Regulation Of Breast Cancer Progression Research Articles

GAS5 regulated by FTO-mediated m6A modification suppresses cell proliferation via the IGF2BP2/QKI axis in breast cancer

BackgroundThe lncRNA growth arrest-specific 5 (GAS5) is involved in regulating breast cancer progression. In this study, we aimed to elucidate the function and mechanism of GAS5 in breast cancer.MethodsThe expressions of GAS5, fat mass and obesity-associated protein (FTO), insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), and Quaking (QKI) were assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot. The m6A modification level of GAS5 was detected using m6A immunoprecipitation assay (MeRIP). The interaction between IGF2BP2 and GAS5 or QKI was detected using RNA immunoprecipitation assay (RIP) and dual luciferase reporter assay. Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) assay. The biological functions of the FTO/GAS5/IGF2BP2/QKI axis was assessed using the tumor xenograft assay.ResultsLncRNA GAS5 expression decreased in breast cancer and was regulated by FTO-mediated m6A modification in an IGF2BP2-dependent manner, resulting in decreased GAS5 stability and expression. GAS5 recruited IGF2BP2 to target QKI and upregulated QKI expression in breast cancer cells. GAS5 suppressed breast cancer growth via IGF2BP2/QKI, and this inhibitory effect was modulated by FTO both in vitro and in vivo.ConclusionsGAS5 regulated by FTO-mediated m6A modification represses the growth of breast cancer via the IGF2BP2/QKI pathway, suggesting that the FTO/GAS5/IGF2BP2/QKI pathway can be a potential target for breast cancer treatment.

Abstract 1409: The role of liver receptor homolog 1<LRH-1>in regulating breast cancer progression by modulating the immune response

Abstract Breast cancer remains the second leading cause of cancer-related death among American women, with the majority of morality being associated with metastatic disease. At this stage, immunotherapy is only approved for a small subset of patients whose tumors are of the triple negative subtype <TNBC> that stain positive for PD-L1. However, the majority of these tumors prove refractory. Thus, it is imperative to develop novel therapeutic targets to improve the immune response in breast cancer. Since cholesterol metabolism is important for myeloid immune cell function, we launched studies to identify proteins involved in cholesterol homeostasis that are amenable to small molecule intervention. Liver Receptor Homolog 1 <LRH-1/NR5A2> is highly expressed in myeloid cells, particularly neutrophils. We have found that elevated LRH-1 expression in breast cancer patients is associated with increased overall and recurrence-free survival rate. Thus, we hypothesized that LRH-1 acts as an immune modulator in myeloid cells, with subsequent effects on breast cancer progression. We initiated a series of experiments to characterize the role of LRH-1 in important aspects of neutrophil function: migration, NETosis, phagocytosis and influence on T cells. Neutrophils and neutrophil NETosis has previously been implicated in breast cancer progression including reemergence from dormancy and metastatic recurrence. Interestingly, we found that LRH-1 inhibits neutrophil migration towards cancer cells, a finding that may have important implications regarding infiltration of immune-suppressive myeloid cells. Furthermore, neutrophils treated with an LRH-1 agonist demonstrated decreased NETosis, while an antagonist resulted in increased NETosis. On the other hand, LRH-1 agonist treatment strongly decreased the neutrophil phagocytotic ability, while treatment with an antagonist did not significantly impact this process. Finally, T cells had increased expansion when cocultured with neutrophils that had been previously treated with an LRH-1 agonist, and reduced proliferation in the presence of neutrophils pretreated with an LRH-1 antagonist or inverse agonist. This was especially apparent in CD4+ helper T cells and later divisions. Ongoing work is aimed at determining the pathological relevance of these findings. Collectively, our data indicate that LRH-1 plays important roles in regulating different neutrophils functions, including migration, NETosis, phagocytosis, and T cell expansion. Overall, these findings suggest that LRH-1 in neutrophils regulates the immune response, and therefore can be a potential therapeutic target for cancer patients. Funding: National Cancer Institute (ERN: R01CA234025) Department of Defense (ERN: BCRP Era of Hope Award) Citation Format: Yu Wang, Bryan Duong, Natalia Krawczynska, Shruti V. Bendre, Claire P. Schane, Erin Weisser, Lara Kockaya, Yifan Fei, Anasuya Das Gupta, Hashni E. Vidana Gamage, Adam T. Nelczyk, Erik R. Nelson. The role of liver receptor homolog 1<LRH-1>in regulating breast cancer progression by modulating the immune response [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 1409.

