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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 3 of 17

Cross-talk between the canonical Wnt/β-catenin and EGFR signaling pathways is evidenced in cancer
cells. For example, the EGFR signaling pathway activates the transcription of canonical Wnt/β-catenin
target genes such as cyclinD1, c-myc, and survivin. In oral and non-small cell lung cancers, there is a link
between EGFR mutations and accumulation of β-catenin within the nucleus [25,26] . The EGFR signaling
pathway is turned on by Wnt ligands through the Fzd receptor, leading to cleavage of EGF-like ligands [27,28] .
EGFR signaling is transactivated in breast cancer patients after knockdown of the Wnt negative regulator
SFRP1. Increased expression of Wnt1 and decreased expression of APC activates EGFR signaling in breast
[30]
cancer [28,29] . Down-regulation of β-catenin decreases levels of EGFR, STAT3 and Akt mRNA . E-cadherin
[31]
regulates EGFR localization and activation . NKD2 is a negative regulator of Dvl-1, which promotes
β-catenin translocation to the nucleus [32,33] . The N-terminal region of NKD2 also contains a moiety capable
of binding to precursor TGF-α (36 kDa), a ligand for the EGFR signaling pathway. This interaction facilitates
the escort of precursor TGF-α to the plasma membrane, whereby it is cleaved into mature TGF-α (17 kDa),
cleaved into a soluble form (6 kDa), and secreted [29,34] .

These previous studies establish a relationship between the EGFR and canonical Wnt/β-catenin signaling
pathways. They also illustrate the complexity involved in this signaling pathway cross-talk. Hyperactivation
of both signaling pathways is a signature of aggressive human breast cancer. NKD2 is involved in a positive
feedback loop in the EGFR signaling pathway and is a known inhibitor of the canonical Wnt/β-catenin
signaling pathway. It is likely that NKD2 acts as a “molecular switch” between these pathways in breast
cancer development and/or progression. The exact mechanism for this communication remains elusive.
We have established a 3D in vitro ESC-microstrand tumor model by encapsulating ESCs in small-diameter
alginate microstrands and co-culturing with metastatic human BCCs. The creation of a 3D in vitro ESC-
microstrand tumor model to probe signaling pathway-related molecules following restoration of signaling
pathway function will provide insight into this complex process [8,10] . This model restricts metastatic BCC
[10]
proliferation, survival, metabolism, invasion, migration, and oncogene expression, as summarized in Table 1 .
To further understand the underlying mechanism for ESCs’ inhibitory effect on BCCs, we hypothesized
that ESC-microstrands will inhibit the EGFR and canonical Wnt/β-catenin signaling pathways in highly
aggressive cancer due to restored signaling pathway regulation. To test this hypothesis, co-cultured BCCs
were treated with EGFR and canonical Wnt/β-catenin signaling pathway inhibitors. Next, co-cultured BCCs
were probed at the molecular level for expression of key molecules related to the EGFR and canonical Wnt/
β-catenin signaling pathways using quantitative reverse transcription polymerase chain reaction (qRT-PCR)
and Western blot analysis. Changes in gene expression would reflect the ability of pluripotent signals from
ESC-microstrands to reverse oncogenic signaling pathway hyperactivation and restore signaling pathway
regulation in metastatic BCCs.

METHODS
Cell culture
Mouse CCE ESCs were purchased from StemCell Technologies (Vancouver, Canada) and cultured in 0.1%
gelatin-coated flasks. The growth medium consisting of DMEM with 4.5 g/L D-glucose supplemented
with 15% (v/v) FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.1 mmol/L MEM non-essential amino
acids, 10 ng/mL murine recombinant LIF (StemCell Technologies, Vancouver, Canada), 0.1 mmol/L
monothioglycerol, 2 mmol/L L-glutamine, and 1 mmol/L sodium pyruvate. Cell media was changed every
day and cells were passaged every 2 to 3 days. Human MDA-MB-231 and MCF7 BCCs were purchased from
ATCC (Manassas, VA). Cells were cultured in growth medium consisting of DMEM supplemented with 15%
(v/v) FBS, 100 U/mL penicillin, 100 µg/mL streptomycin and 2 mmol/L L-glutamine. Human 3T3 fibroblasts
were obtained from the NIH and cultured in growth medium containing DMEM supplemented with 15%
(v/v) FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cell lines were maintained at 37 ºC in a
humidified atmosphere containing 5% CO .
2