Negative feedback loop between p66Shc and ZEB1 regulates fibrotic EMT response in lung cancer cells

The epithelial-to-mesenchymal transition (EMT) program is crucial for the epithelial cancer progression and fibrotic diseases. Our previous work has demonstrated that p66Shc, a focal adhesion-associated adaptor protein, is frequently downregulated in lung cancers and its depletion promotes metastasis behavior through anoikis resistance. However, mechanism underlying loss of p66Shc and EMT response is not fully understood. Here, we showed that p66Shc deficiency enhanced the expression of ZEB1, the known mesenchymal transcription factor and consequently increased Vimentin, and decreased epithelial markers of E-cadherin and β-catenin. p66Shc depletion also increased cell invasion and migration. In addition, ChIP and luciferase assays showed that these effects were directly mediated by ZEB1 repression of p66Shc promoter. Thus, our findings define a critical role of p66Shc in the suppression of fibrotic EMT response with a negative feedback loop between p66Shc and ZEB1 in lung epithelial cancer cells.

Owing to its high metastasis, lung cancer is clinically one of the leading causes of cancer-related death worldwide. 1 Although the exact mechanisms are not well understood, the process of oncogenic epithelial-to-mesenchymal transition (EMT) seems to be involved in these neoplastic processes. [2][3][4] Oncogenic EMT is a developmental program implying a substantial chromatin plasticity and change in gene expression profile for cell reprogramming, 5 and also is an improper occurrence and is associated with the acquisition of a new phenotype, which seems crucial for tumor progression. 6 This process involves loss of cell-cell adhesion and increased cellular mobility, 7 as well as anoikis (detachment-induced cell death) resistance by constitutively activating specific prosurvival signal pathways. [8][9][10][11][12][13] Most of these changes are related to the switch-off or decreased expression of epithelial genes in contrast with a new expression or overexpression of mesenchimal genes and markers, presumably linked to the precise temporal and spatial requirement of EMT in specific cell types at precisely coordinated times during development. 14 Oncogenic EMT induction involves a complex network of signaling molecules and transcription factors, including SNAIL, zinc-finger E-box-binding homeobox 1 (ZEB1) and basic helix-loop-helix transcription factors, as well as a diverse array of cytokines and growth factors including transforming growth factor-β (TGFβ). 6,15 Currently, targeting EMT process remains a very likely candidate for carcinoma metastasis, with opportunities for diagnosis, prognosis, and treatment to be explored. 6 This simplified thought integrates clinically relevant aspects of dormancy, metastatic tendency and therapy resistance. 11 The mammal adaptor proteins Shc1 (p66 Shc , p52 Shc and p46 Shc ) share a C-terminal Src homology 2 domain, a central collagen-homologous (CH1) domain and an N-terminal phosphotyrosine-binding domain and have N termini of different lengths. 16 Despite their high structural similarity, p66 Shc and p52 Shc /p46 Shc are involved in multiple signaling mechanisms to support diversity in cell homeostasis. 17 Of the two Shc1 transcripts and three isoforms, p66 Shc activates pro-apoptosis in response to oxidative stress and functions as a critical regulator of longevity in mice. 18,19 Structurally, an additional 110-amino-acid CH2 domain of p66 Shc at the amino terminal differs from p52 Shc and p46 Shc isoforms. 18 We have previously shown that p66 Shc is also a focal adhesion-associated protein, permits anchorage sensing through a RhoA-dependent mechanosensory test of the environment, mediates anoikis in epithelial cells, and thus has an important role in preventing solid cancer metastasis. 20 In addition, a stress related transcription factor Nrf2 binds to p66 Shc promoter and promotes p66 Shc transcription, which links to reactive oxygen species (ROS) control and tumorigenesis. 19,21 Loss of p66 Shc expression leads to unrestrained k-Ras activation and anoikis resistance, allowing anchorage independence in vitro and aggressive metastasis of lung cancer cells in vivo. 22,23 Anoikis resistance is one of the hallmarks of oncogenic EMT, 24,25 and ZEB1 is required for the neurotrophic tyrosine kinase receptor, TrkBinduced EMT, anoikis resistance and metastasis. 26 Similarly, p66 Shc depletion contributes to anoikis resistance and promotes metastasis in human lung cancer cells. 22 Therefore, it is possible that aberrant expression of p66 Shc may lead to the activation of a developmentally regulated fibrotic EMT response in lung cancer cells.
