Chromatin remodeling underlies the senescence-associated secretory phenotype of tumor stromal fibroblasts that supports cancer progression

Abstract

Age is a major risk factor for the development of cancer. Senescent fibroblasts, which accumulate with age, secrete protumorigenic factors collectively referred to as the senescence-associated secretory phenotype (SASP). Here, we examined the molecular mechanisms that control SASP activation, focusing on the known SASP factor osteopontin (OPN). We found that expression of the canonical SASP members interleukin (IL)-6 and IL-8, but not OPN, were dependent upon a persistent DNA damage response (DDR) as evidenced by ATM and NF-κB activation. Treatment with several histone deacetylase (HDAC) inhibitors robustly activated SASP in the absence of DNA breaks, suggesting that DDR-dependent SASP activation occurs in response to chromatin remodeling rather than physical breaks in DNA. In the setting of HDAC inhibition, IL-6 and IL-8 expression remained dependent upon ATM and NF-κB, while OPN expression remained independent of these factors. Further analysis revealed that HDAC1 inhibition was sufficient to induce OPN expression, which is interesting given that loss of HDAC1 expression correlates with increased OPN expression within the stromal compartment of invasive breast cancers. Importantly, fibroblasts treated with HDAC inhibitors promoted tumor growth in vivo. Our findings therefore indicate that HDAC modulation plays an important role in stromal cell activation, with important implications for the use of HDAC inhibitors in the treatment of cancer.

Figure 1. The p53 and Rb pathways are dispensable for OPN upregulation in senescence

A. Western Blot analysis of BJ fibroblasts stably transduced with a p53 mutant (DD) or CDK4/cyclinD1 (DK) fusion construct that inhibits wildtype p53 and Rb, respectively. β- and γ-actin serve as loading controls for the two cell lines. B. SA-βgal staining of BJ fibroblasts treated with vehicle alone (vehicle) or bleomycin (bleo) and BJ fibroblasts stably transduced with DD or DK treated with bleomycin (DD+Bleo and DK+Bleo, respectively). Scale bar is 100 microns. C. OPN mRNA levels were measured by quantitative PCR (qPCR) in cells described in A following treatment with vehicle or bleomycin. Vehicle-treated cells are defined as 1. The mean ± STDEV is shown (n=3).

Figure 2. Transcriptional regulation of the SASP factors OPN versus IL6 and IL8 is governed by distinct mechanisms

A. NF-κB activity was examined in BJ fibroblasts stably expressing a vector control or IκBα-mut allele. Cells were transiently transfected with an NF-κB-responsive promoter driving Firefly luciferase. Data are presented as percent Renilla Luciferase, a control for transfection efficiency. The mean ± SEM is shown (n=3). B. SA-βgal staining of BJ fibroblasts stably transduced with a vector control or IκBα-mut allele and treated with vehicle (Vector+Vehicle) or bleomycin (Vector+Bleo and IκBα-mut+Bleo). Scale bar is 100 microns. C. The mRNA levels of OPN, IL6, and IL8 as indicated were measured by qPCR in vector control BJ fibroblasts treated with vehicle (Vector) (set to 1) or bleomycin (Vector+Bleo) or BJ fibroblasts expressing the IκBα-mut cDNA and treated with bleomycin (IκBα-mut+Bleo). The mean ± STDEV is shown (n=3). D. ATM mRNA levels were quantified by qPCR in BJ fibroblasts expressing one of two short-hairpin RNAi constructs targeting ATM (shATM-1 or shATM-2) or a control hairpin (shSCR), which was defined as 100 percent. The mean ± STDEV is shown (n=3). E. SA-βgal staining of control hairpin cells treated with vehicle alone (shSCR+Vehicle) or bleomycin (shSCR+Bleo) and shATM expressing BJ fibroblasts treated with bleomycin (shATM-1+Bleo). Scale bar is 100 microns. F. The mRNA levels of OPN, IL6, and IL8 were measured by qPCR in BJ fibroblasts expressing a control hairpin treated with vehicle or bleomycin (shSCR+Vehicle and shSCR+Bleo) or a short-hairpin targeting ATM and treated with bleomycin (shATM-1+Bleo and shATM-2+Bleo, respectively). Expression levels in shSCR+Vehicle cells were set to 1 for each factor measured. The mean ± STDEV is shown (n=3).

Figure 3. HDAC inhibitors activate the SASP

A. Western Blot analysis of BJ fibroblasts treated with vehicle or sodium butyrate (NaB) for 72 hr were analyzed for γ-H2AX. Cells irradiated with 10 Gy (IR) serve as a positive control and β-actin was used as a loading control. B. The mRNA levels of OPN, IL6, and IL8 were measured by qPCR in representative vehicle-treated BJ fibroblasts or BJ fibroblasts treated independently with several HDAC inhibitors as indicated. Expression levels in Vehicle-treated cells were set to 1 for each factor measured. The mean ± STDEV is shown (n=3). C. BJ fibroblasts and AT fibroblasts were treated with vehicle or NaB, and IL6 and IL8 mRNA levels were measured by qPCR. The mean ± STDEV is shown (n=3). Expression levels in vehicle-treated cells were set to 1 for each cell line. D. BJ fibroblasts expressing a vector control (Vector) or an IκBα-mut cDNA (IκBα-mut) were treated with vehicle or NaB. IL6 and IL8 mRNA levels were quantified by qPCR and expression levels of IL6 and IL8 in the Vector+Vehicle were set to 1. The mean ± STDEV is shown (n=3).

Figure 4. Fibroblasts treated with HDAC inhibitors promote preneoplastic cell growth

A. Bioluminescent images (day 10) of representative mice injected in the flanks with HaCaT_CBR cells in combination with vehicle-treated (Vehicle), bleomycin-treated (Bleo), or NaB–treated (NaB) fibroblasts. B. Quantification of the luminescence in panel A (measured as photons/second/cm²/steradian (photons/s/cm²/Sr), vehicle-treated cells are defined as 1. The mean ± SEM is shown, * p < 0.05 (n=8). C. Bioluminescent images (day 12) of representative mice injected in the flanks with a mixture of HaCaT_CBR cells with either vehicle-treated BJ fibroblasts or vorinostat-treated BJ fibroblasts. D. Quantification of the luminescence in the groups described in panel C, vehicle-treated cells are defined as 1. The mean ± SEM is shown, * p < 0.05 (n=8).

Figure 5. Inhibition of HDAC1 leads to OPN upregulation

A. Oncomine expression analysis of HDAC1 levels between normal stroma and invasive breast cancer-associated stroma extracted from the Finak et. al. study (29). Expression values are log transformed and median-centered per array (see Materials and Methods). B. HDAC1 mRNA levels were measured by qPCR in vehicle and bleomycin-treated BJ fibroblasts. The mean ± STDEV is shown (n=3). C. HDAC1 activity was measured in lysates from vehicle or bleomycin-treated cells after immunoprecipitation of HDAC1 with an HDAC1 antibody. The mean ± SEM is shown, * p < 0.05, (n=3). D. OPN, IL6 and IL8 mRNA levels were quantified by qPCR in BJ fibroblasts transduced with a vector control (pBp or pBh) or a dominant negative cDNA of HDAC1 (HDAC-DN) or HDAC3 (HDAC3-DN). The mean ± STDEV is shown (n=3).