Dianne S. Hirsch

Growth and Motility Inhibition of Breast Cancer Cells by Epidermal Growth Factor Receptor Degradation Is Correlated with Inactivation of Cdc42

Overexpression of epidermal growth factor receptor (EGFR) contributes to increased cell proliferation and migration in breast cancer. However, mechanisms of EGFR overexpression remain elusive and often cannot be attributed to gene amplification. In NIH3T3 fibroblasts, active Cdc42 inhibits c-Cbl-regulated EGFR degradation to induce cellular transformation. Here, we use two EGFR-overexpressing breast cancer cell lines, MDA-MB-231 and BT20, as models to test the hypothesis that up-regulated Cdc42 activity impairs c-Cbl-mediated EGFR degradation and contributes to EGFR overexpression. We show that silencing Cdc42 significantly reduces protein levels of EGFR, leading to a marked reduction in cell proliferation and migration, and c-Cbl knockdown increases the levels of EGFR. Expression of c-Cbl-N480, a c-Cbl mutant that is not regulated by Cdc42 and blocks Cdc42-induced transformation but still binds and ubiquitinates EGFR, enhances the rate of EGFR degradation and subsequently inhibits cell proliferation. Moreover, down-regulated EGFR signaling induced by c-Cbl-N480 decreased activity of Cdc42 and Rac1, resulting in inhibition of cell migration. These findings indicate that Cdc42 and c-Cbl are critical components involved in the regulation of EGFR protein levels and that restoration of proper EGFR degradation by disrupting Cdc42 regulation of c-Cbl can reduce cell proliferation and migration in MDA-MB-231 and BT20 cells.

Rac1 contributes to trastuzumab resistance of breast cancer cells: Rac1 as a potential therapeutic target for the treatment of trastuzumab-resistant breast cancer

Although treatment with trastuzumab improves outcomes for women with ErbB2-positive breast cancer, many patients who achieve an initial response to trastuzumab subsequently acquire resistance within 1 year. Rac1, a Ras-like small GTPase, has been implicated in the control of cell growth and morphology and is believed to be associated with breast cancer progression and metastasis. Here, we show that when parental SKBR3 cells become resistant to trastuzumab, Rac1 activity is increased, leading to altered cell morphology, which is accompanied by significant cytoskeleton disorganization. Furthermore, both trastuzumab-mediated down-regulation of ErbB2 and epidermal growth factor–induced down-regulation of epidermal growth factor receptor are impaired in the trastuzumab-resistant SKBR3 cells, indicating that the endocytic down-regulation of ErbB receptors is compromised in the resistant cells. This results in an aberrant accumulation of ErbB2 on the cell surface and enhanced ErbB2 and extracellular signal-regulated kinase activity in trastuzumab-resistant SKBR3 cells. Additionally, overexpression of constitutively active Rac1G12V in parental SKBR3 cells reduces sensitivity to trastuzumab. After reduction of Rac1 activity by NSC23766, a specific Rac1 inhibitor, trastuzumab-resistant SKBR3 cells display a cellular morphology similar to parental SKBR3 cells. Moreover, we show that NSC23766 restores trastuzumab-mediated endocytic down-regulation of ErbB2 and reduces extracellular signal-regulated kinase activity in resistant SKBR3 cells. Our findings highlight an important role for Rac1 in trastuzumab resistance of human breast cancer cells and identify the impaired trastuzumab-mediated endocytic down-regulation of ErbB2 as a novel mechanism of trastuzumab resistance. The significant effects of NSC23766 on trastuzumab-resistant SKBR3 cells warrant further study of NSC23766 as a potential treatment of trastuzumab-resistant breast cancers. [Mol Cancer Ther 2009;8(6):1557–69]

Trastuzumab Alters the Expression of Genes Essential for Cardiac Function and Induces Ultrastructural Changes of Cardiomyocytes in Mice

Treatment with trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of Human Epidermal Growth Factor Receptor 2 (HER2), very successfully improves outcomes for women with HER2-positive breast cancer. However, trastuzumab treatment was recently linked to potentially irreversible serious cardiotoxicity, the mechanisms of which are largely elusive. This study reports that trastuzumab significantly alters the expression of myocardial genes essential for DNA repair, cardiac and mitochondrial functions, which is associated with impaired left ventricular performance in mice coupled with significant ultrastructural alterations in cardiomyocytes revealed by electron microscopy. Furthermore, trastuzumab treatment also promotes oxidative stress and apoptosis in myocardium of mice, and elevates serum levels of cardiac troponin-I (cTnI) and cardiac myosin light chain-1 (cMLC1). The elevated serum levels of cMLC1 in mice treated with trastuzumab highlights the potential that cMLC1 could be a useful biomarker for trastuzumab-induced cardiotoxicity.

