Adipocyte-derived IL-6 and leptin promote breast Cancer metastasis via upregulation of Lysyl Hydroxylase-2 expression
Adipocytes make up the major component of breast tissue, accounting for 90% of stromal tissue. Thus, the crosstalk between adipocytes and breast cancer cells may play a critical role in cancer progression. Adipocyte-breast cancer interactions have been considered important for the promotion of breast cancer metastasis. However, the specific mechanisms underlying these interactions are unclear. In this study, we investigated the mechanisms of adipocyte-mediated breast cancer metastasis.
Breast cancer cells were cocultured with mature adipocytes for migration and 3D matrix invasion assays. Next, lentivirus-mediated loss-of-function experiments were used to explore the function of lysyl hydroxylase (PLOD2) in breast cancer migration and adipocyte-dependent migration of breast cancer cells. The role of PLOD2 in breast cancer metastasis was further confirmed using orthotopic mammary fat pad xenografts in vivo. Clinical samples were used to confirm that PLOD2 expression is increased in tumor tissue and is associated with poor prognosis of breast cancer patients. Cells were treated with cytokines and pharmacological inhibitors in order to verify which adipokines were responsible for activation of PLOD2 expression and which signaling pathways were activated in vitro.
Gene expression profiling and Western blotting analyses revealed that PLOD2 was upregulated in breast cancer cells following coculture with adipocytes; this process was accompanied by enhanced breast cancer cell migration and invasion. Loss-of-function studies indicated that PLOD2 knockdown suppressed cell migration and disrupted the formation of actin stress fibers in breast cancer cells and abrogated the migration induced by following coculture with adipocytes. Moreover, experiments performed in orthotopic mammary fat pad xenografts showed that PLOD2 knockdown could reduce metastasis to the lung and liver. Further, high PLOD2 expression correlated with poor prognosis of breast cancer patients. Mechanistically, adipocyte-derived interleukin-6 (IL-6) and leptin may facilitate PLOD2 upregulation in breast cancer cells and promote breast cancer metastasis in tail vein metastasis assays. Further investigation revealed that adipocyte-derived IL-6 and leptin promoted PLOD2 expression through activation of the JAK/STAT3 and PI3K/AKT signaling pathways.
Our study reveals that adipocyte-derived IL-6 and leptin promote PLOD2 expression by activating the JAK/STAT3 and PI3K/AKT signaling pathways, thus promoting breast cancer metastasis.
KeywordsAdipocytes PLOD2 IL-6 Leptin Breast Cancer Metastasis
Cancer associated fibroblasts
Insulin-like growth factor 1
Monocyte chemotactic protein-1
Prolyl 4-hydroxylase subunit alpha-1
Plasminogen activator inhibitor 1
Lysyl hydroxylase 2
Quantitative real time PCR
Tissue inhibitor of metalloproteinase 1
Tumor necrosis factor
The tumor microenvironment plays a vital role in the initiation and progression of many cancers . Adipocytes comprise approximately 90% of breast tissue. In breast cancer, the crosstalk between cancer cells and “cancer associated adipocytes” (CAAs) promotes breast cancer progression and metastasis [2, 3]. Recent research suggests that adipocytes can act as an endocrine organ, secreting several signaling molecules, such as chemokines and adipokines. Adipocyte-derived factors, such as leptin, interleukin-6 (IL-6), adiponectin, tumor necrosis factor (TNF-α), monocyte chemotactic protein-1 (MCP-1) and endotrophin (ETP), function within the tumor microenvironment to promote tumor progression [4, 5, 6]. These adipokines activate several signaling networks associated with migration, proliferation, angiogenesis, fibrosis and apoptosis, including the JAK/STAT, AKT and ERK1/2 signaling pathways, which are frequently activated in tumor tissues [7, 8].
