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Protein-tyrosine pseudokinase 7 (PTK7) directs cancer cell motility and metastasis.

ABSTRACT: It is well established that widely expressed PTK7 is essential for vertebrate tissue morphogenesis. In cancer, the functionality of PTK7 is selectively regulated by membrane type-1 matrix metalloproteinase (MT1-MMP), ADAMs (a disintegrin domain and metalloproteinases), and γ-secretase proteolysis. Here, we established that the full-length membrane PTK7, its Chuzhoi mutant with the two functional MT1-MMP cleavage sites, and its L622D mutant with the single inactivated MT1-MMP cleavage site differentially regulate cell motility in a two-dimensional versus three-dimensional environment. We also demonstrated that in polarized cancer cells, the levels of PTK7 expression and proteolysis were directly linked to the structure and kinetics of cell protrusions, including lamellipodia and invadopodia. In the functionally relevant and widely accepted animal models of metastasis, mouse and chick embryo models, both the overexpression and knock-out of PTK7 in HT1080 cells abrogated metastatic dissemination. Our analysis of human tissue specimens confirmed intensive proteolysis of PTK7 in colorectal cancer tumors, but not in matching normal tissue. Our results provide convincing evidence that both PTK7 expression and proteolysis, rather than the level of the cellular full-length PTK7 alone, contribute to efficient directional cell motility and metastasis in cancer.

SUBMITTER: Golubkov VS

PROVIDER: S-EPMC4148854 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Protein-tyrosine pseudokinase 7 (PTK7) directs cancer cell motility and metastasis.

Golubkov Vladislav S VS Prigozhina Natalie L NL Zhang Yong Y Stoletov Konstantin K Lewis John D JD Schwartz Phillip E PE Hoffman Robert M RM Strongin Alex Y AY

The Journal of biological chemistry 20140708 35

It is well established that widely expressed PTK7 is essential for vertebrate tissue morphogenesis. In cancer, the functionality of PTK7 is selectively regulated by membrane type-1 matrix metalloproteinase (MT1-MMP), ADAMs (a disintegrin domain and metalloproteinases), and γ-secretase proteolysis. Here, we established that the full-length membrane PTK7, its Chuzhoi mutant with the two functional MT1-MMP cleavage sites, and its L622D mutant with the single inactivated MT1-MMP cleavage site differ ...[more]

PMID: 25006253

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Project description:Background & aimsMetastasis is found in most advanced hepatocellular carcinoma (HCC) patients, and it drives tumor recurrence and systemic failure. There is no effective treatment owing to its complex biological features. Many of the molecular drivers of metastasis are crucial players in normal physiology but behave unconventionally during cancer progression. Targeting these molecular drivers for therapy and differentiating them from a physiological background require a detailed examination of the novel mechanisms involved in their activation during metastasis.MethodsPublicly available transcriptomic data such as that of TCGA-LIHC and Gene Expression Omnibus were utilized to identify novel targets upregulated in advanced and metastatic HCC. Validation of candidates was assisted by immunohistochemistry performed on tissue microarrays derived from more than 100 HCC patients. Expression of protein tyrosine kinase 7 (PTK7) was studied under the treatment of transforming growth factor-β1 and knockdown of SRY-Box Transcription Factor 9 (SOX9) to delineate upstream regulation, while CRISPR-mediated knockout and lentiviral overexpression of PTK7 in HCC cells were performed to study their functional and signaling consequences. Manipulated HCC cells were injected into mice models either by orthotopic or tail-vein injection to observe for any in vivo pro-metastatic effects.ResultsPTK7 was discovered to be the kinase most significantly upregulated in advanced and metastatic HCC, at both transcriptomic and proteomic level. Bioinformatic analyses and functional assays performed in HCC cell lines revealed transforming growth factor-β signaling and SOX9 to be important activators of PTK7 expression. Functionally, enrichment of PTK7 expression could positively regulate metastatic potential of HCC cells in vitro and in lung metastasis models performed in immunodeficient mice. The up-regulation of PTK7 recruited the epithelial-mesenchymal transition components, zinc finger protein SNAI2 (SLUG) and zinc finger E-box-binding homeobox 1 (ZEB1).ConclusionsOur study proposes PTK7 as a novel molecular driver in metastatic HCC, particularly in a transforming growth factor-β-activated microenvironment. The preferential expression of PTK7 resulted in a previously unobserved regulatory effect on the recruitment of epithelial-mesenchymal transition components, which established PTK7 as a potential determinant of specific epithelial-mesenchymal transition status. Therefore, our data support the continual development of PTK7-targeted agents as antimetastatic therapies.

Project description:BackgroundThe 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.ResultsWe 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.ConclusionsOur 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.

Project description:IntroductionT-cell lymphoma invasion and metastasis-inducing protein (Tiam1) is a Ras-related C3 botulinum toxin substrate (Rac)-specific guanine nucleotide exchange factor that was isolated based on its ability to induce a metastatic phenotype. In polarized migrating keratinocytes, Tiam1 is found at the leading edge where it cooperates with the Protease-activated receptor 1 (Par1) complex to establish front-rear polarity. Although a positive correlation has been observed between Tiam1 expression and tumor grade in a variety of human malignancies, including breast, its role in breast cancer cells has not yet been examined.MethodsTiam1 expression and Rac activity were examined in a panel of human breast cancer cell lines which exhibit different degrees of cell motility. The contribution of Tiam1 to cell motility was directly examined using transwell motility, and wound healing assays.ResultsAlthough we observe a striking, positive correlation between Tiam1 expression and cell motility in the panel of breast cancer cell lines, we do not observe a correlation between Tiam1 expression and overall levels of Rac activity. Consistent with this, small interfering ribonucleic acid (siRNA)-mediated suppression of Tiam1 expression limits the motility of cell lines in which Tiam1 expression is high (MDA-MB-231 and MDA-MB-453), but does not substantially alter the overall levels of activated Rac. Tiam1 overexpression is also not sufficient to increase the motility of more poorly motile cells (T-47D) or increase Rac activity. Immunofluorescence and cellular fractionations indicate that Tiam1 is found predominantly in the Golgi of breast cancer cells, and in the latter case Tiam1 was shown to co-fractionate with a limited pool of Rac1. Consistent with this Golgi localization, Tiam1 supports cell motility and Golgi reorientation in response to serum in a wound healing assay using MDA-MB-231and MDA-MB-435S cells.ConclusionsTiam1 expression correlates with cell motility in human breast cancer cells, and is required to support the motile phenotype. Localization of endogenous Tiam1 to the Golgi, and its demonstrated role in Golgi reorientation, suggest that it may support motility through a mechanism that is discrete from its known function in leading edge dynamics.

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Protein-tyrosine pseudokinase 7 (PTK7) directs cancer cell motility and metastasis.

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Protein-tyrosine pseudokinase 7 (PTK7) directs cancer cell motility and metastasis.

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