Epidermal Growth Factor Stimulation Research Articles

Abstract 4389: The role of Rab5 in epithelial growth factor stimulated E-cadherin internalization in spheroid growth of colon cancer cells

Abstract In epithelial based cancer formation, such as colon cancer, the tumor growth is accompanied with malfunction of cell-cell contact inhibition which is regulated by E-cadherin. However, the dynamics of E-cadherin is complicated, the underlying mechanism in tumor nest growth and collective cell migration is still not clear. Rab5 is the endocytic vesicle protein for the membrane receptor internalization into early endosome. Therefore, this study is to investigate the role of Rab5 in E-cadherin turnover of spheroid formation in colon cancer cells. HT-29 colon cancer cells were used in this study. The cells with less anchorage ability were isolated for spheroid formation. The distribution of Rab5, E-cadherin and focal adhesion kinase were observed by immuno-confocal microscopy. The molecular expressions were analyzed by western blot. HT-29 cells showed well defined epithelial phenotype. In the result of immunostaining, Rab5 was distributed in the cytosol and the cell-cell contacts where it colocalized with E-cadherin. Rab5 was also observed in the cell leading edge, however, not much associated with focal adhesions. When HT-29 cell clusters were stimulated with epidermal growth factor, Rab5 was found abundant in the cell-cell contacts. In cell spheroid that derived from HT-29 cells, Rab5 and E-cadherin co-localization at cell-cell contacts was also increased. Furthermore, when cells were treated with Nocodazole, the tubulin inhibitor blocking tubulin-mediated endocytosis, Rab5 accumulated and colocalized with E-cadherin in the cell leading edge. The results of this study demonstrated Rab5 plays the novel role in E-cadherin turnover upon epidermal growth factor stimulation in the progress of spheroid formation and Rab5 may mediate E-cadherin endocytosis through the cell leading edge. Citation Format: Yu-Chun Wang, Yu-Chien Ching, Hui-Chun Chen, Chun-Pu Cheng, Chia-En Ku, Shin-Ru Lee, Wei-Ting Chao. The role of Rab5 in epithelial growth factor stimulated E-cadherin internalization in spheroid growth of colon cancer cells [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 4389.

Abstract 304: The role of DDR1 mediated E-cadherin stability in collective colon cancer cell migration

Abstract In cancer metastasis, the mechanism of single cell migration and collective cell migration are investigated. The dynamics of E-cadherin is critical in collective cell migration as the important mechanism of metastasis. DDR1 (Discoidin Domain Receptor 1) has been shown to promote E-cadherin stability at adherens junction in epithelial cells by regulating endocytosis of E-cadherin. However, in the growth of epithelial cell cluster and collective cell migration, the role of DDR1 in the cell-cell contacts is not clear. In this study, HT-29 colon cancer cells with epithelial phenotype were used in the experiments. Cells were treated with epidermal growth factor to stimulate E-cadherin turnover or applied with lysosome inhibitor chloroquine to interfere cellular vesicle transport. DDR1 and E-cadherin were revealed by immuno-confocal microscopy. The immunostaining results showed DDR1 was localized at cell-cell contacts and co-localized with E-cadherin. When cells were treated with epidermal growth factor, the phosphor MAPK activity was increased and DDR1 was localized at cell-cell contacts intensively. When lysosome activity was blocked by chloroquine treatment. The cellular vesicle transport and collective cell migration was reduced and furthermore inhibited DDR1 translocation to the cell-cell contacts. This study demonstrated DDR1 co-localized with E-cadherin is responsible for epidermal growth factor stimulation in cell cohort. Lysosome activity is required for E-cadherin turnover and affected DDR1 translocation. Chloroquine maybe the potential drug to suppress tumor nest growth and collective cell migration by interfere DDR1 mediated E-cadherin stability. Citation Format: Hui-Chun Chen, Yu-Chun Wang, Yu-Chien Ching, Chun-Pu Cheng, Chia-En Ku, Shin-Ru Lee, Wei-Ting Chao. The role of DDR1 mediated E-cadherin stability in collective colon cancer cell migration [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 304.

Growth factor-induced activation of MSK2 leads to phosphorylation of H3K9me2S10 and corresponding changes in gene expression.

