The function of many living organisms is coupled with a vast array of cells .These units in turn serve as role playing agents for the fundamental physiology that determines the well being of organisms. However, variations in cellular environment are thought to regulate cell growth, division and survival. A network of molecular pathways or signaling events are interlinked with the cellular functions .Alterations in this specific functional aspect could lead to unnecessary complications which may lead to health abnormalities. In such context , the present description is concerned with highlighting about mTOR Signaling in Carcinogenesis.
mTOR stands for mammalian target of rapamycin and TOR pathway has gained attention as it displays sensing potential to cellular growth, multiplication, protein synthesis, cytoskeleton
rearrangement and metabolism. It executes its activity by associating with several pathways that
are instruemental in cancer development and progression. Activation and phosphorylation of mTOR targets and their inhibition effects have been widely studied and described to be instrumental in carcinogenesis.mTOR is activated by hormonal secretions, mitogens and other factors. This feature was exploited by researchers in understanding the signaling events. However, interaction with novel pathway targets like AKT,INK and GOLPH3 was not been thoroughly addressed earlier. The precise cascade of events how mTOR interacts with or mediates several pathways is a matter of further investigation. It was of the opinion that mTOR role could be better understood from diverse cancer studies with emerging technologies.
The statement is that it is unknown whether mTOR signaling is significantly involved in carcinogenesis through interaction with multiple targets.
Basics of mTOR:
Target of rapamycin (TOR) proteins are ser/thr kinases. They are the members of protein kinase (PIKK) family and are thought to be evolutionarily conserved. They were earlier recognized in the budding yeast Saccharomyces cerevisiae (Mennon & Banning,2008). TORs are of two types TOR1 and TOR2 and their genes were recognized in a screening experiment for identifying mutations that induce resistance to rapamycin, antibiotic mTOR signaling was considered as important event as the network works in integration with growth factors and nutrient signals and goes out of control in condition like cancer and genetic tumor syndromes.The occurrence of two TOR proteins in yeast made to infer that TOR proteins occur in two different physically and functionally macromole- cular complexes from yeast to humans (Mennon & Banning,2008).. In addition, core components conserved within these complexes are a)mTOR complex 1 (mTORC1) made of mLST8 (mammalian Lethal with SEC13 protein 8) and Raptor (Regulatory associated protein of mTOR b)mTORC2 made of mLST8, mSIN1 (mammalian Stress- activated protein kinase interacting protein 1) and Rictor (Rapamycin insensitive companion of mTOR) (Mennon & Banning,2008)..
mTOR is regulated by TSC1 and TSC2, referred to as tuberous sclerosis complex that is formed by mutated tumor suppressor genes. mTORC1 regulation is essential for maintaining homeostasis to regulate constructive processes that ensure cell growth and multiplication. In general, tumor cells tend to alter growth-promoting processes from the growth signal perception. This disconnection is thought to contribute to mTORC1 dysregualtion.mTORC1 is activated by presence or absence of speciﬁc growth stimuli signals driven from several oncogenes ,tumor suppressors and other components involved in malignant tumor formation and progression
(Mennon & Banning,2008)
mTORC1 signaling in cancers:
Increased mTORC1 signaling was found in most human cancers at a large proportion (Leary et al., 2013).Researchers intend to score the mTORC1 signaling activation in tumors by testing the phosphorylation levels of its mTOR direct downstream targets, such as S6K1 substrate ribosomal S6, 4E-BP1 and S6K1. It was of common belief that mTORC1 signaling is determined by these phosphorylation events. The activity of mTOR is better indicated by phosphorylation on S-2448(Leary et al., 2013) .In Ovarian cancer,PI3K/Akt/mTOR signaling pathway was reported play important role.The phophatidylinositol 3 Kinase (PI3K) pathway is a signaling network that coordinates several upstream inputs from tyrosine kinase receptors such as IGF1R, EGFR, HER2, growth factors like EGF, heregulin, TGF and with the pathway Ras-Raf-Mek-Erk related to membrane receptors.PI3K/Akt/mTOR signaling pathway gets dysregualted in ovarian