Article30 November 2015Open Access PTPRN2 and PLCβ1 promote metastatic breast cancer cell migration through PI(4,5)P2-dependent actin remodeling Caitlin A Sengelaub Caitlin A Sengelaub Laboratory of Systems Cancer Biology, Rockefeller University, New York, NY, USA Search for more papers by this author Kristina Navrazhina Kristina Navrazhina Laboratory of Systems Cancer Biology, Rockefeller University, New York, NY, USA Search for more papers by this author Jason B Ross Jason B Ross Laboratory of Systems Cancer Biology, Rockefeller University, New York, NY, USA Search for more papers by this author Nils Halberg Nils Halberg Laboratory of Systems Cancer Biology, Rockefeller University, New York, NY, USA Search for more papers by this author Sohail F Tavazoie Corresponding Author Sohail F Tavazoie Laboratory of Systems Cancer Biology, Rockefeller University, New York, NY, USA Search for more papers by this author Caitlin A Sengelaub Caitlin A Sengelaub Laboratory of Systems Cancer Biology, Rockefeller University, New York, NY, USA Search for more papers by this author Kristina Navrazhina Kristina Navrazhina Laboratory of Systems Cancer Biology, Rockefeller University, New York, NY, USA Search for more papers by this author Jason B Ross Jason B Ross Laboratory of Systems Cancer Biology, Rockefeller University, New York, NY, USA Search for more papers by this author Nils Halberg Nils Halberg Laboratory of Systems Cancer Biology, Rockefeller University, New York, NY, USA Search for more papers by this author Sohail F Tavazoie Corresponding Author Sohail F Tavazoie Laboratory of Systems Cancer Biology, Rockefeller University, New York, NY, USA Search for more papers by this author Author Information Caitlin A Sengelaub1, Kristina Navrazhina1, Jason B Ross1, Nils Halberg1 and Sohail F Tavazoie 1 1Laboratory of Systems Cancer Biology, Rockefeller University, New York, NY, USA *Corresponding author. Tel: +1 212 327 208; Fax: +1 212 327 7209; E-mail: [email protected] The EMBO Journal (2016)35:62-76https://doi.org/10.15252/embj.201591973 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Altered abundance of phosphatidyl inositides (PIs) is a feature of cancer. Various PIs mark the identity of diverse membranes in normal and malignant cells. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) resides predominantly in the plasma membrane, where it regulates cellular processes by recruiting, activating, or inhibiting proteins at the plasma membrane. We find that PTPRN2 and PLCβ1 enzymatically reduce plasma membrane PI(4,5)P2 levels in metastatic breast cancer cells through two independent mechanisms. These genes are upregulated in highly metastatic breast cancer cells, and their increased expression associates with human metastatic relapse. Reduction in plasma membrane PI(4,5)P2 abundance by these enzymes releases the PI(4,5)P2-binding protein cofilin from its inactive membrane-associated state into the cytoplasm where it mediates actin turnover dynamics, thereby enhancing cellular migration and metastatic capacity. Our findings reveal an enzymatic network that regulates metastatic cell migration through lipid-dependent sequestration of an actin-remodeling factor. Synopsis The phosphatidylinositol phosphatase activity harboring protein tyrosine phosphatase PTPRN2 and the phospholipase PLCβ1 are overexpressed in breast cancer. Both enzymes reduce plasma membrane PI(4,5)P2 levels, thereby enhancing cofilin-mediated actin remodeling and breast cancer metastasis. Increased expression of PTPRN2 and PLCβ1 clinically correlates with breast cancer metastasis. PTPRN2 and PLCβ1 promote breast cancer cell migration. PTPRN2 and PLCβ1 modulate plasma membrane PI(4,5)P2 levels to promote actin remodeling. Introduction PIs are major determinants of membrane identity and regulators of membrane trafficking in both normal and disease states (Vicinanza et al, 2008; Balla, 2013). The functions of PIs differ greatly depending on the phosphorylation state of the hydroxyl groups on the inositol ring head group, which can be metabolically interconverted to different phosphorylated states by PI kinases and PI phosphatases, or reduced to second messengers through PI hydrolyzers. Through the use of biochemical and cell-biological methods, the cellular roles of various PIs in secretion, endocytosis, actin dynamics, and intracellular signaling have begun to be elucidated. One