One of the first intracellular kinases to be explicated as a protooncogene was c-Src, an upstream mediator of both the PI 3-K and MAPK pathways. The amplified c-Src activity has been implicated in multiple gastrointestinal disorders, such as pancreatic cancer. The Src family of kinases (Src, Fyn, Yes, Lck, Lyn, Hck, Fgr, and Blk) are nonreceptor tyrosine kinases that co-operate with the intracellular domains of growth factor receptors, cytokine receptors, G protein-coupled receptors (GPCRs) and integrins. Src kinase activity is regulated by phosphatases by way of binding to adaptor proteins along with proteasomal degradation.
Potent Src Inhibitors
The potent Src inhibitors A 419259 and KB SRC 4 can suppress the accumulation of several malignant cells in vitro. Additionally, A 419259 has demonstrated an ability to inhibit AML stem cell spread in vitro and in vivo. Another notable compound that targets Src function is pyridostatin, which reduces Src protein levels and decreases Src-dependent motility in breast cancer cells by specifically targeting the SRC gene.
The Bcr-Abl fusion protein (caused by a t(9,22) translocation) known for causing the development of tumors is also associated with the development of chronic myeloid leukemia and has been effectively targeted by tyrosine kinase inhibitors. The powerful multi-kinase and pan-BCRABL inhibitor AP 24534 (ponatinib) may offer further understanding into overcoming mutated forms of BCR-ABL, e.g. BCR-ABLT3151, while bosutinib, the dual Src, and Abl kinase inhibitor, has been proved to control the spread and migration of specific breast and colon cancer cell lines.
Cause of PI 3-K/Akt/mTOR Signaling Dysfunction
PI 3-K/Akt/mTOR signaling dysfunction is often seen in cancers and therefore is a heavily studied pathway. The most common cause of PI 3-K/Akt/mTOR pathway dysfunction in human cancers is unusual RTK regulation, although mutations in the tumor suppressor PTEN and N-Ras have also caused hyperactivation of this pathway. In normal cells, growth factors activate RTKs, which results in PI 3-K recruitment. Activated PI 3-K then catalyzes PIP2 into PIP3, which then activates Akt; Akt then activates mTOR signaling. Dysregulation of this pathway has been shown previously to result in tumor development and promotes cancer progression.
Signaling through all classes of PI 3-K play a part in cell growth and division, but cancer research is chiefly centered on mutations in the PIK3CA gene, which encodes the catalytic subunit of p110α of the class 1A PI 3-K. This has been shown to be commonly mutated in human cancers, including both lung and cervical malignancies.
Abnormal PI 3-K activation from mutations in the genes encoding downstream components of the PI 3-K pathway has been associated with the formation of malignancies such as lymphoma (p85 PI 3-K regulatory subunit), glioma (PTEN), breast cancer (S6K1) and gastric cancer (Akt1). Key compounds for PI 3-K research include LY 294002, a prototypical PI 3-K inhibitor, which is able to inhibit proliferation and bring on cell death in human colon cancer cells in vitro and in vivo.
Studies have shown that selective PI 3-K p110α inhibitor A66 inhibits Akt signaling and tumor growth in ovarian cancer xenografts in mice. Other important compounds range from dual ATP-competitive PI 3-K/mTOR inhibitors such as PF 04691502 to PF 05212384, which effectively inhibit the growth of tumor cells and shows antitumor activity in a number of cancer xenograft models.
Akt (protein kinase B) is an essential mediator of PI 3-K signaling; it encourages glycolysis and promotes the growth of cells and inhibiting cell death. Preclinical research has shown that abnormal Akt signaling is key in malignant transformation by encouraging the survival of cells, angiogenesis, and migration. Additionally, Akt has been shown to play a role in chemoresistance. The inhibition of Akt by the small molecule inhibitors API-1 and API-2, results in antitumor activity in vitro, as well as selectively inhibiting cell growth in mouse models of human cancers overexpressing Akt.
AMPK works in contrast to Akt1. It works as an energy sensor and is stimulated under energetic stress when the ratio of AMP:ATP is increased, or in conditions where oxygen levels are compromised (Figure 4). AMPK activation impedes mTOR, brining on cell death and autophagy. Tumor cells are often found to suppress AMPK signaling, subverting the cellular metabolic shift to oxidative metabolism that is usually mediated by AMPK. Strong AMPK activators and inhibitors such as A 769662 and dorsomorphin respectively are effective tools for investigating the role of AMPK in cancer.
The mechanistic target of rapamycin (mTOR; mammalian target of rapamycin) is a highly conserved serine/threonine-protein kinase. In cells, mTOR operates as distinct multiprotein complexes mTORC1 and mTORC2 (Figure 4) that exhibit individual functionalities.
mTORC1 is implicated in metabolic tissue consumption and cell proliferation and is the main mediator of protein synthesis, by way of regulating the eukaryotic translation initiation factor 4F (eIF4F) complex. mTORC2 plays a key part in mediating cell growth, division, and survival by phosphorylating Akt, along with being an important regulator of glucose and lipid metabolism.
