Prolyl-4-hydroxylase α subunit 2 promotes breast cancer progression and metastasis by regulating collagen deposition
© Xiong et al.; licensee BioMed Central Ltd. 2014
Received: 27 September 2013
Accepted: 26 December 2013
Published: 2 January 2014
Increased collagen deposition provides physical and biochemical signals to support tumor growth and invasion during breast cancer development. Therefore, inhibition of collagen synthesis and deposition has been considered a strategy to suppress breast cancer progression. Collagen prolyl-4-hydroxylase α subunit 2 (P4HA2), an enzyme hydroxylating proline residues in -X-Pro-Gly- sequences, is a potential therapeutic target for the disorders associated with increased collagen deposition. However, expression and function of P4HA2 in breast cancer progression are not well investigated.
Gene co-expression analysis was performed in the published microarray datasets to identify potential regulators of collagen I, III, and IV in human breast cancer tissue. Expression of P4HA2 was silenced by shRNAs, and its activity was inhibited by 1, 4-DPCA, a prolyl-4-hydroxylase inhibitor. Three-dimensional culture assay was used to analyze roles of P4HA2 in regulating malignant phenotypes of breast cancer cells. Reduced deposition of collagen I and IV was detected by Western blotting and immunofluorescence. Control and P4HA2-silenced breast cancer cells were injected into fat pad and tail vein of SCID mice to examine effect of P4HA2 on tumor growth and lung metastasis.
Using gene co-expression analysis, we showed that P4HA2 was associated with expression of Col1A1, Col3A1, and Col4A1 during breast cancer development and progression. P4HA2 mRNA levels were significantly upregulated in breast cancer compared to normal mammary tissue. Increased mRNA levels of P4HA2 correlated with poor clinical outcome in breast cancer patients, which is independent of estrogen receptor status. Silencing P4HA2 expression or treatment with the P4HA inhibitor significantly inhibited cell proliferation and suppressed aggressive phenotypes of breast cancer cells in 3D culture, accompanied by reduced deposition of collagen I and IV. We also found that knockdown of P4HA2 inhibited mammary tumor growth and metastasis to lungs in xenograft models.
These results suggest the critical role of P4HA2 in breast cancer progression and identify P4HA2 as a potential therapeutic target and biomarker for breast cancer progression.
KeywordsTumor microenvironment Breast cancer Collagen deposition Cancer progression Cell proliferation
Extracellular matrix (ECM) is an important component of tumor microenvironment and plays critical roles in cancer development [1–3]. Collagens are the major structural ECM proteins and form fibers or networks in tumor tissue [4–6]. Cell-collagen interaction controls a variety of cellular activities including proliferation, migration, and invasion through integrin and discoidin domain receptor [7–9]. Enhanced expression and deposition of collagens are associated with tumor development and progression [10–12]. Recent studies demonstrate that increased collagen deposition and crosslinking enhance the stiffness and density of mammary tissue [5, 10, 13], which is an important risk factor for breast cancer development. Type I collagen has been identified as a prognosis marker and is associated with cancer recurrence in human breast cancer patients . Collagen VI knockout mice have reduced primary tumor formation and growth , while enhancing collagen deposition and inhibiting collagen degradation significantly enhances tumor initiation and tumor growth [5, 10]. In addition, cancer cell invasion usually occurs at tumor-stromal interfaces with oriented collagen fibers, and aligned collagen fibers can facilitate cell migration and metastasis [5, 10, 11, 15]. These results indicate that increased collagen expression and deposition promotes breast cancer development and progression by enhancing tumor growth and invasion. Therefore, inhibiting collagen synthesis or deposition is a promising strategy to suppress breast cancer progression.
