Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase that is frequently modified by glycosylation post-translationally. In cancer, EGFR amplifications and hotspot mutations such as L858R that promote proliferation have been detected in a significant fraction of non-small cell lung carcinomas and breast adenocarcinomas. Molecular dynamic simulations suggested that glycosylation at asparagine residue 361 (N361) promotes dimerization and ligand binding. We stably expressed glycosylation-deficient mutant EGFR N361A, with or without the oncogenic mutation L858R. Immunofluorescence and flow cytometry demonstrated that the mutants were each well expressed at the cell membrane. N361A decreased proliferation relative to wild-type EGFR as well as decreased sensitivity to ligands. Proximity ligation assays measuring co-localization of EGFR with its binding partner HER2 in cells revealed that N361A mutations increased co-localization. N361A, located near the binding interface for the EGFR inhibitor necitumumab, desensitized cells expressing the oncogenic EGFR L858R to antibody-based inhibition. These findings underline the critical relevance of post-translational modifications on oncogene function.

Gingival fibroblasts are a significant source of paracrine signals required to maintain periodontal homeostasis and to mediate pathological events linked to periodontitis and oral squamous cell carcinomas. Among the potential paracrine signals are stanniocalcin-1 (STC1), involved in oxidative stress and cellular survival; amphiregulin (AREG), a growth factor that mediates the cross-talk between immune cells and epithelial cells; chromosome 11 open reading frame 96 (C11orf96) with an unclear biologic function; and the inflammation-associated prostaglandin E synthase (PTGES). Gingival fibroblasts increasingly express these genes in response to bone allografts containing remnants of injured cells. Thus, the gene expression might be caused by the local release of damage-associated molecular patterns arising from injured cells. The aim of this study is consequently to use the established gene panel as a bioassay to measure the damage-associated activity of oral cell lysates. To this aim, we have exposed gingival fibroblasts to lysates prepared from the squamous carcinoma cell lines TR146 and HSC2, oral epithelial cells, and gingival fibroblasts. We report here that all lysates significantly increased the transcription of the entire gene panel, supported for STC1 at the protein level. Blocking TGF-β receptor 1 kinase with SB431542 only partially reduced the forced expression of STC1, AREG, and C11orf96. SB431542 even increased the PTGES expression. Together, these findings suggest that the damage signals originating from oral cells can change the paracrine activity of gingival fibroblasts. Moreover, the expression panel of genes can serve as a bioassay for testing the biocompatibility of materials for oral application.

Mesenchymal cells within theplacental villi play a crucial role in shaping the morphology of branching structures and driving the development of blood vessels. However, the markers and functions of placental villous pericytes (PVPs) as distinct subgroups of placental villous mesenchymal cells, remain unclear. Therefore, in this study, the markers and functions of PVPs were investigated. Single-cell sequencing data from the first-trimester placental villi was obtained and the Seurat tool was used to identify PVP markers. Gene Ontology (GO) analysis of specific genes was performed using the DAVID database. The Cellchat tool was employed to investigate the interaction signals between the PVPs and other cells. Expression of the PVP markers was confirmed using immunofluorescence. Presence of extracellular vesicles in the placental villous mesenchyme and PVPs was examined by transmission electron microscopy. Our findings revealed that renin (REN) and amphiregulin (AREG)-positive fibroblasts in the placental villi specifically expressed several classic pericyte markers. In the first trimester, certain conserved functions of pericytes were observed and they displayed tissue-specific functions such as in the integrin-mediated signaling pathway and extracellular exosomes. Moreover, the placental villous mesenchyme was found to be rich in extracellular vesicles. AREG is specifically transcribed in the first trimester PVPs, however, its protein was located in syncytiotrophoblasts. These insights contribute to a comprehensive understanding of early placental development and offer new therapeutic targets for placenta-derived pregnancy complications.

