Expression In Triple-negative Breast Cancer Research Articles

Characteristics and transcriptional regulators of spontaneous epithelial–mesenchymal transition in genetically unperturbed patient-derived non-spindled breast carcinoma

BackgroundAlthough tumor cells undergoing epithelial–mesenchymal transition (EMT) typically exhibit spindle morphology in experimental models, such histomorphological evidence of EMT has predominantly been observed in rare primary spindle carcinomas. The characteristics and transcriptional regulators of spontaneous EMT in genetically unperturbed non-spindled carcinomas remain underexplored.MethodsWe used primary culture combined with RNA sequencing (RNA-seq), single-cell RNA-seq (scRNA-seq), and in situ RNA-seq to explore the characteristics and transcription factors (TFs) associated with potential spontaneous EMT in non-spindled breast carcinoma.ResultsOur primary culture revealed carcinoma cells expressing diverse epithelial–mesenchymal traits, consistent with epithelial–mesenchymal plasticity. Importantly, carcinoma cells undergoing spontaneous EMT did not necessarily exhibit spindle morphology, even when undergoing complete EMT. EMT was a favored process, whereas mesenchymal–epithelial transition appeared to be crucial for secondary tumor growth. Through scRNA-seq, we identified TFs that were sequentially and significantly upregulated as carcinoma cells progressed through the EMT process, which correlated with increasing VIM expression. Once upregulated, the TFs remained active throughout the EMT process. ZEB1 was a key initiator and sustainer of EMT, as indicated by its earliest significant upregulation in the EMT process, its exact correlation with VIM expression, and the reversal of EMT and downregulation of EMT-upregulated TFs upon ZEB1 knockdown. The correlation between ZEB1 and vimentin expression in triple-negative breast cancer and metaplastic breast carcinoma tumor cohorts further highlighted its role. The immediate upregulation of ZEB2 following that of ZEB1, along with the observation that the knockdown of ZEB1 or ZEB2 downregulates both ZEB1 and ZEB2 concomitant with the reversal of EMT, suggests their functional cooperation in EMT. This finding, together with that of a lack of correlation of SNAI1, SNAI2, and TWIST1 expression with the mesenchymal phenotype, indicated EMT-TFs have a context-dependent role in EMT. Upregulation of EMT-related gene signatures during EMT correlated with poor patient outcomes, highlighting the biological importance of the model. Elevated EMT gene signatures and increased ZEB1 and ZEB2 expression in vimentin-positive compared to vimentin-negative carcinoma cells within the corresponding primary tumor tissue confirmed ZEB1 and ZEB2 as intrinsic, instead of microenvironmentally-induced, EMT regulators, and vimentin as an in vivo indicator of EMT.ConclusionsOur findings provide insights into the characteristics and transcriptional regulators of spontaneous EMT in primary non-spindled carcinoma.

TRPS1, a sensitive marker for different histological and molecular types of breast cancer

ObjectivesWe explored Trichorhinophalangeal syndrome type 1 (TRPS1) expression in special types of breast carcinoma, and analyzed the correlation between TRPS1 and androgen receptor (AR) expression in triple-negative breast cancer (TNBC).MethodsTRPS1 expression was analyzed in 801 patients with special types of breast carcinoma. A total of 969 TNBC were used to analyze the correlation between the expression of TRPS1 and AR. TRPS1 expression was evaluated in 1975 cases of breast cancer with different molecular types.ResultsA total of 801 special types of breast cancers were stained with TRPS1.TRPS1 was positive in 100% (63/63) of mucinous carcinoma, 100% (7/7) adenoid cystic carcinomas (4 classic adenoid cystic carcinomas and 3 solid-basaloid adenoid cystic carcinomas), 100% (4/4) tubular carcinomas, 100% (2/2) secretory carcinomas, and 99.59% (243/244) invasive lobular carcinomas, 99.26% (267/269) invasive micropapillary carcinomas, 97.44% (38/39) ER-positive neuroendocrine tumors, 94.44% (34/36) metaplastic breast carcinomas (MBCs), 63.73% (65/102) apocrine carcinomas. TRPS1 was negative in all triple-negative neuroendocrine carcinomas (0/7).TRPS1 was positive in 92.86% (26/28) of metastatic special types of breast cancer. TRPS1 and AR expression were analyzed in 969 cases of TNBC. 90.40% were positive for TRPS1, and 42.41% were positive for AR. A significant inverse correlation between TRPS1 and AR expression was shown in TNBC (p < .001). TRPS1 showed a higher positive rate (93.13%) in TNBC compared to GATA binding protein 3 (GATA3), gross cystic disease fluid protein 15 (GCDFP-15) and forkhead box transcription Factor C 1 (FOXC1).ConclusionsIn conclusion, our study demonstrated that TRPS1 is a highly sensitive marker for most special types of breast carcinoma. TRPS1 was positive in 63.73% of apocrine carcinomas. TRPS1 and AR expression was inversely correlated in TNBC.