We report here that metastasis suppressor p66 Shc expression is dependent on cell density in lung cancer cells and represses the oncogenic EMT-activator ZEB1. During oncogenic EMT, p66 Shc is shown to be downregulated, promoting ZEB1 activity, downregulating E-cadherin and β-catenin. Considering the role of p66 Shc in metastasis suppression, we propose that a novel feedback loop between ZEB1 and p66 Shc mediates fibrotic EMT response.

Results
TGFβ1 induces p66 Shc downregulation. TGFβ1 has been traditionally used to investigate the EMT process and fibrotic response in epithelial cancer cells. 2,[27][28][29] As shown in Figures  1a and b, p66 Shc protein level was significantly decreased by time-dependent TGFβ1 treatment in human bronchial epithelial cells (HBECs) and human lung adenocarcinoma A549 cells, respectively. Also, similar tendency was observed in TGFβ1treated hepatocellular cancer HepG2 cells (Supplementary Figure 1). These data indicate that p66 Shc is endogenously regulated for epithelial maintenance and inhibits the TGFβ1induced fibrotic EMT process.
To investigate the function of p66 Shc in fibrotic EMTresponse, we used short hairpin RNA (shRNA) specifically against p66 Shc and created stable p66 Shc knockdown lung cancer A549 cells as performed previously. 30 Immunofluorescence staining of βcatenin showed that knockdown of p66 Shc resulted in a phenotypic change from a 'cobble-stone'-like shape to a fibroblastic shape (Figure 1c, middle panel). A similar cell morphology change with TGFβ1 treatment was observed in Figure 1c, left panel, when compared with the non-treated control cells (Figure 1c, right panel). β-catenin immunostaining showed its dissociation from plasma and its relocalization to the cytosol and nucleus in p66 Shc -depleted cells (Figure 1c, left panel), confirming that p66 Shc was involved in fibrotic EMT response with cell morphological change.
The mammal SHC1 locus contains two alternative promoters, which drive the constitutive transcription of p52/p46 Shc and the differential expression of p66 Shc by epigenetic modification, respectively. 16,23 Previously, we reported that p66 Shc but not p52 Shc expression is increased with cell density in cultured mouse fibroblast NIH 3T3 and MDCK epithelial cells. 20 We hypothesized that p66 Shc may also be regulated by cell density in epithelial lung cancer cells. To test this possibility, we cultured HBECs and A549 cells with different cell density and determined p66 Shc expression by western blot. Indeed, p66 Shc protein level was nearly four fold induction under over confluence when compared to sparse conditions (Figure 1d; Supplementary Figure 2a). Similar results were obtained in neuroendocrine H1155 cells (Supplementary Figure 2b). These findings indicate that the expression level of p66 Shc positively correlates with cell density, which might be closely connected to the cell-cell contact and fibrotic EMT phenotype.
p66 Shc depletion confers the EMT-like phenotype. Given that p66 Shc expression is necessary for maintaining the epithelial phonotype, we then asked whether loss of p66 Shc in cancer cells is related to the EMT-like process. We conducted lentiviral shRNA knockdown of p66 Shc to attenuate its expression in A549 cells and lentiviral overexpression in H1155 cells relative to the empty vector control ( Figure 2a). Depletion of p66 Shc decreased the protein level of E-cadherin and β-catenin and markedly increased the level of the mesenchymal marker Vimentin (Figure 2b). Accordingly, p66 Shc overexpression in H1155 cells showed the opposite tendency compared with the empty vector control (Figure 2b). Exposure of A549 cells to TGFβ1 also decreased the epithelial marker E-cadherin, whereas phosphor-Smad2/3, a downstream target of TGFβ1 pathway increased (Supplementary Figure 3). However, TGFβ1-activated phosphor-Smad2/3 was not activated by p66 Shc depletion in A549 cells or p66 Shc overexpression in H1155 cells ( (Figure 3a), which is consistent with the previous report showing endogenous ZEB1 expression in both A549 and H1155 cells. 31 Less p66 Shc protein level was at least in part related to higher ZEB1 expression level (Figure 3a). In addition, TGFβ1 treatment or ZEB1 overexpression significantly suppressed p66 Shc at the mRNA and protein levels, respectively (Figures 3b and c). We also confirmed that overexpression of ZEB1 bound to the expected ZEB1-binding sites of p66 Shc promoter, and TGFβ1 treatment also induced the similar ZEB1-binding tendency in A549 cells (Figure 3d). Luciferase assay also showed that depletion of ZEB1-binding sites decreased p66 Shc promoter activity in ZEB1overexpressing A549 cells (Figure 3e). Taken together, these data suggest that p66 Shc depletion promotes the EMT-like phenotype, which is at least partially mediated by elevated ZEB1 transcriptional regulation.
