Carlos Martinez-Perez

Novel flavonoids as anti-cancer agents: mechanisms of action and promise for their potential application in breast cancer.

Flavonoids are a large group of ubiquitous polyphenolic secondary metabolites in plants with a wide range of properties, including a widely reported anti-cancer effect. The present review focuses on the different known mechanisms partaking in said anti-tumour effects, with particular emphasis on breast cancer. Their structure and reactivity allows flavonoids to work as antioxidant agents and phyto-oestrogens, modulating oestrogen signalling and metabolism to induce an overall anti-proliferative response. Other effects include the ability of flavonoids to modulate the CYP1 (cytochrome P450 1) and ABC (ATP-binding cassette) protein families, involved in carcinogenesis and drug delivery respectively. They can also induce apoptosis and cell cycle arrest and regulate other signalling pathways involved in the development and progression of cancer. In conclusion, there is accumulating evidence on the versatility of flavonoids and the numerous activities contributing to their anti-tumour effect. The complex, yet effective, mechanism of action of flavonoids, together with their interesting pharmacological properties, is the basis for their potential application in breast and other cancers. This rationale has led to the current interest in the application of flavonoids, including clinical trials currently underway and the development of novel flavonoids with improved properties, which hold great promise for tackling breast cancer.

Antitumour activity of the novel flavonoid oncamex in preclinical breast cancer models

Background:The natural polyphenol myricetin induces cell cycle arrest and apoptosis in preclinical cancer models. We hypothesised that myricetin-derived flavonoids with enhanced redox properties, improved cell uptake and mitochondrial targeting might have increased potential as antitumour agents.Methods:We studied the effect of a second-generation flavonoid analogue Oncamex in a panel of seven breast cancer cell lines, applying western blotting, gene expression analysis, fluorescence microscopy and immunohistochemistry of xenograft tissue to investigate its mechanism of action.Results:Proliferation assays showed that Oncamex treatment for 8 h reduced cell viability and induced cytotoxicity and apoptosis, concomitant with increased caspase activation. Microarray analysis showed that Oncamex was associated with changes in the expression of genes controlling cell cycle and apoptosis. Fluorescence microscopy showed the compound’s mitochondrial targeting and reactive oxygen species-modulating properties, inducing superoxide production at concentrations associated with antiproliferative effects. A preliminary in vivo study in mice implanted with the MDA-MB-231 breast cancer xenograft showed that Oncamex inhibited tumour growth, reducing tissue viability and Ki-67 proliferation, with no signs of untoward effects on the animals.Conclusions:Oncamex is a novel flavonoid capable of specific mitochondrial delivery and redox modulation. It has shown antitumour activity in preclinical models of breast cancer, supporting the potential of this prototypic candidate for its continued development as an anticancer agent.

Inhibition of pH regulation as a therapeutic strategy in hypoxic human breast cancer cells

Hypoxic cancer cells exhibit resistance to many therapies. This study compared the therapeutic effect of targeting the pH regulatory proteins (CAIX, NHE1 and V-ATPase) that permit cancer cells to adapt to hypoxic conditions, using both 2D and 3D culture models. Drugs targeting CAIX, NHE1 and V-ATPase exhibited anti-proliferative effects in MCF-7, MDA-MB-231 and HBL-100 breast cancer cell lines in 2D. Protein and gene expression analysis in 2D showed that CAIX was the most hypoxia-inducible protein of the 3 targets. However, the expression of CAIX differed between the 3 cell lines. This difference in CAIX expression in hypoxia was consistent with a varying activity of FIH-1 between the cell lines. 3D expression analysis demonstrated that both CAIX and NHE1 were up-regulated in the hypoxic areas of multicellular tumor spheroids. However, the induction of CAIX expression in hypoxia was again cell line dependent. 3D invasion assays conducted with spheroids showed that CAIX inhibition significantly reduced the invasion of cells. Finally, the capability of both NHE1 and CAIX inhibitors to combine effectively with irradiation was exhibited in clonogenic assays. Proteomic-mass-spectrometric analysis indicated that CAIX inhibition might be combining with irradiation through stimulating apoptotic cell death. Of the three proteins, CAIX represents the target with the most promise for the treatment of breast cancer.

