Long noncoding RNA LINP1: scaffolding non-homologous end joining.

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

Introduction. Triple negative breast cancer (TNBC), which comprises 15-20% of all breast cancers, is the deadliest and most aggressive subtype. TNBC affects a younger patient population and is associated with an increased risk of distant recurrence, higher rates of metastases, earlier time to recurrence, and worse prognosis after recurrence. AMP-activated protein kinase (AMPK) is a major cellular energy regulator that has recently been implicated in cancer progression. AMPK can act as a tumor suppressor by decreasing anabolism and inducing cell cycle arrest. FND 4b is a novel AMPK activator that has been shown to inhibit growth and induce apoptosis in colon cancer, but its effects have not been studied in breast cancer. The purpose of this project is to test the effects of FND 4b on AMPK activation, cellular proliferation, and apoptosis in several types of breast cancer—with a particular emphasis on TNBC. Methods. (i) To assess signaling pathways, estrogen-receptor positive (ER+) breast cancer cells (MCF-7 and T-47D), TNBC cells (MDA-MB-231 and HCC-1806), and breast cancer stem cells were treated with FND 4b (0, 1, 2.5, 5, 10, and 20 µM) for 24 h. Immunoblot analysis assessed phosphorylated and total forms of AMPK, acetyl CoA carboxylase (ACC), and ribosomal protein S6 as well as cyclin D1 and cleaved PARP. (ii) Growth assays with sulforhodamine B (SRB) were performed by treating ER+ breast cancer cells and TNBC cells with FND 4b (0, 2.5, 5, 10, and 20 µM) for 72 h and then quantifying cellular protein content. Proliferation was also assessed by treating all cell lines with FND 4b (0 or 5 µM) for 72 h and then counting viable cells. (iii) Cell death was assessed by treating ER+ breast cancer cells and TNBC cells with FND 4b (0, 2.5, 5, and 10 µM) for 72 h and then quantifying fragmented DNA in the cytoplasm by ELISA. Results. (i) FND 4b treatment for 24 h increased AMPK activation with concomitant decreases in ACC activity, phosphorylated S6, and cyclin D1 in ER+ breast cancer, TNBC, and breast cancer stem cells. (ii) FND 4b treatment for 72 h (5 µM) decreased proliferation in all breast cancer cells, while dose-dependent decreases in growth were also observed in ER+ breast cancer and TNBC cells. (iii) Increases in apoptosis were found in ER+ breast cancer and one TNBC cell line with FND 4b treatment for 72 h. Conclusions. Our findings indicate that FND 4b can decrease proliferation for a variety of breast cancers by activating AMPK—with notable effects on TNBC. The reductions in growth were mediated through decreases in fatty acid synthesis (ACC), mTOR signaling (S6), and cell cycle flux (cyclin D1). ER+ breast cancer cells were more susceptible to FND 4b-induced apoptosis, but MDA-MB-231 cells still underwent apoptosis with higher dose FND 4b treatment. Further development of FND compounds—with extensions to animal studies—could result in a novel therapeutic agent in the treatment of TNBC. Citation Format: Jeremy Johnson, Piotr Rychahou, Vitaliy M. Sviripa, Heidi L. Weiss, David Watt, B. Mark Evers. FND 4b decreases proliferation and increases apoptosis of triple negative breast cancer through AMPK activation [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 3868.

