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Insights into a problematic side-effect of anti-cancer therapy

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‘Radiation-induced Reprogramming of Breast Cancer Cells’

From Stem Cells 
Commentary by Carla B. Mellough

In solid cancer tumors, such as those found in breast cancer and glioma, the cancer stem cell (CSC) component represents only a small number of cells within the tumor - yet these cells are the most highly tumorigenic, being able to completely regenerate the tumor, are associated with higher risk of metastasis and recurrence, and are more resistant to radiation and chemotherapy than their differentiated progeny. Recent reports indicate that an unfortunate result of anti-cancer therapy is the enrichment of these highly tumorigenic CSCs within the tumor mass following treatment, which has been ascribed either to the selective killing of less tumorigenic progeny, or a shift from asymmetric to symmetric CSC division, resulting in an overall increase in CSCs1-3. To investigate this phenomenon, Lagadek et al.4 from UCLA have studied the activity of breast cancer stem cells (BCSCs) in three patient-derived breast cancer samples and several breast cancer cell lines following exposure to ionising radiation in vitro, and their results give us some insight into a problematic radiation-mediated phenomenon that has huge implications for current anti-cancer therapeutic approaches.

BCSCs exhibit high ALDH1 enzymatic activity, making this a useful marker of this cancer cell type. In their study, Lagadek et al.4 started by purifying then replating non-BCSCs from fresh breast cancer tissue on the basis of their ALDH1 negativity (ALDH1-) by fluorescence-activated cell sorting (FACS) and the following day exposed them to 0, 4 or 8Gy of radiation. Interestingly, analysis of the number of BCSCs within these cultures 5 days later revealed a dose-dependent increase in the number of ALDH1+ cells, with the frequency of these cells indicating that they were unlikely to have arisen from contaminating ALDH1+ cells following FACS. Another explanation for the emergence of ALDH1+ cells could be that some non-tumorigenic cells acquire a BCSC phenotype following exposure to ionizing radiation. To investigate this possibility, Lagadek et al.4 isolated the non-BCSC fragment of three human breast cancer cell lines (SUM159PT, MCF-7 and T47D) for more in depth analysis. In addition to their ALDH1 negativity, non-BCSCs exhibit a CD24+/high/CD44- expression profile and high protease activity. Using a ZsGreen- cODC reporter system as an indicator of protease activity (where high cellular protease activity results in a decrease in fluorescent protein), the authors identified BCSCs on the basis of their low proteasome activity and purged these cells from the population, sorting only the non-tumorigenic cells by combining all phenotypic indicators (CD24+/high/CD44-/ALDH1-/ZsGreen-cODC-). Five days following irradiation these cultures were analysed for their BCSC content which, akin to the results from patient-derived samples, showed a dose-dependent increase in the number of ALDH1+ cells.

As Notch has been implicated in CSC maintenance,5 the authors then repeated these experiments in the presence of a Notch inhibitor which resulted in partial attenuation of the BCSC-forming ability of non-BCSCs after exposure to ionising radiation. Using targeted siRNA, the activity of either Notch1, Notch2, Notch3 or Notch4 receptors in SUM159PT-ZsGreen-cODC cells was blocked, then the non-BCSC (ZsGreen-cODC-) fragment isolated for ionization experiments. This revealed that while downregulation of Notch2, 3 or 4 had little effect on BCSC formation, interference of Notch1 signaling prevented the formation of BCSCs. Interestingly, by mixing StrawberryRed-labelled BCSCs with non-BCSCs, the authors show that radiation-induced BCSC formation capacity was reduced in the presence of pre-existing BCSCs. To demonstrate that radiation-induced BCSCs (iBCSCs) had similar potency to native BCSCs, the authors then performed sphere-forming assays comparing irradiated samples against non-irradiated controls. Sphere formation is a measure of self-renewal capacity and closely related to tumorigenic potential. Consolidating previous results, this work showed that irradiated samples demonstrated enhanced sphere-forming capacity.

To further test the tumorigenic potential of iBCSCs, the authors isolated non-BCSC (SUM159PT-ZsGreen-cODC-) cells, exposed them to 0-8Gy of radiation and subsequently injected them subcutaneously into nude mice. The number of cells required to obtain tumors in 50% of the animals (TD50) was calculated for each radiation dose. The results showed that while TD50 values were high for non-irradiated cells (1.15x105 cells), one single dose of 4Gy radiation reduced this value 32 fold, meaning that less of the irradiated cells were required to form tumors and thus these cells were intrinsically more tumorigenic in nature. The gene expression profile of BCSCs and iBCSC was also remarkably similar, indicating that these cells are governed by a similar set of genes.

To assess whether pluripotency marker expression was reactivated in non-BCSCs following irradiation, the authors studied expression levels of Oct4, Sox2, Nanog, Klf4 and c-Myc and found a dose-dependent increase in all but c-Myc, with levels matching that of native BCSCs. The authors also noticed that radiation had caused an increase in the number of polyploidy cells, an effect which has also been described for inhibition of Notch signaling.6 These cells were ZsGreen-cODC+ and showed low proteasome activity, similar to BCSCs. Using siRNA targeted to Sox2 and Nanog, the authors then demonstrate that downregulation of either of these transcription factors in isolation had no effect on iBCSC induction, whereas targeting both simultaneously significantly attenuated radiation-mediated iBCSC induction.

To determine whether the radiation effect could be mimicked by pharmacological induction of polyploidy, Lagadek et al.4 treated sorted non-tumorigenic cells with Noscapine. Noscapine is a plant-derived non-toxic chemical usually available as a cough medicine, but when used in higher doses has been found to kill human cancer cells in a manner similar to chemotherapy. Indeed, five days after Noscapine treatment the level of polyploidy and number of ALDH1+ cells had significantly risen to levels comparable with irradiated cells. Downregulation of Notch receptors, Sox2 and Nanog with targeted siRNA abrogated this effect, confirming that Notch, Sox2 and Nanog signaling are required for BCSC induction and maintenance.

This work provides new evidence for the unfortunate phenomenon of accelerated cancer growth rates observed during treatment gaps and implicates ionizing radiation treatment as a compounding factor, which can enable some non-tumorigenic cancer cells to adopt a more aggressive CSC phenotype. Results from this study indicate that this unwanted side effect of anti-cancer radiation treatment may arise due to an increased number of Oct4 and Sox2 gene copies in polyploidy cells, and implicates a key role for Notch1 in BCSC formation and maintenance in breast cancer tissue. This important work represents a step forward towards controlling this phenomenon in patients, by potential modulation of Notch signaling following radiation treatment and thus allowing greater efficacy of anti-cancer therapy.

 

References

1. Phillips TM et al. (2006) ‘The response of CD24(-/low)/CD44+ breast cancer-initiating cells to radiation’ J Natl Cancer Inst. 98:1777-1785.

2. Woodward WA et al. (2007) ‘WNT/betacatenin mediates radiation resistance of mouse mammary progenitor cells’ Proc Natl Acad Sci U S A. 104:618-623.

3. Bao S et al. (2006) ‘Glioma stem cells promote radioresistance bypreferential activation of the DNA damage response’ Nature 444:756-760.

4. Lagadek C et al. (2012) ‘Radiation-Induced Reprogramming of Breast Cancer Cells’ Stem Cells 30(5):833-844.

5. Dontu G et al. (2004) ‘Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells’ Breast Cancer Res. 6:R605-615.

6. Baia GS et al. (2008) ‘Notch activation is associated with tetraploidy and enhanced chromosomal instability in meningiomas’ Neoplasia 10:604-612.