Laura Carraresi

Expression, purification and kinetic behaviour of fission yeast low Mr protein-tyrosine phosphatase

A gene named stp1 +, coding for a 17.5‐kDa protein, that rescues cdc25‐22 when overexpressed, has been previously isolated from fission yeast. Here we describe the expression and purification of Stp1 protein as a fusion with the glutathione S‐transferase in E. coli and its kinetic characterisation. Stp1 deduced protein sequence shows an high homology to members of a class of cytosolic low M r protein phosphatase previously known to exist only in mammalian species. Stp1 has a kinetic behaviour that appears to be intermediate with respect to the two isoenzymatic forms of low M r protein tyrosine phosphatases present in mammalian tissues. These differing kinetic characteristics are mainly due to the sequence 45–56 that is spatially close to the active site pocket.

Expression of the small tyrosine phosphatase (Stp1) in Saccharomyces cerevisiae: a study on protein tyrosine phosphorylation.

Small tyrosine phoshatase 1 (Stp1) is a Schizosaccharomyces pombe low‐molecular‐mass phosphotyrosine‐phosphatase 50% identical to Saccharomyces cerevisiae Ltp1. In order to investigate the role of Stp1 in yeast, a mutant was generated having the characteristic of a dominant negative molecule. Changes in protein tyrosine phosphorylation in S. cerevisiae proteome in response to Stp1 or its dominant negative mutant expression were analyzed by high‐resolution two‐dimensional (2‐D) electrophoresis. The most remarkable result is the modification by phosphorylation on tyrosine of several proteins involved in carbohydrate metabolism. Twelve proteins were identified on the basis of their positions in the anti‐phosphotyrosine immunoblot of the 2‐D electrophoresis. Ten of these present tyrosyl residues that are within the consensus sequence for protein kinase CK2 (casein kinase‐2). These data open the possibility for the identification of Stp1 substrates in yeast and provide hints about the nature of tyrosine phosphorylating agents in yeast and in other organisms where bona fide tyrosine kinases are lacking.

The conformational state of hERG1 channels determines integrin association, downstream signaling, and cancer progression

Whether hERG1 potassium channels promote proliferation or metastasis in breast cancer cells depends on channel conformation. Channeling proliferation or metastasis The hERG1 potassium channel is best known for its role in repolarizing excitable cells such as cardiomyocytes, but the abundance of this cardiac channel is aberrantly high in cancer cells. Becchetti et al. investigated the interaction of hERG1 with the β1 integrin subunit, a member of a family of adhesion molecules. Forms of hERG1 with mutations that fixed the channel in the open conformation more weakly interacted with β1 integrin in cells and enhanced proliferation when expressed in breast cancer cells injected into mice. However, K+ flow through open hERG1 channels enhanced the activation of FAK downstream of β1 integrin and promoted metastasis in breast cancer cells injected into mice. Thus, whether hERG1 promotes proliferation or metastasis in cancer cells depends on the conformation of the channel and suggests that hERG1 inhibitors that are tailored to the channel conformation could be used to prevent different aspects of tumor progression. Ion channels regulate cell proliferation, differentiation, and migration in normal and neoplastic cells through cell-cell and cell–extracellular matrix (ECM) transmembrane receptors called integrins. K+ flux through the human ether-à-go-go–related gene 1 (hERG1) channel shapes action potential firing in excitable cells such as cardiomyocytes. Its abundance is often aberrantly high in tumors, where it modulates integrin-mediated signaling. We found that hERG1 interacted with the β1 integrin subunit at the plasma membrane of human cancer cells. This interaction was not detected in cardiomyocytes because of the presence of the hERG1 auxiliary subunit KCNE1 (potassium voltage-gated channel subfamily E regulatory subunit 1), which blocked the β1 integrin–hERG1 interaction. Although open hERG1 channels did not interact as strongly with β1 integrins as did closed channels, current flow through hERG1 channels was necessary to activate the integrin-dependent phosphorylation of Tyr397 in focal adhesion kinase (FAK) in both normal and cancer cells. In immunodeficient mice, proliferation was inhibited in breast cancer cells expressing forms of hERG1 with impaired K+ flow, whereas metastasis of breast cancer cells was reduced when the hERG1/β1 integrin interaction was disrupted. We conclude that the interaction of β1 integrins with hERG1 channels in cancer cells stimulated distinct signaling pathways that depended on the conformational state of hERG1 and affected different aspects of tumor progression.

