Activated RARα counteracts the effects of TACC1v25 on the differentiation and invasion of head and neck squamous cell carcinoma

Introduction

As the most prevalent malignant head and neck tumor, head and neck squamous cell carcinoma (HNSCC), including oral squamous cell carcinoma (OSCC), has a high mortality rate (1). Over the past 30 years, despite some progress in the development of curative treatments for HNSCC, the 5-year survival rate is no more than 50% (2). Therefore, it is essential to further elucidate the potential molecular biological mechanisms of the tumorigenesis of HNSCC.

Alternative splicing plays an essential role in regulating cellular processes such as proliferation, apoptosis, invasion, and metastasis (3). Aberrant alternative splicing patterns are frequently found in cancer, and a previous study has shown that alternative splicing events are common in HNSCC (4). Dysregulated splicing may lead to the production of oncogenic splice isoforms, which could drive malignant progression (5). However, isoforms generated through alternative splicing may play distinct roles in tumor progression (6).

Transforming acidic coiled-coil containing protein 1 (TACC1) is a key regulator of cellular processes, including gene regulation, growth, and differentiation. It interacts with various molecules involved in transcription, centrosome/microtubule dynamics, and messenger RNA (mRNA) processing (7-9). The dysregulation of TACC1 is associated with various malignant tumors (9). The human TACC1 gene contains 13 different exons, producing multiple splice variants, and the different splicing patterns may cause different TACC1 functions (10). Full-length TACC1 contains a large carboxy-terminal coiled-coil domain with binding sites, along with a serine-and proline-rich azurocidin 1 (AZU-1) region and nuclear localization signals, enabling it to interact with nuclear components. Full-length TACC1 has been shown to function as an oncogene by suppressing apoptosis during mammary gland involution and collaborating with PI3K pathway mutations to drive tumorigenesis in murine models (5). In contrast, TACC1 variant 25 (TACC1v25), a short-form splice variant lacking exon 1, retains nuclear localization signals and its ability to interact with certain cellular elements involved in chromatin remodeling and/or RNA processing machinery in the nucleus (7,11). Our previous research found that TACC1v25 was decreased in HNSCC cell lines and tissues, and its ectopic overexpression could significantly inhibit cell proliferation and promote autophagy in the Cal27 and Fadu cells (12).

Retinoic acid receptor alpha (RARα) is a key regulatory molecule that mediates the effects of retinoic acid on cell differentiation and growth (13-15). However, high expression of RARα in laryngeal squamous cell carcinoma (SCC) is associated with poorly differentiated characteristics (16), and is considered an adverse prognostic factor in OSCC (17). RARα is reported as the key receptor in all-trans-retinoic acid (ATRA)-mediated differentiation (14). Although ATRA effectively promotes differentiation in acute promyelocytic leukemia (13,14), its efficacy in HNSCC and oral premalignancies is limited, with high toxicity and frequent relapse (18,19). The mechanism of retinoic acid action in HNSCCs and oral premalignancies differs from that in acute promyelocytic leukemia (13,20), possibly implicating other signaling pathways and factors that require further investigation.

Full-length TACC1 has been shown to bind RARα in the nucleus, acting as a coactivator to enhance RARα activation by ATRA and influencing transcriptional activity on target genes. It can also directly control the nuclear localization of RARα or regulate its transport within chromatin, thereby modulating its effects on target genes (8).

This study confirmed the interaction between TACC1v25 and RARα, revealing that TACC1v25 promoted cell differentiation while inhibiting invasion and migration in HNSCC cells. However, the dissociation of activated RARα from TACC1v25, triggered by ATRA, partially counteracted the effects of TACC1v25 on differentiation, invasion, and migration. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-685/rc).

Methods

Cell culture

The Cal27 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco’s modified Eagle medium (DMEM) containing 10% foetal bovine serum (FBS). The Fadu cells were kindly provided by Dr. Califano at Johns Hopkins University and grown in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% FBS. ATRA (1 µM) (Sigma, St. Louis, MO, USA) was added to the medium for 24 h when needed. Normal human oral keratinocytes (NHOKs) were isolated from the normal oral mucosa of patients undergoing extraction of impacted teeth at the Department of Oral Surgery, Shanghai Ninth People’s Hospital, between 2022 and 2023. The cells were cultured in keratinocyte serum-free (KSF) growth medium and incubated at 37 ℃ in 5% CO₂. All media were supplemented with 1% penicillin/streptomycin. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine (No. SH9H-2019-T112-1) and informed consent was obtained from all individual participants.

