Inhibin and p27 Interact to Regulate Gonadal Tumorigenesis
Sherry C. Cipriano,
Lei Chen,
Kathleen H. Burns,
Andrew Koff and
Martin M. Matzuk
Departments of Pathology (S.C.C., L.C., K.H.B., M.M.M.)
Molecular and Cellular Biology (M.M.M.), and Molecular and Human
Genetics (K.H.B., M.M.M.) Baylor College of Medicine Houston,
Texas 77030
Program in Molecular Biology (A.K.) Memorial
Sloan-Kettering Cancer Center New York, New York 10021
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ABSTRACT
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Tumor suppressors function as antiproliferative
signaling proteins, and defects in these genes lead to uncontrolled
cell proliferation and cancer. For example, absence of the tumor
suppressor p27Kip1, a cyclin-dependent kinase
inhibitor (CKI), results in increased body size, hyperplasia of several
organs including the testes, and cancer in mice. Similarly, lack of
inhibins,
/ß heterodimeric members of the transforming growth
factor-ß (TGFß) superfamily, causes testicular and ovarian tumors
of the granulosa/Sertoli cell lineage beginning at 4 weeks of age and
adrenal tumors in gonadectomized mice. Neither the cell cycle
alterations in the absence of inhibin nor the cause of the increased
testis size in the p27 knockout mice is known. To study the molecular
(cell cycle) changes that result from absence of inhibins, we analyzed
the regulation of cell cycle proteins in gonadal tumors derived from
inhibin
knockout mice (Inha-/-). Northern
blot analyses demonstrate that cyclin-dependent kinase 4 (Cdk4) and
cyclin D2 mRNA levels are elevated, and immunohistochemistry shows that
p27 protein levels are decreased in both ovarian and testicular tumors
from Inha-/- mice. These findings suggest
that increased Cdk4/cyclin D2 (positive) activity and decreased p27
(negative) activity is causal for gonadal tumor formation. To test this
hypothesis, we generated double mutant mice lacking both p27 and
inhibin
to determine whether the tumor suppressors p27 and inhibin
have additive suppressor activity in the gonads. Like
Inha-/- mice,
p27-/-Inha-/- mice
demonstrate elevated serum activin levels, ovarian and testicular
tumors, and a resultant lethal cachexia-like syndrome. However, whereas
95% of the Inha-/- female mice die by 18
weeks of age, 100% of the
p27-/-Inha-/-
female mice are dead by 8 weeks. Similarly, 95% of the
Inha-/- single mutant males die by 13 weeks
while 100% of the p27-/-Inha-/-
male mice die by 10 weeks. Moreover, tumor foci in
p27-/-Inha-/- mice
can be observed as early as 2 weeks of age in males and as early as 4
weeks in females. These findings demonstrate that absence of both
inhibin and p27 in mice causes earlier development of ovarian and
testicular tumors and earlier death compared with absence of inhibin
alone.
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INTRODUCTION
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Cell proliferation is mediated by a delicate balance between
stimulatory and inhibitory signals that function at defined stages of
the cell cycle. Among the proteins that regulate each cell cycle stage
are the cyclins, cyclin-dependent kinases (Cdks), and cyclin-dependent
kinase inhibitors (CKIs). Cell proliferation is dependent upon
activation of cyclin D-Cdk4/6 and cyclin E-Cdk2 complexes (1).
Deregulation of cell cycle events such as overexpression of cyclins
and/or Cdks or underexpression of CKIs can lead to uncontrolled cell
proliferation and malignancy. Two distinct families of CKIs exist, the
Cip/Kip and Ink4 families (2, 3, 4, 5, 6, 7, 8, 9). The Cip/Kip family consists of
p21Cip1 (3), p27Kip1 (9),
and p57Kip2 (7). CKIs have been shown to be
involved in DNA damage checkpoints and can function as rate-limiting
regulators at the G1 to S phase transition by
binding to and inactivating cyclin/Cdk complexes. For example,
p27Kip1 interacts with cyclin D and cyclin E
complexes to inhibit their activity in vitro (10).
Several knockout mouse models lacking cyclins, Cdks, or CKIs have been
created to study cell cycle control in vivo. Among these,
mice lacking cyclin D2 and p27 exhibit gonadal defects. Female cyclin
D2 knockout mice are sterile due to a failure of granulosa cell
proliferation, while male mice have decreased testis size (11).
p27-deficient mice have larger than normal body size and display
intermediate lobe pituitary hyperplasia (12, 13, 14). p27-deficient males
have hyperplastic testes (12, 13, 14) and benign prostatic hyperplasia
(15), yet are fertile; p27-deficient females are infertile due to
defects in cell cycle withdrawal and corpus luteum formation
(12, 13, 14).
Our laboratory is interested in the gonadal growth-regulatory
properties of the inhibins and activins, which inhibit and stimulate,
respectively, pituitary FSH synthesis and secretion in adult mammals
(16, 17). We previously generated transgenic mice lacking inhibin
and therefore functional inhibins (
/ßA or
/ßB heterodimers of
the TGFß superfamily that share ß- subunits with the activins)
(18). Deletion of inhibin
leads to development of ovarian and
testicular granulosa/Sertoli cell tumors in these mice, identifying
inhibin as a critical negative regulator of gonadal cell proliferation.
Tumor development is accompanied by a cancer cachexia-like wasting
syndrome (19) that mimics the cachexia syndromes found in human cancer
patients (20, 21). This wasting syndrome was found to be due to
oversecretion of activins from the tumors (18, 19). Elevated levels of
FSH in inhibin-deficient mice suggested to us that FSH may be a
modulator of the tumorigenic process. Indeed, double knockout mice
deficient in FSH and inhibin demonstrate a delay in tumor formation and
a reduction in the cachexia symptoms compared with mice lacking only
inhibin (22).
Extracellular signals control mammalian cell proliferation principally
during the G1 phase of the cell cycle. TGFßs,
for example, are potent growth inhibitors that suppress cell
proliferation and induce cell cycle arrest by multiple mechanisms
during the G1 phase (23, 24).
p15Ink4b expression and cell cycle arrest are
induced this way in human keratinocytes treated in vitro
with TGFß1 (5). Activin has been implicated in cell cycle arrest in
some cell types. For example, the HepG2 human hepatoma cell line has
been shown to be growth arrested by activin A. In addition, activin A
further induced accumulation of hypophosphorylated Rb in HepG2 cells
preventing progression to the S phase (25). Another study revealed that
in mouse B cell hybridoma cells activin A caused
G1 cell cycle arrest (26). Similarly, pituitary
adenoma cells in primary culture were growth inhibited after treatment
for 24 h with activin, and p21Cip1 was
up-regulated after 4 h treatment in a dose-dependent manner (27).
However, activin function appears to be different in the gonads where
activins can stimulate growth of gonadal tumor cell lines from
inhibin-deficient mice (28). Little is known about the inhibin signal
transduction cascade, how inhibins are linked to cell cycle regulation,
or how inhibins effect growth suppression in the gonads.
In this manuscript, we demonstrate that several cell cycle regulators
are altered in the ovaries and testes in the absence of inhibin, and
that aberrant expression of these factors is key in loss of growth
control and tumor formation. Gonadal tumors that develop in the absence
of inhibin show increases in Cdk4 and cyclin D2 mRNA and a decrease in
p27 protein expression as compared with normal gonads. Furthermore,
absence of the two tumor suppressors, p27 and inhibin, results in
earlier tumor development and death, indicating that inhibin and p27
cooperate in regulating gonadal tumor formation and progression.
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RESULTS
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Cdk4 and Cyclin D2 Are Increased in Gonadal Tumors
To determine whether there are any cell cycle alterations in the
absence of inhibin, we assessed whether up-regulation or
down-regulation of a number of cyclins, Cdks, and/or CKIs was
associated with the uncontrolled proliferation in the gonads of
Inha-/- mice. Northern blot analysis showed
only a slight increase in cyclin D2 mRNA between ovarian tumors from
Inha-/- mice compared with wild-type ovaries
(Fig. 1
). However, we found a 6-fold
increase in cyclin D2 mRNA in testicular tumors of
Inha-/- mice compared with wild-type testes
(Fig. 1
). Cdk4 mRNA levels were elevated 3-fold in ovarian and
testicular tumors from Inha-/- mice when
compared with wild-type ovaries and testes (Fig. 1
). When mRNA levels
of cyclin D2 and Cdk4 in the ovaries and testes of 4-, 8-, and
12-week-old Inha-/- mice were compared, no
temporal difference in either cyclin D2 or Cdk4 expression was found,
suggesting that inhibins effects are early (data not shown). In
contrast, levels of p18Ink4c,
p19Ink4d, cyclin B1, cyclin B2, and cyclin D3
were increased in wild-type testes alone when compared with wild-type
ovaries and gonadal tumors from Inha-/- mice
(data not shown). We were not able to detect any significant change in
mRNA levels of p15Ink4b,
p16Ink4a, p21Cip1,
p27Kip1, p57Kip2, cyclin
A2, cyclin D1, or Cdk2 in gonadal tumors of
Inha-/- mice, as compared with normal tissue
(data not shown). Thus, inhibin may normally act to inhibit cyclin D2
and Cdk4 and thereby regulate gonadal growth and proliferation (see
below).

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Figure 1. Northern Blot Analysis of Cyclin D2 and Cdk4 mRNA
in Mouse Testes and Ovaries
Higher expression of cyclin D2 (cyc D2) and Cdk4 mRNA is present in
ovarian and testicular tumors from individual 12- to 14-week-old
Inha-/- mice when compared with wild-type (WT) ovaries
from adult mice. 18S rRNA was used as control for RNA loading.
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Gonadal Tumors of Inha-/- Mice Have
Decreased p27 Expression
The inhibitory effects of TGFßs on p27 expression are mainly at
the posttranscriptional level (29). Therefore, we analyzed the levels
of p27 protein in the gonads of wild-type and
Inha-/- male and female mice by
immunohistochemistry. In normal adult mouse and human testes, Sertoli
cells (and Leydig cells) stain for p27, which is consistent with the
terminally differentiated state of these cells (30). We hypothesized
that Sertoli cell tumors present in Inha-/-
mice might have reduced p27 protein due to the lack of differentiation
and high proliferative activity of these cells. Indeed, by
immunohistochemical analysis, p27 was found to be low to undetectable
in the Sertoli cell component of Inha-/-
ovarian and testicular tumors (Fig. 2
, B
and C, and E and F). Testes from wild-type males at 6 weeks of age
showed p27 staining in the Sertoli cells and Leydig cells (Fig. 2D
;
Ref. 30). In contrast, p27 expression in Sertoli cell tumor nodules of
Inha-/- male testes had minimal or no p27
protein expression (Fig. 2E
). Even though p27 expression was absent or
barely detectable in Sertoli cell tumors of 6-week-old
Inha-/- testes, we observed p27 staining of
normal Leydig cells in these same tissues (Fig. 2F
; Ref. 30). Staining
of testes from adult p27-/- mice showed no
expression of p27 (data not shown; Ref. 30). The findings in the
ovaries of the Inha-/- mice were also
consistent. As expected, we observed expression of p27 in the
interstitium, primordial follicles, and corpus luteum of 6-week-old
wild-type female mice (Fig. 2A
and data not shown; Ref. 31). We also
observed p27 expression in normal appearing primordial follicles and
the interstitium of ovaries from Inha-/- mice
at 6 weeks of age (Fig. 2B
). However, in these same ovaries,
undifferentiated Sertoli-like tumor cells had very low or undetectable
levels of p27 (Fig. 2C
). Thus, these results suggest a correlation
between granulosa cell and Sertoli cell loss of p27, lack of terminal
differentiation, and uncontrolled proliferation.

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Figure 2. Immunohistochemical Analysis of p27 Expression
A, Wild-type (WT) ovary showed p27 expression in the interstitium and
primordial follicles (expression in the corpus luteum is not depicted).
Terminally differentiated Sertoli cells express high levels of p27.
While p27 expression was detected in normal appearing primordial
follicles and the interstitium of 6-week-old Inha-/- mice
(B), undifferentiated Sertoli-like tumor cells in this same ovary were
not or barely stained for p27 (C). D, Sertoli cells in the testes of
adult wild-type (WT) mice stained for p27. However, Sertoli-like tumor
cells in 6-week-old Inha-/- testes minimally or did not
stain for p27 (E), while normal-appearing Leydig cells in this same
tissue did stain for p27 (F). Magnification is 1040x.
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p27-/-Inha-/-
Mice Develop Cachexia and Have a Decreased Survival Rate
To determine the in vivo relationship between p27
and inhibin in gonadal tumorigenesis, we generated double mutant mice
deficient in both of these genes. An early indicator of ovarian and
testicular tumor development in Inha-/- mice is
severe weight loss and a cachexia-like wasting syndrome that eventually
kills the mice (19). Therefore, to monitor tumor development in
p27-/-Inha-/- mice, we
weighed wild-type, Inha-/-,
p27-/-, and p27-/-
Inha-/- mice weekly for 420 weeks. As
expected, p27-/- male and female mice had
higher weight averages beginning at 410 weeks of age when compared
with wild-type mice (Fig. 3
, A
and B; Ref. 13). Male and female
p27-/-Inha-/- mice
gained weight initially as did wild-type mice up to 6 weeks of age, but
rapidly lost weight thereafter. The cachectic appearance of the
p27-/-Inha-/- mice was
similar to the Inha-/- mice except that it
occurred at an earlier stage and progressed more rapidly (Fig. 3
, A and
B). Serum hematocrit levels of
p27-/-Inha-/- males and
females revealed that they were anemic, similar to
Inha-/- mice (Ref. 18 and data not shown).

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Figure 3. Body Weights of Male (A) and Female (B) Wild-Type,
p27-/-, Inha-/-, and
p27-/-Inha-/- Mice
Mice were weighed weekly on the same day between 4 and 20 weeks of age.
Wild-type, male n = 8, female n = 5; p27-/-,
male n = 19, female n = 16; Inha-/-, male
n = 20, female n = 20;
p27-/-Inha-/-, male n = 17, female
n = 3.
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The p27-/-Inha-/-
male and female mice have decreased survival compared with
p27-/- or Inha-/-
single knockout mice. All of the
p27-/-Inha-/- male mice
were dead by 10 weeks of age (0% survival), whereas at 10 weeks, 56%
of the male Inha-/- mice were still alive (Fig. 4A
). Similarly, all female
p27-/-Inha-/- mice died
by 8 weeks of age (0% survival) vs. a 90% survival of the
female Inha-/- mice at 8 weeks (Fig. 4B
). As
expected, the survival rate for wild-type and
p27-/- male and female mice was 100% during
this 4- to 20-week period (Fig. 4
, A and B, and data not shown). In
total, we have generated 21 male and 9 female
p27-/-Inha-/- mice. The
males have been generated at the expected Mendelian ratio of 1:16.
However, the number of viable
p27-/-Inha-/- female
mice was lower than expected over this same time period, possibly due
to early tumor-induced death before genotype analysis.

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Figure 4. Survival Curves of Wild-Type, p27-/-,
Inha-/-, and p27-/-Inha-/-
Male (A) and Female (B) Mice
Mice were counted once per week between 4 and 20 weeks of age.
Wild-type, male n = 8, female n = 5; p27-/-,
male n = 5, female n = 14; Inha-/-, male n
= 20, female n = 18; p27-/-Inha-/-,
male n = 17, female n = 9. Wild-type mice had a 100%
survival rate from 4 to 20 weeks.
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p27-/-Inha-/-
Mice Develop Bilateral Hemorrhagic Testicular and Ovarian Tumors
To study the cause of the weight loss and reduced survival
of the p27-/-Inha-/-
mice, we performed pathological examination of the
p27-/-Inha-/- male and
female mice at various time points. We found that gonadal tumors
developed sooner and were more progressive in
p27-/-Inha-/- mice
compared with Inha-/- mice. By 7.5 weeks of
age, testes of
p27-/-Inha-/- mice were
grossly enlarged and hemorrhagic compared with wild-type testes (Fig. 5A
). In contrast to
Inha-/- mice, which developed either unilateral
or bilateral tumors, all
p27-/-Inha-/- male
mice analyzed developed bilateral gonadal tumors. Furthermore,
these bilateral gonadal tumors in the
p27-/-Inha-/- mice were
grossly evident by 4 weeks of age in males and 4.5 weeks of age in
females.

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Figure 5. Morphological and Histological Analysis of Testes
from Male Wild-Type and Mutant Mice
A, Morphological comparison of testes from wild-type
(upper part of panel) and 7.5- week-old
p27-/-Inha-/-(lower part of
panel) mice. Testicular tumors were grossly enlarged and contained
areas of hemorrhage (white arrowhead). B, Histological
analysis of testes at high-power magnification from a 2-week-old
p27-/-Inha-/- mouse demonstrated early
Sertoli tumor cell arrangement (starred area) adjacent
to normal appearing seminiferous tubules. C, Testis from a 4- week-old
p27-/-Inha-/- mouse at low power
magnification showed approximately 50% of the tissue is occupied by a
single nodular tumor mass consisting of undifferentiated granulosa
cells. Some hemorrhage (H) is evident. D, Testis at high-power
magnification from a 5.5-week-old
p27-/-Inha-/- mouse showed both
undifferentiated Sertoli (starred area) and granulosa
cell (G) tumor with hemorrhage (H). E, At high power, gland-like
structures were seen (black arrows) among the granulosa
tumor cells. F, Mitotically active (black arrows) granulosa
cell tumor taken from a 8- to 9-week-old
p27-/-Inha-/- mouse is shown at high power.
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Histological examination of the testes of
p27-/-Inha-/- mice
revealed that tumor formation was first evident in some males at 2
weeks of age as focal lesions (Fig. 5B
). Strikingly, by 4 weeks of age,
approximately 4050% of each
p27-/-Inha-/- testis
was composed of single or multiple tumor nodule(s). The masses
consisted of approximately 90% granulosa cell tumor cells (Fig. 5C
).
This is in contrast to testicular tumors in 4-week-old
Inha-/- mice, which had rare, small nodule
lesions of undifferentiated tumor cells (18). While mixed
granulosa/Sertoli cell tumors were seen in some
p27-/-Inha-/- male mice
at 5 weeks of age (Fig. 5D
), most tumors consisted of primarily
granulosa cells (Fig. 5C
). In one 8-week-old
p27-/-Inha-/- male
mouse, we observed gland-like structures in the testicular tumor (Fig. 5E
). This histopathological feature had never been observed in any
Inha-/- male tumors. Similar structures are
present in the gonads of human patients with Peutz-Jeghers syndrome,
which is characterized by oral mucocutaneous hyperpigmentation,
intestinal hamartomas, and an increased risk for intestinal and
extraintestinal malignancies including gonadal tumors (32). The
p27-/-Inha-/-
testicular tumors from 8- to 9-week-old males consisted predominantly
of mitotically active granulosa tumor cells (Fig. 5F
) with limited
areas of normal tubules located at the periphery of the testis (not
shown).
The p27-/-Inha-/-
mutant females were also grossly affected at an early age. In contrast
to wild-type mice or Inha-/- mice [which have
histological evidence of tumors only at an early age (18)], ovaries
from p27-/-Inha-/-
female mice at 4.55.5 weeks of age could show up to a 10-fold
increase in the size of the ovary with areas of punctate hemorrhage
(Fig. 6A
and data not shown).
Histologically, we found no evidence of normal follicles in the ovaries
examined at 5.5 weeks of age (Fig. 6B
) in contrast to
Inha-/- ovaries at the same age (18, 33).
Sertoli cell tumors were a prominent component of the tumor
phenotype in Inha-/- ovaries. Ovaries from
p27-/-Inha-/- mice
consisted of only 010% Sertoli cell tumor with the majority of the
tissue being granulosa cell tumor (Fig. 6C
). Multiple hemorrhagic cysts
(Fig. 6C
) and significant mitotic activity (Fig. 6D
) were also seen in
the p27-/-Inha-/-
ovarian tumors. Thus, absence of p27 increases the rate of development
of testicular and ovarian tumors in inhibin
knockout mice.

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Figure 6. Morphological and Histological Analysis of Ovaries
from Female Mice at 5.5 Weeks of Age
A, Ovaries from a p27-/-Inha-/- mouse
showed gross enlargement (lower part of panel) compared
with wild-type ovaries (upper part of panel). B,
Histological analysis at low power of one ovary from panel A showed no
normal follicles remaining; instead the ovary consists of primarily
granulosa cell tumor with only about 110% being Sertoli-like tubule
tumor cells. Also, note areas of hemorrhage. C, An ovary from a
p27-/-Inha-/- mouse is shown at higher
power demonstrating both Sertoli-like tumor (starred
area) and granulosa (G) tumor cells along with hemorrhage (H). D, At
high power, the undifferentiated granulosa cells are mitotically active
as evident in this ovary from a
p27-/-Inha-/- mouse.
