Ionizing radiation induces stemness in cancer cells

Abstract

The cancer stem cell (CSC) model posits the presence of a small number of CSCs in the heterogeneous cancer cell population that are ultimately responsible for tumor initiation, as well as cancer recurrence and metastasis. CSCs have been isolated from a variety of human cancers and are able to generate a hierarchical and heterogeneous cancer cell population. CSCs are also resistant to conventional chemo- and radio-therapies. Here we report that ionizing radiation can induce stem cell-like properties in heterogeneous cancer cells. Exposure of non-stem cancer cells to ionizing radiation enhanced spherogenesis, and this was accompanied by upregulation of the pluripotency genes Sox2 and Oct3/4. Knockdown of Sox2 or Oct3/4 inhibited radiation-induced spherogenesis and increased cellular sensitivity to radiation. These data demonstrate that ionizing radiation can activate stemness pathways in heterogeneous cancer cells, resulting in the enrichment of a CSC subpopulation with higher resistance to radiotherapy.

Figure 1. Ionizing radiation increases spherogenesis in HCC cells.

A and B HepG2 cells (a) and Huh7 cells (b), were exposed to increasing doses of gamma radiation, and then seeded in stem cell media onto 96-well ultra low attachment plates at 500 cells/well. The sphere numbers were counted after 7 days (top) and 14 days (bottom) of culture, and relative numbers were reported on the graphs. Lower radiation doses of 2 and 4 Gy induced a significant increase in sphere formation in both cell lines compared to untreated samples. Results are presented as mean±SEM of four independent experiments. *p<0.05, **p<0.01 versus untreated control cells. C and D Representative images of HepG2 (c) and Huh7 (d) spheres formed after 14 days of culture in stem cell media. The images were captured using a digital camera (AmScope, iScope Corp., Chino, CA), mounted on a Zeiss Axiovert 25 inverted microscope. Magnification: 100x.

Figure 2. Ionizing radiation increases spherogenesis in the non-Side Population fraction of HCC cells.

A HepG2 cells (left panels) and Huh7 cells (right panels) were stained using Hoechst 33342 with (lower panels) or without (upper panels) Verapamil, and then sorted using a MoFLO2 fluorescence activated cell sorter. The R3 gate identified the Side Population (SP) fraction. Non-Side Population (Non-SP) cells, isolated via the R4 gate, were collected, rinsed in PBS and resuspended in stem cell media. B and C Unsorted, non-SP and PI-sorted HepG2 (b) and Huh7 (c) cells were exposed to 0, 2 or 4 Gy of gamma radiation and then seeded onto 96-well ultra low attachment plates at 500 cells/well. Sphere numbers were then counted after 7 days (top panels) and 14 days (bottom panels) of culture, and relative numbers were reported on the graphs. White bars represent unsorted tumor cells, black bars represent sorted non-side population (non-SP) cells, and hatched bars represent PI-sorted cells. After 14 days of culture in SCM, radiation doses of 2 and 4 Gy induced a significant increase in sphere formation in the bulk tumor population and in the non-SP population of both cell lines compared to untreated samples, while PI-sorted Huh7 cells showed significantly increased sphere formation following 2 Gy of radiation treatment. Results are presented as mean±SEM of five independent experiments (unsorted and non-SP populations) or mean±SEM of two independent experiments (PI-sorted population). *p<0.05, **p<0.01 versus untreated control cells.

Figure 3. Upregulation of stemness genes in HCC cells after radiation treatment.

A and B, E and F HepG2 and Huh7 cells were exposed to 0, 2 or 4 Gy of gamma radiation and total RNA was extracted after 3, 6 or 24 hours. Oct3/4 (a and b) and Sox2 (e and f) mRNA levels in each cell line were then determined by quantitative Real-Time PCR and normalized to GAPDH mRNA levels in each sample. In HepG2 cells, treatment with 2 and 4 Gy of gamma radiation induced a significant increase of Oct3/4 mRNA levels. Sox2 mRNA levels were also strongly upregulated in Huh7 cells following low dose gamma radiation treatment. Results are presented as mean±SEM of four independent experiments. *p<0.05, **p<0.01, ***p<0001 versus t0 sample or untreated samples. The dashed line represents a comparison to the control at the same time point. C and D, G and H HepG2 cells and Huh7 cells, were exposed to 0, 2 or 4 Gy of gamma radiation and Oct4 or Sox2 protein expression was determined by Western blot analysis after 6 hours (for Oct4; c and d), or after 4 hours (for Sox2; g and h). Oct4 and Sox2 protein levels increased following radiation treatment consistent with the increases in mRNA levels for each gene.

Figure 4. Silencing of stemness genes increases the sensitivity of hepatocellular carcinoma cells to gamma radiation.

A and B HepG2 cells (a) and Huh7 cells (b), were transfected with 100 nM of aiRNA targeting GFP, Sox2 or Oct4, and knockdown efficiency was evaluated by Western blot after 48 hours. Sox2 and Oct4 belong to the same regulatory circuit; therefore, single gene knockdown leads to reduced expression levels of both proteins. C and D HepG2 and Huh7 cells transfected with aiRNA targeting GFP, Sox2 or Oct4 were irradiated with 0, 2, 4, 6, 8 or 10 Gy of gamma radiation and equal numbers of cells were plated onto 6-well plates for colony formation assay. On day 7, colonies were counted and the fraction of surviving clonogenic cells expressed as a natural log was plotted. Lines are fitted using a first-order polynomial regression and represent the mean of four independent experiments. *p<0.05, **p<0.01, ***p<0.001 versus GFP-transfected cells.

Figure 5. Downregulation of Sox2 and Oct3/4 expression inhibits sphere formation induced by radiation treatment.

A and B HepG2 cells and Huh7 cells transfected with aiRNA targeting GFP, Sox2 or Oct4 were exposed after 24 hours to 0, 2 or 4 Gy of gamma radiation and then plated onto 96-well ultra low attachment plates at 500 cells/well in stem cell media. Sphere numbers for each knockdown group and radiation treatment were recorded on day 7 of culture in stem cell media. Silencing of Sox2 or Oct4 significantly reduced sphere formation in HepG2 and Huh7 cells treated with low doses of gamma radiation compared to non-irradiated cells, or cells transfected with aiRNA against GFP. Results are presented as mean±SEM of four independent experiments, n = 4. *p<0.05, **p<0.01, versus GFP-transfected cells or non-irradiated control cells (0 Gy).