The miR-423-5p/MMP-2 Axis Regulates Nerve Growth Factor-Induced Promotion of Chondrosarcoma Metastasis

doi:10.21203/rs.3.rs-571776/v1

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The miR-423-5p/MMP-2 Axis Regulates Nerve Growth Factor-Induced Promotion of Chondrosarcoma Metastasis

https://doi.org/10.21203/rs.3.rs-571776/v1

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published 02 Jul, 2021

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Background: The effects of Nerve growth factor (NGF) in chondrosarcoma are not confirmed, although NGF is capable of promoting the progression and metastasis of several different types of tumors. Here we aim to explore the role of NGF in chondrosarcoma and elucidate how NGF acts.

Methods: Immunohistochemistry (IHC)-stained tissue samples from chondrosarcoma patients were stained with NGF and MMP-2 antibodies. Cell migration was examined by Transwell and wound healing assays. The expression levels of MMP-2, microRNA-423-5p (miR-423-5p) were measured by quantitative real-time polymerase chain reaction. MMP-2, FAK, and c-Src protein expression were examined by Western blot assays. The interaction between MMP-2 3’-UTRs and miR-423-5p binding site was explored by luciferase assay. We established the orthotopic in vivo model of chondrosarcoma lung metastasis to further investigate the promoting effects of NGF in metastatic chondrosarcoma.

Results: Here, we found that the levels of NGF and matrix metalloproteinase-2 (MMP-2) correlated with tumor stage in patients with chondrosarcoma. NGF facilitated MMP-2-dependent cellular migration in human chondrosarcoma JJ012 cells, while overexpression of NGF enhanced lung metastasis in a mouse model of chondrosarcoma. NGF promoted MMP-2 synthesis and cell migration by inhibiting miR-423-5p expression through the FAK and c-Src signaling cascades. NGF appears to be a worthwhile therapeutic target in the treatment of metastatic chondrosarcoma.

Conclusions: Our study has identified that NGF promotes MMP-2-dependent cell migration in human chondrosarcoma tissue by inhibiting miR-423-5p synthesis via the FAK and c-Src signaling cascades

Cancer Biology

NGF

Chondrosarcoma

Metastasis

MMP-2

miR-423-5p

Chondrosarcoma is a common tumor of soft tissue and bone that occurs typically in cartilage-enriched bone (e.g., femur, tibia, or pelvis) (1, 2) and has a high propensity to metastasize to distant organs (1). High-grade chondrosarcomas are particularly prone to metastasize to the lungs (3, 4), so therapeutic strategies that delay or inhibit this phenomenon will improve patient survival. The metastatic process involves the secretion of proteolytic enzymes such as matrix metalloproteinases (MMPs) and cathepsins, capable of degrading the extracellular matrix and basement membrane (5, 6). Not only have significantly higher levels of MMP-2 expression been recorded in human chondrosarcoma specimens than in normal cartilage (7), but also, increasingly higher levels of MMP-2 expression in human chondrosarcoma cells stimulate their migratory and metastatic potential [8,9]. Notably, chemokine (C-C motif) ligand 3 (CCL3) has been found to increase MMP-2 expression in human chondrosarcoma cells and thus encourage their migratory abilities, while inhibition of MMP-2 expression abolishes this effect of CCL3 (7). It therefore seems reasonable to speculate that inhibiting MMP-2 expression be a useful therapeutic tactic in chondrosarcoma metastasis.

MicroRNAs (miRNAs) are involved in the cellular processes of different human diseases, including cancer, cardiovascular disease, and arthritic diseases (8-11) where they regulate different activities of the tumor cell, including apoptosis, proliferation, angiogenesis, drug resistance and metastasis (12). The importance of miRNAs in tumorigenesis is underlined by the fact that they perpetuate the process by targeting key metabolic enzymes and protein messenger RNAs (mRNAs) (13). In particular, as regards lung cancer cellular metabolism, researchers have suggested that miRNA mimics or inhibitors of metabolic processes and gene regulatory events could improve overall survival in lung cancer (13). In recently, miRNAs levels have been suggested as serving as potential biomarkers and therapeutic targets in cancer (14). In addition, miRNAs are regulated in different roles of the tumor cell, (12). Interestingly, evidence suggests that suppressing miR-101 and MMP-2 expression in human chondrosarcoma cells inhibits chondrosarcoma metastasis to the lungs (15).

