epigenetic control of mitochondrial fission enables self ... · supplementary figure 1. effects of...

Cell Metabolism, Volume 30

Supplemental Information

Epigenetic Control of Mitochondrial

Fission Enables Self-Renewal of Stem-like

Tumor Cells in Human Prostate Cancer

Gianluca Civenni, Roberto Bosotti, Andrea Timpanaro, Ramiro Vàzquez, JessicaMerulla, Shusil Pandit, Simona Rossi, Domenico Albino, Sara Allegrini, AbhishekMitra, Sarah N. Mapelli, Luca Vierling, Martina Giurdanella, Martina Marchetti, AlyssaPaganoni, Andrea Rinaldi, Marco Losa, Enrica Mira-Catò, Rocco D'Antuono, DiegoMorone, Keyvan Rezai, Gioacchino D'Ambrosio, L'Houcine Ouafik, SarahMackenzie, Maria E. Riveiro, Esteban Cvitkovic, Giuseppina M. Carbone, and Carlo V.Catapano

C

Bo

dy w

eig

ht

(g)

0

10

20

30

40

19 23 27 31 35

OTX015Control

Time (days from injection)

DU145

Bo

dy w

eig

ht

(g)

0

10

20

30

40

14 21 28 35 42

ControlOTX015


PC3

Bo

dy w

eig

ht

(g)

0

10

20

30

40

15 22 29 36 43

ControlOTX015


LNCaP

Bo

dy w

eig

ht

(g)

0

10

20

30

40

16 23 30 37

OTX015Control


22Rv1

D

0

500

1000

1500

2000

2500 Plasma

OT

X0

15

(n

g/m

l)

0

500

1000

1500

2000

2500Tumor

OT

X0

15

(n

g/m

l)

22Rv1 LNCaP

Cell

cycle

sta

ge

(%)

0

50

100

150

500 nM OTX015 48-h drugwashout

* ***

* *

G1 S G2/M

0

50

100

150

500 nM OTX015 48-h drugwashout

***

* * *

G1 S G2/M

DU145 PC3

Cell

cycle

sta

ge

(%)

0

50

100

150

48-h drugwashout

***

500 nM OTX015

G2/MSG1

**

0

50

100

150

48-h drugwashout

* **

* * *

500 nM OTX015

G2/MSG1

B

0

1

2

3

4

5

adh sph

P= 0.077

0

10

20

30

40

50

adh sph

P= 0.001

Fo

ld Δ

ΔC

tF

old

ΔΔ

Ct

0

1

2

3

4

5

adh sph

P= 0.017

0

10

20

30

40

50

adh sph

LNCaP

Fo

ld Δ

ΔC

tF

old

ΔΔ

Ct

0

1

2

3

4

5

adh sph

P= 0.003

0

1

2

3

4

5

adh sph

P= 0.0001

0

2

4

6

8

10

adh sph

P= 0.033

0

2

4

6

8

10

adh sph

P= 0.0001

0

1

2

3

4

5

adh sph

P= 0.012

0

1

2

3

4

5

adh sph

P= 0.025

OC

T4

0

1

2

3

4

5

adh sph

0

1

2

3

4

5

adh sph

NA

NO

G

DU145 PC3

P= 0.002 P=0.004 P= 0.013

Fo

ld Δ

ΔC

t

Fo

ld Δ

ΔC

t

0

1

2

3

4

5

adh sph

P= 0.037

0

2

4

6

8

10

adh sph

22Rv1

P= 0.0003

Fo

ld Δ

ΔC

tF

old

ΔΔ

Ct

Fo

ld Δ

ΔC

t

Fo

ld Δ

ΔC

t

LIN

28

AL

IN2

8B

Fo

ld Δ

ΔC

tF

old

ΔΔ

Ct

Fo

ld Δ

ΔC

tF

old

ΔΔ

Ct

0

1

2

3

4

5

adh sph

P= 0.009

0

2

4

6

8

10

adh sph

P=0.991

Fo

ld Δ

ΔC

tF

old

ΔΔ

Ct

E

Supplementary Figure 1

A

Cell line IC50 (nM) CI95 (nM) Emax %

DU145 386.8 218.8-683.7 95

PC3 96.1 55.3-167.2 86

22Rv1 36.7 13.5-100.2 86

LNCaP 97.8 65.0-147.1 93

Supplementary Figure 1. Effects of OTX015 in cell cultures, tissue distribution and mouse body

