Abstract PD5-1: The type II TGFb receptor neutralizing antibody, TR1, abrogates the tumor-initiating cell (TIC) population in triple negative breast cancer (TNBC) via inhibition of SMAD and non-SMAD pathways
Authors: Bhola N , Arteaga C 
Published: August 2014

Abstract
Tumor-initiating cells (TICs) or cancer stem cells (CSCs) are resistant to chemotherapy and have been associated with metastatic recurrences and poor patient outcome. We have shown that the TGFb pathway plays a crucial role in the expansion of TICs and that inhibition of the type I TGFβ receptor (ALK5) decreases chemotherapy-resistant TICs. Further, a TGFβ-responsive gene signature is enriched in TNBCs upon treatment with chemotherapy and is predictive of poor prognosis (Bhola et al. JCI 2013). Herein we investigated the molecular and antitumor effects of TR1, a TGFβRII neutralizing antibody currently in phase I clinical trials, against TNBC. TR1 recognizes the TGFβ ligand binding epitope in the extracellular domain of TGFβRII; it blocks ligand binding in the nM range. Small molecule TGFβRI kinase inhibitors are another approach to inhibit TGFβ/SMAD signaling. However, they cross-react with other type I receptors (ALK1, 4, 7), some of which are important for normal physiology. Furthermore, TGFβRII can induce EMT in breast cancer cells independent of dimerization with TGFβRI. Therefore, we hypothesized that, by neutralizing TGFβRII, TR1 can abrogate the breast TIC population via inhibition of SMAD and non-SMAD signaling.
Using the pCAGA12-luciferase reporter to assess SMAD-dependent transcriptional activity, TR1 decreased luciferase activity in TNBC cell lines (SUM159 and BT549) and steady-state levels of P-SMAD2, suggesting it inhibited autocrine TGFβ signaling. TR1 decreased the TIC fraction as identified by FACS analysis of cell line-specific TIC markers (ALDH for SUM159; CD44hi/PROCR+ for BT549; PROCR+/ESA+ for MDA231 cells). Although TR1 displayed no effects on TNBC growth in vitro, it decreased tumor growth in vivo. We used a phospho-kinase array (45 kinases) to elucidate the non-SMAD pathways altered by TR1. TR1 decreased phosphorylated EGFR (Tyr1068) and STAT3 (Tyr705) in TNBC cell lines. By flow cytometry, the cell-surface expression of EGFR was also lowered upon treatment with TR1. TR1 also reduced P-ERK1/2 and P-Akt (Ser473) levels, and lowered NFκB transcriptional activity as measured by luciferase reporter assay. Using a cytokine array (120 cytokines) and qRT-PCR we observed that TR1 decreased the expression and release of cytokines involved in TIC enrichment such as IL-6 and IL-8. Inhibition of EGFR (with gefitinib) and MEK1/2 (with selumetinib) decreased the TIC population while augmenting pCAGA luciferase activity. The combination of SMAD2 siRNA with each EGFR or MEK inhibitors significantly decreased the TIC population. We speculate based on these data that inhibition of both SMAD and non-SMAD signaling is necessary for complete abrogation of TICs. This may explain the efficacy of TR1 as a single agent.
These findings suggest that targeting TGFβRII with TR1 exerts its anti-tumor effects via inhibition of both SMAD and non-SMAD (EGFR, ERK) pathways that are important to TIC survival. The results also encourage combination therapies of chemotherapy with TR1 to reduce tumor recurrences in patients with TNBC.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD5-1.