Progression In Breast Cancer Patients Research Articles

Associations between clinical-pathological parameters and biomarkers, HER-2, TYMS, RRMI, and 21-gene recurrence score in breast cancer

BackgroundChemotherapy is the predominant treatment option for patients with breast cancer. Selection of patients according to biomarker will improve chemotherapy efficacy. In the present study, we examined the relations of the expression of candidate genes and 21-recurrence score (RS) results to patients’ demographic characteristics, histopathological factors, and outcomes. MethodsA total of 146 patients were enrolled in this study. The patients underwent 21-gene RS testing. In addition, expressions of candidate genes, TYMS, RRM1, TUBB3, TOP2A, PTEN, were detected. Information was obtained on age, tumor size, TMN stage, tumor grade, and status of Ki-67, HER2, ER and PR. The treatment information on the type of endocrine therapy was also obtained. ResultsResults clearly showed that the 21-gene RS significantly correlated with the TNM stage of breast cancer (P = 0.047). The RS also correlated with the number of sentinel lymph node (P = 0.038). The pathological type of tumors was strongly associated with the expression of RRM1 (P < 0.015), and slightly correlated with TYMS (P = 0.095) and tumor size (P = 0.061). Further analysis showed that TYMS and RRM1 were two independent factors affecting the disease progression of patients. Besides, for HER-2 stain, staining of grade 2 or above significantly increased the risk of disease progression. ConclusionOur studies showed that TNM stage and sentinel lymph node were important clinical parameters correlated with 21-gene RS results. Also, RRM1, TYMS and HER2 expressions were independent factors associated with disease progression in breast cancer patients. Future study is warranted to investigate the usefulness of these genes in treatment efficacy.

Abstract 4904: High levels of microRNA-210-3p are associated with increased risk of disease progression in breast cancer patients treated with docetaxel

Abstract MiR-210-3p is the most hypoxia-sensitive miRNA showing overexpression in different human cancers, but its role as biomarker in breast cancer (BC) is still controversial. In our study, we performed the expression analysis of miR-210-3p in a retrospective cohort of breast cancer patients with a median follow-up of 91 months enrolled according to the REMARK guidelines (n=284). As controls, 13 normal breast tissues (NBTs) from reductive mammoplasty were also profiled. By this analysis, we found that miR-210-3p levels were significantly increased in tumours as compared with NBTs (P=0.021). According to the surrogate molecular classification, the Triple Negative subgroup showed the highest levels of miR-210, followed by Luminal B tumours, whereas Luminal A, and HER2-amplified tumours showed the lowest expression levels (P=0.0137). The evaluation of the association of miR-210-3p with time-to-event outcomes in patients without synchronous metastases (n=268) demonstrated an association between risk of disease progression (HR 2.13, 95%CI 0.33-3.39, P=0.002) and higher levels of miR-210-3p in the subgroup treated with docetaxel in sequential therapy with anthracyclines (ECD). Moreover, a cut-off value of 20.966 as established by ROC curve analysis allowed to discriminate patients who developed distant metastases with an accuracy of 85% at 3-years (AUC 0.87, CI 0.69-1) and 83% at 5-years follow up (AUC 0.832, CI 0.656-1). Instead, the accuracy in discriminating patients who died for the disease was of 79.6% at both 5- (AUC 0.804, CI 0.517-1) and 10-years (AUC 0.804 CI 0.517-1) follow up. Overall, our data support the putative involvement of miR-210-3p in the response to treatments including docetaxel and suggest its eligibility to become a biomarker of early metastases development in ECD-treated breast cancer patients. Citation Format: BARBARA PASCULLI, RAFFAELA BARBANO, MICHELINA RENDINA, ANDREA FONTANA, MASSIMILIANO COPETTI, VANNA MARIA VALORI, MARIA MORRITTI, EVARISTO MAIELLO, VITO MICHELE FAZIO, MANEL ESTELLER, PAOLA PARRELLA. High levels of microRNA-210-3p are associated with increased risk of disease progression in breast cancer patients treated with docetaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4904.

