Y772 phosphorylation of EphA2 is responsible for EphA2-dependent NPC nasopharyngeal carcinoma growth by Shp2/Erk-1/2 signaling pathway

EphA2 is an important oncogenic protein and emerging drug target, but the oncogenic role and mechanism of ligand-independent phosphorylation of EphA2 at tyrosine 772 (pY772-EphA2) is unclear. In this study, we established nasopharyngeal carcinoma (NPC) cell lines with stable expression of exogenous EphA2 and EphA2-Y772A (phosphorylation inactivation) using endogenous EphA2-knockdown cells, and observed that pY772A EphA2 was responsible for EphA2-promoting NPC cell proliferation and anchorage-independent and in vivo growth in mice. Mechanistically, EphA2-Y772A mediated EphA2-activating Shp2/Erk-1/2 signaling pathway in the NPC cells, and Gab1 (Grb2-associated binder 1) and Grb2 (growth factor receptor-bound protein 2) were involved in pY772-EphA2 activating this signaling pathway. Our results further showed that Shp2/Erk-1/2 signaling mediated pY772-EphA2-promoting NPC cell proliferation and anchorage-independent growth. Moreover, we observed that EphA2 tyrosine kinase inhibitor ALW-II-41-27 inhibited pY772-EphA2 and EphA2-Y772A decreased the inhibitory effect of ALW-II-41-27 on NPC cell proliferation. Collectively, our results demonstrate that pY772-EphA2 is responsible for EphA2-dependent NPC cell growth in vitro and in vivo by activating Shp2/Erk-1/2 signaling pathway, and is a pharmacologic target of ALW-II-41-27, suggesting that pY772-EphA2 can serve as a therapeutic target in NPC and perhaps in other cancers.


Introduction
Nasopharyngeal carcinoma (NPC) is a head and neck cancer that shows a distinct endemic distribution with a high prevalence in Southern China and Southeast Asia, and remains one of the leading lethal malignancies in these areas 1 . Radiotherapy is the major therapeutic modality used to treat NPC and NPC patients can be cured if the disease is diagnosed and treated at an early stage. However, most of NPC patients are diagnosed at advanced stages due to nonspecific symptoms and these patients respond poorly to radiotherapy, whose prognosis is dismal 2 . Therefore, it is imperative to elucidate the underlying mechanism of development and progression of NPC, which could offer novel therapeutic targets.
EphA2 is a member of the EphA family of receptor tyrosine kinases (RTKs) and possesses both tumorpromoting role in a ligand-independent manner and tumor -suppressing role in a ligand-dependent manner 3,4 . EphA2 signals from both the receptor (forward signaling) and the ligand (reverse signaling) to form a communication system with critical and diverse roles in health and disease 3,4 . EphA2 activation by a ligand follows a classical pattern of RTK activation. When ligand Ephrin-A1 binds to EphA2, EphA2 occurs oligomer clustering and subsequent trans-and auto-phosphorylation of tyrosine residues in the juxtamembrane region and kinase domain 5 . This ligand-and tyrosine kinase-dependent EphA2 activation (reverse signaling) inhibits cancer cell proliferation, adhesion, and motility and tumor angiogenesis [6][7][8][9] . In contrast, ligand-and tyrosine kinaseindependent EphA2 activation (forward signaling) promotes cancer development and progression 4,10,11 . Previous studies have shown that Serine897 (S897) and Tyrosine 772 (Y772) of EphA2 are important phosphorylated residues 4,6 . Although numerous studies have demonstrated that ligand-independent S897 phosphorylation of EphA2 (pS897-EphA2) promotes cancer development and progression 4,[10][11][12][13][14][15] , the role of ligandindependent Y772 phosphorylation of EphA2 (pY772-EphA2) in cancers is unclear.
We recently used immunoprecipitation and mass spectrometry analysis (IP-MS) to search proteins interacted with EphA2 in NPC cells and found that Shp2, a protein tyrosine phosphatase (PTP), is an interactor of EphA2. PTPs play a crucial role in cancers 16 . Shp2 encoded by PTPN11 (PTP, non-receptor type 11) gene is the first PTP to be identified as an oncogene 17,18 and possesses an oncogenic role in the melanoma, leukemia, and lung and breast cancers [19][20][21][22] . Shp2 is implicated in the transduction of mitogenic, pro-survival, and pro-migratory signals from growth factor receptors 23 , and is required for the activation of Erk-1/2 signaling downstream of most RTKs [24][25][26] . EphA2 overexpression contributes to ErK-1/2 activation and cancer progression has been reported in many types of cancers 27,28 . A recent study indicates that EphA2 phosphorylates Shp2 and subsequently activates Erk-1/2 29 . However, whether ligand-independent pY772-EphA2 mediates EphA2-activating Shp2/Erk-1/ 2 signaling is unknown.
In the present study, we try to determine whether and how ligand-independent pY772-EphA2 promotes NPC growth, and tested whether pY772-EphA2 is a target of ALW-II-41-27. Our results demonstrate that pY772-EphA2 is responsible for EphA2-dependent NPC cell growth both in vitro and in vivo by activating the Shp2/ Erk-1/2 signaling pathway, and that pY772-EphA2 is a pharmacologic target of ALW-II-41-27.
pY772-EphA2 is responsible for EphA2-dependent anchorage-independent growth and in vivo tumorigenicity of NPC cells Soft agar colony formation assay showed that EphA2-WT dramatically increased the anchorage-independent growth of NPC cells, whereas EphA2-YA failed to do it as compared to endogenous EphA2 knockdown (Fig. 1e), indicating that Y772A mutation abolished the effects of EphA2-WT on the anchorage-independent growth of NPC cells. Tumor formation experiment showed that EphA2-WT dramatically increased in vivo tumorigenicity of NPC cells, whereas EphA2-YA slightly did it as compared to endogenous EphA2 knockdown (Fig. 1f, g), indicating that Y772A mutation almost abolished the effects of EphA2-WT on the in vivo tumorigenicity of NPC cells. Together, these results demonstrate that pY772-EphA2 is responsible for EphA2-dependent anchorage-independent growth and in vivo tumorigenicity of NPC cells.