Plasmid construction and transfection

The TACC1v25 coding sequence (NM_001352798.1) was ligated into a lentiviral vector of PGMLV-CMV-MCS1-3xFlag-PGK-Puro (Genomeditech, Shanghai, China). The lentiviral vectors and negative control (NC) PGMLV-CMV-MCS1-PGK-Puro were transfected into the Cal27 and Fadu cells. Stable Cal27-v25/Fadu-v25 and Cal27-NC/Fadu-NC cells were obtained after 5 µg/mL puromycin selection (12).

RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) was used to extract RNA following standard procedures. The PrimeScript™ RT Reagent Kit (TaKaRa, Tokyo, Japan) was used for reverse transcription. The generated complementary DNA (cDNA) was utilized for subsequent qPCR using TB Green Premix Ex Taq (TaKaRa). With glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene, the comparative cycle threshold (CT) (2^−ΔΔCT) method was used for data quantification. The primer sequences were as follows: RARα (forward: 5'-ATTGAGAAGGTGCGCAAAGC-3', reverse: 5'-TGCACTTGGTGGAGAGTTCA-3'); GAPDH (forward: 5'-GGACCTGACCTGCCGTCTAG-3', reverse: 5'-GTAGCCCAGGATGCCCTTGA-3').

Co-immunoprecipitation (Co-IP) assay

The Co-IP assay was performed using the Pierce™ Classic Magnetic IP/Co-IP Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Total protein was incubated with immunoglobulin G (IgG), Flag, or RARα antibodies and then incubated with magnetic beads. Finally, the samples were analyzed by western blot. The antibodies used are listed in Table S1.

Cytoplasmic and nuclear protein extraction

The NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) were used to separate the cytoplasmic and nuclear proteins according to the manufacturer’s protocol. The cytoplasmic fraction and nuclear extracts were confirmed by GAPDH and Lamin A expression, respectively.

Western blot

Radio-immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) was used for cell protein extraction. Then, 7.5–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins, which were then transferred to nitrocellulose membranes. After blocking, the membranes were incubated with primary antibodies against RARα, Flag, K14, K13, p63, p53, p-p53, p21, p-p21, vimentin, PI3K, p-PI3K, AKT, p-AKT, Lamin A, and GAPDH at 4 ℃ overnight. The antibodies used are shown in Table S1.

Immunofluorescence assay

The cells were fixed in paraformaldehyde and permeabilized with Triton X-100. After blocking, the cells were stained with TACC1v25 and RARα antibodies. The cells were then incubated with fluorescently labeled secondary antibodies. 4',6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei before analysis by fluorescence microscopy. The antibodies used are listed in Table S1.

Transwell invasion and migration assays

Transwell assays with or without Matrigel (BD Biosciences, San Jose, CA, USA) were performed to examine the presence of cell invasion and migration ability. Cells (2×10⁵ cells/mL, 200 µL) in a medium without FBS were plated into the upper chamber, and the bottom chamber was supplemented with medium containing 10% FBS for 48 h, followed by fixing and staining with crystal violet. The number of cells was counted manually.

In vivo xenograft model of metastasis

Twelve five-week-old male BALB/c nude mice were randomly divided into the Cal27-v25 group (n=6) and the Cal27-NC group (n=6). 1×10⁶ Cal27-v25/Cal27-NC cells were injected into the tail vein of each nude mouse for 8 weeks. Meanwhile, 2×10⁵ Cal27-v25/Cal27-NC cells were implanted into the right side of the tongue in 12 mice as an orthotopic xenograft model, and the mice were scheduled to be euthanized after 6 weeks (21-23). All mice were maintained in specific pathogen-free conditions. Samples, including the tongues and lungs, were stained for hematoxylin and eosin (H&E) after embedding the slices. A protocol was prepared before the study without registration. All animal experiments were performed under a project license (No. SH9H-2020-A430-1) granted by the Ethics Committee of Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, in compliance with the Guideline for the Care and Use of Laboratory Animals and the institutional laboratory animal welfare guideline.

