Altered Expression of Metastasis-Associated and Regulatory Molecules in Effusions from Breast Cancer Patients

Abstract

Purpose: The aim of this study was to characterize phenotypic alterations along the progression of breast carcinoma from primary tumor to pleural effusion through analysis of the expression of proteases, laminin receptors (LRs), and transcription factors involved in invasion and metastasis.Experimental Design: The material studied consisted of 60 malignant pleural effusions from breast cancer patients and 68 corresponding solid tumors (37 primary and 31 metastatic tumors). Expression of matrix metalloproteinases [MMPs (MMP-1, MMP-2, MMP-9, and MMP-14)], the MMP inhibitor tissue inhibitor of metalloproteinase-2, the MMP inducer EMMPRIN, the 67-kDa LR, the ␣ 6 integrin subunit, and the transcription factors AP-2, Ets-1, and PEA3 was studied using immunohistochemistry, mRNA in situ hybridization, reverse transcription-polymerase chain reaction, zymography, and flow cytometry. Hormone receptor (estrogen receptor and progesterone receptor) status and c-erbB-2 status were also studied.Results: Significantly reduced estrogen receptor (P < 0.001) and progesterone receptor (P ‫؍‬ 0.001) expression was seen in effusions compared with primary tumors, with opposite findings for c-erbB-2 (P ‫؍‬ 0.003). Tumor cell MMP-2 protein expression in effusions was higher than that in primary tumors (P < 0.001) and lymph node metastases (P ‫؍‬ 0.01). In situ hybridization demonstrated higher MMP-2 (P ‫؍‬ 0.007), PEA3 (P ‫؍‬ 0.038), and EMMPRIN (P ‫؍‬ 0.026) mRNA expression in effusions. The time to progression from primary tumor to effusion was significantly shorter for patients whose primary tumors expressed MMP-1 (P ‫؍‬ 0.016) and who expressed the 67-kDa LR protein in primary tumor (P ‫؍‬ 0.007) and effusion (P ‫؍‬ 0.015).Conclusions: Our data provide documented evidence of molecular events that occur during the progression of breast carcinoma from primary tumor to effusion. The coordinated up-regulation of MMP-2 and Ets transcription factors in carcinoma cells in effusions is in full agreement with our previous reports linking these factors to poor prognosis in ovarian cancer. The rapid progression to effusion in cases showing MMP-1 and 67-kDa LR expression in primary tumor cells links aggressive clinical behavior with expression of metastasis-associated molecules in this setting.

The tumor microenvironment disarms CD8+ T lymphocyte function via a miR-26a-EZH2 axis

“…Pleural effusion CTLs served as surrogates for lung TILs since it has been reported that these CTLs have experienced a similar microenvironment as that of tumors and display similar phenotypes as those of TILs. [27][28] Indeed, our functional analysis showed that, in comparison to CTLs collected from PBMCs, CTLs from the pleural effusion of NSCLC cancer patients exhibited a deficiency in granzyme B production (Fig. S1).…”

“…It has been shown by others that the restricted glucose concentration in the TME upregulates miR-26a. 27 We examined whether other factors within the TME could also have such an impact. To evaluate the role of factors directly produced by the tumor cell, we stimulated CD8 C T cells in the presence of lyophilized supernatant from 3T3 cell or tumor cell conditioned media (CM).…”

“…6A and B). Consequently, based on in vitro cytotoxicity assays with gp100 [25][26][27][28][29][30][31][32][33] peptidepulsed EL4 lymphoma cells, the tumor-specific killing capacity of miR-26a Decoy pMel-1 cells was significantly improved (Fig. 6C).…”

“…3A) and reported to be involved in CTL function. 27,28 We took a targeting decoy approach 15 to validate the impact of miR-26a on candidate gene expression. A miR-26a decoy was designed as a tandem of eight different miR-26a binding elements attached to the 3-end of a GFP gene.…”

“…pMel-1 transgenic CD8 C T cells were primed in vitro with their cognate antigen (gp100 [25][26][27][28][29][30][31][32][33] peptide) and transduced with retrovirus expressing mock or miR-26a Decoy . Two days after initial activation, CD8 C T cells were deprived of antigen, or treated with CM (B16 cells derived) for an additional 48 h. As expected, even under optimal priming conditions, miR-26a Decoy was capable of enhancing IFNg and granzyme B protein production by CTLs, based on both population percentage and per-cell production.…”