, Hsp = Hsp90ab1, Eno1 = enolase 1, VCL = vinculin, Oct4 = Oct4 plasmids, and CM = conditioned medium. The double asterisk indicates p < 0.01. (A) Reduction in the size and weight of mammary tumors by the daily intravenous administration of 1 µg/mL Eno1. EO771 mammary tumor cells were inoculated into the mammary fat pad of C57BL/6 female mice (N = 10). (B) Reduction in p-AKT, NFkB p65, p-ERK, and TNFa in 4T1.2 cells in response to Eno1 recombinant proteins. (C) Reduction in PDL1 in 4T1.2 cells by Oct4/cMyc-overexpressing 4T1.2 CM, and Eno1, Hsp, Eef2, and VCL recombinant proteins. (D) Reduction in Kdm3a in 4T1.2 cells in response to Eno1, Hsp90ab1, Eef2, and/or VCL recombinant proteins. (E) Elevation of Kdm3a in 4T1.2 cells by the overexpression of Eno1, Eef2, and VCL. (F) Suppression of RANKL-stimulated osteoclast development by Oct4-overexpressing CM. (G) Reduction in the levels of cathepsin K and NFATc1 in RANKL-stimulated osteoclasts by Oct4-overexpressing CM. (H) Regulatory mechanism by the tumor-suppressive secretomes, which were derived from Oct4- and c-Myc-overexpressing tumor cells.

Fig 3: Prediction of the tumor suppressors in CM by mass spectrometry-based whole-genome proteomics. The single and double asterisks indicate p < 0.05 and 0.01, respectively. CN = control, Oct4 = Oct4 plasmids, c-Myc = c-Myc plasmids, Sox2 = Sox2 plasmids, Klf4 = Klf4 plasmids, and CM = conditioned medium. (A) Summary list of the potential tumor suppressors by mass spectrometry-based whole-genome proteomics. (B) Enolase 1 (Eno1), Hsp90ab1 (HSP), Eef2, and vinculin (VCL) as 4 tumor-suppressor candidates based on MTT-based viability. (C) Upregulation of Eno1, Hsp90ab1, Eef2, VCL, p53, and Trail in 4T1.2 cell-derived CM with the overexpression of Oct4, c-Myc, and the treatment with OAC2. The overexpression of Sox2 and Klf4 did not alter their levels. (D) Alterations in the levels of Eno1 and Hsp90ab1 in Oct4 and c-Myc CM by ELISA. (E) Reduction in MTT-based viability of 4T1.2 cells by the treatment with Eno1 and/or Hsp90ab1 recombinant proteins. (F) Tumor selectivity from the MTT-based viability of tumor cells (4T1.2 mammary tumor cells, EO771 mammary tumor cells, and MDA-MB-231 breast cancer cells) and human epithelium cells (KTB34-hTERT and KTB22-hTERT). Tumor selectivity is defined as a ratio of (MTT-based reduction in tumor cells) to (MTT-based reduction in non-tumor cells).