Triple-negative Breast Cancer Lung Metastasis Research Articles

The incorporation of acetylated LAP-TGF-β1 proteins into exosomes promotes TNBC cell dissemination in lung micro-metastasis

Triple-negative breast cancer (TNBC) stands as the breast cancer subtype with the highest recurrence and mortality rates, with the lungs being the common site of metastasis. The pulmonary microenvironment plays a pivotal role in the colonization of disseminated tumor cells. Herein, this study highlights the crucial role of exosomal LAP-TGF-β1, the principal form of exosomal TGF-β1, in reshaping the pulmonary vascular niche, thereby facilitating TNBC lung metastasis. Although various strategies have been developed to block TGF-β signaling and have advanced clinically, their significant side effects have limited their therapeutic application. This study demonstrates that in lung metastatic sites, LAP-TGF-β1 within exosomes can remarkably reconfigure the pulmonary vascular niche at lower doses, bolstering the extravasation and colonization of TNBC cells in the lungs. Mechanistically, under the aegis of the acetyltransferase TIP60, a non-canonical KFERQ-like sequence in LAP-TGF-β1 undergoes acetylation at the K304 site, promoting its interaction with HSP90A and subsequent transport into exosomes. Concurrent inhibition of both HSP90A and TIP60 significantly diminishes the exosomal burden of LAP-TGF-β1, presenting a promising therapeutic avenue for TNBC lung metastasis. This study not only offers fresh insights into the molecular underpinnings of TNBC lung metastasis but also lays a foundation for innovative therapeutic strategies.

TRIM21-mediated Sohlh2 ubiquitination suppresses M2 macrophage polarization and progression of triple-negative breast cancer

Lung metastasis is the major cause of death in patients with triple-negative breast cancer (TNBC). Tumor-associated macrophages (TAMs) represent the M2-like phenotype with potent immunosuppressive activity, and play a pro-tumor role in TNBC lung metastasis. Sohlh2 belongs to the basic helix-loop-helix transcription factor family. However, its role in macrophages polarization remains unknown, especially in TNBC progression. Here we demonstrated that Sohlh2 overexpression promoted M2 macrophage polarization. Moreover, high expression of Sohlh2 in M2-like macrophage enhanced TNBC cell growth, migration and lung metastasis in vivo and in vitro. Mechanistically, we revealed that Sohlh2 functioned through up-regulating LXRα, ABCA1, ABCG1 expression and disturbing the lipid homeostasis on the membrane of macrophages. Sohlh2 could directly bind to the promoter of LXRα and promote its transcription activity. E3 ubiquitin ligase TRIM21 promoted Sohlh2 ubiquitination and degradation, and suppressed M2 macrophage polarization and TNBC progression. Collectively, our findings suggested that Sohlh2 in macrophage could be a novel therapeutic target for TNBC metastatic treatment.

RGCC-mediated PLK1 activity drives breast cancer lung metastasis by phosphorylating AMPKα2 to activate oxidative phosphorylation and fatty acid oxidation

BackgroundMore than 90% of the mortality of triple-negative breast cancer (TNBC) patients is attributed to cancer metastasis with organotropism. The lung is a frequent site of TNBC metastasis. However, the precise molecular mechanism for lung-specific metastasis of TNBC is not well understood.MethodsRNA sequencing was performed to identify patterns of gene expression associated with lung metastatic behavior using 4T1-LM3, MBA-MB-231-LM3, and their parental cells (4T1-P, MBA-MB-231-P). Expressions of RGCC, called regulator of cell cycle or response gene to complement 32 protein, were detected in TNBC cells and tissues by qRT-PCR, western blotting, and immunohistochemistry. Kinase activity assay was performed to evaluate PLK1 kinase activity. The amount of phosphorylated AMP-activated protein kinase α2 (AMPKα2) was detected by immunoblotting. RGCC-mediated metabolism was determined by UHPLC system. Oxidative phosphorylation was evaluated by JC-1 staining and oxygen consumption rate (OCR) assay. Fatty acid oxidation assay was conducted to measure the status of RGCC-mediated fatty acid oxidation. NADPH and ROS levels were detected by well-established assays. The chemical sensitivity of cells was evaluated by CCK8 assay.ResultsRGCC is aberrantly upregulated in pulmonary metastatic cells. High level of RGCC is significantly related with lung metastasis in comparison with other organ metastases. RGCC can effectively promote kinase activity of PLK1, and the activated PLK1 phosphorylates AMPKα2 to facilitate TNBC lung metastasis. Mechanistically, the RGCC/PLK1/AMPKα2 signal axis increases oxidative phosphorylation of mitochondria to generate more energy, and promotes fatty acid oxidation to produce abundant NADPH. These metabolic changes contribute to sustaining redox homeostasis and preventing excessive accumulation of potentially detrimental ROS in metastatic tumor cells, thereby supporting TNBC cell survival and colonization during metastases. Importantly, targeting RGCC in combination with paclitaxel/carboplatin effectively suppresses pulmonary TNBC lung metastasis in a mouse model.ConclusionsRGCC overexpression is significantly associated with lung-specific metastasis of TNBC. RGCC activates AMPKα2 and downstream signaling through RGCC-driven PLK1 activity to facilitate TNBC lung metastasis. The study provides implications for RGCC-driven OXPHOS and fatty acid oxidation as important therapeutic targets for TNBC treatment.

