Signaling interplay between transforming growth factor-β receptor and PI3K/AKT pathways in cancer  Long Zhang, Fangfang Zhou, Peter ten Dijke  Trends in Biochemical Sciences  Volume 38, Issue 12, Pages 612-620 (December 2013) DOI: 10.1016/j.tibs.2013.10.001 Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 1 Transforming growth factor (TGF)-β/SMAD (SMA- and MAD-related protein) signaling in tumor suppression and tumor progression. (A) TGF-β signals via type II and type I serine/threonine kinase receptors (TβRII and TβRI, respectively). TGF-β binds first to the constitutively active TβRII. The binding event is recognized by TβRI, which mobilizes to form a receptor complex. Upon heteromeric receptor complex formation, TβRII kinase trans-phosphorylates TβRI on serine and threonine residues in a glycine/serine-rich domain. The activated TβRI phosphorylates R-SMAD2 and R-SMAD3. SMAD2 and SMAD3 are recruited to the activated TGF-β receptor complex via the SMAD anchor for receptor activation (SARA), which contains a FYVE domain that interacts with phosphatidylinositol 3-phosphate (PIP3). The R-SMADs form heteromeric complexes with SMAD4. Heteromeric SMAD complexes accumulate in the nucleus. There, in concert with other DNA-binding transcription factors, coactivators, and co-repressors, they bind to target gene promoters to regulate transcription. Inhibitory SMAD7 antagonizes TGF-β/SMAD signaling by competing with R-SMADs for receptor binding and by recruiting E3 ubiquitin ligases (SMURF2 and SMURF1) to the activated TGF-β receptor complex. These SMURFs target the receptor for ubiquitin-mediated proteasomal degradation. The covalent attachment of ubiquitin chains to TβRI is reversible; ubiquitin specific protease (USP)4/15 deubiquitinases can remove ubiquitin chains from TβRI, and thus stabilize the TGF-β receptor. The SMAD7 gene is directly targeted by the TGF-β/SMAD signaling pathway; thus, it participates in a negative feedback loop to regulate the amplitude and duration of TGF-β/SMAD signaling. (B) In normal and premalignant cells, TGF-β enforces homeostasis by inducing tumor-suppressive effects, including cytostasis, differentiation, and apoptosis. TGF-β-induced growth arrest is mediated (in part) through the upregulation of cyclin-dependent kinase inhibitors, p21CIP and p15INK4B, and downregulation of the proto-oncogene, c-Myc. Plasminogen activator-1 (PAI1, encoding an extracellular matrix protein, is a prototypic upregulated target gene of TGF-β. TGF-β exerts tumor suppressive effects by inhibiting expression of inflammation and stroma-derived mitogens. (C) In colorectal and pancreatic carcinomas or in ovarian cancers, frequent mutations occur in TGF-β receptors (TGFβRI/TGFβRII) or in SMAD4, which render cancer cells insensitive to the cytostatic effects of TGF-β. However, cancer cells benefit from elevated tumor production of TGF-β, which induces stromal production of cytokines that promote cell survival. (D) In breast cancer, gliomas, and melanomas, oncogenic mutations frequently do not occur in the central TGF-β receptor/SMAD components; instead, genetic or epigenetic changes selectively impair TGF-β-induced tumor-suppressive responses, such as upregulation of p21CIP1 and p15INK4B and downregulation of c-MYC. Those mutations do not disturb pro-oncogenic responses such as EMT. In this context, cancer cells benefit from a TGF-β-rich environment. They respond to TGF-β activation of both SMAD-mediated and non-SMAD-mediated inductions of target genes. In the case of bone metastasis of breast cancer, TGF-β (which is highly abundant in bone) induces the expression of interleukin (IL)-11 and parathyroid hormone related protein (PTHRP), which participate in a vicious cycle promoting bone resorption and osteolytic metastasis. Trends in Biochemical Sciences 2013 38, 612-620DOI: (10.1016/j.tibs.2013.10.001) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 2 Transforming growth factor (TGF)-β receptor-initiated non-SMAD (SMA- and MAD-related protein) signaling. (A) Direct TGF-β receptor activation of PI3K/AKT pathway. Type II serine/threonine kinase receptor (TβRII) constitutively associates with the phosphoinositide 3-kinase (PI3K) subunit, p85. Upon TGF-β stimulation, type I serine/threonine kinase receptor (TβRI) associates with p85, and this mediates AKT activation, followed by tuberous sclerosis complex (TSC) inhibition and mammalian target of rapamycin complex (mTORC) activation. The interaction of TGF-β receptors with PI3K is not direct, but TβRI and TβRII kinase activity are required. Activation of mTORC1 leads to increases in mRNA translation and cell size during epithelial–mesenchymal transition (EMT), migration, and metastasis. TGF-β induces activation of mTORC2, which subsequently promotes AKT activation. In addition, TGF-β initiates other non-SMAD signaling pathways, including Rho-like GTPases and mitogen-activated protein kinase (MAPK) pathways that are involved in the induction of EMT, invasion, and metastasis. Abbreviations: CDC42, cell division cycle 42; MEK, extracellular signal-regulated kinase kinase; MKK, MAPK kinase; PAK, p21 activated kinase; RAC, RAS-related C3 botulinum toxin substrate 