Biborka Bereczky

Bio: Biborka Bereczky is an academic researcher from Kyushu University. The author has contributed to research in topics: Endocytic cycle & Endocytosis. The author has an hindex of 6, co-authored 6 publications receiving 230 citations.

The EGFR inhibitor gefitinib suppresses ligand-stimulated endocytosis of EGFR via the early/late endocytic pathway in non-small cell lung cancer cell lines.

Abstract: The drug gefitinib (Iressa), which is a specific inhibitor of EGFR tyrosine kinase, has been shown to suppress the activation of EGFR signaling for survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. A recent study demonstrated rapid down-regulation of ligand-induced EGFR in a gefitinib-sensitive cell line and inefficient down-regulation of EGFR in a gefitinib-resistant cell line in the exponential phase of growth; this implies that each cell type employs a different unknown down-regulation mechanism occurs. However, the mechanism of drug sensitivity to gefitinib remains unclear. In this study, to further substantiate the effect of gefitinib on the EGFR down-regulation pathway and to understand the detailed internalization mechanism of gefitinib-sensitive PC9 and gefitinib-resistant QG56 cell lines, we examined the internalization of Texas red-EGF in the absence or presence of gefitinib in both cell lines. The distribution of internalized Texas red-EGF, early endosomes, and late endosomes/lysosomes was then assessed by confocal immunofluorescence microscopy. Here, we provide novel evidence that efficient endocytosis of EGF-EGFR occurs via the endocytic pathway in the PC9 cells, because the internalized Texas red-EGF-positive small punctate vesicles were transported to the late endosomes/lysosomes and then degraded within the lysosomes after 60 min of internalization. Additionally, gefitinib exerted a strong inhibitory effect on the endocytosis of EGFR in PC9 cells, and the internalization rate of EGFR from the plasma membrane via the early endosomes to the late endosomes/lysosomes was considerably delayed. This indicates that gefitinib efficiently suppresses ligand-stimulated endocytosis of EGFR via the early/late endocytic pathway in PC9 cells. In contrast, the internalization rate of ligand-induced EGFR was not significantly changed by gefitinib in QG56 cells because even in the absence of gefitinib, internalized EGFR accumulation was noted in the early and late endosomes after 60 min of internalization instead of its delivery to the lysosomes in QG56 cells. This suggests that the endocytic machinery of EGFR might be basically impaired at the level of the early/late endosomes. Taken together, this is the first report demonstrating that the suppressive effect of gefitinib on the endocytosis of EGFR is much stronger with PC9 cells than QG56 cells. Thus, impairment in some steps of the EGF-EGFR traffic out of early endosomes toward the late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines.

Evidence for efficient phosphorylation of EGFR and rapid endocytosis of phosphorylated EGFR via the early/late endocytic pathway in a gefitinib-sensitive non-small cell lung cancer cell line

Abstract: Gefitinib (Iressa)–a specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase–has been shown to suppress the activation of EGFR signaling required for cell survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. We recently provided novel evidence that gefitinib-sensitive PC9 cells show normal endocytosis of EGFR: internalized EGF-EGFR complexes were transported to late endosomes/lysosomes 15 min after EGF stimulation, and then degraded within the lysosomes. However, gefitinib-resistant QG56 cells showed internalized EGFR accumulation in early endosomes after 60 min of internalization, instead of its trafficking to lysosomes, indicating an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes. Therefore, we postulate that impairment in some steps of EGF-EGFR trafficking from early endosomes to late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines. To further substantiate the detailed internalization mechanism of gefitinib-sensitive and gefitinib-resistant cells, using confocal immunofluorescence microscopy, we examined the endocytic trafficking of phosphorylated EGFR (pEGFR) in the absence or presence of gefitinib. In PC9 and QG56 cells without EGF stimulation, a large number of pEGFR-positive small vesicular structures not colocalized with late endosomes/lysosomes were spread throughout the cytoplasm, and some pEGFR staining was distributed in the nucleus. This implies a novel intracellular trafficking pathway for pEGFR from cytoplasmic vesicles to the nucleus. Furthermore, an aggregated vesicular structure of early endosomes was observed in the perinuclear region of QG56 cells; it was revealed to be associated with SNX1, originally identified as a protein that interacts with EGFR. Therefore, we confirmed our previous data that an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes occurs in QG56 cells. Furthermore, in PC9 cells, efficient phosphorylation of EGFR and rapid internalization of pEGFR was observed at 3 min after EGF stimulation; these internalized pEGFR-positive vesicles were trafficked to late endosomes at 15 min, indicating rapid trafficking of EGF-pEGFR complexes from early to late endosomes in PC9 cells. Gefitinib treatment strongly reduced the phosphorylation level of EGFR, and subsequent endocytosis of EGFR was significantly suppressed in PC9 cells. In contrast, in QG56 cells, EGFR trafficking via the early endocytic pathway was basically impaired; therefore, gefitinib appeared to slightly suppress the internalization of pEGFR. Collectively, our data provide novel evidence that extensive impairment in pEGFR endocytosis via the early endocytic pathway might confer gefitinib-resistance in QG56 cells.

