Cancer stem-like cells represent poorly differentiated multipotent tumor-propagating cells that contribute disproportionately to therapeutic resistance and tumor recurrence. Transcriptional mechanisms that control the phenotypic conversion of tumor cells lacking tumor-propagating potential to tumor-propagating stem-like cells remain obscure. Here we show that the reprogramming transcription factors Oct4 and Sox2 induce glioblastoma cells to become stem-like and tumor–propagating via a mechanism involving direct DNA methyl transferase (DNMT) promoter transactivation, resulting in global DNA methylation- and DNMT-dependent downregulation of multiple microRNAs (miRNAs). We show that one such downregulated miRNA, miRNA-148a, inhibits glioblastoma cell stem-like properties and tumor-propagating potential. This study identifies a novel and targetable molecular circuit by which glioma cell stemness and tumor-propagating capacity are regulated.

Despite our growing molecular-level understanding of glioblastoma (GBM), treatment modalities remain limited. Recent developments in the mechanisms of cell fate regulation and nanomedicine provide new avenues by which to treat and manage brain tumors via the delivery of molecular therapeutics. Here, we have developed bioreducible poly(β-amino ester) nanoparticles that demonstrate high intracellular delivery efficacy, low cytotoxicity, escape from endosomes, and promotion of cytosol-targeted environmentally triggered cargo release for miRNA delivery to tumor-propagating human cancer stem cells. In this report, we combined this nanobiotechnology with newly discovered cancer stem cell inhibiting miRNAs to develop self-assembled miRNA-containing polymeric nanoparticles (nano-miRs) to treat gliomas. We show that these nano-miRs effectively intracellularly deliver single and combination miRNA mimics that inhibit the stem cell phenotype of human GBM cells in vitro. Following direct intratumoral infusion, these nano-miRs were found to distribute through the tumors, inhibit the growth of established orthotopic human GBM xenografts, and cooperatively enhance the response to standard-of-care γ radiation. Co-delivery of two miRNAs, miR-148a and miR-296-5p, within the bioreducible nano-miR particles enabled long-term survival from GBM in mice.

Solid malignancies contain subsets of multipotent cells that grow as spheres and efficiently propagate tumors in xenograft models, reflecting a stem-like, self-renewing and tumor-propagating phenotype. These cancer ‘stem cells (SCs)’ have been shown to maintain tumor growth, contribute to resistance and drive tumor recurrence. Cancer cell sternness is dynamically influenced by epigenetic mechanisms and differentially regulated coding and noncoding RNAs. How these mechanisms specifically contribute to the generation and/or maintenance of cancer SCs remains unclear. This study identifies a novel epigenetically regulated circuit that integrates microRNA, chromatin remodeling and the reprogramming transcription factor Sox2 to regulate glioblastoma (GBM)-propagating SCs. We show that miR-296-5p expression is repressed in a DNA methylation-dependent manner under conditions that promote GBM cell stemness and that miR-296-5p inhibits GBM cell stemness and their capacity to self-renew as spheres and propagate glioma xenografts in vivo. We show that the chromatin remodeling protein HMGA1 functions as a downstream effector of these biological responses to miR-296-5p and regulates Sox2 expression, a master driver of cell stemness, by modifying chromatin architecture at the Sox2 promoter. These results show for the first time that miR-296-5p inhibits transcriptional mechanisms that support GBM SCs and identify a miR-296-5p:HMGA1:Sox2 axis as a novel regulator of GBM SCs and candidate pathway for targeting therapies directed at depleting tumors of their tumor-propagating stem cell subsets.

Pure nucleotide precursor pools are a prerequisite for high-fidelity DNA replication and the suppression of mutagenesis and carcinogenesis. ITPases are nucleoside triphosphate pyrophosphatases that clean the precursor pools of the non-canonical triphosphates of inosine and xanthine. The precise role of the human ITPase, encoded by the ITPA gene, is not clearly defined. ITPA is clinically important because a widespread polymorphism, 94C>A, leads to null ITPase activity in erythrocytes and is associated with an adverse reaction to thiopurine drugs. We studied the cellular function of ITPA in HeLa cells using the purine analog 6-N hydroxylaminopurine (HAP), whose triphosphate is also a substrate for ITPA. In this study, we demonstrate that ITPA knockdown sensitizes HeLa cells to HAP-induced DNA breaks and apoptosis. The HAP-induced DNA damage and cytotoxicity observed in ITPA knockdown cells are rescued by an overexpression of the yeast ITPase encoded by the HAM1 gene. We further show that ITPA knockdown results in elevated mutagenesis in response to HAP treatment. Our studies reveal the significance of ITPA in preventing base analog-induced apoptosis, DNA damage and mutagenesis in human cells. This implies that individuals with defective ITPase are predisposed to genome damage by impurities in nucleotide pools, which is drastically augmented by therapy with purine analogs. They are also at an elevated risk for degenerative diseases and cancer.