RARα expression analysis in HNSCC tissue using The Cancer Genome Atlas (TCGA)

Data on RARα gene expression in HNSCC and normal tissues were extracted from the TCGA database (https://portal.gdc.cancer.gov/), which included 519 HNSCC samples and 44 normal tissue samples. A Gene Expression Profiling Interactive Analysis 2 (GEPIA2) (http://gepia2.cancer-pku.cn/#index) box plot was generated using the criteria of |log₂fold change (FC)| >0.5 and P<0.05.

RNA sequencing (RNA-seq) analysis

The Cal27-v25/Cal27-NC and Fadu-v25/Fadu-NC cells with or without ATRA were analyzed by RNA-seq at NEO-BIO (Shanghai, China). DESeq software was used to determine differentially expressed genes (DEGs) by setting the absolute FC to ≥2 and P<0.05. After screening DEGs, the GOseq R package was used for Gene Ontology (GO) analysis. The adjusted value of P<0.05 was then used as the criterion for significant DEG enrichment. The significantly enriched pathways were determined if at least two associated genes were included and P<0.05. The Search Tool for the Retrieval of Interacting Genes (STRING) database (https://cn.string-db.org/) and Cytoscape software (version 3.7.2) were used to obtain the protein-protein interaction networks.

Statistical analysis

Data were expressed as the mean ± standard deviation (SD) from at least three independent experiments. Statistical comparisons were conducted using Student’s t-test in GraphPad Prism 9.4.1. P<0.05 was deemed statistically significant.

Results

TACC1v25 physically interacts with RARα in HNSCC cells

High RARα expression is considered an adverse prognostic factor for OSCC (17). Therefore, we examined the RARα expression in the HNSCC cell lines and tissues using the TCGA database. In the Cal27 cells, RARα was highly expressed as compared with the NHOK cells (Figure 1A), and its expression was upregulated in the HNSCC tissues as compared with the adjacent normal tissues (Figure 1B).

Figure 1 TACC1v25 physically combines with RARα in the HNSCC cells. (A) Expression of RARα in the Fadu, Cal27, and NHOK cells detected by RT-qPCR (left) and western blot (middle and right). (B) Relative expression of RARα in normal and HNSCC tissues in the TCGA database. (C) Western blot verification of the established Cal27-v25 and Fadu-v25 cells. (D) Physical interaction between TACC1v25 and RARα in the Cal27-v25 and Fadu-v25 cells by Co-IP. The cell lysates were subjected to IP with an anti-Flag or an anti-IgG antibody, followed by western blot with an anti-Flag or an anti-RARα antibody. (E) Physical interaction between TACC1v25 and RARα in the Cal27-v25 and Fadu-v25 cells by Co-IP. The cell lysates were subjected to IP with an anti-RARα or an anti-IgG antibody, followed by western blot with an anti-RARα or an anti-Flag antibody. *, P<0.05. −, negative; +, positive. ATRA, all-trans-retinoic acid; Co-IP, co-immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HNSCC, head and neck squamous cell carcinoma; IgG, immunoglobulin G; IP, immunoprecipitation; mRNA, messenger RNA; NC, negative control; NHOK, normal human oral keratinocytes; RARα, retinoic acid receptor alpha; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; TACC1v25, transforming acidic coiled-coil containing protein 1; TCGA, The Cancer Genome Atlas; v25, variant 25.

Full-length TACC1 physically interacts with RARα in the nucleus (8). To investigate whether TACC1v25 exhibits similar binding properties with RARα, we established the stable Cal27-v25 and Fadu-v25 cell lines (Figure 1C). Our Co-IP results demonstrated that TACC1v25 directly bound to RARα in the Cal27-v25 cells. Similarly, this interaction was observed in the Fadu-v25 cells (Figure 1D,1E). After ATRA treatment, the coprecipitation products of TACC1v25 and RARα exhibited a decreasing trend in the Cal27-v25 cells. However, no similar results were observed in the Fadu-v25 cells (Figure 1D,1E and Figure S1A,S1B).

Subcellular localization of TACC1v25 and RARα in HNSCC cells

Previously, TACC1v25 was found in both the nucleus and cytoplasm (12). As an essential nuclear receptor, RARα shuttles between the nucleus and cytoplasm (24). Therefore, we performed immunofluorescence to determine the subcellular distribution pattern of TACC1v25 and RARα in the Cal27-v25 (Figure 2A) and Fadu-v25 (Figure 2B) cells with or without ATRA treatment. Both proteins were observed in the nucleus and cytoplasm of ATRA-untreated Cal27-v25 cells. Some TACC1v25 and RARα proteins were co-localized (Figure 2A). Similarly, in ATRA-untreated Fadu-v25 cells, TACC1v25 and RARα were also detected in both the nucleus and cytoplasm, with some overlapping regions indicating co-expression (Figure 2B). After ATRA treatment, TACC1v25 increased in the cytoplasm, whereas RARα predominantly translocated to the nucleus (Figure 2A,2B and Figure S2). Our results showed that there was no significant change in the expression of TACC1v25 and RARα after ATRA treatment (Figure S3). Western blot analysis confirmed that TACC1v25 was primarily found in the cytoplasm, while RARα was predominantly located in the nucleus (Figure 2C,2D). After ATRA treatment, the TACC1v25 levels increased both in the cytoplasm and nucleus of Cal27-v25 (Figure 2C) and Fadu-v25 (Figure 2D) cells, while the RARα levels increased in the nucleus of Cal27-v25 (Figure 2C) and Fadu-v25 (Figure 2D) cells, and decreased in the cytoplasm of Fadu-v25 (Figure 2D) cells.

Figure 2 Subcellular localization of TACC1v25 and RARα in the Cal27 and Fadu cells. (A) Immunofluorescence staining of Cal27-v25 cells showing the localization of TACC1v25 (red) and RARα (green) in the nucleus and cytoplasm. DAPI (blue) was used to stain the nuclei. Scale bar, 50 µm. (B) Immunofluorescence staining of Fadu-v25 cells showing the localization of TACC1v25 (red) and RARα (green) in the nucleus and cytoplasm. DAPI (blue) was used to stain the nuclei. Scale bar, 50 µm. (C) Western blot analysis of cytoplasmic and nuclear fractions of Cal27-v25 cells, showing the distribution of TACC1v25 (anti-Flag) and RARα before and after ATRA treatment. GAPDH and Lamin A were used as markers for cytoplasmic and nuclear fractions, respectively. (D) Western blot analysis of cytoplasmic and nuclear fractions of Fadu-v25 cells, showing the distribution of TACC1v25 (anti-Flag) and RARα before and after ATRA treatment. GAPDH and Lamin A were used as markers for cytoplasmic and nuclear fractions, respectively. *, P<0.05; **, P<0.01; ***, P<0.001. −, negative; +, positive. ATRA, all-trans-retinoic acid; Cyto, cytoplasm; DAPI, 4',6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; Nuc, nucleus; RARα, retinoic acid receptor alpha; TACC1v25, transforming acidic coiled-coil containing protein 1; v25, variant 25.

Ectopic overexpression of TACC1v25 induces HNSCC cell differentiation

The TACC3 protein controls cell differentiation by interacting with other transcriptional regulators (25). The expression of K14 and K13, which are tightly linked to epithelial differentiation (26), was examined in TACC1v25-overexpressing HNSCC cells. There was a significant upregulation of K14 in both the Cal27-v25 and Fadu-v25 cells, and a significant increase of K13 expression in the Fadu-v25 cells. A similar trend of increased K13 expression was observed in the Cal27-v25 cells, although this increase was not statistically significant (Figure 3A). We noted that the addition of ATRA significantly counteracted the pro-differentiation effect of TACC1v25 in the Cal27-v25 cells, as evidenced by a decrease in K14 expression levels, but this effect was not observed in the Fadu-v25 cells (Figure 3A).

