Sunali Mehta

Predictive and prognostic molecular markers for cancer medicine

Over the last 10 years there has been an explosion of information about the molecular biology of cancer. A challenge in oncology is to translate this information into advances in patient care. While there are well-formed routes for translating new molecular information into drug therapy, the routes for translating new information into sensitive and specific diagnostic, prognostic and predictive tests are still being developed. Similarly, the science of using tumor molecular profiles to select clinical trial participants or to optimize therapy for individual patients is still in its infancy. This review will summarize the current technologies for predicting treatment response and prognosis in cancer medicine, and outline what the future may hold. It will also highlight the potential importance of methods that can integrate molecular, histopathological and clinical information into a synergistic understanding of tumor progression. While these possibilities are without doubt exciting, significant challenges remain if we are to implement them with a strong evidence base in a widely available and cost-effective manner.

Anti-inflammatory activity of fruit fractions in vitro, mediated through toll-like receptor 4 and 2 in the context of inflammatory bowel disease

Pattern recognition receptors such as Toll-Like Receptor 2 (TLR2) and 4 (TLR4) are important in detecting and responding to stress and bacterial stimuli. Defect or damage in the TLR2 and TLR4 pathways can lead to sustained inflammation, characteristic of inflammatory bowel disease (IBD). The goal of this study was to identify fruit fractions that can be tested further to develop them as complementary therapies for IBD. In order to do this, we identified fruit fractions that mediate their anti-inflammatory response through the TLR4 and TLR2 pathway. Human Embryonic Kidney (HEK)-hTLR4 and hTLR2 cells were stimulated with their respective ligands to induce inflammation. These cells were treated with one of the 12 fractionated fruits and the inflammatory effect measured. 10 of the fruits came up as anti-inflammatory in the hTLR4 assay and nine in the hTLR2 assays. Many of the fruit fractions mediated their anti-inflammatory actions either mainly in their hydrophobic fractions (such as elderberry) or hydrophilic fractions (such as red raspberry), or both. The strongest anti-inflammatory effects were seen for feijoa and blackberry. This study shows that fruits can have multiple fractions eliciting anti-inflammatory effects in a pathway specific manner. This suggests that the compounds found in fruits can act together to produce health benefits by way of reducing inflammation. Exploiting this property of fruits can help develop complimentary therapies for inflammatory diseases.

Susceptibility to chronic inflammation: an update

Abstract Chronic inflammation is defined by the persistence of inflammatory processes beyond their physiological function, resulting in tissue destruction. Chronic inflammation is implicated in the progression of many chronic diseases and plays a central role in chronic inflammatory and autoimmune disease. As such, this review aims to collate some of the latest research in relation to genetic and environmental susceptibilities to chronic inflammation. In the genetic section, we discuss some of the updates in cytokine research and current treatments that are being developed. We also discuss newly identified canonical and non-canonical genes associated with chronic inflammation. In the environmental section, we highlight some of the latest updates and evidence in relation to the role that infection, diet and stress play in promoting inflammation. The aim of this review is to provide an overview of the latest research to build on our current understanding of chronic inflammation. It highlights the complexity associated with chronic inflammation, as well as provides insights into potential new targets for therapies that could be used to treat chronic inflammation and consequently prevent disease progression.

Dietary interactions with the bacterial sensing machinery in the intestine: the plant polyphenol case

There are millions of microbes that live in the human gut. These are important in digestion as well as defense. The host immune system needs to be able to distinguish between the harmless bacteria and pathogens. The initial interaction between bacteria and the host happen through the pattern recognition receptors (PRRs). As these receptors are in direct contact with the external environment, this makes them important candidates for regulation by dietary components and therefore potential targets for therapy. In this review, we introduce some of the main PRRs including a cellular process known as autophagy, and how they function. Additionally we review dietary phytochemicals from plants which are believed to be beneficial for humans. The purpose of this review was to give a better understanding of how these components work in order to create better awareness on how they could be explored in the future.

