Invariant γδTCR natural killer-like effector T cells in the naked mole-rat.

The 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.

The lncRNA MIR99AHG directs alternative splicing of SMARCA1 by PTBP1 to enable invadopodia formation in colorectal cancer cells.

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-ß1 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.

Increased hyaluronan by naked mole-rat Has2 improves healthspan in mice.

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.

A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A.

Sirtuin 6 (SIRT6) is a deacylase and mono-ADP ribosyl transferase (mADPr) enzyme involved in multiple cellular pathways implicated in aging and metabolism regulation. Targeted sequencing of SIRT6 locus in a population of 450 Ashkenazi Jewish (AJ) centenarians and 550 AJ individuals without a family history of exceptional longevity identified enrichment of a SIRT6 allele containing two linked substitutions (N308K/A313S) in centenarians compared with AJ control individuals. Characterization of this SIRT6 allele (centSIRT6) demonstrated it to be a stronger suppressor of LINE1 retrotransposons, confer enhanced stimulation of DNA double-strand break repair, and more robustly kill cancer cells compared with wild-type SIRT6. 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 was 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.

Characterization of naked mole-rat hematopoiesis reveals unique stem and progenitor cell patterns and neotenic traits.

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.

The megakaryocytic transcription factor ARID3A suppresses leukemia pathogenesis.

Given the plasticity of hematopoietic stem and progenitor cells, multiple routes of differentiation must be blocked in the the pathogenesis of acute myeloid leukemia, the molecular basis of which is incompletely understood. We report that posttranscriptional repression of the transcription factor ARID3A by miR-125b is a key event in the pathogenesis of acute megakaryoblastic leukemia (AMKL). AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndrome are at a high risk of developing the disease. The results of our study showed that chromosome 21-encoded miR-125b synergizes with Gata1s to drive leukemogenesis in this context. Leveraging forward and reverse genetics, we uncovered Arid3a as the main miR-125b target behind this synergy. We demonstrated that, during normal hematopoiesis, this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFß-induced apoptosis and cell cycle arrest in complex with SMAD2/3. Although Gata1s mutations perturb erythroid differentiation and induce hyperproliferation of megakaryocytic progenitors, intact ARID3A expression assures their megakaryocytic differentiation and growth restriction. Upon knockdown, these tumor suppressive functions are revoked, causing a blockade of dual megakaryocytic/erythroid differentiation and subsequently of AMKL. Inversely, restoring ARID3A expression relieves the arrest of megakaryocytic differentiation in AMKL patient-derived xenografts. This work illustrates how mutations in lineage-determining transcription factors and perturbation of posttranscriptional gene regulation can interact to block multiple routes of hematopoietic differentiation and cause leukemia. In AMKL, surmounting this differentiation blockade through restoration of the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease.

Ectopic cervical thymi and no thymic involution until midlife in naked mole rats.

Immunosenescence is a hallmark of aging and manifests as increased susceptibility to infection, autoimmunity, and cancer in the elderly. One component of immunosenescence is thymic involution, age-associated shrinkage of the thymus, observed in all vertebrates studied to date. The naked mole rat (Heterocephalus glaber) has become an attractive animal model in aging research due to its extreme longevity and resistance to disease. Here, we show that naked mole rats display no thymic involution up to 11 years of age. Furthermore, we found large ectopic cervical thymi in addition to the canonical thoracic thymus, both being identical in their cell composition. The developmental landscape in naked mole rat thymi revealed overt differences from the murine T-cell compartment, most notably a decrease of CD4+ /CD8+ double-positive cells and lower abundance of cytotoxic effector T cells. Our observations suggest that naked mole rats display a delayed immunosenescence. Therapeutic interventions aimed at reversing thymic aging remain limited, underscoring the importance of understanding the cellular and molecular mechanisms behind a sustained immune function in the naked mole rat.

The stem cell-specific long noncoding RNA HOXA10-AS in the pathogenesis of KMT2A-rearranged leukemia.

