Driver Of Breast Cancer Metastasis Research Articles

Abstract LB433: Targeting of ABI1 via DNA-based PROTACs

Abstract Breast cancer is the most commonly diagnosed non-cutaneous cancer in American women. Although current therapies reduce initial tumor burden, patients often relapse with aggressive tumors which can metastasize. Abelson Interactor 1 (ABI1) is an adaptor protein which is traditionally seen as a WAVE complex component- regulating actin polymerization, cell migration and adhesion. In this role, ABI1 serves as a driver of breast cancer metastasis. Recent studies have discovered that ABI1 contains a homeobox homology region (HHR) which is able to bind DNA and regulate transcription. In order to specifically target and degrade ABI1, we designed a novel PROteolysis-TArgeting Chimera (PROTAC) system using DNA as the bait for ABI1. We hypothesized that by linking ABI1 DNA targets to the ubiquitin system, we would be able to effectively target ABI1 for degradation. Lenalidomide, an E3 ubiquitin ligase ligand, was attached to an oligonucleotide via click chemistry. Cells were then treated with PROTAC systems via direct addition to media or via lipofection reagents, and levels of ABI1 were detected using western blotting. Using this DNA-based PROTAC system in cell-culture models of both breast and ABI1-elevated prostate cancer suggested that the system is able to target and degrade ABI1. Usage of lipofection reagents were able to improve drug delivery and effectiveness. Further research is needed to identify off-target effects and effectiveness in vivo models of disease to reduce metastasis. In conclusion, PROTACs targeting ABI1 may prove to be an effective and novel method to reduce metastasis and improve outcomes for cancer treatment. Citation Format: Kevin M. Lin, Xiang Li, Leszek Kotula. Targeting of ABI1 via DNA-based PROTACs [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 LB433.

Abstract B003: Investigating PHLDA2 as a Genetic Driver of Breast Cancer Metastasis

Abstract Metastasis is cancer’s lethal driver. Metastatic cancer cells leave the primary tumor and travel through the blood stream to distal sites. These cancer cells are aggressive, highly recurring and pose challenges for clinical treatment. There is a critical need to define drivers of metastasis in order to identify novel drug targets and improve patient prognosis. Our previous work identified a potential breast cancer metastasis promoting gene known as PHLDA2. By single cell RNA sequencing, PHLDA2 gene expression was elevated in metastatic lesions compared to primary breast tumor tissue. This gene has not previously been linked to cancer metastasis; therefore, the objective of the current study is to confirm PHLDA2 as a driver of breast cancer metastasis and define the mechanisms by which it exerts its pro-metastatic function. We manipulated multiple breast cancer models to have elevated or reduced PHLDA2 levels to investigate its role in metastasis. We have additionally explored the mechanism of PHLDA2 in metastasis at the RNA level by qPCR, the protein level by western blot analysis, and the organ level using immunofluorescence staining and bulk sequencing. We confirmed using our breast cancer models of elevated or reduced PHLDA2 expression that PHLDA2 has a pro-metastatic function. Bulk RNA sequencing data revealed that PHLDA2 promotes extracellular matrix (ECM) remodeling. This finding is significant as ECM remodeling plays a critical role in cancer progression and metastasis. Investigation of PHLDA2’s pro-metastatic function is necessary to gain deeper insights into the mechanisms of cancer cell spread and reveal novel drug targets to improve patient survival. Citation Format: Paige Halas, Hannah Savage, Dennis Ma, Jacob Insua-Rodriguez, Sharmila Mallya, Connie Chan, Radhika Domadia, Tatyana Lev, Devon Lawson. Investigating PHLDA2 as a Genetic Driver of Breast Cancer Metastasis [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 B003.

Abstract 5828: Functional role of long non-coding RNA YTHDF3-AS1 in breast cancer

