TRAF3-EWSR1 signaling axis acts as a checkpoint on germinal center responses.

The formation of germinal centers (GCs) is crucial for humoral immunity and vaccine efficacy. Constant stimulation through microbiota drives the formation of constitutive GCs in Peyer's patches (PPs), which generate B cells that produce antibodies against gut antigens derived from commensal bacteria and infectious pathogens. However, the molecular mechanism that regulates this persistent process is poorly understood. We report that Ewing Sarcoma Breakpoint Region 1 (EWSR1) is a brake to constitutive GC generation and immunoglobulin G (IgG) production in PPs, vaccination-induced GC formation, and IgG responses. Mechanistically, EWSR1 suppresses Bcl6 upregulation after antigen encounter, thereby negatively regulating induced GC B cell generation and IgG production. We further showed that tumor necrosis factor receptor-associated factor (TRAF) 3 serves as a negative regulator of EWSR1. These results established that the TRAF3-EWSR1 signaling axis acts as a checkpoint for Bcl6 expression and GC responses, indicating that this axis is a therapeutic target to tune GC responses and humoral immunity in infectious diseases.

Dapl1 controls NFATc2 activation to regulate CD8+ T cell exhaustion and responses in chronic infection and cancer.

CD8+ T cells are central mediators of immune responses against infections and cancer. Here we identified Dapl1 as a crucial regulator of CD8+ T cell responses to cancer and infections. Dapl1 deficiency promotes the expansion of tumour-infiltrating effector memory-like CD8+ T cells and prevents their functional exhaustion, coupled with increased antitumour immunity and improved efficacy of adoptive T cell therapy. Dapl1 controls activation of NFATc2, a transcription factor required for the effector function of CD8+ T cells. Although NFATc2 mediates induction of the immune checkpoint receptor Tim3, competent NFATc2 activation prevents functional exhaustion of CD8+ T cells. Interestingly, exhausted CD8+ T cells display attenuated NFATc2 activation due to Tim3-mediated feedback inhibition; Dapl1 deletion rescues NFATc2 activation and thereby prevents dysfunction of exhausted CD8+ T cells in chronic infection and cancer. These findings establish Dapl1 as a crucial regulator of CD8+ T cell immunity and a potential target for cancer immunotherapy.

Matriptase-2/NR4A3 axis switches TGF-ß action toward suppression of prostate cancer cell invasion, tumor growth, and metastasis.

Dysregulation of pericellular proteolysis is strongly implicated in cancer metastasis through alteration of cell invasion and the microenvironment. Matriptase-2 (MT-2) is a membrane-anchored serine protease which can suppress prostate cancer (PCa) cell invasion. In this study, we showed that MT-2 was down-regulated in PCa and could suppress PCa cell motility, tumor growth, and metastasis. Using microarray and biochemical analysis, we found that MT-2 shifted TGF-ß action towards its tumor suppressor function by repressing epithelial-to-mesenchymal transition (EMT) and promoting Smad2 phosphorylation and nuclear accumulation to upregulate two TGF-ß1 downstream effectors (p21 and PAI-1), culminating in hindrance of PCa cell motility and malignant growth. Mechanistically, MT-2 could dramatically up-regulate the expression of nuclear receptor NR4A3 via iron metabolism in PCa cells. MT-2-induced NR4A3 further coactivated Smad2 to activate p21 and PAI-1 expression. In addition, NR4A3 functioned as a suppressor of PCa and mediated MT-2 signaling to inhibit PCa tumorigenesis and metastasis. These results together indicate that NR4A3 sustains MT-2 signaling to suppress PCa cell invasion, tumor growth, and metastasis, and serves as a contextual factor for the TGF-ß/Smad2 signaling pathway in favor of tumor suppression via promoting p21 and PAI-1 expression.

DYRK1a mediates BAFF-induced noncanonical NF-κB activation to promote autoimmunity and B-cell leukemogenesis.

