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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.

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Cancer-associated fibroblasts (CAFs) exert various effects upon biological behaviours of cancer. In this study, we examined the correlation of CAFs with the intra-tumoural immune system in the lung adenocarcinoma microenvironment. We studied 27 and 113 cases of lung adenocarcinoma tentatively as Cohorts 1 and 2, respectively. The patients in Cohort 1 received epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) for recurrent lung adenocarcinoma. α-smooth muscle actin (α-SMA), a surrogate marker for CAFs, was examined by immunohistochemistry. We then examined the effects of CAFs isolated from lung cancer tissues on programmed death ligand 1 (PD-L1) expression in lung adenocarcinoma cell lines. No significant associations were detected between α-SMA status and the ratios of CD8/CD4 and Foxp3/CD8 in Cohort 1. However, α-SMA status was significantly associated with PD-L1 status in both Cohorts 1 and 2. Conditioned medium of CAFs significantly induced PD-L1 expression in lung adenocarcinoma cell lines, A549, PC-9, and H1975. Among the cytokines examined by antibody array, C-X-C motif chemokine ligand 2 (CXCL2) increased PD-L1 mRNA expression in these cell lines. CXCL2 is therefore considered to have a potential to induce PD-L1 expression in lung adenocarcinoma cells as a result of an interaction between carcinoma cells and CAFs. These findings did firstly demonstrate that CAFs indirectly influenced tumour immunity through increasing PD-L1 expression in lung adenocarcinoma cells.

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-α, 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 Res; 70(16); 6659-69. ©2010 AACR.

BackgroundEstrogen 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.MethodsWe 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.ResultsSeventeen 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.ConclusionsThese 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.

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Invasive ductal and lobular carcinomas (IDC and ILC) are the two most common histological types of breast cancer, and have been considered to develop from terminal duct lobular unit but their molecular, pathological, and clinical features are markedly different between them. These differences could be due to different mechanisms of carcinogenesis and tumor microenvironment, especially cancer-associated fibroblasts (CAFs) but little has been explored in this aspect. Therefore, in this study, we evaluated the status of angiogenesis, maturation of intratumoral microvessels, and proliferation of CAFs using immunohistochemistry and PCR array analysis to explore the differences of tumor microenvironment between ILC and IDC. We studied grade- and age-matched, luminal-like ILC and IDC. We immunolocalized CD34 and αSMA for an evaluation of CAFs and CD31, Vasohibin-1, a specific marker of proliferative endothelial cells and nestin, a marker of pericytes for studying the status of proliferation and maturation of intratumoral microvessel. We also performed PCR array analysis to evaluate angiogenic factors in tumor stromal components. The number of CAFs, microvessel density, and vasohibin-1/CD31 positive ratio were all significantly higher in ILC than IDC but nestin immunoreactivity in intratumoral microvessel was significantly lower in ILC. These results did indicate that proliferation of CAFs and endothelial cells was more pronounced in ILC than IDC but newly formed microvessels were less mature than those in IDC. PCR array analysis also revealed that IGF-1 expression was higher in ILC than IDC. This is the first study to demonstrate the differences of tumor microenvironment including CAFs and proliferation and maturation of intratumoral vessels between ILC and IDC.