# TGF-β1-Induced Expression of the Poor Prognosis SERPINE1/PAI-1 Gene Requires EGFR Signaling: A New Target for Anti-EGFR Therapy

Authors: Rohan Samarakoon; Craig E. Higgins; Stephen P. Higgins; Paul J. Higgins
Journal: Journal of Oncology
 (2009)

---

## Abstract

Increased transforming growth factor-β (TGF-β) 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-β 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-β1-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.


---

## Body

## 1. Introduction

Transition  of a normal epithelial cell to
an early malignant phenotype often involves mutation of the p53 and p21ras genes and progressive
increases in autocrine TGF-β1 expression [1–10]. Elevated TGF-β1
production, in fact, typifies advanced pathologies in both mouse and human SCC
[8, 10, 11]. Despite relatively high
concentrations of TGF-β in the immediate tumor microenvironment, some malignant
epithelial cells become refractory to TGF-β1-initiated proliferative arrest likely due to reductions
in either TGF-βRII and/or SMAD4 levels as well as the now recognized p21ras-dependent antagonism of
TGF-β1-mediated
growth inhibition/apoptosis [10–13]. In certain epithelial
malignancies, moreover, resistance to TGF-β1-mediated growth suppression is often coupled with
EGFR amplification or dysregulated EGFR signaling, particularly during the
later stages of tumor development [14–19]. The associated reprogramming of gene expression initiates and
perpetuates TGF-β1-induced
cellular “plasticity” (usually referred to as epithelial-to-mesenchymal
transition or EMT) which facilites tumor invasion and metastasis [8, 20–25].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
mesenchymal-like 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]. The association between PAI-1
expression and tumor “progression” has significant clinical implications. Current data suggest that this serine
protease inhibitor maintains an angiogenic “scaffold,” stabilizes nascent
capillary vessel structure, and facilitates tumor cell invasion through precise
control of the peritumor proteolytic microenvironment [29–31]. Increased
PAI-1 expression is, in fact, an early event in the progression of epidermal
SCC, often localizing to tumor cells and myofibroblasts at the invasive front
[24, 32–36] and, most importantly, is a biomarker with significant prognostic
value [37]. Indeed, two of the
best-validated prognostic indicators (level of evidence [LOE] = 1) in breast
carcinoma are the serine protease urokinase plasminogen activator (uPA) and its
endogenous inhibitor PAI-1 [38]. Certain
PAI-1 tumor thresholds predict both poor prognosis and reduced disease-free
survival in patients with breast, lung, ovarian, and oral SCC [29, 38] with the expression amplitude frequently associated
with the 4G polymorphism at the PE1 E box motif in the PAI-1 promoter [37]. Identification
of PAI-1 in tumor-proximal stromal myofibroblasts, furthermore, implies a more
global involvement in modulating cellular invasive potential [34–36], perhaps
as a matricellular effector of epithelial motility [39], invasion and the
associated angiogenic response [24, 30, 31, 40, 41].Recent findings clearly implicate EGFR/MEK/rho-ROCK signaling as required for PAI-1
expression in TGF-β1-stimulated
cells. E box motifs (CACGTG) in the
PAI-1 PE1/PE2 promoter regions, moreover, are platforms for a MAP
kinase-directed upstream stimulatory factor (USF) subtype switch (USF-1→USF-2)
in response to growth factor addition [42–44] suggesting that the EGFR/MEK/rho-ROCK axis impacts PAI-1 expression
through USF-dependent transcriptional controls. The continued definition of TGF-β1-activated
pathways that influence expression of this important target gene may lead to
therapeutically useful approaches to manage human cancer. This paper,
therefore, reviews data regarding the rapid transactivation of the EGFR in TGF-β1-stimulated
cells suggesting cooperativity between TGF-β1 and EGFR→MAP
kinase pathways in PAI-1 gene expression.


