Hiroyuki Achiwa

Determinants of sensitivity and resistance to gemcitabine: The roles of human equilibrative nucleoside transporter 1 and deoxycytidine kinase in non‐small cell lung cancer

Gemcitabine is one of the most commonly used agents for lung cancer chemotherapy, but the determinants of sensitivity and/or resistance to this agent are not yet fully understood. In this study we used quantitative RT‐PCR to examine the expression levels of human equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (dCK) genes in non‐small cell lung cancer (NSCLC) cell lines in relation to sensitivity and resistance to gemcitabine. The basal expression levels of hENT1 were significantly correlated with the IC50 values for gemcitabine (r=‐0.6769, P=0.0005), whereas dCK expression levels were not. In a highly gemcitabine‐sensitive cell line, NCI‐H23, the sensitivity to gemcitabine was inhibited by nitrobenzylmercaptopurine ribonucleoside (NBMPR), an inhibitor of hENT1, without significant modulation of hENT1 expression. These data suggest that hENT1 is associated with gemcitabine sensitivity in lung cancer. We also continuously exposed NCI‐H23 cells to gemcitabine and subsequently established the drug‐resistant clone H23/GEM‐R, which showed a significant decrease of dCK expression; however, hENT1 expression was not altered in the continuously exposed sublines or in the resistant clone. We conclude that increased hENT1 expression is a determinant of gemcitabine sensitivity, while decreased dCK expression is associated with acquired resistance to gemcitabine in NSCLC cells. Thus, hENT1 and dCK might be useful as predictive markers for efficacy of gemcitabine therapy in NSCLC.

MRP8/ABCC11 directly confers resistance to 5-fluorouracil

Multidrug-resistance–associated protein, MRP8/ABCC11 (ABCC11), is an efflux pump for nucleotide analogues and 5-fluoro-2′-deoxyuridine 5′-monophosphate (FdUMP). To test whether ABCC11 directly confers 5-fluorouracil (5-FU) resistance, we used the 5-FU–resistant subline PC-6/FU23-26 selected from PC-6 human small-cell lung cancer cells by 5-FU and found that it increases the resistance by ∼25-fold. The intracellular FdUMP accumulation was reduced in PC-6/FU23-26 cells concomitant with the overexpression of the ABCC11 gene. These findings suggest that ABCC11 confers 5-FU resistance in the sublines by enhancing the efflux for the active metabolite FdUMP. Previously, methotrexate also increased the efflux by ABCC11, and we found cross-resistance to methotrexate in PC-6/FU23-26 cells. To confirm our hypothesis, we examined whether decreasing the expression of ABCC11 in PC-6/FU23-26 cells by small interfering RNA altered the cytotoxicity to 5-FU and methotrexate and found that this enhanced 5-FU and methotrexate cytotoxicity in PC-6/FU23-26 cells. These data indicate that expression of the ABCC11 gene is induced by 5-FU, and that ABCC11 is directly involved in 5-FU resistance by the efflux transport of the active metabolite FdUMP. [Mol Cancer Ther 2007;6(1):122–7]

PRL-1 Tyrosine Phosphatase Regulates c-Src Levels, Adherence, and Invasion in Human Lung Cancer Cells

Phosphatases of regenerating liver (PRL) constitute a subfamily of the protein tyrosine phosphatases that are implicated in oncogenic and metastatic phenotypes. In this study, we evaluated the role of PRL-1 in cell proliferation and metastatic processes in human lung cancer cells. We stably transfected human A549 lung cancer cells with several short hairpin RNAs for PRL-1 and found decreased invasive activity in the resulting clones compared with control cells. In addition, cells with suppressed PRL-1 exhibited greater adherence and cell spreading on fibronectin and a decreased proliferation rate compared with control cells. To address possible mechanisms for the altered phenotypes, we examined known biochemical regulators of adhesion and invasion. Inhibition of PRL-1 decreased c-Src and p130Cas expression and Rac1 and Cdc42 activation without any apparent modification of focal adhesion kinase (FAK) expression. Total tyrosine FAK phosphorylation and Tyr397 phosphorylation levels were continuously elevated in PRL-1 knockdown cells plated on fibronectin. In immunofluorescence studies, reduction in PRL-1 seemed to decrease cell membrane protrusions with a reduction in actin fiber extensions in spite of continuous phosphorylation of Tyr397 FAK, which could reflect reduced adhesion turnover. Our data implicate PRL-1 in the fundamental process of cell adhesion and migration in human lung cancer cells by affecting Rac1, Cdc42, and c-Src activation. These results support the hypothesis that PRL-1 plays an important role in maintaining the malignant phenotype by exploiting Src activation processes, and that PRL-1 could be a promising therapeutic target for cancer metastasis and cell growth.

