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

Supplementary Data

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

Supplementary Fig. S2. Evaluation of the toxicity of sCA-ICG conjugate in vivo. (A) Body weight loss was evaluated for the PBS control (n = 3), treated with sCA alone (n = 3), ICG (n = 3) and sCA-ICG (n = 4). Mice received intravenous administrations of PBS, sCA, ICG and sCA-ICG on days 0, 2, 5, 7, 9 and 12 via a tail vein injection (arrows indicate days of injections). Data represent mean {plus minus} SD. (B) Blood chemistry tests were performed on day 14. (C) Mice were sacrificed on day 14. Hematoxylin and eosin-stained sections of each organ tissue (heart, lung, liver, spleen, kidney) revealed no particular histological damage in sCA-ICG-treated mice. Bars: 50mm. sCA: super carbonate apatite.

Table S2

Immunohistochemical analysis of primary tumor and lymph node with/without tumor metastasis for PTBP1. In lymph node metastasis, metastatic lesions were positive for PTBP1 to the contrary, no staining was observed in intact lymph node in independent two cases. Scale bar: 100 μm.

PTBP1 was preferentially overexpressed in CRC specimens. (A) qRT-PCR for PTBP1 mRNA levels in CRC specimens (n = 178) and corresponding normal mucosal specimens (n = 80). PTBP1 mRNA expression levels in CRCs were significantly higher than in normal samples. ***, significant difference, P < 0.0001. (B) Representative immunohistochemical analysis for PTBP1: (a) normal mucosa; (b) well-differentiated adenocarcinoma; (c) moderately differentiated adenocarcinoma; and (d) poorly differentiated adenocarcinoma. Scale bar: 100 μm.

(A) Representative immunohistochemical analysis of clinical samples. Scale bar: 100 μm. (B) Correlation between mRNA and protein levels of PTBP1 in clinical specimens. PTBP1 low-expression group determined by immunohistochemistry (n = 5) also had significantly lower PTBP1 mRNA levels than the PTBP1 protein high-expression group (n = 11) (P = 0.012).

Immunohistochemical analysis of primary tumor for PTBP1 (Upper) and CDK11p58 (lower). The expression of PTBP1 and CDK11p58 was significantly correlated (P=0.025).

PDF - 364K, List of microRNA array results.

We previously demonstrated that miR-29b-3p is a hopeful miRNA-based therapy against colorectal cancer. In this study, we aimed to clarify a value of miR-29b-1-5p as a next-generation treatment, especially for KRAS-mutant colorectal cancer. RT-PCR assay showed that the expression of miR-29b-3p was high, and its partner strand, miR-29b-1-5p, level was only negligible in clinical colorectal cancer samples. Mimic-miR-29b-1-5p significantly inhibited proliferation of KRAS-mutant colorectal cancer cell lines DLD1 and SW480 and KRAS wild-type HT29 cells. Proliferative activity was further examined by either miR-29b-1-5p strand or its opposite complementary sequence because miR-29b-1-5p is a passenger miRNA and may have no physiologic function. We found that completely opposite complementary strand to miR-29b-1-5p, but not miR-29b-1-5p, possessed a potent antitumor effect and named this byproduct miRNA sequence “MIRTX.” MIRTX directly targeted the 3′-UTR of CXCR2 and PIK3R1 mRNA and suppressed the NF-κB signaling pathway in KRAS-mutated colorectal cancer cells. MIRTX induced apoptosis in DLD1 with downregulation of antiapoptotic BCL2, BCL-xL, and MCL1 and upregulation of cleaved caspase-3 and cleaved PARP. In mouse xenograft models, systemic administration of MIRTX using a super carbonate apatite as a delivery vehicle significantly inhibited tumor growth of DLD1 and HT29 cells without any particular toxicities. In conclusion, these findings indicate that inhibition of NF-κB signaling by this novel miRNA-based therapeutic could be a promising treatment against refractory KRAS-mutant colorectal cancer and KRAS wild-type colorectal cancer. Mol Cancer Ther; 17(5); 977–87. ©2018 AACR.

