Tumor Progression In Breast Cancer Research Articles

Aberrant promoter hypermethylation inhibits RGMA expression and contributes to tumor progression in breast cancer.

Breast cancer (BC) is the most common cancer in women worldwide, and the exploration of aberrantly expressed genes might clarify tumorigenesis and help uncover new therapeutic strategies for BC. Although RGMA was recently recognized as a tumor suppressor gene, its detailed biological function and regulation in BC remain unclear. Herein, we found that RGMA was downregulated in BC tissues compared with non-tumorous breast tissues, particularly in metastatic BC samples, and that patients with low RGMA expression manifested a poorer prognosis. Furthermore, DNMT1 and DNMT3A were found to be recruited to the RGMA promoter and induced aberrant hypermethylation, resulting in downregulation of RGMA expression in BC. In contrast, RGMA overexpression suppressed BC cell proliferation and colony-formation capabilities and increased BC cell apoptosis. Furthermore, RGMA knockdown accelerated BC cell proliferation and suppressed cellular apoptosis in vitro and in vivo. Reversal of RGMA promoter methylation with 5-Aza-CdR restored RGMA expression and blocked tumor growth. Overall, DNMT1- and DNMT3A-mediated RGMA promoter hypermethylation led to downregulation of RGMA expression, and low RGMA expression contributed to BC growth via activation of the FAK/Src/PI3K/AKT-signaling pathway. Our data thus suggested that RGMA might be a promising therapeutic target in BC.

EGF-SNX3-EGFR axis drives tumor progression and metastasis in triple-negative breast cancers.

Epidermal growth factor receptor (EGFR) has critical roles in epithelial cell physiology. Over-expression and over-activation of EGFR have been implicated in diverse cancers, including triple-negative breast cancers (TNBCs), prompting anti-EGFR therapies. Therefore, developing potent therapies and addressing the inevitable drug resistance mechanisms necessitates deciphering of EGFR related networks. Here, we describe Sorting Nexin 3 (SNX3), a member of the recycling retromer complex, as a critical player in the epidermal growth factor (EGF) stimulated EGFR network in TNBCs. We show that SNX3 is an immediate and sustained target of EGF stimulation initially at the protein level and later at the transcriptional level, causing increased SNX3 abundance. Using a proximity labeling approach, we observed increased interaction of SNX3 and EGFR upon EGF stimulation. We also detected colocalization of SNX3 with early endosomes and endocytosed EGF. Moreover, we show that EGFR protein levels are sensitive to SNX3 loss. Transient RNAi models of SNX3 downregulation have a temporary reduction in EGFR levels. In contrast, long-term silencing forces cells to recover and overexpress EGFR mRNA and protein, resulting in increased proliferation, colony formation, migration, invasion in TNBC cells, and increased tumor growth and metastasis in syngeneic models. Consistent with these results, low SNX3 and high EGFR mRNA levels correlate with poor relapse-free survival in breast cancer patients. Overall, our results suggest that SNX3 is a critical player in the EGFR network in TNBCs with implications for other cancers dependent on EGFR activity.

Combined analysis of PTEN, HER2, and hormone receptors status: remodeling breast cancer risk profiling

BackgroundPhosphatase and tensin homolog (PTEN) loss is associated with tumorigenesis, tumor progression, and therapy resistance in breast cancer. However, the clinical value of PTEN as a biomarker in these patients is controversial. We sought to determine whether the benefit of traditional biomarkers testing is improved by the analysis of PTEN status for the identification of high-risk breast cancer.MethodsA cohort of 608 patients with breast cancer was included in this study. Based on the expression on the neoplastic cells compared to the normal internal controls by immunohistochemistry (IHC), cases were classified as PTEN-low (PTEN-L) or PTEN-retained (PTEN-WT). The former constituted the study group, while the latter the control group. Analysis of gene expression was performed on publicly available genomic data and included 4265 patients from the METABRIC and MSK cohorts retrieved from cBioPortal. The Shapiro-Wilk test was used to analyze the normal distributions of continuous variables. Relationships between PTEN status and the clinicopathologic and molecular features of the patient population were assessed using Fisher’s exact test or Chi-squared/Wilcoxon rank-sum test. Survival curves were built according to the Kaplan-Meier method.ResultsAlteration in PTEN status was significantly different at protein and gene levels, where the reduced protein expression was observed in 280/608 cases (46.1%) from our group, while genetic aberrations in only 315/4265 (7.4%) cases of the METABRIC and MSK cohorts. PTEN-L tumors were significantly enriched for hormone receptors (HR) and HER2 negativity (n = 48, 17.1%) compared to PTEN-WT tumors (n = 22, 6.7%; p = 0.0008). Lack of HR with or without HER2 overexpression/amplification was significantly associated with worse overall survival (OS) in PTEN-L but not in PTEN-WT breast cancers (p < .0001). Moreover, PTEN-L protein expression but not gene alterations was related to the outcome, in terms of both OS and disease-free survival (p = 0.002).ConclusionsThe combined analysis of PTEN, HER2, and HR status offers relevant information for a more precise risk assessment of patients with breast cancer.

