Epidermal Growth Factor Receptor Downstream Pathways Research Articles

Potential role and prognostic value of Globo-H in lung cancer.

e20037 Background: Globo-H (GH), a globo-series carbohydrate antigen highly expressed on multiple epithelial cancers, is a promising target for cancer immunotherapy. Clinical trials have been conducted to evaluate the efficacy of anti-GH vaccine Adagloxad Simolenin/OBI-821 (in Ph 3) and OBI-833/OBI-821 (in Ph 2) in cancer patients. Epidermal growth factor receptor (EGFR) mutations are well known as a frequent class of driver mutations in lung cancer (LC), which are responsible for tyrosine kinase inhibitors. Besides high expression in cancer cells, GH has been implicated in regulation of EGFR signaling pathway. Knockdown of key enzyme involved in GH biosynthesis β1,3-galactosyltransferase V (β3GalT5) induces apoptosis through disruption of EGFR-related signaling protein complex. In addition, GH is reported to associate the EGFR mutant in LC patients. This study aims to further investigate the potential role of GH in LC. Methods: Clinical study: The level of GH expression was evaluated by immunohistochemistry (IHC) using H-score in clinical samples from 141 patients with lung adenocarcinoma. In vitro study: FACS and Western blotting methods were used to measure the levels of total EGFR, phospho-EGFRs or GH in LC cell lines. Microwestern protein array (MWA) analysis was performed in LC NCI-H1975 cells. Results: Clinical study: Positive GH expression (H score≥1; 18.4%) was significantly associated with poor differentiation grade (p = 0.044) in all samples and disease specific survival in patients with metastasis and stage 4 LC (p = 0.033). In vitro study: Inhibition of EGFR by an EGFR-specific shRNA resulted in a decrease of GH expression in LC cells while overexpression of EGFR resulted in an increase of GH levels. β3GalT5 knockout cells and anti-GH-antibody-treated cells showed a reduction of cell proliferation as well as the total level of EGFR and phosphor-EGFRs expression, which is consistent with the results from previous studies. MWA analysis on LC cells with β3GalT5 knockout, GH ceramide-treated β3GalT5 knockout cells and anti-GH Ab-treated LC cells indicated a positive correlation between the level of GH and EGFR-related signaling proteins including phosphor-JNK, phosphor-c-Jun, phosphor-PP2A, phosphor-PP2A, phosphor-AKT, and Cyclin D1 proteins. In addition, JNK signaling inhibitor partially reduced GH expression in NCI-H1975 cells. These results revealed the mechanistic link between GH regulation and tumor promoting signals in LC. Conclusions: GH expression was associated with the worse survival of LC patients in clinical setting. In addition, the level of GH is positively correlated with levels of EGFR and EGFR-related signaling proteins in vitro. Expression of GH can induce the expression of EGFR and vice versa in LC cells. Furthermore, EGFR downstream JNK signaling pathway might involve in GH regulation by EGFR. Taken together, GH-targeting therapy has a potential application for the treatment of LC patients.

Plasma ctDNA biomarker study in patients with non-small cell lung cancer with EGFR exon 20 insertion mutation treated with sunvozertinib.

8563 Background: There are limited reports on biomarker studies of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 20 insertion mutation (exon20ins). Sunvozertinib (DZD9008), an oral, potent, irreversible and selective EGFR tyrosine kinase inhibitor (TKI), has been granted conditional approval in China for the treatment of NSCLC with EGFR exon20ins in the ≥ second-line setting. Here we report biomarker analysis of EGFR exon20ins in large sample size, by pooling data from WU-KONG series of studies. Methods: Serial plasma ctDNA samples were collected from baseline until progressive disease (PD). Next generation sequencing was used to assess EGFR exon20ins in baseline plasma ctDNA, and to analyze genetic alterations in baseline and PD plasma ctDNA to identify potential resistance mechanisms to sunvozertinib. Droplet digital PCR was used to dynamically monitor the quantity of EGFR exon20ins during treatment. In addition, association between baseline EGFR exon20ins and disease characteristics as well as antitumor activity of sunvozertinib was analyzed. Furthermore, in vitro and in vivo experiments were performed to explore potential approach which may overcome the resistance to sunvozertinib. Results: A total of 121 patients were included in this biomarker analysis, among them 81 out of 121 (66.9%) had detectable EGFR exon20ins in baseline plasma ctDNA. There was a positive correlation between detectable EGFR exon20ins and higher number of metastasis sites. In addition, higher EGFR exon20ins abundance was positively correlated with more metastasis sites and brain metastasis. Patients with EGFR exon20ins negative in plasma ctDNA achieved a higher objective response rate (ORR) (68.0% vs. 45.8%) and longer median progression free survival (PFS) (7.4 months vs. 5.5 months) with sunvozertinib treatment. In patients with baseline detectable EGFR exon20ins, the mutant allele decreased over time with treatment in majority of patients (12/14, 85.7%), and the earliest clearance of EGFR exon20ins occurred after one week of treatment. In patients who developed disease progression, 12 of 18 (66.7%) maintained EGFR exon20ins at PD, and among them, 3 concurrently acquired EGFR C797S, all in cis with exon20ins. In addition, EGFR G724S and other genetic aberrations in EGFR downstream signaling pathway, were also detected. Exploratory work on overcoming resistance to sunvozertinib suggests that combination of golidocitinib, a selective JAK1 inhibitor, with chemotherapy could be a potential approach. Conclusions: This study showed that positive EGFR exon20ins in plasma ctDNA is associated with advanced disease characteristics. Sunvozertinib can effectively clear EGFR exon20ins in plasma ctDNA, confirming its direct effect on EGFR pathway. The resistance to sunvozertinib can be through EGFR-dependent and EGFR-independent mechanisms.

