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Current Pharmaceutical Biotechnology, 2012, 13, 000-000 1

1389-2010/12 $58.00+.00 © 2012 Bentham Science Publishers

The Tumor Stroma as Mediator of Drug Resistance - A Potential Target to Improve Cancer Therapy?

Susanne Sebens1,* and Heiner Schäfer2

1Institute of Experimental Medicine, Clinic of Internal Medicine I, UKSH-Campus Kiel, Kiel, Germany,

2Laboratory of

Molecular Gastroenterology & Hepatology, Clinic of Internal Medicine I, UKSH-Campus Kiel, Kiel, Germany

Abstract: Tumors irrespective of their origin are heterogenous cellular entities whose growth and progression greatly de-pend on reciprocal interactions between genetically altered (neoplastic) cells and their non-neoplastic microenvironment. Thus, microenvironmental factors promote many steps in carcinogenesis, e.g. proliferation, invasion, angiogenesis, metas-tasis and chemoresistance. Drug resistance, either intrinsic or acquired, essentially limits the efficacy of chemotherapy in many cancer patients. To some extent, this resistance is maintained by reduced drug accumulation, alterations in drug tar-gets and increased repair of drug-induced DNA damage. However, the pivotal mechanism by which tumor cells elude the cytotoxic effect of chemotherapeutic drugs is their efficient protection from induction and excecution of apoptosis. It is meanwhile well established, that cellular and non-cellular components of the tumoral microenvironment, e.g. myofibro-blasts and extracellular matrix (ECM) proteins, respectively, contribute to the ani-apoptotic protection of tumor cells. Cel-lular adhesion molecules (e.g. L1CAM or CD44), chemokines (e.g. CXCL12), integrins and other ECM receptors which are involved in direct and indirect interactions between tumor cells and their microenvironment have been identified as suitable molecular targets to overcome chemoresistance. Accordingly, several therapeutic strategies based on these targets have been already elaborated and tested in preclinical and clinical studies, including inhibitors and blocking antibodies for CD44/hyaluronan, integrins, L1CAM and CXCL12. Even though these approaches turned out to be promising, the up-coming challenge will be to prove the efficacy of these strategies in improving treatment and prognosis of cancer patients.

Keywords: Chemoresistance, microenvironment, apoptosis, extracellular matrix, desmoplasia, molecular targets.

INTRODUCTION

It is meanwhile widely accepted that cancer cells do not exist and expand as an isolated cell entity but rather in a con-tinuous and reciprocal communication with non-neoplastic cells of the adjacent microenvironment [1]. This applies for hematological malignancies such as leukemias [1-3] as well as for solid tumors, particularly carcinomas [1,4,5]. In the latter, the tumor stroma comprises a non-cellular compart-ment being composed of extracellular matrix (ECM) proteins such as laminin, collagen and vitronectin, and a cellular compartment. This includes fibroblasts, myofibroblasts, en-dothelial cells, pericytes, smooth muscle cells, adipocytes, macrophages, lymphocytes and mast cells [4,6-8]. The com-position and proportion of the tumor stroma is highly vari-able depending on the type and location of the tumor. This indicates that stroma formation depends on a complex set of interactions between the genetically altered (tumor) cells, non-malignant cells and the ECM in a particular tissue. As example, pancreatic ductal adenocarcinoma (PDAC) is char-acterized by a pronounced tumor stroma often representing the bulk of the tumor mass Fig. (1).

This excessive desmoplasia is mainly composed of the ECM proteins collagen (type-1 and type-3) and fibronectin as well as activated fibroblasts (activated stellate cells,

*Address correspondence to this author at the Institute of Experimental Medicine, Clinic of Internal Medicine I, UKHS-Campus Kiel, Arnold-Heller-Str. 3, Haus 6, 24105 Kiel, Germany; Tel: +49-431-597-3835; Fax: +49-431-597-1427; E-mail: [email protected]

myofibroblasts) and immuno-suppressive immune cells [6, 9,10].

Fig. (1). Pronounced tumor stroma in pancreatic ductal adenocarci-noma. S= stroma compartment, T= tumor cells. Scale bar = 100

m. The picture was kindly provided by B. Sipos, Institute of Pa-thology, University Tübingen, Germany.

There are many ways in which tumor cells might interact with and be influenced by its microenvironment: (i) tumor cells can directly interact with the tumoral stroma by adhe-sion to (different) stroma cells, to each other or to the ECM;

2 Current Pharmaceutical Biotechnology, 2012, Vol. 13, No. ?? Sebens and Schäfer

(ii) besides these adhesion-based mechanisms, tumor cells can also communicate with each other and with stroma cells by secretion of soluble factors in an autocrine or paracrine fashion. Owing to the complexicity of the tumor microenvi-ronment involving different malignant and non-malignant cells and a plethora of soluble factors released by and recip-rocally affecting different cells at the same time, investigat-ing the impact of tumor stroma interactions on tumor cell behaviour is challenging. For in vitro studies, different model systems have been used: tumor cells are grown (i) on layers of different ECM proteins [11], (ii) in three-dimensional spheroids alone [12] or together with different stroma cells [13], (iii) in direct coculture with stroma cells in a two-dimensional manner [14] or (iv) under indirect cocul-ture conditions using transwell inserts [15]. Each system offers advantages and disadvantages at the same time but to thoroughly investigate and to better understand the mecha-nisms by which the tumoral stroma impacts on tumorigenesis it is inevitable to focus on a certain approach and/or on a certain interaction.

Recent studies provided compelling evidence that the tumor microenvironment strongly influences the sensitivity of tumor cells towards anti-cancer treatment [15-19]. Thus, the current research in translational oncology intensively works on the identification of molecular targets by which tumor stroma interactions may be abrogated and on the de-velopment of therapeutic strategies to overcome stroma-mediated chemoresistance. Thus, the present review high-lights the impact of the tumor microenvironment on resis-tance towards drug-induced apoptosis in solid tumors. With respect to the complexicity of tumor stroma interactions in a tumor context-specific manner, the authors do not raise a claim on completeness and wish to point to excellent reviews and articles published elsewhere. However, we will outline a panel of molecular determinants and structures that play a pivotal role in stroma-mediated chemoresistance and illus-trate therapeutical approaches to overcome this stroma-mediated drug resistance in cancer therapy.

