OBJECTIVE: Mitomycin-c (MMC) is the most used intravesical adjuvant agent in non-muscle invasive bladder cancer to prevent recurrence. However, a consensus on about appropriate dosage and treatment schedule of MMC is lacking. We, therefore, aimed to evaluate the most appropriate MMC dosage using an in vitro model of high-grade human bladder cancer.
METHODS: UMUC-3 cells, a model for high-grade bladder cancer, were exposed to MMC in different time courses to assess its toxicological effects. XTT cell proliferation kit was used to evaluate the effect of MMC on the proliferation of UMUC-3 cell line. Gene expression analysis for the MDR1, BCL2 and ANXA5 genes was performed by Real-time PCR and flow cytometry analysis were conducted to evaluate the cell death mechanism and acquired resistance after MMC exposure. An ANXA5 kit was used to detect apoptotic cells, and 7-AAD was used to detect necrotic cells.
RESULTS: Cell proliferation was prevented to a large extent (IC50, 0.17-0.081 mg/mL) and cytotoxic effects were observed after 5 μg/mL and 10 μg/mL MMC administrations for 1 and 2-h, after the 4th and 2nd dose cycles, respectively. Moreover, cell death was observed at 5 μg/mL and 10 μg/mL MMC applications for 1-h and 2-h by the sixth and second week, respectively. Flow cytometry exhibits increased subpopulation of drugextruding UMUC-3 cells after a single dose of MMC for 1-h. MMC did not increase the number of apoptotic or necrotic cells; yet, MDR1 (multiple drug resistance) and ANXA5 (apoptotic) expression levels were increased and BCL2 (anti-apoptotic) expression was decreased.
LIMITATIONS: In-vitro nature of the study and working with only one cell culture are inherit limitations of this project.
CONCLUSION: A single dose of MMC administration for 1 or 2-h results in drug-resistance. If maintenance treatment is administered for one hour, it should be continued throughout a 6-week period.

Most high-grade serous ovarian cancer (HGSOC) patients develop resistance to platinum-based chemotherapy and recur, but 15% remain disease free over a decade. To discover drivers of long-term survival, we quantitatively analyzed the proteomes of platinum-resistant and -sensitive HGSOC patients from minute amounts of formalin-fixed, paraffin-embedded tumors. This revealed cancer/testis antigen 45 (CT45) as an independent prognostic factor associated with a doubling of disease-free survival in advanced-stage HGSOC. Phospho- and interaction proteomics tied CT45 to DNA damage pathways through direct interaction with the PP4 phosphatase complex. In vitro, CT45 regulated PP4 activity, and its high expression led to increased DNA damage and platinum sensitivity. CT45-derived HLA class I peptides, identified by immunopeptidomics, activate patient-derived cytotoxic T cells and promote tumor cell killing. This study highlights the power of clinical cancer proteomics to identify targets for chemo- and immunotherapy and illuminate their biological roles.

BACKGROUND: Annexin A5 (ANXA5) is a kind of Ca2+-dependent phospholipid binding protein which is involved in cell membrane dynamics and organization. Recent data showed that ANXA5 might involve in tumorigenesis.
OBJECTIVE: To explore what role ANXA5 play in human uterine cervical carcinoma.
MATERIALS AND METHODS: In this study, a recombined ANXA5 plasmid was constructed and uterine cervical carcinoma cell lines HeLa and SiHa were transfected with it. After ANXA5 overexpression was determined by Western Blot, cell proliferation test was detected by MTT assay and colony formation assay respectively. FACS assay and Hochest33258 staining methods were employed to detect cell apoptosis. To further investigate whether ANXA5 influence cell migration and invasion, wound healing assay and transwell assay were applied. At the same time, the relative mechanism was investigated.
RESULTS: When ANXA5 expression increased, cell proliferation was inhibited by regulating the expression of bcl-2 and bax while cell metastasis was suppressed by regulating E-cadherin and MMP-9 expression.
CONCLUSION: ANXA5 overexpression in the uterine cervical carcinoma might play important roles in cell proliferation and metastasis of uterine cervical cancer cells and act as an anti-cancer gene in uterine cervical cancer.

Formation of fusion genes is pathogenetically crucial in many solid tumors. They are particularly characteristic of several mesenchymal tumors, but may also be found in epithelial neoplasms. Ovarian carcinomas, too, may harbor fusion genes but only few of these were found to be recurrent with a rate ranging from 0.5 to 5%. Because most attempts to find specific and recurrent fusion transcripts in ovarian carcinomas focused exclusively on high-grade serous carcinomas, the situation in the other carcinoma subgroups remains largely uninvestigated as far as fusion genes are concerned. We performed transcriptome sequencing on a series of 34 samples from ovarian tumors that included borderline, clear cell, mucinous, endometrioid, low-grade and high-grade serous carcinomas in search of fusion genes typical of these subtypes. We found a total of 24 novel fusion transcripts. The PCMTDI-CCNL2 fusion transcript, which involves a member of the cyclin family, was found recurrently involved but only in endometrioid carcinomas (4 of 18 tumors; 22%). We also found three additional fusion transcripts involving genes belonging to the cyclin family: ANXA5-CCNA2 and PDE4D-CCNB1 were detected in two endometrioid carcinomas, whereas CCNY-NRG4 was identified in a clear cell carcinoma. The recurrent involvement of CCNL2 in four fusions and of three other genes of the cyclin family in three additional transcripts hints that deregulation of cyclin genes is important in the pathogenesis of ovarian carcinomas in general but of endometrioid carcinomas particularly.

