Protein Kinase D1 Overexpression Potentiates Epidermal Growth Factor Signaling Pathway in Mcf-7 Cells

Protein Kinase D1 Overexpression Potentiates Epidermal Growth Factor Signaling Pathway in Mcf-7 Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Protein Kinase D1 Overexpression Potentiates Epidermal Growth Factor Signaling Pathway in Mcf-7 Cells Christine LEGAY, Sophie DOUBLIER, Sylvie BABAJKO, Jean-Marc RICORT This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1877745/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Feb, 2023 Read the published version in Molecular Biology Reports → Version 1 posted 4 You are reading this latest preprint version Abstract I. Background: Protein kinase D1, PKD1, is a serine-threonine kinase implicated in cell proliferation, migration, invasion, and/or apoptosis and its activation by several growth factors sets this enzyme as a key regulator of tumorigenesis and tumor progression. Despite many studies, its role in the regulation of intracellular signaling pathways remains widely disparate and needs to be clarified. II. Methods and Results: By using human breast cancer cells MCF-7, overexpressing or not PKD1, we demonstrated that PKD1 expression level modulated the tumor growth-promoting epidermal growth factor (EGF) signaling pathway. We also showed that EGF acutely stimulated PKD1 phosphorylation with similar time courses both in control and PKD1-overexpressing cells. However, PKD1 overexpression specifically and markedly increased EGF-induced phosphorylation of Akt (onto T308 and S473 residues) and extracellular-regulated protein kinase (ERK1/2). Finally, pharmacological inhibition of PKD1 activity or lowering its expression level using specific siRNAs drastically reduced EGF-stimulated Akt and ERK phosphorylation in PKD1overexpressing cells, but not in control cells. III. Conclusions: Overall, these results identified the level of PKD1 expression as a key determinant in the regulation of the EGF signaling pathway highlighting its crucial role in a tumorigenic setting. protein kinase D1 cell signaling breast cancer cell epidermal growth factor Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Protein kinase D1 (PKD1) is a serine/threonine kinase with a wide tissue expression which is schematically composed of two domains, an amino-terminal regulatory region and a carboxyl-terminal catalytic region. Based on strong homologies between its catalytic domain and those of Ca 2+ /calmodulin-dependent protein kinases (CaMKs), PKD1 was classified in the CaMKs superfamily [ 1 , 2 ]. The regulatory region of PKD1 contains an alanine- and proline-rich apolar (AP) domain, a diacylglycerol (DAG)/phorbol esters binding domain (C1 domain), an acidic (AC) domain, and a pleckstrin homology (PH) domain. C1 domain allows PKD1 to localize either to the Golgi apparatus or to the plasma membrane upon its interaction with DAG and also plays a role in the nuclear import of PKD1. On the other hand, PH domain regulates the nuclear export of PKD1 and allows PKD1 to interact with molecular partners. Both C1 and PH domains auto-inhibit the catalytic domain and the deletion of one of them generates a constitutively active form of PKD1 (for review see [ 3 ]). PKD1 activation is generally triggered through a phospholipase C- (PLC) and protein kinase C- (PKC) dependent signaling pathway stimulated after the binding of an extracellular ligand to a transmembrane receptor [ 4 ]. Thus, growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) [ 5 ], or mitogenic neuropeptides such as bombesin, vasopressin and angiotensin II [ 6 ], or lysophosphatidic acid (LPA) [ 7 ] were shown to stimulate PLC-dependent signaling pathways and to increase DAG intracellular concentration leading to the recruitment of PKD1 at the plasma membrane and its subsequent phosphorylation by different PKCs [ 8 , 9 ]. The two serine residues of PKD1, 738 and 742 (S738/S742) (human numbering), localized into its activation loop, are the main targets of PKCs. Their phosphorylation increases the kinase activity of PKD1 inducing its subsequent autophosphorylation onto its C-terminal S910 residue [ 9 ]. Moreover, PKC-independent signaling pathways and some stimuli, such as oxidative stress or caspase-induced PKD1 cleavage, were also described to induce the S738/S742 phosphorylation of PKD1 without affecting the phosphorylation state of the S910 residue [ 10 – 12 ]. PKD1 regulates numerous biological functions such as cell proliferation, differentiation, cell-cell interactions, migration, invasion, apoptosis, and survival. This multiplicity of actions is especially due to the ability of PKD1 to regulate several signaling pathways such as the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK)- and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt-dependent signaling pathways that play major roles in cell proliferation and survival, especially in tumor cells. In breast cancer cells, the role of PKD1 remains controversial since we and others demonstrated that PKD1 promotes cell proliferation and that its overexpression strengthens this function, whereas others suggested that PKD1 has anti-proliferative effect (for review see [ 13 ]). However, PKD1 expression level varies from one breast cancer cell type to another, potentially impacting downstream signaling pathways and biological responses. Therefore, in this study, we determine whether and how PKD1 expression levels may modulate the EGF-stimulated MEK/ERK and PI 3-kinase/Akt signaling pathways by using MCF-7 cells overexpressing or not PKD1. 2. Materials And Methods 2.1. Antibodies and materials Anti-Akt1, anti-phospho-ERK1/2 antibodies used for immunoblotting were purchased from Upstate Biotechnology-Millipore (Lake Placid, NY); anti-PKD1, anti-α-actinin and anti-actin from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phospho-PKD1 (S738/S742) and anti-phospho-Akt (T308 and S473) from Cell Signaling (Danvers, MA). EGF was purchased from PeproTech (Rocky Hill, NJ); PKD1-targeting (sc-36245) and control non-targeting (sc-37007) siRNA from Santa Cruz Biotechnology, and Gö6976 from Calbiochem (La Jolla, CA). All other biochemicals were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France), or ICN (Orsay, France). 2.2. Cell culture Stably transfected MCF-7 cells (ATCC HTB22, CVCL_0031), overexpressing PKD1, or not [ 14 ], were grown to 80–85% confluence in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 100 U.mL − 1 penicillin, 100 µg.mL − 1 streptomycin and 1 mg.mL − 1 G418 (Calbiochem, Darmstadt, Germany). Before each experiment, cells were starved in serum free media for 16–24 hours. 2.3. Western Immunoblotting MCF-7 cells were cultured as described and treated for different periods of time with or without EGF (40 ng.mL − 1 ). Cells were then solubilized in lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 100 mM NaF, 10 mM EDTA, 2 mM Na 3 VO 4 , 10 mM pyrophosphate, 1 mM PMSF, 100 U.ml − 1 aprotinin) containing 1% Nonidet P-40 (NP-40), and proteins separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were incubated for 1 hour at room temperature or overnight at 4°C with the primary specific antibodies, then incubated for 1 hour at room temperature with the peroxidase-conjugated secondary antibodies and revealed by enhanced chemiluminescence (Amersham, GE Healthcare, UK). 2.4. Fluorescence microscopy Stably transfected MCF-7 cells were cultured onto coverslips. Cells were rinsed in PBS, fixed for 5 minutes at -20°C with cold acetone and rinsed again with a three-fold excess of PBS before being incubated for 30 min at 4°C in PBS containing 3% BSA. Cells were incubated for 1 hour at 4°C with 10 µg.mL − 1 anti-PKD1 antibody diluted in PBS-BSA 3%, then washed twice for 10 min in PBS-BSA 3% and incubated for 30 min at room temperature with 1 µg.mL − 1 Alexa-488 conjugated goat anti-rabbit immunoglobulin G heavy plus light chains (H + L) (Molecular Probes, Eugene, OR) diluted in PBS-BSA 3%. Cells were rinsed three times with PBS-BSA 3% and mounted with Mowiol antifading reagent (Calbiochem, La Jolla, CA). Microscope analysis was carried out using the TCS NT confocal imaging system (Leica Instruments, Heidelberg, Germany), equipped with a 63× objective (plan apo, numerical aperture = 1.4). For Alexa-488, an argon-krypton ion laser adjusted to 488 nm was used. The signal was integrated over four to eight frames to reduce the noise. The pinhole was adjusted to allow a field depth of about 1 µm, corresponding to the increment between two adjacent sections. At least 20 cells were observed in each condition. 