Updates on Tumor Biology in Vestibular Schwannoma

Introduction

Vestibular schwannomas (VS) are benign intracranial tumors that arise from the Schwann cells of the vestibulocochlear nerve. They occur sporadically as unilateral tumors or exist bilaterally as part of an autosomal dominant tumor disposition syndrome called Neurofibromatosis Type 2 (NF2). VS can cause hearing loss, tinnitus, and dizziness, among other neurological complications¹. In this review, we summarize key pathways in VS tumor biology and the regulation of its microenvironment that contribute to tumorigenesis, tumor progression, and hearing loss.

Discussion

Tumor Biology of VS

Biallelic NF2 inactivation

The NF2 gene is located on chromosome 22q11 and encodes the tumor suppressor merlin². In NF2-associated VS, one NF2 allele is inactivated through a germline mutation¹. Tumorigenesis occurs when the other NF2 allele is lost, acquires a somatic mutation, or silenced by other means. In sporadic VS, however, a Schwann cell acquires de novo, somatic inactivation of both NF2 alleles to initiate tumorigenesis³. Following biallelic NF2 inactivation, merlin is truncated and dysfunctional or expressed at lower levels with minimal effect on intrinsic function^4,5. Truncating mutations are associated with worse disease severity in NF2 patients⁶.

Merlin Function

Merlin is a scaffold protein that links membrane receptors and intracellular effectors to regulate signaling pathways that control cell proliferation and survival². Merlin activity is modulated through conformational changes that are complex⁷. In general, unphosphorylated merlin remains in its closed conformation and acts as a tumor suppressor. When merlin is phosphorylated, it transitions to an open configuration and acts as a scaffolding protein that facilitates cell proliferation through receptor-mediated and intracellular pathways².

Receptor Tyrosine Kinases

Receptor tyrosine kinases (RTK) are plasma membrane proteins that regulate cell proliferation in VS. VS have increased expression of multiple RTKs, including: (1) hepatocyte growth factor (HGF) receptor, also known as c-MET, (2) the ERBB family of RTKs, including epidermal growth factor receptor (EGFR, also known as ErbB1/HER1), ErbB2 (HER2), and ErbB3 (HER3), (3) the platelet-derived growth factor receptor (PDGFR) family including PDGFR-α and PDGFR-β, and (4) the vascular endothelial growth factor receptor (VEGFR) family^8–13.

When unphosphorylated, merlin co-localizes with receptor tyrosine kinases and CD44 (cell surface adhesion receptor) at the plasma membrane, blocking the assembly of Ras (GTP-binding protein) complex and inhibiting downstream signaling through PI3K/Akt/JNK (phosphatidylinositol-3-kinase / Akt / c-Jun N-terminal kinase) and Raf/MEK/ERK (Raf / mitogen-activated protein kinase kinase / extracellular signal-regulated kinase) pathways (Figure 1)². Merlin can also inhibit Src tyrosine kinase-mediated activation of Raf/MEK/ERK signaling and focal adhesion kinase (FAK) signaling, promoting cell proliferation by p53 degradation through ubiquitination (Figure 1)^14,15.

Figure 1. Receptor Tyrosine Kinase Signaling.

Merlin co-localizes with RTKs and inhibits downstream Ras, SRC, and Rac1 signaling to suppress cell proliferation.

Merlin can interact with c-MET and inactivate Rac1, resulting in p21 activated kinase (PAK) inhibition^16,17. In turn, PAK inhibition down regulates Raf/MEK/ERK signaling to block cell proliferation¹⁸. In addition, PAK inhibition down regulates AuroraA and LIM domain kinases, which lead to actin stabilization and blockage of cell proliferation (Figure 1)².

VEGF binds VEGFR RTKs to promote tumor angiogenesis, supporting a microenvironment rich in nutrients and oxygen to sustain growth. VEGF expression has been correlated positively to VS tumor volume and growth¹⁹. Hypoxic VS cells can express hypoxia-inducible factor 1α (HIF-1α), stimulating VEGF expression and angiogenesis²⁰. Semaphorin 3F (SEMA3F) is a secreted protein that regulates angiogenesis. In schwannoma, merlin deficiency activates Rac1, down regulating SEMA3F and increasing angiogenesis through VEGF signaling (Figure 1)²¹.

