We describe here the identification of small molecules that regulate invasive behavior. The initial goal of this research was to generate a system to identify inhibitors of invasion. However, from the primary screen we noticed that cantharidin and paclitaxel increased invasive behavior. Cantharidin is a general protein phosphatase-1 (PP1) and protein phosphatase-2 (PP2A) inhibitor (19
), and likely promotes invadopodia formation by inhibiting the dephosphorylation of key invadopodia components such as ERK-1 and -2 (21
). Analogues of cantharidin have been reported to increase xanthine oxidase activity which can increase intracellular reactive oxygen (ROS) (22
). We have recently demonstrated that ROS are necessary for invadopodia formation (23
), suggesting that cantharidin-mediated induction of intracellular ROS might also promotes invadopodia formation.
The cytoskeleton is the master regulator of several cellular functions, including migration and mitosis. Some of the most clinically successful cancer chemotherapies directly target one component of the cytoskeleton, microtubules (24
). A good example is paclitaxel, which is used to treat patients with many forms of cancer (25
). The anti-tumor effect of paclitaxel is based on its ability to bind and stabilize microtubules and consequently inhibit mitosis and induce apoptosis (26
). However, formation of podosomes is dependent on intact microtubules (27
), and it has recently been shown that mature invadopodia contain microtubules (28
). We show here that paclitaxel treatment promotes the invasive behavior of a number of cancer cells through the stimulation of invadopodia formation. Of note, the concentration of paclitaxel required to promote invadopodia formation, 2µM, is an achievable clinical dose. These results raise the concern that continued treatment with paclitaxel in those cancer patients with refractory tumors, or neo-adjuvant treatment with the drug, may stimulate tumor cell extravasation and dissemination. In this regard, paclitaxel has been shown to increase metastasis formation in animals bearing paclitaxel-resistant leukemic cells (29
). It has also been shown that paclitaxel increases the number of circulating tumor cells more than 1000-fold in breast cancer patients treated with paclitaxel in the neoadjuvant setting (30
), which may be a result of extravasation of tumor cells from the primary tumor. Furthermore, in epithelial ovarian cancer, most patients initially respond to surgery and chemotherapy, but then relapse and become refractory to chemotherapy with more aggressive tumors associated with metastasis into the abdomen (31
). Our observation that paclitaxel did not promote invasive behavior in cells treated with purvalanol suggests that combining paclitaxel with an invadopodia inhibitor might be explored as a means to limit tumor dissemination.
Several of the inhibitors detected in our screen were cyclin-dependent kinase inhibitors. This enzyme family has not been previously associated with control of either invadopodia or podosomes. Several members of the Cdk family act as checkpoint kinases to regulate cell cycle progression (32
). But our EC50
analysis dissociated invadopodia inhibition from cell cycle inhibition. This led us to test Cdk5, which is also potently inhibited by purvalanol A and other Cdk inhibitors in the collection (14
). Cdk5 is highly expressed in neurons and is involved in post-mitotic processes such as neuronal migration and neurite outgrowth (13
). Cdk5 is expressed in many tissues besides neurons (33
). Our finding that Cdk5 regulates invasion is consistent with a previous report that expression of dominant negative Cdk5 in prostate carcinoma cells reduces their ability to metastasize (35
). And Cdk5 expression in glioblastoma cells is higher than in normal astrocytes and appears to play a role in glioblastoma cell migration and invasion (36
). We used an siRNA-mediated knockdown approach to show for the first time that Cdk5 is required for the formation and function of invadopodia, and for invasion, in mouse fibrosarcoma cells, and in several human cancer cells. Very little is known about the regulation of Cdk5 expression in non-neuronal tissues. In the future it will be interesting to investigate the mechanism by which it is frequently over-expressed in human cancers. Interestingly, it was recently shown that the Cdk5-mediated phosphorylation of talin prevents its ubiquitination and degradation (37
), resulting in limited focal adhesion turnover and stabilization of lamellipodia at the leading edge, thus promoting cell migration. Taken together, these studies suggest that Cdk5 promotes cell migration and invasion by optimizing the formation of both focal adhesions and invadopodia.
Cdk5 has been reported to phosphorylate a large number of proteins in vitro and in vivo, many involved in cell morphology and motility, including ezrin (38
) and dynamin (39
), both invadopodia proteins (40
). Here we provide new mechanistic insights into the role of Cdk5 in invadopodia formation, and propose that Cdk5 promotes invasive behavior by phosphorylating and down regulating the invadopodia suppressor caldesmon. In support of this hypothesis, we show that in the absence of caldesmon, inhibition of Cdk5 has little effect on invadopodia formation. We describe for the first time that Cdk5 controls caldesmon protein abundance, predominantly through phosphorylation of serine 527. Caldesmon is an actin-binding protein present in both smooth muscle, where the long form (h-caldesmon) predominates, and non-muscle cells, where only a short form (l-caldesmon) is detectable (43
). In vitro evidence implicates caldesmon in the regulation of smooth muscle and non-smooth muscle contraction and cell motility (45
). Furthermore, caldesmon localizes to the invadopodia in Src-transformed cells (46
), and reduction in the level of caldesmon facilitates podosome (17
) and invadopodia formation and increases invasive behavior (18
). Phosphorylation of caldesmon at S527 has previously been reported to correlate with ERK1/2, Cdc2 and p38 MAPK activity (47
), although these studies used high concentrations of inhibitors, and did not address the consequence of caldesmon phosphorylation. In this study we have provided strong evidence that the primary S527 kinase in cancer cells is Cdk5, and that phosphorylation on this site destabilizes the protein, thus promoting invadopodia formation. This conclusion is supported by the observation that MG132-mediated inhibition of FITC-gelatin degradation only occurred in cells expressing caldesmon. These experiments also provide the first link between protein ubiquitination and ECM degradation.
Our studies demonstrate the feasibility of using high content invadopodia screens to identify invasion regulators, and highlight the importance of both the microtubule and actin-based cytoskeletons in controlling invasive behavior. Invadopodia-related structures, called podosomes, have been observed in monocyte-derived cells, such as macrophages and osteoclasts, in endothelial cells, and in vascular smooth muscle cells (2
). Dysregulation of podosome formation has recently been associated with development of atherosclerosis (9
). These results suggest that a strategy to identify regulators of invadopodia and podosomes might lead to the development of new therapeutic drugs to control metastatic cancer growth, as well as other diseases.