2.5. siRNA transfection siRNA transfection was performed according to the manufacturer’s protocol. Briefly, 3 x 10 5 cells were seeded per well in 2 mL antibiotic-free DMEM supplemented with 10% FBS and incubated for 24 h. For each well, 1 µg siRNA and 8 µL siRNA transfection reagent, each diluted in 100 µL siRNA transfection medium, were combined, incubated for 45 min at room temperature and then applied to the cells in a final volume of 1 mL siRNA transfection medium. After a 7-h incubation at 37°C, DMEM supplemented with 10% FBS was added and cells cultured for an additional 18–24 h at 37°C before analysis. 2.6. Statistical analysis The statistical significance of differences between experimental groups was determined with the Student’s t-test using Prims 5.03 software for Windows (GraphPad Software, San Diego, CA). Differences between values were considered to be highly significant when p ≤ 0.01 (**) or p ≤ 0.001 (***). 3. Results 3.1. EGF-stimulated PKD1 phosphorylation is increased in PKD1 overexpressing cells Before determining whether overexpression of PKD1 modulated the EGF-induced MEK/ERK and PI 3-kinase signaling pathways in MCF-7 cells, we first analyzed the status of EGF-stimulated PKD1 in control (Mock) and PKD1 overexpressing cells. As we previously described [ 14 ], MCF-7 cells stably transfected with PKD1-expressing vector (clones P1 and P2) expressed higher levels of PKD1 (approximately 4.35 ± 1.8- and 3.91 ± 1.15-fold, respectively) than control cells stably transfected with the corresponding empty vector (clones C1 and C2) (Fig. 1A). This result was further confirmed by immunofluorescence staining through the direct detection of PKD1 in living cells (Fig. 1B). EGF stimulated PKD1 phosphorylation onto S738/S742 residues in control cells (C1 and C2) with maximal effect occurring after 30 min of EGF treatment and remaining mostly stable up to 60 min (Fig. 1C). In cells overexpressing PKD1, EGF stimulated PKD1 phosphorylation with a similar time course than in control cells but, whatever the incubation time considered, EGF-stimulated PKD1 phosphorylation was significantly stronger (by almost 1.4- to 1.9-fold) than in control cells. Therefore, these results demonstrated that PKD1 overexpression did not modify PKD1 time course activation in response to EGF but strongly increased the levels of EGF-induced phosphorylated PKD1. 3.2. Overexpression of PKD1 potentiates EGF-stimulated PI 3-kinase signaling pathway We thus analyzed whether increasing PKD1 expression and activity levels may affect the EGF-stimulated PI 3-kinase signaling pathway. To this end, we studied the phosphorylation state of T308 and S473 residues of Akt, a downstream target of PI 3-kinase. In control cells (C), EGF transiently stimulated Akt phosphorylation onto its T308 and S473 residues with maximal effect occurring after 7 min of EGF treatment and decreasing very rapidly to basal levels after 15 min (Fig. 2A and 2B). In contrast, EGF-stimulated Akt phosphorylation was much less transient in PKD1 overexpressing cells and return to basal values was markedly delayed since a significant phosphorylation of the two residues still remained after 30 min of EGF treatment. Moreover, although PKD1 overexpression did not change the maximal phosphorylation level of EGF-induced S473-Akt residue (Fig. 2B, right), it markedly increased the maximal phosphorylation level of EGF-induced T308-Akt residue (by 1.4-fold over control cells) (Fig. 2B, left). Since we previously demonstrated that overexpression of PKD1 did not change the expression level of Akt [ 14 ], these results indicated that PKD1 overexpression markedly affected the EGF-induced Akt phosphorylation level and time course. To determine whether these effects were specifically due to the overexpression of PKD1, cells were first incubated with a pharmacological inhibitor of PKD1, Gö6976, before being stimulated by EGF (Fig. 2C). Gö6976 totally inhibited EGF-stimulated PKD1 phosphorylation in control and PKD1-overexpressing cells (Fig. 2C) without affecting PKD1 expression [ 15 ]. EGF-stimulated T308- and S473-Akt phosphorylation was not affected by Gö6976 in control cells but was strongly impaired (by almost 60%) in PKD1-overexpressing cells reaching similar levels to those observed in EGF-stimulated control cells (Fig. 2C). To further strengthen these results, PKD1 was also inhibited by transfecting cells with siRNAs that specifically target PKD1 mRNA (Fig. 2D). PKD1-targeting siRNA (siP) strongly reduced PKD1 expression and EGF-stimulated PKD1 phosphorylation both in control and PKD1-overexpressing cells (Fig. 2D). EGF-induced T308- and S473-Akt phosphorylation was not significantly changed in control cells upon siP treatment but was profoundly decreased (by 60% and 54%, respectively) in PKD1-overexpressing cells (Fig. 2D). For each condition, we checked that control non-targeting (scrambled) siRNAs (siC) did not affect PKD1 expression (data not shown and [ 14 ]). Taken together, these results indicated that the EGF-stimulated PI 3-kinase signaling pathway is not directly dependent upon endogenous PKD1 in control cells but is strongly potentiated by PKD1 overexpression (data summarized in Fig. 4). 3.3. Overexpression of PKD1 potentiates EGF-stimulated ERK signaling pathway We further investigated whether PKD1 overexpression affected the EGF-stimulated MEK/ERK signaling pathway. MCF-7 cells overexpressing PKD1, or not, were acutely stimulated with EGF for different periods of time and the status of ERK1/2 phosphorylation analyzed. In control cells, EGF transiently stimulated ERK1/2 phosphorylation, with maximal effect occurring after 7 min of EGF treatment, then decreasing rapidly after 15 min and remaining mostly stable up to 30 min (Fig. 3A and 3B). In PKD1-overexpressing cells, maximal ERK1/2 phosphorylation level, measured after 7 min of EGF treatment, was significantly increased (about 1.3-fold compared to control cells). Moreover, the ERK1/2 phosphorylation time course was profoundly changed, becoming much less transient and remaining mostly stable, close to maximal values, up to 30 min of EGF incubation (Fig. 3A and 3B). In order to understand how PKD1 expression may affect the EGF-stimulated MEK/ERK signaling pathway, cells were either treated with a PKD1 pharmacological inhibitor (Fig. 3C) or transfected with siRNAs specifically targeting PKD1 mRNA (Fig. 3D), as previously described. In control cells, inhibition of PKD1, either with a pharmacological inhibitor (Gö6976) or with a PKD1-targeting siRNA (siP), did not affect EGF-induced ERK1/2 phosphorylation (Fig. 3C and 3D). In contrast, in PKD1-overexpressing cells, inhibition of PKD1 drastically decreased EGF-stimulated ERK1/2 phosphorylation (by almost 40%) (Fig. 3C and 3D). These results demonstrated that the EGF-stimulated MEK/ERK signaling pathway is not dependent upon PKD1 in control cells but strongly enhanced by PKD1 overexpression (data summarized in Fig. 4). 