TUMOR PROGRESSION AND APOPTOSIS RESIS-TANCE

One important mechanism by which transformed cells survive and expand during tumorigenesis is by evasion from apoptosis induced by immune cells or by growth-limiting signals in the microenvironment. Thus, malignant cells can evade the attack by innate and adaptive immune cells, e.g. by interfering with the induction of an anti-tumor immune re-sponse in various ways or by exhibiting decreased immuno-genicity [5,20]. Furthermore, tumor cells are enabled to sur-vive unfavourable growth conditions such as deprivation of nutrients, oxygen and/or growth factors, contact inhibition by neighbouring cells or after loss of anchorage from the cell layer [21-23]. Since endogenous and chemotherapy-induced apoptosis share several signalling mediators, these broad anti-apoptotic defense mechanisms primarily promote the survival and expansion of malignant cells resulting in tumor formation and metastasis, but later on these mechanisms also account for drug resistance. On the one hand, tumor cells acquire an apoptosis resistant phenotype by accumulation of genetic alterations targeting genes involved in cell cycle con-trol, proliferation and mainly apoptosis. On the other hand,

tumor cells gain anti-apoptotic protection from exposure to environmental factors released by the tumor microenviron-ment that likewise affect signalling pathways involved in survival and apoptosis.

As reviewed in more detail elsewhere [25,29-31], apop-tosis is a tightly controlled cellular cell death program that can be induced in several ways and that represents a central regulator of normal tissue homeostasis. Under physiological conditions, apoptosis eliminates redundant, damaged and infected cells thereby contributing to the development and integrity of tissues and whole organisms, but several che-motherapeutic drugs in cancer therapy also exert their effects by apoptosis induction. Apoptosis is characterized by a mul-titude of morphological and molecular alterations, e.g. mem-brane blebbing, cell shrinkage, nuclear fragmentation, chro-matin condensation and DNA fragmentation [24]. Induction of apoptosis occurs via two alternative routes depending on the mode of stimulation: (i) the extrinsic or death-receptor pathway and (ii) the intrinsic or mitochondrial pathway. However, the activated signalling cascades of both pathways merge at a common point, namely the activation of caspases which are central executors of apoptotic cell death [25-27].

In brief, the extrinsic pathway is induced by binding of death ligands (e.g. TNF- , TRAIL or CD95L/Fas) to their respective receptors leading to activation of the death-inducing signalling complex (DISC). The Fas-associated death domain (FADD) protein which is part of the DISC recruits and activates the initiator caspase-8 [25,28]. In con-trast, the intrinsic pathway is initiated by cellular stress, e.g. by DNA damage after radio- or chemotherapy. In this case, the homeostatic balance between dimerizing pro- and anti-apoptotic members of the Bcl-2 protein family is shifted to-wards those proteins favoring apoptosis. These lead to the permeabilization of the mitochondrial membrane followed by the release of cytochrome c into the cytosol where it binds to APAF-1 and caspase-9, thereby forming the so called apoptosome [25,29-31]. Both, caspase-8 and caspase-9 acti-vated by either of the above mentioned pathways lead to the activation of caspase-3 and -7 that mediate the execution of the cell death program [32]. Physiologically, this process can be blocked at various steps in the apoptotic signalling cas-cade, e.g. the FLICE inhibitory protein (c-FLIP) is able to prevent the interaction of FADD and caspase-8, thereby blocking apoptosis initiation at a very early point [33]. In addition, execution of the apoptotic program and conse-quently cell death can be prevented at a late stage of the sig-nalling cascade by the inhibitor of apoptosis (IAP) proteins which abolish the activity of caspase-9 and -3 [34]. In the following paragraphs we will outline several examples of how the tumor microenvironment promotes the acquisition of an apoptosis-resistant phenotype in tumor cells and thereby contributes to drug resistance.

THE TUMOR STROMA AND APOPTOSIS RESIS-

TANCE TOWARDS CHEMOTHERAPY

Apoptosis resistance mediated by cell adhesion to various substrates is an emerging concept that may explain the ob-served phenotypic differences between cells grown within a three-dimensional context of a tumor and cells grown in standard monolayer culture conditions in vitro. This mecha-

Tumor Stroma and Drug Resistance Current Pharmaceutical Biotechnology, 2012, Vol. 13, No. ?? 3

nism is referred to as adhesion-mediated apoptosis resistance [17,35]. In general, tumor cells can directly interact with and adhere to the adjacent stroma by three different modes: (i) by adhesion to each other, (ii) by adhesion to stroma cells or (iii) by adhesion to ECM proteins. The latter form the non-cellular compartment of the tumor microenvironment and are produced by both the tumor cells and a variety of stromal cells, particularly myofibroblasts. The role of the ECM within a tumor is not limited to being a barrier against tumor invasion. In fact, it is a reservoir of cell binding proteins and growth factors that essentially affects tumor progression [1,36]. The ECM can be modified by proteases produced by tumor cells as well as stroma cells [1,37,38]. As a result of the activity of these proteases, cell-cell and cell-ECM inter-actions are altered and the bioavailability and activity of many growth factors, growth factor receptors and cytokines are modified.

Several studies provided experimental evidence that the signalling pathways involving MAPK (mitogen activated protein kinase)/ERK (extracellular regulated kinase) kinase (MEK) or the phosphatidylinositol-3-kinase (PI3-kinase) are frequently activated upon cell-matrix interaction and are hence of particular importance for adhesion-mediated sur-vival and apoptosis resistance. Activation of the Raf/MEK/ERK pathway is a highly conserved signalling cascade from the membrane into the nucleus affecting vari-ous cellular functions such as growth factor-induced gene regulation, cell cycle control and differentiation [39]. Clus-tering of integrins or binding of growth factors to their recep-tor initiates a signalling cascade leading to Src-mediated phosphorylation of the focal adhesion kinase (FAK) and its subsequent binding to adapter molecules such as Shc and Grb2 [40,41]. These interact with small GTPases leading to their activation. Thus, the GTPase Ras transmits the signal onto Raf which in turn phosphorylates MAP kinase kinases or MEKs. Finally, these kinases phosphorylate and thereby activate MAP kinases or ERK which then translocate into the nucleus. The precise regulation of this signalling cascade can affect the sensitivity towards apoptotic stimuli either indi-rectly, e.g. by activating cell cycle progression, [42] or di-rectly, e.g. by promoting cell survival via modifying the ex-pression level of Bcl-2 family members. Thus, it could be shown that the phosphorylation and subsequent degradation of the pro-apoptotic protein Bim (Bcl-2 interacting mediator of cell death) can be induced by ERK directly as well as in-directly [43,44]. However, it has been also demonstrated that ERK inhibition results in downregulation of several anti-apoptotic proteins such as Bcl-2 or Bcl-xl [45] indicating that ERK activation is able to inhibit the expression of pro-survival factors. The second pathway which is often acti-vated upon cell-matrix interaction is the PI3-kinase/Akt pathway. Upon ligand binding of integrin receptors, PI3-kinase binds to FAK which phosphorylates membrane bound lipids [46]. Consequently, Akt is recruited to the membrane leading to the activation of different survival promoting events, e.g. the inactivation of pro-apoptotic Bcl-2 family members BAD (BCL2-antagonist of cell death), Bim, Puma or Noxa [47,48]. In addition, activation of the PI3-kinase can result in inhibition of caspase-9 or enhanced DNA repair [48,49].

