The F-box protein FBXL16 up-regulates the stability of C-MYC oncoprotein by antagonizing the activity of the F-box protein FBW7.

F-box proteins, such as F-box/WD repeat-containing protein 7 (FBW7), are essential components of the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. They bind to S-phase kinase-associated protein 1 (SKP1) through the F-box motif and deliver their protein substrate to the E3 ligase complex for ubiquitination and subsequent degradation. F-box and leucine-rich repeat protein 16 (FBXL16) is a poorly studied F-box protein. Because it does not interact with the scaffold protein cullin 1 (CUL1), we hypothesized that FBXL16 might not form a functional SCF-E3 ligase complex. In the present study, we found that FBXL16 up-regulates the levels of proteins targeted by SCF-E3 ligases, such as C-MYC, ß-catenin, and steroid receptor coactivator 3 (SRC-3). Focusing on C-MYC, a well-known oncoprotein overexpressed in most human cancers, we show that FBXL16 stabilizes C-MYC by antagonizing FBW7-mediated C-MYC ubiquitination and degradation. Further, we found that, although FBXL16 does not interact with CUL1, it interacts with SKP1 via its N-terminal F-box domain and with its substrate C-MYC via its C-terminal leucine-rich repeats (LRRs) domain. We found that both the F-box domain and the LRR domain are important for FBXL16-mediated C-MYC stabilization. In line with its role in up-regulating the levels of the C-MYC and SRC-3 oncoproteins, FBXL16 promoted cancer cell growth and migration and colony formation in soft agar. Our findings reveal that FBXL16 is an F-box protein that antagonizes the activity of another F-box protein, FBW7, and thereby increases C-MYC stability, resulting in increased cancer cell growth and invasiveness.

ERK3 is transcriptionally upregulated by ∆Np63α and mediates the role of ∆Np63α in suppressing cell migration in non-melanoma skin cancers.

BACKGROUND: p63, a member of the p53 gene family, is an important regulator for epithelial tissue growth and development. ∆Np63α is the main isoform of p63 and highly expressed in Non-melanoma skin cancer (NMSC). Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) whose biochemical features and cellular regulation are distinct from those of conventional MAPKs such as ERK1/2. While ERK3 has been shown to be upregulated in lung cancers and head and neck cancers, in which it promotes cancer cell migration and invasion, little is known about the implication of ERK3 in NMSCs. METHODS: Fluorescent immunohistochemistry was performed to evaluate the expression levels of ΔNp63α and ERK3 in normal and NMSC specimens. Dunnett's test was performed to compare mean fluorescence intensity (MFI, indicator of expression levels) of p63 or ERK3 between normal cutaneous samples and NMSC samples. A mixed effects (ANOVA) test was used to determine the correlation between ΔNp63α and ERK3 expression levels (MFI). The regulation of ERK3 by ΔNp63α was studied by qRT-PCR, Western blot and luciferase assay. The effect of ERK3 regulation by ΔNp63α on cell migration was measured by performing trans-well migration assay. RESULTS: The expression level of ∆Np63α is upregulated in NMSCs compared to normal tissue. ERK3 level is significantly upregulated in AK and SCC in comparison to normal tissue and there is a strong positive correlation between ∆Np63α and ERK3 expression in normal skin and skin specimens of patients with AK, SCC or BCC. Further, we found that ∆Np63α positively regulates ERK3 transcript and protein levels in A431 and HaCaT skin cells, underlying the upregulation of ERK3 expression and its positive correlation with ∆Np63α in NMSCs. Moreover, similar to the effect of ∆Np63α depletion, silencing ERK3 greatly enhanced A431 cell migration. Restoration of ERK3 expression under the condition of silencing ∆Np63α counteracted the increase in cell migration induced by the depletion of ∆Np63α. Mechanistically, ERK3 inhibits the phosphorylation of Rac1 G-protein and the formation of filopodia of A431 skin SCC cells. CONCLUSIONS: ERK3 is positively regulated by ∆Np63α and mediates the role of ∆Np63α in suppressing cell migration in NMSC.

