Protein Tyrosine Kinase 7 Research Articles

Protein tyrosine kinase 7 is essential for tubular morphogenesis of the Wolffian duct

The Wolffian duct, the proximal end of the mesonephric duct, undergoes non-branching morphogenesis to achieve an optimal length and size for sperm maturation. It is important to examine the mechanisms by which the developing mouse Wolffian duct elongates and coils for without proper morphogenesis, male infertility will result. Here we show that highly proliferative epithelial cells divide in a random orientation relative to the elongation axis in the developing Wolffian duct. Convergent extension (CE)-like of cell rearrangements is required for elongating the duct while maintaining a relatively unchanged duct diameter. The Wolffian duct epithelium is planar polarized, which is characterized by oriented cell elongation, oriented cell rearrangements, and polarized activity of regulatory light chain of myosin II. Conditional deletion of protein tyrosine kinase 7 (PTK7), a regulator of planar cell polarity (PCP), from mesoderm results in loss of the PCP characteristics in the Wolffian duct epithelium. Although loss of Ptk7 does not alter cell proliferation or division orientation, it affects CE and leads to the duct with significantly shortened length, increased diameter, and reduced coiling, which eventually results in loss of sperm motility, a key component of sperm maturation. In vitro experiments utilizing inhibitors of myosin II results in reduced elongation and coiling, similar to the phenotype of Ptk7 knockout. This data suggest that PTK7 signaling through myosin II regulates PCP, which in turn ensures CE-like of cell rearrangements to drive elongation and coiling of the Wolffian duct. Therefore, PTK7 is essential for Wolffian duct morphogenesis and male fertility.

A PTK7/Ror2 Co-Receptor Complex Affects Xenopus Neural Crest Migration

Neural crest cells are a highly migratory pluripotent cell population that generates a wide array of different cell types and failure in their migration can result in severe birth defects and malformation syndromes. Neural crest migration is controlled by various means including chemotaxis, repellent guidance cues and cell-cell interaction. Non-canonical Wnt PCP (planar cell polarity) signaling has previously been shown to control cell-contact mediated neural crest cell guidance. PTK7 (protein tyrosine kinase 7) is a transmembrane pseudokinase and a known regulator of Wnt/PCP signaling, which is expressed in Xenopus neural crest cells and required for their migration. PTK7 functions as a Wnt co-receptor; however, it remains unclear by which means PTK7 affects neural crest migration. Expressing fluorescently labeled proteins in Xenopus neural crest cells we find that PTK7 co-localizes with the Ror2 Wnt-receptor. Further, co-immunoprecipitation experiments demonstrate that PTK7 interacts with Ror2. The PTK7/Ror2 interaction is likely relevant for neural crest migration, because Ror2 expression can rescue the PTK7 loss of function migration defect. Live cell imaging of explanted neural crest cells shows that PTK7 loss of function affects the formation of cell protrusions as well as cell motility. Co-expression of Ror2 can rescue these defects. In vivo analysis demonstrates that a kinase dead Ror2 mutant cannot rescue PTK7 loss of function. Thus, our data suggest that Ror2 can substitute for PTK7 and that the signaling function of its kinase domain is required for this effect.

PTK7 Is Involved in the Chemotherapy Resistance of T-Cell Acute Lymphoblastic Leukemia through Interaction with FADD-Death Domain