4. Discussion PKD1 plays a crucial role in breast cancer proliferation and reported data indicate that its expression level may represent a potential biomarker [16, 17]. Thus, understanding how PKD1 expression level can affect signaling pathways tightly involved in breast cancer progression such as the EGF one represents an interesting challenge to take up. In order to understand PKD1 involvement in EGF promoting breast cancer cell, we used a PKD1-overexpressing cell model previously published [14], and investigated whether and how an increase in PKD1 expression level modulates EGF-stimulated PKD1 activity. We showed that PKD1 overexpression increased the levels of EGF-induced phosphorylated PKD1 but did not modify the pattern of the PKD1 time course activation in response to EGF. Lack of alteration of the PKD1 time course activation in PKD1-overexpressing cells represents an important point because it suggests that the signaling pathways implicated both in the activation and the inactivation of PKD1 are not altered following PKD1overexpression, indicating that the effects observed in PKD1-overexpressing cells are dependent on PKD1 overexpression only. Moreover, it also suggests that the overexpression of PKD1 does not seem to give raise to counter-regulatory signals within this cell model. We should point out, however, that, despite a four-fold PKD1 overexpression factor, the maximum increase in the EGF-induced PKD1 phosphorylation level is about 1.8-fold. Although we did not notice any particular changes in the subcellular localization of the protein in PKD1-overexpressing cells (Figure 1B), these results suggest that the overexpressed proteins could be localized in subcellular compartments in which they cannot be activated upon EGF binding to its receptor. Moreover, one could not also exclude that there is a maximum level of activation of PKD1 in response to EGF in MCF-7 cells which cannot be exceeded whatever the PKD1 levels. We showed that PKD1 is not involved in the PI 3-kinase/Akt and MEK/ERK signaling pathways stimulated by EGF in MCF-7 control cells. In fact, pharmacological inhibition of PKD1 or PKD1-targeting siRNAs do not affect EGF-induced Akt and ERK phosphorylation state in control cells. These data are totally consistent with previous ones in which we showed that pharmacological inhibition of PKD1 by Gö6976 did not affect ERK nor Akt phosphorylation level in MCF-7 control cells cultured in DMEM containing 10% FBS [14]. They indicate that, when PKD1 is expressed at basal level, EGF-stimulated PI 3-kinase/Akt and MEK/ERK signaling pathways are independent of PKD1 in MCF-7 cells. By contrast, overexpression of PKD1 markedly impacts the time courses of EGF-induced Akt and ERK phosphorylation (i.e. activation) increasing both the level of phosphorylated proteins and the activation time duration. Such results are consistent with previous data showing that PKD1 overexpression increased the duration of bombesin-induced ERK signaling in different cell models [6, 18]. Considering the crucial role played by these two signaling pathways in cancer cell proliferation, such quantitative and qualitative effects can have major consequences on the nature and specificity of molecular interactions that occur within signaling pathways. Indeed, an increase in the amount of activated Akt and ERK proteins will allow their interaction with substrates or partners for which these proteins do not interact in control cells due to weaker affinities. In addition, an increase in the duration of the EGF-induced time course activation can also participate in the appearance or an increase in the level of active Akt and ERK proteins in subcellular compartments in which they are not usually found in control conditions and their subsequent interactions with novel partners. In accordance with these hypotheses, PKD1-overexpressing MCF-7 cells were shown to be more resistant to the induction of apoptosis (results not shown) indicating a modulation of intracellular signaling pathways and the acquisition of a more aggressive phenotype [14, 17, 19, 20] following PKD1 overexpression. An increase in the duration of the activation time course of a protein could be the consequence of an inhibition of the mechanisms involved in its inactivation. In the particular case of Akt, its inactivation can proceed from indirect mechanisms through lipid phosphatases such as PTEN or SHIP, or from direct mechanisms implicating protein phosphatases such as PP2A or PHLPP(for review see [21]). Overexpression of PKD1 was shown to enhance the association of PTEN and the regulatory subunit of PI 3-kinase, p85, in intestinal epithelial cells stimulated by G protein-coupled receptor (GPCR) agonists [22]. This suggests that similar mechanisms could occur in PKD1-overexpressing MCF-7 cells leading to the observed modulation of the EGF-stimulated PI 3-kinase/Akt signaling pathway. Although the EGF-induced T308 and S473 residues phosphorylation time course was similarly modulated in PKD1-overexpressing cells, it should be noted that only the level of phosphorylation of T308 residue is significantly increased after 7 minutes of stimulation by EGF. T308 and S473 residues of Akt are phosphorylated by two distinct kinases, PDK1 and mTORC2, respectively suggesting that overexpression of PKD1 in MCF-7 cells specifically modulates a PDK1-dependent signaling pathway without affecting the mTORC2 signaling pathway. To our knowledge, a link has never been demonstrated between PKD1 and PDK1 but it would be interesting to study whether PKD1 is capable of modulating the activity of PDK1. Therefore, taken together, our results highlight the crucial role of PKD1 expression level to regulate the EGF signaling pathway in MCF-7 cells. As summarized in Figure 4, when expressed at its basal level, PKD1 does not mediate the EGF-induced activation of the PI 3-kinase/Akt and MEK/ERK signaling pathways (Figure 4A). However, when overexpressed, PKD1 contributes to an increase in the activation state of these two signaling pathways in response to EGF (Figure 4B). In conclusion, we demonstrated that PKD1 overexpression up-regulates the EGF-stimulated PI 3-kinase/Akt and MEK/ERK pro-proliferative signaling pathways in MCF-7 breast cancer cells. Therefore, these data highlight the importance to consider the PKD1 expression level as a potential interesting biomarker in breast cancers. Declarations Funding: This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) and the Ecole Normale Supérieure Paris-Saclay. Competing interest : The authors have no relevant financial or non-financial interests to disclose. Author Contributions : CL, SD and SB performed experiments and led the project. JMR conceived and directed the project, designed the experiments, supervised the participants, and wrote the manuscript. 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Front Pharmacol 10:1683. https://doi.org/10.3389/fphar.2019.01683 Risso G, Blaustein M, Pozzi B, et al (2015) Akt/PKB: one kinase, many modifications. Biochem J 468:203–214. https://doi.org/10.1042/BJ20150041 Ni Y, Sinnett-Smith J, Young SH, Rozengurt E (2013) PKD1 mediates negative feedback of PI3K/Akt activation in response to G protein-coupled receptors. PLoSOne 8:e73149. https://doi.org/10.1371/journal.pone.0073149 Cite Share Download PDF Status: Published Journal Publication published 17 Feb, 2023 Read the published version in Molecular Biology Reports → Version 1 posted Reviewers agreed at journal 14 Oct, 2022 Reviewers invited by journal 25 Jul, 2022 Editor assigned by journal 21 Jul, 2022 First submitted to journal 20 Jul, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1877745","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":123696969,"identity":"59c72444-0c05-4ea8-a08f-03bdf79887e0","order_by":0,"name":"Christine LEGAY","email":"","orcid":"","institution":"Ecole Normale Superieure Paris-Saclay","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Christine","middleName":"","lastName":"LEGAY","suffix":""},{"id":123696970,"identity":"f06bb4c3-11d3-448c-86a7-d86cb4a3f66f","order_by":1,"name":"Sophie DOUBLIER","email":"","orcid":"","institution":"Université de Paris Faculté de Santé: Universite de Paris Faculte de Sante","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sophie","middleName":"","lastName":"DOUBLIER","suffix":""},{"id":123696971,"identity":"2b492455-2cd3-412a-80e4-30f0d78ab087","order_by":2,"name":"Sylvie BABAJKO","email":"","orcid":"","institution":"Institut national de la santé et de la recherche médicale: INSERM","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sylvie","middleName":"","lastName":"BABAJKO","suffix":""},{"id":123696972,"identity":"a49faad7-f679-4d84-984d-47cd6d5de6b8","order_by":3,"name":"Jean-Marc