function of PIs is to serve as docking lipids for the recruitment of specific proteins to cellular compartments, which results in enhancement or inhibition of their activity. PI(4,5)P2 has previously been shown to regulate cellular migration, a key feature of cancer progression (Clark et al, 2000; Sahai & Marshall, 2002; Condeelis & Segall, 2003; Condeelis et al, 2005; Ling et al, 2006; Luga et al, 2012). Enhanced migratory capacity is a required phenotype of metastatic cells, which must move through surrounding tissue, enter the vasculature, and ultimately arrive at and colonize distal organs (Bissell & Radisky, 2001; Chiang & Massague, 2008; Hanahan & Weinberg, 2011). Identifying enzymes that regulate the levels of this lipid in cancer cells could provide additional insights into the mechanisms of cancer cell migration and reveal potential targets for development against metastatic progression. Here, we identify two enzymes, PTPRN2 and PLCβ1, with activity toward PI(4,5)P2 and that promote cancer cell migration and metastasis in breast cancer. PTPRN2 was initially identified as an auto-antigen in type I diabetes and is predominantly present in neuroendocrine cells (Lan et al, 1996; Lu et al, 1996; Wasmeier & Hutton, 1996). As a transmembrane protein, PTPRN2 shuttles between secretory vesicles and the plasma membrane. Due to its presence in neurosecretory vesicles, PTPRN2 has been implicated in insulin and neurotransmitter exocytosis; however, the precise role of PTPRN2 in the secretory pathway is unknown (Cai et al, 2011). PTPRN2 belongs to the protein tyrosine phosphatase family, but does not exhibit activity against phosphoprotein substrates due to several critical amino acid variations in the PTP domain (Magistrelli et al, 1996). Recently, PTPRN2 was found to exhibit phosphatidylinositol phosphatase (PIP) activity against PI(4,5)P2 and, to a lesser extent, PI3P (Caromile et al, 2010). PLCβ1 belongs to the family of PLC enzymes, which hydrolyze PI(4,5)P2 to generate the second messengers diacylglycerol (DAG) and inositol triphosphate (IP3) (Rhee, 2001). PLCβ1 localizes mainly to the inner leaflet of the plasma membrane, where it is activated by the Gaq family of G proteins, although a subset of the protein is found in the cytoplasm and nucleus (Smrcka et al, 1991; Taylor et al, 1991). Nuclear PLCβ1 has been identified as regulating cellular proliferation and differentiation (Manzoli et al, 1997). The best-characterized member of the PLC family, PLCγ1, has been implicated in oncogenesis through its effects on cell motility and adhesion mediated by IP3 and DAG downstream signaling events (Rebecchi & Pentyala, 2000; Jones et al, 2005). We find that PLCβ1, another member of the PLC family, promotes breast cancer migration by reducing plasma membrane PI(4,5)P2. We identify PTPRN2 and PLCβ1 as enzymes that convergently reduce the abundance of PI(4,5)P2 in the plasma membrane. Through complimentary cell-biological experiments, we find that targeted reduction in plasma membrane PI(4,5)P2 by these enzymes releases plasma membrane-bound cofilin, enhancing actin remodeling in breast cancer cells and increasing their metastatic migration. Our findings reveal novel roles for PTPRN2 and PLCβ1 in cancer cell migration and identify PTPRN2 and PLCβ1 as co-modulators of PI(4,5)P2 in the plasma membrane of cancer cells and as drivers of breast cancer metastasis. Results Given the importance of PI(4,5)P2 in multiple cellular processes, we were intrigued by the finding that transcriptomic profiling of MDA-MB-231 breast cancer cells and their in vivo-selected highly metastatic derivative LM2 subline revealed two genes, PTPRN2 and PLCB1, that possess known enzymatic activity for PI(4,5)P2 to be both upregulated at the transcript and protein levels in LM2 cells (Figs 1A and B, and EV1A and B) (Minn et al, 2005; Tavazoie et al, 2008). We validated the upregulation of these genes in a second independent breast cancer cell line, CN34, and found that both genes exhibited markedly increased expression at the transcript and protein levels in the metastatic CNLM1a derivative subline relative to its parental cell population (Figs 1A and B, and EV1A and B). To functionally test their roles in breast cancer metastasis, we depleted PTPRN2 and PLCβ1 in highly metastatic LM2 cells and performed tail vein metastatic colonization assays. Knockdown of these genes reduced