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INPP4B Is a PtdIns(3,4,5)P 3 Phosphatase That Can Act as a Tumor Suppressor Satoshi Kofuji 1,2 , Hirotaka Kimura 1,2 , Hiroki Nakanishi 1 , Hiroshi Nanjo 3 , Shunsuke Takasuga 2 , Hui Liu 4 , Satoshi Eguchi 2 , Ryotaro Nakamura 2 , Reietsu Itoh 2 , Noriko Ueno 1 , Ken Asanuma 2 , Mingguo Huang 1,5 , Atsushi Koizumi 5 , Tomonori Habuchi 5 , Masakazu Yamazaki 1,6 , Akira Suzuki 7 , Junko Sasaki 1 , and Takehiko Sasaki 1,2

RESEARCH BRIEF

1 Research Center for Biosignal, Akita University, Akita, Japan. 2 Department of Medical Biology, Akita University Graduate School of Medicine, Akita, Japan. 3 Department of Pathology, Akita University Graduate School of Medicine, Akita, Japan. 4 Division of Hematology and Oncology, Beth Israel Deaconess Medical Center, Boston, Massachusetts. 5 Department of Urol-ogy, Akita University Graduate School of Medicine, Akita, Japan. 6 Depart-ment of Cell Biology and Morphology, Akita University Graduate School of Medicine, Akita, Japan. 7 Division of Embryonic and Genetic Engineering, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.

Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).

Corresponding Author: Takehiko Sasaki, Department of Medical Biology, Graduate School of Medicine, Research Center for Biosignal, Akita Univer-sity, 1-1-1 Hondo, 010-8543 Akita, Japan. Phone: 81-188-84-6080; Fax: 81-188-36-2607; E-mail: [email protected]

doi: 10.1158/2159-8290.CD-14-1329

©2015 American Association for Cancer Research.

INTRODUCTION

Thyroid cancer is the most common endocrine malig-

nancy, and its frequency is increasing dramatically in both

men and women ( 1, 2 ). Indeed, thyroid cancer is predicted

to be the fourth leading cancer diagnosis by 2030 ( 3 ). The

histopathology of thyroid cancers is diverse. About 80% of

all malignant thyroid neoplasms are papillary thyroid carci-

noma (PTC), which are usually not aggressive. In contrast,

about 10% to 15% of thyroid neoplasms are follicular thy-

roid carcinoma (FTC), which can invade blood vessels and

metastasize to lung or bone. Follicular variant of papillary

ABSTRACT Inositol polyphosphate 4-phosphatase B (INPP4B) has been identifi ed as a tumor

suppressor mutated in human breast, ovary, and prostate cancers. The molecular

mechanism underlying INPP4B’s tumor-suppressive role is currently unknown. Here, we demonstrate

that INPP4B restrains tumor development by dephosphorylating the PtdIns(3,4,5)P 3 that accumulates

in situations of PTEN defi ciency. In vitro , INPP4B directly dephosphorylates PtdIns(3,4,5)P 3 . In vivo ,

neither inactivation of Inpp4b ( Inpp4b Δ/Δ ) nor heterozygous deletion of Pten ( Pten +/− ) in mice causes

thyroid abnormalities, but a combination of these mutations induces malignant thyroid cancers with

lung metastases. At the molecular level, simultaneous deletion of Inpp4b and Pten synergistically

increases PtdIns(3,4,5)P 3 levels and activates AKT downstream signaling proteins in thyroid cells. We

propose that the PtdIns(3,4,5)P 3 phosphatase activity of INPP4B can function as a “back-up” mecha-

nism when PTEN is defi cient, making INPP4B a potential novel therapeutic target for PTEN-defi cient

or PIK3CA-activated cancers.

SIGNIFICANCE: Although INPP4B expression is reduced in several types of human cancers, our work on

Inpp4B- defi cient mice provides the fi rst evidence that INPP4B is a bona fi de tumor suppressor whose

function is particularly important in situations of PTEN defi ciency. Our biochemical data demonstrate

that INPP4B directly dephosphorylates PtdIns(3,4,5)P 3 . Cancer Discov; 5(7); 730–9. ©2015 AACR.

See related commentary by Vo and Fruman, p. 697.

See related article by Chew et al., p. 740.

Research. on February 7, 2020. © 2015 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst April 16, 2015; DOI: 10.1158/2159-8290.CD-14-1329

http://cancerdiscovery.aacrjournals.org/

JULY 2015�CANCER DISCOVERY | 731

INPP4B Suppresses Tumorigenesis by Degrading PtdIns(3,4,5)P3 RESEARCH BRIEF

thyroid carcinoma (FV-PTC) has a unique histopathology

characterized by the growth pattern of an FTC but the

nuclear features of a PTC ( 4 ). The mechanisms driving

the formation of these thyroid cancer variants are largely

unknown.

Phosphoinositide (PI) signaling is a lipid second messenger

cascade involving the sequential phosphorylation of phos-

phatidylinositol (PtdIns) to generate fi rst PtdIns monophos-

phate (PtdInsP) and then phosphatidylinositol bisphosphate

(PtdInsP 2 ). Phosphorylation of PtdInsP 2 by PI3Ks then gener-

ates phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ].

In mammalian cells, a total of eight PIs (three regioisomers

each for PtdInsP and PtdInsP 2 ) can be interconverted, and

intracellular levels of each class of these lipids are regulated

by the activities of 19 kinases and 29 phosphatases ( 5 ). PIs

function as direct regulators of a broad range of intracellular

proteins with diverse functions ( 6 ). To date, the list of mole cules

that bind to PIs includes protein kinases, phospholipases, ion

channel proteins, scaffold proteins, cytoskeletal proteins, and

regulators of membrane traffi cking. Hence, PIs control cel-

lular responses such as proliferation, inhibition of apoptosis,

motility, secretion, and endocytosis, all of which are altered in

cancer cells. Given the myriad functions of PI target proteins,

it is not surprising that genetic mutations or alterations to

the protein expression of PI kinases and phosphatases cause

a variety of disorders. For instance, the inherited disease

Cowden syndrome/multiple hamartoma syndrome is caused

by either germline heterozygous inactivating mutations in

the PtdIns(3,4,5)P 3 phosphatase PTEN (OMIM #158350,

CWS1), or germline activating mutations or amplifi cations

of the PtdIns(3,4,5)P 3 -synthesizing enzyme PIK3CA (OMIM

#615108, CWS5; ref. 7 ). Patients with Cowden syndrome

show a predisposition to developing breast, thyroid, and

endometrial cancers, and about 70% of these individuals have

benign thyroid abnormalities, such as multinodular goiter,

adenomatous nodules, and follicular adenoma ( 8 ). These

abnormalities are presumably due to the deleterious effects

of excessive intracellular PtdIns(3,4,5)P 3 , although formal

experimental proof is lacking.

