Trial Information

The Roles of Neutrophil Elastase in Lung Cancer

Objectives

1. To demonstrate the presence of NE in vitro and the entrance into lung adenocarcinoma
cells

2. To identify the intracellular mechanisms through which NE mediates its tumor
proliferating effects

3. To verify the activation of NE and its related pathway component in clinical lung
adenocarcinoma specimens

Background of research

Lung Cancer in Hong Kong Lung cancer is the leading cause of cancer death in Hong Kong [1].
Surgical resection for early stage lung cancer is the only curative option and advanced
stage lung cancer is treated with chemoirradiation or molecular targeted agents, one of
which is Epidermal Growth Factor Receptor - Tyrosine Kinase Inhibitor (EGFR-TKI). Recent
advances in molecular biology of lung cancer allowed for development of these targeted
therapy. However, treatment response depends on the presence of activating EGFR gene
mutation, and the median response period is usually maintained for nine to twelve months,
after which the tumor usually become non-responsive or resistant to EGFR-TKI.

EGFR mutation vs EGFR wildtype lung adenocarcinomas

In Asian populations including Hong Kong, EGFR mutations at exons 18 - 21 are present in up
to 50% of lung adenocarcinomas, while the other 50% are EGFR wild type lung adenocarcinomas
and are usually bearing K-ras gene mutations [2]. Even with the presence of activating EGFR
gene mutations at exons 18 - 21, clinical response was observed in only 70% of patients with
tumors bearing EGFR mutations [3]. It is apparent that in a cohort of lung adenocarcinomas,
there can be the following groupings according to their EGFR gene mutation status:

(i) EGFR mutated lung adenocarcinomas (~50%)

1. with response to TKI treatment (~35%)

2. but unresponsive to TKI treatment (~15%) (ii) EGFR wildtype lung adenocarcinomas
(~50%) The presence of group (i)(b) tumors with EGFR but failed response to treatment
would mean suboptimal targeting of EGFR and the presence of group (ii) EGFR wildtype
tumors would imply alternative pathways mediating tumor progression and susceptibility
to tumor therapy in lung cancer. Exploration of molecular pathways in lung
adenocarcinomas may allow for discovery of new molecular targets for therapeutic
development in lung cancer.

Neutrophils vs cancer cells

Neutrophil infiltration, and hence inflammatory infiltrate, is a frequently observed in lung
cancer [4]. Recognized similarities between neutrophils and cancer cells include:

1. ability to circulate as single cells

2. target attachment via vascular system

3. target invasion Neutrophils achieve migration through pulmonary epithelial layers by
secreting NE, which is readily balanced by the local action of secretory leukocyte
peptidase inhibitor (SLPI). SLPI has also been detected in cancer tissues but the
relevant function role in lung cancer has not been defined yet [5]. The major
difference between neutrophils and cancer cells lies in the fact that migrated
neutrophils will undergo apoptosis while cancer cells can escape apoptosis and are able
to proliferate in target organs.

The possible roles of NE in lung cancer This has led to the speculation that neutrophils and
cancer cells may share similar inflammatory cascades by secreting a similar panel of
proteases, and one of these had been demonstrated to be neutrophil elastase (NE). The action
of NE may happen at different cellular levels: (1) action on extracellular matrix to
facilitate migration and tissue invasion; (2) action on cell surface signaling receptors; or
(3) intracellular action on signaling pathways following uptake into signaling endosomes.
There are recent evidence in animal studies that NE secreted by neutrophils actually moved
into lung tumor cells and mediates lung tumor growth via degradation of Insulin receptor
substrate-1 (IRS-1) and subsequent effects on the intracellular PI3K signaling pathways
acting on the intracellular tyrosine kinase domain of the platelet-derived growth factor
receptor (PDGFR)[6]. It is possible that neutrophils infiltrating lung tumor stromal tissues
may be acting in collaboration with cancer cells via the direct and indirect actions of NE.