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FSH Levels in
p27-/-Inha-/- Mice
Are Not Significantly Increased When Compared with
Inha-/- Mice
Inha-/- mice have increased serum FSH due
to loss of local and circulating inhibin (18). Mice lacking both
inhibin and FSH had prolonged survival compared with mice lacking only
inhibin (22), demonstrating that FSH acts as a modifier of tumor
development and the cachexia-like syndrome. To determine whether
the earlier death in our
p27-/-Inha-/- mice
could be due to higher FSH levels or an earlier increase in the FSH
levels, we performed FSH enzyme-linked immunosorbent assays (ELISAs).
FSH serum levels from
p27-/-Inha-/- male mice
at an early time point (4 weeks of age) were 195 ± 30.0 ng/ml
(n = 3), which was comparable to
Inha-/- male mice at the same age (188.1
± 32.3, n = 5) (Table 1
). The
p27-/-Inha-/- male
mice at 79 weeks of age (n = 7) had similar FSH levels of
240.7 ± 22.6 ng/ml when compared with
Inha-/- 8-week-old male level 257.7 ±
28.1 ng/ml; n = 7. These levels were elevated compared with 4- and
8-week-old male p27-/- mice (75.7 ±
10.98, n = 4 and 78.8 ± 39.3, n = 4, respectively). For
unknown reasons, these FSH levels in the p27-/-
male mice were higher when compared with wild-type 8-week-old male mice
(49.25 ± 3.09 ng/ml, n = 8).
The FSH levels in the 4- to 8-week-old
p27-/-Inha-/- female
mice (n = 4) were 216 ± 25.5 ng/ml and were elevated
compared with 6-week-old wild-type females (22.5 ± 6.07 ng/ml,
n = 7) but comparable to Inha-/- females
at 6 weeks of age (242.5 ± 24.0 ng/ml, n = 6) (Table 1
).
Similar to males, 6 week-old female p27-/- mice
had unexplained increases in FSH levels (61.2 ± 7.33, n = 5)
compared with the wild-type control females. Thus, while FSH serum
levels are increased in
p27-/-Inha-/- male and
female mice compared with wild-type mice, they are not
significantly different than age-matched serum FSH levels in
Inha-/- male and female mice. This suggests
that FSH is not directly causal for the earlier death of the
p27-/-Inha-/- mice
compared with Inha-/- mice.
p27-/-Inha-/-
Mice Have Elevated Activin Levels
Gonadal tumors of Inha-/- mice secrete
excess activin A and B, which contributes directly to the wasting
syndrome (19, 34, 35). To assess this feature of cachexia in
p27-/-Inha-/- mice, we
determined the serum activin A levels. At an early timepoint,
4-week-old Inha-/- and
p27-/-Inha-/- male mice
had comparable serum activin A levels that were modestly elevated over
controls (Fig. 7A
). However,
p27-/-Inha-/- male mice
at 79 weeks of age (a timeframe when most of these mice were
cachectic and dying) had significantly elevated levels of circulating
activin A (55.1 ± 12.6 ng/ml) when compared with 8-week-old
wild-type males (0.41 ± 0.10 ng/ml) and 8-week-old
p27-/- males (0.12 ± 0.01 ng/ml) and were
about 2-fold higher than 8-week-old Inha-/-
mice (29.4 ± 8.05 ng/ml) (Fig. 7A
). Similarly,
p27-/-Inha-/- female
mice at 47 weeks of age (also a timeframe when most were cachectic
and dying) had significantly higher activin A levels (18.4 ± 2.1
ng/ml) compared with 6-week-old wild-type (0.28 ± 0.7 ng/ml),
6-week-old p27-/- (0.22 ± 0.06 ng/ml),
4-week-old Inha-/- (4.83 ± 6.0 ng/ml),
and 6-week-old Inha-/- (3.79 ± 0.86
ng/ml) females (Fig. 7B
). As expected, activin A levels of 12- to
14-week-old cachectic Inha-/- male (68.5
± 11.7 ng/ml) and female Inha-/- mice
(84.1 ± 17.4 ng/ml) were also elevated and served as internal
controls (Fig. 7
, A and B; Ref. 18). These findings suggest that the
early onset of gonadal tumors in the
p27-/-Inha-/- mice
results in early increases in activin A levels, earlier and more severe
cachexia, and a resultant earlier death of these mice as compared with
Inha-/- mice (see below).

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Figure 7. Serum Activin A Levels for Male (A) and Female (B)
Mice
Activin A levels for male 8-week-old wild-type (n = 8), 8 week-old
p27-/- (n = 8), 4-week-old Inha-/-
(n = 7), 8-week-old Inha-/- (n = 11), 12- to
14-week-old Inha-/- (n = 8), 4- to 5-week-old
p27-/-Inha-/-(n = 4), and 7- to
9-week-old p27-/-Inha-/-(n = 9) are
shown. Female 6-week-old wild-type (n = 7), 6-week-old
p27-/- (n = 9), 4-week-old Inha-/-
(n = 6), 6-week-old Inha-/- (n = 5), 12- to
14-week-old Inha-/- (n = 8), and 4- to 8-week-old
p27-/-Inha-/-(n = 4) are represented.
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Stomach and Liver Pathology Are Similar in
p27-/-Inha-/- and
Inha-/- Mice
The high serum activin levels in the
Inha-/- mice are a direct cause of severe liver
and stomach pathology (19, 34, 35). This pathology includes liver
atrophy (i.e. destruction of cells around the central vein
and diffuse areas of inflammation) and a block in differentiation of
several cell types in the glandular stomach including parietal cells.
Both livers and stomachs of male and female
p27-/-Inha-/- cachectic
mice had equivalent, but not more severe, pathology (data not shown)
than cachectic Inha-/- mice (19, 34, 35).
Thus, the earlier development of the gonadal tumors in the
p27-/-Inha-/- mice and
the accompanying elevated activin levels cause a similar but
earlier-appearing cachexia-like syndrome compared with mice lacking
only inhibin.
Cdk2 Activity Is Increased in
p27-/-inha-/-
Ovarian Tumors
Levels of p27 in a particular cell type correlate with
proliferative activity. The previous Northern blot and
immunohistochemical data (Figs. 1
and 2
) and the early appearance and
rapid progression of ovarian and testicular tumors in
p27-/-Inha-/- mice
(Figs. 3
and 4
) suggested to us that Cdk activity may be increased as
well. To test this, we first compared levels of gonadal Cdk4 mRNA in
p27-/- vs.
p27-/-Inha-/-
mice. Cdk4 mRNA expression was slightly higher in the testis of
p27-/-Inha-/- males
compared with p27-/- male mice (Fig. 8A
). Due to the low number of mice that
were generated, ovaries from
p27-/-Inha-/- females
were not available for a similar analysis. We next analyzed Cdk kinase
activity from the gonads of these mice to determine whether increased
Cdk activity was stimulating tumor progression. Unfortunately, attempts
to assay Cdk4 and Cdk6 kinase activity in mouse gonadal extracts were
not successful (our unpublished data). However, we were
able to analyze Cdk2 activity using in vitro kinase assays
from individual gonads of male and female wild-type and mutant mice. We
found that Cdk2 activity was increased in ovarian tumors from
5.5-week-old
p27-/-Inha-/- mice when
compared with ovaries from 4-week-old wild-type,
Inha-/-, and p27-/-
mice (Fig. 8B
). However, there appeared to be no difference in Cdk2
activity among testes isolated from wild-type,
Inha-/-, p27-/-, or
p27-/-Inha-/- mice
(Fig. 8B
). Taken together, the Northern blot analysis and in
vitro kinase assays suggest that inhibin and p27 predominantly act
through increased Cdk2 and Cdk4 activity in the ovaries, and through
increased Cdk4 activity in the testes.

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Figure 8. Northern Blot Analysis of Cdk4 Expression and Cdk2
Kinase Activity Assay in Mouse Ovaries and Testes
A, Testicular tumors from 5.5-week-old
p27-/-Inha-/- mice had higher expression of
Cdk4 mRNA when compared with testes from p27-/- mice. 18S
rRNA was used as control for RNA loading. B, Cdk2 activity was higher
in ovarian tumor protein extract from 5.5-week-old
p27-/-Inha-/- mice as compared with adult
ovaries from wild-type (WT), p27-/-, and
Inha-/- mice (top). There was no
detectable difference in Cdk2 activity among testes from wild-type
(WT), -/-, p27-/-, Inha-/-,
and p27-/- Inha-/- mice.
|
|
 |
DISCUSSION
|
---|
Cyclins, Cdks, and Cdk inhibitors have been shown to affect cell
proliferation and differentiation, and mouse models have greatly
contributed to our understanding of the functions of the genes in
vivo. Cyclin D2-deficient female mice are infertile due to the
lack of granulosa cell proliferation in the ovaries upon stimulation by
FSH; cyclin D2-deficient males have hypoplastic testes (11). Cdk
inhibitors have been found to be mutated or deleted in a wide range of
sporadic tumors, and a decrease in p27 expression has been shown to
correlate with poor prognosis in a number of cancers including human
colon and breast carcinomas (36, 37), hyperproliferative responses to
mitogenic stimulation, and pituitary hyperplasia and adenomas (38). It
is possible to study redundancy or cooperativity of gene functions by
generating double knockout mice. For example, mice lacking both tumor
suppressors p18 and p27 were larger in overall size than p27-deficient
mice alone and developed pituitary adenomas at a faster rate (38)
linking p18 and p27 in a cooperative cell proliferation control
pathway. Using a similar approach, we have generated mice lacking both
inhibin and p27 and show that these two tumor suppressors act together
to prevent gonadal tumor formation and progression. In this
study, p27-/-Inha-/-
mice develop tumors faster than mice lacking either inhibin or
p27 alone. Also, the earlier development of the tumors results in
earlier activin production, earlier symptoms of the activin-induced
cachexia syndrome, and earlier death, with all of the
p27-/-Inha-/- females
dying by 8 weeks and all of the males dying by 10 weeks.
