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Involvement of multiple signaling pathways in follicular lymphoma transformation: p38-mitogen-activated protein kinase as a target for therapy
*Department of Pathology,
Associated Regional and University
Pathologists Institute for Clinical and Experimental Pathology,
Department of Mathematics, and
¶Division of Medical Oncology, University of
Utah, Salt Lake City, UT 84132; ||Cross Cancer
Institute, Edmonton, AB, Canada T6G 1Z2; and
**Advanced Therapeutics Program and
Department of Pathology, Sunnybrook
and Women's College Health Sciences Centre, Toronto, ON, Canada M4N 3M5
Edited by Janet D. Rowley, University of Chicago Medical Center, Chicago, IL and approved April 7, 2003 (received for review December 9, 2002)
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-mitogen-activated
protein kinase (MAPK) were differentially expressed in the DLBCLs.
Immunohistochemistry of another blinded set of samples demonstrated expression
of phosphorylated p38MAPK in 6/6 DLBCLs and 1/5 FLs, but not in benign
germinal centers. SB203580 an inhibitor of p38MAPK specifically induced
caspase-3-mediated apoptosis in t(14;18)+/p38MAPK+-transformed FL-derived cell
lines. Lymphoma growth was also inhibited in SB203580-treated NOD-SCID mice.
Our results implicate p38MAPK dysregulation in FL transformation and suggest
that molecular targeting of specific elements within this pathway should be
explored for transformed FL therapy.
The advent of microarray analysis provides an opportunity to assess genomewide perturbations involved in FL transformation. Lossos et al. (14) recently showed that transformed FLs may exhibit germinal center B cell (GCB)-like or activated B cell (ABC)-like profiles analogous to those originally described for de novo DLBCL (15). They also determined that transformed FLs are biologically distinct from de novo DLBCL. However, functional studies were not performed. Nevertheless, that and other studies have established the important role of expression profiling for class prediction (16) and discovery of prognostically relevant classes (15). The next important challenge is the utilization of expression profiles in the discovery of therapeutic targets.
Here, we have used expression profiling for the identification of important genes/pathways involved in FL transformation. We studied an initial cohort of matched pairs of FLs and their transformed counterparts (DLBCLs) occurring within the same individual. We show that dysregulation of several growth factor and cytokine receptors connected to p38-mitogen-activated protein kinase (MAPK) may be relevant to FL transformation. We validated the results by quantitative RT-PCR (qRT-PCR) and immunohistochemistry (IHC) and further demonstrated that selected differentially expressed genes could serve as robust classifiers of a blinded and independent validation set of FL and DLBCL samples. Finally, we showed that pharmacologic targeting of phosphorylated-p38MAPK in t(14;18)+/p38MAPK-overexpressing lymphoma-derived cell lines inhibits their growth in vitro and in vivo, confirming its importance to FL progression.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Cell Culture and Treatment. Cell lines were cultured under standard conditions. SUDHL-4, SUDHL-5, and OCI-LY1 cell lines (1.5 x 105 cells per ml) were treated with SB203580, a selective p38MAPK inhibitor (Upstate Biotechnology, Lake Placid, NY), or U0126, a selective p42/44MAPK inhibitor (Cell Signaling, Beverly, MA) dissolved in DMSO. Cells were collected at specified time points for viability, proliferation, cell cycle, and apoptosis analysis. Control cells were exposed to equal volumes of DMSO and maintained under the same conditions as the treated cells.
Assessment of Clonal Relationships. PCR analysis for Ig heavy chain gene (IgH) rearrangements and for detection of t(14;18)(bcl-2/ JH) were performed as described (18). Fluorescent in situ hybridization for t(14;18) was performed on paraffin-embedded tissues with a probe against t(14;18) (Vysis, Downers Grove, IL).
