Triple-negative Breast Carcinoma Cells Research Articles

Nano-inducer of ferroptosis for targeted chemotherapy of human triple negative breast carcinoma

Triple negative breast carcinoma (TNBC) accounts for 15–20 % of all incident breast cancers (BC) and is known to be highly invasive, has fewer treatment options, and tends to have a worse prognosis. However, due to its biological heterogeneity and diverse clinical and epidemiological behaviors, TNBC lacks a tumor-specific targeted therapy. In the present work we have developed a TNBC-specific targeted nano-delivery agent comprising of a cRGD labeled magneto-liposome (T-LMD) co-encapsulated with oleic acid coated iron oxide nanoparticles (MN-OA) and doxorubicin (Dox) in the liposome bilayer and core, respectively. T-LMD was found to show enhanced uptake and induction of ferroptotic cell death in MDA-MB-231, a TNBC model cell line. Additionally, T-LMD induced ferroptosis was found to be accompanied by release of HMGB1, an immunogenic cell death marker, suggesting its immunogenicity for augmenting the activation of anti-tumor immunity in TNBC. The strategic placement of IONPs in the liposome bilayer of T-LMD facilitates the sensitization of MDA-MB-231 cells to undergo ferroptosis; predominantly via the activation of the iron/lipid metabolism pathway, as validated by use of small molecule ferroptosis inhibitor (ferrostatin-1) and iron chelator (deferoxamine). Activation of ferroptotic cell death was also corroborated by ferroptosis specific-ultrastructural alterations in the shape/size of cellular mitochondria and cell ballooning as observed by transmission electron microscopy and bright field imaging, respectively. Thus, our ferroptosis nano-inducer (T-LMD) can efficiently kill TNBC cells via enhanced LPO and ROS generation leading to membrane damage and consequent release of LDH and HMGB1, induce mitochondrial alterations and enhanced DNA double strand breaks. Altogether, our results suggest significant implications of T-LMD for treatment of TNBC.

Effects of a humanized CD47 antibody and recombinant SIRPα proteins on triple negative breast carcinoma stem cells.

Signal regulatory protein-α (SIRPα, SHPS-1, CD172a) expressed on myeloid cells transmits inhibitory signals when it engages its counter-receptor CD47 on an adjacent cell. Elevated CD47 expression on some cancer cells thereby serves as an innate immune checkpoint that limits phagocytic clearance of tumor cells by macrophages and antigen presentation to T cells. Antibodies and recombinant SIRPα constructs that block the CD47-SIRPα interaction on macrophages exhibit anti-tumor activities in mouse models and are in ongoing clinical trials for treating several human cancers. Based on prior evidence that engaging SIRPα can also alter CD47 signaling in some nonmalignant cells, we compared direct effects of recombinant SIRPα-Fc and a humanized CD47 antibody that inhibits CD47-SIRPα interaction (CC-90002) on CD47 signaling in cancer stem cells derived from the MDA-MB- 231 triple-negative breast carcinoma cell line. Treatment with SIRPα-Fc significantly increased the formation of mammospheres by breast cancer stem cells as compared to CC-90002 treatment or controls. Furthermore, SIRPα-Fc treatment upregulated mRNA and protein expression of ALDH1 and altered the expression of genes involved in epithelial/mesenchymal transition pathways that are associated with a poor prognosis and enhanced metastatic activity. This indicates that SIRPα-Fc has CD47-mediated agonist activities in breast cancer stem cells affecting proliferation and metastasis pathways that differ from those of CC-90002. This SIRPα-induced CD47 signaling in breast carcinoma cells may limit the efficacy of SIRPα decoy therapeutics intended to stimulate innate antitumor immune responses.

Detection of the interactions of tumour derived extracellular vesicles with immune cells is dependent on EV-labelling methods.