Honokiol prevents lung metastasis of triple-negative breast cancer by regulating polarization and recruitment of macrophages

Metastasis is the leading cause of breast cancer-associated death. Lung metastasis commonly occurs in triple-negative breast cancer (TNBC) metastasis, worsening the TNBC prognosis. Considering their role in tumor progression and metastasis, tumor-associated macrophages (TAMs) are essential therapeutic targets in cancer therapy. Previous studies have demonstrated that honokiol inhibits tumor growth and progression. Here we assessed how honokiol inhibits lung metastasis of TNBC by regulating the polarization of macrophages. We found that honokiol decreased the expression of IL-13-triggered M2 markers like CD206, Arg1, and CCL2, preventing the invasion and migration ability of TNBC cells. The activation of signal transducer and activator of transcription STAT6 and STAT3 was significantly suppressed by honokiol in M2 polarized macrophages. Meanwhile, honokiol increased the expression of LPS/IFNγ-induced M1 markers such as CD11c, iNOS, and IL12 by promoting STAT1 phosphorylation. Besides, honokiol decreased both the ratio of M2/M1 macrophages and the expression of the IL-10/IL-12 gene in lung tissues, thereby inhibiting the proliferation and metastasis of murine breast cancer. Moreover, honokiol reduced the infiltration of macrophages to the lung tissue through the CCL2/CCR2 pathways. These results highlight the potential of honokiol in suppressing TNBC tumor progression and lung metastasis by regulating the polarization and recruitment of macrophages.

Lentinan regulates the immune efficacy of macrophage for lung metastasis in triple negative breast

Lentinan (LNT), an immunoregulatory agent, is a compound derived from the mushroom Lentinus edodes. However, the immunological function and antitumor effect of LNT and the underlying mechanism remain poorly understood. In this study, we aimed to investigate the potential mechanism of LNT in treating TNBC lung metastasis by regulating macrophages in vivo and in vitro. In the Luc-4T1 mouse model, LNT inhibited the metastasis of the triple-negative breast cancer (TNBC) cells into the lungs. Additionally, as demonstrated by the immunohistochemical results and down-regulation of the mRNA expression of M2-type macrophage marker genes, LNT substantially inhibited IL-4-induced M2 macrophage polarization. Molecular docking and western blotting data further suggested that LNT is likely to target activation of the JAK2/STAT3 signaling pathway, and siRNA transfection experiments confirmed these findings. These results provide important implications for the use of LNT as a functional food to enhance immune function in the treatment of breast cancer.

FSTL1 Suppresses Triple-Negative Breast Cancer Lung Metastasis by Inhibiting M2-like Tumor-Associated Macrophage Recruitment toward the Lungs.

Immune cell infiltration into the tumor microenvironment is associated with cancer prognosis. Tumor-associated macrophages play essential roles in tumor initiation, progression, and metastasis. Follistatin-like protein 1 (FSTL1), a widely expressed glycoprotein in human and mouse tissues, is a tumor suppressor in various cancers and a regulator of macrophage polarization. However, the mechanism by which FSTL1 affects crosstalk between breast cancer cells and macrophages remains unclear. By analyzing public data, we found that FSTL1 expression was significantly low in breast cancer tissues compared to normal breast tissues, and high expression of FSTL1 in patients indicated prolonged survival. Using flow cytometry, we found that total and M2-like macrophages dramatically increased in the metastatic lung tissues during breast cancer lung metastasis in Fstl1+/- mice. Transwell assay in vitro and q-PCR experimental results showed that FSTL1 inhibited macrophage migration toward 4T1 cells by decreasing CSF1, VEGF-α, and TGF-β secretion in 4T1 cells. We demonstrated that FSTL1 inhibited M2-like tumor-associated macrophage recruitment toward the lungs by suppressing CSF1, VEGF-α, and TGF-β secretion in 4T1 cells. Therefore, we identified a potential therapeutic strategy for triple-negative breast cancer.

