SIRT6 is a target of regulation by UBE3A that contributes to liver … · 2017. 12. 7. ·...

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SIRT6 is a target of regulation by UBE3A that contributes to liver

tumorigenesis in an ANXA2-dependent manner

Saishruti Kohli#, Abhishek Bhardwaj

#, Richa Kumari and Sanjeev Das*

Molecular Oncology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New

Delhi-110067, India.

*Correspondence should be addressed to- Sanjeev Das, Molecular Oncology Laboratory,

National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India. Phone: 91-

11-26703702, Fax: 91-11-26742125, Email: [email protected]

#These authors contributed equally to this work

Running Title: UBE3A downregulates SIRT6 to promote tumorigenesis

Keywords: SIRT6, UBE3A, ubiquitylation, ANXA2, tumour suppressor, transcription

Research. on January 4, 2021. © 2017 American Association for Cancercancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on December 7, 2017; DOI: 10.1158/0008-5472.CAN-17-1692

http://cancerres.aacrjournals.org/

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ABSTRACT

UBE3A is an E3 ubiquitin ligase well known for its role in the proteasomal degradation of p53 in

human papillomavirus (HPV)-associated cancers. Here we report that UBE3A ubiquitylates and

triggers degradation of the tumor suppressive sirtuin SIRT6 in hepatocellular carcinoma. UBE3A

ubiquitylated the highly conserved Lys160 residue on SIRT6. FOXO1-mediated transcriptional

repression of UBE3A was sufficient to stabilize SIRT6 and epigenetically repress ANXA2, a key

mediator of UBE3A oncogenic function. Thus, UBE3A-mediated SIRT6 degradation promoted

the proliferative capacity, migration potential and invasiveness of cells. In mouse models of

hepatocellular carcinoma, SIRT6 downregulation and consequent induction of ANXA2 were

critical for UBE3A-mediated tumorigenesis. Furthermore, in clinical specimens, increased

UBE3A levels correlated with reduced SIRT6 levels and elevated ANXA2 levels in increasing

tumor grades. Overall, our findings show how the tumor suppressor SIRT6 is regulated in

hepatocellular carcinoma and establish the mechanism underlying UBE3A-mediated

tumorigenesis in this disease.




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INTRODUCTION

UBE3A is the founding member of the HECT family of E3 ubiquitin ligases. UBE3A was first

identified by its ability to interact with p53 and target it for proteasomal degradation in

association with the human papillomavirus oncoprotein E6 (1). Besides cervical cancer, UBE3A

has also been implicated in prostate and breast cancer (2-4). However, apart from E6-dependent

degradation of p53, the substrate repertoire of UBE3A involved in tumorigenesis is yet unclear.

The only other substrate linking UBE3A E3 ligase activity to tumorigenesis is tumour suppressor

PML. UBE3A promotes degradation of PML which facilitates Myc driven B-cell

lymphomagenesis (5).

A recent study reported that UBE3A induces Annexin A2 (ANXA2) levels by an

unidentified mechanism which promotes proliferation and invasiveness of cancer cells (6).

ANXA2 has been characterised as a calcium-dependent phospholipid binding protein. Elevated

expression of ANXA2 has been reported in several cancers including liver cancer (7).

Accumulating data suggest that ANXA2 plays a key role in diverse processes linked to

tumorigenesis (8).

SIRT6 is a NAD+-dependent histone deacetylase belonging to the highly conserved

sirtuin family of proteins. Accumulating data now suggest that SIRT6 functions as a tumor

suppressor by modulating cellular transcription program (9-11). SIRT6 was also found to be

downregulated in several cancers including hepatocellular carcinoma (10,12). Though SIRT6 is

implicated in several metabolic pathways and is induced upon metabolic stress (13), the

mechanistic details of its regulation under starvation conditions are yet unclear. SIRT6 has been

reported to be post-translationally stabilized under glucose stress and serum deprivation

conditions (14). UBE3A was identified as a SIRT6 interacting protein in a proteomics screen




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performed previously (10). Our further studies now indicate that UBE3A ubiquitylates SIRT6,

leading to its proteasomal degradation, under normal physiological conditions. However upon

metabolic stress, UBE3A is transcriptionally repressed resulting in SIRT6 stabilisation.

Furthermore, SIRT6 suppresses the expression of ANXA2 through an epigenetic mechanism

resulting in abrogation of UBE3A-mediated tumorigenesis.




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MATERIALS AND METHODS

Cell lines and culture conditions

HepG2, H1299, HCT116, U2OS and 293A cells were grown in DMEM with 10% fetal bovine

serum (Invitrogen) and 100U/ml penicillin and 100g/ml streptomycin at 37C. The cell lines

were obtained from ATCC between 2012 and 2016. The cell lines were authenticated by ATCC

and tested for absence of mycoplasma contamination (MycoAlert, Lonza). The cells used in

experiments were within 10 passages from thawing. Amplification and titration of recombinant

adenoviruses was carried out as described previously (15). Cells were cultured to ~50-70%

confluency followed by infection with recombinant adenovirus at a multiplicity of infection

(MOI) of 10-20. For glucose starvation conditions, cells were cultured to ~50% confluency and

then grown in glucose-free DMEM (Invitrogen) with dialysed fetal bovine serum (Invitrogen)

and 5mM glucose (Sigma). For inducing serum deprivation, cells were cultured to ~50%

confluency and then grown in serum-free DMEM (Invitrogen). Early passage MEFs were

cultured, and subjected to retroviral transduction as described (16).

In vitro ubiquitylation assay

In vitro ubiquitylation assay was carried out using the E2-Ubiquitin Conjugation Kit (Abcam)

according to the manufacturer’s instructions. Briefly, recombinant SIRT6 (Abcam) was

incubated along with His-UBE3A or His-UBE3AC833A

(from E. coli BL21) in a reaction buffer

containing E1, E2 (UBCH7) and ubiquitin, at 37°C for 2 hours. The reaction was terminated by

adding the gel loading buffer. Immunoblotting was performed as described above.

