p73 is required for endothelial cell differentiation,migration and the formation of vascular networks regulating VEGF and TGFβ signaling

Vasculogenesis, the establishment of the vascular plexus and angiogenesis, branching of new vessels from the preexisting vasculature, involves coordinated endothelial differentiation, proliferation and migration. Disturbances in these coordinated processes may accompany diseases such as cancer. We hypothesized that the p53 family member p73, which regulates cell differentiation in several contexts, may be important in vascular development. We demonstrate that p73 deficiency perturbed vascular development in the mouse retina, decreasing vascular branching, density and stability. Furthermore, p73 deficiency could affect non endothelial cells (ECs) resulting in reduced in vivo proangiogenic milieu. Moreover, p73 functional inhibition, as well as p73 deficiency, hindered vessel sprouting, tubulogenesis and the assembly of vascular structures in mouse embryonic stem cell and induced pluripotent stem cell cultures. Therefore, p73 is necessary for EC biology and vasculogenesis and, in particular, that DNp73 regulates EC migration and tube formation capacity by regulation of expression of pro-angiogenic factors such as transforming growth factor-β and vascular endothelial growth factors. DNp73 expression is upregulated in the tumor environment, resulting in enhanced angiogenic potential of B16-F10 melanoma cells. Our results demonstrate, by the first time, that differential p73-isoform regulation is necessary for physiological vasculogenesis and angiogenesis and DNp73 overexpression becomes a positive advantage for tumor progression due to its pro-angiogenic capacity.Vascular system formation is one of the earliest events during organogenesis.¹ The original vascular plexus is established by vasculogenesis, through differentiation and assembly of mesodermal precursors.² The angiogenesis process allows the formation of new blood vessels from the existing vasculature and is perturbed in many diseases, including cancer.³ Although efforts have been made to identify factors that control vascular development, the understanding of the molecular networks remains incomplete.The formation of new capillaries and the remodeling of preexisting blood vessels is linked by signal transduction pathways.⁴ The members of the p53 family (p53, p73 and p63) coordinate cell proliferation, migration and differentiation, and could act as regulators of vascular development. TP73 function in angiogenesis is quite controversial,^{5, 6, 7} and it has never been addressed using developmental models.TP73 has a dual nature that resides in the existence of TA and DNp73 variants. TAp73 is capable of transactivating p53 targets^{8, 9, 10} whereas DNp73 can act as p53 and TAp73 repressor.^{11, 12, 13} TP73 final outcome will depend upon the differential expression of the TA/DNp73 isoforms in each cellular context, as they can execute synergic, as well as antagonist, functions.TP73 role during development is emphasized by the p73-knockout mice (Trp73−/−, p73KO from now on) multiple growth defects.¹⁴ These mice, which lack all p73 isoforms, exhibit gastrointestinal and cranial hemorrhages,¹⁴ suggestive of vascular fragility. Furthermore, TAp73 directly regulates GATA-1,⁸ which is essential for endothelial and hematopoietic differentiation.^{15, 16} This compounded information led us to hypothesize that p73 could be implicated in the regulation of vasculogenesis/angiogenesis.Regulation of these processes involves a broad range of signaling molecules essential for vascular growth and stability,¹⁷ such as vascular endothelial growth factor (VEGF)¹⁸ and transforming growth factor-β (TGF-β).¹⁹ TGF-β operates as a rheostat that controls endothelial cell (EC) differentiation, having an inhibitory effect on EC migration and proliferation by the TGF-β/TGFRI (ALK5)/Smad2/3 pathway, while the TβRII–ALK5/ALK1 complex activates Smad1/5/8, ID1 expression and a pro-angiogenic state.^{20, 21, 22}Regulation of the TGF-β and VEGF pathways by p53 family members has been documented.^{23, 24} However, p73''s function in these pathways during development remains largely unexplored. In this work, we have used mouse embryonic stem cells (mESC) and induced pluripotent stem cells (iPSCs) as models that recapitulate early vascular morphogenesis.^{25, 26, 27} ESC and iPSC form multi-cellular aggregates (embryoid bodies, EBs) that, under appropriate conditions, generate functional EC.²⁸ mESC and iPSC differentiation capacity into ECs has been fully addressed.^{29, 30} We have also performed retinal vascularization analysis to assess vascular processes in vivo.^{31, 32}We demonstrate that p73 deficiency perturbs density and stability of mouse retinal development by affecting VEGF and TGF-β signaling. Furthermore, p73 is necessary for the assembly of vascular structures under physiological conditions in mESC and iPSC. Moreover, DNp73 positively affects angiogenesis through regulation of the TGF-β pathway in human umbilical vein cells (HUVEC) and DNp73-overexpression results in enhanced angiogenic potential of B16-F10 melanoma cells.     

