The Regulatory Mechanism Of TXNIP Interaction With Her-1/Her-2 Mediated Breast Cancer Cell Proliferation

BackgroundBreast cancer, specifically mammary carcinoma, is the most common cause of death from cancer in women worldwide. China is one of the fastest growing incidence of breast cancer with gradually dropping age. Breast cancer has become the number one killer of women in the city. Although people have done a lot of research about breast cancer, the exact mechanism and pathogenesis are still unclear.The thioredoxin (TRX) system, dependent of NADPH, is widely distributed in the body. It has also been reported that the redox balance in cancer cells is disrupted by oxidative stress caused by accelerated cell proliferation and alterations in metabolic activity.Therefore, it is regarded as a therapeutic target for cancer treatment. Thioredoxin interaction protein (TXNIP) (also known as thioredoxin binding protein 2 (TBP-2) or Vitamin D3 up-regulated protein 1 (VDUP-1)), is one member of Alpha rhodopsin inhibit protein family. TXNIP gene is located on human chromosome 1 q21-22, contains 8 exons and is 4174 bp in length. By interacting with the TRX,TXNIP can inhibit TRX activity involved in the regulation of levels of reactive oxygen species (ROS) and mitochondrial function, and induced cell apoptosis. Cadenas et al. reported that in 788 patients with node-negative breast cancer,TXNIP was associated with better prognosis (HR= 0.642; P< 0.001). TXNIP may also be a prognostic marker of breast cancer response to anthracycline-based chemotherapy. They induced Her-2 in MCF-7 cells, which strongly inhibited TXNIP expression, following a strong upregualtion of ROS level. This Her-2-dependent repression of TXNIP expression was interpreted as being part of Her-2-triggered survival program.Her family is one of the first identified important targets of these novel antitumor agents.Her family contain four receptor tyrosine kinases, including Her-1, Her-2, Her-3and Her-4. Dimerizations of members of Her family increased tyrosine kinase activity and generate apotent signaling response mediated primarily through activation of the RAS-Raf-mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathways that control cancer cell proliferation and apoptosis.The target drugs contain Her-1 monoclonal antibody cetuximab, and small molecule tyrosine kinase inhibitors gefitinib, erlotinib and so on. The Her-2-targeted drugs include trastuzumab and pertuzumab. Approximately half of triple-negative breast cancers (TNBC) and inflammatory breast cancers (IBC) overexpress Her-1. Thus, Her-1 inhibitors for treatment of breast cancer have been evaluated in several studies. In particular, Her-2 is overexpressed in ～30% of all breast cancers. Therefore Her family in the treatment of breast cancer patients act as increasingly important roles.As a dual Her-1 and Her-2 tyrosine kinase inhibitor (TKI), lapatinib has been approved for trastuzumab-resistant Her-2 positive advanced breast cancer patients. It has been reported that lapatinib inhibit the activity of Her-1/Her-2 signaling and decrease the expression of cyclin D1, leading to increase expression of p27. As the most important factor in the regulation of cell cycle, p27 could inhibit all most kinase activity of cyclin-CDK complexes, and play a pivotal role in the control of cell proliferation, differentiation and apoptosis. It is reported that p27 could inhibit activity of cyclin D1-CDK4 and the process of cell cycle. Our team have made comprehensive studies about the relationship of p27 and breast cancer. One meta-analysis indicates that reduced p27 is an independent prognostic factor for poor overall and disease-free cancer survival. Lapatinib treatment led to increased FOXO3a expression and enhanced p27 transcription. It also increaed DYRK1B expression, which enhanced p27 phosphorylation at Ser10 and decreased p27 degradation. Subcellular localization is an important way to affect p27 play function, and its stability in the nucleus is the most important prerequisite. c-Jun protein can interact with the p27, leading to the translocation of p27 from the nucleus to cytoplasm and its ubiquitin-proteasome degradation. Binding to the N-terminal activity region of c-Jun, Jabl could enhance the regulation of c-Jun to p27.TXNIP interacts with c-terminal of Jabl protein and blocks Jab1-mediated translocation