ALKBH5 enhances lipid metabolism reprogramming by increasing stability of FABP5 to promote pancreatic neuroendocrine neoplasms progression in an m6A-IGF2BP2-dependent manner

The process of post-transcriptional regulation has been recognized to be significantly impacted by the presence of N6-methyladenosine (m6A) modification. As an m6A demethylase, ALKBH5 has been shown to contribute to the progression of different cancers by increasing expression of several oncogenes. Hence, a better understanding of the key targets of ALKBH5 in cancer cells could potentially lead to the development of new therapeutic targets. However, the specific role of ALKBH5 in pancreatic neuroendocrine neoplasms (pNENs) remains largely unknown. Here, we demonstrated that ALKBH5 was up-regulated in pNENs and played a critical role in tumor growth and lipid metabolism. Mechanistically, ALKBH5 over-expression was found to increase the expression of FABP5 in an m6A-IGF2BP2 dependent manner, leading to disorders in lipid metabolism. Additionally, ALKBH5 was found to activate PI3K/Akt/mTOR signaling pathway, resulting in enhanced lipid metabolism and proliferation abilities. In conclusion, our study uncovers the ALKBH5/IGF2BP2/FABP5/mTOR axis as a mechanism for aberrant m6A modification in lipid metabolism and highlights a new molecular basis for the development of therapeutic strategies for pNENs treatment. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12967-023-04578-6.


Introduction
Neuroendocrine neoplasms (NENs) originate from the neuroendocrine system and frequently manifest in organs including the lungs, pancreas, and pituitary glands [1].Nevertheless, the low incidence rates and the inadequacy of experimental models have impeded our comprehension of the mechanisms underlying NEN development [2].Pancreatic neuroendocrine neoplasms (pNENs) account for 1-2% of pancreatic neoplasms and predominantly arise in the duodenal-pancreatic region, representing the second most frequent pancreatic neoplasm [3].The prevalence of pancreatic neuroendocrine neoplasms (pNENs), once regarded as uncommon heterogeneous neoplasms, has shown an almost twofold increase to 10% in the last two decades [4,5].According to the statistics from SEER, the incidence rate of pNENs exhibited a notable rise of 8.20% between 1996 and 2015.More specifically, the incidence rate of pNET experienced a notable rise of 9.94% among females after 2000, and a significant increase of 7.55% in males following 1993.The upward trend in pNET incidence is expected to persist, with projections indicating that by 2025, the incidence rate will reach 2.23 per 100,000 in females and 2.48 per 100,000 in males [6].The increasing incidence of pNET could also be partly attributed to a higher number of diagnoses made through imaging studies [7].As the most frequent primary sites of NENs, surgery is currently the sole potential curative measure for pNENs [8].However, over half of the patients with pNENs present with locally advanced disease or distant metastasis at the time of diagnosis, resulting in an overall median survival of just 3.6 years [7,9,10].The treatments available for advanced pNENs comprise somatostatin, molecular targeted agents, cytotoxic chemotherapy agents, and immune checkpoint inhibitors [11,12].Nonetheless, the current drug regimens for pNENs have demonstrated limited therapeutic efficacy according to past studies [13].Therefore, research examining the cellular and molecular mechanisms underlying the development of pNENs is urgently required and may pave the way for developing novel therapeutic strategies for patients with advanced diseases.
Metabolic reprogramming is a prevalent hallmark of tumorigenesis, present in many types of cancer, and is considered one of the 14 vital hallmarks [24].Cancer cells harbor unique metabolic characteristics, such as glucose, lipid, and amino acid metabolism, which facilitate the production of biomass that supports cell duplication and other vital hallmarks of cancer [25].For glucose metabolism, to support the rapid growth and proliferation of the tumor, cancer cells exhibit heightened glycolysis levels, even when there is excessive oxygen available; this phenomenon is known as the "Warburg effect, " and it is a hallmark of cancer [26].Additionally, lipid metabolism also plays a crucial role in determining the fate and function of tumor cells, alongside glucose and amino acid metabolism.Although abnormal lipid metabolism in tumor cells has not received as much attention, in recent years, its significance has been increasingly acknowledged.Functioning as lipid transporters, FABPs play an active role in transporting lipids to various cellular components, such as the lipid droplet, endoplasmic reticulum, mitochondria or peroxisome, and nucleus, as well as facilitating autocrine or paracrine signaling outside of the cell [27].FABP5, a petite 15 kDa cytoplasmic protein, mainly participates in the uptake, transport, and metabolism of fatty acids in cell cytoplasm [28].Present studies have revealed that FABP5 plays significant roles in different types of cancers, such as lung cancer [29], breast cancer [30], bladder cancer [31], hepatocellular carcinoma [32], clear cell renal cell carcinoma [33], multiple myeloma [34] and so on [35].However, the role of FABP5 in pNENs remains unclear and needs to be further explored.
In this work, we demonstrated the high expression and carcinogenesis of ALKBH5 in pNENs progression in vitro and in vivo.Further studies suggested that ALKBH5 promoted lipid metabolism by increasing the expression of FABP5 in an m6A-IGF2BP2-dependent manner, which subsequently promoted the malignant behaviors of pNENs.Conversely, the down-regulation of FABP5 could reverse the increase in lipid metabolism and tumor growth.Overall, our findings identified m6A-modified FABP5 as a novel metabolic regulator in pNENs development which may be a potential therapeutic target for pNENs treatment.

