MTMR6 downregulation contributes to cisplatin resistance in oral squamous cell carcinoma

Abstract

Background

The therapeutic effectiveness of cisplatin, a widely used chemotherapy drug for oral squamous cell carcinoma (OSCC), is often compromised by resistance, making it difficult to predict treatment outcomes. The role of myotubularin and myotubularin-related (MTMR) genes in cisplatin resistance remains unclear. We aimed to elucidate the molecular mechanisms underlying MTMR6 with cisplatin resistance in OSCC.

Methods

MTMR6 expression was compared between UMSCC1 and cisplatin-resistant UM-Cis cells. Gain- and loss-of-function experiments involving MTMR6 was performed to evaluate its impact on cisplatin resistance. The regulatory role of hsa-miR-544a on MTMR6 expression was explored via antagomir and miRNA mimic assays. The relationship between MTMR6 protein levels and cisplatin sensitivity was assessed in OSCC patient tissues classified as sensitive or resistant to cisplatin monotherapy. A survival analysis based on The Cancer Genome Atlas (TCGA) head and neck squamous cell carcinoma (HNSCC) dataset was performed to evaluate the correlation between MTMR6 expression and patient outcomes following cisplatin treatment. In vivo cisplatin resistance was examined using mouse xenografts derived from MTMR6-knockdown UMSCC1 cells.

Results

MTMR6 expression was markedly reduced in cisplatin-resistant UM-Cis cells compared to UMSCC1 cells. Functional analyses revealed that modulating MTMR6 activity alters cisplatin resistance. A luciferase assay confirmed that hsa-miR-544a regulates MTMR6 gene expression. Additionally, antagomir and miRNA mimics demonstrated that hsa-miR-544a enhances cisplatin resistance by suppressing MTMR6 expression. In OSCC patient tissues, higher MTMR6 protein levels were associated with cisplatin sensitivity, while cisplatin-resistant tissues had lower MTMR6 expression. Survival analysis of the TCGA HNSCC dataset indicated that low MTMR6 expression correlates with poorer outcomes in cisplatin-treated patients compared to those with high MTMR6 expression. Mouse xenografts derived from MTMR6-knockdown UMSCC1 cells exhibited increased resistance to cisplatin compared to controls.

Conclusion

Assessing mRNA levels of MTMR6 and has-miR-544a in biopsy samples could help predict cisplatin responsiveness in OSCC.

Background

Despite advancements in the treatment of head and neck squamous cell carcinomas (HNSCCs), including oral squamous cell carcinoma (OSCC), the five-year survival rate has seen only modest improvement [1]. The incidence of OSCC, particularly in older populations, has risen due to increasing population aging. A major challenge in treatment is the development of resistance to chemotherapeutic agents, which can lead to disease recurrence in a more aggressive form and remains a leading cause of cancer-related mortality [2]. Platinum-based compounds, such as cisplatin (cis-diamminedichloroplatinum (II); CDDP) and carboplatin, are commonly used in the treatment of a broad range of solid tumors [3,4,5,6]. Cisplatin, either alone or in combination therapies, is part of first-line treatment regimens for approximately 50% of cancer patients, including those with lung, head and neck, breast, testicular, ovarian, cervical, prostate, and bladder cancers [7, 8]. However, its application is constrained by significant side effects and diminished effectiveness, especially in solid tumors such as OSCC. [6, 9]. Therefore, significant research efforts have been focused on uncovering the molecular mechanisms behind cisplatin resistance.

The myotubularin and myotubularin-related (MTMR) protein subfamily is a large group within the tyrosine dual-specificity phosphatase superfamily in eukaryotes. MTMR6 is involved in several regulatory mechanisms, including vacuolar transport, membrane trafficking [10], and apoptosis in mammalian cells [11, 12]. Notably, decreased MTMR6 expression in chronic lymphocytic leukemia cells is related to resistance to irradiation-induced apoptosis [13]. However, we lack reports on the role of MTMR6 in cancer or its effect on anticancer drug efficacy. In a previous study, we established a cisplatin-resistant OSCC cell line (UM-Cis) from original UMSCC1 cells and performed DNA microarray analysis [14]. We observed that MTMR6 expression was significantly downregulated in UM-Cis cells compared to UMSCC1 cells. However, the mechanism by which MTMR6 affects cisplatin resistance remains unknown. This study sought to identify the role of MTMR6 in cisplatin resistance in OSCC. Prediction of response to cisplatin in OSCC patients would substantially improve chemotherapeutic outcomes and benefit patients.

