Aberrant expression of gastrin-releasing peptide and its
receptor by well-differentiated colon cancers in humans
Robert E.
Carroll1,2,
Kristina
A.
Matkowskyj1,2,
Subrata
Chakrabarti3,
Thomas J.
McDonald4, and
Richard V.
Benya1,2
1 Department of Medicine,
University of Illinois at Chicago, and
2 Chicago Veterans Affairs Medical
Center (West Side Division), Chicago, Illinois 60612; and Departments
of 3 Pathology and
4 Medicine, University of Western
Ontario, London, Ontario, Canada N6A 5A5
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ABSTRACT |
Epithelial cells
lining the adult human colon do not normally express gastrin-releasing
peptide (GRP) or its receptor (GRPR). In contrast, approximately
one-third of human colon cancers and cancer cell lines have been shown
to express GRP-binding sites. Because GRPR activation causes the
proliferation of many cancer cell lines, GRP has been presumed to act
as a clinically significant growth factor. Yet GRP has not been shown
to be expressed by colon cancers in humans nor has the effect of GRP
and/or GRPR coexpression on tumor behavior been investigated.
We therefore determined GRP and GRPR expression by immunohistochemistry
in 50 randomly selected colon cancers resected between 1980 and 1997, all 37 associated lymph node and liver metastases, and 20 polyps. Tumor
sections studied were those that contained the margin and adjacent
nonmalignant epithelium. Overall, 84% of cancers aberrantly expressed
GRP or GRPR, with 62% expressing both ligand and receptor, whereas
expression was not observed in adjacent normal epithelium. Consistent
with the previously established mitogenic capabilities of GRP, tissues coexpressing GRP and GRPR were more likely to express proliferating cell nuclear antigen than tissues not expressing both ligand and receptor. Yet GRP/GRPR coexpression was seen with equal frequency in
stage A as in stage D cancers and was only detected in 1 in 37 metastases. Furthermore, Kaplan-Meier analysis did not reveal any
difference in patient survival between those whose tumors did or did
not express GRP/GRPR. In contrast, GRP/GRPR coexpression was found in
all well-differentiated tumor regions, whereas poorly differentiated
tissues never coexpressed GRP/GRPR. Overall, these data indicate that,
although GRP is a mitogen, it is not a clinically significant growth
factor in human colon cancers. Rather, the strong association of
GRP/GRPR coexpression with tumor differentiation raises the possibility
that these proteins primarily act in vivo as morphogens.
adenocarcinoma; bombesin; mitogen; morphogen
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INTRODUCTION |
GASTRIN-RELEASING PEPTIDE (GRP) is the mammalian
homologue of bombesin, a tetradecapeptide originally isolated from the
skin of the frog Bombina bombina (8).
GRP and/or bombesin is important in regulating a
number of normal physiological processes within the gastrointestinal
(GI) system, including modulating secretion of the exocrine pancreas
and other GI peptide hormones as well as altering smooth muscle
contractility and intestinal transit (18). GRP mediates its effects in
humans by binding to a specific seven transmembrane-spanning G
protein-coupled receptor that has been cloned and sequenced (6).
Although GRP receptors (GRPR) are found on intestinal smooth muscle
cells (46), they are not normally expressed by epithelial cells lining
the human colon (9). In contrast, two studies each with relatively few
patients showed that 24% (32) to 40% (36) of surgically resected
colon cancers aberrantly expressed GRP binding sites. Because GRPR
expression has been associated with the proliferation of all human
cancer cell lines in which it is expressed, including those derived
from the lung (7, 24-26), breast (15), prostate (30, 38), and
colon (12, 13, 35, 37), GRP has been proposed to act as an autocrine growth factor. However, except for small-cell lung cancer cell lines
(7), GRP has yet to be shown to be present in any human cancer or
cancer cell line. Furthermore, the clinical contribution of GRP
and/or GRPR expression by any human cancer has not been elucidated.
