cancers

Article A2 Regulates AKT Upon H2O2-Dependent Signaling Activation in Cancer Cells

Stéphanie Anais Castaldo 1,†, Tom Ajime 1, Gisela Serrão 1,Fábio Anastácio 1, Joana Teixeira Rosa 1, Carman Anthony Giacomantonio 2, Alison Howarth 3, Richard Hill 1,3 and Patrícia Alexandra Madureira 1,3,* 1 Centre for Biomedical Research (CBMR), Campus of Gambelas, University of Algarve, Building 8, Room 2.22, 8005-139 Faro, Portugal; [email protected] (S.A.C.); [email protected] (T.A.); [email protected] (G.S.); [email protected] (F.A.); [email protected] (J.T.R.); [email protected] (R.H.) 2 Department of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada; [email protected] 3 Brain Tumour Research Centre of Excellence, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, PO1 2DT Portsmouth, UK; [email protected] * Correspondence: [email protected]; Tel.: +351-28-924-4443 † Current Address: VIB Center for Cancer Biology and KU Leuven Department of Oncology, University of Leuven, 3000 Leuven, Belgium.

 Received: 14 March 2019; Accepted: 3 April 2019; Published: 7 April 2019 

Abstract: Hydrogen peroxide (H2O2) is a main second messenger in oncogenic signaling networks including the Ras and the growth factor receptor pathways. This is achieved predominantly through the oxidation of redox-sensitive cysteine (Cys) residues in resulting in changes to their structure and function. We previously identified annexin A2 (ANXA2) as a redox regulatory that plays an important cellular role during oxidative stress and also promoting tumorigenesis. Here we investigated the role of ANXA2 in the regulation of H2O2-dependent signaling that drives tumor progression. We show that depletion of ANXA2 leads to the enhanced activation of AKT following either EGF/EGFR stimulation or oncogenic Ras transformation. The phosphatase and tensin homologue (PTEN) protein negatively regulates the PI3K/AKT pathway. We demonstrate that ANXA2 via its reactive Cys-8 residue, binds to PTEN and that the co-expression of PTEN and ANXA2, but not ANXA2 Cys-8-Ala mutant, inhibits AKT phosphorylation on Ser 473. These results indicate that ANXA2 is important for PTEN regulation within the PI3K/AKT signaling cascade. Furthermore, we also reveal that ANXA2 inversely regulates the expression of the peroxidase, peroxiredoxin 2, in a reactive oxygen species dependent manner.

Keywords: ANXA2; PTEN; PI3K/AKT; PRDX2; H2O2-dependent signaling

1. Introduction

Hydrogen peroxide (H2O2) plays a key role as a second messenger in cell , regulating cell proliferation, differentiation, migration and apoptosis [1–3]. This occurs primarily through the oxidation of redox-sensitive (reactive) cysteine (Cys) residues within proteins by H2O2 which results in changes to their structure and function [4–6]. The epidermal growth factor receptor (EGFR) signaling is commonly up-regulated in cancer, constituting an important pathway that regulates cell growth, survival, proliferation and differentiation [7,8]. EGFR signaling is initiated by growth factor (e.g., EGF) binding which stimulates dimerization and autophosphorylation of the receptor and subsequent downstream signaling by phosphorylation/activation of tyrosine and the concomitant inhibition of protein tyrosine

Cancers 2019, 11, 492; doi:10.3390/cancers11040492 www.mdpi.com/journal/cancers Cancers 2019, 11, 492 2 of 15 phosphatases and the phosphatase and tensin homologue (PTEN) protein. Particularly, the activation of the EGFR/PI3K/AKT pathway enables Rac to bind to and activate NADPH oxidase (NOX), ultimately leading to the production of H2O2 [9,10]. H2O2 reversibly oxidizes and inactivates the main inhibitor of the PI3K/AKT pathway, PTEN, allowing signal transduction to occur. Another prominent H2O2-dependent signaling network associated with tumorigenesis is the Ras pathway. Ras is a small G-coupled protein, whose activity is regulated by association with GTP (promoted by Guanine Exchange Factors) generating an ON/active protein or GDP (promoted by its own GTPase activity) constituting the OFF/inactive Ras. The constitutive activation of Ras has been shown in approximately 30% of all cancers [11]. This occurs via mutation(s) of critical amino acid residue(s) leading to the loss of Ras GTPase activity [12]. Ras regulates three critical signaling networks, namely the Raf/MEK/ERK, PI3K/AKT and Ral-GDS pathways [12]. Ras/PI3K/AKT signaling induces ROS production through the activation of the NOX regulatory subunit, Rac [13]. To balance the proliferative and survival advantage of moderate reactive oxygen species (ROS) levels versus their damaging effect at high concentrations, cancer cells undergo a redox adaptation process through the induction of the antioxidant response [14]. We identified a novel redox regulatory protein, annexin A2 (ANXA2) that plays a key role in protecting cells from ROS induced death, supporting both tumor growth and chemo-resistance [1,4,15]. ANXA2 can exist in the cells as a monomer or a heterotetramer, where two ANXA2 molecules are bound together by a dimer of the protein, [16,17]. We previously demonstrated that the redox regulatory function of ANXA2 is independent of S100A10 [1,14,15]. Here we show that ANXA2 depleted cells have enhanced phosphorylation of AKT upon EGF/EGFR activation or transformation with oncogenic H-RasV12. We demonstrate that ANXA2, via its reactive Cys-8 residue, can bind to the negative regulator of the PI3K/AKT signaling pathway, PTEN. Our co-expression studies reveal that ANXA2 positively regulates PTEN activity, leading to the inhibition of AKT. We also report for the first time that ANXA2 inversely regulates the peroxidase, PRDX2 in a reactive oxygen species (ROS) dependent manner.

2. Results

2.1. ANXA2 Depleted Cells Show Enhanced AKT Activation Upon EGF/EGFR Stimulation To investigate the role of ANXA2 in the regulation of EGF/EGFR signaling, we established cancer cell lines depleted of ANXA2 (A549, HT1080 and MDA-MB-231 ANXA2 knockdown (KD)) and respective controls (ANXA2 scramble). The treatment of ANXA2 KD cells with 15 nM EGF over time led to a significantly higher activation of the PI3K/AKT signaling pathway, as assessed by enhanced AKT phosphorylation at Thr308 and Ser473, and increased activation of the PI3K/AKT target protein, P-4E-BPI in ANXA2 KD cells compared to their matched controls (Figure1A,B and Supplementary Materials Figure S1). This result was concomitant with increased proliferation of ANXA2 depleted cells compared to their respective control cells following EGF treatment (Figure S2). We noted no significant difference in the activation of the MAPK/ERK 1/2 signaling pathway, evaluated through the phosphorylation of ERK 1/2, or PTEN expression in ANXA2 KD versus matched control cells (Figure1A,B and Supplementary Materials Figure S1). Cancers 2019, 11, 492 3 of 15 Cancers 2019, 11, x 3 of 15

