br Integrins are bidirectional signalling receptors and can
Integrins are bidirectional signalling receptors  and can be activated by proteins binding to their extracellular domain (called outside-in signalling) or to their intracellular domain (named inside-out signal-ling); the signals are transmitted through tilting or pistoning move-ments of the transmembrane domain [10,11]. Integrin β3 in particular is a member of the RGD motif receptor family and plays a crucial pro-metastasis role in lung cancer, breast cancer, colorectal cancer, ST-2825 cancer, melanoma, and glioblastoma [12–17]. Integrin β3 promotes cancer metastasis in part by phosphorylating a series of downstream proteins including Src, FAK, and Syk; this action enhances cell migra-tion and invasion [18,19]. Some microRNAs (e.g. miR-let-7c and miR-30a-5p) can suppress metastasis by down-regulating integrin β3 [14,20]. In addition, a number of studies have revealed that actual participation of integrin β3 in metastasis depends on a series of downstream signalling activation events, aﬀecting for example PI3-K–AKT, NF-κB, MAPK, and STAT1 pathways [19,21,22]. Nevertheless, all the aforementioned cascades belong to ‘inside-out’ transduction. r> ∗ Corresponding author. Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University. #1 Yi-Xue-Yuan Rd., Yu-zhong District, Chongqing, 400016, Chongqing, China.
E-mail address: [email protected] (M. Liu).
Whether a component of a tumour microenvironment, especially CAFs, can facilitate breast cancer invasion and metastasis through integrin β3 in an ‘outside-in’ manner remains unclear.
Interleukin 32 (IL32), also known as NK4, contains an RGD cell attachment sequence and is a member of the cytokine family that can induce pro-inflammatory cytokines such as TNF-α, IL8, and MIP2 . Expression of IL32 has been found in a variety of tissues, including lungs, the liver, colon, and pancreas [24–27]. In contrast to other in-flammatory factors that are expressed predominantly in immune cells, IL32 is expressed in epithelial cells and fibroblasts [28,29]. Although some research has revealed participation of IL32 in immune diseases (e.g. rheumatoid arthritis), infectious diseases (e.g. hepatitis B), in-flammatory reactions, and angiogenesis [30–33], whether IL32 is in-volved in biological behaviours of a tumour, particularly the aggres-siveness mediated by the tumour microenvironment, is largely unknown.
In this study, we demonstrate that CAFs (in contrast to NFs) can promote tumour invasion of integrin β3–positive breast cancer cells. The protein IL32 derived from CAFs binds to integrin β3 at the surface of breast cancer cells, thereby activating the downstream p38 MAPK pathway to enhance fibronectin, N-cadherin, and vimentin expression and strengthens breast cancer cell invasion and metastasis. Therefore, our data shed light on the mechanism underlying the cross-talk of stromal cells with tumour cells and oﬀer a new strategy for the search for stromal therapeutic targets.
2. Materials and methods
2.1. Tissue samples and isolation of primary fibroblasts
Human breast tumour tissues and their corresponding normal breast tissues (at least 5 cm away from a tumour) were obtained from patients with breast tumours resected at the First Aﬃliated Hospital of Chongqing Medical University. All the patients who were involved in this study consented to participate in the study and to publication of its results, did not receive any radiotherapy or chemotherapy previously. The experiments were approved by the Ethics Committee of Chongqing Medical University.
Fibroblasts were isolated as previously described . In brief, tu-mours and non-tumour tissues were minced and digested in type I collagenase (Sigma, USA), then cultured in DMEM with 10% of foetal bovine serum (FBS; GIBCO, USA) in a humidified atmosphere con-taining 5% of CO2 at 37 °C until the fibroblasts got attached to the culture dish. The primary fibroblasts were used before passage 6.
2.2. Cell culture, plasmids, and reagents
Human mammary epithelial cell line MCF10A, breast cancer cells (including cell lines SKBr3, T47D, MDA-MB-453, MCF7, BT474, MDA-MB-468, BT549, and Hs578T), and HEK293T embryonic kidney cells were acquired from the American Type Culture Collection (ATCC). The cells were cultured according to standard protocols. Paired im-mortalised CAFs and NFs have been described elsewhere . Cell lines MCF10A/Twist, MCF10A/Vector, BT549-shCtrl, and BT549-shTwist were established as described previously . The human integrin β3-, integrin β4-, integrin β1-encoding vectors were obtained from Addgene (www.addgene.org) or Origene (www. origene.com.cn). For all the short hairpin RNA (shRNA) oligonucleo-tides, synthetic DNA inserts were cloned into the pGLVH1/GFP vector (GenePharma, China). The target sequences of integrin β3 shRNAs were shRNA-1: 5'-GAATTGTACCTATAAGAAT-3', and shRNA-2: 5'-GAAAAT CCGTTCTAAAGTA-3', the target sequences of IL-32 shRNA were 5′-CTCCTCTACTTGAAAAAGA-3′. The control shRNA, which did not match any known human cDNA, was 5′-TTCTCCGAACGTGTCACGT-3′. Human recombined IL32 (rIL32), TNF-α, MIP2, and IL8 were the pro-ducts of R&D systems (Minnesota, USA); and mutant recombinant IL32