Jun-ichiro Ikeda

MicroRNA-mediated downregulation of mTOR/FGFR3 controls tumor growth induced by Src-related oncogenic pathways.

The tyrosine kinase c-Src is upregulated in various human cancers, but the molecular mechanisms underlying c-Src-mediated tumor growth remain unclear. Here we examined the involvement of microRNAs in the c-Src-mediated tumor growth. Microarray profiling revealed that c-Src activation downregulates a limited set of microRNAs, including miR-99a, which targets oncogenic mammalian target of rapamycin (mTOR) and fibroblast growth factor receptor 3 (FGFR3). Re-expression of miR-99a suppressed tumor growth of c-Src-transformed cells, and this effect was restored by the overexpression of mTOR. The downregulation of miR-99a was also observed in epidermal growth factor- and Ras-transformed cells, and it was suppressed by inhibiting the mitogen-activated protein kinase (MAPK) pathway. Furthermore, miR-99a downregulation is associated with mTOR/FGFR3 upregulation in various human lung cancer cells/tissues. The tumorigenicity of these cells was suppressed by the introduction of miR-99a. These findings suggest that the miR-99a-mTOR/FGFR3 pathway is crucial for controlling tumor growth in a wide range of human cancers that harbor upregulation of the Src-related oncogenic pathways.

Expression of CUB domain containing protein (CDCP1) is correlated with prognosis and survival of patients with adenocarcinoma of lung.

CUB domain containing protein (CDCP1), a transmembrane protein with intracellular tyrosine residues which are phosphorylated upon activation, is supposed to be engaged in proliferative activities and resistance to apoptosis of cancer cells. Expression level of CDCP1 was examined in lung adenocarcinoma, and its clinical implications were evaluated. CDCP1 expression was immunohistochemically examined in lung adenocarcinoma from 200 patients. Staining intensity of cancer cells was categorized as low and high in cases with tumor cells showing no or weak and strong membrane staining, respectively. MIB‐1 labeling index was also examined. There were 113 males and 87 females with median age of 63 years. Stage of disease was stage I in 144 cases (72.0%), II in 19 (9.5%), and III in 37 (18.5%). Sixty of 200 cases (30.0%) were categorized as CDCP1‐high, and the remaining as CDCP1‐low. Significant positive correlation was observed between CDCP1‐high expression and relapse rate (P < 0.0001), poor prognosis (P < 0.0001), MIB‐1 labeling index (P < 0.0001), and occurrence of lymph node metastasis (P = 0.0086). There was a statistically significant difference in disease‐free survival (DFS) (P < 0.0001) and overall survival (OS) rates (P < 0.0001) between patients with CDCP1‐high and CDCP1‐low tumors. Univariate analysis showed that lymph node status, tumor stage, and CDCP1 expression were significant factors for both OS and DFS. Multivariate analysis revealed that only CDCP1 expression was an independent prognostic factor for both OS and DFS. CDCP1 expression level is a useful marker for prediction of patients with lung adenocarcinoma (Cancer Sci 2009; 100: 429–433).

Prognostic implication of types of tumor-associated macrophages in Hodgkin lymphoma

To evaluate roles of tumor-associated macrophages (TAMs) for prognosis of classical Hodgkin lymphoma (CHL). Expression of markers for TAMs, CD68, HLA-DR, CD163, HLA-DR/CD68 (M1), and CD163/CD68 (M2) was immunohistochemically examined in 82 cases with CHL. Positively stained cells were counted and correlation of number of TAMs and patients’ survival time was analyzed. Number of CD163+ cells and M2 cells was significantly correlated with shorter overall survival (P < 0.05), while it was marginally significant for CD68+ cells (P = 0.0827). HLA-DR + cells and M1 cells showed no significant correlation with overall survival. When confined to mixed cellularity subtype, number of M1 cells was correlated with favorable prognosis (P < 0.05), while M2 did not (P = 0.7). Older age and male sex were unfavorable factors for prognosis. At multivariate analysis, number of CD163+ cells, M2+ cells, and age were independent factors for poor overall survival (P = 0.03, 0.02, and 0.01, respectively). CD163+ cells and M2 cells might work to be tumor promotive in CHL. M1 cells might be tumor suppressive in mixed cellularity type.

