Tumor factors stimulate lysosomal degradation of tumor antigens and undermine their cross-presentation in lung cancer.

Activities of dendritic cells (DCs) that present tumor antigens are often suppressed in tumors. Here we report that this suppression is induced by tumor microenvironment-derived factors, which activate the activating transcription factor-3 (ATF3) transcription factor and downregulate cholesterol 25-hydroxylase (CH25H). Loss of CH25H in antigen presenting cells isolated from human lung tumors is associated with tumor growth and lung cancer progression. Accordingly, mice lacking CH25H in DCs exhibit an accelerated tumor growth, decreased infiltration and impaired activation of intratumoral CD8+ T cells. These mice do not establish measurable long-term immunity against malignant cells that undergo chemotherapy-induced immunogenic cell death. Mechanistically, downregulation of CH25H stimulates membrane fusion between endo-phagosomes and lysosomes, accelerates lysosomal degradation and restricts cross-presentation of tumor antigens in the intratumoral DCs. Administration of STING agonist MSA-2 reduces the lysosomal activity in DCs, restores antigen cross presentation, and increases therapeutic efficacy of PD-1 blockade against tumour challenge in a CH25H-dependent manner. These studies highlight the importance of downregulation of CH25H in DCs for tumor immune evasion and resistance to therapy.

Chemotherapy-Induced Changes in the Lung Microenvironment: The Role of MMP-2 in Facilitating Intravascular Arrest of Breast Cancer Cells.

Previously, we showed that mice treated with cyclophosphamide (CTX) 4 days before intravenous injection of breast cancer cells had more cancer cells in the lung at 3 h after cancer injection than control counterparts without CTX. At 4 days after its injection, CTX is already excreted from the mice, allowing this pre-treatment design to reveal how CTX may modify the lung environment to indirectly affect cancer cells. In this study, we tested the hypothesis that the increase in cancer cell abundance at 3 h by CTX is due to an increase in the adhesiveness of vascular wall for cancer cells. Our data from protein array analysis and inhibition approach combined with in vitro and in vivo assays support the following two-prong mechanism. (1) CTX increases vascular permeability, resulting in the exposure of the basement membrane (BM). (2) CTX increases the level of matrix metalloproteinase-2 (MMP-2) in mouse serum, which remodels the BM and is functionally important for CTX to increase cancer abundance at this early stage. The combined effect of these two processes is the increased accessibility of critical protein domains in the BM, resulting in higher vascular adhesiveness for cancer cells to adhere. The critical protein domains in the vascular microenvironment are RGD and YISGR domains, whose known binding partners on cancer cells are integrin dimers and laminin receptor, respectively.

Stress-Inducible Gene Atf3 Dictates a Dichotomous Macrophage Activity in Chemotherapy-Enhanced Lung Colonization.

Previously, we showed that chemotherapy paradoxically exacerbated cancer cell colonization at the secondary site in a manner dependent on Atf3, a stress-inducible gene, in the non-cancer host cells. Here, we present evidence that this phenotype is established at an early stage of colonization within days of cancer cell arrival. Using mouse breast cancer models, we showed that, in the wild-type (WT) lung, cyclophosphamide (CTX) increased the ability of the lung to retain cancer cells in the vascular bed. Although CTX did not change the WT lung to affect cancer cell extravasation or proliferation, it changed the lung macrophage to be pro-cancer, protecting cancer cells from death. This, combined with the initial increase in cell retention, resulted in higher lung colonization in CTX-treated than control-treated mice. In the Atf3 knockout (KO) lung, CTX also increased the ability of lung to retain cancer cells. However, the CTX-treated KO macrophage was highly cytotoxic to cancer cells, resulting in no increase in lung colonization-despite the initial increase in cell retention. In summary, the status of Atf3 dictates the dichotomous activity of macrophage: pro-cancer for CTX-treated WT macrophage but anti-cancer for the KO counterpart. This dichotomy provides a mechanistic explanation for CTX to exacerbate lung colonization in the WT but not Atf3 KO lung.

