Hu et al. Cancer Cell Int (2020) 20:563 https://doi.org/10.1186/s12935-020-01663-4 Cancer Cell International

PRIMARY RESEARCH Open Access CD44 cross‑linking increases malignancy of breast cancer via upregulation of p‑Moesin Song Hu1,3, Xiaoxing Shi2, Yiwen Liu1, Yiqing He1, Yan Du1, Guoliang Zhang1, Cuixia Yang1,3* and Feng Gao1,3*

Abstract Background: CD44 is highly expressed in most cancer cells and its cross-linking pattern is closely related to tumor migration and invasion. However, the underlying molecular mechanism regarding CD44 cross-linking during cancer cell metastasis is poorly understood. Therefore, the purpose of this study was to explore whether disruption of CD44 cross-linking in breast cancer cells could prevent the cells migration and invasion and determine the efects of CD44 cross-linking on the malignancy of the cancer cells. Methods: The expression of CD44, CD44 cross-linking and Moesin phosphorylation in breast cancer cells was assessed by Western Blot assays. Efects of CD44 cross-linking on tumor metastasis were evaluated by Transwell assay. The efects of CD44 cross-linking disruption on cell viability were assessed using CCK-8 assays. The expression of p-Moesin between normal and breast cancer tissues was examined by immunohistochemical staining. Results: High expression of CD44 cross-linking was found in invasive breast cancer cells (BT-549 and MDA-MB-231), which is associated with the malignancy of breast cancer. The expressions of ERM complex in a panel of breast cancer cell lines indicate that Moesin and its phosphorylation may play a signifcant role in cell metastasis. Moesin phospho- rylation was inhibited by CD44 de-crosslinking in breast cancer cells and Moesin shRNA knockdown attenuated the promotion of CD44 cross-linking on cell migration and invasion. Finally, immunohistochemistry results demonstrated that p-Moesin was overexpressed in primary and metastatic cancers. Conclusions: Our study suggested that CD44 cross-linking could elevate p-Moesin expression and further afect migration and invasion of breast cancer cells. These results also indicate that p-Moesin may be useful in future tar- geted cancer therapy. Keywords: Breast cancer, CD44 cross-linking, p-Moesin, Migration, Invasion

Background As one of the adhesion molecules related to tumor Malignant tumor metastasis is usually the leading cause metastasis, CD44, a receptor of hyaluronan (HA), is of death in cancer patients [1]. Breast cancer is a multi- becoming attractive for its role as a stem cell marker state tumor that often has fatal efects by metastasizing [5, 6]. Until now, the function of CD44 in cancers is not to distant organs, such as bones, lungs, and liver [2–4]. fully understood, although data have proved that CD44 In light of this, it is critical to understand the regulatory activities are usually triggered by binding with its ligands, mechanisms driving breast cancers (BrCas) metastasis especially HA. In fact, the interaction of receptors with and discover new therapeutic targets. ligands has always been a focus regarding the cellular behaviors under pathological processes, like infam- mation, immune responses, wound healing and cancer *Correspondence: [email protected]; [email protected] 1 Department of Molecular Biology, Shanghai Jiao Tong University progression [7–9]. As HA is a non-sulfated glycosamino- Afliated Sixth People’s Hospital, Shanghai 200233, China glycan which usually binds with the multiple sites on the Full list of author information is available at the end of the article receptors that can cause CD44 linking together, we and