To specifically investigate the extent and significance of GRP/GRPR
expression in adenocarcinomas of the human colon, we evaluated 50 randomly selected cancers, along with adjacent normal tissue, all 37 associated metastases, as well as 20 polyps. We herein demonstrate that
aberrant expression of GRP and GRPR is common but that our evidence
does not support this peptide hormone acting as a clinically
significant growth factor. Rather, because GRP/GRPR coexpression is
found only in the most well-differentiated tumor regions, irrespective
of cancer stage, we propose the novel hypothesis that GRP may act in an
autocrine fashion as a morphogen in human colon cancer.
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METHODS |
Materials.
Ammonium hydroxide, Harris' modified hematoxylin with acetic acid,
hydrochloric acid, 10% formalin (wt/vol), Permount, and xylene
(histology grade) were purchased from Fisher Scientific (Pittsburgh,
PA). Absolute and 95% anhydrous ethanol were purchased from Pharmco
Products (Brookfield, CT). PBS was purchased from GIBCO BRL (Grand
Island, NY). Unless otherwise indicated, all immunohistochemical
reagents including large volume DAKO LSAB(R)2 kit and DAKO liquid DAB
substrate-chromogen system were from DAKO (Carpenteria, CA), and all
other reagents were obtained from Sigma (St. Louis MO). All reagents
and solvents were used at reagent-grade purity.
GRP expression was determined using a polyclonal rabbit anti-porcine
whole peptide antibody (11), whereas GRPR expression was evaluated
using a polyclonal rabbit anti-peptide antibody (20). The latter
antibody (generously provided by Dr. J. Battey, National Institute of
Diabetes and Digestive and Kidney Diseases, Bethesda, MD) was designed
using a sequence corresponding to the distal third intracellular
loop of the mouse and human GRPR (CVEGNIHVKKQIESRKR). In all instances,
antibodies were used at a 1:250 dilution, predetermined as optimal by
dilution titration using human small cell lung carcinoma tissue as a
positive control. Proliferating cell nuclear antigen (PCNA) was from
Boehringer Mannheim (Indianapolis, IN) and used at 1:200 as directed by
the manufacturer.
Patient and tumor block selection.
The Chicago Veterans Affairs Medical Center (CVAMC) (West Side Branch)
Gastrointestinal Tissue Bank contains surgical pathology specimens from
all patients undergoing surgical resection between 1980 and 1997. From
this data base, we used a random number generator to select hospital
identification numbers to identify 50 patients undergoing colectomy
between 1990 and 1997 [10 Dukes stage A, 10 stage B1, 10 stage
B2, 10 stage C, and 10 stage D, along with all associated
cancer-containing lymph nodes (n = 30)
and liver or peritoneal metastases (n = 7)]. An additional 20 randomly selected polyps resected either
endoscopically (n = 18) or surgically
(n = 2) were included in the study
sample (5 hyperplastic, 5 tubular, 5 villous, 5 villous with high-grade
dysplasia). Pertinent clinical information was obtained from the
Veterans Information System and Technical Architecture computer system.
The University of Illinois-CVAMC combined Institutional Review Board
approved this study.
Tissue preparation and classification.
Specimens previously fixed in paraffin-embedded blocks were freshly
sectioned at a thickness of 5 µm and mounted on
poly-L-lysine-coated slides.
Slides were heat fixed at 75°C for 30 min to promote adherence and
stained with hematoxylin and eosin using standard techniques (2).
Blocks including the tumor margin and containing both cancer and
adjacent normal mucosa were identified and used for immunohistochemical
analyses. In this fashion, all slides contained regions of nonmalignant
epithelium and thus possessed an internal negative control.
Immunohistochemistry.
For GRP and GRPR detection, a standard three-stage indirect
immunoperoxidase technique was used (17). Briefly, fixed tissue sections were rehydrated in graded alcohols and then rinsed in a
running water bath. To quench endogenous peroxidase activity, slides
were preincubated in 3% hydrogen peroxide in a light impermeable chamber. After they were washed in PBS, slides were incubated in
blocking solution [5% skim milk (vol/vol) and 0.15%
H2O2
(vol/vol) in deionized water]. After slides were washed again in
PBS, primary antibody was applied, and the tissue was incubated for 1 h
in a humidity chamber (control tissues were processed similarly except that primary antibody was not applied). To evaluate for PCNA
positivity, we treated slides similarly except that primary antibody
was incubated overnight at 4°C. After being washing again in PBS,
the tissues were incubated with biotinylated anti-rabbit IgG (DAKO) for
15 min. After they were washed in PBS, the slides were incubated with
streptavidin conjugated to horseradish peroxidase (DAKO) for 15 min and
washed again in PBS buffer. Incubating slides with the liquid DAB
substrate-chromogen system (DAKO) for 5 min identified bound antibody.
After a final wash in PBS and distilled water, the slides were
counterstained with either Gill's or Harris' modified hematoxylin for
4 min, dehydrated in graded alcohols, and mounted with a coverslip
using Permount.
Microscopic analysis.
All specimens were evaluated using a Nikon E600 microscope with
Axioplan objectives connected to a Microlumina ultraresolution scanning
digital camera [3,380 × 2,700 pixels (Leaf
Systems, Fort Washington, PA)].
Assessment of tumor differentiation was performed using a three-grade
classification system as previously described (21). 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 geographic extent of staining in each section was determined and
scored independently by three investigators (Benya, Carroll, and
Matkowskyj). Each observer evaluated 10 or more (10+)
high-power fields (hpf) containing tumor as well as an equal number of
normal mucosal fields at ×400 magnification. Fields were
scored as 1+ = <25%, 2+ = 25-50%, 3+ = 50-75%, and
4+= >75% cells/hpf positive for chromogen.
Statistical analysis.
Statistical evaluations were performed using StatView (Abacus Concepts,
Berkeley, CA). Survival distribution was estimated by Kaplan-Meier
analysis; the data were stratified by tumor stage, with censored and
uncensored observations segregated in calculating the hazard function.
Comparisons between groups were otherwise performed by ANOVA or
2 analysis. All data in this
paper are reported as means ± SE.
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RESULTS |
Clinical characteristics of the patient population.
Fifty patients with adenocarcinoma of the colon were randomly selected
by stage from the CVAMC Gastrointestinal Tissue Bank (Table
1). Consistent with a CVAMC population, all
patients were male and of relatively advanced age (mean = 68.9 ± 3.4 yr; range 36-87 yr). Postsurgery, only five patients were lost
to follow-up (at 1, 1.5, 2, 12, and 30 mo postsurgery).
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Table 1.
Clinical characteristics of 50 patients with adenocarcinoma of the
colon evaluated by immunohistochemistry
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Overall, 17 patients died during follow-up (2 stage A, 3 stage B1, 1 stage B2, 2 stage C, and 9 stage D). In 6 of 17 patients, death was
unrelated to their underlying malignancy. One patient each died from
respiratory failure associated with pneumonia (stage A), advanced
chronic obstructive pulmonary disease (stage A), congestive heart
failure associated with coronary artery disease (stage B1), and acute
myocardial infarction (stage B1). In an additional two instances,
deaths were due to the development of new lung cancers unrelated to
their colon primaries [1 small cell lung cancer (stage B1), 1 non-small cell lung cancer (stage D)]. No patient showed evidence
of colon cancer recurrence or progression.
In contrast, 11 of 17 deaths were directly attributable to colon cancer
recurrence or progression. Eight of these deaths were in patients that
had metastatic disease at the time of their initial presentation (i.e.,
stage D). In the three other patients, death was directly due to tumor
recurrence, including one each from malignant bowel obstruction (stage
B2), brain metastasis (stage C), and tumor cachexia associated with
peritoneal carcinomatosis (stage C).
Antibody sensitivity and specificity.
Because small cell lung cancer cells have previously been shown to
express GRP and GRPR, we used paraffin blocks containing this tumor to
establish the optimal dilutions for immunohistochemistry. Optimal
antibody dilution was determined to be 1:250 by dilution titration to
stain tumor tissue but not adjacent noncancerous tissue (Fig.
1,
A-C).

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Fig. 1.
Determination of gastrin-releasing peptide (GRP) and its receptor
(GRPR) antibody specificity in resected human tissues. Optimal dilution
of GRP and GRPR antibody for use in this study was established by using
small cell lung cancer (SCCL) tissue as a positive control. Positive
staining is seen as the brown color indicated by arrowheads.
A: GRP antibody applied to a SCCL
tumor at 1:250 dilution; strong staining of the tumor cells (arrowhead)
is shown. B: SCCL tissue treated
similarly except for the absence of primary antibody as a negative
control. C: GRPR antibody applied to a
SCCL tumor at 1:250 dilution. Similar to
A, tumor cells stain positively
(arrowhead). Treatments of normal human colon epithelium
(D) and colon removed for ischemic
colitis (E) or diverticulitis
(F) with GRP antibody at the same
dilution are also shown. Similar lack of positivity for these colonic
tissues was also observed when treated with GRPR antibody. Tissues were
processed as described in METHODS.
Magnification = ×1,000
(A-C)
and ×400
(D-F).
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The specificities of the antibodies for GRP (11) and GRPR (20) have
been previously shown. We further evaluated antibody specificity by
evaluating a number of negative control tissues not suspected to
express GRP or GRPR. Because we have previously shown that epithelial
cells lining the colon do not normally express GRPR mRNA (9), we tested
our two antibodies on normal colon tissue (Fig.
1D) and on colons resected for
diverticulitis (Fig. 1F) and
ischemic colitis (Fig. 1E). Normal
and diseased colons devoid of underlying malignancy did not show any
evidence of immunostaining for either GRP or GRPR (Fig. 1,
D-F).
Thus GRP/GRPR expression does not occur in nonmalignant disorders nor
does inflammation per se allow for nonspecific binding of the primary antibodies.
Aberrant GRP/GRPR expression in cancer.
In contrast to normal colonic epithelium, markedly increased ligand and
receptor immunostaining was observed in the majority of the
adenocarcinomas we evaluated. Overall, 84% of tumors expressed either
GRP or GRPR, with 35 of 50 (70%) cancers immunopositive for GRP and 38 of 50 (76%) for GRPR (Table 2). This
staining was predominantly cytoplasmic for both (Fig.
2,
A-C).
In contrast, GRP/GRPR expression was not detected in normal tissues
adjacent to the tumor margin (Fig.
2D). Approximately equal numbers of stage A tumors and stage D tumors expressed GRP and/or GRPR
(50-90% vs. 60-70%) (Table 2). Because we studied
consecutive histological sections for both ligand and receptor, we
could assess whether the same tumor regions expressed both proteins.
Overall, 31 of 50 (62%) tumors expressed both GRP and GRPR, with both
proteins always coexpressed in the same histological area. In contrast, 4 of 50 (8%) tumors expressed only GRP and 7 of 50 (14%) expressed only GRPR. In only 8 of 50 (16%) tumors was ligand or receptor not
detected at all (Table 2). Thus aberrant GRP/GRPR expression is common in adenocarcinomas of the colon but show no evidence of
increasing expression as a function of stage, as might be expected if
expression provided tumors with a growth advantage.

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Fig. 2.
GRP and GRPR expression (dark brown staining) in adjacent 5-µm
sections from a moderately differentiated Dukes stage D adenocarcinoma
of the colon. A: treatment of tumor
tissue with GRP antibody. B:
consecutive section treated similarly except for the absence of primary
antibody as a negative control. C:
consecutive section treated with GRPR antibody.
D: evaluation of a stage D tumor,
sectioned across the tumor margin, showing normal tissue at
left and cancer at
right, with antibody for GRPR. Similar
result was obtained when treated with antibody for GRP. Note that only
malignant cells are immunopositive. Tissues processed as described in
METHODS. Magnification = ×400
(A-C)
and ×100 (D).
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Because of the near-equal rates of expression of GRP/GRPR in tumors
irrespective of stage (Table 2), we looked for evidence of receptor or
ligand expression in premalignant lesions. A total of 20 polyps were
examined (5 hyperplastic, 5 tubular adenomas, 5 villous adenomas, and 5 villous adenomas with dysplasia). GRPR immunostaining could not be
identified in any polyp, whereas GRP was detected in two of five
villous adenomas containing regions of high-grade dysplasia.
Significantly, GRP expression was only detected in severely dysplastic
cells (Fig.
3A).