Apoptosis-based therapies have been used as a powerful weapon against hematologic malignancies (Reed JC et al, Blood 2005 15; 408-418). One strategy for attacking cancer is to overcome its resistance to apoptosis via specific targeting molecules. In this study, we are focusing on protein tyrosine kinase 7 (PTK7) as a new target for cancer therapy. PTK7, also known as CCK4, is a member of transmembrane tyrosine kinase family proteins that has been implicated in cell migration, polarity and apoptosis. PTK7 is overexpressed in acute T-cell lymphoblastic leukemia cells, and a recent report indicated that PTK7 expression was associated with poor prognosis. However, the mechanism by which PTK7 elicits its anti-apoptotic effects and directly promotes tumor proliferation has not yet been clarified. In addition, no data has been reported regarding the relationship between doxorubicin resistance and PTK7 expression. In this study, we investigated the role of PTK7 in chemotherapy resistance. Two different shRNA lentiviruses targeting PTK7 were stably infected in Jurkat cells (Jurkat tet-shPTK7#1 and #2), and gene knockdown assays against PTK7 were performed. In the presence of doxycycline, we observed a decrease in the viability of PTK7 knockdown cells following stimulation with αFAS-Ab (Jurkat tet-shPTK7#1: 28.6% ± 5.2%, #2: 8.7% ± 0.3%) compared to control cells (considered to be 100%). Similar results of decreased cell viability were also obtained with TRAIL stimulation (Jurkat tet-shPTK7#1: 22.5% ± 5.9%, #2: 14.7% ± 9.8%) compared to control cells (considered to be 100%). Furthermore, we observed a decrease in the viability of PTK7 knockdown cells following treatment with Doxorubicin compared to control cells treated with scramble siRNA. The reduction of cell viability in PTK7 knockdown cells could not be attributed to PTK7 knockdown itself since annexin-V and PI staining showed limited apoptotic changes in Jurkat tet-shPTK7 cells. These changes were also not the result of Fas receptor (CD95) and Trail receptor (DR4, DR5) expression up-regulation as flow cytometric analysis revealed no differences in the expression rates of Fas receptor and Trail receptor between scramble control cells and PTK7 knockdown cells. Very interestingly, PTK7 knockdown had no effect on viability when non-cancer cell lines were stimulated with Fas, TRAIL and Doxorubicin, suggesting that the synergistic effect of PTK7 knockdown and TNF family cytokine treatment is cancer specific. Next, to identify interaction partners of the PTK7 cytoplasmic domain (PTK7-cyto), we performed a yeast two-hybrid assay, and showed that FADD-DD was the only molecule found to bind to PTK7-cyto in both LEU2 reporter and LacZ reporter assays. This protein interaction was further verified by co-immunoprecipitation assays. Finally, to demonstrate the activation of Caspase-8 and PARP, which is induced by TNF-family death receptors, immunoblotting was performed. The shRNA-mediated silencing of PTK7 in Jurkat cells indeed increased Fas- and TRAIL-induced processing of caspase-8 and PARP compared to control RNA. These data suggest that PTK7 appears to be important for a proximal step in Fas and TRAIL signaling, which is consistent with the selective sensitization of tumor cells to the extrinsic apoptosis pathway. Together, our findings reveal an unexpected role for PTK7 in terms of death receptor-mediated apoptosis, suggesting that PTK7 could provide a new target for cancer therapy. DisclosuresNo relevant conflicts of interest to declare.

SERS-fluorescence dual mode nanotags for cervical cancer detection using aptamers conjugated to gold-silver nanorods

We describe nanotags suitable for both surface enhanced Raman scattering (SERS) and fluorescence detection and imaging. A fluorescently-labeled aptamer conjugated to gold-silver nanorods used for specific and sensitive detection of cervical cancer. NRs with different Au-Ag ratios were synthesized. The Raman reporter 4-aminothiophenol and fluorescently-labeled aptamers were assembled on the surface of NRs via a layer-by-layer process. The fluorescence and SERS signals can be generated independently using different excitation wavelengths, which can avoid the disturbance from each other. The nanotags were proven to be specific to the human protein tyrosine kinase-7 (PTK-7) expressed on Hela (cervical cancer) cells through aptamer-protein interaction. The binding of aptamers towards their targets induced the assembly of nanotags on the cell surface, resulting in strong fluorescence and SERS signals. However, the controls, randomized sequence oligonucleotide conjugated NRs, showed no detectable signal. Fluorescence and SERS mapping images were also performed to confirm targeting ability of the nanotags on the target cell membrane. The success of this method extends the feasibility of the dual mode nanotags for highly sensitive and specific cancer diagnostic.

18F-Labeled Single-Stranded DNA Aptamer for PET Imaging of Protein Tyrosine Kinase-7 Expression.

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. 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. 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. 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.

PTK6 Potentiates Gemcitabine-Induced Apoptosis by Prolonging S-phase and Enhancing DNA Damage in Pancreatic Cancer.

