p-53 INDEPENDENT TRAIL-INDUCTION TRIGGERS TUMOR CELL DEATH

 

Inhibition of cell death or apoptosis has been implicated in the chemotherapeutic resistance of tumor cells. TRAIL (tumor-necrosis-factor-related apoptosis inducing ligand; http://en.wikipedia.org/wiki/TRAIL) is involved with induction of apoptosis in tumor cells thereby represents an attractive target for the development of anticancer therapy. A type II transmembrane protein and a member of tumor necrosis factor family, TRAIL is expressed in wide range of tissues. Even though it is a membrane protein, TRAIL is also expressed in soluble form. Induction of TRAIL-mediated apoptosis involves binding of TRAIL with its receptor TR1 (also known as death receptor 4, DTRAIL signaling resized 600R4) and TR2 (DR5). TRAIL also binds with two other receptors TR3 and TR4. Unlike TR1 and TR2, these receptors have incomplete death domains. Elevated expression of TR3 and TR4 in normal cells is thereby suggested to protect normal cells from TRAIL-induced death signaling. Induction of apoptotic signaling involves binding of TRAIL to DR4 or DR5. This results in homotrimerization and activation of receptors, enabling the receptors’ death domain to recruit the adaptor protein Fas-associated death domain along with pro-caspase- 8 and pro-caspase- 10. These all together then form the multi-protein death-inducing signaling complex, (DISC). Inside the DISC procaspeses become autoactivated and become caspase-8/10. Activated caspase-8/10 then activates downstream effector caspase-3 or caspase-7. Finally, cleavage of downstream substrates by effector caspases results in DNA fragmentation leading to apoptosis.

Several studies assessed efficacy of TRAIL-based therapies in cancer as a single agent or in combination with other anti-cancer agents. In monotherapy, monoclonal antibodies conatumumab, AMG 655, CS-1008, dulanermin, exatumumab, and mapatumumab exhibited responses in follicular lymphoma, lung adenocarcinoma and in a rare form cancer synovial sarcoma with minimal toxicity. In addition, efficacy of TRAIL-monoclonal antibodies has also been evaluated in combination with anti-cancer agents such as paclitaxel, cisplatin, carboplatin, gemcitabine, histone deacetylase inhibitors, proteosome inhibitors, and kinase inhibitors. However, several cases of adverse effects and toxicities were reported in these studies. In addition, recent TRAIL-based therapies are expensive to produce for clinical applications and may also be limited by endogenous TRAIL level as well as dependency on tumor suppressor gene p53 as a regulator of TRAIL gene transcription. Therefore, studies are required to overcome the shortcoming of current TRAIL-based therapies.

A preclinical study published recently in Science Translational Medicine (Allen et al., 2013) reported that small-molecule drug TRAIL-inducing compound 10 (TIC10) can trigger tumor cell death in a variety of cancers including breast, colon, lung, and also gliobalstoma, a type of brain tumor that is very difficult to treat. The anti-tumor activity of TIC10 is mediated by the activation of TRAIL in Foxo3a dependent manner. In their study, Allen et al. observed significant tumor regression in human colon, triple-negative breast, non-small cell lung, and brain cancer xenograft-bearing mice following single-dose treatment of TIC10. Inactivation of both phosphoinositide 3-kinase/Akt (PI3-K/Akt) and mitoHuman glioblastoma bearing mouse recovered following TIC10 treatment compared to untreated.gen-activated protein kinase (MAPK) pathways was noted following TIC10 treatment in this in vivo study that resulted in translocation of the transcription factor Foxo3a into the nucleus, where Foxo3a induced expression of TRAIL gene to activate apoptosis.

In the pre-clinical study, small-molecule drug TIC10 showed potent anti-tumor activity with limited toxicity through sustained induction of TRAIL even in refractory brain malignancy in a p53-independent manner. This revives the hope of TRAIL-based therapies in cancer. However, clinical trials need to be conducted first in order to confirm safety and efficacy of TIC10 before it can ultimately be used within the clinical setting.

 

References:

1.         Allen JE, Krigsfeld G, Mayes PA, et al. Dual Inactivation of Akt and ERK by TIC10 Signals Foxo3a Nuclear Translocation, TRAIL Gene Induction, and Potent Antitumor Effects. Sci Transl Med. 2013;5(171):171ra117.

2.         Hellwig CT, Rehm M. TRAIL signaling and synergy mechanisms used in TRAIL-based combination therapies. Mol Cancer Ther. 2012;11(1):3-13.

3.         Dimberg LY, Anderson CK, Camidge R, Behbakht K, Thorburn A, Ford HL. On the TRAIL to successful cancer therapy? Predicting and counteracting resistance against TRAIL-based therapeutics. Oncogene. 2012.

 

Epithelial to Mesenchymal Transition: the Key to Cancer Metastasis

We have all seen science fiction movies where alien invaders, in search of a fertile planet like earth, start a full scale war against humanity. A common theme in many of these stories, but often overlooked, is the role of the scout. Before any invasion, a solitary member of the invaders, fortified for the harsh journey, is sent on a quest to scout out the new land. As soon as this scout finds a promised land, the colony is formed and the invasion begins. This concept of invasion is seen in nature with viral and bacterial infections, where fortified virus or sporulated bacteria are able to survive harsh conditions and then proliferate in their host system upon arrival. In an ironic display, cancer metastasis follows a similar system; cancer cells are able to leave the primary tumor, travel long distances in harsh conditions, and form colonies in other tissues within an organism. These metastases have grave consequences. In fact in certain cancers, such as melanoma, it is metastasis to vital areas like the brain that makes it life-threatening. One of the biggest areas in cancer biology research is elucidating the mechanisms involved in cancer metastasis, which includes the concept of the epithelial to mesenchymal transition (EMT). It is this process that gives the cancer “scouts” the means to invade vasculature, fortify themselves for a journey to the metastatic site, and resist most therapies at all locations in the tumor-bearing patient. In normal development, the epithelial to mesenchymal transition is a reversible system that is involved in embryonic gastrulation1 and cardiac development1,2. In adulthood, EMT is involved in wound healing3. Unfortunately, EMT is also involved in pathological events such as fibrosis of injured tissue1 and cancer development and progression1-4. Here, we focus on EMT’s role in tumorigenesis, cancer proliferation, treatment resistance, and metastasis.

