Lunatic fringe deficiency cooperates with the Met/Caveolin gene amplicon to induce basal-like breast cancer

Abstract

Basal-like breast cancers (BLBC) express a luminal progenitor gene signature. Notch receptor signaling promotes luminal cell fate specification in the mammary gland, while suppressing stem cell self-renewal. Here we show that deletion of Lfng, a sugar transferase that prevents Notch activation by Jagged ligands, enhances stem/progenitor cell proliferation. Mammary-specific deletion of Lfng induces basal-like and claudin-low tumors with accumulation of Notch intracellular domain fragments, increased expression of proliferation-associated Notch targets, amplification of the Met/Caveolin locus, and elevated Met and Igf-1R signaling. Human BL breast tumors, commonly associated with JAGGED expression, elevated MET signaling, and CAVEOLIN accumulation, express low levels of LFNG. Thus, reduced LFNG expression facilitates JAG/NOTCH luminal progenitor signaling and cooperates with MET/CAVEOLIN basal-type signaling to promote BLBC.

Figure 1. Lfng Is Expressed in Mammary Basal Cells and Its Deletion Causes Increased Proliferation and Expansion of the CD24⁺CD49f^HiCD61⁺Sca1⁻ Population in the Pubescent Mammary Gland

(A) Expression of Lfng, Dll1, and Jagged1 in the mouse mammary gland. Shown are representative photomicrographs of whole-mount (left column) and sectioned X-Gal staining of mammary glands from Lfng^LacZ/+, Dll1^LacZ/+, and Jagged1^β–Geo/+ virgin mice at 6 weeks and 5 months of age. Sections for the TEBs are shown in the second column from left. (B) Mammary hyperplasia in the Lfng^−/− mutant. Shown are representative photomicrographs of whole-mount mammary glands from 6- and 12-week-old virgins. (C) Representative photomicrographs of anti-K8, −K14 immunofluorescence staining in mammary sections from 6-week-old virgins of Lfng^+/+ and Lfng^−/− mice. TEBs and ducts are shown in upper and lower panels, respectively. (D) Ductal elongation at 6 weeks old, measured as the distance between the lymph node and the ductal front line normalized to the distance between the lymph node and the end of the fat pad, are presented as mean values ± standard error. *p < 0.05. Note, the Lfng null allele was generated through expression of Cre in the germline of female Lfng^flox/+; MMTV-Cre mice. Increased mammary epithelial proliferation in Lfng^−/− compared with control Lfng^+/+ littermates. Shown are representative photomicrographs of anti-Ki67 immunostaining in mammary sections same as (C). Numbers of Ki67⁺ cells are normalized to the total number of epithelial cells in TEBs or in ductal areas, presented as mean values ± standard error. **p < 0.005. (E) Representative flow cytometry analyses of lineage-depleted mammary cells from Lfng^−/−; TNR mutants compared with Lfng^+/+; TNR littermates at 6 weeks old. Shown are CD24/CD49f plots and Sca-1/CD61 plots on populations I, II, III gated as in CD24/CD49f plots. Scale bars correspond to 1 mm in left panels of (A), 5 mm in (B), and 50 μm in all others. See also Figure S1.

Figure 2. Deletion of Lfng Induced Mammary Tumors in Mice

(A) Kaplan-Meier mammary tumor-free survival curve for control Lfng^flox/flox and Lfng^flox/flox; MMTV-Cre conditional mutant mice. (B) Representative photomicrographs of H&E stained sections showing three histological types of Lfng^flox/flox; MMTV-Cre mammary tumors. (C) Anti-ERα immunostaining on two main types of Lfng^flox/flox; MMTV-Cre mammary tumors. n: normal tissue; t: tumor. Note positive ERα staining in normal tissue adjacent to the tumor. (D) Representative photomicrographs and quantification of anti-Ki67 immunostaining on two main types of Lfng^flox/flox; MMTV-Cre mammary tumors. Data are derived from two sections in each of three Type I and Type II tumors presented as mean values ± standard error. (E) Representative photomicrographs of anti-K8, −K14 immunofluorescence staining on three types of Lfng^flox/flox; MMTV-Cre mammary tumors. Arrows point to cells showing co-expression of K8 and K14. Scale bars correspond to 100 μm in (B), 50 μm in (C), (D) and (E).

