Co-dependent regulation of p-BRAF and potassium channel KCNMA1 levels drives glioma progression

BRAF mutations have been found in gliomas which exhibit abnormal electrophysiological activities, implying their potential links with the ion channel functions. In this study, we identified the Drosophila potassium channel, Slowpoke (Slo), the ortholog of human KCNMA1, as a critical factor involved in dRafGOF glioma progression. Slo was upregulated in dRafGOF glioma. Knockdown of slo led to decreases in dRafGOF levels, glioma cell proliferation, and tumor-related phenotypes. Overexpression of slo in glial cells elevated dRaf expression and promoted cell proliferation. Similar mutual regulations of p-BRAF and KCNMA1 levels were then recapitulated in human glioma cells with the BRAF mutation. Elevated p-BRAF and KCNMA1 were also observed in HEK293T cells upon the treatment of 20 mM KCl, which causes membrane depolarization. Knockdown KCNMA1 in these cells led to a further decrease in cell viability. Based on these results, we conclude that the levels of p-BRAF and KCNMA1 are co-dependent and mutually regulated. We propose that, in depolarized glioma cells with BRAF mutations, high KCNMA1 levels act to repolarize membrane potential and facilitate cell growth. Our study provides a new strategy to antagonize the progression of gliomas as induced by BRAF mutations. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-023-04708-9.


Introduction
BRAF is a serine/threonine protein kinase in the RAS/ RAF/MEK pathway. It plays an important role in cell proliferation and differentiation [1,2]. Abnormalities in the proto-oncogene BRAF have been found in various tumors [2][3][4][5]. BRAF V600E , the typical oncogenic mutation, accounts for about 90% of BRAF mutation tumors [6,7]. In the central nervous system, BRAF mutations are usually found in gliomas, such as the pilocytic astrocytomas, diffuse midline gliomas, and in high grade astrocytomas with piloid features [8]. In children, pediatric low-grade gliomas (pLGGs) also exhibit high rates of BRAF alteration. BRAF V600E , for example, was found in about 10-20% of all pLGGs [9][10][11][12][13]. Patients with BRAF V600E usually exhibited worse outcomes than those with non-mutated BRAF pLGG [13]. Mechanistically, BRAF gain-of-function mutations [14,15], or the activation of the MAPK pathway [16][17][18], clearly altered the electrophysiological properties of neurons. Brain slices carrying the BRAF V600E mutation showed significant increases of focally spontaneous firing rates, suggesting a relationship between BRAF V600E mutation and seizures [19]. When presented in astrocytes, BRAF V600E also led to hyper-excitable intrinsic properties such as threefold increases in action potential firing rates, low thresholds for the firing of action potentials, depolarized membrane potentials, and elevated hyperpolarization-activated inward currents [20]. Though Shanshan Xie and Chengyan Xu contributed equally. ion-channel dysfunction was strongly implicated in such observations, how ion channels specifically function in BRAF glioma progression currently remained unclear.
KCNMA1 encodes a pore-forming α-subunit of the largeconductance Ca 2+ -activated K + channel [21]. KCNMA1 mediates the out-flow of K + and is regulated by membrane depolarization and intracellular levels of Ca 2+ [22,23]. It plays multi-effect roles in many physiological processes, including membrane potential repolarization [24] and in the control of nerve excitability and neurotransmitter release [25]. Accumulating evidence suggests that K + channels may be involved in tumorigenesis in a variety of tissues [26][27][28][29][30][31]. Differential expression levels of KCNMA1 in different cancer cells may reflect multiple-related functions in tumor growth. slowpoke (slo) is the ortholog of human KCNMA1 in Drosophila [32][33][34]. It is widely expressed throughout excitable tissues where it affects action potential shape, neural excitability, transmitter release, and smooth muscle tone [35]. Studies of slo mutants have revealed the corresponding roles of K + channels in neuronal functions, circadian activity [34,36].
The Drosophila larval brain is a unique system for genetic screening identifying factors that inhibit tumor growth. Drosophila Raf (dRaf) is the only ortholog of human BRAF. Overexpression of a truncated form of dRaf (dRaf GOF ) in glial cells of the larval brains led to glioma phenotypes such as enlarged brain sizes, over-proliferation of glial cells, seizure-like behavior in adult animals, and shortened lifespans [37,38].
In this study, we employed a Drosophila Raf GOF glioma model and identified that Slo was required for tumor progression. dRaf GOF and Slo expression levels were revealed as co-dependent and mutually regulated in glioma brains. This co-dependency was recapitulated in human cells where elevated KCNMA1 was observed in gliomas with BRAF-GOF mutation. In such case knockdown of KCNMA1 reduced p-BRAF levels and inhibited cell growth. Our data suggests that this co-dependent regulation of p-BRAF and KCNMA1 levels is mediated by a membrane potential-sensitive mechanism. Our study may lead to KCNMA1 becoming highlighted as a potential drug target that could be functionallyspecific for tumors induced by BRAF mutations.

