Down-regulation Of Epidermal Growth Factor Receptor Research Articles

Impact of Interleukin-6 Activation and Arthritis on Epidermal Growth Factor Receptor (EGFR) Activation in Sensory Neurons and the Spinal Cord.

In tumor cells, interleukin-6 (IL-6) signaling can lead to activation of the epidermal growth factor receptor (EGFR), which prolongs Stat3 activation. In the present experiments, we tested the hypothesis that IL-6 signaling activates EGFR signaling in peripheral and spinal nociception and examined whether EGFR localization and activation coincide with pain-related behaviors in arthritis. In vivo in anesthetized rats, spinal application of the EGFR receptor blocker gefitinib reduced the responses of spinal cord neurons to noxious joint stimulation, but only after spinal pretreatment with IL-6 and soluble IL-6 receptor. Using Western blots, we found that IL-6-induced Stat3 activation was reduced by gefitinib in microglial cells of the BV2 cell line, but not in cultured DRG neurons. Immunohistochemistry showed EGFR localization in most DRG neurons from normal rats, but significant downregulation in the acute and most painful arthritis phase. In the spinal cord of mice, EGFR was highly activated mainly in the chronic phase of inflammation, with localization in neurons. These data suggest that spinal IL-6 signaling may activate spinal EGFR signaling. Downregulation of EGFR in DRG neurons in acute arthritis may limit nociception, but pronounced delayed activation of EGFR in the spinal cord may be involved in chronic inflammatory pain.

Combination Treatment with EGFR Inhibitor and Doxorubicin Synergistically Inhibits Proliferation of MCF-7 Cells and MDA-MB-231 Triple-Negative Breast Cancer Cells In Vitro.

The role of the epidermal growth factor receptor (EGFR) in tumor progression and survival is often underplayed. Its expression and/or dysregulation is associated with disease advancement and poor patient outcome as well as drug resistance in breast cancer. EGFR is often overexpressed in breast cancer and particularly triple-negative breast cancer (TNBC), which currently lacks molecular targets. We examined the synergistic potential of an EGFR inhibitor (EGFRi) in combination with doxorubicin (Dox) in estrogen-positive (ER+) MCF-7 and MDA-MB-231 TNBC cell lines. The exposure of MDA-MB-231 and MCF-7 to EGFRi produced an IC50s of 6.03 µM and 3.96 µM, respectively. Dox induced MDA-MB-231 (IC50 9.67 µM) and MCF-7 (IC50 1.4 µM) cytotoxicity. Combinations of EGFRi-Dox significantly reduced the IC50 in MCF-7 (0.46 µM) and MBA-MB 231 (0.01 µM). Synergistic drug interactions in both cell lines were confirmed using the Bliss independence model. Pro-apoptotic Caspase-3/7 activation occurred in MCF-7 at 0.1-10 µM of EGFRi and Dox single treatments, whilst 1 μM Dox yielded a more potent effect on MDA-MB-231. EGFRi and Dox individually and in combination downregulated the EGFR gene expression in MCF-7 and MDA-MB-231 (p < 0.001). This study demonstrates EGFRi's potential for eliciting synergistic interactions with Dox, causing enhanced growth inhibition, apoptosis induction, and downregulation of EGFR in both cell lines.

SERPINE2 promotes liver cancer metastasis by inhibiting c-Cbl-mediated EGFR ubiquitination and degradation.