Extracellular signals are transmitted through kinase cascades to modulate gene expression, but it remains unclear how epigenetic changes regulate this response. Here, we provide evidence that growth factor-stimulated changes in the transcript levels of many responsive genes are accompanied by increases in histone phosphorylation levels, specifically at histone H3 serine-10 when the adjacent lysine-9 is dimethylated (H3K9me2S10). Imaging and proteomic approaches show that epidermal growth factor (EGF) stimulation results in H3K9me2S10 phosphorylation, which occurs in genomic regions enriched for regulatory enhancers of EGF-responsive genes. We also demonstrate that the EGF-induced increase in H3K9me2S10ph is dependent on the nuclear kinase MSK2, and this subset of EGF-induced genes is dependent on MSK2 for transcription. Together, our work indicates that growth factor-induced changes in chromatin state can mediate the activation of downstream genes.

RSK/GSK3-mediated phosphorylation of FilGAP regulates chemotactic cancer invasion.

Cell migration plays a crucial role in various biological processes, such as gastrulation, immune response, and cancer metastasis. In response to chemoattractant-like growth factors, cells form protrusions and migrate toward the source of the signal. Rho family small GTPase Rac is a key regulator of cell migration by stimulating actin polymerization to generate lamellipodia, flat membrane protrusions at the leading edge of migrating cells. FilGAP (ARHGAP24), a Rac-specific GTPase-activating protein (GAP), suppresses lamellipodia formation, and controls tumor cell migration. In this study, we found that FilGAP is phosphorylated downstream of epidermal growth factor (EGF) signaling. Upon EGF stimulation, FilGAP is phosphorylated at Ser625 by p90 ribosomal S6 kinase (RSK) and then at Ser621 by glycogen synthase kinase 3 (GSK3). Phosphorylation of FilGAP induces its dissociation from actin filaments. We identified a novel actin-localization domain of FilGAP that is essential for stabilizing cell adhesion. Additionally, we found that phosphorylation of FilGAP inhibits its lamellipodia suppression activity. Finally, we showed the expression of nonphosphorylatable FilGAP mutant, but not wild-type FilGAP, reduced cell migration speed and persistence toward the EGF gradient. Taken together, our results suggest that phosphorylation of FilGAP downstream of EGF-signaling plays a critical role in regulating chemotactic tumor cell migration by controlling cell-matrix adhesion and protrusion formation.

Decreased NMIIA heavy chain phosphorylation at S1943 promotes mitoxantrone resistance by upregulating BCRP and N-cadherin expression in breast cancer cells.

Mitoxantrone (MX) is an effective treatment for breast cancer; however, high efflux of MX that is accomplished by breast cancer resistance protein (BCRP) leads to acquired multidrug resistance (MDR), reducing MX's therapeutic efficacy in breast cancer. Non-muscle myosin IIA (NMIIA) and its heavy phosphorylation at S1943 have been revealed to play key roles in tumor metastasis and progression, including in breast cancer; however, their molecular function in BCRP-mediated MDR in breast cancer remains unknown. In this study, we revealed that the expression of NMIIA heavy chain phosphorylation at S1943 was downregulated in BCRP-overexpressing breast cancer MCF-7/MX cells, and stable expression of NMIIA-S1943A mutant increased BCRP expression and promoted the resistance of MCF-7/MX cells to MX. Meanwhile, NMIIA S1943 phosphorylation induced by epidermal growth factor (EGF) was accompanied by the downregulation of BCRP in MCF-7/MX cells. Furthermore, stable expression of NMIIA-S1943A in MCF-7/MX cells resulted in upregulation of N-cadherin and the accumulation of β-catenin on the cell surface, which inhibited the nucleus translocation of β-catenin and Wnt/β-catenin-based proliferative signaling. EGF stimulation of MCF-7/MX cells showed the downregulation of N-cadherin and β-catenin. Our results suggest that decreased NMIIA heavy phosphorylation at S1943 increases BCRP expression and promotes MX resistance in breast cancer cells via upregulating N-cadherin expression.

Sirtuin 5-mediated deacetylation of TAZ at K54 promotes melanoma development.

