Role of CD82 tetraspanins in regulation of extracellular signals within the tumor microenvironment during transendothelial migration
Open Access
- Author:
- Khanna, Payal
- Graduate Program:
- Bioengineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 10, 2012
- Committee Members:
- Cheng Dong, Dissertation Advisor/Co-Advisor
Peter J Butler, Committee Member
Margaret June Slattery, Committee Member
Costas D Maranas, Special Member
Esther Winter Gomez, Special Member - Keywords:
- tetraspanins
cytokines
melanoma
cadherins
cancer
endothelial - Abstract:
- Melanoma cancer arises in approximately 70,000 new patients each year and nearly 1 in 63 of patients will have in an invasive form of the cancer, which spreads to the heart and lungs (King 2006; Pennacchioli, Tosti et al. 2012). Once melanoma has spread to distant organs, the disease is increasingly difficult to treat and few therapeutic approaches exist to inhibit melanoma metastasis. An important event during cancer metastasis is tumor cell extravasation through blood vessels into neighboring tissues in which the cancer cell adheres to endothelial substrates, degrades and migrates through the underlying extracellular matrix, and then forms a new colony in the invaded tissue. Understanding the underlying mechanisms that facilitate these events will lead to the development of effective cancer therapies. The goal of this work is to investigate the underlying mechanisms by which cancer cells activate endothelial signaling networks to form gaps in endothelial substrates and facilitate the migration of cancer cells into the blood stream. Endothelial gap formation is triggered by adhesion molecules (such as α4β1 integrins) and inflammatory cytokines (such as interleukin (IL)-8), IL-6, and IL-1β) from the tumor microenvironment. The binding of these molecules to endothelial cells allows Rac GTPase to exchange a GTP molecule for GDP and subsequent activation of PAK and p38 mitogen activated protein kinases (MAPKs), which regulate events leading to gaps in endothelial cells. Gaps in the endothelium form through two primary mechanisms: (i) the dissociation of vascular endothelial (VE)-cadherin homodimers and (ii) contractility of endothelial cells involving the phosphorylation of myosin light chain and actin polymerization. In normal physiology, duffy antigen receptors/groups (DARC) on endothelial cells DARC binds to chemokines, in particular, IL-8. DARC receptors on endothelial cells undergo transcytosis of IL-8 to the lumen surface where they are presented on the cell surface to direct recruitment and chemotaxis leukocytes through endothelial monolayers ((Middleton, Patterson et al. 2002)). During tumor induced inflammation, excess secretion of IL-8 within the tumor microenvironment disrupts homeostasis and IL-8 on endothelial surfaces recruits PMN to sites of melanoma cell extravasation. In melanoma cells, IL-8 induced effects promote adhesion of melanoma cells to endothelial cells, facilitating formation of gaps and transendothelial migration of melanoma cells. Similarly, overexpression of VLA-4 adhesion molecules on melanoma cell surfaces result in abnormal activation of signaling pathways in endothelial cells and an increase in invasiveness of melanoma cells. The mutated form of B-Raf is found in nearly 50% of patients with malignant melanomas and is a known target for treating melanoma cancer (Flaherty, Robert et al. 2012). Results have shown that targeting mutant V600EB-Raf reduces melanoma metastasis through reductions in active melanoma cell extravasation through endothelial cells. Mechanistically, reduced melanoma extravasation following inhibition of mutant V600EB-Raf is due to the disruption of downstream NF-κB signaling, which inhibits IL-8 production. While previous data show knocking down V600EB-Raf decreases melanoma cell extravasation, our current data shows that inhibiting V600E or wild-type B-Raf genes in early stage melamoma cancers increases melanoma cell extravasation. B-Raf is upstream of many proteins in the cell membrane, specifically tetraspanins known to have varying functions. Tetraspanins (including CD82) play an important function in regulating adhesion molecules (VLA-4) and secretion of cytokines (IL-8) from melanoma cells. CD82 tetraspanin is one tumor suppressor gene which is expressed in the early stages of melanoma cancer, but disappears as melanoma cancer increases invasive capabilities of melanoma cancer. In this work we show that re-introducing CD82 expression in late stage melanoma cancer suppresses melanoma cell invasion through human blood vessels. We show that CD82 binding to DARC receptors interrupts IL-8 binding to endothelial substrates. While knocking down B-Raf in late stage melanoma cells suppresses metastasis, CD82 expression is still present in early stage melanoma cancers. As a result, inhibiting B-Raf (an upstream mediator of CD82) could result in a decrease in CD82 expression and an increase in melanoma cell invasion. Furthermore, CD82 expression plays a role in suppressing IL-8 secretion and maturation of the β1 subunit of VLA-4 integrins and their binding affinities. Previous work has shown that these two factors within the tumor microenvironment play a key role in cancer invasiveness. Consistent with previous work, we find that IL-8 secretion and VLA-4 binding affinities trigger signaling proteins (such as p38 and PAK) that play a key role in regulating VE-cadherin disassembly and contractility of endothelial cells.