The epithelial to mesenchymal transition occurs in many different solid tumors of epithelial origin. Many tumors are carcinoma based, which implies that they are usually confined from motility by the basement membrane1 but the process of converting toward a cancer cell that has mesenchymal characteristics allows cancer cells to infiltrate the circulatory and lymphatic systems, causing motility that will later lead to metastasis1,5. As a cancer progresses, epithelial features, such as cellular adhesion marker  E-cadherin3 and intracellular adhesion components such as tight junctions, cytokeratins, and desmosomes, are downregulated5. Coordinating with this downregulation is an upregulation of mesenchymal markers such as N-Cadherin, Vimentin, and Fibronectin3. This phenotypic shift is orchestrated by a coordination of many different mechanisms: epigenetic changes, post-translational modifications of proteins, transcriptional silencing by noncoding-RNAs (ncRNAs), and activation of epithelial to mesenchymal transcription factors (EMT-TFs)3. However, while all mechanisms of EMT development are important, this article will focus on the main orchestrators of EMT: the EMT-TFs.

EMT-TFs are many, but the main transcription factors fall into three families: SNAIL, ZEB, and TWIST. SNAIL has three main transcription factors: SNAIL (Snail1), SLUG (Snail2), and SMUC (Snail3)5. These proteins are zinc finger nucleases that are at the crux of EMT phenotype. In fact, it has been noted that SNAIL, and possibly SLUG, directly repress the expression of E-cadherin by binding to its promoter, CDH13. Furthermore, the SNAIL family has been shown to repress desmoplakin, adherens junctions, occulidins, and cytokeratin upon activation5. While not much is known about SMUC’s role in normal or pathological development, SNAIL, unlike SLUG, is so crucial to cancer metastasis that has been implicated in being an independent prognostic factor in the metastatic potential and severity of cancers5. Interestingly, not only does the SNAIL family propagate the characteristics of EMT transition, they also upregulate other EMT-TFs, such as ZEB1 and ZEB2, to further propagate the mechanism of EMT 5.

The Zinc-finger E-box-binding homeobox (ZEB) family are also a family of transcription factors with zinc-finger nuclease properties3. Like SNAIL, ZEB1 and ZEB2 bind to the E-cadherin promoter5. Moreover, ZEB1 and ZEB2 also downregulate tight/gap junctions, desmosomes and markers of polarity5. In addition, the ZEB family has been implicated in repressing P- and R-cadherins, other markers that inhibit the motility of epithelial cells5. ZEB1 is usually not found on non-cancerous cells but is found highly expressed on many cancer types5. In contrast, ZEB2 is expressed on normal epithelial cells, but is vastly upregulated in cancerous cells5.

describe the imageThe final group is the TWIST family, a basic helix-loop-helix transcription factor involved in different steps in embryonic development1. Interestingly, while TWISTs are vital to embryogenesis, they are absent in normal adult epithelium5. As a cancerous cell progresses , TWIST1 and TWIST2 appear and their reactivity increases in correlation with the tumorigenic progress5. Interestingly, while TWIST1 is involved  in regulating some of SLUG’s EMT effects, and directly drives expression of N-cadherin, it is not directly associated with the downregulation of E-cadherin5.

It is through these EMT-TFs that EMT is involved in tumorigenesis and tumor progression. EMT-TFs are involved in suppressing senescence and increasing cell cycle proliferation5. However, EMT-TFs are not enough to push tumorigenesis on their own, but require another event of tumorigenesis3. Therefore, EMT-TFs may act as a facilitator of tumorigenesis, but not be tumorigenic factors by themselves. EMT is also crucial for tumor invasiveness and metastasis. Besides the aforementioned changes in cellular adhesion molecules, activation of the EMT-TFs causes upregulation of matrix-metalloproteinases (MMP), enzymes involved in degradation of the extracellular matrix and invasion of cancer cells3. It has been demonstrated that the SNAIL family activates the expression of MMP1, MMP2, MMP7, and MT1-MP 5. Furthermore, the upregulation of MMPs additionally activates EMT-TFs, thus forming a feed-forward loop3. EMT events may also go beyond extracellular degradation; TWIST1 is known to be involved in the formation of invadopodia which has been correlated with invasiveness3.

The invasion of metastasis is not just due to intrinsic changes but also to changes in the host, and target microenvironment4. For instance, TGF-β, a cytokine that normally acts as a tumor suppressor, can enhance tumor invasion in later-stage tumors2. Furthermore, TGF-β activates the SNAIL and TWIST families of the EMT-TFs2,5. TGF-β may be produced by myeloid derived suppressor cells and CD11b+/F4/80+ tumor associated macrophages (TAMs) in the primary tumor microenvironment, thus perpetuating the EMT phenotype4. TAMs also express the cytokines fibroblast growth factor (FGF), epidermal growth factor (EGF), and macrophage colony stimulating factor (CSF-1) which are involved in EMT-based invasion as well as recruitment of immune cells toward a metastatic-favored microenvironment4. Moreover, TNF-α, usually correlated with  an anti-tumor phenotype, also stabilizes SNAIL expression, thus implicating it in facilitating EMT3. Besides immune cells, other stromal cells are involved in EMT initiating and metastasis. For instance, mesenchymal stem cells (MSCs) and cancer associated fibroblasts (CAFs) are both involved in EMT initiation and propagation4.

One of the more clinically relevant aspects of EMT in cancer progression is the ability of EMT to confer therapy resistance. Resistance to doxorubicin is breast cancer correlates with higher levels of ZEB15 and both ZEB1 and ZEB2 have been shown to guard against cisplatin therapy5. TWIST1 mediates resistance to paciltaxel and TWIST1 and TWIST2 block daunorubicin by inhibiting degradation of the anti-apoptotic protein, Bcl-2, in bladder, ovarian, and prostate cancer5. Along the same lines, the EMT has been shown to confer upon cells a cancer stem cell-like phenotype2,4, a cancer phenotype also known for its therapy resistance5. TGF-β-driven EMT activates protein involved in stem cell phenotype, such as Sox2, PDGFB, and LIF 2. Furthermore, ZEB1 has been shown to be vital in the formation and maintenance of stem cell phenotype in some cancers5. However, while activation of the EMT pathway may confer a cancer stem cell-like phenotype, it is not necessary to get this phenotype3 as EMT-TFs are not necessarily involved in dedifferentiation3. In fact, some reports shine controversy on EMT-TFs’ role in stem cell development: while colorectal cancer spheroids show higher levels of SNAIL, other studies have shown that overexpression of SNAIL and SLUG in ovarian cancer drives these cancers away from a stem cell phenotype5.

cancer patientBesides the difficulty in treatment that EMT poses, one of the main obstacles that is troubling the cancer metastasis field is difficulty in identification of EMT/MET in cancer in vivo4. Because EMT is an orchestration between tumor and it’s microenvironment, researchers have been unable to definitively demonstrate the role of EMT beyond stromal epithelium4. The field needs better phenotypic markers  to identify tumor epithelial cells from normal epithelial cells as well as a way to trace lineages of human cancers in vivo 4. Another is MET, the reverse of EMT and the end result of metastasis. To date, bona fide evidence for MET is only found in vitro studies and xenograft experiments4. MET explains why the phenotype of metastatic tumors mirror the primary site, but it is not an explanation for the system4. Several methods have been proposed to more appropriately study EMT, such as intravital 2-photon microscopy4. However, the sporadic nature of the EMT event makes observation, even in this system, very difficult4. In such a case, we are left to the spontaneous tumor-forming mouse models or studying xenograft models of immortalized cancer lines that are known to be highly metastatic. In addition, since there are no anatomically distinguishable between mesenchymal and epithelial cells, people have proposed the solutions of creating tumor lines the express reporter genes linked to promoters for epithelial/mesenchymal fates4. If one were able to combine an intravital two-photon system with a xenograft of a highly metastatic cancer line transduced with mesenchymal/epithelial reporter constructs, this would be the most feasible model to study EMT in real-time. As technology advances to detect individual cell populations in real-time, the expectation to solidify the mechanism of epithelial to mesenchymal transition in metastasis will increase the reliably of current EMT findings.