The vitamin D analog EB1089 sensitizes triple-negative breast cancer cells to the antiproliferative effects of antiestrogens

PurposePatients bearing estrogen receptor (ER)α-negative breast cancer tumors confront poor prognosis and are typically unresponsive to hormone therapy. Previous studies have shown that calcitriol, the active vitamin D metabolite, can induce ERα expression in ERα-negative cells. EB1089, a calcitriol analog with reduced calcemic effects, exhibits greater potency than calcitriol in inhibiting cancer cell growth. However, the impact of EB1089 on ERα expression in triple-negative breast cancer (TNBC) cells remains unexplored. This study aims to investigate whether EB1089 could induce functional ERα expression in TNBC cell lines, potentially enabling the antiproliferative effects of antiestrogens. Materials and methodsTNBC cell lines HCC1806 and HCC1937 were treated with EB1089, and ERα expression was analyzed using real-time PCR and Western blots. The transcriptional activity of induced ERα was evaluated through a luciferase reporter assay. The antiproliferative effects of tamoxifen and fulvestrant antiestrogens were assessed using the sulforhodamine B assay in the EB1089-treated cells. ResultsOur findings indicated that EB1089 significantly induced ERα mRNA and protein expression in TNBC cells. Moreover, EB1089-induced ERα exhibited transcriptional activity and effectively restored the inhibitory effects of antiestrogens, thereby suppressing cell proliferation in TNBC cells. ConclusionEB1089 induced the expression of functional ERα in TNBC cells, restoring the antiproliferative effects of antiestrogens. These results highlight the potential of using EB1089 as a promising strategy for re-establishment of the antiproliferative effect of antiestrogens as a possible management for TNBC. This research lays the foundation for potential advancements in TNBC treatment, offering new avenues for targeted and effective interventions.

STAT3 Regulates the Redox Profile in MDA-MB-231 Breast Cancer Cells.

Unbalanced redox status and constitutive STAT3 activation are related to several aspects of tumor biology and poor prognosis, including metastasis and drug resistance. The triple-negative breast cancer (TNBC) is listed as the most aggressive and exhibits the worst prognosis among the breast cancer subtypes. Although the mechanism of reactive oxygen species (ROS) generation led to STAT3 activation is described, there is no data concerning the STAT3 influence on redox homeostasis in TNBC. To address the role of STAT3 signaling in redox balance, we inhibited STAT3 in TNBC cells and investigated its impact on total ROS levels, contents of hydroperoxides, nitric oxide (NO), and total glutathione (GSH), as well as the expression levels of 3-nitrotyrosine (3NT), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and nuclear factor kappa B (NF-κB)/p65. Our results indicate that ROS levels depend on the STAT3 activation, while the hydroperoxide level remained unchanged, and NO and 3NT expression increased. Furthermore, GSH levels, Nrf2, and NF-κB/p65 protein levels are decreased in the STAT3-inhibited cells. Accordingly, TNBC patients' data from TCGA demonstrated that both STAT3 mRNA levels and STAT3 signature are correlated to NF-κB/p65 and Nrf2 signatures. Our findings implicate STAT3 in controlling redox balance and regulating redox-related genes' expression in triple-negative breast cancer.

High Caveolin-1 mRNA expression in triple-negative breast cancer is associated with an aggressive tumor microenvironment, chemoresistance, and poor clinical outcome.