Acute myeloid leukaemia (AML) is an aggressive blood cancer characterized by a distinct block in differentiation of myeloid progenitors, recurrent chromosomal translocations and gene mutations of which >50% involve signal transduction through dysregulated kinases and phosphatases. In search for novel protein biomarkers for disease stratification we investigated the phosphoproteome in leukaemic cells from 62 AML patients at time of diagnosis using immobilized metal-affinity chromatography, protein separation by two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry before validation by selected reaction monitoring (SRM). Unsupervised clustering found 27 phosphoproteins significantly discriminating patients according to leukaemic cell differentiation (French-American-British (FAB) classification), cytogenetic and mutational (FLT3, NPM1) status or response to chemotherapy. Monocytic differentiation (FAB M4-M5) correlated with enrichment of proteins involved in apoptosis (MOES, ANXA5 and EFHD2). TALDO, a protein associated with thrombocytopenia if down-regulated, was elevated in patients with wild type NPM1 compared to patients with NPM1 mutation. This study demonstrates the potential of quantitative proteomics in AML classification and risk stratification.
BIOLOGICAL SIGNIFICANCE: Patients diagnosed with AML are currently categorized according to cellular morphology, cytogenetic alterations and mutations, although the majority of these cellular and genetic alterations have no or unsolved impact on therapy selection or prognosis. We therefore explored the phosphoproteome for abundance changes associated with traditional classifiers to unravel patterns that could stratify patients at the protein level. MOES, ANXA5 and EFHD2 were confirmed by SRM to be correlated to monocytic differentiation, whilst TALDO was elevated in NPM1 wild type patients.

OBJECTIVES: The aim of this study was to identify biomarkers for predicting the efficacy of docetaxel, cisplatin, and S-1 (DCS) therapy for advanced gastric cancer using microarrays of biopsy specimens before chemotherapy.
METHODS: Nineteen samples were taken from 19 patients with unresectable metastatic gastric cancer who received DCS as a first-line therapy. Laser capture microdissection was performed, and total cellular RNA was extracted from each microdissected sample. Whole-gene expression was analyzed by microarray, and the difference in mRNA expression observed with the microarrays was confirmed by quantitative real-time PCR. Immunohistochemical staining was performed using clinical tissue sections obtained by endoscopic biopsy.
RESULTS: Eleven patients were identified as early responders and 8 patients as nonresponders to DCS therapy. Twenty-nine genes showed significant differences in relative expression ratios between tumor and normal tissues. A classifier set of 29 genes had high accuracy (94.7%) for distinguishing gene expression between 11 early responders and 8 nonresponders. Decreasing the size of the classifier set to 4 genes (PDGFB, PCGF3, CISH, and ANXA5) increased the accuracy to 100%. Expression levels by real-time PCR for validation were well correlated with those 4 genes in microarrays.
CONCLUSION: The genes identified may serve as efficient biomarkers for personalized cancer-targeted therapy.

OBJECTIVE: We investigated the expression of annexin A5 (ANXA5) in human cholangiocarcinoma (CCA) cell line and its effect on proliferation, migration, and apoptosis of human CCA cells.
MATERIALS AND METHODS: Expression of ANXA5 was detected by fluorescent quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blotting method in 2 human CCA cell lines, QBC939 and RBE. 3 shRNA plasmids for ANXA5 silencing (ANXA5-sh1, ANXA5-sh2, ANXA5-sh3) and 1 negative control plasmid were constructed to infect QBC939 cells. The infection efficiency, expression of ANXA5, apoptosis and cell cycle of QBC939 cell were measured separately.
RESULTS: The expression of ANXA5 in QBC939 cell was significantly higher than RBE cell. Expressed ANXA5 protein in the QBC939-KD cell (QBC939 cell treated by RNAi) was significantly lower than QBC939-BC (QBC939 cell) and QBC939-NC cells (QBC939 cell treated by scramble plasmid). The ratio of G0/1 phase cells and apoptosis rate increased in QBC939-KD cell. The proliferation activity and invasion ability decreased in QBC939-KD cell compared with QBC939-NC and QBC939-BC cells.
CONCLUSIONS: ANXA5 play important role in the migration and apoptosis of CCA cells. Inhibiting the expression of ANXA5 significantly reduce the proliferation, migration and invasion ability of QBC939 cells, and increase the apoptosis of QBC939 cells.

