The Potential Role of Nigella sativa Seed Oil as Epigenetic Therapy of Cancer.

Nigella sativa oil, commonly known as black seed oil (BSO), is a well-known Mediterranean food, and its consumption is associated with beneficial effects on human health. A large number of BSO's therapeutic properties is attributed to its pharmacologically active compound, thymoquinone (TQ), which inhibits cell proliferation and induces apoptosis by targeting several epigenetic players, including the ubiquitin-like, containing plant homeodomain (PHD) and an interesting new gene, RING finger domains 1 (UHRF1), and its partners, DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1). This study was designed to compare the effects of locally sourced BSO with those of pure TQ on the expression of the epigenetic complex UHRF1/DNMT1/HDAC1 and the related events in several cancer cells. The gas chromatographs obtained from GC-MS analyses of extracted BSO showed that TQ was the major volatile compound. BSO significantly inhibited the proliferation of MCF-7, HeLa and Jurkat cells in a dose-dependent manner, and it induced apoptosis in these cell lines. BSO-induced inhibitory effects were associated with a significant decrease in mRNA expression of UHRF1, DNMT1 and HDAC1. Molecular docking and MD simulation showed that TQ had good binding affinity to UHRF1 and HDAC1. Of note, TQ formed a stable metal coordinate bond with zinc tom, found in the active site of the HDAC1 protein. These findings suggest that the use of TQ-rich BSO represents a promising strategy for epigenetic therapy for both solid and blood tumors through direct targeting of the trimeric epigenetic complex UHRF1/DNMT1/ HDAC1.

A key genomic subtype associated with lymphovascular invasion in invasive breast cancer.

BACKGROUND: Lymphovascular invasion (LVI) is associated with the development of metastasis in invasive breast cancer (BC). However, the complex molecular mechanisms of LVI, which overlap with other oncogenic pathways, remain unclear. This study, using available large transcriptomic datasets, aims to identify genes associated with LVI in early-stage BC patients. METHODS: Gene expression data from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort (n = 1565) was used as a discovery dataset, and The Cancer Genome Atlas (TCGA; n = 854) cohort was used as a validation dataset. Key genes were identified on the basis of differential mRNA expression with respect to LVI status as characterised by histological review. The relationships among LVI-associated genomic subtype, clinicopathological features and patient outcomes were explored. RESULTS: A 99-gene set was identified that demonstrated significantly different expression between LVI-positive and LVI-negative cases. Clustering analysis with this gene set further divided cases into two molecular subtypes (subtypes 1 and 2), which were significantly associated with pathology-determined LVI status in both cohorts. The 10-year overall survival of subtype 2 was significantly worse than that of subtype 1. CONCLUSION: This study demonstrates that LVI in BC is associated with a specific transcriptomic profile with potential prognostic value.

Utility of ankyrin 3 as a prognostic marker in androgen-receptor-positive breast cancer.

PURPOSE: Androgen receptor (AR) and AR signaling pathways are thought to play a role in breast cancer (BC) and are potentially related to treatment responses and outcomes. Ankyrin 3 (ANK3) is associated with AR stability in cancer cells. In the present study, we investigated the clinicopathological utility of ANK3 expression with emphasis on AR and its associated signalling pathway at transcriptomic and proteomic phases. PATIENTS AND METHODS: The Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort (n = 1980) and The Cancer Genome Atlas (TCGA) dataset (n = 1039) were used to assess the expression and significance of ANK3 mRNA and other AR signalling pathway-associated gene signature. Using immunohistochemistry, ANK3 protein expression was evaluated in large (n = 982) cohort of early-stage BC with long-term follow-up and compared with clinicopathological characteristics and its prognostic value in the whole cohort and the subgroups stratified by AR protein expression. RESULTS: An AR-related gene signature was developed, comprising 20 genes, which included ANK3. This AR-related gene signature was significantly associated with AR mRNA expression, oestrogen receptor, human epidermal growth factor receptor 2 (HER2) status and the patients' outcomes. In tumours with high AR protein expression (n = 614), high ANK3 protein expression was significantly associated with progesterone receptor positivity and it was independently associated with the good outcomes (p = 0.025). CONCLUSIONS: This study indicates that ANK3 is related to AR signalling pathway and is associated with BC prognosis.