Merlin can regulate cell survival and proliferation by inhibiting PIKE-L, a GTPase responsible for PI3K/Akt activation and downstream mammalian target of rapamycin (mTOR) signaling (Figure 2).

Figure 2. PI3K/Akt/mTOR signaling.

Merlin regulates cell proliferation by suppressing PI3K/Akt/mTOR signaling. HDAC bypasses merlin suppression by releasing PP1 inhibition on Akt.

Histone Deacetylases

Histone deacetylases (HDAC) are enzymes that remove the acetyl groups from the lysine residues of histones and non-histone substrates. HDACs regulate tumorigenesis by repressing expression of tumor suppressor genes or regulating oncogenic signaling. HDAC can bypass merlin suppression of PIKE-L and activate Akt by interacting with PP1 phosphatase and releasing PP1 inhibition of Akt (Figure 2)^2,22. In turn, Akt activates mTORC1 and downstream effects to promote cell proliferation and survival.

Hippo Pathway

Merlin is also an upstream regulator of the Hippo pathway. When merlin binds large tumor suppressor kinases (LATS1/2), mammalian Ste20-like kinases (MST1/2) can phosphorylate the LATS1/2 complex, facilitating cytoplasmic retention and degradation of the transcriptional co-activator Yes-associated protein (YAP) and its homolog TAZ. By preventing YAP/TAZ nuclear localization, TEA domain (TEAD) transcription is halted, thus blocking cell proliferation and survival². Merlin also regulates YAP cytoplasmic localization by interacting with angiomotin (AMOT), which binds YAP and regulates Rac1 signaling². In addition, merlin inhibits E3 ubiquitin ligase CRL4^DCAF1, resulting in LATS1/2 activation, YAP down regulation, and reduced cell proliferation (Figure 3)². RTKs interact extensively with the Hippo pathway, with investigations demonstrating YAP/TAZ as downstream effectors of RTK/RAS-mediated signaling through PI3K/Akt and Raf/MEK pathways. Furthermore, YAP promotes transcription of multiple genes, including amphiregulin, an EGFR ligand²³. VS have demonstrated aberrant YAP/TAZ expression, with TAZ correlating positively to tumor growth. Increased nuclear YAP correlated positively with high Ki-67 proliferative index and low merlin expression²⁴.

Figure 3. Mammalian Hippo Pathway.

Merlin regulates LATS kinases and AMOT to prevent YAP nuclear localization and transcription of cell proliferation genes.

NF-κB Signaling

Merlin also inhibits degradation of IκBα (a member of the IκB kinase family of proteins), which in turn, reduces NF-κB-dependent transcription (Figure 4). Thus, merlin inactivation promotes NF-κB, a transcription factor that regulates inflammation and cell death genes. Aberrant NF-κB signaling is a critical event in VS tumorigenesis²⁵.

Figure 4. NF-κB Pathway.

Merlin inhibits NF-κB signaling by preventing degradation of IκBα.

Thus, merlin inactivation can lead to cell proliferation and tumor progression by dysregulation of several pathways: RTKs including VEGF-VEGFR, Ras-mediated PI3K/Akt/JNK and Raf/MEK/ERK, SRC-mediated Raf/MEK/ERK and FAK, PI3K/Akt/mTOR, Rac1/ PAK, HDAC/Akt, YAP/TAZ, and NF-κB signaling pathways (Figure 5).

Figure 5. Merlin Inactivation.

Merlin inactivation leads to dysregulation of receptor-mediated and intracellular pathways that promote cell proliferation.

