EGF Receptor Research Articles

Tubular microdomains of Rab7-positive endosomes retrieve TrkA, a mechanism disrupted in Charcot-Marie-Tooth disease 2B.

Axonal survival and growth requires signalling from tropomyosin receptor kinases (Trks). To transmit their signals, receptor-ligand complexes are endocytosed and undergo retrograde trafficking to the soma, where downstream signalling occurs. Vesicles transporting neurotrophic receptors to the soma are reported to be Rab7-positive late endosomes and/or multivesicular bodies (MVBs), where receptors localize within so-called intraluminal vesicles (herein Rab7 corresponds to Rab7A unless specified otherwise). Therefore, one challenging question is how downstream signalling is possible given the insulating properties of intraluminal vesicles. In this study, we report that Rab7-positive endosomes and MVBs retrieve TrkA (also known as NTRK1) through tubular microdomains. Interestingly, this phenotype is absent for the EGF receptor. Furthermore, we found that endophilinA1, endophilinA2 and endophilinA3, together with WASH1 (also known as WASHC1), are involved in the tubulation process. In Charcot-Marie-Tooth disease 2B (CMT2B), a neuropathy of the peripheral nervous system, this tubulating mechanism is disrupted. In addition, the ability to tubulate correlates with the phosphorylation levels of TrkA as well as with neurite length in neuronal cultures from dorsal root ganglia. In all, we report a new retrieval mechanism of late Rab7-positive endosomes, which enables TrkA signalling and sheds new light onto how neurotrophic signalling is disrupted in CMT2B. This article has an associated First Person interview with the first author of the paper.

Persicoimidate isolated from Allium ampeloprasum Subsp. Persicum with Apoptotic effects against Breast cancer cell lines

Breast cancer is the most common malignancy among American women and the second leading cause of cancer death after the lung cancer. For this reason, trying to find new drugs for the treatment of this disease is essential. The aim of the present study was to evaluate the cytotoxic effects of three cinnamic acid derivatives isolated from Allium ampeloprasum var. Persicum including Caffeoyl tyramine, Feruloyl tyramine, and Persicoimidate on two breast cancer cell lines, MCF-7 and BT-474. Evaluation the cytotoxic effects of mentioned purified compounds against MCF-7 and BT-474 cells was performed using MTT assay and their IC50 was determined. Finally, flow cytometery analysis on MCF-7 cells was performed and used to determine the cell death mechanism of investigated compounds. The results exhibited that the cytotoxic effects of all three compounds against breast cancer cell lines was concentration-dependent. The IC50 of Caffeoyl tyramine, Feruloyl tyramine, and Persicoimidate was determined to be as 73, 56, and 40 μg/ml for MCF-7 and 100, 54, and 37 μg/ml for BT-474 cells, respectively. So, Perscicoimidate showed the most potent cytotoxic effects against two breast cancer cells. Finally, flow cytometery analysis showed that Persicoimidate caused approximately 48% of apoptosis in concentration of 40 μg/ml. According to the tyrosine kinase inhibitory activity of cinnamic acid derivatives, these compounds has the potential of being cancer drug candidates for complementary studies on breast tumors with highly expression of EGF receptor. However, evaluation of anti-cancer effects of these compounds against other breast cancer cell lines is suggested.

MA03.01 EPICAL Trial. A Phase Ib StudyCombining Anti-Epidermal Growth Factor (EGF) Vaccination With Afatinib in EGFR-Mutant Non-Small Cell Lung Cancer

Stage IIIB-IV non-small cell lung cancer patients with mutations in the EGF receptor gene (EGFR) usually derive clinical benefit from to tyrosine kinase inhibitors (EGFR TKIs) but ultimately relapse. In preclinical studies, we have showed that anti-EGF antibodies generated by vaccination significantly increased the antitumor activity of TKIs in EGFR-mut cell lines, blocking EGFR, Erk1/2, Akt and STAT3 activation and delaying emergence of resistance. Based on these findings, the EPICAL trial was initiated (ClinicalTrials.gov number, NCT03623750) .

