Migration Of Breast Cancer Cells Research Articles

Long non‑coding RNA DANCR aggravates breast cancer through the miR‑34c/E2F1 feedback loop.

Emerging scientific evidence has suggested that the long non‑coding (lnc)RNA differentiation antagonizing non‑protein coding RNA (DANCR) serves a significant role in human tumorigenesis and cancer progression; however, the precise mechanism of its function in breast cancer remains to be fully understood. Therefore, the objective of the present study was to manipulate DANCR expression in MCF7 and MDA‑MB‑231 cells using lentiviral vectors to knock down or overexpress DANCR. This manipulation, alongside the analysis of bioinformatics data, was performed to investigate the potential mechanism underlying the role of DANCR in cancer. The mRNA and/or protein expression levels of DANCR, miR‑34c‑5p and E2F transcription factor 1 (E2F1) were assessed using reverse transcription‑quantitative PCR and western blotting, respectively. The interactions between these molecules were validated using chromatin immunoprecipitation and dual‑luciferase reporter assays. Additionally, fluorescence in situ hybridization was used to confirm the subcellular localization of DANCR. Cell proliferation, migration and invasion were determined using 5‑ethynyl‑2'‑deoxyuridine, wound healing and Transwell assays, respectively. The results of the present study demonstrated that DANCR had a regulatory role as a competing endogenous RNA and upregulated the expression of E2F1 by sequestering miR‑34c‑5p in breast cancer cells. Furthermore, E2F1 promoted DANCR transcription by binding to its promoter in breast cancer cells. Notably, the DANCR/miR‑34c‑5p/E2F1 feedback loop enhanced cell proliferation, migration and invasion in breast cancer cells. Thus, these findings suggested that targeting DANCR may potentially provide a promising future therapeutic strategy for breast cancer treatment.

Identification of PTPN12 Phosphatase as a Novel Negative Regulator of Hippo Pathway Effectors YAP/TAZ in Breast Cancer.

The Hippo pathway plays crucial roles in governing various biological processes during tumorigenesis and metastasis. Within this pathway, upstream signaling stimuli activate a core kinase cascade, involving MST1/2 and LATS1/2, that subsequently phosphorylates and inhibits the transcriptional co-activators YAP and its paralog TAZ. This inhibition modulates the transcriptional regulation of downstream target genes, impacting cell proliferation, migration, and death. Despite the acknowledged significance of protein kinases in the Hippo pathway, the regulatory influence of protein phosphatases remains largely unexplored. In this study, we conducted the first gain-of-functional screen for protein tyrosine phosphatases (PTPs) regulating the Hippo pathway. Utilizing a LATS kinase biosensor (LATS-BS), a YAP/TAZ activity reporter (STBS-Luc), and a comprehensive PTP library, we identified numerous novel PTPs that play regulatory roles in the Hippo pathway. Subsequent experiments validated PTPN12, a master regulator of oncogenic receptor tyrosine kinases (RTKs), as a previously unrecognized negative regulator of the Hippo pathway effectors, oncogenic YAP/TAZ, influencing breast cancer cell proliferation and migration. In summary, our findings offer valuable insights into the roles of PTPs in the Hippo signaling pathway, significantly contributing to our understanding of breast cancer biology and potential therapeutic strategies.

Abstract LB034: First-in-class compounds targeting the MIEN1 signaling pathway: A breakthrough approach for breast and prostate cancer therapy

Abstract We report the identification and complete characterization of first-in-class molecules targeting the MIEN1 signaling pathway in cancer. Migration and invasion enhancer 1 (MIEN1) overexpression characterizes several cancers and facilitates cancer cell migration and invasion. Leveraging conserved ITAM and prenylation motifs within MIEN1, we identified potent anti-cancer peptides. Among them, bioactive peptides LA3IK and RP-7 induced pronounced transcriptomic and protein expression changes at sub-IC50 concentrations. The peptides effectively inhibited genes and proteins driving cancer cell migration, invasion, and EMT pathways, concurrently suppressing EGF-induced NF-kB nuclear translocation in metastatic breast cancer cells. Specifically, peptides targeted the same signal transduction pathway initiated by MIEN1. Molecular docking and circular dichroism spectroscopy indicated the formation of MIEN1-peptide complexes. The third-positioned isoleucine in LA3IK and CVIL motif in RP-7 were crucial for inhibiting breast cancer cell migration. This is evident from the limited migration inhibition observed when MDA-MB-231 cells were treated with scrambled peptides LA3IK-SCR and RP-7-SCR. Additionally, LA3IK and RP-7 effectively suppressed tumor growth in an orthotopic breast cancer model. Notably, mice tolerated high peptide doses of up to 90 mg/Kg well, surpassing significantly lower doses of 5 mg/Kg intravenously (iv) and 30 mg/Kg intraperitoneally (ip) used in both in vivo pharmacokinetic studies and orthotopic mouse model assays. D-isomers of LA3IK and RP-7 showed enhanced anti-cancer activity compared to their L-isomers. D-LA3IK remained stable in mouse plasma for 24 h with 75% remaining, exhibiting superior pharmacokinetic properties over D/L-RP-7. In summary, our findings mark the first report of short peptides based on MIEN1 protein sequence capable of inhibiting cancer signaling pathways, effectively impeding cancer progression both in vitro and in vivo. Citation Format: Amit Kumar Tripathi, Jamboor K. Vishwanatha. First-in-class compounds targeting the MIEN1 signaling pathway: A breakthrough approach for breast and prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB034.

