Drives Breast Cancer Metastasis Research Articles

7881 Using Selective Estrogen Receptor Modulators to Reveal Therapeutic Vulnerabilities in ESR1 Mutant Breast Cancer

Abstract Disclosure: S.W. Fanning: Grant Recipient; Self; Olema Oncology. Hotspot activating somatic mutations to ESR1, the gene for estrogen receptor alpha (ER), enable hormone therapy resistance and drive breast cancer metastasis. The Y537S missense mutation within the ER ligand binding domain is the most activating, therapy resistant, and increases metastatic potential. Next generation antiestrogens including lasofoxifene (laso) and elacestrant (RAD1901) show improved progression-free survival over fulvestrant (fulv), but further progress is needed to achieve durable response. To fully address Y537S ESR1, it is critically important to understand how the mutation boosts metastasis. We have developed a small molecule antiestrogen, T6I-29, that uniquely impacts transcriptional pathways related to cell-cell adhesions and morphology. Two allele-specific phenotypes identified by the Oesterreich Laboratory enhance the metastasis of Y537S ESR1 breast cancer cells. T6I-29-specific pathway effects were driven by a significant downregulation of dickkopf-1 (DKK1), which was also observed but to a lesser extent with fulv, laso, and RAD1901. DKK1 is an immunosuppressive, tumor-secreted glycoprotein and its overexpression correlates with poor outcomes in multiple cancers. However, DKK1 acts as a tumor-suppressor in colorectal cancer, which does not rely on ER. Our preliminary studies suggest that DKK1 plays a role in ER+ breast cancer progression. We measured significantly elevated DKK1 protein levels in the blood plasma of 108 diverse ER+ breast cancer patients compared to 100 matched healthy female donors and levels increase coincident with tumor stage. Unfortunately, we lack ESR1 mutation status and other critical details to make further insights on these patients. DKK1 gene expression and protein secretion is stimulated in WT cells by the pathogenic hormone 17β-estradiol (E2), while it is constitutively expressed in Y537S ESR1 cells. DKK1 is characterized as a WNT antagonist but can also induce PI3K/Akt signaling. Interestingly, DKK1 is elevated and ERα responsive in WT not mutant PI3KCA cell lines. DKK1 knockdown also reduced proliferation of Y537S ESR1 breast cancer cells where elevated DKK1 was observed. Presentation: 6/3/2024

RNA-binding protein LSM7 facilitates breast cancer metastasis through mediating alternative splicing of CD44

AimsThe RNA-binding protein LSM7 is essential for RNA splicing, acting as a key component of the spliceosome complex; however, its specific role in breast cancer (BC) has not been extensively investigated. Materials and methodsLSM7 expression in BC samples was evaluated through bioinformatics analysis and immunohistochemistry. The impact of LSM7 on promoting metastatic tumor characteristics was examined using transwell and wound healing assays, as well as an orthotopic xenograft model. Additionally, the involvement of LSM7 in alternative splicing of CD44 was explored via RNA immunoprecipitation and third-generation sequencing. The regulatory role of TCF3 in modulating LSM7 gene expression was further elucidated using luciferase reporter assays and chromatin immunoprecipitation. Key findingsOur findings demonstrate that LSM7 was significantly overexpressed in metastatic BC tissues and was associated with poor prognostic outcomes in patients with BC. LSM7 overexpression markedly increased the migratory and invasive capabilities of BC cells in vitro and significantly promoted spontaneous lung metastasis in vivo. Furthermore, RIP-seq analysis revealed that LSM7 binded to CD44 RNA, enhancing the expression of its alternatively spliced isoform CD44s, thereby driving BC metastasis and invasion. Additionally, the transcription factor TCF3 was found to activate LSM7 transcription by directly binding to its promoter. SignificanceIn summary, this study highlights the pivotal role of LSM7 in the production of the CD44s isoform and the promotion of breast cancer metastasis. Targeting the TCF3/LSM7/CD44s axis may offer a promising therapeutic strategy for breast cancer treatment.

CCDC88C, an O-GalNAc glycosylation substrate of GALNT6, drives breast cancer metastasis by promoting c-JUN-mediated CEMIP transcription

