Animal models indicate that carcinoma cells located in invasion-inducing microenvironments undergo transient and sometimes stable epigenetic changes similar to those that drive morphogenetic cell movements in the developing embryonic organ [2
]. One of the changes is the increased expression of the actin regulatory protein MenaINV
and decreased expression of Mena11a [8
]. Here we have found that changes in isoform expression from Mena11a to MenaINV
correlate with the loss of epithelial cell-cell contacts and the emergence of a discohesive cell population in the primary tumor. Additionally, previous studies have reported an increase in cell motility in vivo
and lung metastases in orthotopic mammary tumors derived from MenaINV
expressing tumor cells [17
]. Thus, the discohesive primary tumor morphology and discontinuous cell-cell adhesion contacts observed in MenaINV
tumors supports the enhanced migratory phenotype previously reported [17
We demonstrate that expression of Mena11a promotes the maintenance of epithelial cell-cell contacts and a cohesive cell population within a mammary tumor. This is consistent with recent findings showing that expression of Mena11a delays but does not prevent metastatic progression in MTLn3 xenograft mammary tumors [21
]. These results suggest that the expression of Mena11a in tumor cells will support their retention in cohesive regions of tissue with relatively stable cell-cell junctions (). Thus, the FNA smears from the tumors with the high proportion of Mena11a expressing cells show mostly cohesive clusters likely reflecting a less malignant clinical outcome.
Tumor cell behavior in mammary tumors as related to Mena isoform expression
Previous studies have shown that MenaINV
expressing carcinoma cells have increased movement in vivo
] (). Sensitivity of tumor cells to a particular chemoattractant will determine the direction and speed of their movement within the tissue [22
]. Previous studies also show that MenaINV
expressing cells are hypersensitive to EGF during in vivo
]. This sensitivity could therefore promote migration toward, and association with EGF producing perivascular macrophages, resulting in intravasation (). This hypothesis is supported by previous observations of carcinoma cell migration toward blood vessels and intravasation in mouse mammary tumors [39
]. We propose that a high proportion of single cells in FNA smears, and their expression of MenaINV
, is likely to reflect the presence of a migratory, and intravasation competent cell population, and therefore an adverse clinical outcome.
Our results show that the presence of MenaINV
expressing carcinoma cells in FNA samples is correlated with the presence of the anatomical structure called TMEM [9
]. As previously mentioned, TMEM is defined as the direct contact of a perivascular macrophage, an endothelial cell and Mena expressing tumor cell (). In recent findings we show that MenaINV
expressing carcinoma cells have dramatically increased intravasation [21
]. Thus, it is possible that the expression of MenaINV
can provide an advantage to tumor cells in their ability to find macrophages when located in microenvironments with low concentrations of EGF due to the increased sensitivity of MenaINV
tumor cells to EGF [17
]. These findings indicate that increased MenaINV
expression may affect metastatic potential of a particular breast tumor by contributing to macrophage-dependent cell motility throughout the primary tumor (), and intravasation at TMEM sites (). Indeed, our findings in PyMT mice indicate that MenaINV
promotes metastatic progression.
We show here that human invasive ductal carcinomas with high MenaINV
expression in FNA samples correlate with a high TMEM score. However, expression of Mena11a does not correlate with TMEM score and this is consistent with the recent findings that Mena11a expressing tumor cells have no effect on intravasation or metastasis relative to GFP control cells [21
]. The observations in human tumors also suggest that expression of the Mena11a isoform does not affect the late stages of metastasis. These results support the involvement of the MenaINV
isoform in assembly of TMEM, and intravasation in human breast carcinomas.
We recently reported a case-control study demonstrating that TMEM density is associated with increased risk of metastasis of invasive ductal carcinomas of the breast [9
]. That study demonstrated that TMEM count correlates with tumor grade supporting the fact that low grade tumors rarely metastasize. We also found a significant difference in TMEM score between low and high tumor grades in our cohort (p= 0.004). In both the previous and current studies, we did not observe any association between TMEM count and ER, PR, HER2/Neu expression or lymph node metastasis. Since about 10-15% of patients develop metastatic disease within 3 years of diagnosis [4
] we would expect about 5 cases from our cohort of 40 to be metastatic within the same time frame. Indeed, only 5 cases from our cohort (15%) have TMEM values around or above the 90th
percentile (107.6) (Table S1
and ). Three of those cases also have MenaINV
around or above the 90th
In summary, our results indicate that carcinoma cells with an elevated MenaINV expression are discohesive (), while those with elevated Mena11a expression are more cohesive. Our results also suggest that MenaINV expressing cells assemble TMEM, and intravasate at TMEM sites (). It will be important to determine how epigenetic changes may affect isoform expression and if these changes act through control of alternative splicing. Studies are underway to investigate the role of alternative splicing mechanisms in control of Mena isoform expression (FG, unpublished). Future studies will investigate molecular and biochemical mechanisms of action of the MenaINV and Mena11a isoforms, as well as their utility as predictors of breast cancer outcome and as targets for therapy.