Lysyl hydroxylases are encoded by distinct procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD) genes. These enzymes trigger the hydroxylation of collagen lysine residues prior to the formation of triple helical pro-collagen molecules [9, 10]. Over the last several years, PLOD2 deregulation has been observed in sarcoma, breast cancer, glioma, lung cancer and cervical cancer [10, 11, 12, 13, 14]. Additional studies have indicated that HIF-1α enhances expression of PLOD2, which in turn promotes sarcoma metastasis. Breast and lung cancers with high PLOD2 expression display enhanced rates of migration and metastasis. Knockdown of PLOD2 significantly inhibits cancer cell migration and invasion [15, 16]. Previous studies have demonstrated that the PI3K/AKT signaling pathway is involved in the regulation of PLOD2 expression . In addition, it has been shown that adipocyte-derived adipokines act as profibrogenic molecules [18, 19, 20]. Additional research has reported that paracrine signals from cancer-associated fibroblasts enhance PLOD2 expression in lung cancer . Therefore, these studies indicate the potential existence of an intimate crosstalk between adipokines and PLOD2.
However, little is known about how adipocyte-derived factors affect the expression of PLOD2. In this study, we investigate the function of PLOD2 in breast cancer and explore the underlying adipocyte-regulated mechanisms responsible for promoting PLOD2 expression. We used a coculture system to identify which adipokines are involved in PLOD2 expression. Through qRT-PCR analyses and RNAi knockdown experiments, we identified lysyl hydroxylase (PLOD2) as a new player in adipocyte-mediated migration. Furthermore, breast cancer cells cocultured with adipocytes or treated with either IL-6 or leptin displayed increased PLOD2 expression and activated JAK/STAT3 and AKT signaling pathways. Adipocyte-stimulated migration of breast cancer cells toward the lungs was impeded by treatment with a murine IL-6 blocking antibody or depletion of OBR. Inhibition of STAT3 and AKT activity using the pharmacological inhibitors ruxolitinib and LY294002, respectively, decreased PLOD2 expression. Taken together, these findings provide new insights into the connections between adipokines and PLOD2 in breast cancer and support a crucial role for PLOD2 in human breast cancer metastasis. Furthermore, excessive accumulation of fat may contribute to the higher expression of PLOD2 and correlation with breast cancer patient poor prognosis.
The MDA-MB-231 and MDA-MB-468 cell lines were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Medium (DMEM, 12100046, Thermo Fisher Scientific, Waltham, MA, USA). The culture medium contained 10% fetal bovine serum (FBS), penicillin (50 U/mL), and streptomycin (50 U/mL). MDA-MB-231 and MDA-MB-468 were used within 6 months of passaging after purchase. 3 T3-L1 preadipocyte cells, SK-BR-3 human breast cancer cells and 293 T embryonic kidney cells were purchased from The Shanghai Institute of Life Science, Chinese Academy of Science. 3 T3-L1 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal calf serum (FCS), penicillin (50 U/mL), and streptomycin (50 U/mL). SK-BR-3 and 293 T cells were cultured in DMEM/F12 with 10% FBS, penicillin (50 U/mL) and streptomycin (50 U/mL). All cells were maintained in a humidified 5% CO2 atmosphere at 37 °C.
The 3 T3-L1 preadipocyte cell line was differentiated as previously described . At 90% confluency, 3 T3-L1 cells were detached with trypsin-EDTA (SunShine Biotechnology) and seeded in 6-well plates (WHB). After reaching confluency, differentiation was induced by incubating cells in DMEM containing 10% FBS, 0.5 mM IBMX (Sigma), 10 μg/mL insulin (Sigma) and 1 μM dexamethasone (Sigma) for a total of 6 days, with a media change after 3 days. Then, the medium was replaced with an adipocyte-maintaining medium comprised of DMEM enriched with 10% FBS and 10 μg/mL insulin for a total of 6 days, with a media change after 3 days. Oil Red O staining was performed as described previously . Human preadipocyte cells were purchased from ScienCell Research Laboratory and were cultured and differentiated identically to the 3 T3-L1 preadipocytes.
