The identification of novel biomarkers that can help to define individual risk signatures in breast cancer patients are of great clinical value, as they might allow individualized treatments. Previously, we and others have identified candidate molecular gene signatures associated with poor prognosis, including GGH FAAH, PIR and TAF5L [3, 4]. In the present study, we investigated the clinical significance of the candidate biomarkers GGH FAAH, PIR and TAF5L in predicting breast cancer progression.
In the present study, we found that the elevated cytoplasmic protein levels of GGH were mainly localized to the tumor cells in comparison to adjacent non-cancerous cells. In urothelial carcinoma of the bladder, elevated level of GGH was also detected in cancerous cells in comparison with non-cancerous cells . In addition, tumoral GGH protein levels were significantly up-regulated in high histological grade tumors in comparison with low histological grade tumors. Taken together, these finding suggest that the expression of GGH is associated with invasiveness and GGH protein levels may increase as the disease progresses. However, detected GGH protein in the non-cancerous tissues may represents the normal function of GGH in maintaining tissue homeostasis of (anti)folate, glutamates or may predict progression of premalignant lesions . High levels of tumoral GGH were also observed to be significantly associated with ER/PR receptors. Eight-year survival of patients with low expression of GGH was significantly better than those with a higher expression. In addition, the multivariate analysis verified that GGH is an independent negative factor in predicting patient DSS as presented by the fact that hazard ratio (HR) for GGH adjusted for age, histological type, histological grade, ER/PR status, HER2/neu status, pathologic tumor size, and axillary lymph node status remained significant. These findings suggest that GGH may be involved in promoting carcinogenesis. Furthermore, the elevated levels of GGH was found to be correlated with shorter RFS with more than 35 fold increased risk, suggesting that GGH expression may predict the recurrence behavior of breast cancer. Notably, the association between GGH expression and different cancer forms has been previously reported. The elevated levels of GGH were reported to be correlated with poor clinical outcome in pulmonary neuroendocrine tumors . Elevated plasma level of GGH was observed in patients with metastatic breast cancer in comparison to control subjects and to patients with cancer in remission . High GGH expression level was also detected in hepatoma cells compared with rat hepatocytes . Furthermore, GGH expression was found to act as a prognostic biomarker for acute leukemia in response to methotrexate therapy . Consistent with these findings, our data further suggest that the dysfunction of GGH may play an important role in breast cancer progression and GGH may be an amenable therapeutic target in breast cancer.
In tumor tissues, the mRNA expression of GGH was increased specifically in patients with short-term survivor. This increase corresponded to protein accumulation based on IHC, indicating transcriptional activation. However, the GGH gene may be also regulated in tumors at posttranscriptional levels, since the GGH protein in 5 of 62 invasive breast cases was elevated, whereas no increase in mRNA was observed in these samples by qRT-PCR.
The cytoplasmic expression of FAAH was significantly up-regulated in invasive breast tumor tissues compared to the non-cancerous tissues. In addition, the expression levels of FAAH were significantly increased in patients with higher number of axillary lymph node metastases. Up-regulation of FAAH indicates down-regulation of cannabinoids, which play an important role in preventing tumor growth . Taken together, these findings suggest that elevated level of FAAH may promote breast cancer tumors invasion and metastasis. Interestingly, a significant correlation between GGH and FAAH protein expressions was detected in the tumor tissues, whereas no association in the non-cancerous tissues was seen, suggesting that the tumor micro-environmental effects may regulate the expression of GGH and FAAH simultaneously . In addition, GGH accumulation may reflect a functional correlation with FAAH expression, which could play a role in the progression of breast carcinoma. In the current study, expression of PIR in tumor tissues was virtually similar to the non-cancerous tissues. Previous studies reported that some gene expression patterns in the invasive tissues are comparable to their non-invasive breast tissues, suggesting that these signatures may predict progression of early premalignant lesions in non-cancerous tissues [28, 34, 35]. The PIR functions as a transcriptional regulator whose expression is deregulated in several cancer types. Furthermore, high expression of PIR was reported to be essential to overcome the senescence barrier . We also examined the clinical significance of PIR protein expression. The higher expression of PIR was significantly associated with presence of lymph node metastasis, suggesting that the expression of PIR is associated with invasiveness and supports the reported association of PIR expression with enhanced malignant potential [36, 37].
In the present study, the nuclear expression of TAF5L was significantly down-regulated in invasive breast tumor tissues compared to the non-cancerous tissues, which suggests a potential tumor suppressor role in breast cancer. The expression of TAF5L was elevated in patients with low histological grade tumors compared to patients with high histological grade tumors, although the differences were not significant (P = 0.06). Furthermore, high mRNA expression levels of TAF5L were detected in 97% of the analyzed tumors, whereas only 56% of the analyzed tumors expressed TAF5L protein. The reduction of TAF5L protein in the analyzed tissues may contribute to disease progression.
The discordant results detected between mRNA and protein expression of FAAH, PIR and TAF5L, could be due to multiple factors including, tumor heterogeneity, posttranscriptional regulation, differences in mRNA and protein turnover rates or poor specificity of the antibody used for IHC [38, 39].
In the cohort of 80 patients, even though the expression of FAAH, and PIR did not predict DSS and RFS, the expression of these candidate biomarkers were significantly associated with other clinicopathological characteristics. These results need to be further validated by a larger sample size and additional studies are needed to elucidate the molecular mechanisms through which GGH, FAAH, PIR and TAF5L may participate in the development and progression of breast cancer.