TRAIL/Apo-2L death signaling pathway and molecular

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REVISIONES
TRAIL/Apo-2L death signaling pathway
and molecular targeting agents:
implications for chemoresistance
Rafael Rosella, Hernán Cortés-Funesb, Mariano Monzó a, c , Enriqueta Felipd,
Agustí Barnadasa and Miquel Taróna
a
Medical Oncology Service. Hospital Germans Trias i Pujol. Badalona (Barcelona). bMedical Oncology Service.
Hospital 12 de Octubre. Madrid. c Department of Morphologic Sciences. School of Medicine. University of Barcelona.
d
Medical Oncology Service. Hospital Vall d’Hebron. Barcelona.
TRAIL/ Apo-2L (tumor necrosis factor-related apoptosis-inducing ligand or Apo-2 ligand) was discovered by its sequence homology to tumor necrosis
factor (TNF) and CD95 ligand (Fas ligand). Recombinant soluble human TRAIL/ Apo-2L is a candidate for clinical research in cancer therapy because it
induces apoptosis in a broad spectrum of human
cancer cell lines but not in many normal cells. It is
now well-known that either ligands of death receptors or chemotherapeutic drugs can induce apoptosis in tumor cells through a common apoptotic
machinery. Central to this process is a family of
intracellular proteases, known as caspases. During
apoptosis, they can act either as initiators in response to apoptotic signals or as effectors that finally
cleave a number of vital proteins and lead to the
demise of the cell. The activation of caspases is controlled via multiple signaling pathways that are described in this review. There are multiple kinases involved in survival signaling that may be targeted by
novel agents. There are several compounds targeting the protein kinase Akt/ PKB that may inhibit
apoptosis at several levels of the caspase cascade,
which are also described in this review. Akt is the
major kinase which phosphorylates the proapoptotic Bcl-2 member Bad and thereby converts Bad into an anti-apoptotic form that does not induce cytochrome c release. Chemotherapeutic drugs trigger
the death pathway through the release of cytochrome c from damaged mitochondria. Besides TRAIL/
Apo-2L, several novel agents are described that
can lead to extend the therapeutic threshold. Hopefully, clinical trials will be begun very soon to
elucidate the possibility of enhancing the therapeu-
Correspondence: Dr. R. Rosell.
Medical Oncology Service.
Hospital Germans Trias i Pujol.
Ctra. Canyet, s/ n.
08916 Badalona (Barcelona).
Correo electrónico: rrosell@ ns.hugtip.scs.es
Recibido el 4-4-2001.
Aceptado para su publicación el 9-7-2001.
284
tic effect in terms of response and, especially, survival. It is thus essential for clinical investigators to
understand the distinct pathways of apoptosis and
caspase activation when deciding to participate in
these trials.
Key words: TRAIL/ Apo-2L, IAP genes, caspase-8, caspase-9, XIAP, Akt, STI571.
Rev Oncología 2001; 3: 284-291.
TRAIL/ Apo-2L y nuevas dianas
terapéuticas: implicaciones
en quimiorresistencia
El TRAIL/ Apo-2L (tumor necrosis factor-related
apoptosis-inducing ligand o Apo-2 ligando) se descubrió por su homología de secuencia con el TNF y
el CD95 (fas ligando). El TRAIL/ Apo-2L humano
soluble recombinante es un candidato para su investigación clínica del tratamiento del cáncer, ya
que induce apoptosis en un amplio espectro de líneas celulares derivadas de tumores humanos, pero no en la mayoría de líneas celulares normales no
neoplásicas. Actualmente se conoce que los ligandos de los receptores inductores de muerte celular
(death receptors) o los agentes de quimioterapia
para inducir apoptosis en células tumorales a través de un mecanismo común. Un papel central de
este proceso lo desarrolla una familia de proteasas
intracelulares conocidas como caspasas. Durante la
apoptosis pueden actuar tanto como iniciadores de
la respuesta a las señales apoptóticas o como efectores que finalmente degradan un número elevado
de proteínas esenciales conduciendo a la muerte
celular. La activación de las caspasas está controlada por múltiples cascadas de señalización y que se
describen en esta revisión. Existen múltiples kinasas involucradas en las vías de rescate o inhibidoras de la muerte celular programada que pueden
ser dianas para nuevos fármacos. En la actualidad
disponemos de múltiples compuestos que inhiben