Coculture and migration assays
After 12 days of adipocyte induction, the cells were cocultured with MDA-MB-231 and MDA-MB-468 human breast cancer cells using a Transwell culture system (0.4 μm pore size, Corning). 3.0 × 105 MDA-MB-231 and MDA-MB-468 cells were seeded in the top chamber. Breast cancer cells were cocultured with adipocytes for 3 h, 6 h, 12 h, 24 h, 48 h and 72 h. MDA-MB-231 and MDA-MB-468 cells were cultured alone as controls and were evaluated at the same time points. To evaluate the effects of adipocyte coculture on breast cancer tumor cell migration, after 3 days of coculture with MDA-MB-231 and MDA-MB-468 cells, tumor cells were used to conduct migration assays using 24-well Boyden chambers containing inserts (8 μm, pores, BD Biosciences, USA). 5 × 104 cells were cultured in the upper wells of Transwell chambers and allowed to migrate toward 10% FBS in the bottom wells. Similar migration assays were conducted with monocultured tumor cells. After 12 h, migrated cells were stained with Diff-Stain Set (Jiancheng), photographed, and counted using ImageJ software.
Spheroid invasion assays in Matrigel matrix
The hanging-drop method has been previously used to generate spheroids [22, 23]. MDA-MB-231 and MDA-MB-468 breast cancer cells were detached following 3 days of either monoculture or coculture with adipocytes. Drops of 1000 tumor cells each were formed using 20 μL medium supplemented with methylcellulose (20%). Cells were incubated as droplets (25 μL) for 48 h to ensure multicellular aggregation. For the spheroid invasion assays, hybrid aggregates were implanted into Matrigel and cultured for 7 days, after which time tumor cell invasion was apparent.
Colony formation assays
Six-well plates were seeded at 1000 cells/well and then cells were cultured in complete medium for almost 20 days. Cells were fixed with 4% formaldehyde and stained with 0.5% crystal violet. Colonies were counted using Image-Pro Plus 6.0.
Recombinant human IL-6 and leptin proteins
MDA-MB-231 and MDA-MB-468 cells were treated with human recombinant IL-6 protein (5 ng/mL, R&D Systems) and human recombinant leptin protein (50 ng/mL, GeneScript).
Immunofluorescence and immunohistochemistry
Tumor cells were seeded on coverslips and either monocultured or cocultured for 3 days with adipocytes or treated with IL-6 and leptin. Cells were fixed in 4% paraformaldehyde for 30 min at room temperature and then permeabilized with 0.3% Triton X-100 in 3% BSA for 15 min at room temperature . Then, 3% bovine serum albumin in PBS was used to block cells for 1 h. Next, cells were incubated with a primary antibody against type I collagen or P-STAT3 overnight at 4 °C. Cells were incubated with the appropriate secondary antibody and Hoechst stain for 1 h at room temperature, with extensive washing between every step. Images were captured using a confocal microscope.
Paraffin sections were deparaffinized according to a standard protocol. Antigen retrieval was performed in citrate buffer for 2 min at 100 °C. Sections were blocked with 5% BSA and then incubated with a PLOD2 antibody overnight at 4 °C. Sections were then incubated with a biotin-labeled rabbit anti-goat antibody for 30 min at room temperature. Sections were visualized with DAB and counterstained with hematoxylin.
RNA extraction and quantitative real time PCR
Total cellular RNA was isolated with TRIzol® Reagent (Vazyme) and reverse transcribed with the RevertAid™ First Strand cDNA Synthesis Kit (Takara). qRT-PCR assays were performed to analyze relative mRNA levels using a SYBR Green-based system (Applied Biosystems). The amount of mRNA for each gene was normalized to the internal control (18S or GAPDH). The sequences of the primers used in this study are provided in Additional file 1: Table S1.
Dot hybridization for the detection of IL-6 and leptin secretion by adipocytes following coculture with breast cancer cells
Conditioned medium was collected from 3 T3-L1 cells, adipocytes and adipocytes cocultured with breast cancer cells. 20 μL of conditioned medium was added to a PVDF membrane containing 1 μg of protein. The membrane was allowed to dry and was then blocked with 5% bovine serum albumin for 1 h at room temperature. Next, the membrane was incubated with a primary antibody against IL-6 or leptin overnight at 4 °C. Then, the membrane was incubated with the appropriate secondary antibody for 1 h at room temperature, with extensive washing between every step. Membranes were analyzed using an Enhanced Chemiluminescence (ECL) detection system.