Cell culture and tissue samples
The human pNENs cell line QGP-1 was obtained from the JCRB cell bank (JCRB0183), which was cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS, Yeasen, Shanghai, China), and 1% penicillin-streptomycin.In addition, we also isolated the primary human pNENs cells (we named it PNET) from the pNENs tissues of patients diagnosed with pNENs.The primary human pNENs cell (PNET) were cultured in McCoy's 5A medium.All cells were cultured in a humidified incubator with 5% CO 2 at 37 °C.All pNENs tissues and matching adjacent normal tissues were obtained from Jiangsu Province Hospital and were diagnosed as pNENs by Pathology Department of Jiangsu Province Hospital and all consents were signed by every participant.

Cell proliferation assays
To evaluate the cell proliferation ability, Cell Counting Kit-8 (CCK-8, New Cell & Molecular Biotech), colony formation, and EdU assays were performed.For the CCK-8 assay, 5 × 10 3 cells were seeded in a 96-well plate with 5 replicates per well and then incubated at 37 °C for 2 h.The absorbance at 450 nm was monitored continuously for 4 days.For the colony formation assay, 2.0 × 10 4 cells were added to 6-well plates, and the process was repeated thrice.Following 14 days of incubation, the plates were rinsed twice with phosphate-buffered saline (PBS), fixed with paraformaldehyde for 15 min, and stained with 0.1% crystal violet solution for 15 min for subsequent analysis.As for the EdU assay, cells in a 96-well plate were subjected to 50 μM EdU for 2 h and then stained according to the instructions provided (RiboBio, Guangzhou, China).

Cell migration and invasion assays
To determine cell migration and invasion capacity, we employed 24-well cell culture plates with 8-μm micropore inserts.For cell migration assays, 1 × 10 5 cells in serum-free medium were placed in the upper chamber and incubated for 48 h.For cell invasion assays, 2 × 10 5 cells were seeded in the upper chamber with 50 μL of Matrigel (Becton, Dickinson) and incubated for 48 h.In both cases, the lower chamber was filled with a conditioned culture medium containing 30% FBS.After 48 h, cells that had invaded the bottom were fixed with 4% paraformaldehyde and stained with 0.25% crystal violet solution for 30 min.

Assays of lipid metabolism
To quantify the lipid droplets in cells, we performed Nile red staining.pNENs cells that were transfected were seeded in 96 well plates.Once the cells had attained confluency between 60 and 80%, they were fixed in 4% paraformaldehyde for 15 min.Afterward, the cells were incubated with Nile Red working fluid for 20 min, followed by staining with DAPI (Beyotime, Nantong, China) for 20 min at room temperature.The fluorescence intensity imaging of Nile Red and DAPI was acquired using fluorescence microscopy.The fluorescence intensities were quantified using ImageJ software.Quantification of fatty acids (FAs) was performed by using a CheKine ™ Micro Free Fat Acid (FFA) Assay Kit (abbkine).Triglyceride and cholesterol contents were performed using EnzyChrom triglyceride and cholesterol kits (Bioassay Systems).All assays were performed following the manufacturer's instructions.