It is widely inferred that elevated PtdIns(3,4,5)P 3 trig-

gers activation of its targets in precancerous cells, includ-

ing the proto-oncogene product AKT that promotes cell

survival, proliferation, oncogenesis, motility, and metastasis.

Recently, another phosphoinositide phosphatase called inosi-

tol polyphosphate 4-phosphatase B (INPP4B; ref. 9 ), which

dephosphorylates PtdIns(3,4)P 2 , has emerged as a tumor

suppressor. The INPP4B gene locus (4q31.1–3) shows loss

of heterozygosity in breast and ovarian carcinomas ( 10, 11 ),

and expression of INPP4B protein is decreased in prostate

cancer ( 12 ). Currently, however, the mechanisms underlying

the putative tumor-suppressive role of INPP4B are a mystery.

Intriguingly, alterations in INPP4B expression are more fre-

quent in cancer patients who bear PTEN mutations than in

those retaining wild-type (WT) PTEN function ( 10, 11 ). This

dual inactivation of PTEN and INPP4B in cancers implies

functional collaboration between these tumor suppressors,

although how such concomitant aberrations of two lipid

phosphatases would promote tumorigenesis is unclear.

Studies of PTEN-defi cient mice by our laboratory and other

groups have unequivocally shown that PTEN is a highly

effective tumor suppressor in a wide variety of tissues ( 5 ).

Pten +/− mice have a shortened lifespan, with 30% dying within

1 year of birth. Aged Pten +/− survivors develop mammary or

endometrial tumors or T-cell lymphomas, among other malig-

nancies. Interestingly, thyroid cancers, a type of tumor associ-

ated with PTEN mutations in humans, have not been detected

in Pten +/− mice ( 13 ). Our mutants thus provide a unique system

with which to analyze cooperation among gene mutations asso-

ciated with thyroid carcinogenesis. In this study, we generated

gene-targeted mice lacking the phosphatase domain of Inpp4B

( Inpp4B Δ/Δ ). Although these mutants are viable and healthy,

Inpp4B Δ/Δ ; Pten +/− compound mutant mice develop thyroid carci-

nomas with complete penetrance. Moreover, our initial studies

in human tissues suggest that reductions in INPP4B and PTEN

co-occur in human thyroid and endometrial cancers. INPP4B

may therefore be an important regulator of cancer progression,

especially in the context of PTEN insuffi ciency.

RESULTS Loss of INPP4B Reduces the Survival of Pten +/- Mice

To gain insight into the mechanism by which INPP4B

exerts its tumor-suppressive function, we scrutinized the abil-

ity of INPP4B to use each PI as a substrate. We overexpressed

INPP4B in 293T cells and recovered an immunoprecipitate

that was used as an enzyme source in vitro . To our sur-

prise, in addition to PtdIns(3,4)P 2 , INPP4B dephosphorylated

PtdIns(3,4,5)P 3 ( Fig. 1A ). This activity toward PtdIns(3,4,5)P 3

was intrinsic to INPP4B and not due to any proteins that

might have bound to it in the immunoprecipitate because

immunoprecipitates of the INPP4B C842S–mutant protein,

which lacks hydrolase activity, did not dephosphorylate

PtdIns(3,4,5)P 3 (Supplementary Fig. S1A). PtdIns(3,4,5)P 3

dephosphoryation catalyzed by PTEN reached a plateau at

a substrate concentration of 1 mmol/L ( Fig. 1B ), whereas

PtdIns(3,4,5)P 3 dephosphoryation by INPP4B did not. These

results suggest that INPP4B requires a higher PtdIns(3,4,5)P 3

concentration than does PTEN to exert its phosphatase activ-

ity. Indeed, when our plots of reaction velocity versus substrate

concentration were fi tted to the sigmoidal Hill equation,

nonlinear regression analysis revealed that the PtdIns(3,4,5)P 3

concentration needed to obtain a velocity half of maximal

( K half ) for PTEN was 0.27 ± 0.04 mmol/L , whereas the K half for

INPP4B was 0.70 ± 0.09 mmol/L. We confi rmed this activity

using an in-house, purifi ed preparation of PtdIns(3,4,5)P 3

(Supplementary Fig. S1B), excluding the possibility that our

earlier results were due to INPP4B-mediated dephosphoryla-

tion of a contaminant [e.g., PtdIns(3,4)P 2 ] in our commercial

source of PtdIns(3,4,5)P 3 .

The above results raised the possibility that INPP4B might

help to control PtdIns(3,4,5)P 3 levels, particularly when PTEN

is defi cient and intracellular levels of this lipid second mes-

senger rise as a result. To investigate this hypothesis, we gen-

erated Inpp4b Δ/Δ mice lacking Inpp4B exon 21, which encodes

the active site motif in the phosphatase domain ( Fig. 1C ).

Successful disruption of the Inpp4B gene was confi rmed by

Southern blotting and immunoblotting ( Fig. 1D ). Inpp4B Δ/Δ

mice were born at the expected Mendelian ratio, were healthy

and fertile, had a normal lifespan, and showed no gross tissue

abnormalities. These fi ndings stand in sharp contrast to the





Kofuji et al.RESEARCH BRIEF

Figure 1. Concurrent mutations of Inpp4b and Pten result in premature death in mice. A and B, quantitation of in vitro PtdIns(3,4,5)P 3 phosphatase activity of (A) INPP4B and (B) PTEN. Data are the mean activity ± SEM of triplicates and are representative of two experiments. C, scheme for the generation of mice lacking the INPP4B phosphatase active site encoded by exon 21 ( Inpp4b Δ/Δ mice). a, genomic structure of the WT murine Inpp4b allele showing exons 20 and 21 (black rectangles). b, targeting vector in which exon 21 was fl anked by two loxP sequences (black arrowheads), with a third loxP sequence fl anking the Neo r gene. c, the recombined allele containing three loxP sequences and the Neo r gene. d, fl oxed allele resulting from Cre-mediated dele-tion of Neo r . e, deleted allele resulting from Cre-mediated deletion of exon 21. D, top, Southern blot of Hin dIII-digested genomic DNA (15 μg/lane) from tails of WT (+/+) and Inpp4b Δ/Δ mice. Blots were hybridized to the probe indicated in (C,e). Bottom, immunoblot confi rming the lack of INPP4B protein in brain lysates of Inpp4b Δ/Δ mice. E, the Kaplan–Meier cumulative survival analysis of WT (open circles; n = 47), Inpp4b Δ/Δ (triangles; n = 46), Pten +/− (squares; n = 27), and Inpp4b Δ/Δ ;Pten +/− (closed circles; n = 40) mice up to 48 weeks of age.

