Knockdown Of Epidermal Growth Factor Receptor Research Articles

TRAF4 promotes lung cancer development by activating tyrosine kinase of EGFR

Objective: To explore the role of tumor necrosis factor receptor-associated factor 4 (TRAF4) in promoting the abnormal activation of epidermal growth factor receptor (EGFR) and its effect on lung cancer cell proliferation, migration and invasion. Methods: Tumor tissues from patients who underwent lung adenocarcinoma resection at The First Affiliated Hospital of Second Military Medical University, from January 2015 to May 2017 were collected, and the expressions of TRAF4 and Ki-67 in lung cancer tissues were detected by immunohistochemistry, the mRNA levels of Cyclin D and Vimentin were detected by real-time fluorescence quantitative PCR (qRT-PCR). The effect of TRAF4 on the tumor growth ability of lung cancer A549 cells was investigated by the xenograft model, the effect of TRAF4 or EGFR on the tumor proliferation ability was detected by using cell counting kit 8 (CCK8) and BrdU assay, and the migration and invasion abilities of tumor cells were detected by Transwell assay. Different structural domain deletion expression vectors of TRAF4 and EGFR were constructed to transfect cells, and the interaction mode of TRAF4 and EGFR was investigated by immunoprecipitation assay. Results: The expression of TRAF4 in non-small cell lung cancer (NSCLC) tissues was positively correlated with the expressions of Ki-67, cyclin D, and vimentin (r2: 0.438, 0.695, and 0.736, respectively, all P＜0.01). Immunohistochemical assay of tumor tissues from NSCLC patients showed that tissues with high expression of TRAF4 were also high in Ki-67. Patients with high TRAF4 expression (TRAF4 positivity >30%) had a shorter progression-free survival (PFS) time than that of patients with low TRAF4 expression (TRAF4 positivity ≤30%) (median PFS of 12 and 19 months, respectively; P=0.034). Traf4-/- cells had a weakened proliferative capacity than traf4+/+ cells and formed tumors with smaller size (P＜0.05). The expression level of Ki-67 in the tumor tissues formed by traf4-/- cells [(45.6±8.7)%] was lower than that in the tumor tissues formed by traf4+/+ cells [(62.3±10.3)%, P=0.015], the mRNA levels of cyclin D (1.01±0.15) and vimentin (1.01±0.12) in the traf4-/- cells were lower than those of the traf4+/+ cells (3.41±0.32 and 3.12±0.18, respectively, both P＜0.05).The western blot results showed that, with the elevated intracellular expression level of TRAF4, phosphorylation level of EGFR was significantly increased in both wild-type EGFR and activation mutant EGFR-expression cells. The capacities of proliferation, migration and invasion of A549 cells was weakened after EGFR knockdown (all P＜0.01). Immunoprecipitation experiments showed that TRAF4 binds to the peptide segment of the near-membrane region of EGFR through the TRAF structural domain, and the mutual binding between EGFR molecules was enhanced under TRAF4 overexpression conditions. Increasing TRAF4 expression promoted EGFR molecular phosphorylation and activation of downstream signaling. Conclusions: TRAF4 expression is elevated in NSCLC tissues and tumor cells, which promotes tumor proliferation, migration and invasion. TRAF4 directly binds to EGFR molecules, enhances its own phosphorylation and activates the downstream signaling pathway by promoting the interaction between EGFR molecules.

The oncogenic ADAMTS1–VCAN–EGFR cyclic axis drives anoikis resistance and invasion in renal cell carcinoma

BackgroundMetastasis, the leading cause of renal cell carcinoma (RCC) mortality, involves cancer cells resisting anoikis and invading. Until now, the role of the matrix metalloproteinase (MMP)-related enzyme, A disintegrin and metalloprotease with thrombospondin motifs 1 (ADAMTS1), in RCC anoikis regulation remains unclear.MethodsThe clinical significance of ADAMTS1 and its associated molecules in patients with RCC was investigated using data from the Gene Expression Omnibus (GEO) and TCGA datasets. Human phosphoreceptor tyrosine kinase (RTK) array, luciferase reporter assays, immunoprecipitation (IP) assays, western blotting, and real-time reverse-transcription quantitative polymerase chain reaction (RT–qPCR) were used to elucidate the underlying mechanisms of ADAMTS1. Functional assays, including anoikis resistance assays, invasion assays, and a Zebrafish xenotransplantation model, were conducted to assess the roles of ADAMTS1 in conferring resistance to anoikis in RCC.ResultsThis study found elevated ADAMTS1 transcripts in RCC tissues that were correlated with a poor prognosis. ADAMTS1 manipulation significantly affected cell anoikis through the mitochondrial pathway in RCC cells. Human receptor tyrosine kinase (RTK) array screening identified that epidermal growth factor receptor (EGFR) activation was responsible for ADAMTS1-induced anoikis resistance and invasion. Further investigations revealed that enzymatically active ADAMTS1-induced versican V1 (VCAN V1) proteolysis led to EGFR transactivation, which in turn, through positive feedback, regulated ADAMTS1. Additionally, ADAMTS1 can form a complex with p53 to influence EGFR signaling. In vivo, VCAN or EGFR knockdown reversed ADAMTS1-induced prometastatic characteristics of RCC. A clinical analysis revealed a positive correlation between ADAMTS1 and VCAN or the EGFR and patients with RCC with high ADAMTS1 and VCAN expression had the worst prognoses.ConclusionsOur results collectively uncover a novel cyclic axis involving ADAMTS1–VCAN–EGFR, which significantly contributes to RCC invasion and resistance to anoikis, thus presenting a promising therapeutic target for RCC metastasis.

