Purpose: The existence of tumor-initiating cells in breast cancer has profound implications for cancer therapy. In this study, we investigated the sensitivity of tumor-initiating cells isolated from human epidermal growth factor receptor type 2 (HER2)-overexpressing carcinoma cell lines to trastuzumab, a compound used for the targeted therapy of breast cancer. Experimental Design: Spheres were analyzed by indirect immunofluorescence for HER2 cell surface expression and by real-time PCR for HER2 mRNA expression in the presence or absence of the Notch1signaling inhibitor (GSI) or Notch1small interfering RNA. Xenografts of HER2-overexpressing breast tumor cells were treated with trastuzumab or doxorubicin. The sphere-forming efficiency (SFE) and serial transplantability of tumors were assessed. Results: In HER2-overexpressing carcinoma cell lines, cells with tumor-initiating cell properties presented increased HER2 levels compared with the bulk cell population without modification in HER2 gene amplification. HER2 levels were controlled by Notch1 signaling, as shown by the reduction of HER2 cell surface expression and lower SFE following g-secretase inhibition or Notch1 specific silencing. We also show that trastuzumab was able to effectively target tumor-initiating cells of HER2-positive carcinoma cell lines, as indicated by the significant decrease in SFE and the loss of serial transplantability, following treatment of HER2-overexpressing xenotransplants. Conclusions: Here, we provide evidence for the therapeutic efficacy of trastuzumab in debulking and in targeting tumor-initiating cells of HER2-overexpressing tumors. We also propose that Notch signaling regulates HER2 expression, thereby representing a critical survival pathway of tumor-initiating cells.

The Cbl proteins are a family of proteins found in metazoans from nematodes to vertebrates. These proteins have several highly conserved domains including an N-terminal tyrosine kinase binding (TKB) 1 domain and a RING finger (1-9). The three mammalian Cbl proteins,2,[6][7][8], are tyrosine-phosphorylated upon activation of a wide variety of growth factor receptors, and they associate with many signaling proteins via SH2 and SH3 interactions (reviewed in Ref. 10 and 11). These diverse interactions modulate signaling through many pathways (10,11). Recent work has shown that c-Cbl-and Cblb-deficient mice have hyperplastic tissues, consistent with a negative regulatory role in cellular proliferation for Cbl proteins (12-15). Together, these data indicate that the Cbl proteins are important regulators of intracellular signaling and consequently of cell function and development.Cbl proteins are negative regulators of epidermal growth factor receptor (EGFR) signaling. This was first shown by genetic studies in Caenorhabditis elegans, which demonstrated that Sli-1 (the C. elegans Cbl homologue) is a negative regulator of the Let-23 receptor tyrosine kinase (the EGFR homologue) in vulva development (3, 16). The Drosophila Cbl protein (D-Cbl) has been shown to associate with the EGFR, and overexpression of D-Cbl in the eye of Drosophila embryos inhibits EGFR-dependent photoreceptor cell development (4, 5). Several studies have shown that mammalian Cbl proteins become phosphorylated and recruited to the EGFR upon stimulation (11, 17) and that they inhibit EGFR function (7, 18 -20).The mechanism underlying the negative regulation of activated tyrosine kinases by Cbl proteins has recently been described. Cbl proteins function as ubiquitin protein ligases, which mediate the ubiquitination of activated tyrosine kinases including the EGFR and target them for degradation (20 -31). Ubiquitination of proteins occurs via the sequential activation and conjugation of ubiquitin to target proteins by the ubiquitinactivating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin protein ligase (E3) (32). The E3 confers specificity to the ubiquitination process. An increasing number of RING finger proteins has been demonstrated to function as E3 proteins or as part of E3 complexes, and in each of them the RING finger is essential to this activity (33-43). The highly conserved TKB and RING finger domains of Cbl proteins are essential and sufficient for their E3 activity, and together these domains target the ubiquitination of activated tyrosine kinases such as the EGFR (20 -31).Here, we show that EGF activation induces a coordinated degradation of the EGFR, Cbl proteins, and other proteins of the EGFR signaling complex. These results suggest that Cbl proteins regulate degradation of multiple proteins in the active EGFR-signaling complex. EXPERIMENTAL PROCEDURESExpression Constructs-The expression plasmid for HA epitopetagged Cbl-b, c-Cbl, and the control vector (pCEFL) have been previously described (18). HA epitope-tagged C...

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The 67-kDa laminin receptor (67LR) is a nonintegrin cell surface receptor that mediates high-affinity interactions between cells and laminin. Overexpression of this protein in tumor cells has been related to tumor invasion and metastasis. Thus far, only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated. The finding that the cDNA for the 37LRP is virtually identical to a cDNA encoding the ribosomal protein p40 has suggested that 37LRP is actually a component of the translational machinery, with no laminin-binding activity. On the other hand, a peptide of 20 amino acids deduced from the sequence of 37LR/p40 was shown to exhibit high laminin-binding activity. The evolutionary relationship between 23 sequences of 37LRP/p40 proteins was analyzed. This phylogenetic analysis indicated that all of the protein sequences derive from orthologous genes and that the 37LRP is indeed a ribosomal protein that acquired the novel function of laminin receptor during evolution. The evolutionary analysis of the sequence identified as the laminin-binding site in the human protein suggested that the acquisition of the laminin-binding capability is linked to the palindromic sequence LMWWML, which appeared during evolution concomitantly with laminin.