Current treatment trends and the need for better predictive tools in the management of ductal carcinoma in situ of the breast

Ductal carcinoma in situ (DCIS) of the breast represents a group of heterogeneous non-invasive lesions the incidence of which has risen dramatically since the advent of mammography screening. In this review we summarise current treatment trends and up-to-date results from clinical trials studying surgery and adjuvant therapy alternatives, including the recent consensus on excision margin width and its role in decision-making for post-excision radiotherapy. The main challenge in the clinical management of DCIS continues to be the tailoring of treatment to individual risk, in order to avoid the over-treatment of low-risk lesions or under-treatment of DCIS with higher risk of recurring or progressing into invasion. While studies estimate that only about 40% of DCIS would become invasive if untreated, heterogeneity and complex natural history have prevented adequate identification of these higher-risk lesions. Here we discuss attempts to develop prognostic tools for the risk stratification of DCIS lesions and their limitations. Early results of a UK-wide audit of DCIS management (the Sloane Project) have also demonstrated a lack of consistency in treatment. In this review we offer up-to-date perspectives on current treatment and prediction of DCIS, highlighting the pressing clinical need for better prognostic indices. Tools integrating both clinical and histopathological factors together with molecular biomarkers may hold potential for adequate stratification of DCIS according to risk. This could help develop standardised practices for optimal management of patients with DCIS, improving clinical outcomes while providing only the amount of therapy required for each individual patient.

Abstract P5-11-02: Predicting local recurrence in patients treated for ductal carcinoma in situ of the breast (DCIS)

Background: Ductal carcinoma in situ (DCIS) of the breast represents a heterogeneous group of precursor, non-invasive breast lesions. Currently we lack accurate tools to stratify DCIS patients according to inherent risk of in breast tumour recurrence (IBTR) or progression to invasive breast cancer (IBC).Most DCIS patients are treated by breast-conversing surgery (BCS), followed by whole-breast radiotherapy (RT) for the majority of high-grade DCIS. The aim of this study was to identify novel biomarkers which predict recurrence after BCS +/- RT. Methods: A single institution study of 466 consecutive patients (median age 61, range 35-94) with DCIS treated by BCS between 2000 and 2010 was carried out. 271 patients with grade 3 DCIS received RT and 155 with grade 1/2 DCIS did not receive RT. For biomarker discovery, a case-control matched series of 200 patients (mean age = 61, range = 36-84) from the above audit that met the following criteria was selected: · 120 with low/intermediate-grade DCIS treated with BCS alone: 30 have recurred, 90 patients matched 3:1 have not recurred by 10 years. · 80 with high-grade DCIS treated by BCS plus RT: 20 have recurred, 60 patients matched 3:1 have not recurred by 10 years. Median follow-up was 7.4 years. RNA has been extracted and Affymetrix Clariom S whole-genome analysis has been performed and is currently being analysed. Results: In the cohort of 466 patients, 271 patients with high grade DCIS had BCS plus RT. Actuarial IBTR and IBC-IBTR in this group were 10% and 4% at 5 years and 18% and 6% at 10 years, respectively. 155 patients with low/intermediate grade DCIS had BCS alone. Actuarial overall IBTR and IBC-IBTR in this group were 6% and 2% at 5 years and 13% and 2% at 10 years respectively. In the high-grade, RT treated group, lesion size (P Full genomic analysis of the 240 patient case-control matched cohort is underway and will be presented. Discussion: · This is the first DCIS biomarker discovery study using whole genome analysis and the matched cohort design looking separately at BCS + RT for high-grade DCIS and BCS only for low/intermediate grade DCIS. · Clinical parameters alone may have insufficient sensitivity to identify high-grade, RT-treated patients at risk of developing IBC-IBTR. · While recurrence rates in the low/intermediate grade DCIS group are lower than in the high-grade group, some patients do recur and there is a need to develop new tools which can identify low grade patients with a sufficiently high risk of recurrence to warrant additional treatment. Citation Format: Martinez-Perez C, Turnbull AK, Ekatah GE, Arthur LM, Fernando A, Sims AH, Thomas JS, Dixon JM. Predicting local recurrence in patients treated for ductal carcinoma in situ of the breast (DCIS) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-11-02.

