Epidermal Growth Factor Receptor Molecules Research Articles

Disrupting EGFR-HER2 Transactivation by Pertuzumab in HER2-Positive Cancer: Quantitative Analysis Reveals EGFR Signal Input as Potential Predictor of Therapeutic Outcome.

Pertuzumab (Perjeta®), a humanized antibody binding to the dimerization arm of HER2 (Human epidermal growth factor receptor-2), has failed as a monotherapy agent in HER2 overexpressing malignancies. Since the molecular interaction of HER2 with ligand-bound EGFR (epidermal growth factor receptor) has been implied in mitogenic signaling and malignant proliferation, we hypothesized that this interaction, rather than HER2 expression and oligomerization alone, could be a potential molecular target and predictor of the efficacy of pertuzumab treatment. Therefore, we investigated static and dynamic interactions between HER2 and EGFR molecules upon EGF stimulus in the presence and absence of pertuzumab in HER2+ EGFR+ SK-BR-3 breast tumor cells using Förster resonance energy transfer (FRET) microscopy and fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS). The consequential activation of signaling and changes in cell proliferation were measured by Western blotting and MTT assay. The autocorrelation functions of HER2 diffusion were best fitted by a three-component model corrected for triplet formation, and among these components the slowly diffusing membrane component revealed aggregation induced by EGFR ligand binding, as evidenced by photon-counting histograms and co-diffusing fractions. This aggregation has efficiently been prevented by pertuzumab treatment, which also inhibited the post-stimulus interaction of EGFR and HER2, as monitored by changes in FRET efficiency. Overall, the data demonstrated that pertuzumab, by hindering post-stimulus interaction between EGFR and HER2, inhibits EGFR-evoked HER2 aggregation and phosphorylation and leads to a dose-dependent decrease in cell proliferation, particularly when higher amounts of EGF are present. Consequently, we propose that EGFR expression on HER2-positive tumors could be taken into consideration as a potential biomarker when predicting the outcome of pertuzumab treatment.

Dendritic cell vaccination in combination with erlotinib in a patient with inoperable lung adenocarcinoma: a case report

BackgroundSatisfactory treatment for patients with unresectable advanced lung cancer has not yet been established. We report a case of unresectable advanced lung cancer (stage IIIb: T2aN3M0) treated with a total of 15 doses of dendritic cells pulsed with a Wilms’ tumor 1 and mucin 1 vaccine in combination with erlotinib, a small molecule epidermal growth factor receptor tyrosine kinase inhibitor, for more than 699 days without recurrence or metastasis.Case presentationA 63-year-old Korean woman was diagnosed with lung adenocarcinoma by pathology and computed tomography. The adenocarcinoma showed an epidermal growth factor receptor (EGFR) mutation, no anaplastic lymphoma kinase expression, and less than 1% expression of programmed death ligand 1. She received erlotinib alone for approximately 1 month. She then received erlotinib and the dendritic cells pulsed with Wilms’ tumor 1 and mucin 1 vaccine. The diameter of the erythema at the vaccinated sites was 30 mm at 48 hours after the first vaccination. Moreover, it was maintained at more than 20 mm during the periods of vaccination. These results suggested the induction of antitumor immunity by the vaccine. Remarkably, the tumor size decreased significantly to 12 mm, a 65.7% reduction, after combined therapy with eight doses of the dendritic cells pulsed with Wilms’ tumor 1 and mucin 1 vaccine and erlotinib for 237 days based on fluorodeoxyglucose uptake by positron emission tomography/computed tomography and computed tomography. Interestingly, after 321 days of combination therapy, the clinical findings improved, and no tumor was detected based on computed tomography. Validation of the tumor’s disappearance persisted for at least 587 days after treatment initiation, without any indication of recurrence or metastasis.ConclusionStandard anticancer therapy combined with the dendritic cells pulsed with Wilms’ tumor 1 and mucin 1 vaccine may have therapeutic effects for such patients with unresectable lung adenocarcinoma.