Cell-cell communication within the complex tumour microenvironment is critical to cancer progression. Tumor-derived extracellular vesicles (TD-EVs) are key players in this process. They can interact with immune cells and modulate their activity, either suppressing or activating the immune system. Deciphering the interactions between TD-EVs and immune cells is essential to understand immune modulation by cancer cells. Fluorescent labelling of TD-EVs is a method of choice to study such interaction. This work aims to determine the impact of EV labelling methods on the detection by imaging flow cytometry and multicolour spectral flow cytometry of EV interaction and capture by the different immune cell types within human Peripheral Blood Mononuclear Cells (PBMCs). EVs released by the triple-negative breast carcinoma cell line MDA-MB-231 were labelled either with the lipophilic dye MemGlow-488 (MG-488), Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) or through ectopic expression of a MyrPalm-superFolderGFP reporter (mp-sfGFP), which incorporates into EVs during their biogenesis. Our results show that these labelling strategies, although analysed with the same techniques, led to diverging results. While MG-488-labelled EVs incorporate in all cell types, CFSE-labelled EVs are restricted to a minor subset of cells and mp-sfGFP-labelled EVs are mainly detected in CD14+ monocytes which are the main uptakers of EVs and other particles, regardless of the labelling method. Furthermore, our results show that the method used for EV labelling influences the detection of the different types of EV interactions with the recipient cells. Specifically, MG-488, CFSE and mp-sfGFP result in observation suggesting, respectively, transient EV-PM interaction that results in dye transfer, EV content delivery, and capture of intact EVs. Consequently, the type of EV labelling method has to be considered as they can provide complementary information on various types of EV-cell interaction and EV fate.

Abstract 111: Characterizing subpopulation response to doxorubicin in a barcode-labeled triple negative breast carcinoma cell line

Abstract Despite recent advances in diagnosis and late-stage treatment, progression-free survival is not achieved for many cancer patients. Triple negative breast cancer (TNBC) poses a particular challenge as an aggressive and heterogeneous disease lacking the benefit of hormonal therapies. We have utilized a novel genetic lineage-tracking system, ClonMapper in TNBC cells to elucidate the pre-existent and de novo cell states that arise from chemotherapeutic intervention. ClonMapper genetic tags were stably integrated into MDA-MB-231 cells using lentiviral transduction and positively labeled cells were isolated with fluorescence-activated cell sorting to generate a starting library of ~500 uniquely barcoded cells. Barcode-labeled MDA-MB-231 were selected with doxorubicin at 250, 400 and 550 nM for 48 hours and allowed to recover. To determine the contribution of transcriptomic state to cellular drug response we carried out single cell RNA-sequencing (scRNA-seq) on 4 populations: a pretreatment cell library, an intermediary taken 30 h into drug selection, and two recovered populations from the 250 nM group. Importantly, we are able to capture and deconvolve ClonMapper lineage tags from scRNA-seq results and overlay them with transcriptomic state. Following treatment with doxorubicin we found a dose-dependent reduction in the number of unique lineages, with all replicates showing >6-fold decrease. Post-treatment replicates were each significantly comprised (90%) by fewer than three individual lineages. Lineage-resolved transcriptomics revealed two distinct lineage-specific subpopulations within the pretreatment cell library. These subpopulations demonstrated unique survivorship trajectories, beginning 30 h into selection and remaining well-separated following recovery from treatment. Comparing these subpopulations directly we observe enrichment for several pathways with markers genes remaining highly expressed post-treatment. Crucially, both subpopulations harbor a unique set of lineages which are found to survive treatment. However, in the absence of treatment we find that one subpopulation displays a faster growth rate and is likely to outcompete the other in culture conditions. The more proliferative subpopulation is marked by enrichment of Myc targets, compared with enrichment of glycolysis and KRAS signaling in the less proliferative subpopulation. This data demonstrates that subclonal heterogeneity inherent within this TNBC cell model, drives differential cell responses to chemotherapy. Citation Format: Daylin Morgan, Amy Brock. Characterizing subpopulation response to doxorubicin in a barcode-labeled triple negative breast carcinoma cell line [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 111.

Narirutin downregulates lipoxygenase-5 expression and induces G0/G1 arrest in triple-negative breast carcinoma cells

BackgroundTriple-negative breast cancer (TNBC) accounts for 20% of breast cancer that does not express HER2, progesterone and estrogen receptors. It is associated with a high mortality rate, morbidity, metastasis, recurrence, poor prognosis and resistance to chemotherapy. Lipoxygenase-5 (LOX-5), cyclooxygenase-2 (COX-2), cathepsin-D (CATD), ornithine decarboxylase (ODC) and dihydrofolate reductase (DHFR) are involved in breast cancer carcinogenesis; hence, there is a pressing need to identify novel chemicals that targets these enzymes. Narirutin, a flavanone glycoside abundantly present in citrus fruits, is reported to have immune-modulatory, anti-allergic and antioxidant potential. Still, the cancer chemopreventive mechanism against TNBC has not been explored. MethodsIn vitro experiments, enzyme activity, expression analysis, molecular docking and MD simulation were carried out. ResultsNarirutin suppressed the growth of MDA-MB-231 and MCF-7 in a dose-proportional manner. The pronounced effect with >50% inhibition was observed in SRB and MTT assays for MDAMB-231 cells. Unexpectedly, narirutin suppressed the proliferation of normal cells (24.51%) at 100 μM. Further, narirutin inhibits the activity of LOX-5 in cell-free (18.18 ± 3.93 μM) and cell-based (48.13 ± 7.04 μM) test systems while moderately affecting COX-2, CATD, ODC and DHFR activity. Moreover, narirutin revealed a down-regulation of LOX-5 expression with a fold change of 1.23. Besides, MD simulation experiments confirm that narirutin binding forms a stable complex with LOX-5 and improves the stability and compactness of LOX-5. In addition, the prediction analysis demonstrates that narirutin could not cross the blood-brain barrier and did not act as an inhibitor of different CYPs. Conclusions and significanceNarirutin could be a potent cancer chemopreventive lead for TNBC, further paving the way for synthesizing novel analogues.