Abstract 3925: Cancer-associated fibroblast-derived Dickkopf-1 impairs anti-tumor immunity in breast cancer by suppressing NK cell-mediated cytotoxicity

Abstract Despite early diagnosis, 10-20% of breast cancer patients experience metastatic disease within 5-10 years. While it is established that breast cancer cells have the capacity to evade immune responses, the mechanisms driving this evasion remain elusive. Therefore, finding ways to enhance anti-tumor immunity is crucial to improve patient outcomes. Dickkopf-1 (DKK1) is a Wnt inhibitor whose levels correlate with poor prognosis and decreased immune infiltration in various cancers. In this study, we used mice orthotopically injected with the PyMT, EO771, and 4T1 breast cancer lines. In all models, we found elevated DKK1 serum levels, and its neutralization with an αDKK1 Ab resulted in a significant reduction in primary tumor growth but also in metastatic dissemination. To identify the source of DKK1, we examined its expression in the tumor. DKK1 was detected in stromal cells with a fibroblast-like morphology but not in cancer cells or immune infiltrates. Co-staining confirmed colocalization between DKK1 and the CAF marker αSMA. To address the role of CAF-derived DKK1, we specifically deleted DKK1 in CAFs by generating FSP1CreDkk1fl/fl and αSMACreERT2Dkk1fl/fl mice. Both models showed a significant reduction in primary tumor growth while maintaining comparable serum DKK1 levels, indicating locally produced DKK1 supports tumor growth. To understand how DKK1 promotes tumor progression, we conducted bulk RNAseq of tumor cells isolated from IgG or αDKK1 treated mice. While no changes related to cell viability or cell cycle were observed, immune response-related genes were upregulated in αDKK1-treated cells. In support of this, αDKK1 did not reduce tumor growth in NSG immune-compromised mice. To pinpoint the immune cell targeted by DKK1, we depleted T, NK cells, or macrophages in WT tumor-bearing mice treated with αDKK1. Remarkably, only NK cell depletion negated αDKK1 anti-tumor effects. In vitro assays further demonstrated that rDKK1 or the presence of CAFs reduced NK cell cytotoxicity against tumor cells while αDKK1 restored it. To assess whether DKK1 levels correlated with tumor progression and immune suppression in breast cancer patients, we analyzed DKK1 serum levels and circulating NK cells at the diagnosis of metastatic bone disease and 15-18 months after standard care. DKK1 levels were significantly elevated in patients with progressive bone disease compared to those with stable disease. Importantly, NK cells from the progressive disease cohort had reduced perforin and granzyme B expression at the follow-up visit compared to baseline, indicating reduced cytotoxicity. Furthermore, rDKK1 reduced perforin in NK cells from healthy donors. In sum, our data show DKK1 is an important regulator of breast cancer progression, and we report for the first time DKK1 directly impairs NK cell cytotoxicity, creating an immune suppressive environment resistant to treatment. Citation Format: Seunghyun Lee, Biancamaria Ricci, Roberta Faccio. Cancer-associated fibroblast-derived Dickkopf-1 impairs anti-tumor immunity in breast cancer by suppressing NK cell-mediated cytotoxicity [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 3925.

Expression and Clinical Significance of NUDCD1, PI3K/AKT/mTOR Signaling Pathway-Related Molecules and Immune Infiltration in Breast Cancer

BackgroundNUDCD1 (NudC Domain Containing 1) performs an essential function in biological processes such as cell progression, migration, cell cycle, and intracellular material transport. Many solid tumors express it highly, which is a prospective biomarker and therapeutic approach. However, the expression and clinical importance of NUDCD1 across breast cancer is unclear. MethodsThe expressions of NUDCD1 in breast cancers and normal breast tissues were studied utilizing the TIMER database and immunohistochemical analysis. Subsequently, we validate the association between the expression of NUDCD1 and clinicopathologic features and prognosis of breast cancer. The immunohistochemical experiments of pathway-related molecules were done on 214 breast cancer tissue microarrays. The investigation of correlation between NUDCD1 expression and tumor immune infiltration was subsequently conducted. ResultsThrough the utilization of bioinformatics analysis and immunohistochemical experiments, it was determined that NUDCD1 exhibited upregulation within breast cancer. Furthermore, it was discovered that an elevated expression of NUDCD1 may potentially be linked to a worse prognosis in breast cancer. Our study reveals that the PI3K/AKT/mTOR signaling pathway may perform a function in NUDCD1 regulating breast cancer progression via enrichment analysis. Furthermore, the expression of NUDCD1 may be associated with the degree of immunological infiltration. ConclusionThe expression of NUDCD1 was explored to be elevated in breast cancer and was observed to be correlated with a poorer prognosis. p-AKT, PI3K, AKT, mTOR, and p-mTOR expression levels underwent significant elevation in breast cancer. The function of NUDCD1 within breast cancer might be associated with the PI3K/AKT/mTOR signaling pathway.

