Human Triple-negative Breast Cancer Cell Lines Research Articles

ESM1 promotes triple-negative breast cancer cell proliferation through activating AKT/NF-κB/Cyclin D1 pathway.

Triple-negative breast cancer (TNBC) is a subtype of invasive breast cancer that tests negative for PR, ER and excess HER2 protein. TNBC has a greater progression potential with poorer prognosis, compared with other types of breast cancer. Endothelial cell-specific molecule 1 (ESM1), also known as endocan, is overexpressed in various cancers including breast cancer and may play an important role in cancer progression. The online resource of The Cancer Genome Atlas (TCGA) was used for analyzing the expression alteration of ESM1 in breast cancer patient tissues. We examined the changes of various malignant behaviors of TNBC cell and in vivo tumor growth after inhibiting or overexpressing ESM1 in two human TNBC cell lines, MDA-MB-468 and MDA-MB-231. When ESM1 was knocked down or overexpressed in TNBC cell, AKT and p65 phosphorylation and Cyclin D1 expression were analyzed by western blotting. The ESM1-overexpressing TNBC cell was treated with MK-2206 and BAY-117082 at various concentrations. Our analyses show that ESM1 is overexpressed in TNBC cell lines as well as patient tissues, which is correlated to poor prognosis. Our results demonstrate that ESM1 knockdown decreases while overexpression of ESM1 increases in vitro proliferation, migration and invasion of TNBC cell and knockdown of ESM1 inhibits in vivo TNBC tumor growth. Our mechanistic study further discloses that ESM1 promotes the proliferation of TNBC cell through activating an Akt-dependent NF-κB/Cyclin D1 pathway. Our results demonstrate that ESM1 knockdown decreases while overexpression of ESM1 increases in vitro migration, proliferation and invasion of TNBC cell and knockdown of ESM1 inhibits in vivo tumor growth of TNBC in the xenograft mouse model. Our mechanistic study further discloses that ESM1 promotes the proliferation of TNBC cell through activating the Akt-dependent NF-κB/CyclinD1 pathway. Our findings expand the knowledge about the molecular mechanisms underlying TNBC progression and provide rationale for using ESM1 as a therapeutic target or prognostic marker for TNBC.

LncRNA MIR503HG inhibits cell proliferation and promotes apoptosis in TNBC cells via the miR-224-5p/HOXA9 axis

Triple-negative breast cancer (TNBC) is a highly invasive subtype of breast cancer. This study investigated the molecular mechanism and influences of MIR503HG, miR-224-5p, and homeobox A9 (HOXA9) on TNBC cell growth and migration. Dual-luciferase reporter gene and RNA immunoprecipitation were performed to examine the regulation of MIR503HG, miR-224-5p, and HOXA9. Cell proliferation, apoptosis, migration, and invasion were evaluated by colony formation, flow cytometry, and Transwell assays. Finally, nude mice were employed to investigate the influence of MIR503HG on TNBC tumor growth. HOXA9 protein levels were detected by immunohistochemical staining. MIR503HG and HOXA9 expression were reduced in TNBC, while miR-224-5p was increased. Overexpression of MIR503HG or HOXA9 reduced the cell migration ability and proliferation and promoted apoptosis, and knockdown of MIR503HG or overexpression of miR-224-5p exhibited the opposite effects. Furthermore, MIR503HG promoted HOXA9 expression by inhibiting miR-224-5p. Overexpression of miR-224-5p reversed the effects of MIR503HG overexpression on TNBC cells, while overexpression of HOXA9 reversed the effect of MIR503HG knockdown. Additionally, an in vivo study proved that MIR503HG inhibited TNBC tumor growth via the miR-224-5p/HOXA9 axis. MIR503HG inhibited cell proliferation and promoted the apoptosis of TNBC cells via the miR-224-5p/HOXA9 axis, which may function as a novel target for the treatment of TNBC.

Abstract PS19-12: The study of jag1-notch in the extravasation of triple negative breast cancer cells in metastasis

