Over-expression of various PKA subunits is a hallmark of a vast array of human tumors 
), including ovarian cancer 
, and often correlates with more severe pathology and poorer prognosis. In this regard, PKA is classically thought to enhance mitogenic signal transduction and promote early cell cycle transition. However, a number of reports have also established a role for PKA in promoting invasion and metastasis in a number of tumor models 
; however, this line of investigation has not been extended to EOC until now. Our results clearly demonstrate that spatial regulation of PKA activity is a critically important facet of the migration and invasion – and thus, perhaps the metastasis – of EOC cells.
Previous work has demonstrated enrichment of PKA in the leading edge of migrating cells, with the most detailed descriptions being in fibroblasts 
and breast and colon carcinoma cells 
. Our data now extends this paradigm to EOC cells and expands the paradigm in an important and substantive way by demonstrating localization of PKA to the leading edge during three-dimensional matrix invasion. Furthermore, we have also demonstrated the requirement for AKAP function for the migration of EOC cells, as has been demonstrated for other cell types 
. Finally, we have also demonstrated – for the first time - the requirement for PKA anchoring via
AKAPs for cancer cell invasion of a three-dimensional ECM. A number of previous reports have implicated a variety of AKAPs as positive or negative regulators of invasion and metastasis: these include AKAP9/yotiao 
; AKAP12/SSeCKS/gravin 
; AKAP13/AKAP-Lbc 
; the erzin/radixin/moesin and merlin protein family 
; and the WAVE (Wiskott-Aldrich syndrome/verpolin-homologous) -1 and 2 proteins 
. Without exception, however, those reports dealt with gain- or loss-of-function of their respective AKAPs as a whole and did not specifically target only the PKA anchoring domain. This is an important corollary, as the overwhelming majority of known AKAPs – and essentially all of those previously implicated in metastasis – have important functions beyond their ability to anchor PKA 
. Thus, complete gain- or loss-of-function studies, i.e.
over-expression or silencing of an entire AKAP, would affect all of its potential functions. In many cases, the functions other than PKA anchoring are quite powerful – e.g.
guanine nucleotide exchange for Rho family GTPases (AKAP-Lbc/AKAP13); scaffolding other kinases and enzymes (AKAP9, AKAP12); coupling membrane receptors to the cortical actin cytoskeleton (ezrin family proteins); dendritic actin nucleation (WAVE1 and 2) – and could readily contribute to invasion and metastasis in their own right. In the current study, however, we specifically inhibited only the ability of AKAPs to anchor PKA; thus, to our knowledge, ours is the first report to establish a role specifically for PKA anchoring per se
in events related to metastasis.
An important question arises from the current observations: if leading edge PKA activity is mediated by exclusively by RII-AKAP interactions, as reported here, then why are both type-I and type-II PKA anchoring required for migration and invasion? This question is, at the same time, both reassuring and vexing, as both type-I and II PKA have been previously implicated in cell migration 
, but it is becoming increasingly clear that the two types of PKA have non-overlapping targets and functions 
. It is also important to note that the current study uses pmAKAR3, a membrane-tethered biosensor for measuring PKA activity, which – by design – does not faithfully report any cytosolic PKA activity that may exist some distance from the membrane. Thus, it is possible that there may be some leading edge PKA activity, within the thin layer of cytoplasm that is distant from the membrane, that is attributable to RI-AKAP interactions. Perhaps more likely is that, during migration, there is significant PKA activity within the cytosol that is both not detected by the membrane-tethered pmAKAR3 and not as ‘concentrated’ (in terms of specific PKA activity per total cellular protein) as the activity within the leading edge 
]. Thus, while RII-AKAP complexes preferentially regulate events within the leading edge, RI-AKAP complexes may preside over myriad events within the rest of the cell – outside the leading edge – that also ultimately contribute to migration/invasion. Delineating the precise deficits in migration and invasion associated with type-I and type-II PKA would best require a complete catalog of the specific molecular targets for each type of PKA activity and an understanding of what role each target plays in migration/invasion; at present, this is quite unfeasible. More reasonably, however, one could begin to empirically address the matter through more frequent imaging and closer correlation of cell morphology and PKA activity over the entire duration of migration and invasion, and subsequent observation of discrete changes in these parameters in the absence of type-I or type-II anchoring.