Wild-type allele

Targeting vector

Recombined allele

Floxed allele

Deleted allele

WTInpp4b

a

b

c

d

e

0

0.2

0.4

0.6

0.8

1

0 10 20 30 40 50

Age (weeks)INPP4B

Cu

mu

lative

su

rviv

al

PtdIns(3,4,5)P3 (mmol/L)

0.2 0.4 0.8 10 0.60

pm

ol/m

in/μ

g

pm

ol/m

in/μ

g

5

10

15

20

25

26.6 × X 2.66Y =

0.702.66 + X 2.66INPP4BA

C

D E

B

PtdIns(3,4,5)P3 (mmol/L)

312 × X 2.03Y =

0.272.03 + X 2.03PTEN

0

exon 20

5.4 kbp

exon 21

0

100

200

300

0.2 0.4 0.80.6 1

Inpp4b Δ/Δ;Pten+/–

exon21

exon 20

6.2 kbp

6.2 kbp

5.4 kbp

exon 21exon 20

exon 21exon 20

DT-A

probe

Neo r

H H

H

+/+ Δ/Δ

Inpp4b +/+ Δ/Δ

H

H H

H

H

H

Pten +/–

Inpp4b Δ/Δ

Neo r

early embryonic lethality of Pten −/− mice and the tumorigen-

esis occurring in various tissues of aged Pten +/− survivors ( 13 ).

Thus, INPP4B is dispensable for PtdIns(3,4,5)P 3 metabolism,

at least when PTEN is functional.

We then crossed Inpp4b Δ/Δ mice with Pten +/− mice ( 14 )

to generate Inpp4B Δ/Δ ;Pten +/− compound mutants. Although

Inpp4B Δ/Δ ;Pten +/− mice were born at the expected ratio and

appeared healthy at birth, they had signifi cantly shorter

lifespans than Pten +/− mice, with half-lifetime values of only

26.2 weeks ( Fig. 1E ). Furthermore, all double mutants dis-

played hoarseness and signs of respiratory distress before

they died. Gross examination revealed that their airways were

compressed by abnormally large thyroid glands. These data

suggest that the functions of INPP4B and PTEN overlap to

some degree, especially in the thyroid gland.

Double Defi ciency of INPP4B and PTEN Leads to Thyroid Abnormalities

As noted above, genetic mutations and decreased protein

expression of PTEN have been implicated in human thyroid

neoplasia. However, inactivation of PTEN alone is thought to

be insuffi cient to induce thyroid cancers because these malig-

nancies tend to affect older adults, and the increase in life-

time risk for developing thyroid cancer in Cowden syndrome

patients is less than 10% ( 8 ). In mice, heterozygous loss of

Pten results in thyroid follicular hyperplasia, but no tumors

progress to aggressive malignancy ( 13 , 15 ). Thus, genetic

alteration(s) in addition to Pten defi ciency are required for

benign thyroid disorders to evolve into malignant cancers.

Our Inpp4B Δ/Δ ;Pten +/− mice had larger thyroid glands than

WT, Inpp4B Δ/Δ , and Pten +/− mice, prompting us to histologi-

cally examine thyroid cells of these animals at 15 weeks of

age. After hematoxylin and eosin (H&E) staining, Inpp4B Δ/Δ

thyroid follicles were indistinguishable from WT ( Fig. 2Aa,b ).

Consistent with previous studies ( 13 , 15 ), Pten +/− mice had

a tendency to form larger follicles than WT mice, but the

overall architecture and nuclear morphology of these struc-

tures were normal ( Fig. 2Ac ). In sharp contrast to Pten +/− fol-

licles, Inpp4B Δ/Δ ;Pten +/− follicles were fi lled with irregularly

arranged epithelial cells ( Fig. 2Ad,e ), and only a few glandular






Figure 2. Defi ciencies in INPP4B and PTEN cooperate to accelerate thyroid gland tumorigenesis in mice. A, H&E staining of thyroid glands from 26-week-old mice of the indicated genotypes. A, e and f, show magnifi ed views of the areas surrounded by black broken-line rectangles in c and d, respec-tively. Scale bars, 300 μm (a–d) and 100 μm (e and f). Note the altered growth pattern of follicular epithelial cells in Inpp4B Δ/Δ ;Pten +/− thyroid tissues in d and f. Arrows, thyroid follicles; *, tracheas. B, H&E staining of PTC-like nuclear phenotypes of follicular cells in Inpp4B Δ/Δ ;Pten +/− thyroid tissue. a, nuclear overlapping and crowding; b, “ground glass” nuclei; c, nuclear grooves; d, cytoplasmic invaginations; and e, nuclear enlargement. C and D, histologic staining to detect vascular invasion and lung metastasis. Serial sections of thyroid (C) and lung (D) from a 32-week-old Inpp4B Δ/Δ ;Pten +/− mouse were stained with: a, H&E; b, Elastica-Masson; or c, anti-thyroglobulin Ab. Black arrows in C indicate vascular invasion by malignant thyroid epithelial cells. Scale bars, 100 μm.

A

Inpp4b Δ/Δ;Pten+/–

Inpp4b Δ/ΔWTa b

Inpp4b Δ/Δ;Pten+/–d f

Pten+/–c

e Pten+/–

C

D

B a

b

c

d

e

H&E Elastica-Masson Thyroglobulina b c

H&E Elastica-Masson Thyroglobulina b c

* * *

*

lumens displayed recognizable colloid ( Fig. 2Af ). Cells in

Inpp4B Δ/Δ ;Pten +/− follicles also displayed numerous nuclear

abnormalities associated with PTC ( Fig. 2Ba–e ). Importantly,

Elastica-Masson staining and thyroglobulin immunostain-

ing revealed that Inpp4B Δ/Δ ;Pten +/− thyroids exhibited thyro-

globulin-positive satellite nodules surrounded by elastic fi bers

( Fig. 2Ca–c ), demonstrating vascular invasion by carcinomas

of follicular cell origin. Furthermore, double-mutant mice

developed pulmonary metastases of thyroid follicular cells

( Fig. 2Da–c ), whereas no local lymph node metastases were

detected. These fi ndings suggested that the thyroid tumors

in Inpp4B Δ/Δ ;Pten +/− mice resembled FTC. Because of their

PTC-like nuclear morphology but FTC-like histopathology,

Inpp4B Δ/Δ ;Pten +/− thyroid tumors were deemed to fall under

the defi nition of FV-PTC. Similar thyroid histopathologies

and pulmonary metastasis were observed in Inpp4B +/Δ ;Pten +/−

mice (Supplementary Fig. S2), indicating that Inpp4B is

haploinsuffi cient for tumor suppression. Finally, except for

thyroid cancer, there appeared to be no difference in the spec-

trum of tumors arising in Pten +/− versus Inpp4b Δ/Δ Pten +/− mice.

