Abstract:
Cancer is associated with alterations in key cellular pathways, including the aspartate β-hydroxylase (ASPH) pathway. ASPH is an oncogene that is upregulated in various carcinomas and plays a vital role in tumor progression and metastasis. To better understand the mechanisms of ASPH oncogenicity, we examined the effect of ASPH inhibition on human papillomavirus (HPV)-positive (CRL-3240, HeLa) and HPV-negative (FaDu, MCF-7) cell lines. Targeting the ASPH catalytic activity using a second-generation small molecule inhibitor MO-I-1151 resulted in a significant reduction in proliferation, migration, and invasiveness of the tumor cell lines. To investigate the effect of MO-I-1151 on pathway modulation under hypoxic and normoxic conditions, we performed transcriptomic analysis by bulk RNA sequencing. The analysis identified 2387 and 2350 differentially expressed genes in hypoxic and normoxic conditions, respectively, when comparing MO-I-1151-treated samples with their respective controls. The data revealed a significant downregulation of genes (1460 in hypoxia and 1325 in normoxia) that regulate cell cycle, DNA replication, and epithelial-mesenchymal transition. RT-qPCR was used to validate selected key genes involved in various cellular pathways and downregulated in at least 3 cell lines (IL7R, LY6D, LY6E, LY6K, TRIP13, SUV39H1, ELAVL2, and WNT10B). The interleukin 7 (IL7) receptor gene (IL7R) was consistently downregulated in both hypoxic and normoxic conditions in all the cell lines. The IL7 receptor and the thymic stromal lymphopoietin (TSLP) receptor share the IL7Rα chain encoded by IL7R and interact with JAK1/3 and JAK1/2, respectively, through their intracellular domains. To investigate the involvement of IL7 and/or TSLP in the JAK/STAT pathway, cells treated with MO-I-1151 or tofacitinib (a Janus kinase inhibitor targeting IL7 receptor signaling) were subjected to western blot analysis. The treatment with MO-I-1151 or tofacitinib downregulated the production of several JAK/STAT pathway components, including JAK2/3, STAT5, BCL2, MCL1, and cyclin D1, in CRL-3240, FaDu, and MCF-7 cells, which indicates that ASPH inhibition affects tumor cell progression by targeting the JAK/STAT pathway primarily through the IL7 receptor rather than the TSLP receptor pathway. ELISA assay showed that MO-I-1151 and tofacitinib treatment significantly decreased the IL7 and TSLP levels in CRL-3240 and FaDu, but no remarkable effect was observed in HeLa and MCF-7 cells. The study provides a comprehensive insight into the molecular events induced by ASPH inhibition in HPV-positive and -negative cell lines under hypoxic and normoxic conditions and highlights the potential for further investigation of IL7/TSLP receptor signaling intratumoral ASPH activity to improve cancer treatment.
Audience Take Away Notes:
- This study provides insight into the role of ASPH in cancer progression and its potential as a therapeutic target
- The study presents the molecular events induced by ASPH inhibition, including differential gene expression and pathway modulation in hypoxic and normoxic conditions
- The information provided opens avenues for further exploration of the ASPH and IL-7R connection
