Therapeutic Target For Triple-negative Breast Cancer Research Articles

FAM83H regulated by glis3 promotes triple-negative breast cancer tumorigenesis and activates the NF-κB signaling pathway.

Triple-negative breast cancer (TNBC) is a highly aggressive and invasive form of breast cancer (BC) with a high mortality rate and a lack of effective targeted drugs. Family with sequence similarity 83 member H (FAM83H) is critically implicated in tumorigenesis. However, the potential role of FAM83H in TNBC remains elusive. Here, we discovered that FAM83H exhibited high expression in tumor tissues of patients with TNBC and was associated with TNM stage. Gain- or loss-of-function experiments were conducted to explore the biological role of FAM83H in TNBC. Subsequently, functional enrichment analysis confirmed that FAM83H overexpression promoted TNBC cell proliferation, invasion, migration and epithelial-mesenchymal transition (EMT), accompanied by upregulation of cyclin E, cyclin D, Vimentin, N-cadherin and Slug. As observed, FAM83H knockdown showed anti-cancer effects, such as fostering apoptosis and inhibiting tumorigenicity and metastasis of TNBC cells. Mechanistically, FAM83H activated the NF-κB signaling pathway. Moreover, a dual-luciferase reporter assay demonstrated that GLIS family zinc finger 3 (GLIS3) bound to the promoter of FAM83H and enhanced its transcription. Notably, overexpression of GLIS3 significantly stimulated TNBC cell proliferation and invasion, and all of this was reversed by rescue experiments involving the knockdown of FAM83H. Overall, FAM83H exacerbates tumor progression, and in-depth understanding of FAM83H as a therapeutic target for TNBC will provide clinical translational potential for intervention therapy.

MIR4435-2HG: A novel biomarker for triple-negative breast cancer diagnosis and prognosis, activating cancer-associated fibroblasts and driving tumor invasion through EMT associated with JNK/c-Jun and p38 MAPK signaling pathway activation

BackgroundBreast cancer has the highest incidence rate and causes the most fatalities among all female cancers worldwide. Triple-negative breast cancer (TNBC) is known for its strong invasiveness and higher rates of recurrence. In this research, we aimed to identify MIR4435-2HG as a promising long non-coding RNA (lncRNA) biomarker and therapeutic target for TNBC. MethodsUtilizing clinicopathological information and transcriptome data from The Cancer Genome Atlas (TCGA) database, we assessed the clinical relevance of MIR4435-2HG in breast cancer through univariate and multivariate COX regression, receiver operating characteristic (ROC) analysis, as well as Kaplan-Meier survival analysis. To investigate the biological role of MIR4435-2HG in TNBC, we conducted gene set enrichment analysis (GSEA), as well as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Additionally, we constructed and validated a nomogram to predict disease-free survival (DFS). Both the R package “pRRophetic” and the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm were employed to forecast the sensitivity to different therapeutics between the high- and low-MIR4435-2HG groups. We employed single-cell RNA sequencing analysis and tumor microenvironment infiltration analysis to investigate the potential involvement of MIR4435-2HG in the TNBC tumor microenvironment. Cellular biological behaviors were assessed utilizing CCK-8, transwell assays, and wound-healing assays. Furthermore, we performed RNA-seq, qRT-PCR, and western blotting analyses to elucidate and confirm the specific mechanisms underlying the role of MIR4435-2HG in TNBC. ResultsIn our study, we have identified MIR4435-2HG as a significant diagnostic and prognostic factor for TNBC. We observed that MIR4435-2HG is widely expressed and might have a significant impact on the reshaping of the TNBC tumor microenvironment. Patients with TNBC in the high-MIR4435-2HG group may show reduced sensitivity to cisplatin, doxorubicin, and gemcitabine and have an increased propensity for immune escape. Knockdown of MIR4435-2HG inhibits cancer-associated fibroblasts (CAFs) activation. Notably, MIR4435-2HG predominantly enhances the migratory and invasive capabilities of TNBC cells through the epithelial-mesenchymal transition (EMT) process. Mechanistically, we validated that MIR4435-2HG activates the JNK/c-Jun and p38 non-classical MAPK signaling pathway in TNBC cells. ConclusionsOur findings highlight the significant potential of MIR4435-2HG as a highly promising biomarker for TNBC. Targeting MIR4435-2HG could represent an appealing therapeutic approach for TNBC.

Therapeutic Potential of PLK1, KIF4A, CDCA5, UBE2C, CDT1, SKA3, AURKB, and PTTG1 Genes in Triple-Negative Breast Cancer: Correlating Their Expression with Sensitivity to GSK 461364 and IKK 16 Drugs.

The treatment of triple-negative breast cancer (TNBC) has been associated with challenges due to the lack of expression of ER, PR, and HER2 receptors in tumor cells. This study aimed to identify genes with potential therapeutic targets in TNBC. Data from the cancer genome atlas regarding breast cancer (BC) were downloaded. After initial preprocessing, cancer samples were categorized into four groups: TNBC, HER2-positive, luminal A, and luminal B. Gene expression differences between these groups were calculated, focusing on genes that showed differential expression in TNBC. A protein-protein interaction network was conducted to identify hub genes among the candidate genes related to TNBC. The protein expression of candidate genes was assessed using immunohistochemistry data from the human protein atlas. Drug resistance and sensitivity associated with hub genes were identified using data from PharmacoDB. TNBC samples and the RT-qPCR method were used to confirm the results. Our findings revealed that eight genes, namely PLK1, KIF4A, CDCA5, UBE2C, CDT1, SKA3, AURKB, and PTTG1, had significant upregulation at the RNA level in TNBC subgroup compared to other subgroups and could be considered hub genes in TNBC. Compared to other subgroups, their expression level in TNBC samples had high sensitivity and specificity. RT-qPCR results also demonstrated a significant increase in levels of SKA3 and PTTG1 in the TNBC compared to healthy tissue and other subgroups. The protein expression of these genes was notably high in some BC samples. PharmacoDB data showed that some candidate genes were closely linked to drug sensitivity of GSK 461364 and IKK 16. The results of this study showed a significant increase in the expression level of PLK1, KIF4A, CDCA5, UBE2C, CDT1, SKA3, AURKB, and PTTG1 in TNBC compared to other BC subgroups. These genes show considerable promise as therapeutic targets for the TNBC subgroup.

