IL-1RA promotes oral squamous cell carcinoma malignancy through mitochondrial metabolism-mediated EGFR/JNK/SOX2 pathway.

BACKGROUND: Interleukin-1 receptor antagonist (IL-1RA), a member of the IL-1 family, has diverse roles in cancer development. However, the role of IL-1RA in oral squamous cell carcinoma (OSCC), in particular the underlying mechanisms, remains to be elucidated. METHODS: Tumor tissues from OSCC patients were assessed for protein expression by immunohistochemistry. Patient survival was evaluated by Kaplan-Meier curve analysis. Impact of differential IL-1RA expression on cultured OSCC cell lines was assessed in vitro by clonogenic survival, tumorsphere formation, soft agar colony formation, and transwell cell migration and invasion assays. Oxygen consumption rate was measured by Seahorse analyzer or multi-mode plate reader. PCR array was applied to screen human cancer stem cell-related genes, proteome array for phosphorylation status of kinases, and Western blot for protein expression in cultured cells. In vivo tumor growth was investigated by orthotopic xenograft in mice, and protein expression in xenograft tumors assessed by immunohistochemistry. RESULTS: Clinical analysis revealed that elevated IL-1RA expression in OSCC tumor tissues was associated with increased tumor size and cancer stage, and reduced survival in the patient group receiving adjuvant radiotherapy compared to the patient group without adjuvant radiotherapy. In vitro data supported these observations, showing that overexpression of IL-1RA increased OSCC cell growth, migration/invasion abilities, and resistance to ionizing radiation, whereas knockdown of IL-1RA had largely the opposite effects. Additionally, we identified that EGFR/JNK activation and SOX2 expression were modulated by differential IL-1RA expression downstream of mitochondrial metabolism, with application of mitochondrial complex inhibitors suppressing these pathways. Furthermore, in vivo data revealed that treatment with cisplatin or metformin-a mitochondrial complex inhibitor and conventional therapy for type 2 diabetes-reduced IL-1RA-associated xenograft tumor growth as well as EGFR/JNK activation and SOX2 expression. This inhibitory effect was further augmented by combination treatment with cisplatin and metformin. CONCLUSIONS: The current study suggests that IL-1RA promoted OSCC malignancy through mitochondrial metabolism-mediated EGFR/JNK activation and SOX2 expression. Inhibition of this mitochondrial metabolic pathway may present a potential therapeutic strategy in OSCC.

Capsaicin Reduces Cancer Stemness and Inhibits Metastasis by Downregulating SOX2 and EZH2 in Osteosarcoma.

Metastasis of osteosarcoma is an important adverse factor affecting patients' survival, and cancer stemness is the crucial cause of distant metastasis. Capsaicin, the main component of pepper, has been proven in our previous work to inhibit osteosarcoma proliferation and enhance its drug sensitivity to cisplatin at low concentrations. This study aims to further explore the anti-osteosarcoma effect of capsaicin at low concentrations (100[Formula: see text][Formula: see text]M, 24[Formula: see text]h) on stemness and metastasis. The stemness of human osteosarcoma (HOS) cells was decreased significantly by capsaicin treatment. Additionally, the capsaicin treatment's inhibition of cancer stem cells (CSCs) was dose-dependent on both sphere formation and sphere size. Meanwhile, capsaicin inhibited invasion and migration, which might be associated with 25 metastasis-related genes. SOX2 and EZH2 were the most two relevant stemness factors for capsaicin's dose-dependent inhibition of osteosarcoma. The mRNAsi score of HOS stemness inhibited by capsaicin was strongly correlated with most metastasis-related genes of osteosarcoma. Capsaicin downregulated six metastasis-promoting genes and up-regulated three metastasis-inhibiting genes, which significantly affected the overall survival and/or disease-free survival of patients. In addition, the CSC re-adhesion scratch assay demonstrated that capsaicin inhibited the migration ability of osteosarcoma by inhibiting its stemness. Overall, capsaicin exerts a significant inhibitory effect on the stemness expression and metastatic ability of osteosarcoma. Moreover, it can inhibit the migratory ability of osteosarcoma by suppressing its stemness via downregulating SOX2 and EZH2. Therefore, capsaicin is expected to be a potential drug against osteosarcoma metastasis due to its ability to inhibit cancer stemness.

PFKFB3 works on the FAK-STAT3-SOX2 axis to regulate the stemness in MPM.