Abstract 3604: Epithelial Caveolin-1 regulates lung metastasis in advanced stages of breast cancer

Abstract Organotypic metastasis is a well-orchestrated sequence of events that takes place in tumors. The interplay between secretory factors from the primary tumor and the microenvironment of a distant organ determines the fate of metastasis. A significant concern in effective triple-negative breast cancer (TNBC) treatment is lung metastasis. Caveolin-1 (Cav-1), a lipid raft protein, is an important component of the metastatic interplay and is reported to be upregulated in the advanced metastatic stage of breast cancer. Although Cav-1 has been shown to have oncogenic potential, its role in TNBC lung metastasis is not fully understood. Our data illustrates, knocking out Cav-1 in the 4T1 cells (a mouse breast cancer cell line) affects cell morphology, extracellular vesicle formation, and MMP release in vitro. Furthermore, a wound healing/cell migration assay showed reduced migration in Cav-1 KO 4T1 cells when compared to control 4T1 cells, and these findings were validated using Cav-1 siRNA. For investigating the metastatic role, we injected Cav-1 KO 4T1 and control 4T1 cells into the mammary fat pad of female BALB/c mice, a syngeneic mouse model of triple-negative breast cancer. We found no evidence of lung metastasis in Cav-1 KO 4T1 injected mice when compared to control 4T1 injected mice. This was further supported by mRNA profiling of the tumor. We observed 21 epithelial cell migration genes that are differentially expressed in Cav-1 KO tumors when compared to WT tumors. Correlation analysis from Breast Cancer Gene-Expression Miner v4.8 (bc-GenExMiner), a human database, showed a significant correlation between Cav-1 expression and several of the 21 epithelial cell migration genes, along with integrin 3 alpha (ITGα3). Interestingly, in silico protein docking predicted an interaction between Cav-1 and ITGα3, which was later confirmed by co-immunoprecipitation. The role of ITGα3 in metastasis was further corroborated by cell migration assay, which was affected when siRNA was used to silence ITGα3 gene expression. Collectively, these findings provide evidence that Cav-1 KO, along with ITGa3, plays an important role in distant lung metastasis in the 4T1 tumor-induced mice model. Citation Format: Dhirendra Pratap Singh, Rashmi Pathak, Abhishek Pandit, Philip J. Ebenezer, Nithya Jambunathan, Sanjay Kumar, Alexander Du Plooy, Joseph Francis, Mary E. White. Epithelial Caveolin-1 regulates lung metastasis in advanced stages of breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3604.

MUC16 promotes triple-negative breast cancer lung metastasis by modulating RNA-binding protein ELAVL1/HUR

BackgroundTriple-negative breast cancer (TNBC) is highly aggressive with an increased metastatic incidence compared to other breast cancer subtypes. However, due to the absence of clinically reliable biomarkers and targeted therapy in TNBC, outcomes are suboptimal. Hence, there is an urgent need to understand biological mechanisms that lead to identifying novel therapeutic targets for managing metastatic TNBC.MethodsThe clinical significance of MUC16 and ELAVL1 or Hu antigen R (HuR) was examined using breast cancer TCGA data. Microarray was performed on MUC16 knockdown and scramble TNBC cells and MUC16-associated genes were identified using RNA immunoprecipitation and metastatic cDNA array. Metastatic properties of MUC16 were evaluated using tail vein experiment. MUC16 and HuR downstream pathways were confirmed by ectopic overexpression of MUC16-carboxyl-terminal (MUC16-Cter), HuR and cMyc as well as HuR inhibitors (MS-444 and CMLD-2) in TNBC cells.ResultsMUC16 was highly expressed in TNBC and correlated with its target HuR. Depletion of MUC16 showed decreased invasion, migration, and colony formation abilities of human and mouse TNBC cells. Mice injected with MUC16 depleted cells were less likely to develop lung metastasis (P = 0.001). Notably, MUC16 and HuR were highly expressed in the lung tropic TNBC cells and lung metastases. Mechanistically, we identified cMyc as a HuR target in TNBC using RNA immunoprecipitation and metastatic cDNA array. Furthermore, MUC16 knockdown and pharmacological inhibition of HuR (MS-444 and CMLD-2) in TNBC cells showed a reduction in cMyc expression. MUC16-Cter or HuR overexpression models indicated MUC16/HuR/cMyc axis in TNBC cell migration.ConclusionsOur study identified MUC16 as a TNBC lung metastasis promoter that acts through HuR/cMyc axis. This study will form the basis of future studies to evaluate the targeting of both MUC16 and HuR in TNBC patients.

