Kohei Kume

Contrasting expression patterns of histone mRNA and microRNA 760 in patients with gastric cancer

Purpose: Recent studies revealed that both disseminated tumor cells and noncancerous cells contributed to cancer progression cooperatively in the bone marrow. Here, RNA-seq analysis of bone marrow from gastric cancer patients was performed to identify prognostic markers for gastric cancer. Experimental Design: Bone marrow samples from eight gastric cancer patients (stages I and IV: n = 4 each) were used for RNA-seq analysis. Results were validated through quantitative real-time PCR (qRT-PCR) analysis of HIST1H3D expression in 175 bone marrow, 92 peripheral blood, and 115 primary tumor samples from gastric cancer patients. miR-760 expression was assayed using qRT-PCR in 105 bone marrow and 96 primary tumor samples. Luciferase reporter assays were performed to confirm whether histone mRNAs were direct targets of miR-760. miR-760 expression was also evaluated in noncancerous cells from gastric cancer patients. Results: RNA-seq analysis of bone marrow samples from gastric cancer patients revealed higher expression of multiple histone mRNAs in stage IV patients. HIST1H3D expression in the bone marrow, peripheral blood, and primary tumor of stage IV patients was higher than that in stage I patients (P = 0.0284, 0.0243, and 0.0006, respectively). In contrast, miR-760 was downregulated in the bone marrow and primary tumor of stage IV patients compared with stage I patients (P = 0.0094 and 0.0018, respectively). Histone mRNA and miR-760 interacted directly. Furthermore, miR-760 was downregulated in noncancerous mucosa in stage IV gastric cancer patients. Conclusion: Histone mRNA was upregulated, whereas miR-760 was downregulated in the bone marrow and primary tumor of advanced gastric cancer patients, suggesting that the histone mRNA/miR-760 axis had a crucial role in the development of gastric cancer. Clin Cancer Res; 19(23); 6438–49. ©2013 AACR.

Individualized Mutation Detection in Circulating Tumor DNA for Monitoring Colorectal Tumor Burden Using a Cancer-Associated Gene Sequencing Panel

Background Circulating tumor DNA (ctDNA) carries information on tumor burden. However, the mutation spectrum is different among tumors. This study was designed to examine the utility of ctDNA for monitoring tumor burden based on an individual mutation profile. Methodology DNA was extracted from a total of 176 samples, including pre- and post-operational plasma, primary tumors, and peripheral blood mononuclear cells (PBMC), from 44 individuals with colorectal tumor who underwent curative resection of colorectal tumors, as well as nine healthy individuals. Using a panel of 50 cancer-associated genes, tumor-unique mutations were identified by comparing the single nucleotide variants (SNVs) from tumors and PBMCs with an Ion PGM sequencer. A group of the tumor-unique mutations from individual tumors were designated as individual marker mutations (MMs) to trace tumor burden by ctDNA using droplet digital PCR (ddPCR). From these experiments, three major objectives were assessed: (a) Tumor-unique mutations; (b) mutation spectrum of a tumor; and (c) changes in allele frequency of the MMs in ctDNA after curative resection of the tumor. Results A total of 128 gene point mutations were identified in 27 colorectal tumors. Twenty-six genes were mutated in at least 1 sample, while 14 genes were found to be mutated in only 1 sample, respectively. An average of 2.7 genes were mutated per tumor. Subsequently, 24 MMs were selected from SNVs for tumor burden monitoring. Among the MMs found by ddPCR with > 0.1% variant allele frequency in plasma DNA, 100% (8 out of 8) exhibited a decrease in post-operation ctDNA, whereas none of the 16 MMs found by ddPCR with < 0.1% variant allele frequency in plasma DNA showed a decrease. Conclusions This panel of 50 cancer-associated genes appeared to be sufficient to identify individual, tumor-unique, mutated ctDNA markers in cancer patients. The MMs showed the clinical utility in monitoring curatively-treated colorectal tumor burden if the allele frequency of MMs in plasma DNA is above 0.1%.

A compensatory role of NF-κB to p53 in response to 5-FU-based chemotherapy for gastric cancer cell lines.

