Insulin-dependent GLUT4 is a risk factor for cancer in the prostate

doi:10.21203/rs.3.rs-2619954/v1

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Insulin-dependent GLUT4 is a risk factor for cancer in the prostate

https://doi.org/10.21203/rs.3.rs-2619954/v1

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Background: Diabetic men are less likely to suffer prostate cancer, and insulin signalling through insulin receptors has been long considered. However, the role of insulin-dependent glucose transporters has yet to be elucidated. The unique metabolic properties of prostate cancer are attributed to the central role of androgens. Androgen-sensitive tumour cells have higher mitochondrial activity, while castration-resistant cells exhibit aerobic glycolysis. In addition, to glycolysis, one of the hallmarks of cancer metabolism is increased glucose uptake. However, the prostate's oncogenic value of glucose transporters (GLUTs) needs to be better characterized. This research aims to discover the relevance of insulin-dependent glucose transporters to cancer progression and their importance in the protective role of diabetes in prostate cancer.

Methods: Androgen-sensitive LNCaP and androgen-insensitive PC-3 cells were used in vitro. Castration-resistant LNCaP-R cells and cells overexpressing GLUT1 or GLUT4 were established from LNCaP cell line. In addition, TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice and prostatic samples from patients were employed.

Results: We found that androgens stimulate insulin-independent glucose transporters, while androgen independence is associated with GLUT4 overexpression. The ectopic overexpression of GLUT4 promotes the characteristics of a castration-resistant phenotype. Metabolomics confirmed that hormone-resistant prostate cancer cells show an oxidative metabolism with a clear enrichment in amino acid metabolism. Diabetic TRAMP mice showed total tumour regression, while insulin administration restored proliferation and recovered GLUT4 levels. The levels of GLUT4 increase along with tumour progression in TRAMP mice, and it is reduced by castration and streptozotocin-induced diabetes. Finally, the levels of GLUT4 accumulation in tumour tissues compared to normal epithelial in patients' samples showed a clear co-location with nuclear AR.

Conclusion: Here it is confirmed the relevance of insulin-mediated glucose uptake through GLUT4 with prostate cancer progression and its relation to the reduced occurrence of prostate cancer in diabetic men.

Prostate cancer

glucose transporters

insulin

diabetes

GLUT4

First described by Otto Warburg more than 90 years ago, cancer cells usually exhibit an intrinsic metabolic switch that is considered one of the hallmarks of cancer[1]. Tumour cells typically show increased nutrient uptake compared to normal cells, particularly glucose and glutamine, to facilitate their growth[2]. The higher glucose uptake feeds the so-called ‘aerobic glycolysis’ observed in these cells, leading to higher lactate production and release. Consequently, aerobic glycolysis equips tumour cells with the necessary macromolecular precursors for cell growth without compromising energy production[3]. Thus, targeting glucose metabolism is one of oncology’s most promising treatment options[4]. This higher glucose uptake rate observed in cancer compared to normal cells can be explained by changes in protein levels or the membrane location of glucose transporters. The GLUT (glucose transporter)/SLC2A family is related to the transcriptional activation of several oncogenes[5]. Even though such an increase in glucose uptake has been mainly associated with GLUT1 overexpression, it may also involve other members of the GLUT family of transporters, including the structurally related insulin-dependent GLUT4[6]. Why carcinogenesis becomes insulin-sensitive in some tissues is one of the unanswered questions in oncology.

Prostate metabolism is highly specialized to produce citrate in the prostatic fluid by inhibiting the isomerization of citrate to isocitrate. Such inhibition compromises the tricarboxylic acid (TCA) cycle, meaning glycolysis is favoured in the normal prostate epithelium. Zinc accumulates in the prostate epithelia and inhibits the TCA cycle enzyme aconitase (ACO2) that prevents citrate oxidation to isothiocyanate. This characteristic metabolism suggests that prostate cancer also displays a unique metabolism. Oxidative phosphorylation (OXPHOS) is stimulated in the first stages of carcinogenesis[7]. Although like many other tumours, in the later stages, prostate cancer (PCa) tissue is considered glycolytic[8]. Given this unique metabolic profile, glucose metabolism was not considered to be as important as glutamine or lipid metabolism in PCa and has thus received less research attention than in other cancers[9]. Androgens stimulate citrate production and secretion and near all the metabolic machinery that characterize the gland. In prostate tumors, androgens regulate zinc and aspartate transporters[10]. Metabolic shift between glycolysis and oxidative phosphorylation in prostate tumors also relies on AR function during oncogenic transformation. Androgen stimulation increases glucose uptake[11] but also, it drives lipogenesis and oxidative phosphorylation [12–14]. In addition, the transition to a castration-resistant phenotype (CRPC) is accompanied by changes in glucose consumption and biomass production. Clinical imaging using the radiolabelled glucose analogue 18F-fluorodeoxyglucose (FDG) is not suitable to detect local prostate cancer, whereas neuroendocrine prostate cancer is thought glycolytic and detectable on FDG imaging. Which confirms the metabolic shift during transition to androgen independence[15]. During the last decade, the number of studies showing the importance of glucose metabolism in PCa has increased dramatically and several inhibitors of glucose metabolism are in early phase clinical trials, however further investigation is still needed[15].

Molecularly, the mitochondrial pyruvate carrier protein has been shown to be transcriptionally regulated by AR signalling, increasing the glucose flux towards the mitochondria[16] and some reports indicate that androgens regulate the expression of GLUT1 in PCa cells[17, 18]. GLUT1 production has been observed in normal prostate glands as well as in the most aggressive tumours[19] but less is known about other glucose transporters. The reduce prevalence of prostate cancer in diabetic patients suggest a role of insulin glucose transporters in prostate cancer still unknown. Recently, our own group reported the expression and production of insulin dependent GLUT4 glucose facilitative transporter in PCa cells[20, 21], however there are no data regarding its regulation or role in tumour progression yet. Its expression could be somehow linked to the inverse relationship between diabetes and PCa progression[22], but the molecular mechanisms of which are unknown.

Thus, the main aim of this work was to decipher the role of GLUT4 during PCa progression and determine its involvement in the inverse relationship between diabetes and prostate cancer.

Cell culture, transfection, and treatments

Human androgen-sensitive LNCaP cells were purchased from the European Collection of Cell Cultures (catalogue no. 89110211) and cultured in RPMI 1640 medium (Lonza, catalogue no. 12-719F) supplemented with 10% FBS, 2 mM l-glutamine, 15 mM HEPES and a 1% antibiotic–antimycotic cocktail. PC-3 cells, a human androgen-insensitive cell line, were purchased from American Type Culture Collection (catalogue no. CRL-1435) and grown in DMEM/F12 medium (Lonza, catalogue no. BE12-702F) supplemented with 10% FBS, 2 mM l -glutamine and a 1% antibiotic–antimycotic cocktail. Both cell lines were authenticated by short tandem repeat profiling. Cell lines were grown at 37°C in a humidified 5% CO₂ environment.

