Procollagen lysyl hydroxylase 2 is essential for hypoxia-induced breast cancer metastasis

Abstract

Metastasis is the leading cause of death among patients who have breast cancer. Understanding the role of the extracellular matrix (ECM) in the metastatic process may lead to the development of improved therapies to treat patients with cancer. Intratumoral hypoxia, found in the majority of breast cancers, is associated with an increased risk of metastasis and mortality. We found that in hypoxic breast cancer cells, hypoxia-inducible factor 1 (HIF-1) activates transcription of the PLOD1 and PLOD2 genes encoding procollagen lysyl hydroxylases that are required for the biogenesis of collagen, which is a major constituent of the ECM. High PLOD2 expression in breast cancer biopsies is associated with increased risk of mortality. We show that PLOD2 is critical for fibrillar collagen formation by breast cancer cells, increases tumor stiffness, and is required for metastasis to lymph nodes and lungs.

©2013 AACR.

Figures

Figure 1

Knockdown of HIF-1α expression blocks PLOD1 and PLOD2 induction under hypoxic conditions. A, levels of PLOD1 and PLOD2 mRNAs were analyzed by RT-qPCR in MDA-MB-231 subclones, which were stably transfected with empty vector (shEV) or vector encoding HIF-1α shRNA (sh1α), HIF-2α shRNA (sh2α), or HIF-1α + HIF-2α shRNAs (sh1/2α), and exposed to 20% or 1% O2 for 24 hours (mean ± SEM, n = 3); ***P < 0.001, **P < 0.01 versus shEV at 20% O2; ###P < 0.001, ##P < 0.05 vs shEV at 1% O2 (one-way ANOVA with Bonferroni post-test). B, immunoblot assays were performed using lysates prepared from MDA-MB-231 subclones exposed to 20% or 1% O2 for 48 hours. C, immunoblot assays were performed using lysates of parental MDA-MB-231 cells that were exposed to 1% O2 for the indicated time in hours. D, PLOD1 and PLOD2 mRNA levels were analyzed by RT-qPCR in MDA-MB-231 cells exposed to vehicle (DMSO) or 200 nM Digoxin at 20% or 1% O2 for 24 hours. ***P < 0.001 vs DMSO-treated 20% O2; ###P < 0.001 vs DMSO-treated 1% O2 (one-way ANOVA with Bonferroni post-test).

Figure 2

PLOD1 and PLOD2 expression in breast cancer patients. A-B, PLOD1 and PLOD2 gene expression levels (presented as normalized microarray intensity values) in normal breast (n = 7) and breast cancer (n = 40) tissues (A) or in paired adjacent normal and breast cancer tissues from The Cancer Genome Atlas (n = 28) (B) are shown. The box represents the 25th through 75th percentiles and whiskers represent the minimum and maximum range of the data. P values were determined by Student’s t test. C, Kaplan-Meier analysis of disease-specific survival of 159 breast cancer patients stratified by PLOD1 (left) or PLOD2 (right) mRNA expression above the median level (High) or below the median level (Low).

Figure 3

PLOD2 knockdown in MDA-MB-231 cells inhibits local tissue invasion. A, immunoblot assays were performed using lysates prepared from MDA-MB-231 control (shLKO.1) or PLOD2-knockdown (shPL2-1 and shPL2-2) subclones exposed to 20% or 1% O2 for 48 hours. B, proliferation of subclones was determined by trypan blue staining on the indicated days. Crystal violet staining of tissue culture dishes on day 12 is shown (right). C-E, the subclones were injected into the MFP of NOD-SCID mice and tumor volume was plotted versus time (C), final tumor weight (in grams) was determined (D), and human PLOD2 mRNA levels were determined by RT-qPCR (E; mean ± SEM, n = 5, one-way ANOVA with Bonferroni post-test; ***P < 0.001, **P < 0.01 versus shLKO.1). F, immunohistochemical staining of primary tumor sections for PLOD2 is shown. Scale bar = 500 µm. G, immunohistochemical staining of primary tumor sections for HIF-1α and PLOD2 is shown. Images were deconvoluted and pseudocolored to assess colocalization (right). Scale bar = 500 µm. H, hematoxylin staining of primary tumor sections. Invasion of shLKO.1 into adipose tissue (red arrow) or muscle (black arrow) is shown in the upper panel. The boundary between shPL2 cells and normal tissue is indicated by dashed line in the lower panel. Scale bar = 500 µm.

