Triple-negative Breast Cancer Metastasis Research Articles

Exploring the prognostic significance of arm-level copy number alterations in triple-negative breast cancer.

Somatic copy number alterations (SCNAs) are prevalent in cancer and play a significant role in both tumorigenesis and therapeutic resistance. While focal SCNAs have been extensively studied, the impact of larger arm-level SCNAs remains poorly understood. Here, we investigated the association between arm-level SCNAs and overall survival in triple-negative breast cancer (TNBC), an aggressive subtype of breast cancer lacking targeted therapies. We identified frequent arm-level SCNAs, including 21q gain and 7p gain, which correlated with poor overall survival in TNBC patients. Further, we identified the expression of specific genes within these SCNAs associated with survival. Notably, we found that the expression of RIPK4, a gene located on 21q, exhibited a strong correlation with poor overall survival. In functional assays, we demonstrated that targeting Ripk4 in a murine lung metastatic TNBC model significantly reduced tumor burden, improved survival, and increased CD4+ and CD8+ T cell infiltration. RIPK4 enhanced the survival of triple-negative breast cancer cells at secondary sites, thereby facilitating the formation of metastatic lesions. Our findings highlight the significance of arm-level SCNAs in breast cancer progression and identify RIPK4 as a putative driver of TNBC metastasis and immunosuppression.

Shikonin and chitosan-silver nanoparticles synergize against triple-negative breast cancer through RIPK3-triggered necroptotic immunogenic cell death

Necroptotic immunogenic cell death (ICD) can activate the human immune system to treat the metastasis and recurrence of triple-negative breast cancer (TNBC). However, developing the necroptotic inducer and precisely delivering it to the tumor site is the key issue. Herein, we reported that the combination of shikonin (SHK) and chitosan silver nanoparticles (Chi-Ag NPs) effectively induced ICD by triggering necroptosis in 4T1 cells. Moreover, to address the lack of selectivity of drugs for in vivo application, we developed an MUC1 aptamer-targeted nanocomplex (MUC1@Chi-Ag@CPB@SHK, abbreviated as MUC1@ACS) for co-delivering SHK and Chi-Ag NPs. The accumulation of MUC1@ACS NPs at the tumor site showed a 6.02-fold increase compared to the free drug. Subsequently, upon reaching the tumor site, the acid-responsive release of SHK and Chi-Ag NPs from MUC1@ACS NPs cooperatively induced necroptosis in tumor cells by upregulating the expression of RIPK3, p-RIPK3, and tetrameric MLKL, thereby effectively triggering ICD. The sequential maturation of dendritic cells (DCs) subsequently enhanced the infiltration of CD8+ and CD4+ T cells in tumors, while inhibiting regulatory T cells (Treg cells), resulting in the effective treatment of primary and distal tumor growth and the inhibition of TNBC metastasis. This work highlights the importance of nanoparticles in mediating drug interactions during necroptotic ICD.

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.

A novel long non-coding RNA MIR4500HG003 promotes tumor metastasis through miR-483-3p-MMP9 axis in triple-negative breast cancer

Breast cancer (BC) is the most common cancer and the leading cause of cancer-related deaths in women worldwide. The 5-year survival rate is over 90% in BC patients, but once BC cells metastasis into distal organs, it is dramatically decreasing to less than 30%. Especially, triple-negative breast cancer (TNBC) patients usually lead to poor prognosis and survival because of metastasis. Understanding the underline mechanisms of TNBC metastasis is a critical issue. Non-coding RNAs, including of lncRNAs and microRNAs, are non-protein-coding transcripts and have been reported as important regulators in TNBC metastasis. However, the underline mechanisms for non-coding RNAs regulating TNBC metastasis remain largely unclear. Here, we found that lncRNA MIR4500HG003 was highly expressed in highly metastatic MDA-MB-231 TNBC cells and overexpression of MIR4500HG003 enhanced metastasis ability in vitro and in vivo and promoted MMP9 expression. Furthermore, we found MIR4500HG003 physically interacted with miR-483-3p and reporter assay showed miR-483-3p attenuated MMP9 expression. Importantly, endogenous high expressions of MIR4500HG003 were correlated with tumor recurrence in TNBC patients with tumor metastasis. Taken together, our findings suggested that MIR4500HG003 promotes metastasis of TNBC through miR-483-3p-MMP9 signaling axis and may be used as potential prognostic marker for TNBC patients.