weight. A, Inhibition of cell proliferation by OTX015. Mean IC50 and CI95 values determined after 72 h

by cell counting. B, Cell cycle distribution of prostate cancer cells exposed to 500 nM OTX015 for 24,

48 and 72 h continuously or for 72 h followed by 48 h in drug-free medium. Cells were stained with 7-

AAD and analyzed by flow cytometry. C, Body weight in control and OTX015-treated mice (as in

Figure 1B) bearing xenografts of DU145, PC3, 22RV1 and LNCaP cells. D, Plasma (left) and tumor

(right) tissue levels of OTX015 in DU145 and LNCaP xenografts from control and OTX015-treated

mice collected at the end of the experiment and determined by HPLC-MASS. E, Expression of OCT4,

NANOG, LIN28a and LIN28B determined by qRT-PCR in prostate cancel cell lines cultured in

adherent and spheres forming conditions. t test were used for P values. * P < 0.01. Related to Figure 1.

c-M

YC

mR

NA

PC3

*

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 100 1000

LNCaP

*

*

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 100 1000

DU145

*

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 100 1000

22Rv1

*

*

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 100 1000

*

*

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 100 1000

OTX015 (nM)

c-M

YC

mR

NA

*

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 100 1000

OTX015 (nM)

**

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 100 1000

OTX015 (nM)

*

*

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 100 1000

OTX015 (nM)

E

*

0

20

40

60

80

100

120

NF-kB RE

*

0

20

40

60

80

100

120

STAT3 RE

*

0

20

40

60

80

100

120

Rep

ort

er

activity

(% o

f co

ntr

ol)

MYC RE

D

0

20

40

60

80

100

120

0

0.0

01

0.0

1

0.1 1

0

20

40

60

80

100

120

0

0.0

01

0.0

1

0.1 1

NF-kB RE STAT3 RE

Rep

ort

er

activity

(% o

f co

ntr

ol)

OTX015 (M) OTX015 (M)

Adh

TS

Adh

TS

A BPC3

0

2

4

6

8

10

12

14

16

Con

tro

l

OT

X0

15

*

c-M

YC

mR

NA

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Con

tro

l

OT

X0

15

DU145

*

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Con

tro

l

OT

X0

15

LNCaP22Rv1

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Con

tro

l

OT

X0

15

*

c-M

YC

pro

tein

(% o

f p

ositiv

e c

ells)

0

20

40

60

80

100

Con

tro

l

OT

X0

15

*

PC3

0

20

40

60

80

100

Con

tro

l

OT

X0

15

*

DU145 22Rv1

0

20

40

60

80

100

Con

tro

l

OT

X0

15

*

0

20

40

60

80

100

Con

tro

l

OT

X0

15

*

LNCaP

C

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

sh

_C

ntr

l

sh

1_B

RD

4

BR

D4

mR

NA

*

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

sh

_C

ntr

l

sh

1_B

RD

4

*

Myc

mR

NA


Supplementary Figure 2. BRD4 inhibition affects transcription factor activity in tumor-

spheres and bulk tumor cells. A, c-MYC mRNA levels in PC3, DU145, 22Rv1 and LNCaP cells

treated with OTX015 in adherent (top panels) and tumor-sphere (bottom panels) conditions for 24

h. B, c-MYC mRNA (top) and protein (bottom) in tumor xenografts from control and OTX015-

treated mice (as in Figure 1B) assessed by qRT-PCR and immunohistochemistry, respectively. C,

BRD4 and MYC mRNA expression in control and BRD4-depleted DU145 tumor xenografts. D,

NF-κB and STAT3 luciferase reporter activity in DU145 cells treated with OTX015 in adherent

(black) and tumor-sphere (red) conditions. E, c-MYC, NF-κB and STAT3 luciferase reporter

activity in DU145 cells transfected with control (siGL3) and BRD4 targeting (siBRD4) siRNA

for 48 h. P values were determined using t test. * P < 0.01. Related to Figure 3.