Despite of remarkable improvement of postoperative 5-FU–based adjuvant chemotherapy, the relapse rate of gastric cancer patients who undergo curative resection followed by the adjuvant chemotherapy remains substantial. Therefore, it is important to identify prediction markers for the chemotherapeutic efficacy of 5-FU. We recently identified NF-κB as a candidate relapse prediction biomarker in gastric cancer. To evaluate the biological significance of NF-κB in the context of 5-FU–based chemotherapy, we analyzed the NF-κB-dependent biological response upon 5-FU treatment in gastric cancer cell lines. Seven genes induced by 5-FU treatment in an NF-κB-dependent manner were identified, five of which are known p53 targets. Knockdown of RELA, which encodes the p65 subunit of NF-κB, decreased both p53 and p53 target protein levels. In contrast, NF-κB was not affected by TP53 knockdown. We also demonstrated that cell lines bearing Pro/Pro homozygosity in codon72 of p53 exon4, which is important for NF-κB binding to p53, are more resistant to 5-FU than those with Arg/Arg homozygosity. We conclude that NF-κB plays an important role in the response to 5-FU treatment in gastric cancer cell lines, with a possible compensatory function of p53. These results suggest that NF-κB is a potential 5-FU-chemosensitivity prediction marker that may reflect 5-FU-induced stress-response pathways, including p53.

Molecular marker identification for relapse prediction in 5-FU-based adjuvant chemotherapy in gastric and colorectal cancers.

To confirm the clinical significance of NF-κB and JNK protein expression from experimentally identified candidates for predicting prognosis for patients with 5-FU treatment, we evaluated the protein expression of surgically removed specimens. A total of 79 specimens were obtained from 30 gastric and 49 colorectal cancer patients who underwent R0 resection followed by postoperative 5-FU based adjuvant chemotherapy. Immunohistochemical examinations of NF-κB and JNK on tissue microarrays (TMAs) revealed that significantly shorter time-to-relapse (TTR) in both NF-κB(+) and JNK(−) subgroups in both gastric (NF-κB(+), p = 0.0002, HR11.7. 95%CI3 3.2–43.4; JNK(−), p = 0.0302, HR4.4, 95%CI 1.2–16.6) and colon (NF-κB(+), p = 0.0038, HR36.9, 95%CI 3.2–426.0; JNK(−), p = 0.0098, HR3.2, 95%CI 1.3–7.7) cancers. These protein expression patterns also show strong discriminately power in gastric cancer patients for overall survival rate, suggesting a potential utility as prognostic or chemosensitivity markers. Baseline expression of these proteins using gastric cancer cell lines demonstrated the reciprocal patterns between NF-κB and JNK, while 5-FU exposure of these cell lines only induced NF-κB, suggesting that NF-κB plays a dominant role in the response to 5-FU. Subsequent siRNA experiments confirmed that gene knockdown of NF-κB increased 5-FU-specific sensitivity, whereas that of JNK did not affect the chemosensitivity. These results suggest that the expression of these proteins may aid in the decisions involved with adjuvant chemotherapy for gastrointestinal tract cancers.

α-Amanitin Restrains Cancer Relapse from Drug-Tolerant Cell Subpopulations via TAF15.

Cancer relapse occurs with substantial frequency even after treatment with curative intent. Here we studied drug-tolerant colonies (DTCs), which are subpopulations of cancer cells that survive in the presence of drugs. Proteomic characterization of DTCs identified stemness- and epithelial-dominant subpopulations, but functional screening suggested that DTC formation was regulated at the transcriptional level independent from protein expression patterns. We consistently found that α-amanitin, an RNA polymerase II (RNAPII) inhibitor, effectively inhibited DTCs by suppressing TAF15 expression, which binds to RNA to modulate transcription and RNA processing. Sequential administration of α-amanitin and cisplatin extended overall survival in a cancer-relapse mouse model, namely peritonitis carcinomatosa. Therefore, post-treatment cancer relapse may occur through non-distinct subpopulations and may be effectively prevented by α-amanitin to disrupt transcriptional machinery, including TAF15.