The success of siRNA gene therapy requires the availability of safe and efficient delivery systems. In the present study, we investigated poly(vinyl benzyl trimethylammonium chloride) (PVTC) and its block copolymer with poly(oligo(ethyleneglycol) methacrylate) (POEGMA) as delivery vector for siRNA. Small polyplexes ranging from 8 to 25nm in diameter were formed in aqueous solution by spontaneous self-assembly of both the homopolymer and block copolymer with siRNA and the formed particles were stable at physiological ionic strength. It was shown that when human ovarian adenocarcinoma cells were transfected, siRNA polyplexes based on PVTC (40kDa) and PVTC-POEGMA-4 (PP4, 34kDa) efficiently induced luciferase gene silencing to the same extent as the formulation based on a commercial lipid (Lipofectamine

BACKGROUND: The control of breast cell survival is of critical importance for preventing breast cancer initiation and progression. The activity of many proteins which regulate cell survival is controlled by reversible phosphorylation, so that the relevant kinases and phosphatases play crucial roles in determining cell fate. Several protein kinases act as oncoproteins in breast cancer and changes in their activities contribute to the process of transformation. Through counteracting the activity of oncogenic kinases, the protein phosphatases are also likely to be important players in breast cancer development, but this class of molecules is relatively poorly understood. Here we have investigated the role of the serine/threonine protein phosphatase 4 in the control of cell survival of breast cancer cells.
METHODS: The breast cancer cell lines, MCF7 and MDA-MB-231, were transfected with expression vectors encoding the catalytic subunit of protein phosphatase 4 (PP4c) or with PP4c siRNAs. Culture viability, apoptosis, cell migration and cell cycle were assessed. The involvement of phosphoprotein enriched in astrocytes 15kDa (PEA15) in PP4c action was investigated by immunoblotting approaches and by siRNA-mediated silencing of PEA15.
RESULTS: In this study we showed that PP4c over-expression inhibited cell proliferation, enhanced spontaneous apoptosis and decreased the migratory and colony forming abilities of breast cancer cells. Moreover, PP4c down-regulation produced complementary effects. PP4c is demonstrated to regulate the phosphorylation of PEA15, and PEA15 itself regulates the apoptosis of breast cancer cells. The inhibitory effects of PP4c on breast cancer cell survival and growth were lost in PEA15 knockdown cells, confirming that PP4c action is mediated, at least in part, through the de-phosphorylation of apoptosis regulator PEA15.
CONCLUSION: Our work shows that PP4 regulates breast cancer cell survival and identifies a novel PP4c-PEA15 signalling axis in the control of breast cancer cell survival. The dysfunction of this axis may be important in the development and progression of breast cancer.

Exosomes are small membrane vesicles that have important roles in transporting a great variety of bioactive molecules between epithelial compartment and their microenvironment during tumor formation including colorectal adenoma-carcinoma sequence. We tested the mRNA expression of the top 25 exosome-related markers based on ExoCharta database in healthy (n=49), adenoma (n=49) and colorectal carcinoma (n=49) patients using Affymetrix HGU133 Plus2.0 microarrays. Most related genes showed significantly elevated expression including PGK1, PKM, ANXA5, ENO1, HSP90AB1 and MSN during adenoma-carcinoma sequence. Surprisingly, the expression of ALIX (ALG 2-interacting protein X), involved in multivesicular body (MVB) and exosome formation, was significantly reduced in normal vs adenoma (P=5.02 × 10(-13)) and in normal vs colorectal carcinoma comparisons (P=1.51 × 10(-10)). ALIX also showed significant reduction (P<0.05) at the in situ protein level in the epithelial compartment of adenoma (n=35) and colorectal carcinoma (n=37) patients compared with 27 healthy individuals. Furthermore, significantly reduced ALIX protein levels were accompanied by their gradual transition from diffuse cytoplasmic expression to granular signals, which fell into the 0.6-2 μm diameter size range of MVBs. These ALIX-positive particles were seen in the tumor nests, including tumor-stroma border, which suggest their exosome function. MVB-like structures were also detected in tumor microenvironment including α-smooth muscle actin-positive stromal cells, budding off cancer cells in the tumor front as well as in cancer cells entrapped within lymphoid vessels. In conclusion, we determined the top aberrantly expressed exosome-associated markers and revealed the transition of diffuse ALIX protein signals into a MVB-like pattern during adenoma-carcinoma sequence. These tumor-associated particles seen both in the carcinoma and the surrounding microenvironment can potentially mediate epithelial-stromal interactions involved in the regulation of tumor growth, metastatic invasion and therapy response.

AIM: To investigate ANXA5 overexpression on in vitro and in vivo malignancies of murine Hca-P cells.
MATERIALS & METHODS: Hca-P with low lymph node metastasis (LNM) potential was used as cell model. TEM, CCK-8 and Boyden transwell assays were performed for in vitro Hca-P behaviors. Hca-P-transplanted mouse model was established for in vivo experiment.
RESULTS: ANXA5-overexpressing monoclonal Anxa5-Hca-P-1, Anxa5-Hca-P-2 and Anxa5-Hca-P-3 cells were obtained. ANXA5 upregulation alters the proliferation, morphology and rough endoplasmic reticulum of Hca-P cells, enhances in vitro migration and invasions of Hca-P, promotes in vivo malignant degree and LNM rate of Anxa5-Hca-P-3-transplanted mice.
CONCLUSION: As a potential indicator for malignancy and lymphatic metastasis, ANXA5 overexpression increases in vitro migration and invasion of Hca-P cell, promotes in vivo malignancy, LNM rate and level of Hca-P-transplanted mice.