Peroxiredoxin 3 regulates breast cancer progression via ERK-mediated MMP-1 expression

Peroxiredoxin 3 (PRDX3), a mitochondrial hydrogen peroxide scavenger, is known to be upregulated during tumorigenesis and cancer progression. In this study, we provide evidence for the first time that PRDX3 could regulate cellular signaling pathways associated with Matrix Metalloproteinase-1 (MMP-1) expression and activity in breast cancer progression. We show that shRNA-mediated gene silencing of PRDX3 inhibits cell migration and invasion in two triple-negative breast cancer cell lines. Reciprocal experiments show that PRDX3 overexpression promotes invasion and migration of the cancer cells, processes which are important in the metastatic cascade. Notably, this phenomenon may be attributed to the activation of MMP-1, which is observed to be upregulated by PRDX3 in the breast cancer cells. Moreover, immunohistochemical staining of breast cancer tissues revealed a positive correlation between PRDX3 and MMP-1 expression in both epithelial and stromal parts of the tissues. Further pathway reporter array and luciferase assay demonstrated that activation of ERK signaling is responsible for the transcriptional activation of MMP-1 in PRDX3-overexpressed cells. These findings suggest that PRDX3 could mediate cancer spread via ERK-mediated activation of MMP-1. Targeted inhibition of ERK signaling may be able to inhibit tumor metastasis in triple-negative breast cancer.

Neutral ceramidase regulates breast cancer progression by metabolic programming of TREM2-associated macrophages

The tumor microenvironment is reprogrammed by cancer cells and participates in all stages of tumor progression. Neutral ceramidase is a key regulator of ceramide, the central intermediate in sphingolipid metabolism. The contribution of neutral ceramidase to the reprogramming of the tumor microenvironment is not well understood. Here, we find that deletion of neutral ceramidase in multiple breast cancer models in female mice accelerates tumor growth. Our result show that Ly6C+CD39+ tumor-infiltrating CD8 T cells are enriched in the tumor microenvironment and display an exhausted phenotype. Deletion of myeloid neutral ceramidase in vivo and in vitro induces exhaustion in tumor-infiltrating Ly6C+CD39+CD8+ T cells. Mechanistically, myeloid neutral ceramidase is required for the generation of lipid droplets and for the induction of lipolysis, which generate fatty acids for fatty-acid oxidation and orchestrate macrophage metabolism. Metabolite ceramide leads to reprogramming of macrophages toward immune suppressive TREM2+ tumor associated macrophages, which promote CD8 T cells exhaustion.

MiR-103a-3p Promotes Tumorigenesis of Breast Cancer by Targeting ETNK1.

We aimed to elucidate the molecular mechanism of miR-103a-3p regulating breast cancer progression. Firstly, clinical tissues was obtained from 2019-2023 at Yancheng Third People's Hospital, Yancheng, China. miR-103a-3p or ETNK1 expression in clinical tissues or breast cancer cell lines was analyzed with qRTPCR. MDA-MB-231 cells were performed with miR-103a-3p inhibitor or mimic, and OE-ETNK1. The proliferation and apoptosis ability were detected by CCK-8 and TUNEL assay. The xenograft models were established by inoculating transfected MDA-MB-231 cells to BALB/c mice. miR-103a-3p showed an overexpression and was related to poor prognosis in breast cancer. miR-103a-3p-deprived MDA-MB-231 cells displayed weaker levels of cell proliferation and higher rates of apoptosis. In contrast, ETNK1 was downregulated in breast cancer and proved to be a downstream target of miR-103a-3p. Xenograft models subjected to either miR-103a-3p antagomir treatment or ETNK1-knockdown resulted in tumor growth suppression. miR-103a-3p might promote breast cancer progression by inhibiting ETNK1.

