L'expression élevée de la GSTP1 est fréquemment observée dans les cancers et est positivement corrélée à la résistance aux chimiothérapies. Cette enzyme de détoxication de phase II peut aussi réguler l'activité de protéines comme JNK et TRAF2 et, par conséquent, peut moduler les voies de prolifération et de mort cellulaire. Ce projet a donc consisté à étudier le rôle de la GSTP1 dans la prolifération des hépatocytes normaux ou transformés. L'étude de la régénération hépatique chez des souris Gstp1/2‐/‐ a permis de démontrer le rôle des protéines GSTP1 et GSTP2 dans le contrôle de la progression des hépatocytes normaux dans le cycle cellulaire. Après hépatectomie partielle chez les souris Gstp1/2‐/‐, une diminution importante du nombre d'hépatocytes dans les phases S, G2 et M est observée comparativement à des foies de souris contrôle. Cette réduction est associée à des retards d'expression de protéines impliquées dans l'initiation de la prolifération, le contrôle du point de restriction dépendant des mitogènes et dans la transition G1/S. Ces modifications sont associées à une réduction de l'expression de TRAF2 et de l'activation de JNK et ERK, alors que les taux de p21 et de p53 sont élevés. Parallèlement, un décalage dans l'expression d'enzymes qui régulent l'homéostasie redox et participent à l'activation des MAPK est observé. L'utilisation de cellules cancéreuses de différentes origines dont le foie, a également permis de corréler l'absence de GSTP1 à une diminution de prolifération cellulaire sans altération de la suivie cellulaire. Cependant dans ces conditions, nous observons une augmentation de l'expression de TRAF2, pJNK, pATF2, ATF3 associée à une induction de p21. Nous avons également montré que les effets de la GSTP1 sur la prolifération cellulaire sont régulés par l'activation de JNK. L'évidence du lien entre l'expression de la GSTP1 et la prolifération hépatocytaire nous a conduit à analyser l'expression d'enzymes de détoxication dans des carcinomes hépatocellulaires (CHC) et nous avons constaté une induction d'expression de GSTP1 dans le tissu péritumoral des CHC par rapport au foie normal. / Increased GSTP1 expression is frequently observed in cancers and is positively correlated with chemotherapy resistance. This phase II detoxifying enzyme can also regulate JNK and TRAF2 activities and, consequently, can modulate proliferation and cell death pathways. This project aimed at studying the role of GSTP1 during proliferation in normal and transformed hepatocytes. Liver regeneration study in Gstp1/2‐/‐ mice showed the involvement of GSTP1 and GSTP2 proteins in the cell cycle progression control of normal hepatocytes. After partial hepatectomy in Gstp1/2‐/‐ mice, the number of cells in S, G2 and M phases was decreased compared to livers of wildtype mice. This reduction is associated with the delay in the expression of proteins involved in proliferation initiation, mitogen restriction point control and G1/S transition. These modifications are associated with the decrease in TRAF2 expression and the activation of JNK and ERK, whereas p21 and p53 levels are high. Furthermore, expression of enzymes involved in redox homeostasis and MAPK activation is delayed. Study of cells derived from various cancers, including HCC, highlighted a correlation between low expression of GSTP1 and decrease in cell proliferation without cell survival alteration. However in these conditions, we observed the increase in TRAF2, pJNK, pATF2 and ATF3 expression together with the induction of p21. We also showed that GSTP1 effects are regulated by JNK activation. These results showed a link between GSTP1 expression and hepatocyte proliferation and led us to investigate the GSTP1 expression in HCC. We noticed an induction of GSTP1 expression in peritumoral tissue compared to normal liver.