MicroRNA-mediated upregulation of integrin-linked kinase promotes Src-induced tumor progression

The tyrosine kinase c-Src is upregulated in various human cancers; however, the molecular mechanisms underlying c-Src-mediated tumor progression remain unclear. Here we show that downregulation of microRNA (miR)-542-3p is tightly associated with tumor progression via c-Src-related oncogenic pathways. In c-Src-transformed fibroblasts and human cancer cells that overexpress c-Src, miR-542-3p is substantially downregulated, and the ectopic expression of miR-542-3p suppresses tumor growth. We identified the integrin-linked kinase (ILK) as a conserved target of miR-542-3p. ILK upregulation promotes cell adhesion and invasion by activating the integrin–focal adhesion kinase (FAK)/c-Src pathway, and can also contribute to tumor growth via the AKT and glycogen synthase kinase 3β pathways. MiR-542-3p expression is downregulated by the activation of c-Src-related signaling molecules, including epidermal growth factor receptor, K-Ras and Ras/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase. In human colon cancer tissues, downregulation of miR-542-3p is significantly correlated with the upregulation of c-Src and ILK. Our results suggest that the novel c-Src–miR-542-3p–ILK–FAK circuit plays a crucial role in controlling tumor progression.

Expression of aldehyde dehydrogenase 1 (ALDH1) in endometrioid adenocarcinoma and its clinical implications

Aldehyde dehydrogenase 1 (ALDH1) is expressed in stem/progenitor cells, including cancer‐initiating cells (CIC) of various organs. In the present study, ALDH1 expression was immunohistochemically examined in uterine endometrioid adenocarcinoma. The ALDH1 was expressed in a small portion of tumor cells, and these ALDH1‐expressing cells were less mature than ALDH1‐non‐expressing cells. The ALDH1‐expressing (ALDH1‐hi) cells were more tumorigenic, resistant to anti‐cancer agents and more invasive than ALDH1‐lo cells. Culture of the sorted ALDH1‐hi cells yielded both ALDH1‐hi and ALDH1‐lo cells, whereas ALDH1‐lo cells yielded ALDH‐lo cells alone. Clinically, a high‐level of ALDH1 expression in tumor cells was correlated with T category, lymphatic invasion, recurrence and prognosis of patients. Patients with high ALDH1 expression showed poorer prognoses than those with low expression (P = 0.015 for disease‐free survival [DFS] and P = 0.010 for overall survival [OS]), and high ALDH1 expression was an independent factor for poor prognosis. Aldehyde dehydrogenase 1 is a candidate for CIC marker for uterine endometrioid adenocarcinoma. (Cancer Sci 2011; 102: 903–908)

Epigenetic regulation of the expression of the novel stem cell marker CDCP1 in cancer cells.

CDCP1 is a novel stem cell marker that is expressed in several types of cancer. The mechanisms by which CDCP1 expression is regulated, and the clinical implications of this marker, have not been clarified. In this report, we examine the epigenetic regulation of CDCP1 expression in cell lines and clinical samples from patients with breast cancer. Many CpG sequences were localized around the transcription initiation site of CDCP1. These CpG motifs were found to be poorly methylated in cell lines with high levels of CDCP1 expression and heavily methylated in cell lines with low levels of CDCP1 expression. The in vitro methylation of CpG sites decreased CDCP1 promoter activity, and the addition of a demethylating reagent restored activity. In 25 breast cancer samples, an inverse correlation was noted between the CDCP1 expression level and the proportion of methylated to non‐methylated CpG sites. Tumours with high‐level CDCP1 expression showed higher levels of proliferation, as revealed by immunohistochemical detection of the MIB‐1 antigen, than tumours with low‐level CDCP1 expression. These findings indicate that the expression of CDCP1 is regulated by methylation of its promoter region in tumours. CDCP1 expression may prove to be useful in the further characterization of cancers. Copyright

MiR-424/503-Mediated Rictor Upregulation Promotes Tumor Progression

mTOR complex 2 (mTORC2) signaling is upregulated in multiple types of human cancer, but the molecular mechanisms underlying its activation and regulation remain elusive. Here, we show that microRNA-mediated upregulation of Rictor, an mTORC2-specific component, contributes to tumor progression. Rictor is upregulated via the repression of the miR-424/503 cluster in human prostate and colon cancer cell lines that harbor c-Src upregulation and in Src-transformed cells. The tumorigenicity and invasive activity of these cells were suppressed by re-expression of miR-424/503. Rictor upregulation promotes formation of mTORC2 and induces activation of mTORC2, resulting in promotion of tumor growth and invasion. Furthermore, downregulation of miR-424/503 is associated with Rictor upregulation in colon cancer tissues. These findings suggest that the miR-424/503–Rictor pathway plays a crucial role in tumor progression.

