Collective cell migration of thyroid carcinoma cells: a beneficial ability to override unfavourable substrates

Purpose

Tumor cell invasion and metastasis are life threatening events. Invasive tumor cells tend to migrate as collective sheets. In the present in vitro study we aimed to (i) assess whether collective tumor cells gain benefits in their migratory potential compared to single cells and (ii) to identify its putative underlying molecular mechanisms.

Methods

The migratory potential of single and collective carcinoma cells was assessed using video time lapse microscopy and cell migration assays in the absence and presence of seven potential gap junction inhibitors or the Rac1 inhibitor Z62954982. The perturbation of gap junctions was assessed using a dye diffusion assay. In addition, LDH-based cytotoxicity and RT-PCR-based expression analyses were performed.

Results

Whereas single breast, cervix and thyroid carcinoma cells were virtually immobile on unfavourable plastic surfaces, we found that they gained pronounced migratory capacities as collectives under comparable conditions. Thyroid carcinoma cells, that were studied in more detail, were found to express specific subsets of connexins and to form active gap junctions as revealed by dye diffusion analysis. Although all potential gap junction blockers suppressed intercellular dye diffusion in at least one of the cell lines tested, only two of them were found to inhibit collective cell migration and none of them to inhibit single cell migration. In the presence of the Rac1 inhibitor Z62954982 collective migration, but not single cell migration, was found to be reduced up to 20 %.

Conclusions

Our data indicate that collective migration enables tumor cells to cross otherwise unfavourable substrate areas. This capacity seems to be independent of intercellular communication via gap junctions, whereas Rac1-dependent intracellular signalling seems to be essential.

     

MicroRNA in rectal cancer

In rectal cancer, one of the most common cancers worldwide, the proper staging of the disease determines the subsequent therapy. For those with locally advanced rectal cancer, a neoadjuvant chemoradiotherapy (CRT) is recommended before any surgery. However, response to CRT ranges from complete response (responders) to complete resistance (non-responders). To date we are not able to separate in advance the first group from the second, due to the absence of a valid biomarker. Therefore all patients receive the same therapy regardless of whether they reap benefits. On the other hand almost all patients receive a surgical resection after the CRT, although a watch-and-wait procedure or an endoscopic resection might be sufficient for those who responded well to the CRT. Being highly conserved regulators of gene expression, microRNAs (miRNAs) seem to be promising candidates for biomarkers. Many studies have been analyzing the miRNAs expressed in rectal cancer tissue to determine a specific miRNA profile for the ailment. Unfortunately, there is only a small overlap of identified miRNAs between different studies, posing the question as to whether different methods or differences in tissue storage may contribute to that fact or if the results simply are not reproducible, due to unknown factors with undetected influences on miRNA expression. Other studies sought to find miRNAs which correlate to clinical parameters (tumor grade, nodal stage, metastasis, survival) and therapy response. Although several miRNAs seem to have an impact on the response to CRT or might predict nodal stage, there is still only little overlap between different studies. We here aimed to summarize the current literature on rectal cancer and miRNA expression with respect to the different relevant clinical parameters.     

Molecular mechanism of selectivity among G protein-coupled receptor kinase 2 inhibitors

G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-Gβγ complex in the presence of two of these inhibitors. Comparison with the apoGRK2-Gβγ structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.     

