Understanding the etiology of metastasis is very important in clinical perspective, since it is estimated that metastasis accounts for 90% of cancer patient mortality. Metastasis results from a sequence of multiple steps including invasion and migration. The early stages of metastasis are tightly controlled in normal cells and can be drastically affected by malignant mutations; therefore, they might constitute the principal determinants of the overall metastatic rate even if the later stages take long to occur. To elucidate the role of individual mutations or their combinations affecting the metastatic development, a logical model has been constructed that recapitulates published experimental results of known gene perturbations on local invasion and migration processes, and predict the effect of not yet experimentally assessed mutations. The model has been validated using experimental data on transcriptome dynamics following TGF-β-dependent induction of Epithelial to Mesenchymal Transition in lung cancer cell lines. A method to associate gene expression profiles with different stable state solutions of the logical model has been developed for that purpose. In addition, we have systematically predicted alleviating (masking) and synergistic pairwise genetic interactions between the genes composing the model with respect to the probability of acquiring the metastatic phenotype. We focused on several unexpected synergistic genetic interactions leading to theoretically very high metastasis probability. Among them, the synergistic combination of Notch overexpression and p53 deletion shows one of the strongest effects, which is in agreement with a recent published experiment in a mouse model of gut cancer. The mathematical model can recapitulate experimental mutations in both cell line and mouse models. Furthermore, the model predicts new gene perturbations that affect the early steps of metastasis underlying potential intervention points for innovative therapeutic strategies in oncology. Bird Shannyn birdgirl@arcticmail.com University of Nebraska-Lincoln 2017-02-01T12:35:02Z 2017-04-28T14:44:10Z

DNA damage

 2017-04-15T12:10:11Z

Extracellular Matrix

 2017-04-15T12:10:11Z 2017-02-01T15:15:00Z

Name: Vimentin

Gene Name: VIM

UNIPROT ID: P08670

Gene ID: 7431

 2017-02-01T15:10:14Z

Name: Mothers against decapentaplegic homolog 3

Gene Name: SMAD3

UNIPROT ID: P84022

Gene ID: 4088

 2017-02-01T15:08:31Z

Name: Cyclin-dependent kinase inhibitor 1

Gene Name: CDKN1A

UNIPROT ID: P38936

Gene ID: 1026

 2017-02-01T15:02:53Z

Name: Zinc finger E-box-binding homeobox 1

Gene Name: ZEB1

UNIPROT ID: P37275

Gene ID: 6935

 2017-02-01T14:57:59Z

Name: RAC-alpha serine/threonine-protein kinase

Gene Name: AKT1

UNIPROT ID: P31749

Gene ID: 207

 2017-02-01T15:10:14Z

Epithelial to Mesenchymal Transition

Morphological change epithelial cells undergo in order to gain motility.

 2017-02-01T15:15:00Z

Name: RAC-beta serine/threonine-protein kinase

Gene Name: AKT2

UNIPROT ID: P31751

Gene ID: 208

 2017-02-01T15:15:00Z

Metastasis is the final stage of cancer development whereby secondary tumors in distant organs have formed.

 2017-02-01T15:15:14Z

Name: Catenin beta-1

Gene Name: CTNNB1

UNIPROT ID: P35222

Gene ID: 1499

 2017-02-01T15:08:31Z

Name: Cadherin-1

Gene Name: CDH1

UNIPROT ID: P12830

Gene ID: 999

 2017-02-01T15:07:34Z

Name: Zinc finger protein SNAI1

Gene Name: SNAI1

UNIPROT ID: O95863

Gene ID: 6615

 2017-02-01T15:00:57Z

Name: Tumor protein p73

Gene Name: TP73

UNIPROT ID: O15350

Gene ID: 7161

 2017-02-01T15:05:17Z

Name: Cadherin-2

Gene Name: CDH2

UNIPROT ID: P19022

Gene ID: 1000

 2017-02-01T15:08:31Z

Name: Zinc finger E-box-binding homeobox 2

Gene Name: ZEB2

UNIPROT ID: O60315

Gene ID: 9839

 2017-02-01T15:05:17Z

Growth Factors

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Name: Zinc finger protein SNAI2

Gene Name: SNAI2

UNIPROT ID: O43623

Gene ID: 6591

 2017-02-01T15:00:57Z

Name: Twist-related protein 1

Gene Name: TWIST1

UNIPROT ID: Q15672

Gene ID: 7291

 2017-02-01T15:00:57Z

Migration is a stage of cancer development whereby motile cancer cells spread to other parts of the body.

