Age-related gene expression signatures from limb skeletal muscles and the diaphragm in mice and rats reveal common and species-specific changes.

BACKGROUND: As a result of aging, skeletal muscle undergoes atrophy and a decrease in function. This age-related skeletal muscle weakness is known as "sarcopenia". Sarcopenia is part of the frailty observed in humans. In order to discover treatments for sarcopenia, it is necessary to determine appropriate preclinical models and the genes and signaling pathways that change with age in these models. METHODS AND RESULTS: To understand the changes in gene expression that occur as a result of aging in skeletal muscles, we generated a multi-time-point gene expression signature throughout the lifespan of mice and rats, as these are the most commonly used species in preclinical research and intervention testing. Gastrocnemius, tibialis anterior, soleus, and diaphragm muscles from male and female C57Bl/6J mice and male Sprague Dawley rats were analyzed at ages 6, 12, 18, 21, 24, and 27 months, plus an additional 9-month group was used for rats. More age-related genes were identified in rat skeletal muscles compared with mice; this was consistent with the finding that rat muscles undergo more robust age-related decline in mass. In both species, pathways associated with innate immunity and inflammation linearly increased with age. Pathways linked with extracellular matrix remodeling were also universally downregulated. Interestingly, late downregulated pathways were exclusively found in the rat limb muscles and these were linked to metabolism and mitochondrial respiration; this was not seen in the mouse. CONCLUSIONS: This extensive, side-by-side transcriptomic profiling shows that the skeletal muscle in rats is impacted more by aging compared with mice, and the pattern of decline in the rat may be more representative of the human. The observed changes point to potential therapeutic interventions to avoid age-related decline in skeletal muscle function.

The Activin/FLRG Pathway Associates with Poor COVID-19 Outcomes in Hospitalized Patients.

A subset of hospitalized COVID-19 patients, particularly the aged and those with comorbidities, develop the most severe form of the disease, characterized by acute respiratory disease syndrome (ARDS), coincident with experiencing a "cytokine storm." Here, we demonstrate that cytokines which activate the NF-κB pathway can induce activin A. Patients with elevated activin A, activin B, and FLRG at hospital admission were associated with the most severe outcomes of COVID-19, including the requirement for mechanical ventilation, and all-cause mortality. A prior study showed that activin A could decrease viral load, which indicated there might be a risk to giving COVID-19 patients an inhibitor of activin. To evaluate this, the role for activin A was examined in a hamster model of SARS-CoV-2 infection, via blockade of activin A signaling. The hamster model demonstrated that use of an anti-activin A antibody did not worsen the disease and there was no evidence for increase in lung viral load and pathology. The study indicates blockade of activin signaling may be beneficial in treating COVID-19 patients experiencing ARDS.

The Effect of Fluid Flow Shear Stress and Substrate Stiffness on Yes-Associated Protein (YAP) Activity and Osteogenesis in Murine Osteosarcoma Cells.

Osteosarcoma (OS) is an aggressive bone cancer originating in the mesenchymal lineage. Prognosis for metastatic disease is poor, with a mortality rate of approximately 40%; OS is an aggressive disease for which new treatments are needed. All bone cells are sensitive to their mechanical/physical surroundings and changes in these surroundings can affect their behavior. However, it is not well understood how OS cells specifically respond to fluid movement, or substrate stiffness-two stimuli of relevance in the tumor microenvironment. We used cells from spontaneous OS tumors in a mouse engineered to have a bone-specific knockout of pRb-1 and p53 in the osteoblast lineage. We silenced Sox2 (which regulates YAP) and tested the effect of fluid flow shear stress (FFSS) and substrate stiffness on YAP expression/activity-which was significantly reduced by loss of Sox2, but that effect was reversed by FFSS but not by substrate stiffness. Osteogenic gene expression was also reduced in the absence of Sox2 but again this was reversed by FFSS and remained largely unaffected by substrate stiffness. Thus we described the effect of two distinct stimuli on the mechanosensory and osteogenic profiles of OS cells. Taken together, these data suggest that modulation of fluid movement through, or stiffness levels within, OS tumors could represent a novel consideration in the development of new treatments to prevent their progression.

