Roles of GSK-3 and microRNAs on epithelial mesenchymal transition and cancer stem cells

Abstract

Various signaling pathways exert critical roles in the epithelial to mesenchymal transition (EMT) and cancer stem cells (CSCs). The Wnt/beta-catenin, PI3K/PTEN/Akt/mTORC, Ras/Raf/MEK/ERK, hedgehog (Hh), Notch and TP53 pathways elicit essential regulatory influences on cancer initiation, EMT and progression. A common kinase involved in all these pathways is moon-lighting kinase glycogen synthase kinase-3 (GSK-3). These pathways are also regulated by micro-RNAs (miRs). TP53 and components of these pathways can regulate the expression of miRs. Targeting members of these pathways may improve cancer therapy in those malignancies that display their abnormal regulation. This review will discuss the interactions of the multi-functional GSK-3 enzyme in the Wnt/beta-catenin, PI3K/PTEN/Akt/mTORC, Ras/Raf/MEK/ERK, Hh, Notch and TP53 pathways. The regulation of these pathways by miRs and their effects on CSC generation, EMT, invasion and metastasis will be discussed.

Keywords: Akt; GSK-3; PI3K; Wnt/beta-catenin; cancer stem cells.

Figure 1. Regulation of GSK-3 Activity by Kinases and Phosphatases and Types of Substrates of GSK-3

On top side of figure above GSK-3 are various kinases which regulate GSK-3. They are depicted in green ovals. Phosphatases which activate GSK-3 are shown in yellow octagons. Amino acid phosphorylation sites which when phosphorylated result in inactivation of GSK are indicated in yellow hemispheres with red letters. The Y216 phosphorylation site which results in activation of GSK-3 is presented in a yellow hemisphere with green letters. Phosphorylation/dephosphorylation events which result in activation of GSK-3 activity are indicated as green arrows. Phosphorylation events which result in inactivation of GSK-3 activity are indicated with red arrows with closed end. On bottom side of the figure below GSK-3 are examples of some of the proteins phosphorylated by GSK-3. Phosphorylation events that result in inactivation are indicated by yellow circles with a red Ps inside. Phosphorylation events that result in activation are indicated by yellow circles with green Ps inside. Types of proteins phosphorylated by GSK-3 include: proteins involved in Wnt/beta-catenin signaling, (e.g., APC, Axin, and LRP6) in red ovals as they are activated by GSK-3 phosphorylation or beta-catenin indicated in a grey oval as it is inactivation by GSK-3 phosphorylation. Anti-apoptotic models such as Mcl-1, kinases such as FAK and mTORC components such as Rictor which are inactivated by GSK-3 phosphorylation are indicated in grey ovals. Transcription factors which are activated by GSK-3 phosphorylation are indicated by yellow diamonds. Transcription factors which are inactivated by GSK-3 phosphorylation are indicated by grey diamonds. This figure is presented to provide the reader an idea of the multiple kinases and phosphatases which can regulate GSK-3 on multiple phosphorylation residues and substrates which can be phosphorylated and either inactivated or activated by GSK-3 phosphorylation.

