FAT1 Acts As an Upstream Regulator of Oncogenic and Inﬂammatory Pathways, Via PDCD4, in Glioma Cells

ORIGINAL ARTICLE FAT1 acts as an upstream regulator of oncogenic and inﬂammatory pathways, via PDCD4, in glioma cells

B Dikshit1, K Irshad1, E Madan1, N Aggarwal1, C Sarkar2, PS Chandra3, DK Gupta3, P Chattopadhyay1, S Sinha1,4 and K Chosdol1

Glioblastoma multiforme (GBM) is the most aggressive and the commonest primary brain tumor with a tendency for local invasiveness. The pathways of neoplasia, invasion and inflammation are inextricably linked in cancer and aberrations in several regulatory pathways for these processes have been identified. Here we have studied the FAT1 (Homo sapiens FAT tumor-suppressor homolog 1 (Drosophila)) gene to identify its role in the tumorigenecity of the gliomas. The expression of FAT1 was found to be high in grade IV glioma cell lines (U87MG, A172, U373MG and T98G) but low in grade III glioma cell lines (GOS3 and SW1088). Two cell lines (U87MG and A172) with high FAT1 expression were chosen for in vitro FAT1-knockdown studies. FAT1 knockdown by small interfering RNA resulted in decreased migration and invasion of both the cell lines along with increased expression of the tumor- suppressor gene programmed cell death 4 (PDCD4). Increased PDCD4 expression led to the attenuation of activator protein-1 (AP- 1) transcription by inhibiting c-Jun phosphorylation and resulted in concomitant decrease in the expression of AP-1-target genes like MMP3, VEGF-C and PLAU, the pro-inflammatory regulator COX-2 and cytokines IL1b and IL-6. Conversely, simultaneous silencing of PDCD4 and FAT1 in these cells significantly enhanced AP-1 activity and expression of its target genes, resulting in increase in mediators of inflammation and in enhanced migratory and invasive properties of the cells. We also observed a negative correlation between the expression of FAT1 and PDCD4 (P ¼ 0.0145), a positive correlation between the expression of FAT1 and COX-2 (P ¼ 0.048) and a similar positive trend between FAT1 and IL-6 expression in 35 primary human GBM samples studied. Taken together, this study identifies a novel signaling mechanism mediated by FAT1 in regulating the activity of PDCD4 and thereby the key transcription factor AP-1, which then affects known mediators of neoplasia and inflammation.

Oncogene (2013) 32, 3798–3808; doi:10.1038/onc.2012.393; published online 17 September 2012 Keywords: FAT1; inﬂammation; PDCD4; AP-1; COX-2; glioma

INTRODUCTION of both the processes.19,20 However, the therapeutic translation of The signaling pathways propagated from the cell surface, through this knowledge has been limited.21 Here we have studied the role transmembrane receptors, to intracellular regulatory molecules of FAT1, a transmembrane protein, in linking the neoplastic are critical for maintaining cellular homeostasis. Knowledge of phenotype and inflammatory mediators in glial cells and have how these transmembrane proteins regulate the terminal corroborated some of the results in primary human tumors. components of the signaling pathway is crucial for understanding FAT1 is a member of cadherin superfamily22,23 and was first normal as well as aberrant cellular function. The link between identified as a tumor suppressor in Drosophila melanogaster, cancer and inflammation is well established and a set of common acting via the Salvador–Warts–Hippo signaling pathway.24–30 molecules (for example, COX-2, IL-1b, IL-6 and so on) may mediate There are contrasting studies about the role of FAT1 in human both the processes.1–5 Various molecular pathways that promote cancers, and its precise role in development and progression of tumor invasion and expression of pro-inflammatory molecules in human cancer is still being investigated. cancer have been identified6,7 but many more are yet to be Available literature on FAT1 in human cancers points toward its explored. dual role, both as an oncogene as well as tumor suppressor. Glioblastoma multiformes (GBMs) are the most frequent and Overexpression of FAT1 has been reported in invasive breast most malignant form of brain tumors.8,9 Tumor angiogenesis, carcinoma31 and leukemia,32 and loss of FAT1 has been reported invasiveness and rapid growth go in concert in GBM.10–12 The to inhibit migration and invasion in OSCC,33 indicating the oncogenic tumor microenvironment exhibits expression of pro-inflammatory role of FAT1 in these tumors. However, tumors like oral cancer34 molecules that promote migration and invasion of tumor cells.13,14 and Mayer-Rokitansky-Ku¨ster-Hauser (MRKH) syndrome35 have There is increasing evidence of the role of the pro-inflammatory shown deletion/loss-of-heterozygosity of the chromosomal region molecules in making glioma and other tumors more aggressive 4q35 harboring FAT1 gene, suggesting its tumor-suppressor role. and resistant to chemo- and/or radio-therapy.15–17 An initial report from our laboratory on FAT1 in glioma36 had shown The common pathways may promote tumor invasiveness and high loss-of-heterozygosity and low mRNA expression in 33% of the expression of pro-inflammatory molecules in GBM.13,18 Of these, tumors studied and suggested that it may have a tumor-suppressor COX-2 and cytokines like IL-1b and IL-6 are the known mediators role. However, the signaling cascades and cellular processes through

