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Mooney et al. J Cancer Metastasis Treat 2019;5:19 I http://dx.doi.org/10.20517/2394-4722.2018.93 Page 15 of 17

coupled with augmented mature TGF-α expression indicated that the EGFR signaling pathway was being
suppressed, whilst decreased vimentin and β-catenin expression coinciding with increased precursor and
mature E-cadherin levels supported the notion of canonical Wnt/β-catenin signaling pathway suppression.
Importantly, these changes corresponded with an extreme up-regulation of NKD2 at both the mRNA
and protein levels [Figure 7]. A possible explanation for this phenomenon is that co-culture with ESC-
microstrands restores NKD2 expression in metastatic MDA-MB-231 BCCs, which enables it to inhibit the
canonical Wnt/β catenin signaling pathway, but that NKD2 is also prevented from transporting precursor
TGF-α to the membrane for activation of the EGFR signaling pathway.

Altogether, we have demonstrated that exposure of triple negative BCCs to a bioengineered 3D ESC
microenvironment restricts their tumorigenic, invasive, and metastatic features owing to restoration of
EGFR and canonical Wnt/β-catenin signaling pathway regulation. In particular, NKD2 could act as a
“molecular switch” between these pathways in BCCs. Co-culture with ESC-microstrands has up-regulated
NKD2 in triple negative, metastatic BCCs both at mRNA and protein levels. The exact role of NKD2 in this
metastatic phenotype reversal will be the focus of subsequent studies. Exposure of MDA-MB-231 BCCs to
ESC-microstrands may prevent NKD2 myristoylation allowing it to solely interact with Dvl-1 and thwarting
precursor TGF-α transport to the plasma membrane causing its increased degradation. Future work will
elucidate the mechanism leading to NKD2 preference for either EGFR or canonical Wnt/β-catenin signaling
pathway interaction allowing for the targeting of NKD2 to treat triple negative breast cancer. This work is
important because it establishes that the bioengineered 3D ESC model can not only restrict triple negative
breast cancer survival and metastatic potential, but can also be applied to determine the mechanism for this
restriction and identify therapeutic targets to reverse metastatic disease. This is the first example of exploring
the role of NKD2 in aggressive breast cancer as all other studies in literature were performed in colon cancer.
In the future, we may test whether using ESC-microstrand-conditioned media could achieve the same level
of restriction of cancer metastasis. Additionally, the use of a panel of triple negative metastasis breast cancer
cells (e.g., MDA-MB-157, MDA-MB-468) will further validate our findings and confirm the utility of the 3D
ESC model system for understanding cancer metastasis.

DECLARATIONS
Authors’ contributions
Made substantial contributions to conception and design of the study and performed data analysis and
interpretation: Mooney B and Xie Y
Performed data acquisition and provided technical and material support: Mooney B
Created Figure 7 and revised the paper: Tian Y
Assisted in drug resistance experiments and revised the paper: Rousseau E

Availability of data and materials
Not applicable.

Financial support and sponsorship
This work was supported by National Science Foundation (CBET0846270).

Conflicts of interest
All authors declared that there are no conflicts of interest.

Ethical approval and consent to participate
Not applicable.

Consent for publication
Not applicable.