Preparation and Antitumour Properties of the Enantiomers of a Hypoxia‐Selective Nitro Analogue of the Duocarmycins

Racemic 2‐{[1‐(chloromethyl)‐5‐nitro‐3‐{5‐[2‐(dimethylamino)ethoxy]indol‐2‐carbonyl}‐1,2‐dihydro‐3H‐benzo[e]indol‐7‐yl]sulfonyl}aminoethyl dihydrogen phosphate, a synthetic nitro derivative of the duocarmycins, is a hypoxia‐selective prodrug active against radiation‐resistant tumour cells at nontoxic doses in mice. An intermediate in the synthesis of this prodrug was resolved by chiral HPLC and the absolute configuration assigned by X‐ray crystallography. The intermediate was used to prepare the prodrug′s enantiomers, and also the enantiomers of the active nitro and amino metabolites. In vitro analysis in the human cervical carcinoma cell line SiHa showed that both nitro enantiomers are hypoxia‐selective cytotoxins, but the “natural” S enantiomer is at least 20‐fold more potent. Examination of extracellular amino metabolite concentrations demonstrated no enantioselectivity in the hypoxia‐selective reduction of nitro to amino. Low levels of amino derivative were also found in aerobic cell suspensions, sufficient to account for the observed oxic toxicity of the nitro form. At an equimolar dose in SiHa‐tumour bearing animals, the (−)‐R enantiomer of the prodrug was inactive, while the (+)‐S enantiomer caused significantly more hypoxic tumour cell kill than the racemate. At this dose, the combination of (+)‐S‐prodrug and radiation eliminated detectable colony‐forming cells in four out of five treated tumour‐bearing animals.

Abstract B76: Cellular metabolism, murine pharmacokinetics and preclinical antitumor activity of SN29966, a novel hypoxia‐activated irreversible pan‐HER inhibitor

Hypoxia occurs in most human tumors and is associated with disease progression, resistance to conventional therapies and poor patient outcome. Hypoxia can up‐regulate HER1 by several known mechanisms, including increased mRNA translation (Franovic et al., PNAS,2007;104:13092) and delayed receptor endocytosis (Wang et al., Nat Med.,2009;15:319). We have developed SN29966, a hypoxia‐activated prodrug of the irreversible pan‐HER inhibitor SN29926, to target and exploit hypoxic cells and thereby broaden the therapeutic index of this class of agent. Quaternization of the aminebearing Michael acceptor masks activity SN29926, and one‐electron reduction provides selective release under hypoxia. This prodrug design confers 62‐fold deactivation of inhibitor activity with respect to HER1 autophosphorylation in A431 cells (Smaill et al., this meeting). In a panel of HER1/2 expressing cell lines SN29966 showed hypoxia‐dependent inhibition of proliferation (hypoxic/oxic IC50 ratios of 63, 38, 31 and 19 in BT474, A431, SKOV3 and SKBR3 cells, respectively), a property SN29926 lacked (IC50 ratios 0.7–1.1). SN29926 was generated from SN29966 under hypoxic conditions at a rate of 200–500 pmol/hr/106 cells. Oxic production was ∼1 pmol/hr/106 cells. Plasma and A431 tumor pharmacokinetics (PK) of prodrug SN29966 and inhibitor were measured in nude mice by LC/MS/MS detection (with D6 internal standards) following administration at their respective MTDs (133 and 75 umol/kg; ip). Prodrug SN29966 gave a plasma AUC0‐72h of 2016 umol‐h/L, some ∼110‐fold greater than achieved for administration of inhibitor SN29926 (18 umol‐h/L). The latter gave a tumor AUC0‐inf of 100 umol‐h/kg with a half‐life (t½) of 9 h. In contrast the prodrug SN29966 gave a tumor AUC0‐72h of 2245 umol‐h/kg with a stable tumor tissue concentration of ∼ 30 umol/kg out to 72 h, such that a t½ could not be determined. Consistent with this long prodrug residency, SN29926 released from prodrug had a t½ in tumor tissue of >72h, providing an AUC0‐72h of 464 umol‐h/kg. Thus the AUC of SN29926 in A431 tumors was at least 4.6‐fold higher after administration of prodrug SN29966 than following administration of inhibitor SN29926 itself at equivalent toxicity. In A431 tumor growth delay studies, SN29966 (113 umol/kg, q4dx6, ip) induced tumor regressions (12/12) with no recovery of growth by day 36 (tumor volume 44 ±19 mm3), whereas controls grew rapidly (day 8 tumor volume 1043 ±109 mm3). Comparative administration of inhibitor SN29926 (63 umol/kg, q4dx6, ip) provided tumor stasis, but with rapid growth following cessation of treatment (day 36 tumor volume 642 ±191 mm3), a difference that was significant from prodrug (p Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B76.