Figure 3. Lfng^flox/flox; MMTV-Cre Tumors Cluster with Basal-like and Claudin-Low Mouse Mammary Tumor Models

(A) Overview of expression of reference genes in tumors from various mouse models of breast cancer, including 11 tumors from Lfng^flox/flox; MMTV-Cre mice. Colored bars at left correspond to regions shown in (B). (B) Expression of selected genes representing the Claudin gene cluster, luminal gene cluster, proliferation-associated gene cluster, CK14 basal-like gene cluster, CK5 basal-like gene cluster, and EMT gene cluster. Expression data from type I and type II Lfng^flox/flox; MMTV-Cre tumors are contained within yellow boxes. Clusters of tumor models are highlighted at the bottom. DMBA, 7,12-dimethylbenz[a]-anthracene. (C) Enrichment map for gene sets over-represented in list of genes that are differentially expressed comparing Lfng^flox/flox; MMTV-Cre mouse mammary tumors to other mouse models of breast cancer. See also Table S1.

Figure 4. Flow Cytometry and Immunohistological Analysis of Lfng^flox/flox; MMTV-Cre Tumors

(A) Representative flow cytometry contour plots for lineage-depleted mammary tumor cells compared with non-tumor lineage-depleted mammary cells from a Lfng^flox/flox; MMTV-Cre mouse. Shown are CD24/CD49f, CD61/CD49f, and Sca1/CD49f plots from a Type I tumor. (B) Additional analysis of the flow cytometry data as shown in (A): CD24/CD61plots and Sca1/CD49f plots on the CD24⁺CD61⁺ populations as gated in the upper panels. (C) Limiting dilution transplantation assay on CD24⁺CD49f⁺, CD24⁻CD49f⁻ and CD24⁻CD49f⁺ tumor cells as gated in a CD24/CD49f plot. Data are derived from two independent experiments using two donors of type I tumor. TIC: Tumor-Initiating Cells. *p = 0.0307 (CD24⁺CD49f⁺ versus CD24⁻CD49f⁻), **p = 0.0155 (CD24⁺CD49f⁺ versus CD24⁻CD49f⁺) (D) Representative photomicrographs of anti-Aldh1, anti-CD61, anti-Vimentin, anti-Twist and anti-Phospho-Akt immunostaining in two main types of Lfng^flox/flox; MMTV-Cre mammary tumors. Scale bars correspond to 50 μm. See also Figure S2.

Figure 5. Dysregulation of Notch Signaling Pathway and Downstream Target Genes in Lfng^flox/flox; MMTV-Cre Tumors

(A) Expression of selected Notch signaling pathway and downstream target genes in tumors from various mouse models of breast cancer, including 11 tumors from Lfng^flox/flox; MMTV-Cre mice. Expression data from type I and type II Lfng^flox/flox; MMTV-Cre tumors are contained within yellow boxes. Genes with lower expression in Lfng^flox/flox; MMTV-Cre tumors are highlighted in green, with higher expression in red. Clusters of tumor models are highlighted at the bottom. DMBA, 7,12-dimethylbenz[a]-anthracene. (B) Western blot analysis of cleaved Notch1 and cleaved Noch2 in mammary tumors (t) and non-tumor mammary tissue (n) from Lfng^flox/flox; MMTV-Cre mice (#1981, #1982, #8802), as well as in non-tumor mammary tissues from 18-month old Lfng^flox/flox and Lfng^flox/flox; MMTV-Cre littermate mice. β-actin served as loading control. (C) Representative photomicrographs of anti-Jagged1, anti-Cyclin D1 and anti-C-Myc, immunostaining in two main types of Lfng^flox/flox; MMTV-Cre mammary tumors. Scale bars correspond to 50 μm. See also Figure S3.

