Jason H. Gill

GW9662, a potent antagonist of PPARγ, inhibits growth of breast tumour cells and promotes the anticancer effects of the PPARγ agonist rosiglitazone, independently of PPARγ activation

Peroxisome proliferator‐activated receptor gamma (PPARγ), a member of the nuclear receptor superfamily, is activated by several compounds, including the thiazolidinediones. In addition to being a therapeutic target for obesity, hypolipidaemia and diabetes, perturbation of PPARγ signalling is now believed to be a strategy for treatment of several cancers, including breast. Although differential expression of PPARγ is observed in tumours compared to normal tissues and PPARγ agonists have been shown to inhibit tumour cell growth and survival, the interdependence of these observations is unclear. This study demonstrated that the potent, irreversible and selective PPARγ antagonist GW9662 prevented activation of PPARγ and inhibited growth of human mammary tumour cell lines. Controversially, GW9662 prevented rosiglitazone‐mediated PPARγ activation, but enhanced rather than reversed rosiglitazone‐induced growth inhibition. As such, these data support the existence of PPARγ‐independent pathways and question the central belief that PPARγ ligands mediate their anticancer effects via activation of PPARγ.

Phospholipase A2 expression in tumours: a target for therapeutic intervention?

Phospholipase A(2) (PLA(2)) enzymes are involved in lipid metabolism and, as such, are central to several cellular processes. The different PLA(2)s identified to date can be classified into three groups: secreted PLA(2) (sPLA(2)), calcium-independent PLA(2) (iPLA(2)) and calcium-dependent cytosolic PLA(2) (cPLA(2)). In addition to their role in cellular signalling, PLA(2)s have been implicated in diverse pathological conditions, including inflammation, tissue repair and cancer. Elevated levels of sPLA(2) and cPLA(2) have been reported in several tumour types. Here, we summarize the current views on the PLA(2)s, and look at their expression, role in human malignancy and potential as targets for anticancer drug development.

Micro-RNA-31 controls hair cycle-associated changes in gene expression programs of the skin and hair follicle

The hair follicle is a cyclic biological system that progresses through stages of growth, regression, and quiescence, which involves dynamic changes in a program of gene regulation. Micro‐RNAs (miRNAs) are critically important for the control of gene expression and silencing. Here, we show that global miRNA expression in the skin markedly changes during distinct stages of the hair cycle in mice. Furthermore, we show that expression of miR‐31 markedly increases during anagen and decreases during catagen and telogen. Administration of antisense miR‐31 inhibitor into mouse skin during the early‐ and midanagen phases of the hair cycle results in accelerated anagen development, and altered differentiation of hair matrix keratinocytes and hair shaft formation. Microarray, qRT‐PCR and Western blot analyses revealed that miR‐31 negatively regulates expression of Fgf10, the components of Wnt and BMP signaling pathways Sclerostin and BAMBI, and Dlx3 transcription factor, as well as selected keratin genes, both in vitro and in vivo. Using luciferase reporter assay, we show that Krt16, Krt17, Dlx3, and Fgf10 serve as direct miR‐31 targets. Thus, by targeting a number of growth regulatory molecules and cytoskeletal proteins, miR‐31 is involved in establishing an optimal balance of gene expression in the hair follicle required for its proper growth and hair fiber formation.—Mardaryev, A. N., Ahmed, M. I., Vlahov, N. V., Fessing, M. Y., Gill, J. H., Sharov, A. A., and Botchkareva, N. V. Micro‐RNA‐31 controls hair cycle‐associated changes in gene expression programs of the skin and hair follicle. FASEB J. 24, 3869–3881 (2010). www.fasebj.org

Single-Chain TNF, a TNF Derivative with Enhanced Stability and Antitumoral Activity

The inflammatory and proapoptotic cytokine TNF possesses a compelling potential as an antitumoral therapeutic agent. Possible target cells include the malignant cells themselves, the tumor vasculature, or the immune system. As the clinical use of TNF is limited by systemic toxicity, targeting strategies using TNF-based fusion proteins are currently used. A major obstacle, however, is that homotrimeric TNF ligands are prone to activity loss due to dissociation into their monomers. In this study, we report the construction of single-chain TNF molecule, a TNF mutant consisting of three TNF monomers fused by short peptide linkers. In comparison to wild-type TNF, single-chain TNF was found to possess increased stability in vitro and in vivo, displayed reduced systemic toxicity yet slightly enhanced antitumoral activity in mouse models. Creation of single-chain variants is a new approach for improvement of functional activity of therapeutics based on TNF family ligands.

