XIAP was the most potent caspase inhibitor, whose molecular structure was known best in IAP family. There were three important components in XIAP gene structure, including BIR domain, Linker domain and Ring-figure domain which possesed the activity as ubiquitin ligase E3, and XIAP could bind to caspase-3, 7, 9 directly to inhibit their activity. Expression of XIAP protein had been detected in most of carcinoma cells, and overexpression of this protein was correlated with patients'sensitivity to anticancer drugs and prognosis. For instance, overexpression of XIAP and Survivin, another member of IAP family, resulted in severe resistance to Adriamycin, Paclitaxel and Vincristine in several kinds of breast cancer cell[17, 18]. In LNCaP prostate cancer cells, XIAP restrained apoptosis induced by Paclitaxel through cutting down activity of caspase-3 and inhibiting processing of pro-caspase-3 moreover. On the contrary, patients of acute myeloid leukaemia with low expression level of XIAP could get more favorite prognosis.
Smac, which was named by direct IAP-binding protein with low pI (DIABLO) too, was one of the two known negative regulators of XIAP presently. Smac promoted apoptosis through several ways, including firstly interacting with cytochrome c/Apaf-1/pro-caspase-9 complex to activate caspase-9 or downstream effector molecule caspase-3[20, 21], secondly binding to BIR domain of XIAP competively and blocking it's inhibitory effect on caspases, and lastly cooperating with Omi/HtrA2 to promote XIAP to degradation[23, 24]. Previous study reported that total positive ratio of Smac was 62% in several carcinomas, and lack of Smac would lead to down regulation of apoptosis.
Preliminary research showed positive ratio of XIAP in breast cancer were 89.7%, but no related report about Smac. Our data indicated not only positive ratio but also immunoscore of XIAP were more higher than Smac in breast IDC, and semi-quantitation analysis of western blot detection proved it too. But it just could be regarded as a attempt because the fresh samples were so limited in this study. Disturbed balance of expression between XIAP and Smac contributed to progression of renal cell carcinoma and XIAP was an independent prognostic biomarker of clear cell renal cell carcinoma[1, 27]. Similarly, it also could be believed that disturbed balance of expression between XIAP and Smac contributed to carcinogenesis of IDC based on results of the present study. A set of data, consisting with other report, showed that expression status of XIAP/Smac was not correlated with patient age, tumor size, lymph node status, histologic grading, expression of ER and PR. Whereas, Jaffer and his colleagues found a possible role of XIAP in the more aggressive clinical behavior of grade 3, compared with lower-grade ductal carcinomas. These conflicting results need be confirmed in a following large sample study.
In previous reports, XIAP was only detected in cytoplasm[26, 28]. However, we found 44 IDC samples were positive in nucleus and cytoplasm for XIAP simultaneously, but none was for Smac. This difference was maybe caused by using different primary antibodies and tissue specimens from different race. Tissue specimens in present study were all from Chinese female patients, but not from Europe or America. Something different from previous reports was that XIAP nuclear labeling (XIAP-N), but not cytoplasmic staining of XIAP (XIAP-C), was the apoptotic marker which correlated significantly with IDC patients' shortened overall survival. Univariate survival analysis disclosed that patient age, tumor size, lymph node status and XIAP-N had prognostic significance. Nevertheless, it was demonstrated by multivariate survival analysis that only patient age, lymph node status and XIAP-N were independent prognostic factors.
It had been introduced previously that XIAP and Survivin, two important caspase inhibitors of IAP family, were comitantly overexpressing in several kinds of breast cancer cell and oweing to elevated resistance to chemotherapeutics[17, 18]. Even more, Survivin was an independent predictor of short-term survival in poor prognostic breast cancer patients. All results stated above called attention to us that overexpression of XIAP and Survivin were significantly correlated with carcinogenesis, progression and prognosis of breast cancer, and the two molecules played similar role in several aspects in breast cancer. Therefore, we concluded XIAP, like Survivin, probably was a new independent prognostic biomarker of breast cancer although it was different that only XIAP nuclear labeling, but not cytoplasm staining, had prognostic significance. This condition was similar to previous reports that Survivin nuclear labeling was a prognostic biomarker of breast cancer and superficial urothelial carcinoma of urinary bladder[29, 30].
There was another interesting result in our study that immunoscore of Smac was prevalent in HER2 positive group than negative group, and apoptosis index was positively correlated with HER2 protein expression. Someone believed that apoptosis was decreasing in malignant tumors. But in fact, increasing apoptotic tumor cells could usually be found in malignancies, especially for tumors with high proliferating rate. On the other hand, overexpression of oncogene HER2 contributed to breast carcinogenesis. So, it's not difficult for us to understand that apoptosis index was positively correlated with HER2 protein expression in our study. Our result also consisted with Hinnis' study that AI was not correlated with patient age, tumor size, lymph node status, histologic grading and expression of XIAP, Smac, ER and PR protein. At the same time, our following up data revealed similar result with previous reports that AI was not correlated with IDC patients' prognosis[31, 32]. However, why there was prevalent Smac in oncogene HER2 positive group? The relationship between the two parameters had not been reported before, and both mechanism and significance were not clear too. Maybe, this point would be a new target for us to perform a large sample study in the future.
As discussed above, XIAP was a potent protein for apoptosis inhibition and Smac was an important negative regulator of the former. Disturbed balance of expression between XIAP and Smac probably contributed to carcinogenesis and XIAP positive nuclear labeling was a sign of unfavourable prognosis in breast invasive ductal carcinoma. It was important for us to demonstrate the XIAP nuclear staining is genuine, and we were going to practice it in breast cancer cell lines in following study. Further more, Relationship among Smac, HER2 and apoptosis index would be explored in the following study too.