Triple-negative Breast Cancer Progression Research Articles

The DRAP1/DR1 Repressor Complex Increases mTOR Activity to Promote Progression and Confer Everolimus Sensitivity in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Transcriptional dysregulation is a hallmark of cancer, and several transcriptional regulators have been demonstrated to contribute to cancer progression. In this study, we identified an upregulation of the transcriptional corepressor downregulator of transcription 1-associated protein 1 (DRAP1) in TNBC, which was closely associated with poor recurrence-free survival in patients with TNBC. DRAP1 promoted TNBC proliferation, migration, and invasion in vitro and tumor growth and metastasis in vivo. Mechanistically, the downregulator of transcription 1 (DR1)/DRAP1 heterodimer complex inhibited expression of the cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1) and thereby increased activation of mTOR, which sensitized TNBC to treatment with the mTOR inhibitor everolimus. DRAP1 and DR1 also formed a positive feedback loop. DRAP1 enhanced the stability of DR1 by recruiting the deubiquitinase USP7 to inhibit its proteasomal degradation; in turn, DR1 directly promoted DRAP1 transcription. Collectively, this study uncovered a DRAP1-DR1 bidirectional regulatory pathway that promotes TNBC progression, suggesting that targeting the DRAP1/DR1 complex might be a potential therapeutic strategy to treat TNBC. Significance: DR1 and DRAP1 form a positive feedback loop and a repressor complex to cooperatively inhibit cytosolic arginine sensor for mTORC1 subunit 1 transcription and stimulate mTOR signaling, leading to progression and increased everolimus sensitivity in triple-negative breast cancer.

Erianin inhibits the progression of triple-negative breast cancer by suppressing SRC-mediated cholesterol metabolism

Triple-negative breast cancer (TNBC) is highly malignant and lacks effective biotherapeutic targets. The development of efficient anticancer drugs with low toxicity and few side effects is a hotspot in TNBC treatment research. Although erianin is known to have potent antitumor activity, its regulatory mechanism and target in TNBC have not been fully elucidated, hampering further drug development. This study showed that erianin can significantly inhibit TNBC cell proliferation and migration, promote cell apoptosis, and inhibit the growth of transplanted tumors in mice. Mechanistically, through network pharmacology analysis, molecular docking and cellular thermal shift assays, we preliminarily identified SRC as the cellular target of erianin. Erianin potently inhibited the expression of SRC, which mediated the anticancer effect of erianin in TNBC. Moreover, erianin can downregulate the expression of genes related to cholesterol synthesis and uptake by targeting SRC, interfering with cholesterol levels in TNBC, thereby inhibiting the progression of TNBC in vivo and in vitro. Taken together, our results suggest that erianin may inhibit the progression of TNBC by suppressing SRC-mediated cholesterol metabolism, and erianin has the great potential to be an effective treatment for TNBC patients.

MiRNA-200c-3p deficiency promotes epithelial-mesenchymal transition in triple-negative breast cancer by activating CRKL expression

Epithelial-mesenchymal transition (EMT) plays an important role in malignant progression of Triple-negative breast cancer (TNBC). Many studies have confirmed that miRNA-200c-3p is related to EMT. And we found that it is involved in the regulation of EMT, but the exact mechanism is unclear. CRKL is highly expressed in a variety of tumors and plays a role in EMT. In this study, the potential targets of miRNA-200c-3p were searched in miRPathDB, Targetscan and PicTar. And there are 68 potential targets at the intersection of the three databases. Then, bioinformatics and text mining performed by Coremine Medica, and found that among 68 potential targets, CRKL has the strongest correlation with EMT in TNBC. Therefore, we speculated that miRNA-200c-3p involvement in EMT might be related to CRKL. To verify miRNA-200c-3p inhibits the malignant phenotype of TNBC by regulating CRKL, RT‒PCR, western blotting, Clonal formation assays,CCK-8 proliferation assays, transwell invasion assays, Luciferase reporter assay and nude mouse transplantation tumor assay were performed. In this study, we found that miRNA-200c-3p is under-expressed and EMT-related genes are up-regulated in TNBC, and miRNA-200c-3p can inhibit cancer cell proliferation, invasion and the expression of EMT-related genes and proteins in TNBC. Further research confirmed that miRNA-200c-3p could inhibit EMT by inhibiting the expression of CRKL that directly combining CRKL gene.