RICORT","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-3924-0816","institution":"Ecole Normale Superieure Paris-Saclay","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jean-Marc","middleName":"","lastName":"RICORT","suffix":""}],"badges":[],"createdAt":"2022-07-20 11:36:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1877745/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1877745/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-023-08300-z","type":"published","date":"2023-02-17T18:57:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":24527436,"identity":"bb64b26c-60da-4c73-9fd6-597e883f8dbf","added_by":"auto","created_at":"2022-07-29 18:36:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":82051,"visible":true,"origin":"","legend":"\u003cp\u003eEGF increases PKD1 activity in PKD1-overexpressing MCF-7 cells. A) Stable overexpression of PKD1 in MCF-7 cells. Proteins from MCF-7 cells transfected with either empty vector (MCF7-pcDNA3, clones C1 and C2) or PKD1-expressing vector (MCF7-PKD1, clones P1 and P2) were subjected to SDS-PAGE, transferred to nitrocellulose and immunodetected with anti-PKD1 or anti-actin antibodies. The autoradiograms presented are from typical experiments. B) MCF-7 cells overexpressing PKD1 (clone P1), or not (clone C1), growing on coverslips were fixed and permeabilized. Cells were incubated with specific antibodies directed against PKD1 processed with secondary antibodies stained with Alexa-488. The photographs presented are representative of three independent experiments. C) MCF-7 cells overexpressing PKD1 (P1), or not (C1), were incubated for different periods of time with EGF (40 ng.mL\u003csup\u003e-1\u003c/sup\u003e). Cells were lysed and equal amounts of proteins were separated by SDS-PAGE. Proteins were transferred to nitrocellulose and immunodetected with antibodies directed against phospho-PKD1 (P-PKD1) or actin. The results presented are from typical experiments. Graphs represent quantitative analysis of phospho-PKD1, in cells overexpressing PKD1 (P1 and P2 clones), or not (clones C1 and C2), under each set of conditions, corrected for background, and expressed relative to untreated control (clones C1 and C2) cells. The results are presented as the means ± SEM of four independent experiments. **p ≤ 0.01 and ***p ≤ 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1877745/v1/8ef2fa6f73950d432f578d28.png"},{"id":24527053,"identity":"305f2ca9-f91c-4b2d-8172-513f9f19ace0","added_by":"auto","created_at":"2022-07-29 18:31:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":123296,"visible":true,"origin":"","legend":"\u003cp\u003ePKD1 overexpression increases EGF-stimulated PI 3-kinase/Akt signaling pathway. A) MCF-7 cells overexpressing PKD1 (P1), or not (C1), were incubated for different periods of time with EGF (40\u0026nbsp;ng.mL\u003csup\u003e-1\u003c/sup\u003e). Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with either two distinct anti-phospho-Akt (P-Akt (T308) or P-Akt (S473)) antibodies, or anti-α-actinin antibodies. Typical experiments are presented. B) Quantitative analyses of phosphorylated Akt onto T308- (left) and S473-residues (right) under each set of conditions, corrected for background, and expressed as percentages of the maximal activity measured in control cells (clones C1 and C2) after 7 min EGF stimulation. The results are presented as the means ± SEM of three independent experiments. **p ≤ 0.01 and ***p ≤ 0.001. C)\u0026nbsp;MCF-7 overexpressing PKD1 (P1), or not (C1), were pre-incubated with or without Gö6976 (200 nM) for 1 h prior to be stimulated for 15 min with or without EGF (40 ng.mL\u003csup\u003e-1\u003c/sup\u003e). Cells were then lysed and proteins were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-phospho-PKD1, anti-phospho-Akt (P-Akt (T308) or P-Akt (S473)), or anti-α-actinin antibodies. The autoradiograms presented are from typical experiments. D) MCF-7 cells overexpressing PKD1 (P1), or not (C1), were transfected with siRNAs targeting PKD1 mRNA (siP), or scrambled siRNAs used as controls (siC). Three days after transfection, cells were treated for 15\u0026nbsp;min with or without EGF (40\u0026nbsp;ng.mL\u003csup\u003e-1\u003c/sup\u003e), then lysed and proteins separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-PKD1, anti-phospho-PKD1, anti-phospho-Akt (P-Akt (T308) or P-Akt (S473)), or anti-actinin antibodies. Autoradiograms presented are from typical experiments. Histograms represent the quantitative analysis of EGF-stimulated T308- and S473- phosphorylated-Akt. Results are presented as the fold-stimulation of either T308- or S473-phosphorylated-Akt measured in EGF-stimulated control cells transfected with non-targeting siRNA. The results are presented as the means ± SEM of three independent experiments. ***p ≤ 0.001 (calculated between control non-targeting (siC) and PKD1-targeting (siP) siRNA transfected cells).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1877745/v1/a15d8b059261157b0fddcee4.png"},{"id":24527438,"identity":"08dd7a8a-f014-4f51-84f9-9a899b61f648","added_by":"auto","created_at":"2022-07-29 18:36:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":69976,"visible":true,"origin":"","legend":"\u003cp\u003ePKD1 overexpression increases EGF-stimulated ERK1/2 activity. A) MCF-7 cells overexpressing PKD1 (P1), or not (C1), were incubated for different periods of time with EGF (40\u0026nbsp;ng.mL\u003csup\u003e-1\u003c/sup\u003e). Cells were lysed and equal amounts of proteins were separated by SDS-PAGE. Proteins were transferred to nitrocellulose and immunoblotted with either anti-phospho-ERK1/2 (P-ERK1/2) or anti-α-actinin antibodies. Typical autoradiograms are presented. B) Quantitative analyses of phosphorylated ERK1/2 under each set of conditions, corrected for background, and expressed as percentages of the maximal activity measured in control cells (clones C1 and C2) after 7 min EGF stimulation. The results are presented as the means ± SEM of three independent experiments. **p ≤ 0.01 and ***p ≤ 0.001. C) MCF-7 cells overexpressing PKD1 (P1), or not (C1), were pre-incubated with or without Gö6976 (200 nM) for 1 h prior to be stimulated for 15 min with or without EGF\u0026nbsp;(40\u0026nbsp;ng.mL\u003csup\u003e-1\u003c/sup\u003e). Cells were then lysed and proteins were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-phospho-ERK1/2 (P-ERK1/2) or anti-α-actinin antibodies. The autoradiograms presented are from typical experiments. D) MCF-7 cells overexpressing PKD1 (clones P), or not (clones C), were transfected with siRNAs targeting PKD1 mRNA (siP), or scrambled siRNAs used as controls (siC). Three days after transfection, cells were treated for 15\u0026nbsp;min with or without EGF (40 ng.mL\u003csup\u003e-1\u003c/sup\u003e), then lysed and proteins separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-phospho-ERK1/2 (P-ERK1/2). The autoradiograms presented are from typical experiments. The histograms represent the quantitative analysis of phosphorylated-ERK1/2 expressed as the fold-stimulation of phosphorylated-ERK1/2 measured in EGF-stimulated cells transfected with non-targeting siRNA (siC). The results are presented as the means ± SEM of three independent experiments. ***p ≤ 0.001 (calculated between control non-targeting (siC) and PKD1-targeting (siP) siRNA transfected cells).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-1877745/v1/553859807daed42ef9699120.png"},{"id":24527054,"identity":"72facd2f-aa9d-4f59-bd8b-87f49705e728","added_by":"auto","created_at":"2022-07-29 18:31:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":28914,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic model presenting the regulation of the EGF signaling pathway by PKD1. A) In control cells, upon EGF binding, the EGF receptor is activated and stimulates signaling pathways leading to the activation of PI 3-kinase, PKD1 and ERK1/2, respectively. According to our results, the activation of PKD1 by EGF has no significant effect on the EGF-stimulated PI 3-kinase and ERK1/2 signaling pathways. B) In PKD1-overexpressing cells, EGF-stimulated PKD1 activity is significantly increased compared to control cells, and positively regulates the EGF-stimulated PI 3-kinase and MEK/ERK signaling pathways.