CHMP4C Antibody

What is CHMP4C and what are its primary cellular functions?

CHMP4C is a charged polyvesicular protein (CHMP) involved in the composition of the endosomal sorting complex (ESCRT-III) required for transport III. It promotes the necessary separation of daughter cells during cell division . CHMP4C is intrinsically involved in cell cycle regulation, with expression levels directly correlating with cellular proliferation rates across both normal and malignant tissues . The protein has been implicated in various cancer progressions, including prostate cancer, cervical cancer, and lung cancers .

What detection methods are most reliable for CHMP4C expression analysis?

Multiple complementary techniques are recommended for comprehensive CHMP4C detection:

• Western blotting: For quantitative protein expression assessment and size validation

• qRT-PCR: For sensitive detection of CHMP4C mRNA levels

• Immunohistochemical (IHC) staining: For visualization of expression patterns within tissue architecture

• Bioinformatic analysis: RNA-seq data from The Cancer Genome Atlas (TCGA) database and Human Protein Atlas (HPA) database provide reference data for differential expression analysis

Research has validated these approaches for detecting CHMP4C overexpression in lung cancer tissues compared to adjacent normal tissues, with consistent results across protein and mRNA levels .

What is the recommended protocol for CHMP4C immunohistochemical staining?

For optimal CHMP4C detection via IHC, follow this validated protocol:

• Prepare tissue sections through dewaxing and rehydration

• Perform antigen retrieval (methods may vary by antibody manufacturer)

• Block endogenous peroxidase activity with 6% hydrogen peroxide

• Wash three times with PBS

• Incubate with anti-CHMP4C antibodies (e.g., Origene antibody) at 4°C overnight

• Wash three times with PBS

• Incubate with horseradish peroxidase-conjugated secondary antibody for 1 hour

• Counterstain nuclei with hematoxylin solution

This protocol has been successfully employed to differentiate CHMP4C expression between lung adenocarcinoma and normal lung tissues.

What cell lines are recommended as positive controls for CHMP4C studies?

Based on existing research, the following cell lines have demonstrated reliable CHMP4C expression:

Lung squamous carcinoma cell lines:

• NCI-H596

• SK-MES-1

• LK2

• H520

• SW900

• LUDLU1

Lung adenocarcinoma cell lines:

• A549

• NCI-H827

• NCI-H1975

• SPC-A-1

• NCI-H460

• NCI-H1299

• PC9

• NCI-H292

These cell lines have been validated for CHMP4C expression using both qRT-PCR and Western blotting, making them suitable positive controls for antibody validation.

How does CHMP4C affect cell cycle regulation in cancer cells?

CHMP4C plays a significant role in cell cycle regulation, particularly in cancer progression. Experimental evidence shows that CHMP4C knockdown in lung adenocarcinoma cell lines (NCI-H1299 and NCI-H292) significantly prolongs the G0/G1 phase of the cell cycle . This suggests that CHMP4C overexpression facilitates G0/G1 checkpoint progression, potentially contributing to unregulated cellular proliferation.

Functional experiments demonstrate that CHMP4C knockdown significantly inhibits:

• Cell proliferation (confirmed via cell proliferation assays)

• Colony formation capacity

• Cell migration and invasion abilities

Gene Ontology analysis reveals that CHMP4C-related genes are primarily involved in cell proliferation and migration regulation, providing mechanistic insight into its oncogenic functions .

What signaling pathways interact with CHMP4C in tumor progression?

CHMP4C interacts with several critical oncogenic signaling pathways:

• PI3K-Akt signaling pathway: KEGG analysis shows CHMP4C participates in this pathway that regulates cell survival, proliferation, and growth

• Additional enriched pathways (identified through GSEA):

  • Oxidative Phosphorylation

  • Myc Target V1

  • Myc Target V2

  • DNA Repair

  • Unfolded Protein Response

  • Protein Transcription

  • mTORC1 Signaling

  • Glycolysis

These pathway interactions provide potential mechanisms through which CHMP4C contributes to tumorigenesis and cancer progression, offering targets for further mechanistic studies.

How does CHMP4C expression correlate with chemotherapy sensitivity in lung cancer?

Research indicates that CHMP4C expression levels significantly influence chemotherapy sensitivity in lung adenocarcinoma. Using the pRRophetic algorithm and the Cancer Drug Sensitivity Genomics (GDSC) database, studies have demonstrated that patients with high CHMP4C expression show altered sensitivity to at least 12 chemotherapeutic agents .

Most notably, high CHMP4C expression correlates with decreased sensitivity to:

• Acitinib (p = 2.4×10^-7)

• Olaparib (p = 1.6×10^-5)

These findings suggest CHMP4C expression may serve as a predictive biomarker for chemotherapy response. Interestingly, in prostate cancer, patients with high CHMP4C expression showed increased sensitivity to paclitaxel and 5-fluorouracil, highlighting context-dependent effects across cancer types .

What techniques are most effective for CHMP4C knockdown studies?