Design, synthesis and evaluation of nitric oxide releasing derivatives of 2,4-diaminopyrimidine as novel FAK inhibitors for intervention of metastatic triple-negative breast cancer

To search for novel medicines for intervention of triple-negative breast cancer (TNBC), a series of phenylsulfonyl furoxan-based 2,4-diaminopyrimidine derivatives (8a-t) were designed and synthesized based on blocking FAK-mediated signaling pathways through both kinase-dependent and -independent manners. The most active compound 8f not only significantly inhibited FAK kinase activity (IC50 = 27.44 nM), displayed potent inhibitory effects on the proliferation (IC50 = 0.126 μM), invasion and migration of MDA-MB-231 cells, superior to the most widely studied FAK inhibitor, TAE226, bearing 2,4-diaminopyrimidine, but also released high levels of NO, contributing to blockage of FAK mediated-signaling pathways by upregulating of p53 as well as suppressing the Y397 phosphorylation and its downstream effectors, including p-Akt, MMP-2, and MMP-9 via kinase-independent manner, leading to apoptosis induction and decrease of FAs and SFs in TNBC cells. Importantly, 8f inhibited the lung metastasis of TNBC in vivo. Together, 8f may serve as a promising candidate for the treatment of metastatic TNBC.

Resveratrol attenuates TNBC lung metastasis by down-regulating PD-1 expression on pulmonary T cells and converting macrophages to M1 phenotype in a murine tumor model

Triple-negative breast cancer (TNBC) is an invasive breast cancer with the characteristics of easy to develop distant metastasis. Immune escape is one of the main reasons for TNBC growth and metastasis. Enhancement of T cell-mediated anti-tumor activity may benefit to inhibit tumor metastasis and improve the efficacy of cancer therapy. As a natural bioactive substance, resveratrol shows potential capability to prevent or suppress the development of a variety of cancers through direct or indirect effects, including immunoregulatory effect. However, whether resveratrol might affect lung metastasis of TNBC, and whether the effect of resveratrol might be associated with resveratrol-regulated immune responses in tumor microenvironment is still unknown. In this study, by using an experimental metastatic mouse 4 T1 tumor model, we identified that resveratrol may suppress TNBC lung metastasis by elevating local anti-tumor immunity. Indeed, an increase in the cytotoxic activity of CD8+T cells as well as the levels of type 1 cytokine IFN-γ and IL-2 in the lungs of resveratrol-treated tumor bearing mice were observed. The enhanced CD8+T cell activity and Th1 immune responses by resveratrol administration might be related to the down-regulated PD-1 expression on pulmonary CD8+T cells and CD4+T cells. Resveratrol may also convert macrophages to M1 phenotype in the lungs of tumor bearing mice. However, it seems likely resveratrol has no effect on pulmonary myeloid-derived suppressor cell activation. Our results provide an evidence that resveratrol might be a promising candidate agent for adjuvant therapy in the process of TNBC metastasis.

Group 2 innate lymphoid cells promote TNBC lung metastasis via the IL-13-MDSC axis in a murine tumor model

Group 2 innate lymphoid cells (ILC2s) are reportedly associated with the progression of many tumors. However, the role of ILC2s in triple‐negative breast cancer (TNBC) lung metastasis remains unclear. In this study, we found that ILC2s may be a key element in the process of TNBC lung metastasis since the adoptive transfer of pulmonary ILC2s increased the numbers of metastatic lung nodules and reduced the survival of tumor-bearing mice. ILC2-promoted 4 T1 lung metastasis appears to be related to ILC2-derived IL-13. An expansion of IL-13-producing ILC2s and an elevated expression of IL-13 mRNA in pulmonary ILC2s were determined in tumor-bearing mice, in parallel with an increase in the levels of local IL-13 by ILC2 transfer. The neutralization of IL-13 reduced the increased pulmonary metastatic nodules and improved the decreased survival rate caused by ILC2-adoptive transfer. Interestingly, adoptive transfer of ILC2s elevated IL-13Ra1 expression in myeloid-derived suppressor cells (MDSCs). Treatment of ILC2-transferred tumor-bearing mice with anti-IL-13 antibodies significantly diminished the number of pulmonary MDSCs and inhibited MDSC activation. Moreover, when pulmonary MDSCs were cocultured with ILC2s in the presence of an anti-IL-13 mAb, the number and activation of MDSCs were reduced. Depletion of MDSCs may promote the proliferation of CD4+ T cells and CD8+ T cells, but reduce the expansion of regulatory T cells (Tregs) in the lungs of ILC2-transferred tumor-bearing mice. Our results suggest that pulmonary ILC2s may promote TNBC lung metastasis via the ILC2-derived IL-13-activated MDSC pathway.

