Naruji Kugimiya

The c‐MYC‐ABCB5 axis plays a pivotal role in 5‐fluorouracil resistance in human colon cancer cells

c‐MYC overexpression is frequently observed in various cancers including colon cancer and regulates many biological activities such as aberrant cell proliferation, apoptosis, genomic instability, immortalization and drug resistance. However, the mechanism by which c‐MYC confers drug resistance remains to be fully elucidated. In this study, we found that the c‐MYC expression level in primary colorectal cancer tissues correlated with the recurrence rate following 5‐fluorouracil (5‐FU)‐based adjuvant chemotherapy. Supporting this finding, overexpression of exogenous c‐MYC increased the survival rate following 5‐FU treatment in human colon cancer cells, and knockdown of endogenous c‐MYC decreased it. Furthermore, c‐MYC knockdown decreased the expression level of ABCB5, which is involved in 5‐FU resistance. Using a chromatin immunoprecipitation assay, we found that c‐MYC bound to the ABCB5 promoter region. c‐MYC inhibitor (10058‐F4) treatment inhibited c‐MYC binding to the ABCB5 promoter, leading to a decrease in ABCB5 expression level. ABCB5 knockdown decreased the survival rate following 5‐FU treatment as expected, and the ABCB5 expression level was increased in 5‐FU‐resistant human colon cancer cells. Finally, using a human colon cancer xenograft murine model, we found that the combined 5‐FU and 10058‐F4 treatment significantly decreased tumorigenicity in nude mice compared with 5‐FU or 10058‐F4 treatment alone. 10058‐F4 treatment decreased the ABCB5 expression level in the presence or absence of 5‐FU. In contrast, 5‐FU treatment alone increased the ABCB5 expression level. Taken together, these results suggest that c‐MYC confers resistance to 5‐FU through regulating ABCB5 expression in human colon cancer cells.

JAB1 regulates unphosphorylated STAT3 DNA-binding activity through protein–protein interaction in human colon cancer cells

Recent studies have revealed that unphosphorylated STAT3 forms a dimer, translocates to the nucleus, binds to the STAT3 binding site, and activates the transcription of STAT3 target genes, thereby playing an important role in oncogenesis in addition to phosphorylated STAT3. Among signaling steps of unphosphorylated STAT3, nuclear translocation and target DNA-binding are the critical steps for its activation. Therefore, elucidating the regulatory mechanism of these signaling steps of unphosphorylated STAT3 is a potential step in the discovery of a novel cancer drug. However, the mechanism of unphosphorylated STAT3 binding to the promoter of target genes remains unclear. In this study, we focused on Jun activation domain-binding protein 1 (JAB1) as a candidate protein that regulates unphosphorylated STAT3 DNA-binding activity. Initially, we observed that both unphosphorylated STAT3 and JAB1 existed in the nucleus of human colon cancer cell line COLO205 at the basal state (no cytokine stimulation). On the other hand, phosphorylated STAT3 did not exist in the nucleus of COLO205 cells at the basal state. Immunoprecipitation using nuclear extract of COLO205 cells revealed that JAB1 interacted with unphosphorylated STAT3. To investigate the effect of JAB1 on unphosphorylated STAT3 activity, RNAi studies were performed. Although JAB1 knockdown tended to increase nuclear STAT3 expression, it significantly decreased unphosphorylated STAT3 DNA-binding activity. Subsequently, JAB1 knockdown significantly decreased the expression levels of MDR1, NANOG, and VEGF, which are STAT3 target genes. Furthermore, the expression level of nuclear JAB1, but not nuclear STAT3, correlated with unphosphorylated STAT3 DNA-binding activity between COLO205 and LoVo cells. Taken together, these results suggest that nuclear JAB1 positively regulates unphosphorylated STAT3 DNA-binding activity through protein-protein interaction in human colon cancer cell line COLO205.

Hypoxic preconditioning reinforces cellular functions of autologous peripheral blood-derived cells in rabbit hindlimb ischemia model.

