The HSP90/R2TP assembly chaperone promotes cell proliferation in the intestinal epithelium

The R2TP chaperone cooperates with HSP90 to integrate newly synthesized proteins into multi-subunit complexes, yet its role in tissue homeostasis is unknown. Here, we generated conditional, inducible knock-out mice for Rpap3 to inactivate this core component of R2TP in the intestinal epithelium. In adult mice, Rpap3 invalidation caused destruction of the small intestinal epithelium and death within 10 days. Levels of R2TP substrates decreased, with strong effects on mTOR, ATM and ATR. Proliferative stem cells and progenitors deficient for Rpap3 failed to import RNA polymerase II into the nucleus and they induced p53, cell cycle arrest and apoptosis. Post-mitotic, differentiated cells did not display these alterations, suggesting that R2TP clients are preferentially built in actively proliferating cells. In addition, high RPAP3 levels in colorectal tumors from patients correlate with bad prognosis. Here, we show that, in the intestine, the R2TP chaperone plays essential roles in normal and tumoral proliferation.

(e) PCR with primers F5, R5, R6 on genomic DNA from organs shows that recombination occurs only in the small intestine and the colon (upper band corresponding to Rpap3 ∆7 ), but not in the other organs of VilCreER T2 ; Rpap3 flox/flox mouse treated with tamoxifen (n=1). Lower band corresponds to unrecombined Rpap3 flox allele. Note that extracts were prepared from total small intestine and colon, which comprise epithelium (where recombination takes place) and stroma (which does not express VilCreER T2 ). Molecular sizes are indicated on the right.

Specimen characteristics
CRC tissue samples were fixed in 10% buffered formalin for 24 h, dehydrated and paraffin-embedded. TMAs were constructed by extracting 2-mm diameter cores of histologically confirmed neoplastic invasive CRC areas from each original paraffin block and re-embedding these cores into gridded paraffin blocks, using a precision instrument (MTA, Beecher Instruments, WI). Before performing the immunohistochemical staining, 5 micrometer TMA sections were cut using a microtome and mounted onto histological polarized glass slides.

Assay methods
Immunohistochemistry was used to detect the presence of RPAP3 (proprietary 19B11 antibody at 1:10 dilution) and mTOR (Cell Signaling 7C10 antibody; cat. Number #2983, at 1:100 dilution) proteins in primary human CRC specimens. Antigen retrieval was performed by microwave treatment at 750 W (10 min) in 10 mmol/l sodium citrate buffer (pH 6.0). The polymer Envision kit (Agilent) was used for signal amplification. DAB (3,3-Diaminobenzidine) was used as chromogen. The two antibodies used for the immunostainings of CRC human samples have been validated by our previous work (i.e., RPAP3 antibody) and by the manufacturer (i.e., mTOR antibody). The receiver operating characteristic (ROC) curve analysis was applied to obtain the RPAP3 and mTOR positivity thresholds. The maximum Youden index indicated the optimum cut-off: values for RPAP3 and mTOR positive expression were 26% and 10%, respectively. Immunohistochemical analysis was done by a pathologist (RL) who was blinded to the clinical data of the patients. The primary endpoint was tumor recurrence or death of a patient. Disease-free survival (DFS) was defined as the time from surgery to the first one of the following events: recurrence at local or distant sites or intercurrent death without recurrence. Overall survival (OS) was defined as the interval between the date of surgery and the date of death or the last known follow-up. The receiver operating characteristic (ROC) curve analysis was applied to obtain positivity thresholds. The maximum Youden index indicated the optimum cut-off value.

Data
Eligible  Tables 1 -3 Patients and tumor characteristics were summarized in Supplemental Table 1, 2 and 3. 157 out of 177 (88.7%) cases expressed RPAP3 in the tumor cell cytoplasm. The proportion of RPAP3-positive cells was in the range of 4-100%, with a mean ± S.E. of 62.6% ± 2.6.
Analysis and presentation RPAP3 expression negatively correlated with the tumor stage of CRC (p = 0.049). Compared with stage I tumors, the expression of RPAP3 was decreased in stage II tumors (p = 0.049). Furthermore, compared with mucinous carcinoma, the expression of RPAP3 was increased in CRC of the adenocarcinoma type (p = 0.025). In addition, high RPAP3 expression was positively correlated with the occurrence of tumor relapse (p=0.003) and patients' mortality (p= 0.036).
See last section of Results; Figure 9b; Supplementary  Tables 1-3 Kaplan-Meier plots comparing patients with RPAP3 expression levels above and below the cut-off value have been reported in Figure 9b. The analysis shows that patients with RPAP3 high tumors have a lower DFS rate than patients with RPAP3 low tumors (p= 0.037). Multivariate analysis of DFS adjusted for other prognostic factors shows that RPAP3 expression was a significant prognostic parameter influencing disease relapse (HR = 2.7: 95% CI, 1.2-6.5; p= 0.023), but not the overall survival of patients.

DISCUSSION
We provide evidence that high RPAP3-expression levels in CRC tissues are associated with poor patient prognosis.