Triple-negative Cancer Cell Lines Research Articles

Abstract 3940: Validation of TFX1, a first-in-class target for cancer metastasis

Abstract Most of the drugs approved for metastatic epithelial carcinomas treat the proliferation of the tumor and not the biology of metastasis. To date, all clinical trials around metastasis that focused on proteases, kinases, or integrins have failed in clinics. As a result, metastasis biology-focused drug discovery has been deprioritized.We asked the question as to what the rate-limiting steps of metastasis were. To answer it, we created a cell-based functional assay platform, METAssay™, that dissected all metastasis biology steps into thirty cellular assays and characterization steps. The platform identified the functional differences between metastatic and proliferating cells. This platform was normalized with patient tumor cells, and a machine learning algorithm, METSCAN™, was subsequently applied to identify the weighted steps that contributed towards successful metastasis. This led to the identification of four first-in-class targets; two transcription factors, one cytokine receptor and one nuclear hormone receptor.We then validated the relevance of the first target, TFX1, by overexpressing it in both colorectal and triple-negative breast cancer cell lines. Interestingly, apart from the expected increase in migration and invasion, all cell lines showed an increase in ROS, Autophagy, PDL-1, CD-73, quiescence, exosome uptake and a decrease in apoptosis, mitosis, and exosome secretion. Conversely, the downregulation of TFX1 showed the opposite effect. TFX1 was purified, and a biochemical isothermal calorimetry (ITC) assay was standardized with a tool compound with a KD of 107 nM. The compound effectively reduced metastatic properties in the cell lines and in multiple patient samples, as analyzed by the METAssay™ platform. Colorectal cancer cell lines overexpressing TFX1 also showed increased platelet binding, which was reduced by the tool compound, but not triple-negative cancer cell lines. Also, the effect of overexpression in non-metastatic cells was more profound than in metastatic cells, suggesting a saturating effect. Further characterization of the tool compound is currently ongoing to help establish a baseline for a novel discovery programme.The ideal drug to delay metastasis should be given to primary tumor patients with no evidence of clinical metastasis, irrespective of their pathological grading. Therefore, it should have a high bar for safety. Our goal is to collaborate on a discovery journey to identify candidates and create a companion diagnostic that will identify primary tumor patients at high risk of metastasis, thereby selecting the right patient cohort for clinical trials. Citation Format: Arnab Roy Chowdhury, Manoj Pandre, Chinmaya Amarkanth, Debabani Roy Chowdhury, John Ellingboe. Validation of TFX1, a first-in-class target for cancer metastasis. [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 3940.

Isoform Selectivity of HDAC Inhibitors Has a Significant Effect on PD-L1 Expression in the Triple-negative Cancer Cell Line MDA-MB-231

Various reports have been published in recent years on the effects of histone deacetylase (HDAC) inhibitors on programmed death ligand 1 (PD-L1) expression in cancer cells. The combination therapy of immune checkpoint inhibitors and HDAC inhibitors utilizing these effects has attracted attention as a new clinical treatment of triple-negative breast cancers. We investigated how the expression level of PD-L1 changes depending on the type of HDAC inhibitor exposed to triple-negative breast cancer cell line MDA-MB-231. We found that the mRNA expression level of PD-L1 was significantly decreased by Vorinostat and K-32 (pan-HDAC inhibitors) at high concentrations exhibiting low cell viability, while it was increased by high concentrations of K-560 (HDAC1,2 inhibitor) and Entinostat (HDAC1,3 inhibitor). On the other hand, the mRNA level of PD-L1 was increased by all of these HDAC inhibitors at low concentrations showing high cell viability. Of particular note, K-32 induced more PD-L1 mRNA than all the other HDAC inhibitors at the lowest concentration of 0.5 μM. This finding might suggest the usefulness of pan-HDAC inhibitors in clinical treatment in combination with immune checkpoint inhibitors.

