The human small cell lung cancer cell lines NCI-H1048 and NCI-H69, the multidrug-resistant cell line NCI-H69AR, and the human embryonic kidney cell line HEK-293T were purchased from ATCC. H446, H69 and H69AR cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Corning). H1048 cells were cultured in DMEM:F12 (Gibco) supplemented with 10% FBS, 0.005 mg/ml insulin (Sigma), 0.01 mg/ml transferrin (Gibco), 30 nM sodium selenite (Sigma), 10 nM hydrocortisone (Sigma), 10 nM beta-estradiol (Sigma) and 4.5 mM L-glutamine (Gibco). HEK-293T cells were cultured in DMEM (Gibco) supplemented with 10% FBS. Penicillin-streptomycin solution (10,000 U/mL) (Gibco) was added to the prepared culture medium with a 1:100 dilution. Cells were cultured in a humidified incubator at 37°C with 5% CO².

Short hairpin RNA (shRNA) and wild-type plasmids were constructed by SyngenTech company (Beijing). Then, HEK-293T cells were transfected with the recombinant plasmids and packaging plasmids (pLP1, pLP2 and pLP/VSVG; Thermo Fisher Scientific) using Lipofectamine 3000 according to the instructions (Thermo). Forty-eight hours later, lentivirus particles were collected and stored at -80°C. The shRNA sequences used for knockdown were as follows: USP13-shRNA-1 5'-TGATTGAGATGGAGAATAA-3'; USP13-shRNA-2 5'-GCACGAAACTGAAGCCAAT-3'; FASN-shRNA 5'-CCTACTGGATGCGTTCTTCAA-3'.

For stable cell line construction, cells were infected with lentivirus in culture medium supplemented with 5 μg/ml polybrene (Sigma) for 24 hours. The effectively transfected cells were selected with the corresponding antibiotics. The gene expression efficiency was determined by immunoblot analyses.

Total RNA was extracted using RNA-Quick Purification Kit (ES-RN001, YISHAN Biotechnology) according to the manufacturer’s protocol. Purified RNA was used to generate cDNA with TransScript All-in-One First-Strand cDNA Synthesis SuperMix (TransGen Biotech, AT341-01). qRT–PCR was performed with PerfectStart Green qPCR SuperMix (TransGen Biotech, AQ601-01) on an ABI 7900HT Real-Time PCR Thermocycler (Life Technologies). Relative mRNA expression was determined using the 2^-ΔΔCt method, and actin was used as an endogenous reference. The following primers were used: FASN forward 5′- CAACTCACGCTCCGGAAA-3′, reverse 5′-TGTGGATGCTGTCAAGGG-3′; actin forward 5′-ATCAAGATCATTGCTCCTCCTGAG-3′, reverse 5′-CTGCTTGCTGATCCACATCTG-3′.

Cells were lysed in RIPA buffer (89901, Thermo) containing proteinase and phosphatase inhibitor cocktail (78442, Thermo). Then, the cell lysates were quantified with a BCA protein assay kit (Thermo) according to the manufacturer’s protocol. Equal amounts of protein from cell lysates were separated by SDS-PAGE and performed immunoblotting according previous description (26).

Cells were lysed in IP lysis buffer (87787, Thermo) containing proteinase and phosphatase inhibitor cocktail and rotated at 4 °C for 30 min. After centrifugation, the cell lysates were incubated overnight with indicated antibodies or normal IgG at 4 °C with rotary agitation. Protein A/G agarose beads (sc-2003, Santa Cruz Biotechnology) were added to the lysates and incubated for an additional 3 hours at 4 °C. Beads were washed three times with IP lysis buffer and boiled for 10 min in 1.5% SDS buffer. Whole cell lysates and immunoprecipitates were analyzed by immunoblot assay.

Antibodies that recognize USP13 (ab99421, 1:1000 dilution), Nanog (ab109250, 1:1000 dilution) and Oct4 (ab19857, 1:1000 dilution) were purchased from Abcam. Antibodies against FASN (3180, 1:1000 dilution), HA (3726, 1:1000 dilution), ubiquitin (3933, 1:1000 dilution) and CD133 (64326, 1:1000 dilution) were purchased from CST. Antibody against β-actin (A1978, 1:5000 dilution) was purchased from Sigma. Normal rabbit IgG (2729, 1:5000 dilution) and secondary antibodies, including anti-rabbit IgG HRP-linked antibody (7074, 1:3000 dilution) and anti-mouse IgG HRP-linked antibody (7076, 1:3000 dilution), were purchased from CST.