Work done by others Immunoreactive NE, both in free and in complex forms, can be
demonstrated in breast cancer cell lines [7] and lung cancer cell lines [8]. NE activities
has been further demonstrated in human breast cancer tissues [9] but not in human lung
cancer tissues or normal lung tissues. These showed that NE is present at the tissue level
but their intracellular action, whether enzymatically active or signaling active, has not
been delineated clearly yet.

In breast cancer tissues, the higher the amount of extracted tissue NE, the worse was the
prognosis [9] and similar prognostic significance was suggested for NE in non-small cell
lung cancer [10].

When lung cancer cells were inoculated into severe combined immunodeficiency (SCID) mice,
concomitant administration of NE inhibitor, ONO-5046, will completely suppressed tumor
growth and appearance of metastatic foci [11]. The exact mechanism of action of ONO-5046
remains to be clarified.

NE has also been shown to cleave cell surface epidermal growth factor (EGF) and transforming
growth factor-alpha (TGF-α) [12, 13]. This may send aberrant signals to tumor cells EGF
receptors and promote cancer cell proliferation in vitro.

TGF-α also induced NE expression in human cancer cell lines (breast and pancreas)

There were recent works by Houghton et al [6] demonstrating NE could influence lung cancer
growth and development by:

1. permeating into lung tumor cells (A549 lung adenocarcinoma cell line with Kras
mutation) and, via degradation of IRS-1, mediating tumor cell progression.

2. being blocked by specific inhibitors (ONO-5046), leading to aberrant intracellular
signaling and then tumor cell growth arrest.

Work done by our groups NE in airway inflammation Our group had done work related to the
roles of NE in airway inflammation and diseases and we have raised cancer and normal
bronchial bronchial epithelial cell lines from local patients that would be appropriate
models for testing the hypothesis in local patient populations.

Syndecan-1 was found to be able to bind NE in sputum sol of bronchiectasis, supporting the
role of NE complexed with syndecan-1 in balance with antielastase [14]. We have also
demonstrated shed syndecan-1 from human lymphoid cell line was able to bind to NE,
disturbing the NE and anti-elastase balance in the airway environment, and thus could be
playing a role in airway inflammation [15].

We have tried to unopposed NE in the airway fluids in a cigarette smoke-induced model of
COPD in rats. Sulfated maltoheptaose to chelate NE was administered to rats and
bronchoalveolar lavage fluid (BALF) and lung tissues were collected 3 days after
administration that showed significant declines in NE activity and myeloperoxidase activity
as compared to BALF of smoking rats not treated. Significant reduction in neutrophil
accumulation was also observed in BALF and lung tissue sections. Airspace enlargement was
significantly reduced following the treatment. All these results suggested that sulfated
maltohepatose is a potential therapeutic against neutrophilic inflammation in the airways of
patients with COPD. (unpublished data).

We have also tested the NE activity of a lung adenocarcinoma cell line A549 and found that
radio-labelled cells migrate into lung cancer cells and is enzymatically inactive
(unpublished data) Lung cancer cell lines and normal bronchial epithelial cells and clinical
lung cancer tissue bank

We have recently established primary lung cancer and normal bronchial epithelial cell lines
from local Chinese patients and they were characterized with in vitro growth kinetics,
morphological and immunohistochemical studies and gene expression profiling [16]. At the
moment, we have:

1. Four primary lung adenocarcinoma cell lines (HKULC 1 - 4) published [16] and six more
newly established in various phase of characterization.

2. Two new normal bronchial epithelial cell lines immortalized with standard protocols
with cdk4 and hTert infections [17].

These new cell lines were all derived from clinical specimen from the local Hong Kong
Chinese patients and more are becoming established with continued accrual of clinical
collection. The clinical data of the patients, including gender and smoking history, from
which these primary lung adenocarcinoma cell lines originated were known. Together with 10
other ATCC lung adenocarcinoma cell lines and established normal bronchial epithelial lines
(HBEC-KT 1 - 5) from John Minna MD, Tx, USA, these cell lines will provide invaluable
materials to test for in vivo effects of NE on cancer cells.