It is important to note that survival and protection from apoptosis can also be induced independently of ERK and PI3-kinase activity. Thus, it has been shown that in certain PDAC cell lines the resistance towards gemcitabine-induced apoptosis does not depend on PI3-kinase activity but rather on constitutive activation of the transcription factor Nuclear factor-kappa B (NF- B) [50]. NF- B has been shown to be an important mediator of tumor development and progres-sion providing a mechanistic link between inflammation and carcinogenesis [51]. Thus, constitutive activation of NF- B is found in a variety of cancers accounting for enhanced re-sistance towards different kind of apoptosis-inducing stimuli such as chemotherapeutic drugs [52,53].

Decreased apoptosis sensitivity and consequently en-hanced drug resistance can also be caused by adhesion of the tumor cells to the ECM [17,35]. The main receptors mediat-ing cell-matrix but also cell-cell adhesion are the members of the integrin family. These integrin receptors are formed by different combinations of and subunits, each exhibiting particular binding specificities and signalling properties. By binding to the ECM or to their appropriate ligand on adjacent cells, integrins regulate cytoskeleton organization and con-trol adhesion and migration as well as cell survival, prolif-eration and apoptosis [54- 57].

STROMA-MEDIATED DRUG RESISTANCE IN

SMALL CELL LUNG CANCER

Adhesion to the ECM protein laminin can lead to drug resistance of small cell lung cancer (SCLC) cells [58]. In addition, Sethi and collegues have shown that adhesion to laminin, fibronectin or collagen IV also protects SCLC cells from apoptosis induction by a variety of chemotherapeutic agents such as doxorubicin, cyclophosphamide and etoposide [11]. Treatment of adherent SCLC cells with the protein ty-rosine kinase (PTK) inhibitor tyrphostine-25 was able to block the protective effects through fibronectin binding. It was shown that this broad drug resistance in SCLC cells is induced by 1-integrin-mediated adhesion to the ECM lead-ing to the activation of the PTK which subsequently blocks caspase activation [11,59]. Moreover, adhesion-mediated resistance towards etoposide-induced apoptosis cannot only abolished by blocking of 1-integrin but also by pharmacol-ogical and genetic inhibition of the PI3-kinase. Binding of

1-integrin to laminin apparently induces PI3-kinase activa-tion thereby leading to phosphorylation of PKB and GSK-3 . This results in the maintenance of cyclin D, E, A and B, the increased expression of phosphorylated CDK2 and the downregulation of CDK inhibitors p21 and p27 which can-not be reversed by the presence of etoposide [60]. Impor-tantly, this protection from drug-induced apoptosis occurs despite etoposide-induced inhibition of the topoisomerase II and persistant DNA damage [60].


BREAST AND OVARIAN CARCINOMA

Integrin-mediated cell adhesion is also important for sur-vival, apoptosis resistance and malignant transformation in mammary epithelial cells [61]. In adherent mammary epithe-lial cells, the pro-apoptotic protein Bax is maintained in the cytosol, whereas it exists in a conformationally altered form


in cells growing in suspension. This conformational change facilitates the reversible translocation of Bax from the cyto-sol to the mitochondria mediating anoikis [62]. These data indicate that cell adhesion mediates cell survival and apopto-sis protection by maintaining pro-apoptotic molecules such as Bax in a rather non-apoptotic state thereby shifting the balance towards apoptosis resistance. In addition, integrin-mediated binding to ECM proteins seems to play an impor-tant role in drug resistance of mammary carcinoma cells. Thus, an adriamycin-resistant but not the sensitive variant of the mammary carcinoma cell line MCF-7 expresses 4 1- and 5 1-integrins, the latter mediating binding to fi-bronectin [63]. Kerbel et al. could demonstrate that culture of the murine mammary carcinoma cell line EMT-6 in three-dimensional multicellular spheroids confers chemoresis-tance. Reversal of this intrinsic drug resistance could be achieved by cell disaggregation upon treatment with hyaluronidase which increases cell cycle entry of the cells along with a sensitization to cell cycle-dependent drugs such as cyclophosphamide [12]. Reduced proliferation of the cells growing in the spheroids seemed to be mediated by a cell contact-dependent upregulation of the cyclin-dependent kinase inhibitor p27KipI [12]. Overall, these data suggest that ECM-integrin-mediated signalling represents one impor-tant mechanism of stroma-mediated drug resistance and might therefore serve as a predictive marker. Helleman et al. recently performed a pathway analysis on seven published gene-sets that have been associated with resistance to a plati-num-based chemotherapy in ovarian carcinoma. Their analy-sis revealed ECM gene clusters (e.g. fibronectin 1, collagen type5 alpha1) to be highly related to chemotherapy resis-tance in ovarian cancer as well as endocrine resistance of breast cancer and pointed to a key role of transforming growth factor-beta (TGF- ) in regulating ECM gene expres-sion [64]. Ahmed et al demonstrated that loss of the ECM protein TGFBI (transforming growth factor beta induced) whose secretion is induced by TGF- 1 and whose functions include cell adhesion to the ECM and integrin-mediated sig-nalling accounts for resistance towards paclitaxel in ovarian cancer [65]. Paclitaxel belongs to the taxanes and exerts its cytostatic effect by microtubule stabilization leading to mi-totic arrest and apoptosis. They showed that various ovarian and breast cancer cell lines are characterized by low TGFBI expression and transfection of paclitaxel resistant cells with recombinant TGFBI restored sensitivity towards paclitaxel via FAK- and Rho-dependent stabilization of microtubules. Vice versa, knock down of this protein in TGFBI expressing ovarian cancer cell lines resulted in a significant resistance to paclitaxel-induced caspase activation. Supporting these data, a microarray analysis with tumor samples from patients sub-ject to pre-treatment revealed that those patients exhibiting no therapeutical response to paclitaxel had a significantly lower TGFBI expression than therapy responders [65].

