Aggressive Triple-negative Breast Cancer Cells Research Articles

177Lu-Labeled Iron Oxide Nanoparticles Functionalized with Doxorubicin and Bevacizumab as Nanobrachytherapy Agents against Breast Cancer.

The use of conventional methods for the treatment of cancer, such as chemotherapy or radiotherapy, and approaches such as brachytherapy in conjunction with the unique properties of nanoparticles could enable the development of novel theranostic agents. The aim of our current study was to evaluate the potential of iron oxide nanoparticles, coated with alginic acid and polyethylene glycol, functionalized with the chemotherapeutic agent doxorubicin and the monoclonal antibody bevacizumab, to serve as a nanoradiopharmaceutical agent against breast cancer. Direct radiolabeling with the therapeutic isotope Lutetium-177 (177Lu) resulted in an additional therapeutic effect. Functionalization was accomplished at high percentages and radiolabeling was robust. The high cytotoxic effect of our radiolabeled and non-radiolabeled nanostructures was proven in vitro against five different breast cancer cell lines. The ex vivo biodistribution in tumor-bearing mice was investigated with three different ways of administration. The intratumoral administration of our functionalized radionanoconjugates showed high tumor accumulation and retention at the tumor site. Finally, our therapeutic efficacy study performed over a 50-day period against an aggressive triple-negative breast cancer cell line (4T1) demonstrated enhanced tumor growth retention, thus identifying the developed nanoparticles as a promising nanobrachytherapy agent against breast cancer.

Hijacking 5-Fluorouracil Chemoresistance in Triple Negative Breast Cancer via microRNAs-Loaded Chitosan Nanoparticles.

Chemotherapy is still the mainstay of treatment for triple-negative breast cancer (TNBC) patients. Yet only 20% of TNBC patients show a pathologic complete response (pCR) after neoadjuvant chemotherapy. 5-Fluorouracil (5-FU) is a stable cornerstone in all recommended chemotherapeutic protocols for TNBC patients. However, TNBC patients' innate or acquired chemoresistance rate for 5-FU is steeply escalating. This study aims to unravel the mechanism behind the chemoresistance of 5-FU in the aggressive TNBC cell line, MDA-MB-231 cells, to explore further the role of the tumor suppressor microRNAs (miRNAs), miR-1275, miR-615-5p, and Let-7i, in relieving the 5-FU chemoresistance in TNBC, and to finally provide a translational therapeutic approach to co-deliver 5-FU and the respective miRNA oligonucleotides using chitosan-based nanoparticles (CsNPs). In this regard, cellular viability and proliferation were investigated using MTT and BrdU assays, respectively. 5-FU was found to induce JAK/STAT and PI3K/Akt/mTOR pathways in MDA-MB-231 cells with contaminant repression of their upstream regulators miR-1275, miR-615-5p, and Let-7i. Moreover, CsNPs prepared using the ionic gelation method were chosen and studied as nanovectors of 5-FU and a combination of miRNA oligonucleotides targeting TNBC. The average particle sizes, surface charges, and morphologies of the different CsNPs were characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. In addition, the encapsulation efficiency (EE%), drug loading capacity (DLC%), and release manner at two different pH values were assessed. In conclusion, the novel CsNPs co-loaded with 5-FU and the combination of the three miRNA oligonucleotides demonstrated synergistic activity and remarkable repression in cellular viability and proliferation of TNBC cells through alleviating the chemoresistance to 5-FU.

Integrative Modulation of Magnetic Resonance Transverse and Longitudinal Relaxivity in a Cell-Viable Bimagnetic Ensemble, γ-Fe2O3@ZnFe2O4.

