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Melanoma-initiating cells (MICs) contribute to the tumorigenicity and heterogeneity of melanoma. MICs are identified by surface and functional markers and have been shown to display cancer stem cell (CSC) properties. However, the existence of MICs that follow the hierarchical CSC model has been questioned by studies showing that single unselected melanoma cells are highly tumorigenic in xenotransplantation assays. Herein, we characterize cells expressing MIC markers (CD20, CD24, CD133, Sca-1, ABCB1, ABCB5, ALDHhigh) in the B16-F10 murine melanoma cell line. We use flow cytometric phenotyping, single-cell sorting followed by in vitro clonogenic assays, and syngeneic in vivo serial transplantation assays to demonstrate that the expression of MIC markers does not select CSC-like cells in this cell line. Previously, our group showed that heme-degrading enzyme heme oxygenase-1 (HO-1) can be upregulated in melanoma and increase its aggressiveness. Here, we show that HO-1 activity is important for non-adherent growth of melanoma and HO-1 overexpression enhances the vasculogenic mimicry potential, which can be considered protumorigenic activity. However, HO-1 overexpression decreases clone formation in vitro and serial tumor initiation in vivo. Thus, HO-1 plays a dual role in melanoma, improving the progression of growing tumors but reducing the risk of melanoma initiation.

SF3B1 is the most mutated splicing factor (SF) in myelodysplastic syndromes (MDSs), which are clonal hematopoietic disorders with variable risk of leukemic transformation. Although tumorigenic SF3B1 mutations have been extensively characterized, the role of "non-mutated" wild-type SF3B1 in cancer remains largely unresolved. Here, we identify a conserved epitranscriptomic program that steers SF3B1 levels to counteract leukemogenesis. Our analysis of human and murine pre-leukemic MDS cells reveals dynamic regulation of SF3B1 protein abundance, which affects MDS-to-leukemia progression in vivo. Mechanistically, ALKBH5-driven 5' UTR m 6A demethylation fine-tunes SF3B1 translation directing splicing of central DNA repair and epigenetic regulators during transformation. This impacts genome stability and leukemia progression in vivo, supporting an integrative analysis in humans that SF3B1 molecular signatures may predict mutational variability and poor prognosis. These findings highlight a post-transcriptional gene expression nexus that unveils unanticipated SF3B1-dependent cancer vulnerabilities.

Splicing is a central RNA-based process commonly altered in human cancers; however, how spliceosomal components are co-opted during tumorigenesis remains poorly defined. Here we unravel the core splice factor SF3A3 at the nexus of a translation-based program that rewires splicing during malignant transformation. Upon MYC hyperactivation, SF3A3 levels are modulated translationally through an RNA stem-loop in an eIF3D-dependent manner. This ensures accurate splicing of mRNAs enriched for mitochondrial regulators. Altered SF3A3 translation leads to metabolic reprogramming and stem-like properties that fuel MYC tumorigenic potential in vivo. Our analysis reveals that SF3A3 protein levels predict molecular and phenotypic features of aggressive human breast cancers. These findings unveil a post-transcriptional interplay between splicing and translation that governs critical facets of MYC-driven oncogenesis.

Whilst the survival rates of childhood acute lymphoblastic leukemia (ALL) have increased remarkably over the last decades, the therapy resistance and toxicity are still the major causes of treatment failure. It was shown that overexpression of heme oxygenase-1 (HO-1) promotes proliferation and chemoresistance of cancer cells. In humans, the HO-1 gene (HMOX1) expression is modulated by two polymorphisms in the promoter region: (GT)n-length polymorphism and single-nucleotide polymorphism (SNP) A(&minus, 413)T, with short GT repeat sequences and 413-A variants linked to an increased HO-1 inducibility. We found that the short alleles are significantly more frequent in ALL patients in comparison to the control group, and that their presence may be associated with a higher risk of treatment failure, reflecting the role of HO-1 in chemoresistance. We also observed that the presence of short alleles may predispose to develop chemotherapy-induced neutropenia. In case of SNP, the 413-T variant co-segregated with short or long alleles, while 413-A almost selectively co-segregated with long alleles, hence it is not possible to determine if SNPs are actually of phenotypic significance. Our results suggest that HO-1 can be a potential target to overcome the treatment failure in ALL patients.

