Herceptin‐induced inhibition of ErbB2 signaling involves reduced phosphorylation of Akt but not endocytic down‐regulation of ErbB2

Abstract

The anti-proliferative effect of the ErbB2 specific antibody Herceptin in cells overexpressing ErbB2 has previously been explained by endocytic downregulation of ErbB2. However, in the following, we demonstrate that Herceptin inhibited proliferation of ErbB2 overexpressing cells without downregulating ErbB2. Herceptin did also not induce endocytosis of ErbB2. Herceptin was found to blunt proliferation of SKBr3 cells overexpressing EGFR, ErbB2, and ErbB3 and expressing functional PTEN, probably by recruiting PTEN to the plasma membrane. Akt was found to be constitutively phosphorylated both in SKBr3 cells overexpressing EGFR, ErbB2 and ErbB3, and in SKOv3 cells, overexpressing EGFR and ErbB2. However, phosphorylation of Akt was inhibited by Herceptin only in SKBr3 cells. SKOv3 cells, which lack the tumour suppressor protein Ras homolog member I, was found to have constitutively phosphorylated mitogen activated protein kinase and functionally increased Ras activity. SKOv3 cells further had low expression levels of PTEN. We thus confirm that the anti-proliferative effect of Herceptin in SKBr3 cells is due to recruitment of PTEN to the plasma membrane and conclude that Herceptin does not blunt phosphatidyl inositol 3 kinase-induced growth in cells with constitutive Ras activity. We further conclude that endocytic downregulation of ErbB2 does not contribute to Herceptin's antiproliferative effect.

Lapatinib, a HER2 tyrosine kinase inhibitor, induces stabilization and accumulation of HER2 and potentiates trastuzumab-dependent cell cytotoxicity

“…The cells with higher HER2 expression showed significantly higher trastuzumab-mediated cytolysis (data not shown). The results are expressed as mean of three independent experiments , others have not (Austin et al, 2004;Hommelgaard et al, 2004;Longva et al, 2005). Interestingly, in some of the models that have not observed trastuzumabmediated receptor downregulation (Austin et al, 2004;Longva et al, 2005), there is also a lack of trastuzumabinduced HER2 phosphorylation.…”

“…The results are expressed as mean of three independent experiments , others have not (Austin et al, 2004;Hommelgaard et al, 2004;Longva et al, 2005). Interestingly, in some of the models that have not observed trastuzumabmediated receptor downregulation (Austin et al, 2004;Longva et al, 2005), there is also a lack of trastuzumabinduced HER2 phosphorylation. It is therefore plausible that kinase activation is a required step for receptor ubiquitination and degradation and that, as a consequence, lapatinib and other receptor TKIs prevent receptor downregulation.…”

Targeting the function of the HER2 oncogene in human cancer therapeutics

“…While some studies show that trastuzumab downregulates HER2 in HER2-overexpressing tumor cells (Cuello et al, 2001;Citri et al, 2002;Lee et al, 2002;Marches and Uhr, 2004), other studies clearly show that it does not (Austin et al, 2004;Longva et al, 2005). Part of the complexity in this field was resolved when it was determined that trastuzumab binds and internalizes with surface HER2 but re-emerges with HER2 at the surface, merely accompanying HER2 passively along its normal endocytic recycling route (Austin et al, 2004).…”

“…Trastuzumab affects the expression of tumor angiogenic factors and exhibits certain anti-angiogenic properties in mouse models (Izumi et al, 2002). Trastuzumab suppresses Akt signaling in some tumor cell types but not others (Normanno et al, 2002;Yakes et al, 2002;Longva et al, 2005), increases plasma phosphatase and tensin homolog (PTEN) localization and activity in cells (Nagata et al, 2004;Longva et al, 2005), and its antiproliferative and antitumor effects are attenuated by PTEN knockdown (Nagata et al, 2004;Fujita et al, 2006). Consistent with a functional role for PTEN in clinical antitumor efficacy, tumors with reduced or absent PTEN are relatively resistant to trastuzumabcontaining chemotherapy regimens (Nagata et al, 2004;Fujita et al, 2006).…”

Endocytic Down-Regulation of ErbB2 Is Stimulated by Cleavage of Its C-Terminus

“…Little attention has been paid to the role of ErbB2 degradation in cancers, although when compromised it also would lead to increased ErbB2 levels and activity. Several studies have shown that endocytic downregulation of ErbB2 is impaired in cancer cells (Sorkin et al, 1993; Baulida et al, 1996;Wang et al, 1999;Hommelgaard et al, 2004;Austin et al, 2004;Longva et al, 2005), and ErbB2 can even transmit this property to the related epidermal growth factor receptor (EGFR; Muthuswamy et al, 1999;Wang et al, 1999;Worthylake et al, 1999;Haslekas et al, 2005). Inhibition of HSP90 (e.g., with geldanamycin [GA]) leads to increased internalization and lysosomal degradation of ErbB2 in a manner depending on proteasomal activity (Tikhomirov and Carpenter, 2000;Austin et al, 2004;Lerdrup et al, 2006).…”