The Clinical Significance of SPOP Upregulation and Nuclear Accumulation in Head and Neck Squamous Cell Carcinoma

Abstract

The speckle-type BTB/POZ protein (SPOP) is an E3 ubiquitin ligase adaptor typically considered a tumor suppressor, yet its role in head and neck squamous cell carcinoma (HNSCC) remains unclear. This study investigated SPOP expression, arecoline regulation, and its potential as a HNSCC biomarker. SPOP mRNA and its protein were quantified in HNSCC (FaDu, GMN, HSC-3, SAS, and A253) and normal oral epithelial (SG) cell lines via RT-qPCR and Western blot; arecoline’s effect on SG, SAS, and A253 cells was evaluated. SPOP mRNA was analyzed using The Cancer Genome Atlas (TCGA) HNSCC cohort, and protein localization was assessed via immunohistochemistry (IHC) on tissue microarrays. SPOP mRNA was higher in some HNSCC lines; arecoline induced SPOP in SG cells, but not in HNSCC cell lines. TCGA confirmed SPOP mRNA upregulation in tumors correlating with grade. IHC showed SPOP upregulation in HNSCC, particularly in palate and pharynx/hypopharynx sites. The nuclear SPOP-positive ratio shifted from 12.14 ± 9.82% in normal tissues to 61.26 ± 33.03% in tumors (p < 0.0001), differentiating grades and sites better than total expression. SPOP is upregulated in HNSCC and inducible by arecoline. Enhanced nuclear SPOP localization indicates malignancy and progression, identifying it as a potential HNSCC diagnostic and progression biomarker.

1. Introduction

Head and neck squamous cell carcinoma (HNSCC) is a major global health challenge, ranking among the most prevalent cancers worldwide. According to GLOBOCAN estimates, the annual incidence of HNSCC is approximately 940,000 cases, resulting in over 480,000 deaths [1]. High morbidity and mortality rates characterize this malignancy [2], largely driven by primary risk factors such as tobacco use, alcohol consumption, and betel quid chewing—a practice particularly prevalent in Asian regions [3]. Despite advancements in multidisciplinary treatment approaches, the prognosis for advanced-stage HNSCC remains poor. This underscores the urgent necessity for identifying reliable diagnostic biomarkers and understanding the molecular drivers of this disease.

The speckle-type BTB/POZ protein (SPOP) functions as the substrate-recognition adaptor within the CULLIN3/RING-box1 E3 ubiquitin ligase complex [4]. By selectively targeting specific protein substrates for polyubiquitination and subsequent degradation by the 26S proteasome, SPOP regulates a wide array of biological processes, including cell cycle progression, DNA damage response, and apoptosis [5,6,7]. While SPOP is generally classified as a tumor suppressor due to its role in clearing pro-oncogenic proteins, its functional outcome is highly context-dependent, dictated by its expression levels, subcellular localization, and mutation status.

It is best known for loss-of-function mutations in prostate and endometrial cancers, where mutated SPOP fails to degrade key oncoproteins such as the androgen receptor and BRD4 (bromodomain and extraterminal (BET) family member), thereby driving tumorigenesis [8,9,10]. Conversely, SPOP is overexpressed and acts oncogenically in lung, kidney, and breast cancers, reflecting its functional pleiotropy [11]. The mechanisms underlying SPOP’s dual role remain poorly understood, and characterizing how its activity is regulated through transcription and protein stability is essential for identifying the substrate dysregulations that drive cancer.

Characterizing how SPOP activity is regulated through transcription, protein stability, and post-translational modifications is essential to identify the substrate dysregulations driving tumorigenesis. Despite its potential importance, research investigating SPOP expression and its clinical relevance in HNSCC remains scarce and contradictory [12]. Establishing the native expression pattern and subcellular distribution of SPOP is a prerequisite for discerning its true function in oral oncogenesis. Furthermore, the regulatory response of SPOP to environmental carcinogens common in HNSCC, such as arecoline, has not been explored.