Tumor Microenvironment

Considerable progress has been made in understanding merlin signaling; however, less is known about the tumor microenvironment (TME) of VS. The TME is a dynamic entity, consisting of complex intercellular networks where communications between multiple cell types regulate intracellular signaling in VS (Figure 6). Yidian and colleagues performed single cell sequencing on three sporadic VS and found the TME to consist of 6 cell clusters: Schwann cells, myeloid cells, T cells, B cells, endothelial cells, and fibroblasts²⁶. Schwann cells and myeloid cells were the predominant cells. Schwann cells were heterogenous, demonstrating varying degrees of differentiation, proliferation, and immune cell chemotaxis. Myeloid cells were comprised of monocytes and dendritic cells, of which intermediate monocytes could be further differentiated into M1 and M2 phenotypic macrophages. Expression analysis also showed that myeloid cells have prominent roles in chemotaxis and cytokine-cytokine receptor signaling. Furthermore, they identified significant interactions between fibroblasts and immune cells through the chemokine CXCL (C-X-C motif ligand) pathway²⁶.

Figure 6. VS Tumor Microenvironment.

The TME consists of multiple cell types whose intercellular communications occur through cell-to-cell and paracrine signaling.

Tumor-Associated Macrophages

More recent studies in VS have focused on understanding the role of tumor-associated macrophages (TAM) on tumor growth and hearing loss²⁷. Although TAMs exist functionally along a spectrum, they are commonly classified into two broad types that can be differentiated by their surface glycoproteins and the cytokines they secrete²⁸. M1 macrophages are proinflammatory, classically activated macrophages that secrete cytokines, such as TNFα. M2 macrophages are protumorigenic, alternatively activated macrophages that secrete cytokines that promote angiogenesis and facilitate tumor growth²⁸. VS tumor growth has been associated with macrophage infiltration²⁹. Increased CD163 expression (M2 macrophage marker) correlated positively to volumetric tumor growth and microvessel density, supporting the role of M2 macrophages in angiogenesis and tumor progression^30,31. CD163 was also associated with poor hearing outcomes³².

Secretome

The secretome plays an important role in the development of the TME, tumor progression, and hearing loss in VS. Myeloid cells contribute largely to chemotaxis and cytokine signaling²⁶. VS can up regulate multiple cytokines and chemokines, including the CXC family (e.g. CXCL12 and CXCL16), IL-1β, IL-6, IL-34, macrophage colony stimulating factor (M-CSF), macrophage inflammatory protein-1alpha (MIP-1α), TNFα, and TGFβ1^31,33–36. These proteins are involved in the polarization of macrophages or can facilitate the inflammatory activities of TAMs critical in tumor formation and progression³⁷. In ex vivo investigations using cultured VS, Dilwali et al. identified fibroblast growth factor 2 (FGF2) to be otoprotective and TNFα to be ototoxic cytokines that modulate hearing in VS patients^38,39.

Furthermore, VS express other pro-inflammatory proteins, including cyclooxygenase-2 (COX-2) and the NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome^40,41. COX-2 is an enzyme important in arachidonic acid metabolism and biosynthesis of prostaglandin E2 (PGE2). PGE2 can modulate macrophage activity, including the production of other inflammatory cytokines⁴². COX-2 was aberrantly expressed in VS, and PGE2 expression correlated with cell proliferation rates in cultured VS⁴⁰. VS also increases VEGF by up regulating cyclooxygenase-2 (COX-2) through Hippo signaling⁴³. The NLRP3 inflammasome is a large cytosolic protein complex that initiates an inflammatory immune response by cleavage of the inflammatory protease procaspase-1 into its activated form, caspase-1. This process will lead to cleavage of pro-IL-1β and pro-IL-18 into their active forms. VS also upregulate NLRP3-associated genes along with macrophage infiltration, further suggesting the role of TAMs in facilitating the inflammatory response⁴¹.

Matrix Metalloproteinases

Matrix metalloproteinases (MMP) are proteases that are secreted into the extracellular space or anchored on the plasma membrane. They function to degrade extracellular matrix components and are secreted by many cell types, but particularly macrophages⁴⁴. MMP-2 was found in the cyst fluid and wall of cystic VS and may be involved in cyst formation, growth, and adhesion to surrounding structures⁴⁵. Up regulation of MMP-9 has been linked to tumor growth in VS⁴⁶. Plasma MMP-14 correlated positively to the degree of hearing loss and extent of surgical resection⁴⁷. MMP-14 may also enhance VEGF activity, contribute to collagen degradation in Antoni B areas, and promote cyst formation in VS⁴⁸.

