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New pROSpects for PTP1B: micro-ManagingOncogene-Induced Senescence

Robert S. Banh,1,2,3 Yang Xu,1,2,3 and Benjamin G. Neel1,2,*1Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 1L7, Canada2Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 2M9, Canada3Co-first Authors*Correspondence: bneel@uhnresearch.cahttp://dx.doi.org/10.1016/j.molcel.2014.08.015

Oncogene-induced senescence (OIS) provides an important, but incompletely understood, barrier to tumor-igenesis. In this issue, Yang et al. (2014) surprisingly report that inactivation of PTP1B by reactive oxygenspecies is essential for OIS, via effects on AGO2 and microRNA maturation.

Oncogene-induced senescence (OIS)

prevents the progression of preneo-

plastic cells harboring oncogenic muta-

tions. Senescence is characterized by

altered morphology (increased cyto-

plasmic granularity and nuclear/cell size;

presence of senescence-associated het-

erochromatin foci), activation of the p53/

p21CIP1 and/or p16INK4A/pRB path-

ways, and arrest of cell-cycle progres-

sion (Courtois-Cox et al., 2008). The

precise mechanism(s) of OIS has been

intensely studied and oft debated, with

several studies reporting tissue-, spe-

cies-, and oncogene-specific OIS path-

ways. Elevated levels of reactive oxygen

species (ROS) are found in most senes-

cent cells and are thought to play a

causal role in OIS via as yet unclear path-

ways. In this issue ofMolecular Cell, Yang

et al. (2014) report that ROS promote

senescence by inactivating the protein-

tyrosine phosphatase PTP1B, thereby

enhancing tyrosyl phosphorylation of the

miRNA-processing enzyme argonaute 2

(AGO2) and altering miRNA processing.

These exciting findings advance the OIS

field and also raise new questions for

future research.

ROS have long been viewed as toxic

metabolic byproducts but also are impli-

cated in OIS. For example, oxidant stress

or overexpression of ROS-generating en-

zymes (e.g., NADPH oxidases [NOXs]) in-

duces premature senescence (Kodama

et al., 2013). Before the work of Yang

et al. (2014), there were two general, and

nonexclusive, models for how ROS

induce senescence (Figure 1). Excessive

ROS can activate p38MAPK (p38), which,

via PRAK, triggers p53 activation and

senescence. Alternatively, ROS cause

DNA damage, and the DNA damage

response evokes OIS.

Increasing evidence also implicates

ROS, particularly H2O2, as second mes-

sengers in cell signaling. Classical pro-

tein-tyrosine phosphatases (PTPs) contain

a highly reactive cysteine, which not only is

required for catalysis but also causes sus-

ceptibility to reversible oxidation/inhibition

(Tonks, 2013). ROS-catalyzed PTP inacti-

vation canconfer switch-likepositive feed-

back properties to growth-factor-, cyto-

kine-, or integrin-evoked signaling, and

specific PTPs are implicated as ROS tar-

gets. Yang et al. (2014) find that a substan-

tial fraction of PTP1B is oxidized during

HRASV12-induced senescence of human

lung (IMR-90) or mouse embryonic fibro-

blasts (MEFs). They identify Tyr-393 in

AGO2, which is known to regulate miRNA

loading (Shen et al., 2013), as a PTP1B

target. ROS-evoked AGO2 phosphoryla-

tion causes decreased loading of

miRNAs targeted against p21CIP1, leading

to increased p21CIP1 levels and OIS

(Figure 1).

PTP1B (encoded by PTPN1) is perhaps

the best understood classical PTP.

PTP1B is a critical negative regulator of in-

sulin and leptin signaling, as well as other

receptor tyrosine kinases and cytokine re-

ceptors, and is known to undergo revers-

ible oxidation (Feldhammer et al., 2013).

However, a role for PTP1B in miRNA

biogenesis or OIS was unanticipated.

Yang et al. (2014) detect other pro-

teins that undergo oxidation during OIS,

but PTP1B inhibition alone accelerates

HRASV12-induced senescence. Further-

more, OIS is blocked by the antioxidant

N-acetyl cysteine (NAC), but PTP1B inhi-

bition restores senescence. The authors

interpret these data as evidence that

PTP1B is the key, if not the only, ROS

target in OIS.

eptember 4, 2014 ª2014 Elsevier Inc. 651

Figure 1. Mechanisms Underlying H-RASV12-InducedSenescenceSchematic illustrating the pathway proposed by Yang et al.(2014) (red lines). In addition, ROS stimulate the DNA damageresponse and/or the p38/PRAK pathway to activate p53. OtherRAS-evoked signaling components that can promotep53stabi-lizationviaMDM2 inactivation (e.g., theERKandPI3Kpathways)also are implicated in OIS (Courtois-Cox et al., 2008). ERK alsocan stimulate p38 indirectly. Thep16INK4a/pRBpathway is impli-cated in OIS, especially in human cell systems, whereas theARF/p53 pathway is more critical in murine cells (Haferkampetal., 2009).Questionmarksanddashed lines indicatepotential,but unproven, targets/pathways. Indirect effects are indicatedby multiple arrows. CDKs, cyclin-dependent kinases. (Modifiedfrom Yaswen and Campisi, 2007; Cell 128, 233–234.)

Molecular Cell

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Although PTP1B clearly is an

important new component of the

senescence program, it is difficult

to reconcile these conclusions with

studies showing that ROS-induced

p38 induction and/or DNA damage

also are/is critical. Notably, Yang

et al. (2014) still observe oxidized

PTP1B (at 6 days) even in the pres-

ence of NAC, suggesting that some

ROS were produced, perhaps

enough to trigger other pathways.

