TRIM59 upregulation in human prostate cancer TMA: correlation with tumorigenesis and tumor progression by TRIM59 intensity until high grade CaP
We designated TRIM59 as one of the ‘tumorigenesis-associated’ genes correlated with SV40 Tag oncogenesis in mouse prostate cancer (GEM-CaP) models.17
SV40 Tag is essentially only required for the initiation of tumorigenesis, that is, the ‘hit-and-run’ effect, in GEM-CaP, but not for the tumour progression and metastasis directly. The ‘tumorigenesis-associated’ effect is due to the initial binding of the Tag oncogene with retinoblastoma (pRB), p53 proteins and several transcriptional coactivators. Once this process is initiated, the signal transduction will continue on, even without the initiation effectors.
In GEM-CaP models, the TRIM59 protein upregulation correlated with tumorigenesis and progression, and downregulated in the high-grade CaP by IHC.17
We assume this ‘tumorigenesis-associated’ effect of TRIM59 may apply to human cancer studies.
We first characterised that TRIM59 antibody (#72) can cross-react with and recognise specifically human TRIM59 counterpart (for details see online supplementary figure S1). We tested by IHC in a TMA of CaP patients (n=88, 176 cores). TRIM59 IHC signals detected in an automated digital image system were identified mostly in cytoplasm of luminal cells (A), which is different from rapid tumour progression mouse CaP models. The intensity (score=2) in PIN (n=4, A) was higher than in non-tumour area (normal and benign prostatic hyperplasia). Moderate-to-strong expression was observed in Gleason score 6 (3+3, n=25), 7 (4+3, n=15) and 8 (4+4, n=14). In high-grade CaP (score 4+5, n=8, A), TRIM59-IHC signals were lower. As shown in a graph of B, TRIM59 protein IHC signals correlated significantly (p=0.014) with tumorigenesis and progression from PIN to WDCaP (Gleason grade 1–2, scores 2–4) and MDCaP (Gleason grade 2–4, scores 4–8) (graph B), and decreased in high-grade CaP (Gleason score 9–10) with p=0.018, which is similar to GEMs.17
Figure 1 Correlation of TRIM59 immunhistochemistry (IHC) staining in prostate cancer cases in tissue microarray (TMA) assessed by the Gleason grading system. All panels in (A) were shown haematoxylin staining, ×20. Non-tumour: weak or negative, prostate (more ...)
TRIM59 upregulation in human renal cell carcinoma patients: correlation of tumorigenesis and tumour progression with TRIM59 intensity until high-grade RCC
Next, we demonstrated that TRIM59 is not androgen-responsive and likely not prostate tissue-specific, indicating that the tumorigenesis role of TRIM59 may be applied to all or most of human cancers. Online supplementary figure S2 showed this result in detail of IHC test by TRIM59 antibody#72 on a TMA of the LNCaP human CaP xenografts in nude/severe combined immuno deficiency castrated mice.
We extended results from prostate cancer clinical samples to kidney cancer. We started 75 renal cell carcinoma (RCC) patients including all 5 different types of RCC tumours: 43 clear cell carcinoma (representative IHC figures shown in A–D), 11 papillary RCC (E,F), 13 chromophobe RCC (G–I), 2 sarcomatoid RCC (J), and 6 cystic RCC (K,L). RCC cases analysed with Fuhrman grade 1–4 were 4, 38, 28 and 5, respectively. TRIM59-IHC staining in normal area including proximal tubules was negative ( last row). Background staining was eliminated by testing antibody dilutions (1
100, 200, 500, 1200 till 5000) while tumour-specific signals of TRIM59 proteins were noticeable. Endogenous biotin signals were blocked and excluded by additional block reagents (avidin-biotin blocking reagent kit).
Figure 2 Immunohistochemistry analysis of TRIM59 expression in kidney cancer (RCC, renal cell carcinoma) cases: correlation with tumour grade by intensity detecting early tumorigenesis. Five types of RCC with different grades were shown clear cell carcinoma (A–D), (more ...)