While we have clearly demonstrated that PKA and AKAP function are both required for EOC cell migration and invasion, the discrete molecular targets for PKA as well as the specific AKAP(s) responsible for PKA localization in these cells are unknown. Delineation of specific signaling pathways that link PKA to migration and invasion is made difficult by the considerable and growing number of cytoskeleton- and/or migration-associated PKA substrates 
and anchoring proteins 
. It is further complicated by the fact that, as discussed above, inhibition of either type-I or type-II PKA anchoring blocks migration and invasion, likely through different effectors. Furthermore, while not the focus of the current study, it should be noted that, as a kinase regulated by intracellular cAMP, PKA is quite likely to work in concert with the other major cAMP effectors, the Epacs (e
ctivated by c
AMP), which can form multi-component scaffolds with PKA to mediate signaling events in response to cAMP 
and also have considerable influence over cell adhesion, migration and related events 
. This possibility notwithstanding, the current study unequivocally establishes a role for PKA itself, in a paradigm involving its localization through AKAPs, in the regulation of ovarian cancer migration and matrix invasion.
While our data demonstrate the necessity for PKA activity and anchoring during migration as well as invasion, it is not known whether the targets for PKA during migration will be the same as those in invasion. Although the processes are intimately related, migration can occur without invasion, but invasion cannot occur without migration, and the exact mechanism of migration during invasion may differ as a function of cellular phenotype and microenvironmental cues 
. Given our growing appreciation of the differences between cell migration in two-dimensions vs three-dimensions 
and the differences between normal and tumor cell migration 
, it would not be surprising if distinct (but, likely, somewhat overlapping) sets of PKA substrates and anchoring proteins were called upon to specifically regulate the processes involved in EOC cell invasion compared to migration. An intriguing example would be a requirement for localized PKA in regulating the expression, secretion or function of ‘invasion-specific’ proteins such as matrix metalloproteases 
. Nonetheless, the identification of the substrates and anchoring proteins for PKA that are relevant to migration and invasion are an ongoing focus for several laboratories, including our own. Indeed, propitious initial candidates for relevant AKAPs would be those that appear to be up-regulated in various forms of EOC AKAP3/AKAP110 
; AKAP1/AKAP149 and AKAP13/AKAP-Lbc (A.K. Howe, unpublished observations)).
Although it only accounts for approximately 3% of all cancer diagnoses in the United States, ovarian cancer is the 5th
leading cause of death among women 
. This is largely due to the fact that most ovarian cancer patients become symptomatic - and are therefore diagnosed - after the tumor has metastasized. The metastatic spread of EOC, like other cancers, is completely dependent on the acquired ability of tumor cells to undergo invasive cell migration 
. In this work, we have shown that both migrating and invading EOC cells exhibit enriched PKA within their leading edge and that both PKA activity and its localization through AKAPs are required for migration and invasion of EOC cells. This is the first demonstration of an association of localized PKA activity and the process of matrix invasion. More importantly, these observations add important new molecular insight into some of the most clinically relevant cellular behaviors associated with the pathogenesis of EOC. To be certain, the in vitro
mode of matrix invasion assessed in the current study does not recapitulate the full course and associated mechanisms of EOC metastasis in vivo
. Especially relevant in this regard are the complex biology of both the interaction of metastatic EOC cells with their target microenvironment, which consists of mesothelial cells and fibroblasts in addition to ECM components 
, and of multicellular tumor spheroids, regarded as the minimal metastatic unit of EOC 
. Nonetheless, the current studies provide a solid foundation that justifies the further exploration of the role of PKA and AKAP function in higher-order models of EOC metastasis.