Figure 3 TACC1v25 promotes differentiation in HNSCC cells. (A) Western blot analysis of K14 and K13 expressions in the Cal27 and Fadu cells. (B) Western blot analysis of p63/p53/p21 in the Cal27 and Fadu cells. *, P<0.05; **, P<0.01; ***, P<0.001. −, negative; +, positive. ATRA, all-trans-retinoic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HNSCC, head and neck squamous cell carcinoma; NC, negative control; TACC1v25, transforming acidic coiled-coil containing protein 1; v25, variant 25.

The activation of the p53/p21 pathway is known to contribute to cell differentiation (27). p63, a homolog of p53, plays an essential role in cell differentiation (28). Consistent with K14 and K13 expression, the p63, p-p53, and p-p21 expression was increased in both the Cal27-v25 and Fadu-v25 cells as compared with those in the Cal27-NC/Fadu-NC cells. Upon ATRA treatment, the p63 and p-p53 expression was significantly decreased in the Cal27-v25 cells. In contrast, no significant changes in p63 and p-p53 expressions were observed in Fadu-v25 cells following ATRA treatment (Figure 3B).

Ectopic overexpression of TACC1v25 inhibits invasion and migration of HNSCC cells

A previous study has demonstrated that TACC1v25 inhibits cell proliferation in HNSCC (12). We further evaluated the effect of TACC1v25 on the invasion and migration of HNSCC cells. Our results showed that the invasion (Figure 4A,4B) and migration (Figure 4C,4D) abilities were significantly reduced in the Cal27-v25 and Fadu-v25 cells as compared with the Cal27-NC and Fadu-NC cells. Intriguingly, the addition of ATRA partially counteracted the anti-invasive effect of TACC1v25 (Figure 4A-4D).

Figure 4 TACC1v25 inhibits invasion and migration in the HNSCC cells. (A) Effect of TACC1v25 with or without ATRA on invasion assessed in the Cal27-v25 cells by the transwell invasion assay (staining with crystal violet). (B) Effect of TACC1v25 with or without ATRA on invasion assessed in the Fadu-v25 cells by the transwell invasion assay (staining with crystal violet). (C) Effect of TACC1v25 with or without ATRA on migration in the Cal27-v25 cells assessed by the transwell migration assay (staining with crystal violet). (D) Effect of TACC1v25 with or without ATRA on migration in the Fadu-v25 cells assessed by the transwell migration assay (staining with crystal violet). Scale bar, 100 µm. (E) Western blot analysis of vimentin expression in the Cal27 and Fadu cells. (F) Western blot analysis of PI3K and AKT expressions in the Cal27 and Fadu cells. (G) Representative photograph of in vivo analysis by implantation of Cal27-NC and Cal27-v25 cells in the tongue (n=6). Left, (upper) tongue and (lower) lung; right, H&E staining of the tongue and lung in the Cal27-NC and Cal27-v25 groups. Scale bars, 50 µm. *, P<0.05; **, P<0.01; ***, P<0.001. −, negative; +, positive. ATRA, all-trans-retinoic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H&E, hematoxylin and eosin; HNSCC, head and neck squamous cell carcinoma; NC, negative control; TACC1v25, transforming acidic coiled-coil containing protein 1; v25, variant 25.

Vimentin, a protein implicated in the metastatic spread of cancer (29), is a downstream target of AKT1 (30). We found that the protein levels of vimentin were decreased in the Cal27-v25 and Fadu-v25 cells. After RARα activation by ATRA, vimentin expression in the Cal27-v25 and Fadu-v25 cells remained lower than that in Cal27-NC and Fadu-NC cells (Figure 4E). Similarly, the p-AKT protein levels were significantly downregulated in the Cal27-v25 and Fadu-v25 cells as compared with those in the Cal27-NC and Fadu-NC cells. However, ATRA treatment countered the inhibitory effect of TACC1v25 on p-AKT (Figure 4F).