Liver cancer is a malignancy with high morbidity and mortality rates. Serpin family E member 2 (SERPINE2) has been reported to play a key role in the metastasis of many tumors. In this study, we aimed to investigate the potential mechanism of SERPINE2 in liver cancer metastasis. The Cancer Genome Atlas database (TCGA), including DNA methylation and transcriptome sequencing data, was utilized to identify the crucial oncogene associated with DNA methylation and cancer progression in liver cancer. Data from the TCGA and RNA sequencing for 94 pairs of liver cancer tissues were used to explore the correlation between SERPINE2 expression and clinical parameters of patients. DNA methylation sequencing was used to detect the DNA methylation levels in liver cancer tissues and cells. RNA sequencing, cytokine assays, immunoprecipitation (IP) and mass spectrometry (MS) assays, protein stability assays, and ubiquitination assays were performed to explore the regulatory mechanism of SERPINE2 in liver cancer metastasis. Patient-derived xenografts and tumor organoid models were established to determine the role of SERPINE2 in the treatment of liver cancer using sorafenib. Based on the public database screening, SERPINE2 was identified as a tumor promoter regulated by DNA methylation. SERPINE2 expression was significantly higher in liver cancer tissues and was associated with the dismal prognosis in patients with liver cancer. SERPINE2 promoted liver cancer metastasis by enhancing cell pseudopodia formation, cell adhesion, cancer-associated fibroblast activation, extracellular matrix remodeling, and angiogenesis. IP/MS assays confirmed that SERPINE2 activated epidermal growth factor receptor (EGFR) and its downstream signaling pathways by interacting with EGFR. Mechanistically, SERPINE2 inhibited EGFR ubiquitination and maintained its protein stability by competing with the E3 ubiquitin ligase, c-Cbl. Additionally, EGFR was activated in liver cancer cells after sorafenib treatment, and SERPINE2 knockdown-induced EGFR downregulation significantly enhanced the therapeutic efficacy of sorafenib against liver cancer. Furthermore, we found that SERPINE2 knockdown also had a sensitizing effect on lenvatinib treatment. SERPINE2 promoted liver cancer metastasis by preventing EGFR degradation via c-Cbl-mediated ubiquitination, suggesting that inhibition of the SERPINE2-EGFR axis may be a potential target for liver cancer treatment.

Cantharidin induces apoptosis of human triple negative breast cancer cells through mir-607-mediated downregulation of EGFR

BackgroundTriple negative breast cancer (TNBC) is a major subtype of breast cancer, with limited therapeutic drugs in clinical. Epidermal growth factor receptor (EGFR) is reported to be overexpressed in various TNBC cells. Cantharidin is an effective ingredient in many clinical traditional Chinese medicine preparations, such as Delisheng injection, Aidi injection, Disodium cantharidinate and vitamin B6 injection. Previous studies showed that cantharidin had satisfactory pharmacological activity on a variety of tumors. In this study, we aimed to study the therapeutic potential of cantharidin for TNBC treatment by targeting EGFR, and expound its novel regulator miR-607.MethodsThe effect of cantharidin on breast cancer in vivo was evaluated by 4T1 mice model. Then the effects of cantharidin on TNBC cells was assessed by the MTT, colony formation, and AnnexinV-PE/7AAD staining. Cantharidin acts on EGFR were verified using the cell membrane chromatography, RT-PCR, Western blotting, MTT, and so on. Mechanistic studies were explored by dual-luciferase report assay, RT-PCR, western blotting, and immunofluorescence staining assay.ResultsCantharidin inhibited TNBC cell growth and induce apoptosis by targeting EGFR. miR-607 was a novel EGFR regulator and exhibited suppressive functions on TNBC cell behaviors. Mechanistic study showed that cantharidin blocked the downstream PI3K/AKT/mTOR and ERK/MAPK signaling pathway.ConclusionOur results revealed that cantharidin may be served as a potential candidate for TNBC treatment by miR-607-mediated downregulation of EGFR.Graphical

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.

Dose-escalation trial of combination dabrafenib, trametinib, and AT13387 in patients with BRAF-mutant solid tumors.

Combination BRAF and MEK inhibitor therapy is an active regimen in patients who have BRAF V600E-mutated tumors; however, the clinical efficacy of this therapy is limited by resistance. Preclinically, the addition of heat shock protein 90 (HSP90) inhibition improves the efficacy of BRAF inhibitor therapy in both BRAF inhibitor-sensitive and BRAF inhibitor-resistant mutant cell lines. Cancer Therapy Evaluation Program study 9557 (ClinicalTrials.gov identifier NCT02097225) is a phase 1 study that was designed to assess the safety and efficacy of the small-molecule HSP90 inhibitor, AT13387, in combination with dabrafenib and trametinib in BRAF V600E/K-mutant solid tumors. Correlative analyses evaluated the expression of HSP90 client proteins and chaperones. Twenty-two patients with metastatic, BRAF V600E-mutant solid tumors were enrolled using a 3+3 design at four dose levels, and 21 patients were evaluable for efficacy assessment. The most common tumor type was colorectal cancer (N=12). Dose-limiting toxicities occurred in one patient at dose level 3 and in one patient at dose level 4; specifically, myelosuppression and fatigue, respectively. The maximum tolerated dose was oral dabafenib 150mg twice daily, oral trametinib 2mg once daily, and intravenous AT13387 260mg/m2 on days 1, 8, and 15. The best response was a partial response in two patients and stable disease in eight patients, with an overall response rate of 9.5% (90% exact confidence interval [CI], 2%-27%), a disease control rate of 47.6% (90% CI, 29%-67%), and a median overall survival of 5.1months (90% CI, 3.4-7.6 months). There were no consistent proteomic changes associated with response or resistance, although responders did have reductions in BRAF expression, and epidermal growth factor receptor downregulation using HSP90 inhibition was observed in one patient who had colorectal cancer. HSP90 inhibition combined with BRAF/MEK inhibition was safe and produced evidence of modest disease control in a heavily pretreated population. Additional translational work may identify tumor types and resistance mechanisms that are most sensitive to this approach.

