MRCKα Is Dispensable for Breast Cancer Development in the MMTV-PyMT Model.

MRCKα is a ubiquitously expressed serine/threonine kinase involved in cell contraction and F-actin turnover, which is highly amplified in human breast cancer and part of a gene expression signature for bad prognosis. Nothing is known about the in vivo function of MRCKα. To explore MRCKα function in development and in breast cancer, we generated mice lacking a functional MRCKα gene. Mice were born close to the Mendelian ratio and showed no obvious phenotype including a normal mammary gland formation. Assessing breast cancer development using the transgenic MMTV-PyMT mouse model, loss of MRCKα did not affect tumor onset, tumor growth and metastasis formation. Deleting MRCKα and its related family member MRCKß in two triple-negative breast cancer cell lines resulted in reduced invasion of MDA-MB-231 cells, but did not affect migration of 4T1 cells. Further genomic analysis of human breast cancers revealed that MRCKα is frequently co-amplified with the oncogenes ARID4B and AKT3 which might contribute to the prognostic value of MRCKα expression. Collectively, these data suggest that MRCKα might be a prognostic marker for breast cancer, but probably of limited functional importance.

ACK1 is dispensable for development, skin tumor formation, and breast cancer cell proliferation.

Activated Cdc42-associated kinase 1 (ACK1), a widely expressed nonreceptor tyrosine kinase, is often amplified in cancer and has been shown to interact with Cell division cycle 42 (Cdc42), Epidermal growth factor receptor (EGFR), and several other cancer-relevant molecules, suggesting a possible role for ACK1 in development and tumor formation. To directly address this scenario, we generated mice lacking a functional ACK1 gene (ACK1 ko) using CRISPR genome editing. ACK1 ko mice developed normally, displayed no obvious defect in tissue maintenance, and were fertile. Primary ACK1-null keratinocytes showed normal phosphorylation of EGFR, but a tendency toward reduced activation of AKT serine/threonine kinase 1 (Akt) and Mitogen-activated protein kinase 1 (Erk). DMBA/TPA-induced skin tumor formation did not reveal significant differences between ACK1 ko and control mice. Deletion of the ACK1 gene in the breast cancer cell lines MDA-MB-231, 67NR, MCF7, 4T1, and T47D caused no differences in growth. Furthermore, EGF-induced phosphorylation kinetics of Erk, Akt, and p130Cas were not detectably altered in T47D cells by the loss of ACK1. Finally, loss of ACK1 in MDA-MB-231 and T47D breast cancer cells had a very limited or no effect on directed cell migration. These data do not support a major role for ACK1 in Cdc42 and EGFR signaling, development, or tumor formation.

Cancer-associated fibroblasts: how do they contribute to metastasis?

Cancer-associated fibroblasts (CAFs) are activated fibroblasts in the tumor microenvironment. They are one of the most prominent cell types in the stroma and produce large amounts of extracellular matrix molecules, chemokines, cytokines and growth factors. Importantly, CAFs promote cancer progression and metastasis by multiple pathways. This, together with their genetic stability, makes them an interesting target for cancer therapy. However, CAF heterogeneity and limited knowledge about the function of the different CAF subpopulations in vivo, are currently major obstacles for identifying specific molecular targets that are of value for cancer treatment. In this review, we discuss recent major findings on CAF development and their metastasis-promoting functions, as well as open questions to be addressed in order to establish successful cancer therapies targeting CAFs.

RhoA, Rac1, and Cdc42 differentially regulate αSMA and collagen I expression in mesenchymal stem cells.