Resistance to hormone therapy in breast cancer cells promotes autophagy and EGFR signaling pathway.

Breast cancer is the leading cause of cancer deaths for women worldwide. Endocrine therapies represent the cornerstone for hormone-dependent breast cancer treatment. However, in many cases, endocrine resistance is induced with poor prognosis for patients. In the current study, we have developed MCF-7 cell lines resistant to fulvestrant (MCF-7Fulv) and tamoxifen (MCF-7Tam) aiming at investigating mechanisms underlying resistance. Both resistant cell lines exerted lower proliferation capacity in two-dimensional (2-D) cultures but retain estrogen receptor α (ERα) expression and proliferate independent of the presence of estrogens. The established cell lines tend to be more aggressive exhibiting advanced capacity to form colonies, increased expression of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and heterodimerization of ERBB family receptors and activation of EGFR downstream pathways like MEK/ERK1/2 and PI3K/AKT. Tyrosine kinase inhibitors tested against resistant MCF-7Fulv and MCF-7Tam cells showed moderate efficacy to inhibit cell proliferation, except for lapatinib, which concomitantly inhibits both EGFR and HER2 receptors and strongly reduced cell proliferation. Furthermore, increased autophagy was observed in resistant MCF-7Fulv and MCF-7Tam cells as shown by the presence of autophagosomes and increased Beclin-1 levels. The increased autophagy in resistant cells is not associated with increased apoptosis, suggesting a cytoprotective role for autophagy that may favor cells' survival and aggressiveness. Thus, by exploiting those underlying mechanisms, new targets could be established to overcome endocrine resistance.NEW & NOTEWORTHY The development of resistance to hormone therapy caused by both fulvestrant and tamoxifen promotes autophagy with concomitant apoptosis evasion, rendering cells capable of surviving and growing. The fact that resistance also triggers ERBB family signaling pathways, which are poorly inhibited by tyrosine kinase inhibitors might attribute to cells' aggressiveness. It is obvious that the development of endocrine therapy resistance involves a complex interplay between deregulated ERBB signaling and autophagy that may be considered in clinical practice.

LHPP suppresses proliferation, migration, and invasion in hepatocellular carcinoma and pancreatic cancer by inhibiting EGFR signaling pathway.

Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) has been reported to be a new tumor suppressor with a significant inhibitory effect in various cancers. Although LHPP has been repeatedly shown to inhibit the progression of various tumors by inhibiting the phosphorylation of AKT, up to now, the studies on the function and mechanism of LHPP in tumors are insufficient. In this study, LHPP expression was found to be downregulated in both hepatocellular carcinoma (HCC) and pancreatic cancer (PC). Here, we found that LHPP could bind to epidermal growth factor receptor (EGFR) and inhibit its phosphorylation, which thereby inhibited the activation of EGFR downstream pathways ERK, AKT, and STAT3, and then weakening the ability to proliferate, invade, and migrate in HCC and PC. This paper showed a new physiological function of LHPP in inhibiting phosphorylation of EGFR and its potential anti-tumor mechanism and indicated that LHPP was a potential therapeutic target for HCC and PC.

Small-molecule nanoprodrug with high drug loading and EGFR, PI3K/AKT dual-inhibiting properties for bladder cancer treatment.

Bladder cancer (BCa) is one of the most common malignancies worldwide. Although multiple efforts have been made, the 5-year survival rate of patients with BCa remains unchanged in recent years. Overexpression of the epidermal growth factor receptor (EGFR) is found in ≈74% of BCa tissue specimens; however, current EGFR-based targeted therapies show little benefit for BCa patients, as the EGFR downstream pathways appear to be circumvented by other receptor tyrosine kinases (RTKs). In this study, two natural products are identified, namely triptolide (TPL) and hesperidin (HSP), that target and inhibit the EGFR and its downstream PI3K/AKT pathway in BCa. To synergistically combine triptolide and hesperidin, a succinic acid linker was employed to conjugate them and formed an amphiphilic TPL-HSP EGFR-targeting prodrug (THE), which further self-assembled to generate nanoparticles (THE NPs). These NPs allowed the EGFR-targeted delivery of the triptolide and hesperidin, and simultaneous inhibition of the EGFR and PI3K/AKT both in vitro and in vivo. This study provides a promising EGFR-targeted delivery approach with the dual inhibition of the EGFR and PI3K/AKT, while also exhibiting a high drug loading and low toxicity. Our formulation may be a suitable option to deliver natural products for BCa treatment by EGFR-targeted therapy.

