The evolving roles of Wnt signaling in stem cell proliferation and differentiation, the development of human diseases, and therapeutic opportunities.

The evolutionarily conserved Wnt signaling pathway plays a central role in development and adult tissue homeostasis across species. Wnt proteins are secreted, lipid-modified signaling molecules that activate the canonical (ß-catenin dependent) and non-canonical (ß-catenin independent) Wnt signaling pathways. Cellular behaviors such as proliferation, differentiation, maturation, and proper body-axis specification are carried out by the canonical pathway, which is the best characterized of the known Wnt signaling paths. Wnt signaling has emerged as an important factor in stem cell biology and is known to affect the self-renewal of stem cells in various tissues. This includes but is not limited to embryonic, hematopoietic, mesenchymal, gut, neural, and epidermal stem cells. Wnt signaling has also been implicated in tumor cells that exhibit stem cell-like properties. Wnt signaling is crucial for bone formation and presents a potential target for the development of therapeutics for bone disorders. Not surprisingly, aberrant Wnt signaling is also associated with a wide variety of diseases, including cancer. Mutations of Wnt pathway members in cancer can lead to unchecked cell proliferation, epithelial-mesenchymal transition, and metastasis. Altogether, advances in the understanding of dysregulated Wnt signaling in disease have paved the way for the development of novel therapeutics that target components of the Wnt pathway. Beginning with a brief overview of the mechanisms of canonical and non-canonical Wnt, this review aims to summarize the current knowledge of Wnt signaling in stem cells, aberrations to the Wnt pathway associated with diseases, and novel therapeutics targeting the Wnt pathway in preclinical and clinical studies.

A cell-penetrating PHLPP peptide improves cardiac arrest survival in murine and swine models.

Out-of-hospital cardiac arrest is a leading cause of death in the US, with a mortality rate over 90%. Preclinical studies demonstrate that cooling during cardiopulmonary resuscitation (CPR) is highly beneficial, but can be challenging to implement clinically. No medications exist for improving long-term cardiac arrest survival. We have developed a 20-amino acid peptide, TAT-PHLPP9c, that mimics cooling protection by enhancing AKT activation via PH domain leucine-rich repeat phosphatase 1 (PHLPP1) inhibition. Complementary studies were conducted in mouse and swine. C57BL/6 mice were randomized into blinded saline control and peptide-treatment groups. Following a 12-minute asystolic arrest, TAT-PHLPP9c was administered intravenously during CPR and significantly improved the return of spontaneous circulation, mean arterial blood pressure and cerebral blood flow, cardiac and neurological function, and survival (4 hour and 5 day). It inhibited PHLPP-NHERF1 binding, enhanced AKT but not PKC phosphorylation, decreased pyruvate dehydrogenase phosphorylation and sorbitol production, and increased ATP generation in heart and brain. TAT-PHLPP9c treatment also reduced plasma taurine and glutamate concentrations after resuscitation. The protective benefit of TAT-PHLPP9c was validated in a swine cardiac arrest model of ventricular fibrillation. In conclusion, TAT-PHLPP9c may improve neurologically intact cardiac arrest survival without the need for physical cooling.

Melanoma: Molecular genetics, metastasis, targeted therapies, immunotherapies, and therapeutic resistance.

Cutaneous melanoma is a common cancer and cases have steadily increased since the mid 70s. For some patients, early diagnosis and surgical removal of melanomas is lifesaving, while other patients typically turn to molecular targeted therapies and immunotherapies as treatment options. Easy sampling of melanomas allows the scientific community to identify the most prevalent mutations that initiate melanoma such as the BRAF, NRAS, and TERT genes, some of which can be therapeutically targeted. Though initially effective, many tumors acquire resistance to the targeted therapies demonstrating the need to investigate compensatory pathways. Immunotherapies represent an alternative to molecular targeted therapies. However, inter-tumoral immune cell populations dictate initial therapeutic response and even tumors that responded to treatment develop resistance in the long term. As the protocol for combination therapies develop, so will our scientific understanding of the many pathways at play in the progression of melanoma. The future direction of the field may be to find a molecule that connects all of the pathways. Meanwhile, noncoding RNAs have been shown to play important roles in melanoma development and progression. Studying noncoding RNAs may help us to understand how resistance - both primary and acquired - develops; ultimately allow us to harness the true potential of current therapies. This review will cover the basic structure of the skin, the mutations and pathways responsible for transforming melanocytes into melanomas, the process by which melanomas metastasize, targeted therapeutics, and the potential that noncoding RNAs have as a prognostic and treatment tool.

Modeling lung diseases using reversibly immortalized mouse pulmonary alveolar type 2 cells (imPAC2).