Abstract PO-096: Blocking the CXCL12/CXCR4 pathway both radiosensitizes brain metastases in mice and protects against radiation-induced cognitive dysfunction following whole brain irradiation in rats

Abstract We have previously shown using an antagonist of CXCR4 (plerixafor) with brain implanted tumors in mice(1)or by blocking CXCL12 by olaptesed pegol (ola-peg) with chemically-induced brain tumors in rats(2) that the radiation response of the tumors is markedly enhanced by blocking the CXCL12/CXCR4 pathway. In the present study we aimed to test whether a similar radiation enhancement is produced in mice with multiple brain metastases, and in addition, in preparation for a clinical trial of whole brain irradiation (WBI) with plerixafor, we sought to determine whether blocking the CXCL12/CXCR4 pathway would affect the cognitive dysfunction of rats given WBI. In the brain metastasis study we produced multiple brain metastases by injection of the luciferase transduced brain-tropic human breast triple negative cancer cell line MDA-MB-231 B3 into the internal carotid artery of nude mice. Adding the CXCL12 antagonist ola-peg after irradiation produced both a doubling of the regrowth delay (as monitored by bioluminescence imaging) and median survival time compared to irradiation alone. We also showed that similar to the brain implanted human glioblastoma studies that blocking the pathway prevented the radiation-induced influx of tumor associated macrophages (TAMs) into the brain metastases. In preliminary studies with cognitive dysfuction we gave adult Sprague-Dawley rats whole brain irradiation of 5, 10, 15 or 20 Gy and 2 months later measured their short term memory using the novel object recognition (NOR) test. We found that 20 Gy but not lower doses abrogated the rats’ ability to recognize novel objects, a key test of memory. Importantly we showed in a follow-up study that a 4-week infusion of plerixafor started immediately after irradiation (with the same dosing and schedule as our earlier tumor studies) not only did not affect memory in the controls and 15 Gy WBI groups but completely protected against memory loss in the 20 Gy group. In conclusion we showed that blocking the CXCL12/CXCR4 pathway not only potentiates the radiation response of a breast cancer brain metastasis model in mice but also protects against radiation-induced cognitive dysfunction in rats. This has led us to propose that blocking of this pathway which prevents the radiation-induced influx of TAMs into tumors and normal tissues is a novel strategy to enhance the therapeutic ratio of radiotherapy (3). 1. Kioi M, Vogel H, Schultz G, Hoffman RM, Harsh GR, Brown JM. Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice. J Clin Invest 2010; 120:694-705. 2. Liu SC, Alomran R, Chernikova SB, Lartey F, Stafford J, Jang T, et al. Blockade of SDF-1 after irradiation inhibits tumor recurrences of autochthonous brain tumors in rats. Neuro Oncol 2014; 16:21-8. 3. Brown JM, Thomas R, Nagpal S, Recht L. Macrophage exclusion after radiation therapy (MERT): A new and effective way to increase the therapeutic ratio of radiotherapy. Radiother Oncol 2019; 144:159-64. Citation Format: Martin Brown, Mehrdad Shamloo, Sophia Chernikova, Lawrence Recht. Blocking the CXCL12/CXCR4 pathway both radiosensitizes brain metastases in mice and protects against radiation-induced cognitive dysfunction following whole brain irradiation in rats [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-096.

In silico studies of some 2-anilinopyrimidine derivatives as anti-triple-negative breast cancer agents

BackgroundBreast cancer is a major form of health problem on the globe and the second cause of death related to cancer amidst women. A prediction of about 1 to 1.3 million cases on cancer of the breast are detected yearly globally. Triple-negative type of breast cancers (TNBCs) are described by the lack of human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), and progesterone receptor (PR). TNBCs metastasize to the central nervous system and lungs regularly. Such metastatic actions reduce the life expectancy of patients with TNBC than patients with non-TNBC due to non-enhanced inhibitor compounds. The purpose of this research was to explore the anti-proliferative activities of 2-anilinopyrimidine derivatives against triple-negative cancer cell line MDA-MB-468 via in silico studies like QSAR and molecular docking studies to further design and develop new anti-breast cancer drug with high potency and low toxicity.ResultsThe quantitative structure–activity relationship QSAR model predicts the bioactivities of the compounds, and molecular docking studies comprehend the interaction between the derivatives (ligand) and thyroid hormone (TRβ1) (receptor). Model 4 was chosen as the best model from the statistical assessment; R2 = 0.8760, R2adj = 0.8451, Q2 = 0.6141, and R2pred of 0.5390. From the external validation of the QSAR model, the coefficient of the mean effect on the model parameters indicates that decreasing (VR1_Dzv and MOMI-R) and increasing (SpMin1_Bh and C3SP3) would increase the anti-proliferative activities (pIC50) of the compounds. The molecular docking studies revealed that ligands 15 and 18 had the highest docking scores of − 7.3 and − 7.4 kcal/mol with thyroid hormone receptor (TRβ1). The ligands had docking scores better than the standard anti-breast cancer drug gefitinib (− 5.3 kcal/mol).ConclusionsThe results indicate that model 4 can be used in developing new 2-anilinopyrimidine derivatives, with better anti-breast cancer prediction activity and performance. It was proved that some series of 2-anilinopyrimidine derivative compounds bind tightly to the receptor, stabilizing the receptor (TRβ1) which is evident from the receptor–ligand interactions, and these compounds would serve as the most promising inhibitors against TRβ1. This shows a breakthrough for pharmaceutical researchers in designing and developing new anti-triple-negative breast cancer drugs.

