FBXW7 Targets the SPT6‐ΔNp63 Axis for Degradation to Inhibit Esophageal Tumorigenesis Induced by 4‐Nitroquinoline N‐Oxide

TL;DR

FBXW7 prevents esophageal cancer by breaking down SPT6 and ΔNp63, and high SPT6 levels with low FBXW7 are linked to worse patient outcomes.

Contribution

FBXW7 is shown to inhibit esophageal tumorigenesis by targeting the SPT6-ΔNp63 axis for degradation.

Findings

FBXW7 inhibits 4NQO-induced esophageal tumorigenesis but not Pik3CaE545K-induced tumorigenesis.

FBXW7 promotes degradation of SPT6 and ΔNp63, which are pro-proliferative in ESCC cells.

High SPT6 and low FBXW7 levels in ESCC tissues correlate with poorer patient survival.

Abstract

FBXW7 (F‐box and WD repeat domain‐containing 7) is a classic tumor suppressor that promotes ubiquitylation and degradation of various oncoproteins. Although its tumor suppressor role in many types of cancers has been established, whether and how FBXW7 regulates in vivo esophageal tumorigenesis was previously unknown. Here, we report, using genetically modified mouse models, that Fbxw7 inhibits esophageal tumorigenesis induced by the carcinogen 4NQO (4‐nitroquinoline N‐oxide), but not by Pik3CaE545K (phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit alpha), a frequently mutated gene in human esophageal squamous cell carcinoma (ESCC). Mechanistically, FBXW7 depletion causes the accumulation of SPT6 (suppressor of Ty6), a transcriptional elongation factor, which is a novel substrate of FBXW7. SPT6 acts as a transcriptional co‐activator of ΔNp63, which is also a substrate of FBXW7 in ESCC cells. Both SPT6 and ΔNp63, accumulated upon FBXW7 knockdown, contribute to the proliferation of ESCC cells. In clinical human ESCC tissues, the protein levels of SPT6 and FBXW7 are inversely correlated, and high SPT6 levels with low FBXW7 levels predict poorer patient survival. Collectively, FBXW7 acts as a tumor suppressor in ESCC by promoting the degradation of both SPT6 and ΔNp63, and the SPT6‐ΔNp63 axis may serve as a therapeutic target for ESCC.

In normal cells, FBXW7 promotes ubiquitylation and degradation of SPT6 and ΔNp63 to keep their pro‐proliferative functions under control. During esophageal tumorigenesis, FBXW7 is inactivated by mutations or deletion, leading to the accumulation of SPT6 and ΔNp63, with ΔNp63 also being up‐regulated by SPT6. Together with 4NQO‐induced initiation, Fbxw7 deletion‐mediated activation of the SPT6‐ΔNp63 axis promotes ESCC development.

Figures

Fbxw7 deletion promotes the development of ESCC induced by 4NQO. (A) FBXW7 mutation hotspots in human esophageal squamous cell carcinoma (ESCC) tissues were analyzed by the cBioPortal database. (B and C) The genetically modified mouse models: mice with indicated genotypes were sacrificed at the age of 12 months, and the esophagus organs were isolated for photography (B and C). Macroscopic (B) and representative (C) images of the esophagus from the mice are shown. (D) Schematic diagram of the short‐term and long‐term 4NQO‐induced carcinogenesis mouse models of ESCC. (E–I) For the long‐term model, female mice at 6–7 weeks of age were administered 100 µg/mL 4NQO in their drinking water for 16 weeks, followed by tap water for 8 weeks. Esophagi were isolated for photography (E and F), H&E (G), and IHC (H and I) staining. Macroscopic (E) and representative (F) images of the esophagus from the long‐term model are shown. The number of tumors in the esophagus of each mouse is shown in (F).  Representative images of Ki67 staining are shown (H), and the percentage of cells with Ki67‐positive staining from three random fields of the esophagus in each mouse is quantified (I). Scale bars: 1.25 mm (left, G), 100 µm (right, G), 100 µm (H), and 25 µm (inset, H); the insets show enlarged images of the red boxes. Data are shown as mean ± SEM and analyzed by Student's t test (F and I); WT: n = 18, Fbxw7 cKO: n = 22 (F), n = 5 (I); *p < 0.001.

Identification of SPT6 as a new substrate of FBXW7 in ESCC. (A and B) ESCC tumors from mice following long‐term treatment were harvested for mass spectrometry analysis (A). Volcano plot (B) shows differentially expressed genes (DEGs), with downregulated genes in blue and up‐regulated genes in red upon Fbxw7 deletion (Fold change > 1.5, p < 0.05). (C and D) KYSE30 and KYSE150 cells were treated with the indicated concentrations of MLN4924 for 24 h, followed by immunoblotting (IB) (C) and the quantification of SPT6 levels from three independent experiments after normalization with β‐Actin as the loading control (D). (E and F) KYSE30 and KYSE150 cells were transfected with siRNAs targeting FBXW7 (siFBXW7s) or scrambled control siRNA (siNC) for 48 h, followed by IB analysis using the indicated antibodies (Abs) (E) and the quantification of SPT6 levels from three independent experiments after normalization with β‐Actin as the loading control (F). (G and H) KYSE30 and KYSE150 cells were transfected with increasing amounts of FBXW7 plasmids for 48 h, followed by IB analysis with the indicated Abs (G) and the quantification of SPT6 levels from three independent experiments after normalization with β‐Actin as the loading control (H). (I–K) ESCC tumor tissues from WT or Fbxw7 cKO mice following long‐term treatment were subjected to IB analysis (I) and IHC staining (J and K). Representative images of Spt6 staining are shown (J), and the percentage of cells with Spt6 positive staining in three random fields of the esophagus in each mouse was quantified (K). Scale bars: 100 and 25 µm (inset). Data are shown as means ± SEM and analyzed by one‐way ANOVA (D, F, and H) and Student's t test (K); n = 3 (D, F, and H), n = 5 (K); *p < 0.05, p < 0.01, ns: no significance.

FBXW7 interacts with SPT6 via its consensus degron motif. (A and B) KYSE30 (A) and KYSE150 (B) cells were harvested and subjected to immunoprecipitation (IP) with FBXW7 or SPT6 Ab, along with normal control IgG, followed by IB analysis with the indicated Abs. (C and D) Schematic representation of FBXW7 (C) and SPT6 (D) and their truncate mutants. Numbers indicate the amino acid (AA) positions. FBXW7: DD, dimerization domain; F‐box, SKP1 binding region; WD40, substrate recognition and binding region. SPT6: SPT6‐C1, 1056–1726AA, SPT6‐C2, 1323–1726AA. The FBXW7 binding motif (SPNTE: 1143–1147 AA) on SPT6 is indicated (D). (E–H) Cells were transfected with the indicated constructs and then subjected to IP with SPT6 (E and H) or FBXW7 (F and G) Ab, followed by IB analysis with the indicated Abs. FL, full length. The quantification is performed with Image J and expressed as the relative level of FBXW7 binding with SPT6 after normalization to immunoprecipitated SPT6 (H). (I) KYSE30 and KYSE150 cells were co‐transfected with GFP‐FBXW7α and FLAG‐SPT6 for 48 h and stained with FLAG antibody. The images were captured using a fluorescence microscope. Scale bar: 10 µm. LE, longer exposure; SE, shorter exposure; WCE, whole cell extract.

FBXW7 negatively regulates SPT6 stability by promoting its ubiquitylation. (A and B) Cells were transfected with the indicated plasmids and siRNA oligos for 48 h (A). Cells were infected with lentivirus expressing the indicated shRNA and then transfected with the indicated plasmids for 48 h (B). After 6 h of MG132 treatment, cells were harvested for Ni‐NTA purification. Ni‐NTA affinity purified fractions and WCE were analyzed by IB with the indicated Abs. (C–E) KYSE150 cells were transfected with the indicated siRNA (C) or plasmids (D and E) for 48 h and then treated with cycloheximide (CHX) for the indicated time periods, followed by IB analysis. (F and G) KYSE30 and KYSE150 cells were transfected with the indicated siRNAs for 48 h, followed by IB analysis (F) and the quantification of SPT6 levels from three independent experiments after normalization with β‐actin as the loading control (G). (H) KYSE150 cells were transfected with indicated plasmids for 48 h, and then treated with GSK3i‐IX or DMSO for 6 h, followed by IP with FLAG beads and IB analysis with the indicated Ab. (I) KYSE150 cells were transfected with indicated plasmids and siRNAs for 48 h, followed by IP with FLAG beads and IB analysis with the indicated Ab. (J) KYSE150 cells were transfected with indicated plasmids for 48 h, followed by IP with FLAG beads and IB analysis with the indicated Ab. (K) KYSE150 cells were transfected with the indicated siRNAs for 48 h, and then treated with CHX for the indicated time periods, followed by IB analysis. (L) KYSE150 cells were transfected with the indicated plasmids and siRNA and then treated with MG132 and GSK3i‐IX for 6 h, followed by Ni‐NTA purification. Ni‐NTA affinity‐purified fractions and WCE were analyzed by IB with the indicated Abs. (M) KYSE150 cells were infected with lentivirus expressing FBXW7 shRNA and then transfected with the indicated plasmids for 48 h, and then treated with MG132 for 6 h, followed by Ni‐NTA purification. Ni‐NTA affinity‐purified fractions and WCE were analyzed by IB with the indicated Abs. PD, pull down. Data are shown as mean ± SEM and analyzed by one‐way ANOVA (G). n = 3 (G), *p < 0.05, p < 0.01.

SPT6 knockdown inhibits the growth and colony formation of ESCC cells. (A–F) KYSE150 cells were transfected with siNC or siRNA targeting SPT6 (A, C, and D) or FBXW7 (B, E, and F), and then subjected to CCK8 (A and B) and colony formation assays (C–F). Images of representative plates are shown (C and E), and colony numbers were quantified (D and F). (G and H) KYSE150 cells were transfected with the indicated siRNAs for 48 h, followed by flow cytometry analysis. The percentage of cells in the G2/M phase (G) and undergoing apoptosis (H) are plotted. (I) KYSE30 and KYSE150 cells were transfected with the indicated siRNAs for 48 h, followed by IB analysis. (J–M) KYSE150 cells were transfected with the indicated siRNAs for 48 h, and then subjected to IB (J), CCK8 (K), and colony formation (L and M) assays. Images of representative plates are shown (L) and colony numbers are quantified (M). Data are shown as mean ± SEM and analyzed by one‐way ANOVA (A, B, D, F, G, H, K, and M), n = 3 (A, B, D, F, G, H, K, and M). *p < 0.05, p < 0.01, *p < 0.001, ns, no significance.

ΔNp63 is regulated positively by SPT6 and negatively by FBXW7. (A and B) KYSE30 and KYSE150 cells were transfected with the indicated siRNAs for 48 h, followed by IB (A) and qRT‐PCR (B) analyses. (C) KYSE30 and KYSE150 cells were transfected with increasing amounts of SPT6 plasmid for 48 h, followed by IB analysis. (D) KYSE30 and KYSE150 cells were harvested for the ChIP assay with SPT6 antibody, followed by PCR (top) or qRT‐PCR analysis (bottom). (E) KYSE150 cells were transfected with the indicated siRNAs for 48 h, followed by IB analysis. (F and G) ESCC tumor tissues from WT or Fbxw7 cKO mice following long‐term treatment were subjected to IHC staining. Representative images of ΔNp63 staining are shown (F), and the percentage of cells with ΔNp63‐positive staining from three random fields of the esophagus in each mouse was quantified (G). Scale bars: 100 and 25 µm (inset). (H) KYSE150 cells were transfected with the indicated siRNAs and plasmids for 48 h and then harvested for Ni‐NTA purification after 6 h of MG132 treatment. Ni‐NTA affinity‐purified fractions and WCE were analyzed by IB with the indicated Abs. (I‐K) KYSE150 cells were transfected with the indicated siRNAs for 48 h and then subjected to CCK8 (I) and colony formation assays (J and K). Images of representative plates are shown (J), and colony numbers are quantified (K). (L–O) KYSE150 cells were transfected with the indicated siRNAs for 48 h, and then subjected to CCK8 assay (L), IB analysis (M), and colony formation assays (N, O). Images of representative plates are shown (N), and colony numbers are quantified (O). Data are shown as mean ± SEM and analyzed by one‐way ANOVA (B, I, L, K, and O) and Student's t test (D and G); n = 3 (B, D, I, L, K, and O); n = 5 (G). *p < 0.05, p < 0.01, *p < 0.001, ns, no significance.

The FBXW7‐SPT6 axis regulates ESCC tumor growth in vivo and their inverse correlation in ESCC tumor tissues. (A–E) KYSE150 cells infected with lentivirus expressing the indicated shRNA were subcutaneously injected into nude mice. Tumor volume was monitored every 2–3 days (A), tumor masses were photographed (B), and tumor weights were measured (C) at the end of the experiment. Tumors were subjected to IHC staining (D and E). Representative images of SPT6 and ΔNp63 staining are shown (D), and the percentage of cells with positive staining from three random fields in each tumor was quantified (E). Scale bar: 100 µm. Data are shown as mean ± SEM and analyzed by one‐way ANOVA (A, C, and E). n = 3 (A), n = 6 (C), n = 5 (E); *p < 0.05, p < 0.01, *p < 0.001, ns, no significance. (F and G) Human esophageal tumor tissue microarrays containing 86 tumor tissues stained for FBXW7 and SPT6. Representative staining images are shown (F). Quantification of positive staining was performed using ImageJ (G) (r = −0.5036, p < 0.001, Pearson correlation coefficient). Scale bar: 100 µm. (H) Kaplan–Meier analysis of 51 ESCC tissue microarray samples showing an inverse correlation between FBXW7 and SPT6 staining. Lower FBXW7 positive staining, coupled with higher SPT6 positive staining, predicted worse overall patient survival. Log‐rank test, p < 0.01. (I) Working model for how the FBXW7‐SPT6‐ΔNp63 axis regulates the ESCC development (see the text for details).

Tables

TABLE 1. Histology of the esophagi in 4NQO‐treated mice.

Genotype	Number of mice	Dysplasia	Carcinoma in situ	p value
WT	18	10 (55.6%)	8 (44.4%)
Fbxw7 cKO	22	4 (18.2%)	18 (81.8%)	0.014

Full text

Introduction

1

Esophageal cancer (EC) is one of the most prevalent cancers globally and the seventh leading cause of cancer‐related death, with an estimated 511,000 new cases and 445,000 deaths in 2022 [1]. EC is primarily categorized into two histological subtypes: adenocarcinoma (EAC) and squamous cell carcinoma (ESCC). While EAC accounts for approximately two‐thirds of cases in Western countries, ESCC predominates in China, representing approximately 90% of EC cases [2]. Recent multi‐omics analyses have classified ESCC into four distinct subtypes: cell cycle pathway activation, NRF2 oncogenic activation, immune suppression (IS), and immune modulation (IM) [3]. This molecular classification not only deepens our understanding of ESCC biology but also provides potential clinical relevance for effective targeted therapy. Whole‐genome sequencing of 508 ESCC patients has revealed that 5.9% of these patients exhibit high tumor mutation burden, while 15.2% harbor actionable mutations in the Chinese population. Key genes frequently mutated in ESCC include TP53, FAT1 (FAT atypical cadherin 1), NOTCH1 (notch receptor 1), KMT2D (lysine methyltransferase 2D), CDKN2A (cyclin‐dependent kinase inhibitor 2A), FBXW7, and PIK3CA (phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit alpha) [4]. Given the complexity and genetic heterogeneity of ESCC, elucidation of the underlying mechanisms that drive esophageal tumorigenesis is essential for developing more effective treatment strategies.

FBXW7 is a well‐characterized tumor suppressor and a key substrate receptor of Cullin‐RING ligase‐1 (CRL1) [5]. CRL1^FBXW7^ E3 orchestrates the ubiquitylation and degradation of a broad range of oncogenic substrates, such as c‐MYC, NOTCH1, c‐JUN, and MCL‐1 (myeloid cell leukemia 1) [6], thereby inhibiting the growth and survival of cancer cells. Notably, almost all FBXW7 substrates contain an evolutionarily conserved phospho‐motif known as the Cdc4 phospho‐degron (CPD), which facilitates FBXW7 binding and subsequent ubiquitylation for proteasomal degradation [7]. Mutations that inactivate FBXW7 are frequently observed in various human cancers, leading to the accumulation of oncoproteins that drive tumorigenesis [8]. In addition to substrate degradation, our previous study showed that FBXW7 promotes non‐homologous end‐joining repair through the K63‐linked polyubiquitylation of XRCC4 (x‐ray repair cross‐complementing 4) [9]. Moreover, FBXW7 undergoes self‐ubiquitylation and degradation upon binding to LSD1 (lysine demethylase 1A), thereby regulating its activity [10]. Although tissue‐specific deletion of Fbxw7 has been studied in various mouse models, including those of the lung [11], intestine [12], and pancreas [13], its role in esophageal tissues remains elusive. Thus, establishing an esophageal epithelium‐specific Fbxw7 knockout mouse model would provide a physiologically relevant tool for studying the role of Fbxw7 in the development of ESCC.

Transcription in eukaryotes involves four steps: initiation, promoter escape, elongation, and termination [14]. The elongation stage, mediated by RNA polymerase II (RNAPII), plays a central role in regulating gene expression in response to developmental and environmental signals [15]. Dysregulated transcriptional elongation has been linked to developmental defects and various diseases, including cancer [16]. Suppressor of Ty6 (SPT6, or SUPT6H) is a histone chaperone associated with RNAPII that promotes transcription elongation and regulates chromatin structure to ensure the fidelity of transcription initiation, a key step across eukaryotes [17]. SPT6 has been shown to interact with staphylococcal nuclease and Tudor domain‐containing 1 (SND1) to promote colorectal cancer development by targeting human telomerase reverse transcriptase (hTERT) [18]. Additionally, SPT6 loss impairs the self‐renewal, genomic stability, and tumor‐initiating capabilities of glioblastoma stem‐like cells (GSCs) [19]. SPT6 depletion has also been implicated in the spontaneous transdifferentiation of the epidermal cells into an intestinal‐like phenotype owing to RNAPII pausing and loss of epidermal master regulator p63 expression [20, 21]. Despite these findings, the specific role of SPT6 in cancer, particularly ESCC, remains unclear. Furthermore, whether and how SPT6 stability is regulated by the ubiquitin–proteasome system (UPS), which is governed by E3 ligases, remains to be determined.

p63, a member of the p53 family, plays a pivotal role in cancer biology by regulating processes such as DNA damage response, cell cycle arrest, apoptosis, stemness, and tumorigenesis [22]. p63 exists as two main isoforms, TAp63 and ΔNp63, which are produced through alternative promoter usage. TAp63 acts as a tumor suppressor that induces senescence and inhibits tumor progression [23, 24], whereas ΔNp63, the shorter isoform without the transactivation domain, acts as an oncogene by overriding oncogene‐induced senescence and promoting squamous cell carcinoma development [25, 26]. In squamous tissues, such as the skin, vagina, oral mucosa, and esophagus, ΔNp63 is the predominant isoform expressed, driving tumorigenesis [22, 27, 28]. Both isoforms of p63 are degraded by multiple E3 ubiquitin ligases, including NEDD4 (neural precursor cell expressed, developmentally down‐regulated 4), AIP4/ITCH (atrophin‐1 interacting protein 4), PIRH2 (p53‐induced RING‐H2 protein), and FBXW7 [29, 30]. Whether oncogenic ΔNp63 is targeted by the tumor suppressor FBXW7 in ESCC remains elusive.

