High levels of γ-glutamyl hydrolase (GGH) are associated with poor prognosis and unfavorable clinical outcomes in invasive breast cancer

Abstract

Background: Previously, we performed analysis of gene expression in 46 axillary lymph node negative tumors and identified molecular gene signatures that resulted in different clinical outcomes. The aim of this study was to determine the correlation of γ-glutamyl hydrolase (GGH), fatty acid amide hydrolase (FAAH), Pirin (PIR) and TAF5-like RNA polymerase II, p300/CBP-associated factor (PCAF)-associated factor, 65 kDa (TAF5L), selected from identified gene signatures, with clinical outcomes as well as classical clinicopathological characteristics in primary invasive breast cancer patients.

Methods: The protein levels of GGH, FAAH, PIR and TAF5L were assessed by immunohistochemistry (IHC) on a panel of 80 primary invasive breast tumors. Quantitative real-time PCR (qRT-PCR) and western blot analysis were performed to verify the expression levels of the candidate biomarkers. Patient disease-specific survival (DSS) and recurrence-free survival (RFS) were evaluated using the Kaplan-Meier method. The prognostic biomarkers were identified by univariate analysis with a log-rank test and by multivariate analysis with Cox proportional hazards regression models.

Results: The GGH and FAAH protein levels were significantly up-regulated in invasive breast cancer tumors compared with adjacent non-cancerous tissues. Furthermore, the protein levels of GGH and FAAH were significantly correlated in tumor tissues. Tumoral GGH protein expression was significantly correlated with shorter DSS and RFS. Furthermore, the protein expression of GGH was positively correlated with undifferentiated tumors (BRE grade III) and ER/PR expressing tumors. Multivariate regression analysis showed that only GGH protein expression independently predicts DSS. No such correlations were found for FAAH, PIR and TAF5L protein expression. However, elevated protein levels of FAAH were positively associated with high number of lymph node involvement and upregulated levels of PIR were positively related with lymph node metastasis. The TAF5L was pronouncedly down-regulated in primary invasive breast cancer tissues compared to matched adjacent non-cancerous tissues.

Conclusion: These data show for the first time that cytoplasmic GGH might play a relevant role in the development and progression of invasive breast cancer, warranting further investigations. Our findings suggest that GGH serve as a potential biomarker of unfavorable clinical outcomes over short-term follow-up in breast cancer. The GGH may be a very attractive targeted therapy for selected patients.

Figure 1

Detection of GGH protein levels in invasive breast cancer tissues. (A) Immunohistochemical staining of GGH protein expression in primary invasive breast tumors. (B) Western blot analysis of GGH protein levels in 7 primary invasive breast cancer tumors. Note: P = GGH-expressing tumor tissues; N = GGH-negative tumor tissues as assessed by immunohistochemistry; IHC = Immunohistochemistry. GAPDH assesses equal loading.

Figure 2

Immunohistochemical detection of FAAH, PIR and TAF5L protein levels in primary invasive breast tumors (A-C).

Figure 3

Kaplan-Meier depicting disease-specific survival in breast cancer according to expression patterns of GGH, FAAH, PIR and TAF5L (A-D). Dashed line represents patients whose tumors expressed GGH (A), FAAH (B), PIR (C) and TAF5L (D). Solid line represents patients whose tumors did not express GGH (A-D). The p-values for the difference between the curves were calculated using log-rank test.

Figure 4

Kaplan–Meier illustrating the recurrence-free survival of breast cancer patients on the basis of GGH expression levels. Dashed line represents patients whose tumors expressed GGH and solid line represents patients whose tumors did not. The p-values for the difference between the curves were calculated using log-rank test.

Figure 5

The relationship between GGH, FAAH, PIR and TAF5L mRNA levels with their corresponding protein levels in breast cancer patients. The box plots Positive and Negative indicate corresponding gene expression levels for each protein. The mRNA expression of GGH was consistent with the IHC findings. There was no association of FAAH, PIR and TAF5L mRNA levels to their corresponding protein levels.