Currently, there are few treatment-predictive and prognostic biomarkers in triple-negative breast cancer (TNBC). Caveolin-1 (CAV1) is linked to chemoresistance and several important processes involved in tumor progression and metastasis, such as epithelial-mesenchymal transition (EMT). Herein, we report that high CAV1 gene expression is an independent factor of poor prognosis in TNBC. CAV1 gene expression was compared across different molecular features (e.g., PAM50 subtypes). CAV1 expression was assessed in relation to clinical outcomes using Cox regression adjusted for clinicopathological predictors. Differential gene expression and gene set enrichment analyses were applied to compare high- and low-expressing CAV1 tumors. Tumor microenvironment composition of high- and low-expressing CAV1 tumors was estimated using ECOTYPER. Tumor tissue microarrays were used to evaluate CAV1 protein levels in stromal and malignant cells. In the SCAN-B (n = 525) and GSE31519 (n = 327) cohorts, patients with CAV1-high tumors had an increased incidence of early recurrence adjusted HR 1.78 (95% CI 1.12-2.81) and 2.20 (95% CI 1.39-3.47), respectively. In further analysis, high CAV1 gene expression was associated with a molecular profile indicating altered metabolism, neovascularization, chemoresistance, EMT, suppressed immune response, and active tumor microenvironment. Protein levels of CAV1 in malignant and stromal cells were not correlated with CAV1 gene expression. CAV1 gene expression in TNBC is a biomarker that merits further investigation in clinical trials and as a therapeutic target.

KMT2D-mediated H3K4me1 recruits YBX1 to facilitate triple-negative breast cancer progression through epigenetic activation of c-Myc.

Lysine methyltransferase 2D (KMT2D) mediates mono-methylation of histone H3 lysine 4 (H3K4me1) in mammals. H3K4me1 mark is involved in establishing an active chromatin structure to promote gene transcription. However, the precise molecular mechanism underlying the KMT2D-mediated H3K4me1 mark modulates gene expression in triple-negative breast cancer (TNBC) progression is unresolved. We recognized Y-box-binding protein 1 (YBX1) as a "reader" of the H3K4me1 mark, and a point mutation of YBX1 (E121A) disrupted this interaction. We found that KMT2D and YBX1 cooperatively promoted cell growth and metastasis of TNBC cells in vitro and in vivo. The expression levels of KMT2D and YBX1 were both upregulated in tumour tissues and correlated with poor prognosis for breast cancer patients. Combined analyses of ChIP-seq and RNA-seq data indicated that YBX1 was co-localized with KMT2D-mediated H3K4me1 in the promoter regions of c-Myc and SENP1, thereby activating their expressions in TNBC cells. Moreover, we demonstrated that YBX1 activated the expressions of c-Myc and SENP1 in a KMT2D-dependent manner. Our results suggest that KMT2D-mediated H3K4me1 recruits YBX1 to facilitate TNBC progression through epigenetic activation of c-Myc and SENP1. These results together unveil a crucial interplay between histone mark and gene regulation in TNBC progression, thus providing novel insights into targeting the KMT2D-H3K4me1-YBX1 axis for TNBC treatment. YBX1 is a KMT2D-mediated H3K4me1-binding effector protein and mutation of YBX1 (E121A) disrupts its binding to H3K4me1. KMT2D and YBX1 cooperatively promote TNBC proliferation and metastasis by activating c-Myc and SENP1 expression in vitro and in vivo. YBX1 is colocalized with H3K4me1 in the c-Myc and SENP1 promoter regions in TNBC cells and increased YBX1 expression predicts a poor prognosis in breast cancer patients.

Prognostic and Clinical Significance of PD-L1, EGFR and Androgen Receptor (AR) Expression in Triple-Negative Breast Cancer (TNBC) Patients.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and is associated with high recurrence rates, a high incidence of distant metastases and poor overall survival. The aim of this study was to investigate the role of PD-L1, EGFR and AR expression in TNBC promotion and progression. To that end, we analyzed the immunohistochemical expression of these genes in 125 TNBC patients and their relation to clinicopathological parameters and survival. An elevated expression of PD-L1 was significantly correlated with higher tumor and nuclear grade, while a low expression was correlated with loco-regional recurrence without any influence on survival. Contrary to this, the expression of AR showed a positive impact on the DFI and a negative association with tumor grade. Furthermore, PD-L1 and AR demonstrated simultaneous expression, and further co-expression analysis revealed that a positive expression of PD-L1/AR notably correlates with tumor and nuclear grade and has a significant impact on a longer DFI and OS, while a negative PD-L1/AR expression is significantly associated with metastases. Therefore, our results suggest that positive PD-L1/AR expression is beneficial for TNBC patients. In addition, an elevated expression of EGFR contributes to metastases and a worse DFI and OS. In conclusion, we think that low PD-L1/low AR/high EGFR expression followed by high Ki67 expression constitutes a 'high risk' profile of TNBC.