In this study, we used genetically modified mouse models to investigate the role of FBXW7 in esophageal carcinogenesis and elucidated its underlying mechanism. Specifically, while the combination of Fbxw7 deletion in esophageal squamous epithelial cells and Pik3ca^E545K^ mutation had no effect on esophageal tumorigenesis, Fbxw7 deletion significantly promoted carcinogenesis induced by 4NQO, a well‐established carcinogen for the experimental development of ESCC [31, 32]. Mechanistically, we identified and characterized SPT6 as a bona fide substrate for FBXW7. SPT6 promotes the growth and survival of ESCC cells, which was counteracted by FBXW7. We also found that ΔNp63 was positively regulated by SPT6 at the transcriptional level, and negatively regulated by FBXW7 at the post‐translational level as a substrate. Finally, we found a negative correlation between FBXW7 and SPT6 levels, and a positive association between low FBXW7‐high SPT6 levels and poor patient survival in human ESCC tissues. Collectively, our study demonstrates mechanistically how the FBXW7‐SPT6‐ΔNp63 axis modulates the growth and survival of ESCC as well as chemical esophageal carcinogenesis and validated that the SPT6‐ΔNp63 axis is an attractive target for ESCC.

Results

2

Alterations of FBXW7 in Human Esophageal Squamous Cell Carcinoma

2.1

FBXW7, a typical tumor suppressor that promotes the ubiquitylation and proteasomal degradation of a variety of oncogenic proteins, is frequently inactivated in a number of human cancers [33, 34]. To determine the possible involvement of FBXW7 in ESCC, we first examined the genetic alterations of FBXW7 in human ESCC samples using the cBioPortal database (https://www.cbioportal.org), which integrates genomic data from the International Cancer Genome Consortium (ICGC) and UCLA databases [35, 36]. Analysis of 227 ESCC samples revealed that approximately 4% of tumor samples exhibited genetic alterations in FBXW7, with the majority being point mutations (Figure S1A). Notably, mutation hotspots were identified in the WD40 substrate binding domain, including S462F, R465C, and R505C/G (Figure 1A), which have also been implicated in other types of human cancers [34]. We further analyzed the COSMIC database (https://cancer.sanger.ac.uk/cosmic), which provides detailed annotations of somatic mutations across the entire gene, and found that the majority of mutations in FBXW7 occurred again in the WD40 domain, including recurrent mutations in R465H, S462F, R479L, and R505C (Figure S1B). Collectively, FBXW7 is mutated in some ESCC tissues, which could contribute to the development of ESCC.

*Fbxw7 deletion promotes the development of ESCC induced by 4NQO. (A) FBXW7 mutation hotspots in human esophageal squamous cell carcinoma (ESCC) tissues were analyzed by the cBioPortal database. (B and C) The genetically modified mouse models: mice with indicated genotypes were sacrificed at the age of 12 months, and the esophagus organs were isolated for photography (B and C). Macroscopic (B) and representative (C) images of the esophagus from the mice are shown. (D) Schematic diagram of the short‐term and long‐term 4NQO‐induced carcinogenesis mouse models of ESCC. (E–I) For the long‐term model, female mice at 6–7 weeks of age were administered 100 µg/mL 4NQO in their drinking water for 16 weeks, followed by tap water for 8 weeks. Esophagi were isolated for photography (E and F), H&E (G), and IHC (H and I) staining. Macroscopic (E) and representative (F) images of the esophagus from the long‐term model are shown. The number of tumors in the esophagus of each mouse is shown in (F).  Representative images of Ki67 staining are shown (H), and the percentage of cells with Ki67‐positive staining from three random fields of the esophagus in each mouse is quantified (I). Scale bars: 1.25 mm (left, G), 100 µm (right, G), 100 µm (H), and 25 µm (inset, H); the insets show enlarged images of the red boxes. Data are shown as mean ± SEM and analyzed by Student's t test (F and I); WT: n = 18, Fbxw7 cKO: n = 22 (F), n = 5 (I); **p < 0.001.

Fbxw7 Deletion Promotes the Development of ESCC Induced by 4NQO

2.2

A recent study showed that mutations of both FBXW7 and PIK3CA are frequent events in ESCC tissues [4]. However, it is unknown whether they play a causal role during ESCC tumorigenesis. To this end, we inter‐crossed mouse strains of *Pik3ca^LSL‐E545K/+^

• (a gift from Dr. Suzanne Baker) [37], *Fbxw7^fl/fl^
• [38], and Ed‐l2‐Cre (purchased from GemPharmatech Co. Ltd.) to generate two compound mouse strains 1) Pik3ca^LSL‐E545K/+^;Ed‐l2‐Cre+, and 2) Fbxw7^fl/fl^;Pik3ca^LSL‐E545K/+^;Ed‐l2‐Cre+ with Ed‐l2‐Cre driven activation of *Pik3a^LSL‐E545K/+^
• by removing the STOP fragment, or simultaneous *Pik3ca^LSL‐E545K/+^
• activation and Fbxw7 deletion in esophageal squamous epithelial cells [39]. For a period of 1 year under ad libitum feeding conditions, both compound mice showed a normal esophagus (Figure 1B,C), with no obvious pathological alterations in the esophageal epithelial layer (Figure S1C). Thus, neither *Pik3ca^LSL‐E545K/+^
• activation alone nor in combination with Fbxw7 deletion induced esophageal tumorigenesis.

Next, we determined the role of Fbxw7 in esophageal carcinogenesis, induced by 4NQO, a well‐known chemical carcinogen for ESCC [31, 32]. Two protocols were used: the cohorts of Fbxw7^fl/fl^;Ed‐l2‐Cre‐ (control) and Fbxw7^fl/fl^;Ed‐l2‐Cre+ (experimental) mice (n = 6–8) were administered 4NQO (100 µg/mL) in drinking water for 8 weeks, followed by 16 weeks of tap water (short‐term treatment), or for 16 weeks, followed by 8 weeks of tap water (long‐term treatment) (Figure 1D). Visual examination of esophageal tissues from paired mice revealed that (1) as expected, long‐term 4NQO treatment gave rise to a greater number of tumors and size of tumors, compared to short‐term treatment, regardless of genotype; and (2) significantly increased tumor numbers and larger tumor sizes were observed in the esophagus of Fbxw7 deleted mice (Figures 1E,F and S1D). Furthermore, body weight loss was significantly greater in the Fbxw7‐deleted group with long‐term 4NQO treatment (Figure S1E).

Histological analysis of H&E‐stained esophageal tissues, particularly from Fbxw7‐deleted mice with long‐term 4NQO treatment, revealed a range of pathological alterations, including focal hyperplasia, disorganized epithelial layer with evidence of dysplasia and in situ carcinoma, exophytic neoplastic squamous lesions (predominantly papillary), and the formation of keratinized beads, a hallmark of squamous cell carcinoma (Figures 1G and S1F). Given the variations in the pathological stages even in the same specimen, we quantified and scored the tumors in the percentage of dysplasia vs. in situ carcinoma. Obviously, Fbxw7‐deleted mice developed more in situ carcinomas but fewer dysplasia than WT control mice (Table 1). The Ki67 staining of ESCC tissues also showed increased numbers of proliferating cells in the Fbxw7 deleted group (Figures 1H,I and S1G,H). Taken together, Fbxw7 is a tumor suppressor whose deletion accelerates the process of esophageal squamous cell carcinogenesis induced by 4NQO.

Identification of SPT6 as a New Substrate of FBXW7 in ESCC

2.3

To elucidate the mechanism by which FBXW7 inhibits esophageal carcinogenesis induced by 4NQO, we used an unbiased whole‐proteomic approach via mass spectrometry analysis of four pairs of ESCC tumor tissues from mice with either wild‐type or Fbxw7‐null genotypes (Figure 2A). The analysis identified 55 increased and 52 decreased proteins that were statistically different (fold change >1.5) upon Fbxw7 deletion (Figure 2B). We then focused on a list of proteins that are putative candidate(s) for Fbxw7 substrates that accumulated upon Fbxw7 deletion. Given that FBXW7 substrates must contain a Cdc4 phosphodegron (CPD, L/I/P T/S PXX S/T/E/D), which mediates FBXW7 binding [33, 34], we searched for all the upregulated protein candidates with this Cdc4 degron and identified a total of 21 candidates (Table S1). Next, we analyzed the mRNA expression of these 21 upregulated protein candidates using the TCGA and GTEx databases (Figure S2A) and assessed the evolutionary conservation of their CPD motifs (Table S1). This led us to identify several candidate substrates that were overexpressed in ESCC and contained at least one evolutionarily conserved CPD motif, including SPT6, UAP1L1 (UDP‐N‐acetylglucosamine pyrophosphorylase 1 like 1), RBM7 (RNA binding motif protein 7), LSM14A (Sm‐like protein 14 homolog A), PDPK1 (3‐phosphoinositide dependent protein kinase 1), PLEKHF2 (pleckstrin homology and FYVE domain containing 2), EI24 (etoposide‐induced protein 2.4), and WASHC4 (WASH complex subunit 4) (Figures 2B and S2B).

*Identification of SPT6 as a new substrate of FBXW7 in ESCC. (A and B) ESCC tumors from mice following long‐term treatment were harvested for mass spectrometry analysis (A). Volcano plot (B) shows differentially expressed genes (DEGs), with downregulated genes in blue and up‐regulated genes in red upon Fbxw7 deletion (Fold change > 1.5, p < 0.05). (C and D) KYSE30 and KYSE150 cells were treated with the indicated concentrations of MLN4924 for 24 h, followed by immunoblotting (IB) (C) and the quantification of SPT6 levels from three independent experiments after normalization with β‐Actin as the loading control (D). (E and F) KYSE30 and KYSE150 cells were transfected with siRNAs targeting FBXW7 (siFBXW7s) or scrambled control siRNA (siNC) for 48 h, followed by IB analysis using the indicated antibodies (Abs) (E) and the quantification of SPT6 levels from three independent experiments after normalization with β‐Actin as the loading control (F). (G and H) KYSE30 and KYSE150 cells were transfected with increasing amounts of FBXW7 plasmids for 48 h, followed by IB analysis with the indicated Abs (G) and the quantification of SPT6 levels from three independent experiments after normalization with β‐Actin as the loading control (H). (I–K) ESCC tumor tissues from WT or Fbxw7 cKO mice following long‐term treatment were subjected to IB analysis (I) and IHC staining (J and K). Representative images of Spt6 staining are shown (J), and the percentage of cells with Spt6 positive staining in three random fields of the esophagus in each mouse was quantified (K). Scale bars: 100 and 25 µm (inset). Data are shown as means ± SEM and analyzed by one‐way ANOVA (D, F, and H) and Student's t test (K); n = 3 (D, F, and H), n = 5 (K); *p < 0.05, *p < 0.01, ns: no significance.