Low-Molecular Weight Cyclin E Confers a Vulnerability to PKMYT1 Inhibition in Triple-Negative Breast Cancer.

Cyclin E is a regulatory subunit of CDK2 that mediates S phase entry and progression. The cleavage of full-length cyclin E (FL-cycE) to low-molecular weight isoforms (LMW-E) dramatically alters substrate specificity, promoting G1-S cell cycle transition and accelerating mitotic exit. Approximately 70% of triple-negative breast cancers (TNBC) express LMW-E, which correlates with poor prognosis. PKMYT1 also plays an important role in mitosis by inhibiting CDK1 to block premature mitotic entry, suggesting it could be a therapeutic target in TNBC expressing LMW-E. In this study, analysis of tumor samples of patients with TNBC revealed that coexpression of LMW-E and PKMYT1-catalyzed CDK1 phosphorylation predicted poor response to neoadjuvant chemotherapy. Compared with FL-cycE, LMW-E specifically upregulates PKMYT1 expression and protein stability, thereby increasing CDK1 phosphorylation. Inhibiting PKMYT1 with the selective inhibitor RP-6306 (lunresertib) elicited LMW-E-dependent antitumor effects, accelerating premature mitotic entry, inhibiting replication fork restart, and enhancing DNA damage, chromosomal breakage, apoptosis, and replication stress. Importantly, TNBC cell line xenografts expressing LMW-E showed greater sensitivity to RP-6306 than tumors with empty vector or FL-cycE. Furthermore, RP-6306 exerted tumor suppressive effects in LMW-E transgenic murine mammary tumors and patient-derived xenografts of LMW-E-high TNBC but not in the LMW-E null models examined in parallel. Lastly, transcriptomic and immune profiling demonstrated that RP-6306 treatment induced interferon responses and T-cell infiltration in the LMW-E-high tumor microenvironment, enhancing the antitumor immune response. These findings highlight the LMW-E/PKMYT1/CDK1 regulatory axis as a promising therapeutic target in TNBC, providing the rationale for further clinical development of PKMYT1 inhibitors in this aggressive breast cancer subtype. Significance: PKMYT1 upregulation and CDK1 phosphorylation in triple-negative breast cancer expressing low-molecular weight cyclin E leads to suboptimal responses to chemotherapy but sensitizes tumors to PKMYT1 inhibitors, proposing a personalized treatment strategy.

Synergistic Activation of LEPR and ADRB2 Induced by Leptin Enhances ROS Generation in TNBC Cells.

Leptin interacts not only with leptin receptor (LEPR) but also engages with other receptors. While the pro-oncogenic effects of the adrenergic receptor β2 (ADRB2) are well-established, the role of leptin in activating ADRB2 in triple-negative breast cancer (TNBC) remains unclear. The pro-carcinogenic effects of LEPR were investigated using murine TNBC cell lines, 4T1 and EMT6, and a tumor-bearing mouse model. Expression levels of LEPR, NOX4, and ADRB2 in TNBC cells and tumor tissues were analyzed via Western blot and qPCR. Changes in reactive oxygen species (ROS) levels were assessed using flow cytometry and MitoSox staining, while immunofluorescence double-staining confirmed the co-localization of LEPR and ADRB2. LEPR activation promoted NOX4-derived ROS and mitochondrial ROS production, facilitating TNBC cell proliferation and migration, effects which were mitigated by the LEPR inhibitor Allo-aca. Co-expression of LEPR and ADRB2 was observed on cell membranes, and bioinformatics data revealed a positive correlation between the two receptors. Leptin activated both LEPR and ADRB2, enhancing intracellular ROS generation and promoting tumor progression, which was effectively countered by a specific ADRB2 inhibitor ICI118551. In vivo, leptin injection accelerated tumor growth and lung metastases without affecting appetite, while treatments with Allo-aca or ICI118551 mitigated these effects. This study demonstrates that leptin stimulates the growth and metastasis of TNBC through the activation of both LEPR and ADRB2, resulting in increased ROS production. These findings highlight LEPR and ADRB2 as potential biomarkers and therapeutic targets in TNBC.

