Overexpression of the RNA binding protein HuR impairs tumor growth in triple negative breast cancer associated with deficient angiogenesis

Abstract

Interactions between RNA binding proteins (RBPs) and genes are not well understood, especially in regulation of angiogenesis. The RBP HuR binds to the AU-rich (ARE) regions of labile mRNAs, facilitating their translation into protein and has been hypothesized to be a tumor-maintenance gene. Elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR controls the expression of multiple genes involved in angiogenesis including VEGFα, HIF1α and thrombospondin 1 (TSP1). We investigated the role of HuR in estrogen receptor negative (ER(-)) breast cancer. MDA-MB-231 cells with higher levels of HuR have alterations in cell cycle kinetics and faster growth. Unexpectedly, HuR overexpression significantly interfered with tumor growth in orthotopic mouse models. The putative mechanism seems to be an anti-angiogenetic effect by increasing expression of TSP1 but also surprisingly, downregulating VEGF, a target which HuR normally increases. Our findings reveal that HuR may be regulating a cluster of genes involved in blood vessel formation which controls tumor angiogenesis. An approach of modulating HuR levels may overcome limitations associated with monotherapies targeting tumor vessel formation.

Figure 1

Overexpression of HA-HuR in MDA-MB-231 cancer cells increases cell growth and alters cell cycle kinetics in vitro while inhibiting tumor growth in vivo. (A) MDA-MB-231 cells transfected with the pZeoSV2 vector expressing HA-HuR, selected with Zeocin and cloned by limiting dilution express HA-HuR compared to a pZeoSV2 empty vector control. MDA-MB-231 clones 4e1 and 5F1 expressed 42% and 38% more HuR than control as shown in representative western blot. Bar graph represents mean HuR percent overexpression from three different western blots. (B) Both clones expressing HA-HuR proliferated significantly faster than the empty vector control in vitro. (C) Overexpression of HA-HuR increased cells in G₀/G₁ cell cycle, from 57.70% to 67.67%. Overexpression of HA-HuR decreased cells in G₂/M phase by 27.19% to 18.29% but did not significantly alter cells in S phase as measured by DNA content. (D) MDA-MB-231 HA-HuR 4E1 showed significantly reduced tumor volume (mm³) and growth starting at two weeks post-inoculation and continuing for five weeks when compared to empty vector MDA-MB-231 controls as measured by both MRI and calipers. For tumor experiments nine animals per group were used. For both counting assay and tumor volumes data represent mean value ± SEM. p < 0.05. Flow cytometry data represent mean value ± SEM from n = 4 separate experiments done in triplicate. p < 0.05.

Figure 2

Overexpression of HA-HuR in MDA-MB-231 cancer cells inhibits tumor growth in athymic nude mice. (A) Repeat experiments comparing MDA-MB-231 HA-HuR 4E1 with both wild-type MDA-MB-231 and empty vector MDA-MB-231 confirmed HuR overexpression reduced tumor volume (mm³) and growth starting at two weeks post-inoculation and continuing for five weeks as measured by calipers. (B) Tumors overexpressing HA-HuR had significantly less mass after harvest 42 days post-inoculation when compared to the WT or empty vector (EV) controls. (C) MRI comparing largest sections for each tumor showed significantly smaller tumors in the HA-HuR overexpressing tumors when compared to EV control tumors. (D) Representative cross sections of tumors showed that those formed by inoculation with HA-HuR resembled a gelatin-like capsule, were significantly smaller and more homogeneous than those formed by inoculation with empty vector. Hematoxylin and eosin stain revealed poorly differentiated carcinomas with similar morphology and lack of inflammatory cells in both HA-HuR tumors and EV control tumors. Five animals were used per group in HA-HuR, empty vector and wild-type control groups. Experiments were repeated with similar results using a different clone, 5F1, (see Supporting Information Fig. S3). Data represent mean value ± SEM. p < 0.05; in photomicrographs bar = 27 microns.

Figure 3

TSP1 is upregulated in HA-HuR tumor and VEGFα is downregulated. (A) Real-time PCR indicates TSP1 is upregulated in tumors (5.44 fold) and cells in culture (4.88 fold) overexpressing HA-HuR when compared to EV control tumors and cells, consistent with the microarray data. VEGF is downregulated (2.6 fold) in tumors overexpressing HA-HuR when compared to EV controls. HIF1α mRNA levels did not appreciably change. Change in gene expression was determined using the comparative C_T method and is represented as fold change in HA-HuR tumors as compared to empty vector controls. GAPDH was used as an endogenous control. (B) Western blot for TSP1 shows increased protein expression of TSP1 (76%) in the HA-HuR overexpressing tumors when compared to EV control tumors. (C) Western blot for VEGF shows decrease protein expression by 23% in the HA-HuR overexpressing tumors when compared to EV control tumors (representative of two independent sets of tumors). Data represent mean value ± SEM from n = 3 separate mice done in triplicate. p < 0.05.

Figure 4

HuR interacts with both TSP1 and VEGF mRNAs in cells overexpressing HuR. (A) RNA immunoprecipiation indicates both TSP1 and VEGF mRNA are increased in the HuR IP when compared to IgG1 control IP in both HA-HuR overexpressing cells and EV control cells. (B) Actinomycin D mRNA stability assay shows VEGF mRNA half-life was not altered between cells overexpressing HA-HuR and EV control cells. (C) TSP1 mRNA from cells overexpressing HA-HuR has a longer half-life than TSP1 mRNA from EV control cells. For RNA immunoprecipitation, data represents mean value ± SEM. p < 0.05.

Figure 5

Tumors overexpressing HA-HuR have no increases in apoptosis but decreased blood vessel formation compared to control. (A) Annexin V staining reveals similar amounts of cells undergoing apoptosis between cells overexpressing HA-HuR and EV control cells. (B) Caspase 3 staining shows no differences in the amount of apoptosis in the EV control tumors compared to HA-HuR tumors harvested 14 days post-inoculation. In the tumors harvested on day 42 post-inoculation, caspase 3 staining showed more apoptotic cells in the EV control tumors compared to tumors overexpressing HA-HuR. CD34 staining shows fewer blood vessels in tumors overexpressing HA-HuR. (B) Quantitation of blood vessels stained (number of vessels per high power field scored) with CD34 indicated significantly fewer blood vessels in the tumors overexpressing HA-HuR. Error bars ± SEM; p < 0.005; in photomicrographs bar = 27 microns. Representative of n = 5 sets of tumors.