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microRNA 34

 2017-02-01T15:05:17Z

This node represents when a cell has stopped progressing through the cell cycle.

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Name: Dickkopf-related protein 1

Gene Name: DKK1

UNIPROT ID: O94907

Gene ID: 22943

 2017-02-01T13:34:36Z

Name: Tumor protein 63

Gene Name: TP63

UNIPROT ID: Q9H3D4

Gene ID: 8626

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microRNA 200

 2017-02-01T15:10:14Z

Name: Cellular tumor antigen p53

Gene Name: TP53

UNIPROT ID: P04637

Gene ID: 7157

 2017-02-01T15:05:17Z

Intracellular domain of the Notch receptor

Name: Neurogenic locus notch homolog protein 1

Gene: NOTCH1

UniProtKB: P46531

 2017-02-01T15:00:57Z

Name: Transforming growth factor beta-1

Gene Name: TGFB1

UNIPROT ID: P01137

Gene ID: 7040

 2017-02-01T14:53:27Z

Programmed cell death

 2017-02-01T15:05:17Z

Invasion is a stage of cancer development whereby motile cancer cells pass through the basement membrane and extracellular matrix.

 2017-02-01T15:15:00Z

microRNA 203

 2017-02-01T15:02:53Z

SMAD, CDH2, GF, and NICD are all positive regulators of ERK. AKT1 inhibits ERK and expresses dominance over SMAD, CDH2, GF, and NICD.

NICD indirectly activates ERK. NICD activates HES1, which inhibits DUSP1/6, a negative regulator of ERK.

SMAD activates ERK.

Growth factors activate the MAPK signaling pathway.

AKT1 inhibits ERK signalling.

CDH2 activates ERK via FGFR polymerisation.

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VIM is upregulated by both CTNNB1 and ZEB2.

CTNNB1 targets the VIM promoter to upregulate VIM expression.

ZEB2 activates VIM gene transcription.

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SMAD is activated by TGFbeta. SMAD is also inhibited by miR200 and miR203, which are dominant to TGFbeta.

miR200 inhibits SMAD2 and SMAD3.

Binding of TGFbeta to its receptor actviates SMAD.

A range of miRNAs have been found to target SMAD.

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AKT2, p73, p63, and p53 all upregulate p21. SMAD also activates p21 in the presence of NICD. ERK and AKT1 are negative regulators of p21 which express dominance over SMAD, AKT2, p53, p63, and p73.

Inhibition of ERK leads tp p21 upregulation.

In proliferating murine keratinocytes, Notch activation induces p21.

NICD enhances SMAD transcriptional activity.

SMAD induces p21 transcription.

AKT2 inhibits p21 by binding to the p21 promoter and by sequestering p21 in the nucleus in order to induce cell cycle arrest. (Studies performed using mouse myoblasts, human fibroblasts, and human myoblasts.)

AKT1 phosphorylates p21 at Thr 145, which decreases p21 binding to the cyclin-dependent kinases Cdk2 and Cdk4.

p63 is similar to p53 and has been found to regulate some of the same gene targets, such as p21.

In human fibroblasts, p53 initiates cell cycle arrest by inducing the synthesis of p21.

p73 can bind to the consensus DNA-binding motif and activate p53 gene targets, including p21.

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SNAI2, NICD, and CTNNB1 upregulate ZEB1. TWIST1 also activates ZEB1, but only in the presence of SNAI1. miR200 is a negative regulator of ZEB1 which expresses dominance over CTNNB1, NICD, SNAI2, and TWIST1.

miR00 is involved in a double-negative feedback loop with ZEB1.

CTNNB1 binds to and activates the ZEB1 promoter.

SNaI1 and TWIST1 work cooperatively to activate ZEB1 transcription. SNAI1 induces the rapid increase in TWIST protein. TWIST in turn is able to bind to the ZEB1 promoter.

Over-expression of Notch-1 increased expression of ZEB1.

SNAI2 binds to and activates the ZEB1 promoter.

SNaI1 and TWIST1 work cooperatively to activate ZEB1 transcription. SNAI1 induces the rapid increase in TWIST protein. TWIST in turn is able to bind to the ZEB1 promoter.

