Formins nucleate actin and stabilize microtubules (MTs). Expression of the formin Diaphanous homolog 1 (DIAPH1) is increased in malignant colon carcinoma cells, while expression of DIAPH3 is up-regulated in breast and prostate carcinoma cells. Both DIAPH1 isoforms are required to stabilize interphase MTs of cancer cells, and it has been shown that loss of this function decreases the metastatic potential of these cells. Moreover, depletion of DIAPH3 increases the sensitivity of breast and prostate carcinoma cells to taxanes. In contrast with DIAPH1 + 3, DIAPH2 regulates metaphase MTs of tumor cells by stabilizing binding of kinetochore MTs to chromosomes. Depletion of DIAPH2 impairs chromosome alignment, thus proper chromosome segregation during mitosis. In summary, expression of DIAPH formins in tumor cells is essential for stabilizing interphase or metaphase MTs, respectively. Thus, it would be very interesting to analyze if tumor cells exhibiting low DIAPH expression are more sensitive to taxanes than those with high DIAPH expression.
Metastasis is the leading cause of death in cancer patients [1,2]. The first step of metastasis is considered to be the epithelial-to-mesenchymal transition (EMT) during which epithelial tumor cells acquire a motile phenotype to escape from the primary tumor. The cells lose adhesion to neighboring tumor cells and adhere to the extracellular matrix (ECM), which surrounds the primary tumor. Thereafter, metallo-matrix proteinases are secreted, enabling the tumor cells to invade the ECM, migrate into the adjacent tissue and invade into blood vessels. After dissemination into the blood stream, the tumor cells leave the blood vessels, undergo mesenchymal-to-epithelial transition and form new metastasis in organs distant from the primary tumor. This EMT model is described in numerous reviews  and is certainly true for many tumor types. However, in the blood, both mesenchymal and epithelial tumor cells, as well as tumor cell clusters, have been detected ; and in the tissue, many tumor cell types perform collective migration . Thus, it seems that not for all tumor cell types, EMT is necessarily required for invasion and to form distant metastasis.
However, during the whole metastatic cascade, tumor cells change their morphology, a process that is controlled by the cellular cytoskeleton, which consists of F-actin, microtubules (MTs) and intermediate filaments . After epithelial tumor cell clusters or mesenchymal tumor cells have detached from the primary tumor, the next step is the adherence to the ECM. It is important to note that adherence between cells and adherence to the ECM is controlled by different mechanisms. Cell–cell contacts are mainly mediated by desmosomes and E-cadherin , while cell–ECM interaction mainly depends on the interaction between integrins with components of the ECM . Depending on the kind of invasion, the cells lose cell–cell contacts prior to the adherence to the ECM or the cells adhere as cell cluster and perform collective invasion .
In this review, we focus on adhesion of tumor cells to the ECM and discuss the role of Diaphanous-related formins (DIAPHs) on MTs during this process.
During cell adhesion, the cell body becomes flattened, and the cell forms focal adhesions (FAs) to stick to the substrate (Figure 1) [10,11]. We recently demonstrated that inhibition of actin polymerization by cytochalasin D did not alter early adhesion of colon cancer cells to collagen, showing that actin dynamics are not essentially involved in this process . On the other hand, inhibition of MT polymerization by nocodazole significantly inhibited early adhesion of colon cancer cells to collagen. This finding can be explained by the nature of morphological changes during the first step of cell–ECM adhesion. The former round- or rhomboid-shaped cells become flattened in order to spread to the substrate. During this process, MTs spread from the center to the periphery for stabilizing the cell body . In addition, integrin-β1, the main protein of FAs, is transported to the plasma membrane via tubulin-mediated vesicle transport (Figure 2). Binding of ECM proteins to integrin-β1 alters the F-actin structure, resulting in consolidation of adhesion. Thus, both cell spreading and formation of FAs depend on MTs.
Adhesion of tumor cells to the ECM.
DIAPH1 mediates transport of integrin-β1 to the plasma membrane.
