Growth Of Breast Cancer Metastases Research Articles

Abstract 2190: Brain-seeking extracellular vesicles derived from metastatic breast cancer cells modulate brain endothelial cell metabolism

Abstract Breast cancer, particularly the triple-negative subtype, is the primary cause of brain metastasis in women and affects ~10-16% of breast cancer patients. Breast cancer patients diagnosed with brain metastasis have limited therapeutic options and, therefore, a dismal prognosis with a median survival time of ~10 months. Recent studies have suggested that metastatic cells extravasate and grow along the blood-brain barrier (BBB) in a favorable microenvironment called the pre-metastatic niche. However, the early steps leading to the onset of breast-to-brain metastasis are poorly understood, limiting the development of strategies to prevent, detect and treat premetastatic or early-stage lesions. It is now well accepted that extracellular vesicles (EVs), nanoparticles secreted from cancer cells, can travel to distant organs, activate resident cells and promote the remodeling of the extracellular matrix and significantly impact organ-specific metastatic spread. Using our mouse model of breast to brain metastasis, we have demonstrated, for the first time, that EVs derived from brain-seeking breast cancer cells (Br-EVs) breach the intact BBB via transcytosis, are taken up by astrocytes and cause a significantly increased incidence of brain metastasis. Within this context, we are studying the interactions between Br-EVs and brain endothelial cells (ECs), the latter being a key functional component of the BBB. Utilizing primary human ECs and using a transwell assay established in our laboratory as an in vitro model of the BBB, we have identified the mechanism(s) through which Br-EVs modulate EC endocytic metabolism in favor of increased vesicle recycling and transcytosis. Our mechanistic studies demonstrate that ECs treated with Br-EVs show a significant decrease of multiple target proteins, all of which are involved in the long-recycling loop of the endocytic vesicles. Br-EVs mediate, at least in part, their effect through the delivery to the target cells of a specific subset of micro RNAs. As a result, Br-EVs upregulate EC basal transcytosis rate promoting their transfer to the brain parenchyma. These findings indicate that Br-EVs can alter BBB basal metabolism and help to prepare a microenvironment, better known as the pre-metastatic niche, that promotes and sustains the survival and growth of breast cancer metastases in the otherwise hostile brain microenvironment. These results increase our understanding of the early events that facilitate breast-to-brain metastasis and have the potential to identify key regulators in the early steps of brain metastasis formation. Our mechanistic studies will also inform and contribute to the development of EV-based diagnostic and therapeutic strategies for the early management of breast cancer patients with brain metastasis. (This work was supported by NIH R21 CA253051-01 and the Breast Cancer Research Foundation). Citation Format: Sara Busatto, Golnaz Morad, Marsha Moses. Brain-seeking extracellular vesicles derived from metastatic breast cancer cells modulate brain endothelial cell metabolism [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 2190.

Suppression of breast cancer metastasis and extension of survival by a new antiestrogen in a preclinical model driven by mutant estrogen receptors.

Many human breast tumors become resistant to endocrine therapies and recur due to estrogen receptor (ERα) mutations that convey constitutive activity and a more aggressive phenotype. Here, we examined the effectiveness of a novel adamantyl antiestrogen, K-07, in suppressing the growth of breast cancer metastases containing the two most frequent ER-activating mutations, Y537S and D538G, and in extending survival in a preclinical metastatic cancer model. MCF7 breast cancer cells expressing luciferase and Y537S or D538G ER were injected into NOD-SCID-gamma female mice, and animals were treated orally with the antiestrogen K-07 or control vehicle. Comparisons were also made with the antiestrogen Fulvestrant. The development of metastases was monitored by in vivo bioluminescence imaging with phenotypic characterization of the metastases in liver and lung by immunohistochemical and biochemical analyses. These breast cancer cells established metastases in liver and lung, and K-07 treatment reduced the metastatic burden. Mice treated with K-07 also survived much longer. By day 70, only 28% of vehicle-treated mice with mutant ER metastases were alive, whereas all K-07-treated D538G and Y537S mice were still alive. K-07 also markedly reduced the level of metastatic cell ER and the expression of ER-regulated genes. The antiestrogen K-07 can reduce in vivo metastasis of breast cancers and extend host survival in this preclinical model driven by constitutively active mutant ERs, suggesting that this compound may be suitable for further translational examination of its efficacy in suppression of metastasis in breast cancers containing constitutively active mutant ERs.