Abstract PO1-16-04: Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression

Abstract Purpose: Low molecular weight isoforms of cyclin E (LMW-E) have been implicated in various human cancers, including triple negative breast cancer (TNBC), and are associated with a poor prognosis. However, targeted therapies for TNBC based on biomarkers are currently lacking. This study aims to investigate LMW-E as a potential therapeutic target in TNBC and evaluate the efficacy of RP-6306, a selective inhibitor of the Protein Kinase, Membrane Associated Tyrosine/Threonine 1 (PKMYT1), in LMW-E-positive breast tumors. Experimental Design: Immunohistochemical (IHC) analysis was performed on pre-treatment tumor specimens from TNBC patients (n=36) to assess the correlation between LMW-E expression, CDK1 phosphorylation at Threonine 14 (pT14), and pathologic complete response to neoadjuvant chemotherapy. LMW-E inducible human mammary epithelial cells (hMEC) and breast cancer cell lines were used to investigate the regulatory effect of LMW-E on PKMYT1, the kinase responsible for CDK1 phosphorylation at T14, and the response to RP-6306, a first in-class and selective inhibitor of PKMYT1. Patient-derived xenograft (PDX) models and transgenic mouse mammary tumor virus (MMTV) models of TNBC expressing human LMW-E (hLMW-E) were also utilized to assess LMW-E as a biomarker for predicting response to RP-6306. Results. Analysis of TNBC tumor biopsies revealed a significant positive correlation between LMW-E expression and CDK1 pT14, and both biomarkers were associated with a lack of pathological complete response to neoadjuvant chemotherapy. In vitro results using LMW-E inducible hMECs and breast cancer cell lines demonstrated that LMW-E up-regulates PKMYT1 and CDK1 pT14, acting as a PKMYT1 binding protein and enhancing PKMYT1 protein stability. High LMW-E protein levels predicted a favorable response to RP-6306, resulting in the accumulation of sub-G1 and polyploid cells, decreased tolerance to replication stress, increased DNA damage, chromosomal breakage, and apoptosis. In vivo treatment of TNBC PDX models and hLMW-E transgenic tumors with RP-6306 resulted in a significant reduction in tumor volume only in mice harboring high LMW-E tumors, while low cyclin E models showed no response. Immunohistochemical analysis confirmed increased γ-H2AX and decreased CDK1-pT14 and Ki67 levels, indicating the efficacy of RP-6306 in both PDX and transgenic models. Conclusion: This study highlights the regulatory axis from LMW-E to PKMYT1 and its predictive value for pathological complete response in TNBC patients receiving neoadjuvant chemotherapy. The selective PKMYT1 kinase inhibitor RP-6306 consistently induced DNA damage and inhibited tumor growth in in vitro and in vivo pre-clinical breast tumor models. Co-expression of LMW-E and CDK1-pT14 in TNBC can be used to stratify patients whose tumors are likely to respond to RP-6306, emphasizing its therapeutic significance. Citation Format: Mi Li, Amriti Lulla, Cansu Karakas, Spiridon Tsavaschidis, Yan Wang, Tuyen Nguyun, Tuyen Bui, Gary Marshall, Kelly Hunt, Khandan Keyomarsi. Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression [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 PO1-16-04.

Abstract PO2-24-01: Kif11 Inhibition Preferentially Kills TP53-mutant Breast Cancer Cells

Abstract Background Triple-Negative Breast Cancer (TNBC) makes up 15-20% of breast cancer diagnoses and is more common in younger women, those with BRCA mutations, and Black patients. Furthermore, TNBCs are defined by their lack of expression of the targetable receptors: estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and progesterone receptor (PR), resulting in few targeted therapy options for TNBC patients. One potential therapeutic target in TNBC is mutant p53, since 85-90% of TNBCs harbor TP53 mutations. However, the mutant p53 protein is challenging to target directly. Therefore, we have sought to identify survival pathways critical to TP53 mutant cells. Through a combined in vitro and in silico drug screen, we identified that the Kif11 inhibitor SB-743921 differentially kills TP53 mutant breast cancer cells compared to TP53 wild-type cells. Hypothesis Based on this screening data, we hypothesized that Kif11 inhibition causes TP53 mutant cells to undergo mitotic catastrophe resulting in cell death due to failure of activation of p53-mediated cell cycle checkpoints. Methods Clinical data was obtained from cBioPortal from the TCGA and METABRIC cohorts. Expression data analysis and survival analyses were performed using GraphPad Prism. Cell growth assays were conducted by seeding cells in 96 well plates, treating under designated conditions, then staining with Hoechst 33342 for cell counting on the ImageXpress Pico. Cell death was determined by co-staining with Hoechst 33342 and DRAQ7 followed by imaging and analysis on the ImageXpress Pico and by Annexin V and Propidium Iodide Staining followed by flow cytometry analysis. Cell cycle analysis was conducted following synchronization with Lovastatin and Mevalonate release followed by collection of samples, fixation, PI staining, and flow cytometry analysis. Immunofluorescence imaging was conducted by staining with anti-alpha-tubulin-AlexaFluor488 and DAPI and imaging at 20x and 63x on the ImageXPress PICO. In vitro expression experiments were carried out through qPCR and western blotting. < Results In human breast cancers, KIF11 is more highly expressed in TP53 mutant, as compared to wild-type breast cancers, and in TNBCs, as compared to other breast cancer subtypes. Patients with tumors that had higher expression of Kif11 had poorer overall survival outcomes. Inhibition of Kif11 with the small molecule inhibitor SB-743921 induces greater death of TP53 mutant as compared to wild-type breast cancer cells. Kif11 inhibition leads to a cell cycle block in both TP53 mutant and wild-type cells, but TP53 mutant cells then die following this cell cycle block. Kif11 inhibition causes mitotic dysfunction, including monopolar spindle formation, in mutant and wild-type cells, but greater incidence of spindle abnormalities and multinucleated cells in TP53 mutants. Introduction of a TP53 mutation into TP53 wild-type cells also induces cell death following Kif11 inhibition. Conclusions The Kif11 mitotic kinesin is more highly expressed in TP53 mutant and triple-negative breast cancers than in TP53 wild-type breast cancers, and high expression is associated with poorer survival outcomes. In TP53 mutant cells, Kif11 inhibition results in mitotic dysfunction and cell death due to mitotic catastrophe. Acknowledgments Research reported in this publication was supported by the John Charles Cain Endowment and the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Numbers TL1TR003169 and UL1TR003167. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We would also like to thank the MDACC NORTH Campus Flow Cytometry and Cellular Imaging Core Facility for their assistance. Citation Format: Amanda Lanier, William Tahaney, Jing Qian, Cassandra Moyer, Yanxia Ma, Banu Arun, Abhijit Mazumdar, Powel Brown. Kif11 Inhibition Preferentially Kills TP53-mutant Breast Cancer Cells [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 PO2-24-01.