Activation of the YY1-UGT2B7 Axis Promotes Mammary Estrogen Homeostasis Dysregulation and Exacerbates Breast Tumor Metastasis.

Metastasis is the most common pathway of cancer death. The lack of effective predictors of breast cancer metastasis is a pressing issue in clinical practice. Therefore, exploring the mechanism of breast cancer metastasis to uncover reliable predictors is very important for the clinical treatment of breast cancer patients. In this study, tandem mass tag quantitative proteomics technology was used to detect protein content in primary breast tumor tissue samples from patients with metastatic and nonmetastatic breast cancer at diagnosis. We found that the high expression of yin-yang 1(YY1) is strongly associated with poor prognosis in high-grade breast cancer. YY1 expression was detected in both clinical tumor tissue samples and tumor tissue samples from mammary-specific polyomavirus middle T antigen overexpression mouse model mice. We demonstrated that upregulation of YY1 expression was closely associated with breast cancer metastasis and that high YY1 expression could promote the migratory invasive ability of breast cancer cells. Mechanistically, YY1 directly binds to the UGT2B7 mRNA initiation sequence ATTCAT, thereby transcriptionally regulating the inhibition of UGT2B7 expression. UGT2B7 can regulate the development of breast cancer by regulating estrogen homeostasis in the breast, and the abnormal accumulation of estrogen, especially 4-OHE2, promotes the migration and invasion of breast cancer cells, ultimately causing the development of breast cancer metastasis. In conclusion, YY1 can regulate the UGT2B7-estrogen metabolic axis and induce disturbances in estrogen metabolism in breast tumors, ultimately leading to breast cancer metastasis. Disturbances in estrogen metabolism in the breast tissue may be an important risk factor for breast tumor progression and metastasis SIGNIFICANCE STATEMENT: In this study, we propose for the first time a regulatory relationship between YY1 and the UGT2B7/estrogen metabolism axis and explore the molecular mechanism. Our study shows that the YY1/UGT2B7/estrogen axis plays an important role in the development and metastasis of breast cancer. This study further elucidates the potential mechanisms of YY1-mediated breast cancer metastasis and the possibility and promise of YY1 as a predictor of cancer metastasis.

Oleate Promotes Triple-Negative Breast Cancer Cell Migration by Enhancing Filopodia Formation through a PLD/Cdc42-Dependent Pathway.

Breast cancer, particularly triple-negative breast cancer (TNBC), poses a global health challenge. Emerging evidence has established a positive association between elevated levels of stearoyl-CoA desaturase 1 (SCD1) and its product oleate (OA) with cancer development and metastasis. SCD1/OA leads to alterations in migration speed, direction, and cell morphology in TNBC cells, yet the underlying molecular mechanisms remain elusive. To address this gap, we aim to investigate the impact of OA on remodeling the actin structure in TNBC cell lines, and the underlying signaling. Using TNBC cell lines and bioinformatics tools, we show that OA stimulation induces rapid cell membrane ruffling and enhances filopodia formation. OA treatment triggers the subcellular translocation of Arp2/3 complex and Cdc42. Inhibiting Cdc42, not the Arp2/3 complex, effectively abolishes OA-induced filopodia formation and cell migration. Additionally, our findings suggest that phospholipase D is involved in Cdc42-dependent filopodia formation and cell migration. Lastly, the elevated expression of Cdc42 in breast tumor tissues is associated with a lower survival rate in TNBC patients. Our study outlines a new signaling pathway in the OA-induced migration of TNBC cells, via the promotion of Cdc42-dependent filopodia formation, providing a novel insight for therapeutic strategies in TNBC treatment.

Restoration of miR-451a-5p/miR-34a-5p could suppress the proliferation and migration of human breast cancer cells through Wnt/β- catenin and ERK/P-ERK signaling pathways.

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a length of 21-25 nucleotides and play an essential role in the regulation of cancer initiation, development and progression. Breast cancer (BC) is the most commonly detected malignancy in women and one of the leading causes of death worldwide. In this study, the effects of transfection of microRNA-451a-5p and miR-34a-5p (tumor suppressors), individually and in combination on apoptosis, proliferation and migration of breast cancer cells in vitro were investigated. For this study, malignant breast cancer cells (MDA-MB-231) were transfected with the miR-451a-5p and miR-34a-5p mimics. Subsequently cytotoxicity, apoptosis, proliferation, migration protein and gene expression of caspase-3, caspase-8, MMP9, ROCK, vimentin and c-Myc of the cancer cells were analyzed by MTT, flow cytometry, q-RT-PCR (expression level of caspase-3, caspase-8, MMP9, ROCK, vimentin and c-Myc genes), wound healing and Western blot assays. The results showed that miR-34a-5p and miR-451a-5p could additionally induce apoptosis and cell cycle arrest in the sub-G1phase, suppress proliferation and migration in breast cancer cells, and also decrease the expression of β- catenin and ERK/P-ERK proteins . The present data document that restoration of the tumor suppressor miR-451/miR-34 strongly induces programmed cell death in vitro and apparently inhibits cell proliferation and migration in human breast cancer cells. In summary, miR-451a and miR-34a play an important role in breast cancer cell proliferation and migration via the Wnt/β-catenin and ERK/P-ERK signaling pathways. Therefore, the simultaneous restoration of the presented tumor suppressor miRNAs can be proposed as a valuable and potential therapeutic strategy in the treatment of breast cancer. However, further studies should be useful.

