CCN6 downregulation in invasive breast carcinomas: In situ characterization of changes in EMT proteins and metastatic disease

LORENZATTI, GUADALUPE; HUANG, WEI; CABANILLAS, ANA MARIA; KLEER, CELINA

Chicago, Illinois

Congreso; AACR Annual Meeting 2012, March 31-April 4 in.; 2012

AACR

Background: The recognized heterogeneity of invasive breast carcinomas is a result of intrinsic characteristics of the tumor cells and their interaction with the microenvironment. CCN6, an extracellular matricellular protein involved in cell communication and epithelial-stromal interactions has a tumor suppressor role in breast cancer. CCN6 downregulation in benign breast cells induces an epithelial to mesenchymal transition marked by downregulation of E-cadherin and increased levels of the transcriptionalregulators ZEB1 and Snai1. However, the relevance of these studies to human breast cancer needs further investigation.We hypothesized that CCN6 mRNA and protein levels in samples of breast cancer patients may be associated with metastasis and features of EMT.Methods: Forty clinical samples of invasive carcinoma and surrounding normal breast, frozen at the time of operation were collected. We extracted mRNA and protein from each sample and performed real time RT-PCR and Western blots for CCN6, respectively. All frozen samples were immunostained with antibodies against E-cadherin (BD Biosciences, 1:4000), ZEB1 (Santa Cruz Biotechnology, 1:400) and Snai1(Cell Signaling, 1:800). E-cadherin was analyzed as aberrant or normal. ZEB1 and Snai1 proteins were scored as positive or negative, either in the cancer or stromal cells.Results: Of the 40 frozen samples of breast cancer, 23 had high quality RNA and protein for further study. CCN6 mRNA and protein were both decreased in 18/23 (78%). Five of 23 (22%) samples exhibited normal or high expression. Low CCN6 was associated with the presence of distantmetastasis. Four of 18 (22%) invasive carcinomas with low CCN6 had distant metastasis compared to none of the normal CCN6 expressing tumors (p<0.05).Seventeen of 18 (94%) of invasive carcinomas with low CCN6 had aberrant E-cadherin protein expression. We noted that in the tumors with aberrant E-cadherin protein, the expression of ZEB1 or Snai1 was mutually exclusive in all cases (p<0.05). We noted that 80% of the tumors exhibited ZEB1protein in the stromal cells.Conclusions: Our results show a direct link between levels of CCN6 mRNA and protein in breast carcinoma patient samples. A decrease in CCN6 expression in the primary breast cancer is associated with distantmetastasis and decreased E-cadherin. We uncover a novel inverse relationship between ZEB1 and Snai1 proteins in breast cancer, which is under further investigation.