Retroviral transduction of cancer cell lines with the gene encoding Drosophila melanogaster multisubstrate deoxyribonucleoside kinase

Nucleoside kinases from several species are investigated as "suicide genes" for treatment of malignant tumors by combined gene/chemotherapy. We have recently cloned a multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK), and we have shown that the enzyme phosphorylates cytotoxic pyrimidine and purine nucleoside analogs. The broad substrate specificity of the enzyme, as well as its very high catalytic rate, makes it a unique member of the nucleoside kinase enzyme family. In the present study, we evaluated Dm-dNK as a suicide gene by constructing a replication-deficient retroviral vector that expresses the enzyme. The human pancreatic adenocarcinoma cell line MIA PaCa-2 and a thymidine kinase-deficient osteosarcoma cell line were transduced with the recombinant virus. We showed that Dm-dNK can be expressed in human cells, that the enzyme retained its enzymatic activity, and that it is localized in the cell nuclei due to a nuclear localization signal in its C-terminal region. The cells expressing Dm-dNK exhibited increased sensitivity to several cytotoxic nucleoside analogs, such as 1-beta-d-arabinofuranosylcytosine, 1-beta-d-arabinofuranosylthymine, (E)-5-(2-bromovinyl)-2'-deoxyuridine, 2-chloro-2'-deoxyadenosine, and 2',2'-difluorodeoxycytidine. These findings suggest that Dm-dNK may be used as a suicide gene in combined gene/chemotherapy of cancer.     

An Undesired Effect of Chemotherapy: GEMCITABINE PROMOTES PANCREATIC CANCER CELL INVASIVENESS THROUGH REACTIVE OXYGEN SPECIES-DEPENDENT,NUCLEAR FACTOR κB- AND HYPOXIA-INDUCIBLE FACTOR 1α-MEDIATED UP-REGULATION OF CXCR4*

Recently, we have shown that CXCL12/CXCR4 signaling plays an important role in gemcitabine resistance of pancreatic cancer (PC) cells. Here, we explored the effect of gemcitabine on this resistance mechanism. Our data demonstrate that gemcitabine induces CXCR4 expression in two PC cell lines (MiaPaCa and Colo357) in a dose- and time-dependent manner. Gemcitabine-induced CXCR4 expression is dependent on reactive oxygen species (ROS) generation because it is abrogated by pretreatment of PC cells with the free radical scavenger N-acetyl-L-cysteine. CXCR4 up-regulation by gemcitabine correlates with time-dependent accumulation of NF-κB and HIF-1α in the nucleus. Enhanced binding of NF-κB and HIF-1α to the CXCR4 promoter is observed in gemcitabine-treated PC cells, whereas their silencing by RNA interference causes suppression of gemcitabine-induced CXCR4 expression. ROS induction upon gemcitabine treatment precedes the nuclear accumulation of NF-κB and HIF-1α, and suppression of ROS diminishes these effects. The effect of ROS on NF-κB and HIF-1α is mediated through activation of ERK1/2 and Akt, and their pharmacological inhibition also suppresses gemcitabine-induced CXCR4 up-regulation. Interestingly, our data demonstrate that nuclear accumulation of NF-κB results from phosphorylation-induced degradation of IκBα, whereas HIF-1α up-regulation is NF-κB-dependent. Lastly, our data demonstrate that gemcitabine-treated PC cells are more motile and exhibit significantly greater invasiveness against a CXCL12 gradient. Together, these findings reinforce the role of CXCL12/CXCR4 signaling in gemcitabine resistance and point toward an unintended and undesired effect of chemotherapy.     

A Kinome Screen Identifies Checkpoint Kinase 1 (CHK1) as a Sensitizer for RRM1-Dependent Gemcitabine Efficacy

