ONCOSTATIN-M EXPRESSION IN ORAL SQUAMOUS CELL CARCINOMA.

Noha Shaban Shams 1 , louloua Mohamed Fathy 2 and Amal Hassan Abd-El Rahman 2 . 1. Lecturer, Oral and Maxillofacial Pathology Department Faculty of Oral and Dental Medicine Ahram Canadian University. 2. Professor of Oral and Maxillofacial Pathology, Faculty of Oral and Dental Medicine, Cairo University. 3. Professor of Oral and Maxillofacial Pathology, Faculty of Oral and Dental Medicine, Cairo University. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 6(1), 22-28 23 It is now widely accepted that inadequately resolved chronic inflammation may increase the risk of cancer by providing bioactive molecules from cells infiltrating the tumor microenvironment. This includes cytokines; growth factors; chemokines that maintain a sustained proliferative rate; cell survival signals to avoid apoptosis; proangiogenic factors and extracellular matrix-modifying enzymes (9) .
Cytokines are low-molecular-weight proteins that mediate cell-to-cell communication, they bind to their receptors and trigger intracellular signaling events that result in the modulation of gene expression. Most cytokine receptors are comprised of a multi-subunit complex containing a unique and specific ligand binding subunit and a signal transducing subunit. Immune and stromal cells, such as fibroblasts and endothelial cells, synthesize them and they regulate proliferation, cell survival, differentiation, immune cell activation, cell migration, and death. Depending on the TME, cytokines can modulate an antitumoral response, but during chronic inflammation, they can also induce cell transformation and malignancy, conditional on the balance of pro-and anti-inflammatory cytokines, their relative concentrations, cytokine receptor expression content, and the activation state of surrounding cells (26) .
Oncostatin-M (OSM) was first purified and biochemically characterized on the basis of its anti-proliferative activity on the A375 human melanoma cell lines in vitro (25) . Its name was coined based on this inhibitory function on A375 and other melanoma cell lines. The crystal structure of human OSM reveals a typical hematopoietic cytokine topology with up-up-down-down four-helix bundle with specific site 2 and site 3 epitopes predicted to interact with receptor chains on cell surfaces (16) .
In contradiction of other members of this family, OSM binds gp130 with low affinity and as such has slight to no biological activity unless a second receptor chain is recruited, either the LIFRα or the specific OSM Receptor-beta chain (OSMRβ) (15) .
Individual cell populations that express OSM comprise activated macrophages, monocytes, T cells, and dendritic cells (24) . It could be released by neutrophils from preformed stores and these cells could also synthesize OSM upon stimulation by granulocyte colony stimulating factor (5) .
Along with OSM role in inflammation, an impending role in inhibiting cellular proliferation was explored for a while, far along its functions have been further explored in various cancers and tumor systems. Researches done on OSCC are relatively limited thus this study anticipated to explore its expression on protein level (23) .

Materials and Methods:-
A total of 30 specimens of OSCC and 10 normal epithelial tissues were retrieved from the archival paraffin blocks from the pathological files of the Department of Oral and Maxillofacial Pathology, Faculty of Oral and Dental Medicine, Department of General Pathology, Faculty of Medicine Cairo University and from Maxillofacial Surgery Department of Naser Institute. Normal tissues were obtained from normal gingival tissues from crown lengthening procedures. 65% of patients were males and most cases were obtained from tongue, floor of the mouth and palate. This work was approved by ethics committee of Cairo University Tissue sections were stained with Hematoxylin and Eosin stain and were rediagnosed to confirm the previously made diagnosis. Other consecutive sections were cut on the positively charged glass slides (Opti-Plus, Biogenex Laboratory, USA) for imunohistochemical staining.

Methods:-
Formalin-fixed, paraffin-embedded 5-m-thick sections from a representative block in each case were subjected to OSM rabbit polycolonal antibody (Cat.No.NBP1-87768, Bioss, SNF MEDICAL, USA).. Immunohistochemical staining was performed using the The universal immunostaining detection kit (Dako Cytomation Envision+ Dual link system, Peroxidase (DAB+) Code K4065, Carpinteria, CA, USA) first Epitope retrieval: Citrate Buffer Epitope Retrieval method was used in this technique The tissue were incubated for I hour at room tempreture in primary antibody. To confirm the specificity of the primary antibodies, negative control slides were run with every batch 24 using serum buffer substrate at the same concentration as that of the primary antibodies then Secondary antibody in the form of biotinylated horse Anti-Mouse IgG diluted in secondary antibody dilution was added. Finally chromogen/substrate sections were incubated in DAB peroxidase substrate solution followed by counter stain.
In order to provide more précised data on OSM immunoexpression in OSCC, image analyzer was used. The data was obtained using the software (SIS, Germany), and by using the image analyzer, five specific fields showing maximum positivity of OSM expression were used in each case; the mean of five values of each slide was calculated and statistically analyzed. For each field of immunostained section, a measuring frame of area 1920000 pixels and then the area % was calculated. The color of immunostaining was automatically selected according to the positive control sections, converted into grey color then masked the red and white to allow automatic measurements by the computer system. Data obtained were subjected to statistical analysis using (ANOVA) test.