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CTNNB1 activates AKT1 in the presence of either CDH2, GF, TGFbeta, or NICD. CDH1, p53, and miR34 are all negative regulators of AKT1 which express dominance over CTNNB1.

The CTNNB1/Tcf-4 complex activates AKT1 through Tcf-4 binding to the promoter.

TGFbeta activates AKT1 through Mapk.

Notch signalling activates HES, which inhibits PTEN, a negative regulator of AKT.

GF induces AKT phosphorylation.

Introduction of miR34 downregulated AKT1.

CDH2 can activate FGFR, which leads to AKT1 activation.

E-cadherin inhibits ligand binding to EGFR at high cell densities, leading to reduced AKT activation.

p53 upregulates PTEN transcription, which in turn inhibits AKT activation.

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EMT is activated by CDH2 and inhibited by CDH1, which expresses dominance over CDH2.

The presence of mesenchymal markers (CDH2) activates EMT.

The absence of epithelial markers (CDH1) activates EMT.

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When miR203, miR34, and p53 are all inactive, TWIST1 activates AKT2 in conjunction with either CDH2, GF, or TGFbeta.

TGFB association with its receptor and with the p85 subunit activates PI3K.

miR203 inhibits AKT2 expression through mRNA degradation.

GF induces AKT phosphorylation.

miR34 inhibits AKT2 phosphorylation.

CDH2 can activate FGFR, which leads to AKT1 activation.

Twist induces AKT2 transcription.

p53 inhibits AKT2 through activation of PTEN.

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Migration is a positive regulator of Metastasis.

In order for secondary tumors to form at distant sites in the body, migration of cancer cells is required.

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DKK1, p53, AKT1, miR34, miR200, CDH1, CDH2 and p63 are all negative regulators of CTNNB1.

miR200 directly targets CTNBB1 mRNA.

DKK1 is a negative regulator of Wnt signaling.

miR34 binds to the 3′ untranslated regions of CTNBB1.

AKT1 ativates Chibbv, which in turn phosphorylates CTNNB1 in the nucleus. This results in the export of CTNNB1 into the cytosol.

CDH2 inhibits Wnt-signalling by forming a complex with CTNNB1.

p63 inhibits CTNBB1 target gene expression.

E-cadherin binds to and sequesters CTNNB1 at the cytoplasmic membrane, thereby preventing its nuclear translocation.

p53 activates nuclear GSK3, which inhibits CTNBB1.

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AKT2, SNAI1, ZEB1, ZEB2, SNAI2, and TWIST1 all inhibit CDH1.

ZEB1 directly binds to the CDH1 promoter to repress its transcription.

SNAI2 activates ZEB1. The two proteins then cooperatively repress CDH1 expression.

SNAI1 directly binds to the CDH1 promoter to repress its transcription.

Snai1 inhibits CDH1 through AKT2-mediated phosphorylation of histone H3 at Thr45.

AKT2 induces gene expression of Dab2, which has been shown to decrease E-cadherin levels through an unknown mechanism.

Snai1 inhibits CDH1 through AKT2-mediated phosphorylation of histone H3 at Thr45.

SNAI2 directly binds to the CDH1 promoter to repress its transcription.

SNAI2 activates ZEB1. The two proteins then cooperatively repress CDH1 expression.

TWIST1 indirectly inhibits CDH1 by activating SNAI2.

ZEB2 directly binds to the CDH1 promoter to repress its transcription.

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TWIST1 and NICD activate SNAI1. CTNNB1, p53, miR203, and miR34 are negative regulators of SNAI1 which hold dominance over NICD and TWIST1.

CTNNB1 inhibits SNAI1 by activating miR-30e.

miR203 repressed endogenous SNAI1.

NICD upregulates SNAI1 expression.

Suppression of miR34 lead to an increase in SNAI1.

TWIST1 upregulates SNAI1 expression.

p53 induces the degradation of SAI1 via mdm2-mediated ubiquitination.

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DNAdamage upregulates p73. AKT1, AKT2, p53, and ZEB1 are all inhibitors of p73 which express dominance over DNAdamage.

ZEB1 inhibits p73 expression.

AKT2 inhibits p73 through MDM2-mediated degradation.

p73 is important in regulating DNA repair systems after the introduction of DNA damage.

AKT1 inhibits p73 through MDM2-mediated degradation.

p53 inhibits p73 expression.

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TWIST1 is a positive regulator of CDH2.