Figure 1. ANXA2 depleted cancercancer cellscells showshow enhancedenhancedAKT AKTphosphorylation/activation phosphorylation/activation uponupon HH22OO22 dependent signalingsignaling comparedcompared to to control control cells. cells. ( a()a) HT1080 HT1080 or or (b ()b MDA-MB-231) MDA-MB-231 cancer cancer cells cells depleted depleted of ANXA2of ANXA2 (ANXA2 (ANXA2 shRNA1, shRNA1, ANXA2 ANXA2 shRNA2) shRNA2) or controls or controls (ANXA2 (ANXA2 scramble) scramble) were serumwere serum starved starved for 6 h (HT1080)for 6 h (HT1080) or for 16 or h for (MDA-MB-231) 16 h (MDA-MB-231) and either and not either treated not (NT) treated or treated (NT) or with treated 15 nM with EGF 15 fornM the EGF times for indicated.the times indicated. (c) HT1080 (c or) HT1080 (d) MDA-MB-231 or (d) MDA-MB-231 ANXA2 knockdown ANXA2 knockdown (KD) and control (KD) and (scr) control cells expressing (scr) cells H-RasV12expressing orH-RasV12 the empty or vectorthe empty pBABE vector were pBABE grown we inre serum grown free in mediaserum forfree 6 hmedia or 16 for h, respectively,6 h or 16 h, afterrespectively, what cells after were what lysed cells and were 20 lysedµg of and each 20 protein µg of each extract protein was subjectedextract was to subjected SDS-PAGE, to SDS-PAGE, transferred ontotransferred nitrocellulose onto nitrocellulose membranes and membranes analyzed byand western analyzed blotting by western with the blotting antibodies with indicated. the antibodies Results areindicated. representative Results are of at representative least three independent of at least three experiments independent (n ≥ 3). experiments (n ≥ 3).

2.2. ANXA2 Depleted Cells Show Enhanced AKT Activation Upon Oncogenic Ras Transformation We next questioned if ANXA2 could play a role in the regulation of other oncogenic H O We next questioned if ANXA2 could play a role in the regulation of other oncogenic H22O22 inducing signaling pathways. We We transformed HT1080 and MDA-MB-231 ANXA2 depleted and control cells cells with with oncogenic oncogenic H-RasV12 H-RasV12 or orempty empty vector vector (pBABE). (pBABE). Cells Cells deplet depleteded of ANXA2 of ANXA2 and over- and over-expressingexpressing oncogenic oncogenic H-RasV12 H-RasV12 showed showed enhanced enhanced pSer473 pSer473 AKT (Figure AKT (Figure1C,D) compared1C,D) compared to ANXA2 to ANXA2scramble scramble cells over-expressing cells over-expressing H-RasV12 H-RasV12 or pBABE or pBABE expressing expressing cells. cells. The The activation activation of of the MAPK/ERK1/2 signaling signaling pathway pathway was was significantly significantly higher higher in in H-RasV12 H-RasV12 expressing expressing cells compared to controls (pBABE), however no differences were observed between ANXA2 depleted and matched control cells (Figure1 1C,D).C,D). 2.3. ANXA2 Via Cys-8 Residue Interacts with PTEN and Regulates Its Activity 2.3. ANXA2 Via Cys-8 Residue Interacts with PTEN and Regulates Its Activity We observed enhanced activation of AKT in ANXA2 depleted compared to control cells in We observed enhanced activation of AKT in ANXA2 depleted compared to control cells in response to H2O2-dependent signaling. Based on these data we questioned if ANXA2 could response to H2O2-dependent signaling. Based on these data we questioned if ANXA2 could interact interact with PTEN, the main inhibitor of the PI3K/AKT pathway to indirectly regulate AKT activity. with PTEN, the main inhibitor of the PI3K/AKT pathway to indirectly regulate AKT activity. To To investigate this hypothesis we performed co-immunoprecipitation (Co-IP) assays using 293T cells investigate this hypothesis we performed co-immunoprecipitation (Co-IP) assays using 293T cells over-expressing WT or mutant ANXA2 Cys-8-Ala (ANXA2C8A) and PTEN. These results showed that over-expressing WT or mutant ANXA2 Cys-8-Ala (ANXA2C8A) and PTEN. These results showed that ANXA2 and PTEN interact via the reactive Cys-8 residue of ANXA2, since mutation of this residue to ANXA2 and PTEN interact via the reactive Cys-8 residue of ANXA2, since mutation of this residue alanine abolished the ANXA2/PTEN complex (Figure2A). to alanine abolished the ANXA2/PTEN complex (Figure 2A).

Cancers 2019, 11, x 4 of 15

To further investigate if and how ANXA2 regulated PTEN activity, we performed transient transfections using 293T cells, which express very low endogenous levels of ANXA2 and PTEN, with a combination of plasmids expressing either WT or mutant ANXA2 and/or PTEN proteins (Figure 2B,C). These data demonstrated that expression of PTEN alone led to a slight decrease in AKT activity compared to control cells. However, AKT inhibition was more striking in cells over-expressing PTEN and ANXA2 (Figure 2B). Interestingly, expression of ANXA2 in 293T cells did not induce any changes in pSer473 AKT levels compared to control cells (293T pcDNA3) (Figure 2B). ANXA2 contains three cysteine (Cys) residues. To determine which ANXA2 Cys residue(s) were important for PTEN activity regulation we co-expressed PTEN with each mutant ANXA2 and evaluated the level of pSer473 AKT. The expression of ANXA2, ANXA2 Cys-132-Ala (ANXA2C132A) or ANXA2 Cys-334-Ala (ANXA2C334A) mutant proteins in 293 T cells over-expressing PTEN resulted in the inhibition of AKT, measured by reduced pSer473 AKT compared to control 293T cells transfected with empty pcDNA3 vector (Figure 2C). However, co-expression of ANXA2C8A and PTEN did not inhibit pSer473 AKT levels compared to control cells (Figure 2C). Taken together these data indicate that the Cys-8 residue of ANXA2 plays an important role in maintaining PTEN activity. Interestingly, we observed that over-expression of the PTENC124S mutant enhanced pSer473 AKT compared to control cells. These C124S Cancersresults2019 suggest, 11, 492 that PTEN might function as a dominant negative protein, having an opposite4 of 15 effect on AKT regulation compared to its WT counterpart protein.

Figure 2. ANXA2ANXA2 via via its its Cys-8 Cys-8 REDOX REDOX sensitive sensitive residue residue binds binds to PTEN to PTEN and and regulates regulates its activity. its activity. (a) (293Ta) 293T cells cells transfected transfected with with either either empty empty vector vector;; ANXA2 ANXA2 and and PTEN-GFP PTEN-GFP expression expression plasmids plasmids or ANXA2 Cys-8-Ala and PTEN-GFP expression plasmidsplasmids were grown in 6060 mmmm plates.plates. Cells were washed with PBS, lysed with TritonTriton X-100 lysis buffer degassed for 1 h onon ice.ice. 300 µgµg of each cell ◦ lysate was was incubated incubated with with the the indicated indicated antibodies antibodies for for 1 h 1 at h 4 at °C 4 andC and then then 50 µL 50 ofµ Lpre-washed of pre-washed 50% ◦ 50%slurry slurry protein protein G-Sepharose™ G-Sepharose (GE™ Healthcare)(GE Healthcare) was ad wasded added for 1 forh at 1 4 h °C. at 4BeadsC. Beads were werewashed washed four fourtimes times with with300 µL 300 ofµ lysisL of buffer, lysis buffer, resuspended resuspended with 20 with µL 20 2×µ SDS-PAGEL 2× SDS-PAGE loading loading buffer, buffer, boiledboiled for 10 formin, 10 subjected min, subjected to SDS-PAGE to SDS-PAGE and analyzed and analyzed by western by western blotting; blotting; 293T ce 293Tlls over-expressing cells over-expressing (b) (1) (emptyb) (1) emptyvector, vector,(2) PTEN, (2) (3) PTEN, ANXA2 (3) ANXA2or (4) ANXA2 or (4) and ANXA2 PTEN; and or ( PTEN;c) (1) ANXA2 or (c) (1) and ANXA2 PTEN, (2) and ANXA2 PTEN, (2)Cys-8-Ala ANXA2 and Cys-8-Ala PTEN, and(3) ANXA2 PTEN, (3)Cys-132-Ala ANXA2 Cys-132-Ala and PTEN, and(4) ANXA2 PTEN, (4) Cys-334-Ala ANXA2 Cys-334-Ala and PTEN, and (5) PTEN,ANXA2 (5) and ANXA2 PTEN andCys-124-Ser PTEN Cys-124-Ser or (6) pcDNA3 or (6) (empty pcDNA3 vector) (empty were vector) grown were in 60 grown mm plates in 60 mmin DMEM plates incomplete DMEM medium complete for medium 24 h. Cell for lysates 24 h. Cell were lysates prepared were and prepared 20 µg andof each 20 µ proteing of each extract protein was extract subjected was subjectedto SDS-PAGE, to SDS-PAGE, transferred transferred onto nitrocellulose onto nitrocellulose membranes membranes and analyzed and analyzedby western by blotting western with blotting the withantibodies the antibodies indicated. indicated.

