The Role of the Tumor
Microenvironment in Invasion and Metastasis of Pancreatic Cancer
Kelly Foley, Lei
Zheng
Kelly
Foley, The Sidney Kimmel Comprehensive Cancer Center, Department
of Oncology, The Skip Viragh Center for Pancreatic Cancer, The Sol Goldman
Pancreatic Cancer Center, Graduate Program in Cellular and Molecular Medicine,
The Johns Hopkins University School of Medicine, Baltimore, Maryland, the
United States
Lei
Zheng, The Sidney Kimmel
Comprehensive Cancer Center, Department of Oncology, The Skip Viragh Center for
Pancreatic Cancer, The Sol Goldman Pancreatic Cancer Center, Department of
Surgery, The Johns Hopkins University School of Medicine, Baltimore, Maryland,
the United States
Correspondence
to: Lei Zheng, MD, PhD, 488 Bunting Blaustein CRB I, 1650
Orleans Street, Baltimore, MD 21231, Maryland, the United States.
lzheng6@jhmi.edu
Telephone:
+1-410-502-6241 Fax:
+1-410-614-8216
Received: August 15,
2013
Revised: September 27, 2013
Accepted: September 29, 2013
Published
online: November 18, 2013
ABSTRACT
Pancreatic cancer, a devastating disease with a median overall survival of
six months, is unique because the vast majority of the tumor is composed of
stromal cells. The stroma, primarily composed of fibroblast cells, immune cell,
and extracellular matrix proteins, has proven to be a major obstacle in the
treatment of pancreatic cancer because the signaling pathways that promote metastasis
are poorly understood. Recently, the hedgehog (Hh) and semaphorin signaling
pathways have been identified as playing important roles in pancreatic cancer
metastasis. The hedgehog pathway has been shown to regulate both cancer stem
cell (CSCs) maintenance as well as growth factor secretion by stromal cells.
Inhibition of the Hh pathway has been proven to be an effective therapeutic
strategy that results in not only a decrease in the therapeutically problematic
CSCs but also a decrease in the stromal compartment, which results in increased
tumor vascularization and pharmacodelivery. Additionally, the semaphorin genes
undergo frequent copy number alterations in pancreatic cancer. In other
epithelial-derived cancers, semaphorin signaling has been shown to mediate
tumor cell invasion into the nerves and blood vessels, as well as increase the
density of nerves within the tumor. Because of the frequency of perineural
invasion and associated pain in pancreatic cancer, the role of semaphorin
signaling in pancreatic cancer metastasis should be further explored. Further
understanding of the invasion-promoting pathways in the tumor microenvironment
will aid in our understanding of how the tumor microenvironment regulates
pancreatic cancer progression and will help to identify novel targets for
improving therapies.
© 2013 ACT. All rights reserved.
Key words: Pancreatic cancer; Invasion; Metastasis; Tumor
microenvironment; Epithelial-to-mesenchymal transition
Foley K, Zheng L.
The Role of the Tumor Microenvironment in Invasion and Metastasis of Pancreatic
Cancer. Journal of Tumor 2013; 1(6): 32-42 Available from: URL: http://www.ghrnet.org/index.php/JT/article/view/518
INTRODUCTION
Pancreatic cancer
remains the fourth leading cause of cancer-related mortality in the United
States. Thus far, surgical resection is the only potential cure for pancreatic
cancer. However, because of the aggressive nature of this disease, the lack of
early symptoms, and limitations in diagnostic tools, few patients present with
locally resectable disease. Approximately 80% of patients present with locally
advanced or metastatic disease at the time of diagnosis, which results in a
median survival of approximately ten and six months, respectively, following
diagnosis. Despite improvements in surgical techniques and chemotherapy, the
number of cases and deaths from pancreatic cancer has steadily increased over
the last five to ten years with the five-year survival rate for patients with
pancreatic cancer remaining steady at less than 5%[1].
Even if pancreatic cancer is
diagnosed early and is resectable, many patients have already developed distant
micrometastases, and metastatic pancreatic cancer responds poorly to
conventional chemotherapeutics[1]. Therefore, because of the wide
prevalence of metastatic pancreatic cancer, it is imperative that treatment
strategies aimed at preventing and treating pancreatic cancer metastasis be
developed.
Pancreatic cancer is one of
the most stromal-rich cancers. In many cases, only 10-40% of the pancreas tumor
is composed of tumor epithelial cells, while the remainder is composed of
stroma. The stroma is very heterogeneous and consists of pancreatic stellate
cells (PSC), fibroblasts, myofibroblasts, immune cells, blood vessels,
extracellular matrix (ECM), cytokines and growth factors[2]. Because
of the excessive desmoplasia, pancreatic cancers also tend to be hypovascular,
which impedes pharmacodelivery. In addition to drug delivery, the stroma has
proven to be a major obstacle in the treatment of pancreatic cancer because of
limited knowledge on the cross-talk between the tumor and stroma in the tumor
microenvironment.