ISGylation drives basal breast tumour progression by promoting EGFR recycling and Akt signalling

ISG15 is an ubiquitin-like modifier that is associated with reduced survival rates in breast cancer patients. The mechanism by which ISG15 achieves this however remains elusive. We demonstrate that modification of Rab GDP-Dissociation Inhibitor Beta (GDI2) by ISG15 (ISGylation) alters endocytic recycling of the EGF receptor (EGFR) in non-interferon stimulated cells using CRISPR-knock out models for ISGylation. By regulating EGFR trafficking, ISGylation enhances EGFR recycling and sustains Akt-signalling. We further show that Akt signalling positively correlates with levels of ISG15 and its E2-ligase in basal breast cancer cohorts, confirming the link between ISGylation and Akt signalling in human tumours. Persistent and enhanced Akt activation explains the more aggressive tumour behaviour observed in human breast cancers. We show that ISGylation can act as a driver of tumour progression rather than merely being a bystander.

Assessing the use of tumor-specific DARPin-toxin fusion proteins for ex vivo purging of cancer metastases from human ovarian cortex before autotransplantation

To assess the use of tumor-specific designed ankyrin repeat proteins (DARPins) fused to a domain of Pseudomonas aeruginosa exotoxin A for purging of cancer metastases from the ovarian cortex. Experimental study. University medical center. Human ovarian cortex. Ovarian cortex harboring artificially induced breast cancer metastases was treated with DARPins targeted to epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor 2 (HER2). The presence of any remaining cancer cells after purging was analyzed by (immuno)histochemistry and reverse transcriptase polymerase chain reaction. Effects on the viability of the ovarian cortex were determined by (immuno)histology, a follicular viability assay, and an assay to determine the invitro growth capacity of small follicles. After purging with EpCAM-targeted DARPin, all EpCAM-positive breast cancer cells were eradicated from the ovarian cortex. Although treatment had no effect on the morphology or viability of small follicles, a sharp decrease in oocyte viability during invitro growth was observed, presumably due to low-level expression of EpCAM on oocytes. The HER2-targeted DARPins had no detrimental effects on the morphology, viability, or invitro growth of small follicles. HER2-positive breast cancer foci were fully eliminated from the ovarian cortex, and the reverse transcriptase polymerase chain reaction showed a decrease to basal levels of HER2 mRNA after purging. Purging cancer metastases from ovarian cortex without impairing ovarian tissue integrity is possible by targeting tumor cell surface proteins with exotoxin A-fused DARPins. By adapting the target specificity of the cytotoxic DARPin fusions, it should be possible to eradicate metastases from all types of malignancies.

The wound healing effect of local leukocyte platelet-rich plasma after total hip arthroplasty: A randomized controlled trial.

Rapid wound closure is important after arthroplasty procedures to prevent postoperative complications. Platelets are rich in growth factors and leukocytes contribute to innate immunity. We hypothesized that topical leukocyte platelet-rich plasma (L-PRP) derived from the blood of patients would be beneficial to wound healing. In this randomized controlled trial, patients subjected to elective total hip arthroplasty (THA) were assigned by concealed allocation either L-PRP application onto the sutured fascia or no application (control) after the THA intervention. In addition, all patients received 1.5g protein/kg, 5 g L-arginine, 500 mg vitamin C and 44 mg zinc daily over the 4-week postoperative period to obtain optimal nutrition. The primary endpoint was complete healing of the skin incision. The secondary endpoints were blood transfusions, length of hospital stay, pain and wound infections. Sixteen patients in the L-PRP group and 17 patients in the control group completed the trial. L-PRP treatment accelerated complete wound healing after 3 weeks (seven in the L-PRP group vs. zero in the control group, p=0.003) and after 4 weeks (12 in the L-PRP group vs. six in the control group, p=0.037). No postoperative superficial wound infections occurred within 4 weeks, and there were no significant differences in the other secondary outcomes. L-PRP generated in 10 sex-matched healthy volunteers revealed increased concentrations of platelets (5.8-fold) and leukocytes (2.3-fold) compared with those in whole blood. Furthermore, the concentration of keratinocyte mitogen epidermal growth factor in L-PRP (380 ± 130 pg/ml, mean ± SD) was higher (p < 0.001) than that in serum (130 ± 26 pg/ml). In conclusion, a single intraoperative local application of L-PRP promoted wound healing after THA, possibly mediated by EGF receptor agonists.

