Targeted delivery of CD44s-siRNA by ScFv overcomes de novo resistance to cetuximab in triple negative breast cancer
Introduction
The HER receptor tyrosine kinase family comprises four members: epidermal growth factor receptor (EGFR, ErbB1/HER1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4) (Arteaga, 2001; Hu et al., 2015a). EGFR is a 170 kDa transmembrane protein that comprises an extracellular domain, which is also used for ligand binding; a transmembrane region; and an intracellular kinase domain. To date, two ligands that bind and activate EGFR have been identified: epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-α). Ligands bind to the extracellular domain of EGFR, resulting in homo- or heterodimerization of the receptor. This heterodimerization can happen between EGFR and one of the HER family of receptor tyrosine kinases. Dimerization then induces autophosphorylation of the tyrosine kinase domain (McCune and Earp, 1989), which serves as a binding site for the recruitment of signal transducers and activators of intracellular substrates. The major signaling routes for EGFR are the Ras-Raf mitogen-activated protein kinase pathway and the phosphatidyl inositol 3′ kinase and Akt pathway, which control important biologic processes such as cellular proliferation, angiogenesis and inhibition of apoptosis (de Bono and Rowinsky, 2002). Cumulative evidence has shown that the dysregulation of EGFR as well as the HER receptor family plays a significant role in tumorigenesis and the progression of cancer (De Pourcq et al., 2012; Hu et al., 2016). Basic and translational studies of the HER receptor family have focused on the approach for converting therapeutic biomolecules from bench to bedside, including the anti-EGFR antibodies panitumumab and cetuximab, the anti-HER2 antibodies trastuzumab and pertuzumab (Baselga et al., 2012; Sliwkowski and Mellman, 2013) and the EGFR tyrosine kinase inhibitors erlotinib and gefitinib.
Breast cancer is a common cancer in women, and its incidence is increasing worldwide (Kamangar et al., 2006). However, the mortality rate in developed countries has declined during the past three decades owing to the implementation of screening, improvements in the local management of early breast cancer, and, most importantly, the introduction of adjuvant systemic treatments (Andergassen et al., 2017). Moreover, in some subgroups of breast cancer, such as HER+ breast cancer, further improvements will be made in the near future as targeted treatments gradually emerge. Triple-negative breast cancers (TNBCs) belong to a subgroup of breast cancers that do not express ER, PR, or HER2. TNBC accounts for 10–20% of all breast cancers, totaling as many as 40,000 new cases per year in the United States alone (Andergassen et al., 2017). Compared to HER+ or hormone receptor-positive breast tumors, TNBC has a poorer prognosis and shorter progression-free (PFS) and overall survivals (OS) and are at higher risk of relapse even with optimal treatment (Mustacchi and De Laurentiis, 2015).
Among patients diagnosed with resectable (stage I-III) TNBC completing tri-modality therapy (neoadjuvant chemotherapy or surgery ± radiotherapy + adjuvant), as many as 50% of patients undergo disease recurrence, and as many as 37% die in the first 5 years following surgical treatment (Liedtke et al., 2008). Moreover, for patients presenting with metastatic TNBC, cytotoxic chemotherapy such as ixabepilone, capecitabine, anthracyclines and taxanes is the standard-of-care treatment (NCCN, 2016). A retrospective analysis of 111 cases treated with monotherapy or combinations (Kassam et al., 2009) showed that the mean duration of the response was 12 weeks after first-line treatment, 9 weeks after second-line treatment, and 4 weeks after third-line treatment, demonstrating that the duration of response to chemotherapy of TNBC is usually short. So far, TNBC is also the only major type of breast cancer for which no specific FDA-approved targeted therapy is available to improve patient outcomes; it is resistant to targeted therapies such as hormonal therapy and HER2-targeting therapy. The overexpression of EGFR often occurs in TNBC. Although EGFR-targeted therapies show efficacy in patients with solid tumors (e.g., colorectal, head and neck, and non-small cell lung cancer), EGFR-targeted therapies have been developed for TNBC without success despite a preclinical rationale suggesting their efficacy (Bonner et al., 2010; Heinemann et al., 2014; Costa et al., 2017).
In the current report, we show that impaired EGFR degradation is crucial for the resistance to cetuximab in breast cancer cell lines, which is associated with the cell surface molecule CD44. CD44 was recently reported to attenuate endocytosis-mediated EGFR degradation, thus sustaining downstream Akt signaling. To further investigate the role of CD44 in EGFR signaling and to determine the treatment potential of such findings, we designed a targeting fusion protein composed of an anti-EGFR single chain antibody generated from cetuximab and truncated protamine (Ce-tP). In the present study, we provide evidence that siRNA can be effectively delivered to EGFR-positive breast cancer cells using the fusion protein. More importantly, our results also show that the targeted delivery of siRNA specific for CD44 can efficiently overcome resistance to EGFR targeting in TNBC cancer cells both in vitro and in vivo.
Section snippets
Cell lines, antibodies, and animals
MCF-7, BT474, MDA-MB-468, MDA-MB-231, and MDA-MB-157 breast cancer cells were obtained from ATCC (ATCC. Manassas, VA). All the cell lines were authenticated twice by morphological and isoenzyme analyses during the study period. Cell lines were routinely checked for contamination with mycoplasma using Hoechst staining and were consistently found to be negative.
Cells were cultured in DMEM or L15 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) in 5% CO2 at 37.5 °C in a
Impaired EGFR degradation is crucial for the resistance to cetuximab
To evaluate the expression levels of the total and activated (phosphorylated) forms of EGFR and check the signaling activation, we first assessed the breast cancer cell lines by western blot (Fig. 1A). The levels of EGFR detected in MDA-MB-231 cells, MDA-MB-468 cells, MDA-MB-157 cells, and BT474 cells were higher than those in MCF-7 cells, which do not express EGFR. The levels of phosphorylated EGFR, AKT and ERK were also notably increased in TNBC cell lines. As cetuximab (Erbitux®) is a
Discussion
The dysregulation of receptor tyrosine kinase (RTK) as well as its downstream ERK and PI3K/Akt signaling is a common mechanism that drives cancer progression. CD44, also referred to as P-glycoprotein 1, is a transmembrane glycoprotein encoded by a single gene on the chromosome locus 11p13. CD44s is the conserved form with a molecular weight of approximately 85–90 kDa and is made of the transcription of exons 1–5 and 16-20, which are spliced together. CD44 comprises an extracellular domain (or
Authors’ contributions
W.F. and W.J. conceived and designed the study. W.F., H.F, Y.Z., M.C., X.Y., and L.Y. participated in the collection, discussion, and analysis of the data. W.F. and W.J. wrote the paper. All authors read and approved the final manuscript.
Conflicts of interest
The authors disclose no conflicts of interest.
Acknowledgement
This work was supported by the grant from National Natural ScienceFoundation of China (81773093) and a Municipal Human Resources Development Program for Outstanding Leaders in Medical Disciplinesin Shanghai (2017BR028).
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