Non-NF2 Pathways

Although NF2 gene inactivation and merlin deficiency are critical events, not all VS have identifiable NF2 mutations. Approximately 15% to 84% of sporadic VS harbor at least one NF2 mutation on genetic testing⁴⁹. In addition, not all cells in the VS tumor harbor NF2 mutations, as suggested by a variant allelic fraction ranging from 8–69% for the NF2 gene. This suggests that cellular heterogeneity (e.g., infiltration by fibroblasts and immune cells) or clonal evolution may contribute to the genetic complexity²⁶. As such, recent investigations have focused on integrative analyses to broadly assess the cellular composition, genome, epigenome, transcriptome, proteome, and secretome of VS, in an effort to better understand the TME and pathways involved in tumorigenesis, tumor progression and hearing loss (Figure 7)^50–52.

Figure 7. Biological Factors Affecting Tumor Progression and Hearing Loss in VS.

NF2 inactivation and dysregulated non-NF2 genes lead to tumorigenesis and evolution of the TME to facilitate tumor progression and hearing loss in VS.

Non-NF2 Mutations

Scientific advances with next generation sequencing have revealed non-NF2 mutations in VS, suggesting alternative or secondary mechanisms responsible for tumorigenesis. In an investigation by Havik and colleagues, forty-six VS and matched blood samples were analyzed using whole exome sequencing (WES)⁵⁰. Excluding one tumor, 716 mutations affecting 692 genes were identified, with a median of 14 genes per tumor sample. The most common mutation was NF2 (78%), followed by cell division cycle protein 27 (CDC27; 11%) and ubiquitin specific peptidase 8 (USP8; 7%). CDC27 has tumor suppressor functions, while USP8 can inhibit RTK degradation^53,54. Pathway analysis showed clustering of mutations in the axonal guidance canonical pathway, providing insight on potential non-NF2 mechanisms of tumorigenesis⁵⁰. Mutations also linked to pathways upstream and downstream of merlin, including Rac1, CD44, mTOR, and mitogen-activated protein kinase (MAPK) signaling.

Agnohitri et al. performed WES on 13 cranial and 13 spinal schwannomas and found 441 somatic single-nucleotide variants. NF2 mutations were most common (77%). Of the 13 VS, other mutations were identified including mutations of LTZR1 (leucine zipper like transcription regulator 1), ARID1A (SWI-SNF chromatin-remodeling complex), and TSC1 (tuberous sclerosis protein 1), among others. LTZR1 and ARID1A encode proteins with tumor suppressor activity^55,56. The TSC genes encodes hamartin, a protein that inhibits mTOR signal transduction⁵⁷. Pathway analysis identified enrichment of MEK, mTOR, NFκB, ErbB2, TNFα, and inflammatory signaling. Furthermore, they identified and characterized a common event occurring in several VS tumors, i.e., an in-frame fusion involving SH3PXD2A and HTRA1, arising through a balanced translocation on chromosome 10q. The authors postulate that SH3PXD2A-HTRA1 fusion causes upregulation of phosphorylated MEK to promote tumorigenesis⁵¹.

DNA Methylation

Hypermethylation of the CpG islands in the promoter regions can lead to gene silencing and is an important epigenetic mechanism causing tumor suppressor inactivation. Conversely, hypomethylation can cause upregulation of gene products. Up to 61% of VS demonstrate aberrant methylation of the NF2 gene, suggesting an alternate pathway of NF2 inactivation^58–60. Using methylation profiling, Agnihotri and colleagues identified loss of 22q as the only recurrent chromosomal aberration, found in 61% of 125 schwannomas. However, there was aberrant methylation of several non-NF2 genes, with pathway analysis demonstrating enrichment in Ras signaling, among other pathways⁵¹. Other VS studies have found aberrant methylation of genes that encode tumor suppressors, DNA repair enzymes, apoptosis initiators, MMP suppressors, and angiogenesis mediators. Some tumor suppressor genes expressing aberrant methylation were RASSF1 (Ras association domain family member 1) and PTEN (phosphatase and tensin homolog)^59,61. Torres-Martin and colleagues found a trend toward hypomethylation in VS, with upregulation of gene products confirmed for multiple hypomethylated genes, including MET proto-oncogene that encodes c-MET⁶².