G protein-coupled estrogen receptor signaling dependent epidermal growth-like factor expression is required for NPR2 inhibition and meiotic resumption in goat oocytes

G protein-coupled estrogen receptor (GPER), which is different from traditional estrogen nuclear receptors (ERs), mediates the rapid transduction of nongenomic signals in cells, and works by regulating transcription and intracellular second messengers. Studies have shown that GPER may regulate oocyte maturation, but the relevant mechanism is not entirely clear. Here, goat cumulus-oocyte complexes (COCs) were used as a model to explore the regulation and mechanism of GPER on oocyte maturation. Our study showed that 17β-estradiol (E2) significantly reduced cyclic guanosine monophosphate (cGMP) synthesis in COCs and accelerated the meiotic resumption of goat oocytes via GPER. Further investigation found that GPER mediated the downregulation of natriuretic peptide receptor 2 (NPR2) protein expression in goat cumulus cells by E2. In addition, we found that E2 significantly upregulated the mRNA levels of epidermal growth (EGF)-like factors in goat cumulus cells through GPER, and activated the downstream EGF receptor (EGFR) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways. Both AG1478 (EGFR inhibitor) and U0126 (ERK1/2 inhibitor) abolished the inhibitory effect of E2 on the protein expression of NPR2. These results indicate that, through GPER, E2 upregulates the mRNA levels of EGF-like factors in goat cumulus cells and activates the downstream EGF signaling network to suppress the expression of NPR2 protein, which results in a decrease in cGMP synthesis and acceleration of meiotic resumption in goat oocytes.

KCa channel blockers increase effectiveness of the EGF receptor TK inhibitor erlotinib in non-small cell lung cancer cells (A549)

Non-small cell lung cancer (NSCLC) has a poor prognosis with a 5 year survival rate of only ~ 10%. Important driver mutations underlying NSCLC affect the epidermal growth factor receptor (EGFR) causing the constitutive activation of its tyrosine kinase domain. There are efficient EGFR tyrosine kinase inhibitors (TKIs), but patients develop inevitably a resistance against these drugs. On the other hand, KCa3.1 channels contribute to NSCLC progression so that elevated KCa3.1 expression is a strong predictor of poor NSCLC patient prognosis. The present study tests whether blocking KCa3.1 channels increases the sensitivity of NSCLC cells towards the EGFR TKI erlotinib and overcomes drug resistance. mRNA expression of KCa3.1 channels in erlotinib-sensitive and -resistant NSCLC cells was analysed in datasets from Gene expression omnibus (GEO) and ArrayExpress. We assessed proliferation and migration of NSCLC cells. These (live cell-imaging) experiments were complemented by patch clamp experiments and Western blot analyses. We identified three out of four datasets comparing erlotinib-sensitive and -resistant NSCLC cells which revealed an altered expression of KCa3.1 mRNA in erlotinib-resistant NSCLC cells. Therefore, we evaluated the combined effect of erlotinib and the KCa3.1 channel inhibition with sencapoc. Erlotinib elicits a dose-dependent inhibition of migration and proliferation of NSCLC cells. The simultaneous application of the KCa3.1 channel blocker senicapoc increases the sensitivity towards a low dose of erlotinib (300 nmol/L) which by itself has no effect on migration and proliferation. Partial erlotinib resistance can be overcome by KCa3.1 channel blockade. The sensitivity towards erlotinib as well as the potentiating effect of KCa3.1 blockade is further increased by mimicking hypoxia. Our results suggest that KCa3.1 channel blockade may constitute a therapeutic concept for treating NSCLC and overcome EGFR TKI resistance. We propose that this is due to complementary mechanisms of action of both blockers.