THU493 The Role Of Liver Receptor Homolog1 (LRH-1) In Regulating Breast Cancer Progression By Modulating The Immune Response

Abstract Disclosure: Y. Wang: None. N. Krawczynska: None. S. Bendre: None. H. Vidana Gamage: None. A. Nelczyk: None. A. Das Gupta: None. E.R. Nelson: None. Breast cancer is the most common type of cancer and the second leading cause of death among American women. While immunotherapy holds much promise, the current FDA-approved immunotherapies are effective for only a subset of patients lacking expression of estrogen receptor, progesterone receptor and HER2, but positive for PD-L1. Thus, there is strong impetus to develop novel ways to engage the immune system. We have demonstrated that cholesterol metabolism and homeostasis within myeloid cells can either promote or suppress anti-cancer immunity. One of the regulators of cholesterol homeostasis is Liver Receptor Homolog 1 (LRH-1/NR5A2). We have found that elevated LRH-1 expression in breast tumors is associated with an increased survival. LRH-1 is highly expressed in myeloid cells, particularly neutrophils, a major immune population of the tumor microenvironment. Therefore, we hypothesized that LRH-1 plays modulatory roles in myeloid cells, that subsequently impacts breast cancer progression. Specifically, we evaluated the impact of LRH-1 on myeloid cell biology, including NETosis and phagocytosis. Treatment of neutrophils with an agonist of LRH-1, DLPC, resulted in decreased NETosis, a function of neutrophils previously implicated in recurrence and metastasis. Treatment with an antagonist resulted in increased NETosis. Both the agonist and antagonist exhibited dose-depended effects, strongly suggesting that LRH-1 regulates the process of NETosis. Macrophage phagocytosis is an important initial step towards antigen presentation and activation of T cells. Bone marrow derived macrophages treated with the LRH-1 antagonist exhibited decreased phagocytosis activity, in a dose dependent manner. Treatment with the agonist (DLPC) did not alter phagocytosis, but this may be because basal phagocytosis was already very high. Collectively, our data indicate that LRH-1 plays important roles in the regulation of myeloid cell function, including NETosis in neutrophils and phagocytosis in macrophages. Overall, these findings suggest that LRH-1 activity would be beneficial for an anti-cancer immune response, and therefore position LRH-1 as a therapeutic target. Our ongoing studies will assess the role of LRH-1 in other myeloid cell-related functions including efferocytosis, T-cell expansion and differentiation. Presentation: Thursday, June 15, 2023

DNAJB4 suppresses breast cancer progression and promotes tumor immunity by regulating the Hippo signaling pathway

PurposeBreast cancer is the most common cancer worldwide. Low DNAJB4 expression levels are strongly correlated with poor prognosis in breast cancer patients. However, the molecular mechanism by which DNAJB4 regulates breast cancer progression is unclear.MethodsThe expression of DNAJB4 was validated in human breast cancer tissues, normal human breast tissues, and breast cancer cell lines. CCK-8, colony-forming, and wound healing assays were used to assess the biological effect of DNAJB4 overexpression on cell proliferation and migration in MCF-7 cell lines. Bioinformatic analysis was used to identify the DNAJB4 related pathways in breast cancer. Epithelial-mesenchymal transition (EMT)-related biomarkers and Hippo pathway components were quantified by Western blots. Luciferase and Western blot assays were used to validate which miRNA regulates DNAJB4. In addition, the effects of DNAJB4 on in vivo tumor growth were assessed in xenograft models.ResultsDNAJB4 was expressed at low levels in human breast cancer tissues and breast cancer cell lines and correlated with poor prognosis. DNAJB4 overexpression significantly inhibited cell proliferation and migration in vitro by activating the Hippo pathway. The dual-luciferase assay showed that hsa-miR-183-5p targeted DNAJB4. Moreover, the effects of DNAJB4 could be reversed by miR-183-5p. In addition, the expression of DNAJB4 was strongly correlated with immune infiltration levels. Notably, DNAJB4 overexpression markedly enhanced CD4 + and CD8 + T cells and reduced PD-L1 levels in 4T1 tumors via the Hippo pathway, which retarded tumor growth in a subcutaneous xenograft tumor mouse model of 4T1 cells.ConclusionsThe present study demonstrated that DNAJB4 overexpression inhibited the malignant biological behavior of breast cancer by regulating the Hippo pathway and tumor immunosuppressive environment.

LncSNHG3 drives breast cancer progression by epigenetically increasing CSNK2A1 expression level.

