Altered expression of sialyltransferases in squamous cell carcinoma of the cervix and their clinical correlation

• Main Authors: Peng-Hui Wang, 王鵬惠
• Other Authors: Ying-Chieh Tsai
• Format: Others
• Language: en_US
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/67720684815916190882

博士 === 國立陽明大學 === 臨床醫學研究所 === 90 === Sialic acids including a number of their derivatives are ubiquitous at the terminal positions of oligosaccharides of glycoproteins. Due to their acidic nature, they impart a net negative charge to the cell surface and are important in cell-cell or cell-matrix interaction. The transfer of the sialic acids from cystidine-5-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to the terminal position of carbohydrate group of glycoproteins and glycolipids is catalyzed by a family of sialyltransferases (STs). There is a large body of evidence to suggest that tumor cells have changed surface properties from their normal counterparts, and that these changes are partially due to altered sialo-glycoconjugates expressed on the plasma membrane and that altered sialylation (change in glycoprotein expression), which occurs during certain pathological processes, such as oncogenic transformation, tumor metastases, and invasion, is associated with enhanced ST activity. Increased β1,6-branching, increased Sialyl-Lewis epitopes, increased Sialyl-Tn antigen (Sialyl α GalNAc-O-Ser/Thr), or the general increase in sialylation of cell surface glycoproteins are commonly observed in N-lined and O-lined oligosaccharides of carcinoma cells. Carbohydrate changes occur common in breast cancer, colorectal cancer, lung cancer, hepatic carcinoma, gastric carcinoma, head and neck squamous cell carcinoma, brain tumor, choriocarcinoma, and prostate cancer. More specifically, these changes in glycoprotein expression are reported to play important roles in tumor grade, invasion, metastatic ability, and poor clinical outcome. Some of these studies also evaluated the mRNA expression of STs and found that increased mRNA expression of STs is correlated with poor outcome in breast cancers, and colon carcinoma. There are some studies that only evaluated the relationship between the change of ST mRNA expression and their clinical correlation, including colorectal cancer, and gastric cancer. In Taiwan, carcinoma of the cervix is still the most common female cancer in Taiwan with an incidence rate of 32.1 per 100,000. The prognosis is dependent mostly on many factors; among them, the majority is based on histo-pathological parameters. Many pathological factors including bulky tumor size, poor differentiation, presence of lymph-vascular space involvement, presence of parametrial invasion, and deep stromal invasion are related with lymph node metastases and closely respond to disease-free survival. Besides these conventional pathological parameters for predicting prognosis in an early stage cervical cancer, there are many biological factors (cellular molecules), which show close correlation with aggressive and invasive behaviors in tumors. Among these biological factors, sialic acids are one of the most promising molecules because they involve cell-cell and cell-matrix interactions and cellular recognition. However, so far, altered expression of mRNA of STs in gynecological cancers is still unavailable. In addition, in our laboratory (Department of Biochemistry and Institute of Biochemistry, National Yang-Ming University), Dr. Wu guided by the professor Tsai (the Director in the program of my Ph.D. in Institute of Clinical Medicine, National Yang-Ming University) in his Ph.D. program used the screening strategy to find a very specific ST3Gal I inhibitor- soyasaponin I (Ki = 2.3 μM), which is 20-fold and 25-fold higher affinity to the active site enzyme than nature substrate CMP-Neu5Ac (KM = 46 μM) and product CMP (Ki = 55 μM), respectively. Soyasaponin I contains no significant inhibition activity for other glycosyltransferases and glycosidases, respectively. This unique finding provides a new feasible method to directly elucidate the functional roles of STs by inhibition ST activity under in vivo experiments and highlight the possibility of use the biological inhibitor for cancer prevention and/or treatment. Therefore, I selected this promising topic- altered expression of sialyltransferases in cervix squamous cell carcinoma and their clinical correlation as the study project for my Ph.D. program when I entered the Institute of Clinical Medicine, National Yang-Ming University since 1999, partly because the squamous cell carcinoma of the cervix is still the most common female cancer in Taiwan, partly because the clear-cut and long-term transformation from normal tissues of the cervix, to pre-cancer lesion (cervical intraepithelial neoplasia) and further toward the invasive squamous cell carcinoma of the cervix. Of the most importance, we manage two hundred women with the early-stage squamous cell