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By Jamshed Arslan, Pharm. D., PhD.
DNA methylation represses transcription of many genes, including tumor suppressor genes. A protein called UHRF1 recruits DNA methyltransferases (DNMTs) to establish and maintain DNA methylation. UHRF1 has several domains, most notably:
Depleting UHRF1 with or without DNMT inhibition reduces DNA methylation, thereby reactivating tumor suppressor genes and suppressing cancers. Studies on re-establishing DNA methylation by using loss-of-function mutant UHRF1 domains are unable to indicate if such DNA re-methylation is purely anew. So, research institutes in USA and China set out to define the UHRF1 domains that maintain cancerous DNA methylation. Using colorectal cancer cells, the team found that chromatin reader domains of UHRF1 maintain cancerous DNA hypermethylation. They found that both histone- and hemimethylated DNA-reader domains (PHD and SRA, respectively) are crucial for colorectal cancer's aberrant DNA methylation. Disrupting SRA or PHD impairs the cancerous potential of colorectal cancer cells by reversing DNA hypermethylation and reactivating tumor suppressor genes.
DNMT1 was detected in HCT 116 human colorectal carcinoma cell line with Sheep Anti-Human DNMT1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6110) at 10 µg/mL for 3 hours at room temperature. Cells were stained with NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody (red; Catalog # NL010) and counterstained with DAPI (blue). Untreated (left) and treated (right) with 1 μM 5-azacytidine for 24 hours. Specific staining was detected in the nuclei and cytoplasm, and nuclear staining was reduced by 5-azacytidine treatment.
To dissect the role of UHRF1, the researchers exogenously expressed UHRF1 transgene in human colorectal cancer cells while simultaneously blocking endogenous UHRF1 through shRNA. Such replacement of endogenous UHRF1, allowed the team to observe exogenous UHRF1’s DNA methylation maintenance capacity. It turned out that UHRF1 cannot maintain genomic and cancer-specific DNA methylation when loss-of-function or deletion mutants of PHD domain, but not TTD domain, are used. Similarly, SRA domain mutants could not recognize hemimethylated DNA. Most notably, only the PHD or SRA mutants produced DNA methylation patterns similar to endogenous UHRF1 depletion. A dramatic cancer-specific DNA demethylation was visible in PHD or SRA mutants. In other words, cancerous DNA methylation and the consequent silencing of tumor suppressor genes are dependent on PHD or SRA domains.
The next step was to explore the effects of either domain on the oncogenic properties of colorectal cancer cells.
The researchers transplanted colorectal cancer cells with TTD, PHD, TTD-PHD, SRA or RING mutants into immunocompromised mice. TTD-PHD and SRA mutants markedly reduced tumor burden with enhanced tumor latency relative to wild-type or RING mutants. Interestingly, reduction in tumor burden and colorectal cancer cell proliferation in PHD mutants was similar to TTD-PHD double mutants. In other words, SRA and PHD domains are enough to confer oncogenic properties to UHRF1 in colorectal cancer cells. This was corroborated by increased expression of various colorectal cancer metastasis-related genes like ICAM4, PRSS8, JDP2 and FBLN2 in the mutants.
The translational value of these findings was evident when tissue samples from 120 colorectal cancer patients were found to have elevated UHRF1 RNA expression relative to individuals without colorectal cancer. Among the colorectal cancer patients, those with UHRF1 overexpression had reduced progression-free survival.
In sum, UHRF1 maintains cancer-specific DNA methylation for silencing tumor suppressor genes, primarily through its histone reader (PHD) and hemimethylated DNA reader (SRA) domains. This contributes to the malignant potential of colorectal cancer.
Reducing cancerous DNA methylation is important for optimal therapy. Although DNMT inhibitors and aromatic ring of TTD domain have been the focus of therapeutics, this research highlights the prognostic and therapeutic significance of PHD and SRA domains of UHRF1 in human colorectal cancer.
Novus Antibodies Used in this Study |
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Western blot: DNMT3A Antibody (64B1446) [NB120-13888] - Analysis of (A) Dnmt3a transfected 293 cell lysate and (B) untransfected 293 cell lysate using Dnmt3a antibody at 1 ug/mL. |
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Western Blot: Fibulin 2 Antibody [NBP1-33479] - Various whole cell extracts (30 ug) were separated by 5% SDS-PAGE, and the membrane was blotted with Fibulin 2 antibody diluted at 1:1000. |
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Immunohistochemistry-Paraffin: JDP2 Antibody [NBP2-31775] - Staining of human kidney shows strong nuclear positivity in cells in tubules. |
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Western Blot: LXN Antibody (1E10) [NBP2-01382] - HEK293T cells were transfected with the pCMV6-ENTRY control (Left lane) or pCMV6-ENTRY LXN (Right lane) cDNA for 48 hrs and lysed. Equivalent amounts of cell lysates (5 ug per lane) were separated by SDS-PAGE and immunoblotted with anti-LXN. |
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Immunohistochemistry-Paraffin: Prostasin/Prss8 Antibody [NBP1-31592] - Paraffin-embedded Ca922 xenograft. Prostasin antibody [N1C2] d ilution: 1:250. |
Jamshed Arslan, Pharm D, PhD
Dr. Arslan is an Assistant Professor at Dow University of Health Sciences, Pakistan,
where he teaches Pharmacology to future pharmacists.
References
Kong, Xiangqian, et al. "Defining UHRF1 Domains that Support Maintenance of Human Colon Cancer DNA Methylation and Oncogenic Properties." Cancer Cell, vol. 35, no. 4, 2019, pp. 633–648. https://doi.org/10.1016/j.ccell.2019.03.003.
