Target For Breast Cancer Metastasis Research Articles

Abstract 4123: Attenuating breast cancer metastasis with BCL-XL-targeting PROTACs

Abstract B-cell lymphoma X-large (BCL-XL) emerges as a significant player in cancer progression. In our recent findings, we unveil the efficacy of proteolysis-targeting chimeras (PROTACs) directed against BCL-XL, demonstrating their capacity to not only eliminate cancer cells but also target tumor-induced regulatory T cells (TI-Tregs). Notably, BCL-XL's role in promoting breast cancer metastasis extends beyond its anti-apoptotic function. We posit that BCL-XL-targeting PROTACs (BCL-XL-Ps) hold promise for treating metastatic breast cancer. Their potential lies in directly eliminating circulating tumor cells during vulnerable stages and/or triggering anti-cancer immunity through Treg cell depletion. Our experiments underscore the potency of IAP-based PROTACs in facilitating BCL-XL degradation via the ubiquitin-proteasome system (UPS) while mitigating on-target platelet toxicity. Treatment of breast cancer-bearing mice with these compounds demonstrates a tangible reduction in breast cancer metastasis to the lungs. Additionally, our studies reveal a significant depletion of tumor-infiltrating Tregs in mice treated with BCL-XL-Ps. While affirming BCL-XL as a viable therapeutic target for breast cancer metastasis, further exploration into the mechanisms behind BCL-XL-P targeting is imperative. Our future endeavors will be devoted to unraveling whether our BCL-XL-Ps effectively target both tumor-intrinsic BCL-XL, potentially inducing tumor cell death, and tumor-infiltrating Tregs, thereby fostering an adaptive immune response against the tumor cells. Citation Format: Madison E. Carelock, Peiyi Zhang, Mo Jiao, Umasankar De, Zeng Jin, Rachel M. Stump, Sarah G. Williams, Guangrong Zheng, Weizhou Zhang. Attenuating breast cancer metastasis with BCL-XL-targeting PROTACs [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 4123.

Nanoparticle (NP)-mediated APOC1 silencing to inhibit MAPK/ERK and NF-κB pathway and suppress breast cancer growth and metastasis.

Breast cancer is one of the most common malignant tumors with high mortality and poor prognosis in women. There is an urgent need to discover new therapeutic targets for breast cancer metastasis. Herein, we identified that Apolipoprotein C1 (APOC1) was up-regulated in primary tumor of breast cancer patient that recurrence and metastasis by immunohistochemistry (IHC). Kaplan-Meier Plotter database showed that high levels of APOC1 in breast cancer patients were strongly associated with worse overall survival (OS) and relapse-free survival (RFS). Mechanistically, APOC1 silencing significantly inhibits MAPK/ERK kinase pathway and restrains the NF-κB to decrease the transcription of target genes related to growth and metastasis in vitro. Based on this regulatory mechanism, we developed these findings into potential therapeutic drugs, glutathione (GSH) responsive nano-particles (NPs) were used for systemic APOC1 siRNA delivery, NPs (siAPOC1) silenced APOC1 expression, and subsequently resulted in positive anti-tumor effects in orthotopic and liver metastasis models in vivo. Taken together, GSH responsive NP-mediated siAPOC1 delivery was proved to be effective in regulating growth and metastasis in multiple tumor models. These findings show that APOC1 could be a potential biomarker to predict the prognosis of breast cancer patients and NP-mediated APOC1 silencing could be new strategies for exploration of new treatments for breast cancer metastasis.

Abstract P4-08-24: Palmitoylation of Flotillin-1 contributes to Breast Cancer Metastasis

Abstract BACKGROUND Breast Cancer metastasis results in the majority of cancer-related mortality. Flotillin-1 is overexpressed in numerous solid tumors and has been demonstrated to contribute to invasion and metastasis in breast cancer. Though its expression alone has been associated with metastasis, there has yet to be any investigation as to how certain post-translational modifications can affect its metastatic capabilities. Flotillin-1 is modified through palmitoylation, a post-translational modification essential for protein stability, processing, and localization. By generating a palmitoylation defective mutant of flotillin-1, we have demonstrated that flotillin-1 palmitoylation contributes to its stability and metastatic capabilities in both 3D and in vivo models of invasion and metastasis. MATERIALS AND METHODS Flotillin-1 wild type or palmitoylation defective C-34-A mutants were expressed in stable flotillin-1 knockdown MDA-MB-231 and SUM-159 triple negative breast cancer cells. These cells were subjected to 2D invasion chambers and 3D collagen invasion assays. Further, both wild type and C34A expressing cells were used for experimental lung metastases in mice by tail vein injection. 2-bromopalmtiate (50µM) was used as a chemical palmitoylation inhibitor and MG-132 (10µM) for proteasomal inhibition. RESULTS Invasion in stable knockdown cells was restored by re-expression of flotillin-1, but not by follitillin-1 C34A. We further observed a significant decrease in lung metastasis in flotillin-1 C34A cells compared to wild type flotillin-1. Flotillin-1 C34A expression led to a significant decrease in protein stability, which was restored by MG-132. CONCLUSION Flotillin-1 palmitoylation contributes to its stability and subsequent metastatic capabilities in breast cancer cells and experimental metastasis models. Thus, flotillin-1 palmitoylation could be a promising target for breast cancer metastasis. Citation Format: Bryan McClellan. Palmitoylation of Flotillin-1 contributes to Breast Cancer Metastasis [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 P4-08-24.

