Phosphorylation of targeting protein for Xenopus kinesin-like protein 2 (TPX2) at threonine 72 in spindle assembly.

The human ortholog of the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cytoskeletal protein that plays a major role in spindle assembly and is required for mitosis. During spindle morphogenesis, TPX2 cooperates with Aurora A kinase and Eg5 kinesin to regulate microtubule organization. TPX2 displays over 40 putative phosphorylation sites identified from various high-throughput proteomic screenings. In this study, we characterize the phosphorylation of threonine 72 (Thr(72)) in human TPX2, a residue highly conserved across species. We find that Cdk1/2 phosphorylate TPX2 in vitro and in vivo. Using homemade antibodies specific for TPX2 phosphorylated at Thr(72), we show that this phosphorylation is cell cycle-dependent and peaks at M phase. Endogenous TPX2 phosphorylated at Thr(72) does not associate with the mitotic spindle. Furthermore, ectopic GFP-TPX2 T72A preferentially concentrates on the spindle, whereas GFP-TPX2 WT distributes to both spindle and cytosol. The T72A mutant also increases the proportion of cells with multipolar spindles phenotype. This effect is associated with increased Aurora A activity and abnormally elongated spindles, indicative of higher Eg5 activity. In summary, we propose that phosphorylation of Thr(72) regulates TPX2 localization and impacts spindle assembly via Aurora A and Eg5.

Genetic disruption of aurora B uncovers an essential role for aurora C during early mammalian development.

Mitosis is controlled by multiple kinases that drive cell cycle progression and prevent chromosome mis-segregation. Aurora kinase B interacts with survivin, borealin and incenp to form the chromosomal passenger complex (CPC), which is involved in the regulation of microtubule-kinetochore attachments and cytokinesis. Whereas genetic ablation of survivin, borealin or incenp results in early lethality at the morula stage, we show here that aurora B is dispensable for CPC function during early cell divisions and aurora B-null embryos are normally implanted. This is due to a crucial function of aurora C during these early embryonic cycles. Expression of aurora C decreases during late blastocyst stages resulting in post-implantation defects in aurora B-null embryos. These defects correlate with abundant prometaphase figures and apoptotic cell death of the aurora B-deficient inner cell mass. Conditional deletion of aurora B in somatic cells that do not express aurora C results in chromosomal misalignment and lack of chromosome segregation. Re-expression of wild-type, but not kinase-dead, aurora C rescues this defect, suggesting functional overlap between these two kinases. Finally, aurora B-null cells partially arrest in the presence of nocodazole, suggesting that this kinase is not essential for the spindle assembly checkpoint.

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.

Mitochondrial RNA methyltransferase TRMT61B is a new, potential biomarker and therapeutic target for highly aneuploid cancers.

Despite being frequently observed in cancer cells, chromosomal instability (CIN) and its immediate consequence, aneuploidy, trigger adverse effects on cellular homeostasis that need to be overcome by anti-stress mechanisms. As such, these safeguard responses represent a tumor-specific Achilles heel, since CIN and aneuploidy are rarely observed in normal cells. Recent data have revealed that epitranscriptomic marks catalyzed by RNA-modifying enzymes change under various stress insults. However, whether aneuploidy is associated with such RNA modifying pathways remains to be determined. Through an in silico search for aneuploidy biomarkers in cancer cells, we found TRMT61B, a mitochondrial RNA methyltransferase enzyme, to be associated with high levels of aneuploidy. Accordingly, TRMT61B protein levels are increased in tumor cell lines with an imbalanced karyotype as well as in different tumor types when compared to control tissues. Interestingly, while TRMT61B depletion induces senescence in melanoma cell lines with low levels of aneuploidy, it leads to apoptosis in cells with high levels. The therapeutic potential of these results was further validated by targeting TRMT61B in transwell and xenografts assays. We show that TRM61B depletion reduces the expression of several mitochondrial encoded proteins and limits mitochondrial function. Taken together, these results identify a new biomarker of aneuploidy in cancer cells that could potentially be used to selectively target highly aneuploid tumors.

SUMOylation modulates the function of Aurora-B kinase.

