Yuhki Yokoyama

Cancer‐associated upregulation of histone H3 lysine 9 trimethylation promotes cell motility in vitro and drives tumor formation in vivo

Global histone modification patterns correlate with tumor phenotypes and prognostic factors in multiple tumor types. Recent studies suggest that aberrant histone modifications play an important role in cancer. However, the effects of global epigenetic rearrangements on cell functions remain poorly understood. In this study, we show that the histone H3 lysine 9 (H3K9) methyltransferase SUV39H1 is clearly involved in regulating cell migration in vitro. Overexpression of wild‐type SUV39H1, but not enzymatically inactive SUV39H1, activated migration in breast and colorectal cancer cells. Inversely, migration was reduced by knockdown of SUV39H1 or chemical inhibition by chaetocin. In addition, H3K9 trimethylation (H3K9me3) was specifically increased in invasive regions of colorectal cancer tissues. Moreover, the presence of H3K9me3 positively correlated with lymph node metastasis in colorectal cancer patients. Furthermore, overexpression of SUV39H1 drove tumorigenesis in mouse, resulting in a considerable decrease in survival rate. These data indicate that H3K9 trimethylation plays an important role in human colorectal cancer progression, possibly by promoting collective cell invasion.

Clinical significance of circulating galectins as colorectal cancer markers

The utility of CEA and CA19-9 as colorectal carcinoma (CRC) markers is limited and development of additional reliable markers is under investigation. We previously showed that galectin-1 is overexpressed in CRC tissues. If such a protein leaks into the peripheral circulation, it might constitute a tumor marker candidate. Here, we test the hypothesis that the levels of circulating galectins could reflect the presence of CRC and/or its progression state. We constructed sandwich ELISAs for galectin-1/-2/-3/-4/-7 and determined their plasma concentrations in 105 CRC patients and 100 healthy volunteers (control). Matched pair samples of 56 patients pre- and post-surgery were also subjected to ELISA analysis. Circulating levels of galectin-1/-3/-4 in CRC patients were significantly higher compared to those in controls. Galectin-1 and galectin-4 levels significantly decreased after surgery (P<0.01), and the level of galectin-4 in most patients fell below the cut-off value. The levels of circulating galectin-4 significantly increased as the tumor stage progressed (P<0.001), whereas those for galectin-1 were relatively high from an early stage. Combined use of galectin-4 with CEA and/or CA19-9 markedly increased the proportion of CRC patients who were positive for tumor markers (from 33.3 to 59.0% for CEA and from 17.1 to 51.4% for CA19-9). Our data show that galectin-4 may be a tumor marker for use in patient follow-up, while galectin-1 could be used for tumor screening. In particular, galectin-4 can be useful as a complementary marker when combined with CEA/CA19-9 to improve CRC follow-up.

Loss of histone H4K20 trimethylation predicts poor prognosis in breast cancer and is associated with invasive activity.

IntroductionLoss of histone H4 lysine 20 trimethylation (H4K20me3) is associated with multiple cancers, but its role in breast tumors is unclear. In addition, the pathological effects of global reduction in H4K20me3 remain mostly unknown. Therefore, a major goal of this study was to elucidate the global H4K20me3 level in breast cancer tissue and investigate its pathological functions.MethodsLevels of H4K20me3 and an associated histone modification, H3 lysine 9 trimethylation (H3K9me3), were evaluated by immunohistochemistry in a series of breast cancer tissues. Univariate and multivariate clinicopathological and survival analyses were performed. We also examined the effect of overexpression or knockdown of the histone H4K20 methyltransferases, SUV420H1 and SUV420H2, on cancer-cell invasion activity in vitro.ResultsH4K20me3, but not H3K9me3, was clearly reduced in breast cancer tissue. A reduced level of H4K20me3 was correlated with several aspects of clinicopathological status, including luminal subtypes, but not with HER2 expression. Multivariate analysis showed that reduced levels of H4K20me3 independently associated with lower disease-free survival. Moreover, ectopic expression of SUV420H1 and SUV420H2 in breast cancer cells suppressed cell invasiveness, whereas knockdown of SUV420H2 activated normal mammary epithelial-cell invasion in vitro.ConclusionsH4K20me3 was reduced in cancerous regions of breast-tumor tissue, as in other types of tumor. Reduced H4K20me3 level can be used as an independent marker of poor prognosis in breast cancer patients. Most importantly, this study suggests that a reduced level of H4K20me3 increases the invasiveness of breast cancer cells in a HER2-independent manner.

