Human breast cancer is currently the highest diagnosed form of cancer and the second leading cause of cancer-related deaths in American women. Triple negative breast cancer is of the basal subtype and displays poor prognosis owing to its highly metastatic properties. Current treatments focused on eradicating breast tumors in lieu of or following local therapy include chemotherapy, hormonal therapy, and targeted therapy. Hormonal therapy is not an option for triple negative breast cancer as it does not contain hormone receptors, and there are currently no approved biological targeted therapies. Chemotherapy has proven unsuccessful because triple negative breast cancer is highly drug resistant and is associated with high toxicity[
Metastatic breast cancer cells have been linked to embryonic stem cells (ESCs) due to their sharing of certain similar gene signatures and convergence of tumorigenic and embryonic pathways (e.g., EGFR, Wnt/β-catenin, and TGF-β)[
The EGFR and canonical Wnt/β-catenin signaling pathways control many cell properties that contribute to the metastatic phenotype. The EGFR (also known as the ErbB1/HER1) belongs to the ErbB family of tyrosine kinase receptors. Binding of ligands (e.g., EGF, TGF-α) to the EGFR initiates dimerization of the EGFR with one of the other three receptors within the same family. Dimerization activates the intracellular tyrosine kinase domain, which undergoes autophosphorylation. This causes a conformational change which exposes the activation loop and allows for protein binding and phosphorylation of downstream signaling targets[
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[
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
Summarization of inhibitory effects of 3D ESC-microstrands on BCCs[
Cancer cell property | Co-cultured BCC |
---|---|
Proliferation | |
WST-1 assay | ↓ |
Cell cycle analysis-G2/M population | ↓ |
Cell cycle analysis-S population | ↑ |
Apoptosis/necrosis | |
Annexin-V FITC propidium iodide assay | ↑ |
Cell cycle analysis-SubG1 population | ↑ |
Cell metabolism | |
Glycolysis | ↓ |
Oxidative phosphorylation | ↓ |
Epithelial-to-mesenchymal transition | |
E-cadherin protein expression | ↑ |
Vimentin protein expression | ↓ |
Cell migration | ↓ |
Invasiveness | ↓ |
ESC: embryonic stem cell; BCC: breast cancer cell
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
A sodium alginate solution (1.5% w/v alginate dissolved in 0.9% w/v sodium chloride) containing ESCs suspended at a density of 1 × 106 cells/mL was loaded into a 3 mL syringe, which was placed in a New Era NE-1000 syringe pump (New Era, Farmingdale, NY). A 50 mmol/L CaCl2 solution was placed in one well of a 24-well plate at a volume of 2 mL. The sodium alginate-ESC mixture in the syringe was pumped into the CaCl2 solution at a constant rate of 0.1 mL/min. Exposing the divalent cation, Ca2+, to sodium alginate cross-linked the polymer chains. This set-up consistently formed microstrands with an approximate 200 µm diameter
Schematic for co-culture of 3D embryonic stem cell (ESC)-microstrands with human breast cancer cells (BCCs). A: Optical image of mouse ESCs encapsulated in alginate hydrogel microstrands; B: experimental set-up of BCC alone (left), cultured with empty microstrands (middle), and co-cultured with 3D ESC-microstrands (right). No microstrands, empty microstrands, and 3T3 fibroblasts instead of BCCs served as experimental controls
As shown in
Cell proliferation of MDA-MB-231 BCCs was measured using a Premixed WST-1 Cell Proliferation Reagent (Clontech, Mountain View, CA), per manufacturer’s instruction.
MDA-MB-231 BCCs were co-cultured with ESC-microstrands for 48 h. and subsequently treated with 5, 10, or 20 μmol/L of either the EGFR inhibitor Erlotinib, the canonical Wnt/β-catenin inhibitor PNU74654, or both to determine how sensitive these cells were to both chemotherapeutic inhibitors. Controls included non-co-cultured BCCs and non-drug-treated BCCs. After 48 h, the sensitivity of the BCCs to drug treatment was assessed using a WST-1 cell proliferation assay per manufacturer’s instructions. For the drug resistance experiments, cells were treated with chemotherapeutic drugs up to four times for 24 h each and allowed to recover for either 24 or 48 h prior to analysis.
