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The classic mode of G protein‐coupled receptor (GPCR)‐mediated transactivation of the receptor tyrosine kinase epidermal growth factor receptor (EGFR) transactivation occurs via matrix metalloprotease (MMP)‐mediated cleavage of plasma membrane‐anchored EGFR ligands. Herein, we show that the Gαs‐activating GPCR ligands vasoactive intestinal peptide (VIP) and prostaglandin E2 (PGE2) transactivate EGFR through increased cell‐surface delivery of the EGFR ligand transforming growth factor‐α (TGFα) in polarizing madin‐darby canine kidney (MDCK) and Caco‐2 cells. This is achieved by PKA‐mediated phosphorylation of naked cuticle homolog 2 (NKD2), previously shown to bind TGFα and direct delivery of TGFα‐containing vesicles to the basolateral surface of polarized epithelial cells. VIP and PGE2 rapidly activate protein kinase A (PKA) that then phosphorylates NKD2 at Ser‐223, a process that is facilitated by the molecular scaffold A‐kinase anchoring protein 12 (AKAP12). This phosphorylation stabilized NKD2, ensuring efficient cell‐surface delivery of TGFα and increased EGFR activation. Thus, GPCR‐triggered, PKA/AKAP12/NKD2‐regulated targeting of TGFα to the cell surface represents a new mode of EGFR transactivation that occurs proximal to ligand cleavage by MMPs.

As a key process within the tissue microenvironment, integrin signaling can influence cell functional responses to growth factor stimuli. We show here that clustering of integrin α5ß1 at the plasma membrane of colorectal cancer-derived epithelial cells modulates their ability to respond to stimulation by receptor tyrosine kinase (RTK)-activating growth factors EGF, NRG and HGF, through GSK3-mediated suppression of Akt pathway. We observed that integrin α5ß1 is lost from the membrane of poorly organized human colorectal tumors and that treatment with the integrin-clustering antibody P4G11 is sufficient to induce polarity in a mouse tumor xenograft model. While adding RTK growth factors (EGF, NRG and HGF) to polarized colorectal cancer cells induced invasion and loss of monolayer formation in 2D and 3D, this pathological behavior could be blocked by P4G11. Phosphorylation of ErbB family members as well as MET following EGF, NRG and HGF treatment was diminished in cells pretreated with P4G11. Focusing on EGFR, we found that blockade of integrin α5ß1 increased EGFR phosphorylation. Since activity of multiple downstream kinase pathways were altered by these various treatments, we employed computational machine learning techniques to ascertain the most important effects. Partial least-squares discriminant analysis identified GSK3 as a major regulator of EGFR pathway activities influenced by integrin α5ß1. Moreover, we used partial correlation analysis to examine signaling pathway crosstalk downstream of EGF stimulation and found that integrin α5ß1 acts as a negative regulator of the AKT signaling cascade downstream of EGFR, with GSK3 acting as a key mediator. We experimentally validated these computational inferences by confirming that blockade of GSK3 activity is sufficient to induce loss of polarity and increase of oncogenic signaling in the colonic epithelial cells.

Integrin α5 clustering at the lateral membrane restores epithelial polarity in invasive colorectal cancer cells via deposition and polymerization of fibronectin and recruitment of paxillin to cluster sites, followed by tight junction formation., Apicobasolateral polarity is a fundamental property of epithelial cells, and its loss is a hallmark of cancer. Integrin-mediated contact with the extracellular matrix defines the basal surface, setting in motion E-cadherin–mediated cell–cell contact, which establishes apicobasolateral polarity. Role(s) for lateral integrins in this polarization process and the consequences of their disruption are incompletely understood. We show that addition of an integrin β1–activating monoclonal antibody, P4G11, to invasive colorectal cancer cells in three-dimensional type 1 collagen reverts the invasive phenotype and restores apicobasolateral polarity. P4G11 induces clustering of integrin α5β1 at lateral, intercellular surfaces. This leads to deposition and polymerization of fibronectin and recruitment of paxillin to sites of lateral integrin α5β1 clustering and is followed by tight junction formation, as determined by ZO-1 localization. Inducible elimination of integrin α5 abrogates the epithelial-organizing effects of P4G11. In addition, polymerization of fibronectin is required for the effects of P4G11, and addition of polymerized superfibronectin is sufficient to induce tight junction formation and apicobasolateral polarization. In the normal human colon, we show that integrin α5 localizes to the lateral membrane of terminally differentiated colonocytes and that integrin α5 staining may be reduced in colorectal cancer. Thus we propose a novel role for integrin α5β1 in regulating epithelial morphogenesis.

