p53-independent tumor suppression by cell-cycle arrest via CREB/ATF transcription factor OASIS.

CREB/ATF transcription factor OASIS/CREB3L1 is upregulated in long-term-cultured astrocytes undergoing cell-cycle arrest due to loss of DNA integrity by repeated replication. However, the roles of OASIS in the cell cycle remain unexplored. We find that OASIS arrests the cell cycle at G2/M phase after DNA damage via direct induction of p21. Cell-cycle arrest by OASIS is dominant in astrocytes and osteoblasts, but not in fibroblasts, which are dependent on p53. In a brain injury model, Oasis-/- reactive astrocytes surrounding the lesion core show sustained growth and inhibition of cell-cycle arrest, resulting in prolonged gliosis. We find that some glioma patients exhibit low expression of OASIS due to high methylation of its promoter. Specific removal of this hypermethylation in glioblastomas transplanted into nude mice by epigenomic engineering suppresses the tumorigenesis. These findings suggest OASIS as a critical cell-cycle inhibitor with potential to act as a tumor suppressor.

The survival and proliferation of osteosarcoma cells are dependent on the mitochondrial BIG3-PHB2 complex formation.

Previous studies reported the critical role of the brefeldin A-inhibited guanine nucleotide exchange protein 3-prohibitin 2 (BIG3-PHB2) complex in modulating estrogen signaling activation in breast cancer cells, yet its pathophysiological roles in osteosarcoma (OS) cells remain elusive. Here, we report a novel function of BIG3-PHB2 in OS malignancy. BIG3-PHB2 complexes were localized mainly in mitochondria in OS cells, unlike in estrogen-dependent breast cancer cells. Depletion of endogenous BIG3 expression by small interfering RNA (siRNA) treatment led to significant inhibition of OS cell growth. Disruption of BIG3-PHB2 complex formation by treatment with specific peptide inhibitor also resulted in significant dose-dependent suppression of OS cell growth, migration, and invasion resulting from G2/M-phase arrest and in PARP cleavage, ultimately leading to PARP-1/apoptosis-inducing factor (AIF) pathway activation-dependent apoptosis in OS cells. Subsequent proteomic and bioinformatic pathway analyses revealed that disruption of the BIG3-PHB2 complex might lead to downregulation of inner mitochondrial membrane protein complex activity. Our findings indicate that the mitochondrial BIG3-PHB2 complex might regulate PARP-1/AIF pathway-dependent apoptosis during OS cell proliferation and progression and that disruption of this complex may be a promising therapeutic strategy for OS.

Functional genomics for breast cancer drug target discovery.

Breast cancer is a heterogeneous disease that develops through a multistep process via the accumulation of genetic/epigenetic alterations in various cancer-related genes. Current treatment options for breast cancer patients include surgery, radiotherapy, and chemotherapy including conventional cytotoxic and molecular-targeted anticancer drugs for each intrinsic subtype, such as endocrine therapy and antihuman epidermal growth factor receptor 2 (HER2) therapy. However, these therapies often fail to prevent recurrence and metastasis due to resistance. Overall, understanding the molecular mechanisms of breast carcinogenesis and progression will help to establish therapeutic modalities to improve treatment. The recent development of comprehensive omics technologies has led to the discovery of driver genes, including oncogenes and tumor-suppressor genes, contributing to the development of molecular-targeted anticancer drugs. Here, we review the development of anticancer drugs targeting cancer-specific functional therapeutic targets, namely, MELK (maternal embryonic leucine zipper kinase), TOPK (T-lymphokine-activated killer cell-originated protein kinase), and BIG3 (brefeldin A-inhibited guanine nucleotide-exchange protein 3), as identified through comprehensive breast cancer transcriptomics.

The GALNT6­LGALS3BP axis promotes breast cancer cell growth.

