Month: February 2013

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, DR4) 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 mitogen-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.

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 development^1,2. 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 progression^1-4. 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 metastasis^1,5. 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.

The 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-TFs^2,5. 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 phenotype^2,4, 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.

Besides 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).

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.

As 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)

Plasma 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.