Ruth Sánchez-Martínez

Greatwall is essential to prevent mitotic collapse after nuclear envelope breakdown in mammals

Significance Nuclear envelope breakdown (NEB) leads to the exposure of nuclear structures to cytoplasmic activities. Greatwall is a kinase able to inhibit PP2A phosphatases that counteract Cdk-dependent phosphorylation required for mitosis. Here we show that Greatwall, an essential protein in mammals, is exported to the cytoplasm in a Cdk-dependent manner before NEB, thus protecting mitotic phosphates from phosphatase activity. Greatwall is a protein kinase involved in the inhibition of protein phosphatase 2 (PP2A)-B55 complexes to maintain the mitotic state. Although its biochemical activity has been deeply characterized in Xenopus, its specific relevance during the progression of mitosis is not fully understood. By using a conditional knockout of the mouse ortholog, Mastl, we show here that mammalian Greatwall is essential for mouse embryonic development and cell cycle progression. Yet, Greatwall-null cells enter into mitosis with normal kinetics. However, these cells display mitotic collapse after nuclear envelope breakdown (NEB) characterized by defective chromosome condensation and prometaphase arrest. Intriguingly, Greatwall is exported from the nucleus to the cytoplasm in a CRM1-dependent manner before NEB. This export occurs after the nuclear import of cyclin B–Cdk1 complexes, requires the kinase activity of Greatwall, and is mediated by Cdk-, but not Polo-like kinase 1-dependent phosphorylation. The mitotic collapse observed in Greatwall-deficient cells is partially rescued after concomitant depletion of B55 regulatory subunits, which are mostly cytoplasmic before NEB. These data suggest that Greatwall is an essential protein in mammals required to prevent mitotic collapse after NEB.

Combinatorial effects of microRNAs to suppress the Myc oncogenic pathway

Many mammalian transcripts contain target sites for multiple miRNAs, although it is not clear to what extent miRNAs may coordinately regulate single genes. We have mapped the interactions between down-regulated miRNAs and overexpressed target protein-coding genes in murine and human lymphomas. Myc, one of the hallmark oncogenes in these lymphomas, stands out as the up-regulated gene with the highest number of genetic interactions with down-regulated miRNAs in mouse lymphomas. The regulation of Myc by several of these miRNAs is confirmed by cellular and reporter assays. The same approach identifies MYC and multiple Myc targets as a preferential target of down-regulated miRNAs in human Burkitt lymphoma, a pathology characterized by translocated MYC oncogenes. These results indicate that several miRNAs must be coordinately down-regulated to enhance critical oncogenes, such as Myc. Some of these Myc-targeting miRNAs are repressed by Myc, suggesting that these tumors are a consequence of the unbalanced activity of Myc versus miRNAs.

A link between lipid metabolism and epithelial-mesenchymal transition provides a target for colon cancer therapy.

The alterations in carbohydrate metabolism that fuel tumor growth have been extensively studied. However, other metabolic pathways involved in malignant progression, demand further understanding. Here we describe a metabolic acyl-CoA synthetase/stearoyl-CoA desaturase ACSL/SCD network causing an epithelial-mesenchymal transition (EMT) program that promotes migration and invasion of colon cancer cells. The mesenchymal phenotype produced upon overexpression of these enzymes is reverted through reactivation of AMPK signaling. Furthermore, this network expression correlates with poorer clinical outcome of stage-II colon cancer patients. Finally, combined treatment with chemical inhibitors of ACSL/SCD selectively decreases cancer cell viability without reducing normal cells viability. Thus, ACSL/SCD network stimulates colon cancer progression through conferring increased energetic capacity and invasive and migratory properties to cancer cells, and might represent a new therapeutic opportunity for colon cancer treatment.