JDP2 and ATF3 deficiencies dampen maladaptive cardiac remodeling and preserve cardiac function.

c-Jun dimerization protein (JDP2) and Activating Transcription Factor 3 (ATF3) are closely related basic leucine zipper proteins. Transgenic mice with cardiac expression of either JDP2 or ATF3 showed maladaptive remodeling and cardiac dysfunction. Surprisingly, JDP2 knockout (KO) did not protect the heart following transverse aortic constriction (TAC). Instead, the JDP2 KO mice performed worse than their wild type (WT) counterparts. To test whether the maladaptive cardiac remodeling observed in the JDP2 KO mice is due to ATF3, ATF3 was removed in the context of JDP2 deficiency, referred as double KO mice (dKO). Mice were challenged by TAC, and followed by detailed physiological, pathological and molecular analyses. dKO mice displayed no apparent differences from WT mice under unstressed condition, except a moderate better performance in dKO male mice. Importantly, following TAC the dKO hearts showed low fibrosis levels, reduced inflammatory and hypertrophic gene expression and a significantly preserved cardiac function as compared with their WT counterparts in both genders. Consistent with these data, removing ATF3 resumed p38 activation in the JDP2 KO mice which correlates with the beneficial cardiac function. Collectively, mice with JDP2 and ATF3 double deficiency had reduced maladaptive cardiac remodeling and lower hypertrophy following TAC. As such, the worsening of the cardiac outcome found in the JDP2 KO mice is due to the elevated ATF3 expression. Simultaneous suppression of both ATF3 and JDP2 activity is highly beneficial for cardiac function in health and disease.

Chemotherapy-Exacerbated Breast Cancer Metastasis: A Paradox Explainable by Dysregulated Adaptive-Response.

An emerging picture in cancer biology is that, paradoxically, chemotherapy can actively induce changes that favor cancer progression. These pro-cancer changes can be either inside (intrinsic) or outside (extrinsic) the cancer cells. In this review, we will discuss the extrinsic pro-cancer effect of chemotherapy; that is, the effect of chemotherapy on the non-cancer host cells to promote cancer progression. We will focus on metastasis, and will first discuss recent data from mouse models of breast cancer. Despite reducing the size of primary tumors, chemotherapy changes the tumor microenvironment, resulting in an increased escape of cancer cells into the blood stream. Furthermore, chemotherapry changes the tissue microenvironment at the distant sites, making it more hospitable to cancer cells upon their arrival. We will then discuss the idea and evidence that these devastating pro-metastatic effects of chemotherapy can be explained in the context of adaptive-response. At the end, we will discuss the potential relevance of these mouse data to human breast cancer and their implication on chemotherapy in the clinic.

Endothelial Regeneration of Large Vessels Is a Biphasic Process Driven by Local Cells with Distinct Proliferative Capacities.

The cellular and mechanistic bases underlying endothelial regeneration of adult large vessels have proven challenging to study. Using a reproducible in vivo aortic endothelial injury model, we characterized cellular dynamics underlying the regenerative process through a combination of multi-color lineage tracing, parabiosis, and single-cell transcriptomics. We found that regeneration is a biphasic process driven by distinct populations arising from differentiated endothelial cells. The majority of cells immediately adjacent to the injury site re-enter the cell cycle during the initial damage response, with a second phase driven by a highly proliferative subpopulation. Endothelial regeneration requires activation of stress response genes including Atf3, and aged aortas compromised in their reparative capacity express less Atf3. Deletion of Atf3 reduced endothelial proliferation and compromised the regeneration. These findings provide important insights into cellular dynamics and mechanisms that drive responses to large vessel injury.

Electrophilic properties of itaconate and derivatives regulate the IκBζ-ATF3 inflammatory axis.

Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring 1 marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite 2 . Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines 2 , including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase2,3, yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of IκBζ protein induction. The regulation of IκBζ is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17-IκBζ-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI-IκBζ regulatory axis could be an important new strategy for the treatment of IL-17-IκBζ-mediated autoimmune diseases.

Stress-inducible gene Atf3 in the noncancer host cells contributes to chemotherapy-exacerbated breast cancer metastasis.