Small Cajal body-specific RNAs (scaRNAs) guide post-transcriptional modification of spliceosomal RNA and, while commonly altered in cancer, have poorly defined roles in tumorigenesis. Here, we uncover that SCARNA15 directs alternative splicing (AS) and stress adaptation in cancer cells. Specifically, we find that SCARNA15 guides critical pseudouridylation (Ψ) of U2 spliceosomal RNA to fine-tune AS of distinct transcripts enriched for chromatin and transcriptional regulators in malignant cells. This critically impacts the expression and function of the key tumor suppressors ATRX and p53. Significantly, SCARNA15 loss impairs p53-mediated redox homeostasis and hampers cancer cell survival, motility and anchorage-independent growth. In sum, these findings highlight an unanticipated role for SCARNA15 and Ψ in directing cancer-associated splicing programs., Graphical Abstract Graphical AbstractOverexpression of SCARNA15 is associated with MYC alterations in multiple cancer subtypes. Mechanistically, SCARNA15-guided U2 pseudouridylation (Ψ39) directs distinct alternative splicing events to impact cancer cell anchorage independence, motility and survival upon oxidative stress.

Whilst the survival rates of childhood acute lymphoblastic leukemia (ALL) have increased remarkably over the last decades, the therapy resistance and toxicity are still the major causes of treatment failure. It was shown that overexpression of heme oxygenase-1 (HO-1) promotes proliferation and chemoresistance of cancer cells. In humans, the HO-1 gene (HMOX1) expression is modulated by two polymorphisms in the promoter region: (GT)n-length polymorphism and single-nucleotide polymorphism (SNP) A(−413)T, with short GT repeat sequences and 413-A variants linked to an increased HO-1 inducibility. We found that the short alleles are significantly more frequent in ALL patients in comparison to the control group, and that their presence may be associated with a higher risk of treatment failure, reflecting the role of HO-1 in chemoresistance. We also observed that the presence of short alleles may predispose to develop chemotherapy-induced neutropenia. In case of SNP, the 413-T variant co-segregated with short or long alleles, while 413-A almost selectively co-segregated with long alleles, hence it is not possible to determine if SNPs are actually of phenotypic significance. Our results suggest that HO-1 can be a potential target to overcome the treatment failure in ALL patients.

Transfer RNA-derived fragments (tRFs) are emerging small noncoding RNAs that, although commonly altered in cancer, have poorly defined roles in tumorigenesis1. Here we show that pseudouridylation (Ψ) of a stem cell-enriched tRF subtype2, mini tRFs containing a 5′ terminal oligoguanine (mTOG), selectively inhibits aberrant protein synthesis programmes, thereby promoting engraftment and differentiation of haematopoietic stem and progenitor cells (HSPCs) in patients with myelodysplastic syndrome (MDS). Building on evidence that mTOG-Ψ targets polyadenylate-binding protein cytoplasmic 1 (PABPC1), we employed isotope exchange proteomics to reveal critical interactions between mTOG and functional RNA-recognition motif (RRM) domains of PABPC1. Mechanistically, this hinders the recruitment of translational co-activator PABPC1-interacting protein 1 (PAIP1)3 and strongly represses the translation of transcripts sharing pyrimidine-enriched sequences (PES) at the 5′ untranslated region (UTR), including 5′ terminal oligopyrimidine tracts (TOP) that encode protein machinery components and are frequently altered in cancer4. Significantly, mTOG dysregulation leads to aberrantly increased translation of 5′ PES messenger RNA (mRNA) in malignant MDS-HSPCs and is clinically associated with leukaemic transformation and reduced patient survival. These findings define a critical role for tRFs and Ψ in difficult-to-treat subsets of MDS characterized by high risk of progression to acute myeloid leukaemia (AML).

