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Page 14 of 17 Mooney et al. J Cancer Metastasis Treat 2019;5:19 I http://dx.doi.org/10.20517/2394-4722.2018.93

precursor TGF-α, pEGFR, and pERK protein expression. The reduction in EGFR protein levels in the co-
culture after 48 h. is not sustained in the long-term experiment suggesting that EGFR signaling pathway
inhibition in the co-culture is not entirely attributable to modulation of EGFR availability. Both soluble
(6 kDa) and precursor (36 kDa) TGF-α protein levels are elevated in triple negative human breast cancer
[51]
because cleavage of the precursor form is prevented and cleavage of the mature form is augmented .
Possible explanations for the decrease in precursor TGF-α expression include (1) prevention of NKD2 from
either interacting with precursor TGF-α and/or transporting it to the plasma membrane triggering its
degradation; (2) increased cleavage of precursor TGF-α into mature TGF-α within the plasma membrane;
and (3) decreased precursor TGF-α production caused by reduced TGF-α mRNA expression. The increase
in NKD2 expression coinciding with decreased precursor TGF-α expression suggests that precursor TGF-α
is being prevented from interacting with NKD2 and this is triggering its degradation. This would imply that
decreased cleavage of mature TGF-α into its soluble form is the major contributor to the augmented mature
TGF-α expression, as opposed to increased cleavage of precursor TGF-α. The EGFR signaling pathway
utilizes phosphorylation cascades to activate downstream molecules that affect cell behavior. Suppression of
pEGFR and pERK protein expression after 120 h indicates that EGFR signaling pathway activation is being
suppressed in the co-cultured BCCs. Taken together, the changes in relative mRNA and protein expression
in the co-cultured BCCs are extremely promising as they suggest that co-culture is altering the BCCs at both
transcriptional and translational levels, indicating that this model can be applied to study the mechanism(s)
for signaling pathway inhibition and highly aggressive BCC restriction.

The canonical Wnt/β-catenin signaling pathway is hyperactivated in human breast cancer, and this leads to
increased β-catenin expression. In this work, exposing MDA-MB-231 BCCs to ESC-microstrands decreases
β-catenin mRNA and protein expression. The decline in β-catenin protein levels in western blot analysis
signifies its increased ubiquitylation and degradation leading to decreased canonical Wnt/β-catenin signaling
pathway activation. Three other major signs of canonical Wnt/β-catenin inhibition in BCCs co-cultured with
ESC-microstrands are increased E-cadherin protein levels, augmented NKD2 mRNA and protein expression,
and decreased vimentin mRNA and protein levels. E-cadherin is synthesized as a 135 kDa precursor that
[52]
undergoes cleavage to its mature 120 kDa form . β-catenin is responsible for anchoring mature E-cadherin
to the plasma membrane, and this interaction may contribute to the comparable β-catenin protein levels in
the co-culture and controls after 72 h coinciding with the extreme up-regulation of mature E-cadherin in the
co-cultured BCCs. Co-culture may directly prevent cytoplasmic β-catenin from translocating to the nucleus.
The belief that β-catenin translocation to the nucleus involves more than its cytoplasmic concentration has
[53]
gained steam, for example, Fang et al. assert that Akt, which is downstream of EGFR signaling pathway,
promotes β-catenin nuclear translocation through phosphorylation. One possibility is that co-culture leads
to decreased activated Akt, which is allowing β-catenin that has accumulated in the cytoplasm to be used for
cell-cell adhesion. After 48 h, there are no detectable levels of mature or precursor E-cadherin in the MDA-
MB-231 BCCs but there is mature E-cadherin expression in the MCF7 BCCs (data not shown). Interestingly,
after restoring the BCC media to demonstrate the long-term effect of co-culture, there is mature and
precursor E-cadherin present at the 72- and 120-h marks, respectively. NKD2 is a tumor suppressor that
[33]
has a very short half-life of approximately sixty minutes and is hypermethylated in human breast cancer .
The increase in NKD2 mRNA expression in the co-culture denotes a reversal of its methylation status and
its increased protein expression suggests an active role in dvl-1 inhibition. Finally, the decreases in vimentin
protein and mRNA expression levels in co-cultured BCCs are another sign of inhibition of this pathway
because vimentin is transactivated by this pathway.

Our results showed that ESC-microstrands induced changes in gene expression of key regulators at both
the mRNA and protein level in MDA-MB-231 BCCs that signified a reversal of the hyperactivated status of
EGFR and canonical Wnt/β-catenin signaling pathways and restored signaling pathway regulation present
in normal non-tumorigenic cells. Specifically, decreased EGFR, pEGFR, pERK, and precursor TGF-α