Extracts of Feijoa Inhibit Toll-Like Receptor 2 Signaling and Activate Autophagy Implicating a Role in Dietary Control of IBD

Background Inflammatory bowel disease (IBD) is a heterogeneous chronic inflammatory disease affecting the gut with limited treatment success for its sufferers. This suggests the need for better understanding of the different subtypes of the disease as well as nutritional interventions to compliment current treatments. In this study we assess the ability of a hydrophilic feijoa fraction (F3) to modulate autophagy a process known to regulate inflammation, via TLR2 using IBD cell lines. Method Mouse embryonic fibroblasts (MEF) deleted for ATG5, and two intestinal epithelial cells HCT15 and HCT116, were used to test the anti-inflammatory effect of F3 after stimulating the cells with a TLR2 specific ligand PAM3CSK4. Results F3 was able to reduce TLR2 specific inflammation and stimulate autophagy in MEFs and HCT15 cells but not in HCT116 cells. The anti-inflammatory effect was reduced in the MEF cells deleted for ATG5. In addition, the activation of autophagy by F3 was enhanced by PAM3CSK4. Conclusion F3 of feijoa can interact with cells via a TLR2 specific mechanism and reduce Nuclear factor kappa B (NF-κB) activation in part due to stimulation of autophagy. These results suggest that there is potential benefit in using feijoa extracts as part of dietary interventions to manage IBD in patients.

∆133p53 isoform promotes tumour invasion and metastasis via interleukin-6 activation of JAK-STAT and RhoA-ROCK signalling.

Abstract∆122p53 mice (a model of ∆133p53 isoform) are tumour-prone, have extensive inflammation and elevated serum IL-6. To investigate the role of IL-6 we crossed ∆122p53 mice with IL-6 null mice. Here we show that loss of IL-6 reduced JAK-STAT signalling, tumour incidence and metastasis. We also show that ∆122p53 activates RhoA-ROCK signalling leading to tumour cell invasion, which is IL-6-dependent and can be reduced by inhibition of JAK-STAT and RhoA-ROCK pathways. Similarly, we show that Δ133p53 activates these pathways, resulting in invasive and migratory phenotypes in colorectal cancer cells. Gene expression analysis of colorectal tumours showed enrichment of GPCR signalling associated with ∆133TP53 mRNA. Patients with elevated ∆133TP53 mRNA levels had a shorter disease-free survival. Our results suggest that ∆133p53 promotes tumour invasion by activation of the JAK-STAT and RhoA-ROCK pathways, and that patients whose tumours have high ∆133TP53 may benefit from therapies targeting these pathways.Aberrant expression of the Δ133p53 isoform is linked to many cancers. Here, the authors utilise a model of the Δ133p53 isoform that is prone to tumours and inflammation, showing that Δ133p53 promotes tumour cell invasion by activation of the JAK-STAT and RhoA-ROCK pathways in an IL-6 dependent manner.

Abstract A247: Mechanism of action of the hypoxia-activated irreversible pan-HER inhibitor SN29966.

