Investigation of the pathways involved in the acquired radioresistant emt6 mouse mammary carcinoma cells to gamma- ray irradiation: in vitro and in vivo studies

Resistance of breast cancer to radiotherapy is the crucial aspect leading to relapse and low survival rate. Radioresistance is suggested to be linked with epithelial-mesenchymal transition (EMT), a process involved in regulating cancer tissue remodelling, resulting in recurrence and metastasis. The underlying mechanisms in acquiring radioresistance to gamma-ray in mouse mammary cancer cell lines (EMT6) were investigated using both in vitro and in vivo approaches. EMT6 cells were irradiated with gamma-ray at 2 Gy/cycle to initiate the development of radioresistance in vitro. Confirmation of EMT6 cells acquired radioresistance was analysed using migration and clonogenic assays. Next-generation sequencing analysis validated 16 genes of interest (GOI) via real-time polymerase chain reaction (qPCR). The signalling pathways and proteins involved in EMT6 cells acquiring radioresistance were verified by KEGG pathway analysis and western blotting, respectively. EMT6RR_MJI radioresistance cells were developed from parental EMT6 cells after 8 cycles of fractionated gamma-ray irradiation. This is confirmed by irradiation with gamma-ray at 2, 4, and 8 Gy, which resulted in higher survival fractions and migratory rates of EMT6RR_MJI compared to parental cells. Six out of 16 GOIs (PD-L1, IL-6, AXL, GAS6, IGFBP4, and APCDD1) were upregulated at the 8th irradiation cycle in EMT6RR_MJI compared to control. Pathway analysis showed PI3K-AKT and JAK-STAT signalling pathways were the common functional pathways contributing to radioresistance by these six genes. Further investigations focused on PD-1 gene and protein expressions, in which PD-1 is the crucial regulator of PI3K- AKT and JAK-STAT pathways. PD-1 gene and protein expressions were upregulated in EMT6RR_MJI compared to parental cells (p<0.05, p<0.05, respectively). The role of PD-1 in radioresistance was further investigated in vivo. Mice were divided into 3 groups: Group 1 (control), Group 2 (inoculated with parental EMT6 cells), and Group 3 (inoculated with EMT6RR_MJI cells). Groups 2 and 3 were further subdivided into 4 subgroups: Subgroup 1 (control, n=6), Subgroup 2 (gamma-ray irradiation, n=6), Subgroup 3 (combination of gamma-ray and Nivolumab, a PD-1 inhibitor, n=6) and Subgroup 4 (Nivolumab, n=6). Mice in subgroups 3 and 4 were further injected with 10 mg/kg body weight Nivolumab at 3 time points. Once the tumour was palpable and visible in all groups, the mice in subgroups 2 and 3 were treated with gamma-ray at 2 Gy per cycle for 8 cycles. Tumour volumes were measured every 2 days. The mice in all groups were sacrificed 5 days post-irradiation and tumour sections were collected to determine the expression of N-cadherin, E-cadherin, PD-1, and PD-L1 using qPCR, while the PD-1 protein was analyzed by western blot and ELISA. N- cadherin and E-cadherin were upregulated and downregulated, respectively, in both tumours. The tumours were confirmed to be derived from proliferative EMT cells. In the parental EMT6, tumour growth in subgroups 2 (p<0.01), 3 (p<0.001), and 4 (p<0.01) decreased compared to control. Furthermore, PD-1 gene expression was higher in subgroup 2 (p <0.05) compared to the control group. PD-1 protein expression increased in subgroup 4 (p<0.05) but reduced in subgroup 2 (p<0.05) as compared to control. For the EMT6RR_MJI tumour, the growth in subgroup 3 was lower in control (p<0.001) group. PD-L1 gene expression decreased in subgroups 2 and 4 compared to EMT6RR_MJI control (p<0.05). PD-1 protein expression was reduced in subgroups 2 and 3 compared to the control group (p<0.05). In conclusion, EMT6RR_MJI acquired resistance to gamma-ray is associated with the upregulation of PD-1/PD-L1 signalling. However, no conclusive evidence suggests Nivolumab inhibits PD-1 expression. Extending the duration of post-treatment is required to elucidate these possible mechanisms, which may provide knowledge for the potential adjuvant treatment during radiotherapy in reducing resistance and recurrence.