Loss of SNAI1 induces cellular plasticity in invasive triple-negative breast cancer cells

The transcription factor SNAI1 mediates epithelial-mesenchymal transition, fibroblast activation and controls inter-tissue migration. High SNAI1 expression characterizes metastatic triple-negative breast carcinomas, and its knockout by CRISPR/Cas9 uncovered an epithelio-mesenchymal phenotype accompanied by reduced signaling by the cytokine TGFβ. The SNAI1 knockout cells exhibited plasticity in differentiation, drifting towards the luminal phenotype, gained stemness potential and could differentiate into acinar mammospheres in 3D culture. Loss of SNAI1 de-repressed the transcription factor FOXA1, a pioneering factor of mammary luminal progenitors. FOXA1 induced a specific gene program, including the androgen receptor (AR). Inhibiting AR via a specific antagonist regenerated the basal phenotype and blocked acinar differentiation. Thus, loss of SNAI1 in the context of triple-negative breast carcinoma cells promotes an intermediary luminal progenitor phenotype that gains differentiation plasticity based on the dual transcriptional action of FOXA1 and AR. This function of SNAI1 provides means to separate cell invasiveness from progenitor cell de-differentiation as independent cellular programs.

MiR-1205/DNAJB1 reverses docetaxel chemoresistance in human triple negative breast carcinoma cells via regulation of mutp53/TAp63 signaling.

Chemoresistance is the major cause of therapeutic failure in human triple negative breast carcinoma (TNBC). Docetaxel (DOC), a first-line therapeutic drug in TNBC treatment, is limited for long-term use due to the development of chemoresistance. Thus, overcoming chemoresistance of DOC remains an important challenge to improve patient's outcome of TNBC. In this study, we aimed to investigate the molecular mechanism behind DOC chemoresistance and the possible therapeutic effects of miRNAs. Utilizing qRT-PCR analysis, we discovered that miR-1205 is gradually downregulated in human triple negative breast carcinoma MDA-MB-231 and docetaxel-resistant MDA-MB-231 (MDA-MB-231/DOC) cells compared with Hs 578Bst normal human breast fibroblasts. Cell viability, cell cycle and apoptosis assays in MDA-MB-231/DOC cells indicated that miR-1205 overexpression enhances docetaxel sensitivity by reducing cell viability as well as inducing G2/M cell cycle arrest and cell apoptosis. Western blot analysis, dual-luciferase reporter assay, co-immunoprecipitation assay and chromatin immunoprecipitation assay revealed that miR-1205 overexpression disrupts the stable complex formation of DNAJB1, mutp53 and TAp63 by directly reducing DNAJB1 expression, which abates the sequestrating effect of mutp53 on TAp63, thereby leading to the enhanced DOC sensitivity in MDA-MB-231/DOC cells. Our findings demonstrate the role of the miR-1205/DNAJB1 axis in the docetaxel resistance of TNBC, which may offer a promising therapeutic approach to resolve docetaxel resistance in TNBC.

Artesunate-induced Cellular Effects Are Mediated by Specific EPH Receptors and Ephrin Ligands in Breast Carcinoma Cells

