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.     

In vivo induction of resistance to gemcitabine results in increased expression of ribonucleotide reductase subunit M1 as the major determinant

Gemcitabine is a deoxycytidine (dCyd) analogue with activity against several solid cancers. Gemcitabine is activated by dCyd kinase (dCK) and interferes, as its triphosphate dFdCTP, with tumor growth through incorporation into DNA. Alternatively, the metabolite gemcitabine diphosphate (dFdCDP) can interfere with DNA synthesis and thus tumor growth through inhibition of ribonucleotide reductase. Gemcitabine can be inactivated by the enzyme dCyd deaminase (dCDA). In most in vitro models, resistance to gemcitabine was associated with a decreased dCK activity. In all these models, resistance was established using continuous exposure to gemcitabine with increasing concentrations; however, these in vitro models have limited clinical relevance. To develop in vivo resistance to gemcitabine, we treated mice bearing a moderately sensitive tumor Colon 26-A (T/C = 0.25) with a clinically relevant schedule (120 mg/kg every 3 days). By repeated transplant of the most resistant tumor and continuation of gemcitabine treatment for >1 year, the completely resistant tumor Colon 26-G (T/C = 0.96) was created. Initial studies focused on resistance mechanisms known from in vitro studies. In Colon 26-G, dCK activity was 1.7-fold decreased; dCDA and DNA polymerase were not changed; and Colon 26-G accumulated 1.5-fold less dFdCTP, 6 hours after a gemcitabine injection, than the parental tumor. Based on in vitro studies, these relative minor changes were considered insufficient to explain the completely resistant phenotype. Therefore, an expression microarray was done with Colon 26-A versus Colon 26-G. Using independently grown nonresistant and resistant tumors, a striking increase in expression of the RRM1 subunit gene was found in Colon 26-G. The expression of RRM1 mRNA was 25-fold increased in the resistant tumor, as measured by real-time PCR, which was confirmed by Western blotting. In contrast, RRM2 mRNA was 2-fold decreased. However, ribonucleotide reductase enzyme activity was only moderately increased in Colon 26-G. In conclusion, this is the first model with in vivo induced resistance to gemcitabine. In contrast to most in vitro studies, dCK activity was not the most important determinant of gemcitabine resistance. Expression microarray identified RRM1 as the gene with the highest increase in expression in the Colon 26-G, which might clarify its complete gemcitabine-resistant phenotype. This study is the first in vivo evidence for a key role for RRM1 in acquired gemcitabine resistance.     

Gemcitabine sensitivity-related mRNA expression in endoscopic ultrasound-guided fine-needle aspiration biopsy of unresectable pancreatic cancer

Background

The aim of this study was to determine a predictive indicator of gemcitabine (GEM) efficacy in unresectable pancreatic cancer using tissue obtained by endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNA).

Methods

mRNAs extracted from 35 pancreatic tubular adenocarcinoma tissues obtained by EUS-FNA before GEM-treatment were studied. mRNAs were amplified and applied to a Focused DNA Array, which was restricted to well-known genes, including GEM sensitivity-related genes, deoxycytidine kinase (dCK), human equilibrative nucleoside transporter 1 (hENT1), hENT2, dCMP deaminase, cytidine deaminase, 5''-nucleotidase, ribonucleotide reductase 1 (RRM1) and RRM2. mRNA levels were classified into high and low expression based on a cut-off value defined as the average expression of 35 samples. These 35 patients were divided into the following two groups. Patients with partial response and those with stable disease whose tumor markers decreased by 50% or more were classified as the effective group. The rest of patients were classified as the non-effective group. The relationship between GEM efficacy and mRNA expression was then examined by chi-squared test.

Results

Among these GEM sensitivity-related genes, dCK alone showed a significant correlation with GEM efficacy. Eight of 12 patients in the effective group had high dCK expression, whereas 16 of 23 patients in non-effective group had low dCK expressions (P = 0.0398).