Aurora kinases are central regulators of mitotic-spindle assembly, chromosome segregation and cytokinesis. Aurora B is a member of the chromosomal passenger complex (CPC) with crucial functions in regulation of the attachment of kinetochores to microtubules and in cytokinesis. We report here that Aurora B contains a conserved SUMO modification motif within its kinase domain. Aurora B can bind SUMO peptides in vitro when bound to the IN-box domain of its CPC partner INCENP. Mutation of Lys207 to arginine (Aurora B(K207R)) impairs the formation of conjugates of Aurora B and SUMO in vivo. Expression of the SUMO-null form of Aurora B results in abnormal chromosome segregation and cytokinesis failure and it is not able to rescue mitotic defects in Aurora-B-knockout cells. These defects are accompanied by increased levels of the CPC on chromosome arms and defective centromeric function, as detected by decreased phosphorylation of the Aurora-B substrate CENP-A. The Aurora-B(K207R) mutant does not display reduced kinase activity, suggesting that functional defects are probably a consequence of the altered localization, rather than decreased intrinsic kinase activity. These data suggest that SUMOylation of Aurora B modulates its function, possibly by mediating the extraction of CPC complexes from chromosome arms during prometaphase.

Phospholipase Cgamma activates Ras on the Golgi apparatus by means of RasGRP1.

Ras proteins regulate cellular growth and differentiation, and are mutated in 30% of cancers. We have shown recently that Ras is activated on and transmits signals from the Golgi apparatus as well as the plasma membrane but the mechanism of compartmentalized signalling was not determined. Here we show that, in response to Src-dependent activation of phospholipase Cgamma1, the Ras guanine nucleotide exchange factor RasGRP1 translocated to the Golgi where it activated Ras. Whereas Ca(2+) positively regulated Ras on the Golgi apparatus through RasGRP1, the same second messenger negatively regulated Ras on the plasma membrane by means of the Ras GTPase-activating protein CAPRI. Ras activation after T-cell receptor stimulation in Jurkat cells, rich in RasGRP1, was limited to the Golgi apparatus through the action of CAPRI, demonstrating unambiguously a physiological role for Ras on Golgi. Activation of Ras on Golgi also induced differentiation of PC12 cells, transformed fibroblasts and mediated radioresistance. Thus, activation of Ras on Golgi has important biological consequences and proceeds through a pathway distinct from the one that activates Ras on the plasma membrane.

The EGFR-TMEM167A-p53 Axis Defines the Aggressiveness of Gliomas.

Despite the high frequency of EGFR and TP53 genetic alterations in gliomas, little is known about their crosstalk during tumor progression. Here, we described a mutually exclusive distribution between mutations in these two genes. We found that wild-type p53 gliomas are more aggressive than their mutant counterparts, probably because the former accumulate amplifications and/or mutations in EGFR and show a stronger activation of this receptor. In addition, we identified a series of genes associated with vesicular trafficking of EGFR in p53 wild-type gliomas. Among these genes, TMEM167A showed the strongest implication in overall survival in this group of tumors. In agreement with this observation, inhibition of TMEM167A expression impaired the subcutaneous and the intracranial growth of wild-type p53 gliomas, regardless of the presence of EGFR mutations. In the absence of p53 mutations, TMEM167A knockdown reduced the acidification of intracellular vesicles, affecting the autophagy process and impairing EGFR trafficking and signaling. This effect was mimicked by an inhibitor of the vacuolar ATPase. We propose that the increased aggressiveness of wild-type p53 gliomas might be due to the increase in growth factor signaling activity, which depends on the regulation of vesicular trafficking by TMEM167A.

Liver organoids reproduce alpha-1 antitrypsin deficiency-related liver disease.

BACKGROUND AND AIMS: Alpha-1 antitrypsin (AAT) is a product of SERPINA1 gene mainly expressed by hepatocytes. Clinically relevant mutations in the SERPINA1 gene, such as Z (Glu342Lys), results in an expression of misfolded AAT protein having high propensity to polymerize, accumulate in hepatocytes and thus to enhance a risk for hepatocyte damage and subsequent liver disease. So far, the relationship between the Z-AAT accumulation and liver cell damage remains not completely understood. We present three-dimensional organoid culture systems, as a novel tool for modeling Z-AAT-related liver diseases. METHODS: We have established liver organoids from liver biopsies of patients with homozygous (ZZ) and heterozygous (MZ) deficiency and normal (MM) genotypes of AAT. The features of these organoid models were characterized by analyzing AAT protein secretion and intracellular aggregation in MZ and ZZ genotypes as well as SERPINA1 expression in differentiated cultures. RESULTS: Transcriptional analysis of differentiated organoid cultures by RNA-Seq showed hepatocyte-specific gene expression profile. Genes, such as ALB, APOB, CYP3A4 and SERPINA1, were validated and confirmed through quantitative-PCR analysis. The organoids from MZ and ZZ cases showed intracellular aggregation and lower secretion of AAT protein, and lower expression of ALB and APOB, as typically seen in hepatocytes from Z-AAT deficiency patients. Furthermore, organoids responded to external stimulus. Treatment with oncostatin M, a well-known inducer of SERPINA1, increased expression of the full-length transcripts (AAT-1C) as well as the short transcript of AAT (AAT-ST1C4). CONCLUSIONS: Liver organoid model recapitulates the key features of Z-AAT deficiency and provides a useful tool for disease modeling.

