Resveratrol exhibits diverse anti-cancer activities through epigenetic regulation of E-cadherin and p21 in triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) has an aggressive phenotype and poor outcome, however no specific targeted therapy has been established for TNBC lacking germline BRCA1/2 pathogenic variants. To develop a novel therapeutic strategy, we explored the potential of resveratrol (RSV) for TNBC treatment. METHODS: We investigated the effects of RSV on malignant phenotypes of TNBC cells as well as on apoptosis induced by ABT263, a specific inhibitor of BCL-2 and BCL-xL, using morphological observation, migration assay, ß-galactosidase staining, and Hoechst staining. To elucidate the underlying mechanisms of RSV-mediated effects, expression levels and histone acetylation levels of cadherin 1 (CDH1, E-cadherin) and cyclin dependent kinase inhibitor 1A (CDKN1A, p21) were determined by RT-qPCR, western blotting, and chromatin immunoprecipitation. Furthermore, knockdown analysis was conducted to evaluate the involvement of E-cadherin and/or p21 in RSV potentiation on cytotoxic activity of ABT263. RESULTS: RSV treatment induced epithelial-like cellular morphology and suppressed the migration capacity in MDA-MB-231 and BT-549-Luc TNBC cells. ß-galactosidase-positive cells were increased after RSV treatment, indicating the induction of cellular senescence, in MDA-MB-231 cells but not in BT-549-Luc cells. RSV increased the expression and histone acetylation of CDH1 and CDKN1A in both cells. Interestingly, pre-treatment with RSV enhanced the induction of apoptosis in the ABT263-treated MDA-MB-231 and BT-549-Luc cells, and knockdown of CDKN1A decreased ABT263-induced apoptosis in RSV-treated MDA-MB-231 cells. CONCLUSIONS: RSV represses the metastatic capacity and enhances the cytotoxic activity of ABT263 in TNBC cells. Our results suggested that RSV can potentially be used as a repressor of metastasis or a sensitizer to ABT263 for TNBC treatment via up-regulation of CDH1 and CDKN1A through epigenetic mechanisms.

One DNA Methylation Regulates CHIP Gene Expression of Human Breast Cancer and Predicts Recurrence.

BACKGROUND/AIM: Carboxyl terminus of Hsc70-interacting protein (CHIP) is a ubiquitin ligase that induces ubiquitination and degradation of its target proteins including oncoproteins. We reported that its down-regulation is associated with tumor progression and metastasis of breast cancer. However, the mechanism through which CHIP gene affects cancer cells is unclear. MATERIALS AND METHODS: We extracted RNA from 45 primary breast cancer samples and compared CHIP mRNA expression profiles, promoter DNA methylation status, and clinicopathological information. RESULTS: CHIP mRNA expression was significantly correlated with the tumor progression status. In several samples, a pinpoint CpG methylation in the CHIP gene promoter region was significantly correlated with CHIP mRNA expression. When this specific CpG was methylated in estrogen receptor (ER)-positive cases, a significant difference in 5-year recurrence was not found compared with ER-negative cases. CONCLUSION: CpG methylation contributes to the long-term prognosis of ER-positive breast cancer.

Molecular targeted drugs resistance impairs double-strand break repair and sensitizes ER-positive breast cancer to PARP inhibitors.

BACKGROUND: There are various treatments for estrogen-positive breast cancer, mainly hormone therapy and molecular-targeted drugs. Acquiring resistance to these drugs is a major clinical problem. Additionally, little is known about the effect of drug resistance on the DNA repair mechanism. Poly ADP ribose polymerase (PARP) inhibitors currently used for treating HER2-negative metastatic breast cancer with BRCA mutations have been shown to be effective in BRCA-deficient cells with impaired homologous recombination repair. Here, we investigated the effect of drug resistance acquisition on the DNA repair mechanism and the effect of PARP inhibitors on ER (estrogen receptor) -positive breast cancer. METHODS: We investigated changes in the expression of DNA repair mechanism-related factors and repair ability of double-strand breaks (DSB) in various drug-resistant cell lines established in our laboratory. Additionally, PARP inhibitor susceptibility was investigated using olaparib. RESULTS: DSB repairs in MCF-7 and hormone therapy-resistant model cells were normal, and these cells demonstrated low sensitivity to olaparib. The resistant cell lines against CDK4/6 inhibitors, fulvestrant and mTOR/PI3K inhibitors showed decreased DSB repair ability and high olaparib sensitivity. They showed low sensitivity to CDK4/6 inhibitors, a close link between acquiring resistance to CDK4/6 inhibitors and hypersensitivity to olaparib. CONCLUSIONS: Our study suggests some cases of acquiring drug resistance impairs DSB repair ability and sensitizes ER-positive breast cancer to PARP inhibitors.