INPP4B Is Reduced in Human Thyroid and Endometrial Cancers

Decreased expression of INPP4B protein has been docu-

mented in human breast, ovarian, and prostate malignan-

cies ( 10–12 ), but its status in thyroid cancers has yet to be

reported. To extend our fi ndings in mice to humans, we fi rst

mined the recently published papillary thyroid cancer data in

The Cancer Genome Atlas (TCGA) dataset using cBioPortal

( 16, 17 ). Using OncoPrint analysis, we found that INPP4B

expression was downregulated in 30.6% (15 of 49 cases) of

human FV-PTCs (Supplementary Fig. S3). Next, we gener-

ated an anti-INPP4B monoclonal antibody (Ab) suitable for

human immunohistochemistry and immunostained sections






of thyroid tissues from FTC and PTC patients. Noncancerous

thyroid glands from patients with goiter served as controls.

High, medium, and low levels of Ab staining intensity were

established such that the immunostaining of control glands

fi t into the medium group (Supplementary Fig. S4). Anti-

INPP4B staining of thyroid follicular epithelial cells was

reduced compared with noncancerous controls in seven of

eight FTC samples and seven of 39 PTC samples ( Fig. 3A ).

These results are the fi rst demonstration that INPP4B expres-

sion is decreased in human thyroid cancers.

Double immunostaining of our human FTC samples with

anti-INPP4B and anti-PTEN Abs detected concurrent reduc-

tions in INPP4B and PTEN in three FTC cases ( Fig. 3A and

Fig. 3Ba–c ), as has been observed for breast and ovarian carci-

nomas ( 10, 11 ). To extend this fi nding to additional thyroid

cancer samples plus another tumor type, we used cBioPortal

to conduct mutual exclusivity and co-occurrence analyses

of the 511 cases of thyroid carcinoma and 240 cases of

endometrial carcinoma listed in the TCGA database ( 16, 17 ).

We identifi ed strong tendencies toward the co-occurrence

of INPP4B and PTEN mRNA downregulation in thyroid

carcinoma (Supplementary Fig. S5A), and alterations to the

INPP4B and PTEN genes in endometrial carcinoma (Sup-

plementary Fig. S5B). INPP4B mutations also showed a

signifi cant positive association with PIK3CA mutations in

the endometrial dataset. These profi les suggest that loss of

INPP4B function may be advantageous to cancer cells when

they fail to degrade, or overproduce, PtdIns(3,4,5)P 3 .

Loss of INPP4B Leads to Hyperactivation of AKT Signaling

A previous study has shown that genetic alterations of PTEN

in thyroid cancers are associated with AKT hyperactivation ( 15 ).

In line with this observation, we found that AKT phosphor-

ylation was modestly increased in a INPP4B hi PTEN lo human

FTC sample compared with control thyroid gland ( Fig. 3Ca,b ).

However, INPP4B lo PTEN lo FTC samples showed dramatically

increased AKT phosphorylation ( Fig. 3Cc ), suggesting that

INPP4B is a potent negative regulator of AKT activation.

To investigate whether the reduced INPP4B in INPP4B lo

PTEN lo FTC actually caused the observed AKT hyperac-

tivation, we compared the activation status of AKT and

its downstream effectors in thyroid follicular cells of

Inpp4B Δ/Δ ;Pten +/− mice and controls. Immunoblot analyses

of Inpp4B Δ/Δ ;Pten +/− follicular cells revealed hyperphospho-

rylation of PDK1 ( Fig. 3D ), a direct target of PtdIns(3,4,5)P 3

that is recruited to the plasma membrane and phosphor-

ylates AKT. Accordingly, double-mutant thyroids exhibited

increased AKT phosphorylation, just as observed in INPP4B lo

PTEN lo human FTC. Consistent with their activated AKT,

Inpp4B Δ/Δ ;Pten +/− follicular cells showed enhanced phosphor-

ylation of PRAS40, mTOR, and S6 ( Fig. 3D ). Histologic exam-

ination of thyroid serial sections revealed positive staining

for phosphorylated (phospho)-AKT at the plasma membrane

of double-mutant follicular cells ( Fig. 3E ) and for phospho-

S6 in the cytosol ( Fig. 3F ). Low levels of phospho-AKT and

phospho-S6 were present in Pten +/− follicular cells, but stain-

ing intensities were weaker than in Inpp4B Δ/Δ ;Pten +/− cells.

It should be noted that no activation of AKT signaling was

observed in Inpp4B Δ/Δ thyroids, suggesting that INPP4B is

dispensable for downregulating AKT activation in the pres-

ence of normal PTEN. These data demonstrate that INPP4B

has a key role in preventing the hyperactivation of AKT and

its downstream effectors that occurs in situations of PTEN

defi ciency.

PtdIns(3,4,5)P 3 Accumulation and AKT2 Hyperactivation Drive Thyroid Tumorigenesis in Inpp4B D/D ;Pten +/- Mice

Two AKT isozymes occur in thyroid follicular cells: AKT1 and

AKT2. The enhanced activation of AKT signaling we observed in

Inpp4B Δ/Δ ;Pten +/− follicular cells prompted us to examine whether

separate deletion of each AKT isozyme could ameliorate the

thyroid tumorigenesis and metastasis in Inpp4B Δ/Δ ;Pten +/− mice.

We generated triple-mutant mice lacking either AKT1 or AKT2

in the Inpp4B Δ/Δ ;Pten +/− background and found that aggressive

thyroid cancers developed in Akt1 −/− ;Inpp4B Δ/Δ ;Pten +/− mice ( Fig.

4Aa,b ), just as observed in Inpp4B Δ/Δ ;Pten +/− animals ( Fig. 2D ).

In contrast, Akt2 −/− ;Inpp4B Δ/Δ ;Pten +/− mice had a much milder

phenotype, with no cytologic evidence of malignancy or pul-

monary metastasis ( Fig. 4Ac,d ). Consistent with these results,

levels of AKT ( Fig. 4B ) and S6 ( Fig. 4C ) phosphorylation were

signifi cantly lower in mutant cells lacking AKT2 compared with

those lacking AKT1. Over a 48-week observation period, 38%

of Akt2 −/− ;Inpp4B Δ/Δ ;Pten +/− mice remained viable, whereas only

10% of Akt1 −/− ;Inpp4B Δ/Δ ;Pten +/− mice survived this long ( Fig.

4D ). These results suggest that AKT2 hyperactivation in thyroid

follicular cells makes a greater contribution to the mortality of

Inpp4B Δ/Δ ;Pten +/− mice than AKT1 hyperactivation.

