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In colon carcinogenesis, the cytoskeletal protein gelsolin is down-regulated during the transition from adenoma to carcinoma
Human Pathology (2008) 39, 1420–1430
www.elsevier.com/locate/humpath
Original contribution
In colon carcinogenesis, the cytoskeletal protein gelsolin is down-regulated during the transition from adenoma to carcinoma☆ Fabien Gay PhD student a,b,c , Yann Estornes PhD a,b,c , Jean-Christophe Saurin MD, PhD a,b,c , Marie-Odile Joly-Pharaboz MD a,b,c,d , Evelyne Friederich PhD e , Jean-Yves Scoazec MD, PhD a,b,c,d , Jacques Abello PhD a,b,c,⁎ a
Inserm, U865, Lyon, F-69372, France; Inserm, IFR62, Lyon, F-69372, France Université de Lyon, Lyon, F-69003, France c Université de Lyon 1, F-69372 Villeurbanne, France d Hospices Civils de Lyon, Hôpital Edouard Herriot, Service Central d’Anatomie et Cytologie Pathologiques, F-69437 Lyon, France e Department of Life Sciences, University of Luxembourg, L-1511, Luxembourg, Luxembourg b
Received 9 November 2007; revised 7 February 2008; accepted 25 February 2008
Keywords: Gelsolin; Immunohistochemistry; Adenoma; Adenocarcinoma; Colon
Summary The actin-binding protein gelsolin is involved in cell motility via the regulation of actin cytoskeleton, and its expression is modified in several human cancers. However, the potential implication of this protein in colorectal carcinogenesis is debated. By using immunohistochemistry, we studied gelsolin expression in 69 cases of colon adenocarcinomas and in 72 lesions representative of the different stages of colonic tumorigenesis. In addition, we performed Northern blot analysis of gelsolin messenger RNA in 12 paired samples of human colon cancer and normal corresponding mucosa. Gelsolin protein and messenger RNA expressions were severely down-regulated in all adenocarcinomas tested. Moreover, gelsolin protein was down-regulated in a large proportion of high-grade adenomas (14/16) before the acquisition of invasive properties but in only a small proportion of low grade adenomas and serrated adenomas (2/30) and in none of the 9 cases of nonneoplastic hyperplastic polyps tested. Our results therefore demonstrate that gelsolin down-regulation is an early and almost constant event in colon carcinogenesis and is associated with the transition from adenoma to carcinoma. © 2008 Elsevier Inc. All rights reserved.
1. Introduction ☆
This work was supported by grants from the Ligue Nationale contre le Cancer, Comité du Rhône (Lyon, France) and from the Cancéropôle Lyon-Auvergne-Rhône-Alpes (CLARA; Lyon, France). ⁎ Corresponding author. E-mail address: [email protected] (J. Abello). 0046-8177/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2008.02.020
One critical step in the neoplastic process is the acquisition of a motile, invasive phenotype by tumor cells. Invasive cancer cells harbor actin protrusive structures called lamellipodia and filopodia. These extensions are largely dependent on a local dynamic reorganization of the actin
Transition from adenoma to carcinoma cytoskeleton, which, in turn, is finely tuned by multiple actin-binding proteins (ABPs) [1-3]. Gelsolin is a widely distributed ABP present in most vertebrate tissues, which plays a major role in actin cytoskeleton rearrangement by capping and severing Factin, thus modulating filament length and polymerization. These functions are tightly regulated by calcium ions, pH, and polyphosphoinositides [4]. Gelsolin is involved in cell motility, as shown in gelsolin-null mice by the reduced motility observed in dermal fibroblasts [5,6] and by the delayed retraction of filopodia in neurons [7]. Furthermore, overexpression of gelsolin enhances the migration of fibroblasts [8] and promotes invasion into type I collagen of MDCK and HEK293T cells [9]. The expression of gelsolin was reported to be frequently silenced in various cancers [10-17]. The down-regulation of gelsolin in malignancy suggests a possible tumor suppressor function, emphasized by reports demonstrating that overexpression of gelsolin inhibits the growth of bladder and lung cell lines in vitro [18,19] and reduces tumorigenicity in nude mice [20]. Similarly, small interfering RNA gelsolin knockdown enhances cell motility and invasiveness in mammary epithelial cancer cells and induced epithelial-mesenchymal transition [21]. However, no mutation of GSN was identified in any of the tumors investigated, suggesting a preponderance of epigenetic mechanisms in gelsolin silencing [12,13,18,22,23]. Despite the high incidence of colorectal cancer, only few and contradictory data are available regarding the expression of gelsolin in this type of malignancy and its precursor lesions. One immunohistochemical study reported a weak staining of gelsolin in 30 normal human colon mucosa and in 8 adenomas and an increase of gelsolin immunoreactivity in 22 adenocarcinomas [24]. In contrast, another report based on 15 human colon tumors showed a reduced gelsolin expression in about 60% of tumors, by comparison with the normal epithelial tissue [25]. This latter result was supported by DNA microarrays studies showing a reduced expression of gelsolin complementary DNA (cDNA) in colon cancer, as compared with normal colon tissue [26,27]. In the same way, a decrease in gelsolin expression has been observed during epitheliomesenchymal transition in human colon cancer cell lines: this phenotypic alteration is accompanied by the induction of invasive capacities and has been recently shown to be mediated by the transcriptional repressor Snail [28]. Finally, there is no information regarding the pattern of gelsolin expression during the early stages of colon carcinogenesis, especially during the transition from adenoma to carcinoma, which corresponds to the acquisition of invasive properties by neoplastic cells. We were therefore prompted to address the following questions: (a) What is the frequency of gelsolin alterations in colon adenocarcinomas? (b) Is there a correlation between the alterations in gelsolin expression and the clinical and pathologic features known to be associated with major invasive capacities? (c) At which stage of the
1421 adenoma-carcinoma sequence is it possible to detect alterations in gelsolin status? We therefore decided to study the pattern of gelsolin expression in a large series of neoplastic epithelial lesions of the colon, representative of the successive stages of the process of colon carcinogenesis, including adenomas of various grades and adenocarcinomas at various clinical stages.
2. Materials and methods 2.1. Study group All cases of colon carcinoma submitted to surgical resection at Hôpital Edouard Herriot, Lyon, France, between 1994 and 2003 were retrieved from the files of the Department of Pathology. Criteria for inclusion in the study group were: (a) sporadic tumor, without evidence of polyposis or familial predisposition syndrome, (b) available clinical information and tumor stage, (c) available tissue material. Among patients fulfilling these criteria, 69 cases were randomly selected. Cases of nonneoplastic epithelial lesions of the colon and of adenoma resected during the first 6 months of 2003 were randomly selected from the files of the Department of Pathology, Hôpital Edouard Herriot, Lyon, France.
2.2. Tissue material Tissue samples of primary tumors (obtained from endoscopic resections (n = 20) or surgical resections (n = 121)) were fixed in Bouin's fluid (n = 29) or buffered formalin (n = 112) and embedded in paraffin. For 11 cases, lymph node metastases were studied. In 13 cases, samples from resected liver metastases were also available and have been studied. In 12 cases of adenocarcinomas, cryopreserved tissue samples from neoplastic tissue and paired normal adjacent mucosa were available through the tumor tissue bank of Hospices Civils de Lyon (Tumorothèque des Hospices Civils de Lyon, France). Diagnostic criteria for the identification of hyperplastic polyps and serrated adenomas were from international recommendations [29]; only cases of conventional serrated adenomas were included; no case of “sessile serrated polyp” was studied. Neoplastic lesions were classified histologically according to the Vienna classification of digestive epithelial neoplasia [30]. The histologic type of adenocarcinomas was determined according to the World Health Organization classification [29]. The following items were recorded: macroscopical aspect, size, architecture (tubulous or villous), and grade of dysplasia for adenomas; localization, size, and grade of dysplasia for serrated adenomas; localization, histologic classification, pathologic stage, TNM classification, and microsatellite status [31] for adenocarcinomas.
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2.3. Immunohistochemistry A standard 2-step indirect streptavidin-biotin method was applied to 4-μm-thick sections of deparaffinized tissue (StreptABComplex/HRP Duet, Mouse/Rabbit amplification kit, DakoCytomation, Trappes, France). After antigen retrieval (40 minutes at 99°C in sodium citrate buffer, pH 7.3) and peroxidase quenching with 3% hydrogen peroxide for 10 minutes, a 1:1000 dilution of antigelsolin antibody 2C4 (Sigma, St Quentin Fallavier, France) was applied for 30 minutes. Biotinylated goat immunoglobulin G and streptavidin-biotin complex were then applied for 30 minutes each. Diaminobenzidine (DakoCytomation) was used as chromogen. Sections were counterstained with Mayer's hematoxylin (Réactifs RAL, Martillac, France). Primary antibody omission served as negative control. Smooth muscle cells and endothelial cells served as internal positive controls. For evaluation of the percentage of gelsolin-positive cells, the whole section was first scanned to appreciate the overall distribution of gelsolin expression within the tumor and its degree of heterogeneity; then, the percentage of gelsolin-
F. Gay et al. positive cells was determined in representative areas, by examination of 10 randomly selected microscopic fields (magnification ×200) of each tissue section by 2 different observers. A grading system was used to express the proportion of positive cells in each case, as follows: G1, above 90% positive cells per lesion; G2, 90% to 51% positive cells; G3, 50% to 10% positive cells; and G4, less than 10% positive cells per lesion. In each type of lesion (hyperplastic polyp, serrated adenoma, low grade adenoma, high-grade adenoma, adenocarcinoma), results were expressed as the percentage of cases for each grade.