DERMATOLOGICAL TOXICITY OF CANCER PATIENTS RECEIVING SMALL MOLECULE EPIDERMAL GROWTH FACTOR RECEPTOR INHIBITORS – A PROSPECTIVE OBSERVATIONAL STUDY

Objectives: The objectives of the study are as follows: (1) To study the incidence of dermatological toxicity in patients receiving small molecule epidermal growth factor receptor inhibitors. (2) To assess the severity of dermatological toxicity. Methods: This prospective observational study of 66 cancer patients carried out in the Department of Radiotherapy, Government Medical College, Thiruvananthapuram. Patients with histopathological evidence of cancer receiving small molecule epidermal growth factor receptor inhibitors and satisfying inclusion criteria were taken up in the study after obtaining written informed consent. History and clinical examination before initiation of treatment done. Data regarding the occurrence of developing dermatological toxicities were obtained by questioning for symptoms and with the help of structured proforma. Severities of dermatological toxicities were assessed, and toxicity grading was done. Results: Sixty-six cancer patients enrolled in the study. The mean age of the patients was 62.4 years. The prevalence was higher in men (53%). Sixty-three patients (95.5%) developed at least one dermatological toxicity. The most common dermatological toxicity observed was papulopustular rash (68.2%), followed by xerosis (66.7%) and pruritus (59.1%). Conclusion: The present study has thus determined the incidence and severity of dermatological toxicity of cancer patients receiving small molecule epidermal growth factor receptor inhibitors.

Overexpression of EGFR in esophageal squamous cell carcinomas - A new biological target in cancer therapy.

Esophageal squamous cell carcinoma (ESCC) is the predominant type of esophageal cancer in the Asian belt. These cancers show poor prognosis with an overall 5-year survival rate less than 19%. Exploring new molecular therapeutic targets such as epidermal growth factor receptor (EGFR) could be the corner stone of new curative treatment. The present study was done to analyze the overexpression of EGFR in different grades of ESCC and explore its role as a diagnostic and theranostic marker in ESCC. In this retrospective study, 50 formalin-fixed paraffin-embedded blocks of ESCCs diagnosed from 2014 to 2019 were retrieved. The biopsies were subjected to immunohistochemistry staining of EGFR. The intensity of the membrane staining was reviewed and scored. Compared with various intrinsic and extrinsic factors using Chi-square test, scores more than 2+ were considered as overexpression. Majority (84%) specimens demonstrated overexpression of EGFR where high-grade ESCCs had greater overexpression rates compared to low-grade ESCC (P < 0.05). By targeting the EGFR molecules, anti-EGFR drugs could block their signals and stop the growth and spread of ESCCs especially high-grade tumors while harming the normal cells as little as possible. A clinical trial using anti-EGFR monoclonal antibodies will help in the long run to develop immunotherapy drugs.

Epidermal growth factor receptor activation confers resistance to lenvatinib in thyroid cancer cells.

Thyroid cancer is the most common endocrine malignancy. A multitargeted tyrosine kinase inhibitor, lenvatinib, has been used for the treatment of advanced thyroid cancer. To elucidate the mechanism of resistance to lenvatinib in thyroid cancer cells, we established lenvatinib‐resistant sublines and analyzed the molecular mechanisms of resistance. Two thyroid cancer cell lines (TPC‐1 and FRO) were used, and resistant sublines for lenvatinib (TPC‐1/LR, FRO/LR) were established. In TPC‐1/LR, the phosphorylation of epidermal growth factor receptor (EGFR), extracellular signal‐regulated kinase (ERK), and Akt was enhanced whereas in FRO/LR, the phosphorylation of EGFR and downstream signal transduction molecules was not enhanced. The addition of epidermal growth factor decreased sensitivity to lenvatinib in TPC‐1 and FRO. The combination of EGFR inhibitors lapatinib and lenvatinib significantly inhibited the growth of TPC‐1/LR in both in vitro and mouse xenograft models. Short‐term exposure to lenvatinib enhanced the phosphorylation of EGFR in six thyroid cancer cell lines regardless of their histological origin or driver gene mutations; however, phosphorylation of ERK was enhanced in all cells except TPC‐1. A synergistic growth‐inhibitory effect was observed in three thyroid cancer cell lines, including intrinsically lenvatinib‐resistant cells. The results indicate that signal transduction via the EGFR pathway may be involved in the development of lenvatinib resistance in thyroid cancer cells. The inhibition of the EGFR pathway simultaneously by an EGFR inhibitor may have therapeutic potential for overcoming lenvatinib resistance in thyroid cancer.

