Expression of von Hippel-Lindau tumor suppressor and tumor-associated carbonic anhydrases IX and XII in normal and neoplastic colorectal mucosa

Abstract

Aim: To analyze possible relationships between CA IX/CA XII and pVHL expression in normal and neoplastic colorectal mucosa.

Methods: Immunohistochemical staining of 42 tissue specimens obtained from 17 cancer patients was performed to evaluate the distribution and semi-quantitatively assess the levels of CA IX, CA XII and pVHL. VHL mRNAs from 14 fresh-frozen tumors was amplified by RT-PCR and subjected to sequencing. CA9 and CA12 mRNA levels were analyzed by semi-quantitative RT-PCR in comparison with VEGF as an indicator of hypoxia that uncouples the pVHL control.

Results: Tumor tissues were associated with a borderline increase of CA IX staining signal and slight but significant decrease of CA XII immunoreactivity, whereas no association was found for pVHL. Sequence analysis of RT-PCR-amplified VHL mRNAs revealed no deletions/mutations, suggesting that they were VHL-competent. We did not observe any correlation between pVHL and CA IX/CA XII proteins as well as between VEGF and CA9 mRNAs, but the tumor-associated changes in mRNA levels of VEGF and CA12 showed a significant inverse relationship.

Conclusion: Our results indicate that CA9 and CA12 are regulated by different intratumoral factors and that lack of apparent relationship between the levels of CA IX/CA XII and pVHL cannot be fully assigned to uncoupling of negative regulatory function of pVHL by tumor hypoxia signified by induced VEGF transcription. The interplay between the functional pVHL and CA IX/CA XII in colorectal tumors seems rather complex and is not evident merely at the expression levels.

Figure 1

Schematic drawing of pVHL domain composition. Epitope region for the monoclonal antibodies VHL06 and VHL40 used in immunohistochemistry is shown below the scheme[30]. Positions of primers (a = antisense, s = sense) used for the amplification of two overlapping RT-PCR products are indicated above the exon map. Involvement of HIF-α-binding β-domain and elongin C-binding α-domain of pVHL in the negative regulation of CA XII and CA IX is illustrated according to data described by Ivanov et al[6].

Figure 2

Associations of the proteins with colorectal tumors based on semi-quantitative immunohistochemical assessment and analysis of the data using the Mann-Whitney rank test. Each box indicates the range of staining indices from the 25% to the 75% quantile, the horizontal line denotes the median and the whiskers above and below the box show the highest and lowest index. Values of significance are given above the graphs.

Figure 3

Immunohistochemical detection of pVHL (B), CA IX (C) and CA XII (D) in serial sections from the normal colon with details shown on magnified areas in the left corners. Arrows in panel C designate a focal membrane signal specific for CA IX. A shows hematoxylin-eosin (H&E) staining of a parallel section. Original magnification, ×100.

Figure 4

Immunohistochemical staining of pVHL, CA IX and CA XII in parallel sections of (B-D) moderate (lower part) and grave (upper part) adenomas and of (F-H) transitional epithelium (small arrows) located between histologically normal region (arrowhead) and grade I adenocarcinoma (not shown). Corresponding H&E staining is shown in panels A and E. Original magnifications, ×100.

Figure 5

Examples of immunohistochemical staining of pVHL, CA IX and CA XII in parallel sections of adenocarcinoma (grade II) with (A-D) or without (E-H) mucinous component. One crypt remains negative for CA IX (C). Corresponding H&E staining is shown in panels A and E. Original magnifications, ×100.

Figure 6

Graphical illustration of the tumor-associated differences in expression of pVHL, CA IX, CA XII and p53 in individual patients analyzed by immunohistochemistry (the same person is designated by the same number in all graphs). Values of staining indices obtained in normal tissues were subtracted from those obtained in corresponding pathological lesions from the same person (mean value was used when more tumor specimens were available from one patient). Resulting data were shown in histograms on compatible scales demonstrating the range of differences related to each marker and allowing for their visual comparisons. There were no relationships found between the studied proteins.

Figure 7

Graphical illustration of the tumor-associated differences in mRNA levels of CA9, CA12 and VEGF in individual patients analyzed by RT-PCR. Values representing relative amounts of mRNAs expressed in normal tissues were subtracted from those obtained in paired pathological lesions. Resulting data were drawn in histograms on compatible scales allowing for their comparisons. Highly significant inverse relationship was found between CA12 and VEGF by χ² analysis (P = 0.0063).