Rapamycin is a classical inhibitor of mTOR, which complexes with FKBP-12 and binds to mTOR reducing its activity, including impeding IL-2-induced phosphorylation and the activation of p70 S6 kinase. The ATP-competitive mTOR inhibitors torin 1 and torin 2 are good tools for clarifying the function of the mTOR/PI 3-K axis in cancer cell biology. Torin 2 inhibits both mTORC1 and mTORC2 and can display cytotoxic effects across several cancer cell lines, inducing both cell death and the consumption of the body’s tissues in the metabolic process, as well as inhibiting the activation of PI 3-K/Akt.
The effective mTOR inhibitor temsirolimus has been shown to exhibit several antitumor effects in preclinical studies. This compound has been shown to stunt tumor growth and HIF-1α-mediated VEGF production in breast cancer cell lines, as well as suppressing the formation of blood vessels in vivo. Temsirolimus also causes G1 /S cell cycle arrest in multiple cancer cell lines.
mTORC1 regulates the assembly of the eIF4F complex and transcription of the genes transcribing rRNA and tRNA. Activation of the mTOR pathway leads to the separation of 4E-binding proteins (4E-BP) from the eIF4G binding sites on eIF4E, which ultimately ends in the assembly of the eIF4F complex. The small molecule 4E1RCat impedes protein translation by blocking eIF4E:eIF4G and eIF4E:4E-BP1 interactions. Additionally, it has been shown to possess chemosensitizing properties. 4EGI-1 also inhibits eIF4E:eIF4G interactions and has displayed activity against leukemia and lung cancer cells.
The MAPK pathway is another major pathway that has been thoroughly investigated for therapeutic cancer intervention. MAPKs are serine-threonine kinases that regulate a large number of cell functions. There are four main mammalian MAP kinase cascades involving ERK1/2, p38, JNK and ERK5/BMK1.
MAPK pathways transduce signals from growth factors and are vital mediators in regulating differentiation and growth in a wide range of cell types. Mutations in essential components of these cascades have been linked to several different types of cancer. As a result, inhibitors targeting the molecules involved in the Ras-Raf-MEK-ERK cascade are potentially significant for use in therapeutic measures (Figure 4).
Ras is a small GTPase that is subject to activating mutations in a large proportion of cancers and is the most commonly activated oncogene. These mutations enable Ras activation in the absence of ligand-RTK binding. K-Ras mutations signaling in colon and pancreatic cancer. N-Ras mutations are often seen in melanomas, and H-Ras mutations are often seen in cancers of the cervix and bladder.
Prenyltransferases upstream of Ras such as farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) are part of the association of Ras with the plasma membrane, and have been targeted by small molecules to inhibit their activity. The reduction in activity of H-Ras by FTase inhibitors is effective in blocking signaling, but K-Ras and N-Ras can avoid FTase inhibition by exploiting the related GGTase. FTase and GGTase inhibitors like FTI 276 and GGTI 298 are valuable tools for investigating Ras and the oncogenic signaling properties linked to it.
Studies have demonstrated that the CAAZ peptidomimetic GGTase I inhibitor GGTI 298 significantly impedes geranylgeranylated Rap1A processing, with limited effects on farnesylated Ha-Ras processing and causing G0 -G1 cell cycle arrest. This compound also triggers apoptosis in lung cancer cells, along with inhibiting cell invasion and migration in colon cancer cells.
FTI 276 is a selective inhibitor of FTase that displays >100-fold selectivity over GGTase I, but still poses notable effects on the functionality of both enzymes (IC50 values are 0.5 and 50 nM respectively). This compound has been proved to stop human lung carcinomas expressing oncogenic K-Ras from developing in nude mice.
Raf kinases are activated by GTP-bound Ras and recruited to the cell membrane when the growth factor is stimulated. There are three genes in the family: A-Raf, B-Raf, and C-Raf. Activating mutations in the B-Raf proteins have been associated with several different cancers, from skin, thyroid, and ovarian, to pancreatic cancer.
BRAF is the most commonly mutated gene in melanoma cases, with BRAF mutations present in over 65% of malignant melanomas. A missense substitution can in a high number of these BRAF mutations, which generates the B-Raf V600E protein, a constitutively active kinase.
Promising preclinical results have been found in a few of the small molecule B-Raf inhibitors. Among these are the potent B-Raf inhibitors AZ 628, GDC 0879, and SB 590885, which have all shown to be able to inhibit ERK signaling along with inhibiting cell growth in a range of cancer cells possessing the B-Raf V600E mutation in vitro. Additionally, the powerful Raf kinase inhibitor ML 786 is beneficial in vivo research tool as it can reduce tumor growth in melanoma cell xenografts expressing the B-Raf V600E mutation in mice.
Signal transduction through Raf also relies on a range of proteins that are central in cancer research, including 14-3-3 and Hsp90. Hsp90 (90 kDa heat shock protein) is a molecular chaperone that helps protein folding processes and quality control for a large number of ‘client’ proteins. It also acts in tandem with other chaperones such as Hsp70. Other key tumor-associated clients include estrogen receptors and p53.
