Fermitin Family Member 2 Promotes Melanoma Progression by Enhancing the Binding of P-Α-Pix to Rac1 to Activate the MAPK Pathway

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ARTICLE OPEN Fermitin family member 2 promotes melanoma progression by enhancing the binding of p-α-Pix to Rac1 to activate the MAPK pathway

1,2,3,5 2,5 1,5 2 2 1 2 2 Shaobin Huang , Wuguo Deng , Peng Wang , Yue Yan , Chuanbo Xie , Xiaoling✉ Cao , Miao Chen✉ , Changlin Zhang ✉, Dingbo Shi2, Yunxian Dong1, Pu Cheng1, Hailin Xu1, Wenkai Zhu4, Zhicheng Hu 1 , Bing Tang 1 and Jiayuan Zhu 1

We identified fermitin family member 2 (FERMT2, also known as kindlin-2) as a potential target in A375 cell line by siRNA library screening. Drugs that target mutant BRAF kinase lack durable efficacy in the treatment of melanoma because of acquired resistance, thus the identification of novel therapeutic targets is needed. Immunohistochemistry was used to identify kindlin-2 expression in melanoma samples. The interaction between kindlin-2 and Rac1 or p-Rac/Cdc42 guanine nucleotide exchange factor 6(α-Pix) was investigated. Finally, the tumor suppressive role of kindlin-2 was validated in vitro and in vivo. Analysis of clinical samples and Oncomine data showed that higher levels of kindlin-2 predicted a more advanced T stage and M stage and facilitated metastasis and recurrence. Kindlin-2 knockdown significantly inhibited melanoma growth and migration, whereas kindlin-2 overexpression had the inverse effects. Further study showed that kindlin-2 could specifically bind to p-α-Pix(S13) and Rac1 to induce a switch from the inactive Rac1-GDP conformation to the active Rac1-GTP conformation and then stimulate the downstream MAPK pathway. Moreover, we revealed that a Rac1 inhibitor suppressed melanoma growth and metastasis and the combination of the Rac1 inhibitor and vemurafenib resulted in a better therapeutic outcome than monotherapy in melanoma with high kindlin-2 expression and BRAF mutation. Our results demonstrated that kindlin-2 promoted melanoma progression, which was attributed to specific binding to p-α-Pix(S13) and Rac1 to stimulate the downstream MAPK pathway. Thus, kindlin-2 could be a potential therapeutic target for treating melanoma.

Oncogene (2021) 40:5626–5638; https://doi.org/10.1038/s41388-021-01954-8

INTRODUCTION effect of BRAFV600E inhibitors is limited; moreover, acquired Melanoma is one of the most malignant cutaneous cancers. Its resistance to MAPK-targeted therapy results in failure to respond occurrence has increased in the past several decades, especially in to anti-PD-1 therapy. Thus, finding a combination of molecular the Western world. Melanoma with early metastasis can develop targeted therapies that more successfully inhibit the MAPK rapidly and eventually cause death [1–4]. For advanced or signaling pathway is essential [10, 15, 16]. Our previous siRNA recurrent melanoma, surgery alone does not have a good library screening identified numerous new proteins implicated in therapeutic effect; thus, other therapeutic options, including melanoma progression [17]. Among these proteins, fermitin family radiotherapy, immunotherapy, immune checkpoint inhibitor member 2 (FERMT2, also known as kindlin-2) was selected for therapy, and molecular targeted therapy, are needed [5–9]. PD-1 functional evaluation in melanoma growth and metastasis. immune checkpoint blockade therapy has already been used in Kindlin-2, is a member of the kindlin family, which contains three melanoma; however, advanced metastatic melanoma exhibits a members that have a highly conserved FERM domain and usually high rate of innate resistance (60–70%) to this treatment [10]. A function to link the cell membrane to the cytoskeleton and as high percentage of melanomas harbor BRAF mutations and RAS molecular linkers [18]. Recently, some studies have discovered that mutations, which affect the downstream MAPK pathway to kindlin-2 mutation or dysregulation can promote the develop- influence melanoma progression [11, 12]. These results provide ment of certain cancers, including breast cancer, hepatocellular reasonable evidence to support combination therapy with the carcinoma (HCC), esophageal cancer, prostate cancer, gastric BRAF inhibitor dabrafenib and the MEK inhibitor trametinib, and cancer and glioma progression [19–25]. In our study, we have this combination has been proven to improve survival in patients explored the potential molecular mechanisms by which kindlin-2 with BRAF V600E/K mutations [13, 14]. Because of acquired regulates the growth and metastasis of melanoma and, more resistance to BRAFV600E inhibitors, the long-lasting therapeutic importantly, evaluated its clinical significance to identify an