A role of LIM kinase 1/cofilin pathway in regulating endocytic trafficking of EGF receptor in human breast cancer cells

Abstract: We have previously shown that overexpression of LIM kinase1 (LIMK1) resulted in a marked retardation of the internalization of the receptor-mediated endocytic tracer, Texas red-labeled epidermal growth factor (EGF) in low-invasive human breast cancer cell MCF-7. We thereby postulate that LIMK1 signaling plays an important role in the regulation of ligand-induced endocytosis of EGF receptor (EGFR) in tumor cells by reorganizing and influencing actin-filament dynamics. In the present study, we further assessed the effect of wild-type LIMK1, a kinase-deficient dominant negative mutant of LIMK1 (DN-LIMK1) and an active, unphosphorylatable cofilin mutant (S3A cofilin) on internalization of EGF-EGFR in MDA-MB-231, a highly invasive human breast cancer cell line. We demonstrate here that a marked delay in the receptor-mediated internalization of Texas red-labeled EGF was observed in the wild-type LIMK1 transfectants, and that most of the internalized EGF staining were accumulated within transferrin receptor-positive early endosomes even after 30 min internalization. In contrast, the expression of dominant-negative LIMK1 mutant rescued the efficient endocytosis of Texas red-EGF, and large amounts of Texas red-EGF staining already reached LIMPII-positive late endosomes/lysosomal vacuoles after 15 min internalization. We further analyzed the effect of S3A cofilin mutant on EGFR trafficking, and found an efficient delivery of Texas red-EGF into late endosomes/lysosomes at 15-30 min after internalization. Taken together, our novel findings presented in this paper implicate that LIMK1 signaling indeed plays a pivotal role in the regulation of EGFR trafficking through the endocytic pathway in invasive tumor cells.

Clathrin-Independent Pathways of Endocytosis

Abstract: There are many pathways of endocytosis at the cell surface that apparently operate at the same time. With the advent of new molecular genetic and imaging tools, an understanding of the different ways by which a cell may endocytose cargo is increasing by leaps and bounds. In this review we explore pathways of endocytosis that occur in the absence of clathrin. These are referred to as clathrin-independent endocytosis (CIE). Here we primarily focus on those pathways that function at the small scale in which some have distinct coats (caveolae) and others function in the absence of specific coated intermediates. We follow the trafficking itineraries of the material endocytosed by these pathways and finally discuss the functional roles that these pathways play in cell and tissue physiology. It is likely that these pathways will play key roles in the regulation of plasma membrane area and tension and also control the availability of membrane during cell migration.

LIM kinases: function, regulation and association with human disease

Abstract: The LIM kinase family consists of just two members: LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2). With uniquely organised signalling domains, LIM kinases are regulated by several upstream signalling pathways, principally acting downstream of Rho GTPases to influence the architecture of the actin cytoskeleton by regulating the activity of the cofilin family proteins cofilin1, cofilin2 and destrin. Although the LIM kinases are very homologous, particularly when comparing kinase domains, there is emerging evidence that each may be subject to different regulatory pathways and may contribute to both distinct and overlapping cellular and developmental functions. Normal central nervous system development is reliant upon the presence of LIMK1, and its deletion has been implicated in the development of the human genetic disorder Williams syndrome. Normal testis development, on the other hand, is disrupted by the deletion of LIMK2. In addition, the possible involvement of each kinase in cardiovascular disorders as well as cancer has recently emerged. The LIM kinases have been proposed to play an important role in tumour-cell invasion and metastasis; fine-tuning the balance between phosphorylated and non-phosphorylated cofilin may be a significant determinant of tumour-cell metastatic potential. In this review, we outline the structure, regulation and function of LIM kinases and their functions at cellular and organismal levels, as well as their possible contributions to human disease.

Insights into the PX (phox-homology) domain and SNX (sorting nexin) protein families: structures, functions and roles in disease

Abstract: The mammalian genome encodes 49 proteins that possess a PX (phox-homology) domain, responsible for membrane attachment to organelles of the secretory and endocytic system via binding of phosphoinositide lipids. The PX domain proteins, most of which are classified as SNXs (sorting nexins), constitute an extremely diverse family of molecules that play varied roles in membrane trafficking, cell signalling, membrane remodelling and organelle motility. In the present review, we present an overview of the family, incorporating recent functional and structural insights, and propose an updated classification of the proteins into distinct subfamilies on the basis of these insights. Almost all PX domain proteins bind PtdIns3P and are recruited to early endosomal membranes. Although other specificities and localizations have been reported for a select few family members, the molecular basis for binding to other lipids is still not clear. The PX domain is also emerging as an important protein-protein interaction domain, binding endocytic and exocytic machinery, transmembrane proteins and many other molecules. A comprehensive survey of the molecular interactions governed by PX proteins highlights the functional diversity of the family as trafficking cargo adaptors and membrane-associated scaffolds regulating cell signalling. Finally, we examine the mounting evidence linking PX proteins to different disorders, in particular focusing on their emerging importance in both pathogen invasion and amyloid production in Alzheimer's disease.

The nuclear epidermal growth factor receptor signaling network and its role in cancer

Abstract: The epidermal growth factor receptor (EGFR) is a member of the EGFR family of receptor tyrosine kinases (RTKs). EGFR activation via ligand binding results in signaling through various pathways ultimately resulting in cellular proliferation, survival, angiogenesis, invasion, and metastasis. Aberrant expression or activity of EGFR has been strongly linked to the etiology of several human epithelial cancers including but not limited to head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer, pancreatic cancer, and brain cancer. Thus intense efforts have been made to inhibit the activity of EGFR by designing antibodies against the ligand binding domains (cetuximab and panitumumab) or small molecules against the tyrosine kinase domain (erlotinib, gefitinib, and lapatinib). Although targeting membrane-bound EGFR has shown benefit, a new and emerging role for EGFR is now being elucidated. In this review we will summarize the current knowledge of the nuclear EGFR signaling network, including how it is trafficked to the nucleus, the functions it serves in the nucleus, and how these functions impact cancer progression, survival, and response to chemotherapeutics.