CDK12 Is Necessary to Promote Epidermal Differentiation Through Transcription Elongation.

Proper differentiation of the epidermis is essential to prevent water loss and to protect the body from the outside environment. Perturbations in this process can lead to a variety of skin diseases that impacts 1 in 5 people. While transcription factors that control epidermal differentiation have been well characterized, other aspects of transcription control such as elongation are poorly understood. Here we show that of the two cyclin-dependent kinases (CDK12 and CDK13), that are known to regulate transcription elongation, only CDK12 is necessary for epidermal differentiation. Depletion of CDK12 led to loss of differentiation gene expression and absence of skin barrier formation in regenerated human epidermis. CDK12 binds to genes that code for differentiation promoting transcription factors (GRHL3, KLF4, and OVOL1) and is necessary for their elongation. CDK12 is necessary for elongation by promoting Ser2 phosphorylation on the C-terminal domain of RNA polymerase II and the stabilization of binding of the elongation factor SPT6 to target genes. Our results suggest that control of transcription elongation by CDK12 plays a prominent role in adult cell fate decisions.

Inhibition of O-GlcNAcase Inhibits Hematopoietic and Leukemic Stem Cell Self-Renewal and Drives Dendritic Cell Differentiation via STAT3/5 Signaling.

Myeloid differentiation blockage at immature and self-renewing stages is a common hallmark across all subtypes of acute myeloid leukemia (AML), despite their genetic heterogeneity. Metabolic state is an important regulator of hematopoietic stem cell (HSC) self-renewal and lineage-specific differentiation as well as several aggressive cancers. However, how O-GlcNAcylation, a nutrient-sensitive posttranslational modification of proteins, contributes to both normal myelopoiesis and AML pathogenesis remains largely unknown. Using small molecule inhibitors and the CRISPR/Cas9 system, we reveal for the first time that inhibition of either OGA or OGT, which subsequently caused an increase or decrease in cellular O-GlcNAcylation, inhibits the self-renewal and maintenance of CD34+ hematopoietic stem/progenitor cells (HSPCs) and leukemic stem/progenitor cells and drives normal and malignant myeloid differentiation. We further unveiled the distinct roles of OGA and OGT inhibition in lineage-specific differentiation. While OGT inhibition induces macrophage differentiation, OGA inhibition promotes the differentiation of both CD34+ HSPCs and AML cells into dendritic cells (DCs), in agreement with an upregulation of a multitude of genes involved in DC development and function and their ability to induce T-cell proliferation, via STAT3/5 signaling. Our novel findings provide significant basic knowledge that could be important in understanding AML pathogenesis and overcoming differentiation blockage-agnostic to the genetic background of AML. Additionally, the parallel findings in normal HSPCs may lay the groundwork for future cellular therapy as a means to improve the ex vivo differentiation of normal DCs and macrophages.

Anti-TIM3 chimeric antigen receptor-natural killer cells from engineered induced pluripotent stem cells effectively target acute myeloid leukemia cells.

BACKGROUND: Acute myeloid leukemia (AML) is a clonal malignant disorder which originates from a small number of leukemia-initiating cells or leukemic stem cells (LSCs)-the subpopulation that is also the root cause of relapsed/refractory AML. Chimeric antigen receptor (CAR)-T cell therapy has proved successful at combating certain hematologic malignancies, but has several hurdles that limit its widespread applications. CAR-natural killer (NK) cells do not carry the risk of inducing graft-versus-host disease (GvHD) frequently associated with allogeneic T cells, thereby overcoming time-consuming, autologous cell manufacturing, and have relatively safer clinical profiles than CAR-T cells. The present study aimed to generate anti-TIM3 CAR-NK cells targeting LSCs from a clonal master induced pluripotent stem cells engineered with the third-generation anti-TIM3 CAR. METHODS: A clonal master umbilical cord blood NK-derived induced pluripotent stem cell (iPSC) line, MUSIi013-A, was used as a starting cells for engineering of an anti-TIM3 CAR harboring TIM3 scFv fragment (clone TSR-022), CD28, 4-1BB, and CD3ζ signaling (CAR-TIM3). The established CAR-TIM3 iPSCs were further differentiated under serum- and feeder-free conditions into functional CAR-TIM3 NK cells and tested for its anti-tumor activity against various TIM3-positive AML cells. RESULTS: We successfully established a single-cell clone of CAR-TIM3 iPSCs, as validated by genomic DNA sequencing as well as antibody and antigen-specific detection. We performed thorough iPSC characterization to confirm its retained pluripotency and differentiation capacity. The established CAR-TIM3 iPSCs can be differentiated into CAR-TIM3 NK-like cells, which were further proven to have enhanced anti-tumor activity against TIM3-positive AML cells with minimal effect on TIM3-negative cells when compared with wild-type (WT) NK-like cells from parental iPSCs. CONCLUSIONS: iPSCs engineered with CARs, including the established single-cell clone CAR-TIM3 iPSCs herein, are potential alternative cell source for generating off-the-shelf CAR-NK cells as well as other CAR-immune cells. The feasibility of differentiation of functional CAR-TIM3 NK cells under serum- and feeder-free conditions support that Good Manufacturing Practice (GMP)-compliant protocols can be further established for future clinical applications.

