ATM suppresses c-Myc overexpression in the mammary epithelium in response to estrogen.

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.

Supplementation with Fermented Barley Extract Prevents Mammary Epithelial Cell Invasion in an Early Breast Cancer Model.

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.

A nontoxic fungal natural product modulates fin regeneration in zebrafish larvae upstream of FGF-WNT developmental signaling.

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/ß-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.

Eribulin mesylate-induced c-Fos upregulation enhances cell survival in breast cancer cell lines.

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.

Estrogen Induces Mammary Ductal Dysplasia via the Upregulation of Myc Expression in a DNA-Repair-Deficient Condition.

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.

Targeting Phosphorylation of Y-Box-Binding Protein YBX1 by TAS0612 and Everolimus in Overcoming Antiestrogen Resistance.

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.

In silico analysis-based identification of the target residue of integrin α6 for metastasis inhibition of basal-like breast cancer.

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 ß 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.

BRCA1 ensures genome integrity by eliminating estrogen-induced pathological topoisomerase II-DNA complexes.

Women having BRCA1 germ-line mutations develop cancer in breast and ovary, estrogen-regulated tissues, with high penetrance. Binding of estrogens to the estrogen receptor (ER) transiently induces DNA double-strand breaks (DSBs) by topoisomerase II (TOP2) and controls gene transcription. TOP2 resolves catenated DNA by transiently generating DSBs, TOP2-cleavage complexes (TOP2ccs), where TOP2 covalently binds to 5' ends of DSBs. TOP2 frequently fails to complete its catalysis, leading to formation of pathological TOP2ccs. We have previously shown that the endonucleolytic activity of MRE11 plays a key role in removing 5' TOP2 adducts in G1 phase. We show here that BRCA1 promotes MRE11-mediated removal of TOP2 adducts in G1 phase. We disrupted the BRCA1 gene in 53BP1-deficient ER-positive breast cancer and B cells. The loss of BRCA1 caused marked increases of pathological TOP2ccs in G1 phase following exposure to etoposide, which generates pathological TOP2ccs. We conclude that BRCA1 promotes the removal of TOP2 adducts from DSB ends for subsequent nonhomologous end joining. BRCA1-deficient cells showed a decrease in etoposide-induced MRE11 foci in G1 phase, suggesting that BRCA1 repairs pathological TOP2ccs by promoting the recruitment of MRE11 to TOP2cc sites. BRCA1 depletion also leads to the increase of unrepaired DSBs upon estrogen treatment both in vitro in G1-arrested breast cancer cells and in vivo in epithelial cells of mouse mammary glands. BRCA1 thus plays a critical role in removing pathological TOP2ccs induced by estrogens as well as etoposide. We propose that BRCA1 suppresses tumorigenesis by removing estrogen-induced pathological TOP2ccs throughout the cell cycle.

Sal-like 4 protein levels in breast cancer cells are post-translationally down-regulated by tripartite motif-containing 21.

Sal-like 4 (SALL4) is a transcription factor that enhances proliferation and migration in breast cancer cells. SALL4 expression therefore has the potential to promote cancer malignancy. However, the regulatory mechanisms involved in SALL4 protein expression have not been thoroughly elucidated. In this study, we observed that treating MCF-7 and SUM159 breast cancer cell lines with a proteasome inhibitor increases SALL4 protein levels, suggesting that SALL4 is degraded by the ubiquitin-proteasome system. Using immunoprecipitation to uncover SALL4-binding proteins, we identified an E3 ubiquitin-protein ligase, tripartite motif-containing 21 (TRIM21). Using an EGFP reporter probe of the major SALL4 isoform SALL4B, we observed that shRNA-mediated knockdown of TRIM21 increases cellular SALL4B levels. Immunostaining experiments revealed that TRIM21 localizes to the nucleus, and a K64R substitution in the nuclear localization motif in SALL4B increased SALL4B levels in the cytoplasm. These results suggested that TRIM21 is involved in nuclear SALL4 degradation. To identify the amino acid residue that is targeted by TRIM21, we fragmented the SALL4B sequence, fused it to EGFP, and identified Lys-190 in SALL4B as TRIM21's target residue. Amino acid sequence alignments of SALL family members indicated that the region around SALL4 Lys-190 is conserved in both SALL1 and SALL3. Because SALL1 and SALL4 have similar functions, we constructed a SALL1-EGFP probe and found that the TRIM21 knockdown increases SALL1 levels, indicating that TRIM21 degrades both SALL1 and SALL4. Our findings extend our understanding of SALL4 and SALL1 regulation and may contribute to the development of SALL4-targeting therapies.

