Fig 2: Tumor selectivity and the involvement of CD44. A5 = MLO-A5 osteocytes, ß-cat = ß-catenin plasmids, BML = BML284, Eno1 = Enolase 1, Ubc = ubiquitin C. The signal and double asterisk indicates p < 0.01 and p < 0.05, respectively. (A) Comparison of MTT-based viability of four non-tumor cells (KTB34, KTB6, MC3T3, MSC) and four tumor cells (EO771, TRAMP, 4T1.2, PANC-1) in response to ß-catenin-iTS CM and BML-iTS CM in 2 days. (B) Tumor selectivity of EO771 CM, 4T1.2 CM, TRAMP CM, and PANC-1 CM. MTT-based tumor selectivity is defined as the ratio of (reduction in tumor cells) to (reduction in non-tumor cells). The selectivity value above 1 indicates that MTT-based inhibition is more selective to tumor cells than non-tumor cells. Of note, N.D. = not defined since the viability of non-tumor cells is stimulated. (C&D) Tumor selectivity of Enolase 1 and ubiquitin C. (E) CD44 was co-immunoprecipitated with Eno1. The protein extracts of EO771 cells were incubated with anti-Eno1 antibody using the protein A/G beads. Immunoprecipitates and total cell lysates were analyzed by Western blotting with anti-CD44 and anti-Eno1 antibodies as indicated. (F&G) siRNA knockdown of CD44 suppressed Eno1-mediated inhibition of the proliferation of EO771 cells. (H) siRNA knockdown of CD44 suppressed Eno1-mediated downregulation of MMP9, Runx2, and Snail in EO771 cells.

Fig 3: Immunohistochemical analysis of ENO1 and ENO2 in BRAF wild-type and BRAF V600E-mutated CRC. A, Representative images of positive and negative immunohistochemical staining with anti–ENO2 antibody in BRAF wild-type CRC and BRAF V600E-mutated CRC. Scale bar: 50 µm. B, The positive rate of ENO1 and ENO2 expression in BRAF wild-type and BRAF V600E-mutated CRC based on immunohistochemical staining. C, The positive rate of ENO2 expression in BRAF wild-type+nonV600E and BRAF V600E-mutated CRC based on immunohistochemical staining in 121 CRC patient

Fig 4: Effects of enolase 1, ubiquitin C, and iTS CM on the expression of tumor-promoting and tumor-suppressing genes. CM = conditioned medium, CN = control (no CM treatment), ß-cat = ß-catenin plasmids, siEno1 = Enolase 1 siRNA, siUbc = ubiquitin C siRNA, EO = EO771 mammary tumor cells. (A&B) Expression of MMP9, Runx2, Snail, p53, and TRAIL in response to enolase 1 and ubiquitin C in EO771 breast cancer cells. (C&D) Expression of MMP9, Runx2, Snail, p53, and TRAIL in response to ß-catenin-overexpressing iTS CM impaired by siRNAs specific to enolase 1 and ubiquitin C. (E) Expression of PDL1 in EO771 mammary tumor cells in response to ß-catenin-overexpressing iTS CM, enolase 1, and ubiquitin C. (F&G) Expression of MMP9, Runx2, Snail, p53, TRAIL, and caspase 3 in EO771 mammary tumor cells in response to ß-catenin-overexpressing pre-treatment tumor cell-derived CM. (H) Low survival for cancer patients with a high transcript level of MMP9, Runx2, or Snail. (I) Proposed regulatory mechanism to inhibit tumor progression by iTS-CM. According to the mechanism, ß-catenin-overexpressing iTS cells secrete ubiquitin C (Ubc), enolase 1 (Eno1), p53, and Trail. They suppress the progression of tumor cells by downregulating MMP9, Runx2, Snail, and PDL1, while upregulating cleaved-caspase 3. It should be noted that Eno1 interacts with CD44 and inhibits MMP9, Runx2, and Snail.

Fig 5: ENO1 induces PASMC resistance to apoptosis. a The amount of apoptotic markers: PARP (full length and cleaved), caspase 3 (full length and cleaved), caspase 9 (full length and cleaved) were detected by western blotting in shENO1–PASMC. b shENO1 and shCTRL PASMC were subjected to the TUNEL assay (Scale bars, 100 µm) and c the apoptotic rates were calculated (n = 3, **P < 0.0001) with the TUNEL assay in shENO1–PASMC. d PASMC were treated with ENOblock in different concentrations for 24 h, and the LDH assay was performed to detect cell death. e PASMCs were treated with 10 µM ENOblock and hypoxia (1% O2) for 24 h, and western blotting was used to detect apoptotic markers in cell lysate. f Images of the TUNEL assay (Scale bars, 100 µm) and g calculated apoptotic rates (n = 3 per group, **P < 0.0001) in ENOblock-treated PASMCs under normoxic and hypoxic conditions. h PASMCs were transfected with pCMV3-ENO1-GFP and treated with etoposide (in gradient concentrations) for 24 h. Afterwards, the cell viability was measured (n = 3 per group, *P < 0.05). i PASMCs were transfected with pCMV3-ENO1-GFP and treated with 100 µM etoposide for 24 h. The apoptotic markers were detected using western blotting in the cell lysate. j Representative images of the TUNEL assay were shown (Scale bars, 100 µm). k Apoptotic rates in ENO1-overexpressed PASMC under the treatment of etoposide were calculated from images in experiments depicted in j. n = 3 per group, *P = 0.04489. Data represent the mean ± SEM. Student t test and one-way ANOVA were used to compare two and multiple groups. Bonferroni post-tests were carried out after ANOVA