Conclusion

dCK mRNA expression is a candidate indicator for GEM efficacy in unresectable pancreatic cancer. Quantitative mRNA measurements of dCK using EUS-FNA samples are necessary for definitive conclusions.     

Synergistic cytotoxicity and pharmacogenetics of gemcitabine and pemetrexed combination in pancreatic cancer cell lines.

PURPOSE: Gemcitabine is an inhibitor of ribonucleotide reductase (RR) and DNA synthesis and is an effective agent in the treatment of pancreas cancer. The present study investigates whether the multitargeted antifolate pemetrexed would be synergistic with gemcitabine against MIA PaCa-2, PANC-1, and Capan-1 pancreatic cancer cell lines. EXPERIMENTAL DESIGN: Cells were treated with gemcitabine and pemetrexed, and the type of drug interaction was assessed using the combination index. Cytotoxicity of gemcitabine was examined with inhibitors of (a) deoxycytidine kinase (dCK), which activates gemcitabine by phosphorylation, and (b) 5'-nucleotidase (drug dephosphorylation) and cytidine deaminase (drug deamination), the main inactivating enzymes. The effects of gemcitabine and pemetrexed on cell cycle were analyzed by flow cytometry, and apoptosis was examined by fluorescence microscopy. Finally, quantitative, real-time PCR was used to study the pharmacogenetics of the drug combination. RESULTS: Synergistic cytotoxicity and enhancement of apoptosis was demonstrated, mostly with the sequence pemetrexed-->gemcitabine. Pemetrexed increased cells in S phase, the most sensitive to gemcitabine, and a positive correlation was found between the expression ratio of dCK:RR and gemcitabine sensitivity. Indeed, pemetrexed significantly enhanced dCK gene expression (+227.9, +86.0, and +135.5% in MIA PaCa-2, PANC-1, and Capan-1 cells, respectively), and the crucial role of this enzyme was confirmed by impairment of gemcitabine cytotoxicity after dCK saturation with 2'-deoxycytidine. CONCLUSIONS: These data demonstrate that the gemcitabine and pemetrexed combination displays schedule-dependent synergistic cytotoxic activity, favorably modulates cell cycle, induces apoptosis, and enhances dCK expression in pancreatic cancer cells.     

Expression of nucleoside transporters, deoxycitidine kinase, ribonucleotide reductase regulatory subunits, and gemcitabine catabolic enzymes in primary ovarian cancer

Purpose

Gemcitabine (2′,2′-difluorodeoxycytidine) (GEM) is one of the most actively investigated drugs in ovarian cancer. Many molecular mechanisms have been proposed to be involved in GEM sensitivity/resistance including the equilibrative nucleoside transporter-1 (hENT1), the concentrative nucleoside transporter-1 (hCNT1), and deoxycytidine kinase (dCK). The expression of the ribonucleotide reductase regulatory subunits M1 (RRM1) and M2 (RRM2), and the catabolic enzymes 5′nucleotidase (5′NT), and cytidine deaminase (CDA) play also an important role. The aim of the study is to investigate the potential clinical role of hENT1, hCNT1, dCK, RRM1, RRM2, CDA, and 5′NT in a single institutional series of 25 primary ovarian carcinomas.

Methods

The expression levels of the target genes were assayed by means of real-time quantitative PCR. The clinical role of the expression levels of each parameter was analyzed by Kaplan and Meier method and the Cox’s proportional hazards model.

Results

hENT1, hCNT1, dCK, CDA, 5′NT, RRM1, and RRM2 gene expression was documented in all samples; undifferentiated and clear cell carcinoma exhibited higher levels of hENT1, dCK, 5′NT, and RRM1 compared to serous ovarian tumors. As of May 2009, the median follow-up was 32 months (range 10–80), and death of disease were observed in 17 cases (68.0%). A statistically significant direct association of RRM2 expression levels and the relative risk of death was observed (Χ ² = 8.18, P = 0.0043). Moreover, cases with high RRM2 expression had a shorter OS (median OS = 19 months) than cases with low RRM2 levels (median OS = 36 months) (P = 0.0506).