Gene Expression Profiling. Total RNA was extracted by using TRIzol Reagent (Life Technologies, Gaithersburg, MD). cDNA synthesis and linear amplification were performed as described (19). cDNA microarray analysis was performed at the Huntsman Cancer Institute Microarray Core Facility (Salt Lake City) by using a two-color hybridization (Cy5/Cy3) scheme as described (20) and a 6,912-clone set (Research Genetics, Huntsville, AL). Test sample RNAs were hybridized against a reference composed of RNA pooled from five cell lines (Jurkat, SKW3, L428, Raji, and NCEB). Reproducibility of test to reference ratios across four replicates was accomplished by using GENESPRING software (Silicon Genetics, Redwood City, CA). Hierarchical clustering analysis was performed by using CLUSTER and visualized with TREEVIEW (21) (http://rana.lbl.gov). The expression profiles of the FLs and their DLBCL counterparts were compared with those of the purified lymphocyte subpopulations, ABCs, the t(14;18)+ SUDHL-4 cell line, and the ABC-like cell line OCI-LY10 (15). We selected 76 genes in our clone set, including some genes previously shown to distinguish GCB and ABC programs for hierarchical clustering of our FLs and DLBCLs (Table 1, which is published as supporting information on the PNAS web site, www.pnas.org). For the identification of genes involved in FL transformation, we performed (indirect) pairwise analysis of the data obtained from the FLs and DLBCLs, both compared with the reference RNA. The raw data for the indirect FL and the DLBCL experiments are provided at www.path.utah.edu/labs/kojo. We also performed experiments involving (direct) hybridization of RNA obtained from a FL against RNA obtained from the DLBCL from same patient (raw data available at www.path.utah.edu/labs/kojo. The results of the indirect and direct analyses were consistent (Fig. 7, which is published as supporting information on the PNAS web site). Details of our array data processing and a description of our gene discovery algorithm are provided in Supporting Text, which is published as supporting information on the PNAS web site.
Real-Time qRT-PCR. qRT-PCR for C-MET, N-RAS, antioxidant
protein 2/acidic phospholipase A2
(PLA2), 
-1 and erythrocyte membrane
protein 4.1-like 1 (EMP 4.1-L1) was performed by using SYBR Green I
and the LightCycler (Roche Applied Science) (PCR protocols are provided in
Tables 2 and 3, which are published as supporting information on the PNAS web
site). The qRT-PCR to microarray data equivalence comparison and Bayesian
analysis for class assignment using qRT-PCR data are provided in
Supporting Text.
IHC. IHC was performed by using standard protocols with the following antibodies at the dilutions indicated: anti-human phospho-p38MAPK (1:60) and phospho-p42/p44 MAPK (1:200) (Cell Signaling).
In Vitro Cell Viability Assay. Viability of drug-treated and control cells were determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay (22). Three replicates were tested for each experimental condition. Statistical comparisons were made by using the two-tailed Student's t test.
Apoptosis Analysis. Annexin V and propidium iodide staining for detection of early and late apoptosis was analyzed by flow cytometry as described (23). Caspase-3 activity was measured by using the CaspACE Assay System (Promega). Cells (1 x 105 cells per ml) were cultured with and without SB203580 at specified concentrations. The pan-caspase inhibitor Z-VAD-FMK was added at a final concentration of 50 µM. Negative controls were prepared by using untreated cells. We performed terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) for the detection of apoptotic nuclear fragments in the xenografted lymphomas, using the In Situ Cell Death Detection (Peroxidase) kit (Roche Molecular Biochemicals).
Immunoblot Analysis. Western blotting was performed following standard protocols using the following antibodies: p42/ p44MAPK, p38MAPK, phospho-p42/p44MAPK (Thr-202/ Tyr-204) E10 mAb, and phospho-p38MAPK 28B10 mAb (Cell Signaling). Immunoreactive bands were visualized by enzyme chemiluminescence (Amersham Pharmacia).
In Vivo Antitumor Activity. Six- to 8-week-old NOD/SCID mice were implanted s.c. with 2.5 x 106 SUDHL-4 cells in the flanks bilaterally. When the tumor implants were palpable, treatment was started with SB2083580 (three animals) at a dose of 4 µmol/kg or an equal volume of vehicle (three animals) injected i.p. five times a week. Perpendicular tumor diameters were measured, and tumor volumes were calculated by using the formula: 0.5(width2 x length). Four weeks after the initiation of drug treatment, the animals were killed, and the tumor implants were studied by histology and in situ TUNEL assay. The Institutional Animal Care and Use Committee of the University of Utah approved the experiments.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Growth Factor and Cytokine Receptors, Members of the RAS Family, and p38MAPK Are Differentially Expressed in Transformed FL. Our algorithm identified genes that showed consistent increasing or decreasing trends in the transition from FL to DLBCL within each matched pair. It also ranked genes that distinguished the FLs from the DLBCLs. Thus, we identified 113 genes that served as effective classifiers. Of these, 67 showed increased expression, and 46 showed decreased expression in the DLBCLs (Fig. 2 and Fig. 8, which is published as supporting information on the PNAS web site).