PTGER3 knockdown inhibits the vulnerability of triple-negative breast cancer to ferroptosis.

Prostaglandin E receptor 3 (PTGER3) is involved in a variety of biological processes in the human body and is closely associated with the development and progression of a variety of cancer types. However, the role of PTGER3 in triple-negative breast cancer (TNBC) remains unclear. In the present study, low PTGER3 expression was found to be associated with poor prognosis in TNBC patients. PTGER3 plays a crucial role in regulating TNBC cell invasion, migration, and proliferation. Upregulation of PTGER3 weakens the epithelial-mesenchymal phenotype in TNBC and promotes ferroptosis both invitro and invivo by repressing glutathione peroxidase 4 (GPX4) expression. On the other hand, downregulation of PTGER3 inhibits ferroptosis by increasing GPX4 expression and activating the PI3K-AKT pathway. Upregulation of PTGER3 also enhances the sensitivity of TNBC cells to paclitaxel. Overall, this study has elucidated critical pathways in which low PTGER3 expression protects TNBC cells from undergoing ferroptosis, thereby promoting its progression. PTGER3 may thus serve as a novel and promising biomarker and therapeutic target for TNBC.

Abstract 5697: MILIP drives triple-negative breast cancer through complexing with transfer RNAs to promote protein production

Abstract Despite the progress in endocrine therapy, targeted therapy and immunotherapy, the treatment of triple-negative breast cancer (TNBC), one of the most aggressive types of breast cancer, is challenging. This is closely associated with the lack of effective molecular targets in TNBC cells for therapeutic intervention. Here we report that the long noncoding RNA (lncRNA) MILIP drives TNBC cell survival, proliferation and tumorigenicity through complexing with transfer RNAs (tRNAs) to promote protein production, and thus represents a potential therapeutic target in TNBC. MILIP was expressed at high levels in TNBC cells that commonly harbor loss-of-function mutations of the tumor suppressor p53, yet MILIP silencing diminished TNBC cell viability and retarded TNBC xenograft growth, indicating that MILIP functions distinctively in TNBC beyond its well-established role in repressing p53 in other types of cancers. Mechanistical investigations revealed that MILIP interacted with eukaryotic translation elongation factor 1 alpha 1 (eEF1α1) and formed an RNA-RNA duplex with tRNALeu and tRNASer, type II tRNAs, through their variable loops, thus facilitating the binding of eEF1α1 to these tRNAs. Disrupting the interaction between MILIP and eEF1α1 or tRNAs diminished protein production and cell viability, with therapeutic potential revealed where targeting MILIP using Gapmers inhibited TNBC growth and cooperated with the clinically available protein synthesis inhibitor omacetaxine mepesuccinate in vivo. Collectively, these results identified MILIP as an RNA translation elongation factor to promote protein production in TNBC cells and demonstrated that experimental therapy with MILIP Gapmers inhibits TNBC growth, indicating MILIP targeting is a potential avenue for developing improved TNBC treatment through blocking protein synthesis. Citation Format: Yuchen Feng, Simin Zheng, Xiaohong Zhao, Yuanyuan Zhang, Ran Xu, Liang Xu, Thomas Preiss, Lei Jin, Jinnan Gao, Xu Dong Zhang. MILIP drives triple-negative breast cancer through complexing with transfer RNAs to promote protein production [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 5697.

Abstract 6800: The tumor progression mechanism of increasing CXCL16-CXCR6 pathway within the tumor microenvironment in TNBC

Abstract Background: Triple negative breast cancer (TNBC) is characterized rapid progression, higher relapse, and poor prognosis, so the establishment of an effective therapeutic target is required. CXCL16, a chemokine abundantly present in the TNBC tumor microenvironment (TME), is secreted by tumor-associated macrophages (TAM) and is known to promote tumor growth and metastasis. However, the exact mechanism through which CXCL16 facilitates tumor growth and metastasis remains unclear. This study aims to study the role of CXCL16 in the tumor progression and confirm the anti-tumor effects of CXCL16 neutralization in TNBC model. Method: MDA-MB-231 human TNBC cell lines were utilized for in vitro validation, and a syngeneic model involving 4T1 mouse TNBC cell lines and Balb/c mice was used for in vivo studies. Results: Stimulation of TNBC cells with recombinant human CXCL16 protein (rhCXCL16) activates the tumor growth signaling pathways, including pAKT and STAT3, as well as EMT markers. These results shows that CXCL16 can promote tumor growth and metastasis. Treatment with CXCL16 neutralizing antibodies inhibited the promotion of cancer growth and metastasis by rhCXCL16. Additionally, combination treatment of CXCL16 antibodies with PD-1 antibodies in the 4T1 syngeneic model increased the proportion of CD8+ T cells and CD80+ M1 like macrophages. These results shows that neutralization of CXCL16 can regulate polarization of macrophage and proportion of T cells. These regulation and changes of immune cells allow neutralization of CXCL16 control the TME to resist tumor cells, suggesting that optimal condition can be constructed to maximize the effectiveness of anti-cancer drugs. Conclusion: This study confirms that the CXCL16-CXCR6 pathway within the TME triggers tumor growth and metastasis, while neutralization of CXCL16 can inhibit tumor progression. This effect is attributed to the increase in CD8+ T cell and M1 macrophage proportions within the TME. In conclusion, these research findings propose the potential of CXCL16 as a novel biomarker and therapeutic target for TNBC. Citation Format: Yun sun Lee, Cham Han Lee, Seong Yun Ha, Sang Hyeob Lee, Jin Yi Yang, Young Sang Kim, Sung Moo Kim, Seong Keun Kim. The tumor progression mechanism of increasing CXCL16-CXCR6 pathway within the tumor microenvironment in TNBC [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 6800.