Cisplatin is commonly used in ovarian cancer treatment by inducing apoptosis in cancer cells as a result of lethal DNA damage. However, the intrinsic and acquired resistance to cisplatin in cancer cells remains a big challenge for improving overall survival. The cyto-protective functions of autophagy in cancer cells have been suggested as a potential mechanism for chemoresistance. Here, we reported MIR152 as a new autophagy-regulating miRNA that plays a role in cisplatin-resistance. We showed that MIR152 expression was dramatically downregulated in the cisplatin-resistant cell lines A2780/CP70, SKOV3/DDP compared with their respective parental cells, and in ovarian cancer tissues associated with cisplatin-resistance. Overexpression of MIR152 sensitized cisplatin-resistant ovarian cancer cells by reducing cisplatin-induced autophagy, enhancing cisplatin-induced apoptosis and inhibition of cell proliferation. A mouse subcutaneous xenograft tumor model using A2780/CP70 cells with overexpressing MIR152 was established and displayed decreased tumor growth in response to cisplatin. We also identified that ATG14 is a functional target of MIR152 in regulating autophagy inhibition. Furthermore, we found that EGR1 (early growth response 1) regulated the MIR152 gene at the transcriptional level. Ectopic expression of EGR1 enhanced efficacy of chemotherapy in A2780/CP70 cells. More importantly, these findings were relevant to clinical cases. Both EGR1 and MIR152 expression levels were significantly lower in ovarian cancer tissues with high levels of ERCC1 (excision repair cross-complementation group 1), a marker for cisplatin-resistance. Collectively, these data provide insights into novel mechanisms for acquired cisplatin-resistance. Activation of EGR1 and MIR152 may be a useful therapeutic strategy to overcome cisplatin-resistance by preventing cyto-protective autophagy in ovarian cancer.

Single nucleotide-polymorphisms (SNPs) are a source of diversity among human population, which may be responsible for the different individual susceptibility to diseases and/or response to drugs, among other phenotypic traits. Several low penetrance susceptibility genes associated with malignant melanoma (MM) have been described, including genes related to pigmentation, DNA damage repair and oxidative stress pathways. In the present work, we conducted a candidate gene association study based on proteins and genes whose expression we had detected altered in melanoma cell lines as compared to normal melanocytes. The result was the selection of 88 loci and 384 SNPs, of which 314 fulfilled our quality criteria for a case-control association study. The SNP rs6854854 in ANXA5 was statistically significant after conservative Bonferroni correction when 464 melanoma patients and 400 controls were analyzed in a discovery Phase I. However, this finding could not be replicated in the validation phase, perhaps because the minor allele frequency of SNP rs6854854 varies depending on the geographical region considered. Additionally, a second SNP (rs6431588) located on ILKAP was found to be associated with melanoma after considering a combined set of 1,883 MM cases and 1,358 disease-free controls. The OR was 1.29 (95% CI 1.12-1.48; p-value = 4×10-4). Both SNPs, rs6854854 in ANXA5 and rs6431588 in ILKAP, show population structure, which, assuming that the Spanish population is not significantly structured, suggests a role of these loci on a specific genetic adaptation to different environmental conditions. Furthermore, the biological relevance of these genes in MM is supported by in vitro experiments, which show a decrease in the transcription levels of ANXA5 and ILKAP in melanoma cells compared to normal melanocytes.

HPK1, a member of mammalian Ste20-like serine/threonine kinases, is lost in >95% pancreatic cancer through proteasome-mediated degradation. However, the mechanism of HPK1 loss has not been defined. The aims of this study are to identify the ubiquitin ligase and to examine the mechanisms that targets HPK1 degradation. We found that the CUL7/Fbxw8 ubiquitin ligase targeted HPK1 for degradation via the 26 S proteasome. The ubiquitination of HPK1 required its kinase activity and autophosphorylation. Wild-type protein phosphatase 4 (PP4), but not the phosphatase-dead PP4 mutant, PP4-RL, inhibits the interaction of Fbxw8 with HPK1 and Fbxw8-mediated ubiquitination of HPK1. In addition, we showed that Thr-355 of HPK1 is a key PP4 dephosphorylation site, through which CUL7/Fbxw8 ubiquitin ligase and PP4 regulates HPK1 stability. Knockdown of Fbxw8 restores endogenous HPK1 protein expression and inhibits cell proliferation of pancreatic cancer cells. Our study demonstrated that targeted degradation of HPK1 by the CUL7/Fbxw8 ubiquitin ligase constitutes a negative-feedback loop to restrain the activity of HPK1 and that CUL7/Fbxw8 ubiquitin ligase promotes pancreatic cancer cell proliferation. CUL7/Fbxw8 ubiquitin ligase-mediated HPK1 degradation revealed a direct link and novel role of CUL7/Fbxw8 ubiquitin ligase in the MAPK pathway, which plays a critical role in cell proliferation and differentiation.