Erlotinib Promotes Ligand-Induced EGFR Degradation in 3D but Not 2D Cultures of Pancreatic Ductal Adenocarcinoma Cells.

Simple SummaryThe EGFR is a tyrosine kinase receptor that responds to different stresses such as UV irradiation, hypoxia and drug treatment by internalizing into endosomal compartments. Receptor trafficking and degradation due to tyrosine kinase inhibitors has been widely studied in two- dimensional (2D) cell culture systems, but little is known about how cells respond to these types of drugs in more physiologically relevant models such as three-dimensional (3D) cultures, whose nanostructured properties allow cells to grow, proliferate, migrate and extend cellular processes in their 3D space. In this study, we show that EGFR suffers degradation in response to erlotinib treatment in 3D-cultured cancer cells but not in classic 2D culture systems, demonstrating that dimensionality strongly affects cell drug response. This 3D model may pave the way for the development of more physiological culture platforms to obtain mechanistic insights into how cells respond to chemotherapy.The epithelial growth factor receptor (EGFR) is a tyrosine kinase receptor that participates in many biological processes such as cell proliferation. In addition, EGFR is overexpressed in many epithelial cancers and therefore is a target for cancer therapy. Moreover, EGFR responds to lots of stimuli by internalizing into endosomes from where it can be recycled to the membrane or further sorted into lysosomes where it undergoes degradation. Two-dimensional cell cultures have been classically used to study EGFR trafficking mechanisms in cancer cells. However, it has been widely demonstrated that in 2D cultures cells are exposed to a non-physiological environment as compared to 3D cultures that provide the normal cellular conformation, matrix dimensionality and stiffness, as well as molecular gradients. Therefore, the microenvironment of solid tumors is better recreated in 3D culture models, and this is why they are becoming a more physiological alternative to study cancer physiology. Here, we develop a new model of EGFR internalization and degradation upon erlotinib treatment in pancreatic ductal adenocarcinoma (PDAC) cells cultured in a 3D self-assembling peptide scaffold. In this work, we show that treatment with the tyrosine kinase inhibitor erlotinib promotes EGFR degradation in 3D cultures of PDAC cell lines but not in 2D cultures. We also show that this receptor degradation does not occur in normal fibroblast cells, regardless of culture dimensionality. In conclusion, we demonstrate not only that erlotinib has a distinct effect on tumor and normal cells but also that pancreatic ductal adenocarcinoma cells respond differently to drug treatment when cultured in a 3D microenvironment. This study highlights the importance of culture systems that can more accurately mimic the in vivo tumor physiology.

Physiological functions of keratinocyte epidermal growth factor receptors and their role in the development of skin toxicity during targeted cancer therapy

The binding of epidermal growth factor (EGFR) receptors is a good target for the treatment of lung, colon, pancreatic, head and neck cancers. The adverse events that develop as a result of therapy, in the form of lesions of the skin and mucous membranes, is a serious problem for the doctor to choose a long-term treatment strategy. The developing symptoms of skin toxicity, as skin problems in patients are often called, are worrisome and often affect the quality of life and compliance with the treatment regimen. Thus, it is important for doctors to know the prerequisites and ways to manage skin toxicity associated with the use of tyrosine kinase receptor inhibitors of epidermal growth factor.&#x0D; The mechanism and consequences of EGF receptor activation are described to explain the development of undesirable skin toxicity associated with inhibition of the epidermal growth factor receptor.

LGR4: Not Just for Wnt Anymore?

Leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) is best known for its role in regulating the ability of cells to respond to Wnt ligands. In this well-known role, LGR4 serves as a receptor for R-spondins and forms a complex with the ubiquitin E3 ligases ring finger protein 43 (RNF43) and zinc and ring finger 3 (ZNRF3). RNF43 and ZNRF3 ubiquitinate Frizzleds (FZD), which are a family of ten WNT receptors. This ubiquitination decreases FZD receptor levels on the cell surface, reducing Wnt ligands' ability to activate signaling. While there were some previous indications of Wnt-independent functions of LGR4, this WNT-centric view has remained predominant. In this issue of Cancer Research, Yue and colleagues report that LGR4 also functions to regulate signaling through the EGF receptor. This work was stimulated by observing that while high levels of LGR4 expression in breast tumors correlated with poor patient outcomes, LGR4 levels did not correlate with a well-established Wnt-associated gene signature in these same patients. In contrast, high levels of Lgr4 expression strongly correlated with EGFR signaling. Reducing Lgr4 expression also inhibited signaling through the EGFR, potentially via regulation of the Casitas B-lineage lymphoma ubiquitin E3 ligase. Consistent with this model, LGR4 could be coimmunoprecipitated with a complex that contained EGFR and was capable of inhibiting EGFR ubiquitination. The implications of this work and how it challenges our understanding of the contributions of Wnt signaling and EGFR signaling in cancer are discussed as our several interesting future directions.See related article by Yue et al., p. 4441.

Abstract P252: Smooth Muscle At1a Receptor Mediates Perivascular Fibrosis In Angiotensin Ii-infused Mice

Angiotensin II (Ang II) signaling via AT1 receptor has been shown to play a critical role in the pathogenesis of hypertension, cardiovascular hypertrophy and fibrosis. We have demonstrated that ADAM17 expressed in vascular smooth muscle cells (VSMC) mediates EGF receptor activation and promotes cardiac hypertrophy and perivascular fibrosis induced by Ang II. It is conceivable that Ang II signaling in VSMCs specifically initiates cardiovascular remodeling, such as hypertrophy and fibrosis. In a recent study, deficiency of smooth muscle AT1a receptors results in diminished hypertension and protection from cardiac hypertrophy induced by Ang II. However, we have limited understanding whether smooth muscle AT1a receptors affects hypertensive fibrosis in vasculature. Thus, this study was designed to elucidate the roles of the AT1a receptor in VSMCs in cardiovascular remodeling including fibrosis during Ang II stimulation using VSMC AT1a receptor deficient mice. To delete the AT1a receptor from VSMCs, we crossed C57BL/6 transgenic mouse lines expressing Cre recombinase under the control of the sm22α promoter (KIsm22α-Cre). Male AT1a flox/flox KIsm22α-Cre+/- (SMKO) and Controls (AT1a flox/flox KIsm22α-Cre-/-) mice were infused with Ang II (1 μg/kg/min) for 2 weeks via osmotic mini-pump. In Control mice, Ang II infusion for 2 weeks induced cardiac hypertrophy indicated by heart-to-body weight ratio and echocardiogram. After 2 weeks of Ang II infusion, heart-to-body weight ratios were significantly increased in Control mice compared with AT1a SMKO mice (6.04 versus 4.89, respectively, p=0.032). Cardiac wall hypertrophy was seen in Controls after 2 weeks of Ang II infusion, which was attenuated in AT1a SMKOs. Control mice (n=5) showed vascular medial hypertrophy and perivascular fibrosis, whereas these phenotypic changes were attenuated in SMKO mice (n=4). In conclusion, AT1a receptors from VSMC could mediate Ang II-induced cardiovascular hypertrophy and perivascular fibrosis. Whether the data can be fully explained by the prevention of hypertension remains to be determined, the data contrast to the past manuscript showing a protective effect in AT1a flox/flox S100A4-Cre+/- mice (fibroblast silencing) with Ang II infusion.

