Naoki Okumura

PIK3CA Mutations and Copy Number Gains in Human Lung Cancers

We investigated the frequency and function of mutations and increased copy number of the PIK3CA gene in lung cancers. PIK3CA mutations are one of the most common gene changes present in human cancers. We analyzed the mutational status of exons 9 and 20 and gene copy number of PIK3CA using 86 non-small cell lung cancer (NSCLC) cell lines, 43 small cell lung cancer (SCLC) cell lines, 3 extrapulmonary small cell cancer (ExPuSC) cell lines, and 691 resected NSCLC tumors and studied the relationship between PIK3CA alterations and mutational status of epidermal growth factor receptor (EGFR) signaling pathway genes (EGFR, KRAS, HER2, and BRAF). We also determined PIK3CA expression and activity and correlated the findings with effects on cell growth. We identified mutations in 4.7% of NSCLC cell lines and 1.6% of tumors of all major histologic types. Mutations in cell lines of small cell origin were limited to two ExPuSC cell lines. PIK3CA copy number gains were more frequent in squamous cell carcinoma (33.1%) than in adenocarcinoma (6.2%) or SCLC lines (4.7%). Mutational status of PIK3CA was not mutually exclusive to EGFR or KRAS. PIK3CA alterations were associated with increased phosphatidylinositol 3-kinase activity and phosphorylated Akt expression. RNA interference-mediated knockdown of PIK3CA inhibited colony formation of cell lines with PIK3CA mutations or gains but was not effective in PIK3CA wild-type cells. PIK3CA mutations or gains are present in a subset of lung cancers and are of functional importance.

Oncogene Mutations, Copy Number Gains and Mutant Allele Specific Imbalance (MASI) Frequently Occur Together in Tumor Cells

Background Activating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes. Methodology/Principal Findings We determined 1) mutational status, 2) copy number gains (CNGs) and 3) relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20%) in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1) MASI with CNG, either complete or partial; and 2) MASI without CNG (uniparental disomy; UPD), due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75%) and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%), was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival. Conclusions MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.

Cytoglobin, the newest member of the globin family, functions as a tumor suppressor gene.

Cytoglobin (CYGB) is a recently discovered vertebrate globin distantly related to myoglobin with unknown function. CYGB is assigned to chromosomal region 17q25, which is frequently lost in multiple malignancies. Previous studies failed to detect evidence for mutations in the CYGB gene. Recent studies provided preliminary evidence for increased methylation of the gene in lung cancer. Our study was aimed at investigating the role of CYGB as a tumor suppressor gene. By nested methylation-specific DNA sequencing analysis of lung and breast cancer cell lines and bronchial and mammary epithelial cell lines, we identified that methylation of a 110-bp CpG-rich segment of the CYGB promoter was correlated with gene silencing. We specifically targeted this sequence and developed a quantitative methylation-specific PCR assay, suitable for high-throughput analysis. We showed that the tumor specificity of CYGB methylation in discriminating patients with and without lung cancer, using biopsies and sputum samples. We further showed the tumor specificity of this assay with multiple other epithelial and hematologic malignancies. To show tumor suppressor activity of CYGB, we performed the following: (a) RNA interference-mediated knockdown of CYGB gene on colony formation in a CYGB expression-positive lung cancer cell line, resulting in increased colony formation; (b) enforced gene expression in CYGB expression-negative lung and breast cancer cell lines, reducing colony formation; and (c) identification of potential proximate targets down-stream of the CYGB genes. Our data constitute the first direct functional evidence for CYGB, the newest member of the globin family, as a tumor suppressor gene.

