Qi L, Higgins SP, Lu Q, Samarakoon R, Wilkins-Port CE, Ye Q et al.SERPINE1 (PAI-1) is a prominent member of the early G0 G1 transition "wound repair" transcriptome in p53 mutant human keratinocytes. J Invest Dermatol 128:749-753

Journal of Investigative Dermatology (Impact Factor: 7.22). 04/2008; 128(3):749-53. DOI: 10.1038/sj.jid.5701068
Source: PubMed
Abbreviations: FBS, fetal bovine serum


Available from: Rohan Samarakoon
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SERPINE1 (PAI-1) Is a Prominent Member of the Early
Transition ‘‘Wound Repair’’ Transcriptome in p53
Mutant Human Keratinocytes
Journal of Investigative Dermatology (2008) 128, 749–753; doi:10.1038/sj.jid.5701068; publish ed online 20 September 2007
Serum-stimulation of quiescent (G
keratinocytes initiates a temporally
regulated program of transcriptional
activity required for G
transit and
subsequent entry into the proliferative
cycle (Qi and Higgins, 2003). Expres-
sion profiling of such ‘‘activated’’ kera-
tinocytes identified physiologically
relevant subsets of cell cycle/growth
state-regulated genes (Gromov et al.,
2002; Gazel et al., 2003). Indeed, non-
cycling human (HaCaT) keratinocytes
express a differentiated (i.e., super-
Abbreviation: FBS, fetal bovine serum
www.jidonline.org 749
LQiet al.
The Early G
Transition ‘‘Wound Repair’’ Transcriptome
Page 1
basal) genetic signature, whereas
the serum-stimulated transcriptome ap-
proximates that of transient amplifying
cells (Pivarcsi et al., 2001; Lemaitre
et al., 2004). Clearly, the associated
transcriptional responses dictate epi-
dermal cell lineage commitments by
impacting the expression of pathway-
relevant genes (Banno et al., 2004;
Lemaitre et al., 2004).
This report provides early evidence
regarding the comprehensive inventory
of genes expressed by human HaCaT-II4
keratinocytes during the initial stage of
cell-cycle re-entry. Re-introduction of
serum to quiescent HaCaT-II4 cells
stimulates G
exit and residence in a
short-lived ‘‘activated G
(i.e., the kinetically defined G
transition state) (Qi et al., 2006).
Microarray analysis of quiescent and
2 hours fetal bovine serum (FBS)-‘‘acti-
vated’’ HaCaT-II4 cells defined the
transcriptional signature of this early
window. A total of 54,675
expressed sequence-tagged genes were
analyzed with 41,083 directly com-
pared for groups A (quiescent) and B
(2 hours FBS-stimulated) and a total of
35,991 reproducibly assessed for all
three experimental conditions (i.e.,
66% of the total sequences available;
this includes group C [FBS for 2 hours in
the presence of puromycin included as
a first approximation of the immediate-
early response cluster]). Genes exhibiting
statistically significant (analysis of variance)
changes (two-fold increase or decrease)
distributed as follows: 1151 for A versus
B, 1241 for A versus C, and 1319 for B
versus C. Among the most prominently
upregulated mRNA transcripts were
those encoding proteins involved in
the initial growth response (EGR1-4),
extracellular matrix remodeling and
tissue invasion (uPA, uPAR, tPA, SER-
PINE1 (PAI-1), PAI-2, MMP-2, MMP-
12, CYR61), transcription (Myc, Fos,
Jun, KLF4, AT3, p300/pTAF), signal
transduction (DUSP1, -4, -8, -10,
MAPK3, TGF-a), proliferation (GAD-
D45a, GADD45b, CDK7, cyclins),
and apoptosis (CASP9, MCL2) (e.g.,
Figure 1a). Reverse transcription-PCR
and northern blotting validated the
expression data for selected genes.