Mediator complex (MED) 7: a biomarker associated with good prognosis in invasive breast cancer, especially ER+ luminal subtypes.

BACKGROUND: Mediator complex (MED) proteins have a key role in transcriptional regulation, some interacting with the oestrogen receptor (ER). Interrogation of the METABRIC cohort suggested that MED7 may regulate lymphovascular invasion (LVI). Thus MED7 expression was assessed in large breast cancer (BC) cohorts to determine clinicopathological significance. METHODS: MED7 gene expression was investigated in the METABRIC cohort (n = 1980) and externally validated using bc-GenExMiner v4.0. Immunohistochemical expression was assessed in the Nottingham primary BC series (n = 1280). Associations with clinicopathological variables and patient outcome were evaluated. RESULTS: High MED7 mRNA and protein expression was associated with good prognostic factors: low grade, smaller tumour size, good NPI, positive hormone receptor status (p < 0.001), and negative LVI (p = 0.04) status. Higher MED7 protein expression was associated with improved BC-specific survival within the whole cohort and ER+/luminal subgroup. Pooled MED7 gene expression data in the external validation cohort confirmed association with better survival, corroborating with the protein expression. On multivariate analysis, MED7 protein was independently predictive of longer BC-specific survival in the whole cohort and Luminal A subtype (p < 0.001). CONCLUSIONS: MED7 is an important prognostic marker in BC, particularly in ER+luminal subtypes, associated with improved survival and warrants future functional analysis.

Clinicopathological and prognostic significance of Ras association and pleckstrin homology domains 1 (RAPH1) in breast cancer.

BACKGROUND: Ras association and pleckstrin homology domains 1 (RAPH1) is involved in cytoskeleton regulation and re-epithelialisation in invasive carcinoma and, therefore, may play a key role in carcinogenesis and metastasis. We, herein, investigated the biological and clinical significance of RAPH1 in breast cancer using large annotated cohorts. METHODS: The clinicopathological and prognostic significance of RAPH1 was assessed at the genomic and transcriptomic levels using The Cancer Genome Atlas (TCGA) dataset (n = 1039) and the results were validated using the Molecular taxonomy of breast cancer international consortium (METABRIC) cohort (n = 1980). RAPH1 protein expression was evaluated by immunohistochemistry in a large, well-characterised cohort of early-stage breast cancer (n = 1040). RESULTS: In both the TCGA and METABRIC cohorts, RAPH1 mRNA expression and RAPH1 copy number alteration were strongly correlated. RAPH1 mRNA overexpression was significantly correlated with high expression of adhesion and EMT markers including CDH1, TGFß1 and CD44. RAPH1 mRNA overexpression was a significant predictor of a poor prognosis (Hazard ratio 3.88; p = 0.049). High RAPH1 protein expression was associated with higher grade tumours with high proliferation index, triple negative phenotype and high E-cadherin expression. High RAPH1 protein expression was an independent predictor of shorter survival (Hazard ratio 4.37; p = 0.037). CONCLUSIONS: High RAPH1 expression is correlated with aggressive breast cancer phenotypes and provides independent prognostic value in invasive breast cancer.

Clinical and biological roles of Kelch-like family member 7 in breast cancer: a marker of poor prognosis.

