Orla T. Cox

Heme-binding Protein HRG-1 Is Induced by Insulin-like Growth Factor I and Associates with the Vacuolar H+-ATPase to Control Endosomal pH and Receptor Trafficking

Endocytosis and trafficking of receptors and nutrient transporters are dependent on an acidic intra-endosomal pH that is maintained by the vacuolar H+-ATPase (V-ATPase) proton pump. V-ATPase activity has also been associated with cancer invasiveness. Here, we report on a new V-ATPase-associated protein, which we identified in insulin-like growth factor I (IGF-I) receptor-transformed cells, and which was separately identified in Caenorhabditis elegans as HRG-1, a member of a family of heme-regulated genes. We found that HRG-1 is present in endosomes but not in lysosomes, and it is trafficked to the plasma membrane upon nutrient withdrawal in mammalian cells. Suppression of HRG-1 with small interfering RNA causes impaired endocytosis of transferrin receptor, decreased cell motility, and decreased viability of HeLa cells. HRG-1 interacts with the c subunit of the V-ATPase and enhances V-ATPase activity in isolated yeast vacuoles. Endosomal acidity and V-ATPase assembly are decreased in cells with suppressed HRG-1, whereas transferrin receptor endocytosis is enhanced in cells that overexpress HRG-1. Cellular uptake of a fluorescent heme analogue is enhanced by HRG-1 in a V-ATPase-dependent manner. Our findings indicate that HRG-1 regulates V-ATPase activity, which is essential for endosomal acidification, heme binding, and receptor trafficking in mammalian cells. Thus, HRG-1 may facilitate tumor growth and cancer progression.

PDLIM2 regulates transcription factor activity in epithelial-to-mesenchymal transition via the COP9 signalosome

PDLIM2 integrates cytoskeletal signaling with gene expression to enable reversible differentiation of epithelial cancer cells. PDLIM2 associates with the COP9 signalosome and controls its nuclear translocation and the stability of key transcription factors necessary for either a mesenchymal or an epithelial phenotype.

IGF-1 Receptor and Adhesion Signaling: An Important Axis in Determining Cancer Cell Phenotype and Therapy Resistance

IGF-1R expression and activation levels generally cannot be correlated in cancer cells, suggesting that cellular proteins may modulate IGF-1R activity. Strong candidates for such modulation are found in cell-matrix and cell–cell adhesion signaling complexes. Activated IGF-1R is present at focal adhesions, where it can stabilize β1 integrin and participate in signaling complexes that promote invasiveness associated with epithelial mesenchymal transition (EMT) and resistance to therapy. Whether IGF-1R contributes to EMT or to non-invasive tumor growth may be strongly influenced by the degree of extracellular matrix engagement and the presence or absence of key proteins in IGF-1R-cell adhesion complexes. One such protein is PDLIM2, which promotes both cell polarization and EMT by regulating the stability of transcription factors including NFκB, STATs, and beta catenin. PDLIM2 exhibits tumor suppressor activity, but is also highly expressed in certain invasive cancers. It is likely that distinct adhesion complex proteins modulate IGF-1R signaling during cancer progression or adaptive responses to therapy. Thus, identifying the key modulators will be important for developing effective therapeutic strategies and predictive biomarkers.

Essential Function for PDLIM2 in Cell Polarization in Three-Dimensional Cultures by Feedback Regulation of the β1-Integrin–RhoA Signaling Axis

PDLIM2 is a cytoskeletal and nuclear PDZ-LIM domain protein that regulates the stability of Nuclear Factor kappa-B (NFκB) and other transcription factors, and is required for polarized cell migration. PDLIM2 expression is suppressed by methylation in different cancers, but is strongly expressed in invasive breast cancer cells that have undergone an Epithelial Mesenchymal Transition (EMT). PDLIM2 is also expressed in non-transformed breast myoepithelial MCF10A cells and here we asked whether it is important for maintaining the polarized, epithelial phenotype of these cells. Suppression of PDLIM2 in MCF10A cells was sufficient to disrupt cell polarization and acini formation with increased proliferation and reduced apoptosis in the luminal space compared to control acini with hollow lumina. Spheroids with suppressed PDLIM2 exhibited increased expression of cell-cell and cell-matrix adhesion proteins including beta 1 (β1) integrin. Interestingly, levels of the Insulin-like growth factor 1 receptor (IGF-1 R) and Receptor of activated protein kinase C 1 (RACK1), which scaffolds IGF-1R to β1 integrin, were also increased, indicating a transformed phenotype. Focal Adhesion Kinase (FAK) and cofilin phosphorylation, and RhoA Guanosine Triphosphatase (GTPase) activity were all enhanced in these spheroids compared to control acini. Importantly, inhibition of either FAK or Rho Kinase (ROCK) was sufficient to rescue the polarity defect. We conclude that PDLIM2 expression is essential for feedback regulation of the β1-integrin-RhoA signalling axis and integration of cellular microenvironment signals with gene expression to control the polarity of breast epithelial acini structures. This is a mechanism by which PDLIM2 could mediate tumour suppression in breast epithelium.