1The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. 2Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China. 3The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. 4Department of Chemistry, Portland State ✉ University, Portland, OR, USA. 5These authors contributed equally: Shaobin Huang, Wuguo Deng, Peng Wang. email: [email protected]; [email protected]; [email protected] Received: 17 August 2020 Revised: 6 June 2021 Accepted: 9 July 2021 Published online: 28 July 2021 S. Huang et al. 5627 improved option for melanoma treatment. Our results demon- Table. 1. Correlation analyses of kindlin-2 protein expression in strated that kindlin-2 knockdown inhibited cell proliferation and relation to clinicopathologic variables. metastasis by hindering the binding of Rac1 and p-α-Pix; thus, Rac1 could not be activated to promote MAPK pathway signaling. Clinical factor Kindlin-2 expression high Low p Rac1 is a member of the typical Rho guanosine triphosphate Gender phosphohydrolase (GTPase) family and can cycle between an active, guanosine triphosphate (GTP)-bound conformation and an Male 35 13 0.1090 inactive, guanosine diphosphate (GDP)-bound conformation. This Female 19 15 process is regulated by guanine nucleotide exchange factors Age (GEFs), which lead to Rac1 activation, and by GTPase-activating <53 26 18 0.1646 α proteins (GAPs), which inactivate Rac1 [26]. -Pix is a GEF that can ≥ activate p21 Rac1 and Cdc42 but not RhoA [27]. We discovered 53 28 10 that high kindlin-2 expression can promote the binding of p-α-Pix Location to Rac1 and then activate the MAPK pathway. Recent studies Body 26 11 0.4444 showed that kindlin-2 promoted tumor growth and progression The Four limbs 28 17 [28, 29]. In our study, we confirmed the role of kindlin-2 in melanoma progression, and also discovered that Rac1 inhibition T could hinder the growth and metastasis of melanoma. Moreover, T1/T2 6 10 0.0077* the combination of a Rac1 inhibitor and vemurafenib (a drug that T3/T4 48 18 fi targets mutant BRAF kinase but lacks long-lasting ef cacy because N of acquired resistance [30]) suppressed melanoma growth and metastasis to a higher degree than either agent as monotherapy. N0/N1 51 27 0.6925 Thus, we identified kindlin-2 as a potential therapeutic target N2/N3 3 1 for melanoma. M M0 45 28 0.0245* RESULTS M1 9 0

1234567890();,: Association between kindlin-2 expression and TNM clinicopathological features of melanoma patients I/II 38 25 0.0602 We screened an siRNA library targeting 46,000 human genes in III/IV 16 3 fi A375 melanoma cells. Kindlin-2 knockdown by siRNA signi - fi cantly suppressed cell viability by 59.22% (P = 0.000188), *P < 0.05, statistically signi cant. indicating that kindlin-2 might be a potential target in melanoma. Then, the relationship between the clinicopathological characteristics of melanoma patients and kindlin-2 protein proliferation of melanoma cells and promoted colony formation expression was analyzed. Among the 82 patients with melanoma, (Fig. 1E, F). Flow cytometric analysis demonstrated that the 54 showed strong kindlin-2 expression, 28 showed weak kindlin- reduction in kindlin-2 protein expression led to an increased rate 2 expression, and 28 showed negative kindlin-2 expression. The of apoptosis in the melanoma cell lines MeWo and WM35 (Fig. 1G). proportion of scores and representative examples of kindlin-2 Collectively, these results indicate that kindlin-2 plays a key role in expression in melanoma tissues are shown in Figure S1. The data mediating melanoma cell proliferation and growth. in Table 1 indicate that a high kindlin-2 expression level might be related to T3-T4-stage (P <0.05) and M1-stage (P <0.05) Kindlin-2 influenced the cell cycle in melanoma cells disease. However, no evidence of an obvious association Compared with the control groups, groups with kindlin-2 between kindlin-2 protein expression and sex (P = 0.1090), age suppression exhibited an increased proportion of G1 phase cells (P = 0.1646), tumor location (P = 0.4444), N stage (P = 0.6925) or but a decreased proportion of S phase and G2 phase cells. In TNM stage (P = 0.0602) was observed. contrast, kindlin-2 overexpression led to a decrease in the proportion of G1 phase cells and an increase in the proportion Transcription levels of kindlin-2 in patients with melanoma of S phase and G2 phase cells. Thus, kindlin-2 knockdown induced The Oncomine database suggested that patients with metastasis G1 phase cell cycle arrest, while kindlin-2 overexpression had higher kindlin-2 mRNA levels than those without metastasis promoted cell division (Fig. 2A). (Fig. S2A). High kindlin-2 mRNA expression was found to be correlated with M1-stage disease (Fig. S2B). In addition, kindlin-2 Kindlin-2 knockdown inhibited the migration but not the mRNA levels were significantly increased in patients with invasion potential of melanoma cells melanoma recurrence (Fig. S2C). After interference, the MeWo and WM35 cell lines exhibited an apparent decrease in migration but not invasion compared with Kindlin-2 promoted the proliferation of melanoma cells the control cells (P < 0.05), while the A375 and A875 cell lines First, we determined the expression levels of kindlin-2 in human showed increased migration but not invasion after kindlin-2 melanoma cell lines (A375, A875, MeWo, WM35, SK-Mel-2, and SK- overexpression (Fig. 2B-D). The results of anoikis assays showed Mel-28) by Western blotting (Fig. 1A). Kindlin-2 expression was that the anoikis rates were significantly increased in shkindlin-2 detected in the cytoplasm in different cell lines (Fig. 1B, C). Then, cells (MeWo and WM35) in suspension, kindlin-2 overexpression we suppressed kindlin-2 expression in the MeWo and WM35 cell markedly reduced the anoikis rates in the A375 and A875 cell lines lines and overexpressed kindlin-2 in the A375 and A875 cell lines. (Fig. 2E). Analysis of the epithelial markers E-cadherin and ZO-1 Kindlin-2 knockdown via specific shRNAs suppressed its expres- and the mesenchymal markers N-cadherin and vimentin revealed sion, whereas kindlin-2 overexpression significantly increased its that kindlin-2 overexpression reduced E-cadherin and ZO-1 expression (Fig. 1D). Kindlin-2 knockdown obviously inhibited expression and increased N-cadherin and vimentin expression. the proliferation of melanoma cells and resulted in a marked However, kindlin-2 knockdown resulted in the inverse effects decrease in the colony formation rates of MeWo and WM35 cells. (Fig. 2F). Then, we knockdowned the expression of E-cadherin and In contrast, kindlin-2 overexpression dramatically increased the ZO-1 by siRNA, respectively, the expression of kindlin-2 had no

