Rapamycin as immunosuppressive agent

mTOR signaling pathway in human cancer

Signaling pathway of mTOR

Many human tumors occur because of dysregulation of mTOR signaling, and can confer higher susceptibility to inhibitors of mTOR.[17] Deregulations of multiple elements of the mTOR pathway, like PI3K amplification/mutationPTEN loss of function, AKT overexpression, and S6K1, 4EBP1, and eIF4E overexpression have been related to many types of cancers. Therefore, mTOR is an interesting therapeutic target for treating multiple cancers, both the mTOR inhibitors themselves or in combination with inhibitors of other pathways.[1]

Upstream, PI3K/AKT signalling is deregulated through a variety of mechanisms, including overexpression or activation of growth factor receptors, such as HER-2 (human epidermal growth factor receptor 2) and IGFR (insulin-like growth factor receptor), mutations in PI3K and mutations/amplifications of AKT.[1] Tumor suppressor phosphatase and tensin homologuedeleted on chromosome 10 (PTEN) is a negative regulator of PI3K signaling. In many cancers the PTEN expression is decreased and may be downregulated through several mechanisms, including mutationsloss of heterozygositymethylation, and protein instability.[16]

Downstream, the mTOR effectors S6 kinase 1 (S6K1), eukaryotic initiation factor 4E-binding protein 1 (4EBP1) and eukaryotic initiation factor 4E (eIF4E) are related to cellular transformation.[1] S6K1 is a key regulator of cell growth and also phosphorylates other important targets. Both eIF4E and S6K1 are included in cellular transformation and their overexpression has been linked to poor cancer prognosis.[16]

Development of mTOR inhibitors

Since the discovery of mTOR, much research has been done on the subject, using rapamycin and rapalogs to understand its biological functions.[15][18] The clinical results from targeting this pathway were not as straight forward as thought at first. Those results have changed the course of clinical research in this field.[15]

Initially, rapamycin was developed as an antifungal drug against Candida albicansAspergillus fumigatus and Cryptococcus neoformans.[5] Few years later its immunosuppressive properties were detected. Later studies led to the establishment of rapamycin as a major immunosuppressant against transplant rejection, along with cyclosporine A.[2] By using rapamycin in combination with cyclosporin A, it enhanced the rejection prevention in renal transplantation. Therefore, it was possible to use lower doses of cyclosporine which minimized toxicity of the drug.[5]

In the 1980s rapamycin was evaluated by the Developmental Therapeutic Branch of the National Cancer Institute (NCI). It was discovered that rapamycin had an anticancer activity and was a non-cytotoxic agent with cytostatic activity against several human cancer types.[5] However, due to unfavorable pharmacokinetic properties, the development of mTOR inhibitors for the treatment of cancer was not successful at that time.[3] Since then, rapamycin has also shown to be effective for preventing coronary artery re-stenosis and for the treatment of neurodegenerative diseases.[5]

First generation mTOR inhibitors

The development of rapamycin as an anticancer agent began again in the 1990s with the discovery of temsirolimus (CCI-779). This was a novel soluble rapamycin derivative that had a favorable toxicological profile in animals. More rapamycin derivatives with improved pharmacokinetics and reduced immunosuppressive effects have since then been developed for the treatment of cancer.[5] These rapalogs include temsirolimus (CCI-779), everolimus (RAD001), and ridaforolimus (AP-23573) which are being evaluated in cancer clinical trials.[19] Rapamycin analogs have similar therapeutic effects as rapamycin. However they have improved hydrophilicity and can be used for oral and intravenous administration.[4] In 2012 National Cancer Institute listed more than 200 clinical trials testing the anticancer activity of rapalogs both as monotherapy or as a part of combination therapy for many cancer types.[7]

Rapalogs, which are the first generation mTOR inhibitors, have proven effective in a range of preclinical models. However, the success in clinical trials is limited to only a few rare cancers.[20] Animal and clinical studies show that rapalogs are primarily cytostatic, and therefore effective as disease stabilizers rather than for regression.[21]The response rate in solid tumors where rapalogs have been used as a single-agent therapy have been modest. Due to partial mTOR inhibition as mentioned before, rapalogs are not sufficient for achieving a broad and robust anticancer effect, at least when used as monotherapy.[19][20][22]