The aberrant regulation of erythropoietin-producing hepatocellular carcinoma (EPH) receptors and ephrin ligands has been implicated in breast carcinoma, and artesunate has been shown to have anticancer effects. The aim of this study was to characterize the involvement of EPH receptors and ephrin ligands in mediating artesunate (ART)-induced growth suppression of normal breast cells and breast carcinoma cell lines. The normal breast epithelial cells (MCF10A), non-invasive ductal breast carcinoma cells (MCF7), and invasive triple-negative breast carcinoma cells (MDA-MB-231) were grown in the absence or the presence of different concentrations of artesunate. The cells were counted, and total RNA was isolated. The abundance of transcripts corresponding to EPH receptors and ephrin ligands was determined by quantitative polymerase chain reaction. Cell viability was significantly reduced when cells were treated with artesunate, with MDA-MB-231 cells having the highest sensitivity. Artesunate had no significant effect on transcription of EPH/ephrins in MCF10A cells, but markedly increased EPHA8, EPHA10, EPHB6 and ephrin-A2 expression in MCF7 cells, and significantly increased EPHA3 and EPHA10 expression while reducing that of EPHA7 and ephrin-A3 in MDA-MB-231 cells. The relative changes in artesunate-treated MCF7 and MDA-MB-231 cells as compared to similarly treated MCF10A cells allow us to implicate combinatorial expression and receptor interactions for EPH receptor-mediated signal transduction that converges into pathways responsible for cell growth, proliferation, and apoptosis. Specifically, the alterations in EPHA7, EPHA8, EPHA10 and EPHB6 transcripts appear to be important participants in artesunate-mediated cellular effects.

PTEN May Involve in Regulation of PD-L1 Expression in Triple Negative Breast Carcinoma

Triple negative breast carcinoma (TNBC) is a rapid progressive tumor and has a poor overall survival. Therefore, it is crucial to find out effective molecular targets and develop optimal therapeutic strategies for TNBC. In this study, immunohistochemical staining was used to detect expressions of programmed death-ligand 1 (PD-L1) and phosphatase and tensin homolog (PTEN) in 136 breast carcinomas including 50 TNBC. The effect of PTEN on regulation of PD-L1 expression was assessed in vitro in the PTEN knockdown TNBC cells. We found that PD-L1(SP142) positive rate in TNBC (48.0%) was significantly higher than non-TNBC (23.3%). PTEN negative rate was 42% in TNBC. The inverse correlation between PTEN and PD-L1(SP142) expression in TNBC was statistically significant (P<0.05). After PTEN knockdown, PD-L1 expression in TNBC cells increased significantly, and the expression level of AKT increased simultaneously. PTEN knockdown promoted cell proliferation, viability and G1/S switch of TNBC cells. These results suggested that PTEN may involve in regulation of PD-L1 expression, because PTEN loss can upregulate PD-L1 expression in TNBC. Antitumor immunity of PD-L1 could be enhanced in TNBC when targeting PTEN at the same time.

CytoCy5S™, a compound of many structures. in vitro and in vivo evaluation of four near-infrared fluorescent substrates of nitroreductase (NTR)

CytoCy5S™, a quenched, red-shifted fluorescent probe, has been used to exploit the imaging potential of the nitroreductase (NTR) reporter gene platform. Its use has been reported in a number of publications, however there are discrepancies in both the reported structure and its physicochemical properties. Herein, we aim to highlight these discrepancies and to define the best candidate of the four substrates under study for preclinical work in NTR reporting by optical applications.We report the synthesis, purification and characterisation of four NTR substrates, including alternately described structures currently referred by the name CytoCy5S. A comparative NTR enzymatic assay was performed to assess the spectroscopic characteristics of the different reductively activated probes. The NTR expressing triple-negative breast carcinoma cell line, MDA-MB-231 NTR+, was employed to compare, both in vitro and in vivo, the suitability of these fluorescent probes as reporters of NTR activity. Comparison of the reporting properties was achieved by flow cytometry, fluorescence microscopy and optical imaging, both in vivo and ex vivo.This study evaluated the different spectroscopic and biological characteristics of the four substrates and concluded that substrate 1 presents the best features for oncological in vivo preclinical optical imaging.

RIPK1 is a negative mediator in Aquaporin 1-driven triple-negative breast carcinoma progression and metastasis

The triple-negative breast carcinoma (TNBC) is the most aggressive subtype of breast cancer. In TNBC, Aquaporin 1 (AQP1), a water-transporting transmembrane protein, is aberrantly enriched in cytoplasm and causes tumor cell death evasion. However, the carcinogenetic bioactivities of cytoplasmic AQP1 cannot be attributed to the canonical “osmotic engine model”. In the present study, the receptor-interacting protein kinase 1 (RIPK1), a cell death regulator, was identified to negatively mediate AQP1-driven TNBC progression and metastasis. AQP1 overabundance and RIPK1 depletion occurred in TNBC, which were correlated with aggressive oncological features and poor prognosis. AQP1 bound with RIPK1, resulting in the inhibition of RIPK1/RIPK3/MLKL-mediated necroptosis and RIPK1/caspase-8/caspase-3-mediated apoptosis. Genetic inhibition of RIPK1 significantly exacerbated the pro-tumor effect of AQP1, while ectopic expression of RIPK1 notably blunted AQP1 signaling. Mechanistically, AQP1 binds to the D324 site of RIPK1, and facilitates RIPK1 cleavage and inactivation by excessively activating the caspase-8/RIPK1 negative feedback loop. RIPK1D324K overexpression significantly prevented RIPK1 cleavage and weakened the aggressiveness of AQP1-enriched TNBC cells. Overall, our findings clarify the underlying mechanism of cytoplasmic AQP1-driven TNBC progression and metastasis, in which RIPK1 exerts an essential role as a negative mediator and exhibits the potential as a therapeutic target for TNBC.