The potential application of magnetic nanosystems as magnetic resonance imaging (MRI) contrast agents has been thoroughly investigated. This work seeks to attain robust MRI-contrast efficiency by designing an interacting landscape of a bimagnetic ensemble of zinc ferrite nanorods and maghemite nanoparticles, γ-Fe2O3@ZnFe2O4. Because of competing spin clusters and structural anisotropy triggered by isotropic γ-Fe2O3 and anisotropic ZnFe2O4, γ-Fe2O3@ZnFe2O4 undergoes the evolution of cluster spin-glass state as evident from the critical slowing down law. Such interacting γ-Fe2O3@ZnFe2O4 with spin flipping of 1.2 × 10-8 s and energy barrier of 8.2 × 10-14 erg reflects enhanced MRI-contrast signal. Additionally, γ-Fe2O3@ZnFe2O4 is cell-viable to noncancerous HEK 293 cell-line and shows no pro-tumorigenic activity as observed in MDA-MB-231, an extremely aggressive triple-negative breast cancer cell line. As a result, γ-Fe2O3@ZnFe2O4 is a feasible option for an MRI-contrast agent having longitudinal relaxivity, r1, of 0.46 s-1mM-1 and transverse relaxivity, r2, of 15.94 s-1mM-1, together with r2/r1 of 34.65 at 1.41 T up to a modest metal concentration of 0.1 mM. Hence, this study addresses an interacting isotropic/anisotropic framework with faster water proton decay in MR-relaxivity resulting in phantom signal amplification.

RAGE/SNAIL1 signaling drives epithelial-mesenchymal plasticity in metastatic triple-negative breast cancer.

Epithelial/Mesenchymal (E/M) plasticity plays a fundamental role both in embryogenesis and during tumorigenesis. The receptor for advanced glycation end products (RAGE) is a driver of cell plasticity in fibrotic diseases; however, its role and molecular mechanism in triple-negative breast cancer (TNBC) remains unclear. Here, we demonstrate that RAGE signaling maintains the mesenchymal phenotype of aggressive TNBC cells by enforcing the expression of SNAIL1. Besides, we uncover a crosstalk mechanism between the TGF-β and RAGE pathways that is required for the acquisition of mesenchymal traits in TNBC cells. Consistently, RAGE inhibition elicits epithelial features that block migration and invasion capacities. Next, since RAGE is a sensor of the tumor microenvironment, we modeled acute acidosis in TNBC cells and showed it promotes enhanced production of RAGE ligands and the activation of RAGE-dependent invasive properties. Furthermore, acute acidosis increases SNAIL1 levels and tumor cell invasion in a RAGE-dependent manner. Finally, we demonstrate that in vivo inhibition of RAGE reduces metastasis incidence and expands survival, consistent with molecular effects that support the relevance of RAGE signaling in E/M plasticity. These results uncover new molecular insights on the regulation of E/M phenotypes in cancer metastasis and provide rationale for pharmacological intervention of this signaling axis.

Inhibitor titrations reveal low control of glyceraldehyde 3-phosphate dehydrogenase and high control of hexokinase on glycolytic flux in an aggressive triple-negative breast cancer cell line

The glycolytic flux, and in particular lactate production, is strongly increased in cancer cells compared to normal cells, a characteristic often referred to as aerobic glycolysis or the Warburg effect. This makes the glycolytic pathway a potential drug target, in particular if the flux control distribution in the pathway has shifted due to the metabolic reprogramming in cancer cells. The flux response of a drug is dependent on both the sensitivity of the target to the drug and the flux control of the target, and both these characteristics can be exploited to obtain selectivity for cancer cells. Traditionally drug development programs have focused on selective sensitivity of the drug, not necessarily focussing on the flux control of the target. We determined the flux control of two steps that have been suggested to have high control in cancer cells, using two inhibitors, iodoacetic acid and 3-bromopyruvate, and measured a flux control of the glyceraldehyde 3-phosphate dehydrogenase close to zero, while the hexokinase holds 50% of all flux control in glycolysis in an invasive cancer cell line MDA-mb-231.

Ir(III) Compounds Containing a Terdentate Ligand Are Potent Inhibitors of Proliferation and Effective Antimetastatic Agents in Aggressive Triple-Negative Breast Cancer Cells.

Herein, we report a series of new octahedral iridium(III) complexes Ir1-Ir9 of the type [Ir(N^N^N)(C^N)Cl]PF6 (N^N^N = 4'-(p-tolyl)-2,2':6',2″-terpyridine; C^N = deprotonated 2-arylbenzimidazole backbone) to introduce new metal-based compounds for effective inhibition of metastatic processes in triple-negative breast cancer (TNBC). The results show that the structural modifications within the C^N scaffold strongly impact the antimetastatic properties of these complexes in TNBC cells. Furthermore, testing the antimetastatic effects of the investigated Ir complexes revealed that the highest antimetastatic activity in TNBC cells is exhibited by complex Ir1. This result was in contrast to the effects of the clinically used drug doxorubicin used in conventional chemotherapy of TNBC, which conversely promoted metastatic properties of TNBC cells. Thus, the latter result suggests that doxorubicin chemotherapy may increase the risk of metastasis of breast cancer cells, so the search for new drugs to treat breast cancer that would show better antitumor effects than doxorubicin is justified.