High expression of STAT2 in ovarian cancer and its effect on metastasis of ovarian cancer cells

To investigate the expression of signal transduction and activator of transcription 2 (STAT2) in ovarian cancer and its correlation with the prognosis of ovarian cancer patients and explore the role of STAT2 inregulating metastasis of ovarian cancer cells. RT-qPCR was performed to detect the expression of STAT2 mRNA in 62 fresh frozen ovarian cancer tissues and 62 normal ovarian tissues; immunohistochemistry was used to detect STAT2 protein expressions in 95 paraffin-embedded ovarian cancer samples and 33 normal ovarian tissues. Kaplan-Meier method was used to analyze the correlation between the expression of STAT2 and the prognosis of the patients. We also examined the relationship between STAT2 and the patients' prognosis by analyzing the data in Kaplan-Meier Plotter database. Western blotting was performed to detect the expression of STAT2 in different ovarian cancer cell lines. In A2780 cells with the highest STAT2 expression, we examined the effects of STAT2 interference on cell migration and invasiveness using Transwell migration assay and on the expressions of the downstream molecule epidermal growth factor receptor (EGFR). Ovarian cancer tissues expressed significantly higher levels of STAT2 mRNA than normal ovarian tissue. A high STAT2 mRNA expression was correlated with an advanced FIGO stage. Immunohistochemistry showed that 67.4% of the ovarian cancer samples, as compared with 28.3% of normal ovarian tissues, showed high STAT2 expressions. In ovarian cancer patients, a high expression of STAT2 protein was associated with ascites volume, distant metastasis and FIGO stage (P < 0.05). Survival analysis showed that ovarian cancer patients with a high expression of STAT2 protein had poor overall survival (P=0.021) and progression-free survival (P=0.018). STAT2 was overexpressed in all the ovarian cancer cell lines tested, and A2780 cell lines showed the highest expression. Interference of STAT2 significantly suppressed the migration and invasiveness (P < 0.01) and lowered the expression level of EGFR in A2780 cells. STAT2 is overexpressed in ovarian cancer. A high expression of STAT2 is associated with a poor prognosis of ovarian cancer patients. STAT2 may promote the metastasis of ovarian cancer by enhancing the expression of EGFR.

Ang-(1-7)/ MAS1 receptor axis inhibits allergic airway inflammation via blockade of Src-mediated EGFR transactivation in a murine model of asthma.

The angiotensin-(1–7) [Ang-(1–7)]/MAS1 receptor signaling axis is a key endogenous anti-inflammatory signaling pathway. However, the mechanisms by which its mediates the anti-inflammatory effects are not completely understood. Using an allergic murine model of asthma, we investigated whether Ang-1(1–7)/MAS1 receptor axis a): inhibits allergic inflammation via modulation of Src-dependent transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling effectors such as ERK1/2, and b): directly inhibits neutrophil and/or eosinophil chemotaxis ex vivo. Ovalbumin (OVA)-induced allergic inflammation resulted in increased phosphorylation of Src kinase, EGFR, and ERK1/2. In addition, OVA challenge increased airway cellular influx, perivascular and peribronchial inflammation, fibrosis, goblet cell hyper/metaplasia and airway hyperresponsiveness (AHR). Treatment with Ang-(1–7) inhibited phosphorylation of Src kinase, EGFR, ERK1/2, the cellular and histopathological changes and AHR. Ang-(1–7) treatment also inhibited neutrophil and eosinophil chemotaxis ex vivo. These changes were reversed following pre-treatment with A779. These data show that the anti-inflammatory actions of Ang-(1–7)/ MAS1 receptor axis are mediated, at least in part, via inhibition of Src-dependent transactivation of EGFR and downstream signaling molecules such as ERK1/2. This study therefore shows that inhibition of the Src/EGRF/ERK1/2 dependent signaling pathway is one of the mechanisms by which the Ang-(1–7)/ MAS1 receptor axis mediates it anti-inflammatory effects in diseases such as asthma.

Sulfasalazine decreases soluble fms-like tyrosine kinase-1 secretion potentially via inhibition of upstream placental epidermal growth factor receptor signalling

