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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.44 No.2 pp.37-42

Role of proteases, cytokines, and growth factors in bone invasion by oral squamous cell carcinoma

Seung Hwa Son1, Won-Yoon Chung1,2,3*
1Oral Cancer Research Institute, Yonsei University College of Dentistry, Seoul 03722, Republic of Korea
2Department of Oral Biology and BK21 PLUS Project, Yonsei University College of Dentistry, Seoul 03722, Republic of Korea
3Department of Applied Life Science, The Graduate School, Yonsei University, Seoul 03722, Republic of Korea
Correspondence to: Won-Yoon Chung, E-mail:
May 30, 2019 June 17, 2019 June 19, 2019


Oral squamous cell carcinoma (OSCC) is the most common oral malignancy and an increasing global public health problem. OSCC frequently invades the jaw bone. OSCC-induced bone invasion has a significant impact on tumor stage, treatment selection, patient outcome, and quality of life. A number of studies have shown that osteoclastmediated bone resorption is a major step in the progression of bone invasion by OSCC; however, the molecular mechanisms involved in OSCC bone invasion are not yet clear. In this review, we present the clinical types of OSCC bone invasion and summarize the role of key molecules, including proteases, cytokines, and growth factors, in the sequential process of bone invasion. A better understanding of bone invasion will facilitate the discovery of molecular targets for early detection and treatment of OSCC bone invasion.


    Ministry of Health and Welfare
    © The Korean Academy of Oral Biology. All rights reserved.

    This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.


    Cancer of the lip and oral cavity is a rising problem worldwide with around 300,000 new cases per annum [1]. Squamous cell carcinoma (SCC) is detected in most patients with oral cancer [2]. Oral squamous cell carcinoma (OSCC) often happens at the gingiva and tongue and contributes above 90% of all oral cancers [3,4]. Genetic aspects and environmental factors, including alcohol abuse, smoking, viral infection, and chronic inflammation, have been associated with the pathogenesis of OSCC [5,6]. Due to the close anatomical structure of the oral mucosa and jaws, OSCC cells may frequently invade bone tissues. Tumors derived from the floor of the mouth, the retromolar zone, and the tongue invade the mandible in 62%, 48%, and 42%, respectively [7]. The patients with oral cancer generally have severe dysfunctions of speaking, chewing, and/or swallowing. Treatment and rehabilitation are particularly difficult in the patients with bone invasion. Thus, early detection and accurate prediction of bone invasion is important to plan surgical ablation and minimize the spread of tumor cells, especially to induce maxillary or mandibular conservative surgery.

    In general, OSCC bone invasion shows histologically two distinct patterns. One is the less aggressive erosive pattern with a tumor mass that invades on a broad pushing front and is detached from the bone by the connective tissue layer. The other is the invasive pattern in which the connective tissue layer is destroyed and the islands of tumor penetrate the bone [7-9] (Fig. 1). The formation of two patterns is affected by regional anatomic aspects of exposed bone, particularly whether the progressing front of the neoplasm contacts cancellous bone, by the inherent properties of the malignant cells, and by the properties of tumor stroma and unidentified factors [10]. In spite of developments in OSCC treatment, recurrence and mortality rates are continuously increasing in patients with bone invasion [11].

    To promote the quality of life and survival, it is necessary to improve diagnosis and therapy for OSCC patients with bone invasion based on molecular mechanisms by which OSCC invades bone. Therefore, we will focus on critical molecular markers associated with bone invasion by OSCC.


    Bone tissue consists of several distinct cell types and a mostly mineralized bone matrix. Osteocytes are integrated within bone and participate in the bone remodeling process with various cell types, including osteoprogenitor cells, osteoclasts, osteoblasts, marrow fibroblasts, and undifferentiated cells [12,13]. Bone-tropic tumor cells produce a variety of enzymes to directly destroy extracellular matrix (ECM) and then migrate into the surrounding tissues [14]. In addition, tumor-derived proteolytic enzymes stimulate differentiation and maturation of bone cells, especially osteoclast precursors, resulting in osteolysis. Matrix metalloproteinases (MMPs) and cathepsins have been considered pivotal proteases in OSCCmediated bone invasion.