The Chorioallantoic Membrane Xenograft Assay as a Reliable Model for Investigating the Biology of Breast Cancer

The chorioallantoic membrane (CAM) assay is an alternative in vivo model that allows for minimally invasive research of cancer biology. Using the CAM assay, we investigated phenotypical and functional characteristics (tumor grade, mitosis rate, tumor budding, hormone receptor (HR) and HER2 status, Ki-67 proliferation index) of two breast cancer cell lines, MCF-7 and MDA-MB-231, which resemble the HR+ (luminal) and triple-negative breast cancer (TNBC) subgroups, respectively. Moreover, the CAM results were directly compared with murine MCF-7- and MDA-MB-231-derived xenografts and human patient TNBC tissue. Known phenotypical and biological features of the aggressive triple-negative breast cancer cell line (MDA-MB-231) were confirmed in the CAM assay, and mouse xenografts. Furthermore, the histomorphological and immunohistochemical variables assessed in the CAM model were similar to those in human patient tumor tissue. Given the confirmation of the classical biological and growth properties of breast cancer cell lines in the CAM model, we suggest this in vivo model to be a reliable alternative test system for breast cancer research to reduce murine animal experiments.

Targeting Wnt/β-catenin signaling pathway in triple-negative breast cancer by benzylic organotrisulfides: Contribution of the released hydrogen sulfide towards potent anti-cancer activity.

The potent anti-cancer activity of naturally occurring organopolysulfides has attracted wide research attention over the last two decades. Sustained donation of hydrogen sulfide (H2S) from organopolysulfides is found to be beneficial for the treatment of several organ-specific cancers. In the present study, for the first time, the mechanism of action for the potent anti-cancer activity of bis(3,5-dimethoxybenzyl) trisulfide 4 against highly aggressive triple-negative breast cancer cells (MDA-MB-231) is described. Preliminary in vitro studies revealed potent anti-proliferative activity of the trisulfide 4 against triple-negative breast cancer cells with an IC50 value of 1.0μM. Mechanistic studies reveal that the compound exhibited anti-cancer activity, primarily by targeting and suppressing the Wnt/β-catenin signaling pathway. The inactivation of the β-catenin level was associated with the cell cycle arrest in the G2/M phase and the significant down-regulation of downstream signaling genes such as Cyclin D1 and c-Myc expression. Several control experiments with analogous organosulfur compounds and the key enzyme inhibitors reveal that the presence of a trisulfide unit in the compound is crucial for the desired inactivation of β-catenin expression, which is promoted by GSK-3β-induced phosphorylation of β-catenin and its proteasomal degradation. Moreover, the trisulfide unit or the released H2S induced down-regulation of the p53 expression with the possible S-sulfhydration process led to p53-independent up-regulation of p21 expression. Therefore, the key results of this study highlighting the potency of synthetic benzylic organotrisulfide and the released H2S towards the growth inhibition of triple-negative breast cancer via Wnt/β-catenin signaling pathway would certainly be helpful for further studies and developing small-molecule anti-cancer therapeutics in future.

Abstract 108: Insulin-like growth factor binding protein 4 exerts differential effects on breast cancer cell proliferation based on the expression status of pregnancy-associated plasma protein A

