C20ORF20 Human

What is C20ORF20 and what is its molecular structure?

C20ORF20, also known as MRG-binding protein (MRGBP), is a subunit of the TRRAP/TIP60-containing histone acetyltransferase complex. The human recombinant protein is a single, non-glycosylated polypeptide chain containing 224 amino acids (covering positions 1-204 a.a.) with a molecular mass of approximately 24.5 kDa . When analyzed via SDS-PAGE, the protein often appears at a higher molecular weight than predicted. Recombinant versions for research applications typically include a 20 amino acid His-tag at the N-terminus .

What is the normal expression pattern of C20ORF20 in human tissues?

Multiple tissue northern blot analysis has detected a 1.6 kb transcript of C20ORF20 that is readily observable in skeletal muscle, testis, and thyroid tissues. In contrast, important vital organs such as the heart, brain, lung, liver, and kidney show relatively low levels of expression . This differential expression pattern suggests tissue-specific functions of C20ORF20 in normal physiology.

What cellular functions is C20ORF20 associated with?

C20ORF20 functions as part of the TRRAP/TIP60-containing histone acetyltransferase complex, suggesting a role in chromatin modification and transcriptional regulation . Research has demonstrated its interaction with bromodomain containing 8 (BRD8), which appears to be an important downstream target . The protein's role in cell proliferation has been experimentally confirmed, as suppression of C20ORF20 inhibits proliferation in multiple cancer cell types .

How does C20ORF20 contribute to cancer pathogenesis?

Research indicates that C20ORF20 is frequently upregulated in multiple cancer types, particularly colorectal cancer and cutaneous squamous cell carcinoma (cSCC) . In colorectal cancer, elevated expression was observed in 10 out of 15 tumor samples compared to matched non-cancerous mucosa using quantitative PCR, and in 10 out of 14 tumors via Western blot analysis . The overexpression of C20ORF20 appears to promote cancer cell proliferation through its interaction with BRD8 .

Mechanistically, while C20ORF20 knockdown in colorectal cancer cells results in decreased proliferation (shown by a 10% decrease in S-phase replicating cells) without inducing apoptosis, its knockdown in cSCC cells demonstrates a different mechanism - inducing significant apoptotic response without altering cell-cycle parameters . This suggests that C20ORF20 may function through different molecular pathways depending on the cancer type.

What is the relationship between C20ORF20 and BRD8, and how does this interaction impact cellular function?

C20ORF20 directly interacts with bromodomain containing 8 (BRD8), as identified through yeast two-hybrid screening and confirmed by immunoprecipitation analysis . In colorectal cancer cells, endogenous interaction between C20ORF20 and BRD8 was demonstrated using nuclear extracts from SW480 cells that were incubated with anti-MRGBP or anti-p120 (BRD8) antibodies .

The functional significance of this interaction is evidenced by RNA interference experiments. When BRD8 expression is suppressed, proliferation of colorectal cancer cells is inhibited, similar to the effects observed with C20ORF20 knockdown . This suggests that BRD8 is an important downstream effector of C20ORF20, and that the C20ORF20-BRD8 axis plays a critical role in promoting cancer cell proliferation.

What genomic alterations are associated with C20ORF20 in different cancer types?

C20ORF20 has been identified as being frequently amplified in multiple cancer types. Studies have documented its amplification in colorectal cancer, cervical cancer, and most recently its overexpression has been confirmed in colorectal cancer through detailed molecular analyses . In cSCC, C20ORF20 was identified as part of a set of cancer-specific genes that are upregulated compared to normal skin and the benign hyperproliferative condition psoriasis . These findings suggest that genomic amplification may be one mechanism driving C20ORF20 overexpression in cancer.

What techniques are used to measure C20ORF20 expression in tissue samples?

Several complementary techniques have been employed to measure C20ORF20 expression:

• cDNA Microarray Analysis: Initial identification of C20ORF20 upregulation in colorectal cancer was achieved using cDNA microarray containing 23,040 genes .

• Quantitative PCR (qPCR): Elevated expression of C20ORF20 in colorectal cancer tissues was confirmed using quantitative PCR, comparing tumor samples with matched non-cancerous mucosa .

