Molecular profiling of signalling proteins for effects induced by the anti-cancer compound GSAO with 400 antibodies

BMC Cancer Molecular profiling of signalling proteins for effects induced by the anti-cancer compound GSAO with 400 antibodies Verity A Cadd 2 Philip J Hogg 1 Adrian L Harris 0 Stephan M Feller 2 0 Cancer Research UK Growth Factor Group, Weatherall Institute of Molecular Medicine, University of Oxford , UK 1 Centre for Vascular Research, University of New South Wales, Sydney 2052 and Children's Cancer Institute Australia for Medical Research , Randwick 2031 , Australia 2 Cancer Research UK Cell Signalling Group, Weatherall Institute of Molecular Medicine, University of Oxford , UK Background: GSAO (4-[N-[S-glutathionylacetyl]amino] phenylarsenoxide) is a hydrophilic derivative of the protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO). It inhibits angiogenesis and tumour growth in mouse models and may be evaluated in a phase I clinical trial in the near future. Initial experiments have implicated GSAO in perturbing mitochondrial function. Other molecular effects of GSAO in human cells, for example on the phosphorylation of proteins, are still largely unknown. Methods: Peripheral white blood cells (PWBC) from healthy volunteers were isolated and used to profile effects of GSAO vs. a control compound, GSCA. Changes in site-specific phosphorylations, other protein modifications and expression levels of many signalling proteins were analysed using more than 400 different antibodies in Western blots. Results: PWBC were initially cultured in low serum conditions, with the aim to reduce basal protein phosphorylation and to increase detection sensitivity. Under these conditions pleiotropic intracellular signalling protein changes were induced by GSAO. Subsequently, PWBC were cultured in 100% donor serum to reflect more closely in vivo conditions. This eliminated detectable GSAO effects on most, but not all signalling proteins analysed. Activation of the MAP kinase Erk2 was still observed and the paxillin homologue Hic-5 still displayed a major shift in protein mobility upon GSAO-treatment. A GSAO induced change in Hic-5 mobility was also found in endothelial cells, which are thought to be the primary target of GSAO in vivo. Conclusion: Serum conditions greatly influence the molecular activity profile of GSAO in vitro. Low serum culture, which is typically used in experiments analysing protein phosphorylation, is not suitable to study GSAO activity in cells. The signalling proteins affected by GSAO under high serum conditions are candidate surrogate markers for GSAO bioactivity in vivo and can be analysed in future clinical trials. GSAO effects on Hic-5 in endothelial cells may point to a new intracellular GSAO target. - Background The term 'cancer' encompasses a wide variety of distinct, multigenic diseases. Even within a specific tumour type, a remarkable degree of heterogeneity on the level of DNA lesions and affected signalling pathways is apparent. Many cancer relevant signalling molecules, but also many molecular targets of anti-cancer drugs, therefore remain unknown. Prominent examples of signalling protein classes long known to be involved in generating cancer pathologies include GTPases, protein kinases and transcription factors. By contrast, protein phosphatases have only recently entered the stage as known players in cancer development. At least 30 protein phosphatases are now implicated in cancer development and other human diseases [1-3]. In some of these cases, mutational inactivation of a protein phosphatase appears to mimic the constitutive activation of its target kinase(s) [3]. In other cases, hyperactivation or deregulation of a phosphatase may contribute to kinase activation. For example, overexpression of the Cdc25 family phosphatases, which control cell cycle progression, is well documented for a variety of cancers, making the Cdc25 proteins interesting potential targets for anti-cancer therapies [4-7] and references therein). The modulation of specific cellular signalling pathways to treat human cancers has only recently developed into an area of intense clinical research activity. A large number of clinical trials for novel signal transduction modulator (STM) drugs are currently planned or under way. STM drugs often have relatively low toxicity, so determination of the maximum tolerated dose (MTD) may not be a prime goal for phase I clinical trials. Instead, identification of an optimal biologically active dose (OBD) is essential [8]. Rapid determination of the OBD requires that in vivo markers of drug activity are available before or very early during the clinical trial. This study identifies several proteins in PWBC which are affected by the novel anti-cancer compound GSAO (4-[N[S-glutathionylacetyl]amino] phenylarsenoxide) [9] (Figure 1A). They may be useful as clinical surrogate markers to monitor or predict the anti-cancer activity of GSAO and could also help to provide further insight into the biological mechanisms of GSAO action. GSAO has anti-angiogenic activity in vitro and in vivo [10]. Mitochondria and in particular the adenine nucleotide translocator (ANT) have been described as one target of GSAO. However, mitochondria are present in virtually all living cells. Therefore, inhibition of ANT does not per se explain the low toxicity and anti-angiogenic activity of GSAO. ibFCnyihcgreGuemSraAeisceOa1losftPrWuctBuCrepGroStAeOin taynrdosGinSeCpAhoanspdhDoroysleat-idoenpeinndduecnetd Chemical structure GSAO and GSCA and Dosedependent increase of PWBC protein tyrosine phosphorylation induced by GSAO. A and B. The boxes indicate the region of the molecule thought to be primarily responsible for the biological activity of GSAO and the corresponding region in the control GSCA. Oxidation of GSAO is possible. The resulting molecule (GSAA) is thought to be inactive, but may become active again, if reduced in vivo. To avoid oxidation, GSAO solutions are prepared freshly, or stored after snap-freezing at -80C. No significant activity loss of deep-frozen GSAO was observed for up to 6 months. PWBC were treated with GSAO or GSCA where indicated for 24 h and tyrosine phosphorylation detected by western blot. C 50 M GSCA does not alter the protein pTyr levels of PBWC detectably. D Changes in protein pTyr patterns are apparent with as little as 1.5 M GSAO. GSAO-affected proteins are indicated with arrows. Recent studies have revealed that differences in multidrug resistance proteins (MRP1 and 2) and cellular glutathione levels [11] appear to contribute to the preferential in vivo activity of GSAO in endothelial cells. Nevertheless, it is well known that practically all drugs in clinical use have multiple molecular in vivo targets. They depend on the affected cell type and applied doses. Some of these targets are intimately linked to the adverse effects of the drug. In cancer cells, with their heterogeneous genetic lesions, drug targets can even be present in different combinations or at different expression and activity levels when compari (...truncated)