CKMT1A Human

What is CKMT1A and what is its primary function in human cells?

CKMT1A (mitochondrial creatine kinase 1A) is an enzyme primarily located in the mitochondrial intermembrane space where it catalyzes the reversible transfer of phosphate from phosphocreatine to ADP, generating ATP and creatine. This reaction is a critical component of the creatine-phosphocreatine shuttle system that facilitates energy transport from mitochondria to sites of cellular energy consumption. In research contexts, CKMT1A is studied for its role in maintaining energy balance in cells with high and fluctuating energy demands, including cancer cells that require substantial energy for proliferation and survival under challenging conditions such as hypoxia .

How is CKMT1A expression regulated in normal tissues versus cancer tissues?

In normal human physiology, CKMT1A shows tissue-specific expression patterns, with highest levels typically observed in tissues with high energy demands like cardiac muscle, skeletal muscle, and brain. The regulation primarily responds to energy metabolism needs and mitochondrial function.

In contrast, cancer tissues show aberrant CKMT1A expression patterns. According to pan-cancer analysis data, CKMT1A is highly expressed in most cancer types including liver, lung, and breast cancers . This upregulation appears to be driven by both genetic factors and microenvironmental conditions:

• Hypoxia induces CKMT1A expression through HIF-1α (Hypoxia-Inducible Factor 1-alpha) signaling

• Experimental evidence shows that exposing lung cancer cell lines to hypoxic conditions (1% O₂) significantly increases CKMT1A protein expression, with levels peaking at approximately 24 hours of hypoxia

• The regulation involves direct binding of HIF-1α to the CKMT1A promoter region, as confirmed by luciferase reporter assays

This differential expression pattern makes CKMT1A a potential biomarker and therapeutic target in oncology.

What analytical methods are most effective for detecting CKMT1A expression in human samples?

Several complementary analytical methods can be employed to detect and quantify CKMT1A expression in human samples:

RNA-level detection methods:

• Quantitative real-time PCR (qRT-PCR): Provides sensitive measurement of CKMT1A mRNA expression in tissue samples and cell lines

• RNA-sequencing (RNA-seq): Enables comprehensive transcriptomic profiling and comparative analysis across multiple cancer types

• In situ hybridization: Allows visualization of CKMT1A mRNA within tissue sections while preserving spatial information

Protein-level detection methods:

• Western blotting: Quantifies CKMT1A protein levels in cell lysates or tissue homogenates and can detect post-translational modifications

• Immunohistochemistry (IHC): Visualizes CKMT1A protein in tissue sections, providing information about expression levels and localization patterns

• Immunofluorescence: Offers higher resolution imaging of CKMT1A subcellular localization

Functional assays:

• Enzymatic activity assays: Measure the catalytic activity of CKMT1A by detecting the conversion of creatine to phosphocreatine or vice versa

• Metabolomic approaches: Gas or liquid chromatography coupled with mass spectrometry can profile metabolites affected by CKMT1A activity

The choice of method should be determined by the specific research question, sample availability, and whether the goal is to assess expression, localization, or functional activity of CKMT1A.

How does hypoxia regulate CKMT1A expression in cancer cells and what are the molecular mechanisms involved?

Hypoxia is a prominent feature of solid tumors and significantly influences CKMT1A expression through several molecular mechanisms:

HIF-1α-Dependent Regulation:

• Under hypoxic conditions (1% O₂), cancer cells show increased expression of both HIF-1α and CKMT1A proteins

• Time-course experiments in H1650 and H1299 lung cancer cell lines demonstrated that CKMT1A protein levels peak at approximately 24 hours of hypoxia exposure

• HIF-1α directly binds to specific sequences in the CKMT1A promoter region to enhance transcription, as demonstrated by luciferase reporter assays

• When HIF-1α binding sites in the CKMT1A promoter are mutated, hypoxia-induced expression is significantly reduced, though not completely eliminated

HIF-1α-Independent Mechanisms:

• The research indicates that even with mutated HIF-1 binding sites, hypoxia still induced some CKMT1A expression, suggesting additional regulatory mechanisms beyond HIF-1α

• These may include other transcription factors activated under hypoxia, post-transcriptional regulation of mRNA stability, or alterations in protein translation efficiency under hypoxic stress

Experimental Validation Approaches:

• The use of LW6, a specific inhibitor of HIF-1α, confirmed the role of HIF-1α in regulating CKMT1A expression

• This approach provides a methodological strategy for researchers studying hypoxia-induced gene expression

Understanding these regulatory mechanisms may provide insights for developing therapeutic strategies targeting hypoxia-induced metabolic adaptations in cancer.

