XRCC4 and MRE11 Roles and Transcriptional Response to Repair of TALEN-Induced Double-Strand DNA Breaks

Double-strand breaks (DSB) are one of the most lethal forms of DNA damage that, if left unrepaired, can lead to genomic instability, cellular transformation, and cell death. In this work, we examined how repair of transcription activator-like effector nuclease (TALEN)-induced DNA damage was altered when knocking out, or inhibiting a function of, two DNA repair proteins, XRCC4 and MRE11, respectively. We developed a fluorescent reporter assay that uses TALENs to introduce DSB and detected repair by the presence of GFP fluorescence. We observed repair of TALEN-induced breaks in the XRCC4 knockout cells treated with mirin (a pharmacological inhibitor of MRE11 exonuclease activity), albeit with ~40% reduced efficiency compared to normal cells. Editing in the absence of XRCC4 or MRE11 exonuclease was robust, with little difference between the indel profiles amongst any of the groups. Reviewing the transcriptional profiles of the mirin-treated XRCC4 knockout cells showed 307 uniquely differentially expressed genes, a number far greater than for either of the other cell lines (the HeLa XRCC4 knockout sample had 83 genes, and the mirin-treated HeLa cells had 30 genes uniquely differentially expressed). Pathways unique to the XRCC4 knockout+mirin group included differential expression of p53 downstream pathways, and metabolic pathways indicating cell adaptation for energy regulation and stress response. In conclusion, our study showed that TALEN-induced DSBs are repaired, even when a key DSB repair protein or protein function is not operational, without a change in indel profiles. However, transcriptional profiles indicate the induction of unique cellular responses dependent upon the DNA repair protein(s) hampered.

A novel imatinib-upregulated long noncoding RNA plays a critical role in inhibition of tumor growth induced by Abl oncogenes

Background Dysregulation of long noncoding RNAs (lncRNAs) has been linked to various human cancers. Bcr-Abl oncogene that results from a reciprocal translocation between human chromosome 9 and 22, is associated with several hematological malignancies. However, the role of lncRNAs in Bcr-Abl-induced leukemia remains largely unexplored. Methods LncRNA cDNA microarray was employed to identify key lncRNAs involved in Bcr-Abl-mediated cellular transformation. Abl-transformed cell survival and xenografted tumor growth in mice were evaluated to dissect the role of imatinib-upregulated lncRNA 1 (IUR1) in Abl-induced tumorigenesis. Primary bone marrow transformation and in vivo leukemia transplant using lncRNA-IUR1 knockout (KO) mice were further conducted to address the functional relevance of lncRNA-IUR1 in Abl-mediated leukemia. Transcriptome RNA-seq and Western blotting were performed to determine the mechanisms by which lncRNA-IUR1 regulates Bcr-Abl-induced tumorigenesis. Results We identified lncRNA-IUR1 as a critical negative regulator of Bcr-Abl-induced tumorigenesis. LncRNA-IUR1 expressed in a very low level in Bcr-Abl-positive cells from chronic myeloid leukemia patients. Interestingly, it was significantly induced in Abl-positive leukemic cells treated by imatinib. Depletion of lncRNA-IUR1 promoted survival of Abl-transformed human leukemic cells in experiments in vitro and xenografted tumor growth in mice, whereas ectopic expression of lncRNA-IUR1 sensitized the cells to apoptosis and suppressed tumor growth. In concert, silencing murine lncRNA-IUR1 in Abl-transformed cells accelerated cell survival and the development of leukemia in mice. Furthermore, lncRNA-IUR1 deficient mice were generated, and we observed that knockout of murine lncRNA-IUR1 facilitated Bcr-Abl-mediated primary bone marrow transformation. Moreover, animal leukemia model revealed that lncRNA-IUR1 deficiency promoted Abl-transformed cell survival and development of leukemia in mice. Mechanistically, we demonstrated that lncRNA-IUR1 suppressed Bcr-Abl-induced tumorigenesis through negatively regulating STAT5-mediated GATA3 expression. Conclusions These findings unveil an inhibitory role of lncRNA-IUR1 in Abl-mediated cellular transformation, and provide new insights into molecular mechanisms underlying Abl-induced leukemogenesis.

GRWD1-WDR5-MLL2 Epigenetic Complex Mediates H3K4me3 Mark and Is Essential for Kaposi’s Sarcoma-Associated Herpesvirus-Induced Cellular Transformation

By performing a genome-wide CRISPR-Cas9 screening, we have identified cellular epigenetic regulators that are essential for KSHV-induced cellular transformation. Among them, GRWD1 regulates epigenetic active mark H3K4me3 by interacting with WDR5 and MLL2 and recruiting them to chromatin loci of specific genes in KSHV-transformed cells.