HOX genes are highly conserved, and their precisely controlled expression is crucial for normal hematopoiesis. Accordingly, deregulation of HOX genes can cause leukemia. However, despite of intensive research on the coding HOX genes, the role of the numerous long noncoding RNAs (lncRNAs) within the HOX clusters during hematopoiesis and their contribution to leukemogenesis are incompletely understood. Here, we show that the lncRNA HOXA10-AS, located antisense to HOXA10 and mir-196b in the HOXA cluster, is highly expressed in hematopoietic stem cells (HSCs) as well as in KMT2A-rearranged and NPM1 mutated acute myeloid leukemias (AMLs). Using short hairpin RNA- and locked nucleic acid-conjugated chimeric antisense oligonucleotide (LNA-GapmeR)-mediated HOXA10-AS-knockdown and CRISPR/Cas9-mediated excision in vitro, we demonstrate that HOXA10-AS acts as an oncogene in KMT2A-rearranged AML. Moreover, HOXA10-AS knockdown severely impairs the leukemic growth of KMT2A-rearranged patient-derived xenografts in vivo, while high HOXA10-AS expression can serve as a marker of poor prognosis in AML patients. Lentiviral expression of HOXA10-AS blocks normal monocytic differentiation of human CD34+ hematopoietic stem and progenitor cells. Mechanistically, we show that HOXA10-AS localizes in the cytoplasm and acts in trans to induce NF-κB target genes. In total, our data imply that the normally HSC-specific HOXA10-AS is an oncogenic lncRNA in KMT2A-r AML. Thus, it may also represent a potential therapeutic target in KMT2A-rearranged AML.

Endogenous Tumor Suppressor microRNA-193b: Therapeutic and Prognostic Value in Acute Myeloid Leukemia.

Purpose Dysregulated microRNAs are implicated in the pathogenesis and aggressiveness of acute myeloid leukemia (AML). We describe the effect of the hematopoietic stem-cell self-renewal regulating miR-193b on progression and prognosis of AML. Methods We profiled miR-193b-5p/3p expression in cytogenetically and clinically characterized de novo pediatric AML (n = 161) via quantitative real-time polymerase chain reaction and validated our findings in an independent cohort of 187 adult patients. We investigated the tumor suppressive function of miR-193b in human AML blasts, patient-derived xenografts, and miR-193b knockout mice in vitro and in vivo. Results miR-193b exerted important, endogenous, tumor-suppressive functions on the hematopoietic system. miR-193b-3p was downregulated in several cytogenetically defined subgroups of pediatric and adult AML, and low expression served as an independent indicator for poor prognosis in pediatric AML (risk ratio ± standard error, -0.56 ± 0.23; P = .016). miR-193b-3p expression improved the prognostic value of the European LeukemiaNet risk-group stratification or a 17-gene leukemic stemness score. In knockout mice, loss of miR-193b cooperated with Hoxa9/Meis1 during leukemogenesis, whereas restoring miR-193b expression impaired leukemic engraftment. Similarly, expression of miR-193b in AML blasts from patients diminished leukemic growth in vitro and in mouse xenografts. Mechanistically, miR-193b induced apoptosis and a G1/S-phase block in various human AML subgroups by targeting multiple factors of the KIT-RAS-RAF-MEK-ERK (MAPK) signaling cascade and the downstream cell cycle regulator CCND1. Conclusion The tumor-suppressive function is independent of patient age or genetics; therefore, restoring miR-193b would assure high antileukemic efficacy by blocking the entire MAPK signaling cascade while preventing the emergence of resistance mechanisms.

lncRNA MIR100HG-derived miR-100 and miR-125b mediate cetuximab resistance via Wnt/ß-catenin signaling.

De novo and acquired resistance, which are largely attributed to genetic alterations, are barriers to effective anti-epidermal-growth-factor-receptor (EGFR) therapy. To generate cetuximab-resistant cells, we exposed cetuximab-sensitive colorectal cancer cells to cetuximab in three-dimensional culture. Using whole-exome sequencing and transcriptional profiling, we found that the long non-coding RNA MIR100HG and two embedded microRNAs, miR-100 and miR-125b, were overexpressed in the absence of known genetic events linked to cetuximab resistance. MIR100HG, miR-100 and miR-125b overexpression was also observed in cetuximab-resistant colorectal cancer and head and neck squamous cell cancer cell lines and in tumors from colorectal cancer patients that progressed on cetuximab. miR-100 and miR-125b coordinately repressed five Wnt/ß-catenin negative regulators, resulting in increased Wnt signaling, and Wnt inhibition in cetuximab-resistant cells restored cetuximab responsiveness. Our results describe a double-negative feedback loop between MIR100HG and the transcription factor GATA6, whereby GATA6 represses MIR100HG, but this repression is relieved by miR-125b targeting of GATA6. These findings identify a clinically actionable, epigenetic cause of cetuximab resistance.