NDRG4 promoter hypermethylation is a mechanistic biomarker associated with metastatic progression in breast cancer patients

The risk of developing metastatic disease in breast cancer patients is traditionally predictable based on the number of positive axillary lymph nodes, complemented with additional clinicopathological factors. However, since lymph node-negative patients have a 20–30% probability of developing metastatic disease, lymph node information alone is insufficient to accurately assess individual risk. Molecular approaches, such as multigene expression panels, analyze a set of cancer-related genes that more accurately predict the early risk of metastasis and the treatment response. Here, we present N-Myc downstream-regulated gene 4 (NDRG4) epigenetic silencing as a mechanistic biomarker of metastasis in ductal invasive breast tumors. While aberrant NDRG4 DNA hypermethylation is significantly associated with the development of metastatic disease, downregulation of NDRG4 transcription and protein expression is functionally associated with enhanced lymph node adhesion and cell mobility. Here, we show that epigenetic silencing of NDRG4 modulates integrin signaling by assembling β1-integrins into large punctate clusters at the leading edge of tumor cells to promote an “adhesive switch,” decreasing cell adhesion to fibronectin and increasing cell adhesion and migration towards vitronectin, an important component of human lymph nodes. Taken together, our functional and clinical observations suggest that NDRG4 is a potential mechanistic biomarker in breast cancer that is functionally associated with metastatic disease.

Future Aspects for Cannabinoids in Breast Cancer Therapy.

Cannabinoids (CBs) from Cannabis sativa provide relief for tumor-associated symptoms (including nausea, anorexia, and neuropathic pain) in the palliative treatment of cancer patients. Additionally, they may decelerate tumor progression in breast cancer patients. Indeed, the psychoactive delta-9-tetrahydrocannabinol (THC), non-psychoactive cannabidiol (CBD) and other CBs inhibited disease progression in breast cancer models. The effects of CBs on signaling pathways in cancer cells are conferred via G-protein coupled CB-receptors (CB-Rs), CB1-R and CB2-R, but also via other receptors, and in a receptor-independent way. THC is a partial agonist for CB1-R and CB2-R; CBD is an inverse agonist for both. In breast cancer, CB1-R expression is moderate, but CB2-R expression is high, which is related to tumor aggressiveness. CBs block cell cycle progression and cell growth and induce cancer cell apoptosis by inhibiting constitutive active pro-oncogenic signaling pathways, such as the extracellular-signal-regulated kinase pathway. They reduce angiogenesis and tumor metastasis in animal breast cancer models. CBs are not only active against estrogen receptor-positive, but also against estrogen-resistant breast cancer cells. In human epidermal growth factor receptor 2-positive and triple-negative breast cancer cells, blocking protein kinase B- and cyclooxygenase-2 signaling via CB2-R prevents tumor progression and metastasis. Furthermore, selective estrogen receptor modulators (SERMs), including tamoxifen, bind to CB-Rs; this process may contribute to the growth inhibitory effect of SERMs in cancer cells lacking the estrogen receptor. In summary, CBs are already administered to breast cancer patients at advanced stages of the disease, but they might also be effective at earlier stages to decelerate tumor progression.

Projected clinical benefit of surveillance imaging for early detection and treatment of breast cancer metastases.

This article presents current best knowledge to assess the projected outcomes benefit of adding multi-modality surveillance imaging to standard follow-up care for breast cancer patients at high risk (>30%) for developing future metastases. This analysis is motivated by recent preliminary clinical studies that have suggested that augmenting systemic treatment of early-stage metastases with targeted surgery and/or radiosurgery achieves significant overall survival and disease-free survival benefit. Our primary aims are to: (a) describe the clinical motivation and scan parameters needed to identify the early onset of metastatic progression in breast cancer patients for effective surgical or radiosurgical treatment; (b) estimate the anticipated survival benefit for high-risk patients under this recommended protocol; and (c) estimate the radiation risks associated with the repeated body imaging of this protocol.

Interaction between glutathione S-transferase M1-null/present polymorphism and adjuvant chemotherapy influences the survival of breast cancer.

Glutathione S‐transferase M (GSTM) family is concerned with oxidative stress, which is associated with breast carcinogenesis and chemotherapy response. The null polymorphism of GSTM1 gene results in a thorough absence of the enzyme function. Our study was to evaluate the association between GSTM1 null/present polymorphism and chemotherapy treatment outcome in breast cancer patients. A total of unrelated 714 patients with a histologically confirmed breast cancer were randomly selected from two independent cancer centers. Polymerase chain reaction was performed to analyze null/present genotypes of GSTM1 in our study. Our study found that the present genotype of GSTM1 was associated with a better relapse‐free survival (RFS) (P = .03) with adjusted hazard ratio (HR) [95% confidence interval (CI)] of 0.63 (95% CI: 0.42‐0.93). The present genotype of GSTM1 was significantly correlated with a better RFS compared with the null genotype in the nonchemotherapy group (HR = 0.17, 95% CI: 0.06‐0.50; P = 0.001), but no effect was observed in the chemotherapy group (HR = 0.81, 95% CI: 0.52‐1.26; P = 0.35). Moreover, the interaction between the GSTM1‐null/present genotype and adjuvant chemotherapy was significant (P = 0.04) in further analysis. Our study suggests that the GSTM1 polymorphism plays a complex role in influencing the chemotherapy response and breast cancer survival. It is suggested that the GSTM1‐present genotype might prevent progression in breast cancer patients. In the meanwhile, it could damage the benefit of adjuvant chemotherapy as well in certain ways.

