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Our study revealed that HDAC11 is overexpressed in human breast cancer compared to normal breast and it correlates with ER expression levels. Furthermore, HDAC11 expression is controlled by ER signaling, as E2 stimulation increases HDAC11 mRNA levels, while inhibition of the pathway by tamoxifen or fulvestrant decreases HDAC11 transcription. KD of HDAC11 in the ER-positive breast cancer cell lines T47D, MCF7 and J110, decreases cell proliferation, but does not significantly alter proliferation of the ER-negative breast cancer cell lines MDA-MB-231 and SUM159. Moreover, RNA-sequencing experiments revealed a higher number of upregulated than downregulated genes upon HDAC11 KD, underscoring its role as a transcriptional repressor. Interestingly, GSEA analysis suggests that HDAC11 affects the estrogen responsive transcriptional program, as genes normally downregulated upon estrogen signaling are upregulated in HDAC11 KD cells compared to control cells. Furthermore, Kaplan-Meier analysis revealed that HDAC11 has prognostic value for patients who received endocrine therapy, as the probability of RFS and DMFS was decreased for the HDAC11-high group.

HDAC11 affects ER expression levels

We observed a reduction in ER protein and mRNA levels upon HDAC11 KD in T47D as well as in MCF7 cells. As HDAC11 is a transcriptional repressor (Villagra et al., 2009), it is unlikely that HDAC11 induces ESR1 transcription in a direct manner. However, HDAC11 might directly repress genes encoding proteins or miRNAs, which repress ESR1 transcription or target ESR1 mRNA, respectively (Figure 3.12). Indeed, several miRNAs, including miR-206 (Adams et al., 2007), miR-221 and miR-222 (Zhao et al., 2008) negatively regulate ER expression. Future experiments such as ChIP-sequencing, might identify genes directly repressed by HDAC11, and could reveal how HDAC11 affects ER levels.

Additionally, HDAC11 might also control ER protein levels through deacetylation of the ER chaperone Hsp90. Hyperacetylation of Hsp90 in response to HDAC6 inhibition decreases its

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binding to ER, leading to polyubiquitylation and proteasomal degradation of ER (Fiskus et al., 2007). Thus, it would be interesting to investigate the acetylation status of Hsp90 in HDAC11 KD cells and to test if ER ubiquitylation is affected by HDAC11 (Figure 3.12).

HDAC11 controls part of the estrogen-induced transcriptional program

Genes normally downregulated in response to estrogen signaling are upregulated in HDAC11 KD cells compared to control cells. Thus HDAC11 might contribute to the estrogen induced transcriptional program by acting as a transcriptional repressor. However, it remains unknown how HDAC11 mediates gene repression in response to estrogen signaling. Several mechanisms are possible, which might include deacetylation of the ER directly and/or ER-interacting proteins such as transcription factors and coregulatory proteins.

The ER represses genes by recruiting corepressors such as NCoR1 and HDAC1 (Jackson et al., 1997; Kawai et al., 2003) to gene promoters. Thus, HDAC11 might be recruited to specific genes through interaction with ER and induce chromatin condensation and/or alter ER transcriptional activity. We did not observe differences in transcriptional activity of ER measured by an ER- responsive reporter construct, however this does not rule out that ER transcriptional activity is affected by HDAC11 on specific genes. As ER has been shown to be acetylated by the HAT p300, which, depending on the acetylation site, increases (Kim et al., 2006) or decreases (Wang et al., 2001) ER transcriptional activity, HDAC11 might control ER activity through its deacetylase function (Figure 4.1). Therefore, it would be interesting to analyze if HDAC11 and ER interact and if HDAC11 can deacetylate ER at specific sites.

