Prognostic significance of minichromosome maintenance proteins in breast cancer

A role for the minichromosome maintenance (MCM) proteins in cancer initiation and progression is slowly emerging. Functioning as a complex to ensure a single chromosomal replication per cell cycle, the six family members have been implicated in several neoplastic disease states, including breast cancer. Our study aim to investigate the prognostic significance of these proteins in breast cancer. We studied the expression of MCMs in various datasets and the associations of the expression with clinicopathological parameters. When considered alone, high level MCM4 overexpression was only weakly associated with shorter survival in the combined breast cancer patient cohort (n = 1441, Hazard Ratio = 1.31; 95% Confidence Interval = 1.11-1.55; p = 0.001). On the other hand, when we studied all six components of the MCM complex, we found that overexpression of all MCMs was strongly associated with shorter survival in the same cohort (n = 1441, Hazard Ratio = 1.75; 95% Confidence Interval = 1.31-2.34; p <0.001), suggesting these MCM proteins may cooperate to promote breast cancer progression. Indeed, their expressions were significantly correlated with each other in these cohorts. In addition, we found that increasing number of overexpressed MCMs was associated with negative ER status as well as treatment response. Together, our findings are reproducible in seven independent breast cancer cohorts, with 1441 patients, and suggest that MCM profiling could potentially be used to predict response to treatment and prognosis in breast cancer patients.

Abstract 1905: Defining a therapeutic classification of breast cancer by actionable targets

Breast cancer remains a frequent cause of female cancer death despite the great strides in elucidation of biological subtypes and their reported clinical and prognostic significance. We have defined a general cohort of breast cancers in terms of putative actionable targets, involving growth and proliferative factors, the cell cycle, and apoptotic pathways, both as single biomarkers across a general cohort and within intrinsic molecular subtypes.

We identified 293 patients treated with adjuvant chemotherapy. Additional hormonal therapy and trastuzumab was administered depending on hormonal and HER2 status respectively. We performed immunohistochemistry for ER, PR, HER2, MM1, CK5/6, p53, TOP2A, EGFR, IGF1R, PTEN, p-mTOR and e-cadherin. The cohort was classified into luminal (62%) and non-luminal (38%) tumors as well as luminal A (27%), luminal B HER2 negative (22%) and positive (12%), HER2 enriched (14%) and triple negative (25%). Patients with luminal tumors and co-overexpression of TOP2A or IGF1R loss displayed worse overall survival (p=0.0251 and p=0.0008 respectively). Non-luminal tumors had much greater heterogeneous expression profiles with no individual markers of prognostic significance. Non-luminal tumors were characterised by EGFR and TOP2A overexpression, IGF1R, PTEN and p-mTOR negativity and extreme p53 expression.

Our results indicate that only a minority of intrinsic subtype tumors purely express single novel actionable targets. This lack of pure biomarker expression is particular prevalent in the triple negative subgroup and may allude to the mechanism of targeted therapy inaction and myriad disappointing trial results. Utilising a combinatorial biomarker approach may enhance studies of targeted therapies providing additional information during design and patient selection while also helping decipher negative trial results.

Uncovering BRCA1-regulated signalling pathways by microarray-based expression profiling

The introduction of microarray technology to the scientific and medical communities has dramatically changed the way in which we now address basic biomedical questions. Expression profiling using microarrays facilitates an experimental approach where alterations in the transcript level of entire transcriptomes can be simultaneously assayed in response to defined stimuli. We have used microarray analysis to identify downstream transcriptional targets of the BRCA1 (Breast Cancer 1) tumour-suppressor gene as a means of defining its function. BRCA1 has been implicated in the predisposition to early onset breast and ovarian cancer and while its exact function remains to be defined, roles in DNA repair, cell-cycle control and transcriptional regulation have been implied. In the current study we have generated cell lines with tetracycline-regulated, inducible expression of BRCA1 as a tool to identify genes, which might represent important effectors of BRCA1 function. Oligonucleotide array-based expression profiling identified a number of genes that were upregulated at various times following inducible expression of BRCA1 including the DNA damage-responsive gene GADD45 (Growth Arrest after DNA Damage). Identified targets were confirmed by Northern blot analysis and their functional significance as BRCA1 targets examined.

Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1

The breast cancer susceptibility gene BRCA1 encodes a protein implicated in the cellular response to DNA damage, with postulated roles in homologous recombination as well as transcriptional regulation. To identify downstream target genes, we established cell lines with tightly regulated inducible expression of BRCA1. High-density oligonucleotide arrays were used to analyze gene expression profiles at various times following BRCA1 induction. A major BRCA1 target is the DNA damage-responsive gene GADD45. Induction of BRCA1 triggers apoptosis through activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), a signaling pathway potentially linked to GADD45 gene family members. The p53-independent induction of GADD45 by BRCA1 and its activation of JNK/SAPK suggest a pathway for BRCA1-induced apoptosis.