GLUT1- or GLUT4-overexpressing LNCaP cells were obtained by using FuGENE®HD (Promega, catalogue no. E2311) following the manufacturer’s instructions. Plasmids pcDNA3.2/v5-DEST hGLUT1 (Addgene plasmid no. 18085) and pcDNA3.2/v5-DEST hGLUT4 (Addgene plasmid no. 18087) were supplied by Wolf Frommer (Heinrich Heine University Düsseldorf) [23]. Cells were selected by incubation with 300 µg/mL G418 (Merck, catalogue no. A1720).

To inhibit the Pentose Phosphate Pathway (PPP), cells were treated with 10 µM of 6-aminonicotinamide (6AN, Merck, catalogue no. A68203) for 48 hours. Androgen stimulation was performed by growing androgen-sensitive cells in androgen-deprived medium supplemented with dextran–charcoal FBS (FBS_chst). Then, 5 nM of dihydrotestosterone (DHT) (Merck, catalogue no. A8380) was used to stimulate ARs. 20 µM of the antiandrogen bicalutamide (CDX, Merck, catalogue no. B9061) was employed in the presence of DHT. Insulin (Ins, Merck, catalogue no. I9278) was added at 1 µM. For all treatments, cells were left to attach for 48 hours. The glucose concentration was always set at 2 g/L (11 mM). Vehicle alone was added to the control group.

Animal procedure

Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice (C57BL/6-Tg(TRAMP)8247Ng/J) were used (The Jackson Laboratory)[24]. Prostates were classified by a pathologist as well, moderately, or poorly differentiated tumours. Mice were maintained under controlled environmental conditions (12:12 light:dark) with ad libitum access to food and drinking water. Animals were sacrificed in a CO₂ chamber and the genitourinary tract was immediately dissected and frozen in liquid nitrogen or fixed in 10% phosphate-buffered formalin.

Castration was done by radical bilateral orchiectomy after anaesthesia with 100 mg/kg ketamine (Imalgene; Merial) and analgesia with 20 mg/kg xylazine (Rompun; Bayer). For androgen replacement in castrated mice, 5 days after surgery testosterone was injected s.c. (2.5 mg/kg body weight). Animals were sacrificed at the indicated times.

For diabetes induction, 50 mg/kg streptozotocin (STZ, Merck) was administered i.p. daily for 5 days. After two weeks, 0.6 U insulin (Humulin 70:30, Lilly) was injected s.c. daily. Insulin was always administered in the morning. Fasting blood glucose levels were measured with levels above 250 mg/dL for at least three consecutive days indicating diabetes. Mice exhibiting levels over 500 mg/dL were discarded.

Patient samples

Paraffin-embedded tissue samples from 132 patients were collected and their medical records, including data related to relapses, metastases, perineural invasion and PSA levels, were provided (Table 1). Samples were classified by Gleason score by the hospital pathologist. Frozen non-pathologic, hyperplastic and tumour tissues were collected from 20 patients. Patients underwent surgery at Hospital Universitario Central de Asturias, Hospital Valle del Nalón (Asturias) or Hospital Universitario Marqués de Valdecilla (Cantabria).

Table 1

Clinical characteristics of diabetic patients
Patient	Age	T1DM¹	Age at diagnosis	PSA²	Gleason	Tumour Grade	Glycosylated Hb (A1c) %	Glucose³
1	61	Yes	15	15	-	-	7.1	154
2	61	Yes	39	39	6 (3 + 3)	3	8.5	158
3	59	Yes	12	12	7 (3 + 4)	2	6.9	157
4	69	Yes	3	3	7 (3 + 4)	2	9.6	298
¹ T1DM, Type 1 Diabetes Mellitus
² Levels of PSA, in ng/ml
³ Fasting glucose levels, in mg/dl

PAGE and immunoblotting

Protein extracts from culture cells were obtained, separated and electrotransferred as previously described[25]. For tissues, a section of 50 mg representing all the prostatic lobes were cut and embedded in 1 mL of chilled RIPA lysis buffer. Samples were homogenized employing an ULTRA-TURRAX® homogenizer, and same procedure to culture cells was followed. Antibodies (Supplementary Table 1) were visualized by binding horseradish peroxidase-conjugated secondary antibodies and detected with chemiluminescence substrate. Densitometry values were obtained using ImageJ and Image Studio software.

To study N-glycosylation of GLUT4 protein, muscle and prostate tissue from TRAMP mice were homogenized in the protein extraction buffer described by Haga Y. et al [26]. Tissue lysates were incubated in ice for 30 minutes, then they were centrifugated for 15 min at 15000xg and supernatant were collected. Supernatants were concentrated using Amicon Ultra-0.5 centrifugal filter devices (Amicon Ultra 10K device. 100000 BNWL, Millipore) following manufacturer instructions. Concentrated proteins were diluted in 1:4 in DEPC treated Water and stored upon usage. Protein extracts were quantified using Bradford reagent (Merck). To remove glycosylation residues, 20 µg of protein were treated with PNGaseF reagent (New England Biolabs) under denaturing conditions following manufacturer's instructions. Treated extracts were loaded into an SDS-page (7.5% acrylamide) and electrophoresis and western-blot were performed like the other experiments.

Immunocytochemistry and Immunohistochemistry studies

Cells were fixed in phosphate-buffered 2% paraformaldehyde (H 7.4). For IHC, 5-µm thick paraffin-embedded tissue sections were deparaffinised and hydrated in a graded series of EtOH solutions. Antigen retrieval was carried out by microwaving. After blocking with 3% goat serum in TBS (Tris-HCl 20 mM pH 7.4, 150 mM NaCl), samples were incubated for 20 min at room temperature with 0.15% Tween-20 for cytoplasmic permeabilization or 0.1% Triton X-100 for nuclear permeabilization. Immunofluorescence was observed under a Leica TCS SP8 confocal microscope. The antibodies and dilution factors used in immunocytochemistry and immunohistochemistry studies are listed in Supplementary Table 1.