MSigDB Canonical Pathways

Activated in Tumor-sphere CelllsGene Set Name # Genes in Gene Set # Genes in Overlap p-value FDR q-value

REACTOME_METABOLISM_OF_LIPIDS_AND_LIPOPROTEINS 478 44 5.08E-17 6.76E-14

REACTOME_INTERFERON_ALPHA_BETA_SIGNALING 64 18 2.96E-16 1.97E-13

REACTOME_IMMUNE_SYSTEM 933 61 7.06E-16 3.13E-13

KEGG_LYSOSOME 121 21 3.72E-14 1.24E-11

REACTOME_BIOLOGICAL_OXIDATIONS 139 22 6.66E-14 1.77E-11

REACTOME_AMYLOIDS 83 17 5.94E-13 1.22E-10

KEGG_GLUTATHIONE_METABOLISM 50 14 6.44E-13 1.22E-10

NABA_MATRISOME 1028 58 1.80E-12 2.99E-10

REACTOME_PHASE_II_CONJUGATION 70 15 6.81E-12 1.01E-09

KEGG_SYSTEMIC_LUPUS_ERYTHEMATOSUS 140 19 5.41E-11 7.19E-09

REACTOME_INTERFERON_SIGNALING 159 20 6.90E-11 8.35E-09

KEGG_METABOLISM_OF_XENOBIOTICS_BY_CYTOCHROME_P450 70 14 9.20E-11 1.02E-08

REACTOME_PACKAGING_OF_TELOMERE_ENDS 48 12 1.26E-10 1.29E-08

REACTOME_RNA_POL_I_PROMOTER_OPENING 62 13 2.31E-10 2.19E-08

REACTOME_METABOLISM_OF_AMINO_ACIDS_AND_DERIVATIVES 200 21 6.96E-10 6.17E-08

REACTOME_MEIOTIC_RECOMBINATION 86 14 1.61E-09 1.34E-07

REACTOME_DEPOSITION_OF_NEW_CENPA_CONTAINING_NUCLEOSO

MES_AT_THE_CENTROMERE 64 12 4.43E-09 3.46E-07

REACTOME_CYTOKINE_SIGNALING_IN_IMMUNE_SYSTEM 270 23 6.50E-09 4.80E-07

REACTOME_DIABETES_PATHWAYS 133 16 1.05E-08 7.34E-07

KEGG_VALINE_LEUCINE_AND_ISOLEUCINE_DEGRADATION 44 10 1.22E-08 8.08E-07

MSigDB Canonical Pathways

Activated in Bulk Tumor CellsGene Set Name # Genes in Gene Set # Genes in Overlap p-value FDR q-value