IL-12, an immunoregulatory cytokine, plays a key role in the development of cell-mediated immune responses. However, very little is known about the regulation and induction of the other members of this family, particularly IL-23 and IL-27. The regulation of these cytokines was studied in the human primary monocytes and monocyte-derived macrophages (MDMs) as they play a key role in innate and adaptive immune responses. THP-1 promonocytic cells were employed as a model system to confirm the results obtained with monocytes and MDMs. Two stimuli IFN-γ and LPS were used as both are strong inducers of IL-12 family cytokines. My results show that IFN-γ induced the production of IL-12/23p40 and IL-23p19 mRNA as well as IL-12p40 and IL-23 proteins in primary human monocytes isolated by positive selection. IFN-γ-induced IL-23 and IL-12/23p40 expression was positively regulated by the p38 mitogen-activated protein kinases (MAPK), independent of the Janus kinase (Jak)/signal transducers and activators of transcription (STAT) signaling. In contrast, IL-12 and IL-23 were negatively regulated by the Jak/STAT, phosphoinositide-3 kinase (PI3K) and the c-Jun-N-terminal kinase (JNK) MAPKs in IFN-γ-stimulated monocytes. LPS significantly stimulated IL-23p19 and IL-12/23p40 mRNA expression as well as IL-12/23p40 and IL-23 protein production in THP-1 cells, while IFN-γ stimulation alone did not affect IL-23 mRNA or protein levels. THP-1 cells were pre-treated with ERK, JNK or p38 MAPK inhibitors and then stimulated with LPS. LPS-induced IL-12p40 and IL-23 proteins were positively regulated by the p38 and JNK MAPKs and PI3K, whereas LPS-induced IL-23p19 mRNA expression was negatively regulated by these kinases. These results were confirmed using siRNA in LPS-stimulated THP-1 cells. My results also show that IFN-γ/LPS-induced IL-23 expression is not regulated through MAPK or PI3K signaling pathways in human MDMs. My results also show for the first time that IFN-γ alone without any second stimulus induced IL-27p28 gene expression and IL-27 protein production in human monocytic cells. I investigated the signalling pathways governing the regulation of IL-27 protein and its subunit IL-27p28 following stimulation with IFN-γ in primary human monocytic cells. IFN-γ-mediated IL-27 protein, but not IL-27p28 gene expression was positively regulated by JNK MAPK and PI3K, independent of JAK/STAT signaling in primary human monocytes. I also investigated the signalling pathways governing the regulation of IL-27 and its α subunit, IL-27p28 following stimulation with IFN-γ alone or IFN-γ-primed LPS-stimulated macrophages (IFN-γ/LPS) and THP-1 cells. A differential regulation of IL-27p28 and IL-27 in response to stimulation by either IFN-γ or IFN-γ/LPS was observed. IFN-γ- and IFN-γ/LPS induced IL-27 expression was positively regulated by the JNK, p38 MAPK and PI3K, independent of Jak/STAT signaling in human MDMs and THP-1 cells. Taken together, my results show that IL-23 induction is differentially regulated by different pathways in response to different stimuli, whereas IL-27 expression is regulated by JNK, p38 MAPK and PI3K regardless in the stimulus in human myeloid cells. These results may provide additional strategies aimed at targeting disease, autoimmune disorders and cancer.

This work sought to investigate the clinical phenotypes and molecular basis of cardio-facio-cutaneous syndrome (CFC), a germline disorder of the Ras-MAPK pathway, like Noonan syndrome (NS) and neurofibromatosis type I, caused by mutations in genes encoding proteins that act within this signal transduction pathway. CFC is most commonly due to mutation in BRAF, and less commonly MAP2K1, MAP2K2 or KRAS. A proportion of patients currently have no mutation identified. Mutations and clinical features of patients with a molecular diagnosis of CFC were investigated, which demonstrated a wide range of causative mutations, and some unclassified variants. Both known and novel clinical features of CFC were identified. A strong association between severe contractures and the p.(Tyr130Cys) mutation in MAP2K1 was found, which has not previously been reported. In contrast to the large number of patients with a confirmed molecular diagnosis, several with a highly suggestive clinical phenotype have been found to have no mutationin any of the known CFC genes. The molecular basis of these presentations was investigated by conventional Sanger sequencing of candidate genes. Fourteen patients with the p.