 

References:

1          Lim, J. & Thiery, J. P. Epithelial-mesenchymal transitions: insights from development. Development 139, 3471-3486, doi:10.1242/dev.071209 (2012).

2          Massagué, J. TGFβ signalling in context. Nat Rev Mol Cell Biol 13, 616-630, doi:10.1038/nrm3434 (2012).

3          Craene, B. D. & Berx, G. Regulatory networks defining EMT during cancer initiation and progression. Nat Rev Cancer 13, 97-110, doi:10.1038/nrc3447 (2012).

4          Gao, D., Vahdat, L. T., Wong, S., Chang, J. C. & Mittal, V. Microenvironmental regulation of epithelial-mesenchymal transitions in cancer. Cancer Res 72, 4883-4889, doi:10.1158/0008-5472.can-12-1223 (2012).

5          Sánchez-Tilló, E. et al. EMT-activating transcription factors in cancer: beyond EMT and tumor invasiveness. Cell Mol Life Sci 69, 3429-3456, doi:10.1007/s00018-012-1122-2 (2012).

 

T cell dysfunction in cancer: Anergy, Exhaustion, or Senescence?

Immune responses against cancer have been shown to be effective in eliminating tumors.  However anti-tumor immunity is limited by dysfunctional T cells which have been described in cancer patients.  Understanding how dysfunctional T cells arise in cancer and the potential mechanisms for restoring functionality are critical for developing effective immunotherapeutics.  In the current issue of Current Opinion in Immunology, Dr. Weiping Zou’s group, (Crespo. et. al.,) review the various types of T cell dysfunction that occur in the tumor microenvironment.

Anergy, exhaustion, and senescence are three different mechanisms underlying T cell hyporesponsiveness, which share distinguishing phenotypic features but arise by different mechanisms and under different experimental settings.  However, what is the difference between these three types of T cell dysfunction and which contribute to impaired T cell responses in the setting of cancer?

Anergy: T cell anergy generally refers to a hyporesponsive state in T cells induced by triggering the TCR either without adequate concomitant co-stimulation through CD28 or in the presence of high co-inhibitory molecule signaling.  Without both TCR and CD28 signals, IL-2 is not effectively transcribed and instead, anergy-associated genes such as GRAIL are expressed which contribute to impaired TCR signaling via negative feedback.

In the tumor microenvironment cancer, altered expression of B7 family members by APCs leads to an enhanced expression of B7 family co-inhibitory molecules including PD-L1 and a reduction in the B7 family co-stimulatory molecules CD80 and CD86.  Thus, T cell activation in this environment could lead to induction of anergy.

Exhaustion: T cell exhaustion occurs as a result of chronic over-stimulation, such as occurs in the settings of chronic viral infections including hepatitis C virus (HCV) and HIV, autoimmunity, and cancer.  Exhausted T cells progressively lose the ability to express effector cytokines including IL-2, IFNg, and TNFα.  They also express multiple inhibitory receptors including PD-1 and LAG-3, lose cytotoxic and proliferative potential, and may ultimately be driven to apoptosis.

Because anti-tumor T cells are persistently exposed to antigen in the tumor microenvironment, exhaustion is a likely mechanism contributing to T cell dysfunction in cancer patients.  As such, exhausted T cells have been described in patients with melanoma, ovarian cancer and hepatocellular carcinoma.

Senescence:  Senescence is thought to occur due to the natural life span, or aging of cells.  However, senescent T cells have been observed in the settings of chronic inflammation and persistent infection in young individuals, indicating other factors beyond a person’s age, such as DNA damage, drive acquisition of this state.  Senescent T cells are marked by deficient CD28 expression, telomere shortening, expression of regulatory receptors such as TIM-3 and KLRG-1, and inability to progress through the cell cycle.

Cells with features of senescence have been described in patients with lung cancer, head and neck cancer, hepatocellular carcinoma, melanoma and lymphoma.  Thus, senescence may also contribute to T cell dysfunction in cancer patients.

In conclusion, there is evidence that anergy, exhaustion, and senescence may all be contributing toward T cell dysfunction in cancer.  In the review, Crespo. et. al., make the point that distinguishing cells in these states may be complicated as they overlap phenotypically and in expression of various markers.  Furthermore, the mechanisms mediating establishment of these three states is not well defined.  However, it is important to clarify the mechanisms by which T cells gain and maintain dysfunction in cancer in order to best develop effective immunotherapeutics.

 

Further Reading:

 T cell anergy, exhaustion, senescence, and stemness in the tumor microenvironment.  Crespo J, Sun H, Welling TH, Tian Z, Zou W.  Curr Opin Immunol. 2013 Jan 5.

The three main stumbling blocks for anticancer T cells.  Baitsch L, Fuertes-Marraco SA, Legat A, Meyer C, Speiser DE. Trends Immunol. 2012 Jul;33(7):364-72.

Induction of T cell anergy: integration of environmental cues and infectious tolerance. P. Chappert, R.H. Schwartz.  Curr. Opin. Immunol., 22 (2010), pp. 552–559.

T cell exhaustion.  E.J. Wherry.  Nat Immunol, 12 (2011), pp. 492–499.

T-cell senescence: a culprit of immune abnormalities in chronic inflammation and persistent infection.  A.N. Vallejo, C.M. Weyand, J.J. Goronzy.  Trends Mol Med, 10 (2004), pp. 119–124.

Recent Findings in Multiple Sclerosis Treatment

Multiple sclerosis (MS) is believed to be a neurodegenerative autoimmune disorder, in which the body’s immune system attacks its own healthy tissue, specifically the myelin sheath surrounding the axons of the central nervous system (CNS). The word sclerosis refers to the scar tissues or the plaques within brain’s white matter, observed through the Magnetic Resonance Imaging (MRI) of MS patients’ brains. These plaques are results of myelin-degeneration (demyelination) and axonal death. The progression of MS symptoms is directly proportional to the failure of remylination by oligodentrocytes, leading to neurodegeneration; this demyelination disrupts the proper conduction of action potentials from CNS to different target organs and will eventually results in permanent disability caused by chronically demyelinated plaques. Some of the common symptoms include changes in sensation, muscle weakness, abnormal muscle spasms, or difficulty moving and maintaining balance, problems in speech or swallowing, as well as visual problems.

