Potential of targeting signal-transducing adaptor protein-2 in cancer therapeutic applications.

Adaptor proteins play essential roles in various intracellular signaling pathways. Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein that possesses pleckstrin homology (PH) and Src homology 2 (SH2) domains, as well as a YXXQ signal transducer and activator of transcription 3 (STAT3)-binding motif in its C-terminal region. STAP-2 is also a substrate of breast tumor kinase (BRK). STAP-2/BRK expression is deregulated in breast cancers and enhances STAT3-dependent cell proliferation. In prostate cancer cells, STAP-2 interacts with and stabilizes epidermal growth factor receptor (EGFR) after stimulation, resulting in the upregulation of EGFR signaling, which contributes to cancer-cell proliferation and tumor progression. Therefore, inhibition of the interaction between STAP-2 and BRK/EGFR may be a possible therapeutic strategy for these cancers. For this purpose, peptides that interfere with STAP-2/BRK/EGFR binding may have great potential. Indeed, the identified peptide inhibitor successfully suppressed the STAP-2/EGFR protein interaction, EGFR stabilization, and cancer-cell growth. Furthermore, the peptide inhibitor suppressed tumor formation in human prostate- and lung-cancer cell lines in a murine xenograft model. This review focuses on the inhibitory peptide as a promising candidate for the treatment of prostate and lung cancers.

[Multiple myeloma with IgH::MYC and multiple extramedullary lesions].

A 41-year-old woman with right shoulder pain was found to have multiple tumors with osteolysis and M-proteinemia. Abnormal plasma cells (CD38+, CD138+, Igλ≫κ) were detected in 1.4% of bone marrow nucleated cells, and G-banding analysis revealed a 46,XX,t (8;14), (q24;q32) karyotype in 4 of 20 cells analyzed. A biopsy specimen from an extramedullary lesion had a packed proliferation of aberrant plasmacytoid cells with positive IgH::MYC fusion signals on fluorescence in situ hybridization. The patient was diagnosed with symptomatic multiple myeloma and treated with the BLd regimen, which significantly reduced M protein levels. Extramedullary lesions were initially reduced, but increased again after four cycles. The lesions disappeared with subsequent EPOCH chemotherapy and radiation, and complete remission was confirmed. The patient was then treated with high-dose chemotherapy with autologous peripheral blood stem cell transplantation. Complete remission was maintained for over one year with lenalidomide maintenance therapy. A solitary IgH::MYC chromosomal translocation is extremely rare in multiple myeloma and may be associated with high tumor proliferative capacity, multiple extramedullary lesions, and poor prognosis. Combined therapeutic modalities with novel and conventional chemotherapy and radiation might be a promising treatment strategy for patients with this type of multiple myeloma.

STAP-2 facilitates insulin signaling through binding to CAP/c-Cbl and regulates adipocyte differentiation.

Signal-transducing adaptor protein-2 (STAP-2) is an adaptor molecule involved in several cellular signaling cascades. Here, we attempted to identify novel STAP-2 interacting molecules, and identified c-Cbl associated protein (CAP) as a binding protein through the C-terminal proline-rich region of STAP-2. Expression of STAP-2 increased the interaction between CAP and c-Cbl, suggesting that STAP-2 bridges these proteins and enhances complex formation. CAP/c-Cbl complex is known to regulate GLUT4 translocation in insulin signaling. STAP-2 overexpressed human hepatocyte Hep3B cells showed enhanced GLUT4 translocation after insulin treatment. Elevated levels of Stap2 mRNA have been observed in 3T3-L1 cells and mouse embryonic fibroblasts (MEFs) during adipocyte differentiation. The differentiation of 3T3-L1 cells into adipocytes was highly promoted by retroviral overexpression of STAP-2. In contrast, STAP-2 knockout (KO) MEFs exhibited suppressed adipogenesis. The increase in body weight with high-fat diet feeding was significantly decreased in STAP-2 KO mice compared to WT animals. These data suggest that the expression of STAP-2 correlates with adipogenesis. Thus, STAP-2 is a novel regulatory molecule that controls insulin signal transduction by forming a c-Cbl/STAP-2/CAP ternary complex.