Cardiac glycoside ouabain efficiently targets leukemic stem cell apoptotic machinery independent of cell differentiation status.

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by an accumulation of immature leukemic myeloblasts initiating from leukemic stem cells (LSCs)-the subpopulation that is also considered the root cause of chemotherapy resistance. Repurposing cardiac glycosides to treat cancers has gained increasing attention and supporting evidence, but how cardiac glycosides effectively target LSCs, e.g., whether it involves cell differentiation, remains largely unexplored. METHODS: Digoxin, a user-designed digitoxigenin-α-L-rhamnoside (D6-MA), and ouabain were tested against various human AML-derived cells with different maturation phenotypes. Herein, we established two study models to specifically determine the effects of cardiac glycosides on LSC death and differentiation-one allowed change in dynamics of LSCs and leukemic progenitor cells (LPCs), while another maintained their undifferentiated status. Regulatory mechanisms underlying cardiac glycoside-induced cytotoxicity were investigated and linked to cell cycle distribution and apoptotic machinery. RESULTS: Primitive AML cells containing CD34+ LSCs/LPCs were very responsive to nanomolar concentrations of cardiac glycosides, with ouabain showing the greatest efficiency. Ouabain preferentially induces caspase-dependent apoptosis in LSCs, independent of its cell differentiation status, as evidenced by (i) the tremendous induction of apoptosis by ouabain in AML cells that acquired less than 15% differentiation and (ii) the higher rate of apoptosis in enriched LSCs than in LPCs. We sorted LSCs and LPCs according to their cell cycle distribution into G0/G1, S, and G2/M cells and revealed that G0/G1 cells in LSCs, which was its major subpopulation, were the top ouabain responders, indicating that the difference in ouabain sensitivity between LSCs and LPCs involved both distinct cell cycle distribution and intrinsic apoptosis regulatory mechanisms. Further, Mcl-1 and c-Myc, which were differentially expressed in LSCs and LPCs, were found to be the key apoptosis mediators that determined ouabain sensitivity in AML cells. Ouabain induces a more rapid loss of Mcl-1 and c-Myc in LSCs than in LPCs via the mechanisms that in part involve an inhibition of Mcl-1 protein synthesis and an induction of c-Myc degradation. CONCLUSIONS: Our data provide new insight for repurposing cardiac glycosides for the treatment of relapsed/refractory AML through targeting LSCs via distinct cell cycle and apoptosis machinery. Video Abstract.

Generation and Functional Characterization of Anti-CD19 Chimeric Antigen Receptor-Natural Killer Cells from Human Induced Pluripotent Stem Cells.

