HTR7 Promotes Laryngeal Cancer Growth Through Activating PI3K/AKT Pathway

Background: Laryngeal cancer is a common malignancy of the head and neck, G protein-coupled receptors (GPCRs) are easily druggable in diseases. The 5-hydroxytryptamine receptor 7 (HTR7) belongs to the GPCR family; however, its role in laryngeal cancer remains unknown. Methods: MTT, Colony formation assay, BrdU incorporation assay, soft agar growth assay and xenograft tumor in nude mice were used to analyze the effect of HTR7 expression level on laryngeal cancer proliferation and growth. Results: We found that HTR7 was signicantly upregulated in laryngeal cancer tissues and cells, and patients with high HTR7 expression had shorter survival time than those with low HTR7 expression. Univariate and multivariate Cox regression models showed HTR7 was an independent predictive factor for the prognosis of patients with laryngeal cancer. Cell proliferation assays and an animal model showed that HTR7 overexpression promoted laryngeal cancer proliferation and growth, while HTR7 knockdown inhibited laryngeal cancer proliferation and growth. Further analysis showed HTR7 activated the PI3K/AKT pathway, characterized by increased phosphorylation of AKT, luciferase reporter activity of FOXO factors and target expression. Inhibition of the PI3K/AKT pathway in HTR7-overexpressing cells suppressed proliferation and growth, suggesting HTR7 promoted laryngeal cancer proliferation and growth by activating the PI3K/AKT pathway. Conclusions: In summary, HTR7 is not only a target for laryngeal cancer therapy, but also a prognostic factor for with


Background
Laryngeal cancer is a common malignancy of the head and neck, with approximately 11,000 to 13,000 cases of laryngeal cancer being diagnosed annually [1]. In the past decade, laryngeal cancer treatment has been improved through surgery, radiation, and systemic therapy, and novel agents for metastasis [2]; however, the death rate is still high. Therefore, to improve laryngeal cancer therapy, a better understanding of the regulatory mechanism of laryngeal cancer is urgently required.
The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT) pathway is frequently mutated in human cancers, and there are several PI3K families exist in human cells [3]. Class I A PI3Ks, which have been proved to regulate tumor progression, are heterodimers consisting of a p85 regulatory subunit and a p110 catalytic subunit. Growth factor receptor tyrosine kinases, such as EGFR, HER2, MET, and FGFR phosphorylate adaptor proteins to bind p85, which relieves the inhibition of p110 by p85. Then, the p85-p110 heterodimer binds to lipid phosphatidylinositol-4,5-bisphosphate (PIP2) at the plasma membrane. p110 phosphorylates PIP2 to generate phosphatidylinositol-3,4,5-triphosphate (PIP3). PTEN and INPP4B can dephosphorylate PIP3 to inhibit the PI3K/AKT pathway. AKT, SGK, and PDK1 bind to PIP3 at the plasma membrane, and then PDK1 and TORC2 phosphorylate AKT to fully activate AKT. Activated AKT can phosphorylate many proteins involved in cell survival, proliferation, growth, and protein synthesis, such as FOXO family proteins, MDM2, and p27 [4,5]. 5-hydroxytryptamine (serotonin) receptor 7 (HTR7) is a G protein-coupled receptor (GPCR) rst described in 1993, and many transcription factors can bind to its promoter, such as AP2, EGR-1, and MAZ [6,7]. HTR7 is associated with circadian rhythm, anxiety, depression, schizophrenia, nociception, and memory [8][9][10]. However, role of HTR7 in tumor progression has not been studied. In the present study, we aimed to investigate the role of in laryngeal cancer. The results showed that HTR7 promoted laryngeal cancer growth by activating the AKT pathway.

Materials And Methods
Cell culture and specimens Human oral keratinocytes cell and laryngeal cancer cell TU212, FaDu, Hep-2, TU212 and TU686 were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium (GIBCO) supplemented with 10% fetal bovine serum (FBS, BI) at 37℃ in a humidi ed incubator with 5% CO 2 . All the laryngeal cancer tissues and adjacent normal laryngeal tissues were obtained from the LSCC patients who were performed surgical procedure in the Otolaryngology Head & Neck department of Guangdong Provincial People's Hospital. Eight pairs of fresh laryngeal cancer tissues and adjacent normal laryngeal tissues were obtained during the procedures and immediately frozen in liquid nitrogen.
A cohort of 113 laryngeal cancer tissues were obtained, the detailed clinicopathological characteristics were showed in Supplemental Table 1. The Ethics Committee of approved all collections. All patients gave written informed consents.
Quantitative reverse transcription (Q-PCR) Total RNA was isolated using FastPure Cell/Tissue Total RNA isolation Kit (Vazyme) according to the manufacturer's protocols. RNA was reversely transcribed into cDNA according to the instructions of HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme). Q-PCR was performed on the CFX-96 PCR system (Bio-Rad) using AceQ Universal SYBR qPCR Master Mix (Vazyme). The relative quanti cation of mRNA expression was calculated using 2 -ΔΔCt method. GAPDH was used as the negative control for detecting mRNA expression.