TRIM59 IHC staining in tumour areas in RCC () was different from cases of CaP-TMA (mainly cytoplasmic). TRIM59 IHC staining was found in both cytoplasm and nucleus in all RCC grades and types (). We assessed TRIM59-IHC by visual scoring of both intensity (cytoplasmic staining) and extent (% nucleus staining) microscopically. Correlation of TRIM59 IHC signals by scoring the intensity in cytoplasm with grades of all five types of RCC is shown in M. TRIM59 IHC signals were increased with tumour progression from grade 1–3 (p<0.05). All grade 1 tumours (n=4) stained with weak TRIM59 IHC signals in cytoplasm, but with high extent of nuclear staining; while all grade 2 and 3 tumours (n=66) showed moderate-to-strong cytoplasmic staining intensity of TRIM59. All grade 4 tumours (n=5) showed weak-to-moderate intensity in cytoplasm of TRIM59 staining. No correlation between TRIM59 IHC staining in nucleus and tumour grade was found, although low-grade RCC showed higher nuclear staining.
Therefore, by systematic IHC studies in CaP (88 patients, ) and kidney cancer (75 patients, ), we almost exactly repeated results from our mouse model studies on TRIM59.17
We confirmed TRIM59 as an IHC marker able to detect low-grade tumour in early tumorigenesis. We also demonstrated the correlation of tumorigenesis and tumour progression with TRIM59 upregulation until high-grade tumour.
TMA analysis of TRIM59 protein expression demonstrates that TRIM59 is a multiple tumour marker
In the basic research previously using animal GEM-CaP models,17
we have disclosed that TRIM59 upregulation is involved in two oncogene families and two signal pathways of SV40Tag/pRB/p53 and Ras/Raf/MEK/ERK. TRIM59 may function as an early signal transducer in Ras signal pathway with bridging genes in two oncogene pathways.17
While it was rarely reported that SV40 Tag oncogene induced human cancer, Ras mutations are among the most frequent alterations in human cancers (for a review see ref. 22
). We assume that TRIM59 as an early Ras signal pathway effector may possibly act as a multiple tumour marker.
We therefore further extended TRIM59 IHC studies to 35 multiple cancer TMA sections (42 tumours, 126 cores, ). We tested different dilutions (1/300, 1/600, 1/1200 and 1/ 5000) of TRIM59 antibody (see online supplementary figure S3). To further confirm the specificity and reliability of TRIM59 antibody in IHC staining, we compared IHC staining in 35 different tumour-TMA sections with positive (TRIM59 antibody at 1:1200 and 1:5000 dilutions) and negative controls (no antibody added, for details see online supplementary figure S4). As summarised in , TRIM59 expression was significant and tissue-specifically upregulated in most of these 35 tumours. When comparing the relative scores (both intensity and extent) in different tumours, the highest staining was observed in breast, lung, liver, skin (squamous cell carcinoma) and endometrial cancers.
Immunohistochemistry (IHC) analysis of TRIM59 as multiple marker in 35 tumour tissue microarray
Further confirmation of TRIM59 as a tumour marker in patients with eight different tumours
Since the 35 tumour-TMA contained only limited cases in each tumour type, we selected more cases (n=92) of eight different tumour types with different tumour grades, which all showed upregulated expression of TRIM59. IHC staining of TRIM59 in eight tumours are shown: lung (n=4, A–C), breast (n=3, D,E), gastrointestinal (n=2, F,G), female genital tract (n=5, H–J), bladder (n=44, K), prostate (n=27 from UWO, L), head and neck mucosal tumour (squamous cell carcinoma, SCC of mouth, tongue and larynx, n=4, M–O) and parotid gland (n=3, P,Q). Normal areas in lung, breast, colon, endometrial, bladder, larynx and parotid tissues showed very weak or completely negative staining ().
Figure 3 Comparison of TRIM59 expression as a multiple-cancer marker in eight types of tumours in breast, lung, parotid, gastrointestinal, female genital tract, bladder, head and neck mucosal tumour and prostate cancer. Negative TRIM59 staining in normal tissues (more ...)
Since some tumours (eg, prostate) showed mostly cytoplasmic and no nuclear TRIM59-IHC staining, as a comparative study, we assessed their relative scores (combine both intensity and extent scores, see Materials and methods). More tumours from kidney (RCC, n=75) and prostate cancer (n=27) were included as references and all were assessed by relative scores simultaneously, since we already analysed a large cohort of these patients. R shows the comparison of the mean of IHC-TRIM59 relative scores. The highest relative scores were found in SCC of the parotid, mouth, larynx and tongue, followed by lung, breast and female genital tract cancers.