Gemcitabine is among the most efficacious and widely used antimetabolite agents. Its molecular targets are ribonucleotide reductase M1 (RRM1) and elongating DNA. Acquired and de novo resistance as a result of RRM1 overexpression are major obstacles to therapeutic efficacy. We deployed a synthetic lethality screen to investigate if knockdown of 87 selected protein kinases by siRNA could overcome RRM1-dependent gemcitabine resistance in high and low RRM1-expressing model systems. The models included genetically RRM1-modified lung and breast cancer cell lines, cell lines with gemcitabine-induced RRM1 overexpression, and a series of naturally gemcitabine-resistant cell lines. Lead molecular targets were validated by determination of differential gemcitabine activity using cell lines with and without target knock down, and by assessing synergistic activity between gemcitabine and an inhibitor of the lead target. CHK1 was identified has the kinase with the most significant and robust interaction, and it was validated using AZD7762, a small-molecule ATP-competitive inhibitor of CHK1 activation. Synergism between CHK1 inhibition and RRM1-dependent gemcitabine efficacy was observed in cells with high RRM1 levels, while antagonism was observed in cells with low RRM1 levels. In addition, four cell lines with natural gemcitabine resistance demonstrated improved gemcitabine efficacy after CHK1 inhibition. In tumor specimens from 187 patients with non-small-cell lung cancer, total CHK1 and RRM1 in situ protein levels were significantly (p = 0.003) and inversely correlated. We conclude that inhibition of CHK1 may have its greatest clinical utility in malignancies where gemcitabine resistance is a result of elevated RRM1 levels. We also conclude that CHK1 inhibition in tumors with low RRM1 levels may be detrimental to gemcitabine efficacy.     

Altered deoxyribonucleotide pools in T-lymphoblastoid cells expressing the multisubstrate nucleoside kinase of Drosophila melanogaster

The multisubstrate nucleoside kinase of Drosophila melanogaster (Dm-dNK) can be expressed in human solid tumor cells and its unique enzymatic properties makes this enzyme a suicide gene candidate. In the present study, Dm-dNK was stably expressed in the CCRF-CEM and H9 T-lymphoblastoid cell lines. The expressed enzyme was localized to the cell nucleus and the enzyme retained its activity. The Dm-dNK overexpressing cells showed approximately 200-fold increased sensitivity to the cytostatic activity of several nucleoside analogs, such as the pyrimidine nucleoside analogs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 1-beta-d-arabinofuranosylthymine (araT), but not to the antiherpetic purine nucleoside analogs ganciclovir, acyclovir and penciclovir, which may allow this technology to be applied in donor T cells and/or rescue graft vs. host disease to permit modulation of alloreactivity after transplantation. The most pronounced effect on the steady-state dNTP levels was a two- to 10-fold increased dTTP pool in Dm-dNK expressing cells that were grown in the presence of 1 microm of each natural deoxyribonucleoside. Although the Dm-dNK expressing cells demonstrated dNTP pool imbalances, no mitochondrial DNA deletions or altered mitochondrial DNA levels were detected in the H9 Dm-dNK expressing cells.     

Synthesis of a covalent gemcitabine-(carbamate)-[anti-HER2/neu] immunochemotherapeutic and its cytotoxic anti-neoplastic activity against chemotherapeutic-resistant SKBr-3 mammary carcinoma

Gemcitabine is a potent chemotherapeutic that exerts cytotoxic activity against several leukemias and a wide spectrum of carcinomas. A brief plasma half-life in part due to rapid deamination and chemotherapeutic-resistance frequently limit the utility of gemcitabine in clinical oncology. Selective ‘targeted’ delivery of gemcitabine represents a potential molecular strategy for simultaneously prolonging its plasma half-life and minimizing exposure of innocent tissues and organ systems.

Materials and methods

Gemcitabine was combined in molar excess with N-[p-maleimidophenyl]-isocyanate (PMPI) so that the isocyanate moiety of PMPI which exclusively reacts with hydroxyl groups preferentially created a carbamate covalent bond at the terminal C₅-methylhydroxy group of gemcitabine. Monoclonal immunoglobulin with binding-avidity specifically for HER2/neu was thiolated with 2-iminothiolane at the terminal ??-amine group of lysine amino acid residues. The gemcitabine-(carbamate)-PMPI intermediate with a maleimide moiety that exclusively reacts with reduced sulfhydryl groups was then combined with thiolated anti-HER2/neu monoclonal immunoglobulin. Western-blot analysis was utilized to delineate the molecular weight profile for gemcitabine-(carbamate)-[anti-HER2/neu] while cell binding characteristics were determined by cell-ELISA utilizing SKBr-3 mammary carcinoma which highly over-expresses HER2/neu receptors. Cytotoxic anti-neoplastic potency of gemcitabine-(carbamate)-[anti-HER2/neu] between the gemcitabine-equivalent concentrations of 10⁻¹² and 10⁻⁶ M was determined utilizing vitality staining analysis of chemotherapeutic-resistant SKBr-3 mammary carcinoma.

