Maria Sadiq

Hypoxia modulates the activity of a series of clinically approved tyrosine kinase inhibitors

Hypoxia in tumours is known to cause resistance to conventional chemotherapeutic drugs. In contrast, little is known about the effects of hypoxia on targeted anti‐cancer drugs. This study evaluated the effect of hypoxia on a series of clinically approved tyrosine kinase inhibitors (TKIs).

Polysialic acid sustains cancer cell survival and migratory capacity in a hypoxic environment.

Polysialic acid (polySia) is a unique carbohydrate polymer expressed on the surface of NCAM (neuronal cell adhesion molecule) in a number of cancers where it modulates cell-cell and cell-matrix adhesion, migration, invasion and metastasis and is strongly associated with poor clinical prognosis. We have carried out the first investigation into the effect of polySia expression on the behaviour of cancer cells in hypoxia, a key source of chemoresistance in tumours. The role of polysialylation and associated tumour cell migration and cell adhesion were studied in hypoxia, along with effects on cell survival and the potential role of HIF-1. Our findings provide the first evidence that polySia expression sustains migratory capacity and is associated with tumour cell survival in hypoxia. Initial mechanistic studies indicate a potential role for HIF-1 in sustaining polySia-mediated migratory capacity, but not cell survival. These data add to the growing body of evidence pointing to a crucial role for the polysialyltransferases (polySTs) in neuroendocrine tumour progression and provide the first evidence to suggest that polySia is associated with an aggressive phenotype in tumour hypoxia. These results have significant potential implications for polyST inhibition as an anti-metastatic therapeutic strategy and for targeting hypoxic cancer cells.

Hydroxytriazole derivatives as potent and selective aldo-keto reductase 1C3 (AKR1C3) inhibitors discovered by bioisosteric scaffold hopping approach

The aldo-keto reductase 1C3 isoform (AKR1C3) plays a vital role in the biosynthesis of androgens, making this enzyme an attractive target for castration-resistant prostate cancer therapy. Although AKR1C3 is a promising drug target, no AKR1C3-targeted agent has to date been approved for clinical use. Flufenamic acid, a non-steroidal anti-inflammatory drug, is known to potently inhibit AKR1C3 in a non-selective manner as COX off-target effects are also observed. To diminish off-target effects, we have applied a scaffold hopping strategy replacing the benzoic acid moiety of flufenamic acid with an acidic hydroxyazolecarbonylic scaffold. In particular, differently N-substituted hydroxylated triazoles were designed to simultaneously interact with both subpockets 1 and 2 in the active site of AKR1C3, larger for AKR1C3 than other AKR1Cs isoforms. Through computational design and iterative rounds of synthesis and biological evaluation, novel compounds are reported, sharing high selectivity (up to 230-fold) for AKR1C3 over 1C2 isoform and minimal COX1 and COX2 off-target inhibition. A docking study of compound 8, the most interesting compound of the series, suggested that its methoxybenzyl substitution has the ability to fit inside subpocket 2, being involved in π-π staking interaction with Trp227 (partial overlapping) and in a T-shape π-π staking with Trp86. This compound was also shown to diminish testosterone production in the AKR1C3-expressing 22RV1 prostate cancer cell line while synergistic effect was observed when 8 was administered in combination with abiraterone or enzalutamide.

Probing cytochrome P450-mediated activation with a truncated azinomycin analogue

A deactivated alkene precursor (IC50 = 81 μM) to the azinomycin epoxide natural product can be bioactivated by several cytochromes P450 (CYP) to generate antiproliferative metabolites with increased potency (IC50 = 1–30 μM) in CHOwt cells. CYP1A1 and 3A4 were shown to generate exclusively the unnatural and the natural-configured azinomycin epoxide diastereoisomer respectively, while CYP1B1 produced both epoxides in a 3 : 1 mixture. The antiproliferative activity is linked to DNA damage as demonstrated using the comet assay.