We validated these eight putative FBXW7 substrates first by treating four human esophageal cancer cell lines with MLN4924, a small‐molecule inhibitor of NEDD8‐activating enzyme (NAE) that inhibits neddylation of cullins and inactivates all cullin‐RING ligase (CRL) family members, including CRL1^FBXW7^ [40]. Interestingly, MLN4924 treatment caused a dose‐dependent accumulation of SPT6, but not the other seven candidates (Figures 2C,D and S2D,E), with a minor increase in SPT6 mRNA expression only at a high drug concentration (Figure S2C). Consistently, FBXW7 knockdown caused a significant accumulation of SPT6 and minor accumulation of LSM14A and PDPK1, but not the other candidates (Figures 2E,F and S2F,G), whereas FBXW7 overexpression reduced the levels of SPT6 in a dose‐dependent manner (Figure 2G,H). Finally, we measured Spt6 levels in ESCC tissues induced by 4NQO from wild‐type and Fbxw7 knockout mice. Both immunoblotting (IB) and immunohistochemical (IHC) staining showed increased Spt6 levels upon Fbxw7 deletion (Figure 2I–K). Collectively, these results suggest that SPT6 could be a new substrate of FBXW7 in ESCC cells.

FBXW7 Interacts With SPT6 via Its Consensus Degron Motif

2.4

We further characterized SPT6 as a novel FBXW7 substrate by a direct protein‐protein binding assay via co‐immunoprecipitation (IP) and found that exogenously expressed FBXW7 interacts with exogenously expressed SPT6 (Figure S3A). Furthermore, exogenously expressed FBXW7 or SPT6 pulled down endogenous SPT6 or FBXW7, respectively (Figure S3B). Importantly, in both KYSE30 and KYSE150 ESCC cells, endogenous SPT6 bound to endogenous FBXW7 under physiological conditions in reciprocal IP assays (Figure 3A,B).

FBXW7 interacts with SPT6 via its consensus degron motif. (A and B) KYSE30 (A) and KYSE150 (B) cells were harvested and subjected to immunoprecipitation (IP) with FBXW7 or SPT6 Ab, along with normal control IgG, followed by IB analysis with the indicated Abs. (C and D) Schematic representation of FBXW7 (C) and SPT6 (D) and their truncate mutants. Numbers indicate the amino acid (AA) positions. FBXW7: DD, dimerization domain; F‐box, SKP1 binding region; WD40, substrate recognition and binding region. SPT6: SPT6‐C1, 1056–1726AA, SPT6‐C2, 1323–1726AA. The FBXW7 binding motif (SPNTE: 1143–1147 AA) on SPT6 is indicated (D). (E–H) Cells were transfected with the indicated constructs and then subjected to IP with SPT6 (E and H) or FBXW7 (F and G) Ab, followed by IB analysis with the indicated Abs. FL, full length. The quantification is performed with Image J and expressed as the relative level of FBXW7 binding with SPT6 after normalization to immunoprecipitated SPT6 (H). (I) KYSE30 and KYSE150 cells were co‐transfected with GFP‐FBXW7α and FLAG‐SPT6 for 48 h and stained with FLAG antibody. The images were captured using a fluorescence microscope. Scale bar: 10 µm. LE, longer exposure; SE, shorter exposure; WCE, whole cell extract.

Next, we defined the binding domain in each protein by creating truncation mutants, including FBXW7‐ΔF (deletion of AA 278–324), FBXW7‐ΔWD40 (encoding AA 1–358), FBXW7‐WD40 (encoding 351–707) for FBXW7 (Figure 3C); SPT6‐C1 (encoding AA 1056–1726, containing all five FBXW7 binding motifs), and SPT6‐C2 (encoding AA 1323–1726, containing the last four FBXW7 binding motifs) for SPT6 (Figure 3D). The co‐IP assay showed that the FBXW7‐WD40 domain, but not the FBXW7‐ΔWD40 domain, bound to endogenous SPT6 (Figure 3E), indicating that the substrate‐recognizing WD40 domain was responsible for SPT6 binding, as expected. In contrast, SPT6‐C1, but not the SPT6‐C2 fragment, bound to FBXW7 (Figures 3F and S3C,D), indicating that the first FBXW7 binding motif (SPNTE; AA 1143–1147), which is evolutionarily conserved (Figure S3E), is responsible for FBXW7 binding.

To further define the requirement of the SPNTE degron on SPT6 for FBXW7 binding, we generated an SPT6‐C1 mutant resistant to phosphorylation by substituting S/T with A (motif SPNTE mutated to APNTA). The co‐IP assays showed that FBXW7 bound to wild‐type SPT6‐C1, but not the SPT6‐C1 mutant in ESCC KYSE30 cells, or with significantly reduced binding to the SPT6‐C1 mutant in HEK293 cells (Figures 3G and S3F), confirming that degron phosphorylation is critical for FBXW7 binding. Next, we determined the potential binding of SPT6 with two cancer‐derived FBXW7 mutants, FBXW7‐R465H and FBXW7‐R479Q, which are among the most frequently occurring mutations found in various human tumor tissues, including SCC [41]. Both residues in the WD40 substrate‐binding domain and are known to inactivate FBXW7. Our results showed that the binding of these two mutants to endogenous SPT6 was remarkably reduced (Figure 3H), further confirming the essential role of the WD40 domain in mediating the FBXW7‐SPT6 interaction. Finally, we used an immunofluorescence staining assay to confirm the co‐localization of FBXW7 with SPT6 in the nucleus of the two ESCC cell lines (Figure 3I), indicating a functional interaction under physiological conditions. Taken together, these results show that FBXW7 interacts with SPT6 at the molecular level through the WD40 domain of FBXW7 and its consensus degron motif (SPNTE) on SPT6.

FBXW7 Negatively Regulates SPT6 Stability by Promoting Its Ubiquitylation

2.5

Next, we determined whether FBXW7 E3 ligase promotes the ubiquitylation of SPT6 using in vivo ubiquitylation assays with the SPT6‐C1 fragment as a substrate. In both KYSE30 and KYSE150 cells, FBXW7 knockdown inhibited the polyubiquitylation of SPT6‐C1 (Figures 4A and S4A). Likewise, FBXW7 overexpression significantly enhanced polyubiquitylation of wild‐type SPT6‐C1, but not its degron/phosphorylation‐dead mutant SPT6‐C1‐Mut (SPT6‐C1‐S1143A/E1147A) (Figure S4B), whereas overexpression of FBXW7‐ΔF, a ligase‐dead mutant, inhibited SPT6‐C1 polyubiquitylation, acting in a dominant negative manner (Figures 4B and S4C). Note that in these experiments, we first knocked down endogenous FBXW7 using a 5'‐UTR shFBXW7 to show the pure effect of exogenously expressed FBXW7 (Figures 4B and S4B,C).

*FBXW7 negatively regulates SPT6 stability by promoting its ubiquitylation. (A and B) Cells were transfected with the indicated plasmids and siRNA oligos for 48 h (A). Cells were infected with lentivirus expressing the indicated shRNA and then transfected with the indicated plasmids for 48 h (B). After 6 h of MG132 treatment, cells were harvested for Ni‐NTA purification. Ni‐NTA affinity purified fractions and WCE were analyzed by IB with the indicated Abs. (C–E) KYSE150 cells were transfected with the indicated siRNA (C) or plasmids (D and E) for 48 h and then treated with cycloheximide (CHX) for the indicated time periods, followed by IB analysis. (F and G) KYSE30 and KYSE150 cells were transfected with the indicated siRNAs for 48 h, followed by IB analysis (F) and the quantification of SPT6 levels from three independent experiments after normalization with β‐actin as the loading control (G). (H) KYSE150 cells were transfected with indicated plasmids for 48 h, and then treated with GSK3i‐IX or DMSO for 6 h, followed by IP with FLAG beads and IB analysis with the indicated Ab. (I) KYSE150 cells were transfected with indicated plasmids and siRNAs for 48 h, followed by IP with FLAG beads and IB analysis with the indicated Ab. (J) KYSE150 cells were transfected with indicated plasmids for 48 h, followed by IP with FLAG beads and IB analysis with the indicated Ab. (K) KYSE150 cells were transfected with the indicated siRNAs for 48 h, and then treated with CHX for the indicated time periods, followed by IB analysis. (L) KYSE150 cells were transfected with the indicated plasmids and siRNA and then treated with MG132 and GSK3i‐IX for 6 h, followed by Ni‐NTA purification. Ni‐NTA affinity‐purified fractions and WCE were analyzed by IB with the indicated Abs. (M) KYSE150 cells were infected with lentivirus expressing FBXW7 shRNA and then transfected with the indicated plasmids for 48 h, and then treated with MG132 for 6 h, followed by Ni‐NTA purification. Ni‐NTA affinity‐purified fractions and WCE were analyzed by IB with the indicated Abs. PD, pull down. Data are shown as mean ± SEM and analyzed by one‐way ANOVA (G). n = 3 (G), *p < 0.05, *p < 0.01.

We further measured the protein half‐life of SPT6 upon FBXW7 manipulation, using c‐MYC as a positive control, and found that the SPT6 half‐life was significantly (a) extended by FBXW7 knockdown (Figures 4C and S4D,E) (the protein half‐lives of SPT6 for each case are as follows: siNC/8.4 h vs. siFBXW7/46.4 h in KYSE150 cells; siNC/11.5 h vs. siFBXW7/37.5 h in KYSE30 cells), (b) shortened by overexpression of wild‐type FBXW7 (Figures 4D and S4F,G) (the protein half‐lives of SPT6: vector/9.4 h vs. HA‐FBXW7/5.1 h in KYSE150 cells; vector/16 h vs. HA‐FBXW7/6.1 h in KYSE30 cells), and (c) extended by overexpression of FBXW7‐ΔF mutant (Figures 4E and S4H,I) (the protein half‐lives of SPT6: HA‐FBXW7/6.5 h vs. HA‐FBXW7‐ΔF/17.1 h in KYSE150 cells; HA‐FBXW7/4.2 h vs. HA‐FBXW7‐ΔF/10.4 h in KYSE30 cells), which again acted in a dominant negative manner.