la proteín kinasa Akt/ PKB que a su vez puede ac-
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R. ROSELL ET AL — TRAIL/APO-2L DEATH SIGNALING PATHWAY AND MOLECULAR TARGETING AGENTS:
IMPLICATIONS FOR CHEMORESISTANCE
tuar inhibiendo la respuesta apoptótica en distintos
niveles de la cascada de caspasas, los cuales se describen igualmente a continuación. AKT es la principal kinasa que fosforila la proteína BAD (miembro
de la familia de Bcl-2 que actúan como inductores
de la apoptosis), convirtiendo a BAD en una forma
antiapoptótica que impide la inducción de la liberación del citocromo c. Los agentes de quimioterapia inducen las vías de muerte celular a través de la
liberación del citocromo c desde la mitocondria
dañada. Juntamente con el TRAIL/ api-2L se han
descrito multitud de nuevos agentes que pueden
ampiar los umbrales de respuesta de los tratamientos actuales. De forma esperanzadora algunos ensayos clínicos se pondrán en marcha en un breve
período de tiempo y permitirán clarificar la posibilidad de potenciar el efecto terapéutico en términos
de respuesta y, especialmente, de supervivencia. Por
tanto es esencial para los investigadores clínicos entender las diferentes vías de apoptosis y de activación de las caspasas en el momento de decidir su
participación en este tipo de ensayos.
Palabras clave: cTRAIL/Apo-2L, genes IAP, caspasa-8,
caspasa-9, XIAP, Akt, STI571.
In spite of the frequent success of surgical and chemotherapeutic measures in controlling primary tumor growth, metastatic disease is seldom amenable
to surgery, displays resistance to chemotherapy, and
is the major cause of terminal illness. Apoptosis, or
programmed cell death, is a physiologic process in
which cells die after exposure to normal or pathologic stimuli. There is mounting evidence that the ability of cancer cells to evade or subvert signals that
lead to apoptosis plays a major role in tumor survival
in the face of cytotoxic drug regimens. The anticancer
action of cytotoxic drugs is in large part attributable
to their ability to induce apoptosis of tumor cells and
is therefore dependent on the integrity of the cellular
signaling pathways that lead to apoptotic cell death.
Defects in some of these pathways are common in
cancer cells, especially of metastatic origin, and are
therefore likely to underlie tumor resistance to chemotherapy.
By outlining the relevance of new apoptotic signaling
pathways, this review attempts to shed some light on
the mechanism whereby cytotoxic agents cause apoptosis and, more importantly, to highlight novel therapeutic approaches that intervene in downstream signaling pathways. TRAIL/ Apo-2L surfaces as a new
therapeutic agent that can overcome resistance to cytotoxic drugs in many tumors. Oncologists today need
to be fully aware of the progress that is being made
towards understanding these death signaling pathways. Many drugs are being developed to circumvent
chemoresistance and enhance tumor killing, leading
to a new therapeutic threshold. Figures 1 and 2 summarize the points dealt with here.
Recent evidence has implicated the members of the
tumor necrosis factor (TNF) receptor superfamily of
cell surface proteins in tumor cell death. Because
transduction of signals that induce apoptosis is the salient functional property of TNF-R1 and Fas (Apo-1/
CD95), they are frequently referred to as death receptors (DRs) and share cysteine-rich repeats in the ligand-binding extracellular region as well as a stretch
of 80 amino acids in the cytoplasmic domain, termed
the «death domain», that is essential for apoptosis signaling. Recently, another member of the TNF family,
the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), also called Apo-2L, has been
identified1. TRAIL/ Apo-2L interacts with 2 newly discovered DRs, DR4 and DR5. Transfection experi-
Fig. 1. Apoptosis control
by death and decoy receptors. TNF: tumor necrosis
factor; DR: death receptor;
FADD: fas associated death domain; TRADD: TNFR
associated death domain;
TNFR1: tumor necrosis factor receptor 1.
285
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REV ONCOLOGÍA, VOLUMEN 3, NÚMERO 6, NOVIEMBRE-DICIEMBRE 2001
Fig. 2. TRAIL/Apo-2L and
PI3K/AKT survival signaling
pathways.
ments have shown that both DR4 and DR5 can initiate caspase-mediated apoptosis1,2.
Tumor necrosis factor receptor 1 (TNFR1), Fas (Apo-1/
CD95), and the DRs for TRAIL/ Apo-2L regulate programmed cell death (apoptosis). Upon engagement by
their respective ligands, TNFR1 and Fas recruit adaptor molecules and activate a cascade of cysteine proteases (caspases), the proteolytic activity of which induces apoptosis. In summary, the best-characterized
DRs and their cognate ligands thus far include TNFR1/
TNF- α Fas (Apo-1/ CD95)/ FasL (Apo-1L or CD95L),
DR4 and DR5/TRAIL/Apo-2L, and Apo-3 (DR3)/Apo-3L
(table 1). We have reviewed 18 references concerning
TRAIL/ Apo-2L.
TRAIL/ Apo-2L is a 281-amino acid protein, related
most closely to Fas/Apo-1 ligand1. Soluble TRAIL/
Apo-2L induces extensive apoptosis in lymphoid as
well as non-lymphoid tumor cell lines. The effect of
TRAIL/ Apo-2L is not inhibited by soluble Fas/ Apo-1
and TNF receptors. It was found that TRAIL/ Apo-2L
acts via a receptor which is distinct from Fas/ Apo-1
and TNF receptors, and it was suggested that TRAIL/
Apo-2L could function as an extracellular signal that
TABLE 1. Death receptors (DR) and their cognate ligands
TNFR1/TNF-α
Fas (CD95 or Apo-1)/FasL (CD95L or Apo-1L)
DR5 and DR5/TRAIL (Apo-2L)
Apo3 (DR3)/Apo-3L
286
triggers programmed cell death. The CD95 ligand
(CD95L) and TNF are important extracellular activators of apoptosis in the mammalian immune system.
The cognate receptors for these cytokines, Fas (CD95/
Apo-1) and TNFR1, contain cytoplasmic «death domains» (table 1 and fig. 1) that activate the cell’s apoptotic machinery through interaction with the death
domains of the adapter proteins FADD (Fas associated death domain) and TRADD (TNFR associated death
domain). Upon activation by a ligand, Fas (CD95/Apo-1)
recruits FADD. FADD in turn activates the ced-3-related protease MACHα/ FLICE (caspase-8), thereby initiating a series of caspase-dependent events that lead
to cell death2. TRAIL/ Apo-2L activates rapid apoptosis in tumor cell lines, acting independently of Fas
(CD95/Apo-1), TNFR1, or FADD. A receptor for TRAIL,
DR4 activates apoptosis independently of FADD. DR4
exhibits several mRNA transcripts that are expressed
in multiple human tissues, including peripheral blood leukocytes. In 1997, 2 additional TRAIL/ Apo-2L
receptors were identified: DR5, which contain a cytoplasmic death domain and induces apoptosis like
DR4; and the receptor designated decoy receptor 1
(DcR1), which acts like a decoy receptor that inhibits
TRAIL/Apo-2L signaling2. By cross-hybridization with
DcR1, a fourth Apo-2L receptor was identified, named
decoy receptor 2 (DcR2). The DcR2 gene mapped to
human chromosome 8p21, as did the gene encoding
DR4, DR5 and DcR1. Upon overexpression, DcR2 did
not activate apoptosis or nuclear factor-KB; however,
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R. ROSELL ET AL — TRAIL/APO-2L DEATH SIGNALING PATHWAY AND MOLECULAR TARGETING AGENTS:
IMPLICATIONS FOR CHEMORESISTANCE
it reduced cellular sensitivity to TRAIL/ Apo-2L-induced apoptosis3. In 1999, the crystal structure of the
complex between Apo-2L and DR5 was reported4. Fas
(CD95/Apo-1) and TNFR1 trigger apoptosis by recruiting the apoptosis initiator caspase-8 through the
adaptor FADD. Fas binds FADD directly, whereas
TNFR1 binds FADD indirectly, through TRADD. TRAIL/
Apo-2L initiates apoptosis through caspase-8 and
FADD is also involved as a universal adaptor for
DRs5,6 (fig. 1).