Consequences of Lmna Exon 4 Mutations in Myoblast Function.

Laminopathies are causally associated with mutations on the Lamin A/C gene (LMNA). To date, more than 400 mutations in LMNA have been reported in patients. These mutations are widely distributed throughout the entire gene and are associated with a wide range of phenotypes. Unfortunately, little is known about the mechanisms underlying the effect of the majority of these mutations. This is the case of more than 40 mutations that are located at exon 4. Using CRISPR/Cas9 technology, we generated a collection of Lmna exon 4 mutants in mouse C2C12 myoblasts. These cell models included different types of exon 4 deletions and the presence of R249W mutation, one of the human variants associated with a severe type of laminopathy, LMNA-associated congenital muscular dystrophy (L-CMD). We characterized these clones by measuring their nuclear circularity, myogenic differentiation capacity in 2D and 3D conditions, DNA damage, and levels of p-ERK and p-AKT (phosphorylated Mitogen-Activated Protein Kinase 1/3 and AKT serine/threonine kinase 1). Our results indicated that Lmna exon 4 mutants showed abnormal nuclear morphology. In addition, levels and/or subcellular localization of different members of the lamin and LINC (LInker of Nucleoskeleton and Cytoskeleton) complex were altered in all these mutants. Whereas no significant differences were observed for ERK and AKT activities, the accumulation of DNA damage was associated to the Lmna p.R249W mutant myoblasts. Finally, significant myogenic differentiation defects were detected in the Lmna exon 4 mutants. These results have key implications in the development of future therapeutic strategies for the treatment of laminopathies.

Emerging cancer therapeutic opportunities by inhibiting mitotic kinases.

Among cellular kinases, several cell cycle protein kinases play critical roles in mitotic entry and chromosome segregation. Inhibition of these proteins frequently results in dramatic mitotic arrest and subsequent apoptosis. Most drug discovery efforts have been directed against members of the cyclin-dependent kinase (CDK), Aurora and Polo-like kinase families. Inhibition of these proteins with small molecules has emerged as a powerful research tool and their clinical use is currently being tested in phase I and phase II trials for cancer therapy. New unexplored kinases or new protein domains distinct to the kinase pocket are now being evaluated for the next generation of mitotic drugs. The therapeutic value of inhibiting these kinases will improve with the availability of new specific and potent inhibitors, but it will also rely on a better knowledge of the physiological requirement for these proteins in normal and tumor cell cycles.

Targeting cell cycle kinases for cancer therapy.

Many tumor-associated mutations result in the abnormal regulation of protein kinases involved in the progression throughout the cell division cycle. The cyclin-dependent kinase (CDK) family has received special attention due to their function as sensors of the mitogenic signals and their central role in cell proliferation. These kinases are frequently upregulated in human cancer most frequently due to overexpression of their cyclin partners or inactivation of the CDK inhibitors. A plethora of small-molecule CDK inhibitors have been characterized in the last years and some of them are currently under clinical development. Other serine-threonine protein kinases such as the Aurora proteins (mostly Aurora A and B) or Polo-like kinases (PLK1) are receiving increased attention as putative cancer targets. Other less studied mitotic kinases such TTK (MPS1), BUB and NEK proteins might also be relevant candidates as new targets of interest in cancer therapy since they play relevant roles on mitotic progression and the spindle checkpoint. Although targeting cell cycle kinases is an efficient procedure to arrest cell proliferation, the best strategy to potently and specifically inhibit tumor cell proliferation is not obvious yet. Thus, some cell cycle kinases may be of interest as targets to abrogate checkpoints and favor apoptotic cell death in tumor cells. New biochemical and genetic studies are required to clarify the use of these kinases as targets in new opportunities to improve cancer therapy.

Ras activation in Jurkat T cells following low-grade stimulation of the T-cell receptor is specific to N-Ras and occurs only on the Golgi apparatus.

