ThermomiR-377-3p-induced suppression of Cirbp expression is required for effective elimination of cancer cells and cancer stem-like cells by hyperthermia.

BACKGROUND: In recent years, the development of adjunctive therapeutic hyperthermia for cancer therapy has received considerable attention. However, the mechanisms underlying hyperthermia resistance are still poorly understood. In this study, we investigated the roles of cold­inducible RNA binding protein (Cirbp) in regulating hyperthermia resistance and underlying mechanisms in nasopharyngeal carcinoma (NPC). METHODS: CCK-8 assay, colony formation assay, tumor sphere formation assay, qRT-PCR, Western blot were employed to examine the effects of hyperthermia (HT), HT + oridonin(Ori) or HT + radiotherapy (RT) on the proliferation and stemness of NPC cells. RNA sequencing was applied to gain differentially expressed genes upon hyperthermia. Gain-of-function and loss-of-function experiments were used to evaluate the effects of RNAi-mediated Cirbp silencing or Cirbp overexpression on the sensitivity or resistance of NPC cells and cancer stem-like cells to hyperthermia by CCK-8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay, and in subcutaneous xenograft animal model. miRNA transient transfection and luciferase reporter assay were used to demonstrate that Cirbp is a direct target of miR-377-3p. The phosphorylation levels of key members in ATM-Chk2 and ATR-Chk1 pathways were detected by Western blot. RESULTS: Our results firstly revealed that hyperthermia significantly attenuated the stemness of NPC cells, while combination treatment of hyperthermia and oridonin dramatically increased the killing effect on NPC cells and cancer stem cell (CSC)­like population. Moreover, hyperthermia substantially improved the sensitivity of radiation­resistant NPC cells and CSC­like cells to radiotherapy. Hyperthermia noticeably suppressed Cirbp expression in NPC cells and xenograft tumor tissues. Furthermore, Cirbp inhibition remarkably boosted anti­tumor­killing activity of hyperthermia against NPC cells and CSC­like cells, whereas ectopic expression of Cirbp compromised tumor­killing effect of hyperthermia on these cells, indicating that Cirbp overexpression induces hyperthermia resistance. ThermomiR-377-3p improved the sensitivity of NPC cells and CSC­like cells to hyperthermia in vitro by directly suppressing Cirbp expression. More importantly, our results displayed the significantly boosted sensitization of tumor xenografts to hyperthermia by Cirbp silencing in vivo, but ectopic expression of Cirbp almost completely counteracted hyperthermia-mediated tumor cell-killing effect against tumor xenografts in vivo. Mechanistically, Cirbp silencing-induced inhibition of DNA damage repair by inactivating ATM-Chk2 and ATR-Chk1 pathways, decrease in stemness and increase in cell death contributed to hyperthermic sensitization; conversely, Cirbp overexpression-induced promotion of DNA damage repair, increase in stemness and decrease in cell apoptosis contributed to hyperthermia resistance. CONCLUSION: Taken together, these findings reveal a previously unrecognized role for Cirbp in positively regulating hyperthermia resistance and suggest that thermomiR-377-3p and its target gene Cirbp represent promising targets for therapeutic hyperthermia.

Hairy gene homolog increases nasopharyngeal carcinoma cell stemness by upregulating Bmi-1.

