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PDF - 135K, qRT-PCR for miR-340 and miR-542-3p in BM4 in patients with and without liver metastasis.

PDF - 89K, Legends for Supplementary Figures 1 and 2.

PDF - 150K, Over expression of miR-340 suppressed c-Met expression in HCT116 and SW480 in vitro.

PDF - 63K, miR-340 expression and clinicopathologic factors.

PDF - 364K, List of microRNA array results.

Studies have shown the prognostic significance of disseminated tumor cells (DTC) in bone marrow of patients with colorectal cancer. However, the molecular characteristics of DTCs, including their miRNA expression profiles, remain mostly unknown. In this study, we analyzed the miRNA expression of DTCs in bone marrow. EpCAM+ bone marrow cells were collected using immunomagnetic beads after exclusion of CD14+ and CD45+ cells, then subjected to miRNA microarray analysis. Cluster analysis (7 colorectal cancer patients with liver metastasis and 12 colorectal cancer patients without liver metastasis) indicated that miR-340 and miR-542-3p expressions were significantly decreased in EpCAM+ bone marrow cells of patients with liver metastasis (P = 0.019 and 0.037, respectively). We demonstrated that pre-miR-340 administration inhibited growth of colon cancer cells and suppressed c-Met expression in vitro. In clinical samples of colorectal cancer, miR-340 was expressed at significantly lower levels in tumor tissues compared with normal mucosa. Survival analysis in 136 patients with colorectal cancer indicated that low miR-340 expression was correlated with shorter 5-year disease-free survival (P = 0.023) and poor 5-year overall survival (P = 0.046). It was of note that the colorectal cancer group with low miR-340 and high c-Met expression had the worst prognosis. We further demonstrated that systemic pre-miR-340 administration suppressed growth of pre-established HCT116 tumors in animal therapeutic models. These findings indicate that miR-340 may be useful as a novel prognostic factor and as a therapeutic tool against colorectal cancer. Our data suggest that miR-340 in bone marrow may play an important role in regulating the metastasis cascade of colorectal cancer. Mol Cancer Ther; 13(4); 976–85. ©2014 AACR.

PDF - 60K, Clinicopathologic factors of patients analyzed by miRNA array.

Non-LPACs were observed after each treatment, 5-FU, L-OHP and radiation (8Gy), at 0 hour, 24 hours, 48 hours and 72 hours. Non-LPACs never changed to LPACs in each assay. Bar: 100μm.

Purpose: We reported in a retrospective study that the presence of micrometastasis in lymph nodes, when assessed by carcinoembryonic antigen (CEA)-specific RT-PCR, is a significant prognostic factor in stage II colorectal cancer. The aim of this study was to clarify the clinical value of micrometastasis in a prospective multicenter trial.Experimental Design: From November 2001 to December 2005, a total of 419 colorectal cancer cases were preoperatively registered at a central data center. Of them, 315 node-negative stage II colorectal cancer cases were enrolled. After RNA quality check, 304 colorectal cancer cases were analyzed for CEA mRNA in lymph nodes by both conventional RT-PCR (a band method) and quantitative RT-PCR. Long-term prognosis of the patients was determined by each method.Results: A positive band for CEA mRNA was detected in 73 (24.0%) of 304 patients. Postoperative adjuvant chemotherapy was applied in 31 CEA band-positive cases with an oral 5-fluorouracil derivative HCFU (1-hexylcarbamoyl-5-fluorouracil) for 1 year, whereas chemotherapy was not administered to CEA band-negative group. Multivariate Cox regression analyses revealed that a high micrometastasis volume (high MMV, n = 95) was an independent poor prognostic factor for 5-year disease-free survival (DFS; P = 0.001) and 5-year overall survival (OS; P = 0.016).Conclusions: This prospective clinical trial demonstrates that micrometastasis volume is a useful marker in identifying patients who are at high or low risk for recurrence of stage II colorectal cancer. Clin Cancer Res; 22(13); 3201–8. ©2016 AACR.

No significant differences were found between the two groups regarding clinicopathological factors, such as age, gender, tumor location, depth of tumor invasion, lymph node metastasis, histological grade, Dukes' stage, and vessel invasion.