Fly seizure-like behavior test
Analysis of seizure phenotype induced by heat-shock was performed in transparent tubes with 20 flies in each tube. Flies were heat shocked at 37 °C for the given time up to 10 min. Within 10 min after treatment, each occasion a fly dropped from the tube wall and lay upside-down at the bottom was counted as one seizure-like event.

Samples from patients
Five samples were obtained following informed consent from patients. All experimental procedures were performed in accordance with the Research Ethics Board at the Children's Hospital, Zhejiang University School of Medicine. The permit to use the clinical data was approved by the institutional review boards.

Cell culture
DBTRG-05MG was purchased from the American Type Culture Collection (ATCC). T98G and U87-MG (from ATCC), were the gifts from Dr. Du Linyong. The HEK293T cell line (from ATCC) was a gift from Dr. Liang Hongqing. All cell lines were cultured in DMEM (GIBCO) at 37 °C in the presence of 5% CO 2 with 10% fetal bovine serum (GIBCO) and 1% penicillin/streptomycin (Thermo Scientific, 100 U/ml penicillin, 100 µg/ml streptomycin). All lines were routinely checked for STR identification and mycoplasma contamination.

Antibody generation
Slo cDNA encoding aa 636 to 816 was cloned into a pGEX-4T-1 expression vector. The antigen was bacterially produced, and the antibody raised in rabbits according to the published protocol [39].
The same method was used to make the dRaf antigen (aa 285-421). Anti-dRaf was generated in guinea pigs.
Three BRAF-related antibodies were used in this study: anti-BRAF (sc-5284, Santa Cruz) were used to detect the total levels of BRAF; anti-BRAF V600E specifically recognizing mutated amino acid sequence (ab228461, abcam) were used to detect the levels of BRAF V600E ; anti-p-BRAF (S729) specifically recognizing phosphorylated site (ab124794, abcam) were used to detect the levels of BRAF with serine 729 phosphorylation, to measure the BRAF activation levels regardless of the presence of BRAF mutations.

Immunofluorescence staining
For brain immunostaining, late 3rd instar larval brains were dissected in ice-cold PBS (10 mM NaH2PO4/Na2HPO4, 175 mM NaCl, pH7.4) and fixed for 15 min in PBS with 4% paraformaldehyde at room temperature. For cell immunostaining, cells on slides were fixed for 15 min in PBS with 4% paraformaldehyde at room temperature. All samples were incubated with primary antibodies at 4 °C overnight, followed by washes and mixture of the secondary antibodies at room temperature for 2 h [39,40]. All commercial secondary antibodies used were from the Jackson Laboratory. DNA was stained with DAPI (C1002, Beyotime) at 1:2000. Antifade Mounting Medium (P0126, Beyotime) was used to protect the fluorescent signals. Images were obtained using an Olympus FV1000 confocal microscope and processed with Adobe Photoshop.

Quantification of mitotic glial cells
All samples were immunofluorescent stained with primary antibody (anti-Repo, anti-PH3), and appropriate secondary antibodies. The proliferating glial cells were double-labelled by anti-Repo and anti-PH3. Mitotic glial cells were quantified by counting the total numbers of proliferating glial cells in a hemispheric lobe.

Immunohistochemistry of tissue sections
Anti-KCNMA1 was used to stain the paraffin embedded sample sections performed by the Core Facilities, Zhejiang University, School of Medicine. Images were taken using a Nikon ECLIPSE 80i microscope.

Assessment of cell proliferation by CCK8
Cells were seeded (5000/well) and maintained in 96-well plates. For the determination of cell proliferation, a Cell Counting Kit-8 (CCK8; Glpbio.GK10001) was used according to the user's manual. Fluorescence (450 nm) was recorded on a microplate reader (Biotek Synergy H1).

Statistical analysis
All data are expressed as the mean ± SD. All statistical data are processed using unpaired two-tailed Student's t test in GraphPad Prism.