STROMA-MEDIATED DRUG RESISTANCE IN PDAC

PDAC is a tumor characterized by a profound desmo-plastic reaction involving a dense ECM network together with myofibroblasts and tumor-associated macrophages as the predominant cellular component [6,9,10,66]. Miyamoto et al. showed that chemoresistance and proliferation of three PDAC cell lines harbouring different grades of malignancy

are highly dependent on adhesion to ECM proteins [67]. In their experimental approach, MIA PaCa-2 cells (grade 3) responded less towards treatment with cisplatin or doxorubi-cin when adhered to fibronectin, laminin, collagen type I or collagen type IV as well as to treatment with 5-fluoruracil when grown on laminin or collagen type IV. Since an in-creased expression of various integrin subunits could be de-tected (albeit only by RT-PCR analysis) it is likely that adhe-sion-mediated chemoresistance in these cells involves bind-ing of the respective integrin receptors to the ECM protein. Interestingly, adhesion to ECM proteins did not affect the response of either cell line towards treatment with gemcit-abine [67]. In contrast, Huanwen et al. could recently show that adhesion of the PDAC cell line AsPC-1 to laminin in-duced phosphorylation of FAK and Akt in a time-dependent manner resulting in elevated levels of the anti-apoptotic pro-tein survivin and the phosphorylated form of Bad along with a decreased apoptotic response following gemcitabine treat-ment [68]. Besides in PDAC [68,69], elevated expression or activation of FAK has been reported in several other human cancers such as colorectal [70], esophageal, liver or breast cancer [71] and its repression by antisense oligonucleotides or siRNA enhanced sensitivity to different kinds of che-motherapeutic drugs [68,72,73]. Altogether, these findings show that FAK is activated by cell adhesion to the ECM and subsequently functions as a critical intracellular mediator in ECM-integrin-mediated drug resistance thus underscoring the important role of the ECM in stroma-mediated chemore-sistance of tumor cells.

Work by our own group identified myofibroblasts as es-sential modulator of apoptosis resistance in PDAC [15,74,75]. We could demonstrate that culture in the pres-ence of pancreatic myofibroblasts increased the resistance of the human PDAC cell lines T3M4 or PT45-P1 towards etoposide-induced apoptosis [15]. Since an indirect coculture system involving murine myofibroblasts and human tumor cells was used it could be concluded that chemoresistance was mediated by soluble factors and since tumor and stroma cells from different species were used we were able to dis-criminate the cellular origin from which these factors were released. Thus, secretion of IL-1 in T3M4 and PT45-P1 cells was increased by nitric oxide (NO) released by the myofibroblasts. While both tumor cell lines secreted only little NO which was in line with undetectable inducible nitric oxide synthase (iNOS) expression, myofibroblasts exhibited significant iNOS expression and NO secretion that could be further induced by the tumor cells. Finally, blockade of ei-ther the IL-1 receptor or iNOS reversed the stroma-induced chemoresistant phenotype of both cell lines pointing to an important role of these factors [15]. These findings are in line with previous observations that have identified IL- 1 as an essential inducer of NF- B thereby leading to profound chemoresistance in PDAC cells in vitro and in vivo [76,77]. Moreover, long-term coculture with murine and human pan-creatic myofibroblasts, respectively, resulted not only in a chemoresistant phenotype of PDAC cells but also in the non-tumorigenic pancreatic ductal epithelial cell line H6c7 [75,76]. This involved a reduced expression of pro-caspases and their inducing transcription factor STAT1, both caused by diminished gene transcription. The DNA-methylation inhibitor 5-Azadeoxycytidine enhanced caspase and STAT1


expression in cocultured H6c7 and T3M4 cells along with a loss of chemoresistance, indicating a role for CpG DNA-hypermethylation in the downregulation of these crucial apoptosis mediators. Moreover, cocultured H6c7 and T3M4 cells exhibited elevated nuclear levels of DNA-methyltransferase-1 (DNMT1) and silencing of DNMT1 expression by siRNA increased expression of pro-caspases and STAT1, thereby restoring responsiveness to anti-cancer drugs. These data were supported by the finding that tumors arising from coinoculated T3M4 cells and myofibroblasts (co-tumors) in SCID mice, responded less towards chemo-therapy than mono-tumors. Accordingly, co-tumors exhib-ited decreased apoptosis, no remission, and reduced expres-sion of pro-caspases and STAT1 [74]. Thus, stroma-mediated chemoresistance apparently results from intense interactions between epithelial/tumor cells and stromal myo-fibroblasts involving early activation of NF- B by IL-1 and NO [15] as well as epigenetically mediated gene silencing of STAT1 and pro-caspases. Both mechanisms might operate consecutively or in parallel. The observation that stimulation of T3M4 and H6c7 cells with the NO donor SNAP leads to an increased nuclear DNMT1 expression (unpublished ob-servations) indicates that the former mechanism might pave the way for epigenetic alterations, thereby cooperatively me-diating intrinsic chemoresistance.

The adhesion molecule L1CAM (L1, CD171) has been detected in various tumor entities such as colon carcinoma, ovarian carcinoma and glioma often being associated with short survival times and poor prognosis for the patients [78- 81]. PDAC is characterized by an elevated L1CAM expres-sion, too [77,82], but it was found that L1CAM expression is already present in ductal structures surrounded by dense fi-brotic tissue during chronic pancreatitis [75]. Supporting the idea that stromal cells present in chronic pancreatitis as well as in PDAC contribute to L1CAM induction in pancreatic epithelial cells and later on in PDAC cells, TGF- 1 depend-ent upregulation of L1CAM expression was observed in H6c7 cells when cultured in the presence of pancreatic myo-fibroblasts [75]. Moreover, elevated L1CAM expression confers resistance towards TRAIL-induced apoptosis (un-published observations) as well as towards apoptosis induc-tion by cytostatic drugs such as etoposide or gemcitabine [75,83]. Since L1CAM can undergo homophilic binding (L1CAM-L1CAM) as well as heterophilic binding to a vari-ety of ligands such as integrins (e.g. v 3, v 1, 5 1), ECM proteins or neuropilin-1 (NRP-1) [84,85] ligation of L1CAM with either of these ligands might also contribute to drug resistance in L1CAM expressing tumors. Accordingly, the L1CAM ligand 5-integrin was shown to be pivotal for L1CAM-mediated chemoresistance in different PDAC cell lines [86]. Moreover, NRP-1 which is overexpressed in PDAC has been reported to promote constitutive activation of MAPK signalling and chemoresistance [87] and one can speculate that this effect of NRP-1 involves ligation to L1CAM.