Polypeptide N­acetylgalactosaminyltransferase 6 (GALNT6), which is involved in the initiation of O­glycosylation, has been reported to play crucial roles in mammary carcinogenesis through binding to several substrates; however, its biological roles in mediating growth­promoting effects remain unknown. The present study demonstrated a crucial pathophysiological role of GALNT6 through its O­glycosylation of lectin galactoside­binding soluble 3 binding protein (LGALS3BP), a secreted growth­promoting glycoprotein, in breast cancer growth. The Cancer Genome Atlas data analysis revealed that high expression levels of GALNT6 were significantly associated with poor prognosis of breast cancer. GALNT6 O­glycosylated LGALS3BP in breast cancer cells, whereas knockdown of GALNT6 by siRNA led to the inhibition of both the O­glycosylation and secretion of LGALS3BP, resulting in the suppression of breast cancer cell growth. Notably, LGALS3BP is potentially O­glycosylated at three sites (T556, T571 and S582) by GALNT6, thereby promoting autocrine cell growth, whereas the expression of LGALS3BP with three Ala substitutions (T556A, T571A and S582A) in cells drastically reduced GALNT6­dependent LGALS3BP O­glycosylation and secretion, resulting in suppression of autocrine growth­promoting effect. The findings of the present study suggest that the GALNT6­LGALS3BP axis is crucial for breast cancer cell proliferation and may be a therapeutic target and biomarker for mammary tumors.

Biophysical characterization of the breast cancer-related BIG3-PHB2 interaction: Effect of non-conserved loop region of BIG3 on the structure and the interaction.

Brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) interacts with and inhibits the tumor suppressor function of prohibitin-2 (PHB2), and recent in vivo studies have demonstrated that the BIG3-PHB2 interaction is a promising target for breast cancer therapy. However, little biophysical characterization on BIG3 and its interaction with PHB2 has been reported. Here we compared the calculated 8-class secondary structure of the N-terminal domains of BIG family proteins and identified a loop region unique to BIG3. Our biophysical characterization demonstrated that this loop region significantly affects the colloidal and thermodynamic stability of BIG3 and the thermodynamic and kinetic profile of its interaction with PHB2. These results establish a model for the BIG3-PHB2 interaction and an entry for drug discovery for breast cancer.

Frequent downregulation of LRRC26 by epigenetic alterations is involved in the malignant progression of triple-negative breast cancer.

Triple-negative breast cancer (TNBC), defined as breast cancer lacking estrogen- and progesterone­receptor expression and human epidermal growth factor receptor 2 (HER2) amplification, is a heterogeneous disease. RNA-sequencing analysis of 15 TNBC specimens and The Cancer Genome Atlas-TNBC dataset analysis identified the frequent downregulation of leucine-rich repeat-containing 26 (LRRC26), which negatively regulates nuclear factor-κB (NF-κB) signaling, in TNBC tissues. Quantitative polymerase chain reaction and bisulfite pyrosequencing analyses revealed that LRRC26 was frequently silenced in TNBC tissues and cell lines as a result of promoter methylation. LRRC26 expression was restored by 5-aza-2'-deoxycytidine (5'-aza-dC) treatment in HCC1937 TNBC cells, which lack LRRC26 expression. Notably, small interfering RNA-mediated knockdown of LRRC26 expression significantly enhanced the anchorage-independent growth, invasion and migration of HCC70 cells, whereas ectopic overexpression of LRRC26 in BT20 cells suppressed their invasion and migration. Conversely, neither knockdown nor overexpression of LRRC26 had an effect on cell viability in the absence of tumor necrosis factor-α (TNF-α) stimulation. Meanwhile, overexpression of LRRC26 caused the reduction of TNF-α-mediated NF-κB luciferase reporter activity, whereas depleting LRRC26 expression resulted in the upregulation of TNF-α-mediated NF-κB downstream genes [interleukin-6 (IL-6), IL-8 and C-X-C motif chemokine ligand-1]. Taken together, these findings demonstrate that LRRC26 is frequently downregulated in TNBC due to DNA methylation and that it suppresses the TNF-α-independent anchorage-independent growth, invasion and migration of TNBC cells. Loss of LRRC26 function may be a critical event in the aggressiveness of TNBC cells through a TNF-α/NF-κB-independent mechanism.

A DDX31/Mutant-p53/EGFR Axis Promotes Multistep Progression of Muscle-Invasive Bladder Cancer.

The p53 and EGFR pathways are frequently altered in bladder cancer, yet their contributions to its progression remain elusive. Here we report that DEAD box polypeptide 31 (DDX31) plays a critical role in the multistep progression of muscle-invasive bladder cancer (MIBC) through its sequential interactions with mutant p53 (mutp53) and EGFR. In early MIBC cells, nuclear DDX31-bound mutp53/SP1 enhanced mutp53 transcriptional activation, leading to migration and invasion of MIBC. Cytoplasmic DDX31 also bound EGFR and phospho-nucleolin in advanced MIBC, leading to EGFR-Akt signaling activation. High expression of both cytoplasmic DDX31 and p53 proteins correlated with poor prognosis in patients with MIBC, and blocking the DDX31/NCL interaction resulted in downregulation of EGFR/Akt signaling, eliciting an in vivo antitumor effect against bladder cancer. These findings reveal that DDX31 cooperates with mutp53 and EGFR to promote progression of MIBC, and inhibition of DDX31/NCL formation may lead to potential treatment strategies for advanced MIBC.Significance: DDX31 cooperates with mutp53 and EGFR to promote progression of muscle invasive bladder cancer. Cancer Res; 78(9); 2233-47. ©2018 AACR.