RNA binding protein RBMS3 is a common EMT effector that modulates triple-negative breast cancer progression via stabilizing PRRX1 mRNA.

The epithelial-to-mesenchymal transition (EMT) has been recognized as a driving force for tumor progression in breast cancer. Recently, our group identified the RNA Binding Motif Single Stranded Interacting Protein 3 (RBMS3) to be significantly associated with an EMT transcriptional program in breast cancer. Additional expression profiling demonstrated that RBMS3 was consistently upregulated by multiple EMT transcription factors and correlated with mesenchymal gene expression in breast cancer cell lines. Functionally, RBMS3 was sufficient to induce EMT in two immortalized mammary epithelial cell lines. In triple-negative breast cancer (TNBC) models, RBMS3 was necessary for maintaining the mesenchymal phenotype and invasion and migration in vitro. Loss of RBMS3 significantly impaired both tumor progression and spontaneous metastasis in vivo. Using a genome-wide approach to interrogate mRNA stability, we found that ectopic expression of RBMS3 upregulates many genes that are resistant to degradation following transcriptional blockade by actinomycin D (ACTD). Specifically, RBMS3 was shown to interact with the mRNA of EMT transcription factor PRRX1 and promote PRRX1 mRNA stability. PRRX1 is required for RBMS3-mediated EMT and is partially sufficient to rescue the effect of RBMS3 knockdown in TNBC cell lines. Together, this study identifies RBMS3 as a novel and common effector of EMT, which could be a promising therapeutic target for TNBC treatment.

A pilot study on DNA hypermethylation status in promoter region of P16 gene in patients with sporadic breast cancer

BackgroundEpigenetic modification of cancer-related genes plays a role over and above their genetic alterations and contributes to the tumor initiation and progression of breast cancer. Promoter methylation of tumor suppressor genes is one such epigenetic modification, which can be potential biomarker. In this study, promoter methylation status of p16 gene was studied in blood samples of patients with breast carcinoma. MethodsSeventy-five patients, freshly diagnosed with carcinoma of breast and 20 age and sex matched healthy control subjects were recruited for the study. DNA extracted from EDTA blood sample was bisulfite converted and subjected to methylation-specific PCR to amplify the p16 promoter region. ResultsOut of 75 patients, 25 (33%) patients showed hypermethylation in promoter region of p16 gene, which was statistically significant in comparison with the control group (p < 0.05). In subgroup analysis, lymph node involvement, cancer grade, and histopathological finding did not show any difference with methylation status of p16 promoter. ConclusionSignificant hypermethylation of p16 promoter region in the blood of histopathologically proven cases of breast cancer was observed suggesting promoter hypermethylation of p16 may be a possible mechanism accounting for sporadic carcinoma of breast.

Chemotherapy-triggered changes in stromal compartment drive tumor invasiveness and progression of breast cancer

