Restoration of Arpin suppresses aggressive phenotype of breast cancer cells
Introduction
Breast cancer is one of the most frequently diagnosed cancers in females worldwide [1]. Despite advances in therapeutic strategy, the prognosis of breast cancer patients, in particular those with advanced disease is still poor [2]. The phosphoinositide 3-kinase (PI3K)/Akt pathway is aberrantly activated in many types of cancers including breast cancer and represents a potential target for anticancer treatment [3]. The serine-threonine kinase Akt can modulate multiple downstream signaling nodes (e.g. GSK3, FoxO, mTORC1), thus affecting various cellular processes such as cell survival, proliferation, migration, and metabolism [4]. It was reported that activation of Akt signaling contributes to aggressive phenotype of breast cancer cells induced by very low-density lipoprotein (VLDL) and LDL [5]. However, the exact mechanisms governing breast cancer growth and progression are not completely understood.
Arpin is a negative regulator of the actin-related protein-2/3 (Arp2/3) complex, which mediates the assembly of actin filaments and regulates cell migration [6], [7]. Arpin contains a homologous acidic motif to nucleation-promoting factors (NPFs) and can compete with NPFs for binding to the Arp2/3 complex [8]. Arpin participates in steering cell migration by interfering with the Arp2/3 complex-mediated signaling [9]. There is evidence for a link between the Arp2/3 complex and Akt activation [10]. Arp2/3-dependent actin branching is involved in Akt plasma membrane translocation and activation [10].
It has been reported that Arpin is underexpressed and correlates with more aggressive parameters in gastric cancer [11]. Similarly, Arpin mRNA and protein expression is downregulated and predicts poor metastasis-free survival in breast cancer patients [12]. These studies encourage us to hypothesize that Arpin may serve as a repressor of breast cancer progression. To test this hypothesis, we restored the expression of Arpin and investigated its functional consequences on breast cancer cell proliferation, invasion, and tumorigenesis.
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Cell culture
Human breast cancer cells MCF-7, MDA-MB-231, Hs578T, and T-47D were purchased from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). Non-malignant MCF10A mammary epithelial cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FCS; Invitrogen, Carlsbad, CA, USA), penicillin (100 U/mL), and streptomycin (100 μg/mL) in a humidified incubator
Arpin impedes breast cancer cell proliferation and colony formation
Western blot analysis showed that Arpin expression levels were markedly lower in the breast cancer cells tested than in MCF-10A non-malignant breast epithelial cells (Fig. 1A). To determine the function of Arpin in breast cancer progression, we attempted to restore Arpin expression in breast cancer cells. Transfection with the Arpin-expressing plasmid resulted in a 5.1- and 3.8-fold increase in Arpin protein levels in MCF-7 and MDA-MB-231 cells, respectively (Fig. 1B). Compared to
Discussion
Clinical evidence demonstrates that Arpin expression is reduced in breast cancer tissues relative to matched paratumoural tissues and that reduced Aprin expression is significantly associated with aggressive parameters including advanced TNM stage and axillary lymph node metastasis [16]. Two previous studies consistently reported that Arpin downregulation has an independent adverse prognostic impact on the survival of breast cancer patients [12], [16]. In agreement with the clinical studies, we
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Both authors contributed equally to this work.