Combination therapy of lymphatic drug delivery and total body irradiation in a metastatic lymph node and lung mouse model.

Chemotherapy using a lymphatic drug delivery system (LDDS) targeting lymph nodes (LNs) in the early stage of metastasis has a superior antitumor effect to systemic chemotherapy. An LDDS produces a higher drug retention rate and tissue selectivity in LNs. To expand the therapeutic coverage of LDDS from local treatment of metastatic LNs to prevention of distant metastases, the combination of treatment with therapies that enhance systemic tumor immune effects is an important therapeutic strategy. Recently, total body irradiation (TBI) has been shown to activate immune responses and alter the tumor microenvironment. Here we show that combination therapy with TBI and LDDS improves the antitumor effect of metastatic LNs and lung metastasis. Tumor cells were inoculated into the subiliac LN (SiLN) to induce metastasis into the proper axillary LN (PALN) and lung in a mouse model. TBI was carried out on day 4 after inoculation using a gamma irradiator. Lymphatic drug delivery into the accessory axillary LN was used to treat PALN. In vivo bioluminescence imaging, high-frequency ultrasound, and histology showed that combination therapy using TBI (total dose 1.0 Gy once) and the LDDS suppressed tumor growth in LNs and lung metastases and was more effective than using LDDS or TBI alone. Quantitative RT-PCR of spleens after combination therapy revealed increased expression of CD4, CD8, and IL-12b, indicating an activated immune response. The results show that combination therapy with TBI and LDDS is a method to improve the efficacy of LN metastases and distant metastases therapy and is a promising novel approach to treat cancer patients.

TWIST1 is a prognostic factor for neoadjuvant chemotherapy for patients with resectable pancreatic cancer: a preliminary study.

Recent advances in the development of chemotherapies have helped improve the prognosis of pancreatic ductal adenocarcinoma (PDAC). However, predicting factors for the outcomes of chemotherapies (either gemcitabine or S-1) have not yet been established. We analyzed the expression of 4 major epithelial-to-mesenchymal transition-inducing transcription factors in 38 PDAC patients who received adjuvant chemotherapy after radical resection to examine the association with patients' prognoses. The TWIST1-positive group showed a significantly poorer prognosis than the TWIST1-negative group for both the relapse-free survival (median survival time [MST] of 8.9 vs. 18.5 months, P = 0.016) and the overall survival (MST of 15.2 vs. 33.4 months, P = 0.023). A multivariate analysis revealed that TWIST1 positivity was an independent prognostic factor for a poor response to adjuvant chemotherapies (hazard ratio 2.61; 95% confidence interval 1.10-6.79; P = 0.029). These results suggest that TWIST1 can be utilized as an important poor prognostic factor for radically resected PDAC patients with adjuvant chemotherapy, potentially including neoadjuvant therapy using these agents.

Epigenetic inactivation of IRX4 is responsible for acceleration of cell growth in human pancreatic cancer.

Epigenetic gene silencing by aberrant DNA methylation is one of the important mechanisms leading to loss of key cellular pathways in tumorigenesis. Methyl-CpG-targeted transcriptional activation (MeTA) reactivates hypermethylation-mediated silenced genes in a different way from DNA-demethylating agents. Microarray coupled with MeTA (MeTA-array) identified seven commonly hypermethylation-mediated silenced genes in 12 pancreatic ductal adenocarcinoma (PDAC) cell lines. Among these, we focused on IRX4 (Iroquois homeobox 4) because IRX4 is located at chromosome 5p15.33 where PDAC susceptibility loci have been identified through genome-wide association study. IRX4 was greatly downregulated in all of the analyzed 12 PDAC cell lines by promoter hypermethylation. In addition, the IRX4 promoter region was found to be frequently and specifically hypermethylated in primary resected PDACs (18/28: 64%). Reexpression of IRX4 inhibited colony formation and proliferation in two PDAC cell lines, PK-1 and PK-9. In contrast, knockdown of IRX4 accelerated cell proliferation in an IRX4-expressing normal pancreatic ductal epithelial cell line, HPDE-1. Because IRX4 is a sequence-specific transcription factor, downstream molecules of IRX4 were pursued by microarray analyses utilizing tetracycline-mediated IRX4 inducible PK-1 and PK-9 cells; CRYAB, CD69, and IL32 were identified as IRX4 downstream candidate genes. Forced expression of these genes suppressed colony formation abilities for both PK-1 and PK-9. These results suggest that DNA methylation-mediated silencing of IRX4 contributes to pancreatic tumorigenesis through aberrant transcriptional regulation of several cancer-related genes.