MOLECULAR PATHOGENESIS OF PANCREATIC CANCER
The first step in
understanding pancreatic tumor progression and metastasis is to elucidate the
genetic alterations that occur during malignant transformation. Advancements in
modern molecular techniques have made it possible to determine the genetic alterations
that occur at each stage of not only pancreatic cancer development, but also
colon, cervical and breast cancer. It is now widely accepted that pancreatic
ductal adenocarcinoma (PDA) undergoes a distinct set of genetic alterations,
which correlate with histologically well-defined precursor stages, during the
progression from normal pancreatic ducts to invasive PDA. The first stage in
the transformation of pancreatic ducts is the development of a pancreatic
intraepithelial neoplasm (PanIN1A/1B), which is characterized by a G12D
mutation in Kras that occurs in 80-95% of PDA cases and an
overexpression of Her-2/neu in approximately 70% of PDA cases. The
second stage, PanIN2, is characterized by a mutation or loss of heterozygosity
at the p16 locus, which encodes for a tumor suppressor involved in
regulating the cell cycle. The prevalence of p16 loss increases with
increasing levels of atypia. The final pre-invasive stage of PDA, PanIN3, is
characterized by the inactivation of p53 in 50-75% of cases. Other mutations
that occur during this stage in a large percentage of PDA patients include
BRCA2, SMAD4 and DPC4[3].
Recently, it was discovered
that the clonal populations of pancreatic cancer cells that give rise to the
distant metastases are represented in the primary tumor, and metastasis from
the primary tumor occurs late during pancreatic cancer progression. Whole exome
sequencing and copy number analysis was previously performed on 24 invasive
PDAs, and a total of 1562 somatic mutation were detected with 25.5% of the
mutations being synonymous, 62.4% being missense, 3.8% being nonsense, 5.0%
being small insertions and deletions and 3.3% of the mutations occurring in
splice sites or within the untranslated region (UTR). Overall, Jones et al[4]
found approximately 63 genetic alterations per cancer. The majority of the
alterations were point mutations, but amplifications and deletions were also
detected. Not surprisingly, many of the genetic alterations that occur in
pancreatic cancer are in pathways involved in DNA damage control, cell cycle
regulation, Hedgehog (Hh) signaling, homophillic cell adhesion, small GTPase
signaling, transforming growth factor-β (TGF-β) signaling, regulation of invasion and
integrin signaling[5].
Further genetic analysis of
the metastatic lesions from these patients revealed two categories of
mutations. The firstcategory of mutations is termed ‘founder’ mutations, and
they account for approximately 64% of all mutations found in each tumor. The
second category of mutations termed ‘progressor’ mutations included
approximately 36% of all mutations found in each patient and was present in one
or more distant metastases but not the primary tumor[6]. This
suggests that the majority of genetic alterations in pancreatic cancer occur before
metastatic spread of the disease. The genetic alterations that occur in the
parental clone tend to be more deleterious than the alterations found in
distant sites, with many of these mutations contributing to chromosome
instability. Additionally, it was found that all mutations found in metastatic
lesions were clonal. In other words, these mutations were found in almost all
cells located at the distant site, and thus, were present in the cell that
invaded from the primary tumor and established the distant metastatic lesions.
A mathematical model was
used by the authors to estimate the timing of the genetic alterations. Yachida et
al[6] concluded that the time from tumor initiation to the birth
of the cell that gives rise to the parental clone containing all of the
mutations at the primary site is approximately 11.7 years. It takes
approximately another 6.8 years for the parental clone to invade and colonize a
distant site and 2.7 years until the patients’ death. Therefore, metastasis
occurs late during pancreatic tumor development. These findings are of
particular importance because it confirms that there is a large window of
opportunity for diagnosis and treatment before distant metastases occur and
treatment options become limited. The challenges now are to develop early
detection methods, identify metastasis-promoting pathways and develop
treatments to target the metastasis-promoting pathways.
MOLECULAR MECHANISMS OF PANCREATIC CANCER METASTASIS
Migration and invasion
The processes tumor
cells use to migrate to distant tissues are similar to the normal physiological
processes that occur during embryonic development, wound healing and immune
cell trafficking[7]. The process of cell trafficking in both
neoplastic cells as well as non-neoplastic cells involves a continuous cycle of
polarization of the leading edge of the cell, elongation, attachment of the
leading edge, detachment of the trailing edge and contraction of the cell.
Because the ECM acts as the substrate for cell migration, many ECM proteins are
important in this process. In addition, the cancer cell must invade the ECM of
its original tissue in order to enter the blood stream, lymphatics or nearby
nerves and travel to distant organ sites.
The first step in cell
migration is initiated by growth factors and chemokines, such as epidermal
growth factor (EGF), LPA, insulin-like growth factor-1 (IGF-1) and CXCR4/SDF1,
and involves the development of cell protrusions, called pseudopods, containing
filamentous actin and other structural and signaling proteins. Actin
polymerizes into filaments[8,9] at the leading edge of the cell to
stabilize the cell protrusion and allow the cell to contact and bind the
adjacent ECM via adhesion molecules, primarily integrins[10].
Transmembrane integrins couple to the actin cytoskeleton via adaptor proteins
and activate kinases to phosphorylate and dephosphorylate key proteins[10].
The clustering of integrins
and other adhesion molecules leads to the recruitment of cell proteases that
degrade the ECM components enabling the cell to invade into the underlying
blood vessels. Surface proteases cluster at the substrate bindings sites[11]
and cleave collagen, fibronectin and laminins, as well as pro-MMPs to create
active, soluble MMPs such as MMP2[12,13,14]. MMPs degrade
fibronectin, collagen, laminin and proteoglycans. MMPs also break cell-cell and
cell-ECM connections by cleaving adhesion molecules like E-cadherin and
CD44[15,16].
Elongation of the cell
occurs both before and during the formation of focal contacts. Myosin light
chain kinase (MLCK) phosphorylates and activates myosin light chain (MLC) to
activate myosin II. Active myosin II binds actin filaments to generate
contraction of the cell[17,18]. Dephosphorylation of MLC is
performed by MLC phosphatase (MLCtase) to stop the contraction cycle. Assembly
and contraction are regulated by Rho and ROCK[17,19].