Identification of a minority population of LMO2+ breast cancer cells that integrate into the vasculature and initiate metastasis.

Metastasis is responsible for most breast cancer-related deaths; however, identifying the cellular determinants of metastasis has remained challenging. Here, we identified a minority population of immature THY1+/VEGFA+ tumor epithelial cells in human breast tumor biopsies that display angiogenic features and are marked by the expression of the oncogene, LMO2. Higher abundance of LMO2+ basal cells correlated with tumor endothelial content and predicted poor distant recurrence-free survival in patients. Using MMTV-PyMT/Lmo2CreERT2 mice, we demonstrated that Lmo2 lineage-traced cells integrate into the vasculature and have a higher propensity to metastasize. LMO2 knockdown in human breast tumors reduced lung metastasis by impairing intravasation, leading to a reduced frequency of circulating tumor cells. Mechanistically, we find that LMO2 binds to STAT3 and is required for STAT3 activation by tumor necrosis factor-α and interleukin-6. Collectively, our study identifies a population of metastasis-initiating cells with angiogenic features and establishes the LMO2-STAT3 signaling axis as a therapeutic target in breast cancer metastasis.

Abstract 5653: Increased expression of brain-specific serine/threonine-protein kinase BRSK2 contributes to breast cancer survival by enhancing autophagy under nutrient stress

Abstract Aberrant expression of certain cancer-promoting genes is the associative factor of metastasis and cancer-related death. Autophagy is a highly conserved catabolic process that can be regulated by starvation or stress. Among several cellular stresses, hypoxia and nutrient stress can strongly activate autophagy-related genes. Autophagy plays dual roles in cancer; it can be pro-death or pro-survival. The ability of the cancer cells to survive long-term under stress is still a vast question to cancer researchers. The nutrient status of is sensed by LKB1 downstream kinases AMP-activated protein kinase (AMPK). Recent studies revealed that BRSK2 is one of the AMPK related kinases regulated by LKB1 is the activator of WIPI protein function as building blocks for the formation of the autophagosome. Brain-specific kinases 1 and 2 are highly expressed in the mammalian forebrain regulating neuronal polarity and have lower expression in the testis and pancreas. We identified BRSK2 as one of the top genes highly expressed in 10 primary breast cancer tissue with a record of future bone metastasis compared to 10 primary breast tumors with no metastasis record through RNA seq analysis. Our extended analysis using TCGA and METABRIC cohort data unfolded that BRSK2 is highly elevated in breast cancer tumors compared to control breast tissue (p< 0.001), mainly in stage IV patients compared to other stages (p= 0.01). Furthermore, we observed that BRSK2 is differentially expressed in various breast cancer cell lines, in contrast, no expression in nonmalignant breast epithelial cells (MCF10A). We selected MDA-231 and BT474 cell lines having relatively lower BRSK2 expression, and expressed BRSK2 ectopically to understand details about BRSK2-mediated autophagy under nutrient stress. Interestingly, we observed that BRSK2 augmented nutrient starvation mediated autophagic flux in Triple-negative and ER/PR-positive breast cancer cell lines, along with elevated AKT, p-NF-kB, and STAT3 signaling pathways for survival under nutrient stress conditions. We further observed that several inflammatory cytokines CXCL1, CXCL6, TNF-α, and IL1-β, were significantly upregulated with BRSK2 overexpressed cells compared with the control cells. Knockdown of BRSK2 reduces autophagy and enhances apoptosis in breast cancer cells. Altogether, our data revealed that BRSK2 play a role in increasing autophagy to support cell survival under nutrient stress condition through AKT signaling pathways. Blocking of BRSK2 with inhibitor has reduced autophagy and increased apoptosis and cell death. Thus, BRSK2 can be a potential molecule as a therapeutic target for breast cancer metastasis. Citation Format: Nitai Hait, APARNA MAITI, Rongrong Wu, Kazuaki Takabe, Masanori Oshi. Increased expression of brain-specific serine/threonine-protein kinase BRSK2 contributes to breast cancer survival by enhancing autophagy under nutrient stress [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 5653.

