A miR-99a-TGF-β1 feedback circuit drives miR-99a upregulation.

EV-miR-99a targets NLRP2 and activates NF-kB signaling in fibroblasts.

miR-99a associates with CRC progression and prognosis.

EVs from highly metastatic CRC cells promote fibroblast activation.

Safety assessment of LY2584702 in mice.

Inflammation and epithelial-to-mesenchymal transition are hallmarks of cancer progression. A better understanding of the mechanisms driving these processes could help uncover strategies to treat and prevent metastasis. In this study, we found that extracellular vesicle (EV)-mediated cross-talk between colorectal cancer cells and fibroblasts facilitates inflammation and promotes metastasis. Fibroblasts were highly activated in primary tumors from patients with colorectal cancer with metastatic disease, and EVs secreted from highly metastatic colorectal cancer cells promoted fibroblast activation. Mechanistically, EV-packaged miR-99a-5p (EV-miR-99a) specifically targeted NLRP2 mRNA in fibroblasts and activated the proinflammatory NFκB signaling pathway, thereby converting normal fibroblasts into cancer-associated fibroblasts (CAF). EV-miR-99a-activated CAFs enhanced the migratory capacity of colorectal cancer cells by secreting CCL7, which potently induced epithelial-to-mesenchymal transition by increasing the expression of multiple E-cadherin repressors via the CCR5-mTOR-p70S6K pathway. Expression of miR-99a in colorectal cancer cells was upregulated by TGFβ1 secreted from CAFs in an NFκB-dependent manner, forming an miR-99a/TGFβ1 regulatory circuit. The communication between colorectal cancer cells and fibroblasts engendered a proinflammatory niche that facilitated metastasis, which could be abolished by treatment with the p70S6K inhibitor LY2584702. Clinically, EV-miR-99a levels in the plasma correlated with the metastatic status of patients with colorectal cancer. Together, these findings highlight the metastasis-promoting function of an inflammatory fibroblast niche induced by cancer cell-derived EVs and provide potential targets for the prediction and management of colorectal cancer metastasis. SIGNIFICANCE:Extracellular vesicle-mediated cross-talk between colorectal cancer cells and fibroblasts orchestrates a proinflammatory niche that induces and facilitates metastasis and provides potential targets for disease prediction and therapeutic intervention.

Naked mole-rats (NMR) emerged as a powerful model to study healthy aging. NMRs are mouse-sized rodents that have maximum lifespan up to 40 years in captivity, and are resistant to the majority of age-related diseases. However, the developmental properties of NMR stem and progenitor compartments are poorly understood. Previously we compiled the first complete cell atlas of hematopoiesis and the immune system in naked mole-rats, identifying several neotenic features, wherein fetal characteristics of hematopoiesis are preserved in adulthood. In the previous studies we defined the most primitive hematopoietic stem and progenitor cell (HSPC) compartment in NMRs as marked by Lineage-Thy1.1+CD34+ (LTC), as these cells showed highest colony formation and replating capacity in vitro and highest engraftment and multi-lineage potential in vivo, when compared to other Lineage- cell populations. Strikingly, bone marrow (BM) frequencies of NMR LTCs, mouse LIN-Kit+Sca-1+ (LSK) and human LIN-CD34+CD38lo are in a close range of 0.1-0.3% of total BM cells. Here we report a high frequency BM HSPC population, which we termed neoLTC, exclusively found in neonatal animals. Colony formation in semisolid cultures and short term xenotranplantations functionally confirmed presence of HSPCs in this population at similar abundances than in adult LTCs. To characterize transcriptomic properties of neoLTCs, we performed population RNA-Seq and integrated them with an RNA-Seq profile collection of adult and neonate human and mouse HSCs as well as adult NMR LTCs. Remarkably, while their immunophenotype is identical to adult NMR HSPCs, neoLTCs showed a unique transcriptomic profile devoid of many canonical HSPC genes. Using the human hematopoietic stem cell (HSC) ontogeny map (PMID 35418685) to pinpoint functional gene subsets which were differentially expressed, we found that neoLTCs underexpressed 9/15 HSC transcription factors and 5/6 HSC signature genes, whereas they overexpressed 5/7 HSC quiescence genes, 9/11 NMR HSPC genes and 10/22 nascent HSC genes. Moreover, neoLTCs produced multiple secreted matrix components related to neonatal BM niche formation, convergently neoLTCs overexpressed all mesenchymal stem cell (MSC) subset genes from the HSC ontogeny map. However, when compared with freshly isolated NMR MSCs from our most recent publication, neoLTCs clustered separately from MSCs and LTCs, and had substantially more common genesets with LTCs than MSCs. The most striking finding was overexpression of both CXCL12 and its alternative receptor CXCR7, thereby abrogating neoLTC homing capacity, which we confirmed by intra-femural xenotransplantations. Captive NMR colony queens may have a litter survival rate ranging between 20-90%, depending on the individual queen specimen, colony size, non-breeder subordinate age and other factors. Interestingly, the neoLTC expression signature resurfaced in whole marrow isolates from moribund (but not in healthy) neonates, which were marked by up to 10-fold overabundance of neoLTCs, accumulation of a direct neoLTC-derived precursor and depleted marrow granulocytes. We thus hypothesize that the neoLTC cell population protects newborns during the critical transition at birth and shortly beyond by boosting a supply of innate immune cells if needed. In summary, our results identify a transient cell stage during postnatal hematopoiesis unique to naked mole-rats.