REACTOME_CELL_CYCLE 421 63 1.22E-46 1.62E-43

REACTOME_CELL_CYCLE_MITOTIC 325 56 5.30E-45 3.53E-42

REACTOME_DNA_REPLICATION 192 36 1.19E-30 5.29E-28

REACTOME_MITOTIC_M_M_G1_PHASES 172 32 2.87E-27 9.55E-25

PID_AURORA_B_PATHWAY 39 16 1.81E-20 4.81E-18

KEGG_CELL_CYCLE 128 22 2.21E-18 4.91E-16

REACTOME_MITOTIC_PROMETAPHASE 87 19 4.02E-18 7.64E-16

REACTOME_G1_S_TRANSITION 112 19 5.96E-16 9.91E-14

REACTOME_MITOTIC_G1_G1_S_PHASES 137 20 2.09E-15 3.09E-13

KEGG_FOCAL_ADHESION 201 23 3.83E-15 5.09E-13

REACTOME_CELL_CYCLE_CHECKPOINTS 124 18 5.86E-14 7.08E-12

REACTOME_S_PHASE 109 17 8.67E-14 9.61E-12

REACTOME_MITOTIC_G2_G2_M_PHASES 81 15 1.82E-13 1.86E-11

REACTOME_KINESINS 24 10 2.34E-13 2.15E-11

REACTOME_HEMOSTASIS 466 31 2.43E-13 2.15E-11

KEGG_REGULATION_OF_ACTIN_CYTOSKELETON 216 21 1.50E-12 1.25E-10

PID_FOXM1_PATHWAY 40 11 2.97E-12 2.32E-10

PID_ILK_PATHWAY 45 11 1.23E-11 8.60E-10

REACTOME_G2_M_CHECKPOINTS 45 11 1.23E-11 8.60E-10

KEGG_PATHWAYS_IN_CANCER 328 24 1.73E-11 1.15E-09


Supplementary Figure 3. Top pathways differentially activated in tumor-spheres (top) and

bulk tumor (bottom) DU145 cells. Gene expression was examined using Illumina arrays and

enriched pathways examined using Enrichr and the Molecular Signatures Database (MSigDB) for

canonical pathways. Related to Figure 3.

C

Ve

hic

leO

TX

01

5

10

0 n

M

MitoRedMitoGreen Merge

Tumor-spheres (7 days)

Ve

hic

leO

TX

01

5

10

0 n

M


Adherent cells (7days)

E

A


Hoechst MergeMitoGreen MitoRed

Adherent cells (3 days)

Ve

hic

leO

TX

01

5

10

0 n

M

Hoechst MergeMitoGreen MitoRed

Tumor-spheres (3 days)

Ve

hic

leO

TX

01

5

10

0 n

M

B

D

F

Ve

hic

leO

TX

01

5

40X 189X Crop 3X

TOM20 DAPI

sh_Control sh1_BRD4 sh2_BRD4

Ad

he

ren

tT

um

or-

sp

he

re

Supplementary Figure 4. Mitochondrial dynamics in tumor-spheres and bulk tumor cells in

response to BRD4 inhibition. A-B, Mitochondria stained with MitoTracker Green (MitoGreen),

MitoTracker Red (MitoRed) in DU145 cells treated with vehicle or OTX015 (100 nM) for 3 days

in adherent (A) and tumor-sphere forming (B) conditions and examined by confocal microscopy.

Nuclei were stained with Hoechst. Images with single and merged channels are shown. C-D,

Mitochondria stained with MitoGreen and MitoRed in DU145 cells treated with vehicle or

OTX015 (100 nM) for 7 days in adherent (C) and tumor-sphere forming (D) conditions. E,

Mitochondria morphology in control and OTX015-treated DU145 tumor xenografts assessed by

immunohistochemical staining for TOM20. F, Mitochondria in adherent and tumor-sphere cells

from control (sh_Control) and BRD4-depleted (sh1_BRD4, sh2_BRD4) DU145 cells. Cells were

stained with TMRM and Hoechst. Related to Figure 4.

C

A

E

Ve

hic

leO

TX

01

5

10

0 n

M


22Rv1 - Tumor-spheres (3 days)

Ve

hic

leO

TX

01

5

10

0 n

M


22Rv1 - Adherent cells (3 days)

B

D

0

20

40

60

80

100

120

Cell p

rolife

ratio

n

(% o

f th

e c

on

tro

l)

RWPE1

F

Tubulin

DRP1


OT

X0

15

10

0 n

MV

eh

icle

MergeMitoGreen MitoRed

RWPE1 - Adherent cells (3 days)

pcD

NA

3d

nD

RP

1

MitoGreen MitoRed Merge

pcD

NA

3d

nD

RP

1

Mito Green Mito Red Merge

DU145 - Tumor-spheres DU145 - Adherent cells

Supplementary Figure 5. Inhibition of mitochondrial fission in prostate cancer stem cells.