Slo knockdown inhibits Drosophila Raf GOF glioma symptoms
To explore the potential roles of ion channels in tumor growth, we employed a Drosophila glioma model induced by constitutively activated Raf (dRaf GOF ) in the glial cells of larval brains. This model mimicked gliomas in patients with aberrant activations of BRAF [38,41], with enlarged brain lobe sizes as marked by eGFP driven by the glial specific repo-GAL4 (Fig. 1b). We performed knockdown screens of ion channel genes with RNAi lines available, and quantified the brain lobe diameters of the 3rd instar larvae as the primary parameter to evaluate the functions of ion channels on tumor growth. The Drosophila line with repo-GAL4, UAS-eGFP only was used as control in this study. Ameliorative phenotypes were observed for dRaf GOF glioma brains with slo knockdown (Fig. 1a, b). Two independent slo-RNAi lines targeting non-overlapping regions of slo transcript (RNAi1, THU2519.N and RNAi2, THU2246 from Tsinghua Drosophila Center) were crossed into the dRaf GOF glioma background, respectively. Glial cell-specific knockdown of slo in dRaf GOF gliomas completely prevented brain sizes ( Fig. 1a-c).
To eliminate the possibility of off-target effects in RNAi experiments, a slo genetic mutation was generated using the CRISPR/Cas9 method. The mutant harbored a frame shift mutation with a 2 bp deletion in codon 85, resulting in a truncated protein ( Fig. S1a and S1b). We named this slo mutation as slo KO . Homozygous slo KO mutant flies were viable and produced progeny, albeit with a weak locomotion defects as reported earlier [42,43]. When the homozygous slo KO was introduced into the dRaf GOF background, the brain lobe sizes of the 3rd instar larvae were significant smaller than those without such a slo KO introduction. This was consistent with the results from RNAi experiments (Fig.  S1c, Fig. 1c). This observation confirms that the slo RNAi rescue phenotype is not an off-target event. Interestingly, neither knocking down slo in glial cells of wt brains nor the homozygous slo KO animals resulted in any discernible effects on their brain sizes or total numbers of mitotic glial cells as compared with wt brains (Fig. S2a-S2c). The viabilities of slo RNAi groups were not significantly altered, neither (Fig. S2d). These findings suggest that, although Slo plays a very prominent role in dRaf GOF -induced glioma growth, its functions are largely dispensable for normal glial cell proliferation.
Recent studies have shown that the low-grade gliomas with BRAF mutations have been frequently accompanied by epileptic seizures [19,44,45]. Similar spontaneous epileptic phenotype was detected and adult animals lying upsidedown were observed in dRaf GOF glioma flies (Fig. 1d). A quick and well-established method to induce the seizure-like behavior in adult flies is to heat-shock the animals at 37 °C [46][47][48][49]. However, the dRaf GOF glioma flies exhibited seizures at a greatly reduced heat shock time as compared to control flies. Four and half minute heat-shock began inducing weak but reproducible seizure-like activities in dRaf GOF glioma flies (Fig. 1e), and the phenotype continued to be greatly enhanced from this point on. No wt flies showed any corresponding seizures (Fig. 1e). Such seizures were largely avoided by slo RNAi treatments (Fig. 1e). We also performed Kaplan-Meier survival curve analyses. Since male flies showed more pronounced survival rate changes, only male flies were used in this study. Results showed a significantly shorter median survival for dRaf GOF glioma group (17 days, P < 0.0001) as compared with the control group (64 days); while dRaf GOF glioma with slo RNAi groups showing extended median survival to 67 days and 58.5 days, respectively (Fig. 1f).