FES-related tyrosine kinase activates the insulin-like growth factor-1 receptor at sites of cell adhesion

IGF-1 receptor (IGF-1R) and integrin cooperative signaling promotes cancer cell survival, proliferation, and motility, but whether this influences cancer progression and therapy responses is largely unknown. Here we investigated the non-receptor tyrosine adhesion kinase FES-related (FER), following its identification as a potential mediator of sensitivity to IGF-1R kinase inhibition in a functional siRNA screen. We found that FER and the IGF-1R co-locate in cells and can be co-immunoprecipitated. Ectopic FER expression strongly enhanced IGF-1R expression and phosphorylation on tyrosines 950 and 1131. FER phosphorylated these sites in an IGF-1R kinase-independent manner and also enhanced IGF-1-mediated phosphorylation of SHC, and activation of either AKT or MAPK-signaling pathways in different cells. The IGF-1R, β1 Integrin, FER, and its substrate cortactin were all observed to co-locate in cell adhesion complexes, the disruption of which reduced IGF-1R expression and activity. High FER expression correlates with phosphorylation of SHC in breast cancer cell lines and with a poor prognosis in patient cohorts. FER and SHC phosphorylation and IGF-1R expression could be suppressed with a known anaplastic lymphoma kinase inhibitor (AP26113) that shows high specificity for FER kinase. Overall, we conclude that FER enhances IGF-1R expression, phosphorylation, and signaling to promote cooperative growth and adhesion signaling that may facilitate cancer progression.

Abstract 1794: The FES-related tyrosine kinase associates with and activates the insulin-like growth factor 1 receptor at sites of cell adhesion

Insulin-like Growth Factor-1 signaling has a well-described function in facilitating tumourigenesis and promoting tumour growth. Attempts to suppress IGF signals at the level of the IGF-1 Receptor have been disappointing, with kinase inhibitors and blocking antibodies generally showing poor efficacy in clinical trials. To address the mechanisms for this lack of efficacy we sought to identify proteins that modulate the cytotoxic response to IGF-1R tyrosine kinase inhibition using a functional siRNA screen. We identified the non-receptor tyrosine FES-related (FER) kinase as a mediator of sensitivity to the IGF-1R tyrosine kinase inhibitor in MCF-7 cells. We found that FER and the IGF-1R co-locate and can be co-immunoprecipitated from different cell types. Ectopic expression of FER strongly enhanced IGF-1R expression and phosphorylation on the atuophosphorylation sites at tyrosines 950 and 1131. FER phosphorylated these sites in an IGF-1R kinase-independent manner and also enhanced IGF-1-mediated phosphorylation of SHC, and activation of either the AKT or MAPK signaling pathways in breast cancer cell lines. The IGF-1R, β1 Integrin, FER and its substrate cortactin were all observed to be co-located in cell adhesion complexes, the disruption of which reduced IGF-1R expression and activity. High FER expression correlates with phosphorylation of SHC in breast cancer cell lines and with a poor prognosis in patient cohorts. FER and SHC phosphorylation and IGF-1R expression could be suppressed with a known ALK inhibitor that shows high specificity for FER kinase. Overall, we conclude that FER-enhances IGF-1R expression, phosphorylation and signaling to promote cooperative growth and adhesion signaling that may facilitate cancer progression and resistance to IGF-1R inhibition. Citation Format: Joanna Stanicka, Leonie Rieger, Orla T. Cox, Sandra O9Shea, Michael Coleman, Ciara O9Flanagan, Barbara Addario, Nuala McCabe, Richard Kennedy, Rosemary O9Connor. The FES-related tyrosine kinase associates with and activates the insulin-like growth factor 1 receptor at sites of cell adhesion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1794.

Abstract 200: PDLIM2 phosphorylation in determining breast cancer phenotype

Several genes that are involved in cancer progression are induced by Insulin-like Growth Factor-1 (IGF-1). One of them encodes the PDZ-LIM protein PDLIM2, which is localized at the cytoskeleton and nucleus in epithelial and hematopoietic cells. PDLIM2 is repressed in certain cancers including breast cancers, but it is highly expressed in invasive cancer cells in triple negative breast cancer (TNBC), where it has also been associated with poor survival. PDLIM2 regulates the stability and activity of transcription factors including beta catenin, STATs, NFκB and IRFs and has been associated with ubiquitination and destabilization of proteins such as NF-κB, E-cadherin, and p27 KIP1 . However, PDLIM2 itself is also regulated by posttranslational modifications, but how this controls PDLIM2 activity is still largely unclear. Therefore, this study aimed to investigate PDLIM2 phosphorylation and its effect on PDLIM2 sub-cellular location and function in transcription factor regulation. The PDLIM2 protein sequence includes multiple potential phosphorylation sites and it migrates as several distinct phosphatase-sensitive species in SDS PAGE and 2D electrophoresis, indicating phosphorylation at several residues. Using cellular fractionation and immunofluorescence we show that PDLIM2 accumulates in the nucleus in response to cell stimulation with IGF-1 or TGFβ. Moreover, we show that inhibitors of Rho Kinases suppress PDLIM2 phosphorylation in response to these stimuli. In addition, we demonstrate that mutation of the predicted Rho Kinase phosphorylation sites in PDLIM2 reduce protein stability, an effect that can be reversed by application of proteasomal inhibitor. Overall, our results indicate that phosphorylation by Rho kinases plays an important role in the subcellular location and regulation of PDLIM2 function, which opens a new avenue in targeting PDLIM2 activity in invasive cancer cells. Citation Format: Orla T. Cox, Janina Berghoff, Deirdre A. Buckley, Rosemary O’Connor. PDLIM2 phosphorylation in determining breast cancer phenotype. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 200.