Abstract 4363: Effects of the small GTPase RhoC on inflammatory breast cancer metabolism

Abstract Inflammatory breast cancer (IBC) is an extremely aggressive and rare form of cancer that disproportionally affects African American and younger women. While IBC represents 1-5% of breast cancers, it accounts for 10% of all breast cancer deaths annually in the United States. We have previously shown that the metabolic characteristics of IBC, specifically in the triple negative (TN) and inflammatory breast cancer cell line SUM149, are significantly altered from those of normal breast cancer cells. We have shown previously that RhoC, a member of the Ras superfamily of GTPases, contributes to the metastatic IBC phenotype and acts as a regulator of the metabolite N-acetyl aspartate (NAA) in SUM149 cells. NAA is the second most abundant metabolite in the brain (only exceeded by glutamate) and has been used for the diagnosis of neurodegenerative disorders such as the fatal genetic disorder Canavan’s disease, which is characterized by toxic NAA levels due to a deleterious mutation in aspartoacyclase(ASPA). ASPA is the enzyme involved in the catabolism of NAA into aspartate and acetate. NAA has previously been considered a brain specific metabolite and the fundamental role of NAA outside of the central nervous system, especially in the context of cancer, remains elusive but highly intriguing. We have previously shown that NAA levels are significantly higher in the IBC-derived TN SUM149 cells than TN cancer cell line MDA-MB-231. Interestingly, and independently, recent studies have also implicated high levels of tumoral NAA with significantly worse survival rates in ovarian cancer patients. The synthesis of NAA from acetyl-CoA and aspartate is catalyzed by the enzyme Asp-NAT, which is encoded for by the gene NAT8L. Knockdown of RhoC in SUM149 cells using shRNA significantly decreases both NAT8L expression and NAA metabolite levels. To further understand RhoC’s role in the modulation of NAA, we generated RhoC and RhoA knockout cell lines using CRIPSR-Cas9 in a set of inflammatory and noninflammatory breast cancer cell lines. RNA-seq analysis of these show that SUM149 RhoC knockout cells produce the largest amount of differentially regulated genes when compared to wild-type cells. The differentially regulated genes include many of the key genes involved in NAA associated pathways. Furthermore, metabolic studies reveal that the most downregulated metabolites in the SUM149 RhoC knockout cell line are N-acetyl derivates including NAA, N-acetylglutamate(NAG), and N-acetylaspartylglutamate (NAAG). Overexpression and siRNA cell lines for both NAT8L and ASPA have been generated to further elucidate the altered metabolism seen in these cells. Additional molecular studies are ongoing to determine the specific role of NAA in the adapted metabolic pathways of inflammatory breast cancer that may shed light on IBC’s increased metastatic potential and decreased survival. Citation Format: Laura E. Goo, Joel A. Yates, Andrew C. Little, Daniel Kremer, Ilya Kovalenko, Christopher R. Oliver, Trisha Westerhof, Zhifen Wu, Nathalie Vandecan, Liwei Bao, Peter J. Ulintz, Costas A. Lyssiotis, Sofia D. Merajver. Effects of the small GTPase RhoC on inflammatory breast cancer metabolism [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 4363.