A novel long non-coding RNA LINC00524 facilitates invasion and metastasis through interaction with TDP43 in breast cancer.

Breast cancer (BC) remains a significant health concern worldwide, with metastasis being a primary contributor to patient mortality. While advances in understanding the disease's progression continue, the underlying mechanisms, particularly the roles of long non-coding RNAs (lncRNAs), are not fully deciphered. In this study, we examined the influence of the lncRNA LINC00524 on BC invasion and metastasis. Through meticulous analyses of TCGA and GEO data sets, we observed a conspicuous elevation of LINC00524 expression in BC tissues. This increased expression correlated strongly with a poorer prognosis for BC patients. A detailed Gene Ontology analysis suggested that LINC00524 likely exerts its effects through RNA-binding proteins (RBPs) mechanisms. Experimentally, LINC00524 was demonstrated to amplify BC cell migration, invasion and proliferation invitro. Additionally, invivo tests showed its potent role in promoting BC cell growth and metastasis. A pivotal discovery was LINC00524's interaction with TDP43, which leads to the stabilization of TDP43 protein expression, an element associated with unfavourable BC outcomes. In essence, our comprehensive study illuminates how LINC00524 accelerates BC invasion and metastasis by binding to TDP43, presenting potential avenues for therapeutic interventions.

Ginsenoside Re Suppress the Proliferation and Migration of MCF-7 Human Breast Cancer Cells

Ginsenoside Re Suppress the Proliferation and Migration of MCF-7 Human Breast Cancer Cells

Ezetimibe inhibits the migration and invasion of triple-negative breast cancer cells by targeting TGFβ2 andEMT.

The important role of cholesterol in tumor metastasis has been widely studied in recent years. Ezetimibe is currently the only selective cholesterol uptake inhibitor on the market. Here, we explored the effect of ezetimibe on breast cancer metastasis by studying its impact on breast cancer cell migration, invasion, and epithelial-mesenchymal transition (EMT). Differential gene expression analysis and validation were also carried out to compare ezetimibe-treated and untreated breast cancer cells. Finally, breast cancer cells overexpressing TGFβ2 were constructed, and the effect of TGFβ2 on the migration and invasion of ezetimibe-treated breast cancer cells was examined. Our results show that ezetimibe treatment of breast cancer cells inhibited cell migration, invasion, and EMT, and it significantly suppressed the expression of TGFβ2. Overexpression of TGFβ2 reversed the inhibitory effect of ezetimibe on the migration and invasion of breast cancer cells. Taken together, our results suggest that ezetimibe might be a potential candidate for the treatment of breast cancer metastasis.

Abstract 1568: Her2 promotes early dissemination of breast cancer by suppressing the p38 pathway through upregulation of Wip1

Abstract (a)Metastasis is the leading cause of cancer-related deaths. Increasing evidence shows that cancer cells can disseminate from early primary lesions that are impalpable, but the pathways are poorly understood. Her2 promotes breast cancer early dissemination by suppressing p38, but how Her2 downregulates p38 is unclear. In this study, we investigated the mechanism underlying suppression of p38 by Her2 in breast cancer early dissemination using cell models and xenograft models of early lesions of breast cancer. (b)We studied the role of Wip1, a p38 phosphatase, in disseminating phenotypes in cell line models of breast cancer early lesions, including cell motility measured in transwell assays, cell invasion measured by percentage of organoids with outward invading cells, and epithelial-to-mesenchymal transition (EMT) measured by levels of EMT markers in Western blotting analysis and immunofluorescence (IF) staining assays. (c)We demonstrate that high levels of Wip1 correlated with Her2+ status, and reduced phosphorylation of p38 and its downstream effectors MK2 and Hsp27 in tissue samples from early lesions of human breast cancer. In addition, ectopic expression of Wip1 reduced phosphorylation of p38, MK2 and Hsp27 in cell models of breast cancer early lesions.We found that Wip1 promoted migration, invasion and EMT, while Wip1 knockdown abrogated the ability of Her2 to induce these disseminating phenotypes, in cell line models of early lesion breast cancer. Thus, Wip1 induction is both sufficient, and necessary for Her2, to promote migration, invasion and EMT in early lesion breast cancer cells. In an attempt to identify the Wip1 downstream effectors involved in disseminating phenotypes, we found that ectopic expression of the constitutively active mutants of p38 reversed the ability of Wip1 to suppress MK2 and Hsp27 phosphorylation and to induce migration, invasion and EMT in early lesion breast cancer cells. These results suggest that Wip1 promotes breast cancer early dissemination by suppressing p38. We investigate the mechanism by which Her2 induces Wip1 expression. Ectopic expression of Her2 did not alter the level of Wip1 mRNA or the Wip1 protein stability, suggesting that Her2 promote Wip1 expression by regulating its translation. We are currently investigating the mechanism by which Her2 induces Wip1 translation.Moreover, inhibitors of Her2, Wip1 and Skp2 can each reduce the disseminating phenotypes in cells models of breast cancer early lesions ectopically expressing Her2, Wip1 or Skp2. (d)Our findings identify the Her2-Wip1-p38-Mk2-Hsp27 cascade as a novel mechanism mediating breast cancer early dissemination and provide a basis for new therapies targeting early metastatic dissemination in Her2+ breast cancer using inhibitors for Her2, Wip1 or Skp2. Citation Format: Nan Wang. Her2 promotes early dissemination of breast cancer by suppressing the p38 pathway through upregulation of Wip1 [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 1568.

