RETRACTED: Collagen prolyl hydroxylases are essential for breast cancer metastasis

Abstract

The presence of hypoxia and fibrosis within the primary tumor are two major risk factors for metastasis of human breast cancer. In this study, we demonstrate that hypoxia-inducible factor 1 activates the transcription of genes encoding collagen prolyl hydroxylases that are critical for collagen deposition by breast cancer cells. We show that expression of collagen prolyl hydroxylases promotes cancer cell alignment along collagen fibers, resulting in enhanced invasion and metastasis to lymph nodes and lungs. Finally, we establish the prognostic significance of collagen prolyl hydroxylase mRNA expression in human breast cancer biopsies and show that ethyl 3,4-dihydroxybenzoate, a prolyl hydroxylase inhibitor, decreases tumor fibrosis and metastasis in a mouse model of breast cancer.

Figure 1

Knockdown of HIF-1α blocks P4HA1 and P4HA2 induction under hypoxic conditions. A, expression of P4HA mRNAs was analyzed by RT-qPCR in MDA-MB-231 subclones exposed to 20% or 1% O₂ for 24 hours (mean ± SEM, n = 3, ANOVA); ***P < 0.001 vs shEV (20% O₂); ^### P < 0.001 vs shEV (1% O₂). B, immunoblot assays were performed using lysates from MDA-MB-231 subclones exposed to 20% or 1% O₂ for 48 hours. C, immunoblot assays (Supplementary Figure S1D) of tumor lysate were quantified by optical density and normalized to MDA-MB-231-shEV expression (mean ± SEM , n = 5, Student's t test). **P < 0.01, ***P < 0.001 vs shEV. D, immunohistochemical staining of serial sections from shEV and sh1/2α tumors. Scale bar = 150 μm. N = Necrotic region. E, shEV images (in D) were deconvoluted and pseudocolored to assess colocalization. Scale bar = 25 μm.

Figure 2

Knockdown of P4HA1 or P4HA2 expression in MDA-MB-231 cells inhibits tumor growth and metastasis. A, immunoblot assays were performed using lysates prepared from MDA-MB-231 subclones exposed to 20% or 1% O₂ for 48 hours. B, tumor volume measured versus time (mean ± SEM, n = 5, ANOVA) C, final tumor weight (in grams, mean ± SEM, n = 5, ANOVA) D, lung sections (5 × 5 mm (left) or .2 × .2 mm (right)) were stained with hematoxylin and eosin. E, human genomic DNA content in mouse lungs was quantified using qPCR (mean ± SEM, n = 5, ANOVA). F, tumor volume versus time G, human DNA content in mouse lungs determined by qPCR (mean ± SEM, n = 5, ANOVA). H, immunohistochemical staining of axillary lymph node sections for human vimentin. I, vimentin staining quantified by image analysis (mean ± SEM, n = 5, ANOVA). J, immunohistochemical staining of primary tumor sections for P4HA1 or P4HA2. Scale bar = 200 μm. **P < 0.01, ***P < 0.001 vs. shLKO.1.

Figure 3

Collagen prolyl hydroxylase expression is associated with collagen content and tumor stiffness. A-B, immunoblot of collagen 1A1 from conditioned media of MDA-MB-231 subclones exposed to 20% or 1% O₂ for 72 hours. Ponceau S staining was used as a loading control. C-D, picrosirius red staining of control tumor sections imaged under circularly polarized light. The % collagen in perinecrotic regions was compared to bulk tumor regions (white boxes) in (C). Top scale bar = 1 mm; bottom scale bar = 0.1 mm; N = necrotic; M = tumor margin. E, picrosirius red staining quantified by image analysis (mean ± SEM, n = 5, ANOVA). F, the stiffness of freshly dissected control, shP4HA1, or shP4HA2 tumors was determined (mean ± SEM, one-way ANOVA). G, the hydroxyproline content of tumor tissue was determined per mg of total protein (mean ± SEM, n = 5, ANOVA). *P<0.05, **P < 0.01, ***P < 0.001 vs. shLKO.1.

Figure 4

Collagen prolyl hydroxylase expression promotes invasion. A, representative hematoxylin and eosin staining of the tumor/adipose tissue boundary. Scale bar = 200 μm. B, crystal violet staining of MDA-MB-231 cells plated on tissue culture plastic (TC) or ECM-derived from indicated subclones. Scale bar = 100 μm; inset scale bar = 25 μm. C, CMFDA-labeled naïve MDA-MB-231 cells (green) were seeded on tumor sections and stained with picrosirius red to image collagen fibers (red). Scale bar = 100 μm.

Figure 5

Collagen prolyl hydroxylase expression promotes intravasation. A, human rRNA expression from peripheral blood (0.5 mL) from non-tumor-bearing NOD-SCID mice mixed with 0, 100, or 400 MDA-MB-231 cells B, the number of tumor cells from the peripheral blood (0.5 mL) of tumor-bearing mice was determined using the standard curve in panel A (mean ± SEM, n = 8, ANOVA). C, image and quantification of picrosirius red staining of lung sections from tumor-free (TF) or tumor-bearing mice (mean ± SEM, n = 5, ANOVA). D, lung sections were stained with hematoxylin and eosin. E, number of metastatic foci per 4x field (mean ± SEM, n = 10, ANOVA). F, image and quantification of picrosirius red staining of lung sections (mean ± SEM, n = 5, one-way ANOVA). *P<0.05, ***P < 0.001 vs. shLKO.1.

Figure 6

P4HA expression is associated with poor prognosis in breast cancer. A-B, P4HA1 and P4HA2 mRNA levels in normal breast (n = 7) and breast cancer (n = 40) tissues (A) or paired adjacent normal vs breast cancer tissues from The Cancer Genome Atlas (n = 28) (B) are shown. Box displays 25th through 75th percentiles and whiskers represent the range of the data. C, P4HA1 and P4HA2 mRNA levels in breast cancer (stage = 1, 2, 3/4) normalized to the mean level in adjacent normal (stage = TA) from the Cancer Genome Atlas, are plotted as described above (n = 458, Pearson's correlation). D, Kaplan-Meier analysis of patient survival (n = 159) stratified by P4HA1 (left) or P4HA2 (center) or both P4HA1 and P4HA2 mRNA levels (right). High (low) = gene expression above (below) the median level.

Figure 7

Inhibiting collagen prolyl hydroxylase activity using DHB prevents tumor growth, collagen deposition, and metastasis. A, chemical structures of α-ketoglutarate and ethyl 3,4 dihydroxybenzoate (DHB). B, immunoblot assay for collagen 1A1 in conditioned media of MDA-MB-231 cells treated with DHB for 24 hours. Ponc S, Ponceau S staining of membrane. C, tumor volume (mean ± SEM, n = 5, ANOVA) was determined. D-E, tumor sections were stained with picrosirius red and quantified (mean ± SEM, n = 5, Student t test). F, lung sections (3 × 3mm) stained with hematoxylin and eosin G, human genomic DNA content in mouse lungs (mean ± SEM, n = 5, Student's t test). *P < 0.05, **P < 0.01 vs. vehicle treatment. H, hypoxia-induced collagen prolyl hydroxylases expression promotes collagen deposition and breast cancer metastasis.