AR and GLUT-4 immunostaining in human biopsies

Double immunolabeling with a monoclonal mouse anti-AR and a rabbit polyclonal anti-GLUT4 antiserum was carried out in two groups of prostate biopsies from diabetic and non-diabetic human patients (n = 4 for each group). Briefly, the sections were immersed in EDTA pH 9.0 at 95°C for 20 minutes for antigen retrieval and subsequently incubated in the anti-AR antiserum at 4 ºC overnight (1:5000 dilution). The AR immunolabeling was detected using a standard procedure with the Vectastain ABC kit (Vector) and diaminobenzidine (DAB) as chromogen. In previous experiments, this method had proved to be more effective than IF when labelling with this particular antiserum. Due to the amplification of the signal obtained with the ABC method, a highly diluted primary antibody provided a clear signal, with little background staining. Immunofluorescence was then carried out to detect GLUT4 on these same sections. The samples were incubated with the anti-GLUT4 antiserum (1:250 dilution) at 4 ºC overnight and, subsequently, in an AlexaFluor 488 goat-anti-rabbit antiserum to visualize the immunolabeled regions. DAPI was used as a nuclear counterstaining. In order to obtain merged pictures with the double immunostaining, the sections were photographed with a Zeiss Axiocam 712 monochrome camera mounted on a Nikon Eclipse 80i microscope under both bright field and fluorescence conditions. The bright field pictures corresponding to AR-immunolabeling were then inverted, turned red and merged with those of GLUT-4 immunostaining and DAPI counterstaining with Adobe Photoshop CS8. To measure the GLUT-4 immunostaining intensity of each biopsy, 9 pictures were randomly taken within the section using the 20x objective. The average intensity of immunostaining (in a range of 0-255 values in a grayscale) was measured with the ImageJ software.

In a parallel experiment, samples of prostatectomy from 15 patients diagnosed with different Gleason scores (which ranged between 3 + 3 and 4 + 5) were immunostained for GLUT-4 following a protocol like the one detailed above, omitting the initial AR immunostaining.

Tumor regions with different Gleason grades (ranging between 3 and 5) were present in all the samples, which also contained healthy (non-tumour) glands. A minimum of 5 pictures from each (including non-tumour regions) were taken using the 20x objective and the same photographic conditions. To obtain an estimate of the average immunostaining intensity within the prostatic gland epithelia, the stromal areas were manually removed in all photographs with Adobe Photoshop CS8. Next, GLUT-4 immunostaining was measured using the ImageJ software.

Flow cytometry

The cell cycle was studied by staining with 50 µg/ml propidium iodide (PI) in cells fixed with 70% ethanol. Surface GLUT1 levels were monitored as previously reported[27]. Data analysis was performed using FlowJo (version 10.6.2) and Kaluza (version 2.1) software.

Real-time PCR

RNA was isolated using TriReagent® (Merck) and cDNA was synthesised using a High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). qRT-PCR was performed using the Light Cycler 480 SYBR Green I Master Kit (ThermoFisher Scientific). The specific primers are listed in Supplementary Table 1.

Metabolic assays

Glucose uptake was measured as described previously[20, 28]^,. For standardisation, number of cells were previously estimated. ATP/AMP determination was achieved by HPLC. Nucleotide extraction and measurement were performed as previously described[25]. Protein concentration was quantified by Bradford Assay for standardisation. Glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase enzyme activities were measured as previously described[29] and employed to evaluate the activity of PPP.

Stable isotope labelling experiments

Cells were grown in culture media as mentioned above but supplemented with 2 g/L U-¹³C₆-glucose (Merck) for 24 hours. Extraction of the intracellular metabolites with methanol and ultrapure water, derivatisation and GC-MS measurement of metabolite-specific isotopic distributions were carried out as previously reported[30]. Heatmap and clustering analysis were performed using the heatmap3 add-in package with R project open-source software (version 3.4)[31]. Enrichment and pathway analysis were done using MetaboAnalyst (version 4.0)[32].

Extracellular lactate levels

Cells were seeded in 24-well plates and harvested by scraping when reached 70% confluence. After centrifugation to eliminate cell debris, the cell culture medium was collected and deproteinised with 4 M perchloric acid and neutralised with 2 M KOH. The pH was adjusted to 7.4. The lactate determination assay was performed following the manufacturer’s instructions (Bioquochem S.L.).

PSA levels

PSA was measured by ELISA following the manufacturer’s instructions (Human Diagnostics). Total protein concentration was quantified by Bradford Assay for standardisation.

Fasting blood glucose and glucose tolerance test

Fasting blood glucose was measured using a One Touch Ultra Easy™ glucometer (Johnson & Johnson). Blood was taken from the tail vein in mice fasted overnight.

To measure glucose tolerance, mice received an intraperitoneal injection of glucose (2 mg/g body weight) and blood glucose levels were determined after 15 min, 30 min, 1 hour and 2 hours. The AUC was then calculated by the linear trapezoidal method.

Blood insulin levels

Insulin levels were measured in the plasma of overnight-fasted mice. Blood was extracted post-mortem, collected in EDTA-treated tubes and immediately centrifuged at 3000 × g for 10 min at RT. Samples were frozen at -80°C until analysis. Insulin levels were measured by ELISA following the manufacturer’s instructions (Millipore, catalogue no. EZRMI-13K).

Blood testosterone levels

Blood was extracted post-mortem, collected in EDTA-treated tubes, and immediately centrifuged at 3000 × g for 10 min. Samples were frozen at -80°C until analysis. Testosterone levels were measured by ELISA following the manufacturer’s instructions (Cayman).

Statistics

Data are represented as individual values, or the mean and error bars represent the standard error of the mean (SEM). Outliers were identified using the Rout test, while normality was studied using the Shapiro–Wilk or Kolmogorov–Smirnov tests depending on the number of samples. If the samples followed a normal distribution, the significance was calculated using the two-tailed unpaired Student’s t-test for comparison of two groups while one-way ANOVA followed by Fisher’s LSD test was used for comparison of more than two groups. For samples that did not present a normal distribution, we used the non-parametric Kruskal–Wallis test followed by Benjamini, Krieger and Yekutieli’s two-stage linear step-up procedure. The tests were performed using Prism GraphPad (version 8). The significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001

Androgens regulate insulin independent GLUT transporters in androgen dependent LNCaP cells

Androgens regulate glucose metabolism in prostate cancer cells through the modulation of glycolic proteins. However, the role of insulin dependent glucose transporters in prostate cancer progression is still under unknown. To confirm androgen control over energy metabolism in the prostate we studied the effect of DHT treatment on glucose uptake and glucose transporters production androgen sensitive LNCaP cells. Thus, to verify DHT biological activity, first we confirmed DHT incubation significantly increased cell proliferation as expected (Fig. 1a). In addition, we found that DHT increased the uptake of glucose (Fig. 1b), it reduced the production and release of lactate (Fig. 1c) and diminished Pentose Phosphate Pathway (PPP) activity (Fig. 1d). Incubation with the antiandrogen bicalutamide (CDX) restored the levels to those of the control cells, indicating the specificity of androgen signalling in these effects.