To further investigate if and how ANXA2 regulated PTEN activity, we performed transient transfections using 293T cells, which express very low endogenous levels of ANXA2 and PTEN, with a combination of plasmids expressing either WT or mutant ANXA2 and/or PTEN proteins (Figure2B,C). These data demonstrated that expression of PTEN alone led to a slight decrease in AKT activity compared to control cells. However, AKT inhibition was more striking in cells over-expressing PTEN and ANXA2 (Figure2B). Interestingly, expression of ANXA2 in 293T cells did not induce any changes in pSer473 AKT levels compared to control cells (293T pcDNA3) (Figure2B). ANXA2 contains three cysteine (Cys) residues. To determine which ANXA2 Cys residue(s) were important for PTEN activity regulation we co-expressed PTEN with each mutant ANXA2 and evaluated the level of pSer473 AKT. The expression of ANXA2, ANXA2 Cys-132-Ala (ANXA2C132A) or ANXA2 Cys-334-Ala (ANXA2C334A) mutant proteins in 293 T cells over-expressing PTEN resulted in the inhibition of AKT, measured by reduced pSer473 AKT compared to control 293T cells transfected with empty pcDNA3 vector (Figure2C). However, co-expression of ANXA2 C8A and PTEN did not inhibit pSer473 AKT levels compared to control cells (Figure2C). Taken together these data indicate that the Cys-8 residue of ANXA2 plays an important role in maintaining PTEN activity. Interestingly, we observed that over-expression of the PTENC124S mutant enhanced pSer473 AKT compared to control cells. These Cancers 2019, 11, 492 5 of 15 results suggest that PTEN C124S might function as a dominant negative protein, having an opposite effect on AKT regulation compared to its WT counterpart protein.

2.4. ANXA2 Regulates PRDX2 Expression We noted that the same ANXA2 shRNAs led to more efficient knockdown of ANXA2 in MDA-MB-231 cells compared to HT1080 or A549 cell lines (Supplementary Materials Figure S3). Since 0 0 ANXA2 is a H2O2 scavenger we investigated the ROS levels in these cells using 2 ,7 -dichlorofluorescin diacetate (DCF-DA) reagent. We observed higher levels of ROS in HT1080 and A549 ANXA2 KD cells compared to their respective controls; interestingly, this was not observed in the MDA-MB-231 isogenic pair (Supplementary Materials Figure S4). These data suggested that a more efficient depletion of ANXA2 in the HT1080 or A549 cells likely resulted in a survival and/or proliferation disadvantage due to excess ROS. We next investigated the expression of the H2O2 scavengers, PRDX1 and PRDX2, in our isogenic cell lines (Supplementary Materials Figure S5). We observed that ANXA2 depletion led to the upregulation of PRDX2 in MDA-MB-231 cells compared to control cells (Supplementary Materials Figure S5B), but this was not observed in HT1080 ANXA2 KD versus control cells (Supplementary Materials Figure S5A). Of note, we were not able to detect PRDX2 in A549 cells (confirmed in Supplementary Materials Figure S5A). We observed that cells depleted of ANXA2 and over-expressing oncogenic H-RasV12 showed enhanced expression of PRDX2 compared to ANXA2 scramble cells over-expressing H-RasV12 or pBABE expressing cells (Supplementary Materials Figure S5D,E). To investigate if a more efficient depletion of ANXA2 in HT1080 cells could lead to increased expression of PRDX2, we abrogated the ANXA2 in the HT1080 cells by CRISPR/Cas9. We noted an approximately 5-6-fold increase in PRDX2 (but not PRDX1) expression in these cells compared to HT1080 WT cells (Figure3A,B and Supplementary Materials Figure S6A). We also established MDA-MB-231 ANXA2 KO cells and observed the up-regulation of PRDX2 compared to control cells (Supplementary Materials Figure S6B). Importantly, cells which reverted to the WT phenotype by non-homologous recombination maintained similar levels of PRDX2 compared to MDA-MB-231 WT cells (Supplementary Materials Figure S6B, lanes 2,5,6). In contrast, depletion of PRDX2 by shRNA did not lead to up-regulation of ANXA2 in either MDA-MB-231, MCF7 or HCT-116 cancer cells (Supplementary Materials Figure S7A–C).To investigate if the up-regulation of PRDX2 in HT1080 ANXA2 KO cells was regulated by ROS, we treated these cells with the antioxidant N-acetyl cysteine (NAC). We observed the down-regulation of PRDX2 upon NAC incubation, indicative that regulation of PRDX2 was ROS dependent (Figure3C). We next over-expressed ANXA2 in 293T cells and observed the down-regulation of PRDX2 compared to control cells transfected with pcDNA3 (Figure3D). Interestingly, while a 24h exposure with 100 µMH2O2 led to up-regulation of ANXA2, this oxidative stress induced the down-regulation of PRDX2. We further assessed the expression levels of ANXA2 and PRDX2 in a panel of cell lines by western blotting (Figure4A). This analysis showed that cells expressing high levels of ANXA2 have low levels of PRDX2 and vice-versa, in agreement with the data described above. analysis showed that the activation of the PI3K/AKT and MAPK/ERK 1/2 pathways was variable for each cell line and did not show a clear correlation with the expression levels of either ANXA2, PRDX1-4 or PTEN. We analyzed the distribution/localization of ANXA2 and PRDX2 in patient colon cancer tissue sections (Figure4B,D and Supplementary Materials Figure S8A). We noted that regions where the expression of ANXA2 was high showed low to no expression of PRDX2 and vice-versa. Dual staining for the nuclear marker hnRNP A2/B1 and ANXA2 showed the presence of cells in regions that have low or no expression of ANXA2 (Supplementary Materials Figure S8B). Double staining for ANXA2 and S100A10 (ANXA2 binding partner [17]), showed co-localization of these proteins as predicted (Figure4C and Supplementary Materials Figure S8C). Cancers 2019, 11, 492 6 of 15 Cancers 2019, 11, x 6 of 15 Cancers 2019, 11, x 6 of 15

a)HT1080 b) PRDX2 a) b) 10 ANXA2 KO #1ANXA2HT1080 KO #2 WT PRDX2*** 10 ANXA213 KO2 #1ANXA213 2KO #2 12 WT 3 *** 8 *** ANXA2 132 132 12 3 8 *** ANXA2 PRDX1 6 PRDX1 6 PRDX2 4 PRDX2 4 tubulin 2 tubulin aditniyAX2K/WT KO/ ANXA2 intensity 2band 0 c) WT KO/ ANXA2 intensity band ANXA2 ANXA2 WT 0 c) NT NAC ANXA2KO #1ANXA2KO #2 WT NT NAC KO #1 KO #2 1 2 293T 1 2 d) d) pcDNA3293T ANXA2 ANXA2 KOANXA2 # KO #2WT ANXA2 KOANXA2 #1 KOWT # pcDNA3 ANXA2 NTH2O2 NT H2O2 ANXA2 ANXA2 KOANXA2 # KO #2WT ANXA2 KOANXA2 #1 KOWT # NTH O NT H O ANXA2 2 2 2 2 ANXA2 PRDX1 ANXA2 PRDX1 PRDX2 PRDX2 PRDX2 PRDX2 actin tubulin actin tubulin