When more stringent criteria were
applied to data filtering (i.e., set thres-
Act D added
2 hours after stimulation
5 10 20 20
U0126 (
Q+ + + + + +
––1.2 2.5 2.5
10 20 20
AG147 (
Y-27632 (
+Act D
Hours post-FBS
Figure 1. A significant fraction of FBS-induced transcripts encode proteins involved in cell proliferation,
transcriptional reprogramming, and control of pericellular proteolysis. The clustergram plot (a) illustrates
functional groupings for several highly expressed (X10-fold; red to orange shading) relative to more
moderately stimulated (42- to o10-fold; light to dark pink colored) genes (mapped using Ingenuity
Pathways software). (b, c) PAI-1 transcripts (both the 3.0 and 2.2kb species) are low to undetectable in
quiescent (Q) HaCaT-II4 keratinocytes and induced within 2 hours of serum re-introduction (FBS-2 hours) or
EGF, but not by replacement with serum-free medium (medium D). (b, c) PAI-1 expression is effectively
inhibited by prior incubation with actinomycin D (Act D) or the MEK inhibitor PD98059 (PD) but not by
puromycin (Puro). (b) PAI-1 mRNA decay rates in cultures treated with Act D 2 hours after FBS addition were
virtually identical to control mRNA decay profiles suggesting that PAI-1 repression was initiated between 2
and 4 hours post-serum stimulation. (d, e) Y-27632 and U0126 inhibited PAI-1 induction implicating both
the rho GTPase effector ROCK and MEK, respectively, in gene control. (d) Pretreatment of quiescent cells
with the EGFR inhibitor AG1478 similarly blocked PAI-1 expression indicating that EGFR ligands were
major contributors to the serum-responsiveness of the PAI-1 gene. Glyceraldehyde-3-phosphate
dehydrogenase (GAPD) hybridization provided a normalizing signal for (b and c) northern analysis
(Mu et al., 1998; Qi et al., 2006 for details), whereas (d, e) western blots were stripped and re-probed with
antibodies to ERK2 to confirm protein loading levels (described in Providence and Higgins, 2004).
Table 1. Genes exhibiting a X10-fold increase in expression 2 hours after
serum stimulation of quiescent HaCaT-II4 cells
expression Description
SERPINE1 97.7 Plasminogen activator inhibitor type-1
PLAUR 76.6 Urokinase plasminogen activator receptor
PLAUR 70.3 Urokinase plasminogen activator receptor
DTR 67.9 Heparin-binding epidermal growth factor-like precursor
NR4A3 65.7 Nuclear receptor subfamily 4, group A, member 3
CLC 62.0 Cardiotrophin-like cytokine
C8FW 61.1 Phosphoprotein regulated by mitogenic pathways
IL8 56.3 Interleukin 8
CLDN4 49.5 Claudin 4
GEM 48.5 GTP-binding protein overexpressed in skeletal muscle
EREG 48.0 Epiregulin
SPRR2B 46.7 Small proline-rich protein 2B
EGR2 46.3 Early growth response 2 (Krox-20 homolog)
Table 1 continued on the following page
750 Journal of Investigative Dermatology (2008), Volume 128
LQiet al.
The Early G
Transition ‘‘Wound Repair’’ Transcriptome
Page 2
hold of X10-fold increase), 79 genes
were identified (Table 1) of which 75
also partitioned to the puromycin-resis-
tant subset. Rank order analysis indi-
cated that PAI-1 (SERPINE1) and the
uPA receptor (PLAUR) were the most
significantly elevated transcripts. uPA
increased (by 12-fold) as well (by
microarray and northern analyses),
although maximal uPA expression oc-
curred several hours later than PAI-1.
Additional genes that comprise the
‘‘tissue repair’’ subset and that were
upregulated 410-fold within the first
2 hours included DTR, IL8, EREG, HB-
SERPINB1, and TGFA. Northern blot-
ting confirmed that PAI-1 transcripts
were low to undetectable in quiescent
HaCaT-II4 cells, peaked in puromycin-
resistant manner 2 hours after serum
addition (during residence in the initial
activated G
substate; Qi et al., 2006),
and then rapidly decreased (Figure 1b
and c). Expression required EGFR/MEK/
rho-ROCK signaling during the G
transition (Figure 1d and e). Actinomy-
cin chase/mRNA decay and temporal
assessments of mRNA abundance in-
dicated, moreover, that PAI-1 tran-
scripts were substantially reduced
(from a maximum at 2 hours) as early
as 4 hours post-stimulation decreasing
further by 6–8 hours post-stimulation
(i.e., approximately mid-G
) likely due
to E2F1-mediated suppression (Kozic-
zak et al., 2001), consistent with a
narrow window of serum-initiated
transcription and short mRNA half-life
(1.5–2 hours) (Mu et al., 1998; White
et al., 2000).