Identification of Shp2 as a protein that interacted with EphA2 in the NPC cells
To search proteins that interact with EphA2, EphA2 interactors were coimmunoprecipitated with anti-EphA2 antibody from NPC cell extracts, separated on SDSpolyacrylamide gel electrophoresis (PAGE), and stained with Coomassie blue (Fig. 2a). All protein bands were excised and subjected to liquid chromatography and high- throughput MS (LC-MS/MS) analysis, the proteomic data of which are available via ProteomeXchange with identifier PXD015242. Shp2, identified as an interactor of EphA2 (Fig. 2b), was selected for further investigation. Co-IP confirmed that Shp2 interacted with EphA2 in the two NPC cell lines (Fig. 2c). Shp2 was coimmunoprecipitated with exogenous EphA2 from the extracts of HEK293 cells transfected with EphA2 expression plasmid ( Fig. 2c). Immunofluorescent staining showed that Shp2 and EphA2 were colocalized in the NPC cells (Fig. 2d). Moreover, co-IP showed that Y772A mutation did not disturb the interaction of EphA2 and Shp2 in the NPC cells, indicating that interaction of EphA2 and Shp2 is not dependent on pY772-EphA2 (Fig. 2e). Collectively, these data provide evidence that Shp2 interacts with EphA2 in the NPC cells. Shp2 is a protein that interacted with EphA2 in the NPC cells. a IP-mass spectrometry (MS) analysis of the proteins interacted with EphA2. Total cell proteins from 5-8F NPC cells were subjected to co-immunoprecipitation with anti-EphA2 antibody. The coimmunoprecipitated protein complex was resolved on SDS-PAGE and Coomassie blue staining, and then the bands were retrieved and analyzed by MS analysis. b Identification of Shp2 as a protein that interacted with EphA2 by MS analysis. The amino acid sequence of a doubly charged peptide with m/z 396.2391 Da was identified as NAAEIESR and Mascot search showing the peptide matched with Shp2. c Co-IP confirming the interaction of Shp2 and EphA2. Total cell proteins from the 5-8F and CNE2 NPC cells (left) and HEK293 cells ectopically expressing EphA2 (right) were prepared and subjected to immunoprecipitation (IP) with anti-EphA2 antibody followed by immunoblotting with antibodies against Shp2 or EphA2. d Immunofluorescence showing the colocalization of EphA2 (green) and Shp2 (red) in the NPC cells. 5-8F and CNE2 cells were incubated with mouse anti-EphA2 and rabbit anti-Shp2 antibodies followed by staining with DyLight® 488 anti-mouse IgG and DyLight® 594 anti-rabbit IgG, and observed by confocal fluorescence microscopy. Scale bar = 10 μm. e Co-IP showing that EphA2-Y772A mutation does not disturb the interaction of EphA2 and Shp2 in the NPC cells. Total cell proteins from the 5-8F and CNE2 cells expressing EphA2-WT or EphA2-YA were prepared and subjected to immunoprecipitation (IP) with anti-EphA2 antibody followed by immunoblotting with antibodies against Shp2 or EphA2.

