Xi-Xi Cao

Twist1-Mediated Adriamycin-Induced Epithelial-Mesenchymal Transition Relates to Multidrug Resistance and Invasive Potential in Breast Cancer Cells

Purpose: Besides its therapeutic effects, chemotherapeutic agents also enhance the malignancy of treated cancers in clinical situations. Recently, epithelial-mesenchymal transition (EMT) has attracted attention in studies of tumor progression. We aimed to test whether transient Adriamycin treatment induces EMT and apoptosis simultaneously in cancer cells, clarify why the same type of cells responds differentially (i.e., apoptosis, EMT) to Adriamycin treatment, and elucidate the role of Twist1, the master regulator of EMT, in this process. Experimental Design: In unsynchronized MCF7 cells or cells synchronized at different phases, apoptosis, EMT, and concurrent events [multidrug resistance (MDR) and tumor invasion] after Adriamycin or/and Twist1 small interfering RNA treatment were examined in vitro and in vivo. The Adriamycin-induced Twist1 expression and the interaction of Twist1 with p53-Mdm2 were examined by immunoblotting and immunoprecipitation, respectively. Results: We showed in vitro that Adriamycin induced EMT and apoptosis simultaneously in a cell cycle–dependent manner. Only the cells undergoing EMT displayed enhanced invasion and MDR. Twist1 depletion completely blocked the mesenchymal transformation, partially reversed MDR, and greatly abolished invasion induced by Adriamycin. Also, we confirmed in vivo that Twist1 RNA interference improved the efficacy of Adriamycin for breast cancers. Further, Twist1 reduction in Adriamycin-treated cells promoted p53-dependent p21 induction and disrupted the association of p53 with Mdm2. Conclusions: Our studies show the diverse responses to Adriamycin treatment in cells at different phases, suggest an unrecognized role of EMT in regulating MDR and invasion, and show the efficacy of Twist1 RNA interference in Adriamycin-based chemotherapies for breast cancer.

Involvement of NF-κB/miR-448 regulatory feedback loop in chemotherapy-induced epithelial–mesenchymal transition of breast cancer cells

The epithelial–mesenchymal transition (EMT) induced by chemotherapeutic agents promotes malignant tumor progression; however, the mechanism underlying the drug-induced EMT remains unclear. In this study, we reported that miR-448 is the most downregulated microRNA following chemotherapy. Suppression of miR-448 correlated with EMT induction in breast cancer in vitro and in vivo. With the use of chromatin immunoprecipitation-seq analysis, we demonstrated that miR-448 suppression induces EMT by directly targeting special AT-rich sequence-binding protein-1 (SATB1) mRNA, leading to elevated levels of amphiregulin and thereby, increasing epidermal growth factor receptor (EGFR)-mediated Twist1 expression, as well as nuclear factor κB (NF-κB) activation. On the other hand, we also found that the adriamycin-activated NF-κB directly binds the promoter of miR-448 suppressing its transcription, suggesting a positive feedback loop between NF-κB and miR-448. Furthermore, all patients who received cyclophosphamide (CP), epirubicin plus taxotere/CP, epirubicin plus 5-fluorouracil chemotherapy showed miR-448 suppression, an increased SATB1, Twist1 expression and acquisition of mesenchymal phenotypes. These findings reveal an underlying regulatory pathway, in which the autoregulation between NF-κB and miR-448 is important for restrain miR-448 suppression upon chemotherapy and may have a role in the regulation of chemotherapy-induced EMT. Disruption of the NF-κB-miR-448 feedback loop during clinical treatment may improve the chemotherapy response of human breast cancers in which EMT is a critical component.