B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) is overexpressed in various cancer types. We found that Bmi-1 mRNA levels were elevated in nasopharyngeal carcinoma (NPC) cell lines. In immunohistochemical analyses, high Bmi-1 levels were observed in not only 5 of 38 non-cancerous nasopharyngeal squamous epithelial biopsies, but also in 66 of 98 NPC specimens (67.3%). High Bmi-1 levels were detected more frequently in T3-T4, N2-N3 and stage III-IV NPC biopsies than in T1-T2, N0-N1 and stage I-II NPC samples, indicating that Bmi-1 is upregulated in advanced NPC. In 5-8F and SUNE1 NPC cells, stable depletion of Bmi-1 using lentiviral RNA interference greatly suppressed cell proliferation, induced G1-phase cell cycle arrest, reduced cell stemness and suppressed cell migration and invasion. Likewise, knocking down Bmi-1 inhibited NPC cell growth in nude mice. Both chromatin immunoprecipitation and Western blotting assays demonstrated that Hairy gene homolog (HRY) upregulated Bmi-1 by binding to its promoter, thereby increasing the stemness of NPC cells. Immunohistochemistry and quantitative real-time PCR analyses revealed that HRY expression correlated positively with Bmi-1 expression in a cohort of NPC biopsies. These findings suggested that HRY promotes NPC cell stemness by upregulating Bmi-1, and that silencing Bmi-1 can suppress NPC progression.

A real-time pluripotency reporter for the long-term and real-time monitoring of pluripotency changes in induced pluripotent stem cells.

To master the technology of reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs), which will lay a good foundation for setting up a technology platform on reprogramming human cancer cells into iPSCs. Mouse iPSCs (i.e., Oct4-GFP miPSCs) was successfully generated from mouse embryonic fibroblasts (MEFs) harboring Oct4-EGFP transgene by introducing four factors, Oct4, Sox2, c-Myc and Klf4, under mESC (Murine embryonic stem cells) culture conditions. Oct4-GFP miPSCs were similar to mESCs in morphology, proliferation, mESC-specific surface antigens and gene expression. Additionally, Oct4-GFP miPSCs could be cultured in suspension to form embryoid bodies (EBs) and differentiate into cell types of the three germ layers in vitro. Moreover, Oct4-GFP miPSCs could develop to teratoma and chimera in vivo. Unlike cell cycle distribution of MEFs, Oct4-GFP miPSCs are similar to mESCs in the cell cycle structure which consists of higher S phase and lower G1 phase. More importantly, our data demonstrated that MEFs harboring Oct4-EGFP transgene did not express GFP, until they were reprogrammed to the pluripotent stage (iPSCs), while the GFP expression was progressively lost when these pluripotent Oct4-GFP miPSCs exposed to EB-mediated differentiation conditions, suggesting the pluripotency of Oct4-GFP miPSCs can be real-time monitored over long periods of time via GFP assay. Altogether, our findings demonstrate that Oct4-GFP miPSC line is successfully established, which will lay a solid foundation for setting up a technology platform on reprogramming cancer cells into iPSCs. Furthermore, this pluripotency reporter system permits the long-term real-time monitoring of pluripotency changes in a live single-cell, and its progeny.

TZAP plays an inhibitory role in the self-renewal of porcine mesenchymal stromal cells and is implicated the regulation of premature senescence via the p53 pathway.

