Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPβ) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPβ, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction.

The tumor microenvironment (TME) exerts critical pro-tumorigenic effects through cytokines and growth factors that support cancer cell proliferation, survival, motility and invasion. Insulin-like growth factor-1 (IGF-1) and signal transducer and activator of transcription 3 (STAT3) stimulate colorectal cancer development and progression via cell autonomous and microenvironmental effects. Using a unique inhibitor, NT157, which targets both IGF-1 receptor (IGF-1R) and STAT3, we show that these pathways regulate many TME functions associated with sporadic colonic tumorigenesis in CPC-APC mice, in which cancer development is driven by loss of the Apc tumor suppressor gene. NT157 causes a substantial reduction in tumor burden by affecting cancer cells, cancer-associated fibroblasts (CAF) and myeloid cells. Decreased cancer cell proliferation and increased apoptosis were accompanied by inhibition of CAF activation and decreased inflammation. Furthermore, NT157 inhibited expression of pro-tumorigenic cytokines, chemokines and growth factors, including IL-6, IL-11 and IL-23 as well as CCL2, CCL5, CXCL7, CXCL5, ICAM1 and TGFβ; decreased cancer cell migratory activity and reduced their proliferation in the liver. NT157 represents a new class of anti-cancer drugs that affect both the malignant cell and its supportive microenvironment.

Objectives

Little is known about targeting the metabolome in non-cancer conditions. Choline kinase (ChoKα), an essential enzyme for phosphatidylcholine biosynthesis, is required for cell proliferation and has been implicated in cancer invasiveness. Aggressive behaviour of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) led us to evaluate whether this metabolic pathway could play a role in RA FLS function and joint damage.

Methods

Choline metabolic profile of FLS cells was determined by (1)H magnetic resonance spectroscopy ((1)HMRS) under conditions of ChoKα inhibition. FLS function was evaluated using the ChoKα inhibitor MN58b (IC₅₀=4.2 μM). For arthritis experiments, mice were injected with K/BxN sera. MN58b (3 mg/kg) was injected daily intraperitoneal beginning on day 0 or day 4 after serum administration.

Results

The enzyme is expressed in synovial tissue and in cultured RA FLS. Tumour necrosis factor (TNF) and platelet-derived growth factor (PDGF) stimulation increased ChoKα expression and levels of phosphocholine in FLS measured by Western Blot (WB) and metabolomic studies of choline-containing compounds in cultured RA FLS extracts respectively, suggesting activation of this pathway in RA synovial environment. A ChoKα inhibitor also suppressed the behaviour of cultured FLS, including cell migration and resistance to apoptosis, which might contribute to cartilage destruction in RA. In a passive K/BxN arthritis model, pharmacologic ChoKα inhibition significantly decreased arthritis in pretreatment protocols as well as in established disease.

Conclusions

These data suggest that ChoKα inhibition could be an effective strategy in inflammatory arthritis. It also suggests that targeting the metabolome can be a new treatment strategy in non-cancer conditions.