Epidermal Growth Factor Receptor Signaling Research Articles

Brain Metastasis from EGFR-Mutated Non-Small Cell Lung Cancer: Secretion of IL11 from Astrocytes Up-Regulates PDL1 and Promotes Immune Escape.

Patients who have non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations are more prone to brain metastasis (BM) and poor prognosis. Previous studies showed that the tumor microenvironment of BM in these patients is immunosuppressed, as indicated by reduced T-cell abundance and activity, although the mechanism of this immunosuppression requires further study. This study shows that reactive astrocytes play a critical role in promoting the immune escape of BM from EGFR-mutated NSCLC by increasing the apoptosis of CD8+ T lymphocytes. The increased secretion of interleukin 11(IL11) by astrocytes promotes the expression of PDL1 in BM, and this is responsible for the increased apoptosis of T lymphocytes. IL11 functions as a ligand of EGFR, and this binding activates EGFR and downstream signaling to increase the expression of PDL1, culminating in the immune escape of tumor cells. IL11 also promotes immune escape by binding to its intrinsic receptor (IL11Rα/glycoprotein 130 [gp130]). Additional in vivo studies show that the targeted inhibition of gp130 and EGFR suppresses the growth of BM and prolongs the survival time of mice. These results suggest a novel therapeutic strategy for treatment of NSCLC patients with EGFR mutations.

The involvement of epidermal growth factor receptor/protein kinase Bsignaling in the tumor intrinsic PD-L1-induced malignant potential of oral squamous cell carcinoma.

Various antigen-presenting cells and tumor cells-expressing PD-L1 inhibits antitumor immune responses in the tumor microenvironment. Recently, numerous studies have shown that tumor cell intrinsic PD-L1 also plays important roles in tumor growth and progression. On the other hand, oral squamous cell carcinoma (OSCC) cells overexpress epidermal growth factor receptor (EGFR) and EGFR signal pathway exacerbates tumor progression. Therefore, this study assessed whether tumor-intrinsic PD-L1 facilitates malignant potential of OSCC cells through regulation of EGFR signaling. Two OSCC cell lines, SAS and HSC-3, were transfected with PD-L1 and EGFR-specific small interfering RNA (siRNA). Influences of PD-L1 knockdown on malignant potentials of OSCC cells were examined by Cell Counting kit-8 assay, transwell assay, sphere formation assay, flow cytometry, and Western blot. Effects of PD-L1 and EGFR knockdown on each expression were examined by quantitative real-time PCR (qRT-PCR), Western blot, and flow cytometry. Transfection of an PD-L1-siRNA into OSCC cells decreased the abilities of proliferation, stemness, and mobility of these cells significantly. PD-L1 knockdown also decreased EGFR expression through the promotion of proteasome- and lysosome-mediated degradation and following activation of the EGFR/protekin kinase B (AKT)signal pathway. Meanwhile, EGFR knockdown did not influence PD-L1 expression in SAS and HSC-3 cells, but treatment with a recombinant human EGF induced its expression. Treatment with erlotinib and cetuximab suppressed rhEGF-induced PD-L1 expression and localization in the cellular membrane of both OSCC cells. OSCC cells-expressing PD-L1 induced by EGF stimulation may promote malignancy intrinsically via the activation of the EGFR/AKT signaling cascade.

New Directions for Advanced Targeting Strategies of EGFR Signaling in Cancer.

Epidermal growth factor (EGF)-EGF receptor (EGFR) signaling studies paved the way for a basic understanding of growth factor and oncogene signaling pathways and the development of tyrosine kinase inhibitors (TKIs). Due to resistance mutations and the activation of alternative pathways when cancer cells escape TKIs, highly diverse cell populations form in recurrent tumors through mechanisms that have not yet been fully elucidated. In this review, we summarize recent advances in EGFR basic research on signaling networks and intracellular trafficking that may clarify the novel mechanisms of inhibitor resistance, discuss recent clinical developments in EGFR-targeted cancer therapy, and offer novel strategies for cancer drug development.

Structural and molecular insights from dual inhibitors of EGFR and VEGFR2 as a strategy to improve the efficacy of cancer therapy.