Abstract Breast cancer (BrCa) is the most common form of cancer in women worldwide, impacting nearly 2.1 million women each year. Importantly, current treatment options for metastatic breast cancer fail to elicit an anti-tumor response, leading to a 5-year survival rate below 30%. While most classes of therapy focus on inhibiting or targeting specific proteins, long non-coding RNAs (lncRNAs) offer a unique, relatively untapped, source of potential therapeutic targets. Ongoing experiments are evaluating the regulatory role of the lncRNA YTHDF3-AS1 (ENSG00000270673), which has the potential to develop into a novel therapeutic intervention of BrCa. Through genome-wide analysis, we found that YTHDF3-AS1 expression correlates with essential developmental regulatory genes such as CA3, VPS28, EXOSC4, and TM2D2 and according to pathway analysis is likely linked with the Wnt pathway. Additionally, TCGA data reveals that over 5% of patients with BrCa have an increased copy number of YTHDF3-AS1. Although the role of YTHDF3-AS1 has yet to be explored, YTHDF3 expression was recently shown to be a critical driver of breast cancer metastasis. Of note, YTHDF3-AS1 is upstream of the protein-coding region, but within the promoter region of the YTHDF3 gene. Mechanistically, this allows the lncRNA YTHDF3-AS1 to potentially upregulate the expression of the protein-coding YTHDF3 gene, as two known functions of antisense lncRNAs describe the ability to 1) induce nearby gene expression through binding to the promoter region and 2) stabilize the RNA product of the nearby gene through binding to the 5’UTR. Interestingly, this manner of relationship between antisense (or "divergent") lncRNAs and their protein-coding partner was shown to be highly prevalent in pluripotent stem cells, with the downregulation of both the antisense and protein-coding transcripts following differentiation. Together, our preliminary analysis and previous literature suggest that the lncRNA YTHDF3-AS1 may contribute to the upregulation of YTHDF3, which is a known metastatic promoting protein. Our current study aims to determine the functional role of YTHDF3-AS1 in BrCa oncogenesis, with an emphasis on the mechanistic actions of this lncRNA for the development of future therapeutic approaches. Citation Format: Alakesh Bera, Surya Radhakrishnan, Eric Russ, Diya Biswas, Madhan Subramanian, Harvey B. Pollard, Hai Hu, Craig D. Shriver, Roopa Biswas, Meera Srivastava. Functional role of long non-coding RNA YTHDF3-AS1 in breast cancer [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 5828.

Filamin A Is a Potential Driver of Breast Cancer Metastasis via Regulation of MMP-1.

Recurrent metastasis is a major fatal cause of breast cancer. Regretfully, the driving force and the molecular beneath have not been fully illustrated yet. In this study, a cohort of breast cancer patients with locoregional metastasis was recruited. For them, we collected the matched samples of the primary tumor and metastatic tumor, and then we determined the mutation profiles with whole-exome sequencing (WES). On basis of the profiles, we identified a list of deleterious variants in eight susceptible genes. Of them, filamin A (FLNA) was considered a potential driver gene of metastasis, and its low expression could enhance 5 years’ relapse survival rate by 15%. To prove the finding, we constructed a stable FLNA knockout tumor cell line, which manifested that the cell abilities of proliferation, migration, and invasion were significantly weakened in response to the gene knockout. Subsequently, xenograft mouse experiments further proved that FLNA knockout could inhibit local or distal metastasis. Putting all the results together, we consolidated that FLNA could be a potential driver gene to metastasis of breast cancer, in particular triple-negative breast cancer. Additional experiments also suggested that FLNA might intervene in metastasis via the regulation of MMP-1 expression. In summary, this study demonstrates that FLNA may play as a positive regulator in cancer proliferation and recurrence. It provides new insight into breast cancer metastasis and suggests a potential new therapeutic target for breast cancer therapy.

Abstract 5172: A functional genomic screen in vivo identifies CEACAM5 as a clinically relevant driver of breast cancer metastasis