BackgroundChemotherapy remains a standard treatment option for breast cancer despite its toxic effects to normal tissues. However, the long-lasting effects of chemotherapy on non-malignant cells may influence tumor cell behavior and response to treatment. Here, we have analyzed the effects of doxorubicin (DOX) and paclitaxel (PAC), commonly used chemotherapeutic agents, on the survival and cellular functions of mesenchymal stromal cells (MSC), which comprise an important part of breast tumor microenvironment.MethodsChemotherapy-exposed MSC (DOX-MSC, PAC-MSC) were co-cultured with three breast cancer cell (BCC) lines differing in molecular characteristics to study chemotherapy-triggered changes in stromal compartment of the breast tissue and its relevance to tumor progression in vitro and in vivo. Conditioned media from co-cultured cells were used to determine the cytokine content. Mixture of BCC and exposed or unexposed MSC were subcutaneously injected into the immunodeficient SCID/Beige mice to analyze invasion into the surrounding tissue and possible metastases. The same mixtures of cells were applied on the chorioallantoic membrane to study angiogenic potential.ResultsTherapy-educated MSC differed in cytokine production compared to un-exposed MSC and influenced proliferation and secretory phenotype of tumor cells in co-culture. Histochemical tumor xenograft analysis revealed increased invasive potential of tumor cells co-injected with DOX-MSC or PAC-MSC and also the presence of nerve fiber infiltration in tumors. Chemotherapy-exposed MSC have also influenced angiogenic potential in the model of chorioallantoic membrane.ConclusionsData presented in this study suggest that neoadjuvant chemotherapy could possibly alter otherwise healthy stroma in breast tissue into a hostile tumor-promoting and metastasis favoring niche. Understanding of the tumor microenvironment and its complex net of signals brings us closer to the ability to recognize the mechanisms that prevent failure of standard therapy and accomplish the curative purpose.

Landscape of Immune Microenvironment Under Immune Cell Infiltration Pattern in Breast Cancer.

BackgroundIncreasing evdence supports the suggestion that the immune cell infiltration (ICI) patterns play a pivotal role in tumor progression in breast cancer (BRCA). Nonetheless, there has been no comprehensive analysis of the ICI patterns effects on the clinical outcomes and immunotherapy.MethodsMultiomic data for BRCA samples were downloaded from TCGA. ESTIMATE algorithm, ssGSEA method, and CIBERSORT analysis were used to uncover the landscape of the tumor immune microenvironment (TIME). BRCA subtypes based on the ICI pattern were identified by consensus clustering and principal-component analysis was performed to obtain the ICI scores to quantify the ICI patterns in individual tumors. Their prognostic value was validated by the Kaplan-Meier survival curves. Gene set enrichment analysis (GSEA) was applied for functional annotation. Immunophenoscore (IPS) was employed to explore the immunotherapeutic role of the ICI scores. Finally, the mutation data was analyzed by using the “maftools” R package.ResultsThree different immune infiltration patterns with a distinct prognosis and biological signature were recognized among 1,198 BRCA samples. The characteristics of TIME under these three patterns were highly consistent with three known immune profiles: immune- excluded, immune-desert, and immune-inflamed phenotypes, respectively. The identification of the ICI patterns within individual tumors based on the ICI score, developed under the ICI-related signature genes, contributed into dissecting biological processes, clinical outcome, immune cells infiltration, immunotherapeutic effect, and genetic variation. High ICI score subtype, characterized with a suppression of immunity, suggested an immune-exhausted phenotype. Abundant effective immune cells were discovered in the low ICI score patients, which corresponded to an immune-activated phenotype and might present an immunotherapeutic advantage. Immunophenoscore was implemented as a surrogate of immunotherapeutic outcome, low-ICI scores samples obtained a significantly higher immunophenoscore. Enrichment of the JAK/STAT and VEGF signal pathways were activated in the ICI low-score subgroup. Finally, the synergistic effect between the ICI score and the tumor mutation burden (TMB) was confirmed.ConclusionThis work comprehensively elucidated that the ICI patterns served as an indispensable player in complexity and diversity of TIME. Quantitative identification of the ICI patterns in individual tumor will contribute into mapping the landscape of TIME further optimizing precision immunotherapy.

Role of Immune Cell-Specific Hypermethylation Signatures in Classification and Risk Stratification of Breast Cancer.