Abstract Insulin-Like Growth Factor Binding Protein 4 (IGFBP-4) is one of six binding proteins involved in sequestering insulin-like growth factors 1 and 2 (IGFs). IGFBP-4 expression is induced by estrogen receptor α (ERα), and its classical function is to prevent activation of IGF-1 receptor (IGF-1R) by sequestration of IGF-1. However, the aggressive triple-negative breast cancer cell line MDA-MB-231 shows high expression of IGFBP-4 along with its protease, pregnancy-associated plasma protein A (PAPP-A). Further, inhibition of IGFBP-4 in MDA-MB-231 cells is sufficient to reduce migration. To better understand the function of IGFBP-4 as a pro-cancer or anti-cancer protein, its anti-proliferative effects in ERα positive cells must be reconciled with its pro-migratory effects in triple negative cells. The role of IGFBP-4 in proliferation across four breast cancer cell lines representing ERα positive and triple negative subtypes was investigated. Stable transfection with IGFBP-4 expression construct in the MCF-7 and T-47D cell lines, which have silenced the PAPP-A gene, shows a reduction in proliferation. Further, MCF-7 parental cells cultured in conditioned media containing IGFBP-4 also show reduced proliferation, suggesting that the anti-proliferative effect of IGFBP-4 is associated with the absence of PAPP-A. Stable IGFBP-4 transfection of triple-negative MDA-MB-468 cell line, which has low extracellular IGFBP-4 when compared to MDA-MB-231 but does express PAPP-A, was sufficient to increase proliferation. Taken together, these results support the conclusion that proliferative effects of IGFBP-4 are dependent upon the expression status of PAPP-A. Citation Format: Keenan L. Flynn, Kevin D. Houston. Insulin-like growth factor binding protein 4 exerts differential effects on breast cancer cell proliferation based on the expression status of pregnancy-associated plasma protein A [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 108.

Small Molecule Anticancer Compound Modulates Cell Cycle DNA Damage Response Pathway and Inhibit Tumorigenesis in Triple Negative Breast Cancer

ObjectiveTo investigate the mechanism of action and therapeutic benefits of NSC001, a small molecule anticancer compound, in the treatment of triple negative breast cancer (TNBC), a clinical subtype of breast cancer that is more aggressive, has poor prognosis. In TNBC, patients harbor tumors that lack expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (Her2).HypothesisWe hypothesize that NSC001 will have a beneficial therapeutic effect on TNBC cells through the regulation of specific tumor associated signaling pathways in vitro.Material and methodsWe screened 1360 compounds from the National Cancer Institute (NCI) Diversity and Mechanistic Set library using the cell counting kit‐8 (CCK‐8) reagent from Dojindo Molecular Technologies, Inc. The screening was performed by applying 10µM of each candidate drug upon plates harboring the aggressive TNBC cell line, MDA‐MB‐231. Treatment time for all screening was fixed at 24 hours. The efficacy and potential mechanism of action of one of the potent drug candidates identified (NSC001) was further investigated on three TNBC cell lines (MDA‐MB‐231, MDA231‐LM2‐4175, and MDA‐MB‐468) using mass spectrometry and proteomic analysis. Afterwards, the results from the proteomic analysis were validated using molecular methods including cell viability assays, apoptosis assays, cell cycle assays, and western blotting.ResultsWe identified eight compounds that reduced the viability of MDA‐MB‐231 cells by up to 80% following a 24 hour treatment time point. Next, our in vitrodata show that NSC001 reduces the cell viability of TNBC cells in a dose‐and‐time dependent manner. Cell death data show that NSC001 induces potent apoptosis in a dose dependent manner in TNBC cells. Also, the results from colony formation assay show that 100nM [NSC001] is sufficient to inhibit the proliferative capacity of TNBC cells. Mass spectrometry and proteomic data reveals that NSC001 activates the cell cycle G2/M DNA damage checkpoint regulation in all the three TNBC cell lines used. Furthermore, we confirmed this finding in our cell cycle assay where we observed a clear G2/M cell cycle phase arrest. The arrest was potently induced at a dose significantly lower than the IC50 of NSC001. Lastly, we validated our proteomic results using western blotting, which confirmed that this low dose of NSC001 induces expression of p21, a cell cycle checkpoint regulator and tumor suppressor protein which is known to trigger cell cycle arrest in most cancer cell lines.ConclusionOverall, our data suggest that NSC001 may be a potent small molecule anticancer compound which should be further investigated for its potential to be used clinically in the treatment of TNBC patients.

Development of a Prognostic Model Based on the Identification of EMT-Related lncRNAs in Triple-Negative Breast Cancer.

