SOBRE LA EVALUACIÓN

the cisplatin-resistant cells

It has been shown that phosphorylated STAT1 complexes translocate to the nucleus, where they specifically bind to the promoters of certain gene controlled by STAT1 such as IFN-stimulated genes to exert growth inhibition of cultured cells (Bromberg et al.1996). STAT1 phosphorylation has been reported to be prevented by HDACi highlighting the role of HDAC in STAT1 phosphorylation in colon cells (Klampfer et al. 2004). The co-localization of HDAC4 and pSTAT1-Y701 in the nucleus after 24hours of cisplatin treatment was observed (Figure 46). HDAC4 inhibition via siRNA or by using APHA4a prevented the phosphorylation of STAT1 at Y701 following cisplatin treatment in cisplatin-resistant cells. A dynamic regulation of STAT1 phospho-acetyl levels is controlled by HDAC4 activity. STAT1 is activated, phosphorylated and translocated to nucleus following cisplatin treatment in ovarian cancer cells with acquired clinical platinum resistance but not platinum sensitive cells indicating that the over-expression of HDAC4 promotes de-acetylation of STAT1 and this permit STAT1 phosphorylation at Y701 and its subsequent nuclear translocation. STAT1 knockdown in resistant ovarian cancer cells restores cisplatin-sensitivity following exposure to platinum (Figure 58).

Figure 57: SiRNA-mediated silencing of lamin and STAT1 on PEO4 cells followed by Caspase Glo 3/7 based apoptosis assay. Cells were transfected with 100 nM siRNA targeting lamin and STAT1. Cell viability was measured by MTT assay at 24 h. Caspase3/7 data are normalized to MTT data are means ± SEM from three separate experiments, each performed in triplicate. P-value: 0.0002. This experiment was performed by Nona Rama, a member of my group in the laboratory, Imperial College.

In contrast, isogenically matched cisplatin sensitive cells have low HDAC4 level therefore; acetyl-STAT1 is present which might explain the inability of cisplatin treatment to induce phospho-STAT1 Y701 in these cells. Furthermore, the reduction of HDAC4 in platinum resistant cells by increasing the acetylation level of STAT1 which later induce apoptosis following cisplatin treatment is similar to low level of HDAC4 in cisplatin sensitive cells. These data show that the level of HDAC4 expression differentiate between the response of STAT1 in clinical platinum sensitivity and acquired platinum resistance and identify a novel mechanism involving HDAC4 and STAT1 interaction to alter the apoptotic response to platinum in ovarian cancer cells with acquired resistance (Figure 47).

Here, the pharmacological modulation of HDAC4 using the HDAC inhibitors resulted in inhibitory effects on both the STAT1 and HDAC4. Furthermore The inhibition of histone deacetylase (HDAC) activity by butyrate, trichostatin A, and suberoylanilide hydroxamic acid has been shown to prevent STAT1 phosphorylation, its nuclear translocation, as well as STAT1-dependent gene activation (Klampfer et al. 2004). Together these data show that HDAC4 inhibition changes the phosphorylation of STAT1 and nuclear translocation in resistant ovarian cancer cells following cisplatin treatment. In addition, STAT1 acetylation depends on the balance between STAT1-associated HDACs and HATs and inhibition of HDAC4 changes this balance and induces STAT1 acetylation. The exact mechanism that causes the resensitisation to cisplatin via HDAC4/STAT1 is unknown. However, the data show evidence that the acquisition of resistance can be reversed by HDAC inhibitors in ovarian cancer.

4.4 Identification of HDAC4-regulated genes by

microarray:

The data presented in the current work showed that HDAC4 knockdown in platinum-resistant ovarian cancer cells restores the sensitivity to cisplatin by

Therefore, in order to identify HDAC4 signature which may correlate HDAC4 to DNA damage response, either through repression of pro-apoptotic gene expression or activation of anti-apoptotic genes, microarray analyses were carried out using RNA from PEO4 cells transfected with siRNA against HDAC4, Lamin A/C (as control) and untransfected PEO4 cells. Differentially expressed genes were compared between untransfected versus HDAC4 knockdown, untransfected versus Lamin A/C knockdown and HDAC4 knockdown versus Lamin A/C knockdown generated using Genechip Operating System (GCOS) using random variance analysis with a P-value cut off of 0.01. A random variance model for the detection of differential gene expression was used as it is recommended for small microarray experiments to generate more accurate and robust variance estimation (Wright and Simon, 2003). Subsequently, the overlapped genes between untransfected versus HDAC4 knockdown and HDAC4 knockdown versus Lamin A/C knockdown were selected for further investigation. This provides strict criteria for selecting differentially expressed genes but the assumption is that those overlapped genes are the ones which are most significant and are most likely to be of biological interest.

Microarray has been used to generate transcriptional profiles to determine the functional consequence of HDAC inhibition on gene expression (Chambers et

al. 2003; Peart et al. 2005). These studies have reported that about 8-10% of

genes were altered by pan HDAC inhibitors; trichostatin A, SAHA, depsipeptide suggesting that certain genes are regulated by HDACs. Here the analyses identified twenty-four genes that appear to be regulated by HDAC4. The twenty-four differentially expressed genes were those overlapped between HDAC4-knockdown versus Lamin-knockdown array and HDAC4- knockdown versus untransfected array but not present in untransfected versus Lamin-knockdown array. Eight out of the 23 genes were up-regulated and 15 were down-regulated in PEO4 cisplatin resistant cells following HDAC4 knockdown by siRNA compared with controls (untransfected and lamin knockdown). The analysis confirmed HDAC4 knockdown in the array (Table 7).