The C-Terminus Tail Regulates ERK3 Kinase Activity and Its Ability in Promoting Cancer Cell Migration and Invasion.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family. It harbors a kinase domain in the N-terminus and a long C-terminus extension. The C-terminus extension comprises a conserved in ERK3 and ERK4 (C34) region and a unique C-terminus tail, which was shown to be required for the interaction of ERK3 with the cytoskeletal protein septin 7. Recent studies have elucidated the role of ERK3 signaling in promoting the motility and invasiveness of cancer cells. However, little is known about the intramolecular regulation of the enzymatic activity and cellular functions of ERK3. In this study, we investigated the role of the elongated C-terminus extension in regulating ERK3 kinase activity and its ability to promote cancer cell migration and invasion. Our study revealed that the deletion of the C-terminus tail greatly diminishes the ability of ERK3 to promote the migration and invasion of lung cancer cells. We identified two molecular mechanisms underlying this effect. Firstly, the deletion of the C-terminus tail decreases the kinase activity of ERK3 towards substrates, including the oncogenic protein steroid receptor co-activator 3 (SRC-3), an important downstream target for ERK3 signaling in cancer. Secondly, in line with the previous finding that the C-terminus tail mediates the interaction of ERK3 with septin 7, we found that the depletion of septin 7 abolished the ability of ERK3 to promote migration, indicating that septin 7 acts as a downstream effector for ERK3-induced cancer cell migration. Taken together, the findings of this study advance our understanding of the molecular regulation of ERK3 signaling by unraveling the role of the C-terminus tail in regulating ERK3 kinase activity and functions in cancer cells. These findings provide useful insights for the development of therapeutic agents targeting ERK3 signaling in cancer.

Activation loop phosphorylation of ERK3 is important for its kinase activity and ability to promote lung cancer cell invasiveness.

ERK3 is an atypical mitogen-activated protein kinase (MAPK) that has recently gained interest for its role in promoting cancer cell migration and invasion. However, the molecular regulation of ERK3 functions in cancer cells is largely unknown. ERK3 has a single phospho-acceptor site (Ser189) in its activation motif rather than the TXY conserved in conventional MAPKs such as ERK1/2. Although dual phosphorylation of the TXY motif is known to be critical for the activation of conventional MAPKs, the role of Ser189 phosphorylation in ERK3 activity and its function in cancer cells remain elusive. In this study, we revealed that activation loop phosphorylation is important for ERK3 in promoting cancer cell invasiveness, as the S189A mutation greatly decreased the ability of ERK3 to promote migration and invasion of lung cancer cells. Interestingly, a catalytically inactive ERK3 mutant was still capable of increasing migration and invasion, although to a lesser extent compared with WT ERK3, suggesting that ERK3 promotes cancer cell invasiveness by both kinase-dependent and kinase-independent mechanisms. To elucidate how the S189A mutation reduces the invasiveness-promoting ability of ERK3, we tested its effect on the kinase activity of ERK3 toward steroid receptor coactivator 3 (SRC3), a recently identified substrate of ERK3 critical for cancer cell invasiveness. Compared with ERK3, ERK3-S189A exhibited a dramatic decrease in kinase activity toward SRC3 and a concomitantly reduced ability to stimulate matrix metalloproteinase expression. Taken together, our study unravels the importance of Ser189 phosphorylation for intramolecular regulation of ERK3 kinase activity and invasiveness-promoting ability in lung cancer cells.

The atypical MAPK ERK3 potently suppresses melanoma cell growth and invasiveness.

Mitogen-activated protein kinase 6 (MAPK6) represents an atypical MAPK also known as extracellular signal-regulated kinase 3 (ERK3), which has been shown to play roles in cell motility and metastasis. ERK3 promotes migration and invasion of lung cancer cells and head and neck cancer cells by regulating the expression and/or activity of proteins involved in cancer progression. For instance, ERK3 upregulates matrix metallopeptidases and thereby promotes cancer cell invasiveness, and it phosphorylates tyrosyl-DNA phosphodiesterase 2, thereby enhancing chemoresistance in lung cancer. Here we discovered that ERK3 plays a converse role in melanoma. We observed that BRAF, an oncogenic Ser/Thr kinase, upregulates ERK3 expression levels by increasing both ERK3 messenger RNA levels and protein stability. Interestingly, although BRAF's kinase activity was required for upregulating ERK3 gene transcription, BRAF stabilized ERK3 protein in a kinase-independent fashion. We further demonstrate that ERK3 inhibits the migration, proliferation and colony formation of melanoma cells. In line with this, high level of ERK3 predicted increased survival among patients with melanomas. Taken together, these results indicate that ERK3 acts as a potent suppressor of melanoma cell growth and invasiveness.