of p27 from the nucleus to the cytoplasm, thus stabilizing p27 protein to enhance the cell cycle arrest.Taken together, TXNIP may therefore potentially impact cell survival through its interaction with p27.MicroRNAs (miRNAs) are small, noncoding RNAs-22 nucleotides (nt) in length that regulate gene expression. They mediate post-transcriptional regulation of protein-coding genes by binding chiefly to the 3’untranslated region (3’-UTR). It has been estimated that miRNAs regulate～30% of human genes. A single miRNA concurrently could downregulate hundreds of target mRNAs, which designated a network involved the development and treatment of cancers. Several studies have shown that miRNAs play crucial roles in the initiation and progression, invasion and metastasis, multidrug resistance of breast cancer. miR-375, miR-221 and miR-21 are reported to be correlated to resistance to trastuzumab. TXNIP is regulated by miR-17-5p at the post-transcriptional levels. Yet, whether miRNAs are associated with lapatinib and the role of miRNAs in the relationship between TXNIP and Her-l/Her-2 need further studies.However, despite the ongoing evaluation of TXNIP as a therapeutic target, little is known about the the relevance of TXNIP to overall survival (OS) in breast cancer patients and its prognostic value in anti-Her-2 treatment. Here, we investigated TXNIP expression in early stage breast cancer patients and its association with OS by tissue array. Using diverse methods we further evaluated TXNIP expression in response to anti-Her-1/Her-2 treatment, and screen miRNAs and its target gene in the regulation of TXNIP interaction with Her-1/Her-2 pathway, to elaborate regulatory mechanisms of TXNIP interaction with Her-1/Her-2 pathway in breast cancer cells and provide new molecular targets and therapeutic strategies for the prevention and treatment of breast cancer.Materials, Methods and ResultsPart I TXNIP interaction with the Her-1/Her-2 pathway contributes to overall survival in breast cancerMaterials and Methods1. The association of TXNIP and p27 with clinicopathologic characteristics and OS in 150 breast cancer tissues2. To demonstrate the expression of TXNIP and p27 expression in breast cancers compared with NCTs by IHC of breast cancer,which were validated in 101 breast cancer samples. We analyzed the correlation between TXNIP, p27 and Her-2 expression by correlation analysis at the tumor microarray or at tumor tissues, respectively.3. TXNIP and p27 expressions were evaluated by Western blotting after transfection with the TXNIP overexpression plasmid.p27 localization and expression were determined by confcoal. The percentage of Gi phase arrest and apoptosis of BT474 and SK-BR-3 cells was determined via fow cytometry after transfection. The colony formation assay was used to measure the proliferating ability of BT474 and SK-BR-3 cells. In the transfected treated with lapatinib, cell numbers were counted and cell viability was determined by trypan blue staining.4. After treatment with cetuximab, trastuzumab or lapatinib, TXNIP and p27 protein and mRNA expression were determined by Western blotting and qRT-PCR, respectively. A firefly luciferase reporter containing the TXNIP promoter sequence was transfected into BT474 and SK-BR-3 cells along with the treatment of drugs mentioned above. Cells were assayed after transfection using a luciferase assay kit.Results1. Of the 150 tumor tissues, the expression of TXNIP has positively related with TNM stage (x2=4.994, P=0.025), and does not have significantly connection with age (X2=2.252, P=0.133), histological stage (x2=0.161, P=0.777), ER status (x2=0.261, P=0.610) and PR status (x2=0.651,P=0.420).2. TXNIP and p27 expressions were decreased in breast cancer tissue compared with NCTs (x2=42.633, P<0.001; x2=9.500, P=0.023, respectively). We found that high TXNIP or p27 expression was associated with better OS (P=0.001 and p=0.012). We next analyzed the correlation between TXNIP, p27 and Her-2 expression at the tumor microarray or at tumor tissues, respectively. A signifcant negative correlation was found between TXNIP and Her-2 status using the breast cancer tissue array (n=150) (r=-0.334, P<0.001), which was again validated in the external cohort (n=101) (r=-0.422, P <0.001). Similarly, a signifcant negative correlation was found between p27 and Her-2 status using the breast cancer tissue