OGG1 co-inhibition antagonizes the tumor-inhibitory effects of targeting MTH1.

Cancer cells develop protective adaptations against oxidative DNA damage, providing a strong rationale for targeting DNA repair proteins. There has been a high degree of recent interest in inhibiting the mammalian Nudix pyrophosphatase MutT Homolog 1 (MTH1). MTH1 degrades 8-oxo-dGTP, thus limiting its incorporation into genomic DNA. MTH1 inhibition has variously been shown to induce genomic 8-oxo-dG elevation, genotoxic strand breaks in p53-functional cells, and tumor-inhibitory outcomes. Genomically incorporated 8-oxo-dG is excised by the base excision repair enzyme, 8-oxo-dG glycosylase 1 (OGG1). Thus, OGG1 inhibitors have been developed with the idea that their combination with MTH1 inhibitors will have anti-tumor effects by increasing genomic oxidative DNA damage. However, contradictory to this idea, we found that human lung adenocarcinoma with low OGG1 and MTH1 were robustly represented in patient datasets. Furthermore, OGG1 co-depletion mitigated the extent of DNA strand breaks and cellular senescence in MTH1-depleted p53-wildtype lung adenocarcinoma cells. Similarly, shMTH1-transduced cells were less sensitive to the OGG1 inhibitor, SU0268, than shGFP-transduced counterparts. Although the dual OGG1/MTH1 inhibitor, SU0383, induced greater cytotoxicity than equivalent combined or single doses of its parent scaffold MTH1 and OGG1 inhibitors, IACS-4759 and SU0268, this effect was only observed at the highest concentration assessed. Collectively, using both genetic depletion as well as small molecule inhibitors, our findings suggest that OGG1/MTH1 co-inhibition is unlikely to yield significant tumor-suppressive benefit. Instead such co-inhibition may exert tumor-protective effects by preventing base excision repair-induced DNA nicks and p53 induction, thus potentially conferring a survival advantage to the treated tumors.

The Existence of MTH1-independent 8-oxodGTPase Activity in Cancer Cells as a Compensatory Mechanism against On-target Effects of MTH1 Inhibitors.

Investigations into the human 8-oxodGTPase, MutT Homolog 1 (MTH1), have risen sharply since the first-in-class MTH1 inhibitors were reported to be highly tumoricidal. However, MTH1 as a cancer therapeutic target is currently controversial because subsequently developed inhibitors did not exhibit similar cytotoxic effects. Here, we provide the first direct evidence for MTH1-independent 8-oxodGTPase function in human cancer cells and human tumors, using a novel ATP-releasing guanine-oxidized (ARGO) chemical probe. Our studies show that this functionally redundant 8-oxodGTPase activity is not decreased by five different published MTH1-targeting small molecules or by MTH1 depletion. Significantly, while only the two first-in-class inhibitors, TH588 and TH287, reduced cancer cell viability, all five inhibitors evaluated in our studies decreased 8-oxodGTPase activity to a similar extent. Thus, the reported efficacy of the first-in-class MTH1 inhibitors does not arise from their inhibition of MTH1-specific 8-oxodGTPase activity. Comparison of DNA strand breaks, genomic 8-oxoguanine incorporation, or alterations in cellular oxidative state by TH287 versus the noncytotoxic inhibitor, IACS-4759, contradict that the cytotoxicity of the former results solely from increased levels of oxidatively damaged genomic DNA. Thus, our findings indicate that mechanisms unrelated to oxidative stress or DNA damage likely underlie the reported efficacy of the first-in-class inhibitors. Our study suggests that MTH1 functional redundancy, existing to different extents in all cancer lines and human tumors evaluated in our study, is a thus far undefined factor which is likely to be critical in understanding the importance of MTH1 and its clinical targeting in cancer.

Thioredoxin-1 protects against androgen receptor-induced redox vulnerability in castration-resistant prostate cancer.

Androgen deprivation (AD) therapy failure leads to terminal and incurable castration-resistant prostate cancer (CRPC). We show that the redox-protective protein thioredoxin-1 (TRX1) increases with prostate cancer progression and in androgen-deprived CRPC cells, suggesting that CRPC possesses an enhanced dependency on TRX1. TRX1 inhibition via shRNA or a phase I-approved inhibitor, PX-12 (untested in prostate cancer), impedes the growth of CRPC cells to a greater extent than their androgen-dependent counterparts. TRX1 inhibition elevates reactive oxygen species (ROS), p53 levels and cell death in androgen-deprived CRPC cells. Unexpectedly, TRX1 inhibition also elevates androgen receptor (AR) levels under AD, and AR depletion mitigates both TRX1 inhibition-mediated ROS production and cell death, suggesting that AD-resistant AR expression in CRPC induces redox vulnerability. In vivo TRX1 inhibition via shRNA or PX-12 reverses the castration-resistant phenotype of CRPC cells, significantly inhibiting tumor formation under systemic AD. Thus, TRX1 is an actionable CRPC therapeutic target through its protection against AR-induced redox stress.

