LAT4 drives temozolomide induced radiotherapy resistance in glioblastoma by enhancing mTOR pathway activation

Abstract

Background

Glioblastoma multiforme (GBM) represents the most prevalent form of primary malignant tumor within the central nervous system. The emergence of resistance to radiotherapy and chemotherapy represents a significant impediment to advancements in glioma treatment.

Methods

We established temozolomide (TMZ)-resistant GBM cell lines by chronically exposing U87MG cell lines to TMZ, and dimethyl sulfoxide (DMSO) was used as placebo control. In vivo and in vitro experiments verified the resistance of resistant cells to chemotherapy and radiotherapy. LAT4 was identified by transcriptomics to be associated with GBM treatment resistance and relapse. The relationship between LAT4 and mTOR pathway activity was also analyzed. Finally, the effect of BCH (LAT inhibitor) combined with radiotherapy on GBM prognosis was verified in vivo.

Results

We have first confirmed that TMZ not only induces resistance to chemotherapy in GBM cells but also enhances their resistance to radiotherapy, which is a significant finding in the process of building TMZ-resistant U87MG GBM cell lines. We then performed comprehensive transcriptomic analysis and identified amino acid metabolism as a potential key factor in radiotherapy resistance. Specifically, we confirmed that the upregulation of LAT4 following chemotherapy enhances leucine metabolism within tumors in vitro and in vivo, thereby modulating the mechanistic target of mTOR pathway and leading to radiotherapy resistance. Of note, the application of inhibitors targeting leucine metabolism was shown to restore the sensitivity of these cells to radiotherapy, highlighting a potential therapeutic strategy for overcoming resistance in GBM.

Conclusions

Our study links tumor sensitivity to chemotherapy and radiotherapy and highlights the critical role of LAT4 in activating the mTOR pathway and GBM radiotherapy resistance. It suggests ways to improve radiotherapy sensitivity to GBM.

Background

Glioma, a malignancy originating from the glial cells of neuroepithelial tissue, is the most prevalent primary craniocerebral malignant tumor. It is estimated to have an annual incidence ranging from 4.67 to 5.73 per 100,000 individuals [1, 2]. Among its various subtypes, glioblastoma multiforme (GBM) is the most aggressive, accounting for 50.1% of all primary central nervous system malignancies [3]. Despite advances in treatment, the prognosis for GBM patients remains grim, with an average survival duration of merely 14.6 to 17.3 months. The current clinical management strategy, which combines active postoperative radiotherapy with temozolomide (TMZ) chemotherapy, only modestly extends the mean survival by 2.5 months [4]. Moreover, the recurrence rate is alarmingly high, reaching between 75–90% [5, 6].

The emergence of resistance to radiotherapy and chemotherapy represents a significant contributing factor to the poor prognosis of GBM. Post-treatment, cancer cells undergo a multitude of molecular alterations [7]. These changes encompass a number of processes, including DNA damage repair, cell cycle redistribution, epithelial-mesenchymal transition, tumor cell dedifferentiation, alterations in autophagy and cellular metabolism. The activation of signaling pathways, including the phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, the Wnt/β-catenin pathway, and the NF-κB pathway, are closely linked to the sensitivity of radio-chemotherapy [8,9,10,11,12,13]. The PI3K/AKT/mTOR signaling pathway is notably activated in a spectrum of tumors, including GBM [14]. The mTOR, a serine/threonine kinase, plays a pivotal role in tumorigenesis, metastasis, cell cycle regulation, cell growth, metabolism, DNA damage repair, and resistance to radio-chemotherapy [15,16,17,18,19,20].

The L-type amino acid transporters (LAT) family, comprising four members (LAT1-4), is primarily responsible for the transmembrane transport of specific amino acids. LAT4 (also called SLC43A2) has been demonstrated to facilitate the intracellular delivery of neutral amino acids, including leucine, phenylalanine, valine, and methionine [21]. Leucine has been demonstrated to stimulate the mTOR complex 1 (mTORC1) through Rag GTP-dependent mechanisms [22]. Furthermore, leucine activates mTORC1 by enhancing glutamine breakdown and α-ketoglutarate (α-KG) production via its interaction with glutamate dehydrogenase [23, 24]. BCH (2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid) is a known selective and competitive inhibitor of the LAT family. Therefore, it indirectly inhibits the activity of mTORC1 by reducing the intracellular content of leucine.