A-kinase anchoring protein BIG3 coordinates oestrogen signalling in breast cancer cells.

Approximately 70% of breast cancer cells express oestrogen receptor alpha (ERα). Previous studies have shown that the Brefeldin A-inhibited guanine nucleotide-exchange protein 3-prohibitin 2 (BIG3-PHB2) complex has a crucial role in these cells. However, it remains unclear how BIG3 regulates the suppressive activity of PHB2. Here we demonstrate that BIG3 functions as an A-kinase anchoring protein that binds protein kinase A (PKA) and the α isoform of the catalytic subunit of protein phosphatase 1 (PP1Cα), thereby dephosphorylating and inactivating PHB2. E2-induced PKA-mediated phosphorylation of BIG3-S305 and -S1208 serves to enhance PP1Cα activity, resulting in E2/ERα signalling activation via PHB2 inactivation due to PHB2-S39 dephosphorylation. Furthermore, an analysis of independent cohorts of ERα-positive breast cancers patients reveal that both BIG3 overexpression and PHB2-S39 dephosphorylation are strongly associated with poor prognosis. This is the first demonstration of the mechanism of E2/ERα signalling activation via the BIG3-PKA-PP1Cα tri-complex in breast cancer cells.

Stapled BIG3 helical peptide ERAP potentiates anti-tumour activity for breast cancer therapeutics.

Estradiol (E2) and the oestrogen receptor-alpha (ERα) signalling pathway play pivotal roles in the proliferative activity of breast cancer cells. Recent findings show that the brefeldin A-inhibited guanine nucleotide-exchange protein 3-prohibitin 2 (BIG3-PHB2) complex plays a crucial role in E2/ERα signalling modulation in breast cancer cells. Moreover, specific inhibition of the BIG3-PHB2 interaction using the ERα activity-regulator synthetic peptide (ERAP: 165-177 amino acids), derived from α-helical BIG3 sequence, resulted in a significant anti-tumour effect. However, the duration of this effect was very short for viable clinical application. We developed the chemically modified ERAP using stapling methods (stapledERAP) to improve the duration of its antitumour effects. The stapledERAP specifically inhibited the BIG3-PHB2 interaction and exhibited long-lasting suppressive activity. Its intracellular localization without the membrane-permeable polyarginine sequence was possible via the formation of a stable α-helix structure by stapling. Tumour bearing-mice treated daily or weekly with stapledERAP effectively prevented the BIG3-PHB2 interaction, leading to complete regression of E2-dependent tumours in vivo. Most importantly, combination of stapledERAP with tamoxifen, fulvestrant, and everolimus caused synergistic inhibitory effects on growth of breast cancer cells. Our findings suggested that the stapled ERAP may be a promising anti-tumour drug to suppress luminal-type breast cancer growth.

Protein kinase A inhibition facilitates the antitumor activity of xanthohumol, a valosin-containing protein inhibitor.

Xanthohumol (XN), a simple prenylated chalcone, can be isolated from hops and has the potential to be a cancer chemopreventive agent against several human tumor cell lines. We previously identified valosin-containing protein (VCP) as a target of XN; VCP can also play crucial roles in cancer progression and prognosis. Therefore, we investigated the molecular mechanisms governing the contribution of VCP to the antitumor activity of XN. Several human tumor cell lines were treated with XN to investigate which human tumor cell lines are sensitive to XN. Several cell lines exhibited high sensitivity to XN both in vitro and in vivo. shRNA screening and bioinformatics analysis identified that the inhibition of the adenylate cyclase (AC) pathway synergistically facilitated apoptosis induced by VCP inhibition. These results suggest that there is crosstalk between the AC pathway and VCP function, and targeting both VCP and the AC pathway is a potential chemotherapeutic strategy for a subset of tumor cells.

