Yuriko Saiki

LUNG CANCER ASSOCIATED WITH USUAL INTERSTITIAL PNEUMONIA

There were 83 cases of usual interstitial pneumonia (UIP) in 3712 consecutive autopsy cases during 1972 to 1992 in Toranomon Hospital. Primary lung cancer had arisen in 40 cases of UIP in that period. The prevalence of lung cancer (48.2%) in UIP was significantly higher than that of lung cancers (9.1%) in the age‐matched general population without UIP (P < 0.001). The prevalence of association of multiple lung cancer in UIP (20.W0) was also significantly increased. Thus, UIP showed a remarkable potency to develop lung cancers. The lung cancer cases in UIP had obvious smoking habits. Both the rates of smokers and the quantity of smoking were significantly increased in the lung cancer cases in UIP (P < 0.05). There was a distinct anatomical distribution of lung cancer in UIP. Most cancers in UIP (98%) arose in the peripheral area of the lung (P < 0.001, compared to lung cancer cases without UIP) with close relation to the honeycombing lesion. Studies on surgical specimens with small cancers showed that most tumors in UIP arose in the border area between honeycombing and the non‐fibrotic area. Thus, the front of the remodeling of the lung is suspected to be a potential field of developing lung cancer. The chronic inflammatory process resulting in the remodeling of the lung may play an important part in the development of lung cancer in UIP under the circumstance of heavy smoking.

DCK is frequently inactivated in acquired gemcitabine-resistant human cancer cells

Although gemcitabine is the most effective chemotherapeutic agent against pancreatic cancer, a growing concern is that a substantial number of patients acquire gemcitabine chemoresistance. To elucidate the mechanisms of acquisition of gemcitabine resistance, we developed gemcitabine-resistant cell lines from six human cancer cell lines; three pancreatic, one gastric, one colon, and one bile duct cancer. We first analyzed gemcitabine uptake using three paired parental and gemcitabine resistant pancreatic cancer cell lines (PK-1 and RPK-1, PK-9 and RPK-9, PK-59 and RPK-59) and found that uptake of gemcitabine was rapid. However, no DNA damage was induced in resistant cells. We further examined the microarray-based expression profiles of the cells to identify genes associated with gemcitabine resistance and found a remarkable reduction in the expression of deoxycytidine kinase (DCK). DCK is a key enzyme that activates gemcitabine by phosphorylation. Genetic alterations and expression of DCK were studied in these paired parental and derived gemcitabine-resistant cell lines, and inactivating mutations were found only in gemcitabine-resistant cell lines. Furthermore, siRNA-mediated knockdown of DCK in the parental cell lines yielded gemcitabine resistance, and introduction of DCK into gemcitabine-resistant cell lines invariably restored gemcitabine sensitivities. Mutation analyses were expanded to three other different paired cell lines, DLD-1 and RDLD-1 (colon cancer cell line), MKN-28 and RMKN-28 (gastric cancer cell line), and TFK-1 and RTFK -1 (cholangiocarcinoma cell line). We found inactivating mutations in RDLD-1 and RTFK-1 and decreased expression of DCK in RMKN-28. These results indicate that the inactivation of DCK is one of the crucial mechanisms in acquisition of gemcitabine resistance.

S100A4, frequently overexpressed in various human cancers, accelerates cell motility in pancreatic cancer cells

S100A4, a member of the Ca(2+) dependent S100 protein family, is reported to associate with metastasis through regulation of the motility and invasiveness of cancer cells. A high level of S100A4 protein has been reported in a variety of cancers, including pancreatic cancer. However, its biological role in pancreatic carcinogenesis is largely unknown. We previously reported that S100A4 is frequently overexpressed and that RNAi-mediated knockdown induces apoptosis and suppression of cell growth, motility, and invasiveness. In this study, we analyzed the effects of forced expression of S100A4 in pancreatic cancer cell lines without S100A4-upregulation. We used two cell lines without upregulation of S100A4 (PCI-35 and PCI-43) as well as two cell lines with highly upregulated S100A4 as the control (MIA PaCa-2 and PAN-07-JCK). Cells did not show acceleration of their growth and invasiveness after forced expression of S100A4, but remarkable acceleration of cell motility was observed only in PCI-35 and PCI-43. We further performed microarray analyses using PCI-35 and PCI-43 with and without forced expression of S100A4 and identified 72 and 18 genes that were 2-fold or more upregulated or downregulated, respectively, in both cell lines after forced expression of S100A4. Our results suggest that S100A4 is crucial for cell motility in pancreatic cancer and that some downstream genes may play important roles in cell motility.

S100A4 is frequently overexpressed in lung cancer cells and promotes cell growth and cell motility.

