mTOR Signaling Cascade in Psoriatic Disease: Double Kinase mTOR Inhibitor a Novel Therapeutic Target.

In this short communication we are providing insight about the regulatory role of the phosphatidylinositol 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) kinase system in psoriatic disease. This is an upcoming active research field in respect to elucidating the inflammatory and proliferative cascades of psoriatic disease. To provide a new dimension to the understandings of the molecular principles of the pathogenesis of autoimmune diseases, we hypothesized that (i) dysregulation of cytokines and growth factors in autoimmune diseases activate the mTOR signaling system and (ii) the activated mTOR kinase system is a key regulator of the inflammatory/proliferative cascades of the disease process. In support of this hypothesis we have earlier reported that growth factors (nerve growth factor (NGF) and platelet-derived growth factor (PDGF)) and relevant cytokines (interleukin (IL)-17, IL-22) known to be critical for psoriasis, psoriatic arthritis, and rheumatoid arthritis activate the mTOR signaling system. Here, we are providing our latest observations that the mTOR signaling proteins are upregulated in psoriatic skin and further we observed that proliferation of keratinocytes (KC) and synovial cells (synovial fibroblasts (FLS)) of psoriatic arthritis are dependent on the PI3K-AKT-mTOR kinase system. To our knowledge, we are the first to explore whether a double kinase inhibitor of mTOR signal proteins has a therapeutic potential for psoriatic disease. Here we will be sharing our views, our research work in this field and as well we will provide evidences how a double kinase inhibitor of mTOR signal proteins can be an effective therapeutic agent for psoriatic disease.

What was known?

Activation of the PI3K-AKT-mTOR kinase system is critical for growth and survival of human cells, but their role in the pathologic outcomes of psoriasis and psoriatic arthritis such as keratinocyte proliferation and pannus formation in the joint remains unknown.

Growth factors control proliferation/survival by activating the phosphatidylinositol 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) kinase system, which is briefly abbreviated as mTOR kinase cascade. This kinase cascade serves as a signal integrator of several upstream signals, including growth factors, nutrients, energy levels, and stress.[123] Thus, a critical function of the mTOR kinase cascade is to integrate growth factors induced signals into a decision to positively or negatively influence cellular growth and proliferation, in other words, the size and rate of replication. mTOR kinase cascade is regulated by the tumor suppressors tuberous sclerosis complexes 1 and 2 (TSC1 and TSC2) which, in the inactive state, form a complex with each other and with mTOR in the plasma membrane. Growth factor stimulation influences mTOR activity through TSC2-Rheb.[2] Growth factors such as epidermal growth factor (EGF), nerve growth factor (NGF), insulin-like growth factor (IGF)-1, and vascular endothelial growth factor (VEGF) primarily regulate mTOR signaling through PI3K-Akt.[456] Activation of the receptors of these growth factors leads to the activation of PI3K which increases levels of phosphatidylinositol (3,4,5)-triphosphate (PIP3) and phosphorylates Akt [Figure 1]. Upon phosphorylation by Akt and TSC2 is released from the complex to the cytoplasm, which allows mTOR to be phosphorylated (p-mTOR) by the small GTP-binding protein Rheb. P-mTOR forms two distinct multiprotein complexes: mTORC1 and mTORC2.[17] mTORC1 regulates the activation of downstream targets p70S6K and 4E-BP1 resulting in protein synthesis and cell growth. The role of mTORC2 is not fully understood; it regulates actin organization and phosphorylates Akt (Ser473), thus it has positive feedback in this cascade.[7] In addition, S6K has negative feedback on this cascade.[17]

Figure 1

Schematic representation of mammalian target of rapamycin (mTOR) signaling pathway regulation. When various ligands such as nerve growth factor (NGF)/epidermal growth factor (EGF)/interleukin (IL)-17/IL-22 bind to their receptor tyrosine kinases (RTKs) result in phosphorylation of tyrosine sites. The phosphorylated tyrosine residues serve as docking sites for downstream pathways including phosphatidylinositol 3-kinase (PI3K)/AKT. AKT phosphorylates various downstream substrates like mTORC1 which involved in the regulation of a range of cellular functions such as proliferation, survival, activation, differentiation, and migration

Among the diverse functions of the PI3K/Akt/mTOR signaling cascade, cell proliferation/survival is an important one [Figure 1]. The critical role of this pathway in cellular proliferation/survival has been substantiated by the presence of activated or mutated mTOR and its upstream (PTEN, Akt) and downstream (p70S6K, eIF4E) signaling proteins in different malignant disorders, which are characterized by the uncontrolled proliferation/survival of cells.[891011] In addition, Akt exerts an antiapoptotic effect by phosphorylating several proapoptotic proteins, including procaspase, 9 BAD and ASK-1.[121314] Akt also contributes to angiogenesis[151617] and differentiation of cells.[1819]