cancer. This aberration was associated with specific histological subtypes (Leary et al., 2013).Ovarian cancer was reported to be a hetero‐ generous disorder with increased variations in histology, molecular profile, prognosis and chemosensitivity as per the sub type. High grade serous ovarian cancer (HGSOC) was considered as the most common subtype with ubiquitous p53 mutations and high genomic instability leading to deletions or amplifications in somatic copy number very often. Hyperactive PI3K/Akt/mTOR pathway could lead to deregulations upstream in receptor tyrosine kinases (RTKs) in a HGSOC pro portion (Leary et al., 2013) In mice, the activation of pathway through PIK3CA mutations and PTEN loss was reported to induce ovarian tumors and prevention of PI3K/mTOR. This led to a delay in growth of tumor and survival finally giving a proof that this pathway in ovarian cancer is essential as a therapeutic target. The alterations related to PI3K/Akt that were demonstrated from patients with ovarian cancer in vivo and the reliance on this oncogenic pathway in pre‐ clinical models has motivated the researchers to investgate the adavantages of targeting mTOR or PI3K, Akt in ovarian cancer.It was reported that the pathway inhibitors to be described first in clinical study were rapamycin analogs (Leary et al., 2013). These bind to FK506 binding protein-12 of the MTORC1 complex and inhibit the activity of mTOR. Rapamycin is an important immunosuppressant used for inhibiting organ transplant rejections and malignant hematological aberrations. Its adverse effects include hypercholesterolemia, ceridemia, hematologic toxicity, renal toxicity, edema and hypertension (Leary et al., 2013).
In various tumor types, rapamycin analogs with decreased immunosuppressive potential were reported to be active. These include ridaforolimus, everolimus and temsirolimus. The benefits of mTOR in combination with Akt in chemoresistance has led researchers to investigate much on mTORinhibitors (mTORi) .But, in some phase 1 studies on combination therapy have yielded inconclusive results. Because these involved small sample size. So, randomized trials are need to determine the efficacy. Similarly, the combination therapy study involving mTOR and vascular endothelial growth factors (VEGF) inhibitors was done, but certain side effects in study has made to wait for updated results (Leary et al., 2013).
Translation is an important process for carrying out various cellular activities like cell development, survival that are in turn under control of a gene expression.These events are finally contribute to protein synthesis. Cancer progression is much determined by translation events and the associated pathways. It was reported that cellular signaling pathways influence the translational machinery by phosphorylating factors of translation initiation. It is important to note that various hormones, mitogens, growth factors stimulate the PI3K/AKT/mTOR signaling pathway. Here, cap-dependent mRNA translation is a downstream target of this pathway. As such, mTOR facilitates the phosphorylation and activation of S6K and that of 4E-BPs (Eukaryotic initiation factors –binding proteins) (Mamane et al., 2006).
eIF4E and 4E-BPs in translation:
. eIF4E is a translation factor and when over expressed that was reported to promote malignant transformation when overexpressed in cultured cells. 4E-BPs are essential in regulating transformation. They suppress cell growth and tend to reverse the ras or src gene induced cell transformation. The interaction of eIF4E/4E-BP at a an advanced level, contributes to cell size decrease and cell cycle inhibition (Mamane et al., 2006).
S6Ks are two ribosomal proteins, S6K1 and S6K2 in mammalian cells. mTOR targets both these S6Ks as downstream targets.
Much focus on mTOR was that its activity is inhibited by Rapamycin. This antibiotic binds to
FK506-binding protein-12 (FKBP) which then binds to mTOR, prevents its kinase activity and later developed signals to 4E-BP and S6K which serve as downstream targets.
This could indicate that cancer cell growth inhibiting potential of rapamycin is facilitated via 4E-BPs
dephosphorylation of 4E-BPs.mTOR importantly targets 4E-BPs which influence rapamycin sensitivity.The potential of 4E-BPs is that they could make cells sensitive to apoptosis process in Ras-overexpressing cell mediated by rapamycin. Overall, the link between mTOR and4E-BP is that regulate survival of cells. This in turn is controlled by eIF4E a key downstream effector of mTOR through the 4E-BP activity (Mamane et al., 2006).