AbstractThe naked mole-rat (Heterocephalus glaber) is a long-lived rodent species showing resistance to the development of cancer. Although naked mole-rats have been reported to lack natural killer (NK) cells, γδ T cell-based immunity has been suggested in this species, which could represent an important arm of the immune system for antitumor responses. Here, we investigate the biology of these unconventional T cells in peripheral tissues (blood, spleen) and thymus of the naked mole-rat at different ages by TCR repertoire profiling and single-cell gene expression analysis. Using our own TCR annotation in the naked mole-rat genome, we report that the γδ TCR repertoire is dominated by a public invariant Vγ4-2/Vδ1-4 TCR, containing the complementary-determining-region-3 (CDR3)γ CTYWDSNYAKKLF / CDR3δ CALWELRTGGITAQLVF that are likely generated by short-homology-repeat-driven DNA rearrangements. This invariant TCR is specifically found in γδ T cells expressing genes associated with NK cytotoxicity and is generated in both the thoracic and cervical thymus of the naked mole-rat until adult life. Our results indicate that invariant Vγ4-2/Vδ1-4 NK-like effector T cells in the naked mole-rat can contribute to tumor immunosurveillance by γδ TCR-mediated recognition of a common molecular signal.

Abundant high molecular weight hyaluronic acid (HMW-HA) contributes to cancer resistance and possibly longevity of the longest-lived rodent, the naked mole-rat 1, 2 . To study whether the benefits of HMW-HA could be transferred to other animal species, we generated a transgenic mouse overexpressing naked mole-rat hyaluronic acid synthase 2 gene (nmrHAS2). nmrHAS2 mice showed increase in hyaluronan levels in several tissues, and lower incidence of spontaneous and induced cancer, extended lifespan and improved healthspan. The transcriptome signature of nmrHAS2 mice shifted towards that of longer-lived species. The most striking change observed in nmrHAS2 mice was attenuated inflammation across multiple tissues. HMW-HA reduced inflammation via several pathways including direct immunoregulatory effect on immune cells, protection from oxidative stress, and improved gut barrier function during aging. These findings demonstrate that the longevity mechanism that evolved in the naked mole-rat can be exported to other species, and open new avenues for using HMW-HA to improve lifespan and healthspan.

