Stathmin destabilizing microtubule dynamics promotes malignant potential in cancer cells by epithelial-mesenchymal transition

Yu Lu, Chen Liu, Yong-Feng Xu, He Cheng, Si Shi, Chun-Tao Wu and Xian-Jun Yu

Shanghai, China

Stathmin destabilizing microtubule dynamics promotes malignant potential in cancer cells by epithelial-mesenchymal transition

Yu Lu, Chen Liu, Yong-Feng Xu, He Cheng, Si Shi, Chun-Tao Wu and Xian-Jun Yu

Shanghai, China

BACKGROUND:Stathmin is a ubiquitous cytosolic regulatory phosphoprotein and is overexpressed in different human malignancies. The main physiological function of stathmin is to interfere with microtubule dynamics by promoting depolymerization of microtubules or by preventing polymerization of tubulin heterodimers. Stathmin plays important roles in regulating many cellular functions as a result of its microtubuledestabilizing activity. Currently, the critical roles of stathmin in cancer cells, as well as in lymphocytes have been valued. This review discusses stathmin and microtubule dynamics in cancer development, and hypothesizes their possible relationship with epithelial-mesenchymal transition (EMT).

DATA SOURCES:A PubMed search using such terms as "stathmin", "microtubule dynamics", "epithelial-mesenchymal transition", "EMT", "malignant potential" and "cancer" was performed to identify relevant studies published in English. More than 100 related articles were reviewed.

RESULTS:The literature clearly documented the relationship between stathmin and its microtubule-destabilizing activity of cancer development. However, the particular mechanism is poorly understood. Microtubule disruption is essential for EMT, which is a crucial process during cancer development. As a microtubule-destabilizing protein, stathmin may promote malignant potential in cancer cells by initiating EMT.

CONCLUSIONS:We propose that there is a stathminmicrotubule dynamics-EMT (S-M-E) axis during cancer development. By this axis, stathmin together with itsmicrotubule-destabilizing activity contributes to EMT, which stimulates the malignant potential in cancer cells.

(Hepatobiliary Pancreat Dis Int 2014;13:386-394)

stathmin;

microtubule dynamics;

epithelial-mesenchymal transition;

malignant potential;

cancer

Introduction

Stathmin, also known as OP18 (oncoprotein 18), is one of the members of microtubule-destabilizing proteins expressed ubiquitously in vertebrates. Stathmin integrates diverse signaling pathways involved in cell proliferation, differentiation and other cell functions.[1]The microtubule-destabilizing activity of stathmin is mainly modulated by phosphorylation, while recent studies[2,3]also demonstrated that signal transducer and activator of transcription 3 (STAT3) can antagonize stathmin's activity by direct binding to its COOH-terminal. Microtubule constitutes one of the major components of the cytoskeleton of eukaryotic cells and is involved in many essential cellular processes, including cell division, ciliary and flagellar motility and intracellular transport.[4]A large number of researches[5-7]revealed that microtubule dynamics could contribute to oncogenic epithelial-mesenchymal transition (EMT). Stathmin, an important microtubule dynamics regulator, has been linked to the promotion of malignant potentials in cancer cells[8-10]as a result of its overexpression in a wide range of cancers. However, whether stathmin contributes to EMT, which plays a critical role in tumor invasiveness, distant dissemination and drug resistance,[11-13]is not clear. We hypothesize that stathmin-related microtubule dynamics might be associated with the malignant potentials of cancer cellsby promoting EMT in tumors such as the stroma-rich pancreatic cancer.[14,15]In this review, we focus on the relationship between stathmin and its microtubuledestabilizing activity in cancer development, as well as the possible correlation with EMT.

Microtubule dynamics

Microtubules are dynamic tubulin polymers and are required for a wide variety of central cellular functions, including mitosis, intracellular transport, polarity, and motility.[16-21]During the interphase of cell cycle, microtubule mainly contributes to intracellular trafficking of vesicles and organelles. When cell turns into mitosis, microtubules become the major component of mitotic spindle which enables correct chromosome segregation.[22]Microtubules are polarized structures composed of the combination of α/β-tubulin heterodimers.[23]The β-tubulin subunit always exposed to the fast growing plus end of microtubule and α-tubulin subunit exposed to the slow growing minus end.[24]There is a transition between growth and shrinkage within the polar ends of microtubule, which is referred to as dynamic instability. The transition from growth to shrinkage is called catastrophe, while a reverse transition is named rescue (Fig. 1A).[25]As is well-known, the growing plus end of microtubule contains a cap of tubulin with GTP in the β-tubulin unit. When GTPs in the cap hydrolyze into GDPs, catastrophe happens (Fig. 1B).[26,27]Highly regulated microtubule dynamics is essential to diverse of intracellular functions. Unbalanced dynamic properties of microtubule can give rise to abnormal mitotic spindle formation, and to the inability to properly segregate chromosomes, thereby leading to aneuploidy, and possibly tumorigenesis.[28]