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-1877745/v1/b7aff3ba21db9ea6ca5f9340.png"},{"id":44719763,"identity":"992bb81a-542d-4abb-b99e-e5a6a5f7b0c4","added_by":"auto","created_at":"2023-10-16 19:02:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":574000,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1877745/v1/6fcd6fa5-24f9-4ba4-853f-596b4d473901.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eProtein Kinase D1 Overexpression Potentiates Epidermal Growth Factor Signaling Pathway in Mcf-7 Cells\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eProtein kinase D1 (PKD1) is a serine/threonine kinase with a wide tissue expression which is schematically composed of two domains, an amino-terminal regulatory region and a carboxyl-terminal catalytic region. Based on strong homologies between its catalytic domain and those of Ca\u003csup\u003e2+\u003c/sup\u003e/calmodulin-dependent protein kinases (CaMKs), PKD1 was classified in the CaMKs superfamily [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The regulatory region of PKD1 contains an alanine- and proline-rich apolar (AP) domain, a diacylglycerol (DAG)/phorbol esters binding domain (C1 domain), an acidic (AC) domain, and a pleckstrin homology (PH) domain. C1 domain allows PKD1 to localize either to the Golgi apparatus or to the plasma membrane upon its interaction with DAG and also plays a role in the nuclear import of PKD1. On the other hand, PH domain regulates the nuclear export of PKD1 and allows PKD1 to interact with molecular partners. Both C1 and PH domains auto-inhibit the catalytic domain and the deletion of one of them generates a constitutively active form of PKD1 (for review see [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]).\u003c/p\u003e \u003cp\u003ePKD1 activation is generally triggered through a phospholipase C- (PLC) and protein kinase C- (PKC) dependent signaling pathway stimulated after the binding of an extracellular ligand to a transmembrane receptor [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Thus, growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], or mitogenic neuropeptides such as bombesin, vasopressin and angiotensin II [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], or lysophosphatidic acid (LPA) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] were shown to stimulate PLC-dependent signaling pathways and to increase DAG intracellular concentration leading to the recruitment of PKD1 at the plasma membrane and its subsequent phosphorylation by different PKCs [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The two serine residues of PKD1, 738 and 742 (S738/S742) (human numbering), localized into its activation loop, are the main targets of PKCs. Their phosphorylation increases the kinase activity of PKD1 inducing its subsequent autophosphorylation onto its C-terminal S910 residue [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, PKC-independent signaling pathways and some stimuli, such as oxidative stress or caspase-induced PKD1 cleavage, were also described to induce the S738/S742 phosphorylation of PKD1 without affecting the phosphorylation state of the S910 residue [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePKD1 regulates numerous biological functions such as cell proliferation, differentiation, cell-cell interactions, migration, invasion, apoptosis, and survival. This multiplicity of actions is especially due to the ability of PKD1 to regulate several signaling pathways such as the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK)- and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt-dependent signaling pathways that play major roles in cell proliferation and survival, especially in tumor cells. In breast cancer cells, the role of PKD1 remains controversial since we and others demonstrated that PKD1 promotes cell proliferation and that its overexpression strengthens this function, whereas others suggested that PKD1 has anti-proliferative effect (for review see [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]). However, PKD1 expression level varies from one breast cancer cell type to another, potentially impacting downstream signaling pathways and biological responses. Therefore, in this study, we determine whether and how PKD1 expression levels may modulate the EGF-stimulated MEK/ERK and PI 3-kinase/Akt signaling pathways by using MCF-7 cells overexpressing or not PKD1.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Antibodies and materials\u003c/h2\u003e \u003cp\u003eAnti-Akt1, anti-phospho-ERK1/2 antibodies used for immunoblotting were purchased from Upstate Biotechnology-Millipore (Lake Placid, NY); anti-PKD1, anti-α-actinin and anti-actin from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phospho-PKD1 (S738/S742) and anti-phospho-Akt (T308 and S473) from Cell Signaling (Danvers, MA). EGF was purchased from PeproTech (Rocky Hill, NJ); PKD1-targeting (sc-36245) and control non-targeting (sc-37007) siRNA from Santa Cruz Biotechnology, and G\u0026ouml;6976 from Calbiochem (La Jolla, CA). All other biochemicals were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France), or ICN (Orsay, France).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell culture\u003c/h2\u003e \u003cp\u003eStably transfected MCF-7 cells (ATCC HTB22, CVCL_0031), overexpressing PKD1, or not [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], were grown to 80\u0026ndash;85% confluence in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium supplemented with 10% fetal bovine serum (FBS), 100 U.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e penicillin, 100 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e streptomycin and 1 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e G418 (Calbiochem, Darmstadt, Germany). Before each experiment, cells were starved in serum free media for 16\u0026ndash;24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Western Immunoblotting\u003c/h2\u003e \u003cp\u003eMCF-7 cells were cultured as described and treated for different periods of time with or without EGF (40 ng.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Cells were then solubilized in lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 100 mM NaF, 10 mM EDTA, 2 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, 10 mM pyrophosphate, 1 mM PMSF, 100 U.ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e aprotinin) containing 1% Nonidet P-40 (NP-40), and proteins separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were incubated for 1 hour at room temperature or overnight at 4\u0026deg;C with the primary specific antibodies, then incubated for 1 hour at room temperature with the peroxidase-conjugated secondary antibodies and revealed by enhanced chemiluminescence (Amersham, GE Healthcare, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Fluorescence microscopy\u003c/h2\u003e \u003cp\u003eStably transfected MCF-7 cells were cultured onto coverslips. Cells were rinsed in PBS, fixed for 5 minutes at -20\u0026deg;C with cold acetone and rinsed again with a three-fold excess of PBS before being incubated for 30 min at 4\u0026deg;C in PBS containing 3% BSA. Cells were incubated for 1 hour at 4\u0026deg;C with 10 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e anti-PKD1 antibody diluted in PBS-BSA 3%, then washed twice for 10 min in PBS-BSA 3% and incubated for 30 min at room temperature with 1 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Alexa-488 conjugated goat anti-rabbit immunoglobulin G heavy plus light chains (H\u0026thinsp;+\u0026thinsp;L) (Molecular Probes, Eugene, OR) diluted in PBS-BSA 3%. Cells were rinsed three times with PBS-BSA 3% and mounted with Mowiol antifading reagent (Calbiochem, La Jolla, CA). Microscope analysis was carried out using the TCS NT confocal imaging system (Leica