Cancer Research Annual Meeting 2010 EMT and Cancer Progression and Treatment
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Serewko, M. M.
Right arrow Articles by Saunders, N. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Serewko, M. M.
Right arrow Articles by Saunders, N. A.
[Cancer Research 62, 3759-3765, July 1, 2002]
© 2002 American Association for Cancer Research

Molecular Biology and Genetics

Alterations in Gene Expression and Activity during Squamous Cell Carcinoma Development1

Magdalena M. Serewko, Claudia Popa, Alison L. Dahler, Louise Smith, Geoff M. Strutton, William Coman, Anthony J. Dicker and Nicholas A. Saunders2

Epithelial Pathobiology Group, Centre for Immunology and Cancer Research [M. M. S., C. P., A. L. D., L. S., N. A. S.], Department of Pathology [G. M. S.], and ENT Department [W. C.], University of Queensland, Queensland, 4067 Australia; Department of Dermatology, Princess Alexandra Hospital, Woolloongabba, Queensland, Australia 4102 [A. J. D.]; and Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Queensland, Australia 4067 [N. A. S.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study focuses on characterizing the genetic and biological alterations associated with squamous cell carcinoma development. Normal human epidermal keratinocytes (HEKs), cells isolated from a preneoplastic lesion (IEC-1), and two neoplastic cell lines, SCC-25 and COLO-16, were grown as raft cultures, and their gene expression profiles were screened using cDNA arrays. Our data indicated that the expression levels of at least 37 genes were significantly (P <= 0.05; 1.9% of genes screened) altered in neoplastic cells compared with normal cells. Of these genes, 10 genes were up-regulated and 27 genes were down-regulated in the neoplastic cells. In addition, 51% of the genes altered in the neoplastic cells were already altered in the preneoplastic IEC-1 cells. Immunohistochemical staining of patient tumors was used to verify the cDNA array analysis. Our analysis indicated that alterations in genes associated with extracellular matrix production and apoptosis are disrupted in preneoplastic cells, whereas later stages of neoplasia are associated with alterations in gene expression for genes involved in DNA repair or epidermal growth factor (EGF) receptor/mitogen-activated protein kinase kinase (MAPKK)/MAPK/activator protein-1 (AP-1) signaling. Subsequent functional analysis of the alterations in expression of the EGF receptor/MAPKK/MAPK/AP-1 genes suggested they did not contribute to the neoplastic phenotype.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The epidermis is a stratified squamous epithelium consisting of four biochemically and morphologically distinct layers. The basal layer is attached to the basement membrane and consists of a single layer of proliferating keratinocytes. Upon committing to terminal differentiation, the basal cells irreversibly withdraw from the cell cycle and down-regulate proliferation-specific genes such as E2F-1 (1, 2, 3) , cdk-1 (4) , and cytokeratins 5 and 14 (5 , 6) . These "committed" cells then pass into the suprabasal layers to form the differentiated spinous and granular layers that express differentiation-specific genes such as transglutaminase type 1 (7) , cornifin (8) , and cytokeratins 1 and 10 (5 , 9) . Finally, the granular cells apoptose and form the cross-linked envelope characteristic of the stratum corneum. Thus, squamous differentiation requires the coordinated activation and repression of genes specific to the various differentiated compartments (5 , 9) , and disruption of this program accompanies neoplasia (1 , 2 , 10) .

Neoplastic development follows a multistep program of targeted disruptions to genes required for normal cellular processes. These genes may be members of specific functional classes of proteins involved in cellular processes such as growth regulation, apoptosis, angiogenesis, cell migration, extracellular matrix (reviewed in Ref. 11 ), terminal differentiation (12 , 13) , and immune surveillance (14) . However, there is a paucity of data correlating the genetic alterations that accompany neoplasia with the biological attributes of cells derived from the different stages of neoplastic progression.

We have recently isolated cells from a preneoplastic IEC3 (IEC-1) from a patient (12) . IEC (Bowen’s disease) is recognized as a preneoplastic precursor to SCC (15) . IEC-1 cells, in culture, retain biochemical characteristics that are transitional between those of normal keratinocytes and those of fully transformed keratinocytes. Specifically, these cells have: (a) an extended life span; (b) undergo reversible but not irreversible growth arrest when treated with differentiation inducing agents; (c) form dysplastic layers when used as the epidermal equivalent in organotypic raft cultures; and (d) nontumorigenic when injected into nude mice (12) . Thus, the IEC-1 cells provide a unique resource to help characterize the stage-specific alterations in gene expression that accompany neoplastic progression (12) .

In this study, HEKs, cells derived from a preneoplastic intraepidermal carcinoma of the skin and SCC cell lines of the skin (COLO-16) or head and neck (SCC-25) were grown as organotypic raft cultures, and RNA isolated from these raft cultures was used to probe cDNA microarrrays in an attempt to identify likely candidates involved in the various stages of neoplastic progression. The use of raft cultures allowed us to maintain the spatial and temporal relationships of keratinocytes in a similar manner to that of "native" epidermis but in the absence of contaminating cell types (12 , 16, 17, 18) . Recent evidence indicates that this maintenance of "normal" tissue architecture and the presence of appropriate interstitial cells may be a critical determinant of normal and neoplastic tissue behavior (11 , 19 , 20) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
Isolation and culture of primary HEKs, IEC-1 cells, and HDFs have been described (2 , 12 , 21) . The neoplastic cell line SCC-25 was originally derived from a human SCC of the tongue (22) . COLO-16 cells are a cell line derived from a metastatic SCC of the skin (23) . Culture of SCC-25 cells and COLO-16 cells has been described (12) .