Although it has long been assumed that PTEN defi ciency

causes PtdIns(3,4,5)P 3 accumulation in tumor cells in vivo ,

quantitative data validating this assumption is lacking for both

mouse and human cancers. To elucidate the molecular mecha-

nism underlying the functional collaboration between Inpp4B

and Pten in tumor suppression, we quantitated PtdIns(3,4,5)P 3

levels in thyroid tissues by modifying a non radioactive method

of PtdIns(3,4,5)P 3 measurement developed by Clark and col-

leagues ( 18 ). As shown in Fig. 4E , heterozygous deletion of Pten

increased cellular PtdIns(3,4,5)P 3 levels in whole murine thyroid

gland extracts. Consistent with the weak phosphatase activity

of INPP4B toward PtdIns(3,4,5)P 3 in vitro ( Fig. 1A ), disrup-

tion of Inpp4b alone caused only a small (but still statistically

signifi cant) increase in PtdIns(3,4,5)P 3 . However, PtdIns(3,4,5)

P 3 accumulation in the thyroid was enormous when Inpp4B

was deleted in the Pten heterozygous background. These results

clearly demonstrate that INPP4B is critical for dephospho-

rylating the PtdIns(3,4,5)P 3 that accumulates under condi-

tions of PTEN defi ciency. In contrast to PtdIns(3,4,5)P 3 , there

was no difference in PtdIns(3,4)P 2 levels between Pten +/− and

Inpp4B Δ/Δ ;Pten +/− thyroid tissues (Supplementary Fig. S6). Taken

together, our data suggest a model ( Fig. 4F ) in which INPP4B’s

dephosphorylation of PtdIns(3,4,5)P 3 comprises a “back-up”

mechanism that prevents tumorigenic accumulation of this

lipid messenger when PTEN activity is insuffi cient.

DISCUSSION Gene mutation, heterozygous deletion, and decreased

protein expression of INPP4B have been implicated in sev-

eral types of human cancers ( 10–12 , 19 ). However, until






Figure 3. Codefi ciency of INPP4B and PTEN enhances AKT signaling in human and mouse thyroid carcinomas. A, percentages of samples of human FTC ( n = 8) and PTC ( n = 39) showing High, Medium, or Low levels of INPP4B or PTEN expression as determined by multiplying staining intensity with the percentage of immunoreactive cells (see Methods). Scored samples were categorized into the indicated 9 (3 × 3) subgroups. Note that a substantial proportion of human FTC were classifi ed as INPP4B lo PTEN lo . B and C, representative fl uorescent images of serial sections of control human goiter (a; n = 9) and two human FTC samples (b and c; n = 8) immunostained with (B) anti-INPP4B Ab and anti-PTEN Ab, or (C) anti–phospho-AKT (pAKT) Ab. DAPI, counterstaining. White arrows, PTEN-positive leukocytes. Scale bars, 10 μm. D, immunoblot to detect the indicated total and phosphorylated (p) forms of AKT, PDK1, PRAS40, mTOR, and S6 in lysates of thyroid glands from mice of the indicated genotypes ( n > 3/group). Total AKT and Tubulin levels were evaluated as loading controls. Results are representative of at least three trials. E and F, immunostaining of thyroid sections from mice of the indicated genotypes ( n > 3/group) to detect hyperphosphorylation of AKT (E) and S6 (F) in thyroid epithelial cells. Scale bars, 50 μm.

WT Inpp4b Δ/Δ Inpp4b Δ/Δ

Pten+/– Inpp4b Δ/Δ;Pten +/–

WT

Pten+/– Inpp4b Δ/Δ;Pten +/–

INPP4B

PTEN

Tubulin

WT

Inpp4b Δ/Δ

Pten +/–

Inpp4b Δ/Δ;Pten +/–

pAKT (S473)

pAKT (T308)

pPDK1 (S241)

Pan-AKT

pS6 (S235/36)

pS6 (S240/44)

pPRAS40 (T246)

pmTOR (S2448)

40

Follicular carcinomaA

B C D

E F

Papillary carcinoma

(%)

30

20

10

0H

PTEN INPP4B DAPI

a a

b b

c c

pAKT DAPI

MPTEN

LH

M

L

INPP4B

40(%)

30

20

10

0H

MPTEN

LH

M

L

INPP4B






Akt1–/–;Inpp4b Δ/Δ;Pten+/–b

Akt2 –/–;Inpp4b Δ/Δ;Pten+/– Akt1 –/–;Inpp4b Δ/Δ;Pten+/–

Akt1 –/–;Inpp4b Δ/Δ;Pten+/– Akt2 –/–;Inpp4b Δ/Δ;Pten+/–

Akt2 –/–;Inpp4b Δ/Δ;Pten+/–Akt1–/–;Inpp4b Δ/Δ;Pten+/– c

Akt2 –/–;Inpp4b Δ/Δ;Pten+/–d

Cum

ula

tive

surv

ival

Age (weeks)

Inpp4b Δ/Δ;Pten+/–(n = 79)

Akt2 –/–;Inpp4b Δ/Δ;Pten+/–(n = 26)

Akt1–/–;Inpp4b Δ/Δ;Pten+/–(n = 20) P < 0.0001

AKT

mTOR

S6

PTEN

AKT

mTOR

S6

PTEN

AKT

mTOR

S6

PTEN INPP4B

PtdIns(3,4,5)P3 PtdIns(3,4,5)P3

Normal Hyperplasia Cancer

PtdIns(3,4,5)P3 level

INPP4B INPP4B

0

0.2

0.4

0.6

0.8

1

0 10 20 30 40 50

Pten+/–(n = 43)

aA B

C

D E F

PtdIns(3,4,5)P3

0 WT

Inpp4b Δ/Δ

Pten +/–

Inpp4b Δ/Δ;Pten +/–

0.1

0.2

*

*

*

0.3

0.5

Ptd

Ins(3

,4,5

)P3 p

mol/m

g t

issue

0.4

Figure 4. The PtdIns(3,4,5)P 3 –AKT2 axis plays a key role in thyroid tumorigenesis in Inpp4B Δ/Δ ;Pten +/− mice. A, H&E staining of sections of (a and c) thyroid glands and (b and d) lungs from mice of the indicated genotypes ( n > 4/group). Scale bars, 300 μm. Development of (a) neoplastic lesions and metastatic foci (arrows in b) were detected in Akt1 −/− ;Inpp4b Δ/Δ; Pten +/− mice but not in Akt2 −/− ;Inpp4b Δ/Δ ;Pten +/− mice (c and d). B and C, immunostaining to detect phospho-AKT (B) and phospho-S6 (C) in thyroid tissues of mice of the indicated genotypes ( n > 3/group). Scale bars, 100 μm. D, the Kaplan–Meier analysis of cumulative survival of mice of the indicated genotypes. E, quantitation by reverse phase LC/MS-MS of PtdIns(3,4,5)P 3 in thyroid tissues of mice of the indicated genotypes. Results are the mean ± SEM of data from 3 WT, 4 Inpp4b Δ/Δ , 4 Pten +/− , and 3 Inpp4b Δ/Δ; Pten +/− mice (20–28 weeks of age). *, P < 0.05. F, schematic model depicting a potential “back-up” mechanism of tumor suppression by INPP4B. INPP4B is a relatively weak PtdIns(3,4,5)P 3 phosphatase with a higher K half and lower V max than PTEN. Thus, this activity of INPP4B is most important when intracellular PtdIns(3,4,5)P 3 accumulates to high concentrations.

our study, it had not been defi nitively shown that INPP4B

deregulation is not just a consequence of tumorigenesis

but an active contributor. Here, we have used Inpp4B single,

double, and triple mutant mice to establish a cause-and-

effect relationship between loss of INPP4B function and

tumorigenesis.