Collagen biosynthesis is a multistep process that involves several post-transcription modification enzymes, and one of the most important members of these enzymes is collagen prolyl-4-hydroxylase . It catalyzes the formation of 4-hydroxyproline by hydroxylating proline residues in -X-Pro-Gly- sequences [17–20]. Collagen prolyl-4-hydroxylase resides within the lumen of the endoplasmic reticulum (ER)  and its expression is used as a marker for collagen synthesis [21, 22]. Because 4-hydroxyproline residues formed in this reaction are essential for triple helix formation and stabilization of collagen [22–24], inhibiting the prolyl-4-hydroxylases activity efficiently blocks collagen synthesis and deposition. All known vertebrate collagen prolyl-4-hydroxylases are α2β2 tetramers consisting of two α subunits and two β subunits. Each α subunit contains the peptide substrate binding domain and the two catalytic sites of the enzyme, and the β subunits have been identified as protein disulfide isomerases [17, 19, 25]. Three types of collagen prolyl-4-hydroxylases α isoforms (P4HA1, P4HA2 and P4HA3) have been identified in human tissue. P4HA1 is expressed in most cell types; P4HA2 is mainly expressed in chondrocytes, osteoblasts, and capillary endothelial cells; P4HA3 expression is detected in adult and fetal tissues at very low levels compared to P4HA1 and P4HA2 [18, 26]. Increased P4HA2 expression has been detected in many solid tumors, including oral cavity squamous cell carcinoma, papillary thyroid cancer, and breast cancer [27–30], however, the function of P4HA2 in cancer progression largely remains to be determined.
Here, we showed that expression of P4HA2 and collagen genes (Col1A1, Col3A1, and Col4A1) is significantly correlated during breast cancer development and progression, and that increased mRNA levels of P4HA2 are associated with poor prognosis in breast cancer patients. Silencing P4HA2 or treatment with the P4HA inhibitor attenuates cell proliferation and suppresses aggressive 3D phenotypes, tumor growth, and cancer metastasis, which are accompanied by reduced collagen deposition. These results suggest that P4HA2 promotes breast cancer progression by enhancing collagen deposition and it may serve as a potential therapeutic target for breast cancer.
Antibodies and reagents
The Click-iT® EdU Alexa Fluor® 488 Imaging Kit and Alexa Fluor® 594 phalloidin were from Invitrogen. Matrigel (lrECM) and Type I collagen were from BD Bioscience. ShP4HA2 plasmids were purchased from Sigma. 1, 4-DPCA was purchased from Cayman Chemical. Masson’s trichrome stain kit was purchased from Polysciences, Inc. The following antibodies were obtained as indicated: integrin α6 (Millipore); collagen I (Abcam); collagen IV (Abcam); P4HA2 (Santa Cruz); tubulin (Millipore).
Cell culture and virus preparation
HMT-3522 T4-2 cells (a kind gift from Dr. Mina J. Bissell) were maintained on tissue culture plastic as previously described . MDA-MB-231 cells were propagated in DMEM/F12 (Sigma) with 10% fetal bovine serum (Invitrogen). MDA-MB-157 cells and ZR-75-1 cells were propagated in DMEM (Sigma) with 10% fetal bovine serum. ZR-75-1 cells: ER-positive and PR positive; T4-2 cells, MDA-MB-231 cells and MDA-MB-157 cells: ER-negative and PR negative.
3D laminin-rich extracellular matrix (3D lrECM) on-top cultures were prepared by trypsinization of cells from tissue culture plastic, seeding of single cells on top of a thin gel of Engelbreth-Holm-Swarm (EHS) tumor extract (Matrigel: BD Biosciences, 354230), and addition of medium containing 5% EHS. T4-2 cells were seeded at a density of 2.1 × 104 cells per cm2; MDA-MB-157 cells, ZR-75-1 cells, and MDA-MB-231 cells were seeded at 1.4 × 104 cells per cm2. T4-2 cells were maintained in their propagation medium with media change every 2 days. MDA-MB-157 cells, ZR-75-1 cells and MDA-MB-231 cells were maintained in H14 medium with 1% fetal bovine serum. The cell colonies cultured in 3D were imaged and used for immunofluorescence staining at Day 4 after seeding.