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other studies have found that CD44 cross-linking and de- dramatically inhibit cancer cell migration and invasion. crosslinking can deliver opposite signals to cells. Unfor- Notably, phosphorylated Moesin (p-Moesin) may play an tunately, little is known about CD44 self-linking in breast important role in CD44 cross-linking. Tese fndings not malignancy. only provide new mechanistic insights into CD44 cross- CD44 cross-linking was initially described in rat pan- linking in BrCas but also indicate that p-Moesin may be creatic cancer cells, which can improve the ligand HA a potential therapeutic target for treating invasive BrCas. binding capacity [10]. Subsequent researches showed that CD44 cross-linking has been associated with the Materials and methods progression of other human cancers. For example, in Cell culture and antibodies neuroblastoma cells, clusters of CD44 are located at the flamentous pseudopods and focal globular, which facili- Human BrCas cell lines (MDA-MB-453, MCF-7, T-47D, tate migration and invasion into the brain [11]. CD44 MDA-MB-231, BT-549 and Hs-578t) were purchased clustering can also promote BrCas metastasis by relo- from the Cell Bank of the Type Culture Collection of the cating metalloproteinase-9 and up-regulating LFA-1 Chinese Academy of Sciences (Shanghai, China). MDA- and VLA-4 [12, 13]. However, most of the studies were MB-453 cells were cultured in L-15(Gibco, USA), MCF-7 conducted in a CD44 antibody-mediated cross-linking cells were cultured in MEM(Gibco, USA); BT-549 cells manner, so a model that naturally mimics the in vivo were cultured in RPMI-1640 medium(Gibco, USA); and clustering of CD44 is urgently needed for cancer study. MDA-MB-231, T-47D and Hs-578t cells were cultured Our previous studies showed that high molecular weight in high-glucose DMEM (Gibco, USA).All the media were HA can stimulate CD44 cross-linking in BrCas cells supplemented with 10% fetal bovine serum(Gibco, USA) [14] and the HA contents are more abundant in invasive and 100 IU/ml penicillin/streptomycin. Additional insu- BrCas cells [15]. Tese prompted us to further investigate lin with a fnal concentration of 0.01 mg/ml was added to the role of HA dependent CD44 self-linking in BrCas the culture medium of BT-549 and Hs-578t. All cell lines progression and its underlying mechanisms. were cultured at 37 °C in humidifed air with 5% ­CO2 In addition to CD44, the ERM proteins (Ezrin/Radixin/ and 95% air. Primary antibodies against CD44 (Abcam, Moesin) [16], which act as cross-linkers between the ab119348), CD44 (clone IM7, Cat #14-0441-86), ERM actin