Cells were washed once with 0.5 mL of ice cold PBS. Total RNA of MDA-MB-231 BCCs was isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA), per manufacturer’s instruction. The RNA was of high purity with 260/280 values close to 2.0. Starting with 125 ng/mL of total RNA, cDNA was synthesized using an Omniscript first-strand cDNA synthesis kit (Invitrogen, Grand Island, NY). qRT-PCR was executed using a StepOnePlusTM Real Time PCR System (Applied Biosystem, Foster City, CA). The Mastermix contained 1.25 µL of the forward and reverse primers
Forward and reverse primer sequences for qRT-PCR
Target | Forward sequence (5’ → 3’) | Reverse sequence (5’ → 3’) |
---|---|---|
EGFR
|
CTC CCA GTG CCT GAA TAC ATA AA
|
CCG TGG TCA TGC TCC AAT AA
|
qRT-PCR: quantitative reverse transcription polymerase chain reaction
BCCs were washed with ice cold PBS and lysed through exposure to 0.2 mL of ice cold RIPA buffer (Sigma Aldrich) for 20 min while pipetting up and down. Cell lysates were centrifuged at 12,000 rpm for 20 min at 4 ºC. The cell pellet was discarded, and the concentration of total protein was assessed using a BCA assay, per manufacturer’s instructions. The protein samples were boiled for 10 min and loaded into the wells (15 µg) of a NuPage® Novex® 4%-12% Bis-Tris Protein Gel (Thermo Fisher Scientific, Waltham, MA) for separation through electrophoresis (100V for 2 h). The running buffer, 1 × NuPAGE® MOPS SDS (Thermo Fisher Scientific, Waltham, MA), contained 50 mmol/L MOPS, 50 mmol/L Tris Base, 0.1% SDS, and 1 mmol/L EDTA. Prior to loading, cell lysates were mixed with LDS sample buffer (4:1) (PierceTM Chemical, Rockford, IL) containing 0.5 mol/L dithiothreitol (DTT) (10:1) (Invitrogen, Grand Island, NY). RIPA buffer was added to give a final volume of 20 µL per well. β-actin was utilized as a loading control and 10 µL of Precision Plus ProteinTM WesternCTM Standards (BioRad, Hercules, CA) was added to a well as a molecular weight reference. The proteins were wet-transferred onto a nitrocellulose membrane for 1 h at 100V. The transfer buffer consisted of 1 × Tris-glycine buffer with 20% (v/v) methanol. Prior to transfer, the nitrocellulose membrane was incubated for 10 min in 100% methanol for activation. Following wet transfer, the nitrocellulose membrane was washed three times in Tris-buffered saline and Tween 20 (TBST) in 15-min intervals and incubated for 1 h at RT in 5% (w/v) BSA diluted in TBST. The nitrocellulose membrane was incubated overnight at 4 ºC in a primary antibody
Antibodies used for western blot analysis
Target | Species | Company | Dilution/concentration |
---|---|---|---|
EGFR
|
Goat
|
Santa Cruz Biotechnology
|
1:500
|
Data analyses were performed using a one-way ANOVA with multiple comparisons. Data were expressed as the mean ± standard deviation. A value of
Highly aggressive MDA-MB-231 BCCs were treated with the chemotherapeutic drugs Erlotinib and PNU 74654, which inhibit the EGFR and canonical Wnt/β-catenin signaling pathways, respectively. For both drugs, there was a reduction in BCC viability when treated at a dose of 20 µmol/L with a 24-h recovery period
Exposure to embryonic stem cell (ESC)-microstrands overcomes chemotherapeutic drug resistance and reduces metastatic MDA-MB-231 cancer cell survival after drug treatment. A: MDA-MB-231 breast cancer cells (BCCs) were sensitive to the chemotherapeutic drugs Erlotinib and PNU 74654 when treated with 20 µmol/L for 30 min, followed by a 24-h recovery period. Simultaneous treatment with Erlotinib and PNU 74654 resulted in the largest decrease in cell viability suggesting that both pathways play a role in the metastatic phenotype; B: after being co-cultured with ESC-micorstrands, MDA-MB-231 BCCs exhibited significant reduction in cell viability after three cycles of treatment and recovery with both drugs at 20 µmol/L compared to non-co-cultured BCC control. *Statistical significance compared to untreated control (#
PNU 74654 decreased metastatic BCC viability more than Erlotinib, with a reduction exceeding 30%. Combining both drugs further diminished cell viability compared to individual treatment. To examine the effect of ESC-microstrands on chemotherapeutic drug resistance, cells were treated three times with both chemotherapeutic drugs at a dose of 20 µmol/L, and viability was compared to the non-co-cultured BCCs. Three 24-h treatment and 24-h recovery cycles were performed to simulate the drug resistant state caused by multiple chemotherapeutic treatments
To understand the inhibitory effect of exposure to ESC-microstrands on metastatic BCCs, relative mRNA expression levels of EGFR and canonical Wnt/β-catenin signaling pathway-related molecules of MDA-MB-231 BCCs following co-culture with ESC-microstrands were examined using qRT-PCR. We have previously shown that co-culture with ESC-microstrands increases NKD2 and decreases TGF-α mRNA expression after 48 h[
Reversal of gene expression of metastatic MDA-MB-231 breast cancer cells (BCCs) after exposure to embryonic stem cell (ESC)-microstrands at the transcriptional level as revealed by qRT-PCR analysis. A: After 72 h, significantly decreased TGF-α and increased NKD2 gene expression in BCCs co-cultured with ESC-microstrands compared to both empty microstrand and non-co-cultured controls; B: after 72 h, EGFR and vimentin gene expression decreased significantly compared to both controls; C: after 48 h, β-catenin and GAPDH gene expression declined in BCCs co-cultured with ESC-microstrands compared to the both controls (****