Estrone analogs as potential inhibitors targeting EGFR-MAPK pathway in non-small-cell lung cancer.

Lung cancer is the deadliest human cancer globally, with non-small-cell lung cancer (NSCLC) being the most frequent type. Epidermal growth factor receptor (EGFR), a central regulator of tumor progression is frequently overexpressed in NSCLC and is a key drug target along with its downstream pathways. Here, we describe the biological evaluation of previously synthesized estrone analogs as potent inhibitors of NCI-H226 cells. Two of the analogs, MMA307 and MM320, significantly inhibited the proliferation of NCI-H226 cells with IC50 doses of 2.88 ± 0.21 and 9.68 ± 0.24 μM, respectively, compared with the positive control and chemotherapy, sorafenib, IC50 of 20.62 ± 1.32 μM. Exposing NCI-H226 cells to IC50 concentration of MMA307 and MMA320 resulted in the downregulation of EGFR and phospho-EGFR expression levels, and suppression of activated MAPK-ERK1/2 signaling proteins; phospho-B-Raf, phospho-MEK1/2 , and phospho-ERK1/2 . Furthermore, the downregulation of cyclin D1 and concomitant upregulation of phospho-cyclin D1 and p21waf1/cip1 were observed after the compounds' addition to NCI-H226 cells resulting in G1 phase cell cycle arrest. MMA320 but not MMA307 downregulated the expression levels of Dyrk1B, a checkpoint kinase at the G1 -S phase transition of the cell cycle. Additionally, molecular dynamic simulations were performed and found that MMA307 and MMA320 have higher binding affinities than sorafenib in MEK, BRAF, cyclin D1 , and Dyrk1B (dual-specificity tyrosine phosphorylation-regulated kinase 1B). To conclude, the present study is the first to report on the antiproliferative potential of novel estrone analogs and provide evidence that MMA307 and MMA320 are promising novel lead candidates for the development of antilung cancer drugs.

Flotillin-2 regulates epidermal growth factor receptor activation, degradation by Cbl-mediated ubiquitination, and cancer growth

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Cbl (Casitas B-lineage lymphoma proto-oncogene) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. Here, we used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and endosomal trafficking. Furthermore, we determined that FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl-dependent, as co-knockdown of Cbl and Cbl-b restored EGFR levels. Additionally, FLOT2 overexpression decreased EGFR signaling and growth. Overexpression of wild type (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. Together, these data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation in vitro and in vivo.

Proteasomal deubiquitylase activity enhances cell surface recycling of the epidermal growth factor receptor in non-small cell lung cancer.

The epidermal growth factor receptor (EGFR) represents a top therapeutic target in the treatment of non-small cell lung cancer. EGFR expression is intricately modulated by receptor endocytosis, during which EGFR ubiquitylation and deubiquitylation play fundamental roles to govern receptor fate. This study aims to uncover novel aspects of the endocytic regulation of EGFR trafficking by deubiquitylases. The expression and ubiquitylation of EGFR in non-small cell lung cancer cells treated with deubiquitylase inhibitors were assessed by immunoblotting, immunoprecipitation and mass spectrometry analyses. The intracellular EGFR distribution was investigated using immunofluorescence and confocal microscopy assays, and colocalizations with endocytic compartments were examined using GFP-tagged Rab proteins as markers. The influence of the proteasomal deubiquitylase inhibitor b-AP15 on EGF- and HSP90 inhibitor-induced EGFR downregulation was evaluated by immunoblotting. The anticancer effects of b-AP15 were assessed by cell proliferation, colony formation and flow cytometry assays, as well as xenograft animal models. We found that b-AP15 caused a dramatically enhanced ubiquitylation of EGFR in lung cancer cells. Treatment with b-AP15 decreased cell surface EGFR levels and accumulated EGFR on recycling endosomes marked with Rab4A and Rab11A. b-AP15 effectively repressed EGF- and HSP90 inhibitor-induced EGFR degradation. Lung cancer cells exposed to b-AP15 showed markedly reduced cell propagation and significantly increased cell apoptosis. Furthermore, b-AP15 effectively inhibited tumor xenograft growth in nude mice. Proteasomal USP14 and UCHL5 act collectively to promote cell surface recovery of EGFR. Inhibition of proteasomal deubiquitylase activity induces increased EGFR ubiquitylation and retention on recycling endosomes. The USP14 and UCHL5 dual inhibitor b-AP15 elicits potent tumor-suppressive effects to deter cell proliferation and induce apoptotic cell death in lung cancer.

