Mechanisms of Dendrobium officinale polysaccharides in repairing gastric mucosal injuries based on mitogen-activated protein kinases (MAPK) signaling pathway.

The present study aimed to investigate the protective effects and molecular mechanisms of Dendrobium officinale polysaccharides on gastric mucosal injuries. Following one week of continuous intragastric administration, a gastric mucosal injury model was established using intragastric administration of anhydrous ethanol. The area of gastric ulcer was measured, the contents of interleukin- 6 (IL-6), epidermal growth factor receptor (EGFR), and thyroid transcription factor 1 (TFF-1) in serum were detected by enzyme linked immunosorbent assay (ELISA), and the expressions of EGFR, TFF-1, IL-6, Raf-2, MAP kinase kinase 1 (MEK1), MEK2, and ERK1 in the gastric tissue were determined utilizing qPCR, Western blotting and immunohistochemistry. Simultaneously, Dendrobium officinale polysaccharides and anhydrous ethanol were added to the gastric mucosal cells (GES1) cultured in vitro, and the protective effects of Dendrobium officinale polysaccharides on cell viability was detected using Cell Counting Kit (CCK)-8. The addition of Dendrobium officinale polysaccharides markedly improved the gastric epithelial defect, inflammatory cell infiltration, and redness and swelling stemmed from gastric mucosal injuries and greatly reduced the area of gastric ulcer. The inhibition rates of gastric ulcer were 48.12 ± 2.98, 42.95 ± 1.52, and 27.96 ± 2.05% in the high, medium, and low concentration Dendrobium officinale polysaccharide groups, respectively. Dendrobium officinale polysaccharides could increase the expressions of EGFR and TFF-1 and decrease the expressions of IL-6, Raf-2, MEK1, MEK2, and ERK1. Dendrobium officinale polysaccharides could reduce the level of inflammatory factors and protect gastric mucosa by inhibiting the expression of MAPK pathway genes and proteins.

Failure to EGFR-TKI-based therapy and tumoural progression are promoted by MEOX2/GLI1-mediated epigenetic regulation of EGFR in the human lung cancer.

BACKGROUND: Mesenchyme homeobox-2 (MEOX2)-mediated regulation of glioma-associated oncogene-1 (GLI1) has been associated with poor overall survival, conferring chemoresistance in lung cancer. However, the role of MEOX2/GLI1 in resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs)-based therapy remains unexplored in human lung cancer. METHODS: Functional assays using genetic silencing strategy by short hairpin RNAs, as well as cytotoxic (tetrazolium dye MTT) and clonogenic assays, were performed to evaluate MEOX2/GLI1-induced malignancy capacity in lung cancer cells. Further analysis performed includes western blot, qPCR and ChIP-qPCR assays to identify whether MEOX2/GLI1 promote EGFR/AKT/ERK activation, as well as EGFR overexpression through epigenetic mechanisms. Finally, preclinical tumour progression in vivo and progression-free disease interval analyses in patients treated with EGFR-TKI were included. RESULTS: Overexpressed MEOX2/GLI1 in both EGFR wild-type and EGFR/KRAS-mutated lung cancer cells were detected and involved in the activation/expression of EGFR/AKT/ERK biomarkers. In addition, MEOX2/GLI1 was shown to be involved in the increased proliferation of tumour cells and resistance capacity to cisplatin, EGFR-TKIs (erlotinib and AZD9291 'osimertinib'), AZD8542-SMO, and AZD6244-MEKK1/2. In addition, we identified that MEOX2/GLI1 promote lung tumour cells progression in vivo and are clinically associated with poorer progression-free disease intervals. Finally, both MEOX2 and GLI1 were detected to be epigenetically involved in EGFR expression by reducing both repressive markers polycomb-EZH2 and histone H3K27me3, but, particularly, increasing an activated histone profile H3K27Ac/H3K4me3 at EGFR-gene enhancer-promoter sequences that probably representing a novel EGFR-TKI-based therapy resistance mechanism. CONCLUSION: MEOX2/GLI1 promote resistance to cisplatin and EGFR-TKI-based therapy in lung cancer cells, modulating EGFR/AKT/ERK signalling pathway activation, as well as inducing an aberrant epigenetic modulation of the EGFR-gene expression in human lung cancer.