BACKGROUND: The functions of many proteins are tightly regulated with a complex array of cellular functions including ubiquitination. In cancer cells, aberrant ubiquitination may promote the activity of oncogenic pathways with subsequent tumour progression. Kelch-like family member 7 (KLHL7) is involved in the regulation of ubiquitination and may play a role in breast cancer (BC). Present study aims to evaluate the biological and clinical usefulness of KLHL7 in BC utilising large well-characterised cohorts with long-term follow-up. METHODS: The relationships between KLHL7 gene copy number alteration (CNA) and mRNA expression and clinicopathological variables and clinical outcomes were evaluated in 1980 patients from the METABRIC BC cohort. Prognostic significance of KLHL7 mRNA was validated using the Breast Cancer Gene-Expression Miner v4.0 datasets (n = 5206). KLHL7 protein expression was assessed using immunohistochemistry in a large annotated series of early-stage BC (n = 917) with long-term follow-up. RESULTS: KLHL7 CNA was significantly correlated with its mRNA expression. KLHL7 mRNA expression was higher in luminal B and basal-like molecular subtypes and in higher grade tumours. Increased KLHL7 protein expression was significantly correlated with features of aggressive phenotype including lymphovascular invasion, high histological grade, hormonal receptor negativity, high PIK3CA and p53 expression. Outcome analysis showed that high KLHL7 expression is an independent predictor of shorter survival (p = 0.0011). CONCLUSIONS: KLHL7 appears to play an important role in BC progression. High KLHL7 protein expression identified a subgroup of BC with aggressive behaviour and provided independent prognostic information.

Saccharomyces cerevisiae-like 1 (SEC14L1) is a prognostic factor in breast cancer associated with lymphovascular invasion.

Lymphovascular invasion is strongly related to breast cancer metastasis. However, the underlying mechanisms of lymphovascular invasion and its driver molecules in breast cancer remain to be defined. In this study, we explore differential expression of genes in large molecularly characterized and clinically annotated datasets of invasive breast cancer patients (n = 8056) coupled with histological review and strict definition for lymphovascular invasion status. The METABRIC series was used to identify genes associated with lymphovascular invasion, as defined using hematoxylin and eosin staining supplemented by immunohistochemistry, at the genomic/transcriptomic levels. Saccharomyces cerevisiae-like 1 (SEC14L1) was identified as one of the most significant genes associated with lymphovascular invasion. The prognostic significance of SEC14L1 gene copy number and mRNA expression was further investigated in the METABRIC series and externally validated using the Breast Cancer Gene-Expression Miner v4.0. Protein expression of SEC14L1 was also assessed using immunohistochemistry in series of early stage breast cancer using tissue microarrays. SEC14L1 gene copy number gain was significantly associated with high histological grade and poor outcome. SEC14L1 mRNA expression showed positive association with higher grade, lymph node metastasis, and poor outcome. SEC14L1 protein overexpression was significantly associated with lymphovascular invasion (p < 0.0001), higher grade (p = 0.011), HER2 positivity (p = 0.036), and shorter survival (p = 0.00075). Our findings specify SEC14L1 as an independent prognostic factor in breast cancer. Its association, at both transcriptome and protein expression levels, with lymphovascular invasion and outcome could imply an important role in tumor progression. A further mechanistic insight into its molecular roles including potential therapeutic utility is warranted.

Rho-GTPase activating-protein 18: a biomarker associated with good prognosis in invasive breast cancer.

BACKGROUND: The prognostic value of lymphovascular invasion (LVI) in breast cancer (BC) has been demonstrated in several independent studies. However, identification of driver molecules for LVI remains a challenging task. Large-scale transcriptomic profiling of histologically validated LVI can potentially identify genes that regulate LVI. METHODS: Integrative bio-informatics analyses of the METABRIC study were performed utilising a subset of strictly defined LVI using histological and immunohistochemical (IHC) criteria. ARHGAP18 was among the top differentially expressed genes between LVI+ and LVI- BC with a 1.8-fold change. The prognostic impact of ARHGAP18 gene expression was assessed in the METABRIC data set (n=1980) and externally validated using the online BC gene expression data sets utilising bc-GenExMiner v4.0 (n=2016). Subsequently, ARHGAP18 protein expression was assessed on a large cohort of invasive BC (n=959) with long-term follow-up using IHC. RESULTS: Pooled analysis of ARHGAP18 mRNA expression showed that overexpression was associated with better outcome (P<0.001, hazard ratio (HR)=0.82, 95% CI 0.75-0.90). ARHGAP18 protein was expressed in the cytoplasm and nuclei of the tumour cells and its expression was positively associated with good prognostic variables. Lack of cytoplasmic expression showed associations with LVI (P=0.006), epithelial-mesenchymal transition and the HER+ subtype (P=0.01). Loss of nuclear expression was associated with higher grade, HER2+ and high Ki67LI (P=0.001). Cytoplasmic and nuclear expression showed a positive association with improved survival independent of other variables (P=0.01, HR=0.74, 95% CI 0.60-87). CONCLUSIONS: ARHGAP18 expression at transcriptomic and protein levels is associated with improved patients' outcomes whose deregulation may play a role in tumour progression and the development of LVI in BC. Further assessment of its potential therapeutic value in BC is warranted.

