Basal-like Triple-negative Breast Cancer Cell Research Articles

HuR (ELAVL1) Stabilizes SOX9 mRNA and Promotes Migration and Invasion in Breast Cancer Cells.

RNA-binding proteins play diverse roles in cancer, influencing various facets of the disease, including proliferation, apoptosis, angiogenesis, senescence, invasion, epithelial-mesenchymal transition (EMT), and metastasis. HuR, a known RBP, is recognized for stabilizing mRNAs containing AU-rich elements (AREs), although its complete repertoire of mRNA targets remains undefined. Through a bioinformatics analysis of the gene expression profile of the Hs578T basal-like triple-negative breast cancer cell line with silenced HuR, we have identified SOX9 as a potential HuR-regulated target. SOX9 is a transcription factor involved in promoting EMT, metastasis, survival, and the maintenance of cancer stem cells (CSCs) in triple-negative breast cancer. Ribonucleoprotein immunoprecipitation assays confirm a direct interaction between HuR and SOX9 mRNA. The half-life of SOX9 mRNA and the levels of SOX9 protein decreased in cells lacking HuR. Cells silenced for HuR exhibit reduced migration and invasion compared to control cells, a phenotype similar to that described for SOX9-silenced cells.

Abstract P4-08-11: AKT and EZH2 inhibitors kill TNBCs by hijacking mechanisms of involution

Abstract Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype and has the highest rate of recurrence. The predominant standard of care for advanced TNBC is systemic chemotherapy with or without immunotherapy, however responses are typically short-lived. Thus, there is an urgent need to develop more effective treatments. PI3K pathway components represent plausible therapeutic targets, as approximately 40% of TNBCs have PIK3CA/AKT1/PTEN alterations. However, unlike hormone receptor-positive tumors, it is still unclear if or how PI3K pathway inhibitors will be effective in triple-negative disease. Here we identify a promising AKT inhibitor-based therapeutic combination for TNBC. Specifically, we show that AKT inhibitors potently synergize with agents that suppress the histone methyltransferase, EZH2, and promote robust tumor regression in multiple TNBC models in vivo. AKT and EZH2 inhibitors exert these effects by first cooperatively driving basal-like TNBC cells into a more differentiated, luminal-like state, which cannot be effectively induced by either agent alone. More importantly, once differentiated, these agents kill TNBCs by hijacking signals that normally drive mammary gland involution. Together these findings identify a promising therapeutic strategy for this highly aggressive tumor type and illustrate how deregulated epigenetic enzymes can insulate tumors from oncogenic vulnerabilities. These studies also reveal how developmental tissue-specific cell death pathways may be co-opted for therapeutic benefit. Citation Format: Amy Schade, Naiara Perurena, Marina Watanabe, Carrie L. Rodriguez, Patrick Loi, Natalie Pilla, Rachel A. Davis, Kaia Mattioli, Dongxi Xiang, Jason J. Zoeller, Zhe Li, Ana C. Garrido-Castro, Sara Tolaney, Karen Cichowski. AKT and EZH2 inhibitors kill TNBCs by hijacking mechanisms of involution [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-11.

Hypoxia-induced YAP activation and focal adhesion turnover to promote cell migration in mesenchymal TNBC cells.

Hypoxia is commonly characterized by malignant tumors that promote the aggressiveness and metastatic potential of cancer. Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with approximately 46% capacity related to distant metastasis. Transcriptional factor yes-associated protein (YAP), a core component of the Hippo pathway, is associated with poor prognosis and outcome in cancer metastasis. Here, we explored the effect of hypoxia-mediated YAP activation and focal adhesions (FAs) turnover in mesenchymal TNBC cell migration. We characterized the effect of hypoxia on YAP in different breast cancer cell lines using a hypoxia chamber and CoCl2 . Hypoxia-induced YAP nuclear translocation is significantly observed in normal breast epithelial cells, non-TNBC cells, mesenchymal TNBC cells, but not in basal-like TNBC cells. Functionally, we demonstrated that YAP activation was required for hypoxia to promote mesenchymal TNBC cell migration. Furthermore, hypoxia induced the localization of FAs at the leading edge of mesenchymal TNBC cells. In contrast, verteporfin (VP), a YAP inhibitor, significantly reduced the migration and the recruitment of nascent FAs at the cell periphery under hypoxia conditions, which only showed in mesenchymal TNBC cells. Our data support the hypothesis that YAP is novel factor and positively responsible for hypoxia-promoting mesenchymal TNBC cell migration. Our findings provide further evidence and outcomes to help prevent the progression of TNBC.