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive neoplasm and often acquires chemoresistance by increasing stemness in tumour tissue, thereby generating cancer stem cells (CSCs). CSCs escape treatment by deploying metabolic pathways to trigger dormancy or proliferation, also gaining the ability to exit and re-enter the cell cycle to hide their cellular identity. METHODS: We employed various cellular and biochemical assays to identify the role of the glycolytic enzyme PFKFB3, by knocking it down and pharmacologically inhibiting it with PFK158, to determine its anticancer effects in vitro and in vivo by targeting the CSC population in MPM. RESULTS: Here, we have identified PFKFB3 as a strategic player to target the CSC population in MPM and demonstrated that both pharmacologic (PFK158) and genetic inhibition of PFKFB3 destroy the FAK-Stat3-SOX2 nexus resulting in a decline in conspicuous stem cell markers viz. ALDH, CD133, CD44, SOX2. Inhibition of PFKFB3 accumulates p21 and p27 in the nucleus by decreasing SKP2. Lastly, PFK158 diminishes tumour-initiating cells (TICs) mediated MPM xenograft in vivo. CONCLUSIONS: This study confers a comprehensive and mechanistic function of PFKFB3 in CSC maintenance that may foster exceptional opportunities for targeted small molecule blockade of the TICs in MPM.

ΔNp63α mediates sulforaphane suppressed colorectal cancer stem cell properties through transcriptional regulation of Nanog/Oct4/Sox2.

Cancer stem cells (CSCs) play a key role in cancer initiation, development, metastasis, and recurrence. Previously, we found that sulforaphane (SFN), a natural compound obtained from cruciferous vegetables, inhibited colorectal CSCs via the downregulation of TAp63α. However, the role of ΔNp63α, another critical isoform of p63 which has been considered to contribute to cancer progression, in SFN-mediated colorectal CSCs inhibition remains unclear. Here, we showed that ΔNp63α expression was enhanced in sphere-forming colorectal cancer cells. Overexpression of ΔNp63α promoted the properties of CSCs, while downregulation of ΔNp63α suppressed those properties. Besides, ΔNp63α was found to activate the transcription of core CSCs genes including Nanog, Oct4, and Sox2. Furthermore, in vitro and in vivo experiments illustrated the regulatory effects of SFN on ΔNp63α and colorectal CSCs. These findings suggested for the first time that ΔNp63α activated the transcription of Nanog, Oct4, Sox2 and mediated the interventional effects of SFN on colorectal CSCs, thus providing a novel mechanism by which SFN inhibits colorectal CSCs.

Functional Odontoblastic-Like Cells Derived from Human iPSCs.

The induced pluripotent stem cells (iPSCs) have an intrinsic capability for indefinite self-renewal and large-scale expansion and can differentiate into all types of cells. Here, we tested the potential of iPSCs from dental pulp stem cells (DPSCs) to differentiate into functional odontoblasts. DPSCs were reprogrammed into iPSCs via electroporation of reprogramming factors OCT-4, SOX2, KLF4, LIN28, and L-MYC. The iPSCs presented overexpression of the reprogramming genes and high protein expressions of alkaline phosphatase, OCT4, and TRA-1-60 in vitro and generated tissues from 3 germ layers in vivo. Dentin discs with poly-L-lactic acid scaffolds containing iPSCs were implanted subcutaneously into immunodeficient mice. After 28 d from implantation, the iPSCs generated a pulp-like tissue with the presence of tubular dentin in vivo. The differentiation potential after long-term expansion was assessed in vitro. iPSCs and DPSCs of passages 4 and 14 were treated with either odontogenic medium or extract of bioactive cement for 28 d. Regardless of the passage tested, iPSCs expressed putative markers of odontoblastic differentiation and kept the same mineralization potential, while DPSC P14 failed to do the same. Analysis of these data collectively demonstrates that human iPSCs can be a source to derive human odontoblasts for dental pulp research and test bioactivity of materials.

Enhanced OCT4 transcriptional activity substitutes for exogenous SOX2 in cellular reprogramming.

Adenoviral early region 1A (E1A) is a viral gene that can promote cellular proliferation and de-differentiation in mammalian cells, features required for the reprogramming of somatic cells to a pluripotent state. E1A has been shown to interact with OCT4, and as a consequence, to increase OCT4 transcriptional activity. Indeed, E1A and OCT4 are sufficient to revert neuroepithelial hybrids to pluripotency, as demonstrated in previous cell fusion experiments. However, the role that E1A might play in the generation of induced pluripotent stem cells (iPSCs) has not been investigated yet. In this report, we show that E1A can generate iPSCs in combination with OCT4 and KLF4, thus replacing exogenous SOX2. The generated iPSCs are bona fide pluripotent cells as shown by in vitro and in vivo tests. Overall, our study suggests that E1A might replace SOX2 through enhancing OCT4 transcriptional activity at the early stages of reprogramming.

Derivation and cardiomyocyte differentiation of induced pluripotent stem cells from heart failure patients.

