Mika Uchiyama

Krüppel-Like Factor 6 Is Frequently Down-Regulated and Induces Apoptosis in Non-Small Cell Lung Cancer Cells

Krüppel-like factor 6 (KLF6) is a ubiquitously expressed zinc finger transcriptional factor, which has been suggested to be a candidate tumor suppressor gene in prostate cancer and astrocytic glioma. Because KLF6 is located at chromosome 10p15, where non-small cell lung cancers (NSCLCs) also exhibit frequent allelic loss, we hypothesized that the inactivation of KLF6 is also involved in the development of NSCLC. To determine this, we performed mutational analysis for 105 NSCLCs, including 9 cell lines and 96 primary tumors, and Northern blot analysis for 74 NSCLCs, including the 9 cell lines and 65 primary tumors. Although somatic mutations were not detected in the coding sequence of KLF6, expression of KLF6 mRNA was down-regulated in the 9 cell lines and in 55 (85%) of the 65 primary tumors compared with normal lung tissue. Treatment of two cell lines expressing KLF6 at low levels with 5-azacytidine did not induce KLF6 expression, suggesting that KLF6 down-regulation is not due to promoter hypermethylation. We also performed loss of heterozygosity (LOH) analysis using the laser capture microdissection technique, and found that 21 of 62 (34%) informative samples had LOH in the KLF6 gene locus. Comparing the LOH status with mRNA expression of KLF6, we found that 14 of the 14 (100%) samples with LOH showed KLF6 down-regulation, and that even 23 of 31 (74%) samples without LOH also showed this down-regulation. We also studied the expression of the WAF1 gene, a possible downstream gene of KLF6, and detected simultaneous down-regulation of WAF1 and KLF6 mRNA in 6 of 9 (67%) cell lines and 48 of the 55 (87%) primary tumors, although there was not a significant association between loss of KLF6 and WAF1 expression. Furthermore, colony formation assay of two NSCLC cell lines (NCI-H1299 and NCI-H2009) induced a markedly reduced colony formation by KLF6 transfection, and Annexin V staining and terminal deoxynucleotidyl transferase-mediated nick end labeling assays revealed that KLF6 induced apoptosis. Our present studies demonstrated that KLF6 is frequently down-regulated in NSCLC and suppresses tumor growth via induction of apoptosis in NSCLC, which may suggest that KLF6 is a tumor suppressor for NSCLC.

Loss of heterozygosity of chromosome 12p does not correlate with KRAS mutation in non‐small cell lung cancer

Activating mutations of RAS gene families have been found in a variety of human malignancies, including lung cancer, suggesting their dominant role in tumorigenesis. However, several studies have shown a frequent loss of the wild‐type KRAS allele in the tumors of murine models and an inhibition of oncogenic phenotype in tumor cell lines by transfection of wild‐type RAS, indicating that wild‐type RAS may have oncosuppressive properties. To determine whether loss of wild‐type KRAS is involved in the development of human lung cancer, we investigated the mutations of KRAS, NRAS and BRAF in 154 primary non‐small cell lung cancers (NSCLCs) as well as 10 NSCLC cell lines that have been shown to have KRAS mutations. We also determined the loss of heterozygosity status of KRAS alleles in these tumors. We detected point mutations of KRAS in 11 (7%) of 154 NSCLCs, with 10 cases at codon 12 and 1 at codon 61, but no mutations of NRAS or BRAF were found. Using the laser capture microdissection technique, we confirmed that 9 of the 11 tumors and 7 of the 10 NSCLC cell lines retained the wild‐type KRAS allele. Among the cell lines with heterozygosity of mutant and wild‐type KRAS, all of the cell lines tested for expression were shown to express more mutated KRAS than wild‐type mRNA, with higher amounts of KRAS protein also being expressed compared to the cell lines with a loss of wild‐type KRAS allele. In addition, among 148 specimens available for immunohistochemical analysis, 113 (76%) showed positive staining of KRAS, indicating that the vast majority of NSCLCs continue to express wild‐type KRAS. Our findings indicate that the wild‐type KRAS allele is occasionally lost in human lung cancer, and that the oncogenic activation of mutant KRAS is more frequently associated with an overexpression of the mutant allele than with a loss of the wild‐type allele in human NSCLC development.

SURGICAL TREATMENT FOR ADRENAL METASTASIS FROM LUNG CANCER

Several long-term survivors after surgical resection for a solitary adrenal metastasis from non-small cell lung cancer (NSCLC) have been reported in case reports and case series with a small number of patients. We have experienced 6 cases of patients who had adrenalectomy (ADR) for a metastasis from NSCLC. The median survival time (MST) after ADR was 24 months, and there was only 1 case of 3-year survivor. To elucidate the surgical indication and the prognostic factors of patients with a solitary adrenal metastasis from NSCLC, we analyzed 104 patients including our 6 patients who had ADR for a metastasis from NSCLC. The MST after ADR and 5-year survival were 24 months and 31%, respectively. Univariate and multivariate analysis demonstrated that lymph node metastasis at the surgery for primary lung cancer was the only significant and independent predictor of poor survival in patients after ADR. The results suggest that aggressive surgical treatment of a solitary adrenal metastasis from NSCLC may be effective when a patient have N0 disease.