Our work is the fi rst to demonstrate that a loss of Inpp4B

predisposes mice to cancer development, providing reverse-

genetics evidence for a tumor-suppressive function of

INPP4B. Although mice with disruption of Inpp4B alone did

not develop any tumors, all Inpp4B Δ/Δ ;Pten +/− mice exhibited

spontaneous and early-onset formation of metastatic thy-

roid cancers. These double mutants displayed a dramatically

reduced lifespan compared with both Pten +/− mice (this study)

and thyrocyte-specifi c Pten -null mice ( 20 ). Thus, INPP4B

is dispensable for thyroid tumor suppression in WT mice

but is essential for preventing tumorigenesis in this tissue

when PTEN activity is insuffi cient. Studies of the genomes

of human thyroid cancer cells have revealed that these malig-

nancies accumulate various genetic alterations that may pro-

mote thyroid cell dedifferentiation and drive thyroid cancer

initiation and progression ( 1, 2 ). Our results are in line with

this observation and indicate that loss of Inpp4B is one step

of the many driving thyroid cancer progression.

Because Pten and Inpp4b are deleted in a systemic manner

in our mouse model, a concurrent loss of these enzymes in

non-thyroid cells may also contribute to the tumor-prone

phenotype of the thyroid gland. Coincident decreases in

expression of INPP4B and PTEN have been observed in

human breast and ovarian carcinomas ( 10, 11 ). Our muta-

tional profi ling of human thyroid and endometrial cancers

using the TCGA dataset also showed that genetic altera-

tions in INPP4B and PTEN have a strong tendency to co-

occur, as do mutations of INPP4B and PIK3CA. In addition,

although not statistically signifi cant, we detected coincident

decreases in INPP4B and PTEN expression in 38% of human

FTC specimens. Our biochemical data showing that INPP4B

hydrolyzes PtdIns(3,4,5)P 3 with a K half value lower than that

of PTEN may explain such concurrent genetic alterations in

PI-metabolizing enzymes. Furthermore, this fi nding suggests

that loss of INPP4B function may be particularly advanta-

geous to cancer cells when they fail to degrade PtdIns(3,4,5)P 3 , or

overproduce PtdIns(3,4,5)P 3 . Such situations can arise due to

either sporadic or hereditary mutations of PTEN or PIK3CA ,






which presumably cause deleterious increases in intracellular

PtdIns(3,4,5)P 3 . Future study should investigate the position

of phosphate that INPP4B hydrolyzes.

Hyperplasia of the thyroid gland, mammary gland, and

uterus are the most common manifestations of Cowden

syndrome, and these patients have increased risks of develop-

ing cancers of these origins ( 8 ). For example, 66% of Cowden

syndrome patients have benign thyroid abnormalities such as

follicular adenomas, whereas 3% to 10% of Cowden syndrome

patients develop thyroid malignancies. It is tempting to spec-

ulate that the presence or absence of INPP4B could determine

whether the benign lesions in Cowden syndrome patients

undergo malignant transformation. We have demonstrated

in mice that concurrent PTEN and INPP4B defi ciencies syn-

ergistically elevate PtdIns(3,4,5)P 3 in the thyroid gland in vivo

( Fig. 4E ). We therefore propose that there must be a threshold

PtdIns(3,4,5)P 3 concentration above which benign hyperplas-

tic thyroid cells acquire cancerous properties ( Fig. 4F ).

A provocative question arising from our work is whether

INPP4A, a PI phosphatase highly related to INPP4B, also

functions as a tumor suppressor. INPP4A and INPP4B share

37% amino acid sequence identity ( 5 ). Remarkably, the CX 5 R

active site motif in the phosphatase domains of these iso-

zymes is identical (CKSAKDR). As expected, we found that

INPP4A is also capable of dephosphorylating PtdIns(3,4,5)P 3

(Supplementary Fig. S1). We have previously reported that

loss of the Inpp4a gene in mice leads to neurodegeneration

in the central nervous system as a result of enhanced gluta-

mate excitotoxicity ( 21 ). These mutants die within 1 month

of birth, precluding an analysis of tumorigenesis. Neverthe-

less, we speculate that INPP4A may be a potential candidate

for a novel tumor-suppressive phosphatase. Examination of

genetic alterations in endometrial and lung cancers listed

in the TCGA database has revealed several mutations in

INPP4A , in particular a missense mutation that replaces

the arginine residue in the CX 5 R active site motif. Future

experiments with single- and double-mutant mice bearing

conditional Inpp4a deletions should answer this intriguing

question.

In conclusion, our study has established a role for INPP4B

in tumor suppression and demonstrated its critical function

in PtdIns(3,4,5)P 3 metabolism. Our Inpp4B Δ/Δ (and parental

Inpp4B fl ox/fl ox ) mice provide useful models for studying disorders

emanating from aberrant accumulation of PtdIns(3,4,5)P 3 .

Notably, downregulation of INPP4B has been observed

in several aggressive human cancers, including in >80% of

basal-like breast cancers ( 11 ), and so may be an important

molecular signature of malignancy. Future studies in which

the biologic properties of PtdIns(3,4,5)P 3 -driven cancers are

determined by reconstituting the underlying genetic altera-

tions in mice may point toward novel clinical interventions.

Because the INPP4B gene is not lost in human cancers but is

only minimally transcribed, INPP4B is a plausible anticancer

drug target. Our mutants may be useful for testing both PI3K

inhibitors and chemical compounds capable of enhancing

residual INPP4B activity in cancer cells. This approach may

open up new therapeutic strategies for a wide range of hyper-

plasias and cancers that harbor mutations in PTEN and/or

PIK3CA and accumulate deleterious levels of intracellular

PtdIns(3,4,5)P 3 .

METHODS Mice

The conditional targeting vector was constructed to delete a

genomic fragment containing the 21st coding exon of the mouse

Inpp4b gene by homologous recombination ( Fig. 1C ). One loxP site

was introduced into intron 20 and two into intron 21. The LacZ-

PGK-Neo r cassette was inserted in antisense orientation to Inpp4b

transcription between the two lox sites in intron 21. The linearized

construct was electroporated into 1 × 10 7 E14K mouse embryonic

stem (ES) cells ( 22 ). ES cell colonies resistant to G418 (0.3 mg/mL;

Life Technologies) were screened for homologous recombination

by standard PCR. Recombinant clones were confi rmed by stand-

ard Southern blotting of Hin dIII-digested genomic DNA fragments

using a 590 bp probe. Targeted ES cells were injected into C57BL/6J

blastocysts (CLEA Japan). Chimeric male mice were crossed with

C57BL/6JJ females to achieve germline transmission. Deletion of the

selection cassette to generate Inpp4b +/ fl ox mice was achieved by cross-

ing to MeuCre40 transgenic mice ( 23 ). Likewise, Inpp4b +/Δ mice were

obtained by crossing Inpp4b +/ fl ox mice with MeuCre40 mice. Inpp4b Δ/Δ

mice were distinguished from Inpp4b +/Δ and Inpp4b +/+ mice by PCR.