array (n=150) (r=-0.344, P<0.001) that was confirmed in the external cohort (n=101) (r=-0.284, P=0.004). Therefore, TXNIP and p27 are inversely associated with Her-2 status.3. In the transfected SK-BR-3 and BT474 cell lines, We found that pcMV6- TXNIP -transfection enhanced p27 expression. The confocal showed TXNIP also increased p27 localization in the nuclei.Using fow cytometry to explore the role of TXNIP in the cell cycle and cell proliferation of breast cancer, we found that TXNIP overexpression enhanced G1 cell cycle arrest (t=-5.385, P= 0.006; t=-4.778, P= 0.009) and induced apoptosis (t=19.906, P< 0.001; t=5.469, P=0.029) both in BT474 and SK-BR-3 cell lines. To investigate whether TXNIP enhances lapatinib-induced inhibition of cell proliferation, we transiently transfected SK-BR-3 cells with a pcMV6-TXNIP-expressing vector, and treated them with 0.5μM lapatinib for 48 h. We found that TXNIP expression inhibited cell proliferation both in the presence and absence of lapatinib. Taken together, our results suggest that TXNIP may enhance lapatinib-induced inhibition of cell proliferation in Her-1/Her-2 positive breast cancer cell lines.4. Cetuximab, trastuzumab and lapatinib treatment induced TXNIP (t=135.311,P=0.076; t=9.825, P=0.063; t=3.222, P=0.084) and p27 expression (t=14.269, P=0.056; t=11.135, P=0.089;t=4.860,p=0.004) in BT474 cell; these three drugs induced TXNIP (t=328.0, P=0.071;t=8.322, P=0.056;t=5.709, P=0.003) and p27 expression(t=34.352, P=0.084; t=5.904, P=0.002;t=14.302, P=0.005) in SK-BR-3 cell.To study the regulatory mechanism of Her-1/Her-2 dependent inhibition of TXNIP, luciferase activity was assayed after transfecting BT474 and SK-BR-3 cells with a TXNIP-promoter plasmid. After TXNIP promoter plasmid transfection the relative luciferase activities were enhanced in all three treated groups of BT474 cell (t=41.617, p=0.001;t=26.771, P=0.001;t=93.916, P<0.001) and SK-BR-3 cell (t=18.322, P=0.003;t=12.924, P=0.006; t=9.596, P=0.011).TXNIP expression could thus be induced by transcriptional inhibition of Her-1/Her-2. Collectively, our results show that TXNIP transcription is upregulated by inhibition of Her-1/Her-2 signaling.Part Ⅱ miR-1470 mediates lapatinib induced p27 upregulation by targeting c-JunMaterials and Methods1. SK-BR-3 and BT474 cells were treated with 0.5 μM lapatinib for 48 h, and p27 mRNA and protein levels were determined by qRT-PCR and Western blotting. The cellular localization of p27 was detected by immunofluorescence after the cells treated by the same treatment.2. SK-BR-3 and BT474 cells were treated with 0.5μM lapatinib for 48 h. Total protein was harvested and western blotted to analyze the important transduction proteins levels of Her-2 signaling.3. miRNA profile was analyzed using miRNA array in samples from SK-BR-3 and BT474 cells treated with three concentration lapatinib. By TargetScan, we predicted the target genes of miRNA. Through the online database DAVID, signaling pathways that were probably regulated by miRNAs were analyzed.4. Using qRT-PCR to detect expression of selected miRNAs and Western blotting to test the protein expression of predictive target genes.5. Schematic description of the hypothesized duplexes of the conserved selective miRNA binding site within its target gene mRNA 3’-UTR. Plasmid containing the wild and mutated sequence that miRNA interacts with the 2-8 bases of target gene mRNA 3’-UTR binding sites was built. Scrambled ncRNA and miR-1470 mines were also produced. After transfection, the regulation of miRNA to its target gene were proved using luciferase assay, qRT-PCR and Western blotting. Cell cycle distribution was quantified by flow cytometry analysis.Results1. It has been shown that exposure to lapatinib enhanced p27 transcription in the two breast cancer cells compared with control, respectively, lapatinib treatment led to an increased p27 expression compared with the controls in SK-BR-3 and BT474 cells(t=21.238, P=0.002; t=24.497, P=0.002). Moreover, immunofluorescence was performed to determine the cellular localization of p27 in the cells stimulated with lapatinib, shown that p27 staining was predominantly nuclear in treated cells. Taken together, these results suggested that lapatinib could significantly upregulate p27 expression in mRNA and protein levels.2. Immunoblot analysis of protein lysates from SK-BR-3 and BT474 cells revealed