Organotypic Raft Cultures and Poly(A)+ mRNA Isolation.
HEKs, IEC-1, COLO-16, and SCC-25 cell lines were grown as organotypic raft cultures using techniques established previously (12) with slight modification. The dermal component of the rafts for IEC-1, COLO-16, and SCC-25 cells were made with HDFs seeded on the bottom of the plates rather than in the collagen. At the end of a 10-day culture period, the epidermal component of the HEK raft was peeled off the dermis, and the entire raft containing IEC-1, COLO-16, and SCC-25 cells was placed in TRIzol reagent (Invitrogen, Sydney, New South Wales, Australia).

cDNA Probe Preparation, cDNA Array Hybridization, and RT-PCR Analysis.
0.5 µg poly(A)+ mRNA isolated from the raft epidermis (7) was annealed to 1 µl of CDS primer mix (Clontech, Sydney, New South Wales, Australia) and then labeled with [{alpha}-32P]dATP (3000 Ci/mmol; Geneworks, Adelaide, South Australia, Australia) as described in the manufacturer’s protocol (Clontech). Array membranes (human cDNA array 1.2 and human cancer cDNA 1.2K; Clontech) were prehybridized and then hybridized overnight and washed as described in the manufacturer’s protocol. Membranes were exposed to a PhosphorImager screen (Molecular Dynamics, Sydney, New South Wales, Australia). Both the human and cancer membranes (1.2K) were probed three times with the poly(A)+ mRNA extracted from all cell types, whereas the HEK and COLO-16 poly(A)+ mRNA were probed an additional three times on the 588 gene human cDNA array and cancer array (Clontech). In total, 12 membrane arrays were probed. Intermembrane variability was accounted for by ensuring that the replicate probings for each cell type used different membranes. The duplication of certain genes on the various membranes resulted in the majority of genes being analyzed between 4 and 12 times. Semiquantitative RT-PCR analysis of PAI-2, Fra-1, calgranulin A, GADD45, actin, and EGF receptor expression was performed under linear conditions with respect to cycle number and template concentration (24) . Primer sequences were as follows: PAI-2 forward, 5'-ATGGAGCATCTCGTCCACCAT; PAI-2 reverse, 5'-GGAGTTCTAGAATCTGAGCC; Fra-1 forward, 5'-GAATTCGTACCCCGCAGAGCCGCCAG; Fra-1 reverse, 5'-GAATTCGTACCCCGCAGAGCCGCCAG; calgranulin A forward, 5'-CTCTATCATCGACGTCTACAC; calgranulin A reverse, 5'-CTCCTGGAAGTTAACTGCACCATCAGTG; GADD45 forward, 5'-CTCGGCTGGAGAGCAGAAGACCG; GADD45 reverse, 5'-GGCAGGATCCTTCCATTGAGATG; EGF receptor forward, 5'-GCAGCGCTCCTGGCGCTGCTGGCT; and EGF receptor reverse, 5'-TGCTGAAGGGCACGGCGCCATGC, and the sequence of actin primers has been described (24) .

Data Analysis.
The intensity of hybridization for each gene was quantified using ImageQuant (version 5.1) with median background subtraction. The data were then normalized to the housekeeping genes (n = 9) present on each membrane. Normalization was then repeated between cell types (i.e., mean HEK versus mean IEC-1, and others) to normalize for differences between cell types. The means (n = 3–12), SD, and coefficient of variation were then calculated for each gene. Differences in expression between cell types was established with a two-tailed t test, and significance was accepted at the P <= 0.05 level.

Immunohistochemistry.
Deparafinized sections of normal skin, SCC of the oral mucosa, or epidermis were subjected to antigen retrieval by autoclaving the slides (121°C for 20 min) in 10 mM EDTA (pH 7.5) and allowing them to cool to room temperature overnight. After incubation with 3% skim milk for 2 h at room temperature, primary antibody was added and further incubated for 1 h. Primary antibodies from Santa Cruz Biotechnology (Monarch Medical, Brisbane, Queensland, Australia) included: PAI-2 goat polyclonal, (sc-7646, 1:30); FRA-1 rabbit polyclonal (sc-605, 1:100); calgranulin A goat polyclonal (sc-8112, 1:30); 14-3-3{sigma} goat polyclonal (sc-7681, 1:3000); and GADD45 rabbit polyclonal (sc-797, 1:200). Primary antibodies purchased from Sigma-Aldrich (Sydney, New South Wales, Australia) included: EGF receptor mouse monoclonal (E-3138, 1:30); cyclin D1 mouse monoclonal (C-7464, 1:30); involucrin mouse monoclonal (I-9018, 1:3000); and PCNA mouse monoclonal (P-8825, 1:1000). The slides were washed three times in PBST, and secondary antibodies were added for an additional 30 min. Secondary antibodies used were: biotinylated rabbit antimouse immunoglobulins (IgG) from DAKO (Melbourne, Victoria, Australia), E0413 (1:200); biotinylated goat antirabbit IgG from DAKO, E0466 (1:1500) and antigoat IgG-biotinylated from Santa Cruz, sc-2042 (1:3000). Staining was visualized using horseradish peroxidase-conjugated streptavidin (DAKO) and diaminobenzidine. Normal mouse IgG (DAKO; X0931), normal rabbit IgG (Santa Cruz, sc-2027), and a nonspecific goat antiserum donated by Dr. Thomas (University of Queensland) were used as negative controls.

