Examining the Cancer Stem Cell Hypothesis in Human Lung Cancers

The cancer stem cell (CSC) hypothesis, which suggests that
tumors are maintained by a population of cells possessing stem
cell characteristics, has emerged as an attractive explanation for
tumor growth, recurrence, and metastasis. CSCs in human leukemia,
breast, brain, colon, and pancreatic cancers have been
identified in transplantation assays. 1–6 Whereas the incidence
of oncogenic mutations, such as those in K-ras or the epidermal
growth factor receptor (EGFR), in human lung cancers has
been well described, the role of CSCs in lung tumors remains
poorly defined. An important goal for lung cancer research is
now to determine the role of CSCs in lung tumorigenesis. An
improved understanding of the cellular mechanisms of lung
CSC renewal should elucidate new therapeutic approaches for
lung cancer.
THE CANCER STEM CELL HYPOTHESIS
Although many current cancer therapies are based on their ability
to kill most cells within a tumor, it has been recognized for
more than 30 years that not all cells within a tumor are alike.
Studies have shown that only a fraction of cells within a tumor
can be propagated in patients, mouse transplants, or cell cultures.
7–10 These rare clonogenic cancer cells were hypothesized
to arise in a stochastic fashion: although the overall occurrence
is rare, any cell within the tumor is equally likely to exhibit
clonogenic activity. An alternative hypothesis to explain these
findings is that a rare subpopulation exists within tumors, and
that these rare tumor cells have unique biological characteristics
that provide clonogenic activity. In support of the latter
hypothesis, it has been recently demonstrated that several types
of hematopoietic and solid tumors harbor a distinct subpopulation
of cells called CSCs that can propagate the tumor phenotype
in vivo. 1–6,11 These cells are called CSCs because the
same molecular markers used to isolate the normal tissue stem
cells could be used to isolate clonogenic tumor cells in some
tissues, the cells could be passaged serially through mice (demonstrating
their ability to self-renew, a hallmark property of
stem cells), and the isolated tumor cell population gave rise to
a heterogeneous tumor (suggesting an ability to differen tiate, a
second hallmark property of stem cells). 12
The CSC hypothesis has also emerged as an attractive explanation
for tumor resistance to chemotherapy, recurrence,
and metastasis. It has been hypothesized that CSCs have distinct
biological mechanisms that render them more resistant to
chemotherapy than other cancer cells, explaining the refractory
nature of many tumors to treatment. 12–14 Specifically, CSCs
are hypothesized to be resistant to chemotherapy because they
may be quiescent and may efficiently export drugs, like stem
cells in normal tissues. 15 CSCs may also be the cells that are
required to generate metastases. For example, the pathways responsible
for the dissemination and homing of normal stem
cells during development may be aberrantly upregulated in
CSCs. Therapeutic strategies that specifically eliminate the
CSC population may therefore be more effective than standard
means of therapy. 16–19
STRATEGIES FOR IDENTIFICATION
OF CANCER STEM CELLS
Methods used in identification and characterization of stem
cell populations from normal adult tissues have proven to be
useful in uncovering CSC populations. The most widely used
technique for isolating stem cells has been fluorescent-activated
cell sorting (FACS) using a combination of cell-surface
markers that select cells with markers of more primitive cells
and exclude cells of differentiated cell lineages, followed by
transplantation of sorted cancer cells into immunodeficient
mice (Table 11.1). For example, CSCs were first identified in
human acute myeloid leukemias as the cancer cells that had
the same surface marker status as human hematopoietic stem
cells (CD34 CD38 ). 1 CD133, a positive marker of hematopoietic
stem cells and neural stem cells, has been used as
a marker of CSCs from brain and colon cancer. 3–5 CSCs have
also been shown to exhibit similarities to normal stem cells
with regard to their ability to self-renew in serial-plating experiments
in culture 5,20–24 as well as their demonstrated activation
of developmental pathways known to function in normal
stem cells. For example, chronic myelogenous leukemia
CSCs exhibit Wnt pathway activation as determined by elevated
levels of nuclear -catenin, 25 and mixed-lineage leukemia
CSCs are granulocyte-macrophage progenitors that share