In summary, apoptosis is controlled by death and decoy receptors. TRAIL/ Apo-2L can bind to five members of the TNFr family, DR4, DR5, DcR1, DcR2, and
osteoprotogerin. Binding of TRAIL/ Apo-2L to DR4
and DR5 leads to the cleavage and activation of caspase-8 and caspase-10. Processed and actived caspase-8 can also cleave and activate the BH3 domain
containing BID (a pro-apoptotic Bcl-2 member),
which translocates to the mitochondria, triggering the
cytosolic release of cytocrome c (fig. 2). In the cytosol,
cytocrome c and dATP bind to Apaf-1 and cause its
oligomerization. Apaf-1, processes procaspase-9 into
and active caspase, which recruits, cleaves, and activates the executioner caspase-3. This can proteolytically cleave poly (ADP-ribose) polymerase, DNA fragmentation factor 45, resulting in the morphologial
features and DNA fragmentation of apoptosis.
The recombinant soluble human TRAIL/ Apo-2L molecule appears remarkably safe and non-inmunogenic, and it is now being tested in clinical trials. In vitro , soluble TRAIL/ Apo-2L exerted cytostatic or
cytotoxic effects on a variety of tumor cell lines7, but
not on several types of normal cells. In vivo, soluble
TRAIL/ Apo-2L shows antitumor activity in xenograft
models in colon carcinoma cell lines, and synergistic
activity with 5-FU or CPT-11, causing marked regression or complete remission of tumors7.
THE ROLE OF TRAIL/ APO-2L IN DIFFERENT
CHEMORESISTANT TUMORS
In contrast to DR4 and DR5, DcR1 (TRAIL-R3/ TrID)
and DcR2 (TRAIL-R4/ TRUNDD) are unable to transduce death signals and may function as decoy receptors. They may also provide inhibitory signals, possibly through activation of nuclear factor-KB. The
DR4, DR5, DcR1 and DcR2 genes are tightly clustered
on human chromosome 8p22-21, they may have been
derived from a common ancestor by gene duplication8.
A fifth TRAIL/ Apo-2L receptor, osteoprotegerin,
exists in a soluble form and inhibits apoptosis by interfering with the binding of TRAIL/ Apo-2L to the
DR4 and DR5 receptors. In contrast to Fas ligand,
the expression of which is limited to cells of the immune system and a few immune-privileged sites,
TRAIL/ Apo-2L is expressed in a wide range of normal fetal and adult tissues and induces apoptosis only
in transformed and malignant cells, thus constituting
a promising new candidate for cancer treatment.
Restricted DR5 expression, or lack thereof, may be an
important mechanism by which Ewing’s sarcoma family tumors (ESFT) cells become resistant to
TRAIL/ Apo-2L8. TRAIL/ Apo-2L-resistant tumors may
also exhibit functional inactivation of the receptors,
either by lack of expression on the cell surface, as reported for melanomas, or by the presence of structural abnormalities, as in the case of DR5 mutations in
head and neck cancers and of the polymorphism at
codon 441 in the death domain of DR4, which acts in
a dominant-negative fashion (reviewed in Mitsiades
et al) 8. DR5 is up-regulated by chemotherapeutic
agents. Therefore, its absence may have implications
in chemotherapy-related tumor cell responses.
TRAIL/ Apo-2L effectively and specifically kills ESFT
cells by engaging DR4 and DR5 and activating a cascade of caspases that begins at caspase-10. Caspase-8
is also activated, but it is not recruited to the TRASC
(TRAIL-associated signaling complex) 8. Experimental
data suggest that TRAIL/ Apo-2L has excellent therapeutic potential in ESFTs. In general, treatment with
DNA damaging anticancer agents can induce p53
and/ or nuclear factor κB, which in turn can up-regulate DR5 and/ or DR4 expression, thereby enhancing
TRAIL/ Apo-2L-induced apoptotic signaling. In contrast, DcR1 and DcR2 can act as inhibitors of
TRAIL/ Apo-2L-induced apoptotic signaling. Flame-1
(also known as c-FLIP [FADD-like IL-1 β-converting
enzyme]) has a dominant negative effect against caspase-8 and potentially inhibits TRAIL/ Apo-2L induced death signaling (fig. 2) 9. For example, pretreatment with paclitaxel or docetaxel enhances
TRAIL/ Apo-2L-induced apoptosis of prostate cancer
cells by inducing DR4 and DR5 protein levels9. The
levels of IAP family members: cIAP1, cIAP2, X-linked
XIAP, and survivin, may also inhibit TRAIL-induced
apoptosis by specifically binding to and inhibiting
the activities of caspase-3, caspase-9, and caspase-7
(fig. 2). Taxane-induced apoptosis is triggered by mitochondrial ∆ψm (permeability transition), release of
cytocrome c into the cytosol, and induction of Apaf-1mediated caspase-9 and caspase-3 activities (fig. 1 and 2).