MTH1 as a Chemotherapeutic Target: The Elephant in the Room.

Many tumors sustain elevated levels of reactive oxygen species (ROS), which drive oncogenic signaling. However, ROS can also trigger anti-tumor responses, such as cell death or senescence, through induction of oxidative stress and concomitant DNA damage. To circumvent the adverse consequences of elevated ROS levels, many tumors develop adaptive responses, such as enhanced redox-protective or oxidatively-generated damage repair pathways. Targeting these enhanced oxidative stress-protective mechanisms is likely to be both therapeutically effective and highly specific to cancer, as normal cells are less reliant on such mechanisms. In this review, we discuss one such stress-protective protein human MutT Homolog1 (MTH1), an enzyme that eliminates 8-oxo-7,8-dihydro-2'-deoxyguanosine triphosphate (8-oxodGTP) through its pyrophosphatase activity, and is found to be elevated in many cancers. Our studies, and subsequently those of others, identified MTH1 inhibition as an effective tumor-suppressive strategy. However, recent studies with the first wave of MTH1 inhibitors have produced conflicting results regarding their cytotoxicity in cancer cells and have led to questions regarding the validity of MTH1 as a chemotherapeutic target. To address the proverbial "elephant in the room" as to whether MTH1 is a bona fide chemotherapeutic target, we provide an overview of MTH1 function in the context of tumor biology, summarize the current literature on MTH1 inhibitors, and discuss the molecular contexts likely required for its efficacy as a therapeutic target.

Comparative gene expression profiling of human umbilical vein endothelial cells and ocular vascular endothelial cells.

BACKGROUND/AIMS: To investigate the difference between human umbilical vein endothelial cells (HUVEC) and human ocular microvascular endothelial cell (MVEC) gene expression, and to determine if these differences could improve the understanding of ocular angiogenic diseases. METHODS: The gene expression profiles of HUVEC and matched unpassaged human choroidal, retinal and iris endothelial cells were conducted using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Selected differences were confirmed by real time PCR. Functional cell proliferation assays were used to support microarray findings. RESULTS: HUVEC showed enrichment for probe sets involved in embryological development while ocular MVEC demonstrated enrichment for probe sets for MHC classes I and II, immune responses and cell signal transduction. Comparison of human retinal and choroidal endothelial cells demonstrated significant differences in the expression of probe sets encoding insulin-like growth factor 1 (IGF-1) signalling. Cell proliferation assays demonstrated the stimulatory role of IGF-1 on retinal endothelial cells compared with choroidal endothelial cells. CONCLUSIONS: Gene expression profiling has demonstrated that HUVEC are probably not a suitable surrogate for the study of ocular angiogenic disorders. There are also significant differences in the gene expression of human retinal and choroidal endothelial cells, which may be important in the mechanism and treatment of choroidal and retinal neovascularisation.

Comparison of choroidal and retinal endothelial cells: characteristics and response to VEGF isoforms and anti-VEGF treatments.

Neovascular eye diseases such as wet age-related macular degeneration and proliferative diabetic retinopathy are two of the most common causes of irreversible visual loss. Although mediated by vascular endothelial growth factor (VEGF), the mechanisms of these diseases are not fully understood. Molecular inhibitors of VEGF including pegaptanib, ranibizumab and bevacizumab are used as treatments for these diseases. However, there have been very few direct comparisons between these agents, and as dose and treatment regimes differ their relative efficacies are hard to determine. In vitro comparisons tend to use cells from different sites or species, which show heterogeneity in their responses. The aim of this study was to compare the characteristics of primary cultures of isolated human choroidal endothelial cells (hCEC) and retinal endothelial cells (hREC), and their proliferation responses to stimulation with VEGF 121 and 165, and to compare the anti-proliferative effects of these three drugs. hCEC and hREC were positive for the cell markers VEGFR1, VEGFR2, CD31, CD34 and von Willebrand's factor (vWF), with greater expression of CD34 on the hREC compared to hCEC. Contrary to previous assumptions VEGF isoforms 121 and 165 were found to be equally potent in stimulating endothelial cell proliferation. However, hREC exhibited higher proliferation with either VEGF isoform compared to hCEC. The anti-VEGF treatments ranibizumab and bevacizumab were effective in decreasing proliferation of hCEC induced by the two VEGF isoforms, individually and in combination, with ranibizumab being moderately more effective, particularly in hREC. Pegaptanib was effective in controlling the proliferation of hCEC stimulated by VEGF 165, but was ineffective against the stimulatory effect of VEGF 121. There were found to be significant differences in microvascular endothelial cells from the retina and choroid, both in the expression of cell markers and their behaviour in response to growth factors and currently available anti-VEGF agents, highlighting the importance of targeting treatments to specific intraocular vascular beds and/or diseases.