730 | CANCER DISCOVERY�JULY 2015 www.aacrjournals.org
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.
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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
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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
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INPP4B Suppresses Tumorigenesis by Degrading PtdIns(3,4,5)P3 RESEARCH BRIEF
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
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Kofuji et al.RESEARCH BRIEF
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
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INPP4B Suppresses Tumorigenesis by Degrading PtdIns(3,4,5)P3 RESEARCH BRIEF
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
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Kofuji et al.RESEARCH BRIEF
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 ,
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INPP4B Suppresses Tumorigenesis by Degrading PtdIns(3,4,5)P3 RESEARCH BRIEF
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
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Kofuji et al.RESEARCH BRIEF
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
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JULY 2015�CANCER DISCOVERY | 739
INPP4B Suppresses Tumorigenesis by Degrading PtdIns(3,4,5)P3 RESEARCH BRIEF
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.
REFERENCES 1. Network TCGAR . Integrated genomic characterization of papillary
thyroid carcinoma . Cell 2014 ; 159 : 676 – 90 .
2. Kondo T , Ezzat S , Asa SL . Pathogenetic mechanisms in thyroid
follicular-cell neoplasia . Nat Rev Cancer 2006 ; 6 : 292 – 306 .
3. Rahib L , Smith BD , Aizenberg R , Rosenzweig AB , Fleshman JM ,
Matrisian LM . Projecting cancer incidence and deaths to 2030: the
unexpected burden of thyroid, liver, and pancreas cancers in the
United States . Cancer Res 2014 ; 74 : 2913 – 21 .
4. Zhu Z , Gandhi M , Nikiforova MN , Fischer AH , Nikiforov YE . Mole-
cular profi le and clinical-pathologic features of the follicular variant
of papillary thyroid carcinoma. An unusually high prevalence of ras
mutations . Am J Clin Pathol 2003 ; 120 : 71 – 7 .
5. Sasaki T , Takasuga S , Sasaki J , Kofuji S , Eguchi S , Yamazaki M , et al.
Mammalian phosphoinositide kinases and phosphatases . Prog Lipid
Res 2009 ; 48 : 307 – 43 .
6. Vanhaesebroeck B , Stephens L , Hawkins P . PI3K signalling: the path to
discovery and understanding . Nat Rev Mol Cell Biol 2012 ; 13 : 195 – 203 .
7. Orloff MS , He X , Peterson C , Chen F , Chen JL , Mester JL , et al.
Germline PIK3CA and AKT1 mutations in Cowden and Cowden-like
syndromes . Am J Hum Genet 2013 ; 92 : 76 – 80 .
8. Eng C . Cowden syndrome . J Genet Counseling 1997 ; 6 : 181 – 92 .
9. Norris FA , Atkins RC , Majerus PW . The cDNA cloning and charac-
terization of inositol polyphosphate 4-phosphatase type II. Evidence
for conserved alternative splicing in the 4-phosphatase family . J Biol
Chem 1997 ; 272 : 23859 – 64 .
10. Gewinner C , Wang ZC , Richardson A , Teruya-Feldstein J , Etemad-
moghadam D , Bowtell D , et al. Evidence that inositol polyphosphate
4-phosphatase type II is a tumor suppressor that inhibits PI3K signal-
ing . Cancer Cell 2009 ; 16 : 115 – 25 .
11. Fedele CG , Ooms LM , Ho M , Vieusseux J , O’Toole SA , Millar EK , et al.
Inositol polyphosphate 4-phosphatase II regulates PI3K/Akt signal-
ing and is lost in human basal-like breast cancers . Proc Natl Acad Sci
U S A 2010 ; 107 : 22231 – 6 .
12. Hodgson MC , Shao LJ , Frolov A , Li R , Peterson LE , Ayala G , et al.
Decreased expression and androgen regulation of the tumor suppres-
sor gene INPP4B in prostate cancer . Cancer Res 2011 ; 71 : 572 – 82 .
13. Stambolic V , Tsao MS , Macpherson D , Suzuki A , Chapman WB , Mak
TW . High incidence of breast and endometrial neoplasia resembling
human Cowden syndrome in pten +/− mice . Cancer Res 2000 ; 60 : 3605 – 11 .