Role of Signal-Transducing Adaptor Protein-1 for T Cell Activation and Pathogenesis of Autoimmune Demyelination and Airway Inflammation.

Signal-transducing adaptor protein (STAP)-1 is an adaptor protein that is widely expressed in T cells. In this article, we show that STAP-1 upregulates TCR-mediated T cell activation and T cell-mediated airway inflammation. Using STAP-1 knockout mice and STAP-1-overexpressing Jurkat cells, we found that STAP-1 enhanced TCR signaling, resulting in increased calcium mobilization, NFAT activity, and IL-2 production. Upon TCR engagement, STAP-1 binding to ITK promoted formation of ITK-LCK and ITK-phospholipase Cγ1 complexes to induce downstream signaling. Consistent with the results, STAP-1 deficiency reduced the severity of symptoms in experimental autoimmune encephalomyelitis. Single-cell RNA-sequencing analysis revealed that STAP-1 is essential for accumulation of T cells and Ifng and Il17 expression in spinal cords after experimental autoimmune encephalomyelitis induction. Th1 and Th17 development was also attenuated in STAP-1 knockout naive T cells. Taken together, STAP-1 enhances TCR signaling and plays a role in T cell-mediated immune disorders.

STAP-2 negatively regulates BCR-mediated B cell activation by recruiting tyrosine-protein kinase CSK to LYN.

Although signal-transducing adaptor protein-2 (STAP-2) acts in certain immune responses, its role in B cell receptor (BCR)-mediated signals remains unknown. In this study, we have revealed that BCR-mediated signals, cytokine production and antibody production were increased in STAP-2 knockout (KO) mice compared with wild-type (WT) mice. Phosphorylation of tyrosine-protein kinase LYN Y508 was reduced in STAP-2 KO B cells after BCR stimulation. Mechanistic analysis revealed that STAP-2 directly binds to LYN, dependently of STAP-2 Y250 phosphorylation by LYN. Furthermore, phosphorylation of STAP-2 enhanced interactions between LYN and tyrosine-protein kinase CSK, resulting in enhanced CSK-mediated LYN Y508 phosphorylation. These results suggest that STAP-2 is crucial for controlling BCR-mediated signals and antibody production by enhanced CSK-mediated feedback regulation of LYN.

The GPR84 molecule is a mediator of a subpopulation of retinal microglia that promote TNF/IL-1α expression via the rho-ROCK pathway after optic nerve injury.

Resident microglia are important to maintain homeostasis in the central nervous system, which includes the retina. The retinal microglia become activated in numerous pathological conditions, but the molecular signatures of these changes are poorly understood. Here, using an approach based on FACS and RNA-seq, we show that microglial gene expression patterns gradually change during RGC degeneration induced by optic nerve injury. Most importantly, we found that the microglial cells strongly expressed Tnf and Il1α, both of which are known to induce neurotoxic reactive astrocytes, and were characterized by Gpr84high -expressing cells in a particular subpopulation. Moreover, ripasudil, a Rho kinase inhibitor, significantly blunted Gpr84 expression and cytokine induction in vitro and in vivo. Finally, GPR84-deficient mice prevented RGC loss in optic nerve-injured retina. These results reveal that Rho kinase-mediated GPR84 alteration strongly contribute to microglial activation and promote neurotoxicity, suggesting that Rho-ROCK and GPR84 signaling may be potential therapeutic targets to prevent the neurotoxic microglial phenotype induced by optic nerve damage, such as occurs in traumatic optic neuropathy and glaucoma.

STAP-2-Derived Peptide Suppresses TCR-Mediated Signals to Initiate Immune Responses.

Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein that contains pleckstrin and Src homology 2-like domains, as well as a proline-rich region in its C-terminal region. Our previous study demonstrated that STAP-2 positively regulates TCR signaling by associating with TCR-proximal CD3ζ ITAMs and the lymphocyte-specific protein tyrosine kinase. In this study, we identify the STAP-2 interacting regions of CD3ζ ITAMs and show that the STAP-2-derived synthetic peptide (iSP2) directly interacts with the ITAM sequence and blocks the interactions between STAP-2 and CD3ζ ITAMs. Cell-penetrating iSP2 was delivered into human and murine T cells. iSP2 suppressed cell proliferation and TCR-induced IL-2 production. Importantly, iSP2 treatment suppressed TCR-mediated activation of naive CD4+ T cells and decreased immune responses in CD4+ T cell-mediated experimental autoimmune encephalomyelitis. It is likely that iSP2 is a novel immunomodulatory tool that modulates STAP-2-mediated activation of TCR signaling and represses the progression of autoimmune diseases.

Comparison between concentric-only, eccentric-only, and concentric-eccentric resistance training of the elbow flexors for their effects on muscle strength and hypertrophy.

PURPOSE: This study compared concentric-eccentric coupled (CON-ECC), concentric-only (CON), and eccentric-only (ECC) resistance training of the elbow flexors for their effects on muscle strength and hypertrophy. METHODS: Non-resistance-trained young adults were assigned to one of the four groups: CON-ECC (n = 14), CON (n = 14) and ECC (n = 14) training groups, and a control group (n = 11) that had measurements only. The training group participants performed dominant arm elbow flexor resistance training in extended elbow joint angles (0°-50°) twice a week for 5 weeks. The total training volume (dumbbell weight × number of contractions) in CON-ECC (5745 ± 1020 kg) was double of that in CON (2930 ± 859 kg) and ECC (3035 ± 844 kg), because 3 sets of 10 contractions were performed for both directions in CON-ECC. Maximum voluntary isometric (MVC-ISO), concentric (MVC-CON), and eccentric contraction (MVC-ECC) torque of the elbow flexors and biceps brachii and brachialis muscle thickness (MT) were measured at baseline, and 3-9 days post-last training session. RESULTS: No significant changes in any measures were evident for the control group. The CON-ECC and ECC groups showed increases (P < 0.05) in MVC-ISO (12.0 ± 15.7% and 11.3 ± 10.8%, respectively) and MVC-ECC torque (12.5 ± 18.3%, 16.2 ± 11.0%) similarly. Increases in MVC-CON torque (P < 0.05) were evident for the CON-ECC (17.5 ± 13.5%), CON (10.5 ± 12.8%), and ECC (14.2 ± 10.4%) groups without a significant difference among groups. MT increased (P < 0.01) after CON-ECC (10.6 ± 5.4%) and ECC (9.7 ± 7.2%) similarly, but not significantly after CON (2.5 ± 4.8%). CONCLUSIONS: ECC training increased muscle strength and thickness similarly to CON-ECC training, despite the half training volume, suggesting that concentric contractions contributed little to the training effects.

STAP-2 Is a Novel Positive Regulator of TCR-Proximal Signals.

TCR ligation with an Ag presented on MHC molecules promotes T cell activation, leading to the selection, differentiation, and proliferation of T cells and cytokine production. These immunological events are optimally arranged to provide appropriate responses against a variety of pathogens. We here propose signal-transducing adaptor protein-2 (STAP-2) as a new positive regulator of TCR signaling. STAP-2-deficient T cells showed reduced, whereas STAP-2-overexpressing T cells showed enhanced, TCR-mediated signaling and downstream IL-2 production. For the mechanisms, STAP-2 associated with TCR-proximal CD3ζ immunoreceptor tyrosine activation motifs and phosphorylated LCK, resulting in enhancement of their binding after TCR stimulation. In parallel, STAP-2 expression is required for full activation of downstream TCR signaling. Importantly, STAP-2-deficient mice exhibited slight phenotypes of CD4+ T-cell-mediated inflammatory diseases, such as experimental autoimmune encephalomyelitis, whereas STAP-2-overexpressing transgenic mice showed severe phenotypes of these diseases. Together, STAP-2 is an adaptor protein to enhance TCR signaling; therefore, manipulating STAP-2 will have an ability to improve the treatment of patients with autoimmune diseases as well as the chimeric Ag receptor T cell therapy.

Effect of daily 3-s maximum voluntary isometric, concentric, or eccentric contraction on elbow flexor strength.