Elevated Slo expression promotes dRaf GOF glioma cell proliferation
slo encodes a pore-forming K + channel protein involved in electrophysiological processes. An antibody against the fragment of Slo from aa 636 to 816 was generated in rabbits and exhibited antigen specificity against Slo protein. Anti-Slo immunofluorescence staining showed punctate signals in glial cells of Drosophila 3rd instar larval brains (arrowheads, Fig. 2a), which was consistent with previous reports [50,51]. In order to verify Slo membrane locations, we stained cells in the larval salivary glands where the Slo punctate signals were associated with cell membranes (Fig. 2b). The Slo signals were notably denser in dRaf GOF gliomas, suggesting a higher Slo protein levels in tumor cells. Upon slo knockdown, punctate Slo signals were decreased significantly (arrowheads, Fig. 2a). The Western blot results clearly showed that the expression of Slo in dRaf GOF glioma brains was significantly higher than that in the controls (Fig. 2c), Fig. 1 Lack of Slo inhibits Drosophila Raf GOF glioma growth and rescues the glioma phenotype. a Schematic diagram of the Drosophila larval central nervous system including brain lobes (arrowhead) and ventral nerve cord (VNC). The brain lobe contains the central brain (CB) and optic lobe (OL) regions. Measured diameters of larval brain lobes are indicated by two-dotted lines. b Confocal images of the third-instar larval brains of the control, dRaf GOF glioma, and dRaf GOF glioma with slo-RNAi treatments (anti-Repo, red and eGFP, green). The enlarged brain lobes (arrow) of dRaf GOF gliomas were rescued by either of the two slo-RNAi as driven by repo-Gal4. c Statistical analysis of the diameter of the third-instar larval brain lobes of the control, dRaf GOF glioma, dRaf GOF glioma with slo-RNAi treatments, and dRaf GOF glioma with slo KO . The data are plotted as mean ± SD. ***P < 0.001. ns, not significant. d Schematic presentation of Drosophila epileptic phenotype induced by heat shock. e Frequencies of epileptic behaviors of adult flies induced by various heat shock time at 37C. n = 20. f Lifespan of the adult flies of the control, dRaf GOF glioma, and two dRaf GOF glioma with slo-RNAi treatments Fig. 2 Membrane-bound protein Slo is highly expressed in dRaf GOF glioma and associated with cell proliferation. a Confocal images of the third-instar larval brains of the control, dRaf GOF glioma, and dRaf GOF glioma with slo-RNAi treatments (anti-Slo, white; anti-Repo, red and eGFP, green). Arrowheads point to the Slo positive punctate signals. Increased numbers of Slo positive punctate signals were found in dRaf GOF gliomas and such signals were decreased with slo-RNAi treatments. b Confocal images of larval salivary gland labeled by anti-Slo (white) and DAPI (blue). c Western blot results showing protein levels of Slo as elevated in dRaf GOF gliomas as compared to controls and that such elevated protein levels were prevented by slo-RNAi treatment. α-tubulin (α-TUB) was used as internal reference. d Cross section diagram of three glial cell types in third-instar larval brains. Perineural and sub-perineural glial cells function as the brain blood barrier (BBB, between two arrows). e Confocal images of the BBB in third-instar larval brains lobes of the control, dRaf GOF glioma, dRaf GOF glioma with slo-RNAi treatments and dRaf GOF glioma with slo KO (anti-Repo, red; eGFP, green; anti-PH3, white). The distances between two arrows indicate the thickness of the BBB. The arrowheads show the mitotic glia cells double labeled by anti-Repo (red) and anti-PH3 (white). f Statistical analysis of the mitotic glia cells for each half brain lobe of the control, dRaf GOF glioma, dRaf-GOF glioma with slo-RNAi treatments and dRaf GOF glioma with slo KO . The data are plotted as mean ± SD. ****P < 0.0001. g Western blot results showing protein levels of Slo in dRaf GOF glioma and dRaf GOF glioma with slo KO . α-tubulin (α-TUB) was used as internal reference while slo knockdown resulted in a reduction of Slo expression. These results were consistent with the immunofluorescence staining data (Fig. 2a).
The superficial layer of the larval brain lobes mainly contains three glial cell types: namely perineural, subperineural and cortex glial cells. Perineural, sub-perineural glial cells collectively function as the brain blood barrier (BBB, Fig. 2d) [52,53]. Since the BBB is an easily accessible structure in Drosophila larval brains, we examined the glial cells in the BBB. In Confocal cross section images, notably thickened glial cell layers were observed in dRaf GOF glioma brains as compared with those of the wt (between two arrows, Fig. 2d, e). When slo expression was knocked down with RNAi, the thickness of glial cell layers was then restored (Fig. 2e). We assumed that the discrepancy of glial layer thickness among different samples was due to the hyper-proliferation of glioma cells [54][55][56]. Therefore, a mitosis marker, anti-PH3, was used to visualize proliferating cells. Indeed, the total numbers of proliferating glial cells double-labeled by anti-PH3 and anti-Repo were almost doubled in dRaf GOF glioma brains as compared with wt (Fig. 2e, f). However, knockdown of slo resulted in decreased numbers of these mitotic glial cells. These experiments were repeated with the slo KO line and similar results were obtained (Fig. S3, Fig. 2e-g).
To exclude the possibility that apoptosis may contribute to the decrease in total glial cell populations in the slo RNAi experiments, we stained the brains with anti-Cleaved Cas-pase3 to evaluate the potential apoptosis in slo knockdown dRaf GOF glioma brains. No apoptotic signal was detected in the control or glioma brains, or in the glioma brains with slo knockdown (Fig. S4), suggesting that the inhibition of glioma progression by slo knockdown was independent of apoptosis. Based on these data, we conclude that the elevated levels of Slo in glial cells promotes tumor progression in dRaf GOF glioma brains.