CD4+ Resident Memory T Cells Mediate Long-Term Local Skin Immune Memory of Contact Hypersensitivity in BALB/c Mice.

In allergic contact dermatitis (ACD) and contact hypersensitivity (CHS), the healed skin shows greater swelling than the naïve skin in the same individual upon re-exposure to the same hapten. This "local skin memory" (LSM) in healed skin was maintained for a prolonged period of time and mediated by skin CD8+-resident memory T (TRM) cells in C57BL/6 mice. However, the number of CD4+ T cells is elevated in ACD-healed human skin, and the contribution of CD4+ TRM cells to the formation of LSM currently remains unclear. We herein demonstrated that immediately after CHS subsided, the healed skin in BALB/c mice showed an accumulation of hapten-specific CD4+ and CD8+ TRM cells, with a predominance of CD4+ TRM cells. The presence of CD4+ or CD8+ TRM cells in the healed skin was sufficient for the induction of a flare-up reaction upon a re-challenge. The CD4+ and CD8+ TRM cells both produced interferon-γ and tumor necrosis factor early after the re-challenge. Moreover, while CD8+ TRM cells gradually decreased over time and were eventually lost from the healed skin at 40-51 weeks after the resolution of CHS, the CD4+ TRM cell numbers remained elevated during this period. The present results indicate that the long-term maintenance of LSM is mediated by CD4+ TRM cells, and thus CD4+ TRM cells are an important target for the treatment of recurrent human ACD.

Heregulin controls ERα and HER2 signaling in mammospheres of ERα-positive breast cancer cells and interferes with the efficacy of molecular targeted therapy.

Estrogen receptor (ER)α and the human epidermal growth factor receptor (HER) family are inversely expressed in ERα-positive cancer in association with resistance to hormonal therapy, but the mechanism underlying their relationship remains unknown. We analyzed the effect of HER family ligands on the expression of ER and the HER family in ERα-positive MCF-7 and T47D breast cancer cell lines in 3D spheroid culture. Here, we demonstrated for the first time that heregulin-1ß (HRG), a HER3 and HER4 ligand, most effectively regulated ER/HER family expression by decreasing ERα mRNA expression and increasing HER family mRNA expression. HRG treatment attenuated fulvestrant-mediated growth inhibition, and promoted the migration of MCF-7 cells. Moreover, HRG increased the CD44+/CD24- cell fraction and side population cells, both of which are recognized as prospective breast cancer stem cell markers. HRG activated both phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) and mitogen-activated protein kinase (MAPK) pathways. Inhibitors of these pathways reduced the growth of MCF-7 cells, but the addition of HRG has different effects on these pathways. HRG blocked the inhibitory effect of mTOR inhibitors, such as rapamycin and everolimus, on cell growth but not that of a PI3K inhibitor. Furthermore, HRG slightly decreased the inhibitory effect of an AKT inhibitor on cell growth. In contrast, HRG enhanced the MEK inhibitor-induced inhibition of cell growth. These findings suggest that HRG-stimulated signaling pathways allow ERα-positive breast cancer cells to escape from growth inhibition caused by everolimus, via MAPK signaling and/or other signaling pathways. Everolimus improves progression-free survival in combination with exemestane as second-line therapy for metastatic hormone receptor-positive breast cancer. Our study suggests that HRG is a novel target for ERα-positive breast cancer therapy.

Decreased ER dependency after acquired resistance to CDK4/6 inhibitors.