Finally, the trailing end of
the cell must detach. Actin severing proteins such as gelsolin and cofilin
break up the formed actin filaments so that they can be recycled and reform at
the leading edge of the cell. Calpain cleaves the focal contacts between the
integrins and ECM proteins by cleaving the cytoplasmic tail of many integrins (β1 and β2)[20,21].
In addition, proteolytic cleavage also helps to weaken the focal contacts[22].
The major producer of
pro-migration and anti-migration signals in pancreatic cancer is the stroma.
The stroma, particularly fibroblast cells, produces fibroblast growth factor
(FGF), IGF-1 and hepatocyte growth factor (HGF), which all bind receptors on
the tumor cells to enable the cells to proliferate and gain motility[23,24,25].
In particular, HGF stimulation of PDA cells acting through c-Met and
neurophilin-1 results in increased invasion of these cells[26]. Thus,
treatment with an anti-uPAR (urokinase-type plasminogen activator receptor)
antibody to inhibit c-Met and IGF-1 receptor-mediated invasion decreases PDA
metastases in mice[27].
In addition, it has been
shown that activated PSCs produce periostin, which accumulates in the ECM and
promotes PDA cell invasion via α6β4-integrin[28,29].
Additionally, Rachagani et al[30] found that silencing of
MUC4, a type-1 transmembrane mucin, resulted in decreased invasion of PDA cells
through FGFR1 stabilization and pFAK, pMKK7, pJNK, pc-Jun and the PI-3K/Akt
pathway. The resulting decrease in MUC4 expression led to a downregulation in
mesenchymal-associated proteins, such as vimentin, N-cadherin, Twist, Slug and
Zeb1, and an upregulation in epithelial-associated proteins, such as E-cadherin,
Occludin, Cytokeratin-18 and Caspase-9[31].
Additionally, it has been
shown that many surface proteins increase invasion of PDA cells. Specifically,
Marchesi et al[32] found that CXCR4, which is frequently
expressed on the surface of pancreatic tumor cells, promotes motility,
invasion, survival and proliferation of PDA cells. Alternatively, Wey et al[33]
have shown that overexpression of VEGFR-1 induces migration and invasion of PDA
cells in an autocrine fashion. Therefore, because of the known role of VEGF in
angiogenesis, the authors suggest that therapeutic targeting of VEGFR-1 would
not only inhibit tumor migration but also suppress angiogenesis and thus
primary tumor growth[33].
Because of the extensive
stromal compartment present in PDA and the recent discovery that migration
signals originate from lymphocytes and stromal cells in the tumor
microenvironment, it has become increasingly important to understand the
molecular cross-talk between these cells and tumor cells. This knowledge will
not only help to reduce the stromal compartment and enable enhanced delivery of
chemotherapeutics to the tumor site but will also help identify important
targets for reducing further spread of this highly metastatic disease.
Epithelial-mesenchymal transition
One of the more
well-characterized mechanisms of cell movement and invasion is called the
epithelial-mesenchymal transition (EMT)[34]. EMT is a normal
morphogenic process that occurs during embryonic development and is therefore silenced
during adulthood. During tumor progression, however, the polar and basal
membrane anchored epithelial tumor cells can dedifferentiate into cells with
more mesenchymal-like characteristics. The first step in this transition is the
loss of cell-cell junctions, such as desmosomes, tight junctions and adherence
junctions, which can occur through loss-of-function mutations in cadherins or
catenins, upregulation in the expression of proteases that cleave cadherins[34,35,36,37],
gene silencing by cadherin promoter methylation or downregulation of growth
factor receptor expression[34]. In particular, cytokines produced by
the tumor microenvironment, such as HGF, can downregulate cadherins. The
changes in cell adhesive properties are also accompanied by changes in gene
expression. In particular, cells undergoing EMT loose cytokeratin expression as
well as claudin, occuldin, desmoplakin, Laminin I, Type IV collagen and E-cadherin
expression. While these cells loose epithelial marker expression, they begin to
express proteins more characteristic of mesenchymal cells such as N-cadherin,
vimentin, fibronectin, laminin 5, Type I collagen, Tenascin C, α5β1 and αvβ6. All of these
changes are induced by growth factors and cytokines from the tumor
microenvironment acting on the transcription factors slug, snail, Zeb1, Zeb2
and twist that regulate the expression of these proteins[38]. The
signaling pathways that regulate intracellular transcription factor activity
have the ability to orchestrate all of the steps of the invasion-metastasis
cascade, except for colonization[39]. Additionally, during EMT,
tumor cells will lose polarity and undergo cytoskeletal and organelle
rearrangement and redistribution[40].
EMT can also be induced by
activating Kras-mutations or Her2 overexpression[41,42]. In
particular, Botta et al[42] found that constitutive K-RasG12D
activation of ERK2 regulates invasion of PDA cells through MMP-1. In addition,
EMT is induced by ECM factors (e.g. type 1 collagen, hyaluronic acid),
cancer-associated fibroblasts, immune cells and many signaling pathways
including Hh, TGF-β, EGF, FGF, HGF or
IGF in PDA[43,44]. Specifically, Zheng et al[45]
have shown that tyrosine 23 phosphorylated, cell-surface AnxA2 is required for
TGF-β induced,
Rho-mediated EMT, which links AnxA2, a protein involved in GTPase-mediated
cytoskeletal rearrangement, with the process of EMT in pancreatic cancer.