Finally, we determined whether glycogen synthase kinase 3β (GSK3β), a known kinase responsible for the phosphorylation of many FBXW7 substrates, including c‐MYC, c‐JUN, and EGR1 (early growth response 1) [42], prior to FBXW7 binding, played an essential role in FBXW7‐mediated ubiquitylation and degradation of SPT6. Indeed, the FBXW7‐SPT6 binding was significantly inhibited by GSK3β inhibitor GSK3i‐IX (Figure 4H) or by siRNA‐based GSK3β knockdown (Figure 4I), whereas overexpression of constitutively active GSK3β‐S9A led to the phosphorylation of wild‐type SPT6‐C1, but not its phosphorylation‐dead mutant (Figure 4J), indicating that SPT6 is directly phosphorylated by GSK3β. Moreover, GSK3β knockdown caused accumulation of SPT6 protein (Figure 4F,G) by extending its half‐life (Figures 4K and S4L,M) (the protein half‐lives of SPT6: siNC/6.6 h vs. siGSK3β/12.3 h in KYSE150 cells; siNC/ 8.4 h vs. siGSK3β/34.9 h in KYSE30 cells). Similarly, treatment with the GSK3β inhibitor the GSK3i‐IX significantly increased the protein levels of SPT6 with β‐Catenin^pSer33/37/Thr41^, serving as a biomarker for inhibitory effects (Figure S4J,K). Next, we found that both GSK3β knockdown and GSK3i‐IX treatment significantly inhibited polyubiquitylation of SPT6‐C1 (Figures 4L and S4N). Furthermore, FBXW7 overexpression significantly increased the polyubiquitylation of wild‐type SPT6‐C1, but not its degron/phosphorylation‐dead mutant SPT6‐C1‐Mut, while constitutively active GSK3β‐S9A further increased FBXW7‐mediated polyubiquitylation of wild‐type SPT6‐C1, but not that of SPT6‐C1‐Mut (Figure 4M). Finally, constitutively active GSK3β‐S9A further reduced endogenous SPT6 protein levels by wild‐type FBXW7, but not by FBXW7 mutants in the substrate‐binding domain (Figure S4O). Taken together, these results demonstrated that the GSK3β‐FBXW7 axis serves as a kinase–E3 ligase pair to promote SPT6 ubiquitylation and degradation. Thus, SPT6 is a new substrate for FBXW7.

SPT6 Knockdown Inhibits the Growth and Colony Formation of ESCC Cells

2.6

Many FBXW7 substrates are oncogenic proteins [33, 34], and SPT6 appears to be yet another oncogenic substrate, because it is accumulated upon FBXW7 deletion during 4NQO‐induced esophageal carcinogenesis. We first analyzed the TCGA and GTEx databases and found that SPT6 mRNA expression was elevated in ESCC tissues, compared to normal esophageal tissues (Figure S5A).

We next assessed the potential effect of SPT6 on the growth and survival of KYSE150 and KYSE30 esophageal cancer cells and found that SPT6 knockdown significantly inhibited cell growth (Figures 5A and S5B) and colony formation (Figures 5C,D and S5D,E), in contrast to the effects of FBXW7 knockdown, which stimulated growth (Figures 5B and S5C) and clonogenic survival (Figures 5E,F and S5F,G), as expected. The nature of growth suppression by SPT6 knockdown was found to be related to growth arrest at the G2/M phase (Figures 5G and S5H,J) with increased levels of cyclin B1 and PLK1 (Figure 5I), and induction of apoptosis (Figures 5H and S5I,K), with increased levels of cleaved PARP and cleaved caspase 3 (Figure 5I).

*SPT6 knockdown inhibits the growth and colony formation of ESCC cells. (A–F) KYSE150 cells were transfected with siNC or siRNA targeting SPT6 (A, C, and D) or FBXW7 (B, E, and F), and then subjected to CCK8 (A and B) and colony formation assays (C–F). Images of representative plates are shown (C and E), and colony numbers were quantified (D and F). (G and H) KYSE150 cells were transfected with the indicated siRNAs for 48 h, followed by flow cytometry analysis. The percentage of cells in the G2/M phase (G) and undergoing apoptosis (H) are plotted. (I) KYSE30 and KYSE150 cells were transfected with the indicated siRNAs for 48 h, followed by IB analysis. (J–M) KYSE150 cells were transfected with the indicated siRNAs for 48 h, and then subjected to IB (J), CCK8 (K), and colony formation (L and M) assays. Images of representative plates are shown (L) and colony numbers are quantified (M). Data are shown as mean ± SEM and analyzed by one‐way ANOVA (A, B, D, F, G, H, K, and M), n = 3 (A, B, D, F, G, H, K, and M). *p < 0.05, **p < 0.01, **p < 0.001, ns, no significance.

Finally, we tested the causal relationship between FBXW7 targeting SPT6 and growth/survival of ESCC cells using a rescue experiment with simultaneous knockdown of both FBXW7 and SPT6 (Figures 5J and S5L). Indeed, stimulation of growth and survival by FBXW7 knockdown was largely rescued by simultaneous SPT6 knockdown (Figures 5K–M and S5M–O). Taken together, our results demonstrated that SPT6 is essential for the optimal growth and survival of ESCC cells, and its accumulation upon FBXW7 knockdown contributes to the growth‐stimulating effect, thus acting as an oncogenic growth promoter.

ΔNp63 is Regulated Positively by SPT6 and Negatively by FBXW7

2.7

Next, we searched for possible downstream effectors of SPT6. SPT6 is a conserved histone chaperone and transcription elongation factor that primarily functions at the post‐translational level biochemically with oncogenic activity biologically [17, 18, 19]. A previous study reported an unexpected finding that SPT6 loss triggered the spontaneous transdifferentiation of epidermal cells into an intestinal‐like phenotype because of stalled transcription of the master regulator of epidermal fate p63, connecting SPT6 to p63 [20]. As p63 exists as two isoforms, TAp63 and ΔNp63, with opposing functions, we focused on ΔNp63, which is predominantly expressed in tumors and exhibits oncogenic properties [25, 26]. Significantly, siRNA‐based SPT6 knockdown caused a reduction in ΔNp63 at both the protein and mRNA levels (Figure 6A,B), whereas ectopic expression of SPT6 increased ΔNp63 protein and mRNA levels in a dose‐dependent manner (Figures 6C and S6A), indicating that ΔNp63 is subjected to SPT6 upregulation. To validate that SPT6 acts as a transcriptional co‐activator of ΔNp63 expression in ESCC cells, we performed a ChIP‐qPCR assay and confirmed that SPT6 binds to the promoter region of ΔNp63 gene [43] (Figure 6D).

*ΔNp63 is regulated positively by SPT6 and negatively by FBXW7. (A and B) KYSE30 and KYSE150 cells were transfected with the indicated siRNAs for 48 h, followed by IB (A) and qRT‐PCR (B) analyses. (C) KYSE30 and KYSE150 cells were transfected with increasing amounts of SPT6 plasmid for 48 h, followed by IB analysis. (D) KYSE30 and KYSE150 cells were harvested for the ChIP assay with SPT6 antibody, followed by PCR (top) or qRT‐PCR analysis (bottom). (E) KYSE150 cells were transfected with the indicated siRNAs for 48 h, followed by IB analysis. (F and G) ESCC tumor tissues from WT or Fbxw7 cKO mice following long‐term treatment were subjected to IHC staining. Representative images of ΔNp63 staining are shown (F), and the percentage of cells with ΔNp63‐positive staining from three random fields of the esophagus in each mouse was quantified (G). Scale bars: 100 and 25 µm (inset). (H) KYSE150 cells were transfected with the indicated siRNAs and plasmids for 48 h and then harvested for Ni‐NTA purification after 6 h of MG132 treatment. Ni‐NTA affinity‐purified fractions and WCE were analyzed by IB with the indicated Abs. (I‐K) KYSE150 cells were transfected with the indicated siRNAs for 48 h and then subjected to CCK8 (I) and colony formation assays (J and K). Images of representative plates are shown (J), and colony numbers are quantified (K). (L–O) KYSE150 cells were transfected with the indicated siRNAs for 48 h, and then subjected to CCK8 assay (L), IB analysis (M), and colony formation assays (N, O). Images of representative plates are shown (N), and colony numbers are quantified (O). Data are shown as mean ± SEM and analyzed by one‐way ANOVA (B, I, L, K, and O) and Student's t test (D and G); n = 3 (B, D, I, L, K, and O); n = 5 (G). *p < 0.05, **p < 0.01, **p < 0.001, ns, no significance.

Previous studies have reported that MDM2 and FBXW7, two E3 ubiquitin ligases, cooperate to promote p63 ubiquitylation and degradation in melanoma cells upon DNA damage or cellular differentiation [29, 30]. In line with this, we investigated possible FBXW7 targeting ΔNp63 in ESCC cells, and found that FBXW7 directly bound to ΔNp63 under physiological conditions (Figure S6B,C). Both IB (Figures 6E and S6D) and IHC staining (Figure 6F,G) showed that FBXW7 knockdown caused accumulation of ΔNp63 protein, whereas FBXW7 overexpression caused a dose‐dependent reduction (Figure S6E). Consistently, FBXW7 knockdown reduced polyubiquitylation of ΔNp63 (Figure 6H), and extended the protein half‐life of ΔNp63 (Figure S6F,G), whereas FBXW7 overexpression shortened its half‐life (Figure S6H,I). Biologically, ΔNp63 knockdown inhibited both growth (Figures 6I and S6J,K) and colony formation in ESCC cells (Figures 6J,K and S6L,M), and rescued the growth‐promoting effect of FBXW7 knockdown (Figure 6L–O). Taken together, these results demonstrate that in ESCC cells, ΔNp63 is a growth‐essential onco‐protein and a downstream target, subjected to transcriptional upregulation by SPT6 at the mRNA level, and posttranslational downregulation by FBXW7 for targeted degradation at the protein level as a substrate.

