Advanced targeted therapies in cancer: Drug nanocarriers, the future of chemotherapy

Cancer is the second worldwide cause of death, exceeded only by cardiovascular diseases. It is characterized by uncontrolled cell proliferation and an absence of cell death that, except for hematological cancers, generates an abnormal cell mass or tumor. This primary tumor grows thanks to new vascularization and, in time, acquires metastatic potential and spreads to other body sites, which causes metastasis and finally death. Cancer is caused by damage or mutations in the genetic material of the cells due to environmental or inherited factors. While surgery and radiotherapy are the primary treatment used for local and non-metastatic cancers, anti-cancer drugs (chemotherapy, hormone and biological therapies) are the choice currently used in metastatic cancers. Chemotherapy is based on the inhibition of the division of rapidly growing cells, which is a characteristic of the cancerous cells, but unfortunately, it also affects normal cells with fast proliferation rates, such as the hair follicles, bone marrow and gastrointestinal tract cells, generating the characteristic side effects of chemotherapy. The indiscriminate destruction of normal cells, the toxicity of conventional chemotherapeutic drugs, as well as the development of multidrug resistance, support the need to find new effective targeted treatments based on the changes in the molecular biology of the tumor cells. These novel targeted therapies, of increasing interest as evidenced by FDA-approved targeted cancer drugs in recent years, block biologic transduction pathways and/or specific cancer proteins to induce the death of cancer cells by means of apoptosis and stimulation of the immune system, or specifically deliver chemotherapeutic agents to cancer cells, minimizing the undesirable side effects. Although targeted therapies can be achieved directly by altering specific cell signaling by means of monoclonal antibodies or small molecules inhibitors, this review focuses on indirect targeted approaches that mainly deliver chemotherapeutic agents to molecular targets overexpressed on the surface of tumor cells. In particular, we offer a detailed description of different cytotoxic drug carriers, such as liposomes, carbon nanotubes, dendrimers, polymeric micelles, polymeric conjugates and polymeric nanoparticles, in passive and active targeted cancer therapy, by enhancing the permeability and retention or by the functionalization of the surface of the carriers, respectively, emphasizing those that have received FDA approval or are part of the most important clinical studies up to date. These drug carriers not only transport the chemotherapeutic agents to tumors, avoiding normal tissues and reducing toxicity in the rest of the body, but also protect cytotoxic drugs from degradation, increase the half-life, payload and solubility of cytotoxic agents and reduce renal clearance.Despite the many advantages of all the anticancer drug carriers analyzed, only a few of them have reached the FDA approval, in particular, two polymer-protein conjugates, five liposomal formulations and one polymeric nanoparticle are available in the market, in contrast to the sixteen FDA approval of monoclonal antibodies. However, there are numerous clinical trials in progress of polymer-protein and polymer-drug conjugates, liposomal formulations, including immunoliposomes, polymeric micelles and polymeric nanoparticles. Regarding carbon nanotubes or dendrimers, there are no FDA approvals or clinical trials in process up to date due to their unresolved toxicity. Moreover, we analyze in detail the more promising and advanced preclinical studies of the particular case of polymeric nanoparticles as carriers of different cytotoxic agents to active and passive tumor targeting published in the last 5 years, since they have a huge potential in cancer therapy, being one of the most widely studied nano-platforms in this field in the last years. The interest that these formulations have recently achieved is stressed by the fact that 90% of the papers based on cancer therapeutics with polymeric nanoparticles have been published in the last 6 years (PubMed search).Authors acknowledge financial support from University of Salamanca, Spain. AFM work is supported by grants FIS PI13/02846 and RTICC RD12/0036/0001 from Instituto de Salud Carlos III (ISCIII), Spain, and grant SA181U13 from JCyL, Spain. EPH work is supported by grant FS/25-2014 from Fundación en Memoria de D. Samuel Solórzano Barruso, Spain.Peer Reviewe