Proliferation Assay




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Cells were cultured in triplicates in 12-well plates and subjected to trypsinization at the indicated

time points to obtain cell suspensions. 0.4% trypan blue (w/v) solution was mixed well with

equal volume of cell suspension and hemocytometer was used to count the unstained healthy

cells.

Plasmin generation Assay

Cells were detached using an enzyme-free dissociation solution (Sigma). Cells were washed with

PBS (phosphate-buffered saline) and tissue plasminogen activator (1μg/ml; Sigma) was added.

Cells were then subjected to 30 minute incubation on ice, washed with PBS, and 0.4μM glu-

plasminogen (Sekisui Diagnostics, Maidstone, UK) was added together with 10μl of

Spectrozyme (Sekisui Diagnostics). Plasmin production was monitored by measuring absorbance

at 405nm.

Matrix Metalloproteinase (MMP) activity Assay

MMP activity was measured using MMP activity assay kit (Abcam) following the

manufacturer’s instructions. Fluorescence intensity was determined at excitation/emission

490/520nm using a fluorogenic peptide substrate.

Xenograft studies

An Institutional Animal Care and Use Committee approved all the animal studies. The right

flank of female nude mice (nu/nu) was subcutaneously injected with 4x106 cells/0.15ml of

DMEM. The mice were randomly assigned to groups for injecting different cell lines. The tumor

size was evaluated for 30 days. No blinding was used. Tumor volume was measured with a




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caliper and calculated using the formula: (widest diameter X smallest diameter2)/2. Post 30 days,

the mice were euthanized using carbon dioxide, followed by tumor excision and its weight

determination. Tumor extracts were then made and immunoblotting was carried out as described

earlier.

Statistical analysis

All experiments were conducted independently at least three times. Results were expressed as

means ± SD. Statistical analyses were performed by a standard two-tailed Student’s t-test, one-

way ANOVA and two-way ANOVA. P<0.05 was considered significant.




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RESULTS

SIRT6 levels are regulated by the ubiquitin proteasome pathway

To investigate the molecular mechanisms underlying SIRT6 regulation, we examined

SIRT6 levels in HepG2 cells under metabolic stress conditions including glucose and serum

deprivation. We observed that there was a robust induction in SIRT6 protein levels while there

was no significant change in its mRNA levels over the time course of metabolic stress (Fig. 1A

and B; Supplementary Fig. S1A and S1B). Similar observations were made in other cell types

including H1299 and primary MEFs (Supplementary Fig. S1C-S1F). These results indicate a

post transcriptional mechanism of SIRT6 regulation. We next determined whether the increase in

SIRT6 protein levels was due to increased translation or increased stability of SIRT6 protein. In

the presence of cycloheximide, there was a gradual decline in SIRT6 protein levels under

unstressed conditions but under starvation conditions sustained high levels of SIRT6 protein

were observed (Fig. 1C and D). However, there was no significant difference in transcript levels

between unstressed and starvation conditions (Supplementary Fig. S1G). These results suggest

that SIRT6 protein is stabilised upon metabolic stress. Since ubiquitin proteasomal machinery is

frequently involved in regulation of protein stability, we next investigated the effect of

proteasome inhibitor MG132 on SIRT6 levels. We observed that MG132 treatment led to

sustained high levels of SIRT6 protein throughout the time course of starvation without

discernable change in its transcript levels, while in absence of MG132 treatment there was a

gradual increase in SIRT6 protein levels upon metabolic stress with no significant change in

transcript levels (Fig. 1E and F). This was further corroborated by the observation that treatment

with an inhibitor of the ubiquitin activating enzyme (E1) PYR41 resulted in similar high levels

of SIRT6 protein throughout the stress period without any discernable change in transcript levels,

while in absence of PYR41 treatment there was a gradual increase in SIRT6 protein levels upon



http://www.bostonbiochem.com/about/ubiquitin-proteasome-pathway-upp


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metabolic stress with no significant change in mRNA levels (Fig. 1G and H; Supplementary Fig.

S1H and S1I). Similar observations were made in H1299 and primary MEFs (Supplementary

Fig. S1J-S1M) Taken together these results suggest that the ubiquitin proteasome pathway plays

a key role in modulating SIRT6 levels in response to metabolic stress.

UBE3A downregulates SIRT6 protein levels

Since UBE3A, an E3 ubiquitin ligase was identified as a SIRT6 interacting protein in a

proteomics screen described previously (10), it raised the possibility that UBE3A may have a

role in SIRT6 regulation. Thus we examined SIRT6 protein and transcript levels in the presence

or absence of UBE3A in different cell lines. Our results indicated that SIRT6 protein levels

increased markedly upon UBE3A depletion while no significant change in its mRNA levels was

observed (Fig. 2A and B). We further observed that UBE3A coimmunoprecipitated with SIRT6

(Fig. 2C). To investigate SIRT6-UBE3A interaction under physiological settings, we first

determined the effect of metabolic stress on UBE3A. Our results indicated that UBE3A levels

declined concomitant to induction in SIRT6 levels upon metabolic stress (Fig. 2D;

Supplementary Fig. S2A). Similar observations were made in other cell types (Supplementary

Fig. S2B and S2C). These results indicate that the reduction in UBE3A levels upon metabolic

stress could be responsible for SIRT6 induction. We next investigated whether endogenous

SIRT6 and UBE3A interacted under metabolic stress conditions. We observed that SIRT6 and

UBE3A interacted under unstressed conditions and the interaction diminished over the time

course of starvation (Fig. 2E).

