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

https://doi.org/10.1016/j.yexcr.2015.03.010Get rights and content

Highlights

  • Loss of H4K20me3 associates with cell invasiveness via transcriptional regulation of various cancer related genes.

  • SUV420H2 induced histone H4K20 trimethylation immediately upstream of the tensin-3 transcription start site.

  • The enrichment of H4K20me3 at the upstream of tensin-3 gene suppressed transcription.

  • The exogenous delivery of SUV420H2 recovered H4K20me3 level and suppressed tumor cell invasion.

Abstract

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.

Introduction

Aberrant histone modification is a hallmark of cancer ([1], and reviewed in [2]). Genome-wide loss of histone H4 lysine 20 tri-methylation (H4K20me3) and histone H4 lysine 16 acetylation (H4K16ac) is observed in multiple types of human cancers, including breast tumors [1], [3], [4], [5]. A global decrease in H4K20me3 is correlated with a poor disease prognosis [5], [6]. Methylation of H4K20 is catalyzed by several histone methyl transferases, including PR-Set7, SUV420H1, and SUV420H2 [7], [8]. The bulk of H4K20me1 is catalyzed primarily by PR-Set7. H4K20me1 serves as a substrate for the SUV420H1/H2 enzymes responsible for H4K20me2 and H4K20me3. In mouse embryonic fibroblasts, the repressed expression of SUV420H2, but not SUV420H1, is associated with a global loss of H4K20me3, suggesting that SUV420H2 is mainly responsible for H4K20me3 [9]. H3K9me3 and H4K20me3 induce constitutive heterochromatin formation [8], and SUV420H1/H2 deficiency results in telomere elongation and depression of telomere recombination [10]. A decrease in H4K20me3 levels in cancer cells is associated with diminished expression of SUV420H2 [11], [12]. In addition, tumor suppressor retino;blastoma 1 family proteins are involved in stabilizing H4K20me3 [13]. Since H4K20me3 is primarily established as a mark of constitutive heterochromatin and governs genomic integrity, the oncogenic function of loss of H4K20me3 has been attributed to a loss of genomic integrity ([8], [14], reviewed in [15]). However, recent genome-wide chromatin immunoprecipitation (ChIP)-sequencing and ChIP-chIP studies have identified specific promoters that are enriched for H4K20me3 [16], [17].

We and others observed decreased H4K20me3 levels in breast cancer cells and tissues [4], [5], [11]. We reported a negative correlation between H4K20me3 levels and poor prognosis in breast cancer patients, and showed that ectopic expression of SUV420H2 suppressed tumor invasiveness in vitro [5]. Here, we investigated the underlying molecular mechanisms by which increased H4K20me3 levels attenuate tumor invasion, and revealed an association between H4K20me3 levels and transcriptional regulation. One of the most down-regulated genes by SUV420H2 expression is tensin-3. The tensin family comprises four proteins, tensin-1, tensin-2, tensin-3, and c-ten (c-ten, for C-terminal tensin-like protein), which are involved in cell migration and are localized to focal adhesion sites. All four tensin family members contain Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains in their carboxyl termini. Through the PTB domain, tensins interact with the cytoplasmic tail of β integrins, transmembrane cell-adhesion molecules [18]. Tensin-3 is phosphorylated by Src and contributes to tumorigenesis and metastasis both in vitro and in an in vivo mouse model [19].

Section snippets

Plasmids

The HA-tagged human SUV420H2 plasmid (pcDNA3.1-HA-SUV420H2) was previously described [5]. The 5′-terminal fragment of the tensin-3 gene (approximately −2000 to +500) was amplified using the HeLa genome and the primer sets, 5′-TGGGGCATCCCATTCCATTGCTA-3′ (Primer #1 forward in Table S2) and 5′-GACATAGCTGCTCCGGTCTCTG-3′ (downstream region of Primer #5 in Table S2). The PCR product was inserted into the pCDNA 3.3 TOPO vector.

Antibodies

Mouse anti-H4K20me3, anti-H4K20me2, anti-H4K20me1, anti-H3K9me3, and

Over-expression of SUV420H2 suppresses cell invasion

H4K20me3 levels are often reduced in various cancer tissues and cultured cells. Consistently, highly metastatic breast cancer cells exhibit decreased expression of the H4K20-specific methyltransferase, SUV420H2 [11], [12]. MDA-MB-231 is a metastatic human breast cancer cell line that exhibits invasive properties when cultured in vitro and when transplanted into mice, and is widely used to study metastasis and cell invasion. To establish that SUV420H2 stably over-expressing MDA-MB-231 cells,

Discussion

Genome-wide loss of H4K20me3 has been observed in a variety of cancer tissues. Here, we showed the functional significance of H4K20me3 in the transcriptional regulation of cancer-associated genes. The exogenous delivery of the histone H4K20-specific methyltransferase, SUV420H2, induced the selective and specific down- and upregulation of cancer-related genes, including tensin-3, and suppressed the invasion of breast cancer cells. In addition, we showed that H4K20me3 was enriched immediately

Conclusion

This study demonstrates an additional function for the loss of H4K20me3 in tumor cells; loss of H4K20me3 modulates gene expression profiles of cancer-related genes and suppresses cell invasiveness.

Conflicts of interest

The authors disclose no potential conflicts of interest.

Acknowledgments

We gratefully acknowledge Dr. T. Kurokawa and Dr. T. Honda of Wako Pure Chemical Industries Ltd., and Dr. Jun Katahira of Osaka University for valuable discussions. We thank Ms. Junko Imada of Osaka University for technical assistance. This study was performed as part of a joint research program with Wako Pure Chemical Industries Ltd. This work was supported by Grants-in-Aid for Scientific Research (c) (24570213) from the Japan Society for the Promotion of Science, and a Grant-in-Aid for

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    Financial support: This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, and a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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