Recent studies showed that several pseudokinases from the receptor tyrosine kinase family are important players in regulating cancer cell invasion, metastasis, and drug resistance, suggesting that targeting these proteins can play a therapeutic role in cancer treatment. Receptor Tyr kinase-like orphan receptors (RORs), protein Tyr kinase 7 (PTK7) (also called colon carcinoma kinase 4 (CCK4)), and receptor-like Tyr kinase (RYK) are Wnt ligand binding receptors within the non-canonical Wnt signaling, with important roles in development, tissue homeostasis, and organogenesis. At the cellular level, these receptors transduce signals important for cell survival, migration, polarization, and chemotaxis. Considerable progress has been made in the last decade in the field of pseudokinase signaling, improving our understanding of their structure-function mechanisms, and intracellular network of transduction components. Consequently, their role in various diseases, including cancer, is now scrutinized for therapeutic interventions to improve treatment outcome. In this article, we review findings regarding molecular mechanisms and targeted therapies for ROR1, PTK7, and RYK in hematological malignancies.

MicroRNAs (miRNAs) are small non‑coding RNAs, which are critical in a diverse range of biological processes, including development, differentiation, homeostasis, and in the formation of diseases by accelerating and/or inhibiting the translation of mRNAs. The present study aimed to examine the potential role of miRNA (miR)‑205‑5p in the developmental process of colorectal cancer (CRC) through protein‑tyrosine kinase 7 (PTK7). Initially, TargetScan was used to predict the miRNA target sites in the sequence of the PTK7 3'‑untranslated region. It was then found that the mRNA expression level of miR‑205‑5p was lower in CRC cells, determined using reverse transcription‑quantitative polymerase chain reaction analysis, and there was a negative correlation between miR‑205‑5p and PTK7 in CRC tissues. It was also found that miR‑205‑5p regulated the gene transcription of PTK7, determined using a luciferase reporter assay. The results of RT‑qPCR and western blot analyses in human colorectal cancer revealed that miR‑205‑5p suppressed the expression of PTK7. Finally, it was revealed that miR‑205‑5p restricted the proliferation ability of CRC cells through inhibiting PTK7, which was determined using colony forming and 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assays. miR‑205‑5p accelerated cell apoptosis through inhibiting PTK7, demonstrated using Annexin V‑FITC/propidium iodide staining. The results of a Transwell assay indicated that miR‑205‑5p inhibited the migration and invasion abilities of CRC cells through inhibiting PTK7. Therefore, miR‑205‑5p is involved in the proliferation, migration and invasion of CRC through inhibiting PTK7.

Esophageal cancer is one of the most common types of cancer, which is a leading cause of cancer-related death worldwide. Based on histological behavior, it is mainly of two types (i) Esophageal squamous cell carcinoma (ESCC), and (ii) esophageal adenocarcinoma (EAD or EAC). In astronomically immense majority of malignancies, receptor tyrosine kinases (RTKs) have been kenned to play a consequential role in cellular proliferation, migration, and metastasis of the cells. The post-translational modifications (PTMs) including phosphorylation of tyrosine (pY) residue of the tyrosine kinase (TK) domain have been exploited for treatment in different malignancies. Lung cancer where pY residues of EGFR have been exploited for treatment purpose in lung adenocarcinoma patients, but we do not have such kind of felicitously studied and catalogued data in ESCC patients. Thus, the goal of this review is to summarize the studies carried out on ESCC to explore the role of RTKs, tyrosine kinase inhibitors, and their pertinence and consequentiality for the treatment of ESCC patients.

Bromodomain and extraterminal domain inhibitors (BETi) represent promising therapeutic agents for metastatic melanoma, yet their mechanism of action remains unclear. Here we interrogated the transcriptional effects of BETi and identified AMIGO2, a transmembrane molecule, as a BET target gene essential for melanoma cell survival. AMIGO2 is upregulated in melanoma cells and tissues compared to human melanocytes and nevi, and AMIGO2 silencing in melanoma cells induces G1/S arrest followed by apoptosis. We identified the pseudokinase PTK7 as an AMIGO2 interactor whose function is regulated by AMIGO2. Epigenomic profiling and genome editing revealed that AMIGO2 is regulated by a melanoma-specific BRD2/4-bound promoter and super-enhancer configuration. Upon BETi treatment, BETs are evicted from these regulatory elements, resulting in AMIGO2 silencing and changes in PTK7 proteolytic processing. Collectively, this study uncovers mechanisms underlying the therapeutic effects of BETi in melanoma and reveals the AMIGO2-PTK7 axis as a targetable pathway for metastatic melanoma.

BACKGROUND: Overexpression of PTK7 has been found in multiple cancers and has been proposed to serve as a prognostic marker for intrahepatic cholangiocarcinoma. Its role in esophageal cancer, however, remains to be clarified. We hypothesize that PTK7 positively regulates tumorigenesis of esophageal cancer.
METHODS: We examined PTK7 expression pattern in human esophageal squamous carcinoma by Oncomine expression analysis and by immunohistochemistry (IHC) staining. We knocked down PTK7 in two esophageal squamous cell carcinoma cell lines, TE-5, and TE-9, by siRNA, and evaluated cell proliferation, apoptosis, and migration ofPTK7-defective cells. Expressions of major apoptotic regulators and effectors were also determined by quantitative real-time PCR in PTK7-defective cells. We further overexpressed PTK7 in the cell to evaluate its effects on cell proliferation, apoptosis, and migration.
RESULTS: Both Oncomine expression and IHC analyses showed that PTK7 is overexpressed in clinical esophageal squamous cell carcinoma tumors. PTK7 siRNA suppressed cell growth and promoted apoptosis of TE-5 and TE-9. PTK7-defective cells further displayed reduced cellular migration that was concomitant with upregulation of E-cadherin. Conversely, overexpression of PTK7 promotes cell proliferation and invasion, while apoptosis of the PTK7-overexpressing cells is repressed. Notably, major apoptotic regulators, such as p53 and caspases, are significantly upregulated in siPTK7 cells.
CONCLUSIONS: PTK7 plays an oncogenic role in tumorigenesis and metastasis of esophageal squamous carcinoma. PTK7 achieves its oncogenic function in esophageal squamous cell carcinoma partially through the negative regulation of apoptosis.

