| Nodal is a cytokine which belongs to the TGF-β superfamily. In Nodal signal transduction, it binds to Type II membrane receptor ACTRII or ACTRIIB and initiates the recruitment and activation of Type I membrane receptor ALK4 or ALK7, which subsequenctly enables the intracellular Smads to be phosphorylated and translocated into nucleus and regulate the expression of target genes. Nodal was firstly discovered to function in embryonic development, while recently it has been revealed that Nodal also has tumor-promoting functions but the molecular mechanism behind remains to be investigated. So far, the functional relationship and roles of Nodal and its receptor ALK4 or ALK7 in oncogenesis are still unclear, particularly due to the various recognition modes of ligands to the receptors in TGF-β superfamily. Among all malignancies, breast cancer has the highest morbidity and second highest mortality in women, especially in China. In this thesis, we mainly explored the expression patterns, function and clinical significance of Nodal and its receptor ALK4 and ALK7 in breast cancer development. Firstly, we detected the expression of Nodal and its receptor ALK4 and ALK7 in different parts of the breast with cancer including invasive ductal carcinoma, lymph node metastasis, ductal carcinoma in situ, adjacent normal tissue, fibroadenoma and adenosis tissues. Further, Western Blotting was introduced in comparing the expression of the three proteins in fresh infiltrating duct carcinoma and their adjacent pairing normal tissue. Secondly, we analyzed the relationship between clinical parameters and the protein expression patterns of Nodal, ALK4 and ALK7 in breast cancer tissues, and their expression correlation with ductal carcinoma in situ, infiltrating duct carcinoma and lymph node metastasis tissues was revealed by Spearman’s rank test. Thirdly, based on the above, we clarified the function and molecular mechanisms of Nodal in breast cancer cell proliferation, migration and invasion. And finally, we revealed the effects of ALK7 on cell proliferation of breast cancer cells. The main results of this study are summarized as follows:1. Nodal protein expression rates are 22.9%, 34.6%, 13.0%, 34.1%, 66.1% and 40.4% in six kinds of breast tissues: adjacent tissues, adenosis, fibroadenoma, ductal carcinoma in situ, invasive ductal carcinoma, lymph node metastasis, respectively. Nodal protein expression rate in invasive ductal carcinoma is significantly higher than that in adjacent normal tissues(P<0.001), adenosis(P=0.003), fibroadenoma(P<0.001), ductal carcinoma in situ(P=0.001) and lymph node metastases(P=0.001). In the lymph node metastasis, Nodal was significantly more highly expressed than in fibroadenomas(P=0.018), while there was no significant difference in other types of tissues. Western Blotting results also showed that in invasive ductal carcinoma, Nodal protein expression was higher than that in paracancerous tissues. In invasive ductal carcinoma, Nodal protein expression has significant correlations with age(P=0.043), tumor histological grade(P=0.001) and clinical stage(P=0.035), in which Nodal protein was highly expressed in groups with lower age, low degree of tumor differentiation and later stage of TNM, whereas its expression has no relationship with tumor size, lymph node metastasis, estrogen receptor(ER) status or progesterone receptor(PR) status.2. ALK7 protein expression rates are 81.6%, 85.0%, 82.1%, 50.0%, 24.6% and 20.0% in six types of breast tissues: adjacent tissues, adenosis, fibroadenoma, ductal carcinoma in situ, invasive ductal carcinoma, lymph node metastasis, respectively. In ductal carcinoma in situ, ALK7 expression was significantly lower than that in adjacent normal tissues(P=0.006), adenosis(P=0.002) and fibroadenoma(P = 0.005), but higher than that in invasive ductal carcinoma(P=0.004) and lymph node metastases(P=0.004); In invasive ductal carcinoma and lymph node metastasis, ALK7 protein expression was significantly lower than that in adjacent tissues(P <0.001), adenosis(P <0.001) and fibroadenoma(P <0.001) as well as in ductal carcinoma in situ, while there was no significant difference in other types of tissues. ALK7 protein expression in primary invasive ductal carcinoma is consistent with that in paired lymph node metastasis. It