Activation of a wound repair tran-
script profile appears to be a general
response to serum addition (Iyer et al.,
1999; this study). The present findings
indicate, furthermore, that PAI-1 is
the most prominent member of the
keratinocyte ‘‘serum response transcrip-
tome’’. Several SERPINS (i.e., PAI-1,
protease nexin-1), in fact, modulate the
complex process of injury resolution
through control of focalized plasmin-
mediated matrix remodeling, cell mi-
gration, and apoptosis (e.g., Bajou
et al., 2001; Li et al., 2000; Deng
et al., 2001; Degryse et al., 2004;
Rossignol et al., 2004; Wang et al.,
2005). Targeted PAI-1 knockdown/
Table 1. continued
expression Description
HB-EGF 41.3 Heparin-binding epidermal growth factor
PHLDA1 40.0 Pleckstrin homology-like domain, family A, member 1
IL11 38.1 Interleukin 11
CTGF 37.8 Connective tissue growth factor
KRTAP3-1 37.4 Keratin-associated protein 3-1
LIF 36.9 Leukemia inhibitory factor
EDN1 36.6 Endothelin 1
FOSL1 35.8 FOS-like antigen 1
TNFAIP3 34.4 Tumor necrosis factor, a-induced protein 3
TNFAIP3 32.9 Tumor necrosis factor, a-induced protein 3
EGR3 32.9 Early growth response 3
PTGS2 32.5 Prostaglandin-endoperoxide synthase 2
COPEB 31.1 Core promoter element-binding protein
ZFP36 30.6 Zinc finger protein 36
APOBEC3A 30.2 Apolipoprotein B mRNA editing enzyme
COPEB 28.9 Core promoter element-binding protein
DUSPI 28.3 Dual specificity phosphatase 1
NR4A2 28.0 Nuclear receptor subfamily 4, group A, member 2
FOXA1 27.4 Forkhead box A1
EDN1;ET1 26.7 Endothelin 1
DUSP10 25.3 Dual specificity phosphatase 10
IL6 25.1 Interleukin 6
SOCS3 24.8 Suppressor of cytokine signaling 3
KLF4 24.1 Kruppel-like factor 4
NR4A2 23.4 Nuclear receptor subfamily 4, group A, member 2
PTGS2 23.1 Prostaglandin-endoperoxide synthase 2
C20oorf16 21.4 Chromosome 20 open reading frame 16
DUSP4 21.0 Dual specificity phosphatase 4
ATF3 19.3 Activating transcription factor 3
PIM1 19.1 Pim-1 oncogene
MAFF-like 18.6 v-maf-like
JUN 18.3 v-jun sarcoma virus 17 oncogene homolog
NR4A2 18.1 Nuclear receptor subfamily 4, group A, member 2
IL1A 18.0 Interleukin 1, alpha
GADD45B 17.4 Growth arrest and DNA-damage-inducible, beta
HAS3 17.3 Hyaluronan synthase 3
EMP1 16.8 Epithelial membrane protein 1
SLC20A1 16.8 Solute carrier family 20 (phosphate transporter), member 1
GADD45B 16.8 Growth arrest and DNA-damage-inducible, beta
DSCR1 16.7 Down syndrome critical region gene 1
GADD45B 16.6 Growth arrest and DNA-damage-inducible, beta
ARTN 16.6 Artemin
B3GNT5 16.1 UDP-GlcNAc:betaGal b-1,3-N-acetylglucosaminyltransferase 5
Table 1 continued on the following page
www.jidonline.org 751
LQiet al.
The Early G
Transition ‘‘Wound Repair’’ Transcriptome
Page 3
overexpression and protein add-back
approaches, moreover, support the
contention that PAI-1 participates with-
in the global program of injury to
coordinate cycles of cell-to-substrate
adhesion/detachment and/or maintain
a stromal ‘‘scaffold’’ to satisfy the
prerequisites for both G
/S transition
and effective cellular migration (Planus
et al., 1997; Chazaud et al., 2002;
Palmeri et al., 2002; Providence et al.,
2002; Czekay et al., 2003; Providence
and Higgins, 2004). PAI-1 is also
expressed at high levels in senescent
cells where it likely interferes with uPA-
dependent growth factor activation (Mu
et al., 1998; Kortlever et al., 2006).