Nuclear accumulation of YAP/TAZ promotes tumorigenesis in several cancers, including melanoma. Although the mechanisms underlying the nuclear retention of YAP are known, those underlying the retention of TAZ remain unclear. Our study investigates a novel acetylation/deacetylation switch in TAZ, governing its subcellular localization in melanoma tumorigenesis. Immunoprecipitation/Western blot assessed TAZ protein interactions and acetylation. SIRT5 activity was quantified with enzyme-linked immunosorbent assay. Immunofluorescence indicated TAZ nuclear localization. TEAD transcriptional activity was measured through luciferase reporter assays. ChIP detected TAZ binding to the CTGF promoter. Transwell and wound healing assays quantified melanoma cell invasiveness and migration. Metastasis was evaluated using a mouse model via tail vein injections. Clinical relevance was explored via immunohistochemical staining of patient tumors. CBP facilitated TAZ acetylation at K54 in response to epidermal growth factor stimulation, while SIRT5 mediated deacetylation. Acetylation correlated with phosphorylation, regulating TAZ's binding with LATS2 or TEAD. TAZ K54 acetylation enhanced its S89 phosphorylation, promoting cytosolic retention via LATS2 interaction. SIRT5-mediated deacetylation enhanced TAZ-TEAD interaction and nuclear retention. Chromatin IP showed SIRT5-deacetylated TAZ recruited to CTGF promoter, boosting transcriptional activity. In a mouse model, SIRT5 overexpression induced melanoma metastasis to lung tissue following the injection of B16F10 melanocytes via the tail vein, and this effect was prevented by verteporfin treatment. Our study revealed a novel mechanism of TAZ nuclear retention regulated by SIRT5-mediated K54 deacetylation and demonstrated the significance of TAZ deacetylation in CTGF expression. This study highlights the potential implications of the SIRT5/TAZ axis for treating metastatic melanoma.

C-Src regulates δ-secretase activation and truncated Tau production by phosphorylating the E3 ligase Traf6

The accumulation of abnormal Tau protein is a common feature of various neurodegenerative diseases. Truncated Tau, resulting from cleavage by asparaginyl endopeptidase (AEP, δ-secretase), promotes its own phosphorylation and aggregation. Our study focused on understanding the regulatory mechanisms of AEP activation and its interaction with other proteins. We discovered that c-Src plays a critical role in mediating the activation and polyubiquitination of AEP in response to epidermal growth factor (EGF) stimulation. Additionally, we investigated the involvement of tumor necrosis factor receptor-associated factor 6 (Traf6), an E3 ligase, in the regulation of AEP levels and its interaction with c-Src. Knockdown of Traf6 effectively inhibited c-Src-induced AEP activation. To gain further insights into the molecular mechanisms, we employed mass spectrometry (MS) to identify the specific tyrosine residues of Traf6 that are phosphorylated by c-Src. By mutating these phosphorylation sites to phenylalanine, we disrupted Traf6-mediated polyubiquitination and subsequently observed the inactivation of AEP. This finding suggests that the phosphorylation of Traf6 by c-Src is crucial for AEP activation. Pharmacological inhibition of c-Src reduced the phosphorylation of Traf6 and inhibited AEP activation in neurons derived from human induced pluripotent stem cells (hiPSCs). Conditional knockout of Traf6 in neurons prevented c-Src-induced AEP activation and subsequent Tau truncation in vivo. Moreover, phosphorylation of Traf6 is highly correlated with AEP activation, Tau368 and pathological Tau (AT8) in Alzheimer's disease (AD) brain. Overall, our study elucidates the role of c-Src in regulating AEP-cleaved Tau through phosphorylating Traf6. Targeting the c-Src/Traf6 pathway may hold potential for the treatment of AD and other tauopathies.

EGCG attenuate EGF triggered matrix abundance and migration in HPV positive and HPV negative cervical cancer cells.

