Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 69 (13), 5364-73

A Novel Lung Metastasis Signature Links Wnt Signaling With Cancer Cell Self-Renewal and Epithelial-Mesenchymal Transition in Basal-Like Breast Cancer

Affiliations

A Novel Lung Metastasis Signature Links Wnt Signaling With Cancer Cell Self-Renewal and Epithelial-Mesenchymal Transition in Basal-Like Breast Cancer

Theresa A DiMeo et al. Cancer Res.

Erratum in

  • Cancer Res. 2009 Aug 1;69(15):6366. Feng, Chang [corrected to Fan, Cheng]

Abstract

The establishment of metastasis depends on the ability of cancer cells to acquire a migratory phenotype combined with their capacity to recreate a secondary tumor in a distant tissue. In epithelial cancers, such as those of the breast, the epithelial-mesenchymal transition (EMT) is associated with basal-like breast cancers, generates cells with stem-like properties, and enables cancer cell dissemination and metastasis. However, the molecular mechanism(s) that connects stem cell-like characteristics with EMT has yet to be defined. Using an orthotopic model of human breast cancer metastasis to lung, we identified a poor prognosis gene signature, in which several components of the wnt signaling pathway were overexpressed in early lung metastases. The wnt genes identified in this signature were strongly associated with human basal-like breast cancers. We found that inhibiting wnt signaling through LRP6 reduced the capacity of cancer cells to self-renew and seed tumors in vivo. Furthermore, inhibition of wnt signaling resulted in the reexpression of breast epithelial differentiation markers and repression of EMT transcription factors SLUG and TWIST. Collectively, these results provide a molecular link between self-renewal, EMT, and metastasis in basal-like breast cancers.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1
A, orthotopic xenograft model of human breast cancer metastasis. i, phase contrast image of SUM1315 cells in culture. ii, H&E-stained section of SUM1315 xenograft (BV, blood vessel). iii, Vimentin-stained section of SUM1315 xenograft. Human tumor cells are Vimentin positive, whereas surrounding mouse stroma is Vimentin negative. iv, H&E-stained section of mouse mammary tissue. The tumor has invaded and replaced most of the normal lymph node tissue. v, GFP image of nodules in lung tissues resulting from a SUM1315 primary tumor. vi, GFP image of metastasis in brain resulting from a SUM1315 primary tumor. GFP-positive cells are tumor cells. vii, H&E-stained section of lungs harvested from mice bearing SUM1315 tumors. Arrows point to metastases. viii, SUM1315 xenograft primary tumor stained with Slug. B, schematic representation of microarray study. GFP images (top) and H&E-stained sections (bottom) of primary tumor and lung metastasis from SUM1315 injections. C, left, lung metastasis gene list (Supplementary Table S1) was compared with van’t Veer poor prognosis data set and is associated with reduced time to metastasis. Middle, pathway analysis of genes identified in lung metastasis signature using the PANTHER database. Wnt gene list (Supplementary Table S3) was compared with published microarray. Right, Wnt gene list was compared with UNC12 data set (Supplementary Fig. S1) and is significantly correlated with basal-like breast cancers.
Figure 2
Figure 2
DKK1 inhibits primary tumor growth through inhibition of LRP6. A, Western blot analysis of whole-cell lysate and conditioned media collected from control empty vector (EV), SFRP1, or DKK1-overexpressing cells. B, TOPFlash assay of EV-SUM1315, SFRP1-SUM1315, and DKK1-SUM1315 cells in the absence and presence of Wnt1. C, tumor growth curves of SUM1315 cells overexpressing SFRP1, DKK. Error bars, SE. H&E stained of primary tumor and matched lung from mouse injected with SFRP1-SUM1315 cells. m, metastasis; a, alveolus TOPFlash assay of Control-SUM1315, SFRP1-SUM1315, and DKK1-SUM1315 cells stimulated by wnt3a-conditioned media. D, qRT-PCR analysis of RNA extracted from stable Lrp6 knockdowns. Error bar, SD. Western blot analysis of several Lrp6 stable knockdowns compared with the control EV and scrambled control. Tumor growth curves of SUM1315 expressing shLrp6 3405. Error bars, SE.
Figure 3
Figure 3
Wnt signaling is necessary for survival in suspension, but not proliferation. A, growth curve analysis of wnt-inhibited (SFRP1, DKK1, and shLrp6 lines) and control SUM1315 cell lines. B, tumorsphere formation of control-SUM1315, SFRP1-SUM1315, and DKK1-SUM1315 cells. Error bars, SD. C, Western blot for caspase-3. Protein lysates were collected from adherent monolayers (lanes 2 and 3) or cells grown in suspension (lanes 4 and 5). The positive control (lane 1) was SUM1315 cells treated with 5-fluorouracil (1 mmol/L) for 6 d. D, serial CFU assays of SUM1315-EV, SFRP1, and DKK1 cell lines plated at low dilutions. Colonies were defined as having >30 cells.
Figure 4
Figure 4
Wnt signaling represses differentiation. Western blot and qRT-PCR analysis for Slug (A), Twist (B), and SOX17 (C) in SUM1315 cells transfected with DKK1. D, differential gene expression of differentiation genes from wnt-inhibited cell lines. Heatmap Builder Software was used to analyze the data. Starred (*) genes represent those of myoepithelial differentiation, whereas unstarred genes represent luminal differentiation. Western blot analysis of α-smooth muscle actin expression in Control-SUM1315, SFRP1-SUM1315, and DKK1-SUM1315 cells to confirm the qRT-PCR results from left.
Figure 5
Figure 5
Reexpression of Slug and Twist in vivo bypasses wnt inhibition. A, qRT-PCR analysis of DKK1 expression in tumor and injection sites from mammary glands of mice injected with control SUM1315-EV or DKK1-expressing cells. B, Western blot analysis of Twist and Slug expression in tumors and injection sites isolated from mice injected with SUM1315-Control EV or DKK1 expressing cells. C, Western blot analysis of DKK1 and SFRP1 tumors for active β-catenin and Slug. D, schematic model of wnt in breast epithelial differentiation, EMT, and metastasis. In the absence of wnt signaling (left; or in the presence of wnt inhibitors), cells undergo lineage commitment and differentiation. In the presence of wnt signals, β-catenin partners with TCF/LEF to activate target genes, such as SLUG and TWIST, which promote an EMT, repress differentiation, and enhance tumor seeding and metastasis.

Similar articles

See all similar articles

Cited by 173 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback