INPP4B Is a PtdIns(3,4,5)P 3 Phosphatase That Can Act as a ... · 730 | CANCER DISCOVERYJULY 2015 INPP4B Is a PtdIns(3,4,5)P 3 Phosphatase That Can Act as a Tumor Suppressor Satoshi
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
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]
732 | CANCER DISCOVERY�JULY 2015 www.aacrjournals.org
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
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
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.
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 ,