Takaaki Kobayashi

Validation of the histone methyltransferase EZH2 as a therapeutic target for various types of human cancer and as a prognostic marker

The emphasis in anticancer drug discovery has always been on finding a drug with great antitumor potential but few side‐effects. This can be achieved if the drug is specific for a molecular site found only in tumor cells. Here, we find the enhancer of zeste homolog 2 (EZH2) to be highly overexpressed in lung and other cancers, and show that EZH2 is integral to proliferation in cancer cells. Quantitative real‐time PCR analysis revealed higher expression of EZH2 in clinical bladder cancer tissues than in corresponding non‐neoplastic tissues (P < 0.0001), and we confirmed that a wide range of cancers also overexpress EZH2, using cDNA microarray analysis. Immunohistochemical analysis showed positive staining for EZH2 in 14 of 29 cases of bladder cancer, 135 of 292 cases of non‐small‐cell lung cancer (NSCLC), and 214 of 245 cases of colorectal cancer, whereas no significant staining was observed in various normal tissues. We found elevated expression of EZH2 to be associated with poor prognosis for patients with NSCLC (P = 0.0239). In lung and bladder cancer cells overexpressing EZH2, suppression of EZH2 using specific siRNAs inhibited incorporation of BrdU and resulted in significant suppression of cell growth, even though no significant effect was observed in the normal cell strain CCD‐18Co, which has undetectable EZH2. Because EZH2 expression was scarcely detectable in all normal tissues we examined, EZH2 shows promise as a tumor‐specific therapeutic target. Furthermore, as elevated levels of EZH2 are associated with poor prognosis of patients with NSCLC, its overexpression in resected specimens could prove a useful molecular marker, indicating the necessity for a more extensive follow‐up in some lung cancer patients after surgical treatment. (Cancer Sci 2011; 102: 1298–1305)

Histone Lysine Methyltransferase SETD8 Promotes Carcinogenesis by Deregulating PCNA Expression

Although the physiologic significance of lysine methylation of histones is well known, whether lysine methylation plays a role in the regulation of nonhistone proteins has not yet been examined. The histone lysine methyltransferase SETD8 is overexpressed in various types of cancer and seems to play a crucial role in S-phase progression. Here, we show that SETD8 regulates the function of proliferating cell nuclear antigen (PCNA) protein through lysine methylation. We found that SETD8 methylated PCNA on lysine 248, and either depletion of SETD8 or substitution of lysine 248 destabilized PCNA expression. Mechanistically, lysine methylation significantly enhanced the interaction between PCNA and the flap endonuclease FEN1. Loss of PCNA methylation retarded the maturation of Okazaki fragments, slowed DNA replication, and induced DNA damage, and cells expressing a methylation-inactive PCNA mutant were more susceptible to DNA damage. An increase of methylated PCNA was found in cancer cells, and the expression levels of SETD8 and PCNA were correlated in cancer tissue samples. Together, our findings reveal a function for lysine methylation on a nonhistone protein and suggest that aberrant lysine methylation of PCNA may play a role in human carcinogenesis.

Deregulation of the histone demethylase JMJD2A is involved in human carcinogenesis through regulation of the G1/S transition

Although a number of JmjC-containing histone demethylases have been identified and biochemically characterized, pathological roles of their dysfunction in human disease such as cancer have not been well elucidated. Here, we report the Jumonji domain containing 2A (JMJD2A) is integral to proliferation of cancer cells. Quantitative real-time PCR analysis revealed higher expression of JMJD2A in clinical bladder cancer tissues than in corresponding non-neoplastic tissues (P<0.0001). Immunohistochemical analysis also showed positive staining for JMJD2A in 288 out of 403 lung cancer cases, whereas no staining was observed in lung normal tissues. Suppression of JMJD2A expression in lung and bladder cancer cells overexpressing this gene, using specific siRNAs, inhibited incorporation of BrdU and resulted in significant suppression of cell growth. Furthermore, JMJD2A appears to directly transactivate the expression of some tumor associated proteins including ADAM12 through the regulation of histone H3K9 methylation. As expression levels of JMJD2A are low in normal tissues, it may be feasible to develop specific inhibitors targeting the enzyme as anti-tumor agents which should have a minimal risk of adverse reaction.