A, DRP1 protein expression in DU145 cells transfected with a dominant negative mutant of

DRP1K38A (dnDRP1) and empty control (pcDNA3.1) vector. B, Mitochondria morphology in

adherent and tumor-sphere cells generated by DU145 cells transfected with the dominant

negative mutant DRP1K38A (dnDRP1) or empty control (pcDNA3.1) vector and stained with

MitoGreen and MitoRed. Nuclei were stained with Hoechst. C-D, Mitochondria stained with

MitoGreen and MitoRed in 22Rv1 cells treated with vehicle or OTX015 (100 nM) for 3 days in

adherent (C) and tumor-sphere forming (D) conditions. E, Mitochondria stained with

MitoGreen and MitoRed in prostate epithelial RWPE-1 cells treated with vehicle or OTX015

(100 nM) for 3 days in adherent conditions. Pictures were edited with Image-J and maximum

projections are shown as separated and merged channels. Scale bars, 10 µM. F, Proliferation of

RWPE1 cells incubated with vehicle (control) or OTX015 (100 nM) for 3 days. Related to

Figure 5.

B Vanaja Prostate

Prostate Adenocarcinoma vs. Normal

P=0.018

Normal

N = 8

Adenocarcinoma

N = 27

Lapointe Prostate

Prostate Carcinoma vs. Normal

P=0.002

Normal

N = 41

Adenocarcinoma

N = 61

Arredouani Prostate


P=0.012

Normal

N = 8

Carcinoma

N = 13

Luo Prostate 2


P=0.033

Normal

N = 15

Carcinoma

N = 15

C

Best Prostate 2

Prostate Adenocarcinoma -

Hormone Refractory

P=0.001

Hormone naive

N = 10

Hormone refractory

N = 10

Tomlins Prostate

Prostate Carcinoma Metastasis

Epithelia - Hormone Refractory

P=0.002

Hormone naive

N = 3

Hormone refractory

N = 16

10X

Normal tissue Primary tumors

MF

F

A

ED

MITOCHONDRION

En

rich

me

nt

sco

re

0.0

0.7 NES=2.229

P-value<0.001

FDR<0.001

TSS

+87- 12

Fwd Rev

MFF


Supplementary Figure 6. MFF expression in human prostate cancer. A, Enrichment of genes

in mitochondrial biogenesis and dynamic pathways among the genes activated in tumor-spheres

from DU145 cells by gene set enrichment analysis (GSEA). B, Comparison of MFF levels

between normal prostate and primary prostate adenocarcinomas (top and middle panels) and

between hormone-naïve and hormone-refractory prostate adenocarcinomas (bottom panels) in the

indicated gene expression datasets. C, Representative images of immunohistochemistry of MFF

protein in normal prostate and primary prostate tumors. D, BRD4 occupancy of the MFF

promoter by ChIP-sequencing data analysis. E, Map of MFF promoter and primers used for

ChIP-pPCR. Related to Figure 6.

MFF

Tubulin

E F

*

*

0

20

40

60

80

100

120

Tu

mo

r-sp

he

re n

um

be

r

(% o

f co

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ol)

Colo

ny n

um

be

r

(% o

f co

ntr

ol)