The presence of constitutively activated mTOR signaling proteins in various malignant disorders and the efficacy of PI3K/Akt/mTOR inhibitors in preclinical models of malignant diseases substantiate its role in ‘uncontrolled cell proliferation’.[1011] The key pathologic outcomes in psoriasis and psoriatic arthritis (PsA) are also the ‘uncontrolled proliferation’ of keratinocytes (KC) and synovial fibroblasts (FLS), respectively, along with the proliferation of endothelial cells (EC) and T cells. Thus, we proposed mechanisms for the role of the mTOR pathway in psoriasis and psoriatic arthritis-(i) dysregulation of cytokines and growth factors in these inflammatory diseases activates the mTOR signaling system, (ii) the activated mTOR kinase system may be a key regulator of the inflammatory and proliferative cascades of psoriatic disease.[20212223] We have already reported that relevant growth factors (NGF and PDGF) and relevant cytokines (IL-17 and IL-22) activate mTOR signaling proteins in effectors cells (KC, EC, and synovial cells (FLS)) for psoriasis and autoimmune arthritis.[20212223] Recently we have reported that calcitriol, a well-known anti-psoriatic drug, regulates the proliferation/survival of KC by inhibiting the phosphorylation of mTOR and this could be a possible mechanism for its therapeutic efficacy.[22]

Based on these observations, we investigated to identify whether components of the mTOR signaling cascade can be identified in active psoriatic plaques. We evaluated mTOR and p-mTOR signaling protein expression by Western blot, using lysates of the epidermis of lesional (n = 5) and non-lesional (n = 5) psoriatic skin. Western blot was performed as per our earlier standardized reports.[20212223] Higher expression of mTOR and phospo-mTOR in the psoriatic skin [Figure 2a] support our hypothesis of association between mTOR signaling pathway and psoriasis disease pathology. Buerger et al., in their study by direct immunofluorescence studies have observed that mTOR is activated throughout the whole epidermis in lesional psoriatic skin.[24] Our observation that higher expression of mTOR and p-mTOR in the psoriatic epidermis [Figure 2a] substantiates the report of Buerger et al., that there is increase in mTOR expression and its phosphorylation in lesional psoriatic skin compared to that of non-lesional psoriatic skin.[24]

Figure 2

The mTOR signaling cascade is activated and is functionally active in psoriasis. (a) Activation of mTOR in the psoriatic skin. Immunoblot studies from the epidermal lystaes demonstrate higher levels of mTOR and phosphorylated-mTOR (p-mTOR) in the epidermis of psoriatic skin (n = 5) compared to the non-lesional skin (n = 5). (b) Effect of mTOR inhibitors on proliferation of keratinocyte (KC) and synovial fibroblasts (FLS) by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. KC proliferation cultured in KGM (Lonza Walkersville, MD, USA) and FLS proliferation cultured in DMEM supplemented with sodium pyruvate (Mediatech, Manassas, VA, USA) was found to be significantly reduced by rapamycin (10 nm) and NVP-BEZ235 (50 nm), t-test, **P< 0.001

Increased levels of p-mTOR in the lesional skin of psoriatic disease encouraged us to investigate the functional significance of mTOR signaling kinase cascades in psoriatic disease. FLS (20,000/well) of PsA synovium (n = 3), 20,000/well KC of non-lesional psoriatic skin (n = 3) were cultured for 3 days in triplicates in their respective media.[20212223] These cells were cultured with and without mTOR inhibitors (rapamycin (10 nM) and NVP-BEZ235 (50 nM)). NVP-BEZ235 (LC Lab, Boston, MA, USA) is a synthetic quinoline derivative that inhibits PI3K and mTOR kinase activity by binding to the ATP-binding cleft of these enzymes. Cellular proliferation was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Proliferation was significantly reduced (P < 0.001) by the mTOR inhibitors in the both cell types compared to the untreated cells [Figure 1b]. mTOR kinase cascade dependent proliferation of KC and FLS substantiate a regulatory role of the mTOR signaling proteins in the inflammatory and proliferative cascades of psoriatic disease.

Inhibition of mTORC1 by rapalogs results in unopposed activation of mTORC2 and withdrawal of the negative feedback of S6K. This activates the PI3K/Akt pathway and favors cell survival [Figure 1]. Thus, mTORC1 inhibition alone may not be adequate to inhibit this signaling cascade in autoimmune disorders. This may be a reason for the suboptimal efficacy of an mTORC1 inhibitor (rapamycin) in psoriasis.[25] The challenge is to overcome the failure of rapamycin (mTORC1 inhibitor). One approach is to inhibit both mTORC1 and mTORC2; alternatively this cascade can be blocked more proximally by targeting either Akt or PI3K [Figure 1].

For the first time we explored whether a double kinase inhibitors of mTOR signal proteins has a therapeutic potential for psoriatic disease. Figure 2b suggests that NVP-BEZ235 which is a dual kinase PI3K/mTORC1 inhibitor has potent antimitotic effect on keratinocyte and synovial cell proliferation. This opens up a critical issue that the inhibition of upstream dual kinases of the mTOR system can be an effective therapeutic target and provides encouragement to develop treatment for psoriatic and other inflammatory diseases by targeting the mTOR signaling pathway.

What is new?

The mTOR signaling proteins are upregulated in psoriatic skin and proliferation of keratinocytes and synovial cells (FLS) of psoriatic arthritis are dependent on the PI3K-AKT-mTOR kinase system.

We have substantiated that inhibition of upstream dual kinases of the mTOR system can be an effective therapeutic target for psoriasis and other autoimmune diseases.