BIG3 Inhibits the Estrogen-Dependent Nuclear Translocation of PHB2 via Multiple Karyopherin-Alpha Proteins in Breast Cancer Cells.

We recently reported that brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) binds Prohibitin 2 (PHB2) in cytoplasm, thereby causing a loss of function of the PHB2 tumor suppressor in the nuclei of breast cancer cells. However, little is known regarding the mechanism by which BIG3 inhibits the nuclear translocation of PHB2 into breast cancer cells. Here, we report that BIG3 blocks the estrogen (E2)-dependent nuclear import of PHB2 via the karyopherin alpha (KPNA) family in breast cancer cells. We found that overexpressed PHB2 interacted with KPNA1, KPNA5, and KPNA6, thereby leading to the E2-dependent translocation of PHB2 into the nuclei of breast cancer cells. More importantly, knockdown of each endogenous KPNA by siRNA caused a significant inhibition of E2-dependent translocation of PHB2 in BIG3-depleted breast cancer cells, thereby enhancing activation of estrogen receptor alpha (ERα). These data indicated that BIG3 may block the KPNAs (KPNA1, KPNA5, and KPNA6) binding region(s) of PHB2, thereby leading to inhibition of KPNAs-mediated PHB2 nuclear translocation in the presence of E2 in breast cancer cells. Understanding this regulation of PHB2 nuclear import may provide therapeutic strategies for controlling E2/ERα signals in breast cancer cells.

Therapeutic advances in BIG3-PHB2 inhibition targeting the crosstalk between estrogen and growth factors in breast cancer.

Our previous studies demonstrated that specific inhibition of the BIG3-PHB2 complex, which is a critical modulator in estrogen (E2) signaling, using ERAP, a dominant negative peptide inhibitor, leads to suppression of E2-dependent estrogen receptor (ER) alpha activation through the reactivation of the tumor suppressive activity of PHB2. Here, we report that ERAP has significant suppressive effects against synergistic activation caused by the crosstalk between E2 and growth factors associated with intrinsic or acquired resistance to anti-estrogen tamoxifen in breast cancer cells. Intrinsic PHB2 released from BIG3 by ERAP effectively disrupted each interaction of membrane-associated ERα and insulin-like growth factor 1 receptor beta (IGF-1Rß), EGFR, PI3K or human epidermal growth factor 2 (HER2) in the presence of E2 and the growth factors IGF or EGF, followed by inhibited the activation of IGF-1Rß, EGFR or HER2, and reduced Akt, MAPK and ERα phosphorylation levels, resulting in significant suppression of proliferation of ERα-positive breast cancer cells in vitro and in vivo. More importantly, combined treatment with ERAP and tamoxifen led to a synergistic suppression of signaling that was activated by crosstalk between E2 and growth factors or HER2 amplification. Taken together, our findings suggest that the specific inhibition of BIG3-PHB2 is a novel potential therapeutic approach for the treatment of tamoxifen-resistant breast cancers activated by the crosstalk between E2 and growth factor signaling, especially in premenopausal women.

Xanthohumol suppresses oestrogen-signalling in breast cancer through the inhibition of BIG3-PHB2 interactions.

Xanthohumol (XN) is a natural anticancer compound that inhibits the proliferation of oestrogen receptor-α (ERα)-positive breast cancer cells. However, the precise mechanism of the antitumour effects of XN on oestrogen (E2)-dependent cell growth, and especially its direct target molecule(s), remain(s) largely unknown. Here, we focus on whether XN directly binds to the tumour suppressor protein prohibitin 2 (PHB2), forming a novel natural antitumour compound targeting the BIG3-PHB2 complex and acting as a pivotal modulator of E2/ERα signalling in breast cancer cells. XN treatment effectively prevented the BIG3-PHB2 interaction, thereby releasing PHB2 to directly bind to both nuclear- and cytoplasmic ERα. This event led to the complete suppression of the E2-signalling pathways and ERα-positive breast cancer cell growth both in vitro and in vivo, but did not suppress the growth of normal mammary epithelial cells. Our findings suggest that XN may be a promising natural compound to suppress the growth of luminal-type breast cancer.

Early growth response 4 is involved in cell proliferation of small cell lung cancer through transcriptional activation of its downstream genes.

