Targeting Cancer by Using Nanoparticles to Modulate RHO GTPase Signaling.

Functionalized nanomaterials have recently been introduced as efficient vehicles for targeted delivery of drugs and other tailored molecules to cancer cells. They emerge as new opportunities for addressing particular challenging targets such as RHO guanosine triphosphatases (GTPases), a group of signaling molecules involved in the progression of a variety of tumor types. RHO GTPases comprise a subfamily of the Ras superfamily of small GTPases. They are best known for their role in cell migration through the remodeling of the actin cytoskeleton. However, they are also key regulators of a broad number of cellular functions, ranging from proliferation to cell adhesion and differentiation. Not surprisingly, their dysregulation has been implicated in the development and progression of many types of cancer. The RHO GTPase subfamily includes 20 members that can be further separated into typical and atypical RHO GTPases. The typical RHO family members include the classical RHOA, RAC1 and CDC42 proteins, which cycle between an active GTP-bound and inactive GDP-bound conformation, under the coordinated action of three types of regulators: GEFs, GAPs and GDIs. Atypical RHO family members have small changes in key residues that alter their regulatory mechanisms. Nevertheless, both typical and atypical RHO GTPases contribute to cancer progression but, in contrast to Ras proteins, very few mutations have been found in tumors. In most cancers, it is the expression level and/or activity of RHO GTPases that is dysregulated. RHO GTPase signaling has thus long been seen as an attractive target for cancer treatment but their ubiquity and the lack of isoform-specific drugs have posed significant obstacles to the development of viable therapeutic strategies. Based on the success of recent nanomedicine approaches, this chapter reviews representative studies of how functionalized nanoparticles can be designed to target tumor-specific molecules and directly or indirectly modulate the expression and/or activity of particular RHO GTPases in cancer cells.

Pro-Inflammatory Cytokines Trigger the Overexpression of Tumour-Related Splice Variant RAC1B in Polarized Colorectal Cells.

An inflammatory microenvironment is a tumour-promoting condition that provides survival signals to which cancer cells respond with gene expression changes. One example is the alternative splicing variant Rat Sarcoma Viral Oncogene Homolog (Ras)-Related C3 Botulinum Toxin Substrate 1 (RAC1)B, which we previously identified in a subset of V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF)-mutated colorectal tumours. RAC1B was also increased in samples from inflammatory bowel disease patients or in an acute colitis mouse model. Here, we used an epithelial-like layer of polarized Caco-2 or T84 colorectal cancer (CRC) cells in co-culture with fibroblasts, monocytes or macrophages and analysed the effect on RAC1B expression in the CRC cells by RT-PCR, Western blot and confocal fluorescence microscopy. We found that the presence of cancer-associated fibroblasts and M1 macrophages induced the most significant increase in RAC1B levels in the polarized CRC cells, accompanied by a progressive loss of epithelial organization. Under these conditions, we identified interleukin (IL)-6 as the main trigger for the increase in RAC1B levels, associated with Signal Transducer and Activator of Transcription (STAT)3 activation. IL-6 neutralization by a specific antibody abrogated both RAC1B overexpression and STAT3 phosphorylation in polarized CRC cells. Our data identify that pro-inflammatory extracellular signals from stromal cells can trigger the overexpression of tumour-related RAC1B in polarized CRC cells. The results will help to understand the tumour-promoting effect of inflammation and identify novel therapeutic strategies.

Cytotoxicity and genotoxicity of MWCNT-7 and crocidolite: assessment in alveolar epithelial cells versus their coculture with monocyte-derived macrophages.