Chemotherapy is a double-edged sword. It is anticancer because of its cytotoxicity. Paradoxically, by increasing chemoresistance and cancer metastasis, it is also procancer. However, the underlying mechanisms for chemotherapy-induced procancer activities are not well understood. Here we describe the ability of paclitaxel (PTX), a frontline chemotherapeutic agent, to exacerbate metastasis in mouse models of breast cancer. We demonstrate that, despite the apparent benefit of reducing tumor size, PTX increased the circulating tumor cells in the blood and enhanced the metastatic burden at the lung. At the primary tumor, PTX increased the abundance of the tumor microenvironment of metastasis, a landmark microanatomical structure at the microvasculature where cancer cells enter the blood stream. At the metastatic lung, PTX improved the tissue microenvironment (the "soil") for cancer cells (the "seeds") to thrive; these changes include increased inflammatory monocytes and reduced cytotoxicity. Importantly, these changes in the primary tumor and the metastatic lung were all dependent on Atf3, a stress-inducible gene, in the noncancer host cells. Together, our data provide mechanistic insights into the procancer effect of chemotherapy, explaining its paradox in the context of the seed-and-soil theory. Analyses of public datasets suggest that our data may have relevance to human cancers. Thus, ATF3 in the host cells links a chemotherapeutic agent-a stressor-to immune modulation and cancer metastasis. Dampening the effect of ATF3 may improve the efficacy of chemotherapy.

ATF3 expression in cardiomyocytes preserves homeostasis in the heart and controls peripheral glucose tolerance.

AIMS: Obesity and type 2 diabetes (T2D) trigger a harmful stress-induced cardiac remodeling process known as cardiomyopathy. These diseases represent a serious and widespread health problem in the Western world; however the underlying molecular basis is not clear. ATF3 is an 'immediate early' gene whose expression is highly and transiently induced in response to multiple stressors such as metabolic, oxidative, endoplasmic reticulum and inflammation, stressors that are involved in T2D cardiomyopathy. The role of ATF3 in diabetic cardiomyopathy is currently unknown. Our research has aimed to study the effect of ATF3 expression on cardiomyocytes, heart function and glucose homeostasis in an obesity-induced T2D mouse model. METHODS AND RESULTS: We used wild type mice (WT) as well as mutant mice with a cardiac-specific ATF3 deficiency (ATF3-cKO). Mice were fed a high-fat diet (HFD) for 15 weeks. HFD induced high ATF3 expression in cardiomyocytes. Mice were examined for cardiac remodeling processes and the diabetic state was assessed. HFD-fed ATF3-cKO mice exhibited severe cardiac fibrosis, higher levels of heart hypertrophic markers, increased inflammation and worse cardiac function, as compared to WT mice. Interestingly, HFD-fed ATF3-cKO mice display increased hyperglycemia and reduced glucose tolerance, despite higher blood insulin levels, as compared to HFD-fed WT mice. Elevated levels of the cardiac inflammatory cytokines IL-6 and TNFα leading to impaired insulin signalling may partially explain the peripheral glucose intolerance. CONCLUSIONS: Cardiac ATF3 has a protective role in dampening the HFD-induced cardiac remodeling processes. ATF3 exerts both local and systemic effects related to T2D-induced cardiomyopathy. This study provides a strong relationship between heart remodeling processes and blood glucose homeostasis.

Activating transcription factor 3 attenuates chemokine and cytokine expression in mouse skeletal muscle after exercise and facilitates molecular adaptation to endurance training.

Activating transcription factor (ATF)3 regulates the expression of inflammation-related genes in several tissues under pathological contexts. In skeletal muscle, atf3 expression increases after exercise, but its target genes remain unknown. We aimed to identify those genes and to determine the influence of ATF3 on muscle adaptation to training. Skeletal muscles of ATF3-knockout (ATF3-KO) and control mice were analyzed at rest, after exercise, and after training. In resting muscles, there was no difference between genotypes in enzymatic activities or fiber type. After exercise, a microarray analysis in quadriceps revealed ATF3 affects genes modulating chemotaxis and chemokine/cytokine activity. Quantitative PCR showed that the mRNA levels of chemokine C-C motif ligand (ccl)8 and chemokine C-X-C motif ligand (cxcl)13 were higher in quadriceps of ATF3-KO mice than in control mice. The same was observed for ccl9 and cxcl13 in soleus. Also in soleus, ccl2, interleukin (il)6, il1ß, and cluster of differentiation (cd)68 mRNA levels increased after exercise only in ATF3-KO mice. Endurance training increased the basal mRNA level of hexokinase-2, hormone sensitive lipase, glutathione peroxidase-1, and myosin heavy chain IIa in quadriceps of control mice but not in ATF3-KO mice. In summary, ATF3 attenuates the expression of inflammation-related genes after exercise and thus facilitates molecular adaptation to training.-Fernández-Verdejo, R., Vanwynsberghe, A. M., Essaghir, A., Demoulin, J.-B., Hai, T., Deldicque, L., Francaux, M. Activating transcription factor 3 attenuates chemokine and cytokine expression in mouse skeletal muscle after exercise and facilitates molecular adaptation to endurance training.