Ras activation is critical for T-cell development and function, but the specific roles of the different Ras isoforms in T-lymphocyte function are poorly understood. We recently reported T-cell receptor (TCR) activation of ectopically expressed H-Ras on the the Golgi apparatus of T cells. Here we studied the isoform and subcellular compartment specificity of Ras signaling in Jurkat T cells. H-Ras was expressed at much lower levels than the other Ras isoforms in Jurkat and several other T-cell lines. Glutathione S-transferase-Ras-binding domain (RBD) pulldown assays revealed that, although high-grade TCR stimulation and phorbol ester activated both N-Ras and K-Ras, low-grade stimulation of the TCR resulted in specific activation of N-Ras. Surprisingly, whereas ectopically expressed H-Ras cocapped with the TCRs in lipid microdomains of the Jurkat plasma membrane, N-Ras did not. Live-cell imaging of Jurkat cells expressing green fluorescent protein-RBD, a fluorescent reporter of GTP-bound Ras, revealed that N-Ras activation occurs exclusively on the Golgi apparatus in a phospholipase Cgamma- and RasGRP1-dependent fashion. The specificity of N-Ras signaling downstream of low-grade TCR stimulation was dependent on the monoacylation of the hypervariable membrane targeting sequence. Our data show that, in contrast to fibroblasts stimulated with growth factors in which all three Ras isoforms become activated and signaling occurs at both the plasma membrane and Golgi apparatus, Golgi-associated N-Ras is the critical Ras isoform and intracellular pool for low-grade TCR signaling in Jurkat T cells.

Systemic tumor targeting and killing by Sindbis viral vectors.

Successful cancer gene therapy requires a vector that systemically and specifically targets tumor cells throughout the body. Although several vectors have been developed to express cytotoxic genes via tumor-specific promoters or to selectively replicate in tumor cells, most are taken up and expressed by just a few targeted tumor cells. By contrast, we show here that blood-borne Sindbis viral vectors systemically and specifically infect tumor cells. A single intraperitoneal treatment allows the vectors to target most tumor cells, as demonstrated by immunohistochemistry, without infecting normal cells. Further, Sindbis infection is sufficient to induce complete tumor regression. We demonstrate systemic vector targeting of tumors growing subcutaneously, intrapancreatically, intraperitoneally and in the lungs. The vectors can also target syngeneic and spontaneous tumors in immune-competent mice. We document the anti-tumor specificity of a vector that systemically targets and eradicates tumor cells throughout the body without adverse effects.

Mouse p10, an alternative spliced form of p15INK4b, inhibits cell cycle progression and malignant transformation.

The INK4 family of proteins negatively regulates cell cycle progression at the G(1)-S transition by inhibiting cyclin-dependent kinases. Two of these cell cycle inhibitors, p16(INK4A) and p15(INK4B), have tumor suppressor activities and are inactivated in human cancer. Interestingly, both INK4 genes express alternative splicing variants. In addition to p16(INK4A), the INK4A locus encodes a splice variant, termed p12--specifically expressed in human pancreas--and ARF, a protein encoded by an alternative reading frame that acts as a tumor suppressor through the p53 pathway. Similarly, the human INK4B locus encodes the p15(INK4B) tumor suppressor and one alternatively spliced form, termed as p10. We show here that p10, which arises from the use of an alternative splice donor site within intron 1, is conserved in the mouse genome and is widely expressed in mouse tissues. Similarly to mouse p15(INK4B), p10 expression is also induced by oncogenic insults and transforming growth factor-beta treatment and acts as a cell cycle inhibitor. Importantly, we show that mouse p10 is able to induce cell cycle arrest in a p53-dependent manner. We also show that mouse p10 is able to inhibit foci formation and anchorage-independent growth in wild-type mouse embryonic fibroblasts, and that these antitransforming properties of mouse p10 are also p53-dependent. These results indicate that the INK4B locus, similarly to INK4A-ARF, harbors two different splicing variants that can be involved in the regulation of both the p53 and retinoblastoma pathways, the two major molecular pathways in tumor suppression.

The Rgr oncogene induces tumorigenesis in transgenic mice.

To study the oncogenic potential of Rgr in vivo, we have generated several transgenic Rgr mouse lines, which express the oncogene under the control of different promoters. These studies revealed that Rgr expression leads to the generation of various pathological alterations, including fibrosarcomas, when its transgenic expression is restricted to nonlymphoid tissues. Moreover, the overall incidence and latency of fibrosarcomas were substantially increased and shortened, respectively, in a p15INK4b-defective background. More importantly, we also have demonstrated that Rgr expression in thymocytes of transgenic mice induces severe alterations in the development of the thymocytes, which eventually lead to a high incidence of thymic lymphomas. This study demonstrates that oncogenic Rgr can induce expression of p15INK4b and, more importantly, that both Rgr and p15INK4b cooperate in the malignant phenotype in vivo. These findings provide new insights into the tumorigenic role of Rgr as a potent oncogene and show that p15INK4b can act as a tumor suppressor gene.

