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Regular Article |
From the Departments of Pathology,*
Oncology,
and
Gastroenterology,
The Johns Hopkins Medical
Institutions, Baltimore, Maryland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The TGF{beta} genes form part of a large superfamily of extracellular ligands, and the corresponding receptors of each type of ligand also constitute distinct gene families. Signal transduction is initiated when a ligand binds and stabilizes the formation of the corresponding heterodimeric receptor pair. Transforming growth factor-{beta} (TGF-{beta}) has long been studied for its tumor-suppressive properties in a variety of cancer types and a lack of TGF-{beta}-responsiveness has been demonstrated in many tumor lines. The importance of this signaling pathway in neoplasia is most strongly supported by the finding of biallelic inactivating mutations in the TGFBR2 gene in nearly all colorectal carcinomas having microsatellite instability.4-6
A number of Smad genes mediate functions of the TGF-{beta}
superfamily (including those of bone morphogenic protein, activin, and
TGF-{beta}).7-9
For example, Smad2 mediates TGF-{beta} signals
and Smad2 mutations have been identified in occasional
colorectal cancers. Likewise, DPC4 is a common mediator of
multiple TGF-{beta} superfamily pathways. As depicted in Figure 1
, ligand binding to a heterodimeric
receptor activates a serine-threonine kinase functional group. Certain
Smad proteins interact with the receptors transiently, become
phosphorylated, and then form a complex with Dpc4. These activated
complexes are translocated into the nucleus where their binding to DNA
stimulates the transcription of nearby genes.10
There is
now a considerable body of literature concerning experimental systems
that confirms these biochemical relationships.9
It was
therefore attractive to assume that Dpc4 functioned as a tumor
suppressor (ie, it was selectively inactivated in tumors) primarily
because it mediated the TGF-{beta} signals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate xenograft tumors, actively growing cell lines were harvested and resuspended in serum-free McCoy’s 5A medium (Life Technologies, Inc., Grand Island, NY) at 5 x 107 cells per ml. The athymic nude mice (strain nu/nu; Harlan, Indianapolis, IN) were then injected subcutaneously with 0.1 ml HCT116 cells on one flank and with cells from cell line 5-60 or cells from cell line 5-18 on the other. HCT116 cells are known to have a truncating mutation of TGFBR2 but are DPC4+/+.4 In contrast, the cell line 5-60 was prepared from HCT116 cells that have been genetically modified so they not only have a truncating mutation of TGFBR2 but are also DPC4+/-. The cell line 5-18 was prepared from HCT116 cells and they were genetically modified so they lack TGFBR2 and are DPC4-/-. The preparation of these cell lines has been previously described.9 Fourteen days after injection, the animals were sacrificed. The tumors were removed and immediately preserved in 10% buffered formalin. Immunohistochemistry using the anti-Dpc4 antibody was then performed as below on each of the harvested tumors.
Specimen Selection
Slides and blocks of colorectal cancers resected from 13 patients with hereditary nonpolyposis colorectal cancer (HNPCC)12 and from six patients with a medullary tumor phenotype (an appearance that has been associated with mismatch repair defects) were retrieved from the surgical pathology files of The Johns Hopkins Hospital. The 13 patients with HNPCC were previously shown to have DNA mismatch repair defects.12 Medullary tumor phenotype was defined using criteria of Jass and colleagues13 and Kim and colleagues.14 These tumors appeared poorly differentiated with solid sheets of tumor cells, prominent intratumoral lymphocytic infiltration, and an intense peritumoral lymphoid response. Also retrieved and sectioned were materials from 41 sporadic colorectal carcinomas and their associated adenomas (n = 14) and lymph node metastases (n = 9).
Immunohistochemistry
Unstained 5-µm sections were cut from the paraffin blocks and deparaffinized using standard methods. Slides were treated with sodium citrate buffer (HIER buffer; Ventana-Bio Tek Solutions, Tucson, AZ) and steamed at 80°C. After cooling for 5 minutes, slides were labeled with monoclonal antibody to Dpc4 (clone B8; Santa Cruz Biotechnology, Santa Cruz, CA) using the Bio Tek-Mate 1000 automated stainer (Ventana-Bio Tek Solutions). Each slide was labeled with a 1:100 dilution of the antibody. The anti-Dpc4 antibody was detected using a biotinylated secondary antibody and 3,3'-diaminobenzidine as the chromagen with hematoxylin counterstaining.
Slides were reviewed by three of the authors (EM, REW, RHH) and recorded as positive or negative for both nuclear and cytoplasmic labeling as has been described.11 For a slide to be interpreted as negative, no expression could be visible. Focal labeling was interpreted as positive. Normal colonic epithelium served as a positive control, and the primary antibody was omitted in negative controls. Pancreatic carcinomas with known Dpc4 genetic status were also included as positive and negative controls.11
Epstein-Barr Virus (EBV) Testing
In situ hybridization was performed to detect EBV using a fluorescein-conjugated oligonucleotide probe to the EBV early RNA transcripts (Novocastra, Newcastle, UK).15 After overnight incubation, probe binding was detected using an in situ hybridization detection kit (Novocastra) that uses an alkaline-phosphatase-conjugated rabbit F(ab') anti-fluorescein isothiocyanate fragment followed by 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium substrate chromogen. Positive control tissues yielded blue/black nuclear staining.