Quantitative real time-polymerase chain reaction (qRT-PCR) and RNA-seq
Total RNA from cells was isolated using the trizol reagent (Vazyme, Nanjing, China).RNA was quantified using a Nanodrop 2000.Subsequently, 5 µg of RNA was used for reverse transcription with the PrimeScript RT Reagent Kit with gDNA Eraser (Yeasen, Shanghai, China).Realtime PCR was then conducted according to the manufacturer's instructions using ChamQ Universal SYBR qPCR Master Mix.For RNA-seq, RNA samples were sequenced by Lianchuan Biotech (Hangzhou, China) and analyzed using the OmicStudio tools at https:// www.omics tudio.cn/ tool.The primers utilized are specified in Additional file 1: Table S1.

Protein extraction and western blot analysis
Total proteins from cultured cells were lysed with cold NP40 lysis buffer (Beyotime) containing a protease inhibitor cocktail (Roche, Mannheim, Germany).The protein concentration was determined using a BCA Protein Assay Kit (Beyotime).Samples were separated on an 10% gel by SDS-PAGE.Nitrocellulose membranes were blocked with a blocking buffer and incubated with the appropriate primary antibody.The membranes were then blocked with 8% skim milk in TBST, incubated with primary antibodies (listed in Additional file 1: Table S3) overnight at 4 °C, and then incubated with HRP-conjugated secondary antibodies for one hour at room temperature.The blots were imaged with Immobilon ™ Western Chemiluminescent HRP Substrate (Millipore) and the ChemiDoc ™ XRS + imaging system (Bio-Rad).

Plasmid construction
To enable ALKBH5 and FABP5 over-expression, we amplified and cloned the cDNA encoding the ALKBH5 and FABP5 CDS region into the pCDH-CMV-MCS-EF1-Puro lentivirus vector.Similarly, we used recombinant lentiviruses containing sh-ALKBH5, sh-IGF2BP2, or FABP5 in the PLKO1 vector, to construct ALKBH5, IGF2BP2, and FABP5 knockdown stable cell lines (listed in Additional file 1: Table S2).These plasmids were from Genomeditech (Shanghai, China).We accomplished plasmids transfection in 293 T cells using PEI (Polysciences, USA) with serum-free medium, followed by the addition of the corresponding serum after 6 h.Subsequently, we harvested the viral supernatant through a 0.45-μm filter after 48 h and applied it to cells having 50% confluence.Treatment with 2 μg/ml puromycin for 7 days was used to select stable cell lines.

Immunofluorescence
Cells were planted in 96 well plates overnight to reach confluency between 60 and 80%.Next, cells were washed with PBS three times and then fixed with 4% paraformaldehyde for 15 min.Then, cells were incubated with 0.2% Triton X-100 for 15 min and blocked in 3% BSA for 30 min.After three times washing of PBST, the cells were incubated at 4° overnight with primary antibody against ALKBH5 and FABP5.Then cells were washed

RNA stability assay
Cells were treated with actinomycin D (MCE, China) at a final concentration of 5 μg/mL for the indicated time periods and collected.Total RNAs were extracted and analyzed with qRT-PCR.

Quantification of global N6-methyladenosine levels
To determine the global level of RNA N6-methyladenosine (m6A), we employed the EpiQuik m6A RNA Methylation Kit (Epigentek, USA) in our study.Initially, we isolated total RNA from cells or tissues utilizing TRIzol reagent and proceeded to bind the RNA onto strip wells using the RNA high-binding solution.Subsequently, we added capture and detection antibodies in a sequential manner into each well.Finally, we compared and measured the absorbance value at a wavelength of 450 nm to ascertain the relative m6A level.

Methylated RNA immunoprecipitation sequencing (MeRIP-seq)
The target gene was selected by MeRIP using MeRIP m6A Kit (Merck Millipore) following the provider's requirements.Specially speaking, total RNAs were extracted from ALKBH5 knockdown or empty vector QGP-1 cells using Seq-Star Poly(A) mRNA Isolation Kit.Next, the RNA was fragmented and incubated with m6A antibody to deposit FABP5.After the concentration of m6A mRNA fragment and construction of the RNA-seq library for sequencing on the Illumina HiSeq 4000 platform.Subsequently, the abundance of FABP5 was tested by qRT-PCR and normalized to the input mRNAs.