Nanopore Identification of Single Nucleotide Mutations in Circulating Tumor DNA by Multiplexed Ligation.

BACKGROUND: Circulating tumor DNAs (ctDNAs) are highly promising cancer biomarkers, potentially applicable for noninvasive liquid biopsy and disease monitoring. However, to date, sequencing of ctDNAs has proven to be challenging primarily due to small sample size and high background of fragmented cell-free DNAs (cfDNAs) derived from normal cells in the circulation, specifically in early stage cancer. METHODS: Solid-state nanopores (ssNPs) have recently emerged as a highly efficient tool for single-DNA sensing and analysis. Herein, we present a rapid nanopore genotyping strategy to enable an amplification-free identification and classification of ctDNA mutations. A biochemical ligation detection assay was used for the creation of specific fluorescently-labelled short DNA reporter molecules. Color conjugation with multiple fluorophores enabled a unique multi-color signature for different mutations, offering multiplexing potency. Single-molecule readout of the fluorescent labels was carried out by electro-optical sensing via solid-state nanopores drilled in titanium oxide membranes. RESULTS: As proof of concept, we utilized our method to detect the presence of low-quantity ERBB2 F310S and PIK3Ca H1047R breast cancer mutations from both plasmids and xenograft mice blood samples. We demonstrated an ability to distinguish between a wild type and a mutated sample, and between the different mutations in the same sample. CONCLUSIONS: Our method can potentially enable rapid and low cost ctDNA analysis that completely circumvents PCR amplification and library preparation. This approach will thus meet a currently unmet demand in terms of sensitivity, multiplexing and cost, opening new avenues for early diagnosis of cancer.

Translation Regulation by eIF2α Phosphorylation and mTORC1 Signaling Pathways in Non-Communicable Diseases (NCDs).

Non-communicable diseases (NCDs) are medical conditions that, by definition, are non-infectious and non-transmissible among people. Much of current NCDs are generally due to genetic, behavioral, and metabolic risk factors that often include excessive alcohol consumption, smoking, obesity, and untreated elevated blood pressure, and share many common signal transduction pathways. Alterations in cell and physiological signaling and transcriptional control pathways have been well studied in several human NCDs, but these same pathways also regulate expression and function of the protein synthetic machinery and mRNA translation which have been less well investigated. Alterations in expression of specific translation factors, and disruption of canonical mRNA translational regulation, both contribute to the pathology of many NCDs. The two most common pathological alterations that contribute to NCDs discussed in this review will be the regulation of eukaryotic initiation factor 2 (eIF2) by the integrated stress response (ISR) and the mammalian target of rapamycin complex 1 (mTORC1) pathways. Both pathways integrally connect mRNA translation activity to external and internal physiological stimuli. Here, we review the role of ISR control of eIF2 activity and mTORC1 control of cap-mediated mRNA translation in some common NCDs, including Alzheimer's disease, Parkinson's disease, stroke, diabetes mellitus, liver cirrhosis, chronic obstructive pulmonary disease (COPD), and cardiac diseases. Our goal is to provide insights that further the understanding as to the important role of translational regulation in the pathogenesis of these diseases.

Inflammatory Breast Cancer Promotes Development of M2 Tumor-Associated Macrophages and Cancer Mesenchymal Cells through a Complex Chemokine Network.

Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer that displays profound cancer stem cell (CSC) and mesenchymal features that promote rapid metastasis. Another hallmark of IBC is high infiltration of M2 tumor-associated (immune-suppressing) macrophages. The molecular mechanism that drives these IBC phenotypes is not well understood. Using patient breast tumor specimens, breast cancer cell lines, and a patient-derived xenograft model of IBC, we demonstrate that IBC strongly expresses IL8 and growth-regulated oncogene (GRO) chemokines that activate STAT3, which promotes development of high levels of CSC-like cells and a mesenchymal phenotype. We also show that IBC expresses high levels of many monocyte recruitment and macrophage polarization factors that attract and differentiate monocytes into tumor-promoting, immune-suppressing M2-like macrophages. The M2 macrophages in turn were found to secrete high levels of IL8 and GRO chemokines, thereby creating a feed-forward chemokine loop that further drives an IBC epithelial-to-mesenchymal transition. Our study uncovers an intricate IBC-initiated autocrine-paracrine signaling network between IBC cells and monocytes that facilitates development of this highly aggressive form of breast cancer. SIGNIFICANCE: This study uncovers a signaling network in which IBC cells commandeer macrophages to become tumor-promoting, and they in turn drive IBC cells to be more cancer stem-like, mesenchymal, and aggressive.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3360/F1.large.jpg.

Molecular basis for AU-rich element recognition and dimerization by the HuR C-terminal RRM.

Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate-rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3' untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12.

mTORC1/2 inhibition re-sensitizes platinum-resistant ovarian cancer by disrupting selective translation of DNA damage and survival mRNAs.

Platinum resistance is a major cause of treatment failure and mortality in epithelial ovarian cancer. mTORC1/2 inhibitors, which impair mRNA translation, can re-sensitize resistant ovarian cancer cells to platinum chemotherapy but the mechanism remains poorly described. Using platinum-resistant OVCAR-3 cells treated with the selective mTORC1/2 inhibitor INK128/MLN128, we conducted genome-wide transcription and translation studies and analyzed the effect on cell proliferation, AKT-mTOR signaling and cell survival, to determine whether carboplatin resistance involves selective mRNA translational reprogramming, and whether it is sensitive to mTORC1/2 inhibition. Gene ontology and Ingenuity Pathway Analysis (IPA) were used to categorize gene expression changes into experimentally authenticated biochemical and molecular networks. We show that carboplatin resistance involves increased mTORC1/2 signaling, resulting in selective translation of mRNAs involved in DNA damage and repair responses (DDR), cell cycle and anti-apoptosis (survival) pathways. Re-sensitization of ovarian cancer cell killing by carboplatin required only modest mTORC1/2 inhibition, with downregulation of protein synthesis by only 20-30%. Genome-wide transcriptomic and translatomic analyses in OVCAR-3 cells revealed that the modest downregulation of global protein synthesis by dual mTORC1/2 inhibition is associated with greater selective inhibition of DDR, cell cycle and survival mRNA translation, which was confirmed in platinum-resistant SKOV-3 cells. These data suggest a clinical path to re-sensitize platinum resistant ovarian cancer to platinum chemotherapy through partial inhibition of mTORC1/2, resulting in selective translation inhibition of DDR and anti-apoptosis protective mRNAs.

Sox2 is required for tumor development and cancer cell proliferation in osteosarcoma.

The stem cell transcription factor Sox2 is highly expressed in many cancers where it is thought to mark cancer stem cells (CSCs). In osteosarcomas, the most common bone malignancy, high Sox2 expression marks and maintains a fraction of tumor-initiating cells that show all the properties of CSC. Knockdown of Sox2 expression abolishes tumorigenicity and suppresses the CSC phenotype. Here we show that, in a mouse model of osteosarcoma, osteoblast-specific Sox2 conditional knockout (CKO) causes a drastic reduction in the frequency and onset of tumors. The rare tumors detected in the Sox2 CKO animals were all Sox2 positive, indicating that they arose from cells that had escaped Sox2 deletion. Furthermore, Sox2 inactivation in cultured osteosarcoma cells by CRISPR/CAS technology leads to a loss of viability and proliferation of the entire cell population. Inactivation of the YAP gene, a major Hippo pathway effector which is a direct Sox2 target, causes similar results and YAP overexpression rescues cells from the lethality caused by Sox2 inactivation. These effects were osteosarcoma-specific, suggesting a mechanism of cell "addiction" to Sox2-initiated pathways. The requirement of Sox2 for osteosarcoma formation as well as for the survival of the tumor cells suggests that disruption of Sox2-initiated pathways could be an effective strategy for the treatment of osteosarcoma.