metastatic lung colonization (Fig 1C and D, and Appendix Fig S1A and B). Depletion of either PTPRN2 or PLCβ1 in CnLM1a cells also decreased lung metastatic colonization (Appendix Fig S1C and D). Cells with knockdown of either PLCβ1 or PTPRN2 exhibited significantly reduced signal in the lungs at 24 h post-injection compared to control cells, indicating that knockdown of these genes impacts early stages of metastatic progression (Fig EV1C). To investigate the clinical significance of these genes, we quantified the expression levels of PTPRN2 and PLCβ1 in primary tumor cDNA samples derived from patients diagnosed with various stages of breast cancer. Interestingly, expression levels of both genes increased significantly in tumors of patients with advanced (stage IV) metastatic disease (Fig 1E and F). Furthermore, increased expression of both PTPRN2 and PLCB1 associated with significantly worse overall survival (Fig 1G) and worse distal metastasis-free survival (Fig 1H) in two large breast cancer patient cohorts (Gyorffy et al, 2010; Cancer Genome Atlas Network, 2012). These findings establish PTPRN2 and PLCβ1 as clinically relevant and functional promoters of breast cancer metastasis. Figure 1. PTPRN2 and PLCβ1 promote breast cancer metastasis A, B. PTPRN2 (A) and PLCB1 (B) expression levels were determined by qRT–PCR. N = 5. C. Bioluminescence imaging quantification of lung colonization by 40,000 LM2 breast cancer cells transduced with shRNAs targeting PTPRN2 or a control hairpin. For shCntrl, sh1PTPRN2: N = 5 mice/group. For sh2PTPRN2: N = 6 mice. Right, H&E staining of representative lung sections. D. Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells transfected with siRNA targeting PLCβ1 or a control siRNA. For siCntrl: N = 5 mice. For si1PLCβ1, si2PLCβ1: N = 6 mice/group. Right, H&E staining of representative lung sections. E, F. PTPRN2 (E) and PLCB1 (F) levels were analyzed in human breast cancers (stages I-IV) and normal breast tissue from TissueScan qPCR Array Breast Cancer Panels II and III (Origene, N = 97). Expression levels were normalized to levels in normal tissue for each gene. G. Kaplan–Meier curve representing overall survival of a cohort of breast cancer patients (N = 528) as a function of their primary tumor's PTPRN2 and PLCB1 expression levels (data from the TCGA Research Network, Cancer Genome Atlas Network, 2012). Patients whose primary tumors' PTPRN2 and PLCB1 expression levels were higher or lower than the median of the population were classified as low (blue) or high (red) expression. H. Kaplan–Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1,609) as a function of their primary tumor's PTPRN2 and PLCB1 expression levels (data from KMPlot, Gyorffy et al, 2010). Patients' primary tumors' PTPRN2 and PLCB1 expression levels were classified as low (blue) or high (red) expression. Data information: Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. PTPRN2 and PLCβ1 promote breast cancer metastasis A, B. Western blot analysis of MCF 10A, MDA-MB-231, LM2, CN34, and CNLM1a1 cell lysate using anti-PTPRN2 (A) or anti-PLCβ1 (B). GAPDH was used as a loading control. Densitometry analysis below the blots is adjusted for GAPDH levels and normalized to MDA-MB-231 values. C. Bioluminescence imaging quantification of lung colonization 1 day after injection of 40,000 LM2 cells with knockdown of PTPRN2, PLCβ1, or control cells. For shCntrl, sh1PTPRN2: N = 5 mice/group. For sh2PTPRN2: N = 6 mice. For siCntrl: N = 5 mice. For si1PLCb1, si2PLCb1: N = 6 mice/group. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint To identify the mechanism(s) by which PTPRN2 and PLCβ1 mediate metastasis, we investigated several cellular metastatic phenotypes. Depletion of either PTPRN2 or PLCβ1 diminished the ability of cells to invade through Matrigel and to migrate through a porous trans-well insert (Figs 2A and B, and EV2A and B). However, depletion of neither gene affected cellular proliferation rates, viability, cytotoxicity, or caspase 3/7 activity (Fig EV2C–E and Appendix Fig S2A–C). Cells were further tested for their ability to migrate in a scratch assay. Depletion of either PLCβ1 or PTPRN2 significantly decreased the ability of cells to migrate over a 24-h period (Fig 2C). Interestingly, knockdown of both PTPRN2 and PLCβ1 reduced cellular migratory capacity to a greater extent than knockdown of either single gene (Appendix