Hypoxia occurs in most human tumors and is associated with disease progression, treatment resistance and poor patient outcome. We have developed the hypoxia-activated prodrug SN29966, designed to release the irreversible pan-HER inhibitor SN29926, following one-electron reduction by hypoxic cells (Smaill et al, Mol Cancer Ther., 2009; 8(12 Suppl), C46). Pharmacokinetic (PK) studies in nude mice bearing A431 tumor xenografts indicated SN29966 has a long tumor half-life (>3 days) and releases SN29926 in tumors. SN29966 demonstrated single agent activity in nude mice bearing A431 and SKOV3 xenografts, inducing striking tumor regressions in both models (Patterson et al, Mol Cancer Ther., 2009; 8(12 Suppl), B76). PR509 and PR610, clinical candidates developed from SN29966, are currently undergoing comparative evaluation with Phase I trials anticipated in early 2012. The single-agent antitumor activity of SN29966 is arguably counter-intuitive given that it is designed to target hypoxic cells within tumors. This activity may arise from a number of contributing mechanisms including; (i) bioactivity of the unreduced prodrug; (ii) local redistribution of released inhibitor in the tumor; (iii) liver metabolism and circulating inhibitor and (iv) a long tumor half-life allowing for targeting of both chronic and cycling hypoxia. To critically assess the relative contribution of each to the mechanism of action of SN29966 we performed a number of studies. We prepared SN31950, a prodrug of SN29926 designed to be incapable of one-electron fragmentation. In target modulation and anti-proliferative assays SN31950 showed no hypoxia-dependent activity. The murine A431 tumor PK of SN29966 and SN31950 demonstrated that at an equimolar dose (20 μmol/kg, ip), both prodrugs gave comparable tumor exposures (AUC0–72h: SN31950, 50 μmol*h/kg; SN29966, 57 μmol*h/kg). In contrast, the tumor exposure of SN29926 released from each prodrug differed by 40-fold (AUC0–72h: SN29926 from SN31950, 0.3 μmol*h/kg; SN29926 from SN29966, 12 μmol*h/kg). Plasma exposure of each prodrug was comparable, as were levels of SN29926 in plasma (presumed mainly due to hepatic prodrug metabolism). Consistent with the observed lack of inhibitor release in A431 tumors, SN31950 was inactive against A431 tumors in growth delay assays. To confirm the hypoxia-dependent nature of SN29966 inhibitor release in A431 tumors we re-oxygenated tumors in mice breathing 100% oxygen at 2.5 atm in a hyperbaric chamber. Accordingly, mice showed a marked reduction (56%, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A247.

A novel EGR‐1 dependent mechanism for YB‐1 modulation of paclitaxel response in a triple negative breast cancer cell line

Chemotherapy with taxanes such as paclitaxel (PTX) is a key component of triple negative breast cancer (TNBC) treatment. PTX is used in combination with other drugs in both the adjuvant setting and in advanced breast cancer. Because a proportion of patients respond poorly to PTX or relapse after its use, a greater understanding of the mechanisms conferring resistance to PTX is required. One protein shown to be involved in drug resistance is Y‐box binding protein 1 (YB‐1). High levels of YB‐1 have previously been associated with resistance to PTX in TNBCs. In this study, we aimed to determine mechanisms by which YB‐1 confers PTX resistance. We generated isogenic TNBC cell lines that differed by YB‐1 levels and treated these with PTX. Using microarray analysis, we identified EGR1 as a potential target of YB‐1. We found that low EGR1 mRNA levels are associated with poor breast cancer patient prognosis, and that EGR1 and YBX1 mRNA expression was inversely correlated in a TNBC line and in a proportion of TNBC tumours. Reducing the levels of EGR1 caused TNBC cells to become more resistant to PTX. Given that PTX targets cycling cells, we propose a model whereby high YB‐1 levels in some TNBC cells can lead to reduced levels of EGR1, which in turn promotes slow cell cycling and resistance to PTX. Therefore YB‐1 and EGR1 levels are biologically linked and may provide a biomarker for TNBC response to PTX.