Fangfang Shi

MiR‐7, Inhibited Indirectly by LincRNA HOTAIR, Directly Inhibits SETDB1 and Reverses the EMT of Breast Cancer Stem Cells by Downregulating the STAT3 Pathway

Epithelial–mesenchymal transition (EMT) contributes to tumor invasion and metastasis in many cancers and correlates highly with the acquisition of cancer stem cell (CSC) characteristics. EMT also correlates with changes in specific microRNAs (miRNAs) that have already been integrated into tumorigenic programs as either oncogenes or tumor suppressor genes. Here, we show that miR‐7, which was downregulated in breast CSCs (BCSCs) isolated from the human MCF‐7 and MDA‐MB‐231 cell lines, inhibited cell invasion and metastasis, decreased the BCSC population and partially reversed EMT in MDA‐MB‐231 cells by directly targeting the oncogene, SETDB1. The conspicuous epigenetic transition induced by miR‐7 overexpression was found not only in MDA‐MB‐231 cells but also in BCSC xenograft tumors. MiR‐7 inhibited the metastasis of BCSCs in lungs, kidneys, and adrenal glands of NOD/SCID mice. ChIP‐polymerase chain reaction result suggested that the SETDB1 induced STAT3 expression by binding to the promoter of STAT3. MiR‐7‐mediated downregulation of SETDB1 resulted in the suppression of STAT3, which led to the downregulation of c‐myc, twist, and mir‐9. In addition, the downregulation of miR‐7 in BCSCs may be indirectly attributed to lincRNA HOTAIR by modulating the expression of HoxD10 that promotes the expression of miR‐7. These findings demonstrate that miR‐7 was a tumor suppressor and that the overexpression of miR‐7 might serve as a good strategy for treating highly invasive breast cancer. Stem Cells 2014;32:2858–2868

MicroRNA-200c overexpression inhibits tumorigenicity and metastasis of CD117 + CD44 + ovarian cancer stem cells by regulating epithelial-mesenchymal transition

BackgroundCancer stem cells (CSCs) are believed to be ‘seed cell’ in cancer recurrence and metastasis. MicroRNAs (miRNAs) can play an important role in the progression of primary tumor towards metastasis by regulating the epithelial-mesenchymal transition (EMT). The goal of this study was to investigate the effect of miRNA-200c overexpression on the EMT, tumorigenicity and metastasis of epithelial ovarian cancer (EOC) CSCs.MethodsThe EOC CD117+CD44+CSCs were isolated from the human ovarian cancer cell line SKOV3 by using a magnetic-activated cell sorting system, and the lentivirus miR-200c transduced CSCs were then selected for the study. The assays of colony forming, wound healing, cellular migration in vitro and tumor progression in vivo were performed.ResultsThe miR-200c expression was reduced in the CD117+CD44+CSCs compared with the non-CD117+CD44+CSCs. However, the stable overexpression of the miR-200c in the CD117+CD44+CSCs resulted in a significant down-regulation of ZEB-1 and the Vimentin expression, an upregulation of the E-cadherin expression as well as a decrease of colony forming, migratory and invasion in vitro. Importantly, the miR-200c overexpression significantly inhibited the CD117+CD44+CSCs xenograft growth and lung metastasis in vivo in nude mice by inhibition of the EMT. In addition, the down-regulation of ZEB-1 showed the same efficacy as the miR-200c overexpression in the CD117+CD44+CSCs.ConclusionThese findings from this study suggest that the miR-200c overexpression may be considered a critical approach for the EOC CD117+CD44+CSCs in clinical trials.

Observation of ovarian cancer stem cell behavior and investigation of potential mechanisms of drug resistance in three-dimensional cell culture.

