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.     

RNA interference demonstrates a novel role for integrin-linked kinase as a determinant of pancreatic adenocarcinoma cell gemcitabine chemoresistance.

Integrin-linked kinase (ILK) facilitates signal transduction between extracellular events and important intracellular survival pathways involving protein kinase B/Akt. We examined the role of ILK in determining pancreatic adenocarcinoma cellular chemoresistance to the nucleoside analogue gemcitabine. Cellular ILK expression was quantified by Western blot analysis. We examined the effects of overexpression of active ILK and of ILK knockdown induced by RNA interference on gemcitabine chemoresistance. We also examined the effects of modulating ILK expression on gemcitabine-induced caspase 3-mediated apoptosis, phosphorylation status of Akt (Ser473) and glycogen synthase kinase. Overexpression of ILK increased cellular gemcitabine chemoresistance, whereas ILK knockdown induced chemosensitization via increased caspase 3-mediated apoptosis. ILK knockdown attenuated Akt Ser473 and glycogen synthase kinase phosphorylation, whereas overexpression of constitutively active myristoylated Akt was sufficient to induce significant recovery in gemcitabine chemoresistance in the presence of ILK knockdown. Levels of ILK expression affect gemcitabine chemoresistance in pancreatic adenocarcinoma cells. This novel finding suggests that therapies directed against ILK and its downstream signaling targets may have the potential to enhance the efficacy of gemcitabine-based chemotherapy.     

Gemcitabine chemoresistance and molecular markers associated with gemcitabine transport and metabolism in human pancreatic cancer cells

To identify predictive molecular markers for gemcitabine resistance, we investigated changes in the expression of four genes associated with gemcitabine transport and metabolism during the development of acquired gemcitabine resistance of pancreatic cancer cell lines. The expression levels of human equilibrative nucleoside transporter-1 (hENT1), deoxycytidine kinase (dCK), RRM1, and RRM2 mRNA were analysed by real-time light cycler-PCR in various subclones during the development of acquired resistance to gemcitabine. Real-time light cycler-PCR demonstrated that the expression levels of either RRM1 or RRM2 progressively increased during the development of gemcitabine resistance. Expression of dCK was slightly increased in cells resistant to lower concentrations of gemcitabine, but was decreased below the undetectable level in higher concentration-resistant subclones. Expression of hENT1 was increased in the development of gemcitabine resistance. As acquired resistance to gemcitabine seems to correlate with the balance of these four factors, we calculated the ratio of hENT1 x dCK/RRM1 x RRM2 gene expression in gemcitabine-resistant subclones. The ratio of gene expression decreased progressively with development of acquired resistance in gemcitabine-resistant subclones. Furthermore, the expression ratio significantly correlated with gemcitabine sensitivity in eight pancreatic cancer cell lines, whereas no single gene expression level correlated with the sensitivity. These results suggest that the sensitivity of pancreatic cancer cells to gemcitabine is determined by the ratio of four factors involved in gemcitabine transport and metabolism. The ratio of the four gene expression levels correlates with acquired gemcitabine-resistance in pancreatic cancer cells, and may be useful as a predictive marker for the efficacy of gemcitabine therapy in pancreatic cancer patients.     

EphA2: a determinant of malignant cellular behavior and a potential therapeutic target in pancreatic adenocarcinoma

The EphA2 receptor tyrosine kinase is overexpressed in a variety of human cancers. We sought to characterize the role of EphA2 in pancreatic adenocarcinoma and, using RNA interference (RNAi) mediated by small interfering RNA (siRNA), we determined the effects of suppressing EphA2 expression in vitro and in vivo. EphA2 expression in PANC1, MIAPaCa2, BxPC3 and Capan2 cells was assessed by Northern and Western blot. We artificially overexpressed EphA2 by transient transfection and suppressed EphA2 expression using RNAi. Cellular invasiveness was quantified by modified Boyden chamber assay. Anoikis was induced by anchorage-independent polyHEMA culture and caspase 3 activity was quantified fluorometrically. Focal adhesion kinase (FAK) phosphorylation was assessed by immunoprecipitation. EphA2 siRNA treatment was assessed in a nude mouse xenograft model. Pancreatic adenocarcinoma cells differentially express EphA2. Inherent and induced EphA2 overexpression is associated with increased cellular invasiveness and anoikis resistance. EphA2 siRNA suppresses EphA2 expression, cellular invasiveness, anoikis resistance and FAK phosphorylation in vitro and retards tumor growth and inhibits metastasis in vivo. EphA2 is both a determinant of malignant cellular behavior and a potential therapeutic target in pancreatic adenocarcinoma.     