In the current study, we systematically investigated SPOP expression at both the mRNA and protein levels in HNSCC cell lines and clinical tumor tissues. Our primary objective was to determine the expression patterns, subcellular localization, and response to arecoline exposure, thereby evaluating the potential of SPOP—particularly its nuclear accumulation—as a diagnostic and progression biomarker for HNSCC.

2. Results

2.1. SPOP Expression in HNSCC Cell Lines and Its Modulation by Arecoline

To evaluate SPOP expression in oral cancer, we first quantified SPOP mRNA levels in a normal oral epithelial cell line (SG) and five HNSCC cell lines (FaDu, GNM, HSC3, SAS, and A253). SPOP mRNA was significantly upregulated in FaDu, GNM, and HSC3 cells compared to the SG control, whereas levels in SAS and A253 cells remained comparable to or lower than those in SG cells (Figure 1A). We further investigated whether arecoline, a primary betel quid carcinogen, modulates SPOP expression in SG cells and the HNSCC cell lines with low baseline SPOP mRNA (SAS and A253). Arecoline treatment induced a dose-dependent increase in SPOP levels in SG cells, but not in SAS or A253 cells (Figure 1B,D). Interestingly, baseline quantification revealed that SPOP levels were inherently higher in SAS and A253 cells than in SG cells (Figure 1C), indicating a discrepancy between SPOP mRNA and protein expression in these two cancer lines. Furthermore, while arecoline significantly induced SPOP in normal epithelial cells, it failed to elicit a similar response in SAS and A253 cells. These findings suggest that SPOP regulatory mechanisms may be dysregulated or constitutive in established cancer cells, rendering them less sensitive to further carcinogen-induced modulation.

2.2. SPOP mRNA Is Upregulated in Tumor Tissues and Associates with Site and Grade in the TCGA HNSCC Cohort

Analysis of The Cancer Genome Atlas (TCGA) database confirmed that SPOP transcriptional levels are significantly elevated in HNSCC tumor tissues compared to non-malignant tissues (Figure 2A). This upregulation was more pronounced in male patients compared to female patients (Figure 2B). While SPOP mRNA expression positively correlated with tumor histologic grade (Figure 2C), Kaplan–Meier survival analysis indicated that SPOP expression levels were not significantly associated with the overall survival of HNSCC patients (p = 0.6737, Figure 2D).

2.3. SPOP Expression Is Upregulated in HNSCC Clinical Tissues

The regulation of gene transcription and translation is highly complex. Messenger RNA (mRNA) expression can be profoundly affected by factors such as degradation rates, translational repression, and subsequent protein degradation pathways. Therefore, it is critical to clarify whether changes observed in SPOP mRNA are consistent with alterations in SPOP expression in HNSCC. To validate the bioinformatic findings, we performed immunohistochemical (IHC) staining on two tissue arrays (OR208a and ORC1021) comprising 150 tumor and 20 normal samples (

Table 1. Summarizes the clinical and demographic information of two tissue arrays (OR208a and ORC1021).

		Normal	Tumor
Sample size		20	150
Age		41.40 ± 15.41	55.32 ± 11.63
Gender	male	13	115
	female	7	35
Site	tongue	19	75
	gingiva and gum	1	24
	hypopharynx and pharynx		18
	palate		12
	oral cavity and buccal		6
	lower jaw and lower mandible		4
	floor		4
	maxillary sinus		3
	lip		2
	others		2
Grade	NA		13
	I		77
	II		38
	III		22
	IV		0
Stage	NA		91 *
	I		5
	II		38
	III		15
	IVA		1

NA refers to the number of tissues for lacking clinical-related information. * Tissue array ORC1021 did not provide the stage information.

). Table 1 summarizes the clinical and demographic information provided by the manufacturer for two tissue arrays, including age, sex, tumor location, grade, and stage. The cohort exhibited a higher prevalence of male samples, with the top three tumor locations being the tongue, gingiva, and hypopharynx/pharynx. Grade 1 was the most common differentiation grade; however, stage information was unavailable for the ORC1021 array.

