Background: Clear-cell renal cell carcinoma (ccRCC) is the most frequent histotype of renal cancer, accounting for about 70% of all kidney carcinomas (1). This histotype is also the most aggressive being responsible for most deaths in patients with kidney cancer (1). Metastatic kidney carcinoma is a lethal pathology that currently is treated in first-line therapy with tyrosine kinase inhibitors (TKI) in combination with immune checkpoint inhibitors. This combined therapy has led to improvements in overall and progression-free survival as well as objective response rate, but this combination shows an increased toxicity compared with TKI monotherapy, often causing treatment interruption (2). In addition, frequent therapy resistance due to different mechanisms, including the activation of autophagy, have been observed. Autophagy is a process used by cancer cells to produce energy and destroy pharmacological molecules by trapping them in autophagosomes (3). Therefore, the use of autophagy inhibitors should improve the efficacy of conventional therapy. Here, we evaluated the angiogenesis pathway and cell migration after cell treatment with the autophagy inhibitor desmethylclomipramine (DCMI), alone and in combination with the TKI sunitinib. Materials and Methods: Caki-2 and KJ29 ccRCC cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 with 1% fetal bovine serum in the presence of DCMI, alone and in combination with the vascular endothelial growth factor receptor (VEGFR) inhibitor sunitinib for 48 and 120 h. Treated and untreated cells were used to study protein expression related to autophagy and angiogenesis as well as to evaluate cell migration. Hypoxia-inducible factor 1 α (HIF1α), autophagy protein markers p62 and microtubule-associated protein 1A/1B-light chain 3 (LC3), and p-VEGFR protein content was analyzed by western blot and calculated as band intensity ratio between the protein of interest and the housekeeping protein β-actin. Cell migration was performed seeding 250,000 KJ29 cells in six-well plates and culturing them in DMEM/F12 medium supplemented with 10% FBS to confluence. Next, a wound between the cells was generated using a sterile tip and cells were grown for a further 48 and 120 h in DMEM/F12 1% FBS with 5 μM DCMI and 5 μM sunitinib individually or mixed together. Wound healing was detected by comparing images acquired at baseline with those acquired after 48 and 120 h of culture by a phase-contrast microscope equipped with a CCD camera. Statistical analysis was performed by GraphPad Prism software using analysis of variance or t-test as appropriate. Values of p<0.05 were considered statistically significant. Results: Treatment with DCMI significantly enhanced p62 and LC3 expression in both cell lines compared with untreated cells (Figure 1A). Conversely, the application of sunitinib did not change the levels of these proteins. As expected, treatment with sunitinib strongly reduced the phosphorylation of VEGFR in KJ29 cells, while in Caki-2 cells, this effect was milder but still significant (Figure 1B). Surprising, the treatment with DCMI was also able to switch off the activity of VEGFR, mainly in KJ29 cells. Interestingly, a greater inhibition of VEGFR phosphorylation in Caki-2 cells treated with DCMI combined with sunitinib compared with single compounds was observed (Figure 1B). Importantly, the expression of HIF1α was reduced by the combination in KJ29 cells as well as in Caki-2 cells (Figure 1B). As observed for p-VEGFR, the combined treatment with these drugs caused a stronger reduction of HIF1α expression as compared to the single treatment in both cell lines (Figure 1B). The analysis of cell migration by scratch-wound assay shows that treatment with DCMI and sunitinib in cells reduced KJ29 cell migration after 48 and 120 h compared with untreated cells (Figure 1C). Moreover, the combined treatment potentiated the inhibition of cell migration not only after 48 h, but also for much longer (120 h). Discussion and Conclusion: Different mechanisms are associated with drug resistance including the activation of autophagy in kidney cancer cells. Moreover, this process promotes cancer cell proliferation and migration (1). Therefore, the inhibition of autophagy reduces cancer progression and improves conventional therapy response. Our findings show that the application of DCMI effectively inhibited autophagy in two different kidney carcinoma cell lines. The treatment with DCMI prevented the fusion of autophagosomes with lysosomes leading to the accumulation of both p62 and LC3 autophagy proteins (Figure 1A). Interestingly, the inhibition of autophagy enhanced the reduction of angiogenesis analyzed as VEGFR dephosphorylation, suggesting that autophagy is a mechanism involved in the activation of angiogenesis. Consistent with this, the reduction of autophagy by DCMI administration strengthened the downregulation of HIF1α induced by sunitinb (Figure 1B). HIF1α is an upstream effector of angiogenesis that is constitutively activated in both Caki-2 and KJ29 ccRCC cells and promotes the activation of VEGFR. Importantly, the inhibition of autophagy combined with the administration of sunitinib caused a greater reduction of HIF1α expression and in turn the inactivation of VEGFR, leading to the inhibition of the angiogenesis pathway. Furthermore, the combined treatment with DCMI and sunitinib induced a stronger reduction of cell migration compared with the single treatment and for a longer time (Figure 1C). In conclusion, our findings indicate that autophagy is involved in processes linked to angiogenesis, cell migration and therapy resistance. Therefore, the inhibition of autophagy combined with current chemotherapy may represent an intriguing option for improving drug response in advanced kidney carcinoma.
