Xiaoyue Tan

SOX2 promotes tumorigenesis and increases the anti-apoptotic property of human prostate cancer cell

SRY-related HMG-box gene 2 (SOX2) is one of the key regulatory genes that maintain the pluripotency and self-renewal properties in embryonic stem cells. Here we used immunohistochemistry to analyze the expression of SOX2 in human prostate tissues and found it contributed to tumorigenesis and correlated with histologic grade and Gleason score. We further investigated SOX2s function in cell growth and apoptosis process by using a human prostate cancer cell line DU145 with SOX2 overexpression or down-regulation. Cell cycle assay revealed that SOX2 promoted cell growth and increased the percentage of cells in S phase. In vitro and in vivo xenograft experiments in NOD/SCID mice further demonstrated that SOX2 increased the apoptosis-resistant properties of DU145 cells with decreased function of store-operated Ca(2+) entry and reduced expression of Orai1 at both mRNA and protein levels, suggesting a potential mechanism that contributes to the anti-apoptotic property of SOX2. To our knowledge, this study is the first to investigate SOX2s function in tumorigenesis and apoptosis of human prostate cancer and to elucidate its regulatory effect on the activity of store-operated Ca(2+) channels. Our results support the concept that SOX2 has the potential to be a significant marker to evaluate the progression of prostate cancer and serve as a potentially useful target for prostate cancer therapy.

Downregulation of transcription factor Oct4 induces an epithelial-to-mesenchymal transition via enhancement of Ca2+ influx in breast cancer cells.

The stem cell-related transcription factor Oct4 regulates tumor proliferation and apoptosis, but its role in tumor migration and invasion is still undefined. Here, we compared Oct4 expression in MCF-7 and MDA-MB-231 cells, two breast cancer cell lines with similar epithelial origins, but distinct invasive and metastatic characteristics. We found MCF-7 cells to express very high levels of Oct4, while no obvious expression was detected in MDA-MB-231 cells. We then downregulated Oct4 expression using small interfering RNA (siRNA) to explore its effects on migration and invasion. Transwell assays showed that silencing Oct4 in MCF-7 cells improved their migration and invasion capabilities. Reverse-transcriptase PCR and western blots showed that E-cadherin expression decreased, and α-smooth muscle actin expression increased with Oct4 downregulation, which suggests that epithelial-to-mesenchymal transition (EMT) occurred. A potent EMT stimulus, TGF-β1, significantly inhibited Oct4 expression in both dose- and time course-dependent manners. Silencing Oct4 also upregulated expression of two major components of store-operated Ca(2+) entry channels (SOCs), STIM1 and Orai1, and enhanced SOC-directed Ca(2+) influx. Silencing STIM1 blocked the Ca(2+) influx and rescued the EMT initiated by Oct4 downregulation. In conclusion, silencing Oct4 promotes invasion and metastasis in breast cancer cells by inducing EMT. This effect may be related to SOCs-directed enhancement of Ca(2+) influx.

Legumain protease-activated TAT-liposome cargo for targeting tumours and their microenvironment

Specific targeting and cellular internalization are key properties for carriers of antitumor therapeutic agents. Here, we develop a drug carrier through the attachment of substrate of endoprotease legumain, alanine-alanine-asparagine (AAN), to cell-penetrating peptides (TAT, trans-activating factor). The addition of the AAN moiety to the fourth lysine in the TAT creates a branched peptide moiety, which leads to a decrease in the transmembrane transport capacity of TAT by 72.65%. Legumain efficiently catalyses the release of TAT-liposome from the AAN-TAT-liposome and thereby recovers the penetrating capacity of TAT. Doxorubicin carried by the AAN-TAT-liposome led to an increase in the tumoricidal effect of doxorubicin and a reduction in its systemic adverse effects in comparison with doxorubicin carried by a control delivery system. Thus, the specific targeting and high efficiency of this delivery platform offers a novel approach to limit the toxicity of anticancer agents as well as increasing their efficacy in cancer therapy.

Alginic acid-coated chitosan nanoparticles loaded with legumain DNA vaccine: effect against breast cancer in mice.

