-
Mariko Masuda,
Yasuhiro Miki, Shuko Hata,
Kiyoshi Takagi,
Minako Sakurai,
Katsuhiko Ono,
Koyu Suzuki,
Yang Yang,
Eriko Abe,
Hisashi Hirakawa,
Takanori Ishida,
Takashi Suzuki,
Noriaki Ohuchi,
Hironobu Sasano
[show abstract]
[hide abstract]
ABSTRACT: Estrogen plays an important role in the development of estrogen-dependent breast carcinoma. Recently, several studies demonstrated a possible involvement of several micro RNAs (miRNAs) in the development of resistance to endocrine therapy in breast cancer patients, but the correlation between estrogen actions and miRNA expression in breast carcinoma still remains largely unknown. Therefore, in this study, we examined the in vitro effects of estrogen upon miRNA expression profiles in breast carcinoma.
We first screened the miRNA expression profiles induced by 17β-Estradiol (E2) using RT2 miRNA PCR Array in the ER-positive breast carcinoma cell line MCF-7. We identified miR-7 as the important miRNA associated with estrogen actions in these cells and further examined the changes of estrogen-dependent EGFR expression by miR-7 in ER-positive or -negative breast carcinoma cell lines including MCF-7. We also evaluated the correlation between miR-7 and EGFR expression in breast carcinoma cells derived from 21 patients using laser capture microdissection combined with quantitative reverse transcriptase-PCR.
Seventeen miRNAs were significantly induced by E2 treatment in the MCF-7 cell line. Among 17 miRNAs induced by estradiol treatment, only miR-7 expression was significantly decreased by subsequent ICI treatment. The expression of miR-7 was up-regulated 2.94-fold by E2 treatment. miR-7 was reported to suppress epidermal growth factor receptor (EGFR) expression in several human malignancies. Transfection of miR-7 significantly suppressed EGFR mRNA levels in MCF-7 cells. Depletion of E2 from cell culture media also increased the expression level of EGFR mRNA in MCF-7 and T-47D cells but not in ER-negative, MDA-MB-231 and SK-BR-3 cells. We also evaluated the status of miR-7 in breast carcinoma tissues, but the correlation between the status of miR-7 and EGFR in carcinoma cells isolated by laser capture microscopy was not detected.
These results suggest that miR-7 may play a role in the development of resistance to endocrine therapy in breast cancer patients through regulating EGFR expression of carcinoma cells.
Journal of Translational Medicine 09/2012; 10 Suppl 1:S2. · 3.41 Impact Factor
-
Yukiko Shibahara,
Yasuhiro Miki,
Yoshiaki Onodera, Shuko Hata,
Monica Sm Chan,
Christopher Cp Yiu,
Tjing Y Loo,
Yasuhiro Nakamura,
Jun-Ichi Akahira,
Takanori Ishida,
Keiko Abe,
Hisashi Hirakawa,
Louis Wc Chow,
Takashi Suzuki,
Noriaki Ouchi,
Hironobu Sasano
[show abstract]
[hide abstract]
ABSTRACT: Aromatase inhibitors (AIs) are considered the gold standard of endocrine therapy for oestrogen receptor-positive postmenopausal breast cancer patients. AI treatment was reported to result in marked alterations of genetic profiles in cancer tissues but its detailed molecular mechanisms have not been elucidated. Therefore, we profiled miRNA expression before and after treatment with letrozole in MCF-7 co-cultured with primary breast cancer stromal cells. Letrozole significantly altered the expression profiles of cancer miRNAs in vitro. Among the elevated miRNAs following letrozole treatment, computational analysis identified let-7f, a tumour-suppressor miRNA which targeted the aromatase gene (CYP19A1) expression. Quantitative real-time PCR assay using MCF-7 and SK-BR-3 cells as well as clinical specimens of a neoadjuvant study demonstrated a significant inverse correlation between aromatase mRNA and let-7f expression. In addition, high let-7f expression was significantly correlated with low aromatase protein levels evaluated by both immunohistochemistry and the western blotting method in breast cancer cases. Results of 3'UTR luciferase assay also demonstrated the actual let-7f binding sites in CYP19A1, indicating that let-7f directly targets the aromatase gene. Subsequent WST-8 and migration assays performed in let-7f-transfected MCF-7 and SK-BR-3 cells revealed a significant decrement of their proliferation and migration. These findings all demonstrated that let-7f, a tumour suppressor miRNA in breast cancer, directly targeted the aromatase gene and was restored by AI treatment. Therefore, AIs may exert tumour-suppressing effects upon breast cancer cells by suppressing aromatase gene expression via restoration of let-7f.