Methods

Chemicals and reagents

UMSCC1 (mouth floor tumor) cells were obtained from Merck KGaA (Darmstadt, Germany). FaDu (hypopharyngeal tumor) and SCC-15 (tongue tumor) cells were obtained from the American Type Culture Collection (ATCC, MD, USA). YD-8 (tongue tumor) and YD-9 (buccal cheek tumor) cells were purchased from the Korean Cell Line Bank (Seoul, South Korea). YD-8/CIS and YD-9/CIS were kindly provided by professor Jong In Yook (Yonsei University hospital, Department of Oral Pathology). Cell lines were routinely screened for mycoplasma contamination every two months using the CellSafe® Mycoplasma PCR Detection Kit (CellSafe Co., Gyeonggi-do, South Korea). The reagents Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and penicillin–streptomycin were sourced from Invitrogen (Carlsbad, CA, USA). MTT [3-(4, 5-dimethyl-2-thiazolyl)−2, 5-diphenyl-2H-tetrazolium bromide] was obtained from Sigma-Aldrich (St. Louis, MO, USA). Qiazol^™ reagent was purchased from Qiagen (Hilden, CA, USA), and the PCR Master Mix was provided by Takara Bio (Otsu, Japan). Antibodies against MTMR6 and KRT13, developed in rabbits, were supplied by Proteintech (Rosemont, IL, USA), while mouse anti-β-actin (HRP-conjugated) antibodies were sourced from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Additionally, rabbit antibodies for caspase 3 and PARP were procured from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies (mouse, Alexa Fluor 488 conjugated) were acquired from Invitrogen (Carlsbad, CA, USA). Cisplatin was obtained from Sigma-Aldrich. Both the miRNA mimic (MC12414) and antagomir (AM12414) targeting has-miR-544a were purchased from Thermo Fisher Scientific (Waltham, MA, USA), and a mixture of siRNAs, consisting of 2–3 specific oligonucleotides, was sourced from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell cultures

OSCC cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, incubated at 37 °C containing 5% CO₂. For the formation of 3D spheroids, cells were seeded into a 96-well U-bottom ultra-low attachment plate (7007, Corning Inc., NY, USA) at a density of 4000 cells/well. Spheroids were cultured for 2–3 days to achieve uniform sizes across wells, each exceeding 300 μm in diameter. The spheroid sizes were evaluated by measuring the surface area of groups containing 6–8 spheroids using a Cell³iMager scanner CC-5000 (Screen Holdings Co., Kyoto, Japan). At the start of the experiment, the surface area of all spheroids was similar, with a variation margin of less than 5%.

Organoid culture

Organoid cultures were established using mouse xenografts obtained from UMSCC1 or UM-Cis cells, as well as tumor tissues from patients with OSCC. The methodology for tissue processing and organoid culture was adapted from the previous protocols [14,15,16]. Primary tissue samples were rinsed with 45 mL of ice-cold Advanced DMEM/F12 medium, which was supplemented with 1 × GlutaMAX, penicillin–streptomycin, 10 mM HEPES, and 100 μg/mL Primocin. This complete medium is referred to as Advanced DMEM (adDMEM/F12) + / + / + . Tissue samples were minced into small fragments (1–3 mm³), following digestion in TrypLE for less than 1 h. Following centrifugation at 200 × g for 5 min at 4 °C, the resulting pellets were resuspended in 10 mL of adDMEM/F12 + + + medium and filtered through a 100 μm cell strainer. The samples were centrifuged again at 200 ×g for 5 min at 4 °C, and the pellets were resuspended in cold basement membrane extracts. Approximately 10 μL droplets of the suspension were placed on the bottom of culture plates. After the droplets were seeded, the plates were inverted and incubated at 37 °C for 30 min to allow the basement membrane extracts to solidify. Following this, pre-warmed organoid medium was added to the plates. The medium was refreshed every 2–3 days, and organoids were passaged once every 1–2 weeks. Cisplatin effectiveness was monitored over a period of seven days using a Nikon ECLIPSE Ti microscope (Nikon Imaging Japan Inc., Tokyo, Japan).

Cell viability assay following cisplatin treatment

Cisplatin sensitivity was determined at 2D and 3D spheroid or organoid models derived from OSCC cells or primary tissues. Cells were plated in 96-well plates at a density of 1 × 10⁴ cells/well to assess viability under 2D culture conditions. After 24 h, the cells were exposed to cisplatin in fresh media and incubated for another 48 h. Cell viability was determined using an MTT assay, with absorbance recorded at 540 nm using an ELISA reader. The effect of cisplatin on the size of 3D spheroids (assessed by surface area) was also analyzed. After the medium was carefully removed, fresh medium containing cisplatin was added to each well. Spheroid growth was tracked for a period of 14 days. Organoids reached approximately 300 μm in diameter about a month after the culture was initiated. In 24-well plates, experiments were performed with five organoids per well, and their size was measured using phase-contrast microscopy (5 × magnification) after seven days. DMSO (0.1% v/v in PBS) was used as the vehicle control in these experiments.

