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Tumor Biology |
Department of Biochemistry and Molecular Genetics [K. W. F., J. A. E., J. M. R.], Department of Pathology [A. R. F., W. E. G.], Division of Hematology/Oncology, Department of Medicine [P. M-B., J. M. R.], Medical Statistics Section, Department of Medicine [C-Y. L.], and Oral Cancer Research Center and Comprehensive Cancer Center [J. A. E., W. E. G., J. M. R.], University of Alabama at Birmingham School of Medicine, Birmingham, Alabama 35294-3300
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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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By expression cloning we recently identified the zinc finger protein GKLF3 as a novel transforming oncogene when expressed in RK3E cells, a diploid epithelial cell line derived from primary rat kidney cells and immortalized with adenovirus E1A (17) . cDNA libraries were prepared using mRNA from human oral squamous cell or breast carcinoma cell lines, tumor-types not reported to exhibit frequent genetic alterations that activate well-characterized oncogenes such as RAS, GLI, or ß-catenin (18) . Retroviral transduction of these libraries into RK3E cells induced morphologically transformed foci, 11 of which were subsequently attributed to enforced expression of wild-type human c-MYC. Two other transformed foci contained independently derived, wild-type alleles of GKLF. No other genes were identified in the screen, suggesting that only a select subset of all oncogenes are able to transform these cells.
Whereas enforced expression of a human wild-type GKLF transgene in RK3E cells induces morphological transformation in vitro and tumorigenicity in athymic mice, the doubling time of GKLF-expressing cells was considerably longer than for RK3E cells (27 h versus 12 h, respectively; Refs. 17 , 19 ). Similar results were obtained for other oncogenes, including GLI and c-MYC, as cells expressing these genes exhibited doubling times of 18 and 19 h, respectively. These oncogenes may therefore function in epithelial cells by interfering specifically with contact inhibition rather than by inducing a more general increase in the rate of cell division.
In support of a role for GKLF as an oncogene, we observed increased expression of GKLF mRNA during progression of squamous cell carcinomas of the oropharynx. As demonstrated by mRNA ISH analysis of surgical specimens, expression in normal epithelial cells is limited to the differentiating compartment. Expression in dysplastic oral epithelium is increased overall and is found in all cell layers, and GKLF is expressed at similar levels in dysplasia and in invasive carcinoma. These results identified loss of the compartment-specific pattern of GKLF mRNA expression in epithelium as a candidate mechanism of tumor progression in oral cancer (17) .
GKLF encodes a DNA-binding transcription factor with functional domains that mediate activation or repression of transcription (20, 21, 22) . GKLF is essential for the barrier function of skin, because homozygous knockout mice exhibit morphologically normal skin but die postpartum due to dehydration (23) . Transcriptional targets of GKLF that may be relevant to epithelial differentiation have been preliminarily identified (23, 24, 25) . As shown by analysis of normal mouse or human tissues, GKLF is preferentially expressed in differentiating epithelial cells of the skin, gut, oral cavity, and thymus (17 , 20 , 23 , 26 , 27) . In contrast to human oral squamous cell carcinoma, expression of GKLF mRNA was found to be reduced in mouse models of intestinal tumorigenesis or hyperproliferation (28 , 29) . Independently, analysis of human colorectal mucosa and tumors by SAGE confirmed the earlier studies in mice (30 , 31) . Specifically, mRNA from specimens of normal colonic mucosa generated GKLF tags at frequencies of 138 or 99 per million SAGE tags, whereas mRNA from a microdissected tumor generated only 20 tags per million. These results suggested that GKLF expression is regulated during neoplastic progression in a tumor type-specific fashion.
Increased expression of specific oncogenes in tumors can result from genetic alterations and play a causal role in tumor progression. Alternatively, expression of some oncogenes is cell cycle dependent, and increased expression can occur as a consequence of increased proliferation or altered cell cycle occupancy of tumor cells. In multiple normal tissues as well as in certain cell lines, GKLF expression is reduced in actively cycling cells compared with terminally differentiated or growth-arrested cells, and enforced expression of GKLF in cultured cell lines can retard cell cycle progression (26 , 32) . These properties predicted that GKLF expression might be reduced in tumors, as observed in colorectal carcinoma. Increased expression in other tumor-types is therefore somewhat unexpected and may result from specific alterations in the pathways that regulate GKLF transcription in normal cells.