ObjectivesPreeclampsia is a hypertensive disorder of pregnancy with no available medical treatment. We recently reported sulfasalazine, an anti-inflammatory medication, to be a candidate therapeutic for preeclampsia. We showed sulfasalazine decreases placental secretion of soluble Fms-like tyrosine kinase-1 (sFlt-1), an anti-angiogenic factor strongly implicated in the pathogenesis of preeclampsia. However, the cellular mechanism(s) by which sulfasalazine reduces placental sFlt-1 are yet to be determined. Recently we also reported that both the mitochondria and the epidermal growth factor receptor (EGFR) signalling pathways regulate secretion of placental sFlt-1. In this study we sought to assess directly whether sulfasalazine's capacity to reduce sFlt-1 secretion may be mediated via EGFR or the mitochondria. Methods and resultsUsing primary cytotrophoblast cells, we confirmed sulfasalazine reduced sFlt-1 secretion. Interestingly, when we measured the mRNA expression of EGFR, we found a reduction in EGFR expression which closely mirrored the changes in sFlt-1 secretion. At the protein level, sulfasalazine significantly reduced phosphorylated and active EGFR (phosphorylated/total) expression. Additionally, sulfasalazine significantly reduced the protein expression of ERK1/2 and STAT3 which are key adaptor molecules downstream of EGFR. Next, we assessed mitochondrial respiration following sulfasalazine treatment and found no effect on basal respiration, ATP production, proton leak or maximal respiration. ConclusionSulfasalazine reduces EGFR and down-stream signalling molecule expression coincident with reduced sFlt-1 secretion. EGFR signalling is a potential mechanism by which sulfasalazine decreases placental secretion of sFlt-1. Further interrogation of the EGFR may identify new candidate treatments for preeclampsia.

Hypoxia-Induced Autophagy Promotes EGFR Loss in Specific Cell Contexts, Which Leads to Cell Death and Enhanced Radiosensitivity

Treatment failure through radio resistance of tumors is associated with activation of the epidermal growth factor receptor (EGFR). Tumor cell proliferation, DNA-repair, hypoxia and metastases-formation are four mechanisms in which EGFR signalling has an important role. However, the effect of hypoxia on EGFR expression is still controversial. In this study, we evaluated the in vitro and in vivo effects of hypoxia on EGFR and its mechanism, as well as its relationship with radiosensitivity. Treatment failure through radio resistance of tumors is associated with activation of the epidermal growth factor receptor (EGFR). Tumor cell proliferation, DNA-repair, hypoxia and metastases-formation are four mechanisms in which EGFR signalling has an important role. However, the effect of hypoxia on EGFR expression is still controversial. In this study, we evaluated the in vitro and in vivo effects of hypoxia on EGFR and its mechanism, as well as its relationship with radio sensitivity. In a panel of cancer cells with low or high cell density under hypoxia, viable cell counting assay was used to calculate the percentage of cell death. Western blot was used to detect the levels of proteins involved in EGFR signaling and autophagy. The localization of EGFR and autophagy-related molecules was observed by confocal microscopy. The xenograft model in nude mice was used to elucidate the in vivo correlation of hypoxia with EGFR and p62. Colony formation assay was performed to assess the radiosensitivity. In this study, we demonstrated that hypoxia enhanced EGFR expression and sustained cell survival in SiHa, CAL 27 and A549 cells at both low and high cell densities, while in MCF-7, MDA-MB-231 and HeLa cells, EGFR and cell survival were regulated by hypoxic treatment in a cell-density dependent manner: upregulated at low cell density and down regulated at high cell density. In MCF-7 and HeLa xenografts in nude mice, EGFR expression varied inversely with the pimonidazole level that was used as an indicator of hypoxia, accordant with the effect of hypoxia at high cell density in vitro. Hypoxia induced more remarkable cell autophagy at high cell density than at low cell density. Autophagy inhibitor 3MA, rather than proteasome inhibitor MG132 inhibited hypoxia-mediated EGFR loss and shifted cell death to cell survival in HeLa cells. The MCF7 cells’ sensitivity to ionizing radiation (IR) under hypoxia was also conditional on the cell densities when the hypoxia treatment was introduced, inversely associated with the expression levels of EGFR. Hypoxia can decrease EGFR expression in some cell lines by enhancing autophagy at high cell density, leading to cell death and hypersensitivity to radiotherapy. This study may help understand how hypoxia influences EGFR expression and radiosensitivity.

Hypertension Editors' Picks Preeclampsia.

Hypertension Editors' Picks Preeclampsia.