1Department of Biochemistry, All India Institute of Medical Sciences (AIIMS), New Delhi, India; 2Department of Pathology, All India Institute of Medical Sciences (AIIMS), New Delhi, India; 3Department of Neurosurgery, All India Institute of Medical Sciences (AIIMS), New Delhi, India and 4National Brain Research Center (NBRC), Gurgaon, India. Correspondence: Professor S Sinha, National Brain Research Centre (NBRC), Near NSG Campus, Nainwal Mode, Manesar, Gurgaon 122050, India or Dr K Chosdol, Department of Biochemistry, All India Institute of Medical Sciences (AIIMS), New Delhi 110029, India. E-mail: [email protected] or [email protected] or [email protected] or [email protected] Received 2 March 2012; revised 13 June 2012; accepted 16 July 2012; published online 17 September 2012 FAT1 gene in cancer and inflammation B Dikshit et al 3799 which FAT1 acts in different contexts are still being elucidated and (Supplementary Figures S2c and d), indicating that FAT1 knockdown very few functional studies are available on the role of FAT1 in did not affect cell viability and apoptosis. human cancers, including GBM. Programmed cell death 4 (PDCD4) is a known tumor-suppressor gene and it has an essential role in many biological processes like FAT1 knockdown enhances PDCD4 expression regulating cap-dependent translation, apoptosis, modulating In initial screening by microarray (unpublished data) to study various signal transduction pathways and so on.37–39 The altered gene expression after FAT1 knockdown in U87MG cell line, expression of PDCD4 is often decreased in human glioma40 and we identified PDCD4 as one of the upregulated genes. This was many other progressive tumors like lung, breast41,42 and so on, further confirmed by quantitative PCR (q–PCR) and western blot leading to increased invasiveness and metastasis.43–45 In addition, analysis in four GBM cell lines, U87MG, A172, U373MG and T98G. PDCD4 is also reported to suppress induction of inflammatory There was significant (Po0.05) upregulation of about 2.4, 2.8, 3.2 mediators.46,47 PDCD4 is known to inhibit activator protein-1 (AP-1)- and 3.5 fold in PDCD4 mRNA expression (Figure 2a) as well as in mediated transcription,48–50 which has a central role in multiple PDCD4 protein expression (Figure 2b) in siFAT1-treated U87MG, processes involved in tumorigenesis, including proliferation, A172, U373MG and T98G cells, respectively, as compared with migration and invasion,51–57 and AP-1 inhibition had been shown their respective siControl-treated cells. The grade III glioma cell to have anti-invasive and anti-growth effect.58,59 line, GOS3, with low endogenous FAT1 expression (Figure 1a) had InordertoelucidatetheroleofFAT1inhumanglioma,westudied high PDCD4 mRNA expression (Figure 2c), corroborating the its expression in several glioma cell lines followed by knockdown in inverse relationship between FAT1 and PDCD4. two GBM cell lines (U87MG and A172) with high FAT1 expression. In literature, phospho-Akt and PDCD4 are known to negatively There was a marked reduction in cell motility and invasiveness, as well regulate each others expression.37,60,61 However, we observed as upregulation of PDCD4 expression. This in turn reduced AP-1 increased phospho-Akt levels (Supplementary Figure S3) along transcriptional activity, thus affecting transcription of downstream with increased PDCD4 expression (Figures 2a, b) after FAT1 genes, including extra cellular matrix (ECM)-remodeling molecules knockdown in U87MG cells, suggesting that increased PDCD4 (MMP3, PLAU and VEGF-C) and pro-inflammatory markers (COX-2, expression after FAT1 knockdown was independent of the p-Akt IL1b and IL-6). This process was reversed by simultaneous pathway. knockdown of FAT1 and PDCD4, thus validating the link between FAT1 and PDCD4 in regulating cellular motility, invasiveness and inflammatory microenvironment in glioma. These in vitro findings FAT1 knockdown diminishes AP-1-mediated transcription were further supported by the inverse relationship observed PDCD4 is reported to inhibit AP-1-dependent transcription48,62 between mRNA levels of FAT1 and PDCD4 and a positive via suppression of c-Jun phosphorylation.49,50 Because c-Jun correlation between the transcript levels of FAT1 and COX-2, as phosphorylation is required for AP-1 activity, we investigated well as of FAT1 and IL-6 in primary human GBM. Thus, this study the effect of FAT1 knockdown on c-Jun phosphorylation status by identifies a novel function of FAT1 in regulating AP-1-mediated western blotting and found to be significantly decreased along transcription via PDCD4. with reduction in the total c-Jun protein level in U87MGsiFAT1 cells as compared with siControl cells (Figure 3a). The c-jun mRNA level, as checked by q–PCR, was also found to be markedly decreased (0.51±0.12, Po0.01) in U87MGsiFAT1 cells as com- RESULTS pared with U87MGsiControl cells (Figure 3b). This could be due to FAT1 knockdown reduces migration and invasion of glioma cells decreased positive autoregulatory loop of c-jun transcription by FAT1 mRNA expression was checked in a panel of glioma cell lines AP-1.63,64 Hence, the reduction in the total c-Jun protein level in and high FAT1 expression was observed in grade IV glioma (GBM) U87MGsiFAT1 cells may be due to both reduction in the c-jun cell lines (U87MG, A172, U373MG and T98G) as compared with mRNA level and ubiquitination and fast degradation of grade III glioma cell lines (GOS3 and SW1088) (Figure 1a). The cell unphosphorylated c-Jun.50,65,66 However, the exact mechanisms lines U87MG and A172 were studied further to analyze the effect leading to decrease in p-c-Jun levels need to be elucidated further. of FAT1 knockdown. Knockdown efficiency of FAT1 in cells was Further, we performed AP-1 luciferase assay to examine checked by using a set of three FAT1-specific small interfering RNA whether the upregulation of PDCD4 and diminished c-Jun (siRNA) (details in Materials and methods section) from Invitrogen phosphorylation in siFAT1 cells has any effect on AP-1 activity. (Carlsbad, CA, USA), and FAT1 siRNA I (HSS176716) was found to We observed about 60% reduction (40.6±1.2, Po0.01) in have maximum knockdown efficiency (Supplementary Figure S1). AP-1 luciferase activity in U87MGsiFAT1 cells as compared FAT1 siRNA I was used for further experiments. We found X90% with U87siControl cells (Figure 3c). Moreover the mRNA knockdown of FAT1 mRNA expression in FAT1 siRNA-treated cells expression of AP-1-target genes like MMP3, VEGF-C and PLAU (U87MGsiFAT1 and A172siFAT1) as compared with control siRNA- (urokinase) were found to be decreased by 490% treated cells (siControl; Invitrogen), 72 h post transfection (Figure 1b). (Supplementary Figure S4a), aswellasECMproteaseactivity There were significant morphological alterations (cells were more was found to be decreased in U87MGsiFAT1 cells as compared spindly) in siFAT1-treated cells (Figure 1c). with siControl cells (Supplementary Figure S4b). To further We observed significant reduction in migration (about 50% corroborate that PDCD4 regulates the AP-1-dependent transcrip- decrease in U87MGsiFAT1 (Po0.01) and about 75% decrease in tion, PDCD4 expression was knocked down by using siPDCD4 in A172siFAT1 cells (Po0.001)) (Figure 1d) as well as invasion U87MG cells, and we observed about 1.5-fold increase (157±5.4, (about 60% decrease in U87MGsiFAT1 and about 40% decrease Po0.05) in AP-1 luciferase activity (Figure 3d). Thus, confirming in A172siFAT1 cells (Po0.01)) (Figure 1e) upon FAT1 knockdown that increased PDCD4 expression after FAT1 knockdown as compared with siControl cells. There was no variation in the attenuates AP-1 transcriptional activity via reduction in the level distribution of cell population in different phases of cell cycle, as of phosphorylated c-Jun. assessed by fluorescence-activated cell sorting analysis (Supple- mentary Figure S2a), as well as in cell viability as assessed by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Knockdown of FAT1 decreases the expression of COX-2 and other (Supplementary Figure S2b), after FAT1 knockdown. Further, there cytokines was no DNA fragmentation (4’,6-diamidino-2-phenylindole staining) COX-2 is known to be negatively regulated by PDCD4,62,67 and and no cleaved caspase-3 (western blot) after 72 h of transfection aberrant induction of COX-2 with upregulation of prostaglandin