We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor 15-PGDH/HPGD, and the most up-regulated gene in SC was versican (VCAN) in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.

The epithelial-to-mesenchymal transition (EMT) transcriptional program is characterized by repression of E-cadherin (CDH1) and induction of N-cadherin (CDH2), and mesenchymal genes like vimentin (VIM). Placenta-specific 8 (PLAC8) has been implicated in colon cancer; however, how PLAC8 contributes to disease is unknown, and endogenous PLAC8 protein has not been studied. We analyzed zebrafish and human tissues and found that endogenous PLAC8 localizes to the apical domain of differentiated intestinal epithelium. Colon cancer cells with elevated PLAC8 levels exhibited EMT features, including increased expression of VIM and zinc finger E-box binding homeobox 1 (ZEB1), aberrant cell motility, and increased invasiveness. In contrast to classical EMT, PLAC8 overexpression reduced cell surface CDH1 and upregulated P-cadherin (CDH3) without affecting CDH2 expression. PLAC8-induced EMT was linked to increased phosphorylated ERK2 (p-ERK2), and ERK2 knockdown restored cell surface CDH1 and suppressed CDH3, VIM, and ZEB1 upregulation. In vitro, PLAC8 directly bound and inactivated the ERK2 phosphatase DUSP6, thereby increasing p-ERK2. In a murine xenograft model, knockdown of endogenous PLAC8 in colon cancer cells resulted in smaller tumors, reduced local invasion, and decreased p-ERK2. Using MultiOmyx, a multiplex immunofluorescence-based methodology, we observed coexpression of cytosolic PLAC8, CDH3, and VIM at the leading edge of a human colorectal tumor, supporting a role for PLAC8 in cancer invasion in vivo.

Mutations in the PKHD1 gene result in autosomal recessive polycystic kidney disease (ARPKD) in humans. To determine the molecular mechanism of the cystogenesis in ARPKD, we recently generated a mouse model for ARPKD that carries a targeted mutation in the mouse orthologue of human PKHD1. The homozygous mutant mice display hepatorenal cysts whose phenotypes are similar to those of human ARPKD patients. By littermates of this mouse, we developed two immortalized renal collecting duct cell lines with Pkhd1 and two without. Under nonpermissive culture conditions, the Pkhd1(-/-) renal cells displayed aberrant cell-cell contacts and tubulomorphogenesis. The Pkhd1(-/-) cells also showed significantly reduced cell proliferation and elevated apoptosis. To validate this finding in vivo, we examined proliferation and apoptosis in the kidneys of Pkhd1(-/-) mice and their wildtype littermates. Using proliferation (PCNA and Histone-3) and apoptosis (TUNEL and caspase-3) markers, similar results were obtained in the Pkhd1(-/-) kidney tissues as in the cells. To identify the molecular basis of these findings, we analyzed the effect of Pkhd1 loss on multiple putative signaling regulators. We demonstrated that the loss of Pkhd1 disrupts multiple major phosphorylations of focal adhesion kinase (FAK), and these disruptions either inhibit the Ras/C-Raf pathways to suppress MEK/ERK activity and ultimately reduce cell proliferation, or suppress PDK1/AKT to upregulate Bax/caspase-9/caspase-3 and promote apoptosis. Our findings indicate that apoptosis may be a major player in the cyst formation in ARPKD, which may lead to new therapeutic strategies for human ARPKD.