Elevated LAT4 expression has been observed in a variety of tumors and is implicated in the processes of tumorigenesis, tumor progression, and therapeutic resistance [25,26,27]. However, its specific role in GBM remains to be elucidated. In this study, we established TMZ-resistant U87MG cell lines, and subsequently characterized their radio-chemotherapy resistant mechanism in vitro and in vivo. Our findings provide a potential therapeutic strategy for overcoming resistance in GBM.

Method

Cell culture

Human GBM cell line U87MG was obtained from Shanghai Institute of Cell Biology of the Chinese Academy of Sciences and was cultured in high glucose DMEM (Gibco, Carlsbad, CA, USA) medium with 10% fetal bovine serum (FBS, Gibco). U87MG, a chemotherapy resistant cell line, was established by long-term TMZ stimulus and cultured in high glucose DMEM along with 400 µM TMZ (#85622-93-1, Sigma Aldrich). Each cell line identity was verified by short tandem repeat profiling. All cell lines were cultured at 37 ^oC in a humidified atmosphere with 5% CO₂.

Animal studies

All the animal experiments were conducted in accordance with approved protocol by the Animal Ethics Committee of Nanfang Hospital, Southern Medical University. Female BALB/c mice (4–6 weeks of age) were obtained from the Experimental Animal Center, Southern Medical University. The mice were housed at 22–24 ^oC temperature, 60 ± 10% humidity, under the 12 h light/dark cycle and pathogen-free conditions. Standard rodent laboratory diet, water and libitum were provided. Mice were randomly allocated into experimental groups.

Radiation therapy

Radiation treatment of mice and cells was performed using a biological irradiator (Faxitron, MultiRad225, USA), in the central laboratory of Southern Medical University. The radiotherapy dose was 2 Gy each time for mice and 0, 1, 2, 4, 6, and 8 Gy each time for cells.

Immunohistochemistry

Tumor specimens were fixed in neutral buffered 10% formalin solution and embedded in paraffin as per standard procedures. 3-mm-thick tissue sections were deparaffinized by xylenes and rehydrated in a descending alcohol series (95%, 90%, 80% and 70%). Heat-mediated antigen repair was performed using antigen repair solution citrate buffer (10 mM, pH 6) for 15 min. Sections were allowed to cool down to room temperature for 30 min. Endogenous peroxidase was blocked with 3% H₂O₂ for 10 min. The slides were then sealed with 5% bovine serum albumin (BSA) at room temperature for 60 min and then incubated with primary antibody: Anti-LAT4 ( #PA5-54451, Thermo Fisher Scientific), Anti-Phospho-mTOR (#4060, Cell Signaling Technology), anti-mTOR (#2983, Cell Signaling Technology) and Anti-Phospho-Histone H2A.X (#9718,Cell Signaling Technology) at 4℃ overnight. The next day, the sections were incubated with secondary antibody for 1 h and diaminobenzidine (DAB) for 3 min, followed by hematoxylin staining. Sections were then rehydrated in an alcohol gradient (70%, 80%, 90% and 95%) and xylenes. The sections were then dehydrated and sealed in neutral resin.

Immunofluorescence

Cells were grown on glass coverslips in 6-well plates with the corresponding treatment. Then, the cells were fixed with 4% paraformaldehyde for 30 min and incubated with primary antibody: Anti-Phospho-Histone H2A.X (#9718,Cell Signaling Technology), fluorescence dye-conjugated secondary antibodies, and DAPI according to standard protocols. Confocal laser microscope scanning of fixed cells was performed using a laser scanning microscope (Carl Zeiss, LSM 980; Oberkochen, Germany), and the fluorescence foci were counted by ImageJ.

