Apoptotic sensitivity of colon cancer cells to histone deacetylase inhibitors is mediated by an Sp1/Sp3-activated transcriptional program involving immediate-early gene induction

Abstract

Histone deacetylase inhibitors (HDACi) induce growth arrest and apoptosis in colon cancer cells and are being considered for colon cancer therapy. The underlying mechanism of action of these effects is poorly defined with both transcription-dependent and -independent mechanisms implicated. We screened a panel of 30 colon cancer cell lines for sensitivity to HDACi-induced apoptosis and correlated the differences with gene expression patterns induced by HDACi in the five most sensitive and resistant lines. A robust and reproducible transcriptional response involving coordinate induction of multiple immediate-early (fos, jun, egr1, egr3, atf3, arc, nr4a1) and stress response genes (Ndrg4, Mt1B, Mt1E, Mt1F, Mt1H) was selectively induced in HDACi sensitive cells. Notably, a significant percentage of these genes were basally repressed in colon tumors. Bioinformatics analysis revealed that the promoter regions of the HDACi-induced genes were enriched for KLF4/Sp1/Sp3 transcription factor binding sites. Altering KLF4 levels failed to modulate apoptosis or transcriptional responses to HDACi treatment. In contrast, HDACi preferentially stimulated the activity of Spl/Sp3 and blocking their action attenuated both the transcriptional and apoptotic responses to HDACi treatment. Our findings link HDACi-induced apoptosis to activation of a Spl/Sp3-mediated response that involves derepression of a transcriptional network basally repressed in colon cancer.

Figures

Figure 1

(A) Relative response of 30 colon carcinoma cell lines to HDACi-induced apoptosis. Cells lines were treated with 1, 5 or 10 mM butyrate for 72 hours (Mean±SEM, n=3). (B). Clonogenic survival of sensitive and resistant lines following 24h HDACi treatment (n=3–4). (C). Response of 2 sensitive and 2 resistant cell lines to VPA treatment in vivo. (D) Quantitation of colony formation shown in panel (C). Values shown are mean±SEM percentage colony formation following 5 mM butyrate treatment relative to control for the sensitive and resistant cell lines. (E). Quantitation of tumor size following HDACi treatment in vivo (n=4–5, *P<0.05).

Figure 2

(A). Heat map of the 48 genes selectively induced by butyrate in sensitive cell lines following 24h treatment. (B) QPCR validation of select gene expression changes following 24 h treatment with 5 mM butyrate (But 5). (C) Western blot validation of differential induction of select proteins in sensitive (HCT8 and HCT116) and resistant cell lines (LIM2405).

Figure 3

(A) Mean GC content of 48 genes preferentially induced by HDACi in sensitive cell lines and an equal number of control genes. (B). Frequency of KLF4/Sp1/Sp3 binding sites in the promoters of HDACi-induced genes. *indicates P<0.05. (C-D) Quantitative ChIP analysis in untreated HCT116 cells demonstrating Sp1 and Sp3 localization to proximal promoters of HDACi-induced genes (C) but not distal promoter regions (D). (E) ChIP analysis demonstrating induction of histone H3 hyperacetylation in the proximal promoter regions of 3 genes following 24 hour treatment of HCT116 cells with butyrate 5 mM (B5). (F) Preferential induction of Sp1/Sp3 reporter activity in HDACi-sensitive cell lines treated with butyrate, SAHA or VPA for 24h (n=2).

Figure 4

The Sp1/Sp3 inhibitor, mithramycin (Mith, M, 1 μM), attenuates HDACi-induction of (A) target gene expression (HCT116), (B) cell viability (HCT116), (C) sub-diploid DNA content (HCT116 and HCT8) or (D) and PARP cleavage (HCT116). Cells were treated with HDACi (Butyrate 5 mM, TSA 1 μM) for 24 hours.

Figure 5

(A) Silencing efficiency of Sp1 and Sp3 protein expression. (B-C) Silencing of Sp1 and Sp3 in HCT116 cell partially attenuates HDACi-induced apoptosis at 24h as assessed by (B) sub-diploid DNA content and (C) PARP cleavage, and (D-F) HDACi-induced transcriptional response.

Figure 6

Expression of multiple HDACi-induced genes is reduced in primary colon tumors relative to adjacent normal mucosa. (A) The percentage of HDACi-induced and (B) un-induced (control) sequences significantly over or underexpressed in colon tumors. (C) Identity of the 9 genes induced by HDACi and significantly repressed in colon tumors relative to normal colonic epithelium (*P<0.05). (D) Immunohistochemical validation of reduced expression of ATF3 and metallothionein in normal colon and colon tumors. Images downloaded from the publicly available protein atlas database (www.proteinatlas.org) as per the image use policy of protein atlas.