Leveraging innate immunity with AFM24, a novel CD16A and epidermal growth factor receptor (EGFR) bispecific innate cell engager: Interim results for the non-small cell lung cancer (NSCLC) cohort.

2533 Background: Patients with EGFR mutant NSCLC who progress on or after small molecule EGFR inhibitors have only limited treatment options. AFM24 is a tetravalent, bispecific innate cell engager that binds EGFR on solid tumor cells and CD16A on NK cells to redirect and enhance antitumor antibody-dependent cellular cytotoxicity, thus acting independently of the EGFR downstream signaling pathway and EGFR expression levels. The Phase 1 dose escalation phase of the presented study (NCT04259450) established the recommended Phase 2 dose of AFM24 monotherapy at 480 mg based on tolerability, receptor occupancy, and pharmacokinetics (PK). Correlative science data revealed an activation and enrichment of immune cells in the tumor microenvironment. Methods: The Phase 2a expansion phase is evaluating 480 mg AFM24 in patients with EGFR mutant NSCLC, relapsed or refractory to ≥1 prior lines of therapy; EGFR-positivity is defined as positive staining for EGFR in ≥1% of tumor cells per local immunohistochemistry assay. The study follows Simon’s two-stage design. The primary endpoint is overall response rate per local RECIST v1.1; secondary endpoints include safety, PK, and immunogenicity. Efficacy per iRECIST is an exploratory endpoint. AFM24 is administered intravenously once weekly in a 28-day cycle. Tumor assessments are performed at initial screening, cycles 2, 4, 6, 8, and every 3 cycles thereafter. Results: As of December 2022, 14 patients (median [range] age 55 [37–82]; 12 male; 11 Asian, 3 White) were enrolled; the median number of prior therapies was 2.5 (1–12). Median treatment duration with AFM24 was 6.7 weeks (1.0–26.1). Best objective response in 10 evaluable patients was confirmed partial response in 1 patient (change from baseline –45%); 4 patients exhibited stable disease, 1 was confirmed. Tumor shrinkage occurred in 2 patients with stable disease (–6%, –18%). The tumor control rate was 50%. Treatment-related adverse events (TRAEs) were reported in 13 patients; infusion related reactions (IRRs, 12), dermatitis acneiform, neutrophil count decreased, decreased appetite, and myalgia (2 each) were most common. Grade ≥3 TRAEs occurred in 4 patients and included decreased neutrophil count (2), lymphopenia, and IRR (1 each). Grade 5 pneumonitis was observed in 1 patient with disease progression and multiple comorbidities; relation to AFM24 could not be ruled out. As of January 2023, enrollment is ongoing; results of the interim analysis will be presented. Conclusions: AFM24 demonstrated clinical activity and acceptable safety in heavily pretreated patients with EGFR mutant NSCLC. The novel mechanism of action of AFM24 could add to the therapeutic options for patients with EGFR expressing tumors and is under clinical evaluation as a single agent and in combination with atezolizumab or autologous NK cells. Clinical trial information: NCT04259450 .

Abstract 1713: TRMT6-mediated N1-methyladenosine methylation promotes tumorigenesis in colorectal cancer

Abstract Background: N1-methyladenosine (m1A) within tRNAs, an essential and highly conserved RNA modification, has recently emerged as a significant epigenetic modification that plays a pivotal role in tumor progression in some cancers. However, the role of m1A methylation in colorectal cancer (CRC) remains largely unexplored. TRMT6 is the most critical methyltransferase that catalyzes N1-methyladenine modification within tRNA, and early evidence suggests that this TRMT6-mediated m1A methylation is also required for the self-renewal of cancer stem cells (CSCs). Herein, we systematically interrogated the role of TRMT6 in mediating stemness and pathogenesis of CRC. Methods: We first analyzed the expression levels of all m1A regulators in paired tumor and adjacent-normal samples from TCGA and GEO databases. Subsequently, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting (WB) assays were performed to validate the dysregulation of TRMT6 in CRC tissues and cell lines. m1A dot-blot assays were undertaken to detect methylation levels in CRC tissues and RKO and SW620 CRC cell lines. Cell viability, colony formation, spheroid formation, wound healing, and invasion assays were conducted to investigate the biological functions of TRMT6 in CRC. Results: Higher m1A methylation levels were detected in CRC than in normal tissues. By analyzing the expression of ten m1A regulators in public datasets, and subsequent qRT-PCR validation in a CRC clinical cohort, we identified that high TRMT6 expression was significantly associated with advanced tumor stages (III&IV vs. I&II, p<0.05) and poor prognosis (5-year relapse-free survival rates in high vs. low expression groups: 50.9% vs. 65.1%, p<0.05). TRMT6 silencing in CRC cell lines inhibited cell growth, colony formation, and metastatic efficiency (p<0.01-0.001). Furthermore, we discovered that the TRMT6 could enhance stem cell-like properties required for CSC self-renewal. Transcriptomic profiling identified that the epidermal growth factor receptor (EGFR) pathway is one of the critical signaling regulators of TRMT6 in CRC. The TRMT6 knockdown resulted in significant inhibition of p-EGFR and p-ERK, but not p-AKT in CRC cell lines (all p<0.05), which suggested that m1A modification exerts its oncogenic effects in CRC by activating EGFR downstream pathway. Conclusion: Our study elucidates for the first time the oncogenic effects of TRMT6 on catalyzing m1A modification through the EGFR/ERK pathway, which could offer a promising therapeutic strategy in CRC. Citation Format: SILEI SUI, CAIMING XU, Ying Qing, Hideo Baba, Ajay Goel. TRMT6-mediated N1-methyladenosine methylation promotes tumorigenesis in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1713.