    MMPs are the proteolytic enzymes responsible for degradation of fibrillar and non-fibrillar collagens, gelatin, elastin, and proteoglycans. Some MMPs are detected at high levels in cartilage and bone of mammals, including humans and mice, and are able to cleave native, non-denatured collagens by potentially functioning as collagenases in vivo [15]. MMP-1 and MMP-9 were strongly expressed in highly differentiated BHY OSCC cells [16]. In another study, 9 of the 24 buccal SCC with mandibular invasion were intensely stained for active MMP- 7 but not 15 without bone invasion, representing that MMP-7 is connected with mandibular invasion [17]. MMP-7 secreted from breast and prostate cancer cells cleaved the receptor activator of nuclear factor-κB (RANK) ligand (RANKL) to its active soluble form, which triggers osteoclast differentiation and bone resorption [18,19]. In addition, high expression of MMP-9 is detected in the cytoplasm of invading OSCC cells [20].

    Cathepsins are lysosomal proteases that degrade proteins at acidic pH. In normal cells, cathepsins modulate the immune responses and signaling pathways. Abnormal cathepsin activity is commonly implicated for altered immunologic and physiological behavior that is caused by cancers [21,22]. Cathepsins, including cathepsin D, E, K, and L, play a crucial role in bone resorption by osteoclasts [23]. Procathepsin L was secreted from BHY cells in large amounts and the active cathepsin L degraded the demineralized bone matrix [24]. The injected BHY cells on the masseter muscle of nude mice developed into highly differentiated SCC invading the mandible, demonstrating that cathepsin L might support to degrade the bone matrix [24]. Cathepsin B and D were intensely expressed in all 78 SCC of tongue, gingiva, or floor of mouth and the labeling indices of the two cathepsins were closely correlated with the degree of bone invasion [25]. Furthermore, the survival period in patients with high serum levels of cathepsin B and D was short, indicating that these molecules could be useful as prognostic indicators.


    Osteoclasts are actually hematopoietic cells, originated from the macrophage/monocyte lineage. The differentiated osteoclasts attach to the bone matrix and then secrete protons to resorb inorganic components and soluble enzymes to degrade organic components of bone matrix [26]. Two hematopoietic factors are required for differentiation of osteoclast precursors to mature osteoclasts (osteoclastogenesis): RANKL and macrophage colony-stimulating factor (M-CSF). Tumor necrosis factor (TNF) receptor/TNF-like proteins, including RANKL, its receptor RANK, and osteoprotegerin (OPG), regulate osteoclast differentiation [27]. RANKL functions a key inducer of bone resorption by binding to RANK but OPG acts as a soluble decoy receptor [28]. Osteoclastogenesis and activation of boneresorbing osteoclasts appears to be the most important factor in OSCC bone invasion [29,30]. In OSCC-induced bone invasion, tumor-derived cytokines, including TNF-α, can directly or indirectly induce osteoclast formation by stimulating RANKL or M-CSF expression [3,31].

    Interleukins (ILs), a family of pleiotropic cytokines derived from osteoblasts, osteoclasts, and stromal cells, play an important role in controlling the metabolism of bone [12]. Osteoclasts share many regulatory molecules with immune cells, thereby several immune cells differentiate as the osteoclasts in bone microenvironment [32]. The discovery that cultured human peripheral blood leukocytes can absorb bone supports the connection between immune system and bone metabolism [33]. IL-1b has been recognized as a primary mediator [34] but IL-6, IL-11, and IL-15 as well promote bone resorption [35]. Parathyroid hormone-related peptide (PTHrP), another key molecule of bone homeostasis, plays a role in preventing apoptosis of osteoblasts and recruiting osteoclasts, and is upregulated in the tissues of OSCC patients [36]. The binding of PTHrP to its receptors stimulates osteoclast activity and facilitates bone destruction [37]. High expression of PTHrP messenger RNA (mRNA) was detected in BHY cells and PTHrP mRNA was observed from tumor tissues in 7 of 11 patients with the lower alveolar and gingival carcinoma, showing an invasive pattern of bone invasion.

    Chemokines, a superfamily of structurally associated cytokines, are classified into four subgroups based on the variations on a conserved cysteine motif in the protein sequences [38]. The CC and CXC are the larger subgroups, whereas CX3C and XC are smaller subgroups. Chemokines bind to their corresponding G-protein-coupled receptors and induce conformational changes in their transmembrane domain. Some chemokines in the bone matrix play a decisive role in osteoclast activation [39]. Additionally, chemokines produced by tumor cells regulate recruitment and mobilization of osteoclasts [40]. CXCL13 and its receptor CXCR5 promoted RANKL expression in OSCC cells and prevented bone invasion by OSCC through NFATc3 and c-Myc expression [41-43]. CXCL2 was found to be involved in bone destruction by expressing RANKL [44].