The FBXW7‐SPT6 Axis Regulates ESCC Tumor Growth In Vivo and Its Inverse Correlation in ESCC Tumor Tissues

2.8

We next extended our in vitro cell culture study on growth regulation by the FBXW7‐SPT6 axis to an in vivo xenograft tumor model using KYSE150 cells with stable knockdown of FBXW7 and SPT6 (Figure S7A–C). Before doing so, we first validated in cultured cells that stable knockdown of SPT6 indeed suppressed both cell growth and survival, whereas stably FBXW7 knockdown promoted growth and survival, which was largely reversed by concurrent stable SPT6 knockdown (Figure S7D–F). Likewise, in the xenograft model, compared to vehicle controls, knockdown of FBXW7 or SPT6 promoted or inhibited tumor growth, respectively, as measured by tumor volume, size, and weight, whereas double knockdown neutralized the effect of each knockdown (Figure 7A–C), indicating a causal relationship. Importantly, assays using immunohistochemical staining and western blotting showed a negative relationship between FBXW7 vs. SPT6 and ΔNp63, but a positive relationship between SPT6 and ΔNp63 in tumor tissues (Figures 7D,E and S7G).

*The FBXW7‐SPT6 axis regulates ESCC tumor growth in vivo and their inverse correlation in ESCC tumor tissues. (A–E) KYSE150 cells infected with lentivirus expressing the indicated shRNA were subcutaneously injected into nude mice. Tumor volume was monitored every 2–3 days (A), tumor masses were photographed (B), and tumor weights were measured (C) at the end of the experiment. Tumors were subjected to IHC staining (D and E). Representative images of SPT6 and ΔNp63 staining are shown (D), and the percentage of cells with positive staining from three random fields in each tumor was quantified (E). Scale bar: 100 µm. Data are shown as mean ± SEM and analyzed by one‐way ANOVA (A, C, and E). n = 3 (A), n = 6 (C), n = 5 (E); *p < 0.05, **p < 0.01, **p < 0.001, ns, no significance. (F and G) Human esophageal tumor tissue microarrays containing 86 tumor tissues stained for FBXW7 and SPT6. Representative staining images are shown (F). Quantification of positive staining was performed using ImageJ (G) (r = −0.5036, p < 0.001, Pearson correlation coefficient). Scale bar: 100 µm. (H) Kaplan–Meier analysis of 51 ESCC tissue microarray samples showing an inverse correlation between FBXW7 and SPT6 staining. Lower FBXW7 positive staining, coupled with higher SPT6 positive staining, predicted worse overall patient survival. Log‐rank test, p < 0.01. (I) Working model for how the FBXW7‐SPT6‐ΔNp63 axis regulates the ESCC development (see the text for details).

Finally, we evaluated the clinical relevance of the FBXW7‐SPT6 axis in human ESCC tumor tissues using a tumor tissue microarray by IHC staining, and found an inverse correlation in general (Figure 7F). Quantitative analysis of staining revealed a statistically significant inverse correlation between FBXW7 and SPT6 in 86 specimens (Figure 7G). Notably, ESCC tumors from patients with advanced stages (III‐IV) showed a higher percentage of SPT6‐positive staining than those in earlier stages (I‐II) (Figure S7H). We further investigated the relationship between FBXW7 and SPT6 staining and lymph node metastasis in ESCC. In tumors with lymph node metastasis, FBXW7 staining was lower, whereas SPT6 staining was higher than that in tumors without lymph node metastasis (Figure S7I). Finally, we assessed the prognostic value of the FBXW7‐SPT6 axis using Kaplan–Meier survival analysis and found that higher staining of FBXW7 but lower staining of SPT6 was associated with a better patient survival (Figure S7J,K). In a subset of 51 ESCC patients (out of a total of 86 samples in the cohort with complete follow‐up information), which showed an inverse correlation between FBXW7 and SPT6, we found that higher FBXW7 and lower SPT6 staining (n = 26) were associated with better overall survival, whereas lower FBXW7 and higher SPT6 staining (n = 25) were correlated with poorer patient survival (Figure 7H). Taken together, our results strongly suggest that FBXW7 is a tumor suppressor, whereas its substrate SPT6 is an oncogenic protein, and the FBXW7‐SPT6 axis regulates the growth and survival of ESCC cells in vitro, and ESCC tumor growth in vivo, and is associated with the prognosis of ESCC patients.

Discussion

3

ESCC is the predominant subtype of esophageal cancer in China, with a variety of identified risk factors, including smoking, alcohol consumption, and unhealthy dietary habits [44, 45]. Genomic sequencing studies in Chinese patients with ESCC identified FBXW7 as one of the most frequently mutated genes [4]. Although FBXW7 is a well‐established tumor suppressor that promotes ubiquitylation and degradation of oncogenic substrates [33, 34], and FBXW7 loss was reported to cause the accumulation of MAP4 in ESCC cells [46], whether and how FBXW7 contributes to ESCC initiation and/or progression in vivo remains elusive. Here, we used both genetic and chemical carcinogenesis models, and report that Fbxw7 is indeed a tumor suppressor whose deletion promotes ESCC development induced by carcinogen 4NQO, but not in combination with *Pik3ca^LSL‐E545K/+^

• activation. In addition, neither Pik3ca E545K mutation alone nor the combination of *Pik3ca^LSL‐E545K/+^
• activation and Fbxw7 deletion plays a role as an initiator of ESCC tumorigenesis, rather Fbxw7 deletion acts as a promoter to accelerate ESCC development, upon initiation by carcinogen 4NQO, a water‐soluble quinoline derivative that effectively induces DNA adduct formation by substituting adenosine for guanosine [47, 48] and produces reactive oxygen species (ROS) via redox cycling to cause DNA mutations and strand breaks [49, 50].

The PI3K signaling pathway is a well‐known oncogenic driver in human cancers, as well as shown in a number of mouse tumor models. Examples include colon adenocarcinomas induced by transgenic p85 iSH2‐p110 fusion [51] and murine mammary tumors driven by PIK3CA hotspot mutations (e.g., H1047R, E545K) [52, 53]. However, its oncogenic driving role in the pathogenesis of ESCC remains elusive. Very interestingly, we showed here that Fbxw7 deletion promoted ESCC tumorigenesis induced by carcinogen 4NQO, but not by the Pik3ca‐E545K mutant, clearly demonstrating that while Fbxw7 is a suppressor of tumor promotion, Pik3ca‐E545K mutant, unlike 4NQO, is not an oncogenic initiator or driver. While the exact underlying mechanism is unknown, it is speculated that 4NQO‐induced genotoxic stress, rather than Pik3ca‐E545K‐activated PI3K‐mTORC‐AKT signaling, effectively modulates the downstream signal of the SPT6‐ΔNp63 axis during ESCC tumorigenesis.

What are the mechanisms by which FBXW7 acts as a tumor suppressor? To this end, we performed unbiased whole‐proteome analysis using mass spectrometry on tumor tissues from Fbxw7 cKO vs. WT control mice. Surprisingly, among the proteins accumulated upon Fbxw7 deletion, we did not identify any common oncogenic proteins previously known as Fbxw7 substrates. To narrow down the possibility that the accumulated proteins are indeed Fbxw7 substrates with oncogenic potential, we set up two search criteria: (1) candidates must contain an evolutionarily conserved Cdc4 phosphodegron (CPD) motif, which is required for FBXW7 binding, and (2) candidates must be overexpressed in ESCC. These exercises narrowed the list down to eight candidates, including SPT6, UAP1L1, RBM7, LSM14A, PDPK1, PLEKHF2, EI24, and WASHC4. Using pharmacological (MLN4924) and genetic (siRNA knockdown) approaches, we identified SPT6 as a possible substrate of FBXW7.

The follow‐up studies validated that SPT6 is indeed a bona fide substrate of FBXW7 with the following supporting evidence: (1) FBXW7 inactivation or depletion causes SPT6 accumulation; (2) FBXW7 interacts with SPT6 through its WD40 domain, whereas SPT6 binds to FBXW7 via its CPD degron, SPNTE; (3) FBXW7 manipulation controls SPT6 protein half‐life, with overexpression shortening and knockdown extending it; (4) FBXW7 promotes SPT6 ubiquitylation and proteasomal degradation; (5) Inactivation of GSK3β, a kinase required for phosphorylation of FBXW7 substrates including SPT6, reduces FBXW7‐SPT6 binding, prolongs SPT6 protein half‐life, and decreases SPT6 polyubiquitylation; and (6) most importantly, Spt6 accumulates in mouse ESCC tissues with Fbxw7 deletion. Thus, SPT6 is a novel substrate of FBXW7, which plays a causal role in ESCC tumorigenesis upon Fbxw7 deletion, as well as in promoting the growth and survival of ESCC cells.

Few studies have been conducted to elucidate the biological functions of SPT6, particularly in cancer cells. One study showed that SPT6 affects the DNA repair process by transcriptionally up‐regulating BRCA1 (breast cancer gene 1), thereby regulating the self‐renewal, genomic stability, and tumor initiating capacity of GSCs [19]. Another study reported that SPT6 promotes colorectal cancer development by targeting hTERT [18]. However, another study showed that SPT6 regulates the transdifferentiation of epidermal cells into an intestinal‐like phenotype by targeting p63 [20]. Here, we showed that SPT6 was essential for the growth and survival of ESCC cells, as its knockdown significantly inhibited their capacity for proliferation and colony formation. Our rescue experiment revealed that SPT6 is a downstream effector of FBXW7 that mediates, at least in part, its tumor‐suppressive activity. In the context of the 4NQO‐induced ESCC mouse model, Fbxw7 deletion caused accumulation of Spt6 to facilitate the transcription of erroneous DNA strands, further exacerbating genomic instability, thus promoting carcinogenesis.