Western blotting and antibodies

Cells and tumor samples were lysed in RIPA buffer (Sigma, R0278) with 1:100 PMSF and alkaline phosphatase, and protein concentrations were determined using a BCA Protein Assay Kit (Solarbio Life Sciences, PC0020; Beijing, China). The 5×loading buffer and RIPA buffer were used to dilute the protein solution to 40 µg/10 µl. Total protein (40 µg) was separated by SDS‒PAGE on 10–15% gels and electrotransferred to PVDF membranes (Millipore, IPVH00010; Billerica, MA). The membranes were then blocked with 5% skim milk (BD Biosciences, 232100; San Jose, CA) or 5% BSA (Solarbio Life Sciences, A8020) in 0.1% Tween 20 (Sigma, P9416) in TBS, incubated with the primary antibodies: Anti-LAT4 (#PA5-54451, Thermo Fisher Scientific), Anti-Phospho-mTOR (#4060, Cell Signaling Technology), anti-mTOR (#2983, Cell Signaling Technology), Anti-p70 S6 Kinase (#34475, Cell Signaling Technology), Anti-Phospho-p70 S6 Kinase (#9234,Cell Signaling Technology), Anti-Phospho-Histone H2A.X (#9718,Cell Signaling Technology) and Anti-GAPDH (#2118, Cell Signaling Technology) overnight at 4 °C, and then incubated with an HRP-conjugated secondary antibody (CST) for 1 h at room temperature. The band intensities were quantified using the Tanon 5500 Chemiluminescence Imaging System (Tanon Science & Technology; Shanghai, China) with Immobilon ECL Ultra Western HRP Substrate (Millipore, WBULS0500) as the chemiluminescent substrate. GAPDH served as the loading control.

Cell colony formation assay

For the cell colony formation assay, cells (200 cells/well) were seeded into 6-well culture plates and cultured in DMEM supplemented with 10% FBS. The cells were treated with the indicated agents and incubated for 14 days at 37 °C in 5% CO₂. The colonies were then stained with 0.1% crystal violet (Sigma Aldrich) and counted. For each set of cells, three independent assays were carried out.

Cell viability assay

To measure cell viability, 2,000 cells per well were seeded in a 96-well plate 1 day before treatment. Upon treatment with the appropriate drugs as indicated, the medium in each well was replaced with fresh medium containing Cell Counting Kit-8 (CCK8) reagent (#B34304; Bimake). After incubation for 1 h at 37 °C, the plate was analyzed using a BMG microplate reader (BMG Labtech, CLARIOstar, RRID: SCR_019751), and the absorbance of the wells was measured at 450 nm.

siRNA interference

To inhibit gene expression, siRNAs against the target genes were used. LAT4-targeting siRNAs were designed and manufactured by GenePharma, Inc. The siRNAs were added to 125 µL of ribonuclease-free water to a concentration of 20 µmol/L, and 5 µL of the siRNA solution was mixed with transfection reagent and incubated at 25 °C for 20 min. For transfection, 5 × 10⁴ cells were seeded in 6-well plates and incubated overnight at 37 °C in 5% CO₂ to reach 80–90% confluence and were then transfected with siRNA. The medium containing siRNAs was removed after 6 h and replaced with medium containing 10% FBS for further incubation. The total protein was extracted 72 h later.

Statistical analysis

All data from more than three independent experiments are presented as mean ± standard deviation (SD). Statistical comparisons between two indicated groups were performed by two-tailed t-test. Correlations were assessed using Spearman rank-order correlation. Actuarial rates of survival were analyzed and compared using Kaplan–Meier methods and log-rank tests. All statistical analyses were performed using GraphPad Prism 8 (GraphPad Prism Software, Inc., San Diego, CA, USA) with a p-value < 0.05 indicating a statistically significant difference (* p < 0.05, ** p < 0.01, *** p < 0.001).

