Amy Chadwick

Inhibition of monocarboxylate transporter-1 (MCT1) by AZD3965 enhances radiosensitivity by reducing lactate transport

Inhibition of the monocarboxylate transporter MCT1 by AZD3965 results in an increase in glycolysis in human tumor cell lines and xenografts. This is indicated by changes in the levels of specific glycolytic metabolites and in changes in glycolytic enzyme kinetics. These drug-induced metabolic changes translate into an inhibition of tumor growth in vivo. Thus, we combined AZD3965 with fractionated radiation to treat small cell lung cancer (SCLC) xenografts and showed that the combination provided a significantly greater therapeutic effect than the use of either modality alone. These results strongly support the notion of combining MCT1 inhibition with radiotherapy in the treatment of SCLC and other solid tumors. Mol Cancer Ther; 13(12); 2805–16. ©2014 AACR.

JNK1 stress signaling is hyper-activated in high breast density and the tumor stroma: connecting fibrosis, inflammation, and stemness for cancer prevention.

Mammography is an important screening modality for the early detection of DCIS and breast cancer lesions. More specifically, high mammographic density is associated with an increased risk of breast cancer. However, the biological processes underlying this phenomenon remain largely unknown. Here, we re-interrogated genome-wide transcriptional profiling data obtained from low-density (LD) mammary fibroblasts (n = 6 patients) and high-density (HD) mammary fibroblasts (n = 7 patients) derived from a series of 13 female patients. We used these raw data to generate a “breast density” gene signature consisting of >1250 transcripts that were significantly increased in HD fibroblasts, relative to LD fibroblasts. We then focused on the genes that were increased by ≥ 1.5-fold (P < 0.05) and performed gene set enrichment analysis (GSEA), using the molecular signatures database (MSigDB). Our results indicate that HD fibroblasts show the upregulation and/or hyper-activation of several key cellular processes, including the stress response, inflammation, stemness, and signal transduction. The transcriptional profiles of HD fibroblasts also showed striking similarities to human tumors, including head and neck, liver, thyroid, lung, and breast cancers. This may reflect functional similarities between cancer-associated fibroblasts (CAFs) and HD fibroblasts. This is consistent with the idea that the presence of HD fibroblasts may be a hallmark of a pre-cancerous phenotype. In these biological processes, GSEA predicts that several key signaling pathways may be involved, including JNK1, iNOS, Rho GTPase(s), FGF-R, EGF-R, and PDGF-R-mediated signal transduction, thereby creating a pro-inflammatory, pro-proliferative, cytokine, and chemokine-rich microenvironment. HD fibroblasts also showed significant overlap with gene profiles derived from smooth muscle cells under stress (JNK1) and activated/infected macrophages (iNOS). Thus, HD fibroblasts may behave like activated myofibroblasts and macrophages, to create and maintain a fibrotic and inflammatory microenvironment. Finally, comparisons between the HD fibroblast gene signature and breast cancer tumor stroma revealed that JNK1 stress signaling is the single most significant biological process that is shared between these 2 data sets (with P values between 5.40E-09 and 1.02E-14), and is specifically associated with tumor recurrence. These results implicate “stromal JNK1 signaling” in the pathogenesis of human breast cancers and the transition to malignancy. Augmented TGF-β signaling also emerged as a common feature linking high breast density with tumor stroma and breast cancer recurrence (P = 5.23E-05). Similarities between the HD fibroblast gene signature, wound healing, and the cancer-associated fibroblast phenotype were also noted. Thus, this unbiased informatics analysis of high breast density provides a novel framework for additional experimental exploration and new hypothesis-driven breast cancer research, with a focus on cancer prevention and personalized medicine.

Metabolic reprogramming of bone marrow stromal cells by leukemic extracellular vesicles in acute lymphoblastic leukemia

To the editor: Cancer cells produce unique heterogeneous vesicles[1][1] capable of transferring oncogenic material[2][2],[3][3] to other cells,[4][4],[5][5] with the potential of modulating a tumor-supportive environment.[6][6][⇓][7]-[8][8] We have previously reported the presence of lipid-

Expression of NAD(P)H quinone dehydrogenase 1 (NQO1) is increased in the endometrium of women with endometrial cancer and women with Polycystic Ovary Syndrome

Women with a prior history of polycystic ovary syndrome (PCOS) have an increased risk of endometrial cancer (EC).

Abstract 4123: Monocarboxylate transporter 4 expression is a prognostic factor for radiotherapy outcome in squamous cell carcinoma of the head and neck

