Targeting the oncogenic protein kinase Ciota signalling pathway for the treatment of cancer.

PKC (protein kinase C) isoenzymes are key signalling components involved in the regulation of normal cell proliferation, differentiation, polarity and survival. The aberrant regulation of PKC isoenzymes has been implicated in the development of many human diseases including cancer [Fields and Gustafson (2003) Methods Mol. Biol. 233, 519-537]. To date, however, only one PKC isoenzyme, the aPKC [atypical PKCiota (protein kinase Ciota)], has been identified as a human oncogene [Regala, Weems, Jamieson, Khoor, Edell, Lohse and Fields (2005) Cancer Res. 65, 8905-8911]. PKCiota has also proven to be a useful prognostic marker and legitimate target for the development of novel pharmacological agents for the treatment of cancer. The PKCiota gene resides at chromosome 3q26 and is a frequent target of tumour-specific gene amplification in multiple forms of human cancer. PKCiota gene amplification in turn drives PKCiota overexpression in these cancers. Genetic disruption of PKCiota expression blocks multiple aspects of the transformed phenotype of human cancer cells including transformed growth in soft agar, invasion through Matrigel and growth of subcutaneous tumours in nude mice. Genetic dissection of oncogenic PKCiota signalling mechanisms demonstrates that PKCiota drives transformed growth by activating a PKCiota --> Rac1 --> PAK (p21-activated kinase) --> MEK [MAPK (mitogen-activated protein kinase) 1,2/ERK (extracellular-signal-regulated kinase) kinase] 1,2 signalling pathway [Regala, Weems, Jamieson, Copland, Thompson and Fields (2005) J. Biol. Chem. 280, 31109-31115]. The transforming activity of PKCiota requires the N-terminal PB1 (Phox-Bem1) domain of PKCiota, which serves to couple PKCiota with downstream effector molecules. Hence, there exists a strong rationale for developing novel cancer therapeutics that target the PB1 domain of PKCiota and thereby disrupt its interactions with effector molecules. Using a novel high-throughput drug screen, we identified compounds that can disrupt PB1-PB1 domain interactions between PKCiota and the adaptor molecule Par6 [Stallings-Mann, Jamieson, Regala, Weems, Murray and Fields (2006) Cancer Res. 66, 1767-1774]. Our screen identified the gold compounds ATG (aurothioglucose) and ATM (aurothiomalate) as specific inhibitors of the PB1-PB1 domain interaction between PKCiota and Par6 that exhibit anti-tumour activity against NSCLC (non-small-cell lung cancer) both in vitro and in vivo. Structural analysis, site-directed mutagenesis and modelling indicate that ATM specifically targets the PB1 domain of PKCiota to mediate its anti-tumour activity [Erdogan, Lamark, Stallings-Mann, Lee, Pellechia, Thompson, Johansen and Fields (2006) J. Biol. Chem. 281, 28450-28459]. Taken together, our recent work demonstrates that PKCiota signalling is required for transformed growth of human tumours and is an attractive target for development of mechanism-based cancer therapies. ATM is currently in Phase I clinical trials for the treatment of NSCLC.

Mycobacteria, but not mercury, induces metallothionein (MT) protein in striped bass, Morone saxitilis, phagocytes, while both stimuli induce MT in channel catfish, Ictalurus punctatus, phagocytes.

Recent advances in molecular immunology indicate that the expression of inducible pro-inflammatory proteins is increased in vertebrates in response to both infectious disease agents and various xenobiotics. For example, iNOS, COX-2, and CYP1A are induced by both inflammation and AhR ligands. Moreover, the expression of these proteins in response to stimuli varies among individuals within populations. Little is known of the differences among fish in the inducibility of proinflammatory proteins in response to both infectious agents and xenobiotics. Through random screening of a striped bass, Morone saxitilis, peritoneal macrophage cDNA library, a full length metallothionein (MT) gene was cloned and sequenced. MT is a low-molecular weight (6-8 kDa), cysteine-rich metal binding protein. Metals are required by pathogenic bacteria for growth, and by the host defense system by serving as a catalyst for the generation of reactive oxygen intermediates (ROIs) by phagocytes. A recombinant striped bass MT (rMT) was expressed and purified, then used to generate a specific mAb (MT-16). MT protein expression was followed in freshly isolated striped bass and channel catfish, Ictalurus punctatus, phagocytes after in vitro exposure to the naturally occurring intracellular pathogen Mycobacteria fortuitum or to 0.1 and 1 microM mercury (Hg), as HgCl(2). MT expression was increased by 24 h in both channel catfish and striped bass phagocytes as a result of exposure to M. fortuitum cells. On the other hand, MT was induced by Hg in channel catfish cells, but not those of striped bass. These results indicate that metal homeostasis in phagocytes is different between catfish and striped bass. In addition, these data suggest that care should be taken to distinguish between inflammation-induced vs. metal-induced MT when using MT expression as a biomarker of metal exposure.

The effects of tributyltin (TBT) and 3,3',4,4',5-pentachlorobiphenyl (PCB-126) mixtures on antibody responses and phagocyte oxidative burst activity in channel catfish, Ictalurus punctatus.

The organotin tributyltin (TBT) is an antifouling biocide used in marine paints and is a common pollutant in harbor estuaries. We previously demonstrated that the immune system of channel catfish, Ictalurus punctatus, is a sensitive target organ of TBT. Exposure strongly suppresses humoral immune responses. Harbor estuaries often contain polychlorinated biphenyls (PCBs) due to their ubuquitous distribution. The coplanar congener 3,3',4,4'5'-polychlorinated biphenyl (PCB-126) is also immunotoxic to channel catfish, but it suppresses only the innate immune responses and only at high doses. In this study we exposed channel catfish to TBT, PCB-126, or both in mixtures, with canola oil (CO) serving as the carrier control. Antibody responses to Vibrio anguillarum and phagocyte oxidative burst activity were measured after (1) a single dose of 0.01 or 1 mg/kg of each or both in combination, and (2) six injections of 1.7 or 170 microg/kg of each (or in combination) given every 3 days over a 16-day period to yield a cumulative dose of 0.01 or 1 mg/kg, respectively. We measured antibody responses to V. anguillarum 21 days after immunization and oxidative burst activities 14 and 21 days after the final treatment. The highest dose of TBT suppressed antibody responses after a single exposure. The high dose of PCB-126 also suppressed antibody responses. The addition of PCB-126 to TBT doses did not alter the antibody responses beyond the effects of TBT alone. In the repeated exposure group, only the high dose of TBT suppressed antibody responses. In animals exposed to mixtures, high levels of PCB-126 enhanced suppression associated with low levels of TBT, whereas PCB-126 protected against suppression associated with high levels of TBT. Single exposures to TBT or PCB-126 suppressed phagocyte oxidative burst activity. In animals exposed to mixtures, as a single exposure, the addition of a low dose PCB-126 protected against low dose TBT-related oxidative burst activity suppression. In the repeated exposure groups TBT suppressed oxidative burst activity, but only at the highest dose on day 21, while high doses of PCB-126 suppressed activity on day 14. Furthermore, low levels of PCB-126 reversed the suppressed oxidative burst activity associated with high levels of TBT on day 21. Overall, this study demonstrates moderate additivity in terms of the immunotoxicity of TBT and PCB-126 mixtures using these two endpoints of immune function in the channel catfish model.