PCK1 Human

What is PCK1 and what are its primary functions in human cells?

PCK1 (Phosphoenolpyruvate carboxykinase 1, also known as PEPCK-C for its cytosolic localization) is a monomeric enzyme of approximately 67-68 kDa belonging to the PEP carboxykinase family. Human PCK1 consists of 622 amino acids with a kinase domain spanning residues 27-615 and potential acetylation sites at Lys70 .

PCK1 serves multiple metabolic functions:

• Gluconeogenesis: Catalyzes the conversion of oxaloacetate to phosphoenolpyruvate (PEP), a rate-limiting step in glucose synthesis

• Cataplerotic and anaplerotic activities: Removes and replaces elements of the TCA cycle

• Glyceroneogenesis: Creates glycerol-3-phosphate used for reesterification and storage of free fatty acids in adipocytes

The enzyme is predominantly expressed in postnatal cells including hepatocytes, adipocytes (both white and brown), skeletal muscle cells, and mammary epithelium .

How can I detect PCK1 expression in human tissue samples?

Several methodological approaches are available for detecting PCK1:

• Western blotting: Using specific antibodies such as Mouse Anti-Human PCK1 Monoclonal Antibody to detect PCK1 protein (approximately 68 kDa) in tissue lysates. Human liver and kidney tissues show strong PCK1 expression under reducing conditions .

• qPCR: Quantitative PCR is effective for measuring PCK1 mRNA expression levels, as demonstrated in studies comparing primary and metastatic colorectal cancer samples .

• Immunohistochemistry: For visualizing PCK1 expression in tissue sections, which has been used to analyze expression patterns across different cancer stages.

• Metabolic flux analysis: Using 13C-labeled substrates (e.g., glutamine) to track PCK1-dependent metabolic pathways .

What are normal expression patterns of PCK1 across human tissues?

PCK1 shows distinct tissue-specific expression patterns:

• Liver: Highest expression level, crucial for systemic glucose production during fasting

• Kidney: High expression levels similar to liver

• Adipose Tissue: Moderate expression, important for triglyceride synthesis

• Skeletal Muscle: Lower expression than liver and kidney

• Mammary Epithelium: Expression increases during lactation

Western blot analysis reveals strong PCK1 detection at approximately 68 kDa in human liver and kidney tissue lysates, reflecting its important role in gluconeogenesis in these tissues .

How does PCK1 expression change in colorectal cancer metastasis?

PCK1 shows significant expression changes during colorectal cancer progression:

• Multiple independent datasets consistently demonstrate that PCK1 is highly upregulated in CRC liver metastases compared to primary tumors .

• QPCR quantification confirms PCK1 up-regulation in liver metastatic derivatives relative to isogenic parental counterparts .

• This upregulation is observed in both unpaired samples and paired samples (comparing primary tumors and liver metastases from the same patients) .

• Statistical analysis shows significant upregulation (p=0.01, Student's t-test) in various datasets comparing metastatic versus primary CRC samples .

The consistent upregulation across multiple independent studies suggests PCK1 plays a fundamental role in the metastatic process rather than representing a passenger alteration.

What functional evidence links PCK1 to colorectal cancer metastasis?

Strong functional evidence supports PCK1's role in metastasis:

• Knockdown studies: Depletion of PCK1 in SW480 cells by two independent shRNAs significantly impaired (p<0.0001) CRC liver metastatic colonization in NSG mice. Similar effects were observed in LS174T cells .

• Overexpression studies: PCK1 over-expression in SW480 cells significantly increased (p=0.003) liver metastatic colonization .

• Immune-competent models: Pck1 depletion in murine CRC cell line CT26 decreased liver colonization in immunocompetent mice, confirming the effect is not dependent on immune deficiency .

• Timing effects: Using inducible PCK1 shRNA showed that even after initial liver colonization (days 0-4), PCK1 depletion continued to impair (p=0.004) CRC metastatic liver growth .