MicroRNAs have also been
implicated in regulating EMT. In particular, the miR-200 family has been shown
to be a potent negative inhibitor of EMT that works through regulating Zeb1 and
Zeb2 to promote E-cadherin expression[46,47] in lung
adenocarcinoma. In pancreatic cancer, miR-197 induces EMT by targeting p120
catenin[48]. Conversely E-cadherin expression has also been
shown to be regulated by Src kinase, and this has been shown to be correlated
with EMT[49].
TGF-β is believed to be
the major inducer of EMT in PDA cells. TGF-β1 is overexpressed in many patients with
PDA and is associated with poor prognosis[50]. Interestingly, KrasG12D
expressing pancreatic tumors with deletions in the DPC4/Smad4 and TIF1γ genes develop few
metastases. This suggests that Kras-dependent activation of the MEK-ERK
signaling pathway is essential for TGF-β-induced EMT[51].
Additionally, Gordon et al[52] found that loss of the TGF-βRIII was necessary
for EMT and invasion of PDA cells. Furthermore, Smad4 was found to be a
negative regulator of STAT3. Therefore, loss of Smad4, which occurs frequently
during PDA development, results in unregulated STAT3 activation, which
increases that results in increased invasion of PDA cells[53].
Finally, bone morphogenetic
proteins (BMPs) play an important role in the EMT of PDA cells. In particular,
BMP-2, BMP-4 and BMP-7 were shown to induce EMT in the Panc-1 cell line. The
BMP-mediated invasiveness of Panc-1 cells is partly due to increased expression
of MMP-2[54]. Additionally, BMP reduces the expression of the TGF-βRIII, which is an
inhibitory receptor. Smad1, which is required for BMP-induced loss of TGF-βRIII expression and
invasiveness, is partially responsible for BMP-mediated MMP-2 upregulation.
Until recently, the EMT
process has been difficult to study in vitro and has been limited to
evaluating gene expression changes under different condition. However,
three-dimensional cultures were recently developed and are currently being used
for studying other solid tumors such as breast cancer to evaluate the
interaction between tumor cells and cells in the surrounding tumor microenvironment in vitro[55].
This assay is superior to previously utilized two-dimensional systems such as
monolayer cultures or transwell assays because it allows the tumor cells and
supporting stromal cells to directly interact mimicking the actual interaction
that occurs in solid tumors between the tumor and ECM[56]. Thus,
because of the importance of the tumor microenvironment in pancreatic cancer,
three-dimensional cultures are currently being utilized and optimized for
studying EMT in PDA and should help to shed light on the signals required for
these processes.
Intravasation and extravasation
Following EMT, the
tumor cells must leave their site of origin and enter the lumen of blood,
lymphatic and nerve vessels. This begins with tumor-associated macrophages
(TAMs) secreting chemokines and cytokines, such as EGF, to attract the tumor
cell to the vessel[57]. Chemotaxis of the tumor cell towards
lymphatic vessels is primarily facilitated by CCL21 or CXCL12, which are
produced by lymphatic endothelial cells and bind CCR7 or CXCR4 receptors on the
PDA cells[32,58]. Intravasation is achieved either by the production
of membrane extensions through gaps in the endothelial wall or by passively
entering vessels through preexisting entry sites[59]. Once in the
vessel, the tumor cells are subjected to a lot of force and mechanical stress
from the fluids in the vessel as well as immune surveillance. For this reason,
very few tumor cells survive in the vessels[60].
The mechanisms of
extravasation are similar to those of intravasation and diapedesis of
leukocytes at sites of inflammation. Extravasation depends not only on
tumor-endothelial cell interactions but also vascular permeability. The
receptors over expressed on cancer cells, such as CXCR4/CXCR7, CXCR6, CD44, VLA
and AnxA2 must respond to the ligands in the target organ such as SDF-1,
CXCL16, hyaluronic acid, FN, GAS-6 and AnxA2[61]. Similar to
invasion and intravasation, extravasation occurs when the cancer cells extend
pseudopodia, penetrate endothelial cell-cell junctions and degrade the ECM.
Little is known about the
signals responsible for intravasation and extravasation of pancreatic cancer,
because the lack of a good in vitro or in vivo model has limited
research in this area. Recently, our group developed a mouse model of liver
metastasis in which we inject tumor cells into the splenic vasculature of mice,
enabling the cells to directly enter the circulation and home to the liver,
which is the primary site of metastasis in PDA[45]. Using this
model, we are able to manipulate the tumor cells to identify important
molecules on the PDA cells necessary for extravasation and colonization of
distant organs. Additionally, through the use of different knockout mouse
strains, we are able to identify important molecules at the distant sites that
are necessary for extravasation and colonization. Data from these studies is
forthcoming and has identified a pathway unique to PDA that is important for
intravasation, extravasation and colonization of distant organ sites. Finally,
several transgenic mouse models including a Kras, p53, Pdx-1
Cre, RosaYFP mouse have been developed to track
pancreatic epithelial cells during tumor progression. Using this model, the authors
demonstrated that pancreatic epithelial cells undergo the process of EMT
extremely early and are able to invade the basement membrane, enter the blood
stream and metastasize to the liver as early as the PanIN2 stage[62].
The development of mice with labeled tumor and vascular cells will make it
possible to evaluate the signals required for intravasation and extravasation
using in vivo cellular imaging techniques.