The Ellagic Acid Derivative 4,4′-Di-O-Methylellagic Acid Efficiently Inhibits Colon Cancer Cell Growth through a Mechanism Involving WNT16

Ellagic acid (EA) and some derivatives have been reported to inhibit cancer cell proliferation, induce cell cycle arrest, and modulate some important cellular processes related to cancer. This study aimed to identify possible structure-activity relationships of EA and some in vivo derivatives in their antiproliferative effect on both human colon cancer and normal cells, and to compare this activity with that of other polyphenols. Our results showed that 4,4′-di-O-methylellagic acid (4,4′-DiOMEA) was the most effective compound in the inhibition of colon cancer cell proliferation. 4,4′-DiOMEA was 13-fold more effective than other compounds of the same family. In addition, 4,4′-DiOMEA was very active against colon cancer cells resistant to the chemotherapeutic agent 5-fluoracil, whereas no effect was observed in nonmalignant colon cells. Moreover, no correlation between antiproliferative and antioxidant activities was found, further supporting that structure differences might result in dissimilar molecular targets involved in their differential effects. Finally, microarray analysis revealed that 4,4′-DiOMEA modulated Wnt signaling, which might be involved in the potential antitumor action of this compound. Our results suggest that structural-activity differences between EA and 4,4′-DiOMEA might constitute the basis for a new strategy in anticancer drug discovery based on these chemical modifications.

Modulation of estrogen and epidermal growth factor receptors by rosemary extract in breast cancer cells.

Breast cancer is the leading cause of cancer‐related mortality among females worldwide, and therefore the development of new therapeutic approaches is still needed. Rosemary (Rosmarinus officinalis L.) extract possesses antitumor properties against tumor cells from several organs, including breast. However, in order to apply it as a complementary therapeutic agent in breast cancer, more information is needed regarding the sensitivity of the different breast tumor subtypes and its effect in combination with the currently used chemotherapy. Here, we analyzed the antitumor activities of a supercritical fluid rosemary extract (SFRE) in different breast cancer cells, and used a genomic approach to explore its effect on the modulation of ER‐α and HER2 signaling pathways, the most important mitogen pathways related to breast cancer progression. We found that SFRE exerts antitumor activity against breast cancer cells from different tumor subtypes and the downregulation of ER‐α and HER2 receptors by SFRE might be involved in its antitumor effect against estrogen‐dependent (ER+) and HER2 overexpressing (HER2+) breast cancer subtypes. Moreover, SFRE significantly enhanced the effect of breast cancer chemotherapy (tamoxifen, trastuzumab, and paclitaxel). Overall, our results support the potential utility of SFRE as a complementary approach in breast cancer therapy.

Targeting the lipid metabolic axis ACSL/SCD in colorectal cancer progression by therapeutic miRNAs: miR-19b-1 role

An abnormal acyl-CoA synthetase/stearoyl-CoA desaturase (ACSL/SCD) lipid network fuels colon cancer progression, endowing cells with invasive and migratory properties. Therapies against this metabolic network may be useful to improve clinical outcomes. Because micro-RNAs (miRNAs/miRs) are important epigenetic regulators, we investigated novel miRNAs targeting this pro-tumorigenic axis; hence to be used as therapeutic or prognostic miRNAs. Thirty-one putative common miRNAs were predicted to simultaneously target the three enzymes comprising the ACSL/SCD network. Target validation by quantitative RT-PCR, Western blotting, and luciferase assays showed miR-544a, miR-142, and miR-19b-1 as major regulators of the metabolic axis, ACSL/SCD. Importantly, lower miR-19b-1 expression was associated with a decreased survival rate in colorectal cancer (CRC) patients, accordingly with ACSL/SCD involvement in patient relapse. Finally, miR-19b-1 regulated the pro-tumorigenic axis, ACSL/SCD, being able to inhibit invasion in colon cancer cells. Because its expression correlated with an increased survival rate in CRC patients, we propose miR-19b-1 as a potential noninvasive biomarker of disease-free survival and a promising therapeutic miRNA in CRC.

Complementary ACSL isoforms contribute to a non-Warburg advantageous energetic status characterizing invasive colon cancer cells

Metabolic reprogramming is one of cancer hallmarks. Here, we focus on functional differences and individual contribution of acyl coA synthetases (ACSL) isoforms to the previously described ACSL/stearoyl-CoA desaturase (ACSL1/ACSL4/SCD) metabolic network causing invasion and poor prognosis in colorectal cancer (CRC). ACSL4 fuels proliferation and migration accompanied by a more glycolytic phenotype. Conversely, ACSL1 stimulates invasion displaying a lower basal respiratory rate. Acylcarnitines elevation, polyunsaturated fatty acids (PUFA) lower levels, and monounsaturated fatty acids (MUFA) upregulation characterize the individual overexpression of ACSL1, ACSL4 and SCD, respectively. However, the three enzymes simultaneous overexpression results in upregulated phospholipids and urea cycle derived metabolites. Thus, the metabolic effects caused by the network are far from being caused by the individual contributions of each enzyme. Furthermore, ACSL/SCD network produces more energetically efficient cells with lower basal respiration levels and upregulated creatine pathway. These features characterize other invasive CRC cells, thus, ACSL/SCD network exemplifies specific metabolic adaptations for invasive cancer cells.