has been confirmed by Western Blotting that ALK7 protein expression was significantly lower in invasive ductal carcinoma than that in paired adjacent tissues. In invasive ductal carcinoma, ALK7 protein expression has significant correlations with tumor histological grade(P=0.028) and clinical stage(P=0.005), showing that its expression in poorly differentiated group was lower than that in highly or moderately differentiated group, and was lower in clinical stage II, Ⅲ and Ⅳ group than stageⅠgroup. ALK7 protein expression has no correlation with age, tumor size, lymph node metastasis, ER status or PR status.3. ALK4 protein expression rates are 100.0%, 92.3%, 95.7%, 92.9%, 69.1% and 73.5% in adjacent tissues, adenosis, fibroadenoma, ductal carcinoma in situ, invasive ductal carcinoma, lymph node metastasis, respectively. In invasive ductal carcinoma, ALK4 protein expression was significantly lower than that in adjacent tissues(P<0.001), adenosis(P=0.014), fibroadenoma(P = 0.008) and ductal carcinoma in situ(P=0.009). In lymph node metastasis, ALK4 protein expression was significantly lower than that in adjacent tissues(P=0.001), adenosis(P=0.043), fibroadenoma(P=0.026) and ductal carcinoma in situ(P=0.038), while there was no significant difference in other types of tissues. Similar to ALK7, ALK4 protein expression in primary invasive ductal carcinoma is consistent with that in paired lymph node metastasis. And it has also been confirmed by Western Blotting that ALK4 protein expression was significantly lower in invasive ductal carcinomathan that in paired adjacent tissues. In invasive ductal carcinoma, ALK4 protein expression has significant correlations with tumor histological grade(P﹤0.001) and ER(P﹤0.001) and PR(P=0.001)status. ALK4 protein expression in poorly differentiated group was lower than in highly or moderately differentiated group, and was lower in ER or PR negative groups than those of positive. ALK4 protein expression has no correlation with age, tumor size, clinical stage or lymph node metastasis.4. Stable expression of Nodal gene in low-invasive breast cancer cell T47 D which has a low expression level of Nodal, can promote cell epithelial-mesenchymal transition. On the other hand, the cell proliferation, migration and invasion of high-invasive breast cancer cell BT-549 with high level of Nodal expression, can be suppressed by treatment with Nodal receptor inhibitor SB-431542.5. Constitutively activation of ALK7 can inhibit the proliferation of breast cancer cell, and overexpressing ALK7 in low-expressing ALK7 breast cancer cells can restore activin B inhibition on breast cancer cell proliferation.6. Spearman’s rank test showed that the expression of Nodal and ALK4 are negatively correlated(R =-0.378, P = 0.047) in ductal carcinoma in situ, and the expression of ALK4 and ALK7 were positively correlated(R = 0.205, P = 0.009) in invasive ductal carcinoma. In addition, these three proteins were not correlated in lymph node metastasis.This study systematically analyzed the expression patterns, function, clinical significance and expression correlation of Nodal and its receptors ALK4 and ALK7 in different types of breast cance tissues. Nodal was highly expressed in invasive ductal carcinoma, and has a relatively high protein expression rate in the tumor with the lower degree of tumor differentiation and later clinical stage. In vitro experiments also confirmed that Nodal has tumor-promoting functions. These results suggest that increased expression of Nodal in breast cancer is associated with tumor progression and Nodal may serve as a diagnostic marker molecule and therapeutic target of breast cancer. The expression of Nodal receptors ALK4 and ALK7 were reduced in the progression of breast cancer, especially in poorly differentiated breast cancer. Correlation analysis showed that expression of Nodal and its receptors ALK4 or ALK7 was not correlated in invasive ductal carcinoma and lymph node metastasis. These results suggest that reduced expression of ALK4 or ALK7 in breast cancer is associated with tumor progression,and these receptors may be used as a diagnostic marker molecule in breast cancer, but their relationships with tumor-promoting effect of Nodal need further studies. |
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