Transfection of Cells and Reporter Assays.
The AP-1-responsive promoter construct (TGAGTCA), derived from the collagenase gene, driving expression of a chloramphenicol acetyltransferase reporter gene (AP1-CAT) has been described previously (25) . The ß-actin-luciferase reporter gene (4) was used to normalize for transfection efficiency. pCDNA3 was used as a control for the transfections (Invitrogen). The pCMV-FRA1 plasmid was a generous gift from Dr. Paul Dobner. HEKs were transfected using Lipofectamine as described previously (1) . COLO-16 cells and SCC-25 cells were transfected with effectine (1) and genejammer (Life Technologies), respectively, using methods described by the manufacturers. Chloramphenicol acetyltransferase assays and luciferase assays were performed as described (4 , 7) .

Nuclear Extracts and Gel Shift Assay.
Preparation of nuclear extracts from proliferating and confluent HEKs were performed as described previously (2 , 4) . DNA binding reactions were performed as described previously (2 , 4) using the following double-stranded AP-1 oligonucleotide (5'-CGCTTGATGAGTCAGCCGGAA-3'; Promega, Sydney, Australia). Specific competition was performed with 100-fold excess cold AP-1 oligonucleotide, and nonspecific competition used 100-fold excess double-stranded CdxA oligonucleotide (5'-AGATCTGGTACCATTTAAGCCCTCGAGATCTA-3').


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Organotypic Raft Cultures Reflect Attributes of the Epidermis.
To construct gene expression profiles of keratinocytes at different stages of neoplasia, we used an organotypic raft culture system. We chose to look at SCC cells derived from an epidermal tumor (COLO-16) and a SCC of the oral mucosa (SCC-25). This meant that our final analysis would identify SCC-specific changes rather than alterations in gene expression specific for epidermal SCC or oral SCC. We grew normal (HEK), preneoplastic (IEC-1), and neoplastic (SCC-25 and COLO-16) cells as organotypic raft cultures (Fig. 1A)Citation . The HEKs formed a structure similar to that of normal skin, with rafts containing a basal layer of proliferative cells that expressed PCNA and a suprabasal layer that expressed involucrin (Fig. 1B)Citation . Both the preneoplastic IEC-1 cells and the neoplastic (SCC-25 and COLO-16) cells: (a) formed poorly differentiated layers; (b) showed evidence of dysplasia; and (c) lacked a visible stratum corneum (Fig. 1A)Citation .



View larger version (47K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Morphology of normal, preneoplastic, and neoplastic cells grown as raft cultures. A, H&E staining of organotypic raft cultures showing normal differentiation of HEK rafts with stratum corneum (arrows) and the dysplastic morphology of cells in IEC-1, SCC-25, and COLO-16 rafts. B, immunohistochemical staining of normal epidermis and HEK rafts using the differentiation-specific marker involucrin and the proliferation-specific marker, PCNA. Involucrin staining was most intense in the suprabasal layers of both epidermis and raft (sb), and little staining was evident in the basal layer (b). PCNA staining is localized to the nucleus of basal keratinocytes in both the foreskin and HEK raft. x25.

 
Gene Expression Is Altered in a Stage-specific Manner during Neoplastic Progression.
Our analyses indicated that of the 1895 genes screened, 163 (8.6%) genes were significantly altered between the HEKs and COLO-16 cells, 124 (6.5%) between HEKs and SCC-25, and of these altered genes, 37 (1.9%) were common to both COLO-16s and SCC-25s but differed significantly from the HEKs (Fig. 2Citation ; Table 1Citation ; P <= 0.05). Combined, a total of 213 individual genes were altered in SCC-25 and COLO-16 cells compared with HEKs. Of these differences, 17% (37 genes) were common to both cancer cell types. There were 163 (8.6%) genes that differed significantly between the two cancer cell lines (SCC-25 and COLO-16). Furthermore, of the 37 genes altered in both neoplastic cell types, 19 (51%) were significantly altered in the preneoplastic cells (Table 1)Citation . From these data, we can conclude that at least 19 genes are associated with the early stages of transformation from the normal state to the preneoplastic state. Furthermore, our data suggest that alterations in at least 18 other genes are associated with later progression from preneoplasia to neoplasia. It should be noted that decreases in gene expression may simply reflect a block in progress through the differentiation pathway in the neoplastic cells. Conversely, increases in gene expression may reflect an expansion of the proliferative compartment in the neoplastic cells.



View larger version (28K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Scatter plots of gene expression data comparing preneoplastic and neoplastic cells. Plot of gene expression level for HEKs (X axis) versus the expression level for the same gene in the other cell type: A, HEK versus IEC-1; B, HEK versus COLO-16; and C, HEK versus SCC-25. D, Venn diagram showing the number of genes significantly altered in expression between the neoplastic COLO-16 and SCC-25 cells and the normal HEKs. A total of 1895 genes were screened for each cell type.

 

View this table:
[in this window]
[in a new window]

  		Table 1 Summary of biological and genetic differences in keratinocytes during neoplastic progression

The top portion represents observed biological changes in squamous epithelial cells as they progress from normal to neoplastic (12) . The bottom portion illustrates the 37 genes found to have significantly (P <= 0.05) altered expression in both neoplastic cells analyzed (COLO-16 and SCC-25) compared with normal HEKs and the 19 genes found to be already altered in the preneoplastic IEC-1 cells. Fold alterations of gene expression are also shown for each gene. The genes in bold represent those used for immunohistochemical verification in Fig. 3Citation .