Hyperactive mTOR and MNK1 phosphorylation of eIF4E confer tamoxifen resistance and estrogen independence through selective mRNA translation reprogramming.

The majority of breast cancers expresses the estrogen receptor (ER+) and is treated with anti-estrogen therapies, particularly tamoxifen in premenopausal women. However, tamoxifen resistance is responsible for a large proportion of breast cancer deaths. Using small molecule inhibitors, phospho-mimetic proteins, tamoxifen-sensitive and tamoxifen-resistant breast cancer cells, a tamoxifen-resistant patient-derived xenograft model, patient tumor tissues, and genome-wide transcription and translation studies, we show that tamoxifen resistance involves selective mRNA translational reprogramming to an anti-estrogen state by Runx2 and other mRNAs. Tamoxifen-resistant translational reprogramming is shown to be mediated by increased expression of eIF4E and its increased availability by hyperactive mTOR and to require phosphorylation of eIF4E at Ser209 by increased MNK activity. Resensitization to tamoxifen is restored only by reducing eIF4E expression or mTOR activity and also blocking MNK1 phosphorylation of eIF4E. mRNAs specifically translationally up-regulated with tamoxifen resistance include Runx2, which inhibits ER signaling and estrogen responses and promotes breast cancer metastasis. Silencing Runx2 significantly restores tamoxifen sensitivity. Tamoxifen-resistant but not tamoxifen-sensitive patient ER+ breast cancer specimens also demonstrate strongly increased MNK phosphorylation of eIF4E. eIF4E levels, availability, and phosphorylation therefore promote tamoxifen resistance in ER+ breast cancer through selective mRNA translational reprogramming.

Myeloid Zinc Finger 1 and GA Binding Protein Co-Operate with Sox2 in Regulating the Expression of Yes-Associated Protein 1 in Cancer Cells.

The transcription factor (TF) yes-associated protein 1 (YAP1) is a major effector of the tumor suppressive Hippo signaling pathway and is also necessary to maintain pluripotency in embryonic stem cells. Elevated levels of YAP1 expression antagonize the tumor suppressive effects of the Hippo pathway that normally represses YAP1 function. High YAP1 expression is observed in several types of human cancers and is particularly prominent in cancer stem cells (CSCs). The stem cell TF Sox2, which marks and maintains CSCs in osteosarcomas (OSs), promotes YAP1 expression by binding to an intronic enhancer element and YAP1 expression is also crucial for the maintainance of OS stem cells. To further understand the regulation of YAP1 expression in OSs, we subjected the YAP1 intronic enhancer to scanning mutagenesis to identify all DNA cis-elements critical for enhancer function. Through this approach, we identified two novel TFs, GA binding protein (GABP) and myeloid zinc finger 1 (MZF1), which are essential for basal YAP1 transcription. These factors are highly expressed in OSs and bind to distinct sites in the YAP1 enhancer. Depletion of either factor leads to drastically reduced YAP1 expression and thus a reversal of stem cell properties. We also found that YAP1 can regulate the expression of Sox2 by binding to two distinct DNA binding sites upstream and downstream of the Sox2 gene. Thus, Sox2 and YAP1 reinforce each others expression to maintain stemness and tumorigenicity in OSs, but the activity of MZF1 and GABP is essential for YAP1 transcription. Stem Cells 2017;35:2340-2350.

PPARγ agonists promote differentiation of cancer stem cells by restraining YAP transcriptional activity.

Osteosarcoma (OS) is a highly aggressive pediatric bone cancer in which most tumor cells remain immature and fail to differentiate into bone-forming osteoblasts. However, OS cells readily respond to adipogenic stimuli suggesting they retain mesenchymal stem cell-like properties. Here we demonstrate that nuclear receptor PPARγ agonists such as the anti-diabetic, thiazolidinedione (TZD) drugs induce growth arrest and cause adipogenic differentiation in human, mouse and canine OS cells as well as in tumors in mice. Gene expression analysis reveals that TZDs induce lipid metabolism pathways while suppressing targets of the Hippo-YAP pathway, Wnt signaling and cancer-related proliferation pathways. Significantly, TZD action appears to be restricted to the high Sox2 expressing cancer stem cell population and is dependent on PPARγ expression. TZDs also affect growth and cell fate by causing the cytoplasmic sequestration of the transcription factors SOX2 and YAP that are required for tumorigenicity. Finally, we identify a TZD-regulated gene signature based on Wnt/Hippo target genes and PPARγ that predicts patient outcomes. Together, this work highlights a novel connection between PPARγ agonist in inducing adipogenesis and mimicking the tumor suppressive hippo pathway. It also illustrates the potential of drug repurposing for TZD-based differentiation therapy for osteosarcoma.