Our previous laboratory findings suggested the beneficial effects of epigallocatechin gallate (EGCG) against cervical cancer (CC) cells survival. The present study is aimed at identifying the effects of EGCG in preventing the actions of epidermal growth factor (EGF) in human papilloma virus (HPV) 68 positive ME180 and HPV negative C33A CC cells. An elevated level of EGF in tumor micro-environment (TME) is linked to the metastasis of several cancers including CC. We hypothesized that EGCG has the ability to block the actions of EGF. To test this, survival assay was performed in cells treated with or without EGF and EGCG. The mitochondrial activity of cells was ascertained using MTT assay and mitored staining. Protein and non-protein components in the extracellular matrix such as collagen and sulphated glycosaminoglycans (GAGs) were evaluated using sirius red and alcian blue staining, respectively. Matrix metalloproteinase-2 (MMP-2) gene expression and enzymatic activity were assessed using real time-reverse transcriptase-polymerase chain reaction (RT-PCR) and gelatin zymography. Wound healing assay was performed to assess the EGF induced migratory ability and its inhibition by EGCG pre-treatment. Clonogenic assay showed thatEGCG pre-treatment blocked the EGF driven colony formation. In silico analysis performed identified the efficacy of EGCG in binding with different domains of EGF receptor (EGFR). EGCG pre-treatment prevented the epithelial-mesenchymal transition (EMT) and metabolic activity induced by EGF, this is associated with concomitant reduction in the gene expression and enzyme activity of MMP-2. Further, reduced migration and ability to form colonies were observed in EGCG pre-treated cells when stimulated with EGF. HPV positive ME180 cells showed increased migratory and clonogenic ability upon EGF stimulation, whose effects were not much significant in HPV negative C33A cells. EGCG effectively blocked the actions of EGF in both HPV positive and HPV negative conditions and can be advocated as supplementary therapy for the management of EGF driven CC. However, further studies using cell line-derived xenograft(CDX)/patient-derived xenograft(PDX) model system is warranted to validate the therapeutic utility of EGCG.

Girdin regulates both migration and angiogenesis in pancreatic cancer cell lines.

Girdin, an actin‑binding protein, is reportedly involved in the invasion and angiogenesis of various cancers. It has been suggested that the flavonoid Scutellarin (SCU) inhibits Girdin signaling. In the present study, the function and therapeutic applications of Girdin in pancreatic cancer (PaCa) were investigated. Immunohistochemical staining of Girdin in resected PaCa specimens from the Department of Gastroenterological Surgery, Nagoya City University Graduate School of Medical Science showed that high Girdin expression was associated with poor overall survival and relapse‑free survival, as well as with T factor, indicating invasion into the surrounding tissues. On the other hand, Girdin was highly expressed in almost all PaCa cell lines, and the migration ability of Girdin‑knockdown cell lines was decreased even under epidermal growth factor (EGF) stimulation. In addition, SCU suppressed PaCa cell migration by inhibiting the phosphorylation of Girdin. The expression and production of vascular endothelial growth factor A (VEGF‑A) was significantly decreased in Girdin‑knockdown cell lines. Furthermore, in Matrigel tube formation assays performed using culture supernatant, the lumen‑forming ability of vascular endothelial cells was also decreased in Girdin‑knockdown cell lines. However, SCU treatment did not significantly alter the expression or production of VEGF‑A. These results suggested that Girdin is involved in EGF signaling‑mediated migration of PaCa cells, that SCU inhibits PaCa invasion by suppressing Girdin activity, and that Girdin is also involved in angiogenesis via an activation pathway different from the action site of SCU. Girdin may be a prognostic biomarker, and the development of a novel molecular‑targeted drugs for Girdin may improve the prognosis of PaCa in the future.

Chemo-preventive effects and antitumorigenic mechanisms of beer and nonalcoholic beer toward 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) - induced lung tumorigenesis in A/J mice

We investigated the chemopreventive effects of beer, nonalcoholic beers (NABs), and beer-components (glycine betaine (GB)) on NNK-induced lung tumorigenesis in A/J mice, and the possible mechanisms underlying the antitumorigenic effects of beer, NABs, and beer-components. Beer, NABs, and GB reduced NNK-induced lung tumorigenesis. We investigated the antimutagenicity of beer, NABs and beer-components (GB and pseudouridine (PU)) toward the mutagenicity of 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Beer, NABs, and beer components were antimutagenic toward MNNG and NNK in the Ames test using S. typhimurium TA1535. In contrast, MNNG and NNK mutagenicity detected in S. typhimurium YG7108, a strain lacking O6-methylguanine DNA methyltransferases (ogtST and adaST) did not decrease in the presence of beer, NABs, or beer components, suggesting that they may mediate its antimutagenic effect by enhancing DNA damage repair. Phosphorylation of Akt and STAT3, with or without epidermal growth factor stimulation, in lung epithelial-like A549 cells were significantly decreased following beer, NABs, GB and PU. They targeted both the initiation and growth/progression steps of carcinogenesis, specifically via antimutagenesis, stimulation of alkyl DNA-adduct repair, and suppression of Akt- and STAT3- mediated growth signaling. GB and PU may contribute, in part, to the biological effects of beer and NABs via the suppression of Akt and STAT3 phosphorylation.

