Obesity rates are climbing, representing a confounding and contributing factor to many disease states, including cancer. With respect to breast cancer, obesity plays a prominent role in the etiology of this disease, with certain subtypes such as triple-negative breast cancer having a strong correlation between obesity and poor outcomes. Therefore, it is critical to examine the obesity-related alterations to the normal stroma and the tumor microenvironment (TME). Adipocytes and adipose stem cells (ASCs) are major components of breast tissue stroma that have essential functions in both physiological and pathological states, including energy storage and metabolic homeostasis, physical support of breast epithelial cells, and directing inflammatory and wound healing responses through secreted factors. However, these processes can become dysregulated in both metabolic disorders, such as obesity and also in the context of breast cancer. Given the well-established obesity-neoplasia axis, it is critical to understand how interactions between different cell types in the tumor microenvironment, including adipocytes and ASCs, govern carcinogenesis, tumorigenesis, and ultimately metastasis. ASCs and adipocytes have multifactorial roles in cancer progression; however, due to the plastic nature of these cells, they also have a role in regenerative medicine, making them promising tools for tissue engineering. At the physiological level, the interactions between obesity and breast cancer have been examined; here, we will delineate the mechanisms that regulate ASCs and adipocytes in these different contexts through interactions between cancer cells, immune cells, and other cell types present in the tumor microenvironment. We will define the current state of understanding of how adipocytes and ASCs contribute to tumor progression through their role in the tumor microenvironment and how this is altered in the context of obesity. We will also introduce recent developments in utilizing adipocytes and ASCs in novel approaches to breast reconstruction and regenerative medicine.

Metaplastic breast carcinoma (MBC) is a rare breast cancer subtype with rapid growth, high rates of metastasis, recurrence and drug resistance, and diverse molecular and histological heterogeneity. Patient-derived xenografts (PDXs) provide a translational tool and physiologically relevant system to evaluate tumor biology of rare subtypes. Here, we provide an in-depth comprehensive characterization of a new PDX model for MBC, TU-BcX-4IC. TU-BcX-4IC is a clinically aggressive tumor exhibiting rapid growth in vivo, spontaneous metastases, and elevated levels of cell-free DNA and circulating tumor cell DNA. Relative chemosensitivity of primary cells derived from TU-BcX-4IC was performed using the National Cancer Institute (NCI) oncology drug set, crystal violet staining, and cytotoxic live/dead immunofluorescence stains in adherent and organoid culture conditions. We employed novel spheroid/organoid incubation methods (Pu·MA system) to demonstrate that TU-BcX-4IC is resistant to paclitaxel. An innovative physiologically relevant system using human adipose tissue was used to evaluate presence of cancer stem cell-like populations ex vivo. Tissue decellularization, cryogenic-scanning electron microscopy imaging and rheometry revealed consistent matrix architecture and stiffness were consistent despite serial transplantation. Matrix-associated gene pathways were essentially unchanged with serial passages, as determined by qPCR and RNA sequencing, suggesting utility of decellularized PDXs for in vitro screens. We determined type V collagen to be present throughout all serial passage of TU-BcX-4IC tumor, suggesting it is required for tumor maintenance and is a potential viable target for MBC. In this study we introduce an innovative and translational model system to study cell–matrix interactions in rare cancer types using higher passage PDX tissue.