2.4. Northern blot Total RNA was extracted from selected frozen tissues using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Ten micrograms was separated by electrophoresis through 1% agarose-formaldehyde gels and transferred onto HybondN membranes (GE Healthcare, Saclay, France). The subcloned human cytoplasmic gelsolin cDNA was used as a probe and was synthesized with ThermoScript reverse
Fig. 1 Gelsolin immunohistochemistry in normal human colon samples. Gelsolin expression is detectable in normal colon epithelium cells (A and B), as well as in endothelial cells surrounding blood vessels (C) and myocytes of the muscularis propria (D). Note the strong cytoplasmic labeling in goblet cells (arrows). Indirect immunoperoxidase followed by light nuclear counterstaining with Mayer's hematoxylin (original magnifications: A, ×220; B, ×280; C, ×320; D, ×180).
Transition from adenoma to carcinoma transcriptase enzyme (Invitrogen) using 2 μg of total RNA extracted from the human colon cell line Isreco1, which expressed gelsolin messenger RNA (mRNA) and protein (not shown). The resulting gelsolin cDNA was sequenced
1423 (Genome Express, Meylan, France) and compared to the reference sequence (GeneBank accession number NM_198252.1). Human β-actin cDNA (GeneBank accession number NM_001101) was used as control. The probes were
Fig. 2 Gelsolin immunohistochemistry in colorectal adenocarcinomas. When compared to normal epithelium, gelsolin expression is either completely undetectable (A) or much reduced (B, D, and E) in adenocarcinomas. In some cases, clusters of positive cells (B) can be observed. In most cases, there is no gelsolin staining along the infiltrative border of the tumor (C). At the cellular level, gelsolin expression is usually associated with the plasma membrane (B and D); in well-polarized cells, the labeling is detectable along the apical and the basolateral poles (D). In some cells, a cytoplasmic labeling is detectable: in conventional (lieberkuhnian) adenocarcinomas, the cytoplasmic labeling is associated with goblet-like cells (E); in colloid adenocarcinomas, it is present in all mucinous cells (F) (original magnifications: A, ×220; B, ×240; C, F, ×200; D, ×350; E, ×280).
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32 P-labeled
using the RadPrime DNA labeling kit (Invitrogen) and hybridized to the RNA membranes. After washing, membranes were exposed to autoradiography.
2.5. Statistical analysis Results were analyzed using a likelihood χ2 test or 1-way analysis of variance followed by post hoc Fisher comparison. Differences between 2 means with P b .05 were regarded as significant.
3. Results 3.1. Gelsolin is expressed in normal cells of the colon Gelsolin expression was detectable in all normal colon samples examined. In epithelial cells, gelsolin was detected in both enterocytes (Fig. 1A) and goblet cells (Fig. 1B). In enterocytes, gelsolin presented a polarized distribution along the apical and lateral cell membranes; no cytoplasmic or nuclear labeling was detected. In goblet cells, a strong cytoplasmic signal was detected at the basal pole of the cells and in the part of the cytoplasm surrounding the mucus vacuoles. In addition, gelsolin had a broad distribution in nonepithelial cells, endothelial cells (Fig. 1C), muscle cells (Fig. 1D), mesenchymal cells, and lymphoid cells (not shown).
3.2. Gelsolin expression is markedly and constantly decreased in primary adenocarcinomas Gelsolin expression was tested by immunohistochemistry in 69 primitive human colon adenocarcinomas and their paired normal mucosa. Gelsolin expression was decreased in
Fig. 3 Distribution of semiquantitative scores for gelsolin along the colon adenoma-carcinoma sequence on 132 normal epithelium, 12 serrated adenomas, 18 low-grade and 16 high-grade dysplasia adenomas, 17 intramucosal neoplasias, and 69 colon adenocarcinomas.
Fig. 4 Expression of gelsolin mRNA in human normal (N) and tumoral (T) colon epithelium from 12 patients presenting colorectal cancer. Gelsolin transcript is detected by Northern blot using a cDNA probe corresponding to the complete cytoplasmic gelsolin sequence. Blots were subsequently probed with a cDNA for β-actin to control RNA loading. Abbreviation: NA, not assessed in immunohistochemistry.
all tumors analyzed, resulting either in a complete absence of detection in 34.8% of cases (Fig. 2A) or in the focal staining of clusters of tumor cells in 65.2% of cases (Fig. 2B). In all cases, immunostaining of lymphocytes, muscle cells and fibroblasts served as a positive internal control [24]. In cases with clusters of gelsolin-positive cells, the mean percentage of gelsolin-positive tumor cells was 9.7 ± 1.7%. No adenocarcinoma was scored as grade 1 (N90% of positive cells); only 1.4% of cases were classified as grade 2 (90%51% of positive cells); 23.2% were scored as grade 3 (50%10% of positive cells); and 75.4%, as grade 4 (b10% of positive cells) (Fig. 3). Gelsolin-positive cells had no preferential distribution; in some cases, they could be observed along the invasion front, but in most cases, they appeared randomly distributed within the whole neoplastic proliferation without a preferential location along the tumor borders (Fig. 2C). In conventional (lieberkuhnian) adenocarcinomas, gelsolin immunostaining was usually restricted to the plasma membrane of tumor cells (Fig. 2B). Gelsolin expression was usually observed all over the cell surface of tumor cells; it was sometimes restricted to the apical pole of well polarized tumor cells in well and moderately differentiated adenocarcinomas (Fig. 2D). In addition, a cytoplasmic labeling was also observed in scattered goblet-like cells containing large mucus vacuoles (Fig. 2E) or in cells presenting the overall morphology of goblet cells but apparently devoid of mucus vacuoles. In colloid-type adenocarcinomas, a strong cytoplasmic labeling of tumor cells, reminiscent of the strong labeling of normal goblet cells, was present (Fig. 2F). The cytoplasmic distribution pattern was also present in signetring adenocarcinoma cells. Because the cytoplasmic isoform of gelsolin cannot be distinguished from the plasma form of the protein (a secreted
Transition from adenoma to carcinoma
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isoform with a signal peptide which may be present in the tumor vasculature) due to the current lack of suitable antibodies, we did not attempt to confirm our immunohistochemical results by Western blotting. We performed Northern blot experiments in 12 tumor samples, using a probe directed against cytoplasmic gelsolin mRNA. As compared to the adjacent mucosa, the apparent levels of gelsolin mRNA were markedly decreased in 7 of the 12 tumor samples tested; in 4 cases, no gelsolin mRNA could be detected in our technical conditions; in only 1 case, the apparent levels of gelsolin mRNA were comparable between the tumor tissue and the adjacent mucosa (Fig. 4). All the tumors tested showed decreased levels of the corresponding protein at immunohistochemical investigation and were classified as G3 or G4 scores. In only 1 case, there was an
apparent discrepancy between mRNA levels and protein levels. This may be because of the enrichment in gelsolinpositive tumor cells of the tissue sample used for Northern blot analysis because the distribution of such cells is usually focal. Thus, our results demonstrate a major down-regulation of gelsolin expression, at the protein and mRNA levels, in primary human colon adenocarcinomas. The decrease in gelsolin expression in adenocarcinomas is not correlated with the clinicopathologic features. We observed no significant correlation between a decrease or an absence of gelsolin expression and the main clinical and histologic characteristics of the tumors (Table 1), such as the TNM status and the degree of histologic differentiation of the lesion. Especially, there was no correlation between the gelsolin status and the extent of
Table 1 Relationship between gelsolin expression level and epidemiological and clinicopathologic characteristics of 69 human colon adenocarcinomas
Sex Male Female Mean age (SD) Pathology stage ⁎ I II III IV TNM classification ⁎ T1 T2 T3-T4 N+ M1 Histologic differentiation ⁎ Poorly differentiated Moderately differentiated Well differentiated Presence of signet-ring cells a Tumor localization a Distal (left-sided) Proximal (right-sided) Transversal Total colectomy Tumor size b ≤2.0 cm N2.0-5.0 cm N5.0 cm Microsatellite status c MSS MSI
Cases (n = 69)
Reduced gelsolin expression (n = 45) G2-G4 score
No gelsolin expression (n = 24) G4 score
Pd
40 (58.0%) 29 (42.0%) 63.9 (16.9)
26 (57.8%) 19 (42.2%) 63.3 (15.9)
14 (58.3%) 10 (41.7%) 67.9 (18.6)
.96
10 (14.5%) 23 (33.3%) 21 (30.4%) 15 (21.7%)
6 (13.3%) 17 (37.7%) 11 (24.4%) 11 (24.4%)
4 (16.7%) 6 (25.0%) 10 (41.7%) 4 (16.7%)
.42
6 (8.7%) 6 (8.7%) 57 (82.6%) 33 (47.8%) 16 (23.2%)
3 (6.7%) 4 (8.8%) 38 (84.4%) 20 (44.4%) 11 (24.4%)
3 (12.5%) 2 (8.3%) 19 (79.2%) 13 (54.2%) 5 (20.8%)
.71
9 (13.0%) 38 (55.1%) 22 (31.9%) 15 (21.7%)
4 (8.9%) 25 (55.5%) 16 (35.6%) 12 (26.7%)
5 (20.8%) 13 (54.2%) 6 (25.0%) 3 (12.5%)
.32
33 (47.8%) 31 (44.9%) 4 (5.8%) 1 (1.5%)
22 (48.9%) 19 (40.0%) 3 (6.7%) 1 (2.2%)
11 (45.8%) 12 (54.1%) 1 (4.2%) 0 (0.0%)
.81
3 (4.3%) 31 (44.9%) 35 (50.7%)
2 (4.4%) 19 (42.2%) 24 (53.3%)
1 (4.2%) 12 (50.0%) 11 (45.8%)
.82
53 (76.8%) 16 (23.1%)
33 (73.3%) 12 (26.7%)
20 (83.3%) 4 (16.7%)
.23
.29
.39 .96
.17
Abbreviations: M1, presence of distant metastasis; N+, corresponds to N1 (metastasis in 1-3 regional lymph nodes) and N2 (metastasis in 4 or more regional lymph nodes); MSS, microsatellite stability; MSI, microsatellite instability. a According to World Health Organization Classification of Tumors of the colon and rectum. b Tumor circumference or length. c Determined using markers Bat-25, Bat-26, D2S123, D5S346, and D17S250. d Determined using χ2 and analysis of variance tests.