Abstract 3362: Multiscale EGFR mutated NSCLC tumor heterogeneity knowledge-based model predicts tumor growth under gefitinib: An avenue to in silico clinical trial

Abstract Introduction: Non-small cell lung cancer (NSCLC) response to gefitinib depends on epidermal growth factor receptor (EGFR) mutational status. Response differs according to tumor heterogeneity, notably through clonal selection according to genetic background. We developed an in silico EGFR mutated lung adenocarcinoma (EGFR+ LUAD) model topredict the effect of EGFR mutations on tumor size in advanced LUAD patients using a mechanistic representation of tumor progression, including response to gefitinib. Tumor heterogeneity, age, gender, initial clinical stage, and smoking status are included as covariates. Methods: 5-step in silico model development: (i) Model Building: Biology of EGFR+LUAD was characterized by extracting biological features and their functional relationships from literature and translating them into ordinary differential equations (ODEs). Mutational burden, EGFR downstream-pathways, tumor growth and heterogeneity, gefitinib-PK/PD, treatment-induced resistance and clinical outcome were modeled in a computational simulation with 43 variables, 170 parameters and 18 to 83 ODEs reflecting intra-tumor heterogeneity. (ii) Calibration: Published spheroid, xenograft and clinical data were used for stepwise calibration. (iii) Virtual populations (VPOP): VPOPs were generated for validation and benchmarking respectively, adapting baseline characteristics of a real population.(iv) Validation: A VPOP with comparable baseline characteristics was tested against published patient data.(v) Benchmarking against a Bayesian reference model : (1) coverage of experimental interquartile range (IQR) with simulated IQR (precision) assesses model fit with experimental data, (2) coverage of simulated IQR with experimental IQR (overlap) assesses model fit with experimental variability. Results: Our model computed in silico data comparable to the reference model without use of original data for calibration (Figure 1B.2: experimental vs. simulated, precision of 68%, overlap of 91%). The reference model reported precision of 72% and overlap of 86%. Therefore, the ISELA model has a better percentage of the literature data area contained in the simulated one while the Bayesian model presents a better percentage of the simulated data contained in the literature one. Conclusion: We simulated tumor growth and treatment response in advanced EGFR+ LUAD patients and successfully validated results with published data, and compared it to an already published model with the same context of use. Both models successfully provide a reliable description of longitudinal tumor size when compared to each other or to observed data. Our model provides a benchmark for future in silico clinical trials. Citation Format: Michael Duruisseaux, Adèle L'Hostis, Emmanuel Peyronnet, Evgueni Jacob, Ben M.W. Illigens, Jim Bosley, Riad Kahoul, Jean-Louis Palgen, Claudio Monteiro. Multiscale EGFR mutated NSCLC tumor heterogeneity knowledge-based model predicts tumor growth under gefitinib: An avenue to in silico clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3362.

Molecular characteristics and treatment strategy in advanced, EGFR-mutant non-small cell lung cancer with concomitant BRAF variations.

e21021 Background: BRAF variants were reported resistant mechanisms to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC). However, concomitant somatic variations other than BRAF variants and efficacy of subsequent treatment remained unclear. Methods: From October 2016 to May 2020, advanced NSCLC patients who underwent next-generation sequencing and were detected with co-mutation of BRAF and EGFR activating mutations are retrospectively included. Since June 2020, EGFR-mutant patients with acquired BRAF V600E after progression from previous Osimertinib are prospectively arranged to explore efficacy of the EGFR-BRAF co-inhibition. Results: Fifty-eight patients were retrospectively identified and five patients were prospectively included. BRAF variants was acquired after a median time of 22.7 months from initial diagnosis. Variations of TP53, PIK3CA, RB1, MET, LRP1B, APC, CDKN2A, MYC, ERBB2 and SMAD4 were over 10%, which were enriched in cell cycle/p53 pathway, EGFR downstream and bypass pathway. Subsequently, median progression-free survival was 5.0 months for chemotherapy and 2.1 months for TKI treatment without targeting both EGFR and BRAF respectively (p = 0.019). Osimertinib plus vemurafenib (n = 4) or dabrafenib + trametinib (n = 1) was prospectively administrated in five patients. Median PFS was 7.8 months. Grade 3 rash was observed in one patient. Upon disease progression, activation of RAS signaling was observed. Conclusions: Variations of EGFR downstream or bypass pathway were also frequent in patient with co-mutation of EGFR and BRAF. Efficacy of subsequent TKI without targeting both EGFR and BRAF was inferior to chemotherapy. EGFR-BRAF co-inhibition showed improved efficacy. More treatment strategy should be explored in the future.