Analysis of mRNA and protein expression

Reverse Transcription qPCR (RT-qPCR) was utilized to assess mRNA expression levels. RNA was extraction, cDNA was synthesis, and gene expression normalization was carried out according to established protocols. The primers used for the RT-qPCR analysis are detailed in Table S1. For miRNA expression analysis, specific primers and TaqMan probes for miR-544a were employed. Each RT-qPCR reaction was conducted in triplicate for each condition, using an ABI 7600 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Gene expression levels were normalized to GAPDH. The fold change in gene expression was calculated using the delta cycle threshold (ΔCt) method, which involved normalizing the average Ct value of each sample to that of the endogenous GAPDH control and subsequently computing the 2^−ΔΔCt value for each treatment.

For the analysis of protein expression, total protein was extracted. Proteins (20–40 µg) were subjected to 8–10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by transfer to nitrocellulose membranes. After a 60-min blocking step with 5% skim milk, the membranes were incubated at 4 °C for 16 h with the appropriate primary antibodies, using β-actin as a loading control. HRP-conjugated secondary antibodies were applied at a dilution of 1:5000 for 1 h at room temperature, and the membranes were washed three times with Tris-buffered saline containing 0.1% Tween 20. Protein bands were visualized using enhanced chemiluminescence.

Transfection of siRNA or overexpression vector

OSCC cells, along with spheroids and organoids, were transfected with a mixture of siRNA containing 2–3 oligonucleotides that specifically target gene transcripts (Santa Cruz Biotechnology, CA, USA). Cells (1 × 10⁴) were plated in a 96-well plate. The next day, the medium was exchanged for serum-free medium just prior to transfection with either specific siRNA or a control siRNA at a final concentration of 10 nM, using Lipofectamine^® 3000 (Thermo Fisher Scientific, Waltham, MA, USA). After a 24 h, the cells were treated with cisplatin for 2 days, followed by an MTT assay. To investigate the impact of specific siRNA on the cisplatin sensitivity of spheroids and organoids derived from OSCC cells or primary tissues, siRNA was administered at a final concentration of 3 nM per 96-well or 15 nM per 24-well. After 24 h, cells were exposed to cisplatin and monitored for an additional 7 to 14 days. For the exogenous overexpression of MTMR6, the pCMV3-ORF-MTMR6 vector was utilized at final concentrations of 100 ng per 96-well or 500 ng per 24-well (Sino Biological Co., Beijing, China). The pCMV3-ORF vector served as the control.

Luciferase reporter assay for miRNA targeting

UM-Cis cells were seeded the day before transfection and subjected to triplicate transfections using 800 ng of various luciferase target constructs with Lipofectamine 3000. After a 24 h incubation, the luciferase assay was carried out using the Dual-Luciferase Reporter Assay Kit (Promega, Madison, WI, USA), following the manufacturer's guidelines. For the miR-544a-dependent luciferase assay, cells were co-transfected with the pGL3 plasmid, containing either wild-type or mutant MTMR6 3′-UTR, and a miR-544a mimic using Lipofectamine 3000. After 48 h, luciferase activity was measured using the same protocol. PCR primers utilized for the luciferase reporter assay are listed in Table S1.

Immunofluorescence (IF) staining

IF staining was conducted on cultured organoids. Organoids embedded in Matrigel were first transferred to PBS and then fixed with 4% paraformaldehyde for 2 h. After fixation, the samples were treated with a blocking solution composed of 5% serum in 1 × PBS with 0.5% Triton-X100 for 1 h. Primary antibodies were applied, and the samples were left to incubate overnight. The following day, the organoids were rinsed three times in PBS containing 0.5% Triton-X100 and subsequently incubated with the secondary antibody for 2 h. For fluorescence microscopy, the organoids were suspended in VECTASHIELD mounting medium with DAPI and placed in a CoverWell Imaging Chamber for nuclear counterstaining.

