CENPM Human

What is the molecular structure and characteristics of CENPM Human protein?

CENPM Human is a single, non-glycosylated polypeptide chain containing 203 amino acids (1-180) with a molecular mass of approximately 22.0kDa. Recombinant CENPM is typically fused to a 23 amino acid His-tag at the N-terminus for purification purposes. The protein contains specific domains that facilitate interactions with other centromere proteins, forming complexes essential for centromere assembly and kinetochore formation . When expressed in E. coli for research purposes, the protein exists in a sterile filtered colorless solution and requires specific formulation conditions for stability, including 20mM Tris-HCl buffer (pH 8.0), 0.15M NaCl, 1mM DTT, and 30% glycerol .

What is the fundamental role of CENPM in normal cellular processes?

CENPM functions as a critical component of the centromere, a specialized chromatin domain that persists throughout the cell cycle and serves as a platform for kinetochore assembly during mitosis. It interacts with several other centromere proteins, including CENPA, CENPC, CENPI, and CENPH, to form a functional complex that preserves kinetochore and spindle microtubule attachment during metaphase . All active centromeres are characterized by the presence of long arrays of nucleosomes in which CENPA replaces histone H3, and CENPM serves as an additional factor required for proper centromere assembly . This protein is essential for maintaining genomic stability through accurate chromosome segregation during cell division.

What are the known synonyms and alternative nomenclature for CENPM?

Researchers should be aware of several alternative designations for CENPM in the literature and databases:

• Centromere Protein M

• Interphase Centromere Complex Protein 39 (ICEN39)

• Chromosome 22 Open Reading Frame 18 (C22orf18)

• Proliferation Associated Nuclear Element 1 (PANE1)

• bK250D10.2

• CENP-M

Understanding these alternative nomenclatures is crucial when conducting comprehensive literature searches or database queries to ensure no relevant research is overlooked.

What are the optimal conditions for recombinant CENPM protein storage and handling?

For researchers working with recombinant CENPM Human protein, the following conditions are recommended:

• Short-term storage (2-4 weeks): Store at 4°C in the original formulation

• Long-term storage: Store frozen at -20°C

• Buffer composition: 20mM Tris-HCl buffer (pH 8.0), 0.15M NaCl, 1mM DTT, and 30% glycerol

• Stability enhancement: For extended storage periods, addition of a carrier protein (0.1% HSA or BSA) is recommended

• Quality control: Confirm purity (>85%) using SDS-PAGE before experimental use

• Critical consideration: Avoid multiple freeze-thaw cycles that can compromise protein integrity and activity

Researchers should note that any modifications to these conditions may affect protein stability and function, potentially impacting experimental outcomes.

What methodological approaches are recommended for studying CENPM expression in cancer tissues?

For comprehensive analysis of CENPM expression in cancer contexts, researchers should employ a multi-modal approach:

• Transcriptomic analysis:

  • RT-qPCR for targeted mRNA expression quantification

  • RNA-seq for genome-wide expression profiling and correlation studies

  • In situ hybridization for spatial expression patterns in tissue samples

• Protein detection methods:

  • Western blotting for semi-quantitative protein expression analysis

  • Immunohistochemistry for tissue localization and expression patterns

  • Immunofluorescence for subcellular localization studies

• Bioinformatic approaches:

  • Analysis of publicly available databases (TCGA, Oncomine, GEPIA, Human Protein Atlas, Kaplan-Meier Plotter)

  • Correlation analysis with clinical outcomes and pathological parameters

  • Gene co-expression network analysis to identify functional relationships

• Clinical sample handling:

  • Paired analysis of tumor tissues with adjacent non-tumor (ANT) tissues

  • Fresh-frozen samples for optimal nucleic acid preservation

  • Careful documentation of clinicopathological features for correlation studies

This comprehensive approach enables researchers to establish both the expression patterns and potential clinical significance of CENPM across various cancer types.

What are effective strategies for CENPM functional manipulation in cell culture models?

To investigate CENPM function through expression manipulation in cell culture models:

For CENPM knockdown:

• siRNA transfection for transient suppression (typically 48-72 hours)

• shRNA (short hairpin RNA) delivered via lentiviral vectors for stable knockdown

• CRISPR-Cas9 genome editing for complete knockout studies

For CENPM overexpression:

• Plasmid-based expression systems with appropriate promoters

• Viral transduction systems for difficult-to-transfect cell lines

• Inducible expression systems for controlled temporal expression

Post-manipulation validation:

• Confirm knockdown/overexpression at both mRNA (RT-qPCR) and protein levels (Western blot)

• Assess functional outcomes through appropriate assays (cell cycle analysis, proliferation, apoptosis)

• Include appropriate controls (scrambled siRNA, empty vector)

Studies have successfully employed shCENPM to suppress CENPM expression in lung adenocarcinoma cell lines, demonstrating significant effects on cell proliferation, cell cycle progression, migration, invasion, and apoptosis .

