Combination Of HDACi And ATRA Restores RARβ2Expression And Its Therapeutic Potential In Human Cervical Cancer

Cervical cancer is one of the most frequent diseases in women and a considerable cause of morbidity and mortality among them. It is the second more frequent type of cancer in women worldwide, preceded only by breast and colorectal cancer; each year approximately500,000women worldwide are diagnosed with invasive cervical cancer and more than half of them die of this disease. Eighty percent of these deaths occur in developing countries. Surgery is the recommended treatment for early-stage cervical cancer, but in advanced stages, namely recurrent and metastatic cases, surgery has very limited value. Additionally, the tumors at advanced stages are commonly resistant to chemotherapy and these patients also frequently have a poor performance status, which limits use of aggressive chemotherapy or radiation. Thus the majority of patients die as a result of uncontrolled disease. Therefore, new therapeutic approaches with fewer adverse effects are needed for cervical cancer.One targeted class of compounds of particular interest is the histone deacetylase (HDAC) inhibitors. Histone acetylation is a critical regulatory mechanism for gene expression. The aberrant deacetylation of critical genes involved in differentiation, apoptosis or cell cycle arrest has been reported to contribute to the pathogenesis of malignancy. In cervical cancer, it has been reported that HDACs are overexpressed, and the presence of phosphorylated and acetylated forms of histone H3in cytologic smears has demonstrated a marked association of histone H3modifications with the progression of the disease from cervical intraepithelial neoplasia (CIN) I to CIN II and CIN III. HDAC inhibitors are therefore attractive target compounds for cervical cancer treatment.All-trans retinoic acid (ATRA) is a potent regulator of cellular growth and differentiation, including normal epithelial cell differentiation; it can function as a chemopreventive agent and is an effective inhibitor of chemical and viral carcinogenesis. The antineoplastic pathways induced by ATRA are regulated predominantly by RARβ (particularly the β2isoform). However, the expression of RARβ2is frequently decreased or lost in many cancer types, including cervical carcinomas. Recent studies have shown that the loss of RARβ2is attributed to the silencing of its promoter region due to histone hypermethylation and deacetylation. The use of HDAC inhibitors, alone and in combination with ATRA, has been shown to restore the expression of silenced RARβ2, thus HDAC inhibitors enhance the growth inhibitory and proapoptotic actions of ATRA in many types of cancer cells. Therefore, whether the promising combination of VPA and ATRA results in the reexpression of silenced RARβ2, and consequently has additive effects on growth suppression in cervical cancer cells, merits further investigation.In this study, we investigated histone H3acetylation and tumor suppressor genes (TSGs) expression in cervical cancer and its association with clinicopathological parameters. The efficacy of combined treatment with HDACi and ATRA was examed in restoring RARβ2expression and anti-neoplastic activity in cervical cancer. Furthermore, we evaluated the therapeutic potential of the HDACi combined with ATRA in treating a tumour xenograft model derived from human cervical carcinoma.1The expression of AcH3, RARβ2and its downstream genes was downregulated or even silenced. In the65cancerous tissues, the immunoreactivity intensity of these four parameters was strong in well-differentiated tumours and reduced in moderately differentiated carcinoma, with weak or negative labelling in poorly differentiated carcinomas. There was a statistically significant correlation between H3acetylation and the expression of RARβ2, E-cadherin, and β-catenin (r=0.560, r=0.731, and r=0.733,respectively, P<0.01). These indicate that transcriptional silencing by histone deacetylation is one of the well-established mechanisms of tumor suppressor genes inactivation.2The combination of HDACi and ATRA restores RARβ2expression via increasing the level of acetylated histone H3. In our experiments, an immunoblot analysis showed that HDACi, either alone or combined with ATRA, strongly induced the hyperacetylation of histone H3. Intriguely, the expression of RARJ32was significantly induced, with50-to90-fold upregulation in combination-treated cells but not detected in untreated control cells. We did not detect any differences in the expression of RARa and RARy between the different treatments, but both were constitutively expressed in both cell lines after treatment.3HDACi in combination with ATRA restore RARβ2expression through RARβ2-RARE. The hyperacetylation of lysine9on histone H3(H3K9ac) is usually associated with actively transcribed genes. To further understand the mechanism for histone acetylation-induced re-expression of RARβ2, we performed a ChIP assay using an anti-H3K9ac antibody to determine the binding of acetylated histone H3to the RAR(32promoter region. This combined treatment was more effective at inducing histone acetylation at the RARβ2-RARE region than any single drug. These results indicate that the RARE in the RARβ2promoter is functional and that histone acetylation at the RARβ2-RARE region is closely correlated with RAR(32re-expression in cervical cancer cells.4Combined treatment with HDACi and ATRA inhibits the growth of cervical cancer cells.1) Combined treatment with HDACi and ATRA enhances growth inhibition in cervical cancer cells. The growth inhibition rate of the combined treatment was significantly higher than that of each drug used alone(P<0.01). The effect of the combination drug treatment was even superior to that of the cytotoxic drug cisplatin (1mg/L) administered alone (P<0.01). Furthermore, the combination of HDACi and ATRA exerted additive effects on growth inhibition. The inhibition rate of the cell lines transfected with pIRES2-RARβ2-EGFP treated with ATRA only was similar as that treated with the combination. Consistent with the decreased cell proliferation, the expression of Ki67, a marker for cell proliferation, and colony formation were significantly decreased in cervical cancer cells treated with VPA and ATRA after48h of incubation.2) Combined treatment with HDACi and ATRA causes G1phase arrest. To