Our data revealed that DIAPH1 is essentially involved in MT-dependent early adhesion of colon cancer cells. The formin DIAPH1 is mainly known as an actin nucleator [14,15]. It recruits profilin/G-actin by its FH1 domain, while the dimeric FH2 domains form a ring around the plus end of actin filaments, protecting the plus end from capping [14–17]. Recruitment of G-actin to the plus end of F-actin finally results in the formation of linear actin filaments . However, in the absence of extracellular stimuli, DIAPH1 is auto-inhibited, because the N-terminal Diaphanous inhibitory domain and the C-terminal Diaphanous autoregulatory domain interact, thereby blocking the FH1 and the FH2 domains [15,19]. In response to receptor stimulation, binding of RhoA to the Rho-binding domain of DIAPH1 releases the FH1 and the FH2 domains, thus stimulating the actin nucleation activity of DIAPH1 [20–22]. In addition to its actin-nucleating activity, DIAPH formins bind MTs via its FH2 domains and thereby stabilize MTs [23,24]. Furthermore, the FH2 domains bind the MT-stabilizing proteins EB1 and APC, contributing to DIAPH-mediated MT stabilization .
We found that in the absence of extracellular stimuli, actin dynamics were not altered in colon cancer cells with reduced DIAPH1 expression but MTs were less stable and more dynamic . This result indicates that DIAPH1 is able to stabilize MTs, even in unstimulated cells. Accordingly, MT-dependent cellular processes, like vesicle trafficking and transport of integrin-β1 to FAs, were inhibited in DIAPH1-depleted colon cancer cells. From these data, we conclude that, even in its auto-inhibited state, DIAPH1 interacts with MTs, resulting in stabilization of MTs required for vesicle trafficking and transport of integrin-β1 to FAs (, Figure 2). Based on our result, showing that depletion of DIAHP1 also reduced adhesion, we conclude that DIAPH1-mediated MT-stabilizing activity controls early adhesion of colon cancer cells, even in the absence of extracellular stimuli. Since adhesion to the ECM is one of the first steps in the metastatic cascade , subsequent experiments with SCID mice revealed that depletion of DIAPH1 nearly completely blocked metastasis of colon cancer cells to the lung and to the liver . This result demonstrates that DIAPH1 is essential for colon cancer metastasis. Based on the current literature, we assume that the MT-stabilizing activity of DIAPH1 is required in early metastasis (attachment of tumor cells to the ECM), while in response to extracellular stimuli, the actin-nucleating activity of DIAPH1 drives invasion by promoting the formation of invadopodia [21,26].
In addition to the effects of DIAPH1 on MTs, the isoform 3 of DIAPH (DIAPH3 or mDia2) has been also shown to be essential for stabilizing interphase MTs . Morley et al.  revealed that down-regulated DIAPH3 expression in prostate and breast cancer cells reduced polarized force generation and contractility. Interestingly, the authors additionally demonstrated that loss of DIAPH3 increased the sensitivity of prostate and breast cancer cells to taxanes. Thus, it seems that DIAPH-mediated MT stabilization attenuates binding of taxol to MTs.
In contrast with DIAPH1 + 3, which are essential to stabilize interphase MTs, the isoform 2 of DIAPH (DIAPH2 or mDia3) seems to be required for stabilization of metaphase MTs. Yasuda et al.  found that DIAPH2 is associated with the kinetochore and siRNA-induced DIAPH2 knockdown in HeLa cells resulted in metaphase chromosome misalignment. Subsequent mechanistic studies revealed that DIAPH2 is essential for kinetochore MT stabilization in cell division because its MT-binding activity contributes to regulate kinetochore-bound MT dynamics during chromosome alignment [28,29]. It is thus believed that DIAPH2 is essential for proper chromosome segregation during mitosis [28,29]. The finding of Kim et al. , showing that the FH2 domain-binding small molecule inhibitor SMIFH2  impairs spindle formation, supports the conclusion that DIAPH2 is essential for attachment of kinetochore MTs to chromosomes. Once chromosome alignment is impaired, the DNA is unequally distributed to daughter cells, resulting in chromosomal instability (CIN) and causing aneuploidy. In many tumor entities, malignant progression is associated with CIN [31–34], demonstrating that impaired kinetochore MT stabilization can increase malignancy. Since it has been shown that overexpression of the spindle checkpoint kinase Aurora kinase A promotes resistance to taxanes , it would be very interesting to test if low DIAPH2 expression increases the sensitivity to taxanes.
In summary, it seems that the roles of DIAPH isoforms in tumor progression are different but all isoforms affect MT stability. Thus, it would be of high clinical relevance to analyze if the expression of DIAPH isoforms might be a predictor of response to taxanes.
MT, microtubule; DIAPH, Diaphanous-related formins; ECM, extracellular matrix; EMT, epithelial to mesenchymal transition; FAs, focal adhesions; CIN, chromosomal instability.
This work was partially supported by a UCCH grant (University Cancer Center Hamburg) and by the Else-Kröner Fresenius-Stiftung.
The Authors declare that there are no competing interests associated with the manuscript.