Abstract P2-01-04: Epithelial-mesenchymal transition promotes triple negative breast cancer immune suppression
Authors: Williams MM , Christenson JL , O'Neill K , Rogers TJ , Greene LI , Slansky J , Richer JK 
Published: May 2019

Background: Epithelial-Mesenchymal Transition (EMT) is a developmental process co-opted by carcinomas to facilitate metastasis. Triple negative breast cancer (TNBC), which has often undergone at least partial EMT, has a poor prognosis due to frequent and rapid recurrence as metastatic disease when compared to other breast cancer subtypes. To identify drivers of TNBC progression microRNA miR-200c was restored to the human TNBC mesenchymal-like BT549 cell line, which decreased 80% of the pan-EMT mesenchymal signature and restored 60% of the epithelial signature. In addition to decreasing proteins involved in immune-suppression via contact-dependent mechanisms, such as CD274 (PD-L1) and (CD273) PD-L2, Ingenuity Pathway and GSEA analysis revealed reduced expression of genes encoding enzymes, including Tryptophan 2,3-Dioxygenase (TDO2) and Heme Oxygenase-1 (HO-1), that generate immune-suppressive metabolites that function in a non-contact dependent manner. TNBC utilizes these and other immune-suppressive factors made by fetal trophoblasts to suppress the maternal immune system to achieve fetal tolerance during pregnancy, suggesting that TNBC may mimic a physiologically relevant mechanism to promote immune evasion.

Hypothesis: TNBC metabolism is altered as part of an EMT program resulting in generation of immune-suppressive metabolites that promote metastasis by facilitating contact-independent immune evasion.

Methods: Expression and activity of TDO2 and HO-1 were determined by UHPLC-MS for kynurenine (generated by TDO2) and bilirubin (generated by HO-1) in multiple TNBC cell lines. Single cell RNAseq was performed to identify genes co-expressed with these enzymes. To develop an immune competent preclinical model, doxycycline inducible miR-200c (TripZ-200c) was stably introduced into the Met-1 mammary carcinoma cell line derived from the MMTV-PyMT mouse model developed in FVB/NJ mice. Results were confirmed via transient miR-200c expression in 66cl-4 mammary carcinoma cells (from a spontaneous BALB/c tumor).

Results: Restoration of miR-200c decreased TDO2 and HMOX1 (HO-1) by 80% and 40% respectively in Met-1 and 66cl-4 mammary carcinoma cells and reduced secretion of kynurenine and bilirubin. HO-1 was increased in lung metastasis from Met-1 and 66cl-4 cells when compared to cells prior to tail vein injection. ScRNAseq revealed specific gene expression signatures co-expressed with TDO2 and are being analyzed to better detect TDO2 in tumor tissues. In vivo studies are underway to investigate the effects of inhibiting tumor TDO2 and HO-1 on infiltrating lymphocyte composition, the anti-tumor immune response, and tumor progression.

Conclusions: Restoration of miR-200c revealed EMT-associated alterations in tumor metabolism and consequent secretion of immune-suppressive metabolites produced by TDO2 and HO-1. We propose that targeting these contact-independent immune-suppressive factors may complement current contact-dependent therapeutic strategies (checkpoint inhibitors) to boost anti-tumor immune cell function and thereby prevent or decrease TNBC metastasis.

Citation Format: Williams MM, Christenson JL, O'Neill K, Rogers TJ, Greene LI, Slansky J, Richer JK. Epithelial-mesenchymal transition promotes triple negative breast cancer immune suppression [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-04.