Twist1 binds to E-boxes in the CDH2 promoter and upregulates CDH2 transcription.

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SNAI2 activates ZEB2 in conjunction with TWIST1. SNAI1 and NICD also upregulate ZEB2. miR200 and miR203 are inhibitors of ZEB2 which express dominance over SNAI1, SNAI2, and NICD.

miR200 is involved in a double-negative feedback loop with ZEB1.

miR203 targets and inhibits ZEB2.

SNAI1 upregulates ZEB2 expression by preventing the processing of a large intron located in ZEB2's 5′-untranslated region.

Over-expression of Notch-1 increased expression of ZEB1.

TWIST1 and SNAI2 work cooperatively to activate ZEB2 transcription.

TWIST1 and SNAI2 work cooperatively to activate ZEB2 transcription.

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GF and CDH2 activate GF. CDH1 is a negative regulator of GF which expresses dominance over both GF and CDH2.

The cell cycle can produce ligands and thus activate the receptor tyrosine kinase family of receptors that eventually the cell to produce growth factor.

N-cadherin caused the upregulation of fibroblast growth factor receptor expression.

E-cadherin blocks GF signaling.

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CTNNB1, NICD, and TWIST1 activate SNAI2. p53, miR203, and miR200 are inhibitors of SNAI2 which express dominance over TWIST1, NICD, and CTNNB1.

The SNAI2 promoter contains cis-regulatory sequences which respond to CTNNB1 signaling.

SNAI2 is a target of miR200.

miR203 targets SNAI2.

SNAI2 is a direct target of Notch signaling.

TWIST1 induces SNAI1 transcription.

p53 induces the degradation of SAI1 via mdm2-mediated ubiquitination.

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SNAI1, NICD, and CTNNB1 all activate TWIST1.

CTNNB1 activates Twist1 transcription.

SNAI1 is needed for up-regulation of Twist1 mRNA.

NICD activates Twist1 transcription.

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VIM activates Migration in conjunction with Invasion, EMT, ERK, and AKT2. AKT1, miR200, and p63 are all negative regulators of Migration which express dominance over VIM.

miR200 inhibits migration by downregulating fibronectin 1.

ERK is necessary for the induction of migration by inhibiting AKT1.

ERK activates cofilin-mediated migration.

Inhibition of AKT2 compromises migration.

In breast and ovarian cancer cells, AKT2 stimulates migration through thr upregulation of beta integrins.

Overexpression of VIM increases cell motility and directional migration.

AKT1 inhibits migration through degradation of NFAT.

AKT1 inhibits migration through phosphorylation of palladin.

EMT promotes cell migration.

In order for cells to migrate to other parts if the body, they must first be able to breach the basement membrane and extracellular matrix.

Decrease in p63 levels leads to an increase in proteins involved in cell motility.

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When SNAI1, ZEB1, and ZEB2 are inactive, AKT2 activates miR34 in the presence of either p53 or p73. AKT1 and p63 are both inhibitors of miR34 that express dominance over AKT2.

SNAI1 represses miR34 expression through binding to E-boxes in the miR-34 promoter.

ZEB1 represses miR34 expression through binding to E-boxes in the miR-34 promoter.

AKT2 induces microRNAs

AKT1 inhibits microRNAs

p63 inhibits miR34 activity by directly binding to p53-consensus sites in the miR34 regulatory regions.

p53 transcriptionally activates miR34.

p73 induces transcription of miR34.

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ZEB2, p21, miR34, miR200, and miR203 all activate CellCycleArrest. AKT1 is an inhibitor of CellCycleArrest that is dominant over ZEB2, p21, miR34, miR200, and miR203.

miR200 induces cell cycle arrest by inhibiting CCNE2 (cyclin E).

miR203 inhibits cell cycle progression by downregulating CDK6.

miR34 prevents cell cycle progression by inhibiting Cyclin D1 activity.

p21 is a cyclin-dependent kinase inhibitor that induces cell cycle arrest in response to a range of stimuli.

AKT1 is required for cell proliferation.

ZEB2 induces cell cycle arrest by inhibiting Cyclin D.

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DKK1 is activated by CTNNB1 and NICD.

DKK1 is a downstream target of CTNNB1.

Two Notch-response elements were identified in the DKK1 promoter, indicating that DKK1 is a target of Notch signaling.

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p63 is activated by DNAdamage. AKT1, AKT2, miR203, NICD, and p53 are all inhibitors of p63 which express dominance over DNAdamage.

miR203 targets human and mouse p63 3'-UTRs.

