Proteomic analysis of differential protein expression by brain metastases of gynecological malignancies

Abstract

Brain metastases of gynecological malignancies are rare, but the incidence is increasing. Patients with brain metastases have a poor prognosis, therefore early detection and optimal management is necessary. In order to determine a new biomarker, we aimed to identify proteins that associated with brain metastases. We investigated proteins associated with brain metastases of gynecological malignancies in three patients who underwent surgical resection (stage IIb cervical cancer, stage Ib endometrial cancer, and stage IIIb ovarian cancer). Proteomic analysis was performed on formalin-fixed paraffin-embedded (FFPE) samples of the primary tumors and brain metastases, which were analyzed by liquid chromatography with tandem mass spectrometry. Thereafter, candidate proteins were identified by the Scaffold system and Mascot search program, and were analyzed using western blotting and immunohistochemistry. As a result, a total of 129 proteins were identified. In endometrial and ovarian cancers, western blotting revealed that the expression of alpha-enolase (ENO1) and triosephosphate isomerase (TPI-1) was higher and the expression of Transgelin-2 (TAGLN2) was lower in metastatic tumors than in primary tumors. On the other hand, the expression of TPI-1 and TAGLN2 was lower in metastatic tumors than in primary tumors in cervical cancer. Immunohistochemistry confirmed that ENO1 expression was elevated in the metastatic tumors compared with the primary tumors. In conclusion, the present study showed that FFPE tissue-based proteomics analysis can be powerful tool, and these findings suggested that ENO1, TPI-1, and TAGLN2 may have a role in the development and progression of brain metastasis from gynecological malignancies.

Fig. 1

Protein expression in primary and metastatic tumors. a Venn diagram showing the differential expression of 129 proteins by primary and metastatic tumors (76 in primary tumors and 101 in metastatic tumors). b Distribution of proteins related to different biological processes. c Comparison of proteins expressed by primary and metastatic tumors. The expression of proteins related to developmental and multicellular organismal process was lower in metastatic tumors than in primary tumors, whereas the expression of proteins related to metabolic processes was higher in metastatic tumors than in primary tumors

Fig. 2

Western blot analysis of ENO1, TPI-1, and TAGLN2. Proteins from primary and metastatic tumors were separated by SDS-PAGE and transferred to PVDF membranes, followed by detection using the respective primary antibodies and an HRP-conjugated secondary antibody. In endometrial and ovarian cancers, the expression of ENO1 and TPI-1 was higher in metastatic tumors than in primary tumors, and the expression of TAGLN2 was lower in metastatic tumors than in primary tumors. Further, in cervical cancer, the expression of TPI-1 and TAGLN2 was lower in metastatic tumors than in primary tumors. P primary tumors, M metastatic tumors

Fig. 3

Representative immunostaining of ENO1 in primary and metastatic tumors. a, b, e, f, i, and j show primary tumors. c, d, g, h, k, and l show metastatic tumors. a, e, i, c, g, and k show HE staining. b, f, j, d, h, and l show ENO1 staining. ENO1 is overexpressed in metastatic tumors. All images are ×400 magnification. Scale bars 100 mm. EC endometrial cancer (a–d). CeC Uterine cervical cancer (e–h). OC ovarian cancer (i–l)