Several cancer-related gene candidates were identified using microarray data from the four cell lines (HCT116, SW480, DLD1, KM12SM)

LNs were collected within 3 hours after resection of the specimens with a disposable LN sampling kit that was prepared in a RNase-free state. In addition to routine pathological examination of LNs, representative five pericolic nodes adjacent to the tumor were cut into halves and stored in RNA laterTM at -20{degree sign}C. Immediately after pathological tests reported node-negative stage II CRC, RNA was extracted from LNs, checked for RNA quality, and then subjected to conventional RT-PCR for the CEA band and qRT-PCR for CEA mRNA value by the Light CyclerTM. Among seven CRC cases tested, Case No. 3, 4, and 6 were judged positive (Lower panel). Positivity of the CEA band was judged consistently by reference of the simultaneously produced control panel. One microgram of RNA from gastric cancer MKN45 cells and colon cancer LoVo cells was subjected to the RT reaction. Each cDNA was serially diluted at 1x10-1 to 1x10-5. PCR was performed using CEA primers and PBGD primers. A positive CEA band was visible at a 1x10-4 dilution in the MKN45 cells and at a 1x10-3 dilution in the LoVo cells (Upper panel).

Purpose: One of the main reasons for cancer treatment resistance is the existence of cancer stem-like cells (CSCs). Here, we elucidated the relationship between low proteasome activity cells (LPACs) and CSCs.Experimental Design: The human colorectal cancer cell lines HCT116, SW480, DLD1, and KM12SM were engineered to stably express a green fluorescent molecule fused to the degron of ornithine decarboxylase, resulting in an accumulation of the fluorescence in LPACs. LPACs were isolated by flow cytometry. Treatment resistance (radio- and chemotherapy) and the capacity of LPACs to act as CSCs were analyzed. Microarray analysis was performed to reveal genes related to treatment resistance. The prognostic impact of potent genes was examined in 190 patients with colorectal cancer.Results: LPACs had a significantly increased capacity for radioresistance and chemoresistance (5-fluorouracil and oxaliplatin), significantly lower reactive oxygen species activity, and significantly increased sphere formation capacity compared with non-LPACs. The number of cells in the G0–G1 phase was significantly higher among LPACs. Subcutaneous injection of as few as 20 LPACs led to tumor formation in immunologically incompetent mice. Microarray analysis revealed that the expression of EP300-interacting inhibitor of differentiation 3 (EID3) was significantly increased in LPACs. In vitro assay revealed that EID3 positively controlled cell proliferation and treatment resistance. The high expression of EID3 was an adverse prognostic indicator in patients with colorectal cancer (P = 0.0400).Conclusions: LPACs have characteristic treatment resistance and act as CSCs in colorectal cancer. In addition, EID3 is one of the potential regulators of treatment resistance in colorectal cancer and may be a potential therapeutic target. Clin Cancer Res; 22(21); 5277–86. ©2016 AACR.

The standard curves were plotted using the serial dilution of cDNA from MKN45. We confirmed that the CEA value was reproducibly measured in the range of 1.0x10-1 to 1.0x10-4 with standard deviation < 10% in each dilution.

Of 55 node-negative CRCs (9 stage I and 46 stage II), Micrometastais was positive in 59 of 373 LNs (15.8%) at level 1, 16 of 193 LNs (8.3%) at level 2 and 4 of 86 LNs (4.7%) at level 3 through 5 slides inspection by immunostaining using pan-cytokeratin AE1/AE3 antibody (Dako). As results, 27 CRC cases were positive for micrometastasis in LNs. Micromstastasis was found at pericolic nodes adjacent to the tumor (level 1) in 26 cases of 27 CRCs (96.3%). On the other hand, micrometastasis stayed at intermediate nodes along the course of the main blood vessel, e.g., inferior mesenteric artery (level 2) and at central nodes around the root of the main blood vessel (level 3) in 37.0% and 14.8%, respectively. Level 1, pericolic nodes adjacent to the tumor; Level 2, intermediate nodes along the course of the main blood vessel; Level 3, central nodes around the root of the main blood vessel. Level 1, pericolic nodes adjacent to the tumor; Level 2, intermediate nodes along the course of the main blood vessel; Level 3, central nodes around the root of the main blood vessel.

