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Departments of Medicine [R. E. C., K. A. M., M. S. T., R. V. B.], Pathology [K. A. M.], and Pharmacology [R. V. B.] University of Illinois at Chicago, and Chicago Veterans Administration Medical Center (West Side Division) [R. E. C., K. A. M., M. S. T., R. V. B.], Chicago, Illinois 60612, and National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, Maryland 20892 [J. F. B.]
Abstract
Little is known about the factors involved in regulating the appearance, or differentiation, of solid tumors including those arising from the colon. We herein demonstrate that the mitogen gastrin-releasing peptide (GRP) is a morphogen, critically important in regulating the differentiation of murine colon cancer. Although epithelial cells lining the mouse colon do not normally express GRP and its receptor (GRP-R), both are aberrantly expressed by all better differentiated cancers in wild-type C57BL/6J mice treated with the carcinogen azoxymethane. Whereas small tumors in both wild-type and GRP-R-deficient (i.e., GRP-R-/-) mice are histologically similar, larger tumors become better differentiated in the former but degenerate into more poorly differentiated mucinous adenocarcinomas in the latter. This alteration in phenotype is attributable to GRP increasing focal adhesion kinase expression in GRP-R-expressing tumors. Consistent with GRP acting as a mitogen, GRP/GRP-R coexpressing tumors in wild-type animals also contain more proliferating cells than those occurring in GRP-R-/- mice. Yet tumors are similarly sized in animals of either genotype receiving azoxymethane for identical times, a finding attributable to the significantly higher number of apoptotic cells detected in GRP/GRP-R coexpressing cancers. Thus, these findings indicate that although GRP is a mitogen, aberrant expression does not result in increased tumor growth. Rather, the mitogenic properties of GRP are subordinate to it acting as a morphogen, where it and its receptor are critically involved in regulating colon cancer histological progression by promoting a well-differentiated phenotype.
Introduction
The progression of normal colonic epithelium to adenocarcinoma is increasingly well established at the genetic and molecular level. Colon cancer is now appreciated to be attributable to both the mutational inactivation of a series of tumor suppressor genes (1, 2) as well as the activation of specific oncogenes (3) . However, the molecular derangements responsible for the pattern of cellular organization in colon cancer, or tumor grade, are not well understood.
Well-differentiated cancers phenotypically resemble the tissue type from which they originate, whereas poorly differentiated cancers display a chaotic and disorganized histology with few features of their tissue of origin. Most solid tumors display a near-uniform degree of differentiation, with increasing dedifferentiation inversely correlated with patient survival (4) . In contrast, adenocarcinomas of the colon are unusual insofar as multiple regions of varying differentiation are simultaneously present in larger tumors (5–7) . Yet the factor(s) regulating the differentiation of individual tumor cells in colon cancer, along with their contribution to cancer progression, have not been identified.
Neuropeptides have been widely studied as growth factors in many different cancer types, including those arising from the colon. Recent work, however, suggests that some of these peptide hormones may also play a role in the differentiation of normal and malignant tissues. GRP3 is such a hormone that has been shown previously to be important in the normal development of selected tissues such as the lung (8, 9) , as well as plays a role as a growth factor when aberrantly secreted by SCCL cells (10) . Within the gastrointestinal tract, GRP is normally found only in enteric nerve terminals (11–13) , where it acts to regulate intestinal motility by binding to a specific seven-transmembrane-spanning, G protein-coupled receptor (14) . In contrast, GRP and its receptor (GRP-R) are not normally expressed by epithelial cells lining the colon but are aberrantly expressed by many human colon cancers and cancer cell lines (reviewed in Ref. 15 ). All colon cancer cell lines expressing GRP-R proliferate when this receptor is activated (16–20) . Thus, GRP is commonly held to act as an autocrine growth factor when it and its receptor are aberrantly expressed. Yet our recent study of human colon cancers suggested that although GRP has mitogenic properties, this peptide hormone might have a more important role acting in an autocrine fashion as a morphogen (7) . Specifically, we found for the first time that all well-differentiated histological regions within colon cancer, irrespective of tumor stage, coexpressed both GRP and GRP-R. In contrast, GRP/GRP-R coexpression was never detected in any colon cancer region that was poorly differentiated or in any metastatic lesion. Thus, this study suggested that aberrant GRP/GRP-R expression might be important in maintaining colon cancers in a well-differentiated state.
