A novel role for carcinoembryonic antigen-related cell adhesion molecule 6 as a determinant of gemcitabine chemoresistance in pancreatic adenocarcinoma cells

Most patients with pancreatic adenocarcinoma present with surgically incurable disease. Gemcitabine, the principal agent used to treat such patients, has little impact on outcome. Overexpression of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6, a feature of this malignancy, is associated with resistance to anoikis and increased metastasis. The purpose of this study was to determine the role of CEACAM6 in cellular chemoresistance to gemcitabine. CEACAM6 was stably overexpressed in Capan2 cells, which inherently express very low levels of the protein. Suppression of CEACAM6 expression was achieved in BxPC3 cells, which inherently overexpress CEACAM6, by stable transfection of a CEACAM6 small interfering RNA-generating vector. The effects of modulating CEACAM6 expression on gemcitabine-induced cytotoxicity were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay, flow cytometric apoptosis quantification, caspase profiling, and Western analysis of cytoplasmic cytochrome c release. The roles of Akt and c-Src kinases as downstream targets of CEACAM6 signaling were examined. Stable overexpression of CEACAM6 in Capan2 increased gemcitabine chemoresistance, whereas CEACAM6 gene silencing in BxPC3 markedly increased the sensitivity of these cells to gemcitabine. Differential expression of CEACAM6 modulates Akt activity in a c-Src-dependent manner, and CEACAM6 overexpression appears to protect cells from cytochrome c-induced caspase 3 activation and apoptosis.     

RNA interference targeting the M2 subunit of ribonucleotide reductase enhances pancreatic adenocarcinoma chemosensitivity to gemcitabine

Ribonucleotide reductase is emerging as an important determinant of gemcitabine chemoresistance in human cancers. Activity of this enzyme, which catalyses conversion of ribonucleotide 5'-diphosphates to their 2'-deoxynucleotides, is modulated by levels of its M2 subunit (RRM2). Here we show that RRM2 overexpression is associated with gemcitabine chemoresistance in pancreatic adenocarcinoma cells, and that suppression of RRM2 expression using RNA interference mediated by small interfering RNA (siRNA) enhances gemcitabine-induced cytotoxicity in vitro. We demonstrate the ability of systemically administered RRM2 siRNA to suppress tumoral RRM2 expression in an orthotopic xenograft model of pancreatic adenocarcinoma. Synergism between RRM2 siRNA and gemcitabine results in markedly suppressed tumor growth, increased tumor apoptosis and inhibition of metastasis. Our findings confirm the importance of RRM2 in pancreatic adenocarcinoma gemcitabine chemoresistance. This is the first demonstration that systemic delivery of siRNA-based therapy can enhance the efficacy of an anticancer nucleoside analog.     

A novel role for placental leucine aminopeptidase (P-LAP) as a determinant of chemoresistance in endometrial carcinoma cells

In several recent studies, we have shown that P-LAP can be a poor prognostic factor and a factor of chemoresistance in endometrial carcinoma, especially in the advanced patients. In our study, we investigated whether P-LAP alters the expression of apoptosis regulatory proteins as a mechanism of drug resistance. We transfected P-LAP cDNA into A-MEC cells (endometrial adenocarcinoma cell line), and A-MEC-LAP cells displayed a 1.8-fold, 2.0-fold and 1.7-fold increase in IC(50) against paclitaxel, carboplatin and cisplatin respectively. Translational downregulation by siRNA2 to P-LAP on A-MEC-LAP cells demonstrated 60%, 51% and 58% decrease in IC(50). To investigate the mechanism of P-LAP-induced chemoresistance, we also assessed whether P-LAP transfection had an effect on carboplatin-induced apoptotic death of A-MEC cells. A-MEC and A-MEC-pc (transfected with vector alone) cells exhibited a strong apoptotic response to carboplatin, while A-MEC-LAP cells exhibited a weak apoptotic response. In an attempt to identify the mechanism of the inhibitory effect on apoptotic response to carboplatin, we next assessed the expression of cleaved caspases and PARP cleavage. While treatment of A-MEC-pc cells with carboplatin exhibited increased levels of cleaved caspase 3, caspase 7 and caspase 9 compared to that after no treatment, A-MEC-LAP cells did not show any expression of these caspases. These results suggest that P-LAP reduces sensitivity to anticancer drugs via inhibition of mitochondria-mediated apoptosis, and may be a molecular target for conquering anticancer drug resistance.     