Bidirectional epithelial–mesenchymal crosstalk provides self-sustaining profibrotic signals in pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic lung disease with a median survival of 2 to 4 years. Injury to and/or dysfunction of the alveolar epithelium is strongly implicated in IPF disease initiation, but the factors that determine whether fibrosis progresses rather than normal tissue repair occurs remain poorly understood. We previously demonstrated that zinc finger E-box-binding homeobox 1–mediated epithelial–mesenchymal transition in human alveolar epithelial type II (ATII) cells augments transforming growth factor-β–induced profibrogenic responses in underlying lung fibroblasts via paracrine signaling. Here, we investigated bidirectional epithelial–mesenchymal crosstalk and its potential to drive fibrosis progression. RNA-Seq of lung fibroblasts exposed to conditioned media from ATII cells undergoing RAS-induced epithelial–mesenchymal transition identified many differentially expressed genes including those involved in cell migration and extracellular matrix regulation. We confirmed that paracrine signaling between RAS-activated ATII cells and fibroblasts augmented fibroblast recruitment and demonstrated that this involved a zinc finger E-box-binding homeobox 1–tissue plasminogen activator axis. In a reciprocal fashion, paracrine signaling from transforming growth factor-β–activated lung fibroblasts or IPF fibroblasts induced RAS activation in ATII cells, at least partially through the secreted protein acidic and rich in cysteine, which may signal via the epithelial growth factor receptor via epithelial growth factor–like repeats. Together, these data identify that aberrant bidirectional epithelial–mesenchymal crosstalk in IPF drives a chronic feedback loop that maintains a wound-healing phenotype and provides self-sustaining profibrotic signals.

Ovulatory upregulation of angiotensin-converting enzyme 2, a receptor for SARS-CoV-2, in dominant follicles of the human ovary

ObjectiveTo determine the temporal expression of angiotensin-converting enzyme 2 (ACE2), a receptor for SARS-CoV-2, in dominant follicles throughout the periovulatory period in women and the regulatory mechanisms underlying ACE2 expression in human granulosa/lutein cells (hGLC).DesignExperimental prospective clinical study and laboratory-based investigation.SettingUniversity Medical Center and private in vitro fertilization center.Patient(s)Thirty premenopausal women undergoing surgery for tubal ligation and 16 premenopausal women undergoing in vitro fertilization.Intervention(s)Administration of human chorionic gonadotropin (hCG) and harvesting of preovulatory/ovulatory follicles by timed laparoscopy, and collection of granulosa/lutein cells and cumulus cells at the time of oocyte retrieval.Main Outcome Measure(s)Expression and localization of ACE2 in granulosa cells and dominant follicles collected throughout the periovulatory period of the menstrual cycle and in hGLC using quantitative polymerase chain reaction, immunoblotting, and immunohistochemistry.Result(s)ACE2 expression (mRNA and protein) is up-regulated in human ovulatory follicles after administration of hCG. ACE2 expression was higher in cumulus cells than in granulosa cells. hCG increased the expression of ACE2 in primary hGLC cultures; the increase was inhibited by RU486 (an antagonist for progesterone receptor and glucocorticoid receptor) and CORT125281 (a selective glucocorticoid receptor antagonist), but not by AG1478 (an EGF receptor tyrosine kinase inhibitor) or by dexamethasone.Conclusion(s)The hormone-regulated expression of ACE2 in granulosa cells suggests a potential role of ACE2 in the ovulatory process. These data also imply the possible impact of COVID-19 on a vital cyclic event of ovarian function and thus on women's overall reproductive health. However, SAR-CoV-2 infection in ovarian cells in vivo or in vitro has yet to be determined.

Targeting of fluorescent Lactococcus lactis to colorectal cancer cells through surface display of tumour-antigen binding proteins.

