br v and both RGD binding
αvβ3 and α5β1, both RGD-binding integrins, were detected in breast cancer 17650-98-5  (Fig. 1B). To assess their aﬃnity for IL32, sa-turation binding assays were conducted. As illustrated in Figure S2C, the αvβ3 integrin has stronger aﬃnity for IL32 than α5β1 does. Fur-thermore, a loss of integrin β3 notably abrogated rIL32-stimulated cell invasion, suggesting that integrin β3 is essential for IL32-mediated cross-talk between CAFs and tumour cells (Fig S2D).
3.4. The interaction between IL32 and integrin β3 activates p38 MAPK signalling in breast cancer cells r> p38 MAPK, PI3K–AKT, and JAK–STAT cascades have been reported to be the canonical downstream pathways of integrin β3 in various cells [19,21,22]. To elucidate which pathway(s) is/are stimulated by the IL32–integrin β3 axis in breast cancer cells, activation of relevant po-tential signalling molecules was determined by western blotting ana-lysis. During co-culture with CAFs but not NFs, the p38 MAPK pathway, but not PI3K–AKT or JAK–STAT pathway, was activated in BT549 and Hs578T cells (Fig. 5A). Moreover, treatment of BT549 cells with rIL32 raised the phosphorylated-p38 levels in a dose-dependent manner (Fig. 5B). rIL32 (20 ng/ml) stimulated phosphorylation of p38, which
was obviously decreased in integrin β3 knockdown breast cancer BT549 and Hs578T cells (Fig. 5C). In addition, the CAF CM neutralised with an anti-IL32 antibody remarkably attenuated the phosphorylation of the p38 protein in BT549 and Hs578T cells (Fig. 5D). Collectively, these data suggested that the p38 MAPK pathway is downstream of the IL32–integrin β3 axis in BT549 and Hs578T breast cancer cells.
3.5. The IL32–integrin β3–p38 MAPK signalling axis promotes breast cancer cell invasion and EMT
To understand the biological significance of the IL32–integrin β3–p38 MAPK axis, we evaluated the EMT biomarker expression levels during breast cancer cell invasion. Compared with the CM from NFs, CAFs’ CM significantly increased the expression of mesenchymal bio-markers – fibronectin, N-cadherin, and vimentin – in BT549 and Hs578T cells (Fig. 6A). Adding rIL32 into the CM of NFs notably up-regulated mesenchymal proteins in BT549 and Hs578T cells (Fig. 6B). Nonetheless, CAF CM neutralised with the anti–IL-32 antibody blunted the expression of mesenchymal biomarkers (Fig. 6C). Similarly, after the knockdown of integrin β3 in BT549 and Hs578T cells, CAF CM could not stimulate fibronectin, N-cadherin, and vimentin expression, in contrast to the control tumour cells (Fig. 6D). In line with these findings, CM from IL32-deficient CAFs did not promote EMT marker expression (Fig. 6D). Furthermore, SB203580 (a p38 MAPK inhibitor) abrogated the rIL32-stimulated upregulation of mesenchymal markers (Fig. 6E). In agreement with these findings, CAF CM neutralised with the anti–IL-32 antibody significantly decreased invasion abilities of BT549 and Hs578T cells (Fig. 6F). Besides, adding rIL32 to CM from NFs notably increased BT549 and Hs578T cell invasion (Fig. 6G). SB203580 apparently abrogated the rIL32-stimulated invasive abilities of BT549 and Hs578 cells (Fig. 6H). These data indicated that the IL32–integrin β3–p38 MAPK axis facilitates breast cancer cell invasion and EMT.
To determine whether IL32 aﬀects integrin β3 expression, mRNA and protein expression levels of integrin β3 in BT549 cells were mea-sured during rIL32 treatment. Indeed, rIL32 up-regulated mRNA and protein expression of integrin β3 (Fig S3A–S3B). TNF-α, MIP2, and IL8 are the main pro-inflammatory cytokines induced by IL32 ; how-ever, only IL8 (not TNF-α or MIP2) stimulated integrin β3 mRNA and protein expression (Fig S3C and S3D) and BT549 cell invasiveness (Fig S3E). These data indicated that IL32 per se and its target cytokine IL8 also promoted breast cancer cell invasion by up-regulating integrin β3.
3.6. IL32 enhances metastasis of breast cancers in vivo
To evaluate the pro-metastatic eﬀect of the IL32-integrin β3-p38 MAPK signalling axis in vivo, BT549 cells (clones BT549-shNC and BT549-shβ3 (shRNA-2)) mixed with NFs or CAFs (clones CAF-shNC or CAF-shIL32) were subcutaneously injected into nude mice. The mice injected with the mixture of BT549 and CAFs developed the largest tumour; NFs slowed the tumour growth at an early stage but promoted the tumour growth at a late stage. The knockdown of IL32 in CAFs slightly enhanced the tumour growth; the knockdown of integrin β3 in cancer cells decreased the tumour growth (Fig. 7A and B). Ki67 staining revealed similar results in tumour tissue samples (Fig S4A). In line with
Fig. 5. IL32 activates the p38 MAPK pathway by inter-acting with integrin β3. (A) Western blotting to detect the activation of p38 MAPK, PI3K–AKT, and JAK–STAT signal-ling pathways in BT549 and Hs578T with integrin β3 and shNC or parental cells in the presence of the CM from CAFs or NFs. (B) The dose eﬀects of rIL32 (0–40 ng/ml) on activation of p38 MAPK signalling in BT549 cells according to western blotting. (C) rIL32 (20 ng/ml) was added to cultured BT549 and Hs578T cells transfected with shβ3 and to control cells. Activation of p38 MAPK signalling was detected by western blotting. (D) CM from CAFs neutralised with the specific antibody against IL32 (10 μg/ml) was used to culture BT549 and Hs578T cells for 3 h. The activation of p38 MAPK sig-nalling was determined by western blotting. β-Actin served as a loading control in all the western blot analyses. (Legend. P: parental cells; NC: control shRNA; shβ3: shRNA against integrin β3).