Nerve growth factor (NGF) plays a critical role in neuronal cell growth, apoptosis and differentiation (16). The binding of NGF to its receptor tropomyosin receptor kinase A (TrkA) activates intracellular signaling, immune cell proliferation, differentiation and survival (17). Several reports have suggested that NGF plays an integral part in the progression of several types of malignancies, such as ovarian, prostate, and liver cancers (18-20). NGF also mediates metastasis in several types of tumors (20-22). However, the role of NGF in chondrosarcoma is unknown. In this study, we found that NGF promotes chondrosarcoma metastasis in vitro and in vivo. NGF also promotes MMP-2-dependent migration and invasion by inhibiting miR-423-5p expression through the FAK and c-Src signaling cascades.

Materials

NGF, MMP-2, FAK, c-Src and β-actin antibodies were obtained from GeneTex International Corporation (Hsinchu City, Taiwan). The phosphorylated forms of FAK (p-FAK) and c-Src (p-c-Src) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). MMP-2, FAK, c-Src and control ON-TARGETplus siRNAs were obtained from Dharmacon (Lafayette, CO, USA). Quantitative polymerase chain reaction (qPCR) primers and probes, as well as Taqman^® One-Step PCR Master Mix, were supplied by Applied Biosystems (Foster City, CA, USA). Recombinant human NGF was obtained from PerpoTech (Rocky Hill, NJ, USA). All other chemicals used in this study were supplied by Sigma-Aldrich (St. Louis, MO, USA).

Cell culture

The human chondrosarcoma cell line JJ012 was kindly provided by Dr. Sean P. Scully (University of Miami School of Medicine, Miami, FL, USA), while the SW1353 chondrosarcoma cell line was obtained from the American Type Cell Culture Collection (Manassas, VA, USA). JJ012 cells stably expressing the NGF complementary DNA (cDNA) clone (JJ012/NGF cells) were established according to our previous method (23). Cells were cultured 50%/50% in Dulbecco's Modified Eagle Medium (DMEM)/alpha-minimum essential medium (α-MEM) medium, 10% fetal bovine serum (FBS) and antibiotics, then maintained in a humidified incubator at 37°C in 5% CO₂.

Cell migration assay

Chondrosarcoma cells were seeded into the upper chamber of Transwell plates (Costar, NY, USA), while NGF and pharmaceutical inhibitors were added to the lower chamber. After 18 h of incubation, migrated cells were fixed with 3.7% formaldehyde and stained with crystal violet, then counted manually under the microscope (24, 25).

Wound healing assay

The confluent chondrosarcoma monolayer was scratched by a fine pipette tip to create extended scratches in each well. Cells were then treated with the conditions as indicated, migratory activity was evaluated by microscopy after 24 h and the rate of wound closure was quantified (26).

Western blot analysis

After the indicated treatments, chondrosarcoma cells were lysed in RIPA buffer. The extracted proteins were resolved by SDS-PAGE and transferred to Immobilon^® polyvinylidene fluoride (PVDF) membranes. Western blot analysis was performed using the methodology described in our previous reports (27-29).

mRNA and miRNA quantification

Total RNA was extracted from chondrosarcoma cells using TRIzol reagent and RNA concentrations were determined using a NanoVue Plus spectrophotometer (GE Healthcare Life Sciences; Pittsburgh, PA, USA). The M-MLV RT kit (Thermo Fisher Scientific; Waltham, MA, USA) and the Mir-X™ miRNA First-Strand Synthesis kit (Clontech; Mountain View, CA, USA) were used to perform reverse transcription of total RNA into cDNA. Quantitative real-time PCR (qPCR) analysis was performed according to our previous reports (30, 31).

Luciferase assay

The Human MMP-2 luciferase reporter plasmids containing wild-type or mutant sequences of the three prime untranslated region (3’-UTR) encompassing miR-423-5p binding sites were obtained from MDBio, Inc. (Taipei, Taiwan). Chondrosarcoma cells were transfected with the plasmids using Lipofectamine 2000 (Thermo Fisher Scientific; Waltham, MA, USA), then stimulated with NGF for 24 h. Luciferase activity was monitored using a luciferase assay kit (30, 32, 33).