Abstract Metastases are responsible for the vast majority of deaths due to breast cancer. Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by high rates of metastasis and poor prognosis. We are employing patient-derived xenograft (PDX) models of TNBC to identify drivers of metastasis. Tumor samples are obtained from the breast tumors of patients with TNBC and engrafted immediately into the humanized mammary fat pads of immune-compromised mice. Lentiviral transduction was employed to express bioluminescent and fluorescent markers in two independent PDX models of TNBC. Using these models, we demonstrated that human breast tumors are capable of completing all stages of the metastatic cascade in mice, and metastatic lesions are observed in organs normally found in patients with metastatic breast cancer including lung, liver, bone, brain, and lymph nodes. Dynamic and reversible shifts in the epithelial-to-mesenchymal transition (EMT) were observed as tumors metastasized to lung and were re-passaged to recipient mouse mammary fat pads (MFPs). Lung metastases were isolated using bioluminescence imaging and lung metastasis gene expression signatures were generated. Metastasis signatures from two independent PDX models were compared to identify genes that were commonly deregulated in lung metastases relative to corresponding mammary tumors. Comprehensive gain-of-function screens were then conducted in vivo to identify functional drivers of TNBC metastasis. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) was identified as a metastatic driver in this screen. CEACAM5 mRNA and protein levels were elevated in lung metastases relative to corresponding MFP tumors in mice. In addition, we demonstrated that CEACAM5 expression was upregulated in the lung metastases of breast cancer patients, and its expression inversely correlated with patient survival. Our data indicate that the metastatic function of CEACAM5 is to promote growth of breast tumors in the lung by inducing MET (mesenchymal-to-epithelial transition). Citation Format: Emily Powell, Jiansu Shao, Hector Picon, Christopher Bristow, Zhongqi Ge, Caitlin Grzeskowiak, Michael Peoples, Frederick Robinson, Sabrina Jeter-Jones, Christopher Schlosberg, Fei Yang, Yun Wu, Ignacio Wistuba, Stacy Moulder, Fraser Symmans, Kenneth Scott, John Edwards, Han Liang, Timothy Heffernan, Helen Piwnica-Worms. A functional genomic screen in vivo identifies CEACAM5 as a clinically relevant driver of breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5172.

A functional genomic screen in vivo identifies CEACAM5 as a clinically relevant driver of breast cancer metastasis

Tumor cells disseminate early in tumor development making metastasis-prevention strategies difficult. Identifying proteins that promote the outgrowth of disseminated tumor cells may provide opportunities for novel therapeutic strategies. Despite multiple studies demonstrating that the mesenchymal-to-epithelial transition (MET) is critical for metastatic colonization, key regulators that initiate this transition remain unknown. We serially passaged lung metastases from a primary triple negative breast cancer xenograft to the mammary fat pads of recipient mice to enrich for gene expression changes that drive metastasis. An unbiased transcriptomic signature of potential metastatic drivers was generated, and a high throughput gain-of-function screen was performed in vivo to validate candidates. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) was identified as a metastatic driver. CEACAM5 overproduction enriched for an epithelial gene expression pattern and facilitated tumor outgrowth at metastatic sites. Tissues from patients with metastatic breast cancer confirmed elevated levels of CEACAM5 in lung metastases relative to breast tumors, and an inverse correlation between CEACAM5 and the mesenchymal marker vimentin was demonstrated. Thus, CEACAM5 facilitates tumor outgrowth at metastatic sites by promoting MET, warranting its investigation as a therapeutic target and biomarker of aggressiveness in breast cancer.

Lysyl Oxidase-like Protein LOXL2 Promotes Lung Metastasis of Breast Cancer.

The lysyl oxidase-like protein LOXL2 has been suggested to contribute to tumor progression and metastasis, but in vivo evidence has been lacking. Here we provide functional evidence that LOXL2 is a key driver of breast cancer metastasis in two conditional transgenic mouse models of PyMT-induced breast cancer. LOXL2 ablation in mammary tumor cells dramatically decreased lung metastasis, whereas LOXL2 overexpression promoted metastatic tumor growth. LOXL2 depletion or overexpression in tumor cells does not affect extracellular matrix stiffness or organization in primary and metastatic tumors, implying a function for LOXL2 independent of its conventional role in extracellular matrix remodeling. In support of this likelihood, cellular and molecular analyses revealed an association of LOXL2 action with elevated levels of the EMT regulatory transcription factor Snail1 and expression of several cytokines that promote premetastatic niche formation. Taken together, our findings established a pathophysiologic role and new function for LOXL2 in breast cancer metastasis. Cancer Res; 77(21); 5846-59. ©2017 AACR.

Abstract 5314: An oncogenic splice variant of the KLF6 tumor suppressor gene is associated with poor survival and is a potent driver of breast cancer metastasis