Mesenchymal stem cells (MSC) are suggested to be important progenitors of myofibroblasts in fibrosis. To understand the role of Rho GTPase signaling in TGFß-induced myofibroblast differentiation of MSC, we generated a novel MSC line and its descendants lacking functional Rho GTPases and Rho GTPase signaling components. Unexpectedly, our data revealed that Rho GTPase signaling is required for TGFß-induced expression of α-smooth muscle actin (αSMA) but not of collagen I α1 (col1a1). Whereas loss of RhoA and Cdc42 reduced αSMA expression, ablation of the Rac1 gene had the opposite effect. Although actin polymerization and MRTFa were crucial for TGFß-induced αSMA expression, neither Arp2/3-dependent actin polymerization nor cofilin-dependent severing and depolymerization of F-actin were required. Instead, F-actin levels were dependent on cell contraction, and TGFß-induced actin polymerization correlated with increased cell contraction mediated by RhoA and Cdc42. Finally, we observed impaired collagen I secretion in MSC lacking RhoA or Cdc42. These data give novel molecular insights into the role of Rho GTPases in TGFß signaling and have implications for our understanding of MSC function in fibrosis.

A novel regulatory relationship between RIPK4 and ELF3 in keratinocytes.

Keratinocytes are central to the barrier functions of surface epithelia, such as the gingiva and epidermis. RIPK4 is a key regulator of keratinocyte differentiation; however, the signalling pathways in which it functions remain poorly defined. In this study, we identified a regulatory relationship between RIPK4 and ELF3, an ETS family transcription factor. RIPK4 was shown to be important for the upregulation of ELF3 gene expression by the PKC agonist PMA in both oral and epidermal keratinocytes. RIPK4 promotes keratinocyte differentiation in part by phosphorylating and thereby activating the IRF6 transcription factor. Significantly, silencing of IRF6 inhibited the PMA-inducible expression of ELF3. A role for the GRHL3 transcription factor, a downstream target gene of IRF6, in the regulation of ELF3 expression was similarly demonstrated. ELF3 has previously been shown to regulate the expression of SPPR1A and SPRR1B, small proline-rich proteins that contribute to the cornification of keratinocytes. Consistently, RIPK4 and IRF6 were important for the PMA-inducible expression of SPRR1A and SPRR1B. They were also important for the upregulation of TGM1, a transglutaminase that catalyses the cross-linking of proteins, including small proline-rich proteins, during keratinocyte cornification. RIPK4 was also shown to upregulate the expression of TGM2 independently of IRF6. Collectively, our findings position RIPK4 upstream of a hierarchal IRF6-GRHL3-ELF3 transcription factor pathway in keratinocytes, as well as provide insight into a potential role for RIPK4 in the regulation of keratinocyte cornification.

RIPK4 activates an IRF6-mediated proinflammatory cytokine response in keratinocytes.

Keratinocytes of the oral mucosa and epidermis play key roles in host defense. In addition to functioning as a physical barrier, they also produce cytokines to elicit inflammation in response to infection or injury. We recently established that receptor-interacting protein kinase 4 (RIPK4) and interferon regulatory factor 6 (IRF6) function as a cell-intrinsic signaling axis to regulate keratinocyte differentiation. In this study, we have demonstrated a functional relationship between RIPK4 and IRF6 in the control of proinflammatory cytokine expression in keratinocytes. The overexpression of RIPK4 by oral keratinocytes induced the strong expression of CCL5 and CXCL11. In contrast, the expression of other cytokines (e.g. IL8 and TNF) was largely unaffected, thus demonstrating specificity in the induction of proinflammatory cytokine expression by RIPK4. CCL5 and CXCL11 expression were also induced in response to the activation of the PKC pathway, and gene silencing experiments indicated that their inducible expression was dependent on RIPK4 and IRF6. Moreover, gene reporter assays suggested that RIPK4 induces CCL5 and CXCL11 expression by stimulating the transactivation of their promoters by IRF6. Accordingly, our findings suggest that the RIPK4-IRF6 signaling axis plays a multifaceted role in barrier epithelial homeostasis through its regulation of both keratinocyte inflammation and differentiation.

IRF6 Regulates the Expression of IL-36γ by Human Oral Epithelial Cells in Response to Porphyromonas gingivalis.