Analysis of Hierarchical Organization in Gene Expression Networks Reveals Underlying Principles of Collective Tumor Cell Dissemination and Metastatic Aggressiveness of Inflammatory Breast Cancer.

Clusters of circulating tumor cells (CTCs), despite being rare, may account for more than 90% of metastases. Cells in these clusters do not undergo a complete epithelial-to-mesenchymal transition (EMT), but retain some epithelial traits as compared to individually disseminating tumor cells. Determinants of single cell dissemination versus collective dissemination remain elusive. Inflammatory breast cancer (IBC), a highly aggressive breast cancer subtype that chiefly metastasizes via CTC clusters, is a promising model for studying mechanisms of collective tumor cell dissemination. Previous studies, motivated by a theory that suggests physical systems with hierarchical organization tend to be more adaptable, have found that the expression of metastasis-associated genes is more hierarchically organized in cases of successful metastases. Here, we used the cophenetic correlation coefficient (CCC) to quantify the hierarchical organization in the expression of two distinct gene sets, collective dissemination-associated genes and IBC-associated genes, in cancer cell lines and in tumor samples from breast cancer patients. Hypothesizing that a higher CCC for collective dissemination-associated genes and for IBC-associated genes would be associated with retention of epithelial traits enabling collective dissemination and with worse disease progression in breast cancer patients, we evaluated the correlation of CCC with different phenotypic groups. The CCC of both the abovementioned gene sets, the collective dissemination-associated genes and the IBC-associated genes, was higher in (a) epithelial cell lines as compared to mesenchymal cell lines and (b) tumor samples from IBC patients as compared to samples from non-IBC breast cancer patients. A higher CCC of both gene sets was also correlated with a higher rate of metastatic relapse in breast cancer patients. In contrast, neither the levels of CDH1 gene expression nor gene set enrichment analysis (GSEA) of the abovementioned gene sets could provide similar insights. These results suggest that retention of some epithelial traits in disseminating tumor cells as IBC progresses promotes successful breast cancer metastasis. The CCC provides additional information regarding the organizational complexity of gene expression in comparison to GSEA. We have shown that the CCC may be a useful metric for investigating the collective dissemination phenotype and a prognostic factor for IBC.

Abstract 3492: Wnt5a induces ROR1 to associate with DOCK1 and promote growth of breast cancer cells

Abstract Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic protein expressed on breast cancer, but not on virtually all normal adult tissues. Studies have found that high-level expression of ROR1 in tumors of breast cancer patients associated with enhanced cell growth, aggressive disease, and shorter overall survival compared to that of patients with tumors that had low expression of ROR1 (Zhang S., et al., PLOS ONE, 7(3): e31127, 2012). Analysis of ROR1-immune precipitates of primary breast cancer patient-derived-xenograft (PDX) lysates via immunoblot analyses revealed that ROR1 was associated with DOCK1 (Dedicator of Cytokinesis 1, also known as DOCK180) in breast cancer PDX tumors. DOCK1 is a member of the DOCK-A subfamily of guanine exchange factors (GEFs) specific for Rac1 that is expressed in breast cancer PDX tumors or cell lines. DOCK1 also contains a N-terminal SH3 domain. We found that ROR1 dissociated from DOCK1 in PDX cells cultured in serum-free medium, unless the cells were stimulated with exogenous Wnt5a. Wnt5a could induce ROR1 to complex with DOCK1 and cause activation of Rac1; this effect could be inhibited by silencing DOCK1 in PDX cells. Furthermore, these effects of Wnt5a on PDX cells also could be blocked by treatment of the PDX cells with cirmtuzumab, a humanized anti-ROR1 mAb, which is undergoing clinical evaluation in patients with cancer. We corroborated these findings using the breast cancer cell-line MCF7, which also expresses DOCK1, but does not express ROR1. MCF7 cells can be transduced to express ROR1 or various mutant forms of ROR1, allowing us to examine the structure-function relationships required for ROR1-DOCK1 interactions. We confirmed that DOCK1 complexes with ROR1 in response to Wnt5a in MCF7-ROR1 cells. We find that silencing DOCK1 specifically impaired the capacity of Wnt5a to enhance growth of MCF7-ROR1 cells in vitro. We generated truncated forms of ROR1 and found the cytoplastmic proline-rich domain (PRD) of ROR1 was required for ROR1 to complex with DOCK1 and activate Rac1 upon stimulation with Wnt5a. We next introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at positions 784, 808, 826, or 841 in potential SH3-binding sites. In contrast to wild-type ROR1, or other ROR1P-&amp;gt;A mutants, ROR1P(808)A had impaired capacity to recruit DOCK1 to ROR1 in response to Wnt5a. Moreover, unlike MCF7 cells transfected with wild-type ROR1 or ROR1 with P-&amp;gt;A substitutions at positions 784, 826, or 841, MCF7 cells transfected to express ROR1P(808)A did not have a growth advantage over that of MCF7 cells that do not express ROR1. This study reveals that the recruitment of DOCK1 may be critical for the capacity of Wnt5a to enhance breast cancer cell growth, which may contribute to the observed increased tendency for disease progression in breast cancer patients who have tumors that express high-levels of ROR1. Citation Format: Md Kamrul Hasan, Victoria Tripple, George F. Widhopf, Suping Zhang, Barbara A. Parker, Thomas J. Kipps. Wnt5a induces ROR1 to associate with DOCK1 and promote growth of breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3492.

