Gamma-aminobutyric acid (GABA) is a non-protein amino acid and contained in various foods, such as fruits and vegetables. Although various health benefits of GABA have been reported, GABA seems to have unidentified functions. In this study, we first show increase in intracellular GABA level in GABA-treated breast cancer cell lines, MCF-7 and T-47D, in amino acid analyses and immunofluorescence. In our experimental condition, GABA treatment reduced estradiol-stimulated cell growth in these cell lines. In addition, we performed mouse early breast cancer induction model system to investigate GABA function in mammary dysplasia formation. In the results, GABA administration reduced dysplasia formation, suggesting that GABA prevents breast tumorigenesis. This study demonstrates a novel function of GABA to maintain normal breast tissues. ### Competing Interest Statement AN and HH are employees of Sanwa Shurui Co., Ltd. ST has no conflicts of interest associated with this manuscript. HS and JI are managers of COT. MT received research funding from Sanwa Shurui Co., Ltd.

Comparison of body weights of 4 experimental groups on day 20 after inoculation. Body weights graph corresponds to Fig. 5A-D.

The effect of everolimus on expression and phosphorylation of YBX1, ERK/p90RSK and AKT/mTOR signaling molecules in six TNBC cell lines.

YBX1 and pYBX1 (S102) expression of 397 invasive breast cancer patients obtained from TCGA (Breast Invasive Carcinoma (TCGA, Provisional).

Expression of YBX1, pYBX1 and downstream molecules in 6 human breast cancer cell lines.

Supplementary Materials and Methods

ATM gene mutation carriers are predisposed to estrogen-receptor-positive breast cancer (BC). ATM prevents BC oncogenesis by activating p53 in every cell; however, much remains unknown about tissue-specific oncogenesis after ATM loss. Here, we report that ATM controls the early transcriptional response to estrogens. This response depends on topoisomerase II (TOP2), which generates TOP2-DNA double-strand break (DSB) complexes and rejoins the breaks. When TOP2-mediated ligation fails, ATM facilitates DSB repair. After estrogen exposure, TOP2-dependent DSBs arise at the c-MYC enhancer in human BC cells, and their defective repair changes the activation profile of enhancers and induces the overexpression of many genes, including the c-MYC oncogene. CRISPR/Cas9 cleavage at the enhancer also causes c-MYC overexpression, indicating that this DSB causes c-MYC overexpression. Estrogen treatment induced c-Myc protein overexpression in mammary epithelial cells of ATM-deficient mice. In conclusion, ATM suppresses the c-Myc-driven proliferative effects of estrogens, possibly explaining such tissue-specific oncogenesis.

IC50 values of fulvestrant, everolimus, AZD8055 and U0126 in ER(+)/HER2(-) and ER(-)/HER2(+) human breast cancer cell lines.

Overview of pathological diagnosis and treatment status of patients refractory to endocrine therapeutics.

Diet-based prevention of malignant transformation contributes to the maintenance of quality of life by avoiding a battle against cancer. Invasion is one of the features of malignant breast cancer, and the prevention of invasion may reduce breast cancer malignancy. A recently established early breast cancer model system showed mammary ductal dysplasia with invasion in mice. This study utilized the model system and investigated the effect of fermented barley extract (FBE), a food material. The elastic fiber layer is the outermost layer of the mammary duct. A reduction in the elastic fiber layer was observed in the mammary glands of the model system, whereas supplementation with 8% FBE containing water prevented this reduction. Moreover, we found that FBE supplementation prevented mammary epithelial cell invasion. Based on our findings, FBE might be a candidate material for a diet-based prevention of early breast cancer invasion.

Anticancer agents are used for cancer therapy. Studies on the biological response to treatment with an agent facilitate its effective use. Eribulin mesylate (eribulin) is an anticancer agent. In this study, we found that c-Fos is upregulated in response to eribulin treatment in the triple-negative breast cancer cell lines MDA-MB-231 and HCC70, which have low eribulin sensitivity. c-Fos expression was not upregulated in other cell lines investigated, including high eribulin-sensitive cells. We hypothesized that c-Fos upregulation is involved in low eribulin sensitivity and thus used the c-Fos inhibitor, T-5224. In MDA-MB-231 and HCC70 cells, combined treatment with eribulin and T-5224 showed a stronger anticancer effect than treatment with eribulin alone in cell growth assays, cell death assays and a mouse xenograft tumor model, whereas T-5224 alone showed no anticancer effect. These results suggest that T-5224 may enhance the anticancer effect of eribulin. Our findings contribute to the improvement of cancer therapy.