BACKGROUND: Cyclin-dependent kinase (CDK) 4/6 inhibitors represent a significant advancement in the treatment of estrogen receptor (ER)-positive human epidermal growth factor receptor 2-negative advanced breast cancer. However, mechanisms of alterations after acquired resistance to CDK4/6 inhibitors and the optimal treatment options are still not established. METHODS: Abemaciclib-resistant cell lines were established from the models of estrogen deprivation-resistant cell lines which retained ER expression and activated ER function derived from MCF-7 breast cancer cell lines. Ribocilib-resistant cell lines were established in the same method as previously reported. RESULTS: Both abemaciclib- and ribociclib-resistant cell lines showed decreased ER expression. ER transcriptional activity was maintained in these cell lines; however, the sensitivity to 4-hydroxytamoxifen and fulvestrant was almost completely lost. These cell lines did not exhibit any ERα gene mutation. Abemaciclib-resistant cell lines demonstrated low sensitivity to other CDK4/6 inhibitors; sensitivities to PI3K inhibitor, mTOR inhibitor, and chemotherapeutic drugs were maintained. CONCLUSIONS: Dependence on ER signaling appears to decrease after the development of acquired resistance to CDK4/6 inhibitors. Further, CDK4/6 inhibitor-resistant cells acquired cross-resistance to other CDK4/6 inhibitors, PI3K/Akt/mTOR inhibitor therapy and chemotherapeutic drugs might serve as optimal treatment options for such breast cancers.

Characterization of a membrane-associated estrogen receptor in breast cancer cells and its contribution to hormone therapy resistance using a novel selective ligand.

The estrogen receptor (ER) plays a role in the progression of hormone-dependent breast cancer and is a hormone therapy target. Estrogen acts as a transcription factor (genomic action) and also produces a quick non-genomic reaction through intracellular signaling pathways. The membrane associated ER (mER) may regulate both these signals and hormone therapy resistance. However, the details remain unclear because a reliable method to distinguish the signals induced by the estradiol (E2)-mER and E2-nuclear ER complex has not been established. In the present study, we prepared the novel ligand Qdot-6-E2, selective for mER, by coupling E2 with insoluble quantum dot nano-beads. We investigated the characteristics of mER signaling pathways and its contribution to hormone therapy resistance using different cell lines including estrogen depletion resistant (EDR) cells with different mechanisms. Qdot-6-E2 stimulated proliferation of nuclear ER-positive cells, but nuclear ER-negative cells showed no response. In addition, Qdot-6-E2 indirectly activated nuclear ER and increased mRNA expression of target genes. We confirmed that E2 was not dissociated from Qdot-6-E2 using a mammalian one-hybrid assay. We visually demonstrated that Qdot-6-E2 acts from the outside of cells. The gene expression profile induced by Qdot-6-E2-mER was different from that induced by E2-nuclear ER. The effect of anti-ER antibody, the GFP-ER fusion protein localization, and the effect of palmitoyl acyltransferase inhibitor also indicated the existence of mER. Regarding intracellular phosphorylation signaling pathways, the MAPK (Erk 1/2) and the PI3K/Akt pathways were both activated by Qdot-6-E2. In EDR cells, only nuclear ER-positive cells showed increased cell proliferation with increased localization of ERα to the membrane fraction. These findings suggested that Qdot-6-E2 reacts with ERα surrounding the cell membrane and that mER signals help the cells to survive under estrogen-depleted conditions by re-localizing the ER to use trace amounts of E2 more effectively. We expect that Qdot-6-E2 is a useful tool for studying the mER.

The p21 levels have the potential to be a monitoring marker for ribociclib in breast cancer.

Although cyclin-dependent kinase (CDK) 4/6 inhibitors have exhibited remarkable results for patients with estrogen receptor (ER)-positive breast cancer in clinical trials, the mechanism of CDK4/6 inhibitor resistance remains unclear. Thus, this study aimed to investigate the mechanism of CDK4/6 inhibitor resistance using two CDK4/6 inhibitor resistant breast cancer cell lines. We established CDK6 overexpressed cell lines (MCF7-C6) from MCF-7 cells using the stably transfected CDK6 expression vector. Additionally, acquired ribociclib-resistant (RIBR) cell lines were created using ER-positive hormone-resistant cell lines by long-term exposure to ribociclib. CDK6 overexpression and the knockdown of CDK4 experiments highlight the significance of high levels of CDK4 and low levels of CDK6 in CDK4/6 inhibitor sensitivity. Moreover, RIBR cell lines did not exhibit incremental CDK6 compared with ER-positive hormone-resistant cell lines. In MCF7-C6 and RIBR cell lines, p21 levels decreased, and p21 levels were proportional to CDK4/6 inhibitor sensitivity. This study suggests that overexpression of CDK6 is one of the many possible mechanisms of resistance to CDK4/6 inhibitors. Furthermore, p21 levels have the potential to serve as a marker for CDK4/6 inhibitors independent of the resistance mechanism.