Abstract PO2-06-14: Kynurenine induces senescence reprogramming to promote metastasis in young triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC), which has been associated with a high risk of relapse and poor prognosis, has an increasing incidence among young women and ongoing inferior long-term outcomes. Given TNBC arising at a young age are more likely to present at advanced stages and to have aggressive biology, it is necessary to investigate the underlying mechanism and develop multidisciplinary strategies for optimal therapy of young women with breast cancer. Here, we integrated the multiomics data of our large TNBC cohort (n=456) to investigate different molecular mechanism between younger and older patients in the context of genetic and transcriptional subtypes and immune cell infiltration. We discovered that young TNBC patients exhibited a poorer outcome of both 2-year relapse-free survival and 2-year distant disease-free survival. Multi-omics analysis revealed that kynurenine (KYN) was preferentially enriched in young TNBC and activated tumor cellular senescence pathway which was positively correlated with increased metastasis. Mechanistically, KYN activated the aryl hydrocarbon receptor (AHR) signaling pathway in an ARNT2-dependent manner which was critical for cellular senescence, to mediate senescence reprogramming and promote tumor metastasis in young TNBC. Our findings unveil a novel interplay between cancer cell metabolites and cell senescence, which provides valuable insights that can potentially enhance therapeutic strategies for young TNBC patients. Keywords: young TNBC, metastasis, kynurenine, cellular senescence, AHR pathway Citation Format: Cui-Cui Liu, Ke-da Yu. Kynurenine induces senescence reprogramming to promote metastasis in young 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 PO2-06-14.

Abstract PO5-06-07: Unravelling the role of PKC-eta in modulating the Hippo Pathway: a novel therapeutic strategy for triple-negative breast cancer metastasis

Abstract Breast cancer (BC) is the leading cause of cancer-related deaths in women, with metastasis being the primary reason for BC mortality. Triple-Negative Breast Cancer (TNBC) is aggressive and highly metastatic, having fewer treatment options than other types of breast cancer. Protein kinase C eta (PKCη), an anti-apoptotic kinase of the novel PKC subfamily, is associated with poor prognosis in invasive BC patients. In this study, we show the role of PKCη in promoting cell migration and invasion, and we demonstrate that its expression is required to maintain the mesenchymal state during EMT. Furthermore, we show that PKCη activates YAP/TAZ, tumor aggressiveness, and metastasis using in vitro and in vivo models. PKCη promotes metastasis in TNBC cells, showing that the PKCƞ-YAP signaling axis mediates this. Data analysis of BC patients with a high metastatic invasive propensity and low survival BC revealed a positive correlation between the elevated expression levels of PKCη and the expression of YAP. Knockout of PKCη (PKCηKO) in the TNBC cells 4T1 and MDA-MB-231 markedly inhibited their invasion and migration capability. We further show that PKCη enhances cell survival and anoikis prevention ability, which have crucial roles in metastasis formation, allowing cancer cells to circulate and home to distant sites in the body. Further substantiated by in vivo experiments with 4T1 xenografts in NSG mice, demonstrating that 4T1 PKCηKO cells depicted reduced primary tumor size and a significant decrease in size and number of tumor metastases in the lungs, with fewer tumor nodules. MDA-MB-231 PKCηKO xenografts also expressed reduced metastasis in the distant brain, lung, and liver organs. Mechanistically, we show that PKCη regulates epithelial-to-mesenchymal transition (EMT) markers as knockout of PKCη in TNBC cell lines increased expression of E-cadherin, EpCAM, and slug, and decreased expression of vimentin and ZEB1. Further profiling of the Hippo-YAP axis showed that PKCη is a negative regulator of the Hippo pathway that leads to YAP stabilization and is associated with phosphorylation at Ser128, which allows YAP to translocate to the nucleus and contributes to metastasis of TNBC cells. Moreover, our data support direct interaction between PKCη and YAP1 in TNBC cells. Finally, we show that treatment of TNBC cells with uPEP2 (a uORF-encoded peptide of the PKCη transcript) downregulates the PKCη expression, activates the Hippo signaling pathway and promotes YAP degradation. Our findings highlight the importance of PKCη in TNBC metastasis and provide a new avenue for therapeutic intervention in this aggressive and lethal disease. In conclusion, our studies identify a novel role of PKCη as a negative regulator of the Hippo pathway and reveal its function in promoting EMT and metastasis by modulating YAP/TAZ activity in TNBC. Results suggest that PKCη may represent a therapeutic target for this highly lethal and metastatic TNBC. Citation Format: Liju Vijaya Steltar Belsamma, Amitha Muraleedharan, Divya Ram Jayaram, Kamran Waidha, Sankar Jagadeeshan, Rose Sinay, Ekaterina Eremenko, Omer Berner, Moshe Elkabets, Etta Livneh. Unravelling the role of PKC-eta in modulating the Hippo Pathway: a novel therapeutic strategy for triple-negative breast cancer metastasis [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 PO5-06-07.