Cells were lysed in cell lysis buffer to extract total proteins. Proteins were then separated by SDS-PAGE and transferred to a PVDF membrane. Membranes were analyzed using an Enhanced Chemiluminescence (ECL) detection system. The antibodies used in this study are presented in Additional file 1: Table S2.
Transfection and generation of stable cells
shRNAs targeting human PLOD2 (SHC [V2LHS_131378], SHD [V3LHS_306074]) as well as a scrambled shRNA were received from the shRNA and ORFeome Core at the MD Anderson Cancer Center. Lentiviruses were generated with either shRNA or cDNA that had been packaged in 293 T cells using pMD2.G and psPAX2 plasmids. 48 h later, conditioned medium was collected from 293 T cells and used to infect target cells. Stable cell lines were generated by selection with puromycin (10 μg/mL).
Transient transfection with siRNAs
Small-interfering RNAs (siRNAs) against OBR were synthesized by GenePharma (Shanghai, China). The sequences of the OBR and negative control siRNAs are presented in Additional file 1: Table S3. Prior to transfection, MDA-MB-231 and MDA-MB-468 breast cancer cells were plated in 6-well plates at 50% confluency. OBR and negative control siRNAs were transfected into cells using Lipofectamine 2000 Reagent (Life Technologies, USA) according to the manufacturer’s protocol. Cells were collected after 48–72 h for qRT-PCR, Western blotting or coculture experiments.
Orthotopic xenograft tumor model
Five to six week old female NOD SCID mice (MARC, Nanjing University, China) were randomly divided into groups (six mice per group). All animal procedures were approved by the Experimental Animal Care Commission of China Pharmaceutical University. Mice were anesthetized by isoflurane inhalation, then MDA-MB-231-SCR and MDA-MB-231-SHC human breast cancer cells (2 × 106 cells per mouse) were orthotopically injected into the inguinal mammary fat pad. Each injection contained 50 μL cell suspension, including 25 μL Matrigel (Corning). Tumor volume was measured using calipers and calculated as V = (L × W2)/2. After 9 weeks, tumor metastasis was assessed by bioluminescent imaging on the Xenogen In Vivo Imaging System (IVIS, Caliper Life Science, Hopkinton, MA). Mice were then sacrificed and lungs and livers were formalin-fixed and paraffin-embedded for hematoxylin and eosin staining. Liver metastases were quantified in five random low power fields per group and are presented as the mean ± s.d.
Tail vein metastasis assays
MDA-MB-231 cells were monocultured, cocultured with adipocytes, cocultured with adipocytes in the presence of a murine IL-6 blocking antibody, or cocultured with adipocytes following OBR depletion. After 3 days, these cells were harvested, centrifuged, and gently resuspended in Cell Tracker™ blue (CMTPX) (Thermo Fisher Scientific, C2925). This reagent was prewarmed (at room temperature) and diluted to a final concentration of 10 μM according to the manufacturer’s instructions. The cells were then incubated at 37 °C for 30 min, centrifuged again and resuspended in serum free media to a concentration of 2.5 × 105 cells per 100 μL. A total of 5 × 105 cells per 200 μL were injected into the tail vein of each NOD SCID mouse. After 2 weeks, mice were sacrificed and lungs were isolated and sectioned for histological studies by fluorescence microscopy. The rest of the lungs were formalin-fixed and paraffin-embedded for hematoxylin and eosin staining. Lungs from different treatment groups were photographed under a microscope to compare the distribution (and, thus, metastatic capacity) of the cells in the lungs.
Correlations between relapse-free survival (RFS) of breast cancer patients and PLOD2 expression were analyzed using the Kaplan-Meier plotter (http://kmplot.com/analysis/) . The expression level of PLOD2 in triple negative breast cancer (TNBC) and non-triple negative breast cancer (non-TNBC) were analyzed using The Cancer Genome Atlas (TCGA) database.
Tumor tissue microarray
Human breast cancer specimens were obtained from the Shanghai Outdo Biotech Co., Ltd., China. 150 breast cancer patients (ages 31–82) were selected for this experiment. PLOD2, GP130 and OBR expression were detected by immunochemistry.