Minimally invasive treatment is getting more and more important in an aging society. The purpose of this study was to explore the possibility of ICG loaded on super carbonate apatite (sCA) nanoparticles as a novel photodynamic therapy (PDT) against cancers. Using colon cancer cells, ICG uptake and anti-tumor effects were examined between the treatments of ICG and sCA-ICG. Reactive oxygen species (ROS) production and temperature rise were also evaluated to explore the underlying mechanism. Atomic force microscopy revealed that the size of sCA-ICG ranged from 10 to 20 nm. In aqueous solution with 0.5% albumin, the temperature increase after laser irradiation was 27.1°C and 23.1°C in sCA-ICG and ICG, respectively (control DW: 5.7°C). A significant increase in ROS generation was noted in cell cultures treated with sCA-ICG plus irradiation compared with those treated with ICG plus irradiation (P < 0.01). Uptake of ICG in the tumor cells significantly increased in sCA-ICG compared with ICG in vitro and in vivo. The fluorescence signals of ICG in the tumor, liver, and kidney faded away in both treatments by 24 hours. Finally, the HT29 tumors treated with sCA-ICG followed by irradiation exhibited drastic tumor growth retardation (P < 0.01), whereas irradiation of tumors after injection of ICG did not inhibit tumor growth. This study shows that sCA is a useful vehicle for ICG-based PDT. Quick withdrawal of ICG from normal organs is unique to sCA-ICG and contrasts with the other nanoparticles remaining in normal organs for a long time. Mol Cancer Ther; 17(7); 1613–22. ©2018 AACR.

(A) Survival rate after surgery of two groups divided according to PTBP1 mRNA expression. The high PTBP1 mRNA group had significantly poorer prognosis (P = 0.0138, log-rank test). (B) Disease-free rate after curative surgery in the two groups divided according to PTBP1 mRNA expression. The high PTBP1 mRNA group had significantly poorer prognosis (P = 0.0083, log-rank test).

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

Figure S1

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.

Survival rate after surgery of two groups divided according to CD44v8-10 mRNA expression. The high CD44v8-10 expression group had significantly poor prognosis (P = 0.0264).

Representative immunohistochemical observation of the invasive front of early stage CRC specimens. Grouping described in Table 1 was according to these findings. Scale bar: 100 μm.

Fig. S1. mRNA expressions and mutations of crenolanib targeting tyrosine kinase receptors in various cancers. Fig. S2. Cells expressing EGFP under Oct3/4 promoter Table S1. The culture medium used for starvation and experiments for colorectal cancer cell line Table S2. Modified stem cell culture medium Table S3. The culture medium used for starvation and experiments for patients derived organoid Table S4. Primer sequences and universal probe libraries Table S5. Primary antibodies used in western blotting analysis

Primers and probes used for qRT-PCR in this study.

74 candidate target genes of MIRTX in DLD1

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

Correlation between MYC and PTBP1 levels in CRC. (A) qRT-PCR of clinical specimens. Expression of PTPB1 mRNA in the MYC mRNA high-expression group (n = 32) was significantly higher than in the MYC low-expression group (n = 32) (P = 0.018). (B) Western blot for PTBP1 protein levels in control RNA or siMYC of HCT116, DLD1, HT29, and SW480.

(A) Strategy for detecting the CD44 variant region. Arrows indicate primers listed in Supplemental Table S1. (B) RT-PCR followed by electrophoresis for CD44 variant region in CRC cell lines. (C) Sequence analysis.

SI legends

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.

Supplementary Figures S1-S5; Supplementary Table S1.

Additional methods used in this study.

Supplementary Data from Three-Year Follow-Up and Response–Survival Relationship of Nivolumab in Previously Treated Patients with Advanced Esophageal Squamous Cell Carcinoma (ATTRACTION-3)

This figure shows p53 status in ESCC cells and the effect of p53 rerated signals after irradiation and SOCS1 over expression. ESCC cells in this study harbored p53 mutation and p53-related response by IR and/or SOCS1 over expression was not observed in our study.

Expression of DKK1 and DKK3 in public database.

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.

Dickkopf3 (DKK3) is a secretory protein that belongs to the DKK family, but exhibits structural divergence from other family members, and its corresponding receptors remain to be identified. Although DKK3 has been shown to have oncogenic functions in certain cancer types, the underlying mechanism by which DKK3 promotes tumorigenesis remains to be clarified. We show here that DKK3 stimulates esophageal cancer cell proliferation via cytoskeleton-associated protein 4 (CKAP4), which acts as a receptor for DKK3. DKK3 was expressed in approximately 50% of tumor lesions of esophageal squamous cell carcinoma (ESCC) cases; simultaneous expression of DKK3 and CKAP4 was associated with poor prognosis. Anti-CKAP4 antibody inhibited both binding of DKK3 to CKAP4 and xenograft tumor formation induced by ESCC cells. p63, a p53-related transcriptional factor frequently amplified in ESCC, bound to the upstream region of the DKK3 gene. Knockdown of p63 decreased DKK3 expression in ESCC cells, and reexpression of DKK3 partially rescued cell proliferation in p63-depleted ESCC cells. Expression of ΔNp63α and DKK3 increased the size of tumor-like esophageal organoids, and anti-CKAP4 antibody inhibited growth of esophageal organoids. Taken together, these results suggest that the DKK3-CKAP4 axis might serve as a novel molecular target for ESCC.Significance: In esophageal cancer, findings identify DKK3 as a poor prognostic indicator and demonstrate CKAP4 inhibition as an effective therapeutic strategy. Cancer Res; 78(21); 6107–20. ©2018 AACR.

