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Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression.

Epithelial-mesenchymal transition (EMT) is a crucial pathological event in cancer, particularly in tumor cell budding and metastasis. Therefore, control of EMT can represent a novel therapeutic strategy in cancer. Here, we introduce an innovative three-dimensional (3D) high-throughput screening (HTS) system that leads to an identification of EMT inhibitors. For the establishment of the novel 3D-HTS system, we chose NanoCulture Plates (NCP) that provided a gel-free micro-patterned scaffold for cells and were independent of other spheroid formation systems using soft-agar. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-β promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-βreceptor I (TGFβR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid-derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-β-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFβR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor, which we showed also had an EMT-inhibitory activity. The half maximal inhibitory concentration (IC50) of SB-525334 and SU9516 were 0.31 μM and 1.21 μM, respectively, while IC50 of SB431542 was 2.38 μM. Taken together, it was shown that this 3D NCP-based HTS system was useful for screening of EMT-regulatory drugs.

MicroRNAs (miRNAs) are small RNA molecules of 21–25 nucleotides that regulate cell behavior through inhibition of translation from mRNA to protein, promotion of mRNA degradation and control of gene transcription. In this study, we investigated the miRNA expression signatures of cell cultures undergoing osteoblastic and osteocytic differentiation from mesenchymal stem cells (MSC) using mouse MSC line KUSA-A1 and human MSCs. Ninety types of miRNA were quantified during osteoblastic/osteocytic differentiation in KUSA-A1 cells utilizing miRNA PCR arrays. Coincidently with mRNA induction of the osteoblastic and osteocytic markers, the expression levels of several dozen miRNAs including miR-30 family, let-7 family, miR-21, miR-16, miR-155, miR-322 and Snord85 were changed during the differentiation process. These miRNAs were predicted to recognize osteogenic differentiation-, stemness-, epinegetics-, and cell cycle-related mRNAs, and were thus designated OstemiR. Among those OstemiR, the miR-30 family was classified into miR-30b/c and miR-30a/d/e groups on the basis of expression patterns during osteogenesis as well as mature miRNA structures. In silico prediction and subsequent qRT-PCR in stable miR-30d transfectants clarified that context-dependent targeting of miR-30d on known regulators of bone formation including osteopontin/spp1, lifr, ccn2/ctgf, ccn1/cyr61, runx2, sox9 as well as novel key factors including lin28a, hnrnpa3, hspa5/grp78, eed and pcgf5. In addition, knockdown of human OstemiR miR-541 increased Osteopontin/SPP1 expression and calcification in hMSC osteoblastic differentiation, indicating that miR-541 is a negative regulator of osteoblastic differentiation. These observations indicate stage-specific roles of OstemiR especially miR-541 and the miR-30 family on novel targets in osteogenesis.

The target of rapamycin (TOR) is a high molecular weight protein kinase that regulates many processes in cells in response to mitogens and variations in nutrient availability. Here we have shown that mTOR in human tissue culture cells plays a key role in responses to proteotoxic stress and that reduction in mTOR levels by RNA interference leads to increase sensitivity to heat shock. This effect was accompanied by a drastic reduction in ability to synthesize heat shock proteins (HSP), including Hsp70, Hsp90 and Hsp110. As HSP transcription is regulated by heat shock transcription factor 1 (HSF1), we examined whether mTOR could directly phosphorylate this factor. Indeed, we determined that mTOR could directly phosphorylate HSF1 on serine 326, a key residue in transcriptional activation. HSF1 was phosphorylated on S326 immediately after heat shock and was triggered by other cell stressors including proteasome inhibitors and sodium arsenite. Null mutation of S326 to alanine led to loss of ability to activate an HSF1-regulated promoter-reporter construct, indicating a direct role for mTOR and S326 in transcriptional regulation of HSP genes during stress. As mTOR is known to exist in at least two intracellular complexes, mTORC1 and mTOR2 we examined which complex might interact with HSF1. Indeed mTORC1 inhibitor rapamycin prevented HSF1-S326 phosphorylation, suggesting that this complex is involved in HSF1 regulation in stress. Our experiments therefore suggest a key role for mTORC1 in transcriptional responses to proteotoxic stress.

Scavenger receptor associated with endothelial cells I (SREC-I) was shown to be expressed in immune cells and to play a role in the endocytosis of peptides and antigen presentation. As our previous studies indicated that SREC-I required intact Toll-like receptor 4 (TLR4) expression for its functions in tumor immunity, we examined potential interactions between these two receptors. We have shown here that SREC-I became associated with TLR4 on binding bacterial lipopolysaccharides (LPS) in RAW 264.7 and HEK 293 cells overexpressing these two receptors. The receptors then became internalized together in intracellular endosomes. SREC-I promoted TLR4-induced signal transduction through the NF-kB and MAP kinase pathways, leading to enhanced inflammatory cytokine release. Activation of inflammatory signaling through SREC-I/TLR4 complexes appeared to involve recruitment of the receptors into detergent-insoluble, cholesterol-rich lipid microdomains that contained the small GTPase Cdc42 and the non-receptor tyrosine kinase c-src. Under conditions of SREC-I activation by LPS, TLR4 activity required Cdc42 as well as cholesterol and actin polymerization for signaling through NF-kB and MAP kinase pathways in RAW 264.7 cells. SREC-I appeared to respond differently to another ligand, the molecular chaperone Hsp90 that, while triggering SREC-I-TLR4 binding caused only faint activation of the NF-kB pathway. Our experiments therefore indicated that SREC-I could bind LPS and might be involved in innate inflammatory immune responses to extracellular danger signals in RAW 264.7 cells or bone marrow-derived macrophages.