 
Alterations in Gene Expression Are Reflected in Protein Expression in Vivo.
To validate the gene expression data, we examined the immunohistochemical staining pattern for 7 genes (Table 2)Citation . These genes were selected on the basis that they represented important functional classes. Initial confirmation of the array data were performed by RT-PCR (Fig. 3A)Citation . SCCs of the oral mucosa and skin were used, and histologically "normal" tissue adjacent to the oral mucosa or skin lesion was used as our control to avoid interslide variability. All of the immunohistochemical staining showed the same qualitative trend as the microarray data (Fig. 3BCitation and Table 2Citation ). PAI-2 is a serine proteinase inhibitor, and down-regulation of this gene has been linked to tumor metastasis (26) . PAI-2 expression decreases markedly in the preneoplastic (3.8-fold) and neoplastic cells (6.8-fold) compared with that of normal HEKs (Table 2)Citation , and this decrease is also seen immunohistochemically in the neoplastic lesion (Fig. 3B)Citation . FRA-1 is a member of the oncogenic AP-1 transcription factor family (27 , 28) . FRA-1 mRNA expression was increased only in neoplastic cells (5-fold). Interestingly, FRA-1 protein expression was cytosolic in the proliferative basal cells of normal epidermis and was increased in the tumor and localized to the nucleus (Fig. 3B)Citation . Growth arrest and DNA damage inducible protein (GADD45) is involved in signaling apoptosis (29) . GADD45 expression is reduced in tumor cells in both the array data (5-fold; Table 2Citation ) and immunohistochemically (Fig. 3B)Citation . EGF receptor is a tyrosine kinase receptor that signals mitogenesis in epithelial tissues (30) via the ras/MAPKK/MAPK/AP-1 signaling pathway (30) . EGF receptor mRNA expression is increased in tumor cells (3-fold; Table 2Citation ), and this is confirmed immunohistochemically in patient tumors (Fig. 3B)Citation . Calgranulin A (or migration inhibitory factor-related protein 8) is an intracellular calcium-binding protein that is suggested to regulate cytoskeletal changes in skin disease (31) . Our data show that the expression of this gene is significantly reduced in tumors (6-fold), and that this decrease is also evident immunohistochemically in tumor sections (Fig. 3B)Citation . The data generated in the present study did allow us to determine whether there were tissue-specific alterations associated with neoplasia. 14-3-3{sigma} mRNA expression was significantly altered in the epidermis-derived COLO-16 cell line (down 4.4-fold) but not the oral SCC (SCC-25; Table 2Citation ). Immunohistochemical staining of normal skin, or tumors of either the head and neck region or the skin, showed that there was little difference in 14-3-3{sigma} expression between normal skin and the head and neck lesion, but there was a noticeable decrease in the expression of this gene within the epidermal lesion (Fig. 4)Citation . Conversely, cyclin D1 expression was increased in the head and neck lesion (3.3-fold) but not the epidermal lesion (Fig. 4)Citation .


View this table:
[in this window]
[in a new window]

  		Table 2 Fold differences of genes used in immunohistochemical verification

Five genes (of 37) representing different functional groups were used to verify the cDNA array analysis (first five). Another two genes (last two) were also used because they appeared to be specific for either oral mucosa (SCC-25) or epidermal (COLO-16) SCCs.

 


View larger version (33K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Confirmation of differences between normal and neoplastic keratinocytes. A, RT-PCR analysis of mRNA expression for PAI-2, GADD45, calgranulin A, FRA-1, EGF receptor, and actin in HEKs or COLO-16 or SCC-25 cells. B, SCCs, with adjoining normal tissue, were stained for PAI-2, GADD45, calgranulin A, FRA-1, and EGF receptor. Note: FRA-1 expression translocates from a cytosolic location in normal skin (evident only in the basal layer, as indicated by arrows) to a nuclear location in neoplastic cells (arrows). Inset, control staining. x25.

 


View larger version (88K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Immunohistochemical staining of proteins differ in lesions originating form oral or epidermal epithelia. Normal tissue and SCCs originating from lesions of the oral mucosa or skin were stained with two differentially expressed proteins, 14-3-3{sigma} and cyclin D1. Staining of 14-3-3{sigma} within normal tissue and SCC of the oral mucosa show cytosolic localization with little difference in intensity. Staining of the skin lesion using 14-3-3{sigma} show a significant decrease in expression. Cyclin D1 expression is localized to the nucleus in both normal skin and the oral mucosal lesion with significantly more expression within the oral mucosal lesion. Staining of cyclin D1 in the SCC of the skin shows no significant difference in expression compared with normal skin.

 
The Role of FRA-1 in Neoplastic Progression.
We were interested to determine whether the increased FRA-1 (Table 1Citation ; Fig. 3Citation ) expression in SCCs may contribute to progression from preneoplasia to neoplasia or whether it merely reflected the deregulated proliferation of cancer cells. We examined AP-1 activity in proliferating cells and then in cells that have been differentiated by growth to confluence. AP-1 reporter activity (Fig. 5A)Citation and DNA binding activity (Fig. 5B)Citation is reduced in cells grown to confluence, indicating that AP-1 activity is proliferation associated and is lost during differentiation. Similarly, the SCC cells also decreased AP-1 reporter activity upon reaching confluence (Fig. 5A)Citation , which is consistent with their decreased proliferation at confluence (12) . Finally, comparison of AP-1 activity in the neoplastic cells (COLO-16 and SCC-25) to proliferating or confluent HEKs indicates that AP-1 activity is decreased in both the COLO-16 cells and the SCC-25 cells compared with the HEKs (Fig. 5A)Citation . These data indicate that although AP-1 activity is proliferation associated, there was no enhancement of AP-1 activity in the cancer cells. Because there were no significant alterations in the expression of other AP-1 family members (c-fos, FRA-2, c-jun, junB, and junD) in the neoplastic cells (Table 3)Citation , we examined the effects of FRA-1 overexpression on normal keratinocytes (Fig. 5C)Citation . Overexpression of FRA-1 in proliferating cells reduced AP-1 activity in HEKs and COLO-16 cells (Fig. 5C)Citation . These data indicate that FRA-1 can be a negative regulator of AP-1 activity.