Deoxycitidine kinase is associated with prolonged survival after adjuvant gemcitabine for resected pancreatic adenocarcinoma

BACKGROUND:

Gemcitabine (2′,2′‐difluorodeoxycytidine) administration after resection of pancreatic cancer improves both disease‐free survival (DFS) and overall survival (OS). Deoxycytidine kinase (dCK) mediates the rate‐limiting catabolic step in the activation of gemcitabine. The authors of this report studied patient outcomes according to the expression of dCK after a postoperative gemcitabine‐based chemoradiation regimen.

METHODS:

Forty‐five patients with resected pancreatic adenocarcinoma received adjuvant gemcitabine based‐therapy in the context of multicenter phase 2 studies. Their tumors were evaluated retrospectively for dCK protein expression by immunohistochemistry. A composite score based on the percentage of dCK‐positive cancer cells and the intensity of staining was generated, and the results were dichotomized at the median values.

RESULTS:

The median follow‐up was 19.95 months (95% confident interval [CI], 3.3‐107.4 months). The lymph node (LN) ratio and dCK protein expression were significant predictors of DFS and OS in univariate analysis. On multivariate analysis, dCK protein expression was the only independent prognostic variable (DFS: hazard ratio [HR], 3.48; 95% CI, 1.66‐7.31; P = .001; OS: HR, 3.2; 95% CI,1.44‐7.13; P = .004).

CONCLUSIONS:

dCK protein expression was identified as an independent and strong prognostic factor in patients with resected pancreatic adenocarcinoma who received adjuvant gemcitabine therapy. The authors concluded that it deserves prospective evaluation as a predictive biomarker for patient selection. Cancer 2010. © 2010 American Cancer Society.     

Circulating U2 small nuclear RNA fragments as a novel diagnostic biomarker for pancreatic and colorectal adenocarcinoma

Improved non‐invasive strategies for early cancer detection are urgently needed to reduce morbidity and mortality. Non‐coding RNAs, such as microRNAs and small nucleolar RNAs, have been proposed as biomarkers for non‐invasive cancer diagnosis. Analyzing serum derived from nude mice implanted with primary human pancreatic ductal adenocarcinoma (PDAC), we identified 15 diagnostic microRNA candidates. Of those miR‐1246 was selected based on its high abundance in serum of tumor carrying mice. Subsequently, we noted a cross reactivity of the established miR‐1246 assays with RNA fragments derived from U2 small nuclear RNA (RNU2‐1). Importantly, we found that the assay signal discriminating tumor from controls was derived from U2 small nuclear RNA (snRNA) fragments (RNU2‐1f) and not from miR‐1246. In addition, we observed a remarkable stability of RNU2‐1f in serum and provide experimental evidence that hsa‐miR‐1246 is likely a pseudo microRNA. In a next step, RNU2‐1f was measured by qRT‐PCR and normalized to cel‐54 in 191 serum/plasma samples from PDAC and colorectal carcinoma (CRC) patients. In comparison to 129 controls, we were able to classify samples as cancerous with a sensitivity and specificity of 97.7% [95% CI = (87.7, 99.9)] and 90.6% [95% CI = (80.7, 96.5)], respectively [area under the ROC curve 0.972]. Of note, patients with CRC were detected with our assay as early as UICC Stage II with a sensitivity of 81%. In conclusion, this is the first report showing that fragments of U2 snRNA are highly stable in serum and plasma and may serve as novel diagnostic biomarker for PDAC and CRC for future prospective screening studies.     

Targeted delivery of gemcitabine to pancreatic adenocarcinoma using cetuximab as a targeting agent

One of the key challenges in anticancer therapy is the toxicity and poor bioavailability of the anticancer drugs. Nanotechnology can play a pivotal role by delivering drugs in a targeted fashion to the malignant cells that will reduce the systemic toxicity of the anticancer drug. In this report, we show a stepwise development of a nanoparticle-based targeted delivery system for in vitro and in vivo therapeutic application in pancreatic cancer. In the first part of the study, we have shown the fabrication and characterization of the delivery system containing gold nanoparticle as a delivery vehicle, cetuximab as a targeting agent, and gemcitabine as an anticancer drug for in vitro application. Nanoconjugate was first characterized physico-chemically. In vitro targeting efficacy, tested against three pancreatic cancer cell lines (PANC-1, AsPC-1, and MIA Paca2) with variable epidermal growth factor receptor (EGFR) expression, showed that gold uptake correlated with EGFR expression. In the second part, we showed the in vivo therapeutic efficacy of the targeted delivery system. Administration of this targeted delivery system resulted in significant inhibition of pancreatic tumor cell proliferation in vitro and orthotopic pancreatic tumor growth in vivo. Tumor progression was monitored noninvasively by measuring bioluminescence of the implanted tumor cells. Pharmacokinetic experiments along with the quantitation of gold both in vitro and in vivo further confirmed that the inhibition of tumor growth was due to targeted delivery. This strategy could be used as a generalized approach for the treatment of a variety of cancers characterized by overexpression of EGFR.     