With the collaboration of Queen Mary Hospital Cardiothoracic Surgical Department, we have
also collected 40 pairs of resected lung adenocarcinomas and normal lung tissues that will
allow for verification of in vitro findings in clinical specimens. Further collection
accrual of clinical specimens is in progress

The research questions to be addressed (Figure 1) are:

1. What are the roles of NE in lung adenocarcinoma proliferation?

2. How are such roles of NE mediated at the intracellular signaling level? Is
IRS-1/PI3K/PDGFR the most common axis involved in cancer cells? Or there will be
'cross-talk' with pathways such as EGFR or TNF-α in lung adenocarcinoma proliferation?

3. Can we verify such roles of NE in cancer proliferation in human lung adenocarcinoma
tissues?

The aims of the study will be:

1. To determine the mode of entrance and site of action of labeled NE in lung
adenocarcinoma cells

2. To study the activation of IRS-1/PI3K/PDGFR in lung adenocarcinoma cells after
penetrance by NE and if there will be concurrent activation of other signaling pathways
including the EGFR and TNF-α pathways

3. To study the effects of blocking NE with specific NE inhibitors on lung adenocarcinoma
cell proliferation

4. To verify the above mechanisms of actions of NE in clinical lung adenocarcinoma
specimens

Plan of study:

1. EGFR mutation profiling of lung adenocarcinoma cell lines and resected lung
adenocarcinomas Genomic DNA will be extracted from available lung adenocarcinoma cell
lines (20 specimens) as well as microdissected surgical lung adenocarcinoma specimens.
Exons 18 - 21 will be PCR-amplified and directly sequenced to read for EGFR mutation in
exons 18 - 21. This will characterize available lung adenocarcinomas cell lines and
clinical tumor specimens with respect to EGFR gene mutation or EGFR wildtype for
subsequent work, where experimental results will be compared between the two groups.

2. For aim (1) - (3), the mode of entrance and site of action of NE in lung adenocarcinoma
cells will be studied.

Live cell imaging Live cell imaging technique with Alex-Flour-488-labelled NE will be
employed to localize and follow the path of NE entrance into lung adenocarcinoma cells.
Eglin C, a proteolytic inhibitor, will be applied at the same time. Normal bronchial
epithelial cell lines will be used for comparison. The intracellular location of
entrance and possible activated structural changes will be recorded.

NE-specific reverse transcription PCR cDNA amplified from total RNA will be amplified
using RT-PCR with specific primers 5'-GAG GCA ATT CCG TGG ATT AG-3' and 5' ACG ACA TCG
TGA TTC TCC AG-3' Western blotting NE-stimulated cells from (1) above will have
proteins extracted by standard methods followed by immunoblotting with specific
monoclonal antibodies to study related signaling pathways including pAkt, Akt, p85,
IRS-1, IRS-2, PDGF, PDGF-α, pPDGF-α, p44/42 MAPK, phosphor-p44/42 MAPK as well as pEGFR
and TNF-α. Anti-β-actin will be used as endogenous control. Reaction to individual
markers will be done in triplicates Cell growth assay The effect of ONO-5046, a
specific NE inhibitor, on cell growth and proliferation will be studied on lung
adenocarcinoma cell lines compared to control (no drug administration).

3. For aims (4), we would like to study the activation of relevant signaling pathways upon
NE stimulation

1. Immunohistochemistry Immunohistochemical staining will be performed on clinical
lung adenocarcinoma specimens with specific monoclonal antibodies against NE,
PDGF, PDGFR, IRS-1 and EGFR. Concordance of staining pattern will be examined
between IRS-1 and EGFR or PDGF respectively. The proportion of discordant cases
will be compared with the proportion of concordant cases.

2. NE assay in clinical lung cancer specimens from human subjects Clinical lung
cancer specimens will be collected from patients with lung adenocarcinomas and NE
activities assayed.