Modified Glasgow prognostic score in patients with incurable stage IV colorectal cancer

BACKGROUND The modified Glasgow prognostic score is an inflammation-based prognostic score. This study examined whether this score, measured before surgical procedures, could predict postoperative cancer-specific survival. METHODS We retrospectively studied 79 colorectal cancer patients who underwent a surgical procedure for incurable stage IV disease. The modified Glasgow prognostic score (0 to 2) comprises C-reactive protein (≤10 vs >10 mg/L) and albumin (<35 vs ≥35 g/L) measurements. RESULTS In terms of overall survival, univariate analysis revealed significant differences in the status of lung metastasis, peritoneal dissemination, distant metastasis, hemoglobin, C-reactive protein, albumin, tumor resection, adjuvant chemotherapy, and modified Glasgow prognostic score. Multivariate analysis revealed that hemoglobin (P = .019), adjuvant chemotherapy (P = .002), and modified Glasgow prognostic score (0 and 1, low; 2, high) (P = .0001) were significant predictive factors for postoperative mortality. CONCLUSIONS The modified Glasgow prognostic score is simple to obtain and useful in predicting survival in incurable stage IV colorectal cancer patients undergoing surgery.

Efficacy of Gum Chewing on Bowel Movement After Open Colectomy for Left-Sided Colorectal Cancer: A Randomized Clinical Trial.

BACKGROUND: Prolonged intestinal paralysis can be a problem after gastrointestinal surgery. Several systematic reviews and meta-analyses have suggested the efficacy of gum chewing for the prevention of postoperative ileus. OBJECTIVE: The purpose of this study was to examine the efficacy of gum chewing for the recovery of bowel function after surgery for left-sided colorectal cancer and to determine the physiological mechanism underlying the effect of gum chewing on bowel function. DESIGN: This was a single-center, placebo-controlled, parallel-group, prospective randomized trial. SETTINGS: The study was conducted at a general hospital in Japan. PATIENTS: Forty-eight patients with left-sided colorectal cancer were included. INTERVENTIONS: The patients were randomly assigned to a gum group (N = 25) and a control group (N = 23). Four patients in the gum group and 1 in the control group were subsequently excluded because of difficulties in continuing the trial, resulting in the analysis of 21 and 22 patients in the respective groups. Patients in the gum group chewed commercial gum 3 times a day for ≥5 minutes each time from postoperative day 1 to the first day of food intake. MAIN OUTCOME MEASURES: The time to first flatus and first bowel movement after the operation were recorded, and the colonic transit time was measured. Gut hormones (gastrin, des-acyl ghrelin, motilin, and serotonin) were measured preoperatively, perioperatively, and on postoperative days 1, 3, 5, 7, and 10. RESULTS: Gum chewing did not significantly shorten the time to the first flatus (53 ± 2 vs 49 ± 26 hours; p = 0.481; gum vs control group), time to first bowel movement (94 ± 44 vs 109 ± 34 hours; p = 0.234), or the colonic transit time (88 ± 28 vs 88 ± 21 hours; p = 0.968). However, gum chewing significantly increased the serum levels of des-acyl ghrelin and gastrin. LIMITATIONS: The main limitation was a greater rate of complications than anticipated, which limited the significance of the findings. CONCLUSIONS: Gum chewing changed the serum levels of des-acyl ghrelin and gastrin, but we were unable to demonstrate an effect on the recovery of bowel function.