Abstract 573: SLC22A17 as a promising target for triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) represents a highly aggressive subtype of breast cancer, accounting for approximately 15% of all cases of invasive breast cancer globally. The absence of a well-defined cell membrane receptor has significantly limited the therapeutic options available for treating this malignancy. Consequently, there is a compelling need to explore novel receptors that could potentially revolutionize the treatment strategies employed for TNBC. Recent scientific research has unveiled SLC22A17, a cell surface receptor known for its involvement in iron transport, as a substantial contributor to the development of various cancer types. Thus, the primary objective of this study is to comprehensively evaluate the significance of SLC22A17 and its potential as a therapeutic target in TNBC. Experimental Procedures: In this study, we meticulously examined the expression patterns of SLC22A17 across distinct subtypes of breast cancer utilizing immunohistochemistry. Computational tools were employed to delve into the role of SLC22A17 within TNBC datasets. To assess the impact of SLC22A17 on TNBC, we employed various methods, including siRNA-mediated silencing, to investigate its role in cell proliferation, apoptosis, epithelial-mesenchymal transition (EMT), and migration. Additionally, we conducted western blot analyses to assess the expression levels of SLC22A17 and cellular signaling molecules, such as Akt/mTOR and JAK/STAT, within the context of TNBC cells. Results: Our analysis revealed a significant upregulation of SLC22A17 in both TNBC tissues and cell lines, establishing a direct correlation with patient survival outcomes. Silencing the expression of SLC22A17 resulted in reduced cell proliferation, migration, EMT, increased apoptosis, and enhanced autophagy. These effects were accompanied by the inhibition of Akt/mTOR and STAT3 signaling pathways. Moreover, we observed the involvement of the inflammatory cytokine TNF-α, which induced the expression of SLC22A17 in TNBC cell lines and governed various hallmark features of TNBC. Notably, the suppression of SLC22A17 mitigated these processes. Conclusion: In conclusion, this investigation suggests the pivotal role played by SLC22A17 in TNBC and posits its potential as a novel and promising therapeutic target for the management of this formidable disease. Citation Format: Ajaikumar B. Kunnumakkara, Krishan Kumar Thakur. SLC22A17 as a promising target for triple-negative breast cancer [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 573.

Abstract 1476: OSBPL3 facilitates endoplasmic reticulum stress and stemness in triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is a type of aggressive breast cancer with a poor survival. Endoplasmic reticulum (ER) stress, induced by exposed to intrinsic (e.g. oncogenes) and external factors (e.g. chemotherapies), is an important factor in both tumor progression and responses to chemotherapies. Recent studies have demonstrated that the progression of ER stress may result in the emergence of cancer stem cells (CSC), which also arise as a consequence of metastasis. However, the mechanism of targeting ER stress signaling in cancer stem cells remains to be investigated. Aim: (i) To investigate the mechanistic role of Oxysterol binding protein-like 3 (OSBPL3) in TNBC and its links with breast cancer mortality, particularly caused by chemotherapeutic resistance and metastasis; and (ii) to test a hypothesis that OSBPL3 facilitates the abnormal activation of ER stress and cancer stemness, result in the resistance to chemotherapy and metastasis in TNBC. Methods: The expression level of OSBPL3 in TNBC was determined by immunohistochemical staining and the mRNA expression profiles from the TCGA database. Roles of OSBPL3 in cancer cell growth, metastasis, stemness and activation of ER stress were determined by molecular and cell biology methods. Immunoprecipitation was used to examine the binding of proteins and chromatin immunoprecipitation (ChIP), promoter luciferase reporter assay was included for detecting the transcription of OSBPL3 in TNBC. TNBC xenograft nude mice were used to study the inhibition of tumor development. Results: OSBPL3 is a novel TNBC target, which is overexpressed in TNBC tissues and significantly associated with clinical pathology of advanced TNM stage and poor prognosis of tumor patients. Mechanistically, Increased OSBPL3 induced ER stress and ER stress agonist (tunicamycin) treatment results in the translocation of XBP1, an ER stress sensor, into the nucleus to induce OSBPL3 expression through direct binding to the OSBPL3 promoter. In addition, OSBPL3 facilitates the stemness induced by ER stress via phosphorylating GSK3β and transporting accumulated β-catenin to the nucleus to promote the expression of stemness-related transcriptional factors, ultimately resulting in tumor metastasis and resistance to chemotherapy. Furthermore, OSBPL3-specific siRNAs encapsulated by jetPEI nanocarriers prominently inhibit tumor growth and epirubicin-induced resistance in vivo. Conclusion: We identified an important role of OSBPL3-ER stress-XBP1 feedback loop in the stemness and resistance to chemotherapy, representing a future biomarker and therapeutic target of TNBC. Citation Format: Shengyu Pu, Huimin Zhang, Yu Ren, Jianjun He, Na Hao. OSBPL3 facilitates endoplasmic reticulum stress and stemness in triple negative breast cancer [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 1476.

