Blockage of SSRP1/Ets-1/Pim-3 signalling enhances chemosensitivity of nasopharyngeal carcinoma to docetaxel in vitro

Abstract

Nasopharyngeal carcinoma (NPC) is a rare cancer in most parts of the world, but is prevalent in South China area. Besides, therapeutic outcome is still unsatisfactory for patients with refractory and relapsed NPC, even though receiving a second line of docetaxel-based chemotherapy. These reasons require a better understanding of mechanisms underlying the carcinogenesis, malignancy and chemoresistance. In the basis of our previous finding of SSRP1 over-expression in NPC cell lines, this study continuously discovered up-regulated Ets-1, phosphor-Ets-1 and Pim-3 in NPC tissues with immunohistochemistry assay and revealed a close correlation of these up-regulated proteins with NPC proliferation and invasion. Using gene-silencing technology followed by western blot and immunocytochemistry detections, SSRP1 was found to facilitate the translocation of phosphor-Ets-1 from cytoplasm to cell nucleus, but have marginal effect on Ets-1 expression and phosphorylation. Pim-3 was positively regulated by Ets-1. In NPC HNE-1 cells, all SSRP1, Ets-1 and Pim-3 knockdown diminished the cell proliferation, enhanced the apoptosis, as well as inhibited the autophagy, invasion and clonogenicity in the presence or absence of docetaxel at IC25. Exposure of HNE-1 cells to docetaxel (IC25) alone had modest effect on cell proliferation and autophagy, and was not as effective as docetaxel treatment after knockdown of SSRP1, Ets-1 or Pim-3 on induction of the apoptosis and on inhibition of the invasion and clonogenicity. Our data indicate that SSRP1/Ets-1/Pim-3 signalling is tightly associated with the proliferation, apoptosis, autophagy, invasion and clonogenicity of NPC cells, and blockage of this signalling facilitates chemosensitivity of the cells to docetaxel.

Introduction

Nasopharyngeal carcinoma (NPC) is a rare cancer in most parts of the world (the worldwide incidence confines to 1 case per 100,000 per year), but it has predominately high morbidity in South China with a general incidence of 15–25 cases per 100,000 per year (some regions in South China even show a morbidity as high as 30/100,000 to 50/100,000), which induces a great concern in the local medical workers [3], [15]. Genetic, environmental, viral and dietary factors have been extensively associated with the pathogenesis of NPC. Radiotherapy alone or concurrent chemoradiotherapy with cisplatin is currently the most primary treatment for early non-disseminated NPC. Docetaxel-based chemotherapy is commonly used in patients with locally advanced or distantly metastatic NPC, but the treatment outcome is modest. Clinical study shows that only 37% and 13.3% NPC patients achieved partial response and stable disease after receiving docetaxel weekly (30 mg/m² every time) [18]. In another independent study, there were 6.8% complete response and 58.9% partial responses among patients with recurrent or metastatic NPC after treated with intravenous docetaxel (75 mg/m², day 1) and nedaplatin (80 mg/m², day 1) (each cycle repeated every 3 weeks for two cycles) [20]. Therefore, it is of great importance to identify regulatory mechanisms conferring the malignant potential and chemo-resistance to NPC cells that could enhance the understanding of cancer progression and result in the development of therapeutic outcomes.

Structure-specific recognition protein 1 (SSRP1) is one of two subunits of the facilitates chromatin transcription (FACT) complex. FACT is involved in multiple biological processes including chromatin remodeling, as well as DNA transcription, replication, recombination, and repair. Recent report, however, suggests that FACT plays important role in cancer in the basis of following discoveries: FACT is expressed at higher levels in many tumor cells, especially in aggressive and poorly differentiated tumor cells, and elevated FACT expression is observed with the development of mammary carcinomas in transgenic mice expressing the Her2/neu protooncogene, whereas expression of FACT subunits are very low or undetectable in most differentiated cells, and show dramatic reduction upon induction of differentiation of stem and progenitor cells [4], [9], [5]; RNAi-mediated knockdown of FACT or suppression of FACT activity by anticancer compounds (e.g. Curaxins) leads to reduced growth and survival of tumor cells [6]. Although the functional roles of SSRP1 in FACT-related cancer initiation and progression are not well understood, [5] uncovered that SSRP1 is required for transcription driven by numerous transcription factors related to cancer growth, cell dedifferentiation, inflammation and stress responses (to ultraviolet, hypoxia, tumor necrosis factor, or genotoxic drug).