Nuclear envelope-localized EGF family protein amphiregulin activates breast cancer cell migration in an EGF-like domain independent manner.

Amphiregulin (AREG), an EGF family protein, is synthesized as a type I transmembrane precursor (proAREG) and expressed on the cell surface with an extracellular EGF-like domain and an intracellular short cytoplasmic tail. The ectodomain shedding yields a soluble EGF receptor ligand (soluble AREG) which binds to EGF receptor (EGFR) and concomitantly induces migration of unshed proAREG from the plasma membrane to the nuclear envelope (NE). AREG is known to play a potential role in breast cancer and has been intensively investigated as an EGF receptor ligand, while the function of the NE-localized proAREG remains unknown. In this study we used a truncated mutant that mimics NE-localized proAREG without shedding stimuli to discriminate between the functions of NE-localized and plasma membrane-localized proAREG and demonstrate that NE-localized proAREG activates breast cancer cell migration, but suppresses cell growth. Moreover, the present study shows that induction of cell migration by NE-localized proAREG does not require the extracellular growth factor domain or EGF receptor function. Collectively these data demonstrate a novel function mediated by the intracellular domain of proAREG and suggest a significant role for NE-localized proAREG in driving human breast cancer progression.

SUV420H2 suppresses breast cancer cell invasion through down regulation of the SH2 domain-containing focal adhesion protein tensin-3

The genome-wide loss of histone H4 lysine 20 tri-methylation (H4K20me3) is observed in multiple types of cancer, including breast tumors. Since H4K20me3 is preferentially targeted to repetitive elements in the pericentromeric and telomeric heterochromatin and plays a role in chromatin integrity, the pathological effects of disrupted H4K20me3 in tumors have been attributed to genomic instability. However, in this report, we show that loss of H4K20me3 modulates gene expression profiles, leading to increased cell invasion. Reduced H4K20me3 levels in tumor cells are often accompanied by a decrease in the expression of the H4K20-specific methyltransferase, SUV420H2. Exogenous delivery of SUV420H2 into MDA-MB-231 human breast cancer cells induced selective and specific changes in the expression of cancer-related genes. One of the most downregulated genes in response to SUV420H2 expression was the Src substrate, tensin-3, a focal adhesion protein that contributes to cancer cell migration. Depletion of tensin-3 suppressed breast cancer cell invasiveness. Furthermore, silencing of tensin-3 was associated with enrichment of H4K20me3 immediately upstream of the tensin-3 transcription start site, suggesting that the loss of H4K20me3 in tumor cells induced the expression of cancer-promoting genes. These findings connect the loss of H4K20me3 with tumor progression, through the transcriptional activation of cancer-promoting genes.

Platelet-derived growth factor regulates breast cancer progression via β-catenin expression.

Objective: The knowledge on the association between platelet-derived growth factor (PDGF) signaling and epithelial cancers is scarce, although overexpression of PDGF and PDGF receptors has been reported in some human mesenchymal tumors. Thus, we studied the effect of PDGF on breast cancer cells in vitro and the distribution of PDGF in breast cancer tissues. Methods: The effect of PDGF-BB on breast cancer cells was assessed by Western blotting, immunofluorescence, WST and 5-bromo-2-deoxyuridine incorporation experiments. PDGF-B and β-catenin expression was investigated in breast cancer tissues by immunohistochemistry. Results: PDGF-BB induces β-catenin expression in breast cancer cells, and immunohistochemically the distribution of PDGF-B was similar to β-catenin in breast cancer cells. PDGF-B-positive cancer cells were more frequent in cases of ductal carcinoma in situ (87.5%) than invasive carcinoma (61.2%). In addition, PDGF-B staining was stronger in intraductal than invasive cancer cells. PDGF-BB tended to induce nuclear translocation of β-catenin, cell proliferation and DNA incorporation in MDA-MB231 cells, while these results were not found in MCF-7 cells. Conclusion: Our results suggest that PDGF-BB regulates protein expression of β-catenin and is associated with cancer cell behavior.