Legumain-based DNA vaccines have potential to protect against breast cancer. However, the lack of a safe and efficient oral delivery system restricts its clinical application. Here, we constructed alginic acid-coated chitosan nanoparticles (A.C.NPs) as an oral delivery carrier for a legumain DNA vaccine. First, we tested its characteristic in acidic environments in vitro. DNA agarose electrophoresis data show that A.C.NPs protected DNA better from degradation in acidic solution (pH 1.5) than did chitosan nanoparticles (C.NPs). Furthermore, size distribution analysis showed that A.C.NPs tended to aggregate and form micrometer scale complexes in pH<2.7, while dispersing into nanoparticles with an increase in pH. Mice were intragastrically administrated A.C.NPs carrying EGFP plasmids and EGFP expression was detected in the intestinal Peyer’s patches. Full-length legumain plasmids were loaded into different delivery carriers, including C.NPs, attenuated Salmonella typhimurium and A.C.NPs. A.C.NPs loaded with empty plasmids served as a control. Oral vaccination was performed in the murine orthotopic 4T1 breast cancer model. Our data indicate that tumor volume was significantly smaller in groups using A.C.NPs or attenuated Salmonella typhimurium as carriers. Furthermore, splenocytes co-cultured them with 4T1 cells pre-stimulated with CoCl2, which influenced the translocation of legumain from cytoplasm to plasma membrane, showed a 4.7 and 2.3 folds increase in active cytotoxic T lymphocytes (CD3+/CD8+/CD25+) when treated with A.C.NPs carriers compared with PBS C.NPs. Our study suggests that C.NPs coated with alginic acid may be a safe and efficient tool for oral delivery of a DNA vaccine. Moreover, a legumain DNA vaccine delivered orally with A.C.NPs can effectively improve autoimmune response and protect against breast cancer in mice.

Loss of vitamin D receptor in chronic kidney disease: a potential mechanism linking inflammation to epithelial-to-mesenchymal transition

Both peritubular inflammation and tubular epithelial-to-mesenchymal transition (EMT) are critical events during the pathogenesis of renal fibrosis. However, the relationship between these two processes is unclear. Here, we investigated the potential role of the vitamin D receptor (VDR) in coupling peritubular inflammation and EMT. In a mouse model of unilateral ureteral obstruction (UUO), loss of VDR was observed as early as 1 day after surgery. In cultured proximal tubular epithelial HK-2 cells, proinflammatory TNF-α inhibited the expression of VDR in a dose- and time-dependant manner. Treatment with TNF-α sensitized HK-2 cells to EMT stimulated by transforming growth factor (TGF)-β1. Ectopic expression of VDR counteracted the synergistic effect of TNF-α and TGF-β1 on EMT. Furthermore, knockdown of VDR using a small interfering RNA strategy mimicked the effect of TNF-α on facilitating EMT. Either TNF-α treatment or a loss of VDR induced β-catenin activation and its nuclear translocation. The VDR ligand calcitriol reversed the VDR loss and inhibited EMT in the mouse UUO model, and late administration of active vitamin D was effective in restoring VDR expression as well, and reduced collagen accumulation and deposition compared with the vehicle control. β-Catenin expression induced by UUO was also significantly inhibited after the late administration of vitamin D. These results indicate that the early loss of VDR in chronic kidney diseases was likely mediated by proinflammatory TNF-α, which renders tubular cells susceptible to EMT. Our data suggest that loss of VDR couples peritubular inflammation and EMT, two key events in renal fibrogenesis.

Fra-1 Promotes Breast Cancer Chemosensitivity by Driving Cancer Stem Cells from Dormancy

Fra-1 is a member of the Fos transcription factor family that is highly expressed in multiple cancers, playing important roles in transformation, proliferation, and metastasis. In this study, we observed an inverse correlation between the expression of Fra-1 in human stage II breast cancer tissues and the corresponding level of clinical chemoresistance. Extending these findings in vitro, we found that knockdown of Fra-1 in breast tumor cells was sufficient to confer resistance to doxorubicin and cyclophosphamide, whereas enhanced Fra-1 expression could render these cells chemosensitive. The tumor cell side population, which is enriched for cancer stem cells, was found to be associated with chemoresistance. Increased side population fractions were detected among tumor cell lines subjected to Fra-1 knockdown. In contrast, enhanced expression of Fra-1 was correlated with a decreased side population fraction, and significantly, this finding was recapitulated in vivo, where tumors with enhanced expression of Fra-1 were found to have blunted growth. Tumor cells subjected to Fra-1 knockdown grew faster and were larger in size. Taken together, our findings suggest that Fra-1 may be an important prognostic marker for breast cancer therapy.

