BMP signaling signature was upregulated in lewis lung carcinoma bone metastases and indicated poor survival in NSCLC
Lewis lung carcinoma originated from a spontaneous lung adenocarcinoma of a C57/BL6 mouse[28, 29]. We injected the lewis lung carcinoma cells (LLCs) into tail veins of C57/BL6 mice, resulting in lung metastases and bone metastases. RNA-seq was carried for bone metastasis tissues, lung metastasis tissues and parental lewis lung cells to analyze the transcriptome differences. The compiled significant differential expressing genes (DEGs) could be classified into five clusters C0, C1, C2, C3 and C4, indicating different expression patterns (Fig. 1A). The C0 module showed high expressing level in both metastasis tissues, meanwhile C1 and C2 showed higher expressing level in bone metastasis tissues than in lung metastasis tissues and parental cells, and the C3 and C4 module showed the unique highly expressed genes in lung metastasis tissues or parental cells (Fig. 1A). Thus, genes in C1 and C2 were more likely to contribute to lewis lung carcinoma bone metastasis. Consistently, ANGPT1 in C1, and MMPs and EDN1 in C2 had been reported to take part in prostate cancer bone metastasis [21, 34–36]. Significant KEGG pathways were enriched in C0, C2, and C4 modules, shown in Fig. 1B. Enriched KEGG pathways for different modules indicated that Jak-Stat signaling and PI3K-Akt might contribute to lung carcinoma metastasis while VEGF signaling, NF-κB signaling and HIF-1α signaling was enriched in parental lewis lung tissues (Fig. 1B). ECM-receptor interaction and amoebiasis associated gene were upregulated in the C2 module, indicating that cell deformation and invasion might be significant in the formation of bone metastasis lesions (Fig. 1B). Furthermore, we analyzed the homologous gene expression features of human based on TCGA data (NSCLC subtypes: LUSC and LUAD) [33] and found that high expression of bone metastasis associated genes identified in cluster C2 indicated poor survival in human NSCLC (Fig. 1C). What’s more, ANGPT1, MMPs and EDN1, the majorly upregulated genes in C2, were found as downstream targets of BMP signaling [18, 35, 36]. Thus, we came to the hypothesis that BMP signaling might contribute to lewis lung carcinoma bone metastasis. As TGF-β signaling was important in bone metastasis of various types of cancer, like prostate cancer and breast cancer[37], we also analyze the activation of TGF-β signaling in lewis lung carcinoma bone metastasis. To analyzed whether TGF-β or BMP signaling was activated in bone metastasis tissues of lewis lung carcinoma, average expression of TGF-β or BMP signaling targeted genes in LLCs were defined as TGF-β or BMP signaling signatures to characterize the activation of TGF-β or BMP signaling. Interestingly, we found that BMP signaling signature but not TGF-β signaling signature was higher in bone metastasis tissues than in lung metastasis tissues and parental lewis lung cells (Fig. 1D). Besides, up regulation of BMP signaling signature genes also indicated poor survival in human NSCLC (Fig. 1F). Moreover, We conformed that human and mice NSCLC cell lines were respond to BMP signaling in vitro (Fig. 1E). We pre-treated LLCs with vehicle or 20 ng/mL BMP2 for 24 h. After that, we injected the vehicle or 20 ng/mL BMP2 pre-treated LLCs into the tail veins of C57BL/6 mice. Furthermore, we find that lung and bone metastasis of BMP2 LLCs caused mice to start to die less than 20 days but mice injected with WT LLCs could survive for a long time (Fig. 1G). Taken together, activation of BMP signaling was found in lewis lung carcinoma bone metastases and indicated poor survival in NSCLC.
Expression of nuclear Smad1 indicated the activation of BMP signaling in bone metastasis tissues of lewis lung carcinoma.
