Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Ken D. Brandon II - University of Alabama, Huntsville
Co-Author(s): W. Andrew Shockey and Manu O. Platt, Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, Atlanta, GA
Breast cancer represents the highest incidence and second leading cause of cancer death in American women (ACS, 2015). Cathepsins (Ctts) are a type of protease upregulated in cancer that degrade extracellular matrix proteins (ECM), particularly collagen and elastin. In tumors, the majority of ctts expression and activity were by macrophages (Joyce et al. 2004). Degradation of ECM proteins by ctts facilitates the passage of cancerous cells into the circulatory and lymphatic systems, leading to tumor metastasis. Successful inhibition of ctts secretion could significantly reduce the metastatic ability of breast cancer, greatly diminishing its life threatening potential, but for proper therapeutic dosing, rates of ctts synthesis, secretion, and activation must be known. However, these rates are not yet defined. This works seeks to assist this progress by empirically quantifying ctts production, secretion, and activation and investigating differences between ctts L and S, two key members implicated in TAMs and metastasis. Thp-1 monocytes were differentiated into macrophages by treatment overnight with phorbol-12-myristate-13-acetate. After differentiation, protein synthesis was halted by incubation with cycloheximide (CHX), providing a baseline for rates of ctts production. The CHX was washed out by replacing with fresh media, and conditioned media and cell lysates were collected at informed time points of 0, 1, 2, 4, 8, and 24 hours. Ctts L and S were quantified by Western blots and densitometry. Ctts L was completely depleted from the macrophages after halting protein synthesis for 24 hours, but not ctts S; suggesting ctts S is more stable intracellularly. In addition, ctts L turnover plateau indicates steady state in the conditioned media compared to an increasing amount of ctts S, which also suggests that ctts S is more stable extracellularly. Quantitative analysis of protein levels over time revealed that Thp-1 macrophages secrete pro-ctts L at a rate 8X higher than pro-ctts S. Results suggest that pro-ctts L is secreted at a greater rate than pro-ctts S, but that ctts S is a more stable intra- and extracellularly than ctts L. Since ctts L is optimally active at acidic pH while ctts S maintains activity under neutral conditions, the disparity in secretion rates by macrophages could imply that the cell is balancing the extracellular concentrations of the enzymes to maintain homeostasis under neutral conditions. However, the acidic tumor milieu could lead to drastically increased proteolytic activity. Future studies involve creating a computation model of ctts trafficking based on rates of synthesis, secretion, and activation to estimate how cancerous conditions and inhibitors alter these rates.
References: American Cancer Society. Breast Cancer Facts & Figures 2015-2016. Atlanta: ACS, Inc. 2015. Joyce, J. A. et al. (2004). Cancer Cell, 5(5), 443-453.
Funder Acknowledgement(s): I would like to thank Manu Platt and Andrew Shockey for their guidance during my 10-week summer program. Funding was provided by an NSF REU grant to Georgia Tech Summer Undergraduate Research in Engineering/Sciences Program. A generous contribution from Intel Corporation assisted in making this work possible.
Faculty Advisor: Manu Platt, firstname.lastname@example.org
Role: I conducted all of the described research presented in my abstract, which includes the informed time courses, SDS-PAGE, immunoblot, and quantified analysis using densitometry.