& 2013 Macmillan Publishers Limited Oncogene (2013) 3798 – 3808 FAT1 gene in cancer and inﬂammation B Dikshit et al 3800 FAT1/18S 1.2 1 0.8 0.6 0.4 to U87MG

Fold Expression 0.2

of FAT1 as compared 0 U87MG A172 U373MG T98G GOS3 SW1088

Mock siControl siFAT1 FAT1/18S 1.4 1.2 U87MG 1 Mock 0.8 sicontrol Fold 0.6 siFAT1 0.4 0.2 A172 Expression of FAT1 0 U87 A172

Migration Invasion siControl siFAT1 siControl siFAT1

U87MG U87MG

A172 A172

1.2 P<0.01 P<0.01 1.4 P<0.01 P<0.001 1.2 1 1 0.8 0.8 siControl 0.6 siControl 0.6 siFAT1 0.4 0.4 siFAT1 0.2

0.2 Fold Invasion Fold Migration 0 0 U87MG A172 U87MG A172 Figure 1. Knockdown of FAT1 inhibited glioma cell migration and invasion. (a) FAT1 expression was checked in six glioma cell lines by q–PCR, out of which grade IV glioma cell lines U87MG, A172, U373MG and T98G were found to have high FAT1 expression, whereas grade III glioma cell lines GOS3 and SW1088 had low FAT1 expression. 18S was used as internal control. (b) Knockdown of FAT1 in U87MG and A172 cell lines (72 h post transfection) was confirmed by q–PCR using FAT1-specific primers. We found X90% downregulation of FAT1 mRNA in siFAT1 cells as compared with siControl-treated cells. 18S was used as internal control; all experiments were done in triplicates and repeated thrice. (c) Distinct phenotypic changes (72 h post transfection) were observed in U87MGsiFAT1 and A172siFAT1 cells as compared with siControl cells. siFAT1-treated cells were spindle shaped or rounded, scattered and had reduced cell–cell interaction. (d, e) FAT1 knockdown inhibited cell migration and invasion in U87MGsiFAT1 as well as A172siFAT1 cells as compared with siControl cells. Modified Boyden chamber assay/matrigel assay was performed for 24 h (72 h post transfection) to assess cell migration and invasion. Cells that migrated across the membrane or invaded through matrigel were fixed, stained and were counted in five different fields and means were calculated. The number of cells that migrated or invaded in siFAT1 cells were normalized against number of cells that migrated in siControl cells. Each value is expressed as mean±s.d. Each experiment was done in triplicate and repeated twice. Significant differences were observed in siFAT1-treated cells as compared with siControl cells as determined by Student’s t-test. Mock: lipofactamin-treated cells; siControl: Invitrogen universal medium GC control siRNA-treated cells and siFAT1: FAT1-specific siRNA-treated (Invirogen) cells. A full colour version of this figure is available at the Oncogene journal online.

synthesis is reported to have a pivotal role in carcinogenesis.68 with siControl cells (42±2.6, Po0.01) (Figure 4c). Thus, this result COX-2 expression at mRNA and protein level was analyzed by depicts inhibition of COX-2 function leading to diminished PGE2 q–PCR and western blotting. We found significant reduction of synthesis after FAT1 knockdown. COX-2 expression at both mRNA (0.04±0.002, Po0.05) (Figure 4a) COX-2 has also been reported to regulate endogenous cytokine and at protein level (Figure 4b) in U87MGsiFAT1 cells as compared production, and its inhibition markedly reduces the release of pro- with siControl cells. The enzymatic product of COX-2, prostaglan- inflammatory cytokines like IL-1b and IL-6.70 We also observed 69 din E2 (PGE2), has a key role in influencing tumor progression. significant reduction in mRNA expression of COX-2 along with The concentration of PGE2 secreted into the medium was found IL-1b (0.12±0.09, Po0.05) and IL-6 (0.13±0.11, P 0.01) after FAT1 to be reduced by about 60% in U87MGsiFAT1 cells as compared knockdown in U87MG cells (Figure 4a), thus identifying a novel