Abstract In breast cancer, the primary tumor is usually not lethal, while metastatic spread to other organs is a frequent cause of death. Up to 20% of breast cancer diagnoses are triple negative breast cancer (TNBC) makes and late stage metastatic TNBC can have a 5-year survival as low as 11%. TNBC is unresponsive to hormonal and targeted therapies of other types of breast cancer. Surgery and adjuvant chemotherapy are effective treatment options for early stage disease, but there are very few therapeutic options for advanced metastatic TNBC spread. For metastasis to occur, circulating tumor cells (CTCs) must cross the endothelial barrier twice: first to migrate away from the primary tumor and enter systemic circulation (intravasation), and then to exit circulation to colonize other tissues (extravasation). We are currently exploring the role of Jagged-1 (Jag1), a Serrate class Notch ligand, in the metastatic process. Jag1 is expressed in many human TNBC cell lines and its expression is associated with a worse prognosis in the clinic. Our data suggest that Jag1 presented on tumor cells promotes extravasation behavior, particularly TNBC binding to endothelium and subsequent transendothelial migration (TEM). Our preliminary studies have blocked Jag1 function with the Notch decoy N110-24, which utilizes EGF-like repeats of the Notch1 receptor that unproductively bind Jag1. Treatment with N110-24 reduces the ability of D3H2LN cells, a Jag1high derivative of the MDA-MB-231, to attach endothelial cells in vitro. Treatment with N110-24 also impedes the ability of these highly metastatic human TNBC cells to migrate across a monolayer of human primary endothelial cells. We therefore conclude that Jag1-Notch signaling inhibition via N110-24 suppresses activities required for TEM of TNBC cells. However, the mechanistic role of Jag1, however, is not understood in TEM. Both TNBC and endothelial cells express Jag1, and the aforementioned secreted decoys may target Jag1 signaling either within TNBC cells, within endothelial cells, or between cell types. We hypothesize that tumor expression of Jag1 is critical for metastasis. In order to explore the effects of tumor-derived Jag1 on tumor-intrinsic signaling, we have knocked out Jag1 in D3H2LN cells and generated multiple Jag1KO clonal lines, which have been interrogated for downstream transcriptional changes related to TEM. To explore the effects of tumor-derived Jag1 on neighboring endothelium, we are studying the extravasation capabilities of Jag1KO clonal lines using dynamic systems that will track TNBC adhesion, rolling, and TEM under dynamic flow in vitro as well as characterizing their metastatic potential in vivo. Our studies are elucidating a mechanism of Jag1-mediated metastasis via endothelial interactions. Citation Format: Benjamin Gordon, Reyhaan A Chaudhri, Tim Sargis, LA Naiche, Jan K Kitajewski. The study of jag1-notch in the extravasation of triple negative breast cancer cells in metastasis [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 PS19-12.

Abstract PS17-49: Investigating CDK4/6 inhibition in triple negative breast cancer

Abstract The purpose of our study is to evaluate the immunological effects of CDK4/6 inhibitors in pre-clinical models of triple negative breast cancer (TNBC). CDK4/6 inhibitors, including abemaciclib, are currently approved to treat patients with metastatic hormone receptor-positive (HR+), Her2-negative breast cancer. We previously reported in our Her2+ model that CDK4/6 inhibitors promote anti-tumor immunity through inducing tumor cell antigen presentation via activating tumor endogenous retroviral elements, which induce type III interferon production and MHC-I upregulation (Goel, DeCristo, et al., Nature, 2017). We also uncovered important anti-tumor effects on the immune system: CDK4/6 inhibitors decrease T regulatory cell proliferation without affecting cytotoxic CD8 T cell numbers. TNBC has been considered a poor candidate for CDK4/6 inhibitor therapy, as tumors often lose retinoblastoma (Rb) protein expression/function, which is critical for CDK4/6 inhibitor-induced cell cycle arrest. However, Rb mutation or loss occurs in only ~20% of TNBC cases and we found Rb expressing murine and human TNBC cell lines decreased proliferation in vitro in response to abemaciclib. In our preclinical model of TNBC, abemaciclib induced tumor regression. Consistent with previous findings, TNBC tumor cells upregulated MHC-I upon abemaciclib treatment, suggesting increased antigen presentation. Tumor cell-surface PD-L1 was also increased with abemaciclib both in vitro and in vivo, as assessed by flow cytometry and RT-qPCR. These results are encouraging, given that αPD-L1 therapy (Atezolizumab) in combination with chemotherapy (nab-paclitaxel) has recently been approved as standard care for metastatic TNBC and the IMpassion130 trial reported enhanced progression-free and overall survival in patients with PD-L1+ tumors. We also found that abemaciclib increased CD8+ and CD4+ T cells and decreased PD1+ CD8 and CD4 cells in the spleen. Furthermore, total numbers of naïve CD8+ and CD4+ T cells increased with abemaciclib treatment, suggesting a favorable anti-tumor systemic immunological effect. Our data suggest the potential efficacy of CDK4/6 inhibitors in combination with αPD-L1 in the treatment of Rb+ TNBC. Deeper analysis of the mechanisms involved in regulating PD-L1 and enhancing naïve T cells should enable us to evaluate combination therapies using CDK4/6 inhibitors for this particularly deadly breast cancer subtype. Citation Format: Qiuchen Guo, Gregory J Goreczny, Milos Spasic, Adam Maynard, Sandra S McAllister. Investigating CDK4/6 inhibition in 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 PS17-49.

Sulforaphane-cisplatin combination inhibits the stemness and metastatic potential of TNBCs via down regulation of sirtuins-mediated EMT signaling axis