HEK293 FT cells were transfected with scrambled RNA sh-control vector or sh-P4HA2-1 (CCGGGCCGAATTCTTCACCTCTATTCTCGAGAATAGAGGTGAAGAATTCGGCTTTTG), sh-P4HA2-2 (CCGGGCAGTCTCTGAAAGAGTACATCTCGAGATGTACTCTTTCAGAGACTGCTTTTTG) plus packaging lentivector using lipofectamine (Invitrogen). Cancer cells were infected with lentivirus and selected by puromycin 48 h after infection.
Immunofluorescence and Masson’s trichrome staining
Cells in lrECM gel were smeared on slides, dried briefly, and fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Immunostaining was performed as previous described . Stained samples were imaged with a Nikon upright epifluorescence microscope or a confocal system comprised of an Olympus IX81 microscope.
Xenograft tumor sections were de-paraffined and hydrated from xylene, 100% ethanol, 95% ethanol, 85% ethanol and 70% ethanol to distilled water. For Masson’s trichrome staining, slides were re-fixed with Bouin’s solution at 60°C for 60 minutes. Slides were washed in running tap water for 5 minutes and stained in Weigert’s working hematoxyin for 10 minutes. Then they were washed in running tap water for 5 minutes and stained in Biebrich scarlet-acid fuchsin solution for 5 minutes. Slides were rinsed in distilled water and differentiated in phosphomolybdic-phosphotungstic acid solution for 10 minutes, transferred to aniline blue solution and stain for 5 minutes. Slides were rinsed in distilled water and images were taken with a Nikon microscope. The percentage of collagen was quantified by calculating the ratio of blue staining (collagen) area in the total area of the tumor section using Imagescope analysis software .
Western blot analysis
Cells grown on plastic were lysed in situ in 2% SDS in PBS buffer containing phosphatase and protease inhibitor cocktails (Calbiochem). Protein concentration was measured using DC™ protein assay (Bio-Rad). Control and shP4HA2 cells were trypsinized and counted; equal amounts of conditional medium (normalized to cell number) were precipitated by pre-cooled acetone. Equal amounts of protein lysates and cell conditional medium were subjected to SDS gel electrophoresis, immunoblotted, and detected with an ECL system (Pierce). Western blotting results were quantified using AlphaInnotech analysis software.
Transwell invasion assay
The Transwells (Corning) were coated with 60 mL 1 mg/mL Matrigel and incubated for 30 minutes at 37°C. Sh-control or sh-P4HA2 silencing MDA-MB-231 cells (1 × 105 cells in 200 μl medium) were plated on the top of the Transwell filter and incubated in 37°C 5% CO2 for 24 h. The invaded cells on the bottom face of the filter were fixed by methanol and stained with 8% crystal violet. Images were taken with a Nikon microscope and the number of invaded cells was counted.
Female SCID mice (6 weeks old) were randomly grouped and injected with 2 × 106 sh-control or shP4HA2-1 MDA-MB-231/Luc cells at mammary fat pad. Tumor volume was measured using an in vivo imaging system (IVIS). Tumors were measured with a caliper every 4 days for 6 weeks. At the experimental endpoint, tumors were harvested and fixed with 4% PFA for paraffin-embedded section. All procedures were performed within the guidelines of the Division of Laboratory Animal Resources at the University of Kentucky.
Lung metastasis experiment
Female SCID mice (6 weeks old) were randomly grouped and injected with 1 × 106 (in 200 μl PBS) sh-control or sh-P4HA2-1 MDA-MB-231/Luc cells via tail vein. To detect lung metastasis, bioluminescent images were taken day 30 after cancer cells injection with IVIS Spectrum. Mice were sacrificed week 5 after cancer cells injection.
Kaplan Meier survival analysis and other statistical analyses
Kaplan-Meier survival analysis was performed in a large combined breast cancer dataset . Breast cancer patients were grouped by estrogen receptor (ER)-positive (n = 1452) and ER-negative (n = 473), and tumor samples were equally grouped into low and high P4HA2 expression based on the mRNA levels. Significant differences in overall survival time were assessed with the Cox proportional hazard (log-rank) test.
