TPX2 overexpression promotes sensitivity to dasatinib in breast cancer by activating YAP transcriptional signaling.

Chromosomal instability (CIN) is a hallmark of cancer aggressiveness, providing genetic plasticity and tumor heterogeneity that allows the tumor to evolve and adapt to stress conditions. CIN is considered a cancer therapeutic biomarker because healthy cells do not exhibit CIN. Despite recent efforts to identify therapeutic strategies related to CIN, the results obtained have been very limited. CIN is characterized by a genetic signature where a collection of genes, mostly mitotic regulators, are overexpressed in CIN-positive tumors, providing aggressiveness and poor prognosis. We attempted to identify new therapeutic strategies related to CIN genes by performing a drug screen, using cells that individually express CIN-associated genes in an inducible manner. We find that the overexpression of targeting protein for Xklp2 (TPX2) enhances sensitivity to the proto-oncogene c-Src (SRC) inhibitor dasatinib due to activation of the Yes-associated protein 1 (YAP) pathway. Furthermore, using breast cancer data from The Cancer Genome Atlas (TCGA) and a cohort of cancer-derived patient samples, we find that both TPX2 overexpression and YAP activation are present in a significant percentage of cancer tumor samples and are associated with poor prognosis; therefore, they are putative biomarkers for selection for dasatinib therapy.

Acute expression of human APOBEC3B in mice results in RNA editing and lethality.

BACKGROUND: RNA editing has been described as promoting genetic heterogeneity, leading to the development of multiple disorders, including cancer. The cytosine deaminase APOBEC3B is implicated in tumor evolution through DNA mutation, but whether it also functions as an RNA editing enzyme has not been studied. RESULTS: Here, we engineer a novel doxycycline-inducible mouse model of human APOBEC3B-overexpression to understand the impact of this enzyme in tissue homeostasis and address a potential role in C-to-U RNA editing. Elevated and sustained levels of APOBEC3B lead to rapid alteration of cellular fitness, major organ dysfunction, and ultimately lethality in mice. Importantly, RNA-sequencing of mouse tissues expressing high levels of APOBEC3B identifies frequent UCC-to-UUC RNA editing events that are not evident in the corresponding genomic DNA. CONCLUSIONS: This work identifies, for the first time, a new deaminase-dependent function for APOBEC3B in RNA editing and presents a preclinical tool to help understand the emerging role of APOBEC3B as a driver of carcinogenesis.

Human APOBEC3B promotes tumor development in vivo including signature mutations and metastases.

The antiviral DNA cytosine deaminase APOBEC3B has been implicated as a source of mutation in many cancers. However, despite years of work, a causal relationship has yet to be established in vivo. Here, we report a murine model that expresses tumor-like levels of human APOBEC3B. Animals expressing full-body APOBEC3B appear to develop normally. However, adult males manifest infertility, and older animals of both sexes show accelerated rates of carcinogenesis, visual and molecular tumor heterogeneity, and metastasis. Both primary and metastatic tumors exhibit increased frequencies of C-to-T mutations in TC dinucleotide motifs consistent with the established biochemical activity of APOBEC3B. Enrichment for APOBEC3B-attributable single base substitution mutations also associates with elevated levels of insertion-deletion mutations and structural variations. APOBEC3B catalytic activity is required for all of these phenotypes. Together, these studies provide a cause-and-effect demonstration that human APOBEC3B is capable of driving both tumor initiation and evolution in vivo.

FOXM1 is critical for the fitness recovery of chromosomally unstable cells.

Tumor progression and evolution are frequently associated with chromosomal instability (CIN). Tumor cells often express high levels of the mitotic checkpoint protein MAD2, leading to mitotic arrest and cell death. However, some tumor cells are capable of exiting mitosis and consequently increasing CIN. How cells escape the mitotic arrest induced by MAD2 and proliferate with CIN is not well understood. Here, we explored loss-of-function screens and drug sensitivity tests associated with MAD2 levels in aneuploid cells and identified that aneuploid cells with high MAD2 levels are more sensitive to FOXM1 depletion. Inhibition of FOXM1 promotes MAD2-mediated mitotic arrest and exacerbates CIN. Conversely, elevating FOXM1 expression in MAD2-overexpressing human cell lines reverts prolonged mitosis and rescues mitotic errors, cell death and proliferative disadvantages. Mechanistically, we found that FOXM1 facilitates mitotic exit by inhibiting the spindle assembly checkpoint (SAC) and the expression of Cyclin B. Notably, we observed that FOXM1 is upregulated upon aneuploid induction in cells with dysfunctional SAC and error-prone mitosis, and these cells are sensitive to FOXM1 knockdown, indicating a novel vulnerability of aneuploid cells.

