Abstract
BackgroundThe INT6 gene was first discovered as a site of integration in mouse mammary tumors by the mouse mammary tumor virus; however, INT6’s role in the development of human breast cancer remains largely unknown. By gene silencing, we have previously shown that repressing INT6 promotes transforming activity in untransformed human mammary epithelial cells. In the present study, guided by microarray data of human tumors, we have discovered a role of Int6 in stromal fibroblasts.ResultsWe searched microarray databases of human tumors to assess Int6’s role in breast cancer. While INT6 expression levels, as expected, were lower in breast tumors than in adjacent normal breast tissue samples, INT6 expression levels were also substantially lower in tumor stroma. By immunohistochemistry, we determined that the low levels of INT6 mRNA observed in the microarray databases most likely occurs in stromal fibroblasts, because far fewer fibroblasts in the tumor tissue showed detectable levels of the Int6 protein. To directly investigate the effects of Int6 repression on fibroblasts, we silenced INT6 expression in immortalized human mammary fibroblasts (HMFs). When these INT6-repressed HMFs were co-cultured with breast cancer cells, the abilities of the latter to form colonies in soft agar and to invade were enhanced. We analyzed INT6-repressed HMFs and found an increase in the levels of a key carcinoma-associated fibroblast (CAF) marker, smooth muscle actin. Furthermore, like CAFs, these INT6-repressed HMFs secreted more stromal cell–derived factor 1 (SDF-1), and the addition of an SDF-1 antagonist attenuated the INT6-repressed HMFs’ ability to enhance soft agar colony formation when co-cultured with cancer cells. These INT6-repressed HMFs also expressed high levels of mesenchymal markers such as vimentin and N-cadherin. Intriguingly, when mesenchymal stem cells (MSCs) were induced to form CAFs, Int6 levels were reduced.ConclusionThese data suggest that besides enhancing transforming activity in epithelial cells, INT6 repression can also induce fibroblasts, and possibly MSCs as well, via mesenchymal-mesenchymal transitions to promote the formation of CAFs, leading to a proinvasive microenvironment for tumorigenesis.
Highlights
The INT6 gene was first discovered as a site of integration in mouse mammary tumors by the mouse mammary tumor virus; INT6’s role in the development of human breast cancer remains largely unknown
Int6 is reduced in the fibroblasts in human breast cancer To determine whether INT6 may act as a tumor suppressor for breast cancer, we searched Oncomine for gene expression changes by focusing on studies in which normal and tumor tissues were compared
Right: stroma gene expression data in the Finak study available in Oncomine were analyzed to show that INT6 mRNA levels were approximately 42 times higher in the tissue surrounding the normal adjacent ducts than in the stroma in the tumor. (B) Control or INT6-repressed MCF7 cells were analyzed by Western blot or IHC by an anti-Int6 antibody
Summary
The INT6 gene was first discovered as a site of integration in mouse mammary tumors by the mouse mammary tumor virus; INT6’s role in the development of human breast cancer remains largely unknown. Int reduction may induce tumor formation in breast epithelial cells by causing a net increase in the levels of proteins that promote tumorigenesis. In support of this hypothesis, we and others have recently shown that repressing INT6 expression in normal mammary epithelial cells induces a transforming phenotype, which correlates with the stabilization of a potent oncoprotein, Src3/AIB1, and altered translation of the ubiquitin genes as well as of genes controlling the epithelial-mesenchymal transition (EMT) [8,15]
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