Conclusions

We first reported that the most relevant genes involved in gemcitabine metabolism are expressed in ovarian carcinoma, and might be associated with more aggressive histotypes. The assessment of the expression levels of RRM2 as marker of clinical outcome deserves further investigation in a larger series of ovarian cancer patients.     

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.     

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.     

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.     

dCK expression correlates with 5-fluorouracil efficacy and HuR cytoplasmic expression in pancreatic cancer: A dual-institutional follow-up with the RTOG 9704 trial

Deoxycytidine kinase (dCK) and human antigen R (HuR) have been associated with response to gemcitabine in small studies. The present study investigates the prognostic and predictive value of dCK and HuR expression levels for sensitivity to gemcitabine and 5-fluorouracil (5-FU) in a large phase III adjuvant trial with chemoradiation backbone in pancreatic ductal adenocarcinoma (PDA). The dCK and HuR expression levels were determined by immunohistochemistry on a tissue microarray of 165 resected PDAs from the Radiation Therapy Oncology Group (RTOG) 9704 trial. Association with overall survival (OS) and disease-free survival (DFS) status were analyzed using the log-rank test and the Cox proportional hazards model. Experiments with cultured PDA cells were performed to explore mechanisms linking dCK and HuR expression to drug sensitivity. dCK expression levels were associated with improved OS for all patients analyzed from RTOG 9704 (HR: 0.66, 95% CI [0.47–0.93], P = 0.015). In a subset analysis based on treatment arm, the effect was restricted to patients receiving 5-FU (HR: 0.53, 95% CI [0.33–0.85], P = 0.0078). Studies in cultured cells confirmed that dCK expression rendered cells more sensitive to 5-FU. HuR cytoplasmic expression was neither prognostic nor predictive of treatment response. Previous studies along with drug sensitivity and biochemical studies demonstrate that radiation interferes with HuR’s regulatory effects on dCK, and could account for the negative findings herein based on the clinical study design (i.e., inclusion of radiation). Finally, we demonstrate that 5-FU can increase HuR function by enhancing HuR translocation from the nucleus to the cytoplasm, similar to the effect of gemcitabine in PDA cells. For the first time, in the pre-treatment tumor samples, dCK and HuR cytoplasmic expression were strongly correlated (chi-square P = 0.015). This dual-institutional follow up study, in a multi-institutional PDA randomized clinical trial, observed that dCK expression levels were prognostic and had predictive value for sensitivity to 5-FU.     

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.     

siRNA knockdown of mitochondrial thymidine kinase 2 (TK2) sensitizes human tumor cells to gemcitabine

Nucleoside metabolism enzymes are determinants of chemotherapeutic drug activity. The nucleoside salvage enzyme deoxycytidine kinase (dCK) activates gemcitabine (2′, 2′-difluoro-2′-deoxycytidine) and is negatively regulated by deoxycytidine triphosphate (dCTP). Reduction of dCTP in tumor cells could, therefore, enhance gemcitabine activity. Mitochondrial thymidine kinase 2 (TK2) phosphorylates deoxycytidine to generate dCTP. We hypothesized that: (1) TK2 modulates human tumor cell sensitivity to gemcitabine, and (2) antisense knockdown of TK2 would decrease dCTP and increase dCK activity and gemcitabine activation. siRNA downregulation of TK2 sensitized MCF7 and HeLa cells (high and moderate TK2) but not A549 cells (low TK2) to gemcitabine. Combined treatment with TK2 siRNA and gemcitabine increased dCK. We also hypothesized that TK2 siRNA-induced drug sensitization results in mitochondrial damage that enhances gemcitabine effectiveness. TK2 siRNA and gemcitabine decreased mitochondrial redox status, DNA content, and activity. This is the first demonstration of a direct role for TK2 in gemcitabine resistance, or any independent role in cancer drug resistance, and further distinguishes TK2 function from that of other dTMP-producing enzymes [cytosolic TK1 and thymidylate synthase (TS)]. siRNA knockdown of TK1 and/or TS did not sensitize cancer cells to gemcitabine indicating that, among the 3 enzymes, only TK2 is a candidate therapeutic target for combination with gemcitabine.     

