22nd Annual NASA Space Radiation Investigators' Workshop (2011)
7109.pdf
The contribution of non-targeted effects in HZE cancer risk: Impact on mammary stem cells
I. Illa-Bochaca1 , I. Fernandez-Garcia1, M. Gonzalez1, J. Tang, J-H. Mao2, S.V. Costes2 and M.H. Barcellos-Hoff1
1
Radiation Oncology and Cell Biology, New York University School of Medicine, New York, NY 10016
2
Lawrence Berkeley National Laboratory, Berkeley, CA 94720
Cancer is an important long term risk for astronauts exposed to protons and high energy charged particles
during travel and residence on other planets and the moon. There is no direct data of the risk from the extended
exposure to space radiation environment. We hypothesize that ionizing radiation targeted and non-targeted effects
(NTE) contribute to radiation carcinogenesis; NTE acting as a promoter for a greater carcinogenic risk of targeted
effects from high LET irradiation. Targeted effects result from interaction of energy with DNA, leading to DNA
damage response and changes in genomic sequence. On the other hand, poorly understood NTE alter phenotype and
multicellular interactions that could contribute to cancer. Using a radiation/genetic mammary chimera in which the
host is irradiated and the mammary stroma is subsequently transplanted with unirradiated Tp53 null mammary
epithelium, we have shown that NTE elicited by host irradiation significantly accelerates tumor development
(Nguyen, Oketch-Rabah et al. 2011). Interestingly, expression profiles of tumors from irradiated hosts, as well as
irradiated mammary gland, were enriched for a previously described mammary stem cell (MaSC) signature (Lim,
Wu et al. 2010). Stem cells are defined by two fundamental properties, ability to self renewal and multipotency,
which make stem cells prime candidates for neoplastic transformation. Acquisition of stem cell properties have been
previously linked to epithelial to mesenchymal transition (EMT) (Mani, Guo et al. 2008; Chaffer, Brueckmann et al.
2011). We have shown that both low and high LET radiation primes human epithelial cells to undergo TGFβ
mediated EMT in vitro (Andarawewa, Erickson et al. 2007; Andarawewa, Costes et al. 2011).
Non-malignant mammary epithelial cell line MCF10A were irradiated shortly after plating with graded
doses of low or high LET radiation (3-200 cGy) and cultured with transforming growth factor β 1 (TGFβ, 400
pg/ml) in serum-free media for 6-7 days. Under these conditions, the progeny of irradiated epithelial MCF10A cells
exhibit increased motility, aberrant morphogenesis, and an EMT phenotype and expression signature. Importantly,
the radiation dose response was ‘switch-like’, i.e. saturates at about 10 cGy and is radiation quality independent. As
a surrogate of stem cells, cultures were analyzed for the luminal and basal lineage commitment markers cytokeratin
18 (CK18) and cytokeratin 14 (CK14). Cells that express both markers (i.e. bi-potent) significantly increased when
compared with control cultures (i.e. for 10cGy, the frequency of bi-potent cells in the irradiated versus sham cultures
was 25.6% Vs 7.0% for γ-radiation, 31.0% Vs. 6.9% for 350 MeV Si and 26.7% Vs. 6.9% for 600 MeV Fe).
Stem cells biology and the EMT phenotype link together through its association with the Notch pathway.
Notch signaling regulates the mammary gland lineage commitment (Bouras, Pal et al. 2008) while its activation is
also required to induce EMT (Sahlgren, Gustafsson et al. 2008). We found that Notch was activated after HZE
exposure in the cells with the EMT signature. However, unlike EMT phenotype which require the combination of
radiation and TGFβ, Notch signal was induced by radiation itself.
We used gene expression microarray data analysis of mice irradiated with either low LET -radiation (100
cGy), or one of two fluences of 350 MeV/amu Si (11.10 cGy and 30.28 cGy) to identify gene expression patterns as
a function of radiation quality. Using Pavlidis template matching method (p<0.005 and fold change > 1.25) and
Conceptgen, we found highly significant enrichment for the MaSC signature identified by Lim et al.(Lim, Wu et al.
2010) one week after low or high LET radiation exposure. At week 4, the MaSC signature was still significant but
weaker, and at week 12, the MaSC signature was not enriched. In contrast, an EMT signature (Taube, Herschkowitz
et al. 2010), was transiently enriched at 4 weeks post irradiation.
To test whether high LET radiation affects the mammary stem/progenitor cell population, we compared a
group of mice irradiated with Fe (600meV) with a control sham group. Female mice at 3 weeks old were irradiated;
after 6 weeks the 4th mammary glands were collected for single cell extraction. The frequency of mammary stem
cells was assessed using a functional assay and the FACS profiles of CD24/CD49 cell surface markers associated
with repopulation capacity. Mammary epithelial cells from mice irradiated with Fe ions had 40% greater
repopulation capacity compared to cells from sham irradiated mice. We then evaluated lineage commitment
signaling in mammary epithelial tissues after HZE radiation exposure using high content image analysis of epithelial
Notch and β-catenin. Analysis was performed using 4th mammary gland tissues and comparing Sham, low LET
gamma irradiation (1Gy), and two fluences of high LET silicon (350Mev) and iron (600 MeV) groups. Results
showed that double positive population for Notch and -catenin is decreased with irradiation.
These results suggest that ionizing irradiation alters mammary repopulation capacity through activated
Notch and β-catenin, and show commonalities in the response to HZE radiation between our in vitro and in vivo
models as found in the association between stem cells and EMT through Notch activation. NTE contribute via
distinct mechanisms on stem cell regulation and that could promote malignant transformation.