Another reason for the limited success is that there is a feedback loop between mTORC1 and AKT in certain tumor cells. It seems that mTORC1 inhibition by rapalogs fails to repress a negative feedback loop that results in phosphorylation and activation of AKT.[18][23] These limitations have led to the development of the second generation of mTOR inhibitors.[7]

Rapamycin and rapalogs

Rapamycin and rapalogs (rapamycin derivatives) are small molecule inhibitors,[24] which have been evaluated as anticancer agents. The rapalogs have more favorable pharmacokinetic profile compared to rapamycin, the parent drug,[3] despite the same binding sites for mTOR and FKBP12.[5]

Chemical structures of sirolimus and temsirolimus

Sirolimus[edit]

The natural antibiotic, rapamycin or sirolimus,[6] a cytostatic agent, has been used in combination therapy with corticosteroids and cyclosporine in patients who received kidney transplantation to prevent organ rejection both in the US[25] and Europe,[26] due to its unsatisfying pharmacokinetic properties.[3] In 2003, the U.S. Food and Drug Administration approved sirolimus-eluting coronary stents, which are used in patients with narrowing of coronary arteries, or so-called atherosclerosis.[27]

Recently rapamycin has shown effective in the inhibition of growth of several human cancers and murine cell lines.[5] Rapamycin is the main mTOR inhibitor, but deforolimus (AP23573), everolimus (RAD001), and temsirolimus (CCI-779), are the newly developed rapamycin analogs.[2]

Temsirolimus

The rapamycin analog, temsirolimus (CCI-779)[2] is also a noncytotoxic agent which delays tumor proliferation.

Temsirolimus is pro-drug of rapamycin. It is approved by the U.S. Food and Drug Administration (FDA)[25] and the European Medicines Agency (EMA),[28] for the treatment of renal cell carcinoma (RCC). Temsirolimus has higher water solubility than rapamycin and is therefore administrated by intravenous injection.[3][6] It was approved in May 30, 2007, by FDA for the treatment of advanced RCC.[6]

Everolimus

Chemical structure of everolimus

Everolimus is the second novel Rapamycin analog.[2] From March 30, 2009 to May 5, 2011 the U.S. FDA approved everolimus for the treatment of advanced renal cell carcinoma after failure of treatment with sunitinib or sorafenibsubependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis (TS), and progressive neuroendocrine tumors of pancreatic origin(PNET).[29] In July and August 2012, two new indications were approved, for advanced hormone receptor-positive, HER2-negative breast cancer in combination with exemestane, and pediatric and adult patients with SEGA.[29] In 2009 and 2011, it was also approved throughout the European Union for advanced breast cancer, pancreatic neuroendocrine tumours, advanced renal cell carcinoma,[30] and SEGA in patients with tuberous sclerosis.[31]

Ridaforolimus

Chemical structure of ridaforolimus

Ridaforolimus (AP23573, MK-8669), or deforolimus, is the newest rapamycin analog and it is not a prodrug.[2] Like temsirolimus it can be administrated intravenously, and oral formulation is being estimated for treatment of sarcoma.[3] It was not on market in June 2012, since FDA wanted more human testing on it due to its effectiveness and safety.[32]

Second generation mTOR inhibitors

Action point of first and second generation mTOR inhibitors on PI3K/AKT/mTOR pathway

The second generation of mTOR inhibitors is known as ATP-competitive mTOR kinase inhibitors.[7] mTORC1/mTORC2 dual inhibitors are designed to compete with ATP in the catalytic site of mTOR. They inhibit all of the kinase-dependent functions of mTORC1 and mTORC2 and therefore, block the feedback activation of PI3K/AKT signaling, unlike rapalogs that only target mTORC1.[7][18] These types of inhibitors have been developed and several of them are being tested in clinical trials. Like rapalogs, they decrease protein translation, attenuate cell cycle progression, and inhibit angiogenesis in many cancer cell lines and also in human cancer. In fact they have been proven to be more potent than rapalogs.[7]