Rasgrf2 controls dopaminergic adaptations to alcohol in mice

A.C. Easton et al.Alcohol abuse leads to serious health problems with no effective treatment available. Recent evidence suggests a role for ras-specific guanine-nucleotide releasing factor 2 (RASGRF2) in alcoholism. Rasgrf2 is a calcium sensor and MAPK/ERK activating protein, which has been linked to neurotransmitter release and monoaminergic receptor adaptations. Rasgrf2 knock out (KO) mice do not develop a dopamine response in the nucleus accumbens after an alcohol challenge and show a reduced consumption of alcohol. The present study aims to further characterise the role of Rasgrf2 in dopaminergic activation beyond the nucleus accumbens following alcohol treatment. Using in vivo microdialysis we found that alcohol induces alterations in dopamine levels in the dorsal striatum between wildtype (WT) and Rasgrf2 KO mice. There was no difference in the expression of dopamine transporter (DAT), dopamine receptor regulating factor (DRRF), or dopamine D2 receptor (DRD2) mRNA in the brain between Rasgrf2 KO and WT mice. After sub-chronic alcohol treatment, DAT and DRRF, but not DRD2 mRNA expression differed between WT and Rasgrf2 KO mice. Brain adaptations were positively correlated with splenic expression levels. These data suggest that Rasgrf2 controls dopaminergic signalling and adaptations to alcohol also in other brain regions, beyond the nucleus accumbens.This work was supported by the Biomedical Research Council (U.K.) and funds of the Friedrich-Alexander-University Erlangen-Nuremberg. A.E. is a recipient of a studentship from the Medical Research Council, U.K. AFM and ES were supported by grants PI13/02846 and RD12/0036/0001 (MINECO) and SA181U13 (JCyL), Spain.Peer Reviewe

Rasgrf2 controls noradrenergic involvement in the acute and subchronic effects of alcohol in the brain

et al.[Rationale]: Alcohol addiction is a major psychiatric disease, and yet, the underlying molecular adaptations in the brain remain unclear. Recent evidence suggests a functional role for the ras-specific guanine-nucleotide releasing factor 2 (Rasgrf2) in alcoholism. Rasgrf2−/− mice consume less alcohol and show entirely absent dopamine responses to an alcohol challenge compared to wild types (WT). [Objective]: In order to further investigate how Rasgrf2 modifies the acute and subchronic effects of alcohol in the brain, we investigated its effects on the noradrenergic and serotonergic systems. [Methods]: We measured noradrenaline and serotonin activity in the brain by in vivo microdialysis and RNA expression by chip analysis and RT-PCR after acute and sub-chronic alcohol exposure in Rasgrf2−/− and WT mice. [Results]: In vivo microdialysis showed a significantly reduced noradrenergic response and an absent serotonergic response in the nucleus accumbens (NAcc) and caudate putamen (CPu) after an alcohol challenge in Rasgrf2−/− mice. A co-expression analysis showed that there is a high correlation between Rasgrf2 and α2 adrenoceptor RNA expression in the ventral striatum in naïve animals. Accordingly, we further assessed the role of Rasgrf2 in the response of the noradrenergic system to subchronic alcohol exposure. A decrease in β1 adrenoceptor gene expression was seen in Rasgrf2+/+, but not Rasgrf2−/− mice following alcohol exposure. Conversely, alcohol resulted in a decrease in both β2 and α2 adrenoceptor gene expression in knockout but not WT Rasgrf2 mice. [Conclusions]: These findings suggest that adaptations in the noradrenergic system contribute to the Rasgrf2 enhanced risk of alcoholism.This work was supported by funds of the Institute of Psychiatry, King’s College London and the Friedrich-Alexander-University Erlangen-Nuremberg. A.E. is a recipient of a studentship from the Medical Research Council (UK). AFM and ES were supported by FIS and RTICC grants from ISCIII (Spain).Peer Reviewe

Transcriptional profiling reveals functional links between RasGrf1 and Pttg1 in pancreatic beta cells