Bladder cancer is a highly heterogeneous neoplasm. We examined the gene expression profile in 3 bladder cancer stages (Ta, T1, T2) using expression microarray analysis of 40 bladder tumors. Differentially expressed genes were found by the t-test, with <0.005 as the significance threshold. KEGG pathway-enrichment analysis was used to study the signaling pathways of the genes. We found 36 genes that could be used as molecular markers for predicting the transition from Ta-T1 to T1-T2. Among these, 11 overlapped between Ta-T1 and T1-T2 stages. Six genes were down-regulated at the Ta-T1 stage, but were up-regulated at the T1-T2 stage (ANXA5, ATP6V1B2, CTGF, GEM, IL13RA1, and LCP1); 5 genes were up-regulated at the Ta-T1 stage, but down-regulated at the T1-T2 stage (ACPP, GNL1, RIPK1, RAPGEF3, and ZER1). Another 25 genes changed relative expression levels at the T1-T2 stage. These genes (including COL1A1, COL1A2, FN1, ITGA5, LGALS1, SPP1, VIM, POSTN, and COL18A1) may be involved in bladder cancer progression by affecting extracellular matrix-receptor interaction and focal adhesion. The cytokine-cytokine receptor interaction, neuroactive ligand-receptor interaction, and calcium-signaling pathway were associated with bladder cancer progression at both the Ta-T1 and T1-T2 stages.

OBJECTIVE: The aim of this study was to analyze the contribution of the M2 haplotype of ANXA5 gene, previously identified as a risk factor for RPL and thrombophilia related pregnancy complications, to repeated miscarriage observed in PCOS patients.
PATIENTS/METHODS: 100 PCOS patients, 500 fertile women and 533 random population controls were genotyped for M2/ANXA5.
RESULTS: M2 haplotype carriers faced a 3.4 fold elevated RPL risk (odds ratio 5.3, 95% confidence interval 3-9.2) compared to female fertile controls and 2.1 (odds ratio 2.6, 95% confidence interval 1.6-4.3) compared to population controls. The relative population risks in subgroups of PCOS patients with primary and secondary RPL were 2.3 (odds ratio 2.5, 95% confidence interval 1.2-5) and 3.3 (odds ratio 3.6, 95% confidence interval 1.5-8.4) respectively. As compared to the fertile women group, the relative risks equaled 4 (odds ratio 5, 95% confidence interval 2.3-10.8) and 6 (odds ratio 7.2, 95% confidence interval 3-17.7). Estimated relative risks for M2 carriers among PCOS RPL patients matched the values previously obtained for repeated miscarriage populations. The essential phenotypes, clinically defining PCOS, associated neither with RPL in their diagnostically relevant combinations, nor with M2 carriage as RPL risk factor in the PCOS RPL subgroups.
CONCLUSIONS: M2/ANXA5 seems an independent RPL risk factor in PCOS patients that progressively correlates with the number of first trimester pregnancies. From our pilot study in PCOS women it appears relevant to offer M2/ANXA5 diagnostic analysis to such patients with RPL complications, to possibly guide proper therapeutic decisions.

Multidrug resistance (MDR) to anticancer drugs is a major obstacle to successful chemotherapy of tumors. Understanding the molecular basis to chemoresistance is likely to provide better treatment. Cell lines resistant to cis-diamminedichloroplatinum (CNE2/cDDP) were established from human nasopharyngeal carcinoma (NPC) cell lines CNE2. Comparative proteomics involving 2-dimensional gel electrophoresis (2-DE) and ESI-Q-TOF-MS were performed on protein extracted from CNE2 and CNE2/cDDP cell lines to screen drug resistance-related proteins. Keratin 1 (KRT1), cathepsin D (CTSD) and annexin a5 (ANXA5) were identified as three proteins showing higher expression in CNE2/cDDP compared to CNE2. Furthermore, suppression of KRT1 expression by siRNA resulted in decreased MDR in siRNA-CNE2/cDDP cells. And upregulation of KRT1 could result in increased of drug resistance in NPC cell lines. Taken together, KRT1 protein and its activity levels were higher in cDDP-resistant NPC cell lines compared to their parental cell lines. These data clearly linked KRT1 and cDDP resistance mechanisms. KRT1 could serve as a biomarker for chemotherapy sensitivity of NPC.

Mithramycin (MIT) and tolfenamic acid (TA) inhibit the activity of the transcription factor Sp1. In the present study, we investigated whether pancreatic cancer treatment with a combination of these compounds has a synergistic effect on Sp1 activity, tumor growth, and their underlying response mechanisms. Treatment of pancreatic tumor xenografts with MIT and TA produced dose-dependent antitumor activity, and significant antitumor activity of either compound alone was directly associated with systemic side effects. Combination treatment with nontoxic doses of both compounds produced synergistic antitumor activity, whereas treatment with a nontoxic dose of either compound alone lacked a discernible antitumor effect. Synergistic therapeutic effects correlated directly with synergistic antiproliferation and antiangiogenesis in vitro. Moreover, combination treatment resulted in Sp1 protein degradation, drastically downregulating expression of Sp1 and vascular endothelial growth factor. Our findings established that Sp1 is a critical target of TA and MIT in human pancreatic cancer therapy, rationalizing clinical studies to determine the effect of existing pancreatic cancer therapy regimens on Sp1 signaling in tumors and normal pancreatic tissue, and the ability of Sp1-targeting strategies to modify cancer responses.

The control of T-cell survival is of overwhelming importance for preventing leukemia and lymphoma. The present report demonstrates that the serine/threonine protein phosphatase PP4 regulates the survival of both leukemic T-cells and untransformed human peripheral blood T-cells, particularly after treatment with anti-leukemic drugs and other cytotoxic stimuli. PP4-induced apoptosis is mediated, at least in part, through de-phosphorylation of apoptosis regulator PEA-15, previously implicated in the control of leukemic cell survival. PP4 activity significantly affects the mutation rate in leukemic T-cells, indicating that PP4 dysfunction may be important in the development and progression of leukemia.