Abstract P6-09-27: The EA2clin test significantly predicts response and survival in both pre and post-menopausal women with ER-positive breast cancers

Background Identifying breast cancer (BC) patients likely to recur on endocrine therapy (ET) is a challenge. Several methods and tests based on clinical parameters and multi-gene or protein classifiers have been shown to predict those likely to recur. Tests that incorporate baseline and on-treatment markers are likely to be more accurate than tests based on baseline characteristics alone. 4 genes were identified by microarray that predicted for to ET: 2 at diagnosis and 2 at 14 days. The EndoAdjuvant2 (EA2) test uses 2 of these genes: IL6ST at diagnosis and on-treatment MCM4 at transcript level or by graded immunohistochemistry (IHC). The aim of this study was to compare EA2 with currently used clinical parameters in 4 different cohorts of pre and postmenopausal women. Patients The cohorts are (1) 73 post-menopausal women (PMW) with ER+ BC treated with neoadjuvant letrozole (L) then surgery, (2) 39 PMW with ER+ BC treated with neoadjuvant anastrozole (A) then surgery, (3) 108 PMW who received 2-weeks of A or L prior to surgery (4) 25 preMW with ER+ BC who received one 750mg dose of fulvestrant prior to surgery. All had adjuvant ET and 5-10 years follow up. Neoadjuvant response was assessed by periodic 3D ultrasound. Results The 4 gene assay had 96% (training; cohort 1) and 93% (validation; cohort 2) accuracy of response prediction to neoadjuvant L or A. In cohort 1, clinical parameters were available. On univariate regression analysis intrinsic subtype (luminal A/B; defined using PAM50) (P=0.002), histological grade (P=0.033) and HER2 status (P=0.001) significantly predicted clinical response. EA2 out-performed all these in both univariate (P EA2 predicted recurrence free in cohorts 1 and 3 combined: RFS (P=0.0004, HR=17.63 95%CI: 4.95-17.6), BCSS (P=0.0007, HR=16.60: 3.36-45.7). The Nottingham Prognostic Index (NPI) also predicted RFS (P=0.0002) and BCSS (P=0.0017) in univariate analysis but in the Cox analysis NPI was not found to be significant, although in the low risk group there were only 1/46 events compared to 19/62 in the moderate/high risk group. Histological grade (P Conclusions • EA2 predicts clinical response, RFS and BCSS • EA2clin combines NPI and EA2, outperforms either alone and predicts outcome in a new validation cohort of preMW treated with Fulvestrant • EA2clin works in preM and PM women regardless of ET. Citation Format: Turnbull AK, Pearce DA, Arthur LM, Martinez-Perez C, Thomas JS, Fernando A, Renshaw L, Sims AH, Dixon JM. The EA2clin test significantly predicts response and survival in both pre and post-menopausal women with ER-positive breast cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-09-27.

Abstract P4-08-06: Modulation of hypoxia-inducible factors and the HIF transcriptional response to hypoxia by ERBB2 overexpression in the MCF7 breast cancer cell line