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor 2 (VEGFR2) are known as valid targets for cancer therapy. Overexpression of EGFR induces uncontrolled cell proliferation and VEGF expression triggering angiogenesis via VEGFR2 signaling. On the other hand, VEGF expression independent of EGFR signaling is already known as one of the mechanisms of resistance to anti-EGFR therapy. Therefore, drugs that act as dual inhibitors of EGFR and VEGFR2 can be a solution to the problem of drug resistance and increase the effectiveness of therapy. In this review, we summarize the relationship between EGFR and VEGFR2 signal transduction in promoting cancer growth and how their kinase domain structures can affect the selectivity of an inhibitor as the basis for designing dual inhibitors. In addition, several recent studies on the development of dual EGFR and VEGFR2 inhibitors involving docking simulations were highlighted in this paper to provide some references such as pharmacophore features of inhibitors and key residues for further research, especially in computer-aided drug design.

The Impact of Inadequate Exposure to Epidermal Growth Factor Receptor–Tyrosine Kinase Inhibitors on the Development of Resistance in Non-Small-Cell Lung Cancer Cells

Epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC) patients treated with EGFR–tyrosine kinase inhibitors (TKIs) inevitably develop resistance through several biological mechanisms. However, little is known on the molecular mechanisms underlying acquired resistance to suboptimal EGFR-TKI doses, due to pharmacodynamics leading to inadequate drug exposure. To evaluate the effects of suboptimal EGFR-TKI exposure on resistance in NSCLC, we obtained HCC827 and PC9 cell lines resistant to suboptimal fixed and intermittent doses of gefitinib and compared them to cells exposed to higher doses of the drug. We analyzed the differences in terms of EGFR signaling activation and the expression of epithelial–mesenchymal transition (EMT) markers, whole transcriptomes byRNA sequencing, and cell motility. We observed that the exposure to low doses of gefitinib more frequently induced a partial EMT associated with an induced migratory ability, and an enhanced transcription of cancer stem cell markers, particularly in the HCC827 gefitinib-resistant cells. Finally, the HCC827 gefitinib-resistant cells showed increased secretion of the EMT inducer transforming growth factor (TGF)-β1, whose inhibition was able to partially restore gefitinib sensitivity. These data provide evidence that different levels of exposure to EGFR-TKIs in tumor masses might promote different mechanisms of acquired resistance.

“Qi Nan” agarwood restores podocyte autophagy in diabetic kidney disease by targeting EGFR signaling pathway

BackgroundDiabetic kidney disease (DKD) is a microvascular complication of diabetes mellitus, contributing to end-stage renal disease with limited treatment options. The development of DKD is attributed to podocyte injury resulting from abnormal podocyte autophagy. Consequently, the restoration of podocyte autophagy is deemed a practicable approach in the treatment of DKD.MethodsDiabetic mice were induced by streptozotocin and high-fat diet feeding. Following 8 weeks of “QN” agarwood treatment, metrics such as albuminuria, serum creatinine (Scr), and blood urea nitrogen (BUN) were evaluated. Renal histological lesions were evaluated by H&E, PAS, Masson, and Sirius red staining. Evaluation of the effects of “QN” agarwood on renal inflammation and fibrosis in DKD mice through WB, q-PCR, and IHC staining analysis. Cytoscape 3.7.1 was used to construct a PPI network. With the DAVID server, the gene ontology (GO) functional annotation and the Kyoto encyclopedia of genes and genomes (KEGG) signaling pathways of the target enrichment were performed. Molecular docking and binding affinity calculations were conducted using AutoDock, while PyMOL software was employed for visualizing the docking results of active compounds and protein targets.ResultsThe results of this study show that “QN” agarwood reduced albuminuria, Scr, and BUN in DKD mice, and improved the renal pathological process. Additionally, “QN” agarwood was observed to downregulate the mRNA and protein expression levels of pro-inflammatory and pro-fibrotic factors in the kidneys of DKD mice. Network pharmacology predicts that “QN” agarwood modulates the epidermal growth factor receptor (EGFR) signaling pathway. “QN” agarwood can increase the expression of LC3B and Nphs1 in DKD mice while reducing the expression of EGFR.ConclusionThe present study demonstrated that “QN” agarwood ameliorated renal injury in DKD by targeting EGFR and restoring podocyte autophagy.Graphical

Decorin suppresses stemness and migration potential of malignant peripheral nerve sheath tumor through inhibiting epidermal growth factor receptor signaling

