| Colorectal cancer (CRC) is one of the common malignant tumors occurred in the digestive system. Though the people’s living standards and the medical condition greatly raised in China, the incident rate of CRC has been increasing because of the environmental problems following the social development and the change of people’s life style. As there has been a remarkable breakthrough in the early diagnosis and treatment of CRC during recent years. But the therapy of advanced CRC fails to yield gratifying outcomes, while the survival rate of patients with advanced CRC has not been well improved. Therefore, it will provide great significance to further study on the mechanism about initiation, development and metastasis of CRC. And it’s urgent to discover the new molecular markers which can be used in the early diagnosis, preventment and individual therapy of CRC, thus eventually improve the life quality of the patients.As the initiation and development of CRC are extremely complex, the underlying mechanisms remain largely unclear. Using the disease model of colorectal cancer, our lab team has been dedicated to study the mechanism and screen diagnostic or prognostic markers of CRC for many years. In 2010, we found that the growth differentiation factor 15 (GDF15) is a secreted protein specifically expressed in the normal colon tissues by the differential secretome method. Besides, GDF15 level is higher in the culture supernatant and cell lysis of lymph node metastases cell line SW620 than of the same colorectal cancer patient’s primary focal cell line SW480. In brief, GDF15 may play an important role in CRC. We have proceeded a series of studies to further clarify its functions in CRC.GDF15, located at p13.11 on chromosome 19, is a divergent member of transforming growth factor (TGF-β) super family. Our previous study has shown that the expression of GDF15 in the serum is markedly higher in CRC patients than in normal population, and it is especially higher in patients with lymph node metastasis than those without metastasis. It was further confirmed by immunohistochemistry that the expression of GDF15 in the tumor tissues from patients with lymph node metastasis is significantly higher than that from patients without metastasis.All the above results have demonstrated that GDF15 is closely related to the CRC metastasis and can be used as a new marker for the diagnosis of metastasis. But the mechanism of metastasis and the effect of prognosis remain unknown. Processing our study about GDF15, we found that the CDS region of GDF15 gene in different CRC cell lines was variant with C or G at the 604 bases site. After checking related information, we found that it is a polymorphic site of GDF15 named rs 1058587 (C/G). Furthermore, when comparing rs1058587 in tumor tissues and their matched normal tissues, we found an interesting phenomenon that different rs1058587 genotypes exist in the same patient (e.g. CG transformed into CC). Thus we suspected that there should be a mutation more than only a polymorphism at GDF15 rs1058587. The rs1058587 is also known as H6D polymorphism as it can turn the sixth histidine (H) of the mature GDF15 into aspartic acid (D). Here we named its mutation as H6D mutation. Either H6D polymorphism or so-called mutation may lead to a change in the structure and even the function of GDF15 protein.In view of all results above, we wonder whether there exist a correlationship between susceptibility of H6D polymorphism and the occurrence of CRC? Whether its polymorphism or mutation will affect the levels of GDF15 or have a concern in the clinical pathologic features such as metastasis and prognosis? What is the metastatic mechanism of CRC caused by GDF15? All these questions are what we intend to explore in this study.1. The relationship between GDF15 and metastasis or prognosis and the underlying mechanism in colorectal cancerThe expression of GDF15 in 250 tumor tissues was determined by immunohistochemistry. And positive correlations between GDF15 and lymphatic metastasis, distant metastasis, TNM stage and tumor bud number were confirmed. We also found that high expression of GDF15 is a risk for poor prognosis.Next, using recombinant human GDF15 protein in colorectal cancer cell lines HT29, SW480 and LS513, we simulated the paracrine pathway in vitro to study the metastatic mechanism of CRC.Results of transwell assays showed that the migration ability of HT29 and SW480 cells is promoted by GDF15 treatment. In the subsequent search of involving signaling pathway, we found that GDF15 could enhance the expression and phosphorylation of Smad2/3 proteins in HT29 and SW480 cells. Moreover, the expression of Twist and MMP9 were increased in HT29 and LS513, combined with a decreased expression of E-cadherin. In SW480, the expression of Twist, MMP9 and Vimentin were all increased, as E-cadherin decreased. The results indicate that GDF15 may promote migration of CRC cells through Epithelial-Mesenchymal Transition (EMT) procedure.To identify whether the expression of EMT markers were regulated by Smad2/3, we knocked down Smad2 and Smad3 via shRNA in HT29 and SW480 cell lines. The results showed that the function of GDF15 can be reversed, which confirmed that the changes in the expression of EMT markers caused by GDF15 are directly regulated by Smad2/3.Because GDF15 is a member of TGF-β superfamily, which is commonly used to induce EMT via activating the Smad2/3 proteins. And our studies showed that Smad2/3 can also be activated by GDF15. So we proposed a hypothesis that GDF15 promotes cell migration and the process of EMT via TGF-β similar signaling pathway.To confirm this assumption, HT29, SW480 and LS513 cells were pretreated by SB-431542 (a specific inhibitor of TGF-β receptor) and then treated by GDF15. The results showed that the expression of Smad2/3 and E-cadherin were not changed. Furthermore, when HT29 and SW480 cells were treated by SB-431542 and GDF15, phosphorylation of Smad2/3 proteins were also not changed. Thus, all the results illustrated that the expression changes of EMT markers caused by GDF15 depend on the TGF-β receptor-Smads signaling pathway.2. The clinical significance of GDF15 H6D polymorphism or mutation in colorectal cancerWe examined the H6D polymorphic site of GDF15 in the tumor tissues of CRC and their matched normal tissues from 250 patients, as well as the serum samples from 198 normal population by pyrosequencing. Then the data were comprehensively analyzed with the clinical pathologic features and prognosis, the results were showed as bellow:(1) There is no significant correlation between the expression levels of GDF15 and its H6D polymorphism or mutation. (2) There is a correlation between GDF15 H6D polymorphism and the occurrence of colorectal cancer. The people with D allele show increased risk of colorectal cancer. (3) There is a H6D mutation of GDF15 in CRC, and the mutation is prior to occur in a CG genotype. Besides, the mortality of patients with mutation are higher than those without mutation. (4) The male CRC patients with H6D mutation are prior to occur distant metastasis with poor prognosis. (5) In colon cancer, patients with D allele of GDF15 are more likely to occur distant metastasis and have a higher mortality. The homozygote HH is a protective factor for prognosis. (6) In rectal cancer, patients with H6D mutation present the higher risk of lymph node metastasis and advanced TNM stage, which leads to a higher mortality.The following conclusions were drawed according to the above study.GDF15 could promote the metastasis of CRC and the patients with high levels of GDF15 exhibit poor prognoses. GDF15 could promote the occurrence of EMT via the TGF-β receptor signaling pathway and facilitate migration of CRC cells. GDF15 H6D polymorphism or mutation is related with the initiation and development of CRC. D allele is susceptible to the occurrence of CRC and has a positive correlation with distant metastasis of colon cancer, patients with D allele have poor prognosis. In CRC, GDF15 H6D polymorphism exist mutation pattern, and this is positively correlated with distant metastasis of male CRC. |
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