MicroRNA‐708‐3p mediates metastasis and chemoresistance through inhibition of epithelial‐to‐mesenchymal transition in breast cancer

Metastasis and chemoresistance remain major challenges in the clinical treatment of breast cancer. Recent studies show that dysregulated microRNAs (miRNAs) play an important role in metastasis and chemoresistance development in breast cancer. Herein, we identified downregulated expression of miR‐708‐3p in breast cancers. In particular, miR‐708‐3p expression was significantly decreased in specimens from breast cancer patients with metastasis compared to that in specimens from patients with no metastasis. Consistent with clinical data, our in vitro data show that miR‐708‐3p was more significantly decreased in invasive breast cancer cell lines. In addition, our data show that inhibition of miR‐708‐3p significantly stimulated breast cancer cell metastasis and induced chemoresistance both in vitro and in vivo. In contrast, overexpression of miR‐708‐3p dramatically inhibited breast cancer cell metastasis and enhanced the sensitivity of breast cancer cells to chemotherapy both in vitro and in vivo. Furthermore, we identified that miR‐708‐3p inhibits breast cancer cell epithelial‐to‐mesenchymal transition (EMT) by directly targeting EMT activators, including ZEB1, CDH2 and vimentin. Taken together, our findings suggest that miR‐708‐3p acts as a cancer suppressor miRNA and carries out its anticancer function by inhibiting EMT in breast cancer. In addition, our findings suggest that restoration of miR‐708‐3p may be a novel strategy for inhibiting breast cancer metastasis and overcoming the chemoresistance of breast cancer cells.     

p53 activated by AND gate genetic circuit under radiation and hypoxia for targeted cancer gene therapy

Radio‐activated gene therapy has been developed as a novel therapeutic strategy against cancer; however, expression of therapeutic gene in peritumoral tissues will result in unacceptable toxicity to normal cells. To restrict gene expression in targeted tumor mass, we used hypoxia and radiation tolerance features of tumor cells to develop a synthetic AND gate genetic circuit through connecting radiation sensitivity promoter cArG₆, heat shock response elements SNF1, HSF1 and HSE₄ with retroviral vector plxsn. Their construction and dynamic activity process were identified through downstream enhanced green fluorescent protein and wtp53 expression in non‐small cell lung cancer A549 cells and in a nude mice model. The result showed that AND gate genetic circuit could be activated by lower required radiation dose (6 Gy) and after activated, AND gate could induce significant apoptosis effects and growth inhibition of cancer cells in vitro and in vivo. The radiation‐ and hypoxia‐activated AND gate genetic circuit, which could lead to more powerful target tumoricidal activity represented a promising strategy for both targeted and effective gene therapy of human lung adenocarcinoma and low dose activation character of the AND gate genetic circuit implied that this model could be further exploited to decrease side‐effects of clinical radiation therapy.     

RPV‐modified epirubicin and dioscin co‐delivery liposomes suppress non‐small cell lung cancer growth by limiting nutrition supply

Chemotherapy for non‐small cell lung cancer (NSCLC) is far from satisfactory, mainly due to poor targeting of antitumor drugs and self‐adaptations of the tumors. Angiogenesis, vasculogenic mimicry (VM) channels, migration, and invasion are the main ways for tumors to obtain nutrition. Herein, RPV‐modified epirubicin and dioscin co‐delivery liposomes were successfully prepared. These liposomes showed ideal physicochemical properties, enhanced tumor targeting and accumulation in tumor sites, and inhibited VM channel formation, tumor angiogenesis, migration and invasion. The liposomes also downregulated VM‐related and angiogenesis‐related proteins in vitro. Furthermore, when tested in vivo, the targeted co‐delivery liposomes increased selective accumulation of drugs in tumor sites and showed extended stability in blood circulation. In conclusion, RPV‐modified epirubicin and dioscin co‐delivery liposomes showed strong antitumor efficacy in vivo and could thus be considered a promising strategy for NSCLC treatment.     