In addition, DHT increased the production of GLUT1 and GLUT3 both insulin independent glucose transporters. However, it does not affect GLUT4, and it reduced the levels of GLUT12 both insulin dependent glucose transporters. Effects also restored by CDX pre-incubation (Fig. 1e). GLUT1 activity in cells is primarily regulated through translocation of GLUT1 to the plasma membrane. Since GLUT1 appeared to be the main target of androgens in LNCaP cells, we studied the membrane exposure of the transporter after the addition of androgens. We found that androgens removal by charcoal-stripped FBS incubation slightly decreased the presence of GLUT1 in the membrane (Fig. 1f, left pannel) as shown by flow cytometry immunostaining. However, membrane GLUT1 levels were not restored after DHT treatment nor influenced by CDX treatment (Fig. 1f, right pannel).

Master metabolic regulators, AMPK or AKT, were also found to be regulated by androgens (Fig. 1g). The stimulation of LNCaP cells with DHT increased the level of pAMPK^Thr172 by six-fold and significantly decreased the phosphorylation of pAKT^Ser473. Again, CDX recovered their levels showing the specificity of androgens. This increment in AMPK activity might support the increment of glucose uptake since it participates in their translocation to the cell membrane.

To confirm the effects of androgen signalling on glucose transporters, we employed hormone-sensitive and castration-resistant LNCaP cells (LNCaP-R). LNCaP-R were previously established by permanent growth without androgens[33]. First, we noticed that GLUT1 was significantly lower in the castration-resistant LNCaP-R corroborating its dependence of androgens but GLUT3, GLUT4 and GLUT12 were higher in LNCaP-R than in the LNCaP parental cells (Fig. 1h).

Overexpression of GLUT4 favours an androgen-independent phenotype

Glucose uptake is the first step in the metabolic shifting of cancer cells but the effect of glucose transporters on prostate cancer phenotype is scarcely known. We investigated the effects of GLUT1 and GLUT4 overexpression, an insulin independent and dependent glucose transporter, on LNCaP cells. First, overexpression was confirmed by western blotting (Supplementary Fig. 1a). As expected, the overexpression of both transporters increased glucose uptake in hormone-sensitive LNCaP (Fig. 2a), although GLUT1 overexpressing cells grow faster than LNCaP^Mock or LNCaP^GLUT4 (Supplementary Fig. 1b). Interestingly, GLUT1 and GLUT4 overexpressing cells exhibited opposite tendency of lactate production. LNCaP cells overexpressing GLUT1 showed a significant reduction of lactate release, while overexpression of GLUT4 significantly enhanced its into the culture media (Fig. 2b). There were no significant differences in PPP activity in both overexpressing clones and the parental mock-transfected LNCaP cells (Supplementary Fig. 1c). However, GLUT1-overexpressing cells were more sensitive than parental cells to glucose-6-phosphate dehydrogenase (G6PDH) inhibition, the first enzyme in the PPP pathway. (Fig. 2c)

To investigate the role of glucose transporters on AR location and activity, western blot was performed after GLUT1 or GLUT4 overexpression. Though total protein levels did not change, nuclear AR was significantly lower in LNCaP^GLUT4 cells (Fig. 2d) but prostate-specific antigen (PSA) release was significantly increased in LNCaP^GLUT4 cells versus parental LNCaP or LNCaP^GLUT1 (Fig. 2e). A decrease in nuclear AR with an increased release of PSA production might be an indicator of the androgen independence of GLUT4-overexpressing prostate cancer cells. This fact was confirmed by investigating the response of LNCaP^GLUT4 cells to DHT in terms of cell proliferation. DHT treatment increased the proliferation of LNCaP^Mock cells as expected, and this increase was prevented by co-incubation with CDX. However, LNCaP^GLUT4 cells did not show any response to DHT (Fig. 2f). Likewise, the number of cells in the S-phase of the cell cycle was increased in LNCaP^Mock studied by flow cytometry while it was significantly decreased in LNCaP^GLUT4 cells after treatment with DHT (Fig. 2g). All these results point that overexpression of GLUT4 encourages an androgen independence.

Hormone resistant cells have an increase in glucose uptake and a glycolytic metabolism

Prostate metabolism is highly specialised to produce citrate in prostatic fluid, which provides the gland with unique metabolic properties. The normal prostate epithelium favours glycolysis over oxidative phosphorylation under aerobic conditions, given the physiological truncation of the TCA cycle. Metabolic reprogramming in prostate tumours leads to a reduction of glycolysis and enhanced mitochondrial oxidative phosphorylation, which are believed to be regulated by androgen signalling. However, progression to a hormone independent phenotype increase glycolytic metabolism in the tumour prostate.

First, we study differences in mitochondrial metabolism between hormone sensitive LNCaP cells and hormone independent PC-3 cells. As shown in Fig. 3a, hormone-resistant PC-3 cells showed higher levels of glucose uptake, a reduce production of ATP (Fig. 3b) and a lower activity of PPP when compared to LNCaP cells (Fig. 3c) than androgen-sensitive LNCaP cells which implies a glycolytic phenotype. To determine whether androgen signalling influence the TCA cycle in prostate cancer cells, we investigated glucose flux by GC-MS-based ¹³C metabolic analysis. Cells were incubated with ¹³C₆-labelled glucose for 24 hours and the incorporation of ¹³C from the 13-labelled glucose in the metabolites was determined by measuring their isotopologue distribution by GC-MS. Mass isotopologue distributions were employed to calculate the molar fraction of ¹³C incorporated. Thus, m0, m1 and m2 refer to the incorporation of zero, one and two ¹³C atoms in the metabolite, respectively. Lactate, glutamate and TCA pathways allowed us to analyse metabolic flux by calculating the different molar fractions of ¹³C incorporated in each metabolite. The absence of AR in PC-3 reduced the molar fraction of M0 in lactate (30% in comparison with LNCaP cells) and significantly increased the incorporation of ¹³C into citrate. The incorporation of ¹³C in succinate was much faster in LNCaP than in PC-3 (Fig. 3d). Although the differences between LNCaP and PC-3 might be due to different metabolic players, such as TP53, our results suggest that AR signalling has a clear metabolic influence on the TCA cycle and favours glycolysis in prostate cancer cells.