Next, we determined whether TACC1v25 reduced the metastatic abilities of HNSCC cells in vivo. No metastasis was observed in any of the groups by the mouse-tail vein injection model after 2 months (Figure S4). In the orthotopic xenograft model, the tumor rapidly spread throughout the tongue, causing eating difficulties, severe weight loss, and death. There were no signs of metastasis in any group (Figure 4G) up to the day of death. The median survival times were 12 days in the Cal27-v25 group and 13 days in the Cal27-NC group.

Verification of the effects of TACC1v25 on the differentiation in HNSCC by RNA-seq

To verify the effect of TACC1v25 in HNSCC, we performed RNA-seq profiling in the Cal27-v25, Fadu-v25, and their respective control cells, with or without ATRA. The DEG profiles were significantly different between the Cal27-v25, Fadu-v25, and the control cells (Figure 5A,5B). We observed that DEGs involved in cornification were notably enriched in the Cal27-v25 cells, as compared with those in the Cal27-NC cells, with and without ATRA treatment (Figure 5C,5D). However, this enrichment was not observed between the Fadu-v25 and Fadu-NC cells (Figure 5E,5F). Unlike Cal27-v25 cells, which showed no significant changes upon ATRA treatment (Figure 5G), ATRA-treated Fadu-v25 cells showed enrichment for the biological process of “cornification” in their DEGs relative to untreated controls (Figure 5H).

Figure 5 Bio-informative analysis of the RNA-seq profile on TACC1v25-overexpressing HNSCC lines. (A,B) Heat maps analyzing DEGs between the Cal27-v25 and Cal27-NC cells (A), and between the Fadu-v25 and Fadu-NC cells (B) with or without ATRA. (C-H) GO function enrichment analysis of DEGs and networks of the enriched GO terms between the Cal27-v25 and Cal27-NC cells (C), between the ATRA-treated Cal27-v25 and Cal27-NC cells (D), between the Fadu-v25 and Fadu-NC cells (E), between the ATRA-treated Fadu-v25 and Fadu-NC cells (F), between the ATRA-treated and untreated Cal27-v25 cells (G), as well as between the ATRA-treated and untreated Fadu-v25 cells (H). Cornification was involved in DEGs between the Cal27-v25 and Cal27-NC cells (C), between the ATRA-treated Cal27-v25 and Cal27-NC cells (D), and between the ATRA-treated and untreated Fadu-v25 cells (H). ATRA, all-trans-retinoic acid; DEG, differentially expressed gene; GO, Gene Ontology; HNSCC, head and neck squamous cell carcinoma; NC, negative control; RNA-seq, RNA sequencing; TACC1v25, transforming acidic coiled-coil containing protein 1; v25, variant 25.

To gain deeper insights into the effects of TACC1v25 on cornification-related gene expression, we conducted a cluster analysis of the cornification-related sequencing data. KRT13 and KRT14 expression was upregulated in the Cal27-v25 cells as compared with those in the Cal27-NC cells with or without ATRA (Figure 6A), which is consistent with our previous western blot results (Figure 2). Additionally, the expressions of several other keratins, including KRT16, KRT17, KRT14, and KRT5, were downregulated following ATRA treatment in TACC1v25-overexpressing cell lines as compared with the control cells (Figure 6A,6B). To determine the interactions among the cornification-related DEGs, we constructed regulatory networks. Our network analysis indicated that upregulated DEGs, such as KRT1, KRT13, and KRT6B, displayed close interactions with each other in the Cal27-v25 cells (Figure 6C). In addition, the PI3, known to be related to keratinization (31), was found to interact with multiple DEGs in both the Cal27-v25 and Fadu-v25 cells, suggesting a potential role for PI3 in mediating keratinization-related pathways in TACC1v25-overexpressing HNSCC cells (Figure 6C,6D).

Figure 6 Cornification-related DEGs analyzed by heat maps and protein-protein interaction network. (A,B) Heat maps of 118 cornification-related DEGs between the Cal27-v25 and Cal27-NC (A) and Fadu-v25 and Fadu-NC (B) cells with or without ATRA. The red and blue colors represent the upregulated and downregulated genes, respectively. (C,D) The protein-protein interaction networks showing the interactions of the upregulated DEGs in the Cal27-v25 (C) and Fadu-v25 (D) cells as compared with those in the controls. KRT1, KRT13, and KRT6B expressions were upregulated in the Cal27-v25 cells (C). ATRA, all-trans-retinoic acid; DEG, differentially expressed gene; NC, negative control; v25, variant 25.