AGR2 facilitates teratoma progression by regulating glycolysis via the AnXA2/EGFR axis

Anterior gradient-2 (AGR2) is highly expressed in several tumors and plays an important role in tumor development. However, the biological function of AGR2 in teratomas has not yet been thoroughly studied. In this study, AGR2 was found to be upregulated in teratoma tissues and in human testicular teratoma cell lines by Western blotting and qRT-PCR assays. A DNA Methylation-Specific PCR assay demonstrated that AGR2 upregulation resulted from hypomethylated AGR2 in teratoma cells. NCC-IT and NT2-D1 cells were transfected with pcDNA-AGR2 or sh-AGR2 to obtain AGR2-overexpressed or -silenced cells, and cell proliferation, invasion and glycolysis were determined using CCK-8, 5-ethynyl-2′-deoxyuridine (EdU), Transwell assays, and commercial kits. The results revealed that overexpression of AGR2 promoted teratoma cell proliferation and invasion and elevated glycolysis levels evidencing by the increase in lactate secretion, glucose consumption, ATP levels and the expression of glycolysis-related proteins, while knockdown of AGR2 showed the opposite results. The interactions between AGR2 and annexin A2 (AnXA2), as well as between AnXA2 and epidermal growth factor receptor (EGFR) were verified by co-immunoprecipitation assay. Mechanistic studies revealed that AGR2 interacts with AnXA2 and increases the level of AnXA2 to recruit more AnXA2 to EGFR, there by promoting EGFR expression. A series of rescue experiments showed that knockdown of AnXA2 or EGFR weakened the promotional effects of AGR2 overexpression on the proliferation, invasion, and glycolysis of teratoma cells. Finally, tumorigenicity assays were performed using NT2-D1 cells stably transfected with either LV-NC-shRNA or LV-shAGR2. The results showed that AGR2 knockdown significantly inhibited teratoma tumor growth in vivo. In conclusion, our data suggested that AGR2 facilitates glycolysis in teratomas through promoting EGFR expression by interacting with AnXA2, thereby promoting teratoma cells proliferation and invasion.

EGF-like growth factors upregulate pentraxin 3 expression in human granulosa-lutein cells

The epidermal growth factor (EGF)-like factors, comprising amphiregulin (AREG), betacellulin (BTC), and epiregulin (EREG), play a critical role in regulating the ovulatory process. Pentraxin 3 (PTX3), an essential ovulatory protein, is necessary for maintaining extracellular matrix (ECM) stability during cumulus expansion. The aim of this study was to investigate the impact of EGF-like factors, AREG, BTC, and EREG on the expression and production of PTX3 in human granulosa-lutein (hGL) cells and the molecular mechanisms involved. Our results demonstrated that AREG, BTC, and EREG could regulate follicular function by upregulating the expression and increasing the production of PTX3 in both primary (obtained from 20 consenting patients undergoing IVF treatment) and immortalized hGL cells. The upregulation of PTX3 expression was primarily facilitated by the activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway, induced by these EGF-like factors. In addition, we found that the upregulation of PTX3 expression triggered by the EGF-like factors was completely reversed by either pretreatment with the epidermal growth factor receptor (EGFR) inhibitor, AG1478, or knockdown of EGFR, suggesting that EGFR is crucial for activating the ERK1/2 signaling pathway in hGL cells. Overall, our findings indicate that AREG, BTC, and EREG may modulate human cumulus expansion during the periovulatory stage through the upregulation of PTX3.

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.