The severity of Duchenne muscular dystrophy (DMD), an incurable disease caused by the lack of dystrophin, might be modulated by different factors, including miRNAs. Among them, miR-378 is considered of high importance for muscle biology, but intriguingly, its role in DMD and its murine model (mdx mice) has not been thoroughly addressed so far. Here, we demonstrate that dystrophic mice additionally globally lacking miR-378 (double-KO [dKO] animals) exhibited better physical performance and improved absolute muscle force compared with mdx mice. Accordingly, markers of muscle damage in serum were significantly decreased in dKO mice, accompanied by diminished inflammation, fibrosis, and reduced abundance of regenerating fibers within muscles. The lack of miR-378 also normalized the aggravated fusion of dystrophin-deficient muscle satellite cells (mSCs). RNA sequencing of gastrocnemius muscle transcriptome revealed fibroblast growth factor 1 (Fgf1) as one of the most significantly downregulated genes in mice devoid of miR-378, indicating FGF1 as one of the mediators of changes driven by the lack of miR-378. In conclusion, we suggest that targeting miR-378 has the potential to ameliorate DMD pathology., miR-378 deficiency improves the dystrophic phenotype in mdx mice, suggesting that modulation of miR-378 level may be a potential approach to attenuate DMD severity.

RNA based processes are in the limelight due to the current technological advances from the fields of high throughput sequencing, structural probing and super-high resolution imaging. RNA biology is implicated in wide range of processes including embryonal development, maintenance of tissue homeostasis or tumor development and progression. We now gain insights at the unprecedented level on how RNAs are modified epitranscriptionally, what are elements regulating translation, how they are dynamically folded, processed to shorter fragments or spliced to elicit tumorigenic effects. Herein, we discuss the implications of the regulation of different RNA species including messenger, non-coding, transfer and ribosomal RNAs and their downstream effector pathways for regulation of cancerogenesis.

Divalent copper and iron cations have been acknowledged for their catalytic roles in physiological processes critical for homeostasis maintenance. Being redox-active, these metals act as cofactors in the enzymatic reactions of electron transfer. However, under pathophysiological conditions, owing to their high redox potentials, they may exacerbate stress-induced injury. This could be particularly hazardous to the liver - the main body reservoir of these two metals. Surprisingly, the involvement of Cu and Fe in liver pathology still remains poorly understood. Hypoxic stress in the tissue may act as a stimulus that mobilizes these ions from their hepatic stores, aggravating the systemic injury. Since ischemia poses a serious complication in liver surgery (e.g. transplantation) we aimed to reveal the status of Cu and Fe via spectroscopic analysis of mouse ischemic liver tissue. Herein, we establish a novel non-surgical model of focal liver ischemia, achieved by applying light locally when a photosensitizer is administered systemically. Photodynamic treatment results in clear-cut areas of the ischemic hepatic tissue, as confirmed by ultrasound scans, mean velocity measurements, 3D modelling of vasculature and (immuno)histological analysis. For reference, we assessed the samples collected from the animals which developed transient systemic endotoxemic stress induced by a non-lethal dose of lipopolysaccharide. The electron paramagnetic resonance (EPR) spectra recorded in situ in the liver samples reveal a dramatic increase in the level of Cu adducts solely in the ischemic tissues. In contrast, other typical free radical components of the liver EPR spectra, such as reduced Riske clusters are not detected; these differences are not followed by changes in the blood EPR spectra. Taken together, our results suggest that local ischemic stress affects paramagnetic species containing redox-active metals. Moreover, because in our model hepatic vascular flow is impaired, these effects are only local (confined to the liver) and are not propagated systemically., Graphical abstract Image 1, Highlights • Liver ischemia causes local dyshomeostasis in redox-active transition metal ions. • Metal ion-reactive species interaction exacerbates injury of the hepatic tissue. • Copper chelation could aid the removal of reactive species.