We found that LLCs could colonize to both limbs and spines to form bone metastasis lesions (Fig. 2A). When BMP signaling is activated, Smad1/5/8 is phosphorylated and translocated into nucleus with Smad4[38, 39]. In our results, we found that Smad1 was expressed in the nucleus of lewis lung carcinoma cells at the invasive sites, where bone resorption and destruction occurred (Fig. 2B-C). Those results suggested that BMP signaling was activated in bone metastasis tissues of lewis lung carcinoma. CK18, as a kind of cytokeratins, frequently expressed in NSCLC[31–33, 40]. We stained lewis lung carcinoma cells with CK18 and Smad1 to further confirm that BMP signaling was activated in lewis lung carcinoma cells rather than other cell types of bone metastasis tissues. As shown in Fig. 2C, nuclear Smad1 staining was usually found in CK18+ LLCs in bone metastasis tissues, indicating that BMP signaling is activated in lewis lung carcinoma cells when bone metastases occurred.
The activation of BMP signaling was higher in bone metastasis tissues than in lung metastasis tissues of lewis lung carcinoma.
Smad1/5/8 phosphorylation and nuclear localization indicates the activation of BMP signaling[39, 41]. Although Smad1 was expressed both in lung metastasis tissues and bone metastasis tissues, the location of Smad1 within the two metastasis tissues was different (Fig. 3A-B). Smad1 majorly located in the nucleus at the invasive boundaries in bone metastasis tissues, while it diffused in both the cytoplasm and nucleus of carcinoma cells in lung metastasis tissues (Fig. 3A-B). Moreover, we found that, in comparison with parental LLCs and lung metastasis tissues, bone metastasis tissues expressed higher phosphorylated Smad1/5 (Fig. 3C). Therefore, the activation of BMP signaling was higher in bone metastasis tissues than in lung metastasis tissues of lewis lung carcinoma. We pre-treated LLCs with 20 ng/mL BMP2 for 24 h. After that, we injected the 20 ng/mL BMP2 pre-treated LLCs into the tail veins of C57BL/6 mice.What’s more, as shown in Fig. 3D, we found that BMP2 could induce more bone metastasis than lung metastasis in vivo. Our results indicated that BMP2 may probably play a more important roles in bone metastasis than in lung metastasis of NSCLC.
BMP2 signaling enhanced migration and invasion of NSCLC cells.
According to our transcriptome results,ECM-receptor interaction and amoebiasis associated genes were upregulated in bone metastasis tissues, indicating that cell deformation, migration and invasion might paly significant roles in the formation of bone metastasis lesions(Fig. 1B). Therefore, we examined whether BMP2 signaling could enhance migration and invasion of NSCLC cells via the transwell assay. We found that BMP2 could enhance the migration of NSCLC cells, LLC, NCIH-1373 and A549 in vitro (Fig. 4A-C). What’s more, the invasion ability of LLC and A549 was also promoted by BMP2 (Fig. 4D-E). We went further to investigated the roles of BMP signaling playing in the invasion of NSCLC cells in vivo. We pre-treated LLCs with vehicle or 20 ng/mL BMP2 for 24 h. After that, we injected the vehicle or 20 ng/mL BMP2 pre-treated LLCs into the hind leg muscles of C57BL/6 mice to analyze the invasion of lewis lung carcinoma in the hind legs. To retain the BMP2 signaling activation in the hind leg muscles, we repetitively injected 3 µg/kg vehicle or BMP2 into the hind leg muscles per week for three weeks. In the vehicle group, we found that the carcinoma and the muscles or bones grew separately, indicating that lewis lung carcinoma did not invade into muscles and bones (Fig. 4F). Differently, in the BMP2 group, the muscles had been destructed and the carcinoma had invaded into the bones, indicating that BMP2 could enhance the migration and invasion of lewis lung carcinoma cells in vivo (Fig. 4F). The activation of BMP2 signaling was confirmed by nuclear Smad1 staining and the levels of phosphorylated Smad1/5 (Fig. 4G-H).
BMP2 derived from the stroma fibroblasts enhanced migration and invasion of NSCLC cells.