Cell-free 59 kDa immunoreactive integrin-linked kinase: a novel marker for ovarian carcinoma.

PURPOSE: We reported that the expression of integrin-linked kinase (ILK) is up-regulated in ovarian carcinomas and that ovarian cancer cells have high expression of ILK. In this study, we have examined the expression of cell-free 59 kDa immunoreactive (ir)ILK in the serum and peritoneal fluid (PTF) of patients with ovarian cancer and evaluated its potential as a serum biomarker for early-stage screening and for monitoring clinical status of patients after chemotherapy treatment. EXPERIMENTAL DESIGN: Thirty-six serum specimens, including normal (n = 6), benign (n = 6), borderline (n = 4), grade 1 (n = 5), grade 2 (n = 5), and grade 3 (n = 10), were evaluated for the expression of irILK by Western blotting. The expression of irILK was evaluated in PTF (n = 10) and peritoneal washings from women with benign ovarian cysts (n = 4). In addition, tissue-conditioned medium obtained from the cultures of primary ovarian tumors (n = 9) was examined for the presence of irILK. Finally, the potential of serum irILK as a biomarker for ovarian cancer screening was evaluated by comparison with cancer antigen 125 (CA 125) concentrations in cancer patients before and after chemotherapy. RESULTS: irILK expression was present in normal serum and in serum of patients with benign ovarian tumors. irILK expression was 6-9-fold higher in the serum of patients with grade 1, grade 2, and grade 3 ovarian cancer than in the serum of healthy volunteers and patients with benign ovarian tumors (P < 0.01). Enhanced expression of irILK in the serum of ovarian cancer patients correlated with the concentration of CA 125. High expression of irILK was present in all 10 PTF tested. Tissue-conditioned medium prepared from malignant ovarian tumors had 4-fold more irILK expression than conditioned medium obtained from borderline and benign tumors (P < 0.01). irILK expression in serum of cancer patients was reduced to basal normal levels after six cycles of Taxol/carboplatin and was consistent with the change of CA 125 levels before and after chemotherapy. CONCLUSIONS: These data suggest that irILK is an ovarian tumor-associated antigen and implicates its potential not only as a biomarker for early-stage screening but also as a marker for monitoring the clinical condition of patients after treatment.     

Enhancing chemosensitivity to gemcitabine via RNA interference targeting the catalytic subunits of protein kinase CK2 in human pancreatic cancer cells

Background  

Pancreatic cancer is a complex genetic disorder that is characterized by rapid progression, invasiveness, resistance to treatment and high molecular heterogeneity. Various agents have been used in clinical trials showing only modest improvements with respect to gemcitabine-based chemotherapy, which continues to be the standard first-line treatment for this disease. However, owing to the overwhelming molecular alterations that have been reported in pancreatic cancer, there is increasing focus on targeting molecular pathways and networks, rather than individual genes or gene-products with a combination of novel chemotherapeutic agents.     

CEACAM6 is a determinant of pancreatic adenocarcinoma cellular invasiveness

Pancreatic adenocarcinoma is among the most aggressively invasive malignancies. The immunoglobulin superfamily member carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is emerging as an important determinant of the malignant phenotype in a range of cancers. We sought to define the role of CEACAM6 in pancreatic adenocarcinoma cellular invasiveness. CEACAM6 was stably overexpressed in Capan2 cells, which inherently express low levels of CEACAM6. Retrovirally mediated RNA interference was used to silence CEACAM6 expression in BxPC3 cells, which inherently overexpress CEACAM6. Cellular invasiveness was quantified using a modified Boyden chamber assay. Overexpression of CEACAM6 increased Capan2 cellular invasiveness, whereas CEACAM6 knockdown attenuated BxPC3 invasiveness. A role for the c-Src tyrosine kinase in mediating CEACAM6-dependent invasiveness was defined using constitutively active and dominant-negative c-Src expression constructs. c-Src-dependent modulation of matrix metalloproteinase-9 activity contributes significantly to the increased cellular invasiveness induced by CEACAM6 overexpression. Levels of CEACAM6 expression can modulate pancreatic adenocarcinoma cellular invasiveness in a c-Src-dependent manner. This pathway warrants further investigation as a target for therapy.     