Characterization of hERG1 channel role in mouse colorectal carcinogenesis

The human ether‐à‐go‐go‐related gene (hERG)1 K+ channel is upregulated in human colorectal cancer cells and primary samples. In this study, we examined the role of hERG1 in colorectal carcinogenesis using two mouse models: adenomatous polyposis coli (Apcmin/+) and azoxymethane (AOM)‐treated mice. Colonic polyps of Apcmin/+ mice overexpressed mERG1 and their formation was reverted by the hERG1 blocker E4031. AOM was applied to either hERG1‐transgenic (TG) mice, which overexpress hERG1 in the mucosa of the large intestine, or wild‐type mice. A significant increase of both mucin‐depleted foci and polyps in the colon of hERG1‐TG mice was detected. Both the intestine of TG mice and colonic polyps of Apcmin/+ showed an upregulation of phospho‐Protein Kinase B (pAkt)/vascular endothelial growth factor (VEGF‐A) and an increased angiogenesis, which were reverted by treatment with E4031. On the whole, this article assigns a relevant role to hERG1 in the process of in vivo colorectal carcinogenesis.

hERG1 behaves as biomarker of progression to adenocarcinoma in Barrett’s esophagus and can be exploited for a novel endoscopic surveillance

Barretts esophagus (BE) is the only well-known precursor lesion of esophageal adenocarcinoma (EA). The exact estimates of the annual progression rate from BE to EA vary from 0.07% to 3.6%. The identification of BE patients at higher risk to progress to EA is hence mandatory, although difficult to accomplish. In search of novel BE biomarkers we analyzed the efficacy of hERG1 potassium channels in predicting BE progression to EA. Once tested by immunohistochemistry (IHC) on bioptic samples, hERG1 was expressed in BE, and its expression levels increased during progression from BE to esophageal dysplasia (ED) and EA. hERG1 was also over-expressed in the metaplastic cells arising in BE lesions obtained in different BE mouse models, induced either surgically or chemically. Furthermore, transgenic mice which over express hERG1 in the whole gastrointestinal tract, developed BE lesions after an esophago-jejunal anastomosis more frequently, compared to controls. A case-control study was performed on 104 bioptic samples from newly diagnosed BE patients further followed up for at least 10 years. It emerged a statistically significant association between hERG1 expression status and risk of progression to EA. Finally, a novel fluorophore- conjugated recombinant single chain variable fragment antibody (scFv-hERG1-Alexa488) was tested on freshly collected live BE biopsies: it could recognize hERG1 positive samples, perfectly matching IHC data. Overall, hERG1 can be considered a novel BE biomarker to be exploited for a novel endoscopic surveillance protocol, either in biopsies or through endoscopy, to identify those BE patients with higher risk to progress to EA.

Generation and characterization of novel recombinant anti-hERG1 scFv antibodies for cancer molecular imaging

Modern molecular imaging techniques have greatly improved tumor detection and post-treatment follow-up of cancer patients. In this context, antibody-based imaging is rapidly becoming the gold standard, since it combines the unique specificity of antibodies with the sensitivity of the different imaging technologies. The aim of this study was to generate and characterize antibodies in single chain Fragment variable (scFv) format directed to an emerging cancer biomarker, the human ether-à-go-go-related gene-1 (hERG1) potassium channel, and to obtain a proof of concept for their potential use for in vivo molecular imaging. The anti-hERG1scFv was generated from a full length monoclonal antibody and then mutagenized, substituting a Phenylalanine residue in the third framework of the VH domain with a Cysteine residue. The resulting scFv-hERG1-Cys showed much higher stability and protein yield, increased affinity and more advantageous binding kinetics, compared to the “native” anti-hERG1scFv. The scFv-hERG1-Cys was hence chosen and characterized: it showed a good binding to the native hERG1 antigen expressed on cells, was stable in serum and displayed a fast pharmacokinetic profile once injected intravenously in nude mice. The calculated half-life was 3.1 hours and no general toxicity or cardiac toxic effects were detected. Finally, the in vivo distribution of an Alexa Fluor 750 conjugated scFv-hERG1-Cys was evaluated both in healthy and tumor-bearing nude mice, showing a good tumor-to-organ ratio, ideal for visualizing hERG1-expressing tumor masses in vivo. In conclusion, the scFv-hERG1-Cys possesses features which make it a suitable tool for application in cancer molecular imaging.