Abstract 203: Promotion of metastasis of ERBB2-positive breast cancer by mesenchymal stem cells via exosomes-mediated activation of ERBB2/ERBB3→PI3K/Akt singaling pathway

Abstract Elevated expression of ERBB3 receptor correlates with increased distant metastasis of breast cancers with amplification and/or overexpression of ERBB2 (HER2/neu), which the underlying molecular mechanism remains unclear. In recent years, cumulative studies have demonstrated that mesenchymal stem cells (MSCs), one of the important cellular components in tumor microenvironment, play important roles in multiple hallmarks of cancer including invasion and metastasis. However, its role in metastasis of ERBB2-positive breast cancer as well as the precise mechanisms are largely unrevealed. In our current study, we found that co-culture with MSCs or treatment with exosomes derived from MSCs can significantly promote the epithelial-mesenchymal transition (EMT), migration and invasion of ERBB2-positive breast cancer cells. MSCs exhibited significantly higher expression of NRG-1, a known ligand of ERBB3, as compared with ERBB2-positive breast cancer cells. The presence of NRG-1 in exosomes derived from MSCs was also evidenced. Mechanistically, co-culture with MSCs or treatment with exosomes derived from MSCs induced upregulation of ZEB1 and Slug via activation of downstream PI3K/Akt singaling to promote EMT of ERBB2-positive breast cancer cells. In addition, the expression of MMP2 was also upregulated by treatment with MSCs-derived exosomes, which enhanced the migration and invasion of ERBB2-positive breast cancer cells simultaneously. Interestingly, our study further demonstrated that treatment with MSCs-derived exosomes could significantly downregulate the expression of PTEN, the key negative regulator of PI3K/Akt singaling, via transferring three PTEN-targeting miRNAs (miR-21-5p, miR-23a-3p, and miR-148a-3p). Taken together, we presented here that MSCs may, on the one hand, directly activate the ERBB2/ERBB3→PI3K/Akt signaling pathway in ERBB2-positive breast cancer cells through the expression and secretion of NRG-1 via exosomes. Meanwhile, miR-21-5p, miR-23a-3p, and miR-148a-3p targeted delivery by exosomes specifically downregulated the expression of PETN, thus reliefs the negative regulation of PI3K/Akt signaling in ERBB2-positive breast cancer cells. These effects of MSCs act in concert to promote the metastasis of ERBB2-positive breast cancer. Our study lays a theoretical foundation and preliminary experimental basis for the development of novel combined therapies for patient with ERBB2-positive breast cancer through co-targeting MSC-derived NRG-1 and miR-21-5p, miR23a-3p, miR148a-3p/PTEN axis in tumor microenvironment in the future. Citation Format: Yating Wu, Li Chen, Yue Cao, Qinfeng Huang, Huimin Niu, Xiaofeng Lai, Meng Zhao, Huihui Yan, Hui Lyu, Bolin Liu, Shenghang Zhang, Shuiliang Wang. Promotion of metastasis of ERBB2-positive breast cancer by mesenchymal stem cells via exosomes-mediated activation of ERBB2/ERBB3→PI3K/Akt singaling pathway [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 203.

Abstract 1510: Cell-cell contacts regulate HER2 cellular localization and the transitions between migration and proliferation in breast cancer cells