Exosomes are secreted vesicles made intracellularly in the endosomal system. We have previously shown that exosomes are not only made in late endosomes, but also in recycling endosomes marked by the monomeric G-protein Rab11a. These vesicles, termed Rab11a-exosomes, are preferentially secreted under nutrient stress from several cancer cell types, including HCT116 colorectal cancer (CRC) cells. HCT116 Rab11a-exosomes have particularly potent signalling activities, some mediated by the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG). Mutant activating forms of KRAS, a downstream target of EGFR, are often found in advanced CRC. When absent, monoclonal antibodies, such as cetuximab, which target the EGFR and block the effects of EGFR ligands, such as AREG, can be administered. Patients, however, inevitably develop resistance to cetuximab, either by acquiring KRAS mutations or via non-genetic microenvironmental changes. Here we show that nutrient stress in several CRC cell lines causes the release of AREG-carrying Rab11a-exosomes. We demonstrate that while soluble AREG has no effect, much lower levels of AREG bound to Rab11a-exosomes from cetuximab-resistant KRAS-mutant HCT116 cells, can suppress the effects of cetuximab on KRAS-wild type Caco-2 CRC cells. Using neutralising anti-AREG antibodies and an intracellular EGFR kinase inhibitor, we show that this effect is mediated via AREG activation of EGFR, and not transfer of activated KRAS. Therefore, presentation of AREG on Rab11a-exosomes affects its ability to compete with cetuximab. We propose that this Rab11a-exosome-mediated mechanism contributes to the establishment of resistance in cetuximab-sensitive cells and may explain why in cetuximab-resistant tumours only some cells carry mutant KRAS.

UNASSIGNED: Muscle invasive bladder cancer (MIBC) remains a prevalent cancer with limited therapeutic options, obviating the need for innovative therapies. The epidermal growth factor receptor (EGFR) is a linchpin in tumor progression and presents a potential therapeutic target in MIBC. Additionally, the EGFR ligands AREG and EREG have shown associations with response to anti-EGFR therapy and improved progression-free survival in colorectal carcinoma.
UNASSIGNED: We investigated the prognostic significance of EGFR, AREG, and EREG in MIBC. Gene expression and copy number analyses were performed via qRT-PCR on tissue samples from 100 patients with MIBC who underwent radical cystectomy at the University Hospital Mannheim (MA; median age 72, interquartile range [IQR] 64-78 years, 25% female). Results were validated in 361 patients from the 2017 TCGA MIBC cohort (median age 69, IQR 60-77 years, 27% female), in the Chungbuk and MDACC cohort. Gene expressions were correlated with clinicopathologic parameters using the Mann-Whitney test, Kruskal-Wallis- test and Spearman correlation. For overall survival (OS), cancer-specific survival (CSS) and disease-free survival (DFS) gene expression was analyzed with Kaplan-Meier and Cox-proportional hazard models.
UNASSIGNED: Significant gene expression differences in EGFR, AREG, and EREG could be detected in all cohorts. In the TCGA cohort, EGFR expression was significantly elevated in patients with EGFR amplification and KRAS wildtype. High AREG expression independently predicted longer OS (HR = 0.35, CI 0.19 - 0.63, p = 0.0004) and CSS (HR = 0.42, CI 0.18 - 0.95, p = 0.0378) in the MA cohort. In the TCGA cohort, high EGFR, AREG, and EREG expression correlated with shorter OS (AREG: HR = 1.57, CI 1.12 - 2.20, p = 0.0090) and DFS (EGFR: HR = 1.91, CI 1.31 - 2.8, p = 0.0008). EGFR amplification was also associated with reduced DFS.
UNASSIGNED: High EGFR and EREG indicate worse survival in patients with MIBC. The prognostic role of AREG should further be investigated in large, prospective series. Divergent survival outcomes between the four cohorts should be interpreted cautiously, considering differences in analysis methods and demographics. Further in vitro investigations are necessary to elucidate the functional mechanisms underlying the associations observed in this study.

