Somatic reprogramming induced by defined transcription factors is a low efficiency process that is enhanced by p53 deficiency 1-5. To date, p21 is the only p53 target shown to contribute to p53 repression of iPSC (induced pluripotent stem cell) generation 1, 3, suggesting additional p53 targets may regulate this process. Here, we demonstrated that mir-34 microRNAs (miRNAs), particularly miR-34a, exhibit p53-dependent induction during reprogramming. mir-34a deficiency in mice significantly increased reprogramming efficiency and kinetics, with miR-34a and p21 cooperatively regulating somatic reprogramming downstream of p53. Unlike p53 deficiency, which enhances reprogramming at the expense of iPSC pluripotency, genetic ablation of mir-34a promoted iPSC generation without compromising self-renewal and differentiation. Suppression of reprogramming by miR-34a was due, at least in part, to repression of pluripotency genes, including Nanog, Sox2 and Mycn (N-Myc). This post-transcriptional gene repression by miR-34a also regulated iPSC differentiation kinetics. miR-34b and c similarly repressed reprogramming; and all three mir-34 miRNAs acted cooperatively in this process. Taken together, our findings identified mir-34 miRNAs as novel p53 targets that play an essential role in restraining somatic reprogramming.

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.

Endoplasmic reticulum (ER)-associated degradation (ERAD) represents a principle quality control mechanism to clear misfolded proteins in the ER; however its physiological significance and the nature of endogenous ERAD substrates remain largely unexplored. Here we discover that IRE1α, the sensor of unfolded protein response (UPR), is a bona fide substrate of the Sel1L-Hrd1 ERAD complex. ERAD-mediated IRE1α degradation occurs under basal conditions in a BiP-dependent manner, requires both intramembrane hydrophilic residues of IRE1α and lectin protein OS9, and is attenuated by ER stress. ERAD deficiency causes IRE1α protein stabilization, accumulation and mild activation both in vitro and in vivo. Although enterocyte-specific Sel1L-knockout mice (Sel1LΔIEC) are viable and appear normal, they are highly susceptible to experimental colitis and inflammation-associated dysbiosis, in an IRE1α-dependent but CHOP-independent manner. Hence, Sel1L-Hrd1 ERAD serves a distinct, essential function in restraint of IRE1α signaling in vivo by managing its protein turnover.

As bona fide p53 transcriptional targets, miR-34 microRNAs (miRNAs) exhibit frequent alterations in many human tumor types and elicit multiple p53 downstream effects upon overexpression. Unexpectedly, miR-34 deletion alone fails to impair multiple p53-mediated tumor suppressor effects in mice, possibly due to the considerable redundancy in the p53 pathway. Here, we demonstrate that miR-34a represses HDM4, a potent negative regulator of p53, creating a positive feedback loop acting on p53. In a Kras-induced mouse lung cancer model, miR-34a deficiency alone does not exhibit a strong oncogenic effect. However, miR-34a deficiency strongly promotes tumorigenesis when p53 is haploinsufficient, suggesting that the defective p53-miR-34 feedback loop can enhance oncogenesis in a specific context. The importance of the p53/miR-34/HDM4 feedback loop is further confirmed by an inverse correlation between miR-34 and full-length HDM4 in human lung adenocarcinomas. In addition, human lung adenocarcinomas generate an elevated level of a short HDM4 isoform through alternative polyadenylation. This short HDM4 isoform lacks miR-34-binding sites in the 39 untranslated region (UTR), thereby evading miR-34 regulation to disable the p53-miR-34 positive feedback. Taken together, our results elucidated the intricate cross-talk between p53 and miR-34 miRNAs and revealed an important tumor suppressor effect generated by this positive feedback loop.

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.