CCDC69 Human

What is the genomic location and basic function of CCDC69?

CCDC69 is located on chromosome 5q33.1 in humans and plays a critical role in controlling the assembly of central spindles and the recruitment of midzone components during cell division . The protein contains characteristic coiled-coil domains that facilitate protein-protein interactions essential for its cellular functions. Functionally, CCDC69 has been implicated in mitotic processes and has emerged as a potential biomarker in several cancers due to its differential expression between normal and malignant tissues .

How is CCDC69 expression typically measured in research studies?

Research studies employ multiple complementary approaches to assess CCDC69 expression:

• Transcriptional level assessment: Real-time quantitative PCR (RT-qPCR) is commonly used to measure CCDC69 mRNA expression, as demonstrated in studies comparing expression between cisplatin-sensitive and cisplatin-resistant ovarian cancer cells .

• Protein detection methods: Western blotting with specific CCDC69 antibodies (such as Novus NBPI-85,139, 1:200 dilution) is frequently utilized to assess protein expression levels .

• Immunohistochemistry (IHC): For tissue samples, IHC protocols typically involve H₂O₂ blocking of endogenous peroxidase activity, overnight incubation with primary CCDC69 antibody at 4°C, followed by secondary antibody incubation, DAB reagent staining, and hematoxylin counterstaining .

• Bioinformatic analysis: Many studies leverage public databases including TCGA, GENT2 (Gene Expression patterns across Normal and Tumor tissues database), and bc-GenExMiner for large-scale expression analysis across cancer types and correlation with clinical parameters .

What expression patterns of CCDC69 have been observed across different cancer types?

CCDC69 demonstrates distinct expression patterns across cancer types, with predominant downregulation in multiple malignancies:

CCDC69 has been found to be significantly downregulated in 18 different cancer types compared to adjacent normal tissues, including cancers of the lung, blood, brain, breast, skin, colon, ovary, pancreas, esophagus, tongue, adrenal gland, prostate, kidney, bladder, liver, vulva, vagina, and endometrium . This broad downregulation pattern suggests CCDC69 may function as a tumor suppressor in many contexts.

How does CCDC69 expression correlate with immune infiltration in breast cancer?

CCDC69 expression demonstrates significant correlations with tumor immune microenvironment characteristics in breast cancer:

The expression of CCDC69 positively correlates with the presence of various tumor-infiltrating lymphocytes (TILs) in breast cancer samples . Studies using the TIMER and GEPIA databases have confirmed this relationship, suggesting that CCDC69 may influence or be influenced by the immune landscape of breast tumors .

Research has indicated that CCDC69 expression is associated with immunomodulators and chemokines as demonstrated in analyses using the Tumor-Immune System Interactions database (TISIDB) . The expression profile correlates with immune category enrichment scores calculated using single-sample Gene Set Enrichment Analysis (ssGSEA) methodology .

These findings suggest that CCDC69 may serve as a potential biomarker for predicting sensitivity to immunotherapies, particularly PD-1/PD-L1 checkpoint inhibitors in breast cancer patients . This connection between CCDC69 and immune infiltration provides a rationale for investigating combination approaches that might enhance immunotherapy efficacy in breast cancer.

What is the prognostic significance of CCDC69 in breast cancer patients?

CCDC69 has emerged as a significant prognostic marker in breast cancer:

Multivariate Analysis: Cox regression models have confirmed CCDC69 as an independent prognostic factor in breast cancer. Analysis of 1082 breast cancer patients from the TCGA database revealed:

This data demonstrates that high CCDC69 expression is independently associated with improved survival outcomes even after adjusting for other clinical factors .

How does CCDC69 influence chemotherapy resistance in ovarian cancer?

CCDC69 plays a complex role in ovarian cancer chemotherapy response:

Expression in Resistant Cells: CCDC69 is significantly upregulated in cisplatin-resistant ovarian cancer cells. A2780cis cells show a 3.9-fold increase in CCDC69 mRNA and a 2.4-fold increase in protein expression compared to chemosensitive A2780 cells . This upregulation suggests a potential role in the development of resistance mechanisms.