Natural killer (NK) cells are a part of innate immunity that can be activated rapidly in response to malignant transformed cells without prior sensitization. Engineering NK cells to express chimeric antigen receptors (CARs) allows them to be directed against corresponding target tumor antigens. CAR-NK cells are regarded as a promising candidate for cellular immunotherapy alternatives to conventional CAR-T cells, due to the relatively low risk of graft-versus-host disease and safer clinical profile. Human induced pluripotent stem cells (iPSCs) are a promising renewable cell source of clinical NK cells. In the present study, we successfully introduced a third-generation CAR targeting CD19, which was validated to have effective signaling domains suitable for NK cells, into umbilical cord blood NK-derived iPSCs, followed by a single-cell clone selection and thorough iPSC characterization. The established single-cell clone of CAR19-NK/iPSCs, which is highly desirable for clinical application, can be differentiated using serum- and feeder-free protocols into functional CAR19-iNK-like cells with improved anti-tumor activity against CD19-positive hematologic cancer cells when compared with wild-type (WT)-iNK-like cells. With the feasibility of being an alternative source for off-the-shelf CAR-NK cells, a library of single-cell clones of CAR-engineered NK/iPSCs targeting different tumor antigens may be created for future clinical application.

Metabolic sensor O-GlcNAcylation regulates megakaryopoiesis and thrombopoiesis through c-Myc stabilization and integrin perturbation.

Metabolic state of hematopoietic stem cells (HSCs) is an important regulator of self-renewal and lineage-specific differentiation. Posttranslational modification of proteins via O-GlcNAcylation is an ideal metabolic sensor, but how it contributes to megakaryopoiesis and thrombopoiesis remains unknown. Here, we reveal for the first time that cellular O-GlcNAcylation levels decline along the course of megakaryocyte (MK) differentiation from human-derived hematopoietic stem and progenitor cells (HSPCs). Inhibition of O-GlcNAc transferase (OGT) that catalyzes O-GlcNAcylation prolongedly decreases O-GlcNAcylation and induces the acquisition of CD34+ CD41a+ MK-like progenitors and its progeny CD34- CD41a+ /CD42b+ megakaryoblasts (MBs)/MKs from HSPCs, consequently resulting in increased CD41a+ and CD42b+ platelets. Using correlation and co-immunoprecipitation analyses, we further identify c-Myc as a direct downstream target of O-GlcNAcylation in MBs/MKs and provide compelling evidence on the regulation of platelets by novel O-GlcNAc/c-Myc axis. Our data indicate that O-GlcNAcylation posttranslationally regulates c-Myc stability by interfering with its ubiquitin-mediated proteasomal degradation. Depletion of c-Myc upon inhibition of OGT promotes platelet formation in part through the perturbation of cell adhesion molecules, that is, integrin-α4 and integrin-ß7, as advised by gene ontology and enrichment analysis for RNA sequencing and validated herein. Together, our findings provide a novel basic knowledge on the regulatory role of O-GlcNAcylation in megakaryopoiesis and thrombopoiesis that could be important in understanding hematologic disorders whose etiology are related to impaired platelet production and may have clinical applications toward an ex vivo platelet production for transfusion.

Bcl-2 Family Members Bcl-xL and Bax Cooperatively Contribute to Bortezomib Resistance in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin lymphoma with poor prognosis, due to the inevitable development of drug resistance. Despite being the first-in-class proteasome inhibitor for relapsed/refractory MCL, resistance to bortezomib (BTZ) in MCL patients remains a major hurdle of effective therapy, and relapse following BTZ is frequent. Understanding the mechanisms underlying BTZ resistance is, therefore, important for improving the clinical outcome and developing novel therapeutic strategies. Here, we established de novo BTZ-resistant human MCL-derived cells with the highest resistance index of 300-fold compared to parental cells. We provided compelling evidence that both Bcl-xL and Bax are key mediators in determining BTZ sensitivity in MCL cells. Overexpression of antiapoptotic Bcl-xL and depletion of proapoptotic Bax cooperatively protected MCL cells against BTZ-induced apoptosis, causing acquired BTZ resistance, likely by tilting the balance of Bcl-2 family proteins toward antiapoptotic signaling. Bioinformatics analyses suggested that high BCL2L1 (encoded Bcl-xL) and low BAX were, in part, associated with poor prognosis of MCL patients, e.g., when combined with low OGT, which regulates cellular O-GlcNAcylation. Our findings support recent strategies in small molecule drug discovery co-targeting antiapoptotic Bcl-2 family proteins using BH3 mimetics and Bax using Bax activators to overcome cancer drug resistance.