For the FASN protein half-life determination, H1048 cells expressing specific plasmids were treated with 100 µg/ml cycloheximide (CHX, HY-12320, MedChem Express) for different periods of time. The cells were collected, and immunoblot analyses were performed with an anti-FASN antibody.

H1048 cells were cultured on coverslips and then fixed in 4% paraformaldehyde for 20 min, followed by permeabilization with 0.1% Triton X-100 (in PBS) for 5 min. After washing with PBS, cells were blocked with 5% BSA for 1 hour at room temperature. Cells were then incubated with the indicated primary antibodies at 4°C overnight. After washing with PBS three times, the cells were incubated with secondary antibodies for 1 hour at room temperature and stained with DAPI (Invitrogen) by using ProLong™ Gold Antifade Mountant with DAPI (P36935, Invitrogen). The immunofluorescent staining was observed using a confocal microscope (Olympus).

Antibody that recognizes USP13 (sc-48357, 1:200) was purchased from Santa Cruz Biotechnology. Antibody against FASN (3180, 1:200) was purchased from CST. Anti-mouse IgG (H+L) F(ab’)2 Fragment (Alexa Fluor 488 Conjugate) (4408, 1:1000 dilution) and anti-rabbit IgG (H+L) F(ab’)2 Fragment (Alexa Fluor 555 Conjugate) (4413, 1:1000 dilution) were purchased from CST.

USP13 was immunoprecipitated from H1048 cells and separated by SDS–PAGE gel. Proteins in-gel were digested overnight in 12.5 ng/μl trypsin in 25 mM NH4HCO3. The peptides were extracted three times with 60% ACN/0.1% TFA and dried completely in a vacuum centrifuge. LC–MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific) coupled to an Easy nLC instrument (Thermo Fisher Scientific) for 60 min.

MS analysis was performed using the MASCOT engine (Matrix Science, London, UK; version 2.2) embedded in Proteome Discoverer 1.4 (Thermo Electron, San Jose, CA.) against the UniProt Human database and the decoy database. MS data were searched against the UniProt database (https://www.uniprot.org/). The cutoff of the global false discovery rate (FDR) for peptide and protein identification was set to 0.01. LC–MS/MS was performed by Shanghai Applied Protein Technology Co., Ltd (Shanghai, China).

H1048 or H69 cells were seeded into 96-well ultralow attachment plates (Corning) in DMEM/F12 (Gibco) supplemented with B27 (Gibco), 20 ng/mL epidermal growth factor (Gibco), and 20 ng/mL basic fibroblast growth factor (PeproTech) according previous description (27). After 7 days, the number of positive (sphere formation) wells in each group were uploaded and calculated in the ELDA website (28). The images were observed using an inverted fluorescence microscope (Olympus).

The indicated cells were seeded in 6 cm plates and incubated at 37°C with 5% CO² in an incubator. Until appropriate confluence, cellular cholesterol and triglyceride levels were extracted and determined using Cholesterol Quantitation Kit (MAK043, Sigma) and Triglyceride Quantification Kit (MAK266, Sigma) according to the manufacturer’s protocol, respectively. Cholesterol and triglyceride levels were normalized to the protein concentration.

H1048 or H69 cells were digested and separated into a single-cell suspension with PBS, then adjusted to a density of 1×10⁷ cells/ml, and subsequently incubated with APC-conjugated anti-CD133 antibody (1:100 dilution) (397906, Biolegend) on ice for 30 min in the dark. Following two washes with PBS, cells were resuspended in 500 µl PBS and subjected to isolation by flow cytometry (BD, LSRII). Negative control was determined by using equal amounts of APC-conjugated immunoglobulin G (IgG) (M1310G05, Biolegend)-stained cells.