IHC staining revealed SPOP positive cells, indicated by a red chromogen, localized within both the cytoplasm and the nucleus of tumor cells. The SPOP score for each tissue sample was determined by multiplying the staining intensity score by the percentage score of positive cells. For example, on the tissue array ORC1021, representative cores at positions 2G, 2E, 8D, and 11E exhibited intensity scores multiplied by proportion scores of 0 × 0, 1 × 2, 2 × 3, and 3 × 4, respectively (Figure 3A). This corresponded to SPOP scores of 2G = 0, 2E = 2, 8D = 6, and 11E = 12. The total SPOP score was significantly higher in HNSCC tissues than in normal tissues (Figure 3B), with higher scores observed in male patients (Figure 3C). Anatomical site analysis revealed a consistent trend of SPOP upregulation across all sites, reaching statistical significance in the palate and hypopharynx/pharynx (Figure 3D). Analysis of the tumor differentiation grade revealed no clear trend in SPOP expression corresponding to tumor grade changes (Figure 3E). Furthermore, only the tissue array OR208 provided tumor stage records, limiting the tumor tissue sample size to 59 (with one tumor sample missing from the array upon purchase). Total SPOP scores showed no significant correlation with the clinical stage (Figure 3F).

2.4. Nuclear Localization of SPOP Is Significantly Enhanced in HNSCC Tumor Tissues

While SPOP expression was observed in both the cytoplasm and the nucleus, its subcellular distribution exhibited a significant shift in malignant tissues. To quantify this, we utilized ImageJ software to calculate the nuclear SPOP-positive ratio on the OR208 tissue array. Representative tissue cores at positions 2A, 2G, 6F, and 11D showed nuclear SPOP positive ratios of 2.5%, 5%, 53%, and 100%, respectively (Figure 4A). The quantitative analysis demonstrated that the nuclear SPOP positive ratio was significantly enhanced in HNSCC tumor tissues (T, 61.26 ± 33.03%) compared to normal tissues (N, 12.14 ± 9.819%) (Figure 4B, p < 0.0001). Furthermore, the enhanced nuclear SPOP ratio effectively differentiated normal tissues from various tumor anatomical sites (Figure 4C) and histological differentiation grades (Figure 4D). These results suggest that the subcellular localization of SPOP—specifically its nuclear accumulation—is a more robust indicator of the transition from normal epithelium to malignancy and a more reliable biomarker for tumor progression than total protein levels alone.

3. Discussion

While SPOP is conventionally recognized as a tumor suppressor gene frequently inactivated by mutations in malignancies such as prostate and endometrial carcinomas [8,9,10], our multi-level investigation reveals a significant departure from this established role in HNSCC. By integrating bioinformatics, cell line experiments, and clinical tissue analysis, we found that SPOP is significantly upregulated in HNSCC tumor tissues (Figure 3B) and exhibits a striking enhancement in nuclear localization (Figure 4B). Furthermore, the direct induction of SPOP by the environmental carcinogen arecoline in normal oral epithelial cells (Figure 1B,D) suggests a context-specific, complex, and potentially involvement in HNSCC pathogenesis, challenging its universal classification as a tumor suppressor. However, the results do not provide direct functional evidence to definitively classify SPOP as a pro-oncogene or tumor suppressor in the context of HNSCC.

The demonstration that arecoline induces SPOP expression in normal SG epithelial cells (Figure 1B,C) establishes a novel regulatory link. Arecoline is known to induce oxidative stress and DNA damage and activated ataxia telangiectasia mutated (ATM) expression [13,14]. SPOP deficiency has been linked to the accumulation of γ-H2AX in the nucleus [15], while its interaction with ATM facilitates DNA repair following ionizing radiation [16]. Moreover, SPOP modulates the expression of essential DNA repair genes, including ATR, BRCA2, Chk1, and Rad51 [17]. Consequently, the arecoline-induced SPOP expression observed here may initially represent a cellular defense response to genotoxic stress aimed at clearing damaged proteins [17]. However, if this induced SPOP becomes functionally impaired or interferes with homeostatic signal transduction, persistent upregulation may contribute to oncogenesis, providing a mechanism by which chronic carcinogen exposure modulates SPOP levels in a pro-tumorigenic manner. SPOP dysregulation may be a key event in early carcinogen-induced transformation that becomes constitutive or ‘out of control’ once malignancy is established. Further investigations using arecoline-induced animal models and patient cohorts with documented betel nut usage are warranted to definitively link environmental carcinogen exposure to SPOP nuclear translocation in vivo.