Methylation-mediated silencing of the LIM homeobox 6 (LHX6) gene promotes cell proliferation in human pancreatic cancer.

Epigenetic gene silencing by aberrant DNA methylation leads to loss of key cellular pathways in tumorigenesis. DNA methylation-mediated silenced genes in pancreatic cancer were searched for using the methyl-CpG targeted transcriptional activation (MeTA) method, and LHX6 (LIM homeobox 6), a transcription factor involved in embryogenesis and head development, was selected as a strong candidate gene. LHX6 was downregulated in most of the pancreatic cancer cell lines (83%, 10/12), mainly through promoter hypermethylation and histone deacetylation. Furthermore, LHX6 was methylated in primary pancreatic cancer specimens (57%, 16/28) in a tumor-specific manner. Re-expression of LHX6 inhibited colony formation and proliferation in LHX6 low-expressing pancreatic cancer cell lines, PK-1 and PK-9. In contrast, knockdown of LHX6 accelerated cell proliferation in LHX6 high-expressing pancreatic cancer cell lines, PCI-35 and MIA PaCa-2. In order to analyze LHX6 downstream genes, we performed microarray analyses using LHX6 inducible PK-1 and PK-9 and found that LHX6 induction upregulated several genes that had tumor suppressive functions. Among these, we focused on TFPI2 (Tissue factor pathway inhibitor-2) and found that TFPI2 was greatly downregulated in all twelve pancreatic cancer cell lines. Our present results suggest that epigenetic inactivation of LHX6 plays an important role in pancreatic tumorigenesis by promoting cell proliferation through aberrant transcriptional regulation of several cancer-related genes.

S100A10 upregulation associates with poor prognosis in lung squamous cell carcinoma.

S100A10 is one of the members of the S100 protein family and is a key plasminogen receptor. Its upregulation has been reported in many types of tumors. In lung cancer, an association between upregulation of S100A10 and poor prognoses has been reported only in adenocarcinoma. We pursued the possibility of significance in lung squamous cell carcinoma (SCC). We first examined S100A10 protein expression by immunohistochemistry in 120 primary resected lung SCCs; 33 (27.5%) tumors showed strong membranous-immunopositivity particularly at the invasive front, i.e., the cancer-cell surface in contact with the stroma. Expression levels were significantly associated with higher pathological TNM stage (P = 0.0119), tumor size (P = 0.0003), lymphatic invasion (P = 0.0005), lymph node metastasis (P = 0.0006), and poorer prognosis (P = 0.0064). Our present results suggest that high S100A10 expression of the lung SCC cells, particularly adjacent to stroma, plays an important role in tumor progression, probably caused by lymphatic invasion and nodal metastasis.

NDRG2, suppressed expression associates with poor prognosis in pancreatic cancer, is hypermethylated in the second promoter in human gastrointestinal cancers.

Although N-myc downstream regulated gene 2 (NDRG2) is frequently downregulated in various cancers and is considered to be a candidate tumor suppressor gene, molecular mechanisms of the expressional suppression that lead to cancers are largely unknown. Recent studies indicated that epigenetic suppression of NDRG2 involved carcinogenesis and progression in several tumor types, and we demonstrated positive association with NDRG2 suppression and poor prognosis in pancreatic cancer. In this study, we analyzed mRNA and protein expressions of NDRG2 in 26 cancer cell lines (20 colorectal and 6 gastric cancers) and found that many cell lines showed variously reduced NDRG2 expressions. Furthermore, NDRG2 expressions were significantly reduced in primary resected cancer tissues compared to corresponding normal tissues immunohistochemically (19 of 20 colorectal and 14 of 17 gastric cancers). Treatment with 5-Aza-2' deoxycytidine predominantly upregulated NDRG2 expressions in NDRG2 low-expressing cell lines. Bisulfite sequencing analyses and methylation specific PCR revealed that methylation status at one of the two promoters (around exon 2) correlated well with the suppressed expression, and this is the major promoter in colorectal and gastric cancer cell lines. Our present results suggest that hypermethylation in promoter around exon 2 is functioning as essential factors of NDRG2 silencing in gastrointestinal cancers.