Multiple Sclerosis,MS,MRI,Inflammatory

Therefore, in addition to inhibiting the autoimmunity, preventing permanent neurodegeneration as well as functional recovery of oligodentrocytes, are current therapeutic targets in MS clinical research. The focus of this article is to discuss the several recent studies that have reported promising results to this end.

Acid-Sensing Ion Channels (ASICs) are neuronal voltage-insensitive cationic channels activated by extracellular hydrogen ions (H+), and mediate entering and excessive accumulation of sodium (Na+) and calcium ions (Ca2+) inside the neuron’s cytoplasm. This intra-axonal accrual of Na+ and Ca2+ ions causes cellular injury and subsequent neurodegeneration in the CNS. Over-expression of ASIC1 has been observed in acute MS lesions (oligodendrocytes and axons with an injury co-express ASIC1 in chronic MS lesions) and believed to play a role in the development of irreversible tissue damage.

Moreover, amiloride, a potassium sparing diuretic (causes excretion of large amounts of potassium from the body), blocks ASICs by acting as “channel-blocker” and has been used for hypertension and congestive heart failure management. In a recent translational clinical study, effects of ASIC1-inhibition was tested in 14 patients with primary progressive MS, by comparing the rates of brain atrophy and tissue damage before and during amiloride treatment for 3 years. The results of this preliminary study show a significant decrease in the rate of whole-brain atrophy during the amiloride treatment period, which indicates reduced neurodegeneration (cell damage) through ASIC blocking. Although further studies with larger populations are needed to confirm the robustness of these observations, this is a safe, inexpensive promising potential neuroprotective MS treatment that may be utilized in conjunction with anti-inflammatory agents.

MS,Multiple sclerosis,Myelin,autoimmune,axon

As mentioned previously, failure of oligodendrocytes to remyelinate leads to the severe clinical impairments associated with MS, which makes myelin- regeneration a significant therapeutic goal. Even though oligodendrocyte precursor cells are present, they fail to mature and myelinate in MS brain. Some of the key factors stimulating migration, maturation and survival of myelinating oligodendrocytes are components of extracellular matrix (ECM).

The ECM component in areas with MS lesions have significant differences when compared with the healthy adult brain tissue: two of these ECM abnormalities are the increased expression of Laminin, as well as upregulation of Fibronectin molecule that is absent in the normal brain’s white matter. Therefore, it has been speculated that fibronectin expression in the injury environment may inhibit oligodendrocyte maturation, contributing to remyelination failure in MS plaques.

A recent study suggests that the MS inflammation in the CNS causes astrocytes to accumulate fibronectin, which impair remyelination within the chronic lesions. These findings offer new strategic clinical approaches to promote remyelination through inhibiting fibronectin aggregation and its clearance from the inflammatory sites within the parenchyma.

Interestingly, another group have recently demonstrated a strong remyelinating effect of testosterone mediated by its receptor. They propose promotion of remyelination in males with MS, through utilizing synthetic drug which specifically bine the brain androgen receptors, employing testosterone as remyelinating agents.

Although there is no approved method to efficiently treat MS up to date, there are increasing reports on not only the disease’s underlying mechanisms, but also promising clinical strategies that are rapidly moving to human clinical trials. To learn about the role of the Blood Brain Barrier and other 2013 findings in MS, visit http://info.sanguinebio.com/neurology.

 

Multidrug-Resistance in Cancer: ABC-Transporters

Multidrug-resistance (MDR) is the chief limitation to the success of chemotherapy. According to the National Cancer Institute, multidrug-resistance is a phenomenon where cancer cells adopt to anti-tumor drugs in such a way that makes the drugs less effective. Studies have shown that 40% of all human cancers develop MDR. Deaths due to cancer occur in most of the cases when the tumor metastasizes. Chemotherapy is the only choice of treatment in metastatic cancer, and MDR limits that option.

Cancer one resized 600As shown in Figure 1, tumor cells adopt several mechanisms to evade death induced by anti-tumor agents. These include changes in apoptotic pathways and activation of cell-cycle check points to increase DNA repair. Alternatively, cancer cells develop resistance by increased expression of multidrug-resistant proteins and altered anti-tumor drug transport mechanisms. Members of the ABC transporters (ATP-binding cassette) are known to be associated with this phenomenon, as the human genome express over 48 genes in this transporter family alone. These proteins bind ATP in their ATP binding domain and use the energy to transport various molecules across the cell, thus they are known as ABC proteins. Among these proteins, P-glycoprotein (Pgp, ABCB1), multidrug resistance-associated protein (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2) are chiefly responsible for drug resistance in tumor cells. Studies are warranted to determine the role of other members of ABC transporters including MRP2, MRP3, MRP4, MRP5, ABCA2 and BSEP in drug-resistance.

MDR1(Pgp)

cancerPlasma membrane glycoprotein (Pgp) was the first ABC-transporter detected in various cancers exerting resistance to a variety of chemically unrelated cytotoxic agents including anti-tumor drugs such as doxorubicin, vinblastine, ritonavir, indinavir and paclitaxel. It works as an energy-dependent efflux pump and can recognize a wide range of substrates. Even though this protein normally protects us from endogenous and exogenous toxins by transporting them out of the cells, the transporter causes a major problem in the bioavailability of anti-tumor drugs to tumor cells during chemotherapy.

Clinical Significance

Pgp plays an important role in altering the pharmacokinetics of a wide variety of drugs. This efflux pump creates a major physiological barrier in pharmacokinetics of drugs because of its localization at the site of drug absorption and elimination. Tumors with detectable levels of Pgp are 3-4 fold more susceptible to chemotherapeutic failure than Pgp negative tumors. Therefore the role of Pgp in the development of MDR is very significant: it has been used as a potential target for reversing clinical MDR. To overcome Pgp mediated drug resistance several inhibitors are developed and currently in clinical trials which include verapamil, cyclosporin A, quinine, and tamoxifen.

Multidrug Resistance Protein 1 (MRP1)

Like Pgp, MRP1 is also overexpressed in tumor cells and represents a major obstacle to drug delivery. MRP1 is ubiquitiously expressed in the lung, testis, kidney, and peripheral blood mononuclear cells in humans.

Clinical Significance

High levels of expression of MRP1 protein was observed in non-small-cell lung cancer. In breast cancer, there is also a significant expression of this protein which may increase the chance of treatment failure. Studies have also shown that over expression of MRP causes resistance to methotrexate (MTX) and antifoliates such as ZD1694 in colorectal cancer. Development of MRP1 inhibitors is in progress. In preclinical studies, effective inhibition of MRP1 was observed following treatment with MK571 and ethacrynicacid.

