Solid cancer cells commonly enter the blood and disseminate systemically but are highly inefficient at forming distant metastases for poorly understood reasons. We studied human melanomas that differed in their metastasis histories in patients and in their capacity to metastasize in NSG mice. All melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficient metastasizers. Melanoma cells in the blood and visceral organs experienced oxidative stress not observed in established subcutaneous tumours. Successfully metastasizing melanomas underwent reversible metabolic changes during metastasis that increased their capacity to withstand oxidative stress, including increased dependence upon NADPH-generating enzymes in the folate pathway. Anti-oxidants promoted distant metastasis in NSG mice. Folate pathway inhibition using low-dose methotrexate, ALDH1L2 knockdown, or MTHFD1 knockdown inhibited distant metastasis without significantly affecting the growth of subcutaneous tumors in the same mice. Oxidative stress thus limits distant metastasis by melanoma cells in vivo.

The differentiation of tumorigenic cancer stem cells into non-tumorigenic cancer cells confers heterogeneity to some cancers beyond that explained by clonal evolution or environmental differences. In such cancers, functional differences between tumorigenic and non-tumorigenic cells influence response to therapy and prognosis. However, it remains uncertain whether the model applies to many, or few, cancers due to questions about the robustness of cancer stem cell markers and the extent to which existing assays underestimate the frequency of tumorigenic cells. In cancers with rapid genetic change, reversible changes in cell states, or biological variability among patients the stem cell model may not be readily testable.

Lin28A and Lin28B selectively block the expression of let-7 microRNAs and function as oncogenes in a variety of human cancers. Lin28A recruits a TUTase (Zcchc11/TUTase4) to let-7 precursors to block processing by Dicer in the cell cytoplasm. Here we find that unlike Lin28A, Lin28B represses let-7 processing through a TUTase-independent mechanism. Lin28B functions in the nucleus by sequestering primary let-7 transcripts and inhibiting their processing by the Microprocessor. The inhibitory effects of Zcchc11 depletion on the tumorigenic capacity and metastatic potential of human cancer cells and xenografts is restricted to Lin28A-expressing tumors. Furthermore, the majority of human colon and breast tumors analyzed exclusively express either Lin28A or Lin28B. Lin28A is expressed in HER2-overexpressing breast tumors while Lin28B expression characterizes triple-negative breast tumors. Overall our results illuminate the distinct mechanisms by which Lin28A and Lin28B function, and have implications for the development of new strategies for cancer therapy.

Lin28 and Lin28B, two developmentally regulated RNA-binding proteins and proto-oncogenes, selectively inhibit the maturation of let-7 family miRNAs in embryonic stem (ES) cells and certain cancers. Moreover, let-7 precursors (pre-let-7) were previously found to be terminally uridylated in a Lin28-dependent fashion. Here, we identify Zcchc11 (zinc finger, CCHC domain containing 11) as the 3′ terminal uridylyl transferase (TUTase) responsible for Lin28-mediated pre-let-7 uridylation and blockade of let-7 processing in mouse ES cells. We demonstrate that Zcchc11 activity is UTP-dependent, selective for let-7, and recruited by Lin28. Furthermore, knockdown of either Zcchc11 or Lin28, or overexpression of a catalytically inactive TUTase, relieves the selective inhibition of let-7 processing and leads to the accumulation of mature let-7 miRNAs and repression of let-7 target reporter genes. Our results establish a novel role for Zcchc11-catalyzed pre-let-7 uridylation in the control of miRNA biogenesis.

Stem cell fate can be influenced by metabolite levels in culture but it is unknown whether physiological variations in metabolite levels in normal tissues regulate stem cell function in vivo. We developed a metabolomics method for analysis of rare cell populations isolated directly from tissues and used it to compare haematopoietic stem cells (HSCs) to restricted haematopoietic progenitors. Each haematopoietic cell type had a distinct metabolic signature. Human and mouse HSCs had unusually high levels of ascorbate, which declined with differentiation. Systemic ascorbate depletion in mice increased HSC frequency and function, partly by reducing Tet2 function, a dioxygenase tumor suppressor. Ascorbate depletion cooperated with Flt3ITD leukaemic mutations to accelerate leukaemogenesis, though cell-autonomous and possibly non-cell-autonomous mechanisms, in a manner that was reversed by dietary ascorbate. Ascorbate acted cell-autonomously to negatively regulate HSC function and myelopoiesis through Tet2-dependent and Tet2-independent mechanisms. Ascorbate thus accumulates within HSCs to promote Tet function in vivo, limiting HSC frequency and suppressing leukaemogenesis.