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The differentially expressed genes included growth factor and cytokine
receptors such as hepatocyte growth factor receptor (C-MET),
fibroblast growth factor receptor 3 (FGFR3), lymphotoxin
receptor (LTR), and platelet-derived growth factor
receptor (PDGFR), all of which have been described to
effect known growth-promoting activity in various cellular systems
(24–27).
N-RAS and various RAS-related genes were also differentially
expressed in the transformed FL (Fig.
2). In addition, p38MAPK was up-regulated in the DLBCLs
(Fig. 2). This finding is
interesting because p38MAPK is an important mediator of cytokine-induced,
stress-induced, and cellular responses. Interestingly, similarity analysis of
expression patterns revealed that the profiles most similar to p38MAPK
were generated by several of the known p38MAPK target genes such as
ATF-2 and several cytokines/receptors (Fig. 9, which is published as
supporting information on the PNAS web site). Up-regulation of
phosphatidylinositol 3-kinase was also observed in some of the transformed FL
(Fig. 2). We further trimmed
the differentially expressed genes to three genes that effectively separated
the FLs from the DLBCLs in three dimensions, based on the microarray data
obtained from the training set of samples
(Fig. 3a). The
separator genes that emerged included additional genes associated with RAS and
MAPK signaling, i.e., PLA2 (x axis),
PDGFR (y axis), and Rab6 (z axis)
(Fig. 3a). Interactive
3D animation is accessible at
www.path.utah.edu/labs/kojo.
The instructions for the interactive 3D animation are in Supporting
Text.
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Quantitative Fluorescent RT-PCR Validation of Microarray Data. We performed qRT-PCR for quantitation of PLA2, N-RAS, and C-MET to validate our microarray data. The microarray results obtained with the initial test set were corroborated by qRT-PCR (data not shown). We further validated the expression trends observed in the training set of 12 matched pairs of transformed FL by analyzing a validation set consisting of a group of unrelated FLs (n = 20) and DLBCLs (n = 20). The trends observed by microarray were corroborated in the unrelated samples. The calculated transcript levels for our selected genes were significantly higher in the DLBCL, N-RAS (P = 0.000849), antioxidant protein (PLA2) (P = 0.0422), and C-MET (P = 0.102) (Fig. 3b).
qRT-PCR Analysis Using a Minimized But Highly Discriminative Gene Set
Provides Accurate Class Assignment into FL or DLBCL Categories. We
selected PLA2, N-RAS, and
-1 transcripts for qRT-PCR to test the accuracy of class
assignment on an independent "test" set of FL (n = 20),
DLBCL (n = 20), and t(14;18)+ cell lines (n = 5). These
genes were selected based on their high ranking by our algorithm as strong
discriminators of FL from DLBCL. qRT-PCR amplification of these transcripts
yielded a classification accuracy of 100% for the FLs, 70% for the DLBCLs, and
100% for the t(14;18)+ cell lines. One case each within the FL and DLBCL
categories was scored as indeterminate
(Fig. 3c).
Immunohistochemical Validation: p38MAPK Protein Is Activated and Constitutively Expressed in Transformed FLs. We analyzed the expression of phosphorylated p38MAPK and p42/p44MAPK by using IHC of cell-line microarrays containing the five t(14;18)+ cell lines and biopsies from a cohort of FLs (n = 5) and DLBCLs (n = 6) independent from the samples used for microarray analysis. We detected nuclear phosphorylated (active) p38MAPK expression in 6/6 (100%) DLBCLs, 3/5 (60%) t(14;18)+ cell lines, and 1/5 (20%) FLs. We observed no reactivity for phosphorylated p38MAPK in the reactive germinal centers of 6/6 benign lymph nodes (Fig. 4). IHC for phosphorylated p42/44MAPK protein revealed no reactivity in benign GC B cells and 5/5 FLs. Low levels were detectable in one case of DLBCL and 1/5 t(14;18)+ cell lines (data not shown).
