Factor In Triple-negative Breast Cancer Research Articles

Abstract C151: Single and combined inhibition of AURKA and AURKB promotes a decrease in the expression of EMT biomarkers and transcription factors in triple-negative breast cancer in women of African ancestry

Abstract A major health disparity exists between Hispanic/Latino women (H/L) and women of African heritage (non-Hispanic black, NHB, Black H/L, and Caribbean H/L) compared to non-Hispanic White women (NHW) as they are more likely to die from breast cancer. Major contributors to these disparities are socioeconomic status, the detection of breast cancers at later stages, and higher rates of triple-negative breast cancer (TNBC). Molecularly, NHB women have increased expression of mRNAs coding for proteins that regulate the cell cycle, including the cyclins, centrosome, and mitotic kinases. TNBC lacks the estrogen receptor (ER), the progesterone receptor (PR), and the ERBB2 (HER2) receptor, which makes it even more challenging to treat with biological therapies. Mitotic kinases such as AURKA and AURKB are proteins essential for the cell cycle that promote normal cell division, the former involved in centrosome separation and the latter in regulating the spindle assembly checkpoint (SAC). However, when overexpressed, they can activate molecular pathways leading to the activation of centrosome amplification, chromosome instability, and early metastasis (epithelial-to-mesenchymal or EMT biomarkers, cell invasion, and migration) through transcription factors that could lead to a more aggressive breast cancer subtype, such as TNBC. This suggests that these mitotic kinases can drive cancer initiation and evolution. Hence, we hypothesize that co-inhibiting AURKA and AURKB will decrease the expression of EMT biomarkers and transcription factors and therefore, early metastasis. Using The Cancer Genome Atlas database, we identified higher expression of AURKA and AURKB in NHB women compared to NHW women and increased expression of AURKB in NHB women with TNBC. After siRNA-mediated knockdown, immunoblotting and Real-Time qPCR were performed using the MDA-MB-231 cells (NHW) and MDA-MB-157 (NHB) mesenchymal TNBC cells. Preliminary data from the immunoblotting indicates that the expression of AURKA and AURKB decreased in all cell lines compared to the control group, and the EMT biomarker Vimentin decreased in MDA-MB-157 after targeting both mitotic kinases singly and in combination. At a genetic level, EMT biomarkers such as Vimentin, N- Cadherin, and transcription factors such as Slug, Twist, Snail, and Zeb1 decreased in the MDA- MB-157 cell line after single and combined inhibition. These results suggest the role of AURKA and AURKB in TNBC and their potential as therapeutic targets in TNBC. Eventually, we are going to be evaluating cell migration, invasion, tumor growth, and metastasis rates after co- inhibiting AURKA and AURKB in TNBC. Because of their more prevalent expression in breast cancers from NHB women, these kinases may represent new treatment modalities for women of African heritage. Citation Format: Joel A. Orengo-Orengo, Melanie Cruz, Alexandra Aquino, Harold I. Saavedra. Single and combined inhibition of AURKA and AURKB promotes a decrease in the expression of EMT biomarkers and transcription factors in triple-negative breast cancer in women of African ancestry [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C151.

Evaluation of prognostic risk factors of triple-negative breast cancer with 18F-FDG PET/CT parameters, clinical pathological features and biochemical indicators.

Breast cancer is a heterogeneous disease comprising various molecular subtypes, including Luminal A, Luminal B, human epidermal growth factor receptor-2 (HER2) positive, and triple negative types, each with distinct biological characteristics and behaviors. Triple negative breast cancer (TNBC) remains a particularly challenging subtype worldwide. Our study aims to evaluate whether Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (18F-FDG PET/CT) parameters, clinical pathological features, and biochemical indicators serve as prognostic risk factors for TNBC. Additionally, we explore correlations between biochemical indicators and 18F-FDG PET/CT parameters. We conducted a retrospective analysis of 95 TNBC patients who underwent preoperative 18F-FDG PET/CT examinations at Tianjin Medical University Cancer Institute and Hospital from 2013 to 2018. Collected data included 18F-FDG PET/CT parameters, clinical and pathological features, and biochemical indicators. We used Kaplan-Meier survival analysis and multivariate Cox regression analysis to evaluate associations between 18F-FDG PET/CT parameters/biochemical indicators and disease free survival (DFS)/overall survival (OS). The log-rank test determined significant differences in survival curves, and the Spearman correlation coefficient analyzed correlations between quantitative variables. Visualization and analysis were performed using R packages. Among 95 TNBC patients, mean standardized uptake value (SUVmean) was significantly correlated with DFS. Fasting blood glucose (FBG), α- L-fucosylase (AFU) and Creatine kinase (CK) were independent predictors of DFS, while Precursor albumin (PALB) and CK were independent predictors of OS. FBG showed correlations with SUVpeak and SUVmean, and CK was correlated with peak standardized uptake value (SUVpeak). Our results indicated that 18F-FDG PET/CT parameters and biochemical indicators may constitute a new prognostic model for TNBC patients post-surgery. We found that SUVmean, FBG, AFU and CK are predictive factors for DFS in TNBC patients post-surgery, while PALB and CK are predictive factors for OS, which prompts us to pay more attention to these indicators in clinical practice. Also 18F-FDG PET/CT parameters and biochemical indicators have potential utility in constituting a new prognostic model for TNBC patients post-surgery.