Purpuric drug eruptions induced by EGFR tyrosine kinase inhibitors are associated with IQGAP1-mediated increase in vascular permeability.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are used as a treatment for non-small-cell lung cancer. There have been some reports of EGFR-TKIs being associated with vascular adverse events. We found that EGFR-TKIs decreased the proliferation of HMEC-1s (immortalized human dermal microvascular endothelial cells) and HMVECs (human dermal microvascular endothelial cells), and also inhibited the phosphorylation of EGFR and ERK. We examined the mRNA expression profile of erlotinib-treated HMEC-1s and identified IQ motif containing GTPase activating protein 1 (IQGAP1) as the most consistently up-regulated transcript and protein. IQGAP1 was also overexpressed and co-localized with endothelial cells in the lesional skin. Notably, increased IQGAP1 expression was associated with decreased transendothelial electrical resistance and increased vascular permeability in vitro. Erlotinib treatment enriched the staining of IQGAP1 and reduced the intensities of α-catenin at the sites of cell-cell contact. In conclusion, erlotinib induces adherens junction dysfunction by modulating the expression of IQGAP1 in dermal endothelial cells. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

[Efficacy of first generation EGFR-TKIs and chemotherapy as first-line therapy in advanced lung adenocarcinoma patients with uncommon EGFR mutations].

Objective: To investigate the clinical effects of first generation epithelial growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) compared with platinum-based chemotherapy as first-line therapy in advanced lung adenocarcinoma patients with uncommon EGFR mutations. Methods: Clinical data of 4 276 patients diagnosed as advanced lung adenocarcinoma (ⅢB/Ⅳ) underwent EGFR gene detection at the Affiliated Cancer Hospital of Zhengzhou University from January 2012 to February 2018 were collected and 99 cases with uncommon EGFR mutations were selected. The clinical pathological features, treatment outcomes, treatment options and prognosis after first-line treatment of the 99 cases were analysed and compared with other patients with common EGFR mutations. Results: The objective response rates of patients with uncommon EGFR mutations receiving EGFR-TKIs or platinum-based chemotherapy were 33.0% and 27.1%, respectively. The disease control rates were 76.5% and 87.5%, respectively. The progression-free survival (PFS) of patients treated with EGFR-TKIs was 7.2 months, significantly superior than 4.9 months of patients receiving chemotherapy (P=0.009). The overall survival of patients treated with EGFR-TKIs was 14.3 months, significantly worse than 20.7 months of patients receiving chemotherapy (P=0.034). Multivariate analysis showed that distant metastases (P=0.001) and smoking history (P=0.013) were independent prognostic factors for OS of lung adenocarcinoma patients with EGFR uncommon mutations. Conclusions: Compared with chemotherapy, the usage of first generation of EGFR-TKIs as first-line therapy can improve the short-term efficacy of advanced lung adenocarcinoma patients with EGFR uncommon mutations. However, platinum-based chemotherapy shows a longer overall survival.

Pembrolizumab versus chemotherapy for previously untreated, PD-L1-expressing, locally advanced or metastatic non-small-cell lung cancer (KEYNOTE-042): a randomised, open-label, controlled, phase 3 trial.