In the past years, several in vitro studies have addressed the pulmonary toxicity of multi-walled carbon nanotubes (MWCNT) and compared it with that caused by asbestos fibers, but their conclusions have been somewhat inconsistent and difficult to extrapolate to in vivo. Since cell coculture models were proposed to better represent the in vivo conditions than conventional monocultures, this work intended to compare the cytotoxicity and genotoxicity of MWCNT-7 (Mitsui-7) and crocidolite using A549 cells grown in a conventional monoculture or in coculture with THP-1 macrophages. Although a decrease in A549 viability was noted following exposure to a concentration range of MWCNT-7 and crocidolite, no viability change occurred in similarly exposed cocultures. Early events indicating epithelial to mesenchymal transition (EMT) were observed which could explain apoptosis resistance. The comet assay results were similar between the two models, being positive and negative for crocidolite and MWCNT-7, respectively. An increase in the micronucleus frequency was detected in the cocultured A549-treated cells with both materials, but not in the monoculture. On the other hand, exposure of A549 monocultures to MWCNT-7 induced a highly significant increase in nucleoplasmic bridges in which those were found embedded. Our overall results demonstrate that (i) both materials are cytotoxic and genotoxic, (ii) the presence of THP-1 macrophages upholds the viability of A549 cells and increases the aneugenic/clastogenic effects of both materials probably through EMT, and (iii) MWCNT-7 induces the formation of nucleoplasmic bridges in A549 cells.

Signaling Pathways Driving Aberrant Splicing in Cancer Cells.

Aberrant profiles of pre-mRNA splicing are frequently observed in cancer. At the molecular level, an altered profile results from a complex interplay between chromatin modifications, the transcriptional elongation rate of RNA polymerase, and effective binding of the spliceosome to the generated transcripts. Key players in this interplay are regulatory splicing factors (SFs) that bind to gene-specific splice-regulatory sequence elements. Although mutations in genes of some SFs were described, a major driver of aberrant splicing profiles is oncogenic signal transduction pathways. Signaling can affect either the transcriptional expression levels of SFs or the post-translational modification of SF proteins, and both modulate the ratio of nuclear versus cytoplasmic SFs in a given cell. Here, we will review currently known mechanisms by which cancer cell signaling, including the mitogen-activated protein kinases (MAPK), phosphatidylinositol 3 (PI3)-kinase pathway (PI3K) and wingless (Wnt) pathways but also signals from the tumor microenvironment, modulate the activity or subcellular localization of the Ser/Arg rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) families of SFs.

The third dimension: new developments in cell culture models for colorectal research.

Cellular models are important tools in various research areas related to colorectal biology and associated diseases. Herein, we review the most widely used cell lines and the different techniques to grow them, either as cell monolayer, polarized two-dimensional epithelia on membrane filters, or as three-dimensional spheres in scaffold-free or matrix-supported culture conditions. Moreover, recent developments, such as gut-on-chip devices or the ex vivo growth of biopsy-derived organoids, are also discussed. We provide an overview on the potential applications but also on the limitations for each of these techniques, while evaluating their contribution to provide more reliable cellular models for research, diagnostic testing, or pharmacological validation related to colon physiology and pathophysiology.

Phosphorylation of SRSF1 by SRPK1 regulates alternative splicing of tumor-related Rac1b in colorectal cells.

The premessenger RNA of the majority of human genes can generate various transcripts through alternative splicing, and different tissues or disease states show specific patterns of splicing variants. These patterns depend on the relative concentrations of the splicing factors present in the cell nucleus, either as a consequence of their expression levels or of post-translational modifications, such as protein phosphorylation, which are determined by signal transduction pathways. Here, we analyzed the contribution of protein kinases to the regulation of alternative splicing variant Rac1b that is overexpressed in certain tumor types. In colorectal cells, we found that depletion of AKT2, AKT3, GSK3ß, and SRPK1 significantly decreased endogenous Rac1b levels. Although knockdown of AKT2 and AKT3 affected only Rac1b protein levels suggesting a post-splicing effect, the depletion of GSK3ß or SRPK1 decreased Rac1b alternative splicing, an effect mediated through changes in splicing factor SRSF1. In particular, the knockdown of SRPK1 or inhibition of its catalytic activity reduced phosphorylation and subsequent translocation of SRSF1 to the nucleus, limiting its availability to promote the inclusion of alternative exon 3b into the Rac1 pre-mRNA. Altogether, the data identify SRSF1 as a prime regulator of Rac1b expression in colorectal cells and provide further mechanistic insight into how the regulation of alternative splicing events by protein kinases can contribute to sustain tumor cell survival.