Figure 2. Regulation of GSK-3 Activity by Growth Factor Signaling

Two pathways activated by growth factors include the PI3K/PTEN/Akt/mTORC1 and Ras/Raf/MEK/ERK signaling pathways. Some of the regulatory interactions between GSK-3 and PI3K/PTEN/Akt/mTOR and Ras/Raf/MEK/ERK pathways are indicated. A generic growth factor (GF) is indicated in purple and the corresponding growth factor receptor (GFR) are indicated in green and yellow. Ras is indicated by a purple oval. Shc, Grb2 and SOS are indicated in orange ovals. Kinases are indicated in green ovals. The p85 regulatory and the p110 catalytic subunits of PI3K are indicated in green ovals. The PTEN phosphatase which dephosphorylates phosphatidylinositol (3,4,5)-trisphosphate (PIP3) into phosphatidylinositol (4,5)-bisphosphate (PIP2) (yellow ovals) is indicated in a light brown octagon. The PP2A and PP1 phosphatases which may activate GSK-3 by dephosphorylation are indicated in yellow octagons. TSC1 and TSC2 are indicated in light brown semi-circles. mTOR interacting proteins which positively regulate mTOR activity are indicated in yellow ovals. mTOR interacting proteins which negatively regulate mTOR activity are indicated in light brown ovals. Transcription factors activated by either ERK or Akt phosphorylation are indicated in yellow diamonds. The Foxo transcription factor FKHR that is inactivated by Akt phosphorylation is indicated by a light brown diamond. beta-catenin is indicated in light brown oval as it is inactivated after GSK-3 phosphorylation. Some of the types of proteins which are regulated by the beta-catenin pathway are indicated (c-Myc, c-Jun, PPPARδ, DKK, Axin2, VEGF, cyclin D). They are colored in light brown as their expression is inhibited by GSK-3 phosphorylation of beta-catenin. In addition, KRas can induce ERK which can result in activation of Ets and GSK-3 expression which can in turn have effects on IkappaK and NF-kappaB activity. Green arrows indicate activating events in pathways. Red arrows indicate inactivating events in pathway. Activating phosphorylation events are depicted in yellow circles with green Ps inside them. Inactivating phosphorylation events are depicted in yellow circles with red Ps inside them. This figure is provided to give the reader an idea of the multiple interactions of GSK-3 with various signaling molecules in the PI3K/PTEN/Akt/mTOR and Ras/Raf/MEK/ERK pathways.

Figure 3. Substrates of GSK-3 which are Involved in EMT

On top of GSK-3 are transcription factors which are inactivated by GSK-3 phosphorylation that are indicated in grey diamonds. On bottom of GSK-3 are transcription factors which are activated by GSK-3 phosphorylation that are indicated in yellow diamonds. Indicated in red ovals are molecules in the Wnt/beta-catenin pathway which are activated by GSK-3 phosphorylation which are indicated in red ovals and beta-catenin which is indicated in an orange oval. Also indicated in a maroon oval is the Bax pro-apoptotic molecule. The mitochondrial localization of Bax is promoted when it is phosphorylated by GSK-3 and then it promotes apoptosis. Also indicated are some cell cycle and signaling proteins which are inactivated by GSK-3 phosphorylation (PTEN, Cyclin D1 and p21^Cip-1) and beta-catenin which are indicated in grey symbols. This figure is presented to provide the reader an idea of the various transcription factors, kinases, phosphatases, apoptotic molecules, cell cycle regulator molecules which are regulated by GSK-3 phosphorylation and may influence EMT.

Figure 4. Overview of Regulation of Snail Activity by GSK-3 and Wnt/beta-catenin and Cytokine Medicated Signaling Pathways

In Panel A., Wnt signaling can prevent GSK-3 phosphorylation which results in the absence of Snail phosphorylation which can have effects on other transcription factors such as Nanog which can regulate the expression of genes involved in EMT. In Panel B., cytokine mediated signaling can result in the activation of multiple signaling pathways including PKC, PI3K/PTEN/Akt and Ras/Raf/MEK/REK which can result in GSK-3 phosphorylation that inhibits its activity. In the absence of active GSK-3, Snail is not phosphorylated can have effects on gene involved in EMT. This figure is presented to provide the reader an idea of some of the mechanisms which can alter GSK-3 activity which influence Snail activity and EMT.

Figure 5. Overview of the Effects of miRs on sFRP Genes and Pathways Involved in EMT

Various miRs can influence the expression of key genes involved in EMT. These miRs may display altered regulation in various cancers. In Panel A., are presented the effects of miR-1207, that is upregulated in ovarian cancer, on the expression of sFRP1, Axin2 and ICAT mRNAs. These events can result in increased Wnt/beta-catenin signaling that leads to cells with the CSC phenotype. These traits are associated with a poor patient survival in ovarian cancer patients. In Panel B., the effects of miR-744 on sFRP1, GSK-3beta, and TLE3 mRNAs are presented that in turn leads to cells with the CSC phenotype, chemotherapeutic drug resistance and is associated with a poor patient prognosis in pancreatic cancer patients. In Panel C., the effects of miR-942 on sFRP4, GSK-3beta, and TLE1 mRNAs are presented that can lead to cells with the CSC tumor sphere phenotype and are associated with a poor patient prognosis in esophageal cancer patients. This figure is presented to provide the reader an idea of some of the mechanisms in which miRs can regulate the expression of sFRPs that when inhibited can have effects on EMT and cancer development.