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License .[Background]: Our prior characterization of RasGrf1 deficient mice uncovered significant defects in pancreatic islet count and size as well as beta cell development and signaling function, raising question about the mechanisms linking RasGrf1 to the generation of those >pancreatic> phenotypes. [Results]: Here, we compared the transcriptional profile of highly purified pancreatic islets from RasGrf1 KO mice to that of WT control animals using commercial oligonucleotide microarrays. RasGrf1 elimination resulted in differential gene expression of numerous components of MAPK- and Calcium-signaling pathways, suggesting a relevant contribution of this GEF to modulation of cellular signaling in the cell lineages integrating the pancreatic islets. Whereas the overall transcriptional profile of pancreatic islets was highly specific in comparison to other organs of the same KO mice, a significant specific repression of Pttg1 was a common transcriptional alteration shared with other tissues of neuroectodermal origin. This observation, together with the remarkable pancreatic phenotypic similarities between RasGrf1 KO and Pttg1 KO mice suggested the possibility of proximal functional regulatory links between RasGrf1 and Pttg1 in pancreatic cell lineages expressing these proteins. [Conclusions]: The specific transcriptional profile and signaling behavior of RasgGrf1 KO pancreatic islets, together with the dominance of Pttg1 over RasGrf1 with regards to the generation of these phenotypes in mouse pancreas, suggest that RasGrf1 is an important upstream component of signal transduction pathways regulating Pttg1 expression and controlling beta cell development and physiological responses.Work supported by grants FIS PI13/02846 and RTICC RD12/0036/0001 from Instituto de Salud Carlos III (ISCIII), and grant SA181U13 from JCyL, Spain. We are grateful to Dr. Douglas Lowy (National Cancer Institute, Bethesda, MD) for providing plasmid pBK-CMV RasGrf1 and to Dr. Shlomo Melmed (Cedars-Sinai Medical Center, Los Angeles, CA) for providing reporter plasmid pGL3-Pttg1 and the single PTTG1 KO mouse strain used in these studies.Peer Reviewe

C3G-mediated suppression of oncogene-induced focus formation in fibroblasts involves inhibition of ERK activation, cyclin A expression and alterations of anchorage-independent growth

We showed previously that exogenous overexpression of C3G, a guanine nucleotide releasing factor (GEF) for Rap1 and R-Ras proteins, blocks the focus-forming activity of cotransfected, activated, sis, ras and v-raf oncogenes in NIH 3T3 cells. In this report, we show that C3G also interferes with dbl and R-Ras focus-forming activity and demonstrate that the transformation suppressor ability of C3G maps to its Crk-binding region (SH3-b domain). Using full-length C3G and C3GACat mutant, lacking catalytic domain, we showed here that overexpression of cotransfected C3G or C3GACat inhibited oncogenic Hras lys12-mediated phosphorylation of ERK, without altering Ras and Raf-1 kinase activation. We also showed that, overexpressed C3G and C3GΔCat inhibited the viability of oncogenic Ras-induced colonies in soft agar, indicating that C3G interferes with the anchorage-independent growth of Ras-transformed cells in a Rap1-independent manner. Consistent with both observations, overexpression of exogenous C3G and C3GΔCat also caused downregulation of Ras-induced cyclin A expression. Altogether, our results indicate that C3G interferes with at least two separate aspects of oncogenic transformation - cell cycle progression and loss of contact inhibition - and that these inhibitory effects probably account for its transformation suppressor activity.This work was supported by grants SAF00-0069 from MCYT and PI021570 from FIS, Spain. CG was supported by the Ramón y Cajal Program from MCYT.Peer Reviewe

The CSN3 subunit of the COP9 signalosome interacts with the HD region of Sos1 regulating stability of this GEF protein