Epstein–barr virus in diffuse large B‐Cell lymphoma in immunocompetent patients in Japan is as low as in Western Countries

According to previous reports, the frequency of Epstein–Barr virus (EBV) positivity in diffuse large B‐cell lymphoma is higher in East Asia (approximately 9%) than in Western countries. The presence of the EBV genome was examined in diffuse large B‐cell lymphoma patients registered with the Osaka Lymphoma Study Group (OLSG) in Osaka, Japan, situated in East Asia. The EBV‐positive rate was examined with in situ hybridization (ISH) in 484 immunocompetent diffuse large B‐cell lymphoma patients registered with OLSG. The male‐to‐female ratio was 1.29, with ages ranging from 16 to 95 (median, 68) years. ISH with EBV‐encoded small RNAs (EBER) probes revealed positive signals in the nuclei of tumor cells: the frequency of positively stained cells among all tumor cells was almost none in 458 cases, 5–10% in 5, 10–20% in 5, 20–50% in 11, and >50% in 5. When the frequency was >20% or >50%, the EBV‐positive rate in the present series (3.3% or 1.0%) was rather similar to that reported in Western cases. Careful evaluation of patient backgrounds, including age distribution, type of lymphomas, exclusion of immunocompromised patients, and establishment of definite criteria for EBV positivity (>20%, >50%, or almost all tumor cells) are essential in comparing geographical differences. J. Med. Virol. 83:317–321, 2011.

Prognostic significance of CD55 expression in breast cancer.

Purpose: Our recent study revealed that CD55-high population in breast cancer cell line was resistant to apoptosis and formed colonies in vitro more efficiently than CD55-low population. The present study was conducted to examine whether CD55-high population in breast cancer cell line possesses higher tumorigenic potential in vivo and presence of CD55-high cells in breast cancer affects clinicopathologic behavior of patients. Experimental Design: CD55-high and CD55-low population was sorted from breast cancer cell line, injected into immunodeficient mice, and the resultant tumor volume was measured. CD55 expression was immunohistochemically examined in clinical samples from 74 cases with breast cancers, and cases with >1% of tumor cells showing high level of CD55 expression were categorized as CD55 high. Results: The xenotransplanted tumor volume derived from CD55-high population was significantly larger than that from CD55-low population. Fifty (67.6%) of 74 cases of breast cancer were CD55-high. A significant correlation was observed between CD55-high character and relapse rate (P < 0.001). Univariate analysis showed that tumor size (P = 0.005) and CD55 expression (P = 0.005) were unfavorable prognostic factors. Multivariate analysis revealed that the tumor size (P = 0.013) and CD55 expression (P = 0.011) were independent prognostic factors. Conclusions: CD55 play an important role in tumorigenesis of breast cancer, and presence of small population of cells with strong CD55 expression would be sufficient to predict poor prognosis of patients.

Distinct pattern of gene expression in pyothorax-associated lymphoma (PAL), a lymphoma developing in long-standing inflammation.

Pyothorax‐associated lymphoma (PAL) is a unique lymphoma developing in the pleural cavity after long‐standing pyothorax. They are diffuse large B‐cell lymphomas (DLBCLs), frequently with immunoblastic morphology, and show a strong association with Epstein‐Barr virus (EBV) infection. In this study, cDNA microarray analysis was performed in six cases with PAL and 12 with nodal DLBCL. Among 5516 informative genes, 348 displayed more than 2‐fold difference (higher or lower) of expression level between PAL and nodal DLBCL (P>0.001). These genes are known to be involved in apoptosis, interferon response, and signal transduction. One of the most differentially expressed genes, IFI27 (interferon‐α‐inducible protein 27) was subjected to quantitative RT‐PCR analysis, and increased expression of IFI27 was confirmed. Over‐expression of IFI27 was also found in cell lines derived from PAL, but not in other lymphoid cell lines. This study shows that PAL is a distinctive subtype of DLBCL not only in its clinical presentation, but also in its molecular profile.