Novel discoveries involving the evaluation of potential therapeutics are based on newly identified molecular targets for atypical teratoid rhabdoid tumors (ATRT), which are the most common form of infantile brain tumors. Central nervous system ATRTs are rare, aggressive, and fast growing tumors of the brain and spinal cord and carry a very poor prognosis. Currently, the standard of care for ATRT patients is based on surgical resection followed by systemic chemotherapy and radiotherapy, which result in severe side effects. As protein tyrosine kinases have proven to be actionable targets that reduce tumor growth in a number of cancers, we examined how inhibiting tyrosine kinases affected ATRT tumor growth. Here, we examine the therapeutic efficacy of the broad-spectrum tyrosine kinase inhibitor vatalanib in the treatment of ATRT. Vatalanib significantly reduced the growth of ATRT tumor cell lines, both in two-dimensional cell culture and in three-dimensional cell culture using a spheroid model. As vatalanib had a remarkable effect on the growth of ATRT, we decided to use a transcriptomic approach to therapy by examining new actionable targets, such as tyrosine kinases. Next-generation RNA-sequencing and NanoString data analysis showed a significant increase in

Protein tyrosine kinase 7 (PTK7), a member of the catalytically defective receptor protein tyrosine kinase family, is upregulated in various cancers including esophageal squamous cell carcinoma (ESCC). Here, we have explored the molecular mechanism of PTK7-dependent invasiveness in ESCC cells. PTK7 knockdown reduced gelatin degradation and MMP-9 secretion in cultures of ESCC TE-10 cells, and showed reduced levels of MMP9 mRNA using real-time RT-PCR and luciferase reporter assays. PTK7 knockdown decreased not only phosphorylation of NF-κB, IκB, ERK, and JNK, but also nuclear localization of NF-κB and AP-1 consisting of c-Fos and c-Jun. Activation of AP-1 and NF-κB requires PTK7-mediated activation of tyrosine kinases, including Src. In addition, NF-κB activation by PTK7 involves the PI3K/Akt signaling pathway. PTK7-mediated upregulation of MMP9 was also observed in other ESCC cell lines and in three-dimensional cultures of TE-10 cells. Moreover, MMP-9 expression positively correlated with PTK7 expression in ESCC tumor tissue. These findings demonstrate that PTK7 upregulates MMP9 through activation of AP-1 and NF-κB and, thus increases invasive properties of ESCC cells.

Tumor radioresistance is a major reason for decreased efficiency of cancer radiation therapy. Although a number of factors involved in radioresistance have been identified, the molecular mechanisms underlying radioresistance of esophageal squamous cell carcinoma (ESCC) have not been elucidated. In this study, we investigated the role of oncogenic protein tyrosine kinase 7 (PTK7) in the resistance of ESCC to radiation therapy. ESCC cell lines with high PTK7 expression were more refractive to radiation than those with low PTK7 levels. In radioresistant ESCC cells, PTK7 knockdown by specific siRNAs decreased the survival of irradiated cells and increased radiation-induced apoptosis, while in radiosensitive ESCC cells, PTK7 overexpression promoted cell survival and inhibited radiation-induced apoptosis. We hypothesized that PTK7 could regulate the activation of transcription factor NF-kB known for its role in cancer radioresistance. Our results indicated that the inhibition of PTK7 suppressed nuclear translocation of NF-kB subunit p65 induced by radiation, suggesting relevance of PTK7 expression with NF-kB activation in radioresistant ESCC. Furthermore, the levels of inhibitor of apoptosis proteins (IAPs), XIAP, and survivin, encoded by NF-kB-regulated genes, were induced in irradiated radioresistant cells but not in radiosensitive cells, while PTK7 knockdown downregulated IAP expression. Our findings revealed a novel mechanism underlying radioresistance in ESCC, which is associated with PTK7 and NF-kB-dependent apoptosis. These results suggest that the manipulation of PTK7 expression can be instrumental in enhancing ESCC response to radiotherapy. This study demonstrates that PTK7 plays a significant role in ESCC radioresistance via the NF-kB pathway.

Protein tyrosine kinases (PTKs) play key roles in cellular signal transduction, cell cycle regulation, cell division, and cell differentiation. Dysregulation of PTK-activated pathways, often by receptor overexpression, gene amplification, or genetic mutation, is a causal factor underlying numerous cancers. In this study, we have developed a parallel reaction monitoring-based assay for quantitative profiling of 83 PTKs. The assay detects 308 proteotypic peptides from 54 receptor tyrosine kinases and 29 nonreceptor tyrosine kinases in a single run. Quantitative comparisons were based on the labeled reference peptide method. We implemented the assay in four cell models: 1) a comparison of proliferating versus epidermal growth factor-stimulated A431 cells, 2) a comparison of SW480Null (mutant APC) and SW480APC (APC restored) colon tumor cell lines, and 3) a comparison of 10 colorectal cancer cell lines with different genomic abnormalities, and 4) lung cancer cell lines with either susceptibility (11-18) or acquired resistance (11-18R) to the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. We observed distinct PTK expression changes that were induced by stimuli, genomic features or drug resistance, which were consistent with previous reports. However, most of the measured expression differences were novel observations. For example, acquired resistance to erlotinib in the 11-18 cell model was associated not only with previously reported up-regulation of MET, but also with up-regulation of FLK2 and down-regulation of LYN and PTK7. Immunoblot analyses and shotgun proteomics data were highly consistent with parallel reaction monitoring data. Multiplexed parallel reaction monitoring assays provide a targeted, systems-level profiling approach to evaluate cancer-related proteotypes and adaptations. Data are available through Proteome eXchange Accession PXD002706.