IFN regulatory factors (IRFs) help to shape the immune response to pathogens by imparting signaling specificity to individual TLRs. We recently demonstrated that IRF6 provides specificity to TLR2 signaling in oral epithelial cells. TLR2 plays an important role in eliciting inflammation to Porphyromonas gingivalis, a keystone pathogen in periodontitis. Therefore, we investigated a role for IRF6 in mediating the inflammatory cytokine response of oral epithelial cells to P. gingivalis. IRF6 expression was strongly upregulated when human oral epithelial cells were challenged with P. gingivalis. Moreover, gene silencing and gene promoter experiments indicated that IRF6 acts downstream of IL-1R-associated kinase 1 to stimulate the expression of the IL-1 family cytokine IL-36γ in response to P. gingivalis. IRF6 and IL-1R-associated kinase 1 also regulated the stimulation of IL-36γ expression by a TLR2 agonist. IL-36γ was shown to elicit inflammatory responses by human monocyte-derived dendritic cells and macrophages, including the expression of the neutrophil chemokines IL-8 and CXCL1, as well as the Th17 chemokine CCL20. IL-36γ similarly stimulated their expression by human oral epithelial cells. Significantly, the Th17 cytokine IL-17 not only stimulated the expression of important regulators of neutrophil recruitment and survival by oral epithelial cells, but IL-17 also stimulated them to express IL-36γ. Thus, our findings suggest that IRF6 is likely to promote inflammation to P. gingivalis through its regulation of IL-36γ.

Disease-associated mutations in IRF6 and RIPK4 dysregulate their signalling functions.

IRF6 and RIPK4 are critical regulators of keratinocyte differentiation and their mutation cause the developmental syndromes Van der Woude syndrome (VWS) and Bartsocas-Papas syndrome (BPS), respectively. RIPK4 promotes keratinocyte differentiation, in part, by inducing IRF6 transactivator function through the phosphorylation of its C-terminal domain at Ser413 and Ser424. Although more than 200 IRF6 mutations have been identified in VWS, a p.Arg412X nonsense mutation is particularly prevalent. A RIPK4 p.Ser376X nonsense mutation in BPS was also recently identified. Here, we demonstrated for the first time that the truncation of IRF6 at Arg412 causes its rapid proteasome-dependent degradation. The truncation of IRF6 also prevented the induction of its transactivator function by RIPK4. Similarly, the p.Ser376X mutation in RIPK4 impaired its induction of IRF6 transactivator function. The mutation also inhibited the stabilisation of ß-catenin by RIPK4, and thus may additionally impair Wnt signalling. Collectively, our findings provide important mechanistic insight into how the p.Arg412X and p.Ser376X mutations may cause VWS and BPS, respectively.

Receptor-interacting protein kinase 4 and interferon regulatory factor 6 function as a signaling axis to regulate keratinocyte differentiation.

Receptor-interacting protein kinase 4 (RIPK4) and interferon regulatory factor 6 (IRF6) are critical regulators of keratinocyte differentiation, and their mutation causes the related developmental epidermal disorders Bartsocas-Papas syndrome and popliteal pterygium syndrome, respectively. However, the signaling pathways in which RIPK4 and IRF6 operate to regulate keratinocyte differentiation are poorly defined. Here we identify and mechanistically define a direct functional relationship between RIPK4 and IRF6. Gene promoter reporter and in vitro kinase assays, coimmunoprecipitation experiments, and confocal microscopy demonstrated that RIPK4 directly regulates IRF6 trans-activator activity and nuclear translocation. Gene knockdown and overexpression studies indicated that the RIPK4-IRF6 signaling axis controls the expression of key transcriptional regulators of keratinocyte differentiation, including Grainyhead-like 3 and OVO-like 1. Additionally, we demonstrate that the p.Ile121Asn missense mutation in RIPK4, which has been identified recently in Bartsocas-Papas syndrome, inhibits its kinase activity, thereby preventing RIPK4-mediated IRF6 activation and nuclear translocation. We show, through mutagenesis-based experiments, that Ser-413 and Ser-424 in IRF6 are important for its activation by RIPK4. RIPK4 is also important for the regulation of IRF6 expression by the protein kinase C pathway. Therefore, our findings not only provide important mechanistic insights into the regulation of keratinocyte differentiation by RIPK4 and IRF6, but they also suggest one mechanism by which mutations in RIPK4 may cause epidermal disorders (e.g. Bartsocas-Papas syndrome), namely by the impaired activation of IRF6 by RIPK4.