Discussion

The human TACC1 gene generates several transcript variants through alternative splicing, encoding different isoforms (10). Among them, TACC1v25 is downregulated in HNSCC, and its ectopic expression could inhibit proliferation and promote autophagy in HNSCC (12). In this study, we demonstrated that TACC1v25 physically interacted with RARα. When TACC1v25 was overexpressed, it combined with RARα and suppressed the transcriptional activation of RARα, promoting differentiation and inhibiting invasion and migration in HNSCC cells. Upon ATRA treatment, RARα became activated and underwent cytoplasm-to-nucleus translocation, counteracting the effects of TACC1v25. Furthermore, we observed that TACC1v25 inhibited the activation of AKT, subsequently inhibiting RARα transactivation (32). Activated RARα might counteract TACC1v25-induced differentiation via the p63 and p53/p21 pathways while promoting invasion and metastasis by upregulating vimentin and p-AKT.

Alternative splicing plays a critical role in tumorigenesis by generating isoforms with distinct functions. For example, collapsin response mediator protein-1 suppresses tumor metastasis, while its long isoform promotes metastasis in non-small cell lung cancer (6). Similarly, full-length TACC1 has been implicated as an oncogene in tumorigenesis (5), whereas TACC1v25 may function as a tumor suppressor (12). According to GenBank, TACC1v25 lacks exon 1 of full-length TACC1 but retains nuclear localization signals in exons 3 and 4, suggesting that its nuclear function remains intact (7).

TACC1 is mainly localized in the cytoplasm, particularly around the nucleus (7), with its subcellular distribution varying based on cell differentiation status (33). RARα predominantly resides in the nucleus and translocates upon ATRA binding, activating transcription (24). Full-length TACC1 participates in RARα-mediated gene transactivation and modulates nuclear receptor localization (8). In the absence of retinoic acid, full-length TACC1 forms a complex with GCN5 and RARα, repressing transcription. ATRA binding induces TACC1 acetylation via GCN5, leading to TACC1 dissociation and transcriptional activation, which is essential for diencephalon development (34). Moreover, TACC1 loss leads to decreased RARα ligand-dependent transcriptional activity and its nuclear translocation (8). Our study indicated that TACC1v25 interacted with RARα, and ATRA treatment facilitated RARα nuclear accumulation, reducing the nuclear TACC1v25/RARα ratio and diminishing TACC1v25’s repressive role on RARα transcription (34). Consequently, the pro-differentiation and anti-invasion effects of TACC1v25 in HNSCC were partially counteracted by RARα activation.

Differentiation markers, including keratins, play a crucial role in epithelial integrity. K14 expression is concentrated in the basal layer of the normal buccal mucosa. As previously reported, reduced expression levels of differentiation-linked keratins, including K1, K4, and K13, and a tendency for downregulation of K5 and K14 have been found in SCC (26). In cases of oral leukoplakia with mild/severe dysplasia and SCC, a sequential decrease in K14 expression levels was observed (35). K13, as a marker of epithelial stratification and differentiation, is typically detectable in the suprabasal cell layers in the normal oral mucosal epithelium, but it is lost in the HNSCC cells (36). We observed that TACC1v25 upregulated the expression of K14 and K13 in HNSCC cells, suggesting a potential role in promoting differentiation. However, retinoic acid treatment reduced K14 expression in SCC-13 cells (37), and TACC1v25-induced differentiation was partially counteracted after ATRA treatment in HNSCC cells. These findings suggest that ATRA partially counteracts TACC1v25-induced differentiation, highlighting the interplay between TACC1v25 and RARα.