B-cell-activating factor (BAFF) mediates B-cell survival and, when deregulated, contributes to autoimmune diseases and B-cell malignancies. The mechanism connecting BAFF receptor (BAFFR) signal to downstream pathways and pathophysiological functions is not well understood. Here we identified DYRK1a as a kinase that responds to BAFF stimulation and mediates BAFF-induced B-cell survival. B-cell-specific DYRK1a deficiency causes peripheral B-cell reduction and ameliorates autoimmunity in a mouse model of lupus. An unbiased screen identified DYRK1a as a protein that interacts with TRAF3, a ubiquitin ligase component mediating degradation of the noncanonical nuclear factor (NF)-κB-inducing kinase (NIK). DYRK1a phosphorylates TRAF3 at serine-29 to interfere with its function in mediating NIK degradation, thereby facilitating BAFF-induced NIK accumulation and noncanonical NF-κB activation. Interestingly, B-cell acute lymphoblastic leukemia (B-ALL) cells express high levels of BAFFR and respond to BAFF for noncanonical NF-κB activation and survival in a DYRK1a-dependent manner. Furthermore, DYRK1a promotes a mouse model of B-ALL through activation of the noncanonical NF-κB pathway. These results establish DYRK1a as a critical BAFFR signaling mediator and provide novel insight into B-ALL pathogenesis.

Afatinib Exerts Immunomodulatory Effects by Targeting the Pyrimidine Biosynthesis Enzyme CAD.

Current clinical trials of combined EGFR-tyrosine kinase inhibitors (TKI) and immune checkpoint blockade (ICB) therapies show no additional effect. This raises questions regarding whether EGFR-TKIs attenuate ICB-enhanced CD8+ T lymphocyte function. Here we show that the EGFR-TKI afatinib suppresses CD8+ T lymphocyte proliferation, and we identify CAD, a key enzyme of de novo pyrimidine biosynthesis, to be a novel afatinib target. Afatinib reduced tumor-infiltrating lymphocyte numbers in Lewis lung carcinoma (LLC)-bearing mice. Early afatinib treatment inhibited CD8+ T lymphocyte proliferation in patients with non-small cell lung cancer, but their proliferation unexpectedly rebounded following long-term treatment. This suggests a transient immunomodulatory effect of afatinib on CD8+ T lymphocytes. Sequential treatment of afatinib with anti-PD1 immunotherapy substantially enhanced therapeutic efficacy in MC38 and LLC-bearing mice, while simultaneous combination therapy showed only marginal improvement over each single treatment. These results suggest that afatinib can suppress CD8+ T lymphocyte proliferation by targeting CAD, proposing a timing window for combined therapy that may prevent the dampening of ICB efficacy by EGFR-TKIs. SIGNIFICANCE: This study elucidates a mechanism of afatinib-mediated immunosuppression and provides new insights into treatment timing for combined targeted therapy and immunotherapy. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3270/F1.large.jpg.

The E3 ubiquitin ligase Peli1 regulates the metabolic actions of mTORC1 to suppress antitumor T cell responses.

Metabolic fitness of T cells is crucial for immune responses against infections and tumorigenesis. Both the T cell receptor (TCR) signal and environmental cues contribute to the induction of T cell metabolic reprogramming, but the underlying mechanism is incompletely understood. Here, we identified the E3 ubiquitin ligase Peli1 as an important regulator of T cell metabolism and antitumor immunity. Peli1 ablation profoundly promotes tumor rejection, associated with increased tumor-infiltrating CD4 and CD8 T cells. The Peli1-deficient T cells display markedly stronger metabolic activities, particularly glycolysis, than wild-type T cells. Peli1 controls the activation of a metabolic kinase, mTORC1, stimulated by both the TCR signal and growth factors, and this function of Peli1 is mediated through regulation of the mTORC1-inhibitory proteins, TSC1 and TSC2. Peli1 mediates non-degradative ubiquitination of TSC1, thereby promoting TSC1-TSC2 dimerization and TSC2 stabilization. These results establish Peli1 as a novel regulator of mTORC1 and downstream mTORC1-mediated actions on T cell metabolism and antitumor immunity.

Intestinal Epithelial TBK1 Prevents Differentiation of T-helper 17 Cells and Tumorigenesis in Mice.