Insights into the Behavior of Triple-Negative MDA-MB-231 Breast Carcinoma Cells Following the Treatment with 17β-Ethinylestradiol and Levonorgestrel.

Oral contraceptives (OCs) are widely used due to their efficiency in preventing unplanned pregnancies and treating several human illnesses. Despite their medical value, the toxicity of OCs remains a public concern. Previous studies indicate the carcinogenic potential of synthetic sex hormones and their link to the development and progression of hormone-dependent malignancies such as breast cancer. However, little is known about their influence on the evolution of triple-negative breast carcinoma (TNBC), a malignancy defined by the absence of estrogen, progesterone, and HER2 receptors. This study reveals that the active ingredients of modern OCs, 17β-Ethinylestradiol, Levonorgestrel, and their combination induce differential effects in MDA-MB-231 TNBC cells. The most relevant behavioral changes occurred after the 24 h treatment with 17β-Ethinylestradiol, summarized as follows: (i) decreased cell viability (64.32% at 10 µM); (ii) cell roundness and loss of confluence; (iii) apoptotic aspect of cell nuclei (fragmentation, membrane blebbing); and (iv) inhibited cell migration, suggesting a potential anticancer effect. Conversely, Levonorgestrel was generally associated with a proliferative activity. The association of the two OCs exerted similar effects as 17β-Ethinylestradiol but was less effective. Further studies are necessary to elucidate the hormones’ cytotoxic mechanism of action on TNBC cells.

A styrylpyrone dimer isolated from Aniba heringeri causes apoptosis in MDA-MB-231 triple-negative breast cancer cells

The styrylpyrone dehydrogoniothalamin (1) and two of its dimers (2 and 3) were isolated from the leaves of Aniba heringeri (Lauraceae). Compound 3 is new, while 1 and 2 are being reported for the first time in this species. Structures were determined by 1D- and 2D-NMR spectroscopy, mass spectrometry, and optical rotation data. Cytotoxic effects and selectivity indices were evaluated in five neoplastic cell lines—PC-3 (prostate), 786–0 (renal), HT-29 (colon), MCF-7, and MDA-MB-231 (breast)—and a non-neoplastic cell line, (NIH/3T3, murine fibroblast). Compound 1 inhibited cell growth by 50% (GI50) at concentrations in the 90.4–175.7 μM range, while 2 proved active against MCF-7 and MDA-MB-231 breast cells (GI50 = 12.24, and 34.22 μM, respectively). Compound 3 showed strong cytotoxicity (GI50 = 4.4 μM) against MDA-MB-231 (an established basal triple-negative breast carcinoma (TNBC) cell line), with a high selective index of 35. This compound was subsequently evaluated for apoptosis induction in MDA-MB-231 cells, using GI50 and 50% lethal concentrations (LC50). Flow cytometry analysis showed that at LC50 compound 3 induced cell death with phosphatidylserine externalization and caspase-3 activation. Apoptotic genes were measured by RT-qPCR, revealing an upregulation of BAX, with an increase in expression of the BAX/BCL2 ratio in treated cells. Fluorescence microscopy disclosed morphological changes related to apoptosis. Overall, these findings showed compound 3 to be a promising prototype against TNBC cells that tend to respond poorly to conventional therapies.

Cancer stem cell transcriptome landscape reveals biomarkers driving breast carcinoma heterogeneity.