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Fig. 1 Kindlin-2 promoted the proliferation of melanoma cells. A Western blot analysis of the expression of kindlin-2 in various melanoma cell lines. B Immunofluorescence analysis of the expression and localization of kindlin-2 in various melanoma cell lines. C Immunohistochemical analysis of the localization of kindlin-2 in melanoma samples. D The kindlin-2 expression level was measured by western blotting in melanoma cells with kindlin-2 knockdown or overexpression. E The viability of melanoma cells with kindlin-2 knockdown or overexpression was measured by a CCK-8 assay. F Colony formation assay in melanoma cells following kindlin-2 knockdown or overexpression. G Apoptosis in melanoma cells with kindlin-2 knockdown was detected by fluorescence-assisted cell sorting (FACS) analysis, and the relative percent of apoptotic cells was calculated. The data are presented as the means ± SDs of three independent tests. *P < 0.05, **P < 0.01, ***P < 0.001, significant differences between the treatment groups and control groups.

change (Fig. S3). That is, kindlin-2 can mediate cell-matrix spectrometry in A375 cells with an anti-kindlin-2 antibody. Rac1, a adhesion itself, also it can inﬂuence cell-cell adhesion by affecting member of the Rac subfamily of Rho family small GTPases with the expression of E-cadherin and ZO-1. strong evidence of its dysregulation in cancer, was found to be a candidate kindlin-2-interacting protein. Moreover, α-Pix, a GEF Kindlin-2 bound independently to Rac1 and α-Pix (activator) of the Rho family small GTP-binding protein family To identify the proteins that interact with kindlin-2 in melanoma members Rac1 and Cdc42, was also found to be a candidate cells, we performed immunoprecipitation combined with mass (Figs. S4, S5). The interaction between kindlin-2 and Rac1 and the

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Fig. 2 Kindlin-2 influenced the cell cycle and the migration but not the invasion of melanoma cells. A The percentage of melanoma cells in each phase was quantified. B The migration ability of melanoma cells with kindlin-2 overexpression or knockdown was assessed by measuring the average gap width (μm) in a wound-healing assay. C The migration ability of melanoma cells with kindlin-2 overexpression or knockdown was measured by a Transwell assay. D The invasive ability of melanoma cells with kindlin-2 overexpression or knockdown was measured by a Transwell assay. E Anoikis in melanoma cells with kindlin-2 overexpression or knockdown was measured by FACS analysis, and the relative percent of apoptotic cells was calculated. F Levels of epithelial-mesenchymal transition (EMT) markers in melanoma cells with kindlin-2 overexpression or knockdown were measured by Western blots. The data are presented as the means ± SDs of three independent tests. *P < 0.05, **P < 0.01, ***P < 0.001, significant differences between the treatment groups and control groups.

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Fig. 3 Kindlin-2 bound independently to Rac-1 and α-Pix. A The interactions between kindlin-2 and Rac1 and between kindlin-2 and α-Pix were confirmed by co-IP in melanoma cells. B The interactions between kindlin-2 and Rac1 and between kindlin-2 and α-Pix were confirmed by colocalization as observed by confocal microscopy in melanoma cells. C kindlin-2 binding to Rac-1 and α-Pix in vitro, respectively, using the GST pull-down assay. D Binding affinities of Rac-1 and α-Pix for the kindlin-2 determined via Isothermal Titration Calorimetry (ITC). E The interaction between Rac1 and α-Pix was confirmed by co-IP in melanoma cells with kindlin-2 overexpression or knockdown.

