Knockdown of CypA inhibits interleukin-8 (IL-8) and IL-8-mediated proliferation and tumor growth of glioblastoma cells through down-regulated NF-κB

Shan Sun 0 1 2 3 4 5 6 Qiuwei Wang 0 1 2 3 4 5 6 An Giang 0 1 2 3 4 5 6 Cong Cheng 0 1 2 3 4 5 6 Chia Soo 0 1 2 3 4 5 6 Cun-Yu Wang 0 1 2 3 4 5 6 Linda M. Liau 0 1 2 3 4 5 6 Robert Chiu 0 1 2 3 4 5 6 0 C. Soo Department of Orthopaedic Surgery, David Geffen School of Medicine , UCLA, Los Angeles, CA 90095, USA 1 C. Cheng Department of Biological Sciences and Biotechnology, School of Medicine, Tsinghua University , Beijing, China 2 S. Sun Q. Wang A. Giang R. Chiu (&) Dental Research Institute, UCLA School of Dentistry , Los Angeles, CA 90095, USA 3 R. Chiu Jonsson Comprehensive Cancer Center , UCLA, Los Angeles, CA 90095, USA 4 R. Chiu Department of Surgery/Oncology, David Geffen School of Medicine , UCLA, Los Angeles, CA 90095, USA 5 L. M. Liau Department of Neurosurgery, David Geffen School of Medicine , UCLA, Los Angeles, CA 90095, USA 6 C.-Y. Wang Division of Oral Biology and Medicine, UCLA School of Dentistry , Los Angeles, CA 90095, USA Although cyclophilin A (CypA) has been reported to be over-expressed in cancer cells and solid tumors, its expression and role in glioblastomas have not been studied. Herein, we show that expression of CypA in human glioblastoma cell lines and tissues is significantly higher than in normal human astrocytes and normal counterparts of brain tissue. To determine the role of overexpressed CypA in glioblastoma, stable RNA interference (RNAi)-mediated knockdown of CypA (CypA KD) was performed in gliobastoma cell line U87vIII (U87MG DEGFR). CypA KD stable single clones decrease proliferation, infiltration, migration, and anchorage-independent growth in vitro and with slower growth in vivo as xenografts in immunodeficient nude mice. We have also observed that knockdown of CypA inhibits expression of interleukin-8 (IL-8), a tumorigenic and proangiogenic cytokine. Conversely, enforced expression of CypA in the CypA KD cell line, Ud-12, markedly enhanced IL-8 transcripts and restored Ud-12 proliferation, suggesting that CypA-mediated IL-8 production provides a growth advantage to glioblastoma cells. CypA knockdown-mediated inhibition of IL-8 is due to reduced activity of NF-jB, which is one of the major transcription factors regulating IL-8 expression. These results not only establish the relevance of CypA to glioblastoma growth in vitro and in vivo, but also suggest that small interfering RNA-based CypA knockdown could be an effective therapeutic approach against glioblastomas. - Malignant gliomas are among the most devastating of cancers, leading to death in most cases [1]. They present unique challenges for therapy due to their difficult access locations, aggressive biological behavior, and diffuse infiltrative growth. Cyclosporin A (CsA) has been reported to have a chemotherapeutic effect in a variety of cancer cells, including human gliomas [24]. In a human glioblastoma cell line and glioblastoma cells from brain slices, CsA was shown to affect their growth/survival and migration/invasion [3, 4]. The mechanisms of these effects may relate to the biochemical properties of CsA such as inhibition of cyclophilin isomerase and calcineurin phosphatase activities that mediate immunosuppressive effects that can alter several signal pathways. CypA is found in normal cells, comprises *0.6% of total cytosolic protein, and is widely conserved among prokaryotes and eukaryotes [5]. It is a member of the peptidylprolyl isomerase (PPI) family, a group of proteins that catalyze cis-trans isomerization of peptidyl-prolyl bonds during protein folding and/or conformational changes [6, 7]. CypA was first identified as the primary intracellular target of the immunosuppressive drug, CsA [8]. The immunosuppressive activity of CsA is thought to result from engagement of calcineurin by the CsA-CypA complex [9], an observation supported by the finding that CypA knockout mice are resistant to immunosuppression by CsA [10]. Several lines of research have revealed that PPIs such as CypA may function as molecular signaling switches that can act as novel molecular timers to help control the amplitude and duration of a cellular process [11]. Moreover, the role of CypA nuclear translocation or CypAs participation in activation of other factors or their nuclear translocation also impacts various cellular functions [1215]. One recent report demonstrated that knockdown of CypA inhibited signal transducer and activator of transcription 3 (Stat3) interleukin-6-induced tyrosine phosphorylation and nuclear translocation, resulting in altered gene expression in myeloma cell lines [16]. The CypA inhibitor, CsA, exhibited similar effects. Additional data also suggest that CypA may contribute to the pathology of certain human malignancies. Several experiments have demonstrated that CypA is substantially up-regulated in various types of cancers and cancer cell lines [1720]. These over-expressed CypAs mediate multiple cellular processes that can confer growth advantage to tumor cells in the neoplastic microenvironment. This was substantiated by CypA over-expression in the SILEK-transformed small airway epithelial cell line, which showed that increased CypA expression correlated with dramatically faster tumor formation in vivo [21]. Furthermore, CypA over-expression in cancer cells conferred resistance to anticancer drugs and hypoxia-induced cell death [22, 23]. Therefore, targeted CypA and inhibition of its PPI activity have been suggested as novel therapeutic strategies for treatment of human cancers. Over-expression of CypA has also been implicated in other pathological processes such as rheumatoid arthritis [24, 25]. In rheumatoid arthritis patients, over-expressed CypA stimulates production of inflammatory cytokines such as tumor necrosis factor alpha (TNF-a), interleukin1b (IL-1b), IL-8, monocyte chemoattractant protein-1 (MCP-1), and matrix metalloproteinase 9 (MMP-9) [25]. These CypA-stimulated products could arise through a pathway that is dependent on NF-jB activation. However, the means by which CypA mediates signaling to activate NF-jB remains to be investigated. Similarly, stably expressed CypA in the SK-Hep1 cell line revealed that CypA up-regulates the expression of many cytokine-related genes such as IL-8, IL-1b, IL-6, CXCL1, CXCL2, and CXCL3 [22]. These up-regulated cytokines and chemokines may confer tumor cell growth advantage in the neoplastic microenvironment. Therefore, a strategy to reverse these effects might offer attractive options for novel therapeutics. In human gliomas, IL-8 is expressed and secreted at high levels both in vitro and in vivo, and recent experiments suggest it is critical to glial tumor neovascularity and progression [26]. IL-8 could act as an inflammatory chemoattractant as part of the host response to neoplasia. Moreover, it also acts as a more general proinflammatory factor released in response to tissue stress and necrosis, a proangiogenic factor that promotes new vessel growth, or an autocrine growth factor secreted by tumor cel (...truncated)