Revealing Cellular Heterogeneity and Key Regulatory Factors of Triple-Negative Breast Cancer through Single-Cell RNA Sequencing.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC). TNBC has a poor prognosis due to high intratumoral heterogeneity and metastasis, pointing to the need to explore distinct molecular subtypes and gene regulatory networks. The scRNA-seq data of five primary BC samples were downloaded from the Gene Expression Omnibus (GEO) database. Clustering was performed based on filtered and normalized data using the Seurat R package to identify marker genes, which were subsequently annotated to each subset using the CellMarker database. AUCell R package was applied to calculate the hallmark score for each epithelial cell. Marker genes of each subset were screened with FindAllMarkers and their biological functions were analyzed using the Database for Annotation Visualization and Integrated Discovery (DAVID) database. Next, cell-cell communication was performed with the CellChat R package. To identify the key regulatory genes, single-cell regulatory network inference and clustering (SCENIC) analysis was conducted. Finally, the expression and potential biological functions of the key regulatory factors were verified through cellular experiments. A total of 29,101 cells were classified into nine cell subsets, namely, Fibroblasts, Fibroepithelial cells, Epithelial cells 1, Epithelial cells 2, Epithelial cells 3, Endothelial cells, T cells, Plasma B cells and Macrophages. Particularly, the epithelial cells had a higher proportion and higher transforming growth factor-β (TGF-β) activity in the TNBC pathotype as compared to the non-TNBC pathotype. Furthermore, four epithelial cell subsets (marked as Stearoyl-CoA Desaturase (SCD1), marker of proliferation Ki67 (MKI67), Annexin A3 (ANXA3) and aquaporin 5 (AQP5)) were identified as having the greatest impact on the TNBC pathotype. Cell-cell interaction analysis revealed that ANXA3-epithelial cell subset suppressed the T cell function through different mechanisms. C-fos gene (FOS) and X-box binding protein 1 (XBP1) were considered critical regulons involved in TNBC progression. Notably, cellular experiments demonstrated that silencing XBP1 and overexpressing FOS inhibited cancer cell invasion. The four epithelial cell subsets and two critical regulons identified based on the scRNA-seq data could help explore the underlying intratumoral heterogeneity molecular mechanism and develop effective therapies for TNBC.

Development and Characterization of Syngeneic Orthotopic Transplant Models of Obesity-Responsive Triple-Negative Breast Cancer in C57BL/6J Mice.

Obesity is an established risk and progression factor for triple-negative breast cancer (TNBC), but preclinical studies to delineate the mechanisms underlying the obesity-TNBC link as well as strategies to break that link are constrained by the lack of tumor models syngeneic to obesity-prone mouse strains. C3(1)/SV40 T-antigen (C3-TAg) transgenic mice on an FVB genetic background develop tumors with molecular and pathologic features that closely resemble human TNBC, but FVB mice are resistant to diet-induced obesity (DIO). Herein, we sought to develop transplantable C3-TAg cell lines syngeneic to C57BL/6 mice, an inbred mouse strain that is sensitive to DIO. We backcrossed FVB-Tg(C3-1-TAg)cJeg/JegJ to C57BL/6 mice for ten generations, and spontaneous tumors from those mice were excised and used to generate four clonal cell lines (B6TAg1.02, B6TAg2.03, B6TAg2.10, and B6TAg2.51). We characterized the growth of the four cell lines in both lean and DIO C57BL/6J female mice and performed transcriptomic profiling. Each cell line was readily tumorigenic and had transcriptional profiles that clustered as claudin-low, yet markedly differed from each other in their rate of tumor progression and transcriptomic signatures for key metabolic, immune, and oncogenic signaling pathways. DIO accelerated tumor growth of orthotopically transplanted B6TAg1.02, B6TAg2.03, and B6TAg2.51 cells. Thus, the B6TAg cell lines described herein offer promising and diverse new models to augment the study of DIO-associated TNBC.