GLIOBLASTOMA AND COLON CANCER

Glioblastoma is another highly malignant stroma-enriched tumor exhibiting profound resistance towards che-motherapy. Due to the fact that over 90% of tumor recur-

rences in glioblastoma patients occur at the tumor periphery, the chemoresistant phenotype may be a distinct property of tumor cells residing at the margin of the tumor [88]. These findings indicate that certain environmental conditions in this tumor area may foster survival and therapy resistance of tu-mor cells. Supporting this idea, Gladson et al. demonstrated that the glioma-derived ECM protein vitronectin is predomi-nantly expressed in the tumor periphery [89] and that glioma cells express v 3- and v 5-integrins being the cognate receptors for adhesion to vitronectin [89]. Adhesion of the glioma cell lines D54 and U251 to vitronectin conferred re-sistance towards topotecan-induced apoptosis and antibody-mediated blocking of either integrin receptor was able to resensitize these cells towards drug treatment [90]. Along with drug resistance, adhesion to vitronectin led to an in-creased expression of the anti-apoptotic proteins Bcl-2 and Bcl-xl and accordingly to increased ratios of Bcl-2:Bax and Bcl-xl:Bax. An explanation of how binding of vitronectin to its receptor may alter the expression level of Bcl-2 family members came from studies with endothelial cells. In these cells, ligation of v 3-integrin leads to reduced levels of p53 which commonly decreases expression of Bcl-2 and in-creases Bax expression [91,92]. Accordingly, a diminished p53 expression mediated by ligation of the integrin receptor enhances the Bcl-2:Bax ratio favoring cell survival [91]. However, the two glioma cell lines used in the study men-tioned above [90] differed regarding their p53 status indicat-ing that upregulation of Bcl-2/Bcl-xl and chemoresistance upon adhesion to vitronectin are independent of p53. Studies by Westhoff et al. provide experimental evidence that glioblastoma cells are able to alter their mode of adhesion-mediated resistance towards apoptosis inducing stimuli de-pending on whether ECM is provided or not [93]. Thus, these cells can change between a form of drug resistance mediated by cell-substrate binding and another being de-pendent on cell-cell-adhesion. Importantly, inhibition of both forms is required for the sensitization towards apoptosis in-duction [93] indicating that both cell-substrate and cell-cell interactions have to be blocked to achieve chemosensitiza-tion in patients.

Finally, adhesion to stromal-derived ECM proteins also confers protection from etoposide- and camptothecin-induced apoptosis in colon cancer cells although in these cells the involvement of bcl-2 and bcl-xl was excluded be-cause of the missing correlation between the protective ECM effect and expression levels of these anti-apoptotic proteins [94].

As outlined above, chemoresistance can be also induced by soluble factors released by stromal cells, e.g. myofibro-blasts [15,74,75]. However, it should be mentioned that mu-tation of p53 in stromal fibroblasts, a phenomenon found e.g. in human breast and prostate cancer [95-98] can result in an elevated release of growth inhibitory factors which lead to apoptotis induction even of multi-drug resistant tumor cells [99]. Moreover, Lafkas et al. could demonstrate in vivo that p53 mutated fibroblasts are able to sensitize breast and pros-tate cancer cells to chemotherapy with doxorubicin and cis-platinum [100]. Thus, these findings suggest that loss of stromal p53 function may rather promote sensitization of certain types of tumor cells towards different chemothera-pies.


Nevertheless, the majority of present data indicates that stroma-mediated drug resistance essentially depends on cell-cell and cell-matrix adhesion, even though it can also rely on autocrine/paracrine effects of a variety of soluble factors Fig. (2). The latter might confer apoptosis resistance in an adhe-sion-dependent manner, e.g. by altering the expression of cell adhesion molecules, or in an adhesion-independent fash-ion. However, different mechanisms most likely contribute at the same time to stroma-mediated drug resistance in vivo Fig. (2).

ECM receptors: e.g. integrins

adhesion molecules: e.g. CD44, L1CAM

extracellular matrix (ECM): e.g. laminin, fibronectin, collagen, HA

chemokines (e.g. CXCL12), growth factors (e.g. TGF- 1),

d)

ECM

c)

b)

a)

e)tumor/stem-like cell

myofibroblast

myofibroblast

direct

indirect

tumor/stem-like cell

Fig. (2). Simplified illustration of tumor stroma interactions con-tributing to drug resistance in tumor cells highlighted in this review. Stroma cells (e.g. myofibroblasts, MFs) and tumor/stem-like cancer cells can interact indirectly by production and secretion of a) ECM proteins (e.g. hyaluronan, HA) or b) auto-/juxtacrine acting chemokines (e.g. CXCL12), cytokines (e.g. IL-1 ), growth factors (e.g. TGF- 1) or NO. In addition, direct interactions between tumor and stroma may also contribute to stroma-mediated drug resistance by adhesion of the tumor/stem-like cancer cells to c) the ECM (e.g.

4 1-integrin/fibronectin), d) adjacent tumor cells (e.g. L1CAM/L1CAM, L1CAM/NRP-1, L1CAM/ 5-integrin) or e) stromal cells (e.g. L1CAM/integrin).

THE TUMOR STROMA AND DRUG RESISTANCE IN

CANCER STEM CELLS

In recent years, it has been postulated that only a certain population of cancer cells, referred to as cancer stem cells, cancer stem-like cells or tumor-initiating cells, are able to give rise to a tumor. This model implies that tumors similar to normal adult tissues contain a subset of cells - represent-ing the minority of the tumor - that have the exclusive ability of self-renewal and differentiation [101,102]. Like normal stem cells, cancer stem cells are highly dependent on their microenvironment. Factors provided by this “niche” enable these cells to maintain their stem cell properties [101,102]. Moreover, Prince et al. detected cancer stem cells physically adjacent to the stromal compartment in tissues of head and neck squamous carcinoma supporting the idea that cancer stem cells require a close interaction with the tumor micro-environment [103].

These stem-like cancer cells possess a highly malignant phenoptype [104,105,106] in that they exhibit great tumori-genicity in an animal host and are highly metastatic. Moreo-ver, these cells are characterized by anti-apoptotic protection and profound chemoresistance [107]. However, the mecha-nisms by which the protection from any kind of apoptotic stimuli is conferred in these cells are still poorly understood. One hallmark of cancer stem cells is the expression of the hyaluronan receptor CD44 [104]. Both CD44 and hyaluronan have been shown to play a role in chemoresis-tance [108] and cell survival [109], as well as in a malignant phenotype. Increased hyaluronan levels, e.g. produced by myofibroblasts [110] were found to stimulate drug resistance in drug-sensitive cancer cells, whereas disruption of endoge-nous hyaluronan-induced signalling suppresses cellular resis-tance to several drugs including doxorubicin, taxol, vincris-tine and methotrexate [111]. Various reports demonstrate that hyaluronan and CD44 promote drug resistance in a vari-ety of cancer cell types, including breast, lung, pancreas and head and neck carcinomas [108,112-115]. Accordingly, cleavage of hyaluronan by hyaluronidase treatment has been reported to enhance the action of various chemotherapeutic drugs [116]. This and other observations underscore the in-volvement of hyaluronan-CD44 interactions in the malignant and chemotherapy resistant phenotype of cancer cells, and possibly cancer stem cells. Although the anti-apoptotic effect of hyaluronan is likely to contribute to these phenomena, hyaluronan-CD44 interactions also regulate expression of drug transporters, including P-glycoprotein [108], MRP2 [108,113] and BCRP [117].