Mouse body weights and tumor volumes were analyzed by two-way ANOVA. KM curves were plotted and log-rank tests were used to determine statistical significance between the survival rates of patients with high and low PLOD2 expression. The remaining results are presented as the means ± SD from triplicate experiments. Student’s t-test (two-tailed, unpaired) or one-way ANOVA (three-tailed or more, unpaired) was used to compare control and treatment groups. *represents statistical significance with P < 0.05, **represents statistical significance with P < 0.01.
PLOD2 is upregulated during adipocyte-stimulated migration and invasion
Next, we explored how adipocyte promote breast cancer cell migration and invasion. Fischbach and colleagues have revealed that obesity-induced interstitial fibrosis promotes breast tumorigenesis . Previous studies have revealed that adipokines are associated with inflammation and fibrosis . For example, leptin can induce liver fibrosis through the activation of hepatic stellate cells (HSCs) . Children with cystic fibrosis display high serum levels of adipokines, suggesting that adipokines may be involved in fibrotic disease . Over-deposition of collagen is the main cause of fibrosis, and aberrant PLOD2 expression contributes to the progression of collagen-related diseases such as fibrosis and cancer . Therefore, we speculated that a link might exist between adipokines and PLOD2. We first compared collagen distribution patterns in breast cancer cells that were either monocultured or cocultured with adipocytes. The results showed that collagen deposition was significantly increased upon coculture with adipocytes (Additional file 2: Figure S1a). qRT-PCR analysis demonstrated that expression of collagen biogenesis-associated genes (P4HA1, PLOD1, PLOD2) were elevated after coculture with adipocytes. Of these genes, PLOD2 was significantly increased, as shown in Fig. 1e. PLOD2 protein expression was also increased in MDA-MB-231 and MDA-MB-468 breast cancer cells cocultured with adipocytes (Fig. 1f). Similar results were obtained by coculturing MDA-MB-231 and SK-BR-3 breast cancer cells with or without human mammary adipocytes (Fig. 1f, Additional file 2: Figure S1b). Taken together, these results suggest that PLOD2 upregulation may stimulate the enhanced migration of breast cancer cells following coculture with adipocytes.
PLOD2 knockdown attenuates breast cancer migration and metastasis in vitro and in vivo
Adipocyte-derived IL-6 and leptin regulate PLOD2 expression
To further verify that adipokines contribute to adipocyte-mediated effects, we investigated the expression of PLOD2 in breast cancer cells cultured in conditioned medium (CM) obtained from preadipocytes, adipocytes, or adipocytes previously grown in the presence of cancer cells. Strikingly, PLOD2 expression increased in MDA-MB-231 and MDA-MB-468 cells following culturing of these cells in CM obtained from adipocytes and CM obtained from adipocytes previously grown in the presence of cancer cells. Meanwhile, CM from preadipocytes had little effect on the expression of PLOD2 (Additional file 4: Figure S3d). Altogether, these results confirm that adipocyte-derived soluble factors play a key role in adipocyte-induced PLOD2 expression. Further, it is likely that IL-6 and leptin are essential for the observed effects of conditioned medium on PLOD2 expression.
IL-6/GP130 and leptin/OBR mediate adipocyte microenvironment-induced activation of PLOD2 and migration in breast cancer cells
Thus far, we have demonstrated that IL-6 and leptin are increased in adipocytes and/or adipocytes cocultured with breast cancer cells. We next explored whether the expression of IL-6 and leptin receptors, which might mediate PLOD2 expression, was increased in MDA-MB-231 and MDA-MB-468 breast cancer cells after coculture with adipocytes. GP130, a subunit of the IL-6 receptor, plays a critical role in mediating IL-6 signaling [31, 32]. Here, we demonstrated that protein levels of GP130 and PLOD2 were significantly increased in MDA-MB-231 and MDA-MB-468 breast cancer cells cocultured with adipocytes (Fig. 4e). Strikingly, PLOD2 expression decreased when MDA-MB-231 cells and SK-BR-3 cells were cocultured with adipocytes in the presence of a murine IL-6 blocking antibody (Fig. 4g, Additional file 4: Figure S3e). It is known that leptin binds to its receptor (OBR/LEPR) on the cell surface, and can thus activate a number of complex signaling cascades . Thus, we next examined OBR protein levels in breast cancer cells cocultured with adipocyte. Interestingly, adipocyte coculture significantly increased the protein expression levels of both OBR and PLOD2 (Fig. 4f). Inversely, OBR depletion abolished the adipocyte-induced upregulation of PLOD2 (Fig. 4h, Additional file 4: Figure S3f). Taken together, these results indicate that adipocyte-derived IL-6 and leptin promote PLOD2 expression through activation of their respective receptors.