TRAIL/ Apo-2L triggers the molecular events of both
the extrinsic (DR) and intrinsic (mitochondrial) pathway of apoptosis9. Pretreatment with paclitaxel up-regulates DR4 and DR5 expression and enhances
TRAIL/ Apo-2L-induced caspase-8, caspase-10, and
Bid processing and the cytosolic accumulation of cytocrome c. It also down-regulates XIAP, cIAP1 and
survivin levels9.
TRAIL-sensitive ESFT cell lines carry p53 mutations
or deletions suggesting that TRAIL-induced apoptosis
is independent of p538. Wild-type p53 can induce
transcriptional regulation of DR5 TRAIL/ Apo-2L receptor in cells undergoing apoptosis. Ectopic expres-
287
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REV ONCOLOGÍA, VOLUMEN 3, NÚMERO 6, NOVIEMBRE-DICIEMBRE 2001
sion of wild-type p53 can enhance TRAIL-induced
apoptosis in malignant gliomas. Using the TRASC assay, it was shown that caspase-10 is the apical in the
TRAIL-signaling pathway8.
The TRAIL/ Apo-2L-resistant phenotype has also been
investigated in brain tumors. Malignant glioma cells
primarily express DR5. DR5 expression is increased
by DNA-damaging chemotherapeutic agents, thus
enhancing TRAIL/ Apo-2L killing of glioma cells.
TRAIL/ Apo-2L killing of glioma cells is characterized
by caspase-8 and caspase-3 activation, PARP (poly
[ADP-ribose] polymerase) cleavage, and DNA fragmentation10. PED/ PEA-15 (phosphoprotein enriched
in diabetes/ phosphoprotein enriched in astrocytes Mr
15,000) protein expression leads to resistance to
TRAIL-induced apoptosis11. PED/ PEA-V is overexpressed in fibroblasts from type 2 diabetics compared
with non-diabetic individuals, as well as in skeletal
muscle and adipose tissues, 2 major sites of insulin
resistance in type 2 diabetes. Overexpression of the
PED/ PEA-15 gene may contribute to insulin resistance in glucose uptake in type 2 diabetes. PED cloning
shows that it encodes a 15 KDa phosphoprotein identical to the protein kinase c (PKC) substrate PEA-1511.
Transfection of PED antisense in TRAIL-resistant cell
lines converted these lines into sensitive lines, whereas transfection of PED cDNA in TRAIL-sensitive
cell lines rendered the cells resistant. Inhibition of
PKC activity restored TRAIL-induced apoptosis in resistant cells, demonstrating that induction of apoptosis in TRAIL-resistant glioma cells is prevented by
PKC phosphorylation. The critical PKC substrate is
postulated to be PED/ PEA-1510. Apoptosis was induced by combination of CD437 and TRAIL. The synthetic retinoid CD437 is a potent inductor in a variety
of cancer cell types through increased levels of DRs.
Treatment of human lung cancer cells with a combination of suboptimal concentrations of CD437 and
TRAIL/ Apo-2L enhanced induction of apoptosis in tumor cell lines but not in normal lung epithelial cells12.
CD437 upregulates DR4 and DR5 expression. The
CD437/ TRAIL/ Apo-2L combination enhances activation of caspase-3, caspase-7, caspase-8 and caspase-9
and the subsequent cleavage of PARP and DNA fragmentation factor 4512. This combination induces BID
cleavage and increases cytocrome c release from mitochondria (fig. 2).
Anticancer drugs sensitize tumor cells. Doxorubicin
and cisplatin sensitize colon cancer cells to TRAILinduced cleavage of procaspase-8 and PARP and caspase activation. Anticancer drugs increase the ability
of TRAIL/ Apo-2L to trigger caspase-dependent cell
death, also described in breast cancer13.