Granulocyte colony‐stimulating factor (G‐CSF) is used in clinical practice to mobilize cells from the bone marrow to the blood; however, it is not always effective. We show that cobalt protoporphyrin IX (CoPP) increases plasma concentrations of G‐CSF, IL‐6, and MCP‐1 in mice, triggering the mobilization of granulocytes and hematopoietic stem and progenitor cells (HSPC). Compared with recombinant G‐CSF, CoPP mobilizes higher number of HSPC and mature granulocytes. In contrast to G‐CSF, CoPP does not increase the number of circulating T cells. Transplantation of CoPP‐mobilized peripheral blood mononuclear cells (PBMC) results in higher chimerism and faster hematopoietic reconstitution than transplantation of PBMC mobilized by G‐CSF. Although CoPP is used to activate Nrf2/HO‐1 axis, the observed effects are Nrf2/HO‐1 independent. Concluding, CoPP increases expression of mobilization‐related cytokines and has superior mobilizing efficiency compared with recombinant G‐CSF. This observation could lead to the development of new strategies for the treatment of neutropenia and HSPC transplantation., Recombinant G‐CSF is the mobilizing factor used for treating neutropenia and prior to harvesting hematopoietic stem cells for transplantation. This article describes cobalt protoporphyrin IX as a new efficient mobilizing factor upstream of G‐CSF.

Study design A cross-sectional study of 2 groups of patients with scoliosis, and an age-matched control group was conducted. Each of the groups such as patients with adolescent idiopathic scoliosis (AIS) as well as control group were divided additionally into 2 groups: premenarcheal and postmenarcheal girls. Objective The aim of the study was to determine the levels of 25-OH-vitamin D3, calcium and phosphate, parathyroid hormone (PTH), and calcitonin in serum of pre- and postmenarcheal girls with AIS and corresponding groups of scoliosis-free controls. Summary of background data The primary etiology and pathogenesis of AIS remains unknown. It is assumed that vitamin D deficiency and genetic predisposition, for example, polymorphisms of vitamin D receptor, have a great significance. Vitamin D plays a key role in skeletal development and prevents bone atrophy, affects the absorption of calcium, maintains calcium-phosphate homeostasis, and the bone matrix mineralization. Its deficiency can result in a wide variety of skeletal deformities, low bone mass, and then leads to the disappearance of bone. Defects in trabecular bone structure and/or bone mineralization are the main features of scoliosis. Some studies have reported that Vitamin D deficiency is common among patients with AIS. The mechanism of Vitamin D action on scoliosis development is still unclear. Methods Determination of serum 25-OH-D3 levels was performed using high-performance liquid chromatography chromatography; concentrations of calcium and phosphate were measured using colorimetric methods, and concentration of PTH and calcitonin was measured using ELISA system. Results Reduction in the serum levels of 25-OH-D3 and calcitonin in girls with AIS compared with healthy girls was demonstrated. Conclusion The phosphate-calcium balance and PTH level seem to be normal in patients with AIS. The calcitonin level in girls with AIS is 2-fold lower than in healthy subjects. It is possible that the deficiency of vitamin D can be involved in AIS. Level of evidence 4.