Tumour endoproteases: the cutting edge of cancer drug delivery?

Despite progression in anticancer drug development and improvements in the clinical utilization of therapies, current treatment regimes are still dependent upon the use of systemic antiproliferative cytotoxic agents. Although these agents are unquestionably potent, their efficacy is limited by toxicity towards ‘normal’ cells and a lack of tumour selective targeting, resulting in a therapeutic index which is modest at best. Consequently, the development of more tumour selective cancer treatments, with better discrimination between tumour and normal cells is unequivocally an important goal for cancer drug discovery. One such strategy is to exploit the tumour phenotype as a mechanism for tumour‐selective delivery of potent therapeutics. An exciting approach in this area is to develop anticancer therapeutics as prodrugs, which are non‐toxic until activated by enzymes localized specifically in the tumour. Enzymes suitable for tumour‐activated prodrug development must have increased activity in the tumour relative to non‐diseased tissue and an ability to activate the prodrug to its active form. One class of enzyme satisfying these criteria are the tumour endoproteases, particularly the serine‐ and metallo‐proteases. These proteolytic enzymes are essential for tumour angiogenesis, invasion and metastasis, the major defining features of malignancy. This review describes the concept behind development of tumour‐endoprotease activated prodrugs and discusses the various studies to date that have demonstrated the huge potential of this approach for improvement of cancer therapy.

Non‐invasive molecular imaging for preclinical cancer therapeutic development

Molecular and non‐invasive imaging are rapidly emerging fields in preclinical cancer drug discovery. This is driven by the need to develop more efficacious and safer treatments, the advent of molecular‐targeted therapeutics, and the requirements to reduce and refine current preclinical in vivo models. Such bioimaging strategies include MRI, PET, single positron emission computed tomography, ultrasound, and optical approaches such as bioluminescence and fluorescence imaging. These molecular imaging modalities have several advantages over traditional screening methods, not least the ability to quantitatively monitor pharmacodynamic changes at the cellular and molecular level in living animals non‐invasively in real time. This review aims to provide an overview of non‐invasive molecular imaging techniques, highlighting the strengths, limitations and versatility of these approaches in preclinical cancer drug discovery and development.

Loss of tumourigenicity of stably ERβ-transfected MCF-7 breast cancer cells

Proliferation of breast cancer cells is mediated by estrogen receptors (ER)-ERalpha and ERbeta. At present, contradictory observations complicate the understanding of involvement of ERbeta in breast cancer and functional definition of ERbeta as a prognostic marker. A stable expression of full length ERbeta was established in the ERalpha-positive MCF-7 breast carcinoma cell line to evaluate the role for ERbeta in maintenance of cell viability and estrogenic response, as well as proliferation, morphology and cell cycle progression. In order to verify in vivo tumourigenicity of ERbeta transfectants were transplanted into nude mice. Transfection of ERbeta in MCF-7 resulted in a marginal increase of gelsolin protein expression. Constitutive expression of ERbeta resulted in a significant 30% inhibition of cellular growth compared with transfection of the mock vector alone (p=0.043). This reduction in growth was associated a retardation of transition into S-phase of the cell cycle. The in vitro response to 17beta-estradiol was reversed in cells over-expressing ERbeta (p=0.016). However, no difference in response to the antiestrogens tamoxifen and ICI 182,780 was observed in the presence of ERbeta. Importantly, over-expression of ERbeta prevented establishment and growth of tumours as subcutaneous xenografts in immunodeficient mice in vivo. These observations support the notion that ERbeta is a tumour suppressor and is exploitable in terms of cancer prevention, improving therapeutic response or predicting disease progression.

Development of a novel tumor-targeted vascular disrupting agent activated by membrane-type matrix metalloproteinases.

Vascular disrupting agents (VDA) offer a strategy to starve solid tumors of nutrients and oxygen concomitant with tumor shrinkage. Several VDAs have progressed into early clinical trials, but their therapeutic value seems to be compromised by systemic toxicity. In this report, we describe the design and characterization of a novel VDA, ICT2588, that is nontoxic until activated specifically in the tumor by membrane-type 1 matrix metalloproteinase (MT1-MMP). HT1080 cancer cells expressing MT1-MMP were selectively chemosensitive to ICT2588, whereas MCF7 cells that did not express MT1-MMP were nonresponsive. Preferential hydrolysis of ICT2588 to its active metabolite (ICT2552) was observed in tumor homogenates of HT1080 relative to MCF7 homogenates, mouse plasma, and liver homogenate. ICT2588 activation was inhibited by the MMP inhibitor ilomastat. In HT1080 tumor-bearing mice, ICT2588 administration resulted in the formation of the active metabolite, diminution of tumor vasculature, and hemorrhagic necrosis of the tumor. The antitumor activity of ICT2588 was superior to its active metabolite, exhibiting reduced toxicity, improved therapeutic index, enhanced pharmacodynamic effect, and greater efficacy. Coadministration of ICT2588 with doxorubicin resulted in a significant antitumor response (22.6 d growth delay), which was superior to the administration of ICT2588 or doxorubicin as a single agent, including complete tumor regressions. Our findings support the clinical development of ICT2588, which achieves selective VDA targeting based on MT-MMP activation in the tumor microenvironment.