Abstract PO1-07-04: Multiomics analysis reveals the landscape of PD-L1 expression in triple-negative breast cancer

Abstract Background: Tumor-infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) remain controversial in predicting clinical outcomes of triple-negative breast cancer (TNBC), as outcomes do not always correlate with the expression of these biomarkers. Genomic and transcriptomic alterations that might contribute to the expression of these biomarkers remain incompletely uncovered. Methods: We evaluated PD-L1 immunohistochemistry (IHC) scores (SP142 and 28-8) and TILs in our TNBC multiomics dataset and two immunotherapy clinical trial cohorts. Then, we analyzed genomic and transcriptomic alterations correlated with TILs, PD-L1 expression and patient outcomes. Results: Despite TILs serving as a decent predictor for TNBC clinical outcomes, there remained exceptions. Our study revealed that several genomic alterations were correlated with unexpected events. Particularly, PD-L1 expression may cause a paradoxical relationship between TILs and prognosis in certain patients. Consequently, we classified TNBCs into four groups based on PD-L1 and TIL levels. The TIL-PD-L1+ and TIL+PD-L1- groups were not typical “hot” tumors; both were associated with worse prognoses and lower immunotherapy efficacy than TIL+PD-L1+ tumors. Copy number variation of PD-L1 and oncogenic signaling activation were correlated with PD-L1 expression in the TIL-PD-L1+ group, whereas GSK3B-induced degradation might cause undetectable PD-L1 expression in the TIL+PD-L1- group. These factors have the potential to affect the predictive function of both PD-L1 and TILs. Conclusions: Several genomic and transcriptomic alterations may cause paradoxical effects among TILs, PD-L1 expression and prognosis in TNBC. Investigating and targeting these factors will advance precision immunotherapy for TNBC patients. Keywords: Triple-negative breast cancer, PD-L1 expression, Tumor-infiltrating lymphocytes, Multiomics, Immunotherapy Citation Format: Han Wang, Xiao-Hong Ding, Cheng-Lin Liu, Yi Xiao, Ruo-Hong Shui, Yan-Ping Li, Chen Chen, Wen-Tao Yang, Zhi-Ming Shao, Yi-Zhou Jiang. Multiomics analysis reveals the landscape of PD-L1 expression in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-07-04.

Abstract PO4-03-10: HER2 Expression in Triple-Negative Breast Cancer: Clinicopathologic Features and Response to Neoadjuvant Chemotherapy

Abstract BACKGROUND: Half of the primary breast cancers reported as HER-2 negative have low HER-2 expression, defined as an immunohistochemical (IHC) score of 1+ or 2+, with negative in situ hybridization. Among these cases, 37% correspond to the triple-negative immunophenotype. The primary objective of this study was to evaluate HER-2 expression in patients with triple-negative breast cancer and compare the clinicopathologic features and response to neoadjuvant chemotherapy between HER-2 negative (0) and HER-2 low (1+/2+) subtypes. METHODS: We retrospectively analyzed 157 patients with early-stage triple-negative breast cancer who underwent neoadjuvant chemotherapy followed by surgery with curative intent and met the study's inclusion criteria. These patients were treated at the National Institute of Neoplastic Diseases between 2010 and 2021. HER-2 analysis was performed using immunohistochemistry (IHC), and in cases of HER-2 (2+) amplification, in situ hybridization was performed. RESULTS: Of all the included patients, 107 were classified in the HER-2 negative group (68%), and 50 were classified in the HER-2 low group (32%). Seventy-four percent of patients with HER-2 negative were in the age range of 40-64 years, compared to 68% in the HER-2 low group. A significant difference was found between the HER2-negative and HER2-low groups regarding age and Ki67. The median age of HER-2 low patients is significantly higher than the median age of HER-2 negative patients (52 years vs. 45 years, respectively). No significant association was found between HER-2 status and clinical stages or histological grade. However, the median Ki67 percentage of HER-2 negative patients is significantly higher than the median Ki67 of HER-2 low patients (70% vs. 50%, respectively (p=0.035)). Thirty-two percent of patients with HER-2 negative achieved a pathological complete response (pCR) compared to 18% of patients with HER-2 low. There were no differences in the pCR rate in the HER-2 1+ and 2+ subgroups. Among the patients included in both groups, who achieved pCR, no disease progression was observed at the time of analysis. CONCLUSIONS: Patients in the HER-2 negative group had a higher rate of complete pathological response to neoadjuvant chemotherapy compared to those in the HER-2 low group. No differences were observed in the pCR rate between HER-2 1+ and 2+ subgroups, suggesting that HER2 expression levels within the low range may not significantly impact treatment response. Table. Table. Citation Format: Zaida Morante, Iris Otoya, Natalia Valdivieso, Cindy Calle, Hugo Fuentes, Silvia Neciosup, Carlos Castañeda, Tatiana Vidaurre, Martin Falla, Valeria Colomo, Henry Gómez. HER2 Expression in Triple-Negative Breast Cancer: Clinicopathologic Features and Response to Neoadjuvant Chemotherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-03-10.