Results

Gemcitabine-(carbamate)-[anti-HER2/neu] was synthesized at a molar incorporation index of 1:1.1 (110%) and had a molecular weight of 150 kDa that was indistinguishable from reference control immunoglobulin fractions. Cell-ELISA detected progressive increases in SKBr-3 mammary carcinoma associated immunoglobulin with corresponding increases in covalent gemcitabine immunochemotherapeutic concentrations. The in vitro cytotoxic anti-neoplastic potency of gemcitabine-(carbamate)-[anti-HER2/neu] was approximately 20% and 32% at 10⁻⁷ and 10⁻⁶ M (gemcitabine-equivalent concentrations) after a 182-h incubation period.

Discussion

The investigations describes for the first time a methodology for synthesizing a gemcitabine anti-HER2/neu immunochemotherapeutic by creating a covalent bond structure between the C₅-methylhydroxy group of gemcitabine and thiolated lysine amino acid residues of monoclonal antibody or other biologically active protein fractions. Gemcitabine-(carbamate)-[anti-HER2/neu] possessed binding-avidity at HER2/neu receptors highly over-expressed by chemotherapeutic-resistant SKBr-3 mammary carcinoma. Alternatively, gemcitabine can be covalently linked at its C₅-methylhydroxy group to monoclonal immunoglobulin fractions that possess binding-avidity for other receptors and membrane complexes uniquely highly over-expressed by a variety of neoplastic cell types. Compared to chemotherapeutic-resistant SKBr-3 mammary carcinoma, gemcitabine-(carbamate)-[anti-HER2/neu] immunochemotherapeutic is anticipated to exert higher levels of cytotoxic anti-neoplastic potency against other neoplastic cell types like pancreatic carcinoma, small-cell lung carcinoma, neuroblastoma, glioblastoma, oral squamous cell carcinoma, cervical epithelioid carcinoma, or leukemia/lymphoid neoplastic cell types based on their reportedly greater sensitivity to gemcitabine and gemcitabine covalent conjugates.     

N-Acyl-phosphoramidates as potential novel form of gemcitabine prodrugs

Gemcitabine (dFdC) is a cytidine analog remarkably active against a wide range of solid tumors. Inside a cell, gemcitabine is phosphorylated by deoxycytidine kinase to yield gemcitabine monophosphate, further converted to gemcitabine di- and triphosphate. The most frequent form of acquired resistance to gemcitabine in vitro is the deoxycytidine kinase deficiency. Thus, proper prodrugs carrying the 5′-pdFdC moiety may help to overcome this problem. A series of new derivatives of gemcitabine possessing N-acyl(thio)phosphoramidate moieties were prepared and their cytotoxic properties were determined. N-Acyl-phosphoramidate derivatives of gemcitabine have similar cytotoxicity as gemcitabine itself, and have been found accessible to the cellular enzymes. The nicotinic carboxamide derivative of gemcitabine 5′-O-phosphorothioate occurred to be the best inhibitor of bacterial DNA polymerase I and human DNA polymerase α.     

Deoxyribonucleoside kinases belonging to the thymidine kinase 2 (TK2)-like group vary significantly in substrate specificity, kinetics and feed-back regulation.

In eukaryotic cells deoxyribonucleoside kinases belonging to three phylogenetic sub-families have been found: (i) thymidine kinase 1 (TK1)-like enzymes, which are strictly pyrimidine deoxyribonucleoside-specific kinases; (ii) TK2-like enzymes, which include pyrimidine deoxyribonucleoside kinases and a single multisubstrate kinase from Drosophila melanogaster (Dm-dNK); and (iii) deoxycytidine/deoxyguanosine kinase (dCK/dGK)-like enzymes, which are deoxycytidine and/or purine deoxyribonucleoside-specific kinases. We cloned and characterized two new deoxyribonucleoside kinases belonging to the TK2-like group from the insect Bombyx mori and the amphibian Xenopus laevis. The deoxyribonucleoside kinase from B. mori (Bm-dNK) turned out to be a multisubstrate kinase like Dm-dNK. But uniquely for a deoxyribonucleoside kinase, Bm-dNK displayed positive cooperativity with all four natural deoxyribonucleoside substrates. The deoxyribonucleoside kinase from X. laevis (Xen-PyK) resembled closely the human and mouse TK2 enzymes displaying their characteristic Michaelis-Menten kinetic with deoxycytidine and negative cooperativity with its second natural substrate thymidine. Bm-dNK, Dm-dNK and Xen-PyK were shown to be homodimers. Significant differences in the feedback inhibition by deoxyribonucleoside triphosphates between these three enzymes were found. The insect multisubstrate deoxyribonucleoside kinases Bm-dNK and Dm-dNK were only inhibited by thymidine triphosphate, while Xen-PyK was inhibited by thymidine and deoxycytidine triphosphate in a complex pattern depending on the deoxyribonucleoside substrate. The broad substrate specificity and different feedback regulation of the multisubstrate insect deoxyribonucleoside kinases may indicate that these enzymes have a different functional role than the other members of the TK2-like group.     