Abstract 218: AVID100 is an anti-EGFR ADC that promotes DM1-meditated cytotoxicity on cancer cells but not on normal cells

Abstract AVID100 is a novel rationally designed antibody drug conjugate (ADC) that specifically targets the epidermal growth factor receptor (EGFR). EGFR is highly expressed on a variety of cancers making it a promising target for ADCs. However, due to the presence of EGFR on normal skin cells, on-target off-tumor toxicity is a concern. AVID100 is an anti-EGFR-DM1 conjugate exhibiting high potency against cancer cells relative to unconjugated antibodies, while not exhibiting increased toxicity on normal cells. In a series of in vitro and in vivo experiments, we provide mechanistic evidence rationalizing why AVID100 shows higher cytotoxicity on tumor cells compared to normal cells. The antibody moiety of AVID100, denoted MAB100, was shown to have a high affinity for EGFR (approximately 2nM) and to compete with EGF for binding to the EGFR. Also, MAB100 prevented downstream signalling from EGFR. In addition to exhibiting full antagonist activity, MAB100 effectively delivered the microtubule inhibitor DM1 to tumor cells, as demonstrated by an increase in apoptosis in AVID100-treated tumor cells relative to MAB100-treated tumor cells. AVID100 was very effective on tumor cell lines derived from breast, head and neck, and lung cancers with the cytotoxic IC50 values generally correlating with the number of EGFR molecules on the cell surface. Importantly, on keratinocytes, AVID100 did not exhibit increased cytotoxicity relative to MAB100. We hypothesized that this lack of increase in cytotoxicity on keratinocytes is due to the antagonistic effect of MAB100 on EGFR signalling. Blocking the EGFR pathway in keratinocytes strongly inhibits their proliferation, which should protect them against the cytotoxic action of DM1. To address this hypothesis, we compared the effect of AVID100 to that of a non-antagonistic anti-EGFR ADC, huML66-DM1. As expected, both ADCs were highly effective at killing MDA-MB-468 cancer cells, confirming their ability to deliver DM1. In contrast, keratinocytes exhibited a significantly higher level of apoptosis in response to huML66-DM1 as compared to AVID100. These results confirm that the antagonistic nature of the antibody moiety of AVID100 plays a critical role in protecting keratinocytes from the cytotoxic effect of DM1. Finally, the activity of AVID100 was investigated in multiple mouse xenograft studies, including in MDA-MB-468 human breast adenocarcinoma, H292 NSCLC, and FaDu SSCHN models. AVID100 treatment significantly reduced tumor growth and caused tumor regressions in some of the mice, even when administered as a single dose. Toxicology studies in cynomolgus monkeys demonstrated that AVID100 was well tolerated at up to 4 weekly doses of 10mg/kg. AVID100 has completed a Phase 1 clinical trial with the recommended Phase 2 dose having been determined. The Phase 2 trial of AVID100 is ongoing. Citation Format: Michael J. Thwaites, Rene Figueredo, Gilles Tremblay, James Koropatnick, Victor Goldmacher, Maureen O’Connor-McCourt. AVID100 is an anti-EGFR ADC that promotes DM1-meditated cytotoxicity on cancer cells but not on normal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 218.

EGFR (Epidermal Growth Factor Receptor) Signaling and the Mitochondria Regulate sFlt-1 (Soluble FMS-Like Tyrosine Kinase-1) Secretion.

Preeclampsia is a hypertensive disorder of pregnancy characterized by maternal endothelial dysfunction and end-organ damage. The antiangiogenic factor, sFlt-1 (soluble FMS-like tyrosine kinase-1) has been strongly implicated in the pathogenesis of preeclampsia. sFlt-1 is secreted into the maternal circulation where it antagonizes VEGF (vascular endothelial growth factor) and ultimately disrupts vascular homeostasis. However, the upstream mechanisms regulating release of sFlt-1 are poorly characterized. We investigated the roles of key prosurvival pathways, EGFR (epidermal growth factor receptor) signaling, and the mitochondria, in regulating sFlt-1 production. We initially found that the mRNA and protein of EGFR and downstream adaptor molecules were significantly increased in preeclamptic placental tissue relative to normotensive controls. Inhibiting the EGFR signaling cascade using siRNA (small interfering ribonucleic acid) or small molecule inhibitors significantly reduced sFlt-1 release from primary cytotrophoblast (placental cells). Additionally, inhibiting the mitochondrial electron transport chain or activating downstream energy-sensing molecules (AMPK [AMP-activated kinase], SIRT1 [sirtuin 1 ], and PGC1α [PPAR-γ co-activator 1]) also significantly reduced sFlt-1 secretion from cytotrophoblast cells, without affecting EGFR signaling. In vivo, treating pregnant mice with gefitinib (an EGFR inhibitor) or resveratrol (perturbs mitochondrial function) significantly reduced circulating murine sFlt-1 compared with vehicle control. Furthermore, treating primary cytotrophoblasts with therapeutics which have been previously found to reduce sFlt-1 secretion, either reduced EGFR signaling (esomeprazole and statins) or perturbed mitochondrial function (esomeprazole, resveratrol, and metformin). Additionally, targeting both pathways simultaneously produced additive reductions in sFlt-1 secretion. Thus, we have identified 2 key survival pathways that seem to be overactive in preeclampsia and involved in the regulation of placental sFlt-1 secretion.