Importantly, PCK1 depletion did not impact subcutaneous tumor growth in multiple cell lines, suggesting its effects are specific to the metastatic environment .

What is the molecular mechanism through which PCK1 promotes cancer metastasis?

PCK1 promotes metastatic growth through a novel metabolic mechanism:

• Hypoxic adaptation: PCK1 promotes growth under hypoxia—a key feature of the hepatic microenvironment. PCK1 depletion significantly reduced CRC cell growth under hypoxia, but not under normoxia .

• Pyrimidine biosynthesis: Metabolite profiling of highly/poorly metastatic CRC PDX pairs revealed increased abundance of multiple nucleoside base precursors in metastatic samples, particularly metabolites in the pyrimidine biosynthetic pathway (orotate, dihydroorotate, and ureidopropionate) .

• Nucleotide pool maintenance: PCK1 depletion under hypoxia led to significant depletion of nucleosides and nucleotides including uridine, guanine, UMP, CMP, CDP, IMP, GMP, and AMP .

• Reductive carboxylation: 13C glutamine metabolic flux analysis showed that generation of pyrimidine precursors (aspartate and orotate) by reductive carboxylation was significantly decreased upon PCK1 depletion .

Supplementation of PCK1-depleted cells with aspartate and pyruvate rescued the hypoxic growth defect, confirming that PCK1 promotes growth by maintaining aspartate levels for nucleotide synthesis .

What are the optimal in vivo models for studying PCK1 in cancer metastasis?

Based on published research, optimal in vivo approaches include:

For quantitative assessment, bioluminescence imaging allows longitudinal monitoring, while end-point analysis should include liver weight, histological examination, and metastatic nodule counting .

How can metabolic flux analysis be optimized to study PCK1-dependent pathways?

Optimal metabolic flux analysis approaches include:

• Isotope tracing: Use 13C-labeled glutamine to track carbon flow through metabolic pathways. This approach revealed that PCK1 promotes reductive carboxylation to generate pyrimidine precursors under hypoxia .

• Compartment-specific analysis: Distinguish between cytosolic and mitochondrial metabolic pools to accurately assess PCK1's cytosolic activity.

• Hypoxic conditions: Perform metabolic flux experiments under physiologically relevant oxygen tensions (1-2% O2) to capture PCK1's role in hypoxic adaptation .

• Time-course measurements: Collect samples at multiple time points to capture dynamic metabolic changes.

• Complementary approaches: Combine flux analysis with metabolite profiling and enzyme activity assays for comprehensive characterization.

Analysis should focus specifically on reductive carboxylation pathways, TCA cycle intermediates, and nucleotide synthesis pathways, as these are most relevant to PCK1's role in cancer progression .

What genetic approaches are most effective for modulating PCK1 expression in experimental systems?

Effective genetic approaches for PCK1 modulation include:

• Multiple independent shRNAs: Using at least two different shRNA sequences targeting PCK1 controls for off-target effects. Studies have successfully used this approach in SW480, LS174T, and CT26 cell lines .

• Inducible knockdown systems: Doxycycline-inducible shRNA provides temporal control of PCK1 expression, allowing distinction between effects on different stages of metastasis .

• Stable overexpression: PCK1 overexpression in SW480 cells significantly increased liver metastatic colonization, providing complementary evidence to knockdown studies .

• Species-specific approaches: For mouse models, targeting murine Pck1 in cell lines like CT26 allows studies in immunocompetent settings .

When designing genetic interventions, it's critical to validate knockdown or overexpression by both mRNA quantification (qPCR) and protein expression (Western blot) .

How does PCK1 function differ under normoxic versus hypoxic conditions?