The inhibin signaling pathway is not well characterized, and inhibin
receptors have only recently been identified (39, 40). Thus, it was
important for us to determine whether inhibin regulates cellular
proliferation by mechanisms resembling those of other TGFß family
members. We have shown that inhibin acts to negatively regulate p27
protein synthesis similar to the TGFßs. We have also shown that
cyclin D2, which is expressed at high levels in some human ovarian
granulosa cell tumors (11), is also increased slightly in the
Inha-/- ovarian tumors and markedly in the
Inha-/- testicular tumors. At this point, it is
unclear whether the cyclin D2 elevation is due directly to the absence
of inhibin or rather is caused by an elevation in FSH (a positive
regulator of cyclin D2 in granulosa cells) (11). Furthermore, we have
demonstrated that Cdk4 mRNA is modestly elevated in the
Inha-/- ovarian and testicular tumors, and Cdk2
kinase activity is increased in
p27-/-Inha-/- ovaries.
This has led us to formulate a model wherein inhibins, p27, FSH, cyclin
D2, and Cdk2 and/or Cdk4 interact to regulate granulosa cell and
Sertoli cell proliferation in vivo (Fig. 9
).

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Figure 9. A Model for the Role of Inhibin in Cell Cycle
Control
Inhibin is known to inhibit the synthesis and secretion of FSH. FSH has
been shown to stimulate cyclin D2 synthesis in granulosa cells, and
p27Kip1 blocks Cdk activity. We believe that inhibin
functions as a tumor suppressor by promoting the formation of inactive
cyclin-Cdk-CKI complexes. In the absence of inhibin, we have
shown that FSH serum levels increase, cyclin D2 levels increase, Cdk4
mRNA levels increase, and p27 levels decrease in granulosa cells and
Sertoli cells. Absence of both p27 and inhibin increases Cdk2 activity
in the ovary. Combined absence of p27 and inhibin is expected to
increase the formation of active cyclin D2/Cdk4 and cyclin E1/Cdk2
complexes and thereby stimulate granulosa cell and Sertoli cell
proliferation. The cooperative action of inhibin and
p27Kip1 is necessary to ensure appropriate control of the
cell cycle in granulosa cells and Sertoli cells, preclude tumorigenesis
or hyperplasia, and allow for terminal differentiation. FSH is a
modifier of tumorigenesis in male and female inhibin knockouts. It
is unclear whether the increase in cyclin D2 levels in inhibin
knockout mice is a direct effect of absence of inhibin or an indirect
effect of increases in FSH.
|
|
We propose that testicular hyperplasia and infertility in p27-deficient
male and female mice, respectively, and tumor formation in both
Inha-/- and
p27-/-Inha-/-
mice result from similar mechanisms, namely lack of cell cycle
arrest and incomplete differentiation. Withdrawal from the cell cycle
marks differentiation in most cell types, and p27 and inhibin
contribute to the induction and maintenance of cell cycle arrest in the
granulosa/Sertoli cell lineage. Our study shows that while p27
expression is present in normal differentiated cells of the ovary and
testis, protein levels were very low or not detectable in tumor cells
of the ovaries and testes in the Inha-/- mice
(Fig. 2
). This suggests that as tumorigenesis progresses in the absence
of inhibin, p27 levels decrease whereas p27 continues to be expressed
in the adjacent normal-appearing cells.
Interestingly, we found that
p27-/-Inha-/- mice
develop only bilateral tumors and that tumors in both sexes include a
disproportionately large granulosa cell component (as opposed to the
Sertoli cell component predominance in Inha-/-
tumors) (18). The combined absence of both p27 and inhibin may disrupt
important developmental programs that normally direct granulosa/Sertoli
cell fate and ensure gonadal genesis. Perhaps steroid hormones produced
by these tumors affect this process. Estrogen signaling is important in
maintaining granulosa cells, and ovaries of ER
ß double knockouts
show granulosa to Sertoli "transdifferentiation." Markers of
Sertoli cell differentiation, including Müllerian inhibiting
substance, SGP-2, and Sox9, may be differentially expressed in
tumors of Inha-/- mice vs.
p27-/-Inha-/-
(41). Also, compared with the Inha-/- tumors,
ovarian and testicular tumors from
p27-/-Inha-/- mice were
much more hemorrhagic. Perhaps an increase in angiogenic factors in the
absence of both p27 and inhibin also contributes to this hemorrhage and
aggressive tumor growth.
Cdk2 activity was found to be increased in ovarian tumors from
p27-/-Inha-/- mice as
compared with ovaries from wild-type, Inha-/-,
and p27-/- mice. However, there was no
detectable difference in Cdk2 activity from testes of wild-type,
Inha-/-, p27-/-, or
p27-/-Inha-/- mice. The
significance of this discrepancy between the sexes is not clear. It is
possible that this reflects differences in the tissue-specific
functions of this particular Cdk (42). It is also conceivable that
enhanced gonadal Cdk2 activity in
p27-/-Inha-/- females
is measurably deleterious to these mice, contributing to their early
and acute demise even in comparison to male double knockout
counterparts. The increased Cdk2 activity seen in
p27-/-Inha-/-ovarian
tumors could be due to a decrease in p27 and/or increases in cyclin D2
and Cdk2 protein levels.
Since 100% of the inhibin
knockout mice develop tumors, yet all
Sertoli or granulosa cells do not become tumor cells, undetermined
factors most likely affect a cells commitment to a tumorigenic path.