Identification of Poliovirus Receptor-like 3 Protein as a Prognostic Factor in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) represents an aggressive subtype of breast cancer, with a bad prognosis and lack of targeted therapeutic options. Characterized by the absence of estrogen receptors, progesterone receptors, and HER2 expression, TNBC is often associated with a significantly lower survival rate compared to other breast cancer subtypes. Our study aimed to explore the prognostic significance of 83 immune-related genes, by using transcriptomic data from the TCGA database. Our analysis identified the Poliovirus Receptor-Like 3 protein (PVRL3) as a critical negative prognostic marker in TNBC patients. Furthermore, we found that the Enhancer of Zeste Homolog 2 (EZH2), a well-known epigenetic regulator, plays a pivotal role in modulating PVRL3 levels in TNBC cancer cell lines expressing EZH2 along with high levels of PVRL3. The elucidation of the EZH2-PVRL3 regulatory axis provides valuable insights into the molecular mechanisms underlying TNBC aggressiveness and opens up potential pathways for personalized therapeutic intervention.

Genomic characterization reveals distinct mutational landscapes and therapeutic implications between different molecular subtypes of triple-negative breast cancer

Triple-negative breast cancer (TNBC) has high heterogeneity, poor prognosis, and limited treatment success. Recently, an immunohistochemistry-based surrogate classification for the “Fudan University Shanghai Cancer Center (FUSCC) subtyping” has been developed and is considered more suitable for clinical application. Seventy-one paraffin-embedded sections of surgically resected TNBC were classified into four molecular subtypes using the IHC-based surrogate classification. Genomic analysis was performed by targeted next-generation sequencing and the specificity of the subtypes was explored by bioinformatics, including survival analysis, multivariate Cox regression, pathway enrichment, Pyclone analysis, mutational signature analysis and PHIAL analysis. AKT1 and BRCA1 mutations were identified as independent prognostic factors in TNBC. TNBC molecular subtypes encompass distinct genomic landscapes that show specific heterogeneities. The luminal androgen receptor (LAR) subtype was associated with mutations in PIK3CA and PI3K pathways, which are potentially sensitive to PI3K pathway inhibitors. The basal-like immune-suppressed (BLIS) subtype was characterized by high genomic instability and the specific possession of signature 19 while patients in the immunomodulatory (IM) subtype belonged to the PD-L1 ≥ 1% subgroup with enrichment in Notch signaling, suggesting a possible benefit of immune checkpoint inhibitors and Notch inhibitors. Moreover, mesenchymal-like (MES) tumors displayed enrichment in the receptor tyrosine kinase (RTK)-RAS pathway and potential sensitivity to RTK pathway inhibitors. The findings suggest potential treatment targets and prognostic factors, indicating the possibility of TNBC stratified therapy in the future.

Utility of human epidermal growth factor 2 heterogeneity as a prognostic factor in triple-negative breast cancer.

In some cases of human epidermal growth factor 2 (HER2)-negative breast cancer, including triple-negative breast cancer, HER2 expression is sporadically and strongly upregulated, a condition known as HER2 heterogeneity. We investigated the clinicopathological features of patients with HER2 heterogeneity in triple-negative breast cancers treated with neoadjuvant chemotherapy. Thirty-nine patients with triple-negative breast cancer who had undergone preoperative chemotherapy participated in this study. To assess for HER2 heterogeneity, we used dual in situ hybridization slides. We evaluated the association between HER2 heterogeneity and clinicopathological factors such as rates of pathologic complete response (pCR) and of recurrence-free survival. Of the 39 patients, 15 (38.5%) had cancers with HER2 heterogeneity. The pCR rates were 13.3% among patients with HER2 heterogeneity and 20.8% among those with HER2 nonheterogeneity, but the difference was not significant. The recurrence-free survival rate was significantly lower in patients with HER2 heterogeneity than in those without (P = 0.025). HER2 heterogeneity is a significant predictor of poor prognosis in patients with triple-negative breast cancer treated with neoadjuvant chemotherapy.