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p38MAPK Blockade Inhibits the Growth of t(14;18)+ Cell Lines. We investigated the functional consequence of pharmacological inhibition of the MAPK pathway by using selective inhibitors of p38MAPK (SB203580) and p42/p44MAPK (U0126). Treatment of two t(14;18)+/phospho-p38MAPK+ cells (SUDHL-4, OCI-LY1) with SB203580 (1–50 µM) led to a marked dose- and time-dependent decrease in cell viability as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay (Fig. 5a). Significant decrease in cell viability was observed with 50 µM SB203580 as early as 6 h (89.8 ± 1.3% of control; P < 0.05) with increasing viability loss at 24 h (76.3 ± 3.1%; P < 0.05) and 48 h (66.1 ± 3.1%; P < 0.05) for SUDHL-4 cells. Similar results were obtained for OCI-LY1 cells (Fig. 5a). In contrast, the SUDHL-4 cells were resistant to p42/44MAPK blockade using the p42/44-specific inhibitor U0126. Similarly, SB203580 treatment of SUDHL-5 cells that are t(14;18) negative and phospho-p38MAPK negative yielded negligible reduction of cell viability.
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Exposure of SUDHL-4 cells to SB203580 (30 µM) resulted in significant time-dependent induction of caspase-3 activity (12.6% at 6 h and 41% at 24 h; P = 0.02), which was inhibited by the pan-caspase inhibitor Z-VAD-FMK (Fig. 5b). Furthermore, SB203580 treatment also led to increased numbers of annexin V-FITC-positive cells compared with control DMSO-treated cells (15.9% vs. 8.5%), indicative of early apoptosis (Fig. 5c).
Western blot analysis of SUDHL-4 cells revealed high basal levels of phospho-p38MAPK. Treatment with 30 µm of SB203580 resulted in specific decrease in the level of phospho-p38MAPK by 6 h. The levels of total p38MAPK remained unchanged and served as protein loading controls. The levels of total and phospho-p42/44 were also unaffected by SB203580 treatment (Fig. 5d).
In Vivo Inactivation of p38MAPK Inhibits Tumor Growth by Inducing Apoptosis. We tested the efficacy of SB203580 in inhibiting the growth of SUDHL-4 cells xenografted into NOD-SCID mice. Tumors typically developed 2 weeks after s.c. inoculation of 2.5 x 106 cells into each flank. SB203580 was administered i.p. at a dose of 4 µmol/kg five times a week. This dose was chosen to achieve peak levels that were above the 50% growth inhibitory concentration deduced from the in vitro experiments. Our studies showed inhibition of tumor growth in the SB203580-treated animals from days 18 to 30. By contrast, mice receiving vehicle treatment exhibited unabated tumor growth until day 30 when the mice were killed (Fig. 6a). Microscopic examination of xenografts revealed significantly increased levels of apoptosis and karyorrhexis in the SB203580-treated animals as compared with the vehicle-treated animals. Correspondingly, in situ TUNEL assays revealed significantly increased apoptotic signals in the tumors exposed to SB203580 treatment when compared with the vehicle-treated tumors (Fig. 6 b and c).
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Discussion |
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We used a longitudinal approach comparing the global transcriptional
profiles of clonally related FL and DLBCLs occurring within the same
individual. This approach offers significant advantages by focusing on changes
distinctively associated with the transformation process, rather than random
differences associated with comparison of tumors from different hosts. We
observed higher levels of C-MET, FGFR3, LTR, and
PDGFR transcripts in the DLBCLs in comparison to the low-grade
FLs. Dysregulation of these genes have been implicated in the pathogenesis of
a variety of human tumors including primary leukemia and lymphoma cell lines
and multiple myeloma (27,
28). However, our study
suggests a role in FL transformation. Additionally, PDGFR was
differentially up-regulated in our DLBCLs and emerged as a useful
discriminator of FL from DLBCL in the 3D analysis. This finding is interesting
because PDGF is a known mitogen that induces the transcription of IL-6, a
potent promoter of B cell proliferation
(29). Of additional interest
was our observation of up-regulation of phosphatidylinositol 3-kinase, which
is also known to exert antiapoptotic and proliferative effects induced by
specific growth factors and cytokines
(25,
30).
