Isoprenylcysteine carboxyl methyltransferase (ICMT) promotes invadopodia formation and metastasis in cancer cells.

Isoprenyl cysteine carboxyl methyltransferase (ICMT) catalyzes the last step of the prenylation pathway. Previously, we found that high ICMT levels enhance tumorigenesis in vivo and that its expression is repressed by the p53 tumor suppressor. Based on evidence suggesting that some ICMT substrates affect invasive traits, we wondered if this enzyme may promote metastasis. In this work, we found that ICMT overexpression enhanced lung metastasis in vivo. Accordingly, ICMT overexpression also promoted cellular functions associated with aggressive phenotypes such as migration and invasion in vitro. Considering that some ICMT substrates are involved in the regulation of actin cytoskeleton, we hypothesized that actin-rich structures, associated with invasion and metastasis, may be affected. Our findings revealed that ICMT enhanced the formation of invadopodia. Additionally, by analyzing cancer patient databases, we found that ICMT is overexpressed in several tumor types. Furthermore, the concurrent expression of ICMT and CTTN, which encodes a crucial component of invadopodia, showed a significant correlation with clinical outcome. In summary, our work identifies ICMT overexpression as a relevant alteration in human cancer that promotes the development of metastatic tumors.

The p53 tumor suppressor regulates AKR1B1 expression, a metastasis-promoting gene in breast cancer.

Alteration of metabolism in cancer cells is a central aspect of the mechanisms that sustain aggressive traits. Aldo-keto reductase 1 B1 (AKR1B1) catalyzes the reduction of several aldehydes to alcohols consuming NADPH. Nevertheless, the ability of AKR1B1 to reduce different substrates renders difficult to comprehensively ascertain its biological role. Recent evidence has implicated AKR1B1 in cancer; however, the mechanisms underlying its pro-oncogenic function remain largely unknown. In this work, we report that AKR1B1 expression is controlled by the p53 tumor suppressor. We found that breast cancer patients bearing wild-type TP53 have reduced AKR1B1 expression. In cancer cell lines, p53 reduced AKR1B1 mRNA and protein levels and repressed promoter activity in luciferase assays. Furthermore, chromatin immunoprecipitation assays indicated that p53 is recruited to the AKR1B1 promoter. We also observed that AKR1B1 overexpression promoted metastasis in the 4T1 orthotopic model of triple-negative breast cancer. Proteomic analysis of 4T1 cells overexpressing AKR1B1 showed that AKR1B1 exerts a marked effect on proteins related to metabolism, with a particular impact on mitochondrial function. This work provides novel insights on the link between the p53 pathway and metabolism in cancer cells and contributes to characterizing the alterations associated to the pathologic role of AKR1B1.

Synthesis of Structurally Related Coumarin Derivatives as Antiproliferative Agents.

A library of structurally related coumarins was generated through synthesis reactions and chemical modification reactions to obtain derivatives with antiproliferative activity both in vivo and in vitro. Out of a total of 35 structurally related coumarin derivatives, seven of them showed inhibitory activity in in vitro tests against Taq DNA polymerase with IC50 values lower than 250 µM. The derivatives 4-(chloromethyl)-5,7-dihydroxy-2H-chromen-2-one (2d) and 4-((acetylthio)methyl)-2-oxo-2H-chromen-7-yl acetate (3c) showed the most promising anti-polymerase activity with IC50 values of 20.7 ± 2.10 and 48.25 ± 1.20 µM, respectively. Assays with tumor cell lines (HEK 293 and HCT-116) were carried out, and the derivative 4-(chloromethyl)-7,8-dihydroxy-2H-chromen-2-one (2c) was the most promising, with an IC50 value of 8.47 µM and a selectivity index of 1.87. In addition, the derivatives were evaluated against Saccharomyces cerevisiae strains that report about common modes of actions, including DNA damage, that are expected for agents that cause replicative stress. The coumarin derivatives 7-(2-(oxiran-2-yl)ethoxy)-2H-chromen-2-one (5b) and 7-(3-(oxiran-2-yl)propoxy)-2H-chromen-2-one (5c) caused DNA damage in S. cerevisiae. The O-alkenylepoxy group stands out as that with the most important functionality within this family of 35 derivatives, presenting a very good profile as an antiproliferative scaffold. Finally, the in vitro antiretroviral capacity was tested through RT-PCR assays. Derivative 5c showed inhibitory activity below 150 µM with an IC50 value of 134.22 ± 2.37 µM, highlighting the O-butylepoxy group as the functionalization responsible for the activity.