Glioblastoma multiforme (GBM) and other solid malignancies are heterogeneous and contain subpopulations of tumor cells that exhibit stem-like features. Our recent findings point to a de-differentiation mechanism by which reprogramming transcription factors Oct4 and Sox2 drive the stem-like phenotype in glioblastoma, in part by differentially regulating subsets of microRNAs (miRNAs). Currently, the molecular mechanisms by which reprogramming transcription factors and miRNAs coordinate CSC tumor-propagating capacity are unclear. In this study, we identified miR-486-5p as a Sox2-induced miRNA that targets the tumor suppressor genes PTEN and FoxO1 and regulates the GBM stem-like cells. miR-486-5p associated with the GBM stem cell phenotype and Sox2 expression and was directly induced by Sox2 in glioma cell lines and patient-derived neurospheres. Forced expression of miR-486-5p enhanced the self-renewal capacity of GBM neurospheres, and inhibition of endogenous miR-486-5p activated PTEN and FoxO1 and induced cell death by upregulating pro-apoptotic protein BIM via a PTEN-dependent mechanism. Furthermore, delivery of miR-486-5p antagomirs to pre-established orthotopic GBM neurosphere-derived xenografts using advanced nanoparticle formulations reduced tumor sizes in vivo and enhanced the cytotoxic response to ionizing radiation. These results define a previously unrecognized and therapeutically targetable Sox2/miR-486-5p axis that enhances the survival of GBM stem cells by repressing tumor suppressor pathways. SIGNIFICANCE:This study identifies a novel axis that links core transcriptional drivers of cancer cell stemness to miR-486-5p-dependent modulation of tumor suppressor genes that feeds back to regulate glioma stem cell survival.Research.

Glioblastomas (GBM) are highly infiltrated by myeloidderived innate immune cells that contribute to the immunosuppressive nature of the brain tumor microenvironment (TME). CD47 has been shown to mediate immune evasion, as the CD47-SIRPa axis prevents phagocytosis of tumor cells by macrophages and other myeloid cells. In this study, we established CD47 homozygous deletion (CD47 À/À ) in human and mouse GBM cells and investigated the impact of eliminating the "don't eat me" signal on tumor growth and tumor-TME interactions. CD47 knockout (KO) did not significantly alter tumor cell proliferation in vitro but significantly increased phagocytosis of tumor cells by macrophages in cocultures. Compared with CD47 wild-type xenografts, orthotopic xenografts derived from CD47 À/À tumor cells grew significantly slower with enhanced tumor cell phagocytosis and increased recruitment of M2-like tumor-associated microglia/macrophages (TAM). CD47 KO increased tumor-associated extracellular matrix protein tenascin C (TNC) in xeno-grafts, which was further examined in vitro. CD47 loss of function upregulated TNC expression in tumor cells via a Notch pathway-mediated mechanism. Depletion of TNC in tumor cells enhanced the growth of CD47 À/À xenografts in vivo and decreased the number of TAM. TNC knockdown also inhibited phagocytosis of CD47 À/À tumor cells in cocultures. Furthermore, TNC stimulated release of proinflammatory factors including TNFa via a Toll-like receptor 4 and STAT3dependent mechanism in human macrophage cells. These results reveal a vital role for TNC in immunomodulation in brain tumor biology and demonstrate the prominence of the TME extracellular matrix in affecting the antitumor function of brain innate immune cells.Significance: These findings link TNC to CD47-driven phagocytosis and demonstrate that TNC affects the antitumor function of brain TAM, facilitating the development of novel innate immune system-based therapies for brain tumors.

Recent studies have shown that loss of TET1 may play a significant role in the formation of tumors. Because genomic instability is a hallmark of cancer, we examined the potential involvement of 10-11 translocation 1 (TET1) in the DNA damage response (DDR). Here we demonstrate that, in response to clinically relevant doses of ionizing radiation (IR), human glial cells made TET1-deficient with lentiviral vectors displayed greater numbers of colony forming units and lower levels of apoptotic markers compared with glial cells transduced with control vectors; yet, they harbored greater DNA strand breaks. The G/M check point and expression of cyclin B1 were greatly diminished in TET1-deficient cells, and TET1-deficient cells displayed lower levels of γH2A.x following exposure to IR. Levels of DNA-PKcs, which are DNA-PK complex members, were lower in TET1-deficient cells compared with control cell lines. However, levels of ATM were similar in both cell lines. Cyclin B1, DNA-PKcs, and γH2A.x levels were each rescued by reintroduction of the TET1 catalytic domain. Finally, cytosine methylation within intron 1 of PRKDC, the gene encoding DNA-PKcs, was significantly higher upon depletion of TET1. Taken together, this study illustrates the involvement of TET1 in the different arms of the DDR and suggests its loss results in the continued survival of cells with genomic instability.

DNA methylation is a reversible process catalyzed by the ten–eleven translocation (TET) family of enzymes (TET1, TET2, TET3) that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Altered patterns of 5hmC and 5mC are widely reported in human cancers and loss of 5hmC correlates with poor prognosis. Understanding the mechanisms leading to 5hmC loss and its role in oncogenesis will advance the development of epigenetic-based therapeutics. We show that TET2 loss associates with glioblastoma (GBM) stem cells and correlates with poor survival of GBM patients. We further identify a SOX2:miR-10b-5p:TET2 axis that represses TET2 expression, represses 5hmC, increases 5mC levels, and induces GBM cell stemness and tumor-propagating potential. In vivo delivery of a miR-10b-5p inhibitor that normalizes TET2 expression and 5hmC levels inhibits tumor growth and prolongs survival of animals bearing pre-established orthotopic GBM xenografts. These findings highlight the importance of TET2 and 5hmC loss in Sox2-driven oncogenesis and their potential for therapeutic targeting.