Cancer stem cells (CSCs) play pivotal roles in the growth, invasion, metastasis, chemo-resistance in malignant peripheral nerve sheath tumor (MPNST). The current characterization of CSCs in MPNST is not complete. Decorin is a critical regulator of microenvironment, but its expression and function in CSCs of MPNST has not been studied. In the current study, Decorin levels and its relationship with lung and liver metastasis were determined in clinical specimens. Decorin expression in CD133-positive or CD44-positive CSCs was analyzed by RT-qPCR on cytospun MPNST cells after flow cytometry-based cell sorting. Decorin-positive cells were separated from Decorin-negative cells in transfected MPNST cell lines using a designed plasmid expressing red fluorescent protein (RFP) under a Decorin promoter. Tumor sphere formation, tumor growth, cell invasion, cell migration, and the resistance to chemotherapy-induced apoptosis were determined on Decorin-positive versus Decorin-negative MPNST cells. In vivo tumor growth was analyzed in mice receiving subcutaneous transplantation of Decorin-positive versus Decorin-negative MPNSTs. We found that Decorin levels were significantly downregulated in MPNST specimens, compared to non-tumorous adjacent tissue. Significantly lower Decorin levels were detected in MPNSTs with lung or liver metastasis compared to those without. Poorer patient survival was detected in Decorin-low MPNST, compared to Decorin-high subjects. More Decorin-negative cells were detected in CD133-positive MPNST cells than CD133-negative MPNST cells, and in CD44-positive MPNST cells than in CD44-negative MPNST cells. Compared to Decorin-positive MPNST cells, Decorin-negative MPNST cells generated significantly more tumor spheres in culture, were more invasive and migratory, and were more resistant to chemotherapy-induced apoptosis, likely due to the inhibition of epidermal growth factor receptor signaling by Decorin. Decorin-negative MPNST cells grew significantly larger tumor in vivo. Thus, depletion of Decorin may occur in CSCs in MPNSTs, serving possibly as a new therapeutic target.

Role of Epiregulin on Lipopolysaccharide-Induced Hepatocarcinogenesis as a Mediator via EGFR Signaling in the Cancer Microenvironment.

Lipopolysaccharides (LPSs) have been reported to be important factors in promoting the progression of hepatocellular carcinoma (HCC), but the corresponding molecular mechanisms remain to be elucidated. We hypothesize that epiregulin (EREG), an epidermal growth factor (EGF) family member derived from hepatic stellate cells (HSCs) and activated by LPS stimulation, is a crucial mediator of HCC progression with epidermal growth factor receptor (EGFR) expression in the tumor microenvironment. We used a mouse xenograft model of Huh7 cells mixed with half the number of LX-2 cells, with/without intraperitoneal LPS injection, to elucidate the role of EREG in LPS-induced HCC. In the mouse model, LPS administration significantly enlarged the size of xenografted tumors and elevated the expression of EREG in tumor tissues compared with those in negative controls. Moreover, CD34 immunostaining and the gene expressions of angiogenic markers by a reverse transcription polymerase chain reaction revealed higher vascularization, with increased interleukin-8 (IL-8) expression in the tumors of the mice group treated with LPS compared to those without LPS. Our data collectively suggested that EREG plays an important role in the cancer microenvironment under the influence of LPS to increase not only the tumor cell growth and migration/invasion of EGFR-positive HCC cells but also tumor neovascularization via IL-8 signaling.

Abstract LB056: IBI334, a novel ADCC-enhanced B7-H3/EGFR bispecific antibody, demonstrated potent pre-clinical efficacy in solid tumors