Immunohistochemical and genetic evaluation of deoxycytidine kinase in pancreatic cancer: relationship to molecular mechanisms of gemcitabine resistance and survival.

Gemcitabine is considered the standard first-line therapy for patients with advanced pancreatic cancer. More recent strategies have focused on improving the efficacy of gemcitabine by either improving the method of delivery or by combining gemcitabine with other non-cross-resistant chemotherapy agents or with small-molecule drugs. However, the clinical benefits, response rates, and duration of responses have been modest. Deoxycytidine kinase (dCK) is the rate-limiting enzyme involved in the metabolism of gemcitabine. The expression of dCK has been postulated to be correlative of gemcitabine resistance. We determined the relationship of dCK immunohistochemical protein expression and/or genetic status of dCK in a panel of human pancreatic cancer tissues and pancreatic cancer cell lines and determined the relationship of these variables to the clinical outcome of patients treated with gemcitabine. We report that dCK protein expression is expressed in the majority of pancreatic cancers analyzed (40 of 44 cases, 91%) and showed a range of labeling intensities ranging from 1+ (labeling weaker in intensity than normal lymphocytes present in same section) to 3+ (labeling greater in intensity than normal lymphocytes present in same section). When labeling intensity was compared with survival, low dCK expression (1+ labeling) was correlated with both overall survival (P < 0.009) and progression-free survival following gemcitabine treatment (P < 0.04). Low dCK labeling intensity was also significantly correlated with patient age (70.3 +/- 8.1 versus 59.8 +/- 7.4 years; P < 0.0006), suggesting that age-related methylation of the dCK gene may account in part for the observed differences. Sequencing of the entire dCK coding sequence in 17 cell lines and 9 patients' cancer tissues with disease progression while on gemcitabine did not identify any mutations, suggesting that genetic alterations of dCK are not a common mechanism of resistance to gemcitabine for this tumor type. Moreover, dCK labeling showed similar patterns and intensities of labeling among matched pretreatment and post-treatment tissues. In summary, pretreatment levels of dCK protein are most correlated with overall survival following gemcitabine treatment and are stable even after resistance to gemcitabine is clinically documented.     

mTORC2 regulates ribonucleotide reductase to promote DNA replication and gemcitabine resistance in non-small cell lung cancer

Ribonucleotide reductase (RNR) is the key enzyme that catalyzes the production of deoxyribonucleotides (dNTPs) for DNA replication and it is also essential for cancer cell proliferation. As the RNR inhibitor, Gemcitabine is widely used in cancer therapies, however, resistance limits its therapeutic efficacy and curative potential. Here, we identified that mTORC2 is a main driver of gemcitabine resistance in non-small cell lung cancers (NSCLC). Pharmacological or genetic inhibition of mTORC2 greatly enhanced gemcitabine induced cytotoxicity and DNA damage. Mechanistically, mTORC2 directly interacted and phosphorylated RNR large subunit RRM1 at Ser 631. Ser631 phosphorylation of RRM1 enhanced its interaction with small subunit RRM2 to maintain sufficient RNR enzymatic activity for efficient DNA replication. Targeting mTORC2 retarded DNA replication fork progression and improved therapeutic efficacy of gemcitabine in NSCLC xenograft model in vivo. Thus, these results identified a mechanism through mTORC2 regulating RNR activity and DNA replication, conferring gemcitabine resistance to cancer cells.     