Background: Triple-negative breast cancer (TNBC) accounts for 10-15% of all breast cancer cases. A major area of innovation in TNBC is identifying potential treatment targets, especially in TNBC cells which survive chemotherapy. Our previous study showed that TNBC cells displayed deregulated kinase-dependent signaling cascades, and uniquely divergent phospho-circuits could be distinguished between TNBC vs non-TNBC cell lines [2014 SABCS abstract 1672, poster P2-05-09]. We further hypothesized that specific dysfunctional phospho-signaling network played a key role in the early adaptive changes in DNA damage response of TNBC cells exposed to DNA damaging chemotherapeutic agents. Methods: TNBC cell lines MDA-MB-231 and MDA-MB-436 were treated with 5-fluorouracil (5-Fu), carboplatin and doxorubicin at their respective half maximal inhibitory concentrations (ic50s). MiSeq gene sequencing of the untreated vs treated TNBC cells was performed to investigate whether exposure to chemotherapy agents for 3-day's duration would induce additional adaptive genetic mutation. Apoptosis and cell-cycle distribution of the untreated and treated TNBC cells were analyzed with flow cytometry. The functional phospho-signature of each TNBC cell sample was analyzed using a high throughput experimental platform that monitors the level of activity of myriad kinases at once. This technique used over 450 phospho-sensing probes, including over 150 controls in an aqueous-based assay to simultaneously and directly measure the phospho-catalytic activity of phosphorylating enzymes in cell lysates. The kinome activities of the untreated vs treated TNBC cell lines were compared respectively, and the most significantly deranged and functionally altered phospho-signaling cascades and their related kinases were identified as the early adaptive changes of the survived TNBC cells after the 3-day exposure to DNA damage chemotherapies. Results: Using the two TNBC cell lines treated with the three chemotherapies, we made 8 cell line samples, including 6 treated and 2 untreated as the control. MiSeq gene sequencing showed no significant additional adaptive genetic mutations in the treated TNBC cells after the 3-day short-term exposure to 5-Fu, carboplatin and doxorubicin. 36 phospho-signatures were generated and validated for repeatability and robustness. The kinase activity signature of each TNBC sample was analyzed and compared to each other using unsupervised hierarchical clustering. The phospho-sensing assay revealed that phospho-signaling cascades related to CHK1/2 and IKK kinases were differentially altered in the untreated vs treated TNBC cell lines, which, when respectively inhibited by AZD7762 and IKK16, successfully increased growth inhibition and cell death of TNBCs. Conclusions: We identified specific phospho-fingerprints of the early adaption of TNBC cell lines and combinatorial targeted therapies that improve treatment outcome. Our next goal is to identify specific phosphorylation cascades in a broader range of cell lines and tumor tissues, to explore the actionable, kinase-dependent mechanisms critical to the DNA damage-induced adaptive reprogramming of TNBCs and early changes driving drug-resistance. Citation Format: Pan B, Olow A, Sun Q, Mori M, Lee PRE, Hartog M, Wang C, Wolf D, Yau C, van 't Veer L, Coppé J-P. Functional detection and inhibition of the targetable oncogenic kinome of chemotherapy-treated triple-negative breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-08-03.

Extracellular vesicles (EVs) containing proteins, DNA, and RNA facilitate cellular crosstalk within and among tissues and are involved in a variety of physiologic and disease processes. Cancer cells secrete EVs that promote multiple aspects of cancer progression, including epithelial-mesenchymal transition (EMT), angiogenesis, metastasis, and immune suppression. EVs are formed by two different mechanisms, budding from the plasma membrane and through the multivesicular endosome pathway, and these subpopulations of EVs are commonly referred to as microvesicles and exosomes respectively. Notably, exosomes range in size from 50-150 nm, while microvesicles can range from 100-1000 nm. Microtubules are important for the vesicular trafficking involved in exosome formation, cargo loading, and release. Therefore, we hypothesized that microtubule targeting drugs would affect the formation and release of exosomes from triple negative breast cancer (TNBC) cells and alter their cargo. We investigated whether eribulin and paclitaxel differently affect exosome formation and release because they have highly divergent effects on microtubules, in that eribulin causes microtubule loss and paclitaxel increases microtubule density. Exosome formation was evaluated by immunofluorescence of the multivesicular endosome/exosome marker, CD63, in multiple TNBC cell lines. Eribulin and paclitaxel significantly altered the cellular localization of CD63 after 4 h. High-content imaging analysis revealed a significant intracellular accumulation of CD63, suggesting that eribulin and paclitaxel could impair exosome release. Small EVs/exosomes were then isolated from TNBC cells treated for 8 h with concentrations of eribulin and paclitaxel that maximally disrupted CD63 without causing significant mitotic accumulation or cell death. Small EVs/exosomes isolated from cell-conditioned media were characterized by electron microscopy, immunoblotting and nanoparticle tracking analysis (NTA). The results of NTA show that while paclitaxel did not change the number of EVs released after an 8 h treatment, eribulin caused a modest, but consistent decrease in the number of EVs released by MDA-MB-231 cells. Immunoblot analysis further revealed a significant reduction in CD63 content of EVs from eribulin, but not paclitaxel-treated MDA-MB-231 cells. Additional fluorescent NTA using CD63 antibody labeling showed that CD63+ EVs were substantially reduced by eribulin but unchanged by paclitaxel. Interestingly, the CD63+ EVs that were isolated from eribulin-treated MDA-MB-231 cells were significantly larger than those from control or paclitaxel-treated cells, demonstrating that eribulin inhibits the release of small CD63+ EVs, likely exosomes. Taken with the fact that the total concentration of small EV particles released by eribulin-treated cells was only modestly reduced, our results suggest that eribulin promotes the release of small microvesicles while inhibiting the release of CD63+ exosomes. This shift in the population of EVs released by TNBC cells as a result of eribulin treatment could have implications for other EV cargo that impact cancer progression. Studies to confirm these effects in additional TNBC cell lines and with other microtubule targeting drugs, as well as proteomic analysis of EV content are ongoing. Furthering our understanding of the differential effects of eribulin and paclitaxel on EV formation and cargo has the potential to inform on the more targeted use of these drugs in the future. These studies were funded by Eisai Inc. Citation Format: Petra EJ Pederson, Huiyun Liang, Susan L Mooberry. Eribulin and paclitaxel differentially affect exosome formation and release from triple negative breast cancer cells [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 PS16-14.