Abstract Many solid tumors, such as colorectal cancer (CRC) and lung cancer, are dependent on epidermal growth factor receptor (EGFR) pathway for tumor growth. Antibodies that inhibit EGFR signaling, such as Cetuximab, show clinical efficacy but are associated with on-target toxicities that potentially limit the drug efficacy. To enhance the EGFR signaling inhibition and reduce on-target toxicities, we performed bioinformatics analysis for EGFR/TAA co-expression. We found that B7-H3, a member of the B7 family of immune checkpoint proteins, is broadly expressed in many solid tumors and their expression correlates with EGFR expression in multiple cancers, including lung cancer, head and neck cancer, pancreatic cancer and esophageal cancer. Using Innovent’s proprietary Innobody platform, we developed a bispecific antibody, IBI334, against B7-H3 and EGFR. IBI334 has an EGFR arm for signal blockage and is coupled with a fine-tuned B7-H3 arm with optimal affinity and binding domain. With the aid of B7-H3, IBI334 showed enhanced EGFR signal inhibition than Zalutumumab (EGFR mAb) and Amivantamab (c-met/EGFR bsAb). In addition, IBI334 is afucosylated to enhance its antibody-mediated cell cytotoxicity (ADCC) effects as compared to wild type antibody. IBI334 showed significantly better in vivo efficacy than Amivantamab in NCI-H358 (bronchioalveolar carcinoma with KRAS-G12C mutation), NCI-H292 (lymph node metastasis of a pulmonary mucoepidermoid carcinoma), SK-MES1 (lung cancer) and SK-MES1-LTC (lung cancer with EGFR L858R, T790M and C797S mutations) xenograft models and comparable efficacy in NCI-H322-EGFRexon20ins (lung cancer) model. Cetuximab, an EGFR monoclonal antibody, had high on-target toxicity with 50% early mortalities in cynomolgus monkeys at 120/75 mg/kg/week. With the careful design of B7-H3/EGFR bispecific antibody, IBI334 ameliorated the on-target toxicities and showed only slight hardening of the skin at the injection site at 120 mg/kg/week. As a result, IBI334 has a large therapeutic window of >200 folds. In conclusion, IBI334 is a potent B7-H3/EGFR bispecific antibody with broad application in many EGFR-driven solid tumors. The large therapeutic window enables safe and effective cancer treatment at high exposure levels. Citation Format: Jian Guan, Shuaixiang Zhou, Weiwei Wu, Tianyu Zhu, Lei Cao, Ming Wu, Ninghuan Li, Fenggen Fu, Zhengguan Liao, Shuming Lin, Zeyu Liu, Mengjia Zhu, Nana Luo, Ying Zhang, Tiong Sun Chia, Bingliang Chen, Kaijie He. IBI334, a novel ADCC-enhanced B7-H3/EGFR bispecific antibody, demonstrated potent pre-clinical efficacy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB056.

Abstract LB301: BRAFV600E drives WNT signalling in colorectal cancer via aberrant DNA methylation

Abstract Objective: The BRAF V600E mutation drives the generation of an aggressive subtype of colorectal cancer (CRC). Although activation of WNT signalling is a generic hallmark of CRC, mutations in APC are uncommon in BRAF V600E-mutant CRC. Instead, mutations in RNF43 are the suspected WNT signalling driving event in these tumours. Here, we aimed to uncover how the WNT pathway is activated in BRAF V600E-mutant colon cancer. Design: CRISPR-LbCpf1-corrected BRAF (V600E) and RNF43 (P441fs) organoids were analysed for their growth potential in the presence or absence of growth factors, for their ability to form tumours, and by immunoblotting, RNA sequencing, and DNA methylation assays. Results: BRAF E600V organoids regained dependency on epidermal growth factor (EGF) receptor signalling for ERK pathway activation and proliferation, and completely lost tumorigenic potential. Correction of BRAF V600E, but not RNF43 P441fs, caused suppression of WNT target genes, while genes reflecting epithelial differentiation and negative regulators of WNT signalling were upregulated in BRAF E600V organoids. DNA methylation analysis revealed promoter hypermethylation of WNT antagonist genes in BRAF V600E organoids. Gene body hypermethylation, associated with transcriptional upregulation, was observed in key WNT-driven intestinal stem cell marker genes LGR5 and EPHB2, and in the WNT-effector TCF4. Treatment of BRAF V600E organoids with the DNA demethylating agent 5-aza-2′-deoxycytidine resulted in upregulation of WNT antagonist genes and a marked decrease in WNT target gene expression. Conclusions: BRAFV600E, rather than mutant RNF43, is a prominent force behind WNT pathway activation, and is essential for maintaining tumorigenic capacity. BRAFV600E causes widespread dysregulation of DNA methylation leading to activation of WNT signalling. Citation Format: Layla El Bouazzaoui, Jeroen M. Bugter, Emre Küçükköse, André Verheem, Jasmin B. Post, Nicola Fenderico, Inne H. Borel Rinkes, Hugo J. Snippert, Madelon M. Maurice, Onno Kranenburg. BRAFV600E drives WNT signalling in colorectal cancer via aberrant DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB301.

Phosphoproteomic investigation of targets of protein phosphatases in EGFR signaling.

Receptor tyrosine kinases (RTKs) initiate cellular signaling pathways, which are regulated through a delicate balance of phosphorylation and dephosphorylation events. While many studies of RTKs have focused on downstream-activated kinases catalyzing the site-specific phosphorylation, few studies have focused on the phosphatases carrying out the dephosphorylation. In this study, we analyzed six protein phosphatase networks using chemical inhibitors in context of epidermal growth factor receptor (EGFR) signaling by mass spectrometry-based phosphoproteomics. Specifically, we focused on protein phosphatase 2C (PP2C), involved in attenuating p38-dependent signaling pathways in various cellular responses, and confirmed its effect in regulating p38 activity in EGFR signaling. Furthermore, utilizing a p38 inhibitor, we classified phosphosites whose phosphorylation status depends on PP2C inhibition into p38-dependent and p38-independent sites. This study provides a large-scale dataset of phosphatase-regulation of EGF-responsive phosphorylation sites, which serves as a useful resource to deepen our understanding of EGFR signaling.