Breast carcinomas are heterogeneous diseases with distinct clinical outcomes and cancer stem cell (CSC) percentages. Exploring breast carcinoma stem cell landscape could help understand the heterogeneity of such cancers with profound clinical relevance. We conducted transcriptional profiling of CSCs and non-stem cancer cells isolated from three triple-negative breast carcinoma cell lines, analyzed the CSC transcriptome landscape that drives breast carcinoma heterogeneity through differentially expressed gene identification, gene ontology (GO) and pathway enrichment analyses as well as network construction, and experimentally validated the network hub gene. We identified a CSC feature panel consisting of 122 and 381 over-represented and under-expressed genes capable of differentiating breast carcinoma subtypes. We also underpinned the prominent roles of the PI3K-AKT pathway in empowering carcinoma cells with uncontrolled proliferative and migrative abilities that ultimately foster cancer stemness, and revealed the potential promotive roles of ATP6V1B1 on breast carcinoma stemness through functional in vitro studies. Our study contributes in identifying a CSC feature panel for breast carcinomas that drives breast carcinoma heterogeneity at the transcriptional level, which provides a reservoir for diagnostic marker and/or therapeutic target identification once experimentally validated as demonstrated by ATP6V1B1.

Abstract 4301: ADAM9 plays a key role in the aggressive behavior of triple negative breast carcinoma and represents a potential treatment target

Abstract Introduction: A Disintegrin and Metalloproteinase 9 (ADAM9) promotes cancer progression via its metalloproteinase and disintegrin domains, and upregulated ADAM9 is found in pancreatic, prostate and breast carcinomas. The expression of ADAM9 in triple negative breast carcinoma (TNBC), however, is still unclear. We investigated the expression of ADAM9 in TNBC, its role in tumor progression and prognostic implication in TNBC patients. Materials and Methods: The expression of ADAM9 was investigated in TNBC and non-TNBC cell lines as well as TNBC and non-TNBC tumor tissues. ADAM9 mRNA expression data in TNBC cell lines (n = 14) and non-TNBC cell lines (n = 11) were obtained from Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/) database. A total of 24 TNBC and 20 non-TNBC breast tumor specimens were also collected. The Kaplan-Meier (KM) plotter was used to investigate the association between ADAM9 expression level and survival in TNBC patients. Cell proliferation, migration and invasion assays were performed to investigate the role of ADAM9 in TNBC cell line (MDA-MB-231). Results: ADAM9 mRNA expression in TNBC cell lines was significantly higher than that of non-TNBC cell lines (p = 0.038). Strong staining of ADAM9 was detected in primary tumor tissues from TNBC patients, but only weak staining was found in primary non-TNBC tumors. Statistical analysis showed that the ADAM9 staining intensity (p < 0.05) and expression level (p < 0.05) were markedly increased in TNBC group compared with the non-TNBC group. High level of ADAM9 mRNA expression was associated with poorer overall survival (p < 0.01), more distant metastasis-free survival (p < 0.01) and shorter relapse-free survival (RFS) (p < 0.001). Furthermore, high ADAM9 mRNA level was also significantly associated with poor RFS in TNBC (p < 0.01). ADAM9 mRNA expression level was associated with RFS in grade II and III tumors, but not in grade I tumors (p = 0.21). An increased hazard ratio was also found with grade II tumors (p = 0.2) and grade III tumors (p = 0.003). Scrambled siRNA with green fluorescent protein was transfected in MDA-MB-231 cells by lipofectamine 2000. ADAM9 protein and mRNA expression were successfully inhibited in these transfected MDA-MB-231 cells, which lead to suppression in cell proliferation, migration and invasion on functional assays. Conclusion: Our results showed the ADAM9 was overexpressed in TNBC and was associated with poor prognosis in TNBC patients. ADAM9 was found to play a key role and may represent a potential treatment target because inhibition of ADAM9 mRNA and protein expression was associated with suppression of cell proliferation, migration and invasion in TNBC cell line. Keywords: ADAM9, Prognosis, Triple negative breast cancer Citation Format: Rui Zhou, William CS Cho, Victor WS Ma, Wah Cheuk, Grace Yik-ka So, SC Cesar Wong, Lawrence WC Chan. ADAM9 plays a key role in the aggressive behavior of triple negative breast carcinoma and represents a potential treatment target [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 4301.

Abstract A16: The α-arrestin ARRDC3 functions as a metastasis suppressor by regulating GPCR activation of the Hippo pathway