To further explore the role of UBE3A in regulating SIRT6, we examined SIRT6 levels

upon metabolic stress in presence or absence of UBE3A. There was an induction of SIRT6 levels




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over the time course of starvation period in HepG2 control cells. However, in HepG2 UBE3A

knockdown cells, SIRT6 levels were constitutively elevated over the time course of starvation

period (Fig. 2F; Supplementary Fig. S2D). Similar observations were made in H1299 and

primary MEFs (Supplementary Fig. S2E and S2F). Furthermore, ectopic expression of UBE3A

at late phase of starvation (36 hours) led to a sharp reduction in SIRT6 levels while in presence

of E3-ligase dead UBE3A mutant (UBE3AC833A

; (17)) there was no evident decline in SIRT6

levels (Fig. 2G), even though both wild-type UBE3A and UBE3AC833A

could interact with

SIRT6 (Fig. S2G). Thus we concluded that UBE3A binds to SIRT6 and regulates its levels.

UBE3A ubiquitylates SIRT6 at K160 residue

We next investigated whether UBE3A ubiquitylates SIRT6. Our results indicated that wild-type

UBE3A could polyubiquitylate SIRT6 while the E3-ligase dead UBE3A mutant (UBE3AC833A

)

had no effect (Fig. 3A). We further investigated UBE3A-mediated SIRT6 polyubiquitylation

under metabolic stress conditions. Our results indicated that UBE3A polyubiquitylates SIRT6

under unstressed conditions, which was abrogated upon metabolic stress as UBE3A levels were

downregulated (Fig. 3B). Moreover upon UBE3A knockdown SIRT6 polyubiquitylation was

abolished. We also observed UBE3A-mediated polyubiquitylation to be K48-linked (Fig. 3C).

Next, we sought to map the specific SIRT6 domain that undergoes UBE3A-mediated

polyubiquitylation. The GST-SIRT6 fusion protein comprising the amino acids 49-271 was

polyubiquitylated by UBE3A under unstressed conditions while upon metabolic stress this

polyubiquitylation was abrogated as UBE3A levels were downregulated (Fig. 3D). To narrow

down further, we investigated UBE3A-mediated polyubiquitylation of GST-SIRT6 segments

spanning amino acids 49-170 and 171-271. The GST-SIRT6 fusion protein comprising the




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amino acids 49-170 was polyubiquitylated by UBE3A under unstressed conditions while upon

metabolic stress this polyubiquitylation was abrogated (Fig. 3E). To identify the specific site of

polyubiquitylation, we mutated each of the five lysine residues within this region. Our results

indicated that the mutation of K160 residue abrogated UBE3A-mediated K48-linked

polyubiquitylation of SIRT6 under unstressed conditions even though similar to other mutants

SIRT6K160R

could interact with UBE3A (Fig. 3F; Supplementary Fig. S3A). Moreover protein

sequence analysis of SIRT6 region encompassing amino acids 151-169 suggests that K160

residue is conserved across diverse organisms (Fig. 3G). Taken together these results suggest

that UBE3A is responsible for K48-linked polyubiquitylation of SIRT6 at the conserved K160

residue which leads to SIRT6 degradation.

FOXO1 downregulates UBE3A upon metabolic stress

To investigate the mechanistic basis of UBE3A downregulation, we examined UBE3A

transcript levels upon metabolic stress. We observed that there was a decrease in the transcript

levels with increasing duration of metabolic stress (Fig. 4A and B). In silico analysis of the

UBE3A promoter sequence revealed two putative binding sites for FOXO1, an important

member of the FOX (Forkhead box) family of transcription factors, involved in metabolic stress

response (Fig. 4C). To analyse the effect of FOXO1 on UBE3A expression we examined UBE3A

mRNA levels upon metabolic stress, in presence or absence of FOXO1. We observed that the

metabolic stress induced reduction in UBE3A transcript levels was abrogated in HepG2 FOXO1

knockdown cells (Fig. 4D; Supplementary Fig. S3B). Similar observations were made in other

cell types (Supplementary Fig. S3C and S3D). We next performed ChIP assay to examine the

binding of FOXO1 to UBE3A promoter. Upon metabolic stress, increasing levels of FOXO1




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were detected at both the sites of the UBE3A promoter (Fig. 4E and F). As the cellular

distribution of FOXO1 and hence its activity is determined by its phosphorylation status (18), we

analysed the levels of phosphorylated FOXO1 and its cellular localisation upon metabolic stress.

We observed that levels of phosphorylated FOXO1, which is cytoplasmic, declined upon

metabolic stress concomitant to an increase in nuclear FOXO1 levels (Fig. 4G). Since previous

studies suggest that FOXO1 can also function as a transcriptional repressor (19), we next

determined whether FOXO1 can repress UBE3A transcription. We observed that wild-type

FOXO1 can progressively repress UBE3A promoter more robustly over the time course of

starvation period (Fig. 4H). However FOXO1 AAA mutant which is exclusively nuclear (20)

represses UBE3A promoter constitutively while FOXO1 HRAAA mutant which is exclusively

nuclear but lacks DNA binding ability (18) cannot repress UBE3A promoter at all. Furthermore,

deletion of any of the sites attenuates FOXO1-mediated repression of UBE3A promoter while

deletion of both the sites abrogates the repression (Supplementary Fig. S3E).

We further investigated the effect of FOXO1-mediated UBE3A regulation on SIRT6

levels. Our results suggested that in FOXO1 knockdown cells UBE3A levels remained

constitutively elevated over the starvation period as compared to control cells where there was a

gradual decline in UBE3A levels (Fig. 4I). Concomitantly, in FOXO1 knockdown cells, SIRT6

levels were constitutively repressed over the starvation period while there was steady increase in

SIRT6 levels in control cells. Additionally, in FOXO1/UBE3A double knockdown cells SIRT6

levels were constitutively elevated over the starvation period. Furthermore, in FOXO1

knockdown cells, K48-linked SIRT6 polyubiquitylation was observed even upon prolonged

metabolic stress (36 hours), due to the presence of high levels of UBE3A as compared to control

cells. However in FOXO1/UBE3A double knockdown cells SIRT6 was not polyubiquitylated




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even in unstressed conditions (Fig. 4J; Supplementary Fig. S3F). Similar observations were

made in other cell types (Supplementary Fig. S3G and S3H). Taken together, these results

suggest that upon metabolic stress, FOXO1 represses UBE3A resulting in SIRT6 stabilisation.