Cancer cells behave differently in a three-dimensional (3D) cell culture compared with in the conventional two-dimensional (2D) one. Accumulated evidences indicate that the characteristics of cancer stem cells (CSCs) are different from common cancer cells due to their ability to produce tumors and resist chemoradiation. The objective of this work was to observe CSC behavior and investigate the potential mechanisms of CSC drug resistance in 3D versus 2D in vitro environment. We first demonstrated that the CD44(+)CD117(+)cells isolated from the human epithelial ovarian cancer HO8910 cell line have the properties of CSCs that revealed faster growth, larger tumorsphere and stronger survival potential in the hypoxic environment in 3D cell culture as well as more powerful tumorigenicity in a xenograft mice than the HO8910 cells. The CD44(+)CD117(+)CSCs also exhibited high chemoresistance to anticancer drugs when the cells were incubated with 5-fluorouracil, cisplatin and carboplatin, respectively in 3D versus 2D environment. This might be associated with the high expression of ABCG2, ABCB1 and the high expression of MMP-2 and MMP-9 in CD44(+)CD117(+)CSCs. Overall, these results suggest the advantages of using 3D culture model to accurately display CSC behavior in vitro. 3D model may improve the efficacy of screening anticancer drugs for treatment of ovarian CSCs.

Effect of downregulation of ZEB1 on vimentin expression, tumour migration and tumourigenicity of melanoma B16F10 cells and CSCs.

Zinc‐finger E‐box binding homeobox 1 (ZEB1) is a master regulator of epithelial‐mesenchymal transition (EMT) and has been implicated in primary epithelial cancer biological processes, such as invasion and metastasis. However, the role of ZEB1 in progression of melanoma and cancer stem cells (CSCs) remains obscure. In this study, the recombinant plasmids of t3 shRNAs targeting mouse ZEB1 were constructed and transfected into melanoma B16F10 cells. The stable transfected cells were selected and the characteristics of ZEB1 downregulated B16F10 cells was assessed. The tumourigenicity of CD44+CD133+CSCs isolated from B16F10 cells stably transfected with the ZEB1‐shRNA2 recombinant was also assessed. ZEB1‐shRNAs B16F10 showed a lower expression of ZEB1 and vimentin, weaker migration, invasiveness, colony forming, and proliferation, and a lower tumourigenicity than the control cells. The tumourigenicity of the ZEB1‐shRNA2 CD44+CD133+CSCs was also inhibited. In conclusion, ZEB1‐shRNA2‐mediated downregulation of ZEB1 expression in B16F10 cells and CSCs is involved in the inhibition of the EMT process. ZEB1 may be a potential target in melanoma targeted.

Gene therapy of ovarian cancer using IL-21-secreting human umbilical cord mesenchymal stem cells in nude mice

BackgroundThe human umbilical cord mesenchymal stem cells (hUCMSCs) have the ability to migrate into tumors and therefore have been considered as an alternative source of mesenchymal progenitors for the therapy of malignant diseases. The present study was aimed to investigate effect of hUCMSCs as vehicles for a constant source of transgenic interleukin-21 (IL-21) on ovarian cancer in vivo.MethodsThe hUCMSCs were engineered to express IL-21 via lentiviral vector- designated ‘hUCMSCs-LV-IL-21’, and then were transplanted into SKOV3 ovarian cancer xenograft-bearing nude mice. The therapeutic efficacy and mechanisms of this procedure on ovarian cancer was evaluated.ResultsThe isolated hUCMSCs were induced to differentiate efficiently into osteoblast and adipocyte lineages in vitro. The expressed IL-21 in the supernatant from hUCMSCs-LV-IL-21 obviously stimulated splenocyte’s proliferation. The hUCMSCs-LV-IL-21 significantly reduced SKOV3 ovarian cancer burden in mice indicated by tumor sizes compared with control mice. The expressed IL-21 not only regulated the levels of IFN-γ and TNF-α in the mouse serum but also increased the expression of NKG2D and MIC A molecules in the tumor tissues. The down regulation of β-catenin and cyclin-D1 in the tumor tissues may refer to the inhibition of SKOV3 ovarian cancer growth in mice. In addition, hUCMSCs did not form gross or histological teratomas up to 60 days posttransplantation in murine lung, liver, stomach and spleen.ConclusionThese results clearly indicate a safety and usability of hUCMSCs-LV- IL-21 in ovarian cancer gene therapy, suggesting the strategy may be a promising new method for clinical treatment of ovarian cancer.