Role of PI3K/AKT/mTOR signaling in translation:
So , translation initiation was regulated by mTOR signaling several corners through a cascade of events.Here,initially PI3K/Akt signaling cascade was activated by insulin, hormones and growth factors (Mamane et al., 2006). mTOR complex that contains LST8/GbL and rictor and PDK1 phosphorylate Akt. Akt in turn targets TSC2 for phosphorylation and destabilizes the TSC1/2 complex. This facilitates Rheb to activate mTOR. But mTOR gets inhibited by factor REDD1during hpoxic state through the TSC1/2 complex. mTOR receives amino acid signal via Vps34 and this facilitates the important Rheb/mTOR complex.This complex then mediates the phosphorylation of 4E-BP1, S6K1 and eIF4G with LST8/GbL and raptor. S6K1 then targets ribosomal protein S6 and eIF4B upon activation (Mamane et al., 2006).
Researchers have used a novel technology known as ribosome profiling to understand the pharmacological inhibition of mTOR7 that furnishes insights on translational landscape of the cancer genome at a codon-by-codon resolution (Hsieh et al., 2012).This was done because translationally regulated nodes of gene expression at downstream that leads to development of cancer was poorly addressed. So, oncogenic mTOR signaling when studied through this method, a wide range of genes participating in cell proliferation, metabolism and invasion were revealed
mTOR in prostrate cancer:
Researchers have widely reported mTOR deregulation in 100% of advanced human prostate cancers. Genetic studies carried in mouse indicated mTOR hyperactivation playing key role in the initiation of prostate cancer. mTOR was found to get constitutively hyperactivated in PC3 human prostate cancer cells (Hsieh et al., 2012).
So, research was much targeted to understand the effects of complete or partial mTOR inhibition on translationally controlled gene expression networks. Here, in the method, the activities of three primary downstream mTOR effectors 4EBP1, p70S6K1/2 and AKT were inhibited by treating PC3 cells with PP242 ,an mTOR ATP site inhibitor. Nearly, 144 target mRNAs were reveled by ribosome profiling where a 68% were found to contain 5’TOP. The effects of PP242 was found to be high at the translation initiation level but absent at elongation level. The translation control of mRNAs upon mTOR activation were modulated by a novel cis-regulatory element (Hsieh et al., 2012). This came into light when researchers discovered mTOR sensitive genes like YB1,vimentin,MTA1and CD44 all with PRTE.PRTE was mutated in the 59 UTR of YB1 that made YB159 UTR insensitive to 4EBP1 inhibition. Thus the PRTE was found to play role in facilitating translational control (Hsieh et al., 2012).
Role of INK128:
Further, mTOR employed in ribosome profiling were exploited for characterizing novel recognized mTOR-sensitive cell invasion genetic components.To accomplish this, INK128 , mTOR ATP site inhibitor, was used due to its potential, specificity for mTOR, small mlecualr mass and desired pharmaceutical features.In PC3 cells, by employing INK 128, scientists have found a low expression of four gene elements YB1, MTA1, vimentin and CD44 at the protein level. mTOR hyper-activation appeared to positively regulate gene invasion signature. On the other hand researchers have also foscused on mTOR ATP site inhibitor effect to observe prostate cancer cell migration and invasion. INK128 was found to lessen the invasive potential of PC3 prostate cancer cells. But this effect was not observed with rapamycin.INK 28 was also found to suppress cancer cell migration (Hsieh et al., 2012).
mTOR inhibitor INK 28 was found to significantly suppress several prostate cancer aspects by facilitating apoptosis, preventing cell multiplication, invasion an metastastic process. INK28 is more potential due the fact that in prostrate cancer it could reprogram mTOR onco- genic translational program which is not feasible with treatment by rapalog. So, coperative control is considered as the novelty of mTOR translationally controlled genes and the human prostate cancer cells with invasive features that provide a platform for mTOR interventions. The mRNA are subject to translation control mediated by 4EBP1–eIF4Eaxis . Hence, they could be pharmacologically targeted with with potent mTOR ATP site inhibitors that are more specifi towards phosphorylation of mTOR-dependent 4EBP1 targets (Hsieh et al., 2012).