(Ser2Gly) mutation in SHOC2 were identified, with clinical presentations consistent with CFC, NS or CS. Target enrichment and massively parallel sequencing of selected genes was undertaken in ten patients. Mutations in known genes were identified in four patients (including the positive control). Candidate causative variants in novel genes were suggested in two further patients, one of which was confirmed on Sanger sequencing. Whole exome sequencing of patient-parent trios was also undertaken to identify de novo variants. Three trios were analysed, and in one patient with a clinical diagnosis of CFC, a frameshift mutation in NF1 was identified, which was confirmed by Sanger sequencing to be present and de novo. The molecular effects of CFC-associated mutations in BRAF on Ras-MAPK pathway signalling were studied in cell culture systems, using Western blotting for ERK1/2 phosphorylation, in vitro kinase assays and luciferase assays, to assess activity of downstream targets of the Ras-MAPK pathway. Altered pathway activity was demonstrated for novel variants that had not previously been characterised at the molecular level, which was in keeping with the findings of the effects of previously studied mutations. The cardiac phenotype in animal models of CFC, CS and NS/CFC was explored using expression microarrays to identify potentially important genes and pathways in the pathogenesis of hypertrophic cardiomyopathy (a progressive but potentially treatable disease feature) in these conditions. A signature of increased expression of Myh7, the embryonic form of myosin, was identified in the heart of the mouse model of CFC due to a B-Raf mutation at four weeks postnatal age, but comparative analysis suggested significant differences in either the mechanisms causing cardiac phenotypes, or the timescales over which these may exert their effects, in the three models. In summary, the most significant findings of this work were that SHOC2 mutation is a frequent cause of a severe NCFC presentation, and massively parallel sequencing can be an effective means of molecular investigation of this group of disorders. Novel features of CFC syndrome that were identified include severe contractures in association with p.(Tyr130Cys) mutations in MAP2K1. The analysis of mouse models of the NCFCs was hampered by heterogeneity within the expression microarray results, and low levels of expression of the H-Ras mutant allele in the mouse model of Costellosyndrome.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22283号 / 理博第4597号 / 新制||理||1659(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 佐藤 ゆたか, 教授 高橋 淑子, 准教授 秋山 秋梅 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

L’étude d’échantillons humains montre que les voies de signalisation RAS/MAPK et PI3K/AKT/mTOR sont fréquemment activées de manière aberrante dans les tumeurs de la prostate, d’autant plus dans les phases de résistance aux traitements. Ces deux voies de signalisation sont sensibles aux facteurs de croissances et impliquées dans la régulation de processus cellulaires fondamentaux tels que la prolifération, la croissance ou encore la différenciation cellulaire. Ces données suggèrent qu’elles ont un rôle essentiel dans la tumorigenèse prostatique. Cependant, le rôle respectif de chacune de ces voies dans la carcinogenèse prostatique, particulièrement dans les phases précoces, n’est pas clairement établit. L’objectif de ma thèse est donc de définir le rôle possible de ces deux voies dans l’initiation et la progression du cancer de la prostate, ainsi que les mécanismes impliqués dans leur co-dérégulation. Cette étude est réalisée dans un modèle in vivo alternatif, la drosophile, qui possèdent un équivalent fonctionnel de la prostate : les glandes accessoires. La première partie des travaux réalisés montre que seule la suractivation de la voie RAS/MAPK dans la glande accessoire conduit à un processus de tumorigenèse, avec la production de masses cellulaires récapitulant de nombreuses caractéristiques cancéreuses : croissance cellulaire et prolifération incontrôlée, expression de métalloprotéases, perte de l’expression de marqueurs épithéliaux et formation de nouvelles trachées. Cependant, les deux voies de signalisation sont nécessaires à la tumorigenèse, mais avec des rôles différents : la voie RAS/MAPK est activée précocement et est capable de recruter la voie PI3K/AKT/TOR grâce à la mise en place de deux boucles autocrines de régulation. La première dépend de spitz (dEGF) et du récepteur EGFR pour amplifier l’activation de la voie RAS/MAPK. La seconde dépend de l’activation d’ILP6 (dIGF1), produit suite à l’activation de la voie RAS/MAPK, et permet le recrutement de la voie PI3K/AKT/TOR par l’intermédiaire du récepteur à l’insuline InR. La deuxième partie des travaux réalisés montre que l’activation de la voie RAS/MAPK conduit à la production de MMP1 dans les cellules qui seront à l’origine des tumeurs avant leur extravasation hors de l’épithélium. Cette expression temporelle contrôlée correspond à une étape où une réorganisation du cytosquelette a lieu et où le microenvironnement est altéré. Ces données placent donc la dérégulation de la voie RAS/MAPK comme un évènement précoce de la tumorigenèse prostatique, capable de recruter la voie PI3K/AKT/TOR et d’entrainer la production de MMP1, pour in fine conduire à l’extravasation des cellules et à la formation