Estrogen-related receptor α (ERRα) was identified as a gene related to estrogen receptor α (ERα) and belongs to a class of nuclear orphan receptors. ERRα binds to estrogen responsive element(s) (ERE) and is considered to be involved in modulation of estrogenic actions. However, biological significance of ERRα remains largely unknown. Therefore, we examined the expression of ERRα in human breast carcinoma tissues using immunohistochemistry (n = 102) and real-time reverse transcription-PCR (n = 30). ERRα immunoreactivity was detected in the nuclei of carcinoma cells in 55% of breast cancers examined, and relative immunoreactivity of ERRα was significantly (P = 0.0041) associated with the mRNA level. Significant associations were detected between ERα and ERE-containing estrogen-responsive genes, such as pS2 (P < 0.0001) and EBAG9/RCAS1 (P = 0.0214), in breast carcinoma tissues. However, no significant association was detected between ERα and pS2 (P = 0.1415) in the ERRα-positive cases (n = 56) or between ERα and EBAG9/RCAS1 (P = 0.8271) in the ERRα-negative group (n = 46). ERRα immunoreactivity was significantly associated with an increased risk of recurrence and adverse clinical outcome by both uni- (P = 0.0097 and P = 0.0053, respectively) and multi- (P = 0.0215 and P = 0.0118, respectively) variate analyses. A similar tendency was also detected in the group of breast cancer patients who received tamoxifen therapy after surgery. Results from our study suggest that ERRα possibly modulates the expression of ERE-containing estrogen-responsive genes, and ERRα immunoreactivity is a potent prognostic factor in human breast carcinoma.

P62/SQSTM1 is a selective substrate of autophagy, and aberrant accumulation of p62 has been observed in various pathological conditions. To understand the roles p62 plays in non‐small‐cell lung cancer (NSCLC), we carried out immunohistochemical analyses of p62 expression in a cohort of patients with annotated clinicopathological data. As analyses of murine and human hepatocellular carcinomas suggested a correlation between p62 and Nrf2 accumulations, we also examined NRF2 expression in the same cohort. The expression of NRF2 and p62 was examined by immunohistochemical methods in 109 NSCLC cases, which included patients with adenocarcinoma (n = 72), squamous cell carcinoma (n = 31), and large cell carcinoma (n = 6). Accumulation of NRF2 and p62 was detected in 34% and 37% of NSCLC patients, respectively. The accumulations of p62 and NRF2 did not correlate with each other, but both were associated with worse lung cancer‐specific survival (P = 0.0003 for NRF2; P = 0.0130 for p62). NRF2 status had an impact on NSCLC prognosis irrespective of histology types, but p62 status did so particularly in adenocarcinoma (P = 0.037). Multivariate analysis indicated that positive immunoreactivities of NRF2 and p62 were both independent factors predicting worse lung cancer‐specific survival (P < 0.0001 for NRF2 and P = 0.04 for p62). This study revealed that both NRF2 and p62 are independent prognostic factors for NSCLC. The prognostic impact of p62 status was pronounced in adenocarcinoma patients, suggesting that molecular mechanisms underlying cancer evolution differ between adenocarcinoma and squamous cell carcinoma. (Cancer Sci 2012; 103: 760–766)

Background: The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined. Methods: Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR. Results: The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial cells during bleomycin-induced lung fibrosis and human idiopathic pulmonary fibrosis. MicroRNA-21 was also upregulated in the cultured alveolar epithelial cells under the conditions that enhance epithelial-mesenchymal transition. Exogenous administration of a microRNA-21 inhibitor prevented the increased expression of vimentin and alpha-smooth muscle actin in cultured primary mouse alveolar type II cells under culture conditions that induce epithelial-mesenchymal transition. Conclusions: Our experiments demonstrate that microRNA-21 is increased in lung epithelial cells during lung fibrosis and that it promotes epithelial-mesenchymal transition.

Cyclin B1 is translocated to the nucleus from the cytoplasm, and plays an essential role in cell proliferation through promotion of mitosis. Although overexpression of cyclin B1 was previously reported in breast carcinomas, the biological significance of the intracellular localization of cyclin B1 remains unclear. Therefore, in this study, we examined cyclin B1 immunoreactivity in 109 breast carcinomas, according to the intracellular localization, that is, nucleus, cytoplasm or total (nucleus or cytoplasm). Total cyclin B1 was detected in carcinoma cells in 42% of breast carcinomas examined, whereas nuclear and cytoplasmic cyclin B1 were positive in 17 and 35% of the cases, respectively. Total or cytoplasmic cyclin B1 were positively associated with histological grade, mitosis, Ki‐67, p53, c‐myc or 14‐3‐3σ, and inversely correlated with estrogen or progesterone receptor. Nuclear cyclin B1 was significantly associated with tumor size, lymph node metastasis, histological grade, mitosis, Ki‐67 or polo‐like kinase 1. Only nuclear cyclin B1 was significantly associated with adverse clinical outcome of the patients, and multivariate analyses of disease‐free and overall survival demonstrated nuclear cyclin B1 as the independent marker. A similar tendency was detected in the patients receiving adjuvant therapy after surgery. These results suggest that an onocogenic role of overexpressed cyclin B1 is mainly mediated in nuclei of breast carcinoma cells, and the nuclear translocation is regulated by polo‐like kinase 1 and 14‐3‐3σ. Nuclear cyclin B1‐positive breast carcinoma is resistant to adjuvant therapy, and nuclear cyclin B1 immunoreactivity is a potent prognostic factor in breast carcinoma patients. (Cancer Sci 2007; 98: 644–651)

Recent studies have shown that the tumor microenvironment plays a significant role in the progression of solid tumors. As an abundant component of the tumor microenvironment, cancer‐associated fibroblasts (CAFs) have been shown to promote tumorigenesis and cancer aggressiveness, but their molecular characteristics remain poorly understood. In the present study, paired CAFs and normal fibroblasts (NFs) were isolated from five colorectal cancer (CRC) tissues from patients who underwent surgical resection. The gene expression profiles of CAFs and NFs identified by RNA sequencing were compared to understand the complex role of CAFs in cancer progression. Gene Set Enrichment Analysis revealed that the gene sets related to the Wnt signaling pathway were highly enriched in CAFs, as well as TGFβ signaling, which is considered to be a regulator of CAFs. Among the components of this pathway, Wnt2 was specifically expressed. The observations led us to speculate that Wnt2 is extremely involved in regulating CRC progression by CAFs. Thus, we performed immunohistochemical analysis on Wnt2 in 171 patients who underwent surgery for colorectal adenocarcinoma. Positive staining for Wnt2 was mainly observed in cancer stroma, although the immunoreactivity was weak in cancer cells. Wnt2 expression in CAFs was significantly correlated with depth of tumor (P < .001), lymph node metastasis (P = .044), TNM stage (P = .010), venous invasion (P < .001), and recurrence (P = .013). Subsequent in vitro analyses were conducted using conditioned medium (CM) from immortalized CAFs transfected with siRNA targeting Wnt2. As a result, cancer cell invasion and migration were significantly decreased in the CM from immortalized CAFs transfected with siRNA targeting Wnt2. Our findings indicated that Wnt2 protein released from CAFs enhances CRC cell invasion and migration. In conclusion, Wnt2 secreted by CAFs plays a key role in cancer progression and is a potential therapeutic target for CRC.