MiR-141 exacerbates lung ischemia-reperfusion injury by targeting EGFR/β-catenin axis-mediated autophagy.

Some microRNAs (miRNAs) play important roles in lung ischemia-reperfusion injury (LIRI) injury. Here, this study aimed to examine whether miR-141 was related to lung ischemia-reperfusion injury (IRI) via regulating autophagy and the epidermal growth factor receptor (EGFR), and to explore the underlying signal transduction pathways. To this end, we constructed the LIRI cell model and mouse models, separately. According to RT-qPCR and Western blotting (WB) analysis results, miR-141 up-regulation together with β-catenin and EGFR down-regulation within mouse pulmonary microvascular endothelial cells (PMVECs) or lung tissues was related to lung IRI. Besides, we conducted dual-luciferase reporter assay, which suggested the binding of EGFR to miR-141. In addition, we carried out TUNEL staining, HE staining, and flow cytometric analysis to assess the apoptosis of PMVECs and the injury to mouse lung tissues. Furthermore, we performed light-chain immunofluorescence assay to examine autophagosomes within PMVECs. According to our results, miR-141 suppressed β-catenin level through reducing EGFR level. Besides, the miR-141/EGFR/β-catenin axis enhanced autophagy to aggravate LIRI. To sum up, miR-141 suppresses EGFR expression to inhibit β-catenin level, which subsequently aggravates autophagy and complicates LIRI. The above results offer the candidate therapeutic target for the treatment of lung IRI.

Brusatol Inhibits Proliferation, Migration, and Invasion of Nonsmall Cell Lung Cancer PC-9 Cells

Objective: The purpose of this study was to investigate the inhibitory effect of brusatol, a nigakilactone extracted from Brucea javanica, on lung cancer for development of therapeutic drugs. We explored the effects of brusatol on the proliferation, migration, and invasion of lung cancer PC-9 cells in vitro and analyzed the mechanisms involved. Materials and Methods: MTT assay was used to determine the effect of brusatol on the proliferative capacity of PC-9 and H1975 cells in vitro. The half-maximal inhibitory concentrations (IC50) were calculated and used as a reference for subsequent experiments. Variations in the number and size of tumor cell clusters were monitored by the colony formation assay as evidence for the effect of brusatol on cell proliferation. The effect of brusatol on the migration and invasion of PC-9 cells was evaluated using wound healing and transwell assays, respectively. Apoptosis in lung cancer cells was detected using the Annexin V-FITC/propidium iodide assay. The correlated anticancer mechanism was detected using Western blotting. Results: The IC50 values of brusatol acting on PC-9 and H1975 cells were 1.58 ± 0.30 mM and 31.34 ± 2.72 mM, respectively, according to the MTT experiment. In addition, brusatol suppressed PC-9 cell proliferation, migration, and invasion, as well as induced apoptosis, which may be related to the downregulation of epidermal growth factor receptor (EGFR), β-catenin, Akt, and STAT3 expression. Conclusions: Brusatol showed potent anticancer activity against lung cancer PC-9 cells, inhibiting the proliferative capacity and metastatic potential of PC-9 cells. Its anticancer effect may be related to the downregulation of EGFR, β-catenin, Akt, and STAT3.