Abundant high-molecular-mass hyaluronic acid (HMM-HA) contributes to cancer resistance and possibly to the longevity of the longest-lived rodent-the naked mole-rat1,2. To study whether the benefits of HMM-HA could be transferred to other animal species, we generated a transgenic mouse overexpressing naked mole-rat hyaluronic acid synthase 2 gene (nmrHas2). nmrHas2 mice showed an increase in hyaluronan levels in several tissues, and a lower incidence of spontaneous and induced cancer, extended lifespan and improved healthspan. The transcriptome signature of nmrHas2 mice shifted towards that of longer-lived species. The most notable change observed in nmrHas2 mice was attenuated inflammation across multiple tissues. HMM-HA reduced inflammation through several pathways, including a direct immunoregulatory effect on immune cells, protection from oxidative stress and improved gut barrier function during ageing. These beneficial effects were conferred by HMM-HA and were not specific to the nmrHas2 gene. These findings demonstrate that the longevity mechanism that evolved in the naked mole-rat can be exported to other species, and open new paths for using HMM-HA to improve lifespan and healthspan.

Alternative splicing regulates gene expression and functional diversity and is often dysregulated in human cancers. Here, we discovered that the long noncoding RNA (lncRNA) MIR99AHG regulated alternative splicing to alter the activity of a chromatin remodeler and promote metastatic behaviors in colorectal cancer (CRC). MIR99AHG was abundant in invasive CRC cells and metastatic tumors from patients and promoted motility and invasion in cultured CRC cells. MIR99AHG bound to and stabilized the RNA splicing factor PTBP1, and this complex increased cassette exon inclusion in the mRNA encoding the chromatin remodeling gene SMARCA1. Specifically, MIR99AHG altered the nature of PTBP1 binding to the splice sites on intron 12 of SMARCA1 pre-mRNA, thereby triggering a splicing switch from skipping to including exon 13 to produce the long isoform, SMARCA1-L. SMARCA1, but not SMARCA1-L, suppressed invadopodia formation, cell migration, and invasion. Analysis of CRC samples revealed that the abundance of MIR99AHG transcript positively correlated with that of SMARCA1-L mRNA and PTBP1 protein and with poor prognosis in patients with CRC. Furthermore, TGF-β1 secretion from cancer-associated fibroblasts increased MIR99AHG expression in CRC cells. Our findings identify an lncRNA that is induced by cues from the tumor microenvironment and that interacts with PTBP1 to regulate alternative splicing, potentially providing a therapeutic target and predictive biomarker for metastatic CRC.

Using DNA methylation profiles (n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species.

The naked mole rat (NMR) is the longest-lived rodent, resistant to multiple age-related diseases including neurodegeneration. However, the mechanisms underlying the NMR's resistance to neurodegenerative diseases remain elusive. Here, we isolated oligodendrocyte progenitor cells (OPCs) from NMRs and compared their transcriptome with that of other mammals. Extracellular matrix (ECM) genes best distinguish OPCs of long- and short-lived species. Notably, expression levels of CD44, an ECM-binding protein that has been suggested to contribute to NMR longevity by mediating the effect of hyaluronan (HA), are not only high in OPCs of long-lived species but also positively correlate with longevity in multiple cell types/tissues. We found that CD44 localizes to the endoplasmic reticulum (ER) and enhances basal ATF6 activity. CD44 modifies proteome and membrane properties of the ER and enhances ER stress resistance in a manner dependent on unfolded protein response regulators without the requirement of HA. HA-independent role of CD44 in proteostasis regulation may contribute to mammalian longevity.

