Makoto Nagahara

Microarray Analysis of Colorectal Cancer Stromal Tissue Reveals Upregulation of Two Oncogenic miRNA Clusters

Purpose: Cancer stroma plays an important role in the progression of cancer. Although alterations in miRNA expression have been explored in various kinds of cancers, the expression of miRNAs in cancer stroma has not been explored in detail. Experimental Design: Using a laser microdissection technique, we collected RNA samples specific for epithelium or stroma from 13 colorectal cancer tissues and four normal tissues, and miRNA microarray and gene expression microarray were carried out. The expression status of miRNAs was confirmed by reverse transcriptase PCR. Furthermore, we investigated whether miRNA expression status in stromal tissue could influence the clinicopathologic factors. Results: Oncogenic miRNAs, including two miRNA clusters, miR-17-92a and miR-106b-25 cluster, were upregulated in cancer stromal tissues compared with normal stroma. Gene expression profiles from cDNA microarray analyses of the same stromal tissue samples revealed that putative targets of these miRNA clusters, predicted by Target Scan, such as TGFBR2, SMAD2, and BMP family genes, were significantly downregulated in cancer stromal tissue. Downregulated putative targets were also found to be involved in cytokine interaction and cellular adhesion. Importantly, expression of miR-25 and miR-92a in stromal tissues was associated with a variety of clinicopathologic factors. Conclusions: Oncogenic miRNAs were highly expressed in cancer stroma. Although further validation is required, the finding that stromal miRNA expression levels were associated with clinicopathologic factors suggests the possibility that miRNAs in cancer stroma are crucially involved in cancer progression. Clin Cancer Res; 18(11); 3054–70. ©2012 AACR.

Circulating microRNA Biomarkers as Liquid Biopsy for Cancer Patients: Pros and Cons of Current Assays

An increasing number of studies have focused on circulating microRNAs (cmiRNA) in cancer patients’ blood for their potential as minimally-invasive biomarkers. Studies have reported the utility of assessing specific miRNAs in blood as diagnostic/prognostic biomarkers; however, the methodologies are not validated or standardized across laboratories. Unfortunately, there is often minimum limited overlap in techniques between results reported even in similar type studies on the same cancer. This hampers interpretation and reliability of cmiRNA as potential cancer biomarkers. Blood collection and processing, cmiRNA extractions, quality and quantity control of assays, defined patient population assessment, reproducibility, and reference standards all affect the cmiRNA assay results. To date, there is no reported definitive method to assess cmiRNAs. Therefore, appropriate and reliable methodologies are highly necessary in order for cmiRNAs to be used in regulated clinical diagnostic laboratories. In this review, we summarize the developments made over the past decade towards cmiRNA detection and discuss the pros and cons of the assays.

Kinesin 18A expression: Clinical relevance to colorectal cancer progression

Kif18A, a member of the kinesin superfamily of molecular motor proteins, is a microtubule depolymerase and a key regulator of chromosome congregation. Kif18As role in cancer progression has not been well defined. Our hypothesis is that Kif18A has a role in the progression of colorectal cancer (CRC). To investigate this expression of Kif18A, mRNA was assessed by quantitative real‐time PCR in 113 operative specimens of primary CRC. Kif18A was overexpressed and significantly (p < 0.0001) higher in CRC than in normal colon tissue. Kif18A overexpression in CRC significantly correlated with clinicopathologic factors such as tumor stage (p < 0.0001), lymphatic invasion (p = 0.001), lymph node metastasis (p = 0.01), venous invasion (p = 0.002) and peritoneal dissemination (p = 0.02), suggesting that it has a key role in CRC progression. In multivariate analysis, high Kif18A expression had independent significance for poorer overall survival after resection of CRC (p = 0.037). To demonstrate Kif18As role in CRC progression, we performed translational and in situ studies. Using in vitro studies on CRC cell lines, we evaluated Kif18As role in proliferation, migration and invasion. CRC cells transfected with Kif18A cDNA demonstrated significant enhanced migration (p < 0.01) and invasion (p = 0.018) compared to mock‐transfected cells. When Kif18A was targeted with specific small interfering RNA, CRC cells had significantly reduced proliferation (p < 0.01), migration (p < 0.01) and invasion (p < 0.05). The in vitro and translational studies demonstrated that Kif18A expression is related to events of metastasis and is a significant factor for CRC progression.