Mice deficient for N-ras: impaired antiviral immune response and T-cell function.

Ras proteins have a key role in the regulation of several cellular functions, and are involved in a significant percentage of human tumors. However, the specific functions of the different Ras isoforms are poorly understood. In this work, we show for the first time a specific role for N-ras in T-cell function and development. Mice defective for N-ras have low numbers of CD8 single positive thymocytes and decreased thymocyte proliferation in vitro. In Ras signaling and activation assays, KO-N-ras thymocytes showed a defective response to T-cell activation. In turn, these deficiencies resulted in a significant reduction in the production of interleukin 2 on thymocyte activation. We have also detected in vivo the functional consequences of N-ras deficiency. KO-N-ras mice showed an increased sensitivity to influenza infection, especially when low doses of virus were used. Finally, we have detected an abnormal activation pattern of downstream Ras molecules in T-cell receptor-activated KO-N-ras thymocytes that is consistent with the defective T-cell function found in these animals. All of the results derived from this work constitute a significant contribution to the knowledge of N-ras-specific functions.

The N-ras proto-oncogene can suppress the malignant phenotype in the presence or absence of its oncogene.

ras proto-oncogenes have traditionally been associated with the regulation and promotion of cell growth. We have induced thymic lymphomas in N-ras(-/-) mice and in transgenic mice that overexpress wild-type N-ras and found that the lack of wild-type N-ras alleles favors the development of thymic lymphomas,whereas overexpression of wild-type N-ras protects against thymic lymphomagenesis in the presence or absence of its oncogene. To investigate the inhibitory role of wild-type N-ras in in vitro transformation, we introduced wild-type N-ras in N-ras-deficient tumor cells that lack ras activating mutations and found decreased growth in both low serum and soft agar. Taken together, our results indicate that wild-type N-ras has "tumor suppressor" activity, even in the absence of its oncogenic allele.

Aurora A drives early signalling and vesicle dynamics during T-cell activation.

Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3ζ-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal.

Aurora B Overexpression Causes Aneuploidy and p21Cip1 Repression during Tumor Development.

Aurora kinase B, one of the three members of the mammalian Aurora kinase family, is the catalytic component of the chromosomal passenger complex, an essential regulator of chromosome segregation in mitosis. Aurora B is overexpressed in human tumors although whether this kinase may function as an oncogene in vivo is not established. Here, we report a new mouse model in which expression of the endogenous Aurkb locus can be induced in vitro and in vivo. Overexpression of Aurora B in cultured cells induces defective chromosome segregation and aneuploidy. Long-term overexpression of Aurora B in vivo results in aneuploidy and the development of multiple spontaneous tumors in adult mice, including a high incidence of lymphomas. Overexpression of Aurora B also results in a reduced DNA damage response and decreased levels of the p53 target p21(Cip1) in vitro and in vivo, in line with an inverse correlation between Aurora B and p21(Cip1) expression in human leukemias. Thus, overexpression of Aurora B may contribute to tumor formation not only by inducing chromosomal instability but also by suppressing the function of the cell cycle inhibitor p21(Cip1).

Aurora kinase A inhibitors: promising agents in antitumoral therapy.

INTRODUCTION: Aurora proteins are serine/threonine kinases with critical functions during mitosis. Aurora A, one of the members of this family, participates in crucial processes including mitotic entry, DNA damage checkpoint recovery and centrosome and spindle maturation. Aurora A is frequently overexpressed in human cancers and, when inhibited, impairs cell proliferation. AREAS COVERED: Here, we review the preclinical studies that support the use of Aurora A inhibitors in antitumoral strategies. We also discuss past or current clinical trials using Aurora A inhibitors in multiple tumor types. We pay special attention to Alisertib, a potent and selective Aurora A inhibitor currently in Phase III. EXPERT OPINION: The potential of Aurora A inhibitors in the treatment of cancer depends on many factors, mainly related with the molecular status of tumor cells. Yet, we still need to find proper biomarkers to select those patients that better react to Aurora A inhibitors. Furthermore, their effect could significantly improve when used in combination with other drugs. Although some clinical trials are already testing the cooperative effect of different antitumoral drugs, additional preclinical studies are necessary to establish the best combinations. Here, we discuss some possibilities that could be explored in future studies.