Fig 1: Ectopic RUNX2 expression leads to delayed differentiation and a lactation defect. (A) Whole-mount and histological analysis of MMTV-Runx2 glands (n=11) during late pregnancy (D17 pregnant) reveals a reduction in side-branching and alveolar expansion compared with WT (n=14), and failure to form mature alveolar units during lactation (WT n=13; Runx2 n=11). (B) Ki67 staining of WT and MMTV-Runx2 glands at day 12 (D12) pregnancy (n=4 each), late pregnancy (D17P; n=6 each) and day 1 of lactation (D1; WT n=6; Runx2 n=7). (C) Immunohistochemistry of whey acidic protein (WAP) and RUNX2 on serial sections at day 1 lactation in WT (n=6) and MMTV-Runx2 (n=7) glands illustrates their reciprocal expression pattern. (D) Quantification of Wap mRNA with dramatically less Wap in Runx2 glands at late pregnancy (d17P) and lactation (d1L); data are means ± s.d. normalised to HPRT relative to WT virgin. Whole-mounts, 8× magnification; H&Es, 200× magnification; IHC images, 100× magnification in B and 200× magnification in C.
Fig 2: Expression of RUNX2 correlates with ER/PR/HER2-negative human breast cancer. Invasive breast carcinomas from a tumour tissue microarray (TMA-1) were stained for RUNX2. (A) Scatterplot showing the range of positive histoscores. Expression was divided into RUNX2-negative (histoscore 0), -low (histoscore 1–24) or -high (histoscore ≥25) in 416 breast cancers. The dotted line at histoscore 25 demonstrates the cut-off for RUNX2-high patients. Position on the y-axis reflects the order in which samples were analysed. (B) Kaplan-Meier of patient survival for 384 patients in A for which follow-up data was available. Survival is plotted for patients with high-RUNX2 tumours (n=21) and negative/low-RUNX2 tumours (n=363). (C) Examples of individual tumours stained for RUNX2 and ER, depicting the reciprocal expression pattern. Boxed areas are shown at higher magnification. (D) Significantly more RUNX2-high cancers were ER-negative (P=0.005; chi-square) and specifically associated with the triple-negative (ER/PR/HER2-negative) group (P=0.008; chi-square).
Fig 3: Mammary glands of aged MMTV-Runx2 females display abnormal hyperplastic and pre-neoplastic changes. (A) Representative whole-mounts of aged MMTV-Runx2 and WT littermate controls (8× magnification). (B) Representative images of H&E sections showing diffuse hyperplasia in two independent MMTV-Runx2 transgenic glands with evidence of secretory hyperplastic lesions and dilated ducts (100× magnification). (C) Abnormal features observed in aged MMTV-Runx2 glands, such as distorted acini with lobular fibrosis and chronic inflammatory cell infiltrate (I), and alveolar hyperplasia with luminal cells exhibiting large nuclei and prominent nucleoli (II,III) (images shown at 400× magnification). (D) Ductal carcinoma in situ (DCIS) in an MMTV-Runx2 female; middle panel is higher magnification of boxed area. Smooth muscle actin (SMA) staining shows an intact basal/myoepithelial layer. (E) Hyperplastic lesions are negative for ER, PR and HER2 but show positivity for MYC as determined by immunohistochemistry (400× magnification).
Fig 4: RUNX2 potentiates mammosphere-forming potential of HC11 cells.(A) qRT-PCR for Runx2 on HC11 grown in 2D and as mammospheres (mammo). Gene expression is shown as relative expression to Gapdh (mean ± SD); n = 3 for each group. Expression in mammospheres was compared to 2D MMECs; *p = 0.02 (Unpaired t-test with Welch’s correction). (B) Western blot of HC11 cells transfected with empty vector (HC11-CTR) and RUNX2 overexpressing plasmid (HC11-Runx2). GAPDH used as loading control. (C) Growth curve of HC11-CTR and HC11-Runx2 cells grown in 2D culture. Cell numbers were counted daily in triplicate for each time point, for each cell line. Data are expressed as mean +/- SD. Graph is representative of two independent experiments from 2 independent lines of HC11-CTR and 3 independent lines of HC11-Runx2. (D) Mammospheres from HC11-CTR and HC11-Runx2 cells grown in non-adherent conditions for 7 days. Data are expressed as mean +/- SD. Graph representative of four independent experiments from 2 independent lines of HC11-CTR and 3 independent lines of HC11-Runx2. The number of mammospheres formed by HC11-CTR and HC11-Runx2 was compared. *p < 0.0001 (Unpaired t-test with Welch’s correction).
Fig 5: Exogenous WNT activation does not rescue Runx2-depleted mammospheres.Bright field images of mammosphere cultures (A) derived from K14-Cre:Runx2wt/wt (top) and K14-Cre:Runx2fl/fl (bottom) mice, treated for 6 days with either vehicle (Vh) or recombinant WNT3a (100 ng/ml). Scale bars represent 100 µm. The average area of primary mammospheres (B) and number (area cut-off 4000 pixel) of primary spheres (C) of MMECs extracted from K14-Cre:Runx2wt/wt and K14-Cre:Runx2fl/fl mice, treated for 6 days with either vehicle (Vh) or WNT3a. Mammospheres were counted and measured after 6 days in culture. Data are expressed as mean (±SD). The area and number of mammospheres formed by K14-Cre:Runxwt/wt and K14-Cre:Runx2fl/fl MMECs after WNT3a treatment was compared. *p < 0.05; **p < 0.001 (Unpaired t-test with Welch’s correction).
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