SALL4 - KHDRBS3 network enhances stemness by modulating CD44 splicing in basal-like breast cancer.

Understanding the mechanism by which cancer cells enhance stemness facilitates cancer therapies. Here, we revealed that a stem cell transcription factor, SALL4, functions to enhance stemness in basal-like breast cancer cells. We used shRNA-mediated knockdown and gene overexpression systems to analyze gene functions. To evaluate stemness, we performed a sphere formation assay. In SALL4 knockdown cells, the sphere formation ability was reduced, indicating that SALL4 enhances stemness. CD44 is a membrane protein and is known as a stemness factor in cancer. CD44 splicing variants are involved in cancer stemness. We discovered that SALL4 modulates CD44 alternative splicing through the upregulation of KHDRBS3, a splicing factor for CD44. We cloned the KHDRBS3-regulated CD44 splicing isoform (CD44v), which lacks exons 8 and 9. CD44v overexpression prevented a reduction in the sphere formation ability by KHDRBS3 knockdown, indicating that CD44v is positively involved in cancer stemness. In addition, CD44v enhanced anoikis resistance under the control of the SALL4 - KHDRBS3 network. Basal-like breast cancer is an aggressive subtype among breast cancers, and there is no effective therapy so far. Our findings provide molecular targets for basal-like breast cancer therapy. In the future, this study may contribute to the establishment of drugs targeting cancer stemness.

5-Chloro-2,4-dihydroxypyridine, CDHP, prevents lung metastasis of basal-like breast cancer cells by reducing nascent adhesion formation.

A drug for metastasis prevention is necessary. The orally administered anticancer drug S-1 contributes to cancer therapy. In a mouse xenograft model of metastatic breast cancer from our previous study, the administration of S-1 inhibited lung metastasis. However, the mechanism of inhibition remains elusive. S-1 contains 5-chloro-2,4-dihydroxypyridine (CDHP), which does not have the antigrowth activity, but prevents the degradation of 5-fluorouracil, an anticancer reagent. In this study, we found that CDHP treatment shrinks cell morphology in metastatic basal-like breast cancer cell lines. Wound healing assays showed reduced cell migration in CDHP-treated cells. At the molecular level, CDHP treatment reduced the number of nascent adhesions, whereas the number of mature focal adhesions was not changed. These findings indicate that CDHP impairs focal adhesion formation, which results in a reduction in cell migration. For the in vivo metastasis assay, we used a highly lung-metastatic cell line. We xenografted them into immunodeficient mice, and administered CDHP. To determine whether CDHP prevents metastasis, we measured the weights of harvested lungs. The results showed that the lung weights of the CDHP-treated animals were not significantly different compared to the no-tumor controls, whereas the vehicle group showed a number of metastatic foci and an increase in lung weight. These observations indicate that CDHP administration prevents metastasis. This study reveals a novel effect of CDHP for lung metastasis prevention. Our findings may facilitate the establishment of future metastasis prevention therapies.

Y-box binding protein YBX1 and its correlated genes as biomarkers for poor outcomes in patients with breast cancer.

The enhanced expression of the Y-box binding protein YBX1 is consistently correlated with poor outcomes or reduced survival of breast cancer patients. However, the mechanism underlying the association between increased YBX1 expression and poor outcomes has yet to be revealed. We searched a database for the top 500 genes that are positively or negatively correlated with YBX1 and with ESR1 in breast cancer patients. We further examined the association between YBX1-correlated genes and breast cancer outcomes in patients at Kyushu University Hospital. More than 60% of genes that are positively correlated with YBX1 are also negatively correlated with ESR1. The enhanced expression levels of the top 20 positively correlated genes mostly predict negative outcomes, while the enhanced expression levels of the top 20 negatively correlated genes mostly predict positive outcomes. Furthermore, in breast cancer patients at Kyushu University Hospital, the expression levels of YBX1 and YBX1-positively correlated genes were significantly higher and the expression levels of genes negatively correlated with YBX1 were significantly lower in patients who relapsed after their primary surgery than in those who did not relapse. The expression of YBX1 together with the expression of its positively or negatively correlated genes may help to predict outcomes as well as resistance to endocrine therapies in breast cancer patients. Determining the expression of YBX1 and its closely correlated genes will contribute to the development of precision therapeutics for breast cancer.

The Sal-like 4 - integrin α6ß1 network promotes cell migration for metastasis via activation of focal adhesion dynamics in basal-like breast cancer cells.