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Triple-negative breast cancers (TNBCs) tend to become highly invasive early during cancer development. Despite some successes in the initial treatment of patients diagnosed with early-stage localized TNBC, the rate of metastatic recurrence remains high with poor long-term survival outcomes. Here we show that elevated expression of the serine/threonine-kinase, Calcium/Calmodulin (CaM)-dependent protein kinase kinase-2 (CaMKK2), is highly correlated with tumor invasiveness. We determined that genetic disruption of CaMKK2 expression, or inhibition of its activity, disrupted spontaneous metastatic outgrowth from primary tumors in murine xenograft models of TNBC. High-grade serous ovarian cancer (HGSOC), a high-risk, poor-prognosis ovarian cancer subtype, shares many genetic features with TNBC, and importantly, CaMKK2 inhibition effectively blocked metastatic progression in a validated xenograft model of this disease. Probing the mechanistic links between CaMKK2 and metastasis we defined the elements of a new signaling pathway that impacts actin cytoskeletal dynamics in a manner which increases cell migration/invasion and metastasis. Notably, CaMKK2 increases the expression of the phosphodiesterase PDE1A which decreases the cGMP-dependent activity of protein kinase G1 (PKG1). This inhibition of PKG1 results in decreased phosphorylation of Vasodilator-Stimulated Phosphoprotein (VASP), which in its hypophosphorylated state binds to and regulates F-actin assembly to facilitate contraction/cell movement. Together, these data establish a targetable CaMKK2-PDE1A-PKG1-VASP signaling pathway that controls cancer cell motility and metastasis. Further, it credentials CaMKK2 as a therapeutic target that can be exploited in the discovery of agents for use in the neoadjuvant/adjuvant setting to restrict tumor invasiveness in patients diagnosed with early-stage TNBC or localized HGSOC.

Triple-negative breast cancers (TNBCs) tend to become invasive and metastatic at early stages in their development. Despite some treatment successes in early stage localized TNBC, the rate of distant recurrence remains high, and long-term survival outcomes remain poor. In a search for new therapeutic targets for this disease, we observed that elevated expression of the serine/threonine kinase calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) is highly correlated with tumor invasiveness. In validation studies, genetic disruption of CaMKK2 expression or inhibition of its activity with small molecule inhibitors disrupted spontaneous metastatic outgrowth from primary tumors in murine xenograft models of TNBC. High-grade serous ovarian cancer (HGSOC), a high-risk, poor prognosis ovarian cancer subtype, shares many features with TNBC, and CaMKK2 inhibition effectively blocked metastatic progression in a validated xenograft model of this disease. Mechanistically, CaMKK2 increased the expression of the phosphodiesterase PDE1A which hydrolyzed cyclic guanosine monophosphate (cGMP) to decrease the cGMP-dependent activity of protein kinase G1 (PKG1). Inhibition of PKG1 resulted in decreased phosphorylation of vasodilator stimulated phosphoprotein (VASP), which in its hypophosphorylated state binds to and regulates F-actin assembly to facilitate cell movement. Together, these findings establish a targetable CaMKK2-PDE1A-PKG1-VASP signaling pathway that controls cancer cell motility and metastasis by impacting the actin cytoskeleton. Further, it identifies CaMKK2 as a potential therapeutic target that can be exploited to restrict tumor invasiveness in patients diagnosed with early-stage TNBC or localized HGSOC.

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Breast cancer (BC) is the most prevalent type of cancer among women with triple negative breast cancer (TNBC) accounting for 15% of BC. The majority of BCs are hormone sensitive, TNBC, a more aggressive subtype is characterized by its negative expression of ER and PR, and lack of Her2/NEU amplification. Due to its receptor status, TNBC lacks targeted therapy. FK228 (Romidepsin), a class I and II histone deacetylase inhibitor (HDACi) has been approved by the FDA for the treatment of PTCL (peripheral T-cell lymphoma) and is a promising drug for TNBC treatment. FK228 mechanism of action was through cell cycle arrest and apoptosis (1,2). FK228 specifically targets the enzymes HDACs 1 and 2, tumor suppressor and other proteins thereby exerting epigenetic changes on cancer cells resulting in anti-tumor activity. To assess the role of HDACi, TNBC cell lines were treated with FK228 and Panobinostat (LBH, a panHDACi) and analyzed for suppression of cell cycle genes. Similar to PTCL, TNBC cell lines showed an increase in cell cycle arrest genes such as p21 and others subsequent to treatment with both drugs. Using PCR and crystal violet we identified changes in morphology and migration in 2k1, MDA-MB 231 and HS-578t cell lines treated with FK228 under in-vitro conditions. Further studies show a reversal of the epithelial to mesenchymal transition (EMT) in FK228 treated cells; specifically in key EMT genes CDH1 and ZEB2, both directly linked to HDAC1/2 activity. Similar results were replicated in 3D culture mammospheres. We are also further studying the effects of HDACi in vivo using PDXs (patient derived xenografts) in mouse models; the PDX is either injected as cells or implanted directly in the mammary pad. The PDX model is an excellent translational tool used to monitor growth patterns and recurrence, study metastasis and drug response. Thus far, the TU-BcX-2O0 PDX in vivo model demonstrated growth suppression in tumor growth with FK228 treatment. We are also studying FK228 in models TU-BX-4IC, 4M4 and 4QX. Metastasis in the models is monitored by H&E staining lung and liver tissue for further staining and analysis; we are also using IVIS imaging in an in vivo model consisting of GFP/luciferase transfected breast cancer cells MDA-MB-231 and SUM-159 to monitor both the proliferation and spread of disease in mice. Ultimately we aim to characterize the pathways altered by FK228 and the involvement of HDACs in tumorigenesis, metastasis and resistance within TNBC. References: 1.Gisselbrecht, Christian, and David Sibon. “New Perspectives in the Therapeutic Approach of Peripheral T-Cell Lymphoma.” Current Opinion in Oncology, vol. 30, no. 5, 2018, pp. 285-291., doi:10.1097/cco.0000000000000469.2.Yang, L. P. (2011). Romidepsin. Drugs, 71(11), 1469-1480. doi:10.2165/11207170-000000000-00000 Citation Format: Madlin Alzoubi, Khoa Nguyen, Hope Burks, Katherine Hebert, Thomas Cheng, Margarite Matossian, Maryl Wright, Bridgette Collins-Burow, Matthew Burow. The response of histone deacetylase inhibitors in triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-05-07.