Background Triple-negative breast cancer (TNBC) remains the most incurable subtype of breast cancer owing to high heterogeneity, aggressive nature, and lack of treatment options. It is generally acknowledged that epithelial-mesenchymal transition (EMT) is the key step in tumor metastasis. Methods With the application of TCGA and GEO databases, we identified EMT-related lncRNAs by the Cox univariate regression analysis. Optimum risk scores were calculated and used to divide TNBC patients into high-/low-risk subgroups by the median value using the Lasso regression analysis. The Kaplan–Meier and ROC curve analyses were applied for model validation. Then, we assessed the risk model from multi-omic aspects including immune infiltration, drug sensitivity, mutability spectrum, signaling pathways, and clinical indicators. We also analyzed the expression pattern of lncRNAs involved in the model using qRT-PCR in TNBC cell lines and constructed the ceRNA network. Results The risk model was composed of EMT-related long noncoding RNAs (lncRNAs), which seemed to be valuable in the prognostic prediction of TNBC patients. The model could act as an independent prognostic factor of TNBC and showed a robust prognostic ability in the stratification analysis. Further investigation demonstrated that the expression of lncRNAs was different between high aggressive and low aggressive TNBC cell lines, as well as TNBC patients. Conclusions Together, our study successfully established a risk model with great accuracy and efficacy in the prognostic prediction of TNBC patients.

Sophoridine Derivatives Induce Apoptosis and Autophagy to Suppress the Growth of Triple-Negative Breast Cancer through Inhibition of mTOR Signaling.

In order to improve the antitumor potency and therapeutic margins of natural product sophoridine, its novel nitrogen mustard carbamate derivatives were designed and synthesized. In screening their in vitro activity, we found all the tested compounds were more potent against the highly aggressive triple-negative breast cancer cell line MDA-MB-231. Cellular functional assays showed that representative compounds could induce G1-phase arrest and trigger apoptosis, evidenced by the alteration of Bax, Bcl-2, caspase-3 and PARP levels. Furthermore, these compounds significantly enhanced the autophagic flux with increased expression of LC3-II and Beclin-1, as well as decreased level of p62, which may attribute to simultaneously inhibition of the phosphorylation of p70S6K, 4E-BP1 and AKT, the key substrates of the mTOR signaling pathway. In vivo, two compounds revealed potent antitumor activity in mice bearing MDA-MB-231. Altogether, our work describes novel leads to yield more potent chemotherapeutics against triple-negative breast cancers, possibly mesenchymal stem-like subtype.

Abstract 1238: Treating estrogen receptor (ER)-negative and triple-negative breast cancer by targeting STAT3 signaling with putative STAT3 inhibitors

Abstract Approximately 20% to a quarter of breast cancer cases are estrogen receptor (ER)-negative breast cancer (ENBC), including the most aggressive subtype, triple-negative breast cancer (TNBC), which spreads early and shows poor prognosis. ENBCs do not respond to selective ER modulators such as tamoxifen. Therefore, there is an urgent need for non-ER-based therapies for ENBC. Recently, STAT3 was found to play an important role in maintaining cancer stem cells in vitro and in mouse models of human cancers. In addition, during mammary carcinogenesis, STAT3 is highly activated in mammary epithelial cells involving in mammary epithelial cell transformation, tumor initiation, progression, and metastasis. STAT3 has been traditionally considered as non-targetable or undruggable, and there is currently a lack of FDA-approved STAT3 inhibitors for clinical use. We recently developed a putative STAT3 inhibitor, HJC0152, and a series of HJC0152-based analogues as orally bioavailable STAT3 inhibitors. These small molecules have been evaluated and a number of them showed low micro-molar potency in inhibiting the proliferation of a panel of breast cancer cell lines. Compound JMX0804 showed improved anti-proliferative effects against the highly aggressive TNBC cell line MDA-MB-231 and other ER+ and ER- breast cancer cell lines in comparison to HJC0152. JMX0804 inhibited STAT3 phosphorylation at the Tyr705 residue and induced apoptosis in MDA-MB-231 cells. In vivo, mice treated with JMX0804 at a 10 mg/kg dose via i.p. showed reduced growth of xenograft tumors arisen from MDA-MB-231 cells, with minimal impact on general health and body weight. Our current research supports the notion that JMX0804 suppresses breast cancer growth by inhibiting breast cancer cell proliferation and inducing apoptosis via mediating STAT3 signaling. JMX0804 is currently undergoing further characterization for breast cancer metastasis and mechanisms of action as well as optimization to improve drug properties including potency and safety profile. This work is supported by NIH/NCI grant R01 CA231150. Citation Format: Hyejin Kim, Doerte R. Fricke, Jimin Xu, Haiying Chen, Jia Zhou, Qiang Shen. Treating estrogen receptor (ER)-negative and triple-negative breast cancer by targeting STAT3 signaling with putative STAT3 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1238.