SIRT6-mediated H3K9 deacetylation results in ANXA2 repression

A previous report suggests that UBE3A regulates ANXA2 by an unidentified mechanism

(6). Thus, we next examined the effect of metabolic stress on ANXA2 levels. We observed that

upon metabolic stress, induction of SIRT6 levels was accompanied by a reduction in ANXA2

levels (Fig. 5A). ANXA2 transcript levels were also repressed (Fig. 5B). Similar observations

were made in other cell types (Supplementary Fig. S4A and S4B). We next examined ANXA2

mRNA levels upon metabolic stress. In HepG2 UBE3A knockdown cells constitutive repression

of ANXA2 levels was observed over the starvation period unlike control cells where there was a

gradual decline (Fig. 5C; Supplementary Fig. S4C). In UBE3A/SIRT6 double knockdown cells,

ANXA2 mRNA levels were constitutively elevated over the time period of metabolic stress.

Similar observations were made in other cell types (Supplementary Fig. S4D and S4E). We

further investigated whether the deacetylase activity of SIRT6 is responsible for ANXA2

repression. In UBE3A/SIRT6 double knockdown cells subjected to starvation, there was a sharp

decline in ANXA2 levels upon ectopic expression of wild-type SIRT6 while no such decline was

observed in case of deacetylase-dead SIRT6 mutant (SIRT6H133Y

) (Fig. 5D; Supplementary Fig.

S4F). Thus we concluded that SIRT6 could function as a transcriptional corepressor of ANXA2.

ANXA2 has been previously reported to be regulated by HIF1 and c-JUN (21,22),

which are known SIRT6 interacting proteins (23,24). We observed that upon metabolic stress

ANXA2 repression was abrogated in SIRT6 knockdown cells. However upon simultaneous




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knockdown of HIF1 there was no significant change in ANXA2 levels (Supplementary Fig. S5A

and S5B). Similar results were observed for c-JUN (Supplementary Fig. S5C and S5D). These

results indicate that HIF1 or c-JUN were not involved with SIRT6 in repression of ANXA2. To

identify the transcription factor involved with SIRT6 in repressing ANXA2 expression, we

examined ANXA2 promoter sequence for potential transcription factor binding sites. Our analysis

revealed one ELK1 binding site at -1.546Kb position (Fig. 5E). Moreover, a recent study

demonstrated that SIRT6 interacts with ELK1 and function as a transcriptional corepressor (25).

Thus we investigated the role of ELK1 in SIRT6-mediated regulation of ANXA2. We observed

that in HepG2 SIRT6 knockdown cells ANXA2 levels were constitutively elevated over the time

period of starvation. However, upon simultaneous knockdown of ELK1, ANXA2 levels were

constitutively repressed (Fig. 5F; Supplementary Fig. S6A). Similar observations were made in

H1299 and primary MEFs (Supplementary Fig. S6B and S6C). We further observed that ectopic

expression of SIRT6 failed to repress ANXA2 in the absence of ELK1. Moreover ectopic

expression of ELK1 led to significantly higher ANXA2 levels in the absence of SIRT6 as

compared to in presence of SIRT6 (Fig. 5G; Supplementary Fig. S6D). Additionally, ELK1 was

also able to trigger ANXA2 expression in the presence of deacetylase-dead SIRT6 mutant

(SIRT6H133Y

). We also observed that there is no significant change in ELK1 levels during

metabolic stress (Supplementary Fig. S6E). Furthermore, SIRT6 interacts with ELK1 under

metabolic stress conditions.

We next examined the presence of SIRT6 at ANXA2 promoter. Increasing levels of

SIRT6 were detected at the ANXA2 promoter upon metabolic stress, while in the absence of

ELK1 there was no discernable recruitment of SIRT6 at the ANXA2 promoter (Fig. 5H). Similar

observations were made in other cell types (Supplementary Fig. S6F and S6G). However, ELK1




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binds to ANXA2 promoter irrespective of presence or absence of SIRT6 (Supplementary Fig.

S6H). Since SIRT6 exhibits H3K9 and H3K56 deacetylase activity, we investigated H3K9 and

H3K56 acetylation status at ANXA2 promoter. Significant decline in H3K9 acetylation was

observed over the metabolic stress period. However, in both HepG2 SIRT6 knockdown and

ELK1 knockdown cells there were no change in H3K9 acetylation status (Fig. 5I). No significant

change in H3K56 acetylation status at the ANXA2 promoter was observed over the time course of

metabolic stress (Fig. 5J). Thus our results suggest that SIRT6 acts as a corepressor of ELK1 to

suppress ANXA2 expression.

UBE3A-mediated SIRT6 downregulation is critical for its oncogenic functions

Since ANXA2 is a key mediator of UBE3A oncogenic functions, we examined the effect

of SIRT6-dependent ANXA2 repression on UBE3A-mediated neoplastic transformation. Our

results indicated that HepG2 UBE3A knockdown cells, expressing high levels of SIRT6, exhibit

significantly reduced proliferation capacity as compared to HepG2 SIRT6 knockdown cells (Fig.

6A and B). Furthermore, HepG2 SIRT6/ANXA2 double knockdown cells have much reduced

proliferation capacity as compared to HepG2 UBE3A/SIRT6 double knockdown cells which

express high levels of ANXA2. Similar observations were made in H1299 cells (Supplementary

Fig. S7A and S7B). We also determined the effect on malignancy of these cells. We observed

that upon abrogation of UBE3A expression, the invasiveness and migration potential of the cells

was significantly reduced but when SIRT6 expression was abrogated the invasiveness and

migration potential of the cells was notably increased (Fig. 6C and D; Supplementary Fig. S7C).

Additionally, SIRT6/ANXA2 double knockdown cells have much reduced invasiveness and




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migration potential as compared to UBE3A/SIRT6 double knockdown cells. Similar

observations were made in H1299 cells (Supplementary Fig. S7D-S7F).