The present study compared a 3-s isometric maximal voluntary contraction (MVC), concentric MVC and eccentric MVC of the elbow flexors performed daily for 5 days a week for 4 weeks for changes in muscle strength and thickness. Young sedentary individuals were assigned to one of three training groups (n = 13 per group) that performed either 3-s isometric, concentric, or eccentric MVC once a day for 20 days, or to a control group (n = 10) that had measurements without training. The participants in the isometric group performed isometric MVC at 55° (0.96 rad) elbow flexion, and those in the concentric or eccentric group performed concentric MVC or eccentric MVC between 10° (0.17 rad) and 100° (1.75 rad) elbow flexion at 30°/s (0.52 rad/s) on an isokinetic dynamometer. MVC isometric torque at 20° (0.35 rad), 55° (0.96 rad), and 90° (1.57 rad) elbow flexion, MVC concentric and eccentric torque at 30°/s (0.52 rad/s) and 180°/s (3.14 rad/s), and muscle thickness (MT) of biceps brachii and brachialis were measured before and several days after the 20th exercise session. The control group did not show any changes. The eccentric group showed increases (p < 0.01) in isometric (three angle average: 10.2 ± 6.4%), concentric (two velocity average: 12.8 ± 9.6%), and eccentric MVC torque (12.2 ± 7.8%). An increase (p < 0.05) was limited for isometric MVC torque (6.3 ± 6.0%) in the concentric group, and for eccentric MVC torque (7.2 ± 4.4%) in the isometric group. No significant changes in MT were evident for all groups. Performing one 3-s MVC a day increased muscle strength, but eccentric MVC produced more potent effects than isometric or concentric MVC.

Pivotal Role of Signal-Transducing Adaptor Protein-2 in Pathogenesis of Autoimmune Hepatitis.

Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein involved in inflammatory and immune responses, such as inflammatory bowel disease and allergic responses. In this study, we investigated the role of STAP-2 in the pathogenesis of autoimmune hepatitis. After intravenous injection of concanavalin A (ConA), STAP-2 knock out (KO) mice showed more severe liver necrosis along with substantial lymphocyte infiltration compared to wild type (WT) mice. Serum alanine aminotransferase levels were significantly higher in ConA-injected STAP-2 KO mice than in WT mice. Levels of interferon-γ (IFN-γ), an important factor for liver necrosis, were also significantly increased in sera of STAP-2 KO mice compared to WT mice after ConA injection. Statistically significant upregulation of Fas ligand (FasL) expression was observed in the livers of ConA-injected STAP-2 KO mice compared to WT mice. In accordance with these results, apoptotic signals were facilitated in STAP-2 KO mice compared to WT mice after ConA injection. Correctively, these results suggest that STAP-2 is involved in the pathogenesis of autoimmune hepatitis by regulating the expression of FasL and the production of IFN-γ.

Signal-transducing adaptor protein-2 has a nonredundant role for IL-33-triggered mast cell activation.

Signal-transducing adaptor protein (STAP)-2 is one of the STAP family adaptor proteins and ubiquitously expressed in a variety types of cells. Although STAP-2 is required for modification of FcεRI signal transduction in mast cells, other involvement of STAP-2 in mast cell functions is unknown, yet. In the present study, we mainly investigated functional roles of STAP-2 in IL-33-induced mast cell activation. In STAP-2-deficient, but not STAP-1-deficient, mast cells, IL-33-induced IL-6 and TNF-α production was significantly decreased compared with that of wild-type mast cells. In addition, STAP-2-deficiency greatly reduced TLR4-mediated mast cell activation and cytokine production. For the mechanisms, STAP-2 directly binds to IKKα after IL-33 stimulation, leading to elevated NF-κB activity. In conclusion, STAP-2, but not STAP-1, participates in IL-33-induced mast cells activation.

Correction: Expression of signal-transducing adaptor protein-1 attenuates experimental autoimmune hepatitis via down-regulating activation and homeostasis of invariant natural killer T cells.

[This corrects the article DOI: 10.1371/journal.pone.0241440.].