cytoskeleton and intracellular domain of CD44, (CST, 3142S), p-ERM (CST, 3141S), Moesin (Abcam, have been implied in cell adhesion and motility [17]. ab52490) p-Moesin (Abcam, ab177943), p-Merlin(CST, Among them, Moesin is particularly attractive for its 13281S), Merlin (CST, 12888) were used. curial role in organizing membrane domains and recep- tor signaling, as well as regulating the metastasis of Quantitative real‑time PCR (qRT‑PCR) tumor cells. Evidence has proved that CD44-Moesin Total RNA was extracted from cultured cells (MCF-7, interaction promotes human brain tumor proliferation by T-47D, BT-549, and MDA-MB-231) by RNAiso Plus activating the Wnt signaling pathway [18]. Other reports (Takara, Japan). RNA (1 μg) was reverse transcribed with suggested that the expression of Moesin in glioma cells the PrimeScript™ RT Reagent Kit with gDNA Eraser was signifcantly higher than that in normal astrocytes (Takara, Japan). Real-time PCR assays were performed [19] and there was a strong negative correlation with pro- by using SYBR Green Mix (Takara, Japan) according to gression-free survival and overall survival [20]. However, the manufacturer’s protocol. All qRT-PCR values of each no role for Moesin activation in BrCas progression has gene were normalized against that of GAPDH. Te rela- been defned. Terefore, it is reasonable to speculate that tive expression of genes was calculated by the ­2−ΔΔCt CD44 receptors self-linking or aggregations are closely method. associated with the intracellular ERM molecules, and it Te primer sequences for qRT-PCR are: CD44-F: GAC​ is necessary to explore the regulating mechanism of ERM ACC​ATG​GAC​AAG​TTT​TGG, CD44-R: GAC​ACC​ on CD44 interactions. ATG​GAC​AAG​TTT​TGG; Moesin-F, ATG​CCC​AAA​ In this study,we aimed to determine the role of CD44 ACG​ATCA GTGTG, Moesin-R: ACT​TGG​CAC​GGA​ cross-linking on BrCas metastasis and explore the ACT​TAA​AGAG; Ezrin-F: ACC​AAT​CAA TGT​CCG​ underlying mechanisms. We frst analyzed the expres- AGT​TAC​C, Ezrin-R: GCC​GAT​AGT​CTT​TAC​CAC​ sion of CD44 cross-linking in BrCas cell lines, and elu- CTGA; Radixin-F: AA TTG​TGG​CTA​GGT​GTT​GAT​GC, cidated the relations between CD44 clustering status Radixin-R: GGT​GCC​TTT​TTG​TCG​ATT​GGC; Merlin-F: and BrCas metastasis. Ten, we investigated the efect of AGT​GGC​CTG​GCT​CAA​AAT​GG, Merlin-R: TGT​TGT​ CD44 cross-linking on downstream ERM proteins. Our GTG​ATC​TCC​TGA​ ACCA; GAPDH-F: AGC​CTC​AAG​ data showed that CD44 clustering could promote BrCas ATC​ATC​AGC​, GAPDH-R: GAG​TCC​TTC​CAC​ GAT​ malignancy and disruption of CD44 clustering could ACC.​ Hu et al. Cancer Cell Int (2020) 20:563 Page 3 of 10

