PTK7, a Catalytically Inactive Receptor Tyrosine Kinase, Increases Oncogenic Phenotypes in Xenograft Tumors of Esophageal Squamous Cell Carcinoma KYSE-30 Cells

Protein tyrosine kinase 7 (PTK7), a catalytically defective receptor protein tyrosine kinase, is upregulated in tumor tissues and cell lines of esophageal squamous cell carcinoma (ESCC). We showed that PTK7 plays an oncogenic role in various ESCC cell lines. However, its role as an oncogene has not been demonstrated in vivo. Here, we examined the influence of PTK7 on the tumorigenic potential of ESCC KYSE-30 cells, which are known to establish xenograft tumors. Overexpression of PTK7 enhanced the proliferation, adhesion, wound healing, and migration of KYSE-30 cells, and these effects were reversed by the knockdown of PTK7. PTK7 overexpression and knockdown, respectively, increased and decreased the tyrosine phosphorylation of cellular proteins and the phosphorylation of ERK, AKT, and FAK, which are important for cell proliferation, survival, adhesion, and migration. Additionally, PTK7 overexpression and silencing, respectively, increased and decreased the weight, volume, and number of Ki-67-positive proliferating cells in xenograft tumors of KYSE-30 cells. Therefore, we propose that PTK7 plays an important role in the tumorigenesis of ESCC cells in vivo and is a potential therapeutic target for ESCC.


Introduction
Protein tyrosine kinase 7 (PTK7) (also known as colon carcinoma kinase-4, CCK-4) is a catalytically defective receptor protein tyrosine kinase (RPTK) molecule that contains an extracellular domain with seven immunoglobulin-like loops, a transmembrane domain, and a tyrosine kinase domain lacking catalytic activity [1].
Homozygous PTK7 knockout mice are perinatally lethal with severe developmental defects, including defective neural tube closure [2]. The PTK7 knockout mice were phenotypically similar to mice and Xenopus with mutations in the planar cell polarity (PCP) genes. PTK7 is also genetically linked to the PCP gene Vangl2. In addition, PTK7 interacts with canonical Wnt signaling pathway proteins, including β-catenin, and activates genes involved in Xenopus development, such as the formation of Spemann's organizer [3]. Moreover, PTK7 functions in non-canonical Wnt signaling by switching off canonical Wnt signaling [4]. PTK7 interacts with Wnt5A, a non-canonical Wnt ligand, and induces morphogenetic cell movements in Xenopus [5]. These findings suggest that PTK7 regulates the PCP and canonical and noncanonical Wnt signaling pathways during development.
Esophageal cancer (EC) is the seventh most common cancer worldwide and the sixth leading cause of malignancy-related deaths [19]. EC is divided into two major subtypes, namely, esophageal adenocarcinoma (EAC) and ESCC. EAC is the most common subtype in Western countries, and the main risk factors include gastroesophageal reflux disease and obesity [20]. Globally, ESCC is the most common EC, with the highest incidence in East Asia and parts of Africa. Cigarette smoking and alcohol consumption are the main risk factors for ESCC [21,22].
We have shown that PTK7 plays a role in enhancing oncogenic properties in TE-6, 9, 10, and 11 ESCC cells harboring TP53 mutations [24]. However, TE-10 cells, which showed a distinct PTK7-dependent tumorigenic response [6,15], did not induce xenograft tumors in nude mice (unpublished data). KYSE-30 ESCC cells harboring epidermal growth factor receptor (EGFR) overexpression and TP53 mutations [25][26][27] can generate tumors in xenografts of orthotopic and subcutaneous implantation [28,29]. In this study, we analyzed the role of PTK7 in oncogenic phenotypes and the activation of signaling proteins in KYSE-30 cells using PTK7 knockdown and overexpression. We then analyzed the role of PTK7 in tumorigenesis in subcutaneous xenograft mice with KYSE-30 cells carrying knocked-down and overexpressed PTK7.