SRC-3Delta4 mediates the interaction of EGFR with FAK to promote cell migration.

EGF induces signal transduction between EGFR and FAK, and FAK is required for EGF-induced cell migration. It is unknown, however, what factor mediates the interaction between EGFR and FAK and leads to EGF-induced FAK phosphorylation. Here, we identify SRC-3Delta4, a splicing isoform of the SRC-3 oncogene, as a signaling adaptor that links EGFR and FAK and promotes EGF-induced phosphorylations of FAK and c-Src. We identify three PAK1-mediated phosphorylations in SRC-3Delta4 that promote the localization of SRC-3Delta4 to the plasma membrane and mediate the interactions with EGFR and FAK. Importantly, overexpression of SRC-3Delta4 promotes MDA-MB231-induced breast tumor metastasis. Our findings identify phosphorylated SRC-3Delta4 as a missing adaptor between EGFR and its downstream signaling molecule FAK to coordinately regulate EGF-induced cell migration. Our study also reveals that a nuclear receptor coactivator can act in the periphery of a cell to directly mediate activation of an enzyme.

Site-specific acetylation of the proteasome activator REGγ directs its heptameric structure and functions.

The proteasome activator REGγ has been reported to promote degradation of steroid receptor coactivator-3 and cyclin-dependent kinase inhibitors p21, p16, and p19 in a ubiquitin- and ATP-independent manner. A recent comparative analysis of REGγ expression in mouse and human tissues reveals a unique pattern of REGγ in specific cell types, suggesting undisclosed functions and biological importance of this molecule. Despite the emerging progress made in REGγ-related studies, how REGγ function is regulated remains to be explored. In this study, we report for the first time that REGγ can be acetylated mostly on its lysine 195 (Lys-195) residue by CREB binding protein (CBP), which can be reversed by sirtuin 1 (SIRT1) in mammalian cells. Site-directed mutagenesis abrogated acetylation at Lys-195 and significantly attenuated the capability of REGγ to degrade its target substrates, p21 and hepatitis C virus core protein. Mechanistically, acetylation at Lys-195 is important for the interactions between REGγ monomers and ultimately influences REGγ heptamerization. Biological analysis of cells containing REGγ-WT or REGγ-K195R mutant indicates an impact of acetylation on REGγ-mediated regulation of cell proliferation and cell cycle progression. These findings reveal a previously unknown mechanism in the regulation of REGγ assembly and activity, suggesting a potential venue for the intervention of the ubiquitin-independent REGγ proteasome activity.

ERK3 promotes endothelial cell functions by upregulating SRC-3/SP1-mediated VEGFR2 expression.

Despite a regain of interest recently in ERK3 kinase signaling, the molecular regulations of both ERK3 gene expression and protein kinase activity are still largely unknown. While it is shown that disruption of ERK3 gene causes neonatal lethality, cell type-specific functions of ERK3 signaling remain to be explored. In this study, we report that ERK3 gene expression is upregulated by cytokines through c-Jun in endothelial cells; c-Jun binds to the ERK3 gene and regulates its transcription. We further reveal a new role for ERK3 in regulating endothelial cell migration, proliferation and tube formation by upregulating SRC-3/SP-1-mediated VEGFR2 expression. The underlying molecular mechanism involves ERK3-stimulated formation of a transcriptional complex involving coactivator SRC-3, transcription factor SP-1 and the secondary coactivator CBP. Taken together, our study identified a molecular regulatory mechanism of ERK3 gene expression and revealed a previously unknown role of ERK3 in regulating endothelial cell functions.