What is the role of CKMT1A in cancer metabolism and how does it contribute to metabolic reprogramming?

CKMT1A plays several crucial roles in cancer metabolism that support tumor growth and survival:

Metabolic Pathway Involvement:

• Enrichment analysis reveals CKMT1A involvement in "Glycolysis/Gluconeogenesis" and broader "metabolic pathways"

• As a mitochondrial creatine kinase, CKMT1A facilitates energy transport from mitochondria to cytosolic sites of ATP consumption via the creatine phosphate shuttle

Contribution to Metabolic Reprogramming:

• CKMT1A may support the Warburg effect (cancer cells' preference for glycolysis even in the presence of oxygen) by:

  • Ensuring efficient energy transport from mitochondria to cytosolic sites

  • Supporting the integrated function of mitochondrial and glycolytic metabolism

  • Maintaining energy homeostasis when oxidative phosphorylation is compromised

Experimental Evidence:

• Knockdown of CKMT1 in lung cancer cell lines (H1650 and H1299) inhibited proliferation, colony formation, and invasion, suggesting that CKMT1A supports the metabolic requirements for cancer cell growth and metastatic potential

Hypoxia Adaptation:

• Upregulation of CKMT1A under hypoxic conditions indicates its role in metabolic adaptation to low oxygen environments, which is critical for tumor regions experiencing hypoxia due to rapid growth and insufficient vascularization

This metabolic role makes CKMT1A a potential target for therapeutic strategies aimed at disrupting cancer cell metabolism, particularly in tumors that show high expression of this enzyme.

What is the relationship between CKMT1A expression and immune cell infiltration in the tumor microenvironment?

The relationship between CKMT1A expression and immune cell infiltration represents an emerging area of research with important implications for understanding tumor immunology:

Key Correlations with Immune Components:

• CKMT1A expression is negatively correlated with the infiltration of cancer-associated fibroblasts (CAFs) in most tumor types

• More significantly, CKMT1A expression shows negative association with CD8+ T-cell infiltration in several tumor types

• Since CD8+ T cells are critical for anti-tumor immunity, this negative association suggests that CKMT1A might contribute to immune evasion mechanisms

Potential Mechanisms:

• Metabolic competition: CKMT1A's role in cancer cell metabolism might create conditions unfavorable for immune cell function

• Signaling effects: Beyond its metabolic role, CKMT1A might influence pathways that affect cytokine production or immune cell recruitment

• Indirect effects: CKMT1A-mediated hypoxia adaptation might secondarily influence immune cell infiltration and function

Therapeutic Implications:

• The negative correlation with CD8+ T-cell infiltration suggests that targeting CKMT1A might potentially enhance T cell infiltration and function

• Combination approaches targeting both CKMT1A and immune checkpoints could be worth investigating

• Understanding metabolic interactions between CKMT1A-expressing cancer cells and immune cells could inform the development of metabolism-targeted immunotherapies

This emerging area highlights the complex interplay between cancer metabolism and the immune microenvironment, with potential implications for immunotherapy development.

How do genetic alterations of CKMT1A affect its function and what are the implications for cancer research?