LIFU/MMP-2 dual-responsive release of repurposed drug disulfiram from nanodroplets for inhibiting vasculogenic mimicry and lung metastasis in triple-negative breast cancer

BackgroundVasculogenic mimicry (VM), when microvascular channels are formed by cancer cells independent of endothelial cells, often occurs in deep hypoxic areas of tumors and contributes to the aggressiveness and metastasis of triple-negative breast cancer (TNBC) cells. However, well-developed VM inhibitors exhibit inadequate efficacy due to their low drug utilization rate and limited deep penetration. Thus, a cost-effective VM inhibition strategy needs to be designed for TNBC treatment.ResultsHerein, we designed a low-intensity focused ultrasound (LIFU) and matrix metalloproteinase-2 (MMP-2) dual-responsive nanoplatform termed PFP@PDM-PEG for the cost-effective and efficient utilization of the drug disulfiram (DSF) as a VM inhibitor. The PFP@PDM-PEG nanodroplets effectively penetrated tumors and exhibited substantial accumulation facilitated by PEG deshielding in a LIFU-mediated and MMP-2-sensitive manner. Furthermore, upon exposure to LIFU irradiation, DSF was released controllably under ultrasound imaging guidance. This secure and controllable dual-response DSF delivery platform reduced VM formation by inhibiting COL1/pro-MMP-2 activity, thereby significantly inhibiting tumor progression and metastasis.ConclusionsConsidering the safety of the raw materials, controlled treatment process, and reliable repurposing of DSF, this dual-responsive nanoplatform represents a novel and effective VM-based therapeutic strategy for TNBC in clinical settings.Graphical

A Comprehensive Strategy Based on High Clinical Translational Nanosystem for Programmable Immunotherapy of Triple Negative Breast Cancer.

Triple negative breast cancer (TNBCs), known as an immunologically cold tumor, is difficult to completely eliminate with existing monotherapies, let alone metastasis and recurrence. It is urgent to design a rational combination of multiple therapies to programmatically reconstitute tumor microenvironment (TME) and reverse the immune "cold" into "hot" inflammatory tumors to improve the therapeutic effect. Hence, in this work, a multifunctional nanosystem (FeSH NPs) that integrates metal-polyphenol coordination complex as a photothermal agent and polyphenol, salvianolic acid B (SAB) as immunomodulator is designed and fabricated for synergistic photothermal-immunotherapy of TNBCs combined with anti-PD-L1 antibody. Guided by photothermal/photoacoustic dual-mode imaging, photothermal therapy (PTT) caused by FeSH NPs induces immunogenic cell death (ICD) under 808nm laser irradiation. Subsequently, the loaded SAB is released with the addition of deferoxamine mesylate (DFO) to remodel TME, specifically TGF-β inhibition and PD-L1 upregulation, and eliminate the primary tumors. The combination of PTT and TME reprogramming by FeSH NPs further synergizes with anti-PD-L1 antibody to eradicate recurrence and inhibit metastasis of TNBCs concurrently. Given the biosafety of FeSH NPs throughout the lifecycle, this work provides a protocol with high clinical translational promise for comprehensive programmed therapeutics of immunologically cold tumors TNBCs.

