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

doi:10.21203/rs.3.rs-4913708/v1

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

https://doi.org/10.21203/rs.3.rs-4913708/v1

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Background

As the strong adaptive radio-chemoresistance, GBM represents the worst prognosis form of primary malignant tumor within the central nervous system. Previous researches indistinctly focused on the mechanisms of resistance to X-ray or chemo regimen in isolation, however, it is still unknown if there exists the synergistic or interact effect between the above two kinds of resistances.

Methods

We established TMZ-resistant GBM cell lines (TMZ-R) by chronically exposing U87MG cell lines to TMZ, and DMSO was used as placebo control. In vivo and in vitro experiments verified the synergistic resistance of TMZ-R cells to radiotherapy. Cell proliferation and clonogenesis assay were used to detect cell tolerance to chemo- or ratio-treatment, immunofluorescence and comet assay to detect cell damage, and in vivo imaging to measure tumor size. By transcriptomics and series validation tests, LAT4 was identified to be associated with such TMZ induced radiotherapy resistance. 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 surprising discovery during the establishment of TMZ-resistant U87MG GBM cell lines. Comprehensive transcriptomic analysis identified amino acid metabolism as a potential key factor in radiotherapy resistance. It has been validated that the upregulation of LAT4, a member of leucine metabolism, subsequent to chemotherapy modulates the mechanistic target of mTOR pathway and leads to radiotherapy resistance both in vitro and in vivo. Importantly, the application of inhibitors targeting leucine metabolism has been demonstrated to restore the sensitivity of these cells to radiotherapy, highlighting a potential therapeutic strategy for overcoming resistance in GBM.

Conclusions

Our study first discover the synergistic effect between tumor resistance to chemotherapy and radiotherapy. Our study highlights the critical role of LAT4 in activating the mTOR pathway and such resistance interaction. Targeting LAT4 and mTOR pathway will improve treatment sensitivity of GBM, especially in recurrent tumors.

GBM

TMZ

radiotherapy

LAT4

amino acid metabolism

mTOR pathway

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, 5]}. Moreover, the recurrence rate is alarmingly high, reaching between 75–90% ^[6].

The emergence of resistance to radiotherapy and chemotherapy represents a significant contributing factor to the recurrence of GBM. Post-treatment, relapsed GBM 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. 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 pathways, is closely linked to the sensitivity of radiochemotherapy and the recurrence of tumors ^[8]. The PI3K/AKT/mTOR signaling pathway is notably activated in a spectrum of tumors, including GBM ^[9]. 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 chemoradiotherapy ^[10–15].

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, in particular, has been demonstrated to facilitate the intracellular delivery of neutral amino acids, including leucine, phenylalanine, valine, and methionine ^[16]. Leucine has been demonstrated to stimulate the mTOR complex 1 (mTORC1) through Rag GTP-dependent mechanisms ^[17]. Furthermore, leucine activates mTORC–1 by enhancing glutamine breakdown and α-ketoglutarate (α-KG) production via its interaction with glutamate dehydrogenase ^{[18, 19]}. Elevated LAT4 expression has been observed in a variety of tumors and is implicated in the processes of tumorigenesis and tumor progression ^[20]. However, its specific role in GBM remains to be elucidated. In this study, we established TMZ-resistant U87MG cell lines, and subsequently characterized their radiochemotherapy resistance mechanism in vitro and in vivo. Our findings provide a potential therapeutic strategy for overcoming resistance in GBM.

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 DMEM (Gibco, Carlsbad, CA, USA) medium with 10% fetal bovine serum (FBS) (Gibco). U87MG chemotherapy resistance cell line was established by long-term temozolomide stimulus and cultured in 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℃ 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℃ temperature, 60 ± 10% humidity, under the 12 hours light/dark cycle and pathogen-free conditions. Standard rodent laboratory diet and water and libitum were provided. Mice were randomly allocated into experimental groups.

Radiation therapy

Radiation treatment of mice and cells using an 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 minutes. Sections were allowed to cool down to room temperature for 30 min. Endogenous peroxidase was blocked with 3% H2O2 for 10 minutes. The slides were then sealed with 5% bovine serum albumin (BSA) at room temperature for 60 minutes 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 hour and diaminobenzidine (DAB) for 3 minutes, 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 minutes 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), and protein concentrations were determined using a BCA Protein Assay Kit (Solarbio Life Sciences, PC0020; Beijing, China). 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 hour 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 hour 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 shRNAs and 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 minutes. For transfection, 5 × 10⁶ cells were seeded in 6-well plates and incubated overnight at 37°C in 5% CO₂ to 80–90% confluence and were then transfected with siRNA. The medium containing siRNAs was removed after 6 hours and replaced with medium containing 10% FBS for further incubation.

