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FAT1 is a novel upstream regulator of HIF1α and invasion of high grade glioma

The hypoxic microenvironment is an important contributor of glioblastoma (GBM) aggressiveness via HIF1α, while tumour inflammation is profoundly influenced by FAT Atypical Cadherin (FAT1). This study was designed to explore the functional interaction and significance of FAT1 and HIF1α under severe hypoxia‐mimicking tumour microenvironment in primary human tumours. We first identified a positive correlation of FAT1 with HIF1α and its target genes in GBM tumour specimens, revealing the significance of the FAT1‐HIF1α axis in glioma cells. We found that severe hypoxia leads to an increased expression of FAT1 and HIF1α in U87MG and U373MG cells. To reveal the relevance of FAT1 under hypoxic conditions, we depleted endogenous FAT1 under hypoxia and found a substantial reduction in the expression of HIF1α and its downstream target genes like CA9, GLUT1, VEGFA, MCT4, HK2, BNIP3 and REDD1, as well as a significant reduction in the invasiveness in GBM cells. At the molecular level, under hypoxia the FAT1 depletion‐associated reduction in HIF1α was due to compromised EGFR‐Akt signaling as well as increased VHL‐dependent proteasomal degradation of HIF1α. In brief, for the first time, these results reveal an upstream master regulatory role of FAT1 in the expression and role of HIF1α under hypoxic conditions and that FAT1‐HIF1α axis controls the invasiveness of GBM. Hence, FAT1 represents a novel potential therapeutic target for GBM.

Glioblastomas (GBM) are the commonest and most lethal intracranial tumours. The GBMs exhibit a relentless malignant progression characterized by invasiveness and resistance to therapy. 1,2 In GBM, hypoxia is readily recognizable, resulting in the necrotic areas in the core of the tumour and surrounded by evidence of hypoxic response and neovascularisation. 3 It is known that hypoxia contributes to an aggressive phenotype in tumours, including features of resistance to apoptosis, drug resistance, increased invasiveness and tumour heterogeneity and aggressive growth patterns. 4 Survival and proliferation of GBM cells under such adverse condition requires an adaptation to the environment, wherein, hypoxia-inducible factor-1 (HIF1a) is a key adaptive molecule for regulating the cellular response. 5 On activation and stabilisation during hypoxia, HIF1a upregulates target genes involved in the process of angiogenesis, tumour invasion and metastasis, energy metabolism, and adaptive survival in GBM 6,7 and tumours at-large. 8 Overexpression of HIF1a in surgically resected tumour specimens has been shown to have positive correlation with tumour aggressiveness and poor prognosis in different tumours including GBM. 9 HIF1a is known to be regulated by growth factors, 10,11 cytokines 12 and mitogens. 13,14 EGFR/Akt pathway is a known positive regulator of HIF1a 10 and this pathway may function via mTOR 10,11 or independent of it. 15,16 The EGFR/Akt pathway is known to be frequently activated in GBM. 17 FAT1 is a transmembrane protein, mapped on human chromosome 4q34-35. 18 FAT1 is the human orthologue of Drosophila tumour suppressor gene 'fat'. The name FAT was given by Dunne et al. (1995) in their paper as it structurally resembles Drosophila tumour suppressor protein 'fat'. In Drosophila, the protein was named 'fat' as the recessive mutations at the locus lead to fattening or overgrowth of the Drosophila imaginal discs 19 hence, was named as 'fat locus'. FAT1 regulates the invasive and/or migratory potential of the normal 20,21 and cancer cells. 22,23 FAT1 exhibits a dual role as it could act as an oncogene [23][24][25] or tumour suppressor 26,27 in different tumour types. For the first time, our laboratory had demonstrated a role of FAT1 in GBM. 22,28 While on one hand, a high proportion of FAT1 LOH was seen in glioma, on the other hand, we observed that FAT1 overexpression 28 acted like an oncogene and promoted an inflammatory environment in GBM. 22 The underlying mechanism includes stimulation of AP1-mediated transcriptional activation due to the down-regulation of the tumour suppressor gene, PDCD4. 22 More recently, increased FAT1 expression has been noted in hepatocellular carcinoma due to reduced levels of the methyl group donor S-adenosyl-L-methionine under hypoxia. 25 Although FAT1 22 and HIF1a 29,30 have emerged as two independent contributors of adverse phenotypes in glioma, a regulatory link between FAT1 and HIF1a remains unknown. We had previously reported the mRNA levels of FAT1 as well as HIF1a and its target genes in primary human GBM samples from the same cohort group. 22,31 On further analysis of these results, we observed a positive correlation of FAT1 with HIF1a and its target genes in these primary GBM specimens. Consequent to these findings, we have elucidated the molecular interaction between FAT1 and HIF1a under severe hypoxia in GBM cell lines (U87MG and U373MG) and the grade-II glioma cell line (GOS3). In brief, we have identified an upstream regulatory role of FAT1 in the expression of HIF1a, and consequently, its functions in GBM cells under hypoxia and elucidated the underlying mechanism of this regulation. This identifies FAT1 as an upstream regulator of the hypoxic response in GBM.
cDNA synthesis and q-PCR Total RNA was isolated from cells at appropriate time point using TRIzol reagent (Invitrogen, Grand Island, NY), quantified using a Nanodrop ND-1000 spectrophotometer. DNase (Ambion) treatment was given and 1 mg of total RNA was used for cDNA synthesis and q-PCR was done for expression analysis. 22 The detail of the primers used for expression analysis is given in Supporting Information table (Supporting  Information Table SI).