The determinants of sensitivity and acquired resistance to gemcitabine differ in non-small cell lung cancer: a role of ABCC5 in gemcitabine sensitivity

We examined the expression levels of the multidrug resistance protein 5 (ABCC5) gene in non–small cell lung cancer (NSCLC) cell lines to clarify the relationship with the sensitivity to gemcitabine. The expression levels of ABCC5 were inversely correlated with gemcitabine sensitivity significantly (r = 0.628; P < 0.01) in 17 NSCLC cells, whereas the expression of ABCC5 in the gemcitabine-resistant NSCLC cell line H23/GEM-R was the same as that in parental NCI-H23 cells. Treatment with the ABCC5 inhibitor zaprinast altered the sensitivity to gemcitabine in ABCC5-expressing NSCLC cells. In addition, decreasing the expression of ABCC5 by small interfering RNA altered the cytotoxicity to gemcitabine. These results indicate that modulation of ABCC5 activity could be used to increase the gemcitabine sensitivity in NSCLC. Previously, we found a decreased expression of deoxycytidine kinase in H23/GEM-R cells, and further investigation in this study showed an increased expression of ribonucleotide reductase subunit 1 in H23/GEM-R cells. We therefore also examined the effect of modifying the expression of both genes on gemcitabine resistance. We found that using small interfering RNA to decrease the expression of ribonucleotide reductase subunit 1 resulted in a decreased resistance to gemcitabine in H23/GEM-R cells. Furthermore, pretreatment with pemetrexed resulted in an increased deoxycytidine kinase expression concomitant with the alteration of the resistance to gemcitabine in H23/GEM-R cells. The determinants for sensitivity and the acquired resistance in gemcitabine are quite different; nonetheless, modification of these factors may increase the efficacy of gemcitabine in the treatment of NSCLC. [Mol Cancer Ther 2006;5(7):1800–6]

LKB1 gene mutations in Japanese lung cancer patients

Mutation of the LKB1 gene (also known as STK11) is regarded as a cause of Peutz–Jeghers syndrome. In Caucasian patients, LKB1 somatic mutations occur in approximately one‐third of lung adenocarcinomas. The aim of the present study was to examine the LKB1 gene in Japanese patients with lung cancer and to evaluate its clinical and pathological implications. We sequenced the LKB1 gene in 22 lung cancer cell lines and 100 Japanese patients with lung cancer (including 81 adenocarcinomas, 14 squamous cell carcinomas and five other histological types) who had undergone curative pulmonary resection. We also determined expression levels of the LKB1 gene by quantitative real‐time reverse transcription–polymerase chain reaction and correlated these results with the clinical and pathological features of patients. Among the 22 cell lines, four had mutations and three of these were in adenocarcinoma cells. Of 100 primary lung cancers, only three had LKB1 gene mutations (3%). All of them were male smokers with adenocarcinomas. Hence, when confined to this subset of patients, the mutation frequency was 9% (3/33). No significant correlation was observed between the expression level of LKB1 and patient clinicopathological features. In conclusion, LKB1 gene mutations were relatively rare in Japanese patients with lung cancer compared with Caucasian patients. LKB1 gene mutations appear to be frequent in male, smoking patients of Caucasian origin, in contrast to EGFR or HER2 mutations that are frequent in non‐smoking, female patients of Asian origin. (Cancer Sci 2007; 98: 1747–1751)

Role of ABCG2 as a biomarker for predicting resistance to CPT-11/SN-38 in lung cancer