Prognostic Value of "Basal-like" Morphology, Tumor-Infiltrating Lymphocytes and Multi-MAGE-A Expression in Triple-Negative Breast Cancer.

"Basal-like" (BL) morphology and the expression of cancer testis antigens (CTA) in breast cancer still have unclear prognostic significance. The aim of our research was to explore correlations of the morphological characteristics and tumor microenvironment in triple-negative breast carcinomas (TNBCs) with multi-MAGE-A CTA expression and to determine their prognostic significance. Clinical records of breast cancer patients who underwent surgery between January 2017 and December 2018 in four major Croatian clinical centers were analyzed. A total of 97 non-metastatic TNBCs with available tissue samples and treatment information were identified. Cancer tissue sections were additionally stained with programmed death-ligand 1 (PD-L1) Ventana (SP142) and multi-MAGE-A (mAb 57B). BL morphology was detected in 47 (49%) TNBCs and was associated with a higher Ki-67 proliferation index and histologic grade. Expression of multi-MAGE-A was observed in 77 (79%) TNBCs and was significantly associated with BL morphology. Lymphocyte-predominant breast cancer (LPBC) status was detected in 11 cases (11.3%) and significantly correlated with the Ki-67 proliferation index, increased number of intratumoral lymphocytes (itTIL), and PD-L1 expression. No impact of BL morphology, multi-MAGE-A expression, histologic type, or LPBC status on disease-free survival was observed. Our data suggest that tumor morphology could help identify patients with potential benefits from CTA-targeting immunotherapy.

CCAAT enhancer binding protein delta activates vesicle associated membrane protein 3 transcription to enhance chemoresistance and extracellular PD-L1 expression in triple-negative breast cancer

BackgroundChemoresistance and immunosuppression are two major obstacles in the current anti-cancer treatments. This study investigates the involvements of a CCAAT enhancer binding protein delta (CEBPD)/vesicle associated membrane protein 3 (VAMP3) axis in paclitaxel (PTX) resistance and immune evasion in triple-negative breast cancer (TNBC).MethodsPTX resistance-related genes were screened by bioinformatics. CEBPD and VAMP3 expression in clinical TNBC samples was examined by immunohistochemistry. Three PTX-resistant TNBC cell lines (MDA-MB-231/PTX, MDA-MB-468/PTX and MDA-MB-453/PTX) were generated, and their drug resistance was analyzed. Autophagy of cells was analyzed by immunofluorescence staining. Interaction between CEBPD and VAMP3 promoter was identified by immunoprecipitation and luciferase assays. The extracellular expression of programmed cell death-ligand 1 (PD-L1) in TNBC cells was detected. Extracellular vesicles (EVs) from TNBC cells were isolated to examine their effects on CD8+ T cell exhaustion.ResultsCEBPD and VAMP3 were upregulated in chemo-resistant tissue samples and in PTX-resistant TNBC cells. The CEBPD downregulation enhanced PTX sensitivity of cells. However, further upregulation of VAMP3 in cells restored PTX resistance, which was likely due to the activation of autophagy, as the autophagy antagonist chloroquine enhanced PTX sensitivity of cells. CEBPD was found to bind to the VAMP3 promoter to activate its transcription. The CEBPD/VAMP3 axis also increased the PD-L1 expression in the conditioned medium of TNBC cells. The TNBC cell-derived EVs increased the exhaustion of co-cultured CD8+ T cells.ConclusionThis study provides novel evidence that CEBPD plays a key role in enhancing PTX resistance in TNBC cells across various subtypes through VAMP3-mediated autophagy activation. Additionally, the CEBPD/VAMP3 axis also increases extracellular PD-L1 level, delivered by cancer cell-derived EVs, to suppress CD8+ T cell-mediated anti-tumor immune response. These significant observations may provide new insights into the treatment of TNBC, suggesting CEBPD and VAMP3 as promising targets to overcome treatment resistance.