CD133, a cancer stem cell (CSC) marker in tumors, including melanoma, is associated with tumor recurrence, chemoresistance, and metastasis. Patient-derived melanoma cell lines were transduced with a Tet-on vector expressing CD133, generating doxycycline (Dox)-inducible cell lines. Cells were exposed to Dox for 24 h to induce CD133 expression, followed by RNA-seq and bioinformatic analyses, revealing genes and pathways that are significantly up- or downregulated by CD133. The most significantly upregulated gene after CD133 was amphiregulin (AREG), validated by qRT-PCR and immunoblot analyses. Induced CD133 expression significantly increased cell growth, percentage of cells in S-phase, BrdU incorporation into nascent DNA, and PCNA levels, indicating that CD133 stimulates cell proliferation. CD133 induction also activated EGFR and the MAPK pathway. Potential mechanisms highlighting the role(s) of CD133 and AREG in melanoma CSC were further delineated using AREG/EGFR inhibitors or siRNA knockdown of AREG mRNA. Treatment with the EGFR inhibitor gefitinib blocked CD133-induced cell growth increase and MAPK pathway activation. Importantly, siRNA knockdown of AREG reversed the stimulatory effects of CD133 on cell growth, indicating that AREG mediates the effects of CD133 on cell proliferation, thus serving as an attractive target for novel combinatorial therapeutics in melanoma and cancers with overexpression of both CD133 and AREG.

BACKGROUND: Biliary atresia (BA) is a neonatal fibro-inflammatory cholangiopathy with ductular reaction as a key pathogenic feature predicting poor survival. Mucosal-associated invariant T (MAIT) cells are enriched in human liver and display multiple roles in liver diseases. We aimed to investigate the function of MAIT cells in BA.
METHODS: First, we analyzed correlations between liver MAIT cell and clinical parameters (survival, alanine transaminase, bilirubin, histological inflammation and fibrosis) in two public cohorts of patients with BA (US and China). Kaplan-Meier survival analysis and spearman correlation analysis were employed for survival data and other clinical parameters, respectively. Next, we obtained liver samples or peripheral blood from BA and control patients for bulk RNA sequencing, flow cytometry analysis, immunostaning and functional experiments of MAIT cells. Finally, we established two in vitro co-culture systems, one is the rhesus rotavirus (RRV) infected co-culture system to model immune dysfunction of human BA which was validated by single cell RNA sequencing and the other is a multicellular system composed of biliary organoids, LX-2 and MAIT cells to evaluate the role of MAIT cells on ductular reaction.
RESULTS: Liver MAIT cells in BA were positively associated with low survival and ductular reaction. Moreover, liver MAIT cells were activated, exhibited a wound healing signature and highly expressed growth factor Amphiregulin (AREG) in a T cell receptor (TCR)-dependent manner. Antagonism of AREG abrogated the proliferative effect of BA MAIT cells on both cholangiocytes and biliary organoids. A RRV infected co-culture system, recapitulated immune dysfunction of human BA, disclosed that RRV-primed MAIT cells promoted cholangiocyte proliferation via AREG, and further induced inflammation and fibrosis in the multicellular system.
CONCLUSIONS: MAIT cells exhibit a wound healing signature depending on TCR signaling and promote ductular reaction via AREG, which is associated with advanced fibrosis and predictive of low survival in BA.
BACKGROUND: This work was funded by National Natural Science Foundation of China grant (82001589 and 92168108), National Key R&D Program of China (2023YFA1801600) and by Basic and Applied Basic Research Foundation of Guangdong (2020A1515110921).

Amphiregulin (AREG) serves as a ligand for the epidermal growth factor receptor (EGFR) and is involved in vital biological functions, including inflammatory responses, tissue regeneration, and immune system function. Upon interaction with the EGFR, AREG initiates a series of signaling cascades necessary for several physiological activities, such as metabolism, cell cycle regulation, and cellular proliferation. Recent findings have provided evidence for the substantial role of AREG in maintaining the equilibrium of homeostasis in damaged tissues and preserving epithelial cell structure in the context of viral infections affecting the lungs. The development of resistance to influenza virus infection depends on the presence of type 1 cytokine responses. Following the eradication of the pathogen, the lungs are subsequently colonized by several cell types that are linked with type 2 immune responses. These cells contribute to the process of repairing and resolving the tissue injury and inflammation caused by infections. Following influenza infection, the activation of AREG promotes the regeneration of bronchial epithelial cells, enhancing the tissue\'s structural integrity and increasing the survival rate of infected mice. In the same manner, mice afflicted with influenza experience rapid mortality due to a subsequent bacterial infection in the pulmonary region when both bacterial and viral infections manifest concurrently inside the same host. The involvement of AREG in bacterial infections has been demonstrated. The gene AREG experiences increased transcriptional activity inside host cells in response to bacterial infections caused by pathogens such as Escherichia coli and Neisseria gonorrhea. In addition, AREG has been extensively studied as a mitogenic stimulus in epithelial cell layers. Consequently, it is regarded as a prospective contender that might potentially contribute to the observed epithelial cell reactions in helminth infection. Consistent with this finding, mice that lack the AREG gene exhibit a delay in the eradication of the intestinal parasite Trichuris muris. The observed delay is associated with a reduction in the proliferation rate of colonic epithelial cells compared to the infected animals in the control group. The aforementioned findings indicate that AREG plays a pivotal role in facilitating the activation of defensive mechanisms inside the epithelial cells of the intestinal tissue. The precise cellular sources of AREG in this specific context have not yet been determined. However, it is evident that the increased proliferation of the epithelial cell layer in infected mice is reliant on CD4+ T cells. The significance of this finding lies in its demonstration of the crucial role played by the interaction between immunological and epithelial cells in regulating the AREG-EGFR pathway. Additional research is necessary to delve into the cellular origins and signaling mechanisms that govern the synthesis of AREG and its tissue-protective properties, independent of infection.