Small cell lung cancer (SCLC) is aggressive, with rapid growth and frequent bone metastasis; however, its detailed molecular mechanism remains poorly understood. Here, we report the critical role of early growth factor 4 (EGR4), a DNA-binding, zinc-finger transcription factor, in cell proliferation of SCLC. EGR4 overexpression in HEK293T cells conferred significant upregulation of specific splice variants of the parathyroid hormone-related protein (PTHrP) gene, resulting in enhancement of the secretion of PTHrP protein, a known mediator of osteolytic bone metastasis. More importantly, depletion of EGR4 expression by siRNA significantly suppressed growth of the SCLC cell lines, SBC-5, SBC-3 and NCI-H1048. On the other hand, introduction of EGR4 into NIH3T3 cells significantly enhanced cell growth. We identified four EGR4 target genes, SAMD5, RAB15, SYNPO and DLX5, which were the most significantly downregulated genes upon depletion of EGR4 expression in all of the SCLC cells examined, and demonstrated the direct recruitment of EGR4 to their promoters by ChIP and luciferase reporter analysis. Notably, knockdown of the expression of these genes by siRNA remarkably suppressed the growth of all the SCLC cells. Taken together, our findings suggest that EGR4 likely regulates the bone metastasis and proliferation of SCLC cells via transcriptional regulation of several target genes, and may therefore be a promising target for the development of anticancer drugs for SCLC patients.

Brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) is predicted to interact with its partner through an ARM-type α-helical structure.

BACKGROUND: Brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) has been identified recently as a novel regulator of estrogen signalling in breast cancer cells. Despite being a potential target for new breast cancer treatment, its amino acid sequence suggests no association with any well-characterized protein family and provides little clues as to its molecular function. In this paper, we predicted the structure, function and interactions of BIG3 using a range of bioinformatic tools. RESULTS: Homology search results showed that BIG3 had distinct features from its paralogues, BIG1 and BIG2, with a unique region between the two shared domains, Sec7 and DUF1981. Although BIG3 contains Sec7 domain, the lack of the conserved motif and the critical glutamate residue suggested no potential guaninyl-exchange factor (GEF) activity. Fold recognition tools predicted BIG3 to adopt an α-helical repeat structure similar to that of the armadillo (ARM) family. Using state-of-the-art methods, we predicted interaction sites between BIG3 and its partner PHB2. CONCLUSIONS: The combined results of the structure and interaction prediction led to a novel hypothesis that one of the predicted helices of BIG3 might play an important role in binding to PHB2 and thereby preventing its translocation to the nucleus. This hypothesis has been subsequently verified experimentally.

Involvement of B3GALNT2 overexpression in the cell growth of breast cancer.

A number of glycosyltransferases have been identified and biologically characterized in cancer cells, yet their exact pathophysiological functions are largely unknown. Here, we report the critical role of ß1,3-N-acetylgalactosaminyltransferase II (B3GALNT2), which transfers N-acetylgalactosamine (GalNAc) in a ß1,3 linkage to N-acetylglucosamine, in the growth of breast cancer cells. Comprehensive transcriptomics, quantitative PCR and northern blot analyses indicated this molecule to be exclusively upregulated in the majority of breast cancers. Knockdown of B3GALNT2 expression by small interfering RNA attenuated cell growth and induced apoptosis in breast cancer cells. Overexpression of B3GALNT2 in HEK293T cells prompted secretion of the gene product into the culture medium, suggesting that B3GALNT2 is potentially a secreted protein. Furthermore, we demonstrated that B3GALNT2 is N-glycosylated on both Asn-116 and Asn-174 and that this modification is necessary for its secretion in breast cancer cells. Our findings suggest that this molecule represents a promising candidate for the development of a novel therapeutic targeting drug and a potential diagnostic tumor marker for patients with breast cancer, especially TNBC.

Targeting BIG3-PHB2 interaction to overcome tamoxifen resistance in breast cancer cells.

The acquisition of endocrine resistance is a common obstacle in endocrine therapy of patients with oestrogen receptor-α (ERα)-positive breast tumours. We previously demonstrated that the BIG3-PHB2 complex has a crucial role in the modulation of oestrogen/ERα signalling in breast cancer cells. Here we report a cell-permeable peptide inhibitor, called ERAP, that regulates multiple ERα-signalling pathways associated with tamoxifen resistance in breast cancer cells by inhibiting the interaction between BIG3 and PHB2. Intrinsic PHB2 released from BIG3 by ERAP directly binds to both nuclear- and membrane-associated ERα, which leads to the inhibition of multiple ERα-signalling pathways, including genomic and non-genomic ERα activation and ERα phosphorylation, and the growth of ERα-positive breast cancer cells both in vitro and in vivo. More importantly, ERAP treatment suppresses tamoxifen resistance and enhances tamoxifen responsiveness in ERα-positive breast cancer cells. These findings suggest inhibiting the interaction between BIG3 and PHB2 may be a new therapeutic strategy for the treatment of luminal-type breast cancer.