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Fig. 3.
GRP and/or GRPR expression in polyps and lymph nodes.
A: tubulovillous polyp containing
high-grade dysplasia treated with GRP antibody. Immunopositivity is
present only in the most severely dysplastic cells.
Inset: compare immunopositive
dysplastic cells on left vs. the
nonstaining and nondysplastic cells on
right of the crypt. Similar results
were not seen when treated with GRPR antibody. Magnification = ×100 and ×400 (inset).
B: metastatic tumor in a lymph node
from a patient with Dukes stage C cancer treated with GRPR antibody.
Similar result was obtained when treated with antibody for GRP.
Magnification = ×100.
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We likewise evaluated all metastatic lesions from patients with stage C
and D tumors. This included all lymph nodes
(n = 30), liver biopsies
(n = 5), and serosal implants
(n = 2) containing tumors. One lymph
node (7%) was strongly positive for both GRP and GRPR (Fig.
3B), whereas one other node showed
evidence of only GRPR expression (Table 2). These two lymph nodes
originated from primary tumors that were strongly positive for both
ligand and receptor. Of the seven non-lymph node metastases, one to the serosa expressed GRPR and not GRP. Similar to the positive lymph nodes,
this positively staining metastasis arose from a primary tumor that
also stained strongly for both GRP and GRPR. However, 5 of 15 primary
tumors stained for GRP/GRPR with similar intensity but gave rise to
metastases that showed no evidence of expressing either protein. Thus
ligand and receptor expression is uncommon in metastatic disease and
does not necessarily correlate with the degree of immunostaining
detected in the primary tumor.
GRP acts as a mitogen.
Because GRP has been proposed to act as an autocrine growth factor in
cancer (7, 24-26), including those originating in the colon (13,
27, 32, 36, 37), we were interested to see if tumor regions
coexpressing GRP and GRPR were associated with increased amounts of
cell proliferation. To do this, we selected five separate
histologically distinct regions positive for both GRP and GRPR, either
GRP or GRPR, and neither GRP nor GRPR. We then counted the
PCNA-positive nuclei in three different high-powered fields in each of
these areas (a total of 1,545 cells were counted) (Table
3). Whereas 37% of nuclei were PCNA
positive in regions expressing both GRP and GRPR, <15% of nuclei
were positive in regions not expressing both ligand and receptor (Table
3, Fig. 4). Thus these data support a role
for GRP as a mitogen acting in an autocrine manner in human colon
cancer.

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Fig. 4.
Proliferating cell nuclear antigen (PCNA) immunopositivity in malignant
epithelial cells coexpressing GRP and GRPR
(A) and tissues not expressing
either ligand or receptor (B).
Tissues were incubated with PCNA antibody at a dilution of 1:200
overnight at 4°C. Positive and negative nuclei were counted and are
summarized in Table 3. Insets: control
tissues treated similarly except for the absence of primary antibody.
Magnification = ×1,000.
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Survival data.
Because we had failed to find evidence of increasing rates of GRP/GRPR
expression with more advanced and metastatic tumors, we were
particularly interested to determine if this mitogenic peptide hormone
and its receptor had any impact on patient survival. We determined if
GRP/GRPR coexpression influenced patient outcome by performing
Kaplan-Meier analysis on the survival data (Fig. 5). Complete information was available for
45 of 50 patients whose tumors were evaluated, since 5 were lost to
follow-up after surgery. We compared survival of patients whose tumors
expressed both ligand and receptor (n = 29) compared with those whose tumors did not coexpress both proteins
(n = 16). We grouped patients whose
tumors expressed only GRP or GRPR with those whose tumors expressed
neither protein, since we postulated that a difference in survival, if present, should only be seen if tumors coexpressed both ligand and
receptor. Censored data were primarily used, since only 16 deaths
occurred in the statistical sample. Overall, no significant difference
in survival could be detected between either group by log rank
(Mantel-Cox) analysis (P = 0.81) (Fig.
5). Thus patient survival is not altered when tumors coexpress GRP and
GRPR and where an autocrine growth loop could conceivably be present.
