Cathepsin K in lymphangioleiomyomatosis: LAM cell-fibroblast Interactions enhance protease activity by extracellular acidification

Lymphangioleiomyomatosis (LAM) is a rare disease in which clonal ‘LAM’ cells infiltrate the lungs and lymphatics. In association with recruited fibroblasts, LAM cells form nodules adjacent to lung cysts. It is assumed LAM nodule derived proteases lead to cyst formation although, this is uncertain. We profiled protease gene expression in whole lung tissue and observed cathepsin K was 40 fold over-expressed in LAM compared with control lungs (p≤0.0001). Immunohistochemistry confirmed cathepsin K protein in LAM nodules but not control lungs. Cathepsin K gene expression, protein and protease activity was detected in LAM associated fibroblasts but not the LAM cell line 621-101. In lung nodules, cathepsin K immune reactivity was predominantly co-localised with LAM associated fibroblasts. In vitro, extra-cellular cathepsin K activity was minimal at pH 7.5 but significantly enhanced in fibroblast cultures at pH 7 and 6. 621-101 cells reduced extracellular pH by 0.5 units over 24 hours. Acidification was dependent upon 621-101 cell mTOR activity and net hydrogen ion transporters, particularly sodium/bicarbonate co-transporters and carbonic anhydrases which were also expressed in LAM lung tissue. In LAM cell/fibroblast co-cultures, acidification paralleled cathepsin K activity and both were inhibited by sodium bicarbonate co-transporter (p≤0.0001) and carbonic anhydrase inhibitors (p=0.0021). Our findings suggest cathepsin K activity is dependent on LAM cell/fibroblast interactions and inhibitors of extracellular acidification may be potential therapies for LAM

Interrogating Tumor Metabolism with AcidoCEST MRI

Tumor metabolism is a highly dysregulated process that is identified as a unique target for therapy. Current philosophy proposes that tumor metabolism is a plastic and flexible process which sustains proliferative and survival advantages. Tumors employ an anaerobic glycolytic pathway resulting in the overproduction of lactate. Additional thinking suggests that the conversion of pyruvate to lactate regenerates the NAD+ pool in the cell, maintaining a sustainable oxidative environment. Regardless of the reasons for lactate overproduction, its excretion and build up in the microenvironment results in acidic tumor microenvironments. Tumor acidosis has been measured with several different methods, but consistently averages from pH 6.6 to 7.0. Tumor acidity can thus be measured as a biomarker for tumor metabolism. This work examines the commonly explored energy pathways available to the cancer cell and a non-invasive MRI method to measure the efficacy of the tumor metabolism targeting agent. Appendix A is an introduction to tumor metabolism pathways and the large list of candidate therapies in interfering with energy production. Glucose, fatty acid, and glutamine metabolisms are all discussed along with PI3K/AKT/mTOR and HIF growth signals and ion transport. Magnetic resonance imaging and positron emission tomography are examined as imaging methods for non-invasively interrogating tumor acidosis. Appendix B presents the findings in a study where tumor metabolism was targeted with an mTOR inhibitor, where tumor growth rate was initially decreased and accompanied by an early, acute increase in tumor extracellular pH with acidoCEST MRI. Chapter 2 discusses the combination of a lactate dehydrogenase inhibitor in conjunction with doxorubicin in a breast cancer model. Tumor extracellular pH was shown to increase when measured with acidoCEST MRI, and an increase in cell death was measured. Chapter 4 discusses the studies and experimental designs that can be done in the near future

Interrogating tumor energy metabolism with acidocest MRI

Tumor metabolism is a highly dysregulated process that is identified as a unique target for therapy. Current philosophy proposes that tumor metabolism is a plastic and flexible process which sustains proliferative and survival advantages. Tumors employ an anaerobic glycolytic pathway resulting in the overproduction of lactate. Additional thinking suggests that the conversion of pyruvate to lactate regenerates the NAD+ pool in the cell, maintaining a sustainable oxidative environment. Regardless of the reasons for lactate overproduction, its excretion and build up in the microenvironment results in acidic tumor microenvironments. Tumor acidosis has been measured with several different methods, but consistently averages from pH 6.6 to 7.0. Tumor acidity can thus be measured as a biomarker for tumor metabolism. This work examines the commonly explored energy pathways available to the cancer cell and a non-invasive MRI method to measure the efficacy of the tumor metabolism targeting agent. Appendix A is an introduction to tumor metabolism pathways and the large list of candidate therapies in interfering with energy production. Glucose, fatty acid, and glutamine metabolisms are all discussed along with PI3K/AKT/mTOR and HIF growth signals and ion transport. Magnetic resonance imaging and positron emission tomography are examined as imaging methods for non-invasively interrogating tumor acidosis. Appendix B presents the findings in a study where tumor metabolism was targeted with an mTOR inhibitor, where tumor growth rate was initially decreased and accompanied by an early, acute increase in tumor extracellular pH with acidoCEST MRI. Chapter 2 discusses the combination of a lactate dehydrogenase inhibitor in conjunction with doxorubicin in a breast cancer model. Tumor extracellular pH was shown to increase when measured with acidoCEST MRI, and an increase in cell death was measured. Chapter 4 discusses the studies and experimental designs that can be done in the near future

Assessing Metabolic Changes in Response to mTOR Inhibition in a Mantle Cell Lymphoma Xenograft Model Using AcidoCEST MRI

AcidoCEST magnetic resonance imaging (MRI) has previously been shown to measure tumor extracellular pH (pHe) with excellent accuracy and precision. This study investigated the ability of acidoCEST MRI to monitor changes in tumor pHe in response to therapy. To perform this study, we used the Granta 519 human mantle cell lymphoma cell line, which is an aggressive B-cell malignancy that demonstrates activation of the phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin ( mTOR) pathway. We performed in vitro and in vivo studies using the Granta 519 cell line to investigate the efficacy and associated changes induced by the mTOR inhibitor, everolimus (RAD001). AcidoCEST MRI studies showed a statistically significant increase in tumor pHe of 0.10 pH unit within 1 day of initiating treatment, which foreshadowed a decrease in tumor growth of the Granta 519 xenograft model. AcidoCEST MRI then measured a decrease in tumor pHe 7 days after initiating treatment, which foreshadowed a return to normal tumor growth rate. Therefore, this study is a strong example that acidoCEST MRI can be used to measure tumor pHe that may serve as a marker for therapeutic efficacy of anticancer therapies.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]