TRAIL/ Apo-2L-resistant neuroblastomas lack caspase-8. Semiquantitative RT-PCR reveals that DR5
(TRAIL-R2, KILLER) and DcR1 (TRAIL-R3, TRID)
are the main TRAIL-receptors used by neuroblasto-
288
ma cells. Failure to express caspase-8 and/ or caspase-10 could be an important mechanism of resistance to
chemotherapy in neuroblastoma cells. Demethylation
by 5-ADC (5-aza-2’-deoxycytidine) restores caspase-8
and caspase-10 expression and TRAIL/ Apo-2L sensitivity14.
TRAIL/Apo-2L is also active in pancreatic cancer. TRAIL/
Apo-2L caused a sustained profound cell death in several human pancreatic cancer cells lines but not in
Aspc1 cells. The resistance of Aspc1 cells to TRAIL/
Apo-2L was not related to the lack of TRAIL/ Apo-2L receptors. The combination of actinomycin D and TRAIL/
Apo-2L induced an almost complete lysis of Aspc1
cells15. Actinomycin D alone had no effect but inhibited the expression of FLICE15.
STI-571 AND TRAIL/ APO -2L IN BCR-ABL-POSITIVE ACUTE LEUKEM IAS
Bcr-Abl tyrosine kinase inhibitor STI-571 produces a
high rate of remission in chronic myeloid leukemia
(CML), but remissions induced in patients with blast
crisis of CML or Bcr-Abl-positive Abl are not durable.
STI-571 and TRAIL/ Apo-2L significantly enhances
TRAIL/ Apo-2L induced apoptosis and increases the
processing of caspase-9, and -3 and XIAP, without
affecting the levels of DR4, DR5, decoy receptor or
C-FLIPL (fig. 2) 16. Interestingly, STI-571-mediates sensitization of Bcr-Abl-positive leukemia cells to TRAIL/
Apo-2L-induced apoptosis. By inhibiting Bcr-Abl tyrosine kinase, STI-571 inhibits constitutively active PI3
kinase, STAT5 proteins (Signal Transducers and Activators of Transcription) 17 and NFκB in Bcr-Abl positive leukemia blasts16. The result is the lowering of BclXL levels and its antiapoptotic activity. It also has the
capacity to lower the levels of IAP and improve caspase-9 activity. These effects of STI-571 could promote
TRAIL/Apo-2L-induced cytocrome c and SMAC/
DIABLO release from mitochondria and the subsequent
activation of the effector caspase-3 and caspase-7, resulting in apoptosis (reviewed in Nimmanapalli et al) 16.
M ETALLOPROTEINASES IN CANCER
TNF and FasL have potent cytotoxic activity against
many types of tumor cells, however, the application of
these death ligands to cancer therapy has been restricted by their severe toxicity to normal tissues. Although we can not use FasL for treatment, intriguingly, FasL is observed to be proteolytically cleaved
from the surface of some cell types by MMP-7 (matrix
metalloproteinase) 18. Proteolytic cleavage of FasL
could provide at least partial protection against Fasmediated cell deaths (also reviewed in Lacal) 19.
MMPs are zinc-dependent enzymes that help regulate
the turnover of ECM (extracellular matrix) components. They are believed to play an important role in
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R. ROSELL ET AL — TRAIL/APO-2L DEATH SIGNALING PATHWAY AND MOLECULAR TARGETING AGENTS:
IMPLICATIONS FOR CHEMORESISTANCE
embryo development, morphogenesis, and tissue remodeling, as well as in tumor invasion and metastasis. MMPs are thought to induce ECM degradation,
angiogenesis, and possibly, regulation of tumor
growth itself. Invading and metastatic tumor cells typically secrete MMPs and induce MMP production by
surrounding stromal cells that may overwhelm the
local tissue capacity to maintain their proteolytic activity in check.
It was recently found that the proteolytic activity of
MMP-7, which is locally expressed in several tumors,
may contribute to tumor resistance to cytotoxic agents.
Specific inhibition of MMP-7 may provide a potentially effective approach toward increasing the efficacy of chemotherapy18. Development of MMP inhibitors is a very appealing novel anti-cancer approach20.