SOXC Transcription Factors Induce Cartilage Growth Plate Formation in Mouse Embryos by Promoting Noncanonical WNT Signaling.

Growth plates are specialized cartilage structures that ensure the elongation of most skeletal primordia during vertebrate development. They are made by chondrocytes that proliferate in longitudinal columns and then progress in a staggered manner towards prehypertrophic, hypertrophic and terminal maturation. Complex molecular networks control the formation and activity of growth plates, but remain incompletely understood. We investigated here the importance of the SoxC genes, which encode the SOX4, SOX11 and SOX12 transcription factors, in growth plates. We show that the three genes are expressed robustly in perichondrocytes and weakly in growth plate chondrocytes. SoxC(Prx1Cre) mice, which deleted SoxC genes in limb bud skeletogenic mesenchyme, were born with tiny appendicular cartilage primordia because of failure to form growth plates. In contrast, SoxC(Col2Cre) and SoxC(ATC) mice, which deleted SoxC genes primarily in chondrocytes, were born with mild dwarfism and fair growth plates. Chondrocytes in the latter mutants matured normally, but formed irregular columns, proliferated slowly and died ectopically. Asymmetric distribution of VANGL2 was defective in both SoxC(Prx1Cre) and SoxC(ATC) chondrocytes, indicating impairment of planar cell polarity, a noncanonical WNT signaling pathway that controls growth plate chondrocyte alignment, proliferation and survival. Accordingly, SoxC genes were necessary in perichondrocytes for expression of Wnt5a, which encodes a noncanonical WNT ligand required for growth plate formation, and in chondrocytes and perichondrocytes for expression of Fzd3 and Csnk1e, which encode a WNT receptor and casein kinase-1 subunit mediating planar cell polarity, respectively. Reflecting the differential strengths of the SOXC protein transactivation domains, SOX11 was more powerful than SOX4, and SOX12 interfered with the activity of SOX4 and SOX11. Altogether, these findings provide novel insights into the molecular regulation of skeletal growth by proposing that SOXC proteins act cell- and non-cell-autonomously in perichondrocytes and chondrocytes to establish noncanonical WNT signaling crosstalk essential for growth plate induction and control.

SOXC proteins amplify canonical WNT signaling to secure nonchondrocytic fates in skeletogenesis.

Canonical WNT signaling stabilizes ß-catenin to determine cell fate in many processes from development onwards. One of its main roles in skeletogenesis is to antagonize the chondrogenic transcription factor SOX9. We here identify the SOXC proteins as potent amplifiers of this pathway. The SOXC genes, i.e., Sox4, Sox11, and Sox12, are coexpressed in skeletogenic mesenchyme, including presumptive joints and perichondrium, but not in cartilage. Their inactivation in mouse embryo limb bud caused massive cartilage fusions, as joint and perichondrium cells underwent chondrogenesis. SOXC proteins govern these cells cell autonomously. They replace SOX9 in the adenomatous polyposis coli-Axin destruction complex and therein inhibit phosphorylation of ß-catenin by GSK3. This inhibition, a crucial, limiting step in canonical WNT signaling, thus becomes a constitutive event. The resulting SOXC/canonical WNT-mediated synergistic stabilization of ß-catenin contributes to efficient repression of Sox9 in presumptive joint and perichondrium cells and thereby ensures proper delineation and articulation of skeletal primordia. This synergy may determine cell fate in many processes besides skeletogenesis.