Dysbindin as a novel biomarker for pancreatic ductal adenocarcinoma identified by proteomic profiling

Pancreatic adenocarcinoma (PDAC) is known to have a poor prognosis partly because of lack of effective biomarkers. In the test set, we investigated dysbindin (DTNBP1) as a potential biomarker for PDAC by comparing preoperative and postoperative serum mass spectrometry (MS) proteomic profilings. Of the included 50 PDAC patients, 42 (positivity of 84.0%) detected a lower MS peak in postoperative serums than preoperative ones which was then identified as dysbindin. In the verification set, receiver operating characteristics (ROC) were used to assess diagnostic efficiency. 550 participants were included in the verification set [250 with PDAC, 80 with benign biliary obstruction (BBO), 70 with chronic pancreatitis (CP) and 150 healthy donors (HD)]. Dysbindin was increased in PDAC patient sera than in all controls. ROC curves revealed the optimum diagnostic cutoff for dysbindin was 699.16 pg/ml [area under curve (AUC) 0.849 (95% CI 0.812–0.885), sensitivity 81.9% and specificity 84.7%]. Raised concentration of dysbindin in sera could differentiate PDAC from BBO, CP and HD. Moreover, dysbindin maintained its diagnostic accuracy for PDAC patients who were CA19‐9 negative [AUC 0.875 (95% CI 0.804–0.945), sensitivity 83.0%, specificity 89.0%] and for patients with benign biliary obstruction [AUC 0.849 (95% CI 0.803–0.894), sensitivity 82.3%, specificity 84.0%].Our discovery of dysbindin may complement measurement of CA19‐9 in the diagnosis of PDAC and help to discriminate PDAC from other pancreatic diseases or begin biliary obstruction.     

低表达miR-33a诱导胰腺癌细胞对吉西他滨的耐药

背景与目的：胰腺癌是一种恶性程度很高的肿瘤。由于其对一线化疗药物吉西他滨的耐受,往往导致预后较差。MicroRNA(miRNA,miR)是一类非编码小RNA,参与肿瘤的多种生物学功能。miR-33a作为代谢相关的miRNA被广泛研究,而与耐药之间关系的报道较少。该研究通过探讨miR-33a参与胰腺癌吉西他滨耐药及其作用解析,为胰腺癌化疗提供新的理论依据。方法：采用原位杂交方法检测胰腺癌组织中miR-33a的表达情况;采用实时荧光定量PCR(Real-time PCR)检测各胰腺癌细胞系中miR-33a的表达情况。利用SW1990和Miapaca-2胰腺癌亲本细胞株,构建吉西他滨耐药细胞株(SW1990^res,Miapaca-2^res)及miR-33a稳定表达细胞株(SW1990-miR-33a,Miapaca-2-miR-33a、SW1990^res-miR-33a和Miapaca-2^res-miR-33a);采用细胞毒性实验检测miR-33a的表达对胰腺癌细胞对吉西他滨敏感性的影响。结果：miR-33a在胰腺癌组织样本中普遍低表达。与HEK293T正常人胚肾细胞相比,其在各胰腺癌细胞系中均呈低表达。miR-33a过表达可以增加胰腺癌细胞对吉西他滨的药物敏感性,能有效逆转胰腺癌细胞对吉西他滨的耐药。结论：miR-33a在胰腺癌组织中低表达,导致胰腺癌患者对吉西他滨获得性耐药。增加miR-33a表达,从而增强了胰腺癌细胞对吉西他滨的药物敏感性,为开发新型胰腺癌分子靶向治疗药物,联合化疗提供新的理论依据。     

Pharmacodynamic modeling of cell cycle and apoptotic effects of gemcitabine on pancreatic adenocarcinoma cells

Purpose

The standard of care for treating patients with pancreatic adenocarcinomas includes gemcitabine (2′,2′-difluorodeoxycytidine). Gemcitabine primarily elicits its response by stalling the DNA replication forks of cells in the S phase of the cell cycle. To provide a quantitative framework for characterizing the cell cycle and apoptotic effects of gemcitabine, we developed a pharmacodynamic model in which the activation of cell cycle checkpoints or cell death is dependent on gemcitabine exposure.