Figure 4

PLOD2 is essential for lymph node and lung metastasis. A, human genomic DNA content in lungs of tumor-bearing mice was quantified using qPCR with human-specific HK2 gene primers (mean ± SEM, n = 5, one-way ANOVA). B, lung sections (5×5 mm) were stained with hematoxylin and eosin. C, metastatic area was determined by image analysis of 5×5 mm lung sections (mean ± SEM, n = 5, one-way ANOVA). D, the number of metastatic foci per 5×5 mm tumor section was determined (mean ± SEM, n = 5, one-way ANOVA). E, axillary lymph node sections were subjected to immunohistochemistry using an antibody specific for human vimentin. Staining of whole lymph nodes in 3.5×3.5 mm sections with preserved lymph node structure outlined in dashed line on left. High-power field of lymph node follicles is shown on right. Scale bar = 100 µm. F-G, vimentin staining (F) was quantified by image analysis (G; mean ± SEM, n = 5, one-way ANOVA). Bonferroni post-tests were performed for all ANOVAs. **P < 0.01, ***P < 0.001 vs. shLKO.1.

Figure 5

PLOD2 expression decreases fibrillar collagen formation but not total collagen deposition. A, collagen 1A1 protein levels in conditioned media and cell lysate of MDA-MB-231 subclones exposed to 1% O2 for 72 hours were determined by immunoblot assay. Actin was used as a loading control for cell lysate. Ponceau S staining was used as a loading control for conditioned media. B, collagen content of tumors was determined using the collagen hydroxyproline assay. C, tumor sections from the indicated subclones were stained with picrosirius red and imaged under circularly polarized light (top) and collagen fiber staining was thresholded and subjected to image analysis (bottom). D, picrosirius red staining of 3 sections from 5 mice per group was quantified by image analysis to determine the area of the tumor section occupied by collagen fibers (% fibrillar collagen; mean ± SEM, one-way ANOVA). E, the stiffness of freshly dissected control (shLKO.1) or shPLOD2 tumors was determined (mean ± SEM, one-way ANOVA). F, picrosirius red staining to detect crosslinked collagen fibers (left) and immunohistochemical staining to detect PLOD2 (right) were performed on serial control tumor sections. Bonferroni post-test was performed for all ANOVAs. *P<0.05, **P < 0.01, ***P < 0.001 vs. shLKO.1 in panels D and E.

Figure 6

PLOD2 is essential for invasion and metastasis of MDA-MB-435 cells. A, immunoblot assays were performed using lysates prepared from control (shLKO.1) or PLOD2-knockdown (shPL2-1 and shPL2-2) MDA-MB-435 subclones exposed to 20% or 1% O2 for 48 hours. B, the indicated subclones were injected into the MFP of NOD-SCID mice and tumor volume was plotted versus time. C, hematoxylin staining of primary tumor sections. Invasion of shLKO.1 into adipose tissue (red arrow) is shown in the left panel. The well-defined boundary between shPL2 cells and normal tissue is indicated by the dashed line in the right panel. Scale bar = .5 mm. D, lung sections (5×5 mm) were stained with hematoxylin and eosin. E, metastatic area was determined by image analysis (mean ± SEM, n = 5, one-way ANOVA). F, the number of metastatic foci per 5×5 mm tumor section was determined (mean ± SEM, n = 5, one-way ANOVA). G, human genomic DNA content in mouse lungs was quantified using qPCR with human-specific HK2 gene primers (mean ± SEM, n = 5, one-way ANOVA). H, ipsilateral axillary lymph node sections were subjected to immunohistochemistry using an antibody specific for human vimentin. Scale bar = 100 µm. I, vimentin staining was quantified by image analysis (mean ± SEM, n = 5, one-way ANOVA). J, the stiffness of freshly dissected control or shPLOD2 tumors was determined. Bonferroni post-tests were performed for all ANOVAs. *P<0.05, **P < 0.01, ***P < 0.001 vs. shLKO.1.

Figure 7

Hypoxia-induced and HIF-1-dependent expression of PLOD2 and LOX family members mediate crosslinking of collagen fibers, leading to increased invasion and metastasis of breast cancer cells.