Fig 2: Combined targeting of EZH2 and p38 enzymatic activities reduces primary breast cancer growth and metastasis(A) Primary tumor growth curves of NOD/SCID mice orthotopically implanted with MDA-MB-231 cells. When primary tumors reached 100 mm3, mice were treated intraperitoneally with EPZ-6438 (10 mg/kg/day), SB202190 (p38i, 1 mg/kg/day), combination, or control (4% DMSO-30% PEG 300-5% Tween 80), 5 days/week for 56 days (n = 10/group). Primary tumor growth as assessed by caliper measurements, shown as mean ± SEM.(B) Quantification of tumor volume at day 56 shown as mean ± SEM.(C) MDA-MB-231 cells were injected intracardially in nude mice (n = 10/group) and treated as in (A), for 3 weeks. Bars show the number of metastases per mouse in each group on day 21 after heart inoculation ±SEM.(D) Representative H&E-stained sections of lung metastases. Magnification 600x. Scale bar 50 µm.(E) Co-immunoprecipitation and immunoblots of methylated p38a in whole-cell lysates of primary MDA-MB-231 xenograft tumors derived from (A).(F) Immunoblots for the indicated proteins in lysates obtained from the MDA-MB-231 primary orthotopic xenografts in (A). For A-C, *p = 0.05; **p = 0.01; ***p = 0.005; ****p = 0.0001.

Fig 3: Combined pharmacological blockade of EZH2 and p38 enzymatic activities reduces neoplastic functions(A) Immunoblots of MDA-MB-231 cells treated with GSK-343 (3 µM for 48 h), SB202190 (p38i, 20 µM for 48 h), or the combination.(B) Cells in A were subjected to growth assays.(C) Synergistic effect of EZH2 inhibitor and p38 inhibitor in MDA-MB-231 cells incubated with various doses of GSK-343 and SB202190 for 4 d. A matrix for synergy score was calculated (Ianevski et al., 2017).(D and E) Wound healing assay to quantify cell migration (D) and reconstituted Boyden basement membrane-invasive chamber assay of MDA-MB-231 cells treated as in (A). Representative chambers after crystal violet staining are shown above bars. Data for B-E are from at least three independent experiments carried out in at least triplicate. Data for B, (D and E) are presented as mean ± SEM. *p = 0.05; **p = 0.01; ***p = 0.005; ****p = 0.0001.

Fig 4: pEZH2-T367 and p-p38 are significantly upregulated in human breast cancer metastasis compared to matched primary tumors from the same patient(A) Representative images of matched primary human breast carcinomas and metastasis (n = 16 patients) immunostained for pEZH2-T367 and p-p38 (600x). pEZH2-T367 is upregulated in the cytoplasm of metastatic cells compared to the primary tumor. Bars 50 µm.(B) Distribution of pEZH2-T367 protein expression in the 16 primary breast carcinomas and matched metastasis; the number of primary and metastatic breast carcinomas with low and high pEZH2-T367.(C) Association between pEZH2-T367 and p-p38 expression in primary and metastatic carcinomas of the breast; 56.75% of metastasis exhibit high expression of both pEZH2-T367 and p-p38.