Functional significance of VEGFR‐2 on ovarian cancer cells

Vascular endothelial growth factor receptor (VEGFR) has recently been discovered on ovarian cancer cells, but its functional significance is unknown and is the focus of this study. By protein analysis, A2780‐par and HeyA8 ovarian cancer cell lines expressed VEGFR‐1 and HeyA8 A2774, and SKOV3ip1 expressed VEGFR‐2. By in situ hybridization (ISH), 85% of human ovarian cancer specimens showed moderate to high VEGFR‐2 expression, whereas only 15% showed moderate to high VEGFR‐1 expression. By immunofluorescence, little or no VEGFR‐2 was detected in normal ovarian surface epithelial cells, whereas expression was detected in 75% of invasive ovarian cancer specimens. To differentiate between the effects of tumor versus host expression of VEGFR, nude mice were injected with SKOV3ip1 cells and treated with either human VEGFR‐2 specific antibody (1121B), murine VEGFR‐2 specific antibody (DC101) or the combination. Treatment with 1121B reduced SKOV3ip1 cell migration by 68% (p < 0.01) and invasion by 72% (p < 0.01), but exposure to VEGFR‐1 antibody had no effect. Treatment with 1121B effectively blocked VEGF‐induced phosphorylation of p130Cas. In vivo treatment with either DC101 or 1121B significantly reduced tumor growth alone and in combination in the SKOV3ip1 and A2774 models. Decreased tumor burden after treatment with DC101 or 1121B correlated with increased tumor cell apoptosis, decreased proliferative index, and decreased microvessel density. These effects were significantly greater in the combination group (p < 0.001). We show functionally active VEGFR‐2 is present on most ovarian cancer cells. The observed anti‐tumor activity of VEGF‐targeted therapies may be mediated by both anti‐angiogenic and direct anti‐tumor effects. © 2008 Wiley‐Liss, Inc.     

Long noncoding‐RNA component of mitochondrial RNA processing endoribonuclease is involved in the progression of cholangiocarcinoma by regulating microRNA‐217

Cholangiocarcinoma (CCA) is a malignant tumor originating from bile duct epithelium and its incidence is increasing year by year. In recent years, long noncoding RNAs (lncRNAs) have been found to play an important role in the occurrence and progression of malignant tumors. In the present study, for the first time, abnormal expression of lnc‐RNA component of mitochondrial RNA processing endoribonuclease (RMRP) and its possible role in CCA were found. We explored the effects of RMRP on various behaviors of CCA cells in vitro and in vivo. In addition, by second‐generation sequencing, we explored the microRNA expression profiles that RMRP may affect in the HCCC‐9810 cell line. We also validated and explored the role of microRNA‐217 (miR‐217) with high differential expression by in vitro experiments. Our findings indicated that RMRP can play a part in promoting cancer by regulating the expression of miR‐217. RMRP is involved in the progression of CCA and can be a novel indicator of poor prognosis in patients with CCA.     

Tumor‐suppressive microRNA‐145 induces growth arrest by targeting SENP1 in human prostate cancer cells

Prostate cancer (PCa) prevails as the most commonly diagnosed malignancy in men and the third leading cause of cancer‐related deaths in developed countries. One of the distinct characteristics of prostate cancer is overexpression of the small ubiquitin‐like modifier (SUMO)‐specific protease 1 (SENP1), and the upregulation of SENP1 contributes to the malignant progression and cell proliferation of PCa. Previous studies have shown that the expression of microRNA‐145 (miRNA‐145) was extensively deregulated in PCa cell lines and primary clinical prostate cancer samples. Independent target prediction methods have indicated that the 3′‐untranslated region of SENP1 mRNA is a potential target of miR‐145. Here we found that low expression of miR‐145 was correlated with high expression of SENP1 in PCa cell line PC‐3. The transient introduction of miR‐145 caused cell cycle arrest in PC‐3 cells, and the opposite effect was observed when miR‐145 inhibitor was transfected. Further studies revealed that the SENP1 3′‐untranslated region was a regulative target of miR‐145 in vitro. MicroRNA‐145 also suppressed tumor formation in vivo in nude mice. Taken together, miR‐145 plays an important role in tumorigenesis of PCa through interfering SENP1.     