Background: Epigenetic regulation, including DNA methylation, plays a major role in shaping the identity and function of immune cells. Innate and adaptive immune cells recruited into tumor tissues contribute to the formation of the tumor immune microenvironment (TIME), which is closely involved in tumor progression in breast cancer (BC). However, the specific methylation signatures of immune cells have not been thoroughly investigated yet. Additionally, it remains unknown whether immune cells-specific methylation signatures can identify subgroups and stratify the prognosis of BC patients.Methods: DNA methylation profiles of six immune cell types from eight datasets downloaded from the Gene Expression Omnibus were collected to identify immune cell-specific hypermethylation signatures (IC-SHMSs). Univariate and multivariate cox regression analyses were performed using BC data obtained from The Cancer Genome Atlas to identify the prognostic value of these IC-SHMSs. An unsupervised clustering analysis of the IC-SHMSs with prognostic value was performed to categorize BC patients into subgroups. Multiple Cox proportional hazard models were constructed to explore the role of IC-SHMSs and their relationship to clinical characteristics in the risk stratification of BC patients. Integrated discrimination improvement (IDI) was performed to determine whether the improvement of IC-SHMSs on clinical characteristics in risk stratification was statistically significant.Results: A total of 655 IC-SHMSs of six immune cell types were identified. Thirty of them had prognostic value, and 10 showed independent prognostic value. Four subgroups of BC patients, which showed significant heterogeneity in terms of survival prognosis and immune landscape, were identified. The model incorporating nine IC-SHMSs showed similar survival prediction accuracy as the clinical model incorporating age and TNM stage [3-year area under the curve (AUC): 0.793 vs. 0.785; 5-year AUC: 0.735 vs. 0.761]. Adding the IC-SHMSs to the clinical model significantly improved its prediction accuracy in risk stratification (3-year AUC: 0.897; 5-year AUC: 0.856). The results of IDI validated the statistical significance of the improvement (p < 0.05).Conclusions: Our study suggests that IC-SHMSs may serve as signatures of classification and risk stratification in BC. Our findings provide new insights into epigenetic signatures, which may help improve subgroup identification, risk stratification, and treatment management.

Parsing β-catenin’s cell adhesion and Wnt signaling functions in malignant mammary tumor progression

During malignant progression, epithelial cancer cells dissolve their cell-cell adhesion and gain invasive features. By virtue of its dual function, β-catenin contributes to cadherin-mediated cell-cell adhesion, and it determines the transcriptional output of Wnt signaling: via its N terminus, it recruits the signaling coactivators Bcl9 and Pygopus, and via the C terminus, it interacts with the general transcriptional machinery. This duality confounds the simple loss-of-function analysis of Wnt signaling in cancer progression. In many cancer types including breast cancer, the functional contribution of β-catenin's transcriptional activities, as compared to its adhesion functions, to tumor progression has remained elusive. Employing the mouse mammary tumor virus (MMTV)-PyMT mouse model of metastatic breast cancer, we compared the complete elimination of β-catenin with the specific ablation of its signaling outputs in mammary tumor cells. Notably, the complete lack of β-catenin resulted in massive apoptosis of mammary tumor cells. In contrast, the loss of β-catenin's transcriptional activity resulted in a reduction of primary tumor growth, tumor invasion, and metastasis formation in vivo. These phenotypic changes were reflected by stalled cell cycle progression and diminished epithelial-mesenchymal transition (EMT) and cell migration of breast cancer cells in vitro. Transcriptome analysis revealed subsets of genes which were specifically regulated by β-catenin's transcriptional activities upon stimulation with Wnt3a or during TGF-β-induced EMT. Our results uncouple the signaling from the adhesion function of β-catenin and underline the importance of Wnt/β-catenin-dependent transcription in malignant tumor progression of breast cancer.

The Heparan Sulfate Binding Peptide in Tumor Progression of Triple-Negative Breast Cancer.

Angiogenesis is the formation of new vessels from pre-existing vasculature. The heparan sulfate chains from endothelial cell proteoglycans interact with the major angiogenic factors, regulating blood vessels´ formation. Since the FDA´s first approval, anti-angiogenic therapy has shown tumor progression inhibition and increased patient survival. Previous work in our group has selected an HS-binding peptide using a phage display system. Therefore, we investigated the effect of the selected peptide in angiogenesis and tumor progression. The HS-binding peptide showed a higher affinity for heparin N-sulfated. The HS-binding peptide was able to inhibit the proliferation of human endothelial umbilical cord cells (HUVEC) by modulation of FGF-2. It was verified a significant decrease in the tube formation of human endothelial cells and capillary formation of mice aorta treated with HS-binding peptide. HS-binding peptide also inhibited the formation of sub-intestinal blood vessels in zebrafish embryos. Additionally, in zebrafish embryos, the tumor size decreased after treatment with HS-binding peptide.

LOC550643, a Long Non-coding RNA, Acts as Novel Oncogene in Regulating Breast Cancer Growth and Metastasis.

