159P - The impact of kisspeptin pre-treatment towards temozolomide resistance mechanisms and associated differently expressed proteins (DEPs) in human glioblastoma cells

Background

To overcome the common Temozolomide (TMZ) resistance in Glioblastoma (GBM), targeting certain receptors such as GPCRs can be of benefit as they are involved in cancer treatment response signalling pathways. One attractive target is KISS1R, activated by Kisspeptin. RNA-Seq of male GBM samples revealed elevated KISS1R expression and low KISS1 expression, suggesting GBM as a paracrine tumour. By re-modulating KISS1R using exogenous kisspeptin (KP-10), we aimed to positively influence the cytotoxic effect of TMZ and investigate the underlying mechanism(s) in KISS1R-expressing GBM cells.

Methods

KISS1R expression was confirmed using RT-PCR. KP-10/TMZ IC₅₀ was determined using MTT cell viability assay and synergistic combinations were analysed using Isobologram analysis with non-constant ratio. Pathway prediction was done using KEGG, Wikipathways and STITCH. Flow cytometry and live-cell imaging identified molecular mechanisms while relevant markers were assessed via JESS simple Westren. LC-MS/MS was used to identify relevant DEPs with Kaplan-Meier survival analysis to identify clinical relevance.

Results

KISS1R expression was confirmed in A-172 (p53 wild-type) and LN-18 (p53 mutant). KP-10/TMZ treatment demonstrated high synergism in both cells. Pathway analysis of KP-10/TMZ predicted interaction at apoptosis, cytoprotective autophagy and DNA damage. This was further confirmed with elevated autophagy marker p62 in A-172, while increased expression of DNA damage and repair markers PARP and γH2AX in LN-18. The proteomic profiles showed DEPs related to RNA splicing, cytoplasmic translation and ER stress in A-172, while negative regulation of mRNA, glycolysis and MHC class 1 protein binding in LN-18. Ten common DEPs impacted GBM patients’ survival.

Conclusions

The response to KP-10/TMZ treatment varied based on the status of p53 of the GBM cell used and commonly impacted DEPs associated with the cytoplasmic translation of proteins and regulation of mRNA, more seen in LN-18 (p53 mutant). This underscores the significance of delving into the concept of dormant cells within a single tumour to correlate the results from the in-vitro GBM model to actual GBM patients.

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

JCSMHS, Monash University Seed Grand and Monash Merit scholarship and funding for doctoral research.

Disclosure

All authors have declared no conflicts of interest.