Evaluation of chemosensitivity prediction using quantitative dose–response curve classification for highly advanced/relapsed gastric cancer

BackgroundThe use of standard chemotherapy regimens has changed the application of chemosensitivity tests from all chemotherapy-eligible patients to those who have failed standard chemotherapy, which includes patients with highly advanced, relapsed, or chemoresistant tumors.MethodsWe evaluated a total of 43 advanced primary and relapsed gastric cancers for chemosensitivity based on drug dose response curves to improve the objectivity and quality of quantitative measurements. The dose response curves were classified based on seven expected patterns. Instead of a binary chemosensitivity evaluation, we ranked drug sensitivity according to curve shapes and comparison with the peak plasma concentration (ppc) of each drug.ResultsA total of 193 dose response curves were obtained. The overall informative rate was 67.4%, and 85.3% for cases that had a sufficient number of cells. Paclitaxel (PXL)and docetaxel tended to show a higher rank, while cisplatin (CIS) and 5-fluorouracil (5-FU) tended to show resistance, particularly among the 20 cases (46.5%) that had recurrent disease after receiving chemotherapy with CIS and S-1 (5-FU). As such, we speculate that the resistant pattern of the chemosensitivity test suggests that cells with acquired drug resistance were selected by chemotherapy. Indeed, we observed a change in the chemosensitivity pattern of a sample before and after chemotherapy in terms of PXL sensitivity, which was used after primary chemotherapy.ConclusionsThese results suggest that: (i) the dose–response pattern provides objective information for predicting chemosensitivity; and (ii) chemotherapy may select resistant cancer cell populations as a result of the therapy.

Ductular reactions in the liver regeneration process with local inflammation after physical partial hepatectomy

Partial hepatectomy models in mice have been widely used for liver regeneration studies. A typical procedure removes ~2/3 of the liver by lobular ligation without tissue dissection. However, hepatectomy in humans involves physical damage (ie, physical partial hepatectomy, PPHx). Therefore, the liver regeneration process after PPHx should involve reactions to acute local injury followed by systematic remodeling. To clarify the liver regeneration process after PPHx, we used a murine liver injury model that mimics the actual human surgical procedure. A 20–30% PPHx was performed by transection of the left lobe of the liver using an ultrasonically activated scalpel in mice. Gene expression and morphological characteristics were analyzed during the liver regeneration process. Liver weight continuously increased by hypertrophic reaction of hepatocytes, whereas Ki67 staining showed hepatocyte proliferation. At the transected border, emergence of ductular reactions, a representative process of hepatic tissue remodeling that contain liver stem/progenitor cells, were observed. Gene expression of the transected border and non-damaged lobes revealed that inflammatory cytokine- and extracellular matrix-associated genes were significantly upregulated at the transected border. Our PPHx model triggered local extracellular matrix remodeling that resulted in ductular reactions. These processes occurred during the tissue repair process in local inflammatory responses as well as compensatory hepatocyte hypertrophy of the entire liver. These findings may provide insight for elucidating the mechanism of tissue repair and regeneration of the liver after PPHx.

Abstract 5201: MicroRNA 760 regulates gastric cancer progression by downregulation of histone mRNA expression

The presence of isolated tumor cells in the peripheral blood (PB) and bone marrow (BM) is an important factor contributing to the metastasis of solid cancers. Moreover, recent studies have demonstrated that various types of host cells are also involved in cancer development and metastasis. BM microenvironment is thought to be a good reflection of the interplay between tumor cells and cancer-associated host cells in metastatic sites compared to the bloodstream. We performed RNA-seq analysis of the BM samples from 8 gastric cancer (GC) patients (Stages I and IV: n = 4 each) in order to identify candidate prognostic markers, and revealed higher expression of multiple histone mRNAs in Stage IV patients. Results were validated through qRT-PCR analysis of HIST1H3D expression in 175 BM, 92 PB, and 115 primary tumor (PT) samples from GC patients. HIST1H3D expression in the BM, PB, and PT of Stage IV patients was higher than that in Stage I patients (p = 0.0284, 0.0243, 0.0006, respectively). In silico analysis revealed that most of the histone genes upregulated in BM samples from Stage IV GC patients had predictive target sites for microRNA 760 (miR-760) in their 3′ UTRs. miR-760 expression was assayed using qRT-PCR in 105 BM and 96 PT samples. In contrast to histone mRNA expression, miR-760 was downregulated in the BM and PT of Stage IV patients compared to Stage I patients (p = 0.0094 and 0.0018, respectively). Direct interaction between miR-760 and histone mRNA was confirmed by luciferase analysis in a GC cell line, NUGC3. HIST1H3D expression was downregulated in 3 GC cell lines (NUGC3, KE39, and KATOIII) after Pre-miR-760 transfection in both mRNA and protein levels. We also examined miR-760 expression levels of noncancerous host cells in samples from GC patients. In BM samples from another set of 4 GC patients, the CD14 positive fraction exhibited the same degree of miR-760 expression compared to a CD45/EpCAM+ fraction. Moreover, in corresponding noncancerous tissues from primary gastric tumors, miR-760 expression was lower in Stage IV patients than in Stage I patients. These results suggest that changes in miR-760 expression in host noncancerous cells are also associated with GC progression. In conclusion, histone mRNA is upregulated, while miR-760 is downregulated in the BM and PT of advanced GC patients and proposed that the histone mRNA/miR-760 axis may have a crucial role in the development of GC. Citation Format: Takeshi Iwaya, Takeo Fukagawa, Yutaka Suzuki, Tomoya Sudo, Kaoru Ishida, Kohei Kume, Satoshi Nishizuka, Hisae Iinuma, Masaki Mori, Mitsuru Sasako, Go Wakabayashi, Koshi Mimori. MicroRNA 760 regulates gastric cancer progression by downregulation of histone mRNA expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5201. doi:10.1158/1538-7445.AM2014-5201

