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Page 12 of 16 Verkoeijen et al. J Cancer Metastasis Treat 2019;5:51 I http://dx.doi.org/10.20517/2394-4722.2019.06

three independent paxillinS178A MTLn3 clones, resulting in decreased EGF-induced activation of signaling
pathways downstream of EGFR. Given the fact that EGFR is often highly expressed in advanced breast cancer,
and that EGFR antagonists inhibit spontaneous metastasis formation of MTLn3 cells, we anticipated that
the reduced EGFR expression was an essential component of the inhibited cell migration and metastasis
formation. Indeed, re-expression of the wt-EGFR in the paxillinS178A cells did rescue the migratory phenotype
of MTLn3 paxillinS178A cells as well as their metastatic capacity under in vivo conditions. This regulation of
the EGFR by paxillin phosphorylation has not been described before. Curiously, a recent study demonstrated
that knockdown of MCLK in mammary MCF10A cells induces increase in cell migration through enhanced
phosphorylation of paxillin at Ser178 which was linked to an increase in JNK activity and very interestingly to
a significant up-regulation of EGFR at protein level . This is striking and yet would suggest that our findings
[60]
might not be specific to our cell model. In our study, paxillinS178A MTLn3 cells showed reduced c-Jun
transcriptional activity. In MCF7 cells stable overexpression of c-Jun induces an increase in EGFR expression
suggesting that AP-1 transcription factors can regulate EGFR transcription levels ; such a regulation is also
[53]
observed in keratinocytes . Therefore, we propose a role of the JNK-c-Jun signaling pathway in the regulation
[54]
of EGFR expression at transcriptional level. Our own data in MCF7 cells that have increased ectopic expression
of EGFR show that depletion of paxillin results in significant downregulation of EGFR expression as well as
downstream signaling such as AKT and ERK [Supplementary Figure 7]. Thus, an alternative explanation
may be that JNK-mediated phosphorylation of paxillin Ser178 is required for efficient EGFR endocytosis and
recycling, an essential component in its activation . Hence this will affect proper JNK activation and AP-1
[61]
transcription factor activation thereby providing a positive feedback for EGFR expression . Alternatively,
[62]
paxillin may affect the expression of EGFR by its known role in the regulation of gene expression through
its interaction with ERK [63,64] , poly-A-binding protein , Abl [66,67] and steroid receptors, or through its own
[65]
ability to undergo nucleocytoplasmic shuttling [37,38,68-71] .

Our findings indicate that the JNK-paxillin axis modulates the scattered phenotype of MTLn3 cells.
Both inhibition of JNK as well as ectopic expression of paxillinS178A reversed the scattered phenotype
towards a more epithelial-like morphology with the formation of E-cadherin/β-catenin cell-cell junctions.
This morphological switch was observed under serum-starved and EGF-treated conditions. The (in)direct
tyrosine phosphorylation of β-catenin by EGFR and other receptor tyrosine kinases such as c-Met is known
to destabilize its binding to E-cadherin. Because of the low EGFR expression in the paxillin mutant cells,
we anticipate that this results in de-phosphorylation of β-catenin leading to its localization to the cell-cell
contacts. These data suggest that paxillinS178A does not affect the intrinsic molecular components and
machinery required for the scattered phenotype and support the notion that the effect of paxillinS178A is
rather related to the defects in the EGFR signaling pathways. Consequently, the defects in in vivo metastasis
formation of MTLn3 paxillinS178A cells are most likely largely due to defects EGF signaling. This fits with
our observations that ectopic EGFR expression itself is sufficient to again allow metastasis formation of
paxillinS178A cells.

In summary, in MTLn3, engagement of EGF receptors by EGF triggers rapid activation of JNK, leading
to the phosphorylation of paxillin on Ser 178 which facilitates adhesion turnover thus promoting rapid
migration. In the MTLn3 cell lines that ectopically express GFP-paxillin-S178A mutants, we found that the
EGF receptor is downregulated at both the protein and mRNA levels. Paxillin S178A mutant protein might
associate with JNK preventing its activation and consequently affecting AP1 activity (c-Jun phosphorylation)
and consequently EGFR expression. In our model [Figure 7], still some links need to be further determined
such as how exactly paxillin regulates via JNK EGFR expression. Given the essential role of Ser178
phosphorylation in the migration and metastasis formation, monitoring this paxillin phosphorylation in
tumor samples from patients may be indicative of the activation of this pro-metastatic pathway and possibly
predictive for the disease prognosis. Moreover, with the further development of specific JNK inhibitors [72,73]
it is anticipated that novel targeted therapies that antagonize the migratory/invasive behavior of tumor cells
may be used in the clinic in the future. Further work in this area is required.