FigureFigure 3. ANXA23. ANXA2 regulates regulates PRDX2 PRDX2 expression expression in a ROSin a dependentROS dependent way. ( away.) HT1080 (a) HT1080 ANXA2 ANXA2 knockout Figure 3. ANXA2 regulates PRDX2 expression in a ROS dependent way. (a) HT1080 ANXA2 orknockout WT cells or were WT grown cells were in complete grown in media complete until media 80% confluency.until 80% confluency. After what After cells what were cells lysed were and knockout or WT cells were grown in complete media until 80% confluency. After what cells were 20lysedµg of and each 20 protein µg of each extract protein was subjectedextract was to subjec SDS-PAGE,ted to SDS-PAGE, transferred transferred onto nitrocellulose onto nitrocellulose membranes lysed and 20 µg of each protein extract was subjected to SDS-PAGE, transferred onto nitrocellulose andmembranes analyzed and by westernanalyzed blotting by western with blotting the antibodies with the indicated.antibodies Cellsindicated. were Cells loaded were in loaded triplicates; in membranes and analyzed by western blotting with the antibodies indicated. Cells were loaded in (b)triplicates; Quantification (b) Quantification analysis of PRDX2 analysis immunoblots of PRDX2 immunoblots from (a). Quantification from (a). Quantification was performed was performed using Image triplicates; (b) Quantification analysis of PRDX2 immunoblots from (a). Quantification was performed J software.using Image Tubulin J software. immunoblot Tubulin was immunoblot used as a was loading used control as a loading for normalization control for normalization of the quantification. of the using Image J software. Tubulin immunoblot was used as a loading control for normalization of the Errorquantification. bars represent Error Standard bars represent Deviations. Standard Statistical Deviat analysisions. Statistical was evaluated analysis was using evaluated two-tailed using Student’s two- quantification. Error bars represent Standard Deviations. Statistical analysis was evaluated using two- t-test.tailed In Student’s every case t-test. a p In-value every of case less a thanp-value 0.05 of (*),less lessthan than 0.05 (*), 0.01(**) less than and 0.01(**) 0.001 (***) and was0.001 considered (***) was tailedconsidered Student’s statistically t-test. In significant;every case a ( cp)-value HT1080 of lessANXA2 than 0.05knockout (*), less sub-populations than 0.01(**) and (ANXA2 0.001 (***) KO was #1; statistically significant; (c) HT1080 ANXA2 knockout sub-populations (ANXA2 KO #1; ANXA2 KO consideredANXA2 KO statistically #2) or wild-type significant; (WT) cells(c) HT1080 were grown ANXA2 in complete knockout media sub-populations either not supplemented (ANXA2 KO (left#1; #2) or wild-type (WT) cells were grown in complete media either not supplemented (left panel) or ANXA2panel) or KO supplemented #2) or wild-type with (WT) 5 mM cells NAC were (right grown panel) in completefor 48 h. After media what either cells not were supplemented lysed and 20 (left µg supplemented with 5 mM NAC (right panel) for 48 h. After what cells were lysed and 20 µg of each panel)of each or protein supplemented extract waswith subjected 5 mM NAC to SDS-PAGE(right panel), transferred for 48 h. After onto what nitrocellulose cells were lysedmembranes and 20 andµg protein extract was subjected to SDS-PAGE, transferred onto nitrocellulose membranes and analyzed ofanalyzed each protein by western extract blotting was subjected with the to antibodies SDS-PAGE indicated;, transferred (d) 293T onto cells nitrocellulose were transiently membranes transfected and by western blotting with the antibodies indicated; (d) 293T cells were transiently transfected with either analyzedwith either by pcDNA3western blotting(empty) wi vectorth the or antibodies pcDNA3-ANXA2 indicated; plasmids (d) 293T as cells indicated were transiently in the figure. transfected 24 h after pcDNA3with either (empty) pcDNA3 vector (empty) or pcDNA3-ANXA2 vector or pcDNA3-ANXA2 plasmids as plasmids indicated as in indicated the figure. in the 24 hfigure. after transfection24 h after transfection these cells were either not treated or treated with 100 µM of H2O2 for 24 h. Cells were these cells were either not treated or treated with 100 µM of H O for 24 h. Cells were then lysed and transfectionthen lysed theseand 20cells µg were of eithereach proteinnot treated extrac or ttreated was subjected with2 1002 µMto SDS-PAGE, of H2O2 for 24transferred h. Cells wereonto 20 µg of each protein extract was subjected to SDS-PAGE, transferred onto nitrocellulose membranes thennitrocellulose lysed and membranes 20 µg of and each analyzed protein by extrac westernt was blotting subjected with the to antibodiesSDS-PAGE, indicated. transferred Results onto are and analyzed by western blotting with the antibodies indicated. Results are representative of three nitrocelluloserepresentative membranes of three independent and analyzed experiments by western (n bl =otting 3). with the antibodies indicated. Results are independentrepresentative experiments of three independent (n = 3). experiments (n = 3).

Figure 4. Cont. Figure 4. 4. ContCont. .

Cancers 2019, 11, 492 7 of 15 Cancers 2019, 11, x 7 of 15

Figure 4. WesternWestern blot analysis of a panel of cell lines and clinical samples. ( (aa)) MCF7, MCF7, 293T, 293T, A549, A549, MiaPaca2, MDA-MB-231,MDA-MB-231, HT1080,HT1080, HCT116, HCT116, HEK HEK 293, 293, TIME TIME or or U251 U251 cells cells were were lysed lysed and and 20 µ g20 of µg each of eachprotein protein extract extract was subjected was subjected to SDS-PAGE, to SDS-PAGE, transferred transferred onto nitrocellulose onto nitrocellulose membranes membranes and analyzed and analyzedby western by blottingwestern withblotting the with antibodies the antibodies indicated. indicated. This is aThis representative is a representative experiment experiment of n > 3;of Colonn > 3; Colontumor tumor clinical clinical samples samples were flashwere frozen flash frozen and sectioned and sectioned using using a cryostat. a cryostat. (b) Sections (b) Sections were thenwere H&Ethen H&Estained stained or (c, dor) ( fixedc,d) fixed with with methanol methanol and and immunostained immunostained with with the the antibodies antibodies indicated, indicated, followed followed by byimmunofluorescence immunofluorescence staining staining with with secondary secondary antibodies antibodies (ANXA2-green; (ANXA2-green; S100A10-red; S100A10-red; PRDX2-red). PRDX2-red).