BACKGROUND: First-line pembrolizumab monotherapy improves overall and progression-free survival in patients with untreated metastatic non-small-cell lung cancer with a programmed death ligand 1 (PD-L1) tumour proportion score (TPS) of 50% or greater. We investigated overall survival after treatment with pembrolizumab monotherapy in patients with a PD-L1 TPS of 1% or greater. METHODS: This randomised, open-label, phase 3 study was done in 213 medical centres in 32 countries. Eligible patients were adults (≥18 years) with previously untreated locally advanced or metastatic non-small-cell lung cancer without a sensitising EGFR mutation or ALK translocation and with an Eastern Cooperative Oncology Group (ECOG) performance status score of 0 or 1, life expectancy 3 months or longer, and a PD-L1 TPS of 1% or greater. Randomisation was computer generated, accessed via an interactive voice-response and integrated web-response system, and stratified by region of enrolment (east Asia vs rest of world), ECOG performance status score (0 vs 1), histology (squamous vs non-squamous), and PD-L1 TPS (≥50% vs 1-49%). Enrolled patients were randomly assigned 1:1 in blocks of four per stratum to receive pembrolizumab 200 mg every 3 weeks for up to 35 cycles or the investigator's choice of platinum-based chemotherapy for four to six cycles. Primary endpoints were overall survival in patients with a TPS of 50% or greater, 20% or greater, and 1% or greater (one-sided significance thresholds, p=0·0122, p=0·0120, and p=0·0124, respectively) in the intention-to-treat population, assessed sequentially if the previous findings were significant. This study is registered at ClinicalTrials.gov, number NCT02220894. FINDINGS: From Dec 19, 2014, to March 6, 2017, 1274 patients (902 men, 372 women, median age 63 years [IQR 57-69]) with a PD-L1 TPS of 1% or greater were allocated to pembrolizumab (n=637) or chemotherapy (n=637) and included in the intention-to-treat population. 599 (47%) had a TPS of 50% or greater and 818 patients (64%) had a TPS of 20% or greater. As of Feb 26, 2018, median follow-up was 12·8 months. Overall survival was significantly longer in the pembrolizumab group than in the chemotherapy group in all three TPS populations (≥50% hazard ratio 0·69, 95% CI 0·56-0·85, p=0·0003; ≥20% 0·77, 0·64-0·92, p=0·0020, and ≥1% 0·81, 0·71-0·93, p=0·0018). The median surival values by TPS population were 20·0 months (95% CI 15·4-24·9) for pembrolizumab versus 12·2 months (10·4-14·2) for chemotherapy, 17·7 months (15·3-22·1) versus 13·0 months (11·6-15·3), and 16·7 months (13·9-19·7) versus 12·1 months (11·3-13·3), respectively. Treatment-related adverse events of grade 3 or worse occurred in 113 (18%) of 636 treated patients in the pembrolizumab group and in 252 (41%) of 615 in the chemotherapy group and led to death in 13 (2%) and 14 (2%) patients, respectively. INTERPRETATION: The benefit-to-risk profile suggests that pembrolizumab monotherapy can be extended as first-line therapy to patients with locally advanced or metastatic non-small-cell lung cancer without sensitising EGFR or ALK alterations and with low PD-L1 TPS. FUNDING: Merck Sharp & Dohme.

Synthesis and preclinical investigation of 99mTc-p-SCN-Bzl-DTPA-cetuximab for targeting EGFR using head and neck squamous cell carcinoma (HNSCC) xenografts.

To assess the preclinical potential of technetium-99m labelled conjugated para-isothiocyanato-benzyl diethylene triamine penta-acetic acid cetuximab (99mTc-p-SCN-Bzl-DTPA cetuximab) for imaging EGFR in HNSCC mice and rabbits xenografts. Cetuximab, a chimeric monoclonal antibody targeting EGFR, was conjugated with p-SCN-Bzl-DTPA followed by labelling with 99mTc. The labelled conjugate was evaluated for in vitro stability in L-cysteine at 37 °C. The 99mTc-p-SCN-Bzl-DTPA cetuximab was also investigated for immunoreactivity, internationalization kinetics, dose escalation (up to 300 µg) and biodistribution in HNSCC mice xenograft. The suitability of labelled moiety as a specific EGFR radio-tracer was assessed in HNSCC rabbit xenograft. 99mTc-p-SCN-Bzl-DTPA cetuximab exhibited more than 98% radiochemical purity at room temperature. In excess L-cysteine, it showed a stable behaviour at 37 °C up to 4 h p.l. The labelled conjugate was internalized in vitro in FaDu tumor cells up to 19.55%. Significantly higher uptake in tumor (at 10 µg; 34.75 ± 0.38% ID/g: pi) was seen in HNSCC mice xenograft with dose escalation assay from 1 to 300 µg/mouse. Blocking of EGFR with excess cetuximab consequently decreased the uptake of tumor up to 6.80 ± 1.25%. SPECT images of rabbit xenograft confirmed increase in tumor to background ratio after 4 h pi and validated its potential in preclinical trial as a specific FaDu tumor tracer. Our in vitro and in vivo preclinical findings indicate that the 99mTc-p-SCN-Bzl-DTPA cetuximab prepared at optimal dose of cetuximab could become a useful tool for EGFR imaging in HNSCC using SPECT.