In combination with our observation that there is no increase in GRP/GRPR expression as a function of tumor stage, these data suggest that this peptide may not be acting as a clinically important growth
factor.

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Fig. 5.
Kaplan-Meier survival analysis for the 45 colon cancer patients in this
study not lost to follow-up. Survival curves were generated for
patients whose tumors expressed both GRP and GRPR ( ,
n = 29) or those whose tumors
expressed neither protein, GRP alone, or GRPR alone ( ,
n = 16). Data were stratified by tumor
stage and segregated according to whether the observation was censored
or uncensored.
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Receptor/ligand expression and tumor differentiation.
We observed that GRP and GRPR immunostaining was always focal in nature
and was never diffusely observed throughout a tumor. The 50 tumor
sections that we evaluated contained a total of 158 separate and
distinct histological regions comprised of well-differentiated, moderately differentiated, or poorly differentiated cells. Because stage A and B1 tumors tended to contain only a single histological region, this means that there were between 1 and 4.6 separate regions
present within any given section. When these 158 sections were
evaluated independently, we found that the extent of both GRP and GRPR
immunostaining was positively associated with the degree of tumor
region differentiation (Fig. 6). The
greatest extent of immunostaining was observed in well-differentiated
tumor regions (Figs. 6 and 7) irrespective
of tumor stage (Fig. 6). To determine if the converse
applied, we then evaluated regional histology as a function of the
immunopositivity status (Table 4). Tumor
regions expressing either ligand or receptor alone tended to be
moderately or poorly differentiated, although some were well
differentiated (Table 4). In contrast, no region was found to be well
differentiated that expressed neither protein and no region expressing
both proteins was poorly differentiated (Table 3). When moderately
differentiated tumors are excluded from analysis, all tumor regions
expressing both GRP and GRPR were well differentiated and none were
poorly differentiated, whereas all regions expressing neither protein
were poorly differentiated.

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Fig. 6.
Degree of GRP and GRPR immunopositivity as a function of tumor
differentiation. A total of 61 well-differentiated, 54 moderately
well-differentiated, and 43 poorly differentiated tumor regions were
evaluated in the indicated tissues for the extent of GRP or GRPR
immunopositivity. Degree of regional immunopositive intensity was
determined using a 0-4 scale as described in
METHODS. Note that, irrespective of
stage, better differentiated tumors are more immunopositive
for both GRP and GRPR. Mets, metastatic lesions. Data are
means ± SE.
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Fig. 7.
Regional GRPR expression in a Dukes stage D tumor. A tumor section
containing both well-differentiated
(right) and poorly differentiated
cells (left) was treated with GRPR
antibody as described in METHODS. Only
well-differentiated tumor cells stain positively and not the poorly
differentiated cells. A similar result was obtained when treating with
GRP antibody. Magnification = ×100.
Insets: high-power view of the cancer
cells from the well-differentiated region identified by the box.
A shows cells treated with GRPR
antibody, and B shows control tissues
treated similarly except for the absence of primary antibody.
Magnification = ×1,000.
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DISCUSSION |
Epithelial cells lining the human GI tract outside of the gastric
antrum do not normally express GRPR (9). In contrast, previous studies
have shown that GRP binding sites are present in 24-40% of
resected colon cancers (32, 36), whereas approximately one-third of
human colon cancer cell lines expresses functional GRPR (13). Because
it was first reported that GRP causes the growth of most human
small-cell lung cancer cell lines by an autocrine mechanism (7), it has
been assumed that this ligand acts as an autocrine growth factor in all
tumors where its cognate receptor is aberrantly expressed. However,
aside from lung cancer cell lines (7, 24-26), studies
investigating GRPR expression by various tumors have not documented the
presence of ligand. Furthermore, because GRP acts as a mitogen in all
cancer cell lines in which GRPR are expressed, including those derived
from GI tumors (13, 27, 32, 36), it has been assumed but never proven
that these proteins are clinically important for tumor
growth and progression. Indeed, we have previously shown that
introduction of the GRPR alone into a nonmalignant human colon cell
line resulted in receptor constitutive activation and
ligand-independent cell proliferation (10). These findings clearly
indicate that GRP/GRPR can act as mitogens; we therefore set out in
this study to quantify the extent and significance of GRP and GRPR
expression by adenocarcinomas of the colon.