Expression of Slo and dRaf GOF are co-dependent and mutually regulated
It is known that oncogenic BRAF mutations activate downstream MEK (MEK1 and MEK2) and ERK (ERK1 and ERK2) kinases in the BRAF/MAPK pathway (Fig. 3a) [1,2]. To further probe the relationship between Slo and the BRAF/MAPK pathway, the expression levels of these proteins were examined. Western blot results showed that the total protein levels of both MEK and ERK remained unchanged, while levels of phosphorylated MEK (p-MEK) and phosphorylated ERK (p-ERK) were upregulated significantly in dRaf GOF gliomas (Fig. 3b). This suggested that the BRAF/MAPK pathway was hyper-active. However, upon slo-RNAi treatments the activity of the BRAF/MAPK pathway was suppressed, as manifested by the reduced levels of p-MEK and p-ERK (Fig. 3b). This experiment was repeated with the slo KO line and similar results were observed (Fig. 3c). This demonstrates that elevated Slo expression is required for BRAF/MAPK pathway activation in dRaf GOF gliomas and that the knockdown of slo reduces p-MEK and p-ERK levels.
We then set to explore the relationship between Slo and dRaf expressions. An antibody against the fragment of dRaf from aa 331 to 466 was generated in Guinea pigs (Fig. 3d). The dRaf GOF protein contained a truncated sequence with a smaller molecular weight of 39 kDa as compared with the full length dRaf of 88 kDa (Fig. 3d) [57]. Our immunofluorescent staining results showed that the strong dRaf signal co-localized with glial specific GFP in the cytoplasm of dRaf GOF glioma cells (Fig. 3e). Western blot data confirmed that dRaf GOF was highly expressed in glioma brains, while the expression levels of the full length wt dRaf was low (Fig. 3f). Furthermore, insufficiency of slo was accompanied by the downregulation of dRaf GOF levels (Fig. 3f, g), while the levels of full length dRaf (88 kDa) remained unchanged (Fig. 3f, g). It seemed that only truncated dRaf GOF levels had fallen upon slo knockdown. Since the expression of dRaf GOF was driven by repo-GAL4 and the total mRNA levels of dRaf showed no significant changes (Fig. 3h), the downregulation of the dRaf GOF was likely due to its degradation. This leaves it unclear as to why the full length dRaf levels remain unchanged. One possibility is that the full length dRaf is more stable than the truncated form.
We further asked whether overexpression of slo would upregulate dRaf expression in the wt brain, leading to tumorlike phenotypes. A Drosophila line with slo overexpression driven by repo-Gal4 in wt background was used in our study. Raised expression levels of Slo were confirmed by RT-PCR and Western blot (Fig. S5a, S5b). Slo overexpression resulted in not only larger brain sizes but also increased numbers of proliferating glial cells as compared with controls ( Fig. S5c-S5e). Remarkably, dRaf expression was also upregulated, without any significant changes in RNA levels (Fig. S5a, S5b). As expected, the p-MEK levels were also elevated, suggesting that over-expression of Slo promoted the glial cell proliferation via the BRAF/MAPK pathway. More importantly, it showed that elevated Slo alone was able to upregulate dRaf levels, which in turn gave rise to tumor like phenotypes. In summary, Slo protein level is elevated in dRaf GOF glioma cells. Knockdown of slo not only downregulates dRaf GOF expression but also ameliorates tumor phenotypes. Overexpression of Slo elevates dRaf expression in wt glial cells. Thus, expressions of Slo and dRaf GOF are co-dependent and mutually regulated.

KCNMA1 expression is elevated in human glioma with BRAF V600E mutation
Having established that Slo functions on dRaf GOF glioma progression in Drosophila larval brains, it is important to move on to consider if the same mechanism is represented in human patients. Since human BRAF mutations have been often detected in children, five glioma samples, classified as WHO grade I-V types, were collected from pediatric patients. Clinical data of these samples were obtained from the medical records (Table 1). We first estimated the expression of human ortholog of slo, KCNMA1, using a commercial anti-KCNMA1 antibody. The sample P1, which was diagnosed as a hair cell astrocytoma and classified as a WHO grade I glioma, exhibited high levels of KCNMA1 expression while signals from the other four samples were either low or undetected (Fig. 4a).
To validate the raised expression of KCNMA1 in this sample, Western Blot analysis was performed. As expected, the expression of KCNMA1 was highest in sample P1 (Fig. 4b), which was consistent with the immunochemical results. Remarkably, DNA sequencing data showed that the sample P1 carried the BRAF V600E , a well-studied and constitutively active form of BRAF mutation. No BRAF mutations were identified in any of the other four samples (Table 1). We then examined the status of BRAF/MAPK pathway. The extremely high level of p-MEK was reflected the hyperactivated status of the BRAF/MAPK pathway in sample P1 (Fig. 4b). These observations followed our findings in Drosophila that constitutively activated BRAF V600E promoting the ion channel KCNMA1 expression in glioma cells.