MTT
Indicated cells were seeded in a 96-well plate at a density of 4 ×10 3 cells/well, Six replications were prepared for treatment group. Cells were incubated with MTT solution at indicated time points for 4h at 37℃, the DMSO was added to each well. The plates were gently shaken on a horizontal shaker for 10min to dissolve crystals, the optical density (OD) value of each well was read at 570nm in a microplate reader (BioTek). Each experiment was repeated 3 times.

Colony formation assay and BrdU incorporation assay
Cells were plated at 6-well plates at a density of 0.5×10 3 cells/well and cultured for 10 days. Colonies were xed with 10% formaldehyde for 5min and then stained with 1% crystal violet for 30s. BrdU incorporation assay was performed using BrdU Kit (eBioscience).

Soft agar growth assay
Cells were resuspended in 2ml complete medium plus 0.3% agar, the agar-cell mixture was plated on the top of a bottom layer consisting 1% agar in complete medium. 10 days after, colony size was measured using an ocular micrometer and colonies larger than 0.1mm in diameter were counted. The experiment was performed three times for each cell line.

Xenograft tumor in nude mice
Nude mice (4-week age) were purchased from Model Animal Research Center of Nanjing University and randomly assigned into 4 groups with 3 mice in each group: Vector control group, HTR7 overexpression group, Scramble group, HTR7 knockdown group. The dorsal ank of each nude mouse was injected subcutaneously with 1×10 7 cells. The length (L) and width (W) of the tumor were measured with a Vernier caliper after the occurrence of the tumor. The volume of the tumor was calculated as V= W 2 × L × 0.52.
Tumor size were measured weekly. All animal experiments were performed in accordance with National Institutes of Health guidelines and approved by the animal care and use committee of Guangdong Academy of Medical Sciences.

Statistical analysis
All data were processed using SPSS 21.0 statistical software (IBM). Measured data were expressed as mean ± standard deviation. Comparisons between two groups were conducted with independent t-test. One-way analysis of variance (ANOVA) was used to compare difference in multiple groups. P < 0.05 was statistically signi cant.

HTR7 is upregulated in laryngeal carcinoma tissues and cells
We found that HTR7 was signi cantly upregulated in laryngeal cancer tissues compared with that in normal laryngeal tissues in data from The Cancer Genome Atlas (TCGA) database ( Figure 1a). We also con rmed this result using eight pairs of laryngeal cancer tissues and their adjacent normal tissues: Q-PCR and western blotting showed that HTR7 was also upregulated in laryngeal tissues (Figure 1b and   1c). Q-PCR and western blotting further demonstrated that HTR7 was upregulated in laryngeal cells compared with that in normal laryngeal epithelia cells (Figure 1d). Gene Set Enrichment Analysis (GSEA) showed the high HTR7 expression correlated positively with strong tumorigenesis ability ( Figure 1e). Finally, further analysis of TCGA data revealed that patients with high HTR7 expression had a shorter survival time than those with low HTR7 expression (Figure 1f). Taken together, these ndings suggested HTR7 was upregulated in laryngeal cancer tissues and cells, and high HTR7 expression was associated with poor outcome.
HTR7 is an independent predictive factor for prognosis of patients with laryngeal cancer According to above results, we determined the association between HTR7 expression and overall survival of patients with laryngeal cancer using long-rank test and multivariable Cox proportional hazard regression analysis. IHC was used to determine HTR7 expression in cohort of 113 patients with laryngeal cancer (Figure 2a). Kaplan-Meier survival analysis showed that patients with low HTR7 expression had longer survival than those with high HTR7 expression (Figure 2b). To identify independent prognostic factors for the survival of patients with laryngeal cancer, univariate and multivariate Cox regression models were used, which showed that relapse, distant metastasis, and HTR7 expression were signi cant prognostic predictors for overall survival of patients with laryngeal cancer. Relapse and HTR7 expression were identi ed as independent predictive factors for the prognosis of patients with laryngeal cancer (Figure 2c). Taken together, these results showed high HTR7 expression was a signi cant independent prognostic factor for poor prognosis in laryngeal cancer and could be used as a biomarker for prognosis in patients with laryngeal cancer.