## 2. EGFR Signaling Is Required for TGF-β1-Induced PAI-1 Expression

TGF-β1 mobilizes both SMAD-dependent and -independent
signaling [45] although the individual roles of specific cross-pathway
events on PAI-1 expression are not well understood. Several recent studies demonstrated that TGF-β1-induced
rapid EGFR transactivation highlighting cooperativity between TGF-β1 and
EGFR signaling events in vascular, epithelial, and endothelial cells. Indeed,
PAI-1 induction in response to TGF-β1 is significantly attenuated by an EGFR pharmacologic
inhibitor (AG1478), by molecular targeting of EGFR activity (i.e., by
adenoviral delivery of EGFRY721A kinase-dead constructs) and, more importantly, by genetic ablation of the EGFR
in mouse fibroblasts [43, 46, 47] with PAI-1 “rescue” evident in EGFR−/− cells engineered to express an EGFR construct. TGF-β1 treatment, moreover, specifically increased EGFR
phosphorylation at the Y845 src-target
residue; either mutation of this
residue (EGFRY845F) or transfection of a DN pp60c-src construct completely blocked TGF-β1-dependent
PAI-1 induction. Similarly, TGF-β1 failed to stimulate PAI-1 expression in cultured mouse
embryonic fibroblasts (MEFs) genetically deficient in three src family kinases (i.e., c-src, c-yes-, c-fyn- null
fibroblasts;
SYF−/−/−) compared to
identically stimulated wild-type SYF+/+/+ cells. PAI-1 synthesis was restored in SYF−/−/− MEFs engineered to re-express a wild-type pp60c-src [47] providing
proof-of-principle for involvement of this particular src kinase in the inductive response. The highly specific src family kinase inhibitor SU6656, morevover, effectively blocked
TGF-β1-initiated
increases in both pp60c-src and EGFR phosphorylation as
well as pp60c-src and EGFR activation (at the Y416 and Y845 residues,
resp.). pEGFRY845 phosphorylation
in response to TGF-β1 was
evident, furthermore, in wild type but not SYF−/−/− fibroblasts. The
TGF-β1-dependent
formation of EGFR/pp60c-src complexes [46] and EGFRY845 phosphorylation and the inhibition of TGF-β1- (but
not PDGF-) induced PAI-1 expression by the EGFRY845F mutant as well
as a DN-Src construct [47] collectively implicate EGFR/pp60c-src interactions and, in
particular, the EGFRY845 pp60c-src site in the kinase domain activation loop in signal
propagation [48]. The time course of
TGF-β1-initiated
SMAD2/3 activation, in contrast, was similar in both wild type and SYF−/−/− MEFs confirming that, in the context
of either EGFR or src family kinase
deficiency, SMAD2/3 activation occurs but is not sufficient for PAI-1
induction. TGF-β1
stimulated ERK1/2 phosphorylation in EGFR+/+ but not in EGFR−/− cells consistent with prior observations that TGF-β1-dependent
ERK1/2 activation is downstream of EGFR signaling [43, 46]. EGFR−/− MEFs, however, are fully capable of responding to exogenous TGF-β1 as
SMAD2 was effectively activated (i.e., phosphorylated) in both wild type and
EGFR−/− fibroblasts [47].