UNLABELLED: Protein tyrosine kinase-7 (PTK7), a member of receptor tyrosine kinase superfamily initially identified as colon carcinoma kinase-4, is highly expressed in various human malignancies. Its expression was found to correlate with aggressive biologic behaviors such as increased cell proliferation, invasiveness, and migration. Despite the importance and unmet need of imaging PTK7 in vivo, there is currently no clinically relevant method to visualize tumoral PTK7 expression noninvasively such as PET or SPECT. This study aimed to develop a specific, selective, and high-affinity PET radioligand based on single-stranded DNA aptamer to address this challenge.
METHODS: Sgc8, a 41-oligonucleotide that targets to PTK7, was labeled with (18)F using a 2-step radiochemical synthesis, which featured a direct 1-step radiofluorination on the distinctive spirocyclic hypervalent iodine(III) precursor to give (18)F-fluorobenzyl azide followed by copper-mediated click conjugation with Sgc8-alkyne. (18)F-Sgc8 was evaluated in vitro and in vivo in 2 cell lines, HCT116 and U87MG, which express high and low amounts of PTK7, respectively.
RESULTS: Sgc8 was labeled efficiently with (18)F in an isolated radiochemical yield of 62% ± 2%, non-decay-corrected based on (18)F-fluorobenzyl azide. (18)F-Tr-Sgc8 was found to possess high-affinity binding to both cell lines, with binding affinity values of 2.7 ± 0.6 nM for HCT116 and 16.9 ± 2.1 nM for U87MG. In vivo PET imaging clearly visualized PTK7 expression in HCT116 xenografted mice, with tumor uptake of 0.76 ± 0.09 percentage injected dose per gram (%ID/g) at 30 min after injection for the subcutaneous tumor model and greater than 1.5 %ID/g for the liver metastasis model. U87MG xenograft tumors had much lower tracer accumulation (0.13 ± 0.06 %ID/g at 30 min after injection), which was consistent with the lower expression of PTK7 in this tumor model. The labeled aptamer was rapidly cleared from the blood through the kidneys and bladder to give high tumor-to-blood and tumor-to-muscle ratios of 7.29 ± 1.51 and 10.25 ± 2.08, respectively.
CONCLUSION: The (18)F-radiolabeling methodology shown here is a robust procedure for labeling aptamers and similar chemical moieties and can be applied to many different targets. Quantification of PTK7 using (18)F-Tr-Sgc8 may be suitable for clinical translation and might help in the future to select and monitor appropriate therapies.

Biomarkers and novel therapeutic targets are urgently needed in colorectal cancer (CRC). The pseudo tyrosine kinase receptor 7 (PTK7) is involved in planar cell polarity and it is deregulated in various malignancies, including CRC. Yet, little is known about its protein expression in human CRC, or about a possible correlation of its expression with clinical endpoints. Using a clinically annotated Tissue MicroArray (TMA) produced from from 192 consecutive CRC patients treated by initial surgery, we examined PTK7 expression by immunohistochemistry in tumoral tissue and matched normal mucosae, and correlated its expression with clinico-pathological features and patient outcome. PTK7 depletion by specific shRNA in HCT116 and HCT15 CRC cell lines was found to affect cell proliferation, resistance to drugs and cell migration. Tumor growth and metastatic phenotype were investigated in vivo using a xenograft mouse model of CRC cells with modulated expression of PTK7 levels. PTK7 was significantly up-regulated in CRC tissue as compared to matched healthy mucosae, and significant overexpression was found in 34% of patients. PTK7 overexpression was significantly associated with a reduced metastasis-free survival in non-metastatic patients. In HCT116 and HCT15 cells, shRNA PTK7 reduced migration but did not affect cell proliferation and resistance to drugs. In a xenograft mouse of HCT15 cells, downregulation of PTK7 led to reduced tumor growth, whereas its overexpression in PTK7-negative cancer cells led to increased metastatic events. PTK7 expression thus represents a potential prognostic biomarker and a novel therapeutic target in CRC.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the top five causes of cancer-related deaths worldwide. We developed a novel technique to identify cancer-related genes of HCC as follows: triple-combination array analysis, which combines gene expression profiles, single nucleotide polymorphism arrays, and methylation arrays.
MATERIALS AND METHODS: Triple-combination array analysis was performed on one HCC sample from a 68-y-old female patient, and one candidate cancer-related gene was selected. Subsequently, we analyzed the identified gene by quantitative real-time reverse-transcriptase polymerase chain reaction (PCR) and methylation-specific PCR in nine HCC cell lines and in samples from 48 HCC patients. Additionally, we evaluated gene expression by immunohistochemistry and Western blotting.
RESULTS: Using this method, protein tyrosine kinase 7 (PTK7) was detected as a candidate cancer-related gene. PTK7 was revealed to be hypermethylated (methylation value 0.826, range 0-1.0) in cancer tissue, compared with that of adjacent noncancerous tissues (0.047) by methylation array. Of the 48 clinical samples, 30 HCC samples (62.5%) showed PTK7 promoter hypermethylation. Downregulation of PTK7 (expressions in tumor tissues decreased by ≥ 50% compared with the noncancerous tissues) was significantly associated with age >60 y (P = 0.030) and elevation in serum protein induced by vitamin K absence or antagonists-II (P = 0.033). Moreover, patients with downregulation were significantly inferior in overall survival (P < 0.001) than the others.
CONCLUSIONS: Our data imply that PTK7 acts as a cancer-related gene and may be a potent prognostic marker for HCC. Triple-combination array analysis was once again found to be useful in identifying cancer-related genes.

