Relapse-free Survival In Breast Cancer Patients Research Articles

Drug repositioning for immunotherapy in breast cancer using single-cell analysis.

Immunomodulatory peptides, while exhibiting potential antimicrobial, antifungal, and/or antiviral properties, can play a role in stimulating or suppressing the immune system, especially in pathological conditions like breast cancer (BC). Thus, deregulation of these peptides may serve as an immunotherapeutic strategy to enhance the immune response. In this meta-analysis, we utilized single-cell RNA sequencing data and known therapeutic peptides to investigate the deregulation of these peptides in malignant versus normal human breast epithelial cells. We corroborated our findings at the chromatin level using ATAC-seq. Additionally, we assessed the protein levels in various BC cell lines. Moreover, our in-house drug repositioning approach was employed to identify potential drugs that could positively impact the relapse-free survival of BC patients. Considering significantly deregulated therapeutic peptides and their role in BC pathology, our approach aims to downregulate B2M and SLPI, while upregulating PIGR, DEFB1, LTF, CLU, S100A7, and SCGB2A1 in BC epithelial cells through our drug repositioning pipeline. Leveraging the LINCS L1000 database, we propose BRD-A06641369 for B2M downregulation and ST-4070043 and BRD-K97926541 for SLPI downregulation without negatively affecting the MHC complex as a significantly correlated pathway with these two genes. Furthermore, we have compiled a comprehensive list of drugs for the upregulation of other selected immunomodulatory peptides. Employing an immunotherapeutic approach by integrating our drug repositioning pipeline with single-cell analysis, we proposed potential drugs and drug targets to fortify the immune system against BC.

Analysis of TLR2 in Primary Endocrine Resistant of Breast Cancer.

Previous clinical studies have suggested that Toll-like receptor (TLR)2 had predictive function for endocrine resistance in HER2-positive breast cancer (BCa). Nevertheless, it remains unclear whether TLR2 would relate to development of endocrine therapy resistance in triple-positive breast cancer (TPBC). Bioinformatic analysis of TLR2 was carried out through a database. Ten tumor tissues were obtained from TPBC patients who underwent surgery, with five patients displaying primary resistance to tamoxifen (TAM) with the remaining 5 being sensitive. Different levels of proteins were identified through mass spectrometry analysis and confirmed through reverse transcription polymerase chain reaction (RT-PCR) and western blot. TAM-resistant cell lines (BT474-TAM) were established by continuous exposure to TAM, and TAM resistance was assessed via IC50. Additionally, TLR2 mRNA was analyzed through western blot and RT-PCR in BT474, BT474-TAM, MCF-7, and MCF10A cells. Furthermore, TLR2-specific interference sequences were utilized to downregulate TLR2 expression in BT474-TAM cells to elucidate its role in TAM resistance. TLR2 had a correlation with decreased relapse-free survival in BCa patients from the GSE1456-GPL96 cohort, and it was involved in cancer development predominantly mediated by MAPK and PI3K pathways. TLR2 protein expression ranked in the top 5 proteins within the TAM-resistant group, and was 1.9 times greater than that in the sensitive group. Additionally, TLR2 mRNA and protein expression increased significantly in the established TAM-resistant BT474/TAM cell lines. The sensitivity of TAM was restored upon TLR2 downregulation in BT474/TAM cells. TLR2 might have a therapeutic value as it was involved in the TAM resistance in TPBC, with potential to be a marker for primary endocrine resistance.

Delineation of Pathogenomic Insights of Breast Cancer in Young Women.

The prognosis of breast cancer (BC) in young women (BCYW) aged ≤40 years tends to be poorer than that in older patients due to aggressive phenotypes, late diagnosis, distinct biologic, and poorly understood genomic features of BCYW. Considering the estimated predisposition of only approximately 15% of the BC population to BC-promoting genes, the underlying reasons for an increased occurrence of BCYW, at large, cannot be completely explained based on general risk factors for BC. This underscores the need for the development of next-generation of tissue- and body fluid-based prognostic and predictive biomarkers for BCYW. Here, we identified the genes associated with BCYW with a particular focus on the age, intrinsic BC subtypes, matched normal or normal breast tissues, and BC laterality. In young women with BC, we observed dysregulation of age-associated cancer-relevant gene sets in both cancer and normal breast tissues, sub-sets of which substantially affected the overall survival (OS) or relapse-free survival (RFS) of patients with BC and exhibited statically significant correlations with several gene modules associated with cellular processes such as the stroma, immune responses, mitotic progression, early response, and steroid responses. For example, high expression of COL1A2, COL5A2, COL5A1, NPY1R, and KIAA1644 mRNAs in the BC and normal breast tissues from young women correlated with a substantial reduction in the OS and RFS of BC patients with increased levels of these exemplified genes. Many of the genes upregulated in BCYW were overexpressed or underexpressed in normal breast tissues, which might provide clues regarding the potential involvement of such genes in the development of BC later in life. Many of BCYW-associated gene products were also found in the extracellular microvesicles/exosomes secreted from breast and other cancer cell-types as well as in body fluids such as urine, saliva, breast milk, and plasma, raising the possibility of using such approaches in the development of non-invasive, predictive and prognostic biomarkers. In conclusion, the findings of this study delineated the pathogenomics of BCYW, providing clues for future exploration of the potential predictive and prognostic importance of candidate BCYW molecules and research strategies as well as a rationale to undertake a prospective clinical study to examine some of testable hypotheses presented here. In addition, the results presented here provide a framework to bring out the importance of geographical disparities, to overcome the current bottlenecks in BCYW, and to make the next quantum leap for sporadic BCYW research and treatment.