Results

Establishment of TMZ-resistant GBM cells

To clarify the mechanisms underlying therapeutic resistance in GBM, we selected the U87MG cell line, which are widely used for the study of TMZ-resistant cell [28, 29] to develop a TMZ-resistant GBM cell line. The cell line was established by chronically exposing U87MG cell line to TMZ, with DMSO used as a placebo control. From this panel, the TMZ-sensitive placebo control cells (U87MG-sensitive) and the TMZ-resistant cells (U87MG-resistant) were generated. We first performed a cell viability assay to calculate the median inhibitory concentration (IC50) of TMZ and found that the IC50 was 228 µM for the parental U87MG cells, 229 µM for the sensitive cells, and 1531 µM for the resistant cells (Fig. 1A). Next, a colony formation assay was performed to evaluate cell proliferation under treatment with different concentrations of TMZ. It was shown that the resistant cells could maintain stable proliferation even in the presence of 600 µM TMZ (Fig. 1B), and the proliferation rate of the resistant cells was significantly higher than that of the sensitive cells under 200 µM of TMZ (Fig. 1C). Since TMZ acts as an alkylating agent, leading to DNA methylation and subsequent double-strand breaks, nuclear γH2AX expression was detected following TMZ treatment. It is shown that the number of TMZ-induced DNA damage was significantly higher in sensitive cells than that in resistant cells, and the expression level of γH2AX exhibited a time- and dose-dependent pattern in both cell types under TMZ treatment (Supplementary Figure S1). Immunofluorescence analysis further confirmed that the γH2AX signal was significantly more robust in the sensitive cells compared to the resistant cells after 600 µM TMZ treatment for 8 h (Fig. 1D). Consistent with these observations, the comet assay also revealed a greater extent of DNA strand breaks in the sensitive cells than in the resistant cells after TMZ treatment (Fig. 1E), demonstrating the low level of DNA damage in TMZ-resistant cells after TMZ treatment. Finally, we employed nude mice to evaluate the effect of resistance to TMZ in U87MG cells by orthotopically injecting of the sensitive or resistant cells with luciferase into the brains of nude mice. After 14 days of sensitive or resistant cell injection, mice were treated intraperitoneally with TMZ (50 mg/kg) or DMSO on days 15–19. After 42 days of injection, in vivo imaging showed that tumors in the nude mice injected with the resistant cells exhibited remarkable TMZ resistance compared to those injected with the sensitive cells (Fig. 1F). Given the critical role of MGMT in TMZ chemoresistance [30], we also examined the expression of MGMT protein in the resistant cells. However, no MGMT protein expression was observed in the resistant cells (Supplementary Figure S2). Taken together, these results demonstrate the successful establishment of TMZ-resistant GBM cells, which is independent of MGMT expression.

Fig. 1

Effect of TMZ on TMZ-sensitive and -resistant U87MG cells in both in vitro and in vivo. (A) The determination of TMZ sensitivity was conducted by assessing the IC50 values for non-treated parent U87MG cells, TMZ-sensitive cells, and TMZ-resistant cells. (B) The proliferation capacity of TMZ-sensitive or -resistant cells was evaluated with a clonogenic assay under gradient TMZ concentrations (0, 200, 400, 600, 800 µM). (C) Cell viability assay results illustrating the optical density (OD) at 450 nm for TMZ-sensitive and -resistant cells following treatment with either DMSO (control) or TMZ (200 µM) within 96 h. (D) Representative fluorescence microscopy images of DAPI/γH2AX staining in TMZ-sensitive and -resistant cells exposed to TMZ (600 µM) for 8 h are presented. The fluorescence of DAPI (blue) and γH2AX (red) indicate the presence of nucleus DNA damage foci, respectively. (E) A quantitative assessment of the DNA damage response in TMZ-sensitive and -resistant cells treated with TMZ was expressed by the mean tail moment of the comet assay. (F) In vivo imaging of tumors in response to TMZ chemotherapy in tumor-bearing mice that had been injected with either TMZ-sensitive or TMZ-resistant cells. All data from more than three independent experiments are presented as SD. A two-tailed t-test was used for comparisons between the two indicated groups (** p < 0.01, *** p < 0.001)