A clinical analysis of clinicopathological features and prognostic factors of triple-negative breast cancer

Objectives. The aim of this study was to explore the clinicopathological characteristics, prognostic factors, recurrence patterns and survival analysis of triple negative breast cancer patients compared to non-triple negative breast cancer patients. Materials and methods. The cohort included 420 patients who were diagnosed with breast cancer. The patients were evaluated based on the molecular classification and grouped into TNBC and non-TNBC. Data was explored using SPSS Version 29.0.0.0. Patient and tumor characteristics were studied. Univariate and multivariate Cox Regression was used to analyze prognostic factors. Kaplan Meier method with the log-rank test was performed to observe DFS and OS. Outcomes. The triple negative subtype was observed in 57(13.6%) patients. Patients with TNBC had a greater proportion of grade 3 tumors compared to those with non-TNBC (43.9% vs. 5.5%, p<.001). In the univariate analysis pathological type, tumor size, tumor grade, nodal invasion, lymphovascular invasion, perineural invasion and extra-nodal extension were identified as statistically significant prognostic factors. The mean time to relapse for TNBC was lower 60.0 (95% CI, 37.16 to 82.83) compared to the non-TNBC group with a mean time to relapse of 72.0 months (95% CI, 55.81 to 88.18), nevertheless distributions were not statistically significant X2(1)=1.524, p.217. Mean overall survival was 80.53 months (95% CI, 77.75 to 83.32) in non-TNBC compared to TNBC of 80.04 month (95% CI, 66.76 to 93.33) with a non-statistically significantly Log rank test X2(1)=1.252, p.263. Conclusions. Triple negative breast cancer represents a heterogenous molecular and prognostic group of tumors which needs further clinical trials in order to develop targeted therapies.

Current progress and prospects for G protein-coupled estrogen receptor in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a biologically and clinically heterogeneous disease. The G protein-coupled estrogen receptor (GPER) plays a crucial role in mediating the effect of estrogen and estrogen-like compounds in TNBC cells. Compared with other subtypes, GPER has a higher expression in TNBC. The GPER mechanisms have been thoroughly characterized and analyzed in estrogen receptor α (ERα) positive breast cancer, but not in TNBC. Our previous work revealed that a higher expression of GPER mRNA indicates a better prognosis for ERα-positive breast cancer; however, its effects in TNBC differ. Whether GPER could serve as a predictive prognostic marker or therapeutic target for TNBC remains unclear. In this review, we provide a detailed introduction to the subcellular localization of GPER, the different effects of various ligands, and the interactions between GPER and closely associated factors in TNBC. We focused on the internal molecular mechanisms specific to TNBC and thoroughly explored the role of GPER in promoting tumor development. We also discussed the interaction of GPER with specific cytokines and chemokines, and the relationship between GPER and immune evasion. Additionally, we discussed the feasibility of using GPER as a therapeutic target in the context of existing studies. This comprehensive review highlights the effects of GPER on TNBC, providing a framework and directions for future research.

Interleukin (IL)-17A in triple-negative breast cancer: a potent prognostic factor associated with intratumoral neutrophil infiltration.

Triple-negative breast cancer (TNBC) is characterized as highly immunogenic and lacks specific targeted therapies. Interleukin 17A (IL-17A) is a controversial cytokine and is known to have anti-tumor and pro-tumor roles depending on the tumor microenvironment. In addition, IL-17A has been recently implicated in the recruitments of neutrophil into tumor tissues. Although IL-17A is considered tumor-promoting in breast cancer, its significance in the possible regulation of neutrophil infiltration in TNBC is not clearly defined. We immunolocalized IL-17A, CD66b (neutrophil marker), and chemokine (C-X-C motif) ligand 1 (CXCL1, neutrophil chemoattractant) in 108 TNBC specimens and assessed their correlation among each other. The correlation between these markers and clinicopathological parameters was also assessed. We subsequently performed in vitro study to address the possible regulation of CXCL1 by IL-17A using TNBC cell lines, MDA-MB-231 and HCC-38. It was revealed that IL-17A correlated significantly with CXCL1 and CD66b, also CD66b with CXCL1. Furthermore, IL-17A was significantly associated with shorter disease-free and overall survival, especially in a high density CD66b group of patients. In vitro results revealed that IL-17A upregulated CXCL1 mRNA expression in a dose and time dependent manner, and this induction was significantly suppressed by an Akt inhibitor. IL-17A was considered to contribute to neutrophil infiltration by inducing CXCL1 in TNBC tissues and educating neutrophils to promote tumor progression. IL-17A might therefore serve as a potent prognostic factor in TNBC.