EGFR Inhibition by Erlotinib Rescues Desmosome Ultrastructure and Keratin Anchorage and Protects Against Pemphigus Vulgaris IgG-Induced Acantholysis in Human Epidermis.

Pemphigus is a severe blistering disease caused by autoantibodies primarily against the desmosomal cadherins desmoglein (DSG)1 and DSG3 which impair desmosome integrity. Especially for the acute phase, additional treatment options allowing to reduce corticosteroids would fulfill an unmet medical need. Here, we provide evidence that epidermal growth factor receptor (EGFR) inhibition by erlotinib ameliorates pemphigus vulgaris immunoglobulin G (PV-IgG) -induced acantholysis in intact human epidermis. PV-IgG caused phosphorylation of EGFR (Y845) and SRC in human epidermis. In line with that, a phosphotyrosine kinome analysis revealed a robust response associated with EGFR and SRC family kinase signaling in response to PV-IgG but not pemphigus foliaceus autoantibodies. Erlotinib inhibited PV-IgG-induced epidermal blistering and EGFR phosphorylation, loss of desmosomes as well as ultrastructural alterations of desmosome size, plaque symmetry, keratin filament insertion and restored the desmosome midline considered as hallmark of mature desmosomes. Erlotinib enhanced both single molecule DSG3 binding frequency and strength and delayed DSG3 fluorescence recovery supporting that EGFR inhibition increases DSG3 availability and cytoskeletal anchorage. Our data indicate that EGFR is a promising target for pemphigus therapy due to its link to several signaling pathways known to be involved in pemphigus pathogenesis.

Chemical Scaffolds for the Clinical Development of Mutant-Selective and Reversible Fourth-Generation EGFR-TKIs in NSCLC.

In nonsmall cell lung cancer (NSCLC), as well as in other tumors, the targeted therapy is mainly represented by tyrosine kinase inhibitors (TKIs), small molecules able to target oncogenic driver alterations affecting the gene encoding the epidermal growth factor receptor (EGFR). Up to now, several different TKIs have been developed. However, cancer cells showed an incredible adaptive tumor response to the inhibition of the sequentially mutated EGFR (EGFRM+), triggering the need to explore novel pharmacochemical strategies. This Review summarizes the recent efforts in the development of new reversible next-generation EGFR TKIs to fight the resistance against T790M and C797S mutations. Specifically, after giving an overview of the role of the EGFR's signaling pathways in cancer progression, we are going to discuss the most relevant approved drugs and drug candidates in terms of chemical structure, binding modalities, and their potency and selectivity against the mutated EGFR over the wild-type form. This could provide important guidelines and rationale for the discovery and iterative development of new drugs.

The impact of nitric oxide on HER family post-translational modification and downstream signaling in cancer.

The human epidermal growth factor receptor (HER) family consists of four members, activated by two families of ligands. They are known for mediating cell-cell interactions in organogenesis, and their deregulation has been associated with various cancers, including breast and esophageal cancers. In particular, aberrant epidermal growth factor receptor (EGFR) and HER2 signaling drive disease progression and result in poorer patient outcomes. Nitric oxide (NO) has been proposed as an alternative activator of the HER family and may play a role in this aberrant activation due to its ability to induce s-nitrosation and phosphorylation of the EGFR. This review discusses the potential impact of NO on HER family activation and downstream signaling, along with its role in the efficacy of therapeutics targeting the family.

Abstract 516: The role of clathrin-mediated endocytosis of EGFR in glioblastoma cell viability and cell cycle progression