Inhibition of PI3K suppresses propagation of drug-tolerant cancer cell subpopulations enriched by 5-fluorouracil

Drug-tolerant cancer cell subpopulations are responsible for relapse after chemotherapy. By continuously exposing the gastric cancer cell line MKN45 to 5-FU for >100 passages, we established a 5-fluorouracil (5-FU)-tolerant line, MKN45/5FU. Orthotopic xenografts of MKN45/5FU cells in the stomach of nude mice revealed that these cells had a high potential to metastasize to sites such as the liver. Levels of phosphorylated phosphatidylinositide 3-kinase (PI3K) increased both in 5-FU-tolerant subpopulations according to the 5-FU dose, and in gastric submucosal orthotopic xenografts of MKN45/5FU cells. Sequential administration of 5-FU and a PI3K inhibitor, GDC-0941, targeted the downstream ribosomal S6 kinase phosphorylation to significantly suppress 5-FU-tolerant subpopulations and tumor propagation of orthotopic MKN45/5FU xenografts. These results suggest that administration of 5-FU followed by GDC-0941 may suppress disease relapse after 5-FU-based gastric cancer chemotherapy.

Abstract 5683: Evaluation of the utilization of blood collection tubes for cell-free DNA research

Many studies have been conducted investigating cell-free DNA (cfDNA) for disease diagnosis. To prevent cellular DNA release into the plasma, plasma separation within 2 hours of blood collection is required, when utilizing the most commonly used type of tube (BD Vacutainer® CPTTM; CPT). These temporal restrictions render operations requiring plasma separation a laborious task. It has been demonstrated that Cell-Free DNA BCT® tubes (Streck Inc; BCTs) prevented nucleated cell disruption. The use of BCTs may enable us to separate plasma without temporal restrictions. The aim of this study was to evaluate changes in DNA levels in plasma after preservation using two types of collection tubes. Samples were drawn from 7 healthy donors into CPTs and BCTs, and stored at room temperature. Plasma was separated immediately (d0), and on days 3, 6, 9, 12, and 14 after blood collection. The concentrations of DNA in the extracted plasma samples were measured. Blood stored in CPTs showed increases in plasma DNA concentrations over time. The mean DNA concentrations at each time-point were 0.39 (d0), 2.96 (d3), 40.4 (d6), 84.9 (d9), 91.4 (d12), and 94.9 ng/μl (d14). However, plasma DNA levels remained stable in BCTs from 6 days. There were significant differences between the DNA concentrations in BCTs on d0 (0.74 [0.43-1.44] ng/µl) and d14 (1.78 [0.63-4.20] ng/µl) (p = 0.0378). Although significant differences were only observed between d0 and d14 in BCTs, the amount of plasma DNA gradually increased after d6. Furthermore, obvious color changes in the plasma were observed, and the boundary between plasma and PBMCs became unclear over time. These results indicate that BCTs may yield stable cfDNA samples for up to 6 days after blood collection. BCTs may be a novel collection tube for cfDNA research, including liquid biopsies in cancer patients. Citation Format: Fumitaka Endo, Takeshi Iwaya, Takehiro Chiba, Mizunori Yaegashi, Kei Sato, Kohei Kume, Atsuhiro Arisue, Yutaka Nishinari, Ryoko Kawagishi, Takenori Segawa, Satoshi Nishizuka, Akira Sasaki. Evaluation of the utilization of blood collection tubes for cell-free DNA research [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 5683. doi:10.1158/1538-7445.AM2017-5683