Metastatic disease is responsible for over 90% of death in patients with breast cancer. Therefore, identifying the molecular mechanisms that regulate metastasis and developing useful therapies are crucial tasks. Long non-coding RNAs (lncRNAs), which are non-coding transcripts with >200 nucleotides, have recently been identified as critical molecules for monitoring cancer progression. This study examined the novel lncRNAs involved in the regulation of tumor progression in breast cancer. This study identified 73 metastasis-related lncRNA candidates from comparison of paired isogenic high and low human metastatic breast cancer cell lines, and their expression levels were verified in clinical tumor samples by using The Cancer Genome Atlas. Among the cell lines, a novel lncRNA, LOC550643, was highly expressed in breast cancer cells. Furthermore, the high expression of LOC550643 was significantly correlated with the poor prognosis of breast cancer patients, especially those with triple-negative breast cancer. Knockdown of LOC550643 inhibited cell proliferation of breast cancer cells by blocking cell cycle progression at S phase. LOC550643 promoted important in vitro metastatic traits such as cell migration and invasion. Furthermore, LOC550643 could inhibit miR-125b-2-3p expression to promote breast cancer cell growth and invasiveness. In addition, by using a xenograft mouse model, we demonstrated that depletion of LOC550643 suppressed the lung metastatic potential of breast cancer cells. Overall, our study shows that LOC550643 plays an important role in breast cancer cell metastasis and growth, and LOC550643 could be a potential diagnosis biomarker and therapeutic target for breast cancer.

RNA-binding protein SAMD4A inhibits breast tumor angiogenesis by modulating the balance of angiogenesis program.

Tumor‐induced angiogenesis is important for further progression of solid tumors. The initiation of tumor angiogenesis is dictated by a shift in the balance between proangiogenic and antiangiogenic gene expression programs. However, the potential mechanism controlling the expression of angiogenesis‐related genes in the tumor cells, especially the process mediated by RNA‐binding protein (RBP) remains unclear. SAMD4A is a conserved RBP across fly to mammals, and is believed to play an important role in controlling gene translation and stability. In this study, we identified the potential role of SAMD4A in modulating angiogenesis‐related gene expression and tumor progression in breast cancer. SAMD4A expression was repressed in breast cancer tissues and cells and low SAMD4A expression in human breast tumor samples was strongly associated with poor survival of patients. Overexpression of SAMD4A inhibited breast tumor angiogenesis and caner progression, whereas knockdown of SAMD4A demonstrated a reversed effect. Mechanistically, SAMD4A was found to specifically destabilize the proangiogenic gene transcripts, including C‐X‐C motif chemokine ligand 5 (CXCL5), endoglin (ENG), interleukin 1β (IL1β), and angiopoietin 1 (ANGPT1), by directly interacting with the stem‐loop structure in the 3′ untranslated region (3′UTR) of these mRNAs through its sterile alpha motif (SAM) domain, resulting in the imbalance of angiogenic genes expression. Collectively, our results suggest that SAMD4A is a novel breast tumor suppressor that inhibits tumor angiogenesis by specifically downregulating the expression of proangiogenic genes, which might be a potential antiangiogenic target for breast cancer therapy.

The circACTN4 interacts with FUBP1 to promote tumorigenesis and progression of breast cancer by regulating the expression of proto-oncogene MYC

BackgroundRecent studies have revealed that circular RNAs (circRNAs) play significant roles in the occurrence and development of many kinds of cancers including breast cancer (BC). However, the potential functions of most circRNAs and the molecular mechanisms underlying progression of BC remain elusive.MethodHere, Circular RNA microarray was executed in 4 pairs of breast cancer tissues and para-cancer tissues. The expression and prognostic significance of circACTN4 in BC cells and tissues were determined by qRT-PCR and in situ hybridization. Gain-and loss-of-function experiments were implemented to observe the impacts of circACTN4 on the growth, invasion, and metastasis of BC cells in vitro and in vivo. Mechanistically, chromatin immunoprecipitation, luciferase reporter, RNA pulldown, mass spectrum, RNA immunoprecipitation, fluorescence in situ hybridization and co-immunoprecipitation assays were executed.ResultsCircACTN4 was significantly upregulated in breast cancer tissues and cells, its expression was correlated with clinical stage and poor prognosis of patients with BC. Ectopic expression of circACTN4 strikingly facilitated the growth, invasion, and metastasis of breast cancer cells in vitro and in vivo. Whereas knockdown of circACTN4 revealed opposite roles. CircACTN4 was mainly distributed in the nucleus. Further mechanistic research proved that circACTN4 could competitively bind to far upstream element binding protein 1 (FUBP1) to prevent the combination between FUBP1 and FIR, thereby activating MYC transcription and facilitating tumor progression of breast cancer. Furthermore, we found that upstream transcription factor 2 (USF2) might promote the biogenesis of circACTN4.ConclusionOur findings uncover a pivotal mechanism that circACTN4 mediated by USF2 might interact with FUBP1 to promote the occurrence and development of breast cancer via enhancing the expression of MYC. CircACTN4 could be a novel potential target for diagnosis and treatment of breast cancer.