Abstract 4759: Targeting slow-cycling persisters in EGFR mutant non-small cell lung cancer

Abstract Activating mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC) patients are associated with clinical benefit in the metastatic and adjuvant settings when treated with EGFR inhibitors, such as osimertinib. Despite its dramatic efficacy, most patients are partial responders and refractive disease leave limited treatment options. The objective was to dissect the mechanism of osimertinib resistance in EGFR mutant NSCLC models to identify a means of re-sensitization. Imaging mass cytometric analysis of 76 NSCLC patients demonstrated EGFR expression was inversely correlated to CD105 membrane expression. CD105 is a transforming growth factor beta (TGF-β) family co-receptor that promotes bone morphogenic protein (BMP) signaling and inhibits TGF-β signaling. Significant elevation in cell surface CD105 by osimertinib was furthered by EGFR knockdown in two NSCLC lines with EGFR activating mutations. These lines were subjected to a program of increasing osimertinib concentrations to generate isogenic resistant lines. Heterogeneous drug-tolerant persister cells had significantly higher mutational load, increased CD105 cell surface expression as well as greater expression of bypass signaling factors such as ERK, PI3K, and BMP ligands compared to their parental counterparts. Single cell RNA-seq analysis of parental NSCLC revealed a small population of cells that shared elevated endothelial and pyrimidine metabolism, with a slow-cycling signature that was enriched in the osimertinib-resistant cells. Combining osimertinib with a CD105 neutralizing antibody, carotuximab, reduced the slow-cycling population and restored osimertinib sensitivity in resistant cell lines. The knockdown of CD105 had a more pronounced decrease in cell viability when combined with osimertinib. The osimertinib-resistant lines demonstrated more condensed chromatin and glycolytic state compared to the respective parental lines by ATAC-seq and Single Cell ENergetIc metabolism profiling, respectively. The addition of carotuximab reversed the chromatin and metabolic reprograming of osimertinib resistance. Carotuximab was discovered to initiate CD105 interaction with EGFR and its dominant active variant, EGFRv3. Ultimately, combination therapy of carotuximab and osimertinib resulted in significantly reduced tumor expansion compared to mice treated with either drug alone. Paradoxically, the small tumors from combination therapy had greater mitosis compared to the larger tumors of the osimertinib single agent treated mice. Inhibition of CD105 with carotuximab can overcome resistance to osimertinib and inhibit NSCLC tumor progression. This is a first-in-class pre-clinical foundation for a novel synthetic lethality treatment strategy resulting from the observation of global changes in EGFR-antagonist resistance as opposed to individual mutations identified in tumor subpopulations. Citation Format: Manish Thiruvalluvan, Sandrine Billet, Zhenqiu Liu, Joseph Lownik, Gabrielle Gonzales, Hyoyoung Kim, Anton L. Villamejor, Larry Milshteyn, Kamya Sankar, Edwin M. Posadas, Jean Lopategui, Sungyong You, Karen Reckamp, Neil A. Bhowmick. Targeting slow-cycling persisters in EGFR mutant non-small cell lung cancer [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 4759.

Co-delivery of PD-L1- and EGFR-targeting siRNAs by synthetic PEG12-KL4 peptide to the lungs as potential strategy against non-small cell lung cancer

BackgroundSmall interfering RNA (siRNA) holds great promise for treating various lung diseases, but the lack of safe and efficient pulmonary siRNA delivery systems has hindered its advance into the clinics. The epidermal growth factor receptor (EGFR) which promotes cell proliferation, and the programmed cell death ligand 1 (PD-L1) which plays a crucial role in suppressing cytotoxic T cells activity, are two important targets for treating non-small cell lung cancer (NSCLC). Here, we explored the potential of PEG12-KL4, a synthetic peptide, to deliver siRNA to various NSCLC cells and to lung tissues in mice. MethodsPEG12-KL4 was used to transfect siRNAs targeted at both EGFR and PD-L1 into NSCLC cells. Immunoblotting was used to evaluate the siRNA silencing effects in HCC827 and NCI-H1975 NSCLC cells. CD8+ T cell-mediated NSCLC cell killing was employed to demonstrate the functional effects of PD-L1 siRNA knock-down. Fluorescent siRNAs were used to visualise siRNA uptake in cells as well as to enable biodistribution studies in BALB/c mice. ResultsOur results showed that PEG12-KL4 was efficient in mediating siRNA knock-down of EGFR and PD-L1 in various NSCLC cells. Importantly, the PEG12-KL4 peptide enabled significantly better siRNA delivery than the commercial Lipofectamine 2000 reagent. We hypothesised that PEG12-KL4 peptide enabled siRNA to either escape from or bypass endosomal degradation as indicated by confocal fluorescence imaging. Notably, combined knock-down of EGFR and PD-L1 in NCI-H1975 cells resulted in better effector T cell-mediated cancer cell killing than knock-down of PD-L1 alone. Moreover, biodistribution of PEG12-KL4/siRNA complexes following intravenous administration revealed poor lung delivery with the fluorescent siRNA accumulating in the liver. In contrast, intratracheal delivery of PEG12-KL4/siRNA complexes resulted in the fluorescent siRNA to be detected in the lung with retarded renal excretion. ConclusionIn conclusion, we demonstrated that the co-delivery of siRNAs targeting EGFR and PD-L1 using PEG12-KL4 is feasible and represents a promising future strategy to treat NSCLC, whereby pulmonary siRNA delivery is favourable to intravenous administration.

