In this study we utilized the MTLn3 cell line to evaluate the role of the YXXM sites in ErbB3 in breast cancer metastasis. We find that these sites are required for ErbB3-mediated chemotactic responses to HRGβ1 in vitro
and invasion responses both in vitro
and in vivo
. Increased expression of wild-type ErbB3 had no effect on primary tumor growth, but significantly enhanced intravasation and spontaneous metastasis ( and our previous publication (Xue et al., 2006
)). Mutation of the YXXM sites dramatically reduced the enhancement of intravasation and metastasis, while having no effect on primary tumor growth. In an experimental metastasis assay, wild-type ErbB3 overexpression had a significant, albeit slight, effect on the lung seeding process, which also required ErbB3 coupling to PI3K. Similarly, an increase in spontaneous motility and cell polarization in the primary tumor induced by wild-type ErbB3 required the YXXM sites. Treatment of tumor-bearing mice with the p110alpha inhibitor PIK-75 caused a significant decrease in intravital motility and in vivo
invasion, attesting to the important role of PI3K in invasion. Overexpression of ErbB3 in the aggressive MDA-MB-231 human breast cancer model also significantly enhanced the invasiveness of the cells in response to HRGβ1 treatment (Supplementary Figure 7
). Taken together the data presented here indicate that the ability of ErbB3 to couple to the PI3K/Akt pathway confers tumors overexpressing this receptor with an enhanced ability to invade and to metastasize.
Based on the studies reported here, we have identified intravasation as a key stage in metastasis that is enhanced by ErbB3 signaling both in vivo
and in the in vitro
transendothelial migration assay. Intriguingly, EGFR overexpression is not equivalent to ErbB3 overexpression in the in vitro
TEM assay, since the former do not show increased intravasation (data not shown), while as we show here ErbB3 overexpression has a positive impact. These results are consistent with our in vivo
studies using EGFR inhibitors (Kedrin et al., 2009a
), where we show that inhibition of EGFR had a slow inhibitory effect on intravasation and was only evident with a delay of several hours after EGFR inhibition. Conversely, inhibition of ErbB2 rapidly inhibited intravasation (Kedrin et al., 2009b
). Since ErbB3 forms preferred heterodimers with ErbB2, this suggests that an ErbB3/ErbB2 heterodimer is important in enhancing the intravasation step, while EGFR is important for tumor cell invasion from the primary tumor to reach the blood vessels (Kedrin et al., 2009a
). HRGβ1 is present in serum in subnanomolar concentrations (Ky et al., 2009
), and by increasing ErbB3 levels in the tumor cells this might enable them to detect low levels of HRGβ1 and increase their intravasation. Endothelial cells have also been reported to express HRGβ1, which may also stimulate intravasation (Iivanainen et al., 2007
; Kalinowski et al.).
HRGβ1 activated PI3K/Akt in both ErbB3 WT and ErbB3-Mutant cells but with different kinetics. In ErbB3WT cells the pathway was maximally activated after 5min of HRGβ1 stimulation, while it took between 10 and 15min to see the same signal intensity in the ErbB3-Mutant cells. This suggests that early pathway activation, which is dependent upon p85/PI3K binding to HRGβ1 activated ErbB3-containing heterodimers, is responsible for the major effects of the ligand on migration and invasion. The fact that blockade of PI3K or Akt activity using selective inhibitors totally blocked HRGβ1-induced invasion (), while ErbB3-Mutant cells were only blocked in their invasive ability by ~ 60% (), very likely reflects the fact that the PI3K/Akt pathway does get activated in these cells, but with slower kinetics. The importance of the kinetics of signaling pathway activation in response to ErbB ligands has been observed in other systems, (see, e.g. (Olayioye et al., 2001
While previous studies have shown the direct binding of p85 and subsequent signaling from all six YXXM motifs on the ErbB3 C-terminus (Hellyer et al., 2001
; Prigent and Gullick, 1994
), high-throughput methods such as protein microarray studies suggest that other molecules could bind these sites (Jones et al., 2006
). To confirm that the phenotype in the ErbB3-Mutant MTLn3 cells is due to defective PI3K pathway signaling, we used the p110-α selective inhibitor PIK-75, revealing that the in vitro
chemotaxis as well as in vivo
invasion of MTLn3-ErbB3WT cells towards HRGβ1 was impaired following PI3K inhibition. Moreover pretreatment of mice carrying ErbB3WT cell-induced tumors with PIK-75 caused reduced tumor cell motility, as visualized by multiphoton microscopy. Furthermore, the cells in the treated tumors were also more rounded on average as quantified by length/width ratios, thus mimicking the phenotype of the mutant ErbB3 tumors. These experiments support the hypothesis that PI3K pathway activation by ErbB3 is a major contributor to enhancing the initial steps of the metastatic cascade of mammary tumor cells.