Elsevier

Cellular Signalling

Volume 25, Issue 5, May 2013, Pages 1075-1085
Cellular Signalling

Rab35 is required for Wnt5a/Dvl2-induced Rac1 activation and cell migration in MCF-7 breast cancer cells

https://doi.org/10.1016/j.cellsig.2013.01.015Get rights and content

Abstract

The small GTPases regulate many major biological processes in both tumorigenesis and tumor progression such as cell survival, actin cytoskeleton organization, cell polarity and movement. Wnt5a, a non-canonical Wnt family member, is implicated in the activation of small GTPases in breast cancer. We previously demonstrated that Wnt5a signaling stimulates the migration of breast cancer cells MDA-MB-231 via activating RhoA. However, we found here that RhoA activation was not enhanced by Wnt5a in breast cancer cells MCF-7. The conflicting results prompted us to further probe novel small GTPases in response to Wnt5a and investigate the mechanisms whereby cell migration is regulated. We showed here that Wnt5a dose dependently activated Dvl2, Rab35 and Rac1 and subsequently promoted the migration of MCF-7 cells, which was, however, abolished by knocking down Wnt5a expression via small interfering RNA (siRNA) transfection. Dvl2 siRNA significantly decreased background and Wnt5a-induced Rab35/Rac1 activation and, consequently, cell migration. Rab35 short hairpin RNA (shRNA) remarkably inhibited background and Wnt5a-induced Rac1 activation and cell migration. Additionally, blockade of Rac1 activation with Rac1 siRNA suppressed background and Wnt5a-induced cell migration. Co-immunoprecipitation and immunofluorescence assays showed that Dvl2 bound to Rab35 in mammalian cells. Taken together, we demonstrated that Wnt5a promotes breast cancer cell migration via the Dvl2/Rab35/Rac1 signaling pathway. These findings implicate Wnt5a signaling in regulating small GTPases, which could be targeted for manipulating breast cancer cell migration.

Graphical abstract

Highlights

► Wnt5a stimulates MCF-7 breast cancer cell migration. ► MCF-7 cell migration requires Dvl2 activation. ► Wnt5a induces cell migration via Rac1, not RhoA, activation. ► Rab35 acts as a downstream target of Dvl2 and mediates cell migration. ► Dvl2 binds to Rab35 in mammalian cells.

Introduction

The small GTPase superfamily is divided into at least five families, including Ras, Rho, Rab, Arf, and Ran. Its most well-known members are the Ras GTPases and hence they are sometimes called the Ras superfamily GTPases. Together with their effectors, Rabs, which represent the largest group within the Ras superfamily, control many aspects of intracellular trafficking and organelle functions such as vesicle budding, transport, tethering, and fusion [1]. Rab small GTPases function as molecular switches that either drive or terminate membrane trafficking or organelle functions by cycling between a GTP-bound active state and a GDP-bound inactive state [1]. Rab35, as a Rab gene, was first identified and cloned from a human skeletal muscle cDNA library [2]. Rab35 appears not to be directly related to its cognate role in membrane trafficking, but rather is an interacting partner for nucleophosmin-anaplastic lymphoma kinase in human lymphoma cells [3]. In cancer, Rab35, also named Ray or Rab1c, interacts with p53-related protein kinase and distributes in the nucleus, cytosol, and cell membrane of HeLa cells [4]. A clinical investigation demonstrates a significant positive correlation between Rab35 and androgen receptor expression in ovarian cancer [5]. However, the mechanisms whereby Rab35 is involved in oncogenic or tumor suppressive activities have not yet been fully understood.

