The scaffold protein KSR1, a novel therapeutic target for the treatment of Merlin-deficient tumors

Abstract

Merlin has broad tumor-suppressor functions as its mutations have been identified in multiple benign tumors and malignant cancers. In all schwannomas, the majority of meningiomas and 1/3 of ependymomas Merlin loss is causative. In neurofibromatosis type 2, a dominantly inherited tumor disease because of the loss of Merlin, patients suffer from multiple nervous system tumors and die on average around age 40. Chemotherapy is not effective and tumor localization and multiplicity make surgery and radiosurgery challenging and morbidity is often considerable. Thus, a new therapeutic approach is needed for these tumors. Using a primary human in vitro model for Merlin-deficient tumors, we report that the Ras/Raf/mitogen-activated protein, extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) scaffold, kinase suppressor of Ras 1 (KSR1), has a vital role in promoting schwannomas development. We show that KSR1 overexpression is involved in many pathological phenotypes caused by Merlin loss, namely multipolar morphology, enhanced cell-matrix adhesion, focal adhesion and, most importantly, increased proliferation and survival. Our data demonstrate that KSR1 has a wider role than MEK1/2 in the development of schwannomas because adhesion is more dependent on KSR1 than MEK1/2. Immunoprecipitation analysis reveals that KSR1 is a novel binding partner of Merlin, which suppresses KSR1's function by inhibiting the binding between KSR1 and c-Raf. Our proteomic analysis also demonstrates that KSR1 interacts with several Merlin downstream effectors, including E3 ubiquitin ligase CRL4(DCAF1). Further functional studies suggests that KSR1 and DCAF1 may co-operate to regulate schwannomas formation. Taken together, these findings suggest that KSR1 serves as a potential therapeutic target for Merlin-deficient tumors.

Figure 1. KSR1 is overexpressed in schwannomas

Expression of KSR1 at the mRNA level in schwannoma cells (NF2−/−) and normal Schwann cells (NF2+/+) (a) and the protein level (b) Glucose-6-phosphate dehydrogenase (G6PDH) was used as control for RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control for Western blot analysis. Error bars represent the mean ±SEM. Statistical data analysis was carried out using Student’s t-test (* p<0.05; ** p<0.01, n=3 for RT-PCR; n=8 for western blotting). (c) Schwann and schwannoma cells were transfected with pcDNA3-pyo-mKSR1 and then stained with anti-KSR1 (red). DAPI (blue) was used as the nuclear marker. Scale bar = 10 μm. White arrows indicate nuclear regions. (d) Immunohistochemical staining of KSR1 in schwannomas and controls (n=15). Left panel, KSR1 staining in schwannoma tissue and adjacent nerves in a single section, scale bar = 200 μm. Middle and right panel, staining of KSR1 in schwannomas tissue and normal nerves in separated sections, scale bar = 50 μm.

Figure 2. Knockdown of KSR1 reduces ERK1/2 activity in schwannoma cells

(a) Western Blot was used to confirm the knockdown efficiency of KSR1. Schwannoma cells were infected with lentivirus based sh-control (scrambled, blue), KSR1 shRNA-A (red) and C (green), and selected with puromycin for 10 days. (b) Quantification of KSR1 knockdown. (c) The phosphorylation and total protein levels of ERK1/2, JNK1/2 and AKT were compared by western blot among sh-control, shRNA-A and shRNA-C. (d) Quantification of the activity of ERK1/2, JNK and AKT pathways after being corrected with their total protein counterpart and loading control GAPDH. Error bars represent the mean ±SEM. Statistical data analysis was carried out using Student’s t-test (ns: p>0.05; * p<0.05; ** p<0.01; *** p<0.001, n=3).

Figure 3. KSR1 regulates morphology, focal adhesion and adhesion of schwannoma cells

(a) Morphological changes of KSR1 shRNA (A and C), compared to sh-control, 7 days after transduction & selection. (b) Morphological changes in schwannoma cells treated with Sorafenib (PDGFR/Raf inhibitor, 1 μM) and U0126 (MEK1/2 inhibitor, 20 μM) for 72 hours, compared to DMSO control. Schwannoma cells (NF2−/−) were stained with Alexa Fluor® 488 Phalloidin (F-actin, cytoskeleton marker) and DAPI (nuclear marker). (c) Schwannoma cells were infected with adenovirus encoding NF2-GFP and GFP alone (48 hours). The morphology was compared by GFP staining. (d&e) Quantification of the increase in bipolar cells (manually counted) after treatment with shRNA–A, shRNA–C, Sorafenib, U0126, and NF2-GFP (Merlin reintroduction), compared to their controls. (f) Schwann cells (NF+/+) were transfected with mKSR1-WT, and KSR1 feedback-deficient mutants, mKSR1S443A and 4xA for 48 hours and stained with anti-KSR1 antibody (red) and DAPI (blue). The multipolar cells were counted manually and shown in (g). (h) sh-control and KSR1 shRNA-A and C infected schwannoma cells were stained with focal adhesion (FA) marker Paxillin (red) and DAPI (blue). (i) Quantification of focal adhesion (manually counted Paxillin stained FA sites). (J) Adhesion assay (detached cell) on sh-control and KSR shRNA-A and C transduced/selected (7 days), or DMSO/U0126 (20 μM) treated schwannoma cells. Error bars represent the mean ±SEM. Statistical data analysis was carried out using Student’s t-test (non-significant, ns, p>0.05; * p<0.05; ** p<0.01; *** p<0.001, n=3). Scale bar = 10 μm.