Mir-660 is downregulated in lung cancer patients and its replacement inhibits lung tumorigenesis by targeting MDM2-p53 interaction

Lung cancer represents the leading cause of cancer-related death in developed countries. Despite the advances in diagnostic and therapeutic techniques, the 5-year survival rate remains low. The research for novel therapies directed to biological targets has modified the therapeutic approach, but the frequent engagement of resistance mechanisms and the substantial costs, limit the ability to reduce lung cancer mortality. MicroRNAs (miRNAs) are small noncoding RNAs with known regulatory functions in cancer initiation and progression. In this study we found that mir-660 expression is downregulated in lung tumors compared with adjacent normal tissues and in plasma samples of lung cancer patients with poor prognosis, suggesting a potential functional role of this miRNA in lung tumorigenesis. Transient and stable overexpression of mir-660 using miRNA mimics reduced migration, invasion, and proliferation properties and increased apoptosis in p53 wild-type lung cancer cells (NCI-H460, LT73, and A549). Furthermore, stable overexpression using lentiviral vectors in NCI-H460 and A549 cells inhibited tumor xenograft growth in immunodeficient mice (95 and 50% reduction compared with control, respectively), whereas the effects of mir-660 overexpression were absent in H1299, a lung cancer cell line lacking p53 locus, both in in vitro and in vivo assays. We identified and validated mouse double minute 2 (MDM2) gene, a key regulator of the expression and function of p53, as a new direct target of mir-660. In addition, mir-660 expression reduced both mRNA and protein expression of MDM2 in all cell lines and stabilized p53 protein levels resulting in an upregulation of p21^WAF1/CIP1 in p53 wild-type cells. Our finding supports that mir-660 acts as a tumor suppressor miRNA and we suggest the replacement of mir-660 as a new therapeutic approach for p53 wild-type lung cancer treatment.Lung cancer is the leading cause of cancer death worldwide, resulting in >1.4 million deaths/year.¹ Lung tumors are often discovered as locally advanced or metastatic disease, and despite improvements in molecular diagnosis and targeted therapies, the overall 5-year survival rate remains in the 10–20% range. Indeed, nonsmall cell lung cancer (NSCLC) is poorly chemosensitive to most of the available agents with response rates ranging from 10 to 25%.² The discovery of recurrent mutations in the epidermal growth factor receptor (EGFR) kinase,³ as well as gene fusion products involving the anaplastic lymphoma kinase (ALK),⁴ has led to a marked change in the treatment of patients with lung adenocarcinoma, the most common type of lung cancer.^{5, 6} To date, patients with mutations in the EGFR gene, suitable for targeting by EGFR tyrosine kinase inhibitors, represent roughly 10%, whereas the subgroup of tumors with ALK rearrangements, targeted by ALK inhibitors, is only ~5%.⁷ Thus, the majority of lung tumors lack effective treatment and novel therapeutic strategies are still needed.MicroRNAs (miRNAs) are short noncoding RNAs, 20–24 nucleotides long, that have important roles in almost all biological pathways,^{8, 9, 10, 11} and influence cancer-relevant processes, such as proliferation,¹² cell cycle,¹³ apoptosis,¹⁴ and migration.¹⁵ Many studies have reported the critical role of miRNAs in lung cancer pathogenesis and their potential as biomarkers for lung cancer risk stratification,¹⁶ outcome prediction,¹⁷ and classification of histological subtypes.^{18, 19} miRNAs are actively released by various cell types and can be detected in biological fluids, such as plasma and serum, making them suitable as circulating biomarkers in NSCLC.^{20, 21}There is limited evidence of mir-660 deregulation in cancer and little is known about its role in lung tumorigenesis and its putative target genes. Mir-660 has been reported to be upregulated in chronic lymphocytic leukemia^{22, 23} and in leukemic cells after treatment with 4-hydroxynonenal, a compound that induces differentiation and blocks proliferation of leukemic cells.