The pan-HDAC Inhibitor Suberoylanilide Hydroxamic Acid Targets Self Renewal of Breast Cancer Stem Cells.
Authors: Robertson F. , Woodward W. , Bornmann W. , Player A. , Ye Z. , Cristofanilli M. 
Published: December 2010

Abstract
Background: Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer (LABC). Patients with IBC often lack estrogen receptor and present with skin metastasis characterized by the presence of tumor emboli within dermal lymphatics. These metastatic tumor cells aberrantly over-express E-cadherin and have characteristics of stem cells. Based on our initial observations that IBC cells express multiple forms of histone deacetylase (HDAC) enzymes involved in epigenetic modulation of multiple genes associated with critical functions of both embryonic stem cells and cancer cells, the present studies evaluated the effects of the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on self-renewal, expression of transcription factors associated with self renewal, homotypic aggregation, and E-cadherin expression by mammospheres of SUM149 and SUM190 established IBC cell lines and of tumor cells isolated from pleural effusion fluid of patients with IBC and LABC.Methods: Low adherence culture conditions that promote mammosphere formation and clonogenic assays were used to assess effects of SAHA on self renewal. Real time PCR arrays and protein arrays were used to evaluate the effects of SAHA on transcription factors Oct4, Nanog and Sox-2 and breast cancer associated genes including E-cadherin and ER/PR. Flow cytometry was used to document stem cell markers present on breast cancer cells grown under low adherence conditions. Confocal microscopy was used to document the effects of SAHA on E-cadherin localization and homotypic aggregation by mammospheres.Results: SAHA inhibited self-renewal of second and third passage mammospheres as assessed by clonogenic assays, which was associated with loss of Oct4, Nanog and Sox-2 transcription factors associated with stem cell self renewal an pluripotency. SAHA blocked E-cadherin mRNA and protein assessed by PCR and Western blotting and visualized by confocal imaging which demonstrated that loss of E-cadherin between cells comprising mammospheres blocked homotypic aggregation, leading to lack of integrity of the 3-dimensional spheroids. Moreover, SAHA induced ER-/low mammospheres to re-express ER mRNA and protein which was associated with responsiveness to tamoxifen and conversely, SAHA inhibited ER expression in ER+breast tumor cells. The effective inhibitory concentration50 (IC50) of SAHA to induce these effects in both established cell lines and in mammospheres derived from tumor cells isolated from pleural effusion fluid of patients with IBC and LABC was in the range of 0.37-3.31 uM, which is achievable by oral administration of SAHA. Initial in vivo studies indicate that intraperitoneal injection of SAHA effectively inhibits SUM149 breast tumor xenograft growth and formation of metastatic lesions.Conclusions: SAHA promotes differentiation of IBC stem cells and blocks E-cadherin expression, which is a hallmark of IBC skin metastasis. Taken together with the observations that SAHA can re-program ER function depending upon the ER status of the breast cancer cells, this observations suggest that SAHA may provide additional therapeutic opportunities for patients with LABC.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3141.