Abstract Glioblastoma (GBM) is characterized by poor prognosis and overexpression of epidermal growth factor receptor (EGFR). EGFR signaling is regulated through endocytosis, including clathrin-mediated endocytosis. We hypothesized that clathrin-mediated EGFR endocytosis promotes GBM cell viability and proliferation. Analysis of patient data from the Gliovis database revealed decreased overall survival in GBM patients with above-median clathrin expression (n=262) compared to below-median expression (n=263, p<0.05). Immunocytochemistry and Western blot analysis confirmed increased clathrin levels in GBM patient samples compared to normal brain tissue (n=15, p<0.01). To determine the functional effect of inhibiting clathrin-mediated endocytosis, U87 and U251 GBM cells were treated with 15 μM Pitstop-2, a clathrin inhibitor, for 24 hours. Pitstop-2 significantly reduced clathrin levels and cell viability by 40% compared to control (p<0.001, n=3). Cell cycle analysis by flow cytometry revealed Pitstop-2 treatment led to G2/M arrest in both cell lines versus control (p<0.01, n=3). Together, these findings indicate clathrin-mediated endocytosis of EGFR may promote GBM cell viability and proliferation. Further studies on the effects of clathrin inhibition on EGFR signaling and tumor growth in vivo will elucidate the therapeutic potential of targeting this pathway. Citation Format: Aaron Deter, Andrew J. Tsung, Maheedhara R. Guda, Swapna Asuthkar, Kiran Velpula. The role of clathrin-mediated endocytosis of EGFR in glioblastoma cell viability and cell cycle progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 516.

Abstract 3003: Exon skipping for the tumor suppressor Annexin A7 promotes EGFR signaling in stem-like brain tumor initiating cells through receptor recycling

Abstract Background: Glioblastoma (GBM) is one of the most lethal forms of cancer. The majority of GBMs express aberrant receptor tyrosine kinase (RTK) activity, which upon activation, are canonically endocytosed and degraded. This process is disrupted in GBM to promote receptor recycling resulting in hyperactive RTK signaling. We demonstrated that the alternative splicing of the tumor suppressor Annexin A7 (ANXA7) supports the impaired endosomal trafficking of RTKs to favor recycling in GBM. Exon six of ANXA7 can be alternatively spliced to yield two distinct protein isoforms through its inclusion (I1) or exclusion (I2). Methods: We investigated ANXA7 isoform differences for epidermal growth factor receptor (EGFR) trafficking fates first in U251 malignant glioma followed by stem-like brain tumor initiating cells (BTICs) originating from patient-derived xenografts (PDXs). U251s and primary BTICs (JX14) were transduced with lentivirus to overexpress ANXA7 and GFP for enrichment by flow cytometry. JX14 empty vector (EV; I2 only) and I1 (I2 and I1 expressing) cells were plated onto Geltrex and ligand starved overnight in serum-free media to maintain BTICs. We assessed EGFR protein levels following EGF stimulation in a time dependent manner. Immunofluorescence experiments were conducted to measure EGFR-ANXA7 colocalization (Pearson) with markers of the endocytic pathway, which included the early endosomes (EEA1), recycling endosomes (Rab4/Rab11), and lysosomes (LAMP1). EV and I1 cells were also incubated with siRNA targeting both ANXA7 isoforms to determine the impact of ANXA7 loss on endosomal EGFR trafficking. Results: Interestingly, we found drastic differences in endosomal EGFR trafficking in I1 expressing cells. EGFR colocalized with EEA1 in both EV and I1 cells (R2=0.01336, P>0.6275). In EV cells, EGFR was recycled via fast/slow recycling endosomes demonstrated by Rab4/Rab11 colocalization while I1 did not (Rab11: R2=0.9267, P<0.0001). Immunoblotting revealed a significant reduction in EGFR protein in I1 cells only. Furthermore, I1 cells and not EV preferentially degraded EGFR as seen by LAMP1 colocalization (R2=0.9398, P<0.0001). ANXA7 siRNA in EV cells did not alter EGFR trafficking, which implicates I2 as nonfunctional. On the other hand, ANXA7 KD in I1 cells impaired EGFR sorting via a 40% increase (P<0.0010) in total EGFR protein and a 40% reduction (P<0.0001) in colocalization with EEA1. Loss of I1 also shifted the degradation of EGFR back towards recycling. Conclusion: GBMs express I2, which supports EGFR hyperactivity by favoring receptor recycling. I1 overexpression significantly reduced EGFR signaling by promoting degradation. I1-induced reduction in EGFR signaling suggests a new therapeutic vulnerability for BTICs by potentially reducing tumorigenicity or improving the efficacy of EGFR inhibition. Citation Format: Aran Merati, Sindhu Nair, Rajani Rajbhandari, Nicholas Johnson, Markus Bredel. Exon skipping for the tumor suppressor Annexin A7 promotes EGFR signaling in stem-like brain tumor initiating cells through receptor recycling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3003.

