TET2 and Caspase-4: New Potential Diagnostic Markers for Breast Cancer?

From the paper: TET2 Inhibits Tumorigenesis of Breast Cancer Cells by Regulating Caspase-4 (Zhu & Li, 2018)

Each year, over 266,000 women are diagnosed with breast cancer (American Cancer Society, 2018). It is the second most common type of cancer found in women (American Cancer Society, 2018), and may be caused by a number of genetic and environmental factors (Zhu & Li, 2018). One process, DNA methylation, is one such factor (Zhu & Li, 2018). DNA methylation occurs when a methyl group (CH₃) binds to DNA, usually turning off expression of the gene (University of Delaware, 2016). The researchers are also interested in ten eleven translocation (TET)proteins. These proteins undo methylation in order to help suppress tumors (Wu & Zheng, 2014). One TET protein, TET2, has been shown to reduce the spread of breast cancer (Song et al., 2013). Additionally, caspase-4 is a protein involved in apoptosis, or programmed cell death. Because caspase-4 works to promote apoptosis, it stops cancers from forming or spreading in some cancers (Zhu & Li, 2018). However, not much is known about if and how it works in breast cancer (Zhu & Li, 2018).

In their study, the researchers first looked at how cells grow with and without TET2. These groups of cells are called wildtype, or WT, and knockout, or TET2 KO, respectively. The cells used in the study were called MCF-7 cells, a type of breast cancer cell line. After subjecting the cells to various conditions, the researchers found that the TET2 KO cells were better than WT cells at anchorage-independent growth - being able to grow while unattached to anything (Figure 1d). TET2 KO cells also had larger tumors. After some more tests, the researchers found that TET2 works to stop anchorage-independent growth in breast cancer cells (Zhu & Li, 2018).

Figure 1. TET2 loss enhances tumorigenesis of MCF-7 cell. c (not shown in blog), Colony formation assay of MCF-7 (WT, TET2 NO1, TET1 KO2) treated with EtOH or 1 nM E2. This assay was performed in 6-well plate, after 2 weeks, the cell colonies were harvested and stained. Then, the colony number was counted. WT denotes wildtype. d, Statistical analysis of colony number shown in Fig. 1c. (Zhu & Li, 2018)

Next, the researchers wanted to examine if TET2 worked to suppress tumor growth by regulating genes related to apoptosis. They conducted a test (called a real time quantitative polymerase chain reaction, or RT-qPCR) to see which genes may be affected by TET2. They found that mRNA levels of CASP4, the gene that encodes caspase-4, were downregulated by about 60-70% in TET2 KO cells compared to WT cells (Figure 2a). This is evidence that caspase-4 is regulated by TET2 (Zhu & Li, 2018).

Figure 2. Caspase-4 is specifically regulated by TET2. a, RT-qPCR analysis shows mRNA levels of Bcl-2 and caspase family genes in MCF-7 (WT, TET2 KO1, TET2 KO2). WT denotes wildtype. (Zhu & Li, 2018)

The researchers suspected TET2 works to regulate gene expression using demethylation. They used another test called a bisulfite PCR to evaluate DNA methylation levels as the CASP4 promoter, the part of DNA that starts transcription. They found that methylation was lower in the WT than TET KO cells (65% methylated vs. 90 and 91.7% methylated), suggesting that TET2 regulated methylation at the CASP4 promoter (Zhu & Li, 2018). Finally, they researchers wanted to know if caspase-4 had a role in anchorage-independent growth of MCF-7 cells. After some tests, they did find this to be the case. Specifically, caspase-4 works to stop the formation and growth of tumors in MCF-7 cells (Zhu & Li, 2018).

Figure 5. A proposed model for caspase-4 involved in TET2-inhibited tumorigenesis of breast cancer cells (Zhu & Li, 2018).

Above is a proposed pathway for how TET2 works to regulate caspase-4, and ultimately, tumors (Figure 5). Normally, TET2 alters the methylation at the CASP4 gene. This results in more caspase-4 proteins working to stop tumor growth. However, without TET2, you don’t see a lot of caspase-4 proteins, and tumor growth can continue, leading to cancer (Zhu & Li, 2018).

The research presented in this paper is relevant and has important implications. Because of the research, we are learning more about breast cancer, and potentially other types of cancer as well. With this new knowledge of how TET2 works to ultimately regulate tumor growth, we may be able to identify new diagnostic markers for cancer. Additionally, we may use this information in therapeutic strategies.

Finally, many questions were raised by the article in terms of where further research should go. The researchers believe further investigation of more detailed mechanisms, pathways and targets, and potential partner proteins is the next step. I also think researchers should look at the role of TET2 and caspase-4 in other types of tumors. Additionally, they should investigate if introducing more TET2 in patients with cancer would be a helpful treatment strategy.

Works Cited

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https://www.cancer.org/cancer/breast-cancer/about/how-common-is-breast-cancer.html#written_by

Epigenie (n.d.). Ten-eleven Translocation (TET) Enzymes Retrieved from:

https://epigenie.com/key-epigenetic-players/important-dna-methylation-factors/ten-eleven-translocation-tet-enzymes/

European Collection of Authenticated Cell Cultures (n.d.). Cell Line Profile MCF7. Retrieved

from: https://admin.phe-culturecollections.org.uk/media/130237/mcf7-cell-line-profile.pdf

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Yuan, X., Cantley, L. C., Richardson, A. L., & Pandolfi, P. P. (2013). MicroRNA-antagonism regulates breast cancer stemness and metastasis via TET-family-dependent chromatin remodeling. Cell 154(2):311-324

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Apoptotic Pathway and the Caspase Cascade [Video File]. Retrieved from: https://www.youtube.com/watch?v=-vmtK-bAC5E

University of Delaware [University of Delaware]. (2016, December 6). How DNA methylation

works [Video File]. Retrieved from: https://www.youtube.com/watch?v=KYHBbEKap0A

Wu, H. & Zheng, Y. (2014). Reversing DNA methylation: mechanisms, genomics, and biological

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Zhu, X. & Li, S. (2018). TET2 inhibits tumorigenesis of breast cancer cells by regulating caspase-

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