BACKGROUND: A healthy alveolar epithelium is critical to the gas exchange function of the lungs. As the major cell type of alveolar epithelium, alveolar type 2 (AT2) cells play a critical role in maintaining pulmonary homeostasis by serving as alveolar progenitors during lung injury, inflammation, and repair. Dysregulation of AT2 cells may lead to the development of acute and chronic lung diseases and cancer. The lack of clinically relevant AT2 cell models hampers our ability to understand pulmonary diseases. Here, we sought to establish reversibly immortalized mouse pulmonary alveolar type 2 cells (imPAC2) and investigate their potential in forming alveolar organoids to model pulmonary diseases. METHODS: Primary mouse pulmonary alveolar cells (mPACs) were isolated and immortalized with a retroviral expression of SV40 Large T antigen (LTA). Cell proliferation and survival was assessed by crystal violet staining and WST-1 assays. Marker gene expression was assessed by qPCR, Western blotting, and/or immunostaining. Alveolar organoids were generated by using matrigel. Ad-TGF-ß1 was used to transiently express TGF-ß1. Stable silencing ß-catenin or overexpression of mutant KRAS and TP53 was accomplished by using retroviral vectors. Subcutaneous cell implantations were carried out in athymic nude mice. The retrieved tissue masses were subjected to H & E histologic evaluation. RESULTS: We immortalized primary mPACs with SV40 LTA to yield the imPACs that were non-tumorigenic and maintained long-term proliferative activity that was reversible by FLP-mediated removal of SV40 LTA. The EpCAM+ AT2-enriched subpopulation (i.e., imPAC2) was sorted out from the imPACs, and was shown to express AT2 markers and form alveolar organoids. Functionally, silencing ß-catenin decreased the expression of AT2 markers in imPAC2 cells, while TGF-ß1 induced fibrosis-like response by regulating the expression of epithelial-mesenchymal transition markers in the imPAC2 cells. Lastly, concurrent expression of oncogenic KRAS and mutant TP53 rendered the imPAC2 cells a tumor-like phenotype and activated lung cancer-associated pathways. Collectively, our results suggest that the imPAC2 cells may faithfully represent AT2 populations that can be further explored to model pulmonary diseases.

Ferroptosis as a novel form of regulated cell death: Implications in the pathogenesis, oncometabolism and treatment of human cancer.

The treatment of cancer mainly involves surgical excision supplemented by radiotherapy and chemotherapy. Chemotherapy drugs act by interfering with tumor growth and inducing the death of cancer cells. Anti-tumor drugs were developed to induce apoptosis, but some patient's show apoptosis escape and chemotherapy resistance. Therefore, other forms of cell death that can overcome the resistance of tumor cells are important in the context of cancer treatment. Ferroptosis is a newly discovered iron-dependent, non-apoptotic type of cell death that is highly negatively correlated with cancer development. Ferroptosis is mainly caused by the abnormal increase in iron-dependent lipid reactive oxygen species and the imbalance of redox homeostasis. This review summarizes the progression and regulatory mechanism of ferroptosis in cancer and discusses its possible clinical applications in cancer diagnosis and treatment.

SATB2: A versatile transcriptional regulator of craniofacial and skeleton development, neurogenesis and tumorigenesis, and its applications in regenerative medicine.

SATB2 (special AT-rich sequence-binding protein 2) is a member of the special AT-rich binding protein family. As a transcription regulator, SATB2 mainly integrates higher-order chromatin organization. SATB2 expression appears to be tissue- and stage-specific, and is governed by several cellular signaling molecules and mediators. Expressed in branchial arches and osteoblast-lineage cells, SATB2 plays a significant role in craniofacial pattern and skeleton development. In addition to regulating osteogenic differentiation, SATB2 also displays versatile functions in neural development and cancer progression. As an osteoinductive factor, SATB2 holds great promise in improving bone regeneration toward bone defect repair. In this review, we have summarized our current understanding of the physiological and pathological functions of SATB2 in craniofacial and skeleton development, neurogenesis, tumorigenesis and regenerative medicine.

Applications of Biocompatible Scaffold Materials in Stem Cell-Based Cartilage Tissue Engineering.