BACKGROUND & AIMS: Intestinal epithelial cells (IECs) regulate intestinal immune cells, particularly development of T-helper 17 (Th17) cells. Deregulation of this process leads to intestinal inflammation and tumorigenesis, via unknown mechanisms. TANK-binding kinase 1 (TBK1) is expressed by IECs and cells in the innate immune system. We studied the functions of TBK1 in the intestinal immune response and tumorigenesis in mice. METHODS: We performed studies of wild-type mice, mice with conditional disruption of Tbk1 (Tbk1IEC-KO), Tbk1IEC-KO mice crossed with ApcMin/+ mice, and Mt-/- mice crossed with ApcMin/+ mice. Some mice were given intraperitoneal injections of a neutralizing antibody against interleukin 17 (IL17) or IL1ß. Intestine tissues were collected from mice and analyzed by histology, for numbers of adenomas and Th17 cells, and expression of inflammatory cytokines by real-time PCR. IECs were isolated from wild-type and Tbk1IEC-KO mice, stimulated with lipopolysaccharide, co-cultured for with bone marrow-derived macrophages, and analyzed by RNA sequencing and biochemical analyses. RESULTS: Compared to ApcMin/+Tbk1WT mice, ApcMin/+Tbk1IEC-KO mice had significant increases in number and size of intestinal polyps, and significantly more Th17 cells in lamina propria. Administration of an antibody against IL17 reduced the number of intestinal polyps in ApcMin/+Tbk1IEC-KO mice to that observed in ApcMin/+Tbk1WT mice. In culture, TBK1-deficient IECs promoted expression of IL1ß by macrophages, which induced differentiation of naïve CD4+ T cells into Th17 cells. RNA sequencing analysis revealed that the TBK1-deficient IECs had increased expression of metallothionein 1 (MT1), an immune regulator that promotes intestinal inflammation. Intestine tissues from ApcMin/+Mt-/- mice had significant fewer Th17 cells than ApcMin/+Mt+/+ mice, and a significantly lower number of polyps. Analyses of colorectal tumors in the Cancer Genome Atlas found colorectal tumors with high levels of MT1 and IL17 mRNAs to be associated with reduced survival times of patients. CONCLUSIONS: Expression of TBK1 by IECs suppresses expression of MT1 and prevents expression of IL1ß by macrophages and differentiation of Th17 cells, to prevent inflammation and tumorigenesis. Strategies to block this pathway might be developed for colorectal tumorigenesis.

Inhibition of TMPRSS2 by HAI-2 reduces prostate cancer cell invasion and metastasis.

TMPRSS2 is an important membrane-anchored serine protease involved in human prostate cancer progression and metastasis. A serine protease physiologically often comes together with a cognate inhibitor for execution of proteolytically biologic function; however, TMPRSS2's cognate inhibitor is still elusive. To identify the cognate inhibitor of TMPRSS2, in this study, we applied co-immunoprecipitation and LC/MS/MS analysis and isolated hepatocyte growth factor activator inhibitors (HAIs) to be potential inhibitor candidates for TMPRSS2. Moreover, the recombinant HAI-2 proteins exhibited a better inhibitory effect on TMPRSS2 proteolytic activity than HAI-1, and recombinant HAI-2 proteins had a high affinity to form a complex with TMPRSS2. The immunofluorescence images further showed that TMPRSS2 was co-localized to HAI-2. Both KD1 and KD2 domain of HAI-2 showed comparable inhibitory effects on TMPRSS2 proteolytic activity. In addition, HAI-2 overexpression could suppress the induction effect of TMPRSS2 on pro-HGF activation, extracellular matrix degradation and prostate cancer cell invasion. We further determined that the expression levels of TMPRSS2 were inversely correlated with HAI-2 levels during prostate cancer progression. In orthotopic xenograft animal model, TMPRSS2 overexpression promoted prostate cancer metastasis, and HAI-2 overexpression efficiently blocked TMPRSS2-induced metastasis. In summary, the results together indicate that HAI-2 can function as a cognate inhibitor for TMPRSS2 in human prostate cancer cells and may serve as a potential factor to suppress TMPRSS2-mediated malignancy.

Author Correction: The Kunitz Domain I of Hepatocyte Growth Factor Activator Inhibitor-2 Inhibits Matriptase Activity and Invasive Ability of Human Prostate Cancer Cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

TBKBP1 and TBK1 form a growth factor signalling axis mediating immunosuppression and tumourigenesis.

TANK-binding kinase 1 (TBK1) responds to microbial stimuli and mediates the induction of type I interferon (IFN). Here, we show that TBK1 is also a central mediator of growth factor signalling; this function of TBK1 relies on a specific adaptor-TBK-binding protein 1 (TBKBP1). TBKBP1 recruits TBK1 to protein kinase C-theta (PKCθ) through a scaffold protein, CARD10. This enables PKCθ to phosphorylate TBK1 at Ser 716, a crucial step for TBK1 activation by growth factors but not by innate immune stimuli. Although the TBK1-TBKBP1 signalling axis is not required for the induction of type I IFN, it mediates mTORC1 activation and oncogenesis. Conditional deletion of either TBK1 or TBKBP1 in lung epithelial cells inhibits tumourigenesis in a mouse model of lung cancer. In addition to promoting tumour growth, the TBK1-TBKBP1 axis facilitates tumour-mediated immunosuppression through a mechanism that involves induction of the checkpoint molecule PD-L1 and stimulation of glycolysis. These findings suggest a PKCθ-TBKBP1-TBK1 growth factor signalling axis that mediates both tumour growth and immunosuppression.