BackgroundSulforaphane (SFN) is a naturally occurring organosulfur compound found in cruciferous vegetables such as broccoli, brussels sprouts and cabbage. SFN is known for its multiple therapeutic properties, such as HDAC inhibitory, chemo preventive and anti-cancer effects. Cisplatin (CIS) has limited effect against metastatic triple-negative breast cancer (TNBC). Additionally, CIS impose severe side effects to normal cells, and later TNBC cells develops resistance. Studies suggest that the overexpression of sirtuins (SIRTs) promotes CIS resistance and metastasis by activating epithelial-to-mesenchymal transition (EMT) pathway in TNBC. PurposeIn view of the above information, we investigated the therapeutic efficacy of SFN, in combination with CIS against TNBC metastasis and CIS resistance. MethodsThe anti-cancerous effect of SFN-CIS combination on human TNBC cell lines was demonstrated by utilizing MTT assay and, apoptosis and cell cycle assay followed by FACS analysis. The synergistic effect of SFN-CIS combination on the experimental metastasis was demonstrated by utilizing migration, invasion, chemotaxis, mammosphere and colony formation assay on human TNBC MDA-MB-231 and MDA-MB-468 cells. The role of SIRTs-mediated EMT signaling axis in the metastasis and chemoresistance was investigated by western blotting technique as well as sirtuin activity tests. This was further validated by using Chromatin immunoprecipitation (ChIP) analysis. ResultsWe found that SFN-CIS combination synergistically inhibits cellular growth of MDA-MB-231 and MDA-MB-468 cells. More importantly, SFN was found to protect normal kidney cells from CIS-induced toxicity. Further, SFN-CIS combination was found to synergistically inhibit metastatic-events via significantly altering EMT markers which was further associated with the suppression of SIRTs functions in TNBC cells. ChIP analysis validated that SFN-CIS combination suppresses EMT mechanism through altered chromatin modifications at E-cadherin promoter resulting in its re-expression. ConclusionThe results of the current study suggests that CIS when supplemented with SFN, inhibits metastasis and stemness potential of TNBC cells by down regulating SIRTs-mediated EMT cascade. Overall this study affirms that, this novel combination could be a promising strategy against SIRT-mediated TNBC metastasis and CIS-resistance.

Investigation of the antitumor activity and toxicity of cisplatin loaded pH-sensitive-pegylated liposomes in a triple negative breast cancer animal model

Over the last decade, studies showed evidence of enhanced sensitivity of triple negative breast cancer (TNBC) to DNA-damaging agents, such as cisplatin (CIS), compared to other breast cancer subtypes. Despite its high efficacy, CIS has its use limited by dose-dependent side effects, mainly nephrotoxicity and neurotoxicity. In previous studies, it has been demonstrated by our research group that the encapsulation of CIS into pH-sensitive-PEGylated liposomes (pHPL-CIS) might allow to overcome some of its side effects. In the present work, we investigated the antitumor activity and toxicity of pHPL-CIS on Balb/c nude female mice bearing MDA-MB-231 tumor, a human TNBC cell line. Treatments with free-CIS or pHPL-CIS were both administered i.v. once a week for three weeks in cumulative doses of 12 and 30 mg/kg. Treatments in the higher dose allowed for similar tumor regression at the end of the experiment, however, the treatment with pHPL-CIS allowed for an earlier appearance of the antitumor effect. Additionally, animals receiving the higher dose of free-CIS presented weight loss, hepatic degeneration and 20% mortality rate, which was not observed for animals that received the high dose of pHPL-CIS. Thus, pHPL-CIS might be considered a promising candidate for the management of TNBC.

Interleukin 17A promotes cell migration, enhances anoikis resistance, and creates a microenvironment suitable for triple negative breast cancer tumor metastasis.

The aim of this study was to investigate the role of IL-17A in the cancer microenvironment and the recurrence of triple negative breast cancer (TNBC). Using human TNBC cell lines, the role of IL17-A was investigated by knocked down of IL-17A (ΔIL-17A) and by administration of IL-17A into the culture medium. Cell proliferation assays, migration assays, as well as Western blot analysis and real-time PCR, were used to evaluate IL-17A-related signaling. Three types of 4T1 cells were implanted into BALB/c mice, namely wild type (WT), ΔIL-17A, and WT + neutralizing IL-17 antibody (WT + Ab) cells. Tumor weight, necrosis area, and the number of circulating tumor cells (CTCs) were measured. Immunohistochemistry and Western blotting were used to analyze expression of CD34, CD8, and TGF-β1 as well as anoikis resistance. The Kaplan-Meier's method was used to correlate IL-17A expression and patient outcome, including disease-free survival (DFS) and overall survival (OS). Our results demonstrated that IL-17A was able to stimulate the migratory activity, but not the growth rate, of MDA-MB-231/468 cells. In vivo, for the ΔIL-17A group, there was an increase in necrosis area, a decrease in tumor CD34 expression and a reduction in the number of CTCs. Furthermore, in WT + Ab group, there was a decreased in tumor expression of CD34, fewer CD8 ( +) cells, and fewer CTCs, but an increase in expression of TGF-β1 expression. Both of the above were compared to the WT group. Knockdown of IL-17A also decreased anoikis resistance in human TNBC and the murine 4T1 cell lines. Kaplan-Meier analysis disclosed a negative correlation between tumor expression of IL-17A and OS in TNBC patients. We conclude that IL-17A promotes migratory and angiogenic activity in tumors, enhances anoikis resistance, and modulates the immune landscape of the tumor microenvironment such changes favor cancer metastasis.

Lamin B2 promotes the progression of triple negative breast cancer via mediating cell proliferation and apoptosis.

