The Combi-Targeting Concept: Selective Targeting of the Epidermal Growth Factor Receptor- and Her2-Expressing Cancer Cells by the Complex Combi-Molecule RB24

Abstract

Within the context of a new tumor-targeting strategy termed "combi-targeting," we designed RB24 to inhibit epidermal growth factor receptor (EGFR) or Her2 phosphorylation and to further degrade upon hydrolysis to 4-(3Ј-bromophenylamino)-6-aminoquinazoline (RB10; another EGFR/Her2 inhibitor) plus a strong DNA-alkylating species. 6-(3-Acetoxymethyl-3-methyltriazenyl)-4-(3Ј-bromophenylamino)quinazoline (RB24) showed significant antiproliferative activity against human breast cancer cells, and transfection of one such cell line, MDA-MB-435, with ErbB1 or ErbB2 (Her2) dramatically enhanced cell death by apoptosis. RB24 was capable of releasing 2-to 3-fold higher levels of RB10 in the transfectants than in their wild-type counterparts. More importantly, RB10 was abundantly distributed in the perinuclear region of the cells, and its elevated levels in the ErbB transfectants were concomitant with increased levels of DNA lesions in the latter cells. This selectivity could be abolished by coincubation of the cells with exogenous RB10, suggesting that the entire combi-molecule may bind primarily to its cognate perinuclear sites before degradation. This localization may exert a bystander effect, allowing the alkylating species to be abundantly propagated into the nucleus. Cell response to this novel targeting mechanism was mediated by 1) activation of c-Jun NH 2 -terminal kinase in response to DNA damage and 2) down-regulation of Bad through blockade of EGFR tyrosine kinase activity: two events that cooperatively converged into enhancement of apoptosis in the oncogene-transfected cells.Members of the epidermal growth factor (EGF) family of receptors, including EGFR, Her2 (ErbB2), and EGFRvIII (truncated EGFR), are overexpressed in 20 to 40% of breast cancers (Tang et al., 2000). The Her2 receptor, the closest EGFR homolog, is overexpressed in 20 to 25% of overall breast cancers, 60 to 70% of ductal carcinoma in situ, and is often associated with highly aggressive tumor phenotypes and poor disease-free survival (Slamon et al., 1989;Arteaga, 2001). For example, the average survival of breast cancer patients with Her2-positive tumors is 2 to 3 years compared with 6 to 7 years of Her2-negative tumors (Slamon et al., 1987;Borg et al., 1990). More importantly, this dysfunction is also associated with resistance to endocrine therapy and chemotherapy (Slamon et al., 1987). The significant implication of the ErbB family members in tumor progression has made them important targets for breast cancer therapy. Indeed, blockade of EGFR or Her2 has proven highly effective in breast xenograft models and in the clinic (Ciardiello et al., 1999; Molder et al., 2001). Although gefitinib (Iressa, ZD1839) that targets the EGFR tyrosine kinase (TK) has shown moderate activity in breast cancer models, trastuzumab (Herceptin), a monoclonal antibody directed toward Her2, has demonstrated significant activity in advanced

Subcellular Distribution of a Fluorescence-Labeled Combi-Molecule Designed to Block Epidermal Growth Factor Receptor Tyrosine Kinase and Damage DNA with a Green Fluorescent Species

“…This is consistent with our previous observation of selectively high levels of DNA damage induced by the combi-molecule SMA41 in NIH 3T3 cells transfected with EGFR when compared with its wild type (42). A similar observation was made in MDA-MB-435 cells transfected with EGFR or ErbB2 (43). In this study, nuclei of cells transfected with EGFR or its closest homologue ErbB2 emitted higher green and blue fluorescence intensity than those of NIH 3T3 wild type.…”

Target Modulation by a Kinase Inhibitor Engineered to Induce a Tandem Blockade of the Epidermal Growth Factor Receptor (EGFR) and c-Src: The Concept of Type III Combi-Targeting

“…1A , the type I molecule I-Tz was designed to release an EGFR tyrosine kinase inhibitor (I) and a DNA damaging species Tz (step 1). I-Tz was also designed to interact with EGFR as an intact structure (step 2) [ 6 – 8 ]. Conversely, I-Tz in its type II form is designed to inhibit EGFR tyrosine kinase and damage DNA without requirement for hydrolysis ( Fig.…”

Positional Isomerization of A Non‐Cleavable Combi‐Molecule Dramatically Altered Tumor Cell Response Profile

“…Fluorescence study on ZR2003 and ZR2009 cellular distribution ZR2003 and ZR2009 being two fluorescent compounds, we sought to investigate whether their differential reactivity would affect their subcellular distribution. We have shown in previous studies that EGFR inhibitors are preferentially distributed in the perinuclear region of the cells (23).…”