Identification of invasion specific splice variants of the cytoskeletal protein Mena present in mammary tumor cells during invasion in vivo - PubMed (original) (raw)

Identification of invasion specific splice variants of the cytoskeletal protein Mena present in mammary tumor cells during invasion in vivo

Sumanta Goswami et al. Clin Exp Metastasis. 2009.

Abstract

We have studied the gene expression pattern of invasive primary mammary tumor cells using a unique in vivo invasion assay that isolates the invasive tumor cells by chemotaxis. One of the genes upregulated in the invasive tumor cells is Mena, an actin binding protein involved in the regulation of cell motility. There are multiple known splice variants of Mena accounted for by four alternatively included exons, +, ++, +++ and 11a. Using the in vivo invasion assay in rats and mice with mammary tumors we observed that two isoforms of Mena, ++ and +++, are upregulated in the invasive tumor cells and one isoform, 11a, is downregulated. The Mena isoform switching pattern described here may provide a new biomarker for the presence of metastatic cancer cells and for prognosis.

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Figures

Fig. 1

Fig. 1

The expression of transcripts containing Mena ++ and Mena +++ exons are upregulated while those containing Mena 11a are downregulated specifically in the invasive tumor cell population as compared to the APTC. Quantification of Mena isoforms by QRT-PCR in MTLn3 rat allograft model (a), and PyMT mouse transgenic model (b). The levels of transcript observed using pan Mena primers (Mena), and ++ and +++ primers are increased while that observed using 11a primers is greatly reduced in both animal tumor models. *11a Message was undetectable in the PyMT mouse transgenic invasive tumor cells. The error bars show standard errors of mean (SEM) performed on three biological repeats and three technical repeats

Fig. 2

Fig. 2

Strategy for primer design for each of the Mena exons and Smart RACE along with Nucleotide and protein sequences of the ++ and +++ isoforms. a The position of the primers and the strategy for primer design along with both the 3′ and 5′ RACE strategies are shown in this figure. Primer numbers refer to those in Supplementary Table 1. b Sequence alignment for ++ and +++ exons expressed in invasive tumor cells are identical to the published mouse sequences. The ++ exon nucleotide and inferred amino acid sequences are aligned in 1 and the +++ exon nucleotide and inferred amino acid sequences are aligned in 2

Fig. 3

Fig. 3

Expression of the isoforms of Mena characteristic of invasive tumor cells are stably expressed in different stages of metastasis in rat adenocarcinoma (MTLn3) cells. The expression status of Mena ++ and +++ isoforms was determined by QRT-PCR. The isoforms characteristic of invasive tumor cells show 3.5-4 fold over expression in the invasive tumor cells, circulating tumor cells in blood and in tumor cells growing as lung metastasis. The stability of elevated expression during progression suggests that these isoforms of Mena may be markers for the presence of metastatic tumor cells in several sites. The error bars show standard errors of mean (SEM) performed on three biological repeats and three technical repeats

Fig. 4

Fig. 4

Domain organization of Mena and location of alternatelyincluded exons. A schematic of Mena is shown. The EVH1 domain binds to proteins that contain a motif with the consensus: (D/E) FPPPPX(D/E)(D/E); an exception to this rule is TES, which binds to Mena EVH1 through a Lim domain [19]. EVH1-interactions are required for subcellular targeting of Mena and to bring it into signaling complexes. The invasion-specific exon “INV” is included in between the EVH1 domain and an extended “LERER” repeat region of unknown function. “PRO” denotes a proline-rich region that contains binding sites for the actin-monomer binding protein profilin as well as several SH3- and WW-domains. The EVH2 domain contains binding sites for G-actin (“G”), F-actin (“FAB”) and has a coiled-coil (“CC”) that forms tetramers. The “11a” exon which is present in Mena within primary tumors and downregulated in invasive cells is included adjacent to the FAB site

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