Mounting evidence demonstrates that long noncoding RNAs (lncRNAs) have critical roles in the initiation and progression of cancer. Here, we report that small nucleolar RNA host gene 3 (SNHG3) is a key regulator of breast cancer progression. We analyzed RNA sequencing data to explore abnormally expressed lncRNAs in breast cancer. The effects of SNHG3 on breast cancer were investigated via in vitro and in vivo assays (CCK-8 assay, colony formation assay, flow cytometry assay, EdU assay, xenograft model, immunohistochemistry, and Western blot). The mechanism of SNHG3 action was explored through bioinformatics, RNA fluorescence in situ hybridization, luciferase reporter assay, RNA pull-down assay, chromatin immunoprecipitation assay and RNA immunoprecipitation assay. We found that SNHG3 expression was upregulated in breast cancer tissues and that its high expression level was associated with poor survival. We also found that high SNHG3 expression was partly induced by STAT3. Moreover, SNHG3 knockdown significantly repressed breast cancer cell growth both in vitro and in vivo. In the cytoplasm, SNHG3 facilitated the expression of Casein kinase II-A1 (CSNK2A1) by absorbing miR-485-5p and recruiting the HuR protein, participating in the malignant progression of breast cancer. Taken together, our study reveals a SNHG3-based regulatory network, which plays an oncogenic role in breast cancer and suggests that SNHG3 may serve as a potential target for the diagnosis and treatment of breast cancer.

G-protein-coupled receptor 141 mediates breast cancer proliferation and metastasis by regulating oncogenic mediators and the p-mTOR/p53 axis.

Breast cancer morbidity is surging towards the peak in females across the globe. An inherent property of cancer cells is enhanced cell proliferation rate and migration capability, leading to deregulated cell signaling cascades. G-protein-coupled receptors (GPCRs) have recently emerged as a hot-spot target in cancer research. We identify aberrant expression of G-protein-coupled receptor 141 (GPR141) in different breast cancer subtypes that correlate with poor prognosis. However, the molecular mechanism via which GPR141 advances breast cancer remains elusive. Increased GPR141 expression enhances the migratory behavior of breast cancer, driving oncogenic pathways both in vitro and in vivo through activation of epithelial to mesenchymal transition (EMT), oncogenic mediators and regulation of p-mTOR/p53 signaling. Our study unveils a molecular mechanism for p53 downregulation and activation of p-mTOR1 and its substrates in GPR141 overexpressed cells, accelerating breast tumorigenesis. We find that an E3 ubiquitin ligase, Cullin1, partly mediates p53 degradation via proteasomal pathway. Co-immunoprecipitation result shows that the phosphorylated form of 40S ribosome protein S6 (ps6., a p-mTOR1 substrate) forms a complex with Cullin1. These findings suggest an interplay between Cullin1 and p-mTOR1 in GPR141 overexpressed cells that downregulates p53 expression, thus inducing tumor growth. GPR141 silencing restores p53 expression and attenuates p-mTOR1 signaling events, thereby impeding proliferation and migration in breast cancer cells. Our findings describe the role of GPR141 in breast cancer proliferation, and metastasis, as well as in influencing the tumor microenvironment. Modulating GPR141 expression could pave the way for a better therapeutic approach to regulating breast cancer progression and metastasis.

Never in mitosis gene A-related kinase-8 promotes proliferation, migration, invasion, and stemness of breast cancer cells via β-catenin signalling activation

Never in mitosis gene A (NIMA)-related kinase-8 (NEK8) is involved in cell cycle progression, cytoskeleton development, and DNA damage repair. However, its role in breast cancer has not yet been explored. To investigate this, NEK8 was knocked down in MDA-MB-231, BT549, and HCC38 breast cancer cell lines. We observed a decrease in cell proliferation and colony formation owing to regulation of the G1/S and G2/M transitions. Furthermore, the expression of several cell cycle regulatory proteins was altered, including that of cyclin D1, cyclin B1, CDK4, CDK2, and surviving. NEK8 knockdown impaired cell migration and invasion as well as reduced the expression of epithelial-mesenchymal transition markers. Regarding stem-cell characteristics, NEK8 knockdown decreased the tumour sphere formation, aldehyde dehydrogenase activity, and stem-cell marker expression, including that of CD44, Sox2, Oct4a, and Nanog. Further analysis revealed that NEK8 interacts with β-catenin. Also, NEK8 knockdown promoted β-catenin degradation. NEK8-silenced MDA-MB-231 cells inhibited xenograft tumour growth, metastasis, and tumour initiation in vivo. Using the Oncomine and TNMplot public databases, we found a significant correlation between NEK8 overexpression and poor clinical outcomes in breast cancer patients. Thus, NEK8 may be a crucial regulator of breast cancer progression and a potential therapeutic target.

