Study On The Significance Of Macrophage Migration Inhibitory Factor In The Adenoid Cystic Carcinoma Of Salivary Gland

Adenoid cystic carcinoma (ACC) is a malignant epithelial tumor, frequently arising in salivary glands with a poor long-term prognosis, and comprised proximately10%of all epithelial salivary neoplasm. It is characterized by:(1) indolent propensity but high rates of recurrence,(2) aggressive histologic features such as perineural and blood vessel invasion,(3) favorable local control rates,(4) high incidence of distant metastasis, yet a very low incidence of regional metastasis,(5) the possibility of stable metastasis, and (6) the treatment of ACC is based on surgery and adjuvant radiotherapy. The tumor is composed of cells that exhibit either luminal epithelial differentiation or myoepithelial differentiation. Three growth patterns have been described for SACC:solid, tubular and cribriform pattern. At present, the biological mechanisms on the proliferation, differentiation, invasion and metastasis of SACC are still unclear. The current therapies available for the management of patients with ACC are inadequate to achieve local control predictably by the aggressive strategy of surgery and irradiation therapy.Since the activity of macrophage migration inhibitory factor (MIF) was first reported in1966, it has gone from a cytokine activity modulating monocyte and macrophage motility. MIF is a trimeric protein composed of identical12.5-kDa subunits, and ubiquitously expressed in different cells and tissues. MIF has structural similarity to the bacterial tautomerase enzymes. MIF is a highly conserved cytokine with multifunctional pleiotropic effects involved in immune responses and several inflammatory diseases. Apart from the inflammatory and immunological functions, MIF is considered to play an important role in cell proliferation and differentiation, tumorigenesis, evasion of apoptosis, angiogenesis, tissue invasion and metastasis and so forth.Previous studies have documented that MIF mainly exerts its biological functions through receptor-mediated pathways including binding to CD74, CD44, CXCR2and CXCR4. However, the precise mechanisms underlying MIF-modulated pathways and their associations with cancer progression remain elusive. Based on the above considerations, we proposed that MIF possibly played potential roles in the pathogenesis of SACC. We investigated the expression status and functional significance of MIF in ACC tissue and cell line, explored the probable mechanism. Meanwhile, influence of MIF on ACC cell migration and invasion was also evaluated in vitro, so as to provide experimental evidence on searching novel therapeutic targets of ACC. And there are three parts as follows:Part Ⅰ Expression of macrophage migration inhibitory factor in adenoid cystic carcinoma and the correlation with metastasisObjective:To investigate the expression status of MIF in SACC tissue, and compare the expression of MIF in different histopathological patterns of SACC.Methods:Immunohistochemical staining was performed to evaluate the expression level of MIF in SACC tissue (40cases), squamous cell carcinoma tissue of tongue (5cases), and normal salivary gland tissue (5cases), then the immunoreactivity of MIF was assessed according to a semiquantitative score. All patients were followed up.Results:As the immunohistochemical results shown, positive expression of MIF was detected in all of the SACC samples, showing moderate-to-strong staining. MIF was located in cytoplasm and/or nucleus. The mean score of MIF staining in SACC tissues was significantly higher than that in NSG tissues. However, no significant difference was found among the solid, cribriform, and tubular types of SACC samples (P>0.05). The expression of MIF was significantly increased in cases with metastasis compared with those without metastasis(P=0.027<0.05).Conclusions:The overexpression of MIF in SACC tissue suggests that it possibly plays a significant role in the pathophysiology and progression of SACC, and there is no relationship between the MIF and the SACC pathological patterns. MIF overexpression was positively associated with the distant metastasis.Part Ⅱ Expression of the MIF correlative proteins in adenoid cystic carcinomaObjective:To investigate the expression status of MIF correlative proteins:HIF-1α, MMP-9, P53, p-JNK and CD74.Methods:Immunohistochemical staining was performed to evaluate the expression levels of MIF, HIF-la, MMP-9, P53, p-JNK and CD74in SACC tissues.Results:As the immunohistochemical results shown, the proteins were overexpressed in SACC, and all of them were significantly different between NSG and SACC tissues (P<0.05). The expression level of MIF showed a significant negative correlation with p-JNK, but showed no relationship with HIF-la or P53. Importantly, the results from doublelabeling immunofluorescence histochemistry also confirmed the negative correlation between MIF overexpression and p-JNK activation in SACC, for the observation that the ACC cells with high expression of MIF were almost negative for p-JNK nuclear location. The staining also revealed a moderate positive correlation between the staining of MIF and MMP-9(P<0.05, r=0.444).Conclusions:The overexpression of MIF in SACC tissue might contribute to its distant metastasis. Meanwhile, there is a negative correlation between the staining of MIF and p-JNK.Part Ⅲ The effects and mechanisms of MIF on adenoid cystic carcinoma cell line (ACC-2)Objective:To investigate the effect of MIF on ACC, and to explore the mechanisms of MIF in ACC.Methods:In vitro, ACC-2cells were exposed to recombinant human MIF (rMIF), ISO-1or transfected by MIFsiRNA, followed by the detection of cell growth, viability, migration, and invasion, as well as the expression levels of several cellular signals. Cell proliferation and viability were measured by MTT assay, and cell counting assay; cell migration was assessed by the wound healing assay and the migration assay, and the ability of invasion was investigated by invasion assay. Furthermore, the expression levels of the proteins (MIF, P38, MMP-9, P53, HIF-1α, ERK, p-ERK, Akt p-Akt, JNK and p-JNK) were assessed by the western blotting.Results:rMIF (0-200ng/ml) could mildly promote the ACC-2proliferation after72h-treating. And, rMIF enhanced the effects of cell immigration and invasion slightly; The treatment for ACC cells with ISO-1significantly attenuated cell migratory and invasive capacity, as opposed to the promotive effects of rMIF. Different concentration of ISO-1, especially200μM, could weaken the effects of proliferation, immigration and invasion but the viability of the ACC cells in24h. Further analyses showed a significant correlation between the expression of the MIF and p-JNK, MMP-9; Efficiency of MIF siRNAs was analysed by quantitative RT-PCR and Western blottings after cell transfection. From siRNA assays to deplete the endogenous level of MIF in ACC cells, the effects of rMIF on the tumor cells were found that rMIF promoted cell proliferation, migration and invasion significantly in these MIF-depleted cells.Conclusion:The results suggest that MIF is likely to be an important player in the pathogenesis of ACC and may promote cancer cell proliferation and immigration, which possibly involves JNK inactivation. Further investigation of MIF-mediated molecular events may provide novel insights into the treatment for ACC.