Meclofenamic Acid Restores Gefinitib Sensitivity by Downregulating Breast Cancer Resistance Protein and Multidrug Resistance Protein 7 via FTO/m6A-Demethylation/c-Myc in Non-Small Cell Lung Cancer.

Background and ObjectiveGefitinib (GE) is a first-line epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) for patients with advanced non-small cell lung cancer (NSCLC) carrying EGFR activating mutations. However, drug resistance limits the clinical efficacy of gefitinib and ultimately leads to extremely poor clinical benefit. Meclofenamic acid (MA) is a non-steroidal anti-inflammatory drug (NSAID) that relieves moderate and severe pain. In the present study, we aim to determine the MA sensibilization of GE in NSCLC.MethodsMTT assay was conducted to determine the synergistic effect of MA with GE in GE-sensitive and -resistant cell lines based on the Chou–Talalay method. The Annexin V-PI flow cytometry analysis was conducted to evaluate apoptosis. Western blot assay was used to detect alterations of EGFR downstream molecules. Tritium-labeled GE accumulation analysis was used to determine the efflux activity of GE. Dot blot assays were conducted to determine m6A levels after the MA and GE co-administration. Western blot evaluated the expression of FTO, c-Myc, MRP7, BCRP, and apoptotic proteins.ResultsMA showed a significant synergistic effect with GE in GE-resistant NSCLC cells; co-administration of MA with GE induced caspase-related apoptosis in resistant NSCLC cells. Moreover, EGFR downstream molecules, including Akt and MAPKs pathways, were significantly inhibited by the MA-GE combination. Short-term incubation of MA did not alter the efflux of GE; however, after incubation for 24 h, the accumulation of tritium-labeled GE significantly increased. A mechanism study showed that co-administration of MA and GE significantly downregulated BCRP and MRP7 expression in GE-resistant cells; increased N6-methylation was also observed after co-administration. The FTO/c-Myc was determined as target pathways on MA and GE co-administration mechanisms.ConclusionOur findings provide novel therapeutic approaches for GE-resistant NSCLC by combination use with MA through FTO-mediated N6-demethylation.

Epidermal growth factor/epidermal growth factor receptor signaling blockage inhibits tumor cell-derived exosome uptake by oral squamous cell carcinoma through macropinocytosis.

Various cell types secrete exosomes into their surrounding extracellular space, which consequently affect the function and activity of recipient cells. Numerous studies have showed that tumor cell‐derived exosomes play important roles in tumor growth and progression. Although a variety of endocytic pathways are reportedly involved in the cellular uptake of exosomes, detailed mechanisms remain unknown. The present study demonstrated that treatment with recombinant epidermal growth factor (EGF) time‐ and dose‐dependently promoted cellular uptake of oral squamous cell carcinoma (OSCC) cell‐derived exosomes into OSCC cells themselves. Conversely, EGF receptor (EGFR) knockdown and treatment with EGFR inhibitors, including erlotinib and cetuximab, abrogated OSCC cell uptake of exosomes. The macropinocytosis inhibitor 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA) blocked the effects of active EGF/EGFR signaling on uptake of OSCC cell‐derived exosomes. These EGFR inhibitors also suppressed OSCC cell‐derived exosome‐induced proliferation, migration, invasion, stemness, and chemoresistance of OSCC cells. Taken together, the data presented herein suggest that EGFR inhibitors might inhibit the malignant potential of OSCC cells through direct inhibition of not only EGFR downstream signaling pathway but also cellular uptake of OSCC cell‐derived exosomes through macropinocytosis.

P71.05 Use of a Multiscale NSCLC Tumor Heterogeneity Model to Predict Tumor Growth Under Gefitinib

Novel high-throughput techniques, advanced tumor micro-environment analyses and new insights in tumor cell heterogeneity have significantly increased understanding of regulatory processes in Non-small cell lung cancer (NSCLC): NSCLC treatment response to Gefitinib depends on the epidermal growth factor receptor (EGFR), but differs depending on EGFR gene mutations making clinical prognosis challenging. We therefore developed an in silico EGFR+ lung adenocarcinoma (LUAD) model to predict the effect of EGFR-related mutations on tumor size in advanced-stage adenocarcinoma patients (IIIb or higher), using a mechanistic representation of tumor evolution, including response to Gefitinib. Tumor heterogeneity, age, gender, initial clinical stage, and smoking status are included as covariates. 5-step in silico model development: 1. Model Building: Pathophysiology of EGFR+LUAD was characterized by extracting biological features and their functional relationships from literature and translating them into ordinary differential equations (ODEs). Mutational burden, EGFR downstream-pathways, tumor growth and heterogeneity, Gefitinib-PK/PD, treatment-induced resistance and clinical outcome were modeled in a computational simulation with 43 variables, 170 parameters and 18 to 83 ODEs reflecting intra-tumor heterogeneity. 2. Calibration: Published spheroid, xenograft and clinical data were used for stepwise calibration. 3. Virtual populations (VPOP): VPOPs were generated for validation and benchmarking respectively, adapting baseline characteristics of a real population. 4. Validation: A VPOP with comparable baseline characteristics was tested against published patient data[1]. 5. Benchmarking against a Bayesian reference model[2]: (1) coverage of experimental interquartile range (IQR) with simulated IQR (precision) assesses model fit with experimental data, (2) coverage of simulated IQR with experimental IQR (overlap) assesses model fit with experimental variability. Our model computed in silico data comparable to the reference model[2] without use of original data for calibration (Figure 1B.2: experimental vs. simulated, precision of 68%, overlap of 91%). The reference model reported precision of 72% and overlap of 86%. We simulated tumor growth and treatment response in advanced-stage adenocarcinoma patients and successfully validated results with a published study[2]. Access to patient-level data for calibration would have improved precision of our model.

