Abstract CN06-01: Low-dose azacytidine and epigenetic regulation of self-renewal.

Breast cancer is the most commonly diagnosed cancer among women worldwide and the second leading cause of cancer death. Breast cancer patients do not die from their primary tumor, but rather from distant metastasis. Metastases are maintained by self-renewing, cancer stem cells (CSCs) and these cells are believed to be primarily responsible for the tumor recurrence that occurs during failed therapy. CSCs are difficult to target with conventional cancer therapies; however we show that self renewing cells can be successfully inhibited using epigenetic approaches. Epigenetics (heritable changes in gene function that do not involve alterations in DNA sequence) play a major role in the inactivation and silencing of genes in cancer, leading to a loss of gene function that provides a selective advantage to cancer cells. This silencing is associated with promoter, CpG island, DNA hypermethylation. These promoters are largely protected from DNA methylation in all normal cells. DNA methylation can be reversed via the use of epigenetic, de-methylating agents such as decitabine (5-aza-29-deoxycytidine, DAC) and azacitidine (5-azacitidine, AZA); however, the mechanisms underlying the clinical efficacies of these drugs is not clear. Historically, DAC and AZA were administered at high micromolar doses and were considered too toxic to be effective as anti-cancer agents. After reassessment in hematologic malignancies (MDS/AML), these agents were found to be effective and less toxic at lower doses, suggesting that dose de-escalation is key for these de-methylating agents. Using cell lines and primary tissue from breast cancer patients, we show that transient, low nanomolar doses of DAC and AZA, which are not immediately cytotoxic, can impart a long term, anti-tumorigenic response, mediated in part, via decreases in self-renewal, and invasiveness and increases in apoptosis and differentiation. In our ex vivo-mouse model, breast cancer cell lines are treated daily for 3 days in culture with 100 nM DAC, or 500 nM AZA. The drugs are then withdrawn and the cells are permitted to recover in culture for 7 days. At day 10, equal numbers of viable cells are harvested and subcutaneously injected into NOD/SCID mice to generate xenografts. Analysis of tumor xenografts showed that low doses of AZA lead to an increased latency, slower tumor growth and a partial regression of some tumors. The tumor xenograft responses are not indicative of tumor de-bulking, but instead suggest a blunting of self-renewal and/or decrease in tumorigenicity of cancer stem cell populations. Moreover, estrogen receptor positive and HER2+ cancer cell lines were more sensitive to azacytidine than triple negative tumors. Similar, but more dramatic results were observed for primary tumors from metastatic breast cancer patients that were orthotopically transplanted into immunodeficient mice. Pre-established, estrogen receptor positive breast tumors in mice, showed diminished growth in response to systemic, AZA administered subcutaneously, five days a week for three weeks. These data represent a potentially a major step for personalization of epigenetic therapy for breast cancer and are currently under further investigation in a phase I/II trial of azacytidine and entinostat in metastatic breast cancer. In a cell culture model for self renewal, pleural effusions from women with metastatic breast cancer were cultured in suspension to generate mammospheres. Serial passage of mammospheres treated with 500nM AZA led to a progressive decrease in both the size and number of spheres while, with each increased passage, mock treated spheres increased in size and number. Likewise, flow analysis revealed alterations in stem cell markers CD44, CD24 and aldehyde dehydrogenase. These data suggest that treatment with AZA targets self-renewing or stem-like cells in primary patient samples. Agilent gene expression data were assessed by Metacore analyses (GeneGo Inc. St. Joseph, MI) for day 3 of AZA treatment, and day 7 and day 10 of post-treatment. Azacytidine led to sustained alterations in gene expression in several key, anti-tumor pathways in both breast cancer cell lines and primary samples. Many of the anti-tumorigenic pathways identified, are critical for self-renewal, mitosis, invasion, and migration, and suggest that self-renewing tumor cell subpopulations may be among those most heavily affected by these changes. The data obtained from our research will prove to be imperative for understanding how epigenetic therapy strategies work to target cancer stem cells, for the development of biomarkers to personalize the therapies, for the design of new drugs for epigenetic therapy, and, most importantly, for the design of our next immediate clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr CN06-01.