de tumeurs. / Clinical studies have demonstrated that, in prostate cancer, RAS/MAPK and PI3K/AKT/TOR signaling pathways are often aberrantly co-activated in tumors, their activation levels increasing again in resistance phases. These pathways, that are regulated by growth factors, are implicated in fundamental cellular processes regulation such as proliferation, growth and cellular differentiation. These data suggest that they are likely implicated in prostate tumorigenesis. However, the relative implication of each of these two pathways during prostate tumorigenesis, especially during early phases, is not fully understood. Thus, the aim of my thesis is to define the possible implication of these pathways in prostate cancer initiation and progression and which molecular mechanisms are implicated in their co-deregulation. Therefore, we have developed an alternative in vivo model of prostate tumorigenesis in drosophila, where accessory glands are a functional equivalent of the human prostate. The first part of my work shows that only the hyperactivation of the RAS/MAPK pathway in accessory glands can promote tumorigenesis, with the formation of cell masses that recapitulate many cancer hallmarks including uncontrolled cell growth and proliferation, enhanced matrix metalloproteinases expression, loss of epithelial markers expression, neovascularization-like tracheogenesis. However, both pathways are necessary to tumorigenesis, even though they display different roles: the RAS/MAPK pathway is activated earlier and is able to recruit the PI3K/AKT/TOR pathway thanks to the formation of two feedback loops. The first depend on Spitz (dEGF) and EGFR receptor to amplify RAS/MAPK pathway activation. The second depends on ILP6 (dIGF1) activation, produced following RAS/MAPK pathway activation and allow PI3K/AKT/TOR pathway recruitment via insulin receptor InR. The second part of the work shows that RAS/MAPK pathway activation allows MMP1 production restricted to the cells that will be the origin of the tumors, before their actual extravasation. This temporally controlled step of MMP1 expression corresponds to a time window where the cells show strong cytoskeletal reorganization and where microenvironment is disturbed. These data place the RAS/MAPK pathway deregulation as an early event of prostate tumorigenesis, able to recruit the PI3K/AKT/TOR pathway and to induce MMP1 production to allow cell extravasation and tumor formation.

Fam84b is a largely unstudied protein, where its function in eukaryotic cells is unclear. This thesis work presents a FAM84B knockout mouse model and characterises the resulting retinal phenotype in detail. FAM84B KO mice were morphologically assessed by optical coherence tomography and histological processing, revealing dynamic changes stemming from the photoreceptor and pigmented epithelial layers. This potent phenotype progresses with age, spreading inwards towards the inner retinal layers, as well as laterally to adjacent retinal regions. Comparative localisation of standard retinal cell markers demonstrates that FAM84B KO retinal layering becomes increasingly disorganised, together with deformation of the retinal macrostructure. Due to this, KO mice experience reducing responses to light, as demonstrated by electroretinography, where overall retinal efficiency falls. Fam84b shows homology to the HRASLS enzyme family, which are capable of attenuating Ras-associated signalling. To investigate whether Fam84b possesses a similar function, the level of phosphorylated and activated downstream Ras effectors were compared between wild type and FAM84B KO mouse retinal lysates. A reduction of pERK1 (pY204) in KO lysates suggests that Fam84b holds some function related to this pathway downstream of Ras....

La voie de signalisation Ras/MAPK (Ras/mitogen-activated protein kinase) régule une variété de protéines intracellulaires qui jouent un rôle important dans la croissance et la prolifération cellulaire. La régulation inappropriée de cette voie de signalisation conduit au développement de nombreux cancers comme le mélanome, qui est caractérisé par des mutations activatrices au niveau des gènes NRAS et BRAF. La protéine kinase RSK (p90 ribosomal S6 kinase) est un composant central de la voie Ras/MAPK, mais son rôle dans la croissance et la prolifération cellulaire n’est pas bien compris. RSK a été montrée pour participer à la résistance des mélanomes aux chimiothérapies, mais le mécanisme moléculaire reste encore à élucider. Nous montrons à l’aide d’un anticorps phospho-spécifique que MDM2 est phosphorylée en réponse à des agonistes et des mutations oncogéniques activant spécifiquement la voie Ras/MAPK. En utilisant des méthodes in vitro et in vivo, nous avons constaté que RSK phosphoryle directement MDM2 sur les Sérines 166 et 186, ce qui suggère que MDM2 est un substrat de RSK. La mutagénèse dirigée envers ces sites nous indique que ces résidus