Hsp90 plays a key part in certain tumor cell types by stabilizing mutated oncogenic proteins. Good preclinical results in the inhibition of heat shock proteins have been produced, for example, 17-AAG an inhibitor of Hsp90. 17-AAG repressed the activity of oncogenic proteins, including p185ErbB2, N-Ras, Ki-Ras and c-Akt, and demonstrated antitumor effects in vivo. Additionally, the Hsp70 inhibitor VER 155008 impedes the proliferation of multiple human tumor cell lines in vitro.
MEK, alternatively namedmitogen-activated protein kinase or MAP2K, is a dual-specificity kinase that phosphorylates both the tyrosine and threonine residues needed for the activation of the mitogen-activated protein kinases ERK. Although there have been few reports showing oncogenic mutations, the frequent activation of the MAPK pathway in cancer has meant that MEK has been investigated thoroughly as a therapeutic target.
Research shows that inhibiting MEK results in promising antitumor effects in cancer models. For example, the potent MEK1/2 inhibitor PD 0325901 stunts the growth of melanoma cell lines both in vitro and in vivo, triggers cell cycle arrest and cell death in a mouse tumor xenograft, and impedes the production of proangiogenic growth factors such as VEGF. Another notable MEK inhibitor is the selective MEK5 inhibitor BIX 02189, which induces cell death in leukemia tumors.
Use of Inhibitors in Cancer Therapies
There is a growing body of evidence showing that inhibitors of other MAPK signaling pathways, could be useful in cancer therapy. For example, in some cancers, activation of p38 and JNK is linked with apoptosis suppression, with correlations found between increased phosphorylation of p38α and malignant transformation in lymphoma, glioma, lung, breast and thyroid cancers. In a similar vein, activating the JNK pathway with the Bcr-Abl protein (associated with leukemia) has been seen in hematopoietic cells.
The activation of ERK results in the phosphorylation of several transcription factors and other kinases, which can modulate cell cycle progression, protein translation, cell differentiation, and apoptosis. Additionally, ERK activation upregulates the expression of EGFR ligands, promoting an autocrine growth loop that enables tumors to continue to grow. Essential research compounds for studying MAPK pathways include the selective ERK inhibitor FR 180204); the selective ERK5/BMK1 inhibitor XMD 8-92 and the highly potent and selective p38α inhibitor VX 745.
In addition to PI 3-K and MAPK signaling, several other signaling pathways are involved in cancer progression, particularly those linked with cell growth and proliferation. Wnt proteins are secreted glycoproteins that regulate a wide range of developmental processes, such as differentiation, cell migration, and proliferation, both during the formation and development of an embryo and in adult tissues. Wnt is known to be proto-oncogenic and promotes the formation of tumors as well as metastasis. Inactivation of the APC gene (a suppressor of the Wnt/β-catenin pathway) or constitutive action of β-catenin, is regularly seen in colon cancer and is believed to be important in malignant transformation.
The TCF/β-catenin-mediated transcription inhibitor ICG 001, inhibits the growth of tumors in colon carcinoma cell lines and in an APC mouse xenograft model. Reports have also stated that ICG 001 suppresses TGF-β1 induction of EMT as well as α-SMA induction in vitro. Another two types of small molecules have been used to modify Wnt signaling in cancer cells; inhibitors of Wnt response (IWR) and inhibitors of Wnt production (IWP) compounds. Endo-IWR 1 hinders Wnt signaling by bringing on a rise in Axin2 protein levels, promoting β-catenin phosphorylation by stabilizing axin-scaffolded destruction complexes. IWP 2 and IWP 4 hinder Wnt processing and secretion by inactivating PORCN and inhibiting palmitoylation of Wnt.
Sphingosine-1-phosphate receptors (S1PR) play a part in the proliferation, migration, differentiation, and survival of cancer cells. Sphingosine-1-phosphate (S1P) signaling is mediated by five subtypes of related G-protein-coupled receptors of the S1PR family; signaling S1P2, S1P3, S1P4, and S1P5. Because of the complex nature of S1PR signaling, the role that S1PRs play in different types of cancer can vary significantly. For example, overexpression of S1PR1 has been implicated in the progression of certain types of hematological malignancies where increased levels of S1PR1 in glioblastomas are associated with a positive prognosis. All S1PR subtypes have been linked to the formation of tumors or cancer progression, including S1PR4, which interacts with HER2 and is also linked to breast cancer progression, by way of stimulating the ERK pathway.
Compounds that provoke S1P receptors are of interest in the attenuation of hyperproliferative, migratory, and inflammatory phenotypes observed in cancer cells. There are several compounds available for investigating the action of S1P receptor signaling in cancer, including the potent S1P4 antagonist CYM 50358, the highly selective and potent S1P2 antagonist JTE 013, and the high-affinity S1P1 and S1P3 receptor antagonist VPC 23019. Also, there is the S1P3 allosteric agonist CYM 5541, which takes up an alternative space within the ligand-binding pocket of S1P3 than S1P, and may be found to be a valuable compound for clarifying the countless effects resulting from S1P signaling.
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