Abstract Introduction: Epithelial-to-mesenchymal transition (EMT) is widely recognized as the primary mechanism regulating invasion and metastatic dissemination in carcinomas. In contrast, the mechanisms triggering single disseminated cancer cells (DCC) to reacquire their epithelial identity at ectopic sites, initiating metastasis formation, remain unclear. Since we had previously noted that HER2 expression levels and cellular density impact on migration and proliferation, we sought to determine whether they are involved in molecular mechanisms regulating transitions between proliferation and migration. Methods: To control HER2 expression levels we generated inducible cell models. Cell density was assessed by the number of cell-cell contacts or by the number of cells per area. Pathway analyses were performed by gene expression analysis, protein interactions by western blotting and mass spectrometry. Migration and proliferation were analyzed by functional assays including in vitro live cell imaging. Results: Migrating epithelial cells undergo reduced protein trafficking, resulting in a perinuclear deposition of HER2. This was accompanied by increased calcium ion regulation via phospholipase-C in the endoplasmic reticulum (ER). Upon the establishment of cell-cell contacts, particularly between cells expressing desmosome cadherin proteins, HER2 is redirected to the cell surface through direct physical binding with desmoplakin, facilitating proliferation signals. Then, PIP2 to PIP3 is converted through the activation of the PI3K pathway, concomitant with the upregulation of PDPK1. The PH domain of PDPK1 exhibits a high binding affinity to PIP2 molecules. Consequently, membrane localization of HER2 significantly diminishes PIP2 availability for the PLC pathway, redirecting cells towards proliferation. Patient sample analysis confirmed the loss of membrane-bound HER2 in solitary circulating tumor cells (CTCs), which was paralleled by increased plakoglobin, elevated ZEB1, and reduced KI67 protein levels. Conclusion: Cell-cell contacts determine the cellular localization and function of growth factor receptors like HER2. Single disseminating cancer cells therefore need to establish such contacts at metastatic sites to activate proliferation programs. Our data are in line with findings that circulating tumor cell clusters are associated with higher metastatic rates, the impact of specific metastatic niches for outgrowth and have implementation for the targeting of single DCC. Citation Format: Hedayatollah Hosseini, Saba Sameri, Shabnam Shahrivari, Carolina Fisch, Miriam Kokal, Michael Schletter, Paul Sylvester Hahn, Lisa Seider, Marisa Schübel, Durdam Das, Martin Hoffmann, Christian Werno, Kathrin Weidele, Vladan Milosevic, Arne Östman, Shamim Sarhadi, Joachim Wegener, Stefanie Michaelis, Florian Weber, Astrid Bruckmann, Ernst R. Tamm, Yuting Li, Christoph A. Klein. Cell-cell contacts regulate HER2 cellular localization and the transitions between migration and proliferation in breast cancer cells [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 1510.

Abstract 5417: PHGDH expression level impacts tumorigenesis and metastasis in human breast cancer

Abstract Background: Phosphoglycerate dehydrogenase (PHGDH) is a metabolic enzyme, which is associated with the first step of serine synthesis. The gene encoding this enzyme is often elevated in some types of human cancer, including breast cancer. There are some researches that High-PHGDH expression promotes cancer cell proliferation and tumorigenesis. However, latest study showed that although breast tumors with high expression levels of PHGDH had more possibility to undergo primary-tumor growth, were less likely to metastasize than tumors with low expression of PHGDH by using mouse cell derived mouse models and PDX models. Nevertheless, the applicability of these outcomes to human breast cancer cell lines remains uncertain. Methods: The survival analysis according to PHGDH mRNA expression was analyzed in METABRIC datasets of breast cancer and the whole-transcriptome sequencing data of 120 breast cancer patients. To investigate the effect of PHGDH in human TNBC cell lines, PHGDH overexpressing MDA-MB-231 cell lines and PHGDH knockdown MDA-MB-468 cell lines were established. Subsequent functional experiments were performed to explore the influence of PHGDH on breast cancer tumorigenesis and metastasis. Results: In the analysis using METABRIC datasets, overall survival and disease-free survival were reduced in patients with high PHGDH expression and basal type breast cancer showed the highest mRNA level of PHGDH. Using whole-transcriptome sequencing data of 120 breast cancer patients, we also found PHGDH expression level was higher in cancer tissue(n=120) than normal tissue(n=90). In particular, triple-negative breast cancer indicated higher PHGDH level than luminal type breast cancer. We also used this data to perform analysis of overall survival, disease-free survival, metastasis free survival of non-TNBC patient and it resulted in reducing survival rate when patient showed high-PHGDH expression. However, TNBC patients demonstrated diametrical results in the identical analysis. We examined the impact of PHGDH overexpression in MDA-MB-231 cells and PHGDH knockdown in MDA-MB-468 cells, observing increased proliferation and decreased migration in breast cancer cells with elevated PHGDH levels. Conversely, PHGDH knockdown exhibited opposing effects. Subsequently analysis of markers associated with epithelial-mesenchymal transition revealed a correlation with PHGDH expression. Conclusions: Our data suggest that a potential role for PHGDH in breast cancer as a marker of reduced survival in triple-negative breast cancer, and as a regulator of cancer cell proliferation and metastasis. Citation Format: Jihong Baik, Juhee Kim, Sangeun Lee, Ayoung Park, Kyoungseob Shin, Sunghoon Kwon, Hanbyoel Lee, Wonshik Han. PHGDH expression level impacts tumorigenesis and metastasis in human 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 5417.

Abstract 200: OA inhibits the development of breast cancer through K1108 acetylation of Zeb1