Activation of the EGFR/PI3K/AKT pathway limits the efficacy of trametinib treatment in head and neck cancer.

Blocking the mitogen-activated protein kinase (MAPK) pathway with the MEK1/2 inhibitor trametinib has produced promising results in patients with head and neck squamous cell carcinoma (HNSCC). In the current study, we showed that trametinib treatment leads to overexpression and activation of the epidermal growth factor receptor (EGFR) in HNSCC cell lines and patient-derived xenografts. Knockdown of EGFR improved trametinib treatment efficacy both in vitro and in vivo. Mechanistically, we demonstrated that trametinib-induced EGFR overexpression hyperactivates the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. In vitro, blocking the PI3K pathway with GDC-0941 (pictilisib), or BYL719 (alpelisib), prevented AKT pathway hyperactivation and enhanced the efficacy of trametinib in a synergistic manner. In vivo, a combination of trametinib and BYL719 showed superior antitumor efficacy vs. the single agents, leading to tumor growth arrest. We confirmed our findings in a syngeneic murine head and neck cancer cell line in vitro and in vivo. Taken together, our findings show that trametinib treatment induces hyperactivation of EGFR/PI3K/AKT; thus, blocking of the EGFR/PI3K pathway is required to improve trametinib efficacy in HNSCC.

Loss of EGFR contributes to high-fat diet-induced nonalcoholic fatty liver disease.

Using a murine model of high-fat diet (HFD)-induced nonalcoholic fatty liver disease (NAFLD), we found that the expression of the epidermal growth factor receptor (EGFR) significantly decreased in hepatocytes. In vitro, free fatty acid influx decreased EGFR in hepatocytes. In HFD-fed mice, ectopic expression of EGFR alleviated intrahepatic lipid accumulation and reduced serum triglyceride and cholesterol, whereas knockdown of EGFR aggravated hepatic steatosis. Notably, EGFR inhibited the induction of lipogenic genes, including Srebf1, Srebf2, Fasn, Acc1 and Ppara, both invitro and invivo. Mechanistically, EGFR potentiates TGF-β/Smad signalling and augments the inhibitory effects of TGF-β1 on lipogenic genes in hepatocytes. Our findings suggest a hitherto unknown paradigm in the pathogenesis of NAFLD, thereby providing a rational basis for future therapeutic considerations.

Tumor suppressor miR-218 directly targets epidermal growth factor receptor (EGFR) expression in triple-negative breast cancer, sensitizing cells to irradiation

PurposeMicroRNA-218 (miR-218) is a key regulator of numerous processes relevant to tumor progression. In the present study, we aimed to characterize the relationship between miR-218 and the Epidermal Growth Factor Receptor (EGFR) as well as to understand downstream effects in triple-negative breast cancer (TNBC).MethodsWe assessed miR-218 and EGFR expression in cell lines and publicly available primary breast cancer gene expression data. We then overexpressed miR-218 in two TNBC cell lines and investigated effects on EGFR and downstream mitogen-activated protein (MAP) kinase signaling. Luciferase reporter assay was used to characterize a direct binding interaction between miR-218 and EGFR mRNA. Digital holographic microscopy helped investigate cell migration and dry mass after miR-218 overexpression. Cell division and invasion were assessed microscopically, while radiation response after miR-218 overexpression alone or combined with additional EGFR knockdown was investigated via clonogenic assays.ResultsWe found an inverse correlation between EGFR expression and miR-218 levels in cell lines and primary breast cancer tissues. MiR-218 overexpression resulted in a downregulation of EGFR via direct binding of the mRNA. Activation of EGFR and downstream p44/42 MAPK signaling were reduced after pre-miR-218 transfection. Cell proliferation, motility and invasiveness were inhibited whereas cell death and mitotic catastrophe were upregulated in miR-218 overexpressing cells compared to controls. MiR-218 overexpressing and EGFR siRNA-treated cells were sensitized to irradiation, more than miR-218 overexpressing cells alone.ConclusionThis study characterizes the antagonistic relationship between miR-218 and EGFR. It also demonstrates downstream functional effects of miR-218 overexpression, leading to anti-tumorigenic cellular changes.