Abstract Breast cancer is a leading causes of cancer death in women worldwide. It is a heterogeneous and genetically complex disease has left a significant proportion of patients with inadequate treatment options. Since metastasis and drug resistance pose significant challengea in breast cancer treatment, elucidating the mechanisms of these processes is critical for understanding the key drivers of disease progression and for the development of targeted therapies. Accumulating evidence from our laboratory and other groups suggest that the tumor suppressor gene, KLF6, and its oncogenic splice variant, KLF6-SV1, play a role in breast cancer progression, dissemination, and chemoresistance. Here we demonstrate that in multiple independent clinical cohorts of over 1200 breast cancer patients with defined clinical outcome, high KLF6-SV1 mRNA levels in the primary tumor associated with poor survival and disease recurrence. Specifically, upregulated KLF6-SV1 expression in the primary tumors correlated with poor survival independent of disease stage and grade. Thus, we hypothesized that KLF6-SV1 is an early driver/molecular determinant of invasive breast cancer. In order to investigate the functional/biological relevance of KLF6-SV1 in breast cancer development and progression, we performed a series of experiments using retroviral-based overexpression in multiple non-tumorigenic, tumorigenic, and metastatic breast cancer cell lines. We demonstrated that overexpression of KLF6-SV1 increased mesenchymal marker gene expression, cellular survival, invasion, as well as the migratory potential of KLF6-SV1 transduced cells. Interestingly, KLF6-SV1 did not increase growth rate of these mammary epithelial cell lines, similar to the clinical data which showed an absence of a correlation between KLF6-SV1 expression and primary tumor size. In a 3D model KLF6-SV1 upregulation disrupted mammary acinar morphogenesis promoting complex multiacinar structures. To assay whether the KLF6-SV1-induced EMT phenotype conferred increased metastatic potential in vivo, we injected tumorigenic cells expressing high levels of KLF6-SV1 subcutaneously into immunodeficient mice. Strikingly, KLF6-SV1 overexpression alone drove the entire metastatic cascade resulting in dissemination to many organs including the liver, kidney, heart, lung, and spleen. Furthermore, KLF6-SV1 overexpression in a metastatic breast cancer cell line increased metastasis to the lungs and liver in an orthotopic model of the disease. This is consistent with our clinical data in which high KLF6-SV1 expression was correlated with decreased overall survival and metastasis free survival. Together these findings suggest a role for the KLF6-SV1 splice variant as a driver of breast cancer metastasis and validate its potential utility as a novel biomarker and therapeutic target for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5314. doi:1538-7445.AM2012-5314

Abstract 5193: p38 gamma is a novel driver of breast cancer metastasis by modulating cellular motility

Abstract Breast cancer is the second leading cause of cancer-related deaths in women in the US each year. The vast majority of these fatalities are not caused by primary tumor burden but rather by metastases to vital organs. The clinical shift from localized to metastatic breast cancer entails a requirement that cancer cells activate an invasive program and be able to adapt to changing extracellular stimuli. The p38 mitogen activated protein kinase (MAPK) pathway represents a potential signaling switch for the transition from primary to metastatic cancer. p38 is a member of the MAPK family of stress and mitogen-responsive protein kinases and consists of four closely related isoforms: alpha, beta, gamma, and delta. p38 serves as a major signaling hub in the cell, integrating signals from a variety of signaling pathways and channeling these stimuli into cellular responses through an array of effector proteins. The four isoforms show unique expression patterns in normal tissue: alpha, beta, and delta are the most ubiquitously expressed, while gamma is restricted primarily to skeletal muscle. Surprisingly, we found high levels of phosphorylated p38 gamma in a myoepithelial-derived aggressive breast cancer cell line, MDA-MB-231, compared to the non-tumorigenic mammary epithelial cell line MCF-10A, despite both cell lines having similar levels of total p38 gamma. This led us to hypothesize that, based on its normal function in skeletal muscle, p38 gamma may play a role in mediating metastatic behavior in basally-derived breast cancers. To test this hypothesis, we used isoform-specific shRNA to knock down p38 gamma expression in MDA-MB-231. Inhibition of p38 gamma (referred to as shGamma) resulted in decreased cell motility and invasion, two hallmarks of cancer metastasis. Analysis of individual cells revealed a change in the mode of motility from mesenchymal-like in control cells to lamellipodia-driven motility in the shGamma cells. This altered motility appears to be the result of a dramatically modified actin cytoskeleton, characterized by loss of lengthwise stress fibers and bundled, rather than branched, actin in the lamellipodia. When we investigated gene expression changes responsible for this phenotype we discovered that expression of the metastatic oncogene RhoC GTPase is significantly reduced at the protein, but not mRNA, level in shGamma cells due to decreased RhoC protein stability. These findings have great potential clinical significance, as early evidence indicates that p38 gamma is overexpressed in cases of invasive breast cancer, and appears to correlate with RhoC expression in these samples. Our current and future work includes determining which breast cancer subtypes utilize p38 gamma for invasion and metastasis, and characterizing in more detail the dynamic actin cytoskeletal changes affected by p38 gamma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5193.