During metastasis, cancer cell migration is enhanced. However, the mechanisms underlying this process remain elusive. Here, we addressed this issue by functionally analyzing the transcription factor Sal-like 4 (SALL4) in basal-like breast cancer cells. Loss-of-function studies of SALL4 showed that this transcription factor is required for the spindle-shaped morphology and the enhanced migration of cancer cells. SALL4 also up-regulated integrin gene expression. The impaired cell migration observed in SALL4 knockdown cells was restored by overexpression of integrin α6 and ß1. In addition, we clarified that integrin α6 and ß1 formed a heterodimer. At the molecular level, loss of the SALL4 - integrin α6ß1 network lost focal adhesion dynamics, which impairs cell migration. Over-activation of Rho is known to inhibit focal adhesion dynamics. We observed that SALL4 knockdown cells exhibited over-activation of Rho. Aberrant Rho activation was suppressed by integrin α6ß1 expression, and pharmacological inhibition of Rho activity restored cell migration in SALL4 knockdown cells. These results indicated that the SALL4 - integrin α6ß1 network promotes cell migration via modulation of Rho activity. Moreover, our zebrafish metastasis assays demonstrated that this gene network enhances cell migration in vivo. Our findings identify a potential new therapeutic target for the prevention of metastasis, and provide an improved understanding of cancer cell migration.

A homeobox protein, NKX6.1, up-regulates interleukin-6 expression for cell growth in basal-like breast cancer cells.

Among breast cancer subtypes, basal-like breast cancer is particularly aggressive, and research on the molecules involved in its pathology might contribute to therapy. In this study, we found that expression of NKX6.1, a homeobox transcription factor, is higher in basal-like breast cancer than in other subtypes. In loss-of-function experiments on basal-like breast cancer cell lines, NKX6.1-depleted cells exhibited reduced cell growth. Because cytokine interleukin-6 (IL-6) is expressed in basal-like breast cancer, and increases cell growth, we analyzed expression levels of IL6, an IL-6 gene, and observed reduced IL6 expression in NKX6.1-depleted cells. In a reporter assay, IL6 promoter activity was reduced by loss of NKX6.1 function. A pull-down assay showed that NKX6.1 binds to the proximal region in IL6 promoter. These results indicate that NKX6.1 directly up-regulates IL6 expression. To investigate further, we established cells with forced expression of IL-6. We observed that exogenous IL-6 expression restored the reduced cell growth of NKX6.1-depleted cells. Furthermore, orthotopic xenografts showed that NKX6.1-depleted cells lost the capacity for tumor formation. We therefore conclude that NKX6.1 is a factor for IL-6-regulated growth and tumor formation in basal-like breast cancer. Our findings facilitate profound understanding of basal-like breast cancer, and the development of suitable therapy.

An optical labeling-based proliferation assay system reveals the paracrine effect of interleukin-6 in breast cancer.

Proliferation analysis is one of the basic approaches to characterize various cell types. In conventional cell proliferation assays, the same sample cannot be observed over time, nor can a specific group within a heterogeneous population of cells, for example, cancerous cells, be analyzed separately. To overcome these limitations, we established an optical labeling-based proliferation assay system with the Kaede protein, whose fluorescence can be irreversibly photo converted from green to red by irradiation. After a single non-toxic photoconversion event, the intensity of red fluorescence in each cell is reduced by cell division. From this, we developed a simple method to quantify cell proliferation by monitoring reduction of red fluorescence over time. This study shows that the optical labeling-based proliferation assay is a viable novel method to analyze cell proliferation, and could enhance our understanding of mechanisms regulating cell proliferation machinery. We used this newly established system to analyze the functions of secreted interleukin-6 (IL-6) in cancer cell proliferation, which had not been fully characterized. Reduction in proliferation was observed following IL-6 knockdown. However, after co-culturing with IL-6-expressing cells, the proliferation of Kaede-labeled IL-6-knockdown cells was restored. These data indicate that in basal-like breast cancer cells, IL-6 exhibits a paracrine effect to positively regulate cell proliferation. Our results thus demonstrate that cancer cells can secrete signaling molecules, such as IL-6, to support the proliferation of other cancer cells.

Data of a fluorescent imaging-based analysis of anti-cancer drug effects on three-dimensional cultures of breast cancer cells.

Three-dimensional (3D) cell culture is a powerful tool to study cell growth under 3D condition. To perform a simple test for anti-cancer drugs in 3D culture, visualization of non-proliferated cells is required. We propose a fluorescent imaging-based assay to analyze cancer cell proliferation in 3D culture. We used a pulse-labeling technique with a photoconvertible fluorescent protein Kaede to identify non-proliferated cells. This assay allows us to observe change in cell proliferation in 3D culture by simple imaging. Using this assay, we obtained the data of the effects of anti-cancer drugs, 5-fluorouracil and PD0332991 in a breast cancer cell line, MCF-7.