Coiled-coil domain containing 88C (CCDC88C) is a component of non-canonical Wnt signaling, and its dysregulation causes colorectal cancer metastasis. Dysregulated expression of CCDC88C was observed in lymph node metastatic tumor tissues of breast cancer. However, the role of CCDC88C in breast cancer metastasis remains unclear. To address this, the stable BT549 and SKBR3 cell lines with CCDC88C overexpression or knockdown were developed. Loss/gain-of-function experiments suggested that CCDC88C drove breast cancer cell motility in vitro and lung and liver metastasis in vivo. We found that CCDC88C led to c-JUN-induced transcription activation. Overlapping genes were identified from the genes modulated by CCDC88C and c-JUN. CEMIP, one of these overlapping genes, has been confirmed to confer breast cancer metastasis. We found that CCDC88C regulated CEMIP mRNA levels via c-JUN and it exerted pro-metastatic capabilities in a CEMIP-dependent manner. Moreover, we identified the CCDC88C as a substrate of polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6). GALNT6 was positively correlated with CCDC88C protein abundance in the normal breast and breast cancer tissues, indicating that GALNT6 might be associated with expression patterns of CCDC88C in breast cancer. Our data demonstrated that GALNT6 maintained CCDC88C stability by promoting its O-linked glycosylation, and the modification was critical for the pro-metastatic potential of CCDC88C. CCDC88C also could mediate the pro-metastatic potential of GALNT6 in breast cancer. Collectively, our findings uncover that CCDC88C may increase the risk of breast cancer metastasis and elucidate the underlying molecular mechanisms.

Abstract PO5-25-01: Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes

Abstract Background: Although breast cancer liver metastases (BCLM) are common events, with 40-50% of patients with metastatic breast cancer developing liver metastases, their development indicates poor prognosis. Patients with BCLM have a 5-year survival rate of 8%. Specific treatments for liver metastases are virtually non-existent, partly because the key events of early liver metastasis formation remain unknown. The liver is a dense organ, and metastatic breast cancer cells (MCC) must clear space among hepatocytes, the primary cell population in the liver, during metastatic formation. Cell competition is a developmentally conserved process that explains how organs or primary tumors form when cells within those tissues have varying fitness and compete with one another. This competition can result in the survival and expansion of one cell population, and the induction of cell death through apoptosis in the other. We hypothesized that MCC use cell competition with hepatocytes to create space and expand within the liver. Objectives: In this study, we tested the hypothesis that MCC use cell competition with hepatocytes to create space and expand within the liver, resulting in hepatocyte apoptosis. Methods: We develop new in-vitro and mouse lineage-traced models to assay cell competition between MCC and hepatocytes, initially using balb/c derived breast cancer (4T1) and hepatocyte (H2.35) cells and syngeneic mouse models of liver metastasis. These models directly capture MCC and hepatocyte interactions, and we can assess modes of cell competition, including apoptosis, through immunostaining. Because prior literature suggests that the developing mammary gland informs both cell competition and metastasis, we next performed a bioinformatics screen to identify potential signals that contribute to cell competition between MCC and hepatocytes. We generated a new single-cell RNA-seq dataset that incorporates information from the developing mammary gland and breast cancer cells. This dataset includes 3264 primary breast cancer cells across 3 models of aggressive breast cancers, encompassing HER2+ and TNBC subtypes, as well as 526 embryonic mammary gland cells. Using this platform, we identify genes enriched in both breast cancer and the developing mammary gland. Next, we validate genes by testing for their high expression in MCC when compared to hepatocytes. Then, we determine the impact of target genes on cell competition in our models. Results/Discussion: Our models demonstrate that MCC use cell competition to create space in the liver by inducing apoptosis in neighboring hepatocytes. MCC induce apoptosis in neighboring hepatocytes through cell competition in in-vitro co-cultures by 11.7-fold compared to MCC apoptosis. Hydrodynamic tail-vein engraftment in NOD-scid-gamma mice livers with MCC also resulted in increased neighboring hepatocyte apoptosis. Using our bioinformatics screen, we identified 5 potential targets that could drive breast cancer metastasis and cell competition. Using our competition models, perturbation of SerpinE2, a protease inhibitor which was one of the top gene targets, successfully modulated competition between MCC and hepatocytes. Knock-out of SerpinE2 in MCC did not affect their viability or growth but resulted in MCC apoptosis adjacent to hepatocyte neighbors by 16.9-fold when compared to wild type conditions. Further, co-culture of MCC that lacked SerpinE2 with hepatocytes resulted in decreased hepatocyte apoptosis by 2.3-fold compared to hepatocytes co-cultured with wild-type (WT) MCC. These findings suggest that hepatocytes cannot outcompete or kill WT MCC, but they can outcompete and kill MCC that lack SerpinE2. Future work focused on the perturbation of SerpinE2 may lead to novel therapies for patients at risk for or with BCLM. Our findings suggest that restoring the competitiveness of the liver parenchyma could be a new treatment modality for treating liver metastases. Citation Format: Katherine Lake, Sakshi Mohta, Clayton Smith, Venkata Repaka, Lily Xu, Kaitlyn Saunders, Vrushali Pandit, Emily Goff, Christine Zhang, Jacob Pena, Christine Hodgdon, Julia Maues, Elizabeth Chen, Isaac Chan. Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-25-01.

