Yoshinori Kagawa

Cell cycle-dependent Rho GTPase activity dynamically regulates cancer cell motility and invasion in vivo

The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers.

A combination of Wnt and growth factor signaling induces Arl4c expression to form epithelial tubular structures

Growth factor‐dependent epithelial morphological changes and proliferation are essential for the formation of tubular structures, but the underlying molecular mechanisms are poorly understood. Co‐stimulation with Wnt3a and epidermal growth factor (Wnt3a/EGF) induced development of tubes consisting of intestinal epithelial cells by inducing expression of Arl4c, an Arf‐like small GTP‐binding protein, in three‐dimensional culture, while stimulation with Wnt3a or EGF alone did not. Arl4c expression resulted in rearrangement of the cytoskeleton through activation of Rac and inactivation of Rho properly, which promoted cell growth by inducing nuclear translocation of Yes‐associated protein and transcriptional co‐activator with PDZ‐binding motif (YAP/TAZ) in leading cells. Arl4c was expressed in ureteric bud tips and pretubular structures in the embryonic kidney. In an organoid culture assay, Wnt and fibroblast growth factor signaling simultaneously induced elongation and budding of kidney ureteric buds through Arl4c expression. YAP/TAZ was observed in the nucleus of extending ureteric bud tips. Thus, Arl4c expression induced by a combination of growth factor signaling mechanisms is involved in tube formation.

Depletion of JARID1B induces cellular senescence in human colorectal cancer

The global incidence of colorectal cancer (CRC) is increasing. Although there are emerging epigenetic factors that contribute to the occurrence, development and metastasis of CRC, the biological significance of epigenetic molecular regulation in different subpopulations such as cancer stem cells remains to be elucidated. In this study, we investigated the functional roles of the H3K4 demethylase, jumonji, AT rich interactive domain 1B (JARID1B), an epigenetic factor required for the continuous cell growth of melanomas, in CRC. We found that CD44(+)/aldehyde dehydrogenase (ALDH)(+) slowly proliferating immature CRC stem cell populations expressed relatively low levels of JARID1B and the differentiation marker, CD20, as well as relatively high levels of the tumor suppressor, p16/INK4A. Of note, lentiviral‑mediated continuous JARID1B depletion resulted in the loss of epithelial differentiation and suppressed CRC cell growth, which was associated with the induction of phosphorylation by the c‑Jun N‑terminal kinase (Jnk/Sapk) and senescence‑associated β‑galactosidase activity. Moreover, green fluorescent‑labeled cell tracking indicated that JARID1B‑positive CRC cells had greater tumorigenicity than JARID1B‑negative CRC cells after their subcutaneous inoculation into immunodeficient mice, although JARID1B‑negative CRC cells resumed normal growth after a month, suggesting that continuous JARID1B inhibition is necessary for tumor eradication. Thus, JARID1B plays a role in CRC maintenance. JARID1B may be a novel molecular target for therapy‑resistant cancer cells by the induction of cellular senescence.

Circulating miR-199a-3p as a novel serum biomarker for colorectal cancer

Serum microRNAs (miRNAs) have been shown to have potential for cancer diagnosis. The main objective of the present study was to identify a novel serum miRNA biomarker from patients with colorectal cancer (CRC). Microarray analysis of miRNA expression was performed using paired pre-operative and post-operative serum from 10 CRC patients. Expression of two miRNAs (let-7a and miR-199a-3p) was significantly decreased in the post-operative serum when compared to levels in the pre-operative serum (P=0.015 and 0.029, respectively). Quantitative real-time polymerase chain reaction (qRT-PCR) confirmed the decrease in the miRNAs in an extended number (n=30) of paired serum samples. Next, we examined the serum let-7a level in 32 non-cancer patients and 84 CRC patients but we found no significant difference (P=0.120). In contrast, miR199a-3p expression was significantly higher in the CRC patients than that in the non-cancer patients (P=0.016). Furthermore, clinical and pathological survey indicated that high expression of miR-199a-3p was significantly associated with deep wall invasion. Our data suggest that circulating miR-199a-3p could be a novel serum biomarker for CRC.

Significance of Polypyrimidine Tract–Binding Protein 1 Expression in Colorectal Cancer

Polypyrimidine tract–binding protein (PTBP1) is an RNA-binding protein with various molecular functions related to RNA metabolism and a major repressive regulator of alternative splicing, causing exon skipping in numerous alternatively spliced pre-mRNAs. Here, we have investigated the role of PTBP1 in colorectal cancer. PTBP1 expression levels were significantly overexpressed in cancerous tissues compared with corresponding normal mucosal tissues. We also observed that PTBP1 expression levels, c-MYC expression levels, and PKM2:PKM1 ratio were positively correlated in colorectal cancer specimens. Moreover, PTBP1 expression levels were positively correlated to poor prognosis and lymph node metastasis. In analyses of colorectal cancer cells using siRNA for PTBP1, we observed that PTBP1 affects cell invasion, which was partially correlated to CD44 splicing, and this correlation was also confirmed in clinical samples. PTBP1 expression also affected anchorage-independent growth in colorectal cancer cell lines. PTBP1 expression also affected cell proliferation. Using time-lapse imaging analysis, PTBP1 was implicated in prolonged G2–M phase in HCT116 cells. As for the mechanism of prolonged G2–M phase in HCT116 siPTBP1 cells, Western blotting revealed that PTBP1 expression level was correlated to CDK11p58 expression level, which was reported to play an important role on progression to complete mitosis. These findings indicated that PTBP1 is a potential therapeutic target for colorectal cancer. Mol Cancer Ther; 14(7); 1705–16. ©2015 AACR.

