IKKα-deficient lung adenocarcinomas generate an immunosuppressive microenvironment by overproducing Treg-inducing cytokines.

The tumor microenvironment (TME) provides potential targets for cancer therapy. However, how signals originating in cancer cells affect tumor-directed immunity is largely unknown. Deletions in the CHUK locus, coding for IκB kinase α (IKKα), correlate with reduced lung adenocarcinoma (ADC) patient survival and promote KrasG12D-initiated ADC development in mice, but it is unknown how reduced IKKα expression affects the TME. Here, we report that low IKKα expression in human and mouse lung ADC cells correlates with increased monocyte-derived macrophage and regulatory T cell (Treg) scores and elevated transcription of genes coding for macrophage-recruiting and Treg-inducing cytokines (CSF1, CCL22, TNF, and IL-23A). By stimulating recruitment of monocyte-derived macrophages from the bone marrow and enforcing a TNF/TNFR2/c-Rel signaling cascade that stimulates Treg generation, these cytokines promote lung ADC progression. Depletion of TNFR2, c-Rel, or TNF in CD4+ T cells or monocyte-derived macrophages dampens Treg generation and lung tumorigenesis. Treg depletion also attenuates carcinogenesis. In conclusion, reduced cancer cell IKKα activity enhances formation of a protumorigenic TME through a pathway whose constituents may serve as therapeutic targets for KRAS-initiated lung ADC.

Lymph node formation and B cell homeostasis require IKK-α in distinct endothelial cell-derived compartments.

Global inactivation of IκB kinase (IKK)-α results in defective lymph node (LN) formation and B cell maturation, and loss of IKK-α-dependent noncanonical NF-κB signaling in stromal organizer and hematopoietic cells is thought to underlie these distinct defects. We previously demonstrated that this pathway is also activated in vascular endothelial cells (ECs). To determine the physiologic function of EC-intrinsic IKK-α, we crossed IkkαF/F mice with Tie2-cre or Cdh5-cre mice to ablate IKK-α in ECs. Notably, the compound defects of global IKK-α inactivation were recapitulated in IkkαTie2 and IkkαCdh5 mice, as both lacked all LNs and mature follicular and marginal zone B cell numbers were markedly reduced. However, as Tie2-cre and Cdh5-cre are expressed in all ECs, including blood forming hemogenic ECs, IKK-α was also absent in hematopoietic cells (HC). To determine if loss of HC-intrinsic IKK-α affected LN development, we generated IkkαVav mice lacking IKK-α in only the hematopoietic compartment. While mature B cell numbers were significantly reduced in IkkαVav mice, LN formation was intact. As lymphatic vessels also arise during development from blood ECs, we generated IkkαLyve1 mice lacking IKK-α in lymphatic ECs (LECs) to determine if IKK-α in lymphatic vessels impacts LN development. Strikingly, while mature B cell numbers were normal, LNs were completely absent in IkkαLyve1 mice. Thus, our findings reveal that IKK-α in distinct EC-derived compartments is uniquely required to promote B cell homeostasis and LN development, and we establish that LEC-intrinsic IKK-α is absolutely essential for LN formation.

IKKα inactivation promotes Kras-initiated lung adenocarcinoma development through disrupting major redox regulatory pathways.

Lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are two distinct and predominant types of human lung cancer. IκB kinase α (IKKα) has been shown to suppress lung SCC development, but its role in ADC is unknown. We found inactivating mutations and homologous or hemizygous deletions in the CHUK locus, which encodes IKKα, in human lung ADCs. The CHUK deletions significantly reduced the survival time of patients with lung ADCs harboring KRAS mutations. In mice, lung-specific Ikkα ablation (IkkαΔLu ) induces spontaneous ADCs and promotes KrasG12D-initiated ADC development, accompanied by increased cell proliferation, decreased cell senescence, and reactive oxygen species (ROS) accumulation. IKKα deletion up-regulates NOX2 and down-regulates NRF2, leading to ROS accumulation and blockade of cell senescence induction, which together accelerate ADC development. Pharmacologic inhibition of NADPH oxidase or ROS impairs KrasG12D-mediated ADC development in IkkαΔLu mice. Therefore, IKKα modulates lung ADC development by controlling redox regulatory pathways. This study demonstrates that IKKα functions as a suppressor of lung ADC in human and mice through a unique mechanism that regulates tumor cell-associated ROS metabolism.