The chemokine receptor CXCR4 and its ligand CXCL12 (also designated as stromal cell-derived factor-1 (SDF-1)) are pivotal for homing and maintainance of hematopoetic stem cells. Thus, CXCL12 released by reticular stromal cells in the bone marrow attracts CXCR4 expressing hematopoetic stem cells in distinct vascular and endosteal niches where they further develop, differentiate and proliferate [118,119]. Besides its role in the hematopoetic system, the CXCR4/CXCL12 axis seems also to contribute to tumor progression by promoting metastatic spread, angiogenesis, growth and survival of tumor cells [119]. Moreover, CXCR4 has been postulated to be a marker of chemoresistant, stem-like cancer cells [120,121].

While CXCR4 has been detected in a variety of cancer cells, e.g. breast cancer, SCLC or glioma cells, CXCL12 can be released in high amounts by stromal fibroblasts under-scoring the role of this receptor-ligand-system in the cros-stalk between tumor and stroma cells during tumor progres-sion [119]. In addition, chemoresistance of various tumor cells, e.g. of SCLC [122] or glioblastoma [121] seemed to be dependent on CXCR4 and CXCL12. Thus, adhesion of SCLC cells to ECM proteins or stromal cells confers a chemoresistant phenotype. CXCL12 induced activation of

2-, 4-, 5- and 1-integrins and CXCR4 which resulted in increased cell adhesion. Usage of a CXCR4 antagonist re-duced cell adhesion and restored chemosensitivity [122]. These findings indicate that CXCR4 cooperates with in-tegrins to mediate drug resistance in tumor cells and substan-tiate the role of both, soluble factors and adhesion-dependent mechanisms in this process. Interestingly, the adhesion molecule L1CAM that has been shown to mediate chemore-


sistance in PDAC [75,83,86] and ovarian cancer cells [113] is regarded as a stem cell marker in glioma [124]. Genetic inhibition of L1CAM expression in L1CAM+CD133+ glioma cells prior to injection into SCID mice suppressed tumor outgrowth and prolonged survival of tumor-bearing animals [124] suggesting L1CAM as an appropriate target to overcome chemoresistance and to eliminate cancer stem cells. Table 1 summarizes the discussed factors and their mode of action in mediating drug resistance in different tu-mor entities. In the next paragraph potential therapeutic strategies to interfere with stroma-mediated drug resistance will be discussed.

STRATEGIES TO OVERCOME STROMA-MEDIA-

TED DRUG RESISTANCE

Chemoresistance of tumor cells can be mediated in dif-ferent ways of which protection from apoptosis induction probably represents the pivotal mechanism. Accordingly, other strategies beyond those directly converting the chemoresistant phenotype of tumor cells might also increase the efficacy of chemotherapy and patient´s survival. For ex-ample, inhibition of Hedgehog signalling in a mouse model of poorly vascularized PDAC depleted tumoral desmoplasia and increased vascular density which led to an improved delivery and efficacy of cytostatic drugs into the tumor along with disease stabilization [125]. One strategy to interrupt the stroma-induced signalling and the resultant apoptosis resis-tance is the inhibition of downstream targets. Thus, targeting of various types of kinases (e.g. PI-3K), transcription factors (e.g. NF- B) or of pro- or anti-apoptotic molecules (e.g. members of the Bcl-2 family) has been evaluated for the sensitization of tumor cells in order to improve anti-cancer therapy. Regarding these anti-cancer strategies, we would like to refer to the numerous excellent reviews highlighting them in more detail and greater broadness [32,52,53]. In the

present chapter, we will rather focus on a selected panel of therapeutic strategies directly interfering with the tumor stroma interplay Fig. (3)

As outlined above, numerous cellular functions induced by cell-cell or cell-matrix interactions are under control of members of the integrin family which essentially contribute to the initiation, progression and metastasis of solid tumours. Given the importance of integrins in several cell types that affect tumour progression, integrins are an appealing target for cancer therapy.

The v 3-integrin which plays an important role in in-tracellular signalling regulating cell proliferation, migration, and differentiation is involved in tumor-induced angiogene-sis, making v 3-integrin an interesting target in cancer therapy. Early evidence suggested clinical benefit in disease stabilization with the use of anti- v 3-antibodies in the set-tings of colorectal cancer, renal cell carcinoma and mela-noma. Thus, etaracizumab (Abegrin), an IgG1 humanized monoclonal antibody against v 3-integrin has gained ac-cess to numerous phase-I and -II studies with patients suffer-ing from advanced solid tumors and melanoma [126-129]. Likewise, the v 3- and v 5-inhibitor cilengitide [130] has shown encouraging activity in phase-II clinical trials in pa-tients with gliomas and prostate cancer [131,132] and this agent is currently being tested in a phase-III trial in patients with glioblastoma [133].

CNTO 95 is a fully human anti- v-integrin monoclonal antibody and has shown anti-tumor activity when used as a single agent in preclinical studies. CNTO 95 can potentiate the efficacy of fractionated radiation therapy in a variety of human cancer xenograft tumor types in nude mice thus indi-cating its relevance for the treatment of patients with solid tumors [134,135]. Other reports demonstrated the efficacy of the 5 1-integrin antagonist, ATN-161 in solid tumors