Abstract A041: Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes

Abstract Background Although breast cancer liver metastases (BCLM) are common events, with 40-50% of patients with metastatic breast cancer developing liver metastases, their development indicates particularly poor prognosis. Targeted treatments for liver metastases are virtually non-existent, partly because the key events of early liver metastasis formation remain unknown. The liver is a dense organ, and metastatic breast cancer cells (MCC) must first clear space among hepatocytes, the primary cell population in the liver, to form. Cell competition is a process that explains how organs or primary tumors form from two populations of competing cells with varied fitness levels. The expansion of one population of cells creates space through the induction of apoptosis in the other. Objectives In this study, we tested the hypothesis that MCC use cell competition with hepatocytes to create space and expand within the liver, resulting in hepatocyte apoptosis. Methods We develop new in-vitro and mouse lineage-traced models to assay cell competition between MCC and hepatocytes. These models directly capture MCC and hepatocyte interactions and we can assess modes of cell competition, including apoptosis, through immunostaining. Because prior literature suggests that the developing mammary gland informs both cell competition and metastasis, we performed a bioinformatics screen to identify potential signals that contribute to cell competition between MCC and hepatocytes. We generate a new single-cell RNA-seq dataset that incorporates information from the developing mammary gland and breast cancer cells and identify genes enriched in both. Next, we validated genes by testing for their high expression in MCC when compared to hepatocytes. Then, we determined the impact of target genes on cell competition in our models. Results/Discussion Our models demonstrate that MCC use cell competition to create space in the liver by inducing apoptosis in neighboring hepatocytes. MCC induce apoptosis in neighboring hepatocytes through cell competition in in-vitro co-cultures by 11.7-fold compared to MCC apoptosis. Hydrodynamic tail-vein engraftment in NOD-scid-gamma mice with MCC also resulted in increased neighboring hepatocyte apoptosis. Using our bioinformatics screen, we identified SerpinE2, a protease inhibitor, as a target that could drive breast cancer metastasis and cell competition. Knock-out of SerpinE2 in MCC did not affect their viability or growth but resulted in MCC apoptosis adjacent to hepatocyte neighbors by 16.9-fold when compared to wild-type (WT) conditions. Further, co-culture of MCC that lacked SerpinE2 with hepatocytes resulted in decreased hepatocyte apoptosis by 2.3-fold compared to hepatocytes co-cultured with WT MCC. These findings suggest that perturbation of SerpinE2 successfully modulates competition between MCC and hepatocytes: Hepatocytes can selectively outcompete and kill MCC that lack SerpinE2. Future work focused on the perturbation of SerpinE2 may lead to novel therapies for patients at risk for or with BCLM. Citation Format: Katherine E Lake, Sakshi Mohta, Clayton A Smith, Venkata Repaka, Lily Xu, Kaitlyn Saunders, Vrushali Pandit, Emily Goff, Jacob Pena, Luise Chinea, Luisa Froessl, Sangeetha M Reddy, Heather L McArthur, Christine Hodges, Julia Maues, Elizabeth Chen, Isaac S Chan. Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A041.

Cytoskeleton remodeling induced by SMYD2 methyltransferase drives breast cancer metastasis

Malignant forms of breast cancer refractory to existing therapies remain a major unmet health issue, primarily due to metastatic spread. A better understanding of the mechanisms at play will provide better insights for alternative treatments to prevent breast cancer cell dispersion. Here, we identify the lysine methyltransferase SMYD2 as a clinically actionable master regulator of breast cancer metastasis. While SMYD2 is overexpressed in aggressive breast cancers, we notice that it is not required for primary tumor growth. However, mammary-epithelium specific SMYD2 ablation increases mouse overall survival by blocking the primary tumor cell ability to metastasize. Mechanistically, we identify BCAR3 as a genuine physiological substrate of SMYD2 in breast cancer cells. BCAR3 monomethylated at lysine K334 (K334me1) is recognized by a novel methyl-binding domain present in FMNLs proteins. These actin cytoskeleton regulators are recruited at the cell edges by the SMYD2 methylation signaling and modulate lamellipodia properties. Breast cancer cells with impaired BCAR3 methylation lose migration and invasiveness capacity in vitro and are ineffective in promoting metastases in vivo. Remarkably, SMYD2 pharmacologic inhibition efficiently impairs the metastatic spread of breast cancer cells, PDX and aggressive mammary tumors from genetically engineered mice. This study provides a rationale for innovative therapeutic prevention of malignant breast cancer metastatic progression by targeting the SMYD2-BCAR3-FMNL axis.

Tumor-educated Gr1+CD11b+ cells drive breast cancer metastasis via OSM/IL-6/JAK-induced cancer cell plasticity.