Immunohistochemical (IHC) analysis

Tissue sections were initially blocked for 5 min, following a 2 h incubation at room temperature with specific antibodies (a dilution of 1:500). The IHC staining was performed using the UltraTek HRP Anti-Polyvalent Kit (ScyTek Laboratories, AMF080), and the tissues were subsequently counterstained with hematoxylin and eosin. Human tissue samples were sourced from patients diagnosed with OSCC who underwent biopsy or tumor resection at Kyungpook National University Hospital between 2016 and 2022. Details regarding the patients are provided in Table S2. After the sections were dewaxed, they were blocked for 5 min and incubated with specific primary antibodies (diluted 1:500–1:100) for 2 h at room temperature. IHC staining was conducted using the UltraTek Horseradish Peroxidase (HRP) Anti-Polyvalent Kit (ScyTek Laboratories, USA), utilizing 3,3-diaminobenzidine (Dako, USA) as the chromogen. Images were captured using a light microscope at 40×magnification. The expression level of the protein in each specimen was assessed and scored as 0, 1, 2, or 3 (0 = negative, 1 = weak, 2 = intermediate, 3 = strong) based on the intensity of staining.

Mouse xenograft model

We evaluated cisplatin efficacy in mice xenograft (6-week-old male BALB/c, 20 ± 2.5 g; Hyochang Science, Daegu, Korea). The siMTMR6 or control siRNA-transfected UMSCC1 spheroids (100 spheroids per injection, approximately 1 × 10⁶ cells) were subcutaneously injected into the right- and left side of the back flanks of mice using a 22-gauge needle. We used spheroids for this in vivo experiment owing to the improved gene knockdown or exogenous overexpression efficiency with transient transfection in 3D spheroids over cells from 2D cultures [17,18,19]. Animals were included in the study if they met specific health and age criteria and were free from any pre-existing conditions that could interfere with the experimental outcomes. Animals exhibiting any signs of illness or stress were excluded from the study. All experimental procedures were carried out in a specialized animal facility that maintained controlled environmental parameters, including a temperature of 22 ± 2 °C, humidity levels between 50 and 60%, and a 12 h light/dark cycle. After a period of 20 days, tumor formation was noted, and the mice in each group were randomly assigned to one of two subgroups (total tumor-forming groups n = 6). Cisplatin (2.5 mg/kg) or a vehicle control was administered via intraperitoneal injection twice weekly, with the mice being euthanized on the 29th day following the initiation of cisplatin treatment. To evaluate the effectiveness of cisplatin, tumor volume was measured using a caliper. Any animals in which tumors did not form were excluded from the final analysis. Tumor size evaluation followed the ARRIVE guidelines, which recommend euthanizing animals if their body weight decreases by 20% or if the tumor volume reaches or exceeds 10 cm.

Statistical analysis

All in vitro experiments were conducted in duplicate or triplicate. The statistical parameters, including those from in vivo analyses, are detailed in the figure legends. Statistical evaluations were performed using Origin v. 8.0 (OriginLab, Northampton, MA, USA) and R software. One-way analysis of variance (ANOVA) was employed for comparisons involving three or more groups, while unpaired t-tests were used for comparisons between two groups.

The Mann–Whitney U test was used to compare mRNA expression between the cisplatin-sensitive and -resistant tissue groups. We obtained the relevant dataset from cBIOPORTAL, specifically HNSCC, for overall survival analysis (TCGA, Firehose Legacy, n = 518). To evaluate the effect of MTMR6 expression on the prognosis of HNSCC patients receiving cisplatin treatment, we extracted the dataset involving 96 patients with cisplatin treatment history. Subsequently, we conducted a Kaplan–Meier survival analysis using MediCalc. The optimal cut-off values were calculated by the ROC AUC curve with the R package. The threshold for statistical significance was set at p < 0.05. Significant p values are presented in the Figures.

Results

Effect of MTMR6 on cisplatin efficacy in OSCC cell lines and organoids

We compared the mRNA and protein levels of MTMR6 in UMSCC1 and UM-Cis cells and found that both were significantly lower in UM-Cis cells (Fig. 1A). This finding prompted us to investigate whether MTMR6 could serve as an indicator of drug resistance by examining its relationship with cisplatin resistance in OSCC. To determine whether MTMR6 downregulation increases cisplatin resistance of UMSCC1, we pretreated the cells with an MTMR6-specific siRNA and then assessed cisplatin efficacy. UMSCC1 cells transfected with siMTMR6 had decreased mRNA and MTMR6 protein levels (Fig. 1B). Furthermore, pretreatment with siRNA, followed by cisplatin treatment, significantly increased cisplatin resistance (Fig. 1C) and decreased apoptosis (Fig. 1D). The quantification graphs of Western blot bands in Fig. 1, along with their statistical analyses, are provided in Supplemental Fig. S1. We examined the effects of siMTMR6 in UMSCC1-derived 3D spheroids. The knockdown effect of siMTMR6 was maintained during cisplatin treatment for 14 days (Fig. 1E). After 24 h of siMTMR6 pretreatment and subsequent cisplatin treatment for 14 days, cisplatin resistance was notably higher in siMTMR6-transfected spheroids than in control spheroids (Fig. 1F). Similar experiments were conducted using organoids cultured from UMSCC1-derived mouse xenografts. The organoids were confirmed to contain squamous cells due to their strong reactivity to the anti-KRT13 antibody (Fig. 1G). We assessed siMTMR6-mediated knockdown efficiency in the organoids using RT-qPCR and IF analysis with an anti-MTMR6 antibody (Fig. 1H). The results observed with organoids mirrored those with spheroids, with a significant increase of cisplatin resistance pretreatment with siMTMR6 and subsequent cisplatin treatment for 7 days (Fig. 1I).