Effect of LGR4/EGFR signaling on cell growth and cancer stem cell-like characteristics in liver cancer

PurposeHepatocellular carcinoma (HCC) is the most common primary liver cancer. Leucine-rich repeat containing G-protein-coupled receptors 4 (LGR4) participates in tumor progression, invasion, and metastasis. Our study aimed to investigate the effect of LGR4 with epidermal growth factor receptor (EGFR) in HCC cells. MethodsWe employed Hep3B and Huh7 cells to conduct our research. Comprehensive biological activities were characterized by CCK8 and transwell assay. Molecular biology techniques were used to determine the expression of proteins. Hep3B was employed to conduct subcutaneous tumor in mice. The tumor growth and the expression levels of proteins were assessed. ResultsLGR4 overexpression could promote the cells proliferation, migration, and invasion ability, while siLGR4 and siEGFR could inhibit cells biological activities. In addition, LGR4 overexpression promoted the expression levels of RSPO2, β-catenin, EGFR and cancer stem cells (CSCs) markers, whereas silence of LGR4 or EGFR could diminish the expression levels of β-catenin and CSCs markers. Furthermore, knockdown of LGR4 or EGFR also inhibited tumor growth and reduced the expression levels of RSPO2, CD133, CD44, Nanog, β-catenin in vivo. ConclusionOur data suggest that LGR4 /EGFR signaling in HCC leads to induce tumor growth, which then contributes to stem cell characteristics. It maybe a new perspective for the targeted therapy of HCC treatment.

The Potential Tumor-Suppressor DHRS7 Inversely Correlates with EGFR Expression in Prostate Cancer Cells and Tumor Samples.

Simple SummaryProstate cancer is one of the most common malignancies in men. Current therapies are initially effective but resistance often develops, leading to tumor recurrence and death. Further research on new players, mechanisms involved in prostate cancer, and therapy resistance is needed. We studied the role of DHRS7, a potential tumor suppressor with currently unknown physiological function, in prostate cancer cells using proteome and gene expression analyses. Despite the fact that DHRS7 can inactivate 5α-dihydrotestosterone, its effect on prostate cancer cells seems to be unrelated to androgen metabolism. When comparing three widely studied prostate cancer cell lines, we observed a negative correlation between DHRS7 and EGFR expression. DHRS7 knockdown enhanced EGFR expression, while knockdown of EGFR tended to increase DHRS7 expression. Importantly, DHRS7 expression negatively correlates with EGFR expression and positively with survival rates in prostate cancer patients. This study suggests a tumor-suppressor role for DHRS7 by modulating EGFR expression in prostate cancer.Prostate cancer (PCa), one of the most common malignancies in men, typically responds to initial treatment, but resistance to therapy often leads to metastases and death. The dehydrogenase/reductase 7 (DHRS7, SDR34C1) is an “orphan” enzyme without known physiological function. DHRS7 was previously found to be decreased in higher-stage PCa, and siRNA-mediated knockdown increased the aggressiveness of LNCaP cells. To further explore the role of DHRS7 in PCa, we analyzed the proteome of LNCaP cells following DHRS7 knockdown to assess potentially altered pathways. Although DHRS7 is able to inactivate 5α-dihydrotestosterone, DHRS7 knockdown did not affect androgen receptor (AR) target gene expression, and its effect on PCa cells seems to be androgen-independent. Importantly, proteome analyses revealed increased expression of epidermal growth factor receptor (EGFR), which was confirmed by RT-qPCR and Western blotting. Comparison of AR-positive LNCaP with AR-negative PC-3 and DU145 PCa cell lines revealed a negative correlation between DHRS7 and EGFR expression. Conversely, EGFR knockdown enhanced DHRS7 expression in these cells. Importantly, analysis of patient samples revealed a negative correlation between DHRS7 and EGFR expression, both at the mRNA and protein levels, and DHRS7 expression correlated positively with patient survival rates. These results suggest a protective role for DHRS7 in PCa.