DNMT3a-dermatopontin axis suppresses breast cancer malignancy via inactivating YAP

Breast cancer (BC) is the most common malignant tumor in women worldwide, and its recurrence and metastasis negatively affect patient prognosis. However, the mechanisms underlying its tumorigenesis and progression remain unclear. Recently, the influence of dermatopontin (DPT), which is an extracellular matrix protein, has been proposed in the development of cancer. Here we found that DNMT3a-mediated DPT, promoter hypermethylation results in the downregulation of DPT expression in breast cancer and its low expression correlated with poor prognosis. Notably, DPT directly interacted with YAP to promote YAP Ser127 phosphorylation, and restricted the translocation of endogenous YAP from the cytoplasm to the nucleus, thereby suppressing malignant phenotypes in BC cells. In addition, Ectopic YAP overexpression reversed the inhibitory effects of DPT on BC growth and metastasis. Our study showed the critical role of DPT in regulating BC progression, making it easier to explore the clinical potential of modulating DPT/YAP activity in BC targeted therapies.

Transcriptomic and Functional Evidence That miRNA193a-3p Inhibits Lymphatic Endothelial Cell (LEC) and LEC + MCF-7 Spheroid Growth Directly and by Altering MCF-7 Secretome.

MicroRNA 193a-3p (miR193a-3p) is a short non-coding RNA with tumor suppressor properties. Breast cancer (BC) progression is governed by active interaction between breast cancer cells, vascular (V)/lymphatic (L) endothelial cells (ECs), and BC secretome. We have recently shown that miR193a-3p, a tumor suppressor miRNA, inhibits MCF-7 BC cell-driven growth of VECs via direct antimitogenic actions and alters MCF-7 secretome. Since LEC-BC cross-talk plays a key role in BC progression, we investigated the effects of miR193a-3p on MCF-7 secretome and estradiol-mediated growth effects in LECs and LEC + MCF-7 spheroids, and delineated the underlying mechanisms. Transfection of LECs with miR193a-3p, as well as secretome from MCF-7 transfected cells, inhibited LEC growth, and these effects were mimicked in LEC + MCF-7 spheroids. Moreover, miR193a-3p inhibited ERK1/2 and Akt phosphorylation in LECs and LEC + MCF-7 spheroids, which are importantly involved in promoting cancer development and metastasis. Treatment of LECs and LEC + MCF-7 spheroids with estradiol (E2)-induced growth, as well as ERK1/2 and Akt phosphorylation, and was abrogated by miR193a-3p and secretome from MCF-7 transfected cells. Gene expression analysis (GEA) in LEC + MCF-7 spheroids transfected with miR193a-3p showed significant upregulation of 54 genes and downregulation of 73 genes. Pathway enrichment analysis of regulated genes showed significant modulation of several pathways, including interferon, interleukin/cytokine-mediated signaling, innate immune system, ERK1/2 cascade, apoptosis, and estrogen receptor signaling. Transcriptomic analysis showed downregulation in interferon and anti-apoptotic and pro-growth molecules, such as IFI6, IFIT1, OSA1/2, IFITM1, HLA-A/B, PSMB8/9, and PARP9, which are known to regulate BC progression. The cytokine proteome array of miR193a-3p transfected MCF secretome and confirmed the upregulation of several growth inhibitory cytokines, including IFNγ, Il-1a, IL-1ra, IL-32, IL-33, IL-24, IL-27, cystatin, C-reactive protein, Fas ligand, MIG, and sTIM3. Moreover, miR193a-3p alters factors in MCF-7 secretome, which represses ERK1/2 and Akt phosphorylation, induces pro-apoptotic protein and apoptosis in LECs, and downregulates interferon-associated proteins known to promote cancer growth and metastasis. In conclusion, miR193a-3p can potentially modify the tumor microenvironment by altering pro-growth BC secretome and inhibiting LEC growth, and may represent a therapeutic molecule to target breast tumors/cancer.

TMEM189 as a target gene of MiR-499a-5p regulates breast cancer progression through the ferroptosis pathway.

MicroRNA (miR)-499a-5p has been reported to regulate the progression of various tumours. However, the role of miR-499a-5p in breast cancer is unclear. The purpose of this study was to investigate the role and mechanism of miR-499a-5p in breast cancer. The growth effect of miR-499a-5p on breast cancer cells was investigated by the CCK-8 assay, wound healing assay and Transwell invasion assay. The luciferase activity assay was used to verify the downstream targets of miR-499a-5p. The levels of GSH, MDA, and ROS were detected by kits. Quantitative real-time PCR and Western blot were used to determine the expression levels of TMEM189, COX-2, GPX4, and other related genes in cells. miR-499a-5p was down-regulated in MDA-MB-231 cells and was shown to reduced the viability, migration and invasion of MDA-MB-231 cells. Further studies revealed that TMEM189 is a target of miR-499a-5p. miR-499a-5p inhibited breast cancer cell growth by downregulating TMEM189. Furthermore, the down-regulation of TMEM189 promotes ferroptosis in breast cancer cells. The low expression of TMEM189 inhibited the development of breast cancer through the ferroptosis pathway. We have demonstrated for the first time that miR-499a-5p inhibits breast cancer progression by targeting the TMEM189-mediated ferroptosis pathway.