SummaryDevelopment of targeted treatment for colorectal cancer is crucial to avoid side effects. To harness the possibilities offered by microbiome engineering, we prepared safe multifunctional cancer cell‐targeting bacteria Lactococcus lactis. They displayed, on their surface, binding proteins for cancer‐associated transmembrane receptors epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor 2 (HER2) and co‐expressed an infrared fluorescent protein for imaging. Binding of engineered L. lactis to tumour antigens EpCAM and HER2 was confirmed and characterised in vitro using soluble receptors. The proof‐of‐principle of targeting was demonstrated on human cell lines HEK293, HT‐29 and Caco‐2 with fluorescent microscopy and flow cytometry. The highest L. lactis adhesion was seen for the HEK293 cells with the overexpressed tumour antigens, where colocalisation with their tumour antigens was seen for 39% and 67% of EpCAM‐targeting and HER2‐targeting bacteria, respectively. On the other hand, no binding was observed to HEK293 cells without tumour antigens, confirming the selectivity of the engineered L. lactis. Apart from cell targeting in static conditions, targeting ability of engineered L. lactis was also shown in conditions of constant flow of bacterial suspension over the HEK293 cells. Successful targeting by engineered L. lactis support the future use of these bacteria in biopharmaceutical delivery for the treatment of colorectal cancer.

Effects of Avitinib on CYP450 Enzyme Activity in vitro and in vivo in Rats

PurposeAvitinib is the first third-generation epithelial growth factor receptor (EGFR) inhibitor independently developed in China and is mainly used for treating non-small cell lung cancer. However, pharmacokinetic details are limited. This study explored the in vivo and in vitro effects of avitinib on cytochrome CYP450 enzymes metabolic activity.MethodsA rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for determining six probe substrates and their metabolites. Avitinib influence on activity levels of CYP isozymes was examined in vitro using human and rat liver microsomes (HLMs/RLMs). For in vivo studies, rats were pretreated with 30 mg/kg avitinib once daily for 7 days (avitinib multiple-doses group), 30 mg/kg avitinib on day 7 (avitinib single-dose group), or an equivalent amount of CMC-Na once daily for 7 days (control group), followed by intragastrical administration of the probe substrates (1 mg/kg tolbutamide and 10 mg/kg phenacetin, bupropion, chlorzoxazone, dextromethorphan, and midazolam). Plasma pharmacokinetics and IC50 values of the probe substrates were then compared. Pharmacokinetic parameters were determined using non-compartmental analysis implemented in a pharmacokinetic program.ResultsIn vitro experiments revealed different inhibitory effects of avitinib on the six probe substrates with various IC50 values (bupropion, 6.39/22.64 μM; phenacetin, 15.79/48.36 μM; chlorzoxazone, 23.15/57.09 μM; midazolam, 27.64/59.6 μM; tolbutamide, 42.18/6.91 μM; dextromethorphan, 44.39/56.57 μM, in RLMs and HLMs respectively). In vivo analysis revealed significant differences (P <0.05) in distinct pharmacokinetic parameters (AUC(0-t), AUC (0-∞), Cmax, MRT(0-t), MRT (0-∞), and CLz/F) for the six probe substrates after avitinib pretreatment.ConclusionA sensitive and reliable UPLC-MS/MS method was established to determine the concentration of six probe substrates in rat plasma. Avitinib had inhibitory effects on CYP450 enzymes, especially cyp2b1, cyp1a2 in RLMs, CYP2C9 in HLMs, and cyp1a2, cyp2b1, cyp2d1, and cyp2e1 in vivo. Our data recommend caution when avitinib was taken simultaneously with drugs metabolized by CYP450 enzymes.

Regulation of lipid homeostasis by the TBC protein dTBC1D22 via modulation of the small GTPase Rab40 to facilitate lipophagy.

The regulation of lipid homeostasis is not well understood. Using forward genetic screening, we demonstrate that the loss of dTBC1D22, an essential gene that encodes a Tre2-Bub2-Cdc16 (TBC) domain-containing protein, results in lipid droplet accumulation in multiple tissues. We observe that dTBC1D22 interacts with Rab40 and exhibits GTPase activating protein (GAP) activity. Overexpression of either the GTP- or GDP-binding-mimic form of Rab40 results in lipid droplet accumulation. We observe that Rab40 mutant flies are defective in lipid mobilization. The lipid depletion induced by overexpression of Brummer, a triglyceride lipase, is dependent on Rab40. Rab40 mutant flies exhibit decreased lipophagy and small size of autolysosomal structures, which may be due to the defective Golgi functions. Finally, we demonstrate that Rab40 physically interacts with Lamp1, and Rab40 is required for the distribution of Lamp1 during starvation. We propose that dTBC1D22 functions as a GAP for Rab40 to regulate lipophagy.