To better understand the spectrum of tumor-types that exhibit GKLF activation, we obtained samples of breast carcinoma, colorectal carcinoma, and prostatic carcinoma and analyzed expression of GKLF in malignant cells and in adjacent normal-appearing epithelium (i.e., uninvolved epithelium). The results show that levels of GKLF mRNA and protein are each up-regulated before invasion in a majority of cases of breast cancer, but not in tumors of the colorectum or prostate. In neoplastic lesions of the breast as well as in cultured mammary epithelial cells in vitro, increased GKLF expression appears to precede overtly malignant behavior. The potent transforming activity of GKLF in vitro, the tumor type-specific activation of expression in vivo, and activation early during tumor progression identify this oncogene as a potential effector of tumor progression in the breast.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mRNA Expression.
ISH was conducted as described (17)
using sense and
antisense [35S]-labeled riboprobes prepared by
in vitro transcription of a cDNA fragment corresponding to
the 3' untranslated region of human GKLF. A
GAPDH antisense probe corresponding to bases 366–680
(GenBank accession no. M33197) was synthesized using a
commercially available template (Ambion, Inc., Austin, TX). High
stringency washes were in 0.1 x SSC and 0.1% (v/v)
2-mercaptoethanol at 58°C for GKLF or 68°C for
GAPDH. Slides were coated with emulsion and exposed for 14
days. The number of silver grains/nucleus were counted within
representative areas by two individuals, and a score from 0.0 to 4.0
was recorded. A score of 0.0 indicated only nonspecific background, as
determined using the sense control, and 1.0 corresponded to an average
of four grains/nucleus.
Breast adenocarcinoma cell lines were obtained from the American Type Culture Collection (Manassus, MD). HMECs were described previously and were cultured in mammary epithelial basal media (Clonetics Corp., Walkersville, MD; Ref. 33 ). Extracts were prepared from exponentially growing cells at 70% confluence, and total RNA isolation and Northern blot analysis were performed as described (17) .
Isolation of an Anti-GKLF Monoclonal Antibody.
The region of the human GKLF cDNA encoding bases 479-1197
(GenBank accession no. AF105036) was cloned into plasmid pET-32a
and expressed in Escherichia coli BL21(DE3) bacteria as a
histidine-tagged protein. Protein was purified from the bacteria
after induction with
isopropyl-1-thio-ß-D-galactopyranoside
using a His-Trap Ni-agarose column (Amersham Pharmacia Biotech,
Piscataway, NJ) and eluted with 500 mM imidazole.
Purified protein was used to immunize two mice, and lymphocytes were
fused with murine myeloma cells (PX63-Ag8.653) as described previously
(34)
. Hybridomas that were immunoreactive in an ELISA
assay for the purified antigen were cloned and recloned by limiting
dilution. Positive clones were identified by ELISA, and an IgG1
antibody (
GKLF) was purified from ascites on a protein A affinity
column.
Immunohistochemistry.
Tissues were fixed in neutral buffered formalin and embedded in
paraffin. Deparaffinized tissue sections were incubated with GKLF at
a concentration of 1.0 µg/ml for 1 h at room temperature, and
processed as described (35)
. Immunodetection was performed
using a biotinylated secondary antibody, streptavidin-horseradish
peroxidase detection system (Signet Laboratories, Dedham, MA), and the
chromogenic substrate diaminobenzidine (Biogenex, San Ramon, CA).
Sections were counterstained with hematoxylin. Results were scored by
using a 0.0 to 4.0 scoring system where 4.0 corresponds to a saturated
signal (36)
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Statistical Analyses.