Instruments, Heidelberg, Germany), equipped with a 63\u0026times; objective (plan apo, numerical aperture\u0026thinsp;=\u0026thinsp;1.4). For Alexa-488, an argon-krypton ion laser adjusted to 488 nm was used. The signal was integrated over four to eight frames to reduce the noise. The pinhole was adjusted to allow a field depth of about 1 \u0026micro;m, corresponding to the increment between two adjacent sections. At least 20 cells were observed in each condition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. siRNA transfection\u003c/h2\u003e \u003cp\u003esiRNA transfection was performed according to the manufacturer\u0026rsquo;s protocol. Briefly, 3 x 10\u003csup\u003e5\u003c/sup\u003e cells were seeded per well in 2 mL antibiotic-free DMEM supplemented with 10% FBS and incubated for 24 h. For each well, 1 \u0026micro;g siRNA and 8 \u0026micro;L siRNA transfection reagent, each diluted in 100 \u0026micro;L siRNA transfection medium, were combined, incubated for 45 min at room temperature and then applied to the cells in a final volume of 1 mL siRNA transfection medium. After a 7-h incubation at 37\u0026deg;C, DMEM supplemented with 10% FBS was added and cells cultured for an additional 18\u0026ndash;24 h at 37\u0026deg;C before analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical significance of differences between experimental groups was determined with the Student\u0026rsquo;s t-test using Prims 5.03 software for Windows (GraphPad Software, San Diego, CA). Differences between values were considered to be highly significant when p\u0026thinsp;\u0026le;\u0026thinsp;0.01 (**) or p\u0026thinsp;\u0026le;\u0026thinsp;0.001 (***).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1. EGF-stimulated PKD1 phosphorylation is increased in PKD1 overexpressing cells\u003c/h2\u003e \u003cp\u003eBefore determining whether overexpression of PKD1 modulated the EGF-induced MEK/ERK and PI 3-kinase signaling pathways in MCF-7 cells, we first analyzed the status of EGF-stimulated PKD1 in control (Mock) and PKD1 overexpressing cells. As we previously described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], MCF-7 cells stably transfected with PKD1-expressing vector (clones P1 and P2) expressed higher levels of PKD1 (approximately 4.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8- and 3.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15-fold, respectively) than control cells stably transfected with the corresponding empty vector (clones C1 and C2) (Fig.\u0026nbsp;1A). This result was further confirmed by immunofluorescence staining through the direct detection of PKD1 in living cells (Fig.\u0026nbsp;1B). EGF stimulated PKD1 phosphorylation onto S738/S742 residues in control cells (C1 and C2) with maximal effect occurring after 30 min of EGF treatment and remaining mostly stable up to 60 min (Fig.\u0026nbsp;1C). In cells overexpressing PKD1, EGF stimulated PKD1 phosphorylation with a similar time course than in control cells but, whatever the incubation time considered, EGF-stimulated PKD1 phosphorylation was significantly stronger (by almost 1.4- to 1.9-fold) than in control cells. Therefore, these results demonstrated that PKD1 overexpression did not modify PKD1 time course activation in response to EGF but strongly increased the levels of EGF-induced phosphorylated PKD1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Overexpression of PKD1 potentiates EGF-stimulated PI 3-kinase signaling pathway\u003c/h2\u003e \u003cp\u003eWe thus analyzed whether increasing PKD1 expression and activity levels may affect the EGF-stimulated PI 3-kinase signaling pathway. To this end, we studied the phosphorylation state of T308 and S473 residues of Akt, a downstream target of PI 3-kinase. In control cells (C), EGF transiently stimulated Akt phosphorylation onto its T308 and S473 residues with maximal effect occurring after 7 min of EGF treatment and decreasing very rapidly to basal levels after 15 min (Fig.\u0026nbsp;2A and 2B). In contrast, EGF-stimulated Akt phosphorylation was much less transient in PKD1 overexpressing cells and return to basal values was markedly delayed since a significant phosphorylation of the two residues still remained after 30 min of EGF treatment. Moreover, although PKD1 overexpression did not change the maximal phosphorylation level of EGF-induced S473-Akt residue (Fig.\u0026nbsp;2B, right), it markedly increased the maximal phosphorylation level of EGF-induced T308-Akt residue (by 1.4-fold over control cells) (Fig.\u0026nbsp;2B, left). Since we previously demonstrated that overexpression of PKD1 did not change the expression level of Akt [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], these results indicated that PKD1 overexpression markedly affected the EGF-induced Akt phosphorylation level and time course. To determine whether these effects were specifically due to the overexpression of PKD1, cells were first incubated with a pharmacological inhibitor of PKD1, G\u0026ouml;6976, before being stimulated by EGF (Fig.\u0026nbsp;2C). G\u0026ouml;6976 totally inhibited EGF-stimulated PKD1 phosphorylation in control and PKD1-overexpressing cells (Fig.\u0026nbsp;2C) without affecting PKD1 expression [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. EGF-stimulated T308- and S473-Akt phosphorylation was not affected by G\u0026ouml;6976 in control cells but was strongly impaired (by almost 60%) in PKD1-overexpressing cells reaching similar levels to those observed in EGF-stimulated control cells (Fig.\u0026nbsp;2C). To further strengthen these results, PKD1 was also inhibited by transfecting cells with siRNAs that specifically target PKD1 mRNA (Fig.\u0026nbsp;2D). PKD1-targeting siRNA (siP) strongly reduced PKD1 expression and EGF-stimulated PKD1 phosphorylation both in control and PKD1-overexpressing cells (Fig.\u0026nbsp;2D). EGF-induced T308- and S473-Akt phosphorylation was not significantly changed in control cells upon siP treatment but was profoundly decreased (by 60% and 54%, respectively) in PKD1-overexpressing cells (Fig.\u0026nbsp;2D). For each condition, we checked that control non-targeting (scrambled) siRNAs (siC) did not affect PKD1 expression (data not shown and [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]). Taken together, these results indicated that the EGF-stimulated PI 3-kinase signaling pathway is not directly dependent upon endogenous PKD1 in control cells but is strongly potentiated by PKD1 overexpression (data summarized in Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Overexpression of PKD1 potentiates EGF-stimulated ERK signaling pathway\u003c/h2\u003e \u003cp\u003eWe further investigated whether PKD1 overexpression affected the EGF-stimulated MEK/ERK signaling pathway. MCF-7 cells overexpressing PKD1, or not, were acutely stimulated with EGF for different periods of time and the status of ERK1/2 phosphorylation analyzed. In control cells, EGF transiently stimulated ERK1/2 phosphorylation, with maximal effect occurring after 7 min of EGF treatment, then decreasing rapidly after 15 min and remaining mostly stable up to 30 min (Fig.\u0026nbsp;3A and 3B). In PKD1-overexpressing cells, maximal ERK1/2 phosphorylation level, measured after 7 min of EGF treatment, was significantly increased (about 1.3-fold compared to control cells). Moreover, the ERK1/2 phosphorylation time course was profoundly changed, becoming much less transient and remaining mostly stable, close to maximal values, up to 30 min of EGF incubation (Fig.\u0026nbsp;3A and 3B). In order to understand how PKD1 expression may affect the EGF-stimulated MEK/ERK signaling pathway, cells were either treated with a PKD1 pharmacological inhibitor (Fig.\u0026nbsp;3C) or transfected with siRNAs specifically targeting PKD1 mRNA (Fig.\u0026nbsp;3D), as previously described. In control cells, inhibition of PKD1, either with a pharmacological inhibitor (G\u0026ouml;6976) or with a PKD1-targeting siRNA (siP), did not affect EGF-induced ERK1/2 phosphorylation (Fig.\u0026nbsp;3C and 3D). In contrast, in PKD1-overexpressing cells, inhibition of PKD1 drastically decreased EGF-stimulated ERK1/2 phosphorylation (by almost 40%) (Fig.