MOVING FORWARD IN LUNG CANCER STEM CELL BIOLOGY

Designing Experiments to Test the Cancer Stem
Cell Hypothesis: Transplantation and Culture An
important aim of basic lung cancer research is to determine
if human lung adenocarcinomas contain a CSC population.
Following the methodology that has been successful in other
malignancies, one method to test this possibility would be
to examine dissociated cells from human lung adenocarcinomas,
sort cells by FACS, and test the ability of tumor cell
subpopulations to propagate tumors with orthotopic transplantation
assays.
CSCs in human leukemia, melanoma, breast, brain, colon,
and pancreatic malignancies have been shown to be required for
serial rounds of tumor propagation in transplantation assays that
often utilize orthotopic strategies. 1–6,11 For example, the function
of breast CSCs was elucidated by injecting cancer cells into
the mammary gland, thereby mimicking the normal environment
of the tumor cells.
A current literature search indicates that the CSC hypothesis
remains largely untested for lung cancer. Experiments with
human lung cancer cell lines and sphere-forming lung cancer
cells have suggested that cancer cells that exhibit some properties
of stem cells are present within lung cancers. These studies
relied on the use of the Hoechst dye exclusion method to isolate
the side population and the presence of the CD133 cell-surface
marker, respectively. Importantly, fresh lung tumors were shown
to contain a CD133 cell fraction that could propagate lung tumors
when injected subcutaneously in immunodeficient mice. 46
Although the studies described previously constitute an
important beginning in addressing the CSC hypothesis in lung
cancer, several key experiments have not yet been reported.
First, serial transplantation of lung CSCs has not been demonstrated.
Because multiple rounds of propagation of tumors
are typically used to show the self-renewal capacity of putative
CSCs, this key property of stem cells has not been shown for
human lung cancer. Second, the use of subcutaneous injection
to test lung CSC activity does not reflect the normal cellular
milieu in which lung cancers arise, grow, and progress.
Particularly important for future work to develop therapeutic
intervention of CSCs, previous studies have shown that lung
tumor cells growing in subcutaneous regions do not exhibit
the same physiological response to chemotherapy as do tumors
growing in the lung. 59,60 Therefore, experiments to test for
lung CSC activity have not yet been done in the most relevant
tissue setting.
Future experiments should build upon the valuable studies
described previously with human lung cancer cell lines and work
in primary murine lung adenocarcinomas to determine if human
lung adenocarcinomas contain a CSC population. Importantly,
serial transplantations of uncultured primary tumor cells from
human lung and characterization of the resulting tumors are required.
Furthermore, testing the ability of cells sorted by both
the putative lung CSC marker CD133 and novel cell-surface
markers in serial transplantation assays will likely be helpful in
learning how to prospectively identify CSCs. If CSC surface
markers from other tissues are not useful for the lung, a side population
of human lung adenocarcinoma cells might be isolated
by their ability to efflux Hoechst dye; this technique has already
been verified to work well in human lung cancer cell lines. 45
Adenocarcinoma side population cells with CSC activity could
be isolated by FACS and characterized by microarray analysis
to elucidate new lung CSC surface markers. Transplantation of
tumor cells into the lung, where the normal microenvironment
may have important roles on cancer progression, niche effects
on CSCs, and cancer cell response to therapeutics, 61,62 should
be done to more clearly define the role of CSCs in human lung
adenocarcinomas or other types of lung cancer.
In parallel to testing for CSCs using murine transplantation
assays, a culture-based method to determine if a subset
of lung cancer cells exhibits the stem cell properties of
self-renewal and differentiation will be valuable. Self-renewing
sphere colonies were originally observed when primary tumor
cells from human brain were cultured on nonadherent plates
in serum-free media supplemented with EGF and basic fibroblast
growth factor (bFGF), 20 and have been subsequently
established from primary tumor cells of colon, breast, and
melanoma in human. 5,21–23 Tumor sphere culture conditions
could be used to identify human lung cancer cells that can be
propagated over multiple passages, providing a surrogate assay
to complement in vivo cell transplantation studies. The development
of this in vitro cell culture system would likely assist in
the isolation and characterization of human lung adenocarcinoma
CSCs. The establishment of cultures of lung adenocarcinoma
CSCs would also enhance future studies of the pathways
that regulate self-renewal and differentiation of these cells, as
well as for identification of small molecules that inhibit CSC
activity. Importantly, culture assays should only be used if the
culture conditions retain cells that exhibit the ability to propagate
tumors in the lung that are indistinguishable from those
arising from primary cell injections. The combination of in
vivo and in vitro assays for CSCs would provide the repertoire
of tools needed for functional analysis of lung CSCs.