Fig. 1. Microtubule dynamics. A: There is a conformational transition between growth and shrinkage within the microtubule polar ends. The transition from growth to shrinkage is named catastrophe, the opposite event is named rescue. The tip of the growing plus end contains a GTP-cap in the β-tubulin subunit. Hydrolysis of the GTP-cap leads to microtubule catastrophe. B: GTP in α-tubulin subunit never hydrolyzes; GTP in the β-tubulin subunit can hydrolyze into GDP.

Stathmin and its microtubule-destabilizing activity

The dynamics of microtubule polymerization and depolymerization is regulated by two major classes of proteins: microtubule stabilizers and microtubule destabilizers.[29]As microtubule stabilizers, microtubuleassociated proteins were identified to bind the body of microtubule and enhance its stability. Microtubule destabilizers such as stathmin and XKCM1 were capable of binding free tubulin heterodimers or promoting catastrophe frequency.[30]

Stathmin is a 149 amino-acids cytosoluble protein which is highly conserved in vertebrates. There has been a debate over the specific mechanism of how stathmin destabilizing microtubules. Stathmin was initially found to act as a catastrophe promoter in regulating microtubule dynamics.[31]It was demonstrated that stathmin could sequester tubulin heterodimers into a tight complex and thus decrease the amount of free tubulin for microtubule assembly. The reduced free tubulin concentration slows down the growth rate of microtubule and thus, increases the catastrophe frequency within the microtubule polar ends. However, a further study indicated that stathmin sequestering tubulin slowed down the growth rate of microtubule but not promote catastrophes.[32]Later in vitro experiments performed by Howell et al[33]showed that the microtubule regulating activity of stathmin can be influenced by the buffer pH. At a pH of 6.8, stathmin could mainly function as a tubulin sequester. When the pH increases to 7.5, stathmin acts as a catastrophe promoter. At this point, stathmin primarily stimulates GTP hydrolysis by its direct binding to the plus end of microtubule (Fig. 2). Those authors also investigated the two roles of stathmin protein. NH2-terminal promotes catastrophe while COOH-terminal sequesters tubulin.

The microtubule-destabilizing activity of stathmin can be down-regulated in a multiple phosphorylation manner by a variety of kinases.[34]Four phosphorylation serine sites have been identified within the NH2-terminal regulatory domain: Ser16, Ser25, Ser38, and Ser63.[1]Ser16 is mainly phosphorylated by the Ca2+/ calmodulindependent kinase IV/Gr (CaMK IV/ Gr), and Ser25 by members of the mitogen-activated protein kinase (MAPK) family.[35,36]Ser38 is a target for phosphorylation by cyclin-dependent kinases (CDKs) and Ser63 is themajor site for protein kinase A (PKA).[37]It was revealed that phosphorylated Ser16 and Ser63 lead to a stronger impairment of stathmin microtubule-destabilizing activity than Ser25 and Ser38 do.[38]Non-phosphorylated stathmin (S) can sequester free α/β-tubulin (T) and generate a stable and "assembly incompetent" ternary T2S complex.[32,39]By combining tubulins in a curved complex that is not incorporated in microtubules, stathmin degrades the "assembly competent" tubulins[40]so as to prevent the polymerization of tubulin heterodimers or to promote depolymerization of microtubules. Meanwhile, microtubule assembles itself resulting in an increased stathmin phosphorylation. These processes might provide a positive feedback loop for microtubule formation.[41]A recent study[3]indicated that STAT3 could antagonize the tubulin-sequestering activity of stathmin by directly binding to its COOH-terminal (Fig. 3).

Fig. 2.Microtubule-destabilizing activity of stathmin. Stathmin is proposed to bind to free tubulin heterodimers so as to prevent their polymerization. Stathmin could also directly bind to the microtubule plus end and stimulate GTP hydrolysis so as to induce microtubule depolymerization.