PCK1 functions dramatically differently depending on oxygen availability:

• Under normoxia:

  • PCK1 primarily contributes to gluconeogenesis in normal cells

  • Depletion of PCK1 has minimal impact on cancer cell proliferation under normal oxygen conditions

  • Nucleotide levels are not significantly affected by PCK1 depletion

• Under hypoxia:

  • PCK1 is repurposed to support nucleotide biosynthesis

  • PCK1 depletion significantly reduces cancer cell growth

  • PCK1 drives reductive carboxylation to generate aspartate, a critical nucleotide precursor

  • Nucleoside and nucleotide levels (uridine, guanine, UMP, CMP, CDP, IMP, GMP, and AMP) are severely depleted in PCK1-knockdown cells

The observed decreases in nucleotide levels under PCK1 depletion were abrogated under normoxic conditions, confirming the oxygen-dependent nature of this mechanism .

What is the relationship between PCK1 and pyrimidine biosynthesis?

PCK1 plays a previously unrecognized role in pyrimidine biosynthesis:

• Metabolite profiling of highly metastatic tumors shows increased abundance of multiple pyrimidine biosynthetic pathway intermediates (orotate, dihydroorotate, and ureidopropionate) .

• PCK1 promotes reductive carboxylation under hypoxia to produce aspartate, a key precursor for pyrimidine biosynthesis .

• PCK1 depletion significantly reduces the generation of aspartate and orotate by reductive carboxylation .

• Supplementation with aspartate partially rescues the growth defect in PCK1-depleted cells under hypoxia, confirming aspartate limitation as a key mechanism .

• The pyrimidine biosynthetic enzyme DHODH represents a potential therapeutic target in PCK1-high tumors, as inhibition with leflunomide substantially impairs CRC liver metastatic colonization and hypoxic growth .

This connection between PCK1 and nucleotide synthesis represents a novel metabolic adaptation that enables cancer cell growth under hypoxic conditions .

How might the PCK1 pathway be therapeutically targeted in cancer?

Several potential therapeutic strategies emerge from PCK1 research:

• DHODH inhibition: Leflunomide, an FDA-approved arthritis drug that inhibits the pyrimidine biosynthetic enzyme DHODH, substantially impairs CRC liver metastatic colonization and hypoxic growth .

• PCK1 direct targeting: Developing specific PCK1 inhibitors could selectively impair metastatic growth while potentially sparing normal tissue function.

• Metabolic interventions: Strategies that disrupt aspartate availability might preferentially affect PCK1-dependent metastatic cells.

• Combination approaches: Combining PCK1/DHODH-targeting strategies with standard-of-care treatments might prevent metastatic development.

These approaches are supported by epidemiologic evidence showing that anti-gluconeogenic drugs are associated with improved CRC metastasis outcomes, potentially through PCK1-related mechanisms .

What antibodies and detection methods are most reliable for human PCK1 research?

Based on available data, reliable detection methods include:

• Western blot: Mouse Anti-Human PCK1 Monoclonal Antibody (Clone #789114) successfully detects PCK1 at approximately 68 kDa in human liver and kidney tissues under reducing conditions .

• Recommended protocol:

  • Use PVDF membrane

  • Probe with 1 μg/mL of Mouse Anti-Human PCK1 Monoclonal Antibody

  • Use HRP-conjugated Anti-Mouse IgG Secondary Antibody

  • Conduct under reducing conditions using appropriate immunoblot buffer

The antibody specifically recognizes human PCK1, with the epitope corresponding to amino acids Met1-Ile88 of the protein (Accession # P35558) .

How should researchers control for PCK1 specificity in experimental models?

To ensure specificity in PCK1 research:

• Multiple knockdown controls: Use at least two independent shRNA sequences targeting different regions of PCK1 to control for off-target effects .

• Rescue experiments: Perform reconstitution with shRNA-resistant PCK1 to confirm phenotypes are specifically due to PCK1 loss.

• Isoform specificity: Distinguish between PCK1 (cytosolic) and PCK2 (mitochondrial) effects, as they may have distinct functions in cancer progression.