U2AF2-SNORA68 promotes triple-negative breast cancer stemness through the translocation of RPL23 from nucleoplasm to nucleolus and c-Myc expression

BackgroundSmall nucleolar RNAs (snoRNAs) play key roles in ribosome biosynthesis. However, the mechanism by which snoRNAs regulate cancer stemness remains to be fully elucidated.MethodsSNORA68 expression was evaluated in breast cancer tissues by in situ hybridization and qRT‒PCR. Proliferation, migration, apoptosis and stemness analyses were used to determine the role of SNORA68 in carcinogenesis and stemness maintenance. Mechanistically, RNA pull-down, RNA immunoprecipitation (RIP), cell fractionation and coimmunoprecipitation assays were conducted.ResultsSNORA68 exhibited high expression in triple-negative breast cancer (TNBC) and was significantly correlated with tumor size (P = 0.048), ki-67 level (P = 0.037), and TNM stage (P = 0.015). The plasma SNORA68 concentration was significantly lower in patients who achieved clinical benefit. The SNORA68-high patients had significantly shorter disease-free survival (DFS) (P = 0.036). Functionally, SNORA68 was found to promote the cell stemness and carcinogenesis of TNBC in vitro and in vivo. Furthermore, elevated SNORA68 expression led to increased nucleolar RPL23 expression and retained RPL23 in the nucleolus by binding U2AF2. RPL23 in the nucleolus subsequently upregulated c-Myc expression. This pathway was validated using a xenograft model.ConclusionU2AF2-SNORA68 promotes TNBC stemness by retaining RPL23 in the nucleolus and increasing c-Myc expression, which provides new insight into the regulatory mechanism of stemness.

Abstract 2549: Activity-regulated cytoskeleton-associated protein (ARC) gene expression is associated with high infiltration of stromal cells and immune cells, but with less cancer cell proliferation and better overall survival in ER-positive/HER2-negative breast cancers

Abstract Introduction: Recently peritumoral lidocaine infiltration prior to removal was reported to be associated with better survival in early-stage breast cancer (BC). This led us to hypothesize that innervation to the tumor affects its biology and patient survival. Activity-regulated cytoskeleton-associated protein (ARC) gene expression is known to be regulated by neuronal activity. Therefore, we studied the clinical relevance of ARC gene expression as a surrogate of neuronal activity in BC. Methods: Sweden Cancerome Analysis Network-Breast (SCAN-B (GSE96058), n=3273) cohort was analyzed, and the results were validated using The Cancer Genome Atlas (TCGA, n=1069). Results: ER+/HER2- and Luminal A type cancers expressed significantly higher ARC compared to the other subtypes in both cohorts (p&amp;lt;0.005). In the tumor microenvironment, the stromal cells (fibroblasts, endothelial cells and adipocytes) were all found to be significantly infiltrated in high ARC BC (p&amp;lt;0.01). Multiple immune cells were significantly infiltrated in high ARC BC, including CD8, CD4 memory cells, helper type II T cells, regulatory T cells, M1 and M2 macrophages, dendritic cells and B cells (all p&amp;lt;0.03 in both cohorts). In terms of cancer characteristics, there was no difference in silent or nonsilent mutations, single nucleotide variant or indel neoantigens between tumors with low or high ARC expression. However, high ARC BC was significantly associated with less homologous recombination deficiency, intratumor heterogeneity and fraction altered mutation rate compared to low ARC BC in TCGA (p&amp;lt;0.001). High ARC expression was significantly associated with smaller tumor size (p&amp;lt;0.001) and without lymph node metastasis in the SCAN-B cohort (p&amp;lt;0.02), and less Stage IV disease in the TCGA cohort (p&amp;lt;0.02); however, these results were not validated by the other. High ARC BC was significantly associated with lower Nottingham histologic grade and lower MKi67 expression in both cohorts (p&amp;lt;0.001). Cell proliferation-related gene sets in the Hallmark collection (E2F targets, G2M checkpoint, and MTORC1 signaling) were significantly less enriched to high ARC BC consistently in both cohorts. Overall survival (OS) was significantly better in high ARC BC in the ER+/HER2- subtype consistently in both cohorts (p&amp;lt;0.01) and when including all subtypes in the SCAN-B cohort (p&amp;lt;0.001); however this was not validated in TCGA. No significant difference in OS was found between low and high ARC gene expression in triple negative BC in either cohort. Conclusion: ARC gene expression as a surrogate of neuronal activity in BC was associated with high infiltration of stromal cells and immune cells but with less cancer cell proliferation and better overall survival, particularly in the ER+/HER2- subtype. Citation Format: Gabrielle Yee, Rongrong Wu, Takashi Ishikawa, Kazuaki Takabe. Activity-regulated cytoskeleton-associated protein (ARC) gene expression is associated with high infiltration of stromal cells and immune cells, but with less cancer cell proliferation and better overall survival in ER-positive/HER2-negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2549.

