Hsp90 inhibition and co-incubation with pertuzumab induce internalization and degradation of trastuzumab: Implications for use of T-DM1.

The receptor tyrosine kinase HER2 is associated with a number of human malignancies and is an important therapeutic target. The antibody-drug conjugate trastuzumab emtansine (T-DM1; Kadcyla® ) is recommended as a first-line treatment for patients with HER2-positive metastatic breast cancer. T-DM1 combines the antibody-induced effects of the anti-HER2 antibody trastuzumab (Herceptin® ) with the cytotoxic effect of the tubulin inhibitor mertansine (DM1). For DM1 to have effect, the T-DM1-HER2 complex has to be internalized and the trastuzumab part of T-DM1 has to be degraded. HER2 is, however, considered endocytosis-resistant. As a result of this, trastuzumab is only internalized to a highly limited extent, and if internalized, it is rapidly recycled. The exact reasons for the endocytosis resistance of HER2 are not clear, but it is stabilized by heat-shock protein 90 (Hsp90) and Hsp90 inhibitors induce internalization and degradation of HER2. HER2 can also be internalized upon activation of protein kinase C, and contrary to trastuzumab alone, the combination of two or more anti-HER2 antibodies can induce efficient internalization and degradation of HER2. With intention to find ways to improve the action of T-DM1, we investigated how different ways of inducing HER2 internalization leads to degradation of trastuzumab. The results show that although both Hsp90 inhibition and activation of protein kinase C induce internalization of trastuzumab, only Hsp90 inhibition induces degradation. Furthermore, we find that antibody internalization and degradation are increased when trastuzumab is combined with the clinically approved anti-HER2 antibody pertuzumab (Perjeta® ).

Protein kinase C regulates ErbB3 turnover.

ErbB3, which belongs to the epidermal growth factor receptor (EGFR) or ErbB family of receptor tyrosine kinases, is involved in progression of several human cancers and a tight regulation of its expression is crucial. An important mechanism for regulation of ErbB proteins is endocytosis and we recently showed that ErbB3, contrary to other ErbB proteins, like EGFR and ErbB2, is constitutively internalized and degraded. Several studies show that protein kinase C (PKC) can regulate the activation, localization and stability of EGFR and ErbB2. Activation of PKC causes their down-regulation from the plasma membrane, but instead of being degraded the receptors accumulate in an endosomal recycling compartment. Since little is known about possible connections between ErbB3 and PKC, we have in the present study investigated effects PKC activity has on ErbB3 stability and intracellular trafficking. While PKC inhibition tends to increase ErbB3 degradation, activation of PKC causes ErbB3 stabilization. The stabilization was not due to inhibited internalization, on the contrary we find that expression of ErbB3 at the plasma membrane is reduced upon PMA-induced PKC activation. However, while endocytosed ErbB3 under normal conditions and upon PKC inhibition is found in early endosomal antigen 1 (EEA1) positive early endosomes and lysosomal-associated membrane protein 1 (LAMP1) positive late endosomes/lysosomes, indicating that it follows the classic degradative pathway, ErbB3 localizes to EEA1 and LAMP1 negative compartments upon PMA-induced activation of PKC. Altogether this shows that PKC regulates the stability of ErbB3, and knockdown experiments show that PKCδ is essential in this process. A likely explanation is that PKC regulates endosomal sorting of ErbB3 and that activated PKC sorts ErbB3 away from the degradative pathway.

Protein kinase C mediated internalization of ErbB2 is independent of clathrin, ubiquitination and Hsp90 dissociation.

Overexpression of ErbB2 is frequent in cancer and understanding the mechanisms which regulate its expression is important. ErbB2 is considered endocytosis resistant. It has no identified ligand, but upon heterodimerization it is a potent mediator of proliferative signaling. A recent study established a role for protein kinase C (PKC) in internalization and recycling of ErbB2. We have now further investigated the molecular mechanisms involved in PKC-mediated downregulation of ErbB2. We confirm that PMA-induced PKC activation causes ErbB2 internalization, but while the Hsp90 inhibitor 17-AAG induced ErbB2 degradation, PMA had no such effect. When combined with 17-AAG, PMA had additive effect on ErbB2 internalization indicating that Hsp90 inhibition and PKC activation induce internalization by alternative mechanisms. We confirm that while 17-AAG-induced internalization was clathrin-mediated, PMA-induced internalization was clathrin independent. This difference may be explained by while both 17-AAG and PMA reduced the constitutive tyrosine phosphorylation of ErbB2, only 17-AAG induced Hsp90 dissociation, Hsp70 recruitment and ubiquitination of ErbB2. Importantly, since PMA induced internalization of ErbB2, but not dissociation of Hsp90, Hsp90 does not per se retain ErbB2 at the plasma membrane. The morphology of the compartment into which receptors are sorted upon PKC activation has not previously been identified. By immuno-electron microscopy, we show that PMA sorts ErbB2 into a complex tubulovesicular or cisternal organelle resembling a previously described endocytic recycling compartment.

ErbB3 interacts with Hrs and is sorted to lysosomes for degradation.

The ErbB family of receptor tyrosine kinases mediates activation of a wide network of signaling pathways. ErbB3 has weak kinase activity, but its six docking sites for the p85 subunit of phosphoinositide 3-kinase make it an important contributor to proliferative signaling. ErbB3 has a relatively short half-life but the exact mechanisms controlling its turnover are unclear as contradictory reports exist. ErbB-mediated signaling is, however, negatively regulated by endocytosis of the receptors, followed by either recycling or degradation. Our previous studies showed that ErbB3 can be endocytosed and degraded in the absence of its ligand heregulin. However, binding of heregulin increased the degradation rate. In the current study we have investigated in more detail the trafficking and degradation of ErbB3 in the presence or absence of heregulin. We report that ErbB3 is internalized by clathrin-mediated endocytosis both in the presence and absence of heregulin. Moreover, we show that both proteasomal and lysosomal activity regulate ErbB3 degradation. Although steady-state expression of ErbB3 is regulated by proteasomal activity to a large extent, probably linked to a previously identified ER-localized quantity control, the results indicate that internalization, both constitutive and ligand-induced, causes lysosomal degradation of ErbB3. Furthermore, we show that ErbB3 interacts with the ESCRT-0 subunit Hrs both in the presence and absence of heregulin. This indicates an ESCRT-mediated sorting of ErbB3 to late endosomes and lysosomes, and in line with this we show that impaired ESCRT function leads to an endosomal accumulation of ErbB3.