IL-11 Is Essential In Promoting Osteolysis In Breast Cancer Bone Metastasis Via RANKL-independent Activation Of Osteoclastogenesis

Backgroud and aimsBreast cancer is one of the most common malignant cancers in women and a major cause of cancer death due to the development of secondary tumors in vital organs.While surgical therapies for primary tumors are generally effective,metastasis is often refractory.An estimating eighty percent of breast cancer patients develop bone metastasisas the disease advances.Bone metastases from breast cancer are typically osteolytic,involving the overactivation of osteoclasts that cause pathological bone resorption,with intense pain,bone fractures,nerve compression,and hypercalcemia.The development of osteolytic lesions is based on complex interactions between cancer cells and osteoclastsin a vicious cycle of bone destruction and tumor expansion.Breast cancer cells produce a variety of osteolytic factors leading to osteoclastic bone resorption,the subsequent release of bone matrix-embedded transforming growth factor beta(TGF-β)in turn promotes tumor growth.Two important regulating factors,receptor activator of nuclear factor κB ligand(RANKL)and macrophage-colony stimulating factor(M-CSF)are necessary for osteoclast differentiation and survival.Nevertheless,osteoclastogenesis can also be stimulated non-canonically by tumour necrosis family alpha(TNFα),TGF-β,interleukin-6(IL-6),lysyl oxidase(LOX),insulin-like growth factor-I(IGFI)and IGF-II.Breast cancer cell is known to produce numerous osteolytic factors including parathyroid hormone-related protein(PTHrP),IL-1,IL-6,IL-8,IL-11,vascular endothelial growth factor(VEGF),connective tissue growth factor(CTGF),matrix metallopeptidase 1(MMP1),hepatocyte growth factor,etc.Some of these factors activate osteoclastogenesis by increasing RANKL expression from osteoblast while some activate osteoclastogenesis synergistically with RANKL stimulation or in a RANKL independent way.In contrast to our knowledge of the abundant osteolytic factors,less is known about the initiation process prior to the overt vicious osteolytic circle.IL-11 is a member of the IL-6 family,and both cytokines share GP130 as the common signal transducer.Both IL-6 and IL-11 are osteolytic factors produced by breast cancer cells and their expression is associated with the development of bone metastases.Differently,IL-6 induces osteoclastogenesis dependent on osteoblastic cells while IL-11 induces osteoclast differentiation directly.It has been shown the breast cancer cell line overexpressing IL-11 increases tumor burden and osteolytic lesions in mouse bone metastasis model.In addition,human breast cancer tumors expressing IL-11 have higher ratesof bone metastasis occurrences.However,it still remains questionable if IL-11 induces osteoclast differentiation independent of RANKL or just enhances osteoclastogenesis synergistically with RANKL.Materials and methodsFor initial in vivo study,the survival time of the mice was counted after injecting three tumor cells with different invasiveness into the mouse femur;μCT and histological TRAP staining were performed to evaluate the degree of osteolysis at the bone metastasis femur.Meanwhile,preosteoclasts were cultured with conditional medium of three kinds of tumor cells.TRAP staining and FAK staining were performed to evaluate osteoclastogenesis.After the osteolysis related genes of three tumor cells were screened from the results of microarray profiling,we used monoclonal antibodies against VEGF,PTHrP,IL-11 and CTGF to block corresponding osteolytic factors in the CM of BoM-1833 cells in osteoclastogenesis assays.Next,western blotting was performed to detect JAK/STAT3 pathway,target gene c-Myc and specific marker of osteoclast differentiation,respectively.After that,AG-490 and adenovirus were used to inhibit the phosphorylation of STAT3 and the expression of c-Myc.TRAP staining and western blotting were used to further evaluate the activation effect of IL-11 on related pathways.Finally,AG-490 was used to inhibit STAT3 phosphorylation in vivo.Histological TRAP staining,immunohistochemistry andμCT were performed and mice survival days were counted to assess the degree of osteolysis at the femoral metastases and tumor growth.ResultsBoM-1833 with the strongest bone metastasis was selected from the three types of breast cancer cells at the initial stage of in vitro study.Meanwhile,in vivo study found that BoM-1833 had the strongest promoting effect on osteoclast differentiation.Microarray profiling revealed differentially expressed genes in MCF-7,MDA-MB-231 and BoM-1833;four osteolysis related genesIL-11,CTGF,VEGF and PTHrP were found to be the most highly expressed in BoM-1833.Interestingly,it was found that the promotion effect of BoM-1833 on osteoclast differentiation was significantly decreased after the expression ofIL-11 was inhibited,suggesting that IL-11 plays an essential role in the process of osteoclastogenesis promoted by BoM-1833 conditioned medium.Then,we identify IL-11 induces osteoclastogenesis independently of RANKL via JAK1/STAT3 activated c-myc pathway.Blocking of STAT3 phosphorylation in vivo effectively inhibits osteolysis and tumor growth of metastatic breast cancer.ConclusionThis study identified a new understanding of IL-11 in promotingosteoclastogenesis via RANKL-independent way and revealed the essential role and the underlying molecular mechanism of IL-11 in breast cancer bone metastasis mediated osteolysis.STAT3 targeting through AG-490 is a potential therapeutic strategy for mitigating osteolysis and tumor growth of bone metastatic breast cancer.