Inhibition of Src tyrosine kinase reverts chemoresistance toward 5-fluorouracil in human pancreatic carcinoma cells: an involvement of epidermal growth factor receptor signaling

Resistance to chemotherapy is believed to be a major cause of treatment failure in pancreatic cancer. Thus, it is necessary to explore alternative therapeutic modalities to overcome drug resistance in pancreatic cancer treatment. We tested the hypothesis that Src tyrosine kinase inhibition could augment the chemosensitivity of 5-fluorouracil (5-FU)-resistant human pancreatic cancer cells to 5-FU. As detected by MTT proliferation assay, propidium iodide and annexin V staining, a combination of 5-FU+Src kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) reflected the chemotherapeutic sensitivity and restored the 5-FU-induced apoptosis in 5-FU-resistant cells. Furthermore, when small-interfering RNA approach to silence Src gene expression was applied, the degree of 5-FU-induced apoptosis was increased in all cell lines independently of the chemoresistance status. Western blotting and RT-PCR analysis revealed that the expression of thymidylate synthase (TS) was higher in 5-FU-resistant cells, however, decreased significantly after pretreatment with PP2. Furthermore, the combination of 5-FU+PP2 decreased the 5-FU-induced activation of epidermal growth factor receptor (EGFR)-AKT pathway. Finally, PP2 in combination with 5-FU substantially decreased the in vivo tumor growth and inhibited distant metastases. Taken together, 5-FU chemoresistance can be reversed through indirect TS regulation by inhibiting Src tyrosine kinase. A potential mechanism of action of Src kinase inhibitors on 5-FU chemosensitivity might be linked to the inhibition of 5-FU-induced EGFR-AKT activation.     

Involvement of ribonucleotide reductase M1 subunit overexpression in gemcitabine resistance of human pancreatic cancer

Pancreatic cancer is the most lethal of all solid tumors partially because of its chemoresistance. Although gemcitabine is widely used as a first selected agent for the treatment of this disease despite low response rate, molecular mechanisms of gemcitabine resistance in pancreatic cancer still remain obscure. The aim of this study is to elucidate the mechanisms of gemcitabine resistance. The 81-fold gemcitabine resistant variant MiaPaCa2-RG was selected from pancreatic cancer cell line MiaPaCa2. By microarray analysis between MiaPaCa2 and MiaPaCa2-RG, 43 genes (0.04%) were altered expression of more than 2-fold. The most upregulated gene in MiaPaCa2-RG was ribonucleotide reductase M1 subunit (RRM1) with 4.5-fold up-regulation. Transfection with RRM1-specific RNAi suppressed more than 90% of RRM1 mRNA and protein expression. After RRM1-specific RNAi transfection, gemcitabine chemoresistance of MiaPaCa2-RG was reduced to the same level of MiaPaCa2. The 18 recurrent pancreatic cancer patients treated by gemcitabine were divided into 2 groups by RRM1 levels. There was a significant association between gemcitabine response and RRM1 expression (p = 0.018). Patients with high RRM1 levels had poor survival after gemcitabine treatment than those with low RRM1 levels (p = 0.016). RRM1 should be a key molecule in gemcitabine resistance in human pancreatic cancer through both in vitro and clinical models. RRM1 may have the potential as predictor and modulator of gemcitabine treatment.     