S100A4 is frequently overexpressed in lung cancer cells and promotes cell growth and cell motility.

S100A4, a small calcium-binding protein belonging to the S100 protein family, is commonly overexpressed in a variety of tumor types and is widely accepted to associate with metastasis by regulating the motility and invasiveness of cancer cells. However, its biological role in lung carcinogenesis is largely unknown. In this study, we found that S100A4 was frequently overexpressed in lung cancer cells, irrespective of histological subtype. Then we performed knockdown and forced expression of S100A4 in lung cancer cell lines and found that specific knockdown of S100A4 effectively suppressed cell proliferation only in lung cancer cells with S100A4-overexpression; forced expression of S100A4 accelerated cell motility only in S100A4 low-expressing lung cancer cells. PRDM2 and VASH1, identified as novel upregulated genes by microarray after specific knockdown of S100A4 in pancreatic cancer, were also analyzed, and we found that PRDM2 was significantly upregulated after S100A4-knockdown in one of two analyzed S100A4-overexpressing lung cancer cells. Our present results suggest that S100A4 plays an important role in lung carcinogenesis by means of cell proliferation and motility by a pathway similar to that in pancreatic cancer.

Suppressed expression of NDRG2 correlates with poor prognosis in pancreatic cancer.

Pancreatic cancer is a highly lethal disease with a poor prognosis; the molecular mechanisms of the development of this disease have not yet been fully elucidated. N-myc downstream regulated gene 2 (NDRG2), one of the candidate tumor suppressor genes, is frequently downregulated in pancreatic cancer, but there has been little information regarding its expression in surgically resected pancreatic cancer specimens. We investigated an association between NDRG2 expression and prognosis in 69 primary resected pancreatic cancer specimens by immunohistochemistry and observed a significant association between poor prognosis and NDRG2-negative staining (P=0.038). Treatment with trichostatin A, a histone deacetylase inhibitor, predominantly up-regulated NDRG2 expression in the NDRG2 low-expressing cell lines (PANC-1, PCI-35, PK-45P, and AsPC-1). In contrast, no increased NDRG2 expression was observed after treatment with 5-aza-2' deoxycytidine, a DNA demethylating agent, and no hypermethylation was detected in either pancreatic cancer cell lines or surgically resected specimens by methylation specific PCR. Our present results suggest that (1) NDRG2 is functioning as one of the candidate tumor-suppressor genes in pancreatic carcinogenesis, (2) epigenetic mechanisms such as histone modifications play an essential role in NDRG2 silencing, and (3) the expression of NDRG2 is an independent prognostic factor in pancreatic cancer.

DNA methylation in cancer: a gene silencing mechanism and the clinical potential of its biomarkers.

Initiation and progression of human cancer not only depends on genetic alterations but also on epigenetic changes such as DNA methylation and histone modifications. Aberrant DNA hypermethylation in the promoter regions of genes is the most well-defined epigenetic change in tumors and is associated with inappropriate gene silencing. This feature can be utilized to search for tumor-specific DNA methylation biomarkers and to examine candidate DNA biomarkers for clinical use. DNA methylation biomarker is defined as a molecular target that undergoes DNA methylation changes in carcinogenesis. Such a biomarker is useful for early detection of cancer, predicting and/or monitoring the therapeutic response, and detection of recurrent cancer. In this review, we describe the mechanism that establishes and maintains DNA methylation patterns as well as the mechanism of aberrant gene silencing in cancer, and then we introduce methods to isolate the DNA methylation biomarkers. We also summarize the current status of clinical implementation for some of the most widely studied and well-validated DNA methylation biomarkers, including tissue factor pathway inhibitor 2 (TFPI2), septin 9 (SEPT9), glutathione S-transferase pi 1 (GSTP1), and O(6)-methylguanine-DNA methyltransferase (MGMT), and assess the clinical potential of these biomarkers for risk assessment, early diagnosis, prognosis, treatment, and the prevention of cancer. Finally we describe the possible involvement of 5-hydroxymethylcytosine in cancer; this is a recently discovered 5-methylcytosine oxidation derivative and might have a diagnostic potential in certain cancers. Abnormal DNA methylations are leading candidates for the development of specific markers for cancer diagnosis and therapy.