FBXL16 promotes cell growth and drug resistance in lung adenocarcinomas with KRAS mutation by stabilizing IRS1 and upregulating IRS1/AKT signaling.

Lung cancer is the leading cause of cancer-related deaths worldwide. Lung adenocarcinomas (LUADs) are a major subtype of non-small-cell lung cancers (NSCLCs). About 25% of LUADs harbor GTPase KRAS mutations associated with poor prognosis and limited treatment options. While encouraging tumor response to novel covalent inhibitors specifically targeting KRASG12C has been shown in the clinic, either intrinsic resistance exists or acquired therapeutic resistance arises upon treatment. There is an unmet need to identify new therapeutic targets for treating LUADs with activating KRAS mutations, particularly those with resistance to KRASG12C inhibitor(s). In this study, we have revealed that F-box/LRR-repeat protein 16 (FBXL16) is selectively upregulated in LUAD with KRAS mutations. It promotes LUAD cell growth and transforms lung epithelial cells. Importantly, FBXL16 depletion greatly enhances sensitivity to the KRASG12C inhibitor (sotorasib) in resistant cells by downregulating phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB; also known as AKT) signaling. Mechanistically, FBXL16 upregulates insulin receptor substrate 1 (IRS1) protein stability, leading to an increase of IGF1/AKT signaling, thereby promoting cell growth and migration. Taken together, our study highlights the potential of FBXL16 as a therapeutic target for treating LUAD with KRAS activating mutations.

Role of the Atypical MAPK ERK3 in Cancer Growth and Progression.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) whose structural and regulatory features are distinct from those of conventional MAPKs, such as ERK1/2. Since its identification in 1991, the regulation, substrates and functions of ERK3 have remained largely unknown. However, recent years have witnessed a wealth of new findings about ERK3 signaling. Several important biological functions for ERK3 have been revealed, including its role in neuronal morphogenesis, inflammation, metabolism, endothelial cell tube formation and epithelial architecture. In addition, ERK3 has been recently shown to play important roles in cancer cell proliferation, migration, invasion and chemoresistance in multiple types of cancers. Furthermore, accumulating studies have uncovered various molecular mechanisms by which the expression level, protein stability and activity of ERK3 are regulated. In particular, several post-translational modifications (PTMs), including ubiquitination, hydroxylation and phosphorylation, have been shown to regulate the stability and activity of ERK3 protein. In this review, we discuss recent findings regarding biochemical and cellular functions of ERK3, with a main focus on its roles in cancers, as well as the molecular mechanisms of regulating its expression and activity.

ERK3 and DGKζ interact to modulate cell motility in lung cancer cells.

Extracellular signal-regulated kinase 3 (ERK3) promotes cell migration and tumor metastasis in multiple cancer types, including lung cancer. The extracellular-regulated kinase 3 protein has a unique structure. In addition to the N-terminal kinase domain, ERK3 includes a central conserved in extracellular-regulated kinase 3 and ERK4 (C34) domain and an extended C-terminus. However, relatively little is known regarding the role(s) of the C34 domain. A yeast two-hybrid assay using extracellular-regulated kinase 3 as bait identified diacylglycerol kinase ζ (DGKζ) as a binding partner. DGKζ was shown to promote migration and invasion in some cancer cell types, but its role in lung cancer cells is yet to be described. The interaction of extracellular-regulated kinase 3 and DGKζ was confirmed by co-immunoprecipitation and in vitro binding assays, consistent with their co-localization at the periphery of lung cancer cells. The C34 domain of ERK3 was sufficient for binding to DGKζ, while extracellular-regulated kinase 3 bound to the N-terminal and C1 domains of DGKζ. Surprisingly, in contrast to extracellular-regulated kinase 3, DGKζ suppresses lung cancer cell migration, suggesting DGKζ might inhibit ERK3-mediated cell motility. Indeed, co-overexpression of exogenous DGKζ and extracellular-regulated kinase 3 completely blocked the ability of ERK3 to promote cell migration, but DGKζ did not affect the migration of cells with stable ERK3 knockdown. Furthermore, DGKζ had little effect on cell migration induced by overexpression of an ERK3 mutant missing the C34 domain, suggesting DGKζ requires this domain to prevent ERK3-mediated increase in cell migration. In summary, this study has identified DGKζ as a new binding partner and negative regulator of extracellular-regulated kinase 3 in controlling lung cancer cell migration.