PTK7 Increases the Activation of Oncogenic Signaling Proteins
Cell proliferation, adhesion, and migration involve the activation of various signaling pathways, including the MAPK, PI3-kinase/AKT, and FAK pathways [30][31][32]. We investigated the phosphorylation of these signaling proteins in PTK7-overexpressing or knockdown KYSE-30 cells. The tyrosine phosphorylation of cellular proteins and the phosphorylation of ERK, AKT, and FAK were increased by PTK7 overexpression and decreased by PTK7 knockdown (Figure 3). Thus, PTK7 activates the signaling pathways involved in cell proliferation, adhesion, and migration, resulting in increased tumorigenesis of ESCC KYSE-30 cells.
Hematoxylin and eosin staining and immunohistochemical staining for PTK7 and Ki-67 were performed on the tumor sections to assess changes in cell morphology and proliferation. PTK7 expression was elevated in PTK7-FLAG tumors and de-creased in PTK7-KD-6433 and -6434 tumors compared to that in the control tumors ( Figures 4C and 5C). Expression of Ki-67, a proliferation marker in solid tumors, was stronger (252.1 ± 29.2%) in PTK7-overexpressing tumors and weaker (30.6 ± 10.1% in PTK7-KD-6433 and 26.8 ± 4.0% in PTK7-KD-6434) in PTK7-knockdown tumors, compared to that in control tumors ( Figures 4C and 5C). This confirmed that PTK7 promotes tumor progression in ESCC.

