Enhancement of the p27^Kip1‐mediated antiproliferative effect of trastuzumab (Herceptin) on HER2‐overexpressing tumor cells

Abstract

The oncogenic activity of the overexpressed HER2 tyrosine kinase receptor requires its localization in the plasma membrane. The antitumor effect of anti-HER2 antibodies (Abs) is mainly dependent on receptor downregulation and comprises p27 Kip1 -mediated G 1 cell cycle arrest. However, one major limitation of anti-HER2 therapy is the reversibility of tumor growth inhibition after discontinuation of treatment caused by the mitogenic signaling associated with cell surface receptor re-expression. We found that the level of Key words: HER2; receptor internalization; p27Kip1 ; G 1 arrestThe neu (c-ErbB-2, HER2) proto-oncogene, encoding a 185 kDa RTK, is a member of the EGF receptor family, which includes ErbB-1 (HER1, EGFR), ErbB-3 (HER3) and ErbB-4 (HER4). 1 Activation of RTKs is dependent on ligand-mediated homodimerization and subsequent autophosphorylation and transphosphorylation of the intracellular kinase domains of the paired receptors. However, HER2 may be activated even in the absence of a direct ligand because it forms heterodimeric complexes with its family members HER1, HER3 and HER4 2-7 upon ligand binding to the ectodomain of these receptors. Once recruited into these heterodimeric complexes, HER2 can modulate ligand-mediated receptor internalization to the recycling route and amplify signal transduction. 7-10 The transforming ability of HER2 has been associated with enhanced cell survival and mitogenic signaling pathways. Amplification of HER2 is observed in cancers of the breast, 11-15 ovary, 12,16 lung 17 and stomach; 18 and its overexpression predicts poor prognosis in several human cancers. 11,16,19 -23 At the molecular level, HER2 overexpression can deregulate the G 1 -S transition by downregulating p27Kip1 and stimulating the cyclin E-cdk2 complex. 24 The crucial role of p27 Kip1 in HER2-mediated transformation is consistent with clinical observations that a decreased p27Kip1 protein level in breast cancer is associated with poor prognosis. 25 The key role of HER2 in signaling cell proliferation and transformation made it an attractive therapeutic target in HER2-overexpressing human tumors. MAbs directed against the HER2 ectodomain could specifically inhibit the growth of tumor cells overexpressing HER2. 26 -30 The major mechanism of anti-HER2-mediated inhibition of cell transformation is suppression of HER2 expression on the cell surface 28,31-34 due to Ab-simulated endocytosis of the receptor via coated pits. 35,36 Indeed, retention of nascent HER2 in the endoplasmic reticulum reversed the HER2-mediated transformation associated with cell surface localization. 37 One such potent anti-HER2 Ab, 4D5, 38 was humanized; 39 and the resulting Ab, HCT, is clinically used for the therapy of HER2-overexpressing metastatic breast cancers. 40,41 HCT, like its parental Ab 4D5, markedly downregulates HER2 expression by stimulating receptor endocytosis 42 and thereby arrests cells in the G 1 phase of the cycle via p27 Kip1 . 24,43 However, monotherapy with HCT has limited antitumor effects 44 and requir...

Rational combinations of siRNAs targeting Plk1 with breast cancer drugs

“…As previously described, p27 kip1 mediates G 0 /G 1 arrest and antiproliferative effects in BT-474 and SK-BR-3 cells induced by Herceptin (Marches and Uhr, 2004). Here, we raised the question whether the combinatorial treatment with Herceptin and Plk1-specific siRNA enhances the expression of p27 kip1 significantly compared with the application of single agents.…”

“…Although the treatment of HER2-positive cells with Herceptin alone does not induce apoptosis (Xia et al, 2006), the combination with Plk1-specific siRNAs showed apoptosis based on cleavage of caspases 3 and 9. In addition, after pre-treatment of SK-BR-3 cells with Plk1-specific siRNAs, we observed an induction of p27 kip1 which mediates the G 0 /G 1 arrest and the antiproliferative effect of Herceptin (Lane et al, 2000;Marches and Uhr, 2004;Dubska et al, 2005). One experimental approach to overcome Herceptin resistance is the transfection of p27 kip1 which restores Herceptin sensitivity to resistant cell lines (Nahta et al, 2004).…”

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).…”

“…The antiproliferative effect of mAb 4D5 or trastuzumab in cell-culture models is associated with the induction of p27 and G1 block (Lane et al, 2000;Marches and Uhr, 2004;Le et al, 2005). Trastuzumab affects the expression of tumor angiogenic factors and exhibits certain anti-angiogenic properties in mouse models (Izumi et al, 2002).…”

Decreased Survival of Human Breast Cancer Cells Expressing HER2/neuonIn vitroIncubation with an Anti-HER2/neuAntibody Fused to C5a or C5adesArg

“…Loss of HER2/neu from the cell surface is incompatible with long-term growth of cell lines with high levels of HER2/neu expression such as SK-BR-3 (27), whereas cell lines that express low levels of HER2/neu seem to be less dependent on HER2/neu expression for their survival (27). In fact, an increased rate of HER2/neu internalization is considered an important intrinsic mechanism of trastuzumab action in therapy (28,29). Although we did not explore the mechanism through which the fusion proteins decreased tumor cell survival, it is plausible that fusion to C5a or C5a desArg interfered with recycling of the fusion protein-HER2/neu complex at the cell membrane.…”