BACKGROUND: CD44 is a molecular marker associated with molecular subtype and treatment resistance in glioma. More effective therapies will result from approaches aimed at targeting the CD44-high gliomas.
METHODS: Protein tyrosine kinase 7 (PTK7) mRNA expression was analyzed based on The Cancer Genome Atlas glioblastoma dataset. PTK7 expression was depleted through lentivirus-mediated short hairpin RNA knockdown. Terminal deoxynucleotidyl transferase dUTP nick-end labeling was used to evaluate cell apoptosis following PTK7 knockdown. Gene expression analysis was performed on Affymetrix microarray. A nude mice orthotopic tumor model was used to evaluate the in vivo effect of PTK7 depletion.
RESULTS: PTK7 is highly expressed in CD44-high glioblastoma and predicts unfavorable prognosis. PTK7 knockdown attenuated cell proliferation, impaired tumorigenic potential, and induced apoptosis in CD44-high glioma cell lines. Gene expression analysis identified inhibitor of DNA Binding 1 (Id1) gene as a potential downstream effector for PTK7. Overexpression of Id1 mostly restored the cell proliferation and colony formation attenuated by PTK7 depletion. PTK7 enhanced anchorage-independent growth in normal human astrocytes, which was attenuated by Id1 knockdown. Furthermore, PTK7 regulated Id1 expression through modulating TGF-β/Smad signaling, while pharmacological inhibition on TGF-β/Smad signaling or PTK7/Id1 depletion attenuated TGF-β-stimulated cell proliferation. PTK7 depletion consistently reduced Id1 expression, suppressed tumor growth, and induced apoptosis in a murine orthotopic tumor model, which could be translated into prolonged survival in tumor-bearing mice.
CONCLUSIONS: PTK7 regulates Id1 expression in CD44-high glioma cell lines. Targeting PTK7 could be an effective strategy for treating glioma with high CD44 expression.

Ovarian cancer (OC) can be classified into five biologically distinct molecular subgroups: epithelial-A (Epi-A), Epi-B, mesenchymal (Mes), Stem-A and Stem-B. Among them, Stem-A expresses genes relating to stemness and is correlated with poor clinical prognosis. In this study, we show that frizzled family receptor 7 (FZD7), a receptor for Wnt signalling, is overexpressed in the Stem-A subgroup. To elucidate the functional roles of FZD7, we used an RNA interference gene knockdown approach in three Stem-A cell lines: CH1, PA1 and OV-17R. Si-FZD7 OC cells showed reduced cell proliferation with an increase in the G0/G1 sub-population, with no effect on apoptosis. The cells also displayed a distinctive morphologic change by colony compaction to become more epithelial-like and polarised with smaller internuclear distances and increased z-axis height. Immunofluorescence (IF) staining patterns of pan-cadherin and β-catenin suggested an increase in cadherin-based cell-cell adhesion in si-FZD7 cells. We also observed a significant rearrangement in the actin cytoskeleton and an increase in tensile contractility in si-FZD7 OC cells, as evident by the loss of stress fibres and the redistribution of phospho-myosin light chain (pMLC) from the sites of cell-cell contacts to the periphery of cell colonies. Furthermore, there was reciprocal regulation of RhoA (Ras homolog family member A) and Rac1 (Ras-related C3 botulinum toxin substrate 1 (Rho family, small GTP-binding protein Rac1)) activities upon FZD7 knockdown, with a significant reduction in RhoA activity and a concomitant upregulation in Rac1 activity. These changes in pMLC and RhoA, as well as the increased TopFlash reporter activities in si-FZD7 cells, suggested involvement of the non-canonical Wnt/planar cell polarity (PCP) pathway. Selected PCP pathway genes (cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3), prickle homolog 4 (Drosophila) (PRICKLE4), dishevelled-associated activator of morphogenesis 1 (DAAM1), profilin 2 (PFN2), protocadherin 9 (PCDH9), protocadherin α1 (PCDHA1), protocadherin β17 pseudogene (PCDHB17), protocadherin β3 (PCDHB3), sprouty homolog 1 (SPRY1) and protein tyrosine kinase 7 (PTK7)) were found to be more highly expressed in Stem-A than non Stem-A subgroup of OC. Taken together, our results suggest that FZD7 might drive aggressiveness in Stem-A OC by regulating cell proliferation, cell cycle progression, maintenance of the Mes phenotype and cell migration via casein kinase 1ɛ-mediated non-canonical Wnt/PCP pathway.

Protein tyrosine kinase 7 (PTK7) is a catalytically inactive receptor tyrosine kinase that is also known as colon carcinoma kinase-4 (CCK-4). Recent reports have shown that PTK7 plays an important role in carcinogenesis, and it is known to be upregulated in gastric, colon and esophageal cancer, as well as in liposarcoma. However, the role of PTK7 in lung cancer has not been investigated. The aim of the present study was to investigate the expression levels and the role of PTK7 in lung cancer. We found that PTK7 expression was downregulated at the mRNA as well as protein levels in human lung squamous cell carcinoma (LSCC). Upon investigation of the functional role of PTK7 in LSCC, we found that overexpression of PTK7 in LSCC cells resulted in inhibition of cell proliferation, invasion and migration. Furthermore, we confirmed that these phenotypic changes are associated with the inactivation of AKT and ERK. Our findings suggest that PTK7 has different oncogenic roles in organs and target tumors.