10P The clinical actionability of PTEN protein and gene expression analysis in HR- and HER2+ breast cancers

Phosphatase and tensin homologue (PTEN) loss is associated with tumorigenesis, tumor progression, and therapy resistance in breast cancer. However, the clinical value of PTEN as a biomarker requires further studies. We seek to identify clinically relevant subtypes of breast cancer based on PTEN status and other clinicopathologic features.

Positive regulation of PFKFB3 by PIM2 promotes glycolysis and paclitaxel resistance in breast cancer.

BackgroundBreast cancer (BC) is one of the most common female malignancies in the world. Chemotherapeutic resistance is the major cause of BC therapy failure, leading to tumor recurrence and metastasis. Studies have illustrated the close relationship between glycolysis and BC progression and drug resistance. The key glycolysis regulator, PFKFB3 makes a difference during BC progression and drug resistance. However, the mechanism remains to be unknown.MethodsMass spectrometry analyses were used to found that PIM2 was a potential new binding protein of PFKFB3. Co‐immunoprecipitated and western blot were used to verify the interaction between PIM2 and PFKFB3 in BC and the molecular mechanism by which PIM2 phosphorylates PFKFB3 in regulating the protein function. PFKFB3 mutant forms were used to demonstrate the need for PFKFB3 in BC drug resistance.ResultsWe identified that PIM2 is a new binding protein of PFKFB3. We used biochemical methods to determine that PIM2 can directly bind and change the phosphorylation of PFKFB3 at Ser478 to enhance PFKFB3 protein stability through the ubiquitin‐proteasome pathway. Importantly, phosphorylation of PFKFB3 at Ser478 promoted glycolysis, BC cell growth, and paclitaxel resistance together with PIM2 in vitro and in vivo.ConclusionOur study demonstrates that PIM2 mediates PFKFB3 phosphorylation thus regulates glycolysis and paclitaxel resistance to promote tumor progression in BC and provides preclinical evidence for targeting PFKFB3 as a new strategy in BC treatment to battle paclitaxel resistance.

Cancer-associated fibroblasts induce an aggressive phenotypic shift in non-malignant breast epithelial cells via interleukin-8 and S100A8.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment have been associated with tumor progression in breast cancer. Although crosstalk between breast cancer cells and CAFs has been studied, the effect of CAFs on non-neoplastic breast epithelial cells is not fully understood to date. Here, we investigated the effect of CAFs on aggressive phenotypes in non-neoplastic MCF10A breast epithelial cells. CAFs induced epithelial-to-mesenchymal transition (EMT) and invasive phenotype in MCF10A cells. S100A8, a potential prognostic marker in several cancers, was markedly increased in MCF10A cells by CAFs. S100A8 was crucial for CAFs-induced invasive phenotype of MCF10A cells. Among cytokines increased by CAFs, interleukin (IL)-8 induced S100A8through transcription factors p65 NF-κB and C/EBPβ. In a xenograft mouse model with MCF10A cells and CAFs, tumor was not developed, suggesting that coinjection with CAFs may not be sufficient for in vivo tumorigenicity of MCF10A cells. Xenograft mouse tumor models with MDA-MB-231 breast carcinoma cells provided an in vivo evidence for the effect of CAFs on breast cancer progression as well as a crucial role of IL-8 in tumor growth and S100A8 expression in vivo. Using a tissue microarray of human breast cancer, we showed that S100A8 expression was correlated with poor outcomes. S100A8 expression was more frequently detected in cancer-adjacent normal human breast tissues than in normal breast tissues. Together, this study elucidated a novel mechanism for the acquisition of invasive phenotype of non-neoplastic breast cells induced by CAFs, suggesting that targeting IL-8 and S100A8 may be an effective strategy against breast cancer.

CCN5 activation by free or encapsulated EGCG is required to render triple-negative breast cancer cell viability and tumor progression.