The therapeutic potential of circular RNA in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is among the most aggressive subtypes of the disease that does not express estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Circular RNAs (circRNAs) are a type of non-coding RNA with a circular shape formed by non-standard splicing or reverse splicing. Numerous circRNAs exhibit abnormal expression in various malignancies, showing their critical role in the emergence and growth of tumors. Recent studies have shown evidence supporting the idea that certain circRNAs regulate the proliferation and metastasis of TNBC. In addition, circRNAs alter metabolism and the immune microenvironment to promote or inhibit the development of TNBC. Notably, circRNAs may affect the efficacy of clinical drug therapy, serve as therapeutic targets, and be used as molecular biomarkers in the future. Herein, we will first summarize the biogenesis and function of circRNAs. Then, we will explain current research on circRNAs related to TNBC and their potential to serve as therapeutic targets or biomarkers for future drug development, providing a new direction and idea for TNBC therapy.

Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling

BackgroundTriple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and chemotherapy still serves as the cornerstone treatment functioning by inducing cytotoxic cell death. Notably, emerging evidence suggests that dying cell-released signals may induce cancer progression and metastasis by modulating the surrounding microenvironment. However, the underlying molecular mechanisms and targeting strategies are yet to be explored.MethodsApoptotic TNBC cells induced by paclitaxel or adriamycin treatment were sorted and their released extracellular vesicles (EV-dead) were isolated from the cell supernatants. Chemokine array analysis was conducted to identify the crucial molecules in EV-dead. Zebrafish and mouse xenograft models were used to investigate the effect of EV-dead on TNBC progression in vivo.ResultsIt was demonstrated that EV-dead were phagocytized by macrophages and induced TNBC metastasis by promoting the infiltration of immunosuppressive PD-L1+ TAMs. Chemokine array identified CXCL1 as a crucial component in EV-dead to activate TAM/PD-L1 signaling. CXCL1 knockdown in EV-dead or macrophage depletion significantly inhibited EV-dead-induced TNBC growth and metastasis. Mechanistic investigations revealed that CXCL1EV-dead enhanced TAM/PD-L1 signaling by transcriptionally activating EED-mediated PD-L1 promoter activity. More importantly, TPCA-1 (2-[(aminocarbonyl) amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide) was screened as a promising inhibitor targeting CXCL1 signals in EVs to enhance paclitaxel chemosensitivity and limit TNBC metastasis without noticeable toxicities.ConclusionsOur results highlight CXCL1EV-dead as a novel dying cell-released signal and provide TPCA-1 as a targeting candidate to improve TNBC prognosis.Graphical abstract

CK2α-mediated phosphorylation of GRP94 facilitates the metastatic cascade in triple-negative breast cancer

Distant metastasis is a significant hallmark affecting to the high death rate of patients with triple-negative breast cancer (TNBC). Thus, it is crucial to identify and develop new therapeutic strategies to hinder cancer metastasis. While emerging studies have hinted a pivotal role of glucose-regulated protein 94 (GRP94) in tumorigenesis, the exact biological functions and molecular mechanisms of GRP94 in modulating cancer metastasis remain to be elucidated. Our study demonstrated an increased expression of GRP94 in TNBC correlated with metastatic progression and unfavorable prognosis in patients. Functionally, we identified that GRP94 depletion significantly diminished TNBC tumorigenesis and subsequent lung metastasis. In contrast, GRP94 overexpression exacerbated the invasiveness, migration, and lung metastasis of non-TNBC cells. Mechanistically, we found that casein kinase 2 alpha (CK2α) active in advanced breast cancer phosphorylated GRP94 at a conserved serine 306 (S306) residue. This phosphorylation increased the stability of GRP94 and enhanced its interaction with LRP6, leading to activation of canonical Wnt signaling. From a therapeutic standpoint, we found that benzamidine, a novel CK2α inhibitor, effectively suppressed GRP94 phosphorylation, LRP6 stabilization, and metastasis of TNBC. Our results point to the critical role of CK2α-mediated GRP94 phosphorylation in TNBC metastasis through activation of Wnt signaling, highlighting GRP94 as a therapeutic target to impede TNBC metastasis.

Taurine Inhibits Lung Metastasis in Triple-Negative Breast Cancer by Modulating Macrophage Polarization Through PTEN-PI3K/Akt/mTOR Pathway.