Blocking of EGFR Signaling Is a Latent Strategy for the Improvement of Prognosis of HPV-Induced Cancer.

Human papillomavirus (HPV) is a double-stranded DNA (dsDNA) virus, and its high-risk subtypes increase cancer risks. However, the mechanism of HPV infection and pathogenesis still remain unclear. Therefore, understanding the molecular mechanisms and the pathogenesis of HPV are crucial in the prevention of HPV-related cancers. In this study, we analyzed cervix squamous cell carcinoma (CESC) and head and neck carcinoma (HNSC) combined data to investigate various HPV-induced cancer common features. We showed that epidermal growth factor receptor (EGFR) was downregulated in HPV-positive (HPV+) cancer, and that HPV+ cancer patients exhibited better prognosis than HPV-negative (HPV−) cancer patients. Our study also showed that TP53 mutation rate is lower in HPV+ cancer than in HPV− cancer and that TP53 can be modulated by HPV E7 protein. However, there was no significant difference in the expression of wildtype TP53 in both groups. Subsequently, we constructed HPV-human interaction network and found that EGFR is a critical factor. From the network, we also noticed that EGFR is regulated by HPV E7 protein and hsa-miR-944. Moreover, while phosphorylated EGFR is associated with a worse prognosis, EGFR total express level is not significantly correlated with prognosis. This indicates that EGFR activation will induce a worse outcome in HPV+ cancer patients. Further enrichment analysis showed that EGFR downstream pathway and cancer relative pathway are diversely activated in HPV+ cancer and HPV− cancer. In summary, HPV E7 protein downregulates EGFR that downregulates phosphorylated EGFR and inhibit EGFR-related pathways which in turn and consequently induce better prognosis.

Synergy between vinorelbine and afatinib in the inhibition of non-small cell lung cancer progression by EGFR and p53 signaling pathways

Currently, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) were approved for the treatment of non-small cell lung cancer (NSCLC) patients harboring EGFR mutation. However, some lung cancer patients fail to respond and eventually develop drug resistance. Therefore, new therapeutic strategies are needed to improve the outcomes for substantial clinical benefit. Here we aimed to explore the combination of vinorelbine with the second EGFR-TKI afatinib in NSCLC cells with or without EGFR mutation. The three cells of H1975, HCC827, and H460 were assessed for the combination of vinorelbine and afatinib. Vinorelbine combined with afatinib synergistically inhibited the three lung cancer cells growth without aggravating adverse effect on the normal lung cells. The combination of low doses of vinorelbine and afatinib suppressed the cancer cell proliferation by cell colony formation assay and significantly induced cell apoptosis. The anti-apoptotic proteins Bcl-xL and Bcl-2 showed significant reduction after the drug combination treatment, while the pro-apoptotic protein Bax as well as apoptosis indicators cytochrome C and cleaved PARP were observed a notable increasing. EGFR downstream pathways including AKT, ERK, JNK, and p38 were highly active and p53 was inactive in the three lung cancer cells, favoring tumor growth. The low doses of vinorelbine plus afatinib blocked the phosphorylation of AKT, ERK, JNK, and p38, but restored the expression of p53. Our findings suggested that the combination of vinorelbine and afatinib could be recommended as a therapeutic regimen for treatment of NSCLC with or without EGFR mutation.

Fluctuating role of antimicrobial peptide hCAP18/LL‑37 in oral tongue dysplasia and carcinoma.

Oral tongue squamous cell carcinoma (OTSCC), the most common cancer in the oral cavity, is aggressive and its incidence is increasing globally. Human host defense cationic antimicrobial peptide‑18/antimicrobial peptide leucine‑leucine‑37 (hCAP18/LL‑37) plays a complex role in various types of cancers. In the present study, we characterized the effects of exogenous LL‑37 on three OTSCC cell lines and determined the expression of hCAP18/LL‑37 in oral dysplastic and OTSCC patient samples. Our data revealed that LL‑37, especially in high doses, mostly reduced the proliferation of OTSCC cells, but the effect was fluctuating. However, LL‑37 stimulated the migration and invasion of OTSCC cells. The high dose of LL‑37 also increased the amount of total epidermal growth factor receptor (EGFR) probably due to stabilization of the receptor to the plasma membrane. However, activation of EGFR downstream pathways was mostly decreased. Our immunohistochemical analysis showed that the hCAP18/LL‑37 expression was higher in normal/mild dysplasia than in moderate/severe dysplasia and OTSCC. The hCAP18/LL‑37 expression did not correlate with clinicopathological features or outcome of OTSCC patients. Our data suggest that LL‑37 has a fluctuating effect on proliferation, migration and invasion of OTSCC cells, but it does not seem to play a role in the progression of OTSCC.