The Abl/Abi signaling links WAVE regulatory complex to Cbl E3 ubiquitin ligase and is essential for breast cancer cell metastasis

The family of Abelson interactor (Abi) proteins is a component of WAVE regulatory complex (WRC) and a downstream target of Abelson (Abl) tyrosine kinase. The fact that Abi proteins also interact with diverse membrane proteins and intracellular signaling molecules places these proteins at a central position in the network that controls cytoskeletal functions and cancer cell metastasis. Here, we identified a motif in Abi proteins that conforms to consensus sequences found in a cohort of receptor and non-receptor tyrosine kinases that bind to Cbl-tyrosine kinase binding domain. The phosphorylation of tyrosine 213 in this motif is essential for Abi degradation. Double knockout of c-Cbl and Cbl B in Bcr-Abl-transformed leukemic cells abolishes Abi1, Abi2, and WAVE2 degradation. Moreover, knockout of Abi1 reduces Src family kinase Lyn activation in Bcr-Abl-positive leukemic cells and promotes EGF-induced EGF receptor downregulation in breast cancer cells. Importantly, Abi1 depletion impeded breast cancer cell invasion in vitro and metastasis in mouse xenografts. Together, these studies uncover a novel mechanism by which the WRC and receptor/non-receptor tyrosine kinases are regulated and identify Abi1 as a potential therapeutic target for metastatic breast cancer.

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

Abstract Background &amp; 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 &amp; 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.

Abstract LB133: EGFR upregulation in breast cancer cells: A key mechanism of escape from antiestrogen induced death, autophagy, and senescence

Abstract EGFR is upregulated commonly in multiple cancer types, including breast cancer. However, the multiple role(s) of EGFR in breast cancer are not fully elucidated. In this study, we characterized a unique antiestrogen resistant breast cancer subline and provide compelling data that upregulation of the EGFR/MEK1/MAPK1/2 signaling axis during antiestrogen treatment facilitated escape from senescence, autophagy, and BimEL-dependent apoptosis. Our studies were conducted with the antiestrogen resistant TR5 cell line established by our laboratory, using a step-wise drug selection with the antiestrogen 4-hydroxytamoxifen (4-OHT) of the estrogen receptor α (ER+) MCF-7 breast cancer cells. A preliminary microarray comparing mRNA expression between 4-OHT-selected TR5 cells and MCF-7 parent cells identified upregulation of the epidermal growth factor receptor (EGFR). Western blot showed a greater than 5-fold upregulation of EGFR expression and downregulation of estrogen receptor alpha (ERα) compared to the levels in MCF-7 and T47-D (antiestrogen sensitive). Selective inhibition of EGFR, phosphorylated at Tyr 1068, was achieved with erlotinib. Inhibition blocked MEK1/MAPK1/2 signaling, which was also upregulated in 4-OHT selected TR5 cells, with detectable increases in the pro-apoptotic BH3 protein BimEL, and a fifty percent reduction in cell viability. However, a high number of cells survived erlotinib therapy due to autophagy induction determined to be cytoprotective. Autophagy induction was identified by analyzing the expression/turnover of autophagy proteins LC3-II and p62, a standard approach to ascertain autophagy levels in cells. Further, erlotinib treatment induced a 2-fold increase in the number of senescent cells in the surviving population. Senescent cells were identified by β-galactosidase staining of erlotinib-treated cells utilizing confocal microscopy. Interestingly, blockade of autophagy with the lysosomotrophic compound chloroquine enhanced the senescence cell population, suggesting that either autophagy blocked senescence induction or lysosome impairment increased senescence. Mechanistically, we have identified phosphorylated AMP-activated protein kinase (pAMPK) as being elevated under conditions of erlotinib treatment, and reduced in 4-OHT-selected TR5 cells relative to MCF-7 parent cells. This correlation supports the hypothesis that pAMPK was downregulated during 4-OHT selection of TR5 cells by elevated EGFR/MEK/MAPK1/2 signaling to allow surviving cells escape senescence induction and/or anti-proliferative autophagy. Overall we propose that EGFR mediated downregulation of pAMPK is a major regulatory pathway in breast cancer cells surviving endocrine treatment. Our current studies are dissecting the role of EGFR-mediated AMPK regulation with somatic cell-based studies and pharmacological intervention. Citation Format: Mackenzie L. Hagan, Michael McGrath, Carol Joseph, Allison Lewis, John R. Barrett, Muthusamy Thangaraju, David Gewirtz, Patricia V. Schoenlein. EGFR upregulation in breast cancer cells: A key mechanism of escape from antiestrogen induced death, autophagy, and senescence [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 LB133.