Discussion
Analysis of the biological pathways deregulated in ESCC showed that the RPTK-MAPK-PI3K pathway, cell cycle, and epigenetic regulatory mechanisms are frequently dysregulated by multiple molecular abnormalities [33][34][35]. In ESCC tumor tissues, EGFR is frequently overexpressed or often amplified, with activation of the PI3K/AKT signaling pathway due to mutations in the PI3KCA gene and loss of PTEN expression [36][37][38]. TP53 and CDKN2A mutations and CCND1 amplification are additional changes observed in the genes regulating the cell cycle [39,40]. Mutations are often found in genes involved in epigenetic regulation, such as histone H3 lysine-4 mono-methyltransferases (KMT2D/MLL2 and KMT2C/MLL3) and histone acetyltransferases (CREBBP and EP300) [39].
EGFR overexpression and TP53 mutations are common in precancerous ESCC lesions [36,41]. TP53 mutations were also correlated with EGFR overexpression. These findings suggest that patients with ESCC may benefit from EGFR-targeted therapy. Nevertheless, the EGFR-neutralizing antibody cetuximab only reacted to ESCC patient-derived xenografts with high EGFR expression and/or amplification [42]. In addition, gefitinib, a low molecular weight EGFR inhibitor, did not improve overall survival in unselected patients with EC, including ESCC, but provided a palliative effect in a subgroup of difficultto-treat patients with short life expectancy [43]. Another low molecular weight EGFR inhibitor, icotinib, was effective in 17.6% of patients with high EGFR-expressing tumors, but not in patients with moderate EGFR-expressing tumors [44]. EGFR inhibitors are effective for epithelial-like ESCC cells but are ineffective for mesenchymal-like ESCC cells, as EGFR signaling cannot be blocked [45]. EGFR inhibitors alone are, therefore, considered less effective in treating ESCC.
PTK7 is upregulated in various cancer types, including ESCC [6,7]. PTK7 reduces apoptosis and promotes proliferation, survival, migration, invasion, and wound healing in ESCC cells [6,16]. PTK7 knockdown reduced the phosphorylation of Akt, Erk, and FAK [6]. PTK7 upregulates MMP-9 through activation of AP-1 and NF-κB, thus increasing the invasive properties of ESCC cells [15]. These results suggest that PTK7 has potential as a prognostic marker for ESCC and could be a candidate for targeted therapy.
In our analysis, the PTK7 levels in ESCC TE-10 and TE-11 cells were higher than those in TE-5, TE-9, and TE-14 cells [6]. TE-10 cells exhibited a PTK7-dependent tumorigenic response, including cell proliferation, survival, wound healing, and invasion [6,15]. TE-10 cells harbor a TP53 mutation [24] and co-amplified INT-2/FGF3 and HST-1/FGF4. The INT-2/FGF3 and HST-1/FGF4 polypeptides are members of the FGF family, which have mitogenic activity in various cell types [46]. Additionally, PTK7 knockdown reduced not only ligand-free and FGF-induced FGFR1 phosphorylation, but also the interaction of signaling adaptor proteins with FGFR1 and activation of downstream signaling proteins in TE-10 cells [23]. Collectively, these results suggest that the FGF/FGFR signaling pathway, in cooperation with PTK7, plays an important role in the pathogenesis of EC in an autocrine or paracrine manner.
We also wanted to use a mouse xenograft model of ESCC cells to demonstrate the role of PTK7 in tumorigenesis. However, it was unclear whether TE-10 cells could establish xenograft tumors in nude mice. Nishihara et al. reported that xenograft tumors could not be generated [46], while Yang et al. recently reported that xenograft tumors were produced using TE-10 cells [47]. However, we were unable to generate tumors after performing xenografts in nude mice with TE-10 cells (unpublished data).
In this study, we showed that KYSE-30 cells express PTK7 at lower levels relative to TE-10 cells. Consistent with our previous results using other ESCC cells [6,15,50], PTK7 overexpression increased proliferation, adhesion, wound healing, and migration as well as tyrosine phosphorylation of cellular proteins and phosphorylation of ERK, AKT, and FAK in KYSE-30 cells. When PTK7 was silenced in KYSE-30 cells, these effects were reversed. ERK inhibition by PD98059 (MEK inhibitor) and Akt inhibition by LY294002 (PI3K inhibitor) in ESCC TE-10 cells, in which PTK7 expression increases invasion via MMP-9 secretion, decreased MMP-9 secretion [15]. Similarly, ERK inhibition by PD98059 and Akt inhibition by LY294002 significantly suppress migration and invasion in ESCC TE-8 and TE-9 cells [51]. Furthermore, knockdown or inhibition (by defactinib) of FAK decreases proliferation, migration, and invasion in various ESCC cells, including KYSE-30 cells [52,53]. Therefore, PTK7-dependent activation of ERK, Akt and FAK appears to be directly related to the oncogenic phenotypes.
Various studies have shown that PTK7 and FGFR1 are significantly upregulated in ESCC tumor tissues and cell lines [6,23,54]. We also demonstrated that PTK7 plays an important role in FGFR1 activation in various ESCC cells [23]. Therefore, regulation of FGFR1 activity, based on PTK7 expression, could possibly modulate oncogenic phenotypes and signaling pathways. However, considering the high EGFR levels in KYSE-30 cells, it was interesting how PTK7 knockdown could reduce oncogenic processes. It was recently reported that PTK7 is involved in the activation of EGFR and Akt signaling in triple-negative breast cancer cells [55]. These results suggest that counteracting PTK7 can efficiently reduce the tumorigenesis of esophageal squamous cells in ESCC cells by blocking FGFR and EGFR signaling.
Importantly, we could demonstrate that xenograft tumors of KYSE-30 cells with PTK7 overexpression and silencing respectively increased and decreased the weight, volume, and number of Ki-67-positive proliferating cells. This proved that PTK7 expression is positively correlated with the tumorigenic process of ESCC in vivo. In addition, this xenograft tumor model of KYSE-30 cells can be used to analyze anti-cancer agents targeting PTK7 and the role of PTK7 in vivo.
Lysates were subjected to SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. Blots were incubated with the indicated antibodies, and the immunoreactive bands were visualized using West-Q PICO Dura ECL solution (GenDepot, Barker, TX, USA), Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore), and an LAS-3000 imaging system (Fujifilm, Tokyo, Japan).