Serine/Threonine kinase 16 phosphorylates STAT3 and confers a JAK2-Inhibition resistance phenotype in triple-negative breast cancer

Although Janus kinase 2 (JAK2) plays a critical role in the progression of triple-negative breast cancer (TNBC), its inhibitors are incapable of eradicating these tumor cells, implicating drug resistance mechanisms exist. Our evidences show that TNBC cells express high level of Serine/Threonine Kinase 16 (STK16) when JAK2 signaling is blocked. Pharmacological inhibition or silencing of STK16 significantly enhances the sensitivity of TNBC cells to JAK2 inhibition, while over-expression of STK16 alleviates the anti-tumor effect of JAK2-inhibitor. Mechanistically, elevated STK16 expression rescues the phosphorylation status and transcriptional activity of STAT3, as STK16 is able to directly catalyze the phosphorylation of STAT3 at ser-727 residue. Our data indicate that upon JAK2 inhibition, TNBC cells express STK16 to maintain STAT3 transcriptional activity, dual-inhibition of JAK2/STK16 offers a potential way to treat TNBC patients.

Exploring the regulatory role of FBXL19-AS1 in triple-negative breast cancer through the miR-378a-3p/OTUB2 axis.

The regulatory potential of long noncoding RNA (lncRNA) FBXL19-AS1 has been highlighted in various cancers, but its effect on triple-negative breast cancer (TNBC) remains unclear. Here, we aimed to elucidate the role of FBXL19-AS1 in TNBC and its underlying mechanism. RT-qPCR was employed to detect the expressions of FBXL19-AS1 and miR-378a-3p in tissues and cells. Immunohistochemical staining and western blot were utilized to detect the expression levels of proteins. Cell activities were detected using flow cytometry, CCK-8, and transwell assay. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were deployed to investigate interactions of different molecules. Protein-protein interaction (PPI) network, gene ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG) pathways were used to analyze the downstream pathway. In vivo xenograft model was conducted to detect the effect of FBXL19-AS1 on tumor growth. FBXL19-AS1 was overexpressed in TNBC tissues and cell lines compared with counterparts. FBXL19-AS1 knockdown suppressed TNBC cell activities, whereas its overexpression exhibited the opposite effect. Mechanistically, FBXL19-AS1 was found to interact with miR-378a-3p. Further analysis revealed that miR-378a-3p exerted tumor-suppressive effects in TNBC cells. Additionally, miR-378a-3p targeted and downregulated the expression of ubiquitin aldehyde binding 2 (OTUB2), a deubiquitinase associated with TNBC progression. In vivo experiments substantiated the inhibitory effects of FBXL19-AS1 knockdown on TNBC tumorigenesis, and a miR-378a-3p inhibitor partially rescued these effects. The downstream pathway of the miR-378a-3p/OTUB2 axis was explored, revealing connections with proteins involved in modifying other proteins, removing ubiquitin molecules, and influencing signaling pathways, including the Hippo signaling pathway. Western blot analysis confirmed changes in YAP and TAZ expression levels, indicating a potential regulatory network. In summary, FBXL19-AS1 promotes exacerbation in TNBC by suppressing miR-378a-3p, leading to increased OTUB2 expression. The downstream mechanism may be related to the Hippo signaling pathway. These findings propose potential therapeutic targets for TNBC treatment.

CD44v5 domain regulates crosstalk between TNBC cells and tumor-associated macrophages by enhancing the IL-4R/STAT3 axis.

Triple-negative breast cancer (TNBC) has greater infiltration of M2-like macrophages (TAMs), which enhances cancer cell invasion and leads to a poor prognosis. TNBC progression is mediated by both tumor cells and the tumor microenvironment (TME). Here we elucidate the mechanism of the interaction between TNBC cells and TAMs. In this study, we confirmed that CD44v5 is highly expressed in TNBC, which drives TNBC cell metastasis and promotes TAM polarization by co-localizing with IL4Rα and inhibiting its internalization and degradation, thereby promoting activation of the STAT3/IL6 signaling axis. At the same time, TAMs also facilitate TNBC cell metastasis by secreting IL-4, IL-6, and other cytokines, in which the IL-4/IL-4R/STAT3/IL-6 signaling axis plays the same role for TNBC cells responding to TAMs. Moreover, we found that the above progress could be suppressed when the CD44v5 domain was blocked. We demonstrated that the CD44v5/IL-4R/STAT3/IL-6 signaling pathway plays a key role in TNBC cell metastasis, and in TNBC cells inducing TAM polarization and responding to TAMs, promoting metastasis. Collectively, we suggest that the CD44v5 domain may be a promising target for regulating the TME of TNBC as well as treating TNBC.