Vitamin D suppresses CD133+/CD44 + cancer stem cell stemness by inhibiting NF-κB signaling and reducing NLRP3 expression in triple-negative breast cancer.

This study aims to investigate the role of Vitamin D (VD) in regulating the stemness and survival of CD133+/CD44 + breast cancer stem cells, and to explore the role of NLRP3 in this process. Breast cancer tissues were collected for RXRα and VDR expression analysis. A triple-negative breast cancer cell line was cultured and stem-like cells (CD133 + CD44+) isolated using flow cytometry. These cells were treated with VD, analyzing their stem-like properties, apoptosis and proliferation, as well as P65 nuclear expression and NLRP3 expression. After NLRP3 inflammasome activator treatment, the parameters were reassessed. RXRα and VDR interaction was confirmed using co-immunoprecipitation (CoIP). Finally, a subcutaneous xenograft model of triple-negative breast cancer was treated with VD and subsequently analyzed for stem-like properties, proliferation, apoptosis, and NLRP3 expression levels. CD133+/CD44 + stem cells expressed high levels of SOX2 and OCT4. VD treatment resulted in a significant decrease in SOX2 and OCT4 expression, fewer sphere-forming colonies, lower proliferation ability, and more apoptosis. Additionally, VD treatment inhibited NF-κB signaling and reduced NLRP3 expression. The NLRP3 activator BMS-986,299 counteracted the effects of VD in vitro. In vivo, VD inhibited the growth of breast cancer stem cells, reducing both tumor volume and weight, and decreased NLRP3, SOX2, and OCT4 expression within tumor tissues. Findings elucidate that VD mediates the modulation of stemness in CD133+/CD44 + breast cancer stem cells through the regulation of NLRP3 expression. The research represents novel insights on the implications for the application of VD in cancer therapies.

CYR61 confers chemoresistance by upregulating survivin expression in triple-negative breast cancer.

Cysteine-rich angiogenic inducer 61 (CYR61) is a protein from the CCN family of matricellular proteins that play diverse regulatory roles in the extracellular matrix. CYR61 is involved in cell adhesion, migration, proliferation, differentiation, apoptosis, and senescence. Here, we show that CYR61 induces chemoresistance in triple-negative breast cancer (TNBC). We observed that CYR61 is overexpressed in TNBC patients, and CYR61 expression correlates negatively with the survival of patients who receive chemotherapy. CYR61 knockdown reduced cell migration, sphere formation and the cancer stem cell (CSC) population and increased the chemosensitivity of TNBC cells. Mechanistically, CYR61 activated Wnt/β-catenin signaling and increased survivin expression, which are associated with chemoresistance, the epithelial-mesenchymal transition, and CSC-like phenotypes. Altogether, our study demonstrates a novel function of CYR61 in chemotherapy resistance in breast cancer.

Regulation of non-canonical proteins from diverse origins through the nonsense-mediated mRNA decay pathway.

Immunotherapy harnesses neoantigens encoded within the human genome, but their therapeutic potential is hampered by low expression, which may be controlled by the nonsense-mediated mRNA decay (NMD) pathway. This study investigates the impact of UPF1-knockdown on the expression of non-canonical/mutant proteins, employing proteogenomic to explore UPF1 role within the NMD pathway. Additionally, we conducted a comprehensive pan-cancer analysis of UPF1 expression and evaluated UPF1 expression in Triple-Negative Breast Cancer (TNBC) tissue in-vivo. Our findings reveal that UPF1-knockdown leads to increased translation of non-canonical/mutant proteins, particularly those originating from retained-introns, pseudogenes, long non-coding RNAs, and unannotated transcript biotypes. Moreover, our analysis demonstrates elevated UPF1 expression in various cancer types, with notably heightened protein levels in patient-derived TNBC tumors compared to adjacent tissues. This study elucidates UPF1 role in mitigating transcriptional noise by degrading transcripts encoding non-canonical/mutant proteins. Targeting this mechanism may reveal a new spectrum of neoantigens accessible to the antigen presentation pathway. Our novel findings provide a strong foundation for the development of therapeutic strategies aimed at targeting UPF1 or modulating the NMD pathway.