Sos1 is an universal, widely expressed Ras guanine nucleotide-exchange factor (RasGEF) in eukaryotic cells. Its N-terminal HD motif is known to be involved in allosteric regulation of Sos1 GEF activity through intramolecular interaction with the neighboring PH domain. Here, we searched for other cellular proteins also able to interact productively with the Sos1 HD domain. Using a yeast two-hybrid system, we identified the interaction between the Sos1 HD region and CSN3, the third component of the COP9 signalosome, a conserved, multi-subunit protein complex that functions in the ubiquitin-proteasome pathway to control degradation of many cellular proteins. The interaction of CSN3 with the HD of Sos1 was confirmed in vitro by GST pull-down assays using truncated mutants and reproduced in vivo by co-immunoprecipitation with the endogenous, full-length cellular Sos1 protein. In vitro kinase assays showed that PKD, a COP9 signalosome-associated-kinase, is able to phosphorylate Sos1. The intracellular levels of Sos1 protein were clearly diminished following CSN3 or PKD knockdown. A sizable fraction of the endogenous Sos1 protein was found ubiquitinated in different mammalian cell types. A significant reduction of RasGTP formation upon growth factor stimulation was also observed in CSN3-silenced as compared with control cells. Our data suggest that the interaction of Sos1 with the COP9 signalosome and PKD plays a significant role in maintenance of cellular Sos1 protein stability and homeostasis under physiological conditions and raises the possibility of considering the CSN/PKD complex as a potential target for design of novel therapeutic drugs.We thank R Brent for the pJG45-HeLa library and R. Jorge for help with yeast two-hybrid screening. J.M.R. received grant support from MINECO-FEDER (SAF2016-78852-R), ISCIII-MINECO (FIS-Intrasalud PI13/00703) and Spanish Association against Cancer (AECC). E.S. and A.F.M. were supported by grants from ISCIII-MINECO (FIS PI16/02137), JCyL (SA043U16-UIC 076) and Solorzano Foundation. E.S. and J.M.R. were also supported by ISCIII-RETIC (groups RTICC-RD12/0036/0001 and RTICC-RD12/0036/0021, respectively) and by CIBERONC (groups CB16/12/00352 and CB16/12/00273, respectively). Research co-financed by FEDER funds.S

PKD phosphorylation and COP9/Signalosome modulate intracellular Spry2 protein stability

Spry2 is a molecular modulator of tyrosine kinase receptor signaling pathways that has cancer-type-specific effects. Mammalian Spry2 protein undergoes tyrosine and serine phosphorylation in response to growth factor stimulation. Spry2 expression is distinctly altered in various cancer types. Inhibition of the proteasome functionality results in reduced intracellular Spry2 degradation. Using in vitro and in vivo assays, we show that protein kinase D (PKD) phosphorylates Spry2 at serine 112 and interacts in vivo with the C-terminal half of this protein. Importantly, missense mutation of Ser112 decreases the rate of Spry2 intracellular protein degradation. Either knocking down the expression of all three mammalian PKD isoforms or blocking their kinase activity with a specific inhibitor contributes to the stabilization of Spry2 wild-type protein. Downregulation of CSN3, a component of the COP9/Signalosome that binds PKD, significantly increases the half-life of Spry2 wild-type protein but does not affect the stability of a Spry2 after mutating Ser112 to the non-phosphorylatable residue alanine. Our data demonstrate that both PKD and the COP9/Signalosome play a significant role in control of Spry2 intracellular stability and support the consideration of the PKD/COP9 complex as a potential therapeutic target in tumors where Spry2 expression is reduced.JMR-C received grant support from MINECO-FEDER (SAF2016-78852-R), AESI-ISCIII (PI20CIII/00029) and Spanish Association against Cancer (AECC, CGB14143035THOM). ES group was supported by grants from ISCIII-MCUI (FIS PI19/00934), JCyL (SA264P18-UIC-076), Areces Foundation (CIVP19A5942), Solorzano-Barruso Foundation (FS/32–2020) and by ISCIII-CIBERONC (group CB16/12/00352). Funding to AM group was provided by the Agencia Estatal de Investigación (PID2019-104867RB-I00/AEI/10.13039/501100011033) and by ISCIII-CIBERONC (group CB16/12/00273). TI was supported by grant PID2020-115218RB-I00 funded by MCIN/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe” and by ISCIII-CIBERNED. RB received grant support from AESI-ISCIII (PI20CIII/00019). Finally, DP-J and MY groups were supported by grants 1.012.022 (to DP-J), 1.010.929 and 1.400.002 (both to MY) from Fundación Universidad Alfonso X el Sabio (FUAX). All research co-financed by FEDER funds.S

