Preliminary Investigation Of Basement Membrane Alterations In The Carcinogenesis Of Esophageal Squamous Cell Carcinoma

Background and ObjectiveBasement membranes are a component of extracellular matrix, a part of the interstitial microenvironment. The primary functions of basement membranes include: induction and maintenance of cell polarity; the organization of cells into tissues; regulation of cell differentiation; proliferation and apoptosis of epithelial cells. Basement membranes are composed of specialized proteins, including laminin, fibronectin, perlecan, collagen IV, nidogen and heparin sulfate proteoglycan. In the esophagus, laminin is a glycoprotein which binds to basement membrane components and may serve as an attachment factor for esophageal epithelium. Proliferation of esophageal epithelium requires interaction of transmembrane proteins with components of the basement membrane.β1 integrin is an important link between basement membranes and epithelial cells. Integrin-basement membrane interactions activate signaling cascades such as mitogen-activated protein kinases. Altered expression of β1 integrin is an important stimulant of the ERK/MAPK pathway.Many genes and signaling pathways are involved in the pathogenesis of esophageal carcinoma. For example, the ERK/MAPK signaling pathway is a proliferation pathway. Aberrant and deregulated functioning of ERK/MAPK caninitiate and support carcinogenesis.The goals of the present study were to investigate basement membrane alterations during the evolution of dysplasia to cancer in esophageal squamous cell carcinoma. We correlated these changes with expression of pi integrin, activation of the ERK/MAPK signaling pathway, and proliferation of the esophageal epithelium.Materials and MethodsFrom July 2000 to December 2004, 436 surgically-resected specimens of esophageal carcinoma were obtained from the Departments of Pathology of Shantou University Medical College, and from the First and Second Affiliated Hospitals of Shantou University Medical College. In each case, full-thickness sections were taken from the grossly invasive tumor and adjacent mucosa, and from representative apparently normal mucosa at the proximal and distal ends of these specimens. These tissue blockswere used to prepare to histologic sections, tissue microarrays, and for molecular analysis. Three tissue arrays included 26 cases with normal epithelium, 28 with hyperplasia, 18 with dysplasia, 27 with carcinoma in situ and 129 with invasive carcinoma. In addition, 21 cases of hyperplasia, 13 cases of dysplasia, and 13 case of carcinoma in situ were obtained by microdissection of the histologic sections.Hematoxylin and eosin-stained sections were used to evaluate the degree of inflammation and epithelial dysplasia. The periodic acid-Schiff stain (PAS) was used to evaluate the structure of the basement membrane. The expression of laminin, pi integrin, activated ERK and Ki67 were examined using the Envision immunohistochemistry method. Western blot and RT-PCR technologies were used to detect the exprsssion of protein and mRNA of pi integrin.ResultsHistopathological alterations of basement membranesHistopathological evaluation of the basement membrane stained with Periodic acide-Schiff stain showed: a significant relationship between basement membranealterations and inflammation score. The various categories of the basement membrane alterations were: focal point loss (<10Mm); segmental loss (10-50 Hm); zonal loss (loss of basement membrane over a large area, —50-1 OOMm); fragmentation and thinning. Fragmentation and thinning were mainly seen in hyperplastic and dysplastic epithelium. Segmental loss was seen in carcinoma in situ. Zone loss were mainly distributed in invasive carcinoma. The expression of 01 integrinThere was a significant relationship between the extent of basement membrane alteration and the expression of pi integrin (^=0.91, P<0.0\). The expression of pi integrin was highest in epithelial cells associated with zonal loss of the basement membrane. The second highest level of expression was associated with segmental loss. Expression of pi integrin was the lowest in normal epithelium. There was no significant difference in the expression of pi integrin between basement membranes showing thining and fragmention. The expression of pi integrin was highest in invasive carcinoma cells, followed by carcinoma in situ. Expression of pi integrin was the lowest in normal epithelial cells.The expression of activated ERKThere was a significant relationship between the extent of basement membrane alteration and the expression of activated ERK (^=0.871, P<0.01). The expression of activated ERK was highest in epithelial cells associated with zonal loss of the basement membrane. The second highest level of expression was associated with segmental loss. Expression of activated ERK was the lowest in normal epithelium. There was no significant difference in the expression of activated ERK between basement membranes showing thining and fragmention. The expression of activated ERK was highest in invasive carcinoma cells, followed by carcinoma in situ. Expression of activated ERK was the lowest in normal epithelial cells.The expression of Ki67, PCNAThere was a significant relationship between the extent of basement membrane alteration and the expression of Ki67, PCNA (rs=0.95, ^=0.94, P<0.01). The expression of Ki67 was highest in epithelial cells associated with zonal loss of the basement membrane. The second highest level of expression was associated with segmental loss. Expression of Ki67 was the lowest in normal epithelium.There was no significant difference in the expression of Ki67 between basement membranes showing thining and fragmention. The expression of Ki67, PCNA was highest in invasive carcinoma cells, followed by carcinoma in situ. Expression of Ki67, PCNA was the lowest in normal epithelial cells.Conclusions1. There was a direct correlation (rs = 0.87, P<0.01) between the degree of inflammation and alteration in the structure of the basement membranes.2. There was a significant relationship between the extent of basement membrane alteration and the expression of pi integrin (^=0.91, ?<0.01).3. There was direct correlation between the degree dysplasia and expression of p-ERK((F= 14.31, R0. 01)),Ki67(F=6.84, K0. 01).4. Basement membrane alterations also correlated with expression of pi integrin, p-ERK((rs=0.87, P<0. 01)), Ki 67 (r=0. 95, P<0. ODand PCNA(r=0.95, P<0. 01).5. The correlations suggest that there is a direct relationship between basement membrane and programin of dysplasia to cancer in squamous cell carcinoma of the esophagus...