Cancer cell plasticity contributes to therapy resistance and metastasis, which represent the main causes of cancer-related death, including in breast cancer. The tumor microenvironment drives cancer cell plasticity and metastasis, and unraveling the underlying cues may provide novel strategies for managing metastatic disease. Using breast cancer experimental models and transcriptomic analyses, we show that stem cell antigen-1 positive (SCA1+) murine breast cancer cells enriched during tumor progression and metastasis had higher in vitro cancer stem cell-like properties, enhanced in vivo metastatic ability, and generated tumors rich in Gr1hiLy6G+CD11b+ cells. In turn, tumor-educated Gr1+CD11b+ (Tu-Gr1+CD11b+) cells rapidly and transiently converted low metastatic SCA1- cells into highly metastatic SCA1+ cells via secreted oncostatin M (OSM) and IL-6. JAK inhibition prevented OSM/IL-6-induced SCA1+ population enrichment, while OSM/IL-6 depletion suppressed Tu-Gr1+CD11b+-induced SCA1+ population enrichment in vitro and metastasis in vivo. Moreover, chemotherapy-selected highly metastatic 4T1 cells maintained high SCA1+ positivity through autocrine IL-6 production, and in vitro JAK inhibition blunted SCA1 positivity and metastatic capacity. Importantly, Tu-Gr1+CD11b+ cells invoked a gene signature in tumor cells predicting shorter overall survival (OS), relapse-free survival (RFS), and lung metastasis in breast cancer patients. Collectively, our data identified OSM/IL-6/JAK as a clinically relevant paracrine/autocrine axis instigating breast cancer cell plasticity and triggering metastasis.

A positive feedback loop between ZEB2 and ACSL4 regulates lipid metabolism to promote breast cancer metastasis

Lipid metabolism plays a critical role in cancer metastasis. However, the mechanisms through which metastatic genes regulate lipid metabolism remain unclear. Here, we describe a new oncogenic–metabolic feedback loop between the epithelial–mesenchymal transition transcription factor ZEB2 and the key lipid enzyme ACSL4 (long-chain acyl-CoA synthetase 4), resulting in enhanced cellular lipid storage and fatty acid oxidation (FAO) to drive breast cancer metastasis. Functionally, depletion of ZEB2 or ACSL4 significantly reduced lipid droplets (LDs) abundance and cell migration. ACSL4 overexpression rescued the invasive capabilities of the ZEB2 knockdown cells, suggesting that ACSL4 is crucial for ZEB2-mediated metastasis. Mechanistically, ZEB2-activated ACSL4 expression by directly binding to the ACSL4 promoter. ACSL4 binds to and stabilizes ZEB2 by reducing ZEB2 ubiquitination. Notably, ACSL4 not only promotes the intracellular lipogenesis and LDs accumulation but also enhances FAO and adenosine triphosphate production by upregulating the FAO rate-limiting enzyme CPT1A (carnitine palmitoyltransferase 1 isoform A). Finally, we demonstrated that ACSL4 knockdown significantly reduced metastatic lung nodes in vivo. In conclusion, we reveal a novel positive regulatory loop between ZEB2 and ACSL4, which promotes LDs storage to meet the energy needs of breast cancer metastasis, and identify the ZEB2–ACSL4 signaling axis as an attractive therapeutic target for overcoming breast cancer metastasis.

A positive feedback loop between ZEB2 and ACSL4 regulates lipid metabolism to promote breast cancer metastasis

Lipid metabolism plays a critical role in cancer metastasis. However, the mechanisms through which metastatic genes regulate lipid metabolism remain unclear. Here, we describe a new oncogenic–metabolic feedback loop between the epithelial–mesenchymal transition transcription factor ZEB2 and the key lipid enzyme ACSL4 (long-chain acyl-CoA synthetase 4), resulting in enhanced cellular lipid storage and fatty acid oxidation (FAO) to drive breast cancer metastasis. Functionally, depletion of ZEB2 or ACSL4 significantly reduced lipid droplets (LDs) abundance and cell migration. ACSL4 overexpression rescued the invasive capabilities of the ZEB2 knockdown cells, suggesting that ACSL4 is crucial for ZEB2-mediated metastasis. Mechanistically, ZEB2-activated ACSL4 expression by directly binding to the ACSL4 promoter. ACSL4 binds to and stabilizes ZEB2 by reducing ZEB2 ubiquitination. Notably, ACSL4 not only promotes the intracellular lipogenesis and LDs accumulation but also enhances FAO and adenosine triphosphate production by upregulating the FAO rate-limiting enzyme CPT1A (carnitine palmitoyltransferase 1 isoform A). Finally, we demonstrated that ACSL4 knockdown significantly reduced metastatic lung nodes in vivo. In conclusion, we reveal a novel positive regulatory loop between ZEB2 and ACSL4, which promotes LDs storage to meet the energy needs of breast cancer metastasis, and identify the ZEB2–ACSL4 signaling axis as an attractive therapeutic target for overcoming breast cancer metastasis.