Heat shock factor 1 in cancer‐associated fibroblasts is a potential prognostic factor and drives progression of oral squamous cell carcinoma

Heat shock factor 1 (HSF1) is highly expressed in various malignancies and is a potential modulator of tumor progression. Emerging evidence suggests that HSF1 activation in stromal cells is closely related to poor patient prognosis. However, the role of HSF1 in oral squamous cell carcinoma (OSCC) remains elusive. We aimed to investigate the function of HSF1 in cancer‐associated fibroblasts (CAFs) of the tumor microenvironment (TME) and in tumor development. In the present study, we found that HSF1 was highly expressed in both CAFs and tumor cells, and was significantly correlated with poor prognosis and overall survival. Moreover, HSF1 overexpression in CAFs resulted in a fibroblast‐like phenotype of Cal27 cells, induced epithelial‐mesenchymal transition (EMT), and promoted proliferation, migration and invasion in Cal27 cells. HSF1 knockdown attenuated features of CAFs and reduced EMT, proliferation, migration and invasion in Cal27 cells. Furthermore, HSF1 in CAFs promoted tumor growth in nude mice. Taken together, these data suggest that HSF1 expression in CAFs drive OSCC progression, and could serve as an independent prognostic marker of patients with OSCC. Thus, HSF1 is a potent mediator of OSCC malignancy.     

Inhibition of transforming growth factor beta/SMAD signal by MiR‐155 is involved in arsenic trioxide‐induced anti‐angiogenesis in prostate cancer

Prostate cancer is the most common cause of cancer‐related deaths in men. Current practices for treatment of prostate cancer are less than satisfactory because of metastasis and recurrence, which are primarily attributed to angiogenesis. Hence, anti‐angiogenesis treatment is becoming a promising new approach for prostate cancer therapy. In addition to treating acute promyelocytic leukemia, arsenic trioxide (As₂O₃) suppresses other solid tumors, including prostate cancer. However, the effects of As₂O₃ on angiogenesis in prostate cancer cells, and the underlying molecular mechanisms remain unclear. In the present study, As₂O₃ attenuated angiogenic ability through microRNA‐155 (miR‐155)‐mediated inhibition of transforming growth factor beta (TGF‐β)/SMAD signal pathway in human prostate cancer PC‐3 and LNCaP cells in vitro and in vivo. Briefly, As₂O₃ inhibited the activations/expressions of both TGFβ‐induced and endogenous SMAD2/3. Furthermore, As₂O₃ improved the expression of miR‐155 via DNA‐demethylation. MiR‐155, which targeted the SMAD2‐3′UTR, decreased the expression and function of SMAD2. Knockdown of miR‐155 abolished the As₂O₃‐induced inhibitions of the TGF‐β/SMAD2 signaling, the vascular endothelial growth factor secretion and angiogenesis. Through understanding a novel mechanism whereby As₂O₃ inhibits angiogenic potential of prostate cancer cells, our study would help in the development of As₂O₃ as a potential chemopreventive agent when used alone or in combination with other current anticancer drugs.     

ERGIC3, which is regulated by miR‐203a,is a potential biomarker for non‐small cell lung cancer

In a previous study, we found that ERGIC3 was a novel lung cancer‐related gene by screening libraries of differentially expressed genes. In this study, we developed a new murine monoclonal antibody (mAb) against ERGIC3. This avid antibody (6‐C4) is well suited for immunohistochemistry, immunoblotting and solid‐phase immunoassays. Furthermore, we systematically investigated expressions of ERGIC3 in a broad variety of normal human tissues and various types of tumors by immunohistochemistry. In normal human tissues, 6‐C4 reacted only in some epithelial cells, such as hepatocytes, gastrointestinal epithelium, ducts and acini of the pancreas, proximal and distal tubules of the kidney, and mammary epithelial cells; however, most normal human tissues were not stained. Moreover, almost all carcinomas that originated from the epithelial cells were positive for 6‐C4, whereas all sarcomas were negative. Notably, 6‐C4 strongly stained non‐small cell lung cancer (NSCLC) cells but did not react with normal lung tissues. Hence, ERGIC3 mAb could be used in histopathological diagnosis and cytopathological testing to detect early‐stage NSCLC. We also studied the mechanisms of ERGIC3 regulation in vitro and in vivo by means of bioinformatics analysis, luciferase reporter assay, miRNA expression profiling and miRNA transfection. Results showed that miR‐203a downregulation induced ERGIC3 overexpression in NSCLC cells.     