Alternatively, HDAC11 might be recruited to ER target genes by interacting with transcription factors such as AP1, a known ER interacting protein (Kushner et al., 2000). Interestingly, genome wide analysis of ER binding sites revealed that late downregulated ER target genes (at 6 and 12h of E2 stimulation) are more likely to contain AP1 binding sites close to their promoter region, suggesting that AP1 promotes transcriptional repression of these genes (Carroll et al., 2006). AP1 interacts with the corepressor protein NRIP1/RIP140 (Teyssier et al., 2003), whose transcription is induced upon estrogen signaling, and KD of NRIP1 prevents repression of

Discussion and Outlook

several genes normally repressed by estrogen signaling (Carroll et al., 2006). Thus, it would be interesting to test if HDAC11 is recruited to ER target genes by AP1. Co-IP experiments should reveal if these two proteins can interact, and Re-ChIP experiments would be required to test whether HDAC11 and AP1 are found at the same gene promoters. Additionally, HDAC11 might also affect AP1 transcriptional activity, as the class II HDAC SIRT1 has been shown to deacetylate AP1, thereby repressing AP1 transcriptional activity (Figure 4.1) (Zhang et al., 2010c).

Moreover, several ER coregulatory proteins are regulated by acetylation. For example, acetylation of the two ER corepressor proteins NRIP1/RIP140 and HDAC1 (Carroll et al., 2006; Kawai et al., 2003; Lopez-Garcia et al., 2006), leads to loss of gene repression. In the case of NRIP1, acetylation prevents its association with the transcriptional regulator CtBP (Vo et al., 2001), while acetylation of HDAC1 inhibits its deacetylase function (Figure 4.1) (Qiu et al., 2006). Thus, HDAC11 could repress gene transcription by various mechanisms. Future studies will be aimed to identify the genes directly regulated by HDAC11 as well as its interaction partners by ChIP-sequencing and mass spectrometric experiments, respectively.

Figure 4.1 Potential mechanisms through which HDAC11 could mediate gene repression in response to estrogen signaling. HDAC11 could be recruited to ER-target genes by ER, coregulatory proteins (CoReg) or

transcription factors (TF), where it could deacetylate histone proteins leading to chromatin condensation, decreased transcriptional activity of ER and/or other TF, or prevent binding of other coregulatory proteins. Abbreviations: response element (RE), estrogen response element (ERE)

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HDAC11 as a therapeutic target for breast cancer

Searching online available databases revealed that HDAC11 is overexpressed in human breast cancer compared to normal breast, suggesting an oncogenic role for HDAC11. This is supported by our finding that HDAC11 controls proliferation of ER-positive breast cancer cell lines. Furthermore, allograft experiments using the ER-positive murine breast cancer cell line J110 revealed reduced tumor growth of HDAC11 KD cells. However, further experiments are required to test if HDAC11 might be a target for breast cancer therapy. Xenograft experiments using T47D and/or MCF7 control and HDAC11 KD cells could shed light on HDAC11’s potential to affect tumor growth of human cell lines in vivo. Treatment of tumor bearing mice with an HDAC11-inhibitor, might give additional insight if targeting HDAC11 is beneficial, particularly since targeting HDAC11 in immune cells has been shown to decrease anti-tumor immune responses and to cause increased tumor growth (Sahakian et al., 2014). However, this experiment would require the development of HDAC11 specific inhibitors, as inhibitors that target HDAC11, generally also target class I and/or class II HDACs (Mottamal et al., 2015). Kaplan-Meier analysis revealed a decrease in the probability for RFS and DMFS for patients treated with endocrine therapy, expressing high HDAC11 levels. As this group of patients is likely to contain endocrine resistant tumors, HDAC11 might play a role in resistance. In order to investigate if tumors expressing high HDAC11 levels are more likely to develop resistance, IHC for HDAC11 on primary tumors and matched relapsed tumors that developed resistance against endocrine therapies are needed. Furthermore, in vitro studies using tamoxifen resistant cell lines such as T47D-TMR and MCF7-TMR, might give additional insights into HDAC11’s potential role in endocrine resistance.

To conclude, our study suggests that HDAC11 is an estrogen induced transcriptional repressor, promoting ER-positive breast cancer cell proliferation. Furthermore, HDAC11 is overexpressed in human breast cancer and high HDAC11 levels in endocrine treated patients correlates with an increased risk of early disease recurrence. Important areas for future research would be to learn more about HDAC11’s potential role in the development of endocrine resistance and development of more specific HDAC11 inhibitors to test their effects in the clinic.

Discussion and Outlook