Our TCGA analysis confirmed SPOP mRNA upregulation in HNSCC tumors (Figure 2A), aligning with the overall increased SPOP observed in our clinical cohort (Figure 3B). This consistent upregulation—rather than the expected downregulation or mutation-driven loss—is highly characteristic of an oncogene or a gene acquiring a new function. A similar oncogenic shift, similar to that observed in renal cell carcinoma where SPOP overexpression promotes tumorigenesis [7,18]. The site-specific upregulation in the palate and pharynx (Figure 3D) further underscores that SPOP function is highly dependent on the anatomical or etiological context, likely reflecting unique transcriptional or post-translational controls in these HNSCC subtypes. While arecoline-mediated SPOP induction is most contextually relevant to the oral cavity, we maintained a broader HNSCC study population to ensure sufficient statistical power for grade and stage stratification and to remain consistent with the inclusion criteria of the TCGA HNSCC cohort. This integrated approach allows for a direct comparison between bioinformatic data and clinical validation on a unified baseline, while acknowledging that subsite-specific differences remain an important factor in head and neck oncogenesis.

A pivotal finding of this study is the significant nuclear enrichment of SPOP in HNSCC (Figure 4B). SPOP exerts its biological functions within the nucleus as an E3 ubiquitin ligase adaptor. The substantial rise in the nuclear SPOP-positive ratio—from 12% in normal tissues to 61% in HNSCC—suggests that HNSCC progression involves mechanisms that either facilitate SPOP nuclear translocation or enhance its protein stability within the nucleus. Previous research indicated that miR-373 is overexpressed in Chinese patients with oral squamous cell carcinoma (OSCC), where it inhibits SPOP without affecting SPOP mRNA levels [12]. This divergence from our results may stem from ethnic differences or varying risk factors between Asian and Western populations. As our samples in this study may not specifically represent Asian cohorts, future investigations should be expanded to include diverse Asian HNCSS specimens.

Mechanistically, SPOP can bind to and degrade Nup153 (a nuclear pore complex component), potentially altering nucleocytoplasmic transport [19]. SPOP mutations also affect nuclear integrity; for instance, point mutations can reduce levels of lamin B2 (LMNB2), leading to enlarged nuclear size [20,21]. SPOP affects nuclear DNA repair by interacting with ATM and modulating the expression of key repair genes to maintain genomic stability [15,16,17]. The interaction between SPOP and death-associated protein 6 (DAXX) forms specialized nuclear bodies that regulate critical cellular pathways. Furthermore, the SPOP/DAXX axis forms specialized nuclear bodies that regulate critical pathways, including apoptosis and the expression of vascular endothelial cell growth factor receptor 2 (VEGFR2) [22]. Thus, SPOP influence extends to nuclear morphology, membrane function, and DNA repair. This enhanced nuclear SPOP availability may drive HNSCC through two potential pathways: (1) the aberrant degradation of novel tumor-suppressor substrates unique to HNSCC, or (2) the accelerated degradation of existing substrates, promoting survival. The distinct variation in the nuclear SPOP ratio across sites and grades (Figure 4C,D) supports its potential as a more robust biomarker for tumor progression than total SPOP expression. The distinct regulatory patterns observed between normal and malignant cells suggest a complex dysregulation of SPOP stability and translocation; consequently, we have designated SPOP-targeted silencing and downstream mechanistic mapping as the primary focus of our next research phase.