Abstract A028: An isogenic H/N/KRAS-less mouse embryonic fibroblast cell line panel derived from a size sorted diploid clonal parent

Abstract Shortly after the formation of the NCI Ras Initiative, a H/N/KRAS-less mouse embryonic fibroblast (MEF) cell line panel was generated, validated, and shared internationally to aid the research community in its reinvigorated effort to apply new technical advancements to understanding the structure and function of Ras and to ultimately design inhibitors. While this panel has been beneficial to the Ras research community, an inherent variability due in part to the ploidy of the parental pool from which the panel was derived complicated comparisons between isolates and alleles. We have minimized this variation by utilizing a diploid clone derived from the DU1473 MEF; Hras-/-; Nras-/-; Kraslox/lox; RERTert/ert - parental cell line. A diploid cell population was sorted, cloned and clones were cultured continuously for up to 20 passages to identify those that retained a diploid signature. These clones were further screened for p53 mutations and mouse exome sequenced from which a final clone was selected to develop the improved cell line panel. The selected clone was treated with 4-hydroxytamoxifen (4-OHT) to activate the Cre-recombinase which excised a portion of the endogenous Kras located between two loxP sites effectively arresting cells in a senescent G1 state. Molecular verification of Kras removal was performed, and the cells were then transduced with a third generation VSV-G pseudotyped lentiviral vector which delivered different versions of the human Ras transgene as well as an antibiotic selection marker. This transduction restored the proliferative ability of the cells allowing for expansion and selection of the transduced pool. The pools were single cell cloned and subsequent clones underwent a systematic, rigorous quality control evaluation culminating with another round of exome sequencing before clones were selected to be part of the MEF 2.0 panel. Clones contained in this new panel were confirmed to be diploid, had fewer integrations than the initial panel, and performed similarly on proliferation assays as well as epidermal growth factor stimulation signaling assays. The new panel has successfully recapitulated the initial panel, but in a far more stable and reproducible cellular background. Instructions to obtain the cell line panel will be presented on the Ras Initiative website at www.cancer.gov and these cells are scheduled to become available to the Ras research community late in the spring of 2023. Citation Format: William E. Burgan, Nicole Fer, Katie Powell, Bingfang Xu, Mackenzie Meyer, Scott Eury, Abigail Neish, Daniel Soppet, Dominic Esposito, Frank McCormick, Dwight V. Nissley. An isogenic H/N/KRAS-less mouse embryonic fibroblast cell line panel derived from a size sorted diploid clonal parent [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A028.

Development of a micronucleus test using the EpiAirway™ organotypic human airway model

BackgroundThe use of organotypic human tissue models in genotoxicity has increased as an alternative to animal testing. Genotoxicity is generally examined using a battery of in vitro assays such as Ames and micronucleus (MN) tests that cover gene mutations and structural and numerical chromosome aberrations. At the 7th International Workshop on Genotoxicity Testing, working group members agreed that the skin models have reached an advanced stage of maturity, while further efforts in liver and airway models are needed [Pfuhler et al., Mutat. Res. 850–851 (2020) 503135]. Organotypic human airway model is composed of fully differentiated and functional respiratory epithelium. However, because cell proliferation in organotypic airway models is thought to be less active, assessing their MN-inducing potential is an issue, even in the cytokinesis-blocking approach using cytochalasin B (CB) [Wang et al., Environ. Mol. Mutagen. 62 (2021) 306–318]. Here, we developed a MN test using EpiAirway™ in which epidermal growth factor (EGF) was included as a stimulant of cell division.ResultsBy incubating EpiAirway™ tissue with medium containing various concentrations of CB, we found that the percentage of binucleated cells (%BNCs) almost plateaued at 3 μg/mL CB for 72 h incubation. Additionally, we confirmed that EGF stimulation with CB incubation produced an additional increase in %BNCs with a peak at 5 ng/mL EGF. Transepithelial electrical resistance measurement and tissue histology revealed that CB incubation caused the reduced barrier integrity and cyst formation in EpiAirway™. Adenylate kinase assay confirmed that the cytotoxicity increased with each day of culture in the CB incubation period with EGF stimulation. These results indicated that chemical treatment should be conducted prior to CB incubation. Under these experimental conditions, it was confirmed that the frequency of micronucleated cells was dose-dependently increased by apical applications of two clastogens, mitomycin C and methyl methanesulfonate, and an aneugen, colchicine, at the subcytotoxic concentrations assessed in %BNCs.ConclusionsWell-studied genotoxicants demonstrated capability in an organotypic human airway model as a MN test system. For further utilization, investigations of aerosol exposure, repeating exposure protocol, and metabolic activation are required.

