Grants and Contracts Details
During tumorigenesis, an accumulation of cross-linked collagen generates a stiff tumor stroma to drive metastasis and impede the influx of anti-tumor immune cells. High expression of a collagen modifying enzyme lysyl hydroxylase 2 (LH2) promotes stable collagen cross-link formation in tumor stroma, increases tumor stiffness and drives sarcoma, lung and breast cancer metastasis. LH2 is a therapeutic target of interest, but selective LH2 inhibitors are not available, a deficiency due in part to a lack of structural insight into LH2. The objective of my proposal is to elucidate LH2 structural features that promote lung cancer metastasis. We recently described new methods to produce human LH2 protein and assay its activity, and we determined the structure of the catalytic domain of a viral LH homologue, which established a platform to probe LH2’s structure and function relationship. The viral LH homologue revealed a conserved homodimer stabilized by Fe2+-binding. We found that the two active sites flank a deep surface cleft on the dimer interface, suggesting that dimerization creates a collagen binding site, and that basic residues adjacent to the active site are positioned to form salt bridges with telopeptidyl acidic residues on collagen. Site-directed mutagenesis and enzymatic activity assays showed LH2 dimer is critical for its enzymatic activity. Disrupting LH2 dimer in cancer cells using CRISPR/Cas-9-mediated gene editing decreases tumor growth and metastasis in an orthotopic lung cancer model. We also confirmed the importance of these basic residues near active site in LH2’s telopeptidyl-LH activity. From these findings, I hypothesize that LH2’s telopeptidyl-LH activity drives tumor metastasis and its telopeptidyl-LH activity is determined by LH2’s unique structural features that allow it to form Fe2+-stabilized dimeric structures that interact with telopeptidyl lysines on collagen. I will test this hypothesis by completing 2 specific aims. Aim 1: To determine how Fe2+-binding stabilizes LH2 dimer assemblies. Aim 2: To determine whether LH2’s telopeptidyl-LH activity promotes lung tumorigenesis. In sum, my proposal will address the clinical problem of lung cancer metastasis. The novelty rests in preliminary results that provide the first structural insights into a collagen LH, hypotheses that challenge current paradigms, and technologies that facilitate quantification of LH enzymatic activity and evaluation of candidate metastasis drivers.
|Effective start/end date||7/1/20 → 6/30/21|
- KY Cabinet for Health and Family Services: $30,666.00
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