RNA interference MCF-7, T-47D, MDA-MB-231 and BT-549 cells were Te shRNA-carrying lentiviruses against Moesin, and suspended in medium containing 5% FBS after trans- 4 negative control were produced by Genechem (Shang- fection. In addition, 3 × 10 cells were seeded into the hai, China). BT-549 and MDA-MB-231 cells were upper chamber of an 8-μm pore size insert with or infected with concentrated virus according to the man- without Matrigel (BD Biosciences, USA). Te chambers ufacturer’s protocol, and the expression of Moesin was were deposited in a 24-well plate with 600 μl of 20% validated by western blot analysis. FBS medium. After 24 h’s incubation, the cells were fxed with 4% paraformaldehyde (Beyotime, China) Western blotting and stained with crystal violet (Beyotime, China). After removing the cells on the upper surface of the chamber, RIPA bufer (Beyotime, China) was used for protein the penetrated cells were captured and the number of extraction. After the total protein concentration was cells was counted by Image J software at 200 magnif- determined by a bicinchoninic acid protein assay kit × cation in fve random felds under a microscope. (Sigma, USA), 30 μg protein samples were separated by 8% SDS polyacrylamide gels and transferred onto Immunohistochemistry (IHC) and staining evaluation PVDF membranes(Millipore, Billerica, USA). Te membrane was blocked with 5% nonfat milk in TBST Formalin-fxed parafn-embedded human BrCas tis- for 1 h and incubated with the indicated antibody at sues were obtained from Shanghai Superchip Biotech 4 °C overnight. Ten HRP-conjugated secondary anti- (HBreD055CD01). Te expression of p-Moesin in the tis- bodies (1:5000) were added. Bands were subsequently sue chip of the cohort of 55 tissues was examined by IHC. visualized using the enhanced plus chemiluminescence Te tissue chip was incubated with rabbit monoclonal assay (Pierce, USA). Measurement of the bands was antibody against human p-Moesin (Abcam, ab177943). conducted on an ImageQuant LAS 4000 mini. Ten the sections of tissue chip were detected by Outdo Biotech (Shanghai, China). Te expression of Moesin was scored with intensity of Cell proliferation staining and the percentage of the cells of interest stain- In brief, cells were treated with 300 μg/ml hyaluroni- ing. We divided the intensity of staining into 4 groups: dase and control medium. Ten, equal numbers of cells 0 (–), 1 (+), 2 (++), and 3(+++). Te (–), (+), (++), (2000 cells/well) were seeded into 96-well plates for the (+++) were defned as no staining, weak staining, mod- proliferation experiment. Te proliferation of MDA- erate staining and intense staining. Ten we ranked the MB-231 and BT-549 cells was measured via CCK-8 percentage of positive staining into 4 categories: 0 (0%), assay (KeyGen Biotech,China) according to the manu- 1 (1–29%), 2 (30–69%), and 3 (≥ 70%) as shown in Addi- facturer’s protocol. tional fle 1: Figure S4. Te IHC scores are obtained by multiplying the above two scores. Te fnal fgures were CD44 Cross‑linking created by Graphpad 7. Cells were treated with diferent concentrations of hya- luronidase for 0.5 h, then the cells were washed 3 times Statistical analysis with ice-cold PBS (20 mM sodium phosphate, 0.15 M All data are presented as the mean ± SD and were ana- NaCl, pH 8.0). CD44 cross-linking was performed by lyzed with GraphPad Prism 7 and SPSS v23 software. incubation with 2 mM bis (sulfosuccinimidyl) suberate Te student’s t-test was used to identify the diferences ­(BS3) (Pierce, USA) for 30 min and quenched by incu- between the treated groups and their controls. IHC bation with 20 mM Tris, pH 7.6 for 15 min at room scores among three groups were analyzed with the non- temperature. Cells were washed twice with PBS and parametric alternative to ANOVA. A P value < 0.05 was lysed with cell lysis bufer. considered statistically signifcant in the text and fgures For antibody-mediated CD44-crosslinking. BrCas (*P < 0.05, **P < 0.01, ***P < 0.001). cells were incubated for 90 min at 37 °C with rat mon- oclonal CD44 antibody (10 mg/ml) (clone IM7, Cat Results #14-0441-86). After three washes, cells were incubated CD44 cross‑linking in breast cancer with 1 mg/ml of goat anti-rat IgG-Fc (Cat #31226) for Since there are few studies involving CD44 cross-link- 60 min at 37 °C and then subjected to cell lysis. ing abnormalities in BrCas, we selected four BrCas cell lines with diferent degrees of malignancy to explore the Cell migration and invasion assays expression of CD44 and CD44 cross-linking. We found To evaluate the migration and invasion abilities of that the expression of CD44 was relatively higher in inva- BrCas cells, Transwell assays were performed. In brief, sive BrCas cells MDA-MB-231 and BT-549, and lower in Hu et al. Cancer Cell Int (2020) 20:563 Page 4 of 10

