PP2A inhibitors induce apoptosis in pancreatic cancer cell line PANC-1 through persistent phosphorylation of IKKα and sustained activation of the NF-κB pathway

Abstract

Serine/threonine protein phosphatase 2A (PP2A), is thought to be a cancer suppresser, as inhibition of PP2A can induce phosphorylation and activation of substrate kinases, most of which can accelerate growth. Interestingly, cantharidin potently inhibits PP2A but efficiently represses various cancer cells. In the present study, we found that PP2A inhibitors, cantharidin or Okadaic acid, inhibited cell viability and triggered apoptosis in PANC-1 pancreatic cancer cell line dependent on PP2A/IKKα/IκBα/p65 NF-κB pathway. The activation of NF-κB pathway up-regulated downstream pro-apoptotic genes, TNF-α, TRAILR1 and TRAILR2, and triggered apoptosis through the extrinsic pathway, indicating that PP2A is a potential target for pancreatic cancer treatment.

Introduction

Pancreatic cancer is one of the most lethal human cancers, which causes about 30,000 and 40,000 annual deaths in the USA and Europe, respectively, and is now the sixth leading cause of death from malignant disease in China [1], [2]. The collective 1-year survival from the time of diagnosis at any stage is only 26%, while 5-year survival falls down to <5% [3]. The main reason for such poor outcomes is mostly due to the fact that the first diagnosis is made at late stages and its overall resistance to available therapies. Even patients undergoing surgical therapy at early stages often suffer from disease recurrence or metastatic disease [1], [2], [4]. Therefore new treatment against this aggressive neoplasm is urgently needed.

Natural products and derivatives thereof show a notable promise for cancer treatment. For instance, vinca alkaloids, taxanes and camptothecins have now become standard repertoire for cancer chemotherapy [5]. Mylabris is the dried body of the Chinese blister beetle. The use of mylabris as a traditional Chinese medicine can be traced back to more than 2000 years ago, and it is still used as a folk medicine today [6]. The active constituent of mylabris is cantharidin [6]. In recent studies, cantharidin and its derivatives demonstrated strong anti-tumor activity against multiple cancer cells in vitro, such as oral cancer cells [7], hepatoma cells [8], colorectal carcinoma cells [9] and breast cancer cells [10]. In clinical trials, cantharidin and its analogs have shown therapeutic effects against primary hepatoma, including cases at the advanced stage [6], [11], suggesting the potential for applying cantharidin to the treatment of pancreatic cancer, which nearly always has a late diagnosis [1], [2], [4].

PP2A is a multimeric serine/threonine phosphatase which can dephosphorylate several kinases [12], [13]. Mechanistically, cantharidin has been shown to be a potent and selective inhibitor of PP2A [14], suggesting its anti-cancer effect might involve the inhibition of this phosphatase. In contradiction to this finding, inhibition of PP2A has always been thought to be cancer promoting, as inhibition of PP2A could induce phosphorylation and activation of many substrate kinases which can accelerate growth [12], [13], [15]. It is still not quite clear how the putative cancer inducer may also be a potential anti-cancer drug. Recently, some kinase-dependent growth inhibition pathways induced by PP2A inhibitors treatment was implicated [16], [17], suggesting inhibition of PP2A could also exert anti-cancer effects through the activation of kinase pathways.

NF-κB pathway plays a pivotal role in the expression of genes involved in inflammatory and immune responses [18], [19], and also participates in carcinogenesis including pancreatic cancer [3], [20], [21]. According to the canonical NF-κB pathway cascade [18], [19], the phosphorylated IKK (IκB kinase) can further phosphorylate IκB, which is the cytoplasmic inhibitor of the NF-κB complex. When phosphorylated, IκB undergoes ubiquitination and proteasome-mediated degradation rapidly, resulting in the release and nuclear translocation of the NF-κB complex. The prototypical NF-κB complex is a heterodimer composed of p50 and RelA/p65. Once entering the nucleus, p65 engages cognate κB enhancers, which contain one or more κB-enhancer consensus sequences (5′-GGGRNYYYCC-3′, where R is a purine, Y is a pyrimidine and N is any nucleic acid), and regulates the expression of downstream genes. Although NF-κB is generally viewed as anti-apoptotic and oncogenic [21], increasing evidence has shed new light on the role of NF-κB in promoting apoptosis [22], [23].

IKK (IκB kinase), the key regulator of the NF-κB pathway [12], [24], is one of the direct substrates of PP2A [12], [24], [25]. Inhibition of PP2A by Okadiac acid (OA), a classical PP2A inhibitor, can cause sustained NF-κB pathway activation through persistent phosphorylation of IKK [25]. As cantharidin appears to be a selective PP2A inhibitor, it may also phosphorylate IKK and induce subsequent activation of NF-κB, which might further trigger apoptosis [22], [23].