OTHER MOLECULAR TARGETING AGENTS
Recent evidence has suggested that X-linked inhibitors of apoptosis protein (XIAP) is a determinant in
cisplatin sensitivity in ovarian cancer. Cell fate is determined by a balance between cell survival and
apoptotic signaling. Cisplatin decreased XIAP protein
levels and induced AKT cleavage and apoptosis in
chemosensitive, but not in chemoresistant, ovarian
cancer cells. It has been proven that XIAP down-regulation induced AKT cleavage and apoptosis21. In the
presence of the PI3-K (phosphatidylinositol 3-kinase)
inhibitor (LY294002), XIAP overexpression failed to
block cisplatin-induced apoptosis and to induce AKT
phosphorylation, suggesting that the site of action
of XIAP is upstream of AKT in this cell survival
pathway21. AKT (also known as protein kinase B) consists of a family of highly conserved serine/ threonine
kinases. Activated AKT has been shown to mediate
cell survival by phosphorylating several downstream
targets, such as BAD and caspase-9 (fig. 2). In contrast, the tumor suppressor PTEN inhibits PI3K-dependent activation of AKT (reviewed in Tanno et al) 22.
PI3K was discovered as an activity that phosphorylates
phosphoinositols at the D-3’ position of the inositol
ring and produces novel phosphoinositides23. The flavanoid derivative LY 294002, a potent PI3K inhibitor, is
a competitive, reversible inhibitor of the ATP binding
site of the enzyme (reviewed in Hidalgo, Rowinsky) 24.
Rapamycin, a macrolide fungicide, and its analog
CCI-779 have prominent anti-tumor activity. Rapamycin binds intracellularly to members of the immunophilin family of FK506 binding proteins (FKBPs) especially FKBP12. The resultant rapamycin-FKBP12
complex interacts with and inhibits the activity of a
kinase named mammalian target of rapamycin
(mTOR). Experimental data show that mTOR functions downstream of the PI3K/AKT pathway24. Another
major signaling pathway is the MAPK (mitogenactivated protein kinase) pathway.
Similarly to AKT, the phosphorylation of MAPK is
also increased in some lung adenocarcinoma cells
upon detachment, suggesting that both PI3K and
MAPK are similarly regulated in the tumor cells25.
The gene corral displayed by the PI3K-AKT pathway
and TRAIL/ Apo-2L open the gates to novel therapeutic approaches. For instance, the 26s proteasome is
the universal multi-catalytic protease, responsible for
the bulk of protein turnover in all cells. A selective
inhibitor PS-341 has been developed, inhibiting the
activation of NF- κB26. Finally, inhibitors of histone deacetylase (HDAC) like depsipeptide are associated with
apoptosis through suppression of IAP genes. Interestingly, the activity of HDAC inhibitors is enhanced in
the presence of the DNA methyltransferase inhibitor
5-aza-2’-deoxycytidine (DAC) 27. Hence, the effect of
HDAC inhibitors may be intimately linked to the
methylation pattern of tumors, at least in non-smallcell lung cancers28.
