Accelerate cancer research with PI3 kinase pathway-specific antibodies

Authors

Neha Sharma, Janakiram Rayabaram, Priyanka Swamynathan, Anna Cartier, April Livengood

Cells grow, proliferate, and differentiate via proteins that are regulated at the molecular level. When this molecular synchrony deviates from its canonical function, proteins malfunction. This leads to abnormal cell growth which can subsequently give rise to benign and malignant tumours. A benign tumour (such as a skin wart) is restricted to its site of origin. Hence, it does not invade the surrounding tissue. Malignant tumours, on the other hand, can invade the surrounding tissue. They can metastasize to other locations in the body via the circulatory or lymphatic systems. Malignant tumours spread quickly and become resistant to treatment. Hence, they are referred as cancer and pose clinical challenges in treatment.

At a molecular level, cancers are strongly associated with changes in signalling pathways where genetic aberrations distort their normal behaviour, thereby impacting cellular processes negatively which in turn drives cancer progression. This progression is further associated with a complex interplay between proteins of tumour cells, neighbouring non-neoplastic cells, and the extracellular matrix. Cancer studies in recent decades have therefore focussed efforts on decoding these interactions. Antibodies play a key role not only in delineating molecular interactions and disease mechanisms but have also been successfully used in cancer treatments. In this blog, we attempt to cast light on the PI3 kinase/AKT/mTOR pathway that is implicated in several cancers and suggest antibodies that can be used to study its biology.

The PI3 kinase/AKT/mTOR Pathway

The phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is an intracellular signalling pathway. This pathway is crucial for regulating cell cycle and directly impacts cellular proliferation, apoptosis, and cancer. Under normal conditions, PI3K is activated by stimuli such as growth factors, cytokines, and hormones. Activated PI3K catalyses the conversion of PIP2 to PIP3, two phospholipids that are localized to the plasma membrane, through phosphorylation. Phosphorylated PIP3 can then serve as a docking scaffold to recruit lipid binding kinases such as Akt to the cell membrane, and subsequently activate cell growth and survival pathways. The PI3K/AKT/mTOR pathway is upregulated in many cancers including breast and colorectal cancer, and carcinomas of the liver and brain, where it contributes to unchecked tumour development and resistance to anti-cancer therapies (1).

The AKT Component

AKT (also known as Protein Kinase B or PKB), refers to a set of three, highly homologous proteins (ATK1, AKT2 and AKT), which apart from regulating important cellular processes, are also found to be genetically amplified and constitutively active in many human cancers. AKT activation can occur when it binds to PIP3 through its pleckstrin homology (PH) domain, leading to the recruitment of and phosphorylation by its activating kinases. Activated AKT can then regulate the function of several substrates that are involved in a wide range of processes such as cell cycle progression and proliferation, protein synthesis and so on. AKT is an attractive biological target for cancer treatment as it plays a critical role in the PI3 kinase (PI3K)/AKT/mTOR signalling cascade, as shown in Figure 1. Mutations upstream of AKT in the PI3K cascade can lead to carcinogenesis.

Figure 1: The PI3K/AKT/mTOR signalling cascade

The mTOR Component:

The mammalian Target of Rapamycin (mTOR) plays a central regulatory function and is an important downstream effector of PI3K/AKT pathway. mTOR acts as the catalytic subunit of two structurally and functionally distinct protein assemblies – mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (2).

mTORC1 is a master cell growth regulator that is involved in activating protein translation. Its activity can be modulated by rapamycin, insulin, and growth factors. Constitutive mTOR signalling leads to mTORC1-induced upregulation of protein synthesis by activating ribosomal S6 kinase (S6K) and inhibiting translational repression by blocking Eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) activity. This in turn leads to deregulated protein and lipid synthesis, mitochondrial biogenesis, and unchecked cell growth (3, 4), which go on to instigate oncogenesis.

mTORC2 protein complex is known to regulate cell proliferation, cell migration, and cytoskeletal remodelling. In response to growth factors, mTORC2 modulates cellular metabolism and survival through activation of AKT. Uncontrolled mTORC2 activity through mutations, gene amplification, and chronic mTORC2 activation can alter glucose metabolism and lipogenesis, and impair lysosomal function, which are contributing factors in cancers and lupus, respectively.