Enrichment and pathway analyses were performed using MetaboAnalyst 4.0 to compare LNCaP vs. PC-3. The pathway enrichment analysis showed that androgen dependency promoted tryptophan metabolism, gluconeogenesis and Warburg rewiring, as well as increasing the cells’ reductive power by increasing glutathione metabolism (Fig. 3e). The alanine, arginine and proline, glutathione and glutamate metabolism pathways also showed significant differences in both comparisons. The TCA cycle was also shown to be affected by AR presence, confirming that androgen-sensitive and insensitive cells have different energy phenotypes (Fig. 3f).

Facilitative insulin-independent and -dependent GLUT levels increase with PCa progression

The role of insulin-independent and -dependent glucose transporters in cancer progression was studied using TRAMP mouse prostate tissues. Tumours were classified by a pathologist into three categories: well (WD), moderately (MD) or poorly differentiated (PD) and tissues were collected in 24- and 32-week-old animals. The androgen-dependent prostate secretory protein of 94 amino acids (Psp94) gene was chosen as a marker of hormone dependence of tumours since rodents have no counterpart of PSA. As shown in Supplementary Fig. 2a, PD tumours exhibited an androgen-insensitive phenotype since they did not express Psp94. The levels of insulin-independent GLUT1 and GLUT3 transporters increased along with tumour progression, as previously suggested by others[15]. But interestingly, it was found that also insulin-dependent transporter GLUT4 increased in prostate tumour tissues with progression, showing a significant increment in PD tumours. In the case of GLUT12, also considered to be an insulin-responsive glucose transporter, no significant differences were found (Fig. 4a). Immunostaining confirmed that insulin-responsive GLUT4 was found in epithelial prostate tissue the highest levels found within the most aggressive areas of the tumours, and they were increased in PD when compared with WT (Fig. 4b,c). GLUT4 validation was performed by immunostaining of muscle and adipose tissue (Supplementary Fig. 2b). GLUT4 staining concurs with conventional prostate markers BCL-2, AR and Hypoxia-Inducible Factor (HIF)1α, which are all increased in androgen-resistant tumours (Supplementary Fig. 2c).

While production of GLUT1 and GLUT4 is increased during PCa progression, the expression of Scl2a1 and Scl2a4 was significantly reduced in tumour tissues compared to normal tissues (Fig. 4d, e), while there were no differences in Scl2a3 or Scl2a12 (data not shown).

To confirm the effect of androgens in vivo, TRAMP mice were surgically castrated for 5 days and then treated daily with testosterone for a further 5 days. The effect of castration was confirmed by genitourinary (GU) weight/ Body weight (BW) (Supplementary Fig. 3a). Castration significantly reduced GU weight, while testosterone recovered it. GLUT1 levels did not change shortly after castration but they are significantly reduced after testosterone injection (Fig. 5a). Regarding GLUT4, a double band was detected. Significant differences were found in the abundance of upper and lower band. GLUT4 upper band was significantly reduced or even absence after castration (Fig. 5a, b). Double band of GLUT4 might be related with the glycosylation of the transporter (upper band). The glycosylation of GLUT4 is related with a higher activation in the transport of glucose[34]. Intriguingly, the glycosylated form of GLUT4 disappeared and protein levels significantly decreased after castration in TRAMP mice and the upper band was again detected in presence of testosterone (Fig. 5a-c). The treatment of TRAMP prostate protein samples with the amidase PNGase F, which cleaves at N-acetylglucosamine (GlcNAc) and asparagine residues of high mannose oligosaccharides, confirmed the changes in electrophoresis mobility claimed as a demonstration of glycosylation of the transporter. Muscle protein samples, a positive control of GLUT4 glycosylation was employed as positive controls (Fig. 5d). Surprisingly, the transcription of Scl2a1 was upregulated after testosterone administration, perhaps as a consequence of protein reduction (Supplementary Fig. 3b) while Scl2a4 did not change significantly pointing a functional modification by castration more than a transcriptional regulation (Supplementary Fig. 3c). Castration did not alter the levels of pAMPK^Thr172, though a significant increase was found after testosterone treatment vs CON. On the other hand, pAKT^Ser473 was augmented after acute castration, but its levels were not restored by testosterone injection in TRAMP mice (Fig. 5e).

TRAMP mice were also long-term castrated by surgery at 12 weeks of age and further sacrificed when they were 24 or 32 weeks old. The effect of castration was confirmed by the significant reduction of GU weight/BW (Supplementary Fig. 3d). GLUT1 protein levels were not significantly changed after permanent castration of TRAMP mice, although the disappearance of the upper band of GLUT4 was further confirmed (Supplementary Fig. 3e).

TRAMP mice have higher levels of insulin and diabetes slows prostate cancer progression in TRAMP mice

Hyperinsulinemia has been associated with aggressive prostate cancer and with the accelerated growth of LNCaP cell xenografts[35]. Our previous results suggested that the insulin-dependent GLUT4 transporter might be involved in PCa progression and it might be responsible, at least in part, for the deleterious effects of insulin in the prostate since insulin activates AKT and promotes GLUT4 translocation.

First, we confirmed that LNCaP and PC-3 were sensitive to insulin. As shown in Fig. 6a, LNCaP and PC-3 cells increased the uptake of glucose in response to insulin. Then, we studied the levels of insulin in wild-type and TRAMP mice at 24 weeks of age. TRAMP mice showed significantly higher levels of insulin than WT mice (Fig. 6b) and, interestingly, castration reduced blood insulin levels, while testosterone injection recovered it (Fig. 6c). However, there were no differences in the blood glucose levels in 16-week-old WT and TRAMP mice (Fig. 6d). Fasting blood glucose tolerance was the lowest in TRAMP mice, as shown in Fig. 6e. The area under the curve (AUC), which correlates with the ability to eliminate glucose from blood, was also lower in TRAMP mice than in WT and castrated mice. Altogether, these results indicate that TRAMP mice show higher blood insulin levels and better glucose tolerance during the first stages of the disease.

The relative risk of developing prostate cancer is reduced in men with diabetes. To evaluate the impact of diabetes on PCa progression in TRAMP mice, Type 1 Diabetes Mellitus (T1DM) was induced by streptozotocin (STZ) treatment for 5 days. All treated mice showed over 250 mg/dL glucose after 1 week of the treatment, which was the criterion used to consider them as diabetic (Supplementary Fig. 4a). Histological analysis of the pancreas confirmed the loss of eosinophilic, granule-containing insulin-producing beta cells within the islets of Langerhans (Supplementary Fig. 4b). Insulin injection restored glucose blood levels to normal within 2 hours but this effect only lasted 24 hours (Supplementary Fig. 4c). For this reason, diabetic TRAMP mice were treated daily with insulin, started two weeks after the first injection with STZ.