Fig. 1 CD44 cross-linking in breast cancer. a Expression of CD44 in breast cancer cell lines MCF-7, T-47D, BT-549 and MDA-MB-231. On the left, qRT-PCR results of CD44,on the right, western blot analysis of CD44; b Expression of CD44 cross-linking in breast cancer cell lines; c, d Comparison of migration and invasion in breast cancer cell lines

MCF-7 and T-47D cells (Fig. 1a). In addition, to show the selected as the efective concentration in our experiment. CD44 cross-linked status on BrCas cells, we treated the Next, we treated BrCas cells with hyaluronidase to abro- cells with ­BS3, a membrane-impermeable compound that gate CD44 cross-linking (Fig. 2a). Our studies showed cross-link only nearby proteins located within the length that the removal of CD44 cross-linking in BT-549 and of the spacer. After incubation with ­BS3 or control bufer, MDA-MB-231 cells signifcantly reduced cell migration obvious cross-linked CD44 aggregations were showed in and invasion without changing CD44 expression (Fig. 2b MDA-MB-231 and BT-549 cells rather than in MCF-7 and c), suggesting that CD44 cross-linking may play an and T-47D cells (Fig. 1b). At the same time, Transwell important role in the malignancy of BrCas. experimental results showed that MDA-MB-231 and BT-549 presented higher abilities of migration and inva- Moesin phosphorylation was inhibited by CD44 sion than MCF-7 and T-47D (Fig. 1c and d). Tese data de‑crosslinking in breast cancer cells suggest that CD44 expression level and CD44 cross-link- Since CD44 cross-linking is an abnormal result of ing abilities may be accompanied by BrCas metastasis. receptor-ligand interaction, and most of cellular activi- ties are conducted through downstream ERM complex, CD44 cross‑linking is associated with the malignancy we further explore the changes of intracellular cytoskel- of breast cancer etal molecules underneath the CD44 receptor. Firstly, To investigate the efect of CD44 cross-linking on BrCas we screened the expressions of ERM complex in a panel cells, we frst carried out experiments to disconnect the of BrCas cell lines. qRT-PCR and Immunoblot analysis cross-linking using three reagents (oligosaccharides of both showed that the expression of Moesin was relatively hyaluronan, hyaluronidase and Latrunculin B). All of higher in invasive BrCas cells MDA-MB-231, Hs-578t them have been proven to efectively block the CD44 and BT-549, and lower in MDA-MB-453, MCF-7 and cross-linking [14, 21]. Te results showed that hyaluroni- T-47D cells (Fig. 3a and b). Interestingly, p-Moesin was dase has the best dis-connecting efect and is not toxic to overexpressed in invasive breast cancer cells (Fig. 3b). cells (Additional fle 2: Figure S1), in which 300 μg/ml was However, there was no signifcant diference in the Hu et al. Cancer Cell Int (2020) 20:563 Page 5 of 10

Fig. 2 CD44 cross-linking is associated with the malignancy of breast cancer. a The efects of diferent concentrations of hyaluronidase on CD44 cross-linking of BT-549 and MDA-MB-231 cells were detected by Western Blot assay. b, c The efect of 300 μg/ml hyaluronidase on the cell migration and invasion ability of BT-549 and MDA-MB-231 cells; *P < 0.05, ***P < 0.001

expression of the other three actin-membrane crosslink- Additional fle 4: Figure S3A), suggesting that CD44 ers (Ezrin, Radixin and Merlin) (Fig. 3b and Additional cross-linking may induce Moesin activation only, not fle 3: Figure S2). Tese results indicated that Moesin the expression. To further clarify this hypothesis, we and its phosphorylation may play a signifcant role in cell used antibody to re-cross link CD44. Te addition of metastasis. However, to our surprise, when we destroyed the secondary antibody enhanced CD44 cross-linking the CD44 cross-linking in MDA-MB-231 and BT-549, we and resulted in increased p-Moesin expression (Fig. 3d found that it was the phosphorylation of Moesin, rather and Additional fle 4: Figure S3B), which suggested that than Moesin expression, that was inhibited (Fig. 3c and CD44 cross-linking could promote p-Moesin expression. Hu et al. Cancer Cell Int (2020) 20:563 Page 6 of 10

Fig. 3 Moesin phosphorylation can be afected by CD44 cross-linking in breast cancer cells. a Detection of Moesin expression in breast cancer cell lines by qRT-PCR; b Detection of ERM, p-ERM and Merlin expression in breast cancer cell lines by western blot; c Efects of hyaluronidase at diferent concentrations on the expression of ERM and Merlin and their phosphorylated forms; d p-Moesin and ERM expression after CD44 antibody mediated cross-linking in MDA-MB-231 cells

