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.

Even though the involvement of the 67-kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated so far. A pool of recently obtained monoclonal antibodies directed against the recombinant 37LRP molecule was used to investigate the processing that leads to the formation of the 67-kDa molecule. In soluble extracts of A431 human carcinoma cells, these reagents recognize the precursor molecule as well as the mature 67LR and a 120-kDa molecule. The recovery of these proteins was found to be strikingly dependent upon the cell solubilization conditions: the 67LR is soluble in NP-40-lysis buffer whereas the 37LRP is NP-40-insoluble. Inhibition of 67LR formation by cerulenin indicates that acylation is involved in the processing of the receptor. It is likely a palmitoylation process, as indicated by sensitivity of NP-40-soluble extracts to hydroxylamine treatment. Immunoblotting assays performed with a polyclonal serum directed against galectin3 showed that both the 67- and the 120-kDa proteins carry galectin3 epitopes whereas the 37LRP does not. These data suggest that the 67LR is a heterodimer stabilized by strong intramolecular hydrophobic interactions, carried by fatty acids bound to the 37LRP and to a galectin3 cross-reacting molecule.

The interactions between tumor cells and laminin or other components of the extracellular matrix have been shown to play an important role in tumor invasion and metastasis. However, the role of the monomeric 67-kDa laminin receptor (67LR) remains unclear. We analyzed the regulation of 67LR expression under different culture conditions with respect to the expression of other well characterized laminin receptors. In A431 cells treated with laminin for different time periods, the regulation of 67LR expression correlated with expression of the alpha6 integrin subunit but not with the expression of other laminin receptors. Moreover, cytokine treatment resulted in down-modulated expression of the alpha6 integrin subunit and the 67LR. Co-regulation of the expression of the two receptors was further suggested by the observation that specific down-modulation of the alpha6-chain by antisense oligonucleotides was accompanied by a proportional decrease in the cell surface expression of 67LR. Biochemical analyses indicated co-immunoprecipitation of 67LR and the alpha6 subunit with an anti-alpha6 but not an anti-beta1 monoclonal antibody. Co-regulation of 67LR and alpha6 subunit expression, together with the physical association between the two receptors, supports the hypothesis that 67LR is an auxiliary molecule involved in regulating or stabilizing the interaction of laminin with the alpha6beta4 integrin.

Fhit phosphorylation proliferation ͉ src ͉ proteasome inhibition

In some HER2-positive breast tumors, cell surface overexpression of HER2 is not associated with gene amplification but may instead rest in altered gene transcription, half-life, or recycling of the oncoprotein. Here, we show that HER2 overexpression in HER2 2+ carcinomas is associated with neither an increase in gene transcription nor a deregulation in the ubiquitin-dependent pathways, but instead seems to be regulated by protein kinase Calpha (PKCalpha) activity. The stimulation of PKCalpha up-regulated HER2 expression, whereas PKCalpha inhibition by pharmacologic treatments and PKCalpha-specific small interfering RNA led to a dramatic down-regulation of HER2 levels only in breast cancer cells HER2 2+. Consistent with the in vitro data, our biochemical analysis of HER2 2+ human primary breast specimens revealed significantly higher levels of phosphorylated PKCalpha compared with HER2-negative tumors. Inhibition of HER2 activation by the tyrosine kinase inhibitor lapatinib led to decreased levels of PKCalpha phosphorylation, clearly indicating a cross-talk between PKCalpha and HER2 molecules. These data suggest that HER2 overexpression in HER2 2+ carcinomas is due to an accumulation of the recycled oncoprotein to the cell surface induced by activated PKCalpha.