Relevant anatomical changes were found during necropsy in the prostate glands of TRAMP (CON), diabetic TRAMP (STZ) and diabetic TRAMP plus insulin (STZ + Ins) mice, as shown in Fig. 7a. Diabetes reduced the size and histopathology of the prostate glands in TRAMP mice. The prostates of STZ-induced diabetic TRAMP mice did not show any relevant pathology. Shortly after insulin treatment, some of the animals developed a PIN (4/5), showed an area of PD tumour (1/5) or exhibited scattered areas of neuroendocrine carcinoma (1/5). The body and genitourinary tract were weighed at 24 weeks of age. Bodyweight was reduced in diabetic mice, likely due to white adipose tissue (WAT) depletion, and a significant reduction in the GU/BW ratio was found in STZ mice compared to CON. However, insulin treatment did not recover GU weight (Fig. 7b). Even though the levels of circulating testosterone were slightly reduced after diabetes induction, the differences were not significant (Fig. 7c).

Lowering of tumour progression by diabetes was confirmed by western blot of the proliferating cell nuclear antigen (PCNA) (Fig. 7d). STZ reduced PCNA production while insulin recovered its levels. Besides, the pro-apoptotic protein BAX was significantly increased in diabetic compared to control mice, while insulin recovered its levels. No differences were found in other tumour markers, such as TP53, P21/CDKN1 or BCL-2.

To evaluate the role of androgens in the protective effect of diabetes on PCa, we studied AR levels and location. STZ did not induce changes in total AR levels, though after insulin stimulation it seems to be located in some nuclei (Supplementary Fig. 4d). However, Psp94 mRNA levels did not show any significant difference (data not shown).

It is well known that insulin promotes activation of the PI3K/AKT pathway, either by direct interaction with its receptor or by Insulin-like Growth Factor (IGF) signalling, and this pathway is promoted in the most aggressive tumours. Levels of IGF1 Receptor (IGF1R)β and IGF Binding Protein (IGFBP)3 were analysed by western blot. There was no difference in IGF1Rβ between groups, and IGFBP3 in the prostate was somewhat reduced by insulin treatment in diabetic mice (Supplementary Fig. 4e). Instead, AKT phosphorylation was significantly reduced in diabetic mice, while insulin restored its levels (Fig. 7e).

To assess the implication of GLUT transporters in the protection of STZ-induced diabetic TRAMP mice against PCa progression, we studied the mRNA expression and protein production of GLUT4 transporters (data not shown). Induction of diabetes in the prostate reduced Slc2a4 expression (Fig. 7f). Interestingly, although there was no difference in total GLUT4 protein levels between groups, diabetes led to the disappearance of the upper band found in the GLUT4 signal and insulin consistently restored it (Fig. 7g). These results might indicate that GLUT4 is a facilitative glucose transporter involved in the protective role of diabetes in PCa in TRAMP mice.

GLUT4 is increased in prostate cancer tissues

To translate our results to patient samples, we studied the production and location of GLUT4 in prostate tumours. Samples were classified as non-tumour or hyperplasic and according to their Gleason score in the tumour tissues. Tumours were classified as Gleason score < 7 or ≥ 7 according to pathologists. The samples with Gleason scores ≥ 7 were characterised by higher PSA and BCL2 levels (Supplementary Fig. 5a) and decreased levels of GLUT1 protein during the first stages of tumour growth (Supplementary Fig. 5b). However, insulin-responsive GLUT4 increased with tumour progression, showing the double band that indicate its activate status. (Fig. 8a). In addition, GLUT4 tissue distribution was examined. Samples of prostatectomy from 15 patients diagnosed with different Gleason scores (ranged between 3 + 3 and 4 + 5) were immunoassayed for GLUT4. GLUT4 immunolabeling intensity was significantly higher in Gleason grade 3 and 4 regions than in healthy (non-tumour) control areas (Fig. 8b, c). A decrease in GLUT4 intensity was detected in Gleason grade 5 (less differentiated tumour) regions, which did not show significant differences when compared to control ones. Such difference in protein location was evident when these tumour regions were located next to healthy glands (Fig. 8d).

From a database of all patients treated for prostate cancer in the Urology service at the Central University Hospital of Asturias, between January 2016 and November 2018, out of 807 patients, only 4 patients had been previously diagnosed with type 1 diabetes mellitus, all of them diagnosed in adulthood. GLUT levels were also confirmed in patient samples (Table 1). Double immunolabeling with anti-AR and anti-GLUT4 antiserum was carried out in prostate biopsies from diabetic and non-diabetic human patients (n = 4 for each group). All the nuclei of the prostate epithelia, both from diabetic and non-diabetic patients, were stained and the signal was robust in all samples. No differences were observed between both groups. DAB-immunolabeled nuclei acquired little or no DAPI staining (Fig. 9a,b)

From 407 patients of prostate cancer treated at “Hospital Valle del Nalon” Asturias and “Hospital Universitario Marqués de Valdecilla” roughly 10% of the patients included in the study were diabetic. Most of the patients were diagnosed with Type 2 Diabetes Mellitus (T2DM) and only 1 suffered Type 1 Diabetes (T1DM) (Table 2). Most of the patients were treated with metformin alone or in combination with insulin or stimulators of insulin production. From those, relapses and perineural invasion did not show any difference between diabetic and non-diabetic patients (Table 3).

Table 2

Distribution of diabetic prostate cancer patients and treatments
Diabetics	N (%)¹	Treatment	N (%)²
Type 1	1 (0.25)	Insulin	1 (100)
Type 2	41 (10.07)	Diet	4 (9.75)
		Metformin	19 (46.34)
		Metformin + Insulin	3 (7.31)
		Stimulator of Insulin production³	6 (14.63)
		Metformin + Insulin + SIP	3 (7.31)
		Stimulator + Metformin³	5 (12.19)
¹ N = 407. Percentage from the total of Prostate Cancer patients.
² The % is referred to the number of patients with T1DM or T2DM, respectively.
³ Different stimulators of insulin production were employed: Repaglinide, Daonil and Glimepirin.

Table 3

Relapses and perineural invasions in prostate cancer patients
	Distribution of patients (%)
	Non-diabetic (N = 90)	Diabetic (N = 42)
Relapses	31 (34.44)	9 (21.43)
Perineural invasion¹	37 (48.68)	15 (48.38)
¹ Information about perineural invasion was not facilitated for all patients

Altogether, this data confirms that diabetes plays a protective role in prostate cancer and our results indicate that insulin dependent facilitative transporters of glucose, such as GLUT4, is a relevant protein implicated, while until know it was completely ignored.

