Here we use inducible ErbB2 activation system and 3D cell culture to better understand ErbB2 induced biological effects in human mammary epithelial cells. We demonstrate that ErbB2 uses redundant mechanisms to induce cell proliferation, whereas it is more selective in pathways used to disrupt cell polarity and inhibit cell death. Only activation of Y1201 was as potent as wild type ErbB2 in its ability to disrupt apical-basal polarity and only Y1226/7 is sufficient for inhibition of cell death. The ability of Y1226/7 to inhibit cell death required the signaling adaptor molecule Shc. Although Shc is known regulator of cell proliferation pathways, its role in cell death is not well understood. Thus, we have identified specific autophosphorylation residues that are sufficient to trigger cell biological effects relevant to cancer. In addition, we have defined a novel role for Shc as a regulator of cell death pathways in ErbB2 mediated transformation mammary epithelial cells.
Consistent with previous studies, our results obtained using inducible ErbB2 autophosphorylation site mutants demonstrate that phosphorylation of Y1144, Y1201, Y1226/7 or Y1253 residues is sufficient to induce cell proliferation and activate the MAP Kinase and Akt signaling pathways. These observations highlight the robustness with which ErbB2 signals to induce cell proliferation and transform mammary epithelia. It also raises caution about developing strategies to target pathways downstream of ErbB2 that regulates cell proliferation because it is more likely that it will overcome the inhibition by activating alternate pathways to drive cell proliferation.
ErbB2 was selective in its ability to disrupt polarity. Only activation of Y1201 phenocopied wild type ErbB2, however, Y1144, Y1226/7 and Y1253 are all capable of inducing a partial disruption of polarity (). We have recently shown that ErbB2 requires an interaction with Par6/aPKC a polarity complex to disrupt cell polarity (Aranda et al., 2006
). Consistent with partial to complete disruption of polarity we find that all the autophosphorylation sites retain the capacity to interact with Par6/aPKC (data not shown). Interestingly, ErbB2 also requires the Src kinase to disrupt polarity in MDCK cells (Kim et al., 2005
). Src interacts with the kinase domain of ErbB2, likely to Tyr 882 within the activation loop (Kim et al., 2005
). This tyrosine is intact in all of the autophosphorylation mutants used in this study and can thus explain the ability of Y1144, Y1226/7 and Y1253 mutants to disrupt polarity. However, except Y1201, activation of other tyrosine residues resulted in a partial effect suggesting that Y1201 has unique properties in addition to interacting with Src. Y1201 is known to recruit CrkII and Nck, known substrates of Src kinase and regulators of the cytoskeleton (Buday et al., 2002
; Dankort and Muller, 2000
; Dankort et al., 1997
; Feller, 2001
), raising the possibility that in addition to interacting with Src, recruitment of Src specific substrate(s) to the proximity of the receptor is necessary to induce a complete disruption of polarity. Further analysis will be necessary to test this hypothesis.
Recent studies demonstrate that pathways downstream of Y1226/7 are also sufficient to induce migration (Marone et al., 2004
) and neovasculogenesis (Saucier et al., 2004
). Our results, together with these observations suggest that among the signaling pathways activated by ErbB2, those that are downstream of Y1226/7 uniquely regulate biological process relevant to cell transformation and offer an opportunity for therapeutic intervention.
It is likely that understanding how Shc regulates cell death pathways will offer new therapeutic opportunities for controlling paclitaxel resistance. Our results suggest that the Shc mediated inhibition of apoptosis is independent of activation of the Ras/MAPK signaling pathways because all ErbB2 autophosphorylation site mutants retain the ability to activate Ras/MAPK signaling () but lack the ability to inhibit cell death ( and ). Most known mechanisms of paclitaxel resistance depend on altering the dynamics of the microtubule polymerization (Orr et al., 2003
). Shc can regulate microtubule dynamics by at least three independent mechanisms. Shc interacts with SHIP-2 (Habib et al., 1998
) which has recently been shown to deter microtubule stabilization in mast cells (Leung and Bolland, 2007
). It is possible that the Shc – SHIP-2 interaction leads to destabilization of microtubules. Shc has also been shown to interact directly with actin in PC12 cells (Thomas et al., 1995
) and to cause reorganization of the cytoskeleton (Gu et al., 1999
). Since changes in the actin cytoskeleton can increase resistance to anti-microtubule drugs (Verrills et al., 2006
), it is possible that Shc's interaction with the actin cytoskeleton mediates resistance to taxol. Finally, the Mediator of ErbB2 Driven Cell Motility (MEMO), which binds to ErbB2's Y1226/7, possibly through Shc, is necessary for ErbB2 induced microtubule polymerization in T47D and SkBr3 cells (Marone et al., 2004
). It is also possible that Shc is activating general anti-apoptotic pathways, such as upregulation of Bcl-2, Bfl-1 or downregulation of BH3 proteins, Bim, consistent with previous studies where overexpression of Bcl-2 has been shown to inhibit apoptosis in 3D structures (Debnath et al., 2002
). Interestingly, a recent study showed that expression of a phosphorylation site mutant version of Shc (Y313F) increases cell death in a polyoma virus mT mouse model of mammary tumorigenesis, suggesting that the ability of Shc to regulate cell survival pathways is not limited to ErbB2 dependent tumors (Ursini-Siegel et al., 2008
). A deeper analysis to understand how Shc signals to inhibit paclitaxel induced cell death is likely to have broad clinical benefit.
Resistance to ErbB2 directed therapies in multiple clinical settings highlights the need to develop alternative treatment paradigms for ErbB2 positive tumors. Our results suggest that inhibiting cell death pathways, and not cell proliferation pathways, downstream of ErbB2 as a potentially effective therapeutic strategy since ErbB2 uses non-redundant mechanisms to inhibit cell death. Understanding the pathways downstream of the ErbB2-Shc interaction may not only identify novel combination drug targets but also identify biomarkers for predicting response to chemotherapies.