Abstract
Epithelial ovarian cancer (EOC) is the deadliest of the gynecological malignancies, due in part to its clinically occult metastasis. Therefore, understanding the mechanisms governing EOC dissemination and invasion may provide new targets for antimetastatic therapies or new methods for detection of metastatic disease. The cAMP-dependent protein kinase (PKA) is often dysregulated in EOC. Furthermore, PKA activity and subcellular localization by A-kinase anchoring proteins (AKAPs) are important regulators of cytoskeletal dynamics and cell migration. Thus, we sought to study the role of PKA and AKAP function in both EOC cell migration and invasion. Using the plasma membrane-directed PKA biosensor, pmAKAR3, and an improved migration/invasion assay, we show that PKA is activated at the leading edge of migrating SKOV-3 EOC cells, and that inhibition of PKA activity blocks SKOV-3 cell migration. Furthermore, we show that while the PKA activity within the leading edge of these cells is mediated by anchoring of type-II regulatory PKA subunits (RII), inhibition of anchoring of either RI or RII PKA subunits blocks cell migration. Importantly, we also show – for the first time – that PKA activity is up-regulated at the leading edge of SKOV-3 cells during invasion of a three-dimensional extracellular matrix and, as seen for migration, inhibition of either PKA activity or AKAP-mediated PKA anchoring blocks matrix invasion. These data are the first to demonstrate that the invasion of extracellular matrix by cancer cells elicits activation of PKA within the invasive leading edge and that both PKA activity and anchoring are required for matrix invasion. These observations suggest a role for PKA and AKAP activity in EOC metastasis.
Highlights
Epithelial ovarian cancer (EOC) is the fifth most common cause of cancer deaths in women in the United States and has the highest mortality rate of all gynecologic cancers [1]
Given the importance of protein kinase (PKA) and A-kinase anchoring proteins (AKAPs) function for cell migration and their dysregulation in EOC, we investigated the spatial regulation of PKA activity in migrating EOC cells and determined whether PKA activity and anchoring are required for EOC cell migration
To determine if PKA was activated at the leading edge of migrating EOC cells, SKOV-3 human EOC cells were transfected with pmAKAR3, a plasma membrane-directed FRET biosensor for PKA activity [21,36], plated on FN-coated glass coverslips, stimulated with 10 ng/ml EGF for 4-6 hrs, and monitored by live-cell imaging
Summary
Epithelial ovarian cancer (EOC) is the fifth most common cause of cancer deaths in women in the United States and has the highest mortality rate of all gynecologic cancers [1]. The anatomic placement of the ovaries facilitates local spread of EOC, which can occur by direct migration and invasion of tumor cells to and into adjacent organs, as well as through transport of exfoliated tumor cells throughout the peritoneal cavity by normal peritoneal fluid flow [2,3]. Because of this unique and furtive mode of dissemination, efforts in developing methods for early detection of EOC have been largely unsuccessful [4,5,6]. Combination chemotherapies and strategies for determining serum biomarkers, local and disseminated chemoresistant cells persist and eventually flourish, leading to the high recurrence and
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