Rajski et al. has reported that high BMP2 derived from stroma cells indicates poor outcome in lung adenocarcinoma[14]. Moreover, stroma derived BMP2 is important in osteogenesis[42]. Tumor stroma fibroblast cells secret diverse cytokines to enhance cancer progression[43, 44]. Thus, we came to the hypothesis that stroma fibroblasts derived BMP2 could enhance the migration and invasion of NSCLC cells to promote metastasis. We found that MEF (mice embryonic fibroblast) can secret much more BMP2 than LLC and pre-osteoblast cell MC3T3-E1(Fig. 5A). Moreover, the conditional media of MEF could promote the migration of LLC (Fig. 5B-C). Those findings suggested that MEF might enhance migration of LLCs via secreted BMP2. Furthermore, in consistent with the migration phenomena, the conditional media of MEF could enhance the invasion of LLCs (Fig. 5D). In the bone metastasis tissues of lewis lung carcinoma, we also found higher BMP2 expression in α-Sma+ activated fibroblasts than other cell types (Fig. 5E). The results above indicated that fibroblast cells might be an important source of BMP2. BMP2 originated from stroma fibroblasts contributed to the migration and invasion of lewis lung carcinoma cells.
BMP2 contributed to NSCLC bone metastases via both osteolytic and osteoblastic mechanisms
In osteolytic metastases, the destruction of bone is mediated by osteoclasts rather than tumor cells[34, 45]. BMP2 has also been reported to stimulate the activation of osteoclasts to regulate the balance of bone remodeling[36]. Osteoblasts and NSCLC cells in tumor microenvironment can cooperate with each other to induce osteoclasts differentiation from macrophages[45]. Thus, we continued to examine whether BMP2 could contribute to this process. We co-cultured the macrophage cell line RAW264.7 with LLC and MC3T3-E1 or A549 and MC3T3-E1 cells to induce its differentiation for 7 days. We found that without BMP2 stimulation, few macrophages could differentiate into TRAP+ osteoclast cells (Fig Sup 1A-B). However, BMP2 could strongly enhance the differentiation of macrophages to osteoclasts, indicating that BMP2 signaling contributed to osteolytic metastasis (Fig Sup 1A-B). The bi-directional interactions between tumor cells and osteoclasts lead to both osteolysis and tumor growth[34, 45].
However, in bone metastasis tissues of lewis lung carcinoma, we could find the immature bone tissues, indicating that osteoblastic mechanisms might also play roles in bone metastases of lewis lung carcinoma (Fig. 6A). Thanks to the strong osteogenesis inducing ability of BMP2, we co-cultured LLCs and pre-osteoblasts MC3T3-E1 with BMP2 to mimic the neoplastic osteogenesis in vivo. Interestingly, LLCs could secret microvesicles, which subsequently attached to MC3T3-E1 cells under BMP2 treatment (Fig. 6B). Moreover, we cultured LLCs in the Corning cell culture insert with 3.0 µm polycarbonate membranes and found that the microvesichles could pass through the 3.0 µm polycarbonate and attached to the MC3T3-E1 cells below, indicating that the diameter of the microvesichles was less than 3.0 µm (Fig. 6B). Furthermore, we examined the osteoblast differentiation of MC3T3-E1 cells. We found that BMP2 could induce ALP+ differentiated MC3T3-E1 cells, while microvesicles from LLCs could further enhance this process. Our results indicated that microvesicles from LLCs induced by BMP2 could enhance the early osteoblasts differentiation of MC3T3-E1 (Fig. 6C-D). In our study, BMP2 was found to co-localize with the microvesicles from LLCs, indicating that LLCs could collect BMP2 from microenvironment into the microvesicles, further enriching BMP2 to MC3T3-E1 cells (Fig. 6E). Then we examined the proliferation and apoptosis of MC3T3-E1 cells. We found that LLCs could enhance the expression of the proliferation marker, Ki67, in MC3T3-E1 cells and attenuate MC3T3-E1 apoptosis in the absence of BMP2, which might be another mechanism supporting neoplastic osteogenesis (Fig Sup 2A-B). However, the formation of calcium nodes, which indicated the mature osteocytes, was inhibited by LLCs (Fig. 6F-G). It has been reported that cancer cells rely on direct calcium influx from osteogenic cells to form bone metastasis lesions [46], indicating that calcium tended to participate in signaling transduction but not inducing osteogenesis in bone metastasis tissues. Those results above show how NSCLC cells, pre-osteoblasts and BMP2 interact with each other in the formation of immature bone tissues in bone metastases.