Mullerian inhibiting substance inhibits breast cancer cell growth through an NFkappa B-mediated pathway

Müllerian inhibiting substance (MIS), a member of the transforming growth factor-beta superfamily, induces regression of the Müllerian duct in male embryos. In this report, we demonstrate MIS type II receptor expression in normal breast tissue and in human breast cancer cell lines, breast fibroadenoma, and ductal adenocarcinomas. MIS inhibited the growth of both estrogen receptor (ER)-positive T47D and ER-negative MDA-MB-231 breast cancer cell lines, suggesting a broader range of target tissues for MIS action. Inhibition of growth was manifested by an increase in the fraction of cells in the G(1) phase of the cell cycle and induction of apoptosis. Treatment of breast cancer cells with MIS activated the NFkappaB pathway and selectively up-regulated the immediate early gene IEX-1S, which, when overexpressed, inhibited breast cancer cell growth. Dominant negative IkappaBalpha expression ablated both MIS-mediated induction of IEX-1S and inhibition of growth, indicating that activation of the NFkappaB signaling pathway was required for these processes. These results identify the NFkappaB-mediated signaling pathway and a target gene for MIS action and suggest a putative role for the MIS ligand and its downstream interactors in the treatment of ER-positive as well as negative breast cancers.

Mullerian inhibiting substance inhibits breast cancer cell growth through an NFκB-mediated pathway

Mullerian inhibiting substance (MIS), a member of the transforming growth factor-β superfamily, induces regression of the Mullerian duct in male embryos. In this report, we demonstrate MIS type II receptor expression in normal breast tissue and in human breast cancer cell lines, breast fibroadenoma, and ductal adenocarcinomas. MIS inhibited the growth of both estrogen receptor (ER)-positive T47D and ER-negative MDA-MB-231 breast cancer cell lines, suggesting a broader range of target tissues for MIS action. Inhibition of growth was manifested by an increase in the fraction of cells in the G₁ phase of the cell cycle and induction of apoptosis. Treatment of breast cancer cells with MIS activated the NFκB pathway and selectively up-regulated the immediate early gene IEX-1S, which, when overexpressed, inhibited breast cancer cell growth. Dominant negative IκBα expression ablated both MIS-mediated induction of IEX-1S and inhibition of growth, indicating that activation of the NFκB signaling pathway was required for these processes. These results identify the NFκB-mediated signaling pathway and a target gene for MIS action and suggest a putative role for the MIS ligand and its downstream interactors in the treatment of ER-positive as well as negative breast cancers.

The prognostic significance of protein tyrosine phosphatase 4A2 in breast cancer

Although PTP4A3 has been shown to be a very important factor in promoting cancer progression, the role of its close family member PTP4A2 is still largely unknown. Recent reports have shown contradicting results on the role of PTP4A2 in breast cancer progression. Considering this, we aimed to investigate the prognostic value of PTP4A2 in five independent breast cancer data sets (minimum 198 patients per cohort, totaling 1,124 patients) in the Gene Expression Omnibus Database. We found that high expression of PTP4A2 was a favorable prognostic marker in all five independent breast cancer data sets, as well as in the combined cohort, with a hazard ratio of 0.68 (95% confidence interval =0.56-0.83; P<0.001). Low PTP4A2 expression was associated with estrogen receptor-negative tumors and tumors with higher histological grading; furthermore, low expression was inversely correlated with the expression of genes involved in proliferation, including MKI67 and the MCM gene family encoding the minichromosome maintenance proteins. These findings suggest that PTP4A2 may play a role in breast cancer progression by dysregulating cell proliferation. PTP4A2 expression was positively correlated with ESR1, the gene encoding estrogen receptor-alpha, and inversely correlated with EGFR expression, suggesting that PTP4A2 may be involved in these two important oncogenic pathways. Together, our results suggest that expression of PTP4A2 is a favorable prognostic marker in breast cancer.

Androgen receptor driven transcription in molecular apocrine breast cancer is mediated by FoxA1

Breast cancer is a heterogeneous disease and several distinct subtypes exist based on differential gene expression patterns. Molecular apocrine tumours were recently identified as an additional subgroup, characterised as oestrogen receptor negative and androgen receptor positive (ER- AR+), but with an expression profile resembling ER+ luminal breast cancer. One possible explanation for the apparent incongruity is that ER gene expression programmes could be recapitulated by AR. Using a cell line model of ER- AR+ molecular apocrine tumours (termed MDA-MB-453 cells), we map global AR binding events and find a binding profile that is similar to ER binding in breast cancer cells. We find that AR binding is a near-perfect subset of FoxA1 binding regions, a level of concordance never previously seen with a nuclear receptor. AR functionality is dependent on FoxA1, since silencing of FoxA1 inhibits AR binding, expression of the majority of the molecular apocrine gene signature and growth cell growth. These findings show that AR binds and regulates ER cis-regulatory elements in molecular apocrine tumours, resulting in a transcriptional programme reminiscent of ER-mediated transcription in luminal breast cancers.