CD81 Facilitates Tumor Cell Clustering and Metastasis in Triple Negative Breast Cancer

Tumor-initiating cells with reprogramming plasticity and/or de-differentiation attributes have been thought to initiate primary tumor development as well as to regenerate secondary tumors in metastatic organs; however, the molecular mechanisms are not fully understood. We previously found that breast tumor-initiating cell marker, CD44, directs multicellular aggregation and cluster formation of circulating tumor cells (CTCs), which further enhance stemness and survival of such cells, enabling metastatic colonization to the lungs. To further elucidate the molecular network underlying CTC cluster formation, we performed global proteomic profiling and discovered that the tetraspanin protein CD81, which is normally enriched in exosomes (small extracellular vesicles), is a new driver of cancer initiation and metastasis as a facilitator and target of CD44. Loss of CD81 compromises tumorigenicity and mammosphere formation of triple negative breast cancer (TNBC) cells. Assisted by machine learning-based algorithms and mutagenesis approach, we found that CD81 interacts with CD44 on the cellular membrane through their extracellular regions. Notably, genetic knockout of CD44 or CD81 results in loss of both CD81 and CD44 in secreted exosomes, a state which abolishes exosome-induced self-renewal of recipient cells, such as mammosphere formation. In addition, RNA sequencing analysis showed that CD81 knockdown up-regulates expression of a cell differentiation marker, SEMA7a, whose down-regulation partially rescues mammosphere formation inhibition by CD81 depletion. Clinically, CD81 expression was observed in >80% of CTCs and specifically enriched and co-expressed along with CD44 in clustered CTCs of breast cancer patients. Mimicing the phenotypes of CD44 deficiency, loss of CD81 also inhibited tumor cell aggregation and lung metastasis of TNBC in both human and mouse tumor models, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights a new driving role of CD81 in cancer exosome-induced stemness, clustered CTCs, and metastasis initiation of TNBC, reported for the first time to our knowledge.

Decomposable black phosphorus nano-assembly for controlled delivery of cisplatin and inhibition of breast cancer metastasis

Novel platforms for cisplatin delivery with a controllable manner and combinable with other treatment modality to achieve synergistic antitumor effect and inhibition metastasis for treatment of triple negative breast cancer (TNBC) are highly desirable. Herein, we report a black phosphorus (BP) nanosheets-based nano-assembly which consists of cisplatin, BP, polydopamine (PDA) and hyaluronic acid (HA), cisplatin/BP/PDA-HA (CBPH), for controlled delivery of cisplatin and inhibition tumor growth as well as lung metastasis of TNBC. For constructing CBPH, the surface of BP was dual modified by PDA and HA, resulting in enhanced stability, tumor target ability and photothermal efficiency of BP. Cisplatin was released in response both to internal and external stimuli existed in tumor microenvironment, including low pH, hydrogen peroxide and NIR light, as accompanied by decomposition of BP. In vitro experiments demonstrated CBPH-treated 4 T1 cells showed elevated intracellular content of Pt and Pt-DNA adduct, which was further improved when exposure to NIR light, leading to potent antitumor effect in a synergistic pattern. Anti-metastasis studies in 2D monolayers and 3D organoids revealed that CBPH plus NIR light treatment exhibited significantly decreased migration, invasion and regrowth ability of 4 T1 cells. Furthermore, TNBC-bearing mice with systemic administrate of CBPH showed enhanced tumor accumulation of cisplatin and light-triggered inhibition of tumor growth at primary site and lung metastasis, with alleviated toxicity. But CBPH is yet to be optimized for realizing smart cisplatin delivery in response to acidic and redox stimuli in vivo. Collectively, our study demonstrates that this novel BP-based nano-assembly with controllable tumor delivery of cisplatin and metastasis inhibition of breast cancer expand the use of BP in biomedicine field and hold great promise for further development.

Structure-Based Design of a Selective Class I Histone Deacetylase (HDAC) Near-Infrared (NIR) Probe for Epigenetic Regulation Detection in Triple-Negative Breast Cancer (TNBC).

Abnormally high levels of class I histone deacetylases (HDACs) are associated with triple-negative breast cancer (TNBC) proliferation, malignant transformation, and poor prognosis of patients. Herein, we report a near-infrared imaging probe for TNBC detection via visualizing class I HDACs. Conjugating Cy5.5 to a cyclic depsipeptide inhibitor, we obtained the probe (20-Cy5.5) that retained desirable class I HDAC affinity and selectivity. Then, this probe could visualize epigenetic changes by class I HDACs in TNBC MDA-MB-231 cells and in xenograft tumor models in real time. Treatment with suberoylanilide hydroxamic acid (SAHA) significantly reduced the uptake of the probe in tumors, suggesting its potential use in evaluation of therapeutic responses of HDACi-mediated therapy. Moreover, 20-Cy5.5 could detect class I HDAC expression in TNBC lung metastasis. This novel NIR probe that achieves tumor class I HDAC imaging not only leads to a better understanding of epigenetic regulation in tumors but also has great potential for improving the TNBC diagnosis and treatment.