• Western Blot Analysis: Protein-level expression was verified using Western blot analysis in various tumor samples .

• Immunohistochemical Analysis: C20ORF20 protein expression in tissues has been assessed through immunohistochemical staining .

• Multiple Tissue Northern Blot: This technique was used to evaluate C20ORF20 expression across different normal human tissues, detecting a 1.6 kb transcript .

What are the effective methods for C20ORF20 knockdown in experimental settings?

Research has employed several approaches for C20ORF20 knockdown:

• Short Hairpin RNA (shRNA): Studies have used MRGBP shRNA to suppress expression in colorectal cancer cells .

• Small Interfering RNA (siRNA): Multiple studies have effectively used siRNA for targeted knockdown:

  • Pools of four C20ORF20-specific siRNA oligos have been utilized .

  • Specific sequences that have demonstrated efficacy include: 5′-GAGAAUUUGUAGCGGUUAU-3′, 5′-GUGACAUGGAUUAGCGCUA-3′, 5′-ACAAAGUCCUGACCGCAAA-3′, and 5′-CAGGGAAAACCUCGGAUUA-3′ .

  • Knockdown efficiency has been reported between 64.3-89.4% in cSCC cells and 51.9-72.3% in normal primary keratinocytes, as measured by reverse transcription-real-time quantitative PCR .

• In vivo siRNA delivery: Direct injection of C20ORF20-targeting siRNA into established xenograft tumors has been successfully implemented to reduce tumor volume .

How can C20ORF20 protein-protein interactions be studied?

The following experimental approaches have been used to investigate C20ORF20 interactions:

• Yeast Two-Hybrid Screening: This technique was employed to identify BRD8 as an interaction partner of C20ORF20 .

• Immunoprecipitation Analysis: To confirm endogenous interaction between C20ORF20 and BRD8, nuclear extracts from cancer cells were incubated with specific antibodies (anti-MRGBP or anti-p120/BRD8), followed by Protein G-Sepharose beads and SDS-PAGE analysis .

• Co-immunoprecipitation Controls: Normal rabbit IgG was used as a negative control to validate specific interactions .

What statistical approaches are recommended for analyzing C20ORF20 expression data?

Based on the research methodologies described in the literature, the following statistical approaches are recommended:

• Differential Expression Analysis: For RNA sequencing data, tools like the R software with the ggplot2 package have been used for visualization, with thresholds such as Log 2|FC| ≥1.1 and q-value <0.05 for screening differentially expressed genes .

• Pathway Analysis: KEGG and GO analyses using the clusterProfiler package in R software, with p-value <0.05 as statistically significantly enriched .

• Protein-Protein Interaction Network Analysis: Construction of PPI networks using STRING database (version 11.5) and Cytoscape software (version 3.8.0), with minimum required interaction score set to high confidence (0.700) .

• Statistical Significance Testing:

  • Independent-sample t-tests for comparing two groups

  • Kruskal-Wallis test for multiple groups comparison

  • Dunn's multiple comparison test for multiple comparisons

  • p-value <0.05 is typically considered statistically significant

How should researchers interpret contradictory findings regarding C20ORF20 function in different cancer types?

When interpreting contradictory findings regarding C20ORF20 function across cancer types, researchers should consider:

• Tissue-Specific Effects: C20ORF20 knockdown produces different outcomes in different cancer types. For example, in colorectal cancer cell lines (HCT116 and SW480), knockdown resulted in decreased proliferation without apoptosis, whereas in cSCC, knockdown induced significant apoptosis without changing cell-cycle parameters .

• Molecular Context: The protein interaction network surrounding C20ORF20 may vary between tissue types, altering downstream effects of its expression or inhibition.

• Experimental Methodology: Differences in knockdown efficiency, cell culture conditions, or assay sensitivity may contribute to different observed outcomes.

• Cancer Subtype Heterogeneity: Even within a single cancer type, molecular subtypes may respond differently to C20ORF20 modulation.

To reconcile contradictory findings, researchers should perform parallel experiments using standardized methodologies across multiple cell lines representing different cancer types.