Abstract 3003: The effects of autophagy inhibition on HNSCC sensitivity to CTX

Abstract Background: Cetuximab (CTX) is a monoclonal antibody commonly used in the treatment of head and neck squamous cell carcinomas (HNSCCs) that acts by targeting the epidermal growth factor receptor (EGFR). Addition of CTX to standard multi-agent chemotherapy only slightly improves the response rate from 20% to 36%, highlighting the urgent need for improved treatment. Autophagy is a naturally occurring mechanism activated within cells to protect them from harsh conditions or stress but can also provide cancerous cells with protection against further treatment attempts. Here, we examine the efficacy of combining CTX with the autophagy inhibitor, SAR405, as well as investigating the specific mechanism of CTX-induced autophagy. Methods: To measure autophagy, we utilized the Promega LC3 reporter assay, Western blotting for LC3, immunofluorescence for LC3, and an acridine orange assay. We ran our assays using the HNSCC cell lines A253 (HPV-), UM-SCC1 (HPV+), and its CTX-resistant derivative, UM-SCC1-C5 (HPV+). Clonogenic assays were run to measure colony formation, cell counting kit 8 (CCK8) assays to determine cell survival, and flow cytometry to investigate cell cycle status. Results: In this study, we first were able to demonstrate that CTX can activate pro-survival autophagy. CTX-treated cells had higher LC3 flux and more LC3 puncta than control cells as assessed by western blot and immunofluorescence staining, respectively. CTX-resistant cells demonstrated higher basal LC3 flux compared to the CTX-sensitive parental cells. We also discovered that the use of the autophagy inhibitor, SAR405, could help improve tumor response when combined with CTX. The addition of SAR405 to CTX treatment led to a decrease in colony formation and a decrease in cell survival, overcoming resistance to CTX. Interestingly, we discovered that SAR405 alone led to cell cycle arrest in the G1 phase but did not lead to a statistically significant decrease in cell survival compared to the control, showing that SAR405 is able to arrest cell growth but is not cytotoxic. Finally, knockdown of EGFR or LAPTM4B prevented CTX-induced activation of autophagy. Conclusion: Through our work, we have been able to demonstrate that CTX is able to activate autophagy, and that this initiation of autophagy plays a cytoprotective role in HNSCC cells. The addition of the autophagy inhibitor SAR405 improved response to CTX treatment. The promising results obtained thus far open the door to future studies investigating the efficacy of combining CTX with more specific autophagy inhibitors. Citation Format: Samantha T. Bradley, Yong-Syu Lee, Justin H. Skiba, Jaimee Eckers, Adam D. Swick, Rong Hu, Kwang P. Nickel, Zafer Gurel, Pippa F. Cosper, Randall J. Kimple. The effects of autophagy inhibition on HNSCC sensitivity to CTX [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 3003.

Abstract 2612: Depleting EGFR and HER2 overcomes resistance to EGFR inhibitors in colorectal cancer

Abstract Background & Goals: Epidermal growth factor receptor (EGFR), an oncogenic receptor tyrosine kinase, is a major therapeutic target in several types of cancers. Several EGFR inhibitors are used to treat EGFR-positive colorectal cancer (CRC). Unfortunately, primary and acquired resistance to the inhibitors is very common. Current approaches to combating the resistance include combining EGFR inhibitors with agent(s) that targets compensatory signaling molecules (HER2, HER3, IGF1R, MET, KRAS, BRAF and PIK3CA). However, responses to such combination treatments are modest, if any. Overcoming drug resistance remains a major unmet medical need in EGFR-positive CRC. Results: Here, we show the main reason for CRC resistance to EGFR inhibitors and a therapeutic strategy that overcomes it. The sensitivity of CRC cells to EGFR inhibitors correlates with EGFR downregulation. PEPDG278D, a recombinant human protein that induces the degradation of both EGFR and HER2, strongly inhibits oncogenic signaling and growth of CRC in vitro and in vivo (subcutaneous tumors, orthotopic tumors, and patient-derived xenografts), regardless of activating mutations of KRAS, BRAF and PIK3CA. siRNA knockdown of EGFR or HER2 also inhibits CRC cells resistant to EGFR inhibitors. PEPDG278D indirectly suppresses HER3, IGF1R and MET by disrupting their heterodimerization with EGFR or HER2. While excessive tumor-generated EGFR ligands may block target engagement by PEPDG278D, inhibiting ligand shedding by aderbasib enables PEPDG278D to exert strong antitumor activity, and fluorouracil further enhances tumor inhibition. Collectively, our study shows that CRC resistance to EGFR inhibitors result primarily from their inability to eliminate their target and a PEPDG278D-centered combination treatment overcomes the resistance. Conclusions & Therapeutic Relevance: The therapeutic activity of PEPDG278D highlights that EGFR and HER2 remain indispensable to the survival of CRC cells resistant to EGFR inhibitors. The strong therapeutic activity of PEPDG278D-centered combination treatment in preclinical models of CRC, which carry clinically relevant gene mutations that confer resistance to current EGFR inhibitors, provides the scientific premise for potential evaluation of this recombinant human protein in patients with drug-resistant CRC. Citation Format: Lu Yang, Arup Bhattacharya, Yun Li, Sandra Sexton, Xiang Ling, Fengzhi Li, Yuesheng Zhang. Depleting EGFR and HER2 overcomes resistance to EGFR inhibitors in colorectal cancer [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 2612.

AG1478 Elicits a Novel Anti-Influenza Function via an EGFR-Independent, GBF1-Dependent Pathway.

