Naoise C Synnott

Targeting ADAM-17 with an inhibitory monoclonal antibody has antitumour effects in triple-negative breast cancer cells.

Background:Identification and validation of a targeted therapy for triple-negative breast cancer (TNBC), that is, breast cancers negative for oestrogen receptors, progesterone receptors and HER2 amplification, is currently one of the most urgent problems in breast cancer treatment. EGFR is one of the best-validated driver genes for TNBC. EGFR is normally activated following the release of ligands such as TGFα, mediated by the two MMP-like proteases ADAM (a disintegrin and metalloproteinase)-10 and ADAM-17. The aim of this study was to investigate the antitumour effects of a monoclonal antibody against ADAM-17 on an in vitro model of TNBC.Methods:We investigated an inhibitory cross-domain humanised monoclonal antibody targeting both the catalytic domain and the cysteine-rich domain of ADAM17-D1(A12) in the HCC1937 and HCC1143 cell lines.Results:D1(A12) was found to significantly inhibit the release of TGFα, and to decrease downstream EGFR-dependent cell signalling. D1(A12) treatment reduced proliferation in two-dimensional clonogenic assays, as well as growth in three-dimensional culture. Furthermore, D1(A12) reduced invasion of HCC1937 cells and decreased migration of HCC1143 cells. Finally, D1(A12) enhanced cell death in HCC1143 cells.Conclusion:Our in vitro findings suggest that targeting ADAM-17 with D1(A12) may have anticancer activity in TNBC cells.

Mutant p53: A Novel Target for the Treatment of Patients with Triple‐Negative Breast Cancer?

The identification and validation of a targeted therapy for patients with triple‐negative breast cancer (TNBC) is currently one of the most urgent needs in breast cancer therapeutics. One of the key reasons for the failure to develop a new therapy for this subgroup of breast cancer patients has been the difficulty in identifying a highly prevalent, targetable molecular alteration in these tumors. Recently however, the p53 gene was found to be mutated in approximately 80% of basal/TNBC, raising the possibility that targeting the mutant p53 protein product might be a new approach for the treatment of this form of breast cancer. In this study, we investigated the anti‐cancer activity of PRIMA‐1 and PRIMA‐1MET (APR‐246), two compounds which were previously reported to reactivate mutant p53 and convert it to a form with wild‐type (WT) properties. Using a panel of 18 breast cancer cell lines and 2 immortalized breast cell lines, inhibition of proliferation by PRIMA‐1 and PRIMA‐1MET was found to be cell‐line dependent, but independent of cell line molecular subtype. Although response was independent of molecular subtype, p53 mutated cell lines were significantly more sensitive to PRIMA‐1MET than p53 WT cells (p = 0.029). Furthermore, response (measured as IC50 value) correlated significantly with p53 protein level as measured by ELISA (p = 0.0089, r=−0.57, n = 19). In addition to inhibiting cell proliferation, PRIMA‐1MET induced apoptosis and inhibited migration in a p53 mutant‐dependent manner. Based on our data, we conclude that targeting mutant p53 with PRIMA‐1MET is a potential new approach for treating p53‐mutated breast cancer, including the subgroup with triple‐negative (TN) disease.

Vitamin D analogues: Potential use in cancer treatment

The vitamin D receptor (VDR) is a member of the thyroid-steroid family of nuclear transcription factors. Following binding of the active form of vitamin D, i.e., 1,25(OH)2D3 (also known as calcitriol) and interaction with co-activators and co-repressors, VDR regulates the expression of several different genes. Although relatively little work has been carried out on VDR in human cancers, several epidemiological studies suggest that low circulating levels of vitamin D are associated with both an increased risk of developing specific cancer types and poor outcome in patients with specific diagnosed cancers. These associations apply especially in colorectal and breast cancer. Consistent with these findings, calcitriol as well as several of its synthetic analogues have been shown to inhibit tumor cell growth in vitro and in diverse animal model systems. Indeed, some of these vitamin D analogues with low calcemic inducing activity (e.g., EB1089, inecalcitol, paricalcitol) have progressed to clinical trials in patients with cancer. Preliminary results from these trials suggest that these vitamin D analogues have minimal toxicity, but clear evidence of efficacy remains to be shown. Although evidence of efficacy for mono-treatment with vitamin D analogues is currently lacking, several studies have reported that supplementation with calcitriol or the presence of high endogenous circulating levels of vitamin D enhances response to standard therapies.

Preclinical evaluation of the AR inhibitor enzalutamide in triple-negative breast cancer cells.

