ETP-46321, a dual p110α/δ class IA phosphoinositide 3-kinase inhibitor modulates T lymphocyte activation and collagen-induced arthritis.

Class IA phosphoinositide 3-kinases (PI3Ks) are essential to function of normal and tumor cells, and to modulate immune responses. T lymphocytes express high levels of p110α and p110δ class IA PI3K. Whereas the functioning of PI3K p110δ in immune and autoimmune reactions is well established, the role of p110α is less well understood. Here, a novel dual p110α/δ inhibitor (ETP-46321) and highly specific p110α (A66) or p110δ (IC87114) inhibitors have been compared concerning T cell activation in vitro, as well as the effect on responses to protein antigen and collagen-induced arthritis in vivo. In vitro activation of naive CD4(+) T lymphocytes by anti-CD3 and anti-CD28 was inhibited more effectively by the p110δ inhibitor than by the p110α inhibitor as measured by cytokine secretion (IL-2, IL-10, and IFN-γ), T-bet expression and NFAT activation. In activated CD4(+) T cells re-stimulated through CD3 and ICOS, IC87114 inhibited Akt and Erk activation, and the secretion of IL-2, IL-4, IL-17A, and IFN-γ better than A66. The p110α/δ inhibitor ETP-46321, or p110α plus p110δ inhibitors also inhibited IL-21 secretion by differentiated CD4(+) T follicular (Tfh) or IL-17-producing (Th17) helper cells. In vivo, therapeutic administration of ETP-46321 significantly inhibited responses to protein antigen as well as collagen-induced arthritis, as measured by antigen-specific antibody responses, secretion of IL-10, IL-17A or IFN-γ, or clinical symptoms. Hence, p110α as well as p110δ Class IA PI3Ks are important to immune regulation; inhibition of both subunits may be an effective therapeutic approach in inflammatory autoimmune diseases like rheumatoid arthritis.

p38α limits the contribution of MAP17 to cancer progression in breast tumors.

MAP17 is a small, 17-kDa, non-glycosylated membrane protein that is overexpressed in a percentage of carcinomas. In the present work, we have analyzed the role of MAP17 expression during mammary cancer progression. We have found that MAP17 is expressed in 60% human mammary tumors while it is not expressed in normal or benign neoplasias. MAP17 levels increased with breast tumor stage and were strongly correlated with mammary tumoral progression. A significant increase in the levels of reactive oxygen species (ROS) was observed in MAP17-expressing cells, as compared with parental cells. This increase was further paralleled by an increase in the tumorigenic capacity of carcinoma cells but not in immortal non-tumoral breast epithelial cells, which provides a selective advantage once tumorigenesis has begun. Expression of specific MAP17 shRNA in protein-expressing tumor cells reduced their tumorigenic capabilities, which suggests that this effect is dependent upon MAP17 protein expression. Our data show that ROS functions as a second messenger that enhances tumoral properties, which are inhibited in non-tumoral cells. We have found that p38α activation mediates this response. MAP17 triggers a ROS-dependent, senescence-like response that is abolished in the absence of p38a activation. Furthermore, in human breast tumors, MAP17 activation is correlated with a lack of phosphorylation of p38α. Therefore, MAP17 is overexpressed in late-stage breast tumors, in which oncogenic activity relies on p38 insensitivity to induce intracellular ROS.

Inhibiting PI3K as a therapeutic strategy against cancer

Class I PI3K is composed of heterodimeric lipid kinases regulating essential cellular functions including proliferation, apoptosis and metabolism. Class I PI3K isoforms are commonly amplified in different cancer types and the PI3Kalpha catalytic subunit, PIK3CA, has been found mutated in a variable proportion of tumours of different origin. Furthermore, PI3K has been shown to mediate oncogenic signalling induced by several oncogenes such as HER2 or Ras. These facts suggest that PI3K might be a good target for anticancer drug discovery. Today, the rise of PI3K inhibitors and their first in vivo results have cleared much of the path for the development of PI3K inhibitors for anticancer therapy. Here we will review the PI3K pathway and the pharmacological results of PI3K inhibition (AU)

Genetic modelling of the PTEN/AKT pathway in cancer research

The focus on targeted therapies has been fuelled by extensive research on molecular pathways and their role in tumorigenesis. Novel models of human cancer have been created to evaluate the role of specific genes in the different stages of cancer. Currently, mouse modelling of human cancer is possible through the expression of oncogenes, specific genetic mutations or the inactivation of tumour suppressor genes, and these models have begun to provide us with an understanding of the molecular pathways involved in tumour initiation and progression at the physiological level. Additionally, these mouse models serve as an excellent system to evaluate the efficacy of currently developed molecular targeted therapies and identify new potential targets for future therapies. The PTEN/AKT pathway is implicated in signal transduction through tyrosine kinase receptors and heterotrimeric G protein-linked receptors. Deregulation of the PTEN/AKT pathway is a common event in human cancer. Despite the abundant literature, the physiological role of each element of the pathway has begun to be uncovered thanks to genetically engineered mice. This review will summarise some of the key animal models which have helped us to understand this signalling network and its contribution to tumorigenesis (AU)

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S-adenosylhomocysteine hydrolase downregulation contributes to tumorigenesis.

