Hyperthermia (HT) has been widely used as an adjuvant to chemotherapy in the treatment of ovarian cancer, it has pleiotropic effects on cellular processes involved in tumor growth and progression. Inducing cellular stress response through heat-shock proteins (HSPs) expression is one of the main hallmarks of HT. HSPs have been reported to trigger an immune activation. However, antitumor immune responses are often invalidated by immune evasion mechanisms such as the overexpression of the immunosuppressive PD-L1 and the loss of MHC class I expression. In this study, we explore the effects of HT on PD-L1 and the transcriptional activator of MHC class I genes NLRC5.
We tested the viability of two ovarian cancer cell lines (IGROV1 and SKOV3) in response to HT by exposing them to 42°C and 43°C, we then analyzed the time-related expression pattern of HSP70, HSP60, HSP90 and HSP27. Coculture with peripheral blood mononuclear cells (PBMC) was carried out and was subjected to HT. Culture supernatants from heat-shocked cancer cells were added to untreated IGROV1 and SKOV3. Pro-inflammatory cytokines, PD-L1 and NLRC5, expression levels were measured. Knockdown of HSP70 and HSP27 was also performed. The correlation between PD-L1 and NLRC5 expression in ovarian cancer was evaluated using The Cancer Genome Atlas (TCGA) dataset.
The WST-8 assay showed that HT at 43˚C caused a decrease in cell viability in a time-dependent manner, whereas treatment at 42˚C induced a significant increase at 24h post heat shock. This increase could be explained by the development of thermotolerance for which HSPs accumulation is responsible. Both PD-L1 and NLRC5 showed a concomitant significant decrease in their mRNA expression levels in coculture conditions after HT, while the addition of culture medium conditioned by heat-shocked cancer cells led to the opposite effect. Moreover, a positive correlation between NLRC5 and PD-L1 expression (r=0.54, p-value <0.001) was found using TCGA database. After knocking down HSPA1A and HSPB1 genes, preliminary results revealed a significant downregulation of both PD-L1 and NLRC5 following HSP27 silencing.
Our findings revealed that hyperthermia modulates the expression of PD-L1 and NLRC5 in a positively correlated manner, and that HSP27 inhibition could reverse this effect.
Legal entity responsible for the study
Saint Joseph University of Beirut.
Saint Joseph University Research Council.
All authors have declared no conflicts of interest.