Fig. 1

Effect of MTMR6 on cisplatin efficacy in OSCC cells and organoids. A mRNA and protein expression of MTMR6 in UMSCC1 and UM-Cis cell lines by RT-qPCR, Western blot analysis, and IF staining. B UMSCC1 cells were pretreated with siMTMR6 for 24 h, followed by cisplatin treatment for another 2 days. siRNA-mediated knockdown efficiency was measured using RT-qPCR and Western blot analysis. C UMSCC1 cell viability was measured by MTT assay under the same experimental conditions. D Apoptosis was determined under the same condition by Western blot analysis. E Cellular spheroids were formed by culturing in a 96-well U-bottom ultra-low attachment plate for 2–3 days. After transfection with siMTMR6 for 24 h, spheroids were treated with cisplatin for another 14 days. MTMR6 knockdown efficiency was measured using RT-qPCR. F Representative images of the eight spheroids analyzed in each group are displayed. The spheroid size (surface area) was analyzed using Cell³iMager. G UMSCC1-derived mouse xenografts were used for organoid culture. UMSCC1 organoids were confirmed by staining with an anti-KRT13 antibody. H After transfecting with siMTMR6 for 24 h, UMSCC1 organoids were treated with cisplatin for another seven days. siMTMR6 efficiency was evaluated by RT-qPCR and IF staining with anti-MTMR6 antibody. I Representative images in each group are shown. The organoid size (average area) was analyzed using the Nikon NIS-Elements microscope imaging software. Results represent the mean ± standard deviation of three experiments (*p < 0.05, **p < 0.01)

Effect of MTMR6 overexpression on cisplatin efficacy in UM-Cis cells and organoids

In UM-Cis cells, MTMR6 overexpression was maintained for three days (Fig. 2A), during which treatment with cisplatin led to a significant reduction in cell viability compared to the control vector (Fig. 2B). Furthermore, pretreatment with overexpression vector followed by cisplatin treatment increased apoptosis remarkably (Fig. 2C). The quantification graphs of Western blot bands in Fig. 2, along with their statistical analyses, are provided in Supplemental Fig. S2. We examined the effects of MTMR6 overexpression in UM-Cis-derived spheroids. The effect of the overexpression vector was maintained during cisplatin treatment for 14 days (Fig. 2D). After 24 h of overexpression vector pretreatment and subsequent cisplatin treatment for 14 days, cisplatin resistance was significantly decreased in MTMR6 overexpression vector-transfected spheroids compared to the control group (Fig. 2E). Similar experiments were conducted using organoids cultured from UM-Cis-derived mouse xenografts. The organoids were confirmed to contain squamous cells due to their strong reactivity to the anti-KRT13 antibody (Fig. 2F). We assessed MTMR6 overexpression efficiency using RT-qPCR and IF analysis with an anti-MTMR6 antibody (Fig. 2G). Cisplatin resistance was significantly decreased by MTMR6 overexpression (Fig. 2H).

Fig. 2

Effect of MTMR6 overexpression on cisplatin efficacy in UM-Cis. A Cells were pretreated with an MTMR6-overexpression vector for 24 h, followed by cisplatin treatment for 2 days. MTMR6 overexpression was evaluated using RT-qPCR and Western blot analysis. B Cell viability was measured using an MTT assay. C Apoptosis was determined under the same condition by Western blot analysis. D Spheroid formation was performed in a 96-well U-bottom ultra-low attachment plate for 2–3 days. After transfection with over-MTMR6 for 24 h, UM-Cis spheroids were treated with cisplatin for another 14 days. MTMR6 mRNA expression was analyzed using RT-qPCR. E Spheroid size (surface area) was analyzed using Cell³iMager. Representative images of spheroids are shown. F UM-Cis-derived mouse xenografts were used for organoid culture. UM-Cis organoids were characterized by staining with an anti-KRT13 antibody. G After transfection with over-MTMR6 for 24 h, each organoid was treated with cisplatin for another seven days. Over-MTMR6 efficiency was evaluated by RT-qPCR and IF staining with anti-MTMR6 antibody. H Representative images in each group are shown. The organoid size (average area) was analyzed using the Nikon NIS-Elements microscope imaging software. Results represent the mean ± standard deviation of three experiments (*p < 0.05, **p < 0.01)