Dacomitinib versus gefitinib as first-line treatment for patients with EGFR-mutation-positive non-small-cell lung cancer (ARCHER 1050): a randomised, open-label, phase 3 trial.

BACKGROUND: Dacomitinib is a second-generation, irreversible EGFR tyrosine kinase inhibitor. We compared its efficacy and safety with that of the reversible EGFR tyrosine kinase inhibitor gefitinib in the first-line treatment of patients with advanced EGFR-mutation-positive non-small-cell lung cancer (NSCLC). METHODS: In this international, multicentre, randomised, open-label, phase 3 study (ARCHER 1050), we enrolled adults (aged ≥18 years or ≥20 years in Japan and South Korea) with newly diagnosed advanced NSCLC and one EGFR mutation (exon 19 deletion or Leu858Arg) at 71 academic medical centres and university hospitals in seven countries or special administrative regions. We randomly assigned participants (1:1) to receive oral dacomitinib 45 mg/day (in 28-day cycles) or oral gefitinib 250 mg/day (in 28-day cycles) until disease progression or another discontinuation criterion was met. Randomisation, stratified by race and EGFR mutation type, was done with a computer-generated random code assigned by a central interactive web response system. The primary endpoint was progression-free survival assessed by masked independent review in the intention-to-treat population. Safety was assessed in all patients who received at least one dose of study treatment. This study is registered with ClinicalTrials.gov, number NCT01774721, and is ongoing but no longer recruiting patients. FINDINGS: Between May 9, 2013, and March 20, 2015, 452 eligible patients were randomly assigned to receive dacomitinib (n=227) or gefitinib (n=225). Median duration of follow-up for progression-free survival was 22·1 months (95% CI 20·3-23·9). Median progression-free survival according to masked independent review was 14·7 months (95% CI 11·1-16·6) in the dacomitinib group and 9·2 months (9·1-11·0) in the gefitinib group (hazard ratio 0·59, 95% CI 0·47-0·74; p<0·0001). The most common grade 3-4 adverse events were dermatitis acneiform (31 [14%] of 227 patients given dacomitinib vs none of 224 patients given gefitinib), diarrhoea (19 [8%] vs two [1%]), and raised alanine aminotransferase levels (two [1%] vs 19 [8%]). Treatment-related serious adverse events were reported in 21 (9%) patients given dacomitinib and in ten (4%) patients given gefitinib. Two treatment-related deaths occurred in the dacomitinib group (one related to untreated diarrhoea and one to untreated cholelithases/liver disease) and one in the gefitinib group (related to sigmoid colon diverticulitis/rupture complicated by pneumonia). INTERPRETATION: Dacomitinib significantly improved progression-free survival over gefitinib in first-line treatment of patients with EGFR-mutation-positive NSCLC and should be considered as a new treatment option for this population. FUNDING: SFJ Pharmaceuticals Group and Pfizer.

Reoxygenation Reverses Hypoxia-related Radioresistance in Head and Neck Cancer Cell Lines.

BACKGROUND/AIM: Head and neck cancer (HNC) is characterized by epidermal growth factor receptor (EGFR) overexpression and radiotherapy (RT) resistance. Cancer cells are able to survive and proliferate in hypoxic conditions. Hypoxia can be transiently interrupted by phases of reoxygenation. This work aimed to analyze the reoxygenation effect on proliferation in response to radiation in HNC cells. MATERIALS AND METHODS: HNC cell lines CAL33 and CAL166 were subjected to an 8-Gy radiation dose in hypoxia and/or after reoxygenation. Cell proliferation and molecular factors involved in response to treatments were studied. RESULTS: Cytotoxicity test confirmed radioresistance in hypoxia and highlighted that reoxygenation before RT restores sensitivity in both cell lines. Our results showed a similar proliferation inhibition effect and EGFR modulation but a different cell death mechanism in the two cell lines after treatment. CONCLUSION: Reoxygenation before RT rescued radiosensitivity in HNC cells.