Fig S2D). Knockdown of PTPRN2 or PLCβ1 in four other breast cancer cell lines (BT-549, CNLM1a, HCC-1806, and MDA-MB-468) also significantly reduced the migratory capacity of these cells (Appendix Fig S2E–L). Figure 2. PLCβ1 and PTPRN2 drive metastatic migration and invasion A. Matrigel invasion by 50,000 LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. Data normalized to control values. N = 6 inserts/group. B. Migration assay by 100,000 LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. Data normalized to control values. N = 6 inserts/group. Right, representative images of the migration assay. Scale bar, 100 μm. C. Quantification of area covered by cells 24 h after a scratch was made through confluent cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. N = 5 wells/group. Right, representative images of the scratch assay. D, E. MDA-MB-231 cells transduced with PTPRN2, PTPRN2C945A, or control vector were subjected to the Matrigel invasion (D) and migration assays (E). N = 5 inserts/group. F. Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PTPRN2, PTPRN2C945A, or control vector. N = 5 mice/group. Right, H&E staining of representative lung sections. G, H. MDA-MB-231 cells transduced with PLCβ1, PLCβ1H331Q, or control vector were subjected to the Matrigel invasion (G) and migration assays (H). N = 5 inserts/group. I. Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PLCβ1, PLCβ1H331Q, or control vector. For Cntrl and PLCβ1H331Q OE: N = 6 mice/group. For PLCβ1 OE: N = 5 mice. Right, H&E staining of representative lung sections. Data information: Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. PLCβ1 and PTPRN2 drive metastatic migration and invasion A, B. Western blot analysis of LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or a control siRNA using anti-PTPRN2 (A) and anti-PLCβ1 (B) at various time points post-transfection. GAPDH was used as a loading control. Densitometry analysis below the blots is adjusted for GAPDH levels and normalized to siCntrl values on day 1. C, D. Proliferation of 20,000 LM2 cells transfected with siRNA targeting PTPRN2 (C), PLCβ1 (D), or a control siRNA. N = 3 wells/group. E. Proliferation of 20,000 LM2 cells transduced with hairpins targeting PLCβ1, PTPRN2, or a control hairpin. N = 3 wells/group. F, G. Western blot analysis of MDA-MB-231 cells overexpressing PLCβ1, PLCβ1H331Q (F), PTPRN2, PTPRN2C945A (G), or a control vector using anti-PTPRN2 (F) or anti-PLCβ1 (G). GAPDH was used as a loading control. Densitometry analysis below the blots is adjusted for GAPDH levels and normalized to Cntrl values. H. Proliferation of 20,000 MDA-MB-231 cells overexpressing PLCβ1, PLCβ1H331Q, PTPRN2, PTPRN2C945A, or a control vector. N = 3 wells/group. Data information: Error bars represent SEM. Download figure Download PowerPoint To further test the ability of PTPRN2 and PLCβ1 to promote metastasis, we overexpressed these genes in the less metastatic MDA parental breast cancer cells (Fig EV2F and G). Overexpression of either gene was sufficient to increase migration and invasive capacity by at least 50% (Fig 2D–H), without affecting cellular proliferation rates (Fig EV2H). Overexpression of both PTPRN2 and PLCβ1 further increased the migratory capacity of breast cancer cells to a greater extent than overexpression of either gene alone (Appendix Fig S2M). Both PTPRN2 and PLCβ1 are enzymes with previously demonstrated activities for the substrate PI(4,5)P2 (Rebecchi & Pentyala, 2000; Caromile et al, 2010). To test whether the enzymatic capacity of these proteins was necessary for the metastatic phenotypes, we mutated the catalytic domain of each protein to generate enzymatically inactive versions. PTPRN2 contains a C(X)5R catalytic domain, common to protein tyrosine phosphatases (Wasmeier & Hutton, 1996; Barford et al, 1998). Mutation of the catalytic cysteine residue to the structurally similar serine residue abrogates PTPRN2's ability to dephosphorylate PI(4,5)P2 (Caromile et al, 2010); however, this mutation also generates a non-hydrolyzable phospho-serine intermediate in the catalytic domain, trapping the substrate and rendering the enzyme nonfunctional. To dissect the influence of the enzyme's catalytic domain independent of trapping the substrate, we instead mutated PTPRN2's catalytic cysteine residue to an inactive alanine residue to generate PTPRN2C945A. We found that while wild-type PTPRN2 