Whispering Gallery Modes-based biosensors for real-time monitoring and binding characterization of antibody-based cancer immunotherapeutics

Development of novel protein-based drugs such as antibodies or immunocytokines and initial validation of target binding typically occurs in 2D settings with e.g., surface plasmon resonance or reflection interferometry using antigen-coated planar sensors. However, a versatile tool to assess binding characteristics of tumour-targeting therapeutics where specific biochemical interactions can be monitored in real-time and in a more realistic 3D interaction setting is currently lacking. Here, we report on the development of versatile small optical 3D biosensors using protein G-coated spherical microparticles based on Whispering Gallery Modes (WGMs). These sensors allowed for an oriented immobilization of theoretically any immunoglobulin G (IgG) and IgG crystallizable fragment domain (Fc)-tagged protein and have been carefully optimized for specific detection of antigen-antibody interactions, as illustrated using the Epithelial Growth Factor Receptor (EGFR) antibody Cetuximab and Program Death Ligand 1 (PD-L1) antibody Atezolizumab. When both ligand and analyte contained an Fc-fragment, protein G binding capacity saturation followed by a “blocking” step with an irrelevant IgG enabled the label-free detection of Fc-tagged antigens with corresponding cognate ligands, as identified using EGFR-Fc, PD-L1-Fc and different members from the tumour necrosis factor receptor family, such as CD27-Fc and 4−1BB-Fc. Thus, WGMs and developed protein G-coated biosensors provide a widely applicable tool to evaluate binding of immunotherapeutics to their targets on the sensor surface, imitating cell surface properties.

Structural basis of the effect of activating mutations on the EGF receptor.

Mutations within the kinase domain of the epidermal growth factor receptor (EGFR) are common oncogenic driver events in non-small cell lung cancer. Although the activation of EGFR in normal cells is primarily driven by growth-factor-binding-induced dimerization, mutations on different exons of the kinase domain of the receptor have been found to affect the equilibrium between its active and inactive conformations giving rise to growth-factor-independent kinase activation. Using molecular dynamics simulations combined with enhanced sampling techniques, we compare here the conformational landscape of the monomers and homodimers of the wild-type and mutated forms of EGFR ΔELREA and L858R, as well as of two exon 20 insertions, D770-N771insNPG, and A763-Y764insFQEA. The differences in the conformational energy landscapes are consistent with multiple mechanisms of action including the regulation of the hinge motion, the stabilization of the dimeric interface, and local unfolding transitions. Overall, a combination of different effects is caused by the mutations and leads to the observed aberrant signaling.

Quantitative assessment of HER2 gene amplification of breast cancer using droplet digital PCR.

We previously reported the usefulness of droplet digital polymerase chain reaction (ddPCR) for the assessment of Human epithelial growth factor receptor 2 (HER2) gene amplification in breast cancer using formalin-fixed and paraffin-embedded sections. In our previous study, we combined HER2/CEP17 ratio (HER2 gene signals to chromosome 17 signals) with ddPCR and tumor content ratio (TCR) of each sample and determined the HER2 status by adopting a two-dimensional chart. This "ddPCR-TCR method" showed a high concordance with conventional HER2 status. In this study, we updated our method to assess the HER2 status of breast cancer in a more quantitative manner. We combined obtained data of the ddPCR ratio [Rx ] and TCR [x]; we calculated "(Rx - 1)/x + 1" for 41 samples with primary breast cancer and named the value led by this formula as "eHER2 (estimated HER2/CEP17 ratio of a tumor cell)". eHER2 was equivalent to conventional in situ hybridization (ISH) HER2/CEP17 ratio in most cases. eHER2 and ISH ratio showed a strong correlation (Spearman rank correlation ρ = 0.70, p < 0.0001). The obtained results indicated that eHER2 is a potential tool for HER2 status diagnosis in breast cancer.