Abstract PO4-28-07: SLC26A9 promotes triple-negative breast cancer progression by regulating lipid metabolism

Abstract Background: The disturbance of lipid metabolism in the microenvironment plays an important role in the occurrence and development of triple-negative breast cancer(TNBC). SLC26A9, a member of the SLC26A family of anion transporters, is involved in breast oncogenesis. However, the effect of SLC26A9 in triple-negative breast cancer on lipid metabolism in the tumor microenvironment remains unclear. Methods: Bioinformatics, tissue microarrays and TNBC cell lines were used to detect the expression of SLC26A9 and its clinical significance. By altering the expression of SLC26A9 gene in TNBC cells, the effects of SLC26A9 on the regulation of lipid metabolism, cell biological behavior and its related molecular mechanisms in the TNBC microenvironment were explored using adoptive staining techniques, flow cytometric analysis and animal models. Results: We found by tissue microarray (TNBC=105 cases, Nor=67 cases) that the expression of SLC26A9 was significantly up-regulated in BC compared to paracancerous tissues, where SLC26A9 expression was higher in TNBC, and the same results were obtained in BC cell lines. Secondly, we found that SCL26A9 was positively correlated with lipid metabolism status in TNBC microenvironment and possessed a close association with P53 mutation by analyzing GEO and TCGA databases. Therefore, we used two cell lines (MDA-MB-231, MDA-MB-468) in which SCL26A9 was highly expressed in TNBC cell lines. It was demonstrated by oil red O staining and lipid fluorescence staining that knockdown/silencing of SLC26A9 diminished the ability of MDA-MB-231 and MDA-MB-468 cells to uptake and utilize lipids, especially for metabolites such as neutral lipids: triglycerides, lipid droplets, and steroidal substances. Then we demonstrated by using fatty acid flow cytometric analysis that knockdown/silencing of SLC26A9 significantly reduced the utilization and production of fatty acids in MDA-MB-231 and MDA-MB-468 cells, which are the key metabolic indicators of cancer cells as well as important energy-providing substances for cancer progression. Therefore, we further verified functionally that cell proliferation, migration, invasion and anti-apoptosis were significantly inhibited in in vitro after knockdown/silencing of SLC26A9, and in vivo tumorigenesis was significantly inhibited in BALB/c nude mice in in vivo. In addition, the expression of markers of lipid metabolic activity (FASN, ACLY) was reduced after knockdown/silencing of SLC26A9 in MDA-MB-231 cells by Western Blot, and the reduced expression of FASN and ACLY mainly inhibited the ab initio synthesis, uptake and utilization of fatty acids in TNBC cells, which in turn regulated the proliferation and invasion of TNBC. Mechanistically, SLC26A9 silencing/knockdown inhibited the synthesis, uptake, and utilization of multiple metabolites of lipids in TNBC, which combined with the TCGA database results revealed the activation of the key signaling pathway, the JAK-STAT3 signaling pathway, to promote the progression of TNBC. Conclusion(s): SLC26A9 is involved in the disturbance of lipid metabolism in the TNBC microenvironment and regulates the progression of TNBC. The search for new SLC26A9 inhibitors is expected to provide a new direction for the treatment of TNBC, but the exact molecular mechanisms need to be further explored. SLC26A9 upregulation in TNBC leads to a poor prognosis by referencing lipid metabolism in the TNBC microenvironment Wench. SLC26A9 is involved in lipid metabolism disorders and cell biological functions in the TNBC microenvironment in vivo. SLC26A9 is involved in disordered lipid metabolism in the TNBC microenvironment. Citation Format: Yingming Zhou, Hu Wang, Zhengxing Zhou, Xuemei Liu, Biguang Tuo, Taolang Li. SLC26A9 promotes triple-negative breast cancer progression by regulating lipid metabolism [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 PO4-28-07.