Protein Tyrosine Kinase 6 (PTK6) is a non-receptor-type tyrosine kinase known to be expressed in various cancers, including pancreatic cancer. The role of PTK6 in cancer chemoresistance remains unclear. Therefore, it was hypothesized that PTK6 mechanistically regulates gemcitabine resistance in pancreatic cancer. Gemcitabine treatment stimulated endogenous PTK6 overexpression in MIAPaCa2 and Panc1 cells. PTK6 gene silencing increased cell survival after gemcitabine treatment and decreased apoptosis, whereas PTK6 overexpression decreased cell survival and increased apoptosis. Selection for gemcitabine resistance revealed substantially lower PTK6 expression in the gemcitabine-resistant subclones compared with the parental lines, while restoring PTK6 rescued gemcitabine sensitivity. Gemcitabine induced phosphorylation of H2AX (γ-H2AX) and ataxia-telangiectasia mutated kinase (pATM), specific markers for DNA double-strand breaks. Both gemcitabine-induced phosphorylation of H2AX and ATM were reduced by PTK6 knockdown and increased by PTK6 overexpression. PTK6 overexpression also increased the S-phase fraction 48 hours after gemcitabine treatment. Although gemcitabine activated both caspase-8 (CASP8) and caspase-9 (CASP9), the effect of PTK6 on gemcitabine-induced apoptosis required CASP8 but not CASP9. In mouse xenografts, PTK6 overexpression in subcutaneous tumors attenuated tumor growth after gemcitabine treatment. In conclusion, PTK6 prolongs S-phase and increases the ability of gemcitabine to cause DNA damage in vitro and in vivo. PTK6 affects cell cycle and DNA damage, thus making it an important therapeutic target to improve the outcomes of patients with pancreatic cancer.

Abstract LB-059: Opposing roles for protein tyrosine kinase 6 (PTK6) in colon cancer

Abstract Background: Protein Tyrosine Kinase 6 (PTK6, also called BRK) is a non-receptor tyrosine kinase expressed in differentiated epithelial cells of the skin, gastrointestinal tract, and prostate. Disruption of the Ptk6 gene led to impaired intestinal differentiation and increased intestinal proliferation in mice. However, PTK6 also has a tumor-promoting role in the colon, as disruption of the Ptk6 gene impaired carcinogen-induced STAT3 activation and tumorigenesis in mice. PTK6 also promotes survival of human colon cancer cell lines following DNA damaging treatments including γ-irradiation and chemotherapeutic drugs via STAT3 activation. Results: We examined and manipulated colon cancer cell lines to understand these two seemingly contradictory roles for PTK6 in the colon. The human colon adenocarcinoma cell lines SW480 and SW620 represent a primary colon tumor and a metastasis from the same patient, respectively. PTK6 is highly expressed in SW480 cells and significantly reduced in SW620 cells. Active PTK6, phosphorylated on tyrosine residue 342, is not detectable in either cell line. Stable knockdown of PTK6 in SW480 cells results in an epithelial-to-mesenchymal transition (EMT), evidenced by decreased E-cadherin expression and increased ZEB-1 and Vimentin; as well as increased migration, wound-healing, and anchorage-independent growth of cells in culture. Additionally, knockdown of PTK6 led to increased in vivo tumorigenesis of xenograft cells in nude mice. Ectopic expression of wild-type PTK6 in SW620 cells drives the reverse process (MET), demonstrating a rescue of the EMT phenotype. When a constitutively active PTK6 construct is expressed in SW620 cells, it promotes activation of cytoplasmic oncogenic targets and downstream effectors, STAT3, FAK, BCAR1 and ERK5. Analysis of patient tissues demonstrates a downregulation of mRNA and protein levels, as well as a change in protein localization in tumor versus normal tissues; while PTK6 is detectable in the nuclei of differentiating normal cells it is excluded from nuclei in colon tumors. Conclusions: Our studies indicate that PTK6 promotes the epithelial phenotype to antagonize the EMT in a kinase-independent manner and a kinase switch activates PTK6 to promote oncogenic signaling. PTK6 protein localization may function to regulate its specific role. Further examination of the complex role of this kinase could allow for a more nuanced understanding of colon cancer initiation and progression for the development of novel treatments. Citation Format: Priya S. Mathur, Jessica J. Gierut, Rosa M. Xicola, Xavier Llor, Angela L. Tyner. Opposing roles for protein tyrosine kinase 6 (PTK6) in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-059. doi:10.1158/1538-7445.AM2015-LB-059

PTK6 inhibition promotes apoptosis of Lapatinib-resistant Her2(+) breast cancer cells by inducing Bim.