Association of Chemokines and Chemokine Receptor Expression with Monocytic-Myeloid-Derived Suppressor Cells during Tumor Progression.

Myeloid-derived suppressor cells (MDSCs) are highly immunosuppressive myeloid cells that show increased expression in cancer patients; however, the molecular mechanisms underlying their generation and function are unclear. Whereas granulocytic-MDSCs correlate with poor overall survival in breast cancer (BC), the presence and relevance of monocytic (Mo)-MDSCs are unknown. Here, we report for the first time increased chemokine and chemokine receptor production by Mo-MDSCs in BC patients. A clear population of Mo-MDSCs with the typical cell surface phenotype (human leukocyte antigen-antigen D related [HLA-DR]low/− CD11b+ CD33+ CD14+) increased significantly during disease progression. In addition, the chemokine receptor expression level on Mo-MDSCs in patients with invasive BC was the highest. Furthermore, different chemokine receptor expression patterns were noted in Mo-MDSCs between healthy controls (HC) and BC patients. Additionally, CD4 T cells proliferations were significantly decreased in the invasive BC groups compared with the HC group. However, the ductal carcinoma in situ (DCIS) group had no significantly compared with the HC group. Our data suggest that monitoring chemokine and chemokine receptor production by Mo-MDSCs may represent a novel and simple biomarker for assessing disease progression in BC patients.

Predictive and Prognostic Brain Metastases Assessment in Luminal Breast Cancer Patients: FN14 and GRP94 from Diagnosis to Prophylaxis.

FN14 has been implicated in many intracellular signaling pathways, and GRP94 is a well-known endoplasmic reticulum protein regulated by glucose. Recently, both have been associated with metastasis progression in breast cancer patients. We studied the usefulness of FN14 and GRP94 expression to stratify breast cancer patients according their risk of brain metastasis (BrM) progression. We analyzed FN14 and GRP94 by immunohistochemistry in a retrospective multicenter study using tissue microarrays from 208 patients with breast carcinomas, of whom 52 had developed BrM. Clinical and pathological characteristics and biomarkers expression in Luminal and non-Luminal patients were analyzed using a multivariate logistic regression model adjusted for covariates, and brain metastasis-free survival (BrMFS) was estimated using the Kaplan–Meier method and the Cox proportional hazards model. FN14 expression was associated with BrM progression mainly in Luminal breast cancer patients with a sensitivity (53.85%) and specificity (89.60%) similar to Her2 expression (46.15 and 89.84%, respectively). Moreover, the likelihood to develop BrM in FN14-positive Luminal carcinomas increased 36.70-fold (3.65–368.25, p = 0.002). Furthermore, the worst prognostic factor for BrMFS in patients with Luminal carcinomas was FN14 overexpression (HR = 8.25; 95% CI: 2.77–24.61; p = 0.00015). In these patients, GRP94 overexpression also increased the risk of BrM (HR = 3.58; 95% CI: 0.98–13.11; p = 0.054—Wald test). Therefore, FN14 expression in Luminal breast carcinomas is a predictive/prognostic biomarker of BrM, which combined with GRP94 predicts BrM progression in non-Luminal tumors 4.04-fold (1.19–8.22, p = 0.025), suggesting that both biomarkers are useful to stratify BrM risk at early diagnosis. We propose a new follow-up protocol for the early prevention of clinical BrM of breast cancer patients with BrM risk.