Probing cytochrome P450 bioactivation and fluorescent properties with morpholinyl-tethered anthraquinones

Structural features from the anticancer prodrug nemorubicin (MMDX) and the DNA-binding molecule DRAQ5™ were used to prepare anthraquinone-based compounds, which were assessed for their potential to interrogate cytochrome P450 (CYP) functional activity and localisation. 1,4-disubstituted anthraquinone 8 was shown to be 5-fold more potent in EJ138 bladder cancer cells after CYP1A2 bioactivation. In contrast, 1,5-bis((2-morpholinoethyl)amino) substituted anthraquinone 10 was not CYP-bioactivated but was shown to be fluorescent and subsequently photo-activated by a light pulse (at a bandwidth 532-587 nm), resulting in punctuated foci accumulation in the cytoplasm. It also showed low toxicity in human osteosarcoma cells. These combined properties provide an interesting prospective approach for opto-tagging single or a sub-population of cells and seeking their location without the need for continuous monitoring.

Abstract 4093: The impact of the prostate cancer microenvironment on the expression and regulation of aldehyde dehydrogenases

Introduction: Aldehyde dehydrogenase (ALDH) enzymes are involved in the detoxification of specific endogenous and exogenous aldehyde substrates. High ALDH activity is used to identify stem cells and has also been associated with poor prognosis in cancer. In prostate cancer (PCa) high ALDH activity has been associated with elevated clonogenicity, migratory behaviour, tumor progression and metastasis. Despite these observations there exists a poor understanding of the role of selected ALDH isoforms in PCa. In an attempt to understand if the tumor microenvironment impacts on ALDH function, we investigated their expression under hypoxia. Methods: Gene and protein expression analysis of ALDH isoforms -1A1, -1A2, -1A3, -1B1, -2, -3A1 and -7A1 in normal prostate cell line PNT2C2, and a panel of PCa cell lines with different stages of cancer was carried out by quantitative PCR and western blot under normoxic and hypoxic conditions at 24 and 48 hours. Gene expression of the ALDHs in primary prostate cultures was assessed following retinoic acid treatment. siRNA knockdown technology was used to study functional roles of selected ALDH isoforms. Results: Gene expression of ALDH1A3 was increased in Bob cells at 24h under hypoxia, at 48h this was less apparent but the expression of most ALDHs in Bob cells including ALDH1A2, -2, -3A1 and -7A1 was reduced at 48h. In SerBob cells, the gene expression of ALDH1A1 and -1B1 was reduced at both time points, whereas the expression of ALDH1A2, -2, and -7A1 was increased under hypoxia. At protein level, the expression of ALDH1A3 appeared low under hypoxia at both time points in SerBob cells while the expression of ALDH3A1 also appeared low after 24h exposure to hypoxia. In DU145 cells, there was an increase in ALDH1A1, -1A2, 1A3, -2, and -3A1 gene expression under hypoxia at 48h. In LNCAP cells, the gene expression of ALDH2 was increased at 48h under hypoxia whereas the expression of ALDH7A1 was reduced at 48h. The protein expression of ALDH7A1 was higher in PC3 cells under hypoxia at 24h. A significant increase was observed in ALDH1A3 and ALDH3A1 expression in response to retinoic acid. ALDH1A3 knockdown showed a significant reduction of cell viability in PC3 cells. Other investigations underway that will be reported at the conference are focused on how prostate cancer stem-like cells expressing ALDHs are responding to hypoxia. Conclusion: Our data suggests that the expression of certain ALDHs is affected by hypoxia and ALDH1A3 potentially is involved in cell survival in PCa. Future work will investigate if any of these isoforms can be used as biomarkers to distinguish indolent from malignant PCa. Citation Format: Maria Sadiq, Simon J. Allison, Fiona Frame, Mark Sutherland, Roger M. Phillips, Norman J. Maitland, Klaus Pors. The impact of the prostate cancer microenvironment on the expression and regulation of aldehyde dehydrogenases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4093.