PCa is unique from the metabolic point of view. The prostate is specialised for the production and secretion of citrate in prostatic fluid. Androgens stimulate citrate production and regulate all metabolic pathways in the prostate, including glucose metabolism[36]. Here, we try to understand the role of glucose transporters sensitive to insulin in the progression of prostate cancer and their role on the lower incidence prostate cancer in individuals suffering diabetes. In this manuscript, the role of androgen signalling in glucose metabolism is confirmed since glucose uptake is increased after androgen treatment of LNCaP cells while lactate production and PPP activity are decreased.

We showed in our previous work that GLUT1 levels are regulated in response to glucose deprivation in androgen sensitive PCa cells promoting survival and antioxidant pathways with no effects on androgen independent prostate cancer [25]. Here we show that androgens stimulate GLUT1 production, although the location on the cell membrane is not changed, suggesting that its overexpression in another organelle must be considered to explain its role on the survival of prostate cancer cells. Interestingly, androgens stimulate the production of insulin independent glucose transporters GLUT1 and GLUT3 as previously reported. AMPK activity that it is promoted by androgens, and it activates GLUT1, plays a role in mitochondrial metabolism of glucose in androgen-sensitive cells[11]. AMPK is also a regulator of GLUT1 synthesis and trafficking [37]. Thus, GLUT1 stimulation by androgens in vitro can be triggered via AMPK activation, as it is suggested by our results. Interesting, on the contrary DHT does not seem to influence insulin dependent transporters and even more, hormone-resistant LNCaP cells showed increase levels of insulin-sensitive GLUT4 and GLUT12. Though tumour cells often increase glucose uptake the physiologic role of glucose transporters in cancer has been scarcely study. Mostly GLUT1 and GLUT3 have gained attention as relevant factors in the accelerated metabolism of cancer cells[38]

Overexpression of GLUT4 in androgen dependent prostate cancer cells promotes glycolysis and lactate production and more interestingly, it stimulates PSA production in an androgen independent manner.

Metabolomics also confirmed the dependence of prostate cancer metabolism of androgens. Although differences between LNCaP and PC-3 cannot be attributed only to AR, since they also differ in TP53, which plays a principal role in glycolysis[39, 40].

Although glucose uptake is higher in androgen-resistant cells, the levels of labelled lactate and, thus, glycolytic activity, are enhanced in androgen-insensitive cells which indicates the transition from glycolytic to oxidative in hormone resistant phenotypes previously described[36]. In addition to a decrease in aerobic glycolysis, as indicate m + 3 lactate molar fraction, some metabolites of the TCA, like succinate, incorporated lower levels of ¹³C in PC-3 than LNCaP cells according to previous results[41]. A possible explanation for these results is that AR signalling reduces the utilisation of glucose through pyruvate production through the upregulation of PPP. Previous studies have confirmed that AR promotes PPP from glucose[42]. Moreover, pathway analysis showed that the alanine, aspartate, and glutamate metabolism pathway is one of the most affected. This pathway is intricately linked with glycine, serine, and threonine metabolism, which was also found to be significantly impacted[43]. It was recently revealed that serine, that can be produced from the glycolytic metabolite glycerate-3P, and one-carbon metabolism pathways are linked with neuroendocrine PCa[44]. In fact, both pathways should also be considered for androgenic regulation.

Both GLUT1 and GLUT4 are highly produced by poorly differentiated tumours, which suggests their potential as biomarkers in advanced stages of cancer. In any case, only the levels of GLUT4 are reduced by surgical castration and restored by testosterone in TRAMP mice. We observed N-glycosylation of GLUT4 in TRAMP mice tumours that disappeared after castration and could be causally related to GLUT4 stability and trafficking, as proposed by others[45]. GLUT4 is regulated in vivo by the absence of androgens, while it seems to be androgen-independent in vitro. This can be explained by the close connection between testosterone and insulin production. Insulin reduces the production of sex hormone-binding globulin, increasing active testosterone levels[46]. Inversely, testosterone also promotes insulin production in pancreatic beta cells via non-genomic actions of AR[47].

Insulin has been proposed as a risk factor for PCa patients because of its mitogenic and antiapoptotic activity[48]. However, its role in tumour progression is still poorly understood and mechanistically limited. TRAMP mice show higher insulin levels, and in agreement, castrated TRAMP mice show lower blood insulin levels. Interestingly, hormone deprivation therapy in humans leads to insulin resistance[49, 50], which is associated with both the effect found in androgen deprivation therapy and the role of insulin in PCa progression. Perhaps one of the most interesting data in this regard is that the risk of cancer incidence associated with diabetes is decrease in the prostate, being the molecular scenario still discussed. We demonstrated experimentally that T1D protects TRAMP mice of prostate cancer. Diabetic mice induced by STZ treatment reversed completely prostate carcer phenotype. We found that the levels of PCNA, a proliferation marker in PCa[51], was reduced and the pro-apoptotic protein BAX was increased in STZ-treated mice, and that insulin reverted this effect. Insulin stimulates IGF secretion, which is related to PCa aggressiveness[52], and IGFBP has been proposed as a predictive marker in advanced PCa, like PSA[53]. However, in TRAMP mice, IGF-1R or IGFBP3 levels do not change after STZ-induced diabetes or insulin treatment.

One aspect to consider is that T1DM reduces visceral obesity. Periprostatic fat influences cancer progression through the secretion of adipokines, proinflammatory cytokines[54]. Moreover, fatty acids are important nutrients in the prostate[55]. In diabetic TRAMP mice GLUT4 glycosylation is highly reduced. The activation of AKT might be related to the increased production of GLUT4 in tumoral tissue. However, AKT activity is inhibited in diabetic TRAMP mice.

The importance of GLUT4 in cancer has been proposed in oral squamous cell carcinoma patients. Here, GLUT4 was significantly associated with a poor overall survival and recurrence-free survival. In addition, the ectopic overexpression of GLUT4 in cell lines caused a significant increase in migration in vitro and in vivo, whereas GLUT4 silencing reversed the cancer phenotype[56]. Also, it has been shown that GLUT4 plays a major role in basal glucose uptake in breast cancer cells[57], and specific knockdown of GLUT4 in breast cancer cell lines impairs glucose uptake and reduces lactate production and more recently, the involvement of ALKBH5-mediated m⁶A RNA demethylation in control of GLUT4 expression and the sensitization of HER2-targeted therapy in breast cancer.[58]

In conclusion, diabetes and cancer are two aged-associated pathologies more prevalent each year in western countries. Targeting dysregulated glucose metabolism has focused attention on the therapy of cancer. However, challenging both is by itself a new difficulty in clinics. We experimentally demonstrated in this work that T1D induced by STZ injection avoids prostate cancer progression in the TRAMP mice. It seems insulin might promote PCa progression by incrementing GLUT4 independently of androgen signalling. Our results prove for the first time, the relevance of insulin-dependent glucose transporters in the progress of prostate tumours in vivo. They also suggest their value as therapeutic targets, particularly in the advanced stages of the tumour, characterised by increased glucose uptake and a glycolytic phenotype.