Inhibitors of ERp44, PDIA1, and AGR2 induce disulfide-mediated oligomerization of Death Receptors 4 and 5 and cancer cell death

Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8.

T-box transcription factor 19 promotes hepatocellular carcinoma metastasis through upregulating EGFR and RAC1.

The effect of targeted therapy for metastatic hepatocellular carcinoma (HCC) is still unsatisfactory. Exploring the underlying mechanism of HCC metastasis is favorable to provide new therapeutic strategies. T-box (TBX) transcription factor family genes, which are crucial regulators in embryo and organ development, are vital for regulating tumor initiation, growth and metastasis. Here we explored the role of TBX19 in HCC metastasis, which is one of the most upregulated TBX family genes in human HCC tissues. TBX19 expression was markedly upregulated in HCC tissues and elevated TBX19 expression predicted poor prognosis. Overexpression of TBX19 enhanced HCC metastasis through upregulating epidermal growth factor receptor (EGFR) and Rac family small GTPase 1 (RAC1) expression. Downregulation of EGFR and RAC1 inhibited TBX19-mediated HCC metastasis, while upregulation of EGFR and RAC1 restored inhibition of HCC metastasis mediated by TBX19 knockdown. Furthermore, epidermal growth factor (EGF)/EGFR signaling upregulated TBX19 expression via the extracellular signal-regulated kinase (ERK)/nuclear factor (NF)-kB axis. Besides, the combined application of EGFR inhibitor Erlotinib and RAC1 inhibitor NSC23766 markedly inhibited TBX19-mediated HCC metastasis. In HCC cohorts, TBX19 expression was positively associated with EGFR and RAC1 expression. Patients with positive coexpression of TBX19/EGFR or TBX19/RAC1 displayed the poorest prognosis. In conclusion, EGF/EGFR signaling upregulated TBX19 expression via ERK/NF-kB pathway and TBX19 fostered HCC metastasis by enhancing EGFR and RAC1 expression, which formed an EGF-TBX19-EGFR positive feedback loop. Targeting this signaling pathway may offer a potential therapeutic strategy to efficiently restrain TBX19-mediated HCC metastasis.

Root extract of Plumbago zeylanica L. induces cytotoxicity, inhibits cell migration and induces S-phase cell cycle arrest through down regulation of EGFR in HeLa cervical cancer cells

Plumbago zeylanica has been used in the traditional system of medicine from thousands of years owing to its potential therapeutic properties, however the anticancer and anti-metastatic effects are largely unknown against cervical cancer cells. In this study we demonstrated the cytotoxicity activity, inhibition of cell migration, induction of S-phase cell cycle arrest and the down regulation of expression level of epidermal growth factor receptor (EGFR) by ethanol extract of the root of P. zeylanica (ERPZ). The cytotoxicity effects were analyzed by MTT assay the IC50 was determined to be 10.49 ​μg/ml after 72 ​h of incubation. Besides, strong inhibition of wound healing activity was observed at 20 ​μg/ml concentration, where the wound size was reduced to 33%. The ethanol root extract of P. zeylanica also showed significant S-phase cell cycle arrest of 68.9% at 20 ​μg/ml concentration in HeLa cells which was accompanied by the down regulation of EGFR. Ethanol extracts of the root of P. zeylanica (ERPZ) treatment inhibited the growth of cervical cancer cells. The ERPZ arrested cells at the S-phase of the cell cycle. Besides the HRLC-MS analysis of ERPZ identified 15 compounds and among which the five major compounds such as 7,8-dihydroxy-4-methylcoumarin, neodiosmin, diosmetin, hispidulin, and formononetin were reported to possess antioxidant and anticancer activities, could plausibly induce cell death in HeLa cells.

MicroRNA-133b Inhibition Restores EGFR Expression and Accelerates Diabetes-Impaired Wound Healing.

