Mindy I. Davis Ph.D. National Institute of Health

Researchers describe a strategy for treating TNBC that combines noncompetitive antibodies to achieve degradation of EGFR and tumor inhibition.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer that does not currently have any targeted therapeutics available. TNBC is characterized by the lack of expression of human epidermal growth factor receptor 2 (HER2 or EGFR2), estrogen receptor, and progesterone receptor, which are expressed in other types of breast cancers and can be targeted directly in those cases. TNBC cell growth is likely not fueled by estrogen, progesterone, or Her2 (the ligands for the three absent receptors), and therefore hormonal therapy or anti-Her2 treatments are likely to be ineffective.

Current TNBC drug treatments consist primarily of standard systemic chemotherapies. TNBC patients respond differently to the various current treatments, and this indicates that the drivers of cancer for TNBC patients may differ between patients. TNBC cells from some patients appear to lack the ability to repair DNA damage efficiently, so platinum-containing drugs that cause DNA damage are being investigated in several clinical trials.

About 50% of TNBC cells express epidermal growth factor receptor (EGFR), and indeed clinical trials have been undertaken to target EGFR with antibodies or tyrosine kinase inhibitors or with combinations of an anti-EGFR antibody and a standard chemotherapeutic agent. There are currently many ongoing trials, which predominantly focus on combinations of agents to treat TNBC. The authors* here have investigated antibodies against EGFR that can be used concurrently and lead to synergy. They were able to find two EGFR antibodies (panitumumab and mAb111) that are noncompetitive for binding to EGFR (i.e., binding of one does not preclude binding of the other).

The combination increased EGFR ubiquitination, which is often associated with receptor degradation (Figure 1). Additionally, pretreatment with bortezomib or bafilomycin before treatment of the antibody pair was used to try to tease out mechanism. Degradation was inhibited by the inhibitor of lysosomal hydrolases (bafilomycin) but not by an inhibitor of the proteasome degradation system (bortezomib; see Figure 1, panel C).

The effect of single agents versus combinations was also studied (Figure 2). EGFR was degraded by combinations of antibodies but not by the single agents. Additionally, a 50% reduction in cell migration (via an invasion assay) was observed in combination but not with single agents.

Figure 1. Anti-EGFR antibodies enhance receptor ubiquitination and degradation. (A) Serum-starved HeLa cells were incubated with mAbs (10 µg/mL), a combination (each at 5 µg/mL), or with EGF (10 ng/mL), and lysates analyzed using immunoprecipitation (IP) and immunoblotting (IB). (B) HeLa cells transfected with a plasmid encoding an MYC peptide–tagged CBL, or an empty vector, were serum-starved and treated for 3 h with the indicated mAbs (20 µg/mL) or a combination (each at 10 µg/mL). Alternatively, cells were treated for 10 min with EGF (10 ng/mL). Lysates were probed as indicated. (C) HeLa cells that were preincubated (12 h) with bortezomib (2 µM) or bafilomycin (10 nM) were incubated (60 min) with EGF (10 ng/mL) or for 6 h with the indicated combination of mAbs (each at 10 µg/mL). Lysates were subjected to IB and signal quantification. EGF, epidermal growth factor; EGFR, EGF receptor; mAbs, monoclonal antibodies.

The authors point out that there is an ongoing clinical trial for two noncompetitive antibodies that target EGFR but for different indications (squamous cell carcinoma of the head and neck and metastatic colorectal cancer). It will be quite interesting to see whether this combination is effective for those cancer types and by extension may be worth pursuing for TNBC. TNBC is an important target representing ~15%–20% of breast cancer cases and finding the right combination of drugs to increase overall survival is imperative. The authors have provided a good case for trying this new combination of EGFR antibodies in the TNBC patient population who have high levels of EGFR expression.

Figure 2. A combination of antibodies down-regulates EGFR and inhibits invasion of TNBC cells. (A) Whole extracts were prepared from the indicated cell lines after treatment with EGF (10 ng/mL, 1 h) or with the mAbs (20 µg/mL total, 6 h). Lysates were immunoblotted as indicated. (B) BT-549 cells were treated for 48 h with mAbs (20 µg/mL total) and lysates immunoblotted for EGFR and ERK2. (C, D) BT-549 cells were treated with mAbs as in (B). Thereafter, cells were plated in the upper compartment of invasion chambers. The lower compartments were filled with the respective mAb-containing media. Eighteen hours later, the filters were removed, fixed, permeabilized, and stained with methyl violet (0.3%). Cells growing on the upper side of the filter were removed and cells on the bottom side were photographed and quantified.

*Abstract from PNAS USA 2013, Vol. 110: 1815–1820

Breast tumors lacking expression of human epidermal growth factor receptor 2 (HER2) and the estrogen and the progesterone receptors (triple-negative; TNBC) are more aggressive than other disease subtypes, and no molecular targeted agents are currently available for their treatment. Because TNBC commonly displays EGF receptor (EGFR) expression, and combinations of monoclonal antibodies to EGFR effectively inhibit other tumor models, we addressed the relevance of this strategy to treatment of TNBC.

Unlike a combination of the clinically approved monoclonal antibodies, cetuximab and panitumumab, which displaced each other and displayed no cooperative effects, several other combinations resulted in enhanced inhibition of TNBC’s cell growth both in vitro and in animals. The ability of certain antibody mixtures to remove EGFR from the cell surface and to promote its intracellular degradation correlated with the inhibitory potential. However, unlike EGF-induced sorting of EGFR to lysosomal degradation, the antibody-induced pathway displayed independence from the intrinsic kinase activity and dimer formation ability of EGFR, and it largely avoided the recycling route.

In conclusion, although TNBC clinical trials testing EGFR inhibitors reported lack of benefit, our results offer an alternative strategy that combines noncompetitive antibodies to achieve robust degradation of EGFR and tumor inhibition.

Mindy I. Davis, Ph.D., works at the NIH.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a “Literature Search and Review” column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the “Literature Search and Review” column, a paper published in Proceedings of the National Academy of Sciences USA titled “Inhibition of triple-negative breast cancer models by combinations of antibodies to EGFR”. Authors of the paper are Ferraro DA, Gaborit N, Maron R, Cohen-Dvashi H, Porat Z, Pareja F, Lavia S, Lindzen M, Ben-Chetrit N, Sela M, and Yarden Y.

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