Abstract 2912: SFRP2 and CD38 colocalize in triple negative breast cancer microenvironment

Abstract Introduction: A promising target for breast cancer is secreted frizzled-related protein 2 (SFRP2), and a humanized monoclonal antibody to SFRP2 (hSFRP2 mAb) inhibits primary triple negative breast cancer (TNBC) in vivo. CD38 is a known cause of PD-1 inhibitor resistance in some tumors, and in metastatic osteosarcoma hSFRP2 mAb overcomes PD-1 mAb resistance by reducing CD38 in tumor infiltrating lymphocytes (TILs). Could this phenomenon be replicated in TNBC? This is unknown as the expression of CD38 in TNBC has not been studied. This study will establish the presence of SFRP2 and CD38 in the TNBC microenvironment. Methods: Immunohistochemistry (IHC) was performed on an 88 core human TNBC tissue microarray (TMA) with antibodies to SFRP2 and CD38. Spatial analysis software was used to determine percent positivity. Each core was categorized using the scoring system for the estrogen receptor in breast cancer (<1% negative, 1-10% low positive, and >10% positive). Multiplex IHC was performed on 4 human triple TNBCs sections. Tissues were stained with antibodies to CD38, CD68 (macrophage), CD3 (T-cell), CD19 (B-cell), CytoKeratin (tumor), and SFRP2. Percent positivity of each marker was analyzed with a software package. For flow cytometry of tumor associated macrophages (TAMs), 1 million EO771 cells were injected into the mammary fat pad of female C57/BL6 mice. After 4 weeks tumors were extracted and a cell suspension was incubated in a mastermix with antibodies to SFRP2, CD38, PD1, CD11B and F480. Cells were fixed and conjugated to a fluorochrome secondary antibody. TAM gated cells were analyzed for the presence of SFRP2 and CD38. Results: For the TNBC TMA, 83 cores were positive and 4 cores were low positive for SFRP2, 1 not evaluable; 87 cores were positive and 1 low positive for CD38. For multiplex IHC there was a total of 1,753,544 individual cells in all 4 human TNBC tumors that were stained. 94% of cells stained positive for SFRP2, and 65% stained positive for CD38. Of the cells that were positive for SFRP2, 70% were also positive for CD38, 15% were positive for CD68, 1% were positive for CD19, 77% were positive for CytoKeratin, and 10% were positive for CD3. Of the cells that were CD38 positive, 99% were also positive for SFRP2, 15% were positive for CD68, 1% were positive for CD19, 81% were positive for CytoKeratin, and 9% were positive for CD3. Flow cytometry analysis of TAMs confirmed the presence of SFRP2, CD38 on TAMs. TAMs that were negative for PD1 and CD38 (pro-inflammatory) had a 56% reduction in SFRP2 compared to TAMs that were double positive for PD1 and CD38 (anti-inflammatory). Conclusion: SFRP2 and CD38 are prevalent in human TNBC and colocalize in the TNBC microenvironment in tumor cells, TAMs and TILs, but not B-cells. The presence and co-localization of SFRP2 and CD38 in TAMs was confirmed with flow cytometry. This confirms that SFRP2 is a therapeutic target in TNBC and provides a mechanistic rational for combination therapy with PD-1 inhibitor. Citation Format: Lillian Hsu, Patrick Nasarre, Rupak Mukherjee, Eleanor Hilliard, Martin Romeo, Elizabeth O'Quinn, Whitney Christians, Nancy Klauber DeMore. SFRP2 and CD38 colocalize in triple negative breast cancer microenvironment [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 2912.

Abstract 309: AHNAK nucleoprotein 2 tune oxidative stress and resistance to chemotherapy in triple negative breast cancer

Abstract Background: Resistance to chemotherapy is a major reason for the poorerprognosis in patients with triple negative breast cancer (TNBC). Growing evidence suggests that oxidative stress induced by reactive oxygen species (ROS) is an important factor in both tumourprogression and responses to chemotherapies, however, many signalling pathways and key molecular underlying the regulation of cancer cells to the ROS metabolism remains unclear. Aim: (i) To investigate the mechanistic role of AHNAK nucleoprotein 2 (AHNAK2) in TNBC and its links with breast cancer mortality, particularly caused by chemotherapeutic resistance; and (ii) to test a hypothesis that AHNAK2 facilitates the abnormal activation of ROSscavenging system, result in the resistance to chemotherapy in TNBC. Methods: The expression level of AHNAK2 was determined by immunohistochemical staining and analysed the mRNA expression profiles from The Cancer Genome Atlas of triple negative breast tumors. Roles of AHNAK2 in cancer cell growth, metastasis, chemotherapeutic resistanceand activation of oxygen scavenging systemwere determined by molecular and cell biology methods. A jetPEI nanocarrier was used as the vehicle for anti-AHNAK2 siRNAs in mouse models of cancer therapeutics. Results: AHNAK2 is a novel triple negative breast cancer target, which is overexpressed in TNBC tissues and significantly associated with clinical pathology of chemotherapeutic resistance and poor prognosis of tumour patients. Mechanistically, AHNAK2 antagonizes the ROS accumulation induced by epirubicinvia activating JAK-STAT signaling pathway and transportingphosphorylated STAT1 & STAT4 to the nucleus to promote the expression of SOD2, a key gene in ROS metabolism.And epirubicin enhanced AHNAK2 expression by induced the accumulation of ROS,which hence the Sp1-driven transcription of AHNAK2,result inde novo resistance. Furthermore, AHNAK2-specific siRNAs encapsulated by jetPEI nanocarriers prominently inhibit tumor growth and epirubicin-induced resistance in vivo. Conclusions: AHNAK2 plays a key role in resistance to chemotherapyby regulating the oxidative stressand ROS metabolism,representing a future biomarker and therapeutic target of TNBC. Citation Format: Na Hao. AHNAK nucleoprotein 2 tune oxidative stress and resistance to chemotherapy in triple negative breast cancer [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 309.