Effect of MTMR6 on cisplatin efficacy in cisplatin-sensitive and paired-insensitive OSCC cell lines

We conducted similar experiments using two sets of OSCC cell lines with differing responses to cisplatin. We compared MTMR6 mRNA and protein expression levels in the cisplatin-sensitive YD-8 and YD-9 cell lines to their original cisplatin-resistant counterparts, YD-8/CIS and YD-9/CIS. MTMR6 expression was significantly higher in the cisplatin-sensitive cells (Fig. S3A, B). Pretreating YD-8 and YD-9 cells with siMTMR6 reduced their sensitivity to cisplatin (Fig. S3C, E). In contrast, overexpressing MTMR6 in YD-8/CIS and YD-9/CIS increased cisplatin sensitivity (Fig. S3D, F), consistent with the results observed in UMSCC1 and UM-Cis.

Effect of siMTMR6 on cisplatin efficacy in cisplatin-sensitive OSCC cell lines and organoids from patients’ tissues

We performed similar experiments using cisplatin-sensitive FaDu and SCC15 cell lines. Following transfection of the cells with siMTMR6, they were treated with cisplatin for two days. FaDu and SCC15 cells did not respond to cisplatin when pretreated with siMTMR6 (Fig. S4A). Next, we transfected 3D spheroids derived from these OSCC cell lines with siMTMR6 and treated them with cisplatin for 14 days. siMTMR6-mediated knockdown was retained after 15 days of transfection (Fig. S4B). When the spheroids were pretreated with siMTMR6, cisplatin resistance significantly increased in both cell lines (Fig. S4C).

These patterns were reproduced in organoids derived from OSCC tissue samples from patients. After transfecting the primary organoids with siMTMR6, cisplatin was administered for an additional seven days. Cisplatin caused a marked decrease in organoid size and breakage in the siControl-transfected group (Fig. S5A, D). However, the siMTMR6 transfection exhibited a significant increase in organoid size, even after cisplatin treatment. The OSCC-derived organoids exhibited strong immunoreactivity to anti-KRT13 antibody, representing a squamous epithelial cell characteristic (Fig. S5B, E). siMTMR6-mediated MTMR6 knockdown efficiency in the organoids after eight days of transfection persisted in 57–79% of the control group (Fig. S5C, F).

Relation between MTMR6 expression and cisplatin resistance in clinical OSCC samples

We explored the association between the expression levels of MTMR6 protein and cisplatin resistance in patients diagnosed with OSCC. Patients were categorized as either sensitive or insensitive to cisplatin monotherapy based on changes in tumor size and overall survival rates. As shown in Fig. 3A, staining results from six tissue samples in each category were analyzed. A comparison of staining intensity revealed that tissues from cisplatin-sensitive patients exhibited a strong MTMR6 signal, while those from cisplatin-insensitive patients demonstrated a significantly weaker response. Furthermore, patients in the cisplatin-sensitive group currently have a follow-up period of 3.5 to 4 years, and all are alive. In contrast, patients in the cisplatin-insensitive group passed away within a follow-up period of 0.5 to 2.5 years (Table S2). These findings reinforce the positive correlation between MTMR6 expression and sensitivity to cisplatin in OSCC tissues, supporting our hypothesis.

Fig. 3

IHC analysis of OSCC tissues from patients for MTMR6 protein expression. A Tissues from patients with OSCC showing cisplatin-sensitive and cisplatin-insensitive tissues were stained with an anti-MTMR6 antibody (Tissue S1-S6:cisplatin-sensitive, Tissue R1-R6: cisplatin-insensitive). The protein expression level on each specimen was scored as 0, 1, 2, and 3 (0 = negative, 1 = weak, 2 = intermediate, and 3 = strong) according to its staining intensity (*p < 0.05). B Kaplan–Meier survival plots for MTMR6 expression were generated with the TCGA dataset of HNSCC patients (n = 515). C Kaplan–Meier survival plots for MTMR6 were generated with the TCGA dataset of HNSCC patients with cisplatin-treatment history (n = 96)

The association between MTMR6 expression and outcomes in HNSCC patients treated with cisplatin was examined through a survival analysis using data from The Cancer Genome Atlas (TCGA). This analysis included 96 patients who had received cisplatin monotherapy and 422 patients with no prior history of cisplatin treatment. The two cohorts were divided into high- and low-MTMR6 expression groups. Kaplan–Meier analysis revealed no significant association between overall survival and MTMR6 expression in patients with and without cisplatin-treatment history (Fig. 3B). However, in cisplatin-treated patients, high MTMR6 expression was strongly associated with longer patient survival, supporting our results (Fig. 3C). OSCC is indeed a subset of HNSCC, and in this dataset, 66% of the patients were classified as having OSCC. This proportion supports the possibility of making an indirect inference to draw conclusions related to OSCC.

