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
TL;DR: Novel evidence is provided that extensive impairment in pEGFR endocytosis via the early endocytic pathway might confer gefitinib-resistance in NSCLC cell lines, and an aberration in some steps of EGF-EGFR trafficking from the early endsomes to late endosomes/lysosomes occurs in QG56 cells.
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.
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TL;DR: An overview of the PX domain proteins is presented, incorporating recent functional and structural insights, and an updated classification of the proteins into distinct subfamilies is proposed on the basis of these insights.
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.
254 citations
Journal Article•
TL;DR: The current knowledge of the nuclear EGFR signaling network is summarized, including how it is trafficked to the nucleus, the functions it serves inThe nucleus, and how these functions impact cancer progression, survival, and response to chemotherapeutics.
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.
218 citations
TL;DR: Light is shed on the role of nuclear EGFR in the sensitivity of wtEGFR-expressing cancer cells to EGFR tyrosine kinase inhibitors and a putative molecular mechanism contributing to gefitinib resistance through BCRP/ABCG2 expression is deciphered.
177 citations
TL;DR: An improved understanding of the molecular mechanisms of resistance to EGFR inhibitors may provide valuable indications to identify biomarkers that can be used clinically to predict response to EG FR blockade and to establish new treatment options to overcome resistance.
Abstract: Targeted therapy against the epidermal growth factor receptor (EGFR) is one of the most promising molecular therapeutics for head and neck squamous cell carcinoma (HNSCC). EGFR is overexpressed in a wide range of malignancies, including HNSCC, and initiates important signal transduction pathways in HNSCC carcinogenesis. However, primary and acquired resistance are serious problems and are responsible for low single-agent response rate and tumor recurrence. Therefore, an improved understanding of the molecular mechanisms of resistance to EGFR inhibitors may provide valuable indications to identify biomarkers that can be used clinically to predict response to EGFR blockade and to establish new treatment options to overcome resistance. To date, no predictive biomarker for HNSCC is available in the clinic. Therapeutic resistance to anti-EGFR therapy may arise from mechanisms that can compensate for reduced EGFR signaling and/or mechanisms that can modulate EGFR-dependent signaling. In this review, we will summarize some of these molecular mechanisms and describe strategies to overcome that resistance.
80 citations
TL;DR: It is shown that acquired resistance of wtEGFR-expressing cancer cells to an EGFR TKI, gefitinib, is associated with elevated expression of breast cancer resistance protein (BCRP/ABCG2), which in turn leads to gefITinib efflux from cells.
Abstract: Background
The sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) is strongly associated with activating EGFR mutations. Although not as sensitive as patients harboring these mutations, some patients with wild-type EGFR (wtEGFR) remain responsive to EGFR TKIs, suggesting that the existence of unexplored mechanisms renders most of wtEGFR-expressing cancer cells insensitive.
80 citations
Additional excerpts
...mutations [6,7,8,10,11,12,13], suggesting that other unknown...
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References
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TL;DR: Epidermal growth factor is a signaling molecule that stimulates the growth of epidermal tissues during development and throughout life and led to the 1986 Nobel Prize in Physiology or Medicine awarded to Cohen and Rita Levi–Montalcini.
1,548 citations
TL;DR: The role of these active anti-receptor agents in the treatment of patients with cancer is addressed and compounds that directly inhibit receptor tyrosine kinases have shown preclinical activity and early clinical activity has been reported.
Abstract: Human carcinomas frequently express high levels of receptors in the EGF receptor family, and overexpression of at least two of these receptors, the EGF receptor (EGFr) and closely related ErbB2, has been associated with a more aggressive clinical behavior. Further, transfection or activation of high levels of these two receptors in nonmalignant cell lines can lead to a transformed phenotype. For these reasons therapies directed at preventing the function of these receptors have the potential to be useful anti-cancer treatments. In the last two decades monoclonal antibodies (MAbs) which block activation of the EGFr and ErbB2 have been developed. These MAbs have shown promising preclinical activity and 'chimeric' and 'humanized' MAbs have been produced in order to obviate the problem of host immune reactions. Clinical activity with these antibodies has been documented: trastuzumab, a humanized anti-ErbB2 MAb, is active and was recently approved in combination with paclitaxel for the therapy of patients with metastatic ErbB2-overexpressing breast cancer; IMC-C225, a chimeric anti-EGFr MAb, has shown impressive activity when combined with radiation therapy and reverses resistance to chemotherapy. In addition to antibodies, compounds that directly inhibit receptor tyrosine kinases have shown preclinical activity and early clinical activity has been reported. A series of phase III studies with these antibodies and direct tyrosine kinase inhibitors are ongoing or planned, and will further address the role of these active anti-receptor agents in the treatment of patients with cancer.
1,349 citations
"Evidence for efficient phosphorylat..." refers background in this paper
...Background The epidermal growth factor receptor (EGFR) is a prototypical member of the ErbB family of tyrosine kinases and plays an important role in the pathogenesis of different tumors; therefore, therapies directed at inhibiting EGFR function have potential as anticancer treatments [1,2]....
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TL;DR: It is demonstrated that nuclear EGFR is strongly correlated with highly proliferating activities of tissues and associated with promoter region of cyclin D1 in vivo, suggesting that EGFR might function as a transcription factor to activate genes required for highly proliferationating activities.