Regenerative responses after mild heart injuries for cardiomyocyte proliferation in zebrafish.

BACKGROUND: The zebrafish heart regenerates after various severe injuries. Common processes of heart regeneration are cardiomyocyte proliferation, activation of epicardial tissue, and neovascularization. In order to further characterize heart regeneration processes, we introduced milder injuries and compared responses to those induced by ventricular apex resection, a widely used injury method. We used scratching of the ventricular surface and puncturing of the ventricle with a fine tungsten needle as injury-inducing techniques. RESULTS: Scratching the ventricular surface induced subtle cardiomyocyte proliferation and responses of the epicardium. Endothelial cell accumulation was limited to the surface of the heart. Ventricular puncture induced cardiomyocyte proliferation, endocardial and epicardial activation, and neo-vascularization, similar to the resection method. However, the degree of the responses was milder, correlating with milder injury. Sham operation induced epicardial aldh1a2 expression but not tbx18 and WT1. CONCLUSIONS: Puncturing the ventricle induces responses equivalent to resection at milder degrees in a shorter time frame and can be used as a simple injury model. Scratching the ventricle did not induce heart regeneration and can be used for studying wound responses to epicardium.

Sal-like 4 (SALL4) suppresses CDH1 expression and maintains cell dispersion in basal-like breast cancer.

In cell cultures, the dispersed phenotype is indicative of the migratory ability. Here we characterized Sal-like 4 (SALL4) as a dispersion factor in basal-like breast cancer. Our shRNA-mediated SALL4 knockdown system and SALL4 overexpression system revealed that SALL4 suppresses the expression of adhesion gene CDH1, and positively regulates the CDH1 suppressor ZEB1. Cell behavior analyses showed that SALL4 suppresses intercellular adhesion and maintains cell motility after cell-cell interaction and cell division, which results in the dispersed phenotype. Our findings indicate that SALL4 functions to suppress CDH1 expression and to maintain cell dispersion in basal-like breast cancer.

Life-long preservation of the regenerative capacity in the fin and heart in zebrafish.

The zebrafish is a widely used model animal to study the regeneration of organs, such as the fin and heart. Their average lifetime is about 3 years, and recent studies have shown that zebrafish exhibit aging-related degeneration, suggesting the possibility that aging might affect regenerative potential. In order to investigate this possibility, we compared regeneration of the fin and heart after experimental amputation in young (6-12 month old) and old (26-36 month old) fish. Comparison of recovery rate of the caudal fin, measured every two or three days from one day post amputation until 13 days post amputation, show that fins in young and old fish regenerate at a similar rate. In the heart, myocardium regeneration and cardiomyocyte proliferation occurred similarly in the two groups. Moreover, neo-vascularization, as well as activation of fibroblast growth factor signaling, which is required for neo-vascularization, occurred similarly. The epicardial tissue is a thin layer tissue that covers the heart, and starts to express several genes immediately in response to injury. The expression of epicardial genes, such as wt1b and aldh1a2, in response to heart injury was comparable in two groups. Our results demonstrate that zebrafish preserve a life-long regenerative ability of the caudal fin and heart.

Migration of cardiomyocytes is essential for heart regeneration in zebrafish.

Adult zebrafish possess a significant ability to regenerate injured heart tissue through proliferation of pre-existing cardiomyocytes, which contrasts with the inability of mammals to do so after the immediate postnatal period. Zebrafish therefore provide a model system in which to study how an injured heart can be repaired. However, it remains unknown what important processes cardiomyocytes are involved in other than partial de-differentiation and proliferation. Here we show that migration of cardiomyocytes to the injury site is essential for heart regeneration. Ventricular amputation induced expression of cxcl12a and cxcr4b, genes encoding a chemokine ligand and its receptor. We found that cxcl12a was expressed in the epicardial tissue and that Cxcr4 was expressed in cardiomyocytes. We show that pharmacological blocking of Cxcr4 function as well as genetic loss of cxcr4b function causes failure to regenerate the heart after ventricular resection. Cardiomyocyte proliferation was not affected but a large portion of proliferating cardiomyocytes remained localized outside the injury site. A photoconvertible fluorescent reporter-based cardiomyocyte-tracing assay demonstrates that cardiomyocytes migrated into the injury site in control hearts but that migration was inhibited in the Cxcr4-blocked hearts. By contrast, the epicardial cells and vascular endothelial cells were not affected by blocking Cxcr4 function. Our data show that the migration of cardiomyocytes into the injury site is regulated independently of proliferation, and that coordination of both processes is necessary for heart regeneration.