DNMT3B and TET1 mediated DNA methylation of LATS1 regulates BC progression via hippo signaling pathway.

Large tumor suppressor gene 1 (LATS1) is one of the key proteins in the Hippo signaling pathway, which plays a role in inhibiting tumor progression, but its anti-tumor role in tumors is inhibited. Nevertheless, the specific molecular mechanism of its role in breast cancer (BC) are still unclear. The expression of LATS1 in BC was detected by RT-qCR and WB. The biological effect of LATS1 on BC was verified in vitro and in vivo. DNA methylation sequencing and MSP were used to detect the effect of DNMT3B and TET1 on LATS1 methylation. Analysis of the effect of DNMT3B and TET1 on the biological behavior of BC cells via LATS1 by cell function experiment. Results indicated that the expression of LATS1 is lower in BC tumor tissues and BC cell lines. low LATS1 expression BC patients have a shorter overall survival. Increasing LATS1 expression can inhibit the proliferation, migration and invasion of BC. LATS1 found to have high DNA methylation changes in the promoter region. Moreover, DNMT3B was increased in BC tissues, and DNA demethylase TET1 was decreased in BC tissues. Furthermore, DNMT3B and TET1 regulate the methylation of LATS1, and methylation of LATS1 inactivates Hippo signaling pathway. Our results indicate that the methylation of LATS1 is regulated by DNMT3B and TET1 and regulates its protein expression and the function of Hippo signaling pathway.

MiR-27a-3p binds to TET1 mediated DNA demethylation of ADCY6 regulates breast cancer progression via epithelial-mesenchymal transition.

IntroductionAdenylyl cyclase isoform 6 (ADCY6) is a member of membrane-bound adenylate cyclase family that converts adenosine triphosphate (ATP) into cAMP and pyrophosphate. An increasing number of researchers have studied the role of ADCY6 in cancer. However, its specific role in breast cancer remains unknown.MethodsBioinformatics and clinical data were used to analyse the expression of ADCY6 in breast cancer. ADCY6 DNA methylation was analysed using DNA methylation-specific PCR and Bisulfite Sanger sequencing. Using lentiviral stable miRNA transfection together with cell biology functional assays and gene expression/target analysis, we investigated the interaction between miR-27a-3p, TET1 and ADCY6 in breast cancer.ResultsWe found that ADCY6 is expressed at low levels in breast cancer and leads to increases in the proliferation, invasion and migration of breast cancer cells. The low expression of ADCY6 is due to the lower demethylation of ten-eleven translocation methylcytosine dioxygenase 1 (TET1), and the methylation of ADCY6 can be altered by TET1. More importantly, bioinformatics analysis showed that TET1 is regulated by miR-27a-3p and regulates the methylation of ADCY6 to affect the EMT process of breast cancer cells, thereby affecting the malignant biological behaviour of breast cancer.ConclusionsOur study demonstrates that the methylation modification of ADCY6 is regulated by TET1 and leads to ADCY6 activation. miR-27a-3p negatively regulates the expression of TET1 and affects the EMT process of breast cancer through ADCY6, thereby promoting the malignant biological behaviour of breast cancer. Our results may provide new research ideas and directions for DNA methylation and EMT changes in breast cancer.

CircCEP85 upregulates IGF1 expression to promote breast cancer progression via sponging miR-1193

BackgroundIncreasing evidence has suggested that circular RNAs (circRNAs) play critical roles in breast cancer (BC) progression. However, the expression level and potential functional role of circRNA centrosomal protein 85 (circCEP85) in BC remains largely unknown. Here, we aimed to explore the role of circCEP85 in BC.MethodsThe levels of circCEP85, insuline-like growth factor I (IGF1) mRNA and microRNA-1193 (miR-1193) were examined by quantitative real-time polymerase chain reaction. The protein level was measured by Western blot. Cell proliferation, migration, apoptosis, angiogenesis and stemness were assessed by cell counting kit-8, 5-ethynyl-2’-deoxyuridine assay, transwell assay, flow cytometry, tube formation and sphere formation assays. Xenograft mouse models were conducted to evaluate the effect of circCEP85 in BC in vivo. Moreover, dual-luciferase reporter, RNA pull-down, and RNA immunoprecipitation (RIP) assays were preformed to confirm the interaction between miR-1193 and circCEP85 or IGF1.ResultsCircCEP85 was upregulated in BC tissues and cells. Silencing of circCEP85 inhibited proliferation, invasion, angiogenesis and stemness, but promoted apoptosis in BC cells in vitro. In addition, circCEP85 silencing inhibited tumor growth in vivo. Mechanistically, circCEP85 elevated IGF1 expression via sponging miR-1193 to promote breast cancer progression.ConclusionThe circCEP85-miR-1193-IGF1 axis regulated BC progression via the competitive endogenous RNA (ceRNA) mechanism. CircCEP85 might be a prognostic biomarker and therapeutic target for BC.

