AKT signaling pathway

AKT Overview

AKT (PKB or Rac) is a serine/threonine kinase located at the core node of the PI3K/AKT/mTOR signaling pathway (PAM pathway), regulating basic functions such as transcription, translation, proliferation, growth, and apoptosis of cells. AKT has three subtypes: Akt1, Akt2, and Akt3. The PH domain (pleckstrin homology domain) of AKT specifically binds to PIP2 and PIP3, allowing AKT to be localized on the cell membrane. Subsequently, the kinase domain transfers the phosphate group of ATP to the substrate threonine for phosphorylation.

The activation of the AKT pathway is usually initiated by the binding of receptor tyrosine kinase (RTK) on the cell surface to ligands. After RTK activation, P13K is recruited to the cell membrane and catalyzes the conversion of phosphatidylinositol diphosphate (PIP2) to phosphatidylinositol triphosphate (PIP3). PIP3 acts as a second messenger to recruit AKT and its upstream kinase PDK1 to the cell membrane. Subsequently, PDK1 and mTORC2 phosphorylate the Thr308 and Ser473 sites of AKT, respectively, activating AKT kinase activity. In addition, when IGF1 binds to IGF1R, IRS-1 and PI3K are recruited and activated. Activated PI3K converts PIP2 to PIP3, which activates PDK1 and subsequently affects Akt.

Figure 1: The structural domains of AKT1/PKB α, including the PH domain, kinase domain, and hydrophobic motif

AKT signaling network targets and regulates key cellular substrates

AKT regulation of target proteins in normal cells: Under normal circumstances, the PAM pathway moderately promotes basic cellular functions such as survival, proliferation, growth, and metabolism. The phosphorylation of downstream targets mediated by AKT, including BAD, IKK α, FOXO, MDM2, CHK1, p21, p27, GSK-3, and TSC2, determines the regulation of different cellular functions.

AKT regulation of target proteins in cancer cells: Mutations in RTK, Ras, PI3K, PTEN protein phosphatase, AKT, and/or other oncogenes may occur in cancer cells, leading to overexpression of AKT, enhanced inhibition of BAD, FOXO, CHK1, p21, p27, GSK3, and TSC2, as well as increased activity of IKK α and MDM2, resulting in higher survival rates, increased proliferation, enhanced growth, and promotion of metabolism.

Main downstream effectors of AKT pathway

AKT phosphorylates downstream effectors in a sequence dependent manner, typically recognizing substrates containing a common phosphorylation motif R-X-R-X-X-S/T. The downstream effects of Akt signaling are extensive, promoting the survival, proliferation, growth, and metabolism of tumor cells by activating its downstream effectors.

1、 mTOR

MTOR is an atypical member of the PI3K related kinase family, typically assembled into complexes mTORC1 and mTORC2, playing critical roles in many biological processes. The mTORC1 complex consists of mTOR, mLST8, raptor, and PRAS40, and controls cell growth by phosphorylating S6K1 and 4EBP1; MTORC2 consists of mTOR, mLST8, SIN1, and rictor.

Many growth factors and their receptors, such as VEGF and VEGFR, act as positive regulatory factors that transmit signals to mTOR through the PI3K/Akt pathway. This step is limited by the mTOR negative regulatory factor PTEN. Akt activates mTOR through phosphorylation of TSC2.

2、 GSK3

The EGFR/RAS/PI3K/PTEN/Akt/GSK3/mTORC1 pathway is common in cancer, and GSK3 is one of its targets. After Akt induced phosphorylation of Ser21 (α) or Ser9 (β) in response to PI3K mediated signaling, GSK3 (GSH3 α and GSK3 β) becomes inactive and targets proteasomal degradation.

3、 FOXOs

FOXO includes four direct downstream targets of Akt: FOXO1, FOXO3, FOXO4, and FOXO6, which are phosphorylated upon activation by Akt and participate in cell cycle regulation and cell survival.

4、 TSC2

The complex containing TSC1 and TSC2 inhibits mTORC1 activity. TSC2 converts Ras related Rheb GTP (an effective activator of mTORC1) into Rheb GDP, thereby inactivating mTORC1. However, Akt mediated TSC2 phosphorylation reversed this process, preventing TSC1 and TSC2 from negatively regulating the small G protein RHEB, thereby indirectly activating mTORC1 to promote cell growth.

5、 MDM2

MDM2 is an oncogene that induces tumorigenesis, and its mRNA level is controlled by p53 to respond to oxidative stress and DNA damage. As phosphorylation is crucial for the transfer of MDM2 from cytoplasm to nucleus, activated PI3K/Akt can induce nuclear translocation of MDM2. Upon entering the nucleus, MDM2 binds to tumor suppressor p53, inhibiting its transcription and inducing its degradation to suppress cell cycle arrest and apoptosis caused by p53. PTEN's inhibition of the PI3K/Akt pathway protects p53 from MDM2 induced degradation.