Interferon-α acts on the S/G2/M phases to induce apoptosis in the G1 phase of an IFNAR2-expressing hepatocellular carcinoma cell line.

Background: The mode of action of interferon-α has been unknown. Results: Its point of action in the cell cycle was analyzed by single cell tracking using time lapse confocal imaging. Conclusion: Interferon-α activates p63 in S/G2/M and induces apoptosis and cell cycle arrest in the subsequent G1. Significance: Tracking cell cycle progression is crucial for understanding the mechanisms of interferon-α. Interferon-α (IFN-α) is used clinically to treat hepatocellular carcinoma (HCC), although the detailed therapeutic mechanisms remain elusive. In particular, IFN-α has long been implicated in control of the cell cycle, but its actual point of action has not been clarified. Here, using time lapse imaging analyses of the human HCC cell line HuH7 carrying a fluorescence ubiquitination-based cell cycle indicator (Fucci), we found that IFN-α induced cell cycle arrest in the G0/G1 phases, leading to apoptosis through an IFN-α type-2 receptor (IFNAR2)-dependent signaling pathway. Detailed analyses by time lapse imaging and biochemical assays demonstrated that the IFN-α/IFNAR2 axis sensitizes cells to apoptosis in the S/G2/M phases in preparation for cell death in the G0/G1 phases. In summary, this study is the first to demonstrate the detailed mechanism of IFN-α as an anticancer drug, using Fucci-based time lapse imaging, which will be informative for treating HCC with IFN-α in clinical practice.

Jumonji/Arid1b (Jarid1b) protein modulates human esophageal cancer cell growth

Although esophageal cancer is highly heterogeneous and the involvement of epigenetic regulation of cancer stem cells is highly suspected, the biological significance of epigenetically modified molecules that regulate different subpopulations remains to be firmly established. Using esophageal cancer cells, we investigated the functional roles of the H3K4 demethylase Jumonji/Arid1b (Jarid1b) (Kdm5b/Plu-1/Rbp2-h1), an epigenetic factor that is required for continuous cell growth in melanoma. JARID1B knockdown resulted in the suppression of esophageal cancer cell growth, sphere formation and invasion ability and was associated with loss of epithelial marker expression. However, these inhibitory effects observed on tumor formation were reverted subsequent to subcutaneous inoculation of these cells into immune-deficient mice. These results indicated that JARID1B plays a role in maintaining cancer stem cells in the esophagus and justifies the rationale for studying the effects of continuous inhibition of this epigenetic factor in esophageal cancer.

Transumbilical laparoscopic-assisted appendectomy for children and adults

Dear Editor: In 1983, Semm reported the first laparoscopic appendectomy. Since then, several randomized controlled trials and metaanalyses have demonstrated that laparoscopic appendectomy is associated with fewer wound infections, less pain, faster recovery, earlier return to work, and cosmesis compared with open appendectomy. However, the longer operation time and increased hospital costs relative to open appendectomy continue to fuel the debate over these techniques. The use of single-port laparoscopic surgery is steadily increasing and several authors have reported the feasibility of single-port laparoscopic appendectomy. However, this technique is technically demanding and requires special devices that increase costs. Transumbilical laparoscopic-assisted appendectomy (TULAA) was first reported by Pelosi and colleagues in 1992 as “Laparoscopic appendectomy using a single umbilical puncture (minilaparoscopy)”. The authors claimed that, in some cases of appendicitis, the appendix could be pulled out through a small umbilical incision and be resected, and the special laparoscope enabled easy exteriorization of the appendix. However, TULAA is still not routinely performed. One reason is the need for a special laparoscope, in which 5-mm laparoscopic forceps can be inserted through its mounted operative channel. Another reason is the notion that it is difficult to extract the appendix from the umbilicus in adult patients, which has led many surgeons to limit TULAA to children. We modified the TULAA technique by using a usual laparoscope. Additionally, we extended the indication of TULAA to adults and analyzed the relationship between feasibility and body habits. With the patient under general anesthesia, we made a 15−20-mm longitudinal incision in the umbilicus and attached a wound protector. The right lower quadrant abdominal wall was then lifted with a thin surgical retractor to provide a working space in the peritoneal cavity. Pneumoperitoneum was not used, eliminating the need for any ports. We inserted a 5-mm laparoscope and 5-mm laparoscopic forceps through the incision. When the appendix was identified under laparoscopy, it or the mesoappendix was grasped and exteriorized through the incision. The mesoappendix and appendix were then dissected and ligated as in open appendectomy. Finally, we washed the peritoneal cavity with saline and closed the wound cosmetically. We converted to a laparoscopic approach if a complicated inflammatory mass was found or when the appendix and mesoappendix could not be exteriorized because of immobility of the appendix. When a massive abscess and/or severe adhesion were found, the operation was converted to an open appendectomy. All of the surgeons were 3–5-year postgraduates while the first assistants were surgeons with at least 7 years of experience. Yoshinori Kagawa, Shinsuke Hata, Junzo Shimizu, Mitsugu Sekimoto, and Masaki Mori contributed equally to this work. Y. Kagawa (*) :M. Sekimoto :M. Mori Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, 2–2 Yamadaoka, Suita, Osaka 565–0871, Japan e-mail: ykagawa@gesurg.med.osaka-u.ac.jp