Integrity of IKK/NF-κB Shields Thymic Stroma That Suppresses Susceptibility to Autoimmunity, Fungal Infection, and Carcinogenesis.

A pathogenic connection between autoreactive T cells, fungal infection, and carcinogenesis has been demonstrated in studies of human autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) as well as in a mouse model in which kinase-dead Ikkα knock-in mice develop impaired central tolerance, autoreactive T cell-mediated autoimmunity, chronic fungal infection, and esophageal squamous cell carcinoma, which recapitulates APECED. IκB kinase α (IKKα) is one subunit of the IKK complex required for NF-κB activation. IKK/NF-κB is essential for central tolerance establishment by regulating the development of medullary thymic epithelial cells (mTECs) that facilitate the deletion of autoreactive T cells in the thymus. In this review, we extensively discuss the pathogenic roles of inborn errors in the IKK/NF-κB loci in the phenotypically related diseases APECED, immune deficiency syndrome, and severe combined immunodeficiency; differentiate how IKK/NF-κB components, through mTEC (stroma), T cells/leukocytes, or epithelial cells, contribute to the pathogenesis of infectious diseases, autoimmunity, and cancer; and highlight the medical significance of IKK/NF-κB in these diseases.

Overactivation of the NF-κB pathway impairs molar enamel formation.

OBJECTIVE: Hypohidrotic ectodermal dysplasia (HED) is a hereditary disorder characterized by abnormal structures and functions of the ectoderm-derived organs, including teeth. HED patients exhibit a variety of dental symptoms, such as hypodontia. Although disruption of the EDA/EDAR/EDARADD/NF-κB pathway is known to be responsible for HED, it remains unclear whether this pathway is involved in the process of enamel formation. EXPERIMENTAL SUBJECTS AND METHODS: To address this question, we examined the mice overexpressing Ikkß (an essential component required for the activation of NF-κB pathway) under the keratin 5 promoter (K5-Ikkß). RESULTS: Upregulation of the NF-κB pathway was confirmed in the ameloblasts of K5-Ikkß mice. Premature abrasion was observed in the molars of K5-Ikkß mice, which was accompanied by less mineralized enamel. However, no significant changes were observed in the enamel thickness and the pattern of enamel rods in K5-Ikkß mice. Klk4 expression was significantly upregulated in the ameloblasts of K5-Ikkß mice at the maturation stage, and the expression of its substrate, amelogenin, was remarkably reduced. This suggests that abnormal enamel observed in K5-Ikkß mice was likely due to the compromised degradation of enamel protein at the maturation stage. CONCLUSION: Therefore, we could conclude that the overactivation of the NF-κB pathway impairs the process of amelogenesis.

IKKα deficiency disrupts the development of marginal zone and follicular B cells.

Only few genes have been confidently identified to be involved in the Follicular (FO) and Marginal Zone (MZ) B cell differentiation, migration, and retention in the periphery. Our group previously observed that IKKα kinase inactive mutant mice IKKαK44A/K44A have significantly lower number of MZ B cells whereas FO B cell numbers appeared relatively normal. Because kinase dead IKKα can retain some of its biological functions that may interfere in revealing its actual role in the MZ and FO B cell differentiation. Therefore, in the current study, we genetically deleted IKKα from the pro-B cell lineage that revealed novel functions of IKKα in the MZ and FO B lymphocyte development. The loss of IKKα produces a significant decline in the percentage of immature B lymphocytes, mature marginal zone B cells, and follicular B cells along with a severe disruption of splenic architecture of marginal and follicular zones. IKKα deficiency affect the recirculation of mature B cells through bone marrow. A transplant of IKKα knockout fetal liver cells into Rag-/- mice shows a significant reduction compared to control in the B cells recirculating through bone marrow. To reveal the genes important in the B cell migration, a high throughput gene expression analysis was performed on the IKKα deficient recirculating mature B cells (B220+IgMhi). That revealed significant changes in the expression of genes involved in the B lymphocyte survival, homing and migration. And several among those genes identified belong to G protein family. Taken together, this study demonstrates that IKKα forms a vial axis controlling the genes involved in MZ and FO B cell differentiation and migration.