TMZ-resistant GBM cells exhibit radiotherapy resistance

Similarly, as with TMZ, the main mechanism of radiotherapy to inhibit tumors is also through inducing DNA damage. It is therefore plausible that TMZ-resistant cells may also exhibit resistance to radiotherapy. Consequently, to ascertain whether temozolomide resistance is associated with radiation resistance, we evaluated the proliferative capacity of TMZ-resistant and -sensitive GBM cells following exposure to graded doses of radiation. Our observations indicated that the resistant cells were able to form colonies even at 4 Gy of radiation (Fig. 2A). The median effective dose (ED50) of radiation was then determined by analyzing the relative number of colonies, which revealed an ED50 of 0.904 Gy for sensitive cells and 2.743 Gy for resistant cells. The cell viability assay showed that the proliferation rate of the resistant cells was significantly higher than that of the sensitive cells after 2 Gy irradiation (Fig. 2B), indicating that TMZ-resistant GBM cells exhibit enhanced radio-resistance. We further investigated the DNA damage level of the cells after exposure to graded doses of radiation by analyzing the γH2AX expression level. The peak of γH2AX expression level was observed, and in contrast to TMZ-sensitive GBM cells, the resistant cells exhibited lower levels of damage at 8 h after treatment with 4 Gy of radiation (Supplementary Figure S3). Both immunofluorescence (Fig. 2C) and comet assays (Fig. 2D) confirmed that the level of DNA damage was reduced in TMZ-resistant GBM cells compared to TMZ-sensitive cells at 6 h after treatment with 4 Gy of radiation. Finally, we employed nude mice to evaluate the resistance of TMZ-resistant cells to radiotherapy. After 14 days of orthotopic injection of TMZ-sensitive or -resistant cells into the brains of the nude mice, the mice were irradiated with 2 Gy every two days, accumulating a total dose of 10 Gy. After 42 days of injection, in vivo imaging showed that the tumor volume in the nude mice injected with the TMZ-resistant cells exhibited remarkable radiotherapy resistance compared to those injected with the sensitive cells (Fig. 2E). Taken together, these results demonstrate that our TMZ-resistant GBM cells also exhibit resistance to radiotherapy.

Fig. 2

Effect of radiation on TMZ-sensitive and -resistant U87MG cells in both in vitro and in vivo. (A) Analysis of the relative number of cell clones in TMZ-sensitive and -resistant cells following exposure to varying radiation doses (0, 2, 4, 6 Gy). The median effective dose (ED50) was calculated and expressed as a percentage relative to the unirradiated control. (B) The cell viability expressed as optical density (OD) measurements at 450 nm for TMZ-sensitive and -resistant cells under different radiation conditions (0 Gy and 2 Gy) within 96 h. (C) Representative fluorescence microscopy images of DAPI/γH2AX staining in TMZ-sensitive and -resistant cells at 0–6 h after 4 Gy radiation exposure are presented. The fluorescence of DAPI (blue) and γH2AX (red) indicate the presence of nucleus DNA damage and repair foci, respectively. (D) A quantitative assessment of the DNA damage response in TMZ-sensitive and -resistant cells at 0–6 h after 4 Gy radiation exposure was expressed by the mean tail moment of the comet assay. (E) In vivo imaging of tumors in response to radiation in tumor-bearing mice that had been injected with either TMZ-sensitive or TMZ-resistant cells. All data from more than three independent experiments are presented as SD. A two-tailed t-test was used for comparisons between the two indicated groups (** p < 0.01, *** p < 0.001)

LAT4 is associated with GBM therapy resistance

To elucidate the underlying molecular mechanisms of radio-chemotherapy resistance in TMZ-resistant GBM cells, we performed transcriptome analysis of the TMZ-resistant (U-R) and TMZ-sensitive (U-S) cells, or the U-R and U-S cells exhibiting radiotherapy resistance 6 h after 4 Gy radiation exposure (referred to as U-S IR or U-R IR). A comparative analysis of transcriptome profiles between U-R and U-S cells, U-R IR and U-S IR cells, or U-R IR and U-R cells identified 21 upregulated differentially expressed genes (DEGs) that were implicated in both chemotherapy and radiotherapy resistance (Fig. 3A). Gene ontology (GO) enrichment analysis of these genes revealed that the top biological processes were predominantly associated with four key genes: LAT4, TRIM22, STOM, and BTG2 (Fig. 3B). Among those, LAT4, a member of the amino acid transporter LAT family, has been demonstrated to show significant expression in various malignancies [31]. As illustrated in Fig. 3C, data from the Chinese Glioma Genome Atlas (CGGA) indicate that LAT4 is significantly upregulated in high-grade gliomas, suggesting a potential correlation with increased tumor malignancy. Moreover, LAT4 expression is significantly elevated in recurrent GBM relative to primary tumors (Fig. 3D). Glioma patients (Fig. 3E) and GBM patients (Fig. 3F) with elevated LAT4 expression show significantly shorter survival times compared to those with lower expression levels, providing further evidence of its role in GBM therapeutic resistance. Furthermore, the Cancer Genome Atlas (TCGA) database and human GBM samples also confirmed these findings (Supplementary Figure S4A-C). Altogether, these results indicate that LAT4 may play a critical role in the resistance and recurrence of GBM.

