CXCR1 in breast cancer

Scientists from the University of Michigan and the INSERM (France) identified Cxcr1 gene as differentially expressed in cancer stem cells in comparison with other cancer cells in breast malignancies.
CXCR1 is the receptor for interleukin 8 and has been involved in tumour progression and metastasis in several kinds of cancers, such as prostate, glioma, ovarian and breast cancers. Furthermore, IL8 has been implicated in self renewal of stem cells in vitro. French researchers tested some small molecule inhibitors targeting CXCR1 or some antibody against this receptor in order to evaluate the effect on cellular behavior. These inhibitors directly acted versus cancer stem cells and at the same time indirectly induced cell death in bulk tumour. The promising result was that the whole cancer population was eliminated. A possible explanation for this larger effect could be the release of FAS ligand after inhibiting CXCR1 that determined apoptosis in all cancer cells. In animal model CXCR1 inhibitors reduced tumour mass and blocked metastasis, either alone or in combination with other drugs. Another possible approach to block cancer progression by acting on CXCR1 pathway is to interfere with IL8 production. Scientists from the University of Texas demonstrated that siRNA-IL8 reduced tumour weight in comparison to control in animal models. These studies seem really promising but further proof is necessary to validate CXCR1 pathway as a target for therapeutic intervention.

Databases

Two main databases are now available: the EMBL-EBI and the NCBI for Europe and US, respectively. These two databases are connected and all the information present are available in both systems. Another database is provided by a Japanese laboratory and is online at genome.jp.

Main databases contain information about DNA sequence, two examples are EMBL datalibrary and GenBank; all other databases regarding RNA, proteins and polymorphisms or rare diseases are connected to these two ones. Databases are usually checked by operators or software: the difference between these two control systems could be observed in the redundancy because manual control is usually more systematic than these performed by software. About proteins, three secondary databases are currently used: Swiss-Prot, TrEMBL and PIR. In these websites several bioinformatic tools are available to align sequences, predict primary and secondary structure of proteins or determine the isoelectric point, all these information are important especially at the beginning of the study. Other databases like PDB or Modbase offer three-dimensional structures of proteins and prediction of three-dimensional structures, respectively. As well as PROSITE collects information about protein motifs, functional domains and so on. Last but not least, Genome.jp is preparing a new tool, KEGG pathway, useful to retrieve information about enzymes and metabolic pathway. Good work!

Kinase activity profiling

In previous posts we have already discussed about kinases and phosphorylation detection. Today, we would like to focus our attention on one new method to profile kinase activity during cell cycle, pharmacological inhibition, cancer, signalling pathway activation. In June 2009, PNAS journal reported this approach: researchers of the Department of Cell Biology in the Harvard Medical School monitored the activation state of kinases in cell lysate by analysing the phosphorylation of 90 synthetic peptides, known as substrates, through mass spectrometry.

As an internal standard for quantification, they used heavy isotope-labelled peptides. The assay is quite easy to do: they used 96 format and plated total lysate and ATP, then they added the substrate to phosphorylation reaction and measured with liquid chromatography coupled with mass spectrometry. In this way they produced a fingerprint of kinomes of several cell lines of breast cancer, exactly showing which pathways were activated. In the paper they presented a novel Src family site in vivo, but this approach could have other important applications. For instance, it’s possible to compare the activation state of pathway in normal and tumoral cells, indeed in cancer alterations in kinase activity are often reported. Furthermore, the quantification of phosphorylation is important to check inhibitory properties of small molecule kinase inhibitors.

Nanog and pluripotency

In August 2009 Cell journal published an important article about the role of Nanog in pluripotency. Silva and colleagues showed that Nanog played a crucial role in development of pluripotency in mice. Indeed, they generated a KO mouse for nanog gene and they observed a block in reprogramming cells to achieve the full pluripotency; Nanog re-expression allowed the complete restoration of the reprogramming process indicating the central role of this protein in the acquisition of pluripotency.

Researchers were not able to obtain embryonic stem cell culture from nanog null mice because the embryos didn’t form the inner cell mass and so the pluripotent compartment. Molecular explanation of the role of Nanog is probably related to the signalling pathway that involves also Oct4, it seems that Nanog is important to sustain that pathway by inducing a self maintenance of the system. Nanog is also important for the reactivation of chromosome X, another crucial step to reach the pluripotency. This work increases our knowledge to obtain the induction of pluripotency in vitro: this goal is crucial for instance for many applications of regenerative medicine. Defining the factors that control, start and maintain pluripotency will help us to obtain safer and better induced pluripotent stem cell cultures.

Signalling pathways as network of functional modules

Signalling pathways are usually described as a chain of consequential events, where proteins interact and transfer signal from external environment to nucleus. If some mutation occurs, all pathway is deregulated and it’s important to know where the mutation is in order to choose the correct approach to normalize it.

Is it possible to understand where is the mutation from genomic signature? It is! In Nature Reviews Genetics of June 2009 a new vision of signalling pathways is presented. Instead of a chain of reactions, functional modules are considered on basis of gene expression signature. From NCI-60 database authors identified genes related to the core protein of the pathway that showed the same variation of expression as this core gene.

For each pathway/ core protein several signatures have been identified and by relating signatures to mutants that selectively activate a specific downstream effector, it has been possible to assign each functional module (core protein and downstream effector) to genomic signature.

Which are the therapeutic implications of this new approach? For instance, it’s possible to define where is the mutation directly from genomic analysis, in this way a correct therapeutic approach can be chosen. Now, we have to increase our knowledge about the relationship between genomic signature and signalling pathway.