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interaction between kindlin-2 and α-Pix were further confirmed by revealed that kindlin-2 can directly bind to Rac-1 and α-Pix, co-IP and immunofluorescence colocalization analyses in different respectively (Fig. 3C). Also, we utilized isothermal titration calori- melanoma cell lines. The results showed that kindlin-2 and Rac1 metry to quantitatively measured the affinity the affinities of kindlin- were mainly localized in the cytosol and α-Pix was localized in the 2forRac-1andα-Pix. Kindlin-2 was found to bind to Rac-1 with Kd cytoplasm. Rac-1 and kindlin-2, α-Pix and kindlin-2 were co-localized = 14.9 ± 4.2 μM. The Kd value of kindlin-2 and α-Pix is 21.6 ± 3.9 µM in the cytoplasm (Figs. 3A, B, S6). In addition, GST pull-down assay (Fig. 3D). To verify whether kindlin-2 influenced the binding of Rac1

Oncogene (2021) 40:5626 – 5638 S. Huang et al. 5632 Fig. 4 Kindlin-2 activated the MAPK pathway by promoting the binding of Rac-1 to p-α-Pix. A A magnetic bead pulldown assay of Rac1 activity was conducted, and western blotting was used to assess α-Pix expression in melanoma cells with kindlin-2 overexpression or knockdown. B Proteins in the MAPK pathway were assessed by western blotting in melanoma cells with kindlin-2 overexpression or knockdown. C The α-Pix expression level was assessed by western blotting in melanoma cells with α-Pix knockdown. D The magnetic bead pulldown assay for Rac1 activity and western blotting showed the levels of proteins in the MAPK pathway. E The interaction between kindlin-2 and α-Pix was confirmed by co-IP in melanoma cells after treatment with alkaline phosphatase. F A magnetic bead pulldown assay for Rac1 activity was conducted in A375 cells expressing α-Pix with individual mutations of the S13, S19, T9 and T23 phosphorylation sites. G The interaction between kindlin-2 and α-Pix was confirmed by co-IP in A375 cells expressing α-Pix with individual mutations of the S13, S19, T9, and T23 phosphorylation sites. H, I Binding model for kindlin-2-328-499 and α-Pix-4-111. The backbone of α-Pix-4-111 is depicted in light blue. The residues in α-Pix-4-111 are colored in orange. The backbone of kindlin-2-328-499 is depicted in gray. The residues in kindlin-2-328-499 are colored green. The binding domain was confirmed by the direct mutual interaction of p-α-Pix (S13) with different domains of flag-tagged kindlin-2. J, K Binding model between kindlin-2-1-105 and rac1. The backbone of rac1 is depicted in light blue. The residues in rac1 are colored orange. The backbone of kindlin-2-1-105 is depicted in gray. The residues in kindlin-2-1-105 are colored green. The binding domain was confirmed by the direct mutual interaction of Rac1 with different domains of flag-tagged kindlin-2.