Although only LAT4 was identified in our data intersection, we performed an analysis of other members of the LAT family, including LAT1 (SLC7A5), LAT2 (SLC7A8), and LAT3 (SLC43A1). LAT3, which is structurally and functionally similar to LAT4, also shows a relationship with malignancy grade and survival time of glioma patients similar to that of LAT4. However, qPCR analysis revealed that the expression of LAT3 is particularly low and not significantly different in both sensitive and resistant cells (Supplementary Figure S4D). The CGGA database indicates that the expression levels of LAT1 and LAT2 are not significantly correlated with glioma malignancy or patient survival (Supplementary FigureS4E-H). In conclusion, the role of other members of the LAT family in the radiosensitivity of resistant cells has been excluded.

Fig. 3

Correlation of LAT4 mRNA expression with GBM. (A) Venn diagram illustrating the intersection of differentially expressed genes in U-R vs. U-S, U-R IR vs. U-S IR, and U-R IR vs. U-R conditions, resulting in the identification of 21 genes shown in the right list. (B) GO-BP enrichment analysis of 21 overlapping genes. (C, D) Comparative box plot analysis of log2-transformed LAT4 mRNA expression levels across different tumor grades of II, III, and IV (C) with a significant difference observed (IV vs. II, P < 0.001; III vs. II, P < 0.001; Student T-test), or between primary and recurrent tumors with a significant difference observed (P < 0.001, Student pairwise t-tests; D). (E, F) Kaplan-Meier survival curves for patients stratified by high and low LAT4 mRNA expression levels. Both the log-rank p-value and the Wilcoxon p-value are shown, indicating a highly significant correlation between LAT4 expression and patient survival time. mRNA expression and survival data were derived from the CGGA database

LAT4 activates mTOR pathway in TMZ-resistant GBM cells

The LAT family has been observed to involve therapeutic resistance in several tumor types [32,33,34], with particular emphasis on LAT1. Elevated LAT1 levels have been shown to induce leucine accumulation in tumor cells. This accumulation is thought to activate the mTOR pathway, either directly or indirectly through the generation of α-KG, thereby contributing to radio-chemotherapy resistance [35]. While the increase of LAT4 in tumors has been evidenced, the precise mechanisms underlying its contribution to treatment resistance remain unclear. Given the robust leucine transport capacity of LAT4, it is plausible that it may also contribute to mTOR activation. Indeed, we observed that increased LAT4 expression levels and enhanced mTOR phosphorylation and activation in TMZ-resistant cells (Fig. 4A & Supplementary FigureS5A). Moreover, the knockdown of LAT4 in TMZ-resistant cells by siRNA resulted in a significant reduction in phosphorylated mTOR (p-mTOR) and phosphorylated Ribosomaiprotein S6 (p-S6) levels (Fig. 4B & Supplementary Figure S5B). This was accompanied by an increase in the sensitivity of the cells to radiotherapy. However, the change in chemotherapy sensitivity was not statistically significant (Fig. 4C). Previous research has shown that BCH, a selective competitive inhibitor of LATs, can inhibit amino acid uptake, thereby affecting mTOR phosphorylation. Therefore, we treated TMZ-resistant cells with BCH and observed a significant reduction in mTOR phosphorylation levels without altering LAT4 expression (Fig. 4D), thereby enhancing the cellular damage caused by radiotherapy rather than chemotherapy (Fig. 4E & F). In subsequent rescue experiments, MHY1485, a direct activator of mTOR, was used to treat resistant cells with knockdown of LAT4. This resulted in an increase in the levels of p-mTOR and in the radioresistance of the cells (Supplementary Figure S5C & D). Taken together, these findings highlight the role of elevated LAT4 in promoting therapeutic resistance of TMZ-resistant cells through the activation of the mTOR signaling pathway.