GOALS: To gain an insight into the putative role of annexin A5 (ANXA5) in the tumor stage and its clinical outcome.
BACKGROUND: ANXA5 is a calcium-binding protein, which has been implicated in the carcinogenesis of several carcinomas. However, the role of ANXA5 in colorectal cancer (CRC) is unclear.
STUDY: We investigated the expression of ANXA5 in colorectal adenocarcinoma. This study included 207 consecutive patients with sporadic CRC. Paired colorectal tissue samples and corresponding nonmalignant tissues were obtained by surgical resection. ANXA5 mRNA and protein expression in each tissue were assessed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical staining. Data were statistically correlated with pathologic parameters and clinical outcome.
RESULTS: Real-time reverse transcriptase polymerase chain reaction showed that there is an up-regulation in the mRNA level of ANXA5 in tumors (P<0.001). Immunohistochemical study revealed that high ANXA5 expression was present in 40.58% (84 of 207) of tumors. Univariate analysis showed increased ANXA5 expression correlated with pT stage (P=0.008), liver metastasis (P=0.024), pathologic tumor-node-metastasis stage (P=0.015), Dukes' stage (P=0.017), recurrence (P=0.024), cancer-related death (P=0.028), recurrence-free probability (P=0.003), and overall survival (P=0.005). Multivariate analysis showed that ANXA5 expression and liver metastasis significantly correlated with recurrence-free probability (P=0.039 and P=0.048, respectively) and overall survival (P=0.012 and P=0.021, respectively) independent of pT stage and pN stage.
CONCLUSIONS: From these findings ANXA5 expression seems to be related to the tumor stage and clinical outcome of CRC. Thus ANXA5 could serve as a prognostic marker for tumor progression.

PURPOSE: To investigate the differential proteins and related molecular mechanism of CDA-II (cell differentiation agent-II) induced therapy on a human hepatocellular carcinoma model in nude mice with high metastatic potential (LCI-D20).
METHODS: After tumors were transplanted 11 days, mice were intraperitoneally injected with CDA-II (1,800 mg/kg) for 20 days continuously. The tumor growth-inhibitory efficiency in CDA-II treated groups was calculated. Proteins extracted from tumor tissue were separated by two-dimensional gel electrophoresis (2DE) and the differential proteins were identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Western blotting (WB) was performed to verify the expression of certain candidate proteins. Reverse transcription-polymerase chain reaction (RT-PCR) was engaged to study the molecular mechanism of the therapy.
RESULTS: CDA-II suppressed the growth and metastasis of tumor. The tumor growth-inhibitory efficiency was 41.8%. In total, 27 differentially expressed proteins were identified, including HSP27, UGDH, CK8, Hsp60, ENOA and AnxA5, with functions involved in oncogene expression and/or cell differentiation. In addition, apparent alternations of HSP60 and beta-actin expression levels and their different posttranslational modifications (PTMs) were investigated. RT-PCR analysis confirmed that the cancer related genes c-myc, N-ras and MMP-9 were significantly down-regulated.
CONCLUSION: Our results demonstrate that CDA-II presence can change the proteome profiling and favors of the tumor suppression in LCI-D20 cell differentiation. Our results also suggest that the dynamic PTM of HSP60 expression levels could be used to predict HCC and might be a promising and useful biomarker to prognosticate CDA-II therapeutic efficacy.

The zinc finger transcription factor Krüppel-like factor 4 (KLF4) has been implicated in both tumor suppression and progression. However, its function in pancreatic cancer has not been well characterized. Here, we show that pancreatic cancer cell lines expressed various levels of KLF4 RNA and protein. Ectopic expression of KLF4 by FG and BxPC-3 pancreatic cancer cells resulted in cell cycle arrest and marked inhibition of cell growth in vitro and attenuation of tumor growth and metastasis in an orthotopic mouse model. Overexpression of KLF4 also led to significant induction of p27(Kip1) expression, at both the RNA and protein levels, in a dose- and time-dependent manner, indicating that KLF4 transcriptionally regulates the expression of p27(Kip1). Chromatin immunoprecipitation assays consistently showed that KLF4 protein physically interacts with the p27(Kip1) promoter. Promoter deletion and point mutation analyses indicated that a region between nucleotides -435 and -60 of the p27(Kip1) promoter and intact of the three KLF4-binding sites within that region were required for the full induction of p27(Kip1) promoter activity by KLF4. Our findings suggest that KLF4 transactivates p27(Kip1) expression and inhibits the growth and metastasis of human pancreatic cancer.