Novel c-Myc-Targeting Compound N, N-Bis (5-Ethyl-2-Hydroxybenzyl) Methylamine for Mediated c-Myc Ubiquitin-Proteasomal Degradation in Lung Cancer Cells.

Aberrant cellular Myc (c-Myc) is a common feature in the majority of human cancers and has been linked to oncogenic malignancies. Here, we developed a novel c-Myc-targeting compound, N, N-bis (5-ethyl-2-hydroxybenzyl) methylamine (EMD), and present evidence demonstrating its effectiveness in targeting c-Myc for degradation in human lung carcinoma. EMD exhibited strong cytotoxicity toward various human lung cancer cell lines, as well as chemotherapeutic-resistant patient-derived lung cancer cells, through apoptosis induction in comparison with chemotherapeutic drugs. The IC50 of EMD against lung cancer cells was approximately 60 µM. Mechanistically, EMD eliminated c-Myc in the cells and initiated caspase-dependent apoptosis cascade. Cycloheximide chase assay revealed that EMD tended to shorten the half-life of c-Myc by approximately half. The cotreatment of EMD with the proteasome inhibitor MG132 reversed its c-Myc-targeting effect, suggesting the involvement of ubiquitin-mediated proteasomal degradation in the process. We further verified that EMD strongly induced the ubiquitination of c-Myc and promoted protein degradation. c-Myc inhibition and apoptosis induction were additionally shown in hematologic malignant K562 cells, indicating the generality of the observed EMD effects. Altogether, we identified EMD as a novel potent compound targeting oncogenic c-Myc that may offer new opportunities for lung cancer treatment. SIGNIFICANCE STATEMENT: The deregulation of c-Myc is frequently associated with cancer progression. This study examined the effect of a new compound, N, N-bis (5-ethyl-2-hydroxybenzyl) methylamine (EMD), in targeting c-Myc in several lung cancer cell lines and drug-resistant primary lung cancer cells. EMD induced dramatic c-Myc degradation through a ubiquitin-proteasomal mechanism. The promising anticancer and c-Myc-targeted activities of EMD support its use in potential new approaches to treat c-Myc-driven cancer.

A novel TRPM7/O-GlcNAc axis mediates tumour cell motility and metastasis by stabilising c-Myc and caveolin-1 in lung carcinoma.

BACKGROUND: Calcium is an essential signal transduction element that has been associated with aggressive behaviours in several cancers. Cell motility is a prerequisite for metastasis, the major cause of lung cancer death, yet its association with calcium signalling and underlying regulatory axis remains an unexplored area. METHODS: Bioinformatics database analyses were employed to assess correlations between calcium influx channels and clinical outcomes in non-small cell lung cancer (NSCLC). Functional and regulatory roles of influx channels in cell migration and invasion were conducted and experimental lung metastasis was examined using in vivo live imaging. RESULTS: High expression of TRPM7 channel correlates well with the low survival rate of patients and high metastatic potential. Inhibition of TRPM7 suppresses cell motility in various NSCLC cell lines and patient-derived primary cells and attenuates experimental lung metastases. Mechanistically, TRPM7 acts upstream of O-GlcNAcylation, a post-translational modification and a crucial sensor for metabolic changes. We reveal for the first time that caveolin-1 and c-Myc are favourable molecular targets of TRPM7/O-GlcNAc that regulates NSCLC motility. O-GlcNAcylation of caveolin-1 and c-Myc promotes protein stability by interfering with their ubiquitination and proteasomal degradation. CONCLUSIONS: TRPM7/O-GlcNAc axis represents a potential novel target for lung cancer therapy that may overcome metastasis.

Nitric oxide promotes cancer cell dedifferentiation by disrupting an Oct4:caveolin-1 complex: A new regulatory mechanism for cancer stem cell formation.