The non-coding RNA landscape of human hematopoiesis and leukemia.

Non-coding RNAs have emerged as crucial regulators of gene expression and cell fate decisions. However, their expression patterns and regulatory functions during normal and malignant human hematopoiesis are incompletely understood. Here we present a comprehensive resource defining the non-coding RNA landscape of the human hematopoietic system. Based on highly specific non-coding RNA expression portraits per blood cell population, we identify unique fingerprint non-coding RNAs-such as LINC00173 in granulocytes-and assign these to critical regulatory circuits involved in blood homeostasis. Following the incorporation of acute myeloid leukemia samples into the landscape, we further uncover prognostically relevant non-coding RNA stem cell signatures shared between acute myeloid leukemia blasts and healthy hematopoietic stem cells. Our findings highlight the importance of the non-coding transcriptome in the formation and maintenance of the human blood hierarchy.While micro-RNAs are known regulators of haematopoiesis and leukemogenesis, the role of long non-coding RNAs is less clear. Here the authors provide a non-coding RNA expression landscape of the human hematopoietic system, highlighting their role in the formation and maintenance of the human blood hierarchy.

LincRNAs MONC and MIR100HG act as oncogenes in acute megakaryoblastic leukemia.

BACKGROUND: Long non-coding RNAs (lncRNAs) are recognized as pivotal players during developmental ontogenesis and pathogenesis of cancer. The intronic microRNA (miRNA) clusters miR-99a ~ 125b-2 and miR-100 ~ 125b-1 promote progression of acute megakaryoblastic leukemia (AMKL), an aggressive form of hematologic cancers. The function of the lncRNA hostgenes MIR99AHG (alias MONC) and MIR100HG within this ncRNA ensemble remained elusive. RESULTS: Here we report that lncRNAs MONC and MIR100HG are highly expressed in AMKL blasts. The transcripts were mainly localized in the nucleus and their expression correlated with the corresponding miRNA clusters. Knockdown of MONC or MIR100HG impeded leukemic growth of AMKL cell lines and primary patient samples. The development of a lentiviral lncRNA vector to ectopically express lncRNAs without perturbing their secondary structure due to improper termination of the viral transcript, allowed us to study the function of MONC independent of the miRNAs in cord blood hematopoietic stem and progenitor cells (HSPCs). We could show that MONC interfered with hematopoietic lineage decisions and enhanced the proliferation of immature erythroid progenitor cells. CONCLUSIONS: Our study reveals an unprecedented function of lncRNAs MONC and MIR100HG as regulators of hematopoiesis and oncogenes in the development of myeloid leukemia.

miR-99a/100~125b tricistrons regulate hematopoietic stem and progenitor cell homeostasis by shifting the balance between TGFß and Wnt signaling.

Although regulation of stem cell homeostasis by microRNAs (miRNAs) is well studied, it is unclear how individual miRNAs genomically encoded within an organized polycistron can interact to induce an integrated phenotype. miR-99a/100, let-7, and miR-125b paralogs are encoded in two tricistrons on human chromosomes 11 and 21. They are highly expressed in hematopoietic stem cells (HSCs) and acute megakaryoblastic leukemia (AMKL), an aggressive form of leukemia with poor prognosis. Here, we show that miR-99a/100∼125b tricistrons are transcribed as a polycistronic message transactivated by the homeobox transcription factor HOXA10. Integrative analysis of global gene expression profiling, miRNA target prediction, and pathway architecture revealed that miR-99a/100, let-7, and miR-125b functionally converge at the combinatorial block of the transforming growth factor ß (TGFß) pathway by targeting four receptor subunits and two SMAD signaling transducers. In addition, down-regulation of tumor suppressor genes adenomatous polyposis coli (APC)/APC2 stabilizes active ß-catenin and enhances Wnt signaling. By switching the balance between Wnt and TGFß signaling, the concerted action of these tricistronic miRNAs promoted sustained expansion of murine and human HSCs in vitro or in vivo while favoring megakaryocytic differentiation. Hence, our study explains the high phylogenetic conservation of the miR-99a/100∼125b tricistrons controlling stem cell homeostasis, the deregulation of which contributes to the development of AMKL.