Certain ‘‘senescence-associated’’ genes
(i.e., p16
, PAI-1) may actually
function in the wound repair program
by inhibiting proliferation while pro-
moting migration (Chan et al., 2001;
Ploplis et al., 2004; Darbro et al., 2005;
Kortlever et al., 2006; Natarajan et al.,
2006). Indeed, keratinocytes at the
leading edge during wound re-epithe-
lialization are less mitotically active
than cells more displaced from the
motile front and express relatively high
levels of PAI-1 (Garlick and Taichman,
1994; Jensen and Lavker, 1996; Provi-
dence and Higgins, 2004). Collectively,
these data suggest that PAI-1 may
regulate the temporal cadence of cell-
cycle progression in replicatively com-
petent cells as part of the injury repair
The authors state no conflict of interest.
Supported by NIH grants GM57242 and
Li Qi
, Stephen P. Higgins
Qi Lu
, Rohan Samarakoon
Cynthia E. Wilkins-Port
Qunhui Ye
, Craig E. Higgins
Lisa Staiano-Coico
Paul J. Higgins
Center for Cell Biology and Cancer Research,
Albany Medical College, Albany, New York,
Center for Cardiovascular Sciences,
Albany Medical College, Albany, New York,
USA and
Department of Surgery, Weill
Medical College of Cornell University,
New York, New York, USA.
E-mail: higginp@mail.amc.edu
Table 1. continued
expression Description
RGC32 16.0 RGC32 protein
JUN 15.0 v-jun sarcoma virus 17 oncogene homolog
TRIF 14.9 TIR domain containing adaptor-inducing interferon-b
KLF4 14.6 Kruppel-like factor 4
IER3 14.5 Immediate early response 3
SERPINB1 14.4 Serine (or cysteine) proteinase inhibitor, clade B, member 1
RFX2 14.4 Regulatory factor X, 2
SGK 13.5 Serum/glucocorticoid-regulated kinase
ADM 13.5 Adrenomedulin
IL1B 12.9 Interleukin 1-b
SPRR3 12.8 Small proline-rich protein 3
HSPC159 12.6 Human galectin-related protein
EMP1 12.5 Epithelial membrane protein 1
PTGER4 12.3 Prostaglandin E, receptor 4
PLEKHC1 12.2 Pleckstrin homology domain containing, family C, member 1
LM07 12.1 LIM domain only 7
PLEKHC1 11.9 Pleckstrin homology domain containing, family C, member 1
ATF3 11.7 Activating transcription factor 3
PDCD1L1 11.7 Programmed cell death 1 ligand 1
CNK 11.6 Cytokine-inducible kinase
PPP1R15A 11.5 Protein phosphatase 1, regulatory (inhibitor) subunit 15A
FST 11.5 Follistatin
MAFF 11.1 v-maf fibrosarcoma oncogene avian homolog F
IFRD1 11.0 Interferon-related developmental regulator 1
SNARK 10.9 Likely ortholog of rat SNF1/AMP-activated protein kinase
ODC1 10.7 Ornithine decarboxylase 1
SLC2A3 10.4 Solute carrier family 2 (fac ilitated glucose transporter), member 3
TGFA 10.4 Transforming growth factor, alpha
GADD45A 10.3 Growth arrest and DNA-damage -inducible, alpha
B3GNT5 10.2 UDP-GlcNAac:betaGal b-1,3-N-acetylglucosaminyltransferase 5
SLC2A14 10.2 Solute carrier family 2 (facilitated glucose transporter), member 14
DUSP5 10.1 Dual specificity phosphatase 5
CUL1 10.1 Cullin 1
PPP1R3C 10.0 Protein phosphatase 1, regulatory (inhibitor) subunit 3C
ANOVA, analysis of variance; FBS, fetal bovine serum; cDNA, complementary DNA.
RNA isolated from quiescent or 2 hours FBS-stimulated keratinocytes was converted into single-
stranded cDNA using Superscript II reverse transcriptase and the GeneChip T7 promoter primer kit.