Abstract Background:Olive oil has long been considered a healthier edible oil, rich in 75% oleic acid (OA) as its main component. OA is a monounsaturated fatty acid, and recent studies have shown that it is no longer limited to protecting cardiovascular and cerebrovascular systems, but can also reduce the risk of cancer. However, there is limited research on the specific mechanism. After fatty acid is ingested into the body, through fatty acids β-oxidation produces acetyl-CoA, which is a donor of protein acetylation. EMT is an important process to regulate tumor metastasis, and Zeb1 is one of the important EMT transcription factors. Therefore, we explored whether OA could inhibit the progress of breast cancer by affecting the acetylation modification of Zeb1. Methods: Western blotting, CCK8 assay, and Transwell were used to test the impact of gradient doses of OA on breast cancer cells. Immunoprecipitation and Immunoprecipitation mass spectrum were used to determine the acetylation modification sites of Zeb1. Coimmunoprecipitation, IF, and pull-down were used to test and verify the acetylase and deacetylase of Zeb1. Knockdown endogenous ZEB1 and then reconstructed stable cell lines of Zeb1 wild-type (Zeb1K1108WT), acetylation activated (Zeb1K1108Q), and inactivated mutants (Zeb1K1108R) by lentivirus. Detection of Zeb1 acetylation in patient tissue samples and mouse models under OA treatment using Zeb1-specific acetylation-modified antibodies. Results: OA inhibits the proliferation and migration of breast cancer cells in a concentration gradient-dependent manner, and inhibits the expression of Zeb1, N Cadherin, and upregulates E Cadherin. Among numerous fatty acids, only OA can promote the acetylation of Zeb1 by adjusting the ratio of acetyl-CoA and NAD+/NADH. K1108 is a biologically functional acetylation site of Zeb1, Zeb1K1108R exerts the cancer promoting function of Zeb1, while Zeb1K1108Q does not. OA can promote acetylation of Zeb1K1108WT, but cannot promote acetylation of Zeb1K1108R. Conclusions: OA inhibits the development of breast cancer through K1108 acetylation of Zeb1. These results suggest that breast cancer may be ameliorated through dietary interventions. Citation Format: Min Guo, Yanjin Wang, Yi Shi, Shuang Yang. OA inhibits the development of breast cancer through K1108 acetylation of Zeb1 [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 200.

Abstract 7628: Pharmacodynamic biomarkers for Rac and Cdc42 inhibitor efficacy

Abstract Metastatic disease drastically reduces clinical prognosis, and remains the primary cause of mortality from solid cancers, such as breast and pancreatic cancer. Since targeted therapeutic options are limited, we developed small molecule compounds to block activation (GTP loading) of the Rho GTPases Rac and Cdc42. Rac and Cdc42 are ideal molecular targets due to their regulation of cell migration, proliferation, invasion, and signaling in cancer and immunosuppressive cells in the tumor microenvironment. Our lead compound MBQ-167 is a dual Rac and Cdc42 inhibitor with IC50 of ~100 nM for inhibition of Rac/Cdc42 activation and the phosphorylation of their common downstream effector p21-activated kinase (PAK). MBQ-167 is highly effective in mouse models of breast cancer metastasis and is currently undergoing a Phase I clinical trial in advanced breast cancer patients. The purpose of this study is to investigate potential target molecules that can be used as biomarkers predictive of Rac/Cdc42 inhibitor efficacy. We recently published that in addition to inhibiting the growth and migration of breast and pancreatic cancer cells, MBQ-167 inhibits macrophage cell migration, immunosuppressive myeloid cell activation and release of interleukin-6 (IL-6), a pro-inflammatory cytokine associated with cancer. Transcriptomic data of tumors from mice treated with vehicle or MBQ-167 identified a number of immunomodulatory pathways downregulated by MBQ-167, including IL-6 and S100A family signaling, and Chitinase 3-like (CHI3L1/CHI4L1) genes coding for the human YKL-40 protein. We tested for the activation status of PAK (phospho (p)-PAK) as a diagnostic marker for Rac/Cdc42 inhibitor efficacy using breast cancer patient tissue in ex vivo culture treated with vehicle or MBQ-167 and found decreased p-PAK staining following immunohistochemistry of MBQ-167-treated tissue. From flow cytometry, using Alexa-488-tagged anti p-PAK antibody, we found that p-PAK was reduced from peripheral blood mononuclear cells (PBMCs) following MBQ-167. ELISA assays for YKL-40/CHI3L1 proteins after treatment of pancreatic cancer cell lines and macrophage-like cells with Rac/Cdc42 inhibitors show that MBQ-167 and the derivative MBQ-168 reduced CHI3L1 levels from conditioned media. These results demonstrate the potential of p-PAK and YKL-40/CHI3L1 from blood as biomarkers for Rac/Cdc42 inhibitor efficacy. This study impacts pharmacodynamic biomarker design for the planned Phase 2 clinical trials for MBQ-167 in breast and pancreatic cancer. Citation Format: Anamaris Torres-Sanchez, Ailed M. Cruz-Collazo, Michael Rivera-Robles, Olga Katsara, Stephanie Dorta-Estremera, Viviana Negron, Victor P. Carlo, Robert J. Schneider, Suranganie Dharmawardhane. Pharmacodynamic biomarkers for Rac and Cdc42 inhibitor efficacy [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 7628.