Tumor microenvironmental growth factors induce long-term estrogen deprivation resistance in breast cancer.

BACKGROUND: Hormonal therapy is an effective treatment for luminal-like breast cancer. Aromatase inhibitor (AI) is widely used for estrogen receptor-positive, postmenopausal breast cancers. However, resistance is occurred and becomes a serious clinical concern. In general, progression of cancer strongly depends on tumor microenvironment, which may, therefore, also contribute to the development of AI resistance. METHODS: We evaluated tumor microenvironment-derived factors with respect to AI resistance using typical estrogen receptor-positive breast cancer cell lines. We established tumor microenvironment-dependent AI-resistant models and elucidated the underlying mechanisms. RESULTS: T-47D cells had a higher dependence on microenvironment-derived factors, such as estrogen or growth factors, for survival than MCF-7 cells. We, therefore, evaluated tumor microenvironment growth factors with respect to AI resistance using T-47D cells. We established three resistant cell lines (V1, V2, and V3) that survived estrogen deprivation and growth factor-supplemented conditions. These cell lines were deficient in estrogen receptor α expression and estrogen-dependent growth. Among six representative growth factors, epidermal growth factor was the most influential. In these models, HER2 protein was overexpressed without gene amplification and intracellular phosphorylation pathways were activated compared to parental cell lines. Molecular targeting inhibitors revealed that V1 and V2 primarily rely on the PI3 K pathway for survival, whereas V3 relies on the MAPK pathway. CONCLUSIONS: This study demonstrates the importance of tumor microenvironment-derived factors for the development of AI resistance. These resistant models did not utilize the same resistance mechanism, suggesting that flexible strategies are essential in conquering resistance.

Utility of Ki67 labeling index, cyclin D1 expression, and ER-activity level in postmenopausal ER-positive and HER2-negative breast cancer with neoadjuvant chemo-endocrine therapy.

In this study, we investigated the relationships of pathological response after neoadjuvant chemo-endocrine therapy with alterations in the Ki67 labeling index (LI), expression of cyclin D1 (CCND1) and progesterone receptor (PgR), and estrogen receptor (ER) activity in breast cancer. A total of 43 Japanese post-menopausal ER-positive and human epidermal growth factor receptor 2-negative invasive breast cancer patients with tumors >2 cm or positive lymph nodes were enrolled. Exemestane alone was administered for 2 months. Neoadjuvant chemo-endocrine therapy included four cycles of doxorubicin plus paclitaxel followed by weekly paclitaxel. The immunohistochemical expression of Ki67 LI, CCND1, and PgR, and ER activity were evaluated using core needle biopsy samples at the pretreatment and post-exemestane alone stages. ER activity was evaluated through transfection of an adenovirus vector carrying an estrogen-response element-green fluorescent protein gene. In current study, marked pathological responses (including 4.7% with pathological complete response) were obtained in 34.9% of patients. Ki67 LI and PgR expression were significantly decreased after treatment. High Ki67 LI at pretreatment was a significant predictive factor of marked pathological response. At the stage of post-exemestane alone, Ki67 LI was not significantly associated with pathological response; however, high CCND1 expression was significantly correlated with high Ki67 LI. Moreover, low-level ER activity at the post-exemestane alone stage was significantly associated with marked pathological response. In conclusions, pretreatment Ki67 LI was a predictor of response to neoadjuvant chemo-endocrine therapy. CCND1 expression and ER activity at the post-endocrine therapy alone stage may be useful in determining further treatments.

Automated Quantification of Extranuclear ERα using Phosphor-integrated Dots for Predicting Endocrine Therapy Resistance in HR+/HER2- Breast Cancer.

