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  • br tumour growth IL treatment or integrin silencing in BT

    2020-08-28


    tumour growth, IL32 treatment or integrin β3 silencing in BT549 4-Aminophenyl Phosphate slightly slowed down the proliferation of BT549 cells in contrast with control BT549 cells (Fig S4B).
    As expected, the mice injected with BT549 cells mixed with CAFs had more metastatic nodules in the lungs than the other mice did. The loss of IL32 in CAFs or silencing of integrin β3 in cancer cells reduced the metastases in mouse lungs. Compared with the mice injected with BT549 alone, exogenous addition of IL32 remarkably promoted cancer cell metastasis; however, the loss of integrin β3 in cancer cells effi-ciently attenuated the IL32-stimulated metastasis in mouse lungs (Fig. 7C). Consistently with tumour metastasis, the phosphorylated-p38 levels were much higher in the tumour of mice injected with the mix-ture of BT549 cells and CAFs than in tumours of other groups of mice. The knockdown of IL32 in CAFs or silencing of integrin β3 in cancer cells blunted the phosphorylation of p38; IL32 treatment of the tumour-bearing mice injected with BT549 cells alone stimulated p38 signalling in the tumour; however, silencing of integrin β3 in cancer cells almost eliminated the response to IL32 stimulation in tumours (Fig. 7D). The status of p38 signalling in tumour tissues was next confirmed by wes-tern blotting (Fig. 7E). These data suggested that IL32 secreted from CAFs contributed to breast cancer metastasis in vivo. Thus, our data provided evidence that CAFs secrete IL32, which binds to integrin β3 at the tumour cell membrane and activates p38 MAPK signalling to pro-mote EMT and invasiveness of breast cancer cells (Fig. 7F).
    4. Discussion
    The tumour microenvironment is well known to facilitate tumour progression. The coordination between CAFs – as the major type of the stromal cells in a tumour microenvironment – and cancer cells is pivotal for tumour initiation, invasion, metastasis, and drug resistance [47]. Nonetheless, the underlying mechanisms are not well understood. Our previous study has shown that a set of integrins is dysregulated during EMT [39]. In the current work, we found that CAFs (in contrast to NFs) 
    can promote invasion of integrin β3–positive breast cancer cells. IL32, an RDG domain–containing secreted protein, is highly expressed in CAFs and serves as a specific ligand of integrin β3. Of note, IL32 binding to integrin β3 at the tumour cell membrane in vitro and in vivo activates downstream p38 MAPK signalling to up-regulate EMT markers and enhance cancer cell invasion. These biological phenotypes and functions are blunted after either IL32 is suppressed in CAFs or integrin β3 is knocked down in cancer cells. Our findings suggest that the in-teraction of CAF-derived IL32 and integrin β3 at the tumour cell membrane mediates the cross-talk between CAFs and breast tumour cells thereby promoting breast cancer invasion.
    The aberrant expression of integrin β3 is seen in some invasive and/ or metastatic carcinomas such as glioblastoma, melanoma, colorectal cancer, and lung cancer [12,14,16,17]. Integrin β3 recognises ligands containing the RGD motif, a tripeptide which endows the ligand with an ability to interact with specific integrins. In accordance with the sy-nergistic domains that confer integrin specificity and conformation of the RGD motif, the RGD-recognizing integrins have been described to bind to other ECM ligands with various degrees of affinity [46]. Some studies indicate that cytokines such as VEGF and tenascin C (TN-c) have a synergistic effect with integrin β3 in terms of promotion of breast cancer metastasis [21,48]. IL8 and TGF-β can induce cell invasion by up-regulating integrin β3 in lung cancer [49,50] although the me-chanisms have yet to be elucidated. Our work reveals that integrin β3 acts as a receptor directly interacting with IL32 derived from CAFs to enhance breast cancer cell invasion.
    Interleukins belong to a large family of proteins, which was firstly described as originating from leukocytes, and often act as pro-in-flammatory factors regulating an immune response and angiogenesis [32,33]. Besides, some interleukins such as IL8 and IL6 can promote cancer progression as well [51]. IL32, a newly identified signalling protein, plays a crucial role in autoimmune disease responses (e.g. al-lergic rhinitis and rheumatoid arthritis). It has been reported that IL32 can suppress proliferation of vascular smooth muscle cells and
    Fig. 6. IL32 promotes breast cancer cell invasion by stimulating p38 MAPK and EMT. (A) A western blotting assay to quantify the EMT markers in BT549 and Hs578T cells co-cultured with CM from CAFs or NFs. (B) CM from NFs with rIL32 (20 ng/ml) was employed to culture BT549 and Hs578T cells for 24 h, the expression of EMT markers was detected by western blotting. (C) CM from CAFs neutralised with the specific antibody against IL32 (10 μg/ml) was used to culture BT549 and Hs578T cells for 24 h, and western blotting was performed to determine the expression levels of EMT markers. (D) BT549/sh-β3 (shRNA-2) cells (left panel) and Hs578T/sh-β3 (shRNA-2) cells (right panel) were co-cultured with CM derived from NFs, CAFs, or CAFs/sh-IL32, and a western blotting assay was conducted to quantify the EMT markers. (E) The BT549 and Hs578T cells were treated with rIL32 together with or without SB203580 (p38 MAPK inhibitor, 20 μM) for 24 h. Western blotting was performed to determine the levels of EMT markers. (F) Cell culture conditions were similar to those in (C), and the invasive ability was measured by a Transwell assay (*P < 0.05, **P < 0.01). (G) Cell culture conditions were similar to those in (B). Invasive abilities of BT549 and Hs578T cells were determined by a Transwell assay (**P < 0.01). (H) BT549 and Hs578T cells were treated the same as in (E), and the Transwell assay was carried out to evaluate the invasive potentials of tumour cells (**P < 0.01). Legend. FN: fibronectin, N-cad: N-cadherin, Vim: vimentin. An isotype-matched non-specific IgG served as an anti-IL32 antibody control. PBS or DMSO was used as a control of group rIL32 and group SB203580, respectively. β-Actin is the loading control in all the western blotting analyses.