## 3. The PAI-1 Gene Is a Model of TGF-β1-Initiated Cooperative EGFR Signaling

While TGF-β1
receptors phosphorylate SMADs downstream of growth factor engagement, it
appears that the Rho/ROCK pathway modulates the duration of SMAD2/3
phosphorylation [47]. How Rho/ROCK
impact TGF-β1-initiated
SMAD2/3 activation and subcellular localization [49, 50] is not known but this
pathway may function to provide efficient SMAD2/3 activation for extended
periods. Alternatively, Rho/ROCK
signaling may be required to inhibit negative regulation of SMAD2/3 function by
inactivation of SMAD phosphatases sustaining, thereby, SMAD2/3 transcriptional
actions (e.g., [51, 52]). TGF-β1-induced SMAD2 phosphorylation is not altered by EGFR
blockade either pharmacologically (with AG1478), molecularly (by expression of
EGFRY721A or EGFRY845F), or by the genetic absence of EGFR
[47]. Clearly, while SMAD2/3 activation
may be necessary it is not sufficient for TGF-β1-stimulated PAI-1 expression in the absence of EGFR
signaling.It is apparent, therefore, that TGF-β1
stimulates PAI-1 expression through two distinct but cooperating pathways that
involve EGFR/pp60c-src→MEK/ERK signaling and EGFR-independent, but
Rho/ROCK-modulated, TGF-βR-directed
SMAD and ERK activation [47]. Interference with any of the specific individual elements in this dual
cascade (EGFR/pp60c-src/MEK or Rho/p160ROCK) markedly reduced, and
in some cases, completely inhibited PAI-1 expression. One model consistent with the available data [24, 40, 43, 44, 47, 53]
suggests that SMADs and specific MAP kinase-targeted bHLH-LZ factors (such as
USF) occupy their separate binding motifs at the critical TGF-β1-responsive
PE2 region E box in the PAI-1 promoter (Figure 1). Dominant-negative interference
with USF DNA-binding ability significantly reduced TGF-β1-mediated
PAI-1 transcription[43, 44, 53]. Since MAP kinases regulate the DNA-binding and transcriptional activites
of USF [40, 43], TGF-βR
signaling through SMAD2/3 may actually cooperate with EGFR/MEK-ERK-activated
USF to attain high level PAI-1 expression [40, 47]. SMADs are known to interact with E
box-binding HLH-LZ factors such as TFE3 at the PE2 site in the PAI-1 geneat
least in one cell type [54]. There is
evidence, in fact, to suggest that such interacting complexes impact PAI-1 gene
control since USF occupancy of the PAI-1 PE2 region E box site, which is
juxtaposed to three SMAD-recognition elements, modulates transcription in
response to TGF-β1 or
serum [40, 43, 44, 53]. Current data
indicate that recruitment of this multicomponent complex likely requires
participation of the TGF-β1-stimulated
EGFR→MEK/ERK
and Rho/ROCK pathways for the optimal response of the PAI-1 gene to TGF-β1.Figure 1
Model for TGF-β1-induced PAI-1 expression. TGF-β1 activates two distinct signaling
pathways to initiate PAI-1 transcription. Rho/ROCK are required to maintain SMAD
phosphorylation and ERK activation (through to be defined mechanisms) while the
pp60c-src-activated EGFR
(at the Y845 site) signals to MEK-ERK initiating ERK/USF interactions resulting
in USF phosphorylation and a subtype (USF-1→USF-2) switch (e.g., [44]) at the PAI-1 PE1/PE2
E box sites. Collectively, these
promoter-level events stimulate high level PAI-1 expression in response to TGF-βR
occupancy. The actual mechanism
underlying EGFR activation in response to TGF-β1 may involve direct recruitment of src kinases to the EGFR or the
processing/release of a membrane-anchored EGFR ligand (e.g., HB-EGF). Events associated with TGF-β1
stimulation of the RhoA/ROCK pathway are similarly unclear. Rho/ROCK may regulate the activity and/or
function of the SMAD phosphatase PPM1A impacting, thereby, the duration of
SMAD-dependent transcription of target genes such as PAI-1. (modified from [47]).The mechanism of MAP kinase activation in TGF-β1-stimulated cells is just becoming clear. Upon ligand binding, the TGF-βRII undergoes autophosphorylation on three tyrosines
(Y259, Y336, Y424), while Y284 is a target site for src kinases [55]. TGF-βRI is also subject to tyrosine phosphorylation
postreceptor accupancy [56]. Such
phosphorylated tyrosine residues provide docking sites for recruitment of
Grb2/Shc/SOS complexes with subsequent mobilization of the ras-raf-MEK-ERK cascade
[46, 47, 55]. Although ERKs are
prominently activated in response to TGF-β1 [40, 43], perhaps the JNK and p38 MAP kinase
pathways are better characterized targets of TGF-β1-initiated signaling. TGF-β1 rapidly activates JNK through MKK4 and p38 via
MKK3/6 perhaps even in a cell type-specific fashion contributing to the
mechanistic complexity of pathway cross-talk. Each of these kinase systems, moreover, has been implicated in a cell
type-dependency of PAI-1 gene control [40, 43, 55]. Should such pathways prove uniquely or, at
least, preferentially utilized in specific cellular lineages, they may provide
tumor type-specific targets for intervention therapy.


## 4. EGFR as a Potential Therapeutic Target for Regulating PAI-1 Expression

Modulation
of EGFR/HER1 signaling by specific receptor function (kinase domain) inhibitors
or neutralizing antibodies against specific EGFR1 ligands (e.g., HB-EGF
antibodies) can be an attractive therapeutic modality (particularly in the context
of neoplastic diseases associated with elevated TGF-β1 levels). This strategy would likely impact not only
PAI-1 suppression but has the potential to regulate other proinvasive target genes. There is, in fact, increasing evidence that TGF-β1-induced connective tissue growth factor and
fibronectin expression similarly involve EGFR/HER1 cooperative pathways (Samarakoon
and Higgins, unpublished data). Moreover,
PAI-1 repression by EGFR signaling blockade may also suppress tumor
angiogenesis consistent with the well-established role of PAI-1 as an inhibitor
of endothelial apoptosis and neovessel
regression [40]. Combinatorial targeting of PAI-1 function using established
small molecule PAI-1 inhibitors and genetic-based PAI-1 expression attenuation
[40] coupled with disruption of EGFR signaling (e.g., with cetuximab or
erlotinib) may impact, therefore, both cancer invasion and the associated
angiogenic response, particularly in the context of a TGF-β1-rich tumor microenvironment.



---

*Source: 342391-2009-04-09.xml*