Genomic instability contributes to the development and expansion of acute myeloid leukemia (AML). Therefore, identification of factors pivotal for the genome integrity is essential for basic science and translational medicine. We propose that interplay between G-quadruplexes (G4) and heme oxygenase-1 (HO-1) affects the response to DNA damage and switch from normal to malignant hematopoiesis. AML is associated with chromosomal translocations as well as single point mutations. The risk of mutagenesis is increased by guanine-rich DNA sequences, folding into G-quadruplexes (G4), which can abrogate the replication. In inorganic solutions G4 are stabilized by heme. Whether such stabilization occurs in the living cells and how it affects mutagenesis' rate have not been tested so far. Cellular heme is removed by heme oxygenase-1 (HO-1), which evokes differential effects depending on subcellular localization. Here we assessed the impact of HO-1 in conjunction with G4 on genomic instability of AML. To address the function of HO-1 in leukemia we established the MOLM13 cell line engineered to knock-down (KD) HO-1. Rescue studies were performed with wild type (WT) and nuclear export signal (NES)-containing HO-1 constructs. Ex vivo assays were done with murine and human bone marrow cells. Cellular localization of HO-1 and G4 was determined by immunofluorescence in hematopoietic stem and progenitor cells (HSPCs). Co-localization of HO-1 and G4 was further assessed by proximity ligation assay (PLA). DNA damage and viability were determined at the steady state and in response to doxorubicin. Finally, association of HO-1 expression with mutational status and progression of AML was evaluated in cohort of 80 control and AML patients. We demonstrated that in HSPCs HO-1 localizes mainly in the nucleus. In stem cells its expression is low and increases upon differentiation, with the highest amount in the myeloid progenitors. Immunostaining confirmed the presence of G4, relatively scarce in stem cells but more numerous in granulocyte-macrophage progenitors. Importantly, we found for the first time that HO-1 co-localizes with G4 and that it can regulate the abundance of DNA damage at the steady state, with HO-1 KD resulting in increased phosphorylation of histone H2AX, marker of double strand DNA breaks. Strikingly, doxorubicin treatment increased not only DNA damage but also expression of HO-1. Accordingly, in AML patients we found a correlation between lower HO-1 expression, higher incidence of FLT3 mutations and the dismal prognosis. HO-1 protein is present in the nucleus of hematopoietic and leukemic cells, where it can co-localize with G-quadruplexes and evoke important functional effects. HO-1 expression is associated with clinical outcome of AML and with FLT3 mutation rate. Citation Format: Anna Konturek-Ciesla, Anette Radziszewska, Maciej Cieśla, Alicja Czmoczek, Monika Zukowska, Jozef Dulak, Alicja Jozkowicz, Karolina Bukowska-Strakova. Charting the landscape of genomic instability in acute myeloid leukemia: Interaction between G-quadruplexes and heme oxygenase-1 in leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2824.

Summary Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ "writer" PUS7 modifies and activates a novel network of tRNA-derived small fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translation regulation, leading to increased protein biosynthesis and defective germ layer specification. Remarkably, dysregulation of this posttranscriptional regulatory circuitry impairs hematopoietic stem cell commitment and is common to aggressive subtypes of human myelodysplastic syndromes. Our findings unveil a critical function of Ψ in directing translation control in stem cells with important implications for development and disease.

Rhabdomyosarcoma is the most common type of soft tissue sarcoma in children. Two main subtypes of rhabdomyosarcoma with different molecular pattern and distinct clinical behaviour may be identified - embryonal and alveolar rhabdomyosarcoma. All types of rhabdomyosarcoma are believed to be of myogenic origin as they express high levels of myogenesis-related factors. They all, however, fail to undergo a terminal differentiation which results in tumour formation. In the aberrant regulation of myogenesis in rhabdomyosarcoma, microRNAs and epigenetic factors are particularly involved. Indeed, these mediators seem to be even more significant for the development of rhabdomyosarcoma than canonical myogenic transcription factors like MyoD, a master regulatory switch for myogenesis. Therefore, in this review we focus on the regulation of rhabdomyosarcoma progression by microRNAs, and especially on microRNAs of the myo-miRNAs family (miR-1, -133a/b and -206), other well-known myogenic regulators like miR-29, and on microRNAs recently recognized to play a role in the differentiation of rhabdomyosarcoma, such as miR-450b-5p or miR-203. We also review changes in epigenetic modifiers associated with rhabdomyosarcoma, namely histone deacetylases and methyltransferases, especially from the Polycomp Group, like Yin Yang1 and Enhancer of Zeste Homolog2. Finally, we summarize how the functioning of these molecules can be affected by oxidative stress and how antioxidative enzymes can influence the development of this tumour. This article is part of a Directed Issue entitled: Rare Cancers.