\u0026nbsp;3C and 3D). These results demonstrated that the EGF-stimulated MEK/ERK signaling pathway is not dependent upon PKD1 in control cells but strongly enhanced by PKD1 overexpression (data summarized in Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePKD1 plays a crucial role in breast cancer proliferation and reported data indicate that its expression level may represent a potential biomarker\u0026nbsp;[16, 17]. Thus, understanding how PKD1 expression level can affect signaling pathways tightly involved in breast cancer progression such as the EGF one represents\u0026nbsp;an interesting challenge to take up.\u003c/p\u003e\n\u003cp\u003eIn order to understand PKD1 involvement in EGF promoting breast cancer cell, we used a PKD1-overexpressing cell model previously published\u0026nbsp;[14], and investigated whether and how an increase in PKD1 expression level modulates EGF-stimulated PKD1 activity. We showed that PKD1 overexpression increased the levels of EGF-induced phosphorylated PKD1 but did not modify the pattern of the PKD1 time course activation in response to EGF. Lack of alteration of the PKD1 time course activation in PKD1-overexpressing cells represents an important point because it suggests that the signaling pathways implicated both in the activation and the inactivation of PKD1 are not altered following PKD1overexpression, indicating that the effects observed in PKD1-overexpressing cells are dependent on PKD1 overexpression only. Moreover, it also suggests that the overexpression of PKD1 does not seem to give raise to counter-regulatory signals within this cell model. We should point out, however, that, despite a four-fold PKD1 overexpression factor, the maximum increase in the EGF-induced PKD1 phosphorylation level is about 1.8-fold. Although we did not notice any particular changes in the subcellular localization of the protein in PKD1-overexpressing cells (Figure 1B), these results suggest that the overexpressed proteins could be localized in subcellular compartments in which they cannot be activated upon EGF binding to its receptor. Moreover, one could not also exclude that there is a maximum level of activation of PKD1 in response to EGF in MCF-7 cells which cannot be exceeded whatever the PKD1 levels.\u003c/p\u003e\n\u003cp\u003eWe showed that PKD1 is not involved in the PI 3-kinase/Akt and MEK/ERK signaling pathways stimulated by EGF in MCF-7 control cells. In fact, pharmacological inhibition of PKD1 or PKD1-targeting siRNAs do not affect EGF-induced Akt and ERK phosphorylation state in control cells. These data are totally consistent with previous ones in which we showed that pharmacological inhibition of PKD1 by G\u0026ouml;6976 did not affect ERK nor Akt phosphorylation level in MCF-7 control cells cultured in DMEM containing 10% FBS\u0026nbsp;[14]. They indicate that, when PKD1 is expressed at basal level, EGF-stimulated PI 3-kinase/Akt and MEK/ERK signaling pathways are independent of PKD1 in MCF-7 cells.\u003c/p\u003e\n\u003cp\u003eBy contrast, overexpression of PKD1 markedly impacts the time courses of EGF-induced Akt and ERK phosphorylation (i.e. activation) increasing both the level of phosphorylated proteins and the activation time duration. Such results are consistent with previous data showing that PKD1 overexpression increased the duration of bombesin-induced ERK signaling in different cell models\u0026nbsp;[6, 18]. Considering the crucial role played by these two signaling pathways in cancer cell proliferation, such quantitative and qualitative effects can have major consequences on the nature and specificity of molecular interactions that occur within signaling pathways. Indeed, an increase in the amount of activated Akt and ERK proteins will allow their interaction with substrates or partners for which these proteins do not interact in control cells due to weaker affinities. In addition, an increase in the duration of the EGF-induced time course activation can also participate in the appearance or an increase in the level of active Akt and ERK proteins in subcellular compartments in which they are not usually found in control conditions and their subsequent interactions with novel partners. In accordance with these hypotheses, PKD1-overexpressing MCF-7 cells were shown to be more resistant to the induction of apoptosis (results not shown) indicating a modulation of intracellular signaling pathways and the acquisition of a more aggressive phenotype\u0026nbsp;[14, 17, 19, 20]\u0026nbsp;following PKD1 overexpression.\u003c/p\u003e\n\u003cp\u003eAn increase in the duration of the activation time course of a protein could be the consequence of an inhibition of the mechanisms involved in its inactivation. In the particular case of Akt, its inactivation can proceed from indirect mechanisms through lipid phosphatases such as PTEN or SHIP, or from direct mechanisms implicating protein phosphatases such as PP2A or PHLPP(for review see\u0026nbsp;[21]). Overexpression of PKD1 was shown to enhance the association of PTEN and the regulatory subunit of PI 3-kinase, p85, in intestinal epithelial cells stimulated by G protein-coupled receptor (GPCR) agonists\u0026nbsp;[22]. This suggests that similar mechanisms could occur in PKD1-overexpressing MCF-7 cells leading to the observed modulation of the EGF-stimulated PI 3-kinase/Akt signaling pathway.\u003c/p\u003e\n\u003cp\u003eAlthough the EGF-induced T308 and S473 residues phosphorylation time course was similarly modulated in PKD1-overexpressing cells, it should be noted that only the level of phosphorylation of T308 residue is significantly increased after 7 minutes of stimulation by EGF. T308 and S473 residues of Akt are phosphorylated by two distinct kinases, PDK1 and mTORC2, respectively suggesting that overexpression of PKD1 in MCF-7 cells specifically modulates a PDK1-dependent signaling pathway without affecting the mTORC2 signaling pathway. To our knowledge, a link has never been demonstrated between PKD1 and PDK1 but it would be interesting to study whether PKD1 is capable of modulating the activity of PDK1.\u003c/p\u003e\n\u003cp\u003eTherefore, taken together, our results highlight the crucial role of PKD1 expression level to regulate the EGF signaling pathway in MCF-7 cells. As summarized in Figure 4, when expressed at its basal level, PKD1 does not mediate the EGF-induced activation of the PI 3-kinase/Akt and MEK/ERK signaling pathways (Figure 4A). However, when overexpressed, PKD1 contributes to an increase in the activation state of these two signaling pathways in response to EGF (Figure 4B).\u003c/p\u003e\n\u003cp\u003eIn conclusion, we demonstrated that PKD1 overexpression up-regulates the EGF-stimulated PI 3-kinase/Akt and MEK/ERK pro-proliferative signaling pathways in MCF-7 breast cancer cells. Therefore, these data highlight the importance to consider the PKD1 expression level as a potential interesting biomarker in breast cancers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by the Institut National de la Sant\u0026eacute; et de la Recherche M\u0026eacute;dicale (INSERM) and the Ecole Normale Sup\u0026eacute;rieure Paris-Saclay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e: The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e: CL, SD and SB performed experiments and led the project. JMR conceived and directed the project, designed the experiments, supervised the participants, and wrote the manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e: not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e: not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJohannes FJ, Prestle J, Eis S, et al (1994) PKCu is a novel, atypical member of the protein kinase C family. JBiolChem 269:6140\u0026ndash;6148\u003c/li\u003e\n\u003cli\u003eValverde AM, Sinnett-Smith J, Van LJ, Rozengurt E (1994) Molecular cloning and characterization of protein kinase D: a target for diacylglycerol and phorbol esters with a distinctive catalytic domain. ProcNatlAcadSciUSA 