Cancer stem cells (CSCs) are unique populations of cells that can self-renew and generate different cancer cell lineages. Although CSCs are believed to be a promising target for novel therapies, the specific mechanisms by which these putative therapeutics could intervene are less clear. Nitric oxide (NO) is a biological mediator frequently up-regulated in tumors and has been linked to cancer aggressiveness. Here, we search for targets of NO that could explain its activity. We find that it directly affects the stability and function of octamer-binding transcription factor 4 (Oct4), known to drive the stemness of lung cancer cells. We demonstrated that NO promotes the CSC-regulatory activity of Oct4 through a mechanism that involves complex formation between Oct4 and the scaffolding protein caveolin-1 (Cav-1). In the absence of NO, Oct4 forms a molecular complex with Cav-1, which promotes the ubiquitin-mediated proteasomal degradation of Oct4. NO promotes Akt-dependent phosphorylation of Cav-1 at tyrosine 14, disrupting the Cav-1:Oct4 complex. Site-directed mutagenesis and computational modeling studies revealed that the hydroxyl moiety at tyrosine 14 of Cav-1 is crucial for its interaction with Oct4. Both removal of the hydroxyl via mutation to phenylalanine and phosphorylation lead to an increase in binding free energy (ΔGbind) between Oct4 and Cav-1, destabilizing the complex. Together, these results unveiled a novel mechanism of CSC regulation through NO-mediated stabilization of Oct4, a key stem cell transcription factor, and point to new opportunities to design CSC-related therapeutics.

Detachment-induced E-cadherin expression promotes 3D tumor spheroid formation but inhibits tumor formation and metastasis of lung cancer cells.

The epithelial-to-mesenchymal transition is proposed to be a key mechanism responsible for metastasis-related deaths. Similarly, cancer stem cells (CSCs) have been proposed to be a key driver of tumor metastasis. However, the link between the two events and their control mechanisms is unclear. We used a three-dimensional (3D) tumor spheroid assay and other CSC-indicating assays to investigate the role of E-cadherin in CSC regulation and its association to epithelial-to-mesenchymal transition in lung cancer cells. Ectopic overexpression and knockdown of E-cadherin were found to promote and retard, respectively, the formation of tumor spheroids in vitro but had opposite effects on tumor formation and metastasis in vivo in a xenograft mouse model. We explored the discrepancy between the in vitro and in vivo results and demonstrated, for the first time, that E-cadherin is required as a component of a major survival pathway under detachment conditions. Downregulation of E-cadherin increased the stemness of lung cancer cells but had an adverse effect on their survival, particularly on non-CSCs. Such downregulation also promoted anoikis resistance and invasiveness of lung cancer cells. These results suggest that anoikis assay could be used as an alternative method for in vitro assessment of CSCs that involves dysregulated adhesion proteins. Our data also suggest that agents that restore E-cadherin expression may be used as therapeutic agents for metastatic cancers.

Induction of Slug by Chronic Exposure to Single-Walled Carbon Nanotubes Promotes Tumor Formation and Metastasis.

Carbon nanotubes (CNTs) represent a major class of engineered nanomaterials that are being used in diverse fields. However, their use has increasingly become a concern because of their carcinogenic potential. Accumulating evidence has demonstrated that certain types of CNTs are carcinogenic or tumor-promoting in animal models. However, the underlying molecular and cellular mechanisms are unclear. Here, we report that chronic exposure to single-walled (SW) CNTs results in the induction of Slug, a key transcription factor that induces an epithelial-mesenchymal transition (EMT), in human lung epithelial cells. We show that SWCNT-induced Slug upregulation plays a critical role in the aggressive phenotype of SWCNT-exposed cells, which includes increased cell migration, invasion, and anchorage-independent cell growth. Our in vivo studies also show that SWCNT-induced Slug upregulation and EMT activation play a pivotal role in tumor formation and metastasis. Our findings illustrate a direct link between CNT-induced Slug upregulation, EMT activation, and tumor formation and metastasis, and they highlight the potential of CNT-induced Slug upregulation as a target for future risk assessment and prevention of CNT-associated diseases.

Iron induces cancer stem cells and aggressive phenotypes in human lung cancer cells.