Abstract 6241: Macc1 regulates breast cancer progression and metastasis via remodeling tumor immune microenvironment

Abstract Although the established cancer cell lines exhibit less degrees of intratumoral heterogeneity when compared to human solid tumors, studies have suggested that, even within the same cancer cell line, substantial degree of heterogeneity may exist. In this study, we have performed single cell cloning experiment using 4T1 mouse mammary carcinoma cell line and compared the phenotypes of different clones. When compared to the parental 4T1 cell line, one clone (C12) showed markedly different phenotype by exhibiting less aggressive features in vitro and significantly slower tumor growth and metastasis in vivo. The tumor tissue of the C12 showed significant alteration in immune microenvironment as higher number of recruited immune cells including T and B cells. RNA sequencing of the C12 clones and parental 4T1 cells identified Macc1 as a significantly up-regulated gene in parental 4T1 cells. Metastasis-associated colon cancer 1 (MACC1) has been associated with poor prognosis and the promotion of metastasis within several types of cancer, especially colon cancer. The gene encoding the hepatocyte growth factor (HGF) receptor, MET, is a transcriptional target of MACC1. Next, we established Macc1-silenced 4T1 cells using the parental 4T1 cells and observed that Macc1-silencing resulted in a similar phenotype seen in the C12 clone; decreased cell proliferation and migration in vitro and slow tumor growth and metastasis in vivo. To mimic the metastasis in vitro, intravasation was determined as which tumor cells were co-cultured with HUVEC, human umbilical vein endothelial cells. We discovered tumor cell penetration into HUVEC cells was blocked in Macc1-silencing 4T1 group. According to CD45, CD3, and CD19 immunohistochemistry, we also defined immune cells, especially T and B cells, were highly infiltrated in Macc1-silencing tumor like as C12 clone tumor. These results were also reconstructed in EMT6, another TNBC mouse cell line model. In this study, we show that MACC1 can regulate tumor progression and metastasis by remodeling of immune microenvironment. Citation Format: Hye-Youn Son, Woo Hang Heo, Song Bin Li, Mingji Quan, Yu Jeong Her, Kyu-Jin Lee, Ju Hee Kim, Han-Byoel Lee, Wonshik Han, Hyeong-Gon Moon. Macc1 regulates breast cancer progression and metastasis via remodeling tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6241.

FOXF2 oppositely regulates stemness in luminal and basal-like breast cancer cells through the Wnt/beta-catenin pathway

The stemness of cancer cells contributes to tumorigenesis, the heterogeneity of malignancies, cancer metastasis, and therapeutic resistance. However, the roles and regulatory mechanisms maintaining stemness among breast cancer subtypes remain elusive. Our previous studies have demonstrated that ectopic expression and dynamic alteration of the mesenchymal transcription factor forkhead box F2 (FOXF2) differentially regulates breast cancer progression and metastasis organotropism in a cell subtype-specific manner. Here, we reveal the underlying mechanism by which FOXF2 enhances stemness in luminal breast cancer cells but suppresses that in basal-like breast cancer (BLBC) cells. We show that luminal breast cancer and BLBC cells with FOXF2-regulated stemness exhibit partial mesenchymal stem cell properties that toward osteogenic differentiation and myogenic differentiation, respectively. Furthermore, we show that FOXF2 activates the Wnt signaling pathway in luminal breast cancer cells but represses this pathway in BLBC cells by recruiting nuclear receptor coactivator 3 (NCoA3) and nuclear receptor corepressor 1 (NCoR1) to the promoters of Wnt family member 2B (WNT2B) and frizzled class receptor 1 (FZD1) genes to activate and repress their transcription, respectively. We propose that targeting the Wnt signaling pathway is a promising strategy for the treatment of breast cancers with dysregulated expression of FOXF2.