Dental pulp cells (DPCs) are known to be enriched in stem/progenitor cells but not well characterized yet. Small non-coding microRNAs (miRNAs) have been identified to control protein translation, mRNA stability and transcription, and have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In order to characterize dental pulp stem/progenitor cells and its mechanism of differentiation, we herein sorted stem-cell-enriched side population (SP) cells from human DPCs and periodontal ligament cells (PDLCs), and performed a locked nucleic acid (LNA)-based miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP) cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Concomitantly, mRNA levels of DNA methyltransferases (DNMTs), which are known to play crucial factors during stem cell differentiation, were also increased by miR-720 through unknown mechanism. Finally, miR-720 decreased DPC proliferation as determined by immunocytochemical analysis against ki-67, and promoted odontogenic differentiation as demonstrated by alizarin red staining, as well as alkaline phosphatase and osteopontin mRNA levels. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs.

Heat shock factor 1 (HSF1) regulates one of the major pathways of protein quality control and is essential for deterrence of protein-folding disorders, particularly in neuronal cells. However, HSF1 activity declines with age, a change that may open the door to progression of neurodegenerative disorders such as Huntington's disease. We have investigated mechanisms of HSF1 regulation that may become compromised with age. HSF1 binds stably to the catalytic domain of protein kinase A (PKAcα) and becomes phosphorylated on at least one regulatory serine residue (S320). We show here that PKA is essential for effective transcription of HSP genes by HSF1. PKA triggers a cascade involving HSF1 binding to the histone acetylase p300 and positive translation elongation factor 1 (p-TEFb) and phosphorylation of the c-terminal domain of RNA polymerase II, a key mechanism in the downstream steps of HSF1-mediated transcription. This cascade appears to play a key role in protein quality control in neuronal cells expressing aggregation-prone proteins with long poly-glutamine (poly-Q) tracts. Such proteins formed inclusion bodies that could be resolved by HSF1 activation during heat shock. Resolution of the inclusions was inhibited by knockdown of HSF1, PKAcα, or the pTEFb component CDK9, indicating a key role for the HSF1-PKA cascade in protein quality control.

Background Many inducible transcription factors are regulated through batteries of posttranslational modifications that couple their activity to inducing stimuli. We have studied such regulation of Heat Shock Factor 1 (HSF1), a key protein in control of the heat shock response, and a participant in carcinogenisis, neurological health and aging. As the mechanisms involved in the intracellular regulation of HSF1 in good health and its dysregulation in disease are still incomplete we are investigating the role of posttranslational modifications in such regulation. Methodology/Principal Findings In a proteomic study of HSF1 binding partners, we have discovered its association with the pleiotropic protein kinase A (PKA). HSF1 binds avidly to the catalytic subunit of PKA, (PKAcα) and becomes phosphorylated on a novel serine phosphorylation site within its central regulatory domain (serine 320 or S320), both in vitro and in vivo. Intracellular PKAcα levels and phosphorylation of HSF1 at S320 were both required for HSF1 to be localized to the nucleus, bind to response elements in the promoter of an HSF1 target gene (hsp70.1) and activate hsp70.1 after stress. Reduction in PKAcα levels by small hairpin RNA led to HSF1 exclusion from the nucleus, its exodus from the hsp70.1 promoter and decreased hsp70.1 transcription. Likewise, null mutation of HSF1 at S320 by alanine substitution for serine led to an HSF1 species excluded from the nucleus and deficient in hsp70.1 activation. Conclusions These findings of PKA regulation of HSF1 through S320 phosphorylation add to our knowledge of the signaling networks converging on this factor and may contribute to elucidating its complex roles in the stress response and understanding HSF1 dysregulation in disease.

BACKGROUND:Environmental changes, air pollution and ozone depletion are increasing oxidative stress, and global warming threatens health by heat stress. We now face a high risk of simultaneous exposure to heat and oxidative stress. However, there have been few studies investigating their combined adverse effects on cell viability. PRINCIPAL FINDINGS:Pretreatment of hydrogen peroxide (H(2)O(2)) specifically and highly sensitized cells to heat stress, and enhanced loss of mitochondrial membrane potential. H(2)O(2) exposure impaired the HSP40/HSP70 induction as heat shock response (HSR) and the unfolded protein recovery, and enhanced eIF2alpha phosphorylation and/or XBP1 splicing, land marks of ER stress. These H(2)O(2)-mediated effects mimicked enhanced heat sensitivity in HSF1 knockdown or knockout cells. Importantly, thermal preconditioning blocked H(2)O(2)-mediated inhibitory effects on refolding activity and rescued HSF1 +/+ MEFs, but neither blocked the effects nor rescued HSF1 -/- MEFs. These data strongly suggest that inhibition of HSR and refolding activity is crucial for H(2)O(2)-mediated enhanced heat sensitivity. CONCLUSIONS:H(2)O(2) blocks HSR and refolding activity under heat stress, thereby leading to insufficient quality control and enhancing ER stress. These uncontrolled stress responses may enhance cell death. Our data thus highlight oxidative stress as a crucial factor affecting heat tolerance.