To evaluate this hypothesis directly, we studied azoxymethane-induced colon cancers in wild-type GRP-R+/+ C57BL/6J mice and their recently created nullizygous GRP-R-/- counterparts (21) . Similar to what we have described in humans (7) , we show that although GRP and GRP-R are not normally expressed by epithelial cells lining the colon, both are aberrantly expressed by all better differentiated cells within cancers in wild-type mice. Significantly, colon cancers in wild-type mice became better differentiated as they increased in size, whereas tumors in GRP-R-/- mice underwent progressive dedifferentiation. Our findings therefore provide direct evidence that GRP is a morphogen, the first such agent identified to regulate colon cancer differentiation.
Results
Noncolonic Effects of AOM.
Although AOM has been well studied in rats (reviewed in Ref.
22
), no published data exist reporting the effects of this
carcinogen in this strain of mouse. In preliminary experiments, we
attempted using higher concentrations of AOM (15 µg/mg) given weekly
for only 2 weeks. Unfortunately, this dose did not induce the formation
of colon cancers (n = 30; data not shown) but often
resulted in the development of fulminant hepatic failure, as we have
reported previously (23)
. We therefore treated mice with
the alternative protocol of lower dose but weekly s.c. injections of
AOM (7.5 µg/mg). Similar to what has been reported previously for its
chemical precursor dimethylhydrazine (24, 25)
, even
this lower dosing regimen resulted in significant side effects
including intestinal ischemia and peritoneal hemangioendothelioma
formation. The former occurred in 
25% of animals and could be
recognized by the absence of expected weight gain, whereas the latter
occurred in 30%. The large cystic peritoneal hemangioendotheliomas
could be identified by abdominal palpation or, because they often bled
internally, by the presence of white paws reflective of anemia. Both
complications occurred equally in wild-type and
GRP-R-/- mice and with an incidence similar to
that which has been recorded previously for dimethylhydrazine
(24, 25)
. Both conditions contributed to the increasing
difficulty in obtaining tumors in animals who received AOM for >31
weeks, whereas it was impossible to maintain animals for >35 weeks.
Effect of AOM on Colonic Epithelia.
AOM caused equivalent increases in colonic crypt cell proliferation
rates in both wild-type and GRP-R-/- mice.
Specifically, whereas basal crypt proliferation rates were 5
mitoses/HPF/animal in both wild-type and
GRP-R-/- mice, AOM administration approximately
doubled the number of metaphases/HPF/animal observed for as long as
mice received this drug (Table 1
). There was no statistically significant difference in the
proliferation rates of nonmalignant crypts between wild-type and
GRP-R-/- mice for as long as they continued to
receive this drug (Table 1)
.
|
. However, and consistent with prior studies demonstrating that GRP
is a mitogen, tumors developing in wild-type mice had a significantly
higher proliferation index (19.1 ± 1.9 metaphases/HPF/tumor) than
those arising in GRP-R-/- mice (6.5 ± 2.0
metaphases/HPF/tumor; P < 0.05; Table 1
).
Evaluation of Tumors in Wild-Type Mice.
Similar to what we have described previously in humans
(7)
, GRP/GRP-R are not expressed by normal-appearing
epithelial cells lining the colons of wild-type mice (Fig. 1A
). In contrast, both proteins are expressed by all better
differentiated adenocarcinomas developing in wild-type mice (Fig. 1, B and C)
. Similar to what has been observed in
human colon cancer (5–7)
, murine colonic tumors were
histologically nonhomogeneous (Fig. 1D
). Additionally, the
amount of GRP and GRP-R detected immunohistochemically in colon cancers
developing in wild-type mice was directly related to the degree of
tumor cell differentiation (Table 2
). Specifically, GRP/GRP-R expression was essentially undetectable in
the most poorly differentiated tumor regions and was 6–7-fold
higher in well-differentiated tumor cells as compared with moderately
poorly differentiated tumor cells (Table 2)
. Thus, colon cancers in
AOM-treated mice are similar to tumors arising in humans insofar as
both are histologically heterogeneous, with GRP/GRP-R expression
strongly associated with the degree of tumor cell differentiation.
|
|
, top panel). In this manner, we could determine that despite
the fact that GRP-R-/- mice were incapable of
expressing this protein, no difference in histology could be detected
between small and presumably younger tumors developing in mice of
either genotype. For animals of either genotype, small tumors were
primarily moderately differentiated, although some heterogeneity in
overall histopathology was apparent (Fig. 2
, bottom left
panel). In contrast, larger and presumably older cancers in
wild-type mice became significantly better differentiated (Fig. 2
,
bottom right panel; Fig. 3A
), whereas tumors in GRP-R-/- mice
progressively dedifferentiated (Fig. 2
, bottom right panel;
Fig. 3B
), than observed in the smaller/earlier lesion.
|
|
) that were not
observed in tumors developing in wild-type mice (Fig. 3C
).
Although small tumors (i.e., <7.5 mg) from
GRP-R-/- mice did not show evidence of mucinous
glands, 15% of intermediate-sized tumors (7.5–15.0 mg) and 80%
of larger tumors (>15.0 mg) developed these structures.
Tumor Proliferation Rates Vary with the Extent of GRP/GRP-R
Expression.
We investigated whether the change in histomorphology between small and
large cancers from wild-type and GRP-R-deficient mice could be
attributable to alterations in tumor cell proliferation. Although
better differentiated tumors had higher proliferation rates in both
wild-type and GRP-R-/- mice, these rates were
significantly higher in tumors expressing increased amounts of GRP and
GRP-R (Fig. 4
). Thus, GRP/GRP-R expressing cancers possess a growth advantage that
might partially account for their histological improvement as they
increased in size in wild-type mice (Fig. 2)
. The better differentiated
tumor regions in GRP-R-/- mice also contained
more proliferating cells than less well-differentiated tumors (Fig. 4)
,
suggesting the presence of other differentiation-associated mitogens.
These factor(s), however, were not sufficient to provide these
histological regions with a significant growth advantage because tumors
in GRP-R-deficient mice progressively dedifferentiated with increasing
size (Fig. 2
, right panel). Intriguingly, and despite higher
numbers of proliferating cells in tumors from wild-type mice as
compared with tumors in GRP-R-/- mice, no
significant difference in tumor size could be detected (Table 1)
.
|
3-fold increase in apoptosis
was similar to the degree to which tumor cell proliferation was
increased in cancers developing in wild-type as compared with
GRP-R-/- mice (Table 1)
. Thus, the increase in
overall cell proliferation observed in tumors from wild-type mice is
balanced by a similarly increased rate of cell apoptosis, accounting
for the similar size of cancers developing in mice of either genotype
when exposed to AOM for identical lengths of time.
GRP Increases FAK Expression.
To examine a potential mechanism whereby GRP-R could influence tissue
morphology, we investigated expression of FAK. To do this, we performed
quantitative immunohistochemistry to determine the amount of FAK
present in each region of distinct differentiation contained within
each tumor. FAK expression was significantly lower in tumors from
knockout mice as compared with those from wild-type mice (Fig. 5
, left panel), with higher FAK levels seen in better
differentiated tumor regions. This difference was observed even when
regions of similar histology were compared between wild-type and
knockout mice (Fig. 5
, left panel).