AIMS: Myocardial cell replacement therapies are hampered by a paucity of sources for human cardiomyocytes and by the expected immune rejection of allogeneic cell grafts. The ability to derive patient-specific human-induced pluripotent stem cells (hiPSCs) may provide a solution to these challenges. We aimed to derive hiPSCs from heart failure (HF) patients, to induce their cardiomyocyte differentiation, to characterize the generated hiPSC-derived cardiomyocytes (hiPSC-CMs), and to evaluate their ability to integrate with pre-existing cardiac tissue. METHODS AND RESULTS: Dermal fibroblasts from two HF patients were reprogrammed by retroviral delivery of Oct4, Sox2, and Klf4 or by using an excisable polycistronic lentiviral vector. The resulting HF-hiPSCs displayed adequate reprogramming properties and could be induced to differentiate into cardiomyocytes with the same efficiency as control hiPSCs (derived from human foreskin fibroblasts). Gene expression and immunostaining studies confirmed the cardiomyocyte phenotype of the differentiating HF-hiPSC-CMs. Multi-electrode array recordings revealed the development of a functional cardiac syncytium and adequate chronotropic responses to adrenergic and cholinergic stimulation. Next, functional integration and synchronized electrical activities were demonstrated between hiPSC-CMs and neonatal rat cardiomyocytes in co-culture studies. Finally, in vivo transplantation studies in the rat heart revealed the ability of the HF-hiPSC-CMs to engraft, survive, and structurally integrate with host cardiomyocytes. CONCLUSIONS: Human-induced pluripotent stem cells can be established from patients with advanced heart failure and coaxed to differentiate into cardiomyocytes, which can integrate with host cardiac tissue. This novel source for patient-specific heart cells may bring a unique value to the emerging field of cardiac regenerative medicine.

MicroRNA-302 increases reprogramming efficiency via repression of NR2F2.

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression through translational inhibition and RNA decay and have been implicated in the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. In this study, we analyzed global miRNA and mRNA microarrays to predict novel miRNA-mRNA interactions in human embryonic stem cells and induced pluripotent stem cells (iPSCs). In particular, we demonstrate a regulatory feedback loop between the miR-302 cluster and two transcription factors, NR2F2 and OCT4. Our data show high expression of miR-302 and OCT4 in pluripotent cells, while NR2F2 is expressed exclusively in differentiated cells. Target analysis predicts that NR2F2 is a direct target of miR-302, which we experimentally confirm by reporter luciferase assays and real-time polymerase chain reaction. We also demonstrate that NR2F2 directly inhibits the activity of the OCT4 promoter and thus diminishes the positive feedback loop between OCT4 and miR-302. Importantly, higher reprogramming efficiencies were obtained when we reprogrammed human adipose-derived stem cells into iPSCs using four factors (KLF4, C-MYC, OCT4, and SOX2) plus miR-302 (this reprogramming cocktail is hereafter referred to as "KMOS3") when compared to using four factors ("KMOS"). Furthermore, shRNA knockdown of NR2F2 mimics the over-expression of miR-302 by also enhancing reprogramming efficiency. Interestingly, we were unable to generate iPSCs from miR-302a/b/c/d alone, which is in contrast to previous publications that have reported that miR-302 by itself can reprogram human skin cancer cells and human hair follicle cells. Taken together, these findings demonstrate that miR-302 inhibits NR2F2 and promotes pluripotency through indirect positive regulation of OCT4. This feedback loop represents an important new mechanism for understanding and inducing pluripotency in somatic cells.

Differentiation of reprogrammed mouse cardiac fibroblasts into functional cardiomyocytes.

Fibroblasts can be reprogrammed by ectopic expression of reprogramming factors to yield induced pluripotent stem (iPS) cells that are capable of transdifferentiating into diverse types of somatic cell lines. In this study, we examined if functional cardiomyocytes (CMs) can be produced from mouse cardiac fibroblasts (CFs), using iPS cell factor-based reprogramming. CFs were isolated from Oct4-GFP-C57 mice and infected with a retrovirus expressing the Yamanaka reprogramming factors, Oct4, Sox2, Klf4, and c-Myc to reprogram the CFs into a CF-iPS cell line. Primary mouse embryonic fibroblast cells (MEFs) were used as a control. We found that the dedifferentiated CF-iPS cells showed similar biological characteristics (morphology, pluripotent factor expression, and methylation level) as embryonic stem cells (ESs) and MEF-iPS cells. We used the classical embryoid bodies (EBs)-based method and a transwell CM co-culture system to simulate the myocardial paracrine microenvironment for performing CF-iPS cell cardiogenic differentiation. Under this simulated myocardial microenvironment, CF-iPS cells formed spontaneously beating EBs. The transdifferentiated self-beating cells expressed cardiac-specific transcription and structural factors and also displayed typical myocardial morphology and electrophysiological characteristics. CFs can be dedifferentiated into iPS cells and further transdifferentiated into CMs. CFs hold great promise for CM regeneration as an autologous cell source for functional CM in situ without the need for exogenous cell transplantation in ischemic heart disease.