Ki67 expression in invasive breast cancer: the use of tissue microarrays compared with whole tissue sections.

BACKGROUND: Although the prognostic value of Ki67 in breast cancer is well documented, using optimal cut-points for patient stratification, reproducibility of the scoring and interpretation of the results remains a matter of debate particularly when using tissue microarrays (TMAs). This study aims to assess Ki67 expression assessed on TMAs and their matched whole tissue sections (WTS). Moreover, whether the cut-off used for WTS is reproducible on TMA in BC molecular classes and the association between Ki67 expression cut-off, assessed on TMAs and WTS, and clinicopathological parameters and patient outcome were tested. METHOD: A large series (n = 707) of primary invasive breast tumours were immunostained for Ki67 using both TMA and WTS and assessed as percentage staining and correlated with each other, clinicopathological parameters and patient outcome. In addition, MKI67 mRNA expression was correlated with Ki67 protein levels on WTS and TMAs in a subset of cases included in the METABRIC study. RESULTS: There was moderate concordance in Ki67 expression between WTS and TMA when analysed as a continuous variable (Intraclass correlation coefficient = 0.61) and low concordance when dichotomised (kappa value = 0.3). TMA showed low levels of Ki67 with mean percentage of expression of 35 and 22% on WTS and TMA, respectively. MKI67 mRNA expression was significantly correlated with protein expression determined on WTS (Spearman Correlation, r = 0.52) and to a lesser extent on TMA (r = 0.34) (p < 0.001). Regarding prediction of patient outcome, statistically significant differences were detected upon stratification of patients with tumours expressing Ki67 at 10, 15, 20, 25 or 30% in TMA. Using TMA, ≥20% Ki67 provided the best prognostic cut-off particularly in triple-negative and HER2-positive classes. CONCLUSION: Ki67 expression in breast cancer can be evaluated using TMA although different cut-points are required to emulate results from WTS. A cut-off of ≥20% for Ki67 expression in BC provides the best prognostic correlations when TMAs are used.

Further evidence to support bimodality of oestrogen receptor expression in breast cancer.

AIMS: Although oestrogen receptor (ER)-negative breast cancers (BCs) do not respond to hormone therapy, the response of ER-positive BCs is reported to be variable, which may suggest a dose-dependent effect. The aim of this study was to assess the pattern of ER expression in BCs at the protein (immunohistochemistry) and transcriptome (microarray-based gene expression) levels. METHODS AND RESULTS: ER immunohistochemical (IHC) expression was assessed in a large series of BCs, including 3649 core biopsies and 1892 cases prepared as tissue microarrays (TMAs) stained with specific antibodies. ESR1 mRNA expression was assessed in the METABRIC study (1980 cases), by the use of the Linear Models for Microarray Data (limma) software, and the results were compared with protein levels. IHC data confirmed the bimodality of ER expression, with 92.2% and 89.2% of the cases showing completely negative (<1%) or highly positive (≥70%) expression on the cores and TMAs, respectively. Weakly positive cases (1-10%) and intermediately positive (11-69%) cases were infrequent (2.7% and 5.1%, and 1.6% and 9.2%, in cores and TMAs, respectively), and did not show survival difference from ER-negative tumours. When full-face sections of the corresponding excision specimens were immunostained, 47% of the ER-low/intermediate group were deemed to be ER-negative. Transcriptomic data not only showed a significant correlation between ESR1 mRNA and protein expression levels, but also confirmed the bimodality of ER expression at the mRNA level. CONCLUSIONS: Our study provides further evidence that ER expression is bimodal, and that it is observed at both the mRNA and protein levels. The reported poor survival of BC patients with low ER expression in the early clinical trials may be related to the inclusion of ER-negative cases.