The identification of biomarkers indicating the level of aggressiveness of prostate cancer (PCa) will address the urgent clinical need to minimize the general overtreatment of patients with non-aggressive PCa, who account for the majority of PCa cases. Here, we isolated formerly N-linked glycopeptides from normal prostate (n = 10) and from non-aggressive (n = 24), aggressive (n = 16), and metastatic (n = 25) PCa tumor tissues and analyzed the samples using SWATH mass spectrometry, an emerging data-independent acquisition method that generates a single file containing fragment ion spectra of all ionized species of a sample. The resulting datasets were searched using a targeted data analysis strategy in which an a priori spectral reference library representing known N-glycosites of the human proteome was used to identify groups of signals in the SWATH mass spectrometry data. On average we identified 1430 N-glycosites from each sample. Out of those, 220 glycoproteins showed significant quantitative changes associated with diverse biological processes involved in PCa aggressiveness and metastasis and indicated functional relationships. Two glycoproteins, N-acylethanolamine acid amidase and protein tyrosine kinase 7, that were significantly associated with aggressive PCa in the initial sample cohort were further validated in an independent set of patient tissues using tissue microarray analysis. The results suggest that N-acylethanolamine acid amidase and protein tyrosine kinase 7 may be used as potential tissue biomarkers to avoid overtreatment of non-aggressive PCa.

Lung cancer remains the most common cause of cancer-related death worldwide and it continues to lack effective treatment. The increasingly large and diverse public databases of lung cancer gene expression constitute a rich source of candidate oncogenic drivers and therapeutic targets. To define novel targets for lung adenocarcinoma, we conducted a large-scale meta-analysis of genes specifically overexpressed in adenocarcinoma. We identified an 11-gene signature that was overexpressed consistently in adenocarcinoma specimens relative to normal lung tissue. Six genes in this signature were specifically overexpressed in adenocarcinoma relative to other subtypes of non-small cell lung cancer (NSCLC). Among these genes was the little studied protein tyrosine kinase PTK7. Immunohistochemical analysis confirmed that PTK7 is highly expressed in primary adenocarcinoma patient samples. RNA interference-mediated attenuation of PTK7 decreased cell viability and increased apoptosis in a subset of adenocarcinoma cell lines. Further, loss of PTK7 activated the MKK7-JNK stress response pathway and impaired tumor growth in xenotransplantation assays. Our work defines PTK7 as a highly and specifically expressed gene in adenocarcinoma and a potential therapeutic target in this subset of NSCLC.

BACKGROUND: The full-length membrane protein tyrosine kinase 7 (PTK7) pseudokinase, an important component of the planar cell polarity and the Wnt canonical and non-canonical pathways, is a subject of step-wise proteolysis in cells and tissues. The proteolysis of PTK7 involves membrane type-matrix metalloproteinase (MT1-MMP), members of the Disintegrin Domain and Metalloproteinase (ADAM) family, and γ-secretase. This multi-step proteolysis results in the generation of the digest fragments of PTK7. These fragments may be either liberated into the extracellular milieu or retained on the plasma membrane or released into the cytoplasm and then transported into the nucleus.
RESULTS: We employed the genome-wide transcriptional and kinome array analyses to determine the role of the full-length membrane PTK7 and its proteolytic fragments in the downstream regulatory mechanisms, with an emphasis on the cell migration-related genes and proteins. Using fibrosarcoma HT1080 cells stably expressing PTK7 and its mutant and truncated species, the structure of which corresponded to the major PTK7 digest fragments, we demonstrated that the full-length membrane 1-1070 PTK7, the N-terminal 1-694 soluble ectodomain fragment, and the C-terminal 622-1070 and 726-1070 fragments differentially regulate multiple genes and signaling pathways in our highly invasive cancer cell model. Immunoblotting of the selected proteins were used to validate the results of our high throughput assays.
CONCLUSIONS: Our results suggest that PTK7 levels need to be tightly controlled to enable migration and that the anti-migratory effect of the full-length membrane PTK7 is linked to the down-regulation of multiple migration-related genes and to the activation of the Akt and c-Jun pathway. In turn, the C-terminal fragments of PTK7 act predominantly via the RAS-ERK and CREB/ATF1 pathway and through the up-regulation of cadherin-11. In general, our data correlate well with the distinct functionality of the full-length receptor tyrosine kinases and their respective intracellular domain (ICD) proteolytic fragments.

BACKGROUND: The incidence, prevalence, and mortality of intrahepatic cholangiocarcinoma (ICC) are increasing worldwide. Protein tyrosine kinase-7 (PTK7) is upregulated in many common human cancers. However, its expression in ICC has not been studied. The present study aimed to explore the underlying mechanism of PTK7 in ICC.
MATERIALS AND METHODS: The role of PTK7 was studied in vitro by suppressing PTK7 expression in ICC cell lines. The in vivo effect of PTK7 was evaluated using a nude mouse model inoculated with a human ICC cell line. We also examined the role of PTK7 in human ICC samples.
RESULTS: Cells with high PTK7 expression exhibited higher proliferation, DNA synthesis, invasion, and migration abilities than did cells with low PTK7 expression. The knockdown of PTK7 with small interfering RNA (siRNA) in high PTK7 expressing cells resulted in impairment of invasion, migration, and DNA synthesis through the regulation of several cell-cycle-related proteins. It also induced cell apoptosis and decreased phospho-RhoA expression. In a xenograft nude mouse model, PTK7 siRNA resulted in a reduction of the tumor size, compared with scrambled siRNA injection. PTK7 expression was higher in human ICC than in the normal bile duct. Patients with low expression of PTK7 had a longer disease-free survival and overall survival than those with high expression.
CONCLUSIONS: PTK7 expression plays an important role in the invasiveness of ICC cells and leads to a poor prognosis in ICC patients. Thus, PTK7 can be used as a prognostic indicator, and the inhibition of PTK7 expression could be a new therapeutic target for ICC.