Evidence has accumulated in support of the critical impact of cancer stem cells (CSCs) behind the chemotherapeutic failure, cancer metastasis, and subsequent disease recurrence and relapse, but knowledge of how CSCs are regulated is still limited. Redox status of the cells has been shown to dramatically influence cell signaling and CSC-like aggressive behaviors. Here, we investigated how subtoxic concentrations of iron, which have been found to specifically induce cellular hydroxyl radical, affected CSC-like subpopulations of human non-small cell lung carcinoma (NSCLC). We reveal for the first time that subchronic iron exposure and higher levels of hydroxyl radical correlated well with increased CSC-like phenotypes. The iron-exposed NSCLC H460 and H292 cells exhibited a remarkable increase in propensities to form CSC spheroids and to proliferate, migrate, and invade in parallel with an increase in level of a well-known CSC marker, ABCG2. We further observed that such phenotypic changes induced by iron were not related to an epithelial-to-mesenchymal transition (EMT). Instead, the sex-determining region Y (SRY)-box 9 protein (SOX9) was substantially linked to iron treatment and hydroxyl radical level. Using gene manipulations, including ectopic SOX9 overexpression and SOX9 short hairpin RNA knockdown, we have verified that SOX9 is responsible for CSC enrichment mediated by iron. These findings indicate a novel role of iron via hydroxyl radical in CSC regulation and its importance in aggressive cancer behaviors and likely metastasis through SOX9 upregulation.

Induction of stemlike cells with fibrogenic properties by carbon nanotubes and its role in fibrogenesis.

We developed a three-dimensional fibroblastic nodule model for fibrogenicity testing of nanomaterials and investigated the role of fibroblast stemlike cells (FSCs) in the fibrogenic process. We showed that carbon nanotubes (CNTs) induced fibroblastic nodule formation in primary human lung fibroblast cultures resembling the fibroblastic foci in clinical fibrosis and promoted FSCs that are highly fibrogenic and a potential driving force of fibrogenesis. This study provides a predictive 3D model and mechanistic insight on CNT fibrogenesis.

Appalachian mountaintop mining particulate matter induces neoplastic transformation of human bronchial epithelial cells and promotes tumor formation.

Epidemiological studies suggest that living near mountaintop coal mining (MTM) activities is one of the contributing factors for high lung cancer incidence. The purpose of this study was to investigate the long-term carcinogenic potential of MTM particulate matter (PMMTM) exposure on human bronchial epithelial cells. Our results show that chronic exposure (3 months) to noncytotoxic, physiological relevant concentration (1 µg/mL) of PMMTM, but not control particle PMCON, induced neoplastic transformation, accelerated cell proliferation, and enhanced cell migration of the exposed lung cells. Xenograft transplantation of the PMMTM-exposed cells in mice caused no apparent tumor formation, but promoted tumor growth of human lung carcinoma H460 cells, suggesting the tumor-promoting effect of PMMTM. Chronic exposure to the main inorganic chemical constituent of PMMTM, molybdenum but not silica, similarly induced cell transformation and tumor promotion, suggesting the contribution of molybdenum, at least in part, in the PMMTM effects. These results provide new evidence for the carcinogenic potential of PMMTM and support further risk assessment and implementation of exposure control for PMMTM.

Induction of stem-like cells with malignant properties by chronic exposure of human lung epithelial cells to single-walled carbon nanotubes.