Synuclein Proteins in Cancer Development and Progression.

Synucleins are a family of small, soluble proteins mainly expressed in neural tissue and in certain tumors. Since their discovery, tens of thousands of scientific reports have been published about this family of proteins as they are associated with severe human diseases. Although the physiological function of these proteins is still elusive, their relationship with neurodegeneration and cancer has been clearly described over the years. In this review, we summarize data connecting synucleins and cancer, going from the structural description of these molecules to their involvement in tumor-related processes, and discuss the putative use of these proteins as cancer molecular biomarkers.

The Rho guanosine nucleotide exchange factors Vav2 and Vav3 modulate epidermal stem cell function.

It is known that Rho GTPases control different aspects of the biology of skin stem cells (SSCs). However, little information is available on the role of their upstream regulators under normal and tumorigenic conditions in this process. To address this issue, we have used here mouse models in which the activity of guanosine nucleotide exchange factors of the Vav subfamily has been manipulated using both gain- and loss-of-function strategies. These experiments indicate that Vav2 and Vav3 regulate the number, functional status, and responsiveness of hair follicle bulge stem cells. This is linked to gene expression programs related to the reinforcement of the identity and the quiescent state of normal SSCs. By contrast, in the case of cancer stem cells, they promote transcriptomal programs associated with the identity, activation state, and cytoskeletal remodeling. These results underscore the role of these Rho exchange factors in the regulation of normal and tumor epidermal stem cells.

New Functions of Vav Family Proteins in Cardiovascular Biology, Skeletal Muscle, and the Nervous System.

Vav proteins act as tyrosine phosphorylation-regulated guanosine nucleotide exchange factors for Rho GTPases and as molecular scaffolds. In mammals, this family of signaling proteins is composed of three members (Vav1, Vav2, Vav3) that work downstream of protein tyrosine kinases in a wide variety of cellular processes. Recent work with genetically modified mouse models has revealed that these proteins play key signaling roles in vascular smooth and skeletal muscle cells, specific neuronal subtypes, and glia cells. These functions, in turn, ensure the proper regulation of blood pressure levels, skeletal muscle mass, axonal wiring, and fiber myelination events as well as systemic metabolic balance. The study of these mice has also led to the discovery of new physiological interconnection among tissues that contribute to the ontogeny and progression of different pathologies such as, for example, hypertension, cardiovascular disease, and metabolic syndrome. Here, we provide an integrated view of all these new Vav family-dependent signaling and physiological functions.

Verbascoside, synthetic derivatives and other glycosides from Argentinian native plant species as potential antitumoral agents.

A phytochemical study was performed on three native plant species from the central-western zone of Argentina: Buddleja cordobensis Grisebach, Baccharis salicina Torr. & A. Gray and Nepeta cataria L. We could obtain verbascoside (1) from B. cordobensis. From N. cataria, we could obtain 1, 5, 9-epi-deoxyloganic acid (2) L. Finally, we could isolate 2-ß-(L-rhamnopyranosyl)-3-angeloyloxy-15-acetyloxy-7,13(14)-E-dien-ent-labdane (3) and 2-ß-(L-rhamnopyranosyl)-3-α-angeloyloxy-15-hydroxy-7,13(14)-E-dien-ent-labdane (4) from B. salicina. Moreover, three derivatives from 1, and one semi-synthetic derivative from 2, were prepared. PCR reaction was used to analyse the activity against DNA polymerase and cell culture to determine cytotoxicity and antitumoral activity. Verbascoside (1) was strongly active in the nanomolar scale (IC50 = 356 nM) against DNA polymerization. Moreover, verbascoside was also strongly active in the nanomolar scale against human melanoma cell line (IC50 = 256 nM) and human colorectal cell line (IC50 = 320 nM). Furthermore, derivatives 6 and 7 were cytotoxic against both cancer cell lines.

Drug Vulnerabilities and Disease Prognosis Linked to the Stem Cell-Like Gene Expression Program Triggered by the RHO GTPase Activator VAV2 in Hyperplastic Keratinocytes and Head and Neck Cancer.