BALB/c nude mice (4–6 weeks old, female, 14–16 g) were purchased from Beijing HFK Bioscience Co. Ltd. and housed under specific pathogen-free and controlled conditions (25–27, 45–55% humidity, 12 h day/night cycle). The study was approved by the Institutional Animal Care and Use Committee (IACUC) of the National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, and the methods were carried out in accordance with the approved guidelines.

The cells were subcutaneously injected into the flanks of mice to establish a xenograft tumor. When the size of tumors reached approximately 100 mm³, animals were randomly divided into four groups and intraperitoneally injected with 10 mg/kg etoposide (S1225, Selleckchem), or 8 mg/kg TVB-2640 (#S9714, Selleckchem), or etoposide (10 mg/kg) and TVB-2640 (8 mg/kg) combination, or 0.9% saline as control. Tumor length (a) and minor diameter (b) were monitored once a week, and tumor size was calculated using the following formula: volume=a×b²/2.

SCLC samples with paired normal lung tissues were collected from patients who underwent radical resections at the Cancer Hospital of the Chinese Academy of Medical Sciences (Beijing, China) from January 2011 to January 2015. Surgically resected tissues have been pathologically diagnosed and stained with Mayer’s hematoxylin and eosin (HE). After embedding into paraffin, tissue microarray (TMA) was then prepared by Superbiotek, Inc. Ethics approval was granted by the Committee for the Ethics Review of Research Involving Human Subjects of the Cancer Hospital of the Chinese Academy of Medical Sciences. Table 1 summarized the clinical features of the patients.

Immunohistochemistry analyses were performed as previously described (29). Briefly, the human SCLC TMA slide was deparaffinized, rehydrated, autoclaved in 10 mM sodium citrate (pH 6.0) for 30 min to unmask antigens, and then incubated with primary antibodies against USP13 (ab99421, 1:200, Abcam), FASN (3180, 1:200, CST), Nanog (ab109250, 1:100, Abcam), or Oct4 (2750, 1:200, CST) at 4°C overnight. The slides were then incubated with secondary antibody, followed by chromogen diaminobenzidine (DAB) staining for signal amplification and detection. The IHC scores were assessed by two independent authors blinded to the treatment groups. IHC scoring was based on the percentage of positive cells and the staining intensity, as previously described (30).

Oil Red O staining was performed on cryosections (6-10 µm) in thickness. In brief, the slides were fixed with formaldehyde, washed with 60% propylene glycerol, and then stained with 0.5% Oil Red O (Sangon Bio) in propylene glycerol for 10 min at 60°C. After staining, the slides were rinsed, counterstained with hematoxylin and mounted in glycerin. The red lipid droplets were visualized by microscopy.

Public dataset GSE60052 were downloaded from the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo) database.

Statistical analyses were conducted with a two-tailed unpaired Student’s t test. Each experiment was carried out in at least triplicate, and all data are expressed as the mean ± SD. Kaplan–Meier analysis and log-rank tests were applied for survival analysis (31). The correlation between USP13 and FASN levels was analyzed using a Pearson correlation coefficient. P values < 0.05 were considered to be significant. Differences that are statistically significant are labeled with *(p < 0.05), **(p <0.01), or ***(p < 0.001). Statistical analyses were performed by using GraphPad Prism (Version 9).

To identify the important role of deubiquitinase USP13 in the clinical features of SCLC, we analyzed the mRNA expression of USP13 in the published profile GSE60052. Overall survival analysis of USP13 in SCLC patients showed that USP13 was prognostically detrimental ( Figure 1A ), which indicated further necessary research of USP13. We then collected tumor tissues and the paired peripheral normal lung tissues which were surgically resected from patients diagnosed with SCLC. Immunohistochemical (IHC) analyses revealed that the expression of the USP13 protein was increased in SCLC tissues compared with normal lung tissues ( Figures 1B, C ). Next, we found that USP13 was significantly upregulated in the tissues of SCLC patients with lymph node metastasis ( Figure 1D ). Moreover, compared to patients evaluated as stage I, patients in stage II or stage III overexpressed USP13 ( Figure 1E ). Consistent with the previous analysis, Kaplan–Meier survival analysis demonstrated that high USP13 levels were correlated with poor overall survival of SCLC patients ( Figure 1F ). Collectively, these results demonstrated that USP13 is overexpressed in SCLC and predicts poor clinical outcomes.

