DNA methyltransferase 1 is essential for initiation of the colon cancers

doi:10.1016/j.yexmp.2012.10.004

Experimental and Molecular Pathology

Volume 94, Issue 2, April 2013, Pages 322-329

https://doi.org/10.1016/j.yexmp.2012.10.004 Get rights and content

Abstract

DNA methyltransferase 1 (Dnmt1) is essential for the maintenance of hematopoietic and somatic stem cells in mice; however, its roles in human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are still elusive. In the present study, we investigated DNMT1 functions in the maintenance of human colon CSCs/CICs using the human colon cancer cell line HCT116 (HCT116 w/t) and its DNMT1 knockout cell line (DNMT1^−/−). The rates of CSCs/CICs were evaluated by side population (SP) analysis, ALDEFLUOR assay and expression of CD44 and CD24. SP, ALDEFLUOR-positive (ALDEFLUOR⁺) and CD44-positive and CD24-positive (CD44⁺CD24⁺) cell rates were lower in DNMT1^−/− cells than in HCT116 w/t cells. Since CSCs/CICs have higher tumor-initiating ability than that of non-CSCs/CICs, the tumor-initiating abilities were addressed by injecting immune deficient (NOD/SCID) mice. DNMT1^−/− cells showed less tumor-initiating ability than did HCT116 w/t cells, whereas the growing rate of DNMT1^−/− cells showed no significant difference from that of HCT116 cells both in vitro and in vivo. Similar results were obtained for cells in which DNMT1 had been transiently knocked-down using gene-specific siRNAs. Taken together, these results indicate that DNMT1 is essential for maintenance of colon CSCs/CICs and that short-term suppression of DNMT1 might be sufficient to disrupt CSCs/CICs.

Introduction

Cancer tissues are composed from several heterogeneous cancer cells and small population of cancer cells are supposed to have higher tumor-initiating ability. These higher tumorigenic populations are named “cancer stem-like cells (CSCs)” or “cancer initiating cells (CICs)”. CSCs/CICs are defined as small population of cancer cells which has (1) higher tumor-initiating ability, (2) self-renewal, (3) differentiation. (Clarke et al., 2006, Dalerba et al., 2007, Huang et al., 2009, O'Brien et al., 2007, Ricci-Vitiani et al., 2007) CSCs/CICs have been reported to be resistant to chemotherapy, radiotherapy and certain molecular targeting therapies (Dean et al., 2005); thus, elucidation of the molecular mechanisms of the maintenance of CSCs/CICs should be useful for establishing efficient CSC/CIC targeting treatment.

Trowbridge et al. reported that hematopoietic stem cell self-renewal was abrogated by conditional gene knockout of Dnmt1, while the mature differentiated hematopoietic lineage was not affected. (Trowbridge et al., 2009) Dnmt1 is essential for maintenance of the leukemia stem cells of bilinear myeloid-B lymphoid leukemia induced by transduction of c-Myc and Bcl-2. (Broske et al., 2009) Dnmt1 was also shown to be essential for the self-renewal of skin progenitor cells. (Sen et al., 2010) Results of these studies indicate that DNMT1 is essential for self-renewal of progenitor or stem cells. However, no study has been mentioned the relation between DNMT1 and CSCs/CICs in solid tumors including colon cancer.

In the present study, we investigated the functions of DNMT1 in maintenance of colon CSCs/CICs. We investigated DNMT1 functions by using a DNMT1^−/− cell line and also cells in which DNMT1 was transiently knocked-down by siRNAs. Permanent DNMT1 gene knockout and transient DNMT1 gene knock down by specific siRNAs reduced the population of CSCs/CICs. These results of this study suggest that DNMT1 has an essential role in maintenance of CSCs/CICs, and transient inhibition of DNMT1 might be sufficient to eradicate CSCs/CICs.

Section snippets

Cell lines

The colon adenocarcinoma cell lines wild-type HCT116 and SW480 were kind gifts from Dr. K. Imai (Sapporo, Japan). A DNMT1 knockout HCT116 cell line (DNMT1^−/−) that lacks exons 2–5 of DNMT1 (Rhee et al., 2000) was a kind gift from Dr. B. Vogelstein (Baltimore, MD). HCT116 and DNMT1^−/− cells were cultured in McCoy's 5A modified medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Grand Island, NY). SW480 cells were cultured in Dulbecco's

Lower rates of CSCs/CICs in DNMT1^−/− cells

DNMT1 has been described to be essential for self-renewal of progenitor or stem cells. (Trowbridge et al., 2009, Broske et al., 2009, Sen et al., 2010). We therefore hypothesized that DNMT1 has also role in maintenance of human colon CSCs/CICs. CSC/CIC population in HCT116 DNMT1^−/− cells and HCT116 w/t cells were analyzed by SP analysis, ALDEFLUOR assays and expression of cell surface CSC/CIC makers. Putative CSC/CIC including SOX2, POU5F1, LGR5 and PROM1 were analyzed by RT-PCR. (O'Brien et

Discussion

In the present study, we found that DNMT1^−/− cells show much lower tumor-initiating ability than that of HCT116 w/t cells in vivo, whereas the growth rate of DNMT1^−/− cells in vitro was not affected compared to that of HCT116 w/t cells, being consistent with a previous report. (Rhee et al., 2002) To address this controversial phenomenon of tumor-initiating ability and cell growth potential, we hypothesized that the CSC/CIC population is reduced in DNMT1^−/− cells. We evaluated DNMT1^−/− cells by

Declaration of financial disclosure

The authors have no financial conflict of interest.

Acknowledgments

The authors thank Ms. Emiri Nakazawa and Eri Saka for technical assistance. The authors thank Drs. K. Imai and B. Vogelstein for kindly providing cell lines. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant nos. 16209013, 17016061 and 15659097) for Practical Application Research from the Japan Science and Technology Agency, and for Cancer Research (15–17 and 19–14) from the Ministry of

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DNA methyltransferase 1 is essential for initiation of the colon cancers

Abstract

Introduction

Section snippets

Cell lines

Lower rates of CSCs/CICs in DNMT1−/− cells

Discussion

Declaration of financial disclosure

Acknowledgments

American Journal of Pathology

Cell Stem Cell

American Journal of Pathology

Lung Cancer

Blood

Laboratory Investigation

Cell Stem Cell

Prospective identification of tumorigenic breast cancer cells

Proceedings of the National Academy of Sciences of the United States of America

An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors

Nature Genetics

DNA methylation protects hematopoietic stem cell multipotency from myeloerythroid restriction

Nature Genetics

CD44 enhances the epithelial–mesenchymal transition in association with colon cancer invasion

International Journal of Oncology

Cancer stem cells—perspectives on current status and future directions: AACR Workshop on cancer stem cells

Cancer Research

Phenotypic characterization of human colorectal cancer stem cells

Proceedings of the National Academy of Sciences of the United States of America

Abnormal regulation of DNA methyltransferase expression during colorectal carcinogenesis

Cancer Research

Tumour stem cells and drug resistance

Nature Reviews Cancer

Identification of DNMT1 (DNA methyltransferase 1) hypomorphs in somatic knockouts suggests an essential role for DNMT1 in cell survival

Proceedings of the National Academy of Sciences of the United States of America

Expression analysis of DNA methyltransferases 1, 3A, and 3B in sporadic breast carcinomas

Clinical Cancer Research

Lessons from common markers of tumor-initiating cells in solid cancers

Cellular and Molecular Life Sciences

Lower rates of CSCs/CICs in DNMT1^−/− cells