HTR7 promotes laryngeal cancer proliferation in vitro
To determine the function of HTR7 in laryngeal cancer progression, we used GSEA to analysis the relationship between HTR7 expression and the expression of key genes associated with tumor proliferation and found that HTR7 expression correlated positively with CCND1 expression (Figure 3a). CCND1 is a key factor for G1/S transition, and accelerates cell cycle progression [11,12]. Cyclin E promotes the cell cycle, and p21 and p16 inhibit the cell cycle [13][14][15]. We overexpressed HTR7 in the FaDu cell line, which has low HTR7 expression, and knocked down HTR7 expression in TU212, which has high HTR7 expression. Western blotting analysis showed HTR7 overexpression increased cyclin D1 and cyclin E levels and reduced p16 and p21 levels (Figure 3b), while HTR7 knockdown reduced cyclin D and cyclin E levels and increased p21 and p16 levels (Figure 4a). The MTT assay showed that HTR7 overexpression promoted cell proliferation signi cantly (Figure 3c), while HTR7 knockdown inhibited cell proliferation signi cantly (Figure 4b). Colony formation assays showed that HTR7 overexpression increased cell proliferation (Figure 3d), while HTR7 knockdown inhibited cell proliferation (Figure 4c). BrdU incorporation assays revealed that HTR7 overexpression promoted cell proliferation (Figure 3e), while HTR7 knockdown inhibited cell proliferation (Figure 4d). These results suggested that HTR7 promoted the proliferation of laryngeal cancer.
HTR7 promotes laryngeal cancer growth in vivo To con rm above results, we used soft agar growth assays and a mouse model to determine the role of HTR7 in laryngeal cancer growth. Soft agar growth assays demonstrated that HTR7 overexpression promoted tumor anchorage-independent growth signi cantly, while HTR7 knockdown inhibited tumor anchorage-independent growth signi cantly (Figure 5a). The mouse model revealed that HTR7 overexpression increased the tumor volume signi cantly, while HTR7 knockdown reduced the tumor volume signi cantly (Figure 5b). These results suggested that HTR7 promoted laryngeal cancer growth.
HTR7 promotes laryngeal cancer growth by activating the PI3K/AKT pathway To determine the regulatory mechanism of HTR7 in laryngeal cancer growth, we used GSEA to analyze the signaling pathways regulated by HTR7 and found that HTR7 expression levels correlated positively with those of PI3K/AKT pathway targets (Figure 6a). Luciferase reporter assays showed that HTR7 overexpression signi cantly increased the luciferase activity, while HTR7 knockdown signi cantly inhibited the luciferase activity, suggesting that HTR7 increased PI3K/AKT pathway activity (Figure 6b). Western blotting assays showed that HTR7 overexpression increased the level of phosphorylated AKT, while HTR7 knockdown reduced the level of phosphorylated AKT (Figure 6c), suggesting that HTR7 activated the AKT pathway. BCL2L1, BCL2A1, BIRC5, BCL2, XIAP, CCNE2, CCND2, CDK2, CDK4, and BAD are targets of AKT, and are associated with tumor proliferation and inhibition of apoptosis [16,17]. Q-PCR analysis showed that HTR7 overexpression promoted their expression, while HTR7 knockdown inhibited their expression (Figure 6d), which supported the view that HTR7 activated the PI3K/AKT pathway.
To understand whether HTR7 promoted laryngeal cancer growth by activating the AKT pathway, we inhibited PI3K/AKT pathway activation using either small interfering RNAs targeting AKT or the AKT pathway inhibitor perifosine [18] in HTR7-overexpressing laryngeal cancer cells. Colony formation assays showed that inhibition of the PI3K/AKT pathway signi cantly inhibited cell proliferation (Figure 7a), which was con rmed using soft agar growth assays (Figure 7b). These results suggested HTR7 promoted laryngeal cancer growth by activating the PI3K/AKT pathway.
We con rmed these results using clinical samples. Western blotting assays showed that laryngeal cancer tissues with high HTR7 expression had high p-AKT levels, while laryngeal cancer tissues with low HTR7 expression had low p-AKT levels, and the expression of HTR7 and p-AKT levels correlated positively (Figure 7c), suggesting that HTR7 activated the PI3K/AKT pathway in clinical samples.

