Abstract B017: The double-edged sword of cystatin B in luminal-like and triple-negative breast cancers
Authors: Zaccaria Anna , Ratti Chiara , Botti Laura , Sangaletti Sabina , Colombo Mario P. , Chiodoni Claudia 
Published: November 2024

Abstract
The complex protease network of cathepsins and their inhibitors, cystatins, intricately regulates numerous cellular processes, and its dysregulation is frequently implicated in cancer pathogenesis. Abnormal cathepsin activity, subcellular localization, and a disrupted balance between cathepsins and cystatins have been identified in cancer. While diminished cystatin levels typically correlate with more aggressive cancer phenotypes, few findings challenge this paradigm. In this context, our latest study revealed elevated expression of cystatin B (CSTB) in luminal-like breast cancer (BC) patients experiencing relapse within five years post-surgery. Furthermore, computational analysis of publicly available datasets supports our idea of CSTB as a potential negative prognostic biomarker in luminal-like tumors. Intriguingly, a converse relationship is observed in basal-like/triple negative BC patients, where increased CSTB expression seems to be associated with improved prognosis. In addition, the cysteine/cathepsin network intricately intersects with immune functions, adding another layer of complexity. Cathepsins are indeed involved in the induction of an efficient anti-tumor immune response, being key in antigen processing and presentation by antigen-presenting cells and in the cytotoxic activities of NK and T cells. On the other hand, myeloid cells rely on cathepsins for the degradation of ECM proteins that may favor migration and invasion of tumor cells. All these pieces of evidence led to the hypothesis that the functional impact of CSTB may vary depending on the tumor intrinsic characteristics and on its microenvironment and therefore that CSTB might fulfill distinct functional roles in luminal-like and basal-like BC. To start addressing our hypothesis, we silenced CSTB expression in the 4T1 mouse cell line, a TNBC model spontaneously metastasizing to the lungs upon intra-mammary fat pad injection. Preliminary in vivo experiments to investigate the effect on primary tumor growth and metastatic dissemination showed that 4T1 shCSTB lines were indeed characterized by increased tumor growth and metastatic dissemination than the two sh-control lines. Interestingly, expression of stemness- related genes (Nanog, Sox2 and Oct4) were up-modulated upon CTSB silencing, in agreement with a more aggressive phenotype. On the other hand, preliminary in vitro assays with a luminal- like BC cell line support the hypothesis of an opposite effect of CSTB down-modulation in luminal BC subtype. Moreover, CSTB silencing in RAW264.7 macrophagic cell line, impaired M1 in vitro polarization, while it did not appear to affect M2 skewing. Overall, although preliminary, these data suggest that CSTB may affect BC aggressiveness by both interfering with tumor cell-intrinsic features and microenviroment-related traits.
Citation Format: Anna Zaccaria, Chiara Ratti, Laura Botti, Sabina Sangaletti, Mario P. Colombo, Claudia Chiodoni. The double-edged sword of cystatin B in luminal-like and triple-negative breast cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B017.