BACKGROUND: Carbon nanotubes (CNT) hold great promise to create new and better products for commercial and biomedical applications, but their long-term adverse health effects are a major concern. The objective of this study was to address human lung cancer risks associated with chronic pulmonary exposure to single-walled (SW) CNT through the fundamental understanding of cellular and molecular processes leading to carcinogenesis. We hypothesized that the acquisition of cancer stem cells (CSC), a subpopulation that drive tumor initiation and progression, may contribute to CNT carcinogenesis. METHODS: Non-tumorigenic human lung epithelial cells were chronically exposed to well-dispersed SWCNT for a period of 6 months at the physiologically relevant concentration of 0.02 µg/cm2 surface area dose. Chronic SWCNT-exposed cells were evaluated for the presence of CSC-like cells under CSC-selective conditions of tumor spheres and side population (SP). CSC-like cells were isolated using fluorescence-activated cell sorting and were assessed for aggressive behaviors, including acquired apoptosis resistance and increased cell migration and invasion in vitro, and tumor-initiating capability in vivo. Non-small cell lung cancer cells served as a positive control. RESULTS: We demonstrated for the first time the existence of CSC-like cells in all clones of chronic SWCNT-exposed lung epithelial cells. These CSC-like cells, in contrary to their non-CSC counterpart, possessed all biological features of lung CSC that are central to irreversible malignant transformation, self-renewal, aggressive cancer behaviors, and in vivo tumorigenesis. These cells also displayed aberrant stem cell markers, notably Nanog, SOX-2, SOX-17 and E-cadherin. Restored expression of tumor suppressor p53 abrogated CSC properties of CSC-like cells. Furthermore, we identified specific stem cell surface markers CD24low and CD133high that are associated with SWCNT-induced CSC formation and tumorigenesis. CONCLUSIONS: Our findings provide new and compelling evidence for the acquisition of CSC-like cells induced by chronic SWCNT exposure, which are likely to be a major driving force for SWCNT tumorigenesis. Thus, our study supports prudent adoption of prevention strategies and implementation of exposure control for SWCNT. We also suggest that the detection of CSC and associated surface markers may provide an effective screening tool for prediction of the carcinogenic potential of SWCNT and related nanoparticles.

Effect of fiber length on carbon nanotube-induced fibrogenesis.

Given their extremely small size and light weight, carbon nanotubes (CNTs) can be readily inhaled by human lungs resulting in increased rates of pulmonary disorders, particularly fibrosis. Although the fibrogenic potential of CNTs is well established, there is a lack of consensus regarding the contribution of physicochemical attributes of CNTs on the underlying fibrotic outcome. We designed an experimentally validated in vitro fibroblast culture model aimed at investigating the effect of fiber length on single-walled CNT (SWCNT)-induced pulmonary fibrosis. The fibrogenic response to short and long SWCNTs was assessed via oxidative stress generation, collagen expression and transforming growth factor-beta (TGF-ß) production as potential fibrosis biomarkers. Long SWCNTs were significantly more potent than short SWCNTs in terms of reactive oxygen species (ROS) response, collagen production and TGF-ß release. Furthermore, our finding on the length-dependent in vitro fibrogenic response was validated by the in vivo lung fibrosis outcome, thus supporting the predictive value of the in vitro model. Our results also demonstrated the key role of ROS in SWCNT-induced collagen expression and TGF-ß activation, indicating the potential mechanisms of length-dependent SWCNT-induced fibrosis. Together, our study provides new evidence for the role of fiber length in SWCNT-induced lung fibrosis and offers a rapid cell-based assay for fibrogenicity testing of nanomaterials with the ability to predict pulmonary fibrogenic response in vivo.

OGT and OGA gene-edited human induced pluripotent stem cells for dissecting the functional roles of O-GlcNAcylation in hematopoiesis.

Hematopoiesis continues throughout life to produce all types of blood cells from hematopoietic stem cells (HSCs). Metabolic state is a known regulator of HSC self-renewal and differentiation, but whether and how metabolic sensor O-GlcNAcylation, which can be modulated via an inhibition of its cycling enzymes O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT), contributes to hematopoiesis remains largely unknown. Herein, isogenic, single-cell clones of OGA-depleted (OGAi) and OGT-depleted (OGTi) human induced pluripotent stem cells (hiPSCs) were successfully generated from the master hiPSC line MUSIi012-A, which were reprogrammed from CD34+ hematopoietic stem/progenitor cells (HSPCs) containing epigenetic memory. The established OGAi and OGTi hiPSCs exhibiting an increase or decrease in cellular O-GlcNAcylation concomitant with their loss of OGA and OGT, respectively, appeared normal in phenotype and karyotype, and retained pluripotency, although they may favor differentiation toward certain germ lineages. Upon hematopoietic differentiation through mesoderm induction and endothelial-to-hematopoietic transition, we found that OGA inhibition accelerates hiPSC commitment toward HSPCs and that disruption of O-GlcNAc homeostasis affects their commitment toward erythroid lineage. The differentiated HSPCs from all groups were capable of giving rise to all hematopoietic progenitors, thus confirming their functional characteristics. Altogether, the established single-cell clones of OGTi and OGAi hiPSCs represent a valuable platform for further dissecting the roles of O-GlcNAcylation in blood cell development at various stages and lineages of blood cells. The incomplete knockout of OGA and OGT in these hiPSCs makes them susceptible to additional manipulation, i.e., by small molecules, allowing the molecular dynamics studies of O-GlcNAcylation.