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Fig. 5 Gelsolin immunohistochemistry in liver metastases. At low magnification (A), gelsolin is readily visible in tumor cells located along the invasion front (arrows), in contact with the adjacent nontumoral liver (L). At higher magnification (B), tumor cells located at the edge of the metastasis (arrows) and infiltrating normal sinusoids in the nontumoral liver (L) strongly express gelsolin; the labeling is cytoplasmic with a frequent submembranous reinforcement. Note that hepatocytes are negative for gelsolin, whereas Kupffer cells are strongly positive (original magnifications: A, ×200; B, ×320).
local invasion and evidence of lymph node or distant metastasis.
3.3. The expression pattern of gelsolin was not correlated in the primary tumor and its metastases Among the 11 cases of lymph node metastases, 1 was scored as G1, 1 as G2, 4 as G3, and 5 as G4. The expression pattern was comparable between the primary tumor and its lymph node metastases in only 5 cases; in 2 cases, there was an increase in the apparent expression level of gelsolin, whereas, in the remaining cases, the apparent expression level of gelsolin was lower in the lymph node metastasis than in the primary. In the 13 cases of liver metastases examined, 4 were scored as G2, 4 as G3, and 5 as G4. The expression pattern was comparable between the primary tumor and the liver metastasis in 5 cases; in 3 cases, there was an increase in the apparent expression level of gelsolin, whereas in the remaining cases, the apparent expression level of gelsolin
Table 2
was lower in the liver metastasis than in the corresponding primary. In 6 cases, there was a strong expression of gelsolin along the invasion front, contrasting with a low or undetectable expression of the protein in the core of the lesion (Fig. 5). The expression pattern and distribution of gelsolin protein at the cellular level presented the same correlations with the differentiation pattern of tumor cells than in the primary tumors.
3.4. Gelsolin expression is down-regulated early during the adenoma-carcinoma sequence The observation that gelsolin expression is usually lost in human adenocarcinomas prompted us to determine at which stage of the colonic carcinogenesis this event occurs. We therefore studied the pattern of gelsolin expression in 30 adenomas with low grade dysplasia (including 12 cases of serrated adenomas), 16 adenomas with high-grade dysplasia and 17 intramucosal adenocarcinomas (Table 2).
Epidemiological characteristics of 72 human colon preneoplastic lesions Total
No. of cases Sex Male Female Mean age (SD) Localization Distal (left-sided) Proximal (right-sided) Transversal Unknown
72 48 (66.7%) 24 (33.3%) 67.2 (13.0) 34 (47.2%) 28 (38.9%) 6 (8.3%) 4 (5.6%)
Hyperplastic polyps
Serrated adenomas
Low grade dysplasia adenomas
High grade dysplasia adenomas
Intramucosal neoplasia
9 (12.5%)
12 (16.7%)
18 (25.0%)
16 (22.2%)
17 (23.6%)
4 (8.3%) 5 (20.8%) 64.2 (3.8)
8 (16.7%) 4 (16.7%) 59.0 (14.5)
10 (20.8%) 8 (33.3%) 65.7 (14.0)
10 (20.8%) 6 (25.0%) 76.2 (12.7)
16 (33.4%) 1 (4.2%) 66.4 (3.6)
3 (8.8%) 7 (25.0%) 1 (16.6%) 1 (25.0%)
6 (17.6%) 9 (32.2%) 1 (16.6%) 2 (50.0%)
6 2 1 0
(17.7%) (7.1%) (16.6%) (0.0%)
9 7 0 0
(26.5%) (25.0%) (0.0%) (0.0%)
10 3 3 1
(29.4%) (10.7%) (50.0%) (25.0%)
Transition from adenoma to carcinoma In all adenomas of conventional type, gelsolin expression was retained in a variable proportion of neoplastic epithelial cells (Fig. 6). In conventional adenomas, 2 patterns of subcellular distribution were observed. In most cells, gelsolin presented a membrane distribution and was detectable all over the cell surface (Fig. 6A and C). A second pattern was observed in the goblet-like cells scattered within the lesion, identified through the presence of typical mucus vacuoles; in such cells, gelsolin was present in the cytoplasm, as in normal goblet cells. In addition, a cytoplasmic labeling was also present in some cells devoid of identifiable mucus vacuoles but with the typical shape of normal goblet cells (Fig. 6A), characterized by a thin basal pole and a dilated apical pole (Fig. 6B). Cells with a cytoplasmic labeling were usually scattered as single cells within the neoplastic proliferation. In the cases of serrated adenomas included in the study, all of low-grade dysplasia, 2 populations of mucus-secreting cells were observed. Like in conventional adenomas, a population of goblet-like cells was present, with a strong
1427 cytoplasmic labeling. In addition, in the upper part of the crypts and along the surface, a population of mucus-secreting cells resembling pyloric mucus-secreting cells was observed; in contrast to goblet-like cells, these cells displayed a membrane labeling over their whole surface, including their apical pole [32]. During the adenoma-carcinoma sequence, the proportion of gelsolin-positive cells was variable according to the grade of dysplasia and the stage of evolution of the disease. Of the cases of low grade adenomas, 72.2% were scored as grade 1 (Fig. 6A, B, and C [bottom part]; Fig. 3); positive cells presented either a membrane or a cytoplasmic labeling. In contrast, 75% of cases of high-grade adenomas (Fig. 6C, top) were scored as grade 3, whereas 12.5% were scored as grade 2 (Fig. 3); most residual positive cells presented a cytoplasmic labeling and a goblet cell-like morphology. In the same way, in intramucosal adenocarcinomas (Fig. 6D), most cases (64.7%) were scored as grade 3, and a small subset of lesions (17.6%) was even scored as grade 4 (Fig. 3).
Fig. 6 Gelsolin immunohistochemistry in different human preneoplastic lesions. These typical examples of low grade adenomas from conventional (A) or serrated (B) types retain readily detectable amounts of gelsolin staining; the labeling is membranous in absorptive cells and cytoplasmic in goblet cells (arrows). (C) In this adenoma with coexisting areas of low and high grade dysplasia, gelsolin is retained in low grade areas but undetectable in high grade areas. (D) This case of intramucosal adenocarcinoma shows a very low level of gelsolin expression. Original magnification: A, ×260; B, C, ×240; D, ×350.
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Fig. 7 Gelsolin immunohistochemistry in nonneoplastic colonic lesions. These cases of hyperplastic polyps (A, B) show a homogeneous expression of gelsolin; the labeling is membranous in pyloric-like mucus-secreting cells (arrowheads) and cytoplasmic in goblet cells (arrows) (original magnification: A, ×200; B, ×280).
3.5. Gelsolin expression is preserved in nonneoplastic lesions of the colon We studied 9 cases of hyperplastic polyps. In all cases, gelsolin was expressed by all surface epithelial cells. As in serrated adenomas, 2 populations of mucus-secreting cells were observed: (a) a population of goblet-like cells, with a strong cytoplasmic labeling; (b) a population of pyloric-like mucus-secreting cells, with a membrane labeling over the whole surface including the apical pole (Fig. 7). According to our grading system, all hyperplastic polyps were therefore graded as G1.
