SUMMARY microRNAs regulate developmental cell fate decisions, tissue homeostasis and oncogenesis in distinct ways relative to proteins. Here, we show that the tumor suppressor microRNA miR-34a is a cell fate determinant in early stage dividing colon cancer stem cells (CCSCs). In pair-cell assays, miR34a distributes at high levels in differentiating progeny, while low levels of miR34a demarcate self renewing CCSCs. Moreover, miR34a loss of function and gain of function alters the balance between self-renewal and differentiation both in vitro and in vivo. Mechanistically, miR34a sequesters Notch1 mRNA to generate a sharp threshold response where a bimodal Notch signal specifies the choice between self-renewal versus differentiation. In contrast, the canonical cell fate determinant Numb regulates Notch levels in a continuously graded manner. Taken together, our findings highlight a unique microRNA regulated mechanism that converts noisy input into a toggle switch for robust cell fate decisions in CCSCs.

Cancer metastasis accounts for the majority of cancer-related deaths and remains a clinical challenge. Metastatic cancer cells generally resemble cells of the primary cancer, but they may be influenced by the milieu of the organs they colonize. Here, we show that colorectal cancer cells undergo metabolic reprogramming after they metastasize and colonize the liver, a key metabolic organ. In particular, via GATA6, metastatic cells in the liver upregulate the enzyme aldolase B (ALDOB), which enhances fructose metabolism and provides fuel for major pathways of central carbon metabolism during tumor cell proliferation. Targeting ALDOB or reducing dietary fructose significantly reduces liver metastatic growth but has little effect on the primary tumor. Our findings suggest that metastatic cells can take advantage of reprogrammed metabolism in their new microenvironment, especially in a metabolically active organ such as the liver. Manipulation of involved pathways may affect the course of metastatic growth.

SUMMARY Emerging evidence suggests that microRNAs can initiate asymmetric division, but whether microRNA and protein cell fate determinants coordinate with each other remains unclear. Here we show that miR-34a directly suppresses Numb in early-stage colon cancer stem cells (CCSCs), forming an incoherent feedforward loop (IFFL) targeting Notch to separate stem and non-stem cell fates robustly. Perturbation of the IFFL leads to a new intermediate cell population with plastic and ambiguous identity. Lgr5+ mouse intestinal/colon stem cells (ISCs) predominantly undergo symmetric division, but turn on asymmetric division to curb the number of ISCs when proinflammatory response causes excessive proliferation. Deletion of miR-34a inhibits asymmetric division and exacerbates Lgr5+ ISC proliferation under such stress. Collectively, our data indicate that microRNA and protein cell fate determinants coordinate to enhance robustness of cell fate decision, and they provide a safeguard mechanism against stem cell proliferation induced by inflammation or oncogenic mutation.

S tringent response is the main strategy used by bacteria to cope with fluctuating nutrient supplies and metabolic and oxidative stresses 1,2 . This process rapidly redirects energy from cell proliferation toward stress survival by reduction of biosynthesis, conservation of ATP and blockage of GTP production 3 . The stringent response is triggered by the accumulation of the bacterial 'alarmone' (p)ppGpp (guanosine tetra-or penta-phosphate, shortened as ppGpp below) through the regulation of ppGpp synthetases and hydrolases in the RelA and SpoT homologue family 2 .Recent studies suggest that the stringent response may also function in metazoans, as metazoan genomes encode a homologue of bacterial SpoT-MESH1 (Metazoan SpoT Homologue 1, encoded by HDDC3)-that can hydrolyse ppGpp in vitro and functionally complement SpoT in Escherichia coli 4 . Furthermore, Mesh1 deletion in Drosophila displays impaired starvation resistance and extensive transcriptional reprogramming 4 . Despite these supporting lines of evidence, neither ppGpp nor its synthetase has been discovered in metazoans, thus obscuring the genuine function and the relevant substrate(s) of MESH1 in mammalian cells. Here, we have identified NADPH as an efficient substrate of MESH1. MESH1 is a cytosolic NADPH phosphatase that is induced under stress conditions, leading to the NADPH depletion and ferroptosis-a novel form of iron-dependent regulated cell death characterized by lipid peroxidation 5 . Accordingly, MESH1 removal preserves the NADPH level in stressed cells and promotes their ferroptotic survival.Critical to the bacterial stringent response is the rapid relocation of resources from proliferation toward stress survival through the respective accumulation and degradation of (p)ppGpp by RelA and SpoT homologues. While mammalian genomes encode MESH1, a homologue of the bacterial (p)ppGpp hydrolase SpoT, neither (p)ppGpp nor its synthetase has been identified in mammalian cells. Here, we show that human MESH1 is an efficient cytosolic NADPH phosphatase that facilitates ferroptosis. Visualization of the MESH1-NADPH crystal structure revealed a bona fide affinity for the NADPH substrate. Ferroptosisinducing erastin or cystine deprivation elevates MESH1, whose overexpression depletes NADPH and sensitizes cells to ferroptosis, whereas MESH1 depletion promotes ferroptosis survival by sustaining the levels of NADPH and GSH and by reducing lipid peroxidation. The ferroptotic protection by MESH1 depletion is ablated by suppression of the cytosolic NAD(H) kinase, NADK, but not its mitochondrial counterpart NADK2. Collectively, these data shed light on the importance of cytosolic NADPH levels and their regulation under ferroptosis-inducing conditions in mammalian cells.

As patient survival drops precipitously from early-stage cancers to late-stage and metastatic cancers, microRNAs that promote relapse and metastasis can serve as prognostic and predictive markers as well as therapeutic targets for chemoprevention. Here we show that miR-1269a promotes colorectal cancer (CRC) metastasis and forms a positive feedback loop with TGF-β signaling. miR-1269a is upregulated in late-stage CRCs, and long-term monitoring of 100 stage II CRC patients revealed that miR-1269a expression in their surgically removed primary tumors is strongly associated with risk of CRC relapse and metastasis. Consistent with clinical observations, miR-1269a significantly increases the ability of CRC cells to invade and metastasize in vivo. TGF-β activates miR-1269 via Sox4, while miR-1269a enhances TGF-β signaling by targeting Smad7 and HOXD10, hence forming a positive feedback loop. Our findings suggest that miR-1269a is a potential marker to inform adjuvant chemotherapy decisions for CRC patients and a potential therapeutic target to deter metastasis.