An indispensable role of TAZ in anoikis resistance promoted by OTUB1 deubiquitinating enzyme in basal-like triple-negative breast cancer cells

Aggressive cancers, such as triple-negative breast cancer (TNBC), are mostly fatal because of their potential to metastasize to distant organs. Cancer cells acquire various abilities to metastasize, including resistance to anoikis, an apoptotic cell death induced by loss of anchorage to the extracellular matrix. Transcriptional coactivator with PDZ binding motif (TAZ) and Yes-associated protein (YAP), the downstream effectors of the Hippo pathway, regulate cell- and tissue-level architectures by responding to mechanical microenvironments of cells, including the cell–extracellular matrix interaction. The Hippo pathway is frequently disrupted in cancer cells, and TAZ and YAP are irrelevantly activated, potentially resulting in anchorage-independent survival/proliferation of cancer cells and metastatic progression. The study aims to investigate the roles of TAZ and YAP in anoikis resistance in basal-like (BL) TNBC cells, which comprise a major subtype (>70%) of TNBC. We found that TAZ and YAP had nonredundant roles in anchorage-independent cancer cell survival or anoikis resistance. Particularly, TAZ was indispensable for anoikis resistance in BL-TNBC cells but not for survival of non-transformed mammary epithelial cells (MECs). In contrast, YAP, a paralog of TAZ, was indispensable for survival of both non-transformed MECs and cancer cells. Therefore, TAZ might be a preferable therapeutic target against dissemination of aggressive cancer cells without killing normal cells. Interestingly, TAZ was abnormally stabilized in BL-TNBC cells under non-adherent conditions, which promoted anoikis resistance. Furthermore, OTUB1, a deubiquitinating enzyme, was responsible for the stabilization of TAZ in detached BL-TNBC cells. Importantly, simultaneous high expression of TAZ and OTUB1 was associated with poor prognosis in BC. Thus, OTUB1 has emerged as a potentially druggable target. Successful inhibition of OTUB1 enzymatic activity is expected to downregulate TAZ and eventually prevents metastasis of aggressive cancers, such as BL-TNBC.

Abstract LB127: Reduced expression of annexin-a6 induces metabolic reprogramming that favors rapid fatty acid oxidation in triple-negative breast cancer cells

Abstract The ability of cancer cells to alter their metabolism is one of the major mechanisms underlying rapid tumor progression and/or therapeutic resistance in solid tumors, including the hard-to-treat triple-negative breast cancer (TNBC) subtype. Here, we assessed the contribution of the tumor suppressor, Annexin A6 (AnxA6) in the metabolic adaptation of rapidly growing versus invasive TNBC cells as well as in Lapatinib resistant (Lap-R) TNBC cells. AnxA6 expression in cultured TNBC cell lines, Lap-R cells and in crude isolates of mitochondria was determined by Western blotting and/or immunofluorescence assays. The oxygen consumption rates (OCR), extracellular acidification rates (ECAR) and mitochondria activity were assessed using the Seahorse XF Analyzer. Intracellular metabolites were detected by 1H-NMR analysis. Using model proliferative basal-like and invasive mesenchymal-like TNBC cell lines, we show that TNBC cells also exhibit metabolic heterogeneity. Down regulation of AnxA6 in TNBC cells attenuated mitochondrial respiration, glycolytic flux, and cellular ATP production capacity, resulting in a quiescent cellular energy phenotype. We further show that AnxA6-depletion was associated with decreased lipid droplet accumulation and the lipolytic phenotype by enhancing the uptake and mitochondrial fatty acid oxidation for ATP production. Chronic Lapatinib-induced up regulation of AnxA6 in AnxA6-low TNBC cells reversed their lipolytic to a more lipogenic/glycolytic metabolic phenotype with gluconeogenic precursors as additional metabolites. Collectively, these data suggest that the expression status of AnxA6 in TNBC cells underlies significant metabolic adaptation during stress and/or chronic Lapatinib treatment and provide additional insights into the potential of AnxA6 as a biomarker for not only therapeutic intervention but also the metabolic subtyping of TNBC subsets. Citation Format: Stephen D. Williams, Sarrah E. Widatalla, Amos M. Sakwe. Reduced expression of annexin-a6 induces metabolic reprogramming that favors rapid fatty acid oxidation in triple-negative breast cancer cells [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 LB127.

Reduced Expression of Annexin A6 Induces Metabolic Reprogramming That Favors Rapid Fatty Acid Oxidation in Triple-Negative Breast Cancer Cells.