²⁴ In a previous study we demonstrated that mir-660 was one of the 24 miRNAs deregulated in plasma samples of NSCLC patients identified in a low-dose computed tomography (LDCT) screening trial.²⁰The p53 tumor suppressor protein is a key regulator of cell cycle G0/G1 checkpoint, senescence, and apoptosis in response to cellular stress signals.^{25, 26} Mouse double minute 2 (MDM2), a p53–E3 ubiquitin ligase,²⁷ is the principal negative regulator of the expression level and function of p53.^{28, 29} Several studies have illustrated different mechanisms of p53 regulation by MDM2,^{30, 31} such as binding transactivation region of p53,^{32, 33} promoting nuclear export and cytoplasmic accumulation of p53 by monoubiquitination,^{34, 35} and inducing p53 proteosomal degradation by polyubiquitination.³⁶ In addition, MDM2 gene has been reported to be amplified or overexpressed in a variety of human cancers, such as sarcoma,³⁷ lymphoma,³⁸ breast cancer,³⁹ lung cancer,⁴⁰ and testicular germ cell tumor.⁴¹ Several miRNAs targeting MDM2 have been identified, such as the mir-143/mir-145 cluster that can be induced by p53,⁴² as well as mir-25 and mir-32, known to inhibit tumor glioblastoma growth in mouse brain.⁴³In this study, we report that mir-660 is downregulated in tissue and plasma samples of lung cancer patients and demonstrate that mir-660 replacement impairs the functionality of p53 wild-type (wt) lung cancer cells and inhibits in vitro and in vivo tumor growth. We showed that all the effects observed after mir-660 overexpression were absent in p53 ko cells, identified MDM2 as mir-660 direct target gene and indicate impairment of the MDM2/p53 interaction as the mechanism underlying tumor growth inhibition.     

New Evidence for Differential Roles of L10 Ribosomal Proteins from Arabidopsis

The RIBOSOMAL PROTEIN L10 (RPL10) is an integral component of the eukaryotic ribosome large subunit. Besides being a constituent of ribosomes and participating in protein translation, additional extraribosomal functions in the nucleus have been described for RPL10 in different organisms. Previously, we demonstrated that Arabidopsis (Arabidopsis thaliana) RPL10 genes are involved in development and translation under ultraviolet B (UV-B) stress. In this work, transgenic plants expressing Pro_RPL10:β-glucuronidase fusions show that, while AtRPL10A and AtRPL10B are expressed both in the female and male reproductive organs, AtRPL10C expression is restricted to pollen grains. Moreover, the characterization of double rpl10 mutants indicates that the three AtRPL10s differentially contribute to the total RPL10 activity in the male gametophyte. All three AtRPL10 proteins mainly accumulate in the cytosol but also in the nucleus, suggesting extraribosomal functions. After UV-B treatment, only AtRPL10B localization increases in the nuclei. We also here demonstrate that the three AtRPL10 genes can complement a yeast RPL10 mutant. Finally, the involvement of RPL10B and RPL10C in UV-B responses was analyzed by two-dimensional gels followed by mass spectrometry. Overall, our data provide new evidence about the nonredundant roles of RPL10 proteins in Arabidopsis.In eukaryotes, the cytosolic ribosomes consist of large 60S and small 40S subunits. In Arabidopsis (Arabidopsis thaliana), ribosomal protein genes exist as families composed of two to seven members that could be differentially incorporated into the cytosolic ribosome under specific situations (Schmid et al., 2005; Byrne, 2009). In this way, ribosomal heterogeneity would allow selective translation of specific mRNAs under particular cell conditions (Barakat et al., 2001; Szick-Miranda and Bailey-Serres, 2001; Giavalisco et al., 2005; Carroll et al., 2008; Carroll, 2013). Arabidopsis mutants in ribosomal proteins exhibit a large range of developmental phenotypes with extreme abnormalities, including embryonic lethality, suggesting that ribosomes also have specific functions regulating the expression of developmental genes (Van Lijsebettens et al., 1994; Degenhardt and Bonham-Smith, 2008; Byrne, 2009; Horiguchi et al., 2011, 