Abstract 1444: RhoC regulation of inflammatory breast cancer metabolism

Abstract Inflammatory breast cancer (IBC) is an extremely aggressive and lethal type of cancer due to its ability to rapidly metastasize. Our previous reports have shown that the metabolic characteristics of IBC are altered from those of normal breast epithelial cells. Specifically, the levels of the metabolite N-acetylaspartate (NAA) are very high in the IBC-derived triple negative cell line SUM149 relative to the triple negative cancer cell line MDA-MB-231. Recent studies have also correlated high levels of tumoral NAA with significantly lower survival rates in ovarian cancer. N-acetylaspartate (NAA) is the second most abundant metabolite in the brain, only exceeded by glutamate. NAA is synthesized from aspartate and acetyl-CoA by the enzyme Asp-NAT, which is encoded for by the gene NAT8L. The catabolism of NAA to aspartate and acetate is carried out by the enzyme aspartoacyclase (ASPA). In the central nervous system (CNS), altered levels of NAA have been historically used as a marker for neurodegenerative diseases such as Canavan disease, a fatal genetic disorder caused by a deleterious mutation in the ASPA gene. However, the fundamental role of NAA, especially in the context of cancer remains elusive. We seek to understand and describe the role of NAA in the adapted metabolic pathways of inflammatory breast cancer that enable proliferation and metastasis of the disease. Our previous work revealed that the pro-metastatic small GTPase RhoC acts as a regulator of NAA metabolism in SUM149 cells. Knockdown of RhoC using shRNA significantly decreases NAT8L expression. RhoC and RhoA knockout cell lines have been generated using CRIPSR-Cas9 in both SUM149 and MDA-MB-231 cells to further distinguish the relationship between NAA and RhoC in these different cell lines. We have conducted metabolic studies to determine the relative abundance of common metabolites, including NAA, throughout glycolysis, the pentose-phosphate pathway, and the tricarboxylic acid cycle. We have performed similar metabolomics using xenograft mice implanted with these CRISPR knockout cell lines to determine in vivo recapitulation of the NAA phenotypes. Our work ultimately hopes to identify promising therapeutic pathways of NAA as well as further characterize NAA's role as a potential oncometabolite. Citation Format: Laura E. Goo, Joel A. Yates, Liwei Bao, Zhifen Wu, Sofia D. Merajver. RhoC regulation of inflammatory breast cancer metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1444.

Energy Metabolism Drugs Block Triple Negative Breast Metastatic Cancer Cell Phenotype.

To establish alternative targeted therapies against triple negative (TN) breast cancer, the energy metabolism and the sensitivity of cell growth, migration, and invasiveness toward metabolic, canonical, and NSAID inhibitors were analyzed in MDA-MB-231 and MDA-MB-468, two TN metastatic breast cancer cell lines, under both normoxia (21% O2) and hypoxia (0.1% O2). For comparative purposes, the analysis was also carried out in the less-metastatic breast MCF-7 cancer cells. Under normoxia, oxidative phosphorylation (OxPhos) was significantly higher (2-times) in MDA-MB-468 than in MDA-MB-231 and MCF-7, whereas their glycolytic fluxes and OxPhos and glycolytic protein contents were all similar. TN cancer cell lines mainly depended on OxPhos (62-75%), whereas MCF-7 cells equally depended on both pathways for ATP supply. Hypoxia for 24 h promoted a significant increase (>20 times) in the glycolytic transcriptional master factor HIF1-α in its target proteins GLUT-1, HKI and II, and LDH-A (2-4 times) as well as in the glycolytic flux (1.3-2 times) vs normoxia in MDA-MB-468, MDA-MB-231, and MCF-7. On the contrary, hypoxia decreased (15-60%) the contents of COXIV, 2OGDH, ND1, and ATP synthase as well as the OxPhos flux (50-75%), correlating with a high mitophagy level in the three cell lines. Under hypoxia, the three cancer cell lines mainly depended on glycolysis (70-80%). Anti-mitochondrial drugs (oligomycin, casiopeina II-gly, and methoxy-TEA) and celecoxib, at doses used to block OxPhos, significantly decreased TN cancer cell proliferation (IC50 = 2-20 μM), migration capacity (10-90%), and invasiveness (25-65%). The present data support the use of mitochondrially targeted inhibitors for the treatment of TN breast carcinoma.