ZNF503 combined with GATA3 is a prognostic factor in triple-negative breast cancer

Purpose Triple-negative breast cancer (TNBC) is a subtype of breast cancer with poor prognosis. Therefore, there is an urgent need to identify prognostic markers to improve current treatment and therapeutic strategies. The transcriptional factor ZNF503 has been reported to promote aggressive breast cancer development through the down-regulation of GATA3 expression and has been identified as a candidate predictive marker. In this study, we explored whether ZNF503 and GATA3 could serve as prognostic markers independently or in combination. Material and methods We performed a survival analysis of 989 breast cancer patients from The Cancer Genome Atlas (TCGA), and validated the findings in 202 breast cancer patients from tissue microarray (TMA). Results In TCGA database, the mRNA expression of GATA3 and ZNF503 could not predict TNBC prognosis alone, though the ratio index, ZNF503/GATA3 could be a novel prognostic biomarker in TNBC patients. In TMA database, we detected the protein expression of ZNF503 and GATA3 and found that the combination of the two genes, ZNF503-GATA3, significantly improved the predictive ability of clinical outcomes. Conclusions The results indicated that the binding index of ZNF503 and GATA3 could be used as a prognostic biomarker in TNBC.

Influence of advanced age on the prognosis of triple-negative breast cancer patients: A surveillance, epidemiology, and end results-based study.

Age at diagnosis has shown significant effect on the prognosis in breast cancer patients. However, whether age is an independent risk factor remains controversial. Furthermore, population-based estimates of age on the prognosis impact in triple-negative breast cancer are still lacking. The aim of this study was to analyze the influence of age and other factors on the prognosis and survival of triple-negative breast cancer patients. We used the surveillance, epidemiology, and end results program data from 2011 to 2014. A retrospective cohort study was conducted to investigate prognosis factors in triple-negative breast cancer. Patients were divided into two groups according to age at diagnosis: 75 + years (the elderly patients) and < 75 years (reference group). The clinicopathologic characteristics of different age groups were compared using Chi-square tests. Overall survival (OS) and breast cancer-specific survival were analyzed using the Kaplan-Meier method. Prognostic factors were compared using the Cox proportional hazards model. We also analyzed the difference of distant metastasis at initial diagnosis on every group. A total of 21,429 triple-negative breast cancer patients were included in our study. The mean breast cancer-specific survival time of triple-negative breast cancer was 70.5 months for the reference group and 62.4 months for the elderly group. Survival analysis showed that the breast cancer-specific survival rate was 78.9% for the reference group and 67.4% for the elderly group. The mean OS time was 69.0 months for the reference group and 52.3 months for the elderly group. The 5-year OS of triple-negative breast cancer patients was 76.4% for the reference group and 51.3% for the elderly group. The prognosis of elderly patients is much poor than reference group. Univariate Cox regression analysis showed that age, race, marital status, histological grade, stage, T, N, M, surgery, radiotherapy, and chemotherapy were risk factors for triple-negative breast cancer (TNBC) (P < 0.05). Multivariate Cox regression analysis showed that age, race, marital status, histological grade, stage, T, N, M, surgery, radiotherapy, and chemotherapy were independent risk factors for TNBC (P < 0.05). Age is an independent risk factor for the prognosis of TNBC patients. Elderly triple-negative breast cancer patients displayed obvious lower 5-year survival rate compared to reference group, even though they have better grade stage, minor tumor, less lymph node metastasis. The lower rate of marital status, radiotherapy, chemotherapy, surgery, and higher rate of metastasis at diagnosis must contribute to their poor outcome.

Inflammation-Driven Regulation of PD-L1 and PD-L2, and Their Cross-Interactions with Protective Soluble TNFα Receptors in Human Triple-Negative Breast Cancer.

Simple SummaryImmune checkpoint blockades (ICBs) to PD-L1 have led to major breakthroughs in cancer therapy, but in triple-negative breast cancer (TNBC) success rates are rather limited. Following studies suggesting that chronic inflammation may limit ICB efficacy, we found that pro-inflammatory cytokines up-regulated the proportion of TNBC cells co-expressing the inhibitory immune checkpoint PD-L1 and its cognate PD-L2 molecule. Moreover, we demonstrated that in the context of inflammation-driven signals, PD-L1 down-regulated the cell-derived levels of sTNFR1 and sTNFR2, the soluble receptors of tumor necrosis factor α (TNFα); these soluble receptors were found to exert protective/anti-metastatic effects in TNBC cells, manifested by their ability to down-regulate TNFα-induced production of pro-metastatic chemokines by TNBC cells. Our findings possibly testify for a novel mechanism of PD-L1-mediated tumor progression in which PD-L1 prevents the anti-metastatic effects of sTNFR1 and sTNFR2 in TNBC cells. This mechanism may also act in vivo, in parallel to immune suppression under inflammatory conditions.Pro-inflammatory cytokines play key roles in elevating cancer progression in triple-negative breast cancer (TNBC). We demonstrate that specific combinations between TNFα, IL-1β and IFNγ up-regulated the proportion of human TNBC cells co-expressing the inhibitory immune checkpoints PD-L1 and PD-L2: TNFα + IL-1β in MDA-MB-231 cells and IFNγ + IL-1β in BT-549 cells; in the latter cells, the process depended entirely on STAT1 activation, with no involvement of p65 (CRISPR-Cas9 experiments). Highly significant associations between the pro-inflammatory cytokines and PD-L1/PD-L2 expression were revealed in the TCGA dataset of basal-like breast cancer patients. In parallel, we found that the pro-inflammatory cytokines regulated the expression of the soluble receptors of tumor necrosis factor α (TNFα), namely sTNFR1 and sTNFR2; moreover, we revealed that sTNFR1 and sTNFR2 serve as anti-metastatic and protective factors in TNBC, reducing the TNFα-induced production of inflammatory pro-metastatic chemokines (CXCL8, CXCL1, CCL5) by TNBC cells. Importantly, we found that in the context of inflammatory stimulation and also without exposure to pro-inflammatory cytokines, elevated levels of PD-L1 have down-regulated the production of anti-tumor sTNFR1 and sTNFR2. These findings suggest that in addition to its immune-suppressive activities, PD-L1 may promote disease course in TNBC by inhibiting the protective effects of sTNFR1 and sTNFR2.