REGgamma modulates p53 activity by regulating its cellular localization.

The proteasome activator REGγ mediates a shortcut for the destruction of intact mammalian proteins. The biological roles of REGγ and the underlying mechanisms are not fully understood. Here we provide evidence that REGγ regulates cellular distribution of p53 by facilitating its multiple monoubiquitylation and subsequent nuclear export and degradation. We also show that inhibition of p53 tetramerization by REGγ might further enhance cytoplasmic relocation of p53 and reduce active p53 in the nucleus. Furthermore, multiple monoubiquitylation of p53 enhances its physical interaction with HDM2 and probably facilitates subsequent polyubiquitylation of p53, suggesting that monoubiquitylation can act as a signal for p53 degradation. Depletion of REGγ sensitizes cells to stress-induced apoptosis, validating its crucial role in the control of apoptosis, probably through regulation of p53 function. Using a mouse xenograft model, we show that REGγ knockdown results in a significant reduction of tumor growth, suggesting an important role for REGγ in tumor development. Our study therefore demonstrates that REGγ-mediated inactivation of p53 is one of the mechanisms involved in cancer progression.

Conditional ERK3 overexpression cooperates with PTEN deletion to promote lung adenocarcinoma formation in mice.

ERK3, officially known as mitogen-activated protein kinase 6 (MAPK6), is a poorly studied mitogen-activated protein kinase (MAPK). Recent studies have revealed the upregulation of ERK3 expression in cancer and suggest an important role for ERK3 in promoting cancer cell growth and invasion in some cancers, in particular lung cancer. However, it is unknown whether ERK3 plays a role in spontaneous tumorigenesis in vivo. To determine the role of ERK3 in lung tumorigenesis, we created a conditional ERK3 transgenic mouse line in which ERK3 transgene expression is controlled by Cre recombinase. By crossing these transgenic mice with a mouse line harboring a lung tissue-specific Cre recombinase transgene driven by a club cell secretory protein gene promoter (CCSP-iCre), we have found that conditional ERK3 overexpression cooperates with phosphatase and tensin homolog (PTEN) deletion to induce the formation of lung adenocarcinomas (LUADs). Mechanistically, ERK3 overexpression stimulates activating phosphorylations of erb-b2 receptor tyrosine kinases 2 and 3 (ERBB2 and ERBB3) by upregulating Sp1 transcription factor (SP1)-mediated gene transcription of neuregulin 1 (NRG1), a potent ligand for ERBB2/ERBB3. Our study has revealed a bona fide tumor-promoting role for ERK3 using genetically engineered mouse models. Together with previous findings showing the roles of ERK3 in cultured cells and in a xenograft lung tumor model, our findings corroborate that ERK3 acts as an oncoprotein in promoting LUAD development and progression.

A Radioactive in vitro ERK3 Kinase Assay.

Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases that have an important role in signal transduction. Extracellular signal-regulated kinase 3 (ERK3), also known as MAPK6, is an atypical MAPK. Here, we describe in detail an in vitro assay for the kinase activity of ERK3 using myelin basic protein (MBP) or steroid receptor coactivator-3 (SRC-3) as substrates. The assay is carried out in the presence of [y-32P]-ATP which results in radiolabeling of phosphorylated substrates. Separation of the reaction components by gel electrophoresis followed by autoradiography enables detection of the radiolabeled products, and hence determination of the kinase activity of ERK3. This assay can be used for several applications including identification of substrates, determination of the effect of molecules or mutations on kinase activity, and testing specific kinase inhibitors. Furthermore, the protocol outlined here can be adapted to measure the activity of other kinases by using their specific substrates.

ΔNp63α suppresses cells invasion by downregulating PKCγ/Rac1 signaling through miR-320a.