Relationship Between EGFR Expression in Oral Cancer Cell Lines and Cetuximab Antibody-dependent Cell-mediated Cytotoxicity.

Cetuximab treatment targets the epidermal growth factor receptor (EGFR) overexpressed in oral cancer. This study aimed to investigate the anti-tumour activity of cetuximab against oral cancer cell lines with respect to antibody-dependent cell-mediated cytotoxicity (ADCC), and determine the correlation between ADCC and EGFR expression. EGFR expression in oral cancer cells was measured by quantitative flow cytometric analysis and immunohistochemistry. ADCC activity was measured by 4-h calcein release assays. Cetuximab-mediated ADCC against oral cancer cells was detectable at a concentration of 0.1 μg/ml. A high correlation was observed between the number of EGFR molecules on the surface of oral cancer cells and ADCC (correlation coefficient: 0.847; p=0.032). ADCC is an important mechanism underlying the therapeutic effect of cetuximab, and EGFR expression in tumour cells might serve as a predictive marker to evaluate the effect of cetuximab treatment.

Partial agonistic effect of cetuximab on epidermal growth factor receptor and Src kinase activation in triple‑negative breast cancer cell lines.

Cetuximab is a monoclonal antibody developed to inhibit the binding of growth factors and the subsequent activation of epidermal growth factor receptor (EGFR). Triple‑negative breast cancer (TNBC) is resistant to cetuximab treatment. The aim of the present study was to examine the partial agonistic properties of cetuximab, which not only blocks ligand binding, but also partially triggers EGFR activation, which may lead to cetuximab resistance in TNBC. The phosphorylation of growth factor receptors and their signalling pathways were evaluated by determining the phosphorylation of EGFR, insulin‑like growth factor receptor (IGF‑1R), vascular endothelial growth factor receptor (VEGFR)‑2, Src kinase, phosphoinositide‑3‑kinase (PI3K), extracellular signal‑regulated kinase (ERK1/2) and serine/threonine‑specific protein kinase (Akt) and the degradation of EGFR, and by assessing the morphology and proliferation of MDA‑MB‑231 and MDA‑MB‑468 cells. Cetuximab treatment led to the phosphorylation of EGFR, VEGFR‑2, IGF‑1R and downstream signalling molecules, Src kinase and PI3K in these cells, as well as Akt in the MDA‑MB‑231 cells. The cetuximab‑mediated phosphorylation of IGF‑1R, VEGFR‑2 and Akt was inhibited by the EGFR kinase inhibitor, AG1478, and the Src kinase inhibitor, PP2. Cetuximab treatment led to the degradation of EGFR. The cetuximab‑induced phosphorylation and EGFR degradation were less prominent compared with those induced by EGF. Cetuximab partially inhibited EGF‑mediated responses. Cetuximab, similar with EGF, altered cellular morphology in a serum‑free medium. In both cell lines, the Src kinase inhibitor enhanced the cetuximab‑induced anti‑proliferative response. These results indicate that cetuximab exerts a partial agonistic effect on EGFR, which activates Src kinase and subsequently transactivates IGF‑1R and VEGFR‑2. This partial agonistic property is likely one of the mechanisms underlying the resistance of TNBC to cetuximab.

