KRT8 Human, His

What is KRT8 and what is its biological function?

KRT8, also known as Cytokeratin-8, Keratin-8, or CK-8, is a type II cytoskeletal protein that forms intermediate filaments in epithelial cells. It functions as a major structural component of the cytoskeleton, providing cellular support and playing crucial roles in cell migration and signaling . KRT8 typically forms heterotetramer structures with type I keratins and helps link the contractile apparatus to dystrophin at costameres of striated muscle . Beyond its structural role, KRT8 is increasingly recognized for its involvement in various cellular processes including epithelial-mesenchymal transition (EMT), cell migration, and cancer progression .

How is KRT8 expression measured in research settings?

KRT8 can be measured using multiple complementary techniques:

The choice of method depends on research objectives, with ELISA offering precise quantification for liquid samples, while IHC provides spatial information about expression patterns within tissues .

What clinical significance does KRT8 have in human diseases?

KRT8 has significant clinical relevance, particularly in cancer. In lung adenocarcinoma (LUAD), high KRT8 expression correlates with poor survival of patients . Similarly, in clear cell renal cell carcinoma (ccRCC), KRT8 overexpression is associated with aggressive characteristics and predicts poor prognosis .

Clinical associations of KRT8 in ccRCC include:

• Positive correlation with metastasis (p=0.039)

• Association with higher pT stages (p=0.014)

• Correlation with advanced clinical stages (p<0.001)

• Relationship with worse Fuhrman grades (p=0.026)

These findings suggest KRT8 has potential as a biomarker for prognostication and possibly as a therapeutic target in multiple cancer types .

How does KRT8 contribute to tumor progression and metastasis?

KRT8 appears to promote cancer progression through multiple mechanisms:

• EMT Promotion: High KRT8 expression promotes epithelial-mesenchymal transition in lung cancer cells, enhancing their migratory capacity .

• Signaling Pathway Activation:

  • In lung cancer, KRT8 activates NF-κB signaling

  • In ccRCC, KRT8 increases IL-11 expression, triggering IL-11 autocrine induction and STAT3 signaling activation

• Anti-apoptotic Effects: KRT8 knockdown studies demonstrate increased apoptosis, suggesting KRT8 may protect cancer cells from programmed cell death .

• Enhanced Invasion Capacity: Experimental evidence shows KRT8 knockdown significantly suppresses cell migration and invasion both in vitro and in vivo .

These mechanisms collectively contribute to KRT8's role in promoting tumor aggressiveness and metastatic potential .

What is the prognostic value of KRT8 in different cancer types?

KRT8 demonstrates consistent prognostic value across multiple cancer types:

In lung adenocarcinoma (LUAD):

In clear cell renal cell carcinoma (ccRCC):

• Higher KRT8 mRNA levels correlated with shorter progression-free survival (PFS) (p=0.0274) and OS (p=0.0171)

• In multivariate analysis, KRT8 expression was an independent risk factor for PFS (HR 2.918, 95% CI 1.342–6.345, p=0.007) and OS (HR 3.512, 95% CI 1.391-8.867, p=0.008)

• Notably, KRT8 had significant prognostic value even in early-stage localized ccRCC (pT stages I and II)

This consistency across cancer types suggests KRT8 may serve as a broadly applicable prognostic biomarker in epithelial cancers .

How does KRT8 expression differ between primary tumors and metastatic sites?

Research indicates KRT8 expression increases during cancer progression and metastasis. In ccRCC, KRT8 mRNA and protein levels were significantly higher in vein tumor thrombi (VTTs) than in corresponding primary tumor or peritumoral tissues . This elevated expression in metastatic tissues suggests KRT8 may play an important role in the metastatic process.

Additionally, analysis of ccRCC samples showed that KRT8 expression was significantly upregulated in primary metastatic ccRCC tissues compared to non-metastatic ones, with KRT8 overexpression significantly correlated with positive metastasis status (p=0.039) . These findings establish a clear relationship between increased KRT8 expression and metastatic potential in cancer.

What are best practices for KRT8 knockdown or overexpression experiments?

Based on published research, several approaches have proven effective for manipulating KRT8 expression:

For KRT8 knockdown:

• Cell models: ACHN and Caki-1 renal cancer cell lines have been successfully used

• Methodology: ShRNA-mediated knockdown appears most common and effective

• Validation: Always confirm knockdown at both mRNA (qRT-PCR) and protein (Western blot) levels

• Controls: Include appropriate non-targeting shRNA controls

For KRT8 overexpression:

• Cell models: 786-O cells have been utilized for KRT8 overexpression studies

• Verification: Both mRNA and protein level increases should be confirmed

• Expected outcomes: Increased migration, invasion, and activated downstream signaling (e.g., STAT3 phosphorylation)

When designing these experiments, it's essential to include appropriate functional assays (migration, invasion, proliferation, apoptosis) and signaling pathway analyses (NF-κB, STAT3) to comprehensively assess KRT8's effects .

How can researchers effectively measure KRT8-dependent phenotypic changes?

Several methodological approaches can effectively quantify phenotypic changes resulting from KRT8 modulation:

• Cell Migration and Invasion:

  • Transwell assays with or without Matrigel coating

  • Wound healing/scratch assays

  • Time-lapse imaging of cell movement

• EMT Assessment:

  • Western blot and qRT-PCR for EMT markers (E-cadherin, N-cadherin, vimentin)

  • Morphological changes via phase-contrast microscopy

  • Immunofluorescence staining for cellular localization of EMT markers

• Signaling Pathway Activation:

  • Western blot for phosphorylated signaling components (e.g., phospho-STAT3)

  • Reporter assays for transcriptional activity (e.g., NF-κB reporters)

  • Cytokine measurements (e.g., IL-11 ELISA of cell supernatants)

• In vivo Studies:

  • Xenograft models measuring tumor growth and metastasis formation

  • IHC analysis of excised tumors for KRT8 and relevant pathway markers

Combining these methodologies allows for comprehensive characterization of KRT8's functional impact.