Oncogene (2013) 3798 – 3808 & 2013 Macmillan Publishers Limited FAT1 gene in cancer and inﬂammation B Dikshit et al 3801 4 PDCD4/18S siControl 3.5 siFAT1 3 U87MGsiControl 3 2.5 U87MGsiFAT1 2.5 GOS3 2 2 Fold 1.5 1.5 1 1 Expression of PDCD4 0.5 Fold Expression 0.5 0 0 U87MG A172 U373MG T98G FAT1/18S PDCD4/18S

U87MG A172 U373MG T98G siControl siFAT1 siControl siFAT1 siControl siFAT1 siControl siFAT1

PDCD4, 60

-actin, 43

Figure 2. Knockdown of FAT1 upregulated PDCD4 expression. (a) PDCD4 mRNA expression as assessed by q–PCR (72 h post FAT1 siRNA transfection) was found to be increased by more than two fold in all the GBM cell lines (U87MG, A172, U373 and T98) analyzed after FAT1 knockdown as compared with their respective siControl cells. 18 S was used as internal control (b) Similarly PDCD4 protein level was also found to be increased in the above GBM cell lines after FAT1 knockdown as compared with respective siControl cells as assessed by western blot analysis. b-actin was used as loading control. (c) GOS3 (World Health Organization grade III glioma) cell line with low endogenous FAT1 was found to have high PDCD4 mRNA expression, which was comparable with U87MGsiFAT1-treated cells. A full colour version of this ﬁgure is available at the Oncogene journal online.

function of FAT1 in regulating the expression of pro-inflammatory molecules. U87MG c-jun/18S 1.2 AP-1-mediated regulation of COX-2 transcription has been siControl siFAT1 71,72 siControl 1 reported earlier. To investigate whether the regulation of COX- siFAT1 c-Jun, 43 2 expression by FAT1 in U87MG cells is mediated via AP-1, we 0.8 inhibited AP-1 activity by treating cells with specific small- 0.6 Phospho molecule inhibitor of AP-1, SR11302. On treating U87MG cells c-Jun,48 0.4 of c-jun with increasing concentration of SR11302 (0.1–10 mM), a dose- 0.2 dependent decrease in COX-2 luciferase activity was observed, -actin, 43 0 Fold mRNA expression U87MG with a maximum inhibition of about 80% at the dose of 5 and 10 mM concentrations (Supplementary Figure S5). We observed P<0.05 marked decrease in COX-2 luciferase activity after treatment of P<0.01 U87MG cells with FAT1 siRNA (Po0.01) or 10 mM SR11302 180 180 (P 0.05), as well as combined treatment with FAT1 siRNA and 160 o 160 140 SR11302 (Po0.001), as compared with siControl þ DMSO-treated 140 120 U87MG cells (Figure 4d). We also observed significant reduction in 120 100 mRNA expression of COX-2 (0.37±0.09, Po0.01), IL1b (0.42±0.08, 100 P 0.01) and IL-6 (0.29±0.1, P 0.05) after 48 h of SR11302 80 80 o o

Activity (%) 60 treatment (Figure 4e). Our results thus provide further supportive

activity (%) 60 40 40 evidence that AP-1 is a key link in the regulation of expression of

Relative AP1 Luciferase 20 20 Relative AP-1 luciferase COX-2 and other cytokines. 0 0 siControl siFAT1 siControl siPDCD4 Figure 3. Inhibition of AP-1 transcriptional activity after FAT1 knock- Inhibition of PDCD4 reverses the effects of FAT1 knockdown in down. (a) FAT1 knockdown in U87MG cells upregulated PDCD4 FAT1-attenuated cells expression, which in turn inhibits phosphorylation of c-Jun. Western To confirm that the demonstrated effects of FAT1 knockdown blot was performed on cell lysates from U87MGsiFAT1 and siControl were indeed due to the upregulation of PDCD4, we cotransfected cells with phospho-c-Jun (Ser63) and c-Jun antibodies. There was FAT1 and PDCD4 siRNA in U87MG cells and could satisfactorily significant reduction in both the c-Jun and p-c-Jun level. b-actin antibody was used as loading control. (b) The mRNA level of c-jun was knock down levels of both the genes simultaneously (Figure 5a). also found to be markedly reduced after FAT1 knockdown. A Migration and invasion were found to be significantly restored reduction in the total c-Jun protein observed after FAT1 knockdown (Po0.001) in U87MG cells after simultaneous knockdown of FAT1 could be due to decreased positive autoregulation of c-Jun expression and PDCD4 (Figures 5b, c). Moreover, AP-1 luciferase activity by AP-1, as well as the ubiquitination and rapid degradation of the (Po0.01), as well as the mRNA expression of AP-1-target genes, unphosphorylated. (c, d) AP-1 luciferase assay was done to assess AP- was found to be significantly regained with simultaneous knock- 1 transcriptional activity. U87MG cells were independently transfected down of FAT1 and PDCD4 as compared with U87MG cells treated with siFAT1 or siPDCD4 and siControl, respectively. After 24 h, cells with siFAT1 alone (Figure 5d). At protein level, high PDCD4 were transfected with 1 mg of AP-1 reporter plasmid, along with 50 ng expression observed after FAT1 knockdown was found to be of pRL-TK (Renilla luciferase) control plasmid. The luciferase activity was measured after 48 h. The luciferase activity with control siRNA decreased after simultaneous knockdown of FAT1 and PDCD4. was designated as 100% and the difference calculated, we observed However, the expression of p-c-Jun, VEGF-C and COX-2 was significant difference in siFAT1- and siPDCD4-treated cells (Po0.01 regained after simultaneous knockdown of PDCD4 and FAT1 as and Po0.05, respectively) compared with siControl cells. A full colour compared with cells treated with siFAT1 alone (Figure 5f). We also version of this figure is available at the Oncogene journal online. observed low expression of AP-1-regulated transcripts in GOS3 cell

& 2013 Macmillan Publishers Limited Oncogene (2013) 3798 – 3808 FAT1 gene in cancer and inﬂammation B Dikshit et al 3802

