Paracrine Wnt signaling both promotes and inhibits human breast tumor growth - PubMed (original) (raw)

Paracrine Wnt signaling both promotes and inhibits human breast tumor growth

Jennifer L Green et al. Proc Natl Acad Sci U S A. 2013.

Abstract

Wnt signaling in mouse mammary development and tumorigenesis has been heavily studied and characterized, but its role in human breast cancer remains elusive. Although Wnt inhibitors are in early clinical development, it is unclear whether they will be of therapeutic benefit to breast cancer patients, and subsequently, to which ones. To address this, we generated a panel of Wnt reporting human breast cancer cell lines and identified a previously unrecognized enrichment for the ability to respond to Wnt in the basal B or claudin-low subtype, which has a poor prognosis and no available targeted therapies. By co-injecting Wnt3A expressing human mammary fibroblasts with human breast cancer cell lines into mouse mammary fat pads, we showed that elevated paracrine Wnt signaling was correlated with accelerated tumor growth. Using this heterotypic system and a dual lentiviral reporter system that enables simultaneous real-time measurement of both Wnt-responsive cells and bulk tumor cells, we analyzed the outcome of elevated Wnt signaling in patient-derived xenograft (PDX) models. Interestingly, the PDX models exhibited responses not observed in the cell lines analyzed. Exogenous WNT3A promoted tumor growth in one human epidermal growth factor receptor 2-overexpressing PDX line but inhibited growth in a second PDX line obtained from a patient with triple-negative breast cancer. Tumor suppression was associated with squamous differentiation in the latter. Thus, our work suggests that paracrine Wnt signaling can either fuel or repress the growth of human breast cancers depending on yet to be determined aspects of the molecular pathways they express.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Dual lentiviral Wnt reporter system. (A) Schematic of the FRED lentiviral vector (Upper). All FRED-transduced cells express red fluorescence, represented by the cartoon cluster of red cells to the right. (Lower) Magnetic separation of FRED-transduced MB231 cells from parental MB231 cells. Cells were mixed, incubated with magnetic particles coupled to anti-CD4 antibody, subjected to magnetic separation, and then stained with anti-CD4 (FITC). (B) Schematic of the WILMA lentiviral vector (Upper). Only WILMA-transduced cells with active Wnt signaling will be luminescent and green, represented by the cartoon of a luminescent green cell to the right. (Lower) Specificity of the WILMA reporter was determined by a real-time bioluminescence monitoring assay. WILMA-transduced MB231 cells were cocultured with iCHO cells secreting WNT3A (green circles) or Fzd8CRD (blue triangles), or parental iCHO cells (control, red circles), and luminescence was measured every 30 min for 48 h. (C) FACS profiles of transgenic cells. FRED-transduced cells were selected with Puromycin, thus all of the cells express mLumin (Left). To collect WILMA-transduced cells, GFP+ cells were collected 24 h after treatment with the GSK-3 inhibitor XV (Center). Double transgenic cells expressing mLumin and GFP were collected after XV treatment (Right). (D) MB231-FW cells were injected into the third and fourth mammary pads in the quantities indicated. Two weeks later, bioluminescence and fluorescence were measured. Signal intensity within the region of interest is shown in units of Efficiency (luminescence) or Radiance Efficiency (fluorescence).

Fig. 2.

Fig. 2.

Wnt responsiveness of breast cancer cell lines. (A) Real-time bioluminescence monitoring assay. Cells were cocultured with parental (control) or WNT3A-producing iCHO cells, and luminescence was measured every 30 min for 40 hours. To adjust for variable transduction efficiency of WILMA, data were normalized by subtracting absorbance in the presence of control from absorbance in the presence of WNT3A, and the remainder was divided by absorbance in the presence of GSK-3 inhibitor XV (because inhibition of GSK-3 activates the reporter regardless of the cell’s ability to respond to Wnt ligands). The cell lines BT549, SUM190PT, and SUM225CWN were analyzed separately and are presented in

Fig. S2

. (B) Summary of Wnt-responsiveness of the entire cell line panel with indication of estrogen receptor (ER), progesterone receptor (PR), and HER2 status and subset classification.