Lack of KCNMA1 suppresses the proliferation and MAPK pathway in BRAF V600E glioma cells
To study KCNMA1 function required for human glioma progression, the glioblastoma cell line DBTRG-05MG, a heterozygous mutant carrying BRAF V600E , was employed in our study together with other two glioblastoma cell lines (U87-MG and T98G) with wt BRAF as controls. U87-MG carries mutations in NF1 and PTEN, while T98G carries the mutations in PTEN and TP53 (cellosaurus.org). The phosphorylation at serine 729 (S729) of BRAF (p-BRAF) was used as a marker of activated BRAF [58,59]. Western Blot with a specific anti-p-BRAF showed that the level of p-BRAF was significantly higher in DBTRG-05MG cells than that in two control glioma cell lines (Fig. 5a). It was most likely that the protein configuration of the BRAF V600E mutation facilitated BRAF phosphorylation [60]. DBTRG-05MG also showed very high levels of p-MEK and p-ERK, indicating the hyperactive status of BRAF/MAPK pathway (Fig. 5a). Importantly, as judged by the Western blot and immunofluorescence staining, only the DBTRG-05MG cell line showed elevated KCNMA1 expression (Fig. 5a, b). KCNMA1 in mammalian cell lines was also membranebound, correlating to what had been previously observed in Drosophila (Fig. 5b).
To investigate the potential roles of KCNMA1 in the glioma cell line with BRAF V600E mutation, two shRNA targeting non-overlapping regions of KCNMA1 transcript were individually cloned into lentivirus derived vectors and introduced into the DBTRG-05MG cell line. The knockdown efficiency of these two shRNAs was confirmed by RT-PCR and Western blot (Fig. 5c, g). Ki67, a well-known proliferation marker was employed in this study. These two shRNAs showed impeded KCNMA1 expression and significantly reduced glioma cell proliferation in the DBTRG-05MG cell line as compared with the controls (Fig. 5d-f).
In order to uncover the relationship between BRAF/ MAPK activation and elevated KCNMA1 expression, we examined BRAF/MAPK components using Western Blot analyses in the DBTRG-05MG cell line. Notably in the presence of RNAi-mediated knockdown of KCNMA1, marked reductions of BRAF V600E and p-BRAF were observed (Fig. 5g), as well as significant decreases of p-MEK and p-ERK, while total MEK and ERK remained unchanged. These results indicate that a sufficient quantity of KCNMA1 is required to maintain BRAF V600E and p-BRAF levels (Fig. 5g). This is reminiscent of the Slo function in Drosophila Raf GOF glioma cells. Taken together, our data indicates that KCNMA1 expression is upregulated in human BRAF V600E glioma cells. Knockdown of KCNMA1 leads to lower levels of p-BRAF (p-BRAF V600E included), resulting in both down-regulation of cell proliferation and suppression of the BRAF/MAPK pathway. The co-dependency and Western blot analysis of the control, dRaf GOF gliomas, and dRaf GOF gliomas with slo-RNAi treatments, showing the levels of proteins in the BRAF/MAPK pathway. Total levels of MEK and ERK remained unchanged, but p-MEK and p-ERK levels had increased significantly in dRaf GOF gliomas. The elevated p-MEK and p-ERK were suppressed by slo-RNAi treatments. c Western blot analysis of dRaf-GOF gliomas and dRaf GOF glioma with slo KO also showed the same phenomenon in the BRAF/MAPK pathway. d Schematic presentations of the full-length dRaf (dRaf), truncated gain of function dRaf (dRaf GOF ), and dRaf fragment used for antigen. e Confocal images of the labeled third-instar larval brain lobes of the control and dRaf GOF glioma (eGFP, green, anti-dRaf, red and anti-Repo, white). f Western blot analysis of dRaf and dRaf GOF levels of the control, dRaf GOF glioma and dRaf GOF glioma with slo-RNAi treatments. Full-length dRaf levels did not change while dRaf GOF levels decreased upon slo-RNAi treatments. g Western blot analysis of dRaf and dRaf GOF levels of dRaf GOF glioma and dRaf GOF glioma with slo KO . h Quantitative RT-PCR showing that total dRaf and dRaf GOF mRNA levels in glioma is up-regulated compared with control, and had remained unchanged with or without slo KO . rp49 as internal reference. ****P < 0.0001, ns, not significant. α-tubulin (α-TUB) was used as internal reference for all Western blot analyses ◂  mutual regulation of KCNMA1 and p-BRAF levels seems to be therefore confirmed for BRAF V600E glioma cells in a human context.

Mutual regulation of p-BRAF and KCNMA1 levels requires channel functions
To understand the mechanism of the mutual regulation of p-BRAF and KCNMA1 levels, we addressed the question whether the KCNMA1 K + channel function was involved in this process. KCNMA1 encodes the pore-forming alpha subunit of the MaxiK channels that mediate the export of K + . Such channels are activated by membrane depolarization and an increase in cytosolic Ca 2+ [22,23]. Cromolyn sodium, an FDA-approved anti-allergy drug involving in calcium ion transport [61,62], was predicted as a potential inhibitor for KCNMA1 [63]. Correspondingly, we observed decreased cell viability in DBTRG-05MG cells upon cromolyn treatment (Fig. 6a), and unambiguous reductions of p-BRAF, BRAF V600E and p-MEK were detected in the presence of cromolyn inhibition (Fig. 6b). It is interesting to note that blockage of K + channel activity also leads to lower levels of KCNMA1, which could be due to decreased levels of p-BRAF. The result from cromolyn treatment demonstrates the co-dependency of p-BRAF and KCNMA1 levels and confirms that the K + channel function is involved in this process.
In the glioma cell line, p-BRAF activated downstream signaling pathways accompanied by higher KCNMA1 expression (Fig. 5a). However, it remained unclear if the increased KCNMA1 expression depended on the activation of the entire pathway. Therefore, we employed Dabrafenib [64], a selective inhibitor blocking MEK phosphorylation by mutated forms of BRAF kinase in our study. Cell growth assays showed that Dabrafenib significantly suppressed the proliferation of DBTRG-05MG cells (Fig. 6c). Under this condition, the high levels of p-BRAF and KCNMA1 remained unchanged and no p-MEK signal was undetected (Fig. 6d). Thus, we concluded that the mutual regulations of p-BRAF and KCNMA1 were not dependent on p-MEK.