Interferon regulatory factor 6 differentially regulates Toll-like receptor 2-dependent chemokine gene expression in epithelial cells.

Epidermal and mucosal epithelial cells are integral to host defense. They not only act as a physical barrier but also utilize pattern recognition receptors, such as the Toll-like receptors (TLRs), to detect and respond to pathogens. Members of the interferon regulatory factor (IRF) family of transcription factors are key components of TLR signaling as they impart specificity to downstream responses. Although IRF6 is a critical regulator of epithelial cell proliferation and differentiation, its role in TLR signaling has not previously been addressed. We show here that IRF6 is activated by IRAK1 as well as by MyD88 but not by TRIF or TBK1. Co-immunoprecipitation experiments further demonstrated that IRF6 can interact with IRAK1. Gene silencing in epithelial cells along with gene promoter reporter assays showed that IRAK1 mediates TLR2-inducible CCL5 gene expression at least in part by promoting IRF6 activation. Conversely, IRAK1 regulated CXCL8 gene expression independently of IRF6, thus identifying a molecular mechanism by which TLR2 signaling differentially regulates the expression of specific chemokines in epithelial cells. Bioinformatics analysis and mutagenesis-based experiments identified Ser-413 and Ser-424 as key regulatory sites in IRF6. Phosphomimetic mutation of these residues resulted in greatly enhanced IRF6 dimerization and trans-activator function. Collectively, our findings suggest that, in addition to its importance for epithelial barrier function, IRF6 also contributes to host defense by providing specificity to the regulation of inflammatory chemokine expression by TLR2 in epithelial cells.

CSF-1 receptor signalling from endosomes mediates the sustained activation of Erk1/2 and Akt in macrophages.

Colony stimulating factor-1 (CSF-1) mediates its pleiotropic effects on macrophages through the CSF-1 receptor (CSF-1R), a receptor tyrosine kinase. Current models of CSF-1 signalling imply that the CSF-1R activates signalling pathways exclusively at the plasma membrane and the subsequent internalisation of the CSF-1R simply facilitates its lysosomal degradation in order to prevent on-going signalling. Here, we sought to establish if the CSF-1R may in fact continue to signal following its internalisation. Erk1/2, Akt and Stat3 activation were abrogated when the internalisation of the CSF-1R was impaired, with the effects on Stat3 distinct from those for Erk1/2 and Akt. Pharmacologic inhibition of the CSF-1R following its internalisation resulted in less sustained Erk1/2 and Akt activity, whereas Stat3 activity was unaffected. Significantly, the suppressive effects of the CSF-1R inhibitor on the up-regulation of gene expression by CSF-1 (e.g. cyclin D1 and Bcl-xL gene expression) were comparable irrespective of whether the inhibitor was added prior to CSF-1 stimulation or following the internalisation of the CSF-1R. Similarly, pharmacologic inhibition of Erk1/2 (or Akt) activity either prior to CSF-1 stimulation or subsequent to CSF-1R internalisation had comparable effects on the regulation of gene expression by CSF-1. Together, our data argue that key signalling responses to CSF-1 depend on the ability of the CSF-1R to signal from endosomes following its internalisation, thus adding an important spatiotemporal aspect to CSF-1R signalling.