Human APOBEC3B promotes tumor heterogeneity in vivo including signature mutations and metastases.

The antiviral DNA cytosine deaminase APOBEC3B has been implicated as a source of mutation in many different cancers. Despite over 10 years of work, a causal relationship has yet to be established between APOBEC3B and any stage of carcinogenesis. Here we report a murine model that expresses tumor-like levels of human APOBEC3B after Cre-mediated recombination. Animals appear to develop normally with full-body expression of APOBEC3B. However, adult males manifest infertility and older animals of both sexes show accelerated rates of tumorigenesis (mostly lymphomas or hepatocellular carcinomas). Interestingly, primary tumors also show overt heterogeneity, and a subset spreads to secondary sites. Both primary and metastatic tumors exhibit increased frequencies of C-to-T mutations in TC dinucleotide motifs consistent with the established biochemical activity of APOBEC3B. Elevated levels of structural variation and insertion-deletion mutations also accumulate in these tumors. Together, these studies provide the first cause-and-effect demonstration that human APOBEC3B is an oncoprotein capable of causing a wide range of genetic changes and driving tumor formation in vivo .

Unveiling the Local Structure of Palladium Loaded into Imine-Linked Layered Covalent Organic Frameworks for Cross-Coupling Catalysis.

Layered covalent organic frameworks (2D-COFs), composed of reversible imine linkages and accessible pores, offer versatility for chemical modifications towards the development of catalytic materials. Nitrogen-enriched COFs are good candidates for binding Pd species. Understanding the local structure of reacting Pd sites bonded to the COF pores is key to rationalize interactions between active sites and porous surfaces. By combining advanced synchrotron characterization methods with periodic computational DFT modeling, the precise atomic structure of catalytic Pd sites attached to local defects is resolved within an archetypical imine-linked 2D-COF. This material was synthesized using an in situ method as a gel, under which imine hydrolysis and metalation reactions are coupled. Local defects formed in situ within imine-linked 2D-COF materials are highly reactive towards Pd metalation, resulting in active materials for Suzuki-Miyaura cross-coupling reactions.

Rare, functional, somatic variants in gene families linked to cancer genes: GPCR signaling as a paradigm.

Oncodriver genes are usually identified when mutations recur in multiple tumours. Different drivers often converge in the activation or repression of key cancer-relevant pathways. However, as many pathways contain multiple members of the same gene family, individual mutations might be overlooked, as each family member would necessarily have a lower mutation frequency and thus not identified as significant in any one-gene-at-a-time analysis. Here, we looked for mutated, functional sequence positions in gene families that were mutually exclusive (in patients) with another gene in the same pathway, which identified both known and new candidate oncodrivers. For instance, many inactivating mutations in multiple G-protein (particularly Gi/o) coupled receptors, are mutually exclusive with Gαs oncogenic activating mutations, both of which ultimately enhance cAMP signalling. By integrating transcriptomics and interaction data, we show that the Gs pathway is upregulated in multiple cancer types, even those lacking known GNAS activating mutations. This suggests that cancer cells may develop alternative strategies to activate adenylate cyclase signalling in multiple cancer types. Our study provides a mechanistic interpretation for several rare somatic mutations in multi-gene oncodrivers, and offers possible explanations for known and potential off-label cancer treatments, suggesting new therapeutic opportunities.

HIF2α acts as an mTORC1 activator through the amino acid carrier SLC7A5.

The mammalian target of rapamycin (mTOR) pathway, which is essential for cell proliferation, is repressed in certain cell types in hypoxia. However, hypoxia-inducible factor 2α (HIF2α) can act as a proliferation-promoting factor in some biological settings. This paradoxical situation led us to study whether HIF2α has a specific effect on mTORC1 regulation. Here we show that activation of the HIF2α pathway increases mTORC1 activity by upregulating expression of the amino acid carrier SLC7A5. At the molecular level we also show that HIF2α binds to the Slc7a5 proximal promoter. Our findings identify a link between the oxygen-sensing HIF2α pathway and mTORC1 regulation, revealing the molecular basis of the tumor-promoting properties of HIF2α in von Hippel-Lindau-deficient cells. We also describe relevant physiological scenarios, including those that occur in liver and lung tissue, wherein HIF2α or low-oxygen tension drive mTORC1 activity and SLC7A5 expression.

Acute Vhl gene inactivation induces cardiac HIF-dependent erythropoietin gene expression.