Peripheral blood mononuclear cell (PBMNC) is one of powerful tools for therapeutic angiogenesis in hindlimb ischemia. However, traditional approaches with transplanted PBMNCs show poor therapeutic effects in severe ischemia patients. In this study, we used autograft models to determine whether hypoxic pretreatment effectively enhances the cellular functions of PBMNCs and improves hindlimb ischemia. Rabbit PBMNCs were cultured in the hypoxic condition. After pretreatment, cell adhesion, stress resistance, and expression of angiogenic factor were evaluated in vitro. To examine in vivo effects, we autografted preconditioned PBMNCs into a rabbit hindlimb ischemia model on postoperative day (POD) 7. Preconditioned PBMNCs displayed significantly enhanced functional capacities in resistance to oxidative stress, cell viability, and production of vascular endothelial growth factor. In addition, autologous transplantation of preconditioned PBMNCs significantly induced new vessels and improved limb blood flow. Importantly, preconditioned PBMNCs can accelerate vessel formation despite transplantation on POD 7, whereas untreated PBMNCs showed poor vascularization. Our study demonstrated that hypoxic preconditioning of PBMNCs is a feasible approach for increasing the retention of transplanted cells and enhancing therapeutic angiogenesis in ischemic tissue.

Loss of skeletal muscle mass after curative gastrectomy is a poor prognostic factor

Sarcopenia has been reported to relate to poor prognosis in various malignant cancer types. The present study aimed to clarify the prognostic impact of skeletal muscle mass (SMM) loss after curative gastrectomy in patients with gastric cancer. A total of 119 patients who underwent curative gastrectomy for gastric cancer between 2009 and 2016 were analyzed. The SMM loss at 6 months postoperatively compared with the SMM prior to surgery was calculated using the hospital records. The median loss of SMM was 3.8%. Multivariate logistic regression analysis demonstrated that total gastrectomy was a significant and independent risk factor for SMM loss of ≥5% (odds ratio=2.58; P=0.02). Results from multivariate analysis using stepwise Cox proportional hazards regression indicated that the following factors were significantly associated with shorter overall survival after curative gastrectomy: Age [>70 years; hazard ratio (HR)=2.46, P=0.04], TNM stage (≥2; HR=2.65, P=0.04) and loss of SMM (≥5%; HR=2.57, P=0.03). The present findings suggested that loss of SMM after curative gastrectomy for gastric cancer is an independent predictive factor for poor prognosis.

JAB1‑STAT3 activation loop is associated with recurrence following 5‑fluorouracil‑based adjuvant chemotherapy in human colorectal cancer

Jun activation domain-binding protein 1 (JAB1) has been shown to have multiple roles in tumorigenesis, including the degradation of tumor suppressor proteins such as p53, Smad7, Runx3 and the cyclin-dependent kinase inhibitor p27Kip1, and the activation of oncogenic transcription factors, such as c-Jun and hypoxia-inducible factor-1α. In addition, our previous study revealed that JAB1 positively regulates signal transducer and activator of transcription 3 (STAT3) DNA-binding activity in human colon cancer cells. In turn, the oncogenic transcription factor STAT3 positively regulates JAB1 expression, indicative of a positive feedback loop. Furthermore, high JAB1 expression is associated with a poor prognosis in numerous malignant carcinomas. However, the association between JAB1 expression and prognosis in colorectal cancer remains unclear. The aim of the present study was to elucidate the association between JAB1 and STAT3 expression and recurrence in colorectal cancer. In the present study, it was found that high JAB1 expression in primary colorectal cancer tissues is an independent predictor of recurrence following 5-fluorouracil (5-FU)-based adjuvant chemotherapy in colorectal cancer patients, and that high expression of both JAB1 and STAT3 in primary colorectal cancer tissues is associated with a lower recurrence-free survival rate following 5-FU-based adjuvant chemotherapy compared to high expression of only JAB1 or STAT3. Overall, these results suggest that JAB1 is a novel predictive marker of recurrence following 5-FU-based adjuvant chemotherapy in colorectal cancer patients, and that the JAB1-STAT3 activation loop may be a potential therapeutic target in recurrent colorectal cancer following 5-FU-based adjuvant chemotherapy.