Lung adenocarcinoma with sarcomatoid transformation after tyrosine kinase inhibitor treatment and chemotherapy

ObjectivesLung cancers have various acquired resistance mechanisms that lead to treatment failure and disease progression, including secondary epidermal growth factor receptor (EGFR) exon 20 T790 M mutations, EGFR downstream or bypass pathway activation, and histologic transformation from adenocarcinoma to small cell carcinoma, squamous cell carcinoma, or sarcomatoid carcinoma. Materials and MethodsThis study compared the pathological and immunohistochemical characteristics before and after sarcomatoid transformation. Six advanced cases of lung adenocarcinoma that developed sarcomatoid transformation after treatment were collected. ResultsFive cases had classic EGFR mutations and one had a ROS1 rearrangement. The interval from initial diagnosis to sarcomatoid transformation ranged from 9 to 88 mo (median of 31.5 mo). The median survival after sarcomatoid transformation was 2.5 mo (1–16 mo). Before sarcomatoid transformation, all cases demonstrated typical adenocarcinoma features, including acinar, micropapillary, or solid/cribriform patterns, negative or weak focal vimentin staining, and strong E-cadherin expression. Histologic features of sarcomatoid transformation included giant cell features (6/6), loose cellular cohesion (6/6), strong staining for vimentin (6/6), decreased or lost E-cadherin expression (5/6), and high PD-L1 expression (5/6; one case demonstrated high PD-L1 staining at initial diagnosis). High MET expression and MET copy number gain (two samples with high polysomy and three with true amplification) were observed in five cases with EGFR mutation treated with tyrosine kinase inhibitors (TKI). One case exhibited MET amplification prior to the start of TKI treatment. ConclusionSarcomatoid transformation is a type of lung cancer histologic evolution with a poor prognosis and a high proportion of cases with aberrant MET activation and PD-L1 expression.

Quinalizarin, a specific CK2 inhibitor, can reduce icotinib resistance in human lung adenocarcinoma cell lines.

The abnormal activation of the downstream signaling pathways of epidermal growth factor receptor (EGFR) that are independent of EGFR, contribute to the acquisition of EGFR-tyrosine kinase inhibitor (TKI) resistance in non-small cell lung cancer (NSCLC). The serine/threonine protein kinase casein kinase II (CK2) phosphorylates and modulates several members of the EGFR downstream signaling pathways. Thus, the purpose of the current study was to investigate the effects of the addition of quinalizarin (a specific CK2 inhibitor) to icotinib (an EGFR-TKI) on the proliferation and apoptosis of four NSCLC cell lines and its underlying mechanisms. The human lung adenocarcinoma cell lines HCC827, A549, H1650 and H1975 were employed to represent the EGFR-TKI-sensitive EGFR (EGFR-sensitive) mutation, wild-type EGFR and the EGFR-TKI-resistant EGFR (EGFR-resistant) mutations. The cell viability was determined by the MTT assay. Cell apoptosis was detected by flow cytom-etry using the Annexin V-enhanced green fluorescent protein Apoptosis Detection kit. The level of proteins in the EGFR downstream pathway was observed using a western blot assay. The results showed that the cells with the EGFR-sensitive mutation (HCC827, EGFR E716-A750del) were more sensitive to icotinib compared with those possessing the EGFR wild-type (A549) and the EGFR-resistant mutations (H1650, EGFR E716-A750del and PTEN lost; H1975, EGFR L858R+T790M). Quinalizarin inhibited proliferation and promoted apoptosis in the cells with the EGFR wild-type and resistant mutations, and the addition of quinalizarin to icotinib partially restored their sensitivity to icotinib. Quinalizarin and/or icotinib increased the apoptotic rates in the EGFR-TKI resistant cells, and the combination of these reduced the level of protein downstream of EGFR, including phosphorylated (p-AKT) and p-(ERK). In conclusion, quinalizarin may partially sensitize cells to icotinib by inhibiting proliferation and promoting apoptosis mediated by AKT and ERK in EGFR-TKI resistant NSCLC cell lines.

Significance of Phosphorylated Epidermal Growth Factor Receptor and Its Signal Transducers in Human Soft Tissue Sarcoma.

Previous studies have shown that total epidermal growth factor receptor (EGFR) protein is highly expressed in soft tissue sarcoma (STS). We aimed to investigate the significance of phosphorylated-EGFR (pEGFR) and its activated-downstream signal transducers in STS tissue samples. A tissue microarray comprising 87 STS samples was assessed for total EGFR, pEGFR and its phosphorylated signal transducers and expression was correlated with clinicopathlogical parameters including patient outcome. Although the expression of total EGFR was significantly associated with adverse STS histologic grade (p = 0.004) and clinical stage (p = 0.012) similar to pEGFR, phosphorylated protein kinase B (pAkt) and phosphorylated extracellular signal regulated kinase (pERK), it is not a prognostic factor for survival. By contrast, the expression of pEGFR is an independent factor for cancer specific survival, while pERK is an independent prognostic factor for both overall and cancer specific survival in STS (p < 0.05, Cox proportional hazard model and log-rank test) in addition to the recognised factors of tumour grade and clinical stage. pERK and pEGFR are new independent prognostic factors for overall and/or cancer specific survival in STS. The expression of EGFR/pEGFR, and their associated downstream signal transducers, was associated with STS progression, suggesting that EGFR downstream signalling pathways may jointly support STS cell survival.