Abstract: Epidermal growth factor receptor (EGFR) has been detected in the nucleus in many tissues and cell lines. However, the potential functions of nuclear EGFR have largely been overlooked. Here we demonstrate that nuclear EGFR is strongly correlated with highly proliferating activities of tissues. When EGFR was fused to the GAL4 DNA-binding domain, we found that the carboxy terminus of EGFR contained a strong transactivation domain. Moreover, the receptor complex bound and activated AT-rich consensus-sequence-dependent transcription, including the consensus site in cyclin D1 promoter. By using chromatin immunoprecipitation assays, we further demonstrated that nuclear EGFR associated with promoter region of cyclin D1 in vivo. EGFR might therefore function as a transcription factor to activate genes required for highly proliferating activities.
1,051 citations
"Evidence for efficient phosphorylat..." refers background in this paper
...It was also shown that nuclear EGFR levels were increased on treatment with EGF and that the EGFR which accumulated in the nucleus was highly tyrosine phosphorylated; it was further demonstrated that nuclear EGFR acts as a transcription factor for activating gene expression of cyclin D1, a well-known cell growth-promoting factor [26]....
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...In fact, it has recently been reported that nuclear localization of EGFR is detected in the highly proliferating state of human cancer tissues in vivo and human breast cancer cell lines in vitro, supporting the close correlation between nuclear EGFR and tumor tissues with high proliferation [25-27]....
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TL;DR: The complex nature of EGFR biology allows for potential opportunities for EGFR inhibitors in a number of areas of cancer therapy, including proliferative, angiogenic, invasive, and metastatic aspects.
838 citations
"Evidence for efficient phosphorylat..." refers background in this paper
...Gefitinib (Iressa, ZD1839) is a selective EGFR tyrosine kinase inhibitor that functions by competing with ATP for binding to the tyrosine kinase domain of the receptor, and it blocks the signal transduction pathways implicated in the proliferation and survival of cancer cells [6-9]....
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Journal Article•
TL;DR: It is shown that coadministration of ZD1839, as with anti-EGFR, will enhance the efficacy of cytotoxic agents against human vulvar (A431), lung (A549 and SK-LC-16 NSCL and LX-1), and prostate (PC-3 and TSU-PR1) tumors, and potentiated most cytot toxic agents in combination treatment against all of these tumors, irrespective of EGFR status.
Abstract: The blockade of epidermal growth factor receptor (EGFR) function with monoclonal antibodies has major antiproliferative effects against human tumors in vivo. Similar antiproliferative effects against some of these same tumors have also been observed with specific inhibitors of the EGFR-associated tyrosine kinase. One such inhibitor, the p.o. active ZD1839 (Iressa), has pronounced antiproliferative activity against human tumor xenografts. We now show that coadministration of ZD1839, as with anti-EGFR, will enhance the efficacy of cytotoxic agents against human vulvar (A431), lung (A549 and SK-LC-16 NSCL and LX-1), and prostate (PC-3 and TSU-PR1) tumors. Oral ZD1839 (five times daily x 2) and cytotoxic agents (i.p. every 3-4 days x 4) were given for a period of 2 weeks to mice with well-established tumors. On this schedule, the maximum tolerated dose (150 mg/kg) of ZD1839 induced partial regression of A431, a tumor that expresses high levels of EGFR, 70-80% inhibition among tumors with low but highly variable levels of EGFR expression (A549, SKLC-16, TSU-PR1, and PC-3), and 50-55% inhibition against the LX-1 tumor, which expresses very low levels of EGFR. ZD1839 was very effective in potentiating most cytotoxic agents in combination treatment against all of these tumors, irrespective of EGFR status, but dose reduction of ZD1839 below its single-agent maximum tolerated dose was required for optimum tolerance. The pronounced growth inhibitory action of the platinums, cisplatin and carboplatinum, as single agents against A431 vulvar, A549 and LX-1 lung, and TSU-PR1 and PC-3 prostate tumors was increased several-fold when ZD1839 was added, with some regression of A431 and PC-3 tumors. Although the taxanes, paclitaxel or docetaxel, as single agents markedly inhibited the growth of A431, LX-1, SK-LC-16, TSU-PR1, and PC-3, when combined with ZD1839, partial or complete regression was usually seen. Against A549, the growth inhibition of doxorubicin was increased 10-fold (>99%) with ZD1839. The folate analogue, edatrexate, was highly growth inhibitory against A549, LX-1, and TSU-PR1, whereas edatrexate combined with ZD1839 resulted in partial or complete regression in these tumors. Against the A431 tumor, paclitaxel alone either was highly growth inhibitory or induced some regression, but when combined with ZD1839, pronounced regression was obtained. Combination with gemcitabine neither added nor detracted from baseline cytotoxic efficacy, whereas ZD1839 combined with vinorelbine was poorly tolerated. Overall, these results suggest that potentiation of cytotoxic treatment with ZD1839 does not require high levels of EGFR expression in the target tumors. They also suggest significant clinical benefit from ZD1839 in combination with a variety of widely used cytotoxic agents.
787 citations
"Evidence for efficient phosphorylat..." refers background in this paper
...It has exhibited significant antitumor activity against a broad range of mouse tumor xenograft models in vivo [10] and tumor cell lines in vitro [11]....
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