Results:-
Normal epithelial tissues showed faint cytoplasmic expression of OSM among epithelial cells in addition to positive expression in connective tissue stroma especially collagen fibers (Fig.1). In well differentiated OSCC epithelial cells in all specimens showed higher homogenous cytoplasmic OSM expression compared to normal as well as in keratin pearls, while stromal cells showed positive expression in collagen fibers along with tumor associated inflammatory cells especially tumor associated macrophages (Fig.2). Statistically, within the different grades of OSCC, The greatest mean value was recorded in poorly differentiated SCC, with the least value recorded in well-differentiated OSCC. One way analysis of variance (ANOVA) test revealed that the difference between different grades was statistically significant (P<0.0001). Tukey's post hoc test revealed no significant difference between well and moderately differentiated SCC. Moreover, the mean value recorded in control was significantly lower than all grades of OSCC (P<0.0001) Fig. (5).

Discussion:-
It was found that expression of OSM has remarkable roles in inflammation and tumorgenesis as modulator of cellular proliferation, epithelial mesynchymal transition, angiogenesis and metastases, thus further investigations of its expression on protein levels was required (18) .
In the current study, all OSCC specimens showed variable homogenous cytoplasmic OSM immunoexpression in tumor cells. This finding may be attributed to endogenous synthesis of OSM by the tumor cells or it may denote a bound form of OSM and its receptor .Although OSM is predominantly formed by activated T lymphocytes and macrophages, the possibility that OSM is produced by tumor cells was previously supported by studies of OSM synthesis in Kaposi's sarcoma (1) , in OSCC (22) , in prostate carcinoma (19) , in breast cancer (7), in esophageal SCC (12) and lastly in OSCC Chuerduangphui et al suggested that keratinocyte growth factor can stimulate OSM expression in tumor cells (2) .
We detected that the most pronounced OSM immunoexpression was recorded in poorly differentiated OSCC while the least expression was found in well differentiated ones where the difference was statistically significant indicating its role in tumor progression. Kozlowska et al explained OSM role in progression where they reported a potential role for OSM to inhibit the activity of metalloproteinase inhibitor which explained the role of this cytokine in neoplastic metastases (13) . Furthermore, studies done on the role of OSM in breast cancer have shown that a complex set of activities are likely involved. Snyder et al, have shown that OSM induces a STAT3-dependent mechanism and thus contributes to a migratory and invasive phenotype of breast cancer cells by inhibiting apoptosis through induction of bcl-2 or TGF-beta (20) . Furthermore, STAT-3 whenever it is activated by gp130 it induces activation of c-myc gene and up-regulation of cyclins D1, D2, D3, which are responsible for the G1 to S cell-cycle progression signals that are elicited by STAT3 that has been activated by gp130 (10) . In addition, OSM can induce EMT and cancer stem cell-like features in breast cancer cells in vitro (8,24) .
As regards the stromal immunoreactivity of OSM, it was identified in all specimens in fibroblasts and collagen fibers, this was explained by Somasundaram et al who reported that human OSM has been found to bind to collagens I, III, and IV in vitro (21) .
Only two specimens out of thirty OSCC, where the neoplastic cells showed faint OSM immunoexpression while the stromal expression showed intense immunoexpression. Comparable results were observed by Tran et al, they explained this indirectly proportional relationship by the possibility of presence of negative feedback mechanism between the tumor cells and the surrounding infiltrative macrophages (22). Inefficient OSM protein expression in neoplastic cells was also explained by Radtke et al who suggested the presence of abnormalities in various stages of OSMRβ production and activation (17) , furthermore, Lacreusetteet al., 2007 in their study on melanoma, identified epigenetic modifications in the OSMRβ promoter as histone deacetylation (14) . Another potential mechanism was proposed by Kausar et al who detected a spliced variant encoding a soluble form of OSMRβ evident in esophageal cancer tissue which binds with OSM and thus acts as neutralizing receptor for OSM (12) . This hypothesis was also confirmed by Diveu et al in their study on glioblastoma (4) .

Conclusion:-
It might be concluded that, OSM production is modulated through stromal and neoplastic cells which in turn have an important role in OSCC progression and hypothesizes the possibility of its use as biomarker.