Analysis of P4HA2 mRNA levels in normal and malignant tissues was performed in the TCGA breast cancer dataset that was downloaded from Oncomine. The association between mRNA levels of P4HA2 and collagen genes was evaluated by the Spearman correlation analysis. All experiments were repeated at least twice. Results are reported as mean ± S.E.M; the significance of difference was assessed by independent Student’s t-test. P < 0.05 represents statistical significance and P < 0.01 represents sufficiently statistical significance. All reported P values were 2-tailed. Statistical analysis was conducted with SigmaPlot (Systat Software, Inc.) and SAS (version 9.2; SAS Institute Inc.).
Results and discussion
P4HA2 is associated with collagen expression and poor prognosis in human breast cancer
A number of genes encoding collagen proteins have been identified as prognostic markers for human breast cancer [37, 38]. Since expression of P4HA2 and collagen genes is correlated in human breast cancer tissues, we asked whether P4HA2 expression is associated with clinical outcome in human breast cancer patients. Breast cancer patients were divided into two groups based on P4HA2 mRNA levels (low and high). Kaplan-Meier log rank analysis showed that patients whose tumors had high P4HA2 expression levels had a significantly shorter overall survival period (Figure 1G). Moreover, the association of P4HA2 with clinical outcome is ER status independent (see Figure 1G).
Inhibition of P4HA2 suppresses the malignant phenotypes of breast cancer cells in 3D culture
Daniele M. Gilkes et al. reported that knockdown of P4HA2 or treatment MDA-MB-231 cells with hydroxylase inhibitor DHB inhibits tumor growth in vivo, but little inhibitory effect on cell proliferation was detected in 2D culture assay . 3D culture has been considered a better model for testing drugs and investigating cancer biology compared to 2D culture, and different drug responses between these two culture systems have recently been reported [47–49]. For example, MDA-MB-231 cells in 3D culture are more sensitive to MEK inhibition compared to cells in 2D culture . Using the 3D culture model, we showed that reducing P4HA2 expression or inhibiting its activity significantly inhibited cell proliferation and suppressed the malignant phenotypes in multiple breast cancer cell lines.
Reducing P4HA2 expression or inhibiting its activity impairs deposition of collagen I and IV
P4HA2 regulates tumor growth and metastasis in vivo
To determine whether P4HA2 promotes breast cancer lung metastasis in vivo, the control and P4HA2-silenced (sh-P4HA2-1) MDA-MB-231 cells were injected into the tail veins of SCID mice. Lung colonization of the cancer cells was monitored by IVIS imaging. We showed that the mice injected with control cells developed lung metastasis within 6 weeks, while no metastasis was detected in the P4HA2-silenced group (Figure 6F). HE staining further confirmed that knockdown of P4HA2 blocked the lung colonization of MDA-MB-231 cells in SCID mice (Figure 6G).
In the present study, we show that P4HA2 is associated with expression of collagen I, III, and IV during breast cancer progression. Increased mRNA levels of P4HA2 correlate with poor prognosis in human breast cancer patients. Silencing P4HA2 or inhibiting its activity suppresses breast cancer progression by reducing tumor growth and metastasis, and this process is accompanied by reduced collagen deposition. During preparation of this manuscript, Daniele M. Gilkes et al. reported that hypoxia-inducible factor 1 activates the transcription of P4HA1 and 2 during breast cancer development, and this activation enhances collagen fiber alignment and breast cancer progression [33, 55]. Taken together, these findings indicate that P4HA2 is a promising therapeutic target to inhibit ECM-dependent breast cancer progression.
Prolyl-4-hydroxylase α subunit 2
Epidermal growth factor receptor 2, 1,4-DPCA, 1,4-dihydrophenonthrolin-4-one-3-Carboxylic acid
We thank the pathology core facility at Markey Cancer Center for assistance in tissue fixation and section. We thank Ruthie S Fligor for scientific editing. This study was supported by grants from ACS (IRG 85-001-22 to R. Xu) and AHA (12SDG8600000 to R. Xu). This publication was also supported by the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health (Grant UL1TR000117). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
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