EZH2-CCF-cGAS Axis Promotes Breast Cancer Metastasis.

Cytoplasmic chromatin fragments (CCF) are recognized by the cytoplasmic DNA sensor cyclic GMP-AMP synthase (cGAS), which activates the cGAS–STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway and promotes the production of inflammatory factors and breast cancer metastasis. However, the mechanisms by which CCF are formed in tumor cells and CCF activation cGAS promotes breast cancer metastasis remain unclear. Here, we report that the enhancer of zeste homolog 2 (EZH2) can promote the formation of CCF and activate the cGAS–STING pathway to promote breast cancer metastasis. Further research found that the EZH2-mediated CCF formation depended on high mobility group A1 (HMGA1), while the stability of EZH2 required ubiquitin-specific peptidase 7 (USP7), indicating that the EZH2–HMGA1–USP7 complex regulated CCF formation. Moreover, EZH2 can activate cGAS through CCF, requiring USP7 to deubiquitinate cGAS and stabilize cGAS. In vivo experimental results showed that EZH2 could promote breast cancer metastasis through CCF. Our findings highlight a new target for breast cancer metastasis. Targeting the EZH2–CCF–cGAS axis may be a potential therapeutic strategy for inhibiting breast cancer metastasis.

Dissecting RAS Oncogene-Induced Kinome Involved in Breast Cancer Metastasis

Background and Hypothesis:
 The RAS and PI3K-AKT-mTOR signaling pathways are often dysregulated in cancer. RAS pathway alterations, however, are more common in breast cancer metastasis. The laboratory’s recently developed model system demonstrated the ability of RAS but not PIK3CA-induced signals in promoting metastasis of breast cancer. Unbiased kinome analyses of isogenic RAS-transformed primary tumor and metastatic cells and PIK3CA-transformed primary tumor cells enabled identification of RAS-activated kinome, which included FER, PAK4, LIMK1, PIK3CD and Casein Kinase 2 (CK2). We hypothesized that therapeutic targeting of these kinases may reduce breast cancer metastasis. As a proof-of-principle, the effect of the CK2 inhibitor Silmitasertib, which is in clinical trial for COVID-19 and refractory multiple myeloma, was tested.
 Experimental Design:
 The study included four isogenic cell lines: “normal” (KTB34-hTERT), PIK3CA-transformed (TKTB34-PIK3CA), RAS-transformed (TKTB34-RAS), and RAS-transformed cells metastasized to lungs (MKTB34-RAS). Active kinomes in these cells were identified using phospho-proteomics and functional kinome profiling using multiplexed kinase inhibitor beads. Expression levels of FER, PAK4, LIMK1, and PIK3CD kinases were compared through Western Blot using the phospho-antibodies as an indicator of kinase activation. Sensitivity to Silmitasertib was measured using the BrdU Cell Proliferation Assay.
 Results:
 FER, PAK4, LIMK1, and PIK3CD were all overexpressed in the TKTB34-RAS and MKTB34-RAS cells compared to KTB34-hTERT and TKTB34-PIK3CA cells. The tested concentration range for Silmitasertib (500 nM to 5 µM) was ineffective in killing the RAS-transformed cells and was overly toxic to “normal” cells.
 Conclusion and Potential Impact:
 FER, PAK4, LIMK1, PIK3CD, and CK2 are potential therapeutic targets for breast cancer metastasis. However, Silmitasertib may not be a good candidate as it is more toxic to “normal” cells compared to cancer cells. The isogenic “normal” and transformed cell line model system described here may help to discover new targets and drugs that kill cancer but not normal cells.

N6 -Methyladenosine Regulates mRNA Stability and Translation Efficiency of KRT7 to Promote Breast Cancer Lung Metastasis.

The roles of RNA modification during organ metastasis of cancer cells are not known. Here we established breast cancer lung metastasis cells by three rounds of selection of lung metastatic subpopulations in vivo and designated them as BCLMF3 cells. In these cells, mRNA N6 -methyladenosine (m6A) and methyltransferase METTL3 were increased, while the demethylase FTO was decreased. Epi-transcriptome and transcriptome analyses together with functional studies identified keratin 7 (KRT7) as a key effector for m6A-induced breast cancer lung metastasis. Specifically, increased METTL3 methylated KRT7-AS at A877 to increase the stability of a KRT7-AS/KRT7 mRNA duplex via IGF2BP1/HuR complexes. Furthermore, YTHDF1/eEF-1 was involved in FTO-regulated translational elongation of KRT7 mRNA, with methylated A950 in KRT7 exon 6 as the key site for methylation. In vivo and clinical studies confirmed the essential roles of KRT7, KRT7-AS, and METTL3 for lung metastasis and clinical progression of breast cancer. Collectively, m6A promotes breast cancer lung metastasis by increasing the stability of a KRT7-AS/KRT7 mRNA duplex and translation of KRT7. SIGNIFICANCE: This study suggests that N6 -methyladenosine is a key driver and potential therapeutic target in breast cancer metastasis.