Triple negative breast cancer (TNBC) is a more common type of breast cancer with high distant metastasis and poor prognosis. The potential role of lamins in cancer progression has been widely revealed. However, the function of lamin B2 (LMNB2) in TNBC progression is still unclear. The present study aimed to investigate the role of LMNB2 in TNBC. The cancer genome atlas (TCGA) database analysis and immunohistochemistry (IHC) were performed to examine LMNB2 expression levels. LMNB2 short hairpin RNA plasmid or lentivirus was used to deplete the expression of LMNB2 in human TNBC cell lines including MDA-MB-468 and MDA-MB-231. Alterations in cell proliferation and apoptosis in vitro and the nude mouse tumorigenicity assay in vivo were subsequently analyzed. The human TNBC tissues shown high expression of LMNB2 according to the bioinformation analysis and IHC assays. LMNB2 expression was correlated with the clinical pathological features of TNBC patients, including pTNM stage and lymph node metastasis. Through in vitro and in vivo assays, we confirmed LMNB2 depletion suppressed the proliferation and induced the apoptosis of TNBC cells, and inhibited tumor growth of TNBC cells in mice, with the decrease in Ki67 expression or the increase in caspase-3 expression. In conclusion, LMNB2 may promote TNBC progression and could serve as a potential therapeutic target for TNBC treatment.

BCL-XL blockage in TNBC models confers vulnerability to inhibition of specific cell cycle regulators.

Cell cycle regulators are frequently altered in Triple-Negative Breast Cancer (TNBC). Emerging agents targeting these signals offer the possibility to design new combinatorial therapies. However, preclinical models that recapitulate TNBC primary resistance and heterogeneity are essential to evaluate the potency of these combined treatments.Methods: Bioinformatic processing of human breast cancer datasets was used to analyse correlations between expression levels of cell cycle regulators and patient survival outcome. The MMTV-R26Met mouse model of TNBC resistance and heterogeneity was employed to analyse expression and targeting vulnerability of cell cycle regulators in the presence of BCL-XL blockage. Robustness of outcomes and selectivity was further explored using a panel of human breast cancer cells. Orthotopic studies in nude mice were applied for preclinical evaluation of efficacy and toxicity. Alterations of protein expression, phosphorylation, and/or cellular localisation were analysed by western blots, reverse phase protein array, and immunocytochemistry. Bioinformatics was performed to highlight drug's mechanisms of action.Results: We report that high expression levels of the BCL2L1 gene encoding BCL-XL and of specific cell cycle regulators correlate with poor survival outcomes of TNBC patients. Blockage of BCL-XL confers vulnerability to drugs targeting CDK1/2/4, but not FOXM1, CDK4/6, Aurora A and Aurora B, to all MMTV-R26Met and human TNBC cell lines tested. Combined blockage of BCL-XL and CDK1/2/4 interfered with tumour growth in vivo. Mechanistically, we show that, co-targeting of BCL-XL and CDK1/2/4 synergistically inhibited cell viability by combinatorial depletion of survival and RTK/AKT signals, and concomitantly restoring FOXO3a tumour suppression actions. This was accompanied by an accumulation of DNA damage and consequently apoptosis.Conclusions: Our studies illustrate the possibility to exploit the vulnerability of TNBC cells to CDK1/2/4 inhibition by targeting BCL-XL. Moreover, they underline that specificity matters in targeting cell cycle regulators for combinatorial anticancer therapies.

LncIHAT Is Induced by Hypoxia-Inducible Factor 1 and Promotes Breast Cancer Progression.

Hypoxia induces thousands of mRNAs and miRNAs to mediate tumor malignancy. However, hypoxia-induced long noncoding RNA (lncRNA) transcriptome and their role in triple-negative breast cancer (TNBC) have not been defined. Here we identified hypoxia-induced lncRNA transcriptome in two human TNBC cell lines by whole transcriptome sequencing. AC093818.1 was one of 26 validated lncRNAs and abundantly expressed in TNBC in vitro and in vivo. 5'- and 3'-rapid amplification of cDNA ends assays revealed that the isoform 2 was a dominant AC093818.1 transcript in TNBC cells and thus referred to as lncIHAT (lncRNA induced by hypoxia and abundant in TNBC). Hypoxia-inducible factor 1 (HIF1) but not HIF2 bound to the hypoxia response element at the promoter of lncIHAT to activate its transcription in hypoxic TNBC cells. LncIHAT promoted TNBC cell survival in vitro and tumor growth and lung metastasis in mice. Mechanistically, lncIHAT was required for the expression of its proximal neighboring oncogenic genes PDK1 and ITGA6 in TNBC cells and tumors. Reexpression of PDK1 and ITGA6 rescued survival and growth of lncIHAT knockdown TNBC cells in vitro. Collectively, these findings uncovered lncIHAT as a new hypoxia-induced oncogenic cis-acting lncRNA in TNBC. IMPLICATIONS: This study systematically identified hypoxia-induced lncRNA transcriptome in TNBC and sheds light on multiple layers of regulatory mechanisms of gene expression under hypoxia.