The molecular underpinning of geminin-overexpressing triple-negative breast cancer cells homing specifically to lungs.

Triple-negative breast cancer (TNBCs) display lung metastasis tropism. However, the mechanisms underlying this organ-specific pattern remains to be elucidated. We sought to evaluate the utility of blocking extravasation to prevent lung metastasis. To identify potential geminin overexpression-controlled genetic drivers that promote TNBC tumor homing to lungs, we used the differential/suppression subtractive chain (D/SSC) technique. A geminin overexpression-induced lung metastasis gene signature consists of 24 genes was discovered. We validated overexpression of five of these genes (LGR5, HAS2, CDH11, NCAM2, and DSC2) in worsening lung metastasis-free survival in TNBC patients. Our data demonstrate that LGR5-induced β-catenin signaling and stemness in TNBC cells are geminin-overexpression dependent. They also demonstrate for the first-time expression of RSPO2 in mouse lung tissue only and exacerbation of its secretion in the circulation of mice that develop geminin overexpressing/LGR5+-TNBC lung metastasis. We identified a novel extravasation receptor complex, consists of CDH11, CD44v6, c-Met, and AXL on geminin overexpressing/LGR5+-TNBC lung metastatic precursors, inhibition of any of its receptors prevented geminin overexpressing/LGR5+-TNBC lung metastasis. Overall, we propose that geminin overexpression in normal mammary epithelial (HME) cells promotes the generation of TNBC metastatic precursors that home specifically to lungs by upregulating LGR5 expression and promoting stemness, intravasation, and extravasation in these precursors. Circulating levels of RSPO2 and OPN can be diagnostic biomarkers to improve risk stratification of metastatic TNBC to lungs, as well as identifying patients who may benefit from therapy targeting geminin alone or in combination with any member of the newly discovered extravasation receptor complex to minimize TNBC lung metastasis.

Follistatin-like 1 deficiency impairs T cell development to promote lung metastasis of triple negative breast cancer.

Our study aims to detect the underlying mechanism of the suppressive effect of Follistatin-like 1 (FSTL1) on lung metastasis of triple negative breast cancer (TNBC). We found that FSTL1 had no effect on the proliferation and metastasis of 4T1 cells in vitro, while in the tumor-bearing Fstl1 heterozygous (Fstl1+/-) mice, the number of anti-tumor T lymphocytes in the lung was significantly reduced with the increase in lung metastasis. Impaired development of T cells can cause dysfunction of adaptive immune system, which promotes cancer metastasis. Therefore the effect of FSTL1 on T cell development was further investigated.Lower population of T cells in periphery and decreased proliferation of CD4- CD8- double negative (DN) thymocytes and impairment development of T cells were found in Fstl1+/- mice. Furthermore, high expression of FSTL1 in medullary thymus epithelial (mTEC) cells and decreased mRNA expression of inducible costimulator on activated T-cell ligand (Icosl) in mTECsh Fstl1 were detected. Combining other studies that the generation of ICOSL by mTEC cells promotes CD4+ single positive (SP) thymocytes to produce IL-2, which promotes T cell development. Our results indicate FSTL1 deficiency in mTEC cells impairs T cell development to promote the lung metastasis of TNBC.

Abstract PD15-03: LGR4 engages a wnt-independent mechanism to enhance EGFR signaling and promote metastasis of triple-negative breast cancer