14. Suzuki A , de la Pompa JL , Stambolic V , Elia AJ , Sasaki T , del Barco
Barrantes I , et al. High cancer susceptibility and embryonic lethality
associated with mutation of the PTEN tumor suppressor gene in
mice . Curr Biol 1998 ; 8 : 1169 – 78 .
15. Yeager N , Klein-Szanto A , Kimura S , Di Cristofano A . Pten loss in
the mouse thyroid causes goiter and follicular adenomas: insights
into thyroid function and Cowden disease pathogenesis . Cancer Res
2007 ; 67 : 959 – 66 .
16. Cerami E , Gao J , Dogrusoz U , Gross BE , Sumer SO , Aksoy BA , et al.
The cBio cancer genomics portal: an open platform for exploring
multidimensional cancer genomics data . Cancer Discov 2012 ; 2 : 401 – 4 .
17. Gao J , Aksoy BA , Dogrusoz U , Dresdner G , Gross B , Sumer SO , et al.
Integrative analysis of complex cancer genomics and clinical profi les
using the cBioPortal . Sci Signal 2013 ; 6 : pl1 .
18. Clark J , Anderson KE , Juvin V , Smith TS , Karpe F , Wakelam MJ , et al.
Quantifi cation of PtdInsP3 molecular species in cells and tissues by
mass spectrometry . Nat Methods 2011 ; 8 : 267 – 72 .
19. Yuen JW , Chung GT , Lun SW , Cheung CC , To KF , Lo KW . Epigenetic
inactivation of inositol polyphosphate 4-phosphatase B (INPP4B),
a regulator of PI3K/AKT signaling pathway in EBV-associated
nasopharyngeal carcinoma . PLoS ONE 2014 ; 9 : e105163 .
20. Antico-Arciuch VG , Dima M , Liao XH , Refetoff S , Di Cristofano A .
Cross-talk between PI3K and estrogen in the mouse thyroid pre-
disposes to the development of follicular carcinomas with a higher
incidence in females . Oncogene 2010 ; 29 : 5678 – 86 .
21. Sasaki J , Kofuji S , Itoh R , Momiyama T , Takayama K , Murakami H ,
et al. The PtdIns(3,4)P(2) phosphatase INPP4A is a suppressor of
excitotoxic neuronal death . Nature 2010 ; 465 : 497 – 501 .
22. Sasaki T , Irie-Sasaki J , Jones RG , Oliveira-dos-Santos AJ , Stanford WL ,
Bolon B , et al. Function of PI3Kg in thymocyte development, T cell
activation, and neutrophil migration . Science 2000 ; 287 : 1040 – 6 .
23. Leneuve P , Colnot S , Hamard G , Francis F , Niwa-Kawakita M ,
Giovannini M , et al. Cre-mediated germline mosaicism: a new trans-
genic mouse for the selective removal of residual markers from tri-lox
conditional alleles . Nucleic Acids Res 2003 ; 31 : e21 .
24. Asanuma K , Takasuga S , Sasaki J , Sasaki T . Phosphatidylinositol 3,
5-bisphosphate is an essential regulator of lysosome morphology .
Akita J Med 2012 ; 39 :129–37.
25. Maehama T , Taylor GS , Slama JT , Dixon JE . A sensitive assay for
phosphoinositide phosphatases . Anal Biochem 2000 ; 279 : 248 – 50 .
26. Lee J , Johnson J , Ding Z , Paetzel M , Cornell RB . Crystal structure
of a mammalian CTP: phosphocholine cytidylyltransferase catalytic
domain reveals novel active site residues within a highly conserved
nucleotidyltransferase fold . J Biol Chem 2009 ; 284 : 33535 – 48 .
27. Takasuga S , Horie Y , Sasaki J , Sun-Wada GH , Kawamura N , Iizuka R ,
et al. Critical roles of type III phosphatidylinositol phosphate kinase
in murine embryonic visceral endoderm and adult intestine . Proc
Natl Acad Sci U S A 2013 ; 110 : 1726 – 31 .
Research. on February 7, 2020. © 2015 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from
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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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