Targeting IL-3Rα on tumor-derived endothelial cells blunts metastatic spread of triple-negative breast cancer via extracellular vesicle reprogramming

The lack of approved targeted therapies highlights the need for new treatments for triple-negative breast cancer (TNBC) patients. Interleukin-3 (IL-3) acts as an autocrine factor for tumor–endothelial cells (TEC), and exerts pro-angiogenic paracrine action via extracellular vesicles (EVs). IL-3Rα blockade on TEC changes TEC-EV (anti-IL-3R-EV) microRNA (miR) content and promotes the regression of established vessels. As TEC is the doorway for “drug” entry into tumors, we aimed to assess whether IL-3R blockade on TEC impacts tumor progression via its unique EV cargo. First, the expression of IL-3Rα was evaluated in 27 human TNBC samples. It was noticed that, besides TEC and inflammatory cells, tumor cells from 55.5% of the human TNBC samples expressed IL-3Rα. Using human TNBC cell lines for in vitro studies, we found that, unlike native TEC-EVs (nEVs), anti-IL-3R-EVs increase apoptosis and reduced cell viability and migration. In vivo, anti-IL-3R-EV treatment induced vessel regression in established tumors formed of MDA-MB-231 cells, decreased Vimentin, β-catenin, and TWIST1 expression, almost abolished liver and lung metastases from primary tumors, and reduced lung metastasis generated via the intravenous injection of MDA-MB-231 cells. nEVs depleted of miR-24-3p (antago-miR-24-3p-EVs) were effective as anti-IL-3R-EVs in downregulating TWIST1 and reducing metastatic lesions in vivo. Consistent with network analyses of miR-24-3p gene targeting, anti-IL-3R-EVs and antago-miR-24-3p-EVs upregulate SPRY2 in MDA-MB-231 cells. Finally, SPRY2 silencing prevented anti-IL-3R-EV and antago-miR-24-3p-EV-mediated apoptotic cues.Overall, these data provide the first evidence that IL-3Rα is highly expressed in TNBC cells, TEC, and inflammatory cells, and that IL-3Rα blockade on TEC impacts tumor progression.

<p>MicroRNA-139 Suppresses the Tumorigenicity of Triple Negative Breast Cancer Cells by Targeting SOX8</p>

PurposeThe effects of miR-139 on the tumorigenicity of triple negative breast cancer (TNBC) and the underlying mechanisms were investigated.MethodsNormal human breast epithelial (MCF-10A) and TNBC cell lines (HCC1806 and BT549) were used for microRNA (miR)-139 overexpression, SOX8 overexpression, and knockdown studies as in vitro models of TNBC. The expression of SOX8 and miR-139 was detected by reverse transcription-polymerase chain reaction. CCK8 and clone formation assays were used to evaluate cell proliferation ability. Transwell assays and flow cytometry were used to test cell migration and apoptosis, respectively. Cell tumorigenicity was examined by tumor sphere formation assays. The interaction between miR-139 and SOX8 was examined by dual-luciferase reporter assays. The expression of SOX8, cleaved caspase-3, and cleaved caspase-9 was analyzed by Western blotting. The findings were validated in vivo using a nude mouse transplanted tumor model.ResultsSOX8 expression was higher (P < 0.05) and miR-139 expression was lower (P < 0.05) in HCC1806 and BT549 cells than in MCF-10A cells. SOX8 overexpression significantly enhanced cell proliferation and migration, reduced the rate of cell apoptosis, and increased tumor sphere formation (P < 0.05) compared with the control group, whereas SOX8 knockdown had the opposite effect (P < 0.05). Overexpression of miR-139 markedly decreased cell proliferation and migration, increased cell apoptosis in vitro, and decreased tumor angiogenesis and volume in vivo (P < 0.05).ConclusionmiR-139 suppressed the tumorigenicity of TNBC cells by targeting SOX8.

MiR-452 Reverses Abnormal Glycosylation Modification of ERα and Estrogen Resistance in TNBC (Triple-Negative Breast Cancer) Through Targeting UGT1A1.

Background: The breast epithelial cells in patients with triple-negative breast cancer (TNBC) actually have specific estrogen receptor (ER) expression, and the abnormal glycosylation of UGT1A1 in TNBC cells resulted in abnormal expression and function of ERα through regulating the modification of ERα. Therefore, our study targets the role of UGT1A1 expression, then glycosylation modification of ERα (estrogen receptor α) and estrogen resistance in development of TNBC.Methods: The differential expression of mRNA and miRNA in TNBC tissues was tested. Luciferase activity was analyzed in TNBC cells treated with miR-452. Moreover, the human mammary gland and TNBC cell lines were dealt with estrogen and miR-452 or its inhibitors, then proliferation ability was further determined. Moreover, the role of interaction between UGT1A1 and ERα in the glycosylation modification of ERα and UGT activity, and metabolism of estrogen were assessed. The effects of miR-452 on TNBC by improving abnormal glycosylation modification of ERα by targeting UGT1A1 and estrogen resistance were studied in vitro and in vivo.Results: The expression level of UGT1A1 in TNBC tumor tissues was higher than its matched para-tumorous tissues, but the miR-452 expression was opposite. The glycosylation modification site of ERα expressed in TNBC cells was different from that of normal mammary epithelial cells. The estrogen 17β-estradiol (E2) significantly promoted mitotic entry of TNBC cells. The interaction between UGT1A1 and ERα affected the expression level of each other, as well as the UGT enzyme activity and proliferation of TNBC cells. UGT1A1 induced production of intracellular estrogens and TNBC proliferation, but it could be reversed by overexpression of ERα. Upregulation of ERα caused the downregulation of UGT1A1 and marked decrease of intracellular estrogen products, and then suppressed TNBC proliferation. Moreover, UGT1A1 was the target gene of miR-452; miR-452 antagomir restrained TNBC xenograft.Conclusion: Our results demonstrated that estrogen was a positive factor in the proliferation of TNBC cells at onset of mitosis through accentuating the expression and enzyme activity of UGT1A1. However, miR-452 targeted to UGT1A1, then regulated glycosylation modification of ERα, estrogen metabolism, and TNBC development associated with estrogen resistance.