Role of syndecan-4 in angiogenesis and vasculogenic mimicry in triple negative breast cancer cells.

Previously, we found that GST-tagged tumor necrosis factor-related apoptosis inducing ligand preferentially killed triple-negative breast cancer (TNBC) cells with a mesenchymal phenotype by activating death receptor 5 (DR5). The purpose of this study was to explore the sensitivity of breast cancer cell lines to drozitumab, a clinically tested DR5-specific agonist; identify potential biomarkers of drozitumab-sensitive breast cancer cells; and determine if those biomarkers were present in tumors from patients with TNBC. We evaluated viability, caspase activity, and sub-G1 DNA content in drozitumab-treated breast cancer cell lines and we characterized expression of potential biomarkers by immunoblot. Expression levels of vimentin and Axl were then explored in 177 TNBC samples from a publically available cDNA microarray dataset and by immunohistochemistry (IHC) in tumor tissue samples obtained from 53 African-American women with TNBC. Drozitumab-induced apoptosis in mesenchymal TNBC cell lines but not in cell lines from other breast cancer subtypes. The drozitumab-sensitive TNBC cell lines expressed the mesenchymal markers vimentin and Axl. Vimentin and Axl mRNA and protein were expressed in a subset of human TNBC tumors. By IHC, ~15 % of TNBC tumors had vimentin and Axl expression in the top quartile for both. These findings indicate that drozitumab-sensitive mesenchymal TNBC cells express vimentin and Axl, which can be identified in a subset of human TNBC tumors. Thus, vimentin and Axl may be useful to identify TNBC patients who would be most likely to benefit from a DR5 agonist.

In the present study, we investigated the role of β-catenin on aerobic glycolysis in breast cancer cells and further evaluated its effects on triple-negative breast cancer (TNBC) cell migration and invasion. We used two metastatic TNBC cell lines, i.e., MDA-MB-468 and MDA-MB-231, and treated them with β-catenin antagonists (WNT5A and XAV939). Evaluation of glycolytic markers in WNT5A-expressing cell lines showed selective downregulation in the expression of β-catenin, platelet type-phosphofructokinase (PFKP), and lactate secretion, without affecting cell proliferation. Of the three phosphofructokinase isoform, we found that only PFKP expression was associated with decreased survival in breast cancer patients. We also found that the cause of PKFP and lactate downregulation was mediated by inhibition of β-catenin signaling, which is in accordance with the finding that β-catenin signaling positively regulates lactate production. Functionally, we showed that specific downregulation of β-catenin resulted in reduced lactate production accompanied by impaired TNBC cells migration and invasion. Finally, we demonstrated that apart from its inhibitory effect on lactate production, inhibition of β-catenin can also impair TNBC cell migration induced by exogenous lactate via downregulating monocarboxylate transporter 1 (MCT1). Overall, we demonstrated that inhibition of β-catenin-PFKP signaling axis impaired lactate production as well as its uptake from tumor microenvironment (via MCT1), thereby impairing TNBC cells' migration. Citation Format: Chandra P. Prasad, Tommy Andersson. Inhibition of β-catenin impairs triple-negative breast cancer (TNBC) cell migration and invasion by modulating aerobic glycolysis components [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B021.