Abstract Leucine-rich repeat-containing G-protein-coupled receptors 4/5/6 (LGR4/5/6) have crucial functions in embryonic development, adult tissue homeostasis, and diseases. LGR4/5/6 also play important roles in cancer initiation and progression. LGR4 is highly expressed in multiple types of cancer and associated with poor patient outcome. LGR4 promotes tumorigenesis and metastasis and modulates cancer stem cells, while LGR5 and LGR6 mark cancer stem cells and progenitor cells that contribute to tumor initiation. The biological functions of LGR4/5/6 are primarily attributed to their roles in potentiating Wnt signaling, which requires the binding of their four R-spondin ligands (RSPO1-RSPO4). However, it is unclear whether these proteins have Wnt signaling-independent functions. We have previously reported that LGR4 is important in mammary gland development and breast cancer progression and metastasis. Here, we demonstrate that LGR4 enhances triple-negative breast cancer (TNBC) metastasis independently of Wnt signaling. We first found that LGR4 was highly expressed in the TNBC subtype and associated with poor metastasis-free survival in TNBC patients, while a well-validated Wnt signature failed to correlate with metastasis-free survival of this same subset of TNBC patients. These bioinformatic data suggested divergent functions of LGR4 versus Wnt signaling in TNBC progression. Then we showed that depletion of LGR4 inhibited, while inducible expression of LGR4 promoted TNBC cell migration and invasion, without added exogenous ligands and any detectable impact on baseline Wnt signaling. Importantly, blockade of Wnt signaling by Wnt inhibitors or by genetic depletion of Wntless cannot abolish the effect of LGR4 on TNBC cell metastatic ability. Furthermore, we have generated non-RSPO-binding LGR4 mutants that failed to activate Wnt signaling. These LGR4 mutants that are uncoupled from Wnt signaling can still promote TNBC cell migration and invasion in vitro and TNBC lung metastasis and bone metastasis in vivo, as potently as wild-type LGR4. To identify the molecular mechanisms by which LGR4 promotes TNBC metastasis, we did computational analyses of TCGA data to search for candidate proteins associated with LGR4 and proteomics screenings to uncover protein pathways regulated by LGR4 knockdown in TNBC cells. Both in silico and proteomic analyses identified EGFR as a downstream component of LGR4 signaling in TNBC. We confirmed LGR4 enhanced EGFR signaling by immunoblot analyses and found that EGFR was the crucial mediator of LGR4’s role in TNBC metastasis. Ectopic EGFR rescued migration and invasion of LGR4 knockout cells, while EGFR knockdown and the EGFR inhibitor erlotinib attenuated LGR4-induced cell migration and invasion. In an intra-iliac injection model of bone metastasis, erlotinib also suppressed LGR4-induced TNBC bone metastasis in vivo. Mechanistically, LGR4 did not affect EGFR mRNA levels but EGFR protein stability. LGR4 interacted with EGFR and blocked EGFR ubiquitination and degradation, resulting in persistent EGFR activation. Together, these data uncover a new signaling pathway controlled by LGRs with broad implications for cancer progression and targeted therapy. Citation Format: Fei Yue, Weiyu Jiang, Amy T Ku, Adelaide IJ Young, Weijie Zhang, Eric P Souto, Yankun Gao, Zhihan Yu, Yi Wang, Chad J Creighton, Chandandeep Nagi, Tao Wang, Xin-Hua Feng, Shixia Huang, Cristian Coarfa, Xiang H.-F. Zhang, Qingyun Liu, Xia Lin, Yi Li. LGR4 engages a wnt-independent mechanism to enhance EGFR signaling and promote metastasis of triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-03.

Polyglutamic acid-based crosslinked doxorubicin nanogels as an anti-metastatic treatment for triple negative breast cancer

Treatment of triple negative breast cancer (TNBC)-associated metastasis represents an unmet clinical need, and we lack effective therapeutics for a disease that exhibits high relapse rates and associates with poor patient outcomes. Advanced nanosized drug delivery systems may enhance the efficacy of first-line chemotherapeutics by altering drug pharmacokinetics and enhancing tumor/metastasis targeting to significantly improve efficacy and safety. Herein, we propose the application of injectable poly-amino acid-based nanogels (NGs) as a versatile hydrophilic drug delivery platform for the treatment of TNBC lung metastasis. We prepared biocompatible and biodegradable cross-linked NGs from polyglutamic acid (PGA) loaded with the chemotherapeutic agent doxorubicin (DOX). Our optimized synthetic procedures generated NGs of ~100 nm in size and 25 wt% drug loading content that became rapidly internalized in TNBC cell lines and displayed IC50 values comparable to the free form of DOX. Importantly, PGA-DOX NGs significantly inhibited lung metastases and almost completely suppressed lymph node metastases in a spontaneously metastatic orthotopic mouse TNBC model. Overall, our newly developed PGA-DOX NGs represent a potentially effective therapeutic strategy for the treatment of TNBC metastases.

EGFR inhibition blocks cancer stem cell clustering and lung metastasis of triple negative breast cancer.