Identification of residues involved in the specificity and regulation of the highly efficient multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster

In contrast to all known deoxyribonucleoside kinases, a single highly efficient deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) is able to phosphorylate all precursor nucleosides for DNA synthesis. Dm-dNK was mutated in vitro by high-frequency random mutagenesis, expressed in the thymidine kinase-deficient Escherichia coli strain KY895 and clones were selected for sensitivity to the nucleoside analogs 1-beta-d-arabinofuranosylcytosine (AraC, Cytarabine), 3'-azido-2', 3'-dideoxythymidine (AZT, Zidovudine, Retrovir, 2', 3'-dideoxyadenosine (ddA) and 2',3'-dideoxycytidine (ddC, Zalcitabine, Hivid. Thirteen mutants with increased sensitivity compared to the wild-type Dm-dNK were isolated from a relatively small pool of less than 10,000 clones. Eight mutant Dm-dNKs increased the sensitivity of KY895 to more than one analog, and two of these mutants even to all four nucleoside analogs. Surprisingly, the mutations did not map to the five regions which are highly conserved among deoxyribonucleoside kinases. The molecular background of improved sensitivity was characterized for the double-mutant MuD (N45D, N64D), where the LD(100) value of transformed KY895 decreased 316-fold for AZT and more than 11-fold for ddC when compared to wild-type Dm-dNK. Purified recombinant MuD displayed higher K(m) values for the native substrates than wild-type Dm-dNK and the V(max) values were substantially lower. On the other hand, the K(m) and V(max) values for AZT and the K(m) value for ddC were nearly unchanged between MuD and wild-type Dm-dNK. Additionally, a decrease in feedback inhibition of MuD by thymidine triphosphate (TTP) was found. This study demonstrates how high-frequency mutagenesis combined with a parallel selection for desired properties provides an insight into the structure-function relationships of the multisubstrate kinase from D. melanogaster. At the same time these mutant enzymes exhibit properties useful in biotechnological and medical applications.     

Active site mutants of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase.

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) is sequence-related to three human deoxyribonucleoside kinases and to herpes simplex virus type-1 thymidine kinase. Dm-dNK phosphorylates both purine and pyrimidine deoxyribonucleosides and nucleoside analogues although it has a preference for pyrimidine nucleosides. We performed site-directed mutagenesis on residues that, based on structural data, are involved in substrate recognition. The aim was to increase the phosphorylation efficiency of purine nucleoside substrates to create an improved enzyme to be used in suicide gene therapy. A Q81N mutation showed a relative increase in deoxyguanosine phosphorylation compared with the wild-type enzyme although the efficiency of deoxythymidine phosphorylation was 10-fold lower for the mutant. In addition to residue Q81 the function of amino acids N28, I29 and F114 was investigated by different substitutions. All of the mutated enzymes showed decreased efficiency of thymidine phosphorylation in comparison with the wild-type enzyme supporting their importance for substrate binding and/or catalysis as proposed by the recently solved structure of Dm-dNK.     

Cloning and characterization of the multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster.

A Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) was reported to phosphorylate all four natural deoxyribonucleosides as well as several nucleoside analogs (Munch-Petersen, B., Piskur, J., and Sondergaard, L. (1998) J. Biol. Chem. 273, 3926-3931). The broad substrate specificity of this enzyme together with a high catalytic rate makes it unique among the nucleoside kinases. We have in the present study cloned the Dm-dNK cDNA, expressed the 29-kDa protein in Escherichia coli, and characterized the recombinant enzyme for the phosphorylation of nucleosides and clinically important nucleoside analogs. The recombinant enzyme preferentially phosphorylated the pyrimidine nucleosides dThd, dCyd, and dUrd, but phosphorylation of the purine nucleosides dAdo and dGuo was also efficiently catalyzed. Dm-dNK is closely related to human and herpes simplex virus deoxyribonucleoside kinases. The highest level of sequence similarity was noted with human mitochondrial thymidine kinase 2, and these enzymes also share many substrates. The cDNA cloning and characterization of Dm-dNK will be the basis for studies on the use of this multisubstrate nucleoside kinase as a suicide gene in combined gene/chemotherapy of cancer.     