*

*

0

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120

0

20

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Cell p

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(% o

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B

68%

104103102101

0

80

60

40

20

Cou

nts

GFP-MFF

100

H

GFP-MFF - - + -Flag-BRD4 - - - +

pcDNA3 - + - -

Flag-BRD4

Tubulin

GFP-MFF


A

DU145MFF

Tubulin

PC3MFF

Tubulin

22Rv1MFF

Tubulin

LNCaPMFF

Tubulin

MF

F m

RN

A

DU145 PC3

MF

F m

RN

A

MF

F m

RN

A

22Rv1

*

*

0.0

0.2

0.4

0.6

0.8

1.0

1.2

sh

_C

ontr

ol

sh

1_M

FF

sh

2_M

FF

*

*

0.0

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0.4

0.6

0.8

1.0

1.2

sh

_C

ontr

ol

sh

1_M

FF

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2_M

FF

*

*

0.0

0.2

0.4

0.6

0.8

1.0

1.2

sh

_C

ontr

ol

sh

1_M

FF

sh

2_M

FF

* *

0.0

0.2

0.4

0.6

0.8

1.0

1.2

sh

_C

ontr

ol

sh

1_M

FF

sh

2_M

FF

MF

F m

RN

A

LNCaP

* *

0

20

40

60

80

100

120

sh

_C

ontr

ol

sh

1_M

FF

sh

2_M

FF

Tu

mo

r-sp

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um

be

r

(% o

f co

ntr

ol) * *

0

20

40

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80

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120

sh

_C

ontr

ol

sh

1_M

FF

sh

2_M

FF

Tu

mo

r-sp

he

re n

um

be

r

(% o

f co

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ol)

22Rv1 LNCaPC

D

0

20

40

60

80

100

120

sh

_C

ontr

ol

sh

1_M

FF

sh

2_M

FF

Cell p

rolife

ratio

n

(% o

f co

ntr

ol)

22Rv1

**

0

20

40

60

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120

sh

_C

ontr

ol

sh

1_M

FF

sh

2_M

FF

Cell p

rolife

ratio

n

(% o

f co

ntr

ol)

LNCaP

* *

K

Control GFP-MFF

Mito Red Hoechst

Tum

or-

sphere

s

Vehicle OTX015 100 nM Vehicle OTX015 100 nM

TMRM Hoechst

J DU145

MFF

Tubulin

FLAG

Adherent cells Tumor-spheres

MitoG

reen

MitoR

ed

Merg

e

Gsh_Controlsh1_MFF sh_Controlsh1_MFF

Adherent cells

Antimycin/

Rotenone

0

10

20

30

40

Con

tro

l

OT

X0

15

0

20

40

60

80

100

120

0 40 80 120

Time (min)

OC

R (

pm

ol/m

in/c

ell)

Oligomycin FCCP

SR

C (

pm

ol/m

in/c

ell)

- Control

- OTX015

100 nM

I

L

Supplementary Figure 7. Loss and gain of MFF function and mitochondrial dynamics I

bulk and tumor-sphere cells. A-B, Reduced MFF protein (A) and mRNA (B) in PC cells

expressing control (sh_Control) or MFF targeting shRNAs (sh1_MFF, sh2_MFF). C-D,

Tumor-sphere formation (C) and proliferation (D) by 22Rv1 and LNCaP cells after MFF

knockdown by shRNAs. E, MFF protein level in DU145 cells transfected with control

(siGL3) and MFF targeting (si1MFF and si2MFF) siRNA examined by Western blot. F,

Colony forming ability (left), tumor-sphere formation (middle) and cell proliferation (right) in

DU145 cells after siRNA-mediated MFF depletion. G, Mitochondria morphology after MFF

depletion in adherent and tumor-sphere DU145 cells stained with Mitotracker Green and Red.

H-I, Ectopic expression of GFP-MFF and Flag-BRD4 in DU145 cells transfected with the

respective plasmid expression vectors determined by flow cytometry (H) and Western blot (I).

J, Expression of Flag-MFF in DU145 cells infected with lentiviral vector constructs

determined by Western blot. K,Mitochondria morphology and functionality in DU145 tumor-

sphere cells transfected with empty control (pcDNA) or GFP-MFF expressing vector and

treated with OTX015 (100 nM). Cells were stained with TMRM (top) or Mitotracker Red

(bottom). Nuclei were stained with Hoechst. L, Mitochondrial activity determined by

Seahorse assay in adherent DU145 cells stably expressing Flag-MFF and treated with

OTX015 (100 nM, 7days) or vehicle. Related to Figure 7.

epigenetic control of mitochondrial fission enables self ... · supplementary figure 1. effects of...

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