We have recently shown that VAV2, a guanosine nucleotide exchange factor that catalyzes the stimulation step of RHO GTPases, is involved in a stem cell-like (SCL) regenerative proliferation program that is important for the development and subsequent maintenance of the tumorigenesis of both cutaneous (cSCC) and head and neck squamous cell carcinomas (hnSCC). In line with this, we have observed that the levels of the VAV2 mRNA and VAV2-regulated gene signatures are associated with poor prognosis in the case of human papillomavirus-negative hnSCC patients. These results suggest that the SCL program elicited by VAV2 in those cells can harbor therapeutically actionable downstream targets. We have addressed this issue using a combination of both in silico and wet-lab approaches. Here, we show that the VAV2-regulated SCL program does harbor a number of cell cycle- and signaling-related kinases that are essential for the viability of undifferentiated keratinocytes and hnSCC patient-derived cells endowed with high levels of VAV2 activity. Our results also show that the VAV2-regulated SCL gene signature is associated with poor hnSCC patient prognosis. Collectively, these data underscore the critical role of this VAV2-regulated SCL program for the viability of both preneoplastic and fully transformed keratinocytes.

VAV2 signaling promotes regenerative proliferation in both cutaneous and head and neck squamous cell carcinoma.

Regenerative proliferation capacity and poor differentiation are histological features usually linked to poor prognosis in head and neck squamous cell carcinoma (hnSCC). However, the pathways that regulate them remain ill-characterized. Here, we show that those traits can be triggered by the RHO GTPase activator VAV2 in keratinocytes present in the skin and oral mucosa. VAV2 is also required to maintain those traits in hnSCC patient-derived cells. This function, which is both catalysis- and RHO GTPase-dependent, is mediated by c-Myc- and YAP/TAZ-dependent transcriptomal programs associated with regenerative proliferation and cell undifferentiation, respectively. High levels of VAV2 transcripts and VAV2-regulated gene signatures are both associated with poor hnSCC patient prognosis. These results unveil a druggable pathway linked to the malignancy of specific SCC subtypes.

Identification of a CIP4 PKA phosphorylation site involved in the regulation of cancer cell invasiveness and metastasis.

CDC42 interacting protein 4 (CIP4) is a CDC42 effector that coordinates membrane deformation and actin polymerization. The correlation of CIP4 overexpression with metastatic capacity has been characterized in several types of cancer. However, little information exists on how CIP4 function is regulated. CIP4 interacts with A-kinase (PKA) anchoring protein 350 (AKAP350) and CIP4 is also a PKA substrate. Here, we identified CIP4 T225 as the major CIP4 PKA phosphorylation site. In vitro and in vivo experiments using hepatocellular carcinoma (HCC) and breast cancer cells showed that expression of a CIP4(T225E) phosphomimetic mutant increased cancer cell metastatic capacity and that, conversely, expression of a CIP4(T225A) non-phosphorylatable mutant reduced invasive properties. PKA inhibition decreased to CIP4(T225A) cell-levels control but not CIP4(T225E) cell migratory and invasive efficiency. Concomitantly, our studies indicate that CIP4 T225 phosphorylation promotes the formation of functional invadopodia and enhances CIP4 localization at these structures. Our findings further provide mechanistic data indicating that CIP4 T225 phosphorylation facilitates CIP4 interaction with CDC42. Altogether this study identifies a signaling pathway that involves CIP4 phosphorylation by PKA during the acquisition of a metastatic phenotype in cancer cells.

Binucleating Hydrazonic Ligands and Their µ-Hydroxodicopper(II) Complexes as Promising Structural Motifs for Enhanced Antitumor Activity.