ΔNp63α, a member of the p53 family of transcription factors, is overexpressed in a number of cancers and plays a role in proliferation, differentiation, migration, and invasion. ΔNp63α has been shown to regulate several microRNAs that are involved in development and cancer. We identified miRNA miR-320a as a positively regulated target of ΔNp63α. Previous studies have shown that miR-320a is downregulated in colorectal cancer and targets the small GTPase Rac1, leading to a reduction in noncanonical WNT signaling and EMT, thereby inhibiting tumor metastasis and invasion. We showed that miR-320a is a direct target of ΔNp63α. Knockdown of ΔNp63α in HaCaT and A431 cells downregulates miR-320a levels and leads to a corresponding elevation in PKCγ transcript and protein levels. Rac1 phosphorylation at Ser71 was increased in the absence of ΔNp63α, whereas overexpression of ΔNp63α reversed S71 phosphorylation of Rac1. Moreover, increased PKCγ levels, Rac1 phosphorylation and cell invasion observed upon knockdown of ΔNp63α was reversed by either overexpressing miR-320a mimic or Rac1 silencing. Finally, silencing PKCγ or treatment with the PKC inhibitor Gö6976 reversed increased Rac1 phosphorylation and cell invasion observed upon silencing ΔNp63α. Taken together, our data suggest that ΔNp63α positively regulates miR-320a, thereby inhibiting PKCγ expression, Rac1 phosphorylation, and cancer invasion.

Metabolic Profiling of Different Parts of Acer truncatum from the Mongolian Plateau Using UPLC-QTOF-MS with Comparative Bioactivity Assays.

Acer truncatum is an important ornamental, edible, and medicinal plant resource in China. Previous phytochemical research has focused on the leaf (AL) due to its long history as a tea for health. Other parts such as the branch (ABr), bark (ABa), fruit (AF), and root (AR) have drawn little attention regarding their metabolites and bioactivities. The strategy of an in-house chemical library combined with Progenesis QI informatics platform was applied to characterize the metabolites. A total of 98 compounds were characterized or tentatively identified, including 63 compounds reported from this species for the first time. Principal component analysis showed the close clustering of ABr, ABa, and AR, indicating that they share similar chemical components, while AL and AF clustered more distantly. By multiple orthogonal partial least-squares discriminant analyses (OPLS-DA), 52 compounds were identified as potential marker compounds differentiating these different plant parts. The variable influence on projection score from OPLS-DA revealed that catechin, procyanidins B2 or B3, and procyanidins C1 or C2 are the significant metabolites in ABa extracts, which likely contribute to its antioxidant and cytotoxic activities.

The ERBB4/HER4 intracellular domain 4ICD is a BH3-only protein promoting apoptosis of breast cancer cells.

ERBB4/HER4 (referred to here as ERBB4) is a unique member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. In contrast to the other three members of the EGFR family (i.e., EGFR, ERBB2/HER2/NEU, and ERBB3), which are associated with aggressive forms of human cancers, ERBB4 expression seems to be selectively lost in tumors with aggressive phenotypes. Consistent with this observation, we show that ERBB4 induces apoptosis when reintroduced into breast cancer cell lines or when endogenous ERBB4 is activated by a ligand. We further show that ligand activation and subsequent proteolytic processing of endogenous ERBB4 results in mitochondrial accumulation of the ERBB4 intracellular domain (4ICD) and cytochrome c efflux, the essential and committed step of mitochondrial regulated apoptosis. Our results indicate that 4ICD is functionally similar to BH3-only proteins, proapoptotic members of the BCL-2 family required for initiation of mitochondrial dysfunction through activation of the proapoptotic multi-BH domain proteins BAX/BAK. Similar to other BH3-only proteins, 4ICD cell-killing activity requires an intact BH3 domain and 4ICD interaction with the antiapoptotic protein BCL-2, suppressed 4ICD-induced apoptosis. Unique among BH3-only proteins, however, is the essential requirement of BAK but not BAX to transmit the 4ICD apoptotic signal. Clinically, cytosolic but not membrane ERBB4/4ICD expression in primary human breast tumors was associated with tumor apoptosis, providing a mechanistic explanation for the loss of ERBB4 expression during tumor progression. Thus, we propose that ligand-induced mitochondrial accumulation of 4ICD represents a unique mechanism of action for transmembrane receptors, directly coupling a cell surface signal to the tumor cell mitochondrial apoptotic pathway.