and α-Pix, we overexpressed the kindlin-2 protein and found pull down kindlin2 (Fig. S8A). After interference, the expression that the binding of Rac1 and α-Pix strengthened, while suppres- of phosphorylation of the JNK and p38 proteins were sing kindlin-2 expression weakened the binding of Rac1 and α-Pix suppressed when the Rac1 fragment containing residues 178- (Fig. 3E). 192, but the levels of kindlin2, p-α-Pix, JNK and p38 were barely affected (Fig. S8B). After the residues 1-177 of Rac1 was deleted, Kindlin-2 activates the MAPK pathway by promoting the cell growth and cell migration were suppressed, while cell binding of Rac1 to p-α-Pix apoptosis was promoted (Fig. S8C, D, E). We analyzed the activation of Rac1 and found that Rac1 activation was significantly increased after kindlin-2 overexpres- Suppression of RAC1 reversed the overexpression of kindlin-2 sion in the A375 and A875 cell lines but substantially decreased 1A-116, an inhibitor of RAC1, was used to treat A375 cells. The after kindlin-2 suppression in the MeWo and WM35 cell lines. We appropriate dose and treatment time of 1A-116 were 5 µg/ml and also conducted integrin activity assays, and found that over- 12 h, respectively (Fig. S9). After expression interference, the expression of kindlin-2 in A375 cells could promote the integrin expression of RAC1-GTP and the phosphorylation of the JNK and activity, but the GTP-Rac1 level cannot be affect by the inhibition p38 proteins were suppressed, but the levels of total RAC1, JNK of integrin (Fig. S7). As the downstream effect of Rac1-GTP is and p38 were barely affected. Because A375 cells harbor the BRAF MAPK pathway activation, Western blotting was carried out to mutation, therefore, the ERK pathway is constitutively activated at detect alterations in the MAPK pathway. Kindlin-2 knockdown in the high level of the MAPK, irrespective of Rac1 activity (Fig. 5A). MeWo dramatically suppressed the phosphorylation of the JNK, Then, we discovered that 1A-116 blocked p-α-Pix interaction of p38, and ERK proteins, but had nearly no effect on the levels of RAC1 (Fig. S10). Moreover, in the CCK-8 and colony formation total JNK, p38, and ERK, while in WM35 melanoma cells assays, inhibition of Rac1 markedly suppressed the proliferation (harbored the BRAF mutations) with kindlin-2 knockdown, the and growth of A375 cells (Fig. 5B, C). Flow cytometric analysis phosphorylation of the JNK and p38 proteins were decreased, demonstrated that inhibiting Rac1 activation led to an increased but had nearly no effect on the levels of total JNK and p38. rate of apoptosis (Fig. 5D). The proportion of G1 phase cells was In contrast, kindlin-2 overexpression in A375 and A875 cells increased after Rac1 inhibition, while that of S phase and G2 phase significantly increased the phosphorylation levels of the JNK, cells was decreased (Fig. 5E). After treatment with 1A-116, p38, and ERK proteins but, similarly, did not change the levels of melanoma cells exhibited an apparent weakening of migration total JNK, p38, and ERK. These findings indicate that the MAPK but not invasion (Figs. 5F, G, S11). The results of the anoikis assays signaling pathway is likely involved in the kindlin-2-mediated showed that the anoikis rate was significantly increased after the promotion of melanoma growth and metastasis. However, the application of 1A-116 (Fig. 5H). expression of α-Pix was not significantly altered (Fig. 4A, B). Thus, we knocked down α-Pix by siRNA in A375 cells (Fig. 4C). After Kindlin-2 knockdown and RAC1 inhibition suppressed interference, Rac1 activation was markedly decreased; moreover, melanoma growth in a mouse xenograft model the phosphorylation levels of the JNK and p38 proteins were Kindlin-2 knockdown inhibited tumor growth, while kindlin-2 decreased, but the levels of total JNK and p38 were barely overexpression promoted tumor growth; however, this promotion affected (Fig. 4D). To determine whether kindlin-2 can bind only was reversed by the RAC1 inhibitor. Daily treatment of mice with p-α-Pix, alkaline phosphatase was used to dephosphorylate α- compound 1A-116 at a dose of at least 3 mg/kg body weight/day Pix. After phosphatase treatment, the binding of kindlin-2 to α- markedly reduced tumor formation. In addition, none of these Pix was abrogated in melanoma cell lines (Fig. 4E). To investigate treatments significantly affected the body weight of the mice, and the binding mode of kindlin-2 to Rac1 as well as to p-α-Pix, no other signs of acute or delayed toxicity were observed in the docking simulation studies were carried out. First, phosphoryla- mice during treatment (Fig. 5I-Q). In addition, Western blot tion sites on α-Pix and the binding domain of p-α-Pix on kindlin- analysis of cell lysates from the xenografted tumor tissues showed 2 were predicted (Table S1, Table S2). To identify the that silencing kindlin-2 in mice inoculated with kindlin-2- phosphorylation sites on α-Pix, the predicted phosphorylation knockdown tumor cells led to decreased phosphorylation levels sites (S13, S19, T9, and T23) were individually mutated in A375 of the JNK and p38 proteins, while kindlin-2 overexpression led to cells. Unlike wild-type (WT) α-Pix, α-Pix S13A failed to activate increased phosphorylation levels of the JNK and p38 proteins; Rac1 and interact with kindlin-2 (Fig. 4G, G). To discover the however, this increase was inhibited by 1A-116 (Fig. S12). binding domains on kindlin-2 for p-α-Pix and Rac1, kindlin-2 truncation mutants were constructed. The kindlin-2 fragment Kindlin-2 knockdown and RAC1-GTP inhibition suppressed containing residues 328-499 pulled down p-α-Pix (Fig. 4H, I), and melanoma metastasis in a mouse model of metastatic the fragment containing residues 1-105 pulled down Rac1 melanoma (Fig. 4J, K). Moreover, when the residues 1-177 of Rac1 was We found that compared to control animals, mice that received deleted, The Rac1 fragment containing residues 178-192 cannot kindlin-2-knockdown tumor cells (MeWo) exhibited a weakened

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metastatic ability and fewer lung metastatic nodules (Figs. 6A–E, Combination of the Rac1 inhibitor and vemurafenib S13A). However, the metastatic ability was strengthened, strengthened the therapeutic effect of each monotherapy in and the number of lung metastasis nodules was substantially melanoma increased in mice injected with kindlin-2-overexpressing A375 The A375 cell line in our study harbors a BRAF mutation, and we cells; moreover, daily treatment of the mice with compound combined vemurafenib (20 mg/kg) and the Rac1 inhibitor to treat 1A-116 at a dose of at least 2 mg/kg body weight/day melanoma [31]. Compared with vemurafenib or Rac1 inhibitor signiﬁcantly reduced the formation of total metastatic lung treatment alone, the combination of the Rac1 inhibitor and colonies (Figs. 6F–J, S13B). vemurafenib signiﬁcantly reduced melanoma growth (Fig. 7A–D).