Expression and activity of SRC regulate interleukin-8 expression in pancreatic adenocarcinoma cells: implications for angiogenesis

Interleukin-8 (IL-8) is an angiogenic factor that promotes growth of pancreatic tumors. The purpose of this study was to determine if c-Src, a protein tyrosine kinase frequently overexpressed in pancreatic cancer, regulated IL-8 expression and to elucidate the Src-mediated signaling pathways that contribute to angiogenesis in pancreatic adenocarcinoma cells. In a panel of pancreatic cancer cell lines, expression of total and activated Src correlated with IL-8 production. Furthermore, ectopic expression of activated Src in PANC-1 cells with low endogenous Src activity significantly increased IL-8 production (P < 0.005). In contrast, pharmacologic inhibition of endogenous c-Src kinase activity or small interfering RNA-mediated "knockdown" of c-Src expression in L3.6pl cells with high Src expression and activity caused significant decreases in IL-8 production (P < 0.005). Inhibition of c-Src activity resulted in decreased phosphorylation of Akt, p38, and extracellular signal-regulated kinase (Erk)-1/2. Significant (P < 0.005) dose-dependent decreases were observed in IL-8 expression by inhibiting Src-dependent signaling molecules Erk-1/2 and p38 but not phosphatidylinositol 3-kinase. To assess the relevance of Src inhibition to angiogenesis, in vivo gelfoam assays were done. Robust infiltration of vessels was observed in gelfoam saturated with conditioned medium from pancreatic carcinoma cells. This angiogenesis was nearly abrogated in gelfoams saturated with conditioned medium from cells treated with the Src family kinase inhibitor, PP2 (P < 0.001). Thus, c-Src regulates critical "downstream" signaling pathways that contribute to expression of IL-8 in human pancreatic tumor cells, suggesting c-Src may be a target for therapeutic intervention in pancreatic adenocarcinoma.     

siRNA directed against TrkA sensitizes human pancreatic cancer cells to apoptosis induced by gemcitabine through an inactivation of PI3K/Akt-dependent pathway

Previously, we have documented that the aggressive and highly metastatic behavior of pancreatic cancer may be due to the aberrant expression of nerve growth factor (NGF) and its high-affinity receptor, proto-oncogene TrkA. In this study, we sought to determine the effect of suppressing TrkA expression on pancreatic cancer chemosensitivity to gemcitabine. Human pancreatic cancer cell lines PANC-1, MIA-PaCa-2 and ASPC-1 were studied. The expression and kinase activity of TrkA were determined by Western blot analysis and in vitro kinase assay respectively. RNA interference was used to suppress TrkA expression. Gemcitabine-induced cytotoxicity was determined by tetrazolium reduction assay and caspase profiling was performed. The effect of TrkA-specific siRNA on PI3K/Akt activity was also quantified. TrkA expression and kinase activity in cell lines were directly correlated with gemcitabine chemoresistance. TrkA-specific siRNA suppressed TrkA expression and kinase activity, and furthermore increased gemcitabine-induced, caspase-mediated apoptosis. PI3K/Akt activity was decreased by suppression of TrkA expression. Taken together, these data demonstrated that TrkA is a determinant of pancreatic adenocarcinoma chemoresistance and PI3K/Akt is a key signaling component by which NGF activation of the TrkA signal transduction pathway protects pancreatic cancer cells from chemotherapy-induced cell death.     

Disrupted plasma membrane localization of equilibrative nucleoside transporter 2 in the chemoresistance of human pancreatic cells to gemcitabine (dFdCyd)

Although the nucleoside pyrimidine analogue gemcitabine is the most effective single agent in the palliation of advanced pancreatic cancer, cellular resistance to gemcitabine treatment is a major problem in the clinical scene. To clarify the molecular mechanisms responsible for chemoresistance to gemcitabine, mRNA expression of the key enzymes including cytidine deaminase (CDA), deoxycytidine kinase (dCK), 5'-nucleotidase (NT5), equilibrative nucleoside transporter 1 and 2 (ENT1 and ENT2), dCMP deaminase (dCMPK), ribonucleotide reductase M1 and M2 (RRM1 and RRM2), thymidylate synthase (TS) and CTP synthase (CTPS) was examined. The interacellular uptake of gemcitabine was greatly impaired in the chemoresistant cell lines due to dysfunction of ENT1 and ENT2. Protein expression of ENT1 and ENT2 and their protein coding sequences were not altered. Immunohistochemical and western blot analyses revealed that localization of ENT2 on the plasma membrane was disrupted. These data suggest that the disrupted localization of ENT2 is one of causes of the impaired uptake of gemcitabine, resulting in a gain of chemoresistance to gemcitabine.     