p66 Shc depletion increases lung cancer cell invasion and migration. EMT response is well characterized by the loss of cell-cell adhesions and apicobasal polarity, and the transition to a cell type with a more spindle-like morphology that is capable of invading the extracellular matrix (ECM). 32,33 Now that p66 Shc differentially regulates the expression of EMTrelated markers, we inquired whether loss of p66 Shc may modulate cellular motility. As expected, knockdown of p66 Shc in A549 cells increased invasion ability (Figure 4a) as well as migratory potential (Figure 4b). However, overexpression of p66 Shc in A549 cells did not show reverse tendency (Figure 4c). On the other hand, p66 Shc overexpression in H1155 cells resulted in mitigated cell invasion (Supplementary Figure 4). When combined, these data indicate that loss of p66 Shc in lung cancer cells may increase their malignance with increased cell invasion and migration.

Discussion
Here we report the mechanisms of the p66 Shc -maintained epithelial phenotype and demonstrate that depletion of p66 Shc induces fibrotic EMTresponse through ZEB1 activation in lung cancer cells. Significantly, p66 Shc overexpression has a critical role in the establishment of epithelial morphogenesis through upregulating the expression of proteins that are responsible for the formation of cell-cell contacts and cell adhesion. 23 In all of these properties, p66 Shc appears to function as an important suppressor for fibrotic EMT response and its suppressive potential of metastasis in solid tumors. Epithelial plasticity responses depend on a spectrum of intrinsic and/or extrinsic signaling changes and transitions including cell density. 34 Two principle types of metastatic progression have been proposed that are phenotypic plasticity involving transient EMT-MET processes and intrinsic genetic alterations keeping cells in an EMT and stemness state. 11,35 During oncogenic EMT, polarity proteins fail to localize correctly to the membrane, promoting TGFβ signaling, which, in turn, stabilizes the EMT phenotype and the consequent resistance to anoikis. 24,36 The EMT-activator ZEB1, a wellcharacterized EMT transcription factor, is a crucial activator of tumorigenicity and metastasis. 11,37,38 ZEB1 also mediates non-cancer stem cell to cancer stem cell (CSC) conversion, which is consistent with the idea that the EMT generates cells with CSC-like activity. 39,40 The observation that p66 Shc depletion directly upregulates expression of ZEB1 in A549 cells raises the possibility that p66 Shc may function as an upstream or 'master' regulator of fibrotic EMT response. In this context, it is interesting that p66 Shc rather than the other two Shc1 isoforms (Figures 1a  and b) and ZEB1 are all involved in the EMT process, whereas ZEB1 mediates cell fate determination in solid cancer cells. Therefore, p66 Shc and ZEB1 may integrate different environmental cues to guide and maintain the metastatic behavior. ZEB1 is induced in response to TGFβ and WNT proteins as well as TWIST1 cooperatively with SNAIL1 (also known as SNAIL), promoting EMT. 6 Increased ROS induce miR-200c and other miR-200 family members, which targets ZEB1 downregulation; however, in p66 Shc knockout mice skeletal muscle cells, upregulation of miR-200c was inhibited. 41 Furthermore, increased cellular p66 Shc results in further increases in ROS accumulation. 42 Therefore, the integrative role proposed for p66 Shc in normal epithelial makes it a particularly interesting candidate for a role in tumorigenesis by mediating fibrotic EMT response and its targets regulation. Consistent with this scenario, downregualted p66 Shc may increase cancerous stemness as well as fibrotic EMT response in given solid cancer cells. Thus, a negative feedback of p66 Shc and ZEB1 may exist during fibrotic EMT response where p66 Shc has an important role of inside-out signaling of mechanosensory test of anchorage.
