Using a syngeneic p53-null mouse mammary gland tumor model that closely mimics human breast cancer, we have identified, by limiting dilution transplantation and in vitro mammosphere assay, a Lin À

Tumor-initiating cells (TICs) have been shown both experimentally and clinically to be resistant to radiation and chemotherapy, potentially resulting in residual disease that can lead to recurrence. In this study, we demonstrate that TICs isolated from p53 null mouse mammary tumors repair DNA damage following in vivo ionizing radiation more efficiently than the bulk of the tumor cells. Down-regulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was observed both in fluorescence activated cell sorting (FACS)-isolated TICs as compared to non-TICs and in TIC-enriched mammospheres as compared to primary tumor cells depleted of TICs. This effect was accompanied by increased Akt signaling, as well as by the direct activation of the canonical Wnt/β-catenin signaling pathway specifically within the TIC subpopulation by phosphorylation of β-catenin on serine 552. Using limiting dilution transplantation performed on p53 null tumor cells transduced with Wnt reporter lentivirus, we demonstrated that FACS sorting of cells expressing TOP-eGFP resulted in a marked enrichment for TICs. Furthermore, FACS analysis demonstrated that cells with active Wnt signaling overlapped with the TIC subpopulation characterized previously using cell surface markers. Finally, pharmacological inhibition of the Akt pathway in both mammospheres and syngeneic mice bearing tumors was shown to inhibit canonical Wnt signaling as well as the repair of DNA damage selectively in TICs, sensitizing them to ionizing radiation treatment. Thus, these results suggest that pretreatment with Akt inhibitors before ionizing radiation treatment may be of potential therapeutic benefit to patients.

Breast cancer metastasis and disease recurrence are hypothesized to result from residual cancer stem cells, also referred to as tumor-initiating cells, which evade initial treatment. Using both syngeneic mouse and human xenograft models of triple-negative breast cancer, we have demonstrated that a subpopulation enriched in cancer stem cells was more resistant to treatment with 6 gray of ionizing radiation than the bulk of the tumor cells, and accordingly their relative proportion increased 48 to 72 hours after ionizing radiation treatment. In contrast, we achieved a larger reduction in tumor size without a concomitant increase in the percentage of cancer stem cells by treating with local hyperthermia for 20 minutes at 42°C after ionizing radiation using intravenously administered, optically activated gold nanoshells. Forty-eight hours after treatment, cells derived from the tumors treated with ionizing radiation plus hyperthermia exhibited both a marked decrease in tumorigenicity and a more differentiated phenotype than mock- and ionizing radiation–treated tumors. Thus, we have confirmed that these cancer stem cells are responsible for accelerated repopulation in vivo and demonstrated that hyperthermia sensitizes this cell population to radiation treatment. These findings suggest that local hyperthermia delivered by gold nanoshells plus radiation can eliminate radio-resistant breast cancer stem cells.

Recent studies have shown that differentiated cancer cells can de-differentiate into cancer stem cells (CSCs) although to date no studies have reported whether this transition is influenced by systemic anti-cancer agents. Valproic acid (VA) is a histone deacetylase (HDAC) inhibitor that promotes self renewal and expansion of hematopietic stem cells and facilitates the generation of induced pluripotent stem cells from somatic cells and is currently being investigated in breast cancer clinical trials. We hypothesized that HDAC inhibitors reprogram differentiated cancer cells towards the more resistant stem cell-like state. Two highly aggressive breast cancer cell lines, SUM159 and MDA-231, were FACS-sorted based on ALDH activity and subsequently ALDH-negative and ALDH-positive cells were treated with one of two known HDAC inhibitors, VA or SAHA (suberoylanilide hydroxamic acid). In addition, primary tumor cells from patients with metastatic breast cancer were evaluated for ALDH activity following treatment with HDAC inhibitors. We demonstrate that single cell sorted ALDH- negative cells spontaneously generated ALDH-positive cells in vitro. Treatment of ALDH-negative cells with HDAC inhibitors promoted the expansion of ALDH-positive cells and increased mammosphere forming efficiency. Most importantly, it significantly increased the tumor-initiating capacity of ALDH- negative cells in limiting dilution outgrowth assays. Moreover, while HDAC inhibitors upregulated β-catenin expression and significantly increased WNT reporter activity, a TCF4 dominant negative construct abolished HDAC-inhibitor induced expansion of CSCs. These results demonstrate that HDAC inhibitors promote the expansion of breast CSCs through dedifferentiation and have important clinical implications for the use of HDAC inhibitors in the treatment of cancer.