JAB1/CSN5: A Multifunctional Protein in Cancer

c-Jun activation domain-binding protein 1 (JAB1) was originally identified as a c-Jun co-activator and subsequently discovered to be a component of the COP9 signalosome (CSN) complex. JAB1, which is also known as CSN5, affects many partner proteins through protein-protein interaction, which leads to protein degradation and transcriptional activation. Thereby, JAB1/CSN5 functions as a multifunctional protein involved in the regulation of cell cycle, signal transduction, and DNA repair. In particular, JAB1/CSN5 plays an essential role in tumorigenesis by degrading tumor suppressor proteins and activating oncogenic transcription factors. JAB1/CSN5 overexpression has been observed in various types of cancer, and it has been multifunctionally involved in cancer progression. In this review, we provide an overview of the roles of JAB1/CSN5 in tumorigenesis and summarize recent findings that highlight the novel roles of JAB1/CSN5 in this process

Laparoscopic low anterior resection for rectal cancer after Whitehead's hemorrhoidectomy: A case report

A 65‐year‐old man presented with bloody stool. Colonoscopy revealed a raised tumor in the rectum, above the peritoneal reflection. He underwent endoscopic mucosal resection, but the pathological findings suggested the possibility of residual cancer. We performed laparoscopic low anterior resection using a circular stapling instrument for additional curative surgery. However, we could not insert the shaft of the endoscopic circular stapler from the anus because of anal stenosis due to Whiteheads hemorrhoidectomy the patient had undergone 20 years earlier. Therefore, we planned to use a linear stapler to insert an anvil into the rectum. The cartridge‐carrying instrument was inserted from the sigmoidal side, and we performed a side‐to‐end anastomosis. The patient was discharged without anastomotic leakage or defecation disorder. We present this case because laparoscopic low anterior resection for rectal cancer with anal stenosis has not been previously reported.

Abstract 1072: Synergistic effect of gemcitabine and a Dclk1 inhibitor on pancreatic cancer cell survival

Pancreatic cancer has the highest mortality rate of all major cancers and is one of the most lethal malignancies. There is a constant upward trend in the number of patients diagnosed with pancreatic cancer and the number of deaths due to pancreatic cancer. Gemcitabine (GEM) is often used in the treatment of pancreatic cancer (PDAC), but has limited effects. Doublecortin-like kinase 1 (Dclk1) is important in the progression of early pancreatic neoplastic lesions and PDAC. However, the functional role of Dclk1 in PDAC is unknown. To identify the substrate protein phosphorylated by Dclk1, we performed a protein microarray analysis on Dclk1 knockdown cells. The results of this analysis directed our studies toward Chk1, which is known to be a potential regulator of the cell cycle and experiences upregulation of phosphorylation after GEM treatment. In general, GEM treatment results in DNA damage to pancreatic cancer cells, an increase in phosphorylated Chk1 (p-Chk1), and arrests the cell cycle progression to repair the damaged DNA. On the basis of the preliminary data, we hypothesized that the decrease in Chk1 phosphorylation by Dclk1 inhibition circumvents cell cycle arrest and impairs the subsequent DNA repair. The aim of this study was to evaluate the synergistic effect of Dclk1 inhibition and GEM treatment on pancreatic cancer cell survival. We used the human pancreatic cancer cell line MIA PaCa-2 and LRRK2-IN-1 (LRRK) as the Dclk1 inhibitor for this study. First, we examined the effects of GEM or the Dclk1 inhibitor or both on cancer cell proliferation and the expression of p-Chk1. Significantly decreased cell proliferation was observed on co-treatment of GEM and LRRK compared to GEM treatment alone. In addition, the expression of p-Chk1 significantly decreased on co-treatment compared to GEM treatment alone. Second, we used flow cytometry to analyze the cell cycle after treatment with GEM and/or LRRK. Almost all cancer cells treated with GEM alone were arrested in the S phase of the cell cycle. The addition of LRRK allows the cell cycle to proceed in the same manner as untreated control cancer cells do. We also evaluated DNA damage by measuring the intensity of gamma-H2AX. Cancer cells that were co-treated experienced more DNA damage than with GEM treatment alone. The co-treatment induced apoptosis without the repair of DNA damage in the cancer cells. In conclusion, the combined treatment with GEM and a Dclk1 inhibitor decreased the cell survival rate compared to treatment with GEM alone through the suppression of p-Chk1. Targeting Dclk1 in combination with GEM might offer an excellent opportunity for future pancreatic cancer treatments. Citation Format: Daichi Kawamura, Yoshihiro Takemoto, Arata Nishimoto, Toshiki Tanaka, Yukari Hironaka, Kumiko Yoshida, Junichi Murakami, Naruji Kugimiya, Eijiro Harada, Koji Ueno, Tohru Hosoyama, Kimikazu Hamano. Synergistic effect of gemcitabine and a Dclk1 inhibitor on pancreatic cancer cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1072. doi:10.1158/1538-7445.AM2017-1072