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Hypoxia contributes significantly to tumor progression and resistance to radiotherapy, decreasing local tumor control and lowering the rates of disease-free and overall survival. Hypoxic tumor cells utilize the glycolytic pathway for survival, producing vast quantities of lactate. Monocarboxylate Transporters (MCTs) 1 and 4 are key transporters of lactate, enabling sustained high glycolytic rates and maintenance of intra-cellular pH. Aim: To carry out the first study evaluating tumor MCT1 and 4 expression as potential biomarkers of prognosis in patients with head and neck squamous cell carcinoma (HNSCC) undergoing radiotherapy, and to determine the impact of MCT expression on radiation resistance. Methods: 125 histologically confirmed SCC pre-treatment diagnostic oropharyngeal cancer biopsies (tonsil or posterior third of the tongue) were collected retrospectively from diagnostic archives. The biopsies were analyzed immunohistochemically to evaluate MCT1 and 4 membrane expression. MCT expression was assessed in a double blind manner using a semi-quantitative scoring system. Scores were analyzed for possible correlations with clinicopathological data relating to outcome 5 years post diagnosis, where all patients had received radiotherapy to the primary site. FaDu HNSCC cells expressing doxycycline inducible shRNA targeting MCT4 were used to evaluate radiosensitivity of wild-type and MCT4-knockdown cells. Results: A univariate analysis comparing high (top 25% of scores) vs low MCT expression (lower 75%) showed that MCT4, but not MCT1, is a significant adverse prognostic factor for radiotherapy outcome. High MCT4 expression correlates with poor loco-regional control (p = 0.017), reduced cancer-specific survival (p = 0.02) and reduced overall survival (p = 0.055). In a multivariate analysis high MCT4 expression retained prognostic significance for poor loco-regional control (p = 0.007). Confirmation of MCT4 as a novel target for increasing hypoxic radiosensitivity was carried out by clonogenic assay in FaDu wild-type and shMCT4 cell lines, MCT4-knockdown cells showed a marked increase in hypoxic radiosensitivity compared to wild-type cells. Conclusions: The increase in significance from overall survival to loco-regional control is consistent with a hypoxia-regulated marker of radiotherapy resistance. The functional role of MCT4 as a lactate transporter in hypoxia may be of key underlying biological importance to this finding, maintaining intracellular pH in a hypoxic microenvironment. These findings suggest that inhibition of MCT4 may modify hypoxic tumor regions and sensitize tumor cells to radiation treatment. Therefore, MCT4 should be explored further as a novel target and biomarker for prognosis and prediction of benefit from hypoxia-modifying therapy in patients undergoing radiotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4123. doi:10.1158/1538-7445.AM2011-4123

Abstract 46: The impact of monocarboxylate transporter expression on metabolic function in prostate cancer cells

INTRODUCTION The tumor microenvironment is subjected to variations in oxygen tension which results in hypoxic regions. Hypoxia in tumors is associated with radioresistance and a metastatic phenotype. Hypoxic conditions cause tumor cells to favor anaerobic metabolism, however this switch to glycolysis is also found in tumor cells under aerobic conditions, known as the ‘Warburg effect’. An increased reliance on glycolysis results in large quantities of lactate, which needs to be transported out of tumor cells to maintain the intracellular pH. The monocarboxylate transporters, MCT1 and MCT4, regulate the transport of lactate in tumor cells and here we report the effect of overexpression or knockdown of these transporters on metabolism, growth and response to therapy in prostate cancer cell lines. METHODS Human prostate cancer cell lines were stably transfected, using lentiviral vectors, to show overexpression of MCT1 (DU145, LNCaP and PC3), overexpression of MCT4 (LNCaP), or knockdown of MCT4 (DU145 and PC3). Using these stable cell lines, the effect of overexpression or silencing of MCT1 or MCT4 was assessed in vitro. A lactate assay was used to investigate the effect on intra- and extracellular lactate under varying oxygen tensions. Effect on cell cycle progression was assessed using flow cytometry and toxicity of docetaxel was determined using MTT and SRB assays. Finally, a metabolome siRNA screen was carried out to determine if MCT4 expression was related to sensitivity to toxicity resulting from knockdown of a range of metabolic proteins. RESULTS Expression levels of MCT1 and MCT4 were confirmed by western blot and immunofluorescence. Silencing of MCT4 in DU145 and PC3 cell lines increased intracellular lactate under hypoxic conditions. No effect on intra- or extracellular lactate was observed in cell lines with overexpression of MCT1. Changes in expression of MCT1 or MCT4 did not appear to result in sensitization or resistance to docetaxel treatment under either normoxic or hypoxic conditions. Overexpression of MCT1 in PC3 cells caused cell cycle arrest in the G2/M phase in both normoxia and hypoxia. The metabolome screen indicated that, in wild type LNCaP cells, down-regulation of the protein product of the genes encoding 2,4-Dehydrocholesterol Reductase or Pyruvate Dehydrogenase Phosphatase Catalytic Subunit 2 was synthetically lethal. Overexpression of MCT4 resulted in protection against these lethal effects. CONCLUSIONS Altering the expression of MCT1 and MCT4 in human prostate cancer cells had a variety of effects on cell function. Overexpression of MCT1 caused cell cycle arrest in PC3 cells which may impact on radiosensitivity as cells are most vulnerable to radiation treatment when in the G2/M phase. The identification of two proteins that cause toxicity when silenced in LNCaP wild-type cells, but not LNCaP cells showing overexpression of MCT4, is an interesting lead for further investigation. Citation Format: Laura Hutchinson, Amy Boyers, Amy Chadwick, Ian Stratford. The impact of monocarboxylate transporter expression on metabolic function in prostate cancer cells. [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 46.