Very few inorganic antineoplastic drugs have entered the clinic in the last decades, mainly because of toxicity issues. Because copper is an essential trace element of ubiquitous occurrence, decreased side effects could be expected in comparison with the widely used platinum anticancer compounds. In the present work, two novel hydrazonic binucleating ligands and their µ-hydroxo dicopper(II) complexes were prepared and fully characterized. They differ by the nature of the aromatic group present in their aroylhydrazone moieties: while H3L1 and its complex, 1, possess a thiophene ring, H3L2 and 2 contain the more polar furan heterocycle. X-ray diffraction indicates that both coordination compounds are very similar in structural terms and generate dimeric arrangements in the solid state. Positive-ion electrospray ionization mass spectrometry analyses confirmed that the main species present in a 10% dimethyl sulfoxide (DMSO)/water solution should be [Cu2(HL)(OH)]+ and the DMSO-substituted derivative [Cu2(L)(DMSO)]+. Scattering techniques [dynamic light scattering (DLS) and small-angle X-ray scattering] suggest that the complexes and their free ligands interact with bovine serum albumin (BSA) in a reversible manner. The binding constants to BSA were determined for the complexes through fluorescence spectroscopy. Moreover, to gain insight into the mechanism of action of the compounds, calf thymus DNA binding studies by UV-visible and DLS measurements using plasmid pBR322 DNA were also performed. For the complexes, DLS data seem to point to the occurrence of DNA cleavage to Form III (linear). Both ligands and their dicopper(II) complexes display potent antiproliferative activity in a panel of four cancer cell lines, occasionally even in the submicromolar range, with the complexes being more potent than the free ligands. Our data on cellular models correlate quite well with the DNA interaction experiments. The results presented herein show that aroylhydrazone-derived binucleating ligands, as well as their dinuclear µ-hydroxodicopper(II) complexes, may represent a promising structural starting point for the development of a new generation of highly active potential antitumor agents.

Effects of alpha-synuclein post-translational modifications on metal binding.

Parkinson's disease is the second most common neurodegenerative disorder worldwide. Neurodegeneration in this pathology is characterized by the loss of dopaminergic neurons in the substantia nigra, coupled with cytoplasmic inclusions known as Lewy bodies containing α-synuclein. The brain is an organ that concentrates metal ions, and there is emerging evidence that a break-down in metal homeostasis may be a critical factor in a variety of neurodegenerative diseases. α-synuclein has emerged as an important metal-binding protein in the brain, whereas these interactions play an important role in its aggregation and might represent a link between protein aggregation, oxidative damage, and neuronal cell loss. Additionally, α-synuclein undergoes several post-translational modifications that regulate its structure and physiological function, and may be linked to the aggregation and/or oligomer formation. This review is focused on the interaction of this protein with physiologically relevant metal ions, highlighting the cases where metal-AS interactions profile as key modulators for its structural, aggregation, and membrane-binding properties. The impact of α-synuclein phosphorylation and N-terminal acetylation in the metal-binding properties of the protein are also discussed, underscoring a potential interplay between PTMs and metal ion binding in regulating α-synuclein physiological functions and its role in pathology. This article is part of the Special Issue "Synuclein".

Vagal afferents contribute to sympathoexcitation-driven metabolic dysfunctions.

Multiple crosstalk between peripheral organs and the nervous system are required to maintain physiological and metabolic homeostasis. Using Vav3-deficient mice as a model for chronic sympathoexcitation-associated disorders, we report here that afferent fibers of the hepatic branch of the vagus nerve are needed for the development of the peripheral sympathoexcitation, tachycardia, tachypnea, insulin resistance, liver steatosis and adipose tissue thermogenesis present in those mice. This neuronal pathway contributes to proper activity of the rostral ventrolateral medulla, a sympathoregulatory brainstem center hyperactive in Vav3-/- mice. Vagal afferent inputs are also required for the development of additional pathophysiological conditions associated with deregulated rostral ventrolateral medulla activity. By contrast, they are dispensable for other peripheral sympathoexcitation-associated disorders sparing metabolic alterations in liver.

Isoprenylcysteine carboxy methyltransferase (ICMT) is associated with tumor aggressiveness and its expression is controlled by the p53 tumor suppressor.