Figure S5: Impact of chemotherapy in High-MMV group. Post-operative chemotherapy was performed in 29 of 95 High-MMV cases and 2 of 201 Low-MMV cases. Irrespective of chemotherapy applied to High-MMV cases, High-MMV group consistently showed significantly worse 5-year DFS as compared to the Low-MMV group with no treatment (P= 0.009, 0.007). Similar results were observed in analyses for OS (P=0.003, 0.026).; Figure S6:Disease recurrence mode in stage II CRC. Among 296 patients, 51 developed disease recurrence after surgery. The recurrent organ site was 60 in all and are listed here. Like more advanced-stage patients, stage II patients had often hematogenous metastasis to liver and lung.

Described markers expression, CD133, CD166, Lgr5 (Alexa Fluor® 647 channel, BD Biosciences) and EpHB2 (PE channel, R&D systems, Minneapolis, MN, USA), were compared between LPACs and non-LPACs. The LPACs clearly showed up-regulation of several CSC markers including CD133, CD166, Lgr5 and EpHB2. Especially, Lgr5 was enriched in all cell lines (HCT116; 0.7% (non-LPACs) vs 18.1% (LPACs), SW480; 0.2% vs 28.1% and DLD1; 2.3% vs 45.2%).

EID3 was identified by microarray analysis. The expression of EID3 was confirmed by PCR and western blotting. (A) The mRNA expression of EID3 was significantly enriched in LPACs of HCT116, KM12SM and DLD1 compared to non-LPACs, except SW480 which just missed statistical significance (p=0.0555). (B) Western blotting was also performed for checking the expression of EID3 protein in LPACs and non-LPACs using EID3 antibody (Abcam, Cambridge, UK, ab124447), but the evaluation was difficult, because expression of EID3 is low in humans as per previous report (31, 32). * p

Figure S8: Survival analyses of the entire CEA　band-positive group.　 Of the entire CEA band-positive cases, 31 cases eventually received post-operative chemotherapy using HCFU for one year, whereas 41 cases had no treatment. There was no significant difference in 5-year DFS and 5-year OS between the two groups.; Figure S9. Survival curves stratified by High CK19 expression and Low CK19 expression. Survival analyses indicated that the High CK19 group (n=96) had a significantly (A) worse 5-year DFS (P= 0.001), (B) 5-year OS (P= 0.020), and (C) OS (P= 0.014) as compared to the Low CK19 group (n=193). A total of 289 cases with residual preserved RNA were analyzed.

(A) The efficiency of over-expression was validated by expression of mRNA. The expression of EID3 was significantly higher in cells with EID3 　expression vector than cells with control vector. (B) The efficiency of down-regulation was confirmed by mRNA. The expression of EID3 was significantly lower in cells with double-stranded RNA duplexes targeting human EID3 than cells with negative control siRNA. * p

MM-free CRC group (group a, n=90) had a considerably favorable prognosis. (A) 5-year DFS: 5-year DSF was 93.3, 86.5, and 74.7% in group a, group b and group c+d (High-MMV), respectively. (B) 5-year OS: 5-year OS was 97.8, 95.5, and 89.5 % in group a, group b, and group c+d (High-MMV), respectively. (C) Final OS: final OS was 97.8, 93.7, and 86.3% in group a, group b, and group c+d (High-MMV), respectively.