PGE2

1.4 siControl PGE2 1.2 siFAT1 siControl siFAT1 1.0 COX2 0.8 COX-2, 74 0.6 Arachidonate 0.4

Fold Expression PGE2 Assay -actin, 43 120 0.2 P<0.01 100 0.0 80 COX-2 IL6 IL1 60

Conc. 40 20

Relative PGE2 0 Media siControl siFAT1

P<0.01 P<0.05 1.2 P<0.01 1.4 48 hrs DMSO 1.2 1 SR11302 1 0.8 0.8 0.6 0.6

activity 0.4 0.4 Fold Expression 0.2 0.2

Relative COX-2 luciferase 0 COX-2 IL6 IL1β 0 siControl siFAT1 siControl siFAT1 + + + + DMSO DMSO SR 11302 SR 11302 Figure 4. Decrease in COX-2 expression and cytokines synthesis after FAT1 knockdown. (a) Expression of COX-2 and cytokines was found to be decreased after FAT1 knockdown. q–PCR was done to measure the mRNA expression of COX-2, IL-6 and IL-1b and about 90% reduction was observed in U87MGsiFAT1 cells as compared with siControl cells. 18S was used as internal control. (b) FAT1 knockdown was found to inhibit COX-2 protein expression. Western blot showed decreased COX-2 expression in siFAT1 cells as compared with siControl cells. b-actin was used as loading control. (c) PGE2 assay was done to quantify the production of PGE2 after FAT1 knockdown. Synthesis of enzymatic product of COX- 2, PGE2, was decreased significantly by about 60% in U87MGsiFAT1 cells as compared with siControl cells. Experiment was done in triplicate and repeated twice. Each value is expressed as mean±s.d. Significant difference (Po0.01) was observed as determined by Student’s t-test. (d) COX-2 luciferase assay was done in U87MG cells after treatment with siFAT1, SR11302 or both and a significant (Po0.05) reduction in COX- 2 luciferase activity was observed as compared with siControl þ DMSO-treated cells. (e) q–PCR was done to evaluate the mRNA expression of the AP-1-regulated transcripts COX-2, IL-6 and IL1b after treatment of U87MG cells with 10 mM SR11302. There was a marked reduction in the mRNA expression of COX-2, IL-6 and IL1b after treatment with SR11302 as compared with DMSO control. A full colour version of this figure is available at the Oncogene journal online.

line (Supplementary Figure S6), which has low FAT1 and high with P ¼ 0.048. The IL-6 expression in groups A (13.6±25.69) and PDCD4 expression (Figure 2c). Together, these results conﬁrm the D (1.21±2.97) was found to follow a similar trend, however, it was role of PDCD4 in mediating the observed effects of FAT1. not statistically signiﬁcant (P ¼ 0.146).

FAT1 and PDCD4 expression is inversely correlated in primary DISCUSSION GBM samples The role of FAT1 as an apical regulator of the Salvador–Warts– PDCD4 expression is reported to be lost in human glioma and may Hippo pathway and its function as a tumor-suppressor gene is well contribute to the development of tumor.40,73 To study the documented in Drosophila24,28 but there are limited reports relationship between FAT1 and PDCD4 expression in tumors, we regarding the role of FAT1 in human cancers. A study from our checked the mRNA expression of both FAT1 and PDCD4 in 35 laboratory36 had shown extensive variation in FAT1 expression GBM samples by q–PCR (Table 1). The 35 GBM samples were with 33% of glioma samples having low FAT1 mRNA expression arranged according to decreasing FAT1 expression and divided and loss-of-heterozygosity at FAT1 locus in a significant number of into quartiles.74 The PDCD4 expression (mean value±s.d.) in the the glioma samples studied. Another study34 had showed first quartile (group A) with the highest FAT1 expression homozygous deletion of FAT1 gene and low expression in oral (0.586±0.998) was significantly less than that in the fourth carcinoma. Role of FAT1 had also been suggested in development quartile (group D) with the lowest FAT1 expression of fallopian tube cancer in patients of MRKH syndrome.35 In (54.108±51.18), with P ¼ 0.0145. The inverse relationship contrast, high FAT1 expression has been reported in breast observed between the expression of FAT1 and PDCD4 in tumor cancer31 and leukemia where it is considered as an independent samples supports the in vitro results. prognostic marker.32 The role of FAT1 in cell migration is also well Further, we also analyzed the mRNA expression of COX-2 and studied75–78 and its knockdown has been reported to inhibit IL-6 in these GBM samples and observed positive correlation with migration of VSMC79 and OSCC cells.33 FAT1 expression. The COX-2 expression in group A (4.99±4.074) The signaling cascade being regulated by FAT1 gene and the was significantly higher as compared with group D (0.248±0.174) function of FAT1 gene in human cancers including GBM is not

Oncogene (2013) 3798 – 3808 & 2013 Macmillan Publishers Limited FAT1 gene in cancer and inﬂammation B Dikshit et al 3803 1.4 P<0.001 3.5 P<0.001 1.2 3 2.5 1 sicontrol sicontrol 2 0.8 siFAT1+sicontrol siFAT1+sicontrol 1.5 siPDCD4+sicontrol 0.6 siPDCD4+sicontrol 1 siFAT1+siPDCD4 0.4

