Targeting the NRG1/HER3 pathway in tumor cells and cancer-associated fibroblasts with an anti-neuregulin 1 antibody inhibits tumor growth in pre-clinical models of pancreatic cancer

Highlights

• NRG1 is expressed in human pancreatic tumors and cancer-associated fibroblasts.

• NRG1 promotes pancreatic cell growth.

• NRG1 trapping by 7E3 antibody inhibits HER3 activation and downstream signaling.

• Targeting NRG1 inhibits growth of orthotopic pancreatic tumor xenografts.

Abstract

Neuregulin 1 (NRG1), a ligand for HER3 and HER4 receptors, is secreted by both pancreatic tumor cells (PC) and cancer-associated fibroblasts (CAFs), the latter representing the most abundant compound of pancreatic stroma. This desmoplastic stroma contributes to Pancreatic Ductal Adenocarcinoma (PDAC) aggressiveness and therapeutic failure by promoting tumor progression, invasion and resistance to chemotherapies. In the present work, we aimed at disrupting the complex crosstalk between PC and CAF in order to prevent tumor cell proliferation. To do so, we demonstrated the promising tumor growth inhibitory effect of the 7E3, an original antibody directed to NRG1. This antibody promotes antibody dependent cellular cytotoxicity in NRG1-positive PC and CAFs and inhibits NRG1-associated signaling pathway induction, by blocking NRG1-mediated HER3 activation. Moreover, 7E3 inhibits migration and growth of pancreatic cancer cells co-cultured with CAFs, both in vitro and in vivo using orthotopic pancreatic tumor xenografts. Our preclinical results demonstrate that the anti-NRG1 antibody 7E3 could represent a promising approach to target pancreatic stroma and cancer cells, thereby providing novel therapeutic options for PDAC.

Introduction

Pancreatic cancer remains one of the most aggressive tumors with an extremely poor prognosis and is estimated to become the second leading cause of cancer-related death by 2030 [1,2]. This dramatic outcome is related to the lack of efficient therapeutic tools, early diagnostic markers, and high resistance to chemotherapy.

Abundant desmoplastic reaction is a prominent feature of pancreatic cancer and constitutes up to 80% of the total tumor volume. It is characterized by the presence mainly of cancer-associated fibroblasts (CAFs), but also of immune cells, vasculature, and extracellular matrix (ECM). These stroma characteristics contribute to pancreatic cancer aggressivity and therapeutic failure because they promote tumor progression, invasion and resistance to chemotherapies [3,4]. Once activated, CAFs express alpha smooth muscle actin (αSMA) and secrete ECM and soluble factors (e.g., growth and inflammatory factors) that stimulate tumor growth and mediate inflammation. In preclinical studies, stroma disruption, by direct CAF targeting (hedgehog signaling inhibitors) [5] or by ECM enzymatic digestion (hyaluronidase), results in tumor inhibition and increases drug delivery [6]; however, controversial or disappointing results were reported by clinical trials. Moreover, recent studies have questioned CAF role in pancreatic tumor maintenance and highlighted the need of caution when targeting CAFs [[7], [8], [9]]. Indeed, several evidences indicate that there are different CAF subtypes with different functions [10,11]. Therefore, characterization of these subtypes is needed to enable the specific targeting of CAFs with pro-tumor features.

Neuregulins (NRGs) are large polypeptide growth factors that are part of the EGF family of proteins. They are encoded by four genes (NRG1-4) that are subject to extensive alternative mRNA splicing, leading to more than 30 variants grouped in six types (I-VI) with a distinct N-terminal part. NRG1 and 2 are both HER3 and HER4 ligands and have at least two splice alternatives in their EGF-like domains, resulting in α and β isoforms. NRG1 β has higher binding affinity for HER3 through HER2-dependent recruitment [12]. NRG3 and 4 bind only to HER4. Most isoforms are synthetized as transmembrane molecules with the EGF-like domain in the extracellular space and are released as soluble factors by cell surface proteases. Binding to the EGF-like domain of NRG1 promotes HER3 or HER4 conformational changes, leading to their dimerization and activation that result in the phosphorylation of downstream signaling molecules. Several evidences indicate that NRG1 contributes to the development and progression of different tumor types, such as breast, prostate or pancreatic cancer [[13], [14], [15], [16]]. High NRG1 expression has been correlated with poor prognosis in breast cancer, head and neck squamous cell carcinoma and pancreatic cancer [[13], [14], [15]]. In addition, NRG1 downregulation by antisense oligonucleotides, or NRG1 signaling inhibition by competitive binding of a shorter HER3 isoform reduces breast cancer cell proliferation and migration [16,17]. Furthermore, the crosstalk between NRG1 and HER3 is involved in resistance to chemo- and targeted therapies through the maintenance of oncogenic signaling pathways [18].

Several studies reported that not only EGF family growth factors but also their receptors play an important role in pancreatic cancer development. Expression of EGFR, HER2 and HER3 has been examined in pancreatic ductal carcinoma and correlated with advanced disease and poor prognosis [[19], [20], [21], [22]]. HER4 expression has not been examined as extensively and its prognostic value is not known.

Disruption of intracellular communications between stromal and cancer cells could affect tumor proliferation and represents an interesting therapeutic strategy. In this context, we hypothesized that targeting NRG, which we here show to be secreted both by CAFs and tumor cells, could be a novel therapy to overcome CAF-triggered tumor growth in pancreatic cancer. Here, we investigated, in in vivo and in vitro co-culture of pancreatic cancer cells and CAFs, the therapeutic potential of an original monoclonal antibody (7E3) that targets NRG1 Ig-like domain. We show promising tumor growth inhibitory effects of this antibody which promotes antibody dependent cellular cytotoxicity (ADCC) in NRG1-permeated pancreatic cancer cells and in NRG1-positive CAFs and blocks NRG1-mediated HER3 activation and downstream signaling pathway induction.