Simple SummaryThe expression status of Annexin A6 (AnxA6) has been shown to influence tumor growth, metastasis, and resistance to therapeutic intervention, but the contribution of this tumor suppressor in the metabolic adaptation of basal-like (AnxA6-low) versus mesenchymal-like (AnxA6-high) TNBC subsets remains unclear. The downregulation of AnxA6 in TNBC cells attenuated mitochondrial respiration, glycolytic flux, and cellular ATP production capacity, resulting in a quiescent metabolic phenotype. The overexpression or chronic lapatinib induced the expression of AnxA6 in AnxA6-low TNBC cells and reversed the quiescent phenotype to a more lipogenic/glycolytic phenotype. Interestingly, AnxA6-depletion was associated with rapid fatty acid uptake and oxidation (lypolytic metabolic phenotype) but decreased lipid droplet accumulation. Our data suggest that the expression status of AnxA6 in TNBC cells is associated with distinct metabolic adaptations of basal-like and mesenchymal-like TNBC subsets in response to cellular stress and/or therapeutic intervention.The ability of cancer cells to alter their metabolism is one of the major mechanisms underlying rapid tumor progression and/or therapeutic resistance in solid tumors, including the hard-to-treat triple-negative breast cancer (TNBC) subtype. Here, we assessed the contribution of the tumor suppressor, Annexin A6 (AnxA6), in the metabolic adaptation of basal-like (AnxA6-low) versus mesenchymal-like (AnxA6-high), as well as in lapatinib-resistant TNBC cells. Using model basal-like and mesenchymal-like TNBC cell lines, we show that TNBC cells also exhibit metabolic heterogeneity. The downregulation of AnxA6 in TNBC cells generally attenuated mitochondrial respiration, glycolytic flux, and cellular ATP production capacity resulting in a quiescent metabolic phenotype. We also show that AnxA6 depletion in mesenchymal-like TNBC cells was associated with a rapid uptake and mitochondrial fatty acid oxidation and diminished lipid droplet accumulation and altered the lipogenic metabolic phenotype of these cells to a lypolytic metabolic phenotype. The overexpression or chronic lapatinib-induced upregulation of AnxA6 in AnxA6-low TNBC cells reversed the quiescent/lypolytic phenotype to a more lipogenic/glycolytic phenotype with gluconeogenic precursors as additional metabolites. Collectively, these data suggest that the expression status of AnxA6 in TNBC cells underlies distinct metabolic adaptations of basal-like and mesenchymal-like TNBC subsets in response to cellular stress and/or therapeutic intervention and suggest AnxA6 as a biomarker for metabolic subtyping of TNBC subsets.

Permissive role of Na+/H+ exchanger isoform 1 in migration and invasion of triple-negative basal-like breast cancer cells.

In breast cancer, it is the resulting metastasis that is the primary cause of fatality. pH regulatory proteins and the tumor microenvironment play an important role in metastasis of cancer cells and acid-extruding proteins are critical in this process. There are several types of breast cancer and triple-negative breast cancer tends to be more metastatic and invasive and is itself is composed of several types. MDA-MB-468 are a triple-negative breast cancer cell line and are classified as basal-like and basal tumors account for up to 15% of breast cancers. Here we examined the effect of removal of the acid-extruding protein, the Na+/H+ exchanger isoform one, from MDA-MB-468 cells. NHE1 was deleted from these cells using the CRISPR/Cas9 system. Western blotting and measurement of activity confirmed the absence of the protein. In wounding/cell migration experiments, deletion of NHE1 reduced the rate of cell migration in the presence of low- or high-serum concentrations. Anchorage-dependent colony formation was also greatly reduced by deletion of the NHE1 protein. Cell proliferation was not affected by knockout of NHE1. The results demonstrate that NHE1 has an important role in migration and invasion of basal-like triple-negative breast cancer cells.

Identification of nuclear export inhibitor-based combination therapies in preclinical models of triple-negative breast cancer

An estimated 284,000 Americans will be diagnosed with breast cancer in 2021. Of these individuals, 15–20% have basal-like triple-negative breast cancer (TNBC), which is known to be highly metastatic. Chemotherapy is standard of care for TNBC patients, but chemoresistance is a common clinical problem. There is currently a lack of alternative, targeted treatment strategies for TNBC; this study sought to identify novel therapeutic combinations to treat basal-like TNBCs. For these studies, four human basal-like TNBC cell lines were utilized to determine the cytotoxicity profile of 1363 clinically-used drugs. Ten promising therapeutic candidates were identified, and synergism studies were performed in vitro. Two drug combinations that included KPT-330, an XPO1 inhibitor, were synergistic in all four cell lines. In vivo testing of four basal-like patient-derived xenografts (PDX) identified one combination, KPT-330 and GSK2126458 (a PI3K/mTOR inhibitor), that decreased tumor burden in mice significantly more than monotherapy with either single agent. Bulk and single-cell RNA-sequencing, immunohistochemistry, and analysis of published genomic datasets found that XPO1 was abundantly expressed in human basal-like TNBC cell lines, PDXs, and patient tumor samples. Within basal-like PDXs, XPO1 overexpression was associated with increased proliferation at the cellular level. Within patient datasets, XPO1 overexpression was correlated with greater rates of metastasis in patients with basal-like tumors. These studies identify a promising potential new combination therapy for patients with basal-like breast cancer.

