The molecular mechanism of synergistic cytotoxic effect of combined treatment with thymidylate synthase inhibitor and small-molecule epidermal growth factor receptor inhibitor (erlotinib) in human lung squamous cell carcinoma cells = 胸腺嘧啶核甘酸合成酶抑制劑(Thymidylate Synthase inhibitor)與表皮生長因子受體抑制劑 (Erlotinib,得舒緩)合併處理人類扁平上皮細胞肺癌細胞具有加強毒殺效果之分子機轉

Project title
胸腺嘧啶核甘酸合成酶抑制劑(Thymidylate Synthase inhibitor)與表皮生長因子受體抑制劑 (Erlotinib,得舒緩)合併處理人類扁平上皮細胞肺癌細胞具有加強毒殺效果之分子機轉
Internal ID
DOH99-HO-2021
Principal Investigator
Start Date
January 1, 2010
End Date
December 31, 2010
Organizations
Partner Organizations
Ministry of Health and Welfare
Description
Lung cancer remains a disease with a high fatality rate (5-year survival rate of 15%) [1]. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers [1]. The majority of patients with NSCLC present with locally advanced inoperable or metastatic disease[2]. The prognosis is especially poor for patients with advanced NSCLC who have not responded to multiple prior chemotherapy regimens. The goals of therapy for advanced lung cancer include symptom improvement, disease stabilization, and improved quality of life. The management of NSCLC largely depends on the stage of disease at diagnosis. The current first-line therapeutic option for patients with advanced NSCLC includes chemotherapy with a platinum (cisplatin or carboplatin) in combination with a second or third-generation cytotoxic agent (paclitaxel, gemcitabine, vinorelbine, irinotecan or docetaxel) [3]. The use of cytotoxic chemotherapies has resulted in improvements in both median overall survival and 1-year survival rates compared with best supportive care [4]. Advances in the understanding of tumor biology have led to the identification of many of the key molecular pathways that drive tumor growth. One of these is the pathway triggered by activation of the epidermal growth factor receptor (EGFR) [5, 6]. EGFR is a transmembrane glycoprotein belonging to the human EGFR family. It consists of an extracellular ligand-binding domain, a transmembrane region, and a cytoplasmic domain that contains a tyrosine kinase (TK) region [7]. TKs are ubiquitously expressed proteins that are involved in numerous intracellular signaling pathways, including both normal and aberrant cell growth [8]. Ligand binding to the EGFR induces receptor dimerization and activation of the TK activity of the receptor. This step subsequently causes receptor autophosphorylation, initiating signal transduction pathways that lead to cell proliferation, inhibition of apoptosis, cell survival and angiogenesis. The most common signaling pathways activated by EGFR are mitogen-activated protein kinase (MAPK) pathway and phosphatidylinositol 3-kinase (PI3-K)/AKT pathway [6, 9-12]. Dysregulation of these pathways can result in oncogenesis and cancer progression. Inhibitors of TK phosphorylation (TK inhibitors [TKIs]) are small-molecule agents that block EGFR activity by interfering with the adenosine triphosphate-binding site on the intracellular region of the receptor [13]. A variety of TKIs have been developed for advanced NSCLC. Gefitinib (ZD1839,Iressa) and erlotinib (OSI-774, Tarceva) are EGFR-TKIs to be approved by the FDA for treatment of advanced NSCLC [2]. Objective tumor responses to gefitinib occurred more frequently in women than in men, were higher with adenocarcinoma than other histologic types, and were higher in those patients who had never smoked. Three recent reports have identified specific mutant versions of EGFR in NSCLC cells that seem to determine response to treatment with gefitinib [14, 15] and with erlotinib [16, 17]. Sensitivity to erlotinib has been associated with EGFR mutations, most commonly deletions of four to six amino acids in exon 19 or a point mutation (L858R) in exon 21. Increased EGFR gene copy number has also been pointed out as a good predictive marker for erlotinib response [18]. Erlotinib can prolong survival in patients with non-small-cell lung cancer after first-line or second-line chemotherapy in stage IIIB or IV non-small-cell lung cancer [19]. Although the survival benefit from erlotinib was observed in varied subgroups of patients, the radiographic responses were more common in certain patient subgroups, such as women, never-smokers, patients with adenocarcinoma histology, patients of Asian ethnicity, and patients with presence of HER1/EGFR TK domain mutations. However, recent report has shown that a white male former smoker with advancedstage squamous cell non-small-cell lung cancer, who responded to first-line erlotinib. The molecular analysis of the tumor did not reveal HER1/EGFR TK mutations [20]. Erlotinib significantly reduced thymidylate synthase (TS) expression and activity, possibly via E2F-1 reduction, as detected by RT-PCR and Western blot. The combination of erlotinib with pemetrexed decreased TS in situ activity in all NSCLC cells [21]. Pemetrexed disodium is a potent new antifolate which inhibits many folate-dependent reactions that are essential for cell proliferation. Pemetrexed inhibits multiple folate-dependent enzymes involved in both purine and pyrimidine synthesis