Autosomal recessive polycystic kidney disease is caused by mutations in PKHD1, which encodes the membrane-associated receptor-like protein fibrocystin/polyductin (FPC). FPC associates with the primary cilia of epithelial cells and co-localizes with the Pkd2 gene product polycystin-2 (PC2), suggesting that these two proteins may function in a common molecular pathway. For investigation of this, a mouse model with a gene-targeted mutation in Pkhd1 that recapitulates phenotypic characteristics of human autosomal recessive polycystic kidney disease was produced. The absence of FPC is associated with aberrant ciliogenesis in the kidneys of Pkhd1-deficient mice. It was found that the COOH-terminus of FPC and the NH2-terminus of PC2 interact and that lack of FPC reduced PC2 expression but not vice versa, suggesting that PC2 may function immediately downstream of FPC in vivo. PC2-channel activities were dysregulated in cultured renal epithelial cells derived from Pkhd1 mutant mice, further supporting that both cystoproteins function in a common pathway. In addition, mice with mutations in both Pkhd1 and Pkd2 had a more severe renal cystic phenotype than mice with single mutations, suggesting that FPC acts as a genetic modifier for disease severity in autosomal dominant polycystic kidney disease that results from Pkd2 mutations. It is concluded that a functional and molecular interaction exists between FPC and PC2 in vivo.

Mutations of the polycystic kidney and hepatic disease 1 ( PKHD1 ) gene have been shown to cause autosomal recessive polycystic kidney disease (ARPKD), but the cellular functions of the gene product (PKHD1) remain uncharacterized. To illuminate its properties, the spatial and temporal expression patterns of PKHD1 were determined in mouse, rat, and human tissues by using polyclonal Abs and mAbs recognizing various specific regions of the gene product. During embryogenesis, PKHD1 is widely expressed in epithelial derivatives, including neural tubules, gut, pulmonary bronchi, and hepatic cells. In the kidneys of the pck rats, the rat model of which is genetically homologous to human ARPKD, the level of PKHD1 was significantly reduced but not completely absent. In cultured renal cells, the PKHD1 gene product colocalized with polycystin-2, the gene product of autosomal dominant polycystic disease type 2, at the basal bodies of primary cilia. Immunoreactive PKHD1 localized predominantly at the apical domain of polarized epithelial cells, suggesting it may be involved in the tubulogenesis and/or maintenance of duct–lumen architecture. Reduced PKHD1 levels in pck rat kidneys and its colocalization with polycystins may underlie the pathogenic basis for cystogenesis in polycystic kidney diseases.

Cyto-nuclear shuttling of β-catenin is at the epicenter of the canonical Wnt pathway and mutations in genes that result in excessive nuclear accumulation of β-catenin are the driving force behind the initiation of many cancers. Recently, Naked Cuticle homolog 1 (Nkd1) has been identified as a Wnt-induced intracellular negative regulator of canonical Wnt signaling. The current model suggests that Nkd1 acts between Disheveled (Dvl) and β-catenin. Here, we employ the zebrafish embryo to characterize the cellular and biochemical role of Nkd1 in vivo. We demonstrate that Nkd1 binds to β-catenin and prevents its nuclear accumulation. We also show that this interaction is conserved in mammalian cultured cells. Further, we demonstrate that Nkd1 function is dependent on its interaction with the cell membrane. Given the conserved nature of Nkd1, our results shed light on the negative feedback regulation of Wnt signaling through the Nkd1-mediated negative control of nuclear accumulation of β-catenin.

Loss of polycystin-2 (PC2) in mice (Pkd2(-/-)) results in total body edema, focal hemorrhage, structural cardiac defects, abnormal left-right axis, hepatorenal and pancreatic cysts, and embryonic lethality. The molecular mechanisms by which loss of PC2 leads to these phenotypes remain unknown. We generated a model to allow targeted Pkd2 inactivation using the Cre-loxP system. Global inactivation of Pkd2 produced a phenotype identical to Pkd2(-/-) mice with undetectable PC2 protein and perinatal lethality. Using various Cre mouse lines, we found that kidney, pancreas, or time-specific deletion of Pkd2 led to cyst formation. In addition, we developed an immortalized renal collecting duct cell line with inactive Pkd2; these cells had aberrant cell-cell contact, ciliogenesis, and tubulomorphogenesis. They also significantly upregulated beta-catenin, axin2, and cMyc. Our results suggest that loss of PC2 disrupts normal behavior of renal epithelial cells through dysregulation of beta-catenin-dependent signaling, revealing a potential role for this signaling pathway in PC2-associated ADPKD.