Abstract 2998: Regulation of epidermal growth factor receptor signaling by BRCA1-microRNA 146a network

Abstract Inherited defects in the breast cancer susceptibility gene, BRCA1 are associated with an increased risk for hereditary breast, ovarian, and other cancers. BRCA1-associated basal like breast cancers (BLBCs) are high-grade ductal carcinomas that do not express ER, PR or HER2, but frequently overexpress epidermal growth factor receptor (EGFR/ErbB1/HER1). The EGFR-driven signaling network plays a pivotal role in BLBC pathogenesis. EGFR overexpression is associated with tumor progression and metastasis, resistance to radiation and chemotherapy, and poor prognosis. Growth factor receptors are regulated by multiple miRNAs, each having many targets, coordinately controlling the complex pathways of downstream signaling. We have previously reported a post-transcriptional mechanism by which BRCA1 regulates EGFR expression through the induction of miR-146a and provided a rationale for the development of miR-146a based therapeutic strategies. In addition, we have shown that BRCA1 also exerts regulatory control over dozens of important miRNAs that appear to play a critical role in breast neoplasia. From TCGA analysis, we show that low expression of these miRNAs is associated with distinctively poor overall survival of TNBC patients. Using miR-146a loss or gain of function experiments in vitro, we show that BRCA1 and miR-146a regulate EGFR signaling, stemness, epithelial-to-mesenchymal transition (EMT), extracellular matrix (ECM) remodeling and chemoresistance. Using in vivo mouse models, we demonstrate that miR-146a overexpression delays tumor formation leading to better overall survival. BLBC unlike the other subtypes, lacks targeted therapy approaches making cytotoxic chemotherapy essentially the only option for treatment. Though EGFR inhibitors have been used to treat other cancers, clinical trials for breast cancer have so far been unsuccessful due to poor response rates. Since one miRNA can target multiple genes and regulate multiple signaling pathways, our study provides evidence to suggest that restoring miR-146a could suppress EGFR-RAS-MEK signaling and the other compensatory pathways, providing a targeted therapeutic option for BRCA1 associated BLBC. Citation Format: Easwari Kumaraswamy, Raeann M. Koren Shimak, Sumedha Gunewardena, Karen L. Wendt, Dani Alexander, Stacey L. Hembruff, Roy A. Jensen. Regulation of epidermal growth factor receptor signaling by BRCA1-microRNA 146a network [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2998.

Abstract 5535: Intracellular negative regulators of RTK-Ras-ERK signaling alter breast cancer perception of metastatic niche-derived growth factors

Abstract Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype that commonly metastasizes to distant organs, such as the lung, resulting in the poor clinical outcomes. TNBC cells commonly overexpress epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK) which when bound by microenvironmental growth factors, results in the activation of downstream effector kinases ERK-AKT and initiates transcriptional programs that control cell fate. Tumor cells, however, simultaneously receive many growth factor inputs from their respective microenvironments, many of which stimulate RTKs other than EGFR, that converge at ERK and AKT resulting in different transcriptional and cell fate outputs. This raises questions as to how tumor cells perceive, decode, and respond to a complex set of microenvironmental signals and what the relevance of this process may be for disease pathogenesis and treatment. To this end, our study aimed to determine how disseminated breast cancer cells perceive and respond to a complex growth factor milieu upon seeding and engraftment in the lung. Using live-cell microscopy techniques and TNBC cells carrying biosensors for ERK and Akt signaling pathways, we monitored the dynamic signaling behaviors of single tumor cells, in real-time, as they adapted to the environment in living human lung tissue. Strikingly, we found that individual disseminated tumor cells are less responsive to growth factors once seeded in the lung, as compared to outside this tissue, despite the presence of RTK stimulating ligands. We showed that partial activation of EGFR is necessary for complete activation of ERK signaling by other growth factor-RTK pairs, and expression of downstream ERK target genes, whereas AKT signaling was unaffected. Using quantitative modeling and experimentation, we showed that reshaping of ERK signaling response dynamics occurred via dysregulation of negative regulators that are dominantly controlled by EGFR signaling. Together, these results provide novel insight into how tumor cells perceive and respond to complex microenvironmental signals, which has important implications for drug targeting strategies. Citation Format: Vaibhav Murthy, Cemal Erdem, Jeremy Copperman, Marc Birtwistle, Alexander Davies. Intracellular negative regulators of RTK-Ras-ERK signaling alter breast cancer perception of metastatic niche-derived growth factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5535.