4. Discussion Our study, based on a large and representative series of neoplastic epithelial lesions of the colon, shows that (a) gelsolin expression is constantly and dramatically decreased in all colon adenocarcinomas as compared to the normal tissue; (b) this decrease is due to a transcriptional repression of gelsolin expression; (c) it occurs early in the process of colon carcinogenesis, during the transition from adenoma to carcinoma. We first verified that gelsolin was constitutively expressed in the epithelial cells of the colonic mucosa. Interestingly, we observed several patterns of subcellular distribution of gelsolin, depending on the cell differentiation. Gelsolin was found to be expressed along the plasma membrane of enterocytes and absorptive cells, but to present a diffuse cytoplasmic distribution in mucus-secreting goblet cells. This likely corresponds to differences in the organization of the cytoskeleton according to the functional specializations of the corresponding cell lineage. It is noteworthy that, according to our observations, the cytoplasmic distribution of gelsolin remains closely associated with the existence of
mucus-secreting differentiation in neoplastic epithelial cells, such as the goblet-like cells observed in adenomas and some conventional adenocarcinomas and the mucinous cells typical of the colloid-type adenocarcinomas. These observations prompt further research on the possibly different functional roles played by gelsolin and other cytoskeletal proteins in the various intestinal epithelial lineages. We then studied the pattern of gelsolin expression in a large series of colon adenocarcinomas. In our series, gelsolin expression was markedly reduced at the protein level in all cases tested. Moreover, in a selected number of cases, a marked decrease in the levels of the corresponding mRNAs was demonstrated, suggesting that the repression of gelsolin expression takes place at or before the translational level. There is some controversy in the literature regarding the pattern of gelsolin expression in colon adenocarcinomas [24,25]. Our findings are in accordance with one previous study and are strongly reminiscent of the observations performed in several other types of epithelial malignancies, including breast cancer, pancreatic carcinoma, non–small cell lung carcinoma and urothelial carcinoma [11,33-35]. Moreover, as previously described in other types of cancer, such as pancreatic carcinoma, lung carcinoma, and urothelial carcinoma, we could demonstrate that, despite the absence of gelsolin expression in the great majority of colon cancer cells, small clusters of positive cells scattered within an overall negative neoplastic proliferation could be detected in 65% of cases in our series of colon adenocarcinomas. In our experience, such clusters usually represented less than 10% of tumor cells within the neoplastic proliferation. In several types of carcinomas, including oral carcinoma, pancreatic carcinoma, urothelial carcinoma, and non–small cell lung carcinoma, the presence of such clusters of gelsolin-positive cells has been associated with poor prognosis and increased invasive capacities [33-36]. However, in our series, we were not able to demonstrate a correlation between the presence of clusters of gelsolin-positive cells and the major
Transition from adenoma to carcinoma clinicopathologic features of the tumor, especially those indicating major capacities of local invasion and metastatic dissemination. This may indicate that the pattern of gelsolin expression has little relevance in the late stages of the invasion process; however, we cannot exclude a lack of sensitivity of gelsolin as a biomarker, because of the very strong heterogeneity of its distribution, which makes easy to overlook the presence of small, scattered clusters of positive cells. Interestingly, there were some differences in the distribution pattern of gelsolin between primary tumors and liver metastases: in our series, a strong gelsolin expression was frequently observed along the invasion front in liver metastases, whereas this association was not observed in primary tumors. This suggests that the role of gelsolin may be different according to the stage of the invasion process or according to the environment. We finally attempted to determine at which stage of the process of colon carcinogenesis the loss of gelsolin expression occurs. By using a large series of nonneoplastic and early neoplastic lesions, we could demonstrate 2 points. First, the loss of gelsolin expression was found to be restricted to the neoplastic process: in our experience, gelsolin expression was constantly retained in non-neoplastic hyperplastic polyps. Second, we show that the shift to a decreased gelsolin expression takes place at an early stage of the neoplastic process, during the transition from adenoma to carcinoma. Indeed, in our series, most neoplastic cells in low grade adenomas, of both conventional and serrated types, retain a normal pattern of gelsolin expression; in contrast, in high-grade adenomas and early adenocarcinomas, most of neoplastic cells presented no detectable expression of gelsolin. Comparable patterns of alterations in gelsolin expression have been demonstrated in other examples of epithelial carcinogenesis, such as breast carcinogenesis, in which the down-regulation of gelsolin also occurs at the in situ stage, before the acquisition of invasive capacities [10,11]. The concurrent observations performed in various examples of epithelial carcinogenesis, including our findings in colon carcinogenesis, strongly suggest that the loss of gelsolin expression is a general mechanism associated with the transition from the early, in situ, noninvasive stage of the neoplastic process and its late invasive stage. Strong evidence, from both in vitro and in vivo experimental models suggests that the loss of gelsolin expression is associated with the acquisition of motile and invasive capacities by neoplastic cells [11,21]. It remains to be verified that the downregulation of gelsolin has the same functional consequences in vivo. The mechanisms responsible for the silencing of gelsolin expression at this stage of the neoplastic process have to be elucidated. So far, no mutation in the GSN gene has been reported in epithelial tumors; the role of promoter methylation and other epigenetic mechanisms has therefore to be explored [12,13,18,22,23]. It is more difficult to hypothesize on the functional consequences of the expression of gelsolin in a minority of
1429 tumor cells at the late stage of the neoplastic process. This seems to be another general mechanism, reported in many epithelial cancers. It is usually interpreted as the consequence of a reexpression of gelsolin in one or several subclones of neoplastic cells through an adaptive mechanism, selected because of the acquisition of major invasive properties. Although this interpretation is supported by some clinical evidence, it lacks strong experimental evidence. Moreover, other possibilities have to be considered: (a) it has been suggested that gelsolin may be involved in biologic processes other than motility, such as apoptosis; (b) in the particular case of colon adenocarcinoma, we cannot exclude that gelsolin may be an indirect marker of a cell differentiation process, as suggested by the specific patterns of gelsolin expression observed in the various normal epithelial cell lineages of the colon and their neoplastic counterparts. In conclusion, our results in colon carcinogenesis reinforce the concept that the down-regulation of gelsolin expression is a general mechanism at the early phase of the neoplastic process, during the transition from the noninvasive to the invasive stages, and likely to be associated with the acquisition of invasive properties by neoplastic cells.
Acknowledgments The authors are grateful to M Blanc, N Gadot, and F Lepinasse for their helpful technical advices.
References [1] Friedl P, Wolf K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 2003;3:362-74. [2] Yamazaki D, Kurisu S, Takenawa T. Regulation of cancer cell motility through actin reorganization. Cancer Sci 2005;96:379-86. [3] dos Remedios CG, Chhabra D, Kekic M, et al. Actin binding proteins: regulation of cytoskeletal microfilaments. Physiol Rev 2003;83: 433-73. [4] Kwiatkowski DJ. Functions of gelsolin: motility, signaling, apoptosis, cancer. Curr Opin Cell Biol 1999;11:103-8. [5] Witke W, Sharpe AH, Hartwig JH, Azuma T, Stossel TP, Kwiatkowski DJ. Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin. Cell 1995;81:41-51. [6] Azuma T, Witke W, Stossel TP, Hartwig JH, Kwiatkowski DJ. Gelsolin is a downstream effector of rac for fibroblast motility. EMBO J 1998; 17:1362-70. [7] Lu M, Witke W, Kwiatkowski DJ, Kosik KS. Delayed retraction of filopodia in gelsolin null mice. J Cell Biol 1997;138:1279-87. [8] Cunningham CC, Stossel TP, Kwiatkowski DJ. Enhanced motility in NIH 3T3 fibroblasts that overexpress gelsolin. Science 1991;251: 1233-6. [9] De Corte V, Bruyneel E, Boucherie C, Mareel M, Vandekerckhove J, Gettemans J. Gelsolin-induced epithelial cell invasion is dependent on Ras-Rac signaling. EMBO J 2002;21:6781-90. [10] Dong Y, Asch HL, Medina D, Ip C, Ip M, Guzman R, et al. Concurrent deregulation of gelsolin and cyclin D1 in the majority of human and rodent breast cancers. Int J Cancer 1999;81:930-8.
1430 [11] Winston JS, Asch HL, Zhang PJ, Edge SB, Hyland A, Asch BB. Downregulation of gelsolin correlates with the progression to breast carcinoma. Breast Cancer Res Treat 2001;65:11-21. [12] Dosaka-Akita H, Hommura F, Fujita H, et al. Frequent loss of gelsolin expression in non-small cell lung cancers of heavy smokers. Cancer Res 1998;58:322-7. [13] Lee HK, Driscoll D, Asch H, Asch B, Zhang PJ. Downregulated gelsolin expression in hyperplastic and neoplastic lesions of the prostate. Prostate 1999;40:14-9. [14] Rao J, Seligson D, Visapaa H, et al. Tissue microarray analysis of cytoskeletal actin-associated biomarkers gelsolin and E-cadherin in urothelial carcinoma. Cancer 2002;95:1247-57. [15] Visapaa H, Bui M, Huang Y, et al. Correlation of Ki-67 and gelsolin expression to clinical outcome in renal clear cell carcinoma. Urology 2003;61:845-50. [16] Kim JH, Choi YK, Kwon HJ, Yang HK, Choi JH, Kim DY. Downregulation of gelsolin and retinoic acid receptor beta expression in gastric cancer tissues through histone deacetylase 1. J Gastroenterol Hepatol 2004;19:218-24. [17] Noske A, Denkert C, Schober H, et al. Loss of gelsolin expression in human ovarian carcinomas. Eur J Cancer 2005;41:461-9. [18] Tanaka M, Mullauer L, Ogiso Y, et al. Gelsolin: a candidate for suppressor of human bladder cancer. Cancer Res 1995;55:3228-32. [19] Fujita H, Okada F, Hamada J, et al. Gelsolin functions as a metastasis suppressor in B16-BL6 mouse melanoma cells and requirement of the carboxyl-terminus for its effect. Int J Cancer 2001;93:773-80. [20] Sazawa A, Watanabe T, Tanaka M, et al. Adenovirus mediated gelsolin gene therapy for orthotopic human bladder cancer in nude mice. J Urol 2002;168:1182-7. [21] Tanaka H, Shirkoohi R, Nakagawa K, et al. siRNA gelsolin knockdown induces epithelial-mesenchymal transition with a cadherin switch in human mammary epithelial cells. Int J Cancer 2006;118: 1680-91. [22] Mielnicki LM, Ying AM, Head KL, Asch HL, Asch BB. Epigenetic regulation of gelsolin expression in human breast cancer cells. Exp Cell Res 1999;249:161-76. [23] Primeau M, Gagnon J, Momparler RL. Synergistic antineoplastic action of DNA methylation inhibitor 5-AZA-2′-deoxycytidine and histone deacetylase inhibitor depsipeptide on human breast carcinoma cells. Int J Cancer 2003;103:177-84.