With the idea to discover novel genes involved in proliferation, we have performed a genome-wide loss-of-function genetic screen to identify additional putative tumor suppressor genes. We have previously identified five genes belonging to different biochemical families. In this report, we focused on the study of one of these genes designated S-adenosylhomocysteine hydrolase (SAHH), which has also been previously identified in an independent short hairpin RNA screening. SAHH inactivation confers resistance to both p53 and p16(INK4)-induced proliferation arrest. Interestingly, SAHH inactivation inhibits p53 transcriptional activity and impairs DNA damage-induced transcription of p21(Cip1). Given that SAHH downregulation modulates senescence in primary cells, we also studied SAHH expression in human tumors at the messenger RNA (mRNA) and protein levels. SAHH mRNA was lost in 50% of tumor tissues from 206 patients with different kinds of tumors in comparison with normal tissue counterparts. Moreover, SAHH protein was also affected in some colon cancers. Such findings may be of relevance to cancer research, suggesting that SAHH might be a largely unexplored tumor suppressor.

Cellular senescence bypass screen identifies new putative tumor suppressor genes.

Senescence is a mechanism that limits cellular lifespan and constitutes a barrier against cellular immortalization. To identify new senescence regulatory genes that might play a role in tumorigenesis, we have designed and performed a large-scale antisense-based genetic screen in primary mouse embryo fibroblasts (MEFs). Out of this screen, we have identified five different genes through which loss of function partially bypasses senescence. These genes belong to very different biochemical families: csn2 (component of the Cop9 signalosome), aldose reductase (a metabolic enzyme) and brf1 (subunit of the RNA polymerase II complex), S-adenosyl homocysteine hydrolase and Bub1. Inactivation, at least partial, of these genes confers resistance to both p53- and p16INK4a-induced proliferation arrest. Furthermore, such inactivation inhibits p53 but not E2F1 transcriptional activity and impairs DNA-damage-induced transcription of p21. Since the aim of the screen was to identify new regulators of tumorigenesis, we have tested their inactivation in human tumors. We have found, either by northern blot or quantitative reverse transcriptase-PCR analysis, that the expression of three genes, Csn2, Aldose reductase and Brf1, is lost at different ratios in tumors of different origins. These genes are located at common positions of loss of heterogeneity (15q21.2, 7q35 and 14q32.33); therefore,we have measured genomic losses of these specific genes in different tumors. We have found that Csn2 and Brf1 also show genomic losses of one allele in different tumors. Our data suggest that the three genes identified in the genome-wide loss-of-function genetic screen are putative tumor suppressors located at 15q21.2; 7q35 and 14q32.33.

A novel regulatory mechanism of MAP kinases activation and nuclear translocation mediated by PKA and the PTP-SL tyrosine phosphatase.

Protein tyrosine phosphatase PTP-SL retains mitogen-activated protein (MAP) kinases in the cytoplasm in an inactive form by association through a kinase interaction motif (KIM) and tyrosine dephosphorylation. The related tyrosine phosphatases PTP-SL and STEP were phosphorylated by the cAMP-dependent protein kinase A (PKA). The PKA phosphorylation site on PTP-SL was identified as the Ser(231) residue, located within the KIM. Upon phosphorylation of Ser(231), PTP-SL binding and tyrosine dephosphorylation of the MAP kinases extracellular signal-regulated kinase (ERK)1/2 and p38alpha were impaired. Furthermore, treatment of COS-7 cells with PKA activators, or overexpression of the Calpha catalytic subunit of PKA, inhibited the cytoplasmic retention of ERK2 and p38alpha by wild-type PTP-SL, but not by a PTP-SL S231A mutant. These findings support the existence of a novel mechanism by which PKA may regulate the activation and translocation to the nucleus of MAP kinases.

Protein similarities beyond disulphide bridge topology.

Structural superimposition is an important procedure to analyse the relationships between proteins. A new approach and program, KNOT-MATCH, has been developed for automated structural superimposition of proteins by means of their disulphide bridge topology. As a result of the superimposition, regular secondary structures, loops and clusters of residues become correctly aligned. This fact allows us to find out important structural overlaps of residues, sometimes with functional significance, not only among proteins belonging to the same family but also between apparently non-related proteins. Different disulphide-rich protein families, such as EGF-like, defensin-like and plant protease inhibitors, have been self or cross analysed with this approach. Some amino acids that have been experimentally determined to be structural and/or functional key residues for these proteins are conserved in the three-dimensional space after superimposition by KNOT-MATCH. The program can be very useful for finding relationships among proteins that would be hidden to the current alignment methods based on sequence and on main-chain topology.

Potato carboxypeptidase inhibitor, a T-knot protein, is an epidermal growth factor antagonist that inhibits tumor cell growth.

Epidermal growth factor (EGF) and its receptor (EGFR) are involved in many aspects of the development of carcinomas, including tumor cell growth, vascularization, invasiveness, and metastasis. Because EGFR has been found to be overexpressed in many tumors of epithelial origin, it is a potential target for antitumor therapy. Here we report that potato carboxypeptidase inhibitor (PCI), a 39-amino acid protease inhibitor with three disulfide bridges, is an antagonist of human EGF. It competed with EGF for binding to EGFR and inhibited EGFR activation and cell proliferation induced by this growth factor. PCI suppressed the growth of several human pancreatic adenocarcinoma cell lines, both in vitro and in nude mice. PCI has a special disulfide scaffold called a T-knot that is also present in several growth factors including EGF and transforming growth factor alpha. PCI shows structural similarities with these factors, a fact that can explain the antagonistic effect of the former. This is the first reported example of an antagonistic analogue of human EGF.