To further establish the role of ANXA2, we next investigated plasmin levels, as ANXA2

facilitates plasmin generation which promotes extracellular matrix degradation and cell invasion

(26). There was significantly more plasmin production in HepG2 SIRT6 knockdown and

UBE3A/SIRT6 double knockdown cells as compared to HepG2 UBE3A knockdown cells.

However, plasmin production was much reduced in HepG2 SIRT6/ANXA2 double knockdown

cells as compared to HepG2 UBE3A/SIRT6 double knockdown cells (Fig. 6E). Similar

observations were made in H1299 cells (Supplementary Fig. S7G). Since plasmin is a known

activator of matrix metalloproteinases (MMPs), we next investigated MMP activity in these

cells. Our results indicated that HepG2 SIRT6 knockdown and UBE3A/SIRT6 double

knockdown cells exhibited enhanced MMP activity as compared to HepG2 UBE3A knockdown

cells. On the other hand, MMP activity was lower in HepG2 SIRT6/ANXA2 double knockdown

cells as compared to HepG2 UBE3A/SIRT6 double knockdown cells (Fig. 6F). Similar

observations were made in H1299 cells (Supplementary Fig. S7H). Taken together our results

suggest that ANXA2 repression by SIRT6 plays is critical for suppressing UBE3A-mediated

malignant transformation.

To examine the role of SIRT6 in supressing UBE3A-mediated tumorigenesis, the

tumorigenicity of HepG2 UBE3A knockdown, SIRT6 knockdown, UBE3A/SIRT6 double

knockdown and SIRT6/ANXA2 double knockdown cells were examined. Upon abrogation of

UBE3A expression significantly smaller tumors were observed in comparison to control cells

(Fig. 6G and H; Supplementary Fig. S8A). However, much larger tumors were observed upon

SIRT6 knockdown, while upon simultaneous knockdown of UBE3A, there was reduction in




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tumor size. Furthermore, smaller tumors were observed in case of SIRT6/ANXA2 double

knockdown cells as compared to UBE3A/SIRT6 double knockdown cells. These results establish

the significance of SIRT6 downregulation in UBE3A-mediated tumorigenesis.

UBE3A-mediated derepression of ANXA2 promotes aggressive tumor phenotype

We next used an orthotopic liver tumor model to examine the metastatic potential of

HepG2 UBE3A knockdown, SIRT6 knockdown, UBE3A/SIRT6 double knockdown and

SIRT6/ANXA2 double knockdown cells. We observed markedly larger orthotopic tumors in

case of SIRT6 knockdown cells as compared to UBE3A knockdown cells (Fig. 7A and B).

Moreover, in case of UBE3A/SIRT6 double knockdown cells numerous metastatic nodules were

also observed in lungs and pancreas of mice (Supplementary Fig. S8B). This was further

corroborated by the observation that the primary orthotopic tumors exhibited downregulation of

epithelial marker E-cadherin and upregulation of mesenchymal markers Vimentin and

Fibronectin, which is suggestive of increased predisposition of these tumors to metastasize

(Supplementary Fig. S8C). Upon simultaneous knockdown of ANXA2 in SIRT6/ANXA2 double

knockdown cells significantly smaller tumors and no metastasis was observed as compared to

UBE3A/SIRT6 double knockdown cells. These results suggest that UBE3A-mediated SIRT6

downregulation and consequent derepression of ANXA2 promotes tumor metastasis.

Since SIRT6 and ANXA2 have been linked to hepatocellular carcinoma, we investigated

UBE3A, SIRT6 and ANXA2 levels in different grades of hepatocellular carcinoma. Elevated

UBE3A and ANXA2 levels were detected in hepatocellular carcinoma as compared to matched

normal adjacent tissue (Fig. 7C). On the other hand, reduced SIRT6 levels were detected in

hepatocellular carcinoma tissue as compared to matched normal adjacent tissue. Furthermore,




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UBE3A and ANXA2 levels increase with increasing tumor grade while SIRT6 levels decline

(Fig. 7D-F). These observations indicate that UBE3A-mediated SIRT6 downregulation and

consequent ANXA2 upregulation is critical for hepatocellular carcinoma progression.




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DISCUSSION

Several E6 independent substrates of UBE3A have been identified (27-29). However the

role of UBE3A E3 ligase activity in malignant transformation is poorly understood. Here we

report that UBE3A ubiquitylates SIRT6 at the conserved K160 site leading to its proteasomal

degradation. This results in abrogation of SIRT6-mediated epigenetic repression of ANXA2, a

key determinant of UBE3A oncogenic functions. Thus our findings expand the E6-independent

substrate repertoire of UBE3A. Moreover, our results also indicate that even though ANXA2

levels decline in tumors with UBE3A knockdown and SIRT6/ANXA2 double knockdown, the

expression of epithelial marker E-cadherin is lower in tumors with SIRT6/ANXA2 double

knockdown as compared to tumors with UBE3A knockdown, and vice-versa in case of

mesenchymal markers Vimentin and Fibronectin. Thus besides ANXA2, UBE3A could also be

regulating other factors which play a role in determining metastatic progression. Since UBE3A

targets p53 for ubiquitin-mediated proteasomal degradation only in association with the HPV

oncoprotein E6, our results also indicate that p53 is maintained at low levels irrespective of

presence or absence of UBE3A (Supplementary Fig. S8C). Thus the difference in tumor growth

in absence of UBE3A is not due to p53 as there is no significant change in its levels. It will be of

interest to use proteomics based approaches to identify diverse substrates of UBE3A involved in

malignant transformation.

