miR-203 drives breast cancer cell differentiation.

A hallmark of many malignant tumors is dedifferentiated (immature) cells bearing slight or no resemblance to the normal cells from which the cancer originated. Tumor dedifferentiated cells exhibit a higher capacity to survive to chemo and radiotherapies and have the ability to incite tumor relapse. Inducing cancer cell differentiation would abolish their self-renewal and invasive capacity and could be combined with the current standard of care, especially in poorly differentiated and aggressive tumors (with worst prognosis). However, differentiation therapy is still in its early stages and the intrinsic complexity of solid tumor heterogeneity demands innovative approaches in order to be efficiently translated into the clinic. We demonstrate here that microRNA 203, a potent driver of differentiation in pluripotent stem cells (ESCs and iPSCs), promotes the differentiation of mammary gland tumor cells. Combining mouse in vivo approaches and both mouse and human-derived tridimensional organoid cultures, we report that miR-203 influences the self-renewal capacity, plasticity and differentiation potential of breast cancer cells and prevents tumor cell growth in vivo. Our work sheds light on differentiation-based antitumor therapies and offers miR-203 as a promising tool for directly confronting the tumor-maintaining and regeneration capability of cancer cells.

Mammalian CDC14 phosphatases control exit from stemness in pluripotent cells.

Maintenance of stemness is tightly linked to cell cycle regulation through protein phosphorylation by cyclin-dependent kinases (CDKs). However, how this process is reversed during differentiation is unknown. We report here that exit from stemness and differentiation of pluripotent cells along the neural lineage are controlled by CDC14, a CDK-counteracting phosphatase whose function in mammals remains obscure. Lack of the two CDC14 family members, CDC14A and CDC14B, results in deficient development of the neural system in the mouse and impairs neural differentiation from embryonic stem cells (ESCs). Mechanistically, CDC14 directly dephosphorylates specific proline-directed Ser/Thr residues of undifferentiated embryonic transcription Factor 1 (UTF1) during the exit from stemness, triggering its proteasome-dependent degradation. Multiomic single-cell analysis of transcription and chromatin accessibility in differentiating ESCs suggests that increased UTF1 levels in the absence of CDC14 prevent the proper firing of bivalent promoters required for differentiation. CDC14 phosphatases are dispensable for mitotic exit, suggesting that CDC14 phosphatases have evolved to control stemness rather than cell cycle exit and establish the CDK-CDC14 axis as a critical molecular switch for linking cell cycle regulation and self-renewal.

Transient exposure to miR-203 enhances the differentiation capacity of established pluripotent stem cells.

Full differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) induces a transient expression of 2C markers that later results in expanded differentiation potency to multiple lineages, as well as improved efficiency in tetraploid complementation and human-mouse interspecies chimerism assays. Mechanistically, these effects are at least partially mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasure of DNA methylation. These data support the use of transient exposure to miR-203 as a versatile method to reset the epigenetic memory in PSCs, and improve their effectiveness in regenerative medicine.

Detection of novel fusion-transcripts by RNA-Seq in T-cell lymphoblastic lymphoma.

Fusions transcripts have been proven to be strong drivers for neoplasia-associated mutations, although their incidence in T-cell lymphoblastic lymphoma needs to be determined yet. Using RNA-Seq we have selected 55 fusion transcripts identified by at least two of three detection methods in the same tumour. We confirmed the existence of 24 predicted novel fusions that had not been described in cancer or normal tissues yet, indicating the accuracy of the prediction. Of note, one of them involves the proto oncogene TAL1. Other confirmed fusions could explain the overexpression of driver genes such as COMMD3-BMI1, LMO1 or JAK3. Five fusions found exclusively in tumour samples could be considered pathogenic (NFYG-TAL1, RIC3-TCRBC2, SLC35A3-HIAT1, PICALM MLLT10 and MLLT10-PICALM). However, other fusions detected simultaneously in normal and tumour samples (JAK3-INSL3, KANSL1-ARL17A/B and TFG-ADGRG7) could be germ-line fusions genes involved in tumour-maintaining tasks. Notably, some fusions were confirmed in more tumour samples than predicted, indicating that the detection methods underestimated the real number of existing fusions. Our results highlight the potential of RNA-Seq to identify new cryptic fusions, which could be drivers or tumour-maintaining passenger genes. Such novel findings shed light on the searching for new T-LBL biomarkers in these haematological disorders.