BACKGROUND: Human pancreatic cancer over expresses the transcription factor Sp1. However, the role of Sp1 in pancreatic cancer angiogenesis and its use as target for antiangiogenic therapy remain unexplored.
METHODS: Archived human pancreatic cancer specimens were used to assess gene expression and microvessel density (MVD) status by immunohistochemistry: Small-interfering RNA (siRNA) was used to determine the impact of altered Sp1 expression on tumor growth and angiogenesis, and mithramycin A (MIT) was used to evaluate Sp1-targeted antiangiogenic treatment of human pancreatic cancer in animal models.
RESULTS: The expression level of Sp1 was correlated directly with the MVD status (P < .001) and the expression level of vascular endothelial growth factor (VEGF) (P < .05). Knockdown of Sp1 expression did not affect the growth of pancreatic cancer cells in vitro but inhibited their growth and metastasis in mouse models. This antitumor activity was consistent with the in vitro and in vivo antiangiogenic activity resulting from Sp1 knockdown. Subcutaneous and intraperitoneal injection of MIT significantly suppressed the growth of human pancreatic cancer in mouse models. This tumor suppression was correlated with the suppression of Sp1 expression in growing tumors but not in normal tissues. Moreover, treatment with MIT reduced tumor MVD, which was consistent with the down-regulation of VEGF, platelet-derived growth factor, and epidermal growth factor receptor.
CONCLUSIONS: Both clinical and experimental evidence indicated that Sp1 is a critical regulator of human pancreatic cancer angiogenesis and the antitumor activity of MIT is a result, at least in part, of the suppression of Sp1 expression and consequent down-regulation the downstream targets of Sp1 that are key to angiogenesis.

The impact of antiangiogenic therapy on the Sp1/vascular endothelial growth factor (VEGF) pathway and that of alteration of Sp1 signaling on the efficacy of antiangiogenic therapy is unclear, yet understanding their interactions has significant clinical implications. Treatment with bevacizumab, a neutralizing antibody against VEGF, suppressed human pancreatic cancer growth in nude mice. Gene expression analyses revealed that this treatment substantially up-regulated the expression of Sp1 and its downstream target genes, including VEGF and epidermal growth factor receptor, in tumor tissues, whereas it did not have this effect on pancreatic cancer cells in culture. Treatment with mithramycin A, an Sp1 inhibitor, suppressed the expression of Sp1 and its downstream target genes in both cell culture and tumors growing in nude mice. Combined treatment with bevacizumab and mithramycin A produced synergistic tumor suppression, which was consistent with suppression of the expression of Sp1 and its downstream target genes. Thus, treatment with bevacizumab may block VEGF function but activate the pathway of its expression via positive feedback. Given the fact that Sp1 is an important regulator of the expression of multiple angiogenic factors, bevacizumab-initiated up-regulation of Sp1 and subsequent overexpression of its downstream target genes may profoundly affect the potential angiogenic phenotype and effectiveness of antiangiogenic strategies for human pancreatic cancer. Therefore, this study is the first to show the significance and clinical implications of alteration of Sp1 signaling in antiangiogenic therapy for pancreatic cancer and other cancers.

BACKGROUND: Previous studies on bladder cancer have shown nodal involvement to be an independent indicator of prognosis and survival. This study aimed at developing an objective method for detection of nodal metastasis from molecular profiles of primary urothelial carcinoma tissues.
METHODS: The study included primary bladder tumor tissues from 60 patients across different stages and 5 control tissues of normal urothelium. The entire cohort was divided into training and validation sets comprised of node positive and node negative subjects. Quantitative expression profiling was performed for a panel of 70 genes using standardized competitive RT-PCR and the expression values of the training set samples were run through an iterative machine learning process called genetic programming that employed an N-fold cross validation technique to generate classifier rules of limited complexity. These were then used in a voting algorithm to classify the validation set samples into those associated with or without nodal metastasis.
RESULTS: The generated classifier rules using 70 genes demonstrated 81% accuracy on the validation set when compared to the pathological nodal status. The rules showed a strong predilection for ICAM1, MAP2K6 and KDR resulting in gene expression motifs that cumulatively suggested a pattern ICAM1 > MAP2K6 > KDR for node positive cases. Additionally, the motifs showed CDK8 to be lower relative to ICAM1, and ANXA5 to be relatively high by itself in node positive tumors. Rules generated using only ICAM1, MAP2K6 and KDR were comparably robust, with a single representative rule producing an accuracy of 90% when used by itself on the validation set, suggesting a crucial role for these genes in nodal metastasis.
CONCLUSION: Our study demonstrates the use of standardized quantitative gene expression values from primary bladder tumor tissues as inputs in a genetic programming system to generate classifier rules for determining the nodal status. Our method also suggests the involvement of ICAM1, MAP2K6, KDR, CDK8 and ANXA5 in unique mathematical combinations in the progression towards nodal positivity. Further studies are needed to identify more class-specific signatures and confirm the role of these genes in the evolution of nodal metastasis in bladder cancer.