Western blot analysis
Lysates were prepared and blot was developed as mentioned earlier. 22 Equal amounts of protein (60 mg) were resolved on a 10% SDS-PAGE. Enhanced chemiluminescence Western blotting detection reagent (Pierce, Rockford, IL) was used to detect protein levels. Band densities of protein were normalized to b-actin.

Cell viability assay and cell apoptosis analysis
To assess the effect of FAT1 knockdown on glioma cell viability, MTT assay was done.

What's new?
The hypoxic microenvironment is an important contributor of glioblastoma aggressiveness via HIF1a while tumor inflammation is profoundly influenced by FAT Atypical Cadherin (FAT1). This study explores the functional interaction of FAT1 and HIF1a in severe hypoxia-mimicking tumor microenvironments. The results show that FAT1 upregulation is critical for enhancing and maintaining high HIFIa levels in tumors with severe hypoxia. FAT1 both increases HIFIa transcription and decreases HIFIa degradation in glioblastoma multiforme cell lines under hypoxic conditions. With FAT1 regulating the activity of HIF1a under hypoxic condition and the FAT1-HIF1a axis controlling the invasiveness of glioblastoma, FAT1 represents a novel potential therapeutic target for glioblastomas.
For MTT assay, 8 3 10 3 cells were seeded in each well of 96-well flat bottom plates. Twenty-four hour after plating, FAT1 and control siRNAs were transfected as described above and then maintained under normoxia or hypoxia. After 24 hr, 48 hr, 72 hr of siRNA transfection, MTT assay was performed as per manufacturer's protocol. Each experiment was done in triplicates and repeated thrice.

Migration and invasion assay
The migratory/invasive responses of glioma cells after FAT1 knockdown were analysed using the modified Boyden chamber/matrigel assay as described earlier. 22 To assess the effect of FAT1 knockdown on migration/invasion under hypoxia, after siRNA transfection, cells were maintained under hypoxia for 72 hr and cell migration and invasion assay was performed.
HRE (HIF1a response element) luciferase reporter assay U87MG cells (5 3 10 4 cells/well) were plated in 6-well plates in triplicate. Cells were transfected with siFAT1 and siControl and then maintained under normoxic/hypoxic condition. After 24 hr of siRNA transfection, cells were transfected with 1 mg of HIF1a luciferase reporter plasmid with six copies of wild-type-TACGTGAC or mutant-TAAAAGAC hypoxiaresponsive elements (HREs), 32 along with 50 ng of pRL-TK (Renilla luciferase) control plasmid using lipofectamine2000 as the transfection reagent. After 48 hr of reporter plasmid transfection, the cells were lysed using 13 passive-lysis-buffer (Promega) and luciferase activity was assayed as previously described. 33 The luciferase activity observed in cells transfected with pGL3 vector was taken as a reference and in the cells co-transfected with control siRNA/FAT1 siRNA and wildtype promoter construct was expressed as fold change.
Metabolic studies ATP levels. Cells of 8 3 10 3 per well were plated in 96-well plate, after 24 hr of plating, cells were transfected with siFAT1 and siControl followed by hypoxia exposure. After 72 hr of siFAT1 transfection and hypoxic treatment, levels of ATP in the cells were measured using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega Biosciences, CA) following manufacturer's protocol. Luminescence was measured by a microplate luminometer.
Lactate levels. Cells of 8 3 10 3 per well were plated in 96well plate and allowed to adhere for 24 hr. After 72 hr of siFAT1 transfection and hypoxic treatment, lactate levels were measured using the Beckman Coulter Machine at Sri Ganga Ram Hospital, New Delhi and were also confirmed using Biovision's Lactate Colorimetric Assay Kit II (Biovision, San Francisco).