Connecting Cancer Stem Cells and Chemotherapeutic
Resistance Distinct aspects of CSC biology,
such as more active DNA-damage response, may render CSCs
more resistant to therapeutics than other cancer cells. 12–14 In
addition, the quiescent nature of CSCs or their proposed ability
to efficiently export drugs, characteristics that have both
been shown in some stem cells in normal tissues, 63 may impact
the response to therapy. Elucidation of a lung CSC population
and therapeutic strategies that specifically eliminate lung
CSCs may offer more effective means of therapy that can be
combined with current clinical approaches. 12,14,64
Resistance to chemotherapy is a major cause of the
high mortality rate associated with advanced lung cancer.
Chemotherapy is associated with only a 20% to 40% chance
of tumor regression in advanced NSCLC, and thus, for most
patients, chemotherapy is ineffective. 65 Adenocarcinomas
bearing mutations in oncogenic K-ras are among the most
chemorefractory lung tumors, suggesting that mutant K-ras
CSCs in these tumors may be more inherently drug resistant
than other lung cancer CSCs. 66,67 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 The molecular mechanisms of EGFR-inhibitor
resistance have been well characterized in comparison with
other forms of chemotherapy for lung cancer, yet the cellular
nature of these mechanisms is poorly understood. For example,
it is not yet clear whether EGFR-inhibitor resistance
arises from de novo EGFR mutations after treatment or if
rare preexisting chemoresistant subclones expand after initial
treatment response.
CSCs may play an important role in the resistance of lung
adenocarcinomas. More specifically, it is possible that CSCs
are either more numerous or more chemorefractory in K-ras
mutant lung adenocarcinomas than in other forms of adenocarcinoma.
Second, EGFR-mutant NSCLC tumors may recur
during TKI treatments because of the presence of a resistant
CSC population. Evidence supports these ideas in other solid
malignancies. For example, it was recently shown that the percentage
of breast CSCs is significantly increased in the tumors
of patients treated with chemotherapy. 22
Future studies to connect CSCs to therapy response in
lung cancer may be best done by analysis of murine lung
cancers, because they currently offer several advantages over
patient samples for understanding the role of CSCs in adenocarcinoma.
First, the identity of human lung CSCs has
not yet been established, whereas a stem cell population that
appears to be important in tumorigenesis in a mouse model
of adenocarcinoma has been identified (above). Second,
there is typically no clinical basis for biopsy or surgical removal
of recurring or chemoresistant lung adenocarcinomas
from patients. In addition, with the exception of EGFRmutant
tumors, the timing of acquired chemoresistance is
not known in patients. These facts make it challenging to
acquire large numbers of fresh chemoresistant human lung
cancer samples in which to examine CSCs, and even more
difficult to have matched chemonaive and chemoresistant
cancer cells from the same patient. Many mouse models of
lung cancer are now available that accurately recapitulate
the mutational status and chemoresistance of human lung
adenocarcinomas, and one can easily obtain genetically
matched tumors that arise in the absence of chemotherapy
and at specific time points during or after the development
of chemoresistant tumors, providing materials that are simply
not available from patients in the same numbers. When
possible, primary human samples should be used to validate
findings in mice that establish the relationship between
lung CSCs and chemotherapeutic response.
Metastasis and Cancer Stem Cells: Another Missing
Link The CSC hypothesis predicts that stem cell characteristics,
which allow CSCs to disseminate and colonize to new
tissues, are responsible for metastatic disease. Importantly, even
for known CSCs, their role in metastasis is largely unexplored,
and it is possible that cells with stem cell properties cause metastases.
Just as described for matched chemonaive and chemoresistant
human lung cancer samples, it is challenging to obtain
large numbers of matched primary and metastatic lung cancer
samples from patients. In addition, although lung cancer metastases
to the brain are surgically removed, most advanced
stage primary and metastatic lung cancers in these patients are
not surgically removed. There is no documented improvement
of patient survival for operation of these advanced stage lesions.
Therefore, the question of metastasis and stem cells is another
setting ideal for the use of mouse models to obtain genetically
matched primary and metastatic lung adenocarcinoma cells. In
particular, LSL-K-ras p53-lox/lox mutant murine lung tumors
are an ideal model to use for the study of metastatic disease
because of the observed incidence of metastasis in these mice.
