Metastasis-associated protein 1 short form stimulates Wnt1 pathway in mammary epithelial and cancer cells

Abstract

Although Wnt1 downstream signaling components as well as cytoplasmic level of metastatic tumor antigen 1 short form (MTA1s) are elevated in human breast cancer, it remains unknown whether a regulatory cross-talk exists between these two pathways. Here, we provide evidence of a remarkable correlation between the levels of MTA1s and stimulation of the Wnt1 signaling components, leading to increased stabilization of beta-catenin and stimulation of Wnt1 target genes in the murine mammary epithelial and human breast cancer cells. We found that MTA1s influences Wnt1 pathway through extracellular signal-regulated kinase (ERK) signaling as selective silencing of the endogenous MTA1s or ERK, or its target glycogen synthase kinase 3beta resulted in a substantial decrease in beta-catenin expression, leading to the inhibition of Wnt1 target genes. Furthermore, downregulation of beta-catenin in cells with elevated MTA1s level was accompanied by a corresponding decrease in the expression of Wnt1 target genes, establishing a mechanistic role for the ERK/glycogen synthase kinase 3beta/beta-catenin pathway in the stimulation of the Wnt1 target genes by MTA1s in mammary epithelial cells. In addition, mammary glands from the virgin MTA1s transgenic mice mimicked the phenotypic changes found in the Wnt1 transgenic mice and exhibited an overall hyperactivation of the Wnt1 signaling pathway, leading to increased stabilization and nuclear accumulation of beta-catenin. Mammary glands from the virgin MTA1s-TG mice revealed ductal hyperplasia and ductal carcinoma in situ, and low incidence of palpable tumors. These findings reveal a previously unrecognized role for MTA1s as an important modifier of the Wnt1 signaling in mammary epithelial and cancer cells.

Figure 1. MTA1s expression correlates with Wnt1, P-GSK-3β and P-ERK expression in breast cancer cell lines

(A) Western blot analysis of MTA1s, phospho-GSK-3β, GSK-3β, P-ERK, and ERK in the human breast cancer cell lines. (B) Confocal analysis of Wnt1 (red), Phospho-GSK-3β (red), GSK-3β (red), P-ERK (green), ERK (green) and MTA1s in MDA-MB-435, SKBR3, T47D and ZR-75 breast cancer cell lines. Scale bar 10 μm. (C) Expression of Wnt1 and MTA1s in human breast tumors. Breast cancer were immunostained for Wnt1 (a–d), and MTA1s (f–i), and control serum (e,j). Four representative sets shown are derived from consecutive sections of same tumor. Higher magnification is shown as inset.

Figure 2. MTA1s overexpression upregulates Wnt1 signaling pathway in mammary epithelial cells

(A) RT-PCR analysis of Wnt1, Wnt2, Wnt4 and Wnt5a in HC11/pcDNA and HC11/MTA1s stable clones. Analysis of expression of T7-MTA1s, β-catenin and phospho-β-catenin by Western blot in pooled HC11/MTA1s and HC11/pcDNA clones. (B) RT-PCR analysis of Wnt1 target genes after selective knockdown of Wnt1 using Wnt1-siRNA in HC11/pcDNA and HC11/MTA1s clones. (C) Confocal analysis of MTA1s (red), and Cyclin D2 (red), expression in HC11/pcDNA and HC11/MTA1s clones. Scale bar, 10 μm. (D) Transcription status of Top-flash, Fop-flash, in HC11/pcDNA and HC11/MTA1s clones. (E) Western analysis of β-catenin protein expression in cytoplasmic (C) and nuclear (N) fractions in HC11/pcDNA and HC11/MTA1s clones. PARP and paxillin were used as markers of nuclear and cytoplasmic fractions, respectively. (F) Western blot analysis for Wnt1, c-Myc, Cyclin D2, and β-catenin expression and RT-PCR analysis of WISP-1 and Cyclin D2 in HC11/MTA1s clones following transfection with β-catenin siRNA or control siRNA for 48 h.

Figure 3. Status of Wnt1 pathway in ZR-75 cells expressing MTA1s and role of MTA1s in modifying Wnt1 pathway

(A) ZR-75/pcDNA and ZR-75/MTA1s clones were transfected with either Top-flash or Fop-flash and luciferase activity was measured after 24h. (B) Confocal analysis of Wnt1 (red), and Cyclin D2 (red) expression in ZR-75/pcDNA and ZR-75 /MTA1s clones. Scale bar 10 μm. (C) Western blot analysis of Wnt1, P-GSK3-β, GSK3-β, MTA1s, β-catenin and Cyclin D2 expression in MDA-MB-435 cell line following transfection with MTA1s specific or control siRNA for 48h. (D) Western blot analysis of β-catenin expression in MDA-MB-435 cell line following transfection with MTA1s siRNA for 48h and treatment with or without proteosomal inhibitor MG132 (10 μM) for 5h.