Objective: To explore the role of HIF2α in growth factor receptor-driven HIF modulation and investigate the relationship between growth factor- and hypoxia-driven HIF activation. HIF-mediated transcriptional activity is known to drive genes involved in various processes which are associated with cancer pathology such as glycolysis, angiogenesis and metastasis. Therefore, understanding the implications of hypoxia-independent HIF regulation for both HIF1α and HIF2α, may give new insight into the mechanisms by which HIF drives cancer pathology in vivo and a greater understanding of when HIF inhibitory agents may be effective therapies. Methods: We used an ERBB2 overexpressing MCF7 cell line (MCF7-HER2) to investigate the effect of ERBB2 on the HIF-axis. Western blotting was used to assess protein level in these cell lines. HIF protein expression was compared with and without ERBB stimulation by ERBB3 ligand neuregulin 1β. Illumina BeadChip analysis was used to compare mRNA levels between these cell lines in normoxia (20% oxygen), acute hypoxia (0.5% oxygen for 24 hours) and chronic hypoxia (0.5% oxygen for 10 weeks). Differentially expressed genes were identified using rank products analysis with a cut-off P-value of 0.01. This allowed an in-depth comparison of hypoxia responses at the level of transcription between the cell lines to ascertain the effect of ERBB2 overexpression on hypoxia driven transcriptional changes. Results: Immunoblotting shows that HIF1α protein level is comparable between MCF7 and MCF7-HER2 cell lines, and is inducible in normoxia by stimulation with neuregulin 1β. Conversely, HIF2α protein is unaffected, but is constitutively expressed in MCF7-HER2 only. This suggests that both HIF isoforms can be up-regulated in normoxia but by different mechanisms. Microarray data suggests that the constitutively higher HIF2α levels in the MCF7-HER2 cell line may be due, at least in part, to the increased transcription of the HIF2A gene which is higher in normoxia and in response to hypoxia when compared to wild-type MCF7. Overexpression of ERBB2 in MCF7-HER2 cells appears to prime cells for their response to hypoxia, as 14% (N= 591) of the genes which are induced in acute hypoxia are also expressed at significantly higher levels in normoxic MCF7-HER2 cells. However, only 1% are more highly expressed in wild-type MCF7 cells. For chronic hypoxic genes, 18% (N= 514) were more highly expressed in normoxic MCF7-HER2 cells and just 8% in wild-type MCF7 cells. These up-regulated genes include both HIF1 and HIF2 target genes which may have important consequences for glycolysis (ALDOC, PFKFB), tumour cell survival (E4BP4, STC2) and proliferation (FOS, KDM5B). Conclusions: We have demonstrated that both HIF1α and HIF2α can be regulated independently of hypoxia, however these appear to be controlled through distinct mechanisms. Whilst the implications of HIF1 in breast cancer pathology have been appreciated for some time, relatively little is known about the impact of HIF2. Here we show that ERBB2 overexpression can not only increase HIF2α protein levels in normoxia, but may also prime cells for hypoxia by allowing the constitutively higher expression of HIF1 and HIF2 target genes. Citation Format: Jarman EJ, Turnbull AK, Martinez-Perez C, Meehan J, Xintralopoulou C, Ward C, Langdon SP. Modulation of hypoxia-inducible factors and the HIF transcriptional response to hypoxia by ERBB2 overexpression in the MCF7 breast cancer cell line. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-08-06.

Abstract P5-04-05: Targeting the pH regulatory mechanisms of breast cancer cells

Background: The abnormal regulation of H+ ions, leading to a reversed pH gradient in tumor cells in comparison to normal cells, is considered to be one of the hallmarks of cancer. This feature, however, has yet to be exploited as a therapeutic target. The aim of this study was to assess whether targeting proteins (CAIX, NHE1 and V-ATPase) that permit hypoxic cancer cell adaptation to acidosis in the tumor microenvironment can produce an effective therapeutic response in breast cancer, using 2D and 3D models. Method: Western blotting and gene expression analysis were performed on MCF-7, MDA-MB-231 and HBL-100 cancer cells to assess target protein expression in differing O2 conditions in 2D, while IHC was used to measure protein levels in 3D using multicellular tumor spheroids. Sulforhodamine B assays were executed to analyze the effects of inhibitors targeting CAIX, NHE1 and V-ATPase on breast cancer cell proliferation in 2D. 3D invasion assays were performed with MDA-MB-231 spheroids and explant tissue derived from human patients to see if CAIX inhibition had any effect on cancer cell invasion. An MDA-MB-231 xenograft model was used to investigate the effects of CAIX inhibition in vivo. Clonogenic assays were performed with MDA-MB-231 spheroids to evaluate whether any of the drugs combined effectively with irradiation. Results: 2D and 3D expression analysis showed that CAIX levels were extremely responsive to changes in O2 conditions in each of the cell lines, with HBL100 cells exhibiting the largest changes in both mRNA (42-fold increase) and protein (78-fold increase) levels at low (0.5%) O2 concentrations. NHE1 and V-ATPase mRNA/protein levels were, however, much more consistently expressed across the cell lines in different O2 conditions. Drugs targeting CAIX, NHE1 and V-ATPase had anti-proliferative effects on the breast cancer cells in 2D. Normoxic cancer cells were the most sensitive to drug treatment, acute hypoxic cancer cells showed increased resistance to the anti-proliferative effects of these drugs, while chronic hypoxic cells had IC50 values more similar to the normoxic cells. The results for the CAIX inhibitor were unexpected, as we had predicted that the increased levels of CAIX in the acute hypoxic cells would make them more sensitive to treatment. CAIX inhibition did, however, significantly reduce the invasion of cancer cells from both MDA-MB-231 spheroids (p≤0.01) and explant tissue (p≤0.001). Targeting pH regulation was also shown to have an effect in vivo on MDA-MB-231 xenografts, with CAIX inhibition significantly reducing the growth (p≤0.05) and proliferation (p≤0.05) of tumors within mice. Finally, clonogenic assays showed that drugs targeting both CAIX and NHE1 led to a significant reduction in colony number when combined with radiation (p≤0.05), compared to either drug individually or radiation treatment alone. Conclusions: This study shows that drugs targeting pH regulation molecules have potential in the treatment of breast cancer. This is highlighted by their ability to affect the proliferation and invasion of breast cancer cells, along with their ability to be combined with radiation. Of the 3 pH regulatory molecules, CAIX represents the target with the most promise. Citation Format: Meehan J, Ward C, Jarman E, Xintaropoulou C, Martinez-Perez C, Turnbull A, Supuran C, Dixon M, Kunkler I, Langdon SP. Targeting the pH regulatory mechanisms of breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-05.