Purpose:Triple-negative breast cancer is characterized by fast progression with high possible for metastasis and poor survival. Dysfunction of microRNAs plays an important role in the initiation and progression of cancer. Our previous microRNA-seq data indicated the downregulation of miR-331-3p in triple-negative breast cancer tissues compared with that of the noncancer tissues. However, the function of miR-331-3p in triple-negative breast cancer remains largely unknown. Herein, the involvement of miR-331-3p in triple-negative breast cancer was investigated and the therapeutic potential of miR-331-3p was also explored.Methods:Real-time quantitative polymerase chain reaction was performed to detect the expression of miR-331-3p in triple-negative breast cancer tissues and cell lines. The cell proliferation was determined by the cell counting kit-8 assay. Apoptosis of triple-negative breast cancer cells was examined by annexin V/propidium iodide staining. miRDB database was used to predict the potential targets of miR-331-3p. Western blot was performed to examine the expression of the target protein.Results:miR-331-3p was significantly downregulated in triple-negative breast cancer tissues and cell line. Lower miR-331-3p expression was significantly correlated with the tumor size, TNM stage, and lymph node metastasis of patients with triple-negative breast cancer. Functional experiments showed that the overexpression of miR-331-3p inhibited the proliferation and increased apoptosis of triple-negative breast cancer cells. Neuropilin-2 was identified as a target of miR-331-3p, which harbored binding site of miR-331-3p in its 3′-untranslated region. Overexpression of miR-331-3p decreased the messenger RNA and protein levels of neuropilin-2 in triple-negative breast cancer cells. Restoration of neuropilin-2 partially reversed the inhibitory effects of miR-331-3p on the proliferation of triple-negative breast cancer cells.Conclusions:Our results demonstrated the novel function of miR-331-3p/neuropilin-2 signaling in regulating the malignant behaviors of triple-negative breast cancer cells, which suggested miR-331-3p as a potential target for the treatment of triple-negative breast cancer.

Background: Triple-negative breast cancer (TNBC) is heterogenous, which includes basal-like, immunomodulatory, mesenchymal, mesenchymal stem cell and luminal androgen receptor subtypes. In addition to chemotherapy, PARP inhibitors and immune checkpoint inhibitors are effective for TNBC with BRCA-mutated and PD-L1-positive cases. However, overall survival of TNBC remains unfavorable. To explore new targeted therapy in TNBC, we reported previously that SOCS1 (suppressor of cytokine signaling 1), IL-13 and several genes were predicted as interactive targets of miRNA in TNBC from the microarray expression analysis (Agilent Technologies, Inc.) of small RNA extracted from 11 breast cancer specimens (AACR2020, #1709). Thus, we investigated SOCS1-related immune-response under co-culture of TNBC and monocyte cell lines with or without paclitaxel exposure. Materials and Methods: MDA-MB-231, MDA-MB-468, RAW264.7 cell lines were used with co-culture system (Falcon® Cell Culture Inserts, Corning, NY, USA). SOCS1-expression vector and siSOCS1 were transfected into TNBC cell lines with polyethylenimine (PEI Max; Polysciences, Inc., Warrington, PA). RAW264.7 was activated with lipopolysaccaride (5ug/ml) and IFNγ (10ng/ml) for 2 days before co-culture. Relative cell viability of TNBC cell lines with vehicle control (DMSO) or paclitaxel exposure was measured with WST-8 (Cell Counting Kit-8; Dojindo, Japan) using TC20 Automated cell counter (Bio-Rad Laboratories, Inc., Japan) after 2 days’ co-culture of TNBC and RAW264.7 cells under several conditions of SOCS1 overexpression and/or RAW264.7 activation. Results: First, paclitaxel inhibited cell growth of MDA-MB-231 and MDA-MB-468 in a dose-dependent manner (at 1nM~100nM). Second, at paclitaxel exposure of 10 nM and 50 nM, relative cell viability of MDA-MB-468 decreased with co-culture of activated RAW264.7. The tendency was enhanced with SOCS1 overexpression in breast cancer cell lines. Similar results were observed in MDA-MB-231 cells. Discussions: SOCS1 is well known as a negative regulator of JAK-STAT pathway. SOCS1 is also related to suppression of T cell activation and modulation of M1 macrophage. At AACR2020, we reported that SOCS1 gene amplification in cancer tissues after neoadjuvant chemotherapy was relatively lower in 6 cases with pathologically partial or complete response than in 11 cases with no pathological response. In vitro, paclitaxel-toxicity in TNBC cells was augmented by activated monocyte (M1 macrophage) and more sensitized with SOCS1 overexpression in TNBC cells. These results suggest that proinflammatory cytokines from M1 macrophage influence cell viability of TNBC. Conclusion: SOCS1 is a promising molecular target to modulate immune-response in TNBC. Citation Format: Shigeru Imoto, Tomohiro Chiba, Hirohito Seki, Yoshiharu Ishizaka, Ai Tsuchiya, Tomoko Kitaoka. New targeted therapy on SOCS1-related immune-response in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5363.