Discussion
In present study, we found HTR7 was signi cantly upregulated in laryngeal cancer cells and tissues, and patients with high HTR7 expression had a shorter survival time. HTR7 was an independent predictive factor for the prognosis of patients with laryngeal squamous cell cancer. Functional assays showed that HTR7 overexpression promoted laryngeal cancer proliferation and growth. Mechanism analysis showed HTR7 promoted laryngeal cancer proliferation and growth by activating the AKT pathway.
Previously, the PI3K/AKT pathway has been associated with laryngeal cancer, and the phosphorylation of AKT is a marker of PI3K/AKT activation. AKT phosphorylation is associated with treatment failure of head and neck cancer, and inhibition of AKT increased radiosensitivity [19]. Many genes and non-coding RNAs have been showed to regulate laryngeal cancer progression by regulating the PI3K/AKT pathway. For example, the microRNA miR-132 promoted laryngeal cancer proliferation and growth by targeting FOXO1, which activated the PI3K/AKT pathway [20]. TRA2β is associated with poor differentiation, lymph node metastasis, and advanced clinical stage of laryngeal cancer, in which it promotes proliferation, growth, invasion, and migration, and inhibits apoptosis by activating PI3K/AKT [21]. Our ndings also suggested HTR7 promoted laryngeal cancer proliferation and growth through the PI3K/AKT pathway. HTR7 overexpression increased the level of phosphorylated AKT, while HTR7 knockdown reduced the level of phosphorylated AKT. Luciferase reporter analyses suggested that HTR7 overexpression increased FOXO transcription factor-mediated transcription. The expression of PI3K/AKT pathway target gene also increased, further demonstrating that HTR7 activated the PI3K/AKT pathway. Inhibition of PI3K/AKT pathway in HTR7-overexpressing cells inhibited cell proliferation and growth, suggesting that HTR7 promoted laryngeal cancer proliferation and growth by activating the PI3K/AKT pathway.
The role of HTR7 has not been studied in tumor progression until now. HTR7 belongs to the GPCR family, which is one of the most studied pharmacological targets because of their diverse biological functions and druggable sites. GPCRs represent about 34% of drugs approved by FDA, and about 27% in terms of sales [22][23][24], suggesting that HTR7 might be easily druggable to treat laryngeal cancer. Moreover, CAR-T or CAR-NK has been used for tumor therapy, the membrane markers of tumors are important for CAR-T or CAR-NK, HTR7 might be a well marker for laryngeal cancer treatment. we will determine whether HTR7 would be a target for CAR-T or CAR-NK therapy. In summary, HTR7 promoted laryngeal cancer growth by activating the PI3K/AKT pathway, providing a new target for laryngeal cancer prognosis and therapy.

Conclusions
To sum up, our results clearly emphasize the role of a G protein-coupled receptor HTR7 in laryngeal cancer. Patients with high HTR7 expression had shorter survival time than those with low HTR7 expression and was an independent predictive factor for patients with laryngeal cancer. HTR7 overexpression promoted laryngeal cancer proliferation and growth, it promoted laryngeal cancer proliferation and growth by activating the PI3K/AKT pathway. HTR7 is not only a target for laryngeal cancer therapy, but also a prognostic factor for the prognosis of patients with laryngeal cancer.  HTR7 is overexpressed in laryngeal cancer tissues and cells. a. HTR7 was signi cantly upregulated in laryngeal cancer tissues compared with that in normal laryngeal tissues; data were downloaded from the TCGA. b. QPCR analysis of HTR7 expression in laryngeal cancer tissues (T) and adjacent normal tissues (ANT). c. Western blotting analysis of HTR7 levels in laryngeal cancer tissues (T) and adjacent normal tissues (ANT). GAPDH was used as the loading control. d. QPCR and western blotting analysis of HTR7 expression in laryngeal cancer cells and normal laryngeal epithelial cells. GAPDH was used as the loading control for western blot analysis. e. GSEA analysis of the relationship between HTR7 expression levels and tumorigenesis. f. Survival curve analysis of HTR7 expression and clinical outcome. Error bars represent the SEM. *P < 0.05. HTR7 is an independent predictive factor for prognosis of patients with laryngeal cancer. a. Representational IHC gure for low and high HTR7 expression. b. Overall survival analysis for HTR7 expression and patent survival time. c. Univariate and multivariate analyses of relapse, distant metastasis, and HTR7 expression in patients with laryngeal cancer using Cox regression analysis. Error bars represent the SEM. *P < 0.05.     HTR7 activated the AKT pathway. a. GSEA analysis of the correlation between HTR7 expression and the expression of AKT pathway target genes. b. Luciferase reporter analysis of FOXO transcription activity after HTR7 overexpression or knockdown. c. Western blotting analysis of AKT, p-AKT, and HTR7 levels after HTR7 overexpression or knockdown, GAPDH was used as the loading control. d. QPCR analysis of BCL2L1, BCL2A1, BIRC5, BCL2, XIAP, CCNE2, CCND2, CDK2, CDK4, and BAD expression after HTR7 overexpression or knockdown, the results are shown using a heatmap. Error bars represent the SEM. *P < 0.05. Representative images for the soft agar growth assay are showed on the left, the statistical analysis is shown on the right. c. Western blotting analysis of HTR7 and p-AKT levels in laryngeal cancer tissues, and the statistical analysis of the relationship between HTR7 and p-AKT levels. GAPDH was used as the loading control. Error bars represent the SEM. *P < 0.05.

Supplementary Files
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