Our results showed that adipocyte-derived IL-6 and leptin stimulate the migration of tumor cells via upregulation of PLOD2 expression. To confirm the effects of adipocyte-derived IL-6 and leptin on the regulation of distal organ seeding and growth of metastatic tumor cells, we conducted tail vein metastasis assays using the MDA-MB-231 human breast cancer cell line. MDA-MB-231 cells were previously cocultured either with or without adipocytes, cocultured with adipocytes in the presence of a murine IL-6 blocking antibody, or cocultured with adipocytes following OBR depletion. After 3 days, cells were harvested, and resuspended in Cell Tracker™ blue (CMTPX), and intravenously injected into NOD SCID mice. As shown in Fig. 5f, the dissemination of breast cancer cells toward the lungs were enhanced in mice injected with MDA-MB-231 cells previously cocultured with adipocytes compared with mice injected with MDA-MB-231 cells grown alone. Interestingly, when MDA-MB-231 cells were cocultured with adipocytes in the presence of a murine IL-6 blocking antibody, cellular dissemination toward the lungs was abrogated compared with mice injected with the same cells cocultured with adipocytes without the blocking antibody (Fig. 5f). Moreover, depletion of OBR abolished the adipocyte-induced dissemination of MDA-MB-231 cells toward the lungs (Fig. 5f). Histological analysis indicated that both the size and number of metastatic nodules were significantly enhanced in mice injected with adipocyte-cocultured MDA-MB-231 cells compared with MDA-MB-231 cells grown alone (Fig. 5f). Treatment with a murine IL-6 blocking antibody and depletion of OBR significantly decreased both the size and number of the nodules (Fig. 5f).
Therefore, our results clearly suggest that adipocyte-derived IL-6 and leptin promote the invasive phenotype of breast cancer cells via upregulation of PLOD2 both in vitro and in vivo.
Adipocytes promote PLOD2 expression by activating the JAK/STAT3 and AKT signaling pathways
Based on the observation that adipocyte-derived IL-6 and leptin promoted PLOD2 expression in breast cancer cells, we next explored whether IL-6 and leptin activated JAK/STAT and AKT signals in vitro. Stimulation with IL-6 significantly increased the expression of PLOD2 and induced STAT3 tyrosine phosphorylation. PLOD2 expression could be inhibited upon treatment with ruxolitinib (Fig. 6e, Additional file 5: Figure S4e). IL-6-stimulated cells also displayed upregulation of STAT3 phosphorylation and a disorganized nuclear state; these phenotypes could be reversed upon treatment with ruxolitinib (Fig. 6f, Additional file 5: Figure S4f). However, AKT signaling was not activated by stimulation with IL-6 (Additional file 5: Figure S4j). A 3-day exposure to leptin significantly increased PLOD2 expression and activated phosphorylation of STAT3 and AKT in MDA-MB-231 and MDA-MB-468 breast cancer cells (Fig. 6 g, i, Additional file 5: Figure S4 g, i). Following treatment with leptin, pharmacological inhibition of the JAK/STAT and AKT signaling pathways with ruxolitinib and LY294002, respectively, decreased PLOD2 expression (Fig. 6g, i, Additional file 5: Figure S4 g, i). Immunofluorescence experiments also confirmed that leptin treatment promoted P-STAT3 nuclear accumulation in MDA-MB-231 and MDA-MB-468 cells and that this nuclear accrual could be inhibited by treatment with ruxolitinib (Fig. 6h, Additional file 5: Figure S4 h). Together, these data reveal that adipocyte-derived IL-6 and leptin activate the JAK/STAT3 and PI3K/AKT signaling pathways to promote PLOD2 expression.