Musashi-2 in cancer-associated fibroblasts promotes non-small cell lung cancer metastasis through paracrine IL-6-driven epithelial-mesenchymal transition.

BACKGROUND: Lung cancer, the most common cause of cancer-related mortality worldwide, is predominantly associated with advanced/metastatic disease. The interaction between tumor cells and cancer-associated fibroblasts (CAFs) in tumor microenvironment is known to be essential for regulating tumor progression and metastasis, but the underlying mechanisms, particularly the role of RNA-binding protein Musashi-2 (MSI2) in CAFs in promoting non-small cell lung cancer (NSCLC) invasiveness and metastatic spread, remain obscure. METHODS: Genomic and proteomic database analyses were performed to evaluate the potential clinical significance of MSI2 in NSCLC tumor and stromal clinical specimens. Molecular approaches were used to modify MSI2 in CAFs and determine its functional role in NSCLC cell motility in vitro using 2D and 3D models, and in metastasis in a xenograft mouse model using live-cell imaging. RESULTS: MSI2, both gene and protein, is upregulated in NSCLC tissues and is associated with poor prognosis and high metastatic risk in patients. Interestingly, MSI2 is also upregulated in NSCLC stroma and activated fibroblasts, including CAFs. Depletion of MSI2 in CAFs by CRISPR-Cas9 strongly inhibits NSCLC cell migration and invasion in vitro, and attenuates local and distant metastatic spread of NSCLC cells in vivo. The crosstalk between CAFs and NSCLC cells occurs via paracrine signaling, which is regulated by MSI2 in CAFs via IL-6. The secreted IL-6 promotes epithelial-mesenchymal transition in NSCLC cells, which drives metastasis. CONCLUSION: Our findings reveal for the first time that MSI2 in CAFs is important in CAF-mediated NSCLC cell invasiveness and metastasis via IL-6 paracrine signaling. Therefore, targeting the MSI2/IL-6 axis in CAFs could be effective in combating NSCLC metastasis.

Role of YAP in hematopoietic differentiation and erythroid lineage specification of human-induced pluripotent stem cells.

BACKGROUND: In vitro production of hematopoietic stem/progenitor cells (HSPCs) from human-induced pluripotent stem cells (hiPSCs) provides opportunities for fundamental research, disease modeling, and large-scale production of HLA-matched HSPCs for therapeutic applications. However, a comprehensive understanding of the signaling mechanisms that regulate human hematopoiesis is needed to develop a more effective procedure for deriving HSPCs from hiPSCs. METHODS: In this study, we investigate the role of YAP during the hematopoietic differentiation of hiPSCs to HSPCs and erythrocytes using the isogenic YAP-overexpressing (YAP-S5A) and YAP-depleting (YAP-KD) hiPSCs to eliminate the effects of a genetic background variation. RESULTS: Although YAP is dispensable for maintaining the self-renewal and pluripotency of these hiPSCs, it affects the early cell-fate determination and hematopoietic differentiation of hiPSCs. Depleting YAP enhances the derivation efficiency of HSPCs from hiPSCs by inducing the mesodermal lineage commitment, promoting hematopoietic differentiation, and preventing the differentiation toward endothelial lineage. On the contrary, the overexpression of YAP reduced HSPCs yield by inducing the endodermal lineage commitment, suppressing hematopoietic differentiation, and promoting the differentiation toward endothelial lineage. CONCLUSIONS: Expression of YAP is crucial for the differentiation of hiPSC-derived HSPCs toward mature erythrocytes. We believe that by manipulating YAP activity using small molecules, the efficiency of the large-scale in vitro production system for generating hematopoietic stem/progenitor cells for future therapeutic use could be improved.