Role of Cyclin D1b in Inducing Macrophages Toward a Tumor-associated Macrophage-like Phenotype in Murine Breast Cancer

Objective:Tumor-associated macrophages(TAMs)of the M2 phenotype are frequently associated with cancer progression.Invasive cancer cells undergoing epithelial-mesenchymal transition(EMT)have a selective advantage as TAM activators.Cyclin D1b is a highly oncogenic splice variant of cyclin D1.We previously reported that cyclin D1b enhances the invasiveness of breast cancer cells by inducing EMT.However,the role of cyclin D1b in inducing macrophage differentiation toward tumor-associated macrophage-like cells remains unknown.This study aimed to explore the relationship between breast cancer cells overexpressing cyclin Dlb and TAMs.Methods:Mouse breast cancer 4T1 cells were transfected with cyclin D1b variant and co-cultured with macrophage cells in a Transwell coculture system.The expression of characteristic cytokines in differentiated macrophages was detected using qRT-PCR,ELISA and zymography assay.Tumor-associated macrophage distribution in a transplanted tumor was detected by immunofluorescence staining.The proliferation and migration ability of breast cancer cells was detected using the cell counting kit-8(CCK-8)assay,wound healing assay,Transwell invasion assay,and lung metastasis assay.Expression levels of mRNAs were detected by qRT-PCR.Protein expression levels were detected by Western blotting.The integrated analyses of The Cancer Genome Atlas(TCGA)datasets and bioinformatics methods were adopted to discover gene expression,gene coexpression,and overall survival in patients with breast cancer.Results:After co-culture with breast cancer cells overexpressing cyclin D1b,RAW264.7 macrophages were differentiated into an M2 phenotype.Moreover,differentiated M2-like macrophages promoted the proliferation and migration of breast cancer cells in turn.Notably,these macrophages facilitated the migration of breast cancer cells in vivo.Further investigations indicated that differentiated M2-like macrophages induced EMT of breast cancer cells accompanied with upregulation of TGF-β1 and integrinβ3 expression.Conclusion:Breast cancer cells transfected with cyclin D1b can induce the differentiation of macrophages into a tumor-associated macrophage-like phenotype,which promotes tumor metastasis in vitro and in vivo.

Cyclin Dlb Splice Variant Promotes αvβ3-mediated EMT Induced by LPS in Breast Cancer Cells

Epithelial-to-mesenchymal transition （EMT） plays a critical role in cancer metastasis, and is relevant to the inflammatory microenvironment. Lipopolysaccharide （LPS）, a cell wall constituent of gram-negative bacteria, has been reported to induce EMT of cancer cells through TLR4 signal. We previously reported that LPS promoted metastasis of mesenchymallike breast cancer cells with high expression of cyclin Dlb. However, the role of cyclin Dlb in LPS-induced EMT has not been fully elucidated. In the present study, we described that cyclin Dlb augmented EMT induced by LPS in MCF-7 breast cancer cells. Cyclin Dlb markedly amplified integrin αvβ3 expression, which was further up-regulated under LPS stimulation. Our results showed ectopic expression of cyclin Dlb promoted invasiveness of epithelial-like MCF-7 cells under LPS stimulation. Additionally, LPS-induced metastasis and EMT in MCF- 7-Dlb cells might depend on αvβ3 expression. Further exploration indicated that cyclin Dlb cooperated with HoxD3, a transcription factor promoting αvβ3 expression, to promote LPS- induced EMT. Knockout of HoxD3 repressed LPS-induced EMT and αvβ3 over-expression in MCF-7 cells with high expression of cyclin Dlb. Specifically, all these effects were in a cyclin Dla independent manner. Taken all together, LPS up-regulated integrin αvβ3 expression in MCF-7 cells with high expression of cyclin D lb and induced EMT in breast cancer cells, which highlights that cyclin Dlb may act as an endogenous pathway participating in exogenous signal inducing EMT in breast cancer cells.