View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. AP-1 activity in normal and neoplastic cells. Proliferating (Prol) and confluent (Conf) cells were transfected with the AP1-cat + ß-actin luciferase constructs, and then reporter assays were performed 48 h later. A, alterations in AP-1 reporter activity for HEKs, COLO-16 cells, or SCC-25 cells were examined. Differences in AP-1 reporter activity between these cell types in proliferating or confluent cultures are also shown. B, gel shift analysis of AP-1 DNA binding activity used nuclear extracts from proliferating (Prol) or confluent (Conf) HEKs. AP-1 binding was competed for by 100-fold excess unlabeled probe (Sp.) but not by 100-fold excess nonspecific probe (Nonsp.). Free probe is shown at the bottom of the gel, and specific AP-1 binding is indicated. C, in some experiments, HEKs or COLO-16 cells were transfected with AP1-cat + ß-actin luciferase plus either the pCDNA3 vector (-) or the pCDNA3-FRA-1 expression plasmid (+). All data represent means from triplicate determinations in at least two independent experiments; bars, SE. Data are normalized for transfection efficiency.

 

View this table:
[in this window]
[in a new window]

  		Table 3 AP-1 family members expressed on cDNA arrays

Of the seven AP-1 family members, six were represented on the cDNA arrays. The table below indicates the mean densities (arbitrary units) and coefficients of variance (CCVs) for each gene for all cell types analyzed.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Organotypic raft cultures are a robust model that possesses many of the attributes of the differentiating epidermis and provide a pathologically relevant model to study SCC progression. The validity of the organotypic raft culture system used in this study was demonstrated by: (a) the similar immunohistochemical staining profile for the proliferation (PCNA)- and differentiation (involucrin)-specific genes in normal skin sections and that of raft cultures; (b) confirmation of genes shown previously to be altered in neoplastic progression [e.g., EGF receptor (32) and PAI-2 (26 , 33) ]; and (c) all of the genes examined immunohistochemically in patient lesions showed similar alterations in expression to that of the array analysis.

An important component of the genetic profile is the correlation of genetic changes with biological changes during SCC progression. Neoplastic progression represents a graded process in which lesional events predispose to further sequential lesional events, culminating in loss of cellular regulation (10 , 11) . In the present study, we could associate alterations in the expression of 19 known genes with the preneoplastic characteristics of: (a) tissue remodeling; (b) increased life span; and (c) aberrant growth and differentiation control. The preneoplastic lesion, from which the IEC-1 cells were derived, exhibited significant tissue remodeling (12) , which correlates with our finding of increases in the expression of PAI-2 and collagenase (MMP8). Moreover, the increased replicative life span reported for the IEC-1 cells may be attributable to defects in either apoptosis, cell cycle control, or differentiation. Evidence for disruption of all of these events was supported by the array data. For example, suppression of caspase 3 (34) and induction of GSHPx-2 (35) and IEX-1L (36) would all be consistent with suppression of apoptosis. Similarly, disruption of growth could contribute to increased replicative life span and may be attributable to alterations in cell cycle control (10 , 11) as well as alterations in apoptosis. Although we found little evidence for large-scale disruptions in the expression of cell cycle genes common to both neoplastic cell types, there were some cell cycle genes (e.g., cyclin D1) that were disrupted selectively in either epidermal or oral SCCs (data not shown). This is consistent with different mechanisms being involved in growth disruption at the different tissue sites (e.g., skin versus oral cavity). For instance, a recent study showed that cyclin D1 could increase the life span of oral keratinocytes and could immortalize them against a background of p53 inactivation (37) . Similarly, a recent study has shown that loss of 14-3-3{sigma} in epidermal keratinocytes can also lead to an increased life span and immortalization (38) . These findings are strikingly similar to the present study in which the neoplastic cells and patient tumors from the oral mucosa had elevated cyclin D1 levels and no p53 (12) , whereas neoplastic cells from the epidermis had decreased 14-3-3{sigma}. The decreased expression of 14-3-3{sigma} in the preneoplastic IEC-1 cells (bottom portion of Table 2Citation ) is also consistent with the increased replicative life span reported previously for these cells (12) .

Later events in neoplastic development involve increased genomic instability (39) , which can predispose a cell to an accelerated progression to disseminated disease. This study has shown that alterations in expression of DNA repair genes is not altered in preneoplastic cells but does occur later in neoplastic cells. We found that GADD45 expression was suppressed, and we had shown previously that immunoreactive p53 was absent in the neoplastic cells (12) . Thus, we have shown an association between progression from preneoplasia to neoplasia and the expression of genes involved in DNA repair. Furthermore, we found a suppression of a migration inhibitor (calgranulin A) in the neoplastic cells, which is consistent with progression to a more advanced disease state. Combined, these data point to an ordered series of events that are sequentially disrupted as a cell passes from normal, through preneoplasia, to neoplasia. Surprisingly, the same number of genes was altered during progression to preneoplasia as were altered during progression from preneoplasia to neoplasia. This suggests that early events in neoplastic development may be more complex than previously thought and may involve multiple lesions in multiple pathways.