The E2F1-miRNA cancer progression network.

The transcription factor E2F1 exhibits dual properties, acting as a tumor suppressor and oncogene. Cellular stress such as DNA damage or mitogenic signaling leads to the activation of E2F1 as a mediator of apoptosis in the context of a conserved cellular anti-tumorigenic safeguard mechanism. However in highly aggressive chemoresistant tumors like malignant melanoma and prostate/bladder cancer it switches off this role and acts as promoter of cancer progression. Possible reasons for E2F1 mediated aggressiveness are defects in cell death pathways caused by epigenetic inactivation of important tumor suppressor genes, which often occur in late stage cancer and contribute to chemoresistance. Nevertheless exact mechanisms underlying E2Fs role in invasiveness and metastasis are largely unknown. Different reports hint towards the existence of feedback loops between E2F1 and microRNAs (miRNAs or miRs). MiRs are activated by E2F1 and either the transcription factor itself or cellular genes necessary for the growth regulating function of E2F1 are inhibited by different miRNAs. This mutual regulation possibly influences the balance between E2F1s proapoptotic versus prosurvival function. In the following we will summarize some miRNA-E2F1-interactions contributing to a complex regulatory network.

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry.

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.

miRNAs can increase the efficiency of ex vivo platelet generation.

The process of megakaryopoiesis culminates in the release of platelets, the pivotal cellular component for hemostasis and wound healing. The regulatory architecture including the modulatory role of microRNAs, which underlies megakaryocytic maturation and platelet formation, is incompletely understood, precluding the ex vivo generation of sufficient platelet numbers for transfusion medicine. We derived a highly efficient differentiation protocol to produce mature polyploid megakaryocytes and functional platelets from CD34⁺-hematopoietic stem and progenitor cells by comparing previously published approaches. Our megakaryocytic culture conditions using the cytokines SCF, TPO, IL-9, and IL-6 include nicotinamide and Rho-associated kinase (ROCK) inhibitor Y27632 as contextual additives. The potency of our novel megakaryocytic differentiation protocol was validated using cord blood and peripheral blood human hematopoietic stem and progenitor cells. Using this novel megakaryocytic differentiation protocol, we characterized the modulatory capacity of several miRNAs highly expressed in normal megakaryocytic cells or malignant blasts from patients with megakaryoblastic leukemia. Overexpression of candidate microRNAs was achieved by lentiviral transduction of CD34⁺-hematopoietic stem and progenitor cells prior to differentiation. We revealed miR-125b and miR-660 as enhancers of polyploidization, as well as platelet output of megakaryocytes. The oncogene miR-125b markedly expanded the number of megakaryocytes during in vitro culture. Conversely, the miR-23a/27a/24-2 cluster, which is highly expressed in normal megakaryocytes, blocked maturation and platelet formation. Our study on the utilization of microRNAs in conjunction with a highly efficient differentiation protocol constitutes another step towards ex vivo platelet manufacturing on a clinically relevant scale.

Checks and balances: E2F-microRNA crosstalk in cancer control.

Extensive research on the E2F transcription factor family over the past decades has led to numerous insights that revealed the involvement of particularly E2F1 not only in proliferation and tumorigenesis but also in apoptosis and differentiation. Latest reports uncovered an essential role for oncogene signaling in regulating the balance of these events on the road to malignancy. Alterations in E2F functions coincide with poor prognosis in cancers, emphasizing their importance for the clinical cancer phenotype. An intriguing addition to the understanding of E2F crosstalks was the finding that their activity can be regulated by micro-RNAs (miRNAs) whose dysregulation has been implicated in malignancy. In turn, miRNAs themselves are targets of E2F family proteins establishing negative feedback loops. Since individual miRNAs may regulate hundreds of genes, these findings add a new challenging layer of complexity to the E2F network that possibly helps to make cell fate decisions. This review outlines our current understanding of the checks and balances of E2Fs and microRNAs in the context of a seemingly paradoxical role in cancer control.

miR-125b-2 is a potential oncomiR on human chromosome 21 in megakaryoblastic leukemia.