Matthew Simon, Jiping Yang , Jonathan Gigas, Eric J Earley , Eric Hillpot, Lei Zhang, Maria Zagorulya, Greg Tombline, Michael Gilbert, Samantha L Yuen, Alexis Pope, Michael Van Meter, Stephan Emmrich , Denis Firsanov, Advait Athreya , Seyed Ali Biashad , Jeehae Han, Seungjin Ryu , Archana Tare, Yizhou Zhu , Adam Hudgins , Gil Atzmon, Nir Barzilai, Aaron Wolfe , Kelsey Moody, Benjamin A Garcia, Paul D Robbins, Jan Vijg , Andrei Seluanov , Yousin Suh & Vera Gorbunova

Abstract Alternative splicing (AS) of pre-mRNAs is an essential process that regulates gene expression and functional diversity, yet the underlying regulatory mechanisms that control this process during cancer metastasis are not clear. Here, we uncovered that a long non-coding RNA (lncRNA), MIR99AHG, mediates regulation of AS to alter chromatin remodeler function and promote invadopodia formation in colorectal cancer (CRC). We found that MIR99AHG is highly expressed in metastatic CRC cells and patient tissue samples. In vitro and in vivo assays showed that MIR99AHG potently drove CRC cell motility, invasion, and metastasis. MIR99AHG bound to and stabilized the RNA splicing factor PTBP1, which cooperatively increased cassette exon inclusion of the chromatin remodeling gene SMARCA1. Mechanistically, MIR99AHG acted as an address label for PTBP1 to fine-tune its binding pattern on SMARCA1 pre-mRNA, thereby controlling the skipping-to-inclusion splicing switch of SMARCA1 and favouring the production of a long isoform (SMARCA1-L). Canonical SMARCA1 was found to suppress cell migration and invasiveness by inhibiting invadopodia formation, but SMARCA1-L was functionally inert. Clinicaldata revealed that MIR99AHG was positively correlated with PTBP1 and SMARCA1-L in human CRC specimens and predicted patient outcome. Furthermore, we showed that the crosstalk with cancer-associated fibroblasts triggers TGF-β/SAMD signalling in CRC cells and activates MIR99AHG expression. Our findings establish a novel mechanism for a lncRNA that interacts with PTBP1 to regulate AS process, providing a potential therapeutic target and predictive biomarker of CRC.

Naked mole rats (NMRs) are the longest-lived rodents yet their stem cell characteristics remain enigmatic. Here, we comprehensively mapped the NMR hematopoietic landscape and identified unique features likely contributing to longevity. Adult NMRs form red blood cells in spleen and marrow, which comprise a myeloid bias toward granulopoiesis together with decreased B-lymphopoiesis. Remarkably, youthful blood and marrow single-cell transcriptomes and cell compositions are largely maintained until at least middle age. Similar to primates, the primitive stem and progenitor cell (HSPC) compartment is marked by CD34 and THY1. Stem cell polarity is seen for Tubulin but not CDC42, and is not lost until 12 years of age. HSPC respiration rates are as low as in purified human stem cells, in concert with a strong expression signature for fatty acid metabolism. The pool of quiescent stem cells is higher than in mice, and the cell cycle of hematopoietic cells is prolonged. By characterizing the NMR hematopoietic landscape, we identified resilience phenotypes such as an increased quiescent HSPC compartment, absence of age-related decline, and neotenic traits likely geared toward longevity.

Sirtuin 6 (SIRT6) is a deacylase and mono-ADP ribosyl transferase (mADPr) enzyme involved in multiple cellular pathways implicated in the regulation of aging and metabolism. Targeted sequencing identified a SIRT6 allele containing two linked substitutions (N308K/A313S) as enriched in Ashkenazi Jewish (AJ) centenarians as compared to AJ control individuals. Characterization of this SIRT6 (centSIRT6) allele demonstrated it to be a stronger suppressor of LINE1 retrotransposons, confer enhanced stimulation of DNA double strand break repair, and more robust cancer cell killing compared to the wild type. Surprisingly, centSIRT6 displayed weaker deacetylase activity, but stronger mADPr activity, over a range of NAD+ concentrations and substrates. Additionally, centSIRT6 displayed a stronger interaction with Lamin A/C (LMNA), which correlated with enhanced ribosylation of LMNA. Our results suggest that enhanced SIRT6 function contributes to human longevity by improving genome maintenance via increased mADPr activity and enhanced interaction with LMNA.