Paired t tests were used to compare the differences in
expression in breast epithelial cells at various stages of tumor
progression (37)
. Pearson correlation coefficients were
used to compare results obtained by ISH to those obtained for the same
cases using immunohistochemistry.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of GKLF was detected in malignant cells in 21 of 31 cases of
ductal adenocarcinoma (68%, Fig. 1
, Table 1
). For several cases that exhibited no detectable expression of GKLF,
prominent expression of the housekeeping gene GAPDH was
observed, indicating that overall mRNA integrity was maintained and
that failure to identify GKLF transcripts may reflect reduced levels of
expression. GKLF expression was increased in malignant cells of 14 of
19 cases that contained adjacent uninvolved epithelium (Fig. 1A)
. For 7 of these 14 cases, no specific signal was
detected in adjacent uninvolved epithelium. In the other seven cases,
expression was detected in both uninvolved and malignant cells, with
expression of GKLF in malignant cells increased by 3- to 5-fold
compared with uninvolved epithelium. Within tumors, expression of GKLF
was specific to malignant cells, with little or no expression detected
in stromal components (Fig. 1B)
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, Fig. 2
). In contrast to results obtained in breast tumors, examination of
several cases of prostatic carcinoma revealed equal or reduced
expression in tumor cells compared with adjacent uninvolved glandular
epithelial cells (Table 1)
. In summary, the results suggest that GKLF
mRNA expression is activated in approximately two-thirds of breast
carcinomas, and that expression in positive cases is consistently
induced in DCIS before invasion.
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GKLF. Immunoblot analysis of
GKLF-transformed RK3E cells and control cell lines detected a single
protein species of 55 kDa consistent with the predicted size of the
full-length polypeptide (data not shown). Compared with RK3E cells or
control cell lines transformed by other oncogenes, apparent GKLF
abundance was increased by several-fold in each of two cell lines
transformed by the human expression vector. The epitope recognized by
the antibody may be denaturation-sensitive, as a signal was obtained
only after overnight exposure of autoradiographic film using a standard
chemiluminescence protocol. The antibody was not sufficiently sensitive
to detect GKLF by immunoblot analysis of extracts of human tumor cell
lines that express the endogenous GKLF mRNA.
The cell type- and tumor type-specific patterns of GKLF mRNA expression
were used to examine the specificity of GKLF in immunohistochemical
assays. These patterns can be summarized as follows. Human GKLF mRNA is
detected by ISH in differentiating cells of oral epithelium, and is
markedly elevated in oral tumors (17)
. The mRNA is not
detected in morphologically normal basal or parabasal cells,
particularly within epidermal pegs that extend further into the
submucosa. Mouse GKLF mRNA is similarly found to be more highly
expressed in superficial, differentiating cells of the skin and gut,
and is reduced or absent in basal epithelial cells in both tissues
(20
, 23 , 26)
. In contrast to human oral and breast cancer,
GKLF mRNA expression is reduced in mouse colorectal tumors compared
with normal epithelium (29)
, and is similarly reduced in
human colorectal cancer as indicated by SAGE (31)
.
The staining pattern of GKLF exhibited a strict concordance with
detection of GKLF mRNA (Figs. 3
and 4
, Table 2
). In positive tissues, GKLF exhibited a mixed nuclear and
cytoplasmic staining pattern. For uninvolved epithelium, DCIS, and
invasive breast carcinoma alike, the average cytoplasmic staining was
1.8- to 2.5-fold greater than nuclear staining, suggesting that
subcellular localization was not altered during breast tumor
progression in any consistent fashion. Cytoplasmic staining was
subsequently used as a more sensitive indicator of overall expression.
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GKLF bound
specifically to differentiating suprabasal epithelial cells (Fig. 3A)
. Compared with adjacent uninvolved epithelium, staining
was markedly increased in malignant cells for each of several cases of
squamous cell carcinoma with little or no staining of stromal
components of the tumor, as shown previously for the mRNA
(17)
. Likewise, staining was increased in superficial
cells compared with cells deeper within epithelial crypts of the small
bowel (Fig. 3B)
or the large bowel (Table 2
;
P = 0.043). In contrast to oral and breast
tumors, staining was reduced in tumor cells compared with adjacent
superficial epithelial cells for each of four cases of human colorectal
adenoma or carcinoma examined (Fig. 3C
, Table 2
;
P = 0.027).