Long Non-Coding RNAs Associated with Mitogen-Activated Protein Kinase in Human Pancreatic Cancer.

Long non-coding RNAs (lncRNAs) have emerged as a significant player in various cancers, including pancreatic cancer. However, how lncRNAs are aberrantly expressed in cancers is largely unknown. We hypothesized that lncRNAs would be regulated by signaling pathways and contribute to malignant phenotypes of cancer. In this study, to understand the significance of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), which is a major aberrant signaling pathway in pancreatic cancer, for the expression of lncRNAs, we performed comparative transcriptome analyses between pancreatic cancer cell lines with or without activation of MAPK. We identified 45 lncRNAs presumably associated with MAPK in pancreatic cancer cells; among these, LINC00941 was consistently upregulated by MAPK. The immediate genomic upstream region flanking LINC00941 was identified as a promoter region, the activity of which was found to be preferentially associated with MAPK activity via ETS-1 binding site. LINC00941 promoted cell proliferation in vitro. Moreover, TCGA data analysis indicated that high expression of LINC00941 was associated with poor prognosis of patients with pancreatic cancer. Transcriptomes comparing transcriptions between cells with and without LINC00941 knockdown revealed 3229 differentially expressed genes involved in 44 biological processes, including the glycoprotein biosynthetic process, beta-catenin-TCF complex assembly, and histone modification. These results indicate that MAPK mediates the aberrant expression of lncRNAs. LINC00941 is the lncRNA by MAPK most consistently promoted, and is implicated in the dismal prognosis of pancreatic cancer. MAPK-associated lncRNAs may play pivotal roles in malignant phenotypes of pancreatic cancer, and as such might represent both potentially valid therapeutic targets and diagnostic biomarkers.

miR-34a is downregulated in cis-diamminedichloroplatinum treated sinonasal squamous cell carcinoma patients with poor prognosis.

For the purpose of analyzing mechanisms related to the cis-diamminedichloroplatinum resistance in head and neck squamous cell carcinoma, we analyzed RPMI2650 and its derived previously established cis-diamminedichloroplatinum resistant cell line RPMI2650CR. To identify resistant phenotype-related microRNAs, we compared microRNA expressions between RPMI2650CR and RPMI2650 by microarray. One of the microRNAs as downregulated, miR-34a, was further investigated. Decreased expression of miR-34a in RPMI2650CR was confirmed by quantitative reverse transcription-polymerase chain reaction, but introduction of the miR-34a precursor into RPMI2650CR or the inhibitor of miR-34a into RPMI2650 did not change cis-diamminedichloroplatinum sensitivities. However, 24 patients with sinonasal squamous cell carcinomas treated with intra-arterial infusion of cis-diamminedichloroplatinum showed a significant association between decreased expression of miR-34a and poor disease specific survival (P = 0.0015), poor disease free survival (P = 0.0019), and poor local control rates (P = 0.017) (median follow-up period: 53 months). Furthermore, multivariate analyses demonstrated significant associations between miR-34a expression and the hazard ratios of disease free survival at 0.005 (95% confidence interval [CI] 0.00-0.29, P = 0.011) and local control rate at 0.008 (95% CI 0.00-0.44, P = 0.019), although other parameters such as age, gender, treatment method, T and N stages did not show any similar association. These results strongly suggest that miR-34a expression can be an independent prognostic biomarker in patients with sinonasal squamous cell carcinoma who are undergoing treatment with cis-diamminedichloroplatinum.

S100A4, frequently overexpressed in various human cancers, accelerates cell motility in pancreatic cancer cells.