Abstract PO1-06-13: Plasma Exosomes in Obesity-driven Diabetes Exacerbate Progression of Triple Negative Breast Cancer: Insights from Animal Models

Abstract Objective: Our objective is to examine how obesity and type 2 diabetes (T2D) drive systemic and local metabolic changes in adipose tissue to promote progression of triple negative breast cancer (TNBC). We investigate the role of plasma exosomes as significant mediators of gene expression and functional changes, increasing TNBC progression and metastasis. We hypothesize that T2D and obesity alter the payload of plasma exosomes. These modified exosomes, specifically differentially expressed microRNAs in T2D, critically reshape the tumor microenvironment, ultimately promoting epithelial to mesenchymal transition (EMT) and distant metastasis of TNBC cells. Our goal is to gain a deeper understanding of the underlying mechanisms connecting T2D, obesity and TNBC metastasis, through the novel factor of exosome crosstalk. Our findings shed light on novel therapeutic targets and strategies to mitigate the impact of metabolic disorders on cancer outcomes. Methods: We subjected C57BL/6J female mice to high-fat diet (HFD; 60% Kcal fat) for 12 weeks with weight monitoring, whereupon oral glucose tolerance test confirmed glucose intolerance and insulin resistance. Plasma exosomes were then isolated from peripheral blood and used to treat E0771 cells, as a TNBC model, for 72 hours for in vitro and in vivo analysis. To assess gene expression changes, we employed a commercial EMT array kit and performed qPCR. Gene expression data were uploaded to Qiagen software, specifically the Ingenuity Pathway Analysis tool, for analysis. To evaluate cell migration as a functional test of aggressiveness, a migration assay was conducted using 24-hour FBS deprivation, followed by 24-hour migration, utilizing transwell plates with 8μm pores. For in vivo analysis, mice were injected with 50k E0771-GFP cells in the 4th mammary fat pad. Tumors were allowed to grow for 28 days, at which time histology and flow cytometry analyses were performed with harvested brain, lung, and spleen. Results: In-vitro analysis showed that HFD-derived exosomes reprogram gene expression in E0771 cells, driving a strong EMT signature, compared to LFD control or exosome-free negative control. This reprogramming elicited a pro-metastatic phenotype and upregulated signaling pathways associated with metastasis. Furthermore, HFD exosomes upregulated PD-L1 receptor expression compared to negative controls, linking EMT to immune tolerance. In-vivo analysis confirmed these findings, demonstrating increased metastatic burden in both lung and brain in the HFD context. Conclusions: Plasma exosomes derived from HFD fed mice dramatically reprogram EMT gene networks in E0771 cells, exerting genomic alterations that persist for at least 28 days. These induced changes in cellular gene expression likely promote the development of brain metastases and immune evasion. The model suggests that TNBC patients with obesity-driven diabetes should be evaluated for increased risk of distant metastases. Plasma exosomes miRNA profiling may be a valuable biomarker in cancer disparities populations where the prevalence of obesity and diabetes are high. Citation Format: Pablo Llevenes. Plasma Exosomes in Obesity-driven Diabetes Exacerbate Progression of Triple Negative Breast Cancer: Insights from Animal Models [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 PO1-06-13.

Inhibition of protein translational machinery in triple-negative breast cancer as a promising therapeutic strategy

Y-box binding protein-1 (YB-1) is a proto-oncogenic protein associated with protein translation regulation. It plays a crucial role in the development and progression of triple-negative breast cancer (TNBC). In this study, we describe a promising approach to inhibit YB-1 using SU056, a small-molecule inhibitor. SU056 physically interacts with YB-1 and reduces its expression, which helps to restrain the progression of TNBC. Proteome profiling analysis indicates that the inhibition of YB-1 by SU056 can alter the proteins that regulate protein translation, an essential process for cancer cell growth. Preclinical studies on human cells, mice, and patient-derived xenograft tumor models show the effectiveness of SU056. Moreover, toxicological studies have shown that SU056 treatment and dosing are well tolerated without any adverse effects. Overall, our study provides a strong foundation for the further development of SU056 as a potential treatment option for patients with TNBC by targeting YB-1.