Human GBM samples
Correlation of FAT1, HIF1a and hypoxia markers (PGK1 and VEGFA) in 35 human Grade IV GBM samples within the same cohort was done using the data already published. 22,31 Here, we analysed the correlation between FAT1 and hypoxia markers. Tumours were arranged in decreasing order of FAT1 expression and grouped into four quartiles (A, B, C and D). Group A had the highest FAT1 expression and group D the lowest. 22 Expression level of HIF1a, PGK1 and VEGFA were compared between A and D and statistical correlation was done using Student's t test (1-tailed). Similarly the Pearson correlation of FAT1 with HIF1a and its target genes was analysed in these GBM samples using SPSS (1tailed). FAT1 and HIF1a expression was also analysed in 18 grade II astrocytoma samples in a manner similar to that for Grade IV tumours. Again, HIF1a expression in the highest and lowest quartiles based on decreasing order of FAT1 expression was analysed, followed by statistical correlation and regression analysis using SPSS.

Statistical analysis
All in vitro experiments were performed in triplicate and repeated thrice. Differences were determined using Student's t test, and p 0.05 was considered significant. Data values are shown as the mean 6 s.d. Graphs were prepared using Prism3 (Graph Pad). Real time data were analysed using REST software v2.0.1.13

FAT1 status correlates well with the HIF1a expression in primary human GBM tumours
Here, we analysed the correlation status of the mRNA expression of FAT1 with HIF1a and its target genes (PGK1and VEGFA) in the cohort of patients used in our recent study 22,31 by statistical means. We arranged the GBM samples in the decreasing order of FAT1 expression and grouped these as quartiles. 22 The expression of HIF1a (mean value 6 s.d) in the first quartile having the highest FAT1 expression (group A) was compared with that observed in the fourth quartile having lowest FAT1 expression (group D). HIF1a expression was significantly higher (14.811 6 18.894) in the highest FAT1 expressing quartile (group A) as compared to the lowest (3.455 6 3.161) FAT1 expression quartile (group D) ( Table 1). The difference was statistically significant with p 5 0.04. Of the HIF1a targets, the correlation of FAT1 with PGK1 and VEGFA was significant with p 5 0.05 and p 5 0.02, respectively (Table 1). Furthermore, regression analysis (Pearson correlation) of 35 GBM samples for these values, showed that FAT1 mRNA levels were significantly correlated with HIF1a (R 2 5 0.520, p 5 0.001), PGK1 (R 2 5 0.559, p < 0.001) and VEGFA (R 2 5 0.512, p 5 0.001) levels. In addition to this, we have also analysed expression correlation of FAT1 and HIF1a in 18 grade II astrocytoma samples. Similar to GBM, the difference of HIF1a expression between the highest (33.10 6 31.12) and lowest (1.16 6 1.24) quartiles (as determined by FAT1 expression) was statistically significant (p 5 0.049). This was also supported by regression analysis (Pearson coefficient 5 R 2 5 0.793, p < 0.001, n 5 18, the exact values are given in Supporting Information Table  S2). Furthermore, on comparing HIF1a expression in the highest and the lowest quartile based on FAT1 expression in all glioma tumours (grade II and grade IV taken together), we observed statistically significant difference (p 5 0.013) in HIF1a expression mean 5 22.94 in the quartile having high FAT1 expression and mean 5 2.86 in quartile having low FAT1 expression. Moreover, we also observed significant correlation between HIF1a and FAT1, as done by Regression analysis (Pearson coefficient 5 R 2 5 0.506, p < 0.001, n 5 52).  Fig S1). There was no difference in expression between mock and siControl treated cells and hence siControl has been used for better comparison with knockdown experiments shown subsequently. We noted that the basal FAT1 mRNA expression in GBM (U87MG and U373MG) cell lines was observed to be higher as compared to grade-II glioma cell line (GOS3). Levels in GOS3 were 20% of that observed in U87MG (Fig. 1a) under standard culture conditions with 20% oxygen. On exposing the cells to severe hypoxia (0.2% O 2 ), the level of FAT1 mRNA was 7.8, 4.3 and 1.8 folds higher in U87MG, U373MG and GOS3 cells as compared to their respective normoxic counterparts (Fig. 1b). On analysing HIF1a mRNA, it was found to be increased by 2.8, 1.8 and 1.5 folds higher in hypoxic U87MG, U373MG and GOS3 cell lines respectively as compared to their normoxic counterparts (Fig.  1c). A significant increase in HIF1a protein was also observed in U87MG (1.45 fold) and U373MG (1.55 fold) cells but not in GOS3 (Fig. 1d), supporting the notion that hypoxia increases expression of FAT1 and HIF1a in U87MG and U373MG cells.