Data from the NIH National Cancer Institute GDC Data Portal identifies 151 mutation sites in SPOP, with research predominantly concentrated on prostate cancer. In prostate cancer, SPOP mutations occur most frequently within the meprin and TRAF homology (MATH) domain. Patients harboring SPOP hotspot mutations (such as Y87C/N/S, F125C/L, W131C, and F133C/L/V/S) or other variants (E50K, S105F, Q120R, R121P, G148E, and A187T) [4]. Mechanistically, nearly all SPOP substrates contain one or more typical or atypical SPOP-binding consensus motifs that interact directly with the MATH domain. For example, W131G and F133V mutations within this substrate-binding domain disrupt the co-localization of SPOP and DAXX upon co-expression [23]. In our findings, SPOP mRNA and protein expression levels were significantly enhanced in HNSCC (Figure 2A and Figure 3B), a trend that parallels observations in prostate cancer. This suggests that SPOP may play comparable roles in the pathogenesis and progression of these two malignancies, potentially involving analogous mutation profiles or shared regulatory mechanisms, such as X-chromosome modulation [24], mutated variant [23], and SPOP-mediated proteolysis [25]. However, these speculations still require further research and verification.

4. Materials and Methods

4.1. Cell Lines and Arecoline Treatment

The study utilized a normal oral epithelial cell line (SG) and HNSCC cell lines (FaDu, GNM, HSC3, SAS, and A253). FaDu, GNM, HSC3, SAS, and A253 cell lines were derived from the hypopharynx, gingiva, tongue (HSC3 and SAS), and salivary gland, respectively. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in a 5% CO₂ atmosphere. To evaluate the regulatory response, cells were treated with 50 and 100 µg/mL arecoline (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Control cells were treated with an equal volume of the vehicle (DMSO). The cell lines were kindly gifted by Professor Chi-Yuan Chen, (Chang Gung Memorial Hospital), and Professor Hsi-Feng Tu, (National Yang Ming Chiao Tung University). Cell culture conditions were as described previously [26].

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

Total RNA was extracted from cell lines using the TRI Reagent^® (Molecular Research Center, Inc., Cincinnati, OH, USA) according to the manufacturer’s instructions. RNA was reverse-transcribed into cDNA using Oligo(dT) 20 primers. RT-qPCR was performed using the StepOne Plus Real-Time PCR System and PowerUp SYBR Green Master Mix (Applied Biosystems, Waltham, MA, USA). SPOP gene expression levels were normalized to glyceralde-hyde 3-phosphate dehydrogenase (GAPDH) using the 2^−△△Ct method. The primer sequences were as follows: SPOP forward: 5′-AGCAAATGATAAACTGAAAT-3′, reverse: 5′-GTCATCAGGGAGAAGCCCGT-3′; GAPDH forward: 5′-TGGTATCGTGGAAGGACTCATGAC-3′, reverse: 5′-ATGCCAGTGAGCTTCCCGTTCAGC-3′.

4.2. Western Blot Analysis

Following arecoline treatment, cells were harvested and lysed to extract the total protein. Western blot analysis was performed as previously described [27,28]. Briefly, 50 μg of protein was separated via 7.5–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. Membranes were blocked with 5% bovine serum albumin (BSA) for 1 h and incubated overnight at 4 °C with an SPOP polyclonal antibody (Cat. No. sc-377206, Santa Cruz Biotechnology, Dallas, TX, USA; 1:1000 dilution). GAPDH (sc-32233, Santa Cruz Biotechnology; 1:10,000 dilution) served as the internal control. Subsequently, membranes were incubated with Peroxidase AffiniPure^® goat anti-mouse IgG (H+L) (Jackson ImmunoResearch, West Grove, PA, USA; 1:30,000 dilution) for 1 h at room temperature. Signals were visualized using SuperSignal West Femto chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA).

4.3. TCGA Database Analysis

Bioinformatic analysis of SPOP mRNA expression in HNSCC was conducted using publicly available data from The Cancer Genome Atlas (TCGA). SPOP mRNA levels were analyzed in relation to clinical parameters, including gender, tumor grade, and overall survival status.