Abstract 4082: PDLIM2 : A cytoskeleton to nuclear courier protein for the IGF-1, Wnt and TGF beta signalling pathways in Epithelial to Mesenchymal Transition

PDLIM2 is a PDZ-LIM domain protein that regulates the stability and activity of transcription factor families (including NFκB, STATs and beta catenin). PDLIM2 expression is repressed in certain cancers but it is also highly expressed in Triple Negative Basal Breast cancers that are characterized by poor survival. Suppression of PDLIM2 reverses the EMT phenotype, inhibits polarized cell migration, and disrupts formation of polarized epithelial acini in 3D cell cultures. PDLIM2 shuttles from the cytoskeleton to the nucleus, but what mediates this nuclear translocation or activity in transcription factor regulation is unknown. The aim of this study was to identify the mechanisms governing PDLIM2 subcellular localization and nuclear translocation. We found that IGF-1or TGF-β promotes PDLIM2 accumulation in the nucleus. Similarly, WNT3a stimulation enhances PDLIM2 accumulation in the nucleus while inhibition of WNT activity results in PDLIM2 stabilization in the cytoplasm. Cytoplasmic to nuclear translocation is associated with reduced phosphorylation on several serine residues in PDLIM2. The de-phosphorylation and subsequent nuclear translocation of PDLIM2 can be prevented by inhibiting the protein phosphatase PP1. In contrast, PDLIM2 phosphorylation can be enhanced by activation of protein kinase C, which is dependent on the presence of the focal adhesion scaffolding protein RACK1 in a complex with PDLIM2. Overall, the data indicate that PDLIM2 cytoplasmic to nuclear translocation in response to IGF-1, WNT or TGF beta signalling is mediated by serine phosphorylation and de-phosphorylation by cytoskeleton-associated kinases and phosphatases. Thus PDLIM2 acts as a “cytoskeleton to nucleus” courier protein for these signalling pathways to promote cancer cell migration and EMT. Citation Format: Milan Bustamante Garrido, Orla T. Cox, Ciara O9Flanagan, Deirdre A. Buckley, Patrick A. Kiely, Rosemary O9Connor. PDLIM2 : A cytoskeleton to nuclear courier protein for the IGF-1, Wnt and TGF beta signalling pathways in Epithelial to Mesenchymal Transition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4082. doi:10.1158/1538-7445.AM2015-4082

Abstract C18: A new IGF‐I‐regulated micronutrient carrier in cancer cells

Cancer cells are dependent on a continuous supply of nutrients to maintain cell proliferation and migration. Nutrients are acquired through transporters whose transcription, trafficking, and degradation are tightly regulated by growth factors, especially through activity of the Insulin/IGF‐I‐activated PI3K/mTOR signalling pathway. The intracellular trafficking of nutrient transporters requires an acidic endosomal pH that is maintained by the Vacuolar H+ ATPase (V‐ATPase) proton pump. Recent studies have indicated a role for V‐ATPase activity in cancer cells, but how this is regulated is not known. We recently isolated a series of new IGF‐I‐regulated genes whose expression is associated with cellular transformation (1, 2). Among these was a previously uncharacterized endosomal protein, which we determined associates with the V‐ATPase, and which we called EVA. This protein was separately identified in C. elegans as a member of a family of heme transporters (HRG‐1) that control heme homeostasis (3). We found that EVA is present throughout the endosome compartments; in early, recycling, and late endosomes, but not in lysosomes. Upon nutrient withdrawal EVA traffics to the plasma membrane. EVA interacts with the c subunit of the V‐ATPase and enhances V‐ATPase activity in isolated yeast vacuoles. Suppression of EVA expression increases endosomal pH and reduces V‐ATPase holoenzyme assembly. This is accompanied by decreased migration, decreased transferrin receptor trafficking, decreased cellular heme uptake, and cell death. Over‐expressed EVA enhances cellular heme uptake in a V‐ATPase‐dependent manner. We conclude that EVA/HRG‐1 regulates V‐ATPase‐dependent acidification of endosomes necessary for trafficking of heme receptors as well as for heme transport within endosomes. Our data suggest that IGF‐I induces expression of this micro‐nutrient transporter to facilitate the enhanced metabolic requirements of cancer cells. Citation Information: Cancer Res 2009;69(23 Suppl):C18.