Hemoptysis and Acute Respiratory Syndrome (ARDS) as Delayed-Type Hypersensitivity After FOLFOX4 Plus Bevacizumab Treatment

As there have been many multidrug regimens introduced in colorectal cancer treatment, hypersensitivity is more often encountered than in the past. Though most allergic adverse events of oxaliplatin are mainly classified as type I reaction, a limited number of case reports of type IV reaction (delayed-type hypersensitivity) have been reported. A 73-year-old man was hospitalized for receiving the third cycle of FOLFOX4 plus bevacizumab. Forty-two hours after administration, he had dyspnea and hemoptysis. Acute respiratory distress syndrome was suspected, and the patient underwent mechanical ventilation and steroid pulse therapy. Delayed-type hypersensitivity is induced by induction of inflammation via IL-1, TNF-α and IL-6. The serum level of IL-6 in patients with advanced colorectal cancers is usually greater than the normal range. Therefore, delayed-type hypersensitivity may be easily induced in those patients. We should pay special attention to delayed-type hypersensitivity in advanced colorectal cancer patients undergoing FOLFOX treatment.

Neurophysiologic Investigation of Anal Function following Double Stapling Anastomosis

Background and Aim: Manual dilatation of the anal sphincter and transanal introduction of the circular stapling device are required for intraluminal stapling anastomosis. This procedure has been regarded as one of the causes of postoperative evacuatory disorder in low anterior resection. However, there has been no evidence of this matter. Therefore, we conducted this study to clarify the impact of the procedure of stapling anastomosis on postoperative anal function. Methods: Twenty-five cases with sigmoid colon cancer underwent potentially curative sigmoid colectomy with stapling anastomosis (ST group) and 20 cases with hand-sewn anastomosis (non-ST group). The patients were questioned regarding the daily frequency of bowel movement, the presence of urgency and soiling, and Wexner’s incontinence score. Anorectal manomatry and pudendal nerve terminal motor latency were also evaluated. The patients’ questionnaire and physiologic examinations were prospectively obtained before, and 1 and 6 months after the operation. Results: Postoperative bowel habit was graded as satisfied in 92% (23/25 patients) in the ST group and 90% (18/20 patients) in the non-ST group. There was no significant difference between the 2 groups in terms of presence of fecal incontinence, discrimination of gas and stool, and daily frequency of bowel movement. In anal manometry, there was no significant difference between the 2 groups regarding the resting and squeezing anal canal sphincter pressures at 1 and 6 months postoperatively. Pudendal nerve terminal motor latency showed their latency from 2.0 to 2.5 ms throughout the periods, and there was no difference between the 2 groups before, and 1 and 6 months after the operation. Conclusion: Stapling anastomosis does not affect anal function in the early postoperative period.

Rationale of pelvic autonomic nerve preservation in rectal cancer surgery based on immunohistochemical study

PurposePrevious studies revealed that the incidence of cancer cell involvement along the pelvic autonomic nerves ranged from 4 to 14%. However, patients’ profiles and methodologies differed among the studies. This study was conducted to clarify the incidence of cancer cell involvement in and around the pelvic autonomic nerves immunohistochemically.MethodsImmunohistochemical staining was performed on pelvic autonomic nerve specimens resected from 17 patients with p-Stage I–III lower rectal cancers. Antibodies used were pan-cytokeratin (AE1/AE3) for staining cancer cells, S-100 for autonomic nerves, and D2-40 for lymphatic vessels. Lymphatic permeation around the pelvic autonomic nerves was defined as present when AE1/AE3-positive cells were detected in D2-40-stained lymphatic vessels. The presence of metastasis to the interstitial tissue or contaminants was also recorded.ResultsTNM staging was stage I in 1, stage II in 5, and stage III in 11 cases, respectively. No cases had lymphatic permeation or metastasis to the interstitial tissue in and around the pelvic autonomic nerves. Cancer cell contaminants were seen in four cases (23%). In three cases (18%), metastatic nodes were located at the root of the middle rectal artery, very close to the pelvic autonomic nerves.ConclusionsCancer cell involvement was not seen in and around the pelvic autonomic nerves, suggesting that complete pelvic autonomic nerve preservation may be feasible, unless nerves are invaded by the tumor. In some cases, however, metastatic nodes were seen very close to the nerves. Meticulous lymph node dissection along the pelvic autonomic nerves is mandatory.