The relationship between sphingosine-1-phosphate receptor 2 and epidermal growth factor in migration and invasion of oral squamous cell carcinoma

Sphingosine-1-phosphate (S1P) is a lipid mediator and its binding to the S1P receptor 2 (S1PR2) is reported to regulate cytoskeletal organization. Epidermal growth factor (EGF) has been shown to induce migration and invasion in tumour cells. Since binding of S1P to S1PR2 and EGF to the EGF receptors exhibit some overlapping functionality, this study aimed to determine whether S1PR2 was involved in EGF-induced migration and invasion of oral squamous cell carcinoma (OSCC) lines and to identify any potential crosstalk between the two pathways. Migration was investigated using the scratch wound assay while invasion was studied using the transwell invasion and multicellular tumour spheroid (MCTS) assays. Activity of Rac1, a RhoGTPase, was measured using G-LISA (small GTPase activation assays) while S1P production was indirectly measured via the expression of sphingosine kinase (Sphk). S1PR2 inhibition with 10 µM JTE013 reduced EGF-induced migration, invasion and Rac1 activity, however, stimulation of S1PR2 with 10 µM CYM5478 did not enhance the effect of EGF on migration, invasion or Rac1 activity. The data demonstrated a crosstalk between EGF/EGFR and S1P/S1PR2 pathways at the metabolic level. S1PR2 was not involved in EGF production, but EGF promoted S1P production through the upregulation of Sphk1. In conclusion, OSCC lines could not migrate and invade without S1PR2 regulation, even with EGF stimulation. EGF also activated S1PR2 by stimulating S1P production via Sphk1. The potential for S1PR2 to control cellular motility may lead to promising treatments for OSCC patients and potentially prevent or reduce metastasis.

Epidermal growth factor represses differentiation of mouse trophoblast stem cells into spongiotrophoblast cells via epidermal growth factor receptor

The mouse placenta is composed of three different trophoblast layers that are occupied by particular trophoblast subtypes to maintain placental function and pregnancy. Accurate control of trophoblast differentiation is required for proper placental function; however, the molecular mechanisms underlying cell fate decisions in trophoblast stem cells remain poorly understood. Epidermal growth factor (EGF) signaling is involved in multiple biological processes including cell survival, proliferation, and differentiation. The effect of EGF on trophoblast function has been reported in various species; however, the role of EGF signaling in mouse trophoblast specification remains unclear. In this study, we aimed to elucidate the role of EGF signaling in mouse trophoblast differentiation using mouse trophoblast stem cells (mTSCs) in an in vitro culture system. EGF stimulation at the early stage of differentiation repressed mTSC differentiation into spongiotrophoblast cells (SpT). Gene deletion and inhibitor experiments showed that the effect of EGF exposure went through epidermal growth factor receptor (Egfr) activity in mTSCs. EGF stimuli induced acute downstream activation of MAPK/ERK, PI3K/AKT, and JNK pathways, and inhibition of the MAPK/ERK pathway, but not others, alleviated EGF-mediated repression of SpT differentiation. Moreover, expression of Mash2, a master regulator of SpT differentiation, was repressed by EGF stimulation, and MAPK/ERK inhibition counteracted this repression. The Mash2 overexpression recovered SpT marker expression, indicating that the decrease in Mash2 expression was due to abnormal SpT differentiation in EGF-treated mTSCs. Our findings suggest that the EGF-Egfr-MAPK/ERK-Mash2 axis is a core regulatory mechanism for the EGF-mediated repression of SpT differentiation.