Our results show that, whereas normal and nonmalignant colonic
epithelia do not express GRP or GRPR, 84% of colon cancers aberrantly
express either one of these proteins. Because 62% of colon cancers
studied contain regions coexpressing both ligand and receptor and
regions coexpressing these proteins contained greater numbers of
PCNA-positive cells, it might appear that GRP acts as an autocrine
growth factor, as has been previously postulated (13, 27, 32, 36, 37).
Yet, surprisingly, our observations do not support the hypothesis that
GRP/GRPR acts as a clinically significant growth factor in colon
cancer. First, no increase in GRP/GRPR expression as a function of
tumor stage could be detected (Table 2). Second, only 2 of 30 lymph
nodes containing tumor and 1 of 7 liver and peritoneal metastases
expressed either protein (Table 2). If GRP acts as a clinically
significant growth factor, the presence of ligand and its receptor
should provide cancers with a growth advantage such that increased
frequency of expression would be observed with more advanced stage
tumors and in metastases. However, similar levels of GRP/GRPR were
detected in stage A as in stage D cancers, whereas 34 of 37 (92%)
metastases did not express either protein (Table 2). Finally, there was
no difference in survival between patients whose tumors expressed both
GRP and GRPR and those whose tumors did not express both proteins (Fig. 5). In aggregate, therefore, these data indicate that, despite the
ability of GRP to cause cell proliferation in vitro, this peptide
hormone does not act as a clinically significant oncogenic growth
factor in vivo.
Instead, we make two observations in this study regarding aberrant
GRP/GRPR expression that suggest a novel function for these proteins in
colon cancer. First, our data show that GRP/GRPR expression is common
to all colon cancers regardless of stage and occurs early in malignant
transformation. As such, the dedifferentiation associated with tumors
assuming a more primitive intestinal phenotype appears to involve
expression of GRP and GRPR. Evidence for this being the case is found
in fetal rats, the only species so studied, in which epithelial cells
lining the GI tract transiently express GRPR from embryonic
days 13-16 until birth (3, 44).
Although the role of GRP/GRPR in the development of the GI tract has
yet to be determined, the transient nature of this expression suggests a possible role for these proteins in gut differentiation
and/or maturation. Thus expression of GRP/GRPR may well reflect
tumor assumption of a more primitive phenotype, as occurs during
malignant transformation.
Second, and directly related to our first observation, GRP/GRPR
expression was only detected in well-differentiated tumor areas (Figs.
6 and 7, Table 4). Expression of GRP or GRPR alone was as likely to be
expressed by poorly differentiated as by well-differentiated tissues
(Table 4). In contrast, all well-differentiated tumor regions
coexpressed GRP and GRPR, whereas no poorly differentiated tissue
coexpressed both proteins. The association of tissue differentiation and GRP/GRPR coexpression was also independent of tumor stage (Fig. 6).
Because the association with differentiation was only observed when
both ligand and receptor were coexpressed, these findings suggest the
possibility that these proteins act in an autocrine fashion regulating
cellular differentiation.
Differentiation factors are more commonly known as morphogens and were
first described in the regulation of normal embryological development
(reviewed in Ref. 16). More recently, morphogens have been shown to be
important in cancer. In the GI tract, perhaps the best-described
morphogen is hepatocyte growth factor (HGF), important in altering the
behavior of gastric adenocarcinomas (reviewed in Ref. 42). HGF is a
weak mitogen synthesized by stromal tissues that binds to the tyrosine
kinase receptor c-met expressed by
gastric cancer cells (5, 31). When gastric cancers concomitantly
express high levels of E-cadherin, important in regulating cell-to-cell
attachment, HGF causes these cells to adopt a more differentiated
phenotype (23). However, when E-cadherin levels are low, HGF instead
acts as "scatter factor" and causes cancer cell migration (4,
42). Thus HGF in gastric cancer can act as a mitogen, motogen, or
morphogen, depending on the cellular situation. Similar to HGF, GRP is
a mitogen. Furthermore, GRP is known to activate multiple different
intracellular signaling pathways, including those that modulate
cell-to-cell attachment. Depending on the cell type, the GRPR couples
to multiple different G proteins, including members of the
p21ras superfamily (33). GRPR
activation of these G proteins, including p21rho, alters
p125fak phosphorylation and
influences the integrity of focal adhesions (22). Thus GRP is similar
to HGF insofar as a theoretical mechanism exists for it being able to
alter cell-to-cell attachment and thereby act as a morphogen in cancer.