Supplementary Figure 4 from Activation of Insulin-like Growth Factor Signaling Induces Apoptotic Cell Death Under Prolonged Hypoxia by Enhancing Endoplasmic Reticulum Stress Response

Supplementary Table 1 from Activation of Insulin-like Growth Factor Signaling Induces Apoptotic Cell Death Under Prolonged Hypoxia by Enhancing Endoplasmic Reticulum Stress Response

Supplementary Figure 1 from Activation of Insulin-like Growth Factor Signaling Induces Apoptotic Cell Death Under Prolonged Hypoxia by Enhancing Endoplasmic Reticulum Stress Response

Supplementary Figure 2 from Activation of Insulin-like Growth Factor Signaling Induces Apoptotic Cell Death Under Prolonged Hypoxia by Enhancing Endoplasmic Reticulum Stress Response

Supplementary Figure 3 from Activation of Insulin-like Growth Factor Signaling Induces Apoptotic Cell Death Under Prolonged Hypoxia by Enhancing Endoplasmic Reticulum Stress Response

Studies have shown the prognostic significance of disseminated tumor cells (DTC) in bone marrow of patients with colorectal cancer. However, the molecular characteristics of DTCs, including their miRNA expression profiles, remain mostly unknown. In this study, we analyzed the miRNA expression of DTCs in bone marrow. EpCAM+ bone marrow cells were collected using immunomagnetic beads after exclusion of CD14+ and CD45+ cells, then subjected to miRNA microarray analysis. Cluster analysis (7 colorectal cancer patients with liver metastasis and 12 colorectal cancer patients without liver metastasis) indicated that miR-340 and miR-542-3p expressions were significantly decreased in EpCAM+ bone marrow cells of patients with liver metastasis (P = 0.019 and 0.037, respectively). We demonstrated that pre-miR-340 administration inhibited growth of colon cancer cells and suppressed c-Met expression in vitro. In clinical samples of colorectal cancer, miR-340 was expressed at significantly lower levels in tumor tissues compared with normal mucosa. Survival analysis in 136 patients with colorectal cancer indicated that low miR-340 expression was correlated with shorter 5-year disease-free survival (P = 0.023) and poor 5-year overall survival (P = 0.046). It was of note that the colorectal cancer group with low miR-340 and high c-Met expression had the worst prognosis. We further demonstrated that systemic pre-miR-340 administration suppressed growth of pre-established HCT116 tumors in animal therapeutic models. These findings indicate that miR-340 may be useful as a novel prognostic factor and as a therapeutic tool against colorectal cancer. Our data suggest that miR-340 in bone marrow may play an important role in regulating the metastasis cascade of colorectal cancer. Mol Cancer Ther; 13(4); 976–85. ©2014 AACR.

Purpose: Connexin 26 (Cx26) is one of the gap junction–forming family members classically considered to be tumor suppressors. However, recent studies show association of elevated expression of Cx26 with poor prognosis in several human malignancies. Furthermore, Cx26 has been observed to be indispensable to spontaneous metastasis of melanoma cells. Here, we assessed Cx26 expression in primary colorectal cancer (CRC) and the metastatic lesions to elucidate its role in metastasis. Experimental Design: Cx26 expression was assessed in 25 adenomas, 167 CRCs, and normal mucosa, together with the metastatic lesions. Results: Normal mucosa and adenomatous tissue expressed Cx26 mainly in the plasma membrane, whereas cancer cells mostly contained Cx26 in the cytoplasm. The incidence of aberrant Cx26 expression varied widely in CRC (mean, 49.5 ± 35.5%), and the expression levels were confirmed by Western blot and quantitative reverse transcription–PCR. Clinicopathologic survey revealed association of high expression with less differentiated histology and venous invasion (P = 0.0053 and P = 0.0084, respectively). Notably, high Cx26 expression was associated with shorter disease-free survival and shorter lung metastasis–free survival in 154 curatively resected CRC sets (P = 0.041 and P = 0.028, respectively). Survey of metastatic lesions revealed that lung metastasis, but not liver and lymph nodes metastases, expressed higher Cx26 than the CRC series or corresponding primary CRCs (P < 0.0001 and P = 0.0001, respectively). Conclusions: These findings suggest that aberrant expression of Cx26 plays an essential role in lung metastasis. Thus, Cx26 is a promising therapeutic target, particularly for CRC patients who develop lung metastasis.