The most consistently up-regulated gene identified by the pairwise
comparisons of the matched pairs of FL and DLBCLs in our study was
PLA2. Growth factor and cytokine-mediated
activation of cytosolic PLA2 (cPLA2)
plays a critical role in -2 macroglobulin-induced cell proliferation in
macrophages (31). Our study
implicates cPLA2 dysregulation in the pathogenesis of
lymphoma.
A unifying feature of the up-regulated growth factor and cytokine receptors
identified in our study is their connection to the RAS/MAPK pathway (Fig. 10,
which is published as supporting information on the PNAS web site).
Interestingly, our microarray and qRT-PCR studies also revealed up-regulation
of N-RAS in the DLBCLs (Figs.
2 and
3b). The RAS family
proteins are key mediators of cell growth, apoptosis, and responses to
extracellular signals. In addition to being significantly overexpressed in our
validation set of DLBCLs (P = 0.000849), N-RAS, together
with PLA2 and the -1 transcript,
served as a robust classifier of a blinded group of FLs and DLBCLs with 86.7%
accuracy. Our studies also revealed that a RAS-related gene,
Rab6, was down-regulated in the DLBCLs, and in combination with two
other RAS pathway genes provided clear segregation of the FL and
DLBCLs in a 3D model (Fig.
3a). This finding is in keeping with previous
observations that down-regulation of Rab6 in simian virus
40-tranformed keratinocytes correlates with the transformed phenotype
(32). Taken together, our
findings indicate a role for transcriptional dysregulation of RAS
pathway-related genes in FL transformation.
We observed increased levels of p38MAPK in the transformed FLs.
Moreover, we demonstrated that several p38 target genes including growth
factors and cytokines showed coordinately expression profiles across our
paired samples. Importantly, we demonstrated increased expression and nuclear
localization of phosphorylated p38MAPK protein in DLBCLs as compared with the
reactive germinal centers and FLs. In addition to its role in stress and
cytokine-induced cellular responses, p38MAPK activation is central to the
antiapoptotic and growth-promoting effects of many of the up-regulated growth
factor and cytokine receptors identified in this study
(25,
27,
31).
We thus hypothesized that p38MAPK may be a rational pharmacologic target for transformed FL therapy. We demonstrated growth inhibition in two p38MAPK-overexpressing t(14;18)+ cell lines by using SB203580, a selective inhibitor of p38MAPK activity. The growth inhibitory effect of SB203580 was specific and was observed only in p38MAPK-overexpressing cell lines. Furthermore, growth inhibition was preceded by decreased levels of phospho-p38MAPK and was affected via caspase-3-mediated apoptosis. The potential role of inhibition of p38MAPK activity for transformed FL therapy is further strengthened by our demonstration of inhibition of tumor growth in an in vivo model by using NOD-SCID mice xenografted with the t(14;18)+ and p38MAPK-overexpressing cell line.
In summary, our studies implicate dysregulation of several growth factor receptors connected to p38MAPK in FL transformation. Dysregulation of p38MAPK target genes may induce growth factors and cytokine genes and thus exert an autocrine-paracrine effect that further promotes lymphoma progression. Our studies also highlight the powerful applications of microarray analysis in the identification of potential targets for transformed FL therapy.
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Acknowledgements |
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Footnotes |
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Abbreviations: FL, follicular lymphoma; DLBCL, diffuse large B cell lymphoma; GCB, germinal center B cell; ABC, activated B cell; MAPK, mitogen-activated protein kinase; IHC, immunohistochemistry; qRT-PCR, quantitative RT-PCR; IgH, Ig heavy chain gene; PLA, phospholipase A; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.
To whom correspondence should be addressed. E-mail:
kojo.elenitobaj{at}path.utah.edu
or
megan.lim{at}path.utah.edu.
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) ranges
from 0 to 1 (gray bars). Conversely, the estimated probability of
classification of the same sample as DLBCL is 1–