Figure 4. Knockdown of KSR1 reduces proliferation but enhances apoptosis

(a) Schwannoma cells were starved for 24 hours and then cultured in GFM for 72 hours. Ki67 was used as proliferation maker. Pink staining shows Ki67-positive cells. (b) Quantification of the Ki67 index after knockdown of KSR1 and treatments with DMSO/ U0126 (20 μM) in schwannoma (NF2−/−) cells. (c) Schwannoma cells were starved for 24hours then cultured in medium containing PDGF (100 ng/ml) for 72 hours. Ki67 and DPAI staining were compared among cells infected with sh-control and KSR1 shRNA-A and shRNA-C. (d) Quantification of (C). (e) Ki67 proliferation assay was performed on schwannoma cells infected with sh-control, KSR1 shRNA-c on 2, or 4 or 6 days. (f) Schwannoma cells were infected with sh-control, shKSR-C or shKSR-A+C. Caspase-Glo® 3/7 Apoptosis assay was performed. Error bars represent the mean ±SEM. Statistical data analysis was carried out using Student’s t-test (ns: p>0.05; * p<0.05; ** p<0.01; *** p<0.001, n=3).

Figure 5. KSR1 has multiple protein-protein interactions with Merlin and is strongly associated with cancer

293T cells with overexpressed pyo tagged mKSR1 and Merlin were used to immunoprecipitate their complexes, respectively. For KSR IP, non-transfected cells were used as controls. For Merlin, control IP was performed with normal rabbit IgG. Proteins were isolated and identified by LC-MS/MS and quantified by label free quantification (LFQ). The protein fold change in LFQ ratios between the IP and control group of 1.75 was used as a threshold to identify protein-binding partners. (a) The Venn diagram depicts overlap of interactors discovered in the interactomes of Merlin (blue) and KSR1 (red), respectively. The pie chart shows that the majority of shared interactors are localized to the nucleus and cytoplasm. (b) A list of the top 10 shared interactors, including VPRBP/DCAF1, DDB1 and AMOT for Merlin and KSR1. (c) The involvement of Merlin and KSR1 in the developmental disorder, hereditary disorder and cell cycle network. The uncolored nodes in the interaction network identify the molecules absent in the Merlin-KSR1 shared interactor datasets, and the colored nodes identify the molecules that were found to be enriched in both interactomes. The subnetwork of VPRBP, DDB1, HDAC1, EMD, AKP8L and CHMP1A is highlighted in a red circle. (d) IPA biological function results illustrate the enrichment in KSR1 (red) and Merlin (blue) interactomes. The shared interactors were used as representative genes/proteins. The figure was generated through the use of Ingenuity Pathway Analysis (Ingenuity® Systems, www.ingenuity.com).

Figure 6. KSR1 function is inhibited by Merlin but links to DCAF1

(a) mKSR1-WT and Merlin mutants S518A&S518D were co-expressed in 293T cells. The KSR1 complex was immunoprecipitated with Pyo/Glu-Glu Affinity Matrix and blotted with anti-KSR1, anti-Merlin, anti-MEK1/2 and anti-c-Raf antibodies. (b) Cell lysates of untransfected or mKSR1-WT transfected 293T cells were immunoprecipitated with Pyo/Glu-Glu Affinity Matrix then blotted with anti-KSR1, anti-DCAF1, and anti-MEK1/2 antibodies. (c) Co-IP was performed with anti-DCAF1 antibody in a 293T KSR1 stable line. IgG rabbit served as a control for co-IP. DCAF1, KSR1 and Merlin were detected with specific antibodies. (d) Schwannoma cells were transfected with mKSR1-WT and stained with anti-DCAF1 rabbit (green) and anti-KSR1 mouse (red). The co-localization of KSR1 and DCAF1 are shown in the merged (scale bar = 20 μm) and Z-stack picture was taken to confirm colocalisation. (e) Fractionation of KSR1, DCAF1, HDAC1 (nuclear marker) and GAPDH (cytoplasmic marker). (f) Schwannoma cells were infected with shRNA control or shRNA against DCAF1. The protein level of KSR1 and DCAF1 were detected, and GAPDH served as control. (g) Schwannoma cells were transduced with sh-control, single shRNA against either KSR1 (sh-KSR1) or DCAF1 (sh-DCAF1) or double knockdown of KSR1 and DCAF1 (sh-KSR1+DCAF1), the proliferation assays were then carried out. Error bars represent the mean ±SEM. Statistical data analysis was carried out using Student’s t-test (ns: p>0.05; * p<0.05; ** p<0.01; *** p<0.001, n=3).

Figure 7. Hypothetical model of KSR1 regulations in normal and Merlin-deficient tumor cells

In normal cells, Merlin inhibits KSR1 by restricting its localization at the cytoplasm and disturbs the binding of KSR1 and c-Raf. Merlin also limits the availability of integrin and tyrosine kinase receptors, such as PDGF receptors. In the nucleus, Merlin binds directly to the E3 ligase receptor DCAF1 and inhibits its target recruitment. Normal (Schwann) cells are therefore maintained in the quiescent state and exhibit a bipolar shape with highly organized F-actin and disassembled cell-matrix and focal adhesions. In the Merlin-deficient tumor cells, overexpressed KSR1 assembles the Raf/MEK/ERK complex upon receiving the mitogenic signal mediated by PDGF and integrin and facilitates the regulation of focal adhesions and the reorganization of F-actin linked cytoskeleton. KSR1 has MEK1/2-independent role in regulating adhesion. KSR1 together with activated MEK1/2 and ERK1/2 shuttles into the nucleus and binds to CRL4^DCAF1 to potentially regulate HDAC1 and nuclear F-actin. CRL4^DCAF1 drives oncogenic gene expression and therefore enhances the adhesion, proliferation and survival of tumor cells. ECM, extracellular matrix, Paxi: Paxillin.