Molecular Mechanisms Underlying Lymphovascular Invasion in Invasive Breast Cancer.

Lymphovascular invasion (LVI), the presence of malignant cells within lymphovascular channels, is a crucial step in the invasion-metastasis cascade. LVI, when identified morphologically in the peritumoural area, is regarded as an indicator of metastatic potential and is strongly associated with a poor prognosis in many solid tumours, including breast cancer (BC). Although molecular mechanisms associated with the development of LVI have been extensively studied, details of driver genes, and molecular pathways and mechanisms involved in its development in BC, remain poorly defined. Although invasive BC cells have the ability to invade surrounding stroma, only those that can interact with endothelial cells, penetrate the vascular wall and withstand the intravascular stress will develop LVI and complete metastatic dissemination. Identification of additional molecular events associated with LVI in the primary tumour and characterisation of the contribution of the tumour micro-environment to modulating biological processes leading to LVI in BC remain a challenging task. This stems not only from the complexity of the molecular alterations in the primary tumour and the interactions with different components of its micro-environment but also from the subjective nature of LVI assessment in human BC. In this review, we discuss the clinicopathological features and the current knowledge of the molecular mechanisms underlying LVI in BC.

Identification of novel interacts partners of ADAR1 enzyme mediating the oncogenic process in aggressive breast cancer.

Triple-negative breast cancer (TNBC) subtype is characterized by aggressive clinical behavior and poor prognosis patient outcomes. Here, we show that ADAR1 is more abundantly expressed in infiltrating breast cancer (BC) tumors than in benign tumors. Further, ADAR1 protein expression is higher in aggressive BC cells (MDA-MB-231). Moreover, we identify a novel interacting partners proteins list with ADAR1 in MDA-MB-231, using immunoprecipitation assay and mass spectrometry. Using iLoop, a protein-protein interaction prediction server based on structural features, five proteins with high iloop scores were discovered: Histone H2A.V, Kynureninase (KYNU), 40S ribosomal protein SA, Complement C4-A, and Nebulin (ranged between 0.6 and 0.8). In silico analysis showed that invasive ductal carcinomas had the highest level of KYNU gene expression than the other classifications (p < 0.0001). Moreover, KYNU mRNA expression was shown to be considerably higher in TNBC patients (p < 0.0001) and associated with poor patient outcomes with a high-risk value. Importantly, we found an interaction between ADAR1 and KYNU in the more aggressive BC cells. Altogether, these results propose a new ADAR-KYNU interaction as potential therapeutic targeted therapy in aggressive BC.

Chemokine (C-C motif) receptor 7 (CCR7) associates with the tumour immune microenvironment but not progression in invasive breast carcinoma.

Some previous studies have reported that the chemokine (C-C motif) receptor 7 (CCR7) plays a role in breast cancer, is associated with lymph node metastasis and drives the site of distant metastasis. However, the impact of its expression on patient outcome and its association with tumour infiltrating inflammatory cells remain to be validated. We evaluated CCR7 protein expression by immunohistochemistry in a large well characterized cohort (n = 866) of early invasive primary breast cancers. CCR7 was expressed in the cytoplasm and membrane of tumour cells. We observed a weak positive association of high CCR7 expression when in either cellular component, but not both together, with axillary lymph node stage 3 tumours (p = 0.043). Logistic regression analysis of lymph node stage revealed no independent predictive value for CCR7 expression. CCR7 expression was higher in HER2 positive tumours (p = 0.03) and associated with positive CD68+ FOXP3+ tumour infiltrating cells. CCR7 staining was negatively associated with CD3+ cells. There was no significant association of CCR7 expression with breast cancer recurrence or survival. We conclude that while CCR7 is not a useful biomarker for predicting lymph node metastasis, it may reflect altered intra- and inter-cellular signalling related to the immune microenvironment. The subcellular localization of CCR7 appears to affect the nature of these interactions.