F. Gay et al. [24] Porter RM, Holme TC, Newman EL, Hopwood D, Wilkinson JM, Cuschieri A. Monoclonal antibodies to cytoskeletal proteins: an immunohistochemical investigation of human colon cancer. J Pathol 1993;170:435-40. [25] Furuuchi K, Fujita H, Tanaka M, et al. Gelsolin as a suppressor of malignant phenotype in human colon cancer. Tumor Target 1997;2: 277-83. [26] Zhang L, Zhou W, Velculescu VE, et al. Gene expression profiles in normal and cancer cells. Science 1997;276:1268-72. [27] Bertucci F, Salas S, Eysteries S, et al. Gene expression profiling of colon cancer by DNA microarrays and correlation with histoclinical parameters. Oncogene 2004;23:1377-91. [28] De Craene B, Gilbert B, Stove C, Bruyneel E, van Roy F, Berx G. The transcription factor snail induces tumor cell invasion through modulation of the epithelial cell differentiation program. Cancer Res 2005;65:6237-44. [29] WHO classification of tumours—pathology and genetics of tumours of digestive system. Geneva, Switzerland: WHO Press; 2000.. [30] Schlemper RJ, Riddell RH, Kato Y, et al. The Vienna classification of gastrointestinal epithelial neoplasia. Gut 2000;47:251-5. [31] Boland CR, Thibodeau SN, Hamilton SR, et al. A National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 1998;58:5248-57. [32] Yao T, Kouzuki T, Kajiwara M, Matsui N, Oya M, Tsuneyoshi M. ‘Serrated’ adenoma of the colorectum, with reference to its gastric differentiation and its malignant potential. J Pathol 1999;187:511-7. [33] Thompson CC, Ashcroft FJ, Patel S, et al. Pancreatic cancer cells overexpress gelsolin family-capping proteins, which contribute to their cell motility. Gut 2007;56:95-106. [34] Shieh DB, Godleski J, Herndon JE, et al. Cell motility as a prognostic factor in Stage I nonsmall cell lung carcinoma: the role of gelsolin expression. Cancer 1999;85:47-57. [35] Rao J. Targeting actin remodeling profiles for the detection and management of urothelial cancers-a perspective for bladder cancer research. Front Biosci 2002;7:e1-8. [36] Shieh DB, Chen IW, Wei TY, et al. Tissue expression of gelsolin in oral carcinogenesis progression and its clinicopathological implications. Oral Oncol 2006;42:599-606.
www.elsevier.com/locate/humpath
Original contribution
In colon carcinogenesis, the cytoskeletal protein gelsolin is down-regulated during the transition from adenoma to carcinoma☆ Fabien Gay PhD student a,b,c , Yann Estornes PhD a,b,c , Jean-Christophe Saurin MD, PhD a,b,c , Marie-Odile Joly-Pharaboz MD a,b,c,d , Evelyne Friederich PhD e , Jean-Yves Scoazec MD, PhD a,b,c,d , Jacques Abello PhD a,b,c,⁎ a
Inserm, U865, Lyon, F-69372, France; Inserm, IFR62, Lyon, F-69372, France Université de Lyon, Lyon, F-69003, France c Université de Lyon 1, F-69372 Villeurbanne, France d Hospices Civils de Lyon, Hôpital Edouard Herriot, Service Central d’Anatomie et Cytologie Pathologiques, F-69437 Lyon, France e Department of Life Sciences, University of Luxembourg, L-1511, Luxembourg, Luxembourg b
Received 9 November 2007; revised 7 February 2008; accepted 25 February 2008
Keywords: Gelsolin; Immunohistochemistry; Adenoma; Adenocarcinoma; Colon
Summary The actin-binding protein gelsolin is involved in cell motility via the regulation of actin cytoskeleton, and its expression is modified in several human cancers. However, the potential implication of this protein in colorectal carcinogenesis is debated. By using immunohistochemistry, we studied gelsolin expression in 69 cases of colon adenocarcinomas and in 72 lesions representative of the different stages of colonic tumorigenesis. In addition, we performed Northern blot analysis of gelsolin messenger RNA in 12 paired samples of human colon cancer and normal corresponding mucosa. Gelsolin protein and messenger RNA expressions were severely down-regulated in all adenocarcinomas tested. Moreover, gelsolin protein was down-regulated in a large proportion of high-grade adenomas (14/16) before the acquisition of invasive properties but in only a small proportion of low grade adenomas and serrated adenomas (2/30) and in none of the 9 cases of nonneoplastic hyperplastic polyps tested. Our results therefore demonstrate that gelsolin down-regulation is an early and almost constant event in colon carcinogenesis and is associated with the transition from adenoma to carcinoma. © 2008 Elsevier Inc. All rights reserved.
1. Introduction ☆
This work was supported by grants from the Ligue Nationale contre le Cancer, Comité du Rhône (Lyon, France) and from the Cancéropôle Lyon-Auvergne-Rhône-Alpes (CLARA; Lyon, France). ⁎ Corresponding author. E-mail address: [email protected] (J. Abello). 0046-8177/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2008.02.020
One critical step in the neoplastic process is the acquisition of a motile, invasive phenotype by tumor cells. Invasive cancer cells harbor actin protrusive structures called lamellipodia and filopodia. These extensions are largely dependent on a local dynamic reorganization of the actin
Transition from adenoma to carcinoma cytoskeleton, which, in turn, is finely tuned by multiple actin-binding proteins (ABPs) [1-3]. Gelsolin is a widely distributed ABP present in most vertebrate tissues, which plays a major role in actin cytoskeleton rearrangement by capping and severing Factin, thus modulating filament length and polymerization. These functions are tightly regulated by calcium ions, pH, and polyphosphoinositides [4]. Gelsolin is involved in cell motility, as shown in gelsolin-null mice by the reduced motility observed in dermal fibroblasts [5,6] and by the delayed retraction of filopodia in neurons [7]. Furthermore, overexpression of gelsolin enhances the migration of fibroblasts [8] and promotes invasion into type I collagen of MDCK and HEK293T cells [9]. The expression of gelsolin was reported to be frequently silenced in various cancers [10-17]. The down-regulation of gelsolin in malignancy suggests a possible tumor suppressor function, emphasized by reports demonstrating that overexpression of gelsolin inhibits the growth of bladder and lung cell lines in vitro [18,19] and reduces tumorigenicity in nude mice [20]. Similarly, small interfering RNA gelsolin knockdown enhances cell motility and invasiveness in mammary epithelial cancer cells and induced epithelial-mesenchymal transition [21]. However, no mutation of GSN was identified in any of the tumors investigated, suggesting a preponderance of epigenetic mechanisms in gelsolin silencing [12,13,18,22,23]. Despite the high incidence of colorectal cancer, only few and contradictory data are available regarding the expression of gelsolin in this type of malignancy and its precursor lesions. One immunohistochemical study reported a weak staining of gelsolin in 30 normal human colon mucosa and in 8 adenomas and an increase of gelsolin immunoreactivity in 22 adenocarcinomas [24]. In contrast, another report based on 15 human colon tumors showed a reduced gelsolin expression in about 60% of tumors, by comparison with the normal epithelial tissue [25]. This latter result was supported by DNA microarrays studies showing a reduced expression of gelsolin complementary DNA (cDNA) in colon cancer, as compared with normal colon tissue [26,27]. In the same way, a decrease in gelsolin expression has been observed during epitheliomesenchymal transition in human colon cancer cell lines: this phenotypic alteration is accompanied by the induction of invasive capacities and has been recently shown to be mediated by the transcriptional repressor Snail [28]. Finally, there is no information regarding the pattern of gelsolin expression during the early stages of colon carcinogenesis, especially during the transition from adenoma to carcinoma, which corresponds to the acquisition of invasive properties by neoplastic cells. We were therefore prompted to address the following questions: (a) What is the frequency of gelsolin alterations in colon adenocarcinomas? (b) Is there a correlation between the alterations in gelsolin expression and the clinical and pathologic features known to be associated with major invasive capacities? (c) At which stage of the
1421 adenoma-carcinoma sequence is it possible to detect alterations in gelsolin status? We therefore decided to study the pattern of gelsolin expression in a large series of neoplastic epithelial lesions of the colon, representative of the successive stages of the process of colon carcinogenesis, including adenomas of various grades and adenocarcinomas at various clinical stages.