H3K27ac-induced RHOXF2 activates Wnt2/β-catenin pathway by binding to HOXC13 to aggravate the malignant progression of triple negative breast cancer

Triple negative breast cancer (TNBC) is insensitive to conventional targeted therapy and endocrine therapy, and is characterized by high invasiveness and high recurrence rate. This study aimed to explore the role and mechanism of RHOXF2 and HOXC13 on the malignant progression of TNBC. RT-qPCR and western blot were used to detect RHOXF2 and HOXC13 expression in TNBC cells. The proliferation, colony formation, invasion, migration, apoptosis and cell cycle of TNBC cells after transfection were analyzed by CCK-8 assay, colony formation assay, transwell assay, wound healing assay and flow cytometry analysis. Co-Immunoprecipitation and GST pull-down assays were used to analyze the combination between RHOXF2 and HOXC13. ChIP-PCR and luciferase reporter gene assay were used to examine the regulation of H3K27ac on RHOXF2. Besides, the expression of Ki67 and cleaved Caspase3 in tumor tissues of nude mice was determined by immunofluorescence. Results revealed that RHOXF2 and HOXC13 expression was increased in TNBC cells. RHOXF2 knockdown suppressed the proliferation, invasion and migration, as well as induced G0/G1 cell cycle arrest and apoptosis of TNBC cells. Besides, RHOXF2 could bind to HOXC13 and RHOXF2 knockdown suppressed HOXC13 expression in TNBC cells. Furthermore, HOXC13 overexpression reversed the impacts of RHOXF2 downregulation on the proliferation, invasion, migration, G0/G1 cell cycle arrest and apoptosis of TNBC cells. In addition, RHOXF2 silencing limited the tumor volume in nude mice, which was reversed by HOXC13 overexpression. Moreover, RHOXF2 knockdown interfered with Wnt2/β-catenin pathway in vitro and in vivo by binding to HOXC13. Importantly, H3K27ac acetylation could activate the expression of RHOXF2 promoter region. In conclusion, RHOXF2 activated by H3K27ac functioned as a tumor promoter in TNBC via mediating Wnt2/β-catenin pathway by binding to HOXC13, which provided promising insight into exploration on TNBC therapy.

Identification of a Gene Expression Signature to Predict the Risk of Early Recurrence and the Degree of Immune Cell Infiltration in Triple-negative Breast Cancer.

Patients with triple-negative breast cancer (TNBC) have a high rate of recurrence within 3 years of diagnosis and a high rate of death within 5 years compared to other subtypes. The number of clinical trials investigating various new agents and combination therapies has recently increased; however, current strategies benefit only a minority of patients. This study aimed to identify specific genes that predict patients at high risk of recurrence and the immune status of the tumor microenvironment at an early stage, thereby providing insight into potential therapeutic targets to improve clinical outcomes in TNBC patients. We evaluated the prognostic significance of microarray mRNA expression of 20,603 genes in 233 TNBC patients from the METABRIC dataset and further validated the results using RNA-seq mRNA expression data in 143 TNBC patients from the GSE96058 dataset. Eighteen differentially expressed genes (AKNA, ARHGAP30, CA9, CD3D, CD3G, CD6, CXCR6, CYSLTR1, DOCK10, ENO1, FLT3LG, IFNG, IL2RB, LPXN, PRKCB, PVRIG, RASSF5, and STAT4) identified in both datasets were found to be reliable biomarkers for predicting TNBC recurrence and progression. Notably, the genes whose low expression was associated with increased risk of recurrence and death were immune-related genes, with significant differences in levels of immune cell infiltration in the tumor microenvironment between high- and low- expression groups. Genes reported herein may be effective biomarkers to identify TNBC patients who will and will not benefit from immunotherapy and may be particularly important genes for developing future treatment strategies, including immunotherapy.

Hypomethylation of ATP1A1 Is Associated with Poor Prognosis and Cancer Progression in Triple-Negative Breast Cancer.

Dysregulated DNA methylation in cancer is critical in the transcription machinery associated with cancer progression. Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, but no treatment targeting TNBC biomarkers has yet been developed. To identify specific DNA methylation patterns in TNBC, methyl-binding domain protein 2 (MBD) sequencing data were compared in TNBC and the three other major breast cancer subtypes. Integrated analysis of DNA methylation and gene expression identified a gene set showing a correlation between DNA methylation and gene expression. ATPase Na+/K+-transporting subunit alpha 1 (ATP1A1) was found to be specifically hypomethylated in the coding sequence (CDS) region and to show increased expression in TNBC. The Cancer Genome Atlas (TCGA) database also showed that hypomethylation and high expression of ATP1A1 were strongly associated with poor survival in patients with TNBC. Furthermore, ATP1A1 knockdown significantly reduced the viability and tumor-sphere formation of TNBC cells. These results suggest that the hypomethylation and overexpression of ATP1A1 could be a prognostic marker in TNBC and that the manipulation of ATP1A1 expression could be a therapeutic target in this disease.