We performed a more thorough analysis of ROS related in HT1080 ANXA2 KO vs. WT 2 and MDA-MB-231 ANXA2 KD vs scramble scramble cell cell lines lines using using the the Human Human Oxidative Oxidative Stress Stress RT² RT Profiler™Profiler™ (Qiagen, Manchester, UK). The results depicted depicted on on Supplementary Materials Materials Table Table S1 and Figure 55A,BA,B did not show substantial differences in the expres expressionsion of ROS related genes in the ANXA2 depleted versus controlcontrol cells.cells. We We further further validated validated the the expression expression of a subsetof a subset of these of genesthese thatgenes showed that showed modest modestdifferential differential expression expression changes inchanges the ANXA2 in the depleted ANXA2 versus depleted control versus cells control by qRT-PCR cells (Figureby qRT-PCR5C–F). (FigureWe observed 5C–F). a We 1.5 foldobserved up-regulation a 1.5 fold ofup-regulationPRDX2 in HT1080 of PRDX2 and in MDA-MB-231 HT1080 and MDA-MB-231 ANXA2 KO compared ANXA2 KOto control compared cells to (Figure control5 C,E).cells (Figure We observed 5C,E). aWe 1.5–2 observed fold induction a 1.5-2 fold of inductionTrxRD2, of and TrxRD2 down, regulationand down regulationof SCARA-3 of inSCARA-3 MDA-MB-231 in MDA-MB-231 ANXA2 depleted ANXA2 compareddepleted compar to controled to cells control (Figure cells5 E,F).(Figure We 5E,F). also Weinvestigated also investigated the expression the expression of ROS related of proteins.ROS related We didproteins. not observe We did significant not observe differences significant in the differencesexpression ofin thesethe expression proteins in of ANXA2 these proteins depleted in versus ANXA2 control depleted cells, versus with the control exception cells, of with PRDX2 the exception(Figure5G). of Even PRDX2 though (Figure there 5G). were Even modest though differences there were in the modest expression differences of CATALASE in the expressionand TrxRD2 of CATALASE and TrxRD2 genes in MDA-MB-231 ANXA2 KD compared to control cells we did not

Cancers 2019, 11, 492 8 of 15 Cancers 2019, 11, x 8 of 15 genesobserve in MDA-MB-231significant differences ANXA2 KDat the compared protein tolevels control. Of cellsnote wewe didcould not not observe detect significant SCARA-3 differences protein in atour the extracts. protein levels. Of note we could not detect SCARA-3 protein in our extracts.

FigureFigure 5.5. AnalysisAnalysis ofof ROSROS relatedrelated genesgenes andand proteins in ANXA2 depleted versus control cancer cells. ((aa)) HT1080HT1080 ANXA2 ANXA2 KO KO #1; #1; ANXA2 ANXA2 KO KO #2 #2 or or WT WT or (orb) ( MDA-MB-231b) MDA-MB-231 ANXA2 ANXA2 shRNA1; shRNA1; ANXA2 ANXA2 shRNA2 or ANXA2 scramble cells were plated in 100 mm plates for 48 h. After what RNA extraction was performed using the RNeasy mini kit (Qiagen, Manchester, UK) according to the manufacturer′s instructions. A panel of 86 ROS dependent genes was analysed using the RT² Profiler™ PCR Array