Tumor xenograft study

Four-week-old male BALB/c nude mice (8 in each group) were bought from Taipei’s National Laboratory Animal Center and orthotopically injected with JJ012 or JJ012/NGF cells (5 × 10⁶, resuspended in 100 μL of medium containing 50% serum-free DMEM/α-MEM and 50% Matrigel), according to a previous protocol (23). Tumor growth in the tibiae was monitored each week by bioluminescence imaging using a Xenogen IVIS imaging system 200 (PerkinElmer, MA, USA). At 12 weeks, the mice were euthanized by CO₂ inhalation. The lungs were removed and fixed in 10% formalin for further analysis. All animal experiments satisfied the protocols specified by China Medical University’s Institutional Animal Care and Use Committee (IACUC Approval No. 104-154-N).

Immunohistochemistry (IHC) staining

Mouse lung tissues or specimens from a human chondrosarcoma tissue array (Biomax; Rockville, MD, USA) were rehydrated and incubated with primary anti-NGF or MMP-2 antibodies, then treated with biotin-labeled secondary antibody. Finally, the slides were detected using the ABC Kit (Vector Laboratories, CA, USA) and photographed using the microscope.

Statistics

All values are presented as the mean ± standard deviation (SD). Significance testing on the difference between the groups was assessed by the Student’s t-test and considered significant if the p-value was <0.05.

NGF and MMP-2 levels are positively correlated in human chondrosarcoma tissue

NGF is associated with progression and survival in several cancer types (34, 35). MMP-2 reported to control migration and metastasis of chondrosarcoma (7, 36). IHC tissue array results revealed higher levels of NGF and MMP-2 expression in patients with higher grade chondrosarcoma than in those with lower grade disease; the levels of NGF and MMP-2 expression were reflected by tumor stage (Fig. 1A-C). These results are quantified in Figures 1D&E, which illustrate how the levels of NGF and MMP-2 expression were significantly higher in higher-stage tumors (IIA and IIB) than in lower-stage tumors (IA and IB). A positive correlation observed between MMP-2 and NGF staining intensity of human chondrosarcoma tissue (r² = 0.6, Fig. 1F) indicates that the levels of these proteins are associated with the progression of chondrosarcoma disease.

NGF promotes MMP-2-dependent migration via the FAK and c-Src pathways in chondrosarcoma

We first investigated the effects of NGF upon cell motility in chondrosarcoma cell lines SW1353 and JJ012. Treatment of cells with NGF promoted migration ability, according to Transwell and wound healing assay data (Fig. 2A-D). Next, we examined whether MMP-2 plays a role in NGF-regulated migration in chondrosarcoma. Stimulation of cells with NGF enhanced mRNA and protein synthesis of MMP-2 (Fig. 2E&F). Transfection of cells with MMP-2 siRNA diminished NGF-induced promotion of migration (Fig. 2G-I), implying that MMP-2 is critical to NGF-induced chondrosarcoma cell migration.

The FAK and c-Src signaling pathway plays a critical role in chondrosarcoma metastasis (37). Treating cells with a FAK inhibitor or c-Src inhibitor (PP2) significantly reduced NGF-induced stimulation of cell migration and MMP-2 production (Fig. 3A-F and 4A-F). Similar effects were observed when the chondrosarcoma cells were transfected with FAK or c-Src siRNAs (Fig. 3A-G and 4A-F). NGF stimulation time-dependently promoted FAK and c-Src phosphorylation in both cell lines (Fig. 3H & 4H). Treating cells with a FAK inhibitor diminished NGF-induced c-Src phosphorylation (Fig. 4I), indicating that the FAK/c-Src signaling cascade regulates NGF-induced MMP-2 synthesis and chondrosarcoma cell migration.