Differentiated epithelial and endothelial cells sense their location through specific interactions with the ECM as well as neighboring cells, presumably to prevent cellular vagrancy and consequent tissue disorgamization. 43 Protection from detachment-induced cell death, termed anoikis, facilitates metastasis, which is responsible for most of the clinical patients' death. 44,45 Anoikis resistance, the loss of anoikis sensitivity, was reported to accompany EMT, 9,10 and the adaptor protein p66 Shc has been showed to mediate anoikis through Rho A activation. 20 p66 Shc expression is limited to the  Ras-dependent control of proliferation and survival. 22,46 In solid tumor cells, however, the promoter of p66 Shc is hypermethylated and consequently silenced its expression, which may contribute to the invasion and metastasis cascade. 16,21 Furthermore, lung cancer cells survive anchorage deprivation through repression of p66 Shc and other genes by co-option of Aiolos, a hematopoietic transcription factor, which is required for lymphopoiesis. 23 We have also shown that p66 Shc suppression results in cell cycle arrest in normal epithelial cells secondary to the activation of the tumor suppressor pRB. 22 pRB inactivation contributes to deregulation of E-cadherin and induction of cellular phenotypic changes that are characteristic of the EMT, as well as loss of cell proliferation control. 47 Previously, we also observed variability of p66 Shc downregulation in primary human lung cancer tissues. 21,23 This may represent a correlation with the EMT-MET process and the various expression levels of p66 Shc in human lung tumor tissues. 23 Thus, p66 Shc may inhibit fibrotic EMT response and mediate anoikis and consequently suppresses metastasis.
In summary, we have demonstrated that p66 Shc -maintained epithelial phenotype and demonstrate that p66 Shc depletion induces fibrotic EMT response through ZEB1 activation in lung cancer cells. During this response, ZEB1 associates with p66 Shc promoter and suppresses p66 Shc transcription and promotes cancer cells metastasis by bypassing anoikis. On the other hand, depletion of p66 Shc in metastatic cancer cells increases the expression of ZEB1, leading to the reduced p66 Shc transcription level and disruption of cell-cell contacts. Thus, p66 Shc inhibits fibrotic EMT response and mediates anoikis and consequently suppresses metastasis. For these reasons, additional analyses of the mechanism of p66 Shc reactivation and its consequences of metastatic inhibition are warranted.
Cell culture and viral infection. HBECs were immortalized by overexpression of cyclin-dependent kinase 4 and human telomerase reverse transcriptase and were cultured in keratinocyte serum-free medium (Invitrogen, Karlsruhe, Germany) with 5 ng/ml epidermal growth factor and 50 μg/ml bovine pituitary extract. 48 Phoenix-293, HepG2, H358, H1155, H1299 and A549 cells were obtained from American Type Culture Collection (Manassas, VA, USA). For lentiviral transduction, phoenix-293 cells were cotransfected with the transfer constructs and the third-generation packaging plasmids pMD2.VSVG, pMDLg/pRRE, and pRSV-REV, and fresh supernatant was used for infection. After 8 h infection, cells were washed and allowed to recover for 24 h prior to further procedure. Targeting sequences for human p66 Shc are listed in Supplementary Table 1.
ChIP assay. Chromatin immunoprecipitation (ChIP) assay was performed as previously described. 49 Briefly, cells were cross-linked with 1% formaldehyde for 10 min, quenched with 0.125 M glycine, and lysed on ice for 10 min in cell-swelling buffer containing 5 mM Pipes (pH 8.0), 85 mM KCl, 0.5% NP-40, 0.5 mM phenylmethylsulfonyl fluoride, and 100 ng/ml leupeptin and aprotinin. Nuclei were collected and resuspended in sonication buffer with 1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1), 0.5 mM phenylmethylsulfonyl fluoride, and 100 ng/ml leupeptin and aprotinin and incubated on ice for 10 min. Samples were sonicated to an average length of 0.5 kb and antibodies against ZEB1 (Millipore, Darmstadt, Germany), mouse normal IgG (Santa Cruz, Santa Cruz, CA, USA) were added to each aliquot of chromatin and incubated on a rotating platform overnight at 4°C. Antibody-protein-DNA complexes were isolated by immunoprecipitation with protein A agarose beads. Following extensive washing, bound DNA fragments were eluted and analyzed by subsequent PCR. The putative ZEB1-binding sites from ENCODE were indicated as site A(−1961 to − 1955, relative to the transcription start site of p66 Shc ), B(−1378 to − 1373) and C(−1285 to − 1243), respectively. Primer sequences are listed in Supplementary Table 1.
