Deubiquitinase USP35 modulates ferroptosis in lung cancer via targeting ferroportin

Abstract

Background: Ferroptosis is essential to regulate tumor growth and serves as a promising therapeutic target to lung cancer. Ubiquitin-specific protease 35 (USP35) belongs to the deubiquitinases family that is associated with cell proliferation and mitosis. In this research, we aim to elucidate the potential role and molecular basis of USP35 in lung cancer.

Methods: Lung cancer cells were infected with lentiviral vectors to silence or overexpress USP35. Cell viability, colony formation, lipid reactive oxygen species production, intracellular iron metabolism, and other ferroptotic markers were detected. The role of USP35 on ferroptosis and tumor progression were also tested in mouse tumor xenograft models in vivo.

Results: USP35 was abundant in human lung cancer tissues and cell lines. USP35 knockdown promoted ferroptosis, and inhibited cell growth, colony formation, and tumor progression in lung cancer cells. USP35 overexpression did not affect tumorigenesis and ferroptosis under basal conditions, but reduced erastin/RSL3-triggered iron disturbance and ferroptosis, thereby facilitating lung cancer cell growth and tumor progression. Further studies determined that USP35 directly interacted with ferroportin (FPN) and functioned as a deubiquitinase to maintain its protein stability. More importantly, we observed that USP35 knockdown sensitized lung cancer cells to cisplatin and paclitaxel chemotherapy.

Conclusion: USP35 modulates ferroptosis in lung cancer via targeting FPN, and it is a promising therapeutic target to lung cancer.

Keywords: ferroportin; ferroptosis; lung cancer; ubiquitin-specific protease 35.

FIGURE 1

USP35 knockdown inhibits lung cancer cell growth, colony formation and tumor progression. A, Relative USP35 mRNA level in ADC, SCC and corresponding ANT tissues (n = 10). B, Relative USP35 mRNA level in normal human lung epithelial cell lines and lung cancer cell lines (n = 6). C, Representative immunoblots of USP35 and the quantitative data (n = 6). D, Relative USP35 protein level in lung cancer cell lines with or without shUSP35 infection (n = 6). E, Cell viability data from CCK‐8 assay (n = 5). F, Colony formation (n = 6). G, Tumor volumes at indicating times in tumor xenografts models (n = 6). H, Tumor weights at day 25 after cell inoculation (n = 6). Data are shown as mean ± SD, *P < .05 versus the matched group

FIGURE 2

USP35 knockdown promotes ferroptosis in lung cancer cells. A,B, Cell viability data from CCK‐8 assay (n = 5). C, Colony formation (n = 6). D,E, Lipid ROS generation and MDA levels in lung cancer cells with or without USP35 silence (n = 6). F, Relative levels of HETEs released to the medium (n = 6). G,H, GSH levels and GPX4 activities in H460 and H1299 cell lines with or without USP35 silence (n = 6). I,J, Relative LIP and Fe²⁺ levels (n = 6). K,L, Cell viability and colony formation (n = 6). Data are shown as mean ± SD, *P < .05 versus the matched group. NS indicates no significance

FIGURE 3

USP35 overexpression blocks erastin/RSL3‐mediated tumor suppressive effects. A, Relative USP35 mRNA level in lung cancer cell lines with or without USP35 overexpression (n = 6). B,C, Cell viability and colony formation (n = 6). D, Tumor volumes at indicating times in tumor xenografts models (n = 6). E, Tumor weights at day 25 after cell inoculation (n = 6). F,G, Cells were incubated with USP35 at a MOI of 10 or the CTRL for 12 h, and then stimulated with erastin (5 µmol/L) or RSL3 (2 µmol/L) for additional 96 h after the removal of lentiviral vectors. Cell viability and colony formation in lung cancer cell lines were determined (n = 6). H,I, Cells with or without USP35 overexpression were subcutaneously injected into the right dorsal flank of the nude mice. To induce ferroptosis in vivo, the tumor‐bearing mice were treated with erastin (15 mg/kg twice every other day) by intraperitoneal injections or RSL3 (100 mg/kg twice a week) via intratumoral injections from day 18 after cell inoculation. Tumor volumes and weights were determined at day 25 after cell inoculation (n = 6). Data are shown as mean ± SD, *P < .05 versus the matched group. NS indicates no significance