Activation of sphingosine kinase by lipopolysaccharide promotes prostate cancer cell invasion and metastasis via SphK1/S1PR4/matriptase.

Gram-negative bacteria have been found to be a major population in prostatitis and prostate cancer (PCa) tissues. Lipopolysaccharide (LPS), a major compound of Gram-negative bacteria, with stimulatory activities in some cancer types, but has not been fully studied in PCa. In this study, we examined the effect of LPS on the invasion of PCa cells. Interestingly, LPS can enhance the invasiveness of PCa, but had no significant effect on PCa cell viability. Using protease inhibitor screening and biochemical analyses, matriptase, a member of the membrane-anchored serine protease family, is found to play a key role in LPS-induced PCa cell invasion. Mechanistically, Toll-like receptor 4 (TLR4, LPS receptor)-sphingosine kinase 1 (SphK1) signaling underlies LPS-induced matriptase activation and PCa cell invasion. Specifically, LPS induced the S225 phosphorylation of SphK1 and the translocation of SphK1 to plasma membrane, leading to the production of sphingosine 1-phosphate (S1P), ERK1/2 and matriptase activation via S1P receptor 4 (S1PR4). This phenomenon is further validated using the patient-derived explant (PDE) model. Indeed, there is a significant correlation among the elevated SphK1 levels, the Gleason grades of PCa specimens, and the poor survival of PCa patients. Taken together, this study demonstrates a potential impact of LPS on PCa progression. Our results provide not only a new finding of the role of bacterial infection in PCa progression but also potential therapeutic target(s) associated with PCa metastasis.

HAI-2 as a novel inhibitor of plasmin represses lung cancer cell invasion and metastasis.

BACKGROUND: Dysregulation of pericellular proteolysis usually accounts for cancer cell invasion and metastasis. Isolation of a cell-surface protease system for lung cancer metastasis is an important issue for mechanistic studies and therapeutic target identification. METHODS: Immunohistochemistry of a tissue array (n = 64) and TCGA database (n = 255) were employed to assess the correlation between serine protease inhibitors (SPIs) and lung adenocarcinoma progression. The role of SPI in cell motility was examined using transwell assays. Pulldown and LC/MS/MS were performed to identify the SPI-modulated novel protease(s). A xenografted mouse model was harnessed to demonstrate the role of the SPI in lung cancer metastasis. RESULTS: Hepatocyte growth factor activator inhibitor-2 (HAI-2) was identified to be downregulated following lung cancer progression, which was related to poor survival and tumour invasion. We further isolated a serum-derived serine protease, plasmin, to be a novel target of HAI-2. Downregulation of HAI-2 promotes cell surface plasmin activity, EMT, and cell motility. HAI-2 can suppress plasmin-mediated activations of HGF and TGF-ß1, EMT and cell invasion. In addition, downregulated HAI-2 increased metastasis of lung adenocarcinoma via upregulating plasmin activity. CONCLUSION: HAI-2 functions as a novel inhibitor of plasmin to suppress lung cancer cell motility, EMT and metastasis.

The Kunitz Domain I of Hepatocyte Growth Factor Activator Inhibitor-2 Inhibits Matriptase Activity and Invasive Ability of Human Prostate Cancer Cells.

Dysregulation of pericellular proteolysis is often required for tumor invasion and cancer progression. It has been shown that down-regulation of hepatocyte growth factor activator inhibitor-2 (HAI-2) results in activation of matriptase (a membrane-anchored serine protease), human prostate cancer cell motility and tumor growth. In this study, we further characterized if HAI-2 was a cognate inhibitor for matriptase and identified which Kunitz domain of HAI-2 was required for inhibiting matriptase and human prostate cancer cell motility. Our results show that HAI-2 overexpression suppressed matriptase-induced prostate cancer cell motility. We demonstrate that HAI-2 interacts with matriptase on cell surface and inhibits matriptase proteolytic activity. Moreover, cellular HAI-2 harnesses its Kunitz domain 1 (KD1) to inhibit matriptase activation and prostate cancer cell motility although recombinant KD1 and KD2 of HAI-2 both show an inhibitory activity and interaction with matriptase protease domain. The results together indicate that HAI-2 is a cognate inhibitor of matriptase, and KD1 of HAI-2 plays a major role in the inhibition of cellular matritptase activation as well as human prostate cancer invasion.

