Preubiquitinated chimeric ErbB2 is constitutively endocytosed and subsequently degraded in lysosomes.

The oncoprotein ErbB2 is endocytosis-deficient, probably due to its interaction with Heat shock protein 90. We previously demonstrated that clathrin-dependent endocytosis of ErbB2 is induced upon incubation of cells with Ansamycin derivatives, such as geldanamycin and its derivative 17-AAG. Furthermore, we have previously demonstrated that a preubiquitinated chimeric EGFR (EGFR-Ub(4)) is constitutively endocytosed in a clathrin-dependent manner. We now demonstrate that also an ErbB2-Ub(4) chimera is endocytosed constitutively and clathrin-dependently. Upon expression, the ErbB2-Ub(4) was further ubiquitinated, and by Western blotting, we demonstrated the formation of both Lys48-linked and Lys63-linked polyubiquitin chains. ErbB2-Ub(4) was constitutively internalized and eventually sorted to late endosomes and lysosomes where the fusion protein was degraded. ErbB2-Ub(4) was not cleaved prior to internalization. Interestingly, over-expression of Ubiquitin Interaction Motif-containing dominant negative fragments of the clathrin adaptor proteins epsin1 and Eps15 negatively affected endocytosis of ErbB2. Altogether, this argues that ubiquitination is sufficient to induce clathrin-mediated endocytosis and lysosomal degradation of the otherwise plasma membrane localized ErbB2. Also, it appears that C-terminal cleavage is not required for endocytosis.

Pertuzumab Increases 17-AAG-Induced Degradation of ErbB2, and This Effect Is Further Increased by Combining Pertuzumab with Trastuzumab.

ErbB2 is an important oncogenic protein involved in carcinogenesis of, among others, breast, gastric, and ovarian carcinoma. Over-expression of ErbB2 is found in almost 20% of breast cancers, and this results in proliferative and anti-apoptotic signalling. ErbB2 is therefore an important treatment target. Antibodies recognizing full-length ErbB2 are clinically established, and drugs targeting the ErbB2 stabilizing heat shock protein 90 (Hsp90) are under clinical evaluation. We have investigated effects of the ErbB2-binding antibodies trastuzumab and pertuzumab alone and in combination, as well as the effect of the antibodies in combination with the Hsp90 inhibitor 17-AAG. Our results confirm the notion that combination of different ErbB2-binding antibodies more efficiently down-regulates ErbB2 than does one antibody in isolation. Additionally, our data demonstrate that ErbB2 is most efficiently down-regulated upon incubation with anti-ErbB2 antibodies in combination with Hsp90 inhibitors. The combination of anti-ErbB2 antibodies, and especially the combination of antibodies with 17-AAG, did also increase the inhibition of Akt activation of either agent, which could suggest an anti-proliferative effect. In such case, combining these agents could be beneficial in treatment of tumors not responding to trastuzumab only.

Pertuzumab increases epidermal growth factor receptor down-regulation by counteracting epidermal growth factor receptor-ErbB2 heterodimerization.

Epidermal growth factor receptor (EGFR) and ErbB2 readily form heterodimers when both are expressed in the same cell and the EGFR is activated by one of its ligands. Our data show that such heterodimers are constitutively formed also in a ligand-independent manner on overexpression of EGFR and ErbB2 in porcine aortic endothelial cells. Interestingly, cross-linking experiments showed that incubation with the antibody pertuzumab, which has been shown to bind the dimerization arm of ErbB2, resulted in dissolution of EGFR-ErbB2 heterodimers. Incubation with pertuzumab also increased the amount of EGF-induced EGFR homodimers, and under these conditions, endocytosis of radiolabeled EGF was increased. This increase was significant, although slightly more EGF was internalized in cells expressing EGFR only compared with pertuzumab-treated cells expressing both EGFR and ErbB2. By confocal microscopy analysis, more EGF was observed in endosomes on incubation with pertuzumab, and under similar conditions, immunoblotting experiments showed increased EGFR degradation on incubation with both EGF and pertuzumab. These results show that pertuzumab enhanced the endocytic down-regulation of EGFR by counteracting EGFR-ErbB2 heterodimerization. Our previous results showing that ErbB2 counteracts EGFR endocytosis can therefore be explained by tethering of EGFR to ErbB2 at the plasma membrane.