Cancers 2019, 11, 492 9 of 15

shRNA2 or ANXA2 scramble cells were plated in 100 mm plates for 48 h. After what RNA extraction was performed using the RNeasy mini kit (Qiagen, Manchester, UK) according to the manufacturer’s instructions. A panel of 86 ROS dependent genes was analysed using the RT2 Profiler™ PCR Array Human Oxidative Stress (Qiagen, Manchester, UK) according to the manufacturer’s instructions in a LightCycler 96 instrument (Roche, Basel, Switzerland). (c) HT1080 ANXA2 KO #1; ANXA2 KO #2 or WT; (d) HT1080 ANXA2 shRNA1; ANXA2 shRNA2 or ANXA2 scramble; (e) MDA-MB-231 ANXA2 KO #1; ANXA2 KO #2 or WT; (f) MDA-MB-231 ANXA2 shRNA1; ANXA2 shRNA2 or ANXA2 scramble cells were plated in 100 mm plates for 48 h. RNA extraction was performed using the RNeasy mini kit (Qiagen, Manchester, UK) according to the manufacturer´s instructions. The was determined by qRT-PCR using the One-step NZYSpeedy RT-qPCR Green kit (Nzytech, Lisbon, Portugal) according to manufacturer’s instructions. The reaction was carried out using the Lightcycler 96 instrument (Roche, Basel, Switzerland). Gene expression levels were normalized to RPLP0 mRNA using the 2−∆∆CT method (Livak KJ, Schmittgen TD). Error bars represent the standard deviation obtained from three independent experiments. Statistical analysis was evaluated using two-tailed Student’s t-test. In every case a p-value of less than 0.05 (*), less than 0.01(**) and 0.001 (***) was considered statistically significant. (g) HT1080 ANXA2 shRNA1, ANXA2 shRNA2 and scramble (first panel); HT1080 ANXA2 KO #1, ANXA2 KO #2 and WT (second panel); MDA-MB-231 ANXA2 shRNA1, ANXA2 shRNA2 and scramble (third panel); MDA-MB-231 ANXA2 KO #1, ANXA2 KO #2 and WT (fourth panel) cells were lysed and 20 µg of each protein extract was subjected to SDS-PAGE, transferred onto nitrocellulose membranes and analyzed by western blotting with the antibodies indicated. Results are representative of three independent experiments (n = 3). 3. Discussion We previously identified ANXA2 as a redox regulatory protein that plays an important role during oxidative stress and tumorigenesis [1]. With this work, we show for the first time, that depletion of ANXA2 in cancer cells leads to enhanced activation of AKT in response to either EGF/EGFR activation or oncogenic H-RasV12 transformation (Figure1 and Supplementary Materials Figure S1). The main inhibitor of the PI3K/AKT pathway, PTEN, contains reactive Cys residues in its catalytic domain that can be readily oxidized by H2O2 inactivating its phosphatase function [18]. Our co-immunoprecipitation studies revealed that ANXA2 interacts with PTEN via ANXA2 Cys-8 residue (Figure2A). These data in conjunction with the decreased activation of AKT observed in ANXA2-containing compared to ANXA2 depleted cells upon H2O2-dependent signaling (Figure1 and Supplementary Materials Figure S1) suggested that ANXA2 positively regulates PTEN activity. Co-transfection studies using a combination of WT or mutants ANXA2 and PTEN expression plasmids, confirmed that ANXA2 positively regulates PTEN inhibition of pSer473 AKT, in a Cys-8 residue of ANXA2 dependent way (Figure2C,D). Importantly, expression of ANXA2 alone in 293T cells did not inhibit pSer473 AKT compared to 293T control cells (293T transfected with pcDNA3) (Figure2B, lane 3). These data suggest that ANXA2 does not directly regulate AKT activity, but does so via PTEN. In this work we show for the first time that ANXA2 regulates the expression of the peroxidase, PRDX2. In summary, we observed increased expression of PRDX2 upon depletion of ANXA2 in cancer cells (Figure5G and Supplementary Materials Figure S5). Of note, depletion of ANXA2 by shRNA was insufficient to induce PRDX2 up-regulation in HT1080 ANXA2 KD cells or in MDA-MB-231 and HT1080 pBABE cells, whose down-regulation of ANXA2 was less efficient compared to MDA-MB-231 ANXA2 KD cells (Figures S3 and S5). However, ANXA2 gene deletion in HT1080 and MDA-MB-231 cells led to the over-expression of PRDX2 compared to their respective WT cells (Figure3, Figure5 and Figure S6). Together these results suggest that a threshold for ANXA2 depletion is required to trigger PRDX2 up-regulation in cancer cells. We demonstrated that PRDX2 regulation by ANXA2 was ROS dependent (Figure3C). In this sense, a pronounced down-regulation of ANXA2 might be necessary to induce sufficient accumulation of ROS leading to the subsequent up-regulation of PRDX2 to compensate for the REDOX imbalance. In fact, cells over-expressing H-RasV12, which leads to up-regulation of intracellular ROS, and depleted of ANXA2 showed a significantly higher Cancers 2019, 11, 492 10 of 15 expression of PRDX2 compared to cells expressing endogenous levels of ANXA2 and H-RasV12 (Supplementary Materials Figure S5). Analysis of intracellular ROS revealed that HT1080 and A549 ANXA2 KD cells have significantly higher levels of endogenous ROS compared to their respective controls, while this was not observed in MDA-MB-231 ANXA2 KD cells (Figure S4). We observed up-regulation of PRDX2 in the MDA-MB-231 ANXA2 KD compared to control cells, which did not occur in the isogenic HT1080 or A549 cells (Supplementary Materials Figure S4). Taken together these data support that up-regulation of PRDX2 in MDA-MB-231 ANXA2 KD cells leads to REDOX homeostasis. Importantly, over-expression of ANXA2 led to down-regulation of PRDX2 in 293T cells (Figure3D), further supporting that ANXA2 inversely regulates the cellular expression of PRDX2. Interestingly, when we depleted PRDX2 in cancer cells we did not observe up-regulation of ANXA2 (Supplementary Materials Figure S7). Analysis of ANXA2 and PRDX2 protein expression in a panel of cell lines further showed an inverse relationship between ANXA2 and PRDX2 levels (Figure4A). Furthermore, expression patterns of ANXA2 and PRDX2 in colon cancer sections revealed that the distribution of these two proteins is highly distinct. Areas with high ANXA2 expression display little to no PRDX2 and vice-versa (Figure4D and Supplementary Materials Figure S8A). PRDX2 and ANXA2 share many characteristics, including similar cellular distribution, high cytoplasmic abundancy with a small fraction in the nucleus [19,20], calcium-dependent plasma membrane binding [17,21,22], lipid raft co-localization [20,23], ion channels regulation [17,24,25], both directly inactivate H2O2 and are recycled/reduced by the thioredoxin system [1,20] and both protect cellular DNA from oxidative damage [15,26]. However, it is highly likely that ANXA2 and PRDX2 also have distinct functions and interact with different proteins and targets within cells. A study by Cao et al. found that PRDX1 binds to PTEN protecting it from oxidation-induced inactivation and indirectly regulating AKT phosphorylation upon Ras signaling [27]. The authors also show that PRDX2 was not able to bind to PTEN, which is in agreement with our results, demonstrating that PRDX2 up-regulation is not able to impede the enhanced AKT phosphorylation observed upon depletion of ANXA2 in cancer cells. Over-oxidation of PRDXs has been linked to cell signaling through the “floodgate model,” in which PRDXs consume low levels of endogenous peroxides, but enhanced generation leads to over-oxidation/degradation and increase in H2O2 that acts as a second messenger [28]. Compared to PRDX2, ANXA2 seems to be more resistant to oxidation during H2O2-dependent signaling [1,4]. In fact, when we treated 293T cells with a high oxidative insult (100 µM of H2O2) we observed decreased levels of PRDX2, while ANXA2 expression increased (Figure3D). In these experiments we transfected 293T cells with the ANXA2-pcDNA3 plasmid to transiently express ANXA2 in these cells. It has been previously shown that H2O2 induces the CMV promoter [29]. For this reason, it is likely that ANXA2 up-regulation in H2O2 treated cells is due at least in part to H2O2 induced CMV promoter activation. Nevertheless, Madureira et al. have previously shown that the treatment of ANXA2 low expressing cells, 293T and MCF7, with 100 µMH2O2 over a long period of time (two weeks) led to up-regulation of ANXA2 in these cells [1].

4. Materials and Methods

4.1. Cell Culture, Transfections and Cell Lines MDA-MB-231, A549, MCF7, MiaPaca2, HCT 116, U251, HEK 293, HT1080 and 293T cell lines were obtained from ATCC and grown in DMEM (HyClone™, Pittsburgh, PA, USA) supplemented with 10% fetal bovine serum (FBS), 20 mM L-Glutamine and 100 U/mL penicillin/streptomycin. TIME cells were obtained from Dr McMahon and grown in EGM2 medium (Lonza, Basel, Switzerland) supplemented with 2% FBS and 100 U/mL of penicillin/streptomycin. Cells were maintained in a humidified ◦ incubator at 37 C with 5% CO2. ANXA2 depleted cell lines and/or over-expressing H-RasV12 or pBABE were obtained by transfection of Phoenix cells with 4 µg of the plasmids described on table SII, using 8 µL of jetPRIME (Polyplus, Strasbourg, France) transfection reagent according to manufacturers’ Cancers 2019, 11, 492 11 of 15 instructions. 48 h after transfection the target cells were infected with Phoenix supernatants and selected with 2–5 µg/mL of puromycin for pSUPER-retro-puro or pBABE or 1 mg/mL neomycin for pSUPER-retro-neo backbone plasmids. HT1080 and MDA-MB-231 ANXA2 KO cell lines were obtained by tranfection of the WT cells with 3 µg of pANXA2-gRNA1-px459-V2 or pANXA2-gRNA2-px459-V2 using 6 µL of jetPRIME transfection reagent according to the manufacturers’ instructions. 48 h after transfection the cells were selected with 5 µg/mL of puromycin. Serial dilutions were performed to obtain HT1080 and MDA-MB-231 ANXA2 KO sub-populations. For transient transfection and co-immunoprecipitation experiments, 293T cells in 6 wells plates were transfected for 24–48 h with 1 µg of either pcDNA3, pcDNA3-ANXA2, pcDNA3-ANXA-Cys-8-Ala, pcDNA3-ANXA-Cys-132-Ala, pcDNA3-ANXA-Cys-334-Ala, pcDNA3.1-PTEN, pcDNA3.1-PTEN-Cys-124-Ser, or GFP-PTEN plasmids, described below, using 2 µL (single plasmid transfection) or 4 µL (co-transfection) of jetPRIME transfection reagent according to the manufacturers’ instructions.