Abstract 562: Novel combination therapy for treating TNBC using LIFR and HDAC Inhibitors

Abstract Background: Triple-negative breast cancer (TNBC) lacks targeted therapies and represents a disproportional share of the breast cancer (BC) mortality rate. TNBC exhibits autocrine stimulation of the LIF/LIFR axis and overexpression of LIF is associated with poorer relapse-free survival in BC patients. Histone deacetylase inhibitors (HDACIs) are emerging as promising multifunctional agents in TNBC to elicit cytotoxic actions. Recent studies have shown that cancer cells elicit feedback activation of leukemia inhibitory factor receptor (LIFR) which in turn curtails response to HDACIs. We developed a first-in-class inhibitor of LIFR, EC359 that directly interacts with LIFR and effectively blocks LIFR downstream signaling. The objective of this study is to examine the therapeutic efficacy of combination therapy using preclinical and patient-derived xenograft (PDX) models. Methods: We tested utility of combination therapy using multiple HDACIs that are currently in clinical trails along with EC359. The effect of combination therapy was evaluated using MTT, invasion, colony formation, and Caspase3/7 assays. Mechanistic studies were performed using Western blotting, qRT-PCR, and STAT3 reporter assays. The efficacy of combination therapy in vivo was examined using xenograft, PDX, and patient-derived explant (PDEx) models. Results: Immunohistochemical analyses of breast tumors using tissue microarrays revealed significant expression of LIFR in TNBC tissues. Treatment of TNBC model cells with four different HDACIs increased the expression of LIFR. LIFR inhibitor EC359 at nM concentration is additive to HDACIs in reducing cell viability. Knockdown of LIFR or treatment with EC359 significantly enhanced the efficacy of HDACIs in reducing the cell viability, colony formation ability, and invasiveness as well as promoted apoptosis compared to monotherapy in TNBC model cells. On the contrary, treatment with STAT3 inhibitor requires µM concentrations to reduce the cell viability of TNBC cells and is not additive to HDACIs. Mechanistic studies utilizing STAT3 reporter gene assays and biochemical studies using multiple TNBC model cells exhibited activation of the LIFR signaling pathway upon HDACIs treatment but was attenuated by EC359 therapy. Treatment of human TNBC utilizing PDEx assays showed that EC359 enhanced the ability of HDACIs to decrease proliferation (Ki-67 positivity) compared to monotherapy. Using TNBC xenografts and PDX models, we demonstrated that EC359 treatment enhanced the ability of HDACIs to reduce in vivo tumor growth compared to monotherapy. Conclusions: Our results suggest that the combination therapy of HDACIs and EC359 provides therapeutic utility in overcoming the limitation of feedback activation of LIFR observed in the treatment of HDACIs in treating TNBC. Supported by DOD BCRP grant W81XWH-18-1-0016 (R.K. Vadlamudi; K.J. Nickisch) Citation Format: Suryavathi Viswanadhapalli, Mengxing Li, Bindu Santhamma, Uday P. Pratap, Yiliao Luo, Junhao Liu, Kristin A. Altwegg, Xiaonan Li, Ahmed Gulzar, Hui Yan, Zhenming Xu, Andrew Brenner, Gangadhara R. Sareddy, Manjeet K. Rao, Rajeshwar R. Tekmal, Hareesh B. Nair, Klaus J. Nickisch, Ratna K. Vadlamudi. Novel combination therapy for treating TNBC using LIFR and HDAC Inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 562.