The Journal of Pathology 03/2012; 227(3):357-66. · 6.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Breast cancer tissue consists of both carcinoma cells and stromal cells, and intratumoral stroma is composed of various cell types such as fibroblasts, adipocytes, inflammatory including lymphocytes and macrophage and lymphatic and blood capillaries including pericytes and endothelial cells. Recently, cell-cell communications or interactions among these cells have been considered to play an important role to cancer initiation, promotion, and progression. In particular, intratumoral fibroblasts are well known as cancer-associated fibroblast (CAF). CAF is considered to be different from normal fibroblasts in terms of promoting cancer progression through the cytokine signals. Carcinoma cell lines have contributed to the advancement of our understanding of cancer cell biology. Numerous researches have employed these carcinoma cell lines as a single- or mono-culture. However, it is also true that this mono-culture system cannot evaluate interactions between carcinoma and intratumoral stromal cells. Co-culture compositions of two different cell type of cancer tissues i.e., carcinoma cell lines and fibroblasts, were established in order to evaluate cell-cell interactions in these cancer microenvironment. This co-culture condition has the advantage of evaluating cell-cell interactions of cancer microenvironment. Therefore, in this review, we focused upon co-culture system and its application to understanding of various biological phenomenon as an ex vivo evaluation method of cancer microenvironment in breast cancer.
The Journal of steroid biochemistry and molecular biology 01/2012; 131(3-5):68-75. · 2.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A tumor-suppressor gene, let-7 microRNA (miRNA) family, is often inactivated in various human malignancies. LIN28 is a RNA-binding protein that has been well characterized for regulation of let-7 maturation in undifferentiated embryonic stem cells at post-transcriptional level. Oncogenic regulation of let-7 miRNAs has been demonstrated in several human malignancies but their correlation with LIN28 has not been studied in breast cancer. We therefore explored a possible mechanism of tumorigenesis in breast carcinoma tissue via an alternation of let-7 miRNA precursor processing by LIN28 in this study. A total of 26 breast cancer surgical pathology specimens were evaluated for LIN28 and LIN28B expression using immunohistochemistry. We then isolated carcinoma cells in 21 cases using laser capture microdissection, and the miRNAs from these samples were profiled using PCR array analysis. LIN28 status was positively correlated with ERα, PR, and Ki-67 status and inversely correlated with HER2 status. These results suggest the possible involvement of LIN28 in regulation of sex steroid dependent cell proliferation of breast carcinoma cells. We further demonstrated that expression of let-7a, let-7c, let-7d (P=0.026) and let-7f (P=0.016) were inversely correlated with those of LIN28. These results also suggest that LIN28 promotes tumorigenic activity by suppressing let-7 miRNA maturation in breast carcinoma cells.
The Journal of steroid biochemistry and molecular biology 11/2011; 131(3-5):101-6. · 2.66 Impact Factor
-
Yasuhiro Miki,
Takashi Suzuki,
Keiko Abe,
Satoshi Suzuki,
Hiromichi Niikawa,
Shinya Iida, Shuko Hata,
Jun-ichi Akahira,
Kazushige Mori,
Dean B Evans,
Takashi Kondo,
Hisafumi Yamada-Okabe,
Hironobu Sasano
[show abstract]
[hide abstract]
ABSTRACT: Estrogens produced as a result of intratumoral aromatization has been recently shown to play important roles in proliferation of human non-small cell lung carcinomas (NSCLC), but the details have remained largely unknown. Therefore, in this study, we evaluated the possible roles of intratumoral aromatase in NSCLCs as follows: (a) evaluation of intratumoral localization of aromatase mRNA/protein in six lung adenocarcinoma cases using laser capture microdissection combined with quantitative reverse transcriptase-PCR and immunohistochemistry; (b) examination of the possible effects of isolated stromal cells from lung carcinoma tissues on aromatase mRNA transcript expression in lung carcinoma cell lines (A549 and LK87) through a coculture system; and (c) screening of cytokines derived from stromal LK001S and LK002S cells using cytokine antibody arrays and subsequent evaluation of effects of these cytokines on aromatase expression in A549 and LK87. Both aromatase mRNA and protein were mainly detected in intratumoral carcinoma cells but not in stromal cells. Aromatase expression of A549 and LK87 was upregulated in the presence of LK001S or LK002S cells. Several cytokines such as interleukin-6 (IL-6), oncostatin M, and tumor necrosis factor-alpha, all known as inducible factors of aromatase gene, were detected in conditioned media of LK001S and LK002S cells. Treatment of both oncostatin M and IL-6 induced aromatase gene expression in A549 an LK87, respectively. These results all indicated that intratumoral microenvironments, especially carcinoma-stromal cell interactions, play a pivotal role in the regulation of intratumoral estrogen synthesis through aromatase expression in human lung adenocarcinomas.