Abstract 1388: Biological role of novel NQO2 inhibitors in cancer therapeutics

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: NRH: quinone oxidoreductase 2 (NQO2), is a cytosolic flavoprotein that catalyses the two-electron reduction of quinones into hydroquinones. The physiological role of NQO2 in cancer biology is still not well understood. Some studies have suggested that inhibiting NQO2 could have a great potential in chemoprevention. This is based on the observation that NQO2 gene knockdown promotes protection against the toxicity of quinones such as menadione. Aim: To evaluate the biological role of novel NQO2 inhibitors in cancer therapeutics. Novel inhibitors will be used as pharmacological tools to study the physiological role of NQO2 in cancer cells, and to identify the consequences of inhibiting NQO2 on the tumor microenvironment. Methods: Novel compounds with diverse structures have been tested for their inhibition of NQO2 enzymatic activity in a cell-free system using a pure enzyme assay. The functional activity of these compounds as NQO2 inhibitors in the MDA-MB 231 breast cancer cells was also assessed. The effect of these inhibitors on the cellular proliferation of MDA-MB 231 breast cancer cells was tested using an MTT assay. Off-target (DNA binding) effects were analyzed by measuring the DNA thermal temperature. Moreover, the effect of these inhibitors on modulating TNFα-mediated NF-kB transcriptional activity was tested and the mechanism behind this modulation was also examined. Results: The novel inhibitors tested resulted in the inhibition of NQO2 activity in the cell free and the cellular system at nano-molar concentrations .Inhibition of MDA-MB 231 cell proliferation was also witnessed at low micro-molar concentrations. The inhibitors also displayed no DNA binding ability. In addition, the inhibitors were able to attenuate TNFα-mediated NF-kB transcriptional activity by blocking the phosphorylation of IkBα. This has resulted in the suppression of cell growth and promotion of cell apoptosis. Conclusions: This study has demonstrated novel potent inhibitors of NQO2 that are functionally active in the cells at nano-molar concentrations. The effect of the inhibitors on TNFα-mediated NF-kB transcriptional activity raises the possibility that inhibiting NQO2 could modulate the tumor micro-environmental factors, such as metastasis and angiogenesis. Citation Format: Elham Santina, Amy Chadwick, Soraya Al Nabulsi, Sally Freeman, Constantinos Demonacos, Ian Stratford. Biological role of novel NQO2 inhibitors in cancer therapeutics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1388. doi:10.1158/1538-7445.AM2014-1388

Abstract 5635: Analysis of monocarboxylate transporter 4 as a biomarker shows prognostic significance as an indicator of radiotherapy in squamous cell carcinoma of the head and neck

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC BACKGROUND: Hypoxia is known to contribute significantly to tumor progression and resistance to radiotherapy, decreasing local tumor control and lowering the rates of disease free and overall survival. Research to target hypoxia in the clinic has produced varying results; hence the discovery of hypoxia markers has become more significant. Monocarboxylate Transporter 4 (MCT4) is a hypoxia-regulated transporter of lactate out of the cell, preventing its intra-cellular accumulation, enabling sustained high glycolytic rates and maintenance of intra-cellular pH. AIM: To evaluate MCT4 immunohistochemically as a potential biomarker for prognosis of patients with head and neck squamous cell carcinoma (SCC) of tonsil or tongue undergoing radiotherapy, and to determine the impact of MCT4 expression on radiotherapy resistance. METHODS: 155 histologically confirmed SCC pre-treatment diagnostic biopsies, originating from the tonsil or posterior third of the tongue, were collected retrospectively from a diagnostic archive. The biopsies were analyzed immunohistochemically to evaluate MCT4 membrane expression. MCT4 expression was assessed in a double blind study using a semi-quantitative scoring system. Scores were analyzed for possible correlations with clinicopathological data relating to outcome 5 years post diagnosis, where all patients had received radiotherapy to the primary site. siRNA against MCT4 was used in SCC cell lines to evaluate radiosensitivity of wild-type and MCT4-knockdown cells by colony forming assays. RESULTS: A univariate analysis to assess high MCT4 expression (top 25% of scores) vs low MCT4 expression (lower 75%) showed that MCT4 is a significant adverse prognostic factor in the series of biopsies. High MCT4 expression correlates with poor loco-regional control (p = 0.017), reduced cancer-specific survival (p = 0.02) and reduced overall survival (p = 0.055). In a multivariate analysis high MCT4 expression retained prognostic significance for poor loco-regional control (p = 0.007). This was confirmed by clonogenic assay in FaDu and PE/CA-PJ-34 cell lines, MCT4-knockdown cells showed a marked increase in radiosensitivity compared to wild-type cells. CONCLUSIONS: MCT4 is a significant biomarker for prognosis and treatment outcome following radiotherapy in SCC of tonsil and tongue. The increase in significance from overall survival to loco-regional control is consistent with a hypoxia-regulated marker of radiotherapy resistance. The functional role of MCT4 as a lactate transporter in hypoxia may be of key underlying biological importance to this finding, maintaining intracellular pH in an hypoxic microenvironment. This suggests that drug inhibition of MCT4 may potentially sensitize tumor cells to radiation treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5635.