Diabetic foot ulcers (DFUs) are caused by impairments in peripheral blood vessel angiogenesis and represent a great clinical challenge. Although various innovative techniques and drugs have been developed for treating DFUs, therapeutic outcomes remain unsatisfactory. Using the GEO database, we obtained transcriptomic microarray data for DFUs and control wounds and detected a significant downregulation of epidermal growth factor receptor (EGFR) in DFUs. We cultured human umbilical vein endothelial cells (HUVECs) and noted downregulated EGFR expression following high-glucose exposure in vitro. Further, we observed decreased HUVEC proliferation and migration and increased apoptosis after shRNA-mediated EGFR silencing in these cells. In mice, EGFR inhibition via focal EGFR-shRNA injection delayed wound healing. Target prediction analysis followed by dual-luciferase reporter assays indicated that microRNA-133b (miR-133b) is a putative upstream regulator of EGFR expression. Increased miR-133b expression was observed in both glucose-treated HUVECs and wounds from diabetes patients, but no such change was observed in controls. miR-133b suppression enhanced the proliferation and angiogenic potential of cultured HUVECs and also accelerated wound healing. Although angiogenesis is not the sole mechanism affected in DFU, these findings suggest that the miR-133b-induced downregulation of EGFR may contribute to delayed wound healing in diabetes. Hence, miR-133b inhibition may be a useful strategy for treating diabetic wounds.

Downregulation of Pinkbar/pAKT and MMP2/MMP9 Expression in MDA-MB-231 Breast Cancer Cells as Potential Targets in Cancer Therapy by hAMSCs Secretome

Breast cancer is one of the leading causes of cancer-related deaths among women worldwide. Cancer therapy based on stem cells is considered as a novel and promising platform. In the present study, we explored the therapeutic effects of human amniotic mesenchymal stromal cells (hAMSCs) through Pinkbar (planar intestinal- and kidney-specific BAR domain protein), pAKT, and matrix metalloproteinases including MMP2 and MMP9 on MDA-MB-231 breast cancer cells. For this purpose, we employed a co-culture system using Transwell 6-well plates with a pore size of 0.4 μm. After 72 h, the hAMSCs-treated MDA-MB-231 breast cancer cells, the expression of epidermal growth factor receptor (EGFR), and c-Src (a key mediator in EGFR signaling pathway), Pinkbar, pAKT, MMP2, and MMP9 were analyzed using quantitative real time PCR and western blot methods. Based on 2D and 3D cell culture models, significant reduction of tumor cell growth and motility through downregulation of EGFR, c-Src, Pinkbar, pAKT, MMP2, and MMP9 were found in MDA-MB-231 breast cancer cells. Moreover, induction of cellular apoptosis was also reported. Our finding indicates that the hAMSCS secretome has therapeutic effects on cancer cells. To identify the details of the molecular mechanisms, more experiments will be required.

Multiomic study of skin, peripheral blood, and serum: is serum proteome a reflection of disease process at the end-organ level in systemic sclerosis?

BackgroundSerum proteins can be readily assessed during routine clinical care. However, it is unclear to what extent serum proteins reflect the molecular dysregulations of peripheral blood cells (PBCs) or affected end-organs in systemic sclerosis (SSc). We conducted a multiomic comparative analysis of SSc serum profile, PBC, and skin gene expression in concurrently collected samples.MethodsGlobal gene expression profiling was carried out in skin and PBC samples obtained from 49 SSc patients enrolled in the GENISOS observational cohort and 25 unaffected controls. Levels of 911 proteins were determined by Olink Proximity Extension Assay in concurrently collected serum samples.ResultsBoth SSc PBC and skin transcriptomes showed a prominent type I interferon signature. The examination of SSc serum profile revealed an upregulation of proteins involved in pro-fibrotic homing and extravasation, as well as extracellular matrix components/modulators. Notably, several soluble receptor proteins such as EGFR, ERBB2, ERBB3, VEGFR2, TGFBR3, and PDGF-Rα were downregulated. Thirty-nine proteins correlated with severity of SSc skin disease. The differential expression of serum protein in SSc vs. control comparison significantly correlated with the differential expression of corresponding transcripts in skin but not in PBCs. Moreover, the differentially expressed serum proteins were significantly more connected to the Well-Associated-Proteins in the skin than PBC gene expression dataset. The assessment of the concordance of between-sample similarities revealed that the molecular profile of serum proteins and skin gene expression data were significantly concordant in patients with SSc but not in healthy controls.ConclusionsSSc serum protein profile shows an upregulation of profibrotic cytokines and a downregulation of soluble EGF and other key receptors. Our multilevel comparative analysis indicates that the serum protein profile in SSc correlates more closely with molecular dysregulations of skin than PBCs and might serve as a reflection of disease severity at the end-organ level.