Separation of breast cancer and organ microenvironment transcriptomes in metastases

BackgroundThe seed and soil hypothesis was proposed over a century ago to describe why cancer cells (seeds) grow in certain organs (soil). Since then, the genetic properties that define the cancer cells have been heavily investigated; however, genomic mediators within the organ microenvironment that mediate successful metastatic growth are less understood. These studies sought to identify cancer- and organ-specific genomic programs that mediate metastasis.MethodsIn these studies, a set of 14 human breast cancer patient-derived xenograft (PDX) metastasis models was developed and then tested for metastatic tropism with two approaches: spontaneous metastases from mammary tumors and intravenous injection of PDX cells. The transcriptomes of the cancer cells when growing as tumors or metastases were separated from the transcriptomes of the microenvironment via species-specific separation of the genomes. Drug treatment of PDX spheroids was performed to determine if genes activated in metastases may identify targetable mediators of viability.ResultsThe experimental approaches that generated metastases in PDX models were identified. RNA sequencing of 134 tumors, metastases, and normal non-metastatic organs identified cancer- and organ-specific genomic properties that mediated metastasis. A common genomic response of the liver microenvironment was found to occur in reaction to the invading PDX cells. Genes within the cancer cells were found to be either transiently regulated by the microenvironment or permanently altered due to clonal selection of metastatic sublines. Gene Set Enrichment Analyses identified more than 400 gene signatures that were commonly activated in metastases across basal-like PDXs. A Src signaling signature was found to be extensively upregulated in metastases, and Src inhibitors were found to be cytotoxic to PDX spheroids.ConclusionsThese studies identified that during the growth of breast cancer metastases, there were genomic changes that occurred within both the cancer cells and the organ microenvironment. We hypothesize that pathways upregulated in metastases are mediators of viability and that simultaneously targeting changes within different cancer cell pathways and/or different tissue compartments may be needed for inhibition of disease progression.

Abstract 946: Suppression of breast cancer metastasis and extension of host animal survival by a new adamantyl antiestrogen, K-07, in a preclinical breast cancer metastasis model driven by constitutively active mutant estrogen receptors

Abstract Over one-third of human breast tumors that become resistant to endocrine therapies have acquired mutations in the estrogen receptor (ER alpha) that result in constitutive receptor activity in the absence of estrogen, and these tumors are much less sensitive to suppression by current standard-of-care antiestrogens such as tamoxifen and fulvestrant. We reported recently that the novel adamantyl antiestrogen, K-07, was effective in inhibiting the proliferation of breast tumor xenografts containing the two most frequent constitutively active mutant forms of the ER, Y537S ER and D538G ER, and that this antiestrogen reduced ER levels and the expression of ER-regulated genes (Zhao et al., Cancer Research 77(20):5602-5613, 2017). Because mutant ERs are usually enriched in recurrent, metastatic breast cancers compared to the primary breast cancer, we have now examined the effectiveness of compound K-07 in preventing the growth of breast cancer metastases and in extending survival in a metastatic tumor model. MCF-7 breast cancer cells expressing luciferase and Y537S ER or D538G ER (ca. 50% mutant ER and 50% wild type ER) were injected i.v. by the tail vein into NOD-SCID-gamma (NSG) female mice. The constitutively active ER-containing breast cancer cells established metastases in liver, bone and brain that increased in number and size over time (day zero – 80 days) as monitored by IVIS imaging and immunohistochemical (IHC) analysis. Daily oral treatment with K-07 (80 mg/kg orally for 30 days and then 40 mg/kg) versus oral vehicle greatly reduced the metastasis of mutant ER-containing breast cancer cells. Notably, mice with mutant ER-containing metastases treated with K-07 survived much longer than mice given daily control vehicle. In fact, by day 60, only 25% of vehicle treated mice with mutant ER breast cancers were alive whereas all K-07 treated D538G mice and 80% of Y537S mice were still alive. Hence, the findings indicate that this antiestrogen can reduce the in vivo metastasis of breast cancers driven by constitutively active mutant ERs and extend host survival. The findings suggest that the antiestrogen K-07 may be suitable for further translational and clinical examination of its efficacy in suppression of metastasis in patients with breast cancers containing constitutively active mutant ERs. Citation Format: Mary J. Laws, Sung Hoon Kim, Jian Min, Yuechao Zhao, Yvonne Ziegler, David Chu, Ben H. Park, John A. Katzenellenbogen, Benita S. Katzenellenbogen. Suppression of breast cancer metastasis and extension of host animal survival by a new adamantyl antiestrogen, K-07, in a preclinical breast cancer metastasis model driven by constitutively active mutant estrogen receptors [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 946.

If You Don’t Look, You Won’t See: Intravital Multiphoton Imaging of Primary and Metastatic Breast Cancer

A fundamental hallmark of cancer is progression to metastasis and the growth of breast cancer metastases in lung, bone, liver and/or brain causes fatal complications. Unfortunately, the cellular and biochemical mechanisms of the metastatic process remain ill-defined. Recent application of intravital multiphoton microscopy (MP-IVM) to image fluorescently labeled cells in mouse models of cancer has allowed dynamic observation of this multi-step process at the cellular and subcellular levels. In this article, we discuss the use of MP-IVM in studies of breast cancer metastasis, as well as surgical techniques for exposing tumors prior to imaging. We also describe a versatile multiphoton microscope for imaging tumor-stroma interactions.