Abstract PS17-04: Immune checkpoint blockade reprograms tumor microenvironment and systemic immune landscape in obesity associated triple negative breast cancer

Abstract Few targeted therapies exist for triple negative breast cancer (TNBC), an aggressive and deadly subtype. Obesity exacerbates poor outcomes in TNBC due to elevated invasion and metastasis leading to increased mortality. Obesity has paradoxically been shown to improve immune checkpoint therapies in other cancers, yet the underlying mechanisms are unclear. Despite being an inflammatory state, obesity increases immune checkpoint ligands PD-1 and PD-L1 which drives immunosuppression. Thus, we tested the hypothesis that obesity-driven changes to the immune milieu will improve ICB efficacy in TNBC. Syngeneic tumors were generated via orthotopic engraftment of E0771 basal-like TNBC cell line into age-matched immune-competent C57BL/6J female littermates on obesogenic or low fat diets. Mice were treated with anti-mouse anti-PD-1 or isotype control (IgG2a) every 3rd day until sacrifice. Obese mice gained almost 2-fold great body weight and 3.6-fold greater adiposity compared to lean mice and immunotherapy did not impact weights or body composition. Obesity led to immunosuppression systemically in bone marrow and spleen in tumor-free mice, which was exacerbated in tumor-bearing mice. Obese mice had significantly greater tumor progression and fewer regressed tumors at endpoint vs. lean mice. Anti-PD-1 significantly reduced tumor progression in obese mice with 4.2-fold reduction in volume and 5.7-fold reduction in tumor weights in obese mice vs. isotype controls. Anti PD-1 significantly reduced immunosuppressive cells including M2-like tumor associated macrophages and monocytic and granulocytic myeloid derived suppressor cells (MDSC) and raised anti-tumor M1-like macrophages, cytotoxic CD8+ T cells, and dendritic cells. Last, the microbiome has potent effects on responses to anti-tumor therapies such as chemotherapy and immune checkpoint blockade. Obesity is a major regulator of the gut microbiome. We found that beneficial bacteria belonging to genus Bifidobacterium was higher in lean compared to obese mice, which could limit tumor progression and lead to greater regression through robust anti-tumor immunity. In obese mice, beneficial bacteria belonging to genus Ruminococcus, Adlercreutzia, Corpococcus that promote anti-tumor immunity increased with anti PD-1 immunotherapy. In sum, we show for the first time that obesity-induced systemic and microenvironmental immunosuppression augmented tumor incidence and tumor progression. Furthermore, anti-PD-1 immune checkpoint blockade successfully reduced tumor progression in obese mice through reprogramming not only the TME but systemic immune milieu as well. Immunosuppressive targets unique to the obese TME could be targeted in concert with checkpoint inhibitors in future interventions to enhance durable anti-tumor immunity. Citation Format: Ajeeth K Pingili, Mehdi Chaib, Laura M Sipe, Emily J Miller, Bin Teng, Rahul Sharma, Qusai AI Abdallah, Deidra Daria, Radhika Sekhri, Heejoon Jo, Mims S Tahliyah, D. Neil Hayes, Joseph F Pierre, Liza Makowski. Immune checkpoint blockade reprograms tumor microenvironment and systemic immune landscape in obesity associated triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-04.

Possible Beneficial Effects of N-Acetylcysteine for Treatment of Triple-Negative Breast Cancer.

N-acetylcysteine (NAC) is a widely used antioxidant with therapeutic potential. However, the cancer-promoting effect of NAC observed in some preclinical studies has raised concerns regarding its clinical use. Reactive oxygen species (ROS) can mediate signaling that results in both cancer-promoting and cancer-suppressing effects. The beneficial effect of NAC may depend on whether the type of cancer relies on ROS signaling for its survival and metastasis. Triple-negative breast cancer (TNBC) has aggressive phenotypes and is currently treated with standard chemotherapy as the main systemic treatment option. Particularly, basal-like TNBC cells characterized by inactivated BRCA1 and mutated TP53 produce high ROS levels and rely on ROS signaling for their survival and malignant progression. In addition, the high ROS levels in TNBC cells can mediate the interplay between cancer cells and the tissue microenvironment (TME) to trigger the recruitment and conversion of stromal cells and induce hypoxic responses, thus leading to the creation of cancer-supportive TMEs and increased cancer aggressiveness. However, NAC treatment effectively reduces the ROS production and ROS-mediated signaling that contribute to cell survival, metastasis, and drug resistance in TNBC cells. Therefore, the inclusion of NAC in standard chemotherapy could probably provide additional benefits for TNBC patients.

Identified the novel resistant biomarkers for taxane-based therapy for triple-negative breast cancer.