Colonization
The site of tumor
cell colonization depends largely on the molecules expressed on the tumor
cells, the endothelial cells of the distant organ as well as the
microenvironment at the distant site. For example, the CXCL12 ligand expressed
in the lung, liver, bone marrow and lymph nodes binds the CXCR4 receptor
expressed on breast cancer cells. Therefore, the lung, liver, bone marrow and
lymph nodes are all common sites of metastasis for breast cancer[63].
While the expression of various surface proteins determines
the distant site the tumor cell will travel to, the tumor microenvironment
determines if the tumor cell will survive once there. Once cancer cells
extravasate they can undergo apoptosis, proliferate or remain dormant. Most
cells die quickly after extravasation[64]. Of the few that remain,
only 0.01% are able to proliferate and form macrometastases. The major factor
preventing proliferation of these cells is a sufficient blood supply. Many
tumor cells will remain dormant for years until they acquire mutations that
allow them to proliferate in a hypoxic environment, explaining why many
patients experience remission after many successful rounds of chemotherapy[65].
Thus, organs that promote angiogenesis are common sites of metastasis.
STEM CELLS IN PANCREATIC CANCER METASTASIS
Cancer stem cells
(CSCs) have recently emerged as a new potential therapeutic target for the
treatment of cancer, including pancreatic cancer. CSCs are a phenotypically
distinct subtype of cells found in all tumors and are characterized by enhanced
tumor-initiating potential, self-renewal and the ability to form any of the
cells found in the primary tumor[66]. Because of these properties,
CSCs are readily able to colonize distant organs. It is therefore suggested
that circulating tumor cells may in fact be CSCs. Additionally, CSCs are of
particular interest because they tend to be highly resistant to
chemotherapeutics (including gemcitabine), they have inherent metastatic
potential[67] and their presence in the tumor is associated with
poor survival[68].
One of the major
controversies and challenges surrounding CSCs, also referred to as
tumor-initiating cells, is establishing an accurate method to identify and
characterize these cells. Mouse models and colony forming assays have
previously been used to identify CSCs[69]. However, the preferred
method for identifying these cells would be through the use of cell surface
markers because this would allow for the cells to be isolated from the whole
tumor and characterized functionally.
The markers used to identify
pancreatic CSCs in vivo include CD44+, CD24+, ESA+, CD133+, ALDH+ and c-Met+[67,68,70,71].
For many of these markers, it is unclear if they are important functionally to
the CSC or are simply a phenotypic marker. Interestingly, because these markers
are not necessarily expressed together on CSCs, depending on the markers used a
different subset of CSCs will be isolated[68,72]. Therefore, it may
be more important to determine the functional role of these cells in order to
determine the best therapeutic strategy for eliminating these cells from the
tumor. Perhaps subsets of CSCs are important in tumor-initiation, while
different subsets are important for metastasis to distant sites. Hermann PC et
al[66] found that a subpopulation of CD133+ CXCR4+ CSCs in
pancreatic tumors were essential for tumor metastasis, such that, when these
cells were eliminated from the tumor, all metastatic potential was lost. Not
only is the function of these CSC subsets important, but their interaction with
distant niches may be equally if not more important. Therefore, it is important
that these subsets be further examined and characterized.
Recent studies have found a
correlation between CSCs and the EMT associated with invasion and metastasis.
In particular, ALDH+ cells in pancreatic cancer tend to be more invasive than
the remaining ALDH- subpopulation, and these cells also have characteristics of
EMT[67,72].
Several signaling pathways,
including the Hh pathway, have been implicated in regulating the self-renewal
properties of normal stem cells and CSCs[73]. Therefore, these
pathways may serve as excellent targets for eliminating CSCs in tumors. A
recent Phase II clinical trial using gemcitabine in combination with IPI-926, a
novel inhibitor of the Hh pathway, in patients with metastatic pancreatic
cancer demonstrated enhanced responses to cytotoxic chemotherapy[74].
Additional pre-clinical studies have shown a decrease in the frequency of CSCs
and decreased tumor formation and metastasis following inhibition of the Hh
pathway[75,76]. However, the direct mechanism of action is unclear
from these studies.
CSCs have emerged as a new
potential therapeutic target to prevent and treat metastatic pancreatic cancer.
However, further characterization of these cells and the pathways that promote
invasion and metastasis is warranted. In addition, it is important to
understand the interaction of CSCs and tumor cells in the tumor
microenvironment in order to target this niche within the primary tumor as well
as in distant organ sites to prevent further metastatic spread.
THE ROLE OF THE TUMOR MICROENVIRONMENT IN PANCREATIC CANCER
METASTASIS
In addition to the
role of the CSC compartment in the progression of pancreatic cancer, the tumor
microenvironment has gained considerable attention in recent years. A hallmark
of pancreatic cancer is the extensive desmoplastic reaction that occurs during
tumor progression. This dense desmoplastic reaction consists of primarily
fibroblasts, PSCs and an extensive deposition of ECM proteins (collagen I,
collagen III, fibronectin), as well as endothelial cells, immune cells, nerves
and lymphatic vessels. Clinically, the desmoplastic reaction is associated with
a less favorable outcome. It is believed that signaling from the tumor
microenvironment helps promote tumor growth, metastatic spread and drug
resistance[77]. Thus, it is important to understand tumor-stroma
interactions in order to develop therapies and improve patient outcomes.