How does CENPM expression vary across different cancer types?

CENPM expression shows significant variation across cancer types, with consistent upregulation compared to normal tissues:

• Comprehensive analyses have identified significant upregulation of CENPM mRNA in at least 14 different types of human cancer

• The degree of overexpression varies by cancer type, with particularly notable expression in breast cancer, lung adenocarcinoma, hepatocellular carcinoma, pancreatic cancer, melanoma, and bladder cancer

• In lung adenocarcinoma, CENPM upregulation correlates with higher pathological stages, suggesting its potential role in disease progression

• Multiple database analyses (Oncomine, GEPIA, Human Protein Atlas) consistently confirm elevated CENPM expression patterns across diverse malignancies

This widespread upregulation across multiple cancer types suggests a fundamental role for CENPM in tumorigenesis that transcends tissue-specific cancer biology.

What molecular mechanisms link CENPM overexpression to cancer progression?

Several interconnected molecular mechanisms explain how CENPM overexpression contributes to cancer progression:

• Chromosome segregation disruption:

  • Overexpression of CENPM leads to unequal numbers of chromosomes during cell division

  • Affected cells can exit mitosis, survive despite chromosomal abnormalities, and contribute to aneuploidy

  • Aneuploidy is found in 65-90% of breast cancer cells and accelerates tumorigenesis by causing chromosomal instability

• Cell cycle dysregulation:

  • CENPM interacts with at least nine genes participating in cell cycle regulation

  • This interaction network influences cell cycle checkpoints and progression

  • Cell cycle analysis confirms that CENPM manipulation affects cell cycle distribution in cancer cells

• AKT1/mTOR pathway activation:

  • In lung adenocarcinoma, CENPM promotes cell proliferation, migration, and invasion through regulation of the AKT1/mTOR signaling pathway

  • This pathway is central to cancer cell growth, survival, and metastatic potential

• Anti-apoptotic effects:

  • CENPM inhibits apoptosis in cancer cells, allowing them to evade programmed cell death

  • This effect has been demonstrated through terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays

These mechanisms collectively contribute to genomic instability, enhanced proliferation, and resistance to cell death—hallmarks of aggressive cancer behavior.

What prognostic significance does CENPM expression have in human cancers?

CENPM expression demonstrates significant prognostic value across several cancer types:

The consistency of these findings across diverse cancer types suggests CENPM expression could serve as a broadly applicable prognostic biomarker, potentially informing treatment decisions and risk stratification.

How do protein-protein interactions contribute to CENPM's role in tumorigenesis?

CENPM's tumorigenic potential is significantly influenced by its interactions with other proteins within complex cellular networks:

• Centromere complex interactions:

  • CENPM interacts with CENPA, CENPC, CENPI, and CENPH to form functional centromere complexes

  • Disruption of these interactions affects kinetochore assembly and chromosome segregation

  • Aberrant interactions can lead to genomic instability that drives cancer progression

• Cell cycle regulatory network:

  • Protein interaction network analysis using STRING and cBioPortal databases has identified at least nine cell cycle-associated genes that interact with CENPM

  • These interactions are crucial for CENPM's function in controlling mitotic progression

  • The network includes proteins involved in spindle assembly, chromosome condensation, and cell cycle checkpoints

• Signaling pathway interactions:

  • CENPM's interaction with components of the AKT1/mTOR pathway directly influences cellular proliferation and survival

  • These interactions represent potential therapeutic vulnerabilities

Advanced techniques for studying these interactions include co-immunoprecipitation, proximity ligation assays, FRET (Fluorescence Resonance Energy Transfer), and mass spectrometry-based proteomics to comprehensively map the CENPM interactome in both normal and cancer contexts.

What experimental approaches can identify synthetic lethal interactions with CENPM in cancer cells?

Identifying synthetic lethal interactions with CENPM provides opportunities for targeted therapeutic strategies:

• Genome-wide screening approaches:

  • CRISPR-Cas9 knockout/knockdown screens in CENPM-high versus CENPM-low cells

  • shRNA library screens to identify genes whose inhibition selectively kills CENPM-overexpressing cells

  • Small molecule compound screens to identify chemical sensitizers

• Targeted pathway analysis:

  • Focus on known vulnerabilities in cells with chromosomal instability

  • Investigate DNA damage response pathways, as cells with CENPM-induced aneuploidy may have compromised repair mechanisms

  • Test inhibitors of cell cycle checkpoints that might prevent adaptation to CENPM-induced chromosomal abnormalities

• Validation methodologies:

  • Genetic validation through orthogonal knockdown/knockout methods

  • Pharmacological validation using available inhibitors

  • In vivo validation in xenograft models with manipulated CENPM expression

• Clinical correlation:

  • Mining cancer genomics databases to identify co-occurring molecular features with CENPM overexpression

  • Retrospective analysis of treatment responses in patients with varying CENPM levels

This systematic approach can identify context-specific vulnerabilities in CENPM-overexpressing cancers, potentially leading to novel therapeutic combinations with enhanced selectivity for cancer cells.