4.4. Tissue Array and Inclusion Criteria

Two HNSCC tissue microarrays (TMAs), OR208a and ORC1021 (TissueArray.Com LLC, Derwood, MD, USA), were used for immunohistochemistry (IHC). The study received ethical approval from the Institutional Review Board in Changhua Christian Hospital (Approval number: 200501).The OR208a array originally contained 60 carcinoma cases (28 from tongue tissue, 15 from gingiva, 5 from oral cavity, 3 from lower jaw, 2 form soft palate, 2 from maxillary sinus, 2 from cavioris bucca, 2 from lip, 1 from palate,) and nine normal tongue tissue; however, positions I10, I11, and I12 were missing upon purchase. The ORC1021 contained 73 cases of oral cavity cancer, 18 cases of pharynx or hypopharynx cancer and 11 cases of normal oral cavity tissues. In total, 20 adjacent normal tissues (N) and 150 HNSCC tumor tissues (T) were evaluated in this study.

4.5. Immunohistochemistry (IHC)

To prevent tissue detachment during antigen retrieval, the HNSCC tissue microarrays (OR208a and ORC1021) were baked at 60 °C for 60 min. The slides were then deparaffinized in xylene, rehydrated through a graded series of ethanol, and subjected to heat-induced antigen retrieval using Tris-EDTA buffer (pH 9.0) at 95 °C for 20 min. Sections were incubated with the SPOP polyclonal antibody (sc-377206; 1:100 dilution) at 4 °C for 16 h. Subsequently, the sections were incubated with a HRP labeling biotinylated goat anti-rabbit secondary antibody (Dako, Glostrup, Denmark) for 1 h at room temperature (RT). Immunoreactivity was visualized using 3,3′-diaminobenzidine (DAB) substrate (Dako) for 3 min at RT, followed by counterstaining with hematoxylin for 2 min. Brown precipitates localized in the cytoplasm, nucleus, or both (co-expression) were identified as positive signals, whereas blue-stained (hematoxylin) structures were considered negative. To ensure staining reliability, internal positive controls were used within each tissue section, with normal oral epithelium serving as a baseline for SPOP expression. Furthermore, external positive controls—adrenal gland pheochromocytoma in L10 position on OR208a tissue array, and skin melanoma in H13 position on ORC1021 tissue array—were employed to validate antibody performance and specificity. For negative controls, the primary antibody was replaced with non-immune serum to confirm the absence of non-specific binding. The cells exhibiting SPOP expression in the cytoplasm, nucleus, or both (co-expression) were all considered “positive” for the calculation of the total SPOP score. The positive intensity was correlating to protein expression levels. Positive cells were quantified as a percentage of the total. SPOP expression was independently evaluated by two experienced pathologists blinded to clinical data. The IHC SPOP score was calculated by multiplying the intensity score (0: none; 1: weak; 2: moderate; 3: strong) by the percentage score (the ratio of positive cells, 0: none; 1: <10%; 2: 10–50%; 3: 51–80%; 4: 81–100%). The resulting SPOP score ranged from 0 to 12. Additionally, recognizing the significance of SPOP translocation in HNSCC, the nuclear-positive ratio was quantified as a distinct parameter to differentiate its clinical relevance from total cellular expression. ImageJ V 1.8.0 software (National Institutes of Health, Bethesda, MD, USA) was used to calculate the nuclear SPOP-positive ratio on the OR208 tissue array.

4.6. Statistical Analysis

Data were analyzed using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Differences between multiple groups were determined using One-way Analysis of Variance (ANOVA). Differences between the variants were considered significant when p < 0.05.

5. Conclusions

In summary, SPOP is upregulated in HNSCC at both the mRNA and protein levels, and its enhanced nuclear localization is a defining feature of HNSCC tumor progression. These data support the utility of SPOP as a diagnostic and potentially prognostic biomarker for HNSCC. Future studies should focus on identifying specific nuclear substrates targeted by SPOP in HNSCC, particularly under arecoline exposure, to fully delineate its mechanism and validate its potential as a therapeutic target.