Role of inhibitor of yes‐associated protein 1 in triple‐negative breast cancer with taxol‐based chemoresistance

Triple‐negative breast cancer (TNBC) is highly clinically aggressive and taxol‐based chemoresistance remains a big TNBC therapeutic problem to be solved. Verteporfin, a small molecular yes‐associated protein 1 (YAP1) inhibitor, is little known as an antitumor drug for TNBC. Our data showed that YAP1 expression was associated with early relapse in tissue samples of patients with TNBC taxol chemoresistance (P < .001). Verteporfin reduced migration and enhanced apoptosis or autophagy of a taxol‐resistant MDA‐MB‐231 cell line in vitro. Knockdown of YAP1 increased epithelial‐mesenchymal transition response in a taxol‐resistant TNBC cell line. In an in vivo experiment, we found that verteporfin was able to shrink tumor weight and volume and decreased Ki67 expression in a taxol‐resistant mouse model. Our results provide evidence that verteporfin could be a chemosensitizer for TNBC patients with taxol‐based treatment.     

MT95‐4, a fully humanized antibody raised against aminopeptidase N,reduces tumor progression in a mouse model

Aminopeptidase N (APN/CD13) is involved in tumor cell invasion and tumor angiogenesis and is considered a promising therapeutic target in the treatment of cancer. To develop a novel monoclonal antibody‐based cancer therapy targeting APN/CD13, we established a fully humanized anti‐APN/CD13 monoclonal antibody, MT95‐4. In vitro, MT95‐4 inhibited APN/CD13 enzymatic activity on the tumor cell surface and blocked tumor cell invasion. B16 mouse melanoma cells stably expressing human APN/CD13 were also established and were inoculated s.c. or injected i.v. into nude mice. We found that expression of human APN/CD13 in murine melanoma cells increased the size of subcutaneous tumors, extent of lung metastasis and degree of angiogenesis in the subcutaneous tumors; these tumor‐promoting and angiogenesis‐promoting characteristics were reduced by the i.p. administration of MT95‐4. To further verify the specificity of MT95‐4 for neutralization of APN/CD13 activity, MT95‐4 was administered into NOD/SCID mice inoculated s.c. with H1299 or PC14 cells, which exhibit high expression of APN/CD13, or with A549 cells, which exhibit weak expression of APN/CD13. MT95‐4 reduced tumor growth and angiogenesis in mice bearing H1299‐derived and PC14‐derived tumors, but not in mice bearing A549‐derived tumors. These results suggested that the antitumor and anti‐angiogenic effects of MT95‐4 were dependent on APN/CD13 expression in tumor cells. Given that MT95‐4 is the first fully humanized monoclonal antibody against APN/CD13, MT95‐4 should be recognized as a promising candidate for monoclonal antibody therapy against tumors expressing APN/CD13.     

Influence of the prodrugs 5‐fluorocytosine and CPT‐11 on ovarian cancer cells using genetically engineered stem cells: tumor‐tropic potential and inhibition of ovarian cancer cell growth

Recent studies have shown that genetically engineered stem cells (GESTECs) to produce suicide enzymes that convert non‐toxic prodrugs to toxic metabolites selectively migrate toward tumor sites and reduce tumor growth. In the present study, we evaluated whether these GESTECs were capable of migrating to human ovarian cancer cells and examined the potential therapeutic efficacy of the gene‐directed enzyme prodrug therapy against ovarian cancer cells in vitro. The expression of cytosine deaminase (CD) or carboxyl esterase (CE) mRNA of GESTECs was confirmed by RT‐PCR. A modified transwell migration assay was performed to determine the migratory capacity of GESTECs to ovarian cancer cells. GESTECs (HB1.F3.CD or HB1.F3.CE cells) engineered to express a suicide gene (CD or CE) selectively migrated toward ovarian cancer cells. A [³H] thymidine incorporation assay was conducted to measure the proliferative index. Treatment of human epithelial ovarian cancer cell line (SKOV‐3, an ovarian adenocarcinoma derived from the ascites of an ovarian cancer patient) with the prodrugs 5‐fluorocytosine (5‐FC) or camptothecin‐11 (CPT‐11) in the presence of HB1.F3.CD or HB1.F3.CE cells resulted in the inhibition of ovarian cancer cell growth. Based on the data presented herein, we suggest that GESTECs expressing CD/CE may have a potent advantage to selectively treat ovarian cancers. (Cancer Sci 2010; 101: 955–962)     

Verteporfin suppresses cell survival,angiogenesis and vasculogenic mimicry of pancreatic ductal adenocarcinoma via disrupting the YAP‐TEAD complex