Breast Cancer Resistance Protein (BCRP)

BCRP belongs to a novel branch of the ABC-transporter family. The members of this subfamily are about half the size of the full-length ABC transporters, thus known as half-transporters. Overexpression of BCRP was reported in the plasma membrane of  drug-resistant ovary, breast, colon, gastric cancer, and fibrosarcoma cell lines. Even though the normal physiological function of BCRP has not been determined, it is possible that BCRP plays an important role in drug disposition. The overexpression of this protein causes reduced accumulation of chemotherapeutic agents such as mitoxantrone, irinotecan, SN-38, topotectan, and flavopiridol.

Clinical Significance

Since BCRP is expressed in the gastrointestinal tract, it is thought that this protein may affect the bioavailability of the drugs. Its overexpression in several types of cancer makes it a relevant target of strategies aimed at defeating multidrug-resistance. Some of the potent inhibitors of BCRP are Fumitremorgin C, reserpine and tryprostatin A.

Cancer defends itself against chemotherapeutic regimes by several mechanisms including MDR. Therefore, a detail understanding of ABC-transporters mediated drug resistance would help to formulate strategies to overcome this problem. Screening of novel inhibitors of ABC-transporters which are not effluxed by these transporters is currently in progress. One of these drugs, ixabepilone, has been approved in the United States for the treatment of breast cancer patients pretreated with an anti-tumor agent. Ongoing efforts to circumvent MDR also include development of potentially effective alternative strategies (e.g drug delivery using liposomes or nanoparticles, inhibition of expression of MDR associated ABC-transporters using monoclonal antibodies). Promising experimental data on these strategies suggest their potentialily to overcome important causes of MDR to significantly improve cancer treatment.

 

References:

1.         Türk D, Hall MD, Chu BF, et al. Identification of compounds selectively killing multidrug-resistant cancer cells. Cancer Res. 2009;69(21):8293-8301.

2.         Litman T, Druley TE, Stein WD, Bates SE. From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance. Cell Mol Life Sci. 2001;58(7):931-959.

3.         Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer. 2002;2(1):48-58.

4.         Gottesman MM, Ludwig J, Xia D, Szakács G. Defeating drug resistance in cancer. Discov Med. 2006;6(31):18-23.

5.         Nobili S, Landini I, Mazzei T, Mini E. Overcoming tumor multidrug resistance using drugs able to evade P-glycoprotein or to exploit its expression. Med Res Rev. 2012;32(6):1220-1262.

Upcoming Immunology Conferences: March – June, 2013

antibodiesI previously posted about 2013 Conferences in Tumor Immunology and Cancer ImmunotherapyThis listing will include other upcoming Immunology-related conferences.

 

Keystone Symposium: Understanding Dendritic Cell Biology to Improve Human Disease

March 3 – 8, 2013.

Keystone, Colorado, USA.

Registration Deadline: March 3, 2013.

 

Gordon Research Conference: Cell Biology of Megakaryocytes & Platelets

March 10 – 15, 2013.

Galveston, Texas, USA

Application Deadline: February 10, 2013.

 

World Immune Regulation Meeting (WIRM) VII: Innate and Adaptive Immune Response and Role of Tissues in Immune Regulation

March 13 – 16, 2013.

Congress Center, Davos, Switzerland.

Registration is still open online.

 

Keystone Symposium: Host Response in Tuberculosis

This is a joint meeting with the Keystone meeting on Tuberculosis: Understanding the Enemy

March 13 – 18, 2013.

Whistler, British Columbia, Canada.

Registration Deadline: March 3, 2013.

 

Keystone Symposium: Tuberculosis: Understanding the Enemy

This is a joint meeting with the Keystone meeting on Host Response in Tuberculosis.

March 13 – 18, 2013.

Whistler, British Columbia, Canada.

Registration Deadline: March 3, 2013.

 

Keystone Symposium: Immune Activation in HIV Infection: Basic Mechanisms and Clinical Implications

April 3 – 8, 2013.

Breckenridge, Colorado, USA.

Registration Deadline: April 3, 2013.

 

Canadian Society for Immunology 26th Annual Spring Meeting.

April 5 – 8, 2013.

TELUS Whistler Conference Centre, Whistler, British Columbia, Canada.

Registration is open online.  Early registration ends March 1, 2013.

Abstract Submission Deadline: March 1, 2013.

 

Keystone Symposium: Immunopathology of Type 1 Diabetes

April 4 – 9, 2013.

Whistler, British Columbia, Canada.

Registration Deadline: April 4, 2013.

 

Keystone Symposium: Advances in the Knowledge and Treatment of Autoimmunity

April l4 – 9, 2013.

Whistler, British Columbia, Canada.

Registration Deadline: April 4, 2013.

 

Molecular Pattern Recognition Receptors

April 11 – 13, 2013.

Boston, Massachusetts, USA.

Early Registration Deadline: March 1, 2013.

 

Clinical Immunology Society Annual Meeting: Regulation and Dysregulation of Immunity

April 25 – 28, 2013.

Miami, Florida, USA.

Pre-Registration Deadline: April 3, 2013.

 

T cell Function and Modulation Meeting

April 28 – May 1, 2013.

Makena Beach & Golf Resort, Maui (Makena), HI.

Registration can be submitted online and is limited to the first 125 attendees.

 

IMMUNOLOGY 2013, AAI Annual Meeting and Centennial Celebration

May 3 – 7, 2013.

Hawaii Convention Center, Honolulu, Hawaii, USA.

Registration is open online.  Early registration ends March 18, 2013.

Abstract Submission Deadline: February 13, 2013.

 

Keystone Symposium: The Innate Immune Response in the Pathogenesis of Infectious Disease

May 5 – 10, 2013.

A Universidade Federal de Ouro Preto, Ouro Preto, Brazil.

Early Registration Deadline: March 5, 2013.

 

Cell Symposia: Microbiome and Host Health

May 12 – 14, 2013.

Lisbon, Portugal.

Abstract Submission Deadline: February 8, 2013.

Early Registration Deadline: March 9, 2013.

 

30 Years of HIV Science: Imagine the Future

May 21 – 23, 2013.

The International Research Centre in Paris, the Institut Pasteur Conference Centre, Paris, France.

Abstract Submission Deadline: March 1, 2013.

Early Registration Deadline: March 20, 2013.

 

Abcam: Allergy & Asthma 2013

May 23 – 24, 2013.

Bruges, Belgium.

Oral Abstract Submission Deadline: February 22, 2013.

Poster Abstract Submission Deadline: March 25, 2013.

Early Registration Deadline: March 25, 2013.

 

ISIR 2013 – Building Bridges in Reproductive Immunology.

May 28 – June 1, 2013.