Robust expression of tumor suppressor miRNA\u2019s let-7 and miR-195 detected in plasma of Saudi female breast cancer patients

BackgroundFemale breast cancer is frequently diagnosed at a later stage and the leading cause of cancer deaths world-wide. Levels of cell-free circulating microRNAs (miRNAs) can potentially be used as biomarkers to measure disease progression in breast cancer patients in a non-invasive way and are therefore of high clinical value.MethodsUsing quantitative RT-PCR, circulating miRNAs were measured in blood samples collected from disease-free individuals (n = 34), triple-negative breast tumours (TNBC) (n = 36) and luminal tumours (n = 57). In addition to intergroup comparisons, plasma miRNA expression levels of all groups were analyzed against RNASeq data from cancerous breast tissue via The Cancer Genome Atlas (TCGA).ResultsA differential set of 18 miRNAs were identified in the plasma of breast cancer patients and 10 miRNAs were uniquely identified based on ROC analysis. The most striking findings revealed elevated tumor suppressor let-7 miRNA in luminal breast cancer patients, irrespective of subtype, and elevated miR-195 in plasma of TNBC breast cancer patients. In contrast, hsa-miR-195 and let-7 miRNAs were absent from cancerous TCGA tissue and strongly expressed in surrounding non-tumor tissue indicating that cancerous cells may selectively export tumor suppressor hsa-miR-195 and let-7 miRNAs in order to maintain oncogenesis.ConclusionsWhile studies have indicated that the restoration of let-7 and miR-195 may be a potential therapy for cancer, these results suggested that tumor cells may selectively export hsa-miR-195 and let-7 miRNAs thereby neutralizing their potential therapeutic effect. However, in order to facilitate earlier detection of breast cancer, blood based screening of hsa-miR-195 and let-7 may be beneficial in a female patient cohort.

543O - Predictive value of the improvement in tumor microenvironment for progression in breast cancer patients treated with neoadjuvant chemotherapy

543O - Predictive value of the improvement in tumor microenvironment for progression in breast cancer patients treated with neoadjuvant chemotherapy

Elevated levels of extracellular vesicles are associated with therapy failure and disease progression in breast cancer patients undergoing neoadjuvant chemotherapy

ABSTRACTExtracellular vesicles (EVs) have been discussed as a diagnostic tool for minimal residual disease (MRD) evaluation in breast cancer (BC) in addition to the analysis of circulating tumor cells (CTCs). Therefore, we investigated circulating EV levels as surrogate markers for disease monitoring and prediction of prognosis in primary, non-metastatic, locally advanced BC patients.EVs were enriched from blood samples of BC patients before and after neoadjuvant chemotherapy (NACT) and from healthy females. EV marker expression analysis was performed and EV sizes and concentrations were determined by nanoparticle tracking analysis. The results were associated with disease status, outcome and CTC presence, evaluated by gene expression analysis after enrichment.We demonstrated that i) the EV concentration was 40-fold higher in BC patients compared to healthy females, ii) the EV concentration increased during therapy, iii) an increased EV concentration pre-NACT was associated with therapy failure and iv) an elevated EV concentration post-NACT was associated with a reduced three-year progression-free and overall survival. Of note, residual stem cell-like and/or resistant CTCs after therapy were associated with a lower EV concentration post-NACT.Our study highlights that the concentration of EVs within BC blood samples may serve as a complementary parameter reflecting the status of MRD as well as therapy and disease outcome in parallel with CTC investigation.