Acidic microenvironment induction of interleukin-8 expression and matrix metalloproteinase-2/-9 activation via acid-sensing ion channel 1 promotes breast cancer cell progression.

The cancer microenvironment exhibits local acidosis compared with the surrounding normal tissue. Many reports have shown that acidosis accelerates the invasiveness and metastasis of cancer, yet the underlying molecular mechanisms remain unclear. In the present study, we focused on acid-induced functional changes through acid receptors in breast cancer cells. Acidic treatment induced interleukin (IL)-8 expression in MDA-MB-231 cells and promoted cell migration and invasion. The acidic microenvironment elevated matrix metalloproteinase (MMP)-2 and MMP-9 activity, and addition of IL-8 had similar effects. However, inhibition of IL-8 suppressed the acid-induced migration and invasion of MDA-MB-231 cells. MDA-MB-231 cells express various acid receptors including ion channels and G protein-coupled receptors. Interestingly, acidic stimulation increased the expression of acid-sensing ion channel 1 (ASIC1), and acid-induced IL-8 was significantly decreased by ASIC1 knockdown. Moreover, phosphorylation of nuclear factor (NF)-κB was induced by acidic treatment, and inhibition of NF-κB activation reduced acid-induced IL-8 expression. These results suggest that IL-8 induction by an acidic microenvironment promotes breast cancer development and that ASIC1 might be a novel therapeutic target for breast cancer metastasis.

Gremlin-1 Promotes Metastasis of Breast Cancer Cells by Activating STAT3-MMP13 Signaling Pathway.

Gremlin-1 (GREM1), one of the bone morphogenetic protein (BMP) antagonists, can directly bind to BMPs. GREM1 is involved in organogenesis, tissue differentiation, and organ fibrosis. Recently, numerous studies have reported the oncogenic role of GREM1 in cancer. However, the role of GREM1 in metastasis of breast cancer cells and its underlying mechanisms remain poorly understood. The role of GREM1 in breast cancer progression was assessed by measuring growth, migration, and invasion of breast cancer cells. An orthotopic breast cancer mouse model was used to investigate the role of GREM1 in lung metastasis of breast cancer cells. GREM1 knockdown suppressed the proliferation of breast cancer cells, while its overexpression increased their growth, migration, and invasion. Cells with Grem1-knockdown showed much lower tumor growth rates and lung metastasis than control cells. GREM1 enhanced the expression of matrix metalloproteinase 13 (MMP13). A positive correlation between GREM1 and MMP13 expression was observed in breast cancer patients. GREM1 activated signal transducer and activator of transcription 3 (STAT3) transcription factor involved in the expression of MMP13. Our study suggests that GREM1 can promote lung metastasis of breast cancer cells through the STAT3-MMP13 pathway. In addition, GREM1 might be a promising therapeutic target for breast cancer metastasis.

Methylation of EZH2 by PRMT1 regulates its stability and promotes breast cancer metastasis.

Enhancer of zeste homolog 2 (EZH2), a key histone methyltransferase and EMT inducer, is overexpressed in diverse carcinomas, including breast cancer. However, the molecular mechanisms of EZH2 dysregulation in cancers are still largely unknown. Here, we discover that EZH2 is asymmetrically dimethylated at R342 (meR342-EZH2) by PRMT1. meR342-EZH2 was found to inhibit the CDK1-mediated phosphorylation of EZH2 at T345 and T487, thereby attenuating EZH2 ubiquitylation mediated by the E3 ligase TRAF6. We also demonstrate that meR342-EZH2 resulted in a decrease in EZH2 target gene expression, but an increase in breast cancer cell EMT, invasion and metastasis. Moreover, we confirm the positive correlations among PRMT1, meR342-EZH2 and EZH2 expression in the breast cancer tissues. Finally, we report that high expression levels of meR342-EZH2 predict a poor clinical outcome in breast cancer patients. Our findings may provide a novel diagnostic target and promising therapeutic target for breast cancer metastasis.

Arginine methylation of SHANK2 by PRMT7 promotes human breast cancer metastasis through activating endosomal FAK signalling.