Downregulated lincRNA HOTAIR expression in ovarian cancer stem cells decreases its tumorgeniesis and metastasis by inhibiting epithelial-mesenchymal transition

BackgroundEmerging evidence indicates that dysregulated long intervening non-coding RNA (lincRNA) HOTAIR correlates highly with tumor invasion and metastasis but a link between the high expression of HOTAIR and the metastatic cascade of cancer stem cells (CSCs) needs to be further studied. The purpose of this study was to investigate the effect of down-regulated HOTAIR expression on tumorgeniesis and metastasis of epithelial ovarian cancer (EOC) CSCs. CD117+CD44+CSCs were isolated from human EOC SKOV3 cell line by using a magnetic-activated cell sorting system, and were then transfected with the expression vector-based small hairpin RNA targeting HOTAIR; the stably transfected cells were selected for the study. Colony-forming, wound-healing, cellular metastasis and tumorigenicity assays were performed.ResultsThe results demonstrated that the HOTAIR expression in clinical EOC tissues and SKOV3 CD117+CD44+CSCs was higher than in SKOV3 tumor tissues and non-CD117+CD44+CSCs. The CD117+CD44+-shHOTAIR showed an inhibited HOTAIR expression, reduced cell migration and invasion than CD117+CD44+- scramble, suggesting the inhibition of an epithelial-mesenchymal transition. Moreover, the downregulated HOTAIR expression in CD117+CD44+ CSCs significantly decreased the tumor growth and lung metastasis in xenograft mice.ConclusionOur findings demonstrated the shHOTAIR-mediated down-regulation of the HOTAIR expression in CD117+CD44+ CSCs can be a promising new opportunity for future clinical trials.

Target therapy of multiple myeloma by PTX-NPs and ABCG2 antibody in a mouse xenograft model.

Multiple myeloma (MM) remains to be an incurable disease. The purpose of this study was to evaluate the effect of ABCG2 monoclonal antibody (McAb) combined with paclitaxel (PTX) conjugated with Fe3O4 nanoparticles (NPs) on MM progressed from cancer stem cells (CSCs)in non-obese-diabetic/severe-combined-immunodeficiency (NOD/SCID) mouse model. Mice were injected with MM CSCs as marked by CD138−CD34− phenotypes through tail veins. The developed MM mice were examined by micro-computer tomography scanning, ultrasonography and enzyme-linked immunosorbent analysis. These mice were then intravenously treated with different combinations of NPs, PTX, McAb, PTX-NPs and melphalan/prednisone once a week for four weeks. The injected mice developed characteristic MM-associated syndromes, including lytic bone lesions, renal damages and proteinuria. All the treated mice showed decrease in bone lesions, renal damages and anemia but increase in apoptosis compared with the mice treated with NPs only. In particular, the treatment with ABCG2 McAb plus PTX-NPs induced the strongest therapeutic response and had an efficacy even better than that of melphalan/prednisone, a conventional regimen for MM patients. These data suggest that PTX-NPs with ABCG2 McAb can be developed into potential treatment regimens for patients with relapsed/refractory MM.

Regulation gene expression of miR200c and ZEB1 positively enhances effect of tumor vaccine B16F10/GPI-IL-21 on inhibition of melanoma growth and metastasis

BackgroundGenetically modified cells have been shown to be one of the most effective tumor vaccine strategies. However, in many cases, such as in melanoma, induction of a potent immune responses against the disease still remains a major challenge. Thus, novel strategies to reinforce tumor vaccine efficacy are needed. Using microRNA (miR) and Zinc-finger E-box binding homeobox (ZEB) have received much attention for potentially regulating tumor progression. To elicit a potent antitumor efficacy against melanoma, we used tumor vaccine in combination with miR200c overexpression or ZEB1 knockdown to assess the efficacy of treatment of murine melanoma.MethodsB16F10 cell vaccine expressing interleukin 21 (IL-21) in the glycosylpho- sphatidylinositol (GPI)-anchored form (B16F10/GPI-IL-21) were developed. The vaccine was immunized into mice challenged by B16F10 cells or B16F10 cells stably transduced with lentiviral-miR200c (B16F10/miR200c) or transfected with the ZEB1-shRNA recombinant (B16F10/shZEB1) or the B16F10/GPI-IL-21 vaccine. The immune responses, tumorigenicity and lung metastasis in mice were evaluated, respectively.ResultsThe vaccination with B16F10/GPI-IL-21 markedly increased the serum cytokine levels of IFN-γ, TNF-α, IL-4 and decreased TGF-β level as well as augmented the cytotoxicity of splenocytes in immunized mice compared with control mice. In addition, the tumor vaccine B16F10/GPI-IL-21 significantly inhibited the tumor growth and reduced counts of lung metastases in mice challenged by B16F10/GPI-IL-21, B16F10/shZEB1 and B16F10/miR200c respectively compared with the control mice challenged by B16F10 cells. The efficacy mechanisms may involve in reinforcing immune responses, increasing expression of miR200c, E-cadherin and SMAD-7 and decreasing expression of TGF-β, ZEB1, Vimentin and N-cadherin in tumor tissues from the immunized mice.ConclusionsThese results indicate that the tumor vaccine B16F10/GPI-IL-21 in combination with miR200c overexpression or ZEB1 knockdown effectively inhibited melanoma growth and metastasis a murine model. Such a strategy may, therefore, be used for the clinical trials.