Generation of induced pluripotent stem cells from human Tenon's capsule fibroblasts.

PURPOSE: This study aimed to develop a feasible and efficient method for generating embryonic stem cell (ESC)-like induced pluripotent stem (iPS) cells from human Tenon's capsule fibroblasts (HTFs) through the expression of a defined set of transcription factors, which will have significant application value for ophthalmic personalized regenerative medicine. METHODS: HTFs were harvested from fresh samples, and reprogramming was induced by the exogenous expression of the four classic transcription factors, OCT-3/4, SOX-2, KLF-4, and C-MYC. The HTF-derived iPS (TiPS) cells were analyzed with phase contrast microscopy, real-time PCR, immunofluorescence, FACS analysis, alkaline phosphatase activity analysis, and a teratoma formation assay. Human ESC colonies were used as the positive control. RESULTS: The resulting HTF-derived iPS cell colonies were indistinguishable from human ESC colonies regarding morphology, gene expression levels, pluripotent gene expression, alkaline phosphatase activity, and the ability to generate all three embryonic germ layers. CONCLUSIONS: This study presents a simple, efficient, practical procedure for generating patient-tailored iPS cells from HTFs. These cells will serve as a valuable and preferred candidate donor cell population for ophthalmological regenerative medicine.

Generation of human melanocytes from induced pluripotent stem cells.

Epidermal melanocytes play an important role in protecting the skin from UV rays, and their functional impairment results in pigment disorders. Additionally, melanomas are considered to arise from mutations that accumulate in melanocyte stem cells. The mechanisms underlying melanocyte differentiation and the defining characteristics of melanocyte stem cells in humans are, however, largely unknown. In the present study, we set out to generate melanocytes from human iPS cells in vitro, leading to a preliminary investigation of the mechanisms of human melanocyte differentiation. We generated iPS cell lines from human dermal fibroblasts using the Yamanaka factors (SOX2, OCT3/4, and KLF4, with or without c-MYC). These iPS cell lines were subsequently used to form embryoid bodies (EBs) and then differentiated into melanocytes via culture supplementation with Wnt3a, SCF, and ET-3. Seven weeks after inducing differentiation, pigmented cells expressing melanocyte markers such as MITF, tyrosinase, SILV, and TYRP1, were detected. Melanosomes were identified in these pigmented cells by electron microscopy, and global gene expression profiling of the pigmented cells showed a high similarity to that of human primary foreskin-derived melanocytes, suggesting the successful generation of melanocytes from iPS cells. This in vitro differentiation system should prove useful for understanding human melanocyte biology and revealing the mechanism of various pigment cell disorders, including melanoma.

Induced pluripotent stem cells at nanoscale.

Reprogramming of mouse and human somatic cells into induced pluripotent stem (iPS) cells has been made possible with the expression of the transcription factor quartet Oct4, Sox2, c-Myc, and Klf4. Here, we compared iPS cells derived from mouse embryonic fibroblasts with the 4 factors to embryonic stem cells by electron microscopy. Both cell types are almost indistinguishable at the ultrastructural level, providing further evidence for the similarity of these 2 pluripotent stem cell populations.

Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) have great promise for cellular therapy, but it is unclear if they have the same potential as human embryonic stem cells (hESCs) to differentiate into specialized cell types. Ocular cells such as the retinal pigmented epithelium (RPE) are of particular interest because they could be used to treat degenerative eye diseases, including age-related macular degeneration and retinitis pigmentosa. We show here that iPSCs generated using Oct4, Sox2, Nanog, and Lin28 can spontaneously differentiate into RPE cells, which can then be isolated and cultured to form highly differentiated RPE monolayers. RPE derived from iPSCs (iPS-RPE) were analyzed with respect to gene expression, protein expression, and rod outer segment phagocytosis, and compared with cultured fetal human RPE (fRPE) and RPE derived from hESCs (hESC-RPE). iPS-RPE expression of marker mRNAs was quantitatively similar to that of fRPE and hESC-RPE, and marker proteins were appropriately expressed and localized in polarized monolayers. Levels of rod outer segment phagocytosis by iPS-RPE, fRPE, and hESC-RPE were likewise similar and dependent on integrin alpha v beta 5. This work shows that iPSCs can differentiate into functional RPE that are quantitatively similar to fRPE and hESC-RPE and further supports the finding that iPSCs are similar to hESCs in their differentiation potential.