In addition to genomic signaling, Estrogen receptor alpha (ERα) is associated with cell proliferation and survival through extranuclear signaling contributing to endocrine therapy (ET) resistance. However, the relationship between extranuclear ERα and ET resistance has not been extensively studied. We sought to measure extranuclear ERα expression by immunohistochemistry using phosphor-integrated dots (IHC-PIDs) and to assess its predictive value for ET resistance. After quantitative detection of ERα by IHC-PIDs in vitro, we developed "the nearest-neighbor method" to calculate the extranuclear ERα. Furthermore, tissue sections from 65 patients with HR+/HER2- BC were examined by IHC-PIDs, and the total ERα, nuclear ERα, extranuclear ERα PIDs score, and ratio of extranuclear-to-nuclear ERα (ENR) were measured using the novel method. We demonstrate that quantification of ERα using IHC-PIDs exhibited strong correlations to real-time qRT-PCR (r2 = 0.94) and flow cytometry (r2 = 0.98). High ERα ENR was significantly associated with poor overall survival (p = 0.048) and disease-free survival (DFS) (p = 0.007). Multivariate analysis revealed that the ERα ENR was an independent prognostic factor for DFS [hazard ratio, 3.8; 95% CI, 1.4-11.8; p = 0.006]. Our automated measurement has high accuracy to localize and assess extranuclear ERα. A high ERα ENR in HR+/HER2- BC indicates decreased likelihood of benefiting from ET.

Cancer stem-like properties of hormonal therapy-resistant breast cancer cells.

BACKGROUND: Presently, hormonal therapy targeting estrogen receptors is the most effective treatment available for luminal breast cancer. However, many patients relapse after the therapy. It has been suggested that cancer stem-like cells are involved with hormonal therapy resistance; in the present study, we evaluated this hypothesis. METHODS: In the present study, we used our previously established hormonal therapy-resistant cell lines, including aromatase inhibitor (AI)-resistant cells (Type 1 and Type 2) and fulvestrant-resistant cells (MFR). RESULTS: AI-resistant cell lines expressing ER (Type 1 V1 and V2) showed high cancer stemness in terms of their CD44/CD24 expression and side populations, which were stimulated by the addition of estrogen and inhibited by fulvestrant. However, ALDH activity was lower than in the ER-negative resistant cells, suggesting that the stemness of luminal cells is distinct from that of basal-like breast cancer cells. The migration and invasion activity of the ER-positive Type 1 V1 and V2 cells were higher than in the ER-negative cell lines, Type 2 and MFR. CONCLUSIONS: Fractionation of parental cells based on CD44/CD24 expression and colony formation assay indicated that CD44+/CD24+ cells might be the origin of hormonal therapy-resistant cells. This population reconstituted various other subpopulations under estrogen deprivation. These results indicate that hormonal therapy resistance is closely related to the cancer stem cell-like properties of luminal breast cancer.

Compensatory role of insulin-like growth factor 1 receptor in estrogen receptor signaling pathway and possible therapeutic target for hormone therapy-resistant breast cancer.

BACKGROUND: Hormone therapy targeting the estrogen receptor (ER) pathway is the most common treatment used for ER-positive breast cancer. However, some patients experience de novo or acquired resistance, which becomes a critical problem. Activation of the insulin-like growth factor (IGF) pathway allows breast cancer cells to proliferate and is associated with the ER pathway. Little is known about the role of the IGF pathway in hormone therapy and resistance; therefore, we investigated whether the inhibition of this pathway may represent a novel therapeutic target for overcoming hormone therapy resistance in ER-positive breast cancers. METHODS: Crosstalk between the ER and IGF pathways was analyzed in breast cancer cell lines by inhibiting or stimulating either one or both pathways. We studied the effect of insulin-like growth factor one receptor (IGF1R) inhibition in aromatase inhibitor-resistant breast cancer cell lines and fulvestrant-resistant cell lines which were uniquely established in our laboratory. RESULTS: Under normal conditions, IGF signaling is controlled by ER signaling to promote cell growth. Temporary disruption of the estrogen supply results in attenuated ER signaling, and IGF-1 dramatically increased relative growth compared with normal conditions. In addition, IGF1R inhibitor strongly suppressd cell growth in hormone-resistant breast cancer cells where ER remains than cells where ER decreased or was almost lost. CONCLUSIONS: Our study suggests that inhibition of the IGF pathway may be an effective strategy for ER-positive breast cancer therapy, even in hormone therapy-resistant cases.