Also we can see that Ezrin, Radxin and Merlin and their e). Tese results suggest that p-Moesin expression plays phosphorylated forms do not show signifcant difer- an important role in afecting the migration and invasion ences (Fig. 3c and Additional fle 4: Figure S3A). Taken of BrCas cells after CD44 cross-linking. together, these results suggested that CD44 cross-linking may regulate tumor cell migration and invasion by alter- p‑Moesin was overexpressed in primary and metastatic ing the phosphorylation of Moesin. cancers As our data show that p-Moesin may play a role in BrCas Moesin shRNA knockdown attenuated the promotion metastasis, we next try to investigate whether p-Moesin of CD44 cross‑linking on cell metastasis and invasion could be used in the clinicopathologic detection in BrCas. To further clarify that CD44 cross-linking indeed regu- We analyzed the p-Moesin expression levels in 55 sam- lates tumor cell migration and invasion by afecting ples, including 34 BrCas (8 ductal breast carcinoma in situ p-Moesin in BrCas, we reduced Moesin expression in and 26 invasive ductal breast carcinoma), 2 benign breast MDA-MB-231 and BT-549 by RNA interference. West- disease, 13 lymph nodes and 6 adjacent normal tissues by ern blot analysis showed that the down-regulation of IHC staining assays. Te data showed that p-Moesin level Moesin could simultaneously reduce the expression of was signifcantly higher in ductal breast carcinoma in situ p-Moesin (Fig. 4a). Trough Transwell experiment, we than those in adjacent normal tissues (Fig. 5a). Accord- analyzed the efect of p-Moesin down-regulation on cell ingly, the expression of p-Moesin in metastase tissues was migration and invasion. Te results showed that after also higher than that in primary foci of invasive ductal p-Moesin was down-regulated in BrCas cells, cell migra- breast carcinoma (Fig. 5b). To further identify the signif- tion and invasion were inhibited despite the presence of cances between p-Moesin and Moesin, we carried on a CD44 cross-linking (Fig. 4b and c), and the same results TCGA database method to analyze the expression levels of were observed in cell invasion experiments (Fig. 4d and Moesin in BrCass. We found that there were no signifcant Hu et al. Cancer Cell Int (2020) 20:563 Page 7 of 10

Fig. 4 Moesin shRNA knockdown attenuated the promotion of CD44 cross-linking on cell metastasis and invasion. a p-Moesin and Moesin expression of BT-549 and MDA-MB-231 and Moesin knockdown cells after treatment with hyaluronidase. b, c Migration of BT-549 and MDA-MB-231 and Moesin knockdown cells after treatment with hyaluronidase; d, e Invasion of BT-549 and MDA-MB-231 and Moesin knockdown cells after treatment with hyaluronidase; ***P < 0.001, NS no signifcance

diferences in the expression of Moesin between cancer activated that afect cell function [22–24]. CD44, the and adjacent tissues (Fig. 5c). Similarly, there was no dif- main receptor for HA, is a well-accepted molecular ference in survival between patients with diferent Moesin marker for cancer stem cells. Data have stated that expression (Fig. 5d). Tese data suggested that the phos- CD44 is involved in tumor-initiating that includes phorylated form of Moesin not Moesin is correlated with CD44 self-interaction and downstream signal activation tumor progression and metastasis and may be applied in [25]. However, the mechanism of CD44 clustering in the future clinicopathologic detection (Additional fle 5: cancer progression is not well illustrated. In this study, Figure S5). we demonstrate for the frst time that CD44 cross- linking promotes BrCas cell aggression by regulating Discussion the downstream ERM proteins, particularly through Receptor cross-linking is often considered to be a hall- p-Moesin. Correspondingly, the decrease of p-Moesin mark of malignant tumor initiation and progression, could inhibit the BrCas cells migration and invasion during which downstream signal pathways are usually after disrupting CD44 cross-linking, suggesting a CD44 Hu et al. Cancer Cell Int (2020) 20:563 Page 8 of 10