Overexpression of cyclin D2 and Cdk4, coupled with inactivation of p27,
could result in sequestration and functional deficiencies of other
Cip/Kip inhibitors. Our studies are important as they define for the
first time a link between inhibin and cell cycle proteins in the
pathogenesis of ovarian and testicular cancers.
 |
MATERIALS AND METHODS
|
---|
Generation of Mice and Genotyping
Generation of
-inhibin heterozygous
(Inha+/-), inhibin homozygous
(Inha-/-) (18), and p27 heterozygous
(p27+/-) (13) mice have been described.
Inha+/- mice were crossed with
p27+/- mice to generate offspring that were
homozygous mutant for both p27 and inhibin
(p27-/-Inha-/-). p27 is
the product of the Cdkn1b gene. The inhibin
genotype of
these offspring was determined using PCR amplification of tail DNA as
described (43). The p27 genotype was determined using either Southern
blot analysis or PCR. Primers used in the PCR reactions were specific
for the targeted p27 allele (primer1: 5'-AGGTGAGAGTGTCTAACGG-3';
primer2: 5'-AGTGCTTCTCCAAGTCCC-3'; primer3:
5'-GCGAGGATCTCGTCGTGAC-3'). All three primers were added to each
PCR reaction yielding either wild-type (130 bp), p27 mutant (450 bp),
or both bands.
Weight Data and Serum Analysis
Mice were weighed once per week for a period of 4 to 20 weeks.
To determine the significance of the survival rates, a confidence
interval test was used. For serum analyses, mice were anesthetized
using Metofane (Schering Plough Animal Health Corp.,
Union, NJ), and serum was collected into Microtainer tubes
(Becton Dickinson and Co., Franklin Lakes, NJ) using
cardiac puncture. Activin A (44) and FSH (43) mouse serum levels were
determined using ELISA methods. A one-way ANOVA test was used to
determine the statistical significance of these results. Values are
reported as mean ± SEM.
Morphological, Histological, and Immunohistochemical Analysis
Tissues were fixed overnight in 10% neutral buffered formalin,
embedded, sectioned, and stained with hematoxylin and eosin or periodic
acid Schiff as described (44). Morphological and histological
analyses were performed on at least 21 male and 5 female
p27-/-Inha-/- mice.
Immunohistochemical analyses of p27 expression in mouse ovaries (31)
and testes (30) were performed as described previously. Tissue sections
of 45 µm were used for these studies.
Northern Blot Analysis
Total RNA was isolated from testes and ovaries of wild-type,
p27-/-, Inha-/-, and
p27-/-Inha-/- mice
using the TRI-Reagent method (45). Fifteen micrograms of total RNA were
run in each lane, and blots were hybridized (45) with their respective
probes. Blots were stripped by boiling in a solution of 0.5x SSC and
1.0% SDS for 15 min and then rehybridized with an 18S rRNA control
probe. Signals detected on these blots were quantified using the
Molecular Dynamics, Inc. photodensitometer (Sunnyvale,
CA).
Kinase Assay
Individual mouse ovaries and testes were snap frozen in liquid
nitrogen and homogenized on ice with a Dounce homogenizer. Protein
lysates were harvested by centrifugation at 4 C at 10,000 rpm for 15
min. Protein concentration was determined as described previously (46).
Kinase activity was determined by immunoprecipitation of protein with
either polyclonal anti-Cdk2, anti-Cdk4, or anti-Cdk6 antibodies
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed
by Histone H1 (Roche Molecular Biochemicals, Indianapolis,
IN) phosphorylation assay for Cdk2, or RB (Santa Cruz Biotechnology, Inc.) phosphorylation assay for Cdk4 and Cdk6
(46). Signals detected on blots were quantified using the
Molecular Dynamics, Inc. photodensitometer (Sunnyvale,
CA).
 |
ACKNOWLEDGMENTS
|
---|
The authors would like to thank the following investigators for
their generous gifts: Dr. Steven Elledge for pBSmcyclinD1,
pBSmcyclinD2, pBSmcyclinD3, and pBSmp27; Dr. Charles Sherr for pBSmp15
and pBSmp16; and Dr. Hideyo Yasuda for pmCdk2 and pmCdk4. We also thank
Ms. Michele Park for excellent advice and critical discussions
regarding this manuscript and Dr. T. Rajendra Kumar for critical review
of the manuscript. We thank Drs. Anthony Lau and Hua Chang for
assistance with computer graphics and Ms. Shirley Baker for assistance
in manuscript preparation.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Martin M. Matzuk, M.D., Ph.D., Professor and Stuart A. Wallace Chair, Department of Pathology, One Baylor Plaza, Baylor College of Medicine, Houston, Texas 77030. E-mail:
mmatzuk{at}bcm.tmc.edu
This work was supported in part by NIH Grant CA-60651 (to M.M.M.) and a
National Research Service Award 1F32-CA-8174601 from the National
Cancer Institute (to S.C.C.). K.H.B. is a student in the Medical
Scientist Training Program and is supported in part by NIH Grant
T32GM-07330 and Grant T32EY07102 from The National Eye Institute.
Received for publication January 3, 2001.
Revision received February 23, 2001.
Accepted for publication March 8, 2001.
 |
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