Abstract 820: Nuclear receptor TLX inhibits cancer cell intrinsic properties required for triple-negative breast cancer progression

Abstract The primary purpose of our work was to characterize the effects of the nuclear receptor TLX (NR2E1) in triple-negative breast cancer (TNBC) in order to evaluate its potential therapeutic value in a subtype of breast cancer that has proven particularly challenging to treat, primarily due to the lack of targeted modes of intervention. Unfortunately, breast cancer continues to be the second deadliest form of cancer among women in the United States. Considering the substantial public health implications, development of new therapeutic strategies for highly aggressive breast cancer subtypes, such as TNBC, is imperative. While the function of nuclear receptors such as the estrogen and androgen receptors has been extensively characterized in cancers of the breast and prostate respectively, due in part to their amenable nature to ligand modulation, it is likely that other nuclear receptors may represent therapeutic targets for these malignancies. Indeed, probing of available clinical datasets demonstrated that nuclear receptor TLX is most highly expressed in basal and estrogen receptor (ER)-negative breast cancer patients, and that ER-negative patients with higher TLX expression had increased relapse-free and overall survival. Therefore, we hypothesized that TLX could influence the pathophysiology of TNBC. Utilizing a stable overexpression model in the TNBC cell lines, MDA-MB-231 and MDA-MB-468, we have shown that TLX can inhibit critical oncogenic properties in the in vitro setting, including proliferation, migration and invasion. Furthermore, xenograft and lung colonization studies demonstrated that TLX continued to exert anti-oncogenic effects in the in vivo environment. In agreement with these results, transcriptomic analysis of TLX-overexpressing cells and xenograft tumors showed that TLX can regulate genes and pathways that are known to play crucial roles in the growth and metastatic dissemination of cancer. As previously published works have identified several putative TLX ligands, our findings demonstrating that TLX functions as an anti-cancer factor in TNBC provides a strong rationale for future research aimed at therapeutically targeting this nuclear receptor. Citation Format: Adam T. Nelczyk, Hashni E. Vidana Gamage, Liqian Ma, Michael T. McHenry, Madeline A. Henn, Mohammed Kadiri, Yu Wang, Anasuya Das Gupta, Natalia Krawczynska, Sisi He, Michael J. Spinella, Erik R. Nelson. Nuclear receptor TLX inhibits cancer cell intrinsic properties required for triple-negative breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 820.

Reversing the Genomic, Epigenetic, and Triple-Negative Breast Cancer-Enhancing Effects of Obesity.

Obesity is an established risk and progression factor for triple-negative breast cancer (TNBC). Given rising global rates of obesity and TNBC, strategies to reduce the burden of obesity-driven TNBC are urgently needed. We report the genomic, epigenetic, and procancer effects of obesity are reversible by various calorie restriction regimens.

Radiomic features quantifying pixel-level characteristics of breast tumors from magnetic resonance imaging predict risk factors in triple-negative breast cancer.