IKKα is required for the homeostasis of regulatory T cells and for the expansion of both regulatory and effector CD4 T cells.

It was reported that TNF receptor type II signaling, which has the capacity to stimulate CD4+ forkhead box P3+ (Foxp3+) regulatory T cells (Tregs), activated the noncanonical NF-κB pathway in an IKKα-dependent manner. Therefore, we studied the role of IKKα in the homeostasis of Treg population. To this end, we generated a mouse strain with conditional knockout of IKKα in CD4 cells (Ikkα(f/f):CD4.Cre) that showed a >60% reduction in the number of Tregs in the thymus and peripheral lymphoid tissues, whereas the number of Foxp3- effector T cells (Teffs) remained at a normal level. The function of Tregs deficient in IKKα was examined using Rag1(-/-) mice cotransferred with naive CD4 cells (nCD4s). Although wild-type (WT) Tregs inhibited colitis induced by transfer of WT nCD4s, IKKα-deficient Tregs failed to do so, which was associated with their inability to reconstitute Rag1(-/-) mice. Furthermore, nCD4s deficient in IKKα also failed to reconstitute Rag1(-/-) mice and were defective in proliferative responses in vitro and in vivo. Thus, our study reveals a novel role of IKKα in the maintenance of a normal Treg population and in the control of expansion of CD4 T cells. These properties of IKKα may be exploited as therapeutic strategies in the treatment of major human diseases.

IKKα-mediated signaling circuitry regulates early B lymphopoiesis during hematopoiesis.

Multiple transcription factors regulate B-cell commitment, which is coordinated with myeloid-erythroid lineage differentiation. NF-κB has long been speculated to regulate early B-cell development; however, this issue remains controversial. IκB kinase-α (IKKα) is required for splenic B-cell maturation but not for BM B-cell development. In the present study, we unexpectedly found defective BM B-cell development and increased myeloid-erythroid lineages in kinase-dead IKKα (KA/KA) knock-in mice. Markedly increased cytosolic p100, an NF-κB2-inhibitory form, and reduced nuclear NF-κB p65, RelB, p50, and p52, and IKKα were observed in KA/KA splenic and BM B cells. Several B- and myeloid-erythroid-cell regulators, including Pax5, were deregulated in KA/KA BM B cells. Using fetal liver and BM congenic transplantations and deleting IKKα from early hematopoietic cells in mice, this defect was identified as being B cell-intrinsic and an early event during hematopoiesis. Reintroducing IKKα, Pax5, or combined NF-κB molecules promoted B-cell development but repressed myeloid-erythroid cell differentiation in KA/KA BM B cells. The results of the present study demonstrate that IKKα regulates B-lineage commitment via combined canonical and noncanonical NF-κB transcriptional activities to target Pax5 expression during hematopoiesis.

The role of Irf6 in tooth epithelial invagination.

Thickening and the subsequent invagination of the epithelium are an important initial step in ectodermal organ development. Ikkα has been shown to play a critical role in controlling epithelial growth, since Ikkα mutant mice show protrusions (evaginations) of incisor tooth, whisker and hair follicle epithelium rather than invagination. We show here that mutation of the Interferon regulatory factor (Irf) family, Irf6 also results in evagination of incisor epithelium. In common with Ikkα mutants, Irf6 mutant evagination occurs in a NF-κB-independent manner and shows the same molecular changes as those in Ikkα mutants. Irf6 thus also plays a critical role in regulating epithelial invagination. In addition, we also found that canonical Wnt signaling is upregulated in evaginated incisor epithelium of both Ikkα and Irf6 mutant embryos.

TNFR1 and TNFR2, Which Link NF-κB Activation, Drive Lung Cancer Progression, Cell Dedifferentiation, and Metastasis.