2. Materials and methods 2.1. Study group All cases of colon carcinoma submitted to surgical resection at Hôpital Edouard Herriot, Lyon, France, between 1994 and 2003 were retrieved from the files of the Department of Pathology. Criteria for inclusion in the study group were: (a) sporadic tumor, without evidence of polyposis or familial predisposition syndrome, (b) available clinical information and tumor stage, (c) available tissue material. Among patients fulfilling these criteria, 69 cases were randomly selected. Cases of nonneoplastic epithelial lesions of the colon and of adenoma resected during the first 6 months of 2003 were randomly selected from the files of the Department of Pathology, Hôpital Edouard Herriot, Lyon, France.
2.2. Tissue material Tissue samples of primary tumors (obtained from endoscopic resections (n = 20) or surgical resections (n = 121)) were fixed in Bouin's fluid (n = 29) or buffered formalin (n = 112) and embedded in paraffin. For 11 cases, lymph node metastases were studied. In 13 cases, samples from resected liver metastases were also available and have been studied. In 12 cases of adenocarcinomas, cryopreserved tissue samples from neoplastic tissue and paired normal adjacent mucosa were available through the tumor tissue bank of Hospices Civils de Lyon (Tumorothèque des Hospices Civils de Lyon, France). Diagnostic criteria for the identification of hyperplastic polyps and serrated adenomas were from international recommendations [29]; only cases of conventional serrated adenomas were included; no case of “sessile serrated polyp” was studied. Neoplastic lesions were classified histologically according to the Vienna classification of digestive epithelial neoplasia [30]. The histologic type of adenocarcinomas was determined according to the World Health Organization classification [29]. The following items were recorded: macroscopical aspect, size, architecture (tubulous or villous), and grade of dysplasia for adenomas; localization, size, and grade of dysplasia for serrated adenomas; localization, histologic classification, pathologic stage, TNM classification, and microsatellite status [31] for adenocarcinomas.
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2.3. Immunohistochemistry A standard 2-step indirect streptavidin-biotin method was applied to 4-μm-thick sections of deparaffinized tissue (StreptABComplex/HRP Duet, Mouse/Rabbit amplification kit, DakoCytomation, Trappes, France). After antigen retrieval (40 minutes at 99°C in sodium citrate buffer, pH 7.3) and peroxidase quenching with 3% hydrogen peroxide for 10 minutes, a 1:1000 dilution of antigelsolin antibody 2C4 (Sigma, St Quentin Fallavier, France) was applied for 30 minutes. Biotinylated goat immunoglobulin G and streptavidin-biotin complex were then applied for 30 minutes each. Diaminobenzidine (DakoCytomation) was used as chromogen. Sections were counterstained with Mayer's hematoxylin (Réactifs RAL, Martillac, France). Primary antibody omission served as negative control. Smooth muscle cells and endothelial cells served as internal positive controls. For evaluation of the percentage of gelsolin-positive cells, the whole section was first scanned to appreciate the overall distribution of gelsolin expression within the tumor and its degree of heterogeneity; then, the percentage of gelsolin-
F. Gay et al. positive cells was determined in representative areas, by examination of 10 randomly selected microscopic fields (magnification ×200) of each tissue section by 2 different observers. A grading system was used to express the proportion of positive cells in each case, as follows: G1, above 90% positive cells per lesion; G2, 90% to 51% positive cells; G3, 50% to 10% positive cells; and G4, less than 10% positive cells per lesion. In each type of lesion (hyperplastic polyp, serrated adenoma, low grade adenoma, high-grade adenoma, adenocarcinoma), results were expressed as the percentage of cases for each grade.
2.4. Northern blot Total RNA was extracted from selected frozen tissues using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Ten micrograms was separated by electrophoresis through 1% agarose-formaldehyde gels and transferred onto HybondN membranes (GE Healthcare, Saclay, France). The subcloned human cytoplasmic gelsolin cDNA was used as a probe and was synthesized with ThermoScript reverse
Fig. 1 Gelsolin immunohistochemistry in normal human colon samples. Gelsolin expression is detectable in normal colon epithelium cells (A and B), as well as in endothelial cells surrounding blood vessels (C) and myocytes of the muscularis propria (D). Note the strong cytoplasmic labeling in goblet cells (arrows). Indirect immunoperoxidase followed by light nuclear counterstaining with Mayer's hematoxylin (original magnifications: A, ×220; B, ×280; C, ×320; D, ×180).
Transition from adenoma to carcinoma transcriptase enzyme (Invitrogen) using 2 μg of total RNA extracted from the human colon cell line Isreco1, which expressed gelsolin messenger RNA (mRNA) and protein (not shown). The resulting gelsolin cDNA was sequenced
1423 (Genome Express, Meylan, France) and compared to the reference sequence (GeneBank accession number NM_198252.1). Human β-actin cDNA (GeneBank accession number NM_001101) was used as control. The probes were
Fig. 2 Gelsolin immunohistochemistry in colorectal adenocarcinomas. When compared to normal epithelium, gelsolin expression is either completely undetectable (A) or much reduced (B, D, and E) in adenocarcinomas. In some cases, clusters of positive cells (B) can be observed. In most cases, there is no gelsolin staining along the infiltrative border of the tumor (C). At the cellular level, gelsolin expression is usually associated with the plasma membrane (B and D); in well-polarized cells, the labeling is detectable along the apical and the basolateral poles (D). In some cells, a cytoplasmic labeling is detectable: in conventional (lieberkuhnian) adenocarcinomas, the cytoplasmic labeling is associated with goblet-like cells (E); in colloid adenocarcinomas, it is present in all mucinous cells (F) (original magnifications: A, ×220; B, ×240; C, F, ×200; D, ×350; E, ×280).
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32 P-labeled
using the RadPrime DNA labeling kit (Invitrogen) and hybridized to the RNA membranes. After washing, membranes were exposed to autoradiography.
2.5. Statistical analysis Results were analyzed using a likelihood χ2 test or 1-way analysis of variance followed by post hoc Fisher comparison. Differences between 2 means with P b .05 were regarded as significant.
3. Results 3.1. Gelsolin is expressed in normal cells of the colon Gelsolin expression was detectable in all normal colon samples examined. In epithelial cells, gelsolin was detected in both enterocytes (Fig. 1A) and goblet cells (Fig. 1B). In enterocytes, gelsolin presented a polarized distribution along the apical and lateral cell membranes; no cytoplasmic or nuclear labeling was detected. In goblet cells, a strong cytoplasmic signal was detected at the basal pole of the cells and in the part of the cytoplasm surrounding the mucus vacuoles. In addition, gelsolin had a broad distribution in nonepithelial cells, endothelial cells (Fig. 1C), muscle cells (Fig. 1D), mesenchymal cells, and lymphoid cells (not shown).
3.2. Gelsolin expression is markedly and constantly decreased in primary adenocarcinomas Gelsolin expression was tested by immunohistochemistry in 69 primitive human colon adenocarcinomas and their paired normal mucosa. Gelsolin expression was decreased in
Fig. 3 Distribution of semiquantitative scores for gelsolin along the colon adenoma-carcinoma sequence on 132 normal epithelium, 12 serrated adenomas, 18 low-grade and 16 high-grade dysplasia adenomas, 17 intramucosal neoplasias, and 69 colon adenocarcinomas.
Fig. 4 Expression of gelsolin mRNA in human normal (N) and tumoral (T) colon epithelium from 12 patients presenting colorectal cancer. Gelsolin transcript is detected by Northern blot using a cDNA probe corresponding to the complete cytoplasmic gelsolin sequence. Blots were subsequently probed with a cDNA for β-actin to control RNA loading. Abbreviation: NA, not assessed in immunohistochemistry.