It is well known that apoptotic and necrotic cells are a
major source for plasma DNA in cancer patients29. Also biochips can be used to test RNA array expression
in blood. cDNA arrays of 170 genes were analyzed in
blood samples from 26 breast cancer patients and 22
healthy controls. Cluster analysis identified a group of
12 genes that were elevated in the blood of cancer patients. Hierarchical clustering identified 5 core genes:
CD44, maspin, gro-alpha, ß-tubulin and N30,31. A bloodbased assay could be a tool to define personalized
chemotherapy or at least classified patterns of chemoresistance by hierarchical cluster analysis. A cDNA
microarray containing 7,075 genes was used to compare gene expression profiles of 3 non-small-cell lung
cancer cell lines and to examine changes after exposure to paclitaxel. There were 27 differentially expressed genes in the chemoresistant line in comparison to
the parental line. Of these, 12 genes were increased
whereas 15 were repressed. We can foresee an array
analysis of gene expression in peripheral blood (table 2) in patients with stage IV before starting chemotherapy and after 3 cycles at the time of radiographic assessment of clinical remission. A hierarchical
set of apoptotic-resistant genes to selective chemotherapy combinations could then be identified, and this
TABLE 2. Chemotherapy-mediated regulation of some
genes related to apoptosis
DR4
DR5
BID
Caspase-10
Caspase-8
Caspase-3
c-IAP1
XIAP
Survivin
Ribonucleotide reductase
DcR1
DcR2
Osteoprotegerin
PI3K
AKT
NF-κB
Bcl-XL
BAD
β-tubulin
STAT5
Caspase-9
SMAC/DIABLO
Hsp70
Hsp90
MMP-7
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REV ONCOLOGÍA, VOLUMEN 3, NÚMERO 6, NOVIEMBRE-DICIEMBRE 2001
TABLE 3. Targeted therapies
Target
Activation of caspase-8,
caspase-3
Inhibits IAP genes
Upregulates DR4 and DR5
New drugs
TRAIL/Apo-2L (Genentech Inc)
Retinoid CD437
Targretin? (Ligand Pharmaceuticals)
Inhibits Abl tirosine kinase acti- STI571 (for signal-transduction
vity, PDGF receptor and cinhibitor) (Novartis)
kit tyrosine kinases
Inhibits SCF-mediated kit activation
Blocks the phosphorylation of
Akt
Inhibits XIAP through P13K
LY294002 (flavonoid derivative)
inhibition
(Lilly)
Inhibits mTOR
Rapamycine (Wyeth)
Inhibits IAP genes
Inhibitors of HDAC, CI-994
(Pfizer)
EGF-receptor inhibitor
ZD1839 (Iressa) (AstraZeneca)
Inhibits the M2 subunit of ribo- Triapine (Vion)
nucleotide reductase
chemoresistance could —at least in some instances—
be overcome by the addition of very promising novel
therapeutic agents like TRAIL/ Apo-2L.
Table 3 summarizes novel therapeutic approaches
based on exploiting new crucial targets in several
apoptotic pathways as well as on inhibiting tyrosine
kinases. In addition to TRAIL/ Apo-2L STI571 stands
out because of its intricate action. STI571 is a phenylaminopyrimidine derivative that selectively inhibits
the enzymatic activity of several tyrosine kinases:
ABL; the BCR-ABL fusion protein of chronic myeloid
leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia; platelet-derived growth
factor receptor; and the product of the c-kit gene.
It has demonstrated excellent activity and tolerability
in patients with chronic myeloid leukemia in whom
treatment with interferon alfa had failed32. Responses
have also been observed in 55% of patients with a
myeloid-blast crisis33. At least 70% of small-cell lung
cancers express the kit receptor tyrosine kinase and
its ligand, stem cell factor (SCF). This coexpression
constitutes a functional autocrine loop, suggesting
that inhibitors of kit tyrosine kinase activity could
have therapeutic efficacy34. It was demonstrated that
STI571 efficiently blocked SCF-mediated activation of
mitogen-activated protein kinase and Akt but did not
affect insulin-like growth factor 135. Aberrant expression of c-kit and/ or SCF has been reported in several
breast, lung, prostate, and colorectal tumors. In gastrointestinal tissues, c-kit expression has been observed in interstitial cells of Cajal in the small intestine.
It has been suggested that c-kit activation plays a role
in gastrointestinal stromal tumors derived from Cajal
cells either through overexpression or mutations of
290
c-kit35. Recently it has been shown that gastrointestinal
stromal tumors are highly responsive to STI571 because they uniformly express c-kit36. Also data indicates that STI571 blocks the phosphorylation of Akt,
which is downstream from c-kit37. It is possible that
STI571 can be active in other tumors such as non-small
cell lung cancer and melanoma. Clinical trials have
been started in lung, prostate and brain tumors38.
CONCLUSIONS
Physicians caring for cancer patients need to become
aware of the rapid development of science today, and
medical oncologists faced with a plethora of clinical
trials with new agents must be able to discern which
is the best combination for their patients. The Spanish
Lung Cancer Group is committed to developing pharmacogenetic studies to elucidate which genetic markers can help to distinguish different subsets of
patients that may be more responsive to specific
therapeutic approaches. By including combinations
with some of the new drugs, we may finally be able to
break through the ceiling of therapeutic results obtained with current chemotherapy regimens.
Acknowledgments
The authors thank Beatriz Rodrigo for technical assistance and Renée O’Brate for assistance with the manuscript.
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