Obesity is associated with an increased risk of, and a poor prognosis for, postmenopausal (PM) breast cancer (BC). Our goal was to determine whether diet-induced obesity (DIO) promotes 1) shorter tumor latency, 2) an escape from tumor dormancy, and 3) an acceleration of tumor growth and to elucidate the underlying mechanism(s). We have developed in vitro assays and PM breast tumor models complemented by a noninvasive imaging system to detect vascular invasion of dormant tumors and have used them to determine whether obesity promotes the escape from breast tumor dormancy and tumor growth by facilitating the switch to the vascular phenotype (SVP) in PM BC. Obese mice had significantly higher tumor frequency, higher tumor volume, and lower overall survival compared with lean mice. We demonstrate that DIO exacerbates mammary gland hyperplasia and neoplasia, reduces tumor latency, and increases tumor frequency via an earlier acquisition of the SVP. DIO establishes a local and systemic proangiogenic and inflammatory environment via the up-regulation of lipocalin-2 (LCN2), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) that may promote the escape from tumor dormancy and tumor progression. In addition, we show that targeting neovascularization via a multitargeted receptor tyrosine kinase inhibitor, sunitinib, can delay the acquisition of the SVP, thereby prolonging tumor latency, reducing tumor frequency, and increasing tumor-free survival, suggesting that targeting neovascularization may be a potential therapeutic strategy in obesity-associated PM BC progression. This study establishes the link between obesity and PM BC and, for the first time to our knowledge, bridges the dysfunctional neovascularization of obesity with the earliest stages of tumor development.

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Triple-negative breast cancer (TNBC), an aggressive breast cancer subtype, accounts for ~15% of all breast cancers and is associated with a poor prognosis and high rates of recurrence and distant metastases. Patients diagnosed with TNBC have limited treatment options and are often restricted to chemotherapy-based regimens, in stark contrast to patients with other breast cancer subtypes where the development of targeted therapeutics have significantly improved patient outcomes. TNBC is characterized by the loss of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. This and the genetic and molecular tumor heterogeneity of TNBC has not only limited advances in the development of targeted therapy but also in the identification of biomarkers that can predict disease status and monitor treatment response. Advances in next-generation sequencing technologies have led to the identification of promising clinically actionable mutations however these tests often require invasive procedures to obtain tumor tissue and lack the ability to monitor treatment efficacy or detect recurrence, highlighting the need for non-invasive markers. A number of serum protein biomarkers have been evaluated in TNBC however none have yet to be successfully deployed in the clinic as indicators of disease. We have previously established that the urinary proteome can serve as a source of highly specific and sensitive biomarkers that can detect disease status and stage of multiple cancer types. Within the context of our global proteomics approach to cancer biomarker discovery, we have utilized the SomaScan discovery platform to identify 281 proteins that are differentially expressed in the urine of patients with TNBC compared to the urine of age- and sex-matched controls. The gene expression profiles of these proteins in TNBC tumor tissue were compared to those of normal breast tissue utilizing the R2 Genomics Platform. Proteins whose gene expression profile was consistent with the proteomic data were validated for the ability to distinguish between TNBC and non-TNBC diagnoses using mono-specific ELISAs of patient urine samples. These results demonstrate that a global proteomics approach coupled with clinical gene expression data can be effectively utilized to identify potential biomarkers for TNBC. These findings identify clinically relevant proteins that should be further explored as potential biomarkers in the development of non-invasive diagnostics for TNBC. (This work was supported by the Breast Cancer Research Foundation and the Nile Albright Research Foundation). Citation Format: Michael N. Lombardo, Roopali Roy, Susan Pories, Meg Lotz, Rama Aldakhlallah, Simon T. Dillon, Towia A. Libermann, Marsha A. Moses. Identification of urinary proteome signatures associated with triple-negative breast cancer. [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 5315.

Introduction: Breast cancer (BC) is a complex heterogenous disease and is a leading cause of death in women. Following primary BC therapy ~40% of patients will develop metastatic BC (MBC) and 60-70% of the recurrent disease will occur at distant sites. Median survival of MBC patients with distant recurrence is ~2-3 yr. While the early detection and monitoring of BC progression are essential to improve prognosis and reduce BC-related mortality, there is a lack of surveillance strategies for monitoring patients for distant recurrence of MBC. The aim of our study was to identify urinary biomarkers for detection and monitoring of MBC. Method: We have conducted a comparative label-free LC-MS/MS analysis of the urinary proteome of patients with MBC and healthy age-matched controls (HC). A hybrid quadrupole time of flight (Q-Tof™) mass spectrometer was used for urine analysis via liquid chromatography (LC) with tandem mass spectrometry (MS/MS). Three different database search algorithms were used for peptide identification including MASCOT, Protein Lynx Global Server and Ion Accounting software. Only peptides detected in at least two out of three replicated LC-MS experiments were counted towards protein identification. Identified candidate biomarkers were validated via ELISA assays. Results: Using this approach, we identified ~400 urinary proteins of which ~104 and 89 were unique to the MBC and HC groups, respectively. Upregulated proteins in the MBC cohort were associated with angiogenesis, apoptosis, proteolysis, ECM regulation, and cell adhesion pathways. Using bioinformatic and in silico approaches, we identified a specific metastasis signature comprised of CALB1, S100A8, ZAG, VTN and TN. Diagnostic accuracies of these candidate markers were validated using independent training and validation sets according to the REMARK criteria. Urinary VTN (uVTN) and uTN levels correlated significantly with disease status in MBC patients when compared to patients with locally invasive BC and HC. Interestingly, a small subset of patients with bone metastasis also had significantly higher uVTN levels. A multiplexed marker panel of uVTN and uTN could discriminate between HC and MBC groups (AUC=0.831, P<0.001). Blinded testing indicated that uVTN levels (cutoff>1018 ng/ml) could discriminate (AUC=0.782, P<0.006) between MBC and HC. Longitudinal analysis of samples from MBC patients indicated a strong correlation between uVTN levels and disease status. Conclusions: Our findings suggest that uVTN and uTN have the potential to serve as biomarkers for the detection of MBC. While validation in larger cohorts is necessary, these results may be useful in the development of noninvasive diagnostic tests for monitoring BC progression and recurrence. [Supported by: the Advanced Medical Research Foundation, the Jo Ann Webb Fund for Angiogenesis Research and the S. Elizabeth O’Brien Trust] Citation Format: Roopali Roy, Elisa Schunkert, Petra Olivova, Martin Gilar, Scott Geromanos, Guo-Zhong Li, John Gebler, Adelle Dagher, Andrew El-Hayek, Rama Aldakhlallah, David Zurakowski, Susan Pories, Marsha A. Moses. Global proteomic analysis of urine using a label-free LC-MS/MS approach identifies potential biomarkers of metastatic breast cancer [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 3433.