Functional Impact of CCDC69 Knockout:

• CRISPR/Cas9-mediated knockout of CCDC69 in cisplatin-resistant ovarian cancer cells (A2780cis and SKOV3) significantly increased their sensitivity to cisplatin .

• Mechanistically, CCDC69 knockout led to:

  • Abrogation of G1 and G2/M cell cycle arrest

  • Increased levels of cleaved caspase-3 and caspase-8 (apoptosis markers)

  • Greater mitochondrial membrane potential loss

  • Enhanced Bax mitochondrial redistribution

  • Increased p53 acetylation at the K382 site

Rescue Experiments: Restoring CCDC69 expression in knockout cells attenuated their sensitivity to cisplatin, confirming the direct role of CCDC69 in mediating resistance .

These findings collectively suggest that CCDC69 confers cisplatin resistance in ovarian cancer through modulation of apoptotic pathways and cell cycle regulation, positioning it as a potential therapeutic target for overcoming chemoresistance.

What methodological approaches have been used to manipulate CCDC69 expression in research models?

Researchers have employed several sophisticated techniques to modulate CCDC69 expression:

CRISPR/Cas9 Gene Editing:

• The CRISPR/Cas9 system has been successfully used to generate stable CCDC69 knockout cell lines.

• The methodology typically involves:

  • Design of guide RNAs (gRNAs) targeting exon 1 of CCDC69

  • Creation of specific primer sets for the targeted genomic region

  • Confirmation of insertions by PCR and sequence analysis

  • Validation of knockout at the protein level by Western blotting

Epigenetic Modification:

• 5-Aza-dC treatment has been used to modulate CCDC69 expression, suggesting epigenetic regulation through DNA methylation.

• In ovarian cancer cells, heavy CpG methylation (73.1% and 74.3%) was observed in A2780 and A2780cis cells, with expression restored after 5-Aza-dC treatment .

Transient Transfection:

• Restoration of CCDC69 expression in knockout cells has been achieved through transient transfection approaches to confirm functional significance .

These methodological approaches provide researchers with tools to investigate CCDC69's function and potential as a therapeutic target in different cancer contexts.

What are the key considerations when assessing CCDC69 as a potential biomarker in clinical samples?

When evaluating CCDC69 as a biomarker, researchers should consider:

Standardized Detection Methods:

• For immunohistochemistry: Utilize validated antibodies (e.g., Novus NBPI-85,139 at 1:200 dilution) with appropriate controls .

• For mRNA analysis: Employ standardized RT-qPCR protocols with suitable reference genes (e.g., GAPDH) .

Sample Considerations:

• Tumor heterogeneity: Multiple sampling sites within tumors may be necessary.

• Cell type specificity: Consider the cellular composition of samples when interpreting results.

• Matched normal-tumor pairs: Essential for determining differential expression.

Integration with Immune Parameters:

• Assess correlation with immune infiltration metrics and immunotherapy response markers .

• Consider multiplex IHC approaches to simultaneously evaluate CCDC69 and immune cell markers in tissue contexts.

How can researchers effectively investigate the functional mechanisms of CCDC69 in cancer cells?

To elucidate CCDC69's functional roles, researchers should consider these approaches:

Pathway Analysis:

• Gene Set Enrichment Analysis (GSEA) using software such as GSEA v4.0.3 with appropriate reference datasets (e.g., c5.all.v7.0.symbols.gmt from MsigDB) .

• Weighted enrichment statistics methods with sufficient permutations (≥1000) to ensure statistical robustness .

Protein Interaction Studies:

• Co-immunoprecipitation to identify binding partners.

• Proximity ligation assays to confirm protein-protein interactions in situ.

• Protein domain mapping to identify functional regions within the coiled-coil domains.