Arginine methyltransferase PRMT7 is associated with human breast cancer metastasis. Endosomal FAK signalling is critical for cancer cell migration. Here we identified the pivotal roles of PRMT7 in promoting endosomal FAK signalling activation during breast cancer metastasis. PRMT7 exerted its functions through binding to scaffold protein SHANK2 and catalyzing di-methylation of SHANK2 at R240. SHANK2 R240 methylation exposed ANK domain by disrupting its SPN-ANK domain blockade, promoting in co-accumulation of dynamin2, talin, FAK, cortactin with SHANK2 on endosomes. In addition, SHANK2 R240 methylation activated endosomal FAK/cortactin signals in vitro and in vivo. Consistently, all the levels of PRMT7, methylated SHANK2, FAK Y397 phosphorylation and cortactin Y421 phosphorylation were correlated with aggressive clinical breast cancer tissues. These findings characterize the PRMT7-dependent SHANK2 methylation as a key player in mediating endosomal FAK signals activation, also point to the value of SHANK2 R240 methylation as a target for breast cancer metastasis.

Abstract 2603: Single cell RNA sequencing-based identification of molecular drivers in circulating tumor cell cluster formation and lung metastasis

Abstract Introduction: Circulating tumor cells (CTCs) mediate metastases, which account for 90% of solid tumor-related mortality. Compared to single cells, clustered CTCs mediate metastasis at a 20-100 times higher efficiency and are associated with lower overall survival in breast cancer (BC). We have recently identified a new mechanism of CTC cluster formation through cellular aggregation instead of cohesive shedding, and demonstrated that CTC clusters have enhanced stemness (Cancer Discovery, 2019). However, the cellular heterogeneity and molecular mechanisms underlying CTC cluster formation and polyclonal metastasis have yet to be fully elucidated. We hypothesize that molecular drivers of metastasis initiation enhance cancer stemness and CTC cluster formation, and serve as a novel therapeutic target for BC metastasis. Methods: Using single cell RNA sequencing, we compared tumor cells from the primary breast tumor site and lung metastases of BC patient-derived xenografts. We identified genes specifically expressed in the lung metastases and determined their functional importance in CTC clustering, cancer stemness, and lung colonization. We performed proteomic and transcriptomic analyses as well as machine learning to elucidate the downstream signaling pathways and protein structural basis involved in CTC cluster formation and lung metastasis. Finally, we explored therapeutic intervention options in blocking CTC cluster formation and lung metastasis. New results: Compared to the primary breast tumor cells, we identified a stemness gene signature enriched in a subpopulation of the CD44+ lung metastases, with 30-60 fold higher expression of CD34, CD36, ICAM1, VCAM1, ZEB1, ALDH1A1, TGFBR2 and TSPAN8. Analysis of patient blood samples (N=40) revealed that CD44 and many of these new candidate proteins were enriched in CTC clusters in comparison to single CTCs. We then examined the CSC-related properties of these tumor cells, such as tumorigenesis, sphere formation, and lung metastasis. Knockdown of selected surface molecules (e.g. ICAM1) significantly reduced the efficiency of lung metastasis of BC cells in vivo. A subset of ICAM1+/CD44+ tumor cells had increased stemness and tumor growth upon orthotopic implantation in vivo. Knockdown of ICAM1 dramatically reduced the self-renewal ability in mammosphere formation of breast tumor cells in vitro. In addition, our studies also revealed that these tumor cells cluster through CD44 and other surface protein-mediated homophilic binding between two neighboring tumor cells. Neutralizing antibodies significantly blocked tumor cluster formation and lung colonization. Conclusions: We identified new molecular mediators of CTC aggregation and lung metastasis in BC. We anticipate that specific blockade of tumor clustering could decrease cancer progression and improve survival of BC patients. Citation Format: Rokana Taftaf, Xia Liu, Salendra Singh, Yuzhi Jia, David Scholten, Youbin Zhang, Andrew Davis, Carolina Reduzzi, Yue Cao, Yang Shen, Massimo Cristofanilli, William A. Muller, Vinay Varadan, Huiping Liu. Single cell RNA sequencing-based identification of molecular drivers in circulating tumor cell cluster formation and lung metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2603.

