Up-regulation Of TROP-2 Expression By Tumor Necrosis Factor-α And Its Role And Significance In Colon Cancer Cell Migration And Invasion

Colon cancer is one of the common gastrointestinal malignant tumor.The incidence is increasing in recent years, and the 5-year survival rate is still low. Tumor invasion and metastasis are the main cause of death in colon cancer patients. However,but the specific mechanism in the metastasis and invasion of cancer cells is unclear. Chronic inflammation is now being recognized as a major driving force in the development of about one-third of all the known cancers. In recent years, several studies have shown that inflammatory microenvironment is closely related to the tumor invasion and metastasis. Tumor associated macrophage(TAMs) is an important inflammatory cells and tumor necrosis factor alpha(TNF-α) is one of the important pro-inflammatory cytokines in tumor inflammatory microenvironment. They are all involved in involved in a variety of malignant transformation, cell proliferation and metastasis in the stage of tumor progression and promotion.Tumor-associated calcium signal transducer-2(TROP-2), is a trans-membrane glycoprotein,has been found higher expression in different kind of tumor tissues. The role of TROP-2 is closely related to invasion, metastasis and prognosis of tumor cell. However it is still unknown whether TAMs and TNF-a contribute to higher expression of TROP-2 in colon cancer. The role of TNF-a in regulating TROP-2 expression in the metastasis and invasion of colon cancer cell need to be investigated.Thus, we will detect TAMs, TNF-a, and TROP-2 expression in colon cancers tissues. We will explore the effect of TNF-a on TROP-2 expression on human HCT-116 colon cancer cells, and get insight of the role of TNF-a/TROP-2 involved in invasion and metastasis of colon cancer HCT-116 cells.Part 1 The expression and clinical significance of TROP-2 protein in colon cancerObjective: To explore the expression of TROP-2 protein in colon cancer tissues and cell line, and investigate its clinical significance in the prognosis of colon cancer.Method: The fresh specimens and pathological paraffin sections were collected from Hebei united university affiliated hospital. Immunohistochemical staining method was used to detect the expression of TROP-2 protein in colon cancer tissues, tumor-adjacent tissues and normal colon tissues. By reviewing the clinical data of patients, we analyzed the correlation between TROP-2 expression and clinical pathological features and clinical prognosis. We detected TROP-2 protein expression in fresh tissue specimens and HCT-116 cell line by using Western blot method. Furthermore, we applied immunofluorescence staining to observe TROP-2 protein localization.Results:1 Immunohistochemical staining: Among 82 cases, positive expression of TROP-2 were detected in 75 cases(91.5%), while 55cases(67.1%) were high expression and 20cases(24.4%) were low expression. In tumor-adjacent tissues, only 10 cases(12.2%) showed positive expression of TROP-2 protein and 2 cases(2.4%) were high-expression. The expression level of TROP-2 in colon cancer tissues is significantly higher than that in tumor-adjacent tissues and normal tissues(P<0.01).2 The correlation between TROP-2 expression and clinical pathological parameters: There was no correlation between TROP-2 expression and gender,age and tumor differentiation(P>0.05). TROP-2 expression were significantly correlated with lymph node metastasis and Dukes stage(P<0.05). The positive rate of TROP-2 in colon cancer with lymph node metastasis was significantly higher than that without lymph node metastasis(P<0.05). With the progress of tumor Dukes stage, the positive rate of TROP-2 ncreased gradually. Interestingly,the positive rate of TROP-2 in stage C+D was obviously higher than that in stage A+B, and the difference was statistically significant(P <0.01).3 Immunefluorescence staining: Positive expression of TROP-2 mainly located on tumor cell membrane. Within the cytoplasm and the nucleus, there was a small amount of positive staining. However,there is no positive staining in normal colon tissue. With the progress of the Dukes stage, the quantity of green fluorescence for TROP-2 protein in colon cancer tissues increased gradually.4 Western blotting: The expression of TROP-2 in colon cancer tissues was significantly higher than that in normal tissues and tumor-adjacent tissues(P<0.01). TROP-2 expression levels were significantly different in groups in regards to lymphatic metastasis and Dukes stage. The expression quantity of TROP-2 protein in colon cancer tissues with lymph node metastasis was obviously higher than those without lymphatic metastasis(P<0.01). With the progress of the Dukes stage, the expression quantity of TROP-2 protein also increased gradually(P<0.05). What’s more,a clearly expression of TROP-2 protein was observed in colon cancer HCT-116 cell line.5 The correlation between TROP-2 expression and prognosis :Kaplan–Meier survival