all tumors analyzed, resulting either in a complete absence of detection in 34.8% of cases (Fig. 2A) or in the focal staining of clusters of tumor cells in 65.2% of cases (Fig. 2B). In all cases, immunostaining of lymphocytes, muscle cells and fibroblasts served as a positive internal control [24]. In cases with clusters of gelsolin-positive cells, the mean percentage of gelsolin-positive tumor cells was 9.7 ± 1.7%. No adenocarcinoma was scored as grade 1 (N90% of positive cells); only 1.4% of cases were classified as grade 2 (90%51% of positive cells); 23.2% were scored as grade 3 (50%10% of positive cells); and 75.4%, as grade 4 (b10% of positive cells) (Fig. 3). Gelsolin-positive cells had no preferential distribution; in some cases, they could be observed along the invasion front, but in most cases, they appeared randomly distributed within the whole neoplastic proliferation without a preferential location along the tumor borders (Fig. 2C). In conventional (lieberkuhnian) adenocarcinomas, gelsolin immunostaining was usually restricted to the plasma membrane of tumor cells (Fig. 2B). Gelsolin expression was usually observed all over the cell surface of tumor cells; it was sometimes restricted to the apical pole of well polarized tumor cells in well and moderately differentiated adenocarcinomas (Fig. 2D). In addition, a cytoplasmic labeling was also observed in scattered goblet-like cells containing large mucus vacuoles (Fig. 2E) or in cells presenting the overall morphology of goblet cells but apparently devoid of mucus vacuoles. In colloid-type adenocarcinomas, a strong cytoplasmic labeling of tumor cells, reminiscent of the strong labeling of normal goblet cells, was present (Fig. 2F). The cytoplasmic distribution pattern was also present in signetring adenocarcinoma cells. Because the cytoplasmic isoform of gelsolin cannot be distinguished from the plasma form of the protein (a secreted
Transition from adenoma to carcinoma
1425
isoform with a signal peptide which may be present in the tumor vasculature) due to the current lack of suitable antibodies, we did not attempt to confirm our immunohistochemical results by Western blotting. We performed Northern blot experiments in 12 tumor samples, using a probe directed against cytoplasmic gelsolin mRNA. As compared to the adjacent mucosa, the apparent levels of gelsolin mRNA were markedly decreased in 7 of the 12 tumor samples tested; in 4 cases, no gelsolin mRNA could be detected in our technical conditions; in only 1 case, the apparent levels of gelsolin mRNA were comparable between the tumor tissue and the adjacent mucosa (Fig. 4). All the tumors tested showed decreased levels of the corresponding protein at immunohistochemical investigation and were classified as G3 or G4 scores. In only 1 case, there was an
apparent discrepancy between mRNA levels and protein levels. This may be because of the enrichment in gelsolinpositive tumor cells of the tissue sample used for Northern blot analysis because the distribution of such cells is usually focal. Thus, our results demonstrate a major down-regulation of gelsolin expression, at the protein and mRNA levels, in primary human colon adenocarcinomas. The decrease in gelsolin expression in adenocarcinomas is not correlated with the clinicopathologic features. We observed no significant correlation between a decrease or an absence of gelsolin expression and the main clinical and histologic characteristics of the tumors (Table 1), such as the TNM status and the degree of histologic differentiation of the lesion. Especially, there was no correlation between the gelsolin status and the extent of
Table 1 Relationship between gelsolin expression level and epidemiological and clinicopathologic characteristics of 69 human colon adenocarcinomas
Sex Male Female Mean age (SD) Pathology stage ⁎ I II III IV TNM classification ⁎ T1 T2 T3-T4 N+ M1 Histologic differentiation ⁎ Poorly differentiated Moderately differentiated Well differentiated Presence of signet-ring cells a Tumor localization a Distal (left-sided) Proximal (right-sided) Transversal Total colectomy Tumor size b ≤2.0 cm N2.0-5.0 cm N5.0 cm Microsatellite status c MSS MSI
Cases (n = 69)
Reduced gelsolin expression (n = 45) G2-G4 score
No gelsolin expression (n = 24) G4 score
Pd
40 (58.0%) 29 (42.0%) 63.9 (16.9)
26 (57.8%) 19 (42.2%) 63.3 (15.9)
14 (58.3%) 10 (41.7%) 67.9 (18.6)
.96
10 (14.5%) 23 (33.3%) 21 (30.4%) 15 (21.7%)
6 (13.3%) 17 (37.7%) 11 (24.4%) 11 (24.4%)
4 (16.7%) 6 (25.0%) 10 (41.7%) 4 (16.7%)
.42
6 (8.7%) 6 (8.7%) 57 (82.6%) 33 (47.8%) 16 (23.2%)
3 (6.7%) 4 (8.8%) 38 (84.4%) 20 (44.4%) 11 (24.4%)
3 (12.5%) 2 (8.3%) 19 (79.2%) 13 (54.2%) 5 (20.8%)
.71
9 (13.0%) 38 (55.1%) 22 (31.9%) 15 (21.7%)
4 (8.9%) 25 (55.5%) 16 (35.6%) 12 (26.7%)
5 (20.8%) 13 (54.2%) 6 (25.0%) 3 (12.5%)
.32
33 (47.8%) 31 (44.9%) 4 (5.8%) 1 (1.5%)
22 (48.9%) 19 (40.0%) 3 (6.7%) 1 (2.2%)
11 (45.8%) 12 (54.1%) 1 (4.2%) 0 (0.0%)
.81
3 (4.3%) 31 (44.9%) 35 (50.7%)
2 (4.4%) 19 (42.2%) 24 (53.3%)
1 (4.2%) 12 (50.0%) 11 (45.8%)
.82
53 (76.8%) 16 (23.1%)
33 (73.3%) 12 (26.7%)
20 (83.3%) 4 (16.7%)
.23
.29
.39 .96
.17
Abbreviations: M1, presence of distant metastasis; N+, corresponds to N1 (metastasis in 1-3 regional lymph nodes) and N2 (metastasis in 4 or more regional lymph nodes); MSS, microsatellite stability; MSI, microsatellite instability. a According to World Health Organization Classification of Tumors of the colon and rectum. b Tumor circumference or length. c Determined using markers Bat-25, Bat-26, D2S123, D5S346, and D17S250. d Determined using χ2 and analysis of variance tests.
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Fig. 5 Gelsolin immunohistochemistry in liver metastases. At low magnification (A), gelsolin is readily visible in tumor cells located along the invasion front (arrows), in contact with the adjacent nontumoral liver (L). At higher magnification (B), tumor cells located at the edge of the metastasis (arrows) and infiltrating normal sinusoids in the nontumoral liver (L) strongly express gelsolin; the labeling is cytoplasmic with a frequent submembranous reinforcement. Note that hepatocytes are negative for gelsolin, whereas Kupffer cells are strongly positive (original magnifications: A, ×200; B, ×320).
local invasion and evidence of lymph node or distant metastasis.
3.3. The expression pattern of gelsolin was not correlated in the primary tumor and its metastases Among the 11 cases of lymph node metastases, 1 was scored as G1, 1 as G2, 4 as G3, and 5 as G4. The expression pattern was comparable between the primary tumor and its lymph node metastases in only 5 cases; in 2 cases, there was an increase in the apparent expression level of gelsolin, whereas, in the remaining cases, the apparent expression level of gelsolin was lower in the lymph node metastasis than in the primary. In the 13 cases of liver metastases examined, 4 were scored as G2, 4 as G3, and 5 as G4. The expression pattern was comparable between the primary tumor and the liver metastasis in 5 cases; in 3 cases, there was an increase in the apparent expression level of gelsolin, whereas in the remaining cases, the apparent expression level of gelsolin
Table 2
was lower in the liver metastasis than in the corresponding primary. In 6 cases, there was a strong expression of gelsolin along the invasion front, contrasting with a low or undetectable expression of the protein in the core of the lesion (Fig. 5). The expression pattern and distribution of gelsolin protein at the cellular level presented the same correlations with the differentiation pattern of tumor cells than in the primary tumors.
3.4. Gelsolin expression is down-regulated early during the adenoma-carcinoma sequence The observation that gelsolin expression is usually lost in human adenocarcinomas prompted us to determine at which stage of the colonic carcinogenesis this event occurs. We therefore studied the pattern of gelsolin expression in 30 adenomas with low grade dysplasia (including 12 cases of serrated adenomas), 16 adenomas with high-grade dysplasia and 17 intramucosal adenocarcinomas (Table 2).
Epidemiological characteristics of 72 human colon preneoplastic lesions Total
No. of cases Sex Male Female Mean age (SD) Localization Distal (left-sided) Proximal (right-sided) Transversal Unknown
72 48 (66.7%) 24 (33.3%) 67.2 (13.0) 34 (47.2%) 28 (38.9%) 6 (8.3%) 4 (5.6%)
Hyperplastic polyps
Serrated adenomas
Low grade dysplasia adenomas
High grade dysplasia adenomas
Intramucosal neoplasia
9 (12.5%)
12 (16.7%)
18 (25.0%)
16 (22.2%)
17 (23.6%)
4 (8.3%) 5 (20.8%) 64.2 (3.8)
8 (16.7%) 4 (16.7%) 59.0 (14.5)
10 (20.8%) 8 (33.3%) 65.7 (14.0)
10 (20.8%) 6 (25.0%) 76.2 (12.7)
16 (33.4%) 1 (4.2%) 66.4 (3.6)
3 (8.8%) 7 (25.0%) 1 (16.6%) 1 (25.0%)
6 (17.6%) 9 (32.2%) 1 (16.6%) 2 (50.0%)
6 2 1 0
(17.7%) (7.1%) (16.6%) (0.0%)
9 7 0 0
(26.5%) (25.0%) (0.0%) (0.0%)
10 3 3 1
(29.4%) (10.7%) (50.0%) (25.0%)
Transition from adenoma to carcinoma In all adenomas of conventional type, gelsolin expression was retained in a variable proportion of neoplastic epithelial cells (Fig. 6). In conventional adenomas, 2 patterns of subcellular distribution were observed. In most cells, gelsolin presented a membrane distribution and was detectable all over the cell surface (Fig. 6A and C). A second pattern was observed in the goblet-like cells scattered within the lesion, identified through the presence of typical mucus vacuoles; in such cells, gelsolin was present in the cytoplasm, as in normal goblet cells. In addition, a cytoplasmic labeling was also present in some cells devoid of identifiable mucus vacuoles but with the typical shape of normal goblet cells (Fig. 6A), characterized by a thin basal pole and a dilated apical pole (Fig. 6B). Cells with a cytoplasmic labeling were usually scattered as single cells within the neoplastic proliferation. In the cases of serrated adenomas included in the study, all of low-grade dysplasia, 2 populations of mucus-secreting cells were observed. Like in conventional adenomas, a population of goblet-like cells was present, with a strong
1427 cytoplasmic labeling. In addition, in the upper part of the crypts and along the surface, a population of mucus-secreting cells resembling pyloric mucus-secreting cells was observed; in contrast to goblet-like cells, these cells displayed a membrane labeling over their whole surface, including their apical pole [32]. During the adenoma-carcinoma sequence, the proportion of gelsolin-positive cells was variable according to the grade of dysplasia and the stage of evolution of the disease. Of the cases of low grade adenomas, 72.2% were scored as grade 1 (Fig. 6A, B, and C [bottom part]; Fig. 3); positive cells presented either a membrane or a cytoplasmic labeling. In contrast, 75% of cases of high-grade adenomas (Fig. 6C, top) were scored as grade 3, whereas 12.5% were scored as grade 2 (Fig. 3); most residual positive cells presented a cytoplasmic labeling and a goblet cell-like morphology. In the same way, in intramucosal adenocarcinomas (Fig. 6D), most cases (64.7%) were scored as grade 3, and a small subset of lesions (17.6%) was even scored as grade 4 (Fig. 3).