Genetic alterations of CKMT1A can significantly impact its function and contribute to cancer development:

Types of Genetic Alterations:

Functional Consequences:

• Alterations may affect:

  • Catalytic activity through mutations in the active site

  • Protein stability and folding

  • Subcellular localization, particularly mitochondrial targeting

  • Protein-protein interactions, especially octamer formation and association with mitochondrial membrane proteins

Methodological Approaches for Study:

• Site-directed mutagenesis to introduce cancer-specific alterations

• Expression of wild-type and mutant CKMT1A in cellular models

• Enzymatic activity assays to assess functional consequences

• Protein localization studies using fluorescently tagged constructs

• CRISPR-Cas9 genome editing to introduce or correct alterations

Implications for Cancer Research:

• Specific alterations might serve as biomarkers for prognosis or treatment response

• Understanding how alterations affect function can inform targeted therapeutic approaches

• CKMT1A alterations might create specific vulnerabilities that could be therapeutically exploited through synthetic lethality approaches

Understanding the implications of CKMT1A genetic alterations contributes to a more complete picture of its role in cancer and may reveal new opportunities for therapeutic intervention.

What experimental models are most suitable for studying CKMT1A function in cancer research?

Selecting appropriate experimental models is crucial for studying CKMT1A function, with each model offering distinct advantages:

Cell Culture Models:

• Cancer cell lines with varying CKMT1A expression

  • H1650 and H1299 lung cancer cell lines have been successfully used

  • Advantages: Easy to manipulate, cost-effective, allows for high-throughput screening

  • Applications: Gene knockdown/overexpression studies, drug screening, basic mechanism studies

• 3D culture systems (spheroids, organoids)

  • Advantages: Recapitulate tumor architecture, allow for development of hypoxic cores

  • Particularly valuable for studying CKMT1A in the context of hypoxia adaptation

  • Can be combined with oxygen sensors to correlate CKMT1A expression with oxygen gradients

Genetic Manipulation Approaches:

• RNA interference (siRNA, shRNA) has been successfully used to study CKMT1A function in cancer cell lines

• CRISPR-Cas9 gene editing allows for complete knockout or introduction of specific mutations

• Overexpression systems for studying gain-of-function effects and structure-function relationships

Animal Models:

• Xenograft models: Useful for studying CKMT1A's role in tumor growth, metastasis, and microenvironment interactions

• Genetically engineered mouse models (GEMMs): Allow study of CKMT1A in de novo tumor development with intact immune system

• Patient-derived xenografts (PDXs): Maintain tumor heterogeneity and more closely recapitulate human disease

Ex Vivo Models:

• Tissue slice cultures: Maintain original tumor architecture while allowing experimental manipulation

• Explant cultures: Enable direct testing of therapies targeting CKMT1A on patient-derived material

Model Selection Considerations:

• Match the model to specific aspects of CKMT1A biology being investigated

• Consider cancer type specificity and endpoint measures

• The most robust approaches often combine multiple model systems to validate findings and address limitations of individual models

How can CKMT1A be targeted in cancer therapy and what experimental approaches have been evaluated?

Targeting CKMT1A represents a promising therapeutic approach based on its role in cancer metabolism and upregulation in various tumor types:

Gene Silencing Approaches:

• RNA interference (RNAi): Studies have used siRNA or shRNA to knockdown CKMT1 expression in cancer cell lines

• Experiments demonstrate that CKMT1 knockdown inhibits proliferation, colony formation, and invasion in non-small cell lung cancer cells

• CRISPR-Cas9 gene editing provides an alternative approach for more complete knockout

Small Molecule Inhibitors:

• Several creatine kinase inhibitors have been developed, though many lack specificity for CKMT1A

• Cyclocreatine analogs function as alternative substrates that can disrupt the creatine-phosphocreatine shuttle

• Structure-based drug design approaches use the crystal structure of CKMT1A to develop specific inhibitors

Combination Therapy Approaches:

• Combining CKMT1A inhibition with hypoxia-targeted therapies may be particularly effective since CKMT1A is upregulated under hypoxic conditions

• Potential synergy with glycolysis inhibitors, based on CKMT1A's involvement in glycolysis/gluconeogenesis pathways

Biomarker Development:

Challenges and Considerations:

• Ensuring specificity for CKMT1A without affecting other creatine kinase isoforms

• Developing effective delivery systems that reach tumor cells

• Identifying and addressing potential resistance mechanisms

• Understanding impacts on normal tissues with high energy demands

These approaches provide a framework for developing CKMT1A-targeted cancer therapies, though more research is needed to fully evaluate their clinical potential.