Dual inhibition of MEK1/2 and MEK5 suppresses the EMT/migration axis in triple-negative breast cancer through FRA-1 regulation.

Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) when cells adopt a motile and invasive phenotype through loss of epithelial markers (CDH1), and acquisition of mesenchymal markers (VIM, CDH2). AlthoughMAPK/ERK1/2 kinase inhibitors (MEKi) are useful antitumor agents in a clinical setting, including the Food and Drug Administration(FDA)-approved MEK1,2 dual inhibitors cobimetinib and trametinib, there are limitations to their clinical utility, primarily adaptation of the BRAF pathway and ocular toxicities. The MEK5 (HGNC: MAP2K5) pathway has important roles in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast, and elevated levels of ERK5 expression in breast carcinomas are linked to a worse prognoses in TNBC patients. The purpose of this study is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells.Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide a rationale for the combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance.

Secreted Factors from Adipose Tissue Reprogram Tumor Lipid Metabolism and Induce Motility by Modulating PPARα/ANGPTL4 and FAK

Recent studies indicate that adipose tissue in obesity promotes breast cancer progression by secreting protumorigenic chemokines, growth factors, and fatty acids. However, the detailed mechanisms by which hypertrophic adipose tissue influences breast cancer cells are still not well understood. Here we show that co-culture with adipose tissue from high-fat diet induced obese C57BL/6 mice alters transcriptome profiles in triple-negative breast cancer (TNBC) cells, leading to upregulation of genes involved in inflammation and lipid metabolism, such as IL1B, PLIN2, and ANGPTL4. Similar results were obtained by treating TNBC cells with adipose tissue conditioned media (ACM) generated from fat tissue of obese female patients. Many of the upregulated genes were activated by PPAR nuclear receptors, as shown by pathway analyses and gene expression experiments using PPAR agonists and antagonists. Metabolic analysis revealed that TNBC cells cultivated with ACM had significantly higher levels of β-oxidation. Furthermore, ACM-treated TNBC cells displayed a pronounced aggressive cell phenotype, with enhanced wound healing, proliferation, and invasion capabilities. ACM-induced invasion was dependent on the PPAR-target ANGPTL4 and activated FAK signaling, as shown by ANGPTL4 depletion and FAK inhibition. Together, our data suggest that factors released by adipose tissue change PPAR-regulated gene expression and lipid metabolism and induce a more aggressive TNBC cell phenotype. These effects are, at least in parts, mediated by fatty acids provided by the adipose tissue. IMPLICATIONS: Adipose tissue provides factors for increased progression of TNBC cells, identifying PPAR- and FAK-signaling as potential novel targets for treatment of TNBC, especially in obese women.

The cell-surface anchored serine protease TMPRSS13 promotes breast cancer progression and resistance to chemotherapy.

Breast cancer progression is accompanied by increased expression of extracellular and cell-surface proteases capable of degrading the extracellular matrix as well as cleaving and activating downstream targets. The type II transmembrane serine proteases (TTSPs) are a family of cell-surface proteases that play critical roles in numerous types of cancers. Therefore, the aim of this study was to identify novel and uncharacterized TTSPs with differential expression in breast cancer and to determine their potential roles in progression. Systematic in silico data analysis followed by immunohistochemical validation identified increased expression of the TTSP family member, TMPRSS13 (transmembrane protease, serine 13), in invasive ductal carcinoma patient tissue samples compared to normal breast tissue. To test whether loss of TMPRSS13 impacts tumor progression, TMPRSS13 was genetically ablated in the oncogene-induced transgenic MMTV-PymT tumor model. TMPRSS13 deficiency resulted in a significant decrease in overall tumor burden and growth rate, as well as a delayed formation of detectable mammary tumors, thus suggesting a causal relationship between TMPRSS13 expression and the progression of breast cancer. Complementary studies using human breast cancer cell culture models revealed that siRNA-mediated silencing of TMPRSS13 expression decreases proliferation, induces apoptosis, and attenuates invasion. Importantly, targeting TMPRSS13 expression renders aggressive triple-negative breast cancer cell lines highly responsive to chemotherapy. At the molecular level, knockdown of TMPRSS13 in breast cancer cells led to increased protein levels of the tumor-suppressive protease prostasin. TMPRSS13/prostasin co-immunoprecipitation and prostasin zymogen activation experiments identified prostasin as a potential novel target for TMPRSS13. Regulation of prostasin levels may be a mechanism that contributes to the pro-oncogenic properties of TMPRSS13 in breast cancer. TMPRSS13 represents a novel candidate for targeted therapy in combination with standard of care chemotherapy agents in patients with hormone receptor-negative breast cancer or in patients with tumors refractory to endocrine therapy.