Two Closed Conformations of CRAF Require the 14-3-3 Binding Motifs and Cysteine-Rich Domain to be Intact in Live Cells

The protein rapidly accelerated fibrosarcoma (RAF) is a kinase downstream of the membrane protein RAS in the cellular signal transduction system. In the structure of RAF, the N- and C-terminus domains are connected with a flexible linker. The open/close dynamics and dimerization of RAF are thought to regulate its activity, although the details of these conformations are unknown, especially in live cells. In this work, we used alternating laser excitation to measure cytosolic CRAF in live HeLa cells and obtained single-molecule Förster resonance energy transfer (smFRET) distributions of the structural states. We compared the results for wild-type (WT)-CRAF before and after epidermal growth factor (EGF) stimulation, with mutations of the 14-3-3 binding sites and cysteine-rich domain, and an N-terminus truncation. The smFRET distributions of full-length CRAFs were analyzed by global fitting with three beta distributions. Our results suggested that a 14-3-3 dimer bound to two sites on a single CRAF molecule and induced the formation of the autoinhibitory closed conformation. There were two closed conformations, which the majority of WT-CRAF adopted. These two conformations showed different responsiveness to EGF stimulation.

Chemopreventive effects and anti-tumorigenic mechanisms of Actinidia arguta, known as sarunashi in Japan toward 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)- induced lung tumorigenesis in a/J mouse

Previously, we reported the inhibitory effect of Actinidia arguta juice, known as sarunashi juice (sar-j) in Japan, on mutagenesis, inflammation, and mouse skin tumorigenesis. The components of A. arguta responsible for the anti-mutagenic effects were identified to be water-soluble, heat-labile phenolic compounds. We proposed isoquercetin (isoQ) as a candidate anticarcinogenic component. In this study, we sought to investigate the chemopreventive effects of A. arguta juice and isoQ on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice, and identify the possible mechanisms underlying the anti-tumorigenic effects of A. arguta. The number of tumor nodules per mouse lung in the group injected with NNK and administered A. arguta juice orally was significantly lower than that in the group injected with NNK only. Oral administration of isoQ also reduced the number of nodules in the mouse lungs. As expected, the mutagenicity of NNK and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) detected using S. typhimurium TA1535 decreased in the presence of sar-j. However, NNK and MNNG mutagenicity detected using S. typhimurium YG7108, a strain lacking the O6-methylguanine DNA methyltransferases (ogtST and adaST) did not decrease in the presence of sar-j suggesting that sar-j may mediate its antimutagenic effect by enhancing the DNA damage repair by ogtST and adaST. Phosphorylation of Akt, with or without epidermal growth factor stimulation, in A549 cells was significantly decreased following sar-j and isoQ treatment, indicating that components in sar-j including isoQ suppressed the PI3K/AKT signaling pathways. Sar-j and isoQ reduced NNK-induced lung tumorigenesis. Sar-j targets both the initiation and growth/progression steps during carcinogenesis, specifically via anti-mutagenesis, stimulation of alkyl DNA adduct repair, and suppression of Akt-mediated growth signaling. IsoQ might contribute in part to the biological effects of sar-j via suppression of Akt phosphorylation, but it may not be the main active ingredient.

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.

Endothelin A (ETA) and Endothelin B (ETB) Receptor Subtypes Potentiate Epidermal Growth Factor (EGF)-Mediated Proliferation in Human Asthmatic Bronchial Airway Smooth Muscle.