The androgen receptor (AR) is present in approximately 80% of invasive breast cancer patients and in up to 30% of patients with triple-negative breast cancer (TNBC). Therefore, our aim was to investigate the targeting of AR as a possible hormonal approach to the treatment of TNBC. Analysis of 2091 patients revealed an association between AR expression and poor overall survival, selectively in patients with the basal subtype of breast cancer, the vast majority of which are TNBC. IC50 values for the second-generation anti-androgen enzalutamide across 11 breast cancer cell lines varied from 4 µM to >50 µM. The activity of enzalutamide was similar in TN and non-TN cell lines but was dependent on the presence of AR. Enzalutamide reduced clonogenic potential and cell growth in a 3D matrix in AR-positive cells. In addition, enzalutamide also inhibited cell migration and invasion in an AR-dependent manner. Enzalutamide appeared to mediate these processes through down-regulation of the transcription factors AP-1 and SP-1. The first-generation anti-androgen flutamide similarly blocked cell growth, migration and invasion. AR-positive TNBC cells clustered separately from AR-negative cells based on an androgen-related gene expression signature, independently of TNBC subtype. We conclude that targeting of the AR with drugs such as enzalutamide may provide an alternative treatment strategy for patients with AR-positive TNBC.

Vitamin D receptor as a target for breast cancer therapy.

Considerable epidemiological evidence suggests that high levels of circulating vitamin D (VD) are associated with a decreased incidence and increased survival from cancer, i.e., VD may possess anti-cancer properties. The aim of this investigation was therefore to investigate the anti-cancer potential of a low calcaemic vitamin D analogue, i.e., inecalcitol and compare it with the active form of vitamin D, i.e., calcitriol, in a panel of breast cancer cell lines (n = 15). Using the MTT assay, IC50 concentrations for response to calcitriol varied from 0.12 µM to >20 µM, whereas those for inecalcitol were significantly lower, ranging from 2.5 nM to 63 nM (P = 0.001). Sensitivity to calcitriol and inecalcitol was higher in VD receptor (VDR)-positive compared to VDR-negative cell lines (P = 0.0007 and 0.0080, respectively) and in ER-positive compared to ER-negative cell lines (P = 0.043 and 0.005, respectively). Using RNA-seq analysis, substantial but not complete overlap was found between genes differentially regulated by calcitriol and inecalcitol. In particular, significantly enriched gene ontology terms such as cell surface signalling and cell communication were found after treatment with inecalcitol but not with calcitriol. In contrast, ossification and bone morphogenesis were found significantly enriched after treatment with calcitriol but not with inecalcitol. Our preclinical results suggest that calcitriol and inecalcitol can inhibit breast cancer cell line growth, especially in cells expressing ER and VDR. As inecalcitol is significantly more potent than calcitriol and has low calcaemic potential, it should be further investigated for the treatment of breast cancer.

Mutant p53 in breast cancer: potential as a therapeutic target and biomarker

ObjectiveThe aim of this article is to discuss mutant p53 as a possible therapeutic target and biomarker for breast cancer.ResultsTP53 (p53) is the most frequently mutated gene in invasive breast cancer. Although mutated in 30–35% of all cases, p53 is mutated in approximately 80% of triple-negative (TN) tumors (i.e., tumors negative for ER, PR, and HER2). Because of this high prevalence, mutated p53 is both a potential biomarker and therapeutic target for patients with breast cancer, especially for those with the TN subtype. Although several retrospective studies have investigated a potential prognostic and therapy predictive role for mutant p53 in breast cancer, the results to date are mixed. Thus, at present, mutant p53 cannot be recommended as a prognostic or therapy predictive biomarker in breast cancer. In contrast to the multiple reports on a potential biomarker role, few studies had until recently, investigated mutant p53 as a potential target for breast cancer treatment. In the last decade, however, several compounds have become available which can reactivate mutant p53 protein and convert it to a conformation with wild-type properties. Some of these compounds, especially PRIMA-1, APR-246 PK11007, and COTI-2, have been found to exhibit anticancer activity in preclinical models of breast cancer.ConclusionSince p53 is mutated in the vast majority of TN breast cancers, compounds such as APR-246, PK11007, and COTI-2 are potential treatments for patients with this subform of the disease. Further research is necessary to identify a potential biomarker role for mutant p53 in breast cancer.

p53 in cancer: ready for therapeutic targeting?