Developing treatment strategies for triple-negative breast cancer (TNBC) has become an important clinical challenge. Currently, taxane-based chemotherapy is one of the standard treatments for TNBC. However, determining the key factor of taxane-resistance is urgently in need for clinical treatment for breast cancer. We used GEO data to generate paclitaxel resistance in two basal-like TNBC cell lines (SUM149 and MDA-MB-468). Seventy-one common upregulated differentially expressed genes (DEGs) and 11 downregulated DEGs were found to be related to paclitaxel resistance. By constructing protein-protein interactions, 28 hub proteins with a degree cutoff criterion of ≥1 were found. Nine hub genes (COL4A6, COL4A5, IL6, PDGFA, LPAR1, FYB, IL20, IL18R1 and INHBA) are involved in important signaling pathways. We found that upregulated PDGFA and downregulated COL4A6 were significantly associated with an insensitive response to neoadjuvant paclitaxel-based therapy. A Kaplan-Meier plot was created to check the prognostic values of 11 hub DEGs in terms of recurrence-free survival. High expressions of PDGFA and LAMB3 were correlated with poor recurrence-free survival, while low levels of FYB, IL18R1, and RASGRP1 indicated poorer relapse-free survival. Our results suggest that PDGFA, COL4A6, LPAR1, FYB, COL4A5, and RASGRP1 might be candidate target genes for taxane-based therapy in basal-like TNBC.

Correction: Targeting of PYK2 Synergizes with EGFR Antagonists in Basal-like TNBC and Circumvents HER3-Associated Resistance via the NEDD4-NDRG1 Axis.

Triple-negative breast cancer (TNBC) is a highly aggressive, heterogeneous disease with poor prognosis and no effective targeted therapies. EGFR is highly expressed in basal-like TNBC and is considered as a potential therapeutic target. However, EGFR targeting exerts only marginal clinical benefits, possibly due to activation of compensatory signaling pathways, which are frequently associated with HER3 upregulation. Here we show that concomitant targeting of EGFR and the nonreceptor tyrosine kinases PYK2/FAK synergistically inhibits the proliferation of basal-like TNBC cells in vitro and attenuates tumor growth in a mouse xenograft model. Dual targeting of EGFR and PYK2/FAK inhibited complementary key growth and survival pathways mediated by AKT, S6K, STAT3, and ERK1/2 activation. PYK2 inhibition also abrogated HER3 upregulation in response to EGFR antagonists, thereby circumventing HER3-associated drug resistance. Mechanistically, PYK2 inhibition facilitated the proteasomal degradation of HER3 while inducing upregulation of NDRG1 (N-myc downstream regulated 1 gene). NDRG1 enhanced the interaction of HER3 with the ubiquitin ligase NEDD4, while PYK2, which interacts with NEDD4 and HER3, interfered with NEDD4–HER3 binding, suggesting that the PYK2–NDRG1–NEDD4 circuit has a critical role in receptor degradation, drug response, and resistance mechanism. Our studies offer a preclinical proof of concept for a strategy of cotargeting the EGFR and PYK2/FAK kinases to improve TNBC therapy. Cancer Res; 77(1); 86–99. ©2016 AACR.

The orphan nuclear receptor estrogen-related receptor beta (ERRβ) in triple-negative breast cancer.

Triple-negative breast cancer (TNBC)/basal-like breast cancer (BLBC) is a highly aggressive form of breast cancer. We previously reported that a small molecule agonist ligand for the orphan nuclear receptor estrogen-related receptor beta (ERRβ or ESRRB) has growth inhibitory and anti-mitotic activity in TNBC cell lines. In this study, we evaluate the association of ESRRB mRNA, copy number levels, and protein expression with demographic, clinicopathological, and gene expression features in breast tumor clinical specimens. ESRRB mRNA-level expression and clinical associations were analyzed using RNAseq data. Array-based comparative genomic hybridization determined ESRRB copy number in African-American and Caucasian women. Transcription factor activity was measured using promoter-reporter luciferase assays in TNBC cell lines. Semi-automatic quantification of immunohistochemistry measured ERRβ protein expression on a 150-patient tissue microarray series. ESRRB mRNA expression is significantly lower in TNBC/BLBC versus other breast cancer subtypes. There is no evidence of ESRRB copy number loss. ESRRB mRNA expression is correlated with the expression of genes associated with neuroactive ligand-receptor interaction, metabolic pathways, and deafness. These genes contain G/C-rich transcription factor binding motifs. The ESRRB message is alternatively spliced into three isoforms, which we show have different transcription factor activity in basal-like versus other TNBC cell lines. We further show that the ERRβ2 and ERRβsf isoforms are broadly expressed in breast tumors at the protein level. Decreased ESRRB mRNA expression and distinct patterns of ERRβ isoform subcellular localization and transcription factor activity are key features in TNBC/BLBC.

IGFBP-3 interacts with NONO and SFPQ in PARP-dependent DNA damage repair in triple-negative breast cancer.