Anthraquinones as Inhibitors of SOS RAS-GEF Activity

Recent breakthroughs have reignited interest in RAS GEFs as direct therapeutic targets. To search for new inhibitors of SOS GEF activity, a repository of known/approved compounds (NIH-NACTS) and a library of new marine compounds (Biomar Microbial Technologies) were screened by means of in vitro RAS-GEF assays using purified, bacterially expressed SOS and RAS constructs. Interestingly, all inhibitors identified in our screenings (two per library) shared related chemical structures belonging to the anthraquinone family of compounds. All our anthraquinone SOS inhibitors were active against the three canonical RAS isoforms when tested in our SOS GEF assays, inhibited RAS activation in mouse embryonic fibroblasts, and were also able to inhibit the growth of different cancer cell lines harboring WT or mutant RAS genes. In contrast to the commercially available anthraquinone inhibitors, our new marine anthraquinone inhibitors did not show in vivo cardiotoxicity, thus providing a lead for future discovery of stronger, clinically useful anthraquinone SOS GEF blockers

Ras in Cancer and Developmental Diseases

Somatic, gain-of-function mutations in ras genes were the first specific genetic alterations identified in human cancer about 3 decades ago. Studies during the last quarter century have characterized the Ras proteins as essential components of signaling networks controlling cellular proliferation, differentiation, or survival. The oncogenic mutations of the H-ras, N-ras, or K-ras genes frequently found in human tumors are known to throw off balance the normal outcome of those signaling pathways, thus leading to tumor development. Oncogenic mutations in a number of other upstream or downstream components of Ras signaling pathways (including membrane RTKs or cytosolic kinases) have been detected more recently in association with a variety of cancers. Interestingly, the oncogenic Ras mutations and the mutations in other components of Ras/MAPK signaling pathways appear to be mutually exclusive events in most tumors, indicating that deregulation of Ras-dependent signaling is the essential requirement for tumorigenesis. In contrast to sporadic tumors, separate studies have identified germline mutations in Ras and various other components of Ras signaling pathways that occur in specific association with a number of different familial, developmental syndromes frequently sharing common phenotypic cardiofaciocutaneous features. Finally, even without being a causative force, defective Ras signaling has been cited as a contributing factor to many other human illnesses, including diabetes and immunological and inflammatory disorders. We aim this review at summarizing and updating current knowledge on the contribution of Ras mutations and altered Ras signaling to development of various tumoral and nontumoral pathologies

The reversed intra- and extracellular pH in tumors as a unified strategy to chemotherapeutic delivery using targeted nanocarriers

Solid tumors are complex entities, comprising a wide variety of malignancies with very different molecular alterations. Despite this, they share a set of characteristics known as “hallmarks of cancer” that can be used as common therapeutic targets. Thus, every tumor needs to change its metabolism in order to obtain the energy levels required for its high proliferative rates, and these adaptations lead to alterations in extra- and intracellular pH. These changes in pH are common to all solid tumors, and can be used either as therapeutic targets, blocking the cell proton transporters and reversing the pH changes, or as means to specifically deliver anticancer drugs. In this review we will describe how proton transport inhibitors in association with nanocarriers have been designed to block the pH changes that are needed for cancer cells to survive after their metabolic adaptations. We will also describe studies aiming to decrease intracellular pH in cancer using nanoparticles as molecular cages for protons which will be released upon UV or IR light exposure. Finally, we will comment on several studies that have used the extracellular pH in cancer for an enhanced cell internalization and tumor penetration of nanocarriers and a controlled drug delivery, describing how nanocarriers are being used to increase drug stability and specificity