Kit-independent mast cell adhesion mediated by Notch.

Kit/CD117 plays a crucial role in the cell-cell and cell-matrix adhesion of mammalian mast cells (MCs); however, it is unclear whether other adhesion molecule(s) perform important roles in the adhesion of MCs. In the present study, we show a novel Kit-independent adhesion mechanism of mouse cultured MCs mediated by Notch family members. On stromal cells transduced with each Notch ligand gene, Kit and its signaling become dispensable for the entire adhesion process of MCs from tethering to spreading. The Notch-mediated spreading of adherent MCs involves the activation of signaling via phosphatidylinositol 3-kinases and mitogen-activated protein kinases, similar to Kit-mediated spreading. Despite the activation of the same signaling pathways, while Kit supports the adhesion and survival of MCs, Notch only supports adhesion. Thus, Notch family members are specialized adhesion molecules for MCs that effectively replace the adhesion function of Kit in order to support the interaction of MCs with the surrounding cellular microenvironments.

Acquired resistance to everolimus in aromatase inhibitor-resistant breast cancer.

We previously reported the establishment of several types of long-term estrogen-depleted-resistant (EDR) cell lines from MCF-7 breast cancer cells. Type 1 EDR cells exhibited the best-studied mechanism of aromatase inhibitor (AI) resistance, in which estrogen receptor (ER) expression remained positive and PI3K signaling was upregulated. Type 2 EDR cells showed reduced ER activity and upregulated JNK-related signaling. The mTOR inhibitor everolimus reduced growth in cells similar to Type 1 EDR cells. The present study generated everolimus-resistant (EvR) cells from Types 1 and 2 EDR cells following long-term exposure to everolimus in vitro. These EvR cells modeled resistance to AI and everolimus combination therapies following first-line AI treatment failure. In Type 1 EvR cells, everolimus resistance was dependent on MAPK signaling; single agents were not effective, but hormonal therapy combined with a kinase inhibitor effectively reduced cell growth. In Type 2 EvR cells, ER expression remained negative and a JNK inhibitor was ineffective, but a Src inhibitor reduced cell growth. The mechanism of acquired everolimus resistance appears to vary depending on the mechanism of AI resistance. Strategies targeting resistant tumors should be tailored based on the resistance mechanisms, as these mechanisms impact therapeutic efficacy.

Overcoming aromatase inhibitor resistance in breast cancer: possible mechanisms and clinical applications.

Estrogen plays crucial roles in the progression of hormone-dependent breast cancers through activation of nuclear estrogen receptor α (ER). Estrogen is produced locally from circulating inactive steroids and adrenal androgens in postmenopausal women. However, conversion by aromatase is a rate-limiting step in intratumoral estrogen production in breast cancer. Aromatase inhibitors (AIs) inhibit the growth of hormone-dependent breast cancers by blocking the conversion of adrenal androgens to estrogen and by unmasking the inhibitory effect of androgens, acting via the androgen receptor (AR). AIs are thus a standard treatment option for postmenopausal hormone-dependent breast cancer. However, although initial use of AIs provides substantial clinical benefit, some breast cancer patients relapse because of the acquisition of AI resistance. A better understanding of the mechanisms of AI resistance may contribute to the development of new therapeutic strategies and aid in the search for new therapeutic targets and agents. We have investigated AI-resistance mechanisms and established six AI-resistant cell lines. Some of them exhibit estrogen depletion-resistance properties via constitutive ER-activation or ER-independent growth signaling. We examined how breast cancer cells can adapt to estrogen depletion and androgen superabundance. Estrogen and estrogenic androgen produced independently from aromatase contributed to cell proliferation in some of these cell lines, while another showed AR-dependent cell proliferation. Based on these findings, currently proposed AI-resistance mechanisms include an aromatase-independent estrogen-producing pathway, estrogen-independent ER function, and ER-independent growth signaling. This review summarizes several hypotheses of AI-resistance mechanisms and discusses how existing or novel therapeutic agents may be applied to treat AI-resistant breast cancers.