Downregulation of specific FBXW7 isoforms with differential effects in T-cell lymphoblastic lymphoma.

FBXW7 is a driver gene in T-cell lymphoblastic neoplasia acting through proteasome degradation of key proto-oncogenes. FBXW7 encodes three isoforms, α, ß and γ, which differ only in the N-terminus. In this work, massive sequencing revealed significant downregulation of FBXW7 in a panel of primary T-cell lymphoblastic lymphomas characterised by the absence of mutations in its sequence. We observed that decreased expression mainly affected the FBXW7ß isoform and to a lesser extent FBXW7α and may be attributed to the combined effect of epigenetic changes, alteration of upstream factors and upregulation of miRNAs. Transient transfections with miRNA mimics in selected cell lines resulted in a significant decrease of total FBXW7 expression and its different isoforms separately, with the consequent increment of critical substrates and the stimulation of cell proliferation. Transient inhibition of endogenous miRNAs in a T-cell lymphoblastic-derived cell line (SUP-T1) was capable of reversing these proliferative effects. Finally, we show how FBXW7 isoforms display different roles within the cell. Simultaneous downregulation of the α and γ isoforms modulates the amount of CCNE1, whilst the ß-isoform alone was found to have a prominent role in modulating the amount of c-MYC. Our data also revealed that downregulation of all isoforms is a sine qua non condition to induce a proliferative pattern in our cell model system. Taking these data into account, potential new treatments to reverse downregulation of all or a specific FBXW7 isoform may be an effective strategy to counteract the proliferative capacity of these tumour cells.

Phosphorylation of FOXO Proteins as a Key Mechanism to Regulate Their Activity.

Phosphorylation of FOXO transcription factors is one of the key mechanisms involved in the regulation of the activity, nucleo-cytosolic shuttling, and stability of this family of proteins. Here, we describe several experimental approaches allowing analysis of changes in the phosphorylation of these proteins upon exposure to different stimuli.

Optimization of a preclinical therapy of cannabinoids in combination with temozolomide against glioma.

Glioblastoma multiforme (GBM) is the most frequent and aggressive form of brain cancer. These features are explained at least in part by the high resistance exhibited by these tumors to current anticancer therapies. Thus, the development of novel therapeutic approaches is urgently needed to improve the survival of the patients suffering this devastating disease. Δ9-Tetrahydrocannabinol (THC, the major active ingredient of marijuana), and other cannabinoids have been shown to exert antitumoral actions in animal models of cancer, including glioma. The mechanism of these anticancer actions relies, at least in part, on the ability of these compounds to stimulate autophagy-mediated apoptosis in tumor cells. Previous observations from our group demonstrated that local administration of THC (or of THC + CBD at a 1:1 ratio, a mixture that resembles the composition of the cannabinoid-based medicine Sativex®) in combination with Temozolomide, the benchmark agent for the treatment of GBM, synergistically reduces the growth of glioma xenografts. With the aim of optimizing the possible clinical utilization of cannabinoids in anti-GBM therapies, in this work we explored the anticancer efficacy of the systemic administration of cannabinoids in combination with TMZ in preclinical models of glioma. Our results show that oral administration of Sativex-like extracts (containing THC and CBD at a 1:1 ratio) in combination with TMZ produces a strong antitumoral effect in both subcutaneous and intracranial glioma cell-derived tumor xenografts. In contrast, combined administration of Sativex-like and BCNU (another alkylating agent used for the treatment of GBM which share structural similarities with the TMZ) did not show a stronger effect than individual treatments. Altogether, our findings support the notion that the combined administration of TMZ and oral cannabinoids could be therapeutically exploited for the management of GBM.

Therapeutic relevance of the PP2A-B55 inhibitory kinase MASTL/Greatwall in breast cancer.