|
, right panel). Yet in
contrast to what was observed for FAK, similar high levels of c-Src
were detected in better differentiated cancer regions in tumors
developing in mice of either genotype. Thus, these data suggest, but
admittedly do not prove, that FAK expression may not simply be a
function of tumor cell differentiation but rather that FAK expression
might be regulated as a function of GRP receptor expression. Discussion
GRP and its pharmacological homologue bombesin are well known to cause the proliferation of a wide range of cell types including those derived from colon cancers (16–20) , normal human bronchial epithelial cells (27) , pancreatic adenocarcinomas (28) , and a number of breast cancer cell lines (29–31) . Arguably the best studied are those derived from human SCCL, most of which secrete GRP, express GRP-R, and respond to exogenous agonist by proliferating in a concentration-dependent manner (10, 32, 33) . The action of GRP as an autocrine growth factor in these cells is specific as antibodies and antagonists block their growth both in vitro and in vivo (10) . Similarly, antagonists can attenuate the growth of gastrointestinal cancer cell lines expressing GRP-R (16–20) . Thus, these studies clearly demonstrate that GRP causes cell proliferation. Yet increasing evidence suggests that the mitogenic properties of GRP may be subordinate to it acting as a morphogen in the few cancers in which this peptide hormone has been studied carefully.
Most, but not all, SCCLs express GRP-R. Recent studies indicate that GRP-Rs are only present in well-differentiated (or typical), but not in poorly differentiated (or variant), small cell carcinomas (34–36) . Likewise, GRP-Rs are not normally found in prostatic tissues (37) but are up-regulated in prostate cancer, with highest receptor concentrations observed in well-differentiated as compared with poorly differentiated tumors (38) . Finally, we showed recently in human colon cancer that GRP/GRP-R coexpression occurs independently of tumor stage and is related to the degree of tumor differentiation. Although colon cancers differ from most solid tumors insofar as they are histologically heterogeneous, we observed that these two proteins were only coexpressed by well-differentiated tumor regions whereas poorly differentiated tissues never coexpressed GRP/GRP-R (7) . In aggregate, therefore, these observations suggested the possibility that GRP and its receptor might be important in regulating the differentiation of various cancers.
The role for GRP acting as a morphogen in colon cancer is conclusively demonstrated in the present study of wild-type C57BL/6J mice and GRP-R-/- mice. We show that whereas small, early tumors in mice of either genetic background were moderately differentiated, they became progressively better differentiated in wild-type mice but degenerated into poorly differentiated mucinous adenocarcinomas in GRP-R-/- mice. This is particularly interesting because mucinous adenocarcinomas are relatively rare in humans, where they are associated with a poor prognosis (39) and have a higher metastatic potential (40) , than nonmucinous tumors.
Yet our data also support GRP acting to cause cell proliferation,
albeit weakly and without affecting overall tumor size. Tumors
developing in wild-type mice contained 3-fold more metaphase
complexes than those in GRP-R-/- mice (Table 1)
, with the degree of cell proliferation related to the amount of
GRP/GRP-R expressed (Fig. 4)
. Yet this increase in proliferation was
almost precisely balanced by an increase in cellular apoptosis rates in
tumors developing in wild-type as compared with
GRP-R-/- mice. As a consequence, there was no
difference in the size of tumors developing in mice of either genotype
(Table 1)
. Indeed, if anything, mean tumor weight was greater in
GRP-R-/- mice than in wild-type mice, although
this difference was not statistically significant.
To identify a potential mechanism whereby GRP/GRP-R could act to
regulate cancer appearance, we studied tumor cell FAK expression
because recent studies have suggested that this enzyme is important in
regulating the differentiation of cancers arising in the human
gastrointestinal tract (41–43)
. Specifically, whereas
epithelial cells lining the human gastrointestinal tract do not
normally express FAK, FAK expression is observed in cancers arising
from the stomach and colon (43)
. Although not directly
commented upon in that particular study, FAK was only present in
well-differentiated regions within any individual colon cancer. This
observation is supported by recent in vitro investigations,
where FAK expression in human colon cancer cell lines increased as they
became better differentiated (41, 42)
. This is
particularly important because GRP has been shown to increase FAK
expression in a number of cell lines (44–46)
. In the
current study, we showed that FAK expression was significantly
decreased in tumors from GRP-R-/- mice, whereas
such expression varied as a function of differentiation and GRP/GRP-R
expression in tumors from wild-type mice (Fig. 5)
. This finding raises
the possibility that the morphogenic capabilities of GRP may be
mediated via GRP-R activation of FAK.
