Abstract. Histone deacetylase (HDAC) activity is one of the widely used and well-established mechanisms for regulation of various genes in cancer. To identify which subtype of class I HDACs are overexpressed in cancers, we analyzed the expression of class I HDAC isotypes composed of HDAC1, 2, 3 and 8 in several cell lines and human cancer tissues, including cancer of the stomach, esophagus, colon, prostate, breast, ovary, lung, pancreas and thyroid. The results showed that >75% of human cancer tissues and their corresponding non-cancerous epithelium showed high expression of these class I HDACs. However, the immunoreactivity of HDAC8 in both prostatic cancer tissue and non-cancerous prostate glands was lower than that in other cancer tissues. Furthermore, 5-40% of cancer tissues overexpressed class I HDACs, when compared with normal epithelium. The results suggest the potential usefulness of HDAC inhibitors for the treatment of a wide variety of human cancers.

Cap43 has been identified as a nickel-and calcium-induced gene, and is also known as N-myc downstream-regulated gene 1 (NDRG1), Drg-1 and rit42. It is also reported that overexpression of Cap43 suppresses metastasis of some malignancies, but its precise role remains unclear. In this study, we asked how Cap43 could modulate the tumor growth of pancreatic cancer. Stable Cap43 cDNA transfectants of pancreatic cancer cells with Cap43 overexpression showed similar growth rates in culture as their control counterparts with low Cap43 protein level. By contrast, Cap43 overexpression showed a marked decrease in tumor growth rates in vivo. Moreover, a marked reduction in tumor-induced angiogenesis was observed. Gelatinolytic activity by matrix metalloproteinase-9 and invasive ability in Matrigel invasion activity were markedly decreased in pancreatic cancer cell lines with high Cap43 expression. Cellular expression of matrix metalloproteinase-9 and two major angiogenic factors, vascular endothelial growth factor and interleukin-8, were also significantly decreased in cell lines with Cap43 overexpression as compared with their parental counterparts. Immunohistochemical analysis of specimens from 65 patients with pancreatic ductal adenocarcinoma showed a significant association between Cap43 expression and tumor microvascular density (P = 0.0001) as well as depth of invasion (P = 0.0003), histopathologic grading (P = 0.0244), and overall survival rates for patients with pancreatic cancer (P = 0.0062). Thus, Cap43 could play a key role in the angiogenic on-or offswitch of tumor stroma in pancreatic ductal adenocarcinoma.

Gefitinib (Iressa) and erlotinib (Tarceva), which target the epidermal growth factor receptor (EGFR), are approved for treatment of patients with advanced non-small cell lung cancer (NSCLC). Patients whose tumors harbor mutations in the EGFR gene, including delE746-A750 in exon 19 and L858R in exon 21, may benefit in particular from gefitinib treatment. However, acquired resistance to gefitinib has been a serious clinical problem, and further optimization is needed for application of EGFR-targeted drugs in lung cancer patients. In this study, we established gefitinib-resistant NSCLC cells from PC-9 cell line, which harbors the delE746-A750 mutation, by exposing the cell line to gefitinib for over 7 months. Gefitinib-resistant PC-9/GEFs cell lines showed a marked downregulation of PTEN expression and increased Akt phosphorylation. In revertant, gefitinibsensitive clones (PC-9/Rev) derived from PC-9/GEF1-1 and PC-9/GEF2-1, PTEN expression, as well as sensitivity to gefitinib and erlotinib, was restored. Knockdown of PTEN expression using small interfering RNA specific for PTEN in PC-9 cells resulted in drug resistance to gefitinib and erlotinib. Nuclear translocation of the EGR1 transcription factor, which regulates PTEN expression, was shown to be suppressed in resistant clones and restored in their revertant clones. Reduced PTEN expression was also seen in tumor samples from a patient with gefitinib-refractory NSCLC. This study thus strongly suggests that loss of PTEN expression contributes to gefitinib and erlotinib resistance in NSCLC. Our findings reinforce the therapeutic importance of PTEN expression in the treatment of NSCLC with EGFR-targeted drugs. Cancer Res; 70(21); 8715-25. ©2010 AACR.

Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.