Current options for preventing or treating influenza are still limited, and new treatments for influenza viral infection are urgently needed. In the present study, we serendipitously found that a small-molecule inhibitor (AG1478), previously used for epidermal growth factor receptor (EGFR) inhibition, demonstrated a potent activity against influenza both in vitro and in vivo. Surprisingly, the antiviral effect of AG1478 was not mediated by its EGFR inhibitory activity, as influenza virus was insensitive to EGFR blockade by other EGFR inhibitors or by siRNA knockdown of EGFR. Its antiviral activity was also interferon independent as demonstrated by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout approach. Instead, AG1478 was found to target the Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1)–ADP-ribosylation factor 1 (ARF1) system by reversibly inhibiting GBF1 activity and disrupting its Golgi-cytoplasmic trafficking. Compared to known GBF1 inhibitors, AG1478 demonstrated lower cellular toxicity and better preservation of Golgi structure. Furthermore, GBF1 was found to interact with a specific set of viral proteins including M1, NP, and PA. Additionally, the alternation of GBF1 distribution induced by AG1478 treatment disrupted these interactions. Because targeting host factors, instead of the viral component, imposes a higher barrier for developing resistance, GBF1 modulation may be an effective approach to treat influenza infection.

Trimethyl-Chitosan Coated Gold Nanoparticles Enhance Delivery, Cellular Uptake and Gene Silencing Effect of EGFR-siRNA in Breast Cancer Cells.

Purpose: Despite the promising therapeutic effects of gene silencing with small interfering RNAs (siRNAs), the challenges associated with delivery of siRNAs to the tumor cells in vivo, has greatly limited its clinical application. To overcome these challenges, we employed gold nanoparticles modified with trimethyl chitosan (TMC) as an effective delivery carrier to improve the stability and cellular uptake of siRNAs against epidermal growth factor receptor (EGFR) that is implicated in breast cancer. Methods: AuNPs were prepared by the simple aqueous reduction of chloroauric acid (HAuCl4) with ascorbic acid and coated with synthesized TMC. EGFR-siRNA was then complexed with the AuNPs-TMC via electrostatic interaction to make AuNPs-TMC/EGFR-siRNA with a w/w ratio of 10:1. Nanoparticles were assessed for physicochemical characteristics and in vitro cellular behavior on MCF-7 breast cancer cell line. Results: Spherical and positively charged AuNPs-TMC (67 nm, +45 mV) were successfully complexed with EGFR-siRNA (82 nm, +11 mV) which were able to retard the gene migration completely. Confocal microscopy and flow cytometry analysis demonstrated complete cellular uptake of Cy5 labeled AuNPs-TMC in the MCF-7 cells after 4 h incubation. MTT test after 48 h incubation showed that the AuNPs-TMC were safe but when combined with EGFR-siRNA exert significant cytotoxicity while the cell viability was about 50%. These nanocomplexes also showed a high gene expression knockdown (86%) of EGFR and also a high apoptosis rate (Q2 + Q3 = 18.5%) after 24 h incubation. Conclusion: This study suggests that the simply synthesized AuNPs-TMC are novel, effective, and promising nanocarriers for siRNA delivery, and AuNPs-TMC/EGFR-siRNA appears to be a potential therapeutic agent for breast cancer treatment.

Circular RNA ZNF609 promotes laryngeal squamous cell carcinoma progression by upregulating epidermal growth factor receptor via sponging microRNA-134-5p

ABSTRACT Emerging evidence has revealed that aberrantly expressed circular RNAs (circRNAs) play vital roles in tumorigenesis and progression of diverse human malignancies. CircZNF609 was found to be involved in hepatocellular carcinoma, but the role and underlying mechanism of circZNF609 in laryngeal squamous cell carcinoma (LSCC) remain unclear. This study aimed to explore the molecular mechanism of circZNF609 in LSCC. qRT-qPCR was performed to detect the expression of circZNF609 and microRNA-134-5p (miR-134-5p) in LSCC. Colony formation assay, CCK-8 assay, BrdU incorporation assay, clone formation assay, transwell invasion assay and Western blot analysis were performed to evaluate LSCC cell proliferation, as well as the expression of proliferating cell nuclear antigen (PCNA) and MMP-2. Luciferase reporter assay, target gene prediction and screening were used to validate downstream target genes of circZNF609 and miR-134-5p. EGFR expression was detected by Western blot analysis and RT-qPCR. Nude mice were used to detect tumor changes. CircZNF609 was upregulated in LSCC and associated with poor survival of LSCC patients. Knockdown of circZNF609 inhibited LSCC proliferation, invasion and the expression of PCNA and matrix matalloproteinases-2 (MMP-2). CircZNF609 can regulate miR-134-5p to upregulate epidermal growth factor receptor (EGFR). In addition, knockdown of EGFR or overexpression of miR-134-5p could reverse the tumor-promoting effects of circZNF609 in LSCC. In LSCC tissues, circZNF609 was negatively correlated with miR-134-5p and positively correlated with EGFR. CircZNF609 promotes the progression of LSCC via the miR-134-5p/EGFR axis, which might be the therapeutic target of LSCC.