Von Hippel Lindau (Vhl) gene inactivation results in embryonic lethality. The consequences of its inactivation in adult mice, and of the ensuing activation of the hypoxia-inducible factors (HIFs), have been explored mainly in a tissue-specific manner. This mid-gestation lethality can be also circumvented by using a floxed Vhl allele in combination with an ubiquitous tamoxifen-inducible recombinase Cre-ER(T2). Here, we characterize a widespread reduction in Vhl gene expression in Vhl(floxed)-UBC-Cre-ER(T2) adult mice after dietary tamoxifen administration, a convenient route of administration that has yet to be fully characterized for global gene inactivation. Vhl gene inactivation rapidly resulted in a marked splenomegaly and skin erythema, accompanied by renal and hepatic induction of the erythropoietin (Epo) gene, indicative of the in vivo activation of the oxygen sensing HIF pathway. We show that acute Vhl gene inactivation also induced Epo gene expression in the heart, revealing cardiac tissue to be an extra-renal source of EPO. Indeed, primary cardiomyocytes and HL-1 cardiac cells both induce Epo gene expression when exposed to low O(2) tension in a HIF-dependent manner. Thus, as well as demonstrating the potential of dietary tamoxifen administration for gene inactivation studies in UBC-Cre-ER(T2) mouse lines, this data provides evidence of a cardiac oxygen-sensing VHL/HIF/EPO pathway in adult mice.

Oxidative phosphorylation is impaired by prolonged hypoxia in breast and possibly in cervix carcinoma.

It has been assumed that oxidative phosphorylation (OxPhos) in solid tumors is severely reduced due to cytochrome c oxidase substrate restriction, although the measured extracellular oxygen concentration in hypoxic areas seems not limiting for this activity. To identify alternative hypoxia-induced OxPhos depressing mechanisms, an integral analysis of transcription, translation, enzyme activities and pathway fluxes was performed on glycolysis and OxPhos in HeLa and MCF-7 carcinomas. In both neoplasias exposed to hypoxia, an early transcriptional response was observed after 8h (two times increased glycolysis-related mRNA synthesis promoted by increased HIF-1alpha levels). However, major metabolic remodeling was observed only after 24h hypoxia: increased glycolytic protein content (1-5-times), enzyme activities (2-times) and fluxes (4-6-times). Interestingly, in MCF-7 cells, 24h hypoxia decreased OxPhos flux (4-6-fold), and 2-oxoglutarate dehydrogenase and glutaminase activities (3-fold), with no changes in respiratory complexes I and IV activities. In contrast, 24h hypoxia did not significantly affect HeLa OxPhos flux; neither mitochondria related mRNAs, protein contents or enzyme activities, although the enhanced glycolysis became the main ATP supplier. Thus, prolonged hypoxia (a) targeted some mitochondrial enzymes in MCF-7 but not in HeLa cells, and (b) induced a transition from mitochondrial towards a glycolytic-dependent energy metabolism in both MCF-7 and HeLa carcinomas.

A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery.

BACKGROUND: Several human pathologies, including neoplasia and ischemic cardiovascular diseases, course with an unbalance between oxygen supply and demand (hypoxia). Cells within hypoxic regions respond with the induction of a specific genetic program, under the control of the Hypoxia Inducible Factor (HIF), that mediates their adaptation to the lack of oxygen. The activity of HIF is mainly regulated by the EGL-nine homolog (EGLN) enzymes that hydroxylate the alpha subunit of this transcription factor in an oxygen-dependent reaction. Hydroxylated HIF is then recognized and ubiquitinilated by the product of the tumor suppressor gene, pVHL, leading to its proteosomal degradation. Under hypoxia, the hydroxylation of HIF by the EGLNs is compromised due to the lack of oxygen, which is a reaction cosubstrate. Thus, HIF escapes degradation and drives the transcription of its target genes. Since the progression of the aforementioned pathologies might be influenced by activation of HIF-target genes, development of small molecules with the ability to interfere with the HIF-regulatory machinery is of great interest. RESULTS: Herein we describe a yeast three-hybrid system that reconstitutes mammalian HIF regulation by the EGLNs and VHL. In this system, yeast growth, under specific nutrient restrictions, is driven by the interaction between the beta domain of VHL and a hydroxyproline-containing HIFalpha peptide. In turn, this interaction is strictly dependent on EGLN activity that hydroxylates the HIFalpha peptide. Importantly, this system accurately preserves the specificity of the hydroxylation reaction toward specific substrates. We propose that this system, in combination with a matched control, can be used as a simple and inexpensive assay to identify molecules that specifically modulate EGLN activity. As a proof of principle we show that two known EGLN inhibitors, dimethyloxaloylglycine (DMOG) and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamide (S956711), have a profound and specific effect on the yeast HIF/EGLN/VHL system. CONCLUSION: The system described in this work accurately reconstitutes HIF regulation while preserving EGLN substrate specificity. Thus, it is a valuable tool to study HIF regulation, and particularly EGLN biochemistry, in a cellular context. In addition, we demonstrate that this system can be used to identify specific inhibitors of the EGLN enzymes.