Up‐regulation of CD147 and matrix metalloproteinase‐2, ‐9 induced by P‐glycoprotein substrates in multidrug resistant breast cancer cells

Treatment of animals bearing multidrug resistant (MDR) tumor cells with P‐glycoprotein (P‐gp) substrates could worsen host survival. It is assumed that this is due to increased tumor metastasis. To clarify the mechanism(s) underlying this observation, the MDR human breast cancer cell line, MCF‐7/AdrR, and its sensitive parental line, MCF‐7, was treated with various concentrations of P‐gp substrate drugs (vincristine, paclitoxel, adriamycin) and a P‐gp non‐substrate drug (bleomycin) in serum‐free media. Increased production of CD147, and matrix metalloproteinases (MMP)‐2, ‐9 was observed only in MDR cancer cells exposed to P‐gp substrates, as determined using real‐time polymerase chain reaction, western blotting and zymography. Correspondingly, P‐gp substrates significantly enhanced the in vitro invasion abilities of MCF‐7/Adr cells. It was also found that the drug‐induced promotion of CD147, and MMP‐2, ‐9 was consistent with increased expression of epidermal growth factor receptor (EGFR) and that inhibition of either EGFR or P‐gp activity could significantly interrupt the downstream effects, and so inhibit in vitro invasion abilities motivated by P‐gp substrates. These results imply that treatment of MDR tumors with P‐gp substrates could adversely affect therapeutic outcomes through modulating the production of CD147, MMP‐2, ‐9, and EGFR, and suggest that this effect may be initiated by the transporter function of P‐gp. (Cancer Sci 2007; 98: 1767–1774)

RACK1: A superior independent predictor for poor clinical outcome in breast cancer.

We aimed to investigate the expression of RACK1 in breast cancer, evaluate its role in predicting prognosis and compare with commonly used biomarkers: Ki67, ER, PR and HER‐2 for patients with breast cancer. The RACK1 expression and its clinical significance were examined in 160 breast carcinoma patients using immunohistochemistry. Correlations of RACK1 expression with other commonly used biomarkers and survival analyses were assessed. Immunohistochemistry results showed that the number of RACK1 cases scoring 0, 1, and 2 were 66, 54, and 40, respectively. RACK1 staining was strongly related to clinical stage, histological grade, Ki67, ER, PR and HER‐2 (all p < 0.05). Consistently, all of the cases exhibiting RACK1 staining score 0 were survivors, whereas the majority (55.0%) of those exhibiting RACK1 staining score 2 were deaths. Kaplan‐Meier survival analysis of 160 cases revealed a correlation between higher RACK1 expression levels and shorter overall survival times (p < 0.001). Univariate and multivariate analyses revealed that RACK1, tumor size, lymph node metastasis, and HER‐2 were independent prognostic factors (all p < 0.05). Interestingly, receiver operator characteristic (ROC) curves showed that the ROC areas for RACK1, Ki67, ER, PR and HER‐2 were 0.833, 0.766, 0.446, 0.387, and 0.689, respectively, and the superiority of RACK1 in sensitivity and specificity as biomarker was demonstrated. To our knowledge, it is the first time to investigate the expression of RACK1, and identified that RACK1 is a superior independent biomarker for diagnosis and prognosis comparing with currently widely used diagnostic index in breast carcinoma.

Involvement of CD147 in regulation of multidrug resistance to P‐gp substrate drugs and in vitro invasion in breast cancer cells

Multidrug resistant (MDR) cancer cells overexpressing P‐glycoprotein (P‐gp) display variations in invasive and metastatic behavior. We aimed to clarify the mechanism(s) underlying this observation and transfected vectors carrying CD147, a glycoprotein enriched on the surface of tumor cells that stimulates the production of matrix metalloproteinases (MMPs), and specific shCD147 into MCF7 and MCF7/Adr cells, respectively. Using quantitative real‐time polymerase chain reaction and Western blot, we found that overexpression of CD147 in MCF7 cells up‐regulated MDR1, MMP2, and MMP9 on both transcription and expression levels, which promoted tumor cells metastasis and conferred them multidrug resistance to P‐gp substrate drugs, as determined by in vitro invasion assay and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. On the other hand, silencing of CD147 in MCF7/Adr cells led to the opposite effect. Moreover, Erk1/2 in CD147‐overexpressing clones were observed to be highly activate and after treatment with U0126, an Erk1/2‐specific inhibitor, the expression of MDR1, MMP2 and MMP9 were decreased significantly. Thus, CD147 may assume a dual role, since it had intrinsic stimulative effects on tumor invasion in vitro as well as increasing resistance to P‐gp substrate drugs. (Cancer Sci 2007; 98: 1064–1069)