Abstract G protein-coupled receptors (GPCRs) are key regulators of the Hippo pathway, a well-established mediator of tumorigenesis and cancer progression. The Hippo pathway is a promising therapeutic target, yet developing inhibitors for the core pathway components has proven difficult. As critical regulators of the Hippo pathway, GPCRs represent a potential upstream therapeutic target for cancer. However, despite the success and promise of GPCRs as therapeutic targets, there are currently no FDA-approved drugs targeting GPCRs for cancer. Protease-activated receptor-1 (PAR1) is a GPCR that has been shown to activate the Hippo pathway and also promote breast cancer progression. PAR1 is overexpressed in breast cancer patient tissue biopsies and in breast carcinoma cell lines and correlates with increased rates of metastasis and poor prognosis and increased invasion and metastatic potential, respectively. One mechanism that leads to PAR1 aberrant overexpression is defective lysosomal trafficking and degradation of the receptor, leading to persistent signaling. We recently showed that arrestin domain containing protein-3 (ARRDC3), an adaptor protein for E3 ubiquitin ligases, functions as a tumor suppressor by controlling proper PAR1 trafficking and degradation in highly aggressive, triple-negative breast carcinoma cells. The dysregulation of PAR1 trafficking due to loss of ARRDC3 expression leads to persistent signaling and promotes breast cancer invasion. However, the mechanism by which ARRDC3 specifically regulates PAR1-stimulated Hippo signaling to promote breast cancer metastasis remains unknown. Here, we show that ARRDC3 regulates GPCR activation of the Hippo pathway in basal-like breast cancer, where the transcriptional coactivators YAP and TAZ play differential roles in GPCR-mediated signaling and invasive capacity. ARRDC3 re-expression in invasive breast carcinoma cells attenuates PAR1-mediated activation of the Hippo pathway through regulation of TAZ but not YAP activation. Furthermore, siRNA knockdown of TAZ inhibits PAR1-stimulated Hippo signaling and invasion, whereas YAP siRNA knockdown has no effect. Our studies suggest a crucial role for ARRDC3 and TAZ in PAR1-Hippo pathway signaling in breast carcinoma invasion and metastasis. A better understanding of the mechanisms by which the Hippo pathway is regulated by GPCRs may lead to new potential therapeutic targets for the treatment or prevention of metastatic breast cancer. Citation Format: Aleena K.S. Arakaki, Wen-An Pan, Helen Wedegaertner, JoAnn Trejo. The α-arrestin ARRDC3 functions as a metastasis suppressor by regulating GPCR activation of the Hippo pathway [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A16.

Enhanced apoptogenesis and oncogene regulatory mechanism of troxerutin in triple negative breast cancer cells.

Triple negative breast carcinoma (TNBC) is an aggressive form of cancer, with high rates of morbidity, mortality, poor prognosis and limited therapeutic options. The objective of the present study was to elaborate the anticancer activity of Troxerutin (TXN) in TNBC/MDA-MB-231 cells. Herein, we demonstrated the inhibitory effects of TXN on the breast cancer cell growth via induction of apoptosis. Mitochondrial membrane potential (∆Ψm), DNA damage and apoptotic nuclear changes were analyzed by flowcytometry, AO/EtBr and Hoechst staining, respectively. Furthermore, apoptotic protein and gene expressions were analyzed by western blot and reverse transcription polymerase chain reaction (RT-PCR), respectively. Our results indicated that TXN induces apoptosis as evidenced by inhibit the cell proliferation, enhanced apoptotic activation, altered mitochondrial membrane potential and elevated level of DNA damage in TNBC cells. Furthermore, the TXN inhibit anti-apoptotic protein expression with the subsequent upregulation of Cytochrome c, Caspase-9 and Caspase-3. Thus, TXN induces apoptosis in TNBC cells through inducing nuclear damage and altered apoptotic marker expressions. Therefore, TXN might be used as a potential therapeutic agent for the treatment of triple negative breast cancer.

Regulation of GPCR activation of the Hippo pathway in metastatic breast cancer

G protein‐coupled receptors (GPCRs) regulate the Hippo signaling pathway, a developmental pathway involved in tumorigenesis and metastasis. Although GPCRs promote cancer progression and are highly druggable, there are currently no FDA‐approved drugs targeting GPCRs for cancer treatment. Protease‐activated receptor‐1 (PAR1) is a GPCR that both activates the Hippo pathway and promotes breast cancer progression. PAR1 is overexpressed in breast cancer patient tumor biopsies and in invasive breast carcinoma cell lines, and correlates with increased metastasis and poor prognosis. We found that PAR1 overexpression is due to defective lysosomal trafficking and results in persistent signaling observed in multiple triple‐negative breast carcinoma cell lines including the model invasive MDA‐MB‐231 cells. We recently showed that the α‐arrestin domain containing protein‐3 (ARRDC3), a tumor suppressor, is reduced or lost in highly aggressive triple‐negative breast carcinoma and this is responsible for aberrant PAR1 lysosomal trafficking and persistent signaling, leading to increased invasion. However, the link between ARRDC3 expression and PAR1‐stimulated Hippo pathway regulation in invasive breast cancer is not known and was examined. Here, we show that ARRDC3 regulates GPCR activation of the Hippo pathway in triple‐negative breast cancer, where the transcriptional co‐activators YAP and TAZ display distinct functions. ARRDC3 re‐expression in invasive breast carcinoma cells attenuates GPCR‐stimulated Hippo signaling and invasion that is mediated by activation of TAZ but not YAP. Furthermore, siRNA‐targeted depletion of TAZ, but not YAP inhibits GPCR‐induced Hippo signaling and invasion. An understanding of the mechanisms by which the Hippo pathway is regulated by GPCRs may lead to new potential therapeutic targets for the treatment or prevention of metastatic breast cancer.Support or Funding InformationFunding for this project was provided by the HHMI Gilliam Fellowship for Advanced Study, the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through NIH General Medical Sciences, T32 GM007752 and the UCSD Tribal Membership Initiative (Aleena Arakaki) and by the National Institute of Health NIGMS R01 GM090689 and R35 GM127121 (JoAnn Trejo).