91:8572\u0026ndash;8576\u003c/li\u003e\n\u003cli\u003eRozengurt E (2011) Protein kinase D signaling: multiple biological functions in health and disease. Physiology(Bethesda) 26:23\u0026ndash;33. https://doi.org/10.1152/physiol.00037.2010\u003c/li\u003e\n\u003cli\u003eWang QJ (2006) PKD at the crossroads of DAG and PKC signaling. Trends PharmacolSci 27:317\u0026ndash;323\u003c/li\u003e\n\u003cli\u003eWong C, Jin ZG (2005) Protein kinase C-dependent protein kinase D activation modulates ERK signal pathway and endothelial cell proliferation by vascular endothelial growth factor. JBiolChem 280:33262\u0026ndash;33269. https://doi.org/10.1074/jbc.M503198200\u003c/li\u003e\n\u003cli\u003eZhukova E, Sinnett-Smith J, Rozengurt E (2001) Protein kinase D potentiates DNA synthesis and cell proliferation induced by bombesin, vasopressin, or phorbol esters in Swiss 3T3 cells. J BiolChem 276:40298\u0026ndash;40305\u003c/li\u003e\n\u003cli\u003ePaolucci L, Sinnett-Smith J, Rozengurt E (2000) Lysophosphatidic acid rapidly induces protein kinase D activation through a pertussis toxin-sensitive pathway. Am J Physiol Cell Physiol 278:C33-39. https://doi.org/10.1152/ajpcell.2000.278.1.C33\u003c/li\u003e\n\u003cli\u003eRey O, Reeve JR, Zhukova E, et al (2004) G protein-coupled receptor-mediated phosphorylation of the activation loop of protein kinase D: dependence on plasma membrane translocation and protein kinase Cepsilon. J Biol Chem 279:34361\u0026ndash;34372. https://doi.org/10.1074/jbc.M403265200\u003c/li\u003e\n\u003cli\u003eSteinberg SF (2012) Regulation of protein kinase D1 activity. MolPharmacol 81:284\u0026ndash;291. https://doi.org/10.1124/mol.111.075986\u003c/li\u003e\n\u003cli\u003eEndo K, Oki E, Biedermann V, et al (2000) Proteolytic cleavage and activation of protein kinase C [micro] by caspase-3 in the apoptotic response of cells to 1-beta -D-arabinofuranosylcytosine and other genotoxic agents. J BiolChem 275:18476\u0026ndash;18481\u003c/li\u003e\n\u003cli\u003eV\u0026aacute;ntus T, Vertommen D, Saelens X, et al (2004) Doxorubicin-induced activation of protein kinase D1 through caspase-mediated proteolytic cleavage: identification of two cleavage sites by microsequencing. Cell Signal 16:703\u0026ndash;709. https://doi.org/10.1016/j.cellsig.2003.11.009\u003c/li\u003e\n\u003cli\u003eD\u0026ouml;ppler H, Storz P (2017) Mitochondrial and Oxidative Stress-Mediated Activation of Protein Kinase D1 and Its Importance in Pancreatic Cancer. Front Oncol 7:41. https://doi.org/10.3389/fonc.2017.00041\u003c/li\u003e\n\u003cli\u003eYoussef I, Ricort J-M (2019) Deciphering the Role of Protein Kinase D1 (PKD1) in Cellular Proliferation. Mol Cancer Res 17:1961\u0026ndash;1974. https://doi.org/10.1158/1541-7786.MCR-19-0125\u003c/li\u003e\n\u003cli\u003eKaram M, Legay C, Auclair C, Ricort JM (2012) Protein kinase D1 stimulates proliferation and enhances tumorigenesis of MCF-7 human breast cancer cells through a MEK/ERK-dependent signaling pathway. ExpCell Res 318:558\u0026ndash;569. https://doi.org/10.1016/j.yexcr.2012.01.001\u003c/li\u003e\n\u003cli\u003eKaram M, Lassarre C, Legay C, Ricort JM (2012) Phosphatidylinositol 3-kinase and protein kinase D1 specifically cooperate to negatively regulate the insulin-like growth factor signaling pathway. BiochimBiophysActa 1823:558\u0026ndash;569. https://doi.org/10.1016/j.bbamcr.2011.12.007\u003c/li\u003e\n\u003cli\u003eSpasojevic C, Marangoni E, Vacher S, et al (2018) PKD1 is a potential biomarker and therapeutic target in triple-negative breast cancer. Oncotarget 9:23208\u0026ndash;23219. https://doi.org/10.18632/oncotarget.25292\u003c/li\u003e\n\u003cli\u003eGe J, Zhang X, Zhang B, et al (2020) Higher tumor protein kinase D1 correlates with increased tumor size, BCLC stage, CA199 level, AFP level and worse overall survival in hepatocellular carcinoma patients. Clin Res Hepatol Gastroenterol S2210-7401(20)30337\u0026ndash;5. https://doi.org/10.1016/j.clinre.2020.11.004\u003c/li\u003e\n\u003cli\u003eZhang L, Li Z, Liu Y, et al (2018) Analysis of oncogenic activities of protein kinase D1 in head and neck squamous cell carcinoma. BMCCancer 18:1107. https://doi.org/10.1186/s12885-018-4965-6\u003c/li\u003e\n\u003cli\u003eKaram M, Bieche I, Legay C, et al (2014) Protein kinase D1 regulates ERalpha-positive breast cancer cell growth response to 17beta-estradiol and contributes to poor prognosis in patients. JCell MolMed 18:2536\u0026ndash;2552. https://doi.org/10.1111/jcmm.12322\u003c/li\u003e\n\u003cli\u003eMerzoug-Larabi M, Youssef I, Bui AT, et al (2019) Protein Kinase D1 (PKD1) Is a New Functional Non-Genomic Target of Bisphenol A in Breast Cancer Cells. Front Pharmacol 10:1683. https://doi.org/10.3389/fphar.2019.01683\u003c/li\u003e\n\u003cli\u003eRisso G, Blaustein M, Pozzi B, et al (2015) Akt/PKB: one kinase, many modifications. Biochem J 468:203\u0026ndash;214. https://doi.org/10.1042/BJ20150041\u003c/li\u003e\n\u003cli\u003eNi Y, Sinnett-Smith J, Young SH, Rozengurt E (2013) PKD1 mediates negative feedback of PI3K/Akt activation in response to G protein-coupled receptors. PLoSOne 8:e73149. https://doi.org/10.1371/journal.pone.0073149\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"protein kinase D1, cell signaling, breast cancer cell, epidermal growth factor","lastPublishedDoi":"10.21203/rs.3.rs-1877745/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1877745/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eI. Background: Protein kinase D1, PKD1, is a serine-threonine kinase implicated in cell proliferation, migration, invasion, and/or apoptosis and its activation by several growth factors sets this enzyme as a key regulator of tumorigenesis and tumor progression. Despite many studies, its role in the regulation of intracellular signaling pathways remains widely disparate and needs to be clarified.\u003c/p\u003e\u003cp\u003eII. Methods and Results: By using human breast cancer cells MCF-7, overexpressing or not PKD1, we demonstrated that PKD1 expression level modulated the tumor growth-promoting epidermal growth factor (EGF) signaling pathway. We also showed that EGF acutely stimulated PKD1 phosphorylation with similar time courses both in control and PKD1-overexpressing cells. However, PKD1 overexpression specifically and markedly increased EGF-induced phosphorylation of Akt (onto T308 and S473 residues) and extracellular-regulated protein kinase (ERK1/2). Finally, pharmacological inhibition of PKD1 activity or lowering its expression level using specific siRNAs drastically reduced EGF-stimulated Akt and ERK phosphorylation in PKD1overexpressing cells, but not in control cells.\u003c/p\u003e\u003cp\u003eIII. Conclusions: Overall, these results identified the level of PKD1 expression as a key determinant in the regulation of the EGF signaling pathway highlighting its crucial role in a tumorigenic setting.\u003c/p\u003e","manuscriptTitle":"Protein Kinase D1 Overexpression Potentiates Epidermal Growth Factor Signaling Pathway in Mcf-7 Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-29 18:31:41","doi":"10.21203/rs.3.rs-1877745/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2022-10-14T11:49:55+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-07-25T09:45:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-07-22T03:23:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2022-07-20T07:35:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e078d28e-8fd6-4d14-9abf-3ebe6dff0977","owner":[],"postedDate":"July 29th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T18:59:13+00:00","versionOfRecord":{"articleIdentity":"rs-1877745","link":"https://doi.org/10.1007/s11033-023-08300-z","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2023-02-17 18:57:10","publishedOnDateReadable":"February 17th, 2023"},"versionCreatedAt":"2022-07-29 18:31:41","video":"","vorDoi":"10.1007/s11033-023-08300-z","vorDoiUrl":"https://doi.org/10.1007/s11033-023-08300-z","workflowStages":[]},"version":"v1","identity":"rs-1877745","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1877745","identity":"rs-1877745","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}