Correlated expression of CD47 and SIRPA in bone marrow and in peripheral blood predicts recurrence in breast cancer patients

Purpose: CD47 plays a variety of roles in intercellular signaling. Herein, we focused on the clinicopathologic significance of CD47 expression in human breast cancer. Our data suggest that the correlation between CD47 and signal regulatory protein α (SIRPA) expression may play a key role in the progression of breast cancer. Experimental Design: Quantitative real-time PCR was used to evaluate CD47 mRNA and SIRPA mRNA expression in bone marrow and in peripheral blood from 738 cases of breast cancer. Results: In patients with high levels of CD47 expression in the bone marrow, survival was significantly poorer compared with patients with low levels of CD47 expression [disease-free survival (DFS), P = 0.0035; overall survival (OS), P = 0.015]. Furthermore, high CD47 expression group in a multivariate analysis showed significance as an independent variable for poorer prognosis in DFS (P = 0.024). In the peripheral blood, however, high CD47 expression in patients was not an independent and significant prognostic factor for DFS and OS in a multivariate analysis. CD47 expression was strongly correlated with SIRPA expression in both the bone marrow (P < 0.0001) and peripheral blood (P < 0.0001) of breast cancer patients. Conclusions: This is one of the first studies to show that a host factor in bone marrow confers prognostic importance. CD47 is an important biomarker in breast cancer, and functions as a prognostic factor for DFS. Moreover, we suggest that the poor prognosis of breast cancer patients with high expression of CD47 is due to an active CD47/SIRPA signaling pathway in circulating cells. Clin Cancer Res; 16(18); 4625–35. ©2010 AACR.

Lymphovascular invasion of colorectal cancer is correlated to SPARC expression in the tumor stromal microenvironment

As an integral component of the microenvironment in colorectal cancer (CRC), stromal cells can influence tumor progression. Found in the extracellular matrix of CRC, secreted protein acidic and rich in cysteine (SPARC) is expressed in stromal and CRC cells. While SPARCs influence on CRC is not clear, we hypothesized that epigenetically regulated SPARC expression in the microenvironment stromal cells of CRC can affect primary CRC progression and is influenced by lymphovascular invasion (LVI). Quantitative immunohistochemistry (IHC) analysis of paraffin-embedded (n=72) from 37 LVI-positive and 35 LVI-negative primary CRCs was performed. MassARRAY sequencing was performed to assess the methylation status of the promoter region in 22 LVI-positive and 20 LVI-negative CRC and to identify specific CpG island(s) regulating SPARC expression. SPARC in CRC cells was not correlated with LVI, whereas SPARC in the microenvironment stromal cells was inversely related to LVI (P < 0.0001). There was a direct relationship between LVI and 6 specific CpG site methylation in the SPARC promoter region of stromal cells (P = 0.017) but not in CRC cells. Stromal SPARC expression inversely correlated with VEGF-A expression in CRC (P = 0.003) and positively correlated with HSP27 expression (P = 0.009). The results suggested that the epigenetic regulation of SPARC expression in tumor cells versus stromal cells of CRC is significantly different. Stromal cell SPARC expression is epigenetically influenced by LVI of CRC tumors, and may play a significant role in primary CRC progression.

Periostin suppression induces decorin secretion leading to reduced breast cancer cell motility and invasion

The ability of cancer cells to metastasize is dependent on the interactions between their cell-surface molecules and the microenvironment. However, the tumor microenvironment, especially the cancer-associated stroma, is poorly understood. To identify proteins present in the stroma, we focused on phyllodes tumors, rare breast tumors that contain breast stromal cells. We compared the expression of proteins between phyllodes tumor and normal tissues using an iTRAQ-based quantitative proteomic approach. Decorin was expressed at reduced levels in phyllodes tumor tissues, whereas periostin was upregulated; this result was validated by immunohistochemical analysis of phyllodes tumors from 35 patients. Additionally, by immunoprecipitation and mass spectrometry, we confirmed that decorin forms a complex with periostin in both phyllodes tumors and BT-20 breast cancer cells. Following siRNA-mediated knockdown of periostin in T-47D cells, secreted decorin in the culture medium could be detected by multiple reaction monitoring (MRM). Furthermore, periostin knockdown in BT-20 cells and overexpression of decorin in MDA-MB-231 cells inhibited cell motility and invasion. Our results reveal the molecular details of the periostin–decorin complex in both phyllodes tumor tissues and breast cancer cells; this interaction may represent a novel target for anti-cancer therapy.

Clinicopathologic Relevance of Claudin 5 Expression in Breast Cancer

OBJECTIVES Claudins are major adhesion molecules in tight junctions and are strongly expressed in various cancers. We thus investigated the expression of claudin 5, a member of the claudin family, in breast cancer. METHODS A total of 193 patients with breast cancer were identified based on their pathologic diagnosis. The expression of each claudin 5 was analyzed in the tumor by immunohistochemical staining. Parametric correlations were done between claudin 5 expression and the clinicopathologic findings. RESULTS Claudin 5 expression in patients with recurrent breast cancer was statistically significantly higher (P = .004). In addition, analysis of the correlation with other clinicopathologic factors showed statistically significant differences with respect to lymphatic invasion (P = .014), venous invasion (P = .048), estrogen receptor status (P = .002), and human epidermal growth factor 2 status (P = .007). Multivariate analysis revealed that claudin 5 expression was an independent predictive factor in the recurrence for relapse-free survival (RFS) (P = .020). Kaplan-Meier analysis showed that the RFS rate was significantly lower in the high claudin 5 expression group (P = .001). CONCLUSIONS Patients with breast cancer with high claudin 5 expression had a significantly lower RFS rate. Our findings suggest that claudin 5 may be useful as a new biomarker of a risk factor.