Assessment of TGF-{beta} Receptor Mutations
Sufficient archival material was available from three of the HNPCC colorectal cancers with nuclear Dpc4 expression for DNA sequencing. DNA was isolated from formalin-fixed paraffin-embedded tissues as previously described.16 Evidence for mutations of the polyA tract of the TGFBR2 gene was determined by polymerase chain reaction amplification and end-labeling of the 5' primer. The radiolabeled 73-bp polymerase chain reaction products were resolved on a 6% polyacrylamide gel. Constitutional DNA from the duodenum of two patients with pancreatic cancer served as negative controls and two pancreatic cancers with known polyA tract mutations17 served as positive controls.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The tumors prepared from HCT116 cells (which are known to have a
truncating mutation of TGFBR2 but are
DPC4+/+) had both nuclear and cytoplasmic
labeling for Dpc4 (Figure 2A)
. Those
prepared from cell line 5-60 cells (lacking intact TGFBR2
but DPC4+/-) also had both nuclear and
cytoplasmic labeling with the Dpc4 antibody (Figure 2B)
. Xenografted
tumors prepared from cell line 5-18 cells (lacking intact TGFBR2,
DPC4-/-) showed neither cytoplasmic nor nuclear
labeling with the Dpc4 antibody (Figure 2C)
. This result confirmed
unambiguously that the nuclear labeling was because of Dpc4 protein
rather than cross-reacting antigen.
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All 13 (100%) carcinomas obtained from patients with HNPCC
(Figure 3A)
and all six carcinomas having
medullary morphology showed both nuclear and cytoplasmic labeling for
Dpc4. To assure that the six carcinomas with medullary morphology were
not Epstein-Barr virus driven (a finding sometimes associated with
medullary phenotype), we also performed in situ
hybridization for Epstein-Barr virus, which was negative in all cases.
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Of the 41 sporadic cancers, 39 labeled with the Dpc4 antibody, and
two (two of 41; 5%) completely lacked expression of the gene product
by immunohistochemistry (Figure 3B)
. Internal controls (normal colon
epithelium and stroma) labeled in all cases. Fourteen associated
sporadic adenomas all expressed the gene product. Two lymph node
metastases, associated with the carcinomas that did not express Dpc4,
were also negative for gene product, whereas the lymph node metastases
from Dpc4-positive carcinomas (Figure 3C)
were also positive.
TGFBR2 Gene Sequencing
Sufficient material was available from three of the 13 carcinomas
from patients with HNPCC for TGFBR2 gene sequencing. All
three cases tested had bi-allelic mutations of the polyA tract of
TGFBR2. In all three cases, immunolabeling revealed intact
nuclear localization of Dpc4 (Figure 3A)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is also known that Dpc4 can be influenced by other upstream signaling inputs. For example, developmental systems have principally concerned BMP-related ligands, not TGF-{beta} itself. Similarly, the somatic knockout model of the DPC4 gene was not only TGF-{beta}-unresponsive but also activin-unresponsive.9
Indeed, there is considerable emerging evidence that raises doubts
regarding, or at least complicates, the TGF-{beta}-Dpc4 relationship. For
example, DPC4-null pancreatic cancer and colorectal cancer
cell lines do not, as a rule, lose TGF-{beta} responsiveness, irrespective
of whether one looks at transcriptional responses or growth
suppression.20,21
The ras genes, the MAP kinase
pathway, and the MKK4-mediated stress-activated protein kinase pathway
are implicated in these responses and would not involve the Dpc4
protein.20-22
DPC4-null transgenic mouse cells
have classes of TGF-{beta} responses that remain intact.23
Both pancreatic and colorectal tumors show instances in which there is
co-existence of genetic inactivation of both a TGF-{beta} receptor gene
and the DPC4 gene.24
This would not be expected
if inactivation of one were sufficient to obviate the function of the
other. These data together suggest considerable branching of the
pathways uniting TGF-{beta} receptors and Dpc4 (Figure 1)
. Furthermore,
preliminary immunohistochemical studies of pancreatic cancer also
demonstrate nuclear localization of Dpc4 in the rare tumors having
genetic inactivation of the TGF-{beta} receptors (RE Wilentz, unpublished
data).
The immunohistochemical labeling results in the current study raise the question of whether TGF-{beta} responsiveness is necessary for Dpc4 function. We found intact nuclear localization of Dpc4 in human carcinomas with inactivating mutations in a TGF-{beta} receptor gene and confirmed these observations using xenografted tumors from appropriate human cell lines. Although the mere presence of Dpc4 in the nucleus does not necessarily imply the existence of conditions sufficient for its tumor-suppressive function, there is evidence from studies of engineered cell lines that the direct manipulation of Dpc4 to relocalize to the nucleus, in the absence of exogenous administration of ligand, is sufficient to cause apoptosis and cell cycle inhibition.25
The accumulated data therefore suggest that TGF-{beta} signaling is not always required for the major tumor-suppressive functions of the DPC4 gene. It would now be important to determine whether there is a dominant signaling input to Dpc4 and what this input might be. On such an understanding would rest the better comprehension of some of the key driving forces in tumorigenesis. This, in turn, would lead to more precise identification of the molecular targets on which we might base hopes for the development of rational therapy for human cancer. Undoubtedly, additional direct observational studies of human tumors will be critical to the evaluation of competing hypotheses.
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Footnotes |
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Supported in part by CA62924 and CA68228 from the National Institutes of Health.
Accepted for publication September 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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