Objective: This phase II trial was performed to evaluate the efficacy and tolerability of tegafur/uracil (UFT) and oral leucovorin (LV) in elderly patients with advanced or metastatic colorectal cancer who had not received prior chemotherapy. Methods: Patients aged ≥70 years were eligible. UFT and LV were taken orally on days 1-28 of the cycle at doses of 300 mg/m2/day and 75 mg/body/day, respectively. Treatment was administered on an outpatient basis every 35 days and consisted of at least two cycles until disease progression. Results: A total of 30 patients were enrolled in this study. The median age of the patients was 81.5 years (range: 74-88 years). The observed overall response rate was 17.9%. The estimated median overall survival time was 23.5 months. Two patients (7%) experienced toxicities with a worst grade of 3, and one patient (4%) experienced toxicities with a worst grade of 4. There were no treatment-related deaths. No patients experienced grade 3 or 4 hematological adverse events. Conclusions: Although the response rate to UFT/LV was moderate, a favorable survival time and good tolerability were observed. UFT/LV can be one of the optional treatments for elderly colorectal cancer patients. Citation Format: Kohei Murata, Hirofumi Yamamoto, Mutsumi Fukunaga, Takeshi Kato, Tadashi Ohnishi, Yoshio Uemura, Hirofumi Ohta, Fumihiko Kimura, Masayuki Ohue, Riichiro Nezu, Mitsugu Sekimoto, Masataka Ikeda, Tsunekazu Mizushima, Yuichiro Doki, Masaki Mori, Clinical Study Group of Osaka University (CSGO), Colorectal Group. Phase II study of first line chemotherapy with tegafur/uracil and oral leucovorin in elderly patients with advanced or metastatic colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4682. doi:10.1158/1538-7445.AM2013-4682

(Background and Aim) Oligosaccharides are one of the most important factors in the posttranslational modification of proteins and lipids. Glycomics, the systematic study of glycans and glycan-binding proteins in various biological systems, is an emerging field in the post-genomics and post-proteomics era. In particular, fucosylation is one of the most important glycosylation involved in cancer and inflammation. Fucosylation, which comprises the transfer of a fucose residue to oligosaccharides and proteins, is regulated by many kinds of molecules, including several kinds of fucosyltranferases, GDP-fucose synthetic enzymes, and GDP-fucose transporter(s). Fucosylation is increased during carcinogenesis of many cancers through the up-regulation of expression of fucosylation-regulatory genes as described above. In contrast, we found a colon cancer cell line HCT116, which had complete loss of fucosylation due to the mutation of a GDP-fucose synthetic enzyme, GMDS (GDP-mannose-4, 6-dehydratase) a few years ago. While knockout mice of a GDP-fucose synthetic enzyme are lethal (Smith et al J Cell Biol, 158, 2002), HCT116 cells showed more aggressive pheno-type, including in vivo tumor growth and metastasis. The underlying mechanisms were found to be an escape from TRAIL-mediated immuno-surveillance of NK cells (Moriwaki et al, Gastroenterology 137, 2009). In the present study, we investigated whether or not the GMDS mutation was really found in clinical samples of colorectal cancer. (Subjects and Methods) Thirteen cases of liver metastasis, 6 cases of lymph node metastasis and 81 cases of the original cancer tissues derived from patients with colorectal cancer were included in this study. All projects were approved by the ethical committee of Osaka University. GMDS mutation was analyzed by RT-PCR using primers, which include all exons of GMDS gene. Immunohistochemical experiments were performed using AAL lectin, which recognizes all types of fucosylation. (Results and Discussion) Approximately 10-15% of GMDS mutation was found in the original cancer tissue as well as their metastatic liver cancer and lymph node metastasis. However no mutation was found in normal colon tissue around cancer. Most GMDS mutation was a hetero-type, but not a homo-type, which was found in HCT 116 cells. Immunohistochemical analysis showed complete loss of fucosylation in a few cases of metastatic cancer. These data indicate the involvement of GMDS mutation in the progression of colorectal cancer. Citation Format: Kotarosumitomo Nakayama, Kenta Moriwaki, Mayuka Shimomura, Hironobu Fujii, Shinji Takamatsu, Yoshihiro Kamada, Kohei Murata, Eiji Miyoshi. Clinical significance of GDP-mannose-4,6-dehydratase mutation and loss of fucosylation in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2684. doi:10.1158/1538-7445.AM2013-2684