Isoprenyl cysteine carboxyl methyltransferase (ICMT) plays a key role in post-translational regulation of prenylated proteins. On the basis of previous results, we hypothesized that the p53 pathway and ICMT expression may be linked in cancer cells. Here, we studied whether WT p53 and cancer-associated p53 point mutants regulate ICMT levels and whether ICMT overexpression affects tumor progression. Studying the effect of p53 variants on ICMT mRNA and protein levels in cancer cells, we found that WT p53 and p53 mutants differentially affect ICMT expression, indicating that p53 status influences ICMT levels in tumors. To investigate the underlying mechanisms, we constructed ICMT-luciferase reporters and found that WT p53 represses ICMT transcription. In contrast, p53 mutants showed a positive effect on ICMT expression. Promoter truncation analyses pinpointed the repressive effect of WT p53 to the -209 and -14 region on the ICMT promoter, and ChIP assays indicated that WT p53 is recruited to this region. Instead, a different promoter region was identified as responsible for the mutant p53 effect. Studying the effect of ICMT overexpression on tumor-associated phenotypes in vitro and in vivo, and analyzing breast and lung cancer databases, we identified a correlation between p53 status and ICMT expression in breast and lung cancers. Moreover, we observed that ICMT overexpression is correlated with negative clinical outcomes. Our work unveils a link between postprenylation protein processing and the p53 pathway, indicating that the functional interplay between WT and mutant p53 alters ICMT levels, thereby affecting tumor aggressiveness.

Vav proteins maintain epithelial traits in breast cancer cells using miR-200c-dependent and independent mechanisms.

The bidirectional regulation of epithelial-mesenchymal transitions (EMT) is key in tumorigenesis. Rho GTPases regulate this process via canonical pathways that impinge on the stability of cell-to-cell contacts, cytoskeletal dynamics, and cell invasiveness. Here, we report that the Rho GTPase activators Vav2 and Vav3 utilize a new Rac1-dependent and miR-200c-dependent mechanism that maintains the epithelial state by limiting the abundance of the Zeb2 transcriptional repressor in breast cancer cells. In parallel, Vav proteins engage a mir-200c-independent expression prometastatic program that maintains epithelial cell traits only under 3D culture conditions. Consistent with this, the depletion of endogenous Vav proteins triggers mesenchymal features in epithelioid breast cancer cells. Conversely, the ectopic expression of an active version of Vav2 promotes mesenchymal-epithelial transitions using E-cadherin-dependent and independent mechanisms depending on the mesenchymal breast cancer cell line used. In silico analyses suggest that the negative Vav anti-EMT pathway is operative in luminal breast tumors. Gene signatures from the Vav-associated proepithelial and prometastatic programs have prognostic value in breast cancer patients.

Interaction of Cu(i) with the Met-X3-Met motif of alpha-synuclein: binding ligands, affinity and structural features.

The identity of the Cu(i) binding ligands at Met-X3-Met site of AcαS and its role into the affinity and structural properties of the interaction were elucidated by NMR spectroscopy. We provide evidence that the source of ligands for Cu(i) binding to the Met-X3-Met site comes from the N-terminal acetyl group and the Met-1, Asp-2 and Met-5 residues. From the study of site-directed mutants and synthetic peptide models of αS we demonstrated the critical role played by Met-1 and Met-5 residues on the binding affinity of the Cu(i) complex, acting as the main metal anchoring residues. While having a more modest impact in the affinity features of Cu(i) binding, as compared to the Met residues, the N-terminal acetyl group and Asp-2 are important in promoting local helical conformations, contributing to the stabilization of these structures by favoring Cu(i) binding.

Repositioning of Anti-parasitic Drugs in Cyclodextrin Inclusion Complexes for Treatment of Triple-Negative Breast Cancer.

Drug repositioning refers to the identification of new therapeutic indications for drugs already approved. Albendazole and ricobendazole have been used as anti-parasitic drugs for many years; their therapeutic action is based on the inhibition of microtubule formation. Therefore, the study of their properties as antitumor compounds and the design of an appropriate formulation for cancer therapy is an interesting issue to investigate. The selected compounds are poorly soluble in water, and consequently, they have low and erratic bioavailability. In order to improve their biopharmaceutics properties, several formulations employing cyclodextrin inclusion complexes were developed. To carefully evaluate the in vitro and in vivo antitumor activity of these drugs and their complexes, several studies were performed on a breast cancer cell line (4T1) and BALB/c mice. In vitro studies showed that albendazole presented improved antitumor activity compared with ricobendazole. Furthermore, albendazole:citrate-ß-cyclodextrin complex decreased significantly 4T1 cell growth both in in vitro and in vivo experiments. Thus, new formulations for anti-parasitic drugs could help to reposition them for new therapeutic indications, offering safer and more effective treatments by using a well-known drug.

Differential Role of the RasGEFs Sos1 and Sos2 in Mouse Skin Homeostasis and Carcinogenesis.