Abstract 7653: Role of ITGB2 in patients with luminal B1 breast cancer

Abstract Purpose: Luminal-type breast cancers are the most commonly diagnosed in women. Among them, the luminal B1 subtype exhibits higher proliferation rates and a worse prognosis compared to the luminal A subtype. Despite significant advancements in detection and therapies, recurrence still occurs after primary treatment. In this study, our aim is to identify genomic alterations in early recurrent luminal B1 breast cancer. Experimental Design: In this VGHTPE cohort, we recruited a total of 42 patients diagnosed with stage I-III luminal B1 breast cancer, comprising 15 subjects who experienced recurrence within five years and 27 subjects who did not. Genomic DNA was extracted from formalin-fixed paraffin-embedded samples and subjected to targeted next-generation sequencing. We assessed cell aggressiveness through in vitro transwell migration, invasion, and sphere assays. Results: Luminal B1 breast cancer patients with advanced stage and poorly differentiated tumors were associated with recurrence within five years. Driver gene mutation analysis revealed that TP53 mutations were present in 46.7% of patients who experienced recurrence within five years, compared to 29.6% in the non-recurrence group. In the analysis of copy number variations, the most common amplifications/gains observed in luminal B1 breast cancer patients were in MCL1, MYC, and KLF6. Remarkably, patients who experienced recurrence within five years showed a higher occurrence of gain in ITGB2, the gene encoding the integrin β chain, compared to those who remained recurrence-free. Patients with ITGB2 amplifications/gains had a shorter 5-year recurrence-free survival compared to those with a neutral status. In vitro studies revealed that the knockdown of ITGB2 suppressed cell migration, invasion, and sphere formation in estrogen receptor-positive HER2-negative breast cancer cells. Conclusion: Our study has identified ITGB2 amplifications/gains as a potential biomarker for early recurrence in luminal B1 breast cancer. Citation Format: Chun-Yu Liu, Ji-Lin Chen, Yi-Fang Tsai, Ta-Chung Chao, Wan-Lun Wang, Pei-Ju Lien, Chih-Yi Hsu, Jiun-I Lai, Chi-Cheng Huang, Jen-Hwey Chiu, Ling-Ming Tseng. Role of ITGB2 in patients with luminal B1 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 7653.

Abstract 5163: Determining the role of septin expression in response of breast cancer patients to microtubule targeted chemotherapy

Abstract Drugs that disrupt the microtubule cytoskeleton, including both microtubule stabilizing taxanes and destabilizers like eribulin, are regularly used in the treatment of metastatic breast cancer. However, there are currently no molecular biomarkers to guide their use. Septins, a class of small GTPases, interact with both microtubules and actin to functionally link these cytoskeletal components and regulate cell biological processes shown to be critical for the invasion and migration of cancer cells. In particular, septin 9 (SEPT9) directly interacts directly with microtubules through an N-terminal domain on the longest isoform, SEPT9i1, which is sufficient to promote migration and invasion of breast cancer cells in vitro. We hypothesized that the relative expression of septin9 isoforms in breast tumors could play a role in their response to treatment with microtubule-targeted chemotherapy, particularly in the metastatic setting. A retrospective investigation of the relationship between septin expression and response to taxane chemotherapy was conducted utilizing full transcriptome sequencing data from biopsies of 75 breast cancer patients treated at our NCI-Designated Mays Cancer Center who received molecular profiling through Caris Life Sciences. Levels of total SEPT9 expression as well as the expression of specific variants that encode mechanistically distinct septin9 isoforms, SEPT9v1, v2, and v3, were determined for each sample and correlated with demographic, treatment, and outcome data for these patients. The oncogenic sept9_v1 variant (which produces the septin9_i1 isoform) was detected in over half of tumors and those with the highest total septin9 expression also expressed significantly higher levels of this isoform. Strikingly, the expression of total septin9, as well as the v3 variant, was significantly increased in taxane-treated patients who survived over a year after diagnosis with metastatic breast cancer, suggesting that septin9 expression could be a molecular correlate of response to taxane chemotherapy. We found that the expression of total septin9 and its variants was not significantly correlated with race, ethnicity, or cancer type suggesting that our findings would apply to our patient population as a whole, which is over 50% Hispanic. These data were used to support an ongoing prospective study to determine effect of septin expression on response to taxane chemotherapy in the neoadjuvant setting and determine the effect of taxane treatment on septin expression by comparing samples pre and post treatment. Together this work supports our overall goal of identifying predictive biomarkers to facilitate the use of microtubule targeted chemotherapy in a more personalized manner. Citation Format: Tamarah Aldawoodi, Jacob Nathaniel Essif, Amna Naqvi, Lauren D. Boyle, Daniela Urueta Portillo, Kate Lathrop, April L. Risinger. Determining the role of septin expression in response of breast cancer patients to microtubule targeted chemotherapy [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 5163.

Abstract 7220: Emerging combination strategy: FANCI suppression induces PARP1 redistribution to enhance efficacy of PARP inhibitors in breast cancer