FAT1 is an upstream regulator of HIF1a expression
As hypoxia increased the expression of both FAT1 and HIF1a, we next investigated the possibility of a regulatory role of FAT1 in influencing the level of HIF1a by depleting the endogenous FAT1 using FAT1 specific siRNA 21 Fig S2). We found that FAT1 depletion was accompanied by 65% and 37% reduction in  On FAT1 knockdown, 60% decrease (p 5 0.01) in HIF1a promoter luciferase activity was observed in comparison to siControl treated cells, as determined by Student's t test. The experiment was repeated thrice and results are expressed as mean 6 standard deviation. As mentioned earlier, the first two lanes for GOS3 in Figure 2c are also depicted in Figure 1d.

Molecular Cancer Biology
the levels of HIF1a mRNA in U87MG and U373MG, as compared to cells treated with control siRNA (Fig. 2b). Similarly, HIF1a protein levels were also decreased significantly in U87MG and U373MG cell lines on FAT1 knockdown (Fig. 2c). In grade-II glioma cell line (GOS3) with low endogenous FAT1 expression, the effect of FAT1 on HIF1a was minimal (Fig. 2b). To confirm the effect of FAT1 knockdown on HIF1a expression, the U87MG cell line was exposed to severe hypoxia for different time periods before analysing the status of HIF1a. We observed a rapid induction of HIF1a protein in cells treated with control siRNA, while depletion of FAT1 impaired the ability of hypoxia to induce HIF1a (Supporting Information Fig S3), revealing a regulatory role of FAT1 on HIF1a expression. To study whether HIF1a, conversely, has a role in regulating the expression of FAT1 under hypoxia, HIF1a was knocked down using HIF1a specific siRNA and analysed the expression of FAT1. We observed a comparable expression of FAT1 in both siHIF1a and siControl treated cells with no effect on FAT1 expression after HIF1a knockdown (Fig. 2d). HIF2a expression was also observed to be upregulated under hypoxia in U87MG cell line (Supporting Information Fig S4). HIF2a knockdown showed no effect on the expression of FAT1 (Supporting Information Fig S4). Next we examined the effect of FAT1 on HIF1a activity using a HIF1a luciferase reporter assay. U87MG cell line was maintained under hypoxia and co- transfected with HIF1a reporter plasmid (wild-type HIF1a promoter) or control plasmid (PGL3 empty vector) 32 in cells with or without depleted FAT1. The results demonstrated 60% decrease in the HIF1a luciferase activity in siFAT1 treated cells (p 5 0.01), in comparison to siControl treated cells (Fig. 2e), reflecting the loss of positive regulatory effect of FAT1 after its knockdown on HIF1a activity under hypoxic condition. As expected, HIF1a luciferase activity in cells transfected with HRE mutated HIF1a promoter was undetectable (Supporting Information Fig S5). Collectively, these results validate a positive regulatory role of FAT1 on HIF1a expression and activity under hypoxic conditions in GBM cells.