Comprehensive analysis of the prognostic value and immune function of chemokine-CXC receptor family members in breast cancer

Recently, immune checkpoint inhibitors (ICIs) have been successfully used for treating melanoma. Unfortunately, many breast cancer (BC) patients show low response to ICIs due to the lack of infiltrating immune cells. Previous studies revealed that chemokine-CXC receptors (CXCRs) play a crucial role in leukocyte infiltration and promote cancer cell proliferation, migration, metastasis, and angiogenesis. However, the underlying functions of CXCRs in cancer-immunity cycle remain unclear. In this study, we firstly found that in comparison to normal tissues, BC tissues, especially basal-like BC, showed increased mRNA levels of CXCR3/4/5/6/8, but decreased CXCR1/2/7 expression using UALCAN and TIMER database. Interestingly, it’s was found that the mRNA levels of CXCR3/4/5/6 were decreased in lymphocyte depleted of the BC immune subtype. Subsequently, functional enrichment analysis of distinct CXCRs indicated that CXCR3/4/5/6 were strongly associated to immune-related biological functions. Therefore, further analysis using TIMER and TISIDB database suggested that CXCR3/4/5/6 expression were strongly correlated with tumor-infiltrating lymphocytes (TILs) and immune checkpoints in BC. Finally, Kaplan–Meier Plotter analysis indicated that high mRNA expression of CXCR4 predicted worse relapse-free survival (RFS), whereas CXCR3/5/6 indicated better RFS in BC patients. These findings suggest a therapeutic value for CXCR3/4/5/6 in combination with ICIs for the treatment of BC.

Metabolic classification of circulating tumor cells as a biomarker for metastasis and prognosis in breast cancer

BackgroundCirculating tumor cells (CTCs) has been demonstrated as a promising liquid biopsy marker for breast cancer (BC). However, the intra-patient heterogeneity of CTCs remains a challenge to clinical application. We aim at profiling aggressive CTCs subpopulation in BC utilizing the distinctive metabolic reprogramming which is a hallmark of metastatic tumor cells.MethodsOncomine, TCGA and Kaplan–Meier plotter databases were utilized to analyze expression and survival relevance of the previously screened metastasis-promoting metabolic markers (PGK1/G6PD) in BC patients. CTCs detection and metabolic classification were performed through micro-filtration and multiple RNA in situ hybridization using CD45 and PGK1/G6PD probes. Blood samples were collected from 64 BC patients before treatment for CTCs analysis. Patient characteristics were recorded to evaluate clinical applications of CTCs metabolic subtypes, as well as morphological EMT subtypes classified by epithelial (EpCAM/CKs) and mesenchymal (Vimentin/Twist) markers.ResultsPGK1 and G6PD expressions were up-regulated in invasive BC tissues compared with normal mammary tissues. Increased tissue expressions of PGK1 or G6PD indicated shortened overall and relapse-free survival of BC patients (P < 0.001). Blood GM+CTCs (DAPI+CD45−PGK1/G6PD+) was detectable (range 0–54 cells/5 mL) in 61.8% of tCTCs > 0 patients. Increased GM+CTCs number and positive rate were correlated with tumor metastasis and progression (P < 0.05). The GM+CTCs ≥ 2/5 mL level presented superior AUC of ROC at 0.854 (95% CI 0.741–0.968) in the diagnosis of BC metastasis (sensitivity/specificity: 66.7%/91.3%), compared with that of tCTCs (0.779) and CTCs-EMT subtypes (E-CTCs 0.645, H-CTCs 0.727 and M-CTCs 0.697). Moreover, GM+CTCs+ group had inferior survival with decreased 2 years-PFS proportion (18.5%) than GM+CTCs− group (87.9%; P = 0.001).ConclusionsThis work establishes a PGK1/G6PD-based method for CTCs metabolic classification to identify the aggressive CTCs subpopulation. Metabolically active GM+CTCs subtype is suggested a favorable biomarker of distant metastasis and prognosis in BC patients.

Hyperglycemia during Adjuvant Chemotherapy as a Prognostic Factor in Breast Cancer Patients without Diabetes.