Double-stranded cDNA was prepared, biotinylated cRNA generated, hydrolyzed to 35–200 base
fragments, and hybridized to the Affymetrix Human Genome U133 Plus 2.0 oligonucleotide array.
Arrays were washed, stained with streptavidin-phycoerythrin, scanned and images analyzed
qualitatively with Affymetrix GCOS software; probe signal outputs (pivot tables) were imported as
text files into GeneSpring v6.1. Values below 0.01 were set to 0.01 and each was divided by the 50th
percentile of all measurements in that sample. Individual gene data points, in each of the experimental
groups, were divided by the median value in the corresponding control sets. ANOVA analysis (95%
confidence) compared groupings as follows: B versus A, C versus A, and B versus C. The statistically
significant genes (in triplicate assessments) were filtered to obtain lists based on expression levels (for
this paper, 10 increased relative to the corresponding control). Signal reproducibility is evidenced by
expression level values for duplicated genes on each array (indicated by color coding in bold font).
752 Journal of Investigative Dermatology (2008), Volume 128
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Journal of Investigative Dermatology (2008) 128, 753–757; doi:10.1038/sj.jid.5701119; publish ed online 25 October 2007
Burn-wound healing is a dynamic,
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Abbreviation: 2D, two-dimensional
www.jidonline.org 753
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Fetuin-A Promotes ‘‘Wound Closure’’
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    • "NFkB also emerged to be a possible transcriptional regulator of 13 out of 40 genes according to the reported informations [35], data which might further indicate that a growth factor-dependent NFkB signaling is activated in a subset of EOC. It is noteworthy that IL6 and 19 of the 40 correlated genes were found up-modulated upon 2 hr serum stimulation of quiescent keratinocytes [36]. We can therefore argue that the activation of growth factor activated signaling can either directly or indirectly induce the expression of IL6 and genes which likely play a role in the growth of EOCs. "
    [Show abstract] [Hide abstract] ABSTRACT: Epithelial ovarian cancer (EOC) is one of the most lethal gynecological cancers; the majority of EOC is the serous histotype and diagnosed at advanced stage. IL6 is the cytokine that has been found most frequently associated with carcinogenesis and progression of serous EOCs. IL6 is a growth-promoting and anti-apoptotic factor, and high plasma levels of IL6 in advanced stage EOCs correlate with poor prognosis. The objective of the present study was to identify IL6 co-regulated genes and gene network/s in EOCs. We applied bioinformatics tools on 7 publicly available data sets containing the gene expression profiles of 1262 EOC samples. By Pearson's correlation analysis we identified, in EOCs, an IL6-correlated gene signature containing 40 genes mainly associated with proliferation. 33 of 40 genes were also significantly correlated in low malignant potential (LMP) EOCs, while 7 genes, named C5AR1, FPR1, G0S2, IL8, KLF2, MMP19, and THBD were IL6-correlated only in advanced stage EOCs. Among the 40-gene signature EGFR ligand HBEGF, genes of the EGR family members and genes encoding for negative feedback regulators of growth factor signaling were included. The results obtained by Gene Set Enrichment and Ingenuity Pathway Analyses enabled the identification, respectively, of gene sets associated with 'early growth factor response' for the 40-gene signature, and a biological network related to 'thrombosis and cardiovascular disease' for the 7-gene signature. In agreement with these results, selected genes from the identified signatures were validated in vitro by real time RT-PCR in serous EOC cell lines upon stimulation with EGF. Serous EOCs, independently of their aggressiveness, co-regulate IL6 expression together with that of genes associated to growth factor signaling, arguing for the hypothesis that common mechanism/s driven by EGFR ligands characterize both advanced-stage and LMP EOCs. Only advanced-stage EOCs appeared to be characterized by a scenario that involves genes which are so far associated with thrombosis and cardiovascular disease, thus suggesting that this pathway is implicated in the growth and/or spread of more aggressive tumors. We have discovered novel activated signaling pathways that drive the expression of IL6 and of co-regulated genes and are possibly involved in the pathobiology of EOCs.