Cutaneous tissue damage induces long-lasting nociceptive sensitization and regulation of cellular stress- and nerve injury-associated genes in sensory neurons.

Tissue damage is one of the major etiological factors in the emergence of chronic/persistent pain, although mechanisms remain enigmatic. Using incision of the back skin of adult rats as a model for tissue damage, we observed sensitization in a nociceptive reflex enduring to 28days post-incision (DPI). To determine if the enduring behavioral changes corresponded with a long-term impact of tissue damage on sensory neurons, we examined the temporal expression profile of injury-regulated genes and the electrophysiological properties of traced dorsal root ganglion (DRG) sensory neurons. The mRNA for the injury/stress-hub gene Activating Transcription Factor 3 (ATF3) was upregulated and peaked within 4 DPI, after which levels declined but remained significantly elevated out to 28 DPI, a time when the initial incision appears healed and tissue-inflammation largely resolved. Accordingly, stereological image analysis indicated that some neurons expressed ATF3 only transiently (mostly medium-large neurons), while in others it was sustained (mostly small neurons), suggesting cell-type-specific responses. In retrogradely-traced ATF3-expressing neurons, Calcium/calmodulin-dependent protein kinase type IV (CAMK4) protein levels and isolectin-B4 (IB4)-binding were suppressed whereas Growth Associated Protein-43 (GAP-43) and Neuropeptide Y (NPY) protein levels were enhanced. Electrophysiological recordings from DiI-traced sensory neurons 28 DPI showed a significant sensitization limited to ATF3-expressing neurons. Thus, ATF3 expression is revealed as a strong predictor of single cells displaying enduring pain-related electrophysiological properties. The cellular injury/stress response induced in sensory neurons by tissue damage and indicated by ATF3 expression is positioned to contribute to pain which can occur after tissue damage.

Activating transcription factor 3 regulates canonical TGFß signalling in systemic sclerosis.

BACKGROUND: Activating transcription factor 3 (ATF3), a member of the ATF/cAMP-responsive element binding (CREB) family of transcription factors, regulates cellular response to stress including oxidative stress. The aim of this study was to analyse the role of ATF3 in fibroblast activation in systemic sclerosis (SSc). METHODS: ATF3 was analysed by reverse transcription quantitative PCR, western blot and immunohistochemistry. ATF3 knockout fibroblasts and mice were used to study the functional role of ATF3. Knockdown experiments, reporter assays and coimmunoprecipitation were performed to study the effects of ATF3 on Smad and activation protein 1 (AP-1) signalling. The role of c-Jun was analysed by costaining, specific inactivation and coimmunoprecipitation. RESULTS: Transforming growth factor-ß (TGFß) upregulates the expression of ATF3 in SSc fibroblasts. ATF3-deficient fibroblasts were less sensitive to TGFß, whereas ectopic expression of ATF3 enhanced the profibrotic effects of TGFß. Mechanistically, ATF3 interacts with Smad3 directly on stimulation with TGFß and regulates Smad activity in a c-Jun-dependent manner. Knockout of ATF3 protected mice from bleomycin-induced fibrosis and fibrosis induced by overexpression of a constitutively active TGFß receptor I. Reporter assays and analyses of the expression of Smad target genes demonstrated that binding of ATF3 regulates the transcriptional activity of Smad3. CONCLUSIONS: We demonstrate for the first time a key role for ATF3 in fibrosis. Knockout of the ATF3 gene reduced the stimulatory effect of TGFß on fibroblasts by interfering with canonical Smad signalling and protected the mice from experimental fibrosis in two different models. ATF3 might thus be a candidate for molecular targeted therapies for SSc.

Islet Brain 1 Protects Insulin Producing Cells against Lipotoxicity.

Chronic intake of saturated free fatty acids is associated with diabetes and may contribute to the impairment of functional beta cell mass. Mitogen activated protein kinase 8 interacting protein 1 also called islet brain 1 (IB1) is a candidate gene for diabetes that is required for beta cell survival and glucose-induced insulin secretion (GSIS). In this study we investigated whether IB1 expression is required for preserving beta cell survival and function in response to palmitate. Chronic exposure of MIN6 and isolated rat islets cells to palmitate led to reduction of the IB1 mRNA and protein content. Diminution of IB1 mRNA and protein level relied on the inducible cAMP early repressor activity and proteasome-mediated degradation, respectively. Suppression of IB1 level mimicked the harmful effects of palmitate on the beta cell survival and GSIS. Conversely, ectopic expression of IB1 counteracted the deleterious effects of palmitate on the beta cell survival and insulin secretion. These findings highlight the importance in preserving the IB1 content for protecting beta cell against lipotoxicity in diabetes.