The developmentally regulated RNA-binding protein Lin28 blocks processing of let-7 family microRNAs (miRNAs) in embryonic cells. The molecular basis for this selective miRNA processing block is unknown. Here we find that Lin28 selectively binds the terminal loop region of let-7 precursors in vitro and that the loop mediates miRNA processing inhibition in vivo. Additionally, we identify the domains of Lin28 required for this inhibition. These findings establish a regulatory role for the terminal loop of precursors in miRNA maturation and provide insight into the mechanism by which Lin28 negatively regulates let-7 processing. MicroRNAs (miRNAs)2 comprise a large family of short regulatory RNAs that repress the expression of target messenger RNAs and have many important roles in development (1). In addition to the requirement of miRNAs for normal development, it is emerging that altered miRNA expression is a hallmark of various cancers (2). Several examples of miRNAs with oncogenic or tumor suppressor properties have been reported. Notably, let-7 miRNA has been reported to play a tumor suppressor role by repression of oncogenes including Hmga2, Ras,. Reduced expression of let-7 miRNA in human lung cancers is associated with shortened postoperative survival (7), and in a mouse lung cancer model, let-7g inhibits tumor development (8). Additionally, low let-7 expression is important for the self-renewal and tumorigenicity of breast cancer initiating cells (9).Hundreds of miRNAs have now been identified, many of which are expressed in a tissue-and developmental stage-specific manner. Under most conditions, control of their expression occurs at the transcriptional level. The miRNA biogenesis pathway involves the sequential processing of primary miRNA transcripts (pri-miRNAs) by the Microprocessor complex (comprising the RNaseIII enzyme Drosha and the doublestranded RNA-binding protein DGCR8) to release 60 -70-nt precursor miRNAs (pre-miRNAs) that are subsequently cleaved by the Dicer complex to yield mature ϳ22 nt miRNAs (10 -13). Emerging evidence indicates that miRNA biogenesis can also be regulated posttranscriptionally (14 -18).The developmentally regulated RNA-binding protein Lin28 was recently identified as a selective inhibitor of miRNA processing in embryonic stem cells and embryonal carcinoma cells (18). Lin28 inhibits the maturation of the let-7 family but not other miRNAs, yet a mechanistic explanation for this selectivity is unknown. We sought to gain insight into the mechanism by which Lin28 selectively blocks the processing of let-7 family miRNAs. Using in vitro and in vivo assays, we explored the RNA sequence and structural requirements for Lin28-mediated regulation and found that Lin28 specifically binds the terminal loop region of let-7 precursors. Furthermore, we demonstrated that the loop mediates miRNA processing inhibition in vivo and identified the domains of Lin28 required for this inhibition. EXPERIMENTAL PROCEDURESElectromobilty Shift Assays (EMSA)-EMSA was conducted using ϳ2 ϫ 10 5 cpm 5Ј-end-l...

The pluripotency factor Lin28 recruits a 39 terminal uridylyl transferase (TUTase) to selectively block let-7 microRNA biogenesis in undifferentiated cells. Zcchc11 (TUTase4/TUT4) was previously identified as an enzyme responsible for Lin28-mediated prelet-7 uridylation and control of let-7 expression. Here we investigate the protein and RNA determinants for this interaction. Biochemical dissection and reconstitution assays reveal the TUTase domains necessary and sufficient for Lin28-enhanced prelet-7 uridylation. A single C2H2-type zinc finger domain of Zcchc11 was found to be responsible for the functional interaction with Lin28. We identify Zcchc6 (TUTase7) as an alternative TUTase that functions with Lin28 in vitro, and accordingly, we find Zcchc11 and Zcchc6 redundantly control let-7 biogenesis in embryonic stem cells. Our study indicates that Lin28 uses two different TUTases to control let-7 expression and has important implications for stem cell biology as well as cancer.

Reactive oxygen species (ROS) are highly reactive molecules that arise from a number of cellular sources, including oxidative metabolism in mitochondria. At low levels they can be advantageous to cells, activating signaling pathways that promote proliferation or survival. At higher levels, ROS can damage or kill cells by oxidizing proteins, lipids, and nucleic acids. It was hypothesized that antioxidants might benefit high-risk patients by reducing the rate of ROS-induced mutations and delaying cancer initiation. However, dietary supplementation with antioxidants has generally proven ineffective or detrimental in clinical trials. High ROS levels limit cancer cell survival during certain windows of cancer initiation and progression. During these periods, dietary supplementation with antioxidants may promote cancer cell survival and cancer progression. This raises the possibility that rather than treating cancer patients with antioxidants, they should be treated with pro-oxidants that exacerbate oxidative stress or block metabolic adaptations that confer oxidative stress resistance.