Figure 6. Effects of Sox17 on miR-371-5q Expression and EMT

Upon demethylation of the Sox17 gene promoter region, the Sox17 transcription factor is expressed that can induce the transcription of the miR-371-5q miR that can in turn suppress Sox2 and other genes involved in EMT, Wnt/beta-catenin signaling and stemness. This figure is presented to provide the reader an idea of some of the mechanisms by which the Sox17 transcription factor can regulate miRs expression which can regulate in turn the expression of other Sox transcription factors which when inhibited can effects on EMT and cancer development.

Figure 7. Effects of WIF-1 on mIRs and Genes involved in Cancer Progression

Panel A. When the WIF1 promoter region is hypermethylated, the WIF1 gene is off. Upon demethylation of the WIF1 promoter region, the WIF1 gene is turned on and there is expression of WIF1 mRNA and protein. WIF1 serves to induce the expression of Let-7a and miR-200c that results in decreased expression of genes associated with pluripotency and stemness and results in more differentiation and cellular senescence. Panel B. When the WIF1 gene is on, it can induce miR-200C expression that can in turn effect the expression of genes involved in EMT such as BMI1, ZEB1 and ZEB2. This can result in increased E-cadherin signaling, adult stem cell renewal and influence multi-lineage differentiation. This figure is presented to provide the reader an idea of some of the mechanisms the WIF1 factor on genes involved in pluripotency, stemness and EMT and the roles that miRs may play in these processes.

Figure 8. Effects of TP53 and miR-34 on the Wnt/beta-catenin Pathway

Panel A. GSK-3 can phosphorylate and activate TIP60 which is a histone acetyl transferase. Activated TIP60 acetylates TP53 which results in its activation. Activation of TP53 can result in transcription of miR-34. miR-34 can suppress many genes in the Wnt/beta-signaling and other signaling pathways. One consequence of inhibition of Axin2 is that GSK-3 may be active. Active GSK-3 can phosphorylate Snail which leads to its inactivation. These events can lead to decreased Wnt/beta-catenin signaling, EMT, invasion and metastasis. Panel B. Effects of demethylation of the miR-34 promoter region by either 5AzaC or SAHA on miR-34 expression and the regulation of gene involved in cell proliferation, self-renewal, invasion, EMT, E-cadherin and N-cadherin expression. In addition, miR-34 expression can induce TP53 expression which can influence genes involved in cell cycle regulation and apoptosis such as p21^Cip-1, p27^Kip-1 and Puma. This figure is presented to provide the reader an idea of some of the mechanisms that TP53 and miR-34 play on regulation of genes involved in Wnt/beta-catenin signaling and EMT and how the TP53 protein may be regulated by GSK-3-induced TIP60 acetylation. Furthermore, the promoter region of the miR-34 gene is regulated by methylation that may be altered by 5AzaC or SAHA treatment.

Figure 9. Effects of miR-34 and miR-504 on TP53-Regulated GSK-3 and Snail Expression

Panel A. Effects of TP53 on miR-34 expression that can control the expression of Snail and Axin2 expression. When Axin2 is suppressed, GSK-3 can be activated that results in suppression of Snail and has negative effects on downstream Nanog and other mRNAs associated with EMT. Panel B. in contrast, if miR-504 is expressed, it can suppress TP53 expression the Snail is expressed that has positive effects on EMT. This figure is presented to provide the reader an idea of some of the mechanisms that the TP53 protein can regulate Snail, Axin2 and GSK-3 expression which will have effects on EMT.

