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Protein arginine methyltransferase 5 is a potential oncoprotein that upregulates G1 cyclins/cyclin-dependent kinases and the phosphoinositide 3-kinase/AKT signaling cascade - PubMed

. 2012 Sep;103(9):1640-50.

doi: 10.1111/j.1349-7006.2012.02367.x. Epub 2012 Aug 8.

Affiliations

Protein arginine methyltransferase 5 is a potential oncoprotein that upregulates G1 cyclins/cyclin-dependent kinases and the phosphoinositide 3-kinase/AKT signaling cascade

Tong-You W Wei et al. Cancer Sci. 2012 Sep.

Abstract

Increasing evidence suggests that PRMT5, a protein arginine methyltransferase, is involved in tumorigenesis. However, no systematic research has demonstrated the cell-transforming activity of PRMT5. We investigated the involvement of PRMT5 in tumor formation. First, we showed that PRMT5 was associated with many human cancers, through statistical analysis of microarray data in the NCBI GEO database. Overexpression of ectopic PRMT5 per se or its specific shRNA enhanced or reduced cell growth under conditions of normal or low concentrations of serum, low cell density, and poor cell attachment. A stable clone that expressed exogenous PRMT5 formed tumors in nude mice, which demonstrated that PRMT5 is a potential oncoprotein. PRMT5 accelerated cell cycle progression through G1 phase and modulated regulators of G1; for example, it upregulated cyclin-dependent kinase (CDK) 4, CDK6, and cyclins D1, D2 and E1, and inactivated retinoblastoma protein (Rb). Moreover, PRMT5 activated phosphoinositide 3-kinase (PI3K)/AKT and suppressed c-Jun N-terminal kinase (JNK)/c-Jun signaling cascades. However, only inhibition of PI3K activity, and not overexpression of JNK, blocked PRMT5-induced cell proliferation. Further analysis of PRMT5 expression in 64 samples of human lung cancer tissues by microarray and western blot analysis revealed a tight association of PRMT5 with lung cancer. Knockdown of PRMT5 retarded cell growth of lung cancer cell lines A549 and H1299. In conclusion, to the best of our knowledge, we have characterized the cell-transforming activity of PRMT5 and delineated its underlying mechanisms for the first time.

© 2012 Japanese Cancer Association.

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Figures

Figure 1
Figure 1
PRMT

5 induces cell proliferation at low serum concentrations and under anchorage‐independent conditions. (a) Stable 293

T

clonal cell lines that expressed

EGFP

or

EGFP

‐

PRMT

5 at higher (clone 2,

H

) or lower levels of expression (clone 8,

L

) were subjected to western blotting with an antibody against

PRMT

5. (b) Clonal cell lines that expressed

EGFP

or

PRMT

5 were subjected to the

MTT

assay in 5% (upper) or 0.5% (lower) serum for 1–4 days. (c) Clonal cell lines that expressed

EGFP

or

PRMT

5 were subjected to a cell foci formation assay in 5% or 0.5% serum. Cell foci were fixed and stained with

G

iemsa solution. The number of foci were counted and plotted. (d) Clonal cell lines that expressed

EGFP

or

PRMT

5 were subjected to a polyhema‐based anchorage‐independent growth assay. The cells were allowed to grow for 2 days on polyhema plates and subsequently were trypsinized and counted with a hemocytometer. The number of cells in each group at day 2 was normalized against the number of cells that were seeded originally (i.e. 5 × 105). *, **, and *** indicate statistical significance by

S

tudent's t‐test with P < 0.05, 0.01 and 0.001 respectively.

Figure 2
Figure 2

The effects of

PRMT

5 sh

RNA

on cell growth in 293

T

cells. (a) 293

T

cells harboring nothing (

M

ock),

EGFP

,

EGFP

‐

PRMT

5,

PRMT

5 sh

RNA

or scrambled sh

RNA

were applied to

W

estern blot adopting anti‐

PRMT

5 antibody. (b) 293

T

cells harboring nothing,

EGFP

,

EGFP

‐

PRMT

5,

PRMT

5 sh

RNA

or scrambled sh

RNA

were applied to

MTT

‐based cell growth assay in 5% or 0.5% serum for 1–4 days. (c) 293

T

cells harboring

PRMT

5 sh

RNA

or scrambled control were applied to focus formation assay in 10% or 0.5% serum. (d) 293

T

cells harboring

PRMT

5 sh

RNA

or scrambled control were subjected to polyhema‐based anchorage‐independent growth assay. (e) 293

T

cells harboring

PRMT

5 sh

RNA

or scrambled control were analyzed by flowcytometer. The percentage cells residing in each specific phase were calculated and plotted. *, **, and *** indicate statistical significance by

S

tudent's t‐test with P < 0.05, 0.01 and 0.001 respectively.