Fold Expression siFAT1+siPDCD4 0.5 0.2 0 FAT1/18S PDCD4/18S 0 Fold Migration Fold Invasion

siControl siFAT1+siControl siPDCD4+siControl siFAT1+siPDCD4 Migration Invasion

2.50 sicontrol 2.0 P<0.01 siFAT1+sicontrol 1.5 2.00 siPDCD4+sicontrol siFAT1+siPDCD4 1.0 1.50

activity (%) 0.5 1.00 Fold Expression Relative AP1 luciferase 0.0 0.50 PGL3 siControl siFAT1 siPDCD4 siFAT1 + + + 0.00 siControl siControl siPDCD4 MMP3 VEGF PLAU COX2 IL6 IL1β

siControl +++- siFAT1 --++ siPDCD4 --+ +

PDCD460, 60

p-c-Jun, 43

VEGF-C, 47

COX-2, 74

-actin, 43

Figure 5. Simultaneous knockdown of FAT1 and PDCD4 reverses the effects of FAT1 knockdown. (a)FAT1andPDCD4mRNAexpressionwas analyzed by q–PCR in U87MG cells treated with siFAT1 and siPDCD4 alone, as well as both the siRNAs treated simultaneously. Treatment with siFAT1 þ siControl was found to upregulate PDCD4 expression, whereas treatment with siFAT1 þ siPDCD4 downregulated the PDCD4 expression to the level of siControl-treated cells alone. (b, c) Simultaneous knockdown of FAT1 and PDCD4 in U87MG cells restored their migratory and invasive properties comparable to that of siControl-treated cells. There was significant increase in cell migration and invasion in U87MG cells treated with siFAT1 þ siPDCD4 as compared with cells treated with siFAT1 þ siControl. Cells were counted in five different fields. Each value is mean±s.d. Experiment was put up in triplicate and repeated twice. (d) AP-1 luciferase activity significantly increased after PDCD4 knockdown in U87MGsiFAT1 cells. The luciferase activity with siControl is designated as 100%. There was a significant increase in AP-1 luciferase activity in U87MG cells treated with siFAT1 þ siPDCD4 as compared with siFAT1 þ siControl. And the luciferase activity in cells treated with siFAT1 þ siPDCD4 is comparable to siControl-treated cells. The experiment was repeated thrice following each of three independent transfections and representative data are shown. Results are expressed as mean±s.d. (e) The mRNA expression of AP-1-target genes increased in U87MG cells treated with siFAT1 þ siPDCD4 as compared with siFAT1 þ siControl. 18S was used as internal control and experiments were done in triplicate. (f) PDCD4 knockdown in U87MGsiFAT1 cells revert back the protein expression of p-c-Jun, VEGF-C and COX-2 comparable to siControl-treated cells alone. Lysates from indicated cells were probed with respective antibodies. b-actin was used as control antibody. A full colour version of this figure is available at the Oncogene journal online.

& 2013 Macmillan Publishers Limited Oncogene (2013) 3798 – 3808 FAT1 gene in cancer and inflammation B Dikshit et al 3804 Table 1. Expression analysis of FAT1, PDCD4, COX-2 and IL-6 in human mRNA expression in grade IV glioma cell lines like U87MG, A172, GBM samples by q-PCR U373MG and T98G and decreased expression in grade III glioma cell lines (GOS3 and SW1088). Knockdown of FAT1 in U87MG and Group Samples FAT1/ PDCD4/ COX-2/ IL-6/ A172 led to marked reduction in migratory and invasive properties 18S 18S 18S 18S of the cells along with high expression of PDCD4. Increased PDCD4 expression was also observed upon FAT1 knockdown in Group A GBM10 70.560 3.160 1.765 80.171 U373MG and T98 cell lines. PDCD4 is known to inhibit migration GBM35 34.844 0.774 6.821 18.189 GBM11 19.990 0.438 8.168 5.152 and invasion in various cancers like breast, ovarian, colon and so on.49,82 Loss of PDCD4 expression has been linked with cell GBM8 19.490 0.430 1.905 0.020 40,73,83 GBM24 13.990 0.004 2.346 3.238 proliferation and unfavorable prognosis in gliomas. GBM30 13.707 0.056 0.006 0.067 Decreased migration and invasion observed in U87MG and A172 GBM25 8.138 0.002 0.387 1.597 cell lines after FAT1 knockdown in this study might be attributed GBM6 5.980 0.303 14.929 12.862 to increased PDCD4 expression. High PDCD4 expression after FAT1 GBM5 5.290 0.112 0.337 1.834 knockdown was not found to induce apoptosis or alter cell-cycle distribution in both U87MG and A172 cell lines, which is in Group B GBM12 4.700 0.555 0.742 25.020 accordance to a similar study in NIH3T3 cells by Shibahara et al.84 GBM7 4.660 10.754 1.778 4.302 85 GBM31 4.000 0.176 0.056 0.034 but in contrast to report on breast cancer cell lines. This difference GBM33 3.949 0.290 0.143 0.155 can be due to molecular cross-talk between different signaling GBM2 2.479 0.100 0.100 0.372 cascades in cell lines affecting the functioning of the cells in a GBM32 1.548 0.195 0.158 0.509 context-dependent manner. In our study, we observed that high GBM29 1.500 134.809 0.001 3.340 PDCD4 levels after FAT1 knockdown mainly regulate migration and GBM28 1.300 1.372 0.001 0.963 invasion in glioma cells. Decreased PDCD4 is known to have an GBM27 1.200 0.333 1.892 2.514 important role in tumor progression86–88 and in remodeling ECM during invasion.89,90 To further confirm the link between FAT1 and Group C GBM1 0.034 0.000 0.014 1.279 PDCD4, we simultaneously knocked down PDCD4 and FAT1 GBM23 0.007 0.003 0.002 0.003 GBM21 0.006 0.001 0.001 74.028 expression and found reversal of migratory and invasive GBM34 0.006 0.002 0.010 73.262 properties of the cells, which further confirmed that the observed GBM4 0.003 0.000 0.007 3.494 effects of FAT1 knockdown are mainly mediated by PDCD4. GBM13 0.003 0.000 0.702 4.332 Because PDCD4 is known to attenuate AP-1-mediated transcrip- GBM3 0.002 0.000 0.611 3.399 tion by inhibiting c-Jun phosphorylation,49,50 we checked the GBM22 0.002 0.002 0.010 0.081 effect of FAT1 knockdown on the AP-1 activity by luciferase assay and observed diminished AP-1 transcriptional activity and Group D GBM9 0.001 0.000 1.347 0.232 decreased expression of AP-1-regulated transcripts like ECM- GBM14 0.001 0.000 0.030 8.545 remodeling molecules MMP3, VEGF-C, PLAU, inflammatory GBM15 0.001 0.000 0.004 0.838 GBM16 0.001 43.633 0.120 0.041 mediator COX-2 and cytokines IL1b and IL-6. Overexpression of pro-inflammatory molecules in GBM and their role in glioma GBM17 0.001 40.818 0.058 0.007 13 GBM18 0.001 106.912 0.743 0.226 progression is well known. Moreover, the role of various GBM19 0.001 82.124 0.009 0.004 signaling molecules in promoting inflammatory response in GBM20 0.001 69.487 0.140 0.009 gliomas has also been convincingly demonstrated.18,20,91,92 The GBM26 0.001 143.998 0.288 0.017 reduced AP-1 activity observed in glioma cell lines after FAT1 Abbreviations: GBM, glioblastoma multiforme; q–PCR, quantitative knockdown could be attributed to inhibition of c-jun transcription PCR.GBM samples were divided into four groups (groups A, B, C and D) and c-Jun phosphorylation by increased PDCD4 expression. These based on quartiles showing decreasing FAT1 expression. The PDCD4 effects were further confirmed by dual knockdown of FAT1 and expression (mean±s.d.) in the groups A and D were calculated PDCD4 in both U87MG and A172 cell lines. Also, the increased (0.586±0.998 and 60.856±50.209, respectively) and the difference in the PDCD4 expression after FAT1 knockdown in glioma cells was two groups were found to be statistically significant (P ¼ 0.0145). Similarly, found to be independent of the Akt pathway. The observation of on comparing the expression of COX-2 in groups A and D, there was a FAT1 in regulating PDCD4 expression and its downstream effect significant positive correlation between FAT1 and the COX-2 expression, on tumor migration and invasion is a novel finding with ± ± with the mean s.d. of groups A and D being 4.99 4.074 and implications for glioma biology. 0.248±0.174, respectively (P ¼ 0.048). For IL-6, there was a similar positive trend between group A and D, however, it was not statistically significant We have shown that FAT1 is linked to COX-2 via PDCD4 and AP-1. (P ¼ 0.146). The downregulation of COX-2 expression and pro-inflammatory cytokines via PDCD4 upregulation after FAT1 knockdown has considerable significance in the regulation of inflammatory responses in glioma. Decreased COX-2 expression after FAT1 knockdown led to reduced PGE2 production and decreased mRNA expression of IL-6 and IL-1b, thus providing a crucial link between known. On the basis of the loss-of-heterozygosity analysis and the tumor progression and inflammation. COX-2 expression is known expression analysis in various tumors, FAT1 gene may have a dual to be regulated by AP-1 binding to cyclic adenosine monopho- role, both as tumor suppressor and oncogene, but no functional sphate response element in the COX-2 promoter.71 We have studies are available to support these notions. Genes like TGFb,80 shown that inhibition of AP-1 by SR11302 led to significant STAT381 and so on have also been reported to have a dual role in reduction in COX-2 promoter luciferase activity as well as mRNA tumorigenesis, depending upon varying cellular contexts. expression of the AP-1-regulated genes COX-2, IL-6 and IL-1b. The Therefore, proper functional studies need to be done to support role of PDCD4 in downregulating COX-2 is known62,67 and and identify the precise role of FAT1 gene in tumors including suppression of PDCD4 is found to increase invasive activity of GBM. COX-2 in breast cancer cells.82 COX-2 overexpression is also Here we investigated the functional role of FAT1 by analyzing reported in high-grade gliomas93–95 and many other tumors with its expression in glioma cell lines followed by siRNA knockdown of potential for targeting it as anti-cancer modality.69,70,96–100 Our FAT1 in cells with high FAT1 expression. We observed high FAT1 finding of reduced COX-2 expression and decreased synthesis of