Next, we investigated the downstream effector of SPT6, mainly focusing on p63, since the loss of SPT6 causes stalled transcription of p63 [20]. Indeed, we found that SPT6 is an upstream positive regulator of ΔNp63, the predominant isoform in ESCC, since SPT6 manipulation positively affects ΔNp63 at both mRNA and protein levels. On the other hand, while MDM2 and FBXW7 cooperate to promote p63 ubiquitylation and degradation in melanoma cells upon DNA damage or cellular differentiation [29, 30], we found that FBXW7 directly interacts with ΔNp63 under physiological conditions, and FBXW7 promotes the ubiquitylation and degradation of ΔNp63 and shortens its half‐life. Biologically, ΔNp63 knockdown negatively regulated the growth and survival of ESCC cells, and a rescue experiment with simultaneous knockdown of both FBXW7 and ΔNp63 demonstrated that FBXW7 and ΔNp63 are functionally connected in the regulation of growth and survival of ESCC cells. Collectively, FBXW7 controls ΔNp63 levels directly by targeting it for degradation or indirectly by targeting SPT6 for degradation, thus reducing ΔNp63 transcription. Upon FBXW7 deletion, both SPT6 and ΔNp63 were activated to promote ESCC development. Now, an open question is whether the role of the FBXW7‐SPT6‐ΔNp63 axis in ESCC is conserved in other types of squamous cell carcinomas (SCCs), such as head and neck (HNSCC) or lung (LUSC). It is indeed an interesting subject for future investigation.

Does SPT6 degradation by FBXW7 affect transcriptional programs beyond ΔNp63? It certainly does. As a central elongation factor and histone chaperone, SPT6 is essential for productive transcription and chromatin integrity [54]. Its depletion is known to cause transcriptional dysregulation, including aberrant activation of cryptic intragenic promoters [54]. However, in this study, SPT6 accumulation upon FBXW7 loss promoted ESCC tumorigenesis, rather than ensuring transcription and chromatin fidelity, indicating SPT6 acts as an oncogenic promoter in this context, in which the induction of ΔNp63 is a valid contributor. Future studies employing transcriptomics may identify additional contributors to the process of ESCC.

Finally, we assessed the clinical relevance of our findings using IHC staining of ESCC tumor tissue microarray and found a negative correlation between FBXW7 and SPT6 at the protein level. Likewise, higher FBXW7 coupled with lower SPT6 was associated with better survival, whereas lower FBXW7 coupled with higher SPT6 was correlated with worse survival. Thus, the FBXW7‐SPT6 axis may serve as an important prognostic marker for ESCC.

Nevertheless, there are two limitations of the study. (1) We found that SPT6 upregulates ΔNp63 at the transcriptional level, which contributes to its pro‐proliferative activity. However, given that SPT6 is a histone chaperone associated with RNAPII that promotes transcription elongation, thus upregulating numerous downstream targets, ΔNp63 is just one of the targets to mediating SPT6 function. We did not pursue other possible contributors, since it is out of the scope of this study; (2) Although we did detect SPT6 accumulation in Fbxw7‐null ESCC tissues (Figure 2I–K), we did not perform a long‐term rescue experiment by simultaneous Spt6 cKO. Instead, the in vivo rescue experiment was conducted using a nude mouse xenograft tumor model.

In summary, our study revealed that FBXW7 acts as a tumor suppressor in ESCC by targeting ubiquitylation and degradation of SPT6 and ΔNp63. Our study fits the following working model. In normal cells, FBXW7 promotes ubiquitylation and degradation of SPT6 and ΔNp63 to keep their pro‐proliferative function under the control. During esophageal tumorigenesis, FBXW7 is inactivated by mutations or deletion which caused the accumulation of SPT6 and ΔNp63, with ΔNp63 also being up‐regulated by SPT6. Together with 4NQO‐induced initiation, Fbxw7 deletion mediated activation of the SPT6‐ΔNp63 axis promotes ESCC development (Figure 7I). Thus, the SPT6‐ΔNp63 axis may serve as a therapeutic target for ESCC treatment.

Materials and Methods

4

Animal Work

4.1

For the 4NQO‐induced ESCC model, female *Fbxw7^fl/fl^ *; Ed‐l2‐Cre‐ and *Fbxw7^fl/fl^ *;Ed‐l2‐Cre+ mice, aged 6–7 weeks and with a C57BL/6J background, were used. For the genetically induced ESCC model, *Pik3ca^LSL‐E545K/+^

• [37];Ed‐l2‐Cre+ (GemPharmatech Co. Ltd, catalog no. T005634) and *Fbxw7^fl/fl^
• [38]; *Pik3ca^LSL‐E545K/+^ *;Ed‐l2‐Cre+ mice, with a C57BL/6J background, were used. For the 4NQO‐induced ESCC model, mice were administered 100 µg/mL 4NQO in their drinking water for either 8 weeks (short‐term) or 16 weeks (long‐term), followed by tap water for 16 weeks (short‐term) or 8 weeks (long‐term). For the xenograft model, female BALB/c nude mice were purchased from Shanghai SLAC Laboratory Animal Center. Six‐week‐old mice were subcutaneously inoculated with 1×10^6^ KYSE150 cells. Tumor growth was measured using calipers, and tumor volumes were calculated as 1/2 × length × width^2^. All animal procedures were approved by and conducted in accordance with the guiding principles of the Laboratory Animal Ethics Committee of the Second Affiliated Hospital, Zhejiang University School of Medicine, China. All the mice were housed under specific pathogen‐free conditions at the Laboratory Animal Center of Zhejiang University.

LC‐MS/MS Analysis

4.2

Mass spectrometry analysis of mouse esophageal tumors was performed using PTM‐Bio (Hangzhou, China). Briefly, the tryptic peptides were dissolved in solvent A and directly loaded onto a home‐made reversed‐phase analytical column (25‐cm length, 100 µm i.d.). The mobile phase consisted of solvent A (0.1% formic acid, 2% acetonitrile in water) and solvent B (0.1% formic acid, 90% acetonitrile/in water). Peptides were separated using the following gradient: 0–68 min, 6%–23%B; 68–82 min, 23%–32%B; 82–86 min, 32%–80%B; 86–90 min, 80%B, and all at a constant flow rate of 500 nL/min on a Vanquish UPLC system (ThermoFisher Scientific). The separated peptides were analyzed using an Orbitrap Exploris 480 instrument with a nano‐electrospray ion source. The electrospray voltage applied was 2300 V. The FAIMS compensation voltage (CV) was set to −45 and −65 V, and the precursors and fragments were analyzed using the Orbitrap detector. The full MS scan resolution was set to 60,000 for a scan range of 400–1200 m/z. The MS/MS scan was fixed at 110 m/z at a resolution of 15,000 with TurboTMT set to off. Up to the 25 most abundant precursors were selected for further MS/MS analyses with 20 s dynamic exclusion. The HCD fragmentation was performed at a normalized collision energy (NCE) of 27%. Automatic gain control (AGC) target was set at 100%, with an intensity threshold of 50,000 ions/s and a maximum injection time of auto.

Database Search

4.3

The resulting MS/MS data were processed using the Proteome Discoverer search engine (v.2.4). Tandem mass spectra were searched against the Mus_musculus_10090_SP_20230103.fasta (17,132 entries) concatenated with a reverse decoy and contaminants database. Trypsin (Full) was specified as the cleavage enzyme, allowing up to two missing cleavages. Min. peptide length was set at six and max. peptide length at 50. The number of modifications per peptide was set to three. The mass error was set to 10 ppm for the precursor ions and 0.02 Da for the fragment ions. Carbamidomethyl on Cys was specified as a fixed modification. Oxidation of Met, acetylation on the protein N‐terminal, met‐loss on Met and Met‐loss+acetyl on Met were specified as variable modifications. False discovery rate (FDR) of protein, peptides, and PSM was adjusted to < 1%.

Bioinformatic Analysis

4.4

For normal esophageal tissues, expression data were obtained from the GTEx expression matrix (https://xena.ucsc.edu/). For esophageal cancer, the fragments per kilobase million (FPKM) expression matrix was extracted from TCGA database (https://portal.gdc.cancer.gov/). Differential analysis was conducted using R (version 4.2.0) and R packages, and the results are presented as box plots.

Cell Culture and Transfection

4.5

Human embryonic kidney HEK293 cells were obtained from the American Type Culture Collection, and human esophageal cancer KYSE30, KYSE150, KYSE450, and EC109 cells were kind gifts from Dr. Lijun Jia's Lab (Nanjing University of Chinese Medicine). HEK293 cells, KYSE30, KYSE450, and EC109 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) and 1% penicillin–streptomycin. Human esophageal cancer KYSE150 was cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin–streptomycin. Cells were incubated at 37°C in a humidified incubator with 5% CO_2_. Cells were transfected with various plasmids using PolyJet In Vitro DNA Transfection Reagent (SignaGen Laboratories) or with siRNA oligos using Genmute siRNA Transfection Reagent (SignaGen Laboratories) or Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions.

Antibodies, Chemical Reagents

4.6

Antibodies were used as follows: FBXW7 (Bethyl, A301‐720A and A301‐721A), SPT6 (Santa Cruz, sc‐393920, and Novus, NB100‐2582), RBM7 (Proteintech, 21896‐1‐AP), LSM14A (Proteintech, 18336‐1‐AP), UAP1L1 (Proteintech, 25262‐1‐AP), WASHC4 (Proteintech, 51101‐1‐AP), PLEKHF2 (Proteintech, 25424‐1‐AP), PDPK1 (Proteintech, 17086‐1‐AP), EI24 (Proteintech, 20456‐1‐AP), NEDD8 (Abcam, ab81264), GSK3α/β (Cell Signaling Technology, 5676), c‐MYC (Cell Signaling Technology, 5605), p‐Ser/Thr‐Pro (05‐368, Upstate), FLAG (Sigma‐Aldrich, F1804), HA (Sigma‐Aldrich, A2095), β‐Actin (HUABIO, R1207‐1), GFP (ABclonal, AE012), cleaved‐NOTCH1 (Cell Signaling Technology, 4147), Cyclin B1 (Cell Signaling Technology, 12231P), PLK1 (Proteintech, 10305‐1‐AP), PARP (Cell Signaling Technology, 9542S), Cleaved PARP (Cell Signaling Technology, 9541S), caspase3 (Cell Signaling Technology, 9662S), cleaved caspase3 (Cell Signaling Technology, 9661S), ΔNp63 (Abcam, ab203826) and Ki67 (Abcam, ab16667). The following chemicals were obtained from commercial sources: cycloheximide (MedChem Express, HY‐12320), 4‐Nitroquinoline‐1‐oxide (Sigma‐Aldrich, N8141), MLN4924 (ApexBio, B1036), and MG132 (MedChem Express, HY‐13259).