Oncogene (2021) 40:5626 – 5638 S. Huang et al. 5634 Fig. 5 Suppression of RAC1 reversed the effect of kindlin-2 overexpression, kindlin-2 knockdown and RAC1 inhibition suppressed melanoma growth. A After the overexpression of kindlin-2 or the treatment of 1A-116 with kindlin-2 overexpression, a magnetic bead pulldown assay of Rac1 activity was conducted, and western blotting was used to assess the expression of proteins in the MAPK pathway. B The viability of A375 cells after the overexpression of kindlin-2 or the treatment of 1A-116 with kindlin-2 overexpression was measured by a CCK-8 assay. C Colony formation assay after the overexpression of kindlin-2 or the treatment of 1A-116 with kindlin-2 overexpression in A375 cells. D Apoptosis in A375 cells after the overexpression of kindlin-2 or the treatment of 1A-116 with kindlin-2 overexpression was detected by FACS analysis and the relative percent of apoptotic cells was calculated. E The percentage of A375 cells in each phase was quantified after the overexpression of kindlin-2 or the treatment of 1A-116 with kindlin-2 overexpression F The migration ability of A375 cells after the overexpression of kindlin-2 or the treatment of 1A-116 with kindlin-2 overexpression was assessed by measuring the average gap width (μm) in a wound-healing assay. G The migration ability of A375 cells after the overexpression of kindlin-2 or the treatment of 1A-116 with kindlin-2 overexpression was measured by a Transwell assay. H Anoikis of A375 cells after the overexpression of kindlin-2 or the treatment of 1A-116 with kindlin-2 overexpression was measured by FACS analysis, and the relative percent of apoptotic cells was calculated. I-K The morphology, weight and volume of the tumor from each mouse at sacrifice (MeWo cells with or without kindlin-2 knockdown). L The tumor volume in each mouse was measured and recorded every three days throughout the course of the experiment MeWo cells with or without kindlin-2 knockdown). M–O The morphology, weight and volume of the tumors from each mouse at sacrifice (A375 cells with or without kindlin-2 overexpression or with the treatment of 1A-116 at different doses after kindlin-2 overexpression). P,Q The tumor volume and body weight of each mouse were measured and recorded every three days throughout the course of the experiment (A375 cells with or without kindlin-2 overexpression or with the treatment of 1A-116 at different doses after kindlin-2 overexpression). The data are presented as the means ± SDs of three independent tests. **P < 0.01, ***P < 0.001, significant differences between the treatment groups and control groups.

In addition, none of these treatments, whether monotherapy or phosphorylated at other sites—most likely, α-Pix can bind to combination therapy, significantly affected the body weight of the kindlin-2 only when the conformation of α-Pix transitions after mice (Fig. 7F). Regarding the metastatic capacity, the inhibitory phosphorylation at S13. Moreover, we revealed that the effect in the combination treatment group was better than that in fragment of kindlin-2 containing residues 328-499 can bind to the vemurafenib treatment group (Figs. 7F-J, S14). p-α-Pix and that the fragment of kindlin-2 containing residues 1-105 can bind to Rac1. In addition, our clinical data and Oncomine database strengthened the evidence that higher DISCUSSION kindlin-2 expression predictsmoreadvancedTstageandM Recently, progress has been made in understanding the stage and facilitates metastasis and recurrence. However, it structure and biological functions of the kindlin-2 protein. made more sense to compare the expression of kindlin-2 in the Certain cancers, such as breast cancer, HCC, esophageal cancer, N stages or M stages within the stage III and IV sub-cohorts. prostate cancer and gastric cancer, as well as glioma, have been Therefore, more detailed patient information was needed for found to be related to kindlin-2 dysfunction. The responsible analysis. mechanisms include the following: enhancing cell proliferation To the best of our knowledge, this study is the first to and migration by stabilizing DNMT1 or driving cancer progres- document that kindlin-2 plays a role in the growth and sion by activating the TGF-β/CSF-1 signaling axis in breast metastasis of melanoma by strengthening the binding of Rac1 cancer, promoting invasion and metastasis by activating Wnt/ and p-α-Pix (S13) and subsequently stimulating the downstream β-catenin signaling in HCC, activating the β-catenin/YB-1/EGFR MAPK pathway. Furthermore, we attempted to determine the pathway to promote progression in glioma, facilitating inva- clinical therapeutic significance of our findings.First,theRac1 siveness via the NF-κB pathway to upregulate MMP-9 and MMP- inhibitor 1A-116 was used to treat kindlin-2-overexpressing 2 expression in prostate cancer, and accelerating invasion in A375 cells, and this treatment greatly reversed the effects of gastric cancer by phosphorylating integrin β1andβ3[19–25]. kindlin-2 overexpression on melanoma cell growth and metas- In our study, we found that kindlin-2 can affect the proliferation tasis. In animal studies, further evidence was obtained to and migration of melanoma cells, which was consistent with demonstrate that kindlin-2 knockdown can dramatically sup- some research. However, our results did not discover that press the growth and metastasis of melanoma. However, kindlin-2 affected melanoma cell invasion, considering that this kindlin-2 overexpression definitely promoted the growth and was related to slight differences in the grouping and statistical metastasis of melanoma, but this promotive effect was hindered methods of the experiments. Many physiological processes, by the Rac1 inhibitor. including mesenchymal stem cell differentiation, podocyte The RAS-RAF-MEK-ERK pathway is a kinase cascade that morphology acquisition, cell spreading, vascular barrier func- controls multiple vital cellular processes, such as cell cycle tion, cardiac function and chondrogenesis, are affected by progression, survival and migration [38, 39]. Drugs targeting this kindlin-2 mutation or dysregulation [32–37]. In malignant pathway have achieved effective outcomes in the treatment of melanoma. kindlin-2 was identified as a candidate target from certain cancers with BRAF mutations, including colorectal cancer, an siRNA library screen, and we found that in melanoma, hairy cell leukemia, melanoma, thyroid cancer, non-small cell kindlin-2 knockdown inhibited cell proliferation, promoted lung cancer, etc. [40–44]. Vemurafenib was discovered as a apoptosis, and suppressed growth and metastasis, while highly specific BRAFV600 kinase inhibitor and produced a kindlin-2 overexpression resulted in the inverse effects. Further notable response in advanced melanoma patients with the analysis showed that kindlin-2 overexpression promoted BRAFV600 mutation [45–47]. However, tumor resistance occurs the phosphorylation of proteins (p-p38, p-JNK, and p-ERK) in rapidly in some patients; unfortunately, searching for the the MAPK pathway; the responsible molecular mechanism is appropriate combination therapy for melanoma remains challen- the strengthening of Rac1 and p-α-Pix (S13) binding mediated ging, and widespread metastasis is acknowledged as the main by high kindlin-2 expression. Then, the inactive, GDP-bound cause of death in melanoma patients [48, 49]. In our research, Rac1 conformation undergoes a switch to the active, GTP-Rac1 kindlin-2 was found to enhance the binding of Rac1 and α-Pix conformation and stimulates the downstream MAPK pathway, and subsequently stimulate the MAPK pathway, and the Rac1 which leads to the growth and metastasis of melanoma. inhibitor was proven to significantly affect the growth and, more Importantly, we discovered that kindlin-2 can bind to only importantly, the metastasis of melanoma both in vivo and p-α-Pix (S13) and not to unphosphorylated α-Pix or α-Pix in vitro. Thus, we tried to determine whether the combination of