Background: Programmed death ligand 1 (PDL1) is commonly expressed on the surface of many tumor cells, including breast cancer. The activity of tumor-infiltrating lymphocytes (TIL) is inhibited by PDL1. High PD-L1 basal cells (particularly basal B) overexpress genes involved in invasion, motility, and chemoresistance (Soliman et al., 2014). Targeting and blocking PD-L1 may enhance eradication of aggressive breast cancer cells by the immune system. Verteporfin (VP), a photosensitizer used to treat macular degeneration, was found to inhibit PD-L1 expression in a high-throughput screen. Studies demonstrated VP inhibits YAP activation by disrupting YAP-TEAD interactions and preventing YAP induced oncogenic growth (Zhang et al., 2016). Here we demonstrate the inhibitory effect of VP on PD-L1 expression in TNBC cell lines. Methods: MDA-MB-231 cells co-cultured with human PBMCs were treated with VP (Sigma) in presence of concanavalin A (ConA) and analyzed using flow cytometry to study levels of CD8+IFNg+ cells. TNBC cell lines (MDA-MB-231, BT-20, HCC-1143, Hs-578T) were treated with VP at doses ranging from 1µM-10µM for 24 hrs. The cells were processed for flow cytometry, Western blot and RT-PCR to check PD-L1 in mechanistic studies looking at manipulation of YAP pathway genes. Results: When MDA-MB-231 cells were co-cultured with normal human PBMCs in the presence of ConA, the CD8+IFNg+ stained cells were reduced compared to PBMC + ConA alone. Interestingly, in the group treated with VP, rescue of CD8+IFNg+ cells was observed. Moreover, MDA-MB-231, BT-20, HCC-1143 and Hs-578T TNBC cells treated with VP showed a significant dose-dependent inhibition of PD-L1 expression by flow cytometry. Western blot analysis also showed complete clearance of PD-L1 protein band with the lowest dose (1µM) used. However, RT-PCR analysis did not show a significant fold change in mRNA levels of PD-L1 in MDA-MB-231 treated cells. Surprisingly, mechanistic studies performed by silencing YAP1, E2F1, and TBK1 in MDA-MB-231, BT-20 and HS-578 T showed a decline in PD-L1 in E2F1 silenced cells, highlighting a plausible role of E2F1-PDL1 signaling axis. However, no change in PD-L1 expression was seen in cells silenced with YAP1 and TBK1. Further, chromatin-immunoprecipitation assay demonstrated E2F1 binding to PD-L1 promoter. Conclusion: Our data so far demonstrate that verteporfin treatment leads to inhibition of PD-L1 in TNBC cell lines and improvement in CD8+IFNg+ cells, indicating that VP might have potential for treatment approaches. Our study warrants further attention towards understanding the mechanism of action of VP in inhibiting PD-L1 and the role of E2F1 in the process. Citation Format: Fatema Khambati, Neha Jaiswal, Srikumar Chellappan, Hatem Soliman. Verteporfin inhibits surface PD-L1 expression in triple-negative breast cancer (TNBC) cells [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 4701.