Cell Proliferation Assay
Cell proliferation assays were performed as previously described [58]. Briefly, cells

Cell Adhesion Assay
The cell adhesion assay was performed as previously described [59]. Briefly, cells were incubated in serum-free DMEM/F12 medium for 24 h. Detached cell suspensions (3 × 10 4 cells/0.1 mL for overexpression analysis or 5 10 4 cells/0.1 mL for knockdown analysis) were loaded onto 96-well plates, which were precoated with rat-tail type I collagen (1 µg/well) overnight and were incubated in DMEM/F12 medium with 1% FBS for 1 h. Cells were fixed with 3.7% paraformaldehyde in PBS and stained with 0.005% crystal violet. The stained cells were lysed with 1% SDS, and the absorbance was measured at 600 nm.

Wound Healing and Chemotactic Migration Assay
A wound was introduced by scraping the monolayer with a micropipette tip. Cells were incubated for 24 h in DMEM/F12 medium containing 2% FBS and evaluated by light microscopy [59]. The chemotactic migration assay was performed as previously described [50]. Briefly, detached cell suspensions (7 × 10 4 cells/0.1 mL serum-free DMEM/F12 medium) were loaded into the upper compartment of transwell chambers (Corning, Tewksbury, MA, USA). The bottom surface of each transwell was coated with 10 µL of 0.1% gelatin. The lower compartment of each well was filled with 0.65 mL DMEM/F12 medium, with 10% FBS as a chemoattractant. The chamber was then incubated at 37 • C for 24 h. After incubation, the remaining cells in the upper compartment were removed using a cotton swab. Cells that migrated to the bottom surface of the filter were fixed with 3.7% paraformaldehyde in PBS and stained with 0.005% crystal violet. The stained cells were solubilized with 1% SDS, and the absorbance was measured at 600 nm.

Xenograft Mouse Model
Four to five-week-old male immune-deficient athymic nude (BALB/c nu/nu) mice were purchased from Orient Bio Inc. (Gyeonggi, Korea). To investigate the effect of PTK7 on tumorigenesis in a xenograft mouse model, 1 × 10 6 KYSE-30 cells expressing either PTK7-FLAG or PTK7 shRNA (PTK7-KD-6433 and 6434) were resuspended in growth-factorreduced Matrigel (Corning) and subcutaneously injected into the backs of mice. Tumor growth in each group was evaluated by measuring the tumor size twice per week using calipers (length × width × depth/2). Mice were sacrificed 6 weeks after cell injection. The xenograft tumors were recovered to measure tumor weight, fixed in formalin, and were paraffin embedded for histological and immunohistochemical analyses [60].

Ethics Statement
All animal experiments were approved and performed in accordance with the Institutional Animal Care and Use Committee (IACUC) review board of the National Cancer Center, which is an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) accredited facility that abides by the Institute of Laboratory Animal Resources guide (NCC-20-561).

Statistical Analysis
Statistical analyses were performed using Microsoft Excel (Microsoft Corp., Redmond, WA, USA). Statistical significance was analyzed using the Student's t test. For all tests, p values less than 0.05 were considered statistically significant.

Conclusions
We previously showed that PTK7 expression correlates with the promotion of oncogenic properties in several ESCC cells. In this study, we analyzed the effect of PTK7 on tumorigenicity at the cellular level and in vivo using KYSE-30 cells, which are known to produce xenograft tumors in nude mice. KYSE-30 cells expressed PTK7 at a relatively lower level than TE-10 cells. Overexpression of PTK7 increased proliferation, adhesion, wound healing, and migration, as well as tyrosine phosphorylation of cellular proteins and phosphorylation of ERK, AKT, and FAK in KYSE-30 cells. PTK7 silencing in KYSE-30 cells was reversed by PTK7 overexpression. PTK7 overexpression and silencing increased and decreased, respectively, the weight, volume, and number of Ki-67-positive proliferating cells in xenograft tumors of KYSE-30 cells. The results showed that the expression of PTK7 in vivo correlates positively with the oncogenic process of ESCC. Furthermore, a xenograft tumor model using KYSE-30 cells can be used to analyze PTK7-targeted anti-cancer drugs and the role of PTK7 in vivo.

Conflicts of Interest:
The authors declare no conflict of interest.