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Fig. 6 Kindlin-2 knockdown and RAC1 inhibition suppressed melanoma metastasis. A Tumor metastasis was monitored using a small animal imaging system. B Lung metastasis was detected by HE staining; ×20 and ×40 magnification. C Number of metastatic nodules in the lungs. D Luciferase signal intensities in the mice. E The number of GFP-labeled CTCs was determined by flow cytometry of whole blood samples. (a-e. MeWo cells with or without kindlin-2 knockdown). F Tumor metastasis was monitored using a small animal imaging system. G Lung metastasis was detected by HE staining; ×20 and ×40 magnification. H Number of metastatic nodules in the lungs. I Luciferase signal intensities in the mice. J The number of GFP-labeled CTCs was determined by flow cytometry of whole blood samples. (F–J. A375 cells with or without kindlin-2 overexpression or with the treatment of 1A-116 at different doses after kindlin-2 overexpression). *P < 0.05, **P < 0.01, ***P < 0.001, statistically significant. the Rac1 inhibitor and vemurafenib could improve outcomes. the inactive, Rac1-GDP conformation to the active, Rac1-GTP Encouragingly, the combination therapy group exhibited a conformation. In turn, the downstream MAPK pathway is stimulated markedly greater therapeutic effect than the vemurafenib and to promote the growth and metastasis of melanoma. Furthermore, Rac1 inhibitor monotherapy groups. Dysregulation of Rac1 we revealed that Rac1 inhibition can reverse melanoma growth and activity has been found in certain cancers, including melanoma, metastasis caused by high kindlin-2 expression, and combination breast cancer, colorectal cancer, gastric cancer, etc., and Rac1 has therapy with a Rac1 inhibitor and vemurafenib can result in a better been discovered to influence cell proliferation, adhesion, therapeutic outcome than monotherapy in melanoma patients with migration, and invasion in the progression of certain cancers high kindlin-2 expression and BRAF mutations (Fig. 8). Collectively, [50–57]. As previously reported, activation of Rac1 can promote these findings indicate that kindlin-2 is a potential diagnostic MAPK pathway signaling; thus, we speculated that the combina- biomarker and therapeutic target for melanoma. tion of the Rac1 inhibitor and vemurafenib, which provided a double hit to the MAPK pathway, might result in improved therapeutic outcomes, and the results of our animal studies MATERIALS AND METHODS validated this hypothesis. Thus, melanomas with high kindlin-2 Cell culture expression and BRAF mutations can be treated more effectively Six melanoma cell lines (A375, A875, MeWo, WM35, SK-Mel-2, and SK-Mel- with the combination of a Rac1 inhibitor and vemurafenib than 28) were cultured for the following experiments. The BRAF mutational with either monotherapy. status of melanoma cell lines were displayed in Table S3. In summary, our study demonstrated a substantial potential oncogenic role of kindlin-2 in melanoma. kindlin-2 can bind Rac1 activation assay with magnetic bead pulldown specifically to p-α-Pix (S13) and Rac1 and subsequently enhance the The following protocol was followed: cells were cultured and rinsed twice binding of p-α-Pix (S13) to Rac1 to induce the switch of Rac1 from with ice-cold PBS. Then, ice-cold leupeptin (0.5 ml per 150-mm tissue