MAF amplification licenses ERα through epigenetic remodelling to drive breast cancer metastasis

MAF amplification increases the risk of breast cancer (BCa) metastasis through mechanisms that are still poorly understood yet have important clinical implications. Oestrogen-receptor-positive (ER+) BCa requires oestrogen for both growth and metastasis, albeit by ill-known mechanisms. Here we integrate proteomics, transcriptomics, epigenomics, chromatin accessibility and functional assays from human and syngeneic mouse BCa models to show that MAF directly interacts with oestrogen receptor alpha (ERα), thereby promoting a unique chromatin landscape that favours metastatic spread. We identify metastasis-promoting genes that are de novo licensed following oestrogen exposure in a MAF-dependent manner. The histone demethylase KDM1A is key to the epigenomic remodelling that facilitates the expression of the pro-metastatic MAF/oestrogen-driven gene expression program, and loss of KDM1A activity prevents this metastasis. We have thus determined that the molecular basis underlying MAF/oestrogen-mediated metastasis requires genetic, epigenetic and hormone signals from the systemic environment, which influence the ability of BCa cells to metastasize.

NUCB2/Nesfatin-1 drives breast cancer metastasis through the up-regulation of cholesterol synthesis via the mTORC1 pathway

BackgroundReprogramming lipid metabolism for tumor metastasis is essential in breast cancer, and NUCB2/Nesfatin-1 plays a crucial role in regulating energy metabolism. Its high expression is associated with poor prognosis in breast cancer. Here, we studied whether NUCB2/Nesfatin-1 promotes breast cancer metastasis through reprogramming cholesterol metabolism.MethodsELISA was employed to measure the concentration of Nesfatin-1 in the serum of breast cancer patients and the control group. Database analysis suggested that NUCB2/Nesfatin-1 might be acetylated in breast cancer, which was confirmed by treating the breast cancer cells with acetyltransferase inhibitors. Transwell migration and Matrigel invasion assays were conducted, and nude mouse lung metastasis models were established to examine the effect of NUCB2/Nesfatin-1 on breast cancer metastasis in vitro and in vivo. The Affymetrix gene expression chip results were analyzed using IPA software to identify the critical pathway induced by NUCB2/Nesfatin-1. We evaluated the effect of NUCB2/Nesfatin-1 on cholesterol biosynthesis through the mTORC1-SREBP2-HMGCR axis by utilizing mTORC1 inhibitor and rescue experiments.ResultsNUCB2/Nesfatin-1 was found to be overexpressed in the breast cancer patients, and its overexpression was positively correlated with poor prognosis. NUCB2 was potentially acetylated, leading to high expression in breast cancer. NUCB2/Nesfatin-1 promoted metastasis in vitro and in vivo, while Nesfatin-1 rescued impaired cell metastasis induced by NUCB2 depletion. Mechanistically, NUCB2/Nesfatin-1 upregulated cholesterol synthesis via the mTORC1 signal pathway, contributing to breast cancer migration and metastasis.ConclusionsOur findings demonstrate that the NUCB2/Nesfatin-1/mTORC1/SREBP2 signal pathway is critical in regulating cholesterol synthesis, essential for breast cancer metastasis. Thus, NUCB2/Nesfatin-1 might be utilized as a diagnostic tool and also used in cancer therapy for breast cancer in the future.

Abstract 5897: Tumor-derived bacteria drive breast cancer metastasis

Abstract Metastasis is a major barrier to long-term survival and therapeutic options for aggressive, metastatic forms of breast cancer remain limited. Studies using patient samples have identified tumor-resident bacteria that preferentially associate with specific breast cancer types, however, it is not yet understood how intratumoral bacteria directly contributes to disease progression and metastatic propensity independent of other prognostic factors. It is therefore the goal of the Dedhar and Finlay labs to identify how specific bacteria within metastatic breast cancer control immune and tumor cell functions to regulate metastatic potential and determine the outcome of disease progression. Using the syngenic, immunocompetent 4T1 and 67NR breast cancer models of metastatic and non-metastatic disease, we found microbiome depletion significantly reduces primary tumor growth of highly metastatic 4T1 tumors specifically. We also found bacterial depletion reduces metastatic burden and extends survival time compared to microbiome-replete controls. Microbiome depletion also induces changes in immune cell function that occur specifically in the metastatic 4T1 tumors, revealing differential microbial-based regulation of metastatic versus non-metastatic disease. To identify bacteria that control metastasis in microbiome-replete controls, we plated surgically resected tumor suspensions on bacterial growth media and compared bacteria from the 4T1 and 67NR primary tumors. We identified several species of the Bacillus genus that were unique to 4T1 tumors and were present both within the primary tumor as well as metastatic nodules. We then designed several in vivo model systems to directly test the ability of the isolated bacteria to promote metastasis. Using an orthotopic inoculation model with 4T1 or EMT6 cells, we found that following intratumoral injection, the 4T1-derived Bacillus species was actually able to augment metastasis when introduced directly back into primary tumors. To determine the specificity of this phenomenon, we then compared the effects of the 4T1 and 67NR-isolated bacteria on metastasis by injecting 4T1 cells that had been co-cultured with either bacteria prior to injection. Interestingly, we found that while the 67NR-derived bacteria had little effect on metastasis, the 4T1-derived Bacillus species significantly enhanced metastatic tumor burden compared to all other groups including those cultured with the 67NR-derived bacteria. These data demonstrate the ability of certain bacteria to promote metastatic disease. Based on these findings, we hypothesize specific bacteria play a causative role in augmenting metastatic propensity, and seek to determine functional differences between intratumoral bacteria to identify mechanistic targets for prevention of metastasis. We also seek to expand this work into clinical models to identify potential prognostic factors as well as mechanistic targets for disease treatment. Citation Format: Zachary J. Gerbec, Antonio Serapio-Palacios, Sarah Woodward, Jorge Pena-Diaz, B Brett Finlay, Shoukat Dedhar. Tumor-derived bacteria drive breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5897.

