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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 13 of 17

Deregulation of canonical Wnt/β-catenin and EGFR signaling is implicated in various forms of tumorigenesis
causing colorectal, lung, breast, ovarian, prostate, liver, and brain cancers [12,44] . For the canonical Wnt/β-catenin
signaling pathway, cancer also arises from overexpression of Wnt ligand genes, DNA hypermethylation of
negative regulators such as p16 and E-cadherin, and overexpression of R-spondins that enhance Wnt ligand
function [23,44] . In BCC lines and primary tumors, Wnt ligands and Fzd receptors are expressed, and β-catenin
[44]
is often localized within the nucleus . Inhibiting EGFR in cancer cells either directly or with cDNA
[45]
transfections in vitro and in vivo causes a decrease in proliferation . Signaling pathways downstream of the
EGFR are deregulated through EGFR mutations, constitutive activation, autocrine/paracrine signaling loops,
enhanced ligand production, up- or down-regulation of signaling mediators, and prolonged interactions
of EGFR with ErbB2 that either increase EGFR expression, activate EGFR, or prevent its deactivation [14,46] .
Activation of Rho GTPases downstream of EGFR signaling reduces E-cadherin expression and actin
[45]
reorganization . Certain tumors contain mutations that delete the extracellular binding domain of the
EGFR, which constitutively activates the receptor. In glioblastoma, there is frequently an in-frame deletion of
exon 2-7 of the EGFR. Ligand-independent activation of the EGFR is caused by overexpression of urokinase-
type plasminogen activator receptor and its association with the integrin α5β1 [11,29,47] . Prolonged interaction
of ErbB2 with EGFR fosters breast CSC self-renewal through activation of the PI3K/Akt signaling cascade.
Increased cellular stress and radiation have been shown to down-regulate phosphatases responsible for the
[48]
EGFR signal abrogation . Downstream of the EGFR signaling pathway, the PI3K/Akt/mTOR pathway
activates protein kinase B through loss of PTEN, a tumor suppressor and negative regulator of PI3K/Akt
signaling. The result is stabilization of β-catenin through phosphorylation and inactivation of GSK3. As
demonstrated by the previous examples, deregulated canonical Wnt/β-catenin and EGFR signaling play an
enormous role in many types of aggressive cancer, which highlights the importance of understanding their
contribution to aggressive breast cancer progression. As such, our first aim was to examine whether exposure
of aggressive MDA-MB-231 BCCs to ESC-microstrands increases sensitivity to chemotherapeutic drugs
known to specifically target these two pathways.

Highly aggressive MDA-MB-231 BCCs were treated with the chemotherapeutic drugs Erlotinib and
PNU74654. Erlotinib specifically targets the EGFR signaling pathway by binding to the ATP-binding site
of the EGFR and preventing phosphorylation by its tyrosine kinase. PNU 74654 targets the canonical Wnt/
β-catenin signaling pathway by preventing the interaction of β-catenin with the transcription factor T-cell
factor/lymphoid enhancer factor. The canonical Wnt/β-catenin signaling pathway may play a larger role in
MDA-MB-231 BCC aggressiveness as these cells were more sensitive to PNU74654. However, both signaling
pathways are involved in the aggressive phenotype because combining both drugs significantly increases
sensitivity. Co-culturing the MDA-MB-231 BCCs with ESC-microstrands prior to dual chemotherapeutic
drug treatment coupled with the addition of multiple treatment and recovery periods, demonstrated that co-
culture with ESC-microstrands increases BCC sensitivity to chemotherapeutic drugs.

Co-culture with ESC-microstrands reduced GAPDH mRNA expression. GAPDH is an enzyme responsible
for catalyzing the sixth step of glycolysis. The original intention was to employ GAPDH as a housekeeping
gene for this study, though recent works have confirmed GAPDH up-regulation in human breast cancer. A
[49]
subsequent study by Maltseva et al. identified five reliable housekeeping genes in human breast cancer and
β-actin, which was used in this study, was one of the top choices. In our model, GAPDH mRNA expression
[10]
was decreased in the co-cultured MDA-MB-231 cells, which may explain their decreased glycolytic rate .
The PI3/Akt pathway lies downstream of the EGFR signaling pathway. GAPDH stabilizes activated Akt
allowing oncogenic cells to circumvent caspase-independent cell death. Interestingly, Akt kinase is one of
the main contributors to the Warburg effect and Akt also prevents the nuclear accumulation of GAPDH
[50]
necessary for caspase-dependent cell death .

In this work, inhibition of the EGFR signaling pathway following co-culture is demonstrated through
reduced EGFR and TGF-α mRNA expression, increased mature TGF-α protein expression, and decreased