TNFR1 and TNFR2, encoded by TNFRSF1A and TNFRSF1B, respectively, are the most well-characterized members among the TNFR superfamily. TNFR1 is expressed in most cell types, while TNFR2 has been reported to be preferentially expressed in leukocytes. Lung cancer remains the leading cause of cancer mortality worldwide but TNFRs' activities in lung cancer development have not been fully evaluated. Recently, overexpressed TNFR1 was reported in a large proportion of human lung squamous cell carcinomas. Increased TNFR1 coupled with increased UBCH10 caused lung SCC cell dedifferentiation with epithelial-mesenchymal transition features and the metastasis in a combined spontaneous lung SCC and TNFR1 transgenic mouse model. UBCH10, an E2 ubiquitin-conjugating enzyme that is an oncogene, increased Sox2, c-Myc, Twist1, and Bcl2 levels. Increased TNFR1 upregulated UBCH10 expression by activating c-Rel and p65 NF-κB. Lung SCC patients overexpressing TNFRSF1A and one of these target genes died early compared to lung SCC patients expressing lower levels of these genes. Recently, we also revealed that TNFR2 was required for lung adenocarcinoma progression, delivering a signaling pathway of TNF/TNFR2/NF-κB-c-Rel, in which macrophage-produced ROS and TNF converted CD4 T cells to Foxp3 Treg cells, generating an immunosuppressive tumor microenvironment and promoting lung ADC progression. In human lung ADC cohorts, TNFRSF1B expression was highly correlated with TNF, FOXP3, and CD4 expression. Of note, TNF stimulated the activities of TNFR1 and TNFR2, two membrane-binding receptors, which accelerate tumorigenesis through diverse mechanisms. This review focuses on these new findings regarding the roles of TNFR1 and TNFR2 in lung SCC and ADC development in humans and mice, and highlights the potential therapeutic targets of human lung cancers.

Characterization and Validation of ceRNA-Mediated Pathway-Pathway Crosstalk Networks Across Eight Major Cardiovascular Diseases.

Pathway analysis is considered as an important strategy to reveal the underlying mechanisms of diseases. Pathways that are involved in crosstalk can regulate each other and co-regulate downstream biological processes. Furthermore, some genes in the pathways can function with other genes via the relationship of the competing endogenous RNA (ceRNA) mechanism, which has also been demonstrated to play key roles in cellular biology. However, the comprehensive analysis of ceRNA-mediated pathway crosstalk is lacking. Here, we constructed the landscape of the ceRNA-mediated pathway-pathway crosstalk of eight major cardiovascular diseases (CVDs) based on sequencing data from ∼2,800 samples. Some common features shared by numerous CVDs were uncovered. A fraction of the pathway-pathway crosstalk was conserved in multiple CVDs and a core pathway-pathway crosstalk network was identified, suggesting the similarity of pathway-pathway crosstalk among CVDs. Experimental evidence also demonstrated that the pathway crosstalk was functioned in CVDs. We split all hub pathways of each pathway-pathway crosstalk network into three categories, namely, common hubs, differential hubs, and specific hubs, which could highlight the common or specific biological mechanisms. Importantly, after a comparison analysis of the hub pathways of networks, ∼480 hub pathway-induced common modules were identified to exert functions in CVDs broadly. Moreover, we performed a random walk algorithm on the hub pathway-induced sub-network and identified 23 potentially novel CVD-related pathways. In summary, our study revealed the potential molecular regulatory mechanisms of ceRNA crosstalk in pathway-pathway crosstalk levels and provided a novel routine to investigate the pathway-pathway crosstalk in cardiology. All CVD pathway-pathway crosstalks are provided in http://www.licpathway.net/cepathway/index.html.

A TNFR1-UBCH10 axis drives lung squamous cell carcinoma dedifferentiation and metastasis through a cell-autonomous signaling loop.

