Diet-induced obesity links to ER positive breast cancer progression via LPA/PKD-1-CD36 signaling-mediated microvascular remodeling - PubMed (original) (raw)
. 2017 Apr 4;8(14):22550-22562.
doi: 10.18632/oncotarget.15123.
Ye Yuan 1 2, Cynthia Opansky 1, Yiliang Chen 1, Irene Aguilera-Barrantes 3, Shiyong Wu 2, Rong Yuan 1, Qi Cao 4, Yee Chung Cheng 5, Daisy Sahoo 5, Roy L Silverstein 1 5, Bin Ren 1 5
Affiliations
- PMID: 28186980
- PMCID: PMC5410244
- DOI: 10.18632/oncotarget.15123
Diet-induced obesity links to ER positive breast cancer progression via LPA/PKD-1-CD36 signaling-mediated microvascular remodeling
Liuyi Dong et al. Oncotarget. 2017.
Abstract
Obesity increases cancer risk including breast cancer (BC). However, the direct regulatory mechanisms by which obesity promotes BC progression remain largely unknown. We show that lysophosphatidic acid/protein kinase D1 (LPA/PKD-1)-CD36 signaling is a bona fide breast cancer promoter via stimulating microvascular remodeling in chronic diet-induced obesity (DIO). We observed that the growth of an estrogen receptor (ER) positive breast cancer was markedly increased when compared to the lean control, and specifically accompanied by increased microvascular remodeling in a syngeneic BC model in female DIO mice. The tumor neovessels in DIO mice demonstrated elevated levels of alpha smooth muscle actin (α-SMA), vascular endothelial growth factor receptor 2 (VEGFR 2) and endothelial differentiation gene 2/LPA receptor1 (Edg2/LPA1), enhanced PKD-1 phosphorylation, and reduced CD36 expression. Tumor associated endothelial cells (TAECs) exposed to LPA demonstrated sustained nuclear PKD-1 phosphorylation, and elevated mRNA levels of ephrin B2, and reduced mRNA expression of CD36. TAEC proliferation also increased in response to LPA/PKD-1 signaling. These studies suggest that the LPA/PKD-1-CD36 signaling axis links DIO to malignant progression of BC via stimulation of de novo tumor arteriogenesis through arteriolar remodeling of microvasculature in the tumor microenvironment. Targeting this signaling axis could provide an additional novel therapeutic strategy.
Keywords: CD36; lysophosphatidic acid; microvascular remodeling; phospholipid; protein kinase D.
Conflict of interest statement
CONFLICTS OF INTEREST
The authors declare no conflicts of interests.
Figures
Figure 1. Diet-induced obesity promotes breast cancer progression
(A) Female mice were maintained on a high fat (HF diet, n = 14) and on a control (CTL, n = 15) diet for the indicated time, and body weight was recorded. (B) E0771 cells were implanted subcutaneously close to the fourth mammary pad, and tumor volumes were measured and calculated. (C) Tumors were extracted and weighed at day 21 or earlier. (D) Tumor sections were stained with an anti-Ki67 antibody (Millipore) using VECTASTAIN®ABC with an alkaline phosphatase enzyme detection system. Images were acquired with a Nikon Eclipse E600 microscope, and Ki67 staining density was analysed with NIH Image J software. Bar = 50 μm in representative images.
Figure 2. Diet-induced obesity promotes tumor angiogenic and metastatic potential
(A) Paraffin-embedded tumor tissues were sectioned and stained by Trichrome. Inserts are magnified images of areas containing blood vessels (left panel). The number of blood vessels in the periphery of each tumor was quantified under a microscope, and the average number was compared in the tumor tissues between mice fed with HF and CTL diets (right panel). Bar = 200 μm in representative images or 50 μm in inserted images. (B) Tumors grown in DIO mice showed increased collagen accumulation in tumor-associated stroma as shown by blue/green color in the Trichrome staining. Inserts are magnified images, and bar = 200 μm in representative images. (C) H & E and Trichrome stain showed BC invasion into the fat tissues in DIO mice. Representative color images of H & E (upper panel, 20 X ) or Trichrome (lower panel) stained sections show many fat vacuoles in the BC tissues in the DIO mice, bar = 500 μm (lower left panel) and 200 μm (lower right panel).