Fig. 5 p-Moesin was overexpressed in primary and metastatic cancers. a, b Representative images from a microarray of human breast tissue that includes tumor and adjacent normal tissues stained by IHC for p-Moesin, IHC scores of p-Moesin were on the right; c Expression levels of Moesin in cancer and adjacent normal tissues of breast cancer in TCGA database; d Moesin expression has no signifcant correlation with patient overall survival in TCGA database,*P < 0.05

clustering-p-Moesin pathway that regulates BrCas that the functions of CD44 cross-linking are diverse and malignancy (Additional fle 5: Figure S5). unknown details are needed to be explored. Tumor cell metastasis is one of the features of most As reported, CD44 molecules are dispersed over the lethal tumors [26]. Studies have found that CD44 is cell surface under homeostatic conditions, whereas clus- overexpressed in metastatic BrCas [27] and its multiple tering could be induced by aberrant HA deposition in spliced forms have also been shown to be closely associ- pathological microenvironments [14, 29]. Intriguingly, ated with BrCas cell metastasis [28]. Besides overproduc- such receptors self-linking could be reversed following tion, CD44 cross-linking is also believed to induce cancer the degradation process by abnormal metabolites, such as cell adhesion and migration [13]. However, a recent hyaluronidase. Te signifcances of CD44 receptors self- study stated that CD44 clustering in breast epithelial aggregation and de-cross- linking are being investigated. cells could reduce cell growth [29]. Tese studies suggest Some observations have suggested that the disruption Hu et al. Cancer Cell Int (2020) 20:563 Page 9 of 10

of CD44 self-linking could inhibit BrCas cells migration Conclusions and invasion [30, 31]. However, the mechanisms involved Our study demonstrated a novel fnding of CD44 cross- are not elucidated thoroughly, including the downstream linking with BrCas cells malignancy. Te downstream molecules closely connected to CD44 or intracellular mechanism revealed that a CD44 associated cytoskel- skeletal complex beneath the CD44 receptors. etal protein, Moesin, was responsible for CD44 aggre- It is well known that ERM complex (Ezrin, Radixin and gation during BrCas metastasis when phosphorylation Moesin) are cytoskeletal molecules connected to CD44 occurred. Further, we suggested that p-Moesin but not C-terminal domain and are believed to be closely related Moesin is overexpressed in primary BrCas and may be to cancer malignancy. Due to their structure homology, useful in future targeted cancer therapy. the three members are often studied as a whole [32]. However, a growing body of evidence proves that ERM Supplementary information proteins perform distinct functions during diferent bio- Supplementary information accompanies this paper at https​://doi. logical processes [19, 33–35]. In our study, we found that org/10.1186/s1293​5-020-01663​-4. CD44 cross-linking could cause Moesin phosphorylation without changing its expression. At the same time, Ezrin Additional fle 1: Figure S4. Representative images and quantifcation of p-Moesin staining. The intensity of IHC staining were ranked into 4 grades: and Radixin, two other components of the ERM complex 0 (–), 1 ( ), 2 ( ), and 3 ( ) as indicated. + ++ +++ showed no alterations in quantity and activity, suggesting Additional fle 2: Figure S1. Efect of hyaluronidase on CD44 crosslinking that CD44 cross-linking plays its downstream role mainly and cell proliferation. (A) Efects of oHA, hyaluronidase and Lat B on CD44 through p-Moesin in BrCas. Previous fndings indicated cross-linking; (B) BT-549 and MDA-MB-231 were treated with 300 μg/ml hyaluronidase, and their proliferation capacity was detected by CCK-8 that HA can signifcantly enhance the invasiveness of gli- assay. oma cells by promoting the binding of CD44 and Moesin Additional fle 3: Figure S2. Expression of Ezrin, Radixin and Merlin in [36]. Nevertheless, in our current study, we found that breast cancer cell lines. (A-C) Expression levels of Ezrin, Radixin and Merlin HA increases BrCas malignancy through inducing CD44 in six breast cancer cell lines. receptors self-linking, thus triggering downstream Moes- Additional fle 4: Figure S3. Alteration of CD44 cross-linking status on