Stem-like Cells from Invasive Breast Carcinoma Cell Line MDA-MB-231 Express a Distinct Set of Eph Receptors and Ephrin Ligands.

Breast cancer cell lines consist of bulk tumor cells and a small proportion of stem-like cells. While the bulk cells are known to express a distinct combination of Eph receptors and ephrin ligands, the transcript profiles of stem-like cells in these cell lines have not been adequately characterized. The aim of this study was to determine Eph receptor/ephrin ligand profiles of cancer stem cells specific to a triple negative breast carcinoma cell line. The normal breast cell line MCF10A and the invasive breast carcinoma cell line MDA-MB-231 were used to isolate CD24+/CD24- cell populations. The profiles of Eph receptors and ephrin ligands were determined by real-time PCR and the relative abundance in bulk and stem cells were compared. Based on the mean ΔCT values, the descending order of abundance was as follows. Ephrin-A5 > EPHA2 > (EPHA8, EPHB2) > ephrin-B2 > (EPHA7, EPHB4, ephrin-A4) > ephrin-A3 > ephrin-A1 > (EPHB3, ephrin-B1) > EPHA4 > EPHA1 > EPHA10. EPHA6 and ephrin-A2 transcripts were not detectable in stem cells from either cell line. The expression of EPHA4, EPHA7, EPHA8, and ephrin-A5 in MDA-MB-231 stem cells was up-regulated by 12, 20, ~500, and 6.5-fold respectively. The up-regulation of transcripts for EPHA8 and its cognate ligand, ephrin-A5, in the stem cells isolated from MDA-MB-231, suggest their involvement in the invasiveness of this cell line. Based on literature reports, we propose the role of EPHA8 and ephrin-A5 in MDA-MB-231 stem cells via the PI3K-AKT-mTOR pathway.

Synthesis of the first mixed ligand Mn (II) and Cd (II) complexes of 4‐methoxy‐pyridine‐2‐carboxylic acid, molecular docking studies and investigation of their anti‐tumor effects in vitro

The first mixed ligand Mn (II) and Cd (II) complexes containing 4‐methoxy‐pyridine‐2‐carboxylic acid (4‐mpic) and 4,4′‐dimethyl‐2,2′‐bipyridine (dmbpy) were synthesized in this study. The geometric structures of [Mn(4‐mpic)2(dmbpy)] (complex 1) and [Cd(4‐mpic)2(dmbpy)] (complex 2) were determined by single crystal X‐Ray diffraction method. FT‐IR and UV–Vis spectra were also recorded to investigate vibrational and electronic properties of complexes 1 and 2. Density functional theory (DFT) calculations were also carried out to provide a deep understanding in geometric, spectroscopic, electronic and nonlinear optical (NLO) properties of complexes 1 and 2. The first‐order hyperpolarizibility (β) parameter calculated as 332.9736 × 10−30 esu demonstrated that complex 1 is an extremely promising candidate to NLO materials. Natural bond orbital (NBO) analysis not only verified the distorted octahedral geometries of central metal ions, but also found out the high‐energy interactions responsible for biological activities for complexes 1 and 2. Anti‐cancer activities of complexes 1 and 2 were tested on human breast carcinoma cell line MCF‐7 (ER and PR positive, HER2 negative) and the triple negative breast carcinoma cell line MDA‐MB 231 (ER, PR and HER2 negative). Dose–response relationship derived from MTT assays indicates that complexes 1 and 2 are showing concentration‐dependent effects, which could suggest a potential use for these drug combinations in cancer cell lines.