The p53/p21 pathway is active during ependymal differentiation, with p53 and p21 loss inhibiting this process (27). TP53 expression is tightly correlated with K14 expression (38), while p63, a p53 homolog essential for epithelial maintenance, is required for K14 expression (28,39). Furthermore, K13 expression is downregulated in p53-null papillomas (40). During the differentiation of human embryonic stem cells into vocal fold epithelial-like cells, the expression of both p63 and K13 is induced (41). Our results showed that TACC1v25 overexpression upregulated K14, K13, p-p53, p-p21, and p63, suggesting a role in differentiation via the p63 and p53/p21 pathways (38-41). However, ATRA treatment reduced K14, p-p53, and p63 expression in certain HNSCC cells, consistent with RARα-mediated p53 suppression in lung cancer cells (32).

Vimentin, a key epithelial-mesenchymal transition marker, is upregulated in HNSCC (42), promoting invasion and migration (29). Its expression is regulated by AKT (30,43). Our data showed that TACC1v25 overexpression downregulated vimentin and p-AKT in Cal27-v25/Fadu-v25 cells, suggesting that TACC1v25 may inhibit invasion and migration through the PI3K/AKT pathway. Conversely, ATRA promotes vimentin expression in mesenchymal stem cells (44), enhancing invasion and survival in lung cancer cells (32). In our study, ATRA treatment increased p-AKT levels in all cells and partially counteracting TACC1v25’s anti-invasion effects. However, vimentin upregulation was not observed in TACC1v25-overexpressing cells post-ATRA treatment, indicating that ATRA’s effects may not primarily rely on vimentin regulation.

The Cal27 and Fadu cell lines used in this study showed differences, particularly in the differentiation impact of ATRA treatment. In Cal27-v25 cells, ATRA significantly reduced p63, p-p53, and K14 expression, whereas Fadu-v25 cells showed no significant changes (Figure 3). The Fadu cells contain a 248 mutation (G → T) in p53, with p53 expression at half the level of normal mucosal cells, while Cal27 cells possess a 193 mutation (A → T) in p53 (45). ATRA also promotes AKT activation by the interaction between RARα and AKT, thereby inhibiting the expression of p53 (32). These discrepancies may arise from differences in the molecular characteristics of the cell lines (46). Furthermore, Cal27 and Fadu cells originate from different sites (tongue vs. pharynx), and HNSCCs from distinct anatomical locations exhibit unique gene expression patterns, supporting the notion of inter-/intratumor heterogeneity (47).

One limitation of this study is the inability to fully validate TACC1v25’s inhibitory effects on invasion and migration in vivo due to rapid tumor growth and local health constraints in mice. In addition, future studies should further analyze RNA-seq data to uncover additional regulatory pathways and compare DEGs between the Cal27-v25/Fadu-v25 and Cal27-NC/Fadu-NC cells to identify shared regulators, providing deeper insights into TACC1v25’s role in HNSCC differentiation.

HNSCC remained a significant clinical challenge due to poor tumor differentiation, therapy resistance, and high invasiveness, which contributed to unfavorable patient prognosis (48). Our previous and current studies demonstrated that TACC1v25 promoted differentiation and autophagy in HNSCC cells while inhibiting their proliferation and invasion (12). Modulating the expression or activity of TACC1v25 showed promise for improving tumor differentiation status and reducing invasiveness, thereby enhancing the efficacy of existing treatment modalities. However, the clinical application of TACC1v25 as a target for therapy requires further validation using in vivo models and clinical samples to establish its safety and effectiveness. Future researches should focus on elucidating the regulatory mechanisms of TACC1v25 to translate these findings into clinical practice.

Conclusions

In summary, our data revealed the physical interaction between TACC1v25 and RARα. TACC1v25 promoted differentiation through the p63 and p53/p21 signaling pathways while inhibiting cell invasion and migration through the PI3K/AKT pathway. However, the activation of RARα by ATRA partially counteracted the effects of TACC1v25, suggesting a complex regulatory mechanism that warrants further investigation.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-685/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-685/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-685/prf

Funding: This work was supported by the National Natural Science Foundation of China (No. 81671036).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-685/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine (No. SH9H-2019-T112-1) and informed consent was obtained from all individual participants. All animal experiments were performed under a project license (No. SH9H-2020-A430-1) granted by the Ethics Committee of Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, in compliance with the Guideline for the Care and Use of Laboratory Animals and the institutional laboratory animal welfare guideline.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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