As CSCs contribute to tumorigenesis, we examined the role of USP13 in SCLC stemness maintenance. The expression of stemness-related factors Oct4 and Nanog was dramatically decreased after depletion of USP13 with two different short hairpin RNAs (shRNAs) in H1048 and H69 cells ( Figure 2A ), which indicated an important role of USP13 in stemness maintenance. Consistently, silencing USP13 significantly inhibited sphere formation ability ( Figure 2B , Supplementary Figure 1A ). Conversely, forced expression of WT USP13 but not the catalytically inactive USP13 (C345A) mutant significantly promoted Oct4 and Nanog expression ( Figure 2C ) and sphere formation ability ( Figure 2D , Supplementary Figure 1B ). CD133⁺ cells are widely considered to be SCLC stem-like cells (32). To validate the expression levels of USP13 in SCLC CSCs, we enriched CD133⁺ subpopulations from H1048 cells with an anti-CD133 antibody. As shown in Figure 2E and Supplementary Figure 1C , USP13 expression level in CD133⁺ subpopulation was substantially higher than that in the CD133^- subpopulation. Together, these results support a critical role of USP13 which depends on its catalytic function in promoting SCLC stemness.

We next determined the role of USP13 in tumor growth by subcutaneously injecting H1048 cells with or without USP13 depletion, or USP13 depletion combined with WT USP13 or catalytically inactive USP13 (C345A) mutant overexpression ( Supplementary Figure 1D ) in mice. The results showed that mice inoculated with USP13-deficient cells evidently formed smaller tumor masses than those inoculated with control cells, and this reduction was abrogated by reconstituted expression of shRNA-resistant WT USP13 but not the catalytically inactive USP13 (C345A) mutant ( Figure 2F ). Moreover, IHC staining analysis in tumor tissues showed that Nanog and Oct4 expression decreased after USP13 depletion, and this decrease was rescued by reconstituted expression of WT USP13 but not catalytically inactive USP13 (C345A) mutant ( Figures 2G, H ). Hence, our data strongly indicated that USP13 promotes SCLC tumor growth.

To verify mechanisms involved in USP13 regulated SCLC stemness, immunoprecipitation (IP) was performed with an anti-USP13 antibody and mass spectrometry (MS) were used to identify proteins that interacts with USP13. MS analysis revealed 2 unique peptides identical to FASN ( Supplementary Figure 2A ), which is a critical enzyme for the synthesis of palmitate (precursor of cholesterol and triglyceride) from acetyl-CoA and malonyl-CoA and contributes to CSC maintenance (33). We confirmed the interaction between endogenous USP13 and FASN by the co-immunoprecipitation (Co-IP) of H1048 cell lysates ( Figure 3A ). Consistently, USP13 and FASN were shown to interact physically by immunofluorescence (IF) analyses ( Figure 3B ). To determine the importance of USP13 in the regulation of FASN, we then analyzed FASN expression with or without USP13 depletion or overexpression. As shown in Figures 3C, D , USP13 downregulation decreased ( Figure 3C ), whereas USP13 overexpression increased FASN protein levels ( Figure 3D ) without affecting FASN mRNA levels ( Supplementary Figure 2B ). To detect the role of USP13-FASN axis in regulating cancer stemness and lipogenesis in SCLC, we stably depleted FASN after WT USP13 reconstitution in USP13 knockdown cells. USP13 depletion in SCLC cells decreased the expression levels of Oct4 and Nanog ( Figure 3E ), reduced the sphere formation ability ( Figure 3F ) and cellular cholesterol and triglyceride levels ( Figure 3G ). In contrast, overexpression of reconstituted WT USP13 in SCLC increased Oct4 and Nanog expression ( Figure 3E ), promoted sphere formation ability ( Figure 3F ) and increased cellular cholesterol and triglyceride levels ( Figure 3G ). Importantly, the effects of USP13 on SCLC cancer stemness maintenance and lipogenesis were abrogated by FASN depletion ( Figures 3E–G ). In addition, IHC analysis and oil red O staining of tumor tissues indicated that USP13-dependent FASN expression increases lipogenesis ( Supplementary Figures 2C, D ). These results indicated USP13 promotes cancer stemness and lipogenesis mainly through FASN.