Although the generation of BCR-ABL is the molecular hallmark of chronic myeloid leukemia (CML), the comprehensive molecular mechanisms of the disease remain unclear yet. Growth arrest specific 2 (GAS2) regulates multiple cellular functions including cell cycle, apoptosis and calpain activities. In the present study, we found GAS2 was up-regulated in CML cells including CD34+ progenitor cells compared to their normal counterparts. We utilized RNAi and the expression of dominant negative form of GAS2 (GAS2DN) to target GAS2, which resulted in calpain activity enhancement and growth inhibition of both K562 and MEG-01 cells. Targeting GAS2 also sensitized K562 cells to Imatinib mesylate (IM). GAS2DN suppressed the tumorigenic ability of MEG-01 cells and impaired the tumour growth as well. Moreover, the CD34+ cells from CML patients and healthy donors were transduced with control and GAS2DN lentiviral vectors, and the CD34+ transduced (YFP+) progeny cells (CD34+YFP+) were plated for colony-forming cell (CFC) assay. The results showed that GAS2DN inhibited the CFC production of CML cells by 57±3% (n = 3), while affected those of normal hematopoietic cells by 31±1% (n = 2). Next, we found the inhibition of CML cells by GAS2DN was dependent on calpain activity but not the degradation of beta-catenin. Lastly, we generated microarray data to identify the differentially expressed genes upon GAS2DN and validated that the expression of HNRPDL, PTK7 and UCHL5 was suppressed by GAS2DN. These 3 genes were up-regulated in CML cells compared to normal control cells and the growth of K562 cells was inhibited upon HNRPDL silence. Taken together, we have demonstrated that GAS2 is up-regulated in CML cells and the inhibition of GAS2 impairs the growth of CML cells, which indicates GAS2 is a novel regulator of CML cells and a potential therapeutic target of this disease.

Protein Tyrosin Kinase 7 (PTK7) is upregulated in several human cancers; however, its clinical implication in breast cancer (BC) and lymph node (LN) is still unclear. In order to investigate the function of PTK7 in mediating BC cell motility and invasivity, PTK7 expression in BC cell lines was determined. PTK7 signaling in highly invasive breast cancer cells was inhibited by a dominant-negative PTK7 mutant, an antibody against the extracellular domain of PTK7, and siRNA knockdown of PTK7. This resulted in decreased motility and invasivity of BC cells. We further examined PTK7 expression in BC and LN tissue of 128 BC patients by RT-PCR and its correlation with BC related genes like HER2, HER3, PAI1, MMP1, K19, and CD44. Expression profiling in BC cell lines and primary tumors showed association of PTK7 with ER/PR/HER2-negative (TNBC-triple negative BC) cancer. Oncomine data analysis confirmed this observation and classified PTK7 in a cluster with genes associated with agressive behavior of primary BC. Furthermore PTK7 expression was significantly different with respect to tumor size (ANOVA, p = 0.033) in BC and nodal involvement (ANOVA, p = 0.007) in LN. PTK7 expression in metastatic LN was related to shorter DFS (Cox Regression, p = 0.041). Our observations confirmed the transforming potential of PTK7, as well as its involvement in motility and invasivity of BC cells. PTK7 is highly expressed in TNBC cell lines. It represents a novel prognostic marker for BC patients and has potential therapeutic significance.

BACKGROUNDS AND OBJECTIVES: We previously examined the amplification status of 10 kinase genes (PIK3CA, EPHB3, TNK2, PTK7, EGFR, MET, ERBB2, HCK, SRC, and AURKA) in gastric cancer (GC). This study aimed to determine the prognostic significance of these gene amplifications in GC.
METHODS: A survival analysis was performed for GC patients. Since TNK2 amplification was identified as a prognostic marker in the analysis, we also examined the functional effect of TNK2 overexpression on gastric cells.
RESULTS: A Kaplan-Meier analysis showed that the prognosis of patients with GC exhibiting TNK2 or AURKA amplification was significantly poorer than the prognosis of patients with GC without TNK2 or AURKA amplification. A further multivariate analysis revealed that TNK2 amplification was an independent predictor of a poor survival outcome among patients with GC (hazard ratio, 3.668; 95% confidence interval, 1.513-7.968; P = 0.0056). TNK2-overexpressing GC cells showed an increase in cell migration and non-anchored cell growth. Finally, microarray and pathway analyses revealed the aberrant regulation of some cancer-related pathways in TNK2-overexpressing GC cells.
CONCLUSIONS: These results suggested that TNK2 amplification is an independent predictor of a poor prognosis in patients with GC and leads to an increase in the malignant potential of GC cells.

Esophageal squamous cell carcinoma (ESCC) is a common subtype of esophageal cancer that is particularly prevalent in East Asian countries. Our previous expression profile analysis showed that the gene encoding protein tyrosine kinase 7 (PTK7) is upregulated in ESCC tissues. Here, we aimed to validate PTK7 as a prognostic factor and a candidate target for molecular treatment of ESCC. Both RT-PCR and Western blot analysis of tissues from ESCC patients revealed that PTK7 was significantly upregulated in tumor tissue samples of ESCC. Immunohistochemical staining of PTK7 showed that increased expression of PTK7 was inversely correlated with overall survival (P = 0.021). In vitro knockdown of PTK7 inhibited proliferation, survival, wound healing, and invasion of ESCC cells. In addition, PTK7 knockdown decreased phosphorylation of Akt, Erk, and focal adhesion kinase (FAK), important determinants of cell proliferation, survival, and migration. Therefore, our findings suggest that PTK7 has potential as a prognostic marker for ESCC and might also be a candidate for targeted therapy in the treatment of ESCC.