Children with trisomy 21/Down syndrome (DS) are at high risk to develop acute megakaryoblastic leukemia (DS-AMKL) and the related transient leukemia (DS-TL). The factors on human chromosome 21 (Hsa21) that confer this predisposing effect, especially in synergy with consistently mutated transcription factor GATA1 (GATA1s), remain poorly understood. Here, we investigated the role of Hsa21-encoded miR-125b-2, a microRNA (miRNA) overexpressed in DS-AMKL/TL, in hematopoiesis and leukemogenesis. We identified a function of miR-125b-2 in increasing proliferation and self-renewal of human and mouse megakaryocytic progenitors (MPs) and megakaryocytic/erythroid progenitors (MEPs). miR-125b-2 overexpression did not affect megakaryocytic and erythroid differentiation, but severely perturbed myeloid differentiation. The proproliferative effect of miR-125b-2 on MEPs accentuated the Gata1s mutation, whereas growth of DS-AMKL/TL cells was impaired upon miR-125b repression, suggesting synergism during leukemic transformation in GATA1s-mutated DS-AMKL/TL. Integrative transcriptome analysis of hematopoietic cells upon modulation of miR-125b expression levels uncovered a set of miR-125b target genes, including DICER1 and ST18 as direct targets. Gene Set Enrichment Analysis revealed that this target gene set is down-regulated in DS-AMKL patients highly expressing miR-125b. Thus, we propose miR-125b-2 as a positive regulator of megakaryopoiesis and an oncomiR involved in the pathogenesis of trisomy 21-associated megakaryoblastic leukemia.

E2F1 in melanoma progression and metastasis.

Metastases are responsible for cancer deaths, but the molecular alterations leading to tumor progression are unclear. Overexpression of the E2F1 transcription factor is common in high-grade tumors that are associated with poor patient survival. To investigate the association of enhanced E2F1 activity with aggressive phenotype, we performed a gene-specific silencing approach in a metastatic melanoma model. Knockdown of endogenous E2F1 via E2F1 small hairpin RNA (shRNA) expression increased E-cadherin expression of metastatic SK-Mel-147 melanoma cells and reduced their invasive potential but not their proliferative activity. Although growth rates of SK-Mel-147 and SK-Mel-103 xenograft tumors expressing E2F1 shRNA or control shRNA were similar, mice implanted with cells expressing E2F1 shRNA had a smaller area of metastases per lung than control mice (n = 3 mice per group; 5% vs 46%, difference = 41%, 95% confidence interval = 15% to 67%; P = .01; one-way analysis of variance). We identified epidermal growth factor receptor as a direct target of E2F1 and demonstrated that inhibition of receptor signaling abrogates E2F1-induced invasiveness, emphasizing the importance of the E2F1-epidermal growth factor receptor interaction as a driving force in melanoma progression that may serve as a paradigm for E2F1-induced metastasis in other human cancers.

Antisense gapmers selectively suppress individual oncogenic p73 splice isoforms and inhibit tumor growth in vivo.

BACKGROUND: Differential mRNA splicing and alternative promoter usage of the TP73 gene results in the expression of multiple NH2-truncated isoforms that act as oncogenes. Abundant levels of these p73 variants in a variety of human cancers correlated with adverse clinical prognosis and response failure to conventional therapies, underscoring their relevance as marker for disease severity and target for cancer intervention. With respect to an equally important role for amino-truncated p73 splice forms (DeltaTAp73) and DeltaNp73 (summarized as DNp73) in the tumorigenic process, we designed locked nucleic acid (LNA) antisense oligonucleotide (ASO) gapmers against individual species that were complementary to DeltaEx2 and DeltaEx2/3 splice junctions and a region in exon 3B unique for DeltaN' and DeltaN. RESULTS: Treatment of cancer cells with these ASOs resulted in a strong and specific reduction of tumorigenic p73 transcripts and proteins, importantly, without abolishing the wild-type p73 tumor suppressor form as observed with p73-shRNA. The specific antisense oligonucleotides rescued cells from apoptosis inhibition due to overexpression of their corresponding amino-truncated p73 isoform and decreased tumor cell proliferation. Furthermore, ASO-116 against DeltaEx2/3 coupled to magnetic nanobead polyethyleneimine (MNB/PEI) carriers significantly inhibited malignant melanoma growth, which correlated with a shift in the balance between endogenous TAp73 and DeltaEx2/3 towards apoptotic full-length p73. CONCLUSION: Our study demonstrates the successful development of LNA-ASOs that selectively differentiate between the closely related p73 oncoproteins, and provide new tools to further delineate their biological properties in different human malignancies and for therapeutic cancer targeting.