Fig. 4

Correlation of LAT4expression with the mTOR activation and radiotherapy sensitivity of TMZ-resistant cells. (A) Western blot analysis was conducted to determine the expression levels of LAT4, p-mTOR, and p-S6 in both TMZ-sensitive and -resistant cells. (B) The effect of LAT4 knockdown on the expression levels of p-mTOR and pS6 in TMZ-resistant cells. (C) Cell viability assay results illustrating the OD at 450 nm for TMZ-sensitive and -resistant cells within 96 h, following treatment with either DMSO (control) or TMZ (200 µM), under different radiation conditions (0 Gy and 4 Gy). (D) The expression level of LAT4, p-mTOR, and p-S6 protein in TMZ-resistant cells under the treatment of BCH. (E, F) Representative fluorescence microscopy images of DAPI/γH2AX staining in TMZ-sensitive and -resistant cells within 96 h, after 4 Gy radiation exposure for 0–6 h are presented (E), or treatment with either DMSO or TMZ (200 µM) (F). The fluorescence of DAPI (blue) and γH2AX (red) indicate the presence of nucleus DNA damage and repair foci, respectively. All data from more than three independent experiments are presented as SD. A two-tailed t-test was used for comparisons between the two indicated groups (** p < 0.01, *** p < 0.001)

BCH enhances radiotherapy sensitivity in vivo

We then employed nude mice to evaluate the modulatory role of BCH in the sensitivity of TMZ-resistant tumor cells to radiotherapy in vivo. Following the orthotopic injection of TMZ-sensitive or -resistant cells into the brains of nude mice for a period of 14 days, the mice were treated intraperitoneally with BCH at a dosage of 200 mg/kg of body weight. This was administered concurrently with a radiotherapy regimen of 2 Gy, with treatments administered every two days for a total of five sessions (Fig. 5A). Following a 42-day course of injections, in vivo imaging analysis revealed a slight reduction in tumor volume in tumor-bearing mice that had been injected with either TMZ-sensitive or TMZ-resistant cells in conjunction with BCH treatment (Fig. 5B). The combination of BCH and radiotherapy resulted in a significant reduction in tumor volume. This intervention ultimately resulted in an increase in survival time in nude mice that had been injected with TMZ-resistant cells following the combination of BCH and radiotherapy (Fig. 5C). Immunohistochemical analysis of tumor tissues in tumor-bearing mice revealed that, following BCH treatment, the levels of mTOR phosphorylation were significantly reduced in mice that had been injected with TMZ-resistant cells, whereas the expression of LAT4 remained unaltered (Fig. 5D). These in vivo results further suggest that BCH treatment may be a promising strategy for modulating the response of TMZ-resistant tumors to radiotherapy.

Fig. 5

Evaluation of BCH and radiotherapy on the tumor formation in mice injected with TMZ-resistant cells. (A) Schematic the U87MG cell injection, BCH and/or radiation treatment paradigm. A total of 1 × 10⁶ sensitive cells were orthotopically injected into the skull of nude mice without thymus, followed by treatment with BCH and/or radiation 14 days post-inoculation. (B) Tumor size was monitored via in vivo imaging at the 42-days of tumor formation after the mice injected with cells, allowing for the assessment of treatment efficacy. A two-tailed t-test was used for comparisons between the two indicated groups (** p < 0.01and *** p < 0.001) (C) A survival curve was plotted to evaluate the prognosis of mice subjected to different treatment modalities 42 days after mice injected with cells. The statistical analysis revealed a significant difference (p = 0.002) in survival between the Resisitant-IR group and the Resistant-BCH + IR group. (D) Immunohistochemical analysis of LAT4 and p-mTOR expression in tumors from mice injected with TMZ-resistant cells and treated with IR or IR combined with BCH