carcinoma of the cervix (FIGO IB-IIA), which is treated with radical hysterectomy and pelvic lymph node dissection in our department (Department of Obstetrics and Gynecology, National Yang-Ming University based Taipei Veterans General Hospital annually. Based on the basic support from the Institute of Biochemistry (The director; Professor Tsai) and the clinical support from the Department of Obstetrics and Gynecology (The director: Doctor Yuan & The Section Chief: Doctor Chao), I can study the change of different-type sialyltransferases in cervical normal tissues and cervical squamous cell carcinoma first. Then, I can compare the difference between the normal tissues and cancer tissues to search for candidate type of sialyltransferases, and use the same strategy to evaluate the clinical correlation between their changes and the tumor behavior such as deep stromal invasion, lymph-vascular space involvement, parametrial invasion and lymph node metastases in clinical practice. Our goal was to clarify their roles in tumor carcinogenesis of cervical squamous cell carcinoma, and their correlation in clinically poor prognosis. We attempted to use these biochemical parameters combining with histo-pathological parameters to identify the high-risk women with recurrence or therapeutic failure. After confirming their roles in cervical cancer, we can highlight the possible role in cancer prevention and treatment using these specific and selective ST inhibitors in the future. The thesis is divided into four parts: first, to set up the study method including using substrate-enzyme assay for sialyltransferase activity, and letting blotting for measuring amounts of sialic acids on the cell line; second, to use semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) for evaluating the altered mRNA of sialyltransferases in cervical squamous cell carcinoma compared to those in normal cervical tissue; third, to use the above-mentioned methods to evaluate their changes in cervical squamous cell carcinoma to clarify their relationship between conventionally clinico-pathological parameters and expression of sialyltransferases and set up the possible value for clinical practice in the future. In the program part one, we successfully detected the activities of each subtype of sialyltransferases using Galβ1,3GalNAc-acetyl-lactosamine)-Obzl (acceptor for ST3Gal I), Galβ1,3GlcNAcβ1,3Galβ1,4GlcNAc (acceptor for ST3Gal III), Galβ1,4GlcNAc (acceptor for ST3Gal IV), asialo-bovine submaxillary mucin (acceptor for ST6GalNAc I), asialo-fetuin (acceptor for ST6GalNAc II), and fetuin (acceptor for ST6GalNAc III), respectively; we also successfully detected the amounts of sialic acids using fluorescein-conjugated Sambucus nigra agglutinin (SNA) specific for α2,6-sialic acids and fluorescein-conjugated Maackia Amurensis agglutinin (MAA) specific for α2,3-sialic acids. We found that increasing enzyme activity of ST3Gal I and ST6GalNAc II might be important in various kinds of gynecological cancers. More specifically, enhanced activity of sialyltransferases involving α2,6-sialic acid sugar chains might be more important in cancer development. Future studies will investigate whether the enzyme activity of these sialyltransferases can be helpful for clinical practice. Based on these findings, we entered the program part two. We directly switched to study the difference of mRNA expression of 4-type sialyltransferases (ST3Gal I, ST3Gal III, ST3Gal IV and ST6Gal I) between the cervical squamous cell carcinoma and the normal cervical tissue using semiquantitative reverse transcription-polymerase chain reaction method and found that sialyltransferase ST6Gal I expression was enhanced in squamous cell carcinoma of the cervix (p=0.026, Mann-Whitney U test), but mRNA expression from the other three sialyltransferases (ST3Gal I, ST3Gal III, and ST3Gal IV) was significant down-expression in squamous cell carcinoma of the cervix compared to the normal cervix (p=0.003, p<0.001, p=0.001, respectively). High ST6Gal I expression was associated with more invasive property of cervical cancer, such as deep stromal invasion, lymph-vascular space involvement, and poor differentiation (p=0.010, p<0.001, p<0.001, respectively). The conclusion of the study shows that combination of enhanced sialyltransferase ST6Gal I mRNA expression and decreased mRNA expression from ST3Gal I, ST3Gal III, and ST3Gal IV might be important processes in cervical cancer. Therefore, our goal of the next study, the program part three, will investigate whether RT-PCR detection of the expression of these enzymes can be helpful for prognostic purposes. In the program part three, this study further investigates their changes in mRNA expression of the four STs in FIGO stage IB1 squamous cell carcinoma to assess the extent of sialylation associated with lymph node metastases. We evaluated the alterations in ST mRNA expression in FIGO IB1 cervical squamous cell