INTRODUCTION: Canonical and non-canonical Wnt pathways are involved in the genesis of multiple tumors; however, their role in pituitary tumorigenesis is mostly unknown.
OBJECTIVE: This study evaluated gene and protein expression of Wnt pathways in pituitary tumors and whether these expression correlate to clinical outcome.
MATERIALS AND METHODS: Genes of the WNT canonical pathway: activating ligands (WNT11, WNT4, WNT5A), binding inhibitors (DKK3, sFRP1), β-catenin (CTNNB1), β-catenin degradation complex (APC, AXIN1, GSK3β), inhibitor of β-catenin degradation complex (AKT1), sequester of β-catenin (CDH1), pathway effectors (TCF7, MAPK8, NFAT5), pathway mediators (DVL-1, DVL-2, DVL-3, PRICKLE, VANGL1), target genes (MYB, MYC, WISP2, SPRY1, TP53, CCND1); calcium dependent pathway (PLCB1, CAMK2A, PRKCA, CHP); and planar cell polarity pathway (PTK7, DAAM1, RHOA) were evaluated by QPCR, in 19 GH-, 18 ACTH-secreting, 21 non-secreting (NS) pituitary tumors, and 5 normal pituitaries. Also, the main effectors of canonical (β-catenin), planar cell polarity (JNK), and calcium dependent (NFAT5) Wnt pathways were evaluated by immunohistochemistry.
RESULTS: There are no differences in gene expression of canonical and non-canonical Wnt pathways between all studied subtypes of pituitary tumors and normal pituitaries, except for WISP2, which was over-expressed in ACTH-secreting tumors compared to normal pituitaries (4.8x; p = 0.02), NS pituitary tumors (7.7x; p = 0.004) and GH-secreting tumors (5.0x; p = 0.05). β-catenin, NFAT5 and JNK proteins showed no expression in normal pituitaries and in any of the pituitary tumor subtypes. Furthermore, no association of the studied gene or protein expression was observed with tumor size, recurrence, and progressive disease. The hierarchical clustering showed a regular pattern of genes of the canonical and non-canonical Wnt pathways randomly distributed throughout the dendrogram.
CONCLUSIONS: Our data reinforce previous reports suggesting no activation of canonical Wnt pathway in pituitary tumorigenesis. Moreover, we describe, for the first time, evidence that non-canonical Wnt pathways are also not mis-expressed in the pituitary tumors.

To test the feasibility of using bacterial artificial chromosomes (BAC) containing kinases for pathological diagnosis using fluorescence in situ hybridization (FISH), 10 BAC probes containing a gene amplified in 5% or more of a pilot cohort were selected from a previous survey using arbitrarily selected BAC clones harboring 100 kinases. In this report, we describe the prevalence and association with the clinicopathological profile of these selected 10 BAC probes in 365 gastric cancer tissues. FISH analyses using these 10 BAC probes containing loci encoding EGFR, ERBB2(HER2), EPHB3, PIK3CA, MET, PTK7, ACK1, STK15, SRC, and HCK showed detectable amplifications in paraffin-embedded tissue in 2.83% to 13.6% of the gastric cancer tissues. Considerable numbers of the cases showed the co-amplification of two or more of the probes that were tested. BAC probes located within a genome neighborhood, such as PIK3CA, EPHB3, and ACK1 at 3q26-29 or HCK, SRC, and STK15 at 20q11-13.1, were often co-amplified in the same cases, but non-random co-amplifications of genes at distant genomic loci were also observed. These findings provide basic information regarding the creation of a strategy for personalizing gastric cancer therapy, especially when using multiple kinase inhibitors.

PURPOSE: To profile different tyrosine kinase (TK) expression patterns in clear cell renal carcinoma (ccRCC).
METHODS: We analysed mRNA expression levels of 89 receptor and non-receptor TK in corresponding cancer and normal renal tissue from 5 patients with ccRCC using the TaqMan Low-Density Array technology. In order to confirm aberrant TK expressions, a subsequent analysis of 25 ccRCC and corresponding normal renal tissues was performed, applying quantitative real-time PCR. To confirm mRNA expression levels on protein level, we studied ERBB4 and HCK using immunohistochemistry.
RESULTS: A total of 12 TK were significantly upregulated in ccRCC (ABL2, FLT1, BTK, HCK, JAK3, CSF1R, MET, JAK1, MATK, PTPRC, FYN and CSK), coherently 7 TK demonstrated a down-regulation (ERBB4, PDGFRA, NRTK3, SYK, ERBB2, FGFR3 and PTK7). These findings were validated by the utilization of RT-PCR for ABL2, FLT1 BTK, HCK, JAK3, CSF1R, MET, JAK1, MATK and vice versa for ERBB4 and PDGFRA. Immunohistochemistry revealed ERBB4 expression to be significantly lower in ccRCC in comparison to papillary RCC, chromophobe RCC, renal oncocytoma and normal renal tissue (P < 0.001). HCK protein expression was reduced in ccRCC in contrast to papillary RCC (P < 0.001) or oncocytoma (P = 0.023), but similar to chromphobe RCC (P = 0.470), sarcomatoid RCC (P = 0.754) and normal renal tissue (P = 0.083). Neither ERBB4 nor HCK were correlated (P > 0.05) with clinical-pathological parameters.
CONCLUSION: TK constitute valuable targets for pharmaceutical anti-cancer therapy. ERBB4 and HCK depict significantly lower expression levels in renal cancer tissues.