S100A4, a member of the Ca(2+) dependent S100 protein family, is reported to associate with metastasis through regulation of the motility and invasiveness of cancer cells. A high level of S100A4 protein has been reported in a variety of cancers, including pancreatic cancer. However, its biological role in pancreatic carcinogenesis is largely unknown. We previously reported that S100A4 is frequently overexpressed and that RNAi-mediated knockdown induces apoptosis and suppression of cell growth, motility, and invasiveness. In this study, we analyzed the effects of forced expression of S100A4 in pancreatic cancer cell lines without S100A4-upregulation. We used two cell lines without upregulation of S100A4 (PCI-35 and PCI-43) as well as two cell lines with highly upregulated S100A4 as the control (MIA PaCa-2 and PAN-07-JCK). Cells did not show acceleration of their growth and invasiveness after forced expression of S100A4, but remarkable acceleration of cell motility was observed only in PCI-35 and PCI-43. We further performed microarray analyses using PCI-35 and PCI-43 with and without forced expression of S100A4 and identified 72 and 18 genes that were 2-fold or more upregulated or downregulated, respectively, in both cell lines after forced expression of S100A4. Our results suggest that S100A4 is crucial for cell motility in pancreatic cancer and that some downstream genes may play important roles in cell motility.

DCK is frequently inactivated in acquired gemcitabine-resistant human cancer cells.

Although gemcitabine is the most effective chemotherapeutic agent against pancreatic cancer, a growing concern is that a substantial number of patients acquire gemcitabine chemoresistance. To elucidate the mechanisms of acquisition of gemcitabine resistance, we developed gemcitabine-resistant cell lines from six human cancer cell lines; three pancreatic, one gastric, one colon, and one bile duct cancer. We first analyzed gemcitabine uptake using three paired parental and gemcitabine resistant pancreatic cancer cell lines (PK-1 and RPK-1, PK-9 and RPK-9, PK-59 and RPK-59) and found that uptake of gemcitabine was rapid. However, no DNA damage was induced in resistant cells. We further examined the microarray-based expression profiles of the cells to identify genes associated with gemcitabine resistance and found a remarkable reduction in the expression of deoxycytidine kinase (DCK). DCK is a key enzyme that activates gemcitabine by phosphorylation. Genetic alterations and expression of DCK were studied in these paired parental and derived gemcitabine-resistant cell lines, and inactivating mutations were found only in gemcitabine-resistant cell lines. Furthermore, siRNA-mediated knockdown of DCK in the parental cell lines yielded gemcitabine resistance, and introduction of DCK into gemcitabine-resistant cell lines invariably restored gemcitabine sensitivities. Mutation analyses were expanded to three other different paired cell lines, DLD-1 and RDLD-1 (colon cancer cell line), MKN-28 and RMKN-28 (gastric cancer cell line), and TFK-1 and RTFK -1 (cholangiocarcinoma cell line). We found inactivating mutations in RDLD-1 and RTFK-1 and decreased expression of DCK in RMKN-28. These results indicate that the inactivation of DCK is one of the crucial mechanisms in acquisition of gemcitabine resistance.

Identification of epigenetically silenced genes in human pancreatic cancer by a novel method "microarray coupled with methyl-CpG targeted transcriptional activation" (MeTA-array).

Identification and characterization of epigenetically silenced genes is important for cancer research, because information from hypermethylated genes provides clues to understand roles of epigenetics in tumorigeneses and genes frequently methylated in a tumor-specific manner can be used as tumor markers. Here, we describe the identification of transcriptionally silenced hypermethylated genes in pancreatic cancer cells by using a novel method called "microarray coupled with methyl-CpG targeted transcriptional activation" (MeTA-array for short), which can effectively reactivate genes containing the stringent criteria of CpG islands at promoter regions. Three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2 and PANC-1, with a normal pancreatic ductal epithelial cell line HPDE as a control, were examined with this method, and 19 genes were upregulated twofold or more in all the three cancer cell lines after MeTA; 16 of these 19 genes have not been detected previously when using a conventional DNA demethylating agent, 5-aza-2'-deoxycytidine. Among these 16 genes, CSMD2, SLC32A1, TMEM204 and TRH were further analyzed by methylation-specific PCR, and we found that 90% (19/21) of CSMD2, 100% (21/21) of SLC32A1, 95% (20/21) of TMEM204 and 100% (21/21) of TRH were methylated in our series of pancreatic cancer cell lines. Furthermore, CSMD2, SLC32A1 and TRH were also hypermethylated in primary pancreatic cancers in a tumor-specific manner. These results suggest that MeTA-array is a highly efficient method for identifying methylation-mediated transcriptionally silenced genes in human pancreatic cancer and that this method can be applied to other types of human cancer.