An oligo primer (5′-CCTGCCATGGGTAGATTTCT-3′) common to

the Inpp4A + and Inpp4A Δ alleles, a primer specifi c for the Inpp4A +

allele (5′-GTTTACATTTGACAGGGTGGTTGG-3′), and a primer

specifi c for the Inpp4A Δ allele (5′-TGCTGTCGCCGAAGAAGTTA-3′), were combined in the same PCR reaction. Inpp4b +/Δ Pten +/− mice

were obtained by crossing Inpp4b +/Δ mice with Pten +/− mice ( 14 ).

To obtain Inpp4b Δ/Δ Pten +/− mice, Inpp4b +/Δ Pten +/− mice were crossed

with Inpp4b +/Δ mice. Triple mutants were obtained by crossing

Inpp4b +/Δ Pten +/− mice with Akt1 −/− mice or Akt2 −/− mice, both of which

were purchased from The Jackson Laboratory. All experimental pro-

tocols were reviewed and approved by the Akita University Institu-

tional Committee for Animal Studies.

Malachite Green Phosphatase Assay FLAG-tagged INPP4A, INPP4B, or PTEN was expressed in 293T

cells and purifi ed using anti-FLAG antibody (Sigma-Aldrich) as previ-

ously described ( 24 ). Recombinant FLAG-INPP4A or FLAG-INPP4B

(0.4 μg) was incubated for 30 minutes at 37°C with 100 to 1,000 μmol/L

1,2-dioctanoyl-PtdIns(3,4,5)P 3 (Cayman Chemical) in 50 mmol/L

Tris–HCl (pH7.4) plus 10 mmol/L EDTA and 200 mmol/L NaCl.

Released phosphate was determined using the malachite green assay

( 25 ). V max and K half values were determined by nonlinear curve fi t-

ting analysis using the GraphPad Prism6.0 software tool (GraphPad

Software, Inc.). The initial velocity (pmol/min/μg) versus substrate

concentration data were fi t to the sigmoidal Hill equation: v = ( V max

[S] n )/( K n + [S] n ), where v = reaction rate; V max = maximum reaction

rate; [S] = substrate concentration; K = the Hill constant, that is,

the substrate concentration producing half-maximal velocity; and

n = Hill coeffi cient ( 26 ).

Preparation of Radiolabeled PI(3,4,5)P 3 Radiolabeled [D3- 32 P]PI(3,4,5)P 3 was prepared enzymatically by

incubating dipalmitoyl-phosphatidylinositol 4,5-bisphosphate

[PI(4,5)P 2 ] (Cayman) with [γ- 32 P]ATP (NEN) in the presence of an

immunoprecipitate containing class IA PI3-kinases that was pre-

pared by incubating mouse liver lysates with anti-p85 Ab (Cell

Signaling Technology). The kinase reaction was quenched by the

addition of chloroform/methanol/8% HClO 4 (5:10:4). After vigorous

vortexing, chloroform/HClO 4 (1:1) was added to isolate the organic

phase, which was washed three times in chloroform-saturated 1%

HClO 4 before drying. Dried lipids were resolved in chloroform/

methanol (95:5) and spotted onto a silica gel 60 plate (MERCK)

that had been pretreated with 1.2% ( w / v ) potassium oxalate in

methanol/water (2/3). PI(3,4,5)P 3 was fractionated by thin layer






chromatography (TLC) in chloroform/methanol/acetone/acetic acid/

water (7/5/2/2/2). The corresponding spot was scraped off of the

silica gel plate and extracted using the Bligh–Dyer method. The

resultant radioactive PI(3,4,5)P 3 (4,000 cpm) was mixed with non-

radioactive dipalmitoyl-PI(3,4,5)P3 (3 nmol, Cayman) in 15-μL

assay buffer [50 mmol/L Tris–HCl (pH 7.5), 0.2% Triton X-100, and

2 mmol/L dithiothreitol] . Purifi ed recombinant FLAG-tagged phos-

phatase (5–10 μg/15 μL) was added to the substrate and incubated at

30°C for 20 hours. After reaction completion, lipids were extracted

by the Bligh–Dyer method and isolated by TLC as described above.

Radiolabeled PI(3,4,5)P 3 was quantitated by autoradiography using

Typhoon FLA 9500 (GE Healthcare).

Antibodies Anti-INPP4B antiserum used for immunoblotting was kindly pro-

vided by Dr. Jean Vacher (Clinical Research Institute of Montreal,

Canada). INPP4B-specifi c rat monoclonal Ab (mAb) against a puri-

fi ed recombinant human INPP4B fragment (2–235 amino acids)

was raised for immunohistochemistry. The mAbs against PTEN,

pan-AKT, phospho-AKT (S473), phospho-AKT (T308), phospho-

PDK1 (S241), phospho-PRAS40 (T246), phospho-mTOR (S2448),

phospho-S6 (S235/236), and phospho-S6 (S240/244) were all from

Cell Signaling Technology. Anti-thyroglobulin mAb was from DAKO.

Anti–α-tubulin polyclonal Ab was from Medical & Biological Labo-

ratories. Antibodies conjugated to Alexa Fluor 488 or 568 were from

Invitrogen, and other secondary antibodies were from DAKO or

Vector Laboratories.

Immunoblotting Thyroid tissue was homogenized in lysis buffer [50 mmol/L Tris–

HCl (pH 8.0), 100 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100,

1 mmol/L DTT, 1 mmol/L Na 3 VO 4 , 30 mmol/L sodium pyrophos-

phate, 50 mmol/L sodium fl uoride, and protease inhibitor cocktail

(Roche Applied Science)]. After centrifugation at 20,000 × g for 15

minutes, supernatants (10 μg total protein) were subjected to stand-

ard SDS-PAGE and immunoblotting as described previously ( 21 ).

Histologic Analyses Mouse thyroid glands were fi xed in 10% formalin neutral buffer

solution and embedded in paraffi n. Sections (3–5 μm) were cut and

stained with H&E or Elastica-Masson in accordance with stand-

ard procedures. For immunohistochemistry, deparaffi nized sections

were incubated in 10 mmol/L citrate buffer (pH 6.0) at 121°C for 10

minutes, treated with 3% H 2 O 2 /methanol for 30 minutes, blocked

with 10% BSA, and incubated overnight at 4°C with primary Ab rec-

ognizing thyroglobulin, phospho-AKT S473, phospho-S6 S235/236,

or PTEN (all at 1:100 dilution). Immunostained sections were incu-

bated with peroxidase-conjugated secondary Abs that were detected

using DAB solution (WAKO).