IntroductionProtein tyrosine kinase 6 (PTK6) is a non-receptor tyrosine kinase that is highly expressed in Human Epidermal Growth Factor 2+ (Her2+) breast cancers. Overexpression of PTK6 enhances anchorage-independent survival, proliferation, and migration of breast cancer cells. We hypothesized that PTK6 inhibition is an effective strategy to inhibit growth and survival of Her2+ breast cancer cells, including those that are relatively resistant to Lapatinib, a targeted therapy for Her2+ breast cancer, either intrinsically or acquired after continuous drug exposure.MethodsTo determine the effects of PTK6 inhibition on Lapatinib-resistant Her2+ breast cancer cell lines (UACC893R1 and MDA-MB-453), we used short hairpin ribonucleic acid (shRNA) vectors to downregulate PTK6 expression. We determined the effects of PTK6 downregulation on growth and survival in vitro and in vivo, as well as the mechanisms responsible for these effects.ResultsLapatinib treatment of “sensitive” Her2+ cells induces apoptotic cell death and enhances transcript and protein levels of Bim, a pro-apoptotic Bcl2 family member. In contrast, treatment of relatively “resistant” Her2+ cells fails to induce Bim or enhance levels of cleaved, poly-ADP ribose polymerase (PARP). Downregulation of PTK6 expression in these “resistant” cells enhances Bim expression, resulting in apoptotic cell death. PTK6 downregulation impairs growth of these cells in in vitro 3-D MatrigelTM cultures, and also inhibits growth of Her2+ primary tumor xenografts. Bim expression is critical for apoptosis induced by PTK6 downregulation, as co-expression of Bim shRNA rescued these cells from PTK6 shRNA-induced death. The regulation of Bim by PTK6 is not via changes in Erk/MAPK or Akt signaling, two pathways known to regulate Bim expression. Rather, PTK6 downregulation activates p38, and pharmacological inhibition of p38 activity prevents PTK6 shRNA-induced Bim expression and partially rescues cells from apoptosis.ConclusionsPTK6 downregulation induces apoptosis of Lapatinib-resistant Her2+ breast cancer cells by enhancing Bim expression via p38 activation. As Bim expression is a critical biomarker for response to many targeted therapies, PTK6 inhibition may offer a therapeutic approach to treating patients with Her2 targeted therapy-resistant breast cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0594-z) contains supplementary material, which is available to authorized users.

Structure-Switching Aptamer Triggering Hybridization Chain Reaction on the Cell Surface for Activatable Theranostics.

The ability to probe low-abundance biomolecules or transport a high-load drug in target cells is essential for biology and theranostics. We develop a novel activatable theranostic approach by using a structure-switching aptamer triggered hybridization chain reaction (HCR) on the cell surface, which for the first time creates an aptamer platform enabling real-time activation and amplification for fluorescence imaging and targeting therapy. The aptamer probe is designed not to initiate HCR in its free state but trigger HCR on binding to the target cell via structure switching. The HCR not only amplifies fluorescence signals from a fluorescence-quenched probe for activatable tumor imaging but also accumulates high-load prodrugs from a drug-labeled probe and induces its uptake and conversion into cisplatin in cells for selective tumor therapy. An in vitro assay shows that this approach affords efficient signal amplification for fluorescence detection of target protein tyrosine kinase-7 (PTK7) with a detection limit of 1 pM. Live cell studies reveal that it provides high-contrast fluorescence imaging and highly sensitive detection of tumor cells, while renders high-efficiency drug delivery into tumor cells via an endocytosis pathway. The results imply the potential of the developed approach as a promising platform for early stage diagnosis and precise therapy of tumors.