Molecular features of triple negative breast cancer cells by genome-wide gene expression profiling analysis.

Triple negative breast cancer (TNBC) has a poor outcome due to the lack of beneficial therapeutic targets. To clarify the molecular mechanisms involved in the carcinogenesis of TNBC and to identify target molecules for novel anticancer drugs, we analyzed the gene expression profiles of 30 TNBCs as well as 13 normal epithelial ductal cells that were purified by laser-microbeam microdissection. We identified 301 and 321 transcripts that were significantly upregulated and downregulated in TNBC, respectively. In particular, gene expression profile analyses of normal human vital organs allowed us to identify 104 cancer-specific genes, including those involved in breast carcinogenesis such as NEK2, PBK and MELK. Moreover, gene annotation enrichment analysis revealed prominent gene subsets involved in the cell cycle, especially mitosis. Therefore, we focused on cell cycle regulators, asp (abnormal spindle) homolog, microcephaly-associated (Drosophila) (ASPM) and centromere protein K (CENPK) as novel therapeutic targets for TNBC. Small-interfering RNA-mediated knockdown of their expression significantly attenuated TNBC cell viability due to G1 and G2/M cell cycle arrest. Our data will provide a better understanding of the carcinogenesis of TNBC and could contribute to the development of molecular targets as a treatment for TNBC patients.

Identification of genes potentially involved in bone metastasis by genome-wide gene expression profile analysis of non-small cell lung cancer in mice.

Lung cancer is commonly associated with multi-organ metastasis, and the bone is a frequent metastatic site for lung cancer. However, the molecular mechanism of organ-specific metastasis remains poorly understood. To elucidate this issue, we analyzed in this study genome-wide gene expression profiles of 15 metastatic lesions from three organs (bone, lung and liver) in a mouse model with multi-organ metastasis properties of human non-small cell lung cancer cells (ACC-LC319/bone2), using a combination of laser-microbeam microdissection and DNA microarrays. We identified 299 genes that could potentially be involved in the organ-selective nature of lung cancer metastasis. Among them, 77 were bone-specifically expressed elements, including genes involved in cell adhesion, cytoskeleton/cell motility, extracellular matrix remodeling and cell-cell signaling as well as genes already known to be involved in the bone metastasis of breast cancers. Quantitative RT-PCR confirmed the specific upregulation of eight genes in bone metastasis tumors, suggesting that these genes may be involved in bone metastasis. Our findings should be helpful for a better understanding of the molecular aspects of the metastatic process in different organs, and could lead to molecular target-based anticancer drugs and prevention of metastasis, especially bone metastasis.

FcepsilonRI beta-chain ITAM amplifies PI3K-signaling to ensure synergistic degranulation response via FcepsilonRI and adenosine receptors.

Simultaneous stimulation with antigen and adenosine in mast cells induces a synergistic degranulation response at a low antigen dose that is insufficient to cause secretion by itself. This kind of stimulation is thought to be relevant to the immediate asthmatic response upon bronchial challenge with low-dose allergen. In this context, FcepsilonRI- and adenosine receptor-mediated signalings cooperate to increase degranulation in mast cells. In the present study, we prepared mast cells that have mutations (Y219F/Y225F/Y229F) in three tyrosine residues of the FcepsilonRI beta-chain (FcRbeta)-ITAM in order to elucidate the molecular mechanisms of degranulation response synergistically elicited by costimulation with low-dose antigen and adenosine. Introduction of mutations in the FcRbeta-ITAM abolished the synergistic degranulation response. Upon costimulation with low-dose antigen and adenosine, tyrosine phosphorylation of Grb2-associated binder 2, which is located upstream of PI3K-signaling, was significantly increased, but severely diminished in FcRbeta-ITAM mutant cells. These findings indicate that FcRbeta acts as a critical element in mast cell synergistic degranulation response through FcepsilonRI and adenosine receptors, and that PI3K-signaling through FcRbeta-ITAM is a crucial participant in augmentation of FcepsilonRI-mediated degranulation by adenosine.