Also, while PTP1B inhibition acceler-

ates senescence, inhibition alone

cannot induce OIS, indicating that a

cooperating pathway(s), which

could be ROS-dependent or -inde-

pendent, is required.

AGO2 is a component of the RISC

complex and is involved in miRNA

maturation and gene silencing. Spe-

cific miRNAs are implicated in OIS,

but the role of AGO2 is complicated.

Yang et al. (2014) identify AGO2 as a

negative regulator of OIS. Yet earlier

work showed that AGO2, with let-7,

inhibits transcription of RB1/E2F-

target genes. In that report, AGO2

knockdown delayed, whereas AOG2

overexpression induced, HRASV12-in-

duced senescence (Benhamed et al.,

2012). Such apparently contradictory

effects might be reconciled by topog-

raphy, temporality, or different cell sys-

tems/experimental conditions. Silencing

occurs in the nucleus; indeed, Benhamed

et al. (2012) observed nuclear transloca-

tion of AGO2 during OIS. Conversely,

miRNA maturation (and effects on

p21CIP1) transpires in the cytoplasm.

Yang et al. (2014) study IMR-90 cells

maintained in 20% oxygen, whereas Ben-

hamed et al. (2012) used WI-38 cells

grown in physiological oxygen (3%).

Hence, future studies are needed to

clarify the kinetics and intracellular loca-

tion of AGO2 tyrosyl phosphorylation, its

intercellular variability and oxygen sensi-

tivity, as well as its effects on AGO2-

induced silencing.

Yang et al. (2014) identify CDKN1ACIP1

as a major target of PTP1B-regulated

AGO2. However, it might not be the only/

most important miRNA target, as

p21CIP1-deficient MEFs remain sensitive

to RAS-induced senescence. By contrast,

p53 or p16 deficiency prevents OIS in

MEFs, although not in IMR-90 fibroblasts

652 Molecular Cell 55, September 4, 2014 ª2

(Haferkamp et al., 2009). Thus, senes-

cence-effector molecules can be cell

type, species, or oncogene specific. Inter-

estingly, Yang et al. (2014) also find miR-

106b and miR-20a associated with

AGO2. These miRNAs target WEE1,

RB1, and RBL1/2 in addition to CDKN1A

(p21CIP1); their role in the PTP1B/AGO2

OIS pathway clearly merits investigation.

Alternatively, as PTP1B inactivation is

necessary but not sufficient, for OIS,

p21CIP1 might cooperate with a parallel

pathway(s) to promote OIS.

Another intriguing detail is the delay be-

tween HRASV12 expression, ROS accu-

mulation, and PTP1B oxidation. Because

redox state is determined by the local

concentration of oxidants and antioxi-

dants, delayed PTP1B oxidation could

indicate indirect effects of RAS on the

expression of oxidases/reductases (e.g.,

NOX4 is upregulated by H-RASV12 in

IMR-90 cells) (Kodama et al., 2013) of

the glutathione/thioredoxin system to a

more oxidized state before PTP1B is

affected significantly. The source of

ROS for PTP1B oxidation also warrants

future study. An early report found

increased levels of mitochondrial ROS in

during H-RASV12-induced senescence

014 Elsevier Inc.

(Lee et al., 1999). By contrast,

more recent work showed that inhi-

bition of mitochondrial ROS by the

complex I inhibitor rotenone did not

prevent OIS, whereas NOX1 and

NOX4 were upregulated (Kodama

et al., 2013). However, complex III

can bypass complex I, via complex

II, to generate mitochondrial ROS,

so conceivably, NOXs and mito-

chondria, or even the ER, generate

ROS that promote PTP1B oxidation

and OIS. Because NOXs are local-

ized to different cell compartments,

and RAS-induced NOX expression

is cell line dependent, oxidation

of other PTPs also might contribute

to OIS in a context-dependent

manner.

Yang et al.’s work has potential

implications for human disease.

PTPN1 is amplified, overexpressed,

and/or required for breast, ovarian,

and prostate tumorigenesis (Feld-

hammer et al., 2013). PTP1Boverex-

pression might decrease the pool of

oxidized PTP1B during transforma-

tion, preventing senescence induc-

tion in response to initiating pro-onco-

genic events and promoting tumor

progression. Indeed, Yang et al. (2014)

show that PTP1B overexpression can

overcome RAS-induced senescence in

IMR-90 cells, establishing the plausibility

of this mechanism. Obviously, it also will

be important to assess the role of the

PTP1B/AGO2 pathway in other forms of

senescence. Furthermore, AGO2, miR-

NAs, and PTP1B are implicated in Type II

diabetes: AGO2 levels are higher in the

pancreatic islets of insulin-resistant mice

and diabetes patients due to reduced

levels of miR-184, which targets AGO2.

HigherAGO2 levels, viamiR-375, increase

b cell proliferation and size to accommo-

date the elevated insulin demand seen in

insulin-resistant states (Tattikota et al.,

2014). By contrast, insulin secretion is

decreased in pancreas-specific Ptpn1�/�

mice (Liu et al., 2014). Finally, ROS are

implicated in replicative senescence,

raising the possibility that the PTP1B/

AGO2 pathway might play an important

role in aging and its associated disorders.

Studies over the next few years should

reveal the importance of this novel ROS/

PTP1B/AGO2 pathway in health and

disease.

Molecular Cell

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