Figure 6. Met/Caveolin Amplicon and Signaling of Met and IgfR in Lfng^flox/flox; MMTV-Cre Mammary Tumors

(A) aCGH analysis of DNA isolated from four Lfng^flox/flox; MMTV-Cre mouse tumors and one Lfng^flox/+; MMTV-Cre tumor showing a commonly amplified locus among all four Lfng^flox/flox; MMTV-Cre tumors on chromosome 6. Red bar corresponds to the overlapping region containing 13 genes. (B) Expression of the commonly amplified genes as shown in (A) in 11 Lfng^flox/flox; MMTV-Cre tumors in comparison with expression of these genes in other mouse models of breast cancer. Expression data from type I and type II Lfng^flox/flox; MMTV-Cre tumors are contained within yellow boxes. (C) Western blot analysis of Met and phospho-Met in mammary tumors (t) and non-tumor mammary tissue (n) from Lfng^flox/flox; MMTV-Cre mice (#1981, #1982, #8802), as well as in mammary tissue from 18-month old Lfng^flox/flox and Lfng^flox/flox; MMTV-Cre littermate mice. β-actin served as loading control. (D) Representative photomicrographs of anti-Caveolin-1 immunostaining in two main types of Lfng^flox/flox; MMTV-Cre mammary tumors. Red arrows indicate positive staining in blood vessels. Black arrow points to a mammary duct adjacent to the tumor. Note, non-tumor mammary tissues show strong staining in adipocytes and weak staining in myoepithelial cells. Scale bars correspond to 50 μm. (E) Western blot analysis of Caveolin-2 and Phospho-IRS-1/2 in mammary tumors (t) and non-tumor mammary tissue (n) from Lfng^flox/flox; MMTV-Cre mice. See also Figure S4.

Figure 7. Human Basal-like and A Subset of Claudin-Low Breast Cancers Exhibit Low Levels of LFNG, HES1, and HEY1 Gene Expression, but High Levels of NOTCH1 and MYC Gene Expression

(A) Expression of selected Notch pathway genes and proliferation-associated Notch target genes in 320 breast tumors and 17 normal breast samples from the North Carolina data set. 5 subtypes of human breast cancers are highlighted. Expression of LFNG, HES1, HEY1, MYC and NOTCH1 in basal-like breast cancers are boxed in white and shown in enlarged view (bottom of panel A). Expression levels for these five genes in a subset of Claudin-low breast cancers (with lower LFNG levels) are boxed in yellow. Green bars mark individual tumors showing decreased expression of all three genes (LFNG, HES1, HEY1). Red bars mark tumors with increased expression of both NOTCH1 and MYC. (B) Mean expression values for LFNG and NOTCH1 from the North Carolina data set. BL, basal-like; CL, Claudin-low; H2, HER2-positive; LA, luminal A; LB, luminal B; NBL, normal breast-like. p values were calculated by comparing expression means across all subtypes. See also Figure S5.

Figure 8. Model of Cooperation between Lfng Deficiency and Met/Caveolin Amplification/Overexpression in Basal-like Breast Cancer

Lfng modifies Notch receptor to inhibit its activation by Jagged ligand and enhance its activation by Dll ligand. Loss of Lfng results in increased Jagged1-mediated Notch activation and upregulation of c-Myc, Cyclin D1, Igf1R and uPA, leading to increased proliferation. Loss of Lfng may decrease Dll1-mediated Notch activation in bipotent mammary progenitor cells, causing expansion of basal cells. Decreased expression of Hes/Hey Notch target genes may de-repress the Met promoter. Selected as cooperative event in Lfng deficiency induced mammary tumorigenesis, the Met/caveolin amplicon increases abundance of Met and Caveolin1/2, the latter proteins are predicted to enhance signaling through Igf-1R and IRS. Signaling through Met and Igf-1R stimulate proliferation. TGN, trans Golgi network.