LncRNA BREA2 promotes metastasis by disrupting the WWP2-mediated ubiquitination of Notch1

Notch has been implicated in human cancers and is a putative therapeutic target. However, the regulation of Notch activation in the nucleus remains largely uncharacterized. Therefore, characterizing the detailed mechanisms governing Notch degradation will identify attractive strategies for treating Notch-activated cancers. Here, we report that the long noncoding RNA (lncRNA) BREA2 drives breast cancer metastasis by stabilizing the Notch1 intracellular domain (NICD1). Moreover, we reveal WW domain containing E3 ubiquitin protein ligase 2 (WWP2) as an E3 ligase for NICD1 at K1821 and a suppressor of breast cancer metastasis. Mechanistically, BREA2 impairs WWP2-NICD1 complex formation and in turn stabilizes NICD1, leading to Notch signaling activation and lung metastasis. BREA2 loss sensitizes breast cancer cells to inhibition of Notch signaling and suppresses the growth of breast cancer patient-derived xenograft tumors, highlighting its therapeutic potential in breast cancer. Taken together, these results reveal the lncRNA BREA2 as a putative regulator of Notch signaling and an oncogenic player driving breast cancer metastasis.

Abstract A047: Tumor-derived bacteria drive breast cancer metastasis

Abstract Metastasis is a major barrier to long-term survival and therapeutic options for aggressive, metastatic forms of breast cancer remain limited. Studies using patient samples have identified tumor-resident bacteria that preferentially associate with specific breast cancer types including highly aggressive TNBC. However, it is not yet understood how intratumoral bacteria directly contributes to disease progression and metastatic propensity independent of other prognostic factors. It is therefore the goal of the Dedhar and Finlay labs to identify how specific bacteria within metastatic breast cancer control immune and tumor cell functions to regulate metastatic potential and determine the outcome of disease progression. Using the syngenic, immunocompetent 4T1 and 67NR breast cancer models of metastatic and non-metastatic disease, we found microbiome depletion significantly reduces primary tumor growth highly metastatic 4T1 tumors specifically. We also found bacterial depletion reduces metastatic burden and extends survival time compared to microbiome-replete controls. Along with alterations in disease progression, microbiome depletion induces changes in immune cell function that occur specifically in the metastatic 4T1 tumors, revealing differential microbial-based regulation of metastatic versus non-metastatic disease. To identify bacteria that control metastasis in microbiome-replete controls, we plated surgically resected tumor suspensions on bacterial growth media and compared bacteria from the 4T1 and 67NR primary tumors. We identified several species of the Bacillus genus that were unique to 4T1 tumors and were present both within the primary tumor as well as metastatic nodules. To determine how these bacteria effect disease progression, we designed several in vivo model systems to directly test the ability of the isolated bacteria to promote metastasis. Using an orthotopic inoculation model with 4T1 or EMT6 cells, we found that following intratumoral injection, the 4T1- derived Bacillus species was actually able to augment metastasis when introduced directly back into primary tumors. To determine the specificity of this phenomenon, we then compared the effects of the 4T1 and 67NR-isolated bacteria on metastasis by injecting 4T1 cells that had been co-cultured with either bacteria prior to injection. Interestingly, we found that while the 67NR-derived bacteria had little effect on metastasis, the 4T1-derived Bacillus species significantly enhanced metastatic tumor burden compared to all other groups including those cultured with the 67NR-derived bacteria. These data demonstrate the ability of certain bacteria to promote metastatic disease. Based on these findings, we hypothesize specific bacteria play a causative role in augmenting metastatic propensity, and seek to determine functional differences between intratumoral bacteria to identify mechanistic targets for prevention of metastasis. We also seek to expand this work into clinical models to identify potential prognostic factors as well as mechanistic targets for disease treatment. Citation Format: Zachary J. Gerbec, Antonio Serapio-Palacios, Sarah E. Woodword, Jorge Pena Diaz, Brett Finlay, Shoukat Dedhar. Tumor-derived bacteria drive breast cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A047.

CECR2 drives breast cancer metastasis by promoting NF-κB signaling and macrophage-mediated immune suppression.