a gene-expression program with hematopoietic stem cells. 24
Several pathways known to be crucial for development, such
as the Wnt, Hedgehog, Notch, and Polycomb-group protein
pathways, have been implicated in adult stem cell selfrenewal,
and dysregulation of these pathways contributes to
many types of cancer. 26,27
NON–SMALL CELL LUNG CANCER
Lung cancer remains the major cause of death from cancer
worldwide, 28 and relatively little is known about the molecular
heterogeneity of the cells within lung cancers. Lung cancer can be
divided into two histopathological groups: 80% are non–small
cell lung cancers (NSCLCs), and 20% exhibit neuroendocrine
features. NSCLCs can be further subdivided into adenocarcinomas
(50% to 60%), squamous cell carcinomas (20% to 25%),
and large cell carcinomas. The average 5-year survival rate for
NSCLC is only 16% because most lung cancers are refractory
to chemotherapeutics or quickly become resistant to therapeutic
response. 29 Also contributing to lung cancer morbidity, most
NSCLC patients already have advanced diseases at the time of
diagnosis; 21% of diagnosed cases have distant metastases in the
brain, bone, liver, or adrenal glands. 29–31 Surgery or therapies
that treat primary lung tumors rarely prevent metastases. For
example, 72% of patients who had NSCLC tumors surgically
removed eventually develop distant metastases, most commonly
in the bone or brain. 32 Lung CSCs may be responsible for these
observations, and an improved understanding of the cellular
mechanisms operating in lung cancers should elucidate new
therapeutic approaches.
Therapeutic Resistance of Lung Adenocarcinomas
Harboring Epidermal Growth Factor Receptor
Mutations The recent treatment success of gefitinib
(Iressa) and erlotinib (Tarceva), two small molecule inhibitors
of EGFR, in a fraction of patients with NSCLC has solidified
the premise that EGFR is an important molecule in the
pathogenesis of lung cancer (see Chapter 49). Several groups
have independently identified frequent somatic mutations in
the kinase domain of the EGFR gene in lung adenocarcinoma.
These occurred in up to 10% of lung adenocarcinoma specimens
sequenced in the United States and up to 30% of those
sequenced in Asia. The mutations are associated with sensitivity
to both gefitinib and erlotinib, explaining in part the
rare and dramatic clinical responses to treatment with these
agents. 33–35 Subsequent studies by multiple groups have now
identified EGFR kinase domain mutations from more than
600 lung cancer patients. These mutations cluster in four
groups or regions: exon 19 deletions, exon 20 insertions, and
point mutations at G719S and L858R. Exon 19 deletions and
exon 21 L858R point mutations account for more than 85%
of all EGFR kinase domain gefitinib- and erlotinib-sensitizing
mutations.
In the clinical setting, although these EGFR-mutant
NSCLCs initially respond rapidly and dramatically to gefitinib
and erlotinib, tumors eventually become refractory to treatment,
and nearly all patients who initially respond to these
drugs subsequently relapse. 36–38 Three studies identified EGFR
T790M mutations in approximately 50% of the tumors from
patients who relapsed. 39–41 These mutants, when combined
with sensitizing EGFR kinase domain mutation, permit the
continued growth of tumor cells in the presence of erlotinib
and gefitinib. Structural studies suggest that the T790M mutation
introduces a bulky methionine residue in the EGFR kinase
domain, which sterically hinders tyrosine kinase inhibitor
(TKI) binding. 36–38 Whereas gefitinib and erlotinib are reversible
inhibitors that mimic adenosine triphosphate (ATP), irreversible
inhibitors such as HKI-272 or BIBW2992 mimic ATP
and covalently bind to EGFR, enabling them to inhibit EGFR
kinase activity even in the presence of T790M. 42–44 Although
irreversible inhibitors are currently being tested in clinical trials,
animal models suggest that tumors will eventually become
refractory to these treatments as well. 44 Thus, it is crucial to
determine the cellular basis of lung adenocarcinoma resistance
to treatment and develop new therapeutic strategies that will
not be susceptible to resistance mechanisms.
Evidence for Non–Small Cell Lung Cancer Stem
Cells Several pieces of evidence suggest that NSCLC tumors,
including adenocarcinomas, contain a rare population
of cells with stem cell characteristics. The initial sensitivity of
human adenocarcinomas with activating EGFR mutations to
EGFR TKIs and the acquired resistance to these treatments
suggest that drug-resistant CSCs may be present in these tumors.
