The role of adiposity in enhancing LIF/LIFR signaling pathway in endometrial cancer

Objectives: The incidence of endometrial cancer (EC) is estimated to increase by 1-2% annually. More than half of all EC diagnoses are currently attributable to obesity, which is an independent risk factor for this cancer. Obesity is suspected to function as a potent inducer of leukemia inhibitory factor (LIF), the most pleiotropic member of the interleukin-6 family of cytokines. Our recent studies utilizing public databases revealed high levels of LIF and its receptor LIFR are associated with poor survival in EC patients (KM plotter). The objective of this study is to determine whether alterations in the LIF/LIFR signal cascade occur in EC cells under conditions of adiposity; and whether this pathway could be a target for therapy. Methods: All patient samples, EC tissue, serum, and adipose tissue, were collected through the UT Health San Antonio tumor registry under IRB approval. Expression of LIF in EC patient samples was analyzed using immunohistochemistry (IHC) using a UT Health generated tumor tissue array (TMA) and scored by UT Health Pathology. Primary EC tissue derived cells and established EC cell lines were treated with primary adipocyte conditioned medium and non-obese patient serum or obese patient serum. The effect of adiposity on EC cells was determined using MTT cell viability assay and colony formation assay, in addition to utilization of organoids derived from primary EC tissue. Mechanistic studies were conducted using Western blotting, RT-qPCR, and reporter assays thus confirming the activation of LIF/LIFR downstream pathways including STAT3, mTOR, Akt, and MAPK. The recently developed LIFR inhibitor, EC359, was used to elucidate the therapeutic utility of targeting the enhanced LIF/LIFR pathway generated by high adiposity. Results: Treatment of established and primary EC cells with adipose environment increased the expression of LIFR protein, enhanced LIFR reporter activity, and increased cellular proliferation. Western blot and RT-qPCR analyses confirmed that increased expression of LIFR correlated with enhanced downstream LIFR signaling and subsequent activation of LIFR target genes. Treatment of EC cells with the LIFR inhibitor EC359 significantly reduced cell viability under adipose conditions. EC359 treatment also decreased adipose environment mediated organoid growth. IHC results using TMA that contained normal tissue (n=33) and EC tumors (n=45) with BMI >30 showed significantly increased expression of LIF in EC compared to normal endometrium. Further, LIF expression was upregulated in EC from obese patients compared to non-obese EC control patients. Conclusions: Collectively, our results suggest that increased adiposity contributes to accelerated EC cell growth via upregulation of the LIF/LIFR pathway. The LIF/LIFR axis represents a potential therapeutic target for adiposity driven EC and the LIFR inhibitor EC359 may be useful in modulating LIF/LIFR signaling in obese EC patients.