Discussion
EphA2 is an important oncogenic protein and emerging drug target 3,40,41 , but the ligand-independent oncogenic role and mechanism of pY772-EphA2 are unclear. To determine the role of pY772-EphA2 in NPC, we established NPC cell lines expressing the equal levels of exogenous EphA2-WT and EphA2-Y772A using endogenous EphA2 knockdown cells, an appropriate cell model for comparing the differences of biological functions in the EphA2-WT and EphA2-Y772A. Using the established cell lines, we observed that Y772A mutation abolished the effects of EphA2 on NPC cells growth in vitro and in vivo. Our results for the first time demonstrate that pY772-EphA2 is responsible for EphA2-dependent NPC cells growth in vitro and in vivo.
The signals downstream of pY772-EphA2 underlying its growth promotion are completely unclear. Our targeted proteomics analysis found that Shp2 interacts with EphA2 in the NPC cells. Previous studies have demonstrated that Shp2 is required for the activation of Erk-1/2 signaling  Total cell proteins from the infected cells were prepared and subjected to immunoprecipitation immunoprecipitation (IP) with anti-EphA2 antibody followed by immunoblotting with antibodies against p-Shp2 (Y542/Y580), Shp2, or EphA2. c Co-IP analysis showing the levels of Grb2 bound to Shp2 in the EphA2-WT-expressed NPC cells infected with the lentiviral vector expressing Gab1 shRNA, and their control cells. Total cell proteins from the infected cells were prepared and subjected to immunoprecipitation (IP) with anti-Shp2 antibody followed by immunoblotting with antibodies against Grb2 or Shp2. d Co-IP showing the levels of p-Gab1, Gab1, and Grb2 bound to Shp2 in the NPC cells expressing EphA2-WT or EphA2-YA and their control cells. Total cell proteins from the indicated cells were prepared and subjected to immunoprecipitation with anti-Shp2 antibody followed by immunoblotting with antibodies against p-Gab1, Gab1, Grb2, or Shp2. e A model for pY772-EphA2-dependent NPC cell growth. EphA2 is phosphorylated at Y772 by growth factors in a ligand-independent manner and phosphorylation of EphA2 at Y772 (pY772-EphA2) activates Shp2/ Erk-1/2 signaling, and both Gab1 and Grb2 are involved in pY772-EphA2-activating Shp2/Erk-1/2 activation, which promotes NPC cell growth. EphA2 tyrosine kinase inhibitor ALW-II-41-27 inhibits pY772-EphA2, which blocks NPC cell growth.
Whether and how Gab1 and Grb2 are involved in EphA2-activating Shp2/Erk-1/2 signaling pathway in a ligand-independent manner are still unclear 29 . Our results showed that Gab1 is required for EphA2-activating Shp2/ Erk-1/2 signaling pathway possibly by increasing the association of Shp2 with EphA2 and Grb2, and Y772 mutation abolished EphA2-WT-promoting the association of Shp2 with p-Gab1 and Grb2. The results indicate that Gab1 and Grb2 are involved in pY772-EphA2activating Shp2/Erk-1/2 signaling pathway in the NPC cells.
Next, we determined whether Shp2/Erk-1/2 signaling pathway mediates pY772-EphA2-promoting NPC cell proliferation and anchorage-independent growth. Using lose and gain of function approaches, we demonstrate that Shp2/Erk-1/2 signaling mediates pY772-EphA2promoting NPC cell proliferation and anchorageindependent growth. Our data suggest that pY772-EphA2/Shp2/Erk-1/2 signaling axis has an important oncogenic function, highlighting the potential of this signaling axis for treating NPC.
The last question is what phosphorylates EphA2 at Y772 in the NPC cells. It has been reported that fetal bovine serum (FBS) induces pS897-EphA2 in cancer cells by a ligand-independent mechanism 4,13,14,37 . Therefore, we investigated whether FBS also induces pY772-EphA2 in the NPC cells and observed that FBS induced the phosphorylation of EphA2 at Y772 and its downstream Shp2 and Erk-1/2 in the 5-8F and CNE2 NPC cells ( Supplementary Fig. 2), indicating that growth factors in the serum induces ligand-independent pY772-EphA2 in the NPC cells, which is consistent with FBS inducing pS897-EphA2.
In summary, we found that ligand-independent pY772-EphA2 is responsible for EphA2-dependent NPC cell growth in vitro and in vivo by activating the Shp2/Erk-1/ 2 signaling pathway and pY772-EphA2 is a target of ALW-II-41-27 in the NPC cells (Fig. 7e). Our data suggest that pY772-EphA2 can serve as a therapeutic target in NPC and perhaps in other cancers.
Cell lines and culture 5-8F and CNE2 NPC cell lines and 5-8F-shEphA2 and CNE2-shEphA2 NPC cell lines with stable knockdown of endogenous EphA2 by shRNA have been described previously by us 37 . Cells were cultured in RPMI-1640 medium supplemented with 10% FBS (Life Technologies) at 37°C in 5% CO 2 . The cell lines were authenticated by short tandem repeat profiling prior to use and were routinely tested negative for mycoplasma contamination using 4,6-diamidino-2-phenylindole staining.