Introduction: Acquisitions of metastasis-associated phenotypes (MA) by Triple Negative Breast Cancer (TNBC) cells are imparted by genetic alterations which cause deregulation of different signaling pathways associated to those phenotypes. We have recently reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the salient genetic features of TNBC and demonstrated that the WP signaling in TNBC is associated with poor prognosis and metastasis (Dey et al., 2013). We have also identified that the functional upregulation of secreted-MMP7, a transcriptional target of WP in TNBC is associated to the functional loss/absence of PTEN gene (Dey et al., 2013), the most common first event associated with basal-like subtype (Martins et al., 2012). Aim: In order to understand the relevance of WP pathway in the biology of metastasizing TNBC tumor cells, here we have undertaken a study in which the involvement of WP is tested in the context of MA phenotypes including (1) integrin-directed migration, (2) matrigel-invasion and (3) clonogenic growth and proliferation in 15 TNBC cell lines. Methods: CHIR9902, Wnt-C59, XAV939, sulindac sulfide, beta-catenin SiRNA as well as physiological stimulation of the WP (LWnt3A CM) were used for the study. Podia-parameters were studied using confocal microscopy for architecture of filamentous actin while matrigel invasion was studied by MMP7 specific caesin-zymography. Results: The collective% of alterations in CTNNB1, APC and DVL1 genes among total breast invasive carcinomas (TCGA 2012) were 17% in contrast to 46% breast invasive carcinomas, PAM50 Basal. A similar trend was observed among subtypes of BC from brca/tcga/pub2015 (cBioPortal). Wnt-C59, XAV939, sulindac sulfide and beta-catenin SiRNA (1) inhibited fibronectin-directed migration (2) decreased maximum relative distance from the origin and average velocity of the movement by real-time video microscopy, (3) altered cytoskeletal organization of the filamentous-actin and decreased the podia parameters, (4) decreased matrigel-invasion, and (6) inhibited cell proliferation and 3D colony formation. Mechanistically the treatment with sulindac sulfide and beta-catenin SiRNA decreased cellular levels of beta-catenin, active beta-catenin and MMP7 in the same TNBC cell lines wherein fibronectin-directed migration, invasion, and colony formation were found inhibited. LWnt3ACM stimulated cell proliferation, clonogenicity, migration and invasion was perturbed by WP modulators, sulindac sulfide and PI3K pathway inhibitor in brain-metastasis specific TNBC cells. Conclusion: Here we present the first conclusive evidence to demonstrate that the WP activation is functionally responsible for MA phenotypes in TNBC. Our data explains the why different components of the WP is upregulated in TNBC and how WP activation is associated with poor prognosis and metastasis in this subtype. Citation Format: Nandini Dey, Jennifer H. Carlson, Pradip De, Brian R. Leyland-Jones. Metastasis-associated tumor cell phenotypes in TNBC is regulated by Wnt-beta-catenin pathway signals. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4605.