CRMP2 is a therapeutic target that suppresses the aggressiveness of breast cancer cells by stabilizing RECK

Metastatic breast cancer is characterized by high mortality and limited therapeutic target. During tumor metastasis, cytoskeletal reorganization is one of the key steps in the migration and invasion of breast cancer cells. Collapsin response mediator protein 2 (CRMP2) is a cytosolic phosphoprotein that plays an important role in regulating cytoskeletal dynamics. Previous researches have reported that altered CRMP2 expression is associated with breast cancer progression, but the underlying mechanism remains poorly understood. Here, we show that CRMP2 expression is reduced in various subtypes of breast cancers and negatively correlated with lymphatic metastasis. Overexpression of CRMP2 significantly inhibits invasion and stemness in breast cancer cells, while downregulation of CRMP2 promotes cell invasion, which is not required for tubulin polymerization. Mechanistic studies demonstrate that CRMP2 interacts with RECK, prevents RECK degradation, which, in turn, blocks NF-κB and Wnt signaling pathways. Furthermore, we find that phosphorylation of CRMP2 at T514 and S522 remarkably abolishes its functions to bind with RECK and to inhibit cell invasion. Pharmacologic rescue of CRMP2 expression suppressed breast cancer metastasis in vitro and in vivo and stimulated a synergetic effect with FN-1501 that induces CRMP2 dephosphorylation. Collectively, this study highlights the potential of CRMP2 as a therapeutic target in breast cancer metastasis and reveals a distinct mechanism of CRMP2.

Long non coding RNA OIP5‑AS1 promotes metastasis of breast cancer via miR‑340‑5p/ZEB2 axis.

Breast cancer is the most common invasive cancer in women with the highest number of related deaths which is caused by distal metastasis. Recently, integrated analysis of gene expression profile suggested widespread gene dysregulation in various types of cancer. Research in the past decade has focused on long non-coding RNAs (lncRNAs), particularly in cell proliferation, tumor progression and metastasis. OPA-interacting protein 5 antisense transcript 1 (OIP5-AS1) is an evolutionarily conserved long non-coding RNA that has been linked to oncogenesis in multiple cancers. In breast cancer, dysregulation of OIP5-AS1 was reported but the precise role in cancer development and progression remains unclear. In the present study, using small interfering RNA (siRNA) targeting OIP5-AS1, it was shown that knockdown of OIP5-AS1 was associated with alteration of EMT markers and suppressed migration and invasion of breast cancer cells. Among the EMT-related transcription factors, ZEB1 and ZEB2 were significantly downregulated with OIP5-AS1 knockdown. Computational analysis and a dual-luciferase reporter system identified miR-340-5p was the target gene for OIP5-AS1. Further experiments verified the function of OIP5-AS1 in cell invasion was dependent on miR-340a-5p through regulating target gene ZEB2. In vivo study demonstrated that overexpressing OIP5-AS1 in breast cancer cells promoted lung metastasis in nude mice. The findings of the present study revealed the mechanism of OIP5-AS1 in breast cancer metastasis. Overall, our study may provide a potential therapeutic target for breast cancer metastasis.

Malic Enzyme 1 Indicates Worse Prognosis in Breast Cancer and Promotes Metastasis by Manipulating Reactive Oxygen Species.

PurposeMalic enzyme 1 (ME1) catalyzes malate to pyruvate and thus promotes glycolysis. Its function in breast cancer remains to be fully clarified. The aim of this work was to investigate the prognostic value of ME1 and its possible mechanism in breast cancer.MethodsWe evaluated ME1 expression in 220 early breast cancer patients with tissue microarray-based immunohistochemistry and explored the relationships between ME1 expression and clinicopathological features. Survival analyses were further performed to determine its prognostic value. The public database was used to confirm tissue microarray results. Further, cell proliferation, migration, invasion ability and reactive oxygen species (ROS) were examined in breast cancer cells.ResultsIn breast cancer tissues, high ME1 expression was significantly associated with larger tumor size, higher incidence of lymph node metastasis and higher incidence of lymph-vascular invasion. High ME1 expression significantly correlated with worse recurrence-free survival (RFS), and was an independent prognostic factor for RFS, which was confirmed by mRNA results in the public database. In vitro, upregulation of ME1 by transfecting MCF-7 cells with virus vector remarkably enhanced viability, motility and epithelial–mesenchymal transition (EMT) and decreased ROS levels, whereas knockdown in MDA-MB-468 cells produced totally opposite effects as expected. When pretreated with oxidizing agent, MCF-7 cells overexpressing ME1 lost its motility, whereas MDA-MB-468 cells with knockdown of ME1 restored its motility when pretreated with antioxidant.ConclusionTo our knowledge, these clinical and experiment works first suggested that ME1 may be a novel biomarker and potential therapeutic target for breast cancer metastasis, and its biological effect is mainly controlled by manipulating ROS.