Metastasis is the major cause of cancer-related deaths due to the lack of effective therapies. Emerging evidence suggests that certain epigenetic and transcriptional regulators drive cancer metastasis and could be targeted for metastasis treatment. To identify epigenetic regulators of breast cancer metastasis, we profiled the transcriptomes of matched pairs of primary breast tumors and metastases from human patients. We found that distant metastases are more immune inert with increased M2 macrophages compared to their matched primary tumors. The acetyl-lysine reader, cat eye syndrome chromosome region candidate 2 (CECR2), was the top up-regulated epigenetic regulator in metastases associated with an increased abundance of M2 macrophages and worse metastasis-free survival. CECR2 was required for breast cancer metastasis in multiple mouse models, with more profound effect in the immunocompetent setting. Mechanistically, the nuclear factor κB (NF-κB) family member v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA) recruits CECR2 to increase chromatin accessibility and activate the expression of their target genes. These target genes include multiple metastasis-promoting genes, such as TNC, MMP2, and VEGFA, and cytokine genes CSF1 and CXCL1, which are critical for immunosuppression at metastatic sites. Consistent with these results, pharmacological inhibition of CECR2 bromodomain impeded NF-κB-mediated immune suppression by macrophages and inhibited breast cancer metastasis. These results reveal that targeting CECR2 may be a strategy to treat metastatic breast cancer.

Abstract PS16-07: Blocking H3K27me3 methyltransferase or demethylase activity impacts epithelial-mesenchymal plasticity, suppressing tumor growth and metastasis

Abstract Background: Epithelial-mesenchymal plasticity (EMP) is key feature driving breast cancer metastasis. Induction of epithelial-mesenchymal transition (EMT) increases the migratory and invasive capabilities associated with metastatic competence and endows tumors with stem cell properties necessary for initiating tumor growth in the metastatic site. Mesenchymal-epithelial transition (MET) has been increasingly recognized as an integral part of the latter stages of the metastatic cascade as it is required for re-epithelialization, proliferation, and expansion of micro-metastases into macro-metastases with a histopathology similar to that of the primary tumor. Interconversion between epithelial and mesenchymal states requires remodeling of the epigenome–including addition and erasure of H3K27me3, especially near gene promoter and enhancers. Deposited by EZH1 or EZH2, members of the PRC2 complex, H3K27me3 is associated with gene silencing. However, gene activation occurs upon H3K27me3 demethylation to H3K27me1, mediated by KDM6A or KDM6B, members of the Compass-like complex. Results: Based upon our prior work, which showed EMT-driven re-distribution of H3K27me3 onto and off of genes that are critical regulators of cell fate and differentiation, we asked whether inhibition of H3K27me3-directed methyltransferases and demethylases could impact breast cancer. We found that the EZH2 inhibitor, EPZ011989, reduced EMP by preventing recovery of epithelial trails in cells exposed to TGFβ, but did not, on its own, induce EMT. However, we found that either inhibition of the H3K27me3-directed demethylases, KDM6A and KDM6B, using GSK-J4 or shRNA knockdown, induced EMT and promoted cellular migration. Thus, manipulation of either EZH2 or KDM6A/B affected the capacity of cells to repress or activate expression of genes known to exhibit dynamic gain or loss of H3K27me3 during EMT. Given the effects of inhibitors of H3K27-targeted enzymes on EMP, we sought to ascertain their impact on tumor growth and metastasis. Using a GFP-expressing patient-derived xenograft (PDX) model of ER-positive breast cancer, we observed a dose-dependent suppression of primary tumor growth and fewer disseminated tumor cells (DTCs) in response to small molecules targeting H3K27me3 regulators. Conclusions: These results indicate that addition and removal of H3K27me3 is critical for maintenance of cell fate and plasticity. Furthermore, targeting this pathway suppresses breast cancer growth and metastasis in a mouse model of ER-positive breast cancer. Citation Format: Joseph Taube, Provas Das, Kelsey Johnson, John Landua, Lacey Dobrolecki, Michael Lewis. Blocking H3K27me3 methyltransferase or demethylase activity impacts epithelial-mesenchymal plasticity, suppressing tumor growth and metastasis [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 PS16-07.

Opioids drive breast cancer metastasis through the δ-opioid receptor and oncogenic STAT3

The opioid crisis of pain medication bears risks from addiction to cancer progression, but little experimental evidence exists. Expression of δ-opioid receptors (DORs) correlates with poor prognosis for breast cancer patients, but mechanistic insights into oncogenic signaling mechanisms of opioid-triggered cancer progression are lacking. We show that orthotopic transplant models using human or murine breast cancer cells displayed enhanced metastasis upon opioid-induced DOR stimulation. Interestingly, opioid-exposed breast cancer cells showed enhanced migration and strong STAT3 activation, which was efficiently blocked by a DOR-antagonist. Furthermore, opioid treatment resulted in down-regulation of E-Cadherin and increased expression of epithelial-mesenchymal transition markers. Notably, STAT3 knockdown or upstream inhibition through the JAK1/2 kinase inhibitor ruxolitinib prevented opioid-induced breast cancer cell metastasis and migration in vitro and in vivo. We conclude on a novel mechanism whereby opioid-triggered breast cancer metastasis occurs via oncogenic JAK1/2-STAT3 signaling to promote epithelial-mesenchymal transition. These findings emphasize the importance of selective and restricted opioid use, as well as the need for safer pain medication that does not activate these oncogenic pathways.