A Multicenter Clinical Phase II Study of FOLFOXIRI Plus Bevacizumab as First-line Therapy in Patients With Metastatic Colorectal Cancer: QUATTRO Study

Background FOLFOXIRI (Fluorouracil, folinate, oxaliplatin, and irinotecan) plus bevacizumab improved progression‐free survival (PFS) and overall survival in patients with metastatic colorectal cancer (mCRC), compared with FOLFIRI (fluorouracil, folinate, and irinotecan) plus bevacizumab, but significantly increased the incidences of adverse events. The efficacy and safety profiles of FOLFOXIRI plus bevacizumab in ethnic Asian patients have not been established yet. Patients and Methods This study was an open‐label, single‐arm, multi‐centered phase II prospective clinical trial in patients with mCRC who received FOLFOXIRI plus bevacizumab. The primary endpoint was the PFS rate at 10 months. Secondary endpoints included overall survival, response rate, and safety. Results A total of 69 patients received FOLFOXIRI plus bevacizumab as induction therapy and were assessed for efficacy and safety. The PFS rate at 10 months was 75.2% and the median PFS was 13.3 months. Complete response and partial response were achieved in 2 (2.9%) and 47 patients (69.1%), respectively. Grade 3 and 4 adverse events with incidence rates exceeding 20% were neutropenia (72.5%), hypertension (34.8%), leucopenia (33.3%), and febrile neutropenia (21.7%). Significantly more patients with grade 4 neutropenia had single‐heterozygous UGT1A1*1/*6 or *1/*28 (46.2%) than UGT1A1 wild‐type genotype (*1/*1) (13.3%) (P = .004). Conclusions FOLFOXIRI plus bevacizumab is considered an effective first‐line regimen that improves the outcome of patients with mCRC regardless of ethnicity. In Asian patients, utmost attention should be paid to the possible onset of severe neutropenia or febrile neutropenia attributed to different types of UGT1A1*6 and *28 polymorphism, when FOLFOXIRI plus bevacizumab is administered. Micro‐Abstract FOLFOXIRI plus bevacizumab is an effective for Asian metastatic colorectal cancer patients and the safety profile is manageable by adopting appropriate measures, even if severe neutropenia and febrile neutropenia develop at a higher frequency in ethnic Asian patients with UGT1A1*6 and *28 polymorphism.

A prospective Phase II study to examine the relationship between quality of life and adverse events of first-line chemotherapy plus cetuximab in patients with KRAS wild-type unresectable metastatic colorectal cancer: QUACK trial

A prospective trial has not been performed to investigate associations between quality of life (QOL), adverse events (AEs), and overall survival (OS) in the first‐line treatment with cetuximab plus standard chemotherapy for advanced/metastatic colorectal cancer (mCRC). Associations between patient outcome and health‐related QOL (HRQOL) together with skin toxicity‐related QOL were prospectively evaluated using EORTC QLQ‐C30 and DLQI questionnaires. One hundred and forty mCRC patients were analyzed in this study, and 87.8% received pre‐emptive skin treatment. Skin toxicity had no clinical impact on HRQOL or skin‐related QOL during the first 8 weeks and throughout the study period. An early skin reaction with a grade ≥2 at 8 weeks was significantly associated with a favorable OS compared with a grade of ≤1 (HR, 0.50; 95% CI, 0.24‐0.95; P = .035) and was confirmed to be an independent predictor of OS (HR, 0.48; 95% CI, 0.21‐0.97; P = .040). Patients symptomatic at baseline who responded to treatment had improved HRQOL compared to nonresponding patients. Severe mucositis/stomatitis had a statistically significant and clinically meaningful negative impact on HRQOL (mean changes from baseline throughout the study period in global health status were −12.64 for a grade of ≥2 vs −0.35 for a grade of 0 or 1 (P = .005)). In conclusion, severe early skin reactions predict favorable OS for patients treated with cetuximab plus chemotherapy without impairing QOL. In addition, mucositis/stomatitis was the most substantial AE compromising both QOL and treatment compliance.