• Metabolic validation: Confirm PCK1 modulation affects expected metabolic pathways through metabolite profiling or flux analysis .

• Tissue-specific controls: When studying PCK1 in cancer, compare expression to the appropriate normal tissue counterpart.

These controls are essential given that PCK1 functions can vary dramatically by tissue type, oxygen conditions, and disease state .

What are the key considerations when analyzing PCK1 expression data from patient samples?

When analyzing PCK1 expression in clinical samples:

• Paired vs. unpaired analysis: When possible, analyze paired samples (primary tumor and metastasis from the same patient) to control for inter-patient variability. Studies have shown significant PCK1 upregulation (p=0.01) in CRC liver metastases in datasets containing paired samples .

• Heterogeneity considerations: PCK1 expression may vary within tumors, particularly between hypoxic and well-oxygenated regions.

• Correlation with hypoxia markers: Co-analyze PCK1 with established hypoxia markers to understand contextual relevance.

• Metastatic site specificity: PCK1 upregulation appears particularly important in liver metastases, so site-specific analysis is recommended .

• Statistical approaches: For comparison between primary tumors and metastases, Student's t-test has been effectively used to demonstrate significant differences (p<0.0001) .

Patient-derived xenograft (PDX) models can provide valuable insights, as they maintain the expression patterns of the original tumor and their metastatic behavior in mice correlates with patient outcomes .

What are the emerging questions about PCK1's role beyond cancer and metabolism?

Several emerging research areas deserve investigation:

• Immune system interactions: How does PCK1-driven metabolism in cancer cells affect the tumor immune microenvironment?

• Stem cell biology: Does PCK1 play a role in stem cell maintenance through metabolic regulation?

• Aging: Could PCK1's role in metabolic adaptation influence longevity and age-related diseases?

• Drug resistance: Does PCK1 contribute to therapy resistance by enabling metabolic adaptation to treatment-induced stress?

• PCK1 regulation: What mechanisms control PCK1 upregulation in metastatic cells, and can these be targeted therapeutically?

These questions represent important frontiers that build upon the established roles of PCK1 in metabolism and cancer progression .

How does PCK1 interact with other metabolic enzymes in cancer progression?

PCK1 likely functions within a coordinated metabolic network:

• Relationship with DHODH: PCK1 and DHODH work in concert to support pyrimidine nucleotide biosynthesis under hypoxia, with DHODH inhibition substantially impairing CRC liver metastatic colonization .

• Interaction with PKM2: Previous studies suggest PCK1 may operate in reverse when pyruvate kinase is impaired, potentially converting phosphoenolpyruvate to oxaloacetate. Highly metastatic CRC cells upregulate PKLR, which inhibits PKM2 activity, potentially facilitating this reverse reaction .

• Coordination with reductive glutamine metabolism: PCK1 promotes reductive carboxylation of glutamine-derived metabolites, suggesting coordination with glutaminolysis pathways .

Understanding these enzyme interactions may reveal vulnerable nodes in cancer metabolism that could be targeted therapeutically.

What is the potential clinical utility of PCK1 as a biomarker or therapeutic target?

PCK1 shows promise for clinical applications:

• As a biomarker: Consistent upregulation in liver metastases suggests PCK1 could serve as a biomarker for metastatic potential in primary CRC .

• Target validation: Functional studies demonstrate that PCK1 knockdown significantly impairs metastatic growth in multiple models, validating it as a potential therapeutic target .

• Drug repurposing: DHODH inhibitors like leflunomide (already FDA-approved for arthritis) could be repurposed for metastatic CRC based on their ability to disrupt the PCK1-dependent pyrimidine synthesis pathway .

• Patient stratification: PCK1 expression levels could potentially identify patients most likely to benefit from metabolic-targeted therapies.

The specificity of PCK1's effects on metastatic growth rather than primary tumor growth suggests potential as a metastasis-specific therapy with potentially reduced systemic toxicity .