MTMR6 downregulation by hsa-miR-544a in UM-Cis cells

Analysis with the TargetScan (http://www.targetscan.org/) search tool revealed hsa-miR-544a as a candidate with the highest probability of regulating MTMR6 expression. Notably, the expression pattern of miR-544a was opposite to that of MTMR6 in RT-qPCR analysis (Fig. 4A). The putative interaction site between miR-544a and the 3′-UTR of MTMR6 mRNA is shown in Fig. 4B. To investigate the possible interaction between miR-544a and MTMR6 mRNA, luciferase reporter constructs containing the wild-type (WT) and mutant-type (MT) 3′-UTR of MTMR6 were generated and transfected into UM-Cis cells. Luciferase activity in UM-Cis cells transfected with WT constructs was significantly lower than in UM-Cis cells transfected with MT constructs (Fig. 4C). Following pretreatment of UMSCC1 cells with a miR-544a mimic, mRNA and MTMR6 protein expression was significantly downregulated (Fig. 4D), and simultaneously cisplatin resistance was significantly increased (Fig. 4E). Furthermore, the miR-544a mimic markedly decreased cisplatin-dependent apoptosis (Fig. 4F). To confirm the regulatory effect of miR-544a on MTMR6 expression, UM-Cis cells were treated with anti-miR544a. As shown in Fig. 4G, mRNA and protein expression of MTMR6 increased in UM-Cis cells, and cisplatin resistance significantly decreased under the same conditions (Fig. 4H). The quantification graphs of Western blot bands in Fig. 4, along with their statistical analyses, are provided in Supplementary Fig. S6.

Fig. 4

hsa-miR-544a downregulated MTMR6 expression. A miR-544a mRNA expression level was compared in UMSCC1 and UM-Cis cells by RT-qPCR. B Predicted miR-544a binding sites in 3′-UTR of MTMR6 mRNA are shown. Mutations in the MTMR6 3′-UTR are shown in red. Luciferase reporter constructs were generated with the wild-type (WT) and mutant (MT) 3′-UTRs of MTMR6. C Dual luciferase reporter activity demonstrates the target relationship between miR-544a and MTMR6 mRNA. The activity was measured in UM-Cis cells transfected with the WT and MT 3′-UTR MTMR6 luciferase constructs. The activity was normalized to that of Renilla luciferase. D UMSCC1 cells were transfected with miR-544a mimic for 24 h, and cisplatin was treated for another 2 days. MTMR6 mRNA and protein expression were analyzed using RT-qPCR and Western blot analysis. E Cell viability was measured under the same conditions using an MTT assay. F Apoptosis was measured under the same experimental condition using Western blot analysis. G UM-Cis cells were transfected with anti-miR-544a for 24 h, and cisplatin was treated for another 2 days. The effect of anti-miR544a on MTMR6 mRNA and protein expression was analyzed using RT-qPCR and Western blot analysis. H Cisplatin efficacy was evaluated under the same conditions via MTT assay. Results represent the mean ± standard deviation of three experiments (*p < 0.05, **p < 0.01)

Effect of MTMR6 knockdown on cisplatin sensitivity in UMSCC1-derived mouse xenografts

We further evaluated the effect of MTMR6 on cisplatin efficacy in mouse xenografts. UMSCC1 spheroids transfected with siMTMR6 were subcutaneously implanted in mice and allowed to grow as xenografts for 21 days, following cisplatin administration for 18 days. There was no significant size difference between siControl- and siMTMR6-transfected tumors on day 21 (Fig. 5A). However, following cisplatin treatment for another 18 days, the tumors developed from siMTMR6-transfected UMSCC1 cells grew significantly larger, suggesting that MTMR6 downregulation caused cisplatin resistance in xenografts. We further evaluated the tumor tissues using IHC. Figure 5B presents the results of anti-KRT13 immunostaining in xenograft samples. Notably, strong immunoreactivity to the anti-MTMR6 antibody was observed only in the siControl tumors (Fig. 5C). The reduction of MTMR6 expression was sustained in the tumor tissues extracted from both the cisplatin-treated and vehicle control groups (Fig. 5D).