4.2. Plasmids and Transfections Construction of shRNA and CRISPR/Cas9 plasmids are described on Supplementary Materials Table S2. Construction of pcDNA3-ANXA2, pcDNA3-ANXA2-Cys-8-Ala, pcDNA3-ANXA2-Cys- 132-Ala and pcDNA3-ANXA2-Cys-334-Ala plasmids is described in [1]. PTEN-Wt and PTEN-Cys- 124-Ser cDNAs were cloned into pcDNA3.1 vector at restriction sites BamHI/NotI to generate pcDNA3.1-PTEN and pcDNA3.1-PTEN-Cys-124-Ser these plasmids were kindly provided by Dr. Wolfgang Link. GFP-PTEN was a gift from Dr. Alonzo Ross (Addgene plasmid # 13039). The plasmids pBabe-puro (1764) and pBabe-puro H-RasV12 (39526) were purchased from Addgene, Watertown, MA, USA. ANXA2 depleted cell lines and/or over-expressing H-RasV12 or pBABE were obtained by transfection of Phoenix cells with 4 µg of the plasmids described on Supplementary Materials Table S2, using 8 µL of jetPRIME (Polyplus) transfection reagent according to manufacturers’ instructions. 48 h after transfection the target cells were infected with Phoenix supernatants and selected with 2–5 µg/mL of puromycin for pSUPER-retro-puro or pBABE or 1 mg/mL neomycin for pSUPER-retro-neo backbone plasmids. HT1080 and MDA-MB-231 ANXA2 KO cell lines were obtained by tranfection of the WT cells with 3 µg of pANXA2-gRNA1-px459-V2 or pANXA2-gRNA2-px459-V2 using 6 µL of jetPRIME transfection reagent according to the manufacturers’ instructions. 48 h after transfection the cells were selected with 5 µg/mL of puromycin. Serial dilutions were performed to obtain HT1080 and MDA-MB-231 ANXA2 KO sub-populations. For transient transfection and co-immunoprecipitation experiments, 293T cells in 6 wells plates were transfected for 24–48 h with 1 µg of either pcDNA3, pcDNA3-ANXA2, pcDNA3-ANXA-Cys-8-Ala, pcDNA3-ANXA-Cys-132-Ala, pcDNA3-ANXA-Cys-334-Ala, pcDNA3.1-PTEN, pcDNA3.1-PTEN-Cys-124-Ser, or GFP-PTEN plasmids, described below, using 2 µL (single plasmid transfection) or 4 µL (co-transfection) of jetPRIME transfection reagent according to the manufacturers’ instructions.

4.3. Antibodies Antibodies are listed in Supplementary Materials Table S3.

4.4. Immunoprecipitation Assays For co-immunoprecipitations, cells were washed with PBS, lysed with Triton X-100 lysis buffer (50 mM Tris pH 7.5, 0.2% Triton X-100, 15 mM NaCl, 2 mM EGTA, 1 mM EDTA, inhibitors, 5 mM NaVO4, 10 mM NaF) degassed for 1 h. Cell lysates were incubated with specific antibodies for 1 h at 4 ◦C and then pre-washed 50% slurry protein G-Sepharose™ (GE Healthcare, Pittsburgh, PA, USA) was added for 1 h at 4 ◦C. Immunoprecipitates were washed four times with 300 µL of lysis buffer, resuspended with 20 µL 2× SDS-PAGE loading buffer, boiled for 10 min, subjected to SDS-PAGE and analyzed by western blotting. Cancers 2019, 11, 492 12 of 15

4.5. Western Blotting Cells were treated as described in the results section and extracts were prepared with lysis buffer (2 mM EGTA, 1 mM EDTA, 50 mM Tris pH 7.5, 120 mM NaCl, 0.1% Na deoxycholate, 1% NP-40, 10 mM NaF, 5 mM NaVO4, protease inhibitors). For western blotting, 20 µg of each cell lysate was subjected to SDS-PAGE, transferred onto a nitrocellulose membrane, incubated with appropriate antibodies and visualized using ChemiDoc™ XRS (BioRad®, Watford, UK).

4.6. qRT-PCR RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. The gene expression was measured by qRT-PCR using the One-step NZYSpeedy RT-qPCR Green kit (Nzytech, Lisbon, Portugal) according to manufacturer´s instructions in a Lightcycler 96 instrument (Roche, Basel, Switzerland). Gene expression levels were normalized to RPLP0 mRNA using the 2−∆∆CT method [30]. Error bars represent the standard deviation obtained from average of triplicate samples from three independent experiments. The primers used for qRT-PCR are on Supplementary Materials Table S4.

4.7. Immunofluorescence Studies Snap frozen tumors were sectioned on a cryostat and mounted on slides. Slides were fixed for 30 min using −20 ◦C cold methanol. Slides were incubated in a humidified chamber at 37 ◦C for 30 min with swine serum (Dako, Glostrup, Denmark). The serum was removed and the slides were incubated with anti-ANXA2 (dil 1:500) and anti-PRDX2 (dil 1:500) for 1 h in a humidified chamber at 37 ◦C. After washing three times with PBS for 10 min at RT, slides were incubated with the appropriate FITC-conjugated secondary antibodies in a humidified chamber at 37 ◦C for 1 h. After washing as described above, chamber slides were mounted on glass cover slips using Vector mounting solution for fluorescence (Vector Laboratories, UK) and viewed using a Zeiss LSM 510 META -Laser Scanning Confocal Microscope (Carl Zeiss Inc., Oberkochen, Germany.

4.8. H&E Staining Snap frozen tumors were sectioned using a cryostat and mounted on slides. Samples were incubated in 95% alcohol for 2 min and 70% alcohol for 2 min. Afterwards they were washed briefly in distilled water. Slides were immersed in Harris hematoxylin solution for 8 min and then washed in running tap water for 5 min. After washing, slides were incubated in 1% acid alcohol for 30 s and washed in running tap water for 1 min. Slides were incubated in 0.2% ammonia water for 45 s and washed again in running tap water for 5 min. Slides were rinsed with 95% alcohol and counterstained in eosin-phloxine B solution 1 min. Following incubation samples were dehydrated with 95% alcohol for 5 min and two changes of absolute alcohol for 5 min. Slides were cleared by 2 changes of xylene for 5 min each. Slides were mounted with xylene based mounting medium (DAKO, Glostrup, Denmark) and visualized using an optical microscope (Axiovert 40C, Carl Zeiss Inc., Oberkochen, Germany).

4.9. Clinical Samples Collection Colon carcinoma samples were obtained from patients at the time of surgery. Patients´ consent was obtained and the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. Ethical approval was obtained for each procedure from the Research Ethics Board, (VG Hospital, Halifax, Nova Scotia, Canada; ethical code: CDHA-RS/2010-111, 17 September 2010).