overexpression was sufficient to increase invasion, migration, and metastatic lung colonization in mice, these effects were dependent on PTPRN2's catalytic activity as equivalent overexpression of PTPRN2C945A failed to enhance these phenotypes (Fig 2D–F). PLCβ1, as a member of the PLC enzymatic family, contains a highly conserved catalytic domain including a catalytic histidine residue. Mutation of PLCβ1's catalytic H331 residue has been previously shown to abrogate its ability to hydrolyze PI(4,5)P2 (Ramazzotti et al, 2008). Overexpression of catalytically inactive PLCβ1H331Q failed to increase migration, invasion, and metastasis by breast cancer cells (Fig 2G–I). These findings establish the catalytic activities of PTPRN2 and PLCβ1 as necessary for their pro-metastatic phenotypes. Given that the enzymatic activities of PTPRN2 and PLCβ1 were required to promote migration and invasion, we next investigated the role of their enzymatic substrate, PI(4,5)P2. PI(4,5)P2 is predominantly present in the plasma membranes of cells, where it has been implicated in various cellular processes (Martin, 2001; Vicinanza et al, 2008). Both PTPRN2 and PLCβ1 also demonstrated localization to the plasma membrane in breast cancer cells, in addition to some cytoplasmic localization (Fig EV3A). These enzymes act to reduce the levels of PI(4,5)P2 through two independent mechanisms. PTPRN2 dephosphorylates PI(4,5)P2, while PLCβ1 hydrolyzes PI(4,5)P2 to generate inositol triphosphate (IP3) and diacyclglycerol (DAG). We first quantified the abundance of PI(4,5)P2 in the plasma membrane of cancer cells using immunocytochemical techniques previously demonstrated to accurately reflect changes in PI(4,5)P2 mass (Hammond et al, 2009, 2012). Interestingly, highly metastatic LM2 cells exhibited lower levels of PI(4,5)P2 in their plasma membranes relative to their less metastatic parental cell population, consistent with increased levels of PTPRN2 and PLCB1 in these cells (Fig 3A). We next tested the functional relationship between plasma membrane PI(4,5)P2 levels and metastatic capacity. Addition of exogenous PI(4,5)P2 (Ozaki et al, 2000) to LM2 cells prior to intravenous injection reduced the ability of these cells to colonize the lungs of mice relative to cells treated with carrier alone (Figs 3B and EV3B). Addition of exogenous PI(4,5)P2 would be expected to only transiently increase PI(4,5)P2 levels and thus impact early stages of metastatic colonization. Consistent with this, breast cancer cells treated with exogenous PI(4,5)P2 demonstrated reduced metastatic lung signal as early as 24 h post-injection compared to cells treated with carrier alone (Appendix Fig S3A). Click here to expand this figure. Figure EV3. PLCβ1 and PTPRN2 regulate membrane PI(4,5)P2 levels Representative images of MDA-MB-231 cells retrovirally transduced with PTPRN2-FLAG, PLCβ1-FLAG, or control vector and immunostained with anti-FLAG (red) and DAPI (blue). Scale bar, 10 μm. Mean fluorescence intensity of membrane PI(4,5)P2 was analyzed in LM2 cells treated with carrier incubated with PI(4,5)P2 or carrier alone. Cells were immunostained with anti-PI(4,5)P2 antibody (red) and DAPI (blue) and analyzed using fluorescence microscopy. N = 50 cells/group. Scale bar, 10 μm. Error bars represent SEM. ***P < 0.001. Representative fluorescence images of MDA-MB-231 overexpressing PTPRN2, PLCβ1, or control vector immunostained with anti-PI(4,5)P2 antibody (red) and DAPI (blue). Scale bar, 10 μm. Download figure Download PowerPoint Figure 3. PLCβ1 and PTPRN2 regulate membrane PI(4,5)P2 levels A. Mean fluorescence intensity of membrane PI(4,5)P2 was analyzed in MDA-MB-231 and LM2 cells immunostained with anti-PI(4,5)P2 antibody using fluorescence microscopy. N = 50 cells/group. Scale bar, 10 μm. B. Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells treated with carrier incubated with PI(4,5)P2 or carrier alone for 1 h and then immediately injected. Right, H&E staining of representative lung sections. N = 6 mice/group. C, D. MDA-MB-231 cells overexpressing PTPRN2, PLCβ1, or a control vector (C) or LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or a control siRNA (D) were immunostained for PI(4,5)P2 levels and analyzed by fluorescence microscopy. Mean fluorescence intensity of plasma membrane levels of the lipid was quantified. N = 50 cells/group. Left, representative immunofluorescence images of cells stained with anti-PI(4,5)P2 antibody (red) and 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 10 μm. Data information: Error bars represent SEM. **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Consistent with the activities of PTPRN2 and PLCβ1, overexpression of either PLCβ1 or PTPRN2 reduced membrane levels of PI(4,5)P2 (Figs 3C and EV3C). Conversely, depleting either enzyme in cancer cells increased the membrane levels of PI(4,5)P2 (Fig 3D). We further confirmed our immunofluorescence data by quantifying PI(4,5)P2 levels using an enzyme-linked immunosorbent assay (ELISA). Quantification of PI(4,5)P2 abundance in membrane fractions of cells with overexpression of PTPRN2 or PLCβ1 revealed reduced amounts of the lipid, while depletion of either enzyme increased PI(4,5)P2 quantity in membrane fractions (Appendix Fig S3B). We further tested the impact of PLCβ1 and PTPRN2 modulation on plasma membrane PI(4,5)P2 levels in two additional breast cancer cells lines. Depletion of PTPRN2 or PLCβ1 in MDA-MB-468 and CNLM1a1 breast cancer cells also significantly increased plasma membrane PI(4,5)P2 levels (Appendix Fig S3C and D). These data indicate that PTPRN2 and PLCβ1 regulate plasma membrane levels of PI(4,5)P2 in breast cancer cells. To test the functional significance of PI(4,5)P2 plasma membrane levels in metastatic migration, we manipulated the levels of this phosphoinositide in cancer cells using two methods. We first sought to determine whether the migration defect of PTPRN2- or PLCβ1-depleted cells could be rescued by decreasing the plasma membrane PI(4,5)P2 levels of these cells. To selectively deplete plasma membrane PI(4,5)P2, we used a rapamycin-induced dimerization system previously developed for this purpose (Heo et al, 2006; Varnai et al, 2006). In this system, inositol polyphosphate-5-phosphatase E (INPP5E), which depletes PI(4,5)P2 by dephosphorylating it, is fused to FKBP and recruited to the plasma membrane by the constitutively membrane-inserted protein Lyn11 fused to FRB. Cancer cells transfected with these constructs and treated with rapamycin showed reduced plasma membrane PI(4,5)P2 staining compared to cells treated with DMSO (Appendix Fig S4A). Adding rapamycin to PTPRN2-depleted or PLCβ1-depleted cells, which displayed greater levels of plasma membrane PI(4,5)P2, significantly enhanced their migratory capacity relative to control cells—effectively rescuing the phenotype (Fig 4A and B). Figure 4. Altered PI(4,5)P2 levels modulate metastatic migration and invasive capacity A, B. LM2 cells transfected with siRNA targeting PTPRN2 (A), PLCβ1 (B) or a control siRNA were transfected with Lyn11-FRB and INPP5E-FKBP, treated with either DMSO or 100 nM rapamycin and subjected to the migration assay. N = 5 inserts/group. C, D. MDA-MB-231 cells overexpressing PTPRN2 (C), PLCβ1 (D) or control vector were treated with carrier alone or carrier incubated with PI(4,5)P2 for 1 h and then immediately subjected to the migration assay. N = 5 inserts/group. E. Kaplan–Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1,609) as a function of their primary tumor's mean PIP5K1A, PIP5KB, and PIP5KC expression levels (data from KMPlot, Gyorffy et al, 2010). Patients' primary tumors' combined PIP5K expression levels were classified as low (blue) or high (red) expression. F, G. Migration (F) and Matrigel invasion (G) of LM2 cells transduced with a retroviral vector overexpressing PIP5K1A or control vector. Data normalized to control values. N = 5 inserts/group. Data information: Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint In a second set of experiments, we increased plasma membrane PI(4,5)P2 levels by adding exogenous PI(4,5)P2. Addition of PI(4,5)P2 using a lipid carrier system increased plasma membrane PI(4,5)P2 levels by 25% (Fig EV3B) (Ozaki et al, 2000). Increasing plasma membrane PI(4,5)P2 abrogated the effects of PTPRN2 and PLCβ1 overexpression on migration (Fig 4C and D). These effects were specific since addition of exogenous PI4P, another lipid present in the plasma membrane (D'Angelo et al, 2008), had no effect on migratory capacity (Appendix Fig S4B). These data reveal that PTPRN2/PLCβ1-mediated decrease in plasma membrane levels of PI(4,5)P2 correlates with increased metastatic migration capacity. Given the importance of plasma membrane PI(4,5)P2 abundance on metastatic capacity, we investigated the