Evidence for the transforming activity of a truncated Int6 gene, in vitro

Abstract

Int6/eIF3-p48 was ®rst identi®ed as a common integration site for MMTV in mouse mammary tumors. In all cases, the MMTV integration event resulted in an interruption of the normal Int6 transcript from one allele leaving the second allele intact and operative. We hypothesize that insertion of MMTV into Int6 results in a mutated allele that encodes a shortened Int6 mRNA and protein (Int6sh), which either modi®es normal Int6 function or possesses a new independent function. To con®rm the transforming potential of the mutation and its dominant function, we transfected two mammary epithelial cell lines, MCF10A (human), and HC11 (mouse), with Int6sh under the control of the elongation factor-1a (eEF1A) promoter. Expression of Int6sh in MCF10A and HC11 mammary epithelial cells leads to anchorage-independent growth in soft agar indicative of a transformed phenotype. Colonies selected from agar exhibited high levels of mutated Int6sh and wild type Int6 RNA transcripts by RT ± PCR and Northern blot analysis. In addition, Int6sh transformed MCF10A and HC11 cells formed nodular growths, in vivo, in immune compromised hosts. NIH3T3 cells, mouse embryo ®broblasts, were also transformed to anchorage-independent growth in vitro by Int6sh expression. These observations provide direct evidence that the Int6 mutations observed in MMTV-induced tumors and hyperplasia contribute to the malignant transformation of the mammary epithelial cells. Oncogene (2001) 20, 5291 ± 5301.

Silencing of human Int-6 impairs mitosis progression and inhibits cyclin B–Cdk1 activation

“…One point that remains unclear is whether MMTV insertion acts by generating haploinsufficiency or truncated proteins that would behave as dominantnegative mutants. In favour of this latter hypothesis, it has been reported that expression of a shortened form of Int-6 can transform mouse and human mammary epithelial cell lines (Rasmussen et al, 2001), as well as NIH 3T3 cells (Mayeur and Hershey, 2002). However, endogenous Int-6 is an abundant protein and the putative C-terminally truncated forms have never been detected so far by immunoblot.…”

A novel oncogenic role for the miRNA-506-514 cluster in initiating melanocyte transformation and promoting melanoma growth

“…Moreover, many previous publications have utilized the soft agar assay to demonstrate the transformation capacity of oncogenes (Rasmussen et al, 2001;Lu et al, 2008;Syed et al, 2008;Monsel et al, 2010), as this assay is well documented to be predictive of in vivo tumorigenicity (Rhim, 1983;Trainer et al, 1988), malignant potential (Montesano et al, 1977) and drug response (Zirvi et al, 1983;Scholz et al, 1990). Taken together, these observations increase confidence in the oncogenic potential of the miR-506-514 cluster.…”

An oncogenic role of eIF3e/INT6 in human breast cancer

“…Individual overexpression of any of five subunits of human eIF3 (eIF3a, b, c, h or i) promoted malignant transformation of immortal fibroblasts (Ahlemann et al, 2006;Savinainen et al, 2006;Zhang et al, 2007). By contrast, studies of eIF3e/INT6 have suggested various roles for this accessory eIF3 subunit either as an oncoprotein or a tumor suppressor (Marchetti et al, 2001;Rasmussen et al, 2001;Buttitta et al, 2005;Chen et al, 2007).…”