High PLOD2 expression correlates with poor prognosis of breast cancer and closely relates to GP130 and OBR in clinical samples
The significance of PLOD2 expression in breast cancer was further confirmed by immunohistochemistry in a tumor tissue microarray. The staining index (SI) of PLOD2 was calculated based on staining intensity and the proportion of positive cells, and defined as score 0, 1, 2 and 3. We excluded 35 broken specimens, leaving 105 specimens total in this experiment. We found that PLOD2 was upregulated in breast cancer tissues compared with para-cancerous tissues. We observed significantly increased PLOD2 expression in basal-like tumors, which exists same characteristics with TNBC (Fig. 7b, c). Taken together, these findings reveal that high PLOD2 expression may serve as a clinical biomarker for poor prognosis in breast cancer patients.
Furthermore, we examined the correlations between relapse-free survival (RFS) of different stages status breast cancer and PLOD2 expression were analyzed by Kaplan-Meier plotter. The results showed that the expression of PLOD2 was positively correlated with the stage of breast cancer, and in particular in breast cancer patients at stage III but not at stage I and II (Fig. 7d). The clinical significance of PLOD2 in samples from different stages status was further evaluated by IHC analysis in a tumor tissue microarray. As shown in Fig. 7e, PLOD2 was positively correlated with the stage of breast cancer, respectively, strongly expressed in breast cancer at stage II and III. In addition, above results indicated that adipocyte-derived IL-6 and leptin promote PLOD2 expression through activation of their respective receptors. And further coexpression analysis indicated that GP130 and PLOD2 or OBR and PLOD2 were simultaneously expressed in breast cancer tissue microarray (Fig. 7f, g). Taken together, these results indicated that GP130 and PLOD2, OBR and PLOD2 exhibited a positive correlation. And PLOD2 is positively correlated with the stage of breast cancer.
Several studies have suggested that PLOD2 is dysfunctional in multiple cancer types, including sarcoma, lung cancer, breast cancer, glioblastoma, cervical cancer and bladder cancer [11, 12, 14, 17, 34]. Recent studies have shown that PLOD2 is closely related to cancer metastasis . Mechanistic studies have revealed that HIF-1α, TGF-β, microRNA-26a/b and EGF are involved in the modulation of PLOD2 expression [34, 35]. In addition, paracrine signals from cancer-associated fibroblasts (CAFs) can also upregulate PLOD2 expression in lung cancer . CAFs play a vital role in the tumor microenvironment and are known to secrete several chemokines and cytokines that are involved in cancer progression . Therefore, we speculated that paracrine signals from other stromal cells, such as adipocytes, might also play a role in regulating PLOD2 expression. Adipocytes are a major component of breast tissue and are known to secrete several factors, including adipokines and cytokines, which play a pivotal role in breast cancer progression [37, 38]. Therefore, in this study we investigated whether adipocyte-derived adipokines could promote breast cancer metastasis by regulating PLOD2 expression. The crosstalk between adipocytes and breast cancer cells may have potential clinical significance.
IL-6 and leptin were identified as important factors involved in cancer progression in a study examining the over-production of inflammatory cytokines due to chronic low-grade inflammation. IL-6 and leptin have been shown to be indispensable for the proliferation, metastasis and initiation of cancer and are significantly associated with poor prognosis in human cancers [42, 43, 44, 45, 46]. Our results demonstrated that, of the adipocyte-derived adipokines, IL-6 and leptin were highly expressed after coculture with breast cancer cells. Interestingly, the receptors for IL-6, leptin and PLOD2 were all upregulated upon adipocyte-stimulated migration. Treatment with a murine IL-6 blocking antibody or depletion of OBR abrogated both the expression of PLOD2 and adipocyte-stimulated metastasis, indicating that PLOD2 plays an important role in mediating breast cancer metastasis via adipocyte-derived IL-6 and leptin. Many other adipokines were expressed in this coculture system, in addition to IL-6 and leptin. Thus, we could not exclude the influence of other adipokines on the expression of PLOD2. However, in our study we confirmed that IL-6 and leptin were responsible for the adipocyte-mediated upregulation of PLOD2 in breast cancer. Previous research has suggested that PLOD2 promotes metastasis both directly and also indirectly through the induction of collagen cross-link switch or collagen fiber alignment [12, 13, 14]. Further research has suggested that PLOD2 is involved in TGF-β-induced EMT in cervical cancer . In this study, we confirmed that PLOD2 mediates adipocyte-stimulated breast cancer metastasis and promotes the epithelial-mesenchymal transition (EMT) of breast cancer cells, suggesting that PLOD2 directly regulates breast cancer metastasis. However, our study did not confirm whether the adipocyte-induced upregulation of PLOD2 could account for collagen reorganization. Our findings indirectly indicate, however, that adipocyte-derived IL-6 and leptin might influence collagen reorganization, further promoting cancer metastasis, though confirmation of this hypothesis will require further experimentation.