A COMBINATION OF TYROSINE KINASE AND AUTOPHAGY INHIBITORS STRENGHTHENS THE INHIBITION OF ANGIOGENIC PATHWAY IN KIDNEY CARCINOMA CELL LINES
Carmelo IppolitoMembro del Collaboration Group
;Gianluca Aguiari
Writing – Review & Editing
2024
Abstract
Background: Clear-cell renal cell carcinoma (ccRCC) is the most frequent histotype of renal cancer, accounting for about 70% of all kidney carcinomas (1). This histotype is also the most aggressive being responsible for most deaths in patients with kidney cancer (1). Metastatic kidney carcinoma is a lethal pathology that currently is treated in first-line therapy with tyrosine kinase inhibitors (TKI) in combination with immune checkpoint inhibitors. This combined therapy has led to improvements in overall and progression-free survival as well as objective response rate, but this combination shows an increased toxicity compared with TKI monotherapy, often causing treatment interruption (2). In addition, frequent therapy resistance due to different mechanisms, including the activation of autophagy, have been observed. Autophagy is a process used by cancer cells to produce energy and destroy pharmacological molecules by trapping them in autophagosomes (3). Therefore, the use of autophagy inhibitors should improve the efficacy of conventional therapy. Here, we evaluated the angiogenesis pathway and cell migration after cell treatment with the autophagy inhibitor desmethylclomipramine (DCMI), alone and in combination with the TKI sunitinib. Materials and Methods: Caki-2 and KJ29 ccRCC cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 with 1% fetal bovine serum in the presence of DCMI, alone and in combination with the vascular endothelial growth factor receptor (VEGFR) inhibitor sunitinib for 48 and 120 h. Treated and untreated cells were used to study protein expression related to autophagy and angiogenesis as well as to evaluate cell migration. Hypoxia-inducible factor 1 α (HIF1α), autophagy protein markers p62 and microtubule-associated protein 1A/1B-light chain 3 (LC3), and p-VEGFR protein content was analyzed by western blot and calculated as band intensity ratio between the protein of interest and the housekeeping protein β-actin. Cell migration was performed seeding 250,000 KJ29 cells in six-well plates and culturing them in DMEM/F12 medium supplemented with 10% FBS to confluence. Next, a wound between the cells was generated using a sterile tip and cells were grown for a further 48 and 120 h in DMEM/F12 1% FBS with 5 μM DCMI and 5 μM sunitinib individually or mixed together. Wound healing was detected by comparing images acquired at baseline with those acquired after 48 and 120 h of culture by a phase-contrast microscope equipped with a CCD camera. Statistical analysis was performed by GraphPad Prism software using analysis of variance or t-test as appropriate. Values of p<0.05 were considered statistically significant. Results: Treatment with DCMI significantly enhanced p62 and LC3 expression in both cell lines compared with untreated cells (Figure 1A). Conversely, the application of sunitinib did not change the levels of these proteins. As expected, treatment with sunitinib strongly reduced the phosphorylation of VEGFR in KJ29 cells, while in Caki-2 cells, this effect was milder but still significant (Figure 1B). Surprising, the treatment with DCMI was also able to switch off the activity of VEGFR, mainly in KJ29 cells. Interestingly, a greater inhibition of VEGFR phosphorylation in Caki-2 cells treated with DCMI combined with sunitinib compared with single compounds was observed (Figure 1B). Importantly, the expression of HIF1α was reduced by the combination in KJ29 cells as well as in Caki-2 cells (Figure 1B). As observed for p-VEGFR, the combined treatment with these drugs caused a stronger reduction of HIF1α expression as compared to the single treatment in both cell lines (Figure 1B). The analysis of cell migration by scratch-wound assay shows that treatment with DCMI and sunitinib in cells reduced KJ29 cell migration after 48 and 120 h compared with untreated cells (Figure 1C). Moreover, the combined treatment potentiated the inhibition of cell migration not only after 48 h, but also for much longer (120 h). Discussion and Conclusion: Different mechanisms are associated with drug resistance including the activation of autophagy in kidney cancer cells. Moreover, this process promotes cancer cell proliferation and migration (1). Therefore, the inhibition of autophagy reduces cancer progression and improves conventional therapy response. Our findings show that the application of DCMI effectively inhibited autophagy in two different kidney carcinoma cell lines. The treatment with DCMI prevented the fusion of autophagosomes with lysosomes leading to the accumulation of both p62 and LC3 autophagy proteins (Figure 1A). Interestingly, the inhibition of autophagy enhanced the reduction of angiogenesis analyzed as VEGFR dephosphorylation, suggesting that autophagy is a mechanism involved in the activation of angiogenesis. Consistent with this, the reduction of autophagy by DCMI administration strengthened the downregulation of HIF1α induced by sunitinb (Figure 1B). HIF1α is an upstream effector of angiogenesis that is constitutively activated in both Caki-2 and KJ29 ccRCC cells and promotes the activation of VEGFR. Importantly, the inhibition of autophagy combined with the administration of sunitinib caused a greater reduction of HIF1α expression and in turn the inactivation of VEGFR, leading to the inhibition of the angiogenesis pathway. Furthermore, the combined treatment with DCMI and sunitinib induced a stronger reduction of cell migration compared with the single treatment and for a longer time (Figure 1C). In conclusion, our findings indicate that autophagy is involved in processes linked to angiogenesis, cell migration and therapy resistance. Therefore, the inhibition of autophagy combined with current chemotherapy may represent an intriguing option for improving drug response in advanced kidney carcinoma.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