KRas induces a Src/PEAK1/ErbB2 kinase amplification loop that drives metastatic growth and therapy resistance in pancreatic cancer

Early biomarkers and effective therapeutic strategies are desperately needed to treat pancreatic ductal adenocarcinoma (PDAC), which has a dismal 5-year patient survival rate. Here, we report that the novel tyrosine kinase PEAK1 is upregulated in human malignancies, including human PDACs and pancreatic intraepithelial neoplasia (PanIN). Oncogenic KRas induced a PEAK1-dependent kinase amplification loop between Src, PEAK1, and ErbB2 to drive PDAC tumor growth and metastasis in vivo. Surprisingly, blockade of ErbB2 expression increased Src-dependent PEAK1 expression, PEAK1-dependent Src activation, and tumor growth in vivo, suggesting a mechanism for the observed resistance of patients with PDACs to therapeutic intervention. Importantly, PEAK1 inactivation sensitized PDAC cells to trastuzumab and gemcitabine therapy. Our findings, therefore, suggest that PEAK1 is a novel biomarker, critical signaling hub, and new therapeutic target in PDACs.     

吉西他滨耐药的A549和NCI-H460细胞中耐药相关基因的表达和RRMl(-)37A/C基因多态性

目的 研究吉西他滨(Gem)耐药的A549和NCI-H460细胞中胞啶脱酰氨酶(CDA)、核糖核苷酸还原酶M1亚单位(RRMl)、10q丢失的与张力蛋白同源的磷酸酶基因(PTEN)、切除修复交叉互补基因1(ERCC1)和脱氧胞苷酸酶(dCK)的表达以及RRMl(-)37A/C基因多态性.方法 采用药物临床血浆峰浓度冲击与逐步增加剂量相结合的诱导方法,成功诱导抗Gem的细胞系A549/Gem和NCI-H460/Gem.在药物诱导前、诱导过程中和诱导成功后,采用实时荧光定量PCR法检测其CDA、RRM1、PTEN、ERCC1和dCKmRNA的表达,并对其RRM1(-)37位点进行基因分型.结果 A549/Gem和NCI-H460/Gem细胞在药物诱导过程中,耐药指数(RI)随诱导时间的延长分别升至163.228和181.684.但达到一定峰值后逐渐降低,直至产生稳定的耐药性,RI分别为115.297和129.783.RRM1、PTEN、ERCC1和CDA mRNA表达量随着对Gem耐受程度的变化增高和降低,而dCK mRNA表达变化不明显.RRM1(-)37等位基因野生型引物能扩增出诱导各阶段A549/Gem和NCI-H460/Gem细胞的基因组DNA,而突变型引物则不能,说明A549/Gem和NCI-H460/Gem细胞目前的基因型与其亲代细胞一样,仍均为野生型.结论 与亲代细胞比较,A549/Gem和NCI-H460/Gem细胞的CDA、RRM1、PTEN和ERCC1表达升高,dCK表达变化不明显;RRM1(-)37位点单核苷酸无突变,其基因型与亲代细胞一样为野生型.     

Inhibition of Src reduces gemcitabine-induced cytotoxicity in human pancreatic cancer cell lines

In the present study, we examined the role of Src in gemcitabine-induced cell growth suppression in human pancreatic cancer cell lines. In two human pancreatic cancer cell lines, PK-9 and MIA PaCa-2, we found that a selective Src protein tyrosine kinase inhibitor, PP2, inhibited gemcitabine-induced cell growth suppression. When dominant negative src cDNA was constitutively expressed in PK-9 cells (PK-9-Src-DN), the degree of gemcitabine-induced cell growth suppression was decreased compared with that of mock-transfected PK-9 cells. The mechanism of the inhibitory effect of gemcitabine-induced cytotoxicity was found to be the suppression of apoptosis, which was downregulated in PK-9-Src-DN cells. These results indicate that Src mediates signals that culminate in suppressing cell growth and survival in the presence of gemcitabine, at least in particular cell lines.     