Overexpression and involvement of special AT-rich sequence binding protein 1 in multidrug resistance in human breast carcinoma cells

Special AT‐rich sequence binding protein (SATB) 1 has been proposed to act as a determinant for the acquisition of metastatic activity by controlling expression of a specific set of genes that promote metastatic activity. Here we found that SATB1 expression is upregulated in multidrug‐resistant breast cancer cells that exhibit higher invasive potential than the parental cells. Apart from accelerating metastasis and inducing epithelial–mesenchymal transition, SATB1 was demonstrated to confer resistance to both P‐glycoprotein‐related and P‐glycoprotein‐non‐related drugs on MCF7 cells, which was accompanied by decreasing accumulation of adriamycin in SATB1‐overexpressing transfectants. SATB1 depletion could partially reverse the multidrug resistance (MDR) phenotype of MCF7/ADR in vitro and in vivo. The SATB1‐induced P‐glycoprotein‐mediated MDR could be reversed by treatment with anti‐P‐glycoprotein mAb. Moreover, SATB1 plays an important role in anti‐apoptotic activity in MCF7/ADR cells in response to adriamycin treatment, which suggests another mechanism contributing to SATB1‐related MDR of breast cancers. These data provide new insights into the mode by which breast tumors acquire the MDR phenotype and also imply a role for SATB1 in this process. (Cancer Sci 2009)

DIXDC1 targets p21 and cyclin D1 via PI3K pathway activation to promote colon cancer cell proliferation.

DIXDC1 is the human homolog of Ccd1, a recently identified DIX domain containing protein in zebrafish. It is a positive regulator in the Wnt signaling pathway functioning downstream of Wnt and upstream of Axin. Since Wnt pathway activation is correlated with human colon cancer formation and progression, the biological role of DIXDC1 in human colon cancer was examined. In the current study, up‐regulation of DIXDC1 protein was detected in human colorectal adenocarcinoma tissues and was found to be correlated well with high cell proliferation index. Ectopic over‐expression of DIXDC1 resulted in increased cell proliferation in vitro and accelerated tumorigenesis on nude mice in vivo. We also showed that DIXDC1 promoted G0/G1 to S phase transition concomitantly with up‐regulation of cyclin D1 and down‐regulation of p21 protein. DIXDC1 over‐expression cells showed activation of the PI3K/AKT pathway. Both siRNA knockdown of DIXDC1 and blocking the PI3K pathway using a specific inhibitor caused G1/S phase arrest, as well as down‐regulation of cyclin D1 and up‐regulation of p21 in DIXDC1 over‐expression colon cancer cells. Collectively, this study demonstrates that over‐expression of DIXDC1 might target p21 and cyclin D1 to promote colon cancer cell proliferation and tumorigenesis at least partially through activation of the PI3K/Akt pathway. (Cancer Sci 2009; 100: 1801–1808)

Interaction between CD147 and P-glycoprotein and their regulation by ubiquitination in breast cancer cells.