S100A4, a small calcium-binding protein belonging to the S100 protein family, is commonly overexpressed in a variety of tumor types and is widely accepted to associate with metastasis by regulating the motility and invasiveness of cancer cells. However, its biological role in lung carcinogenesis is largely unknown. In this study, we found that S100A4 was frequently overexpressed in lung cancer cells, irrespective of histological subtype. Then we performed knockdown and forced expression of S100A4 in lung cancer cell lines and found that specific knockdown of S100A4 effectively suppressed cell proliferation only in lung cancer cells with S100A4-overexpression; forced expression of S100A4 accelerated cell motility only in S100A4 low-expressing lung cancer cells. PRDM2 and VASH1, identified as novel upregulated genes by microarray after specific knockdown of S100A4 in pancreatic cancer, were also analyzed, and we found that PRDM2 was significantly upregulated after S100A4-knockdown in one of two analyzed S100A4-overexpressing lung cancer cells. Our present results suggest that S100A4 plays an important role in lung carcinogenesis by means of cell proliferation and motility by a pathway similar to that in pancreatic cancer.

The Expression of S100A4 in Human Pancreatic Cancer Is Associated With Invasion

Objectives Pancreatic cancer is one of the most lethal malignancies; its poor prognosis is strongly associated with invasion and metastasis. Expression of S100A4 has been reported to correlate with poor prognosis in various cancers. We have investigated the role of S100A4 in pancreatic cancer tumorigenesis and its clinicopathologic significance. Methods Protein expression of S100A4 was examined by Western blot in pancreatic cancer cell lines and a human pancreatic ductal epithelium cell line, HPDE-6. Then the expressions of S100A4, TP53, and CD133 were examined immunohistochemically in resected specimens from 83 patients with pancreatic cancer to clarify their clinicopathologic significance. Survival analyses were performed using the Kaplan-Meier method and the Mantel-Cox method. Results Forty-eight (58%) of 83 patients with pancreatic cancer positively expressed S100A4, and 50 (60%) and 29 (36%) patients positively expressed TP53 and CD133, respectively. S100A4 expression was significantly correlated with perineural invasion (P = 0.029) and invasion pattern (P = 0.001). Neither TP53 nor CD133 expression showed significant correlations with any other parameters. Conclusions Our present results suggest that S100A4 plays an important role in the invasiveness, particularly with perineural invasion and invasion pattern, of pancreatic cancer. Development of new strategies targeting S100A4 or its downstream effectors is warranted.

Molecular pathology of pancreatic cancer

By genomic and epigenomic screening techniques, substantial progress has been made in our understanding of pancreatic cancer. The comprehensive studies of the pancreatic cancer genome have revealed that most genetic alterations are identified to be associated with specific core signaling pathways including high‐frequency mutated genes such as KRAS, CDKN2A, TP53, and SMAD4 along with several low‐frequency mutated genes. Three types of histological precursors of pancreatic cancer: pancreatic intraepithelial neoplasia, mucinous cystic neoplasm, and intraductal papillary mucinous neoplasm, had been recognized by morphological studies and the recent genomic screening techniques revealed that each of these precursor lesions were associated with specific molecular alterations. In the familial pancreatic cancer cases, several responsible genes were discovered. Epigenetic changes also play an important role in the progression of pancreatic cancer. Several tumor suppressor genes were silenced due to aberrant promoter CpG island hypermethylation. Several genetically engineered mouse models, based on the Kras mutation, were created, and provided reliable tools to identify the key molecules responsible for the development or progression of pancreatic cancer.

Single-dose rosuvastatin ameliorates lung ischemia-reperfusion injury via upregulation of endothelial nitric oxide synthase and inhibition of macrophage infiltration in rats with pulmonary hypertension.

OBJECTIVE Lung ischemia-reperfusion (IR) injury during cardiopulmonary surgery is associated with postoperative morbidity and mortality, particularly in patients with pulmonary hypertension (PH). Using a rat model for monocrotaline-induced PH, we investigated the protective effect of rosuvastatin against IR injury in lungs affected by PH and attempted to elucidate its mechanism of action. METHODS Male Sprague-Dawley monocrotaline-treated rats were divided into 4 groups (n = 8-9): sham, control + IR, statin + IR, and statin + mevalonolactone + IR. Lung ischemia was induced by left pulmonary artery occlusion (1 hour), followed by reperfusion (4 hours). Rosuvastatin (2 mg/kg) was injected 18 hours before reperfusion and mevalonolactone (1 mg/kg) was injected immediately before reperfusion. The arterial oxygen tension/inspired oxygen fraction ratio was used as a measure of lung oxygenation. Left lung tissue was analyzed for the wet-to-dry lung weight ratio and protein expression of endothelial nitric oxide synthase (eNOS) and phospho-eNOS. Macrophage recruitment was assessed by CD68 immunostaining. RESULTS Our results showed that rosuvastatin decreased IR lung injury (control + IR vs statin + IR) in terms of the arterial oxygen tension/inspired oxygen fraction ratio (272 ± 43 vs 442 ± 13), wet-to-dry ratio (5.7 ± 0.7 vs 4.8 ± 0.6), and macrophage infiltration (8.0 ± 0.6/field vs 4.0 ± 0.5/field) (P < .05 for all). eNOS and phospho-eNOS were downregulated by IR, which was blocked by rosuvastatin. Effects of rosuvastatin were blunted by mevalonolactone. CONCLUSIONS Single-dose rosuvastatin decreased IR injury in lungs affected by PH via 2 anti-inflammatory mechanisms: preserving eNOS function and inhibiting macrophage infiltration.

miR-34a is downregulated in cis-diamminedichloroplatinum treated sinonasal squamous cell carcinoma patients with poor prognosis.