Mechanically stimulated osteocytes promote the proliferation and migration of breast cancer cells via a potential CXCL1/2 mechanism

Bone represents the most common site for breast cancer metastasis. Bone is a highly dynamic organ that is constantly adapting to its biophysical environment, orchestrated largely by the resident osteocyte network. Osteocytes subjected to physiologically relevant biophysical conditions may therefore represent a source of key factors mediating breast cancer cell metastasis to bone. Therefore, we investigated the potential proliferative and migratory capacity of soluble factors released by mechanically stimulated osteocytes on breast cancer cell behaviour. Interestingly the secretome of mechanically stimulated osteocytes enhanced both the proliferation and migration of cancer cells when compared to the secretome of statically cultured osteocytes, demonstrating that mechanical stimuli is an important physiological stimulus that should be considered when identifying potential targets. Using a cytokine array, we further identified a group of mechanically activated cytokines in the osteocyte secretome, which potentially drive breast cancer metastasis. In particular, CXCL1 and CXCL2 cytokines are highly expressed, mechanically regulated, and are known to interact with one another. Lastly, we demonstrate that these specific factors enhance breast cancer cell migration independently and in a synergistic manner, identifying potential osteocyte derived factors mediating breast cancer metastasis to bone.

Decellularized extracellular matrix scaffolds identify full-length collagen VI as a driver of breast cancer cell invasion in obesity and metastasis.

The extracellular matrix (ECM), a major component of the tumor microenvironment, promotes local invasion to drive metastasis. Here, we describe a method to study whole-tissue ECM effects from disease states associated with metastasis on tumor cell phenotypes and identify the individual ECM proteins and signaling pathways that are driving these effects. We show that decellularized ECM from tumor-bearing and obese mammary glands drives TNBC cell invasion. Proteomics of the ECM from the obese mammary gland led us to identify full-length collagen VI as a novel driver of TNBC cell invasion whose abundance in tumor stroma increases with body mass index in human TNBC patients. Last, we describe the mechanism by which collagen VI contributes to TNBC cell invasion via NG2-EGFR cross-talk and MAPK signaling. Overall, these studies demonstrate the value of decellularized ECM scaffolds obtained from tissues to identify novel functions of the ECM.

LINC02273 drives breast cancer metastasis by epigenetically increasing AGR2 transcription

BackgroundThe majority of breast cancer patients die of metastasis rather than primary tumors, whereas the molecular mechanisms orchestrating cancer metastasis remains poorly understood. Long noncoding RNAs (lncRNA) have been shown to regulate cancer occurrence and progression. However, the lncRNAs that drive metastasis in cancer patients and their underlying mechanisms are still largely unknown.MethodslncRNAs highly expressed in metastatic lymph nodes were identified by microarray. Survival analysis were made by Kaplan-Meier method. Cell proliferation, migration, and invasion assay was performed to confirm the phenotype of LINC02273. Tail vein model and mammary fat pad model were used for in vivo study. RNA pull-down and RIP assay were used to confirm the interaction of hnRNPL and LINC02273. Chromatin isolation by RNA purification followed by sequencing (ChIRP-seq), RNA-seq, ChIP-seq, and luciferase reporter assay reveal hnRNPL-LINC02273 regulates AGR2. Antisense oligonucleotides were used for in vivo treatment.ResultsWe identified a novel long noncoding RNA LINC02273, whose expression was significantly elevated in metastatic lesions compared to the primary tumors, by genetic screen of matched tumor samples. Increased LINC02273 promoted breast cancer metastasis in vitro and in vivo. We further showed that LINC02273 was stabilized by hnRNPL, a protein increased in metastatic lesions, in breast cancer cells. Mechanistically, hnRNPL-LINC02273 formed a complex which activated AGR2 transcription and promoted cancer metastasis. The recruitment of hnRNPL-LINC02273 complex to AGR2 promoter region epigenetically upregulated AGR2 by augmenting local H3K4me3 and H3K27ac levels. Combination of AGR2 and LINC02273 was an independent prognostic factor for predicting breast cancer patient survival. Moreover, our data revealed that LINC02273-targeting antisense oligonucleotides (ASO) substantially inhibited breast cancer metastasis in vivo.ConclusionsOur findings uncover a key role of LINC02273-hnRNPL-AGR2 axis in breast cancer metastasis and provide potential novel therapeutic targets for metastatic breast cancer intervention.