The comparison of relative scores on low and high grades separately was done (data not shown). Cases of grade 1 lung cancer (bronchoalveolar, adenocarcinoma, squamous cell carcinoma (SCC) and large cell carcinoma) and breast cancer (invasive lobular and invasive mammary carcinoma) all showed the strongest staining as compared with other tumours. In endometrial cancer, the TRIM59 relative scores were moderate in grade 1 and moderate to strong in grade 2. The three tumours of SCC from mouth, tongue and larynx with different grades (M–O) also showed high relative scores (both intensity and extent).
As a comparative study (R, ), we tested 44 bladder cancer cases with 38 low-grade and 6 high-grade tumours. The mean value of relative scores was 1.6, that is, weak to moderate in bladder cancer cases. In 27 prostate cancer cases (from UWO only) tested, the relative scores of TRIM59 (cytoplasmic staining only) from PIN through Gleason scores of 10 were actually relatively weak (R), although in Gleason score 4, 6 and 8 were weak to moderate separately.
So far, we identified that TRIM59 upregulation is ‘tumour-specific’. First, we demonstrated the correlation of TRIM59-enhanced IHC signals with tumorigenesis and progression, which were statistically significant in this report with 291 cases and 37 tumour type analyses. Second, although TRIM59 is a normal gene involved in CDC (cell cycle division) regulation from G1 to S-phase and involved in DNA S-phase and cell growth,17
we demonstrated that in normal or non-tumour areas in all tested 37 different kinds of cancers, TRIM59 IHC staining signals were mostly negative or very low (–3). By moderating antibody dilutions and testing various blocking reagents (see online supplementary figure S4), we demonstrated that TRIM59 induces tumorigenesis/oncogenesis only when it is abnormally upregulated.
In most of human cancers tested in this clinical IHC study, strong TRIM59 expression in tumour were identified in epithelial cancers: lung, breast, skin, which all associated with epithelium originations (very rare from mesenchymal tumours, for details see ).
Furthermore, we also confirmed that TRIM59 expression involved in multiple tissue expression even in embryo development. We carried out IHC of mouse embryo sections by double-staining TRIM59. Cytokeratin (keratin), a family of proteins that are primarily found in epithelial cells was used as reference. Online supplementary figure S5 illustrated confocal microscope images of IHC staining of TRIM59 in different organs/tissues of mouse embryo (14.5 days postconception). TRIM59 was highly expressed in cytokeratin-expressing cells in the lung (first row), skin (second row), and kidney (not shown) of mouse embryos. TRIM59 staining in mouse embryos revealed the same pattern of the epithelium origin as in human tumours, which the TRIM59 gene were found highly upregulated in those tumour types as well (see table 2).
Given our previous experiments suggesting TRIM59 functions in the Ras pathway, we tested if TRIM59 upregulation was correlated with the BRAF, an early signal effector Ras signal pathway (for a review see ref. 22
). We selected 24 RCC patients, which previously were confirmed with upregulation of TRIM59 expression. Three antibodies were used: B-Raf Antibody (mAb) and Raf-l Antibody (Ab-259) testing the total Raf protein and Raf-l Antibody (Phospho-Ser259
pAb) testing the activated phosphorylated B-Raf. Some of the serial slides were stained in parallel on each patient by different antibodies. As shown in online supplementary figure S6A first two columns, in all 12 clear cell carcinoma of RCC samples, there were no or very weak B-Raf IHC signals in all three used Raf antibodies. In papillary RCC (see online supplementary figure S6B) and chromophobe RCC samples (see online supplementary figure S6C) (15 samples of 24 samples or 62% of all RCC samples tested), there were higher IHC signals in all three antibodies used for staining in cancer areas specifically, showing higher intensity and extent than clear cell carcinoma (see online supplementary figure S6A). Nuclear signals were found only by B-Raf P-Ser antibody in papillary and chromophobe tumours. Online supplementary table S1 summarises the results. It is intriguing that in those TRIM59 upregulated kidney cancers (RCC), neither total nor phosphorylated BRaf were detected in clear RCC (as a control), but were all highly positive in other two RCCs (papillary and chromophobe) tumours. We could not definitely confirm that TRIM59 was acting along the Ras pathway in all cases where it was detected.