Fig. 3.Structure and regulating activity of stathmin. Stathmin can be phosphorylated at its four phosphorylation serine sites have been identified within its NH2-terminal domain. Stathmin can sequester free α/β-tubulin and generate a stable and "assembly incompetent" T2S complex by its COOH-terminal domain. Phosphorylated stathmin or STAT3-binding stathmin is incapable to sequester free α/β-tubulin.

Expression of stathmin in cancer

In recent years, many studies have confirmed that stathmin was overexpressed in different human malignancies, such as leukemia,[42,43]lymphoma,[44]cervical cancer,[45,46]breast cancer,[47-49]prostate cancer,[50,51]lung cancer,[9]osteosarcoma,[52]and gastrointestinal tumors.[53,54]In cervical carcinoma tissues, the expression levels of stathmin mRNA and protein were significantly higher than those in adjacent non-cancerous margin samples. Besides, a high stathmin expression was significantly related to the diameter of the primary tumor, the clinical TNM classification and the long-term survival rate, suggesting that stathmin was involved in cervical carcinogenesis and tumor progression. Those results indicated that stathmin could be used as a prognostic indicator in patients with cervical carcinoma.[46]Likewise, in breast carcinoma,[48]localized upper urinary tract urothelial carcinoma,[55]hepatocellular carcinoma,[56]oral squamous-cell carcinoma,[57]medulloblastoma[58]and adenoid cystic carcinoma of the salivary glands,[59]high stathmin expression is associated with cancer progression and prognosis.

Stathmin and cell cycle

Stathmin plays an critical role in the cell cycle progress.[60]In interphase, microtubule nucleation rate from centrosome was greatly influenced by stathmin. High stathmin level impaired nucleation, whereas low stathmin level enhanced this process.[61]At the beginning of mitotic phase, phosphorylated stathmin was significantly increased in the K562 erythroleukemia cells.[62]A recent study[63]showed that stathmin phosphorylation is remarkably lower in silent cells than that in proliferating cells. They also demonstrated that phosphorylation of stathmin can be regulated by P34cdc2, a dominating protein kinase controlling the entry of mitosis in eukaryotic cells. Marklund et al[64]investigated the effects of stathmin overexpression of wild type and P34cdc2-target site deficient mutant on the morphological changes of the mitotic spindle, the results demonstrated that induced expression of wild-type stathmin mainly results in microtubule depolymerization in interphase. Phosphorylation switches off stathmin microtubuledepolymerizing activity and promotes the assembly of the mitotic spindle at the onset of mitosis.[64,65]All those observations suggested that stathmin is inactivatedby phosphorylation which plays an important role in the regulation of mitotic spindle formation during cell cycle progression. Mistry and Atweh[66]examined the effects of okadaic acid, an inhibitor of serine/threonine protein phosphatases, on parameters of mitosis with or without inhibition of stathmin. They found that dephosphorylation of stathmin is necessary for mitotic spindle disassembly and the proper exit from mitosis.

Recent reports[67-69]also illustrated that stathmin was related to p53 in regulating cell cycles. In HCT116 cell lines and Hela cells, depletion of stathmin by siRNA or shRNA leads to a G2/M blockade while depletion of both p53 and stathmin displayed a blockade in G2 phase. It is worth noting that up-regulated p53 restrains stathmin expression and induces the G2/M delay,[70]both stathmin and p53 are necessary for the G2/M cell cycle progression.

Stathmin and cell proliferation/differentiation

High expression of stathmin was found in undifferentiated multipotent cells or initial stage tissues.[71-73]However, stathmin expression decreased dramatically in most adult tissues. Similar results were found in the liver regeneration model.[74,75]Stathmin was rarely expressed in mature liver tissue; partial hepatectomy significantly increases the level of stathmin. At a peek around 60 hours postoperation, the expression decreased in the next few days. It appears that stathmin expression prevents uncontrolled cell growth before initiation of cell differentiation. The finding seems to be inconsistent with the fact that stathmin was overexpressed in high proliferative tumors. With the theories above, we propose that the expression of stathmin may prevent the normal cell from overproliferation, this effect is not sufficient enough in cancer cells. The specific molecular mechanism remains unclear. Recent studies[45,76,77]revealed a relationship between stathmin phosphorylation and the PI3K pathway, which is involved in cellular functions such as cell growth, proliferation/differentiation and apoptosis. Stathmin may be a component of the PI3K pathway in cell proliferation and differentiation.