e12612 Background: Computationally derived quantitative imaging (radiomic) features that describe tumor phenotypes at the pixel level have demonstrated associations with clinical characteristics in early investigations of other cancers. This implies that molecular differences among tumors may be reflected in their structure on the scales probed by 3D magnetic resonance imaging (MRI). We investigated whether radiomic features computed over tumor volumes from pre-treatment breast MRI could predict risk factors in triple-negative breast cancer (TNBC). Methods: We evaluated breast tumors on pre-treatment, post-contrast T1-weighted MRI from 156 patients with non-metastatic TNBC who underwent neoadjuvant chemotherapy. Tumor regions of interest were segmented by a convolutional neural network algorithm, with validation by breast radiologists. Features quantifying tumor shape and texture were extracted for the largest tumor present in each patient. We identified 23 principal components (PCs) describing these data within the original 364-dimensional feature space for further analysis. Tumor volume was also extracted for comparison with the shape and texture PCs, clinical variables and outcomes, but was kept separate from other radiomic features, since it directly correlates with clinical stage. We compiled for the cohort clinical variables including demographics, stage, grade, and, where available, absolute lymphocyte count (ALC) and Ki-67, a cellular proliferation index routinely used in clinical practice. We then performed a series of univariate and multivariate regression analyses to identify radiomic PCs and clinical variables that significantly predict patient outcomes, and radiomic PCs that predict established risk factors. Our multivariate analyses utilized 5-fold cross-validation and Monte-Carlo determination of p-values (based on 3000 random samplings from the null hypothesis), to ensure statistical rigor in identifying predictive relationships while correcting for multiple hypothesis testing. Results: Our univariate analyses confirmed expected correlations between: overall survival and pre-treatment tumor volume (p = 0.010); survival and ALC (p = 0.002); and clinical stage and tumor volume (p = 1.2⨉10-7). From our multivariate analysis, shape and texture radiomic features were predictive of: tumor volume (p &lt; 0.001); clinical stage (p &lt; 0.001); and Ki-67 (p = 0.02). We confirmed that Ki-67 was predictive of post-treatment residual cancer (p = 0.014), as has been previously reported. Conclusions: Radiomic features predict breast cancer risk factors that are significant for determining outcomes for TNBC patients. Combinations of radiomic shape and texture features track closely with tumor volumes, stage, and proliferative activity, potentially reflecting underlying molecular evolution.

Quantitative Apparent Diffusion Coefficients From Peritumoral Regions as Early Predictors of Response to Neoadjuvant Systemic Therapy in Triple-Negative Breast Cancer.

Pathologic complete response (pCR) to neoadjuvant systemic therapy (NAST) in triple-negative breast cancer (TNBC) is a strong predictor of patient survival. Edema in the peritumoral region (PTR) has been reported to be a negative prognostic factor in TNBC. To determine whether quantitative apparent diffusion coefficient (ADC) features from PTRs on reduced field-of-view (rFOV) diffusion-weighted imaging (DWI) predict the response to NAST in TNBC. Prospective. A total of 108 patients with biopsy-proven TNBC who underwent NAST and definitive surgery during 2015-2020. A 3.0 T/rFOV single-shot diffusion-weighted echo-planar imaging sequence (DWI). Three scans were acquired longitudinally (pretreatment, after two cycles of NAST, and after four cycles of NAST). For each scan, 11 ADC histogram features (minimum, maximum, mean, median, standard deviation, kurtosis, skewness and 10th, 25th, 75th, and 90th percentiles) were extracted from tumors and from PTRs of 5 mm, 10 mm, 15 mm, and 20 mm in thickness with inclusion and exclusion of fat-dominant pixels. ADC features were tested for prediction of pCR, both individually using Mann-Whitney U test and area under the receiver operating characteristic curve (AUC), and in combination in multivariable models with k-fold cross-validation. A P value < 0.05 was considered statistically significant. Fifty-one patients (47%) had pCR. Maximum ADC from PTR, measured after two and four cycles of NAST, was significantly higher in pCR patients (2.8 ± 0.69 vs 3.5 ± 0.94 mm2 /sec). The top-performing feature for prediction of pCR was the maximum ADC from the 5-mm fat-inclusive PTR after cycle 4 of NAST (AUC: 0.74; 95% confidence interval: 0.64, 0.84). Multivariable models of ADC features performed similarly for fat-inclusive and fat-exclusive PTRs, with AUCs ranging from 0.68 to 0.72 for the cycle 2 and cycle 4 scans. Quantitative ADC features from PTRs may serve as early predictors of the response to NAST in TNBC. 1 TECHNICAL EFFICACY: Stage 4.