MicroRNA‐661 modulates redox and metabolic homeostasis in colon cancer

Cancer cell survival and metastasis are dependent on metabolic reprogramming that is capable of increasing resistance to oxidative and energetic stress. Targeting these two processes can be crucial for cancer progression. Herein, we describe the role of microRNA‐661 (miR661) as epigenetic regulator of colon cancer (CC) cell metabolism. MicroR661 induces a global increase in reactive oxygen species, specifically in mitochondrial superoxide anions, which appears to be mediated by decreased carbohydrate metabolism and pentose phosphate pathway, and by a higher dependency on mitochondrial respiration. MicroR661 overexpression in non‐metastatic human CC cells induces an epithelial‐to‐mesenchymal transition phenotype, and a reduced tolerance to metabolic stress. This seems to be a general effect of miR661 in CC, since metastatic CC cell metabolism is also compromised upon miR661 overexpression. We propose hexose‐6‐phosphate dehydrogenase and pyruvate kinase M2 as two key players related to the observed metabolic reprogramming. Finally, the clinical relevance of miR661 expression levels in stage‐II and III CC patients is discussed. In conclusion, we propose miR661 as a potential modulator of redox and metabolic homeostasis in CC.

The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

Glycosyltransferase enzyme GCNT3, has been proposed as a biomarker for prognosis in colorectal cancer (CRC). Our study goes in depth into the molecular basis of GCNT3 role in tumorigenesis and drug resistance, and it explores its potential role as biomarker in epithelial ovarian cancer (EOC). High levels of GCNT3 are associated with increased sensibility to 5-fluoracil in metastatic cells. Accordingly, GCNT3 re-expression leads to the gain of anti-carcinogenic cellular properties by reducing cell growth, invasion and by changing metabolic capacities. Integrated transcriptomic and proteomic analyses reveal that GCNT3 is linked to cellular cycle, mitosis and proliferation, response to drugs and metabolism pathways. The vascular epithelial growth factor A (VEGFA) arises as an attractive partner of GCNT3 functions in cell invasion and resistance. Finally, GCNT3 expression was analyzed in a cohort of 56 EOC patients followed by a meta-analysis of more than one thousand patients. This study reveals that GCNT3 might constitute a prognostic factor also in EOC, since its overexpression is associated with better clinical outcome and response to initial therapy. GCNT3 emerges as an essential glycosylation-related molecule in CRC and EOC progression, with potential interest as a predictive biomarker of response to chemotherapy.

Thrombocytopenia-associated mutations in Ser/Thr kinase MASTL deregulate actin cytoskeleton dynamics in platelets

MASTL, a Ser/Thr kinase that inhibits PP2A-B55 complexes during mitosis, is mutated in autosomal dominant thrombocytopenia. However, the connections between the cell-cycle machinery and this human disease remain unexplored. We report here that, whereas Mastl ablation in megakaryocytes prevented proper maturation of these cells, mice carrying the thrombocytopenia-associated mutation developed thrombocytopenia as a consequence of aberrant activation and survival of platelets. Activation of mutant platelets was characterized by hyperstabilized pseudopods mimicking the effect of PP2A inhibition and actin polymerization defects. These aberrations were accompanied by abnormal hyperphosphorylation of multiple components of the actin cytoskeleton and were rescued both in vitro and in vivo by inhibiting upstream kinases such as PKA, PKC, or AMPK. These data reveal an unexpected role of Mastl in actin cytoskeletal dynamics in postmitotic cells and suggest that the thrombocytopenia-associated mutation in MASTL is a pathogenic dominant mutation that mimics decreased PP2A activity resulting in altered phosphorylation of cytoskeletal regulatory pathways.