Fig. 3.

Fig. 3.

Orthotopic heterotypic recombinations. (A) Schematic of orthotopic heterotypic recombination assay. Transgenic human breast cancer cells (red) were coinjected into the mouse mammary fat pad with human reduction mammary fibroblasts (green) that were engineered to overexpress WNT3A. (B) Schematic of the WNT3A expression lentiviral vector (pJG071) (Upper); the control vector (pJG070) has eGFP in the place of WNT3A. Transgenic RMFs were cocultured with HEK293A cells carrying the SUPERTOPFLASH Wnt reporter; luminescence was measured 24 h later (Lower). (C) Orthotopic heterotypic recombination pilot experiment. WNT3A-producing or control (GFP) RMFs were coinjected with MB231-FW cells into the left and right number four mouse mammary fat pads, in duplicate (representative cage is shown).

Fig. 4.

Fig. 4.

WNT3A promotes tumorigenesis of breast cancer cell lines. Orthotopic heterotypic recombination experiments as in Fig. 3_C_ using MB231-FW (Left) or MB468-FW (Right) cell lines, n = 8 (MB231-FW) or n = 10 (MB468-FW). (A) Bioluminescence was measured twice per week for 3–4 wk. (B) Tumors were harvested at day 27 (MB231-FW) or day 33 (MB468-FW), and luminescence was measured ex vivo. Data were normalized to the lowest value. (C) Ex vivo red fluorescence (mLumin) intensity of the harvested tumors, as in B.

Fig. 5.

Fig. 5.

Wnt signaling in PDX lines. Orthotopic heterotypic recombination experiments as in Fig. 4 using the transgenic PDX lines BCM-3963-FW and BCM-4272-FW, n = 10. Tumors were harvested at day 19 (BCM-3963-FW) or day 21 (BCM-4272-FW), and luminescence and fluorescence were measured ex vivo. The entire experiment was repeated with both PDX lines (

Fig. S3

).

Fig. 6.

Fig. 6.

Representative histopathology and immunohistochemistry of PDX tumors. (A) Anti-HER2 staining of BCM-4272-FW, (TNBC), and BCM-3963-FW, derived from a HER2-overexpressing breast cancer. Both tumors were from the eighth passage after transduction. Counterstained with Mayer’s hematoxylin for nuclei. (B) H&E staining of BCM-4272-FW coinjected with RMF-GFP (Upper) or RMF-WNT3A (Lower). Eight different tumors are shown, harvested at day 17 or at 4 wk. (C) Anti-CK10 staining of BCM-4272-FW tumors harvested at day 17; four different tumors are shown. (D) Anti-Ki67 staining of BCM-4272-FW tumors harvested at day 17; four different tumors are shown.

References

    1. Clevers H, Nusse R. Wnt/β-catenin signaling and disease. Cell. 2012;149(6):1192–1205. - PubMed
    1. Nusse R, Varmus HE. Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome. Cell. 1982;31(1):99–109. - PubMed
    1. Ayyanan A, et al. Increased Wnt signaling triggers oncogenic conversion of human breast epithelial cells by a Notch-dependent mechanism. Proc Natl Acad Sci USA. 2006;103(10):3799–3804. - PMC - PubMed
    1. Incassati A, Chandramouli A, Eelkema R, Cowin P. Key signaling nodes in mammary gland development and cancer: β-catenin. Breast Cancer Res. 2010;12(6):213. - PMC - PubMed
    1. Jönsson M, Borg A, Nilbert M, Andersson T. Involvement of adenomatous polyposis coli (APC)/beta-catenin signalling in human breast cancer. Eur J Cancer. 2000;36(2):242–248. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources