Mesenchymal-like Cells Research Articles

Regulatory effects of lncRNAs and miRNAs on the crosstalk between autophagy and EMT in cancer: a new era for cancer treatment.

Autophagy and EMT (epithelial-mesenchymal transition) are the two principal biological processes and ideal therapeutic targets during cancer development. Autophagy, a highly conserved process for degrading dysfunctional cellular components, plays a dual role in tumors depending on the tumor stage and tissue types. The EMT process is the transition differentiation from an epithelial cell to a mesenchymal-like cell and acquiring metastatic potential. There is evidence that the crosstalk between autophagy and EMT is complex in cancer. In recent years, more studies have shown that long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are involved in autophagy, EMT, and their crosstalk. Therefore, accurate understanding of the regulatory mechanisms of lncRNAs and miRNAs in autophagy, EMT and their interactions is crucial for the clinical management of cancers. An extensive literature search was conducted on the Google Scholar and PubMed databases. The keywords used for the search included: autophagy, EMT, crosstalk, lncRNAs, miRNAs, cancers, diagnostic biomarkers, and therapeutic targets. This search provided relevant articles published in peer-reviewed journals until 2021. Data from these various studies were extracted and used in this review. The results showed that lncRNAs/miRNAs as tumor inhibitors or tumor inducers could regulate autophagy, EMT, and their interaction by regulating several molecular signaling pathways. The lncRNAs/miRNAs involved in autophagy and EMT processes could have potential uses in cancer diagnosis, prognosis, and therapy. Such information could help find and develop lncRNAs/miRNAs based new tools for diagnosing, prognosis, and creating anti-cancer therapies.

Microscopical observations on the regenerating tail of tsinling dwarf skink (Scincella tsinlingensis)

Although the key steps of tail regeneration in lizards are well understood, further investigations involving skinks can provide the field of regeneration research with additional information. In order to characterize the cytoarchitecture of tail regeneration in Scincella tsinlingensis, an endemic species in China, its histological events and growth trends are investigated. The rate of tail regeneration varies with the season: it proceeds faster in summer and autumn than it does in winter and spring. Tail regeneration of S. tsinlingensis is summarized as wound healing, blastema formation, cell differentiation and tail growth, which can be subdivided into seven stages. Wound healing following tail loss, begins with an obvious outgrowth undergoing re-epithelialization. Numerous proliferating mesenchymal-like cells aggregate near the distal end of the severed spinal cord to form the blastema. The expanding blastema is invaded by blood vessels, nerves and ependyma. A cartilaginous skeleton is formed around the ependymal tube and the muscle starts to differentiate. The keratinization of epidermis coincides with scale formation. Pigmentation eventually occurs in the regenerated tail. Tail regeneration in S. tsinlingensis is an epimorphic kind of regeneration that is also known as blastema-mediated. Structure and composition of the regenerated tail, including its cytoarchitecture, represent a conserved pattern of regeneration also known from other lizards.

The Roles of S100A4 and the EGF/EGFR Signaling Axis in Pulmonary Hypertension with Right Ventricular Hypertrophy.

Simple SummaryPulmonary hypertension (PH) is a condition characterized by increased pulmonary arterial pressure. PH can further lead to right ventricular hypertrophy (RVH) and, consequently, heart failure. Endothelial-to-mesenchymal transition (EndMT) was identified as a key process in PH pathology. Nonetheless, the exact systemic and local levels of EndMT factors have not been comprehensively studied so far. Here, we quantified S100A4, epidermal growth factor (EGF), and EGF receptor (EGFR) in serum and tissue samples of two classes of PH with RVH. Patients with left heart disease and healthy volunteers served as controls. Serum S100A4 was decreased in the PH groups investigated, while EGF levels were increased in one PH cohort. EGFR was diminished in all groups compared to healthy controls. Surgical treatment of PH showed no effect on systemic EndMT marker levels. Furthermore, we observed a positive correlation between advanced PH stages and S100A4. Together, these findings help to deepen our understanding of the complex molecular events contributing to PH pathology and disease progression.Pulmonary hypertension (PH) is characterized by increased pulmonary arterial pressure caused by the accumulation of mesenchymal-like cells in the pulmonary vasculature. PH can lead to right ventricular hypertrophy (RVH) and, ultimately, heart failure and death. In PH etiology, endothelial-to-mesenchymal transition (EndMT) has emerged as a critical process governing the conversion of endothelial cells into mesenchymal cells, and S100A4, EGF, and EGFR are implicated in EndMT. However, a potential role of S100A4, EGF, and EGFR in PH has to date not been elucidated. We therefore quantified S100A4, EGF, and EGFR in patients suffering from chronic thromboembolic pulmonary hypertension (CTEPH) and idiopathic pulmonary arterial hypertension (iPAH). To determine specificity for unilateral heart disease, the EndMT biomarker signature was further compared between PH patients presenting with RVH and patients suffering from aortic valve stenosis (AVS) with left ventricular hypertrophy. Reduced S100A4 concentrations were found in CTEPH and iPAH patients with RVH. Systemic EGF was increased in CTEPH but not in iPAH, while AVS patients displayed slightly diminished EGF levels. EGFR was downregulated in all patient groups when compared to healthy controls. Longitudinal data analysis revealed no effect of surgical therapies on EndMT markers. Pulmonary thrombo-endarterectomized samples were devoid of S100A4, while S100A4 tissue expression positively correlated with higher grades of Heath–Edwards histopathological lesions of iPAH-derived lung tissue. Histologically, EGFR was not detectable in CTEPH lungs or in iPAH lesions. Together, our data suggest an intricate role for S100A4 and EGF/EGFR in PH with right heart pathology.

In vitro preparation of human Dental Pulp Stem Cell grafts with biodegradable polymer scaffolds for nerve tissue engineering.

Human Dental Pulp Stem Cells (hDPSCs) are one of the most promising stem cell sources for tissue engineering and regeneration, due to their extraordinary multi-lineage differentiation ability, ease of extraction from biological waste in dental clinics, safe non-tumorigenic phenotype, immune-tolerance upon in vivo transplantation, and great possibilities of application in autologous tissue reconstruction. The in vitro manipulation of hDPSCs paves the way for drug screening and tailor-made regeneration of damaged tissues, in the context of personalized medicine. The neural crest phenotype of these stem cells gives them the capacity to differentiate to a large variety of cell types, including neural-lineage cells. In this chapter, we describe various culture methods to generate different cellular phenotypes from hDPSCs, which can not only grow as mesenchymal-like plastic adherent cells, but also as non-adherent neurogenic dentospheres in serum-free medium. Floating dentospheres can be used to generate large populations of mature neuronal and glial marker expressing cells, which may be cultured over a substrate of nanopatterned scaffold based on biodegradable poly(lactide-co-caprolactone) (PLCL) to induce a controlled alignment of neurites and cell migration, to generate in vivo biocompatible constructs for nerve tissue bioengineering.

Integrin-β4 regulates the dynamic changes of phenotypic characteristics in association with epithelial-mesenchymal transition (EMT) and RhoA activity in airway epithelial cells during injury and repair.

Background: In airway disease such as asthma a hyperactive cellular event of epithelial-mesenchymal transition (EMT) is considered as the mechanism of pathological airway tissue remodeling after injury to the airway epithelium. And the initiation of EMT in the airways depends on the epithelial disruption involving dissolution and/or destabilization of the adhesive structures between the cells and ECM. Previously, we have shown that integrin-β4, an epithelial adhesion molecule in bronchial epithelium is an important regulator of cell proliferation and wound repair in human airway epithelial cells. Therefore, in this study we aimed to investigate whether integrin-β4 also regulates EMT phenotypes during injury and repair in airway epithelial cells of both wild type/integrin-β4-/- mice in vivo and cultured cells treated with integrin-β4/nonsense siRNA in vitro.Methods: We induced injury to the airway epithelial cells by either repeated exposure to ozone and mechanical scratch wound, and subsequently examined the EMT-related phenotypic features in the airway epithelial cells including biomarkers expression, adhesion and cytoskeleton reorganization and cell stiffness.Results: The results show that in response to injury (ozone exposure/scratch wound) and subsequent spontaneous repair (ozone withdrawal/wound healing) both in vivo and in vitro, the airway epithelial cells underwent dynamic changes in the epithelial and mesenchymal biomarkers expression, adhesion and cytoskeleton structures as well as cell stiffness, all together exhibiting enhanced EMT phenotypic features after injury and reversal of the injury-induced effects during repair. Importantly, these injury/repair-associated EMT phenotypic changes in airway epithelial cells appeared to be dependent on integrin-β4 expression. More specifically, when integrin-β4 was deficient in mice (integrin-β4-/-) the repair of ozone-injured airway epithelium was impaired and the recovery of ozone-enhanced EMT biomarkers expression in the airway epithelium was delayed. Similarly, in the scratch wounded airway epithelial cells with integrin-β4 knockdown, the cells were impaired in all aspects related to EMT during wound and repair including cell proliferation, wound closure rate, adhesion and cytoskeleton protein expression (vinculin and vimentin), mesenchymal-like F-actin reorganization, cell stiffness and RhoA activation.Conclusion: Taken together, these results suggested that integrin-β4 may be essential in regulating the effects of injury and repair on EMT in airway epithelial cells via influencing both the cell adhesion to ECM and cells' physical phenotypes through RhoA signaling pathway.

The Role of Dielectrophoresis for Cancer Diagnosis and Prognosis.

Simple SummaryDielectrophoresis (DEP) is a label-free cell manipulation technique based on electrical differences that can be applied to liquid biopsies in the context of cancer prognosis and early diagnosis. In fact, it can overcome the current limitations of other platforms in detecting circulating tumor cells (CTC) in prostate cancer (PC), such as the low number of CTCs detected (less than one CTC/mL of peripheral blood), high-cost and time-consuming means for detection, quantification, isolation and characterization. The aim of this review is to give deep insights into the role of DEP for the diagnosis and prognosis of cancer.Liquid biopsy is emerging as a potential diagnostic tool for prostate cancer (PC) prognosis and diagnosis. Unfortunately, most circulating tumor cells (CTC) technologies, such as AdnaTest or Cellsearch®, critically rely on the epithelial cell adhesion molecule (EpCAM) marker, limiting the possibility of detecting cancer stem-like cells (CSCs) and mesenchymal-like cells (EMT-CTCs) that are present during PC progression. In this context, dielectrophoresis (DEP) is an epCAM independent, label-free enrichment system that separates rare cells simply on the basis of their specific electrical properties. As compared to other technologies, DEP may represent a superior technique in terms of running costs, cell yield and specificity. However, because of its higher complexity, it still requires further technical as well as clinical development. DEP can be improved by the use of microfluid, nanostructured materials and fluoro-imaging to increase its potential applications. In the context of cancer, the usefulness of DEP lies in its capacity to detect CTCs in the bloodstream in their epithelial, mesenchymal, or epithelial–mesenchymal phenotype forms, which should be taken into account when choosing CTC enrichment and analysis methods for PC prognosis and diagnosis.

LncCRLA Enhanced Chemoresistance in Lung Adenocarcinoma That Underwent EpithelialMesenchymal Transition.

EMT confers increased metastatic potential and the resistance to chemotherapies to cancer cells. However, the precise mechanisms of EMT-related chemotherapy resistance remain unclear. c-Src-mediated caspase 8 phosphorylation essential for EMT in lung adenocarcinoma cell lines preferentially occurs in cells with the mesenchymal phenotype, resulting in chemoresistance to cisplatin plus paclitaxel in patients with resectable lung adenocarcinoma and a significantly worse 5-year PFS. Cisplatin killed lung adenocarcinoma cells regardless of caspase 8. Paclitaxel-triggered necroptosis in lung adenocarcinoma cells was dependent on the phosphorylation or deficiency of caspase 8, during which FADD interacted with RIPK1 to activate the RIPK1/RIPK3/MLKL signaling axis. Accompanied with c-Src-mediated caspase 8 phosphorylation to trigger EMT, a novel lncRNA named lncCRLA was markedly upregulated and inhibited RIPK1-induced necroptosis by impairing RIPK1–RIPK3 interaction via binding to the intermediate domain of RIPK1. Dasatinib mitigated c-Src-mediated phosphorylation of caspase 8-induced EMT and enhanced necroptosis in mesenchymal-like lung adenocarcinoma cells treated with paclitaxel, while c-FLIP knockdown predominantly sensitized the mesenchymal-like lung adenocarcinoma cells to paclitaxel + dasatinib. c-Src–caspase 8 interaction initiates EMT and chemoresistance via caspase 8 phosphorylation and lncCRLA expression, to which the dasatinib/paclitaxel liposome + siFLIP regimen was lethal.

Spontaneous formation and spatial self-organization of mechanically induced mesenchymal-like cells within geometrically confined cancer cell monolayers

There is spatiotemporal heterogeneity in cell phenotypes and mechanical properties in tumor tissues, which is associated with cancer invasion and metastasis. It is well-known that exogenous growth factors like transforming growth factor (TGF)-β, can induce epithelial-mesenchymal transition (EMT)-based phenotypic transformation and the formation of EMT patterning on geometrically confined monolayers with mechanics heterogeneity. In the absence of exogenous TGF-β stimulation, however, whether geometric confinement-caused mechanics heterogeneity of cancer cell monolayers alone can trigger the EMT-based phenotypic heterogeneity still remains mysterious. Here, we develop a micropattern-based cell monolayer model to investigate the regulation of mechanics heterogeneity on the cell phenotypic switch. We reveal that mechanics heterogeneity itself is enough to spontaneously induce the emergence of mesenchymal-like phenotype and asymmetrical activation of TGF-β-SMAD signaling. Spatiotemporal dynamics of patterned cell monolayers with mesenchymal-like phenotypes is essentially regulated by tissue-scale cell behaviors like proliferation, migration as well as heterogeneous cytoskeletal contraction. The inhibition of cell contraction abrogates the asymmetrical TGF-β-SMAD signaling activation level and the emergence of mesenchymal-like phenotype. Our work not only sheds light on the key regulation of mechanics heterogeneity caused by spatially geometric confinement on regional mesenchymal-like phenotype of cancer cell monolayers, but highlights the key role of biophysical/mechanical cues in triggering phenotypic switch.

The matricellular protein CCN6 differentially regulates mitochondrial metabolism in normal epithelium and in metaplastic breast carcinomas.

Metaplastic breast carcinoma (MBC) is an aggressive subtype of triple negative breast cancer with undefined precursors, limited response to chemotherapy, and frequent distant metastasis. Our laboratory has reported that CCN6/WISP3, a secreted protein that regulates growth factor signaling, is downregulated in over 85% of MBCs. Through generation of a mammary epithelial cell-specific Ccn6 knockout mouse model (MMTV-cre;Ccn6fl/fl) we have demonstrated that CCN6 is a tumor suppressor for MBC; MMTV-cre;Ccn6fl/fl mice develop tumors recapitulating the histopathology and proteogenomic landscape of human MBC, but the mechanisms need further investigation. In this study, we report that preneoplastic mammary glands of 8-week-old MMTV-Cre;Ccn6fl/fl female mice show significant downregulation of mitochondrial respiratory chain genes compared to controls, which are further downregulated in MBCs of MMTV-Cre;Ccn6fl/fl mice and humans. We found that CCN6 downregulation in non-tumorigenic breast cells reduces mitochondrial respiration and increases resistance to stress-induced apoptosis compared to controls. Intracellular ectopic CCN6 protein localizes to the mitochondria in MDA-MB-231 mesenchymal-like breast cancer cells, increases mitochondrial respiration and generation of reactive oxygen species, and reverses doxorubicin resistance of MBC cells. Our data highlight a novel function of CCN6 in the regulation of redox states in preneoplastic progression and suggest potential preventative and treatment strategies against MBC based on CCN6 upregulation.

Atorvastatin preferentially inhibits the growth of high ZEB-expressing canine cancer cells.

The epithelial-to-mesenchymal transition (EMT) is fundamental in cancer progression and contributes to the acquisition of malignant properties. The statin class of cholesterol-lowering drugs exhibits pleiotropic anticancer effects in vitro and in vivo, and many epidemiologic studies have reported a correlation between statin use and reduced cancer mortality. We have shown previously that sensitivity to the anti-proliferative effect of statins varies among human cancer cells and statins are more effective against mesenchymal-like cells than epithelial-like ones in human cancers. There have only been few reports on the application of statins to cancer therapy in veterinary medicine, and differences in statin sensitivity among canine cancer cells have not been examined. In this study, we aimed to clarify the correlation between sensitivity to atorvastatin and epithelial/mesenchymal states in 11 canine cancer cell lines derived from mammary gland, squamous cell carcinoma, lung, and melanoma. Sensitivity to atorvastatin varied among canine cancer cells, with IC50 values ranging from 5.92 to 71.5μM at 48 h, which were higher than the plasma concentrations clinically achieved with statin therapy. Atorvastatin preferentially attenuated the proliferation of mesenchymal-like cells. In particular, highly statin-sensitive cells were characterized by aberrant expression of the ZEB family of EMT-inducing transcription factors. However, ZEB2 silencing in highly sensitive cells did not induce resistance to atorvastatin. Taken together, these results suggest that high expression of ZEB is a characteristic of highly statin-sensitive cells and could be a molecular marker for predicting whether cancers are sensitive to statins, though ZEB itself does not confer statin sensitivity.

Statins affect cancer cell plasticity with distinct consequences for tumor progression and metastasis.

SummaryStatins are among the most commonly prescribed drugs, and around every fourth person above the age of 40 is on statin medication. Therefore, it is of utmost clinical importance to understand the effect of statins on cancer cell plasticity and its consequences to not only patients with cancer but also patients who are on statins. Here, we find that statins induce a partial epithelial-to-mesenchymal transition (EMT) phenotype in cancer cells of solid tumors. Using a comprehensive STRING network analysis of transcriptome, proteome, and phosphoproteome data combined with multiple mechanistic in vitro and functional in vivo analyses, we demonstrate that statins reduce cellular plasticity by enforcing a mesenchymal-like cell state that increases metastatic seeding ability on one side but reduces the formation of (secondary) tumors on the other due to heterogeneous treatment responses. Taken together, we provide a thorough mechanistic overview of the consequences of statin use for each step of cancer development, progression, and metastasis.

TCOF1 upregulation in triple-negative breast cancer promotes stemness and tumour growth and correlates with poor prognosis

BackgroundTriple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor prognosis. By performing multiomic profiling, we recently uncovered super-enhancer heterogeneity between breast cancer subtypes. Our data also revealed TCOF1 as a putative TNBC-specific super-enhancer-regulated gene. TCOF1 plays a critical role in craniofacial development but its function in cancer remains unclear.MethodsOverall survival and multivariant Cox regression analyses were conducted using the METABRIC data set. The effect of TCOF1 knockout on TNBC growth and stemness was evaluated by in vitro and in vivo assays. RNA-seq and rescue experiments were performed to explore the underlying mechanisms.ResultsTCOF1 is frequently upregulated in TNBC and its elevated expression correlates with shorter overall survival. TCOF1 depletion significantly inhibits the growth and stemness of basal-like TNBC, but not of mesenchymal-like cells, highlighting the distinct molecular dependency in different TNBC subgroups. RNA-seq uncovers several stem cell molecules regulated by TCOF1. We further demonstrate that KIT is a downstream effector of TCOF1 in mediating TNBC stemness. TCOF1 expression in TNBC is regulated by the predicted super-enhancer.ConclusionsTCOF1 depletion potently attenuates the growth and stemness of basal-like TNBC. Expression of TCOF1 may serve as a TNBC prognostic marker and a therapeutic target.

Melatonin enhances cisplatin-induced cell death through inhibition of DERL1 in mesenchymal-like CD44high OSCC cells.

Melatonin is a hormone that is primarily produced in the pineal gland and is involved in wide range of biological functions. However, the impact of melatonin on chemotherapy-induced cell death remains to be elucidated in oral squamous cell carcinoma (OSCC) cells. The objective of this study was to clarify the role of melatonin in cisplatin-induced cytotoxicity in CD44high OSCC cells. CD44high OSCC cells were cultured on fibronectin-coated hydrogel. A lactate dehydrogenase cytotoxicity assay was performed to evaluate cisplatin-induced cell death. The effect of melatonin on cisplatin-induced cell death and Derlin-1 (DERL1) endoplasmic reticulum membrane protein expression was investigated. CD44high OSCC cells exhibited mesenchymal-like features when cultured on fibronectin-coated hydrogel. Mesenchymal-like CD44high OSCC cells demonstrated strong resistance to cisplatin-induced cell death compared with epithelial-like CD44high OSCC cells. DERL1mRNA and DERL1 protein expression levels were significantly higher in mesenchymal-like CD44high cells compared with epithelial-like CD44high cells. Cisplatin-induced cell death was significantly enhanced after DERL1siRNA knockdown, suggesting that DERL1 is involved in resistance to cisplatin-induced cell death. Melatonin significantly inhibited DERL1 expression and enhanced cisplatin-induced cell death in mesenchymal-like CD44high cells. miR-181c-5p expression was significantly upregulated in the presence of melatonin. Furthermore, melatonin-inhibited DERL1 expression was significantly recovered by miR-181c-5p inhibitor. In addition, melatoninenhanced cisplatin-induced cell death was attenuated by miR-181c-5p inhibitor. These results suggest that melatonin-induced miR-181c-5p enhances cisplatin-induced cell death through inhibition of DERL1 in mesenchymal-like CD44high cells. Melatonin plays a vital role in promoting cisplatin-induced cytotoxicity in mesenchymal-like CD44high OSCC cells.

Disulfiram Sensitizes a Therapeutic-Resistant Glioblastoma to the TGF-β Receptor Inhibitor.

Despite neurosurgery following radiation and chemotherapy, residual glioblastoma (GBM) cells develop therapeutic resistance (TR) leading to recurrence. The GBM heterogeneity confers TR. Therefore, an effective strategy must target cancer stem cells (CSCs) and other malignant cancer cells. TGF-β and mesenchymal transition are the indicators for poor prognoses. The activity of aldehyde dehydrogenases (ALDHs) is a functional CSC marker. However, the interplay between TGF-β and ALDHs remains unclear. We developed radiation-resistant and radiation-temozolomide-resistant GBM models to investigate the underlying mechanisms conferring TR. Galunisertib is a drug targeting TGF-β receptors. Disulfiram (DSF) is an anti-alcoholism drug which functions by inhibiting ALDHs. The anti-tumor effects of combining DSF and Galunisertib were evaluated by in vitro cell grow, wound healing, Transwell assays, and in vivo orthotopic GBM model. Mesenchymal-like phenotype was facilitated by TGF-β in TR GBM. Additionally, TR activated ALDHs. DSF inhibited TR-induced cell migration and tumor sphere formation. However, DSF did not affect the tumor growth in vivo. Spectacularly, DSF sensitized TR GBM to Galunisertib both in vitro and in vivo. ALDH activity positively correlated with TGF-β-induced mesenchymal properties in TR GBM. CSCs and mesenchymal-like GBM cells targeted together by combining DSF and Galunisertib may be a good therapeutic strategy for recurrent GBM patients.

Tinzaparin inhibits VL30 retrotransposition induced by oxidative stress and/or VEGF in HC11 mouse progenitor mammary cells: Association between inhibition of cancer stem cell proliferation and mammosphere disaggregation.

Tinzaparin is an anticoagulant and antiangiogenic drug with inhibitory properties against tumor growth. VEGF stimulates angiogenesis, while an association between reactive oxygen species (ROS) and angiogenesis is involved in tumor progression. The present study aimed to investigate the effect of tinzaparin on VL30 retrotransposition-positive mouse HC11 mammary stem-like epithelial cells, previously reported to be associated with induced mammosphere/cancer stem cell (CSC) generation and tumorigenesis. Under 24 h serum starvation, 15.2% nominal retrotransposition frequency was increased to 29%. Additionally, while treatment with 3–12 ng/ml VEGF further induced retrotransposition frequency in a dose-dependent manner (up to 40.3%), pre-incubation with tinzaparin (2 IU/ml) for 0.5–4 h reduced this frequency to 18.3% in a time-dependent manner, confirmed by analogous results in NIH3T3 fibroblasts. Treatment with 10–40 pg/ml glucose oxidase (GO) for 24 h induced HC11 cell retrotransposition in a dose-dependent manner (up to 82.5%), while a 3 h pre-incubation with tinzaparin (1 or 2 IU/ml) elicited a 13.5 or 25.5% reduction in retrotransposition, respectively. Regarding tumorigenic VL30 retrotransposition-positive HC11 cells, treatment with 2 IU/ml tinzaparin for 5 days reduced proliferation rate in a time-dependent manner (up to ~55%), and after 3 weeks, disaggregated soft agar-formed foci, as well as low-adherent mammospheres, producing single mesenchymal-like cells with a ~50% reduced retrotransposition. With respect to the VL30 retrotransposition mechanism: While 12 ng/ml VEGF increased the level of VL30 and endogenous reverse transcriptase (enRT) transcripts ~1.41- and ~1.16-fold, respectively, subsequent tinzaparin treatment reduced both endogenous/ROS- and VEGF-induced levels 1.15- and 0.40-fold (VL30) and 0.60- and 0.52-fold (enRT), respectively. To the best of our knowledge, these data demonstrate for the first time, the novel inhibition activity of tinzaparin against ROS- and VEGF-induced VL30 retrotransposition, and the proliferation and/or aggregation of mouse HC11 mammosphere/tumor-initiating CSCs, thus contributing to the inhibition of VL30 retrotransposition-induced primary tumor growth.

Cyclin D1b induces changes in the macrophage phenotype resulting in promotion of tumor metastasis.

In breast cancer, tumor-associated macrophages with activated phenotypes promote tumor invasion and metastasis. The more aggressive mesenchymal-like breast cancer cells have a selective advantage, skewing macrophages toward the more immunosuppressive subtype. However, the mechanism underlying this shift is poorly understood. Cyclin D1b is a highly oncogenic variant of cyclin D1. Our previous study showed that non-metastatic epithelial-like breast cancer cells were highly metastatic in vivo when cyclin D1b was overexpressed. The present study determined whether cyclin D1b contributed to the interaction between breast cancer cells and macrophages. The results showed that cyclin D1b promoted the invasion of breast cancer cells in vitro. Specifically, through overexpression of cyclin D1b, breast cancer cells regulated the differentiation of macrophages into a more immunosuppressive M2 phenotype. Notably, tumor cells overexpressing cyclin D1b activated macrophages and induced migration of breast cancer cells. Further investigations indicated that SDF-1 mediated macrophage activation through breast cancer cells overexpressing cyclin D1b. These results revealed a previously unknown link between aggressive breast cancer cells and Tumor-associated macrophages, and highlighted the importance of cyclin D1b activity in the breast cancer microenvironment.

Macrophages in Atherosclerosis, First or Second Row Players?

Macrophages represent a cell type that has been widely described in the context of atherosclerosis since the earliest studies in the 17th century. Their role has long been considered to be preponderant in the onset and aggravation of atherosclerosis, in particular by participating in the establishment of a chronic inflammatory state by the release of pro-inflammatory cytokines and by uncontrolled engorgement of lipids resulting in the formation of foam cells and later of the necrotic core. However, recent evidence from mouse models using an elegant technique of tracing vascular smooth muscle cells (VSMCs) during plaque development revealed that resident VSMCs display impressive plastic properties in response to an arterial injury, allowing them to switch into different cell types within the plaque, including mesenchymal-like cells, macrophage-like cells and osteochondrogenic-like cells. In this review, we oppose the arguments in favor or against the influence of macrophages versus VSMCs in all stages of atherosclerosis including pre-atherosclerosis, formation of lipid-rich foam cells, development of the necrotic core and the fibrous cap as well as calcification and rupture of the plaque. We also analyze the relevance of animal models for the investigation of the pathophysiological mechanisms of atherosclerosis in humans, and discuss potential therapeutic strategies targeting either VSMCs or macrophage to prevent the development of cardiovascular events. Overall, although major findings have been made from animal models, efforts are still needed to better understand and therefore prevent the development of atherosclerotic plaques in humans.

Systematic Analysis of Cytostatic TGF-Beta Response in Mesenchymal-Like Hepatocellular Carcinoma Cell Lines.

Hepatocellular carcinoma (HCC) is one of the most challenging malignancies, with high morbidity and mortality rates. The transforming growth factor-β (TGF-β) pathway plays a dual role in HCC, acting as both tumor suppressor and promoter. A thorough understanding of the mechanisms underlying its opposing functions is important. The growth suppressive effects of TGF-β remain largely unknown for mesenchymal HCC cells. Using a systematic approach, here we assess the cytostatic TGF-β responses and intracellular transduction of the canonical TGF-β/Smad signaling cascade in mesenchymal-like HCC cell lines. Nine mesenchymal-like HCC cell lines, including SNU182, SNU387, SNU398, SNU423, SNU449, SNU475, Mahlavu, Focus, and Sk-Hep1, were used in this study. The cytostatic effects of TGF-β were evaluated by cell cycle analysis, BrdU labeling, and SA-β-Gal assay. RT-PCR and western blot analysis were utilized to determine the mRNA and protein expression levels of TGF-β signaling components and cytostatic genes. Immunoperoxidase staining and luciferase reporter assays were performed to comprehend the transduction of the canonical TGF-β pathway. We report that mesenchymal-like HCC cell lines are resistant to TGF-β-induced growth suppression. The vast majority of cell lines have an active canonical signaling from the cell membrane to the nucleus. Three cell lines had lost the expression of cytostatic effector genes. Our findings reveal that cytostatic TGF-β responses have been selectively lost in mesenchymal-like HCC cell lines. Notably, their lack of responsiveness was not associated with a widespread impairment of TGF-β signaling cascade. These cell lines may serve as valuable models for studying the molecular mechanisms underlying the loss of TGF-β-mediated cytostasis during hepatocarcinogenesis.

Epigenetic Silencing of HER2 Expression during Epithelial-Mesenchymal Transition Leads to Trastuzumab Resistance in Breast Cancer.

HER2 receptor tyrosine kinase (encoded by the ERBB2 gene) is overexpressed in approximately 25% of all breast cancer tumors (HER2-positive breast cancers). Resistance to HER2-targeting therapies is partially due to the loss of HER2 expression in tumor cells during treatment. However, little is known about the exact mechanism of HER2 downregulation in HER2-positive tumor cells. Here, by analyzing publicly available genomic data we investigate the hypothesis that epithelial-mesenchymal transition (EMT) abrogates HER2 expression by epigenetic silencing of the ERBB2 gene as a mechanism of acquired resistance to HER2-targeted therapies. As result, HER2 expression was found to be positively and negatively correlated with the expression of epithelial and mesenchymal phenotype marker genes, respectively. The ERBB2 chromatin of HER2-high epithelial-like breast cancer cells and HER2-low mesenchymal-like cells were found to be open/active and closed/inactive, respectively. Decreased HER2 expression was correlated with increased EMT phenotype, inactivated chromatin and lower response to lapatinib. We also found that induction of EMT in the HER2-positive breast cancer cell line BT474 resulted in downregulated HER2 expression and reduced trastuzumab binding. Our results suggest that ERBB2 gene silencing by epigenetic regulation during EMT may be a mechanism of de novo resistance of HER2-positive breast cancer cells to trastuzumab and lapatinib.

Activation of Pancreatic Stellate Cells Is Beneficial for Exocrine but Not Endocrine Cell Differentiation in the Developing Human Pancreas.

Pancreatic stellate cells (PaSCs) are non-endocrine, mesenchymal-like cells that reside within the peri-pancreatic tissue of the rodent and human pancreas. PaSCs regulate extracellular matrix (ECM) turnover in maintaining the integrity of pancreatic tissue architecture. Although there is evidence indicating that PaSCs are involved in islet cell survival and function, its role in islet cell differentiation during human pancreatic development remains unclear. The present study examines the expression pattern and functional role of PaSCs in islet cell differentiation of the developing human pancreas from late 1st to 2nd trimester of pregnancy. The presence of PaSCs in human pancreata (8–22 weeks of fetal age) was characterized by ultrastructural, immunohistological, quantitative RT-PCR and western blotting approaches. Using human fetal PaSCs derived from pancreata at 14–16 weeks, freshly isolated human fetal islet-epithelial cell clusters (hIECCs) were co-cultured with active or inactive PaSCs in vitro. Ultrastructural and immunofluorescence analysis demonstrated a population of PaSCs near ducts and newly formed islets that appeared to make complex cell-cell dendritic-like contacts. A small subset of PaSCs co-localized with pancreatic progenitor-associated transcription factors (PDX1, SOX9, and NKX6-1). PaSCs were highly proliferative, with significantly higher mRNA and protein levels of PaSC markers (desmin, αSMA) during the 1st trimester of pregnancy compared to the 2nd trimester. Isolated human fetal PaSCs were identified by expression of stellate cell markers and ECM. Suppression of PaSC activation, using all-trans retinoic acid (ATRA), resulted in reduced PaSC proliferation and ECM proteins. Co-culture of hIECCs, directly on PaSCs or indirectly using Millicell® Inserts or using PaSC-conditioned medium, resulted in a reduction the number of insulin+ cells but a significant increase in the number of amylase+ cells. Suppression of PaSC activation or Notch activity during the co-culture resulted in an increase in beta-cell differentiation. This study determined that PaSCs, abundant during the 1st trimester of pancreatic development but decreased in the 2nd trimester, are located near ductal and islet structures. Direct and indirect co-cultures of hIECCs with PaSCs suggest that activation of PaSCs has opposing effects on beta-cell and exocrine cell differentiation during human fetal pancreas development, and that these effects may be dependent on Notch signaling.