Abstract 7340: Investigating the function and impact of UBASH3B in head and neck squamous cell carcinoma and its implications for racial health disparities

Abstract Head and Neck Squamous Cell Carcinoma (HNSCC) is one of the top cancers with significant cancer health disparities among racial and ethnic groups. African Americans (AAs) with HNSCC demonstrate worse overall survival compared to European Americans (EAs). While the reasons behind this disparity are multifaceted, recent findings suggest that biological features in HNSCC may contribute to differences in disease progression. However, race-related biological factors in HNSCC have been relatively little explored. We have identified the gene UBASH3B, a protein tyrosine phosphatase that plays a role in the stabilization of the Epidermal Growth Factor Receptor (EGFR). High expression of UBASH3B in AAs is associated with significantly worse overall survival compared to high expression in EAs and low expression in AAs. This study seeks to examine the role of UBASH3B in HNSCC and determine its impact on tumorigenesis. We hypothesize that the upregulation of UBASH3B in AAs contributes to the survival disparity by promoting tumor progression through EGFR signaling. Preliminary data using The Cancer Genome Atlas (TCGA) shows overexpression of UBASH3B in tumor samples of both AAs and EAs HNSCC samples. Plus, the overexpression of UBASH3B is associated with perineural invasion. Additionally, among AA-HNSCC patients, advanced T-stage is associated with increased UBASH3B expression. Elucidating the role UBASH3B plays in tumorigenesis will give us a further understanding of HNSCC in African Americans and can serve as a potential therapeutic target for new treatment options, as well as the biomarkers for the detection of the high-risk group. Citation Format: Madeleine Ndahayo, Vera Mukhina, Ishita Gupta, Daria A. Gaykalova. Investigating the function and impact of UBASH3B in head and neck squamous cell carcinoma and its implications for racial health disparities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7340.

Abstract 6956: Role of soluble CD109 in the progression of head and neck squamous cell carcinoma

Abstract Introduction: About five percent of global cancer deaths are attributed to Head and Neck Squamous Cell Carcinoma (HNSCC), and its annual incidence is predicted to skyrocket by thirty percent by 2030 (according to GLOBOCAN estimates). HNSCC originates from squamous cells in the oral cavity, lips, tongue, larynx, pharynx, and salivary glands. Despite the comparatively easy tumor diagnosis, the number of patients diagnosed at an advanced stage with metastatic spread is substantial. Multiple cell-signaling pathways have been proven to be dysregulated in HNSCC, including the epidermal growth factor receptor (EGFR). Our group has identified CD109, a GPI-anchored membrane-associated protein, as a TGF-β co-receptor that inhibits TGF-β signaling. CD109 expression is upregulated in SCC, and its elevated expression in premalignant lesions is associated with an increased risk of metastatic progression. Recent studies by our team have revealed that CD109 promotes tumor growth in vivo, likely by enhancing EGFR signaling. Interestingly, we have also discovered that membrane CD109 (mCD109) is endogenously released from the cell surface; however, the mechanism of action of the released/soluble form of CD109 (sCD109) remains unknown. In the current study, we examined whether sCD109 opposes the mCD109/EGFR-mediated pro-tumorigenic signaling and HNSCC progression. Research Questions: 1.Does sCD109 regulate EGF-induced EGFR signaling and downstream stem cell marker expression?2.Does sCD109 regulate EGFR expression levels on the HNSCC cell surface?3.Does sCD109 affect the nuclear localization of EGFR?4.Does sCD109 promote EGFR internalization and degradation? Methodology: HNSCC cells and patient-derived primary cells were treated with/without sCD109 recombinant protein (1nM, 5nM, and 10nM) without and with the EGF induction (10ng/ml for 15 mins) and were analyzed by western blotting, immunofluorescence, and immunocytochemistry. The role of sCD109 in regulating membrane EGFR levels was assessed by flow cytometry and surface protein biotinylation assay. Results: Our findings demonstrated that sCD109 inhibited EGF-induced phosphorylation of EGFR (Y1068), STAT3, AKT, and ERK. The sCD109 suppressed the stem cell marker expression (Nanog, OCT4, SOX2). Additionally, sCD109 hindered the nuclear localization of activated EGFR. The sCD109 decreased the membrane EGFR expression levels and activated the phosphorylation of EGFR (Y1045), suggesting a role for sCD109 in promoting EGFR degradation. Conclusion: Our team has previously reported that mCD109 is pro-tumorigenic, and our current findings suggest that sCD109 opposes the pro-tumorigenic effects of mCD109. This study aimed to determine how sCD109 regulates cellular signaling pathways and will provide key insights into the molecular mechanisms underlying the progression of HNSCC, a highly prevalent malignancy. Citation Format: Varsha Reddy Durgempudi, Tenzin Kungyal, Anie Philip. Role of soluble CD109 in the progression of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6956.