Expression of asporin reprograms cancer cells to acquire resistance to oxidative stress.

Asporin (ASPN), a small leucine-rich proteoglycan expressed predominantly by cancer associated fibroblasts (CAFs), plays a pivotal role in tumor progression. ASPN is also expressed by some cancer cells, but its biological significance is unclear. Here, we investigated the effects of ASPN expression in gastric cancer cells. Overexpression of ASPN in 2 gastric cancer cell lines, HSC-43 and 44As3, led to increased migration and invasion capacity, accompanied by induction of CD44 expression and activation of Rac1 and MMP9. ASPN expression increased resistance of HSC-43 cells to oxidative stress by reducing the amount of mitochondrial reactive oxygen species. ASPN induced expression of the transcription factor HIF1α and upregulated lactate dehydrogenase A (LDHA) and PDH-E1α, suggesting that ASPN reprograms HSC-43 cells to undergo anaerobic glycolysis and suppresses ROS generation in mitochondria, which has been observed in another cell line HSC-44PE. By contrast, 44As3 cells expressed high levels of HIF1α in response to oxidant stress and escaped apoptosis regardless of ASPN expression. Examination of xenografts in the gastric wall of ASPN-/- mice revealed that growth of HSC-43 tumors with increased micro blood vessel density was significantly accelerated by ASPN; however, ASPN increased the invasion depth of both HSC-43 and 44As3 tumors. These results suggest that ASPN has 2 distinct effects on cancer cells: HIF1α-mediated resistance to oxidative stress via reprogramming of glucose metabolism, and activation of CD44-Rac1 and MMP9 to promote cell migration and invasion. Therefore, ASPN may be a new therapeutic target in tumor fibroblasts and cancer cells in some gastric carcinomas.

Propolis suppresses cytokine production in activated basophils and basophil-mediated skin and intestinal allergic inflammation in mice.

BACKGROUND: Propolis is a resinous mixture produced by honey bees that contains cinnamic acid derivatives and flavonoids. Although propolis has been reported to inhibit mast cell functions and mast cell-dependent allergic responses, the effect of propolis on basophil biology remains unknown. This study aimed to investigate the inhibitory effect of propolis on FcεRI-mediated basophil activation. METHODS: To determine the inhibitory effect of propolis on basophil activation in vitro, cytokine production and FcεRI signal transduction were analyzed by ELISA and western blotting, respectively. To investigate the inhibitory effect of propolis in vivo, IgE-CAI and a food allergy mouse model were employed. RESULTS: Propolis treatment resulted in the suppression of IgE/antigen-induced production of IL-4, IL-6 and IL-13 in basophils. Phosphorylation of FcεRI signaling molecules Lyn, Akt and ERK was inhibited in basophils treated with propolis. While propolis did not affect the basophil population in the treated mice, propolis did inhibit IgE-CAI. Finally, ovalbumin-induced intestinal anaphylaxis, which involves basophils and basophil-derived IL-4, was attenuated in mice prophylactically treated with propolis. CONCLUSIONS: Taken together, these results demonstrate the ability of propolis to suppress IgE-dependent basophil activation and basophil-dependent allergic inflammation. Therefore, prophylactic treatment with propolis may be useful for protection against food allergic reactions in sensitive individuals.

Expression of signal-transducing adaptor protein-1 attenuates experimental autoimmune hepatitis via down-regulating activation and homeostasis of invariant natural killer T cells.