Figure 3
Figure 3
PRMT

5 accelerates progression through

G

1 and upregulates regulators of

G

1 phase. (a) The

EGFP

and

PRMT

5 clonal cell lines were analyzed by flow cytometry, and the percentage of cells in each specific phase was calculated and plotted. (b) The protein level of various cell cycle regulators in the

EGFP

and

PRMT

5 clonal cell lines was analyzed by western blotting. Antibodies against cyclins

A

,

B

1,

D

1–

D

3,

E

1, and

E

2;

CDK

2, 4, and 6; p19;

R

b protein; phospho‐

R

b protein; and β‐actin were used for western blotting. (c) The kinase activities of various

CDK

s were assayed by performing in vitro kinase reaction where

CDK

2,

CDK

4 or

CDK

6 was isolated by immunoprecipitation employing specific antibodies from cells harboring nothing,

EGFP

empty vector,

EGFP

‐

PRMT

5,

PRMT

5 sh

RNA

, or scrambled sh

RNA

. Recombinant

H

istone

H

1 or

R

b was used as substrate and incubated with

CDK

s in kinase reaction buffer in the presence of [γ‐

P

32]‐

ATP

. *Statistical significance by

S

tudent's t‐test with P < 0.05.

Figure 4
Figure 4
PRMT

5 induces tumor formation in nude mice. (a–c)

PRMT

5 clonal cells formed tumors in nude mice.

EGFP

clonal cells or

EGFP

‐

PRMT

5 (clone 2) clonal cells were injected subcutaneously into nude mice in the absence (a) or presence (b) of

M

atrigel. Tumor size was measured weekly (c). (d,e) Cells from the

PRMT

5‐induced tumor grew in the absence of serum.

PRMT

5‐induced tumor cells taken from nude mice were subjected to the

MTT

assay (d) or counted with a hemocytometer (e) in serum‐free medium. *, **, and *** represent statistical significance by

S

tudent's t‐test with P < 0.05, 0.01 and 0.001, respectively.

Figure 5
Figure 5
PRMT

5 activates

AKT

but suppresses

JNK

signaling cascades.

EGFP

and

PRMT

5 clonal cells were subjected to western blotting using antibodies against (a)

AKT

signaling molecules, including phospho‐ and total

PI

3

K

p85,

PI

3

K

p110, phospho‐

PDK

1, phospho‐ and total

PTEN

, phospho‐ and total

AKT

, m

TOR

, phospho‐m

TOR

, el

F

4

E

, phospho‐

I

κ

B

‐α,

NF

‐κ

B

p65, phospho‐

GSK

‐3β, c‐

R

af, phospho‐c‐

R

af and β‐actin, and (b)

MAPK

signaling factors including phospho‐

MEK

1/2, phospho‐ and total

ERK

, phospho‐ and total p38, phospho‐ and total

JNK

, phospho‐ and total c‐

J

un, and β‐actin. (c) An inhibitor of

PI

3

K

suppressed the growth of

PRMT

5 clonal cells.

EGFP

and

PRMT

5 clonal cells were subjected to the

MTT

‐based cell proliferation assay in the absence or presence of the

PI

3

K

inhibitor

LY

294002 for 1–4 days. (d) Overexpression of

JNK

1 or

JNK

2 did not alter the rate of cell proliferation induced by

PRMT

5.

EGFP

and

PRMT

5 clonal cells transfected with

HA

‐

JNK

1 or

HA

‐

JNK

2 were subjected to

W

estern blot adopting anti‐

HA

, ‐pan

JNK

or ‐actin antibody, or the

MTT

assay for 1–4 days. *Statistical significance by

S

tudent's t‐test with P < 0.05.

Figure 6
Figure 6

PRMT5 is overexpressed in patients with lung cancer. (a) The level of PRMT5 m

RNA

in samples from 29 patients with lung cancer was determined by microarray analysis. Overexpression of PRMT5 was observed in all three probesets (217786_at, 1564521_x_at, and 1564520_s_at). The numbers on the X axis represent the codes of the patients. (b) Box plot shows the distribution of data in grouping classification and a statistically significant difference (P < 0.0001) between tumor tissues and adjacent non‐tumor tissues from the same lung cancer patients.

T

, tumor tissue;

N

, adjacent non‐tumor tissue.

Figure 7
Figure 7

Protein expression of

PRMT

5 in 32 samples of lung cancer tissue. (a) Biopsies from paired lung tumor (

T

) and adjacent normal tissues (

N

) were subjected to western blotting using an antibody against

PRMT

5 or actin. The numbers on the X axis represent the codes of the patients. Actin served as a loading control. (b) The levels of

PRMT

5 and actin were quantified by densitometry. The level of

PRMT

5 protein in each paired tissue was normalized against that of actin. The ratio of normalized

PRMT

5 in the tumor to that in normal tissue was calculated and plotted.

Figure 8
Figure 8

Effects of

PRMT

5 sh

RNA

on the cell growth of

A

549 and

H

1299 cells. (a)

A

549 cells or

H

1299 cells harboring

PRMT

5 sh

RNA

or scrambled control were applied to

W

estern blot adopting anti‐

PRMT

5 antibody. (b)

A

549 or

H

1299 cells harboring

PRMT

5 sh

RNA

or scrambled control were applied to

MTT

‐based cell growth assay in 10% or 0.5% serum for 1–4 days. (c)

A

549 or

H

1299 cells harboring

PRMT

5 sh

RNA

or scrambled control were subjected to polyhema‐based anchorage‐independent growth assay. (d)

A

549 or

H

1299 cells harboring

PRMT

5 sh

RNA

or scrambled control were applied to focus formation assay in 10% or 0.5% serum. * and ** represent statistical significance by

S

tudent's t‐test with P < 0.05 and 0.01 respectively.

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