curves showed that patients with high-expression of TROP-2 had poorer overall survival compared to those with low-expression of TROP-2(P<0.01). TROP2 protein up-regulation maybe identified as an independent predictive factor for poor prognosis.Summary: Increased expression of TROP-2 was observed in colon cancer tissues and cell line. Upregulation of TROP-2 is related to lymph node metastasis and Dukes stage. TROP-2 may play an important role in colon cancer metastasis and invasion and could be used as an independent predictive factor for poor prognosis.Part 2 The correlation between Tumor-associated macrophages, Tumor necrosis factor alpha and TROP-2 in colon cancerObjective: To investigate the expression of TAMs and TNF-a in colon cancer tissues as well as their correlation with TROP-2 expression and clinical pathological features. We aimed to provide the theoretical insight in the metastasis and invasion.Method: 82 pathological paraffin sections were collected from Hebei united university affiliated hospital. Immunohistochemical staining method was used to detect the expression of TAMs with an antibody against CD68 and TNF-a in colon cancer tissues and normal colon tissues. By reviewing the clinical data of patients, we analyzed the correlation between TAMs infiltration, TNF-a expression and clinical pathological features. Finally,we explored the relationship between TAMs, TNF-a and TROP-2 in colon cancer tissues.Results:1 TAMs infiltration in colon cancer tissues:All of 82 cases were visible for CD68 positive macrophages. CD68 positive TAMs mainly located in tumor stroma,while no positive cells were observed in normal colon tissues. The numbers of TAMs in colon cancer tissues were higher than that in normal tissues(P<0.05). With the progress of tumor Dukes stage, the numbers of TAMs also increased gradually. According to the median TAMs numbers,all cases were divided into two groups:high density group and low density group.2 The correlation between TAMs infiltration and clinical pathological parameters:There was no correlation between TAMs infiltration and gender, age(P>0.05). TAMs infiltration was significantly correlated with tumor differentiation, lymph node metastasis and Dukes stage(P<0.05). The numbers of TAMs in colon cancer with lymph node metastasis were significantly higher than those in the colon cancer without lymph node metastasis(P<0.05). With the progress of tumor Dukes stage, the numbers of TAMs also increased gradually. The difference between stage Band stage C was of no statistically significant(P>0.05). But the differences among other groups were statistically significant(P< 0.01).3 The expression of TNF-a in colon cancer tissues:TNF-a positive xpression were detected in 70 cases(85.3%). Among those,70 cases with TNF-a positive expression, 48 cases showed higher expression of TNF-a and 22 cases(26.8%) showed lower expression. Few TNF-a positive cells weredetected in normal colon tissues. The TNF-a positive rate of colon cancer tissues was obviously higher than that of normal colon tissues(P<0.01).4 The correlation between TNF-a expression and clinical pathological parameters:There was no correlation between TNF-a expression and gender, age(P>0.05). TNF-a expression was significantly correlated with tumor differentiation, lymph node metastasis and Dukes stage(P<0.05). The expression level of TNF-a in colon cancer with lymph node metastasis were significantly higher than those without lymph node metastasis(P<0.05). With the progress of tumor Dukes stage, the expression level of TNF-a also increased gradually. The difference among four groups was statistically significant(P<0.01).5 The correlation between TAMs infiltration, TNF-a expression and prognosis:Kaplan–Meier survival curves showed that patients with TNF-a high-expression and TAMs high-infiltration had poorer overall survival compared to those with TNF-a low-expression and TAMs low-infiltration(P<0.001). TAMs infiltration and TNF-a expression were related to the prognosis,but they were not identified as an independent predictive factor for poor prognosis through multi-factor Cox regression analysis.6 The relationship between TROP-2 expression and TAMs infiltration and TNF-a expression :The positive rate of TROP-2 in group with TAMs high infiltration was 85.7%(36/42). The positive rate of TROP-2 in group with TAMs low infiltration was only 47.5%(19/40). With the increase of TAMs number, the TROP-2 expression level also increased. So there was a positive correlation between TROP-2 and TAMs in colon cancer tissues(rs=0.569,P<0.001). The positive rate of TROP-2 in group with TNF-a high expression was 94.3%(66/70), however, that in group with TNF-a low expression was 66.7%(8/12). With the increase of TNF-a expression, the TROP-2 expression increased too. There was also a positive correlation etween TROP-2 and TNF-a(rs=0.592,P<0.001).Summary: There were high-infiltration of TAMs and higher level expression of TNF-a in colon cancer tissues. Increased TAMs infiltration