Trastuzumab regulates IGFBP-2 and IGFBP-3 to mediate growth inhibition: implications for the development of predictive biomarkers for trastuzumab-resistance

Activation of insulin-like growth factor-I receptor (IGF-IR) signaling is an important mechanism for trastuzumab resistance. IGF-binding proteins (IGFBP) modulate IGF-IR signaling and play important roles in the control of breast cancer progression. In this article, we report that trastuzumab treatment enhances the expression and secretion of IGFBP-3 in SKBR3 cells, a trastuzumab-sensitive breast cancer cell line, and that this upregulation of IGFBP-3 induced by trastuzumab correlates with trastuzumab-mediated growth inhibition. We describe a new role for IGFBP-3 in the regulation of IGF-I–mediated cross-talk between IGF-IR and ErbB2 signaling pathways. In particular, treatment of SKBR3 cells with recombinant IGFBP-3 blocks IGF-I–induced activation of IGF-IR and ErbB2, and stable expression of IGFBP-3 inhibits SKBR3 cell growth. We find an inverse relationship in the levels of secreted IGFBP-3 such that high levels of IGFBP-3 are associated with trastuzumab-sensitive breast cancer cells (SKBR3 and BT-474), whereas low levels of IGFBP-3 are found in trastuzumab-resistant cells (clone 3 and JIMT-1). In contrast to IGFBP-3, the secretion and expression of IGFBP-2 are upregulated in trastuzumab-resistant SKBR3 cells. Furthermore, we show that IGFBP-2 stimulates activation of ErbB2 and that trastuzumab reduces IGFBP-2–stimulated ErbB2 activation. Based on our data, we propose a novel mechanism of action whereby trastuzumab enhances the expression and secretion of IGFBP-3, which interferes with IGF-I–mediated mitogenic signaling via autocrine and paracrine mechanisms and reduces IGFBP-2–induced ErbB2 activation to mediate growth inhibition. Changes in secretion profiles of IGFBP-2 and IGFBP-3 in trastuzumab-sensitive and trastuzumab-resistant cells may promote the development of IGFBP-2 and IGFBP-3 as predictive biomarkers for trastuzumab resistance. Mol Cancer Ther; 10(6); 917–28. ©2011 AACR.

Trastuzumab-induced recruitment of Csk-homologous kinase (CHK) to ErbB2 receptor is associated with ErbB2-Y1248 phosphorylation and ErbB2 degradation to mediate cell growth inhibition

The inhibitory effect of trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of ErbB2, is associated with its ability to induce ErbB2-Y1248 phosphorylation, and the status of phosphorylated ErbB2-Y1248 (ErbB2-pY1248) may correlate with the sensitivity of breast cancers to trastuzumab. The mechanisms of which remain unclear. Here, we show that binding of trastuzumab to ErbB2 activates ErbB2 kinase activity and enhances ErbB2-Y1248 phosphorylation in trastuzumab-sensitive breast cancer cells. This in turn increases the interaction between ErbB2 and non-receptor Csk-homologous kinase (CHK), leading to growth inhibition of breast cancer cells. Overexpression of CHK mimics trastuzumab treatment to mediate ErbB2-Y1248 phosphorylation, Akt downregulation, and growth inhibition of trastuzumab-sensitive breast cancer cells. CHK overexpression combined with trastuzumab exerts an additive effect on cell growth inhibition. We further demonstrate that positive ErbB2-pY1248 staining in ErbB2-positive breast cancer biopsies correlates with the increased trastuzumab response in trastuzumab neoadjuvant settings. Collectively, this study highlights an important role for ErbB2-pY1248 in mediating trastuzumab-induced growth inhibition and trastuzumab-induced interactions between CHK and ErbB2-pY1248 is identified as a novel mechanism of action that mediates the growth inhibition of breast cancer cells. The novel mechanistic insights into trastuzumab action revealed by this study may impact the design of next generation of therapeutic monoclonal antibodies targeting receptor tyrosine kinases, as well as open new avenues to identify novel targets for the treatment of ErbB2-positive cancers.