Discussion

GBM continues to present a significant challenge in neuro-oncology due to its aggressive nature and resistance to radiotherapy and chemotherapy. The objective of our study was to elucidate the mechanisms underlying the resistance to TMZ and radiotherapy in GBM, with a particular focus on the role of LAT4 and its impact on the sensitivity of radio-chemotherapy. Our findings demonstrate that TMZ-resistant GBM cells also exhibit a significant radiotherapy resistance both in vitro and in vivo. This resistance is manifested by a higher rate of proliferation and a lower response to DNA damage after receiving radiation. The DNA damage response, as indicated by γH2AX expression levels, was notably higher in sensitive cells, suggesting that TMZ-resistant cells might possess enhanced DNA repair mechanisms or reduced DNA damage induction. This observation is consistent with previous studies that have implicated DNA repair pathways in the development of resistance to DNA-damaging agents [36, 37].

The results of our transcriptomic analysis revealed that LAT4 may play a pivotal role in the radio-chemotherapy resistance of GBM. LAT4, a member of the L-type amino acid transporter family, is known to facilitate the uptake of essential amino acids, such as leucine, which is a potent activator of the mTOR pathway [21]. The results demonstrate that the upregulation of LAT4 in radio-chemotherapy-resistant cells leads to the activation mTOR pathway and contributes to radiotherapy resistance. This finding is consistent with previous research indicating a correlation between LAT1-mediated leucine uptake and mTOR activation in diverse cancers [22]. The utilization of inhibitors targeting LAT4, such as BCH, has been demonstrated to diminish the activation of mTOR and augment the sensitivity of resistant cells to radiotherapy. These findings suggest that the inhibition of LAT4 or its downstream metabolic pathways may represent a promising therapeutic strategy for overcoming radio-chemotherapy resistance in GBM. The in vivo experiments provide further corroboration of this hypothesis, demonstrating that BCH treatment significantly enhanced the sensitivity of resistant cells to radiotherapy and extended the survival of mice bearing these tumors.

Interestingly, the transcriptome analysis revealed an enrichment of processes associated with viral infection (Fig. 3B). Reviewing our data, only two genes were involved in these pathways: TRIM22 and STOM. The TRIM22 gene encodes an E3 ubiquitin ligase that has been shown to participate in GBM chemotherapy resistance by activating NF-kB pathway [38]. The STOM gene encodes a membrane protein involved in viral replication, protein membrane localization, and ion channel regulation, and has been reported to be associated with non-small cell lung cancer metastasis [39]. Their role in radio-chemotherapy resistance in GBM may be further investigated.

While our study provides valuable insights into the role of LAT4 in the radio-chemotherapy resistance in GBM, there are several limitations that should be acknowledged. Firstly, our study has identified radiation resistance in GBM cells following TMZ treatment, shedding light on the underlying mechanisms contributing to the failure of GBM radiotherapy. While this discovery may be particularly relevant to patients who develop TMZ resistance and subsequently encounter radiation therapy failure, the findings could be further validated by incorporating a broader range of cell lines and primary tumor cells in future research.Secondly, it should be noted that the study was conducted primarily with only one type of GBM cell line and limited human samples, which may not fully represent the heterogeneity of GBM observed in clinical settings. It would be beneficial for future studies to include more diverse range of GBM cell lines and patient-derived xenograft to validate these findings. Thirdly, the mechanisms by which LAT4 mediates resistance are complex and may involve additional pathways beyond the mTOR pathway. Further research is required to fully elucidate these mechanisms and identify potential synergistic targets for combination therapies.

Conclusion

In conclusion, our study confirmed that chemotherapy resistance caused radiotherapy resistance in TMZ-resistant U87MG GBM cell line and highlighted the potential role of LAT4 in the radiotherapy resistance of GBM. Our findings suggest that targeting LAT4 or its associated metabolic pathways may be a viable strategy to improve radio-chemotherapy outcomes. Further research should concentrate on the clinical application of these findings and investigate the potential for combining LAT4 inhibitors with other therapeutic approaches to improve the efficacy of GBM treatment.