DNA methylation-independent reversion of gemcitabine resistance by hydralazine in cervical cancer cells

Background

Down regulation of genes coding for nucleoside transporters and drug metabolism responsible for uptake and metabolic activation of the nucleoside gemcitabine is related with acquired tumor resistance against this agent. Hydralazine has been shown to reverse doxorubicin resistance in a model of breast cancer. Here we wanted to investigate whether epigenetic mechanisms are responsible for acquiring resistance to gemcitabine and if hydralazine could restore gemcitabine sensitivity in cervical cancer cells.

Methodology/Principal Findings

The cervical cancer cell line CaLo cell line was cultured in the presence of increasing concentrations of gemcitabine. Down-regulation of hENT1 & dCK genes was observed in the resistant cells (CaLoGR) which was not associated with promoter methylation. Treatment with hydralazine reversed gemcitabine resistance and led to hENT1 and dCK gene reactivation in a DNA promoter methylation-independent manner. No changes in HDAC total activity nor in H3 and H4 acetylation at these promoters were observed. ChIP analysis showed H3K9m2 at hENT1 and dCK gene promoters which correlated with hyper-expression of G9A histone methyltransferase at RNA and protein level in the resistant cells. Hydralazine inhibited G9A methyltransferase activity in vitro and depletion of the G9A gene by iRNA restored gemcitabine sensitivity.

Conclusions/Significance

Our results demonstrate that acquired gemcitabine resistance is associated with DNA promoter methylation-independent hENT1 and dCK gene down-regulation and hyper-expression of G9A methyltransferase. Hydralazine reverts gemcitabine resistance in cervical cancer cells via inhibition of G9A histone methyltransferase.     

Let-7 Sensitizes KRAS Mutant Tumor Cells to Chemotherapy

KRAS is the most commonly mutated oncogene in human cancers and is associated with poor prognosis and drug resistance. Let-7 is a family of tumor suppressor microRNAs that are frequently suppressed in solid tumors, where KRAS mutations are highly prevalent. In this study, we investigated the potential use of let-7 as a chemosensitizer. We found that let-7b repletion selectively sensitized KRAS mutant tumor cells to the cytotoxicity of paclitaxel and gemcitabine. Transfection of let-7b mimic downregulated the expression of mutant but not wild-type KRAS. Combination of let-7b mimic with paclitaxel or gemcitabine diminished MEK/ERK and PI3K/AKT signaling concurrently, triggered the onset of apoptosis, and reverted the epithelial-mesenchymal transition in KRAS mutant tumor cells. In addition, let-7b repletion downregulated the expression of β-tubulin III and ribonucleotide reductase subunit M2, two proteins known to mediate tumor resistance to paclitaxel and gemcitabine, respectively. Let-7 may represent a new class of chemosensitizer for the treatment of KRAS mutant tumors.     

Nucleoside-catabolizing Enzymes in Mycoplasma-infected Tumor Cell Cultures Compromise the Cytostatic Activity of the Anticancer Drug Gemcitabine

The intracellular metabolism and cytostatic activity of the anticancer drug gemcitabine (2′,2′-difluoro-2′-deoxycytidine; dFdC) was severely compromised in Mycoplasma hyorhinis-infected tumor cell cultures. Pronounced deamination of dFdC to its less cytostatic metabolite 2′,2′-difluoro-2′-deoxyuridine was observed, both in cell extracts and spent culture medium (i.e. tumor cell-free but mycoplasma-containing) of mycoplasma-infected tumor cells. This indicates that the decreased antiproliferative activity of dFdC in such cells is attributed to a mycoplasma cytidine deaminase causing rapid drug catabolism. Indeed, the cytostatic activity of gemcitabine could be restored by the co-administration of tetrahydrouridine (a potent cytidine deaminase inhibitor). Additionally, mycoplasma-derived pyrimidine nucleoside phosphorylase (PyNP) activity indirectly potentiated deamination of dFdC: the natural pyrimidine nucleosides uridine, 2′-deoxyuridine and thymidine inhibited mycoplasma-associated dFdC deamination but were efficiently catabolized (removed) by mycoplasma PyNP. The markedly lower anabolism and related cytostatic activity of dFdC in mycoplasma-infected tumor cells was therefore also (partially) restored by a specific TP/PyNP inhibitor (TPI), or by exogenous thymidine. Consequently, no effect on the cytostatic activity of dFdC was observed in tumor cell cultures infected with a PyNP-deficient Mycoplasma pneumoniae strain. Because it has been reported that some commensal mycoplasma species (including M. hyorhinis) preferentially colonize tumor tissue in cancer patients, our findings suggest that the presence of mycoplasmas in the tumor microenvironment could be a limiting factor for the anticancer efficiency of dFdC-based chemotherapy. Accordingly, a significantly decreased antitumor effect of dFdC was observed in mice bearing M. hyorhinis-infected murine mammary FM3A tumors compared with uninfected tumors.     