Taurine (Tau) has been found to inhibit triple-negative breast cancer (TNBC) invasion and metastasis. However, its effect on tumor-promoting macrophages and tumor suppressor macrophages in breast cancer progression remains unknown. In this study, we investigated the effects of Tau on macrophage polarization and its role in TNBC cell growth, invasion, and metastasis. We induced human THP-1 monocytes to differentiate into M2 macrophages through exogenous addition of interleukin-4. We used the TNBC cell lines MDA-MB-231 and BT-549 cultured in a conditioned medium from M2 macrophages to investigate the effect of Tau on tumor growth and invasion. We analyzed macrophage subset distribution, M1 and M2 macrophage-associated markers, and mRNA expression by quantitative polymerase chain reaction. We also detected the PTEN-PI3K/Akt/mTOR signaling pathway that mediates M1 macrophage to suppress tumor invasion using western blotting. Our results showed that Tau inhibits breast cancer metastasis to the lungs in vivo and cell invasion by altering the polarization of tumor-associated macrophage in vitro. In addition, Tau can up-regulate PTEN expression, suppress the PI3K-Akt signaling pathway, and promote the M1 polarization of macrophages, which ultimately inhibits the metastasis of TNBC cells. Our findings suggest that Tau inhibits the activation of the PI3K-Akt-mTOR signaling pathway by up-regulating PTEN, promotes the proportion of M1 macrophages in tumor-associated macrophage, and suppresses the invasion and metastasis of TNBC. This provides a potential therapeutic approach to influence cancer progression and metastasis.

Ultrabright quantum dots assisted in vivo NIR-II fluorescence microscopic imaging for brain metastases in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive and often fatal disease, especially since the brain metastasis of TNBC has been a particularly severe manifestation. However, brain metastasis in TNBC at early stages often lacks noticeable symptoms, making it challenging to detect. Near-infrared II (NIR-II) fluorescence microscopic imaging obtains long wavelength, which enables reduced scattering, high spatial resolution and minimal autofluorescence, it is also a favorable imaging method for tumor diagnosis. PbS@CdS quantum dots (QDs) are one of the popular NIR-II fluorescence nanoprobes for well brightness. In this study, NIR-II emissive PbS@CdS QDs were utilized and further encapsulated with thiol-terminated poly(ethylene oxide) (SH-PEG, MW = 5000) to form PbS@CdS@PEG QDs nanoparticles (NPs). The obtained PbS@CdS@PEG QDs NPs were then characterized and further studied in detail. The PbS@CdS@PEG QDs NPs had large absorption spectra, exhibited strong NIR-II fluorescence emission at approximately 1300[Formula: see text]nm, and possessed good NIR-II fluorescence properties. Then, the mice model of early-stage brain metastases of TNBC was established, and the PbS@CdS@PEG QDs NPs were injected into the tumor-bearing mice for NIR-II fluorescence microscopic bioimaging. The brain vessels and tumors of the living mice were detected with high spatial resolution under the NIR-II fluorescence microscopic imaging system with irradiation of 808[Formula: see text]nm laser. The tumor tissues were further restricted and prepared as thin slices. The NIR-II fluorescence signals were collected from the tumor slices with high spatial resolution and signal-to-background ratio (SBR). Thus, the PbS@CdS@PEG QDs NPs-assisted NIR-II fluorescence microscopic system can effectively achieve targeting brain metastases of TNBC imaging, offering a novel and promising approach for TNBC-specific diagnosis.

Abstract SY34-01: Plasticity, dormancy, and glycosylation of circulating tumor cell clusters in response to therapy

Abstract Cellular plasticity and stemness properties enable dynamic changes of circulating tumor cells (CTCs) during cancer dissemination, such as aggregation or cohesion of single CTCs into multicellular CTC clusters with 20-100 times higher metastatic propensity than the singles. Our work demonstrated that stemness glycoproteins on breast cancer stem cells, such as CD44, CD81, and ICAM1 drive CTC aggregation in metastatic breast cancer, such as triple negative breast cancer (TNBC). To determine the glycosylation patterns in CTCs and its association with clinical outcomes, we established multiple CTC analysis approaches including CellSearch, flow cytometry, and immunohistochemistry using patient blood and tissue sections collected at the pre-treatment baseline and after-therapy timepoints. We found that chemotherapy-evasive CTC clusters are relatively quiescent with a specific loss of terminal sugar residues α2,6-sialic acids in glycoproteins. CTCs showed dynamic hypo-sialylation in the blood with loss of the sialyl-transferase ST6GAL1, promoting CTC cluster formation with cellular quiescence (proliferative dormancy) and evading chemotherapy in breast cancer. Seeded tumor cells regained ST6GAL1 to enable metastatic colonization. Many adhesion proteins and stemness drivers as glycoprotein substrates of ST6GAL1 drive CTC clustering and metastatic seeding. Neutralizing antibodies against these clustering drivers inhibit CTC cluster formation and improve therapy response, thereby blocking lung metastasis in TNBC. Citation Format: Huiping Liu. Plasticity, dormancy, and glycosylation of circulating tumor cell clusters in response to therapy [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 SY34-01.