Abstract 3969: AF1q/Mllt11 regulates the expression of intercellular adhesion molecule-1 in breast cancer

Abstract Intercellular adhesion molecule-1 (ICAM-1) is a cell surface glycoprotein in the immunoglobulin superfamily. ICAM-1 expression is frequently observed on many cell types including endothelial cells and different cancer cell entities. Experimental data strongly indicate that ICAM-1 can activate intracellular signaling pathways in cancer cells leading to enhanced cell motility, invasion and metastasis. In clinical investigation, however, ICAM-1 expression was negatively correlated to tumor size, lymph node metastasis, and tumor infiltration. Also, there was improved relapse-free and overall survival in patients with ICAM-1 positive tumors. Yet, the function of ICAM-1 expression during malignant progression in breast cancer patients is not understood clearly. However, ICAM-1 is still considered as a breast cancer target and biomarker may lead to the development of a new strategy and platform for breast cancer patients. We observed AF1q, a metastasis enhancer, is only expressed in metastatic cells, MDA-MB-231LN (invasive sub line from MDA-MB-231), not in the primary tumor cells. To investigate whether AF1q has a responsibility in acquisition of metastatic phenotype, we performed RNA-Seq and applied the Metacore network building algorithm. Intriguingly, most genes were directly linked with Intercellular Adhesion Molecule-1 (ICAM-1). Likewise, we identified that ICAM-1 expression is attenuated in MDA-MB-231LN compared to MDA-MB-231. Moreover, the expression of ICAM-1 was negatively regulated, when AF1q was overexpressed in MDA-MD-231LN. Suppressed endogenous AF1q by shRNA could effectively increase the ICAM-1 expression. Attenuation of ICAM-1 by AF1q on tumor cells would disadvantage host anti-tumor defenses by trafficking of lymphocytes, which affect tumor progression and metastasis. We observed that NF-κB activity which regulates ICAM-1 expression is attenuated in response to AF1q expression in breast cancer cells. Published reports shown that Wnt/β-catenin negatively regulates NF-κB activity through protein-protein interaction in colon and breast cancer cells. Previously, we reported that AF1q enhance the TCF7/LEF/β-catenin complex binding affinity as a cofactor. Structurally, AF1q has highly acidic peptide regions highly conserved between species that fulfill the criteria for an acidic blob, a typical feature for cofactors. When we performed immunoprecipitation with NF-κB antibody in MDA-MB-231LN overexpressed AF1q, we observed that β-catenin and AF1q were pulled down together. However, it is not clear yet whether AF1q promote protein interaction between β-catenin and NF-κB. These results suggest that activated β-catenin by AF1q would archive higher affinity to bind with NF-κB and thereby attenuate ICAM-1 expression. Citation Format: Jino Park, Soojin Kim, William Tse. AF1q/Mllt11 regulates the expression of intercellular adhesion molecule-1 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3969. doi:10.1158/1538-7445.AM2017-3969

Demethylation of Repressor Element-1 Silencing Transcription (REST) Suppresses the Malignant Phenotype of Breast Cancer via MMP9.

Breast cancer is the leading cause of cancer deaths in females all over the world, mainly resulting from metastasis. Previous studies have revealed that repressor element-1 (RE-1) silencing transcription (REST) acted as a tumor suppressor in breast cancer. However, the mechanism by which REST is regulated remains unknown, and its role in the metastasis in breast cancer cells remains unclear. In the present study, we showed that the expression of REST was lower in breast cancer samples than that of adjacent samples by immunohistochemical analysis, which may be due to hypermethylation of the REST promoter. Low REST levels are significantly associated with malignant progression in breast cancer patients. Additionally, we elucidated the functions of REST on proliferation and invasion in breast cancer cells. Lentivirus transfection was used to overexpress REST in human breast MDA-MB-231 cells. Then the biologic consequences of overexpressing REST in regard to cell proliferation, apoptosis, and invasion were determined. Furthermore, we also determined matrix metalloproteinase-9 (MMP9) as a target of REST. These results demonstrate that downregulation of REST, a tumor suppressor in breast cancer, is associated with hypermethylation. Induced REST expression is capable of attenuating invasion ability of breast cancer cells, which may be a novel strategy for metastatic breast cancer treatment.

Abstract P6-15-02: Phenotypically distinct HRG positive cancer cells impact standard of care therapies in metastatic breast cancer models