Boston Park Plaza Hotel, Boston, Massachusetts, USA.

Registration is open online.

Abstract Submission Deadline: February 15, 2013.

 

78th Cold Spring Harbor Symposium on Quantitative Biology: Immunity & Tolerance

May 29 – June 3, 2013.

Cold Spring Harbor Laboratory, New York, USA

Abstract and Registration Deadline: March 15, 2013.

 

6th International Singapore Symposium of Immunology.

June 5 – 6, 2013.

Matrix Level 2 Auditorium, Biopolis, Singapore.

Registration is open online.

Abstract Submission Deadline: April 5, 2013.

 

Cell Symposium: Immunometabolism: From Mechanisms to Therapy

June 9 – 11, 2013.

The Sheraton Centre Toronto Hotel, Toronto, Canada.

Abstract Submission Deadline: February 22, 2013.

Early Registration Deadline: April 5, 2013.

 

Gordon Research Conference: Mucosal Health & Disease

June 9 – 14, 2013.

Stonehill College, Easton, Massachusetts, USA.

Application Deadline: May 12, 2013.

 

Gordon Research Conference: Phagocytes

June 9 – 14, 2013.

Waterville Valley, New Hampshire, USA.

Application Deadline: May 12, 2013.

 

European Academy of Allergy & Clinical Immunology and World Allergy Organization: World Allergy & Asthma Congress

June 22 – 26, 2013.

Milan, Italy.

Early Registration Deadline: February 20, 2013.

 

Aegean Conference: 10th International Conference on Innate Immunity

June 23 – 28, 2013.

Kos, Greece.

Early Registration and Abstract Submission Deadline: March 15, 2013.

Gordon Research Conference: Apoptotic Cell Recognition & Clearance

June 23 – 28, 2013.

University of New England, Biddeford, Maine, USA.

Application Deadline: May 26, 2013.

 

Abcam: Inflammasomes in Health and Disease

June 24 – 25, 2013.

Boston, Massachusetts, USA.

Oral Abstract Submission Deadline: April 26, 2013.

Poster Abstract Submission Deadline: May 17, 2013.

Early Registration Deadline: April 26, 2013.

 

FOCIS 2013

June 27 – 30, 2013.

Boston, Massachusetts, USA.

Late-breaking Abstract Submission Deadline: April, 2, 2013.

Registration Deadline for Poster Presenters:  March 28, 2013.

Websites that list upcoming Conferences & Events in Immunology, Tumor Immunology, and Cancer Immunotherapy:

The American Association of Immunologists (AAI) Meetings and Events Calendar

Nature Reviews Immunology’s list of conferences

Cancer Immunity Journal’s List of Conferences

A novel role of TPO in the regulation of HSC DNA repair

HSC transplantation is routinely used to treat patients with malignant and non-malignant disorders of the blood and immune system, but its therapeutic application is often restricted by difficulties in in vitro maintenance and expansion of HSCs.  Studies aimed at understanding the mechanisms governing self-renewal of HSCs within hematopoietic tissues identified a set of growth factors and cytokines that can augment in vitro expansion of HSCs, including stem cell factor (SCF), fms-like tryrosine kinase 3 ligand (FLT3l) and thrombopoietin (TPO) 1.  TPO and its receptor, Mpl, are primarily known for their role in megakaryopoiesis, but TPO has also been shown to support HSC quiescence during adult hematopoiesis, with the loss of signaling associated with bone marrow failure and thrombocytopenia 2.

Recently, in Cell Stem Cell, de Lavel et al. identified a novel role of TPO in the regulation of DNA repair in HSCs 3.  Exposure to genotoxic agents, such as ionizing radiation (IR), induces DNA damage comprised of double-strand breaks (DSBs).  DNA damage is repaired through two main pathways: homologous recombination (HR) and nonhomologous end-joining (NHEJ).  DNA repair is essential for cell survival, and studies have shown that NHEJ is necessary for HSC maintenance 4,5.  In their study, de Lavel et al. found that γH2AX foci, a marker of DSB formation, were significantly increased in Mpl-deficient HSCs and in their progenitors following IR exposure.  Moreover, a TPO injection into mice prior to IR reduced the number of γH2AX foci in HSCs in vivo, while HSCs exposed to IR in the absence of TPO demonstrated an increased number of γH2AX foci.  Other experiments showed that TPO modulates the efficiency of the NHEJ pathway by increasing the phosphorylation of the DNA-PK catalytic subunit, a major enzyme involved in NHEJ.  Pharmacological or genetic inhibition of DNA-PK abrogated TPO-mediated DNA repair.  Interestingly, the other cytokines involved in HSC maintenance and expansion, SCF and FLT3l, did not have the same effects as TPO, suggesting that DNA repair activity is a specific function of TPO.

clinical trialIn short, TPO regulates NHEJ-mediated DNA repair of DSBs by stimulating DNA-PK activity in HSCs.  This is the first demonstration that a cytokine involved in HSC maintenance may also regulate DSB repair machinery.  Since TPO treatment prior to IR exposure reduces DNA damage, TPO agonists could potentially be given to patients prior to receiving chemotherapy to reduce the risk of developing oncogenic mutations and defects in HSC function.  Romiplostim, a TPO peptide mimetic, and eltrombopag, a non-peptide TPO mimetic, have been successfully used for the treatment of immune thrombocytopenic purpura (ITP) and are approved by the FDA 6.  de Lavel et al showed that injection of romiplostim prior to IR exposure also completely abolished persistent DNA damage in HSCs, similar to TPO.  Thus, these TPO agonists might also be suited for clinical applications involving protection of normal HSC from DNA-damaging agents.

 

References 

1          Ohmizono, Y. et al. Thrombopoietin augments ex vivo expansion of human cord blood-derived hematopoietic progenitors in combination with stem cell factor and flt3 ligand. Leukemia 11, 524-530 (1997).

2          Ballmaier, M., Germeshausen, M., Krukemeier, S. & Welte, K. Thrombopoietin is essential for the maintenance of normal hematopoiesis in humans: development of aplastic anemia in patients with congenital amegakaryocytic thrombocytopenia. Ann N Y Acad Sci 996, 17-25 (2003).

3          de Laval, B. et al. Thrombopoietin-Increased DNA-PK-Dependent DNA Repair Limits Hematopoietic Stem and Progenitor Cell Mutagenesis in Response to DNA Damage. Cell Stem Cell 12, 37-48, doi:10.1016/j.stem.2012.10.012 (2013).

4         Rossi, D. J. et al. Deficiencies in DNA damage repair limit the function of haematopoietic stem cells with age. Nature 447, 725-729, doi:10.1038/nature05862 (2007).

5          Nijnik, A. et al. DNA repair is limiting for haematopoietic stem cells during ageing. Nature 447, 686-690, doi:10.1038/nature05875 (2007).