Upregulation of FGF9 and NOVA1 in cancer-associated fibroblasts promotes cell proliferation, invasion and migration of triple negative breast cancer.

Cancer-associated fibroblasts (CAFs) play a pivotal role in cancer progression. This study aimed to explore the roles of CAFs-derived Fibroblast growth factor 9 (FGF9) and Neuro-oncological ventral antigen 1 (NOVA1) in triple negative breast cancer (TNBC) progression. MDA-MB-231 and BT-549 cells were cocultured with CAF conditioned-medium (CAF-CM) or normal fibroblasts conditioned-medium (NF-CM). MTT, EdU, colony formation, wound healing, transwell migration, and invasion assays were employed to determine cell proliferation, migration and invasion, respectively. Western blot and RT-qPCR were carried out to examine the protein and mRNA expression of FGF9 and NOVA1. Xenograft tumor experiments were conducted to evaluate the effects of CAFs, FGF9, and NOVA1 on tumor growth in vivo. Our results showed that CAFs significantly promoted the proliferation, invasion, and migration of TNBC cells. FGF9 and NOVA1 were significantly upregulated in TNBC CAFs, tissues and cells. CAF-CM also could increase the expression of FGF9 and NOVA1 in TNBC cells. Knockdown of FGF9 or NOVA1 could hamper cell proliferation, invasion, migration, and EMT of TNBC cells. Moreover, CAFs with FGF9/NOVA1 knockdown also could inhibit TNBC progression. Besides, CAFs significantly accelerated tumor growth in vivo, which was blocked by FGF9/NOVA1 knockdown in nude mice. In conclusion, our results indicated the tumor-promoting role of CAFs in TNBC progression. FGF9 and NOVA1 upregulation in CAFs induced cell proliferation, migration and invasion in vitro, and facilitated tumor growth in vivo in TNBCdevelopment.

Type II Interleukin-4 Receptor Activation in Basal Breast Cancer Cells Promotes Tumor Progression via Metabolic and Epigenetic Modulation.

Interleukin-4 (IL4) is a Th2 cytokine that can signal through two different receptors, one of which-the type II receptor-is overexpressed by various cancer cells. Previously, we have shown that type II IL4 receptor signaling increases proliferation and metastasis in mouse models of breast cancer, as well as increasing glucose and glutamine metabolism. Here, we expand on those findings to determine mechanistically how IL4 signaling links glucose metabolism and histone acetylation to drive proliferation in the context of triple-negative breast cancer (TNBC). We used a combination of cellular, biochemical, and genomics approaches to interrogate TNBC cell lines, which represent a cancer type where high expression of the type II IL4 receptor is linked to reduced survival. Our results indicate that type II IL4 receptor activation leads to increased glucose uptake, Akt and ACLY activation, and histone acetylation in TNBC cell lines. Inhibition of glucose uptake through the deletion of Glut1 ablates IL4-induced proliferation. Additionally, pharmacological inhibition of histone acetyltransferase P300 attenuates IL4-mediated gene expression and proliferation in vitro. Our work elucidates a role for type II IL4 receptor signaling in promoting TNBC progression, and highlights type II IL4 signaling, as well as histone acetylation, as possible targets for therapy.

PYCR3 modulates mtDNA copy number to drive proliferation and doxorubicin resistance in triple-negative breast cancer

Triple-negative breast cancer (TNBC) poses significant challenges in treatment due to its aggressive nature and limited therapeutic targets. Understanding the underlying molecular mechanisms driving TNBC progression and chemotherapy resistance is imperative for developing effective therapeutic strategies. Thus, in this study, we aimed to elucidate the role of pyrroline-5-carboxylate reductase 3 (PYCR3) in TNBC pathogenesis and therapeutic response. We observed that PYCR3 is significantly upregulated in TNBC specimens compared to normal breast tissues, correlating with a poorer prognosis in TNBC patients. Knockdown of PYCR3 not only suppresses TNBC cell proliferation but also reverses acquired resistance of TNBC cells to doxorubicin, a commonly used chemotherapeutic agent. Mechanistically, we identified the mitochondrial localization of PYCR3 in TNBC cells and demonstrated its impact on TNBC cell proliferation and sensitivity to doxorubicin through the regulation of mtDNA copy number and mitochondrial respiration. Importantly, Selective reduction of mtDNA copy number using the mtDNA replication inhibitor 2′, 3′-dideoxycytidine effectively recapitulates the phenotypic effects observed in PYCR3 knockout, resulting in decreased TNBC cell proliferation and the reversal of doxorubicin resistance through apoptosis induction. Thus, our study underscores the clinical relevance of PYCR3 and highlight its potential as a therapeutic target in TNBC management. By elucidating the functional significance of PYCR3 in TNBC, our findings contribute to a deeper understanding of TNBC biology and provide a foundation for developing novel therapeutic strategies aimed at improving patient outcomes.