Abstract 2406: Novel thienopyrimidine analog TPH104 induces immunogenic cell death in TNBC cells

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with high propensity for metastasis and recurrence, accounting for one-fourth of all breast cancer deaths. Despite being chemo-sensitive to anthracyclines and taxanes, TNBCs have poor prognosis due to development of resistance often due to the overexpression of efflux transporters, increased DNA repair, and occurrence of genetic mutations that decrease the likelihood of apoptosis. Recently, we discovered a novel class of thieno-pyrimidin-hydrazinyl (TPH) compounds that in the nanomolar range, selectively elicited a unique, caspase-independent cell death in TNBC cells. The lead compound, TPH104, selectively activates receptor interacting protein kinase 1 (RIPK1), thus inducing a necroptosis like cell death. Since necroptosis is known to release endogenous immunogenic markers, we wanted to study whether TPH104 can promote immunogenic cell death (ICD) in TNBC cells and activate cells in the tumor immune milieu. ICD is characterized by presence of damage-associated molecular patterns (DAMPs) such as cell surface exposure of calreticulin (CRT), secretion of ATP, and release of high-mobility group box protein B1 (HMGB1) from dying tumor cells. Methods: Human TNBC cell line MDA-MB-231 cells were treated with TPH104 and cell survival was examined by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT), clonogenic and live-cell imaging assays. Western blot analysis was used to assess death signal transducers. DAMPs were examined by ELISA, luminescence-based assay systems and immunofluorescence. Activation of mouse bone marrow derived dendritic cells (BMDCs) in response to conditioned media (CM) harvested after treatment of MDA-MB-231 cells with TPH104 (TPH104-CM) and/or mitoxantrone (MX, positive control) for 48h was evaluated using flow cytometry analysis of maturation markers (CD86, MHCII) expressed on CD11C+ BMDCs. Inflammatory gene expression was evaluated by q-PCR. Results: The results demonstrated that TPH104 reduced cell survival and induced death of MDA-MB-231 cells in a dose-dependent pattern. TPH104 induced ICD, as evidenced by increased release of ATP, as well as surface exposure of CRT. Flow cytometric analysis revealed TPH104-CM treatment increased CD11C+CD86+MHC+ BMDCs as robustly as MX-CM. Further, TPH104 posed no toxicity to mice in a single dose toxicity study. Conclusion: TPH104 induced selective ICD in MDA-MB-231 cells. TPH104 similar to MX primed cells to ICD and increased extracellular ATP levels and surface exposure of CRT. TPH104 promoted maturation of CD11C+ BMDCs and increased inflammatory gene expression. Our study demonstrated TPH104 as an ICD-inducer and immunomodulator in TNBC. Citation Format: Diwakar Bastihalli Tukaramrao, Siddharth Saraiya, Ross A. Hanely, Saloni Malla, Amit K. Tiwari. Novel thienopyrimidine analog TPH104 induces immunogenic cell death in TNBC cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2406.

Abstract 1761: Examination of therapeutic potential of epigenetic modulation by dual HDAC-LSD1 inhibition in murine models of triple negative breast cancer (TNBC)