2DG: Deoxyglucose; 6AN: 6-aminonicotinamide; 6GPD: 6-phosphogluconate dehydrogenase; ACO2: Aconitase; AR: Androgen Receptor; AUC: Area Under the Curve; BD: Body Weight; CAS: Castrated; CDX: Bicalutamide; CON: Control; CRPC: Castration-Resistant Prostate Cancer; DAB: Diaminobenzidine; DHT: Dihydrotestosterone; FBS: Fetal Bovine Serum; FBS_CHST: Charcoal-stripped FBS; FDG: Fluorodeoxyglucose; G6PDH: Glucose-6-phosphate dehydrogenase; GlcNAc: N-acetylglucosamine; GLUT: Glucose Transporter; GU: Genitourinary tract; HDAC: Histone deacetylase; HIF: Hypoxia-Inducible Factor; IF: Immunofluorescence; IGF: Insulin-like Growth Factor; IGF1R: IGF1 Receptor; IGFBP: Insulin-like Growth Factor Binding Protein; IHC: Immunocytochemistry; Ins: Insulin; MD: Moderately-differentiated tumor; OXPHOS: Oxidative phosphorylation; PCa: Prostate Cancer; PCNA: Proliferating Cell Nuclear Antigen; PD: Poorly-differentiated tumor; PNGase: N-glycosidase; PPP: Pentose Phosphate Pathway; PSA: Prostate Specific Antigen; SEM: Standard Medium Error; STZ: Streptozotocin; T1DM: Type 1 Diabetes Mellitus; T2DM: Type 2 Diabetes Mellitus; TCA: Tricarboxylic Acid Cycle; TES: Testosterone; TRAMP: Transgenic Adenocarcinoma of Mouse Prostate; WD: Well-differentiated tumor

Ethics approval and consent to participate

This work was carried out in accordance with the World Medical Association Declaration of Helsinki for the ethical principles for Medical Research involving human subjects. All patients were informed, and consent was obtained prior to sample collection and the protocol was approved by “Comite de Etica de la Investigacion del Principado de Asturias”. Experiments and procedures with mice were conducted in accordance with European Directive 2012/63/UE and were approved by the Ethical Committee Board for Animal Experiments at the University of Oviedo.

Consent for publication

All authors consent to publication

Availability of the data and material

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Rosa M Sainz ([email protected]).

Competing interests

The authors declare no competing interests.

Funding

This work was supported by the “Agencia Estatal de Investigacion” (MINECO-BFU2016-79139-R, and MCI-20-PID2019-111418RB-I00) and co-funded by the European Regional Development Fund (FEDER) and by the “Gobierno del Principado de Asturias, Programa de Grupos de Investigacion 2018-2020” (IDI/2018/000239). The EU is acknowledged for the provision of FEDER funds for the purchase of the GC-MS/MS instrument.

Authors' contributions

P.G.M., J.C.M. and R.M.S. designed the study. P.G.M. performed the experiments. A.M.A study GLUT4 production and activation. R.C.C. performed confocal microscopy studies and morphologic analysis. A.A.A. assisted with animal experimentation. D.H., P.R.G. and J.I.G.A. performed mass spectrometry analysis and metabolomics. R.A.A. assisted with flow cytometry. M.A.M., M.D.E. collected and oversaw prostate cancer patients’ and C.L. gather and supervised diabetic patients. P.G.M, J.C.M., D.H, P.R., J.I.G.A. and R.M.S interpreted and discussed the data. P.G.M and R.M.S conceived, analysed the data and wrote the manuscript with assistance from the other authors. All authors critically reviewed the manuscript.

Acknowledgements

P.G.M. thanks the finantial support of “Ministerio de Universidades de España, Subvenciones para la Recualificacion del Sistema Universitario Español María Zambrano (MU-21-UP2021-030). A.M.A was funded by “Asociacion Española Contra el Cancer” (SV-19-AECC-FPI-2). A.A.A. was supported by the University of Oviedo “Ayudas predoctorales para la realización de tesis doctorales modalidad A” (PAPI-18-PF-06). C.L is recipient from a Margarita Salas Post-doctoral grant from the University of Barcelona. We thank Sandrina Kinet and Naomi Taylor for their helpful assistance in the labelling and detection of GLUT1. We thank Marta Alonso-Guervos and Ana Salas Bustamante for their technical support with the confocal microscopy and flow cytometry assays. We thank Scientific and Technical Services (SCTs) from the University of Oviedo for their technical support, including biological, biotechnical, and biomedical testing, optical microscopy and image processing, mass spectrometry, and the animal facility. We also thank the Molecular Histopathology in Animal Models of Cancer of the IUOPA for the processing and analysis of prostate samples.

Materials & correspondence

Rosa M. Sainz, PhD. Department of Morphology and Cell Biology, University Institute of Oncology of Asturias (IUOPA). School of Medicine, C/Julian Claveria 6, 33006 Oviedo, SPAIN. Phone # 34 985103610, e-mail: [email protected]

Pedro Gonzalez-Menendez, PhD. Department of Morphology and Cell Biology, University Institute of Oncology of Asturias (IUOPA). School of Medicine, C/Julian Claveria 6, 33006 Oviedo, SPAIN. Phone # 34 985103610, e-mail: [email protected]

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Insulin-dependent GLUT4 is a risk factor for cancer in the prostate

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Abstract

Figures

Background

Methods

Cell culture, transfection, and treatments

Animal procedure

Patient samples

PAGE and immunoblotting

Immunocytochemistry and Immunohistochemistry studies

AR and GLUT-4 immunostaining in human biopsies

Flow cytometry

Real-time PCR

Metabolic assays

Stable isotope labelling experiments

Extracellular lactate levels

PSA levels

Fasting blood glucose and glucose tolerance test

Blood insulin levels

Blood testosterone levels

Statistics

Results

Androgens regulate insulin independent GLUT transporters in androgen dependent LNCaP cells

Overexpression of GLUT4 favours an androgen-independent phenotype

Hormone resistant cells have an increase in glucose uptake and a glycolytic metabolism

Facilitative insulin-independent and -dependent GLUT levels increase with PCa progression

TRAMP mice have higher levels of insulin and diabetes slows prostate cancer progression in TRAMP mice

GLUT4 is increased in prostate cancer tissues

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

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