Using Sos1 knockout (Sos1-KO), Sos2-KO, and Sos1/2 double-knockout (Sos1/2-DKO) mice, we assessed the functional role of Sos1 and Sos2 in skin homeostasis under physiological and/or pathological conditions. Sos1 depletion resulted in significant alterations of skin homeostasis, including reduced keratinocyte proliferation, altered hair follicle and blood vessel integrity in dermis, and reduced adipose tissue in hypodermis. These defects worsened significantly when both Sos1 and Sos2 were absent. Simultaneous Sos1/2 disruption led to severe impairment of the ability to repair skin wounds, as well as to almost complete ablation of the neutrophil-mediated inflammatory response in the injury site. Furthermore, Sos1 disruption delayed the onset of tumor initiation, decreased tumor growth, and prevented malignant progression of papillomas in a DMBA (7,12-dimethylbenz[α]anthracene)/TPA (12-O-tetradecanoylphorbol-13-acetate)-induced skin carcinogenesis model. Finally, Sos1 depletion in preexisting chemically induced papillomas resulted also in decreased tumor growth, probably linked to significantly reduced underlying keratinocyte proliferation. Our data unveil novel, distinctive mechanistic roles of Sos 1 and Sos2 in physiological control of skin homeostasis and wound repair, as well as in pathological development of chemically induced skin tumors. These observations underscore the essential role of Sos proteins in cellular proliferation and migration and support the consideration of these RasGEFs as potential biomarkers/therapy targets in Ras-driven epidermal tumors.

Binding Modes of Phthalocyanines to Amyloid ß Peptide and Their Effects on Amyloid Fibril Formation.

The inherent tendency of proteins to convert from their native states into amyloid aggregates is associated with a range of human disorders, including Alzheimer's and Parkinson's diseases. In that sense, the use of small molecules as probes for the structural and toxic mechanism related to amyloid aggregation has become an active area of research. Compared with other compounds, the structural and molecular basis behind the inhibitory interaction of phthalocyanine tetrasulfonate (PcTS) with proteins such as αS and tau has been well established, contributing to a better understanding of the amyloid aggregation process in these proteins. We present here the structural characterization of the binding of PcTS and its Cu(II) and Zn(II)-loaded forms to the amyloid ß-peptide (Aß) and the impact of these interactions on the peptide amyloid fibril assembly. Elucidation of the PcTS binding modes to Aß40 revealed the involvement of specific aromatic and hydrophobic interactions in the formation of the Aß40-PcTS complex, ascribed to a binding mode in which the planarity and hydrophobicity of the aromatic ring system in the phthalocyanine act as main structural determinants for the interaction. Our results demonstrated that formation of the Aß40-PcTS complex does not interfere with the progression of the peptide toward the formation of amyloid fibrils. On the other hand, conjugation of Zn(II) but not Cu(II) at the center of the PcTS macrocyclic ring modified substantially the binding profile of this phthalocyanine to Aß40 and became crucial to reverse the effects of metal-free PcTS on the fibril assembly of the peptide. Overall, our results provide a firm basis to understand the structural rules directing phthalocyanine-protein interactions and their implications on the amyloid fibril assembly of the target proteins; in particular, our results contradict the hypothesis that PcTS might have similar mechanisms of action in slowing the formation of a variety of pathological aggregates.

A Paradoxical Tumor-Suppressor Role for the Rac1 Exchange Factor Vav1 in T Cell Acute Lymphoblastic Leukemia.

Rho guanine exchange factors (GEFs), the enzymes that stimulate Rho GTPases, are deemed as potential therapeutic targets owing to their protumorigenic functions. However, the understanding of the spectrum of their pathobiological roles in tumors is still very limited. We report here that the GEF Vav1 unexpectedly possesses tumor-suppressor functions in immature T cells. This function entails the noncatalytic nucleation of complexes between the ubiquitin ligase Cbl-b and the intracellular domain of Notch1 (ICN1) that favors ICN1 ubiquitinylation and degradation. Ablation of Vav1 promotes ICN1 signaling and the development of T cell acute lymphoblastic leukemia (T-ALL). The downregulation of Vav1 is essential for the pathogenesis of human T-ALL of the TLX+ clinical subtype, further underscoring the suppressor role of this pathway.