Androgen-Induced TMPRSS2 Activates Matriptase and Promotes Extracellular Matrix Degradation, Prostate Cancer Cell Invasion, Tumor Growth, and Metastasis.

Dysregulation of androgen signaling and pericellular proteolysis is necessary for prostate cancer progression, but the links between them are still obscure. In this study, we show how the membrane-anchored serine protease TMPRSS2 stimulates a proteolytic cascade that mediates androgen-induced prostate cancer cell invasion, tumor growth, and metastasis. We found that matriptase serves as a substrate for TMPRSS2 in mediating this proinvasive action of androgens in prostate cancer. Further, we determined that higher levels of TMPRSS2 expression correlate with higher levels of matriptase activation in prostate cancer tissues. Lastly, we found that the ability of TMPRSS2 to promote prostate cancer tumor growth and metastasis was associated with increased matriptase activation and enhanced degradation of extracellular matrix nidogen-1 and laminin ß1 in tumor xenografts. In summary, our results establish that TMPRSS2 promotes the growth, invasion, and metastasis of prostate cancer cells via matriptase activation and extracellular matrix disruption, with implications to target these two proteases as a strategy to treat prostate cancer.

Curcumin-targeting pericellular serine protease matriptase role in suppression of prostate cancer cell invasion, tumor growth, and metastasis.

Curcumin has been shown to possess potent chemopreventive and antitumor effects on prostate cancer. However, the molecular mechanism involved in curcumin's ability to suppress prostate cancer cell invasion, tumor growth, and metastasis is not yet well understood. In this study, we have shown that curcumin can suppress epidermal growth factor (EGF)- stimulated and heregulin-stimulated PC-3 cell invasion, as well as androgen-induced LNCaP cell invasion. Curcumin treatment significantly resulted in reduced matrix metalloproteinase 9 activity and downregulation of cellular matriptase, a membrane-anchored serine protease with oncogenic roles in tumor formation and invasion. Our data further show that curcumin is able to inhibit the induction effects of androgens and EGF on matriptase activation, as well as to reduce the activated levels of matriptase after its overexpression, thus suggesting that curcumin may interrupt diverse signal pathways to block the protease. Furthermore, the reduction of activated matriptase in cells by curcumin was also partly due to curcumin's effect on promoting the shedding of matriptase into an extracellular environment, but not via altering matriptase gene expression. In addition, curcumin significantly suppressed the invasive ability of prostate cancer cells induced by matriptase overexpression. In xenograft model, curcumin not only inhibits prostate cancer tumor growth and metastasis but also downregulates matriptase activity in vivo. Overall, the data indicate that curcumin exhibits a suppressive effect on prostate cancer cell invasion, tumor growth, and metastasis, at least in part via downregulating matriptase function.

Matriptase is involved in ErbB-2-induced prostate cancer cell invasion.

Deregulation of both ErbB-2 signaling and matriptase activity has been associated with human prostate cancer (PCa) progression. In this communication, we investigated the roles of both ErbB-2 signaling in matriptase zymogen activation and matriptase in ErbB-2-induced PCa malignancy. In a human PCa cell progression model, we observed that advanced PCa C-81 LNCaP cells exhibited an aggressive phenotype with increased cell migration and invasion capacity; these cells concurrently showed both enhanced ErbB-2 phosphorylation and increased matriptase zymogen activation compared with parental C-33 LNCaP cells. Moreover, ErbB2 activation, both ligand-dependent (eg, epidermal growth factor treatment) and ligand-independent (eg, overexpression), was able to induce matriptase zymogen activation in this cell line. Inhibition of ErbB-2 activity by either the specific inhibitor, AG825, in epidermal growth factor-treated C-33 LNCaP cells or ErbB-2 knockdown in C-81 LNCaP cells, reduced matriptase activation. These observations were confirmed by similar studies using both DU145 and PC3 cells. Together, these data suggest that ErbB-2 signaling plays an important role in matriptase zymogen activation. ErbB-2-enhanced matriptase activation was suppressed by a phosphatidylinositol 3-kinase inhibitor (ie, LY294002) but not by a MEK inhibitor (ie, PD98059). Suppression of matriptase expression by small hairpin RNA knockdown in ErbB-2-overexpressing LNCaP cells dramatically suppressed cancer cell invasion. In summary, our data indicate that ErbB-2 signaling via the phosphatidylinositol 3-kinase pathway results in up-regulated matriptase zymogen activity, which contributes to PCa cell invasion.