Bone allografts are widely used as osteoconductive support to guide bone regrowth. Bone allografts are more than a scaffold for the immigrating cells as they maintain some bioactivity of the original bone matrix. Yet, it remains unclear how immigrating cells respond to bone allografts. To this end, we have evaluated the response of mesenchymal cells exposed to acid lysates of bone allografts (ALBA). RNAseq revealed that ALBA has a strong impact on the genetic signature of gingival fibroblasts, indicated by the increased expression of IL11, AREG, C11orf96, STC1, and GK-as confirmed by RT-PCR, and for IL11 and STC1 by immunoassays. Considering that transforming growth factor-β (TGF-β) is stored in the bone matrix and may have caused the expression changes, we performed a proteomics analysis, TGF-β immunoassay, and smad2/3 nuclear translocation. ALBA neither showed detectable TGF-β nor was the lysate able to induce smad2/3 translocation. Nevertheless, the TGF-β receptor type I kinase inhibitor SB431542 significantly decreased the expression of IL11, AREG, and C11orf96, suggesting that other agonists than TGF-β are responsible for the robust cell response. The findings suggest that IL11, AREG, and C11orf96 expression in mesenchymal cells can serve as a bioassay reflecting the bioactivity of the bone allografts.

BACKGROUND: With advanced maternal age, abnormalities during oocyte meiosis increase significantly. Aneuploidy is an important reason for the reduction in the quality of aged oocytes. However, the molecular mechanism of aneuploidy in aged oocytes is far from understood. Histone acetyltransferase 1 (HAT1) has been reported to be essential for mammalian development and genome stability, and involved in multiple organ aging. Whether HAT1 is involved in ovarian aging and the detailed mechanisms remain to be elucidated.
METHODS: The level of HAT1 in aged mice ovaries was detected by immunohistochemical and immunoblotting. To explore the function of HAT1 in the process of mouse oocyte maturation, we used Anacardic Acid (AA) and small interfering RNAs (siRNA) to culture cumulus-oocyte complexes (COCs) from ICR female mice in vitro and gathered statistics of germinal vesicle breakdown (GVBD), the first polar body extrusion (PBE), meiotic defects, aneuploidy, 2-cell embryos formation, and blastocyst formation rate. Moreover, the human granulosa cell (GC)-like line KGN cells were used to investigate the mechanisms of HAT1 in this progress.
RESULTS: HAT1 was highly expressed in ovarian granulosa cells (GCs) from young mice and the expression of HAT1 was significantly decreased in aged GCs. AA and siRNAs mediated inhibition of HAT1 in GCs decreased the PBE rate, and increased meiotic defects and aneuploidy in oocytes. Further studies showed that HAT1 could acetylate Forkhead box transcription factor O1 (FoxO1), leading to the translocation of FoxO1 into the nucleus. Resultantly, the translocation of acetylated FoxO1 increased the expression of amphiregulin (AREG) in GCs, which plays a significant role in oocyte meiosis.
CONCLUSIONS: The present study suggests that decreased expression of HAT1 in GCs is a potential reason corresponding to oocyte age-related meiotic defects and provides a potential therapeutic target for clinical intervention to reduce aneuploid oocytes.