PP2A is a major tumor suppressor whose inactivation is frequently found in a wide spectrum of human tumors. In particular, deletion or epigenetic silencing of genes encoding the B55 family of PP2A regulatory subunits is a common feature of breast cancer cells. A key player in the regulation of PP2A/B55 phosphatase complexes is the cell cycle kinase MASTL (also known as Greatwall). During cell division, inhibition of PP2A-B55 by MASTL is required to maintain the mitotic state, whereas inactivation of MASTL and PP2A reactivation is required for mitotic exit. Despite its critical role in cell cycle progression in multiple organisms, its relevance as a therapeutic target in human cancer and its dependence of PP2A activity is mostly unknown. Here we show that MASTL overexpression predicts poor survival and shows prognostic value in breast cancer patients. MASTL knockdown or knockout using RNA interference or CRISPR/Cas9 systems impairs proliferation of a subset of breast cancer cells. The proliferative function of MASTL in these tumor cells requires its kinase activity and the presence of PP2A-B55 complexes. By using a new inducible CRISPR/Cas9 system in breast cancer cells, we show that genetic ablation of MASTL displays a significant therapeutic effect in vivo. All together, these data suggest that the PP2A inhibitory kinase MASTL may have both prognostic and therapeutic value in human breast cancer.

Programmed mitophagy is essential for the glycolytic switch during cell differentiation.

Retinal ganglion cells (RGCs) are the sole projecting neurons of the retina and their axons form the optic nerve. Here, we show that embryogenesis-associated mouse RGC differentiation depends on mitophagy, the programmed autophagic clearance of mitochondria. The elimination of mitochondria during RGC differentiation was coupled to a metabolic shift with increased lactate production and elevated expression of glycolytic enzymes at the mRNA level. Pharmacological and genetic inhibition of either mitophagy or glycolysis consistently inhibited RGC differentiation. Local hypoxia triggered expression of the mitophagy regulator BCL2/adenovirus E1B 19-kDa-interacting protein 3-like (BNIP3L, best known as NIX) at peak RGC differentiation. Retinas from NIX-deficient mice displayed increased mitochondrial mass, reduced expression of glycolytic enzymes and decreased neuronal differentiation. Similarly, we provide evidence that NIX-dependent mitophagy contributes to mitochondrial elimination during macrophage polarization towards the proinflammatory and more glycolytic M1 phenotype, but not to M2 macrophage differentiation, which primarily relies on oxidative phosphorylation. In summary, developmentally controlled mitophagy promotes a metabolic switch towards glycolysis, which in turn contributes to cellular differentiation in several distinct developmental contexts.

Fueling the Cell Division Cycle.

Cell division is a complex process with high energy demands. However, how cells regulate the generation of energy required for DNA synthesis and chromosome segregation is not well understood. Recent data suggest that changes in mitochondrial dynamics and metabolic pathways such as oxidative phosphorylation (OXPHOS) and glycolysis crosstalk with, and are tightly regulated by, the cell division machinery. Alterations in energy availability trigger cell-cycle checkpoints, suggesting a bidirectional connection between cell division and general metabolism. Some of these connections are altered in human disease, and their manipulation may help in designing therapeutic strategies for specific diseases including cancer. We review here recent studies describing the control of metabolism by the cell-cycle machinery.

Dihydroceramide accumulation mediates cytotoxic autophagy of cancer cells via autolysosome destabilization.

Autophagy is considered primarily a cell survival process, although it can also lead to cell death. However, the factors that dictate the shift between these 2 opposite outcomes remain largely unknown. In this work, we used Δ9-tetrahydrocannabinol (THC, the main active component of marijuana, a compound that triggers autophagy-mediated cancer cell death) and nutrient deprivation (an autophagic stimulus that triggers cytoprotective autophagy) to investigate the precise molecular mechanisms responsible for the activation of cytotoxic autophagy in cancer cells. By using a wide array of experimental approaches we show that THC (but not nutrient deprivation) increases the dihydroceramide:ceramide ratio in the endoplasmic reticulum of glioma cells, and this alteration is directed to autophagosomes and autolysosomes to promote lysosomal membrane permeabilization, cathepsin release and the subsequent activation of apoptotic cell death. These findings pave the way to clarify the regulatory mechanisms that determine the selective activation of autophagy-mediated cancer cell death.