Supplementary Figures S1-S3; Supplementary Table S1.

Supplementary Table from Three-Year Follow-Up and Response–Survival Relationship of Nivolumab in Previously Treated Patients with Advanced Esophageal Squamous Cell Carcinoma (ATTRACTION-3)

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

This text is Figure Legends of Supplementary Figure S1 and S2.

Purpose:Limited long-term data are available on immune checkpoint inhibitor use in patients with advanced esophageal squamous cell carcinoma (ESCC). We report 3-year follow-up data from our study of nivolumab versus chemotherapy (paclitaxel or docetaxel) in patients with previously treated ESCC.Patients and Methods:ATTRACTION-3 was a randomized, multicenter, open-label, phase III trial. Overall survival (OS), time from randomization to death from any cause, was the primary endpoint. An exploratory subanalysis assessed OS according to the best overall response (BOR) with and without landmark at 4 months.Results:Of the enrolled patients, 210 received nivolumab and 209 received chemotherapy. With a minimum follow-up of 36.0 months, OS was longer in the nivolumab versus the chemotherapy group (median, 10.9 vs. 8.5 months; HR, 0.79; P = 0.0264), with 3-year OS rates of 15.3% and 8.7%, respectively. The median OS was longer with nivolumab versus chemotherapy irrespective of the BOR (complete response/partial response: 19.9 vs. 15.4 months; stable disease: 17.4 vs. 8.8 months; and progressive disease: 7.6 vs. 4.2 months). Grade 3 or higher treatment-related adverse events were reported in 40 patients (19.1%) in the nivolumab group and 133 patients (63.9%) in the chemotherapy group.Conclusions:Nivolumab as second-line therapy demonstrated clinically meaningful long-term improvement in OS compared with chemotherapy in previously treated patients with advanced ESCC. The OS was consistently improved in the nivolumab group compared with the chemotherapy group regardless of BOR. Nivolumab was well tolerated over the 3-year follow-up.See related commentary by Yoon et al., p. 3173

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).

Supplementary Figure legend

Purpose: This study aimed to identify novel hypoxia-inducible and prognostic markers in vivo from hypoxic tumor cells.Experimental Design: Using carbonic anhydrase 9 and CD34 as a guide for hypoxic tumor cells, laser capture microdissection was used to isolate colorectal cancer (CRC) liver metastases. The samples were analyzed by microarray analysis, in parallel with five CRC cell lines cultured under hypoxic conditions. To evaluate the prognostic impact of the expression of certain genes, samples from a total of 356 CRC patients were analyzed by microarray or quantitative reverse transcription-PCR. In vitro mechanistic studies and in vivo therapeutic experiments were also done about a histone H3 Lys9 demethylase, Jumonji domain containing 1A (JMJD1A).Results: Several candidate genes were identified by microarray analysis of liver metastases and culturing of CRC cells under hypoxic conditions. Among them, we found that JMJD1A was a novel independent prognostic factor for CRC (P = 0.013). In vitro assays revealed that loss of JMJD1A by small interfering RNA treatment was associated with a reduction of proliferative activity and decrease in invasion of CRC cell lines. Furthermore, treatment with an adenovirus system for antisense JMJD1A construct displayed prominent therapeutic effects when injected into established tumor xenografts of the CRC cell lines HCT116 and DLD1.Conclusions: JMJD1A is a useful biomarker for hypoxic tumor cells and a prognostic marker that could be a promising therapeutic target against CRC. Clin Cancer Res; 16(18); 4636–46. ©2010 AACR.

Univariate (Supplementary Table S3) and multivariate (Supplementary Table S4) analysis of overall survival.

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.

PDF file, 87KB, Tissue section stained with hematoxylin and eosin before (left) and after (right) laser microdissection in six representative samples of colorectal cancer. (Part II)

Supplementary Figure S1 and Supplementary Tables S1-S3.

Relationship between DKK3 and CKAP4 expression and clinicopathological characteristics of ESCC.