BACKGROUND: Mesenchymal stromal cells (MSCs) were originally characterized by the ability to differentiate into different mesenchymal lineages in vitro, and their immunomodulatory and trophic functions have recently aroused significant interest in the application of MSCs in cell-based regenerative medicine. However, a major problem in clinical practice is the replicative senescence of MSCs, which limits the cell proliferation potential of MSCs after large-scale expansion. Telomeric zinc finger-associated protein (TZAP), a novel specific telomere-binding protein, was recently found to stimulate telomere trimming and prevent excessive telomere elongation. The aim of this study was to elucidate the role of TZAP in regulating MSCs senescence, differentiation and proliferation. METHOD: Primary porcine mesenchymal stromal cells (pMSCs) were isolated from the bone marrow of Tibet minipigs by a noninvasive method in combination with frequent medium changes (FMCs). The deterioration of the pMSCs' proliferation capacity and their resultant entry into senescence were analyzed by using CCK8 and EdU incorporation assays, SA-ß-gal staining and comparisons of the expression levels of cellular senescence markers (p16INK14 and p21) in pMSC cell lines with TZAP overexpression or knockout. The effects of TZAP overexpression or knockout on the differentiation potential of pMSCs were assessed by alizarin red S staining after osteogenic induction or by oil red O staining after adipogenic induction. The effect of TZAP overexpression and the involvement of the p53 signaling pathway were evaluated by detecting changes in ARF, MDM2, P53 and P21 protein levels in pMSCs. RESULTS: TZAP levels were significantly elevated in late-passage pMSCs compared to those in early-passage pMSCs. We also observed significantly increased levels of the senescence markers p16INK4A and p21. Overexpression of TZAP reduced the differentiation potential of the cells, leading to premature senescence in early-passage pMSCs, while knockout of TZAP led to the opposite phenotype in late-passage pMSCs. Furthermore, overexpression of TZAP activated the P53 pathway (ARF-MDM2-P53-P21WAF/CDKN1A) in vitro. TZAP also downregulated the expression levels of PPARγ and Cebpα, two key modulators of adipogenesis. CONCLUSIONS: This study demonstrates that the level of TZAP is closely related to differentiation potential in pMSCs and affects cellular senescence outcomes via the p53 pathway. Therefore, attenuation of intracellular TZAP levels could be a new strategy for improving the efficiency of pMSCs in cell therapy and tissue engineering applications.

Direct conversion of pig fibroblasts to chondrocyte-like cells by c-Myc.

Unexpectedly, we found that c-Myc-expressing porcine embryonic fibroblasts (PEFs) subcutaneously implanted into nude mice formed cartilage-like tissues in vivo, while previous studies revealed the direct conversion of mouse and human somatic cells into chondrocytes by the combined use of several defined factors, including c-Myc, which prompted us to explore whether PEFs can be reprogrammed to become pig induced chondrocyte-like cells (piCLCs) via ectopic expression of c-Myc alone. In this study, c-Myc-expressing PEFs, designated piCLCs, which exhibited a significantly enhanced proliferation ability in vitro, displayed a chondrogenic phenotypes in vitro, as shown by the cell morphology, toluidine blue staining, alcian blue staining and chondrocyte marker gene expression. Additionally, piCLCs with a polygonal chondrocyte-like morphology were readily and efficiently converted from PEFs by enforced c-Myc expression within 10 days, while piCLCs maintained the chondrocytic phenotype and normal karyotype during long-term subculture. piCLC-derived single clones with a chondrogenic phenotype in vitro exhibited homogeneity in cell morphology and staining intensity compared with mixed piCLCs. Although the mixtures of cartilaginous tissues and tumorous tissues accounted for ~12% (6/51) of all xenografts (51), piCLCs generated stable, homogenous, hyaline cartilage-like tissues without tumour formation at 45 out of the 51 injected sites when subcutaneously injected into nude mice. The hyaline cartilage-like tissues remained for at least 16 weeks. Taken together, these findings demonstrate for the first time the direct induction of chondrocyte-like cells from PEFs with only c-Myc.

Gambogic Acid Efficiently Kills Stem-Like Colorectal Cancer Cells by Upregulating ZFP36 Expression.

BACKGROUND/AIMS: Gambogic acid (GA), the main active compound of Gamboge hanburyi, has been reported to be a potential novel antitumor drug. Whether GA inhibits putative cancer stem cells (CSCs), which are considered to be the major cause of cancer treatment failure, remains largely unknown. This study investigated whether GA inhibits the CSCs of colorectal cancer (CRC) and its possible mechanisms. METHODS: We performed CCK8 and tumor sphere formation assays, percentage analysis of both side population and CD133+CD44+ cells, and the detection of stem cells markers, in order to assess the role of GA in inhibiting the stem celllike features of CRC. An mRNA microarray was performed to identify the downstream gene affected by GA and rescue assays were performed to further clarify whether the downstream gene is involved in the GA induced decrease of the stem cell-like CRC population. CRC cells were engineered with a CSC detector vector encoding GFP and luciferase (Luc) under the control of the Nanog promoter, which were utilized to investigate the effect of GA on putative CSC in human tumor xenograft-bearing mice using in vivo bioluminescence imaging. RESULTS: Our results showed that GA significantly reduced tumor sphere formation and the percentages of side population and CD133+CD44+ cells, while also decreasing the expression of stemness and EMT-associated markers in CRC cells in vitro. GA killed stem-like CRC cells by upregulating the expression of ZFP36, which is dependent on the inactivation of the EGFR/ ERK signaling pathway. GFP+ cells harboring the PNanog-GFP-T2A-Luc transgene exhibited CSC characteristics. The in vivo results showed that GA significantly inhibited tumor growth in nude mice, accompanied by a remarkable reduction in the putative CSC number, based on whole-body bioluminescence imaging. CONCLUSION: These findings suggest that GA significantly inhibits putative CSCs of CRC both in vitro and in vivo by inhibiting the activation of the EGFR/ ERK/ZFP36 signaling pathway and may be an effective drug candidate for anticancer therapies.

Recognition and killing of cancer stem-like cell population in hepatocellular carcinoma cells by cytokine-induced killer cells via NKG2d-ligands recognition.

There is an urgent need for more potent and safer approaches to eradicate cancer stem cells (CSCs) for curing cancer. In this study, we investigate cancer-killing activity (CKA) of cytokine-induced killer (CIK) cells against CSCs of hepatocellular carcinoma (HCC). To visualize CSCs in vitro by fluorescence imaging, and image and quantify CSCs in tumor xenograft-bearing mice by bioluminescence imaging, HCC cells were engineered with CSC detector vector encoding GFP and luciferase controlled by Nanog promoter. We found that CIK cells have a strong CKA in vitro against putative CSCs of HCC, as shown by tumorsphere formation and time-lapse imaging. Additionally, time-lapse recording firstly revealed that putative CSCs were attacked simultaneously by many CIK cells and finally eradicated by CIK cells, indicating the necessity of achieving sufficient effector-to-target ratios. We firstly illustrated that anti-NKG2D antibody blocking partially but significantly inhibited CKA of CIK cells against putative CSCs. More importantly, intravenous infusion of CIK cells remarkably delayed tumor growth in mice with a significant decrease in putative CSC number monitored by bioluminescence imaging. Taken together, these findings demonstrate CKA of CIK cells against putative CSCs of HCC, at least in part, by NKG2D-ligands recognition.

Hes1 triggers epithelial-mesenchymal transition (EMT)-like cellular marker alterations and promotes invasion and metastasis of nasopharyngeal carcinoma by activating the PTEN/AKT pathway.

Overexpression of the transcriptional factor Hes1 (hairy and enhancer of split-1) has been observed in numerous cancers, but the precise roles of Hes1 in epithelial-mesenchymal transition (EMT), cancer invasion and metastasis remain unknown. Our current study firstly revealed that Hes1 upregulation in a cohort of human nasopharyngeal carcinoma (NPC) biopsies is significantly associated with the EMT, invasive and metastatic phenotypes of cancer. In the present study, we found that Hes1 overexpression triggered EMT-like cellular marker alterations of NPC cells, whereas knockdown of Hes1 through shRNA reversed the EMT-like phenotypes, as strongly supported by Hes1-mediated EMT in NPC clinical specimens described above. Gain-of-function and loss-of-function experiments demonstrated that Hes1 promoted the migration and invasion of NPC cells in vitro. In addition, exogenous expression of Hes1 significantly enhanced the metastatic ability of NPC cells in vivo. Chromatin immunoprecipitation (ChIP) assays showed that Hes1 inhibited PTEN expression in NPC cells through binding to PTEN promoter region. Increased Hes1 expression and decreased PTEN expression were also observed in a cohort of NPC biopsies. Additional studies demonstrated that Hes1-induced EMT-like molecular changes and increased motility and invasion of NPC cells were mediated by PTEN. Taken together, our results suggest, for what we believe is the first time, that Hes1 plays an important role in the invasion and metastasis of NPC through inhibiting PTEN expression to trigger EMT-like phenotypes.

