Leucine-rich repeat containing 4 act as an autophagy inhibitor that restores sensitivity of glioblastoma to Temozolomide

Temozolomide(TMZ) insensitivity and resistance are major causes of treatment failure and poor prognosis for GBM patients. Here, we identify LRRC4 as a novel autophagy inhibitor that restores the sensitivity of GBMs to temozolomide.

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Leucine-rich repeat containing 4 (LRRC4), also called NGL-2 (netrin-G ligand-2), belongs to a member of the leucine-rich repeat (LRR) superfamily. We firstly cloned LRRC4 gene on human chromosome 7q31-32 (1). It is well know that LRRC4 plays a central role in early nervous system development and differentiation, especially during synapse formation. it have been shown that LRRC4 regulates auditory responses (2), excitatory synapse development, synapse maintenance and restoration in the retina (3) and autistic-like behaviours that are responsive to NMDAR modulation (4) by using lrrc4 knockout mice.

Our team has investigated the role of LRRC4 in glioma over fifteen years, since we first revealed the function of LRRC4 as a tumour suppressor for glioma (5). We have reported that LRRC4 reduced the activity of the Ras/c-Raf/ERK/MAPK signalling pathways and inhibited glioblastoma cell proliferation and invasion (6). Mechanistically, LRRC4 abolished ERK1/2 activation and inhibited ERK1/2 nuclear translocation through direct interaction with ERK1/2 and then inhibited ERK1/2 binding to MEK (7). Moreover, LRRC4 also plays an important role in the GBM immunemicroenvironment. LRRC4 bound to phosphoinositide-dependent protein kinase 1 (PDPK1) and HSP90 to promote NF-κB translocation and cytokine production in GBM cells and influenced the infiltration of Treg cells in the GBM microenvironment (8). Furthermore, to investigate the new function of LRRC4 in glioma, we used co-immunoprecipitation (CoIP) combined with mass spectrometry to identify its interaction partners. Among those molecules, DEPTOR (DEP domain containing MTOR interacting protein) was identified as a potential LRRC4 interaction partner and we demonstrated that LRRC4 binds to DEPTOR directly through the C-terminal PDZ binding domain of LRRC4 and the PDZ domain of DEPTOR. DEPTOR is a naturally occurring inhibitor of mTOR that directly binds to both mTORC1 and mTORC2 (9). DEPTOR is subject to proteasome-dependent degradation (10), and the degradation of DEPTOR contributes to mTOR activation, thus inhibiting the cell autophagy pathway. We found that LRRC4 induces the degradation of DEPTOR by directly interacting with DEPTOR, thus leads to inhibit the GBM cell autophagy. Meanwhile, it is well known that autophagy activation contribute to drug resistance. We also identified GBM cells within LRRC4 expression restores TMZ sensitivity in vitro and in vivo by inhibiting autophagy and promoting apoptosis. All in all, this study revealed a novel mechanism of LRRC4 in GBM: LRRC4, which is frequently deregulated in GBM, directly binds to DEPTOR and induces its degradation to activate mTOR, thereby inhibiting cell autophagy. Moreover, autophagy inhibition increased the treatment efficacy of TMZ in GBM, and LRRC4-expressing cells underwent increased apoptosis with TMZ treatment. Importantly, in clinical GBM samples, LRRC4 was also negatively associated with DEPTOR and LC3 expression. Combined LRRC4 expression and TMZ treatment could be an effective strategy for GBM therapy. Thus, the expression of LRRC4 is likely to have significant potential as a therapeutic marker and target for TMZ treatment in GBM patients.

References

1. Ru WJ, Qian J, Li D, Ling LX, Chen T, Jiang LI et al. Identification of LRRC4, a Novel Member of Leucine-rich Repeat (LRR) Superfamily, and Its Expression Analysis in Brain Tumor. Progress in Biochemistry & Biophysics.2002;29:233-239.

2. Zhang W, Rajan I, Savelieva KV, Wang CY, Vogel P, Kelly M et al. Netrin-G2 and netrin-G2 ligand are both required for normal auditory responsiveness. Genes Brain Behav.2008;7:385-392.

3.    Soto F, Zhao L, Kerschensteiner D. Synapse maintenance and restoration in the retina by NGL2. Elife:(c) 2018, Soto et al.2018;7.

4.    Um SM, Ha S, Lee H, Kim J, Kim K, Shin W et al. NGL-2 Deletion Leads to Autistic-like Behaviors Responsive to NMDAR Modulation. Cell Rep.2018;23:3839-3851.

5. Wu M, Huang C, Gan K, Huang H, Chen Q, Ouyang J et al. LRRC4, a putative tumor suppressor gene, requires a functional leucine-rich repeat cassette domain to inhibit proliferation of glioma cells in vitro by modulating the extracellular signal-regulated kinase/protein kinase B/nuclear factor-kappaB pathway. Mol Biol Cell.2006;17:3534-3542.

6. Li P, Xu G, Li G, Wu M. Function and mechanism of tumor suppressor gene LRRC4/NGL-2. Mol Cancer.2014;13:266

7. Wang Z, Guo Q, Wang R, Xu G, Li P, Sun Y et al. The D Domain of LRRC4 anchors ERK1/2 in the cytoplasm and competitively inhibits  MEK/ERK activation in glioma cells. J Hematol Oncol.2016;9:130.

8. Li P, Feng J, Liu Y, Liu Q, Fan L, Liu Q et al. Novel Therapy for Glioblastoma Multiforme by Restoring LRRC4 in Tumor Cells: LRRC4 Inhibits Tumor-Infitrating Regulatory T Cells by Cytokine and Programmed Cell Death 1-Containing Exosomes. Frontiers in Immunology.2017;8:1748.

9.    Peterson TR, Laplante M, Thoreen CC, Sancak Y, Kang SA, Kuehl WM et al. DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival. Cell.2009;137:873-886. 10.  Zhao Y, Xiong X, Sun Y. DEPTOR, an mTOR inhibitor, is a physiological substrate of SCF(betaTrCP) E3 ubiquitin ligase and regulates survival and autophagy. Mol Cell.2011;44:304-316.


Minghua Wu

professor, central South university

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