Overexpression of KLHL22 correlates with poor prognosis in patients with triple-negative breast cancer.

Kelch-like family member 22 (KLHL22) is a protein-coding gene that is responsible for several Mendelian diseases and has been reported to promote tumorigenesis and aging. The purpose of this study was to investigate its expression in triple-negative breast cancer (TNBC) and its prognostic significance. Immunohistochemistry (IHC) was performed to examine the expression levels of KLHL22 in 146 patients with TNBC. The Chi-squared test was used to analyze the correlations between KLHL22 expression level and clinicopathological features, and the Kaplan-Meier survival analysis and Cox multivariate regression model were used to analyze the prognostic significance of KLHL22 in patients with TNBC. The results of immunohistochemical analysis showed that the high expression rate of KLHL22 protein in TNBC was 56.85% (83/146). Further analysis revealed a significantly positive correlation (P<0.05) between KLHL22 expression and primary tumor and regional lymph node status, clinical stage, and relapse. Kaplan-Meier survival analysis revealed that patients with low KLHL22 expression had a longer mean survival time than those with high KLHL22 expression (147.93 vs. 90.1 months; P<0.05). In the multivariate analysis, KLHL22 level, P53 expression, and clinical stage were found to be independent prognostic factors for overall survival (P<0.05), while clinical stage and KLHL22 level were independent prognostic factors for progression-free survival (P<0.05). The present study concludes that KLHL22 may serve as a biomarker for poor prognosis in patients with TNBC.

The Role of IMP3 Immunohistochemistry Expression in Triple-negative Breast Cancer as a Prognostic Factor

Introduction: Triple-negative breast cancer (TNBC) accounts for 12%–18% of the breast malignancies. An insulin-like growth factor-2 mRNA-binding protein 3 (IMP3) is linked to a number of aggressive malignancies like TNBC. The aim of the study was to evaluate the correlation between IMP3 expression and histopathological parameters in TNBC. Materials and Methods: A prospective and retrospective study between January 2019 and June 2023 was done on 48 TNBC subjects with IMP3 expression. The Chi-square test is applied to investigate the correlations between categorical variables. The Kaplan–Meier (KM) method is applied for survival analysis. Results: Out of 48 subjects, 23 (47.9%) had negative staining, whereas 25 (52.1%) of them expressed strong staining to IMP3. The KM curve for disease-free survival showed that was almost same up to 35 months in both IMP3 negative and cases having strong staining features which was also found to be statistically significant. Conclusion: According to our findings, IMP3 can be employed as a risk predictor for TNBC. Focussing the IMP3 expression could be a viable strategy to managing TNBC with novel immune treatments that are currently unavailable.

Diagnostic biopsy does not accurately reflect the PD-L1 expression in triple-negative breast cancer

PD-L1 expression is known to predict the benefits of immune checkpoint inhibitor therapy for triple-negative breast cancer (TNBC). We examined whether the PD-L1 expression evaluated in biopsy specimens accurately reflects its expression in the whole tumor. Immunohistochemistry was performed on 81 biopsy and resection specimens from patients with TNBC to determine their PD-L1 status. We found PD-L1-positive tumors in 23 (28%) biopsy specimens and primarily PD-L1-negative tumors in 58 (72%). The PD-L1 status was reevaluated in matching postoperative specimens of primarily PD-L1-negative tumors. Of them, 31% (18/58) were positive, whereas 69% (40/58) were negative. Considering the pre- and postoperative analyses, 41 (51%) patients had PD-L1-positive tumors, while 40 had PD-L1-negative tumors. We found 18 (22%) more PD-L1-positive tumors while examining the resection specimens compared to biopsies, and the difference was statistically significant (p = 0.0038). Diagnostic biopsies do not fully reflect the PD-L1 expression in TNBC. Our results suggest that a significant subset of TNBC patients may be misclassified as PD-L1-negative and disqualified from anti-PD-L1 therapy.

475P Dynamics of TROP2 expression in triple-negative breast cancer

475P Dynamics of TROP2 expression in triple-negative breast cancer