Abstract P4-07-12: Re-introduction of tumor suppressor miR-34a harbors therapeutic efficacy in triple negative breast cancer

Abstract Place Holder Abstract Date Expected for Obtaining Final Information for Abstract: 9/14 - 10/14 Triple negative breast cancer accounts for a disproportionate share of the total breast cancer morbidity because of its aggressive behavior, increased incidence in younger women, and lack of effective targeted therapies. MicroRNAs are global regulators of survival and proliferation pathways important in cancer development and tumor maintenance. MicroRNAs function as tumor suppressors and oncogenes, and are highly dysregulated in cancer. Here, we identified miR-34a to be aberrantly lost in triple negative cancer cell lines when compared to both a luminal cancer subtype as well as normal breast cells. Re-introduction of miR-34a in triple negative cancer lines results in inhibition of cell proliferation, reactivation of senescence, and enhances sensitivity to apoptotic-inducing agents. Furthermore, intratumoral delivery of miR-34a into subcutaneously implanted tumors in nude mice delays tumor growth as compared to a scrambled control. In conclusion re-introduction of miR-34a in triple negative breast cancer promotes anti-tumorigenic phenotypes both in vitro and in vivo, and could therefore be used as a therapeutic agent to treat the disease. Citation Format: Brian D Adams, Lajos Pusztai, David L Rimm, Frank J Slack. Re-introduction of tumor suppressor miR-34a harbors therapeutic efficacy in triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-07-12.

Downregulation of Smurf2, a tumor-suppressive ubiquitin ligase, in triple-negative breast cancers: Involvement of the RB-microRNA axis

BackgroundThe HECT family ubiquitin ligase Smurf2 regulates cell polarity, migration, division, differentiation and death, by targeting diverse substrates that are critical for receptor signaling, cytoskeleton, chromatin remodeling and transcription. Recent studies suggest that Smurf2 functions as a tumor suppressor in mice. However, no inactivating mutation of SMURF2 has been reported in human, and information about Smurf2 expression in human cancer remains limited or complicated. Here we demonstrate that Smurf2 expression is downregulated in human breast cancer tissues, especially of the triple-negative subtype, and address the mechanism of Smurf2 downregulation in triple-negative breast cancer cells.MethodsHuman breast cancer tissues (47 samples expressing estrogen receptor (ER) and 43 samples with triple-negative status) were examined by immunohistochemistry for the expression of Smurf2. Ten widely-studied human breast cancer cell lines were examined for the expression of Smurf2. Furthermore, microRNA-mediated regulation of Smurf2 was investigated in triple-negative cancer cell lines.ResultsImmunohistochemical analysis showed that benign mammary epithelial cells expressed high levels of Smurf2, so did cells in ductal carcinomas in situ. In contrast, invasive ductal carcinomas showed focal or diffuse decrease in Smurf2 expression, which was observed more frequently in triple-negative tumors than in ER-positive tumors. Consistently, human triple-negative breast cancer cell lines such as BT549, MDA-MB-436, DU-4475 and MDA-MB-468 cells showed significantly lower expression of Smurf2 protein, compared to ER + or HER2+ cell lines. Studies using quantitative PCR and specific microRNA inhibitors indicated that increased expression of miR-15a, miR-15b, miR-16 and miR-128 was involved in Smurf2 downregulation in those triple-negative cancer cell lines, which have mutations in the retinoblastoma (RB) gene. Forced expression of RB increased levels of Smurf2 protein with concomitant decreases in the expression of the microRNAs.ConclusionsThis study provides evidence of posttranscriptional downregulation of Smurf2 in triple-negative breast cancers, and demonstrates that the loss of RB function is involved in microRNA-mediated interference with Smurf2 translation. The new link from RB inactivation to Smurf2 downregulation is likely to play a role in malignant phenotypes of triple-negative breast cancer cells.

Synthesis of new 2-galactosylthiazolidine-4-carboxylic acid amides. Antitumor evaluation against melanoma and breast cancer cells

A set of 2-galactosylthiazolidine-4-carboxylic acid amides was synthesized with different length for the carbon chain amide moiety. The cytotoxicity of the molecules was evaluated against A375 melanoma and MCF7 breast cancer cell lines. For the derivatives tested, the one that contains a C16 amide carbon chain is the most active with an IC50 of 17.0 μM for A375 and 5.8 μM for MCF7. This compound also shows cytotoxicity in the triple negative cancer cell line HCC1806. The selectivity of the compounds was assessed by comparing the cytotoxicity in cancer cell line versus in a fibroblast cell line. Flow cytometry studies show the activation of apoptotic pathways and also DNA damages with blockage of the cell cycle in the S-phase and appearance of peaks in G0/G1-phase.