    Full-text · Article · Jul 2013 · BMC Genomics
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    • "Expression of genes for the major Has enzymes (Has1-3) and HA-degrading enzymes (Hyal1-3) was measured in injured REK 3-D cultures, and Has2 and Has3 were upregulated whereas expression of the hyaluronidases remained unchanged after injury (Fig. 6). Interestingly, the fact that Has3 is induced more than Has2 in our system is consistent with reports that Has3 is preferentially induced over Has2 in human keratinocytes exposed to cytokines (Sayo et al., 2002) or to serum in order to stimulate the " wound repair transcriptome " (Qi et al., 2008). From these data we conclude that the soluble factor (HB-EGF) induces overall HA production by increasing Has gene expression. "
    [Show abstract] [Hide abstract] ABSTRACT: Hyaluronic acid (HA), a glycosaminoglycan located between keratinocytes in the epidermis, accumulates dramatically following skin wounding. To study inductive mechanisms, a rat keratinocyte organotypic culture model that faithfully mimics HA metabolism was used. Organotypic cultures were needle-punctured 100 times, incubated for up to 24 hours, and HA analyzed by histochemical and biochemical methods. Within 15 minutes post-injury, HA levels had elevated two-fold, increasing to four-fold by 24 hours. HA elevations far from the site of injury suggested the possible involvement of a soluble HA-inductive factor. Media transfer experiments (from wounded cultures to unwounded cultures) confirmed the existence of a soluble factor. From earlier evidence, we hypothesized that an EGF-like growth factor might be responsible. This was confirmed as follows: (1) EGFR kinase inhibitor (AG1478) completely prevented wounding-induced HA accumulation. (2) Rapid tyrosine-phosphorylation of EGFR correlated well with the onset of increased HA synthesis. (3) A neutralizing antibody that recognizes heparin binding EGF-like growth factor (HB-EGF) blocked wounding-induced HA synthesis by > or =50%. (4) Western analyses showed that release of activated HB-EGF (but neither amphiregulin nor EGF) occured after wounding. In summary, rapid HA accumulation after epidermal wounding occurs through a mechanism requiring cleavage of HB-EGF and activation of EGFR signaling.
    Preview · Article · Feb 2009 · Journal of Investigative Dermatology
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    • "Microarray of the EMT transcriptome in several clinically relevant model systems has provided insights into the specific repertoire of " plasticity " genes. Plasminogen activator inhibitor type-1 (PAI-1; SERPINE1), the major physiologic regulator of the pericellular plasmin-generating cascade, is a prominent member of the subset of TGF-β1-induced, EMT-associated genes in human malignant keratinocytes [21] [26] [27]. In epithelial cells undergoing a mesenchymallike conversion in response to the E-cadherin transcriptional repressors Snail, Slug or E47, PAI-1 upregulation appears to be an essential characteristic of the plastic phenotype [28]. "
    [Show abstract] [Hide abstract] ABSTRACT: Increased transforming growth factor-beta (TGF-beta) expression and epidermal growth factor receptor (EGFR) amplification accompany the emergence of highly aggressive human carcinomas. Cooperative signaling between these two growth factor/receptor systems promotes cell migration and synthesis of stromal remodeling factors (i.e., proteases, protease inhibitors) that, in turn, regulate tumor invasion, neo-angiogenesis and inflammation. ranscript profiling of transformed human cells revealed that genes encoding wound healing, matrix remodeling and cell cycle proteins (i.e., the "tissue repair" transcriptome) are significantly up-regulated early after growth factor stimulation. The major inhibitor of plasmin generation, plasminogen activator inhibitor-1 (PAI-1), is among the most highly induced transcripts during the phenotypic transition initiated by TGF-beta maximal expression requires EGFR signaling. PAI-1 induction occurs early in the progression of incipient epidermal squamous cell carcinoma (SCC) and is a significant indicator of poor prognosis in epithelial malignancies. Mouse modeling and molecular genetic analysis of complex systems indicates that PAI-1 regulates the temporal/spatial control of pericellular proteolysis, promotes epithelial plasticity, inhibits capillary regression and facilitates stromal invasion. Defining TGF-beta1-initiated signaling events that cooperate with an activated EGFR to impact the protease-protease inhibitor balance in the tumor microenvironment is critical to the development of novel therapies for the clinical management of human cancers.
    Full-text · Article · Feb 2009 · Journal of Oncology
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