enzyme upstream of this lipid, PIP5K. PIP5K generates PI(4,5)P2 from PI4P in the plasma membrane (van den Bout & Divecha, 2009). Consistent with our findings that high levels of PI(4,5)P2 decrease the migratory capacity of cancer cells and reduce lung metastasis, breast cancer patients whose tumors expressed high levels of the three isoforms of PIP5K (PIP5K1A, PIP5K1B, and PIP5K1C) experienced increased distal metastasis-free survival compared to patients whose tumors expressed low levels of PIP5K isoforms (Fig 4E) (Gyorffy et al, 2010). We focused on the PIP5K1A isoform since this isoform showed reduced expression in highly metastatic LM2 cells compared to poorly metastatic MDA-MB-231 cells (Appendix Fig S4C and D). Overexpression of PIP5K1A in LM2 cells increased plasma membrane PI(4,5)P2 and reduced the ability of these cells to migrate and invade (Fig 4F and G, and Appendix Fig S4E and F). Taken together, these data indicate that the plasma membrane levels of PI(4,5)P2 negatively impact metastatic capacity and that PTPRN2 and PLCβ1 govern the levels of this lipid in breast cancer cells. Plasma membrane PI(4,5)P2 regulates cellular processes through binding effector proteins, either to recruit these proteins to the plasma membrane or to modulate their activity. Several proteins involved in actin dynamics are known to be inhibited by plasma membrane PI(4,5)P2, including gelsolin, profilin, twinfilin, capping proteins, and cofilin (Saarikangas et al, 2010). Of these genes, only the increased expression of cofilin was found to significantly correlate with worse distal metastasis-free survival in a cohort of breast cancer patients (Fig 5A and Appendix Fig S5A–E) (Gyorffy et al, 2010). Increased cofilin expression has previously been implicated in breast cancer progression, as well as in oral squamous cellular carcinoma, renal cell carcinoma, and ovarian cancer progression (Martoglio et al, 2000; Unwin et al, 2003; Wang et al, 2004, 2007; Turhani et al, 2006). Cofilin binds to plasma membrane PI(4,5)P2, and its membrane binding prevents its ability to bind actin (Yonezawa et al, 1991; Ojala et al, 2001; Gorbatyuk et al, 2006). When PI(4,5)P2 is hydrolyzed, cofilin is released from the plasma membrane and acts in the cytoplasm as an actin severing protein to promote migration (Ghosh et al, 2004; Andrianantoandro & Pol
The keystone perforator flap design has been gaining popularity for reconstruction of cutaneous defects due to its robust vascular supply and high rates of flap survival. However, the design requires significant tissue mobilization relative to the defect and is consequently technically demanding, time intensive, and has associated morbidity. We present a novel, simplified modification of the keystone flap that may increase its reconstructive applications.A retrospective chart review was conducted of patients who underwent V-Y hemi-keystone advancement flap reconstruction of cutaneous defects by a single surgeon. Outcomes of interest included wound healing complications.Eighty-six consecutive V-Y hemi-keystone advancement flaps were performed with an overall complication rate of 7% (6/86). Reconstruction sites included lower extremities (75/86, 87.2%), upper extremities (9/86, 10.5%), and the trunk (2/86, 2.3%). Mean follow-up time was 26.3 weeks. Four out of 5 surgical site infections occurred on lower extremity wounds. There were no cases of complete or partial flap loss.The current series presents a simplification of the traditional keystone flap that decreases surgical complexity and time required for successful reconstruction of cutaneous defects, especially in challenging wounds on the lower extremities. The complication rates were similar, or lower, than previously reported series of keystone flap reconstructions. The consistently favorable outcome of this technique supports the integration of the V-Y hemi-keystone advancement flap into reconstructive surgery.
International Dermatology Outcome Measures (IDEOM) is a non-profit organization whose mission is to improve the availability of evidence-based, consensus-driven outcome measures for dermatological diseases. IDEOM facilitates collaboration between stakeholders from various backgrounds, including researchers, patients, physicians, and industry representatives, to develop objective benchmark metrics that enable better treatment and management of dermatologic conditions.