6          Kuter, D. J. New thrombopoietic growth factors. Clin Lymphoma Myeloma 9 Suppl 3, S347-356, doi:10.3816/CLM.2009.s.034 (2009).

Generation of Dendritic Cells from Peripheral Monocytes

describe the imagePBMCs are not just a source of many different circulating immune cell types, but also a source of potential cells that one can generate in vitro. One excellent and long-standing example of this is the generation of dendritic cells (DCs) from monocytes.  Monocyte derived DCs (mDCs) are an excellent tool for researchers to do immunological assays requiring a source of professional antigen presenting cells (APCs). While circulating B cells are capable of antigen presentation and T cell activation, they do not offer the robust response that DCs do. The generation of mDCs is a relatively simple protocol that anyone can do with just a source of PBMCs, a few important cytokines, and, of course, some media and incubator space. After this protocol, you will have obtained immature mDCs that can then be matured for use as APCs in your assay.

The first step in generating mDCs is to decide how you would like to isolate the monocyte population from your PBMCs, which serve as your precursor cells for DCs. The easiest and cheapest way is to simply plate your PBMCs on a cell culture dish and let the inherent qualities of monocytes go to work. Monocytes are unique amongst other PBMC cells in their tendency to stick to plastic. An incubation period between 1-24 hours will allow your monocytes to adhere to the dish and let you gently wash off any other PBMCs. The alternative to the adherence method for isolating monocytes is to use a magnetic antibody based technology of your choice. Several companies, such as Miltenyi Biotec, Life Technologies, and Stem Cell Technologies, offer excellent kits for this. While the adherence method is cheaper, antibody based kits give you higher monocyte recovery and purity, which may or may not matter depending on your downstream assays.

Once you have your monocytes isolated from your PBMCs, you can begin the 7 day culture to generate mDCs. Monocytes can be plated in a standard cell culture media along with two important cytokines, GM-CSF and IL-4 (50ng/mL and 100ng/mL). GM-CSF will push the monocytes down a DC differentiation pathway. IL-4 will inhibit the monocytes from differentiating into macrophages, thereby insuring they become DCs. Continue the culture for 6-8 days and be sure to refresh your cytokines every other day.

As the monocytes differentiate over the culture period, note their progress by examining them with your tissue culture room microscope. The cells should appear as fairly round and are generally 2-3 times the size of lymphocytes. It is important to note that the mDCs will not appear like the elongated cartoon DCs with long extensions you see in text books. Those DC characteristics are generally only found in tissues and not in vitro.  While you may see some cells that resemble this, those are more likely to be somewhat of a natural stromal layer, made up of cells including macrophages, that the monocyte culture develops to support cell growth. In fact, the immature mDCs will have very few if any, cytoplasmic protrusions.

DC2 resized 600Once the culture period has finished, between 6-8 days, the mDCs can be collected. The exact day is not critical, as long as you remain consistent in the day you pick for your following experiments. To collect the mDCs, gently wash the culture dishes with several streams of media by pipetting up and down. The mDCs, which are currently immature, will be somewhat floating and only loosely adherent. Because of their loose adherence, they require several rounds of gentle pipetting, but do not require cell scraping, EDTA, or trypsin treatment. Note that the culture dishes will still contain some adherent cells. Do not worry about these cells, since these are not the loosely adherent DCs we are interested in.

After completion of these steps, you should have a nice population of immature mDCs, which express CD11c, CD1c, and are CD123-. In my next post, I will cover some tips and tricks for analyzing these cells by flow cytometry. Importantly, I will also cover ways to mature the immature mDCs for use as APCs.

Colt EgelstonColt Egelston is currently a post-doctoral fellow at the Beckman Research Institute of the City of Hope, in Duarte, CA. He received his Ph.D. from Rush University in Chicago and is interested in all things immunology.

 

 






Human PBMC T cell immediate early activation markers: What are they and what do they do?

melanoma dividing cellsThere are many strategies for assessing the function of T cells from human peripheral blood mononuclear cells (PBMC).  T cells that have recently been activated through their T cell receptor (TCR) will express a series of activation markers at different time points following activation.   Activation markers include receptors such as chemokine and cytokine receptors, adhesion molecules, co-stimulatory molecules, and MHC-class II proteins.  Some of these molecules have established functions in T cell biology, while the relevance or function of others remains elusive.  Flow cytometry is the method of choice for evaluating various types of surface or intracellular markers that indicate the activation status of T cells.  However, what are these markers, what is their function in T cell biology, what T cell populations will express them, and when can they be assessed are key questions to address when deciding which markers are best for a given assay and question of interest.

In this article, the first of a short series, I will discuss two of the most commonly used immediate early activation markers for assessing the activation status of human PBMC T cells: CD69 and CD40L.

Immediate Early Activation Markers:

CD69 (AIM, Leu23, MLR3) is a signaling membrane glycoprotein involved in inducing T cell proliferation. CD69 is expressed at very low levels on resting CD4+ or CD8+ T cells in PBMC (<5-10%), and is one of the earliest assessable activation markers, being rapidly upregulated on CD4+ or CD8+ T cells within 1 hour of TCR stimulation or other T cell activators such as phorbol esters via a protein kinase C (PKC) dependant pathway.  Expression of CD69 peaks by 16-24 hours and then declines, being barely detectable 72 hours after the stimulus has been withdrawn.

The inability to upregulate CD69 following TCR activation may be associated with T cell dysfunction.  For instance, Critchley-Thorne et. al, showed that PBMC T cells from metastatic melanoma patients with lower responsiveness to interferons had reduced CD69 upregulation compared with healthy controls, and this corresponded with multiple other functional defects in T cells from these patients.  Thus CD69 expression may be a measure of T cell dysfunction in human disease.

CD40L (CD154) is a member of the TNF-receptor superfamily that functions as a co-stimulatory molecule by binding CD40 which is constitutively expressed on antigen presenting cells (APCs).  The CD40L-CD40 ligation results in the activation of multiple downstream pathways including the MAPK (JNK, p38, ERK1/2), NF-ĸB, and STAT3 transcription factors.  CD40L expression is quickly upregulated within 1-2 hours after TCR stimulation via the transcription factors NFAT and AP-1.  CD40L expression peaks near 6 hours after stimulation, and declines by 16-24hrs. CD40L expression however is biphasic, and the addition of anti-CD28 or IL-2 along with TCR stimulation leads to sustained expression for several days (Snyder et. al., 2007).

Expression of CD40L on resting PBMC CD4+ or CD8+ T cells from healthy donors is very low (<1%).  However this percentage has been shown to be significantly increased on up to 17% of CD4+ T cells and 21% of CD8+ T cells in patients with active SLE, and these differences between healthy and SLE patients were also seen following anti-CD3 stimulation of PBMCs (Desai-Mehta, et. al, 1996).  The review below by Daoussis et. al, discusses the role of CD40L expression in several other human diseases.