Inhibiting epidermal growth factor receptor signalling potentiates mesenchymal-epithelial transition of breast cancer stem cells and their responsiveness to anticancer drugs.

The recurrence of breast cancer in patients is a persistent challenge to the medical fraternity. Breast tumor contains a small population of cells with high tumor initiating and metastatic potential, known as cancer stem cells (CSCs), which are resistant to existing chemotherapeutics. CSCs contribute to the aggressiveness of triple negative breast cancers (TNBCs), thereby necessitating the identification of molecular targets on breast CSCs. TNBC cell line MDA-MB-231, in comparison with MCF-7, demonstrated a higher expression of epidermal growth factor receptor (EGFR). Thus, the naturally occurring flavanone, chrysin, with limited potential as a chemotherapeutic agent, was structurally modified by designing an analog with EGFR binding affinity using a molecular docking approach and subsequently synthesised. Chrysin analog CHM-09 and known EGFR inhibitors demonstrated a comparable anti-proliferative, anti-migratory activity along with the induction of apoptosis and cell cycle arrest in MDA-MB-231. Furthermore, sorted CD24- /CD44+ -breast CSCs and CD24+ -breast cancer cells from MDA-MB-231 demonstrated a markedly high expression of EGFR in the former than in the latter. CHM-09 and EGFR inhibitors could perturb EGF-induced EGFR signalling of breast CSC proliferation, migration, mammosphere formation and mesenchymal tri-lineage differentiation. CHM-09 or EGFR inhibitors not only led to inactivation of EGFR downstream signalling pathways such as Akt, extracellular signal regulated kinase and signal transducer and activator of transcription 3, but also induction of mesenchymal-epithelial transition as confirmed by decreased N-cadherin and increased E-cadherin expression. Finally, combinatorial treatment of EGFR inhibitors and doxorubicin led to significant increase in breast CSCs responsiveness to a chemotherapeutic drug. The results of the present study suggest that EGFR is a therapeutic target in breast CSCs and that abrogation of EGFR signalling along with chemotherapeutic drugs is an effective approach against breast cancer.

Abstract A90: Mesothelin confers tumor cell vulnerabilities for gefitinib treatment

Abstract Combination therapy for pancreatic adenocarcinoma (PDAC) has recently had two successful stage III trials with FOLFIRINOX (combination folinic acid, fluorouracil, irinotecan, and oxalplatin) and combination gemcitabine and paclitaxel. Because of the heterogeneity of PDAC, the benefits of combination therapy are immediately apparent since it targets multiple facets of cancer simultaneously. However, FOLFIRINOX and gemcitabine + paclitaxel are highly toxic and affect all dividing cells, making apparent the need for better understanding of the cell profiles in PDAC. The epidermal growth factor receptor (EGFR) is involved in the pathogenesis and progression of many cancers, with EGFR or its ligands often overexpressed or mutated. Recently we have found that EGFR expression is correlated to mesothelin (MSLN) expression in PDAC. MSLN, a cell surface glycoprotein, has been shown by us and other labs to be overexpressed in 80 to 90% of all PDAC cases. To examine the relationship between MSLN and EGFR, we modulated MSLN expression by generating a MSLN overexpressing cell line from MIA-PaCa2 cells, and a MSLN knockdown cell line from CAPAN-2 cells. The change in EGFR signaling mimicked that of MSLN, with MSLN overexpression significantly increasing EGFR expression and MSLN knockdown significantly decreasing EGFR expression. Furthermore, high MSLN (and therefore high EGFR expression) showed increased sensitivity to gefitinib, an EGFR ATP binding site inhibitor, at concentrations as low as 100 nM, while MSLN low expressing cells did not exhibit any effect from gefitinib at concentrations below 10 µM. Cell Cycle analysis demonstrated that gefitinib increased the number of cells in G1 phase after 24 hours in MSLN high expressing cells, but had no effect on MSLN low expressing cells. In addition, western blot analysis of EGFR downstream pathways showed an increase in phosphorylated phospholipase C-Υ (PLC-Υ) in MSLN high expressing cells, which could be mitigated by treatment with gefitinib. Finally, combination therapy with gemcitabine and 100 nM gefitinib showed an additive effect in MSLN high expressing cells, while in MSLN low expressing cells combination gemcitabine and gefitinib had no additional effect over gemcitabine alone. This study demonstrates the correlation between MSLN expression and EGFR expression and indicates that gefitinib could be a potential addition to combination therapy for PDAC in patients with high MSLN expression. Citation Format: Ethan Poteet, Zhengdong Liang, Youwen Wang, Changyi Chen, Qizhi Yao.{Authors}. Mesothelin confers tumor cell vulnerabilities for gefitinib treatment. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A90.