Increasing evidence demonstrates that the JAK/STAT3, PI3K/AKT and MEK/ERK pathways are frequently activated by adipokines, such as IL-6 and leptin and that these pathways are often altered in several types of cancer [42, 47, 48]. Therefore, we explored whether the expression of PLOD2 is regulated by any of these three classical regulatory pathways. In our coculture system, PLOD2 was regulated by the JAK/STAT3 and PI3K/AKT signaling pathways but not by the MEK/ERK signaling pathway. Treatment with IL-6 increased PLOD2 expression by activating the JAK/STAT3 signaling pathway, while stimulation with leptin activated both the JAK/STAT3 and PI3K/AKT signaling pathways to enhance PLOD2 expression. Together, our results suggest that pharmacological inhibition of IL-6 and leptin as well as the downstream JAK/STAT3 and PI3K/AKT signaling pathways can decrease PLOD2 expression and suppress breast cancer metastasis.
Over the past several years, PLOD2 has been implicated as a protumorigenic agent in multiple cancers, with a specifically pivotal role in cancer metastasis. We are the first to report that adipocyte-derived adipokines activate PLOD2 expression via activation of the JAK/STAT3 and PI3K/AKT signaling pathways, thus promoting cancer migration and metastasis. Thus, our research reveals a novel interaction between adipocytes and breast cancer and lays the way for clinical study aimed at exploring the interaction between adipocytes and PLOD2 in obese breast cancer patients. Furthermore, our results may indicate that obese breast cancer patients more likely to show high expression of PLOD2 which predict a poor prognosis. This assumption still need to be confirmed in human model. Further research may provide new insights leading to new therapeutic targets.
This study provides novel evidence that adipocytes promote breast cancer metastasis via activation of PLOD2. Inhibition of PLOD2 may be sufficient to suppress migration and invasion in vitro and to alleviate lung and liver metastasis of breast cancer cells in vivo. Further investigation revealed that adipocyte-derived IL-6 and leptin promote PLOD2 expression, thus facilitating breast cancer metastasis. PLOD2 upregulation is regulated by the JAK/STAT3 and PI3K/AKT signaling pathways. Therefore, our results suggest that PLOD2 plays a vital role in adipocyte-dependent invasive activity and high PLOD2 expression may predict poor survival in breast cancer patients.
We thank Dr. Li Sun and Shengtao Yuan for designing and supporting this experiment. And we thank Dr. Li Sun for her critical reading of the manuscript.
This work was supported by the National Natural Science Foundation of China (Grant numbers 81573456 and 81773766).
Availability of data and materials
All results of this study are presented in this article and additional files.
JH, XW, LS and SY conceived and designed this project. JH and XW contributed equally to this work, performed the experiments and analyzed the data. SL, XK, ZL and LW performed Western blotting and qRT-PCR. JH, XW, YL, XS and HH performed the animal experiments. All authors read and approved the final manuscript.
Li Sun is an Associated Researcher at China Pharmaceutical University whose research interests center around the tumor microenvironment, oncology, and pharmacology. Li Sun has received several research grants from the National Nature Science Foundation of China.
Ethics approval and consent to participate
Animal experiments were approved by the Experimental Animal Care Commission of China Pharmaceutical University. All samples were encoded to protect patient confidentiality. All patients signed an informed consent form, which was approved by the Institutional Review Board of the Human Subject Research Ethics Committee of Southeast University, Nanjing, China.
Consent for publication
The authors declare that they have no competing interests.
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