Cartilage, especially articular cartilage, is a unique connective tissue consisting of chondrocytes and cartilage matrix that covers the surface of joints. It plays a critical role in maintaining joint durability and mobility by providing nearly frictionless articulation for mechanical load transmission between joints. Damage to the articular cartilage frequently results from sport-related injuries, systemic diseases, degeneration, trauma, or tumors. Failure to treat impaired cartilage may lead to osteoarthritis, affecting more than 25% of the adult population globally. Articular cartilage has a very low intrinsic self-repair capacity due to the limited proliferative ability of adult chondrocytes, lack of vascularization and innervation, slow matrix turnover, and low supply of progenitor cells. Furthermore, articular chondrocytes are encapsulated in low-nutrient, low-oxygen environment. While cartilage restoration techniques such as osteochondral transplantation, autologous chondrocyte implantation (ACI), and microfracture have been used to repair certain cartilage defects, the clinical outcomes are often mixed and undesirable. Cartilage tissue engineering (CTE) may hold promise to facilitate cartilage repair. Ideally, the prerequisites for successful CTE should include the use of effective chondrogenic factors, an ample supply of chondrogenic progenitors, and the employment of cell-friendly, biocompatible scaffold materials. Significant progress has been made on the above three fronts in past decade, which has been further facilitated by the advent of 3D bio-printing. In this review, we briefly discuss potential sources of chondrogenic progenitors. We then primarily focus on currently available chondrocyte-friendly scaffold materials, along with 3D bioprinting techniques, for their potential roles in effective CTE. It is hoped that this review will serve as a primer to bring cartilage biologists, synthetic chemists, biomechanical engineers, and 3D-bioprinting technologists together to expedite CTE process for eventual clinical applications.

Notch signaling: Its essential roles in bone and craniofacial development.

Notch is a cell-cell signaling pathway that is involved in a host of activities including development, oncogenesis, skeletal homeostasis, and much more. More specifically, recent research has demonstrated the importance of Notch signaling in osteogenic differentiation, bone healing, and in the development of the skeleton. The craniofacial skeleton is complex and understanding its development has remained an important focus in biology. In this review we briefly summarize what recent research has revealed about Notch signaling and the current understanding of how the skeleton, skull, and face develop. We then discuss the crucial role that Notch plays in both craniofacial development and the skeletal system, and what importance it may play in the future.

A one-step construction of adenovirus (OSCA) system using the Gibson DNA Assembly technology.

Adenovirus (Ad) is a non-enveloped linear double-stranded DNA virus with >50 serotypes in humans. Ad vectors have been used as gene delivery vehicles to express transgenes, small interfering RNAs (siRNAs) for gene silencing, or CRISPR/Cas and designer nucleases for genome editing. Although several methods are used to generate Ad vectors, the Ad-making process remains technically challenging and time consuming. Moreover, the Ad-making techniques have not been improved for the past two decades. Gibson DNA Assembly (GDA) technology allows one-step isothermal DNA assembly of multiple overlapping fragments. Here, we developed a one-step construction of Ad (OSCA) system using GDA technology. Specifically, we first engineered several adenoviral recipient vectors that contain the ccdB suicide gene flanked with two 20-bp unique sequences, which serve as universal sites for GDA reactions in the Ad genome ΔE1 region. In two proof-of-principle experiments, we demonstrated that the GDA reactions were highly efficient and that the resulting Ad plasmids could be effectively packaged into Ads. Ad-mediated expression of mouse BMP9 in mesenchymal stem cells was shown to effectively induce osteogenic differentiation both in vitro and in vivo. Collectively, our results demonstrate that the OSCA system drastically streamlines the Ad-making process and should facilitate Ad-based applications in basic, translational, and clinical research.

Identification of a prognostic signature based on copy number variations (CNVs) and CNV-modulated gene expression in acute myeloid leukemia.

OBJECTIVES: Acute myeloid leukemia (AML) is caused by multiple genetic alterations in hematopoietic progenitors, and molecular genetic analyses have provided useful information for AML diagnosis and prognostication. This study aimed to integratively understand the prognostic value of specific copy number variation (CNV) patterns and CNV-modulated gene expression in AML. METHODS: We conducted integrative CNV profiling and gene expression analysis using data from the Therapeutically Applicable Research To Generate Effective Treatments (TARGET) and The Cancer Genome Atlas (TCGA) AML cohorts. CNV-related genes associated with survival were identified using the TARGET AML cohort and validated using the TCGA AML cohort. Genes whose CNV-modulated expression was associated with survival were also identified using the TARGET AML cohort and validated using the TCGA AML cohort, and patient bone marrow samples were then used to further validate the effects of CNV-modulated gene expression on survival. CNV and mRNA survival analyses were conducted using proportional hazards regression models (Cox regression) and the "survminer" and "survival" packages of the R Project for Statistical Computing. Genes belonging to the Kyoto Encyclopedia of Genes and Genomes (KEGG) cancer panel were extracted from KEGG cancer-related pathways. RESULTS: One hundred two CNV-related genes (located at 7q31-34, 16q24) associated with patient survival were identified using the TARGET cohort and validated with the TCGA AML cohort. Among these 102 validated genes, three miRNA genes (MIR29A, MIR183, and MIR335) were included in the KEGG cancer panel. Five genes (SEMA4D, CBFB, CHAF1B, SAE1, and DNMT1) whose expression was modulated by CNVs and significantly associated with clinical outcomes were identified, and the deletion of SEMA4D and CBFB was found to potentially exert protective effects against AML. The results of these five genes were also validated using patient marrow samples. Additionally, the distribution of CNVs affecting these five CNV-modulated genes was independent of the risk group (favorable-, intermediate-, and adverse-risk groups). CONCLUSIONS: Overall, this study identified 102 CNV-related genes associated with patient survival and identified five genes whose expression was modulated by CNVs and associated with patient survival. Our findings are crucial for the development of new modes of prognosis evaluation and targeted therapy for AML.