Revelation of FAT1 regulation of EGFR-Akt signaling under hypoxia
Because the EGFR-Akt pathway augments HIF1a expression under hypoxia, 10 we next analysed the effect of FAT1 knockdown on the expression of EGFR as well as the expression and phosphorylated state of Akt in glioma cell lines under hypoxia. On FAT1 knockdown, there was 75% reduction in the level of EGFR transcription in U87MG cells (Fig. 3a) and significant reduction of EGFR protein in U87MG and U373MG (Fig. 3a) as compared to their respective siControl treated hypoxic cells. There was marked reduction in the levels of p-Akt in both U87MG and U373MG cells (Fig. 3b) after FAT1 knockdown as compared to their respective hypoxia control cells. p-mTOR and p-S6K1 levels under hypoxia in both U87MG and U373MG cells was found to be low as compared to normoxic counterparts (Fig. 3b). On FAT1 knockdown, there was further reduction in their levels (Fig.  3b). Hence, it appears that FAT1 regulates HIF1a expression via EGFR/Akt/mTOR pathway as previously reported. 15,16 FAT1 is a negative regulator of VHL (Von Hippel-Lindau) expression under hypoxia VHL regulates HIF1a level in tissues by promoting its proteasomal degradation. 34 Normally, VHL levels are high under normoxia and low under hypoxia. 35 In glioma cells exposed to hypoxia, the level of VHL was observed to be low in comparison to its normoxic counterpart (Fig. 3c). On FAT1 knockdown, there was no significant increase of VHL mRNA (Fig. 3c); however, the levels of VHL protein were increased in both U87MG and U373MG cell lines (Fig. 3c). To further validate FAT1 regulated proteasomal degradation of HIF1a, glioma cells were treated with MG132 (proteasomal inhibitor) with and without FAT1 knockdown under hypoxia. Here, U87MG cell line was maintained under either normoxia or hypoxia followed by treatment with MG132 for 48 hr and levels of the HIF1a protein was determined by Western blot. Under normoxia, on treatment with MG132, there was a three fold increase in the level of HIF1a as compared to the untreated cells (Fig. 3d). Under hypoxia, HIF1a protein was found to be four fold higher than their normoxic counterparts and the level was similar in both MG132 treated and untreated cells (Fig. 3d), indicating the expected lack of proteasomal degradation of HIF1a under hypoxia. Next, we knocked-down FAT1 and simultaneously treated with MG132. In hypoxic conditions, the HIF1a protein was found to be 4.5 folds higher after FAT1 knockdown with MG132 treatment, as compared to FAT1 knockdown-MG132 untreated cells (Fig. 3d). This indicates that even under hypoxia, glioma cells with FAT1 knockdown show increased proteasomal degradation of HIF1a. These findings suggest a regulatory effect of FAT1 on the status of HIF1a via proteasomal degradation in addition to the decreased synthesis of HIF1a via EGFR/Akt/mTOR pathway.