BACKGROUND:The regeneration of larvae zebrafish fin emerged as a new model of regeneration in the last decade. In contrast to genetic tools to study fin regeneration, chemical probes to modulate and interrogate regeneration processes are not well developed.RESULTS:We set up a zebrafish larvae fin regeneration assay system and tested activities of natural product compounds and extracts, prepared from various microbes. Colomitide C, a recently isolated product from a fungus obtained from Antarctica, inhibited larvae fin regeneration. Using fluorescent reporter transgenic lines, we show that colomitide C inhibited fibroblast growth factor (FGF) signaling and WNT/β-catenin signaling, which were activated after larvae fin amputation. By using the endothelial cell reporter line and immunofluorescence, we showed that colomitide C did not affect migration of the blood vessel and nerve into the injured larvae fin. Colomitide C did not show any cytotoxic activities when tested against FGF receptor-amplified human cancer cell lines.CONCLUSION:Colomitide C, a natural product, modulated larvae fin regeneration likely acting upstream of FGF and WNT signaling. Colomitide C may serve as a template for developing new chemical probes to study regeneration and other biological processes.

Abstract Nuclear expression of Y-box–binding protein (YBX1) is closely correlated with clinical poor outcomes and drug resistance in breast cancer. Nuclear translocation of YBX1 is facilitated by YBX1 phosphorylation at serine 102 by AKT, p70S6K, and p90RSK, and the phosphorylated YBX1 (pYBX1) promotes expression of genes related to drug resistance and cell growth. A forthcoming problem to be addressed is whether targeting the phosphorylation of YBX1 overcomes antiestrogen resistance by progressive breast cancer. Here, we found that increased expression of pYBX1 was accompanied by acquired resistance to antiestrogens, fulvestrant and tamoxifen. Forced expression of YBX1/S102E, a constitutive phosphorylated form, resulted in acquired resistance to fulvestrant. Inversely, YBX1 silencing specifically overcame antiestrogen resistance. Furthermore, treatment with everolimus, an mTORC1 inhibitor, or TAS0612, a novel multikinase inhibitor of AKT, p70S6K, and p90RSK, suppressed YBX1 phosphorylation and overcame antiestrogen resistance in vitro and in vivo. IHC analysis revealed that expression of pYBX1 and YBX1 was augmented in patients who experienced recurrence during treatment with adjuvant endocrine therapies. Furthermore, pYBX1 was highly expressed in patients with triple-negative breast cancer compared with other subtypes. TAS0612 also demonstrated antitumor effect against triple-negative breast cancer in vivo. Taken together, our findings suggest that pYBX1 represents a potential therapeutic target for treatment of antiestrogen-resistant and progressive breast cancer.

Mammary ductal dysplasia is a phenotype observed in precancerous lesions and early-stage breast cancer. However, the mechanism of dysplasia formation remains elusive. Here we show, by establishing a novel dysplasia model system, that estrogen, a female hormone, has the potential to cause mammary ductal dysplasia. We injected estradiol (E2), the most active form of estrogen, daily into scid mice with a defect in nonhomologous end joining repair and observed dysplasia formation with cell proliferation at day 30. Moreover, we found that isoflavones inhibited E2-induced dysplasia formation. Our dysplasia model system provides insight into the mechanistic understanding of breast tumorigenesis, and the development of breast cancer prevention.

Metastasis causes death in breast cancer patients. To inhibit breast cancer metastasis, we focused on integrin α6, a membrane protein that contributes to cell migration and metastasis. According to in silico analysis, we identified Asp‐358 as an integrin α6‐specific vertebrate‐conserved residue and consequently as a potential therapeutic target. Because Asp‐358 is located on the surface of the β propeller domain that interacts with other molecules for integrin α6 function, we hypothesized that a peptide with the sequence around Asp‐358 competitively inhibits integrin α6 complex formation. We treated basal‐like breast cancer cells with the peptide and observed reductions in cell migration and metastasis. The result of the immunoprecipitation assay showed that the peptide inhibited integrin α6 complex formation. Our immunofluorescence for phosphorylated paxillin, a marker of integrin‐regulated focal adhesion, showed that the peptide reduced the number of focal adhesions. These results indicate that the peptide inhibits integrin α6 function. This study identified the functional residue of integrin α6 and designed the inhibitory peptide. For breast cancer patients, metastasis inhibition therapy may be developed in the future based on this study.

Mammary ductal dysplasia is a phenotype observed in precancerous lesions and early-stage breast cancer. However, the mechanism of dysplasia formation remains elusive. Here we show, by establishing a novel dysplasia model system, that estrogen, a female hormone, has the potential to cause mammary ductal dysplasia. We injected estradiol (E2), the most active form of estrogen, daily into scid mice with a defect in non-homologous end joining repair and observed dysplasia formation with cell proliferation at day 30. The protooncogene Myc is a downstream target of estrogen signaling, and we found that its expression is augmented in mammary epithelial cells in this dysplasia model. Treatment with a Myc inhibitor reduced E2-induced dysplasia formation. Moreover, we found that isoflavones inhibited E2-induced dysplasia formation. Our dysplasia model system provides insights into the mechanistic understanding of breast tumorigenesis and the development of breast cancer prevention.