To identify novel methylation-silenced genes in gastric cancers, we carried out a chemical genomic screening, a genome-wide search for genes upregulated by treatment with a demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC). After 5-aza-dC treatment of a gastric cancer cell line (AGS) 579 genes were upregulated 16-fold or more, using an oligonucleotide microarray with 39,000 genes. From these genes, we selected 44 known genes on autosomes whose silencing in gastric cancer has not been reported. Thirty-two of these had CpG islands (CGI) in their putative promoter regions, and all of the CGI were methylated in AGS, giving an estimated number of 421+/-75 (95% confidence interval) methylation-silenced genes. Additionally, we analyzed the methylation status of 16 potential tumor-related genes with promoter CGI that were upregulated four-fold or more, and 14 of these were methylated in AGS. Methylation status of the 32 randomly selected and 16 potential tumor-related genes was analyzed in 10 primary gastric cancers, and 42 genes (ABHD9, ADFP, ALDH1A3, ANXA5, AREG, BDNF, BMP7, CAV1, CDH2, CLDN3, CTSL, EEF1A2, F2R, FADS1, FSD1, FST, FYN, GPR54, GREM1, IGFBP3, IGFBP7, IRS2, KISS1, MARK1, MLF1, MSX1, MTSS1, NT5E, PAX6, PLAGL1, PLAU, PPIC, RBP4, RORA, SCRN1, TBX3, TFAP2C, TNFSF9, ULBP2, WIF1, ZNF177 and ZNF559) were methylated in at least one primary gastric cancer. A metastasis suppressor gene, MTSS1, was located in a genomic region with frequent loss of heterozygosity (8q22), and was expressed abundantly in the normal gastric mucosa, suggesting its role in gastric carcinogenesis. (Cancer Sci 2006; 97: 64 -71).

The specific signaling connections between the mitogen-activated protein kinases (MAPK) such as c-Jun N-terminal kinase (JNK-1) and phosphatases PP4 and M3/6, affecting the family of early nuclear factors, is complex and remains poorly understood. JNK-1 regulates cellular differentiation, apoptosis and stress responsiveness by up-regulating early nuclear factors such as c-Jun, a member of the activating protein (AP-1) family, and the Early Growth Factor (EGR-1). C-Jun, when phosphorylated by c-Jun N-terminal kinase (JNK-1) associates with c-Fos to form the AP-1 transcription factor that activates gene expression. We have investigated the regulation of the JNK-1 kinase by co-transfecting phosphatases PP4 and M3/6 in prostate cancer cell lines PC-3 and LNCaP, which have been previously stimulated with human EGF or cisplatin. Co-transfections of plasmids expressing the JNK-1 and the serine/threonine phosphatases PP4 resulted in a significant increase in JNK-1 activity in both PC3 and LNCaP cells. In contrast, co-transfection of JNK-1 with the dual specific phosphatase serine/threonine M3/6 showed only a marginal effect in JNK-1 activity. The phosphatase M3/6 also failed in blocking the induction of JNK-1 activity observed in presence of PP4. The higher activity of JNK-1 was associated with increased activities of the factors c-Jun/AP-1 and EGR-1. This suggests that JNK-1 activity in PC-3 and LNCaP cells requires not only active PP4 for stable maintenance but also suggests that the relative degree of phosphorylation of multiple cellular components is the determinant of JNK-1 stability.

Inducible nitric oxide (NO) synthase (NOS) II has been implicated in macrophage-mediated antitumor activity. However, use of the NOS II gene in cancer therapy is problematic because of the double-edged nature of NO action. Herein we show that adenoviral vectors mediated effective NOS II gene transfer into various human tumors. Production of NO significantly up-regulated multiple angiogenic molecules. However, the NO-producing tumor cells did not form tumors or metastases in ectopic or orthotopic xenograft nude mouse models. The dramatic loss of malignancy was due to NO-mediated apoptosis. We also generated a series of adenoviral vectors harboring mutant NOS II genes that expressed mutant NOS II proteins with defined levels of enzymatic activity. Tumor cells transduced with these NOS II genes produced NO at different levels, which directly correlated with the antitumor activity in vitro and in vivo. This demonstration using a relevant biological system shows that NO produces dose-dependent antitumor activity in vitro and in vivo, regardless of its up-regulation of protumor factors.

Butyrate induces differentiation and alters cell proliferation in intestinal-epithelial cells by modulation of the expression of several genes. Annexins are a superfamily of ubiquitous proteins characterized by their calcium-dependent ability to bind to biological membranes; their involvement in several physiological processes, such as membrane trafficking, calcium signaling, cell motility, proliferation, and differentiation has been proposed. Thus, we have analyzed changes in annexin A1 (AnxA1), annexin A2 (AnxA2), and annexin A5 (AnxA5) levels and localization in human colon adenocarcinoma cells differentiated by butyrate treatment or by culture in glucose-free inosine-containing medium. The acquired differentiated phenotype increased dipeptidyl peptidase-IV (DPP-IV) expression and alkaline phosphatase (ALP) activity, two well known brush border markers. Butyrate induces cell differentiation and growth arrest in BCS-TC2, BCS-TC2.2, HT-29, and Caco-2 cells, increasing the levels of AnxA1 and AnxA5, whereas AnxA2 decreases except in Caco-2 cells. Inosine-differentiated cells present increased amounts of the three studied annexins, as occurs in spontaneously differentiated Caco-2 cells. AnxA2 down-regulation is not due to proteasome activation and seems to be related to the butyrate-induced cell proliferation arrest; AnxA1 and AnxA5 expression is growth-state independent. AnxA1 and AnxA5 are mainly found in the cytoplasm while AnxA2 is localized underneath the plasma membrane in cell-to-cell contacts. Butyrate induces changes in subcellular localization towards a vesicle-associated pattern. Human colon adenocarcinoma cell differentiation is associated with an up-regulation of AnxA1, AnxA2, and AnxA5 and with a subcellular relocation of these proteins. No correlation between annexin levels and tumorigenicity was found. Up-regulation of AnxA1 could contribute to the reported anti-inflammatory effects of butyrate in colon inflammatory diseases.