Fig. 5

Effect of MTMR6 knockdown on cisplatin sensitivity in UMSCC1-derived mouse xenografts. A UMSCC1 spheroids were formed in 96-well U-bottom ultra-low attachment plates and transfected with siMTMR6 for 24 h. The spheroids were then subcutaneously injected into mice on the right and left side of the backs of six mice. (100 per tumor, approximately 1 × 10⁶ cells). After tumor formation, the mice were divided into cisplatin injection (n = 3) and vehicle control (n = 3) in each siControl and siMTMR6 group. Cisplatin (2.5 mg/kg) or DMSO (0.1% v/v in PBS) vehicle control was intraperitoneally injected twice a week and sacrificed on the 18th day after cisplatin administration. Tumor volume was measured using a caliper till sacrifice. Results represent the mean ± standard deviation (*p < 0.05; **p < 0.005). B IHC staining for KRT13 and MTMR6 in mice xenograft tissues. H&E staining images are presented as controls. C MTMR6-positive cells were counted in the immunostained mice tumor tissues (***p < 0.001). D MTMR6 mRNA expression was measured in mice tumor tissues using RT-qPCR analysis. The fold-change of each mRNA is represented as the mean ± standard deviation (**p < 0.005)

Discussion

This study identified specific genes involved in cisplatin resistance in OSCC through cisplatin-resistant cell lines. We examined MTMR6 gene expression in patient-derived OSCC tissue samples in which the efficacy of cisplatin monotherapy was determined and confirmed that the lack of MTMR6 gene expression could be clinically involved in drug resistance. When 96 patients who received cisplatin monotherapy were extracted from 518 HNSCC patients and survival analysis was performed on MTMR6 expression, there was a positive correlation between MTMR6 expression and survival period with cisplatin chemotherapy. A comprehensive and faithful representation of the biological context of a tumor is likely to benefit the choice of personalized chemotherapeutic methods for patients. Furthermore, drug resistance and adverse side effects can be minimized by increasing drug efficacy, thereby improving cancer therapy outcomes. MTMR6 showed marked downregulation in UM-Cis cells compared to parental UMSCC1 cells. Previous studies have shown that MTMR6 induces macropinocytosis in OSCC cells [20], resulting in increased intracellular uptake of cisplatin [21]. These data support our results that MTMR6 upregulates apoptosis under cellular stress conditions.

The present study confirms the effect of MTMR6 on cisplatin resistance using organoids cultured from OSCC cell-derived mouse xenografts and tissues from patients as ex vivo models. Three-dimensional tumor structures generate various physical and chemical gradients that produce phenotypic heterogeneity within the tumors [22, 23]. Furthermore, 3D cell culture models are superior experimental models due to cell–cell interactions and cell-ECM interactions that mimic in vivo structures [24]. Limited studies have used organoid cultures to analyze OSCC compared to other major solid cancers. This study used various experimental models to investigate the efficacy of cisplatin more accurately. In addition, our previous studies reported the long-term persistence of siRNA effects or exogenous overexpression in 3D models [14, 25]. This study evaluated the effect of MTMR6 on cisplatin efficacy in OSCC using specific siRNAs or overexpression vectors. The efficacy of genetic manipulation of MTMR6 (both downregulation and overexpression) was retained for the 7 − 14-day duration of the experiment. Tumor development was initiated over a three-week period in mouse xenografts generated from UMSCC1 spheroids transfected with siMTMR6, after which cisplatin was administered for 18 days. Notably, siMTMR6-transfected xenografts exhibited significantly increased cisplatin resistance. Furthermore, IHC analysis using a specific MTMR6 antibody revealed significantly decreased protein expression in mouse xenografts derived from siMTMR6-transfected UMSCC1 spheroids. Therefore, 3D spheroids or organoids derived from OSCC tumors represent an effective model for evaluating the function of specific genes through genetic manipulation.

This study has certain limitations, primarily the small sample size of only 12 patient tissues used to confirm the clinical correlation between MTMR6 expression and cisplatin resistance. To address this, additional experiments involving a larger cohort of tissue samples are currently in progress. Moreover, organoid models directly derived from OSCC tissues are being developed to further validate these findings. Despite these limitations, the potential of MTMR6 as a predictive marker for cisplatin resistance in OSCC patients remains promising and warrants further investigation.

Conclusion

Identifying the optimal drug regimen tailored to an individual's tumor profile could significantly enhance therapeutic outcomes and minimize drug-related toxicities. Personalized treatment approaches, guided by the genetic profiling of cancer tissues, have shown promise in determining the most effective drug combinations for patients. [26, 27]. Accurately predicting cisplatin resistance in OSCC using biopsy samples could have substantial benefits for patient management. Our findings suggest that MTMR6 mRNA or protein expression holds potential as a biomarker to forecast cisplatin efficacy in OSCC patients, offering a promising tool for personalizing treatment and improving clinical outcomes.