36–38 Side population cells , isolated by their ability to efflux
Hoechst dye, were identified in six human NSCLC cell lines.
These cells exhibit several stem cell characteristics, including increased
drug-exporting transporter expression, enriched tumorinitiating
capacity, and resistance to multiple chemotherapies. 45
Additionally, CD133 cells from human lung tumors were
recently shown to form self-renewing spheres in culture that
could propagate tumors when transplanted subcutaneously into
immunodeficient mice. 46 Importantly, although these studies
support the likelihood of CSCs in lung cancers, the isolation
and characterization of a population of human lung cancer cells
that can serially passage the lung tumor phenotype in the lung
microenvironment has not been reported, and the operation of
pathways that regulate stem cells in advanced lung tumors has
not been understood.
Identification of Normal Lung Stem Cells Recent identification
of putative endogenous and extrinsic lung stem cell
populations has added to the diversity of the respiratory system.
47–52 The pulmonary system contains various epithelial
cell populations. Each population resides in a distinct anatomical
location, or niche. 53 Basal cells, secretory Goblet cells, submucosal
glands, and ciliated cells line the trachea and upper
airways. The nonciliated columnar Clara cells that line the
bronchioles and terminal bronchioles secrete surfactants to
aide in oxygen exchange and provide a protective epithelial
barrier in the airways. The alveolar epithelium is composed of
alveolar type II (AT2) cells, the cuboidal epithelial cells that
produce surfactants and the resulting surface tension required
for gas exchange, as well as alveolar type I (AT1) cells, the flat
epithelial cells that deliver oxygen to the blood. Human and
murine lung adenocarcinomas most frequently arise in the distal
lung where AT2 and Clara cells reside, and these tumors are
frequently positive for molecular markers of either AT2 cells
or Clara cells.
Our laboratory determined that cells expressing both
the AT2 cell marker, prosurfactant protein-C (SP-C), and
the Clara cell marker (CCSP [also known as CCA], CC10,
utergloblin, Scgb1a1) are present in normal murine lung, and
that they constitute a stem cell population in the distal lung
epithelium. These cells, named bronchioalveolar stem cells
(BASCs), reside in the bronchioalveolar duct junction (BADJ)
in terminal bronchioles, which is the last portion of the airway
before the alveolar space. BASCs can be isolated from mouse
lung using a FACS methodology based on the presence of the
surface markers (stem cell antigen-1 [Sca-1] and CD34) and
the absence of the hematopoietic and endothelial cell markers
(CD45 and CD31), respectively. BASCs self-renew over multiple
passages and give rise to bronchiolar and alveolar cells
in culture, providing evidence that they are a stem cell population.
Further supporting the hypothesis that BASCs are
stem cells, they are quiescent in normal lung and proliferate
in response to lung injury. 47 Notably, Sca-1 cannot be used
to isolate human cell populations; separate studies are underway
in our laboratory to identify additional markers of murine
BASCs that may help in identification of human BASCs and
human lung CSCs.
Role of Bronchioalveolar Stem Cells in Murine Lung
Tumorigenesis To examine lung stem cells and their role in
cancer, we have used a mouse model that accurately recapitulates
human lung adenocarcinoma. K-ras, a component of the
Ras signal transduction pathway, functions in multiple aspects of
growth control and is mutated to an oncogenic form in 15% to
50% of human lung adenocarcinomas. 54–56 In “Lox-Stop-Lox”
K-ras (LSL-K-ras) mice, expression of oncogenic K-ras is spatially
and temporally controlled by a removable transcriptional termination
(stop) element. Intranasal infection with a recombinant
adenovirus-carrying Cre recombinase (AdenoCre) results in deletion
of the stop element, producing the Lox-K-ras allele that
expresses oncogenic K-ras G12D from the endogenous K-ras
promoter. These mice develop epithelial hyperplasia that appear
to progress to adenomas and overt adenocarcinomas. 57 The tumors
recapitulate the histopathological and molecular signature
of human lung adenocarcinomas. 57,58 Interestingly, BASCs were
also detected in these lung adenocarcinomas, indicating that they
may contribute to tumor growth and progression. 57 This raises
the possibility that BASCs in these murine lung cancers are the
CSC population