AMPK and PFKFB3 mediate glycolysis and survival in response to mitophagy during mitotic arrest.

Blocking mitotic progression has been proposed as an attractive therapeutic strategy to impair proliferation of tumour cells. However, how cells survive during prolonged mitotic arrest is not well understood. We show here that survival during mitotic arrest is affected by the special energetic requirements of mitotic cells. Prolonged mitotic arrest results in mitophagy-dependent loss of mitochondria, accompanied by reduced ATP levels and the activation of AMPK. Oxidative respiration is replaced by glycolysis owing to AMPK-dependent phosphorylation of PFKFB3 and increased production of this protein as a consequence of mitotic-specific translational activation of its mRNA. Induction of autophagy or inhibition of AMPK or PFKFB3 results in enhanced cell death in mitosis and improves the anti-tumoral efficiency of microtubule poisons in breast cancer cells. Thus, survival of mitotic-arrested cells is limited by their metabolic requirements, a feature with potential implications in cancer therapies aimed to impair mitosis or metabolism in tumour cells.

Activation of the endomitotic spindle assembly checkpoint and thrombocytopenia in Plk1-deficient mice.

Polyploidization in megakaryocytes is achieved by endomitosis, a specialized cell cycle in which DNA replication is followed by aberrant mitosis. Typical mitotic regulators such as Aurora kinases or Cdk1 are dispensable for megakaryocyte maturation, and inhibition of mitotic kinases may in fact promote megakaryocyte maturation. However, we show here that Polo-like kinase 1 (Plk1) is required for endomitosis, and ablation of the Plk1 gene in megakaryocytes results in defective polyploidization accompanied by mitotic arrest and cell death. Lack of Plk1 results in defective centrosome maturation and aberrant spindle pole formation, thus impairing the formation of multiple poles typically found in megakaryocytes. In these conditions, megakaryocytes arrest for a long time in mitosis and frequently die. Mitotic arrest in wild-type megakaryocytes treated with Plk1 inhibitors or Plk1-null cells is triggered by the spindle assembly checkpoint (SAC), and can be rescued in the presence of SAC inhibitors. These data suggest that, despite the dispensability of proper chromosome segregation in megakaryocytes, an endomitotic SAC is activated in these cells upon Plk1 inhibition. SAC activation results in defective maturation of megakaryocytes and cell death, thus raising a note of caution in the use of Plk1 inhibitors in therapeutic strategies based on polyploidization regulators.

CDK6 as a key regulator of hematopoietic and leukemic stem cell activation.

The cyclin-dependent kinase 6 (CDK6) and CDK4 have redundant functions in regulating cell-cycle progression. We describe a novel role for CDK6 in hematopoietic and leukemic stem cells (hematopoietic stem cells [HSCs] and leukemic stem cells [LSCs]) that exceeds its function as a cell-cycle regulator. Although hematopoiesis appears normal under steady-state conditions, Cdk6(-/-) HSCs do not efficiently repopulate upon competitive transplantation, and Cdk6-deficient mice are significantly more susceptible to 5-fluorouracil treatment. We find that activation of HSCs requires CDK6, which interferes with the transcription of key regulators, including Egr1. Transcriptional profiling of HSCs is consistent with the central role of Egr1. The impaired repopulation capacity extends to BCR-ABL(p210+) LSCs. Transplantation with BCR-ABL(p210+)-infected bone marrow from Cdk6(-/-) mice fails to induce disease, although recipient mice do harbor LSCs. Egr1 knock-down in Cdk6(-/-) BCR-ABL(p210+) LSKs significantly enhances the potential to form colonies, underlining the importance of the CDK6-Egr1 axis. Our findings define CDK6 as an important regulator of stem cell activation and an essential component of a transcriptional complex that suppresses Egr1 in HSCs and LSCs.