Oncogene (2013) 3798 – 3808 & 2013 Macmillan Publishers Limited FAT1 gene in cancer and inflammation B Dikshit et al 3805 prostanoids (PGE2) and cytokines, IL1b and IL-6 after FAT1 no.12935-112) were purchased from Invitrogen Life technologies (Grand knockdown points toward the role of FAT1 in regulating Island, NY, USA). For simultaneous knockdown, on-target plus siRNA oncogenic and inflammatory properties in gliomas and this may against FAT1(J-010513-07-0020) and PDCD4(J-004438-08-0020) and on- also hold true for other tumors. Thus, targeting FAT1 would have target plus non-targeting pool(D-001810-10-20) as control siRNA from strong advantages for managing both tumorigenicity and the Dharmacon (Lafayette, CO, USA). AP-1 luciferase reporter plasmid (AP-1 7 Â ) was purchased from Stratagene, and COX-2 promoter luciferase inflammatory microenvironment of gliomas. construct was a kind gift from Dr Miguel A Iniguez,Centro de Biologıá To further validate the in vitro findings about the relationship Molecular Universidad Autońoma de Madrid, Spain. AP-1 inhibitor SR11302 between FAT1 and PDCD4, we studied the expression of FAT1 and was purchased from Tocris Biosciences (Bristol, UK).103 Normal brain RNA PDCD4 in 35 primary GBM samples by q–PCR and found a was procured from Ambion (Austin, TX, USA), Life Technologies (Carlsbad, statistically significant inverse correlation between the two. CA, USA) and Clontech (Mountain view, CA, USA). Antibodies used for Further, we also observed a positive correlation between FAT1 western blots, like PDCD4, COX-2, p-c-Jun, c-Jun and b-actin were from Cell and COX-2 expression and a similar trend for IL-6 expression in Signaling Technology (Beverly, MA, USA), VEGF and Caspase-3 were from GBM samples studied. These findings further support the in vitro Abcam (Cambridge, UK). Primers were designed using Primer3 software results about the inverse relationship between FAT1 and PDCD4 and ordered from MWG Biotech (Ebersberg, Germany). and their influence on the expression of AP-1-mediated transcripts like COX-2, IL-6 and so on. Cell culture and siRNA transfection We therefore demonstrate for the first time that FAT1 has an The human glioma cell lines U87MG, U373, A172, T98G, GOS3 and SW1088 important role in modulating PDCD4 expression, which in turn were obtained from ATCC (Rockville, MD, USA) cultured in Dulbecco’s regulates AP-1-dependent transcription, controlling processes Modified Eagle Medium (Sigma-Aldrich, St Louis, MO, USA) supplemented crucial for migration and invasion in glioma cells, as well as the with 10%(v/v) fetal calf serum (Sigma-Aldrich), 3.7 g/l sodium bicarbonate (Sigma-Aldrich), ciprofloxacin 10mg/ml and 5%CO2 at 37 1C. For transfec- induction of a pro-inflammatory microenvironment in glioma. The 5 2 study illustrates a link between inflammation and cancer in glioma tion, 2 Â 10 cells were seeded per 25-cm flask. After 24 h, cells were transfected with siFAT1 and siControl(Invitrogen) according to the and raises the possibility that FAT1 might be a master regulator in manufacturer’s protocol with a final concentration of 20 nM of siRNA using this process. This work highlights the importance of FAT1 in the Lipofectamine-2000 (Invitrogen) and Opti-Mem media (Invitrogen). After induction of the cellular pathways of migration and invasion, 4 h of siRNA transfection, the media was supplemented with Dulbecco’s proteolysis of the ECM and the expression of pro-inflammatory Modified Eagle Medium containing 2 Â fetal calf serum. The ability of each molecules leading to a favorable microenvironment for tumor siRNA to knockdown FAT mRNA expression was confirmed using progression. The possible signaling pathway being regulated by quantitative real-time PCR. Photomicrographs of the cells were taken FAT1 is shown in Figure 6 and has been illustrated in a set of using an inverted phase contrast microscope (Nikon TMS, Japan). For glioma cell lines with corroborative evidence from primary human simultaneous knockdown of FAT1 and PDCD4, siRNA from Dharmacon GBM. Till now, targeting pro-inflammatory molecules or the were used. Transfection was performed as earlier with 50 nM of siFAT1 along with 50 nM of siPDCD4 or 50 nM of siControl. related pathways as therapeutic strategies to prevent or treat cancers has had a limited success and there is considerable interest in focusing on such molecules.101,102 The transmembrane cDNA synthesis and q–PCR molecule, FAT1, thus promises to be one such novel target for Total RNA was isolated from cells at appropriate time point using TRIzol therapeutic intervention in cancer and inflammation as well as the reagent (Invitrogen), quantified using a Nanodrop ND-1000 spectro- link between the two processes. photometer. DNase(Ambion) treatment was given and 1 mg of total RNA was used for cDNA synthesis, done by Fermentas RevertAid First Strand cDNA Synthesis Kit using random decamers. PCR reactions were carried in MATERIALS AND METHODS 10-ml reaction volumes (2.5 ml of 1:5 diluted cDNA, 0.5 ml of primer mix, 1 ml of 10 Â Taq Buffer-A, 0.5 U Taq polymerase (Bangalore-Genei, Bangalore, Reagents and antibodies India), 1 ml Syto9(Invitrogen), 0.25 mlof10mM dNTPs (MBI-Fermentas; Set of three siRNAs for FAT1 knockdown, FAT Stealth RNAi siRNA- Thermo Fisher Scientific, Rockford, IL, USA) and 4.6 ml of NFW (Ambion)) I(HSS103567) FAT Stealth RNAi siRNA-II(HSS103568) and FAT Stealth RNAi on a RotorGene6000 Real-Time PCR Corbett Life Science (Mortlake, NSW, siRNA-III(HSS176716) and universal medium GC control siRNA (cat. Australia) in triplicates. The detail of the primers used is given in Supple- mentary Table S1. DCt values were defined as target gene Ct minus 18 S RNA Ct and averaged for each triplicate sample. Statistical significance was determined by Student’s t-test analysis (Po0.05).