Notch represses p63 expression.

AKT2 inhibits p63 through MDM2-mediated degradation.

p63 is important in regulating DNA repair systems after the introduction of DNA damage.

AKT1 inhibits p63 through MDM2-mediated degradation.

p53 initiates cell cycle arrest by inducing the synthesis of p21.

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p53, p63, and p73 all upregulate miR200. AKT2, SNAI1, SNAI2, ZEB1, and ZEB2 all inhibit miR200 and express dominance over p53, p63, and p73.

ZEB1 is involved in a double-negative feedback loop with miR200.

miR200 promoter activity significantly decreased upon 12 h of SNAI1 induction.

Cell expressing AKT2 observed a decrease in miR200 abundance.

SNAI2 inhibits miR200 expression.

p63 activates transcription of the miR200 family.

p53 activates transcription of miR200c.

ZEB2 is involved in a double-negative feedback loop with miR200.

p73 activates transcription of the miR200 family.

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CTNNB1, DNAdamage, miR34, and NICD all activate p53. SNAI2, p73, AKT1, and AKT2 are all negative regulators of p53 which express dominance over CTNNB1, DNAdamage, miR34, and NICD.

CTNNB1 upregulates p53 by inhibiting MDM2 through activation of p14ARF.

NICD activates myc. This in turn activates p14ARF, which inhibits MDM2-mediated degradation of p53.

AKT1 inhibits p53 via MDM2-mediated degradation.

MiR34a upregulates p53 expression by inhibiting sirt1, a negative regulator of p53.

AKT1 inhibits p53 via MDM2-mediated degradation.

p53 is activated in response to DNA damage.

SNAI2 inhibits p53 transcription.

The truncated form of p73 is able to repress p53 activity. p73 can bind to and activate MDM2, leading to the degradation of p53.

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ECM activates NICD. miR34, miR200, p53, p63, and p73 all express dominance over ECM and inhibit NICD.

miR200 targets Notch pathway components, such as Jagged1.

miR34 inhibits Notch1, which in turn inhibits NICD. (Observed in cervical carcinoma and chroiocarcinoma cells.)

p63 activates Jag. Cis-interaction between Jag ligands inhibits Notch signalling within the same cell.

p53 inhibits PSEN, which activates NICD.

p63 activates Jag. Cis-interaction between Jag ligands inhibits Notch signalling within the same cell.

ECM can upregulate NICD through laminin 411-mediated activation of Notch signaling.

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ECM and NICD are positive regulators of TGFbeta. CTNNB1 downregulates TGFbeta and is dominant to ECM and NICD.

CTNNB1 activates Bambi, which inhibit TGFbeta.

Notch activates Nodal, which activates TGFbeta.

The mechanical properties of the ECM determines TGFbeta activity and availability.

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p53, p63, p73, miR200, and miR34 are all activators of Apoptosis. ERK, AKT1, and ZEB2 are inhibitors of Apoptosis which express dominance over p53, p63, p73, miR200, and miR34.

miR200 inhibits apoptosis be downregulating XIAP.

ERK inhibits apoptosis by upegulating the expression of anti-apoptotic proteins such as Bcl2, Bcl-XL, and Mcl-2.

miR34 activates apoptosis by inhibiting the anti-apoptotic gene BCL2.

AKT1 suppresses anoikis (detachment-induced apoptosis).

p63 suppresses anoikis (detachment-induced apoptosis) by downregulating integrin beta4.

p53 induces anoikis (detachment-induced apoptosis).

Downregulation of ZEB2 induces apoptosis through the activation of caspase-3.

p73 is a p53 homolog and regulates some of the same gene targets as p53. Overexpression of p73 can also induce apoptosis, like p53.

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Invasion is actived by both CTNNB1 and SMAD in the presence of CDH2.

CTNNB1 induces invasion by activating transcription of MMPs.

N-cadherin promotes cancer invasion, potentially through the increase in production of MMP-9 production in cells expressing N-cadherin.

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miR203 is activated by p53. SNAI1, ZEB1, and ZEB2 all inhibit miR203 and express dominance over p53.

SNAI1 inhibits miR203 expression.

ZEB1 inhibit miR203 promoter activity.

p53 activates transcription of miR203 in keratinocytes.

ZEB2 inhibit miR203 promoter activity.

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