Loss of APC is an early event in almost all sporadic colon cancers, and in most of these cases, the remaining, mutated APC protein is unable to interact with the microtubule cytoskeleton normally. Here, we show that one consequence of such APC loss is a decrease in the overall stability of microtubules and cell migration. Migration of cells is crucial for the normal maintenance of gut epithelium because constant proliferation, coupled with active, directed migration is required to replenish cells that are constantly exfoliated (Näthke, 2004
). A decrease in efficient cell migration induced by loss of APC may lead to the accumulation of cells in the toxic environment of the gut, allowing additional mutations to accumulate. This might provide an effective mechanism for the transcriptional changes that accompany APC loss to persist and propagate to produce malignancies.
A role for APC in cell migration has previously been suggested by the observation that inactivating APC in mouse tissue leads to an abrupt decrease in cell migration of enterocytes in situ (Sansom et al., 2004
). However, in this situation the inactivation of APC is also accompanied by changes in differentiation, making it difficult to dissect the main cause for the decrease in migration. Using our cell systems, we were able to show directly for the first time that loss of APC immediately results in defects in cell migration. This was detected in cells that constitutively lack APC but also in those where APC loss was induced conditionally using a Cre–Flox system or by RNAi. Migration of whole populations of cells in a monolayer () but also movements of single cells () was reduced by APC depletion.
The relative difference between control and APC-deficient cells was highest for cells constitutively lacking APC (B) and lowest for epitheliod cells immediately after APC loss (B). This suggests that long-term changes resulting from APC loss, like alterations in the transcriptional profile, may further contribute to a decrease in migration.
We detected two differences in APC-deficient cells that related to changes in microtubules. First, we found that the ability of cells to induce protrusions was strongly reduced in all three APC-deficient cell systems (A). This change correlated with the loss of APC-clusters near the periphery of extending cell margins (, B and C). Because cellular protrusions are also dependent on F-actin dynamics and their coordination with microtubules, this observation raises the possibility that loss of APC not only affected microtubules but also produced changes in F-actin. Indeed, N-terminal regions of APC can interact with regulatory proteins for F-actin, including Asef, a Rac-specific guanine nucleotide exchange factor that is stimulated by APC and thus stimulates F-actin (Kawasaki et al., 2000
; Kawasaki et al., 2003
), and IQGAP to form a ternary complex with Rac1/cdc42 (Watanabe et al., 2004
). Furthermore, the link that has been established among mDIA, cdc42, and APC may also contribute to changes in F-actin (Wen et al., 2004
; Yamana et al., 2006
Importantly, we found that the isolated N-terminal domain of APC that contains the full complement of binding sites for IQGAP, Asef, and Kap3 is unable to cluster in cellular protrusions (Zumbrunn et al., 2001
) and does not induce protrusions when overexpressed (B), suggesting that these protein interactions on their own are not responsible for this effect of APC.
A second change that was common to all APC-deficient cells was a change in the pattern of posttranslationally modified microtubules. Acetylation on lysine residue 78 of α-tubulin is a common modification that occurs in microtubules as they persist, so it is often used as an indicator of microtubule lifetime (age) (Westermann and Weber, 2003
). Cells lacking APC contained fewer acetylated microtubules (; conversely, microtubules decorated with exogenous GFP-APC were usually acetylated (data not shown). In migrating control cells, acetylated microtubules were always localized to the leading front of cells, whereas in APC-deficient cells, acetylated microtubules were more randomly distributed and often clustered around the nucleus. This suggested that in the short term, loss of APC led to a decrease in stable microtubules specifically in the cellular periphery. Indeed, the presence of endogenous APC protein at microtubule plus ends in the periphery correlates with their longevity (Kita et al., 2006
). This suggests that overall APC can contribute to microtubule stability and helps to orient more stable microtubules toward the direction of migration.
We further confirmed that loss of APC rendered microtubules less stable in cells by comparing the sensitivity of microtubules to depolymerizing agents in cells before and after APC inactivation (Supplemental Figure 2). In these experiments, we found that in APC-deficient cells, microtubules depolymerized more readily than in control cells. In addition, we found that the resistant microtubules were usually acetylated, confirming their increased stability. This observation is in contrast to previously published data that showed no correlation between acetylation and increased stability against microtubule-depolymerizing agents (Palazzo et al., 2003
). It is possible that this effect is cell type specific or that only specific concentration ranges of microtubule drugs reveal such differences. In contrast, our data are consistent with previously published report that showed a decrease in microtubule stability in cells expressing a dominant fragment of EB1 that can sequester APC away from microtubules (Wen et al., 2004
Consistent with the ability of APC to affect cell shape by modulating microtubule stability, we found that expressing full-length APC induced the formation of cellular asymmetry by creating longer cellular protrusions (). Only full-length APC but not individual fragments had this effect. Most important for this effect was the ability of the exogenous protein to cluster at the end of membrane protrusions, suggesting that locally induced changes were involved in enhancing cellular asymmetry.
In conclusion, we found that loss of wild-type APC, the situation faced in cells at early stages of tumorigenesis, compromised overall microtubule stability and cell migration in monolayers as well as in single cells. This effect was an immediate consequence of APC depletion. These findings could have important implications for therapeutic considerations in APC-deficient tumor cells. The possible weakness created by a decrease in microtubule stability in tumor cells lacking APC, i.e., the majority of colorectal cancers, should prompt the reconsideration of microtubule poisons for treatment of colorectal cancers, particularly recently developed microtubule poisons that are less sensitive to multiple drug resistance (Bacher et al., 2001
; Goodin et al., 2004
), which minimized the usefulness of this type of agent for treating colorectal cancer in the past.