PurposeBreast cancer treatments, including chemotherapy, administered in combination with glucocorticoids can induce hyperglycemia. This study aimed to investigate the effect of hyperglycemia during adjuvant chemotherapy on the prognosis of breast cancer patients without a known history of diabetes.MethodsIn this study, 936 patients who underwent breast cancer surgery from 2010 to 2015 were initially selected as participants. Chemotherapy-related hyperglycemia was defined as fasting plasma glucose levels ≥ 100 mg/dL or random blood glucose levels ≥ 140 mg/dL during 2 or more cycles of adjuvant chemotherapy. After dividing the patients into the euglycemia and hyperglycemia groups, univariate and multivariate analyses were performed, and survival outcomes were analyzed by propensity score matching.ResultsThe mean age of the patients was 47.4 ± 7.7 years, and the median follow-up period was 70.1 months. Eighty-two patients (19.4%) were diagnosed as having hyperglycemia. There were significant differences between the euglycemia and hyperglycemia groups with respect to age, hypertension, body mass index, axillary surgery extents, nodal stage, and total steroid dosage. T stage, vascular invasion, and hyperglycemia were identified as prognostic factors of relapse-free survival (RFS). The 5-year RFS rates were 92.0% and 82.3% in the euglycemia and hyperglycemia groups, respectively, and there was a statistically significant difference between the 2 groups (p = 0.011). The 5-year overall survival rates were 94.6% and 92.0% in the euglycemia and hyperglycemia groups, respectively, showing no statistically significant difference between the 2 groups (p = 0.113).ConclusionThese data suggest that hyperglycemia during adjuvant chemotherapy is a prognostic factor for RFS in breast cancer patients without diabetes.

Abstract P2-06-02: Development of a first-in-class small molecule inhibitor (EC359) targeting oncogenic LIF/LIFR signaling for the treatment of triple negative breast cancer

Abstract Background: Leukemia inhibitory factor (LIF) and its receptor LIFR are over-expressed in multiple solid tumors and play a key role in tumor growth, progression, and resistance to standard anti-cancer treatments. Triple-negative breast cancer (TNBC) lacks targeted therapies and represents a disproportional share of breast cancer (BCa) mortality. TNBC exhibits autocrine stimulation of the LIF/LIFR axis and overexpression of LIF is associated with poorer relapse-free survival in BCa patients. LIF signaling also promotes maintenance of stem cells. Therefore, targeting the LIF/LIFR axis may have therapeutic utility in TNBC. Methods: We rationally designed a small organic molecule (EC359) that emulates the LIF/LIFR binding site and functions as a LIFR inhibitor from a library of compounds. In silico docking studies were used to identify the putative interaction of the EC359 and LIF/LIFR complex. Direct binding of EC359 to LIFR was confirmed using surface plasmon resonance (SPR) and microscale thermophoresis technique (MST) assays. In vitro activity was tested using Cell-Titer Glo, MTT, invasion, and apoptosis assays. Mechanistic studies were conducted using Western blot, reporter gene assays, and RNA-seq analysis. Xenograft, patient-derived xenograft (PDX), and patient-derived explant (PDEX) models were used for preclinical evaluation and toxicity. Results: Molecular docking studies showed that EC359 interacts at the LIF/LIFR binding interface. SPR and MST studies confirmed direct interaction of EC359 to LIFR. EC359 reduced the growth of TNBC cells with high potency (IC50 50-100nM) and promoted apoptosis. Further, EC359 treatment reduced invasion and stemness of TNBC cells. EC359 activity is dependent on the expression levels of LIFR and showed little or no activity on TNBC cells that have low levels of LIFR or ER+ve BCa cells. Further, EC359 significantly reduced the viability of cisplatin and taxane-resistant TNBC cells and enhanced the efficacy of HDAC inhibitors. Mechanistic and biochemical studies showed that EC359 interacts with LIFR and effectively blocking LIF/LIFR interactions. EC359 also blocked LIFR interactions with other LIFR ligands such as oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1. EC359 treatment attenuated the activation of LIF/LIFR driven pathways including STAT3, mTOR, AKT, and MAPK. RNA-seq analysis identified regulation of apoptosis as one of the important pathway modulated by EC359. In TNBC xenograft and PDX assays, EC359 significantly reduced tumor progression. Further, using human primary BCa PDEX cultures, we demonstrated that EC359 has the potential to substantially reduce the proliferation of human BCa. Pharmacologically, EC359 exhibited high oral bioavailability and long half-life with a wide therapeutic window. Conclusions: EC359 is a novel targeted therapeutic agent that inhibits LIF/LIFR oncogenic signaling in TNBC via a unique mechanism of action. EC359 has the distinct pharmacologic advantages of oral bioavailability, in vivo stability, and is associated with minimal systemic side effects. (DOD BCRP grant #BC170312) Citation Format: Viswanadhapalli S, Luo Y, Sareddy GR, Santhamma B, Zhou M, Li M, Pratap UP, Altwegg KA, Li X, Srinivasan U, Ma S, Chang A, Riveros AC, Zhang KY, Dileep KV, Pan X, Murali R, Bajda M, Raj G, Brenner A, Manthati V, Rao M, Tekmal RR, Nair HB, Nickisch KJ, Vadlamudi RK. Development of a first-in-class small molecule inhibitor (EC359) targeting oncogenic LIF/LIFR signaling for the treatment of triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-02.