Ligand-activated epidermal growth factor receptor (EGFR) signaling governs endocytic trafficking of unliganded receptor monomers by non-canonical phosphorylation

The canonical description of transmembrane receptor function is initial binding of ligand, followed by initiation of intracellular signaling and then internalization en route to degradation or recycling to the cell surface. It is known that low concentrations of extracellular ligand lead to a higher proportion of receptor that is recycled and that non-canonical mechanisms of receptor activation, including phosphorylation by the kinase p38, can induce internalization and recycling. However, no connections have been made between these pathways; i.e. it has yet to be established what happens to unbound receptors following stimulation with ligand. Here we demonstrate that a minimal level of activation of epidermal growth factor receptor (EGFR) tyrosine kinase by low levels of ligand is sufficient to fully activate downstream mitogen-activated protein kinase (MAPK) pathways, with most of the remaining unbound EGFR molecules being efficiently phosphorylated at intracellular serine/threonine residues by activated mitogen-activated protein kinase. This non-canonical, p38-mediated phosphorylation of the C-tail of EGFR, near Ser-1015, induces the clathrin-mediated endocytosis of the unliganded EGFR monomers, which occurs slightly later than the canonical endocytosis of ligand-bound EGFR dimers via tyrosine autophosphorylation. EGFR endocytosed via the non-canonical pathway is largely recycled back to the plasma membrane as functional receptors, whereas p38-independent populations are mainly sorted for lysosomal degradation. Moreover, ligand concentrations balance these endocytic trafficking pathways. These results demonstrate that ligand-activated EGFR signaling controls unliganded receptors through feedback phosphorylation, identifying a dual-mode regulation of the endocytic trafficking dynamics of EGFR.

Lapatinib Inhibits Breast Cancer Cell Proliferation by Influencing PKM2 Expression.

Pyruvate kinase type M2, which is expressed in multiple tumor cell types and plays a key role in aerobic glycolysis, also has nonglycolytic functions and can regulate transcription and cell proliferation. The results of this study show that epidermal growth factor receptor activation induces pyruvate kinase type M2 nuclear translocation. To further determine the relationship between pyruvate kinase type M2 and epidermal growth factor receptor, we analyzed pathological data from mammary glands and performed epidermal growth factor receptor/human epidermal growth factor receptor 2 knockdown to reveal that pyruvate kinase type M2 is associated with epidermal growth factor receptor and human epidermal growth factor receptor 2. Lapatinib is a small molecule epidermal growth factor receptor tyrosine kinase inhibitor that can inhibit epidermal growth factor receptor and human epidermal growth factor receptor 2, though its effect on pyruvate kinase type M2 remains elusive. Accordingly, we performed Western blotting and reverse transcription polymerase chain reaction and analyzed pathological data from mammary glands, with results suggesting that lapatinib inhibits pyruvate kinase type M2 expression. We further found that the antitumor drug lapatinib inhibits breast cancer cell proliferation by influencing pyruvate kinase type M2 expression, as based on Cell Counting Kit-8 analyses and pyruvate kinase type M2 overexpression experiments. Signal transducer and activator of transcription 3, which is a transcription factor-associated cell proliferation and the only transcription factor that interacts with pyruvate kinase type M2, we performed pyruvate kinase type M2 knockdown experiments in Human breast cancer cells MDA-MB-231 and Human breast cancer cells SK-BR-3 cell lines and examined the effect on levels of Signal transducer and activator of transcription 3 and phosphorylated Signal transducer and activator of transcription 3. The results indicate that pyruvate kinase type M2 regulates Signal transducer and activator of transcription 3 and phospho-Stat3 (Tyr705) expression. Together with previous reports, our findings show that lapatinib inhibits breast cancer cell proliferation by influencing pyruvate kinase type M2 expression, which results in a reduction in both Signal transducer and activator of transcription 3 and phosphorylated Signal transducer and activator of transcription 3.

Production of a germline-humanized cetuximab scFv and evaluation of its activity in recognizing EGFR- overexpressing cancer cells

ABSTRACTThe aim of this study was to produce a humanized single chain antibody (scFv) as a potential improved product design to target EGFR (Epidermal Growth Factor Receptor) overexpressing cancer cells. To this end, CDR loops of cetuximab (an FDA-approved anti-EGFR antibody) were grafted on framework regions derived from type 3 (VH3 and VL3 kappa) human germline sequences to obtain recombinant VH and VL domainslinked together with a flexible linker [(Gly4Ser)3] to form a scFv. Codon optimized synthetic gene encoding the scFv (with NH2-VH-linker-VL-COOH orientation) was expressed in E. coli Origami™ 2(DE3) cells and the resultant scFv purified by using Ni-NTA affinity chromatography. The scFv, called cet.Hum scFv, was evaluated in ELISA and immunoblot to determine whether it can recognize EGFR. The scFv was able to recognize EGFR over-expressing cancer cells (A-431) but failed to detect cancer cells with low levels of EGFR (MCF-7 cells). Although the affinity of the scFv forA-431 cells was 9 fold lower than that of cetuximab, it was strong enough to recognize these cells. Considering its ability to bind EGFR molecules, the scFv may exhibit a potential application for the detection of EGFR-overexpressing cancer cells.