FAT1 regulation of HIF1a targets and metabolic activity in GBM cells
As HIF1a is known to regulate the expression of various genes including GLUT1, MCT4, REDD1 and HK2 which are known to be involved in anaerobic glycolytic pathway 36 ; here, we analysed the effect of FAT1 on the expression of HIF1a target genes as well as the HIF1a regulated metabolic activity in gliomas. First, the expression of HIF1a target genes (CA9, VEGFA, GLUT1, MCT4, HK2, BNIP3 and REDD1) was analysed after FAT1 knockdown in cells maintained under hypoxia. The HIF1a target genes expression was found to be significantly upregulated under hypoxia in U87MG and U373MG cells; however, on FAT1 knockdown, there was significant reduction in the expression of all target genes in both the cell lines (Fig. 4a). In general, effects of FAT1 knockdown on HIF1a target genes were modest in GOS3 cells as compared to GBM cell lines (Fig. 4a).
As there was significant reduction in the expression of HIF1a target genes in glioma cell lines after FAT1 knockdown under hypoxia, we measured the levels of lactate as well as ATP in these cell lines (U87MG, U373MGand GOS3). Significant decrease in the level of lactate (p 0.03) and ATP (p 0.02) after FAT1 knockdown was observed in U87MG and U373MG cells as compared to their respective control cells (Fig. 4b).The decrease in the level of lactate and ATP molecules corroborated with the decreased expression of HIF1a regulated metabolic genes (CA9, GLUT1, MCT4 and HK2) observed after FAT1 knockdown under hypoxia (Fig. 4a). In GOS3 cell line, as the basal expression of FAT1 was low as compared to GBM cell lines, the effect of FAT1 on HIF1a and its target genes was found to be minimal with no significant difference in the level of lactate and ATP with or without FAT1 knockdown under hypoxia (Fig. 4b).
These results suggest the importance of overexpressed FAT1 in regulating the expression of HIF1a and its target genes which further regulates the metabolic activity of GBM cells under hypoxia. Thus, the positive correlation between the mRNA expression of FAT1, HIF1a and its target genes observed in primary human GBM samples was further supported by our physiologically relevant models, establishing a positive regulatory effect of FAT1 on HIF1a activity under hypoxia.

FAT1 status control migration and invasion of glioma cells under hypoxia
We next analysed the effect of FAT1 on migration and invasion of glioma cells under hypoxia. We observed significant increase in the migratory (p < 0.01) as well as invasive (p < 0.05) properties of cells under hypoxia, as compared to their normoxic counterparts (Figs. 5a and 5b). However, on FAT1 knockdown, significant reduction in both migratory (p < 0.01) and invasive ability (p < 0.02) of glioma cells (U87MG and U373MG) was observed as compared to their respective controls under hypoxia (Figs. 5a and 5b). In GOS3, with low endogenous FAT1 expression, the effect of FAT1 knockdown on migratory and invasive ability was significant but the fold effect was not as pronounced as was observed in U87MG and U373MG, with high endogenous FAT1 expression (Figs. 5a and 5b). This observation points to the important and critical role of FAT1 in cell migration and invasion under severe hypoxia. On analysing the effect of FAT1 knockdown on cell viability in U87MG cell line under severe hypoxia, we observed no difference in cell viability with and without FAT1 knockdown under hypoxia (Fig. 5c). This finding reflects that transient FAT1 knockdown does not affect the glioma cell viability under hypoxia, similar to our earlier observation under normoxia. 22

Discussion
Several mechanisms regulate the stability and activity of HIF1a protein in both oxygen-dependent and -independent manner. HIF1a is known to be mainly regulated by affecting its proteasomal degradation and translation. The oxygen dependent regulation of the HIF-a subunit through its ubiquitin-proteasomal degradation by the pVHL pathway has been well studied. 37,38 In addition, studies have reported oxygen independent degradation of HIF1a by other factors like SSAT2, 37 os-9, 39 where these factors help in stabilising interaction of VHL with HIF1a, PHD, elongin C and so forth, thereby promoting HIF1a degradation. HIF1a is also known to be regulated by other mechanisms independent of VHL, involving heat shock protein (Hsp90), receptor of activated protein kinase (RACK1), MDM2 and so forth. [40][41][42] In addition, numerous reports indicated that activation of EGFR/ PI3K/Akt pathway (-dependent and -independent of mTOR) is responsible for increased HIF1a translation under both normoxia and hypoxia. 11,16 Other signaling pathways like raf-1-MEK1-ERK, Insulin-like growth factors, EGFR and so forth 10 and cytokines like IL1b and TNFa 43 are also known to upregulate the level of HIF1a protein.
Here, we studied the regulatory role of FAT1 on HIF1a by knocking down FAT1 expression by siRNA in GBM cell lines under severe hypoxia. The indication that FAT1 could be linked to HIF1a regulation came from our analysis on primary GBM specimen were we observed statistically Figure 5. (a,b) Effect of FAT1 knockdown on migration and invasion of glioma cells: (a) On FAT1 knockdown, significant reduction in migratory ability of U87MG (72%, p 5 0.008), U373MG (70%, p 5 0.001) and GOS3 (36%, p 5 0.001) cells was found. Migration assay was done using Modified Boyden chamber assay. (b) The invasiveness of glioma cells transfected with FAT1 siRNA was reduced significantly in U87MG (74%, p 5 0.016), U373MG (73%, p 5 0.013) and GOS3 (55%, p 5 0.021) cell lines. Invasion assay was done using Matrigel assay. As compared to GBM cell lines (U87MG and U373MG) the fold migration and invasion was lower in GOS3 cell line. Significant differences were observed in siFAT1 treated cells as compared to siControl cells as determined by Student's t test. (c) Cell viability after FAT1 knockdown: There was no change in cell viability as assessed by MTT after 24 hr, 48 hr and 72 hr of FAT1 knockdown under hypoxia; although minimal decrease observed in cell viability in both siControl and siFAT1 treated hypoxic cells at 72 hr could be due to the hypoxic stress. Each experiment was done in triplicates and repeated twice (d) Proposed FAT1 regulated signalling pathway affecting HIF1a expression under hypoxia in glioma cells: Under hypoxic condition, on FAT1 knockdown there was inhibition of EGFR/Akt/mTOR pathway and stimulation of VHL levels leading to decreased HIF1a level and its activity which in-turn led to decreased HIF1a target genes expression in glioma cell lines.