News in the studies of multidrug resistance of breast cancer cells

Breast cancer (BC) is the most common cancer among women in Russia. One of the main treatment methods of BC is systemic chemotherapy. Multidrug resistance of tumor cells (MDR) is the important hindrance on the way to successful chemotherapy. The new data concerning molecular mechanisms of MDR will be presented in this review. The recent data concerning some new biological prognostic markers will be also discussed. There are data showing that transporters of ABC family (ABC transporters) influence tumor progression not only by MDR induction but also by the influence on the traits of malignancy in tumor cells. The results of the studies of ABC transporters, participation in the processes of accumulation of tumor stem cells under the influence of chemotherapy will be discussed. The problem of the participation of ABC transporters in the phenomenon of influence of PI3K/AKT/PTEN signal transduction pathway on the MDR regulation is discussed. The results of the studies of the role of microRNA deregulation in breast cancer drug resistance as well as studies of some epigenetic mechanisms of MDR regulation will be considered. Protein phosphatase 2A (PP2A, serine/threonine phosphatase), PTK7 (protein tyrosine kinase 7). fascin (an actin bundling cytoskeletal protein) multifunctional YB-1 protein will considered as new BC prognostic markers. The perspectives of MDR studies will be discussed as well.

News in the studies of multidrug resistance of breast cancer cells

Breast cancer (BC) is the most common cancer among women in Russia. One of the main treatment methods of BC is systemic chemotherapy. Multidrug resistance of tumor cells (MDR) is the important hindrance on the way to successful chemotherapy. The new data concerning molecular mechanisms of MDR will be presented in this review. The recent data concerning some new biological prognostic markers will be also discussed. There are data showing that transporters of ABC family (ABC transporters) influence tumor progression not only by MDR induction but also by the influence on the traits of malignancy in tumor cells. The results of the studies of ABC transporters, participation in the processes of accumulation of tumor stem cells under the influence of chemotherapy will be discussed. The problem of the participation of ABC transporters in the phenomenon of influence of PI3K/AKT/PTEN signal transduction pathway on the MDR regulation is discussed. The results of the studies of the role of microRNA deregulation in breast cancer drug resistance as well as studies of some epigenetic mechanisms of MDR regulation will be considered. Protein phosphatase 2A (PP2A, serine/threonine phosphatase), PTK7 (protein tyrosine kinase 7). fascin (an actin bundling cytoskeletal protein) multifunctional YB-1 protein will considered as new BC prognostic markers. The perspectives of MDR studies will be discussed as well.

PTK7+ Mononuclear Cells Express VEGFR2 and Contribute to Vascular Stabilization by Upregulating Angiopoietin-1.

In angiogenesis, circulating mononuclear cells are recruited to vascular lesions; however, the underlying mechanisms are poorly understood. Here, we characterize the functional role of protein tyrosine kinase 7 (PTK7)-expressing CD11b(+) mononuclear cells in vitro and in vivo using a mouse model of angiogenesis. Although the frequencies of PTK7(+)CD11b(+) cells in the bone marrow remained similar after vascular endothelial growth factor-A-induced neovascularization, we observed an 11-fold increase in the cornea. Importantly, vascular endothelial growth factor-A-induced chemotaxis of PTK7(+) cells was mediated by vascular endothelial growth factor receptor 2. In a coculture with endothelial cells, PTK7(+)CD11b(+) cells stabilized the vascular network for 2 weeks by expressing high levels of angiopoietin-1. The enhanced vascular stability was abolished by knockdown of angiopoietin-1 in PTK7(+)CD11b(+) cells and could be restored by angiopoietin-1 treatment. We conclude that PTK7 expression in perivascular mononuclear cells induces vascular endothelial growth factor receptor 2 and angiopoietin-1 expression and thus contributes to vascular stabilization in angiogenesis.

