Determine How Collagen Lysyl Hydroxylase 2 Drives Metastasis

TECHNICAL ABSTRACT Background: Lung cancer is the primary cause of cancer death in western countries, including the United States. Kentucky leads the nation both in lung cancer incidence and mortality. Lung cancer-related mortality is mainly due to metastasis. During metastasis, malignant tumors accumulate over modified and aligned collagen fibrils to drive cancer cell dissemination and impede the influx of anti-tumor immune cells. We recently identified a prometastatic collagen glucosyltransferase (GGT) named lysyl hydroxylase 2 isoform b (LH2b). We found that LH2’s GGT but not its LH activity is the major driver of metastasis. LH2b’s GGT and prometastatic activities are determined by an alternatively spliced exon 13a. High mRNA expression levels of LH2b with exon 13a predict a worse prognosis, while the levels of LH2a without exon 13a are not prognostic. However, how LH2b drives metastasis is not known. Objective/hypothesis: This study seeks to define how LH2b drives tumor progression and identify LH2b’s druggable structural features. Our central hypothesis is that cancer- and stroma- expressed LH2b utilizes its unique structural features to modify collagen subtypes and drive tumor progression. Specific aims: 1) Determine LH2b’s unique structural features critical for type V collagen (Col5) glucosylation; 2) Define to what extent cancer-expressed LH2b drives Col5 secretion and LUAD progression; 3) Determine to what extent CAFs-expressed LH2b promotes LUAD progression. Study design: In Aim 1, we will use protein biochemistry and crystallography to determine LH2b’s unique structural features critical for collagen glucosylation and cancer progression. In Aim 2, we will use collagen matrix analysis techniques (e.g., LC-QTOF-MS, HPLC) to determine the collagen defects in LH2b mutant cancer cells. In aim 3, we will use orthotopic mouse models and 3-dimensional cultures to study the roles of stroma LH2b in tumor progression. Cancer relevance: LH2 is a therapeutic target of interest in multiple cancer types. However, how LH2 drives cancer metastasis is unclear and there is no available LH2 inhibitor. These deficiencies can be addressed only by gaining mechanistic insight into LH2’s structure-function. The findings from our proposed work will fill in that knowledge gap to inform how metastasis occurs and identify unique druggable structural features. GENERAL AUDIENCE SUMMARY Lung cancer is the leading cause of cancer-related death worldwide because it is common, spreads to other parts of the body easily and is difficult to treat with existing therapies. Although the direct killing of cancer cells by drugs (such as chemotherapy) remains a major area of cancer research, the benefit from these therapeutic approaches is limited owing to the cancer cells’ ability to rapidly acquire resistance to anti-cancer drugs. Even if a drug can eliminate 99.9% of all cancer cells in a patient, the remaining cancer cells that were resistant to the treatment can multiply and repopulate a tumor within a few months, reversing the outstanding progress made during treatment. Thus, addressing the clinical problems of cancer will require new therapeutic approaches. This reality has caused many of us in the cancer research community to rethink our approach to cancer therapy. As cancer progresses, it causes problems that are lethal or reduce the quality of life, such as stroke, bleeding, infection, pain, and loss of organ function. For cancer cells to grow and spread to new locations, they must learn to hide from the immune system and survive in foreign environments. Our laboratory found that lung cancer progression requires cancer cells to produce a collagen-modifying enzyme called LH2 (lysyl hydroxylase 2). LH2 builds a dense collagen network that surrounds the cancer cells, allowing the cancer cells to hide from the immune system and anti-cancer drugs, survive and thrive in the primary site, and spread to new sites. If strategies were developed to block LH2, then cancers might be shrunk and eradicated by the immune system. However, we do not know what LH2 looks like structurally, and LH2 antagonist is not available. In this application, we have established new methods to generate LH2 protein and measure LH2’s cancer-promoting activities, which will be used to screen small molecule libraries to identify LH2 antagonists. We will learn about how LH2 promotes tumor progression and determine the unique druggable structural features that distinct LH2 from other human proteins. These findings will be used to optimize the LH2-specific antagonists that are trying to identify. Since LH2-driven collagen network formation forms a physical barrier for anti-cancer therapeutics, LH2 antagonists may remove the barrier and help boost the effects of commonly used cancer treatments and anti-tumor immunity. LH2 antagonists could be a ground-breaking treatment for multiple cancer types because LH2 is shown to promote the progression of lung cancer, breast cancer, and sarcoma.