Fibroblasts and pancreatic stellate
cells
PSCs, believed to
arise from mesenchymal, endodermal and neuroectodermal cell origins, express
glial fibrillary acidic protein and desmin in normal tissues. However, in the
case of cancer, these cells, derived from quiescent PSCs, fibroblasts, bone
marrow-derived cells or EMT, become activated, acquire characteristics of
myofibroblasts and begin to express α-smooth muscle actin (α-SMA). The primary
mechanism by which PSCs promote tumorigenesis and metastasis is through the
secretion of various cytokines, such as TGF-β, HGF, FGF, EGF, IGF-1 and Tenascin C
(TnC). In particular, co-culture of tumor cells and their associated
fibroblasts or with conditioned media from fibroblast cultures has been shown
to promote invasiveness of the tumor cells[78], which is at least in
part mediated by AnxA2[45]. Co-culture of tumor cells and stromal
cells results in an upregulation of the expression of several different gene
families including members of the CXC/CC chemokine family, hyaluronan synthase
2 (HAS2) and matrix metalloproteinases (MMPs), which are involved in tumor
invasion, angiogenesis and regulation of the ECM, respectively.
Hedgehog signaling
In addition to
maintaining the CSC compartment, Hh signaling has been implicated in the tumor
microenvironment of pancreatic cancer. Although, Hh signaling has not been
shown to contribute to normal differentiation of the pancreas, several reports
have demonstrated that the Hh pathway is misregulated during the development
and progression of pancreatic cancer[79]. In particular, sonic
hedgehog (SHH) is upregulated in pancreatic cancer. Bailey et al[80]
found that SHH expression correlated with the desmoplastic reaction that is
characteristic of pancreatic cancer. Overexpression of SHH results in the
presence of infiltrating fibroblasts with elongated nuclei that express
collagen I and fibronectin. However, tumor-bearing mice treated with the SHH
neutralizing antibody 5E1 did not have a desmoplastic response. In particular,
they found that SHH acted as a chemoattractant to recruit fibroblasts, which
enhances tumor progression. In addition, SHH can induce the differentiation and
proliferation of PSCs, which contribute to the desmoplastic reaction that
occurs in pancreatic cancer.
As previously mentioned,
Olive et al[75] demonstrated a decrease in the stromal
compartment following co-administration of gemcitabine and IPI-926, a Hh
antagonist. By decreasing the stromal compartment, they were able to
transiently increase the vascularization of the tumor, which correlated with
increased levels of gemcitabine within the tumor and resulted in transient
stabilization of the disease.
An additional study by
Feldmann et al[76] demonstrated a downregulation of snail
and an upregulation of E-cadherin expression, which was consistent with
inhibition of EMT in pancreatic cancer cells following inhibition of Hh
signaling with cyclopamine. More importantly, inhibition of EMT in these cells
resulted in a noticeable reduction in the invasion capacity of these cells in
vitro. Conversely, if Gli1 was overexpressed, the cells no longer
expressed E-cadherin and were remarkably invasive. Hh inhibition
inhibited metastatic spread in an orthotopic xenograph model, and in
combination with gemcitabine, significantly reduced the size of the primary
implant. In addition, cyclopamine preferentially decreased ALDH positive cells.
Based upon the results of
several Hh studies in mice, it appeared that inhibition of the Hh pathway was
an effective therapeutic strategy that resulted in not only a decrease in the
therapeutically problematic CSCs but also a decrease in the stromal
compartment, which resulted in increased tumor vascularization and
pharmacodelivery. In addition, inhibition of Hh signaling was shown to reduce the
size of the primary tumor as well as inhibit metastatic spread in mice.
However, disappointingly, these results were not observed in clinical trials.
Infinity pharmaceuticals conducted a double-blind, randomized,
placebo-controlled phase 2 clinical trial comparing IPI-926 and gemcitabine to
gemcitabine alone in 122 previously untreated patients with metastatic
pancreatic cancer and followed patient survival as their primary endpoint.
Preliminary analysis showed that the median survival for patients receiving
gemcitabine plus IPI-926 was less than the historical median survival for
single-agent gemcitabine of approximately six months[1,81]. While no
toxicities were reported, the results of this trial are extremely disappointing
given the promising results seen in preclinical studies, and it is still
unclear why this trial was unsuccessful.
Conversely, Kim et al[82]
at the University of Michigan saw promising results using GDC-0449 in
treatment-naïve patients with advanced pancreatic cancer. Pancreatic cancer
patients were treated with GDC-0449 for three weeks with gemcitabine being
added to the treatment on the second week. This group found a 50%
progression-free survival at three months with five patients achieving a
partial response and another four patients having stable disease. It is
possible that this trial was more successful because patients were treated
first with a Hh antagonist to disrupt the desmoplastic stroma and improve the
subsequent delivery of chemotherapy. However, because of the limited number of
patients treated in this trial, it is unclear if these results would be
significant when compared with the control arm[82]. Additional
studies treating patients with metastatic pancreatic cancer with LDE225,
gemcitabine and nab-Paclitaxel (clinical trial number NCT01431794) or GDC-0449,
erlotnib and gemcitabine (clinical trial number NCT00878163) are on-going and
the results of these studies are currently unknown[83,84]. Although
all current clinical trials have evaluated the use of Hh antagonists in
advanced pancreatic cancer, it may also be interesting to include patients with
resectable PDA to see if inhibiting Hh in the primary tumor is able to prevent
metastatic spread of the disease.
Extracellular matrix
Activated stromal
fibroblasts produce ECM components that alter tumor progression by directly
binding with surface proteins on the tumor cells[85]. Specifically,
many MMPs are frequently over expressed in pancreatic cancers and bind to and
interact with collagen I to promote invasion of these cells[86,87,88].