Bioinformatics analysis and clinical significance of NRP-1 in triple-negative breast cancer

PurposeThis study aimed to investigate the diagnostic and prognostic values of neuropilin-1 (NRP-1) in triple-negative breast cancer (TNBC) and analyze its immune function in the tumor microenvironment. MethodsBased on The Cancer Genome Atlas (TCGA), Gene Expression Omnibus, Genotype Tissue Expression, Immune Cell Abundance Identifier (ImmuCellAI), Reactome, and Genomics of Drug Sensitivity in Cancer databases, the cancer tissues from 50 patients with TNBC and corresponding adjacent noncancerous tissues from 10 patients (tissue microarrays were purchased from Shanghai Xinchao Biotechnology Co., Ltd.) were collected for validation. Bioinformatics combined with immunohistochemistry was used to analyze the relationship among NRP-1 expression, prognosis, tumor immune cell infiltration, immune genes, and drug resistance so as to investigate the role of NRP-1 in the development of TNBC. ResultsA significant difference in NRP-1 gene expression was found between the cancerous and noncancerous tissues (p-value < 0.05); NRP-1 expression was high in carcinoma. No significant correlation was found between NRP-1 protein expression levels and each stage in the TCGA database. Prognostic expression survival analysis showed that the survival probability of patients with high NRP-1 expression was significantly lower than that of patients with low NRP-1 expression (p-value < 0.05), suggesting that the gene might be a pro-oncogene. The data from 50 clinical samples also confirmed that the NRP-1 expression was significantly higher in triple-negative breast cancer (TNBC) tissues than in adjacent noncancerous tissues. The NRP-1 expression significantly correlated with the tumor diameter and pathological grade (p-value < 0.05), but not with age, stage, and ki67 (p-value > 0.05). The Kaplan–Meier survival curves suggested that the median overall survival was significantly shorter in patients with high NRP-1 expression than in those with low NRP-1 expression (13.6 months vs 15.2 months, p-value < 0.05). The 300 genes most significantly positively associated with this gene were selected for Gene Ontology (including Biological Process, Molecular Function, and Cellular Component groups) and Kyoto Encyclopedia of Genes and Genomics enrichment analysis. The findings showed that NRP-1 was involved in immune regulation in TNBC. In addition, the NRP-1 expression in TNBC positively correlated with a variety of immune cells and checkpoints. ConclusionNRP-1 can be used as a potential biomarker and therapeutic target in TNBC.

Identification of biomimetic nanoplatform-mediated delivery of si-ISG15 for treatment of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is recognized as the most malicious form of breast cancer and exhibits an alarming tendency for recurrence, a heightened propensity for metastasis, and an overwhelmingly grim prognosis. Therefore, effective therapy approaches for TNBC are urgently required. In this study, the interferon-stimulated gene 15 (ISG15) expression level was analyzed by bioinformatics and verified by Western blot analysis. The effects of ISG15 on the proliferation and metastasis of TNBC cells were assessed using MTT, Colony formation, EdU, Transwell, and Flow cytometry assays. We also developed a cancer cell-biomimetic nanoparticle delivery system and evaluated its therapeutic efficacy in vivo. In this study, we reported that ISG15 was upregulated in TNBC, and its high expression level correlated with an increased risk of tumorigenesis. Through in vitro and in vivo studies, we discovered that ISG15 knockdown drastically suppressed cell proliferation, invasion, and migration and induced apoptosis in TNBC cells. Our findings revealed that ISG15 was a candidate therapeutic target in TNBC because of its key role in malignant growth and invasion. Moreover, co-immunoprecipitation showed that ISG15 exerted oncogenic functions through its interaction with ATP binding cassette subfamily E member 1 and activated the Janus kinase/signal transducers and activators of the transcription signaling pathway. Furthermore, we created a nanoparticle-based siRNA camouflaged using a cancer cell membrane vesicle delivery system (the CM@NP complex) and confirmed its therapeutic effects in vivo. Our findings confirmed that ISG15 may play a pivotal oncogenic role in the development of TNBC and that CM@siRNA-NP complexes are an effective delivery system and a novel biological strategy for treating TNBC.

A MAP1B-cortactin-Tks5 axis regulates TNBC invasion and tumorigenesis.

The microtubule-associated protein MAP1B has been implicated in axonal growth and brain development. We found that MAP1B is highly expressed in the most aggressive and deadliest breast cancer subtype, triple-negative breast cancer (TNBC), but not in other subtypes. Expression of MAP1B was found to be highly correlated with poor prognosis. Depletion of MAP1B in TNBC cells impairs cell migration and invasion concomitant with a defect in tumorigenesis. We found that MAP1B interacts with key components for invadopodia formation, cortactin, and Tks5, the latter of which is a PtdIns(3,4)P2-binding and scaffold protein that localizes to invadopodia. We also found that Tks5 associates with microtubules and supports the association between MAP1B and α-tubulin. In accordance with their interaction, depletion of MAP1B leads to Tks5 destabilization, leading to its degradation via the autophagic pathway. Collectively, these findings suggest that MAP1B is a convergence point of the cytoskeleton to promote malignancy in TNBC and thereby a potential diagnostic and therapeutic target for TNBC.