p-BRAF and KCNMA1 levels are linked to membrane depolarization
We then wanted to know whether any co-dependency of p-BRAF and KCNMA1 levels also occurs in cells with wt BRAF. In the human embryonic kidney cell line HEK293T (wt BRAF), basal levels of p-BRAF and KCNMA1 were detectable (Fig. 7a). p-BRAF was then noted as clearly downregulated upon KCNMA1 knockdown, while the total expressions of BRAF remained unchanged (Fig. 7a) Concurrent p-MEK decreases were also observed, confirming decreased p-BRAF activity (Fig. 7a). We also raised KCNMA1 expression to see whether p-BRAF levels would follow. Indeed, the overexpression of KCNMA1 also resulted in elevated levels of p-BRAF, BRAF expression, and a corresponding increase in cell proliferation (Fig. 7b, c). These observations confirmed the co-dependency of p-BRAF and KCNMA1 levels in HEK293T cells.
It has been reported that the addition of KCl in cell culture medium depolarizes the membrane potential and activates the BRAF/MAPK pathway [65,66]. HEK293T cells were therefore treated with 20 mM KCl to induce membrane depolarization. As expected, p-BRAF levels were  (Fig. 7d) and, interestingly, the KCNMA1 protein levels were also increased (Fig. 7d). We note that the depolarization of membrane also activates KCNMA1 K + channels [22,23]. We then wanted to know whether the KCNMA1 was required for the activation of BRAF/MAPK pathway by depolarization. Knockdown of KCNMA1 in the presence of 20 mM KCl failed to elevate p-BRAF or p-MEK (Fig. 7d). This result suggests that the KCNMA1 channel is necessary for the activation of the BRAF/ MAPK pathway upon membrane depolarization induced by KCl.
We further explored the physiological significance of the co-dependency of p-BRAF and KCNMA1 regulation in HEK293T cells. The cultured cells were treated with 20 mM KCl, with untreated cells as control. Cell growth was then monitored. In the presence of KCl alone, the proliferation of HEK293T cells was almost unaffected, while with additional knockdown of KCNMA1, cell proliferation was dramatically suppressed (Fig. 7e). These results uncover the robust nature of the codependency of p-BRAF and KCNMA1 levels and suggest that both are regulated by membrane potential changes. In summary, p-BRAF levels show strong positive correlation with depolarization status. It is KCNMA1, as a K + channel, that functions to provide compensatory repolarization of the membrane which is required to maintain optimal cell growth conditions.