Biphasic effect of PTK7 on KDR activity in endothelial cells and angiogenesis

Protein tyrosine kinase 7 (PTK7) is a member of the defective receptor protein tyrosine kinase family which lacks catalytic activity. Expression of PTK7 is increased in various cancers but its role in carcinogenesis is not well understood. We previously showed that disruption of PTK7 function suppresses VEGF-induced angiogenic phenotypes in HUVECs and mice. Here, we investigated molecular mechanisms for modulating VEGF-induced physiological effects by PTK7. Treatment with a high concentration of extracellular domain of PTK7 (soluble PTK7; sPTK7) or knockdown of PTK7 inhibited VEGF-induced phosphorylation of kinase insert domain receptor (KDR) but did not inhibit phosphorylation of fms-related tyrosine kinase 1 (FLT-1) in HUVECs. PTK7, more specifically sPTK7, interacted with KDR but not with FLT-1 in HUVECs and HEK293 cells. In vitro binding assay showed that sPTK7 formed oligomers with the extracellular domain of KDR (sKDR) up to an approximately 1:3 molar ratio, and vice versa. sPTK7 at lower molar ratios than sKDR enhanced the binding of VEGF to sKDR. At the same or higher molar ratios, it reduced the binding of VEGF to sKDR. Increasing concentrations of sPTK7 or increasing levels of PTK7 expression first increased and then decreased VEGF-induced KDR phosphorylation, migration, and capillary-like tube formation of HUVECs, as well as in vivo angiogenesis. Taken together, our data demonstrates that PTK7 regulates the activity of KDR biphasically by inducing oligomerization of KDR molecules at lower concentrations and by surrounding KDR molecules at higher concentrations.

Silver decahedral nanoparticles-enhanced fluorescence resonance energy transfer sensor for specific cell imaging.

We report on a silver decahedral nanoparticles (Ag10NPs)-based FRET (fluorescence resonance energy transfer) sensor for target cell imaging. Fluorophores-functionalized aptamers (Sgc8-FITC) were bound with Ag10NPs via the SH group on the aptamer to form Ag10-Sgc8-FITC. Then, quencher-carrying strands (BHQ-1) were hybridized with Sgc8-FITC to form a Ag10NPs-based FRET sensor (Ag10-Sgc8-F/Q). The sensor interacted with membrane protein tyrosine kinase-7 (PTK-7) on the CCRF-CEM (CCL-119, T-cell line, human acute lymphoblastic leukemia) cell surface to attain fluorescence imaging of CCRF-CEM cells. The addition of CCRF-CEM cells resulted in many sensors binding with cells membrane and the displacement of BHQ-1, thus disrupting the FRET effect and the enhanced fluorescence intensity of FITC. It was found that Ag10NPs largely enhanced the fluorescence intensity of FITC. The results also showed that the Ag10NPs-based FRET sensor (Ag10-Sgc8-F/Q) was not only superior to the bare FRET sensor (Sgc8-F/Q) and sensor Ag-Sgc8-F/Q but also highly sensitive and specific for CCRF-CEM cells imaging.

Protein tyrosine kinase 7: a hepatocellular carcinoma-related gene detected by triple-combination array

BackgroundHepatocellular 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 methodsTriple-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. ResultsUsing 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. ConclusionsOur 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.

Downregulated expression of PTK6 is correlated with poor survival in esophageal squamous cell carcinoma.

To investigate the clinical prognostic value of protein tyrosine kinase 6 (PTK6) in patients with esophageal squamous cell carcinoma (ESCC), quantitative RT-PCR and Western blotting were utilized to measure the mRNA and protein expression levels of PTK6 in 29 and eight pairs of ESCC and peritumoral normal esophageal tissues, respectively. Furthermore, the expression of PTK6 protein in 210 ESCCs was examined with immunohistochemistry (IHC), and its clinical value was analyzed using Kaplan-Meier plots and the Cox proportional hazards regression model. The results found that the expression levels of both PTK6 mRNA and protein in ESCC tissues were significantly lower than those in peritumoral normal esophageal tissues. Regarding the IHC analysis of ESCC, the cytoplasmic expression of PTK6 was significantly correlated with tumor grade (P < 0.001). Compared with patients with low PTK6 expression, ESCC patients with overexpression of PTK6 displayed preferable disease-free survival (DFS) and overall survival (OS) (P < 0.001 and P = 0.001, respectively), especially in stage II disease (P = 0.002 and P = 0.021, respectively). PTK6 was evaluated as an independent prognostic factor for ESCC using multivariate Cox regression analysis. All data demonstrated that the expression level of PTK6 is an independent prognostic factor in ESCCs. Low expression of PTK6 is correlated with poor DFS and OS in ESCCs.