Background: Fucosylation is a crucial oligosaccharide modification in cancer and inflammation. Total cellular proteins of cancer cells and the sera of patients with cancer both show increased fucosylation levels. Certain kinds of fucosylated proteins can be applied as novel cancer biomarkers in glyco-proteomic analyses. We previously identified fucosylated haptoglobin (Fuc-Hpt) as a serologic marker for pancreatic cancer, and recently developed a lectin-antibody enzyme-linked immunosorbent assay system for quantifying Fuc-Hpt. In the present study, we investigated the clinical outcome of Fuc-Hpt levels in patients with colorectal cancer (CRC), and examined the mechanisms underlying Fut-Hpt production using a murine tumor transplantation model. Methods: The relationship between Fuc-Hpt levels and clinical parameters was investigated in 77 patients with CRC, all of whom underwent primary resection. Serum Fuc-Hpt levels were examined in athymic nude mice injected with colon cancer cell lines that lacked fucosylation. Results: Fuc-Hpt levels were significantly associated with overall and relapse-free survival, distant metastasis, clinical stage, and curability. Multivariate analysis revealed that distant metastasis was an independent factor for increased Fuc-Hpt levels. The combination of Fuc-Hpt and CEA might be a better serologic marker to predict prognosis. Fuc-Hpt levels were higher in mice with direct injection of tumor cells into the spleen than in those injected subcutaneously. Conclusions: Fuc-Hpt might be a useful marker for the prognosis of CRC. Fuc-Hpt could be produced by the tissue surrounding tumor cells, which might be the mechanism underlying Fuc-Hpt elevation associated with distant metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4531. doi:1538-7445.AM2012-4531

Background: Incorporation of molecular targeting agents such as bevacizumab, cetuximub, and panitumumab, into cytotoxic chemotherapeutic regimens has increased survival in colorectal cancer patients, resulted in provision of a promising means of individualization of these therapies. Nonetheless, the clinical efficacy and toxicity of basic regimens, such as FOLFIRI and FOLFOX, remain unpredictable. Thus, identification of molecular indicators of individual response to the basic regimens is urgently required. Methods: This prospective study was conducted to determine potent predictive markers of individual response to mFOLFOX6, in terms of efficacy and toxicity. Chemonaive patients with stage IV colorectal cancer were eligible for this trial after a palliative operation, and received mFOLFOX6 treatment. Along with a search for KRAS gene somatic mutations, analyses of mRNA expression levels in cancerous portions were conducted for 10 candidate marker genes, namely DPYD, TYMS, ERCC1, ERCC2, XRCC1, GSTP1, EGFR, VEGF, TYMP and TNFRSF1B, by means of real-time RT-PCR. In addition, some acknowledged polymorphisms of DPYD, TYMS, ERCC1, ERCC2, XRCC1, GSTP1 and EGFR genes were also determined. Results: Fifty pts received a total of 424 treatment cycles. Overall response rate was 60.4% (1CR and 28 PRs), and median overall and progression survival were 930 days and 234 days, respectively. The most common grade 3/4 toxicity was neutropenia (40%), followed by anorexia (6%), anemia (4%), leucopenia (4%), etc. The therapeutic response was not related to KRAS somatic mutational status, and no genotype-toxicity association was observed, thus far tested. On the other hand, ERCC1, ERCC2, and TYMS gene expression levels were significantly higher in patients with PD than in those with CR/PR/SD (P=0.017, 0.028, and 0.032, respectively). The expression levels of TYMS were highly correlative with best tumor response rate (R=0.414, P=0.004). Overall survival was significantly better in patients with low ERCC2 expression (P=0.039). Furthermore, combined expression levels of ERCC1, ERCC2, and TYMS were found to be associated with PFS: Those patients with high expression levels of all of these 3 genes showed poorer survival than the others (P=0.0029). Conclusions: Expression levels of ERCC1, ERCC2, and TYMS genes in tumor may be predictable for individual response to mFOLFOX6. A large-scale prospective study to validate their predictive values is now under planning. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4134. doi:10.1158/1538-7445.AM2011-4134