Autosomal dominant (ADPKD) and autosomal recessive (ARPKD) polycystic kidney disease are caused by mutations in Pkd1/Pkd2 and Pkhd1, which encode polycystins (PCs) and fibrocystin/polyductin (FPC). Our recent study reported that a deficiency in FPC increases the severity of cystic disease in Pkd2 mutants and down-regulates PC2 in vivo, but the precise molecular mechanism of these effects is unknown (Kim, I., Fu, Y., Hui, K., Moeckel, G., Mai, W., Li, C., Liang, D., Zhao, P., Ma, J., Chen, X.-Z., George, A. L., Jr., Coffey, R. J., Feng, Z. P., and Wu, G. (2008) J. Am. Soc. Nephrol. 19, 455–468). In this study, through the use of deletion and mutagenesis strategies, we identified a PC2-binding domain in the intracellular C terminus of FPC and an FPC-binding domain in the intracellular N terminus of PC2. These binding domains provide a molecular basis for the physical interaction between PC2 and FPC. In addition, we also found that physical interaction between the binding domains of PC2 and FPC is able to prevent down-regulation of PC2 induced by loss of FPC. In vivo, we generated a mouse model of ADPKD with hypomorphic Pkd2 alleles (Pkd2nf3/nf3) and show that PC2 down-regulation is accompanied by a phenotype similar to that of Pkhd1–/– mice. These findings demonstrate a common mechanism underlying cystogenesis in ADPKD and ARPKD and provide insight into the molecular relationship between PC2 and FPC.

Naked family members ( Drosophila Naked Cuticle and mammalian Naked1 and Naked2) have been identified as inducible antagonists of canonical Wnt signaling. We recently reported that Naked2, but not Naked1, interacts with the cytoplasmic tail of TGF-α, thereby coating TGF-α-containing exocytic vesicles and directing these vesicles to the basolateral corner of polarized epithelial cells. Here, we show that Naked2 is a short-lived protein with a half-life of 60 min caused by its rapid ubiquitin-mediated proteasomal degradation. Overexpression of TGF-α stabilizes Naked2 protein in an EGF receptor (EGFR)-independent manner; a physical interaction between the cytoplasmic tail of TGF-α and Naked2 is necessary and sufficient for this protection. We have identified a RING finger protein, AO7/RNF25, as a ubiquitin ligase for Naked2, and we have shown that overexpression of TGF-α reduces binding of AO7 to Naked2. These results identify an EGFR-independent action of TGF-α, in which it protects Naked2 from proteasomal degradation, thus ensuring its delivery to the basolateral surface of polarized epithelial cells.

Wnt and EGFR signaling play key roles in embryonic development and cell proliferation. It is well documented that dysregulation of these two pathways often leads to tumorigenesis with poor prognosis. However, the possible crosstalk between the two pathways in cancer development is largely unknown. Although some reports show that EGFR might antagonize Wnt signaling during development in Drosophila, an increasing body of evidence indicates that Wnt and EGFR signaling crosstalk and transactivate one another in development and cancer. This review summarizes recent studies on the crosstalk between Wnt and EGFR signaling in cancers and points out several possible convergence points. Wnt ligands can activate EGFR signaling through their 7-transmembrane domain receptor Frizzled while EGFR can activate β-catenin via receptor tyrosine kinase-PI3K/Akt pathway; EGFR has been shown to form a complex with β-catenin and increase the invasion and metastasis of cancer cells. NKD2, a Wnt antagonist by interacting with Dishevelled, also escorts TGFα-containing exocytic vesicles to the basolateral membrane of polarized epithelial cells. Down-regulation of NKD2 causes Wnt activation and TGFα misdelivery, suggesting its functions in cell homeostasis and prevention of tumorigenesis.