BackgroundAsthma is a chronic disease characterized by chronic inflammation, reversible airway obstruction, airway hyperresponsiveness (AHR), and airway remodeling. One of the important features of asthma is airway remodeling, which plays a central role in airflow limitation. Airway remodeling involves numerous changes in the bronchial walls, including airway smooth muscle (ASM) cell hypertrophy and hyperplasia. Studies have shown that ASM hyperplasia in asthma is mediated by the increased production of mitogens. Endothelin-1 (ET-1) has been shown to induce proliferation and function as a co-mitogen in vascular and ASM. In patients with asthma, plasma and bronchoalveolar lavage fluid have been shown to have elevated ET-1 levels, which have been linked to airway remodeling and airflow obstruction in severe asthma. This study investigates the role of ET-1 in proliferation, the receptor subtype mediating its effect, and the signaling pathway.MethodologyNormal and asthmatic bronchial airway smooth muscle (BASM) cells were seeded into 5 × 103 cells/well. Cell proliferation was assayed using 5-bromo-2’-deoxyuridine (BrdU) incorporation. Confluent cells were treated with different concentrations of ET-1 in the presence or absence of the epidermal growth factor (EGF). Signaling pathways were explored using pretreatment of BASM with antagonists 15 minutes before ET-1/EGF stimulation.ResultsIn asthmatic BASM, ET-1 (0.1 nM) functions as a co-mitogen in the presence of EGF (10 nM), showing a significantly greater effect on asthmatic BASM proliferation compared with normal BASM. The ETA receptor antagonist BQ-123 (10-1,000 nM) significantly reduced the proliferative effect of ET-1/EGF on asthmatic BASM more than normal BASM. Moreover, the effect of ETB antagonist BQ-788 (1,000 nM) or pretreatment with the ETB agonist S6C (1-10 nM) followed by co-treatment with EGF in asthmatic BASM showed a small but significant decrease when pretreated with the inhibitor and increased with the agonist, thereby suggesting that the co-mitogenic effect of ET-1 is mainly via the activation of ETA receptors, with a small contribution by the ETB receptors in asthmatic BASM. Finally, pertussis toxin (PTX) pretreatment (25 and 50 ng/mL) showed that EGF and ET-1/EGF mitogenic and co-mitogenic signaling utilizes Gi/0-mediated transactivation by EGF and ET receptors, especially in asthmatic BASM, leading to the activation of Ras-ERK-PI3K pathways. Enhanced ERK and PI3K effects on proliferation suggested that these kinases modulate the co-mitogenic effect of ET-1 in asthmatic BASM. Enhanced cross-talk between ET and EGF receptors may be a potential mechanism contributing to airway remodeling in asthmatic BASM.ConclusionsET-1 enhances the mitogenic effect of EGF predominantly via the ETA receptor in asthmatic BASM with the activation of Ras, ERK, and PI3K. The cross-talk mechanism between ET and EGF receptors may be a potential therapeutic target to prevent the progression of airway remodeling in ASM in patients with asthma.

Optimized Workflow for Enrichment and Identification of Biotinylated Peptides Using Tamavidin 2-REV for BioID and Cell Surface Proteomics.

Chemical or enzymatic biotinylation of proteins is widely used in various studies, and proximity-dependent biotinylation coupled to mass spectrometry is a powerful approach for analyzing protein-protein interactions in living cells. We recently developed a simple method to enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. However, the level of biotinylated proteins in cells is low; therefore, large amounts of cellular proteins were required to detect biotinylated peptides. In addition, the enriched biotinylated peptide solution contained many contaminant ions. Here, we optimized the workflow for efficient enrichment of biotinylated peptides and removal of contaminant ions. The efficient recovery of biotinylated peptides with fewer contaminant ions was achieved by heat inactivation of trypsin, prewashing Tamavidin 2-REV beads, clean-up of biotin solution, mock elution, and using optimal temperature and salt concentration for elution. The optimized workflow enabled identification of nearly 4-fold more biotinylated peptides with higher purity from RAW264.7 macrophages expressing TurboID-fused STING (stimulator of interferon genes). In addition, sequential digestion with Glu-C and trypsin revealed biotinylation sites that were not identified by trypsin digestion alone. Furthermore, the combination of this workflow with TMT labeling enabled large-scale quantification of cell surface proteome changes upon epidermal growth factor (EGF) stimulation. This workflow will be useful for BioID and cell surface proteomics and for various other applications based on protein biotinylation.

Glucose Increases STAT3 Activation, Promoting Sustained XRCC1 Expression and Increasing DNA Repair.

Dysregulation of DNA repair is a hallmark of cancer, though few cancer-specific mechanisms that drive the overexpression of DNA repair proteins are known. We previously identified STAT3 as a novel transcriptional regulator of X-ray cross-complementing group 1 (XRCC1), an essential scaffold protein in base excision repair in triple-negative breast cancers. We also identified an inducible response to IL-6 and epidermal growth factor stimulation in the non-tumorigenic embryonic kidney cell line HEK293T. As IL-6 and EGF signaling are growth and inflammatory-inducible responses, we examined if glucose challenge can increase STAT3 activation, promoting adaptive changes in XRCC1 expression in different cell types. Acute high glucose exposure promoted XRCC1 expression through STAT3 activation, increasing the repair of methyl methanesulfonate-induced DNA damage in HEK293T cells and the osteosarcoma cell line U2OS. Sustained exposure to high glucose promoted the overexpression of XRCC1, which can be reversed upon glucose restriction and down-regulation of STAT3 activation. Thus, we have identified a novel link between XRCC1 expression and STAT3 activation following exogenous exposures, which could play a critical role in dictating a cancer cell’s response to DNA-damaging agents.