Stathmin and cell motility

Studies[21,78]showed that microtubule stability takes part in the regulation of cell motility. Therefore, as a critical microtubule-destabilizing protein, stathmin is involved in the regulation of cell motility, particularly in cancer cells. Belletti et al[10]evaluated the stathmin expression in human sarcomas and demonstrated that stathmin stimulated cell motility via extracellular matrixin vitroand contributed to the local invasion and distant dissemination. Besides, a range of cellular proteins were reported to be in concert with stathmin to influence the cell motility-stimulating activity.[8]Singer et al[9]considered the far upstream sequence element-binding protein-1 as a key molecule in the induction of different stathmin family members. Their studies indicated that far upstream sequence elementbinding protein-1 and overexpression of microtubuledestabilizing factors such as stathmin is a pivotal process to destabilize microtubule dynamics and further increases proliferative and motile ability of malignant cells.

Stathmin might contribute to the activation and migration of T cells. T cell activation and polarization were involved in intracellular material transportations, promoting the formation of the immunological synapse and/or facilitating the directed cytokines secretion.[79-81]Reposition of microtubule-organizing center is critical for the activation of T cells. Filbert et al[82]demonstrated that activated extracellular signal-regulated kinase which located in immunological synapses could contribute to microtubule-organizing center polarization as a result of inducing phosphorylation of stathmin. Deletion of stathmin leads to detention of microtubuleorganizing center reposition and thus impairs cytotoxic T lymphocyte cytolysis. Verma et al[2]reported that STAT3 is critical for T cell migration by interacting with stathmin and interfering its microtubule-destabilizing activities.

Stathmin and drug resistance

Anti-cancer chemotherapeutics used in the clinic such as taxanes and vinca alkaloids are classified as microtubule stabilizers or destabilizers.[83-88]These drugs inhibit mitosis through a similar mechanism of slowing microtubule dynamics, resulting in mitotic arrest and apoptosis, thus limiting the cell proliferation. As a microtubule-destabilizing protein, stathmin plays a special role in the drug resistance. Using a set of human breast cancer cell lines, Alli et al[89]found that there are at least two different ways for stathmin to influence the action of anti-microtubule chemotherapy drugs, i.e. directly interfere drug-tubulin binding and growth arrest at G2/M phase during a cell cycle. Combining antisense to target certain proteins and chemotherapeutic agents, Benner and colleagues demonstrated that some proteins are essential for the malignant phenotype.[90]

A hypothetical S-M-E axis

Overexpression of stathmin and its microtubule-destabilizing activity contribute to cell proliferation, differentiation, motility and drug resistance during cancer development. However, the underlying mechanism is still poorly understood.

Microtubule is an essential component of cytoskeleton. Highly organized microtubules are required for many cellular functions including the maintenance of cell morphology, intracellular material transportation, cell motility and differentiation.[10]Microtubule disruption leads to the break-down of basement membrane, which is the first step of EMT process.[5]EMT is a crucial mechanism for embryogenesis, forming fibroblasts and/or mesenchymal cells in injured tissues, and activating metastasis of epithelial cancer cells.[91]Abnormal activation of EMT contributes to pathologic changes, including fibrosis, neoplasia and cancer development.[92-96]During the EMT process, epithelial cells usually lose the capability of intercellular adhesion and acquire a mesenchymal phenotype,[97-99]which requires the modification in cellular architecture, morphology, adhesion, and migration capacity.[100,101]The key point of EMT is the down-regulation of E-cadherin expression, a crucial step reducing the cell-cell adhesion and leading to remolding of the cytoskeleton.[102,103]Moreover, the acquisition of a motile and invasiveness may result in the tumor resistance to chemotherapy.[104-106]For instance, pancreatic cancer is characterized by an abundant tumor stroma resulted from EMT. Tumor stroma contributes to a majority of malignant biological features, such as dissemination at early stages and resistance to chemotherapeutics.[13-15]As a microtubule-destabilizing protein that involved with various oncogenic functions, stathmin might promote the EMT process through regulating microtubule dynamics in the development of cancer.