The role of PTEN as a regulator of the PI3 kinase/AKT/mTOR pathway

Phosphatase and tensin homolog (PTEN) is a key molecule within the PI3K/AKT/mTOR pathway. It is a negative regulator of the pathway and functions by dephosphorylating PIP3. Thus it inhibits the activation of AKT in this pathway. In many cancers, PTEN activity is downregulated, leading to constitutive activation of the PI3K cascade. Loss of PTEN activity has been implicated in brain, breast, and prostate cancers (5).

Understanding the mechanisms that lead to hyper-activation of PI3 kinase/AKT and subsequently, mTOR signalling, is vital to identify and develop therapeutic targets against cancer. Genetic factors such as mutations and chromosomal translocations play an important role in cancers. Antibodies are essential research tools that can help scientists unravel these mechanisms at a molecular level. However, poor antibody specificity can significantly affect the ability to obtain reproducible results, questioning the reliability of experiments. Therefore, it is vital to choose antibodies that bind to the right target and work in the desired application.

Antibodies to study the PI3K/AKT/mTOR pathway

Thermo Fisher Scientific provides antibodies against a wide array of proteins important to the PI3 kinase/AKT/mTOR pathway, tested in applications including western blot and immunofluorescence. These antibodies further undergo advanced verification for target specificity in cell models modified using siRNA or CRISPR-Cas9 gene editing systems, where elimination of the target protein from the cell model and the corresponding absence of signal from the antibody, provides a high degree of confidence in the antibody. Figure 2 exhibits some examples.

Figure 2: Antibodies specific to the PI3K/AKT/mTOR signal transduction pathway.

• In figures A, C-F, antibody specificity was demonstrated using CRISPR-Cas9 stable KO lines generated using Lenti Array^TM CRISPR libraries.
• In figure B, antibody specificity was determined by siRNA knockdown generated using Silencer® Select siRNA (Cat. No. s11094).
• The blots were probed with the following antibodies:

A. Invitrogen^TM PI3KCA Monoclonal Antibody (H.843.0) (Cat. No. MA5-14870)

B. Invitrogen^TM AKT Pan Monoclonal Antibody (J.314.4) (Cat. No. MA5-14916)

C. Invitrogen^TM mTOR Monoclonal Antibody (GT649) (Cat. No. MA5-31505)

D. Invitrogen^TM Phospho-p70 S6 Kinase (Thr389) Monoclonal Antibody (R.566.2) (Cat. No. MA5-15202)

E. Invitrogen^TM 4EBP1 Monoclonal Antibody (E.992.6) (Cat. No. MA5-15005)

F. Invitrogen^TM PTEN Monoclonal Antibody (2F4C9) (Cat. No. 32-5800)

• In all experiments, the lysates were electrophoresed using Invitrogen^TM NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Cat. No. NP0322BOX) or Invitrogen^TM NuPAGE^TM 3-8% Tris-Acetate Mini Protein Gel (Cat. No. EA0378BOX).
• Resolved proteins were transferred onto Invitrogen^TM iBlot^TM 2 Transfer Stacks, nitrocellulose, regular size membranes ( No. IB23001) using the iBlot® 2 Dry Blotting System (Cat. No. IB21001).
• Chemiluminescent detection was performed using Invitrogen^TM Novex® ECL Chemiluminescent Substrate Reagent Kit (Cat. No. WP20005) or Thermo Scientific^TM SuperSignal^TM West Atto Ultimate Sensitivity Substrate (Cat. No. A38556) and the blots were imaged using the Invitrogen^TM iBright FL 1000 (Cat. No. A32752).

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References

1. Ningni Jiang, Qijie Dai, Xiaorui Su, Jianjiang Fu, Xuancheng Feng, Juan Peng Mol Biol Rep. 2020; 47(6): 4587–4629. Published online 2020 Apr 24. doi: 10.1007/s11033-020-05435-1 PMCID: PMC7295848
2. Angelia Szwed, Eugene Kim, Estela Jacinto Physiol Rev. 2021 Jul 1; 101(3): 1371–1426. Published online 2021 Feb 18. doi: 10.1152/physrev.00026.2020 PMCID: PMC8424549
3. Tian Tian, Xiaoyi Li, Jinhua Zhang Int J Mol Sci. 2019 Feb; 20(3): 755. Published online 2019 Feb 11. doi: 10.3390/ijms20030755 PMCID: PMC6387042
4. Cedric Magaway, Eugene Kim, Estela Jacinto Cells. 2019 Dec; 8(12): 1584. Published online 2019 Dec 6. doi: 10.3390/cells8121584 PMCID: PMC6952948
5. Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, Puc J, Miliaresis C, Rodgers L, McCombie R, et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science. 1997;275:1943–7.