Two limitations of this study are that we only assessed the contribution of the GRP-R to tumor cell differentiation, and that this study did not determine the contribution(s) GRP/GRP-R make to other aspects of tumor behavior, including metastasis and animal survival/outcome. With respect to the first criticism, there are at least three different receptors in the bombesin family, and there is some evidence that subtypes other than the GRP-R can be up-regulated in selected cancers, such as those developing in the lung (47) . However, we are unaware of any prior study systematically investigating the expression of non-GRP bombesin receptor subtypes in colon cancer. Although we do not exclude the possibility that other bombesin receptors may also be important in regulating colon cancer differentiation, the present data do show that expression of at least the GRP-R subtype regulates tumor grade.
With respect to the second criticism, it should be observed that this
study was not designed to study the ability of GRP/GRP-R to alter tumor
behavior, such as metastasis or alter animal outcome, when aberrantly
expressed in colon cancer. This is attributable to the fact that AOM,
similar to its chemical precursor dimethylhydrazine, is associated with
significant side effects including intestinal ischemia and peritoneal
hemangioendotheliomas. These two complications occurred in 50% of
mice of either genotype treated with AOM and developed with increasing
time of exposure to this carcinogen. Because tumors did not reliably
develop until mice had been exposed to AOM for >25 weeks, it simply
was not possible to determine whether the ability to express GRP-R
altered important clinical end points, such as tumor metastases and/or
animal survival. Because tumor differentiation inversely correlates
with clinical outcome in cancers that are histologically homogeneous
(4)
, our findings suggest the possibility that GRP/GRP-R
expression may be beneficial because these proteins promote the
assumption of a well-differentiated phenotype. As such, aberrant
GRP/GRP-R expression may prove to be of favorable prognostic
significance.
Materials and Methods
Materials.
Wild-type C57BL/6J and GRP-R-/- mice were
obtained by establishing breeding pairs as described previously
(21)
. To minimize potential hormonal influences, only male
mice were studied. Because the murine GRP-R gene is located
on the X chromosome (14)
, male heterozygous (+/-) animals
could not be evaluated. The University of Illinois at Chicago
Animal Care Committee approved this study, and all care was provided in
accordance with institutional guidelines. The source and use of
antibodies for GRP and GRP-R were as described previously
(7)
; antibody for FAK was from Upstate Biotechnology (Lake
Placid, NY); antibody for c-Src was from Santa Cruz Biotechnology
(Carpinteria, CA), whereas all other immunohistochemical materials were
from Dako (Santa Cruz, CA). All other reagents including AOM and
vincristine were from Sigma Chemical Co. (St. Louis, MO).
Colon Cancer Induction and Determination of Cell Proliferation.
Wild-type and GRP-R-/- mice received weekly
s.c. injections of AOM at a dose of 7.5 µg/mg in normal saline as
described previously (22)
. Crypt and tumor proliferation
rates were determined by administering 1 µg/mg vincristine 2 h
prior to sacrifice. The stathmokinetic effects of vincristine are
attributable to its arresting dividing cells in metaphase so that they
can be readily identified and counted (48)
. Crypt
proliferation rates were determined by staining a segment of colon in
Carnoy’s fixative for 4 h, dissecting out 20 separate colonic
crypts, and counting the total metaphases as described previously
(49)
. The tumor proliferation index was determined by
averaging the number of metaphases for 10 separate HPFs (x400),
scoring only tumor sections meeting the criteria of Baak
(50)
.
Digital Image Capture and Assessment of Tumor Differentiation
Status.
Digital images were acquired using a Microlumina Ultra Resolution
Scanning Digital Camera [3380 x 2700 pixels (Leaf Systems, Fort
Washington, PA)] attached to a Nikon E600 microscope system.