Enhancement of gemcitabine sensitivity in pancreatic cancer by co-regulation of dCK and p8 expression

The purpose of this study was to improve the gemcitabine sensitivity in pancreatic cancer by adenovirus-mediated co-regulation of dCK and p8 expression. Firstly, we analyzed the sensitivity of three human pancreatic tumor cell lines (Capan-2, Panc-1 and BxPc-3) to gemcitabine using MTT assays, and found Panc-1 to be relatively resistant to gemcitabine. Further, we investigated the expression of dCK and p8 in different pancreatic cancer cell lines using real-time PCR and Western blot analysis, and found that the expression levels of these two genes were related to the gemcitabine sensitivity of pancreatic cancer cells. We constructed recombinant adenovirus vectors, Ad-dCK and Ad-p8-siRNA, that overexpressed dCK and knocked down p8, respectively. Using MTT assays, we observed that combined infection using Ad-dCK and Ad-p8-siRNA in vitro led to a significant decrease in the gemcitabine IC50 with an increase in apoptosis and caspase-3 activity in Panc-1 cells, which are relatively resistant to gemcitabine. Furthermore, in established subcutaneous pancreatic cancer models in nude mice, the tumor inhibition was markedly enhanced accompanied by elevation of the apoptosis index after intratumoral injection of Ad-dCK and Ad-p8-siRNA on the basis of intraperitoneal gemcitabine chemotherapy. Taken together, the present findings suggest that, dCK and p8 may be the important factors in the regulation of gemcitabine sensitivity in pancreatic cancer cells. Moreover, co-regulation of the two factors achieved better effects than regulation of either one alone.     

The long non-coding RNA HOTTIP enhances pancreatic cancer cell proliferation,survival and migration

HOTTIP is a long non-coding RNA (lncRNA) transcribed from the 5′ tip of the HOXA locus and is associated with the polycomb repressor complex 2 (PRC2) and WD repeat containing protein 5 (WDR5)/mixed lineage leukemia 1 (MLL1) chromatin modifying complexes. HOTTIP is expressed in pancreatic cancer cell lines and knockdown of HOTTIP by RNA interference (siHOTTIP) in Panc1 pancreatic cancer cells decreased proliferation, induced apoptosis and decreased migration. In Panc1 cells transfected with siHOTTIP, there was a decrease in expression of 757 genes and increased expression of 514 genes, and a limited gene analysis indicated that HOTTIP regulation of genes is complex. For example, Aurora kinase A, an important regulator of cell growth, is coregulated by MLL and not WDR5 and, in contrast to previous studies in liver cancer cells, HOTTIP does not regulate HOXA13 but plays a role in regulation of several other HOX genes including HOXA10, HOXB2, HOXA11, HOXA9 and HOXA1. Although HOTTIP and the HOX-associated lncRNA HOTAIR have similar pro-oncogenic functions, they regulate strikingly different sets of genes in Panc1 cells and in pancreatic tumors.     

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.     

Roles for hENT1 and dCK in gemcitabine sensitivity and malignancy of meningioma

BackgroundHigh-grade meningiomas are aggressive tumors with high morbidity and mortality rates that frequently recur even after surgery and adjuvant radiotherapy. However, limited information is currently available on the biology of these tumors, and no alternative adjuvant treatment options exist. Although we previously demonstrated that high-grade meningioma cells were highly sensitive to gemcitabine in vitro and in vivo, the underlying molecular mechanisms remain unknown.MethodsWe examined the roles of hENT1 (human equilibrative nucleoside transporter 1) and dCK (deoxycytidine kinase) in the gemcitabine sensitivity and growth of meningioma cells in vitro. Tissue samples from meningiomas (26 WHO grade I and 21 WHO grade II/III meningiomas) were immunohistochemically analyzed for hENT1 and dCK as well as for Ki-67 as a marker of proliferative activity.ResultshENT1 and dCK, which play critical roles in the intracellular transport and activation of gemcitabine, respectively, were responsible for the high gemcitabine sensitivity of high-grade meningioma cells and were strongly expressed in high-grade meningiomas. hENT1 expression was required for the proliferation and survival of high-grade meningioma cells and dCK expression. Furthermore, high hENT1 and dCK expression levels correlated with stronger tumor cell proliferative activity and shorter survival in meningioma patients.ConclusionsThe present results suggest that hENT1 is a key molecular factor influencing the growth capacity and gemcitabine sensitivity of meningioma cells and also that hENT1, together with dCK, may be a viable prognostic marker for meningioma patients as well as a predictive marker of their responses to gemcitabine.