Cancer Research 08/2010; 70(16):6659-69. · 7.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The metabolism of drugs, xenobiotic compounds, and other endogenous/exogenous substrates generally begins with their oxidation through cytochrome P450 (CYP). The results of recent pharmacogenetic analyses have demonstrated CYP's polymorphisms to be related to individual differences in metabolism, but only a limited number of CYP3A4 and CYP2E1 variant alleles influence enzymatic activities. Therefore, CYP gene expression profiling of both normal and pathological human livers should provide critical information for an evaluation of the biological significance of CYPs.
In our present study, we first characterized the individual differences in CYP3A4 and CYP2E1 expression levels among Japanese normal or non-pathological liver tissue obtained from autopsy or surgery using immunohistochemistry and quantitative RT-PCR array of phase I metabolic enzymes with combined laser capture microscopy and qPCR analysis.
Both CYP3A4 and CYP2E1 mRNA and proteins were predominantly detected in hepatocytes surrounding central veins in normal liver, but there were marked individual differences in both CYP3A4 and CYP2E1 mRNA and proteins among the 23 Japanese subjects examined. Individual differences in CYP3A and CYP2E1 subtypes were also detected in the livers obtained from monozygotic neonatal Japanese female twins with different survival periods. CYP3A and CYP2E1-positive cells were decreased in number in non-pathological hepatocytes of diseased livers compared to those in disease-free livers from autopsy.
The above results suggest that individual differences in CYP3A4 and CYP2E1 exist among normal human liver tissues and in non-pathological hepatocytes between diseased and normal liver, and these differences may be important in evaluating the pharmacodynamics of various substances.
Life sciences 03/2010; 86(11-12):393-401. · 2.56 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Steroid and xenobiotic receptor (SXR) is a nuclear receptor activated by diverse exogenous and endogenous compounds and has been demonstrated to play a pivotal role in detoxification through its regulation of various metabolizing enzymes and transporters. Recent studies also demonstrated the potential roles of SXR in the regulation of apoptosis and inflammation in various carcinoma cells, but the status of SXR in human esophageal squamous cell carcinoma (ESCC) has not been examined. Therefore, in this study, we performed immunohistochemical and quantitative RT-PCR evaluations in human ESCC in order to clarify its biological and clinical significance. We first immunolocalized SXR in 73 human ESCC cases. SXR immunoreactivity was detected in the nuclei, or in both nuclei and cytoplasm of carcinoma cells (98%, 20% of cases, respectively). The status of nuclear SXR immunoreactivity was inversely correlated with histological grade, lymph node status, ki67/MIB1 labeling index, and positively correlated with retinoid X receptor alpha status. In addition, high nuclear SXR expression was significantly correlated with favorable clinical outcome of the patients. Multivariate analysis further demonstrated SXR status in carcinoma cells as an independent favorable prognostic factor of the patients. Results of quantitative RT-PCR study demonstrated that SXR mRNA expression was detected in three of five cases, and was marked higher in the cancerous tissue than non-neoplastic tissue of these patients. This is the first study to demonstrate the status of SXR in human ESCC and the results suggest that SXR is a potent favorable prognostic factor of human ESCC.
Cancer Science 10/2009; 101(2):543-9. · 3.33 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: SERMs bind to both estrogen receptor (ER)alpha and beta, resulting in tissue dependent estrogen agonist or antagonist responses. Both raloxifene and tamoxifen are most frequently used SERMs and exert estrogen agonistic effects on human bone tissues, but the details of their possible direct effects on human bone cells have remained largely unknown. In our present study, we examined the comparative effects of raloxifene, tamoxifen, and native estrogen, estradiol on human osteoblast cell line, hFOB in vitro. Both the cell numbers and the ratio of the cells in S phase fraction were significantly increased by the treatment of raloxifene or tamoxifen as well as estradiol treatments in hFOB. Gene profile patterns following treatment with raloxifene, tamoxifen, and estradiol demonstrated similar patterns in a microarray/hierarchal clustering analysis. We also examined the expression levels of these genes detected by this analysis using quantitative RT-PCR. MAF gene was induced by raloxifene treatment alone. GAS6 gene was induced by raloxifene and tamoxifen as well as estradiol. An estrogen receptor blocker, ICI 18, 286, inhibited an increase of GAS6 gene expression but not the levels of MAF gene mRNA expression. Results of our present study demonstrated that raloxifene exerted direct protective effects on human osteoblasts in both estrogen receptor dependent and independent manners.
The Journal of steroid biochemistry and molecular biology 03/2009; 113(3-5):281-9. · 2.66 Impact Factor