Moreover, hyaluronan (HA), a component of the ECM that is secreted by
pancreatic tumor cells[89], may signal through hyaladherins such as
CD44 to regulate receptor tyrosine kinases and small GTPases involved in EMT
and angiogenesis[90]. In addition to promoting invasion of tumor
cells, HA sequesters water in the tumor microenvironment leading to increased
interstitial fluid pressure (IFP) that, when coupled with inadequate lymphatic
drainage, leads to vascular collapse[91]. This contributes to the
chemoresistance that is characteristic of pancreatic cancer. Jacobetz et al[92]
demonstrated that enzymatic targeting of HA improves vascular blood flow and
drug delivery in a mouse model of PDA. Thus, targeting HA, or perhaps any
component of the ECM, may improve drug delivery in patients with PDA.
Recently, Halozyme developed
a PEGylated recombinant human hyaluronidase (PEGPH20) that has shown promising
results in patients with stage IV metastatic pancreatic cancer. In preclinical
studies, PEGPH20 has been shown to deplete the HA-rich matrix that surrounds
pancreatic cancer, possibly enabling better chemotherapy delivery to the tumor.
In a recent single-arm Phase 1b trial, 28 stage IV treatment naïve PDA patients
were treated with PEGPH20 and gemcitabine. The overall response rate (complete
response+partial response) by RECIST 1.1 criteria was 42 percent (95% CI 22-62)
for those treated at therapeutic dose levels of PEGPH20 (1.6 and 3.0 µg/kg) as
assessed by an independent radiology review (n=24). Additionally, in
subjects with high levels of HA,
the overall response rate was 64%. Because of the promising results, a Phase 2
randomized clinical trial has been initiated in patients with stage IV metastatic
pancreatic cancer to compare the efficacy of gemcitabine and nab-paclitaxel
either with or without PEGPH20. The primary outcome measure for this study will
be progression-free survival[93].
Immune cells
The immune reaction
in PDA is largely immunosuppressive and pro-tumorigenic, and it begins in the
earliest stages of pre-invasive disease and rarely results in an effective
response[94]. Therefore, it is presumed that inflammatory cells help
contribute to the progression and development of pancreatic cancer. Support for
this presumption comes from extensive evidence suggesting that chronic
inflammation of the pancreas, as in the case of pancreatitis, is a risk factor
for PDA[95]. In addition, several pro-inflammatory cytokines such as
IL-6, IL-8, IL-10 and IL-1 receptor antagonists are elevated in the serum of
patients with PDA, and these cytokines are associated with poor survival[96].
Interestingly, TAMs are one of the most abundant types of immune cells in the
tumor microenvironment[97]. TAMs are recruited to tumors by various
chemokines, VEGF, colony stimulating factor 1 (CSF-1), stromal-derived factor 1
(SDF-1) and thrombospondin-1 (TSP-1)[98,99,100]. Once activated,
TAMs secrete various factors such as PDGF, a potent mediator of PSCs
proliferation, into the tumor microenvironment, which contributes to the
desmoplastic reaction[101,102]. In addition, macrophages secrete
TGF-β to stimulate the
synthesis of type I collagens and fibronectin by PSCs[103,104].
Thus, the immune reaction within the tumor microenvironment further contributes
to the desmoplastic reaction that induces invasion of PDA cells.
Perineural invasion
The normal pancreas
is rich in ganglia and myelinated and unmyelinated nerve cells, and invasive
cancer cells are able to spread into the nerve sheath as an alternate route for
dissemination[105]. Therefore, pancreatic cancer has one of the
highest incidences of perineural invasion among all cancer types[106].
In PDA patients, the size and density of the pancreatic nerves are increased
compared to a normal pancreas. Interestingly, undifferentiated tumors tend to
have more perineural invasion[107]. The exact mechanism by which
perineural invasion occurs is unclear, but it appears to be mediated by an
interaction between molecules on the cancer cell and peripheral nerves. These
include many signaling pathways involved in pain as well as TGF-α, EGFR, the NGF
family and their receptors, the GDNF family and their receptors, chemokines and
their receptors (CX3CR1, Sema3A, PlxnA1, NRP1 and MMPs) as well as other
surface molecules and their receptors[106]. In particular, it was
demonstrated that the neurotrophic factor artemin can influence perineural
invasion of PDA cells[108]. Additionally, norepinephrine has been
shown to promote perineural invasion of PDA by increasing levels of phosphorylated
STAT3. Inhibition of STAT3 inhibited norepinephrine-induced expression of NGF,
MMP2 and MMP9 to decrease invasion of PDA cells[109]. Given that
perineural invasion is an indicator of metastatic spread, patients with
perineural invasion have a worse prognosis.
Semaphorins
Axon guidance genes
have recently been implicated in perineural invasion of tumor cells during
metastatic spread. In particular, the expression of semaphorins, which are
primarily involved in axon guidance and blood vessel formation during
development, has been shown to influence the invasiveness of many types of
cancer. In addition, semaphorins are important in the development of the
nervous, immune, cardiovascular, hepatic, gastrointestinal and musculoskeletal
systems, but the expression of this family of proteins is dramatically reduced
in the adult[110]. Because of the many roles that semaphorins play
during development such as cell adhesion, migration, angiogenesis and immune
system development, it is understandable that these pathways can become
reactivated in cancer to regulate cell proliferation, invasion and
apoptosis. Emerging evidence
suggests that semaphorins and plexins are important mediators of invasion and
metastasis of cancer. Recently, the semaphorins have been found to be
misregulated in a variety of different cancer types. Semaphorins can be
pro-tumorigenic or tumor suppressive depending on the type of semaphorin and
the type of cancer.