including thymidylate synthase (TS), dihydrofolate reductase, glycinamide ribonucleotide formyltransferase, and aminoimidazole carboxamide ribonucleotide formyltransferase [22]. Its primary target is thymidylate synthase but it also inhibits folate-dependent enzymes involved in purine synthesis [23]. As a single agent pemetrexed has shown good activity against non-small-cell lung cancer, squamous-cell carcinoma of head and neck, colon cancer, and breast cancer, and it appears to be particularly active in combination with cisplatin against non-small-cell lung cancer and mesothelioma [24]. Pemetrexed is registered, at the dose of 500 mg/m2 on day 1 of a 3-week schedule, in combination with cisplatin, for first-line therapy and, as single-agent, for second-line treatment of patients with non-squamous NSCLC [25]. Recent report has revealed that pemetrexed induced an epidermal growth factor receptor-mediated activation of the phosphatidylinositol 3-kinase/AKT pathway, which was inhibited by erlotinib and a specific phosphatidylinositol 3-kinase inhibitor, LY294002. The combination of pemetrexed and erlotinib has synergistic cytotoxic effect in NSCLC in vitro if exposure to erlotinib before pemetrexed is avoided [26]. Recent study also has shown that the synergistic cytotoxicity was found in all NSCLC cells, most pronounced with pemetrexed + erlotinib (24 h) --> erlotinib (48 h) sequence, which was associated with a significant induction of apoptosis. Pemetrexed increased EGFR phosphorylation and reduced Akt phosphorylation, which was additionally reduced by drug combination with erlotinib [21]. The up-regulation of the expression of the TS gene may have an important role in the acquired resistance to pemetrexed. Knockdown of TS expression using siRNA enhanced pemetrexed cytotoxicity in PC6/MTA-4.0 cells (small cell lung cancer cell line). In addition, TS may be a predictive marker for pemetrexed sensitivity in lung cancer [27]. Thymidylate synthase, the main molecular target of pemetrexed, has higher mRNA and protein expression in squamous- and small-cell lung cancer compared with adenocarcinoma. This differential expression might well molecularly explain the differential clinical activity of pemetrexed in the various histotypes of lung cancer, including the marginal activity in small-cell lung cancer [28]. Xeloda (capecitabine), a thymidine phosphorylase activated fluoropyrimidine carbamate, is currently the only universally approved orally administered 5-fluorouracil (5-FU) prodrug. It belongs to a newer generation of orally administered fluoropyrimidines. Capecitabine is not a cytotoxic drug in itself, but via a three-step enzymatic cascade, it is converted to 5-FU mainly within human cancer cells. It has been found effective in colorectal, breast, and recently nasopharyngeal carcinomas [29]. Lymphoepithelioma-like carcinoma (LELC) of lung has previously demonstrated good clinical response to 5-fluorouracil containing chemotherapy regimen [30]. Recent report has revealed a patient with metastatic lung adenocarcinoma with high levels of LDH and CEA with clear partial response to capecitabine after several lines of chemotherapy. The increase in thymidine phosphorylase (TP) expression in NSCLC could provide a rationale for the use of capecitabine in this tumour [31]. The fluoropyrimidine anticancer agent 5-fluorouracil (5-FU) is active in a wide range of solid tumors, particularly gastric, colorectal, and head and neck cancers. The oral administration of 5-FU itself is not feasible owing to the high activity of dihydropyrimidine dehydrogenase (DPD) in the gut wall, which causes rapid metabolism of the drug and results in decreased and erratic absorption of 5-FU. To bypass this problem, oral fluoropyrimidine derivatives were developed either in the form of 5-FU prodrugs (i.e., tegafur, doxifluridine or capecitabine), or as enzyme inhibitors (i.e., eniluracil) administered with 5-FU, or as both prodrugs and enzyme inhibitors (i.e., S-1, UFT or BOF-A2). The oral fluoropyrimidine S-1, which consists of the 5-FU prodrug tegafur (ftorafur, FT) and two enzyme inhibitors, CDHP (5-chloro-2,4-dihydroxypyridine) and OXO (potassium oxonate), in a molar ratio of 1(FT):0.4 (CDHP):1(OXO). Phase II trials have demonstrated that S-1, as a single agent, is active for the treatment of gastric, colorectal, head and neck, breast, non-small cell lung, and pancreatic cancers [29]. The aims of this study are to evaluate the combined effect of erlotinib with TS inhibitors or si-TS RNA transfection on the cytotoxicity in NSCLC cells, especially in squamous cell lung cancer cells, and to evaluate the molecular mechanism of this cytotoxic effect, including the influence on the ERK1/2 and PI3K/AKT signaling pathways and on the DNA repair proteins, such as ERCC1 and Rad51, We hope that this study can provide the new therapeutic direction to overcome the drue resistance of squamous cell lung cancer to the small-molecule tyrosine kinase inhibitor, especially erlotinib, in order to prolong the survival of patients with squamous cell lung cancer.

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