Host JDP2 expression in the bone marrow contributes to metastatic spread.

The c-Jun Dimerization Protein 2, JDP2, is a basic leucine zipper protein member of the activator protein-1 (AP-1) family of transcription factors. JDP2 typically suppresses gene transcription through multiple mechanisms and plays a dual role in multiple cellular processes, including cell differentiation and proliferation which is dependent on AP-1 function. Whereas the role of JDP2 expression within cancer cells has been studied, its role in stromal cells at the tumor microenvironment is largely unknown. Here we show that mice lacking JDP2 (JDP2-/-) display a reduced rate of metastasis in Lewis lung carcinoma (LLC) and polyoma middle T-antigen (PyMT) breast carcinoma mouse models. The replacement of wild-type bone marrow derived cells (BMDCs) with JDP2-deficient BMDCs recapitulates the metastatic phenotype of JDP2-/- tumor-bearing mice. In vitro, conditioned medium of wild-type BMDCs significantly potentiates the migration and invasion capacity of LLC cells as compared to that of JDP2-/- BMDCs. Furthermore, wild-type BMDCs secrete CCL5, a chemokine known to contribute to metastasis, to a greater extent than JDP2-/- BMDCs. The supplementation of CCL5 in JDP2-/- BMDC conditioned medium was sufficient to potentiate the invasion capacity of LLC. Overall, this study suggests that JDP2-expressing BMDCs within the tumor microenvironment contribute to metastatic spread.

ATF3 mediates inhibitory effects of ethanol on hepatic gluconeogenesis.

Increases in circulating glucagon during fasting maintain glucose balance by stimulating hepatic gluconeogenesis. Acute ethanol intoxication promotes fasting hypoglycemia through an increase in hepatic NADH, which inhibits hepatic gluconeogenesis by reducing the conversion of lactate to pyruvate. Here we show that acute ethanol exposure also lowers fasting blood glucose concentrations by inhibiting the CREB-mediated activation of the gluconeogenic program in response to glucagon. Ethanol exposure blocked the recruitment of CREB and its coactivator CRTC2 to gluconeogenic promoters by up-regulating ATF3, a transcriptional repressor that also binds to cAMP-responsive elements and thereby down-regulates gluconeogenic genes. Targeted disruption of ATF3 decreased the effects of ethanol in fasted mice and in cultured hepatocytes. These results illustrate how the induction of transcription factors with overlapping specificity can lead to cross-coupling between stress and hormone-sensitive pathways.

Atf3 negatively regulates Ptgs2/Cox2 expression during acute inflammation.

By generating prostaglandins, cyclooxygenase-2 (Cox-2/Ptgs2) plays a critical role in regulating inflammatory responses. While several inflammatory stimuli have been shown to increase Ptgs2 expression, less is known about how the transcription of this gene is terminated. Here we show that stimulation of macrophages with yeast zymosan, a TLR2/6 and dectin-1 agonist, causes a transient increase in the expression of Ptgs2 accompanied by a simultaneous increase in the expression of the transcriptional repressor, activating transcription factor-3 (Atf3). The expression of Ptgs2 was significantly higher in resident peritoneal macrophages isolated from Atf3(-/-) mice than that from Atf3(+/+) mice and was associated with higher prostaglandin production upon stimulation with zymosan. In activated macrophages, Atf3 accumulated in the nucleus and chromatin-immunoprecipitation analysis showed that Atf3 is recruited to the Ptgs2 promoter region. In acute peritonitis and in cutaneous wounds, there was increased leukocyte accumulation and higher levels of prostaglandins (PGE2/PGD2) in inflammatory exudates of Atf3(-/-) mice compared with WT mice. Collectively, these results demonstrate that during acute inflammation Atf3 negatively regulates Ptgs2 and therefore dysregulation of this axis could potentially contribute to aberrant Ptgs2 expression in chronic inflammatory diseases. Moreover, this axis could be a new therapeutic target for suppressing Ptgs2 expression and the resultant inflammatory responses.

ATF3 is a novel regulator of mouse neutrophil migration.