Tumor necrosis factor receptor 1 (TNFR1), encoded by TNFRSF1A, is a critical transducer of inflammatory pathways, but its physiological role in human cancer is not completely understood. Here, we observed high expression of TNFR1 in many human lung squamous cell carcinoma (SCCs) samples and in spontaneous lung SCCs derived from kinase-dead Ikkα knock-in (KA/KA) mice. Knocking out Tnfrf1a in KA/KA mice blocked lung SCC formation. When injected via tail vein, KALLU+ lung SCC cells that highly expressed TNFR1/TNF, Sox2, c-Myc, Twist1, Bcl2, and UBCH10, generated dedifferentiated spindle cell carcinomas with epithelial-mesenchymal transition markers in mouse lungs. In contrast, KALLU+ cells with silenced TNFR1 and KALLU- cells that expressed low levels of TNFR1 generated well-differentiated lung SCCs and were less tumorigenic and metastatic. We identified a downstream effector of TNFR1: oncogenic UBCH10, an E2 ubiquitin-conjugating enzyme with targets including Twist1, c-Myc, and Sox2, which enhanced SCC cell dedifferentiation. Furthermore, Tg-K5.TNFR1;KA/KA mice, which expressed transgenic TNFR1 in keratin 5-positve epithelial cells, developed more poorly differentiated and metastatic lung SCCs than those found in KA/KA mice. These findings demonstrate that an overexpressed TNFR1-UBCH10 axis advances lung carcinogenesis and metastasis through a dedifferentiation mechanism. Constituents in this pathway may contribute to the development of differentiation-related therapies for lung SCC.

Reduction of IKKalpha expression promotes chronic ultraviolet B exposure-induced skin inflammation and carcinogenesis.

Ultraviolet B light (UVB) is a common cause of human skin cancer. UVB irradiation induces mutations in the tumor suppressor p53 gene as well as chronic inflammation, which are both essential for UVB carcinogenesis. Inhibitor of nuclear factor kappaB kinase-alpha (IKKalpha) plays an important role in maintaining skin homeostasis, and expression of IKKalpha was found to be down-regulated in human and murine skin squamous cell carcinomas. However, the role of IKKalpha in UVB skin carcinogenesis has not been investigated. Thus, here we performed UVB carcinogenesis experiments on Ikkalpha(+/+) and Ikkalpha(+/-) mice. Ikkalpha(+/-) mice were found to develop a twofold greater number of skin tumors than Ikkalpha(+/+) mice after chronic UVB irradiation. In addition, tumor latency was significantly shorter and tumors were bigger in Ikkalpha(+/-) than in Ikkalpha(+/+) mice. At an early stage of carcinogenesis, an increase in UVB-induced p53 mutations as well as macrophage recruitment and mitogenic activity, and a decrease in UVB-induced apoptosis, were detected in Ikkalpha(+/-) compared with those in Ikkalpha(+/+) skin. Also, reduction of IKKalpha levels in keratinocytes up-regulated the expression of monocyte chemoattractant protein-1 (MCP-1/CCL2), TNFalpha, IL-1, and IL-6, and elevated macrophage migration, which might promote macrophage recruitment and inflammation. Therefore, these findings suggest that reduction of IKKalpha expression orchestrates UVB carcinogen, accelerating tumorigenesis.

Role of IKKα in skin squamous cell carcinomas.

Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are two major types of skin cancer derived from keratinocytes. SCC is a more aggressive type of cancer than BCC in humans. One significant difference between SCC and BCC is that SCC development is generally associated with cell dedifferentiation and morphological changes. When SCC is converted to spindle cell carcinoma, the latest stage of cancer, the tumor cells change to a fibroblastic cell morphology (epithelial-to-mesenchymal transition) and lose their differentiation markers. Recently, several laboratories have reported altered IκB kinase α (IKKα) protein localization, downregulated IKKα, and IKKα gene deletions and mutations in human SCCs of the skin, lung, esophagus, and neck and head. In addition, IKKα reduction promotes chemical carcinogen- and ultraviolet B-induced skin carcinogenesis, and IKKα deletion in keratinocytes causes spontaneous skin SCCs, but not BCCs, in mice. Thus, IKKα emerges as a bona fide skin tumor suppressor. In this article, we will discuss the role of IKKα in skin SCC development.

Attribution of NF-κB Activity to CHUK/IKKα-Involved Carcinogenesis.