PTK7 as a potential prognostic and predictive marker of response to adjuvant chemotherapy in breast cancer patients, and resistance to anthracycline drugs.

Biomarkers predicting resistance to particular chemotherapy regimens could play a key role in optimally individualized treatment concepts. PTK7 (protein tyrosine kinase 7) belongs to the receptor tyrosine kinase family involved in several physiological, but also malignant, cell behaviors. Recent studies in acute myeloid leukemia have associated PTK7 expression with resistance to anthracycline therapy. PTK7 mRNA expression in primary tumor tissue (PTT) and corresponding lymph node tissue (LNT) were retrospectively measured in 117 patients with early breast cancer; PTK7 expression was available in 103 PTT and 108 LNT samples. Median age was 60 years (range, 27–87 years). At a median follow-up of 28.5 months, 6 deaths and 16 recurrences had occurred. PTK7 expression correlations with clinicopathological features were computed and PTK7 expression effects on patient outcome were analyzed in three cohorts defined by adjuvant treatment: anthracycline-based treatment, other chemotherapy regimens (including taxane or other substances), or no chemotherapy. Association of PTK7 expression with clinicopathological features was seen only for age in PTT and nodal stage in LNT. High LN PTK7 was associated with poorer disease-free survival (DFS) in the total population (3-year DFS: low [81.7%] versus high [70.4%]; P=0.016) and in patients without adjuvant chemotherapy (3-year DFS: low [91.7%] versus high [22.3%]; P<0.001), but not in patients receiving adjuvant chemotherapy (P=0.552). DFS stratified by PTK7 expression was compared in treatment cohorts: In patients with low LN PTK7 expression, neither chemotherapy cohort showed significantly better survival than the no-chemotherapy cohort. In patients with high LN PTK7 expression, those receiving chemotherapy, including substances other than anthracyclines, but not those receiving only anthracycline-based chemotherapy, showed significantly better DFS than those receiving no chemotherapy (P=0.001). Our results support earlier findings that PTK7 may be a prognostic and predictive marker associated with resistance to anthracycline-based chemotherapy. Further investigations are needed to validate these findings in breast cancer.

Abstract 2100: Regulation of breast tumor kinase (Brk) expression in triple negative breast cancer integrates cell stress and cortisol signaling pathways

Abstract Triple negative breast cancers (TNBC) lack expression of the molecules currently exploited for targeted therapy (estrogen receptor, progesterone receptor, and HER2). These tumors frequently respond well to chemotherapy, but emergence of chemotherapy-resistance is a common clinical problem. Thus, identification of drivers of TNBC that may be exploited for targeted therapy is of great importance. Breast tumor kinase (Brk), also known as protein tyrosine kinase 6 (PTK6), is a soluble tyrosine kinase that is absent from normal mammary epithelial cells but inappropriately expressed in 85% of breast cancers. Previous studies from our lab have shown Brk to be a critical regulator of breast cancer cell migration in vitro and a potent driver of basal-type mammary tumors in vivo. Notably, we recently identified a novel mechanism of upregulation of Brk expression in TNBC cells. Brk expression is induced following exposure to cell stress, including hypoxia, oxidative damage, and nutrient starvation. Moreover, we found that Brk is a direct target gene of both hypoxia-inducible factor 1 alpha (HIF-1α) and HIF-2α. It is becoming increasingly evident that cortisol signaling via activation of the glucocorticoid receptor (GR) leads to enhanced survival and chemoresistance in tumors of epithelial origin, such as breast cancer. In fact, GR expression in TNBC predicts poor outcome. Herein, we sought to investigate crosstalk between cell stress pathways (HIFs) and cortisol signaling (GR) that may influence expression of Brk in TNBC. Brk mRNA and protein were induced in response to the GR ligand, dexamethasone (dex), in both normoxia and hypoxia, and blocked following treatment with RU486, a GR antagonist. Brk mRNA and protein failed to be induced following dex treatment in cells lacking HIF-1α and HIF-2α, suggesting that even in normoxia, GR regulation of Brk requires HIF-1α/2α. Chromatin immunoprecipitation (ChIP) assays showed HIF and GR recruitment to multiple regions of the Brk promoter in response to either hypoxia (a ligand-independent GR action) or treatment with dex, indicating that Brk is a direct GR/HIF target gene. These data show that cortisol signaling via GR is a novel mechanism of upregulation of Brk expression in TNBC. Additionally, GR upregulation of Brk is HIF-dependent, revealing molecular linkage between cell stress pathways and stress hormone signaling. Collectively, our studies suggest that GR and HIF cross talk may promote aggressive tumor behavior, in part via upregulation of Brk. Breast cancer patients are routinely given dex (a GR agonist) to alleviate the inflammatory side effects of chemotherapy. This treatment may inadvertently promote tumor progression via induction of Brk expression. Citation Format: Tarah M. Regan Anderson, Andrea R. Daniel, Carol A. Lange. Regulation of breast tumor kinase (Brk) expression in triple negative breast cancer integrates cell stress and cortisol signaling pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2100. doi:10.1158/1538-7445.AM2014-2100