Western blotting analyses Cells were lysed in triple detergent buffer containing protease and phosphatase inhibitors (Sigma-Aldrich), lysates were quantified by BCA kit (Pierce, Thermo Fisher Scientific, Rockford, IL, USA) and electro- phoresed in 10% SDS–PAGE and electroblotted on nitrocellulose membrane (Millipore, Billerica, MA, USA). Membrane was blocked with 5% bovine serum albumin in 1 Â Tris–buffered saline at 4 1C O/N. Primary and secondary antibodies were diluted in 5% bovine serum albumin and 0.1% tween-20 in 1 Â Tris-buffered saline. Blot was incubated in primary antibody overnight at 4 1C followed by secondary antibody incubation for 2 h at room temperature. Blot was developed by BCIP-NBT (Promega, Madison, WI, USA) and captured using Alpha Imager EP software (Alpha Figure 6. The proposed signaling pathway downstream of FAT1 Innotech Corp, San Leandro, CA, USA). regulating AP-1-dependent transcription. Knockdown of FAT1 expression releases its inhibitory effect on PDCD4 and increase the expression of PDCD4. Increased PDCD4 expression in turn Migration and invasion assays inhibits the phosphorylation of c-Jun, thus decreasing phospho-c- The modified Boyden chamber/matrigel assay was performed according to Jun levels. Because phospho-c-Jun is required for AP-1-dependent the manufacturer’s directions (BD Biosciences, Bedford, MA, USA). For transcription, there was inhibition of AP-1 transcriptional activity in vitro migration assays, we used chambers with control inserts that lacked and downregulation of target genes like COX-2, MMP3, VEGF-C, the matrigel coating. Transfections were carried out in U87MG and A172 PLAU, IL-6 and IL-1b. A full colour version of this figure is available at cells and 48 h later 3 Â 104 U87MG cells per well and 7 Â 104 A172 cells per the Oncogene journal online. well were resuspended in serum-free Dulbecco’s Modified Eagle Medium

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