Metabonomics study of the effects of single copy mutant KRAS in the presence or absence of WT allele using human HCT116 isogenic cell lines

Introduction KRAS was one of the earliest human oncogenes to be described and is one of the most commonly mutated genes in different human cancers, including colorectal cancer. Despite KRAS mutants being known driver mutations, KRAS has proved difficult to target therapeutically, necessitating a comprehensive understanding of the molecular mechanisms underlying KRAS-driven cellular transformation. Objectives To investigate the metabolic signatures associated with single copy mutant KRAS in isogenic human colorectal cancer cells and to determine what metabolic pathways are affected. Methods Using NMR-based metabonomics, we compared wildtype (WT)-KRAS and mutant KRAS effects on cancer cell metabolism using metabolic profiling of the parental KRASG13D/+ HCT116 cell line and its isogenic, derivative cell lines KRAS+/– and KRASG13D/–. Results Mutation in the KRAS oncogene leads to a general metabolic remodelling to sustain growth and counter stress, including alterations in the metabolism of amino acids and enhanced glutathione biosynthesis. Additionally, we show that KRASG13D/+ and KRASG13D/− cells have a distinct metabolic profile characterized by dysregulation of TCA cycle, up-regulation of glycolysis and glutathione metabolism pathway as well as increased glutamine uptake and acetate utilization. Conclusions Our study showed the effect of a single point mutation in one KRAS allele and KRAS allele loss in an isogenic genetic background, hence avoiding confounding genetic factors. Metabolic differences among different KRAS mutations might play a role in their different responses to anticancer treatments and hence could be exploited as novel metabolic vulnerabilities to develop more effective therapies against oncogenic KRAS. Graphical abstract

Targeting Mitochondrial Protein Expression as a Future Approach for Cancer Therapy

Extensive metabolic remodeling is a fundamental feature of cancer cells. Although early reports attributed such remodeling to a loss of mitochondrial functions, it is now clear that mitochondria play central roles in cancer development and progression, from energy production to synthesis of macromolecules, from redox modulation to regulation of cell death. Biosynthetic pathways are also heavily affected by the metabolic rewiring, with protein synthesis dysregulation at the hearth of cellular transformation. Accumulating evidence in multiple organisms shows that the metabolic functions of mitochondria are tightly connected to protein synthesis, being assembly and activity of respiratory complexes highly dependent on de novo synthesis of their components. In turn, protein synthesis within the organelle is tightly connected with the cytosolic process. This implies an entire network of interactions and fine-tuned regulations that build up a completely under-estimated level of complexity. We are now only preliminarily beginning to reconstitute such regulatory level in human cells, and to perceive its role in diseases. Indeed, disruption or alterations of these connections trigger conditions of proteotoxic and energetic stress that could be potentially exploited for therapeutic purposes. In this review, we summarize the available literature on the coordinated regulation of mitochondrial and cytosolic mRNA translation, and their effects on the integrity of the mitochondrial proteome and functions. Finally, we highlight the potential held by this topic for future research directions and for the development of innovative therapeutic approaches.

K15 promoter-driven enforced expression of NKIRAS exhibits tumor suppressive activity against the development of DMBA/TPA-induced skin tumors

NKIRAS1 and NKIRAS2 (also called as κB-Ras) were identified as members of the atypical RAS family that suppress the transcription factor NF-κB. However, their function in carcinogenesis is still controversial. To clarify how NKIRAS acts on cellular transformation, we generated transgenic mice in which NKIRAS2 was forcibly expressed using a cytokeratin 15 (K15) promoter, which is mainly activated in follicle bulge cells. The ectopic expression of NKIRAS2 was mainly detected in follicle bulges of transgenic mice with NKIRAS2 but not in wild type mice. K15 promoter-driven expression of NKIRAS2 failed to affect the development of epidermis, which was evaluated using the expression of K10, K14, K15 and filaggrin. However, K15 promoter-driven expression of NKIRAS2 effectively suppressed the development of skin tumors induced by treatment with 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA). This observation suggested that NKIRAS seemed to function as a tumor suppressor in follicle bulges. However, in the case of oncogenic HRAS-driven cellular transformation of murine fibroblasts, knockdown of NKIRAS2 expression drastically suppressed HRAS-mutant-provoked cellular transformation, suggesting that NKIRAS2 was required for the cellular transformation of murine fibroblasts. Furthermore, moderate enforced expression of NKIRAS2 augmented oncogenic HRAS-provoked cellular transformation, whereas an excess NKIRAS2 expression converted its functional role into a tumor suppressive phenotype, suggesting that NKIRAS seemed to exhibit a biphasic bell-shaped enhancing effect on HRAS-mutant-provoked oncogenic activity. Taken together, the functional role of NKIRAS in carcinogenesis is most likely determined by not only cellular context but also its expression level.

