Genetic ablation of the fatty acid-binding protein FABP5 suppresses HER2-induced mammary tumorigenesis

Abstract

The fatty acid-binding protein FABP5 shuttles ligands from the cytosol to the nuclear receptor PPARβ/δ (encoded for by Pparδ), thereby enhancing the transcriptional activity of the receptor. This FABP5/PPARδ pathway is critical for induction of proliferation of breast carcinoma cells by activated epidermal growth factor receptor (EGFR). In this study, we show that FABP5 is highly upregulated in human breast cancers and we provide genetic evidence of the pathophysiologic significance of FABP5 in mammary tumorigenesis. Ectopic expression of FABP5 was found to be oncogenic in 3T3 fibroblasts where it augmented the ability of PPARδ to enhance cell proliferation, migration, and invasion. To determine whether FABP5 is essential for EGFR-induced mammary tumor growth, we interbred FABP5-null mice with MMTV-ErbB2/HER2 oncomice, which spontaneously develop mammary tumors. FABP5 ablation relieved activation of EGFR downstream effector signals, decreased expression of PPARδ target genes that drive cell proliferation, and suppressed mammary tumor development. Our findings establish that FABP5 is critical for mammary tumor development, rationalizing the development of FABP5 inhibitors as novel anticarcinogenic drugs.

Figure 1. FABP5 is upregulated in human breast tumors

a) Levels of FABP5 mRNA in samples of normal human breast and invasive breast carcinoma reported in: The Cancer Genome Atlas - Invasive Breast Carcinoma Gene Expression Data (https://tcga-data.nci.nih.gov/tcga/) and (52). Data were obtained from OncomineTM (Compedia Bioscience, Ann Arbor, Michigan). b) Analysis of TissueScanTM tissue qPCR array consisting of cDNA derived from samples of normal breast and denoted stages of breast tumors (OriGene). FABP5 expression was normalized to 18s and calibrated to the mean mRNA level in normal tissue (grey bars).

Figure 2. FABP5 promotes cell proliferation and invasion

a) Expression levels of FABP5 in normal human breast epithelial cells (HMEC) and in NaF mammary carcinoma cells, derived from a tumor that arose in the MMTV-neu mouse model of breast cancer. b) Immunoblot demonstrating reduced level of FABP5 in NaF cells stably expressing FABP5shRNA. c) Proliferation of NaF cells stably expressing empty vector (e.v.) or FABP5shRNA. d) Levels of FABP5 mRNA in HMEC cells transfected with e.v. or a vector encoding FABP5. Inset: Immunoblot demonstrating higher levels of FABP5 in HMEC cells overexpressing FABP5. e) Rate of growth of HMEC cells expressing e.v. or vector encoding FABP5. f) Levels of FABP5 mRNA in NIH3T3 fibroblasts expressing e.v., and in two NIH3T3 cell lines stably over-expressing FABP5 (F5-1, F5-2). Inset: immunoblots demonstrating expression levels of FABP5 in the NIH3T3 lines. g) Proliferation of NIH3T3 cells stably expressing varying levels of FABP5, measured by MTT assays. h) Representative images of zebrafish embryos injected into the yolk sac (YS) with NaF cells stably expressing control shRNA (Ctrlsh, upper panels) or shRNA directed towards FABP5 (FABP5shRNA, lower panels). Cells were stained with the fluorescent cell membrane dye CM-DiO and injected into the yolk sac of 2 dpf dechorionated zebrafish embryos (Marques et al., 2009). Fish were imaged 4 days later (see Experimental Procedures). Left panels: merge of light and fluorescent images of a whole zebrafish. Middle panels: merge of light and fluorescent images of zebrafish tails. Right panels: fluorescent images of zebrafish tails. The images show massive invasion into the tail by control cells (arrow in upper panels). Cells with reduced expression of FABP5 remained in the yolk sac and did not metastasize. Experiments were repeated four times with 10-15 fish per condition. Magnification 5x. i) Left: “Wound healing” assays using 3T3 cells stably expressing varying levels of FABP5. Images were obtained immediately following and 24 h. after scratching. Right: wound widths, quantified using image J software. Data are mean±S.D. (n=3).