    Growth Factors

    Many growth factors influence function of osteoblasts and osteoclasts during bone remodeling. In the progression of the OSCC, osclast-mediated bone resorption induces secretion of growth factors from reservoirs within mineralized bone matrix, including transforming growth factor (TGF), epidermal growth factor, or connective tissue growth factor, which function as local managers of tumor growth and bone destruction [45]. Various growth factors advance the local microenvironment around tumor cells that express receptors for growth factors, promote tumor cell proliferation, and suppress their apoptosis [46-48]. TGF-β1 not only induces epithelial-mesenchymal transition to increase OSCC invasion capacity but also upregulates factors for prolonged osteoclast survival [49]. Recent studies have shown that TGF-β1 overexpression promoted invasiveness, migration, and angiogenesis of SCC9 cells by activating slug/MMP-9 axis [50].


    OSCC bone invasion is an extremely coordinated process that can described in ‘vicious cycle’ of early, resorption, and ultimate stages, which is stimulated by interaction between OSCC cells, osteoblasts, osteoclasts, and bone matrix-derived factors (Fig. 2). In the early stage, proteases degrade the ECM of the adjacent soft tissues and promote the invasion of OSCC into the bone. The next step in bone invasion is the resorption stage, where osteoclasts play a major role in absorbing the mineral components of the bone. Osteoclast-mediated resorption leads to ultimate stage of bone invasion by secreting growth factors from bone matrix and consequently exacerbate OSCC bone invasion by promoting the growth of tumor cells.

    Previous studies have reported molecular mechanisms associated with OSCC invasion, based on OSCC characteristics. Although many researchers are exploring molecular mechanisms of OSCC bone invasion, novel targeted markers are still needed to quickly predict bone invasion and to treat patients. The useful target molecules that can predict and serve to treat OSCC bone invasion will increase early diagnostic and therapeutic success and effectively reduce the morbidity and mortality associated with bone invasion by OSCC.


    This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI15C1901).



    Distinct patterns of oral squamous cell carcinoma (OSCC) bone invasion. (A) OSCC without bone invasion. (B) Erosive pattern of bone invasion with clear interface between mandible and OSCC. (C) Invasive pattern of bone invasion with irregular front and residual bone islands within OSCC.


    Coordinated process of oral squamous cell carcinoma (OSCC) bone invasion. In the early stage, proteases degrade the surrounding extracellular matrix and stimulate OSCC cells to invade the bone tissue. In the resorption stage, cytokines regulate osteoclastogenesis and osteoclastmediated bone resorption. In the ultimate stage, growth factors released from bone matrix promote OSCC growth. The bone invasion deteriorates by continuously repeating these stages.