The miR-423-5p/MMP-2 axis controls NGF-induced stimulation of chondrosarcoma cell migration

miRNA-associated regulation of MMP-2 expression is critical mechanism in the development, progression, migration and metastasis of cancer cells (38). Five online databases for miRNA target prediction indicated that the 3’-UTR region of MMP-2 mRNA contains 13 promising candidate miRNAs (Fig. 5A&B). Treatment of JJ012 cells with NGF (100 ng/ml) significantly reduced miR-423-5p expression (Fig. 5B) and at the concentrations of 30, 50, or 100 ng/ml, significantly inhibited miR-423-5p synthesis in both chondrosarcoma cell lines, in a concentration-dependent manner (Fig. 5C). Transfection of cells with miR-423-5p mimic significantly reduced NGF-induced stimulation of cell migration and MMP-2 mRNA expression in both chondrosarcoma cell lines (Fig. 5D-F). Next, we investigated whether FAK and c-Src signaling regulate NGF-induced suppression of miR-423-5p synthesis. FAK and c-Src inhibitors, and their respective siRNAs, all reversed NGF-induced inhibition of miR-423-5p expression (Fig. 5G&H). Analyses of the MMP-2 3’-UTR luciferase plasmids revealed that NGF increased luciferase activity of the wild-type, but not mutant, MMP-2 3’-UTRs (Fig. 5I&J). These results indicate that miR-16-5p controls MMP-2 expression by anchoring to the 3’-UTR region of the human MMP-2 gene via the FAK/c-Src pathway.

Overexpression of NGF facilitates chondrosarcoma metastasis to lungs in mice

We used the orthotopic in vivo model of chondrosarcoma lung metastasis to further investigate the promoting effects of NGF in metastatic chondrosarcoma (23). JJ012 and JJ012/NGF cells were orthotopically implanted into the right leg tibia and tumor size was monitored each week by the IVIS system (Fig. 6A&B). Overexpression of NGF significantly increased tumor growth in the tibia (Fig. 6A&B). At 12 weeks, metastasis to the lung was significantly more likely with JJ012/NGF cells than with JJ012 cells (Fig. 6C&D). IHC results revealed significant increases in the levels of NGF and MMP-2 expression in the JJ012/NGF orthotopic model (Fig. 6E), confirming that NGF facilitates the metastasis of chondrosarcoma to mouse lung.

Chondrosarcoma is a malignant bone neoplasm that constitutes almost one-third (~26%) of all bone cancers (39). Chemotherapy and radiotherapy have very limited effectiveness, so treatment with surgery is therefore the major management modality for chondrosarcoma. This malignancy is notorious for its aggressive clinical course and propensity to metastasize (40). An effective adjuvant therapy is urgently needed to suppress the metastasis of chondrosarcoma (1, 3). NGF plays an important role in tumor cell proliferation, migration and survival (34, 35). The effect of NGF in chondrosarcoma metastasis is uncertain. Our investigation has found that levels of NGF and MMP-2 expression are positively correlated with tumor staging in patients with chondrosarcoma. We also confirmed that NGF facilitates MMP-2-dependent chondrosarcoma cell migration and metastasis by inhibiting miR-423-5p synthesis via FAK/c-Src signaling.

NGF acts not only on the central and peripheral nervous systems, but also on non-neuronal tissues and cancer cells (34). NGF plays a multi-functional role in the tumor environment, exerting effects on tumor cell proliferation, survival, apoptosis, angiogenesis and metastasis (34, 41, 42). Our study is the first to describe an association between NGF levels and tumor stage in chondrosarcoma tissue specimens. The evidence from us in vitro and in vivo results suggest that NGF facilitates chondrosarcoma metastasis. The binding of NGF with the neurotrophin receptor TrkA mediates NGF control in the development of cancer (43). Knockdown of TrkA receptor suppresses the progression of liver cancer (44) and a Trk receptor inhibitor antagonizes NGF-induced cell motility in prostate cancer (45). These results suggest that inhibition of the TrkA receptor reduces NGF-mediated cancer development. In this study, we did not examine TrkA receptor levels in patients with chondrosarcoma. Whether TrkA inhibition antagonizes NGF-mediated chondrosarcoma metastasis is yet to be confirmed.

