Expression And Regulation Of Urokinase-type Plasminogen Activator In Supraglottic Laryngeal Cancer

ObjectiveLaryngeal carcinoma is a very common malignant tumor in head and neck, and 93% -99% tissue type of this disease is laryngeal squamous cell carcinoma (LSCC). It is paid great attention to in recent years, because the morbidity of LSCC has been increasing gradually. Clinical data showed; the morbidity of LSCC has great difference in different areas in our country, and is highest in northeast area, especially the supraglottic carcinoma is much common. Because of anatomic position, the supraglottic larynx has rich lymphatic drainage, resulting in a high incidence of cervical lymph node metastasis and occult cervical lymph node metastasis, which led to poor prognosis in patients with supraglottic carcinoma. Therefore, it is significant for improving effective treatment of LSCC to analyze mechanism of invasion and cervical lymph node metastasis.Tumor invasion and metastasis is a multistep process. Many kinds of proteases released from cancer cells degrade proteins in extracellular matrix (ECM) and basement membrane is a prerequisite for cancer invasion and metastasis. U-rokinase - type plasminogen activator ( uPA) catalyzes the proteolytic conversion of the inactive zymogen plasminogen to the active proteinase plasmin, which in turn degrade ECM, plays an important role in tumor invasion and metastasis. uPA receptor (uPAR) and type I plasminogen activator inhibitor ( PAI - 1) have modulatory effect on plasminogen activation. uPA, uPAR and PAI - 1 areoverexpressed in many kinds of human malignant tumor, and they are closely associated with tumor invasion and metastasis. Mitogen activated protein kinase (MAPK) is one of most important membrane - to - nucleus signaling mechanisms and extracellular signal regulated kinase (ERK) pathway including ERK1 and ERK2 is a key and classic pathway of the system. It has been indicated that deregulation activation of the signal pathway facilitate up - regulating expression of the proteolytic enzymes associated with tumor. Epigenetic modification is an important regulation mechanism of gene expression. Recent researches have shown methylation status of uPA promoter modulation expression involved in tumor invasion and metastasis.Starting from DNA, mRNA and protein levels, the aim of this research was to investigate the correlation between uPA expression and invasion and metastasis of supraglottic carcinoma. We also analyzed if abnormal activation of ERK signal pathway can promote up - regulating uPA expression. This study will provide new information for research on the mechanism of invasion and metastasis of LSCC.Materials and Methods1. Tissue sample; 40 cases of fresh supraglottic carcinoma tissues were obtained from 29 male and 11 female patients ( median age 63. 2years; range, 46-81years) underwent surgical resection in the first and the second affiliated hospital of China Medical University from February in 2003 to March in 2004. 10 cases of normal peripheral tissues around cancer were obtained from total lar-yngectomy as control.2. Reverse transcription polymerase chain reaction ( RT - PCR) was applied to detection of uPA, uPAR and PAI - lmRNA expression in supraglottic carcinoma and normal peripheral tissues.3. Western blot was introduced to detect ERK1/2 protein expression; and in the meanwhile the change of ERK1/2 activity was examined to measure its efficiency of phosphate transferase in supraglottic carcinoma.4. Immunohistochemsty was applied to detection of uPA protein expressionin supraglottic carcinoma.5. Methylation specific PCR (MSP) was used to detection methylation status of uPA promoter in supraglottic carcinoma.Results1. Positive expression rate of uPA, uPAR and PAI - lmRNA in LSCC was respectively 65% (26/40) , 55% (22/40) and 60% (24/40). There was a significant positive linear association among uPA, uPAR and PAI - 1 mRNA positive expression, and also for level of expression( P <0.05). Neither T - category , nor grade was significantly related with positive expression and level of expression of uPA, uPAR and PAI - lmRNA. There were significant correlation between uPA, uPAR and PAI - 1 mRNA positive expression or level of expression and TNM stage or cervical lymph node status ( P <0.05). There was obviously increased in cases with clinical late stage or cervical lymph node metastasis. LSCC cases with concourse positive expression of uPA, uPAR and PAI -lmRNA revealed significant higher tendency of cervical lymph node metastasis than those with concourse negative expression ( P <0. 05). The result of Logistic regression analysis showed expression of uPAmRNA was associated with cervical lymph node metastasis in LSCC ( Wald = 4. 372, P = 0.037).2. In laryngeal carcinoma, level of ERK1 protein expression appeared to be correlated with level of ERK2 protein expression(r = 0. 467, P = 0. 002). Neither level of ERK1 or ERK2 protein expression was significantly related with T -category, grade, TNM stage and cervical lymph node status. Level of ERK1/2 activity was neither correlated with level of ERK1 nor ERK2 protein expression (r = 0. 175 , P = 0. 280; r = 0. 183 , P = 0. 258 ). Neither T - category, nor grade was significantly related with level of ERK1/2 activity. There was significant correlation between level of ERK1/2 activity and TNM stage or cervical lymph node status(P <0.05). There was obviously increased in cases with clinical late stage or cervical lymph node metastasis. Neither T - category, nor grade was significantly related with level of uPA protein expression. There was significant correlation between level of uPA protein expression and TNM stage orcervical lymph node status ( P <0. 05 ). There was obviously increased in cases with clinical late stage or cervical lymph node metastasis. Level of uPA protein expression was neither correlated with level of ERK1 nor ERK2 protein expression (r, = 0.248, P = 0. 123; ra = 0. 199, P = 0. 219). There was a significant positive linear association between level of uPA protein expression and level of ERK1/2 activity (r8 =0. 504, P =0. 001). In the cases of uPA protein strong positive expression, the level of ERK1/2 activity was 1.28 times higher than in the cases of uPA protein weak positive expression (t =2.531, P =0.019).3. Methylation of uPA promoter was observed in 8 (20% ) of 40 cases of la-ryngeal carcinoma. In the cases of uPA protein positive expression, methylation of uPA promoter was apparently fewer than in the cases of uPA protein expression deletion(P <0. 05 ). Neither T - category, nor grade was significantly related with methylation of uPA promoter. There was significant correlation between methylation of uPA promoter and TNM stage or cervical lymph node status (P <0. 05). There was obviously decreased in cases with clinical late stage or cervical lymph node metastasis. Neither T - category, nor grade was significantly related with uPA protein expression. There was significant correlation between uPA protein expression and TNM stage or cervical lymph node status ( P < 0. 05). There was obviously increased in cases with clinical late stage or cervical lymph node metastasis.Conclusions1. Abnomal transcription activation of gene resulting in up - regulation of uPA, uPAR and PAI - lmRNA expression which are related with carcinogenesis and development of LSCC.2. Up -regulation of uPA, uPAR and PAI - lmRNA expression and positive linear association among them which may contribute significantly to LSCC invasion and metastasis.3. Concourse positive expression of uPA, uPAR and PAI - lmRNA in LSCC is much associated with cervical lymph node metastasis which indicated poor prognosis.