SIRT6 has been reported to be targeted for proteasomal degradation by E3 ligases

including MDM2, ITCH and CHIP. Using exogenous overexpression based studies, MDM2 has

been reported to ubiquitylate SIRT6 and target it for proteasomal degradation (30). But the

pertinence of MDM2-mediated ubiquitylation in SIRT6 regulation under stress conditions is yet

unclear. ITCH is a HECT-type E3 ligase predominantly localised in late endosomal




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compartments and lysosomes (31). Loss of ITCH downregulates SIRT6 ubiquitylation and

induces SIRT6 protein levels (32). However, molecular mechanism of ITCH-mediated regulation

of SIRT6 is yet unclear. Our studies indicate that there is no significant change in ITCH levels

upon metabolic stress (Supplementary Fig. S9A). Neither its mutants nor wild-type SIRT6

exhibited discernable interaction with ITCH under unstressed and starvation conditions

(Supplementary Fig. S3A and S9B). Presence or absence of ITCH had no effect on SIRT6

polyubiquitylation under unstressed conditions while under metabolic stress conditions, even in

presence of ITCH SIRT6 polyubiquitylation was abrogated as UBE3A levels were

downregulated (Supplementary Fig. S9C). Thus our data suggests UBE3A plays a key role in

determining SIRT6 levels irrespective of presence or absence of ITCH. Interestingly, CHIP

noncanonically ubiquitylates SIRT6 resulting in stabilisation of SIRT6 protein (33). CHIP-

dependent SIRT6 stabilisation promotes DNA repair. Our studies indicate that UBE3A

polyubiquitylates SIRT6 at K160 residue, which targets SIRT6 for proteasomal degradation.

However, across diverse cell types the induction in SIRT6 protein levels upon abrogation of

UBE3A expression varies, which indicates that, in addition to UBE3A, there could be other

factors playing in role in determining SIRT6 levels. Thus it would be interesting to use

proteomics based approaches to identify other factors which play a role in regulating SIRT6

levels.

There is mounting evidence indicating that SIRT6 functions as a tumor suppressor while

ANXA2 has been associated with increased proliferation and invasiveness. SIRT6 levels are

reported to exhibit a stepwise reduction from pre-neoplastic stages of hepatocellular carcinoma

(HCC) (34), whereas ANXA2 levels are upregulated in HCC (6,7). Our studies indicate that

SIRT6 represses ANXA2 expression in association with transcription factor ELK1. SIRT6-




21

mediated repression of ANXA2 is a key determinant of its tumor suppressor functions. In

conclusion, our results provide insights on the significance of SIRT6 degradation for UBE3A-

mediated tumorigenesis.




22

ACKNOWLEDGMENTS

We thank the members of the Molecular Oncology Laboratory for helpful discussions. We are

also thankful to Dr. Vivek Rao, CSIR-Institute of Genomics & Integrative Biology (IGIB) for

helping with live animal imaging. The authors would also like to acknowledge the financial

support received from NII Core Fund. S.K. is supported by a fellowship from the Council for

Scientific and Industrial Research, Government of India and A.B. was supported by a fellowship

from the Department of Biotechnology, Government of India. This work was supported by a

grant from the Department of Biotechnology, Government of India (BT/

PR12875/BRB/10/1394/2015) to S.D.




23

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28

FIGURE LEGENDS

Figure 1.

SIRT6 protein levels are upregulated upon metabolic stress. A and B, HepG2 cells were

subjected to glucose starvation for the indicated time points. Western blotting and (B) RT-qPCR

were then performed. Ct method was used to analyse RT-qPCR data with 18S rRNA as

internal control and relative mRNA levels were determined w.r.t 0 hour time point. Error bars are

means ± SD of three independent experiments with triplicate samples. C and D, HepG2 cells

were starved for 24 hours. The cells were then subjected to Cycloheximide chase (15µg/ml) for

the indicated time points under unstressed (25mM) or glucose starvation conditions (5mM).

Western blotting was then performed, and (D) SIRT6 protein levels were quantitated from 3

independent experiments. ***P<0.001. E and F, HepG2 cells were subjected to glucose

starvation for indicated time points. The cells were also treated with MG132 (10 μM) for the last

6 hours of every time point as indicated. Western blotting and (F) RT-qPCR were then

performed. Ct method was used to analyse RT-qPCR data with 18S rRNA as internal control

and relative mRNA levels were determined w.r.t control (untreated with MG132) cells at 0 hour

time point. Error bars are means ± SD of three independent experiments with triplicate samples.

G and H, HepG2 cells were subjected to glucose starvation for indicated time points. The cells

were also treated with PYR41 (40µM) for the last 6 hours of every time point as indicated.

Western blotting, and (H) RT-qPCR were then performed. Ct method was used to analyse

RT-qPCR data with 18S rRNA as internal control and relative mRNA levels were determined

w.r.t control (untreated with PYR41) cells at 0 hour time point. Error bars are means ± SD of

three independent experiments with triplicate samples.




29

Figure 2.

UBE3A interacts with SIRT6 and suppresses its levels. A and B, HepG2, H1299, HCT116,

U2OS and HEK293 cells were transfected with control (scrambled) or UBE3A shRNA. 36 hours

post-transfection, western blotting and (B) RT-qPCR were performed. Error bars are means ± SD

of three independent experiments with triplicate samples. C, HepG2 cells were infected with Ad-

GFP or Ad-SIRT6 for 24 hours. Cells were treated with MG132 (10 M) for the last 6 hours.

Immunoprecipitations were performed followed by western blotting. D, HepG2 cells were

subjected to glucose starvation for the indicated time points. Western blotting was then

performed. E, HepG2 cells were subjected to glucose starvation for the indicated time points and

treated with MG132 (10µM) for the last 6 hours of every time point. Immunoprecipitations were

performed followed by western blotting. F, HepG2 cells were stably transfected with control

(scrambled) or UBE3A shRNA. HepG2 control (HepG2) and UBE3A knockdown (HepG2

UBE3Akd) cells were subjected to glucose starvation for the indicated time points. Western

blotting was then performed. G, HepG2 cells were subjected to glucose starvation for the

indicated time points and infected with indicated adenoviruses during the last 24 hours prior to

the end of the 36 hour time point as indicated. Western blotting was then performed.

Figure 3.