For CHMP4C knockdown studies, the following approaches have demonstrated efficacy:

siRNA Transfection:

• Validated siRNAs: si-CHMP4C#1 and si-CHMP4C#2

• Transfection duration: 48 hours

• Validation method: qRT-PCR and western blotting

• Functional assessment: Cell proliferation, colony formation, wound healing, migration and invasion assays

Experimental design considerations:

• Include appropriate controls (si-CHMP4C#NC)

• Validate knockdown efficiency at both mRNA and protein levels

• Perform multiple functional assays to comprehensively assess phenotypic effects

• Analyze cell cycle changes using flow cytometry

In published studies, CHMP4C knockdown successfully reduced both mRNA and protein levels in NCI-H1299 and NCI-H292 cells, resulting in significant inhibition of malignant behaviors.

How can CHMP4C expression patterns help differentiate between lung cancer subtypes?

CHMP4C expression analysis shows diagnostic value in differentiating lung cancer subtypes:

For lung squamous cell carcinoma (LUSC):

• ROC curve analysis from TCGA database showed average AUC of 0.829

• ROC from GSE19188 database showed average AUC of 0.708

• Logistic regression model achieved:

  • Average AUC: 0.823

  • Accuracy: 0.898

  • Precision: 0.912

  • Recall: 0.983

  • F1-score: 0.946

For lung adenocarcinoma (LUAD):

• CHMP4C is significantly upregulated compared to normal tissues

• Expression correlates with clinical progression and prognosis

These findings suggest CHMP4C expression could serve as a biomarker to distinguish between different lung cancer subtypes, potentially complementing traditional histopathological assessments.

What are the challenges in targeting CHMP4C for cancer therapeutics?

Despite CHMP4C's potential as a therapeutic target, several challenges exist:

• Expression in normal cells: CHMP4C is detectable in normal human cells, indicating potential off-target effects that could limit the therapeutic window

• Functional complexity: CHMP4C is involved in fundamental cellular processes like cell division, complicating targeted approaches

• Context-dependent effects: CHMP4C's role varies between cancer types, with different effects on chemosensitivity observed across cancers

• Early detection limitations: The association of CHMP4C with increased cell numbers complicates early detection and does not significantly enhance current scanning and biopsy methods

Potential approaches to overcome these challenges include:

• Combination therapies targeting CHMP4C alongside conventional chemotherapeutics

• Development of more specific CHMP4C inhibitors

• Exploration of synthetic lethality approaches

How might CHMP4C serve as a biomarker for early cancer detection?

While CHMP4C shows promise as a diagnostic biomarker, current research suggests its greatest utility may be in:

• Prognostic assessment: High CHMP4C expression correlates with poor prognosis in lung adenocarcinoma patients

• Treatment stratification: CHMP4C expression levels could guide treatment decisions, particularly regarding chemotherapy selection

• Liquid biopsy development: Further investigation of CHMP4C expression in blood samples may enable less invasive detection methods

Research limitations include the complexity of distinguishing cancer-specific CHMP4C overexpression from normal cellular proliferation. Current evidence suggests CHMP4C should be considered as part of a multi-biomarker panel rather than as a standalone diagnostic marker.

What molecular docking studies have been conducted for CHMP4C inhibition?

Recent molecular docking studies have predicted potential small molecule compounds that may target CHMP4C in lung adenocarcinoma . These computational approaches have identified:

• At least 10 key drugs with potential binding affinity to CHMP4C

• Compounds that may modulate CHMP4C's function in cell proliferation and migration

• Structures that could serve as starting points for drug development

These findings provide promising directions for developing CHMP4C-targeting therapeutics, though further experimental validation is needed to confirm binding affinity, specificity, and functional effects.

What are the optimal cell culture conditions for studying CHMP4C expression?

For consistent and reliable CHMP4C expression studies, the following culture conditions have been validated:

For LUAD cell lines:

• NCI-H827, NCI-H1975, SPC-A-1, NCI-H460, NCI-H1299, PC9, NCI-H292: RPMI-1640 medium (Gibco)

• BEAS-2B and A549: DMEM (Gibco)

• All media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin

• Culture environment: 37°C with 5% CO₂

For sample preparation:

• Cell lysis buffers should include protease inhibitors to prevent CHMP4C degradation

• Standardized protein quantification is essential for comparative analyses

• Consider subcellular fractionation to detect compartment-specific localization

Maintaining consistent culture conditions is critical for reproducible CHMP4C expression analyses, particularly when comparing across multiple cell lines.

How should researchers interpret conflicting CHMP4C expression data?

When encountering inconsistent CHMP4C expression data, consider these methodological factors:

• Detection method sensitivity: Western blotting may show different results than IHC due to epitope accessibility and antibody specificity

• Sample preparation variables: Fixation methods and duration significantly impact antibody binding and epitope preservation

• Antibody selection: Different antibodies target distinct CHMP4C epitopes, potentially yielding variable results

• Heterogeneity within samples: Tumor heterogeneity may result in different expression patterns across regions of the same sample

• Context-dependent regulation: CHMP4C expression may fluctuate based on cell cycle phase, stress conditions, or microenvironment

Validation strategies include:

• Using multiple antibodies targeting different epitopes

• Employing complementary detection methods

• Including appropriate positive and negative controls

• Correlating protein with mRNA expression

• Analyzing larger sample cohorts when possible