pp60c-Src Phosphorylates and Activates Vacuolar Protein Sorting 34 to Mediate Cellular Transformation

Vacuolar protein sorting 34 (VPS34) contributes to the regulation of the mammalian target of rapamycin complex 1/S6 kinase 1 pathway downstream of nutrient signaling. However, intracellular mechanisms leading to VPS34 activation remain unclear. Here, we report that Src directly phosphorylates VPS34, and that this phosphorylation activates VPS34 lipid kinase activity, leading to Src-Y527F-mediated cellular transformation. Silencing endogenous VPS34 specifically inhibits Src-Y527F-induced colony formation in soft agar, but not Ras-G12V-induced colony formation. We have identified two novel hVPS34 mutations, which either eliminate lipid kinase activity (kinase-dead mutant) or reduce tyrosine phosphorylation by Src-Y527F. When kinase-dead mutant of hVPS34 is stably expressed in Src-Y527F-transformed cells, transformation activities are blocked, indicating that the lipid kinase activity of hVPS34 is essential for Src-mediated cellular transformation. Furthermore, stable expression of this hVPS34 kinase-dead mutant causes an increased number of binucleate and multinucleate cells, suggesting that the kinase activity of hVPS34 is also required for cytokinesis. Moreover, when the hVPS34 mutant that has reduced tyrosine phosphorylation by Src is stably expressed in Src-Y527F-transformed cells, Src-Y527F-stimulated colony formation is also reduced. Data presented here provide important evidence that VPS34 lipid kinase activity could be positively regulated by Src-mediated tyrosine phosphorylation in mammalian cells. This finding highlights a previously unappreciated relationship between VPS34, a class III phosphatidylinositol-3-kinase, and Src non-receptor tyrosine kinase. Additionally, we find that the levels of VPS34 expression and tyrosine phosphorylation are correlated with the tumorigenic activity of human breast cancer cells, indicating that Src to VPS34 signaling warrants further investigation as a pathway contributing to the development and progression of human cancers.

Small GTPase Rho regulates R-cadherin through Dia1/profilin-1.

R-cadherin is a member of the classical cadherins. Though much is known about E-cadherin in adherens junction formation in epithelial cells, the role of R-cadherin in epithelial cells remains elusive. This study examines regulation of R-cadherin adherens junctions by the small GTPase Rho and its downstream effectors in MDA-MB-231 breast cancer cells, MDA-MB-231 cells stably expressing the N-terminus of c-Cbl, and MCF10A normal breast epithelial cells. We find that the small GTPase Rho regulates R-cadherin adherens junction formation via Dia1 (also known as p140mDia) and profilin-1-mediated signaling pathway. The role played by Rho in regulating R-cadherin is underscored by the fact that constitutively active RhoA(Q63L) induces R-cadherin junction formation in MDA-MB-231 cells. Importantly, R-cadherin adherens junction formation facilitates a mesenchymal to epithelial-like transition in MDA-MB-231 cells. Additionally, our data suggest an inverse relationship between EGFR signaling and R-cadherin adherens junction formation. Taken together, results from this study indicate that R-cadherin is a critical regulator of epithelial phenotype.

Insulin activation of vacuolar protein sorting 34 mediates localized phosphatidylinositol 3-phosphate production at lamellipodia and activation of mTOR/S6K1

The class III phosphatidylinositol 3-kinase, VPS34, phosphorylates the D3 hydroxyl of inositol generating phosphatidylinositol 3-phosphate (ptdins(3)p). Initial studies suggested that ptdins(3)p solely functioned as a component of vesicular and endosomal membranes and that VPS34 did not function in signal transduction. However, VPS34 has recently been shown to be required for insulin-mediated activation of S6 kinase 1 (S6K1). Whether VPS34 activity is directly regulated by insulin is unclear. It is also not known whether VPS34 activity can be spatially restricted in response to extracellular stimuli. Data presented here demonstrate that in response to insulin, VPS34 is activated and translocated to lamellipodia where it produces ptdins(3)p. The localized production of ptdins(3)p is dependent on Src phosphorylation of VPS34. In cells expressing VPS34 with mutations at Y231 or Y310, which are Src-phosphorylation sites, insulin-stimulated VPS34 translocation to the plasma membrane and lamellipodia formation are blocked. mTOR also colocalizes with VPS34 and ptdins(3)p at lamellipodia following insulin-stimulation. In cells expressing the VPS34-Y231F mutant, which blocks lamellipodia formation, mTOR localization at the plasma membrane and insulin-mediated S6K1 activation are reduced. This suggests that mTOR localization at lamellipodia is important for full activation of S6K1 induced by insulin. These data demonstrate that insulin can spatially regulate VPS34 activity through Src-mediated tyrosine phosphorylation and that this membrane localized activity contributes to lamellipodia formation and activation of mTOR/S6K1signaling.