Oncolytic adenovirus encoding apolipoprotein A1 suppresses metastasis of triple-negative breast cancer in mice

BackgroundDysregulation of cholesterol metabolism is associated with the metastasis of triple-negative breast cancer (TNBC). Apolipoprotein A1 (ApoA1) is widely recognized for its pivotal role in regulating cholesterol efflux and maintaining cellular cholesterol homeostasis. However, further exploration is needed to determine whether it inhibits TNBC metastasis by affecting cholesterol metabolism. Additionally, it is necessary to investigate whether ApoA1-based oncolytic virus therapy can be used to treat TNBC.MethodsIn vitro experiments and mouse breast cancer models were utilized to evaluate the molecular mechanism of ApoA1 in regulating cholesterol efflux and inhibiting breast cancer progression and metastasis. The gene encoding ApoA1 was inserted into the adenovirus genome to construct a recombinant adenovirus (ADV-ApoA1). Subsequently, the efficacy of ADV-ApoA1 in inhibiting the growth and metastasis of TNBC was evaluated in several mouse models, including orthotopic breast cancer, spontaneous breast cancer, and human xenografts. In addition, a comprehensive safety assessment of Syrian hamsters and rhesus monkeys injected with oncolytic adenovirus was conducted.ResultsThis study found that dysregulation of cholesterol homeostasis is critical for the progression and metastasis of TNBC. In a mouse orthotopic model of TNBC, a high-cholesterol diet promoted lung and liver metastasis, which was associated with keratin 14 (KRT14), a protein responsible for TNBC metastasis. Furthermore, studies have shown that ApoA1, a cholesterol reverse transporter, inhibits TNBC metastasis by regulating the cholesterol/IKBKB/FOXO3a/KRT14 axis. Moreover, ADV-ApoA1 was found to promote cholesterol efflux, inhibit tumor growth, reduce lung metastasis, and prolonged the survival of mice with TNBC. Importantly, high doses of ADV-ApoA1 administered intravenously and subcutaneously were well tolerated in rhesus monkeys and Syrian hamsters.ConclusionsThis study provides a promising oncolytic virus treatment strategy for TNBC based on targeting dysregulated cholesterol metabolism. It also establishes a basis for subsequent clinical trials of ADV-ApoA1 in the treatment of TNBC.

17β-Estradiol promotes metastasis in triple-negative breast cancer through the Calpain/YAP/β-catenin signaling axis.

β-catenin is an important regulator of malignant progression. 17β-Estradiol (E2), an important sex hormone in women, promotes the growth and metastasis of triple-negative breast cancer (TNBC). However, whether β-catenin is involved in E2-induced metastasis of TNBC remains unknown. In this study, we show that E2 induces the proliferation, migration, invasion, and metastasis of TNBC cells. E2 induces β-catenin protein expression and nuclear translocation, thereby regulating the expression of target genes such as Cyclin D1 and MMP-9. The inhibition of β-catenin reversed the E2-induced cell malignant behaviors. Additionally, E2 activated Calpain by increasing intracellular Ca2+ levels and reducing calpastatin levels. When Calpain was inhibited, E2 did not induce the proliferation, migration, invasion, or metastasis of TNBC cells. In addition, E2 promoted translocation of YAP into the nucleus by inhibiting its phosphorylation. Calpain inhibition reversed the E2-induced YAP dephosphorylation. Inhibition of YAP transcriptional activity reversed the effects of E2 on the proliferation, migration, invasion, and β-catenin of TNBC cells. In conclusion, we demonstrated that E2 induced metastasis-related behaviors in TNBC cells and this effect was mediated through the Calpain/YAP/β-catenin signaling pathway.