Is conversion therapy possible in stage IV gastric cancer: the proposal of new biological categories of classification

Conversion therapy for gastric cancer (GC) has been the subject of much recent attention. It is defined as a surgical treatment aiming at an R0 resection after chemotherapy for tumors that were originally unresectable or marginally resectable for technical and/or oncological reasons. However, the indications for resection remain to be clarified. In the present review, we focus on the biology and heterogeneous characteristics of stage IV GC and propose new categories of classification. Stage IV GC patients can be divided based on the absence (categories 1 and 2) or presence (categories 3 and 4) of macroscopically detectable peritoneal dissemination, which has a different biological outcome compared to hematological metastasis. Category 1 is defined oncologically as stage IV but the metastasis is technically resectable. Category 2 includes a marginally resectable metastasis or patients for whom the operation would not necessarily be the best choice. Category 3 includes a potentially unresectable metastasis of peritoneal dissemination that is only macroscopically detectable. Category 4 includes noncurable metastasis with peritoneal and other organ metastasis. The indications for conversion therapy might include the patients from category 2, some patients from category 3 and a very small number of patients from category 4. The longer survival can be expected for patients corresponding to categories 1, 2 and, to a lesser extent, 3, while the treatment of other patients focuses on “care.” The provision of conversion therapy for stage IV GC patients might be one of the main roles of surgical oncologists in the near future.

The Roles of Surgical Oncologists in the New Era – Minimally Invasive Surgery for Early Gastric Cancer and Adjuvant Surgery for Metastatic Gastric Cancer

In the new era of technical development in surgery, operative devices, molecular targeting and chemotherapeutic agents, surgical oncologists have two main roles in the treatment of gastric cancer. One is to provide patients with minimally invasive surgery, including laparoscopy- or robot-assisted surgery in early gastric cancer patients, and the new concept of surgical intervention toward advanced and metastatic disease. Since recently, laparoscopy-assisted distal gastrectomy has become prevalent in Japan as a surgery which is minimally invasive for the patients and provides them with a good quality of life afterwards. However, the provision of advanced surgical techniques, including lymph node dissection and reconstruction, is more important for patient survival. The second role of surgical oncologists is to evaluate the significant values of the aggressive treatment which we term ‘adjuvant surgery’ for stage IV gastric cancer patients who have successfully responded to initial chemotherapy for curative intent. Stage IV gastric cancer patients are now being informed about the possibility of longer survival with the new chemotherapeutic and surgical strategic approach.

Estradiol Stabilizes p53 Protein in Breast Cancer Cell Line, MCF–7

Overexpression of the oncoprotein MDM2, an important regulator of the p53 tumor suppressor protein, is often observed in breast cancer tissues and cell lines, particularly in those which express estrogen receptor a (ERa). In MCF–7 breast cancer cell line possessing wild‐type p53, ERa, and overexpressing MDM2, p53 accumulation was stimulated by 17β‐estradiol (E2) in a concentrationdependent manner. On the other hand, E2 caused no change of the expression of p53 mRNA, indicating that E2 affects p53 at the post‐transcriptional level. To analyze the mechanism of p53 accumulation by E2, the stability of p53, ERa and MDM2 proteins was analyzed in the presence of cycloheximide under an E2–supplemented or‐depleted condition. E2 significantly extended the half‐life of p53 protein, but shortened that of ERa in MCF–7 cells. E2 significantly decreased the stability of p90(MDM2) and p60(MDM2) in MCF–7. Interestingly, E2 increased the ratio p60(MDM2)/p90(MDM2) inversely proportionally to the degradation of p53. These results suggest that the ratio of the two MDM2 proteins, p90(MDM2) and p60(MDM2), may affect the accumulation of wild‐type p53 protein in response to E2.

Vascular endothelial growth factor protects hepatoma cells against oxidative stress‐induced cell death

Background:  The aim of the present study was to examine coordination of the vascular endothelial growth factor (VEGF) and VEGF receptor (Flk‐1) system and to study control of VEGF expression by oxidative stress, which is considered a model for chronic liver disease.

Laparoscopic technique and safety experience with barbed suture closure for pelvic cavity after abdominoperineal resection.