Epigallocatechin‐3‐gallate (EGCG) has been considered an anticancer agent despite conflicting and discrepant bioavailability views. EGCG impairs the viability and self‐renewal capacity of triple‐negative breast cancer (TNBC) cells and makes them sensitive to estrogen via activating ER‐α. Surprisingly, the mechanism of EGCG’s action on TNBC cells remains unclear. CCN5/WISP‐2 is a gatekeeper gene that regulates viability, ER‐α, and stemness in TNBC and other types of cancers. This study aimed to investigate whether EGCG (free or encapsulated in nanoparticles) interacts with the CCN5 protein by emphasizing its bioavailability and enhancing its anticancer effect. We demonstrate that EGCG activates CCN5 to inhibit in vitro cell viability through apoptosis, the sphere‐forming ability via reversing TNBC cells’ stemness, and suppressing tumor growth in vivo. Moreover, we found EGCG‐loaded nanoparticles to be functionally more active and superior in their tumor‐suppressing ability than free‐EGCG. Together, these studies identify EGCG (free or encapsulated) as a novel activator of CCN5 in TNBC cells and hold promise as a future therapeutic option for TNBC with upregulated CCN5 expression.

Systematic Characterization of Expression Profiles and Prognostic Values of the Eight Subunits of the Chaperonin TRiC in Breast Cancer.

BackgroundChaperonin-containing TCP-1 (TRiC or CCT) was demonstrated to be involved in oncogenesis of cancers carcinogenesis and development of various malignancies. Increasing experimental evidence indicated that dysregulation of TRiC was implicated in the tumor progression of breast cancer (BCa). However, few definitive studies have addressed the diverse expression patterns and prognostic values of eight TRiC subunits. Thus, we aimed to investigate the clinical significance of TRiC subunit expression and prognostic values for their possible implications in diagnosis and treatment of BCa.MethodsBased on updated public resources and comprehensive bioinformatics analysis, we used some online databases (e.g., UALCAN, GEPIA, cBioPortal, TIMER, BC-GenExMiner, metascape, and GeneMANIA) to comprehensively explore the expression levels and the prognostic effects of eight TRiC subunits in patients with BCa.ResultsThe transcriptional levels of most subunits of the Chaperonin TRiC (CCT2, CCT3, CCT4, CCT5, CCT6A, and CCT7) were significantly elevated compared with normal breast tissues, whereas TCP1, CCT4, and CCT6B were lower in BCa tissues than in normal tissues. Besides, copy-number alterations (CNA) of eight TRiC subunits positively regulated their mRNA expressions. Furthermore, high mRNA expression of TCP1/CCT2/CCT4/CCT5/CCT6A/CCT7/CCT8 was significantly associated with poor overall survival (OS) in BCa patients. The eight subunits of the chaperonin TRiC was related to tumor purity and immune infiltration levels of BCa. Co-expression analysis showed CCT6B was negatively associated with other subunits of TRiC and other subunits of TRiC were positively correlated with each other. Additionally, TRiC and their interactive proteins were correlated with positive regulation of biological process, localization, and biological regulation.ConclusionThis study systematically illustrated the expression profiles and distinct prognostic values of chaperonin TRiC in BCa, providing insights for further investigation of subunits of the chaperonin TRiC as novel therapeutic targets and potential prognostic biomarkers in BCa.

Abstract IA004: Genetic variants in the tumor microenvironment alter radiation responses in breast cancer