Statistical analysis

All data represent the mean ± standard deviation (SD) of more than three separate experiments to ensure accuracy. 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) and p-value < 0.05 indicated a statistically significant difference (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Establishment of TMZ-resistant GBM cells

To elucidate the mechanisms underlying the recurrence and treatment resistance of GBM, we developed TMZ-resistant GBM cell lines. These lines were established by chronically exposing U87MG cell lines to TMZ, and dimethyl sulfoxide (DMSO) was used as placebo control, from this panel the temozolomide-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 (Fig. 1A), and determined that the IC50 is 228 µM for the parental U87MG cells, 229 µM for the sensitive cells, and 1531 µM for the resistant cells. Next, 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 γH2A.X were detected following TMZ treatment, the results demonstrated 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 exhibits a time- and dose-dependent pattern in both cells under TMZ treatment (Supplementary Figure S1). Immunofluorescence analysis further confirmed that the γH2A.X signal was significantly more robust in the sensitive cells compared to the resistant cells after 600 µM TMZ treatment for 8 hours (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 resistant effect of the U87MG cells to TMZ 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. In vivo imaging demonstrated that the tumors in the nude mice injected with the resistant cells exhibited remarkable TMZ resistance compared to those injected with the sensitive cells (Fig. 1F). Together, these results demonstrate the successful establishment of TMZ-resistant GBM cells.

TMZ-resistant GBM cells exhibit radiotherapy resistance

Similar with that of TMZ, the main mechanism of radiotherapy to inhibit tumors is also by causing DNA damage, it is plausible that TMZ-resistant cells may also exhibit resistance to radiotherapy. Therefore, to find out whether temozolomide resistance is associated with radiation resistance, we evaluated the proliferative capacity of TMZ-resistant and sensitive GBM cells after exposure to graded doses of radiation and observed 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), highlighting the enhanced radio-resistance of the TMZ-resistant GBM cells. We further investigated the DNA damage level of the cells after exposuring 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 after treatment with 4 Gy of radiation for a period of 8 hours (Supplementary Figure S2). Both immunofluorescence (Fig. 2C) and comet assays (Fig. 2D) confirmed that the level of DNA damage in TMZ-resistant GBM cells was reduced compared to TMZ-sensitive cells after treatment with 4 Gy of radiation for 6 hours. Finally, we employed nude mice to evaluate the resistant effect of TMZ-resistant cells to radiotherapy. After 14 days of orthotopically injecting 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. In vivo imaging demonstrated 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.

LAT4 is associated with GBM therapy resistance

To elucidate the underlying molecular mechanisms of chemoradiotherapy 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 hours after 4 Gy radiation exposure (referred to as U-S IR or U-R IR). 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, 21 upregulated differentially expressed genes (DEGs) implicated in both chemotherapy and radiotherapy resistance were identified (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, is known to its significant expression in various malignancies ^[21]. Data from the Chinese Glioma Genome Atlas (CGGA) show that LAT4 is significantly upregulated in high-grade gliomas, suggesting a potential correlation with increased tumor malignancy (Fig. 3C). Furthermore, LAT4 expression is significantly higher in recurrent GBM than in primary tumors, supporting its involvement in GBM recurrence (Fig. 3D), while GBM patients with elevated LAT4 expression have significantly shorter survival times compared to those with lower expression levels (Fig. 3E). Altogether, these findings suggest that LAT4 may play a critical role in the resistance and recurrence of GBM.

LAT4 activates mTOR pathway in TMZ-resistant GBM cells

LAT family has been observed to involve therapeutic resistance in several tumor types, 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 chemoradiotherapy resistance ^[22]. While the increase of LAT4 in tumors has been demonstrated, the precise mechanisms underlying its contribution to treatment resistance and tumor recurrence are not fully understood. Given the robust leucine transport capacity of LAT4's, 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). Moreover, knockdown of LAT4 in TMZ-resistant cells by siRNA significantly reduced phosphorylated mTOR (p-mTOR) and phosphorylated Ribosomaiprotein S6 (p-S6) levels (Figs. 4B), concomitantly increased the sensitivity of the cells to radiotherapy, well the change of chemotherapy sensitivity was not significant (Figs. 4C). Previous research has shown that BCH (2-amino-2-norbornanecarboxylic acid), a selective competitive inhibitor of LATs, can inhibit amino acid uptake and 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 increasing the cellular damage caused by radiation therapy rather than chemotherapy (Fig. 4E & F). Taken together, these findings underscore the role of elevated LAT4 in promoting therapeutic resistance of TMZ-resistant cell through activation of the mTOR signaling pathway.