régulent l’ubiquitination de MDM2, suggérant que RSK régule la stabilité de MDM2 et de p53. De plus, nous avons observé que l’inhibition de RSK conduit à une augmentation du niveau protéique de p53 après un dommage à l’ADN dans les cellules de mélanomes. En conclusion, nos travaux suggèrent un rôle important de la protéine kinase RSK dans la régulation de MDM2 et de sa cible, p53. L’étude de ces mécanismes moléculaires aidera à mieux définir le rôle de RSK dans la croissance tumorale, mais également dans la résistance aux agents chimiothérapeutiques. / The Ras/mitogen-activated protein kinase (Ras/MAPK) signaling cascade regulates various intracellular targets involved in growth and proliferation. Inappropriate regulation of this pathway leads to many types of cancer, including melanomas, which are characterized by activating mutations in NRAS and BRAF. The protein kinase RSK (p90 ribosomal S6 kinase) is a central component of the Ras/MAPK pathway, but its role in cell growth and proliferation is not well understood. RSK has also been shown to participate in the resistance of melanoma cells to chemotherapy, but the mechanisms involved remain elusive. We show that MDM2 becomes phosphorylated in response to agonists and oncogenes of the Ras/MAPK pathway. Using in vitro and in vivo approaches, we found that RSK directly phosphorylates MDM2 at Ser166 and Ser186, suggesting that MDM2 is a bona fide RSK substrate. Site-directed mutagenesis indicated that these residues regulate MDM2 ubiquitination, suggesting that RSK regulates p53 function in an MDM2-dependent manner. Overexpression of active and inactive mutants of RSK revealed that this kinase regulates p53 stability, suggesting a role for RSK in the DNA damage response. Taken together, our results suggest an important role for RSK in the regulation of MDM2 and its target p53. In view of the role of p53 in the response to DNA-damaging agents, our results provide a potential mechanism involved in melanoma chemoresistance.

Amiodarone is a potent antidysrhythmic agent that can cause potentially life-threatening pulmonary fibrosis. Accumulating evidence has demonstrated that myofibroblast differentiation is related to the pathogenesis of pulmonary fibrosis. In the present study, we treated human embryonic lung fibroblasts (HELFs) with amiodarone, and investigated the relative molecular mechanism of amiodarone-induced pulmonary fibrosis and pathway determinants PD98059 (extracellular signal-regulated kinase (ERK) inhibitor) and SB203580 (p38 mitogen-activated protein kinase (MAPK) inhibitor). Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8). The secretion of collagen Ⅰ was detected by ELISA. The expressions of α-smooth muscle actin (α-SMA), vimentin, phosphorylated ERK1/2 (p-ERK1/2), ERK1/2, phosphorylated p38 MAPK (p-p38), and p38 MAPK were investigated using Western blot analysis. The levels of α-SMA and vimentin were also determined by immunofluorescence and qRT-PCR. We report that amiodarone promoted cell proliferation and collagen Ⅰ secretion, induced α-SMA and vimentin protein and mRNA expression accompanied by increased phosphorylation of ERK1/2 and p38 MAPK, and furthermore, PD98059 and SB203580 remarkably reduced the proliferative response of HELFs compared with amiodarone group and greatly attenuated α-SMA, vimentin and collagen Ⅰ protein production induced by amiodarone. Taken together, our study suggests that amiodarone regulates cell proliferation and myofibroblast differentiation in HELFs through modulating ERK1/2 and p38 MAPK pathways, and these signal pathways may therefore represent an attractive treatment modality in amiodarone-induced pulmonary fibrosis.

We examined the role of Tollip in the hypertrophic response of cardiomyocytes. C57BL/6 mice were subjected to transverse aortic constriction (TAC) for 2 weeks and age-matched sham surgical operated mice served as control. TAC significantly reduced the association of Tollip with IRAK-1 by 66.4 percent and increased NF-kappaB binding activity by 86.5 percent and the levels of phosphop38 by 114.6 percent in the myocardium compared with sham control, respectively. In vitro experiments showed that IL-1beta stimulation also significantly reduced the association of Tollip with IRAK-1 and increased NFkappaB binding activity in neonatal cardiomyocytes. Tollip overexpression by transfection of cardiac myocytes significantly attenuated the IL-1beta-induced hypertrophic response of cardiac myocytes as evidenced by reduced cell size (16.4 percent) and decreased ANP expression (33.3 percent). Overexpression of Tollip also reduced NFkappaB binding activity by 30.7 percent and phospho-p38 by 47.1 percent, respectively. The results suggest that Tollip could be a negative regulator during the development of cardiac hypertrophy. The negative regulation of cardiac hypertrophy by Tollip may involve downregulation of the MyD88-dependent NF-kappaB activation pathway.