Sirtuin 1 facilitates chemoresistance of pancreatic cancer cells by regulating adaptive response to chemotherapy‐induced stress

Chemotherapy drugs themselves may act as stressors to induce adaptive responses to promote the chemoresistance of cancer cells. Our previous research showed that sirtuin 1 (SIRT1) was overexpressed in pancreatic cancer patients and deregulation of SIRT1 with RNAi could enhance chemosensitivity. Thus, we hypothesized that SIRT1 might facilitate chemoresistance in pancreatic cancer cells through regulating the adaptive response to chemotherapy‐induced stress. In the present study, SIRT1 in PANC‐1, BXPC‐3, and ASPC‐1 cells was upregulated after treatment with gemcitabine. Moreover, the decrease in SIRT1 activity with special inhibitor EX527 had a synergic effect on chemotherapy with gemcitabine in PANC‐1 and ASPC‐1 cell lines, which significantly promoted apoptosis, senescence, and G₀/G₁ cycle arrest. Western blot results also showed that SIRT1, acetylated‐p53, FOXO3a, and p21 were upregulated after combined treatment, whereas no obvious change was evident in total p53 protein. To further confirm the role of SIRT1 in clinical chemotherapy, SIRT1 was detected in eight pancreatic cancer tissues acquired by endoscopy ultrasonography guided fine needle aspiration biopsy before and after chemotherapy. Compared to before chemotherapy, SIRT1 was significantly increased after treatment with gemcitabine in six cases. Thus, our results indicated a special role for SIRT1 in the regulation of adaptive response to chemotherapy‐induced stress, which is involved in chemoresistance. Moreover, it indicates that blocking SIRT1 activity with targeting drugs might be a novel strategy to reverse the chemoresistance of pancreatic cancer.     

NDRG1下调对吉西他滨干预人胰腺癌细胞 PANC －1增殖及凋亡的影响

目的：观察 siNDRG1干扰对吉西他滨干预的胰腺癌细胞增殖、细胞周期及凋亡的影响并探讨其机制。方法：以 Western blot 法对 PANC －1、BXPC －3、CAPAN －2、SW1990细胞中 NDRG1的表达进行检测;构建靶向 NDRG1的干扰质粒 siNDRG1,转染 PANC －1细胞,以 Western blot 法及（qRT）－PCR 法检测 NDRG1干扰效率;对 siRNA 干扰细胞给予吉西他滨干预,采用 MTT 法检测细胞增殖,PI 法检测细胞周期,流式细胞术检测细胞调亡。结果：Western blot 显示,NDRG1在四组细胞株中均有表达,低分化细胞（PANC －1）中表达量较中分化（BXPC －3）及高分化细胞株（CAPAN －2、SW1990）中高（P ＜0．05）;MTT 显示,吉西他滨干预后 PANC －1细胞在 siNDRG1转染组细胞生长抑制率高于 siNC 组,差异有显著性（P ＜0．01）。PI 法显示,转
　　染 siNDRG1的 PANC －1细胞停留在 G1期比例减少,G2及 S 期比例升高,与 siNC 组对比,差异具有显著性（t＝4．21,P ＜0．05;t ＝3．54,P ＜0．05;t ＝3．29,P ＜0．05）。经吉西他滨处理后,siNDRG1较 siNC 组 G1期及 G2期细胞比例明显减少,S 期细胞比例升高,差异具有显著性（t ＝14．65,P ＜0．01;t ＝13．28,P ＜0．01;t ＝15．17, P ＜0．01）。流式细胞仪检测经吉西他滨处理后 siNDRG1组凋亡率高于 siNC 组,差异有显著性（t ＝22．42,P＜0．001）。结论：对胰腺癌细胞 NDRG1表达进行下调可以增强吉西他滨化疗敏感性,抑制细胞增殖,促进凋亡,可作为胰腺癌治疗新的靶向候选基因。     

Inhibition of SRC tyrosine kinase as treatment for human pancreatic cancer growing orthotopically in nude mice.