Angiopoietin-2, an Angiogenic Regulator, Promotes Initial Growth and Survival of Breast Cancer Metastases to the Lung through the Integrin-linked Kinase (ILK)-AKT-B Cell Lymphoma 2 (Bcl-2) Pathway

The early onsets of breast cancer metastasis involve cell retention, survival, and resistant to apoptosis and subsequent growth at target vascular beds and tissues in distant organs. We previously reported that angiopoietin-2 (Ang2), an angiogenic regulator stimulates MCF-7 breast tumor metastasis from their orthotopic sites to distant organs through the α(5)β(1) integrin/integrin-linked kinase (ILK)/Akt pathway. Here, by using an experimental tumor metastasis model and in vitro studies, we further dissect the underlying mechanism by which Ang2 promotes the initial growth and survival of MCF-7 breast cancer metastasis in the lung of animals. We show that Ang2 increases cell survival and suppresses cell apoptosis through ILK-induced phosphorylation of Akt1, Akt2, and up-regulation of Bcl-2 in breast cancer cells. Inhibition of ILK, Akt1, and Akt2, and their effector Bcl-2 diminishes Ang2-stimulated breast cancer cell survival and Ang2-attenuated apoptosis in vitro, and initial survival and growth of breast cancer metastasis in the lung of animals. Additionally, siRNA knockdown of endogenous Ang2 in three human metastatic breast cancer cell lines also inhibits phosphorylation of Akt, expression of Bcl-2, and tumor cell survival, migration, and increases cell apoptosis. Since increased expression of Ang2 correlates with elevated potential of human breast cancer metastasis in clinic, our data underscore the importance that up-regulated Ang2 not only stimulates breast cancer growth and metastasis at late stages of the process, but is also critical at the initiating stages of metastases onset, thereby suggesting Ang2 as a promising therapeutic target for treating patients with metastatic breast cancer.

Parathyroid Hormone–Related Protein Protects against Mammary Tumor Emergence and Is Associated with Monocyte Infiltration in Ductal Carcinoma In situ

Parathyroid hormone-related protein (PTHrP) is required for mammary gland development and promotes the growth of breast cancer metastases within bone. However, there are conflicting reports of the prognostic significance of its expression in primary breast cancers. To study the role of PTHrP in early breast cancer, the effect of conditional deletion of PTHrP was examined in the context of neu-induced mammary tumorigenesis. Loss of PTHrP resulted in a higher tumor incidence. Transcriptional profiling of the tumors revealed that PTHrP influenced genes relevant to heterotypic cell signaling, including regulators of monocyte recruitment. Immunohistochemical analysis of human breast cancers revealed that PTHrP expression was associated with both HER-2/neu expression and macrophage infiltration in preinvasive ductal carcinoma in situ. The gene expression signature associated with loss of PTHrP expression in vivo correlated with poorer outcome in human breast cancer. Together, these data indicate that loss of PTHrP accelerates mammary tumorigenesis possibly by a non-cell-autonomous tumor suppressor pathway.

Death receptor 5 agonist TRA8 in combination with the bisphosphonate zoledronic acid attenuated the growth of breast cancer metastasis

Bone metastasis affects the majority of patients with advanced breast cancer and no adequate therapy exists. Bisphosphonates, like zoledronic acid, inhibit the osteolytic component of tumor growth in osseous tissues, but these drugs are not curative. The current study evaluated the combination of zoledronic acid with death receptor 5 agonists in an animal model of breast cancer bone metastasis. Materials and Methods. Female athymic nude mice (age 4-6 weeks, n=35) were inoculated with 200,000 luciferase-positive MDA- MB-435 cells by injection into the left ventricle. Animals were immediately imaged by bioluminescence technique and placed into one of the following therapy groups: Saline, hTRA8, hTRA8 + zoledronic acid, mTRA8, mTRA8 + zoledronic Acid, or zoledronic acid monotherapy. DR5 agonists were given at 200 µg/dose and zoledronic acid 5 µg /dose, with mice treated biweekly for 4.5 weeks and imaged weekly. Results. Combination therapy containing either hTRA8 or mTRA8 with zoledronic acid significantly reduced the number of secondary lesions (7.67+2.2 and 7.5+1.7 lesions/mouse, respectively) compared to saline treated controls (12.1+/-1.56 lesions/mouse) as assessed by bioluminescence imaging (p