Luciferase assay. A549 cells grown in 24-well plates were cotransfected with 100 ng of reporter construct, 50 ng of expression vector, and 5 ng of internal control Renilla construct (Promega, Madison, WI, USA) using Lipofectamine 2000 (Invitrogen). Thirty-six hours after transfection, luciferase activity was monitored using the Dual-Luciferase Reporter Assay System and a luminometer (Promega).
Transwell invasion and boyden chamber cell migration assay. Cells (1 × 10 5 ) were plated without serum on 8-μm pore size Transwell filters (Corning, Corning, NY, USA) in the Matrigel invasion assay as described. 23 The Boyden chamber cell migration assay was performed in Boyden chambers without Matrigel as described. 31 Assays were stained and quantified after cells migrated for 48 h.
Wound-healing assay. Migration into wounds was examined on fibronectincoated coverslips. At least five wounded fields per coverslip were analyzed on six coverslips per condition, and identical fields were photographed under phase at 0, 36, and 72 h.
Real-time qRT-PCR. RNA was isolated using RNeasy Mini kit (Qiagen, Hilden, Germany) and used for real-time quantitative reverse transcription (qRT)-PCR using SYBR Green in an ABI PRISM 7500 sequence detection system with a 96-block module and automation accessory (Applied Biosystem, Karlsruhe, Germany). Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control gene. All samples were analyzed in triplicate. The primer sequences are listed in Supplementary Table 1.   Table 1) as indicated by black dots. Vertical red arrows indicate three ZEB1-binding sites, namely A, B and C, respectively (see Materials and methods). Bar graphs show fold enrichment of ZEB1 binding. Data represent the mean ± S.E.M. of three experiments. **Po0.01 as compared with the mouse IgG control. (e) Repressor activity of ZEB1 on p66 Shc promoter in A549 cells. ZEB1 expression plasmid was cotransfected with luciferase reporter containing wild type or p66 Shc promoter with ZEB1-binding site deletion (ΔA, B and C, respectively). Activity (relative luminescence unit, RLU) was normalized to that present in cells transfected with control empty vector. Mean ± S.E.M. of three determinations is shown. *Po0.05 as compared with the empty vector ZEB1 suppresses p66 Shc X Li et al formaldehyde for 15 min, permeabilized with 0.5% Triton X-100 for 10 min, blocked with 10% goat serum for 1 h, incubated with proper primary antibodies for 1 h, with secondary antibodies conjugated with fluorescence dye (Alexa Fluor 555, Invitrogen) for 1 h, and counterstained with 4ʹ,6-diamidino-2-phenylindole for 10 min. At least 50 cells from more than 10 fields were counted for statistical analysis.
Statistical analysis. Data are expressed as mean ± S.E.M. from at least three independent experiments. Statistical analysis was performed with ANOVA for multiple variables and with t-tests for comparison of two groups with normal distribution.

Conflict of Interest
The authors declare no conflict of interest. Figure 4 p66 Shc depletion increases cell invasion and migration. (a) Boyden chamber assay for A549 cells transduced with control or p66 Shc shRNA as described in Materials and methods. Scale bars, 100 μm. Quantification of invasion change are shown in the right panel. **Po0.01 as compared with the cells treated with control shRNA alone. (b) A549 cells were transduced as in a and plated on fibronectin-coated coverslips. Phase-contrast images were obtained immediately after wounding and at 36 and 72 h after wounding. Quantification of mean speed of cells migrating into the wound shown in the right panel. Error bars represent S.E.M. *Po0.05 as compared with the cells treated with control shRNA alone, based on three independent experiments. (c) A549 p66 Shc -overexpressing cells were plated on fibronectin-coated coverslips. Phase-contrast images were obtained immediately after wounding and at 24 and 48 h after wounding as in b, respectively ZEB1 suppresses p66 Shc X Li et al