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive human malignancies. The Yes‐associated protein‐1 (YAP) plays a critical role in cell proliferation, apoptosis and angiogenesis. Verteporfin is a photosensitizer used in photodynamic therapy and also a small molecular inhibitor of the Hippo‐YAP pathway. However, little is known about whether verteporfin could inhibit YAP activity in PDAC cells. Our present results showed that verteporfin suppressed the proliferation of human PDAC PANC‐1 and SW1990 cells by arresting cells at the G1 phase, and inducing apoptosis in dose‐ and time‐dependent manners. Verteporfin also inhibited the tumor growth on the PDAC xenograft model. Treatment with verteporfin led to downregulation of cyclinD1 and cyclinE1, modulation of Bcl‐2 family proteins and activation of PARP. In addition, verteporfin exhibited an inhibitory effect on angiogenesis and vasculogenic mimicry via suppressing Ang2, MMP2, VE‐cadherin, and α‐SMA expression in vitro and in vivo. Mechanism studies demonstrated that verteporfin impaired YAP and TEAD interaction to suppress the expression of targeted genes. Our results provide a foundation for repurposing verteporfin as a promising anti‐tumor drug in the treatment of pancreatic cancer by targeting the Hippo pathway.     

Targeting tumor‐infiltrating Ly6G+ myeloid cells improves sorafenib efficacy in mouse orthotopic hepatocellular carcinoma

Sorafenib, a multikinase inhibitor with antiangiogenic activity, is an approved therapy for hepatocellular carcinoma (HCC). It is unclear whether the proinflammatory and immunosuppressive mechanisms may limit the therapeutic efficacy of sorafenib in HCC. We used a syngeneic mouse liver cancer cell line to establish orthotopic liver or subcutaneous tumors to study how proinflammatory and immunosuppressive mechanisms impact on the efficacy of sorafenib. We found sorafenib exhibited a potent therapeutic effect in subcutaneous tumors, but a less potent effect in orthotopic liver tumors. The protein levels of interleukin‐6 (IL‐6) and vascular endothelial growth factor A (VEGF‐A) were persistently elevated in orthotopic liver tumors, but not in subcutaneous tumors, treated with sorafenib. Likewise, the tumor‐infiltrating Ly6G⁺ myeloid‐derived suppressor cells (MDSCs) and immune suppressors were increased in orthotopic liver tumors, not in subcutaneous tumors, treated with sorafenib. The tumor‐infiltrating Ly6G⁺ MDSCs of sorafenib‐treated orthotopic liver tumors significantly induced IL‐10 and TGF‐β expressing CD4⁺ T cells, and downregulated the cytotoxic activity of CD8⁺ T cells. IL‐6, but not VEGF‐A, protected Ly6G⁺ MDSCs from sorafenib‐induced cell death in vitro. The combination of anti‐Ly6G antibody or anti‐IL‐6 antibody with sorafenib significantly reduced the cell proportion of Ly6G⁺ MDSCs in orthotopic liver tumors, enhanced the T cells proliferation and improved the therapeutic effect of sorafenib synergistically. Modulating tumor microenvironment through targeting tumor‐infiltrating Ly6G⁺MDSCs represents a potential strategy to improve the anti‐HCC efficacy of sorafenib.     

Mammary tissue microenvironment determines T cell‐dependent breast cancer‐associated inflammation

Although the importance of the host tissue microenvironment in cancer progression and metastasis has been established, the spatiotemporal process establishing a cancer metastasis‐prone tissue microenvironment remains unknown. In this study, we aim to understand the immunological character of a metastasis‐prone microenvironment in a murine 4T1 breast tumor model, by using the activation of nuclear factor‐κb (NF‐κB) in cancer cells as a sensor of inflammatory status and by monitoring its activity by bioluminescence imaging. By using a 4T1 breast cancer cell line stably expressing an NF‐κB/Luc2 reporter gene (4T1 NF‐κB cells), we observed significantly increased bioluminescence approximately 7 days after metastasis‐prone orthotopic mammary fat‐pad inoculation but not ectopic s.c. inoculation of 4T1 NF‐κB cells. Such in vivo NF‐κB activation within the fat‐pad 4T1 tumor was diminished in immune‐deficient SCID or nude mice, or T cell‐depleted mice, suggesting the requirement of host T cell‐mediated immune responses. Given the fat‐pad 4T1 tumor expressed higher inflammatory mediators in a T cell‐dependent mechanism compared to the s.c. tumor, our results imply the importance of the surrounding tissue microenvironment for inflaming tumors by collaborating with T cells to instigate metastatic spread of 4T1 breast cancer cells.