The case for GRP/GRPR acting as a morphogen in colon cancer is
strengthened by recent reports indicating that these proteins are
important in normal fetal organogenesis. In mice, mRNA for GRPR is
observed in lung buds starting at embryonic day
12 (19). Branching of explanted buds, a marker of
increasing lung differentiation, was significantly increased in the
presence of bombesin, a pharmacological homologue of GRP (19).
Likewise, in rabbits, GRP is synthesized by pulmonary neuroendocrine
cells and acts on GRPR expressed by distal airway epithelial tubes only
at the time of peak airway growth and differentiation (45).
Interestingly, in both cases, administration of GRP/bombesin also
increased airway cell proliferation (19, 45). Thus, during at least
normal lung development, GRP acts as both a mitogen and a morphogen,
suggesting that these two properties are linked.
Because in normal development many morphogens act via heptaspanning
receptors (16), it is not surprising that some have now been shown to
perform this role in cancer. Of the heptaspanning receptors associated
with differentiation in cancer, the best described is vasoactive
intestinal peptide (VIP). Similar to GRP, VIP has been shown to act as
an autocrine growth factor in various cancer cell lines, including
neural crest tumors such as neuroblastomas (28). Yet VIP also induces
neuroblastoma cell line differentiation in vitro (29), whereas
expression has been shown to correlate with the presence of more
differentiated neuroblastomas (34) and other neural tumors (1) in vivo.
In contrast to our data, however, VIP expression by these neural tumors
is associated with improved patient survival.
Unlike other GI tumors, the prognosis for patients with colon cancer
does not correlate with the tumor differentiation status (40, 41). This
is probably due to the fact that, unlike other GI tumors, colon cancers
contain multiple, different histological regions. In this study, larger
tumors contained on average 4.6 histologically distinct regions,
irrespective of tumor stage. When the pathologist describes a colon
cancer's stage of differentiation, they are providing an overview of
the predominant tumor histology and are not stating that such
differentiation is exclusively present. Thus a
"well-differentiated" tumor may also contain regions of moderately and/or poorly differentiated cells (an example of
this is shown in Fig. 7). Because there is no way to know with
certainty which cells in a primary tumor give rise to the metastatic
lesion, it is not surprising that the predominating histology does not convincingly correlate with patient outcome in colon cancer (40, 41).
The association of GRP/GRPR expression with tumor differentiation does
not, of course, prove that differentiation is due to the aberrant
expression of these proteins. At this point, our observations serve
only to 1) question whether GRP acts
as a clinically significant growth factor in colon cancer and
2) suggest the possibility that this
peptide hormone acts in a completely novel fashion as a morphogen. The
association of GRP/GRPR coexpression with tumor differentiation is
novel and serves to underscore the need for additional studies into the
normal and abnormal roles of these proteins in the GI tract.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Robert T. Jensen (Digestive Diseases Branch, National
Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD)
for insights arising from the careful reading of this manuscript and
Dr. Robert Mrtek (University of Illinois at Chicago) for assistance
with our statistical evaluations.
 |
FOOTNOTES |
This work was supported by an American Digestive Health Foundation
(ADHF)/Astra Merck Advanced Research Fellowship award (to R. E. Carroll) and by an ADHF/American Gastroenterological Association Industry Research Scholar Award, National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-51168, and a Veterans Affairs
Merit Review (to R. V. Benya). The contributions of the first
two authors are equal.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. V. Benya,
Dept. of Medicine, Univ. of Illinois at Chicago, 840 South Wood St.
(M/C 787), Chicago, IL 60612 (E-mail: rvbenya{at}uic.edu).
Received 16 September 1998; accepted in final form 18
November 1998.
 |
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