Research ArticleAlpha-2 Heremans Schmid Glycoprotein (AHSG) Modulates Signaling Pathways in Head and Neck Squamous Cell Carcinoma Cell Line SQ20B
Graphical Abstract
Introduction
Head and neck squamous cell carcinoma (HNSCC) includes cancers of the lips, oral cavity, salivary glands, nasal cavity, sinus, throat and larynx and is the 6th leading cause of cancer mortality in the world [1]. In 2012, it was estimated that approximately 37,000 Americans would be diagnosed with oral or pharyngeal cancer, with the preponderance diagnosed as late stages III and IV disease [2]. African-Americans are more likely diagnosed in advanced stages after adjustments for socioeconomic status, insurance status and other confounding factors [3]. The overall 5-year survival rate is 59%, although African-Americans have a 5-year survival rate of 39.5% which is broken down to 34% for African-American males and 52% for African American females [3], [4]. There are no prognostic markers or biomarkers for HNSCC and treatment is problematic due to a propensity for reoccurrence and loco-regional metastasis [5], [6]. Early diagnosis and an understanding of the progression of HNSCC would yield better results in treatment. In an attempt to identify serum biomarkers for HNSCC, we demonstrated that AHSG was elevated almost three times in the serum proteome of late stage HNSCC patients compared to controls and immunoblot analysis confirmed the presence of AHSG in primary tumors (Yarbrough and Marshall, unpublished data).
AHSG is a 63-kDa glycoprotein synthesized by the liver and secreted into the serum. The most widely accepted physiological function of AHSG is bone remodeling and inhibition of unwanted systemic ectopic calcification [7]. AHSG is associated with brain development and immune function including functioning as an anti-inflammatory mediator that participates in the inhibition of apoptosis of vascular smooth muscle cells [8]. It is a negative acute phase response protein [9], whose production decreases as disease load increases. We have shown, using in vivo animal models, that AHSG promotes breast cancer progression [9] and Lewis Lung Carcinoma tumorigenesis [10]. AHSG has been shown to be TGF-β receptor mimic in that its TRHI ( TGF-β receptor II homology domain I) motif closely resembles the TGF-β receptor II in structure. Therefore the level of AHSG expression or secretion can significantly alter TGF-β signaling in tumor cells. For example in intestinal tumors where TGF-β drives tumorigenicity, more tumors were observed in AHSG (fetuin-A) knockout mice [9]. Lastly we demonstrated that AHSG is capable of stabilizing matrix metalloproteinases in solution and preventing their degradation by autolysis [11]. We therefore followed both TGF-β signaling and the expression of MMPs in these sublines of HNSCC and questioned whether these molecules altered the growth of the cells.
For decades, debate raged as to whether fetuin-A (the bovine homolog of AHSG) was the major adhesion protein in serum, particularly fetal bovine serum that is generally used to supplement cell growth media in vitro [12]. We recently demonstrated using highly purified fetuin-A that it was the major attachment factor [13]. In the present study, we questioned whether AHSG, the human homolog of fetuin-A also supported attachment and growth of tumor cells. We also analyzed TGF-β signaling in the three sub-clones with different levels of AHSG expression.
In addition to these associations, AHSG has been shown to be a competitive inhibitor of TGF-β [11], [12], [14]. The TRHI motif in AHSG mimics TGF-β receptor II and therefore high expression and secretion of AHSG has the potential to down regulate TGF-β signaling. We therefore hypothesized that high expression of AHSG in EV and AH50 sublines would diminish TGF-β growth inhibitory properties but somehow reduce the growth of AH20 which express very low levels of AHSG.
Section snippets
Materials
Polyclonal antibody to AHSG was purchased from Meridian (Cincinnati, OH, USA). Monoclonal antibodies to total SMAD, pSMAD 2/3, total Erk, pERK1/2 and GAPDH were purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA). Monoclonal antibodies to MMP-9, β-actin and α-tubulin were obtained from Cell Signaling (Danvers, MA, USA). Unless otherwise indicated, cell culture reagents were purchased from Invitrogen.