The CD44 standard isoform contributes to radioresistance of pancreatic cancer cells

Abstract Resistance to chemoradiotherapy is one reason for the increased recurrence rate of pancreatic cancer after these therapies. These cells change the expression levels of several proteins, such as epithelial–mesenchymal transition (EMT), while acquiring the chemo- or radio-resistance. In this study, we focused on CD44, a pancreatic cancer stem cell marker. CD44 has isoforms with different functions: standard isoform (CD44s) and several variant isoforms (CD44v). However, little is known about the roles of these isoforms after ionizing irradiation. The purpose of this study was to investigate the role of CD44 isoforms in radioresistance of pancreatic cancer cells. AsPC-1 (a human pancreatic cancer cell line) was irradiated with 4 MV X-rays. The mRNA and protein levels of CD44s were strongly upregulated, dose dependently, compared with CD44v after irradiation. Thus, we further investigated CD44s at the point of cell proliferation. We evaluated cell proliferation and survival, using CD44s knockdown cells. CD44s knockdown did not change the proliferation rate for up to 72 h after the irradiation, but decreased cell viability in the colony formation assay. As one of the reasons for these effects, we found downregulation of phosphorylated extracellular signal–regulated kinase (Erk; which is involved with cell proliferation) by CD44s knockdown, time dependently. Moreover, radiation-induced EMT-like expression changes were detected and suppressed by CD44s knockdown. In conclusion, our work demonstrated that CD44 standard isoform was especially upregulated after high-dose X-ray irradiation in several isoforms of CD44 and contributed to longer-term cell survival after the irradiation through the maintenance of Erk phosphorylation and radiation-induced EMT.

Photodynamic Therapy Using Indocyanine Green Loaded on Super Carbonate Apatite as Minimally Invasive Cancer Treatment

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.

Aberrant expression of disintegrin-metalloprotease proteins in the formation and progression of uterine cervical cancer.

Objective: Dysregulated expression of disintegrin-metalloprotease proteins [a disintegrin and metalloproteases (ADAMs) and ADAMs with thrombospondin motif (ADAMTSs)] has been reported in many types of cancers and is believed to play an important role in cancer formation and metastasis. However, little is known about the expression of ADAMs and ADAMTSs in the development of human cervical cancer. Methods: Reverse transcriptase polymerase chain reaction and immunoblotting were performed to assess the expression of several disintegrin-metalloproteases and tissue inhibitors of metalloproteinases (TIMPs) in squamous-type cervical cancer cells and oncogenically modified keratinocytes (immortalized human cervical keratinocytes transduced with human papilloma virus-16 E6/E7 proteins with or without oncogenes). Immunohistochemistry of ADAM-9, ADAM-10 and TIMP-3 was performed on 31 primary human cervical tissue specimens of preinvasive and invasive cervical carcinoma. Results: mRNA levels of ADAM-9, ADAM-10, ADAM-12, TIMP-2 and TIMP-3 were upregulated as cervical cells progressed from dysplastic to malignant lesions compared to normal cervical cells. These results were corroborated at the protein level by Western blot analysis and immunohistochemistry. Conclusion: The expression of disintegrin-metalloproteases and their endogenous regulators was dysregulated during cervical carcinogenesis. The aberrant expression of ADAMs might contribute to the pathogenesis of cervical cancer formation and progression.

A miR-29b Byproduct Sequence Exhibits Potent Tumor Suppressive Activities via Inhibition of NF-κB Signaling in KRAS Mutant Colon Cancer Cells

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.