Plasmids, siRNAs, and shRNA Oligos

4.7

Plasmid constructs expressing FLAG‐tagged and HA‐tagged FBXW7 were used as described previously [55]. GFP‐tagged FBXW7 and truncations were provided by Dr. Qiang Zhang (University of Michigan). FLAG‐tagged GSK3β‐S9A was provided by Dr. Zhao Yongchao (Zhejiang University). FLAG‐tagged SPT6 and ΔNp63 were purchased from FenghBio. siRNA or shRNA oligos were synthesized by GenePharma (Shanghai, China). The shRNA sequences were as follows: shSPT6: 5’‐GAG CTG AGC TGT CGA TAT ATT‐3’; shFBXW7‐1#: 5’‐ACA GGA CAG TGT TTA CAAA‐3’; shFBXW7‐2#: 5’‐CCA ATT GTG TAG ACG ATA TAC‐3’. The sequences of siRNA oligos are as follows: siFBXW7‐1#: 5’‐UGA UAC AUC AAU CCG UGU UUG‐3’; siFBXW7‐2#: 5’‐ACA GGA CAG UGU UUA CAA A‐3’; siFBXW7‐3#: 5’‐GCU GUG UUC AAU AUG AUG GC‐3’; siSPT6‐1#: 5’‐GCA UGA AGC CGC AGC AAU U‐3’; siSPT6‐2#: 5’‐GAA AGG GCA CUG AGG GAU A‐3’; siGSK3β‐1#: 5’‐CAU GAA AGU UAG CAG AGA CAA‐3’; siGSK3β‐2#: 5’‐AGC AAA UCA GAG AAA UGA AC‐3’; siΔNp63‐1#: 5’‐GCG UAU UAU GAA AGC UGG AAU‐3’; siΔNp63‐2#: 5’‐GAA GAC GAC UUA UAC UAU U‐3’.

Quantitative Real‐Time PCR Assay

4.8

Total RNA was extracted using TRIzol reagent (Invitrogen, 15596018) and transcribed into cDNA using the PrimeScript first Strand cDNA Synthesis Kit (RR037A, TaKaRa). Quantitative RT‐PCR was performed using TB Green Premix Ex Taq (RR420A, TaKaRa) according to the manufacturer's instructions. The relative expression of target genes was calculated using the 2^−ΔΔCT^ method. The primer sequences were as follows: SPT6‐F: 5’‐TCA CCA CCC CTC AGT ACC AC‐3’; SPT6‐R: 5’‐CTG CAT GGC TGT TGG ACT T‐3’; ΔNp63‐F: 5’‐GAA AAC AAT GCC CAG ACT CAA‐3’; ΔNp63‐R: 5’‐TGC GCG TGG TCT GTG TTA‐3’; β‐Actin‐F: 5’‐TCA CCC ACA CTG TGC CCA TCT AC‐3’; β‐Actin‐R: 5’‐GGA ACC GCT CAT TGC CAA TG‐3’.

Immunoblotting and Immunoprecipitation

4.9

For immunoblotting (IB) analysis, cells or esophageal tissues, were lysed in lysis buffer with protease inhibitors (Roche), and then subjected to IB analysis as previously described [55]. For immunoprecipitation (IP) analysis, cells were lysed in IP lysis buffer (50 mM Tris, pH 7.5, 0.15 M NaCl, 1% NP‐40, 0.1% SDS, 1 mM EDTA, 1 mM DTT) with protease inhibitors (Roche). The cell lysates were incubated with FLAG beads (Sigma‐Aldrich, A2220) or indicated antibodies in a rotating incubator overnight at 4°C. Following this, the samples incubated with primary antibodies were further incubated with Protein G Sepharose beads (GE Healthcare) for 4–5 h. The immunoprecipitates were washed five times with IP lysis buffer and subjected to IB analysis.

H&E and Immunohistochemical Staining

4.10

Mouse tissues were fixed in 10% formalin and embedded in paraffin. Sections were prepared for hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining. For IHC staining, the sections were incubated with antibodies against SPT6 (Novus, NB100‐2582), ΔNp63 (Abcam, ab203826), and Ki67 (Abcam, ab16667) overnight at 4°C. Following this, the sections were incubated with an HRP‐conjugated secondary antibody and stained with 0.05% 3,3‐diaminobenzidine tetrahydrochloride (DAB). Human esophageal tumor tissue microarrays consisting of 86 tumors were purchased from Shanghai Outdo Biotech, China. The study was approved by the Ethics Committee of the Shanghai Outdo Biotech Company. The slides were then incubated with SPT6 (Novus, NB100‐2582) or FBXW7 (Bethyl, A301‐721A) antibodies. The images were captured using a Virtual Slide Scanner (KFBIO, KF‐FL‐020, China) and analyzed using ImageJ software.

In Vivo Ubiquitylation Assay

4.11

Cells were harvested following treatment with 20 µM MG132 for 6–8 h. Cells were then lysed in a buffer containing 6 M guanidinium‐HCl, 10 mM Tris‐HCl (pH 8.0), 0.1 M Na_2_HPO_4_/NaH_2_PO_4_, and 10 mM β‐mercaptoethanol, followed by sonication. The lysates were incubated with Ni‐NTA agarose beads (30210, Qiagen) for 4–5 h at room temperature. The beads were washed sequentially with buffer A (6 M guanidinium‐HCl, 0.1 M Na_2_HPO_4_/NaH_2_PO_4_, 10 mM imidazole, pH 8.0) and buffer B (8 M urea, 10 mM Tris‐HCl, pH 6.3, 0.1 M Na_2_HPO_4_/NaH_2_PO_4_, 10 mM β‐mercaptoethanol) containing 0.2% Triton X‐100, followed by buffer B containing 0.1% Triton X‐100. Proteins were eluted using an elution buffer containing 200 mM imidazole, 0.15 M Tris‐HCl (pH 6.8), 30% glycerol, 0.72 M β‐mercaptoethanol, and 5% SDS, and then analyzed by IB.

Immunofluorescent Staining

4.12

Cells were fixed in 10% formalin for 20 min, and then washed three times with PBS. Next, cells were permeabilized with 0.5% Triton X‐100 for 10 min. Following blocking with PBS containing 5% goat serum for 30 min, cells were incubated with anti‐FLAG antibody (1:500) overnight at 4°C, followed by incubation with fluorescent‐dye‐conjugated secondary antibodies (1:500, Thermo, A10036) for 1 h at room temperature. Cellular nuclei were stained with DAPI (1:500, Beyotime, C1002) for 30 min. Images were acquired using a Nikon A1‐Ti confocal microscope (Nikon, Japan).

Cell Growth and Colony Formation Assays

4.13

Cells were seeded into 96‐well plates (3,000 cells/well) in triplicate and cultured in complete medium for the indicated time periods. Cell growth was assessed using the Cell Counting Kit‐8 (CCK8, MedChem Express) according to the manufacturer's instructions. For the colony formation assay, cells were seeded into 6‐well plates in triplicate and cultured at 37°C for 10–14 days. Colonies were stained with Coomassie Brilliant Blue and counted.

Flow Cytometry

4.14

Cells were fixed in ice‐cold 75% ethanol, stained with propidium iodide and Annexin V (BD Biosciences, 556547), according to the manufacturer's instructions, and then analyzed on a CytoFLEX flow cytometer (Beckman Coulter).

ChIP Assay

4.15

The ChIP assay was performed using the SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads, Cell Signaling Technology, 9003, USA) according to the manufacturer's instructions. After reverse cross‐linking and DNA purification, the immunoprecipitated DNA was amplified by PCR using the following primers: ΔNp63‐p‐F, 5’‐GGA GTC CAG GTG GAA GTT GA‐3’; ΔNp63‐p‐R, 5’‐CTT CTG GCT CCA GGA TTT TG‐3’.

Statistical Analysis

4.16

Student's t‐test and one‐way ANOVA were performed using data from three independent experiments. For categorical variables, Chi‐square test was used. Kaplan–Meier survival curves were generated and compared using log‐rank tests. GraphPad Prism software (version 8.0) and SPSS software (version 20.0) were used for the analysis. *p *< 0.05 was considered statistically significant.

Author Contributions

J. Z., L.Y., X. X., and Y. S. conceived of and designed the study. J. Z. and L. Y. performed most of the experiments and analyzed the data. X. Y. performed some animal experiments. J. Z. drafted the manuscript. Y. S. and X. X. analyzed the data and revised the manuscript. Y. S. finalized the manuscript. Y. S. supervised the study. The manuscript was approved by all the authors.

Funding

This work was supported in part by the National Natural Science Foundation of China (U22A20317 and 92253203 to Y.S.; 81974429 and 82172898 to X.X.), the National Key Research and Development Program of China (2022YFC3401500 and 2021YFA1101000 to Y.S.), and the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang, China (2022R01002 to Y.S.).

Ethics Statement

All animal procedures were approved and conducted in accordance with the guiding principles of the Laboratory Animal Ethics Committee of the Second Affiliated Hospital, Zhejiang University School of Medicine, China (approval no. 2024–263). The study of human esophageal tumor tissue microarrays was approved by the Ethics Committee of the Shanghai Outdo Biotech Company (approval no. YBM‐05‐02 and SHYJS‐BC‐2310001).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Fig. S1: Fbxw7 deletion promotes the development of ESCC induced by 4NQO. Fig. S2: FBXW7 negatively regulates SPT6 in ESCC. Fig. S3: FBXW7 interacts with SPT6 via its consensus degron motif. Fig. S4: FBXW7 negatively regulates SPT6 stability by promoting its ubiquitylation. Fig. S5: SPT6 knockdown suppresses ESCC cell growth and colony formation. Fig. S6: ΔNp63 is regulated positively by SPT6 and negatively by FBXW7. Fig. S7: The FBXW7‐SPT6 axis regulates ESCC tumor growth in vivo and their inverse correlation in ESCC tumor tissues. Table S1: A list of 21 CPD‐containing candidates significantly increased in Fbxw7 KO vs. WT ESCC tumors.