Ischemia-Reperfusion Injury and Immunosuppressants Promote Polyomavirus Replication Through Common Molecular Mechanisms.

BackgroundBK polyomavirus (BKPyV)-associated nephropathy (BKPyVAN) causes renal allograft dysfunction and graft loss. However, the mechanism of BKPyV replication after kidney transplantation is unclear. Clinical studies have demonstrated that immunosuppressants and renal ischemia–reperfusion injury (IRI) are risk factors for BKPyV infection. Studying the pathogenic mechanism of BKPyV is limited by the inability of BKPyV to infect the animal. Mouse polyomavirus (MPyV) is a close homolog of BKPyV. We used a model of MPyV infection to investigate the core genes and underlying mechanism of IRI and immunosuppressants to promote polyomavirus replication.Materials and MethodsOne-day-old male C57BL/6 mice were intraperitoneally injected with MPyV. At week 9 post-infection, all mice were randomly divided into IRI, immunosuppressant, and control groups and treated accordingly. IRI was established by clamping the left renal pedicle. Subsequently, kidney specimens were collected for detecting MPyV DNA, histopathological observation, and high-throughput RNA sequencing. Weighted gene correlation network analysis (WGCNA), protein–protein interaction network analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to screen for core genes and common signaling pathways involved in promoting MPyV replication by IRI and immunosuppressants.ResultsAfter primary infection, MPyV established persistent infection in kidneys and subsequently was significantly increased by IRI or immunosuppressant treatment individually. In the IRI group, viral loads peaked on day 3 in the left kidney, which were significantly higher than those in the right kidney and the control group. In the immunosuppressant group, viral loads in the left kidney were significantly increased on day 3, which were significantly higher than those in the control group. Protein–protein interaction network analysis and WGCNA screened complement C3, epidermal growth factor receptor (EGFR), and FN1 as core genes. Pathway enrichment analysis based on the IRI- or immunosuppressant-related genes selected by WGCNA indicated that the NF-κB signaling pathway was the main pathway involved in promoting MPyV replication. The core genes were further confirmed using published datasets GSE47199 and GSE75693 in human polyomavirus-associated nephropathy.ConclusionsOur study demonstrated that IRI and immunosuppressants promote polyomavirus replication through common molecular mechanisms. In future studies, knockdown or specific inhibition of C3, EGFR, FN1, and NF-κB signaling pathway will further validate their critical roles in promoting polyomavirus replication.

The effectiveness of EGFR knockdown by RNAi lines varies depending on the tissue.

In Drosophila , the Epidermal growth factor receptor (EGFR) signaling pathway is known to be critically involved in multiple stages of development. We induced a loss of function phenotype in the eyes, wings, and somatic follicle cells using four EGFR RNAi lines: HMS05003 and JF02283, which produce short hairpin RNAs, as well as JF01368 and KK100051, which produce long hairpin RNAs. Using these four lines, we completed a systematic comparison of the ability of short hairpin vs long hairpin RNAi lines to produce loss-of-function phenotypes in the above-mentioned tissues. Tissue specific knockdown was achieved by using Gal4 drivers specific to the three tissues being studied. In the eyes and wings, the KK100051 line induced the most severe phenotype, while the JF01368 line was the least severe, but in the somatic follicle cells, the KK100051 line was the least effective, while the JF01368 and JF02283 lines were comparable with respect to phenotypic severity. We conclude that there is significant tissue specific variability exhibited by the different RNAi lines.

The unique Akt inhibitor SC66 suppressed AMPK activity and abolished autophagy through the EGFR-p62 pathway.

Akt is usually considered to be a negative regulator of both autophagy and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling. In the present study, we found that SC66, a pyridine-based allosteric Akt inhibitor, suppressed basal and H2 O2 -induced autophagy concurrent with decreased phosphorylation and activity of AMPK. SC66 treatment led to the formation of a high molecular weight (HMW) form of SQSTM1/p62 (p62), which is an autophagic substrate and is essential for selective autophagy. Moreover, we observed that SC66 inhibited the binding of p62 and microtubule-associated protein light chain 3 (LC3). The immunoprecipitation results revealed the interaction between p62 and epidermal growth factor receptor (EGFR), and knockdown of EGFR reversed SC66-mediated autophagy inhibition without affecting the phosphorylation of acetyl-CoA carboxylase (ACC), a well-known substrate of AMPK. SC66 increased the interaction between EGFR and Beclin 1 and markedly decreased the association of EGFR with VPS34, a critical protein for autophagy induction. Collectively, the data presented here indicate that EGFR-p62 pathway plays a critical role in Akt-mediated positive regulation of autophagy.