Effect of targeted ovarian cancer immunotherapy using ovarian cancer stem cell vaccine

BackgroundAccumulating evidence has shown that different immunotherapies for ovarian cancer might overcome barriers to resistance to standard chemotherapy. The vaccine immunotherapy may be a useful one addition to conditional chemotherapy regimens. The present study investigated the use of vaccine of ovarian cancer stem cells (CSCs) to inhibit ovarian cancer growth.MethodsCD117+CD44+CSCs were isolated from human epithelial ovarian cancer (EOC) SKOV3 cell line by using a magnetic-activated cell sorting system. Pre-inactivated CD117+CD44+CSC vaccine was vacccinated into athymic nude mice three times, and then the mice were challenged subcutaneously with SKOV3 cells. The anti-tumor efficacy of CSC vaccine was envaluated by in vivo tumorigenicity, immune efficient analysis by flow cytometer, and enzyme-linked immunosorbent assays, respectively.ResultsThe CD117+ CD44+CSC vaccine increased anti-ovarian cancer efficacy in that it depressed ovarian cancer growth in the athymic nude mice. Vaccination resulted in enhanced serum IFN-γ, decreased TGF-β levels, and increased cytotoxic activity of natural killer cells in the CD117+ CD44+CSC vaccine immunized mice. Moreover, the CSC-based vaccine significantly reduced the CD117+CD44+CSC as well as the aldehyde dehydrogenase 1 positive cell populations in the ovarian cancer tissues in the xenograft mice.ConclusionThe present study provided the first evidence that human SKOV3 CD117+ CD44+CSC-based vaccine may induce the anti-ovarian cancer immunity against tumor growth by reducing the CD117+CD44+CSC population.

Inhibitory effect of epirubicin-loaded lipid microbubbles with conjugated anti-ABCG2 antibody combined with therapeutic ultrasound on multiple myeloma cancer stem cells

Abstract Ultrasound-targeted microbubble destruction (UTMD) technique is thought to improve the chemotherapeutic agent delivery from microbubbles (MBs) in tumor tissues and reduce the side effects in non-tumor tissues. Multiple myeloma (MM) is a bone marrow cancer and remains to be an incurable disease. In this study, we used the UTMD technique to investigate the inhibitory effect of our developed novel reagent on MM cancer stem cells (CD138−CD34−MM CSCs) that are MM cells with CD138−CD34− phenotypes, responsible for MM-initiating potential, drug resistance and eventual relapse. The preparatory steps of novel reagent was first epirubicin (EPI)-loaded in the lipid MBs that was consisted of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]-biotin, dipalmitoyl-phosphatidylglycerol and 25-NBD-cholesterol, then anti-ABCG2 monoclonal antibody (mAb) was conjugated onto the MB surface to form EPI-MBs+mAb. CD138−CD34−MM CSCs were isolated from human MM RPMI 8226 cell line by the magnetic associated cell sorting method. The results showed that the attenuated proliferation, migration and invasion ability, and increased apoptosis were observed when MM CSCs were incubated with a various agents. EPI-MBs+mAb combined with therapeutic ultrasound significantly promoted the MM CSC apoptosis compared with EPI, EPI-MBs alone or EPI-MBs+mAb without ultrasound exposure. These results suggest that the developed EPI-MBs+mAb combined with therapeutic ultrasound remarkably induced MM CSC apoptosis in vitro.