2012; Szakonyi and Byrne, 2011). Furthermore, it has been recently demonstrated that ribosomal proteins control auxin-mediated developmental programs by translational regulation of auxin response factors (Rosado et al., 2012). In addition, the characterization of single, double, and, in certain cases, triple mutants as well as complementation by paralog genes have demonstrated full, partial, and no redundancy between members of ribosomal protein families (Briggs et al., 2006; Guo and Chen, 2008; Guo et al., 2011; Horiguchi et al., 2011; Stirnberg et al., 2012).RIBOSOMAL PROTEIN L10 (RPL10) was initially identified in humans as a putative suppressor of Wilms’ tumor (Dowdy et al., 1991). Since then, RPL10 has been studied in different organisms from archaea and bacteria to eukaryotes such as mammals, insects, yeast, and plants (Marty et al., 1993; Mills et al., 1999; Hwang et al., 2000; Zhang et al., 2004; Wen et al., 2005; Singh et al., 2009). A remarkable property of this protein is its high degree of amino acid conservation, suggesting fundamental and critical conserved functions of RPL10 in different organisms (Farmer et al., 1994; Eisinger et al., 1997; Hofer et al., 2007; Nishimura et al., 2008). Likewise, the crystallographic structural similarity observed among RPL10 orthologs in eukaryotes, bacteria, and archaea (called L16) established the conservation of this universal ribosomal protein family and provided evidence of the inalterability of the ribosome during evolution (Spahn et al., 2001; Nishimura et al., 2008). Nevertheless, besides being a constituent of ribosomes and participating in protein translation, additional extraribosomal functions have been described for RPL10 (Mills et al., 1999; Hwang et al., 2000; Chávez-Rios et al., 2003; Zhang et al., 2004; Singh et al., 2009). In yeast, RPL10 is essential for viability, organizes the union site of the aminoacyl-tRNA, and its incorporation into the 60S subunit is a prerequisite for subunit joining and the initiation of translation (West et al., 2005; Hofer et al., 2007). Extensive analysis of the in vivo assembly of ribosomes revealed that RPL10 is loaded to the ribosome in the cytosol with the assistance of its chaperone suppressor of QSR1 truncations (Hedges et al., 2005; West et al., 2005).Arabidopsis has three genes encoding RPL10 proteins, AtRPL10A, AtRPL10B, and, AtRPL10C. Recently, we demonstrated that Arabidopsis RPL10 genes are differentially regulated by UV-B radiation: RPL10B is down-regulated, RPL10C is up-regulated, while RPL10A is not UV-B regulated. Arabidopsis single mutants showed that RPL10 genes are not functionally equivalent. Heterozygous rpl10a mutant plants are translation deficient under UV-B conditions, knockout rpl10A mutants are not viable, and knockdown homozygous rpl10B mutants show abnormal growth. Conversely, knockout homozygous rpl10C mutants do not exhibit any visible phenotype. Overall, RPL10 genes are involved in development and translation under UV-B stress (Falcone Ferreyra et al., 2010b). Furthermore, coimmunoprecipitation studies showed an association of RPL10 with nuclear proteins, suggesting that at least one of the RPL10 isoforms could have an extraribosomal function in the nucleus (Falcone Ferreyra et al., 2010a).The aim of this work was to further investigate the contribution of each Arabidopsis RPL10 to plant development and UV-B responses. We examined the spatiotemporal expression of each AtRPL10 using transgenic plants expressing Pro_RPL10:GUS fusions. By AtRPL10-GFP fusions, we analyzed the subcellular localization of each RPL10, demonstrating that the three isoforms are mainly localized in the cytosol but also in the nucleus. In order to investigate the functional redundancy between AtRPL10 genes in more detail, we generated and characterized double rpl10 mutants. We also here demonstrate that the three AtRPL10 genes can complement a yeast RPL10 mutant. Finally, the involvement of RPL10B and RPL10C in UV-B responses was analyzed by two-dimensional (2D) gels followed by mass spectrometry. Overall, our data provide new insights into the role of each RPL10 in Arabidopsis.