Cytokine-induced killer cells efficiently kill stem-like cancer cells of nasopharyngeal carcinoma via the NKG2D-ligands recognition.

Cancer stem cells (CSCs) are considered to be the root cause for cancer treatment failure. Thus, there remains an urgent need for more potent and safer therapies against CSCs for curing cancer. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against putative CSCs of nasopharyngeal carcinoma (NPC) was fully evaluated in vitro and in vivo. To visualize putative CSCs in vitro by fluorescence imaging, and image and quantify putative CSCs in tumor xenograft-bearing mice by in vivo bioluminescence imaging, NPC cells were engineered with CSC detector vector encoding GFP and luciferase (Luc) under control of Nanog promoter. Our study reported in vitro intense tumor-killing activity of CIK cells against putative CSCs of NPC, as revealed by percentage analysis of side population cells, tumorsphere formation assay and Nanog-promoter-GFP-Luc reporter gene strategy plus time-lapse recording. Additionally, time-lapse imaging firstly illustrated that GFP-labeled or PKH26-labeled putative CSCs or tumorspheres were usually attacked simultaneously by many CIK cells and finally killed by CIK cells, suggesting the necessity of achieving sufficient effector-to-target ratios. We firstly confirmed that NKG2D blockade by anti-NKG2D antibody significantly but partially abrogated CIK cell-mediated cytolysis against putative CSCs. More importantly, intravenous infusion of CIK cells significantly delayed tumor growth in NOD/SCID mice, accompanied by a remarkable reduction in putative CSC number monitored by whole-body bioluminescence imaging. Taken together, our findings suggest that CIK cells demonstrate the intense tumor-killing activity against putative CSCs of NPC, at least in part, by NKG2D-ligands recognition. These results indicate that CIK cell-based therapeutic strategy against CSCs presents a promising and safe approach for cancer treatment.

MicroRNA-19 triggers epithelial-mesenchymal transition of lung cancer cells accompanied by growth inhibition.

The miR-19 family (miR-19a and miR-19b-1) are key oncogenic components of the miR-17-92 cluster. Overexpression of miR-19 is strongly associated with cancer invasion and metastasis, and poor prognosis of cancer patients. However, the underlying mechanisms remain largely unknown. In the present study, we found that enforced expression of miR-19 including miR-19a and miR-19b-1 triggered epithelial-mesenchymal transition (EMT) of lung cancer cells A549 and HCC827 as shown by mesenchymal-like morphological conversion, downregulation of epithelial proteins (e.g., E-cadherin, ZO-1 (zona occludens 1), and α-catenin), upregulation of mesenchymal proteins (e.g., vimentin, fibronectin 1, N-cadherin, and snail1), formation of stress fibers, and reduced cell adhesion. In addition, enhanced migration and invasion were observed in the cancer cells A549 and HCC827 undergoing EMT. In contrast, silencing of endogenous miR-19 reversed EMT and reduced the migration and invasion abilities of A549 and HCC827 cells. DNA microarray results revealed significant changes of the expression of genes related to EMT, migration, and metastasis of miR-19-expressing A549 cells. Moreover, siRNA-mediated knockdown of PTEN, a target of miR-19, also resulted in EMT, migration, and invasion of A549 and HCC827 cells, suggesting that PTEN is involved in miR-19-induced EMT, migration and invasion of lung cancer cells. Furthermore, lung cancer cells undergoing EMT induced by miR-19 demonstrated reduced proliferation in vitro and in vivo, and enhanced resistance to apoptosis caused by TNF-α. Taken together, these findings suggest that miR-19 triggers EMT, which has an important role in the invasion and migration of lung cancer cells, accompanied by the reduced proliferation of cells.