How might CENPM function as a therapeutic target, and what challenges need to be addressed?

Developing CENPM-targeted therapies presents both promising opportunities and significant challenges:

Therapeutic opportunities:

• Direct targeting approaches:

  • Small molecule inhibitors disrupting CENPM's protein-protein interactions

  • Degrader technologies (PROTACs) targeting CENPM for proteasomal degradation

  • RNA interference-based therapeutics (siRNA, antisense oligonucleotides)

• Pathway-based interventions:

  • Targeting the AKT1/mTOR pathway in CENPM-overexpressing tumors

  • Exploiting vulnerabilities created by CENPM-induced chromosomal instability

  • Combination strategies with existing chemotherapeutics or targeted agents

Challenges requiring resolution:

• Target specificity concerns:

  • CENPM is expressed in normal dividing cells, raising potential for off-target toxicities

  • Identifying cancer-specific vulnerabilities is essential for therapeutic window

• Delivery and bioavailability:

  • Nuclear localization of CENPM presents delivery challenges

  • Development of appropriate drug delivery systems for different cancer types

• Resistance mechanisms:

  • Potential compensatory pathways that might emerge upon CENPM inhibition

  • Need for combination strategies to prevent resistance development

• Patient selection:

  • Development of companion diagnostics to identify patients most likely to benefit

  • Standardization of CENPM assessment methods for clinical decision-making

Addressing these challenges requires integrated approaches combining structural biology, medicinal chemistry, and translational research to develop effective CENPM-targeted therapeutic strategies.

What cell-based assays are most informative for studying CENPM's impact on cell cycle and proliferation?

To comprehensively evaluate CENPM's effects on cell cycle progression and proliferation, researchers should employ multiple complementary assays:

• Cell cycle analysis techniques:

  • Flow cytometry with propidium iodide staining for DNA content analysis

  • BrdU incorporation assays to measure S-phase entry

  • Phospho-histone H3 (Ser10) staining to identify mitotic cells

  • Combined with CENPM knockdown or overexpression conditions

• Proliferation assays:

  • Cell Counting Kit-8 (CCK-8) assays for measuring proliferation rates

  • Colony formation assays to assess long-term growth potential

  • Real-time cell analysis systems for continuous monitoring of proliferation dynamics

• Apoptosis detection methods:

  • Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

  • Annexin V/PI staining for flow cytometry

  • Caspase activity assays to assess apoptotic pathway activation

• Migration and invasion assays:

  • Transwell migration assays to assess cell motility

  • Matrigel invasion assays to evaluate invasive capacity

  • Wound healing assays for directional migration assessment

• In vivo validation:

  • Xenograft models with CENPM-manipulated cells

  • Analysis of tumor growth, proliferation markers, and apoptotic indices

  • Correlation with CENPM expression levels

These methodologies have successfully demonstrated CENPM's role in promoting cell proliferation, altering cell cycle progression, enhancing migration and invasion, and inhibiting apoptosis in multiple cancer cell lines .

What techniques can detect CENPM-induced chromosomal instability and aneuploidy?

Effective assessment of CENPM-related chromosomal instability requires multiple complementary approaches:

• Cytogenetic techniques:

  • Metaphase spread preparation and chromosome counting

  • Fluorescence in situ hybridization (FISH) using chromosome-specific probes

  • Spectral karyotyping for comprehensive chromosomal abnormality detection

• Flow cytometry-based methods:

  • DNA content analysis to identify aneuploid populations

  • Combined DNA/protein analysis to correlate aneuploidy with cell cycle markers

  • Sorting of aneuploid cells for further characterization

• Molecular analysis:

  • Array comparative genomic hybridization (aCGH) to detect copy number variations

  • Single-cell whole-genome sequencing for detailed aneuploidy characterization

  • Assessment of chromosomal instability markers and signatures

• Functional consequences:

  • Analysis of gene expression changes resulting from aneuploidy

  • Assessment of cellular stress responses activated by chromosome imbalance

  • Correlation with phenotypic changes (proliferation, migration, drug sensitivity)

• Time-course experiments:

  • Tracking aneuploidy development following CENPM manipulation

  • Assessment of selection pressures favoring specific aneuploid karyotypes

  • Correlation with malignant phenotype acquisition

These methods can establish the direct relationship between CENPM overexpression and the generation of chromosomal instability, a hallmark feature of many aggressive cancers.