Functional Assays:

• For chemoresistance studies: MTT/MTS assays, apoptosis assessment (Annexin V/PI staining, caspase activity) .

• Cell cycle analysis: Flow cytometry with propidium iodide staining .

• Mitochondrial function: Membrane potential measurements, Bax translocation assays .

Mechanistic Investigations:

• Assess effects on p53 acetylation and activity .

• Examine impact on central spindle assembly using immunofluorescence microscopy.

• Investigate potential roles in recruiting midzone components during cell division.

What factors should be considered when interpreting contradictory data on CCDC69 expression across different cancer types?

When reconciling seemingly contradictory findings about CCDC69 in cancer research:

Cancer Type Specificity:

• CCDC69 shows distinct expression patterns across cancer types—predominantly downregulated in 18 cancer types but upregulated in specific contexts like cisplatin-resistant ovarian cancer .

• Consider tissue-specific regulatory mechanisms and cellular functions that may explain these differences.

Methodological Variations:

• Assess differences in detection methods (antibodies, primers, platforms) and normalization strategies.

• Evaluate sample preparation techniques that may affect measurement outcomes.

• Consider the cellular composition of samples (tumor purity, stromal/immune content).

Context-Dependent Functional Roles:

• CCDC69 may serve as a tumor suppressor in primary malignancies but acquire different functions during treatment resistance or disease progression.

• In chemoresistant contexts, CCDC69 upregulation appears to promote survival through specific pathways like p53 regulation .

• In immunotherapy contexts, CCDC69 correlates with immune infiltration, suggesting context-specific interactions .

Statistical Considerations:

What are the most promising avenues for therapeutic targeting of CCDC69 in cancer?

Based on current knowledge, several therapeutic approaches warrant investigation:

Chemosensitization Strategies:

• Development of CCDC69 inhibitors to overcome cisplatin resistance in ovarian cancer, potentially through restoring p53 function and apoptotic sensitivity .

• Combination approaches that target CCDC69-regulated pathways alongside standard chemotherapeutics.

Immunotherapy Enhancement:

• Exploration of CCDC69 as a biomarker for patient selection in immunotherapy trials, particularly for PD-1/PD-L1 checkpoint inhibitors in breast cancer .

• Investigation of combination approaches targeting CCDC69 and immune checkpoints simultaneously.

Epigenetic Modulators:

• Given the evidence of CpG methylation controlling CCDC69 expression, development of epigenetic therapies to modulate its expression in context-appropriate ways .

Structure-Based Drug Design:

• Characterization of CCDC69's coiled-coil domains and protein interaction surfaces to develop specific inhibitors that disrupt key protein-protein interactions.

Patient Stratification Approaches:

• Implementation of CCDC69 expression assessment in clinical trials to stratify patients and identify those most likely to benefit from specific treatment modalities.

How might single-cell analysis advance our understanding of CCDC69's role in the tumor microenvironment?

Single-cell approaches offer powerful opportunities to elucidate CCDC69's functions:

Cellular Heterogeneity:

• Single-cell RNA sequencing (scRNA-seq) can reveal cell type-specific expression patterns of CCDC69 within the complex tumor microenvironment.

• This approach could help identify specific cell populations (cancer cells, fibroblasts, immune cells) where CCDC69 expression is most relevant.

Spatial Context:

• Spatial transcriptomics techniques can map CCDC69 expression within tissue architecture, revealing potential relationships with tumor borders, immune-rich regions, or hypoxic zones.

Trajectory Analysis:

• Pseudo-time analyses of single-cell data could elucidate how CCDC69 expression changes during cancer progression, treatment response, or resistance development.

Cellular Interaction Networks:

• Cell-cell communication analyses using single-cell data might reveal how CCDC69-expressing cells interact with immune populations, potentially explaining the observed correlations with immune infiltration .

Treatment Response at Single-Cell Resolution:

• Pre- and post-treatment single-cell profiling could identify cell populations where CCDC69 expression changes in response to therapy, providing insights into resistance mechanisms.