BackgroundBetween April 2005 and December 2012, we performed laparoscopic colorectal resection with regional lymph node dissection on 273 cases of colorectal cancer patients. However, Laparoscopic rectal cancer surgery requires a high degree of skill. Any surgeon who is going to embark on these difficult resections should have at a minimum laparoscopic suturing skills in order to be able to close the peritoneal defect.MethodsIn laparoscopic surgery for rectal cancer, the intracorporeal suture technique required to close the pelvic cavity is very difficult. Barbed sutures have recently been proposed to facilitate laparoscopic suturing. Two patients with rectal cancer who underwent laparoscopic abdominoperineal resection (APR) with intracorporeal closure of the pelvic cavity from September to October 2012 were enrolled in this study.ResultsWe present our initial experience of two consecutive cases of intracorporeal closure of the pelvic cavity by totally laparoscopic APR. After clinical follow-up, the two patients have no complaints and have shown no signs of recurrence.ConclusionsWe hypothesized that barbed sutures could potentially improve the efficiency of intracorporeal closure of the pelvic cavity after laparoscopic APR. Further, we expect that use of the V-Loc™ will reduce intra-operative stress on the endoscopic surgeon.

Decreased FANCJ caused by 5FU contributes to the increased sensitivity to oxaliplatin in gastric cancer cells

BackgroundOxaliplatin is effective against many types of cancer, and the combination of 5-fluorouracil (5FU) and oxaliplatin is synergistically effective against gastric cancer, as well as colon cancer. The FANCJ protein is one of the Fanconi anemia (FA) gene products, and its interaction with the tumor suppressor BRCA1 is required for DNA double-strand break (DSB) repair. FANCJ also functions in interstrand crosslinks (ICLs) repair by linking to mismatch repair protein complex MLH1-PMS2 (MutLα). While oxaliplatin causes ICLs, 5FU is considered to cause DSBs. Therefore, we investigated the importance of FANCJ in the synergistic effects of oxaliplatin and 5FU in MKN45 gastric cancer cells and the derived 5FU-resistant cell line, MKN45/F2R.MethodsMKN1, TMK1, MKN45, and MKN45/F2R (5FU-resistant) gastric cancer cells were treated with 5FU and/or oxaliplatin. The signaling pathway was evaluated by a western blotting analysis and reverse transcription polymerase chain reaction (RT-PCR). Drug resistance was evaluated by the 3-(4,5-dimethyl-2-tetrazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay.ResultsIn MKN45 cells, the combination of 5FU and oxaliplatin had synergistic effects. DSBs appeared when the cells were treated with 5FU. FANCJ was down-regulated, and BRCA1 was induced in a dose- and time-dependent manner. MKN45 cells showed increased sensitivity to oxaliplatin when FANCJ was knocked down by short interfering (si) RNA. However, these findings were not observed in MKN45/F2R 5FU-resistant cells.ConclusionThese results strongly suggest that the decrease in FANCJ caused by 5FU treatment leads to an increase in sensitivity to oxaliplatin, thus indicating that the FANCJ protein plays an important role in the synergism of the combination of 5FU and oxaliplatin.

Strategy for synchronous and multiple liver metastasis.

BACKGROUND/AIMS Surgical indications for resection of synchronous metastasis from colorectal cancer (CRC) and the optimal timing of hepatectomy are still controversial and widely debated. METHODOLOGY Synchronous and multiple metastatic liver tumors were detected in 57 patients since May 2005. Our treatment policy was to perform hepatectomy if the resection could be done with no limit on size and number of tumors. However, if curative resection could not be done, chemotherapy was begun and timing for the possibility of a radical operation was planned immediately. RESULTS In 37 patients whose tumors were located only in the liver, primary tumor resection was performed in 16 patients and after tumor-decreasing by chemotherapy, in 7 patients. In 20 patients in whom chemotherapy was performed first, after controlling the distant metastasis, hepatectomy was performed in 3 patients and staged hepatectomy was performed in 10. Recurrence was detected after hepatectomy in 75.0% of simultaneous resection cases and in 70.0% of staged cases. In the recurrence cases, early detection after tumor resection occurred in 58.3% of the simultaneous and 14.2% of the staged. CONCLUSIONS The present data show that neoadjuvant chemotherapy does not increase the risk of postoperative complications or the surgical difficulties of hepatectomy for colorectal metastases.