PURPOSE: The Src family comprises a family of nonreceptor intracellular tyrosine kinases that mediate a variety of cellular pathways. Src kinases are overexpressed in a variety of human tumors, including cancer of the colon, breast, and pancreas, and they are an integral part of tumor cell signaling pathways associated with migration, proliferation, adhesion, and angiogenesis. EXPERIMENTAL DESIGN: We investigated whether the blockade of Src kinase by daily oral administration of the novel Src tyrosine kinase inhibitor AZM475271 [kindly provided by AstraZeneca (Macclesfield, United Kingdom)], alone or in combination with intraperitoneal gemcitabine, can inhibit growth and metastasis of orthotopically implanted human pancreatic carcinoma cells in nude mice. RESULTS: Treatment with AZM475271 alone reduced the primary pancreatic tumor volume by approximately 40%, whereas AZM475271 plus gemcitabine reduced tumor volume by 90%. Furthermore, treatment with AZM475271 and gemcitabine significantly reduced metastasis: none of eight animals who received the combination treatment had lymph node or liver metastases, compared with five of five and three of five animals, respectively, in the control group (P = 0.001). Src inhibition by AZM475271 (alone or with gemcitabine) was associated with significantly reduced tumor cell proliferation, decreased tumor microvessel density, and increased apoptosis in vivo. Moreover, these effects were all significantly increased when gemcitabine was combined with AZM475271 compared with gemcitabine alone. CONCLUSIONS: Src inhibition by AZM475271, either alone or in combination with gemcitabine, demonstrated significant antitumor and antimetastatic activity in an orthotopic nude mouse model for human pancreatic cancer. The combination of AZM475271 with gemcitabine sensitized tumor cells to the cytotoxic effect of gemcitabine.     

Inhibition of PDGFR phosphorylation and Src and Akt activity by GN963 leads to therapy of human pancreatic cancer growing orthotopically in nude mice

GN963 is a tyrosine kinase inhibitor with activity against platelet-derived growth factor receptor (PDGFR) and Src kinases. We determined whether oral administration of GN963, alone or in combination with gemcitabine produces therapy against L3.6pl human pancreatic cancer cells growing orthotopically in nude mice. The optimal biological dosage of oral GN963 was determined to be 100 mg/kg every 48 h. Seven days after injection of L3.6pl cells into the pancreas of nude mice, mice (n=10) were treated with vehicle (control), thrice-weekly oral GN963 (100 mg/kg), twice-weekly intraperitoneal gemcitabine (100 mg/kg), or GN963 plus gemcitabine. Treatment with gemcitabine did not significantly differ from control. In contrast, treatment with GN963 (100 mg/kg) or GN963 plus gemcitabine produced a 52% and 81% decrease in tumor volume, respectively. GN963 plus gemcitabine completely inhibited the incidence of liver metastasis. Administration of GN963 inhibited PDGFR phosphorylation in both tumor and tumor-associated endothelial cells, decreased Src and Akt kinase activity in tumor cells, decreased microvessel density, and decreased tumor cell proliferation, while increasing apoptosis of tumor and tumor-associated endothelial cells. Collectively, these data indicate that targeting PDGFR, Src, and Akt on tumor and tumor-associated endothelial cells may be an effective therapy for human pancreatic carcinoma.     

Ribonucleotide reductase subunits M1 and M2 mRNA expression levels and clinical outcome of lung adenocarcinoma patients treated with docetaxel/gemcitabine

Ribonucleotide reductase subunits M1 (RRM1) and M2 (RRM2) are involved in the metabolism of gemcitabine (2',2'-difluorodeoxycytidine), which is used for the treatment of nonsmall cell lung cancer. The mRNA expression of RRM1 and RRM2 in tumours from lung adenocarcinoma patients treated with docetaxel/gemcitabine was assessed and the results correlated with clinical outcome. RMM1 and RMM2 mRNA levels were determined by quantitative real-time PCR in primary tumours of previously untreated patients with advanced lung adenocarcinoma who were subsequently treated with docetaxel/gemcitabine. Amplification was successful in 42 (79%) of 53 enrolled patients. Low levels of RRM2 mRNA were associated with response to treatment (P< 0.001). Patients with the lowest expression levels of RRM1 had a significantly longer time to progression (P=0.044) and overall survival (P=0.02) than patients with the highest levels. Patients with low levels of both RRM1 and RRM2 had a significantly higher response rate (60 vs 14.2%; P=0.049), time to progression (9.9 vs 2.3 months; P=0.003) and overall survival (15.4 vs 3.6; P=0.031) than patients with high levels of both RRM1 and RRM2. Ribonucleotide reductase subunit M1 and RRM2 mRNA expression in lung adenocarcinoma tumours is associated with clinical outcome to docetaxel/gemcitabine. Prospective studies are warranted to evaluate the role of these markers in tailoring chemotherapy.     

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.