Abstract B42: Identification of molecular drivers in circulating tumor cell cluster formation and lung metastasis

Abstract Introduction: Breast cancer (BC) is the leading type of cancer and the second leading cause of cancer-related deaths among women in the US. Metastasis accounts for 90% of solid tumor-related mortality and is mainly mediated by hematogenous spread of circulating tumor cells (CTCs). Compared to single cells, clustered CTCs mediate metastasis at a 20-100 times higher efficiency and are associated with lower overall survival. We have recently identified a new mechanism of CTC cluster formation through cellular aggregation instead of cohesive shedding and demonstrated that CTC clusters have enhanced stemness (Cancer Discovery 2019). However, the cellular heterogeneity and molecular mechanisms underlying CTC cluster formation and polyclonal metastasis have yet to be fully elucidated. We hypothesize that molecular drivers of metastasis initiation enhance cancer stemness and CTC cluster formation and serve as a novel therapeutic target for BC metastasis. Methods: Using single-cell RNA sequencing, we compared tumor cells from the primary breast tumor site and lung metastases of breast cancer patient-derived xenograft. We identified genes with differential expression levels in the lung mets and determined their functional importance in CTC clustering, cancer stemness, and lung colonization. Upon the candidate gene modulation, we performed proteomic and transcriptome analyses to elucidate the downstream signaling pathways involved in CTC cluster formation and lung metastasis. Finally, we explored therapeutic intervention options in blocking CTC cluster formation and lung metastasis. New Results: Compared to the primary breast tumor cells, we identified a stemness gene signature enriched in a subpopulation of the CD44+ lung metastases, with 30- to 60-fold higher expression of CD34, CD36, VCAM1, ZEB1, ALDH1A1, TGFBR2, TSPAN8, and ICAM1. Patient blood analyses (N=40) revealed that CD44 and many of these new candidate proteins are enriched in CTC clusters in comparison to single CTCs. We then examined the CSC-related properties of these tumor cells, such as tumorigenesis, sphere formation, and lung metastasis. Knockdown of selected surface molecules from the signature genes significantly reduced the efficiency of lung metastasis of BC cells in vivo. A subset of these tumor cells had increased stemness and highest tumor growth upon orthotopic implantation in vivo. Knockdown of the signature genes dramatically reduced the self-renewal ability in mammosphere formation of breast tumor cells in vitro. In addition, our studies also revealed that these tumor cells cluster through CD44 and other surface protein-mediated homophilic binding between two neighboring tumor cells. Neutralizing antibodies significantly blocked tumor cluster formation and lung colonization. Conclusion: We identified new molecular mediators of CTC aggregation and lung metastasis in breast cancer. We anticipate that a specific blocking of tumor clustering could decrease cancer progression and improve survival of breast cancer patients. Citation Format: Rokana Taftaf, Xia Liu, Salendra Singh, Yuzhi Jia, David Scholten, Massimo Cristofanilli, William A. Muller, Vinay Varadan, Huiping Liu. Identification of molecular drivers in circulating tumor cell cluster formation and lung metastasis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B42.

Role of E2F3 and shugoshin I in epithelial-to-mesenchymal transition and cell invasion in breast cancer.

e13100 Background: Triple-negative breast cancer (TNBC) is the most aggressive and poorly prognostic breast cancer subtype, yet there are currently no biological therapies against this subtype. Our laboratory is finding the sources of novel biological targets in TNBC by studying the E2F transcription factors, which are essential for cellular proliferation and maintenance of genomic stability. While the deregulated Rb/E2F pathway signals the epithelial-to-mesenchymal transition (EMT), the underlining mechanism of how E2Fs drive EMT in TNBC remains unknown. We recently published that the E2F transcriptional activators (E2Fs) are overexpressed in the vast majority of TNBC and that their overexpression upregulates mitotic kinases such as TTK, which we have shown to induce EMT and invasion in TNBC cells. We also demonstrated that the E2Fs maintain genomic integrity in part through Shugoshin I (SGO1), which normally controls chromosome cohesion; however, the role of SGO1 in EMT in breast cancer is unknown. Our hypothesis is that E2F3 and SGO1 are highly expressed in TNBC and that their overexpression modulates EMT genes, thus promoting cell invasion. Methods: To test our hypothesis, we conducted siRNA transfection to knockdown E2F3 and SGO1 in MDA-MB-231 and Hs578t, which are TNBC cells. After 48 hours, we evaluated mRNA levels of EMT-related genes after E2F3 or SGO1 depletion using RT-PCR analysis. We also evaluated the effects of SGO1 depletion in protein localization by immunofluorescence. Furthermore, we evaluated the invasive behavior of MDA-MB-231 and Hs578t cells after SGO1 depletion using a Boyden Chamber Assay. Results: Our results demonstrate that E2F3 and SGO1 depletion decrease MMP3 mRNA levels. Moreover, E2F3 and SGO1 depletion restore E-cadherin expression and localization. Furthermore, E2F3 and SGO1 depletion significantly reduce cell invasion in MDA-MB-231 and Hs578t cells. Conclusions: Our results suggest that SGO1 is a promising drug target for breast cancer metastasis since EMT and invasion are essential early steps in breast cancer metastasis and E2F3 is presently undruggable.