and TNF-a expression related with the progress of colon cancer and could serve as markers to predict the prognosis. There was a positive correlation between TROP-2 and TAMs and TNF-a, which indicating that TNF-a may play an important role in up-regulating TROP-2 expression in colon cancer.Part 3 Tumor necrosis factor-a up-regulates the expression of TROP-2 and enhances the capability of migration and invasion in colon cancer cellObjective: To explore the effect of TNF-a on the expression of TROP-2 as well as the role of TROP-2 involved in the metastasis and invasion in colon cancer HCT-116 cells.Method: The concentration and time of TNF-a treatment was detected in colon cancer HCT-116 cells by MTT test. The expression of TROP-2 protein in HCT-116 cells treated with TNF-a(20ng/m L) were detected by western blot. The effects of TNF-a on cell migration and invasion were investigated by wound healing assay and Transwell method. Furthermore, small interfering RNA(si RNA) targeting TROP-2 gene was used to knock down endogenous TROP-2 expression. The expression of TROP-2 in HCT-116 cells treated with TROP-2 si RNA were detected by q RT-PCR and Western blot methods. Finally,the migratory and invasive capability in HCT-116 cells treated with TROP-2 si RNA were also detected by wound healing assay and Transwell method.Results:1 The effect of different concentration TNF-a on cell proliferation:Low concentration of TNF-a(≤50ng/m L) had no obvious effects on cell proliferation within 24 and 48 hours. But high concentration of TNF-a(≥100ng/m L)led to remarkable proliferation inhibition in HCT-116 cells.2 The expression level of TROP-2 protein in HCT-116 cells with different oncentration of TNF-a:After different concentration of TNF-a(0、10、20、30、50、100、200ng/m L) treatment for 24 hours,the expression of TROP-2 protein were detected by Western blot. Low concentration of TNF-a(≤50ng/m L) led to an increase of TROP-2 expression, while 100ng/m L TNF-a inhibited TROP-2 expression. The highest expression of TROP-2 protein was achieved by 20ng/m L TNF-a treatment.3 The effects of TNF-a on cell migration and invasion:The treatment with 20ng/m L TNF-a promoted the migratory and invasive capability in colon cancer HCT-116 cells(P<0.05).4 The effect of TROP-2siRNA on the expression level of TROP-2 gene and protein : The results of q RT-PCR and Western blot showed that TROP-2si RNA significantly down-regulated the TROP-2m RNA and protein expression in colon cancer HCT-116 cells.5 The effects of TNF-a on cell migration and invasion in HCT-116 cells with TROP-2si RNA transfection: Compared with control group, blocking TROP-2 expression by TROP-2si RNA inhibited the migratory and invasive capability in HCT-116 cells treated by TNF-a(P<0.05). The results indicate that TROP-2 play a critical role in TNF-a-induced migration and invasion in colon cancer HCT-116 cells.Summary: The mechanism that low concentration of TNF-a promoted HCT-116 cells migration and invasion by up-regulating the expression of TROP-2.Part 4 The molecular mechanism of TNF-a up-regulating the expression of TROP-2 in colon cancer HCT-116 cellsObjective: To explore the possible signal pathway of low concentration of TNF-a in up-regulating the expression of TROP-2 and promoting colon cancer HCT-116 cells migration and invasion.Method: Colon cancer HCT-116 cells were cultured with TNF-a(20ng/m L). The expression of p-p38, p-JNK and p-ERK1/2, which were the hree key proteins of MAPK signal pathway, were detected by Western blot. The colon cancer HCT-116 cells were pretreated with specific inhibitors for 30 minutes,and then the cells were treated with TNF-a. The expression of TROP-2 protein and the key protein of MAPK signal pathways were detected by Western blot. The migratory and invasive capability were detected by wound healing and Transwell assay.Results: Higher level expression of p-ERK1/2 was induced by TNF-a significantly. However, TNF-a had no effect on the expression of p-p38 and p-JNK. Thus, the results indicate that ERK1/2 signal pathway may contribute increased TROP-2 expression induced by TNF-a. We applied PD98059, the specific inhibitor of p-ERK1/2, to block the ERK1/2 signal pathway in HCT-116 cells. Increased expression of TROP-2 by TNF-a was significantly inhibited by PD98059. Blocking ERK1/2 pathway significantly inhibited cell migration and invasion in HCT-116 cells treated with TNF-a.Summary: Low concentration of TNF-a up-regulated the expression of TROP-2 to promote colon cancer HCT-116 cells migration and invasion by activating the ERK1/2 signal pathway.Conclusion: Higher expression of TROP-2 was observed in colon cancer tissues. With the progress of Dukes stage,the expression of TROP-2 increases gradually. TROP-2 may be used as an independent predictive factor for poor prognosis. Chronic inflammation plays a critical role in colon cancer cell invasion and metastasis. TNF-a up-regulating TROP-2 expression contributes to cell migration and invasion by activating the ERK1/2 signal pathway.