BackgroundDouble-strand breaks (DSBs) are primarily repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). Given that DSBs are highly cytotoxic, PARP inhibitors (PARPi), a prominent class of anticancer drugs, are designed to target tumors with HR deficiency (HRD), such as those harboring BRCA mutations. However, many tumor cells acquire resistance to PARPi, often by restoring HR in HRD cells through the inactivation of NHEJ. Therefore, identifying novel regulators of NHEJ could provide valuable insights into the mechanisms underlying PARPi resistance.MethodsCellular DSBs were assessed using neutral comet assays and phospho-H2AX immunoblotting. Fluorescence-based reporter assays quantified repair via NHEJ or HR. The recruitment of proteins that promote NHEJ and HR to DSBs was analyzed using immunostaining, live-cell imaging following laser-induced microirradiation, and FokI-inducible single DSB generation. Loss-of-function experiments were performed in multiple human cancer cell lines using siRNA-mediated knockdown or CRISPR-Cas9 gene knockout. Cell viability assays were conducted to evaluate resistance to PARP inhibitors. Additionally, bioinformatic analyses of public databases were performed to investigate the association between TLK expression and BRCA1 status.ResultsWe demonstrate that human tousled-like kinase (TLK) orthologs are essential for NHEJ-mediated repair of DSBs and for PARPi sensitivity in cells with BRCA1 mutation. TLK1 and TLK2 exhibit redundant roles in promoting NHEJ, and their deficiency results in a significant accumulation of DSBs. TLKs are required for the proper localization of 53BP1, a key factor in promoting the NHEJ pathway. Consequently, TLK deficiency induces PARPi resistance in triple-negative breast cancer (TNBC) and ovarian cancer (OVCA) cell lines with BRCA1 deficiency, as TLK deficiency in BRCA1-depleted cells, impairs 53BP1 recruitment to DSBs and reduces NHEJ efficiency, while restoring HR.ConclusionsWe have identified TLK proteins as novel regulators of NHEJ repair and PARPi sensitivity in BRCA1-depleted cells, suggesting that TLK repression may represent a previously unrecognized mechanism by which BRCA1 mutant cancers acquire PARPi resistance.

Background: Triple-negative breast cancer (TNBC) is defined by lack of estrogen and progesterone receptors and absence of HER2 amplification. Compared with other breast cancer subtypes, TNBC is more aggressive with more frequent metastasis especially involving visceral and central nervous system. Currently, the only available standard adjuvant treatment is chemotherapy. However, mortality rate remains high despite chemotherapy. To improve treatment, it is crucial to develop novel strategies to predict and overcome drug resistance. Our pervious study showed that CD44/CD24 expressions were associated with drug sensitivity and CD44+/CD24+ TNBC was shown to be resistant to docetaxel. In this study, we aimed to investigate the regulatory mechanism involved in CD24-mediated taxane resistance in TNBC. Methods: In this study, we used flow cytometry to determine the CD24 expressions in 7 TNBC cell lines. Western blot was used to demonstrate the levels of CD24 and LC3B-II (a marker for cellular autophagy activity) expressions in TNBC cell lines. CD24 expressions in TNBC cell lines were manipulated by target-specific shRNA and gene overexpression approaches. Cell apoptosis assay was used to determine docetaxel drug sensitivity in cells with manipulated CD24 expression. Using the same assay, autophagy inhibitors were used to further evaluate the involvement of autophagy in CD24-mediated drug resistance. To investigate the possible regulatory mechanism, autophagy related signaling pathway Akt/mTOR was studied in CD24 modified TNBC cells. Results: In this study, the relationship between CD24 expression and autophagy activities in TNBC was evaluated. LC3B-II protein expression was used to determinate autophagy activities. We have shown that TNBC cell lines with high CD24 expressions are associated with increased LC3B-II expression. Suppression of autophagy in CD24 high TNBC cells improves drug sensitivities toward docetaxel. We further demonstrated that knocking down CD24 in TNBC cells suppressed LC3B-II expression and improved docetaxel sensitivity. In addition, gene overexpression of CD24was found to enhance docetaxel resistance in TNBC cells. Our study has also shown that docetaxel increases the activation of Akt/mTOR signaling pathway in drug-resistant TNBC cells. Knocking down CD24 increased Akt/mTOR activation, and decreased the expression of PP2A and LC3B-II in TNBC. Conclusions: CD24 expression is associated with docetaxel sensitivity and autophagy activity in TNBC cells. Drug sensitivity of CD44+CD24+/high TNBC cells is regulated through autophagy related signaling pathway. Taken together, CD24 mediated TNBC drug resistance is at least partially through autophagy activity. Citation Format: Lihong Huo, Xinyu Deng, Xiang Huang, Helena R Chang. Role of autophagy in CD24-mediated TNBC drug resistance [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-16.

Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited effective treatment options. New therapeutic approaches are urgently needed to improve the prognosis of TNBC. Reactive oxygen species (ROS) are inherent byproducts of oxidative metabolism, and forced stimulation of glucose oxidation in cancer cells raises oxidative stress and sensitizes cells to different stresses. Therefore, targeting the antioxidant capacity of cancer cells has become a promising anticancer strategy. As a redox modulator, selenocystine (SeC) has received a great deal of attention and has been shown effective against human melanoma, hormone receptor-positive breast cancer, and cancers of liver, lung and cervical in vitro. However, whether SeC exerts an anticancer effect on TNBC cells has never been explored. Methods: The dose-response effects and time course of effects of SeC on three different TNBC cell lines, MDA-MB-231, MDA-MB-436 and MDA-MB-468, were investigated in this study. Cellular viability was determined by the CCK-8 assay and cell morphology were recorded under a light microscope. Cellular apoptosis was detected using Annexin V/PI staining assay and cell cycle distribution was analyzed by flow cytometry. Results: SeC induced cell growth inhibition in all three TNBC cell lines. For 24, 48 and 72 hours of SeC treatments, the IC50 values were 40.8, 12.8 and 9.2 μM for MDA-MB-231 cells; 14.6, 5.4 and 3.0 μM for MDA-MB-436 cells; and 69.6, 29.3 and 19.9 μM for MDA-MB-468 cells. The changes of cellular morphology of TNBC cells in response to SeC treatment were similar to those cells undergoing apoptotic pathway. This result was confirmed by Annexin V/PI staining assays (Table 1). Cell cycle analysis further revealed that SeC also induced S-phase arrest in a dose-dependent manner (Table 2). Conclusion: In summary, SeC inhibited TNBC cell viability in a dose- and time-dependent manner which was attributed to the induction of apoptosis and S-phase arrest. Our finding indicates that SeC is a potential therapeutic agent for TNBC. Table 1. Apoptotic rate of TNBC cells after SeC treatment.Conc.(μM)MDA-MB-231MDA-MB-436MDA-MB-46805.8 %18.1 %26.7 %1025.5 %45.8 %60.7 %2040.1 %67.1 %70.9 %4054.7 %70.7 %74.5 % Table 2. SeC induces S-phase arrest in TNBC cells in a dose-dependent manner. Conc.(μM)G0/G1 (%)S (%)G2/M (%)MDA-MB-231016.655.927.5 1050.139.310.6 2045.548.16.4 4041.753.05.3MDA-MB-436042.127.630.3 1036.335.028.7 2034.340.725.0 4032.445.422.2MDA-MB-468059.123.717.2 1054.929.315.8 2050.135.714.2 4043.843.113.1 Citation Format: Long M, Qiu W, Wu J, Liu R, Su H. Selenocystine inhibits triple-negative breast cancer cell proliferation by inducing cell apoptosis and S-phase arrest. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-12.

The basal-A subtype of triple-negative breast cancer is characterized by high levels of ΔNp63. Various functions have been proposed for p63 in breast cancer initiation and growth, and p63 mediates chemotherapeutic response in a subset of triple-negative breast cancers. We investigated the signaling pathways that are controlled by ΔNp63 in basal-A triple-negative breast cancer. Human basal-A triple-negative breast cancer cell lines with ΔNp63α induction or inhibition were studied, along with primary human triple-negative breast cancer tissues. Proteomic, phospho-kinase array, mRNA measurements, and immunohistochemistry were employed. Global phosphoproteomics identified increased EGFR phosphorylation in MDA-MB-468 cells expressing ΔNp63α. ΔNp63α expression increased EGFR mRNA, total EGFR protein, and phospho-EGFR(Y1086), whereas silencing endogenous ΔNp63 in HCC1806 cells reduced both total and phospho-EGFR levels and inhibited the ability of EGF to activate EGFR. EGFR pathway gene expression analysis indicated that ΔNp63 alters EGFR-regulated genes involved in cell adhesion, migration, and angiogenesis. Addition of EGF or neutralizing EGFR antibodies demonstrated that EGFR activation is responsible for ΔNp63-mediated loss of cellular adhesion. Finally, immunohistochemical staining showed that p63-positive triple-negative breast cancers were more likely to express high levels of EGFR than p63-negative cancers, corroborated by in silico analysis of gene expression profiling data. These data identify EGFR as a major target for ΔNp63 regulation that influences cancer cell adhesion in basal-like triple-negative breast cancer.