Significant prognostic values of aquaporin mRNA expression in breast cancer.

IntroductionAquaporins (AQPs), also called water channels, have been shown to have functions in the migration, invasion, and proliferation of human breast tumor cells. Most AQP mRNA expression levels were tested by cell lines, mouse models, and even human breast tissues. However, the mRNA expression of individual AQPs in different clinicopathologic characteristics and prognostic values according to different kinds of classifications of breast cancer patients remains unclear.Materials and methodsIn the current study, we used the Oncomine database, Breast cancer Gene-Expression Miner v4.1, Kaplan–Meier Plotter, and cBioPortal to investigate the expression distribution and prognostic values of AQPs in breast cancer patients.ResultsOur study revealed that the mRNA expression levels of AQP8, AQP9, and AQP10 were upregulated, while those of AQP3, AQP4, AQP5, and AQP7 were downregulated in breast cancer. The clinical database showed that lower mRNA levels of AQP1 were associated with a high Scarff–Bloom–Richardson grade, but AQP9 showed the opposite trend. Further survival analyses indicated that high mRNA expression levels of AQP0, AQP1, AQP2, AQP4, AQP6, AQP8, AQP10, and AQP11 were significantly associated with better relapse-free survival (RFS). Conversely, AQP3 and AQP9 were associated with worse RFS in breast cancer patients, suggesting that these two genes might be potential targets in future chemotherapy.DiscussionThese significant AQP members might be further explored as new biomarkers for breast cancer prognosis, but this needs further study.

PERK-Phosphorylated eIF2α Pathway Suppresses Tumor Metastasis Through Downregulating Expression of Programmed Death Ligand 1 and CXCL5 in Triple-Negative Breast Cancer.

Endoplasmic reticulum (ER) stress has been reported to be associated with metastasis in many malignant tumors. PKR-like ER kinase-phosphorylated eukaryotic translation initiation factor 2α (PERK-p-eIF2α) pathway is one of the three main signal pathways in ER stress, however, its mechanism in regulating breast cancer (BC) relapse or metastasis was still not completely understood. Besides, drug resistance was an important factor influencing the effect of tumor treatment and whether PERK-p-eIF2α pathway was involved in the drug resistance to BC treatment also needs to be explored. The authors conducted survival analysis of ER stress-related genes in the The Cancer Genome Atlas (TCGA) database to find the candidate molecule and found that eIF2α was significantly correlated with relapse-free survival in BC patients, especially in the triple-negative BC (TNBC) patients. Furthermore, BC cell lines were used to study the downstream target of PERK-p-eIF2α. In this study, p-eIF2α could negatively regulate the expression of programmed death ligand 1 (PDL1) and C-X-C motif chemokine ligand 5 (CXCL5), which were important ligands of the immune cells such as T cells and myeloid-derived suppressor cells in the tumor microenvironment. Besides, p-eIF2α expression in highly metastatic human TNBC cells after treatment of carboplatin was significantly decreased. The data indicated the possible novel immune-related mechanism of PERK-p-eIF2α in regulating TNBC metastasis and drug resistance of carboplatin in highly metastatic TNBC.

An early biomarker and potential therapeutic target of RUNX 3 hypermethylation in breast cancer, a system review and meta-analysis.

Runt-related transcription factor 3 (RUNX3) methylation plays an important role in the carcinogenesis of breast cancer (BC). However, the association between RUNX3 hypermethylation and significance of BC remains under investigation. The purpose of this study is to perform a meta-analysis and literature review to evaluate the clinicopathological significance of RUNX3 hypermethylation in BC. A comprehensive literature search was performed in Medline, Web of Science, EMBASE, Cochrane Library Database, CNKI and Google scholar. A total of 10 studies and 747 patients were included for the meta-analysis. Pooled odds ratios (ORs) with corresponding confidence intervals (CIs) were evaluated and summarized respectively. RUNX3 hypermethylation was significantly correlated with the risk of ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC), OR was 50.37, p < 0.00001 and 22.66, p < 0.00001 respectively. Interestingly, the frequency of RUNX3 hypermethylation increased in estrogen receptor (ER) positive BC, OR was 12.12, p = 0.005. High RUNX3 mRNA expression was strongly associated with better relapse-free survival (RFS) in BC patients. In summary, RUNX3 methylation could be a promising early biomarker for the diagnosis of BC. High RUNX3 mRNA expression is correlated to better RFS in BC patients. RUNX3 could be a potential therapeutic target for the development of personalized therapy.