Baicalein promotes KDM4E to induce BICD1 and inhibit triple-negative breast cancer progression by blocking PAR1 signaling.

Baicalein has been implicated in the chemotherapy overcoming triple-negative breast cancer (TNBC). However, many unanswered questions remain regarding its role in treating TNBC. Here, we sought to demonstrate the molecular pathway mediated by baicalein in TNBC. Lysine-specific demethylase 4E (KDM4E), reduced in TNBC cells, was identified as a target protein of baicalein, and baicalein enhanced the protein expression and stability of KDM4E in TNBC cells. Knockdown of KDM4E attenuated the inhibitory effect of baicalein on TNBC cell activity, as demonstrated by intensified mobility, viability, and apoptosis resistance in TNBC cells. KDM4E activated protein bicaudal D homolog 1 (BICD1) expression by reducing the deposition of histone H3 lysine 9 trimethylation (H3K9me3) in its promoter, whereas BICD1 promoted protease-activated receptor-1 (PAR1) endocytosis and blocked PAR1 signaling through physical interaction with PAR1. Knockdown of KDM4E strengthened the PAR1-dependent activity of TNBC cells in response to thrombin activation, whereas TNBC progression activated by PAR1 signaling was blocked by combined overexpression of BICD1. Taken together, our data indicate that baicalein-promoted KDM4E enhanced the expression of BICD1 and activated the inhibitory effect of BICD1 on PAR1 signaling, thereby inhibiting TNBC progression.

COL5A1 promotes triple-negative breast cancer progression by activating tumor cell-macrophage crosstalk.

Triple-negative breast cancer (TNBC) is an exceptionally aggressive subtype of breast cancer. Despite the recognized interplay between tumors and tumor-associated macrophages in fostering drug resistance and disease progression, the precise mechanisms leading these interactions remain elusive. Our study revealed that the upregulation of collagen type V alpha 1 (COL5A1) in TNBC tissues, particularly in chemoresistant samples, was closely linked to an unfavorable prognosis. Functional assays unequivocally demonstrated that COL5A1 played a pivotal role in fueling cancer growth, metastasis, and resistance to doxorubicin, both in vitro and in vivo. Furthermore, we found that the cytokine IL-6, produced by COL5A1-overexpressing TNBC cells actively promoted M2 macrophage polarization. In turn, TGFβ from M2 macrophages drived TNBC doxorubicin resistance through the TGFβ/Smad3/COL5A1 signaling pathway, establishing a feedback loop between TNBC cells and macrophages. Mechanistically, COL5A1 interacted with TGM2, inhibiting its K48-linked ubiquitination-mediated degradation, thereby enhancing chemoresistance and increasing IL-6 secretion. In summary, our findings underscored the significant contribution of COL5A1 upregulation to TNBC progression and chemoresistance, highlighting its potential as a diagnostic and therapeutic biomarker for TNBC.