Theoretically, the most important advantages of these mTOR inhibitors is the considerable decrease of AKT phosphorylation on mTORC2 blockade and in addition to a better inhibition on mTORC1.[15] However, some drawbacks exist. Even though these compounds have been effective in rapamycin-insensitive cell lines, they have only shown limited success in KRAS driven tumors. This suggests that combinational therapy may by necessary for the treatment of these cancers. Another drawback is also their potential toxicity. These facts have raised concerns about the long term efficacy of these types of inhibitors.[7]

The close interaction of mTOR with the PI3K pathway has also led to the development of mTOR/PI3K dual inhibitors.[7]Compared with drugs that inhibit either mTORC1 or PI3K, these drugs have the benefit of inhibiting mTORC1, mTORC2, and all the catalytic isoforms of PI3K. Targeting both kinases at the same time reduces the upregulation of PI3K, which is typically produced with an inhibition on mTORC1.[15] The inhibition of the PI3K/mTOR pathway has been shown to potently block proliferation by inducing G1 arrest in different tumor cell lines. Strong induction of apoptosis and autophagy has also been seen. Despite good promising results, there are preclinical evidence that some types of cancers may be insensitive to this dual inhibition. The dual PI3K/mTOR inhibitors are also likely to have increased toxicity.[7]

Mechanism of action

The studies of rapamycin as immunosuppressive agent enabled us to understand its mechanism of action.[5] It inhibits T-cell proliferation and proliferative responses induced by several cytokines, including interleukin 1 (IL-1)IL-2IL-3IL-4IL-6IGFPDGF, and colony-stimulating factors (CSFs).[5] Rapamycin inhibitors and rapalogs can target tumor growth both directly and indirectly. Direct impact of them on cancer cells depend on the concentration of the drug and certain cellular characteristics. The indirect way, is based on interaction with processes required for tumor angiogenesis.[5]

Effects in cancer cells

Effects of Rapamycin and rapalogs in cancer cells

Rapamycin and rapalogs crosslink the immunophilin FK506 binding protein, tacrolimus or FKBP-12, through its methoxy group. The rapamycin-FKBP12 complex interferes with FRB domain of mTOR.[5][6] Molecular interaction between FKBP12, mTOR, and rapamycin can last for about three days (72 hours). The inhibition of mTOR blocks the binding of the accessory protein raptor (regulatory-associated protein of mTOR) to mTOR, but that is necessary for downstreamphosphorylation of S6K1 and 4EBP1.[5][22]

As a consequence, S6K1 dephosphorylates, which reduces protein synthesis and decreases cell motality and size. Rapamycin induces dephosphorylation of 4EBP1 as well, resulting in an increase in p27 and a decrease in cyclin D1 expression. That leads to late blockage of G1/S cell cycle. Rapamycin has shown to induce cancer cell death by stimulating autophagy or apoptosis, but the molecular mechanism of apoptosis in cancer cells has not yet been fully resolved. One suggestion of the relation between mTOR inhibition and apoptosis might be through the downstream target S6K1, which can phosphorylate BAD, a pro-apoptotic molecule, on Ser136.[5]That reaction breaks the binding of BAD to BCL-XL and BCL2, a mitochondrial death inhibitors, resulting in inactivation of BAD[5] and decreased cell survival.[6] Rapamycin has also shown to induce p53-independent apoptosis in certain types of cancer.[5]

Effects on tumor angiogenesis

Effects of Rapamycin and rapalogs on endothelial and tumor cells.

Tumor angiogenesis rely on interactions between endothelial vascular growth factors which can all activate the PI3K/AKT/mTOR in endothelial cells, pericytes, or cancer cells. Example of these growth factors are angiopoietin 1 (ANG1), ANG 2, basic fibroblast growth factor (bFGF)ephrin-B2vascular enothelial growth factor (VEGF), and members of the tumor growth factor-β (TGFβ) superfamily. One of the major stimuli of angiogenesis is hypoxia, resulting in activation of hypoxia-inducible transcription factors (HIFs) and expression of ANG2, bFGF, PDGF, VEGF, and VEGFR. Inhibition of HIF1α translation by preventing PDGF/PDGFR and VEGF/VEGFR can result from mTOR inhibition. A G0-G1 cell-cycle blockage can be the consequence of inactivation of mTOR in hypoxia-activated pericytes and endothelial cells.[5]

There are some evidence that extended therapy with rapamycin may have effect on AKT and mTORC2 as well.[2][33]

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