Immunofl uorescence Analyses For immunofl uorescent examination of INPP4B, PTEN, and phos-

pho-AKT S473 in human thyroid tissues, paraffi n-embedded human

thyroid tissue arrays (thyroid cancer–normal) were purchased from

SuperBioChips Laboratories. Deparaffi nized sections were boiled in

10 mmol/L citrate buffer (pH 6.3) and incubated with primary mAb

recognizing PTEN or phospho-AKT S473, or with anti-INPP4B. Bind-

ing was visualized using secondary Abs conjugated to Alexa Fluor

488 or 568. For counterstaining, DAPI was added at 1 μg/mL for 5

minutes. Fluorescent images were acquired on a LSM 780 microscope

(Carl Zeiss) and processed with ZEN software (Carl Zeiss).

To score expression levels of INPP4B and PTEN in human thyroid

tissue samples, the intensity of Ab staining (0, +1, +2) was multiplied

by the percentage of immunoreactive cells to generate three groups

(Low: <50; Medium: 50–150; High: >150). For each section, staining

intensities were determined for 100 cells in at least three distinct

areas of the slide. This method allowed classifi cation of nine control

(goiter/adenoma) samples in the “Medium” groups of INPP4B and

PTEN expression. Samples were then categorized into nine (3 × 3)

subgroups representing all combinations of low, medium, and high

INPP4B and PTEN expression.

Phosphoinositide Measurement For PtdIns(3,4,5)P 3 measurement, acidic phospholipids were

extracted from thyroid tissues by the Bligh–Dyer method as described

previously ( 21 ). PtdIns(3,4,5)P 3 was measured by modifying a method

developed by Clark and colleagues (ref. 18 ; Nakanishi and colleagues,

manuscript in preparation ). An UltiMate 3000 LC system (Thermo

Fisher Scientifi c) equipped with HTC PAL autosampler (CTC Analytics)

was used for column chromatography. TSQ-Vantage (Thermo Fisher

Scientifi c) was used for electrospray ionization MS/MS analysis. For

PtdIns(3,4)P 2 measurement, mice were intravenously injected with 2

mCi [ 32 P]-phosphorus (PerkinElmer) and thyroid glands were isolated

4 hours later. Total lipids were extracted, deacylated, and subjected to

high-performance liquid chromatography (HPLC ) analysis using a Par-

tisphere SAX column (Whatman) as previously described ( 27 ).

Concurrence/Mutual Exclusivity Analyses cBioPortal, the open platform for exploring multidimensional

cancer genomics data, was used to obtain summary statistics on

mutual exclusivity and co-occurrence of genomic alterations in

human INPP4B and PTEN ( 16, 17 ). The portal calculated odds ratios

that indicated the likelihood that the mutations in each pair of genes

were mutually exclusive or concurrent. The 511 cases of PTC and

the 240 cases of uterine corpus endometrial carcinoma listed in the

TCGA database were analyzed for mutual exclusivity of INPP4B and

PTEN alterations. P values were determined by the Fisher exact test,

with the null hypothesis that the frequency of occurrence of a pair

of alterations in two genes was proportional to their uncorrelated

occurrence in each gene ( 17 ).

Among the 511 cases of PTC, 96 samples were coded as “8340/3.”

According to the International Classifi cation of Disease for Oncology

(third edition ICD-O-3), 8340/3 specifi es “Papillary carcinoma, fol-

licular variant” (FV-PTC). Sample IDs for all FV-PTC were obtained

and the “OncoPrint” results were analyzed on the basis of the Inter-

national Classifi cation of Disease for Oncology histology code. The

percentages of FV-PTC and non–FV-PTC samples with decreased

INPP4B protein were calculated.

Statistical Analyses Statcel2 (OMS, Japan) was used to perform the Kaplan–Meier

cumulative mouse survival analyses. For PtdIns(3,4,5)P 3 measure-

ments, data were analyzed using the unpaired Student t test. P < 0.05

were considered signifi cant.

Disclosure of Potential Confl icts of Interest No potential confl icts of interest were disclosed.

Authors’ Contributions Conception and design: J. Sasaki, T. Sasaki

Development of methodology: H. Nakanishi, T. Sasaki

Acquisition of data (provided animals, acquired and managed

patients, provided facilities, etc.): S. Kofuji, H. Kimura, H. Nakanishi,

H. Nanjo, S. Takasuga, S. Eguchi, R. Nakamura, N. Ueno, K. Asanuma,

M. Huang, A. Koizumi, M. Yamazaki, J. Sasaki

Analysis and interpretation of data (e.g., statistical analysis,

biostatistics, computational analysis): S. Kofuji, H. Kimura,

H. Nakanishi, H. Nanjo, S. Takasuga, H. Liu, S. Eguchi, R. Nakamura,

N. Ueno, K. Asanuma, M. Huang, A. Koizumi, M. Yamazaki, T. Sasaki






Writing, review, and/or revision of the manuscript: S. Kofuji,

T. Sasaki

Administrative, technical, or material support (i.e., reporting

or organizing data, constructing databases): R. Itoh, M. Huang,

A. Suzuki, J. Sasaki, T. Sasaki

Study supervision: T. Habuchi, T. Sasaki

Acknowledgments The authors thank K. Kofuji, At. Kato, Ak. Kato, S. Kumagai, Y.

Kadowaki, Y. Sugihara, H. Takahashi, Y. Tsuya, T. Ayukawa, K. Asa-

numa, C. Horie, Y. Horie, Y. Moribayashi, S. Nishino, Y. Kuribayashi,

S. Ooi, and K. Yanase for critical discussions and technical support,

and H. Watanabe for tissue processing and histological analyses.

Grant Support This work was supported in part by research grants from Japan

Science and Technology Agency (JST); the Ministry of Education,

Culture, Sports, and Technology of Japan; the Japan Society for the

Promotion of Science (JSPS); Astellas Foundation for Research on

Metabolic Disorders; Ono Medical Research Foundation; Uehara

Memorial Foundation; and Takeda Science Foundation. T. Sasaki

and J. Sasaki received funding from Core Research for Evolutional

Science and Technology (CREST; JST) and Precursory Research for

Embryonic Science and Technology (PRESTO; JST).

The costs of publication of this article were defrayed in part by

the payment of page charges. This article must therefore be hereby

marked advertisement in accordance with 18 U.S.C. Section 1734

solely to indicate this fact.

Received November 10, 2014; revised March 27, 2015; accepted

April 1, 2015; published OnlineFirst April 16, 2015.

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2015;5:730-739. Published OnlineFirst April 16, 2015.Cancer Discovery Satoshi Kofuji, Hirotaka Kimura, Hiroki Nakanishi, et al. Suppressor

Phosphatase That Can Act as a Tumor3INPP4B Is a PtdIns(3,4,5)P

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