Studies analyzing human cancer genome sequences and genetically modified mouse models have extensively expanded our understanding of human tumorigenesis, even challenging or reversing the dogma of certain genes as originally characterized by in vitro studies. Inhibitor-κB kinase α (IKKα), which is encoded by the conserved helix-loop-helix ubiquitous kinase (CHUK) gene, is first identified as a serine/threonine protein kinase in the inhibitor-κB kinase complex (IKK), which is composed of IKKα, IKKß, and IKKγ (NEMO). IKK phosphorylates serine residues 32 and 36 of IκBα, a nuclear factor-κB (NF-κB) inhibitor, to induce IκBα protein degradation, resulting in the nuclear translocation of NF-κB dimers that function as transcriptional factors to regulate immunity, infection, lymphoid organ/cell development, cell death/growth, and tumorigenesis. NF-κB and IKK are broadly and differentially expressed in the cells of our body. For a long time, the idea that the IKK complex acts as a direct upstream activator of NF-κB in carcinogenesis has been predominately accepted in the field. Surprisingly, IKKα has emerged as a novel suppressor for skin, lung, esophageal, and nasopharyngeal squamous cell carcinoma, as well as lung and pancreatic adenocarcinoma (ADC). Thus, Ikkα loss is a tumor driver in mice. On the other hand, lacking the RANKL/RANK/IKKα pathway impairs mammary gland development and attenuates oncogene- and chemical carcinogen-induced breast and prostate tumorigenesis and metastasis. In general, NF-κB activation leads one of the major inflammatory pathways and stimulates tumorigenesis. Since IKKα and NF-κB play significant roles in human health, revealing the interplay between them greatly benefits the diagnosis, treatment, and prevention of human cancer. In this review, we discuss the intriguing attribution of NF-κB to CHUK/IKKα-involved carcinogenesis.

NLRP3 Inhibition Ameliorates Severe Cutaneous Autoimmune Manifestations in a Mouse Model of Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy-Like Disease.

Patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy show diverse endocrine and nonendocrine manifestations initiated by self-reactive T cells because of AIRE mutation-induced defective central tolerance. A large number of American patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy suffer from early-onset cutaneous inflammatory lesions accompanied by an infiltration of T cells and myeloid cells. The role of myeloid cells in this setting remains to be fully investigated. In this study, we characterize the autoinflammatory phenotypes in the skin of both autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy-like kinase-dead Ikkα knockin mice and patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. We found a marked infiltration of autoreactive CD4 T cells, macrophages, and neutrophils; elevated uric acid; and increased NLRP3, a major inflammasome component. Depleting autoreactive CD4 T cells or ablating Ccl2/Cxcr2 genes significantly attenuated the inflammasome activity, inflammation, and skin phenotypes in kinase-dead Ikkα knockin mice. Importantly, treatment with an NLRP3 inhibitor reduced skin phenotypes and decreased infiltration of CD4 T cells, macrophages, and neutrophils. These results suggest that increased myeloid cell infiltration contributes to autoreactive CD4 T cell-mediated skin autoinflammation. Thus, our findings reveal that the combined infiltration of macrophages and neutrophils is required for autoreactive CD4 T cell-mediated skin disease pathogenesis and that the NLRP3-dependent inflammasome is a potential therapeutic target for the cutaneous manifestations of autoimmune diseases.

Oral-Gut Microbiome Axis in Gastrointestinal Disease and Cancer.

It is well-known that microbiota dysbiosis is closely associated with numerous diseases in the human body. The oral cavity and gut are the two largest microbial habitats, playing a major role in microbiome-associated diseases. Even though the oral cavity and gut are continuous regions connected through the gastrointestinal tract, the oral and gut microbiome profiles are well-segregated due to the oral-gut barrier. However, the oral microbiota can translocate to the intestinal mucosa in conditions of the oral-gut barrier dysfunction. Inversely, the gut-to-oral microbial transmission occurs as well in inter- and intrapersonal manners. Recently, it has been reported that oral and gut microbiomes interdependently regulate physiological functions and pathological processes. Oral-to-gut and gut-to-oral microbial transmissions can shape and/or reshape the microbial ecosystem in both habitats, eventually modulating pathogenesis of disease. However, the oral-gut microbial interaction in pathogenesis has been underappreciated to date. Here, we will highlight the oral-gut microbiome crosstalk and its implications in the pathogenesis of the gastrointestinal disease and cancer. Better understanding the role of the oral-gut microbiome axis in pathogenesis will be advantageous for precise diagnosis/prognosis and effective treatment.

Cancer cells escape autophagy inhibition via NRF2-induced macropinocytosis.