Triple-negative breast cancer (TNBC) is one of the most aggressive and metastatic breast cancer subtypes lacking targeted therapy. Our recent work demonstrated that circulating tumor cell (CTC) clusters and polyclonal metastasis of TNBC are driven by aggregation of CD44+ cancer stem cells (CSC) and associated with an unfavorable prognosis, such as low overall survival. However, there is no existing therapeutic that can specifically block CTC or CSC cluster formation.Methods: Using patient-derived xenograft (PDX) models, we established an ex vivo tumor cell clustering assay for a pilot screening of blockade antibodies. After identifying EGFR as a target candidate, we modulated the gene expression and inhibited its kinase activity to determine its functional importance in tumor cell clustering and therapeutic inhibition of lung metastasis. We also examined the molecular regulation network of EGFR and a potential connection to CSC marker CD44 and microRNAs, which regulate CTC clustering.Results: We report here that EGFR inhibition successfully blocks circulating CSC (cCSC) clustering and lung metastasis of TNBC. EGFR enhances CD44-mediated tumor cell aggregation and CD44 stabilizes EGFR. Importantly, blocking EGFR by a novel anti-EGFR monoclonal antibody (clone LA1) effectively blocked cell aggregation in vitro and reduced lung metastasis in vivo. Furthermore, our data demonstrated that the tumor suppressor microRNA-30c serves as another negative regulator of cCSC clustering and lung metastasis by targeting CD44 as well as its downstream effector EGFR.Conclusion: Our studies identify a novel anti-EGFR therapeutic strategy to inhibit cCSC aggregation and therefore abolish cCSC cluster-mediated metastasis of TNBC.

Abstract P2-15-03: Liposomal annamycin inhibition of lung localized breast cancer

Abstract Background and Rationale: Annamycin is an anthracycline antibiotic currently in clinical studies in AML patients. Our earlier studies showed that Annamycin is not cross-resistant with doxorubicin (DOX) and is a poor substrate for P-glycoprotein 1 (P-gp) [aka ATP-binding cassette sub-family B member 1 (ABCB1) or multidrug resistance protein 1 (MDR1)], a major mechanism of doxorubicin resistance in different types of cancer. Annamycin, in contrast to DOX, achieves relatively high levels of cellular accumulation, especially in multidrug resistant (MDR) cell lines, and induces significant DNA damage in MDR cancer cells. Clinically, Annamycin is administered in a liposomal formulation (L-Annamycin) the in vivo activity of which has been demonstrated in different tumor models. IND aimed preclinical studies revealed that in sharp contrast to DOX, Annamycin displays dramatically reduced to no cardiotoxicity. Subsequent pharmacokinetic and organ distribution studies revealed surprisingly high levels of Annamycin in lung tissue. Annamycin levels (AUC, 24 h) in lungs were over 10-fold greater than in plasma. In addition, when compared with DOX, lung levels of Annamycin were 6-fold higher than those achievable with DOX. Thus, studies aimed to assess efficacy of L-Annamycin in lung localized tumor models were initiated. Objective: The objective of this study was to assess the in vitro and in vivo activity of L-Annamycin in 4T1, a highly aggressive triple negative breast cancer (TNBC) lung colonization model. Methods: In vitro efficacy of Annamycin was investigated in a panel of TNBC cell lines including murine 4T1 and human MDA-MB 231, MDA-MB-468, BT20, and SUM149 cell lines. Uptake and subcellular distribution of Annamycin was assessed using fluorescent microscopy and FACS. Biodistribution of Annamycin was tested in naïve CD-1 mice after intravenous administration of the drug. Efficacy of L-Annamycin was tested in a syngeneic TNBC lung model, specifically, in immune-competent Balb/c mice injected intravenously with 4T1 cells expressing firefly luciferase. Results: Annamycin shows consistently high cytotoxicity across all tested cell lines with IC50s in the low nanomolar range. Uptake analysis performed on 4T1 cells indicated significantly higher uptake of the drug as compared to DOX. Annamycin shows distinct subcellular distribution with high cytosolic deposition, while DOX was mostly confined to the nucleus. In vivo efficacy studies in mice treated with L-Annamycin showed significantly increased survival when compared to vehicle treated animals. Delayed tumor progression was assessed by bioluminescent imaging and correlated with increased survival. Conclusion: Our in vitro studies demonstrate the high potency of L-Annamycin in vitro and in vivo. Its unique intracellular uptake and distribution might in part be responsible for L-Annamycin’s activity profile and lack of cardiotoxicity. The observed high in vivo efficacy of L-Annamycin in an aggressive TNBC lung metastasis model coupled with consistently high accumulation of L-Annamycin in lung parenchyma, may prove to open new therapeutic options for patients with tumors metastatic to the lung. Citation Format: Rafal Zielinski, Krzysztol Grela, Stanislaw Skora, Izabela Fokt, Matthew Sander, Adam Didluch, Waldemar Priebe. Liposomal annamycin inhibition of lung localized breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-15-03.