Molecular classification of non-invasive breast lesions for personalised therapy and chemoprevention

Breast cancer screening has led to a dramatic increase in the detection of pre-invasive breast lesions. While mastectomy is almost guaranteed to treat the disease, more conservative approaches could be as effective if patients can be stratified based on risk of co-existing or recurrent invasive disease.Here we use a range of biomarkers to interrogate and classify purely non-invasive lesions (PNL) and those with co-existing invasive breast cancer (CEIN). Apart from Ductal Carcinoma In Situ (DCIS), relative homogeneity is observed. DCIS contained a greater spread of molecular subtypes. Interestingly, high expression of p-mTOR was observed in all PNL with lower expression in DCIS and invasive carcinoma while the opposite expression pattern was observed for TOP2A.Comparing PNL with CEIN, we have identified p53 and Ki67 as predictors of CEIN with a combined PPV and NPV of 90.48% and 43.3% respectively. Furthermore, HER2 expression showed the best concordance between DCIS and its invasive counterpart.We propose that these biomarkers can be used to improve the management of patients with pre-invasive breast lesions following further validation and clinical trials. p53 and Ki67 could be used to stratify patients into low and high-risk groups for co-existing disease. Knowledge of expression of more actionable targets such as HER2 or TOP2A can be used to design chemoprevention or neo-adjuvant strategies. Increased knowledge of the molecular profile of pre-invasive lesions can only serve to enhance our understanding of the disease and, in the era of personalised medicine, bring us closer to improving breast cancer care.

A BRCA1 deficient, NFκB driven immune signal predicts good outcome in triple negative breast cancer

Triple negative (TNBCs) and the closely related Basal-like (BLBCs) breast cancers are a loosely defined collection of cancers with poor clinical outcomes. Both show strong similarities with BRCA1-mutant breast cancers and BRCA1 dysfunction, or 'BRCAness', is observed in a large proportion of sporadic BLBCs. BRCA1 expression and function has been shown in vitro to modulate responses to radiation and chemotherapy. Exploitation of this knowledge in the treatment of BRCA1-mutant patients has had varying degrees of success. This reflects the significant problem of accurately detecting those patients with BRCA1 dysfunction. Moreover, not all BRCA1 mutations/loss of function result in the same histology/pathology or indeed have similar effects in modulating therapeutic responses. Given the poor clinical outcomes and lack of targeted therapy for these subtypes, a better understanding of the biology underlying these diseases is required in order to develop novel therapeutic strategies.We have discovered a consistent NFκB hyperactivity associated with BRCA1 dysfunction as a consequence of increased Reactive Oxygen Species (ROS). This biology is found in a subset of BRCA1-mutant and triple negative breast cancer cases and confers good outcome. The increased NFκB signalling results in an anti-tumour microenvironment which may allow CD8+ cytotoxic T cells to suppress tumour progression. However, tumours lacking this NFκB-driven biology have a more tumour-promoting environment and so are associated with poorer prognosis. Tumour-derived gene expression data and cell line models imply that these tumours may benefit from alternative treatment strategies such as reprogramming the microenvironment and targeting the IGF and AR signalling pathways.

ARB: Phase II window of opportunity study of short-term preoperative treatment with enzalutamide in ER-positive and triple-negative breast cancer

The androgen receptor (AR) is expressed in 60-80% of breast cancers (BC) across all molecular phenotypes, with a higher incidence in oestrogen receptor positive (ER+) BC compared to ER negative tumours. In ER+ disease, AR-expression has been linked to endocrine resistance which might be reversed with combined treatment targeting ER and AR. In triple negative BCs (TNBC), preclinical and clinical investigations have described a subset of patients that express the AR and are sensitive to androgen blockade, providing a novel therapeutic target. Enzalutamide, a potent 2nd generation anti-androgen, has demonstrated substantial preclinical and clinical anti-tumour activity in AR+ breast cancer. Short-term preoperative window of opportunity studies are a validated strategy for novel treatments to provide proof-of-concept and define the most appropriate patient population by directly assessing treatment effects in tumour tissue before and after treatment. The ARB study aims to assess the anti-tumour effects of enzalutamide in early ER+ breast cancer and TNBC, to identify the optimal target population for further studies and to directly explore the biologic effects of enzalutamide on BC and stromal cells. Methods: ARB is an international, investigator sponsored WOO phase II study in women with newly diagnosed primary ER+ BC or AR+ TNBC of ≥ 1cm. The study has two cohorts. In the ER+ cohort, postmenopausal patients will be randomised 2:1 to receive either enzalutamide (160mg OD) plus exemestane (50mg OD) or exemestane (25mg OD). In the TNBC cohort, AR+ will receive single agent treatment with enzalutamide (160mg OD). Study treatment is planned for 15–29 days, followed by surgery or neo-adjuvant therapy. Tissue and blood samples are collected before treatment and on the last day of study treatment. The primary endpoint is inhibition of tumour-cell proliferation, as measured by change in Ki67 expression, determined centrally by 2 investigators. Secondary endpoints include induction of apoptosis (Caspase3), circulating hormone levels and safety. ARB aims to recruit ≈235 patients from ≈40 sites in the UK, Germany, Spain and USA. The study is open to recruitment.