Women with triple-negative breast cancer (TNBC) are generally treated by chemotherapy but their responsiveness may be blunted by DNA double-strand break (DSB) repair. We previously reported that IGFBP-3 forms nuclear complexes with EGFR and DNA-dependent protein kinase (DNA-PKcs) to modulate DSB repair by non-homologous end-joining (NHEJ) in TNBC cells. To discover IGFBP-3 binding partners involved in chemoresistance through stimulation of DSB repair, we analyzed the IGFBP-3 interactome by LC-MS/MS and confirmed interactions by coimmunoprecipitation and proximity ligation assay. Functional effects were demonstrated by DNA end-joining in vitro and measurement of γH2AX foci. In response to 20µM etoposide, the DNA/RNA-binding protein, non-POU domain-containing octamer-binding protein (NONO) and its dimerization partner splicing factor, proline/glutamine-rich (SFPQ) formed complexes with IGFBP-3, demonstrated in basal-like TNBC cell lines HCC1806 and MDA-MB-468. NONO binding to IGFBP-3 was also shown in a cell-free biochemical assay. IGFBP-3 complexes with NONO and SFPQ were blocked by inhibiting EGFR with gefitinib or DNA-PKcs with NU7026, and by the PARP inhibitors veliparib and olaparib, which also reduced DNA end-joining activity and delayed the resolution of the γH2AX signal (i.e. inhibited DNA DSB repair). Downregulation of the long noncoding RNA in NHEJ pathway 1 (LINP1) by siRNA also blocked IGFBP-3 interaction with NONO-SFPQ. These findings suggest a PARP-dependent role for NONO and SFPQ in IGFBP-3-dependent DSB repair and the involvement of LINP1 in the complex formation. We propose that targeting of the DNA repair function of IGFBP-3 may enhance chemosensitivity in basal-like TNBC, thus improving patient outcomes.

Mutant TP53 modulates metastasis of triple negative breast cancer through adenosine A2b receptor signaling.

PurposeThe identification of genes with synthetic lethality in the context of mutant TP53 is a promising strategy for the treatment of basal-like triple negative breast cancer (TNBC). This study investigated regulators of mutant TP53 (R248Q) in basal-like TNBC and their impact on tumorigenesis.Experimental DesignTNBC cells were analyzed by RNA-seq, and synthetic-lethal shRNA knock-down screening, to identify genes related to the expression of mutant TP53. A tissue microarray of 232 breast cancer samples, that included 66 TNBC cases, was used to assess clinicopathological correlates of tumor protein expression. Functional assays were performed in vitro and in vivo to assess the role of ADORA2B in TNBC.ResultsTranscriptome profiling identified ADORA2B as up-regulated in basal-like TNBC cell lines with R248Q-mutated TP53, with shRNA-screening suggesting the potential for a synthetic-lethal interaction between these genes. In clinical samples, ADORA2B was highly expressed in 39.4% (26/66) of TNBC patients. ADORA2B-expression was significantly correlated with ER (P < 0.01), PgR (P = 0.027), EGFR (P < 0.01), and tumor size (P = 0.037), and was an independent prognostic factor for outcome (P = 0.036). In line with this, ADORA2B-transduced TNBC cells showed increased tumorigenesis, and ADORA2B knockdown, along with mutant p53 knockdown, decreased metastasis both in vitro and in vivo. Notably, the cytotoxic cyclic peptide SA-I suppressed ADORA2B expression and tumorigenesis in TNBC cell lines.ConclusionsADORA2B expression increases the oncogenic potential of basal-like TNBC and is an independent factor for poor outcome. These data suggest that ADORA2B could serve as a prognostic biomarker and a potential therapeutic target for basal-like TNBC.

Tumor-stroma interactions differentially alter drug sensitivity based on the origin of stromal cells.

Due to tumor heterogeneity, most believe that effective treatments should be tailored to the features of an individual tumor or tumor subclass. It is still unclear, however, what information should be considered for optimal disease stratification, and most prior work focuses on tumor genomics. Here, we focus on the tumor microenvironment. Using a large‐scale coculture assay optimized to measure drug‐induced cell death, we identify tumor–stroma interactions that modulate drug sensitivity. Our data show that the chemo‐insensitivity typically associated with aggressive subtypes of breast cancer is not observed if these cells are grown in 2D or 3D monoculture, but is manifested when these cells are cocultured with stromal cells, such as fibroblasts. Furthermore, we find that fibroblasts influence drug responses in two distinct and divergent manners, associated with the tissue from which the fibroblasts were harvested. These divergent phenotypes occur regardless of the drug tested and result from modulation of apoptotic priming within tumor cells. Our study highlights unexpected diversity in tumor–stroma interactions, and we reveal new principles that dictate how fibroblasts alter tumor drug responses.