Abstract While polyADP-ribose polymerase (PARP) inhibitors have made advancements in the treatment of breast cancer, challenges such as chemotherapy resistance and limited application persist. Fanconi AnemiaComplementation Group I (FANCI), a DNA repair protein associated with breast cancer development, represents a potential target for novel combination therapeutic strategies. However, the role of FANCI in breast cancer and its impact on the efficacy of PARP inhibitors require further investigation. In our study, we analyzed FANCI expression in breast cancer tissues and cell lines, and its correlation with clinical parameters and patient prognosis. Lentiviral vectors were utilized and functional assays were performed to evaluate the effects of FANCI modulation on breast cancer cell growth and migration. Co-immunoprecipitation assays and protein interaction analysis were conducted to identify the interaction between FANCI and PARP1 and determine the specific binding region. The functionality and nuclear distribution of PARP1 were assessed upon FANCI modulation. Finally, the sensitivity of breast cancer cells to the PARP inhibitor talazoparib upon FANCI knockdown was evaluated in vitro and in vivo. Our findings demonstrated that FANCI was overexpressed in breast cancer and associated with poor prognosis. FANCI significantly promoted breast cancer cell proliferation both in vitro and in vivo. We identified the interaction between FANCI and PARP1, specifically at the FANCI helical domain 2 binding site. FANCI knockdown led to reduced nuclear localization of PARP1 and decreased PARP1 activity. Importantly, combination treatment with FANCI knockdown and talazoparib significantly inhibited cancer growth in vitro and in vivo. Additionally, we found that the cyclin-dependent kinase (CDK) 4/6 inhibitor palbociclib, which effectively suppresses FANCI protein expression, exhibited a robust synergistic effect with talazoparib both in vitro and in vivo. In conclusion, FANCI is a novel therapeutic target for breast cancer. Suppression of FANCI regulates PARP1 redistribution and activity, making breast cancer cells more responsive to PARP inhibitors. This combination therapeutic strategy shows potential in enhancing the effectiveness of PARP inhibitors for breast cancer treatment, regardless of BRCA mutations. Citation Format: Yu-zhou Huang, Ming-Yi Sang, Pei-Wen Xi, Ruo-Xi Xu, Meng-Yuan Cai, Zi-Wen Wang, Jian-Yi Zhao, Yi-Han Li, Ji-Fu Wei, Qiang Ding. Emerging combination strategy: FANCI suppression induces PARP1 redistribution to enhance efficacy of PARP inhibitors in 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 7220.

Abstract 294: The HOXB7 protein promotes breast cancer cell growth through activation of the CCL5/CCR5 pathway in the crosstalk of adipocyte

Abstract Homeobox genes are regulatory genes that encode nuclear proteins acting as transcription factors during normal development and differentiation. One such gene, HOXB7, plays a role in various developmental processes, including hematopoietic differentiation, lymphoid development, and mammary gland development. However, the role of HOX genes in breast cancer development remains largely unexplored. Our previous studies revealed that HOXB7 expression was significantly elevated in primary cancer and distant metastasis. HOXB7 overexpression promoted cell proliferation in SKBR3 breast cancer cells, leading to robustly vascularized xenografts in immunodeficient mice. Furthermore, HOXB7 enhanced tumor growth through TGF beta signaling and recruitment of macrophages, indicating the role of HOXB7 in the crosstalk of stromal components. We found evidence that HOXB7 overexpression in ER+ breast cancer cells promotes tumor cell growth through increased expression and signaling of CCL5/CCR5 in crosstalk with adipocytes. By screening secreted factors in adipocytes induced by TCM of MCF-7-HOXB7 cells, it was also found that CCL5 is highly expressed in HOXB7-overexpressing MCF-7 cells. The CCR5 inhibitor, maraviroc, sensitized MCF-7-HOXB7 cells compared to control cells. These findings suggest that blocking the interaction between CCL5 and CCR5 signaling significantly inhibits ER+ breast cancer with HOXB7 overexpression. We aim to further investigate if HOXB7 overexpression in ER+ breast cancer cells promotes tumor cell growth through increased expression and signaling of CCL5/CCR5 in crosstalk with adipocytes and the role of CCL5 in breast cancer cell growth and migration. Citation Format: Ahone Gina Akume, Kideok Jin. The HOXB7 protein promotes breast cancer cell growth through activation of the CCL5/CCR5 pathway in the crosstalk of adipocyte [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 294.

Abstract 3332: Kalkitoxin thiol amide suppresses the breast cancer metastasis to bone and brain

Abstract Introduction: Breast cancer is the primary frequently diagnosed cancer worldwide, with a high fatality rate. Metastasis resulting from advanced breast cancer increases mortality in patients. MDA-MB-231 is triple-negative breast cancer that does not have any of the receptors that are commonly found in breast cancer. The lipopeptide toxin kalkitoxin thiol amide (KTA), generated from the marine cyanobacterium Moorena producens (formerly Lyngbya majuscule), has a wide range of biological and pharmacological effects. Its promise as a therapeutic agent for treating bone metastases from breast cancer is still uncertain. This study aimed to determine whether KTA could prevent breast cancer from metastasis to mice's bones and to elucidate any potential mechanisms causing this inhibition. Experimental Procedures: MDA-MB-231 cells were transfected with pGL4.50 (Luc2/CMV/Hygro) vector using LipofectamineTM 2000. Positive clones of MDA-MB-231 cells were selected after treatment with 300 µg/ml hygromycin for one week. After cancer implantation to the heart, bioluminescent imaging of femurs was measured upto 5 weeks and signal intensity of KTA-treated groups was compared with the vehicle group. Summary: KTA decreased breast cancer cell proliferation, migration, and invasion. KTA prevented MDA-MB-231 cells from breast cancer metastasizing to the bone and brain via inhibiting MAPK and STAT signaling. The epithelial-mesenchymal transition markers, which are responsible for distant metastasis, are highly opposed by KTA. Compared to mice in the control group, mice receiving KTA displayed considerably less bone and brain metastasis, a lower tumor burden, and less bone destruction in the murine breast cancer bone metastasis model. Conclusion: KTA has new inhibitory effects on cancer cell invasion and migration both in vitro and in vivo. From these findings, KTA may have therapeutic promise for treating breast cancer patients with distant metastases. Citation Format: Yunjo Soh, Saroj Kumar Shrestha, Se woong Kim. Kalkitoxin thiol amide suppresses the breast cancer metastasis to bone and brain [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 3332.