Discussion
In this study, we have focused on the dependency between glioma growth and ion channel function. The Drosophila Raf GOF tumor model mimics gliomas that carries BRAF mutations [38] and show corresponding phenotypes such as glial cell over-proliferation, seizure-like behavior, and shortened life span. Using the Drosophila Raf GOF glioma model, together with patient samples and cell lines, we have uncovered the mechanisms of how K + channels slo/ KCNMA1 and p-BRAF strongly enhance glioma growth.
KCNMA1 has been noted as highly expressed in glioblastomas (high grade gliomas). Inconsistent conclusions have been drawn linking the KCNMA1 levels with glioblastoma malignancy [67][68][69]. Such inconsistencies could be due to the different genetic backgrounds of these gliomas. Based on our investigation, we have concluded that KCNMA1 expression is not simply biased toward high grade gliomas, but is linked more specifically to the BRAF mutations that enhance tumor proliferation. It would be interesting to look at more samples from patients in future study to strengthen this observation.
The trans-membrane protein Slo exhibits a low expression in the wt Drosophila brain. We have shown that dRaf GOF promotes Slo expression and that the knockdown of slo brings down dRaf GOF in dRaf GOF gliomas. In addition, the overexpression of Slo in wt background also leads to an elevated dRaf level, resulting in BRAF/MAPK pathway activation. The glioblastoma cell line DBTRG-05MG with the BRAF V600E mutation [70,71] showed high levels of both p-BRAF and KCNMA1 with a correspondingly hyper-activated BRAF/MAPK pathway. The co-dependency of p-BRAF and KCNMA1 levels was therefore confirmed for this cell line. The same co-dependency of p-BRAF and KCNMA1 levels was then also recognized in the HEK293T cell line, which was derived from nontumor tissue with wt BRAF. This p-BRAF and KCNMA1 level codependency in mammalian cells was highly reminiscent of that previously observed in Drosophila Raf GOF glioma brains. Thus, we believe that the co-dependency of dRaf GOF /p-BRAF and Slo/KCNMA1 is a conserved phenomenon occurring in many different cells and across various species.
It is known that the DBTRG-05MG cells are heterozygous for BRAF V600E and contain a copy of wt BRAF [70,71]. BRAF V600E is highly phosphorylated as compared with its wt copy [6], which again resembles that observed in the Drosophila Raf GOF glioma model [38,72]. In particular, we noticed that the in slo or KCNMA1 knockdown experiments, dRaf GOF or BRAF V600E levels decreased obviously while the endogenous wt counterparts remained unchanged. Therefore, it is likely that the p-dRaf GOF or p-BRAF V600E is more susceptible for protein degradation. When the channel protein is knocked down, it is p-dRaf-GOF or p-BRAF V600E , rather than their wt counterparts that degrade quickly.
Two inhibitor experiments have uncovered interesting features of mutual regulations of p-BRAF and KCNMA1 levels in DBTRG-05MG cells. We found that the p-BRAF and KCNMA1 levels remained unaffected even when MEK phosphorylation was inhibited by Dabrafenib [64]. Thus we propose that an unknown mechanism exists to relay the signals from p-BRAF to establish the KCNMA1 levels in glioma cells, and that this occurs without the involvement of p-MEK. Furthermore, in the presence of cromolyn, K + channel activity was decreased [63], leading to lower levels of p-BRAF and diminished glioma cell viability. This result indicates that the K + channel function is involved in the mutual regulation of p-BRAF and KCNMA1 levels and suggests that the KCNMA1 channel could be a potential drug target specific for tumors that are induced by BRAF mutations.
The study here on HEK293T cells cultured with KCl provides a further clue why p-BRAF and KCNMA1 levels are codependent. Treatment of KCl leads to membrane depolarization [65,66], which in turn activates KCNMA1 channel activity [22,23]. The fact that cells are able to cope with these membrane potential changes in the presence of KCNMA1 may be due to KCNMA1 being able to compensate for depolarization by promoting repolarization of the membrane potential [25]. Correspondingly, poor cell growth was then observed when such repolarization is inhibited by KCNMA1 knockdown in these depolarized cells. Based on this, we propose that the depolarization by BRAF mutations [20] may be an unhealthy condition for cell growth and that KCNMA1 acts to counter this and repolarize the membrane potential to re-optimize conditions for these cells. In this scenario, there must be a membrane potential-mediated mechanism that co-regulates p-BRAF and KCNMA1 levels for optimal membrane potential. In this relationship, high levels of p-BRAF will therefore also require elevated expressions of KCNMA1, and vice versa. However, the exact nature of this membrane potential-mediated feedback loop remains to be elucidated.
In conclusion, our study reveals that p-BRAF and KCNMA1 levels are mutually regulated and co-dependent (Fig. 7f). In glioma cells, high levels of p-BRAF V600E leads to membrane depolarization and elevated KCNMA1 expression. Raised KCNMA1 then acts to repolarize the membrane potential and sustain p-BRAF levels, which promotes tumor cell proliferation. When KCNMA1 is knocked down, insufficient amounts of KCNMA1 are available to repolarize the membrane potential. Cell growth is therefore inhibited and p-BRAF V600E becomes degraded. Therefore, the co-dependency of KCNMA1 and p-BRAF is highlighted as critical for BRAF-mutated glioma progression and, due to this, KCNMA1 could be a valuable potential therapeutic drug target for the treatment of tumors with BRAF mutations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Fig. 7 The mechanisms of co-dependency of p-BRAF and KCNMA1. a Western blot analysis of HEK293T cell showing moderate levels of KCNMA1 and p-BRAF, and other BRAF/MAPK pathway components. shRNA1 transfections suppressed not only KCNMA1 but also p-BRAF and p-MEK. b Western blot analysis indicating that KCNMA1 overexpression (KCNMA1 OE ) in HEK293T cells leads to not only increased KCNMA1 expression, but also higher levels of BRAF, p-BRAF and p-MEK. c The proliferation rates of HEK293T cells with KCNMA1 overexpression (KCNMA1 OE ) were examined with CCK-8 assay (***P < 0.001). d In the presence of 20 mM KCl in the culture medium, levels of KCNMA1, p-BRAF and p-MEK were raised slightly. The effects of 20 mM KCl were completely blocked in the presence of KCNMA1 shRNA1. e Summary of HEK293T cells proliferation rates of control, shRNA1 treated, and subsequent treatments with or without 20 mM KCl with CCK-8 assay (ns, not significant. ****P < 0.0001). f Diagram depicts the proposed mechanism of the co-dependent regulation of p-BRAF and KCNMA1 levels. In gliomas, the oncogenic BRAF mutations lead to membrane depolarization and elevated KCNMA1 expression. Raised KCNMA1 repolarizes the membrane potential and sustains p-BRAF levels. This then promotes tumor cell proliferation (left). When KCNMA1 is knocked down, insufficient amount of KCNMA1 is then available to repolarize the membrane potential. Thus, cell growth is inhibited and p-BRAF V600E is degraded (right) ◂