Abstract ErbB3 is a member of the human epidermal growth factor receptor (ErbB or HER) family which is comprised of four receptors (ErbB1-4). A defining feature of the ErbB network is that two members of the family, ErbB2 and ErbB3, are non-autonomous. ErbB2 lacks the capacity to interact with a growth-factor ligand, whereas the kinase activity of ErbB3 is defective. Heregulin (HRG), the ErbB3 ligand, has been identified as a potent driver of proliferation and enhanced survival. HRG expression leads to a distinct tumor cell phenotype characterized by an inability to respond to the effects of numerous Standard of Care (SOC) therapies, including chemotherapies, anti-hormonal agents and other targeted therapeutics. In surveys of HRG expression, we have shown the presence of HRG+ cells in approximately 50% of the cases of most solid tumor types. We hypothesize that these HRG+ cells are protected from the effects of SOC therapy and continue to proliferate even in the presence of SOC, resulting in limited clinical benefit. In this model, if HRG activity is blocked, HRG+ cells become susceptible to SOC, resulting in enhanced clinical benefit. Seribantumab is a fully human anti-ErbB3 monoclonal antibody designed to block HRG activity by inhibiting the binding of HRG to ErbB3. In the presence of seribantumab, HRG+ tumor cells are predicted to be able to respond to co-administered SOC therapy. For hormone receptor positive (HR+) breast cancer, hormone deprivation strategies have proven clinical benefit in the adjuvant and metastatic settings. Unfortunately, clinical benefit from these therapies can be short-lived in some patients. Optimal clinical management of these patients requires a comprehensive molecular understanding of the drivers of rapid clinical progression. We and others have found that HRG mRNA expression measured in tumor samples defines a subgroup of patients who derive only limited clinical benefit from SOC when compared to patients whose tumors do not express HRG. This was observed in a previously published Phase 2 clinical study with exemestane, and preclinically with multiple classes of anti-hormonal agents, including letrozole and fulvestrant -- treatments that currently represent the mainstay of treatment options for HR+, HER2 negative (HER2-) advanced breast cancer. Here we will present data supporting the hypothesis that phenotypically distinct HRG+ cells in breast cancer models persist despite treatment with SOC and various novel classes of therapy. We will also show that the addition of the anti-ErbB3 antibody seribantumab to these other therapies promotes sustained treatment responses. Continued expansion of HRG+ cells could be the key to rapid clinical progression in breast cancer patients treated with SOC therapy. These findings support the development of seribantumab in combination with anti-hormonal agents in a planned Phase 3 clinical trial in HR+, HER2- advanced breast cancer. Citation Format: Finn GJ, Zhang H, Blois A, Mathews SE, Kudla AJ, Baum JS, Demars NA, Cieslewicz MJ, Czibere A. Phenotypically distinct HRG positive cancer cells impact standard of care therapies in metastatic breast cancer models [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 P6-15-02.

A novel application of E1A in combination therapy with EGFR-TKI treatment in breast cancer.

Epidermal growth factor receptor (EGFR) is commonly overexpressed in breast cancer and is associated with poor clinical outcomes; however, an increasing number of patients have shown a poor effective response to EGFR tyrosine kinase inhibitors (EGFR-TKI). Here, we found that AXL expression was positively correlated with poor progression in breast cancer patients. Suppression of AXL by an anti-tumor protein, E1A, enhanced EGFR-TKI (gefitinib, erlotinib and lapatinib) sensitization, resulting in significant inhibition of tumor growth in breast cancer cells. Additionally, AXL overexpression dramatically impaired E1A-mediated EGFR-TKI sensitization. These findings show that downregulation of AXL expression by E1A contributes to sensitization to EGFR-TKI in breast cancer, suggesting that combinatorial therapy of AXL inhibitors or E1A gene therapy with EGFR-TKI may be a potential therapeutic strategy for treatment of breast cancer patients.

Blocking Interleukin-4 Receptor α Using Polyethylene Glycol Functionalized Superparamagnetic Iron Oxide Nanocarriers to Inhibit Breast Cancer Cell Proliferation.

PurposeThe specific targeting of interleukin-4 receptor α (IL4Rα) receptor offers a promising therapeutic approach for inhibition of tumor cell progression in breast cancer patients. In the current study, the in vitro efficacy of superparamagnetic iron oxide nanoparticles conjugated with anti-IL4Rα blocking antibodies (SPION-IL4Rα) via polyethylene glycol polymers was evaluated in 4T1 breast cancer cells.Materials and MethodsCell viability, reactive oxygen species generation, and apoptosis frequency were assessed in vitro in 4T1 cancer cell lines following exposure to SPION-IL4Rα alone or combined with doxorubicin. In addition, immunofluorescence assessments and fluorimetrywere performed to confirm the specific targeting and interaction of the developed nanocarriers with IL4Rα receptors in breast cancer cells.ResultsBlocking of IL4Rα receptors caused a significant decrease in cell viability and induced apoptosis in 4T1 cells. In addition, combined treatment with SPION-IL4Rα+doxorubicin caused significant increases in cell death, apoptosis, and oxidative stress compared to either SPION-IL4Rα or doxorubicin alone, indicating the enhanced therapeutic efficacy of this combination. The decrease in fluorescence intensity upon immunofluorescence and fluorimetry assays combined with increased viability and decreased apoptosis following the blocking of IL4Rα receptors confirmed the successful binding of the synthesized nanocarriers to the target sites on murine 4T1 breast cancerous cells.ConclusionThese results suggest that SPION-IL4Rα nanocarriers might be used for successfulreduction of tumor growth and inhibition of progression of metastasis in vivo.