Abstract This study examines the effects of the epigenetic modulator 4SC-202, a dual histone deacetylase (HDAC) /lysine-specific histone demethylase 1A (LSD1) small molecule inhibitor, on tumor growth and metastasis in a mouse model of breast cancer. 4SC-202 differs from clinically available HDAC inhibitors such as Vorinostat (SAHA) by specifically targeting class I HDACs including HDAC1, HDAC2, and HDAC3 and the histone demethylase LSD1. The hypothesis to be examined is that epigenetic modulation via dual inhibition of HDAC1 and LSD1 improves disease control in breast cancer. In vitro data indicates that 4SC-202 is significantly cytotoxic and cytostatic to the highly metastatic murine Triple Negative Breast Cancer (TNBC) murine model 4T1 and the human TNBC cell line MDA-MB-231 and reduces cancer cell migration. In vivo studies conducted in the syngeneic 4T1 model which closely mimics human TNBC in terms of sites of metastasis, show increased necrosis in 4T1 tumors, reduced tumor burden in vivo as compared to Vorinostat treatment, and significantly reduced metastasis. Furthermore, multiple studies in different cell types indicate that the mechanism of action of 4SC-202 may involve effects on cancer stem cells, which are able to self-renew and form metastatic lesions. It has been shown that a marker of cancer stem cells (CSC's) in breast cancer is high aldehyde dehydrogenase (ALDH) activity and that ALDH high cells mediate metastasis. In the 4T1 syngeneic mouse model of breast cancer, inhibition of ALDH activity suppresses stem-like cell properties. Preliminary data suggests 4SC-202 substantially reduces the ALDH high 4T1 cancer stem population. Transcriptome bulk RNA -sequencing analysis from 4T1 tumors reveal changes in metastatic related pathways in 4SC-202 treated tumors, including significant changes to the expression levels of genes implicated in focal adhesion and ECM-receptor interaction pathways. Furthermore, 4SC-202 appears to alter expression of cytokines which serve as chemokines, potentially leading to increased expression of tumor infiltrating lymphocytes. In summary, treatment of a highly metastatic murine model of TNBC with 4SC-202 significantly reduces metastasis, and warrants further preclinical studies in conjunction with standard of care chemotherapy treatment. Citation Format: Shanta M. Messerli, Mariah M. Hoffman, Jessica S. Zylla, Etienne Gnimpieba, Keith W. Miskimins. Examination of therapeutic potential of epigenetic modulation by dual HDAC-LSD1 inhibition in murine models of triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1761.

Shikonin is a novel and selective IMPDH2 inhibitor that target triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is heterogeneous disease with a poor prognosis. It is therefore important to explore novel therapeutic agents to improve the clinical efficacy for TNBC. The inosine 5'-monophosphate dehydrogenase 2 (IMPDH2) is a rate-limiting enzyme in the de novo synthesis of guanine nucleotides. It is always overexpressed in many types of tumors, including TNBC and regarded as a potential target for cancer therapy. Through screening a library of natural products, we identified shikonin, a natural bioactive component of Lithospermum erythrorhizon, is a novel and selective IMPDH2 inhibitor. Enzymatic analysis using Lineweaver-Burk plot indicates that shikonin is a competitive inhibitor of IMPDH2. The interaction between shikonin and IMDPH2 was further investigated by thermal shift assay, fluorescence quenching, and molecular docking simulation. Shikonin treatment effectively inhibits the growth of human TNBC cell line MDA-MB-231, and murine TNBC cell line, 4T1 in a dose-dependent manner, which is impaired by exogenous supplementation of guanosine, a salvage pathway of purine nucleotides. Most importantly, IMPDH2 knockdown significantly reduced cell proliferation and conferred resistance to shikonin in TNBC. Collectively, our findings showed the natural product shikonin as a selective inhibitor of IMPDH2 with anti-TNBC activity, impelling its further study in clinical trials.

Myeloid-derived suppressor cell depletion therapy targets IL-17A-expressing mammary carcinomas

Triple-negative breast cancer (TNBC) is an invasive subtype of breast cancer but paradoxically associated with increased tumor-infiltrating leukocytes. The molecular and cellular mechanisms underlying TNBC immunobiology are incompletely understood. Interleukin (IL)-17A is a pro-inflammatory cytokine that has both pro- and anti-tumor effects and found in 40–80% of TNBC samples. We report here that IL-17A mRNA and protein are detectable in some human TNBC cell lines and further upregulated by IL-23 and LPS stimulation. Furthermore, the impact of tumor-derived IL-17A in host immune response and tumor growth was examined using murine TNBC 4T1 mammary carcinoma cells transduced with an adenoviral vector expressing IL-17A (AdIL-17A) or control vector (Addl). Compared to Addl-transduction, AdIL-17A-transduction enhanced 4T1 tumor growth and lung metastasis in vivo, which was associated with a marked expansion of myeloid-derived suppressor cells (MDSCs). However, AdIL-17A-transduction also induced strong organ-specific and time-dependent immune activation indicated by dynamic changes of NK cells, B cells, CD4, and CD8 T cells in peripheral blood, lung, and tumor site, as well as the plasma levels of IFNγ. Such findings highlight that tumor-associated IL-17A induces concurrent immune activation and immune suppression. Administration of anti-Gr1 or anti-G-CSF antibody effectively depleted MDSCs in vivo, markedly reducing the growth of AdIL-17A-transduced 4T1 tumors, and eliminating lung metastasis. Collectively, our study demonstrates that MDSC depletion is an effective and practical approach for treating IL-17A-enriched mammary carcinomas.

Rhophilin-associated tail protein 1 promotes migration and metastasis in triple negative breast cancer via activation of RhoA.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with high motile and invasive capacity that contributes to metastasis. Understanding the mechanisms for the motility of TNBC might provide novel targetable vulnerabilities of the tumors. Herein, we find that Rhophilin-associated tail protein 1 (ROPN1) is selectively overexpressed in human TNBC cell lines and tissues. Overexpression of ROPN1 promotes, while silencing of ROPN1 inhibits the robust migration, invasion, and in vivo metastasis of TNBC cells. Moreover, we find that ROPN1 activates RhoA signaling via rhophilin-1 (RHPN1), leading to enhanced actin stress fibers formation in TNBC cells. RhoA signaling is demonstrated to be essential for ROPN1-mediated migration and metastasis of TNBC cells. Finally, we find that high levels of ROPN1 are significantly associated distant metastasis and predicted poor prognosis in patients with breast cancer. These findings reveal a novel mechanism for the high motility and metastasis of TNBC cells, suggesting that ROPN1 might be a potential prognostic marker and therapeutic target.