The Effect of Salvianolic Acid A on Tumor-Associated Macrophage Polarization and Its Mechanisms in the Tumor Microenvironment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with a high degree of malignancy and poor prognosis. Tumor-associated macrophages (TAMs) have been identified as significant contributors to the growth and metastasis of TNBC through the secretion of various growth factors and chemokines. Salvianolic acid A (SAA) has been shown to have anti-cancer activities. However, the potential activity of SAA on re-polarized TAMs remains unclear. As there is a correlation between the TAMs and TNBC, this study investigates the effect of SAA on TAMs in the TNBC microenvironment. For that purpose, M2 TAM polarization was induced by two kinds of TNBC-conditioned medium (TNBC-TCM) in the absence or presence of SAA. The gene and protein expression of TAM markers were analyzed by qPCR, FCM, IF, ELISA, and Western blot. The protein expression levels of ERK and p-ERK in M2-like TAMs were analyzed by Western blot. The migration and invasion properties of M2-like TAMs were analyzed by Transwell assays. Here, we demonstrated that SAA increased the expression levels of CD86, IL-1β, and iNOS in M2-like TAMs and, conversely, decreased the expression levels of Arg-1 and CD206. Moreover, SAA inhibited the migration and invasion properties of M2-like TAMs effectively and decreased the protein expression of TGF-β1 and p-ERK in a concentration-dependent manner, as well as TGF-β1 gene expression and secretion. Our current findings for the first time demonstrated that SAA inhibits macrophage polarization to M2-like TAMs by inhibiting the ERK pathway and promotes M2-like TAM re-polarization to the M1 TAMs, which may exert its anti-tumor effect by regulating M1/M2 TAM polarization. These findings highlight SAA as a potential regulator of M2 TAMs and the possibility of utilizing SAA to reprogram M2 TAMs offers promising insights for the clinical management of TNBC.

Abstract 1549: Molecular mechanism of CD146 nanobody in tumor metastasis inhibition mediating by IL-6/JAK/STAT3 signaling in triple-negative breast cancer

Abstract Background: CD146, an adhesion molecule involved in inflammatory regulation, is linked to increased aggressiveness in Triple Negative Breast Cancer (TNBC). Yet, the molecular mechanisms of CD1146-driven TNBC metastasis in an inflammatory context remain elusive. We synthesized CD146 nanobody (anti-CD146 #112-2), which degraded CD146 protein specifically, inhibiting IL-6-mediated JAK/STAT3 activation and suppressing TNBC metastasis. This study aims to elucidate the mechanisms of CD146 degradation by 112-2 and the role of CD146 in mediating inflammatory responses leading to TNBC metastasis. Methods: CD146 knockout and overxpression cell models were created using CRISPR/Cas9 or lentiviral techniques. Lung metastasis was evaluated by injecting luciferase-labeled cells into the tail veins of Balb/c-nu mice, monitored with the IVIS Kinetic system. The impact of CD146 on TNBC metastasis in an inflammatory environment was explored through cell-adhesion assays and trans-endothelial migration assays using CD146 knockout models and HUVEC cells. Transwell assays assessed the influence of IL-6 and 112-2 on TNBC. CD146 mutant vectors with specific truncations were generated to confirm the binding sites between CD146 and 112-2. ELISA and immunofluorescence assays examined CD146 protein internalization. CD146 degradation by 112-2 was confirmed using proteasome inhibitor MG132 and lysosome inhibitor Bafilomycin A1, along with western blotting and immunofluorescence experiments. Results: Stimulation of TNBC cells with IL-6 increased CD146 expression and tumor metastasis. Knocking down CD146 significantly reduced IL-6 secretion by TNBC cells, leading to reduced tumor-endothelial cell adhesion and trans-endothelial migration. CD146 knockout or 112-2 treatment inhibited IL-6-mediated JAK/STAT3 activation and tumor metastasis. Experiments with CD146 mutants and molecular docking identified the specific 112-2 binding site at leucine positions 331-332 of CD146 protein. 112-2 treatment led to CD146 protein internalization and cleavage, resulting in an 80 kDa fragment recognized by both 112-2 and C-terminus-specific CD146 antibodies, ultimately inhibiting the activation of IL-6/JAK/STAT3 signaling. Notably, the inhibition of proteasome or lysosome activity failed to reverse the 112-2-induced reduction in CD146 protein levels. However, inhibiting lysosomal acidification resulted in significant co-localization of CD146 with lysosomes, suggesting a potential role of lysosomes in 112-2-mediated cleavage of CD146. Conclusion: CD146 promotes TNBC metastasis by modulating IL-6/JAK/STAT3 activation. The nanobody anti-CD146 #112-2 specifically binds to CD146 extracellular domain induces the proteolysis of CD146 protein, thereby inhibiting the tumor-promoting effect of IL-6. Citation Format: Kaiyuan Huang, Hexing Sun, Chengyu Wu, Danping Lin, Jinling Chen, Yanhan Chen, Xinqiang Fang, Shuzhao Chen, Yuanke Liang, Haoyu Lin. Molecular mechanism of CD146 nanobody in tumor metastasis inhibition mediating by IL-6/JAK/STAT3 signaling 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 1549.