OBJECTIVE: Signal-transducing adaptor protein (STAP) family members function as adaptor molecules and are involved in several events during immune responses. Notably however, the biological functions of STAP-1 in other cells are not known. We aimed to investigate the functions of STAP-1 in invariant natural killer T (iNKT) cells and iNKT cell-dependent hepatitis. METHODS: We employed concanavalin A (Con A)-induced hepatitis and α-galactosylceramide (α-GalCer)-induced hepatitis mouse models, both are models of iNKT cell-dependent autoimmune hepatitis, and STAP-1 overexpressing 2E10 cells to investigate the role of STAP-1 in iNKT cell activation in vivo an in vitro, respectively. RESULTS: After Con A- or α-GalCer-injection, hepatocyte necrotic areas and plasma alanine aminotransferase elevation were more severe in STAP-1 knockout (S1KO) mice and milder in lymphocyte-specific STAP-1 transgenic (S1Tg) mice, as compared to wild-type (WT) mice. Two events that may be related to Con A-induced and/or α-GalCer-induced hepatitis were influenced by STAP-1 manipulation. One is that iNKT cell populations in the livers and spleens were increased in S1KO mice and were decreased in S1Tg mice. The other is that Con A-induced interleukin-4 and interferon-γ production was attenuated by STAP-1 overexpression. These effects of STAP-1 were confirmed using 2E10 cells overexpressing STAP-1 that showed impairment of interleukin-4 and interferon-γ production as well as phosphorylation of Akt and mitogen-activated protein kinases in response to Con A stimulation. CONCLUSIONS: These results conclude that STAP-1 regulates iNKT cell maintenance/activation, and is involved in the pathogenesis of autoimmune hepatitis.

Persistent Na+ influx drives L-type channel resting Ca2+ entry in rat melanotrophs.

Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca2+ concentration ([Ca2+]i) remain unknown. We analyzed mechanisms regulating resting [Ca2+]i in dissociated rat melanotrophs by Ca2+-imaging and patch-clamp techniques. Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca2+ channels (VGCCs) as well as removal of extracellular Ca2+ resulted in a rapid and reversible decrease in [Ca2+]i, indicating constitutive Ca2+ influx through L-type VGCCs. Reduction of extracellular Na+ concentration (replacement with NMDG+) similarly decreased resting [Ca2+]i. When cells were champed at -80 mV, decrease in the extracellular Na+ resulted in a positive shift of the holding current. In cell-attached voltage-clamp and whole-cell current-clamp configurations, the reduction of extracellular Na+ caused hyperpolarisation. The holding current shifted in negative direction when extracellular K+ concentration was increased from 5 mM to 50 mM in the presence of K+ channel blockers, Ba2+ and TEA, indicating cation nature of persistent conductance. RT-PCR analyses of pars intermedia tissues detected mRNAs of TRPV1, TRPV4, TRPC6, and TRPM3-5. The TRPV channel blocker, ruthenium red, shifted the holding current in positive direction, and significantly decreased the resting [Ca2+]i. These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca2+]i in rat melanotrophs.

Macrophage-mediated transfer of cancer-derived components to stromal cells contributes to establishment of a pro-tumor microenvironment.

Tumor-derived extracellular vesicles (TEVs) secreted into the blood create a pre-metastatic niche in distant organs; however, it is unclear how TEVs are delivered and how they affect stromal cells in the tumor microenvironment. Tumor-associated macrophages (TAMs) have pivotal roles in cancer progression by interacting with cancer cells and other stromal cells. Here, we report a novel function of TAMs: delivery and transmission of TEV contents. TEV-incorporating macrophages (TEV-MΦs) showed increased invasiveness and were disseminated widely. Upon contact with host stromal cells (peritoneal mesothelial cells (PMCs), fibroblasts, and endothelial cells), TEV-MΦs released membrane blebs containing TEVs, a process dependent upon localized activation of caspase-3 in MΦs. Scattered blebs were incorporated into stromal cells, leading to transfer of cancer-derived RNA and proteins such as TGF-ß, activated Src, Wnt3, and HIF1α. TEV-MΦ-secreted blebs containing cancer-derived components contributed to myofibroblastic changes in recipient stromal cells. TEVs delivered by MΦs penetrated deep into the parenchyma of the stomach in TEV-injected mice, and transmitted TEVs to PMCs lining the stomach surface; this process induced PMCs to undergo mesothelial-mesenchymal transition. PMCs infiltrated the gastric wall and created a niche, thereby promoting tumor invasion. Depletion of MΦs prevented these events. Moreover, TEV-MΦs created a pro-metastatic niche. Taken together, these results suggest a novel function for TAMs: transfer of cancer-derived components to surrounding stromal cells and induction of a pro-tumor microenvironment via an increase in the number of CAF-like cells.