Phase I Trial of Radiotherapy Concurrent with Twice-Weekly Gemcitabine for Head and Neck Cancer: Translation From Preclinical Investigations Aiming to Improve the Therapeutic Ratio

BACKGROUND: Once-weekly gemcitabine concurrent with radiotherapy was highly effective in the treatment of head and neck cancer (HNC) but limited by high mucosal toxicity. Pre-clinical investigations suggested that delivering gemcitabine at substantially lower doses twice weekly during radiotherapy improved the therapeutic ratio. We sought to translated these preclinical findings to a phase I trial. METHODS: Twenty-five patients with non-resectable HNC were scheduled to receive gemcitabine twice weekly during the last 2 weeks (total 5 infusions) of hyperfractionated radiotherapy delivering 1.2 Gy twice daily to total 76.8 Gy. Tumor biopsies to measure active intracellular (phosphorylated) gemcitabine were planned after the first drug delivery. Patients were assigned to escalating dose cohorts using the Continuous Reassessment Method. RESULTS: Twenty-one patients evaluable for toxicity were divided into cohorts receiving twice weekly treatment with 10, 20, 33, or 50 mg/m² gemcitabine. Dose-limiting toxicity was grade 3-4 confluent mucositis/pharyngitis, and the maximally tolerated dose (MTD) was 20 mg/m². Median survival was 20 months, with no difference between cohorts receiving lower (10, 20 mg/m²) or higher (33, 50 mg/m²) gemcitabine doses. Tumor biopsies after the first drug delivery showed only a minority of tumor cells in the specimens. CONCLUSION: These findings validate preclinical models that show that gemcitabine is radiation sensitizer at doses far below those used for systemic chemotherapy. However, the improvement in the therapeutic ratio predicted from the preclinical study did not translate into a substantial relative increase in the MTD of the drug in the clinical phase I trial.     

Tumor BRCA1, RRM1 and RRM2 mRNA Expression Levels and Clinical Response to First-Line Gemcitabine plus Docetaxel in Non-Small-Cell Lung Cancer Patients

Background

Overexpression of RRM1 and RRM2 has been associated with gemcitabine resistance. BRCA1 overexpression increases sensitivity to paclitaxel and docetaxel. We have retrospectively examined the effect of RRM1, RRM2 and BRCA1 expression on outcome to gemcitabine plus docetaxel in advanced non-small-cell lung cancer (NSCLC) patients.

Methodology and Principal Findings

Tumor samples were collected from 102 chemotherapy-naïve advanced NSCLC patients treated with gemcitabine plus docetaxel as part of a randomized trial. RRM1, RRM2 and BRCA1 mRNA levels were assessed by quantitative PCR and correlated with response, time to progression and survival. As BRCA1 levels increased, the probability of response increased (Odds Ratio [OR], 1.09: p = 0.01) and the risk of progression decreased (hazard ratio [HR], 0.99; p = 0.36). As RRM1 and RRM2 levels increased, the probability of response decreased (RRM1: OR, 0.97; p = 0.82; RRM2: OR, 0.94; p<0.0001) and the risk of progression increased (RRM1: HR, 1.02; p = 0.001; RRM2: HR, 1.005; p = 0.01). An interaction observed between BRCA1 and RRM1 allowed patients to be classified in three risk groups according to combinations of gene expression levels, with times to progression of 10.13, 4.17 and 2.30 months (p = 0.001). Low BRCA1 expression was the only factor significantly associated with longer time to progression in 31 patients receiving cisplatin-based second-line therapy.

Conclusions

The mRNA expression of BRCA1, RRM1 and RRM2 is potentially a useful tool for selecting NSCLC patients for individualized chemotherapy and warrants further investigation in prospective studies.