SIRT6 is a UBE3A E3 ligase substrate. A, Ubiquitylation assay was performed using

recombinant SIRT6, wild-type UBE3A and mutant UBE3A as indicated. B, HepG2 control

(HepG2) and UBE3A knockdown (HepG2 UBE3Akd) cells were subjected to glucose starvation

for the indicated time points and treated with MG132 (10µM) for the last 6 hours of each time

point. Immunoprecipitations were performed followed by western blotting. C, HepG2 cells were




30

subjected to glucose starvation for the indicated time points and treated with MG132 (10µM) for

the last 6 hours of each time point. Immunoprecipitations were performed followed by western

blotting. D, HepG2 cells were transfected with constructs expressing full- length or different

domains of SIRT6 as GST fusion protein. 24 hours post-transfection, the cells were subjected to

glucose starvation for the indicated time points and treated with MG132 (10µM) for the last 6

hours of starvation period. GST pull-down was performed followed by immunoblotting. E,

HepG2 cells were transfected with the indicated GST-tagged SIRT6 subdomains. 24 hours post-

transfection, the cells were subjected to glucose starvation for the indicated time points and

treated with MG132 (10µM) for the last 6 hours of starvation period. GST pull-down was

performed followed by immunoblotting. F, HepG2 cells were transfected with the indicated HA-

tagged SIRT6 mutants. 24 hours post-transfection, the cells were subjected to glucose starvation

for the indicated time points and treated with MG132 (10µM) for the last 6 hours of starvation

period. Immunoprecipitations were performed using anti-HA antibody followed by western

blotting. G, Amino acid sequence comparison of SIRT6 region spanning residues 151-169 from

different organisms.

Figure 4.

FOXO1 is a transcriptional repressor of UBE3A. A, HepG2 cells were subjected to glucose

starvation for the indicated time points. RT-qPCR was then performed. Error bars are means ±

SD of three independent experiments with triplicate samples. **P<0.01. B, HepG2 cells were

subjected to serum starvation for the indicated time points. RT-qPCR was then performed. Error

bars are means ± SD of three independent experiments with triplicate samples. **P<0.01. C,

Schematic representation of FOXO1 binding sites at the UBE3A promoter. D, HepG2 cells were




31

stably transfected with control (scrambled) or FOXO1 shRNA. HepG2 control (HepG2) and

FOXO1 knockdown (HepG2 FOXO1kd) cells were subjected to glucose starvation for the

indicated time points. RT-qPCR was performed (left panel). Error bars are means ± SD of three

independent experiments with triplicate samples. FOXO1 knockdown was confirmed by

performing western blotting (right panel). **P<0.01. E, HepG2 cells were subjected to glucose

starvation for indicated time points. ChIP assay was then performed. Error bars are means ± SD

of three independent experiments with triplicate samples. **P<0.01. F, HepG2 cells were

subjected to glucose starvation for indicated time points. ChIP assay was then performed. Error

bars are means ± SD of three independent experiments with triplicate samples. **P<0.01. G,

HepG2 cells were subjected to glucose starvation for the indicated time points. Western blotting

was then performed from the nuclear and cytoplasmic fractions. H, HepG2 FOXO1 knockdown

cells were co-transfected with UBE3A-Luc construct along with indicated FOXO1 constructs. 24

hours post-transfection, cells were subjected to glucose starvation for indicated time points and

luciferase assay was then performed (left panel). Error bars are means ±SD of three independent

experiments with duplicate samples. Equal expression of different FOXO1 constructs was

confirmed by subjecting HepG2 cells transfected with the individual FOXO1 constructs to

glucose starvation for 0 and 24 hours, followed by western blotting (right panel). **P<0.01. I,

HepG2 control (HepG2), FOXO1 knockdown (HepG2 FOXO1kd) and FOXO1/UBE3A double

knockdown (HepG2 FOXO1kd/UBE3Akd) cells were subjected to glucose starvation for

indicated time points. Western blotting was then performed. J, HepG2 control (HepG2), FOXO1

knockdown (HepG2 FOXO1kd) cells and FOXO1/UBE3A double knockdown (HepG2

FOXO1kd/UBE3Akd) cells were subjected to glucose starvation for indicated time points. Cells




32

were treated with MG132 for the last 6 hours of each time point. Immunoprecipitations were

performed followed by western blotting.

Figure 5.

SIRT6 downregulates ANXA2 levels. A, HepG2 cells were subjected to glucose starvation for the

indicated time points. Western blotting was then performed. B, HepG2 cells were subjected to

glucose starvation for the indicated time points. RT-qPCR was performed. Error bars are means

± SD of three independent experiments with triplicate samples. *P<0.05. C, HepG2 control

(HepG2), UBE3A knockdown (HepG2 UBE3kd) and UBE3A/SIRT6 double knockdown

(HepG2 UBE3Akd/SIRT6kd) cells were subjected to glucose starvation for indicated time

points. RT-qPCR was then performed. Error bars are means ± SD of three independent

experiments with triplicate samples. *P<0.05. D, HepG2 UBE3A/SIRT6 double knockdown

cells (HepG2 UBE3Akd/SIRT6kd) were subjected to glucose starvation for 36 hours. The cells

were infected with indicated adenoviruses during the last 12 hours of starvation. RT-qPCR was

then performed. Error bars are means ± SD of three independent experiments with triplicate

samples. ***P<0.001. E, Schematic representation of ELK1 binding sites at the Annexin A2

promoter. F, HepG2 control (HepG2), SIRT6 knockdown (HepG2 SIRT6kd), and SIRT6/ELK1

double knockdown (HepG2 SIRT6kd/ELK1kd) cells were subjected to glucose starvation for the

indicated time points. RT-qPCR was then performed. Error bars are means ± SD of three

independent experiments with triplicate samples. *P<0.05. G, HepG2 SIRT6/ELK1 double

knockdown cells were stably transfected with dual expression plasmid encoding wild-type

SIRT6, ELK1, and ELK1 along with wild-type SIRT6 or SIRT6H133Y

as indicated. Control

represents HepG2 SIRT6/ELK1 double knockdown cells stably transfected with empty vector.