As a deubiquitinase, we hypothesized that endogenous USP13 could regulate FASN stability. We treated USP13 knockdown cells with the proteasome inhibitor MG-132 and found that inhibition of FASN expression by USP13 depletion was clearly blocked by MG-132 treatment in H1048 and H69 cells ( Figure 4A ). To determine whether USP13 stabilizes the FASN protein through deubiquitination, we first examined FASN protein turnover by using cycloheximide (CHX) and traced the protein levels. Indeed, the FASN protein was gradually degraded with CHX treatment. As anticipated, the FASN protein half-life was decreased after depletion of USP13 ( Figure 4B ), while ectopic WT USP13 but not catalytically inactive USP13 (C345A) mutant expression largely increased FASN stability ( Figure 4C ). Moreover, we found that depletion of USP13 enhanced the ubiquitination level of endogenous FASN ( Figure 4D ). In contrast, forced expression of WT USP13 but not the catalytically inactive USP13 (C345A) mutant reduced the ubiquitination of FASN ( Figure 4E ). Overall, our experiments indicate that USP13 promotes FASN protein stability by decreasing polyubiquitination of FASN and thus prevents its degradation through the proteasome pathway.

To determine the clinical significance of FASN expression, we performed IHC analyses of SCLC tissue specimens. IHC staining showed FASN was highly upregulated in SCLC tissues than in the paired adjacent normal tissues ( Figures 5A, B ). More importantly, the expression of FASN was stage-dependent ( Figure 5C ). In addition, patients with high FASN expression had shorter survival duration than those with low FASN expression ( Figure 5D ). Consistent with previous results, FASN protein expression levels were positively correlated with USP13 expression levels ( Figure 5E ). These results strongly suggested that USP13-stabilized FASN expression promotes clinical aggressiveness of SCLC.

Since FASN is a key enzyme involved in USP13-promoted cancer stemness, we further examined whether FASN inhibitor could be a favorable treatment strategy. TVB-2640, a highly potent and selective FASN inhibitor, was designed to reduce hepatic fat in nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (34, 35). More importantly, with compelling support as an oncology therapeutic drug, TVB-2640 was the first highly selective FASN inhibitor to enter clinical studies (36). We found that TVB-2640 treatment decreased cellular cholesterol and triglyceride levels and reduced sphere formation ability in a dose- and time-dependent manner in both H1048 and H69 cells ( Figures 6A–D , Supplementary Figures 3A, B ). Given that FASN expression is associated with chemoresistance (37), targeting lipid metabolism might be a new potential therapy for SCLC patients with acquired drug-resistance. We subcutaneously injected multi-drugs resistant H69AR cells into mice (38), then treated with VP16 (etoposide) or TVB-2640 for monotherapy, or concurrent therapy with VP16 and TVB-2640. Consistent with previous study (38), tumors in the VP16 treatment group displayed slight shrinkage ( Figure 6E ). Importantly, TVB-2640 treatment dramatically suppressed tumor growth ( Figure 6E ). In addition, the group treated with both VP16 and TVB-2640 showed an improved better response than the group treated with TVB-2640 alone ( Figure 6E ), indicated synergistic treatment effect. IHC staining analysis confirmed that lipogenesis inhibition by TVB-2640 repressed SCLC stemness features, reflected by decreased Nanog expression, and in combination with classical chemotherapeutics induced a dramatic synergistic effect ( Supplementary Figure 3C ). To further study whether TVB-2640 treatment can reverse USP13-mediated tumor formation, we subcutaneously injected H1048 cells with or without USP13 depletion, or H1048 USP13-depleted cells with reconstituted expression of shRNA-resistant WT USP13 with or without TVB-2640 treatment. Consistent with previous result, USP13 depletion dramatically suppressed tumor growth, and forced overexpression of WT HA-rUSP13 rescued the decreased tumor volume ( Figure 6F ). Importantly, TVB-2640 treatment can significantly reduce the tumorigenesis caused by USP13 overexpression. Together, these data show that the FASN inhibitor TVB-2640 can be used as a potential chemotherapy combination anti-SCLC drug.