Direct proof on the contribution of stathmin to EMT is difficult to conduct. Li et al[7]performed a series of researches utilizing Siva1 to examine the role of stathmin and microtubule dynamics in promoting EMT. They proved that Siva1 counteracts stathmin by impeding the interaction between stathmin and tubulin and/or promoting CaMK II-mediated phosphorylation of stathmin at Ser16. They further investigated the role of Siva1 in EMT and found that Siva1 overexpression suppresses cell migration and EMT, whereas knockdown of Siva1 reverses this effect. However, this EMT-regulating activity of Siva1 vanished when stathmin was knocked-out. These findings suggest that Siva1 may suppress EMT by attenuate the activity of stathmin. This is an indirect proof of the role of stathmin in promoting EMT.

Fig. 4.The hypothetical S-M-E axis. Stathmin may promote microtubule disruption by its microtubule-destabilizing activity; microtubule disruption causes the depolarization of cell shape, cell locomotion which is involved in EMT process thereby contributing to high invasive and metastatic characteristics of cancer. The dashed line with arrow indicates the progress is putative or involves multiple steps.

We suppose that there is a stathmin-microtubule dynamics-EMT (S-M-E) axis in the development of cancer (Fig. 4). The axis might work mainly in three steps. First, up-regulated non-phosphorylated stathmin sequesters tubulin heterodimers so as to promote depolymerization of microtubules or prevent polymerization of tubulin heterodimers. Second, such microtubule destabilizing activity of stathmin promotes EMT in cells. Third, EMT contributes to the aggressive features of malignant cells, including high invasiveness and drug resistance.

S-M-E axis as a potential target for cancer therapy

Besides the high invasiveness and metastasis propensity, the poor response to existing treatments contributes a large part to the lethality of human malignancies.[107]As mentioned above, stathmin was overexpressed in many cancers and significantly related to the prognosis of cancer patients. Stathmin and its microtubuledestabilizing activity contribute to malignant potentials in cancer cells. All those facts strongly suggest that stathmin is a promising target for more effective therapeutic interventions.[108-110]Microtubule disruption contributes greatly to cellular morphological changes, and to the EMT process, which is involved in the development of diverse solid human malignancies.[111-113]As a result, targeting microtubule and EMT may also provide novel anti-cancer therapies. Clinically, multiple anti-cancer drugs like taxanes and vinca alkaloids targeting microtubule dynamics were widely used.[83-88]The microtubule-destabilizing activity of stathmin contributes to the resistance of tumors to chemotherapy,[89]and can be targeted by numerous ofchemotherapy agents. The S-M-E axis was supposed to be an important pathway in cancer development, including the invasiveness and drug resistance. Consequently, we speculate that the S-M-E axis may be a potential target in anti-cancer therapies.

Conclusion and perspectives

The microtubule-destabilizing protein stathmin was involved in cancer development. However, the specific molecular mechanism remains unclear. We reviewed the available studies and summarized the role of stathmin and its microtubule destabilizing activity in the process of cancer development. Microtubule disruption causes the depolarization of cell shape, cell locomotion which is involved in EMT process. All of these suggest that stathmin destabilizes microtubule which promotes malignant potential in cancer cells. We proposed a stathmin-microtubule dynamics-EMT (S-M-E) axis: stathmin and its microtubule-depolymerizing activity lead to the EMT progress during cancer development. The S-M-E axis can be involved in tumor invasion, metastasis and drug resistance. Further studies are required to investigate the specific mechanism of this axis. Targeting the S-M-E axis may provide novel therapy for human malignancies.

Contributors:LY and LC wrote the main body of the article and contributed equally to this article. YXJ revised the manuscript. All authors contributed to the design and interpretation of the study and to further drafts. YXJ is the guarantor.

Funding:This work was supported by grants from the National Natural Science Foundation of China (81172276, 81001058, 8110156, Sino-German GZ857) and the Shanghai Committee of Science and Technology, China (11JC1402500).

Ethical approval:Not needed.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Received April 1, 2013

Accepted after revision September 23, 2013

Author Affiliations: Pancreatic Cancer Institute, Fudan University; Department of Pancreatic and Hepatobiliary Surgery, Fudan University Shanghai Cancer Center; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China (Lu Y, Liu C, Xu YF, Cheng H, Shi S, Wu CT and Yu XJ)

Xian-Jun Yu, MD, PhD, Pancreatic Cancer Institute, Fudan University; Department of Pancreatic and Hepatobiliary Surgery, Fudan University Shanghai Cancer Center, Shanghai 200032, China (Tel: +86-21-64175590ext1307; Fax: +86-21-64031446; Email: yuxianjun88@hotmail.com)

? 2014, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(14)60038-2

Published online March 27, 2014.