FIGURE 4

USP35 overexpression reduces erastin/RSL3‐triggered iron disturbance and ferroptosis. A,B, Lipid ROS generation and MDA levels in lung cancer cells (n = 6). C,F, Statistical data about the releases of arachidonic acid metabolites (5‐HETE, 11‐HETE, 12‐HETE, and 15‐HETE) in the cell culture medium using LC–MS/MS analysis (n = 6). G,H, GSH levels and GPX4 activities in H460 and H1299 cell lines (n = 6). I,J, Relative LIP and Fe²⁺ levels (n = 6). Data are shown as mean ± SD, *P < .05 versus the matched group. NS indicates no significance

FIGURE 5

USP35 modulates cell growth, colony formation, tumor progression, and ferroptosis in EGFR mutated H1650 cells. A, Relative USP35 mRNA level in H1650 cells with or without USP35 silence (n = 6). B, Cell viability and colony formation (n = 6). C, Tumor volumes and tumor weights at day 25 after cell inoculation (n = 6). D, Lipid ROS generation and MDA levels in H1650 cells with or without USP35 silence (n = 6). E, GSH levels and GPX4 activities in H1650 cells with or without USP35 silence (n = 6). F, Relative LIP and Fe²⁺ levels (n = 6). G, Relative USP35 mRNA level in H1650 cells with or without USP35 overexpression (n = 6). H,I, Cell viability and colony formation (n = 6). J, Tumor volumes and tumor weights at day 25 after cell inoculation (n = 6). K,L, Lipid ROS generation and MDA levels in H1650 cells (n = 6). M, Relative LIP and Fe²⁺ levels (n = 6). Data are shown as mean ± SD, *P < .05 versus the matched group. NS indicates no significance

FIGURE 6

USP35 modulates ferroptosis via targeting FPN. A‐D, Representative immunoblots and the quantitative data (n = 6). E, Cystine uptake levels (n = 6). F, Relative LIP and Fe²⁺ levels (n = 6). G, Lipid ROS generation (n = 6). Data are shown as mean ± SD, *P < .05 versus the matched group. NS indicates no significance

FIGURE 7

USP35 overexpression inhibits RSL3‐induced ferroptosis and tumor progression via targeting FPN. A, Relative LIP and Fe²⁺ levels (n = 6). B,C, Lipid ROS generation and MDA levels in lung cancer cells (n = 6). D‐F, Cell viability and colony formation (n = 6). G‐H, Tumor volumes and tumor weights in tumor xenografts models (n = 6). I, Representative immunoblots and the quantitative data (n = 6). Data are shown as mean ± SD, *P < .05 versus the matched group

FIGURE 8

USP35 is required for FPN protein stability in lung cancer cells. A, Relative ubiquinated levels of FPN in lung cancer cells with or without USP35 manipulation (n = 6). B, FPN expression in the whole lysates or cell membrane from shUSP35‐infected cells with or without MG132 treatment (n = 6). C, Endogenous interaction between USP35 and FPN via the IP assay (n = 4). Data are shown as mean ± SD, *P < .05 versus the matched group

FIGURE 9

USP35 knockdown enhanced the chemotherapeutic sensitivity of lung cancer cells. A‐C, Cell viability and colony formation (n = 6). D‐E, Tumor volumes and tumor weights in tumor xenografts models (n = 6). F‐G, Cell viability and colony formation of the H460 or H1299 cell lines (n = 6). H, Tumor volumes and tumor weights in tumor xenografts models of the H460 or H1299 cell lines (n = 6). I, Cell viability and colony formation of the H1650 cells (n = 6). J, Tumor volumes and tumor weights in tumor xenografts models of the H1650 cells (n = 6). Data are shown as mean ± SD, *P < .05 versus the matched group