Expression of epidermal growth factor receptor or ErbB3 facilitates geldanamycin-induced down-regulation of ErbB2.

Overexpression of the epidermal growth factor receptor (EGFR), ErbB2, and ErbB3 promotes growth and antiapoptotic signaling. Overexpression of ErbB2 in breast cancer is associated with poor clinical outcome, and ways of down-regulating ErbB2 are important as therapeutic approaches. In contrast to EGFR, ErbB2 has been shown to be endocytosis deficient. However, down-regulation of ErbB2 can be induced by incubation of cells with geldanamycin and geldanamycin derivatives, counteracting the stabilizing function of heat shock protein 90 on ErbB2. In the present study, we have made use of stably transfected isogenic cell lines expressing ErbB2 only or ErbB2 together with EGFR and/or ErbB3. We now show that whereas ErbB2 can be down-regulated by incubation with geldanamycin in cells expressing ErbB2 only, the rate of geldanamycin-induced down-regulation increases significantly when the cells additionally express EGFR and/or ErbB3. This increase does, however, not correlate with activation/phosphorylation of ErbB2. The potential of heterodimer formation in ErbB2-positive breast cancer cells could thus turn out to be prognostically predictive with respect to outcome of treatment with geldanamycin derivatives.

Epsin 1 is involved in recruitment of ubiquitinated EGF receptors into clathrin-coated pits.

Epsin consists of an epsin NH(2)-terminal homology domain that promotes interaction with phospholipids, several AP-2-binding sites, two clathrin-binding sequences and several Eps15 homology domain-binding motifs. Epsin additionally possesses ubiquitin-interacting motifs (UIMs) and has been demonstrated to bind ubiquitinated cargo. We therefore investigated whether epsin promoted clathrin-mediated endocytosis of the ubiquitinated EGF receptor (EGFR). By immunoprecipitation, we found that epsin 1 interacted with ubiquitinated EGFR and that functional UIMs were essential for complex formation. Furthermore, RNA interference-mediated knockdown of epsin 1 was found to inhibit internalization of the EGFR, while having no effect on endocytosis of the transferrin receptor. Additionally, upon knockdown of epsin 1, translocation of the EGFR to central parts of clathrin-coated pits was inhibited. This supports the contention that epsin 1 promotes endocytosis of the ubiquitinated EGFR.

Dysregulation of Ack1 inhibits down-regulation of the EGF receptor.

The protein tyrosine kinase Ack1 has been linked to cancer when over-expressed. Ack1 has also been suggested to function in clathrin-mediated endocytosis and in down-regulation of the epidermal growth factor (EGF) receptor (EGFR). We have studied the intracellular localization of over-expressed Ack1 and found that Ack1 co-localizes with the EGFR upon EGF-induced endocytosis in cells with moderate over-expression of Ack. This co-localization is mainly observed in early endosomes. Furthermore, we found that over-expression of Ack1 retained the EGFR at the limiting membrane of early endosomes, inhibiting sorting to inner vesicles of multivesicular bodies. Down-regulation of Ack1 in HeLa cells resulted in reduced rate of (125)I-EGF internalization, whereas internalization of (125)I-transferrin was not affected. In cells where Ack1 had been knocked down by siRNA, recycling of internalized (125)I-EGF was increased, while degradation of (125)I-EGF was inhibited. Together, these data suggest that Ack1 is involved in an early step of EGFR desensitization.

Characterization of the Bacillus cereus Nhe enterotoxin.

The non-haemolytic enterotoxin (Nhe) is one of two three-component enterotoxins responsible for the diarrhoeal food-poisoning syndrome caused by Bacillus cereus. Nhe is composed of NheA, NheB and NheC. The three genes encoding the Nhe components constitute an operon, and the transcriptional start site is located 61 bp upstream of the nheA translational start site. The nhe genes were cloned separately, and expressed in either Bacillus subtilis or Escherichia coli. Separate expression showed that all three components were required for biological activity. In addition, NheA and NheB were purified from B. cereus culture supernatants. As NheC seems to be expressed in only small amounts by B. cereus, NheC was expressed and purified as a histidine-tagged fusion protein. The maximum cytotoxic activity was obtained when the molar ratio between NheA : NheB : His6-NheC was 10 : 10 : 1, and it was shown that NheB was the binding component of the enterotoxin complex.