Expression of GKLF Protein Is Increased during Neoplastic
Progression in the Breast.
Eighteen cases were tested for GKLF expression by
immunohistochemistry (Table 2
, Fig. 4
). Nuclear and cytoplasmic staining of normal breast epithelium, DCIS,
and invasive carcinoma were semiquantitatively assessed. Low-level
staining of tumor cells was observed for 6 cases (e.g.,
cytoplasmic staining ranging from 0.20 to 0.85), with 11 cases
exhibiting higher-level staining (e.g., cytoplasmic staining
ranging from 1.00 to 1.75). These results are consistent with detection
of the mRNA in approximately two-thirds of tumors by ISH. For cases
23–31, which were analyzed by both ISH and immunohistochemical
staining, results of the two methods exhibited a close correlation that
reached statistical significance for invasive carcinoma cells
(N = 8; coefficient = 0.77;
P = 0.024). In DCIS, the correlation was
moderate, although the sample number was small (N = 7; coefficient = 0.43). Perhaps because of the
overall lower level of expression in uninvolved tissue, the correlation
was weakest in uninvolved ducts. Minor differences observed for the two
methods may be attributed to differences in sensitivity and
specificity, to false negative results attributable to partial
degradation of mRNA in some surgical samples, or to analysis of
nonserial sections of the same tissue block. As observed in uninvolved
tissue adjacent to tumors, staining was low or undetectable for each of
five cases of reduction mammoplasty (data not shown).
Apparent GKLF expression as determined by nuclear or cytoplasmic
immunostaining was increased in both DCIS and invasive carcinoma
compared with uninvolved ducts (Table 2
, Fig. 5
). For morphologically normal ducts, staining of myoepithelial cells was
not significantly different from that of luminal epithelial cells
(P = 0.303, data not shown). However,
staining of neoplastic cells in DCIS was significantly increased
compared with myoepithelial cells within the same ducts
(P = 0.0001), which was consistent with other
studies indicating similarities between tumor cells and luminal
epithelial cells (39)
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. GKLF expression was high in MCF7 and ZR75-1; intermediate in BT474,
BT20, MDAMB361, and SKBR3; and reduced in MDAMB453 and MDAMB231. Thus,
expression in six of eight breast tumor-derived cell lines was
increased relative to 184 cells, an HMEC population of finite life span
derived from normal breast tissue after reduction mammoplasty
(Lane 1). Expression was similarly increased in 184A1 cells
(33)
. These immortalized cells were derived from 184 cells
by treatment with benzo(a)pyrene. They are wild-type for p53
and p105Rb and are anchorage-dependent and
nontumorigenic in animals. The results obtained for breast tumor cell
lines support the conclusion that GKLF expression is up-regulated at
the mRNA level in most breast tumors, whereas activation in 184A1 cells
is consistent with identification of GKLF induction as an early event.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GKLF encodes a zinc finger transcription factor of the GLI-Krüppel family (46) and is distinct from many other oncogenes in that expression in normal tissue is observed in terminally differentiating epithelial cells. In addition, expression is induced in association with cell growth-arrest in vitro (26) . As predicted by these observations, expression in certain tumor-types is reduced compared with the relevant normal epithelia. Thus, GKLF expression is reduced in colorectal tumors, a result supported by multiple approaches including analysis of RNA extracted from tissues (29) , SAGE (31) , and immunohistochemical analysis of human tissues (this work). ISH analysis of several prostatic tumors likewise indicates that GKLF is expressed in normal prostatic epithelium, and that expression can be lost during tumor progression.