Comprehensive Proteomic Characterization Reveals Subclass-Specific Molecular Aberrations within Triple-negative Breast Cancer.

SummaryTriple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer lacking targeted therapies. This is attributed to its high heterogeneity that complicates elucidation of its molecular aberrations. Here, we report identification of specific proteome expression profiles pertaining to two TNBC subclasses, basal A and basal B, through in-depth proteomics analysis of breast cancer cells. We observed that kinases and proteases displayed unique expression patterns within the subclasses. Systematic analyses of protein-protein interaction and co-regulation networks of these kinases and proteases unraveled dysregulated pathways and plausible targets for each TNBC subclass. Among these, we identified kinases AXL, PEAK1, and TGFBR2 and proteases FAP, UCHL1, and MMP2/14 as specific targets for basal B subclass, which represents the more aggressive TNBC cell lines. Our study highlights intricate mechanisms and distinct targets within TNBC and emphasizes that these have to be exploited in a subclass-specific manner rather than a one-for-all TNBC therapy.

Aqueous leaf extract of Clinacanthus nutans inhibits growth and induces apoptosis via the intrinsic and extrinsic pathways in MDA-MB-231 human breast cancer cells

Background: Clinacanthus nutans possesses several reported biological activities against different human cancer cells. However, reports on the growth-inhibitory effect of C. nutans leaf extract on the aggressive triple-negative breast cancer cells and the mechanisms of induced-cell death in these cells are limited. Objectives: The study aimed to assess the anticancer efficacy and associated mechanisms of the crude aqueous extract of C. nutans leaves (cCN) in MDA-MB-231 triple-negative human breast cancer cells. Materials and Methods: The metabolic viability of the MDA-MB-231 cells following respective treatments with cCN was measured using an adenosine triphosphate luminescent assay. The mode of cell death in MDA-MB-231 cells induced by cCN was examined using a luminescence- and fluorescence-based assay and the mechanisms involved were evaluated by comparative analysis of gene expression by reverse transcription–quantitative polymerase chain reaction. Results: Dose- and time-dependent growth inhibition of MDA-MB-231 cells by cCN was observed (IC50: 191.20 μg/mL). cCN also induced apoptotic cell death in the treated cells via the intrinsic and extrinsic apoptosis pathways by affecting the mRNA expression levels of Bad, Bax, Bcl-2, Bcl-xL, and FasL. Conclusion: These results suggest that C. nutans can be used as a potential agent in the treatment and prevention of breast cancer.

Abstract 1116: The role of ADAM9 and PI3K-dependent NF-kappa B pathway in promoting metastasis of triple-negative breast cancer