Expression of the activating transcription factor 3 (ATF3) gene is induced by Toll-like receptor (TLR) signaling. In turn, ATF3 protein inhibits the expression of various TLR-driven proinflammatory genes. Given its counter-regulatory role in diverse innate immune responses, we defined the effects of ATF3 on neutrophilic airway inflammation in mice. ATF3 deletion was associated with increased lipopolysaccharide (LPS)-driven airway epithelia production of CXCL1, but not CXCL2, findings concordant with a consensus ATF3-binding site identified solely in the Cxcl1 promoter. Unexpectedly, ATF3-deficient mice did not exhibit increased airway neutrophilia after LPS challenge. Bone marrow chimeras revealed a specific reduction in ATF3(-/-) neutrophil recruitment to wild-type lungs. In vitro, ATF3(-/-) neutrophils exhibited a profound chemotaxis defect. Global gene expression analysis identified ablated Tiam2 expression in ATF3(-/-) neutrophils. TIAM2 regulates cellular motility by activating Rac1-mediated focal adhesion disassembly. Notably, ATF3(-/-) and ATF3-sufficient TIAM2 knockdown neutrophils, both lacking TIAM2, exhibited increased focal complex area, along with excessive CD11b-mediated F-actin polymerization. Together, our data describe a dichotomous role for ATF3-mediated regulation of neutrophilic responses: inhibition of neutrophil chemokine production but promotion of neutrophil chemotaxis.

ßIV-Spectrin and CaMKII facilitate Kir6.2 regulation in pancreatic beta cells.

Identified over a dozen years ago in the brain and pancreatic islet, ßIV-spectrin is critical for the local organization of protein complexes throughout the nervous system. ßIV-Spectrin targets ion channels and adapter proteins to axon initial segments and nodes of Ranvier in neurons, and ßIV-spectrin dysfunction underlies ataxia and early death in mice. Despite advances in ßIV-spectrin research in the nervous system, its role in pancreatic islet biology is unknown. Here, we report that ßIV-spectrin serves as a multifunctional structural and signaling platform in the pancreatic islet. We report that ßIV-spectrin directly associates with and targets the calcium/calmodulin-dependent protein kinase II (CaMKII) in pancreatic islets. In parallel, ßIV-spectrin targets ankyrin-B and the ATP-sensitive potassium channel. Consistent with these findings, ßIV-spectrin mutant mice lacking CaMKII- or ankyrin-binding motifs display selective loss of expression and targeting of key protein components, including CaMKIIδ. ßIV-Spectrin-targeted CaMKII directly phosphorylates the inwardly-rectifying potassium channel, Kir6.2 (alpha subunit of KATP channel complex), and we identify the specific residue, Kir6.2 T224, responsible for CaMKII-dependent regulation of KATP channel function. CaMKII-dependent phosphorylation alters channel regulation resulting in KATP channel inhibition, a cellular phenotype consistent with aberrant insulin regulation. Finally, we demonstrate aberrant KATP channel phosphorylation in ßIV-spectrin mutant mice. In summary, our findings establish a broader role for ßIV-spectrin in regulation of cell membrane excitability in the pancreatic islet, define the pathway for CaMKII local control in pancreatic beta cells, and identify the mechanism for CaMKII-dependent regulation of KATP channels.

Transcription factor ATF3 links host adaptive response to breast cancer metastasis.

Host response to cancer signals has emerged as a key factor in cancer development; however, the underlying molecular mechanism is not well understood. In this report, we demonstrate that activating transcription factor 3 (ATF3), a hub of the cellular adaptive response network, plays an important role in host cells to enhance breast cancer metastasis. Immunohistochemical analysis of patient tumor samples revealed that expression of ATF3 in stromal mononuclear cells, but not cancer epithelial cells, is correlated with worse clinical outcomes and is an independent predictor for breast cancer death. This finding was corroborated by data from mouse models showing less efficient breast cancer metastasis in Atf3-deficient mice than in WT mice. Further, mice with myeloid cell-selective KO of Atf3 showed fewer lung metastases, indicating that host ATF3 facilitates metastasis, at least in part, by its function in macrophage/myeloid cells. Gene profiling analyses of macrophages from mouse tumors identified an ATF3-regulated gene signature that could distinguish human tumor stroma from distant stroma and could predict clinical outcomes, lending credence to our mouse models. In conclusion, we identified ATF3 as a regulator in myeloid cells that enhances breast cancer metastasis and has predictive value for clinical outcomes.