in’s phosphorylation, rather than simply binding to CD44. the expression level of P-Moesin in BT-549 cells. (A) Efects of hyaluroni- dase at diferent concentrations on the expression of ERM and Merlin and Moreover, our loss-and-gain-of-function experiment their phosphorylated forms in BT-549 cells; (B) p-Moesin and ERM expres- proved that removing ligand (HA) with hyaluronidase to sion after CD44 antibody mediated cross-linking in BT-549 cells. destroy CD44 cross-linking obviously inhibited p-Moesin Additional fle 5: Figure S5. Working model of the CD44 cross-linking : expression, while p-Moesin was rescued following the p-Moesin regulatory axis in breast cancer. addition of an antibody that re-induced CD44 cross-link- ing. Since CD44 clustering does not afect the expression Abbreviations of Moesin, we knocked down Moesin to downregulate HA: Hyaluronic acid; DMEM: Dulbecco’s modifed Eagle medium; PBS: Phos- phate bufered solution; TCGA:​ The cancer genome atlas; qRT-PCR: Quantita- p-Moesin. We observed that the promoting efect of tive real time polymerase chain reaction; IHC: Immunohistochemistry; PVDF: CD44 clustering on BrCas cell migration could be halted Polyvinylidene fuoride; CCK-8: Cell counting kit-8; shRNA: Short hairpin RNA; after the Moesin knockdown. Taken together, these data GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; ERM: Ezrin/Radixin/ Moesin. suggested that p-Moesin may be indispensable in BrCas cell aggression caused by CD44 cross-linking and may Acknowledgements have potential clinical application value. This work was supported by the National Natural Science Foundation of China (81572821, 81672843, 81702852, 81872357, 81974445 and 81974446), To further verify the clinical signifcance of p-Moesin in Shanghai Municipal Education Commission-Gaofeng Clinical Medicine BrCas, we determined its expression level with a sample Grant Support (20171924), Program of Shanghai Leading Talents (2013-038), cohort. Our IHC results showed that p-Moesin is highly Doctor Innovation Fund of Shanghai Jiaotong University School of Medicine (BXJ201944) and Shanghai Pujiang Program (2019PJD037). expressed in primary BrCas compared with normal tis- sue. Additionally, we also observed that the p-Moesin Authors’ contributions expression in metastases is higher than that in primary SH, CYand FG designed the study. SH, XS, YL, YH, YD and GZ performed the study. SH and YD analyzed the data and wrote the paper. All authors read and tumors, suggesting that p-Moesin is clinically associated approved the fnal manuscript. with the induction of metastasis in BrCas. Furthermore, through analyzing the TCGA database, we found that Availability of data and materials The datasets used and/or analysed during the current study are available from there was no signifcant diference in Moesin expression the corresponding author on reasonable request. between BrCas and adjacent tissues, as well as no correla- tion of Moesin with BrCas patients’ poor prognosis. Col- Ethics approval and consent to participate This study had been permitted by the Ethical Review Committee of The Six lectively, our study highlighted that p-Moesin rather than Afliated Hospital of Shanghai jiaotong University. Moesin, could be applied as a potential target for BrCas therapy. Hu et al. Cancer Cell Int (2020) 20:563 Page 10 of 10

Consent for publication 18. Zhu X, Morales FC, Agarwal NK, Dogruluk T, Gagea M, Georgescu MM. Not applicable. Moesin is a glioma progression marker that induces proliferation and Wnt/beta-catenin pathway activation via interaction with CD44. Can- Competing interests cer Res. 2013;73(3):1142–55. The authors declare that they have no competing interests. 19. Wang Q, Lu X, Zhao S, Pang M, Wu X, Wu H, et al. Moesin expression is associated with glioblastoma cell proliferation and invasion. Anticancer Author details Res. 2017;37(5):2211–8. 1 Department of Molecular Biology, Shanghai Jiao Tong University Afliated 20. Wu M, Liu DY, Yuan XR, Liu Q, Jiang XJ, Yuan D, et al. The expression of Sixth People’s Hospital, Shanghai 200233, China. 2 Department of Labora- moesin in astrocytoma: correlation with pathologic grade and poor tory Medicine, Shanghai Wujing General Hospital, Shanghai 201103, China. clinical outcome. 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