Different phenotypes in human prostate cancer: alpha6 or alpha3 integrin in cell-extracellular adhesion sites

Abstract

The distribution of alpha6/alpha3 integrin in adhesion complexes at the basal membrane in human normal and cancer prostate glands was analyzed in 135 biopsies from 61 patients. The levels of the polarized alpha6/alpha3 integrin expression at the basal membrane of prostate tumor glands were determined by quantitative immunohistochemistry. The alpha6/alpha3 integrin expression was compared with Gleason sum score, pathological stage, and preoperative serum prostate-specific antigen (PSA). The associations were assessed by statistical methods. Eighty percent of the tumors expressed the alpha6 or alpha3 integrin and 20% was integrin-negative. Gleason sum score, but not serum PSA, was associated with the integrin expression. Low Gleason sum score correlated with increased integrin expression, high Gleason sum score with low and negative integrin expression. Three prostate tumor phenotypes were distinguished based on differential integrin expression. Type I coexpressed both alpha6 and alpha3 subunits, type II exclusively expressed alpha6 integrin, and type III expressed alpha3 integrin only. Fifteen cases were further examined for the codistribution of vinculin, paxillin, and CD 151 on frozen serial sections using confocal laser scanning microscopy. The alpha6/alpha3 integrins, CD151, paxillin, and vinculin were present within normal glands. In prostate carcinoma, alpha6 integrin was colocalized with CD 151, but not with vinculin or paxillin. In tumor phenotype I, the alpha6 subunit did not colocalize with the alpha subunit indicating the existence of two different adhesion complexes. Human prostate tumors display on their cell surface the alpha6beta1 and/or alpha3beta1 integrins. Three tumor phenotypes associated with two different adhesion complexes were identified, suggesting a reorganization of cell adhesion structures in prostate cancer.

Figure 1

Immunoperoxidase localization of β4, α3, and α6 integrins in normal and malignant prostate glands. Frozen serial sections of prostate tissue containing normal glands (N) and Gleason sum score (3+3=6) infiltrating carcinoma were stained with H&E (A) or immunoreacted with specific antibodies to β4 (B), α3 (C), or α6 (D) integrins as indicated. All three integrins were expressed in the epithelial basal membrane in the normal glands. Some weak staining with anti-β4 integrin antibody was detected within the malignant epithelial cytoplasm, but none in the basal membrane of malignant epithelia. Note β4 expression in periglandular vascular basement membranes. Both antibodies specific for α3 and α6 integrins focally outlined the basal membranes (arrows) of malignant glands. Arrowheads: blood vessels; N: normal glands. Bar, 20 µm.

Figure 2

Expression profile of α6 and α3 integrins polarized at the basal membranes of human prostate carcinoma epithelium. One hundred and thirty-five needle biopsy containing carcinoma obtained from 61 patients were analysed for the expression of the α6 and/or α3 integrin subunit and the intensity of the immunoreaction was scored: 2 to 12=all expression; 2 to 8=low expression; 9 to 12=high expression. Graph (A) demonstrates the percentage of all biopsies analyzed (135=100%) that expressed α6 and/or α3 integrin. The group of cases with the expression levels 2 to 12 included the cases with high expression levels 9 to 12, which also were analyzed separately (A). Graph (B) shows the expression profile of the integrin subunits in association with the tumor grading (Gleason sum score). The definitions of the descriptors are: α6, all cases which expressed α6 integrin; α3, all cases which expressed α3 integrin; integrin-negative, all cases which were integrin-negative. None of the tumors expressed polarized β4 integrin. The Gleason sum score was found to be inversely associated with the integrin expression.

Figure 3

Distribution pattern of tumor phenotypes as compared to Gleason sum score (A) and pathological stage (B). (A) The distribution pattern of the three phenotypes in relation to the Gleason sum score. A total number of 94 biopsies (100%) were graded with Gleason sum score ≤6, and 41 biopsies (100%) with Gleason sum score ≥7. (B) The distribution of the three phenotypes in relation to the pathological staging within the biopsy group with Gleason sum score ≤6. Of the 135 biopsies examined, 94 were graded with Gleason sum score ≤6 and were correlated after prostatectomy to pathological staging as follows: T1a to T2c=29 biopsies; T3a to T3b=38 biopsies; and T3c=27 biopsies. (C) The average expression intensity (2–12) of the individual integrin subunits in each phenotype compared to the pathological stage and Gleason sum score ≤6. (D) The distribution of the three phenotypes in relation to the pathological stage T3a to T3b within the biopsy group (26 biopsies) with Gleason sum score ≥7 (for pathological stages T1a to T2c and T3c, the number of cases available for examination was too low to be representative).

Figure 4

Confocal laser scanning micrographs of double-label immunofluorescence microscopy of α6 integrin (B, C, E, F; green) with vinculin (A, C; red) and paxillin (D, F; red) in human normal prostate glands. Overlays are shown in (C) and (F). Note that in most of the areas, the red was resolved from the green. A few areas showed yellow indicating some colocalization of the α6 integrin with either vinculin or paxillin. Note that vinculin is also present in smooth muscle cells. Bars, 10 µm (A), 2 µm (D).

Figure 5

Confocal laser scanning micrographs of simultaneous detection of α6 integrin (A, D, G, J; red) and cytokeratins 5 and 14 (B; green), paxillin (E; green) or vinculin (H, K; green) in the malignant prostate epithelium of two patients (patient 1: Gleason grade 3 +3, pathological stage T3c, shown in A–C; patient 2: Gleason grade 3+3, pathological stage T3c, shown in D–L). Overlays are shown in (C, F, I, L). Malignant glands were distinguished from normal glands by the absence of cytokeratins 5 and 14 (A–C). Note the abundant staining of the basal membrane in normal glands, whereas the basal membranes in malignant glands were outlined by an attenuated staining. In serial sections (D–I), the distribution of α6 was compared to paxillin (D–F) and vinculin (G–L). The field within the white frame in (I) is shown at high magnification in (J–L), revealing that α6 was not distributed to focal adhesions in malignant prostate glands lacking the β4 integrin subunit. Both paxillin and vinculin strongly stained smooth muscle cells. Vinculin was also expressed in the apical junctional zone of the luminal cells (arrows). N, normal gland; C, cancer; C1, cancer gland 1 indicating the same gland in two serial sections; L, lumen of gland. Bars, 10 µm (B), 5 µm (E, F), 2 µm (K).

Figure 6

Simultaneous detection of α6 integrin (red) with CD151 (green) in human malignant prostate glands (Gleason grade 3+3, pathological stage T3c) as viewed by indirect immunofluorescence and laser scanning confocal microscopy. CD151 (A, B, D: green) and α6 integrin (A, B, C: red) colocalized (yellow) at the interface of epithelium to basal membrane. (A) Shows field of malignant prostate glands. Malignant gland 1 is shown at higher magnification in (B–D). N, normal gland. Bars, 10 µm (A), 5 µm (B).

Figure 7

Confocal laser scanning micrographs of α6 (green; E) and α3 integrin (red; D) in human normal (A) and neoplastic prostate (Gleason grade 3+3, pathological stage T3a; A–E). (A) Field with normal (N) and cancer glands. Cancer gland 1 is shown at higher magnification in (B, D, and E). (C) Shows high resolution field of basal membrane in cancer gland 1. The monoclonal antibody specific for α3 integrin subunit showed some cross-reaction with the nuclei. N, normal glands; V, vessel. Bars, 50 µm (A), 20 µm (D), 2 µm (C).