Stem Cell-Friendly Scaffold Biomaterials: Applications for Bone Tissue Engineering and Regenerative Medicine.

Bone is a dynamic organ with high regenerative potential and provides essential biological functions in the body, such as providing body mobility and protection of internal organs, regulating hematopoietic cell homeostasis, and serving as important mineral reservoir. Bone defects, which can be caused by trauma, cancer and bone disorders, pose formidable public health burdens. Even though autologous bone grafts, allografts, or xenografts have been used clinically, repairing large bone defects remains as a significant clinical challenge. Bone tissue engineering (BTE) emerged as a promising solution to overcome the limitations of autografts and allografts. Ideal bone tissue engineering is to induce bone regeneration through the synergistic integration of biomaterial scaffolds, bone progenitor cells, and bone-forming factors. Successful stem cell-based BTE requires a combination of abundant mesenchymal progenitors with osteogenic potential, suitable biofactors to drive osteogenic differentiation, and cell-friendly scaffold biomaterials. Thus, the crux of BTE lies within the use of cell-friendly biomaterials as scaffolds to overcome extensive bone defects. In this review, we focus on the biocompatibility and cell-friendly features of commonly used scaffold materials, including inorganic compound-based ceramics, natural polymers, synthetic polymers, decellularized extracellular matrix, and in many cases, composite scaffolds using the above existing biomaterials. It is conceivable that combinations of bioactive materials, progenitor cells, growth factors, functionalization techniques, and biomimetic scaffold designs, along with 3D bioprinting technology, will unleash a new era of complex BTE scaffolds tailored to patient-specific applications.

Leptin Potentiates BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells Through the Activation of JAK/STAT Signaling.

Mesenchymal stem cells (MSCs) are multipotent progenitors that have the ability to differentiate into multiple lineages, including bone, cartilage, and fat. We previously demonstrated that the least known bone morphogenetic protein (BMP)9 (also known as growth differentiation factor 2) is one of the potent osteogenic factors that can induce both osteogenic and adipogenic differentiation of MSCs. Nonetheless, the molecular mechanism underlying BMP9 action remains to be fully understood. Leptin is an adipocyte-derived hormone in direct proportion to the amount of body fat, and exerts pleiotropic functions, such as regulating energy metabolism, bone mass, and mineral density. In this study, we investigate the potential effect of leptin signaling on BMP9-induced osteogenic differentiation of MSCs. We found that exogenous leptin potentiated BMP9-induced osteogenic differentiation of MSCs both in vitro and in vivo, while inhibiting BMP9-induced adipogenic differentiation. BMP9 was shown to induce the expression of leptin and leptin receptor in MSCs, while exogenous leptin upregulated BMP9 expression in less differentiated MSCs. Mechanistically, we demonstrated that a blockade of JAK signaling effectively blunted leptin-potentiated osteogenic differentiation induced by BMP9. Taken together, our results strongly suggest that leptin may potentiate BMP9-induced osteogenesis by cross-regulating BMP9 signaling through the JAK/STAT signaling pathway in MSCs. Thus, it is conceivable that a combined use of BMP9 and leptin may be explored as a novel approach to enhancing efficacious bone regeneration and fracture healing.

Genetic and epigenetic control of RKIP transcription.

Raf kinase inhibitory protein (RKIP) is known to modulate key signaling cascades and regulate normal physiological processes such as cellular proliferation, differentiation, and apoptosis. The expression of RKIP is found to be downregulated in several cancer metastases and the repressed RKIP expression can be reactivated on treatment with chemotherapeutic agents. RKIP is a proven tumor metastasis suppressor gene and investigating the mechanisms of transcriptional regulation of RKIP is therefore of immense clinical importance. In this review, we discuss the basal expression of RKIP in various tissues and the genetic aspects of the RKIP chromosomal locus including the structure of the RKIP promoter as well as gene regulatory elements such as enhancers. We also review the genetic and epigenetic modulation of RKIP transcription through EZH2, a component of the polycomb repressive complex 2 (PRC2) and sequence specific transcription factors (TFs) BACH1 and Snail. Emerging experimental evidence supports a unifying model in which both these TFs repress RKIP transcription in cancers by recruiting the EZH2 containing repressive complex to the proximal RKIP promoter. Finally, we review the known mechanisms employed by different types of chemotherapeutic agents to activate RKIP expression in cancer cells.