Background: Multidrug-resistant cancer cells overexpressing P-glycoprotein (P-gp) display variations in invasive and metastatic ability through the upregulation of the extracellular matrix metalloproteinase (MMP) inducer (CD147). However, the direct linkage between these two proteins is still unclear. Methods: We used immunoprecipitation, immunofluorescence analysis, migration and invasion assays, drug sensitivity assay and Western blot to measure the physical and functional interaction between P-gp and CD147. Then we transfected vectors carrying ubiquitin C-terminal hydrolase L1 (UCH-L1) or UCH-L1 siRNA into MCF7 and MCF7/Adr cells, respectively, and investigated the role of UCH-L1 in the regulation of the expression and degradation of P-gp, CD147 and MMP-1, MMP-2, and MMP-9 by quantitative real-time polymerase chain reaction, Western blot and immunoprecipitation. Results: In this paper, we showed that P-gp and CD147 interacted with each other, and that the ubiquitin-proteasome pathway played an important role in the turnover of them. In addition, we found that inhibition of N-glycosylation increased the ubiquitination and degradation of P-gp and CD147, and affected their function. UCH-L1 not only regulated the expression of P-gp, CD147 and MMP-1, MMP-2, and MMP-9, but also the ubiquitination and degradation of P-gp and CD147 in breast cancer cells. Conclusion: Our results demonstrate a mechanism underlying the linkage between multidrug resistance and tumor metastasis, and suggest for the first time that modulating the ubiquitination of P-gp and CD147 might be a novel method for tumor therapy.

The role of P-glycoprotein/cellular prion protein interaction in multidrug-resistant breast cancer cells treated with paclitaxel

Abstract.We previously reported that treatment with P-glycoprotein (P-gp) substrates promotes in vitro invasion in multidrug-resistant (MDR) breast cancer cells. This effect is initiated by the P-gp pump function and mediated by interaction of P-gp with some unknown component(s). However, the underlying mechanism(s) remains poorly understood. Here we confirm a novel physical interaction between P-gp and cellular prion protein (PrPc). Blocking P-gp activity or depletion of PrPc inhibited paclitaxel (P-gp substrate)- induced invasion. Paclitaxel further facilitated the formation of P-gp/PrPc clusters residing in caveolar domains and promoted the association of P-gp with caveolin-1. Both caveolin-1 and the integrity of caveolae were required for the drug-induced invasion. In addition, the P-gp/PrPc complex also played an important role in anti-apoptotic activity of MCF7/Adr cells.These data provide new insights into the mode by which MDR breast cancers evade cytotoxic attacks from P-gp substrates and also suggest a role for P-gp/ PrPc interaction in this process.

The effect of TGF-β1 and Smad7 gene transfer on the phenotypic changes of rat alveolar epithelial cells.

The aim of this study was to investigate whether transforming growth factor-β1 (TGF-β1) could induce alveolar epithelial-mesenchymal transition (EMT) in vitro, and whether Smad7 gene transfer could block this transition. We also aimed to elucidate the possible mechanisms of these processes. The Smad7 gene was transfected to the rat type II alveolar epithelial cell line (RLE-6TN). Expression of the EMT-associated markers was assayed by Western Blot and Real-time PCR. Morphological alterations were examined via phase-contrast microscope and fluorescence microscope, while ultrastructural changes were examined via electron microscope. TGF-β1 treatment induced a fibrotic phenotype of RLE-6TN with increased expression of fibronectin (FN), α-smooth muscle actin (α-SMA) and vimentin, and decreased expression of E-cadherin (E-cad) and cytokeratin19 (CK19). After transfecting the RLE-6TN with the Smad7 gene, the expression of the mesenchymal markers was downregulated while that of the epithelial markers was upregulated. TGF-β1 treatment for 48 h resulted in the separation of RLE-6TN from one another and a change into elongated, myofibroblast-like cells. After the RLE-6TN had been transfected with the Smad7 gene, TGF-β1 treatment had no effect on the morphology of the RLE-6TN. TGF-β1 treatment for 48 h resulted in an abundant expression of α-SMA in the RLE-6TN. If the RLE-6TN were transfected with the Smad7 gene, TGF-β1 treatment for 48 h could only induce a low level of α-SMA expression. Furthermore, TGF-β1 treatment for 12 h resulted in the degeneration and swelling of the osmiophilic multilamellar bodies, which were the markers of type II alveolar epithelial cells. TGF-β1 can induce alveolar epithelialmesenchymal transition in vitro, which is dependent on the Smads signaling pathway to a certain extent. Overexpression of the Smad7 gene can partially block this process