Correction: Ono, S.; Lam, S.; Nagahara, M.; Hoon, D.S.B. Circulating microRNA Biomarkers as Liquid Biopsy for Cancer Patients: Pros and Cons of Current Assays. J. Clin. Med. 2015, 4, 1890–1907

The authors wish to make the following corrections to this paper [1]:[...].

Abstract 4870: Proteomics of phyllodes tumor revealed that decorin increase in the extracellular matrix by periostin deficiency decreased cancer cell motility and invasion

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The ability of cancer cells to metastasize is dependent on the interactions of their cell surface molecules with microenvironment. However the tumor microenvironment, especially cancer-associated stroma, is poorly understood. To search for proteins which are present in the stroma, we investigated the phyllodes tumor, which contains breast stromal tissue, specific expression proteins compared with normal tissue by using iTRAQ-based quantitative proteomic approach. Periostin and versican core protein were up-regulated in phyllodes tumor. Decorin, mimecan, hemoglobin subunit alpha, hemoglobin subunit beta and ketatin, type1 cytoskeletal 19 were up-regulated in normal tissue. Periostin and decorin are one of component of the extracellular matrix. Periostin upregulation has been reported in many cancer types and is consequently defined as a tumor-enhancing factor. On the other hand, decorin upregulation inhibits tumor growth by antagonizing tumor angiogenesis. Periostin upregulation in phyllodes tumor and decorin upregulation in normal tissue was validated by immunohistochemical analysis from phyllodes tumor thirty five patients. We determined the interaction between decorin and periostin in phyllodes tumor tissues and breast cancer cell line BT-20 cells, by using immunoprecipitation and mass spectrometry analysis. Furthermore, we uncovered the plasma membrane translocation of decorin from cytoplasm in BT-20 or T-47D cells by siRNA-mediated knockdown of periostin, and detected the secreted decorin to the extracellular medium using Multiple Reaction Monitoring (MRM) method. The periostin siRNA-treated BT-20 cells motility and invasion were prevented by the secreted decorin into the extracellular medium. The overexpression of decorin suppressed the metastatic breast cancer cell line, MDA-MB-231 cells motality and invasion. These results demonstrated the secreted decorin by periostin deficiency decreased cancer cells motality and invasion. This molecular mechanism may become the new target for anti-cancer therapy. Citation Format: Toshiyuki Ishiba, Akira Nakanishi, Takanobu Sato, Tsuyoshi Nakagawa, Makoto Nagahara, Ryo Oono, Hiroyuki Uetake, Kenichi Sugihara, Yoshio Miki. Proteomics of phyllodes tumor revealed that decorin increase in the extracellular matrix by periostin deficiency decreased cancer cell motility and invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4870. doi:10.1158/1538-7445.AM2014-4870

Abstract C95: Correlated intrinsic breast cancer subtypes and expression of CD47 in BM predicts recurrence

Purpose: The intrinsic breast cancer subtypes have shown the prognostic features. Focused on CD47 expression in bone marrow (BM), we previously demonstrated that CD47 is an important biomarker in breast cancer, and functions as a prognostic factor for DFS. The purpose of this study is to identify the correlation between breast cancer subtypes and CD47 expression in BM, which may indicate clinical implications. Methods: Quantitative real-time PCR was used to evaluate CD47 mRNA expression in bone marrow (BM) and in peripheral blood (PB) from 738 cases of breast cancer. ER, PR, and Her2 scores were obtained from immunohistochemistry (IHC) staining, and Her2 FISH analysis conducted for IHC 2+. Results: According to ER, PR, and Her2 statuses, the groups of Her2 enriched (ER- and PR- and Her2 3+) and triple negative(ER- and PR- and Her2-) were divided (n=38 and 72). The 5 years disease free survival rates in each subgroup were as followed: p=0.0004; ER+ and PR+ and Her2- 0.94, ER+ and/or PR+ or- and/or Her2+ or- 0.16, Her2 enriched 0.23, triple negative 0.34). In patients with high levels of CD47 expression in the BM, survival was significantly poorer compared to patients with low levels of CD47 expression (DFS of Her2 enriched group: P=0.003, DFS of triple negative group: P=0.002). Furthermore, high CD47 expression group in a multivariate analysis showed significance as an independent variable for poorer prognosis in DFS (P = 0.02). In the PB, however, high CD47 expression in patients was not an independent and significant prognostic factor for DFS and OS in a multivariate analysis. Conclusions: We confirmed that overexpression of CD47 in BM correlated with the aggressiveness of Her2 enriched and triple negative subgroups. CD47 is a useful prognostic biomarker for predicting survival of Her2 enriched and triple negative subgroups in breast cancer. We suggest that high CD47 expression in BM may represent tumor dormancy in breast cancer involved in postsurgical recurrence and metastasis of Her2 enriched and triple negative subtypes. Citation Format: Makoto Nagahara, Kenichi Sugihara, Masaki Mori. Correlated intrinsic breast cancer subtypes and expression of CD47 in BM predicts recurrence. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C95.