    1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015;136:E359-86.
    2. Warnakulasuriya S. Global epidemiology of oral and oropharyngeal cancer. Oral Oncol 2009;45:309-16.
    3. Jimi E, Shin M, Furuta H, Tada Y, Kusukawa J. The RANKL/RANK system as a therapeutic target for bone invasion by oral squamous cell carcinoma (Review). Int J Oncol 2013;42:803-9.
    4. Attar E, Dey S, Hablas A, Seifeldin IA, Ramadan M, Rozek LS, Soliman AS. Head and neck cancer in a developing country: a population-based perspective across 8 years. Oral Oncol 2010;46:591-6.
    5. Choi S, Myers JN. Molecular pathogenesis of oral squamous cell carcinoma: implications for therapy. J Dent Res 2008;87:14-32.
    6. Petti S. Lifestyle risk factors for oral cancer. Oral Oncol 2009;45:340-50.
    7. Brown JS, Lowe D, Kalavrezos N, D’Souza J, Magennis P, Woolgar J. Patterns of invasion and routes of tumor entry into the mandible by oral squamous cell carcinoma. Head Neck 2002;24:370-83.
    8. Jimi E, Furuta H, Matsuo K, Tominaga K, Takahashi T, Nakanishi O. The cellular and molecular mechanisms of bone invasion by oral squamous cell carcinoma. Oral Dis 2011;17:462-8.
    9. Lubek JE, Magliocca KR. Evaluation of the bone margin in oral squamous cell carcinoma. Oral Maxillofac Surg Clin North Am 2017;29:281-92.
    10. Ito M, Izumi N, Cheng J, Sakai H, Shingaki S, Nakajima T, Oda K, Saku T. Jaw bone remodeling at the invasion front of gingival squamous cell carcinomas. J Oral Pathol Med 2003;32:10-7.
    11. Shaw RJ, Brown JS, Woolgar JA, Lowe D, Rogers SN, Vaughan ED. The influence of the pattern of mandibular invasion on recurrence and survival in oral squamous cell carcinoma. Head Neck 2004;26:861-9.
    12. Raggatt LJ, Partridge NC. Cellular and molecular mechanisms of bone remodeling. J Biol Chem 2010;285:25103-8.
    13. Georges S, Ruiz Velasco C, Trichet V, Fortun Y, Heymann D, Padrines M. Proteases and bone remodelling. Cytokine Growth Factor Rev 2009;20:29-41.
    14. Woodward JK, Holen I, Coleman RE, Buttle DJ. The roles of proteolytic enzymes in the development of tumourinduced bone disease in breast and prostate cancer. Bone 2007;41:912-27.
    15. Krane SM, Inada M. Matrix metalloproteinases and bone. Bone 2008;43:7-18.
    16. Erdem NF, Carlson ER, Gerard DA, Ichiki AT. Characterization of 3 oral squamous cell carcinoma cell lines with different invasion and/or metastatic potentials. J Oral Maxillofac Surg 2007;65:1725-33.
    17. Chuang HC, Su CY, Huang HY, Huang CC, Chien CY, Du YY, Chuang JH. Active matrix metalloproteinase-7 is associated with invasion in buccal squamous cell carcinoma. Mod Pathol 2008;21:1444-50.
    18. Thiolloy S, Halpern J, Holt GE, Schwartz HS, Mundy GR, Matrisian LM, Lynch CC. Osteoclast-derived matrix metalloproteinase- 7, but not matrix metalloproteinase-9, contributes to tumor-induced osteolysis. Cancer Res 2009;69:6747-55.
    19. Lynch CC, Hikosaka A, Acuff HB, Martin MD, Kawai N, Singh RK, Vargo-Gogola TC, Begtrup JL, Peterson TE, Fingleton B, Shirai T, Matrisian LM, Futakuchi M. MMP-7 promotes prostate cancer-induced osteolysis via the solubilization of RANKL. Cancer Cell 2005;7:485-96.
    20. Quan J, Johnson NW, Zhou G, Parsons PG, Boyle GM, Gao J. Potential molecular targets for inhibiting bone invasion by oral squamous cell carcinoma: a review of mechanisms. Cancer Metastasis Rev 2012;31:209-19.
    21. Gocheva V, Joyce JA. Cysteine cathepsins and the cutting edge of cancer invasion. Cell Cycle 2007;6:60-4.
    22. Olson OC, Joyce JA. Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response. Nat Rev Cancer 2015;15:712-29.
    23. Goto T, Yamaza T, Tanaka T. Cathepsins in the osteoclast. J Electron Microsc (Tokyo) 2003;52:551-8.
    24. Kawamata H, Nakashiro K, Uchida D, Harada K, Yoshida H, Sato M. Possible contribution of active MMP2 to lymphnode metastasis and secreted cathepsin L to bone invasion of newly established human oral-squamous-cancer cell lines. Int J Cancer 1997;70:120-7.
    25. Kawasaki G, Kato Y, Mizuno A. Cathepsin expression in oral squamous cell carcinoma: relationship with clinicopathologic factors. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;93:446-54.
    26. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 2003;423:337-42.