Cell lines
The HNSCC cell lines SQ20B, FaDu and UMSCC47 were kindly donated by Dr. Wendell
AHSG expression in HNSCC cell lines
Initially we evaluated three human cell lines, SQ20B, FaDu and UMSCC47 for the presence of AHSG message and protein. Varying amounts of AHSG mRNA were detected, with SQ20B and FaDu showing greater expression than UMSCC47 (Fig. 1A and B). Immunoblot analysis confirmed the correlation between the AHSG message and protein (Fig. 1C). We hypothesized that AHSG enhances cell proliferation in AHSG-expressing HNSCC cell lines.
Fetal bovine serum (FBS), routinely used as a growth supplement for human
Discussion
The present study demonstrates that a number of established HNSCC cell lines synthesize AHSG and depletion of AHSG significantly decreases proliferation, adhesion, migration and invasion in vitro. Our data suggest that AHSG plays a role in tumorigenic properties of HNSCC. The liver produces most of the AHSG that is present in blood, which then is incorporated into cells [9], [14], [18]. For the first time we report that HNSCC cell lines synthesize AHSG and some of these cell lines also secrete
Conclusion
In conclusion, our data show multifaceted functions of AHSG in the progression of HNSCC. By depleting AHSG in the SQ20B cell line, we show decreases in adhesion, proliferation, migration and invasion. In future studies, transcriptomic and proteomic analyses of the depleted AHSG cell lines will be helpful in elucidating some of the effects associated with AHSG. This AHSG involvement with HNSCC may provide better insight into possible targets for therapeutic treatment.
Acknowledgments
This work was supported by grants from the SC1 CA134018-01 (JO); DOD W81XWH-07-1-0254 (JO); and U54 CA091408 (subproject DM) and 5 T32 HL007735-15 (SA). MMC Morphology Core is supported in part by NIH Grants U54MD007593, U54CA091408, G12MD007586, and S10RR0254970.
We thank Brandee Brown (VU), Joan Smith (MMC), and Kurt Watson (MMC) for their assistance with the production of this data.
References (35)
- et al.
Hypoxia-induced autophagic response is associated with aggressive phenotype and elevated incidence of metastasis in orthotopic immunocompetent murine models of head and neck squamous cell carcinomas (HNSCC)
Exp. Mol. Pathol.
(2011) - et al.
Structure of the gene encoding human alpha 2-HS glycoprotein (AHSG)
Gene
(1997) - et al.
Lack of fetuin-A (alpha2-HS-glycoprotein) reduces mammary tumor incidence and prolongs tumor latency via the transforming growth factor-beta signaling pathway in a mouse model of breast cancer
Am. J. Pathol.
(2010) - et al.
Members of the cystatin superfamily interact with MMP-9 and protect it from autolytic degradation without affecting its gelatinolytic activities
Biochim. Biophys. Acta
(2003) - et al.
Fetuin/alpha2-HS glycoprotein is a transforming growth factor-beta type II receptor mimic and cytokine antagonist
J. Biol. Chem.
(1996) - et al.
Fetuin-A ({alpha}2HS-glycoprotein) is a major serum adhesive protein that mediates growth signaling in breast tumor cells
J. Biol. Chem.
(2010) - et al.
Anchorage-independent growth of breast carcinoma cells is mediated by serum exosomes
Exp. Cell. Res.
(2009) - et al.
Fetuin-A triggers the secretion of a novel set of exosomes in detached tumor cells that mediate their adhesion and spreading
FEBS Lett.
(2012) - et al.
Fetuin-A: a major fetal serum protein that promotes “wound closure” and scarless healing
J. Invest. Dermatol.
(2008) - et al.
Characterization of 3 oral squamous cell carcinoma cell lines with different invasion and/or metastatic potentials
J. Oral Maxillofac. Surg.
(2007)
Prognostic value of MMP-2, -9 and TIMP-1,-2 immunoreactive protein at the invasive front in advanced head and neck squamous cell carcinomas
Pathol. Res. Pract.
TGF-beta1 induced MMP-9 expression in HNSCC cell lines via Smad/MLCK pathway
Biochem. Biophys. Res. Commun.
Portable oral cancer detection using a miniature confocal imaging probe with a large field of view
J. Micromech. Microeng.
Determinants of head and neck cancer survival by race
Head Neck
African American and poor patients have a dramatically worse prognosis for head and neck cancer: an examination of 20,915 patients
Cancer
Head and neck cancer
N. Engl. J. Med.
Head and neck tumors
Cancer Manage.
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