Table 1. Factors Induced by Tumor Stroma Interactions and their Mode of Action in Mediating Drug Resistance in Various Tu-

mor Entities

Molecular Factor Mechanism of Action Tumor Entity

CD44/Hyaluronan drug transporter expression (P-glycoprotein, MRP2, BCRP)

growth factor receptor interaction (ERBB2, EGFR, IGF-1R, PDGFR, c-

MET)

breast, lung, pancreas,

head & neck carcinoma

L1CAM ECM, neuropilin-1 (NRP1) and 5i interactions

PI3K/Akt, MAPK activation

>> caspase activity

colon, endometrium, ovary & pancreas

carcinoma, glioma

1-i / laminin

4 1-i , 5 1-i / fibronectin

PI3K/Akt, GSK-3 activation

>> Cyclin D, E ,B , A and CDK2 expression

>> p21, p27 expression

>> p21, p27 expression

NSCLC

Breast

TGFBI / ECM FAK- and Rho-activation >> caspase activation breast & ovarian

ECM adhesion / fibronectin,

laminin, collagen type I or IV

FAK and Akt activation

>> survivin expression

>> Bad phosphorylation

pancreas carcinoma

CXCL12 / CCR4 2-, 4-, 5- and 1-i activation breast, lung (SCLC) carcinoma glioma


[136,137]. For example, ATN-161 was applied in combina-tion with 5-fluorouracil infusion yielding reduced liver me-tastases formation and improved survival in a colon cancer model [137]. However, the presence of an U-shaped dose-response curve yet presents a significant challenge to identi-fying a biologically active dose of ATN-161 [138].

d)

ECM

c)

b)

a)

e)tumor/stem-like cell

myofibroblast

myofibroblast

direct

indirect

tumor/stem-like cell

anti-IL-1b, IL1RA

CXCR4 antagonists

hyaluronidase

integrin inhibitors

anti-L1CAM, CD44 antagonists

integrin inhibitors

Fig. (3). Therapeutic strategies to overcome stroma-mediated drug resistance outlined in this review.

Other developments include the novel oral anti-cancer agent E7820 - an aromatic sulfonamide derivative - which blocks the 2-integrin subunit [139,140] or the chimeric monoclonal antibody volociximab [141] that specifically binds to 5 1-integrin. Owing to the absence of severe tox-icities and preliminary activity at the highest dose level [142], usage of volociximab in further disease-directed stud-ies is appealing. As can be appreciated from the exciting clinical developments with integrin inhibitors and blocking antibodies, a detailed understanding of how integrin inhibi-tion affects the tumor and its microenvironment is greatly needed.

Another strategy that has revealed promising results in vitro and in vivo is the inhibition of the adhesion molecule L1CAM. As outlined above, this molecule mediates chemoresistance of PDAC [75,83,86] and ovarian cancer cells [123] and inhibition of L1CAM either by siRNA-mediated knock down or by using blocking antibodies sensi-tized tumor cells towards apoptosis induction by different kinds of chemotherapy [75,83,86,123]. Moreover, treatment of tumor-bearing mice with L1CAM blocking antibodies markedly reduced tumor growth and dissemination of tumor cells [143]. Owing to the putative role of L1CAM as cancer stem cell marker [124] one can speculate that targeting L1CAM will lead to the elimination of tumor- initiating cells. In fact, identification of targets by which cancer stem cells can be eradicated is supposed to be the most effective strategy to fight against even therapy resistant tumors.

Based on the considerable experimental evidence on the role of hyaluronan and CD44 in cancer stem cells and tu-morigenesis using animal models, approaches have been employed for the perturbation of endogenous hyaluronan-protein interactions. For example, the soluble ectodomain of CD44 competitively displaces hyaluronan from its endoge-nous cell surface receptor, and overexpression of the CD44 ectodomain in mouse mammary carcinoma cells or in human

malignant melanoma cells has been shown to inhibit growth, local invasion and metastasis in animal models [144-147]. Most likely, these effects arise from the induction of apopto-sis [145] and cell cycle arrest [146] in vivo. Likewise, ad-ministration of antibodies that block hyaluronan binding to CD44 inhibits tumor growth and invasion [147,148]. A solu-ble form of Rhamm, another hyaluronan receptor, induces cell cycle arrest and inhibits metastasis [149] and a soluble hyaluronan-binding complex derived from cartilage inhibits both tumor growth and metastasis [150]. In addition, treat-ment with small hyaluronan oligosaccharides (oligomers) retards growth of several tumor types in vivo [117,151,152].

Aberrant activities of growth factor receptors, especially members of the ERBB family, as well as their downstream signalling pathways such as the MAP kinase or PI3-kinase/Akt pathway have been implicated in the progression of numerous types of human cancers and have been shown to be linked to the endogenous hyaluronan-CD44 interaction.

Thus, elevated levels of hyaluronan enhance ERBB2 phosphorylation in cells that normally exhibit low levels of ERBB2 activity [151]. Furthermore, stimulation of hyaluronan production induces assembly of a constitutive, lipid raft-associated, signalling complex containing phos-phorylated ERBB2, CD44, ezrin, PI3-kinase, as well as the chaperone molecules HSP90 and CDC37. Accordingly, the inhibition of endogenous hyaluronan-CD44 interactions- e.g. by hyaluronan oligomers, soluble hyaluronan-binding pro-teins and siRNA against CD44 - causes disassembly of this complex and inactivation of ERBB2 [153]. Similar effects of constitutive hyaluronan-CD44 interaction occur on other receptor tyrosine kinases, i.e. EGFR, IGF-1R, PDGFR and c-MET [154], and corresponding effects have been shown for downstream anti-apoptotic and proliferation pathways known to be regulated by these receptor kinases. For exam-ple, increased hyaluronan production stimulates the PI3-kinase and MAP kinase pathways whereas antagonists of hyaluronan interactions suppress these pathways [111,155]. As shown by a recent report, inhibition of the CD44 expres-sion disrupted a hyaluronan/CD44-pErbB2-Cox-2 interaction pathway in the Apc Min/-mice model thereby reducing ade-noma number and growth [156].

With respect to all these functions of hyaluronan and CD44 in tumorigenesis and therapy resistance of many types of cancer, one could expect that antagonists of the hyaluronan-CD44 interaction, e.g. small hyaluronan oli-gomers, may be useful in therapeutic strategies aimed at pre-venting tumor recurrence from therapy-resistant subpopula-tions within malignant cancers.