Poor Glycemic Control Is a Risk Factor for Triple-Negative Breast Cancer in Patients With Type 2 Diabetes [A323

INTRODUCTION: We sought to investigate the relationship between glycemic control, race, and the incidence of triple-negative breast cancer (TNBC) among patients with type 2 diabetes (T2D). METHODS: A retrospective cohort study evaluating 1,416 patients with T2D diagnosed with breast cancer between 2015 and 2020 was conducted. Tumor subtype was categorized as either hormone receptor-positive (ER+, PR+, or both) or triple-negative (ER/PR- and HER2/neu-). Based on Hgb A1c measured within 3 months of cancer diagnosis, patients were assigned to one of three categories: well-controlled (Hgb A1c less than 7.0), moderately controlled (Hgb A1c 7.0-9.4), or poorly controlled (Hgb A1c greater than or equal to 9.5) T2D. RESULTS: A significant increase in the percentage of non-Hispanic White patients with TNBC across the three levels of glycemic control was observed (P=.037). When racial comparisons of cancer subtype within categories of glycemic control were performed, a significant (P=.001) increase in the percentage of Black patients with TNBC compared with non-Hispanic White patients was only observed among those with well-controlled T2D. CONCLUSION: Although previous studies have shown that Black patients are 2 to 3 times more likely to develop TNBC than their non-Hispanic White counterparts, a statistically significant racial difference was only noted among our patients with well-controlled T2D. Thus, our data not only suggest that T2D may serve as a modifiable risk factor for the development of TNBC but also that the risk conferred by poor glycemic control may bear more significance for non-Hispanic White patients than Black patients.

ERα36-High Cancer-Associated Fibroblasts as an Unfavorable Factor in Triple-Negative Breast Cancer.

Simple SummaryThe tumor microenvironment, as an important constituent of neoplastic tissue, has been a promising target for cancer therapy. Triple-negative breast cancer accounts for around 10–20% of invasive breast cancers. This study describes a new cancer-associated fibroblast subtype characterized by high ERα36 levels that secretes HGF, which can impact triple-negative breast cancer. The data enlighten the importance of the stromal effect on the disease course and underlines the significance of further research on the tumor microenvironment and its role in the progression of cancer.Background: Cancer-associated fibroblasts (CAFs) are the most abundant cell type in the tumor microenvironment (TME). Estrogen receptor alpha 36 (ERα36), the alternatively spliced variant of ERα, is described as an unfavorable factor when expressed in cancer cells. ERα can be expressed also in CAFs; however, the role of ERα36 in CAFs is unknown. Methods: Four CAF cultures were isolated from chemotherapy-naïve BC patients and characterized for ERα36 expression and the NanoString gene expression panel using isolated RNA. Conditioned media from CAF cultures were used to assess the influence of CAFs on triple-negative breast cancer (TNBC) cells using a matrigel 3D culture assay. Results: We found that ERα36high CAFs significantly induced the branching of TNBC cells in vitro (p < 0.001). They also produced a set of pro-tumorigenic cytokines compared to ERα36low CAFs, among which hepatocyte growth factor (HGF) was the main inducer of TNBC cell invasive phenotype in vitro (p < 0.001). Tumor stroma rich in ERα36high CAFs was correlated with high Ki67 expression (p = 0.041) and tumor-associated macrophages markers (CD68 and CD163, p = 0.041 for both). HGF was found to be an unfavorable prognostic factor in TCGA database analysis (p = 0.03 for DFS and p = 0.04 for OS). Conclusions: Breast cancer-associated fibroblasts represent distinct subtypes based on ERα36 expression. We propose that ERα36high CAFs could account for an unfavorable prognosis for TNBC patients.

NRG1 regulates Fra-1 transcription and metastasis of triple-negative breast cancer cells via the c-Myc ubiquitination as manipulated by ERK1/2-mediated Fbxw7 phosphorylation.

Neuregulin 1 (NRG1), an EGF family member, is expressed in most breast cancers. It promotes breast cancer growth and metastasis in HER2 receptor expressing breast cancer. However, its role in triple-negative breast cancer (TNBC) has not been extensively investigated. In this study, we observed that NRG1 knockdown resulted in the suppression of TNBC cells (MDA-MB-231 cell and MDA-MB-468 cell) metastasis and downregulation of Fra-1 (FOS-like 1, AP-1 transcription factor subunit, which is an overexpressed transcription factor in TNBC and acts as a coordinator of metastasis). In addition, the transcriptional regulation of Fra-1 by NRG1 was mediated by ERK1/2-induced recruitment of c-Myc (MYC proto-oncogene, transcription factor) to the promoter of Fra-1. Furthermore, c-Myc was targeted by an E3 ligase Fbxw7 and its ubiquitination and degradation by Fbxw7 was regulated by NRG1 expression and ERK1/2-mediated Fbxw7 phosphorylation that results in the dissociation and nuclear import of c-Myc. Taken together, the results of our study demonstrated that NRG1 regulates the Fra-1 expression to coordinate the TNBC metastasis via the novel ERK1/2-Fbxw7-c-Myc pathway and targeting NRG1 expression could be a potential therapeutic strategy for TNBC.