Aberrant Hypermethylation-Mediated Suppression of PYCARD Is Extremely Frequent in Prostate Cancer with Gleason Score ≥ 7.

Epigenetic gene silencing by aberrant DNA methylation leads to loss of key cellular pathways in tumorigenesis. In order to analyze the effects of DNA methylation on prostate cancer, we established LNCaP-derived human prostate cancer cells that can pharmacologically induce global reactivation of hypermethylated genes by the methyl-CpG targeted transcriptional activation (MeTA) method. The MeTA suppressed the growth of LNCaP-derived cells and induced apoptosis. Microarray analysis indicated that PYCARD (PYD and CARD domain containing) encoding an apoptosis-inducing factor was upregulated by 65-fold or more after treatment with MeTA. We analyzed DNA methylation statuses using 50 microdissected primary prostate cancer tissues and found an extremely high frequency of tumor-specific promoter hypermethylation of PYCARD (90%, 45/50). Moreover, DNA methylation status was significantly associated with Gleason score (P = 0.0063); the frequency of tumor-specific hypermethylation was 96% (44/46) in tumors with Gleason score ≥ 7, whereas that in tumors with Gleason score 6 was 25% (1/4). Immunohistochemical analyses using these 50 cases indicated that only 8% (4/50) of cancerous tissues expressed PYCARD, whereas 80% (40/50) of corresponding normal prostate epithelial and/or basal cells expressed PYCARD. In addition, there was no relationship between PYCARD immunostaining and the Gleason score in cancerous tissue and surrounding normal tissue. Inducible expression of PYCARD inhibited cell proliferation by induction of apoptosis. These results suggest that aberrant methylation of PYCARD is a distinctive feature of prostate cancers with Gleason score ≥ 7 and may play an important role in escaping from apoptosis in prostatic tumorigenesis.

Development of a system combining comprehensive genotyping and organoid cultures for identifying and testing genotype-oriented personalised medicine for pancreatobiliary cancers.

BACKGROUND: Pancreatobiliary cancer is a highly aggressive tumour with a dismal prognosis. Personalised medicine represents a promising and effective therapeutic approach for this intractable disease. In this study, we aimed to establish a system for identifying and testing genotype-oriented targeted drugs for pancreatobiliary cancers by combining exome sequencing and organoid culture of primary tumours. METHODS: Tumour cells isolated from resected tumours were subjected to organoid cultures based on published protocols with modifications. Exome sequencing was performed on the primary tumours. Histopathological and molecular features of the primary tumours were validated in the corresponding organoids. Genotype-oriented candidate targeted drugs were identified from exome sequencing, and their efficacies were tested in the organoids. RESULTS: Organoid cultures succeeded in 30 of 54 (55.6%) cases. Six primary cancers of the biliary tract and gall bladder were subjected to exome sequencing, which revealed a variety of somatic mutations of genes involved in signalling pathways, epigenetic modifiers, genome maintenance and metabolic enzymes. Most of the organoids of these 6 cases showed identical histopathological features and genomic aberrations as those of the primary tumours. Some of the aberrations were candidates for targeted therapies. Integrin-linked kinase (ILK) was one such candidate target, and an ILK inhibitor was confirmed to suppress proliferation of patient-derived organoids. CONCLUSIONS: By combining exome sequencing and organoid culture, our model enabled to identify genotype-oriented targets for personalised medicine and to test efficacies of candidate targeted drugs in the organoids. The current proof-of-concept approach could increase therapeutic opportunities for patients with pancreatobiliary cancers.

Methyl-CpG targeted recruitment of p300 reactivates tumor suppressor genes in human cancer cells.