Another approach to interfere with the tumor stroma in-terplay and to sensitize tumor cells towards chemotherapy is by inhibition of the CXCR4/CXCL12 axis [118]. CXCR4 antagonists have been already discovered in the early 1990s for the treatment of HIV-1 infection although the exact mechanisms by which these agents exert its activity was un-known [157,158]. Meanwhile, four major classes of CXCR4 antagonists and agonists are available: i) small peptide CXCR4 antagonists (e.g. T140 and its analog TN14003), ii) non-peptide CXCR4 antagonists (e.g. AMD3100), iii) CXCR4 specific blocking antibodies and iv) modified ago-nists and antagonists for SDF-1. Treatment of SCLC cells


with T140 or either of its analogs TC14012 or TN14003 en-hanced the apoptotic response towards etoposide treatment in vitro [122]. Moreover, antagonizing CXCR4 showed activity in numerous preclinical studies e.g. involving animal models for breast or colorectal cancer by inhibiting tumor growth and metastasis [122,159,160]. To extend the effect of CXCR4 inhibition to its chemosensitizing ability, further studies are required to evaluate combinations of CXCR4 antagonists and chemotherapy. Regarding the clinical appli-cation, one has to keep in mind that a long-term or perma-nent inhibition of the CXCR4/CXCL12 axis in patients would probably favour immunological dysfunctions because of the involvement of CXCR4 in trafficking and homing of various lymphocyte subsets to different lymphoid organs during development and immunosurveillance [161,162]. Another concern regarding the application in cancer patients is the mobilization of hematopoetic stem cells from their protective microenvironmental niche into the blood where they could be exposed to the effects of other therapeutic agents, e.g. cytotoxic drugs. The first proof-of-principle stud-ies to evaluate the efficacy of CXCR4 antagonists in leuke-mia patients are currently underway.

Owing to its widely demonstrated role in tumorigenesis as well as its known contribution to stroma-induced chemoresistance in cancer [15,163,164], IL-1 is considered as molecular target since many years [165]. Numerous ani-mals studies proved the impact of IL-1 blockade by either the naturally occurring IL-1 receptor antagonist (IL-1RA/ anakinra), a blocking IL-1 antibody (canakinumab) or a soluble IL-1 receptor Fc-derivative (rilonacept) on metasta-sis formation, tumor burden and tumor vascularization. Moreover, a recent clinical trial in patients with smoldering or indolent myeloma at high risk for multiple myeloma re-vealed a blocking effect by anakinra treatment on myeloma progression [166].

Given the availability of these therapeutic agents for lim-iting IL-1 activity - as already applied in the treatment of chronic and acute inflammatory diseases - as well as their safety and clear benefit in animal models, clinical trials of IL-1 blockade in cancer patients seem to be highly promis-

ing. In particular, canakinumab exhibiting IL-1 selectivity and longer plasma half-life time would be an attractive measure to antagonize the tumorigenic and chemoresistance inducing effect of IL-1 in cancer patients [165].

Table 2 summarizes the outlined therapeutic strategies to overcome stroma-mediated drug resistance in different tumor entities.

CONCLUSIONS

Altogether, the rising number of studies strongly suggests that anti-stromal agents may offer a reasonable strategy to overcome chemoresistance and to improve cancer treatment of even therapy-refractory tumors. Based on current knowl-edge on the impact of the microenvironment on tumor pro-gression in general and on chemoresistance of tumor cells in particular, one can appreciate that targeting just the tumor cells in stroma-rich tumors would be insufficient to fully eradicate the tumor mass and to prevent tumor recurrences. Regardless of the considerable progress in concepts and strategies for improved cancer therapy, there are several tu-mors that still prove to be highly therapy-refractory, e.g. glioblastoma or PDAC. Thus, our knowledge on the molecu-lar and cellular mechanisms underlying tumor stroma inter-actions needs to be deepened and new therapeutical concepts combining stroma-related molecular targets and chemothera-peutic drugs have to be pursued. These efforts will bring us ahead to improve therapeutic options and prognosis of pa-tients with still highly malignant and incurable tumors. Moreover, high-throughput screening of anti-cancer drug efficacy in the context of tumor stroma interactions in vitro [167] on the one hand and the generation of an individual-ized predictive signature [13] on the other hand will help to identify individual resistance towards certain cytostatic drugs and to design optimized therapy concepts for each patient.

ACKNOWLEDGMENTS

This work was supported by grants from the Deutsche Forschungsgemeinschaft, Werner und Klara Kreitz Stiftung, Hensel-Stiftung and Else-Kröner-Fresenius Stiftung.

Table 2. Molecular Targets, the Respective Targeting Drug and its Application in Preclinical or Clinical Studies

Molecular Target Targeting Drug Application in Clinical Trials or Preclinical

Studies

CD44/Hyaluronan blocking antibodies, CD44 ectodomain, soluble

Rhamm, hyaluronan oligosaccharides

preclinical [144-147,149-152,155,156]

L1CAM / ECM, 5-i, NRP1 blocking antibodies, siRNA preclinical [143]

v 3-i etaracizumab (Abegrin) phase-I and -II trials, advanced solid tumors &

melanoma [126-129]

v 3- and v 5 cilengitide phase-II, gliomas & prostate cancer [131,132]

v-i, 5 1-i CNTO 95, ATN-161 preclinical [139,140,142], phase-I trial [141]

2-i, 5 1-i E7820, volociximab preclinical [163,164]

IL-1 / IL-1R canakinumab, rilonacept anakinra high risk smoldering myeloma [166]

CXCL12 / CCR4 T140, TN14003, AMD3100 preclinical [157-160]


ABBREVIATIONS

APAF-1 = Apoptotic protease activating factor-1

BAD = Bcl-2 antagonist of cell death

Bcl-2 = B-cell lymphoma 2

Bim = Bcl-2 interacting mediator of cell death

BRCP = Breast cancer resistance protein

DISC = Death inducing signalling complex

DNMT1 = DNA-methyltransferase-1

ECM = Extracellular matrix

EGFR = Epidermal growth factor receptor

ERBB2 = Human Epidermal growth factor Receptor 2

ERK = Extracellular regulated kinase

FADD = Fas-associated death domain

FAK = Focal adhesion kinase

FLICE = FADD-like interleukin-1 beta-converting enzyme

c-FLIP = FLICE inhibitory protein

GSK-3 = Glycogen Synthase Kinase-3 beta

HIV-1 = Human immunodeficiency virus-1

IGF-1R = Insulin-like growth factor 1 receptor

iNOS = Inducible nitric oxide synthase

MAPK = Mitogen activated protein kinase

MRP2 = Multidrug resistance protein 2

NF- B = Nuclear factor-kappa B

NO = Nitric oxide

NRP-1 = Neuropilin-1

PI3-kinase = Phosphatidylinositol 3-kinase

PDAC = Pancreatic ductal adenocarcinoma

PDGFR = Platelet-derived growth factor receptor

PTK = Protein tyrosine kinase

SCID = Severe combined immunodeficiency

SCLC = Small cell lung cancer

SDF-1 = Stromal cell-derived factor-1

STAT1 = Signal transducer and activator of transcrip-tion-1

TGFBI = Transforming growth factor beta induced

TNF- = Tumor necrosis factor-alpha

TRAIL = Tumor Necrosis Factor-related apoptosis-inducing ligand

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