Breast cancer is the second most common malignancy in the world, accounting for over 1.7 million new diagnoses and an estimated 500,000 deaths per year (1). Overexpression of the receptor tyrosine kinase ErbB2, also known as Her2 or Neu, occurs in over 30% of breast cancers and correlates with metastasis, poor prognosis, and decreased survival (1, 2). Although therapeutics targeting ErbB2 show clinical efficacy, many patients display no initial response or develop drug resistance over time (2). A deeper understanding of the molecular basis of ErbB2-driven tumorigenesis is thus required for the development of improved therapeutic strategies. In vitro experiments suggest that activation of the c-Jun NH2-terminal kinase (JNK) pathway, a mitogen-activated protein kinase pathway, promotes proliferation, cellular invasion, and stem cell expansion in ErbB2-driven breast cancer (3, 4). Furthermore, unpublished data from our lab using mammary epithelial cells expressing activated ErbB2 show that JNK is required for acinus formation in in vitro 3D cultures. In contrast to these studies showing a tumorigenic role for the JNK pathway, other data from our lab show that JNK loss results in accelerated breast tumor growth, suggesting a tumor suppressive role (5, 6). However, these studies were performed in p53 knockout mice with or without a Kras mutation, where the latter required extensive aging and genomic instability to occur before differences in tumor growth were observable. To date, limited in vivo studies exist to confirm the role of JNK in more biologically relevant breast tumor models, such as in ErbB2-mediated cancer, which accounts for over 30% of all human breast cancers. In addition, the molecular mechanisms by which JNK signaling promotes ErbB2-driven tumorigenesis remains poorly understood. To address the discrepancy in JNK function between the in vitro ErbB2-driven breast cancer data and the in vivo p53 knockout tumor data, I began the development of an in vivo murine model to confirm the role of JNK in ErbB2-driven breast cancer. This mouse model will also allow us to test a potential mechanism by which JNK regulates tumorigenesis. Studies show that ErbB2-mediated secretion of the inflammatory cytokine IL6 promotes transformation and tumor growth by activation of the STAT3 transcription factor, triggering an IL6/STAT3 autocrine signaling loop (7,8). A major regulator of Il6 gene expression includes activator protein 1 (AP-1), a transcription factor composed of downstream JNK targets in the Jun protein family (9). In vitro experiments using ErbB2-overexpressing mammary epithelial cell lines show that chemical inhibition of JNK suppresses secreted IL6 protein levels, supporting a role for the JNK pathway in IL6 regulation (7). Thus, I hypothesize that JNK drives ErbB2-driven breast cancer by promoting IL6-mediated tumor progression. Addressing this will increase our understanding of the role of JNK in ErbB2-driven breast cancer and reveal a potentially new mechanism by which JNK functions in tumor progression. Additionally, I began the development of a mouse model that will allow us to investigate the role of JNK in macrophage polarization as an alternative mechanism by which JNK regulates ErbB2-driven breast cancer. In addition to promoting STAT3-dependent tumor growth, IL6 can indirectly drive tumorigenesis by promoting expression of the IL4 receptor in macrophages, triggering STAT6-mediated macrophage polarization towards the pro-tumorigenic M2 phenotype (10, 11). Unlike classically activated M1 macrophages, which promote inflammation and anti-tumor immunity, alternatively activated M2 macrophages function in immunosuppression and metastasis and correlate with advanced stages of breast cancer (12, 13). Further evidence supporting a role for the JNK pathway in macrophage polarization includes a recent study suggesting that JunB, a downstream JNK target and component of the AP-1 complex, plays a crucial role in the induction of M2 macrophage polarization in human alveolar macrophages (13). I hypothesize that activation of the JNK signaling pathway induces IL6-dependent macrophage polarization towards the pro-tumorigenic M2 phenotype. Addressing this hypothesis will determine for the first time whether JNK functions in regulating macrophage polarization within the tumor microenvironment, offering a potentially new mechanism by which JNK can promote ErbB2-driven breast cancer. Determining the role of JNK in ErbB2-mediated breast cancer will have direct therapeutic relevance, as targeting JNK has the potential to inhibit ErbB2-driven breast cancer and other IL6-mediated diseases. Investigating the underlying mechanisms by which JNK functions in ErbB2-positive breast cancer can also offer new molecular targets and further contribute to effective drug design.