To examine the mechanism of resistance to 7‐ethyl‐10‐hydroxycamptothecin (SN‐38) in lung cancer, we continuously exposed the non‐small‐cell lung cancer (NSCLC) cell line NCI‐H23 to SN‐38 and selected the SN‐38‐resistant clone H23/SN‐38. After 2 months of culturing in SN‐38‐free conditions, H23/SN‐38 cells recovered their sensitivity to SN‐38 and were subsequently established as the revertant H23/SN‐38REV cell line. Because H23/SN‐38 cells show cross resistance to certain anticancer drugs, such as topotecan, etoposide, doxorubicin and mitoxantrone, we examined the gene and protein expression levels of drug efflux transporters of the ATP‐binding cassette (ABC) family. We found that both gene and protein expression of ABCG2/BCRP (ABCG2) in H23/SN‐38 cells was increased compared with that in NCI‐H23 cells and H23/SN‐38REV cells. The cellular accumulation of topotecan in H23/SN‐38 cells was decreased compared with that in NCI‐H23 and H23/SN‐38REV cells, and treatment with reserpine (an inhibitor of ABCG2) increased the cellular accumulation of topotecan in H23/SN‐38 cells. Furthermore, treatment with reserpine also altered the sensitivity of H23/SN‐38 cells to SN‐38. These results indicate that the upregulation of ABCG2 was functional, and related to the resistance of H23/SN‐38 cells to SN‐38. Moreover, we found that gene expression levels of ABCG2 were significantly correlated with the concentration of SN‐38 for 50% cell survival in 13 NSCLC cells (r = 0.592, P < 0.05). The present results indicate that the induction of ABCG2 by SN‐38 does confer acquired resistance to CPT‐11/SN‐38, but the induction of ABCG2 and subsequent drug resistance are reversible. However, the expression level of ABCG2 may be a useful indicator of CPT‐11/SN‐38 activity in lung cancer. (Cancer Sci 2006; 97: 192–198)

Efficacy of docetaxel as a second-line chemotherapy for thymic carcinoma.

Thymic carcinoma is a rare and aggressive tumor, and the efficacy of second-line chemotherapy is still unclear. Here, we reported a case of thymic carcinoma that responded well to the administration of docetaxel alone as a second-line chemotherapy. A 64-year-old woman was diagnosed with thymic carcinoma (squamous cell type) with bone metastasis, and she, therefore, received nedaplatin combined with etoposide and ifosfamide. She responded partially, after which she received irradiation for bone metastasis. Two months after chemotherapy, the thymic carcinoma exhibited gradual regrowth and she experienced shoulder pain. We treated this with docetaxel alone (60 mg/m2 every 4 weeks). After three courses of docetaxel, we observed a partial response and her shoulder pain disappeared. This case demonstrated that docetaxel is effective as a second-line chemotherapy for thymic carcinoma.

Analysis of β-tubulin gene alteration in human lung cancer cell lines

We examined lung cancer cell lines for the presence of β-tubulin gene alteration based on a previous report of a relationship between frequent β-tubulin gene mutation in non-small-cell lung cancer and clinical response to taxanes as well as the prognosis. The mutation was defined by analyzing genomic DNA from 31 lung cancer cell lines by direct genomic sequencing using specific primers for the β-tubulin class I gene. We detected only three genetic alterations at nonsplice sites in two introns, and a silent genetic alteration at codon 217 in exon 4. The mutation of the β-tubulin gene was rare; moreover, these genetic alterations were predicted to evoke no biological alteration of the cancer cells. Our data suggest that the β-tubulin gene mutation does not play a major role in the genetic mechanism of resistance to taxanes. In addition, the presence of a closely related family of β-tubulins or pseudogenes was thought to hinder accurate evaluation of the β-tubulin gene.

p185HER-2/neu and p21CIP1/WAF1 Expression in Primary Tumors and Lymph Node Metastases in Non-small Cell Lung Cancer

p185HER‐2/neu, a tyrosine kinase receptor, is one of the target molecules for cancer therapy, and its expression may reduce the sensitivity of tumor cells to anti‐cancer drugs. p21CIP1/WAF1 is a cyclindependent kinase inhibitor, and its expression may also be involved in chemoresistance. Non‐small cell lung cancer (NSCLC) is a potentially systemic disease, and systemic therapies play an important role in its treatment. However, there have been no studies comparing the expression of these molecules between primary and metastatic tumors. We investigated the expression of p185HER‐2/neu and p21CIP1/WAF1 in 57 paired samples of primary NSCLC tumors and corresponding lymph node metastases by immunohistochemistry. Expression of each of p185HER‐2/neu and p21CIP1/WAF1 was highly correlated between primary tumors and lymph node metastases, and similar correlations were also obtained when adenocarcinoma and squamous cell carcinoma cases were analyzed individually. However we failed to detect any correlation between p185HER‐2/neu and p21CIP1/WAF1 expression. Our results suggested that expression of both p185HER‐2/neu and p21CIP1/WAF1 is concordant between primary and metastatic tumors.