Methods

Three pancreatic adenocarcinoma cell lines (AsPC-1, BxPC-3, and MiaPaca-2) were exposed to varying concentrations (0–100,000 ng/mL) of gemcitabine over a period of 96 h in order to quantify proliferation kinetics and cell distributions among the cell cycle phases. The model assumes that the drug can inhibit cycle-phase transitioning in each of the 3 phases (G1, S, and G2/M) and can cause apoptosis of cells in G1 and G2/M phases. Fitting was performed using the ADAPT5 program.

Results

The time course of gemcitabine effects was well described by the model, and parameters were estimated with good precision. Model predictions and experimental data show that gemcitabine induces cell cycle arrest in the S phase at low concentrations, whereas higher concentrations induce arrest in all cell cycle phases. Furthermore, apoptotic effects of gemcitabine appear to be minimal and take place at later time points.

Conclusion

The pharmacodynamic model developed provides a quantitative, mechanistic interpretation of gemcitabine efficacy in 3 pancreatic cancer cell lines, and provides useful insights for rational selection of chemotherapeutic agents for combination therapy.     

RNA干扰--肿瘤研究的新工具

RNA干扰（RNA interference,RNAi）是由双链RNA分子介导的序列特异性转录后基因静默过程,为双链RNA分子在mRNA水平上关闭相关基因表达的过程,是一项新兴的基因阻断技术。RNAi有望成为分析人类基因组功能的有力工具,在肿瘤病因、免疫机制及治疗等方面的研究上有广阔的发展前景。     

Ascites-derived pancreatic ductal adenocarcinoma primary cell cultures as a platform for personalised medicine

Background:

Challenges in developing drugs for pancreatic ductal adenocarcinoma (PDAC) include obtaining metastatic cancer tissue for research and validating biomarkers predicative for personalised therapeutic decisions. We have recently developed a novel therapeutic model for PDAC to address these challenges based on the isolation of viable PDAC cells derived from ascites fluid.

Methods:

Ascites fluid was obtained from PDAC patients undergoing palliative paracentesis. Ascites-derived PDAC primary cells were isolated, cultured and characterised in ovo and in vitro.

Results:

We successfully established ascites-derived primary cell cultures within 2–7 days from 92% (93 out of 101) of the ascites fluid samples obtained (from 36 different patients). Homogeneous epithelial PDAC-enriched cell cultures were identified and characterised. We observed a wide range in doubling times and migration properties among the different patient-derived cell cultures. The diverse nature of each individual patient''s cell cultures was further demonstrated by differences in therapeutic susceptibility and resistance. The tumorigenicity and invasiveness of the cells were demonstrated in vivo using chicken chorioallantoic membrane grafts.

Conclusions:

We have developed a unique ascites-derived PDAC primary cell culture model. This model has the potential to study signalling pathways in PDAC progression and to evaluate targeted therapies for the individual patient expeditiously, thereby supporting personalised treatment decisions.     

Intrinsic chemoresistance to gemcitabine is associated with decreased expression of BNIP3 in pancreatic cancer.

PURPOSE: Although chemotherapy with gemcitabine is a common mode of treatment of pancreatic cancer, 75% of patients do not benefit from this therapy. It is likely that the sensitivity of cancer cells to gemcitabine is determined by a number of different factors. EXPERIMENTAL DESIGN: To identify genes that might contribute to resistance to gemcitabine, 15 pancreatic cancer cell lines were subjected to gemcitabine treatment. Simultaneously, gene expression profiling using a cDNA microarray to identify genes responsible for gemcitabine sensitivity was performed. RESULTS: The pancreatic cancer cell lines could be classified into three groups: a gemcitabine "sensitive," an "intermediate sensitive," and a "resistant" group. Microarray analysis identified 71 genes that show differential expression between gemcitabine-sensitive and -resistant cell lines including 27 genes relatively overexpressed in sensitive cell lines whereas 44 genes are relatively overexpressed in resistant cell lines. Among these genes, 7 genes are potentially involved in the phosphatidylinositol 3-kinase/Akt pathway. In addition to this major signaling pathway, Bcl2/adenovirus E1B 19 kDa protein interacting protein (BNIP3), a Bcl-2 family proapoptotic protein, was identified as being expressed at lower levels in drug-resistant pancreatic cancer cell lines. In an analysis of 21 pancreatic cancer tissue specimens, more than 90% showed down-regulated expression of BNIP3. When expression of BNIP3 was suppressed using small interfering RNA, gemcitabine-induced cytotoxicity in vitro was much reduced. CONCLUSIONS: These results suggest that BNIP3 and the phosphatidylinositol 3-kinase/Akt pathway may play an important role in the poor response to gemcitabine treatment in pancreatic cancer patients.