Head-to-Head Comparison of [18F]PSMA-1007 and [18F]FDG PET/CT in Patients with Triple-Negative Breast Cancer

Background: Triple-negative breast cancer (TNBC) exhibits high aggressiveness and a notably poorer prognosis at advanced stages. Nuclear medicine offers new possibilities, not only for diagnosis but also potentially promising therapeutic strategies. This prospective study explores the potential of prostate-specific membrane antigen (PSMA) as a diagnostic and therapeutic target in TNBC. Methods: the research investigates PSMA expression in vivo among TNBC patients using [18F]PSMA-1007 PET/CT and compares it head-to-head with the standard-of-care [18F]FDG PET/CT. Results: The study involves 10 TNBC patients, revealing comparable uptake of [18F]PSMA-1007 and [18F]FDG in primary and metastatic lesions. Nodal metastases were found in eight patients, showing similar SUVmax values in both modalities. Two patients had uncountable lung metastases positive in both [18F]FDG and [18F]PSMA-1007 scans. PET-positive bone metastases were identified by 18F-PSMA in four patients, while elevated [18F]FDG uptake was found only in three of them. Distant metastases displayed higher SUVmax values in the [18F]PSMA-1007 PET/CT, as compared to [18F]FDG. Additionally, brain metastases were exclusively detected using [18F]PSMA-1007. Conclusions: the findings provide valuable insights into the expression of PSMA in TNBC and underscore the potential clinical significance of [18F]PSMA-1007 PET/CT in enhancing both diagnostic and therapeutic approaches for this aggressive breast cancer subtype.

Novel exosomal circEGFR facilitates triple negative breast cancer autophagy via promoting TFEB nuclear trafficking and modulating miR-224-5p/ATG13/ULK1 feedback loop

Triple-negative breast cancer (TNBC) cells are in a more hypoxic and starved state than non-TNBC cells, which makes TNBC cells always maintain high autophagy levels. Emerging evidence has demonstrated that circular RNAs (circRNAs) are involved in the progress of tumorigenesis. However, the regulation and functions of autophagy-induced circRNAs in TNBC remain unclear. In our study, autophagy-responsive circRNA candidates in TNBC cells under amino acid starved were identified by RNA sequencing. The results showed that circEGFR expression was significantly upregulated in autophagic cells. Knockdown of circEGFR inhibited autophagy in TNBC cells, and circEGFR derived from exosomes induced autophagy in recipient cells in the tumor microenvironment. In vitro and in vivo functional assays identified circEGFR as an oncogenic circRNA in TNBC. Clinically, circEGFR was significantly upregulated in TNBC and was positively associated with lymph node metastasis. CircEGFR in plasma-derived exosomes was upregulated in breast cancer patients compared with healthy people. Mechanistically, circEGFR facilitated the translocation of Annexin A2 (ANXA2) toward the plasma membrane in TNBC cells, which led to the release of Transcription Factor EB (a transcription factor of autophagy-related proteins, TFEB) from ANXA2-TFEB complex, causing nuclear translocation of TFEB, thereby promoting autophagy in TNBC cells. Meanwhile, circEGFR acted as ceRNA by directly binding to miR-224-5p and inhibited the expression of miR-224-5p, which weakened the suppressive role of miR-224-5p/ATG13/ULK1 axis on autophagy. Overall, our study demonstrates the key role of circEGFR in autophagy, malignant progression, and metastasis of TNBC. These indicate circEGFR is a potential diagnosis biomarker and therapeutic target for TNBC.

The use of MFAP2 for diagnosis, prognosis and immunotherapy of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is characterized by significant heterogeneity, presenting a formidable challenge with a poor prognosis and a deficiency of efficacious treatment options. In this comprehensive study, we investigated the multifaceted role of Microfibril-associated glycoprotein 2 (MFAP2) in TNBC using a combination of bioinformatics analysis involving Gene Expression Omnibus (GEO), OncoDB, UALCAN, Human Protein Atlas (HPA), TIMER, STRING, DAVID, and GSCA databases and in vitro experiments, such as cell culture, MFAP2 gene knockdown, RT-qPCR, western Blot, colony formation, Cell counting kit-8, and wound healing assays. Our findings demonstrated a significant up-regulation of MFAP2 mRNA in TNBC cell lines, emphasizing its potential as a diagnostic biomarker. Validation across multiple datasets further affirmed the elevated expression of MFAP2 in TNBC tissues, underscoring its prognostic relevance. Notably, our study revealed a correlation between MFAP2 expression and immune cell infiltration, suggesting its role in shaping the tumor microenvironment. STRING analysis unveiled interactions with proteins involved in elastic fibers and extracellular matrix constituents. Furthermore, KEGG pathway analysis highlighted enrichment in the TGF-beta signaling pathway, implicating MFAP2 in key cancer-related processes. Drug sensitivity analysis identified potential therapeutic targets, supporting MFAP2's utility in personalized treatment strategies. In vitro experiments corroborated the oncogenic impact of MFAP2, demonstrating its influence on TNBC cell proliferation and migration. These comprehensive findings position MFAP2 as a promising biomarker and therapeutic target in TNBC, offering valuable insight for future research and clinical application.