Molecular Cancer Biology
significant positive correlation of FAT1 with HIF1a (p 5 0.04) and its target genes: PGK1 (p 5 0.05) and VEGFA (p 5 0.02) as well as our observation of augmented expression of both FAT1 and HIF1a on exposure of GBM (U87MG and U373MG) cell lines to severe hypoxia. We have also analysed the expression of FAT1 and HIF1a in 18 grade II astrocytoma samples. Similar to GBM, there is statistically significant positive correlation between FAT1 and HIF1a mRNA (Pearson coefficient: R 2 5 0.793, p < 0.001) in grade II astrocytomas, further strengthening the association between the two, irrespective of tumour grades.
Next, we carried out in vitro culture of GBM cell lines (U87MG and U373MG) under severe hypoxia (0.2% O 2 ) where we observed increased expression of both FAT1 and HIF1a. HIF1a induction was observed as early as at 0.5 hr of severe hypoxia exposure at protein level (Supporting Information Fig S3), although this falls by about 6 hr. However, there is another rise in protein levels, at 48 hr which continued till 72 hr (Supporting Information Fig S6). As regards mRNA, while there was a small early rise of 1.24 folds at 0.5 hr, the levels started to decline by about 4 hr (Supporting Information Fig S3). However, there was a second rise (>2 folds) at 48 hr and 72 hr in HIF1a mRNA on long term hypoxia. We have not investigated the mechanisms behind this variable expression of HIF1a.
The mechanism of FAT1 over-expression has not been studied in here. Negative expression of FAT1 has been correlated to gene deletion and promoter hypermethylation in oral cancer. 27 A study in hepatocellular carcinoma by Valletta et al. 25 have shown increased FAT1 expression under hypoxia and linked that to increased HGF (hepatocellular growth factor) and hypoxia related altered promoter methylation status.
Further confirmation of this hypothesis was carried out by knocking down FAT1 expression by siRNA in GBM (U87MG and U373MG) and grade-II glioma (GOS3) cell lines. Knockdown of FAT1, under hypoxia, led to marked decrease in the level of HIF1a protein and HIF1a promoter activity in GBM (U87MG and U373MG) cell lines. Moreover, we also observed decreased expression of HIF1a mRNA on FAT1 knockdown under hypoxia. Although, studies have reported the role of miRNAs and other factors like Nacetyltransferases (NAT) in regulating HIF1a mRNA expression, [44][45][46] the mechanism involved in regulating HIF1a mRNA by FAT1 is yet to be elucidated. In addition, there was also marked decrease in expression of HIF1a-regulated molecules like CA9, VEGFA, GLUT1, MCT4, HK2, BNIP3 and REDD1 on FAT1 knockdown under severe hypoxia. In GOS3, with low endogenous level of FAT1 with minimal effect of hypoxia on its expression, the differences were not as distinct as was observed in the GBM cell lines. However, the converse was not true, as HIF1a knockdown had no effect on FAT1 level.
In addition to HIF1a, HIF2a expression was also observed to be upregulated under hypoxia (Supporting Information Fig S4). To confirm whether HIF2a has any role in regulating FAT1 expression, HIF2a was knocked down in U87MG cell line and checked for the expression of FAT1. We observed no regulatory effect of HIF2a on FAT1 expression (Supporting Information Fig S4). Thus, ruling out the regulatory effect of HIF2a on FAT1 expression.