Abstract 4058: Race-associated breast cancer disparities: The role of CRYBB2 and its pseudogene

Abstract Research demonstrates young African American (AA) women have higher mortality rates for triple-negative breast cancer (TNBC), despite lower incidence rates, when compared to Caucasian American (CA) women. These disparate poor patient outcomes may be attributed to access to quality care, socioeconomic status, education, or geographic location. However, numerous other studies have demonstrated that gene expression differences may also contribute to the higher mortality rates experienced by AA women, suggestive of biological differences among these two patient populations. Consequently, CRYBB2 is one gene that has consistently been associated with AA race in normal and malignant tissues. Although, its basic function in these tissues is currently unknown, higher expression of CRYBB2 is significantly correlated with poor outcome in breast, colorectal and prostate cancers in AAs. We analyzed all publically available gene expression datasets from studies focused on breast cancer health disparities and discovered that all probes detected CRYBB2 as well as its pseudogene, CRYBB2P1. It is becoming increasingly clear that pseudogenes have the ability to modify the mechanisms of their functional parental genes. However, it remains unclear whether the high expression of CRYBB2, CRYBB2P1, or both, affect cell behaviors to alter disease progression. Herein, we investigated the biological consequences of CRYBB2 and CRYBB2P1 in three basal-like TNBC cell lines (SUM159, MDA-MB-231, Hs578t) using CRISPR/Cas9 and overexpression technologies. The functional consequence of CRYBB2 and CRYBB2P1 expression were evaluated using in vitro and biochemical assays, as well as in vivo tumorigenesis studies. Our findings demonstrate overexpression of CRYBB2 and CRYBB2P1 alter cell proliferation, cytokine and growth factor production, and tumorigenesis. This data highlights the potential molecular mechanisms by which CRYBB2 and CRYBB2P1 contribute to poor patient outcome. Citation Format: Maya A. Barrow, Megan E. Martin, Denise K. Reaves, Breanna Jeffcoat, Ginger R. Smith, Michael Tarpley, Kevin P. Williams, Jodie M. Fleming. Race-associated breast cancer disparities: The role of CRYBB2 and its pseudogene [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 4058.

Abstract 789: Nuclear IGFBP-3 is a potential biomarker for response to EGFR-sphingosine kinase targeted therapy in basal-like triple-negative breast cancer (TNBC)

Abstract Background: TNBC comprises about 15% of breast cancer cases and, with no approved targeted therapy, is commonly treated with adjuvant chemotherapy. We have described a novel targeted therapy for basal-like TNBC combining EGFR and sphingosine kinase (SphK) inhibitors, which induce a strongly synergistic cytostatic effect in six TNBC cell lines (Martin et al, Breast Cancer Res 19:90, 2017). IGFBP-3, which is highly expressed in basal-like TNBC and prognostic for poor recurrence-free survival, initiates an oncogenic pathway involving SphK1 and EGFR activation. Since IGFBP-3 downregulation in vitro prevents the synergistic cytostasis caused by EGFR and SphK inhibitors, we aimed in this study to evaluate nuclear IGFBP-3 as a response biomarker in TNBC tumors after treatment with the inhibitor combination, with or without doxorubicin (dox), a common component of TNBC chemotherapy. Methods: Human basal-like TNBC cell lines HCC1806 and MDA-MB-468 were grown as orthotopic xenograft tumors in female BALB/c nude mice, and treated when tumors reached 100 mm3 with the SphK inhibitor and S1P receptor modulator, fingolimod, plus the EGFR kinase inhibitor, gefitinib (F+G) ± dox at the maximum tolerated dose of 2 mg/Kg weekly. Tumors were analyzed by immunohistochemistry (IHC). Cell proliferation was studied in vitro by IncuCyte live-cell imaging. Results: In both cell lines in vitro, F+G at doses that caused minimal cytostasis, acted synergistically with a sub-cytostatic dox dose (10 nM), to cause almost complete inhibition of proliferation. In contrast, in xenograft mouse models, in which F+G administered 3x weekly significantly inhibited tumor growth and enhanced mouse survival, dox administration for 6 weeks showed minimal growth inhibition alone, and no significant incremental effect with F+G. Examined by IHC, TNBC tumor IGFBP-3 staining showed predominantly nuclear localization, was positively correlated with Ki67, and was significantly downregulated by F+G treatment, with no added dox effect. By Kaplan-Meier analysis, high (&amp;gt; median) nuclear IGFBP-3 IHC scores were strongly associated with worse mouse survival, similar to high Ki67 scores, while high apoptosis (cleaved caspase-3) scores were associated with better survival. Conclusion: The synergism between dox and combination F+G, observed in vitro, was not seen in vivo, possibly because of dose-limiting dox toxicity. This might be improved by alternative dox delivery methods. However, the F+G combination alone is highly inhibitory to basal-like TNBC tumor growth in both xenograft models. IGFBP-3 is known to interact with nuclear hormone receptors and DNA damage response intermediates in the nucleus. Nuclear IGFBP-3 staining may have utility as a biomarker of treatment response in TNBC, alone or together with Ki67 and cleaved caspase-3. Supported by Cancer Council NSW. Citation Format: Sohel M. Julovi, Janet L. Martin, Robert C. Baxter. Nuclear IGFBP-3 is a potential biomarker for response to EGFR-sphingosine kinase targeted therapy in basal-like triple-negative breast cancer (TNBC) [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 789.