Cell type-specific pharmacological kinase inhibition for cancer chemoprevention

Safety is prerequisite for preventive medicine, but non-toxic agents are generally ineffective as clinical chemoprevention. Here we propose a strategy overcoming this challenge by delivering molecular-targeted agent specifically to the effector cell type to achieve sufficient potency, while circumventing toxicity in the context of cancer chemoprevention. Hepatic myofibroblasts drive progressive fibrosis that results in cirrhosis and liver cancer. In a rat model of cirrhosis-driven liver cancer, a small molecule epidermal growth factor receptor inhibitor, erlotinib, was delivered specifically to myofibroblasts by a versatile nanoparticle-based system, targeting platelet-derived growth factor receptor-beta uniquely expressed on their surface in the liver. With systemic administration of erlotinib, tumor burden was reduced to 31%, which was further improved to 21% by myofibroblast-targeted delivery even with reduced erlotinib dose (7.3-fold reduction with equivalent erlotinib dose) and less hepatocyte damage. These findings demonstrate a strategy, cell type-specific kinase inhibition, for more effective and safer precision cancer chemoprevention.

Gd2O3-doped silica @ Au nanoparticles for in vitro imaging cancer biomarkers using surface-enhanced Raman scattering

There has been an interest in developing multimodal approaches to combine the advantages of individual imaging modalities, as well as to compensate for respective weaknesses. We previously reported a composite nano-system composed of gadolinium-doped mesoporous silica nanoparticle and gold nanoparticle (Gd-Au NPs) as an efficient MRI contrast agent for in vivo cancer imaging. However, MRI lacks sensitivity and is unsuitable for in vitro cancer detection. Thus, here we performed a study to use the Gd-Au NPs for detection and imaging of a widely recognized human cancer biomarker, epidermal growth factor receptor (EGFR), in individual human cancer cells with surface-enhanced Raman scattering (SERS). The Gd-Au NPs were sequentially conjugated with a monoclonal antibody recognizing EGFR and a Raman reporter molecule, 4-meraptobenzoic acid (MBA), to generate a characteristic SERS signal at 1075cm−1. By spatially mapping the SERS intensity at 1075cm−1, cellular distribution of EGFR and its relocalization on the plasma membrane were measured in situ. In addition, the EGFR expression levels in three human cancer cell lines (S18, A431 and A549) were measured using this SERS probe, which were consistent with the comparable measurements using immunoblotting and immunofluorescence. Our SERS results show that functionalized Gd-Au NPs successfully targeted EGFR molecules in three human cancer cell lines and monitored changes in single cell EGFR distribution in situ, demonstrating its potential to study cell activity under physiological conditions. This SERS study, combined with our previous MRI study, suggests the Gd-Au nanocomposite is a promising candidate contrast agent for multimodal cancer imaging.

A Dual Functional Scaffold Tethered with EGFR Antibody Promotes Neural Stem Cell Retention and Neuronal Differentiation for Spinal Cord Injury Repair.

Neural stem cells (NSCs) transplantation is a promising strategy to restore neuronal relays and neurological function of injured spinal cord because of the differentiation potential into functional neurons, but the transplanted NSCs often largely diffuse from the transplanted site and mainly differentiate into glial cells rather than neurons due to the adverse microenviornment after spinal cord injury (SCI). This paper fabricates a dual functional collagen scaffold tethered with a collagen-binding epidermal growth factor receptor (EGFR) antibody to simultaneously promote NSCs retention and neuronal differentiation by specifically binding to EGFR molecule expressed on NSCs and attenuating EGFR signaling, which is responsible for the inhibition of differentiation of NSCs toward neurons. Compared to unmodified control scaffold, the dual functional scaffold promotes the adhesion and neuronal differentiation of NSCs in vitro. Moreover, the implantation of the dual functional scaffold with exogenous NSCs in rat SCI model can capture and retain NSCs at the injury sites, and promote the neuronal differentiation of the retained NSCs into functional neurons, and finally dedicate to improving motor function of SCI rats, which provides a potential strategy for synchronously promoting stem cell retention and differentiation with biomaterials for SCI repair.