Many cancers, including pancreatic ductal adenocarcinoma (PDAC), depend on autophagy-mediated scavenging and recycling of intracellular macromolecules, suggesting that autophagy blockade should cause tumor starvation and regression. However, until now autophagy-inhibiting monotherapies have not demonstrated potent anti-cancer activity. We now show that autophagy blockade prompts established PDAC to upregulate and utilize an alternative nutrient procurement pathway: macropinocytosis (MP) that allows tumor cells to extract nutrients from extracellular sources and use them for energy generation. The autophagy to MP switch, which may be evolutionarily conserved and not cancer cell restricted, depends on activation of transcription factor NRF2 by the autophagy adaptor p62/SQSTM1. NRF2 activation by oncogenic mutations, hypoxia, and oxidative stress also results in MP upregulation. Inhibition of MP in autophagy-compromised PDAC elicits dramatic metabolic decline and regression of transplanted and autochthonous tumors, suggesting the therapeutic promise of combining autophagy and MP inhibitors in the clinic.

Arabidopsis guard cell CO2/HCO3 - response mutant screening by an aequorin-based calcium imaging system.

BACKGROUND: The increase in atmospheric CO2 is causing a number of changes in plant growth such as increases in leaf area and number, branching, plant size and biomass, and growth rate. Despite the importance of stomatal responses to CO2, little is known about the genetic and molecular mechanisms that mediate stomatal development and movement in response to CO2 levels. Deciphering the mechanisms that sense changes in CO2 and/or HCO3 - concentration is critical for unraveling the role of CO2 in stomatal development movement. In Arabidopsis, CO2-induced stomatal closure is strongly Ca2+-dependent. To further dissect this signaling pathway and identify new components in the CO2 response pathway, we recorded [Ca2+]cyt changes in mutagenized Arabidopsis leaves and screened for mutants with abnormal guard cell behavior in response to CO2/HCO3 -. RESULTS: We observed that 1 mM HCO3 - induces [Ca2+]cys transient changes in guard cells and stomatal closure both in light and darkness. The changes in [Ca2+]cys induced by HCO3 - could be detected by an aequorin-based calcium imaging system. Using this system, we identified a number of Arabidopsis mutants defective in both [Ca2+]cyt changes and the stomatal response to CO2/HCO3 -. CONCLUSIONS: We provide a sensitive method for isolating stomatal CO2/HCO3 - response genes that function early in stomatal closure and that have a role in regulating [Ca2+]cyt. This method will be helpful in elucidating the Ca2+-dependent regulation of guard cell behavior in response to CO2/HCO3 -.

C-CBL is required for inhibition of angiogenesis through modulating JAK2/STAT3 activity in ROP development.

PURPOSE: The incidence of retinopathy of prematurity (ROP) has increased continuously in recent years. However, the therapeutic effects of current treatments still remain undesired. This study aims to investigate the role of C-CBL in retinal angiogenesis in ROP and its potential as a therapeutic target. METHODS: Mouse retina microvascular endothelial cells (mRMECs) and induced experimental ROP/ oxygen-induced retinopathy (OIR) mice were employed to investigate the role of C-CBL in angiogenesis with combined molecular and cellular approaches, and histopathology methods. OIR mouse pups at postnatal day 12 (P12) were either injected intravitreally with adenovirus overexpressing c-Cbl or c-Cbl siRNA. Retinal neovascularization and avascular status were evaluated by retinal immunofluorescence (IF) staining, whole-mounts and hematoxylin and eosin (H&E) staining. RESULTS: C-CBL inhibits neovascularization by negatively regulating JAK2/STAT3/VEGF signaling axis in a ubiquitination-dependent manner. Knockdown of c-Cbl by siRNA reduced ubiquitin-mediated JAK2 degradation and increased levels of p-JAK2, p-STAT3, VEGF, and neovascularization in mRMECs, which can be reversed by JAK2 inhibitor treatment. While knockdown of c-Cbl significantly increased neovascular (NV) zone in the retinas, c-Cbl overexpression inhibited neovascularization in the retinal tissues in OIR mice. CONCLUSION: We found that C-CBL is required for anti-neovascularization process in ROP development by inhibiting JAK2/STAT3-dependent angiogenesis. Thus, our finding strongly suggest that C-CBL may be a potential novel therapeutic target for treating ROP.