Single-cell transcriptomics reveals the intra-tumoral heterogeneity and SQSTM1/P62 and Wnt/β-catenin mediated epithelial to mesenchymal transition and stemness of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is characterized by the complex tumor microenvironment (TME) consisting of an abundance of mesenchymal stem cells (MSCs), which is known to facilitate epithelial-to-mesenchymal transition (EMT). The development of single-cell genomics is a powerful method for defining the intricate genetic landscapes of malignancies. In this study, we have employed single-cell RNA sequencing (scRNA-seq) to dissect the intra-tumoral heterogeneity and analyze the single-cell transcriptomic landscape to detect rare consequential cell subpopulations of significance. The scRNA-seq analysis of TNBC and Normal patient derived samples revealed that EMT markers and transcription factors were most upregulated in MSC population. Further, exploration of gene expression analysis among TNBC and Normal patient-derived MSCs ascertained the role of SQSTM1/P62 and Wnt/β-catenin in TNBC progression. Wnt/β-catenin and Wnt/PCP signaling pathways are prominent contributors of EMT, stemness, and cancer stem cell (CSC) properties of TNBC. SQSTM1/P62 cooperates with the components of the Wnt/PCP signaling pathway and is critically involved at the interface of autophagy and EMT.Moreover, siRNA targeting SQSTM1/P62 and inhibitor of Wnt/β-catenin (FH535) in conjunction was used to explore molecular modification of EMT and stemness markers. Although SQSTM1/P62 is not crucial for cell survival, cytotoxicity assay revealed synergistic interaction between the siRNA/inhibitor. Modulation of these important pathways helped in reduction of expression of genes and proteins contributing to CSC properties. Gene and protein expression analysis revealed the induction of EMT to MET. Moreover, co-treatment resulted in inactivation of non-canonical Wnt VANGL2-JNK signaling axis. The synergistic impact of inhibition of SQSTM1/P62 and Wnt/β-catenin signaling facilitates the development of a potential therapeutic regimen for TNBC.

SF3A2 promotes progression and cisplatin resistance in triple-negative breast cancer via alternative splicing of MKRN1.

Triple-negative breast cancer (TNBC) is the deadliest subtype of breast cancer owing to the lack of effective therapeutic targets. Splicing factor 3a subunit 2 (SF3A2), a poorly defined splicing factor, was notably elevated in TNBC tissues and promoted TNBC progression, as confirmed by cell proliferation, colony formation, transwell migration, and invasion assays. Mechanistic investigations revealed that E3 ubiquitin-protein ligase UBR5 promoted the ubiquitination-dependent degradation of SF3A2, which in turn regulated UBR5, thus forming a feedback loop to balance these two oncoproteins. Moreover, SF3A2 accelerated TNBC progression by, at least in part, specifically regulating the alternative splicing of makorin ring finger protein 1 (MKRN1) and promoting the expression of the dominant and oncogenic isoform, MKRN1-T1. Furthermore, SF3A2 participated in the regulation of both extrinsic and intrinsic apoptosis, leading to cisplatin resistance in TNBC cells. Collectively, these findings reveal a previously unknown role of SF3A2 in TNBC progression and cisplatin resistance, highlighting SF3A2 as a potential therapeutic target for patients with TNBC.

Abstract LB314: Organ-specific roles of CD83+ macrophages in breast cancer

Abstract The purpose of this study is to understand how heterogeneity in organ-specific immune infiltrates can lead to Triple Negative Breast Cancer (TNBC) progression and immunotherapies resistance in metastasis. Both innate and adaptive immune responses are essential for breast cancer progression. Using single-cell multiplexing technology on human and murine models of primary and metastatic breast cancers, we investigated the pattern of immune cell infiltration in bone and lung metastases in a temporal manner. The parallel profiling of metastasis sites revealed a strong involvement of the innate immunity in restraining tumor progression with an acute increase in Mrc1+ Cd83+ tumor-associated macrophages at the early stage of lung colonization. Interestingly, the increase was modest in bone metastasis and remained consistent overtime despite bone metastasis progression. In contrast, we saw an increase in the number of Ly6G+ Lrg1+ granulocytes. Profiling of paired TNBC patient samples from the primary and metastatic sites have shown an immunologically quiescent tumor microenvironment in metastasis as compared to the primary tumors. Hence, to further explore the role of Mrc1+ Cd83+ macrophages on therapeutic resistance, we profiled the orthotopic claudin-low TNBC models, T11 and T12, following their exposure to chemotherapy. While responsive tumors displayed a decreased infiltration of Mrc1+ Cd83+ macrophages, the opposite phenotype was observed in non-responders, suggesting their involvement in tumor progression. Overall, our results suggest differential responses in untreated lung/bone metastases and primary tumors indicating divergent roles for Cd83+ TAMs in restraining or promoting tumor progression, while also indicating some crosstalk between neutrophils and stromal cells that aid in bone metastatic progression. These site-specific differences in immune cell phenotype indicate that there is a great need to thoroughly characterize the tumor immune compartment in both the primary and the metastatic setting to improve treatment responses. Considering the inhibitory effect of macrophages on T cells in these models, this study highlights a tissue-specific reprogramming of immune cells in breast cancer metastasis. Citation Format: Swarnima Singh, Igor Bado. Organ-specific roles of CD83+ macrophages in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB314.