Abstract A65: Evaluating an under-studied kinase as a potential druggable target for breast cancer

Abstract Breast cancer is the most frequently diagnosed cancer among women, accounting for 23% of all cancer cases. Triple-negative breast cancer (TNBC) is a very aggressive type of breast cancer that occurs in African-American women at three-fold higher frequency compared to Hispanic and Caucasian women. TNBC has a lower 5-year survival rate compared to other subtypes of breast cancer and no targeted therapeutics. In order to identify novel targets for TNBC, replicate kinome-wide RNAi screens were performed with HCC1806, a basal-like TNBC cell line, using a 4-day cell viability assay. Among the RNAi hits, we identified the COASY gene, encoding Coenzyme A synthase (CoAsy), as an under-studied kinase. Thus, we sought to confirm this RNAi hit and evaluate CoAsy for its potential as a novel druggable target for TNBC. CoAsy is a bifunctional enzyme that carries out the last two steps in the coenzyme A (CoA) biosynthetic pathway, including the phosphorylation of dephospho-CoA to generate CoA. Our hypothesis is that TNBC cells require a higher level of CoA synthase activity for cell proliferation compared to normal cells and thus inhibiting CoA synthase represents a novel therapeutic strategy for TNBC. Using four different individual RNAi targeting COASY, we used transient RNAi transfection of a panel of breast cancer cell lines and a non-transformed breast cell line to determine the effect of CoAsy knock down on cell viability and proliferation. Knockdown of CoAsy protein by RNAi was demonstrated by Western blot. Transient COASY RNAi transfection inhibited the cell viability/proliferation assay for several TNBC cell lines, but not for a normal breast cell line. Thus, these data suggest that knockdown of CoAsy may preferentially inhibit cell proliferation of breast cancer cell lines. In addition, Western blot was performed to determine and compare the expression of CoAsy in a panel of breast cancer cell lines and nontransformed cell lines. Ongoing studies are investigating the dependence of cell line viability and proliferation on the expression level of CoAsy by creating stable inducible shRNA cell lines. Citation Format: Hamzah Kharabsheh, Xiyou Zhou, John Scott. Evaluating an under-studied kinase as a potential druggable target for breast cancer [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr A65.

Nuclear Insulin-Like Growth Factor Binding Protein-3 As a Biomarker in Triple-Negative Breast Cancer Xenograft Tumors: Effect of Targeted Therapy and Comparison With Chemotherapy

Triple-negative breast cancer (TNBC) typically has a worse outcome than other breast cancer subtypes, in part owing to a lack of approved therapeutic targets or prognostic markers. We have previously described an oncogenic pathway in basal-like TNBC cells, initiated by insulin-like growth factor binding protein-3 (IGFBP-3), in which the epidermal growth factor receptor (EGFR) is transactivated by sphingosine-1-phosphate (S1P) resulting from sphingosine kinase (SphK)-1 activation. Oncogenic IGFBP-3 signaling can be targeted by combination treatment with the S1P receptor modulator and SphK inhibitor, fingolimod, and the EGFR kinase inhibitor, gefitinib (F + G). However, the interaction of this treatment with chemotherapy has not been documented. Since we observed nuclear localization of IGFBP-3 in some TNBC tumors, this study aimed to evaluate the prognostic significance of nuclear IGFBP-3 in pre-clinical models of basal-like TNBC treated with F + G and doxorubicin. Orthotopic xenograft tumors were grown in nude mice from the human basal-like TNBC cell lines MDA-MB-468 and HCC1806, and were treated with gefitinib, 25 mg/Kg, plus fingolimod, 5 mg/Kg, 3-times weekly. In some studies, doxorubicin was also administered once weekly for 6 weeks. Tumor tissue proteins were quantitated by immunohistochemistry (IHC). Interaction between doxorubicin and F + G was also studied in proliferation assays in vitro. In both tumor models, tissue staining for IGFBP-3 was predominantly nuclear. Combination of F + G significantly enhanced mouse survival, decreased nuclear IGFBP-3 and Ki67 staining, and increased apoptosis (cleaved caspase-3) staining. Kaplan–Meier survival analysis showed that a high tumor IGFBP-3 IHC score (>median), like a high Ki67 score, was significantly associated with shorter survival time, whereas a high apoptosis score was associated with prolonged survival. Studied in vitro in both cell lines, low-dose doxorubicin that had little effect alone, strongly enhanced the cytostatic effect of low-dose F + G combination. However, in both in vivo models, doxorubicin at maximum-tolerated dose neither inhibited tumor growth when administered alone, nor enhanced the significant inhibitory effect of F + G. We conclude that doxorubicin may not add benefit to the inhibitory effect of F + G unless its dose-limiting toxicity can be overcome. Nuclear IGFBP-3 appears to have potential as a prognostic marker in TNBC and could be evaluated for clinical utility.