Triple-negative breast cancers (TNBC) 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. Furthermore, 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. Significance: CaMKK2 regulates actin cytoskeletal dynamics to promote tumor invasiveness and can be inhibited to suppress metastasis of breast and ovarian cancer, indicating CaMKK2 inhibition as a therapeutic strategy to arrest disease progression.

Alterations in the tumor extracellular environment and matrix stiffness promote tumor progression. Furthermore, correlational studies have identified enrichment of extracellular matrix (ECM) proteins (glycoproteins, collagens) in breast tumors. Despite these findings, there has yet to be an interdisciplinary analysis of both ECM composition and structural architecture in rare breast tumors, such as metaplastic breast cancer. Here, we explored changes in ECM protein expression and architecture in a triple-negative breast cancer (TNBC) metaplastic tumor through SEM, proteomics, and RNA sequencing. SEM revealed that the tumor pore size was larger compared to the control adipose tissue. Oscillating rheometry demonstrated increased ECM stiffness in the tumor compared to the control adipose breast adipose. Proteomic analysis of the metaplastic TNBC tumor showed significant enrichment for ECM proteins, notably glycoproteins compared to the control adipose. Interestingly, these samples showed no observed changes in expression for major fibrillar collagens COL1A1 and COL1A2, and a reduced expression of COL3A1. To determine the impact of less characterized ECMs in metaplastic TNBC, we overexpressed MFAP2 in primary metaplastic breast cancer cells and performed RNA sequencing. MFAP2 overexpression was associated with upregulation of epithelial-to-mesenchymal transition-related genes. Overall, our results establish an extracellular signature and onco-architecture for the metaplastic triple-negative tumor type.

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Individuals with a high body mass index (BMI) have an increased risk of developing many adult cancers, including breast cancer. In triple negative breast cancer (TNBC), a clinically aggressive subtype of breast cancer, increased BMI is associated with more aggressive tumor types and a higher risk of recurrence. The differences in outcomes between obese and non-obese breast cancer patients is a consequence of the complex interplay between social, environmental, and physiological factors that contribute to the etiology of breast cancer. Therefore, understanding the complex signaling events in the obese tumor microenvironment is essential. Adipose stem cells (ASCs) are a component of the breast microenvironment, and are a subset of mesenchymal stem cells. ASCs from obese patients (obASCs) secrete higher levels various cytokines and adipokines that induce a more invasive phenotype in triple negative breast cancer cells compared to ASCs from lean individuals. Leptin, an adipokine that is expressed proportionally to fat mass, has been implicated in many cancers. Increased leptin and leptin receptor expression is associated with worse prognosis. This study seeks to examine the role of leptin signaling in triple negative breast cancer. Previous work in conjunction with a collaborating lab has shown that leptin signaling promotes metastasis and increased expression of epithelial-mesenchymal transition (EMT) markers in triple negative breast cancer. This project expands upon this work through using both patient-derived cell lines and and patient-derived xenografts (PDX), and examines the role of leptin signaling both in vitro and in vivo. To determine the effects of obesity upon a PDX model, a high fat diet was used to induce obesity in vivo. Exposure to conditioned media harvested from obASCs increased the percentage of TNBC cells that expressed cancer stem cell markers, whereas exposure to a small peptide antagonist of the leptin receptor decreased the percentage of cells with CSC markers. PDX tumors implanted into diet-induced obesity mice had an increased volume compared to tumors implanted into lean controls. Conclusions: These molecular differences may contribute to the differences in cancer outcomes between obese and lean individuals with breast cancer, and further study of the crosstalk between obASCs and TNBC is critical. Citation Format: Courtney Brock, Maryl Wright, Khoa Nguyen, Katherine Hebert, Madlin Alzoubi, Thomas Cheng, Bridgette Collins-Burow, Matthew Burow. Examining the role of obesity and leptin signaling in triple negative 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 3200.