assess the specific impact of the combination of HDACi and ATRA on cell proliferation, a flow cytometric analysis of cellular DNA content was performed. An evident G1arrest was observed in cervical cancer cell lines treated with HDACi and ATRA for48h. To determine the causes of the G1phase arrest, we examined the expression of cell cycle regulators (Fig.3B). We observed that VPA combined with ATRA induced an evident up-regulation of P21and P53but a significantly decreased level of p-Stat3expression. However, the expression of cyclin D1was only moderately changed.3) HDACi potentiates ATRA-induced differentiation in cervical cancer cells. Immunoblotting further confirmed that the combinatorial treatment definitely led to cell differentiation but not apoptosis in the cervical cancer cell lines. Consistent with Annexin V labeling, HDACi alone or in combination with ATRA at the designated concentration induced no major changes in the expression of Bcl-2or Caspase-3and its cleavage products. However, HDACi potentiated ATRA-induced differentiation even at a pharmacological dose of ATRA (1μmol/L) as revealed by the expression of the differentiation markers Filaggrin, Loricrin, and Involucrin.4) E-cadherin engagement and the activation of the PI3K/Akt pathway determine the differentiation of cervical cancer cells. The above results suggested that when exposed to a combination of HDACi and ATRA, cervical cancer cells exit the cell cycle and undergo differentiation. E-cadherin and phosphorylated Akt was strongly induced by the combination of drugs and continued to increase with up to48h of exposure, which indicates that PI3K/Akt signaling plays a role in the control of cervical cancer cell differentiation. Blocking the PI3K/Akt pathway by either LE135(selective antagonist of RAR02), the PI3K pharmacological inhibitor LY294002, or specific siRNA for E-cadherin and Aktl/2, caused a significantly decreased expression of the terminal differentiation markers Filaggrin, Loricrin, and Involucrin.5Combined treatment with HDACi and ATRA inhibits the progression of tumor xenografts. Consistently, the combination of VPA and ATRA in vivo caused a statistically significant reduction in tumor volume compared with the single-drug treatments (P<0.01). Importantly, the average tumor volume in the mice that received VPA and ATRA was not significantly different from that of the mice treated with cisplatin alone (P>0.05), suggesting the valid chemotherapeutic potential of this drug combination in cervical cancer. Further, we also found that VPA combined with ATRA induced histone H3hyperacetylation, restored RARβ2expression, and up-regulated P53and P21, whereas this treatment down-regulated p-Stat3in vivo. A pathological examination showed a significant repression of Ki67and the greatest level of induction of the differentiation signature in the combination-treated tumors.6To evaluate the therapeutic effect of HDACi combined with ATRA on the tumour xenografts derived from human donors.1) Combined treatment of HDACi and ATRA inhibits the progression of tumour xenografts. To determine the effect of VPA and ATRA on tumor xenograft growth, animals received vehicle as a control, VPA (300mg/kg/d i.p.), ATRA (15mg/kg/d i.g.), or the combination treatment for28days as the tumors were established. After28days, the mean tumor volume for the combination treatment group was66.84±19.10mm3, while the tumor volumes for mice treated with vehicle, VPA, and ATRA were316.65±94.58mm3,119.83±7.74mm3, and208.64±76.61mm3, respectively. These data indicate that the xenografts were suppressed by59.39%,37.58%, and73.33%(percentage of tumor-growth inhibition,%TGI) for the VPA, ATRA, and combined treatments, respectively.2) Combined treatment of HDACi and ATRA induces cell differentiation and part apoptosis in the tumour xenografts. Importantly, the histological appearance showed that VPA and the combination treatment improved the extent of histological differentiation in the tumor xenografts. Cancer cells from xenografts treated with VPA alone or in combination with ATRA have abundant eosinophilic keratinized cytoplasm, while mitotic figures are absent or noted only occasionally. However, cancer cells in the tissues from both the control and ATRA-treated groups were poorly differentiated. Mitotic figures were common (2-4per high-power field in the control group, but≤2per high-power field in ATRA-treated group), and the cytoplasm was eosinophilic to amphophilic, while keratinization was minimal or absent, similar to the original carcinoma. These observations were further verified by immunohistochemical staining for involucrin and loricrin (markers of terminal epithelium differentiation) in tissues from tumor xenografts. Treatment with VPA in combination with ATRA led to a significant decrease in Ki67-positive cells. In addition to inducing differentiation, VPA also promotes apoptosis. To explore this effect, we performed TUNEL staining to evaluate apoptosis in tumor xenografts. There was a significant increase in apoptotic cells in the combination-treated tumors.3) HDACi and ATRA restore RARβ2expression via epigenetic modification and sequentially inhibit tumor growth. VPA treatment significantly increased the level of acetylated H3compared to the control and the ATRA group. When combined with ATRA, VPA exhibited an evident additive effect in histone epigenetic modification. The ChIP assay showed that VPA combined with ATRA caused a significant increase in the level of histone acetylation at the RARβ2-promoter region. Therefore, the combination treatment reactivated RARβ2expression via epigenetic modification, sequentially enhancing the expression of target genes, including E-cadherin, Involucrin, and Loricrin. Furthermore, a concomitant increase in the levels of P21CIP1, P53, and activated caspase3as well as a decrease in the levels of p-Stat3and Bcl2were noted.In conclusion, HDACi in combination with ATRA has additive effects on the inhibition of cell proliferation and on retarding rumor growth in cervical cancer by restoring RARβ2expression via epigenetic modulation and sequentially promoting differentiation through the PI3K/Akt pathway. Based on these results, a therapeutic regimen combining HDACi and ATRA could be useful in the treatment of cervical cancer, but clinical trials are needed to further evaluate the potential of this combination therapy.