    27. Martin CK, Dirksen WP, Shu ST, Werbeck JL, Thudi NK, Yamaguchi M, Wolfe TD, Heller KN, Rosol TJ. Characterization of bone resorption in novel in vitro and in vivo models of oral squamous cell carcinoma. Oral Oncol 2012;48:491-9.
    28. Takayama Y, Mori T, Nomura T, Shibahara T, Sakamoto M. Parathyroid-related protein plays a critical role in bone invasion by oral squamous cell carcinoma. Int J Oncol 2010;36:1387-94.
    29. Dougall WC, Chaisson M. The RANK/RANKL/OPG triad in cancer-induced bone diseases. Cancer Metastasis Rev 2006;25:541-9.
    30. Cochran DL. Inflammation and bone loss in periodontal disease. J Periodontol 2008;79(8 Suppl):1569-76.
    31. Jimi E, Kokabu S, Matsubara T, Nakatomi C, Matsuo K, Watanabe S. NF-κB acts as a multifunctional modulator in bone invasion by oral squamous cell carcinoma. Oral Sci Int 2016;13:1-6.
    32. Nakashima T, Takayanagi H. The dynamic interplay between osteoclasts and the immune system. Arch Biochem Biophys 2008;473:166-71.
    33. Bar-Shavit Z. The osteoclast: a multinucleated, hematopoietic-origin, bone-resorbing osteoimmune cell. J Cell Biochem 2007;102:1130-9.
    34. Dewhirst FE, Stashenko PP, Mole JE, Tsurumachi T. Purification and partial sequence of human osteoclastactivating factor: identity with interleukin 1 beta. J Immunol 1985;135:2562-8.
    35. Walsh MC, Kim N, Kadono Y, Rho J, Lee SY, Lorenzo J, Choi Y. Osteoimmunology: interplay between the immune system and bone metabolism. Annu Rev Immunol 2006;24:33-63. doi: 10.1146/annurev.immunol.24.021605.090646.
    36. Datta NS, Abou-Samra AB. PTH and PTHrP signaling in osteoblasts. Cell Signal 2009;21:1245-54.
    37. Soki FN, Park SI, McCauley LK. The multifaceted actions of PTHrP in skeletal metastasis. Future Oncol 2012;8:803-17.
    38. Panda S, Padhiary SK, Routray S. Chemokines accentuating protumoral activities in oral cancer microenvironment possess an imperious stratagem for therapeutic resolutions. Oral Oncol 2016;60:8-17.
    39. Bonfil RD, Chinni S, Fridman R, Kim HR, Cher ML. Proteases, growth factors, chemokines, and the microenvironment in prostate cancer bone metastasis. Urol Oncol 2007;25:407-11.
    40. Gronthos S, Zannettino AC. The role of the chemokine CXCL12 in osteoclastogenesis. Trends Endocrinol Metab 2007;18:108-13.
    41. Sambandam Y, Sundaram K, Liu A, Kirkwood KL, Ries WL, Reddy SV. CXCL13 activation of c-Myc induces RANK ligand expression in stromal/preosteoblast cells in the oral squamous cell carcinoma tumor-bone microenvironment. Oncogene 2013;32:97-105.
    42. Yuvaraj S, Griffin AC, Sundaram K, Kirkwood KL, Norris JS, Reddy SV. A novel function of CXCL13 to stimulate RANK ligand expression in oral squamous cell carcinoma cells. Mol Cancer Res 2009;7:1399-407.
    43. Pandruvada SN, Yuvaraj S, Liu X, Sundaram K, Shanmugarajan S, Ries WL, Norris JS, London SD, Reddy SV. Role of CXC chemokine ligand 13 in oral squamous cell carcinoma associated osteolysis in athymic mice. Int J Cancer 2010;126:2319-29.
    44. Oue E, Lee JW, Sakamoto K, Iimura T, Aoki K, Kayamori K, Michi Y, Yamashiro M, Harada K, Amagasa T, Yamaguchi A. CXCL2 synthesized by oral squamous cell carcinoma is involved in cancer-associated bone destruction. Biochem Biophys Res Commun 2012;424:456-61.
    45. Matsuo K, Irie N. Osteoclast-osteoblast communication. Arch Biochem Biophys 2008;473:201-9.
    46. Goda T, Shimo T, Yoshihama Y, Hassan NM, Ibaragi S, Kurio N, Okui T, Honami T, Kishimoto K, Sasaki A. Bone destruction by invading oral squamous carcinoma cells mediated by the transforming growth factor-beta signalling pathway. Anticancer Res 2010;30:2615-23.
    47. Papadimitrakopoulou VA, Brown EN, Liu DD, El-Naggar AK, Jack Lee J, Hong WK, Lee HY. The prognostic role of loss of insulin-like growth factor-binding protein-3 expression in head and neck carcinogenesis. Cancer Lett 2006;239:136-43.
    48. Partridge M, Kiguwa S, Luqmani Y, Langdon JD. Expression of bFGF, KGF and FGF receptors on normal oral mucosa and SCC. Eur J Cancer B Oral Oncol 1996;32B:76-82.
    49. Quan J, Elhousiny M, Johnson NW, Gao J. Transforming growth factor-β1 treatment of oral cancer induces epithelial- mesenchymal transition and promotes bone invasion via enhanced activity of osteoclasts. Clin Exp Metastasis 2013;30:659-70.
    50. Gao J, Ma Y, Shen J, Yao H. TGF-β promotes invasion and angiogenesis of oral squamous cell carcinoma SCC9 cells by upregulation of slug signal. Int J Clin Exp Pathol 2017;10:5325-33.