Activation of the FAK/c-Src pathway is important in the regulation of different cellular functions (46, 47). This signaling pathway also regulates the expression of MMP-mediated cancer motility (48, 49). Here, our results show that NGF promotes phosphorylation of FAK and c-Src, while FAK and c-Src pharmacological inhibitors suppress NGF-induced promotion of MMP-2 expression and chondrosarcoma migration. This phenomenon is confirmed by similar effects observed with genetic siRNAs of FAK and c-Src. However, c-Src has been reported to be an upstream molecule of FAK, with the capacity to regulate cell motility (50, 51). Here, we found that a FAK inhibitor curtailed NGF-promoted phosphorylation of c-Src, indicating that FAK activates c-Src.

MiRNAs post-transcriptionally regulate gene expression (52). During tumor metastasis, aberrant miRNA expression mediates cancer cell migration and invasion (53). Our analysis of five open-source databases identified that 13 miRNAs potentially interfere with MMP-2 transcription. NGF significantly lowered miR-423-5p expression. We enhanced miR-423-5p levels in chondrosarcoma cells by transfecting them with a specific miR-423-5p mimic, which also reduced MMP-2 synthesis and the migratory capacity of the cells. miR-423-5p expression was negatively correlated with MMP-2 expression and cell migratory activity in chondrosarcoma cells. Thus, our evidence has identified novel antimetastatic properties of miR-423-5p.

In conclusion, our study has identified that NGF promotes MMP-2-dependent cell migration in human chondrosarcoma tissue by inhibiting miR-423-5p synthesis via the FAK and c-Src signaling cascades (Fig. 7). It appears to be worth targeting NGF expression in metastatic chondrosarcoma.

NGF, Nerve growth factor; MMP-2, matrix metalloproteinase-2; CCL3, chemokine (C-C motif) ligand 3; miRNAs, MicroRNAs; mRNAs, messenger RNAs; TrkA, tropomyosin receptor kinase A; qPCR, quantitative polymerase chain reaction; cDNA, complementary DNA; DMEM, Dulbecco's Modified Eagle Medium; α-MEM, alpha-minimum essential medium; FBS, fetal bovine serum; PVDF, polyvinylidene fluoride; 3’-UTR, three prime untranslated region; IHC, Immunohistochemistry; SD, standard deviation.

Ethics approval and consent to participate

The authors declare that the data supporting the findings of this study are available within the article. All animal experiments satisfied the protocols specified by China Medical University’s Institutional Animal Care and Use Committee (IACUC Approval No. 104-154-N).

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author for reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by grants from the Ministry of Science and Technology of Taiwan (MOST 107-2320-B-039-019-MY3, MOST 109-2320-B-039-065) and China Medical University Hospital, Taichung, Taiwan (DMR-110-158, DMR-110-180).

Authors’ contributions

HET, SLL, and CHT conceived and designed the experiments. LAT, CYK, JFL, and CYL performed the experiments. HET, SLL, and LAT contributed to perform in vitro and in vivo experiments. LAT and CYL contributed to analyze experiment data. YCF and CHT contributed to the writing of the manuscript. All authors approved for publication.

Acknowledgments

We would like to thank Iona J. MacDonald from China Medical University for her English language revision of this manuscript.

Author details

¹Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan. ²Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan. ³Division of Hematology/Oncology, Department of Medicine, Taipei Medical University Hospital, Taiwan. ⁴School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan. ⁵Translational Medicine Research Center, China Medical University Hospital, Taichung, Taiwan. ⁶International Master Program of Biomedical Sciences, China Medical University, Taichung, Taiwan. ⁷Department of Orthopedic Surgery, China Medical University Hospital, Taichung, Taiwan. ⁸School of Oral Hygiene, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan. ⁹Department of Pharmacology, School of Medicine, China Medical University, Taichung, Taiwan. ¹⁰Department of Sports Medicine, College of Health Care, China Medical University, Taichung, Taiwan. ¹¹Department of Orthopedic Surgery, China Medical University Beigang Hospital, Yunlin, Taiwan. ¹²Department of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan. ¹³Chinese Medicine Research Center, China Medical University, Taichung, Taiwan.

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The miR-423-5p/MMP-2 Axis Regulates Nerve Growth Factor-Induced Promotion of Chondrosarcoma Metastasis

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Figures

Background

Materials And Methods

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Status:

Journal Publication

Version 1