Abstract Objectives: As a zinc-dependent proteinase family member, A Disintegrin and Metalloproteinase 9 (ADAM9) has the metalloproteinase domain that performs proteolytic function to liberate or shed factors, such as bFGF, VEGF, EGF, TGF-α and IGF, from cell surface and make them more accessible to tumor cells. It was shown in previous studies that the upregulation of ADAM9 in breast cancer is associated with metastasis. In this study, we examined the effect of ADAM9 on the cell proliferation, migration and invasion and elucidated the molecular mechanism in TNBC. We hypothesize that ADAM9 sheds Tumor Necrosis Factor alpha (TNF-α), which activates TNF receptor and then triggers PI3K-dependent NF-kappa B pathway, affecting metastatic phenotypes of triple negative breast cancer (TNBC). Materials and Methods: Correlation of the levels of ADAM9 expression and survival time in triple negative breast cancer patients was analysed by the Kaplan-Meier (KM) plotter integrative data analysis tool. The TNBC subjects were divided into two groups of high and low ADAM9 expression levels with respect to the median. We also compared the protein expression levels of ADAM9 in a highly aggressive, invasive and poorly differentiated TNBC cell line, MDA-MB-231, and a non-tumorigenic epithelial cell line, MCF-10A. ADAM9 was knocked down in MDA-MB-231 using siRNA. In comparison to negative and blank controls, we examined the cell proliferation, migration and invasion, and the phosphorylation levels of AKT subject to ADAM9 knockdown. We also profiled the mRNA expression levels of ADAM9 and NF-kappa B in breast cancer patients. Results: As shown in Figure 1(a), the survival curves of these two groups were plotted and compared using Kaplan-Meier survival analysis, indicating that lower ADAM9 expression is linked to better survival rate (p=0.0038). Figure 1(b) illustrates significantly higher ADAM9 protein level in MDA-MB-231, compared with MCF-10A (p=0.0019). It is shown in Figure 2 and Figure 3(a) that the cell proliferation, migration and invasion, and the phosphorylation of AKT are significantly suppressed by ADAM9 knockdown in the aggressive TNBC cell line. Figure 3(b) shows significant association between ADAM9 and NF-kappa B in the mRNA expression level (p<0.001). Conclusions: The results clearly showed the activation of PI3K-dependent NF-kappa B pathway triggered by ADAM9. This suggests a novel molecular mechanism that the stable homotrimers of TNF-α are released from the membrane through the proteolytic cleavage by ADAM9 and activate the signaling pathway leading to metastatic phenotype of TNBC. Acknowledgments: This project was supported by the National Basic Research Program of China (973 program, 2014CB744505). 1 Citation Format: Rui Zhou, Hong Zhang, Chi Shing Cho, Sze Chuen Wong, Mei Tian, Wing Chi Chan. The role of ADAM9 and PI3K-dependent NF-kappa B pathway in promoting metastasis of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1116.

Abstract 5196: Vorinostat exhibits anticancer effects through modulation of nuclear receptors and tumor suppressor genes in sub-types of triple negative breast cancer cells

Abstract Aberrantly expression of histone deacetylases (HDAC) play a critical role in tumorigenesis. The expression of individual HDACs has been shown to correlate to decreases in both, disease-free and overall survival, thus targeting HDACs is emerging as a new therapy for cancer. Histone acetylation is associated with elevated transcription, while deacetylation is associated with gene repression. Emerging evidence suggests their role in triple negative breast cancer (TNBC) progression. TNBC is an aggressive type of cancer that has poor prognosis and molecular studies have shown several subtypes within TNBC. These cancers lack targeted treatments and several options used such as, PARP inhibitors, angiogenesis inhibitors, androgen receptor inhibitors and others have shown limited efficacy, however histone deacetylase inhibitors are showing promising results in clinical trials. Vorinostat, a histone deacetylase inhibitor, also known as suberanilohydroxane acid (SAHA) was approved by the FDA for treatment of cutaneous T cell lymphoma. In this study, vorinostat was investigated in subtypes of TNBC cell lines, MB231(MSL) and two basal-like 2 TNBC cell lines, HCC70 and HCC1806. Cells were treated with vorinostat for 24 hrs. Vorinostat significantly up-regulated the tumor suppressor gene NR4A1 by 20 fold, NR4A3 by 10 fold and AR5A by 5 fold. RORα gene had the highest up-regulation, 60 fold, whereas PPARγ 4.5 fold. Estrogen related receptor A, ESRRA was also up-regulated in HCC70 cells. A decrease in expression was noted in DDX5, which is known to be elevated in basal-like tumors. A different profile was noted in the MB231 TNBC cells. Vorinostat also decreased growth and cell invasion, which shows its functional effects on these aggressive TNBC cell lines These changes suggest that vorinostat, which is currently in clinical trials for TNBC may be exerting its effect through modulation of orphan nuclear receptors and reactivation of tumor suppressor genes through epigenetic mechanisms. Further studies are needed to determine other signaling pathways modulated by this HDAC inhibitor. Citation Format: Fatemeh NouriEmamzadeh, Beverly Word, Ebony Cotton, Kai Littlejohn, Gustavo Miranda-Carboni, Beverly Lyn-Cook. Vorinostat exhibits anticancer effects through modulation of nuclear receptors and tumor suppressor genes in sub-types of triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5196.