LOXL2-mediated matrix remodeling in metastasis and mammary gland involution

Abstract

More than 90% of cancer patient mortality is attributed to metastasis. In this study, we investigated a role for the lysyl oxidase-related enzyme lysyl oxidase-like 2 (LOXL2) in breast cancer metastasis, in both patient samples and in vivo models. Analysis of a published microarray data set revealed that LOXL2 expression is correlated with metastasis and decreased survival in patients with aggressive breast cancer. In immunocompetent or immunocompromised orthotopic and transgenic breast cancer models we showed that genetic, chemical or antibody-mediated inhibition of LOXL2 resulted in decreased metastasis. Mechanistic investigations revealed that LOXL2 promotes invasion by regulating the expression and activity of the extracellular proteins tissue inhibitor of metalloproteinase-1 (TIMP1) and matrix metalloproteinase-9 (MMP9). We found that LOXL2, TIMP1, and MMP9 are coexpressed during mammary gland involution, suggesting they function together in glandular remodeling after weaning. Finally, we found that LOXL2 is highly expressed in the basal/myoepithelial mammary cell lineage, like many other genes that are upregulated in basal-like breast cancers. Our findings highlight the importance of LOXL2 in breast cancer progression and support the development of anti-LOXL2 therapeutics for the treatment of metastatic breast cancer.

Figure 1. LOXL2 correlates with metastasis and survival

Kaplan-Meier survival curves based on analysis of a previously published microarray dataset from breast cancer patients(n=295) showing (A)overall survival and (B)metastasis-free survival of patients with ER− tumors(n=72) separated into low and high LOXL2 expression.

Figure 2. LOXL2 enables metastases in vivo

(A) Quantification of lung metastases formed after orthotopic injection of 4T1control (cont; wild-type or expressing a scrambled control shRNA) and shLOXL2 cells. Mice were either untreated or treated with D-penicillamine(D-pen). At least n=9 mice per group. Representative images are shown in right panel. Scale bar, 200μm. (B) Quantification of liver metastases in mice bearing 4T1control or shLOXL2 orthotopic tumors. Representative images are shown in right panel. Scale bar, 200μm. (C) Quantification of bone metastases in mice bearing 4T1control or shLOXL2 orthotopic tumors. Representative CT images are shown in right panel. (D) Left panel: Quantification of lung metastases formed after orthotopic injection of 4T1 cells. Mice were treated with IgG or LOXL2-specific antibody. N=5 mice per group. Right panel: Quantification of lung metastases formed in MMTV-PyMT transgenic mice. Mice were either untreated or treated with D-pen. N=14 mice per group.

Figure 3. LOXL2 increases in vitro invasion

Quantification of 4T1 or MDA-MB-231control (wild-type or expressing a scrambled control shRNA) and shLOXL2 breast cancer cells invading through (A) collagen or (B) Matrigel towards a chemoattractant.

Figure 4. LOXL2 regulates TIMP1

(A) Western blot analysis of LOXL2 protein expression and TIMP1 in concentrated CM from 4T1/MDA-MB-231 cells expressing control or LOXL2 shRNA constructs, or transfected with siLOXL2 (compared with mock-transfected control). (B) Immunofluorescent images of tumor sections from mice bearing 4T1/MDA-MB-231control or shLOXL2 tumors stained with TIMP1(green) and DAPI(blue). Scale bar, 100μm. (C) Invasive branching structures formed by MDA-MB-231control or siLOXL2 cells grown in 3D Matrigel, treated ±recombinant human TIMP1. Scale bar, upper panels; 500μm, lower panels; 200μm.

Figure 5. High LOXL2 expression and activity during mammary gland involution

(A) QRT-PCR analysis of LOXL2 mRNA expression levels in FVB mouse mammary gland samples at different stages of development (wV=week virgin, dP=day pregnancy, dL=day lactation, dI=day involution). N=3 mice per time-point. (B) Western blot analysis of LOXL2 and TIMP1 protein expression in lysates prepared from mice in(A). (C) LOXL2 enzymatic activity in blood serum from mice in(A). N=3 mice per time-point.

Figure 6. LOXL2 is expressed in stromal and basal epithelial cells

(A) Mammary gland sections from 8 week-old virgin and 2 day involution FVB mice stained with LOXL2 antibody and counterstained with haematoxylin. Scale bar, 50μm. (B) QRT-PCR analysis of LOXL2 and K14 mRNA expression in sorted mammary epithelial cell populations (basal/myoepithelial=CD24^low/Sca-1⁻; luminal ER+=CD24^high/Sca-1⁺; luminal ER−=CD24^high/Sca-1⁻). (C) Mammary gland sections from 18 day pregnant(18dP) and 8 day lactating(8dL) FVB mice stained with LOXL2(green) and K14(red) antibodies and counterstained with DAPI(blue). Scale bar, 30μm. (D) 4T1 tumor sections stained with K14 antibody and counterstained with haematoxylin. Scale bar, 200μm.