The versatile role of HuR in Glioblastoma and its potential as a therapeutic target for a multi-pronged attack

Abstract

Glioblastoma (GBM) is a malignant and aggressive brain tumor with a median survival of ∼15 months. Resistance to treatment arises from the extensive cellular and molecular heterogeneity in the three major components: glioma tumor cells, glioma stem cells, and tumor-associated microglia and macrophages. Within this triad, there is a complex network of intrinsic and secreted factors that promote classic hallmarks of cancer, including angiogenesis, resistance to cell death, proliferation, and immune evasion. A regulatory node connecting these diverse pathways is at the posttranscriptional level as mRNAs encoding many of the key drivers contain adenine- and uridine rich elements (ARE) in the 3' untranslated region. Human antigen R (HuR) binds to ARE-bearing mRNAs and is a major positive regulator at this level. This review focuses on basic concepts of ARE-mediated RNA regulation and how targeting HuR with small molecule inhibitors represents a plausible strategy for a multi-pronged therapeutic attack on GBM.

Keywords: Adenine- and Uridine-rich elements; Cytoplasmic translocation; GBM; HuR multimerization; Posttranscriptional regulation; Small molecule inhibitors.

Figure 1:. General principles of ARE-mediated posttranscriptional regulation and the role of HuR.

In the nucleus, HuR binds to ARE regions of some newly transcribed pre-mRNAs and participates in splicing and mRNA maturation. HuR can help translocate to the cytoplasm some ARE-bearing mRNAs, and generally promotes the stabilization and translation of bound mRNAs. Factors that promote mRNA decay, which include microRNAs and ARE-binding proteins TTP, KSRP and AUF1, are blocked from binding to the mRNA. If HuR is retained in the nucleus, has impaired RNA binding, or is sequestered by non-coding RNAs, RNA destabilizers and/or the microRNA/RISC (miRISC) complex gain access to the mRNA which is then transported to exosomes or P bodies for degradation.

Figure 2:. Structural features of HuR.

Schematic domain organization of HuR. The N-terminal RRM and central RRM 2 domains are separated by a short linker, while C-terminal RRM3 is connected to the tandem domains by a long intrinsically disordered hinge region (upper panel). Each of the RRMs has a cognate topology of β1α1β2β3α2β4, where the antiparallel β sheets are arranged against 2 α helices that are perpendicularly oriented (lower left panel). The N-terminal cysteine (cys13) residue, and/or C-terminal tryptophan residues (trp261, trp261 and trp271) are essential for multimerization of HuR which fine-tunes its function (lower right panel) [, –38].

Figure 3:. HuR is upregulated in malignant glioma and correlates with poor overall survival.

(A) Comparison of HuR mRNA levels in GBM and lower grade (LG) glioma tissue with normal control brain tissue. The sample numbers are shown in parentheses. * P < 10–9. (B) Overall survival in patients with malignant glioma (GBM and LG gliomas) with high HuR mRNA levels (upper 50%, n = 338) compared to those with low HuR mRNA levels (lower 50%, n = 338). Hazard ratio for the high group is 3.8. 95% CI are shown. Data obtained from GEPIA (Gene Expression Profiling Interactive Analysis) (cancer-pku.cn).

Figure 4:. HuR positively regulates ARE mRNAs in GSCs, GTCs and TAMs to drive tumor progression.

(A) Immunohistochemistry of biopsies obtained from the edge and core of a GBM tumor showing extensive HuR immunostaining in tumor cells and Iba1+ TAMs both in the tumor (Tu) and the surrounding brain parenchyma (Br). There is extensive translocation of HuR in Iba1+ cells as indicated by a merged signal with HuR (yellow). Dashed line indicates approximate border between Br and Tu. Scale bars, 100 μm. (B) HuR functions within each of the three major cellular components of GBM to promote intrinsic and secreted factors that drive GBM progression. GSCs, glioma stem cells; GTC,s glioma tumor cell; TAMs, tumor-associated microglia and macrophages; CTLs, Cytotoxic T lymphocytes; PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells;

Figure 5:. Development of small molecules that block HuR multimerization.

(A) Based on studies with primary GBM tissues, there is extensive multimerization of cytoplasmic HuR which is an essential component to its oncogenic effect [38, 96]. (B) A split-luciferase assay was developed by Filippova and Nabors to detect HuR multimerization [38]. The N- and C-terminal halves of firefly luciferase were fused to HuR in separate plasmids which were then transfected into malignant glioma cells. Upon HuR dimerization, there is reconstitution of the luciferase protein as indicated by luminescence after addition of luciferin to the culture media. MS-444, a previously identified inhibitor of HuR dimerization [180] and lethal to malignant glioma cells [97], potently attenuates luminescence (shown to the right). A control plasmid expressing full-length luciferase is not affected. Likewise DHTS, which was shown by others to inhibit HuR-TNF-α ARE complex formation [124], also suppresses dimerization. The assay was then used in a high-throughput screen where a new class of dimerization inhibitors was discovered with SRI-42127 being the prototype [96].