TP53 ( p53 ) is one of the oldest and best studied genes implicated in cancer formation or progression. Although originally identified as an oncogene (1,2), it has been known for several decades that wild-type (WT) p53 functions as a tumor suppressor gene. Suppression of cancer formation involves the binding of p53 to specific DNA response elements, followed by the induction of genes involved in one or more of the following processes, apoptosis, cell cycle arrest, senescence, DNA repair, metabolism or reactive oxygen species (ROS) modulation (3). Because of its ability to prevent cancer formation, p53 has been referred to as the “Guardian of the Genome” (4).

The Mutant p53-Targeting Compound APR-246 Induces ROS-Modulating Genes in Breast Cancer Cells

TP53 is the most frequently mutated gene in human cancer and thus an attractive target for novel cancer therapy. Several compounds that can reactive mutant p53 protein have been identified. APR-246 is currently being tested in a phase II clinical trial in high-grade serous ovarian cancer. We have used RNA-seq analysis to study the effects of APR-246 on gene expression in human breast cancer cell lines. Although the effect of APR-246 on gene expression was largely cell line dependent, six genes were upregulated across all three cell lines studied, i.e., TRIM16, SLC7A11, TXNRD1, SRXN1, LOC344887, and SLC7A11-AS1. We did not detect upregulation of canonical p53 target genes such as CDKN1A (p21), 14-3-3σ, BBC3 (PUMA), and PMAIP1 (NOXA) by RNA-seq, but these genes were induced according to analysis by qPCR. Gene ontology analysis showed that APR-246 induced changes in pathways such as response to oxidative stress, gene expression, cell proliferation, response to nitrosative stress, and the glutathione biosynthesis process. Our results are consistent with the dual action of APR-246, i.e., reactivation of mutant p53 and modulation of redox activity. SLC7A11, TRIM16, TXNRD1, and SRXN1 are potential new pharmacodynamic biomarkers for assessing the response to APR-246 in both preclinical and clinical studies.

PO-037 Targeting mutant p53 with COTI-2: a new approach for the treatment of patients with triple-negative breast cancer?

Introduction The identification of a targeted therapy for triple-negative breast cancer (TNBC) is one of the most urgent needs in breast cancer therapeutics. Since the p53 gene is mutated in approx. 80% of TNBC tumours, it is an attractive target. COTI-2 is a clinical stage, small molecule which claims to target p53. The aim of this study was to investigate COTI-2 as a new treatment for TNBC. Material and methods Cell viability was determined by MTT assay. p53 protein levels were quantified by ELISA and immunofluorescent staining. P53 binding kinetics were measured by Surface Plasmon Resonance. Apoptosis was measured using Annexin V-FITC Apoptosis Detection Kit. Caspase 3/7 was measured by CellEventCaspase-3/7Green Flow Cytometry Assay Kit. CI values were calculated using Calcusyn software. Results and discussions Using a panel of 18 breast cell lines, TNBC cell lines were more responsive to COTI-2 than non-TNBC cells (p=0.04). Lower IC50 values for COTI-2 were found in p53 mutant vs p53 WT cells (p=0.001). Additionally, the higher the endogenous p53 protein, the more sensitive the cell line was to COTI-2 (p=0.035, r=−0.51, n=18). By staining with antibodies specific for folded WT p53 (PAb1620) or unfolded mutant p53 (PAb240), we showed that COTI-2 can induce refolding of mutant p53. Moreover, by SPR, we showed that COTI-2 binds to fl-mut-p53 protein, in a concentration dependent manner. In addition to inhibiting proliferation, COTI-2 induced apoptosis in a concentration dependent manner. Furthermore, inhibition of caspase activity with Z-VAD-FMK reduced apoptosis, suggesting that COTI-2 induces caspase-dependent apoptosis. Accordingly, COTI-2 induced a significant increase in caspase 3/7. In an effort to enhance response, COTI-2 was combined with a number of cytotoxic agents. Highly synergistic growth inhibition, i.e. CI <1, was found when COTI-2 was combined with doxorubicin in 6 different cell lines. In addition, COTI-2 plus docetaxel or eribulin was synergistic in 4/6 cell lines, plus carboplatin was synergistic in 3/6, while plus cisplatin was synergistic in 2/6. Finally, we compared response to COTI-2 with that of APR-246, the best studied p53 reactivating compound. Overall, the mean COTI-2 IC50 value was 64 fold lower than that for APR-246 (p=0.0006). Furthermore, no correlation was seen between response to COTI-2 or APR-246, suggesting that the compounds act differently in inhibiting cell growth. Conclusion We conclude that targeting mutant p53 with COTI-2 is a potential new approach for treating p53-mutated TNBC.