In vivo brain microdialysis: advances in neuropsychopharmacology and drug discovery

INTRODUCTION: Microdialysis is an important in vivo sampling technique, useful in the assay of extracellular tissue fluid. The technique has both pre-clinical and clinical applications but is most widely used in neuroscience. The in vivo microdialysis technique allows measurement of neurotransmitters such as acetycholine (ACh), the biogenic amines including dopamine (DA), norepinephrine (NE) and serotonin (5-HT), amino acids such as glutamate (Glu) and gamma aminobutyric acid (GABA), as well as the metabolites of the aforementioned neurotransmitters, and neuropeptides in neuronal extracellular fluid in discrete brain regions of laboratory animals such as rodents and non-human primates. AREAS COVERED: In this review we present a brief overview of the principles and procedures related to in vivo microdialysis and detail the use of this technique in the pre-clinical measurement of drugs designed to be used in the treatment of chemical addiction, neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and as well as psychiatric disorders such as attention-deficit/hyperactivity disorder (ADHD) and schizophrenia. This review offers insight into the tremendous utility and versatility of this technique in pursuing neuropharmacological investigations as well its significant potential in rational drug discovery. EXPERT OPINION: In vivo microdialysis is an extremely versatile technique, routinely used in the neuropharmacological investigation of drugs used for the treatment of neurological disorders. This technique has been a boon in the elucidation of the neurochemical profile and mechanism of action of several classes of drugs especially their effects on neurotransmitter systems. The exploitation and development of this technique for drug discovery in the near future will enable investigational new drug candidates to be rapidly moved into the clinical trial stages and to market thus providing new successful therapies for neurological diseases that are currently in demand.     

A genomic strategy for the functional validation of colorectal cancer genes identifies potential therapeutic targets

Genes that are highly overexpressed in tumor cells can be required for tumor cell survival and have the potential to be selective therapeutic targets. In an attempt to identify such targets, we combined a functional genomics and a systems biology approach to assess the consequences of RNAi‐mediated silencing of overexpressed genes that were selected from 140 gene expression profiles from colorectal cancers (CRCs) and matched normal mucosa. In order to identify credible models for in‐depth functional analysis, we first confirmed the overexpression of these genes in 25 different CRC cell lines. We then identified five candidate genes that profoundly reduced the viability of CRC cell lines when silenced with either siRNAs or short‐hairpin RNAs (shRNAs), i.e., HMGA1, TACSTD2, RRM2, RPS2 and NOL5A. These genes were further studied by systematic analysis of comprehensive gene expression profiles generated following siRNA‐mediated silencing. Exploration of these RNAi‐specific gene expression signatures allowed the identification of the functional space in which the five genes operate and showed enrichment for cancer‐specific signaling pathways, some known to be involved in CRC. By comparing the expression of the RNAi signature genes with their respective expression levels in an independent set of primary rectal carcinomas, we could recapitulate these defined RNAi signatures, therefore, establishing the biological relevance of our observations. This strategy identified the signaling pathways that are affected by the prominent oncogenes HMGA1 and TACSTD2, established a yet unknown link between RRM2 and PLK1 and identified RPS2 and NOL5A as promising potential therapeutic targets in CRC.     

In tumor cells regulation of DNA double strand break repair through EGF receptor involves both NHEJ and HR and is independent of p53 and K-Ras status

Purpose

The purpose of this study was to examine whether the epidermal growth factor receptor (EGFR) may be used as a general target to modulate DNA double strand break (DSB) repair in tumor cells.

Material and methods

Experiments were performed with human tumor cell lines A549, H1299 and HeLa and primate cell line CV1. EGF, ARG and TGFα were used for EGFR activation, cetuximab or erlotinib for inhibition. Overall DSB repair was assessed by γH2AX/53BP1 co-immunostaining and non-homologous end-joining (NHEJ) and homologous recombination (HR) by using NHEJ and HR reporter cells; cell cycle distribution was determined by flow cytometry and protein expression by Western blot.

Results

EGFR activation was found to stimulate overall DSB repair as well as NHEJ regardless of the ligand used. This stimulation was abolished when EGFR signaling was blocked. This regulation was found for all cell lines tested, irrespective of their p53 or K-Ras status. Stimulation and inhibition of EGFR were also found to affect HR.

Conclusions

Regulation of DSB repair by EGFR involves both the NHEJ and HR pathway, and appears to occur in most tumor cell lines regardless of p53 and K-Ras mutation status.