Assessment of tumor differentiation was performed using a five-grade
classification system based on what has been described previously
(51)
. Well-differentiated tumors were defined by the
presence of well-formed glands containing malignant columnar cells
displaying small regular nuclei. The complete absence of gland
formation or the presence of bizarrely shaped glands identified poorly
differentiated tumors. Moderately differentiated tumors possessed
well-formed glands, but the cells were less columnar or frankly
cuboidal, with reduced cell polarity and more dysplastic nuclei than
those observed in well-differentiated tumors. The intermediate grades
of moderately well and moderately poor shared histological features of
both well and moderate differentiation, or of moderate and poor
differentiation, respectively.
Three representative 5-µm-thick sections were taken from the part of each tumor containing the largest cross-sectional area. After staining with H&E according to standard techniques, the entire tumor section was photographed at x100 using a 3380 x 2700 pixel Microlumina Ultra Resolution digital scanning camera (Leaf Systems). The number of pixels in each region of indicated histology in the entire tumor was determined using the Magic Wand function in Photoshop (Adobe, San Jose, CA) and then expressed relative to the area (i.e., in pixels) occupied by the entire tumor section. Each section was evaluated separately by two individuals (R. E. C., K. A. M.) at different times, and their values were averaged.
Quantitative Immunohistochemistry.
All immunohistochemistry was performed using a standard three-stage
indirect immunoperoxidase technique on 5-µm-thick tissue sections. In
all instances, sections were stained with the indicated antibody,
incubated with biotinylated anti-rabbit IgG, followed by Streptavidin
conjugated to horseradish peroxidase. Incubating slides with Liquid DAB
Substrate-Chromogen System for 2 min identified bound antibody. All
sections were counterstained using a 50% dilution of Gill’s
hematoxylin.
Chromogen quantification was performed as described previously
(52)
. In brief, chromogen abundance was determined by
calculating the cumulative signal strength within three regions for
each separate histological region/tumor. For any image file of
dimensions N1 by
N2 pixels, and where
n1 and
n2 identify the specific position of
the data within the digital image file, the cumulative signal strength
(or mathematical energy, E) is defined as:
 |
Tumor Apoptosis Rates.
Cellular apoptosis rates were determined using the Dead End
Colorimetric Apoptosis Detection System (Promega Corp., Madison, WI), a
modified terminal deoxynucleotidyl transferase-mediated nick end
labeling assay. Briefly, tumor sections were permeabilized using
proteinase K, followed by labeling of DNA strand breaks with
biotinylated nucleotide mix. After quenching background with 0.3%
H2O2, tissues were treated
with Streptavidin HRP and developed by exposing to
3,3'-diaminobenzidine for 10 min. Apoptotic cells/HPF (x400) were
counted and averaged for a minimum of 10 HPFs/tumor.
Footnotes
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.
1 This work was supported by NIH Grant CA-80360
(to R. E. C.) and NIH Grant DK-51168 and a Veterans Affairs Merit
Review Award (to R. V. B.). 
2 To whom requests for reprints should be
addressed, at Department of Medicine, University of Illinois at
Chicago, 840 South Wood Street (M/C 787), Chicago, IL 60612. Phone:
(312) 666-6500, extension 3439; Fax: (312) 455-5877; E-mail: rvbenya{at}uic.edu
3 The abbreviations used are: GRP,
gastrin-releasing peptide; GRP-R, GRP receptor; AOM, azoxymethane; HPF,
high-powered field; FAK, focal adhesion kinase; SCCL, small cell cancer
of the lung.
Received for publication 3/16/00. Revision received 5/12/00. Accepted for publication 5/15/00.
References
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) within the box straddling the lower panels
identifies the mean differentiation, with closed symbols
for wild-type tumors and open symbols for GRP-R knockout
tumors. Data expressed are means; bars, SE.
) and GRP-R-/- (
) mice.
Individual tumor cells were manually counted and graded in 15 separate
tumors of varying size. Metaphase complexes were identified using the
criteria of Baak 