For the purpose of analyzing mechanisms related to the cis‐diamminedichloroplatinum resistance in head and neck squamous cell carcinoma, we analyzed RPMI2650 and its derived previously established cis‐diamminedichloroplatinum resistant cell line RPMI2650CR. To identify resistant phenotype‐related microRNAs, we compared microRNA expressions between RPMI2650CR and RPMI2650 by microarray. One of the microRNAs as downregulated, miR‐34a, was further investigated. Decreased expression of miR‐34a in RPMI2650CR was confirmed by quantitative reverse transcription‐polymerase chain reaction, but introduction of the miR‐34a precursor into RPMI2650CR or the inhibitor of miR‐34a into RPMI2650 did not change cis‐diamminedichloroplatinum sensitivities. However, 24 patients with sinonasal squamous cell carcinomas treated with intra‐arterial infusion of cis‐diamminedichloroplatinum showed a significant association between decreased expression of miR‐34a and poor disease specific survival (P = 0.0015), poor disease free survival (P = 0.0019), and poor local control rates (P = 0.017) (median follow‐up period: 53 months). Furthermore, multivariate analyses demonstrated significant associations between miR‐34a expression and the hazard ratios of disease free survival at 0.005 (95% confidence interval [CI] 0.00–0.29, P = 0.011) and local control rate at 0.008 (95% CI 0.00–0.44, P = 0.019), although other parameters such as age, gender, treatment method, T and N stages did not show any similar association. These results strongly suggest that miR‐34a expression can be an independent prognostic biomarker in patients with sinonasal squamous cell carcinoma who are undergoing treatment with cis‐diamminedichloroplatinum.

A Human Head and Neck Squamous Cell Carcinoma Cell Line with Acquired cis-Diamminedichloroplatinum-Resistance Shows Remarkable Upregulation of BRCA1 and Hypersensitivity to Taxane

Recently, an inverse relationship between resistance to platinum-based chemotherapeutic agents and taxanes has been implicated in breast and ovarian cancers, and a possible pivotal role for BRCA1 has also been suggested. Because cis-diamminedichloroplatinum (CDDP) and taxanes are the most active antitumor agents against head and neck squamous cell carcinoma (HNSCC), we analyzed the sensitivity of nine HNSCC cell lines and their previously established derived CDDP-resistant cell lines to two representative taxanes: docetaxel and paclitaxel. None of the nine original cell lines showed any cross resistance between CDDP and taxanes, but one of the CDDP-resistant cell lines, RPMI2650CR, demonstrated hypersensitivity to both taxanes when compared to the parental cell line, RPMI2650. Furthermore, RPMI2650CR exhibited increased expression of BRCA1. These data suggest that (i) taxanes are a good candidate for a second-line therapeutic drug for HNSCC patients with acquired CDDP resistance and (ii) BRCA1 can be a candidate marker for predicting an inverse CDDP/taxane sensitivity phenotype in HNSCC.

Acquisition of chemoresistance to gemcitabine is induced by a loss-of-function missense mutation of DCK.

The anti-tumor activity of gemcitabine (GEM) has been clinically proven in several solid tumors, including pancreatic cancer, biliary tract cancer, urinary bladder cancer, and non-small cell lung cancer. However, problems remain with issues such as acquisition of chemoresistance against GEM. GEM is activated after phosphorylation by deoxycytidine kinase (DCK) inside of the cell; thus, DCK inactivation is one of the important mechanisms for acquisition of GEM resistance. We previously investigated the DCK gene in multiple GEM resistant cancer cell lines and identified frequent inactivating mutations. In this study, we identified two crucial genetic alteration in DCK. (1) A total deletion of DCK in RTGBC1-TKB, an acquired GEM resistant cell line derived from a gall bladder cancer cell line TGBC1-TKB. (2) An E197K missense alteration of DCK in MKN28, a gastric cancer cell line; its acquired GEM resistant cancer cell line, RMKN28, showed a loss of the normal E197 allele. We introduced either normal DCK or altered DCK_E197K into RMKN28 and proved that only the introduction of normal DCK restored GEM sensitivity. Furthermore, we analyzed 104 healthy volunteers and found that none of them carried the same base substitution observed in MKN28. These results strongly suggest that (1) the E197K alteration in DCK causes inactivation of DCK, and that (2) loss of the normal E197 allele is the crucial mechanism in acquisition of GEM resistance in RMKN28.