Protective effect of hsa-miR-570-3p targeting CD274 on triple negative breast cancer by blocking PI3K/AKT/mTOR signaling pathway.

To find out the role of hsa-miR-570-3p targeting CD274 in triple negative breast cancer (TNBC) via PI3K/AKT/mTOR signaling pathway. Hsa-miR-570-3p and CD274 expressions in 175 TNBC patients were detected by qRT-PCR and immunohistochemistry respectively. The human TNBC cell lines (MDA-MB-468 and MDA-MB-231) were used to verify the targeting relationship between hsa-miR-570-3p and CD274 via dual-luciferase reporter gene assay. Then, MDA-MB-468 and MDA-MB-231 cells were divided into Blank, miR-NC, miR-570-3p mimics, NC siRNA, CD274 siRNA, and miR-570-3p inhibitors + CD274 siRNA groups. Next, the biological activities of cells were detected by MTT, Cell-Light EdU, Annexin-V-FITC/PI, wound healing and Transwell invasion assays. Western blotting was conducted to detect protein expressions.MiR-570-3p expression was lower in tumor tissues than that in adjacent normal tissues, which was more obvious in CD274-positive TNBC patients, which targeted CD274 in TNBC cell lines. MiR-570-3p inhibited cell proliferation, invasion and migration, but induced cell apoptosis accompanying the upregulation of apoptotic proteins and downregulation of anti-apoptotic protein. CD274 siRNA had the similar results of miR-570-3p mimics, which could be reversed by miR-570-3p inhibitors. Besides, both miR-570-3p mimics and CD274 siRNA blocked PI3K/AKT/mTOR signaling pathway in TNBC cell lines. Hsa-miR-570-3p was downregulated and CD274 was upregulated in TNBC patients. Besides, hsa-miR-570-3p targeted CD274 to inhibit cell proliferation, invasion, migration, and induce cell apoptosis, which may be related to the suppression of PI3K/AKT/mTOR pathway.

Regorafenib sensitizes human breast cancer cells to radiation by inhibiting multiple kinases and inducing DNA damage

Purpose Triple-negative breast cancer (TNBC) is the most challenging and aggressive subtype of breast cancer with limited treatment options because of tumor heterogeneity, lack of druggable targets and therapy resistance. TNBCs are characterized by overexpression of growth factor receptors such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and platelet derived growth factor receptor (PDGFR) making them promising therapeutic targets. Regorafenib is an FDA approved oral multi-kinase inhibitor that blocks the activity of multiple protein kinases including those involved in the regulation of tumor angiogenesis [VEGFR1-3, TIE2], tumor microenvironment [PDGFR-β, FGFR] and oncogenesis (KIT, RET, RAF-1, BRAF). In the current study, we examined the radiosensitizing effects of Regorafenib on TNBC cell lines and explored the mechanism by which Regorafenib enhances radiosensitivity. Methods MDA-MB-231 and SUM159PT (human TNBC cell lines) and MCF 10a (human mammary epithelial cell line) were treated with Regorafenib, ionizing radiation or a combination of both. Following treatment with Regorafenib and radiation we conducted clonogenic assay to determine radiosensitivity, immunoblot analysis to assess the effect on key signaling targets, tube formation to evaluate effect on angiogenesis and comet assay as well as western blot for γH2AX to assess DNA damage response (DDR). Results Regorafenib reduced cell proliferation and enhanced radiosensitivity of MDA-MB-231 and SUM159PT cell lines but had no effect on the MCF 10a cells. Clonogenic survival assays showed that the surviving fraction at 2 Gy for both MDA-MB-231 and SUM159PT was reduced from 66.4 ± 8.9 and 88.2 ± 1.7 in controls to 38.1 ± 4.9 and 75.1 ± 1.1 following a 24 hr pretreatment with 10 μM and 5 μM Regorafenib, respectively. A marked reduction in the expression of VEGFR, PDGFR, EGFR and the downstream target, ERK, was observed with Regorafenib treatment alone or in combination with radiation. We also observed a significant inhibition of VEGF-A production in the TNBC cell lines following treatment with Regorafenib. Further, the addition of conditioned medium from Regorafenib-treated tumor cells onto human umbilical vein endothelial cells (HUVEC) suppressed tube formation, indicating an inhibition of tumor angiogenesis. Regorafenib also decreased migration of TNBC cells and suppressed radiation-induced DNA damage repair in a time-dependent manner. Conclusions Our findings demonstrate that Regorafenib enhanced radiosensitivity of breast cancer cells by inhibiting the expression of multiple receptor tyrosine kinases, VEGF-mediated angiogenesis and DNA damage response in TNBC. Therefore, combining Regorafenib with radiation and antiangiogenic agents will be beneficial and effective in controlling TNBC.