Breast cancer is the most prevalent type of cancer among women. Most breast cancers are hormone sensitive, however triple negative breast cancer (TNBC), a more aggressive subtype of breast cancer is characterized by its negative expression of estrogen and progesterone receptors, and lack of Her2/NEU amplification. Because of its receptor status, hormone receptor targeted treatments are ineffective against TNBC making it difficult to treat. FK228, known as Romidepsin, is a histone deacetylase inhibitor (HDACi) that specifically targets the enzymes HDACs 1 and 2, tumor suppressor and other proteins exerting epigenetic changes on tumor cells resulting in anticancer activity. FK228 has not been studied in TNBC, but has been approved by the FDA for the treatment of peripheral T-cell lymphoma (PTCL), where the mechanism of action has demonstrated cell cycle arrest and apoptosis [1,2]. To assess the preliminary role of FK228 in breast cancer, a number of TNBC cell lines were treated with the drug and analyzed for suppression of cell cycle genes. Similar to PTCL, TNBC cell lines showed an increase in cell cycle arrest genes such as p21 and others subsequent to treatment. Moving forward, to recapitulate the tumor microenvironment we have utilized 2D and 3D culture, while PDXs (patient derived xenografts), an excellent translational tool. We first identified changes in morphology and migration in 2k1, MDA-MB 231 and HS-578t cell lines treated with FK228 under in-vitro conditions. Additional molecular studies also show a reversal of the epithelial to mesenchymal transition (EMT) in FK228 treated BC cell lines; specifically in relation to EMT genes CDH1 and ZEB2, both directly connected to HDAC1/2 activity. To further study the effects of FK228, PDXs are implanted into mice to study growth patterns, recurrence, metastatic potential and response to FK228 in different patient tumor models. FK228 showed a drastic suppression in tumorigenesis in model TU-BCX-2O0, prompting the study of several other models in tumorigenesis including TU-BX-4IC, 4M4 and 4QX. Metastasis was also monitored by H&E staining lung and liver tissue for analysis for each model, and using an in vivo model consisting of GFP/luciferase transfected breast cancer cells MDA-MB-231 and SUM-159 to monitor both the proliferation and spread of disease in mice. Ultimately this study aims to characterize the FK228 alteration of pathways involved in tumorigenesis, metastasis and resistance within TNBC. Citation Format: Madlin Alzoubi, Khoa Nguyen, Katherine Hebert, Margarite Matossian, Hope Burks, Bridgette Collins-Burow, Matthew Burow. Potential therapeutic effects of HDACi FK228 on TNBC using various models [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 PS16-11.