For example, Sema3C has been
associated with multi-drug resistance in cancer, and its expression is
upregulated following treatment with several chemotherapeutic agents[111].
In addition, Sema3C overexpression has been associated with poor survival in
ovarian cancer[112] and invasion in prostate cancer[113].
Sema3E expression has been
shown to be upregulated in breast, melanoma, colorectal and ovarian cancers.
Overexpression of Sema3E correlates with increased metastatic ability[114]
and EMT-like characteristics in ovarian cancer[115]. Additionally,
Sema5A has been found to be overexpressed in prostate, gastric and pancreatic
cancer, and its overexpression correlates with invasion and metastasis
regulated by Plexin-B3 signaling[116].
Finally, expression Sema3A
was found to be correlated with poor prognosis in PDA patients[117]
but suppresses tumor growth and metastasis formation in a mouse model of
melanoma[118]. While it is unclear the exact mechanisms by which
semaphorins promote or inhibit metastasis of cancer cells, it is clear from
their role in development that these proteins are important in cell
trafficking.
Although the mechanism is
not entirely clear, it is known that semaphorins can influence invasion at
several levels. Sema3E induces focal adhesion disassembly and integrin
endocytosis and reduces cell adhesion[115,119]. Sema3E has also been
shown to promote an EMT phenotype in ovarian cancer by inducing the
translocation of snail into the nucleus in a MAPK and PI3K-dependent manner[120].
Sema3C has been shown to decrease E-cadherin levels in prostate cancer[113],
but overexpression of Sema3C correlates with metastasis of lung adenocarcinoma[121].
Sema3A-NP1 signaling
increases adhesion and decreases metastasis by increasing the expression of a
metastatic suppressor, α2β1, through a GSK-3β-dependent pathway[122].
However, autocrine Sema3A signaling can mediate glioma cell migration, invasion
and metastasis[123] through NP2, an atypical receptor for Sema3A[124].
One of the better
characterized signaling pathways in cancer is Sema4D-PlexinB1. When Sema4D
binds to and induces transphosphorylation of PlexinB1, PlexinB1 recruits Grb2
and couples with p190RhoGAP[125]. RhoGAP then inactivates RhoA to
inhibit cell migration[125]. Conversely, Sema4D-PlexinB1 signaling
promotes perineural invasion in a RhoA/ROC-dependent manner. PlexinB1
overexpression in the tumor causes the tumor cells to be attracted to nerves
that express high levels of Sema4D. Interestingly, the authors also noted that
the nerves were stimulated to produce dendritic like projections toward tumors
that overexpressed PlexinB1. In addition, tumors that secrete Sema4D have a
high density of nerves[126].
Additionally, secretion of Sema5A has been shown to be
upregulated in pancreatic cancer, and its upregulation is correlated with
increased invasiveness and metastatic spread of pancreatic cancer[110].
In particular, Sadanandam et al[127] found that the
extracellular domain of Sema5A is secreted from pancreatic tumor cells, which
correlates with increased invasion to the liver through enhanced ERK
phosphorylation. Furthermore, they demonstrated an increase in microvessel
density following culture with conditioned media from pancreatic tumor cells
that secrete Sema5A suggesting that the increase in metastasis to the liver is
due to increased angiogenesis, which is mediated through IL-8 and VEGF.
PROSPECTIVE
Pancreatic
cancer-related deaths continue to increase annually despite an overall decrease
in cancer-related deaths in the United States and in the world. The unique
tumor microenvironment of PDA, composed primarily of dense stroma, supports the
rapid growth and invasion of PDA cells making it an important target for
effective therapies. Although, interactions between tumor cells and stromal
cells have been identified in other epithelial-derived cancers, the mechanism
by which the stroma mediates invasion of PDA is largely unknown. Investigating
invasion-promoting mechanisms within the tumor microenvironment will aid in our
understanding of how the tumor microenvironment regulates pancreatic cancer
progression and will help to identify novel targets for improving therapies.
ACKNOWLEDGMENTS
This review is part
of a thesis dissertation written by Kelly Foley. We would like to thank Dr.
Elizabeth Jaffee for her careful reading of this review and helpful
suggestions. This work was supported in part by National Institutes of Health
Grant K23 CA148964-01 (L.Z.), the NCI SPORE in Gastrointestinal Cancers P50
CA062924-14 (L.Z.), Johns Hopkins School of Medicine Clinical Scientist Award
(L.Z.), the National Pancreas Foundation (L.Z.), Lefkofsky Family Foundation (L.Z.),
Lustgarten Foundation (L.Z.), Viragh Foundation and the Skip Viragh Pancreatic
Cancer Center at Johns Hopkins (L.Z.), and the Sol Goldman Pancreatic Cancer
Center (L.Z.).
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Peer reviewers: Kenoki
Ohuchida, MD, PhD, Department of Surgery and Oncology, Kyusyu university, 3-1-1
Maedashi Fukuoka, 812-8582, Japan; Kun Chun Chiang, General Surgery Department,
Chang Gung Memorial Hospital, 222, Mai-Chin Road, Keelung, Taiwan; Zhou Yuan
MD, Department of General Surgery, the 6th Affiliated Hospital of
Shanghai Jiaotong University, 600 Yishan St, Shanghai 200233, China.
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