In contrast to colorectal and prostatic carcinoma, GKLF expression is activated in both invasive carcinoma and preinvasive neoplastic lesions during progression of most breast carcinomas and virtually all oropharyngeal squamous cell carcinomas. Breast and oral cancers share a number of additional molecular alterations. Loss-of-function mutations frequently affect p53 and p16/CDKN2, whereas a smaller proportion of tumors (5–20%) exhibit gene amplification of c-MYC, cyclin D1, erbB-family members including the EGF receptor and erbB-2/HER-2/neu, or others (47, 48, 49, 50, 51) . Unlike carcinomas of the GI tract or skin, neither breast nor oral carcinoma is reported to exhibit frequent genetic alterations that activate known transforming oncogenes such as RAS, ß-catenin, c-MYC, or GLI. By analogy with oncogenes in other tumor types, disruption of the pathways that control GKLF mRNA expression in breast epithelial cells and in oral mucosa represents a potential mechanism of tumor initiation or progression in vivo.
The pattern of GKLF expression in normal epithelia may provide clues as to how GKLF functions in tumor progression. Stratified squamous epithelium contains at least four functionally distinct compartments (52 , 53) . The stem cell compartment is composed of cells within the basal cell layer that exhibit a capacity for self-renewal, but which rarely divide. The transit-amplifying compartment is composed of cells within the basal or parabasal cell layers that exhibit rapid cell division but a reduced capacity for self-renewal. Differentiation occurs within the prickle cell layer that contains identifiable desmosomes, leading to the outermost, keratinized superficial layer. Whereas mechanisms regulating transitions from one compartment to the next remain poorly understood, c-MYC activation can induce stem cells to enter the highly proliferative transit-amplifying compartment (40) . Because self-renewal and rapid cell division occur in distinct cell types, the organization of compartments enables the rapid turnover of epithelial cells while minimizing the possibility of sustaining permanent genetic damage in stem cells.
The observation that GKLF functions normally in the prickle cell layer suggests that each of the three compartments (stem cell, transit-amplifying, and prickle layer) expresses a transforming activity or a critical function (e.g., self-renewal or proliferation) that may contribute to the progression of carcinoma. These compartments appear to be intermingled in dysplastic stratified squamous epithelium, with prickle layer markers including GKLF misexpressed in the basal layers, whereas other basal or parabasal markers are misexpressed in superficial layers. Loss of compartment-specific patterns of gene expression may result in coexpression of the properties of several compartments in a single cell. For example, specific properties of the prickle cell layer, such as reduced cellular adhesion to basement membranes, altered adhesion to other cells, and/or loss of the cellular mechanisms that mediate contact inhibition could confer invasive or metastatic properties to oral carcinomas.
To better understand the mechanism of transformation, we are characterizing transcriptional alterations induced by GKLF when expressed in epithelial cells in vitro. In the future, identification of upstream regulators of GKLF transcription in epithelial cells may elucidate the pathways that regulate GKLF and the mechanism of deregulation of GKLF in specific tumor-types.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by USPHS Grant R29CA65686-05 (to
J. M. R.), the University of Alabama at Birmingham Oral Cancer
Research Center P50 DE11910-04 (to J. M. R., W. E. G., and
J. A. E.), Grants P50 DE08228-10S3 (to J. A. E.) and NIGMS 5T32
GM08361-08 (to K. W. F.), and a gift to the Comprehensive Cancer
Center from the Avon Products Foundation Breast Cancer Research and
Care Program. 
2 To whom requests for reprints should be
addressed, at Department of Medicine, Room 570 Wallace Tumor
Institute, University of Alabama at Birmingham School of
Medicine, Birmingham, AL 35294-3300. Phone: (205) 975-0556; Fax:
(205) 934-9573; E-mail: mruppert{at}uab.edu
3 The abbreviations used are: GKLF, gut-enriched
Krüppel-like factor; DCIS, ductal carcinoma in
situ; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HMEC,
human mammary epithelial cell; IDC, internal ductal carcinoma; ISH,
mRNA in situ hybridization; SAGE, serial analysis
of gene expression.
Received 4/12/00. Accepted 9/12/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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