Certain pathway mutations reported in sporadic human cancer could significantly influence mTOR activity and signaling events. For instance, PI3K/AKT and RAS oncogenic signalling modules undergo mutation in cancer cells (Astle et al., 2012). The importance of this pathway relevant to mTOR study is that mutations in PI3K were reported in 30% of sporadic tumours in humans. Hrypeactivation of AKT, a key mediator of PI3K signaling was reported in 40–70% of melanomas, 50% of prostate cancers, 60% of lung cancers and 20–55% of breast cancers. Most importantly, AKT serve as a core point in pathways stimulated by oncogenic mutation in genes that encode RAS, LKB1 and EGFR (Astle et al., 2012).
These pathways were shown to improve growth of tumors and their multiplication. However, they could promote cell senescence in healthy human cells upon activation. It was shown that AKT- driven senescence relies on p-53 gene translation and stabilization regulated by mTORC.
This is because AKT expressing cells when treated with allosteric mTORC1 inhibitor rapamycin
decreased p53 and even p21 to normal levels and senescence was avoided (Astle et al., 2012)
So, researchers believe that mTORC targeting in tumors with activated PI3K/AKT signalling could promote lethal effects due to senescence activation on cancer promoting cells (Astle et al., 2012).
Next, researchers have dissected key association between Golgi protein, GOLPH3 and mTOR
(Scott et al, 2009).In several cancer types, researcher have identified a frequent 5p13 amplification through genome-wide copy number analyses. In the regulation of cancer signaling, golgi apparatus role was anticipated due to a finding that cytoplasmic membrane oncoproteins,like RAS could provide signals when located in the apparatus.Added to that, researchers recognized a Golgi protein, GOLPH3, as an important target for amplification and also as a oncogene from gain- and loss-of-function analysis. In human cancer cells, growth-factor-induced mTOR signaling activity was reported to be improved by GOLPH3 (Scott et al, 2009) . This component was also shown to change the responses in vivo to mTOR inhibitor. There is a growing body of interest on GOLPH3 targetting for amplification as it is able to alter responses to cancer drug,rapamycin.So, mTOR signaling , with its complex interactions, when activated could provide a fundamental molecular rationale for GOLPH3’s oncogenic activity.The interaction between retromer complex and GOLPH3, is thought to be essential for trafficking of proteins between trans-golgi network (TGN) and endosomes. This biological process was implicated in cancer for the first time by researchers. As GOLPH3 activates mTOR signaling, GOLPH3 expression levels could help in predicting sensitivity to mTOR inhibitors and hence could also serve as predictive markers of rapamycin sensitivity (Scott et al, 2009).
So, mTOR could appear very crucial for living organisms,especially humans, right from developing embryos till adult stage that is characterized by aging and lifespan events. Its implication in tumor types has made it an ideal target for research studies and therapeutic area (Beauchamp & Platanias, 2013).
mTOR is a potent molecular target that has interactions with diverse molecular networks. Emerging technologies appear to evolve with the goal of dissecting the key molecular interactions associated with mTOR signaling pathways. Associations with factors like AKT,INK and GOLPH3 has furnished much insights on mTOR involvement in cancer. Added to that, mTOR fundamental role in cell metabolism and other functions has made it an important target
in cancer therapeutics. Phosphorylation of various molecular protein factors involved in pathways has led to an increased emphasis on mTOR signalling. Further, dysregualtion of mTOR ,pathway effectors located downstream/ upstream in various cancers has stimulated to investigate on inventing mTOR targeting anticancer drugs. Overall, mTOR signaling appears a complex network characterized by effects from translation to inhibition. Thus, mTOR signaling is significantly involved in carcinogenesis through interaction with multiple targets. There is a need of evidence based research strategies to assess the mTOR significance in carcinogenesis and continue studies on mTOR with good number of cancer patient samples.
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