Noncovalent Mutant Selective Epidermal Growth Factor Receptor Inhibitors: A Lead Optimization Case Study.

Because of their increased activity against activating mutants, first-generation epidermal growth factor receptor (EGFR) kinase inhibitors have had remarkable success in treating non-small-cell lung cancer (NSCLC) patients, but acquired resistance, through a secondary mutation of the gatekeeper residue, means that clinical responses only last for 8-14 months. Addressing this unmet medical need requires agents that can target both of the most common double mutants: T790M/L858R (TMLR) and T790M/del(746-750) (TMdel). Herein we describe how a noncovalent double mutant selective lead compound was optimized using a strategy focused on the structure-guided increase in potency without added lipophilicity or reduction of three-dimensional character. Following successive rounds of design and synthesis it was discovered that cis-fluoro substitution on 4-hydroxy- and 4-methoxypiperidinyl groups provided synergistic, substantial, and specific potency gain through direct interaction with the enzyme and/or effects on the proximal ligand oxygen atom. Further development of the fluorohydroxypiperidine series resulted in the identification of a pair of diastereomers that showed 50-fold enzyme and cell based selectivity for T790M mutants over wild-type EGFR (wtEGFR) in vitro and pathway knock-down in an in vivo xenograft model.

Renal effects of targeted anticancer therapies.

The use of novel targeted anticancer agents has led to overall improvement in the prognosis of many patients affected by various malignancies, but has also been associated with an increased risk of poorly characterized toxic effects to different organs, including the kidneys. The high prevalence of kidney impairment in the general population complicates the issue further. Nephrologists most frequently work with patients with cancer when they are asked to investigate kidney function to assess the need for dose adjustments in anticancer therapy. A thorough knowledge of the renal safety profile of novel life-prolonging anticancer therapies, specific features of their metabolism, and pharmacokinetic and pharmacodynamic properties (under normal circumstances as well as in the setting of renal replacement therapy) is, therefore, necessary to preserve kidney function as far as possible and to ensure optimum treatment. In this Review we summarize the present knowledge of renal toxic effects from novel targeted anticancer agents and discuss whether the management of patients' treatment needs to be modified. We also advocate the development of a new onconephrology subspeciality.

Brain natriuretic peptide modulates calcium homeostasis and epidermal growth factor receptor gene signalling in asthmatic airways smooth muscle cells.

The airway epithelium acts as a barrier and provides a critical interface between the body and the external environment. Brain natriuretic peptide (BNP) plays an important role in several bronchial functions, including relaxation. BNP relaxes airways by binding and activating natriuretic peptide receptor-A expressed from the airway epithelium. Although relaxation effect has been extensively investigated, less is known about BNP-regulated intracellular biomolecular pathways leading to bronchial relaxation. To this aim, we investigated BNP effects on gene signalling of airway smooth muscle cells (ASM) obtained from donors with asthma by using a RT(2) profiler™ PCR array. When compared with control, treatment for 2 h with supernatant from BNP-treated (1 µM) bronchial epithelial cells (BEAS-2B) induced in asthmatic ASM cells a rapid reduction of transcription of EGFR and genes involving in actin and calcium homeostasis, as those of Protein kinase C (PKC) and RhoA-ROCK gene pathways. Immunofluorescence and western blotting did not shown any difference comparing control and ASM cells treated with conditioned medium from BNP-treated BEAS-2B. This study provides evidence that the effect of BNP on relaxing bronchial in ASM cells is mediated from epithelium and associates to rapid changes of EGFR and calcium homeostasis-associated gene levels.

Phase II study of afatinib, an irreversible ErbB family blocker, in EGFR FISH-positive non-small-cell lung cancer.