The differences between BCCs co-cultured with ESC-microstrands and both the empty microstrand and non-co-cultured controls were statistically significant. For both 48 h (data not shown) and 72 h, there was a decline in both EGFR and vimentin mRNA levels in the BCCs co-cultured with ESC-microstrands
We further examined expression of EGFR, β-catenin, NKD2, and TGF-α in metastatic MDA-MB-231 BCCs at protein level after 48 h of co-culture with ESC-microstrands, in comparison with non-invasive MCF7 BCCs and 3T3 fibroblasts
Protein expression of key signaling pathway molecules in metastatic MDA-MB-231 breast cancer cells (BCCs) after co-culture with embryonic stem cell (ESC)-microstrands for 48 h in comparison to non-aggressive MCF7 BCCs and 3T3 fibroblasts. Western blots show reduced EGFR, β-catenin, and mature TGF-α protein expression in the co-cultured MDA-MB-231 BCCs (CC: co-cultured with ESC-microstrands; E: co-cultured with empty microstrands; C: non-co-cultured control) (A); densitometry of protein expression of EGFR (B); β-catenin (C); mature TGF-α (D); NKD2 (E) (***
Since co-culture with ESC-microstrands decreased EGFR and β-catenin expression in MCF7 and MDA-MB-231 BCCs following 48 h of co-culture, ESC-microstrands and media were removed and replaced with BCC media to see if there are other long-term signaling pathway-related changes in protein expression in MDA-MB-231 BCCs. The BCCs recovered for either one day or three days, which are denoted as 72 h and 120 h, respectively.
Western blot analysis of long-term protein expression of key EGFR signaling pathway molecules. Metastatic MDA-MB-231 breast cancer cells (BCCs) were co-cultured with embryonic stem cell (ESC)-microstrands for 48 h, followed by removing ESC-microstrands, replacing BCC media, and analyzing protein expression at 72 h and 120 h. Western blots for protein expression in metastatic MDA-MB-231 BCCs (CC: co-cultured with ESC-microstrands; E: co-cultured with empty microstrands; C: non-co-cultured control) (A); densitometry of expression of EGFR (B), precursor TGF-α (C), mature TGF-α (D), pEGFR (E), and pERK (F) (***
Immunoblots for molecules related to the canonical Wnt/β-catenin signaling pathway are provided in
Western blot analysis of long-term protein expression of key canonical Wnt/β-catenin signaling pathway molecules. Metastatic MDA-MB-231 breast cancer cells (BCCs) were co-cultured with embryonic stem cell (ESC)-microstrands for 48 h, followed by removing ESC-microstrands, replacing BCC media, and analyzing protein expression at 72 h and 120 h. Western blots for protein expression in metastatic MDA-MB-231 BCCs (CC: co-cultured with ESC-microstrands; E: co-cultured with empty microstrands; C: non-co-cultured control) (A); densitometry of expression of β-catenin (B), NKD2 (C), precursor E-cadherin (D), mature E-cadherin (E), and vimentin (F) (***
This work has demonstrated the utility of a bioengineered 3D ESC microenvironment (so-called ESC-microstrands) for the study of cross-talk of signaling pathways in cancer cells. It reveals that inhibitory effects of the ESC microenvironment on triple negative, metastatic MDA-MB-231 BCCs is attributable to restoration of EGFR and canonical Wnt/β-catenin signaling pathway regulation. Simultaneous treatment of metastatic BCCs with EGFR and canonical Wnt/β-catenin signaling inhibitors suppresses growth more than individual treatment, suggesting that both pathways play a role in the restriction of metastatic phenotype.
The microenvironments of ESCs play a fundamental role in providing cells with appropriate signaling to induce cell proliferation, differentiation, or death. ESCs secrete soluble factors or exosomes that could reprogram malignant cancer cells to benign phenotype and suppress tumorigenesis[
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[
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 subsequent study by Maltseva
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 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 because cleavage of the precursor form is prevented and cleavage of the mature form is augmented[
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 undergoes cleavage to its mature 120 kDa form[
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 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
Schematic representation of the effects of co-culture with embryonic stem cell (ESC)-microstrands on expression of key signaling pathway molecules in MDA-MB-231 breast cancer cells (BCCs). Left Panel: BCC alone; Right Panel: co-cultured breast cancer cells with ESC-microstrands. Changes in protein and mRNA expression after co-culture with ESC-microstrands indicated inhibition of the canonical Wnt/β-catenin (above the dash line) and EGFR (below the dash line) signaling pathways. Co-culture with ESC-microstrands causes NKD2 up-regulation at both the mRNA and protein levels, and this coincides with dual signaling pathway inhibition. It suggested restored ability of NKD2 to inhibit the canonical Wnt/β-catenin signaling pathway and reduced ability to promote the EGFR signaling pathway following co-culture
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.
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
Assisted in drug resistance experiments and revised the paper: Rousseau E
Not applicable.
This work was supported by National Science Foundation (CBET0846270).
All authors declared that there are no conflicts of interest.
Not applicable.
Not applicable.
© The Author(s) 2019.