Liver kinase B1 restoration promotes exosome secretion and motility of lung cancer cells.

Liver kinase B1 (LKB1) regulates a variety of cellular functions, including cell polarity, energy metabolism and cell growth, by targeting multiple signaling pathways such as AMPK/mTOR and p53. LKB1 functions as a tumor suppressor in sporadic cancers including lung cancer. Extracellular vesicles such as exosomes secreted by cancer cells modulate the tumor microenvironment and progression by targeting both tumor cells (autocrine actions) and other types of cells associated with tumors (paracrine actions). While the roles of LKB1 in cellular signaling in general is well-studied, its specific role in exosome-mediated signaling remains to be explored. To this purpose, we reintroduced LKB1 into H460 and A549 lung cancer cells that are endogenously deficient in LKB1 expression. Notably, we found that while restoration of LKB1 significantly reduced lung cancer cell growth as expected, it greatly promoted cell motility and enhanced the release of exosomes. In addition, exosomes isolated from H460 cells with stable restoration of LKB1 had much higher ability in stimulating lung cancer cell migration than did those from H460 cells lacking LKB1. Mechanistically, restoration of LKB1 in H460 cells inhibited cellular expression and exosomal secretion of migration-suppressing microRNAs (miRNAs), including miR-125a, miR-126 and let7b. Taken together, the present study revealed a new role for LKB1 in promoting cell motility by downregulating migration-suppressing miRNA expression and exosome secretion.

Regulation of Pathogenic T Helper 17 Cell Differentiation by Steroid Receptor Coactivator-3.

T helper 17 (Th17) cell development is programmed by the orphan nuclear receptor RORγt, but the underlying mechanism is not well understood. Nuclear receptor-mediated transcriptional activation depends on coactivators. Here, we show that steroid receptor coactivator-3 (SRC-3) critically regulates Th17 cell differentiation. Reduced incidence of experimental autoimmune encephalitis (EAE) associated with decreased Th17 cell generation in vivo was observed in mice with SRC-3 deletion specifically in T cells. In vitro, SRC-3 deficiency did not affect TGF-ß/IL-6-induced Th17 cell generation but severely impaired pathogenic Th17 differentiation induced by IL-1/IL-6/IL-23. Microarray analysis revealed that SRC-3 not only regulates IL-17A but also IL-1R1 expression. SRC-3 bound to Il17a and Il1r1 loci in a RORγt-dependent manner and was required for recruitment of the p300 acetyltransferase. Thus, SRC-3 is critical for RORγt-dependent gene expression in Th17 cell-driven autoimmune diseases.

L290P/V mutations increase ERK3's cytoplasmic localization and migration/invasion-promoting capability in cancer cells.

Protein kinases are frequently mutated in human cancers, which leads to altered signaling pathways and contributes to tumor growth and progression. ERK3 is an atypical mitogen-activated protein kinase (MAPK) containing an S-E-G activation motif rather than the conserved T-X-Y motif in conventional MAPKs such as ERK1/2. Recent studies have revealed important roles for ERK3 in cancers. ERK3 promotes cancer cell migration/invasion and tumor metastasis, and its expression is upregulated in multiple cancers. Little is known, however, regarding ERK3 mutations in cancers. In the present study, we functionally and mechanistically characterized ERK3 L290P/V mutations, which are located within ERK3's kinase domain, and are shown to exist in several cancers including lung cancer and colon cancer. We found that in comparison with wild type ERK3, both L290P and L290V mutants have greatly increased activity in promoting cancer cell migration and invasion, but have little impact on ERK3's role in cell proliferation. Mechanistically, while they have no clear effect on kinase activity, L290P/V mutations enhance ERK3's cytoplasmic localization by increasing the interaction with the nuclear export factor CRM1. Our findings suggest that L290P/V mutations of ERK3 may confer increased invasiveness to cancers.