Figure 3. FABP5 binds GW0742 and RA but not TTNPB

a) Recombinant mFABP5 expressed in E. coli and purified was resolved by SDS-PAGE and visualized by Coomassie-blue staining. b) Fluorescence titration of FABP5. Protein (1 μM) was titrated with ANS and probe fluorescence was monitored. Data were fit to obtain Kd. c) Association of FABP5 with RA, GW0742 and TTNPB was assessed by fluorescence competition titrations. FABP5 was pre-complexed with ANS and titrated with non-fluorescent ligands.

Figure 4. FABP5 promotes oncogenic properties in NIH3T3 fibroblasts by enhancing the transcriptional activity of PPARβ/δ

a) Proliferation of NIH3T3 cells stably expressing varying levels of FABP5 and treated with the PPARβ/δ-selective agonist GW0742 (1 μM, 4 days), assessed by MTT assays. b) Migration assays using NIH3T3 cells stably expressing varying levels of FABP5 and treated with GW0742 (1 μM, 24 h). c) Invasion assays using NIH3T3 cells stably expressing varying levels of FABP5 and treated with GW0742 (1 μM, 24 h). d)-f) proliferation (d) migration (e) and invasion (f) of 3T3 cell lines treated with retinoic acid (RA) or the pan-RAR agonist TTNPB (1 μM) as in panels b)-d). *p denotes values vs. non-treated 3T3 cells expressing an empty vector (e.v.). g) Levels of Pdpk1 and Plin2 mRNA in 3T3 cells stably expressing varying levels of FABP5 in the absence or presence of the PPARδ antagonist PTS58 (2 μM, 6h). ‡p=0.03; ‡‡p= 0.00004; #p=0.006; ##p=0.000009. h) Proliferation of 3T3 cells stably expressing varying levels of FABP5 and treated with denoted ligands (1 μM, 4 days) in the presence or absence of PTS58 (2 μM), assessed by MTT assays. Data are mean±SD from 3 independent experiments. Statistical analyses were carried out using two-tailed Student's t-test.

Figure 5. Ablation of FABP5 suppresses mammary tumor growth in vivo

a) Time course of tumor development in M5^+/+ (n=15), M5^+/− (n=14), and M5^−/− (n=14) mice. Development of the first tumor to appear in each mouse was monitored by measuring its length and width, and tumor volume calculated as length × width²/2. Data are mean±S.E.M. Inset: immunoblots of FABP5 in tumors that developed in three individual M5^−/−, M5^+/− and M5^+/+ mice. b) Kaplan-Meier analysis of survival of M5^+/+, M5^+/−, and M5^−/− mice. Mice were sacrificed when their largest tumors reached a volume of 1200 mm³. Surviving M5^+/−, and M5^−/− mice had no measurable tumors at termination of the experiments. c) Scatter plots of volume of tumors measured as in (c) at age 59 weeks. Horizontal grey lines indicate the mean in each group. d) Scatter plots of the number of tumors/mouse found at termination of the experiment as indicated in (d). Horizontal lines indicate the mean in each group. e) Immunostaining of the proliferation marker Ki67 and the apoptosis marker cleaved caspase 3 in tumors that arose in M5^+/+, M5^+/−, and M5^−/− mice. (Scale bars: 50 μm).

Figure 6. Ablation of FABP5 diminishes ErbB2-induced proliferative signaling and attenuates the transcriptional activity of PPAR □In vivo

a)-c) Upper panels: immumoblots demonstrating phosphorylation status of ERK (a), phosphorylation status of AKT (b), and expression level of CNX26 (c) in tumors of three individual M5^+/+, M5^+/−, and M5^−/− mice. Lower panels: quantitation of immunoblots. Data are mean±S.D. p values for differences between denoted groups and respective M mice were calculated by a two-tail Student's t-test. d) Levels of mRNA for the direct PPAR~target gene VEGFa in tumors of three individual M5^+/+, M5^+/−, and M5^−/− mice. Mean±S.D. p values vs. M5^+/+ tumors are shown. e) Left: immunoblots of PDPK1, encoded by the direct PPAR~target gene pdpk1, and PTEN, whose expression is suppressed by PPARβ/δ, in tumors of three individual M5^+/+, M5^+/−, and M5^−/− mice. Right: quantitation of immunoblots. Mean±S.D. *p vs. respective M mice, and **p vs. M5^+/− mice are shown.