Although earlier work, including our own, indicates that the promoters of differentiation-specific genes contain functional AP-1 sites (9 , 40, 41, 42) , only a few studies have examined AP-1 activity in differentiating keratinocytes (41 , 43 , 44) , and the results of these studies remain unclear (43 , 44) . In the present study, we found that: (a) AP-1 reporter activity and DNA binding activity are decreased during differentiation; (b) AP-1 activity is proliferation associated; (c) FRA-1 is a negative regulator of AP-1 activity in keratinocytes and carcinoma cells; and (d) AP-1 activity is modestly decreased in squamous carcinoma cells. AP-1 activity is the downstream effector for many signaling pathways, including the mitogenic EGF receptor/MAPKK/MAPK signaling pathway (9 , 45) . Thus, it was noteworthy that in the present study we found increased expression for EGF receptor and FRA-1 and decreased expression of MAPKK 3 and MAPK 2 (Table 1)Citation . Although the overexpression of EGF receptor would be predicted to be mitogenic and activate AP-1, the decreases in MAPKK and MAPK gene expression and the increase in FRA-1 would be predicted to suppress EGF receptor-mediated stimulation of this pathway. Moreover, the switch in FRA-1 from a cytosolic location in normal keratinocytes to a nuclear location in cancer cells would also be predicted to suppress AP-1 activity (FRA-1; Fig. 3Citation ). These data suggest that despite overexpression of the EGF receptor, AP-1 activity remains equal or less than that of normal keratinocytes because of the counterbalancing inhibitory response by MAPKK, MAPK, and FRA-1. We contend that this is a deliberate response by the cancer cells to counteract deregulation of mitogenic signaling by the overexpressed EGF receptor. An important implication of this is that the lesion controlling deregulated proliferation in cancer cells must lie downstream of AP-1. These data also indicate that SCCs can arise in the absence of gross alterations in AP-1 activity. Although these data are pertinent to the neoplastic cells examined in this study, it does not exclude the possibility that disruption of other AP-1 members such as c-fos (46) may occur at later neoplastic stages.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the critical comments on the manuscript from Dr. Nick Hayward (Queensland Institute of Medical Research). We thank Prof. Paul Dobner (University of Massachusetts Medical School, Worcester, MA) for the generous gift of the FRA-1 expression plasmid. We also thank Prof. M. Karin (University of California, San Diego, CA) for the gift of the AP-1 reporter construct. We greatly acknowledge the assistance of Prof. Peter Parsons and Dr. Glen Boyle of the Queensland Institute of Medical Research for constructive dialogue when initiating the array screening.


   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 Queensland Cancer Fund 98/QCFN001G, The Garnett Passe and Rodney Williams Memorial Foundation, The Association for International Cancer Research, The Australian National Health and Medical Research Committee Grant 142906, and the Princess Alexandra Hospital Foundation. N. A. S. is sponsored by a Lions Medical Research Foundation Senior Research Fellowship, and C. P. is supported by an Australian Postgraduate Award. M. M. S. is supported by a postgraduate scholarship awarded by the Garnett Passe and Rodney Williams Memorial Foundation. Back

2 To whom requests for reprints should be addressed, at Centre for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Ipswich Road, Woolloongabba, Queensland, Australia 4102. Phone: (07) 3240 5936; Fax: (07) 3240 5946; E-mail: nsaunders{at}medicine.pa.uq.edu.au Back

3 The abbreviations used are: IEC, intraepidermal carcinoma; SCC, squamous cell carcinoma; HEK, human epidermal keratinocyte; RT-PCR, reverse transcription-PCR; TPA, 12-O-tetradecanoylphorbol-13-acetate; HDF, human dermal fibroblast; PAI, plasminogen activator inhibitor; FRA-1, fos-related antigen-1; AP-1, activator protein-1; EGF, epidermal growth factor; MAPK, mitogen activated protein kinase; MAPKK, MAP kinase kinase; PCNA, proliferating cell nuclear antigen; GADD, growth arrest and DNA-damage inducible. Back