Figure 10. Effects of GSK-3 on EMT and Drug Resistance

Panel A. miR-451 can suppress c-Myc expression and miR-451 is down-regulated in docetaxel-resistant lung adenocarcinoma cells. c-Myc overexpression can induce ERK activation which in turn results in GSK-3 phosphorylation and its inactivation. This can result in Snail stabilization which in turn leads to chemoresistance. Panel B. GSK-3 is involved in the survival of GMB-CSCs. Suppression of GSK-3 with GSK-3 inhibitors can result in inhibition of PP2A activity, cell growth, colony formation and induced programmed cell growth. This figure is presented to provide the reader an idea of some of the mechanisms that GSK-3, c-Myc and miR-451 can be involved in cell growth and chemoresistance.

Figure 11. Effects of GSK3 on HIF-1alpha, Motility and Metastasis

Panel A. GSK-3 can result in phosphorylation HIF-1alpha that results in its inactivation. Suppression GSK-3 can result in HIF-1alpha mRNA stabilization and inhibition of invasion and cell polarity. Panel B. Anthocyanins can activate 5′AMP protein kinase (AMPK) that can in turn lead to GSK-3 activation and suppression of beta-catenin. Panel C. Effects of suppression of PTEN on Akt activation and inhibition of GSK-3 which can lead to PC3 xenograft formation upon injection with PTEN-deficient PC3 cells. Src can phosphorylate GSK-3 on Y216 which result in GSK-3 activation. Inhibition of Src or GSK-3 with Desatinib or SB415286 respectively, can have effects on PC3 xenograft formation, PC3 motility, cell proliferation and colony formation. This can lead to suppression of proliferation and metastasis of HCC cells. This figure is presented to provide the reader an idea some of the mechanisms that GSK-3, can be involved in angiogenesis, tumor formation and metastasis.

Figure 12. Effects of TNF on GSK-3, NF-kappaB and Snail Activities and EMT

TNF can activate multiple signaling pathways including: Akt, p38MAPK and ERK that can phosphorylate and inactivate GSK-3 that can promote Snail activity. Snail can prevent the expression of the E-cadherin gene and contribute to EMT. TNF can also activate NF-kappaB which can stimulate Snail gene expression which also promotes EMT. This figure is presented to provide the reader an idea of some of the mechanisms that TNF-alpha can regulate GSK-3, Snail and NF-kappa-activity and EMT.

Figure 13. Effects of miRs and FZD on Drug Resistance, Invasion and Metastasis

Panel A. The FZD5 gene can influence the expression of the MDR1 gene which can result in chemotherapeutic drug resistance. Panel B. GSK-3 can suppress FZD8 which has effects on Wnt/beta-catenin signaling, invasion and metastasis. miR-100 can also suppress FZD8 expression which has effects on Wnt/beta-catenin signaling, invasion and metastasis. This figure is presented to provide the reader an idea of some of the mechanisms that FZD can regulate proteins involved in drug resistance and how they can be regulated by miRs.

Figure 14. Effects on TP53 and ∆Np63 on FZD Gene Expression and Wnt/beta-catenin Signaling

Panel A. TP53 can induce the CD82 gene that can suppress metastasis. CD82 can also induce expression of miR-203A and inhibit the expression of miR-338-3p. miR-203A can suppress FZD2 expression which in turn resulted in inhibition Wnt/beta-catenin signaling. Panel B. ∆Np63 can result in FZD7 expression that can have effects on Wnt/beta-catenin signaling and is associated with a poor prognosis in TNBC. miR-199A can inhibit FZD7 expression which in turn inhibits Wnt/beta-catenin expression. This figure is presented to provide the reader an idea of some of the mechanisms that TP53 and ∆Np63 can regulate FZD expression that affects EMT.

Figure 15. Effects of miRs on Notch Signaling

Panel A. Ectopic expression of miR-34c can result in suppression of Notch4 expression which blocks self renewal and EMT. Panel B. Overexpression of miR-34a can block Notch1 expression and inhibit mammosphere formation, migration, proliferation and drug resistance. Notch1 can serve as a balance between self-renewal and differentiation and is regulated by miR-34a. Panel C. Notch1 overexpression can influence EMT and pancreatospheres as well as miRs. These events can influence cell growth, clonogenicity and EMT. This figure is presented to provide the reader an idea of some of the mechanisms that Notch and miRs can regulate important genes involved in self renewal, differentiation and EMT.