Abstract P6-02-08: Novel flavonoid Anto-028 shows promising antitumor activity in preclinical models of breast cancer

New drugs are required to treat both endocrine resistant and triple negative breast cancers (TNBC). Among new structures being investigated are flavonoids, which are natural polyphenolic compounds with antitumor properties. Clinical trials are underway to assess their application as chemopreventatives and as sensitisers to chemotherapy. Among flavonoids, myricetin has shown particular promise, inducing cell cycle arrest and mitochondrial-dependent apoptosis in cancer cell line models. In this study we evaluated a novel series of myricetin-derived flavonoids with improved antioxidant and mitochondrial targeting properties. We hypothesised that these compounds might have potential in breast cancer management and in particular to treat estrogen receptor positive (ER+) tumors, in which estrogen controls tumor growth at least partially through oxidative stress-related mitochondrial signalling. We first assessed the effect of 8 flavonoids on 4 breast cancer cell lines (MDA-MB-231, BT-549, MCF-7 and HBL-100) and 3 MCF-7-derived cell lines with reduced sensitivity to endocrine therapy (LCC-1/LCC-2/LCC-9). The novel flavonoids were designed to assess the involvement of redox potential and mitochondrial targeting through selective structure-activity changes. Anto-028 was the most potent compound identified with 4 to 140-fold lower IC50 values than myricetin, as shown by sulforhodamine B (SRB) assays applied to assess antiproliferative effects. Although endocrine-resistant cell lines were less sensitive, Anto-028 exerted a strong, ER-independent antiproliferative effect on both ER+ and TNBC cell lines, with IC50 values in the low micromolar range. Treatment for 8 hours exerted dose-dependent reduction in cell viability and induction of cytotoxicity and apoptosis, with a 2 to 5-fold increase in caspase activation, as detected by luminescence-based plate assays. The involvement of reactive oxygen species (ROS) regulation in these effects was demonstrated by plate-based assays and microscopic detection of fluorescent probes for mitochondrial hydrogen peroxide and superoxide. Results indicated that different species of ROS are sequentially generated by treatment with a range of concentrations of Anto-028, suggesting a possible biphasic effect of the drug at different concentrations. Experiments in mice implanted with subcutaneous MDA-MB-231 xenografts showed that Anto-028 can significantly inhibit tumour growth in vivo with associated changes in percentage of viable areas within the xenografts (from 56% in controls to 37% in mice treated with 25mg/kg/day of Anto-028) and reduced Ki67 proliferation index (from 60% in controls to 36% in treated xenografts). Current studies are assessing combinatorial effects with TRAIL and chemotherapy which have produced synergistic effects in ovarian cancer models, with nanomolar concentrations of Anto-028 sensitising resistant cells to induce strong, significant antiproliferative effects. In conclusion, Anto-028 is a novel flavonoid with promising antitumor activity in preclinical models of breast cancer. Citation Format: Carlos Martinez-Perez, Carol Ward, Peter Mullen, Graeme Cook, Donald McPhail, Arran K Turnbull, David J Harrison, Simon P Langdon. Novel flavonoid Anto-028 shows promising antitumor activity in preclinical models of breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-02-08.