The transcription factor v-ets avian erythroblastosis virus E26 oncogene homolog 1 (Ets-1) has been closely associated with the carcinogenesis, invasion, and progression of various tumors tumor aggressiveness. High level of Ets-1 has been identified in breast cancer, ovarian cancer, cervical carcinoma and acute lymphoblastic leukemia [17], [1]. Of note, a broad range of genes and proteins which are involved in cell survival (e.g. c-Met, p53 and dual specificity phosphatase 6), cell cycle (e.g. cyclins D1, D3, and E2), invasion [e.g. Metal matrix proteinase 1 (MMP1), MMP9, u-PA and integrin-β3] and epithelial-mesenchymal transition (e.g. E-cadherin and N-cadherin) are regulated by Ets-1 [10], [1], [11]. Besides, Ets-1 is responsible for chemoresistance to antifolate in both acute lymphoblastic leukemia cells and acute myeloid leukemia cells [21]. The critical important role of Ets-1 in diverse properties of cancer stirred up our strong interest in its function in NPC.

Pim-3 is a member of serine/threonine protein kinases, belonging to provirus integration site for Moloney murine leukemia virus (Pim) family and harbors serine/threonine kinase activity [16]. Ongoing research showed that Pim-3 over-expression is a common event in a wide variety of tumors, particularly those of endoderm-derived organs such as the stomach, liver, pancreas, and colon [25]. Silencing Pim-3 expression retards cell proliferation of hepatocellular, pancreatic, and colon carcinoma cell lines in vitro by promoting cell apoptosis [12]. Using Pim-3 kinase inhibitor effectively interrupted the cancer cell proliferation in nude mice that were injected with human pancreatic cancer cells [12], [22]. Pim-3 has also been found to be concentrated at the cellular lamellipodia and co-localized with focal adhesion [29]. Further research unveiled that Pim-3 is implicated in spreading and migration of vascular endothelial cells and in angiogenesis [29], [27]. These evidence suggests that Pim-3 is an important contributor for the proliferation, metastasis and invasion of tumors. However, the expression of Pim-3 in NPC and the relationship between Pim-3 and NPC have not been reported yet.

Our recent data (ready to be published in another journal) manifested that SSRP1 is up-regulated in NPC cell lines (including CNE-1, HNE-1, SUNE-1, 5–8F and 6–10 B cells) compared with that in human immortalized nasopharyngeal epithelial cell line (NP69 cells), and indentified that SSRP1 is tightly associated with the invasion and epithelial-mesenchymal transition of NPC cells, while the mechanisms underlying SSRP1 involvement in NPC remains incompletely understood. A report of Garcia et al. implied an interaction of SSRP1 with Ets-1, by which SSRP1 may control expression of some SSRP1-associated/-enriched genes [5]. Besides, a study shows Ets-1 can target Pim-3 and affect its expression and activity in pancreatic cancer cells [13]. These recent data prompted us to examine whether there is cross-talk among SSRP1, Ets-1, Pim-3 in NPC, and how there proteins impact on NPC properties, including proliferation, apoptosis, autophagy, invasion, clonogenicity and chemoresistance to docetaxel. The results of the present study contribute to an improved understanding of the precise molecular mechanisms underlying diverse processes of NPC and lay a theoretical foundation for the development of therapeutic outcomes in the treatment with docetaxel.