Additive impact of HER2-/PTK6-RNAi on interactions with HER3 or IGF-1R leads to reduced breast cancer progression in vivo

The human epidermal growth factor receptor 2 (HER2) and the protein tyrosine kinase 6 (PTK6) are often co- and over-expressed in invasive breast cancers. At early diagnosis, only distinct groups, such as HER2-or hormone receptor-positive benefit from a targeted therapy. However, a part of these tumours develops resistance within a year of administration of the drug but the majority of the patients depends on general therapies with severe side effects. A PTK6-directed approach does not yet exist.In our present study, we successfully demonstrate, in vitro and in vivo, a significantly additive reduction of tumourigenesis of breast cancer cells simultaneously depleted of both HER2 and PTK6. In comparison with single RNAi approaches, the combined RNAi (co-RNAi) led to a stronger reduced phosphorylation of tumour-promoting proteins. Moreover, the co-RNAi additively decreased cell migration as well as two and three dimensional cell proliferation in vitro. The in vivo experiments showed an additive reduction (p < 0.00001) in the growth of xenografts due to the co-RNAi compared with HER2 or PTK6 RNAi alone. Interestingly, the complexes of HER2 or PTK6 with tumour-relevant interaction partners, such as HER3 or the insulin-like growth factor receptor 1 (IGF-1R), respectively, were also reduced in xenografts although their protein expression levels were not affected following the co-RNAi of HER2 and PTK6. Our present study reveals the potential of using combined HER2- and PTK6- knockdown as a powerful strategy for the treatment of breast cancers. Therefore, the combined inhibition of these proteins may represent an attractive tool for efficient therapy of breast cancers.

The PTK7-related transmembrane proteins off-track and off-track 2 are co-receptors for Drosophila Wnt2 required for male fertility.

Wnt proteins regulate many developmental processes and are required for tissue homeostasis in adult animals. The cellular responses to Wnts are manifold and are determined by the respective Wnt ligand and its specific receptor complex in the plasma membrane. Wnt receptor complexes contain a member of the Frizzled family of serpentine receptors and a co-receptor, which commonly is a single-pass transmembrane protein. Vertebrate protein tyrosine kinase 7 (PTK7) was identified as a Wnt co-receptor required for control of planar cell polarity (PCP) in frogs and mice. We found that flies homozygous for a complete knock-out of the Drosophila PTK7 homolog off track (otk) are viable and fertile and do not show PCP phenotypes. We discovered an otk paralog (otk2, CG8964), which is co-expressed with otk throughout embryonic and larval development. Otk and Otk2 bind to each other and form complexes with Frizzled, Frizzled2 and Wnt2, pointing to a function as Wnt co-receptors. Flies lacking both otk and otk2 are viable but male sterile due to defective morphogenesis of the ejaculatory duct. Overexpression of Otk causes female sterility due to malformation of the oviduct, indicating that Otk and Otk2 are specifically involved in the sexually dimorphic development of the genital tract.