Abstract 1570: Lipid metabolism alterations promote TNBC metastasis and immunotherapy resistance via PD-L1 internalization and signaling

Abstract Programmed death-ligand 1, PD-L1 (CD274), is an immune checkpoint protein upregulated by cancer cells to facilitate immune evasion. PD-L1 also performs oncogenic functions within cancer cells, but the underlying mechanisms of PD-L1 internalization and signaling remain largely unknown. Here, we report a new mechanism whereby altered ceramide metabolism via ceramide synthase 4 (CerS4) drives PD-L1 internalization to potentiate a TGFβ/Shh/β-catenin signaling axis to drive triple-negative breast cancer (TNBC) migration and metastasis. In response to altered ceramide metabolism, we observed that PD-L1 is internalized via clathrin-mediated endocytosis and depends on S278/S279 in its cytoplasmic domain. Once internalized, PD-L1 directly interacts with Caprin-1, an RNA-binding protein previously unassociated with PD-L1 signaling. Through our analysis of clinical specimens, we established a positive association between PD-L1/Caprin-1 interaction and metastatic progression in human TNBC. In terms of mechanism, we determined that the PD-L1/Caprin-1 complex plays a pivotal role in mediating CerS4-dependent cell migration. This complex selectively binds and stabilizes mRNA, resulting in heightened TGFβ/Shh/β-catenin signaling. Conversely, CerS4 restoration in metastatic TNBC cells impaired cellular migration and the interaction between PD-L1 and Caprin-1. Genetic depletion of CerS4 enhanced lung metastasis derived from 4T1 orthotopic allografts and transgenic MMTV-PyMT breast tumors concomitant with PD-L1/Caprin-1 interaction and TGFβ/Shh/β-catenin signaling. Bioinformatics analysis of clinical datasets was used to validate the impact of the CerS4/PD-L1/Caprin-1 axis on metastatic progression in human TNBC. In alignment with enhanced tumor progression, CerS4 depletion in tumors promoted regulatory T-cell infiltration, suggesting an immunosuppressive tumor microenvironment (TME). Treatment with sonidegib, an FDA-approved Hedgehog (Hh) inhibitor, effectively prevented PD-L1 internalization and its interaction with Caprin-1. We hypothesized that sonidegib could synergize with immune checkpoint blockade to sensitize TNBC to immunotherapy, which typically has low response rates. Sonidegib, in combination with αPD-L1 therapy, was significantly more effective in controlling TNBC primary and metastatic tumor growth, especially when Cers4 was downregulated, compared to αPD-L1 monotherapy. Our data also suggested that this potent anti-tumor effect was due to immunotherapy sensitization after combined flow cytometry, multiplexed immunofluorescence, and bulk RNA-sequencing analyses. Altogether, these findings demonstrate a mechanism to define intracellular PD-L1 signaling in response to altered ceramide metabolism, providing a new therapeutic strategy to target TNBC metastasis and improve response to immunotherapy. Citation Format: Wyatt O. Wofford, Jisun Kim, Dosung Kim, Alhaji H. Janneh, Han Gyul Lee, F. Cansu Atilgan, Natalia Oleinik, Elizabeth Hill, Ozge Saatci, Salih Gencer, Paramita Chakraborty, Shikhar Mehrotra, Ozgur Sahin, Besim Ogretmen. Lipid metabolism alterations promote TNBC metastasis and immunotherapy resistance via PD-L1 internalization and signaling [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 1570.

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.