4.10. Oxidative Stress RT2 PCR Profiler Array 1 × 106 cells were grown in 100 mm plates for 24 h. After what RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Manchester, UK) according to the manufacturer´s instructions. RNA Cancers 2019, 11, 492 13 of 15 quality was analyzed using an RNA 6000 Nano Kit chip assay (Agilent, Santa Clara, CA, USA) in a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) to ensure RNA extracts had an integrity index value greater than 9.0. Two µg of each RNA was used for cDNA synthesis using the RT2 first strand kit (Qiagen, Manchester, UK) according to the manufacturer’s instructions. The RT2 Profiler™ PCR Array HumanCancers 2019 Oxidative, 11, x Stress; ref: PAHS-065Z (Qiagen, Manchester, UK) was performed according13 to of the 15 manufacturer’s instructions in a LightCycler 96 instrument (Roche, Basel, Switzerland). 4.11. Statistical Analysis 4.11.The Statistical statistical Analysis significance was evaluated using two-tailed Student’s t-test. The p-values were calculatedThe statistical from at least significance three independent was evaluated experiments using two-tailed (n ≥ 3). Student’s t-test. The p-values were calculated from at least three independent experiments (n ≥ 3). 4.12. Supplementary Materials and Methods 4.12. Supplementary Materials and Methods Materials and Methods related to supplementary Figures are described in Appendix A. Materials and Methods related to supplementary Figures are described in AppendixA. 5. Conclusions 5. Conclusions Our data allowed us to propose the model depicted in Figure 6. In conclusion, we provide mechanisticOur data evidence allowed that us ANXA2 to propose via its Cys-8 the model residue depicted has a key in role Figure in positively6. In conclusion,regulating PTEN we provideto inhibit mechanistic AKT phosphorylation evidence that ANXA2at Ser473 via itsactivation Cys-8 residuesite. The has aco-expression key role in positivelyand co- regulatingimmunoprecipitation PTEN to inhibit studies AKT in conjunction phosphorylation with our at Ser473observation activation that ANXA2 site. The depleted co-expression cells show and co-immunoprecipitationenhanced phosphorylation studies of AKT in conjunction following withH2O2 our-dependent observation signaling that ANXA2 compared depleted to control cells show cells enhancedsupport that phosphorylation direct regulation ofAKT of PTEN following by AN H2XA2O2-dependent might constitute signaling an comparedimportant tomechanism control cells in supportregulating that AKT direct phosphorylation regulation of PTENfollowing by ANXA2H2O2-dependent might constitute signaling an (e.g important., EGF/EGFR; mechanism Ras V12) in regulating(Figure 6A). AKT However phosphorylation we cannot rule following out the H possib2O2-dependentility that other signaling molecular (e.g., EGF/EGFR;mechanisms Rasmight V12) be (Figureinvolved6A). in Howeverthe regulation we cannot of AKT rule activity out the bypossibility ANXA2 during that other H2O2 molecular-dependent mechanisms signaling. Activation might be involvedof the EGF/PI3K/AKT in the regulation or RAS/PI3K/AKT of AKT activity bynetworks ANXA2 enable during Rac H2 Oto2 -dependentbind to and signaling. activate NOX. Activation This ofultimately the EGF/PI3K/AKT drives the orincrease RAS/PI3K/AKT in intracellular networks H2O enable2 and Racpotently to bind induces to and activatethe levels NOX. of Thisthe ultimatelyperoxiredoxin, drives PRDX2 the increase to compensate in intracellular for the H RE2ODOX2 and unbalance potently induces and buffer/inactivate the levels of the the peroxiredoxin, excess H2O2 PRDX2(Figure to6B). compensate for the REDOX unbalance and buffer/inactivate the excess H2O2 (Figure6B).

Figure 6.6.Proposed Proposed model model for for ANXA2 ANXA2 regulation regulation of AKT of activation.AKT activation. (a) ANXA2 (a) ANXA2 binds to andbinds positively to and

regulatespositively the regulates activity the of PTEN.activity This of PTEN. mechanism This me mightchanism play might a role inplay the a regulationrole in the ofregulation H2O2-dependent of H2O2- signaling,dependent e.g., signaling, EGFR/EGF e.g., orEGFR/EGF RAS, leading or RAS, to inactivation leading to of inactivation the PI3K/AKT of signalingthe PI3K/AKT pathway. signaling (b) In thepathway. absence (b of) ANXA2,In the absence PTEN activity of ANXA2, is inhibited PTEN leading activity to enhancedis inhibited AKT leadin activation.g to enhanced The activation AKT ofactivation. the EGFR/PI3K/AKT The activation orof the RAS/PI3K/AKT EGFR/PI3K/AKT signaling or RAS/PI3K/AKT pathways enable signaling Rac pathways to bind and enable activate Rac

NADPHto bind and oxidase activate (NOX), NADPH ultimately oxidase leading (NOX), to theultimately production leading of H to2O the2. Increaseproduction in intracellular of H2O2. Increase H2O2 levelsin intracellular induce PRDX2 H2O2 levels up-regulation. induce PRDX2 up-regulation.

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: ANXA2 depleted cancer cells show enhanced AKT phosphorylation/activation upon EGF treatment compared to control cells, Figure S2: ANXA2 depleted cells show higher proliferation rate compared to matched control cells following EGF treatment, Figure S3: Analysis of ANXA2 shRNA cancer cell lines, Figure S4: Analysis of intracellular ROS levels in ANXA2 depleted versus control cells, Figure S5: Analysis of PRDX2 expression in ANXA2 KD cancer cells, Figure S6: HT1080 and MDA-MB-231 ANXA2 knockout cells show enhanced PRDX2 expression compared to control cells, Figure S7: PRDX2 depleted cancer cells show identical levels of ANXA2 expression compared to control cells, Figure S8: Immunofluorescence staining of ANXA2 and PRDX2 in colon

Cancers 2019, 11, 492 14 of 15

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/11/4/492/s1, Figure S1: ANXA2 depleted cancer cells show enhanced AKT phosphorylation/activation upon EGF treatment compared to control cells, Figure S2: ANXA2 depleted cells show higher proliferation rate compared to matched control cells following EGF treatment, Figure S3: Analysis of ANXA2 shRNA cancer cell lines, Figure S4: Analysis of intracellular ROS levels in ANXA2 depleted versus control cells, Figure S5: Analysis of PRDX2 expression in ANXA2 KD cancer cells, Figure S6: HT1080 and MDA-MB-231 ANXA2 knockout cells show enhanced PRDX2 expression compared to control cells, Figure S7: PRDX2 depleted cancer cells show identical levels of ANXA2 expression compared to control cells, Figure S8: Immunofluorescence staining of ANXA2 and PRDX2 in colon clinical samples. Table S1: Analysis of ROS related genes in ANXA2 depleted versus control cancer cells, Table S2: List of shRNA and CRISPR/Cas9 plasmids, Table S3: Antibodies list, Table S4: qRT-PCR primers list. Author Contributions: Conceptualization, P.A.M.; methodology, S.A.C. and P.A.M.; validation, S.A.C., T.A. and P.A.M.; formal analysis, S.A.C., R.H. and P.A.M.; investigation, S.A.C., T.A., G.S., F.A., J.T.R., A.H., C.A.G., R.H. and P.A.M.; resources, C.A.G. and P.A.M.; writing—original draft preparation, S.A.C., T.A. and P.A.M.; writing—review and editing, R.H. and P.A.M.; supervision, S.A.C. and P.A.M.; project administration, P.A.M.; funding acquisition, P.A.M. Funding: P.A.M. is funded by FCT Investigator contract from the Foundation for Science and Technology (FCT), Portugal (ref:IF/00614/2014) and FCT exploratory grant, ref:IF/00614/2014/CP12340006. CBMR is financed by FCT Research Center Grant ref:UID/BIM/04773/2013CBMR1334. S.A.C. was recipient of FCT research fellowship (ref: WELCOMEII/57/UALG/1050/2011). T.A. was recipient of ERASMUS Mundus fellowship (EMQAL), ref: FPAnr2013-0225. A.H. is sponsored (and grateful to) the Ollie Young Foundation, charity number 1148511. R.H. is funded by Brain Tumour Research. Conflicts of Interest: The authors declare no conflict of interest.

Appendix A Intracellular ROS Measurement: 2 × 104 cells/well were seeded in 96 wells plates and incubated overnight at 37 ◦C. Cell medium was removed and cells were incubated with serum free medium for 16 h (MDA-MB-231, A549) or 6 h (HT1080). Cells were then incubated with 50 µM DCF-DA (SIGMA) ◦ for 30 min at 37 C, 5% CO2. After what fluorescence was measured (Excitation: 488 nm, Emission: 535 nm) using a fluorometer plate reader. MTS Assay: 1 × 104 cells/well were seeded in 96 wells plates and incubated overnight at 37 ◦C. After what cells were washed with PBS and incubated overnight (16 h) with serum free DMEM media. Cells were then either not treated or treated with 15 nM EGF for 24 h. Cell proliferation was determined using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

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