IP and MS analysis
Total proteins were extracted from NPC 5-8F cells using NP-40 lysis buffer and 1.2 mg of total proteins were incubated with 30 μl Protein A/G-Sepharose™ 4B for 4 h at 4°C followed by centrifugation for 5 min at 4°C. The clarified supernatants were immunoprecipitated with 2 μg anti-EphA2 antibody and 30 μl Protein A/G-Sepharose 4B overnight at 4°C, and centrifuged for 5 min at 4°C. The beads were washed three times with PBS at 4°C and boiled in 2× SDS-PAGE loading buffer for 5 min to elute protein complexes. The elutants were separated on SDS-PAGE gel followed by Coomassie brilliant blue G250 staining and then all protein bands were excised from the gels, in-gel trypsin digested, and subjected to NanoLC-MS/MS analysis with the Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific) to get MS/MS spectra as previously described 42 . Data of MS/MS were searched against Swiss-Prot database (Homo sapiens). Individual ions scores > 35 and unique peptide ≥ 1 indicate identity or extensive homology (P < 0.05) and were considered significant. All proteins deemed to be high confidence interactors of EphA2 were identified by at least two times of three replicates.
Establishment of NPC cell lines expressing exogenous EphA2 or EphA2-Y772A using endogenous EphA2knockdown cells 5-8F-shEphA2 and CNE2-shEphA2 NPC cells were infected with the lentiviral vector GV341 expressing EphA2-Y772A cDNA following the manufacturer's instruction, or transfected with pBabepuro-EphA2 plasmid using Lipofectamine 2000 (ThermoFisher Scientific). Cells were selected using puromycin (ThermoFisher Scientific) for 2 weeks, and 5-8F and CNE2 cell lines with the stable expression of exogenous EphA2 or EphA2-Y772A and their control cell lines were obtained.

Tumor formation assay in nude mice
Nude male mice (BALB/c nu/nu) that were 4 weeks old were obtained from the Laboratory Animal Center of Central South University and maintained in pathogen-free conditions. All animal experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Xiangya Hospital, Central South University, with the approval of the Institutional Animal Ethics Committee.
The mice were randomly divided into the indicated groups before inoculation and 5 × 10 6 cells resuspended in 200 μl of medium without serum were subcutaneously injected into the flanks of mice (n = 5 mice each). The mice were monitored daily for palpable tumor formation and tumor volume (in mm 3 ) was measured by a vernier caliper every 3 days and calculated by using the modified ellipse formula (volume = length × width 2 /2). After 21 days, the mice were killed by cervical dislocation and their tumors were excised, weighed, and embedded in paraffin.

Western blotting
Western blotting was performed as described previously by us 37 . Briefly, proteins were exacted from cells or tissues using RIPA lysis buffer. An equal amount of protein in each sample was subjected to SDS-PAGE separation, followed by blotting onto a PVDF membrane. After blocking, blots were incubated with primary antibody overnight at 4°C, followed by incubation with HRPconjugated secondary antibody for 1 h at room temperature. The signal was visualized with chemiluminescence detection reagent (Millipore).

IP and immunoblotting (Co-IP)
Co-IP was performed to detect protein interaction. In brief, whole cell lysates were incubated with indicated antibodies and Protein G/A-Sepharose 4B overnight at 4°C. After five times wash with RIPA buffer, beads were boiled in 2× SDS-PAGE loading buffer for 5 min to elute protein complexes, followed by SDS-PAGE separation and immunoblotting with specific antibodies.

CCK-8 assay
Cell proliferation was measured using a CCK-8 kit as described previously by us 43 . The assay was performed three times in triplicate.

Plate clone formation assay
Plate colony formation assay was performed to detect cell proliferation described previously by us 43 . The assay was performed three times in triplicate.

EdU incorporation assay
EdU incorporation assay was performed to detect cell proliferation as described previously by us 43 . The assay was performed three times in triplicate.

Soft agar colony formation assay
Soft agar colony formation assay was performed to detect cell anchorage-independent growth as described previously by us 43 . Cells were allowed to grow in the soft agar cultures for 12 days and colonies consisting of >50 cells were counted under the microscope. The assay was performed three times in triplicate.
Staining intensity was categorized as follows: absent staining as 0, weak as 1, moderate as 2, and strong as 3. The percentage of stained cells (examined in at least 500 cells) was categorized as no staining = 0, <30% of stained cells = 1, 30~60% = 2, and >60% = 3. The staining score (ranging from 0 to 6) for each tissue was calculated by adding the area score and the intensity score. A combined staining score of ≤3 was considered to be low expression and >3 was considered to be high expression.

Statistical analysis
Statistical analysis was performed using IBM SPSS statistical software package 22. Data are presented as means ± SD. Qualitative variables were compared by the Student's t-test or χ 2 -test. All statistical tests were twosided and p-values < 0.05 were considered statistically significant.