In summary, CD69 and CD40L are both rapidly induced following T cell activation and both exert important functions in T cell biology. Expressions of these markers have both been shown to be altered in various human diseases.  Understanding the biology of T cell activation markers will allow for the best application of these markers to specific experimental questions and assay types.

 

Additional Reading:

Multiparametric flow cytometric analysis of the kinetics of surface molecule expression after polyclonal activation of human peripheral blood T lymphocytes. Biselli R, Matricardi PM, D’Amelio R, Fattorossi A. Scand J Immunol. 1992 Apr;35(4):439-47.

Surface markers of lymphocyte activation and markers of cell proliferation.  Shipkova M, Wieland E.  Clin Chim Acta. 2012 Sep 8;413(17-18):1338-49.

Flow cytometric analysis of activation markers on stimulated T cells and their correlation with cell proliferation.  Caruso A, Licenziati S, Corulli M, Canaris AD, De Francesco MA, Fiorentini S, Peroni L, Fallacara F, Dima F, Balsari A, Turano A.   Cytometry. 1997 Jan 1;27(1):71-6.

T cell activation via Leu-23 (CD69).  Testi R, Phillips JH, Lanier LL. J Immunol. 1989 Aug 15;143(4):1123-8.

A whole-blood assay for qualitative and semiquantitative measurements of CD69 surface expression on CD4 and CD8 T lymphocytes using flow cytometry.  Lim LC, Fiordalisi MN, Mantell JL, Schmitz JL, Folds JD. Clin Diagn Lab Immunol. 1998 May;5(3):392-8.

Utility of flow cytometric detection of CD69 expression as a rapid method for determining poly- and oligoclonal lymphocyte activation.  P E Simms and T M Ellis.  Clin Diagn Lab Immunol. 1996 May; 3(3): 301–304.

Down-regulation of the interferon signaling pathway in T lymphocytes from patients with metastatic melanoma.  Critchley-Thorne RJ, Yan N, Nacu S, Weber J, Holmes SP, Lee PP. PLoS Med. 2007 May;4(5):e176.

Direct inhibition of CD40L expression can contribute to the clinical efficacy of daclizumab independently of its effects on cell division and Th1/Th2 cytokine production.  Snyder JT, Shen J, Azmi H, Hou J, Fowler DH, Ragheb JA. Blood. 2007 Jun 15;109(12):5399-406.

Targeting CD40L: a Promising Therapeutic Approach.  D. Daoussis, A.P. Andonopoulos, and S. C. Liossis. Clin Diagn Lab Immunol. 2004 July; 11(4): 635–641.

Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production. J. Clin. Investig. 97:2063-2073. Desai-Mehta, A., L. Liangjun, R. Ramsey-Goldman, and S. Datta. 1996.

Photo credit: wellcome images / Foter.com / CC BY-NC-ND

Klotho’s Potential to Reverse MS Demyelination

Klotho is a type I transmembrane protein, expressed in the brain, kidneys and reproductive organs; it is named after “Clotho”, a goddess from Greek mythology who “spins the thread of life” (the length of the threat is determinant of how long a certain individual will live). This denomination is due to the direct positive correlation between Klotho’s expression levels and life span/anti-aging phenotypes.

Klotho protein exists in two forms, membrane Klotho and secreted Klotho, and each form is associated with distinct functions. Some of Klotho’s age-suppressing functions include regulation of fibroblast growth factor-23 (FGF23) signaling, inhibition of insulin/insulin-like growth factor (IGF-1) and Wnt signaling pathways, and regulation of multiple cell-surface ion channels and growth factor receptors.

In contrast to other organ systems, the downstream effects of Klotho have not been as extensively studied within the central nervous system (CNS). This is surprising, considering that not only Klotho is present in serum and cerebrospinal fluid (CSF), but also is highly expressed in the hippocampus, choroid plexus and neurons. Disruption of the myelin sheath, either by activated pro-inflammatory cells or by protein defection within the oligodendrocytes has been previously described in aging brain, but the underlying factor stimulating the disruption was not clear. However, In 2008, Abraham’s group reported the significance of Klotho in age-related cognitive decline (ARCD), showing reduced expression of Klotho in regions of brain white matter as a function of age.

Multiple Sclerosis,White matter,Brain,Alzheimer's,cognition

 

In January 2013, Abraham’s group reported their new findings regarding the effects of Klotho in oligodendrocyte maturation and CNS myelination, and its relation to ARCD. They showed Klotho‘s role in inducing oligodendrocytic progenitor cells (OPCs) maturation, by enhancing the expression of myelin proteins, such as myelin-associated glycoprotein (MAG), myelin basic protein (MBP), oligodendrocyte-specific protein (OSP/Claudin11), and proteolipid protein (PLP). Based on their in vivo studies, the loss of Klotho expression is correlated to defects in myelin that result in similar progressive axonal degeneration observed in hypomyelinating and demyelinating diseases, such as multiple sclerosis (MS).

Previous studies have shown Klotho’s role in facilitating removal of reactive oxygen species (ROS) and increasing resistance to oxidative stress. Furthermore, Nagai’s team observed impaired cognitive function in Klotho-deficient mice, as well as improved cognition upon treatment with the α-tocopherol antioxidant. Thus, Abraham’s group concluded that Klotho protein may function as a neuroprotective factor in the CNS through its antioxidative stress effect.

Together, these results provide strong evidence for Klotho protein as a key player, not only in myelin maintenance, but also in supporting oligodendrocyte and OPC function in the CNS; this makes Klotho an important member of the family of proteins responsible for neuron-oligodendrocyte communication. Abraham’s group hypothesized that downregulation of Klotho may be accountable for the observed white matter myelin degeneration-mediated ARCD, hence increasing Klotho protein expression can potentially prevent damage/protect myelin in the aging brain.

Brain,memory loss,Alzheimer's,MS

Abraham’s new findings are an exciting and important initial step towards development of new neuroprotective therapeutic strategies, such as induction of endogenous remyelination, for treatment of CNS diseases characterized by oligodendrocyte cell loss, such as MS and Schizophrenia. Additionally, early defects of insulin/IGF-1 receptor signaling in Alzheimer’s disease (AD), including the deficit of glucose metabolism that anticipates cognitive decline, may be partially due to deficiencies in Klotho levels. Further investigation on the precise mechanisms involved in Klotho’s regulation within the CNS seems promising for the future of neurodegenerative disease therapy.

 

Further Readings:

The Antiaging Protein Klotho Enhances Oligodendrocyte Maturation and Myelination of the CNS

Gene Profile Analysis Implicates Klotho As An Important Contributor To Aging Changes In Brain White Matter Of The Rhesus Monkey

The Putative Role of The Antiageing Protein Klotho in Cardiovascular and Renal Disease