Received 11/ 5/01. Accepted 5/ 7/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Dicker A. J., Popa C., Dahler A. L., Serewko M. M., Hilditch-Maguire P. A., Frazer I. H., Saunders N. A. E2F-1 induces proliferation-specific genes and suppresses squamous differentiation-specific genes in human epidermal keratinocytes. Oncogene, 19: 2887-2894, 2000.[Medline]
  2. Jones S. J., Dicker A. J., Dahler A. L., Saunders N. A. E2F as a regulator of keratinocyte proliferation: implications for skin tumor development. J. Investig Dermatol., 109: 187-193, 1997.[Medline]
  3. Saunders N. A., Jetten A. M. Control of growth regulatory and differentiation-specific genes in human epidermal keratinocytes by interferon {gamma}. Antagonism by retinoic acid and transforming growth factor ß1. J. Biol. Chem., 269: 2016-2022, 1994.[Abstract/Free Full Text]
  4. Dahler A. L., Jones S. J., Dicker A. J., Saunders N. A. Keratinocyte growth arrest is associated with activation of a transcriptional repressor element in the human cdk1 promoter. J. Cell. Physiol., 177: 474-482, 1998.[Medline]
  5. Fuchs E. Epidermal differentiation: the bare essentials. J. Cell Biol., 111: 2807-2814, 1990.[Free Full Text]
  6. Irvine A. D., McLean W. H. Human keratin diseases: the increasing spectrum of disease and subtlety of the phenotype-genotype correlation. Br. J. Dermatol., 140: 815-828, 1999.[Medline]
  7. Saunders N. A., Bernacki S. H., Vollberg T. M., Jetten A. M. Regulation of transglutaminase type I expression in squamous differentiating rabbit tracheal epithelial cells and human epidermal keratinocytes: effects of retinoic acid and phorbol esters. Mol. Endocrinol., 7: 387-398, 1993.[Abstract/Free Full Text]
  8. Marvin K. W., George M. D., Fujimoto W., Saunders N. A., Bernacki S. H., Jetten A. M. Cornifin, a cross-linked envelope precursor in keratinocytes that is down-regulated by retinoids. Proc. Natl. Acad. Sci. USA, 89: 11026-11030, 1992.[Abstract/Free Full Text]
  9. Eckert R. L., Welter J. F. Transcription factor regulation of epidermal keratinocyte gene expression. Mol. Biol. Rep., 23: 59-70, 1996.[Medline]
  10. Hartwell L. H., Kastan M. B. Cell cycle control and cancer. Science (Wash. DC), 266: 1821-1828, 1994.[Abstract/Free Full Text]
  11. Hanahan D., Weinberg R. A. The hallmarks of cancer. Cell, 100: 57-70, 2000.[Medline]
  12. Dicker A. J., Serewko M. M., Dahler A. L., Khanna K., Kaur P., Li A., Strutton G. M., Frazer I. H., Saunders N. A. Functional characterisation of cultured cells derived from an intraepidermal carcinoma of the skin (IEC-1). Exp. Cell Res., 258: 352-360, 2000.[Medline]
  13. Saunders N. A., Smith R. J., Jetten A. M. Regulation of guanylate-binding protein expression in interferon-{gamma}-treated human epidermal keratinocytes and squamous cell carcinoma cells. J. Investig. Dermatol., 112: 977-983, 1999.[Medline]
  14. Nishigori C., Yarosh D. B., Donawho C., Kripke M. L. The immune system in ultraviolet carcinogenesis. J. Investig. Dermatol. Symp. Proc., 1: 143-146, 1996.[Medline]
  15. Kao G. F. Carcinoma arising in Bowen’s disease. Arch. Dermatol., 122: 1124-1126, 1986.[Abstract/Free Full Text]
  16. Regnier M., Darmon M. Human epidermis reconstructed in vitro: a model to study keratinocyte differentiation and its modulation by retinoic acid. In Vitro Cell Dev. Biol., 25: 1000-1008, 1989.[Medline]
  17. Kopan R., Fuchs E. The use of retinoic acid to probe the relation between hyperproliferation-associated keratins and cell proliferation in normal and malignant epidermal cells. J. Cell Biol., 109: 295-307, 1989.[Abstract/Free Full Text]
  18. Brown L. J., Geesin J. C., Rothnagel J. A., Roop D. R., Gordon J. S. Retinoic acid suppression of loricrin expression in reconstituted human skin cultured at the liquid-air interface. J. Investig. Dermatol., 102: 886-890, 1994.[Medline]
  19. Liotta L. A., Kohn E. C. The microenvironment of the tumour-host interface. Nature (Lond.), 411: 375-379, 2001.[Medline]
  20. Shekhar M. P., Werdell J., Santner S. J., Pauley R. J., Tait L. Breast stroma plays a dominant regulatory role in breast epithelial growth and differentiation: implications for tumor development and progression. Cancer Res., 61: 1320-1326, 2001.[Abstract/Free Full Text]
  21. Brinkmann H., Dahler A. L., Popa C., Serewko M. M., Parsons P. G., Gabrielli B. G., Burgess A. J., Saunders N. A. Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts. J. Biol. Chem., 276: 22491-22499, 2001.[Abstract/Free Full Text]
  22. Rheinwald J. G., Beckett M. A. Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultures from human squamous cell carcinomas. Cancer Res., 41: 1657-1663, 1981.[Abstract/Free Full Text]
  23. Moore G. E., Merrick S. B., Woods L. K., Arabasz N. M. A human squamous cell carcinoma cell line. Cancer Res., 35: 2684-2688, 1975.[Abstract/Free Full Text]
  24. Popa C., Dicker A. J., Dahler A. L., Saunders N. A. Cytochrome P450, CYP26AI, is expressed at low levels in human epidermal keratinocytes and is not retinoic acid-inducible. Br. J. Dermatol., 141: 460-468, 1999.[Medline]
  25. Angel P., Imagawa M., Chiu R., Stein B., Imbra R. J., Rahmsdorf H. J., Jonat C., Herrlich P., Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell, 49: 729-739, 1987.[Medline]
  26. Yoshino H., Endo Y., Watanabe Y., Sasaki T. Significance of plasminogen activator inhibitor 2 as a prognostic marker in primary lung cancer: association of decreased plasminogen activator inhibitor 2 with lymph node metastasis. Br. J. Cancer, 78: 833-839, 1998.[Medline]
  27. Shaulian E., Karin M. AP-1 in cell proliferation and survival. Oncogene, 20: 2390-2400, 2001.[Medline]
  28. Eckert R. L., Crish J. F., Banks E. B., Welter J. F. The epidermis: genes on—genes off. J. Investig. Dermatol., 109: 501-509, 1997.[Medline]
  29. Zhan Q., Bae I., Kastan M. B., Fornace A. J., Jr. The p53-dependent gamma-ray response of GADD45. Cancer Res., 54: 2755-2760, 1994.[Abstract/Free Full Text]
  30. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell, 103: 211-225, 2000.[Medline]
  31. Clark B. R., Kelly S. E., Fleming S. Calgranulin expression and association with the keratinocyte cytoskeleton. J. Pathol., 160: 25-30, 1990.[Medline]
  32. Porter A. C., Vaillancourt R. R. Tyrosine kinase receptor-activated signal transduction pathways which lead to oncogenesis. Oncogene, 17: 1343-1352, 1998.[Medline]
  33. Chen C. S., Lyons-Giordano B., Lazarus G. S., Jensen P. J. Differential expression of plasminogen activators and their inhibitors in an organotypic skin coculture system. J. Cell Sci., 106: 45-53, 1993.[Abstract]
  34. Wolf B. B., Green D. R. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J. Biol. Chem., 274: 20049-20052, 1999.[Free Full Text]
  35. Lu Y. P., Lou Y. R., Yen P., Newmark H. L., Mirochnitchenko O. I., Inouye M., Huang M. T. Enhanced skin carcinogenesis in transgenic mice with high expression of glutathione peroxidase or both glutathione peroxidase and superoxide dismutase. Cancer Res., 57: 1468-1474, 1997.[Abstract/Free Full Text]
  36. Wu M. X., Ao Z., Prasad K. V., Wu R., Schlossman S. F. IEX-1L, an apoptosis inhibitor involved in NF-{kappa}B-mediated cell survival. Science (Wash. DC), 281: 998-1001, 1998.[Abstract/Free Full Text]
  37. Opitz O. G., Suliman Y., Hahn W. C., Harada H., Blum H. E., Rustgi A. K. Cyclin D1 overexpression and p53 inactivation immortalize primary oral keratinocytes by a telomerase-independent mechanism. J. Clin. Investig., 108: 725-732, 2001.[Medline]
  38. Dellambra E., Golisano O., Bondanza S., Siviero E., Lacal P., Molinari M., D’Atri S., De Luca M. Downregulation of 14-3-3{sigma} prevents clonal evolution and leads to immortalization of primary human keratinocytes. J. Cell Biol., 149: 1117-1130, 2000.[Abstract/Free Full Text]
  39. Yuspa S. H. The pathogenesis of squamous cell cancer: lessons learned from studies of skin carcinogenesis. J. Dermatol. Sci., 17: 1-7, 1998.[Medline]
  40. Medvedev A., Saunders N. A., Matsuura H., Chistokhina A., Jetten A. M. Regulation of the transglutaminase I gene. Identification of DNA elements involved in its transcriptional control in tracheobronchial epithelial cells. J. Biol. Chem., 274: 3887-3896, 1999.[Abstract/Free Full Text]
  41. Welter J. F., Crish J. F., Agarwal C., Eckert R. L. Fos-related antigen (Fra-1), junB, and junD activate human involucrin promoter transcription by binding to proximal and distal AP1 sites to mediate phorbol ester effects on promoter activity. J. Biol. Chem., 270: 12614-12622, 1995.[Abstract/Free Full Text]
  42. Rossi A., Jang S. I., Ceci R., Steinert P. M., Markova N. G. Effect of AP1 transcription factors on the regulation of transcription in normal human epidermal keratinocytes. J. Investig. Dermatol., 110: 34-40, 1998.[Medline]
  43. Briata P., D’Anna F., Franzi A. T., Gherzi R. AP-1 activity during normal human keratinocyte differentiation: evidence for a cytosolic modulator of AP-1/DNA binding. Exp. Cell Res., 204: 136-146, 1993.[Medline]
  44. Rutberg S. E., Saez E., Glick A., Dlugosz A. A., Spiegelman B. M., Yuspa S. H. Differentiation of mouse keratinocytes is accompanied by PKC-dependent changes in AP-1 proteins. Oncogene, 13: 167-176, 1996.[Medline]
  45. Watts R. G., Huang C., Young M. R., Li J. J., Dong Z., Pennie W. D., Colburn N. H. Expression of dominant negative Erk2 inhibits AP-1 transactivation and neoplastic transformation. Oncogene, 17: 3493-3498, 1998.[Medline]
  46. Ozanne B. W., McGarry L., Spence H. J., Johnston I., Winnie J., Meagher L., Stapleton G. Transcriptional regulation of cell invasion: AP-1 regulation of a multigenic invasion programme. Eur. J. Cancer, 36: 1640-1648, 2000.