Figure 3. LPA/PKD-1 signaling in arteriogenic gene expression
(A) Tumor sections were co-stained with anti-α-SMA antibody and anti-VEGFR2 antibody (e-Bioscience) followed by FITC (Santa Cruz) or biotin-labeled CYR3 secondary antibody and mounted with DAPI. Images were acquired and overlayed using an EVOS®FL cell imaging system. Bar = 100 μm in representative images. (B) Tumor sections were stained with anti-CD36, or anti-VEGFR2 antibodies as described in A. Images were acquired and overlayed using an EVOS®FL cell imaging system. Bar = 200 μm in representative images. Note: the obvious green staining of cells within the ER+ tumor tissues appeared to be CD36 positive cancer cells. (C) G-protein coupled receptor LPA1 expression increased in the tumor endothelium in DIO mice. Tumor sections were stained with anti-VEGFR2 and anti-LPA1 antibodies (Cayman Chemical) followed by appropriate secondary antibodies. Images were acquired as in Figure 1. (D) Phospho-PKD-1 levels increased in tumor vessels in DIO mice. Tumor sections were stained with phospho-PKDSer744-748 antibodies and assessed using the VECTASTAIN®ABC with an alkaline phosphatase enzyme detection system. Images were acquired as in Figure 1D. Representative images are shown, in which vascular staining of phosphorylated PKD-1 within tissues are denoted by arrows (bar = 50 μm), and inserts are modified magnified images with arbitrary increase of contrast and magnification.
Figure 3. LPA/PKD-1 signaling in arteriogenic gene expression
(A) Tumor sections were co-stained with anti-α-SMA antibody and anti-VEGFR2 antibody (e-Bioscience) followed by FITC (Santa Cruz) or biotin-labeled CYR3 secondary antibody and mounted with DAPI. Images were acquired and overlayed using an EVOS®FL cell imaging system. Bar = 100 μm in representative images. (B) Tumor sections were stained with anti-CD36, or anti-VEGFR2 antibodies as described in A. Images were acquired and overlayed using an EVOS®FL cell imaging system. Bar = 200 μm in representative images. Note: the obvious green staining of cells within the ER+ tumor tissues appeared to be CD36 positive cancer cells. (C) G-protein coupled receptor LPA1 expression increased in the tumor endothelium in DIO mice. Tumor sections were stained with anti-VEGFR2 and anti-LPA1 antibodies (Cayman Chemical) followed by appropriate secondary antibodies. Images were acquired as in Figure 1. (D) Phospho-PKD-1 levels increased in tumor vessels in DIO mice. Tumor sections were stained with phospho-PKDSer744-748 antibodies and assessed using the VECTASTAIN®ABC with an alkaline phosphatase enzyme detection system. Images were acquired as in Figure 1D. Representative images are shown, in which vascular staining of phosphorylated PKD-1 within tissues are denoted by arrows (bar = 50 μm), and inserts are modified magnified images with arbitrary increase of contrast and magnification.
Figure 4. Sustained LPA/PKD-1 signaling may be involved in TAEC differentiation
(A) TAECs were exposed to LPA (10 μM) for 24 hours. Cytosol and nuclear fraction were isolated, and phospho-PKD-1 Ser744-748 levels were detected by Western blots. α-tubulin and histone 3 respectively represent cytosol and nuclear components. Representative images from three separate experiments are shown. (B) TAECs were treated as described in A, and cytosol and nuclear fractions were isolated for the detection of HDAC7. Representative images and quantified densitometry data from three separate experiments are shown. (C) TAECs were treated with LPA (10 μM), and cytosol and nuclear fractions were isolated for the detection of FoxO1 as indicated in A. Representative images from three separate experiments are shown. (D) TAECs were transfected with plasmids expressing Halo-tagged FoxO1 for 24 hours followed with LPA (10 μM) for an additional 24 hours for Halo pull-down assays.
Figure 5. LPA/PKD-1 signaling in the expression of CD36 and ephrin B2 and endothelial cell proliferation
(A) TAECs were treated with vehicle or LPA (10 μM) for 24 hours, and CD36 mRNA levels were assayed by real time qPCR (left panel); Cardiac ECs starved for 6 hours, were infected by lentivirus human CD36:GFP for 72 hours, with addition of LPA (10 uM) at the last 24 hours. Images were acquired using an EVOS®FL cell imaging system. (B) TAECs were exposed to LPA (10 μM) or LPA (10 μM) and PKD inhibitor CID 755673 (25 μM) for 48 hours, and total RNA was extracted for real time qPCR. A representative result is shown. (C) TAECs transduced with GFP were exposed to LPA (10 μM) or LPA (10 μM) and CID755673 (25 μM) for 24 hours for proliferation assays.
Figure 6. LPA/PKD-1-CD36 signaling axis in de novo tumor arteriogenesis in obesity
A working model that shows the mechanisms by which LPA/PKD-1-CD36 signaling axis promotes arteriolar remodeling of microvasculature in BC for malignant progression via G-protein coupled receptor LPA1 in chronic DIO. ATX: autotaxin; LPC: lysophosphatidylcholine. Dash line means that mechanisms are unclear.
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