How should researchers design experiments to investigate CENPM's role in the AKT1/mTOR signaling pathway?

To rigorously investigate CENPM's regulatory role in the AKT1/mTOR pathway, researchers should follow these experimental design principles:

• Expression manipulation studies:

  • Compare AKT1/mTOR pathway activation in cells with CENPM knockdown, overexpression, and controls

  • Western blot analysis for phosphorylated forms of key pathway components (p-AKT, p-mTOR, p-S6K, p-4EBP1)

  • Assess both total protein levels and phosphorylation status to distinguish between expression and activation effects

• Pathway inhibition studies:

  • Use specific inhibitors (e.g., LY294002 for PI3K) to block the pathway

  • Determine whether CENPM-induced phenotypes are rescued by pathway inhibition

  • Test combination effects of CENPM manipulation with pathway inhibitors

• Mechanistic investigations:

  • Co-immunoprecipitation to detect physical interactions between CENPM and pathway components

  • Proximity ligation assays to confirm interactions in situ

  • siRNA-mediated knockdown of individual pathway components to identify essential mediators

• Functional readouts:

  • Proliferation, migration, invasion, and apoptosis assays with and without pathway inhibition

  • Analysis of downstream transcriptional targets of the AKT1/mTOR pathway

  • Assessment of metabolic changes associated with pathway activation

• In vivo validation:

  • Xenograft models with pathway inhibition in CENPM-manipulated cells

  • Immunohistochemistry for pathway activation markers in tumor samples

  • Correlation with CENPM expression levels in patient samples

This systematic approach can establish whether CENPM's oncogenic effects are primarily mediated through the AKT1/mTOR pathway or involve additional signaling mechanisms.

How can CENPM expression be effectively evaluated in clinical samples?

Standardized methods for CENPM evaluation in clinical samples should include:

• Tissue sample considerations:

  • Paired analysis of tumor and adjacent non-tumor tissues when possible

  • Consistent collection and preservation protocols to minimize pre-analytical variables

  • Documentation of tumor region, necrosis status, and stromal content

• Protein expression analysis:

  • Immunohistochemistry with validated antibodies and standardized scoring systems

  • Western blot analysis with appropriate loading controls

  • Tissue microarrays for high-throughput analysis across multiple samples

• mRNA expression analysis:

  • RT-qPCR with validated reference genes for normalization

  • RNA-seq for genome-wide expression profiling

  • In situ hybridization for spatial expression analysis in heterogeneous tumors

• Standardization considerations:

  • Inclusion of positive and negative controls in each batch

  • Inter-laboratory validation to ensure reproducibility

  • Clear threshold definitions for "high" versus "low" expression based on clinical outcomes

• Data integration:

  • Correlation with clinicopathological features and patient outcomes

  • Multivariate analysis to assess independent prognostic value

  • Integration with other molecular markers for comprehensive profiling

These standardized approaches enable reliable assessment of CENPM expression for potential clinical applications in diagnosis, prognosis, and treatment selection.

What is the potential for CENPM as a biomarker in cancer diagnostics and prognostics?

CENPM demonstrates significant potential as a cancer biomarker based on multiple lines of evidence:

The consistency of CENPM's prognostic significance across diverse cancer types suggests it could serve as a broadly applicable biomarker, though additional prospective validation studies are needed before clinical implementation.

How might CENPM research inform future clinical trials and therapeutic development?

CENPM research has multiple implications for future clinical research and therapy development:

• Patient stratification strategies:

  • CENPM expression levels could identify patients for specific clinical trials

  • High CENPM expression may identify patients with more aggressive disease requiring intensified therapy

  • Correlation of CENPM with other biomarkers could define molecular subtypes for targeted approaches

• Novel therapeutic targets:

  • Direct targeting of CENPM or its interactions with other centromere proteins

  • Exploiting synthetic lethal interactions in CENPM-high tumors

  • Targeting the AKT1/mTOR pathway in CENPM-overexpressing cancers

• Rational combination approaches:

  • Combining CENPM-targeted therapies with existing chemotherapeutics

  • Exploiting vulnerabilities created by chromosomal instability

  • Sequential therapy approaches based on CENPM-associated pathway dependencies

• Resistance mechanisms:

  • Understanding how CENPM contributes to therapy resistance

  • Identifying biomarkers of response to CENPM-targeted approaches

  • Developing strategies to overcome resistance mechanisms

• Trial design considerations:

  • Incorporating CENPM assessment in correlative studies

  • Adaptive trial designs based on CENPM expression or downstream effects

  • Utilizing liquid biopsies to monitor CENPM expression during treatment

These approaches could accelerate the translation of CENPM research findings into clinically meaningful advances for patients with CENPM-overexpressing cancers.