Cancer Biology and Signal Transduction ERBB2 Overexpression Establishes ERBB3- Dependent Hypersensitivity of Breast Cancer Cells to Withaferin A Wenjun Liu 1 , Annalise R. Barnette 2 , Samita Andreansky 3,4 , and Ralf Landgraf 1,4 Abstract The catalytically deficient ERBB3 strongly synergizes with the receptor tyrosine kinase ERBB2, and elevated levels represent an overall risk factor for unfavorable disease outcomes in breast cancer. Although itself not a target of pan-ERBB kinase inhibitors, it contributes to resistance in ERBB2-targeted treatment regi- ments. The steroidal lactone Withaferin A (WA) has established broad anticancer properties through several modes of action and was shown to be effective against triple-negative breast cancers at elevated concentrations. We found that ERBB2 overexpression does render cells hypersensitive to WA. Although ERBB2 down- regulation is one aspect of WA treatment at high concentrations, it is not causal for the elevated sensitivity at lower dosages. Instead, WA targets the ability of ERBB3 to amplify ERBB2 signaling. ERBB3 receptor levels, constitutive phosphorylation of both ERBB3 and ERBB2, as well as signaling through AKT are elimi- nated by WA treatment. By targeting ERBB2/ERBB3 as a functional unit, it is also effective in cases in which ERBB2-directed inhibi- tors, such as lapatinib, alone show reduced potency. Hence, WA or derivatives thereof may present a low toxicity addition to ERBB2-targeting therapeutics, especially in cases in which ERBB3 involvement is driving resistance or reduced overall sensitivity. Mol Cancer Ther; 15(11); 2750–7. Ó2016 AACR. Introduction Overexpression of the receptor tyrosine kinase ERBB2 (ErbB2, HER2) occurs in various solid tumors but most prominently in breast cancer, with approximately one third of patients displaying high ERBB2 levels due to gene amplification. Inconsistent response rates to therapeutic antibodies and ERBB family–direct- ed kinase inhibitors as well as disease reoccurrence remain a problem for a large percentage of patients. ERBB3, the most potent heterodimerization partner of ERBB2, has emerged as a critical factor in both the initial oncogenicity as well as the emergence of resistance to ERBB2-directed therapies (1). When targeted for inhibition, residual levels of active ERBB2 can drive the elevation of both ERBB3 and pERBB3 (1, 2). Impaired in its own kinase activity (3), ERBB3 acts as an allosteric activator of its heterodimerization partners (4), and elevated levels of ERBB3 are correlated with progression of several solid tumors (5–10). With its six docking sites for activated PI3K, much of the ERBB2- amplifying phenotype of ERBB3 relates to its exceptionally potent activation of the PI3K/AKT pathway (11, 12). This has prompted an intense search for therapeutics that target either ERBB3 directly, such as mAbs, or its downstream signals, especially PI3K/AKT. Given the pronounced supporter role of ERBB3, treatment approaches will almost certainly involve a combination with drugs targeting the primary oncoprotein, such as ERBB2. For such approaches, the ability to complement the primary treatments at low intrinsic toxicity is highly desirable. Although often limited in their utility as stand-alone therapeutics, natural compounds with known medicinal utility can meet this requirement. In fact, the very same complex mode of action that is often associated with their limitations as classic therapeutics may prove beneficial for their use as a sensitizers or supplementary treatment. Beyond their direct use, chemically well characterized natural products, such as Withaferin A (WA), can provide lead compounds for subsequent structure–activity relationship–based drug development. WA is a steroidal lactone isolated from winter cherry (Withania somnifera). The purified compound was first shown to suppress Ehrlich ascites carcinoma in treated mice and increase disease-free survival when administered (continuously) posttreatment (13, 14). Since then, WA has been studied extensively as a prototype withanolide for anticancer treatment (reviewed in ref. 15). In addition to its broad and likely multicausal anticancer activity, observed generally in the high micromolar concentration range, WA can exhibit selective and cell type-specific functions at lower concentrations. It inhibits human umbilical vein endothe- lial cell proliferation and exerts potent antiangiogenic activity in FGF-2 Matrigel mouse models of angiogenesis with an IC 50 of 12 nmol/L (16). This antiangiogenic property is linked to the cova- lent and degradation-enhancing modification of vimentin by WA (17). The complex mode of action of WA is a limiting factor in the utilization as a stand-alone treatment. On the other hand, the 1 Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, Miami, Florida. 2 Department of Micro- biology and Immunology, Miller School of Medicine, University of Miami, Miami, Florida. 3 Department of Pediatrics, Miller School of Medicine, University of Miami, Miami, Florida. 4 Sylvester Comprehen- sive Cancer Center, Miller School of Medicine, University of Miami, Miami, Florida. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). S. Andreansky and R. Landgraf contributed equally to this article. Corresponding Author: Ralf Landgraf, Department of Biochemistry and Molec- ular Biology, University of Miami, Box 016129 (R-629), Miami, FL 33101-6129. Phone: 305-243-5815; Fax: 305-243-3955; E-mail: RLandgraf@med.miami.edu doi: 10.1158/1535-7163.MCT-15-0932 Ó2016 American Association for Cancer Research. Molecular Cancer Therapeutics Mol Cancer Ther; 15(11) November 2016 2750 Downloaded from http://aacrjournals.org/mct/article-pdf/15/11/2750/1850817/2750.pdf by guest on 26 June 2022