VDR Status Arbitrates the Prometastatic Effects of Tumor-Associated Macrophages

The relationship between tumor-associated macrophages (TAM) and epithelial-to-mesenchymal transition (EMT) during the initiation and progression of metastasis is still unclear. Here, a role for the vitamin D receptor (VDR) in metastasis was identified, as well as a role in the relationship between TAMs and EMT. First, the expression level of VDR was examined in clinical tissue from human patients with breast cancer or a mouse model of breast cancer with differential metastasis. These results revealed that VDR expression negatively correlates with metastasis in breast cancer. Second, coculture of VDR-overexpressing breast cancer cells with a macrophage cell line demonstrated that overexpression of VDR alleviated the prometastatic effect of cocultured macrophages on breast cancer cells. Furthermore, VDR overexpression abrogated the induction of EMT in breast cancer cells by cocultured macrophage cells, as measured by a loss of E-cadherin (CDH1) and induction of α-smooth muscle actin (α-SMA). TNFα in macrophage conditioned media inhibited VDR expression, whereas downregulation of VDR further mediated the promotion of TGFβ-induced EMT by TNFα. In addition, β-catenin expression was inhibited in VDR-overexpressing breast cancer cells and tumor xenografts. Finally, administration of calcitriol [1,25-(OH)2D3], an active vitamin D metabolite, exerted similar antimetastatic effects in breast cancer cells in vitro and a mouse model of breast cancer in vivo with preservation of VDR and suppression of β-catenin. Implications: VDR suppression by TNFα mediates the prometastatic effect of TAMs through enhancement of the β-catenin pathway. Mol Cancer Res; 12(8); 1181–91. ©2014 AACR.

Yes-associated protein (YAP) increases chemosensitivity of hepatocellular carcinoma cells by modulation of p53

The yes-associated protein (YAP) transcription co-activator has been reported either as an oncogene candidate or a tumor suppressor. Liver tissue chips revealed that about 51.4% human hepatocellular carcinoma (HCC) samples express YAP and 32.9% HCC samples express phosphorylated YAP. In this study, we found that chemotherapy increased YAP protein expression and nuclear translocation in HepG2 cells, as well as p53 protein expression and nuclear translocation. However, little is known about YAP functions during chemotherapy. Our results show that overexpression of YAP increases chemosensitivity of HepG2 cells during chemotherapy. Dominant negative transfection of Flag-S94A (TEAD binding domain mutant) or Flag-W1W2 (WW domain mutant) to HepG2 cells decreases p53 expression/ nuclear translocation and chemosensitivity when compared with control HepG2 cells. Furthermore, rescue transfection of Flag-5SA-S94A or Flag-5SA-W1W2, respectively to HepG2 cells regains p53 expression/nuclear translocation and chemosensitivity. These results indicate that YAP promotes chemosensitivity by modulating p53 during chemotherapy and both TEAD and WW binding domains are required for YAP-mediated p53 function. ChIP assay results also indicated that YAP binds directly to the p53 promoter to improve its expression. In addition, p53 could positively feedback YAP expression through binding to the YAP promoter. Taken together, our current data indicate that YAP functions as a tumor suppressor that enhances apoptosis by modulating p53 during chemotherapy.

M2 tumour-associated macrophages contribute to tumour progression via legumain remodelling the extracellular matrix in diffuse large B cell lymphoma

Effects of M2 tumour-associated macrophages on the pathogenesis of diffuse large B cell lymphoma (DLBCL) are still controversial. Our data showed that the number of CD163-positive M2 macrophages correlated negatively with DLBCL prognosis. Macrophage depletion by clodronate liposomes significantly suppressed tumour growth in a xenograft mouse model of DLBCL using OCI-Ly3 cells. Moreover, M2 polarization of macrophages induced legumain expression in U937 cells. Exogenous legumain promoted degradation of fibronectin and collagen I, which was abolished by administration of a legumain inhibitor RR-11a. Overexpression of legumain in Raw 264.7 cells also induced tube formation of endothelial cells in matrigel. In the xenograft mouse model of DLBCL, decreased fibronectin and collagen I, as well as increased legumain expression and angiogenesis were found at the late stage tumours compared with early stage tumours. Co-localization of legumain and fibronectin was observed in the extracellular matrix of tumour tissues. Administration of the legumain inhibitor to the xenograft DLBCL model suppressed tumour growth, angiogenesis and collagen deposition compared with the control. Taken together, our results suggest that M2 tumour-associated macrophages affect degradation of the extracellular matrix and angiogenesis via overexpression of legumain, and therefore play an active role in the progression of DLBCL.

Activating transcription factor 4 increases chemotherapeutics resistance of human hepatocellular carcinoma

It has been reported that activating transcription factor 4 (ATF4) increases the processes of tumor growth, metastasis and drug resistance. However, the role played by ATF4 in chemoresistance of hepatocellular carcinoma (HCC) remains unknown. Clarification of this role of ATF4 in HCC could greatly benefit the efficacy of clinical treatment of HCC. In this study, we found that ATF4 was overexpressed in about 50.7% of HCC tissues. In fact knockdown of ATF4 significantly increased the cytotoxicity of cisplatin in both in vitro and in vivo assays, while overexpression of this molecule dramatically decreased the sensitivity of HCC cell lines to cisplatin. Additionally, we found that synthesis of glutathione was significantly reduced in HCC cell lines subjected to ATF4 knockdown. Taken together, these results demonstrate that ATF4 can increase resistance to cisplatin in HCC by increased biosynthesis of glutathione, and that this may be a potent novel target for the future development of anti-HCC drugs.