Downregulation of miR-129-2 by promoter hypermethylation regulates breast cancer cell proliferation and apoptosis.

Aberrant expression of the miR-129 family has been found in several types of cancer, yet its expression and potential biologic role in breast cancer remain largely unknown. In the present study, we found that miR-129-2 was consistently downregulated in the breast cancer specimens and cell lines. Overexpression of miR-129-2-3p markedly suppressed breast cancer cell proliferation and induced its apoptosis. In addition, a luciferase reporter assay revealed that miR-129-2-3p suppressed BCL2L2 expression. Furthermore, BCL2L2 was able to reverse miR-129-2-3p-mediated cell apoptosis, indicating that BCL2L2 plays a crucial role in mediating the tumor-suppressive role of miR-129-2-3p. Moreover, bisulfite DNA sequencing PCR (BSP) analysis identified that promoter hypermethylation was responsible for the downregulation of miR-129-2 in breast cancer. Collectively, our findings indicate that miR-129-2 is downregulated in breast cancer cells by promoter hypermethylation. Moreover, downregulation of miR-129-2 results in BCL2L2 overexpression and disease progression in breast cancer patients.

P6: Prognostic significance of immunohistochemical and genetic markers in Russian breast cancer patients treated with adjuvant tamoxifen

Background Tamoxifen, a selective estrogen receptor modulator, remains a standard of endocrine therapy for estrogen receptor alpha (ER α ) positive breast cancer (Goldhirsch et al., 2013). Despite well-known advantages of tamoxifen treatment, approximately one third of patients experience a relapse or disease progression due to tamoxifen resistance (Osborne et al., 2011). Identification of additional molecular markers associated with both ER genomic and non-genomic pathways could be very useful to help identify patients who will likely benefit from such endocrine treatment. The aim of the present study was to evaluate the influence of the distribution pattern of ER α expression as well as ESR1, TGF-βR1 and IGF-1R single nucleotide polymorphisms (SNPs) and the expression of growth factors receptors on disease progression in breast cancer patients of Russian Western Siberian population treated by adjuvant tamoxifen. Materials and methods Formalin-fixed paraffin-embedded tumor tissue samples were retrospectively collected from 97 hormone receptor-positive breast cancer patients treated with adjuvant tamoxifen at the Tomsk Cancer Research Institute. Genotypes for ESR1 +30T>C (rs2077647), ESR1 2014G>A (rs2228480), IVS7+24G > A (rs334354) and IGF-1R (rs2016347) SNPs were detected by a TaqMan assay. The distribution patterns of ER α expression and EGFR, TGF- β R1, IGF-1R protein expression were determined using the immunohistochemistry. Patients who developed distant metastasis or recurrence after tamoxifen treatment during the follow-up period were defined as tamoxifen resistance (TR) group, while distant metastasis-free patients were analyzed as tamoxifen sensitive (TS) group. Results We found that the heterogeneous distribution of ER α expression was statistically significant related with poor prognosis of tamoxifen treated patients ( p = 0.021). Similarly, we showed high EGFR expression in TR group compared to TS patients (80.0% vs. 41.9% respectively, p = 0.009). Additionally, EGFR expression and distribution pattern of ER α expression were significantly associated with response to tamoxifen and this association remained significant in both univariate and multivariate analysis. Our results demonstrate that the ESR1 2014A mutant allele carriers were more prevalent in TR patients than in TS group (26.3% vs. 8.0%, respectively, p = 0.009). Concerning the IGF-1R polymorphism, we found that TT homozygous carriers were more frequent in the TS group compared with TR group ( p = 0.043). A significant association between IVS7+24G > A SNP and high TGF- β R1 protein expression was observed ( p = 0.044). Nonetheless, among all studied polymorphisms only ESR1 2014G>A SNP was correlated with a heterogeneous distribution of ER α expression ( r = 0.353, p = 0.016). Conclusion These data suggest that the distribution pattern of ER α expression, EGFR expression and ESR1 2014G>A genetic variation could be useful additional prognostic markers for hormone receptor-positive breast cancer patients treated with adjuvant tamoxifen.