As, EGFR/PI3K/Akt pathway is known to be a potentially unifying pathway for oxygen independent HIF1a activation, 10 we studied the effect of FAT1 knockdown on this pathway. On hypoxia exposure, we observed increased REDD1 mRNA levels and decreased p-mTOR levels as compared to normoxic counterparts (Fig. 3b). The reason for low mTOR levels under hypoxia could be due to known negative feedback loop between REDD1 and mTOR. 47,48 However, here, we have not conducted further detailed study in relation to this. On FAT1 knockdown, we observed decreased level of EGFR, p-Akt and further reduction in p-mTOR levels in GBM (U87MG and U373MG) cell lines under hypoxia. The reduction of p-m-TOR level on FAT1 knockdown (Figs. 3a and 3b) could be due to decreased EGFR and p-Akt levels, leading to decreased HIF1a level on FAT1 knockdown, reflecting the regulatory role of FAT1 on HIF1a via EGFR/AKT/ mTOR signalling.
In addition to the effect of FAT1 on synthetic pathway (EGFR/Akt/mTOR) of HIF1a, we also observed FAT1 to negatively regulate VHL level under hypoxia. Normally, VHL level is high under normoxia and is responsible for maintaining HIF1a protein at a low level by enhancing proteosomal degradation. Under hypoxia, VHL level remains low and thereby HIF1a is stable. In our study we observed higher VHL protein on knockdown of FAT1, abrogating the hypoxia induced reduction of VHL, thus resulting in the low HIF1a levels. The effect of FAT1 knockdown on VHL and HIF1a was further substantiated using the proteosomal inhibitor MG132 along with FAT1 knockdown, where we observed accumulation of HIF1a protein in cells treated with both siFAT1 and MG132 as compared to cells treated with siFAT1 alone. Thus, the reduced HIF1a level and activity observed under hypoxia on FAT1 depletion can be attributed to both decreased synthesis as well as increased proteasomal degradation of HIF1a.
In addition, other important hypoxia related cellular properties are also affected by FAT1. The effect of FAT1 in metabolic adaptation is evident from the reduction in ATP and lactate levels following FAT1 knockdown in hypoxia. This is possibly an effect of the modulation of HIF1a target genes GLUT1, HK2, MCT4 and REDD1, which are the known regulators of the metabolic (glucose) activity. 36 The FAT1 induced increase in cellular migration and invasion under hypoxia was observed to be reduced on FAT1 knockdown in GBM cell lines.
Taken together, the results indicate that at least in glial tumours with upregulated FAT1, there is a major role of this atypical cadherin in the upregulation of HIF1a during severe hypoxia. This further affects metabolic adaptation and augments the invasive properties of the tumour cells. On the basis of these findings we conclude that FAT1 is a novel regulator of HIF1a under severe hypoxia via both EGFR/Akt/ mTOR axis (synthetic) and VHL (proteasomal degradation) in GBM. This is the first report on the important role of FAT1 in modulating HIF1a expression and activity representing a novel pathway, essential for glioma progression and aggressiveness. This study raises the possibility of FAT1 acting as a regulator of the master regulator HIF1a (Fig. 5d) and forming a link between hypoxia and cancer in glioma. This along with the positive association observed between FAT1 and HIF1a in GBM tumour tissues which initially suggested the regulatory role of FAT1 points to a clinical relevance for this cadherin in those GBMs where it is upregulated. While we have studied this pathway only in glioma, it is possible that this FAT1-HIF1a axis is relevant in other tumour types as well. Till now, targeting HIF1a or other hypoxia markers/pathways as therapeutic strategies to treat cancers has had a limited success. 49 The FAT Atypical Cadherin could possibly be a novel target for therapeutic intervention in gliomas with high FAT1 expression and hypoxic microenvironment.