that Her-2, phosphorylated-Her-2 (p-Her-2), p38, p-p38 MAPK, p44/42 MAPK, p- p44/42MAPK expression were downregulated after treated with 0.5 μM lapatinib for 48 h, suggesting Her-2 involved in the lapatinib induced p27 upregulation.3. Lapatinib significantly altered the miRNA expression profile in human breast cancer cell lines BT474 and SK-BR-3. There were 7 miRNAs upregulated in both SK-BR-3 and BT474 cells after either 0.5μM or 2μM lapatinib treatment. It contained miR-219-1-3P, miR-126, miR-26A-1*, miR-1470, miR-126*, miR-1908-P and miR-1208. In order to determine the probable biological function of the differentlly expressed miRNAs, we predicted the putative targets of the union set of differently expressed miRNAs via TargetScan online software. KEGG pathway analysis showed that the dominant pathway those miRNAs involved in was the ErbB signaling pathway. Among the 7 miRNAs upregulated by lapatinib, miR-1470, miR-126 and miR-1208 were involved in ErbB signaling pathway.4. Our qRT-PCR data validated that lapatinib treatment could upregulate the expression of the three miRNAs in SK-BR-3(t=19.118, P=0.003;t=38.645, P=0.001;t=27.411, P=0.001) and BT474(t=36.647, P=0.001; t=39.172,p=0.001;t=25.076, P=0.002) cells. In the TargetScan software, the predicted target genes of miR-1470, miR-126, and miR-1208 were c-Jun, Crk and Src respectively. Western blotting analysis showed only the expression of c-Jun protein was reduced, which was consisted with the qRT-PCR data. After treated with lapatinib, the Crk and Src expressions were increased. Taken together, those results suggested that lapatinib treatment suppressed c-Jun expression through induction of miR-1470.5. The predicted interactions between miR-1470 and its target sites in the c-Jun 3’-UTR were illustrated, which showed that there was one potential miR-1470 target site in the 3’-UTR of the c-Jun mRNA sequence. Moreover, perfect base pairing between the seed region (the core sequence that encompasses the first 2-8 bases of the mature miRNA), and the miR-1470 binding sequences in the c-Jun 3’-UTR were highly conserved across species. To determine whether the negative regulatory effects miR-1470 exerted on c-Jun expression were mediated through the binding of miR-1470 to the presumed sites in the 3’-UTR of the c-Jun mRNA, we fused part of the c-Jun 3’-UTR, which included the predicted miR-1470 binding sites, to the downstream of the luciferase reporter plasmid. As expected, overexpression of miR-1470 resulted in a significant decrease in the luciferase reporter activity, when compared to cells treated with the scrambled ncRNA(t=7.059, P=0.009; t=13.093, P=0.006). Furthermore, we introduced point mutations into the corresponding seed complementary sites in the c-Jun 3’-UTR to eliminate the predicted miR-1470 binding site. Mutations in the complementary seed sites almost fully rescued the repression of the c-Jun activity caused by the expression of miR-1470mincs. After transfected with ncRNA and miR-1470 for 48 h, cell cycle distribution was quantified by flow cytometry analysis. We detected the expression level of miR-1470 and c-Jun. Upregulation of miR-1470 led to downregulation of c-Jun and cyclin D1, which enhanced p27-dependent G1 cell cycle arrest (t=-4.019,P=0.051; t=-3.572,P=0.003) in SK-BR-3 and BT474 cells.Conclusions1. TXNIP expressions were decreased in breast cancer tissue compared with NCTs. A signifcantly negative correlation was found between TXNIP and Her-2 status. High TXNIP was associated with better OS.2. In vivo, overexpression of TXNIP resulted in G1 cell cycle arrest,apoptosis and inhibition of cell proliferation.3. Her-1/Her-2 inhibitors could increase TXNIP expression.4. Lapatinib increased p27, and inhibited the activity of Her-1/Her-2 downstream signaling pathway, including expression of Her-2, phosphorylated-Her-2 (p-Her-2), p38, p-p38MAPK, p44/42MAPK, p-p44/42MAPK.5. Lapatinib could up-regulate miRNAs expression involved in the ErbB signaling pathway, including miR-1470, miR-126 and miR-1208. miR-1470 regulated p27 expression via inhibition of c-Jun, to enhanced p27-dependent G1 cell cycle arrest and inhibit cell proliferation.6. Taken together, TXNIP may therefore potentially impact cell survival through its interaction with p27 in the Her-1/Her-2 pathway, and may be correlated with the Her-1/Her-2 inhibitors treatment of breast cancer.