Figure 4. Effect of MTA1s on ERK and GSK-3β-pathway in mammary epithelial cells

(A) Western blot analysis of P-ERK, ERK, P-GSK-3β and GSK-3β in HC11/pcDNA and HC11/MTA1s clones. LE-Long exposure; SE-Short exposure. (B) Confocal analysis of phospho-ERK (red) and Phospho-GSK-3β (red) in HC11/pcDNA and HC11/MTA1s clones. Scale bar, 10 μm. (C) Effect of ERK inhibitor U0126 (10 μM) on the levels of phospho-GSK-3β, GSK-3β, phospho-ERK, ERK, β-catenin and Cyclin D2 in HC11/pcDNA and HC11/MTA1s clones. LE-Long exposure; SE-Short exposure. (D) RT-PCR analysis of the Wnt1 target genes WISP-1 and WISP-2 following treatment with the ERK inhibitor U0126 (10 μM) for 2 h in HC11/pcDNA and HC11/MTA1s clones. (E) Transcription status of Top-flash in HC11/MTA1s clones following treatment with the ERK inhibitor U0126 (10 μM) for 2 h. (F) Western blot analysis of β-catenin expression in HC11/MTA1s cells following transfection with either ERK-siRNA or GSK-3β–siRNA for 48h. (G) Transcription status of Top-flash in HC11/MTA1s clones following treatment with the ERK -siRNA for 48h.

Figure 5. MTA1s expression enhances the expression of biologically active Wnt1

(A) Western blot analysis of Wnt1 expression in conditioned medium (CM) from HC11/pcDNA (con-CM) and HC11/MTA1s cells (MTA1s-CM). (B) Confocal analysis of Wnt1 (green), P-GSK-3β (green), β-catenin (green), and cyclin D2 (red) expression in HC11/pcDNA clones following treatment with con-CM or MTA1s-CM for 24 h. Scale bar, 10 μm. (C) MTA1s-CM stimulates WISP-1, and Cyclin D2 promoter-luc activity in HC11/pcDNA cells. *p<0.05.

Figure 6. MTA1s stimulate the Wnt1 pathway in MTA1s-TG mice

(A) The MMTV-MTA1s transgene. (B) Southern blot detection of the MTA1s transgene in the tail genomic DNA of transgenic (TG) and wild-type (WT) littermates of F1 from lines 3, 11 and 17. (C) Representative PCR analysis of genomic DNA from founder line mice. Lanes marked 10 and 1 show respective copy number equivalents of control MTA1s cDNA plasmid. (D) Confocal analysis of T7-MTA1s (red) expression using anti-T7 antibodies in the mammary gland from 12-week-old virgin MTA1s-TG and WT-mice. Scale bar, 10μm. Note that the transgene is mainly localized in the cytoplasm with few nuclear localization in luminal epithelial cells. (E) Carmine Red-stained whole mounts of inguinal mammary glands obtained from WT mice and MTA1s-TG mice at 12 weeks of age. The images in the middle and right panels are the images in the left panels at increased magnification. (F) Hematoxylin and eosin-stained sections of mammary glands of 12-week-old WT mice and MTA1s-TG mice. Dilated ducts (a, b) and lobuloalveolar structures (c) in the mammary glands of the wild type mice. Dilated ducts (d, e) and lobuloalveolar structures (f) can be seen in the mammary glands of the MTA1s-TG mice. (G) BrdU incorporation into the nuclei and its quantitation in mammary epithelial cells obtained from WT and virgin MTA1s-TG mice at 12 weeks of age. We counted 5000 cells per mouse and examined six mice per line. (H, I) Western blot showing expression of β-casein, β-catenin and Cyclin D2 in mammary glands of 12-week-old WT and virgin MTA1s-TG mice.

Figure 7. Development of mammary tumors in MTA1s-TG mice

(A) Example of ductal hyperplasia in mammary glands from virgin MTA1s-TG mice and higher magnification is shown in bottom panel. (B) Example of ductal carcinoma in situ in mammary glands from virgin MTA1s-TG mice and higher magnification is shown in bottom panel. (C) Example of focal hyperplastic nodules in mammary glands from virgin MTA1s-TG mice and higher magnification is shown in bottom panel. (D) MTA1s-TG mice founder line 3 (upper panel) and 17 (lower panel), with large tumor on the thoracic mammary gland. (E) Hematoxylin and eosin staining showing mammary adenocarcinomas in MTA1s-TG (upper panels, a-e) and immunohistochemical analysis of Wnt1 (f–j), β-catenin (k–o), and smooth muscle actin (p–t) in mammary tumors from MTA1s-TG mice. Higher magnification is shown as an inset.