INTRODUCTION: Afatinib, an oral irreversible ErbB Family Blocker, has demonstrated efficacy and safety in epidermal growth factor receptor (EGFR) mutation-positive advanced lung adenocarcinoma. It is unknown whether such activity also occurs in patients with EGFR gene overexpression, regardless of mutation status. This phase II study investigated the activity and safety of afatinib in advanced non-small-cell lung cancer with increased EGFR gene copy number and/or gene amplification by fluorescence in situ hybridization (FISH), with or without EGFR mutation. METHODS: EGFR gene overexpression was assessed by FISH analysis; patients with high polysomy or gene amplification were considered FISH positive. Patients received daily afatinib less than or equal to 50 mg (monotherapy). Endpoints included objective response rate (ORR; primary), disease control rate (DCR), progression-free survival (PFS), overall survival (OS), and safety. RESULTS: Of 223 patients screened, 69 patients were FISH-positive and met eligibility criteria for treatment. The ORR was 13.0% overall (n =9 of 69). Higher ORRs were observed in patients with gene amplification (20.0%; n =5 of 25) and EGFR mutation-positive tumors (25.0%; n =3 of 12). The DCR was 50.7% overall (n = 35 of 69; median duration: 24.9 weeks) with higher DCRs observed in patients with gene amplification 64.0%; (n = 16 of 25), and in patients with EGFR mutation-positive tumors 66.7% (n = 8 of 12). In the overall population, median PFS was 8.4 weeks and median OS was 50.4 weeks. The most common afatinib-related adverse events were rash/acne (83%) and diarrhea (78%). CONCLUSIONS: First- or second-line afatinib demonstrated preliminary activity and manageable safety in EGFR FISH-positive patients with advanced non-small-cell lung cancer.

Identification and optimization of new dual inhibitors of B-Raf and epidermal growth factor receptor kinases for overcoming resistance against vemurafenib.

Epidermal growth factor receptor (EGFR) amplification has been demonstrated to be critical for the inherent and/or acquired resistance against current B-Raf(V600E) inhibitor therapy for melanoma and colorectal cancer patients. We describe the discovery and structure-activity relationship study of a series of 1H-pyrazolo[3,4-b]pyridine-5-carboxamide analogues as novel dual inhibitors of EGFR and B-Raf(V600E) mutant. One of the most promising compounds, 6a, potently inhibited both of the kinases with IC50 values of 8.0 and 51 nM, respectively. The compound also strongly suppressed the proliferation of a panel of intrinsic and acquired resistant melanoma and/or colorectal cancer cells harboring overexpressed EGFR with submicromolar IC50 values. Further mechanism investigation revealed that 6a could sustainably inhibit the activation of the MAPK path way in the resistant SK-MEL-28 PR30 melanoma cancer cells and WiDr colorectal cancer cells with EGFR amplification. Our results support the hypothesis that the EGFR/B-Raf(V600E) dual inhibition might be a tractable strategy to overcome the intrinsic and acquired resistance of melanoma and/or colorectal cancers against the current B-Raf(V600E) inhibitor therapy.

Novel cancer therapies: treatments driven by tumour biology.

The traditional view is that treatments within oncology largely consist of chemotherapy, which aims to maximise damage to the rapidly dividing cancer cells but often at the expense of normal cells and overall quality of life for the patient. The development of anticancer drugs has changed from the serendipitous discoveries of the past, to today's purposeful targeting of cancer cells which takes advantage of novel technological developments and a greater understanding of tumour biology. The aim of these new treatments is to affect the essential function of the cancer cell while sparing normal cells, and limiting side effects. The phenotypic characteristics of tumours, such as unregulated growth signalling, development of new vascular systems and the evasion of immune destruction are used to identify potential drug targets. Here we review the clinical evidence and molecular mechanisms for novel therapies that are currently in use and those that are in development.

Shifting paradigms in non-small cell lung cancer: an evolving therapeutic landscape.

Globally, lung cancer is the leading cause of cancer-related mortality among both men and women, and while mortality associated with the disease has demonstrated relative stability over the years, evidence has suggested an increasing incidence and prevalence of the disease. Unfortunately, the diagnosis of lung cancer is often made late in the course of the disease, with almost 70% of patients presenting with locally advanced or metastatic disease at initial diagnosis. Non-small cell lung cancer (NSCLC) is the most common form of the malignancy, occurring in up to 85% of cases. There are 3 subtypes of NSCLC: squamous-cell carcinoma, adenocarcinoma, and large-cell carcinoma. Enhanced understanding of the pathophysiology of NSCLC has led to substantial improvements in diagnostic, prognostic, and therapeutic interventions for NSCLC. The discovery of targetable molecular alterations in genes, such as epidermal growth factor receptor (EGFR), has driven the evolution of targeted therapies for NSCLC and shifted treatment paradigms for the disease. This article will summarize the epidemiology and pathophysiology of NSCLC, its associated gene mutations and biomarkers, and the approach to treatment, with a focus on patients whose tumors harbor EGFRactivating mutations.