Fig. 6 Gelsolin immunohistochemistry in different human preneoplastic lesions. These typical examples of low grade adenomas from conventional (A) or serrated (B) types retain readily detectable amounts of gelsolin staining; the labeling is membranous in absorptive cells and cytoplasmic in goblet cells (arrows). (C) In this adenoma with coexisting areas of low and high grade dysplasia, gelsolin is retained in low grade areas but undetectable in high grade areas. (D) This case of intramucosal adenocarcinoma shows a very low level of gelsolin expression. Original magnification: A, ×260; B, C, ×240; D, ×350.
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Fig. 7 Gelsolin immunohistochemistry in nonneoplastic colonic lesions. These cases of hyperplastic polyps (A, B) show a homogeneous expression of gelsolin; the labeling is membranous in pyloric-like mucus-secreting cells (arrowheads) and cytoplasmic in goblet cells (arrows) (original magnification: A, ×200; B, ×280).
3.5. Gelsolin expression is preserved in nonneoplastic lesions of the colon We studied 9 cases of hyperplastic polyps. In all cases, gelsolin was expressed by all surface epithelial cells. As in serrated adenomas, 2 populations of mucus-secreting cells were observed: (a) a population of goblet-like cells, with a strong cytoplasmic labeling; (b) a population of pyloric-like mucus-secreting cells, with a membrane labeling over the whole surface including the apical pole (Fig. 7). According to our grading system, all hyperplastic polyps were therefore graded as G1.
4. Discussion Our study, based on a large and representative series of neoplastic epithelial lesions of the colon, shows that (a) gelsolin expression is constantly and dramatically decreased in all colon adenocarcinomas as compared to the normal tissue; (b) this decrease is due to a transcriptional repression of gelsolin expression; (c) it occurs early in the process of colon carcinogenesis, during the transition from adenoma to carcinoma. We first verified that gelsolin was constitutively expressed in the epithelial cells of the colonic mucosa. Interestingly, we observed several patterns of subcellular distribution of gelsolin, depending on the cell differentiation. Gelsolin was found to be expressed along the plasma membrane of enterocytes and absorptive cells, but to present a diffuse cytoplasmic distribution in mucus-secreting goblet cells. This likely corresponds to differences in the organization of the cytoskeleton according to the functional specializations of the corresponding cell lineage. It is noteworthy that, according to our observations, the cytoplasmic distribution of gelsolin remains closely associated with the existence of
mucus-secreting differentiation in neoplastic epithelial cells, such as the goblet-like cells observed in adenomas and some conventional adenocarcinomas and the mucinous cells typical of the colloid-type adenocarcinomas. These observations prompt further research on the possibly different functional roles played by gelsolin and other cytoskeletal proteins in the various intestinal epithelial lineages. We then studied the pattern of gelsolin expression in a large series of colon adenocarcinomas. In our series, gelsolin expression was markedly reduced at the protein level in all cases tested. Moreover, in a selected number of cases, a marked decrease in the levels of the corresponding mRNAs was demonstrated, suggesting that the repression of gelsolin expression takes place at or before the translational level. There is some controversy in the literature regarding the pattern of gelsolin expression in colon adenocarcinomas [24,25]. Our findings are in accordance with one previous study and are strongly reminiscent of the observations performed in several other types of epithelial malignancies, including breast cancer, pancreatic carcinoma, non–small cell lung carcinoma and urothelial carcinoma [11,33-35]. Moreover, as previously described in other types of cancer, such as pancreatic carcinoma, lung carcinoma, and urothelial carcinoma, we could demonstrate that, despite the absence of gelsolin expression in the great majority of colon cancer cells, small clusters of positive cells scattered within an overall negative neoplastic proliferation could be detected in 65% of cases in our series of colon adenocarcinomas. In our experience, such clusters usually represented less than 10% of tumor cells within the neoplastic proliferation. In several types of carcinomas, including oral carcinoma, pancreatic carcinoma, urothelial carcinoma, and non–small cell lung carcinoma, the presence of such clusters of gelsolin-positive cells has been associated with poor prognosis and increased invasive capacities [33-36]. However, in our series, we were not able to demonstrate a correlation between the presence of clusters of gelsolin-positive cells and the major
Transition from adenoma to carcinoma clinicopathologic features of the tumor, especially those indicating major capacities of local invasion and metastatic dissemination. This may indicate that the pattern of gelsolin expression has little relevance in the late stages of the invasion process; however, we cannot exclude a lack of sensitivity of gelsolin as a biomarker, because of the very strong heterogeneity of its distribution, which makes easy to overlook the presence of small, scattered clusters of positive cells. Interestingly, there were some differences in the distribution pattern of gelsolin between primary tumors and liver metastases: in our series, a strong gelsolin expression was frequently observed along the invasion front in liver metastases, whereas this association was not observed in primary tumors. This suggests that the role of gelsolin may be different according to the stage of the invasion process or according to the environment. We finally attempted to determine at which stage of the process of colon carcinogenesis the loss of gelsolin expression occurs. By using a large series of nonneoplastic and early neoplastic lesions, we could demonstrate 2 points. First, the loss of gelsolin expression was found to be restricted to the neoplastic process: in our experience, gelsolin expression was constantly retained in non-neoplastic hyperplastic polyps. Second, we show that the shift to a decreased gelsolin expression takes place at an early stage of the neoplastic process, during the transition from adenoma to carcinoma. Indeed, in our series, most neoplastic cells in low grade adenomas, of both conventional and serrated types, retain a normal pattern of gelsolin expression; in contrast, in high-grade adenomas and early adenocarcinomas, most of neoplastic cells presented no detectable expression of gelsolin. Comparable patterns of alterations in gelsolin expression have been demonstrated in other examples of epithelial carcinogenesis, such as breast carcinogenesis, in which the down-regulation of gelsolin also occurs at the in situ stage, before the acquisition of invasive capacities [10,11]. The concurrent observations performed in various examples of epithelial carcinogenesis, including our findings in colon carcinogenesis, strongly suggest that the loss of gelsolin expression is a general mechanism associated with the transition from the early, in situ, noninvasive stage of the neoplastic process and its late invasive stage. Strong evidence, from both in vitro and in vivo experimental models suggests that the loss of gelsolin expression is associated with the acquisition of motile and invasive capacities by neoplastic cells [11,21]. It remains to be verified that the downregulation of gelsolin has the same functional consequences in vivo. The mechanisms responsible for the silencing of gelsolin expression at this stage of the neoplastic process have to be elucidated. So far, no mutation in the GSN gene has been reported in epithelial tumors; the role of promoter methylation and other epigenetic mechanisms has therefore to be explored [12,13,18,22,23]. It is more difficult to hypothesize on the functional consequences of the expression of gelsolin in a minority of
1429 tumor cells at the late stage of the neoplastic process. This seems to be another general mechanism, reported in many epithelial cancers. It is usually interpreted as the consequence of a reexpression of gelsolin in one or several subclones of neoplastic cells through an adaptive mechanism, selected because of the acquisition of major invasive properties. Although this interpretation is supported by some clinical evidence, it lacks strong experimental evidence. Moreover, other possibilities have to be considered: (a) it has been suggested that gelsolin may be involved in biologic processes other than motility, such as apoptosis; (b) in the particular case of colon adenocarcinoma, we cannot exclude that gelsolin may be an indirect marker of a cell differentiation process, as suggested by the specific patterns of gelsolin expression observed in the various normal epithelial cell lineages of the colon and their neoplastic counterparts. In conclusion, our results in colon carcinogenesis reinforce the concept that the down-regulation of gelsolin expression is a general mechanism at the early phase of the neoplastic process, during the transition from the noninvasive to the invasive stages, and likely to be associated with the acquisition of invasive properties by neoplastic cells.
Acknowledgments The authors are grateful to M Blanc, N Gadot, and F Lepinasse for their helpful technical advices.
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