carcinomas using RT-PCR and substrate-enzyme assay methods. We found that both ST6Gal I mRNA and ST3Gal III mRNA expressions were significantly increased in patients with lymph node metastases compared to those without lymph node metastases (p=0.002 and p=0.001, respectively, Mann-Whitney U test). Using receiver operating characteristic (ROC) curves of ST ratio index for accuracy comparison of lymph node metastases, ST3Gal III and ST6Gal I were observed to be fairly interchangeable (area under the curve (AUC) of 3Gal I=0.810; AUC of 6Gal I=0.786, significance of difference between AUC=0.810). High ST6Gal I expression was associated with other invasive properties of cervical cancer, such as deep stromal invasion and presence of lymph-vascular space involvement. ST6Gal I expression seemed to be more enhanced in bigger tumors. Our conclusion suggested that ST3Gal III and ST6Gal I were of importance for the lymph node metastases in FIGO IB1 cervical cancer patients; more specifically, over-expression of ST6Gal I was of crucial relevance for the presence of poor-prognostic factors, such as deep stromal invasion and lymph-vascular space involvement and lymph node metastases. In the program part four, based on the fact that increasing messenger ribonucleic acid (mRNA) expression of ß-galactosideα2,6-sialyltransferase I (ST6Gal I) is important in squamous cell carcinoma (SCC) of the cervix. In many tissues, ST6Gal I is transcriptionally regulated through the use of different promoters that originate in the mRNAs which diverge in the 5’-untranslated regions. In this study, to clarify the roles of ST6Gal I mRNA species in cervical SCC, we investigated their expression including a “constitutive” promoter- placental form (Y + Z form), “hepatic” promoter and a specific lymphoblastic promoter (X form) in normal and SCC tissues of the cervix using real-time quantitative reverse-transcription-polymerase chain reaction (RT-PCR). Expression of the ST6Gal I species was investigated in normal cervices (n = 38) and FIGO IB1 cervical SCC (n = 38) by real-time quantitative RT-PCR, using primers designed for amplification of a portion of the coding region common to all mRNA species, or ones for amplification of the placental transcript (Y + Z form), the hepatic transcript (H form) or lymphoblastic transcript (X form). Our results showed that ST6Gal I mRNA expression was significantly increased in cancerous tissues compared to that in normal tissues (p = 0.004, Mann-Whitney U test). Expressions of the Y+Z form did not appear to be affected by cancer transformation, since it was detected at comparable levels in normal and cancerous tissues (p = 0.13), but the H form expression was significantly enhanced in cancerous tissues compared to that in normal tissues (p < 0.0001). Surprisingly, the X form could be detected in a portion of patients either with or without cancer, but the detection rate was significantly higher in patients with cancers compared to those without cancers (86.8% vs. 52.6%, p= 0.021, Fisher’s exact test), although the X transcript was only detected at a very low level compared to the H and Y + Z transcripts. Therefore, we concluded that an increased levels of hepatic transcripts might be very important in cancer transformation because it contributes to enhanced ST6Gal I expression in cancerous tissues. A high percentage of the X form transcript in cancerous tissues might resulted from lymphocyte infiltration, and may involve a host-tumor interaction, although the clinical evidence for this is not clear yet. According to all findings in this project of Ph.D.- altered expression of sialyltransferases in cervix squamous cell carcinoma and their clinical correlation, we can clearly identify the important and significant role of altered sialyltransferase expression in the cervix squamous cell carcinoma. ST6Gal I overexpression is a very important process during the establishment of cervix SCC. With progressively growth and invasive process, enhanced ST6Gal I expression became more importance. This ST6Gal I overexpression mainly results from the activating 5’ untranslated H form promoter although both Y + Z and H transcripts contribute the total pool of ST6Gal I. When entering the metastatic status, initiated overexpression of ST3Gal III should be occurred, because overexpression of both ST6Gal I and ST3Gal III could predict pelvic lymph node metastases accurately in FIGO IB1 sqamous cell carcinoma of the cervix. We highlight the vision that, with advancing biotechnology, more specific molecules against different kinds of STs might be found in the near future, just like soyasaponin I, which is a potent and specific sialyltransferase inhibitor for ST3Gal I and is first identified in our laboratory. Using this strategy might provide a vision of possibly synergistic therapy in cervical cancer patients or offer an effective tool in cancer prevention, especially for cervix squamous cell carcinoma.