ErbB1 epidermal growth factor receptor is a valid target for reducing the effects of multiple inhibitors of axonal regeneration.

Pharmacological inhibitors of epidermal growth factor receptor (ErbB1) attenuate the ability of CNS myelin to inhibit axonal regeneration. However, it has been claimed that such effects are mediated by off-target interactions. We have tested the role of ErbB1 in axonal regeneration by culturing neurons from ErbB1 knockout mice in the presence of various inhibitors of axonal regeneration: CNS myelin, chondroitin sulfate proteoglycans (CSPG), fibrinogen or polyinosinic:polycytidylic acid (poly I:C). We confirmed that ErbB1 was activated in cultures of cerebellar granule cells exposed to inhibitors of axonal regeneration and that ErbB1 kinase inhibitors promoted neurite outgrowth under these conditions. In the presence of myelin, fibrinogen, CSPG and poly I:C ErbB1 -/- neurons grew longer neurites than neurons expressing ErbB1. Furthermore, inhibitors of ErbB1 kinase did not improve neurite outgrowth from ErbB1 -/- neurons, ruling out an off-target mechanism of action. ErbB1 kinase activity is therefore a valid target for promoting axonal elongation in the presence of many of the molecules believed to contribute to the failure of axonal regeneration in the injured CNS.

Efficacy and mechanism of action of Proellex, an antiprogestin in aromatase overexpressing and Letrozole resistant T47D breast cancer cells.

Aromatase inhibitors (AI) are considered as a first line therapy for ER+PR+ breast cancers. However, many patients acquire resistance to AI. In this study, we determined the response of antiprogestin CDB-4124 (Proellex) on the aromatase overexpressing and Letrozole resistant cell lines and also studies its mechanism of action in inhibition of breast cancer cell proliferation. For these studies we generated aromatase overexpressing T47D (T47Darom) and respective control (T47Dcon) breast cancer cell lines by stable transfection with plasmid containing CYP19A1 gene, or empty vector respectively. Letrozole resistant cell line (T47DaromLR) was generated by incubating T47Darom for 75 weeks in the presence of 10 µM Letrozole. Cell proliferation was determined by MTT or crystal violet assays. Gene expressions were quantified by QRT-PCR whereas proteins were identified by western blot analyses, flow cytometry and immunofluorescence staining. Aromatase activity was determined by estradiol ELISA. The effects of Proellex on the anchorage independent growth were measured by soft agar colony formation. Statistical differences between the various groups were determined by Student's 't' test or ANOVA followed by Bonferroni's post hoc test. Results showed that T47Darom and T47DaromLR cell lines had significantly higher aromatase expression (mRNA; 80-90 fold and protein) and as a result exhibited increased aromatization of testosterone to estradiol as compared to T47Dcon. Both these cell lines showed enhanced growth in the presence of Testosterone (50-60%). In T47DaromLR cells increased PR-B and EGFR expression as compared to T47Dcon cells was observed. Proellex and other known aromatase inhibitors (Letrozole, Anastrozole, and Exemestane) inhibited testosterone induced cell proliferation and anchorage independent growth of T47Darom cells. Cell growth inhibition was significantly greater when cells were treated with Proellex alone or in combination with other AIs as compared to AIs alone. Proellex inhibited mRNA and protein levels of PR-B, reduced PRB/p300 complex formation in the nuclei and significantly reduced EGFR expression in T47Darom cells. Our results in the present study indicate that antiproliferative effect of Proellex is probably due to PR-B/EGFR modulation in ER+PR+, aromatase expressing cells. Overall these results suggest that antiprogestin, Proellex can be developed as a possible treatment strategy for aromatase overexpressing ER+/PR+ breast cancer patients as well as for aromatase inhibitor resistant breast cancer patients.