Abstract P6-07-06: Shugoshin I signals epithelial-mesenchymal transition and promotes cell invasion in breast cancer

Abstract Despite being the most aggressive and poorly prognostic breast cancer subtype, there are currently no biological therapies against triple-negative (TN, or ER-PR-Her2-) breast cancers. Our laboratory is finding the sources of novel biological targets in TNBC by studying the E2F transcription factors, which are essential for cellular proliferation and maintenance of genomic stability. We recently published that the E2F transcriptional activators are overexpressed in the vast majority of TNBC and can dictate the high percentage of mitotic cells in mammary tumors. We also demonstrated that that the E2Fs control multiple mitotic regulators and maintain genomic integrity through Shugoshin I (SGO1) and BubR1. SGO1 is essential for chromosome segregation by preventing premature dissociation of the cohesion complex and sister chromatids after prophase. Until recently, SGO1 research was restricted to embryogenesis; however, recent studies have shown a role for SGO1 in tumorigenesis and epithelial-mesenchymal transition (EMT), but none have been done in breast cancer. Thus, we investigated SGO1 expression and genetic alteration in open databases such as KM-Plotter and cBioPortal and found that SGO1 is highly expressed in triple-negative breast cancer subtype and that its expression correlates with poor prognostic factors. Therefore, we decided to investigate the role of SGO1 in breast cancer EMT and cell invasion. Our hypothesis is that SGO1 modulates EMT genes, thus promoting cell invasion. To test our hypothesis, we conducted siRNA transfection to knockdown SGO1 in MDA-MB-231 and Hs578t, which are TNBC cells. After 48 hours, we evaluated mRNA levels of EMT-related genes after SGO1 depletion using RT-PCR analysis. We also evaluated the effects of SGO1 depletion in protein localization by immunofluorescence analysis. Furthermore, we evaluated the invasive behavior of MDA-MB-231 and Hs578t cells after SGO1 depletion using a Boyden Chamber Assay. Our results demonstrate that SGO1 depletion signals EMT by decreasing N-cadherin and increasing E-cadherin mRNA levels. SGO1 depletion also restores protein expression of E-cadherin in MDA-MB-231 cells. Strikingly, SGO1 depletion significantly reduces invasion in MDA-MB-231 and Hs578t cells. Our results suggest that SGO1 is a promising drug target for breast cancer metastasis since EMT and invasion are essential early steps in breast cancer metastasis. Currently, we are evaluating SGO1 expression in tissue microarrays derived from breast cancer patients (including Luminal and TNBC subtypes), in order to determine the percentage of those patients expressing Sgo1, as well as to establish important correlations with surrogate markers of prognosis (EMT, proliferation, mitosis). Our results may help establish new druggable targets in order to prevent and reduce metastasis in TNBC patients. Citation Format: Shirley Jusino, Harold I. Saavedra. Shugoshin I signals epithelial-mesenchymal transition and promotes cell invasion in breast cancer [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 P6-07-06.

HER2 Upregulates ATF4 to Promote Cell Migration via Activation of ZEB1 and Downregulation of E-Cadherin.

HER2 (human epidermal growth factor receptor 2) activation is critical in breast cancer development. HER2 promotes cell proliferation, angiogenesis, survival, and metastasis by activation of PI3K/Akt, Ras/MEK/ERK, and JAK/STAT pathways. However, beyond these signaling molecules, the key proteins underlining HER2-mediated metastasis remain elusive. ATF4 (Activating transcription factor 4), a critical regulator in unfolded protein response (UPR), is implicated in cell migration and tumor metastasis. In this study, we demonstrate that HER2 upregulated ATF4 expression at both mRNA and protein levels, resulting in cell migration increased. In addition, ATF4 upregulated ZEB1 (Zinc finger E-box-binding homeobox 1) and suppressed E-cadherin expression resulting in promoting cell migration. Restoration of E-cadherin expression effectively inhibited HER2- or ATF4-mediated cell migration. In addition, upregulated expression of ATF4 was found in HER2-positive breast cancer specimens. Together, this study demonstrates that ATF4-ZEB1 is important for HER2-mediated cell migration and suggests that ATF4-ZEB1 may be potential therapeutic targets for breast cancer metastasis.