Wnt signaling has historically been divided into two categories, namely, the canonical and the non-canonical signaling pathways. The canonical Wnt signaling pathway has been implicated in promoting malignant transformation and tumor progression [6], while very limited studies have been done on the role and mechanisms of non-canonical Wnt signaling in tumor progression. Wnt5a, a representative of Wnt proteins that activates non-canonical Wnt signaling, is involved in such cellular functions as cell growth, proliferation, differentiation, motility and survival, all of which could impact cancer development. In breast cancer, we have previously reported that Wnt5a stimulates the migration of breast cancer cells MDA-MB-231 [7]. Other studies have found that Wnt5a secreted by macrophages is proved to be essential for macrophage-induced invasiveness of breast cancer cells [8]. Wnt5a is frequently upregulated in breast tumors in comparison with surrounding tissues, and is shown to promote the invasion of MCF-7 cells via the Jun N-terminal kinase (JNK) pathway [8], [9]. In contrast to the hypothesis that Wnt5a functions as an oncogene, Wnt5a can inhibit breast epithelial cell migration, whose expression positively correlates with disease-free survival and blocks breast cancer cell invasion as well [10], [11], [12], [13].

Those complicated viewpoints of Wnt5a in breast cancer progression led us to further elucidate the function of Wnt5a and investigate the mechanisms whereby cell migration is regulated. We previously demonstrated that RhoA signaling facilitates Wnt5a-induced acceleration of cell motility via the activation of Dishevelled 2 (Dvl2) and Dishevelled-associated activator of morphogenesis 1 (Daam1) in MDA-MB-231 cells [7]. Wnt5a signaling can activate the small GTPases; however, we have not yet understood whether RhoA or novel small GTPases could be triggered by Wnt5a in other breast cancer cells. Here, we demonstrated that Rab35 and Rac1, but not RhoA, mediate Wnt5a-induced migration of breast cancer cells MCF-7. Wnt5a signaling, in need of Dvl2, directly activated Rab35 and Rac1.

Section snippets

Plasmids and transient transfections

The human Dvl2 construct was generously provided by Dr. Marc Fiedler (MRC Laboratory of Molecular Biology, Cambridge, UK). Full-length Dvl2 was generated by PCR and sub-cloned in pCMV-HA vector. The GFP-Rab35 construct was kindly given by Dr. Mikael Simons (Max Planck Institute for Experimental Medicine, University of Göttingen, Germany). Cell lines MCF-7, MDA-MB-231 and HEK-293T (ATCC, Manassas, VA) were grown in Dulbecco's modified Eagle's medium (DMEM, high glucose) (Hyclone, Thermo

Wnt5a stimulates MCF-7 breast cancer cell migration in vitro

To assess the effect of Wnt5a on breast cancer cell migration, we treated MCF-7 cells with different doses of recombinant Wnt5a (rWnt5a) and measured the migration rate by using Boyden chamber assay and wound-healing assay. We found that rWnt5a (100 ng/mL) caused a remarkable increase in the migration of MCF-7 cells compared with untreated cells (Fig. 1A and B). MCF-7 cells express higher levels of Wnt5a than other breast cancer cell lines [9]. We used siRNAs to knock down Wnt5a expression in

Discussion

In spite of advances in early diagnosis and adjuvant and neoadjuvant therapy of breast carcinoma, this disease still remains the most common malignancy among women worldwide [14]. Aggressive breast cancers are generally recalcitrant to current anti-cancer therapies and prone to early recurrence, and metastasize to distant organs such as the brain and lungs [15]. For this reason, elucidating the signaling pathways involved in the metastatic cascade is a key goal for developing novel and

Conclusions

In summary, we present the first direct evidence here that Wnt5a promotes breast cancer cell migration via Dvl2/Rab35/Rac1 signaling. Those findings highlight the presence of a molecular pathway linking Wnt5a signaling with Rab35 in cell motility, which may represent a rational molecular target for manipulating breast cancer.

The following is the supplementary data related to this article.

Conflict of interest

These authors claim no conflict of interest.

Acknowledgments

This work was supported by grant from the National Natural Science Foundation of China (81172002) and research grants from State Key Laboratory of Reproductive Medicine, Nanjing Medical University to Luo Gu, the National Natural Science Foundation of China (81101999) to Yichao Zhu, the Natural Science Foundation of Jiangsu Province (BK2012893) and the Postdoctoral Science Foundation of China (2011M501254 and 2012T50484) to Jun Du, and a Project Funded by the Priority Academic Program

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