Archived human samples could also be examined to determine
if pathways identified in mouse studies are useful prognostic
markers of metastatic disease in patients.
Searching for Molecular Targets in Cancer Stem
Cells The CSC hypothesis predicts that the same pathways,
which are essential for promoting self-renewal of normal stem
cells, are also important for propagating the growth of cancer
cells. In fact, many cancer mutations cause upregulation of
pathways that promote self-renewal. 26 In addition, it has been
shown that CSCs, which are identified by markers distinct
from markers of normal tissue stem cells, can exhibit shared
gene-expression signatures with stem cells. 24 Thus, stem cell
pathways may be upregulated in chemoresistant lung adenocarcinomas
compared to chemonaive tumors.
A logical place to start examining the importance of
stem cell regulatory molecules in lung cancer are the Wnt,
Hedgehog (Hh), and Bmi-1 pathways, because they have been
implicated in stem cell self-renewal and lung biology. In addition,
evidence suggests that dysregulation of these pathways
contributes to NSCLC. Wnt pathways play a crucial role in
lung embryonic development, and more specifically, in cell
fate decisions and differentiation. 68–70 -catenin levels are
eleva ted in some NSCLCs; however, unlike many other cancers,
APC and -catenin mutations are rare in NSCLC. 70–74
The mechanisms leading to elevated -catenin levels in primary
NSCLCs, therefore, most likely occur upstream of -
catenin, for example, via overexpression of Wnt effectors or
repression of Wnt antagonists. 70–75 The Hh pathways play a
crucial role in lung embryonic development. 76 Most NSCLC
tumors exhibit increased expression of Hh-signaling proteins
compared to normal lung tissues, and 87% of lung adenocarcinomas
have increased levels of GLI1. 77 Moderate-to-high
levels of Bmi-1 are expressed in most NSCLC tumors, and
Bmi-1 status is a useful prognosis factor of lung adenocarcinoma
tumor metastasis and patient survival. 78,79 We have also
recently found that Bmi-1 is required for lung tumorigenesis
in the K-ras mouse model of lung cancer and is also required
for BASC self-renewal, suggesting that the Bmi-1 pathway is
one molecular link between stem cell function and tumorigenesis
in the lung. 80 Future studies will likely elucidate the
connections between these important pathways and others to
test for activation in lung cancer.
In addition to the pathways discussed previously, the CSC
hypothesis also predicts that the pathways responsible for the
dissemination and homing of normal stem cells during development
and wound healing are upregulated in metastatic
tumor cells. In support of this idea, adhesion, chemokine, and
cytokine receptors have been implicated in metastasis. 81 The
SDF-1/chemokine (C-X-C motif) receptor 4 (CXCR4) axis
plays a crucial role in hematopoietic stem cell (HSC) trafficking
and has been hypothesized to be harnessed by CSCs during
metastasis. 82 During embryogenesis, a chemokine SDF-1
gradient secreted by the bone marrow attracts HSCs expressing
CXCR4 from the fetal liver, and in adults, the gradient is also
crucial for retention of the HSCs in the adult bone marrow. 82
The SDF-1/CXCR4 axis is crucial in CSC-driven pancreatic
metastasis, whereas in CD133 pancreatic CSCs propagated
primary tumors, only CD133 cells that were CXCR4
were capable of producing metastases. 83 Although the role of
CXCR4 in tumor metastasis is the most well defined, other
chemokine receptors are also involved in invasion and migration:
CCR7 is involved in the dissemination of many types of
tumor cells to the lymph nodes; CCR10 is involved in homing
of melanoma cells to the skin 84,85 ; and chemokines such as
CCL2, CCL5, and CXCL8, can stimulate the migration of
some types of tumor cell lines. 84
Lung CSCs with chemokine receptors may initiate lung
cancer invasion and migration. Many types of human tumors
that exhibit high CXCR4 expression levels, including NSCLCs,
are prone to metastasis. 84,86 In NSCLC, recent studies have
shown that high primary tumor CCR7 and CXCL8 mRNA
levels are correlated with the presence of lymph node metastases.
87,88 Thus, it is likely that other chemokine receptors play
a role in lung cancer metastasis and that a more global survey
of chemokine receptors in adenocarcinoma could provide additional
molecules to examine in lung CSC studies.