Prevalent overexpression of prolyl isomerase Pin1 in human cancers

Abstract

Phosphorylation of proteins on serine or threonine residues preceding proline (pSer/Thr-Pro) is a major regulatory mechanism in cell proliferation and transformation. Interestingly, the pSer/Thr-Pro motifs in proteins exist in two distinct cis and trans conformations, whose conversion rate is normally reduced on phosphorylation, but is catalyzed specifically by the prolyl isomerase Pin1. Pin1 can catalytically induce conformational changes in proteins after phosphorylation, thereby having profound effects on catalytic activity, dephosphorylation, protein-protein interactions, subcellular location, and/or turnover of certain phosphorylated proteins. Recently, it has been shown that Pin1 is overexpressed in human breast cancer cell lines and cancer tissues and plays a critical role in the transformation of mammary epithelial cells by activating multiple oncogenic pathways. Furthermore, Pin1 expression is an excellent independent prognostic marker in prostate cancer. However, little is known about Pin1 expression in other human normal and cancerous tissues. In the present study, we quantified Pin1 expression in 2041 human tumor samples and 609 normal tissue samples as well as normal and transformed human cell lines. We found that Pin1 was usually expressed at very low levels in most normal tissues and its expression was normally associated with cell proliferation, with high Pin1 levels being found only in a few cell types. However, Pin1 was strikingly overexpressed in many different human cancers. Most tumors (38 of 60 tumor types) have Pin1 overexpression in more than 10% of the cases, as compared with the corresponding normal controls, which included prostate, lung, ovary, cervical, brain tumors, and melanoma. Consistent with these findings, Pin1 expression in human cancer cell lines was also higher than that in the normal cell lines examined. These results indicate that Pin1 overexpression is a prevalent and specific event in human cancers. Given previous findings that Pin1 expression is an excellent prognostic marker in prostate cancer and that inhibition of Pin1 can suppress transformed phenotypes and inhibit tumor cell growth, these findings may have important implications for the pathogenesis, diagnosis, and treatment of human cancers.

Figure 1

Immunocytochemical staining of Pin1 in cultured cells. Cells were incubated with anti-Pin1 antibodies, followed by secondary biotinylated antibody and ABC (avidin and biotinylated horseradish peroxidase complex)-diaminobenzidine reaction. A: WI38 (normal fibroblast) cell line; B: HUV-EC-C (normal epithelial) cell line; C: DU 145 (prostate cancer) cell line; D: RPMI 7951 (melanoma) cell line; E: SW620 (colon cancer) cell line; F: PC3 (prostate cancer) cell line. Original magnifications, ×40.

Figure 2

Immunoblotting analysis of Pin1 expression of cell extracts. Total cell lysates prepared from different cell lines were subjected to immunoblotting analysis with monoclonal Pin1 and actin antibodies. The locations of Pin1 and actin are indicated. The average Pin1concentrations as determined from the Pin1:actin ratio from a minimum of three Western blots in cell lines is shown in Table 1. Purified protein standards were used to determine the linear range for both proteins. Immunohistochemistry of these cell lines is shown in Figure 1.

Figure 3

Immunohistochemistry of Pin1 in cancer tissues on conventional tissue sections. Sections from paraffin-embedded tissues were subjected to immunostaining with anti-Pin1 antibodies. A: Breast cancer; B: lung cancer; C: ovary cancer; D: lymphoma. Original magnifications: ×20; ×40 (insets).

Figure 4

Immunoblotting and immunohistochemistry analyses of Pin1 expression in normal and cancerous prostate tissues. Prostate tissue lysates were subjected to immunoblotting analysis with monoclonal Pin1 and actin antibodies. Tumor lysates are identified by patient number. Lysates 722, 874, and 910 are Gleason score 8. Lysates 959, 913, and 855 are Gleason score 9. Both normal and cancer tissue were obtained from patients 874 and 910. The locations of Pin1 and actin are indicated. The average Pin1concentration as determined from the Pin1:actin ratio from multiple Western blots in cell lines is shown in Table 3. Tissue samples from 041 and 941 (normal) and 855 and 959 (cancer) were also subjected to immunostaining with monoclonal Pin1 antibody. Original magnifications, ×20.

Figure 5

Evaluation of Pin1 immunostaining by ACIS and its correlation with manual evaluation. A: An example of quantitative evaluation for Pin1 immunostaining on tissue microarrays using ACIS. Three small circles were used to selectively measure tumor cell area and obtained the mean of the intensity and percentage of positive tumor cells. B: Correlation between manual evaluation and automated analysis in brain tumors (n = 104).

Figure 6

Comparison of Pin1 expression in tumor tissues and corresponding normal tissues on tissue microarrays. Tissue microarray sections were subjected to immunostaining with anti-Pin1 antibodies. A: Normal prostate tissue; B: prostate cancer; C: normal lung tissue; D: lung cancer; E: lung cancer staining negative for Pin1; F: normal ovary tissue; and G: ovary cancer. Original magnifications, ×10.

Figure 7

Overexpression of Pin1 in human cancers. Pin1 immunostaining was evaluated quantitatively using ACIS, with the Pin1 score = staining intensity + percentage of positive immunostaining. The percentage of total cases showing elevated levels of Pin1 = the numbers of tumor samples with score larger than the score of the highest normal case divided by the total number of tumor samples.

Figure 8

Pin1 levels in three representative categories of tumor tissues in comparison with corresponding normal tissues. Pin1 immunostaining was evaluated quantitatively using ACIS with the Pin1 score = staining intensity + percentage of positive immunostaining. Overexpression of Pin1 in prostate cancers (A) and small cell lung cancers (B). No change of Pin1 in skin squamous cell cancers (C) and underexpression of Pin1 in kidney cancers (D).