33

24 hours post-transfection RT-qPCR was then performed. Error bars are means ± SD of three

independent experiments with duplicate samples. **P<0.01, ***P<0.001. H, HepG2 control

(HepG2) and ELK1 knockdown (HepG2 ELK1kd) cells were subjected to glucose starvation for

indicated time points. ChIP assay was then performed. Error bars are means ± SD of three

independent experiments with triplicate samples. *P<0.05. I, HepG2 control (HepG2), SIRT6

knockdown (HepG2 SIRT6kd) and ELK1 knockdown (HepG2 ELK1kd) cells were subjected to

glucose starvation for indicated time points. ChIP assay was then performed. Error bars are

means ± SD of three independent experiments with triplicate samples. *P<0.05. J, HepG2

control (HepG2), SIRT6 knockdown (HepG2 SIRT6kd) and ELK1 knockdown (HepG2

ELK1kd) cells were subjected to glucose starvation for indicated time points. ChIP assay was

then performed. Error bars are means ± SD of three independent experiments with triplicate

samples.

Figure 6.

SIRT6 downregulation facilitates UBE3A oncogenic functions. A, HepG2 control (HepG2),

UBE3A knockdown (UBE3Akd), SIRT6 knockdown (SIRT6kd), UBE3A/SIRT6 double

knockdown (UBE3Akd/SIRT6kd) and SIRT6/ANXA2 double knockdown

(SIRT6kd/ANXA2kd) cells were harvested and immunoblotting was performed. B, HepG2

control (HepG2), UBE3A knockdown (UBE3Akd), SIRT6 knockdown (SIRT6kd),

UBE3A/SIRT6 double knockdown (UBE3Akd/SIRT6kd) and SIRT6/ANXA2 double

knockdown (SIRT6kd/ANXA2kd) cells were cultured in complete medium and counted at the

indicated time points. Error bars are means ± SD of three independent experiments with

duplicate samples. **P<0.01, ***P<0.001. C, In vitro invasion of HepG2 control (HepG2),




34



(SIRT6kd/ANXA2kd) cells was measured as percentage of cells migrating to chambers with 10

% FBS. Error bars are means ± SD of three independent experiments with duplicate samples.

**P<0.01, ***P<0.001. D, Migration potential of HepG2 control (HepG2), UBE3A knockdown

(UBE3Akd), SIRT6 knockdown (SIRT6kd), UBE3A/SIRT6 double knockdown

(UBE3Akd/SIRT6kd) and SIRT6/ANXA2 double knockdown (SIRT6kd/ANXA2kd) cells was

measured as percentage of cells migrating to chambers with 10 % FBS. Error bars are means ±

SD of three independent experiments with triplicate samples. **P<0.01, ***P<0.001. E, Plasmin

production in HepG2 control (HepG2), UBE3A knockdown (UBE3Akd), SIRT6 knockdown

(SIRT6kd), UBE3A/SIRT6 double knockdown (UBE3Akd/SIRT6kd) and SIRT6/ANXA2

double knockdown (SIRT6kd/ANXA2kd) cells was determined. Error bars are means ± SD of

three independent experiments with duplicate samples. ***P<0.001. F, General MMP activity

was determined for HepG2 control (HepG2), UBE3A knockdown (UBE3Akd), SIRT6

knockdown (SIRT6kd), UBE3A/SIRT6 double knockdown (UBE3Akd/SIRT6kd) and

SIRT6/ANXA2 double knockdown (SIRT6kd/ANXA2kd) cells. Error bars are means ± SD of

three independent experiments with triplicate samples. ***P<0.001. G, HepG2 control (HepG2),



(SIRT6kd/ANXA2kd) cells were subcutaneously injected into nude mice. Tumor volume was

measured on indicated days. The data shown are representative of three independent experiments

(n = 5 mice per group) and error bars represent means ± SD (n=5 mice per group). H, At the end

of 30 days, tumors were excised and weighed. The data shown are representative of three




35

independent experiments (n = 5 mice per group) and error bars represent means ± SD (n=5 mice

per group).

Figure 7.

Role of ANXA2 in UBE3A-mediated tumorigenesis. A, HepG2Luc2

cells were stably transfected

with UBE3A shRNA, SIRT6 shRNA, and SIRT6 shRNA along with UBE3A shRNA or ANXA2

shRNA as indicated. HepG2Luc2

cells stably transfected with empty vector was used as control

(Control). These cells were injected into the liver of nude mice. Bioluminescence imaging was

performed weekly and representative images are shown. The data shown are representative of

three independent experiments (n = 5 mice per group). B, Bioluminescence quantification (7A

above) was performed at the indicated time points. The data shown are representative of three

independent experiments (n = 5 mice per group). Error bars represent means ± SD from five

individual mice. **P<0.01, ***P<0.001. C, Representative image of immunostaining of SIRT6,

ANXA2 and UBE3A in different grades of human liver carcinoma (HCC) and matched normal

adjacent tissue (NAT) sections. DAPI was used to counter stain nucleus. D, Quantitation of

UBE3A levels in different grades of human liver carcinoma normalized with respect to matched

normal adjacent tissue. The data shown are representative of three independent experiments.

Error bars are means ± SD. E, Quantitation of ANXA2 levels in different grades of human liver

carcinoma normalized with respect to matched normal adjacent tissue. The data shown are

representative of three independent experiments. Error bars are means ± SD. F, Quantitation of

SIRT6 levels in different grades of human liver carcinoma normalized with respect to matched

normal adjacent tissue. The data shown are representative of three independent experiments.

Error bars are means ± SD.




Published OnlineFirst December 7, 2017.Cancer Res Saishruti Kohli, Abhishek Bhardwaj, Richa Kumari, et al. liver tumorigenesis in an ANXA2-dependent mannerSIRT6 is a target of regulation by UBE3A that contributes to

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