Aberrant hypermethylation of gene promoters is a major mechanism associated with inactivation of tumor suppressor genes (TSGs) in cancer. We have previously shown that the methyl-CpG targeted transcriptional activation (MeTA) that allows re-expression of TSGs in human cancer cells is accomplished by combining a methyl-CpG binding domain (MBD) with a NFkappaB transcriptional activation domain (AD), accompanied by histone H3K9/K14 acetylation. Herein we demonstrate that p300 histone acetyltransferase (HAT), one of the NFkappaB (AD)-associated coactivators, reactivates epigenetically silenced MLH1 in 293T cells. Interestingly, the HAT domain of p300 is not essential for the reactivation of MLH1; instead, the C-terminal transactivation domain (C-TAD) but not the N-terminal one (N-TAD) reactivates MLH1. Furthermore, all ten of the cancer-related genes analyzed in three types of cancer cells were reactivated by the effect of p300 linked to MBD. These results demonstrate that it is possible to reactivate epigenetically silenced TSGs in human cancer cells by direct targeting of a transcriptional coactivator at highly methylated promoters.

Expression of SNAIL in accompanying PanIN is a key prognostic indicator in pancreatic ductal adenocarcinomas.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer, mainly because of its invasive and metastatic characteristics. Pancreatic intraepithelial neoplasia (PanIN) is one of the major precursor lesions of PDAC. Although epithelial-to-mesenchymal transition (EMT) is known to play an important role for these malignant behaviors, the association between PanIN and EMT has not been clearly understood. Therefore, we explored possible molecules for regulation of EMT immunohistochemically. Using surgically resected specimens from 71 PDAC patients, expressions of SNAIL, SLUG, TWIST1, and ZEB1 were investigated in high-grade PanIN (HG-PanIN) and PDAC. Results demonstrated that PDAC accompanied by SNAIL-positive HG-PanIN showed a significantly better relapse-free survival (RFS) (median survival time (MST) of 11.3 months vs 4.4 months, P < 0.001) and overall survival overall survival (OS) (MST of 25.2 months vs 13.6 months, P < 0.001). In PDAC accompanied by SLUG-positive HG-PanIN, RFS and OS (P = 0.09 and P = 0.05) tended to have a better prognosis. In contrast, we could not find any significant prognostic benefits in the expression of TWIST1 or ZEB1 in PDAC accompanied by HG-PanIN. Our present results suggest that (1) EMT may play an important role in the development of PDAC from HG-PanIN, and (2) SNAIL may predict a distinct subgroup that shows a better prognosis.

CD45+CD326+ Cells are Predictive of Poor Prognosis in Non-Small Cell Lung Cancer Patients.

PURPOSE: The epithelial-to-mesenchymal transition, the major process by which some cancer cells convert from an epithelial phenotype to a mesenchymal one, has been suggested to drive chemo-resistance and/or metastasis in patients with cancer. However, only a few studies have demonstrated the presence of CD45/CD326 doubly-positive cells (CD45/CD326 DPC) in cancer. We deployed a combination of cell surface markers to elucidate the phenotypic heterogeneity in non-small cell lung cancer (NSCLC) cells and identified a new subpopulation that is doubly-positive for epithelial and non-epithelial cell-surface markers in both NSCLC cells and patients' malignant pleural effusions. EXPERIMENTAL DESIGN: We procured a total of 39 patients' samples, solid fresh lung cancer tissues from 21 patients and malignant pleural effusion samples from 18 others, and used FACS and fluorescence microscopy to check their surface markers. We also examined the EGFR mutations in patients with known acquired EGFR mutations. RESULTS: Our data revealed that 0.4% to 17.9% of the solid tumor tissue cells and a higher percentage of malignant pleural effusion cells harbored CD45/CD326 DPC expressing both epithelial and nonepithelial surface markers. We selected 3 EGFR mutation patients and genetically confirmed that the newly identified cell population really originated from cancer cells. We also found that higher proportions of CD45/CD326 DPC are significantly associated with poor prognosis. CONCLUSIONS: In conclusion, varying percentages of CD45/CD326 DPC exist in both solid cancer tissue and malignant pleural effusion in patients with NSCLC. This CD45/CD326 doubly-positive subpopulation can be an important key to clinical management of patients with NSCLC.