MicroRNA

MiRNAs are small non-coding RNAs that are complementary to target mRNAs. They function by binding mRNA and inhibiting translation into their respective proteins. VS tumors demonstrate aberrant expression of several miRNAs, some associated with rapid tumor growth^52,63,64. In a study by Torres-Martin et al., 174 deregulated miRNAs were identified, among which global up-regulation of a miRNA cluster in the chromosomal region of 14q32 indicate a potential role in VS tumorigenesis⁵². Increased expression of miR-19, miR-21, and miR-221 were common findings, whose expression can decrease tumor suppressor PTEN in various cell types^63,65,66. Targeting miRNAs is a novel therapeutic approach to VS tumor control, and Torres-Martin et al. demonstrated that miR-21 down regulation reduced cell proliferation and induced apoptosis and autophagy in VS cells⁵².

Transcriptome

The culmination of these mutations, epigenetic modifications, and miRNA-associated transcriptional changes of NF2 and non-NF2 genes ultimately impacts the VS transcriptome. Transcriptome analysis can provide insight into dysregulated signaling pathways that lead to tumorigenesis. In brief, transcriptional analyses of VS demonstrated increased expression of: (1) several tumor suppressor genes including CAV1 and PTEN, (2) RTKs and their associated ligands, (3) downstream effectors involved with merlin (e.g. PI3K, Akt1), and (4) angiogenesis mediators, such as VEGF^{8,9,25,51,67–71}. The osteopontin gene (SPP1) which is involved in merlin protein degradation, was also upregulated⁸. Pathway analysis of dysregulated genes link to merlin-associated signaling, including EGFR, VEGFR, mTOR, and FAK, among others^25,51,71.

Summary

In summary, the mechanisms involved in VS tumorigenesis and progression is complex and involve thorough understanding of NF2 and non-NF2 signaling pathways and the dynamic interplay of cell types and secreted proteins in the TME. Understanding these molecular and cellular events will provide important insight into potential therapies for tumor control and hearing protection in VS.

Clinical Care Points

• VS tumorigenesis occurs following biallelic NF2 inactivation.

• In VS without NF2 mutations, dysregulation of non-NF2 genes can promote tumorigenesis.

• Tumor progression and hearing loss in VS are governed by VS tumor biology and the TME.

Synopsis.

Vestibular schwannomas (VS) are benign tumors that develop after biallelic inactivation of the NF2 gene that encodes the tumor suppressor merlin. Merlin inactivation leads to cell proliferation by dysregulation of receptor tyrosine kinase signaling and other intracellular pathways. In VS without NF2 mutations, dysregulation of non-NF2 genes can promote pathways favoring cell proliferation and tumorigenesis. The tumor microenvironment (TME) of VS consists of multiple cell types that influence VS tumor biology through complex intercellular networking and communications. Understanding the VS tumor biology and the TME will provide insight into therapeutic targets that mediate tumor progression and hearing loss in VS.

Key Points.

• Vestibular schwannomas (VS) arise from biallelic inactivation of the NF2 gene that encodes tumor suppressor merlin.

• Merlin inactivation causes cell proliferation by dysregulation of receptor- and non-receptor-mediated pathways.

• In VS without NF2 mutations, dysregulation of non-NF2 genes promotes cell proliferation and tumorigenesis.

• The VS TME is dynamic with multiple cell types engaging in intercellular communications that act in conjunction with VS intracellular signaling to modulate tumor progression and hearing loss.