Modulation of Breast Cancer Stem Cell Response to Radiation

Institution: University of California, Los Angeles
Investigator(s): Frank Pajonk, M.D., Ph.D. -
Award Cycle: 2007 (Cycle 13) Grant #: 13IB-0003 Award: $149,377
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure

Initial Award Abstract (2007)
A major reason for failures in cancer therapy is an incomplete elimination of a special type of cell, termed cancer stem cells (CSCs). The CSC model argues that tumors arise from cells that retain the properties of adult stem cells, particularly for their ability to self-renew and differentiate into multiple cell types, often seen as the heterogeneity of cell types in patient samples. In addition, CSCs persist in tumors as a distinct population and they account for disease relapse and metastasis. The CSCs represent only 1-2% of the total tumor cell population, but are thought to be highly resistant to conventional chemotherapy and radiotherapy. However, the exact phenotype of breast CSCs is still undefined, and their mechanisms for escaping effective therapy continue to elude researchers.

In breast cancer, a breast cancer initiating cell population (BCIC), highly enriched for CSCs, can be identified and propagated from patient samples and from established breast cancer cell lines. Therefore, we will use BCICs with the understanding that our results will include CSCs and other early progenitor cells at the same time. We hypothesize that: (1) CSCs exhibit increased radiation resistance based on increased levels of radical scavengers, (2) BCICs create and restore their own stem cell niche, and (3) the size of the stem cell pool and the fate of these cells is determined by co-dependency with the signaling stem cell niche, which provides a potential target for improving radiotherapeutic outcome. For our studies cancer stem cells will be isolated from MCF-7 and T-47D breast cancer cell lines and from ER+ breast cancer patient specimens. The cells will be propagated under serum-free conditions as hollow balls of cells, called mammospheres. Radiation response will be determined by flow cytometry, TUNEL-assays to measure programmed cell death (apoptosis), and clonogenic assays (i.e., studying the effectiveness of specific agents on the proliferation of cells). Radiation-induced signal transduction in breast cancer stem cells involving Notch, a membrane protein controlling cell differentiation, will be monitored using flow cytometry and Western blotting. The goal is to compare the radiosensitivity of breast cancer stem cells to non-stem cells from established cell lines and patient specimens, and develop a mechanistic explanation for these differences with the goal of pointing the way for new approaches to increase radiation sensitivity specific to CSCs.

Currently, radiation is a safe and efficient breast cancer therapy and co-determines treatment success and cosmetic outcome. However, despite technical advances in radiation therapy, disease recurrence remains almost unchanged over the last three decades. We hope that studying the response of breast CSCs to therapy will lead to improvements in treatment.

Final Report (2009)
Note: Dr. Pajonk was awarded two years of additional CBCRP in 2009 to continue this research.

High-risk breast cancer patients benefit radiation therapy, but 10-year disease-free survival rates are still disappointing. In general, an incomplete elimination of tumor cells causes local recurrence of cancer after radiation therapy. The ability of tumorgenicity and self-renewal causing recurrence of cancer is attributed to a small population of surviving cancer stem cells (CSCs). We hypothesized that breast CSCs (BCSCs) exhibit increased radioresistance based on increased levels of radical scavengers, that BCSCs create and restore their own stem cell niche and that the size of the stem cell pool and the fate of these cells is determined by co-dependency with the signaling stem cell niche, which provides a potential target for improving radiotherapeutic outcome.

In our study we investigated mechanisms of radiation resistance of BCSCs, how they change in numbers in response to fractionated, extent and kinetics of activation the developmental Notch pathway and DNA-damage dependent cell signaling. We initially planned to perform these experiments in BCSCs derived from established cell lines and primary breast cancer samples but we were unable to establish and maintain BCSCs from patients samples in-vitro or in-vivo. Therefore, we reduced the scope of our work using established breast cancer lines. We were able to successfully verify the extended ability of BCSC to scavenge radiation-induced free radicals and their increased radiation resistance in both, clonogenic survival assays and apoptosis assays. We demonstrated that BCSCs increase in numbers after fractionated radiation, which resulted in increased self-renewal of BCSCs. Furthermore, our data does not indicate increased levels of quiescence in BCSCs populations. The developmental Notch signaling pathway was activated differentially in BCSCs and non-tumorigenic cells, and expression of the different Notch ligand and receptor family members differed in their in response to radiation. Finally, BCSCs did not activate DNA-damage dependent signaling to the same extend as non-tumorigenic cells in response to irradiation, which was consistent with a reduced level of DNA damage through increased free radical scavenging in BCSCs.

We struggled using established markers to reliably predict the amount of BCSCs in our samples. A major accomplishment of our study was the discovery of a novel BCSC marker that allows for identification, tracking, and targeting of BCSCs in vitro and in vivo and the demonstration that activation of the developmental Notch pathway was part of the radiation response of BCSCs. In future studies will use our novel marker system to explore the connection the role of this signaling for the radiation resistance of BCSCs.

Radiation responses of cancer stem cells.
Periodical:Journal of Cellular Biochemistry
Index Medicus: J Cell Biochem.
Authors: Vlashi E, McBride WH, Pajonk F
Yr: 2009 Vol: 108 Nbr: 2 Abs: Pg:339-42

In vivo imaging, tracking, and targeting of cancer stem cells.
Periodical:Journal of the National Cancer Institute
Index Medicus: J Natl Cancer Inst
Authors: Vlashi E, Kim K, et al, and Pajonk F
Yr: 2009 Vol: 101 Nbr: 5 Abs: Pg:350-9