At a Crossroads to Cancer: How p53-Induced Cell Fate Decisions Secure Genome Integrity

P53 is known as the most critical tumor suppressor and is often referred to as the guardian of our genome. More than 40 years after its discovery, we are still struggling to understand all molecular details on how this transcription factor prevents oncogenesis or how to leverage current knowledge about its function to improve cancer treatment. Multiple cues, including DNA-damage or mitotic errors, can lead to the stabilization and nuclear translocation of p53, initiating the expression of multiple target genes. These transcriptional programs may be cell-type- and stimulus-specific, as is their outcome that ultimately imposes a barrier to cellular transformation. Cell cycle arrest and cell death are two well-studied consequences of p53 activation, but, while being considered critical, they do not fully explain the consequences of p53 loss-of-function phenotypes in cancer. Here, we discuss how mitotic errors alert the p53 network and give an overview of multiple ways that p53 can trigger cell death. We argue that a comparative analysis of different types of p53 responses, elicited by different triggers in a time-resolved manner in well-defined model systems, is critical to understand the cell-type-specific cell fate induced by p53 upon its activation in order to resolve the remaining mystery of its tumor-suppressive function.

Microbiota and Colorectal Cancer: From Gut to Bedside

Colorectal cancer (CRC) is a complex condition with heterogeneous aetiology, caused by a combination of various environmental, genetic, and epigenetic factors. The presence of a homeostatic gut microbiota is critical to maintaining host homeostasis and determines the delicate boundary between health and disease. The gut microbiota has been identified as a key environmental player in the pathogenesis of CRC. Perturbations of the gut microbiota structure (loss of equilibrium and homeostasis) are associated with several intestinal diseases including cancer. Such dysbiosis encompasses the loss of beneficial microorganisms, outgrowth of pathogens and pathobionts and a general loss of local microbiota diversity and richness. Notably, several mechanisms have recently been identified how bacteria induce cellular transformation and promote tumour progression. In particular, the formation of biofilms, the production of toxic metabolites or the secretion of genotoxins that lead to DNA damage in intestinal epithelial cells are newly discovered processes by which the microbiota can initiate tumour formation. The gut microbiota has also been implicated in the metabolism of therapeutic drugs (conventional chemotherapy) as well as in the modulation of radiotherapy responses and targeted immunotherapy. These new findings suggest that the efficacy of a given therapy depends on the composition of the host’s gut microbiota and may therefore vary from patient to patient. In this review we discuss the role of host-microbiota interactions in cancer with a focus on CRC pathogenesis. Additionally, we show how gut bacteria can be exploited in current therapies and how mechanisms directed by microbiota, such as immune cell boost, probiotics and oncolytic bacteria, can be applied in the development of novel therapies.

MAPK15 Controls Hedgehog Signaling in Medulloblastoma Cells by Regulating Primary Ciliogenesis

In medulloblastomas, genetic alterations resulting in over-activation and/or deregulation of proteins involved in Hedgehog (HH) signaling lead to cellular transformation, which can be prevented by inhibition of primary ciliogenesis. Here, we investigated the role of MAPK15 in HH signaling and, in turn, in HH-mediated cellular transformation. We first demonstrated, in NIH3T3 mouse fibroblasts, the ability of this kinase of controlling primary ciliogenesis and canonical HH signaling. Next, we took advantage of transformed human medulloblastoma cells belonging to the SHH-driven subtype, i.e., DAOY and ONS-76 cells, to ascertain the role for MAPK15 in HH-mediated cellular transformation. Specifically, medullo-spheres derived from these cells, an established in vitro model for evaluating progression and malignancy of putative tumor-initiating medulloblastoma cells, were used to demonstrate that MAPK15 regulates self-renewal of these cancer stem cell-like cells. Interestingly, by using the HH-related oncogenes SMO-M2 and GLI2-DN, we provided evidences that disruption of MAPK15 signaling inhibits oncogenic HH overactivation in a specific cilia-dependent fashion. Ultimately, we show that pharmacological inhibition of MAPK15 prevents cell proliferation of SHH-driven medulloblastoma cells, overall suggesting that oncogenic HH signaling can be counteracted by targeting the ciliary gene MAPK15, which could therefore be considered a promising target for innovative “smart” therapies in medulloblastomas.