Purpose: Therapeutic responses of non-small cell lung carcinoma (NSCLC) to epidermal growth factor receptor (EGFR)-targeted drugs, such as gefitinib and erlotinib, are closely associated with activating EGFR mutations. The most common mutations are delE746-A750 in exon 19 and L858R in exon 21, accounting for ∼90% of all EGFR mutations. Recently, EGFR mutation-specific antibodies were developed and did well in immunohistochemical analysis, giving a sensitivity of ∼90%. We have investigated whether this method detects activating EGFR mutations with sensitivity comparable with direct DNA sequencing, which is used to detect these mutations in NSCLC.Experimental Design: We used antibodies specific for the E746-A750 deletion mutation in exon 19 and the L858R point mutation in exon 21 in Western blot analysis and immunohistochemistry to determine the presence of these mutations in NSCLC cell lines. We also examined these EGFR mutations in NSCLC tumor samples from 60 patients by immunohistochemically and direct DNA sequencing.Results: We were able to identify EGFR mutations in NSCLC tumor samples immunohistochemically with a sensitivity of 79% using the anti-delE746-A750 antibody and 83% using the anti-L858R antibody. Additional DNA sequencing markedly improved the sensitivity obtained by immunohistochemistry.Conclusions: This simple and rapid assay for detecting EGFR mutations, even in the small bronchial biopsies obtained in stage IV NSCLC patients, will be useful for diagnosing responsiveness to EGFRtargeted drugs in patients with NSCLC. Combining this with DNA sequencing is recommended for the development of improved personalized EGFR-targeted therapeutics.

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In our present study, we examined whether nuclear localization of Y-box binding protein-1 (YB-1) is associated with the expression of epidermal growth factor receptors (EGFR), hormone receptors, and other molecules affecting breast cancer prognosis. The expression of nuclear YB-1, clinicopathologic findings, and molecular markers [EGFR, HER2, estrogen receptor (ER)A, ERB, progesterone receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), phosphorylated Akt, and major vault protein/lung resistance protein] were immunohistochemically analyzed. The association of the expression of nuclear YB-1 and the molecular markers was examined in breast cancer cell lines using microarrays, quantitative real-time PCR, and Western blot analyses. Knockdown of YB-1 with siRNA significantly reduced EGFR, HER2, and ERA expression in ERA-positive, but not ERAnegative, breast cancer cell lines. Nuclear YB-1 expression was positively correlated with HER2 (P = 0.0153) and negatively correlated with ERA (P = 0.0122) and CXCR4 (P = 0.0166) in human breast cancer clinical specimens but was not correlated with EGFR expression. Nuclear YB-1 expression was an independent prognostic factor for overall (P = 0.0139) and progression-free (P = 0.0280) survival. In conclusion, nuclear YB-1 expression might be essential for the acquisition of malignant characteristics via HER2-Akt-dependent pathways in breast cancer patients. The nuclear localization of YB-1 could be an important therapeutic target against not only multidrug resistance but also tumor growth dependent on HER2 and ERA. [Cancer Res 2008;68(5):1504-12]

Y-box binding protein-1 (YB-1) plays pivotal roles in acquisition of global drug resistance and cell growth promotion through transcriptional activation of genes for both drug resistance and growth factor receptors. In this study, we investigated whether YB-1 is involved in regulation of the cell cycle and cell proliferation of human cancer cells. Treatment with YB-1 siRNA caused a marked suppression of cell proliferation and expression of a cell cycle related gene, CDC6 by cancer cells. Of cell cycle of cancer cells, S phase content was specifically reduced by knockdown of YB-1. The overexpression of CDC6 abrogated this inhibition of both cell proliferation and S phase entry. ChIP assay demonstrated that YB-1 binds to a Y-box located in the promoter region of the CDC6 gene. Expression of cyclin D1, CDK1 and CDK2 was also reduced with increased expression of p21(Cip1) and p16(INK4A) when treated with YB-1 siRNA. Furthermore, the nuclear YB-1 expression was significantly associated with the level of CDC6 nuclear expression in patients with breast cancer. In conclusion, YB-1 plays an important role in cell cycle progression at G1/S of human cancer cells. YB-1 thus could be a potent biomarker for tumour growth and cell cycle in its close association with CDC6.