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Fig. 7 Combination of the Rac1 inhibitor and vemurafenib strengthened the therapeutic effect of each monotherapy. A–C The morphology, weight and volume of the tumors from each mouse at sacrifice. D, E The tumor volume and body weight of each mouse were measured and recorded every three days throughout the course of the experiment. F Tumor metastasis was monitored using a small animal imaging system. G Lung metastasis was detected by HE staining; ×20 and ×40 magnification. H Number of metastatic nodules in the lungs. I Luciferase signal intensities in the mice. J The number of GFP-labeled CTCs was determined by flow cytometry of whole blood samples. K A schematic model of the functions of kindlin-2 in melanoma growth and metastasis. (A–K A375 cells with or without kindlin-2 overexpression or with the treatment of 1A-116 after kindlin-2 overexpression or with the treatment of vemurafenib after kindlin-2 overexpression or with the combination of vemurafenib and 1A-116 after kindlin-2 overexpression). *P < 0.05, **P < 0.01, ***P < 0.001, statistically significant.

culture plate) was added, and cells were detached by scraping and lysed. PBS) were injected subcutaneously into the left flank of female athymic Next, the cell lysates were transferred to microfuge tubes on ice, and 0.5 ml nude mice aged 3–4 weeks, and tumors developed at the injection sites of each cell extract was aliquoted to a microfuge tube. A total of 10 µl after 1 week. When the formed tumors reached 100 mm3, the animals were (10 µg) of Rac1/Cdc42 Assay Reagent (PAK-1 PBD-conjugated magnetic divided randomly into different groups with 6 mice per group. Certain beads) was added to each tube and incubated for 45 min at 4 °C. The groups were treated with different concentrations (1, 2, 3, 4, 5, and 6 mg/ beads were pelleted by centrifugation (10 s, 14,000×g, 4 °C), and the kg) of 1A-116 or with vemurafenib (20 mg/kg) daily via intratumoral supernatant was removed and discarded. The beads were washed 3 times injection [58]. The experiment was terminated 18 days after tumor cell with leupeptin and resuspended in 40 µl of 2× Laemmli buffer. Next, 2 µl of inoculation. Tumor sizes were measured using Vernier calipers every three 1 M dithiothreitol was added and boiled for 5 min, and the beads were days, and tumor volumes were calculated as follows: V = (width2 × length)/ pelleted by centrifugation. The supernatant and beads were mixed, and 2. At the termination of the experiment, the mice were sacrificed, and the 20 µl of the mixture was loaded on a polyacrylamide gel for SDS-PAGE. tumors were excised from each mouse and weighed. A portion of the After that, the proteins were transferred to a membrane. After the above tumors was fixed with 10% formalin and used to prepare tumor tissue steps, the membranes were blocked with 5% skim milk (w/v) at room lysates for western blot analysis. No blinding was done. temperature for 1 h and incubated overnight at 4 °C with an anti-Rac1 antibody (diluted to 1 µg/ml). Secondary antibodies were then added and incubated for 1 h at room temperature. Protein-antibody complexes were Metastasis model then detected by chemiluminescence (Pierce ECL Western Blotting A tail vein injection model was used for lung colonization assays. All female athymic nude mice aged 3-4 weeks were divided randomly into different Substrate, Thermo, USA). groups with six mice per group. All mice were injected via the tail vein with 1×105 luciferase and GFP-positive melanoma cells in 0.1 ml of serum-free Xenograft animal experiment and tissue processing medium. On the day of cell inoculation, bioluminescence imaging using All animal procedures were performed in accordance with the Guide for the IVIS@ Lumina II system (Caliper Life Sciences, Hopkinton, MA) was the Care and Use of Laboratory Animals (NIH publication No. 80-23, revised performed to verify a fluorescence signal in all mice after intraperitoneal 1996) and the Institutional Ethical Guidelines for Animal Experiments injection of 4.0 mg of luciferin (Gold Biotech) in 50 µl of saline. The developed by Sun Yat-sen University. Melanoma cells (5 × 106 in 100 μlof metastases were monitored using the IVIS@ Lumina II system (Caliper Life

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Fig. 8 Inﬂuence of kindlin-2 in melanoma growth and metastasis, and treatment targeted to kindlin-2 and BRAF mutation. Kindlin-2 can bind speciﬁcally to p-α-Pix (S13) and Rac1, enhance the binding of p-α-Pix (S13) to Rac1 to induce the switch of Rac1 from the inactive, Rac1- GDP conformation to the active, Rac1-GTP conformation. Then, the downstream MAPK pathway is stimulated to promote the growth and metastasis of melanoma. The combination of Rac1 inhibitor and vemurafenib can reverse melanoma growth and metastasis caused by high kindlin-2 expression.

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Long GV, Stroyakovskiy D, Gogas H, Levchenko E, de Braud F, Larkin J, et al. article’s Creative Commons license and your intended use is not permitted by statutory Flaherty combined BRAF and MEK inhibition versus BRAF inhibition alone in regulation or exceeds the permitted use, you will need to obtain permission directly melanoma. N Engl J Med. 2014;371:1877–88. from the copyright holder. To view a copy of this license, visit http://creativecommons. 50. Halaban R. RAC1 and melanoma. Clin Ther. 2015;37:682–5. org/licenses/by/4.0/. 51. Morrison Joly M, Williams MM, Hicks DJ, Jones B, Sanchez V, Young CD, et al. Two distinct mtorc2-dependent pathways converge on rac1 to drive breast cancer metastasis. Breast Cancer Res. 2017;19:74. © The Author(s) 2021

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