Correlation between cell aggregation and antibody production in IgE-producing plasma cells.

Allergic conditions result in the increase of immunoglobulin (Ig)E-producing plasma cells (IgE-PCs); however, it is unclear how IgE production is qualitatively controlled. In this study, we found that IgE-PCs in spleen of immunized mice formed homotypic cell aggregates. By employing IgE-producing hybridomas (IgE-hybridomas) as a model of IgE-PCs, we showed that these cells formed aggregates in the presence of specific antigens (Ags). The formation of the Ag-induced cell aggregation involved secreted IgE and Fcγ receptor (FcγR)II/FcγRIII, but not FcεRs. Ag-induced cell aggregation plus lipopolysaccharide signaling resulted in an enhancement of IgE production in aggregated IgE-hybridomas. Furthermore, the administration of anti-FcγRII/FcγRIII antagonistic monoclonal antibody to immunized mice tended to reduce the splenic IgE-PC aggregation as well as the serum IgE levels. Taken together, our results suggested that Ag-IgE complexes induced IgE-PCs aggregation via FcγRII/FcγRIII, leading to the enhancement of IgE production. These findings suggest the presence of a novel mechanism for regulation of IgE production.

Single CpG site methylation controls estrogen receptor gene transcription and correlates with hormone therapy resistance.

Hormone therapy is the most effective treatment for patients with estrogen receptor α-positive breast cancers. However, although resistance occurs during treatment in some cases and often reflects changed estrogen receptor α status, the relationship between changes in estrogen receptor α expression and resistance to therapy are poorly understood. In this study, we identified a mechanism for altered estrogen receptor α expression during disease progression and acquired hormone therapy resistance in aromatase inhibitor-resistant breast cancer cell lines. Subsequently, we investigated promoter switching and DNA methylation status of the estrogen receptor α promoter, and found marked changes of methylation at a single CpG site (CpG4) in resistant cells. In addition, luciferase reporter assays showed reduced transcriptional activity from this methylated CpG site. This CpG region was also completely conserved among species, suggesting that it acts as a methylation-sensitive Ets-2 transcription factor binding site, as confirmed using chromatin immunoprecipitation assays. In estrogen receptor α-positive tumors, CpG4 methylation levels were inversely correlated with estrogen receptor α expression status, suggesting that single CpG site plays an important role in the regulation of estrogen receptor α transcription.

Different epigenetic mechanisms of ERα implicated in the fate of fulvestrant-resistant breast cancer.

Approximately 70% of breast cancers express estrogen receptor α (ERα), which plays critical roles in breast cancer development. Fulvestrant has been effectively used to treat ERα-positive breast cancer, although resistance remains a critical problem. To elucidate the mechanism of resistance to fulvestrant, we established fulvestrant-resistant cell-lines named MFR (MCF-7 derived fulvestrant resistance) and TFR (T-47D derived fulvestrant resistance) from the ERα-positive luminal breast cancer cell lines MCF-7 and T-47D, respectively. Both fulvestrant-resistant cell lines lost sensitivity to estrogen and anti-estrogens. We observed diminished ERα expression at both the protein and mRNA levels. To address the mechanism of gene expression regulation, we examined epigenetic alteration, especially the DNA methylation level of ERα gene promoters. MFR cells displayed high methylation levels upstream of the ERα gene, whereas no change in DNA methylation was observed in TFR cells. Hence, we examined the gene expression plasticity of ERα, as there are differences in its reversibility following fulvestrant withdrawal. ERα gene expression was not restored in MFR cells, and alternative intracellular phosphorylation signals were activated. By contrast, TFR cells exhibited plasticity of ERα gene expression and ERα-dependent growth; moreover, these cells were resensitized to estrogen and anti-estrogens. The difference in epigenetic regulation among individual cells might explain the difference in the plasticity of ERα expression. We also identified an MFR cell-activating HER/Src-Akt/MAPK pathway; thus, the specific inhibitors effectively blocked MFR cell growth. This finding implies the presence of multiple fulvestrant resistance mechanisms and suggests that the optimal therapies differ among individual tumors as a result of differing epigenetic mechanisms regulating ERα gene expression.