Thromboxane A2 receptor (TBXA2R) is a potent survival factor for triple negative breast cancers (TNBCs)

Triple Negative Breast Cancer (TNBC) is defined by the lack of ERα, PR expression and HER2 overexpression and is the breast cancer subtype with the poorest clinical outcomes. Our aim was to identify genes driving TNBC proliferation and/or survival which could represent novel therapeutic targets. We performed microarray profiling of primary TNBCs and generated differential genelists based on clinical outcomes following the chemotherapy regimen FEC (5-Fluorouracil/Epirubicin/Cyclophosphamide -‘good’ outcome no relapse > 3 years; ‘poor’ outcome relapse < 3 years). Elevated expression of thromboxane A2 receptor (TBXA2R) was observed in ‘good’ outcome TNBCs. TBXA2R expression was higher specifically in TNBC cell lines and TBXA2R knockdowns consistently showed dramatic cell killing in TNBC cells. TBXA2R mRNA and promoter activities were up-regulated following BRCA1 knockdown, with c-Myc being required for BRCA1-mediated transcriptional repression. We demonstrated that TBXA2R enhanced TNBC cell migration, invasion and activated Rho signalling, phenotypes which could be reversed using Rho-associated Kinase (ROCK) inhibitors. TBXA2R also protected TNBC cells from DNA damage by negatively regulating reactive oxygen species levels. In summary, TBXA2R is a novel breast cancer-associated gene required for the survival and migratory behaviour of a subset of TNBCs and could provide opportunities to develop novel, more effective treatments.

Targeting treatment resistant breast cancer stem cells with FKBPL and its peptide derivative, AD-01

FKBPL and its peptide derivative, AD-01, have already demonstrated well-established inhibitory effects on breast cancer growth and CD44 dependent anti-angiogenic activity1, 2, 3. Since breast cancer stem cells (BCSCs) are CD44 positive, we wanted to explore if AD-01 could specifically target BCSCs. FKBPL stable overexpression or AD-01 treatment were highly effective at reducing the BCSC population measured by inhibiting mammosphere forming efficiency (MFE) in cell lines and primary breast cancer samples from both solid breast tumours and pleural effusions. Flow cytometry, to assess the ESA+/CD44+/CD24- subpopulation, validated these results. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed across three generations of mammospheres, where mammospheres were completely eradicated by the third generation (p<0.001). Clonogenic assays suggested that AD-01 mediated BCSC differentiation, with a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones. In support of this, the stem cell markers, Nanog and Oct4 were significantly reduced following AD-01 treatment, whilst transfection of FKBPL-targeted siRNAs led to an increase in these markers and in mammosphere forming potential, highlighting the endogenous role of FKBPL in stem cell signalling. The clinical relevance of this was confirmed using a publically available microarray data set (GSE7390), where, high FKBPL and low Nanog expression were independently associated with improved overall survival in breast cancer patients (log rank test p=0.03; hazard ratio=3.01). When AD-01 was combined with other agents, we observed synergistic activity with the Notch inhibitor, DAPT and AD-01 was also able to abrogate a chemo- and radiotherapy induced enrichment in BCSCs. Importantly, using ‘gold standard’ in vivo limiting dilution assays we demonstrated a delay in tumour initiation and reoccurrence in AD-01 treated xenografts. In summary, AD-01 appears to have dual anti-angiogenic and anti-BCSC activity which will be advantageous as this agent enters clinical trial.

Activation of STING-Dependent Innate Immune Signaling By S-Phase-Specific DNA Damage in Breast Cancer

BACKGROUND: Previously we identified a DNA damage response-deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance.

METHODS: We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided.

RESULTS: We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher's exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response-proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response-proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle-specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner.

CONCLUSIONS: We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint-based therapies.