This article has been cited by other articles:


Home page
 Cancer Res.Home page
A. Banning, A. Kipp, S. Schmitmeier, M. Lowinger, S. Florian, S. Krehl, S. Thalmann, R. Thierbach, P. Steinberg, and R. Brigelius-Flohe
Glutathione Peroxidase 2 Inhibits Cyclooxygenase-2-Mediated Migration and Invasion of HT-29 Adenocarcinoma Cells but Supports Their Growth as Tumors in Nude Mice
Cancer Res., December 1, 2008; 68(23): 9746 - 9753.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Walshe, M. M. Serewko-Auret, N. Teakle, S. Cameron, K. Minto, L. Smith, P. C. Burcham, T. Russell, G. Strutton, A. Griffin, et al.
Inactivation of Glutathione Peroxidase Activity Contributes to UV-Induced Squamous Cell Carcinoma Formation
Cancer Res., May 15, 2007; 67(10): 4751 - 4758.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
W. Yan and X. Chen
GPX2, a Direct Target of p63, Inhibits Oxidative Stress-induced Apoptosis in a p53-dependent Manner
J. Biol. Chem., March 24, 2006; 281(12): 7856 - 7862.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
A. Banning, S. Deubel, D. Kluth, Z. Zhou, and R. Brigelius-Flohe
The GI-GPx Gene Is a Target for Nrf2
Mol. Cell. Biol., June 15, 2005; 25(12): 4914 - 4923.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
P. Mrass, M. Rendl, M. Mildner, F. Gruber, B. Lengauer, C. Ballaun, L. Eckhart, and E. Tschachler
Retinoic Acid Increases the Expression of p53 and Proapoptotic Caspases and Sensitizes Keratinocytes to Apoptosis: A Possible Explanation for Tumor Preventive Action of Retinoids
Cancer Res., September 15, 2004; 64(18): 6542 - 6548.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
F.-F. Chu, R. S. Esworthy, P. G. Chu, J. A. Longmate, M. M. Huycke, S. Wilczynski, and J. H. Doroshow
Bacteria-Induced Intestinal Cancer in Mice with Disrupted Gpx1 and Gpx2 Genes
Cancer Res., February 1, 2004; 64(3): 962 - 968.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Serewko, M. M.
Right arrow Articles by Saunders, N. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Serewko, M. M.
Right arrow Articles by Saunders, N. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online