BCH enhances radiotherapy sensitivity in vivo

We finally employed nude mice in vivo to evaluate the modulatory role of BCH in the sensitivity of TMZ-resistant tumor cells to radiotherapy. Following the orthotopical 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 each treatment occurring every two days for a total of five treatments (Fig. 5A). 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 following treatment with BCH (Fig. 5B). However, 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 in modulating the response of TMZ-resistant tumors to radiotherapy.

Glioblastoma multiforme (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 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 ^[8].

The results of our transcriptomic analysis revealed that LAT4 plays a pivotal role in the chemoradiotherapy 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 ^[16]. The results demonstrate that the upregulation of LAT4 in radio-chemotherapy-resistant cells leads to enhanced leucine uptake, thereby activating the mTOR pathway and contributing to radiotherapy resistance. This finding is consistent with previous research indicating a correlation between LAT1-mediated leucine uptake and mTOR activation in diverse cancers ^[17]. The utilization of inhibitors targeting leucine metabolism, 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 of 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. These findings are promising and suggest the need for further investigation into the potential clinical applications of LAT4 inhibitors in the treatment of GBM.

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, it is should be noted that the study was conducted primarily with one type of GBM U87MG cell line, 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 xenografts to validate these findings. Secondly, 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.

In conclusion, our study confirmed chemotherapy resistance caused radiotherapy resistance in TMZ-resistant U87MG GBN cell line and highlighted the critical role of LAT4 in the radiotherapy resistance of GBM. Our findings suggest that targeting LAT4 or its associated metabolic pathways could be a viable strategy to improve chemoradiotherapy 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.

GBM

Glioblastoma multiforme

TMZ

Temozolomide

DMSO

Dimethyl sulfoxide

LAT

L-type amino acid transporters

BCH

2-amino-2-norbornanecarboxylic acid

mTOR

Mammalian target of rapamycin

Standard deviation

Declaration of competing interest

The authors declare that they have no conflicts of interest.

Funding

This work was funded by grants from the Fund of the National Nature Science Foundation of China (81902887, 82373398);the President Funding of Nanfang hospital, Southern Medical University(2023A027). Medical Scientific Research Foundation of Guangdong Province, China (A2024301, B2022237); General Guidance Project on Health Science and Technology in Guangzhou, Guangdong Province, China (20221A010067).

Author Contribution

Authors’ contributionsConception and design: Y.T.L, J.J.L, W.R.ZDevelopment of methodology: W.R.Z, J.J.L;Acquisition of data (provided animals, provided facilities, etc.): Y.W.L, J.J.Z, C.Y.L, J.K.Z, Q.Z, L.C, Z.Y.W; Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.J.L, W.R.Z, Y.F.C, W.L.Z;Acquisition: J.J.L, Y.T.L, Y.F.C. Supervision: J.J.L and Y.T.L.Writing, review, and/or revision of the manuscript: W.R.Z, J.J.L, Y.T.L.All authors reviewed the manuscript.

Acknowledgement

AcknowledgementsWe sincerely thanks for kindly help from Dr. Dongying Zheng from department of Neurosurgery, Guangdong Provincial People’s Hospital, Guangdong Province, China.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Background

Method

Cell Culture

Animal Studies

Radiation therapy

Immunohistochemistry

Immunofluorescence

Western blotting and antibodies

Cell colony formation assay

Cell viability assay

siRNA interference

Statistical analysis

Results

Establishment of TMZ-resistant GBM cells

TMZ-resistant GBM cells exhibit radiotherapy resistance

LAT4 is associated with GBM therapy resistance

LAT4 activates mTOR pathway in TMZ-resistant GBM cells

BCH enhances radiotherapy sensitivity in vivo

Discussion

Conclusion

Abbreviations

Declarations

Declaration of competing interest

Funding

Author Contribution

Acknowledgement

Availability of data and materials

References

Additional Declarations

Supplementary Files

Status:

Version 1