What evidence supports C20ORF20 as a potential therapeutic target in cancer?

Several lines of evidence support C20ORF20 as a potential therapeutic target:

• Differential Expression: C20ORF20 is overexpressed in cancer tissues but shows low expression in normal vital organs, suggesting a favorable therapeutic window .

• Functional Effects of Knockdown:

  • siRNA-mediated knockdown of C20ORF20 consistently reduces cell viability in cSCC cells without significantly affecting normal keratinocytes .

  • In colorectal cancer cells, C20ORF20 knockdown inhibits proliferation .

• In Vivo Efficacy: C20ORF20-targeting siRNA injected into established xenograft tumors reduced tumor volume compared to non-targeting siRNA controls . Histological examination revealed that treated tumors had marked reduction in tumor keratinocytes and appeared hollow .

• Cancer-Specific Effects: The apoptotic effect of C20ORF20 knockdown appears to be selective for cancer cells, as normal cells were not significantly affected under similar conditions .

What challenges exist in developing C20ORF20-targeted therapies?

Several challenges must be addressed in developing effective C20ORF20-targeted therapies:

• Delivery Method: While siRNA approaches have shown efficacy in vitro and in localized xenograft models, systemic delivery of RNA interference therapies remains challenging. Researchers need to develop effective delivery vehicles that can protect siRNA from degradation and ensure targeted delivery to tumor cells.

• Specificity: Given that C20ORF20 is expressed at low levels in normal tissues, therapies must maintain specificity to avoid off-target effects. The differential response between cancer cells and normal cells to C20ORF20 knockdown suggests this may be achievable .

• Resistance Mechanisms: As with many targeted therapies, cancer cells may develop resistance mechanisms. Understanding potential compensatory pathways that might be activated upon C20ORF20 inhibition will be crucial.

• Combination Approaches: Determining optimal combination strategies with existing therapies will require extensive preclinical investigation. Given the role of C20ORF20 in cell proliferation and survival, combinations with cell cycle inhibitors or apoptosis inducers may be particularly effective.

What are promising areas for future research on C20ORF20?

Based on current knowledge, several research directions warrant further investigation:

• Mechanistic Studies: Further elucidation of the precise molecular mechanisms by which C20ORF20 promotes cancer cell proliferation and survival, particularly exploring why the effects of knockdown differ between cancer types (apoptosis in cSCC vs. cell cycle arrest in colorectal cancer) .

• Expanded Cancer Profiling: Comprehensive analysis of C20ORF20 expression across a broader range of cancer types to identify additional malignancies where it may serve as a therapeutic target.

• Structural Biology: Determination of the three-dimensional structure of C20ORF20 and its complexes with interacting partners like BRD8 to facilitate structure-based drug design.

• Small Molecule Inhibitors: Development of small molecule inhibitors targeting either C20ORF20 directly or its interaction with BRD8.

• Biomarker Development: Evaluation of C20ORF20 expression as a prognostic or predictive biomarker in various cancer types.

• Improved Delivery Methods: Development of more effective in vivo delivery methods for C20ORF20-targeting therapeutics, particularly for systemic administration.

How might C20ORF20 research integrate with other emerging cancer biology concepts?

Integration of C20ORF20 research with other emerging areas in cancer biology could yield valuable insights:

• Epigenetic Regulation: Given C20ORF20's role in the histone acetyltransferase complex, exploring its contribution to cancer epigenetics and potential interactions with other epigenetic regulators could reveal new therapeutic opportunities.

• Cancer Metabolism: Investigating whether C20ORF20 influences metabolic reprogramming in cancer cells, particularly in relation to proliferation and survival pathways.

• Immune Microenvironment: Examining if C20ORF20 expression in cancer cells affects tumor-immune interactions or response to immunotherapies.

• Precision Medicine Approaches: Determining whether C20ORF20 expression levels or mutations could serve as predictive biomarkers for response to specific therapies, enabling more personalized treatment strategies.

• Combinatorial Therapeutic Strategies: Testing C20ORF20-targeted approaches in combination with established therapies or other emerging targets, such as PLK1 inhibitors, which have shown efficacy in similar cancer models .