What sample preparation considerations are important for KRT8 analysis?

Sample handling can significantly impact KRT8 detection and analysis:

For tissue samples:

• Fresh frozen samples are optimal for RNA and protein extraction

• Formalin fixation time affects KRT8 antigenicity in IHC; standardize fixation protocols

• Antigen retrieval methods should be optimized for KRT8 detection

For liquid biopsies:

• Serum and plasma samples should be collected using standardized protocols

• Sample storage temperature and freeze-thaw cycles can affect KRT8 stability

• Consider detection range (0.156-10 ng/ml) and sensitivity (0.087 ng/ml) of ELISA kits

For cell culture:

• Cell density and growth conditions can affect KRT8 expression levels

• Collection timing (for supernatants) should be standardized when measuring secreted factors like IL-11

• Lysis buffers should preserve cytoskeletal proteins effectively for Western blot analysis

Maintaining consistent sample handling protocols is essential for reproducible KRT8 research results.

How does KRT8 interact with signaling networks in cancer progression?

KRT8 interfaces with multiple signaling networks, with two pathways particularly well-documented:

• IL-11/STAT3 Pathway in ccRCC:

  • KRT8 knockdown significantly decreases IL-11 mRNA and protein levels

  • KRT8 overexpression increases IL-11 secretion and STAT3 phosphorylation

  • IL-11 mRNA levels are significantly higher in ccRCC tumor tissues

  • This KRT8-IL-11-STAT3 axis appears critical for ccRCC metastasis

• NF-κB Signaling in Lung Cancer:

  • KRT8 knockdown significantly inhibits NF-κB signaling

  • This inhibition may underlie the reduced migration and invasion observed after KRT8 depletion

  • The mechanism suggesting KRT8 may regulate lung carcinogenesis through NF-κB modulation

Understanding these interactions presents opportunities for targeted interventions that could disrupt KRT8-mediated cancer progression through precision targeting of downstream effectors.

What molecular mechanisms explain KRT8's role in epithelial-mesenchymal transition?

While the complete mechanistic understanding remains under investigation, current evidence suggests KRT8 promotes EMT through:

• Signaling pathway modulation: KRT8 activates signaling cascades known to drive EMT, including NF-κB and STAT3 pathways

• Cytoskeletal reorganization: As an intermediate filament protein, KRT8 likely participates in the cytoskeletal remodeling required for the morphological changes during EMT

• Transcriptional regulation: Through its signaling effects, KRT8 may influence the expression of EMT-associated transcription factors

Research demonstrates that KRT8 knockdown suppresses EMT and migration in lung cancer cells , confirming its functional role in this critical cancer progression process.

How might therapeutic targeting of KRT8 be achieved in cancer treatment?

Based on current understanding, several approaches could potentially target KRT8 for cancer therapy:

• RNAi-based approaches: siRNA or shRNA delivery systems targeting KRT8 mRNA could reduce expression, as demonstrated in experimental models showing reduced invasion and metastasis after KRT8 knockdown

• Disruption of KRT8-dependent signaling: Small molecule inhibitors targeting the downstream pathways activated by KRT8 (e.g., STAT3 or NF-κB inhibitors) could mitigate its pro-oncogenic effects

• Blocking KRT8-protein interactions: Development of peptides or small molecules that interfere with KRT8's interactions with signaling components

• Antibody-drug conjugates: Given KRT8's specific upregulation in certain cancers, antibodies against KRT8 conjugated to cytotoxic drugs could provide targeted therapy

What are common challenges in KRT8 detection and quantification?

Researchers frequently encounter several technical challenges when working with KRT8:

• Antibody specificity: Due to structural similarities between keratins, ensuring antibody specificity is crucial. Cross-reactivity with other keratin family members can lead to false positives

• Variable expression levels: KRT8 expression can vary substantially between different cell types and tissues, requiring careful assay optimization and appropriate positive controls

• Post-translational modifications: Phosphorylation and other modifications can affect antibody recognition of KRT8, potentially leading to underestimation of total KRT8 levels

• Sample preparation effects: The intermediate filament nature of KRT8 can make protein extraction challenging, with different lysis buffers yielding variable efficiency

• Quantification standardization: For IHC analyses, standardizing scoring methods is essential for reliable comparison between studies, as seen in the TMA analyses of ccRCC and LUAD samples

Addressing these challenges requires rigorous experimental controls and validation of detection methods.

How can researchers interpret conflicting KRT8 experimental results?

When facing contradictory findings regarding KRT8 function or expression:

A systematic approach comparing methodologies, models, and context can often reconcile apparently contradictory results.

What quality control measures are essential for KRT8 research?

To ensure reliable and reproducible KRT8 research:

• Antibody validation:

  • Confirm specificity using KRT8 knockdown/overexpression controls

  • Test multiple antibody clones where possible

  • Validate across different applications (Western blot, IHC, IF)

• Expression analysis controls:

  • Include appropriate positive and negative tissue/cell controls

  • Use housekeeping genes/proteins optimized for the specific experimental context

  • Consider multiple reference genes for qRT-PCR normalization

• Functional studies:

  • Include multiple KRT8 knockdown/overexpression clones to control for off-target effects

  • Rescue experiments to confirm specificity of observed phenotypes

  • Use multiple cell lines to ensure findings aren't cell-line specific

• Clinical correlations:

  • Validate findings across independent patient cohorts

  • Consider multivariate analyses to account for confounding factors

  • Standardize scoring methods for IHC/TMA