Antitumor activity of a duocarmycin analogue rationalized to be metabolically activated by cytochrome P450 1A1 in human transitional cell carcinoma of the bladder.

We identify cytochrome P450 1A1 (CYP1A1) as a target for tumor-selective drug development in bladder cancer and describe the characterization of ICT2700, designed to be metabolized from a prodrug to a potent cytotoxin selectively by CYP1A1. Elevated CYP1A1 expression was shown in human bladder cancer relative to normal human tissues. RT112 bladder cancer cells, endogenously expressing CYP1A1, were selectively chemosensitive to ICT2700, whereas EJ138 bladder cells that do not express CYP1A1 were significantly less responsive. Introduction of CYP1A1 into EJ138 cells resulted in 75-fold increased chemosensitivity to ICT2700 relative to wild-type EJ138. Negligible chemosensitivity was observed in ICT2700 in EJ138 cells expressing CYP1A2 or with exposure of EJ138 cells to CYP1B1- or CYP3A4-generated metabolites of ICT2700. Chemosensitivity to ICT2700 was also negated in EJ138-CYP1A1 cells by the CYP1 inhibitor α-naphthoflavone. Furthermore, ICT2700 did not induce expression of the AhR-regulated CYP1 family, indicating that constitutive CYP1A1 expression is sufficient for activation of ICT2700. Consistent with the selective activity by CYP1A1 was a time and concentration-dependent increase in γ-H2AX protein expression, indicative of DNA damage, associated with the activation of ICT2700 in RT112 but not EJ138 cells. In mice-bearing CYP1A1-positive and negative isogenic tumors, ICT2700 administration resulted in an antitumor response only in the CYP1A1-expressing tumor model. This antitumor response was associated with detection of the CYP1A1-activated metabolite in tumors but not in the liver. Our findings support the further development of ICT2700 as a tumor-selective treatment for human bladder cancers. Mol Cancer Ther; 12(1); 27–37. ©2012 AACR.

Peroxisome proliferator-activated receptor (PPAR) alpha-regulated growth responses and their importance to hepatocarcinogenesis

Peroxisome proliferators (PPs) are a class of non-genotoxic rodent hepatocarcinogens that act by perturbing liver growth regulation. We have demonstrated previously that PPs suppress both spontaneous rat hepatocyte apoptosis and that induced by exogenous stimuli such as transforming growth factor-beta1 (TGF beta1). More recently, we have demonstrated that PPs can suppress apoptosis induced by more diverse stimuli such as DNA damage or ligation of Fas, a receptor related to the tumour necrosis factor alpha (TNF alpha) family of cell surface receptors. PPs transcriptionally activate the peroxisome proliferator activated receptor-alpha, PPAR alpha, a member of the nuclear hormone receptor superfamily. We investigated whether activation of PPAR alpha mediates the suppression of rat hepatocyte apoptosis induced by PPs. We isolated a naturally occurring variant form of PPAR alpha (hPPAR alpha-6/29) from human liver by PCR cloning. hPPAR alpha-6/29 shared the ability of mPPAR alpha to bind to DNA but, unlike mPPAR alpha, could not be activated by PPs. Furthermore, hPPAR alpha-6/29 could act as a dominant negative regulator of PPAR-mediated gene transcription. When introduced into primary rat liver cell cultures by transient transfection, hPPAR alpha-6/29 prevented the suppression of hepatocyte apoptosis by the PP nafenopin, but not that seen in response to phenobarbitone (PB), a non-genotoxic carcinogen whose action does not involve PPAR alpha. The suppression of hepatocyte apoptosis was abrogated completely even though only 30% of hepatocytes were transfected, suggesting the involvement of a soluble factor. Recent data have suggested that TNF alpha, perhaps released by liver Kupffer cells in response to PPs, may play a key role in mediating the effects of PPs on hepatocyte growth regulation.