Identification of a region on hypoxia-inducible-factor prolyl 4-hydroxylases that determines their specificity for the oxygen degradation domains.

HIFs [hypoxia-inducible (transcription) factors] are essential for the induction of an adaptive gene expression programme under low oxygen partial pressure. The activity of these transcription factors is mainly determined by the stability of the HIFalpha subunit, which is regulated, in an oxygen-dependent manner, by a family of three prolyl 4-hydroxylases [EGLN1-EGLN3 (EGL nine homologues 1-3)]. HIFalpha contains two, N- and C-terminal, independent ODDs (oxygen-dependent degradation domains), namely NODD and CODD, that, upon hydroxylation by the EGLNs, target HIFalpha for proteasomal degradation. In vitro studies indicate that each EGLN shows a differential preference for ODDs, However, the sequence determinants for such specificity are unknown. In the present study we showed that whereas EGLN1 and EGLN2 acted upon any of these ODDs to regulate HIF1alpha protein levels and activity in vivo, EGLN3 only acted on the CODD. With the aim of identifying the region within EGLNs responsible for their differential substrate preference, we investigated the activity and binding pattern of different EGLN deletions and chimaeric constructs generated by domain swapping between EGLN1 and EGLN3. These studies revealed a region of 97 residues that was sufficient to confer the characteristic substrate binding observed for each EGLN. Within this region, we identified the minimal sequence (EGLN1 residues 236-252) involved in substrate discrimination. Importantly, mapping of these sequences on the EGLN1 tertiary structure indicates that substrate specificity is determined by a region relatively remote from the catalytic site.

Analysis of HIF-prolyl hydroxylases binding to substrates.

Hypoxia inducible transcription factors (HIF) are mainly regulated by a group of proline hydroxylases (EGLNs) that, in the presence of oxygen, target HIF for degradation. HIFalpha contains two independent oxygen degradation domains (N-ODD and C-ODD) that are substrates for these enzymes. In this work, we employed the yeast two-hybrid assay to study the sequence determinants required for the binding of EGLN1 and 3 to HIF1alpha in a cellular context. Our results demonstrate that, while EGLN1 is able to recognize both ODDs within full length HIF1alpha protein, EGLN3 only binds to CODD. The analysis of the residue substitutions within CODD uncovered novel critical determinants for EGLN1 and 3 binding. In addition, our results show that both enzymes have a very similar, albeit not identical, residue preference at specific positions in their substrate sequences.

Brote de gastroenteritis por clostridium perfringens en un comedor institucional

Se describe un brote de gastroenterititis de origen alimentario por Clostridium perfringens tipo A en un comedor institucional de Buenos Aires, Argentina. El menú consistió en ensaladas de papas con carne de cubitos (Salpicón) y ensalada de frutas. De las 53 personas en riesgo encuenstadas 27(51%) desarrollaron la enfermedad. Los porcentajes de los signos y síntomas fueron: cólicos abdominales (85%), diarrea (85%), naúseas (35%), 13,30 h (rango 03:00-20:30h) y la mediana de duración de los síntomas 03:00h. La prueba de Chi2 (p < 0.001) mostró una franca asociación con el consumo de las ensaladas de papas con carne (salpicon). El recueto de C. perfringens en un trozo de carne (2,5-3,0 Kg) del mismo lote, fue 2.4 x 10**4 UFC/g. El principal factor determinante del brote fue el enfriamiento inadecuado de los cortes luego de la cocción incompleta, circunstancia que permitió la activación de las esporas del agente y la posterior multiplicación. Es prbable también, que algunas células vegetativas de C. perfringens hayan sobrevivido a la cocción incompleta. Tanto la cocción como el enfriamiento son puntos críticos que deben vigilarse. Se recomienda 1) usar cortes pequeños de carne (2,5Kg o menores) para lograr una cocción completa y facilitar el enfriamiento. 2) enfriar rapidamente los cortes de carne. 3) evitar, en lo posible, preparar los alimentos un día o más antes del consumo. 4) mantener en óptimas condiciones de funcionamiento e higiene los equipos culinarios, y evitar sobrecargar los regrigeradores