1; RAF, rapidly accelerated fibrosarcoma; RAS, RAS sarcoma; RHO, RAS homolog gene family; ROCK; Rho kinase; TAB, TAK1 binding protein; TAK, TGF-β activated kinase. (B and C) Indirect TGF-β receptor activation of PI3K/AKT pathway. (B) TGF-β signaling stimulates miR-216a and miR-217 expression, which inhibits PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SMAD7 transcription, thereby potentiating PI3K/AKT and TGF-β signaling. This results in an amplification of the oncogenic signal. (C) TGF-β induces miR-21 to target PTEN transcripts. This lifts the PTEN inhibition of the AKT/mTORC1 signaling cascade, which promotes mesangial cell hypertrophy and matrix protein synthesis. Not indicated in Figure 3B and C are the mechanisms by which TGF-β induces PI3K/AKT by stimulating the expression or shedding of growth factors that activate receptor tyrosine kinases. Trends in Biochemical Sciences 2013 38, 612-620DOI: (10.1016/j.tibs.2013.10.001) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 3 In premalignant states, PI3K/AKT activation antagonizes cytostatic and proapoptotic effects of transforming growth factor (TGF)-β/SMAD (SMA- and MAD-related protein) pathway. (A) AKT interacts directly with unphosphorylated SMAD3 to sequester it outside the nucleus; this prevents TGF-β-induced SMAD3/SMAD4 heteromeric complex formation and nuclear translocation, which inhibits TGF-β-induced apoptosis. (B) AKT phosphorylates forkhead transcription factor (FOXO), which mediates its nuclear export; this interferes with the formation of the nuclear FOXO/SMAD transcriptional complex, which is required to dock on the p15INK4B and p21CIP1 promoters to mediate a TGF-β-induced cytostatic response. (C) PTEN (phosphatase and tensin homolog deleted on chromosome 10) loss induces hyperactivation of AKT in the premalignant state. Prostate cancer progression is promoted when PTEN loss is combined with SMAD4 inactivation, through either SMAD4 gene deficiency or SMAD4 inhibition by nuclear receptor COUP transcription factor II (COUP-TFII). Trends in Biochemical Sciences 2013 38, 612-620DOI: (10.1016/j.tibs.2013.10.001) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 4 PI3K/AKT activation switches transforming growth factor (TGF)-β/SMAD (SMA- and MAD-related protein) into a tumor-promoting signal: potential mechanisms. (A) Increase in the matrix rigidity switches the cellular response to TGF-β by favoring PI3K/AKT activation. (B) TGF-β-induced AKT activation (in cooperation with growth factor signals or oncogenic mutations) stabilizes SNAIL protein through inactivation of glycogen synthase kinase (GSK)-3β (left) and indirectly induces both SNAIL gene expression and SNAIL protein stabilization through nuclear factor (NF)-κB activation (right). SNAIL suppresses the expression of E-Cadherin (CDH1). (C) TGF-β-induced AKT activation (in cooperation with growth factor signals or oncogenic mutations) mediates phosphorylation of TWIST to promote TGF-β2 expression and phosphorylates ubiquitin-specific protease (USP)4 to enhance membrane type I serine/threonine kinase receptor (TβRI) stability. These factors enhance TGF-β/SMAD signaling to drive epithelial–mesenchymal transition (EMT), invasion, and metastasis. Trends in Biochemical Sciences 2013 38, 612-620DOI: (10.1016/j.tibs.2013.10.001) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 5 Schematic representation of therapeutic approaches for targeting both the TGF-β/SMAD (SMA- and MAD-related protein) and the PI3K/AKT pathways for treating cancer progression. (Left) TGF-β/SMAD signaling can be intracellularly inhibited by: type I serine/threonine kinase receptor (TβRII) or type I serine/threonine kinase receptor (TβRI) kinase inhibitors (SB-431542, SB-505124, SB-525334, LY2109761, LY2157299, LY580276, LY364947, and GW788388), and SMAD transcriptional inhibitory peptide aptamers (TRX-LEF and TRX-FOXH1B). For details and other strategies, see the previously published review [51]. (Right) Inhibitors are shown that target multiple steps in the PI3K/AKT pathway. Indicated are inhibitors for: PIP3 (phosphatidylinositol 3, 4, 5-trisphosphate), that is, BKM120, XL147, GDC0941, and GSK1059615; AKT, that is, perifosine, MK2206, VqD-002, XL418, and mammalian target of rapamycin (mTOR), that is, rapamycin, CCl-779, RAD001, and AP23573. Abbreviations: EGFR, epidermal growth factor receptor; GPCR, G-protein-coupled receptor; HER2, human epidermal growth factor receptor 2 (also known as ERBB2); MET, MNNG HOS transforming gene; RTK, receptor tyrosine kinase; VEGFR, vascular endothelial growth factor receptor. For details and other strategies, see the previously published review [50]. Trends in Biochemical Sciences 2013 38, 612-620DOI: (10.1016/j.tibs.2013.10.001) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure I PI3K/AKT pathways in growth control and cell survival. Upon RTK and GPCR activation PI3K can catalyze the conversion of PIP2 to PIP3. PIP3 mediates recruitment to membrane of AKT, where it gets phosphorylated and induces proliferation and survival signals. Trends in Biochemical Sciences 2013 38, 612-620DOI: (10.1016/j.tibs.2013.10.001) Copyright © 2013 Elsevier Ltd Terms and Conditions