Liposarcomas are the most common type of soft tissue sarcoma but their genetics are poorly defined. To identify genes that contribute to liposarcomagenesis and serve as prognostic candidates, we undertook expression profiling of 140 primary liposarcoma samples, which were randomly split into training set (n = 95) and test set (n = 45). A multigene predictor for distant recurrence-free survival (DRFS) was developed by the supervised principal component method. Expression levels of the 588 genes in the predictor were used to calculate a risk score for each patient. In validation of the predictor in the test set, patients with low risk score had a 3-year DRFS of 83% versus 45% for high risk score patients (P = 0.001). The HR for high versus low score, adjusted for histologic subtype, was 4.42 (95% CI, 1.26-15.55; P = 0.021). The concordance probability for risk score was 0.732. In contrast, the concordance probability for histologic subtype, which had been considered the best predictor of outcome in liposarcoma, was 0.669. Genes related to adipogenesis, DNA replication, mitosis, and spindle assembly checkpoint control were all highly represented in the multigene predictor. Three genes from the predictor, TOP2A, PTK7, and CHEK1, were found to be overexpressed in liposarcoma samples of all five subtypes and in liposarcoma cell lines. RNAi-mediated knockdown of these genes in liposarcoma cell lines reduced proliferation and invasiveness and increased apoptosis. Taken together, our findings identify genes that seem to be involved in liposarcomagenesis and have promise as therapeutic targets, and support the use of this multigene predictor to improve risk stratification for individual patients with liposarcoma.

Protein tyrosine kinase-7 (PTK7) is a catalytically inactive receptor tyrosine kinase (RTK). PTK7 is upregulated in many common human cancers, including colon cancer, lung cancer, gastric cancer and acute myeloid leukemia. The reason for this up-regulation is not yet known. To explore the functional role of PTK7, the expression of PTK7 in HCT 116 cells was examined using small interference (siRNA)-mediated gene silencing. Following transfection, the siRNA successfully suppressed PTK7 mRNA and protein expression. Knocking down of PTK7 in HCT 116 cells inhibited cell proliferation compared to control groups and induced apoptosis. Furthermore, this apoptosis was characterized by decreased mitochondrial membrane potential and activation of caspase-9 and -10. Addition of a caspase-10 inhibitor totally blocked this apoptosis, suggesting that caspase-10 may play a critical role in PTK7-knockdown-induced apoptosis, downstream of mitochondria. These observations may indicate a role for PTK7 in cell proliferation and cell apoptosis and may provide a potential therapeutic pathway for the treatment of a variety of cancers.

PTK7 is an essential component of the Wnt/planar cell polarity (PCP) pathway. We provide evidence that the Wnt/PCP pathway converges with pericellular proteolysis in both normal development and cancer. Here, we demonstrate that membrane type-1 matrix metalloproteinase (MT1-MMP), a key proinvasive proteinase, functions as a principal sheddase of PTK7. MT1-MMP directly cleaves the exposed PKP(621)↓LI sequence of the seventh Ig-like domain of the full-length membrane PTK7 and generates, as a result, an N-terminal, soluble PTK7 fragment (sPTK7). The enforced expression of membrane PTK7 in cancer cells leads to the actin cytoskeleton reorganization and the inhibition of cell invasion. MT1-MMP silencing and the analysis of the uncleavable L622D PTK7 mutant confirm the significance of MT1-MMP proteolysis of PTK7 in cell functions. Our data also demonstrate that a fine balance between the metalloproteinase activity and PTK7 levels is required for normal development of zebrafish (Danio rerio). Aberration of this balance by the proteinase inhibition or PTK7 silencing results in the PCP-dependent convergent extension defects in the zebrafish. Overall, our data suggest that the MT1-MMP-PTK7 axis plays an important role in both cancer cell invasion and normal embryogenesis in vertebrates. Further insight into these novel mechanisms may promote understanding of directional cell motility and lead to the identification of therapeutics to treat PCP-related developmental disorders and malignancy.

Osteosarcoma is a primary malignant tumor of bone arising from primitive bone-forming mesenchymal cells and accounts for approximately 60% of malignant bone tumors. Our comparative genomic hybridization (CGH) studies have identified frequent amplification at 6p12-p21, 12q13-q15, and 17p11.2 in osteosarcoma. Of these amplified regions, 6p12-p21 is particularly interesting because of its association with progression and poor prognosis in patients with osteosarcoma. In an attempt to identify aberrantly expressed gene(s) mapping to the 6p12-p21 amplicon, a region-specific array was generated using 108 overlapping BAC and P1 clones covering a 28.8-Mb region at 0.26-Mb intervals. Based on array CGH analysis, the 6p amplicon was refined to 7.9 Mb between the clones RP11-91E11 and RP1-244F2 and 10 amplified clones, with possible target genes, were identified. To study the expression pattern of the target genes from the hotspot amplicon and known candidate genes from 6p12-21, we did quantitative reverse transcription-PCR analysis of MAPK14, MAPK13, CDKN1A, PIM1, MDGA1, BTB9, DNAH8, CCND3, PTK7, CDC5L, and RUNX2 on osteosarcoma patient samples and seven cell lines. The combined array CGH and quantitative reverse transcription-PCR analysis identified amplification and overexpression of CDC5L, CCND3, and RUNX2. We screened these three genes for protein expression by Western blotting and immunohistochemistry and detected overexpression of CDC5L. Furthermore, we used an in vivo assay to show that CDC5L possesses potential oncogenic activity. These results indicate that CDC5L, a cell cycle regulator important for the G2-M transition, is the most likely candidate oncogene for the 6p12-p21 amplicon found in osteosarcoma.