Abstract Objectives: The tumor microenvironment (TME) can impact breast cancer tumor growth, progression, and treatment responses. Data suggests that genetic variants in not only breast cancer cells, but also in the TME, can also alter these processes. We have utilized a Consomic Xenograft Model (CXM), which maps germline variants that impact only the TME, as well as a species-specific RNA-seq (SSRS) protocol which allows detection of expression changes in the malignant and nonmalignant cellular compartments of tumor xenografts, in parallel to identify genetic variants in the TME that affect radiation sensitivity. Materials/Methods: Human triple negative breast cancer MDA-MD-231 cells were implanted into immunodeficient (IL2RG KO) consomic rat strains that are genetically identical except for chromosome 3 is inherited from a separate strain (SS and SS.BN3 strains). On day 10, tumors were treated with 3 daily ionizing radiation (IR) treatments of 4 Gy or sham, and tumor growth was monitored. Tumors were also harvested for hypoxia staining using pimonidazole or for RNA-seq. RNA-Seq was performed and a custom SSRS protocol was used to align both rat and human transcripts. This yielded transcript and gene level estimated fold-change and adjusted p-values for human- and rat-derived transcripts separately. E077 mammary tumor cells were implanted into adult female immune competent C57/Bl6 mice. On day 5, tumors were treated with 5 daily IR treatments of 5 Gy or sham. Either vehicle or a mAb to the Notch ligand Dll4 (Genentech) was given twice weekly. Chi-square, Fisher’s exact, and Kolmogorov-Smirnov tests and empirical cumulative distribution plots for differential expression significance values were performed. Results: Using CXM, we discovered that BN strain-derived genetic variant(s) on rat chromosome 3 are important for tumor IR sensitivity, as human breast cancer xenografts in the consomic strain (SS.BN3) were significantly more IR sensitive than SS rat strain tumors (supra-additive). Vascular gene pathways were differentially expressed, and tumor vascular phenotypes were distinct, with SS.BN3 tumors with increased but poorly functioning blood vessels. Hypoxia was similar at baseline, but increased in SS.BN3 tumors following IR. These results were consistent with less Dll4 expression in the SS.BN3 TME. The use of a Dll4-targeted mAb in mice demonstrated that targeting Dll4 enhanced mammary tumor IR responses. Conclusion: CXM demonstrated TME genetic variants can affect IR sensitivity of genetically identical tumor cells. Using SSRS, we identified candidate genes on rat chromosome 3 that may potentially influence IR sensitivity, and our studies ultimately led to identification of the Notch ligand Dll4 as a target to enhance breast cancer IR responses. Future studies will investigate the possibility of the Dll4 pathway as a therapeutic target, as well as interrogate other pathways responsible for changes in IR sensitivity seen in the CXM model. Determining TME factors that affect the IR sensitivity will allow more tailored and effective treatments. Citation Format: Carmen Bergom, Michael W. Straza, Amy Rymaszewski, Anne Frei, Angela Lemke, Rachel A. Schlaak, Shirng-Wern Tsaih, Michael J. Flister. Genetic variants in the tumor microenvironment alter radiation responses in breast cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA004.

Overexpression of EZH2/NSD2 Histone Methyltransferase Axis Predicts Poor Prognosis and Accelerates Tumor Progression in Triple-Negative Breast Cancer.

Two histone methyltransferases, enhancer of zeste homolog 2 (EZH2) and nuclear SET domain-containing 2 (NSD2), are aberrantly expressed in several types of human cancers. However, the regulatory relationship between EZH2 and NSD2 and their prognostic values in breast cancer (BC) have not been fully elucidated. In this study, we demonstrated that EZH2 and NSD2 were overexpressed in BC compared with benign lesions and normal tissues using tissue microarray, immunohistochemistry, and bioinformatic databases. Both EZH2 and NSD2 expression were associated with pathological grade of tumor and lymph node metastasis. A comprehensive survival analysis using Kaplan-Meier Plotter database indicated that EZH2 expression was negatively correlated with relapse-free survival (RFS), overall survival (OS), distant metastasis-free survival (DMFS), and postprogression survival (PPS) in 3951 BC patients, and NSD2 expression was negatively correlated with RFS and DMFS. Notably, EZH2 and NSD2 expression were coordinately higher in triple-negative breast cancer (TNBC) than that in other subtypes. Stable knockdown of EZH2 using lentiviral shRNA vector significantly reduced the proliferation, migration and invasion abilities of TNBC cell line MDA-MB-231 and MDA-MB-468, and downregulated NSD2 expression as well as the levels of H3K27me3 and H3K36me2, two histone methylation markers catalyzed by EZH2 and NSD2, respectively. By contrast, overexpression of EZH2 using adenovirus vector displayed an inverse phenotype. Furthermore, knockdown of NSD2 in EZH2-overexpressing cells could dramatically attenuate EZH2-mediated oncogenic effects. Bioinformatic analysis further revealed the function and pathway enrichments of co-expressed genes and interactive genes of EZH2/NSD2 axis, suggesting that EZH2/NSD2 axis was associated with cell division, mitotic nuclear division and transition of mitotic cell cycle in TNBC. Taken together, EZH2/NSD2 axis may act as a predictive marker for poor prognosis and accelerate the progression of TNBC.