Figure 2: Schematic diagram of AKT pathway activation

AKT pathway: a key target of breast cancer

Cancer, as a major disease that threatens human health, has always been an important topic and challenge in medical research for its treatment. With the development of molecular biology and medicinal chemistry, targeted therapy targeting key signaling pathways within cancer cells has gradually become a research hotspot. In cancer, the AKT pathway is often abnormally activated, which may be caused by mutations in upstream growth factor receptors and Ras proteins, inactivation of the inhibitor PTEN, and AKT self amplification or domain mutations. Activated AKT can regulate a variety of downstream effectors, including inhibiting apoptosis (such as BAD), promoting protein synthesis (activating S6K through mTORC1), interfering with cell cycle inhibitors (such as p21 and p27), and inhibiting tumor suppressor genes (such as FOXO), which enables cancer cells to escape apoptosis, rapidly proliferate, and gain metabolic advantages in breast cancer. In addition, the AKT pathway has a complex cross regulatory network with other key signaling pathways such as mTOR, NF-κB, and GSK-3β, which further enhances its multiple roles in cancer cells.

AKT inhibitors: emerging anti-tumor drugs

Due to the importance of the AKT signaling pathway, it has become a hot topic in the development of anti-cancer drugs. The current drug development strategy mainly focuses on two directions: ATP competitive inhibitors and allosteric inhibitors.

1、  ATP competitive inhibitor

ATP competitive inhibitors competitively bind to the ATP binding site of AKT kinase, exhibiting higher binding affinity to the activated AKT kinase's AKT binding site and inhibiting its catalytic activity, thereby blocking downstream signaling and suppressing cancer cell proliferation and survival.

◉ GDC-0068： GDC-0068 is a highly selective oral AKT inhibitor that targets AKT1, AKT2, and AKT3. In the IPAUnity130 clinical study, GDC-0068 combined with paclitaxel and paclitaxel alone had no statistically significant difference in PFS among breast cancer patients with PIK3CA or AKT1 or PTEN mutations (median PFS were 7.4 and 6.1 months, respectively), which did not reach the primary endpoint of the study. However, for patients with advanced breast cancer who failed to be treated with CDK4/6 inhibitors in the past, GDC-0068 combined therapy can obtain better clinical efficacy in some patients.

◉ GSK2110183：GSK2110183 is also a broad-spectrum AKT inhibitor, and is currently conducting clinical research on HR+/HER2 breast cancer, triple negative breast cancer, metastatic castration resistant prostate cancer and other indications. Research has shown that GSK2110183 can significantly inhibit the proliferation of tumor cells and induce cell apoptosis, especially when used in combination with chemotherapy drugs, exhibiting a synergistic effect.

◉ GSK2141795：GSK2141795, as a small molecule oral AKT inhibitor, has anti-tumor and anti proliferative effects both in vitro and in vivo. GSK2141795 binds to AKT kinase and inhibits substrate phosphorylation activity, which may lead to downstream signal transduction inhibition of the PI3K/AKT pathway and induce tumor cell death. In clinical trials, GSK2141795 has shown anti-tumor activity in various advanced solid tumors, and the side effects are tolerable.

2、  Allosteric AKT inhibitors

Inhibitors that bind and interact outside the ATP binding site. The loss of AKT conformational changes after binding with allosteric inhibitors promotes the loss of Thr308 phosphorylation, leading to incorrect localization of hydrophobic motifs and resulting in dephosphorylation of Ser473 resin. Secondly, the binding of inhibitors can block upstream steric hindrance activation of phosphorylated Ser473, thereby completely inhibiting AKT activation.

◉ KRX-0401：KRX-0401 mainly acts on the cell membrane, blocking signal transduction and inhibiting the ability of AKT kinase on the cell membrane. In addition, KRX-0401 also inhibits proteins such as MAPK, JNK, and NF kB that are associated with cell growth, differentiation, survival, and apoptosis. Some clinical studies have shown that its safety in the treatment of breast cancer remains to be verified, but its unique mechanism of action provides valuable experience for subsequent development.

◉ MK-2206：MK-2206 is an oral nonsteroidal AKT inhibitor that targets AKT1/AKT2 and AKT3, inhibiting their downstream signaling pathways. Clinical trials have shown that MK-2206, when used in combination with other targeted drugs or chemotherapy drugs, can improve the sensitivity of targeted drugs and effectively inhibit the growth of xenografts in breast cancer. Some cell lines with PTEN/PIK3CA mutations have drug resistance.

3、  AKT inhibitors based on natural products

◉ In addition to synthetic small molecule drugs, some natural products have also been found to have inhibitory effects on AKT. For example, natural product Solenopsin and its derivatives have been found to inhibit AKT kinase activity. Solenopsin is an alkaloid extracted from fire ants that inhibits its activity by binding to AKT. In recent years, drug design based on Solenopsin and derivatives screened through pharmacological calculations have provided new ideas for the development of AKT inhibitors.