Mechanism of E-cadherin lysosomal degradation

We read with great interest the Review by Yaron Mosesson, Gordon B. Mills and Yosef Yarden1 (Derailed endocytosis: an emerging feature of cancer. Nature Rev. Cancer 8, 835– 850 (2008)) as it is highly relevant to our research. Experimental evidence indicates that epithelial (E)-cadherin ubiquitylation and degradation is an important mechanism contributing to the metastasis of epitheliumderived cancers. We would like to bring to your attention three articles that provide experimental evidence in support of the model of E-cadherin lysosomal degradation that is proposed in FIG. 3 of this Review. In the legend to FIG. 3, the authors state that, following growth factor-induced activation of SRC, “SRC tags E-cadherin with phosphotyrosine. This is followed by ubiquitylation of E-cadherin by an E3 ligase called Hakai, thereby biasing degradation of E-cadherin and diminishing recycling through RAB11-containing recycling endosomes.” Within the text, the authors properly cited Fujita et al.2, who originally identified Hakai as an E3 ligase that ubiquitylates E-cadherin, leading to E-cadherin endocytosis. However, key studies that support the authors’ proposed endocytic mechanisms underlying lysosomal degradation of E-cadherin leading to disruption of adherens junctions and loss of tumour cell polarity were not cited. These recent publications have significantly contributed to defining the mechanisms of lysosomal degradation of internalized E-cadherin. Palacios et al.3 provided evidence that, following SRC expression, E-cadherin is internalized and then shuttled to the lysosomes for degradation resulting in reduced recycling of E-cadherin back to the lateral membrane. This study also showed that E-cadherin ubiquitylation is essential for the shuttling of E-cadherin to lysosomes. The studies by Janda et al.4 indicate that early stages of epithelial–mesenchymal transition involve cooperative, post-translational downregulation of E-cadherin. Recently, Shen et al.5 reported that CDC42, a Ras family small GTP-binding protein, regulates E-cadherin ubiquitylation and lysosomal degradation through a pathway mediated by epidermal growth factor receptor followed by SRC. Data from this study support a model that activation of CDC42 contributes to the development of a mesenchyme-like phenotype in the well-differentiated breast cancer cells by targeting E-cadherin for lysosomal degradation. Taken together, these studies established the machinery of lysosomal degradation of internalized E-cadherin in response to mitogens and oncogenic signals and should be cited to provide experimental evidence to support the model proposed by Mosesson et al. in their Review.

VPS34 regulates TSC1/TSC2 heterodimer to mediate RheB and mTORC1/S6K1 activation and cellular transformation

VPS34 is reported to activate S6K1 and is implicated in regulating cell growth, the mechanisms of which remain elusive. Here, we describe novel mechanisms by which VPS34 upregulates mTOR/S6K1 activity via downregulating TSC2 protein and activating RheB activity. Specifically, upregulation of VPS34 lipid kinase increases local production of ptdins(3)p in the plasma membrane, which recruits PIKFYVE, a FYVE domain containing protein, to ptdins(3)p enriched regions of the plasma membrane, where VPS34 forms a protein complex with PIKFYVE and TSC1. This in turn disengages TSC2 from the TSC1/TSC2 heterodimer, leading to TSC2 ubiquitination and degradation. Downregulation of TSC2 promotes the activation of RheB and mTOR/S6K1. When VPS34 lipid kinase activity is increased by introduction of an H868R mutation, ptdins(3)p production at the plasma membrane is dramatically increased, which recruits more PIKFYVE and TSC1 molecules to the plasma membrane. This results in the enhanced TSC2 ubiquitination and degradation, and subsequent activation of RheB and mTORC1/S6K1, leading to oncogenic transformation. The role played by VPS34 in regulating mTOR/S6K1 activity and cellular transformation is underscored by the fact that the VPS34 kinase dead mutant blocks VPS34-induced recruitment of PIKFYVE and TSC1 to the plasma membrane. This study provides mechanistic insight into the cellular function of VPS34 in regulating oncogenic transformation and important indications for identifying VPS34 specific mutations in human cancers.