RNA immunoprecipitation (RIP) assays
In accordance with the manufacturer's instructions, the Magna RIP Kit (17-700, Millipore, MA) was utilized to perform the RIP assay.Specifically, 5 μg of anti-ALKBH5 (Abcam, USA), anti-IGF2BP2 (Abcam, USA), or anti-N6-methyladenosine (m6A) (Abcam, USA) and anti-rabbit IgG (Millipore, Germany) were incubated with 40 μL magnetic beads, prior to the addition of cell lysates (approximately 5 × 10 7 cells per sample).Next, the RNA-protein IP complexes were washed six times.Following treatment with proteinase K, the RNAs of interest were extracted and purified from the immuno precipitated complex for further qRT-PCR analysis.The relative enrichment was normalized with the input.

Mouse xenograft model
For tumor xenograft models, QGP-1cells (5 × 10 6 ) with ALKBH5 over-expression, ALKBH5 over-expression with FABP5 knockdown, and negative control were subcutaneously injected into the right axilla of female BALB/c nude mice (4-6 weeks).After 4 weeks, the mice were sacrificed via a form of euthanasia.The tumors were weighed, imaged, and fixed in 4% paraformaldehyde or frozen for further analysis.Tumor volume was measured by the following formula: volume = length × width 2 × 1/2.All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University.

Statistical analysis
Statistics were analyzed using GraphPad Prism 8.0 (GraphPad, Inc., USA).Comparisons between different groups were calculated using Student's t-test.Experiments were independently repeated at least three times.Representative data was exhibited as the means ± SD. p-values for every result were labeled on figures, and p < 0.05 was reckoned as statistically significant.

ALKBH5 is increased in pancreatic neuroendocrine neoplasms
To investigate the potential role of ALKBH5 in pNENs, we initially examined the expression of ALKBH5 in tumor tissues and normal tissues by using an immunohistochemistry (Fig. 1A).Subsequently, we analyzed the expression of ALKBH5 protein in four pairs of pNENs and found that the most of them exhibited significantly higher expression levels than the corresponding normal adjacent tissues (Fig. 1B).Consistent with these results of tissues, qRT-PCR and western blot analyses revealed that pNENs cell lines also displayed elevated levels of ALKBH5 mRNA and protein expression compared to normal pancreatic cells (Fig. 1C, D).Furthermore, the (See figure on next page.)Fig. 3 ALKBH5 regulates lipid metabolism.A Enrichment analysis of KEEG and GO signal pathways.B Individual GSEA plots of fatty acid elongation, glycerolipid metabolism, and biosynthesis of unsaturated acid pathway in RNA-seq data from QGP-1 cells with ALKBH5 knockdown.C, D LDs (lipid droplets) were detected using Nile red in indicated cells with ALKBH5 over-expression, magnification: ×200, along with the results of relative fluorescence intensity.E-G The relative amounts of free fatty acids, triglycerides, and cholesterol were measured in cells with ALKBH5 over-expression.H, I The mRNA expression of dysregulation of genes involved in fatty acid synthesis (ACSL1, ACSL3, FABP5, SCD1, ACACA, MLYCD), fatty acid uptake (CD36), cholesterol biosynthesis (SREBF2 and HMGCR ) in QGP-1 cells with ALKBH5 knockdown and over-expression.*p < 0.01, **p < 0.01, ***p < 0.001 results of the immunofluorescence assay indicated the altered expression of ALKBH5 in pNENs cell lines compared to the normal pancreatic cells (Fig. 1E).To investigate the potential involvement of m6A modification in pNENs, we also compared the global m6A levels in pNENs cell lines to those of normal pancreatic cells.The findings suggest that the overall levels of global m6A expression were significantly lower in pNENs compared to the normal pancreatic cells (Fig. 1F).Finally, we also examined the protein expression of some common m6A regulators (such as FTO, ALKBH5, METTL3, WTAP, METTL14, and YTHDC1) which indicated the role of the m6A modification in the progression in pNENs (Additional file 1: Figure S1A).The results showed the m6A writers highly expressed while m6A erasers showed lower expression which indicated the high m6A modification in pNENs.Taken together, these results highlight the possible indispensable role of ALKBH5 in the development and progression of pNENs.

ALKBH5 promotes the malignant progression of pNENs in vitro and in vivo
To explore the potential promoting effects of ALKBH5 on tumor development, lentiviral transfection technology was utilized to construct the stabled transfected cell lines with ALKBH5 knockdown and ALKBH5 overexpression in QGP-1 cells (Fig. 2A, B).ALKBH5 inhibition led to a significant reduction in proliferation and colony formation in pNENs cells, as indicated by the results of the CCK8 assay and colon formation, while ALKBH5 overexpression resulted in the opposite effects.(Fig. 2C-F).Furthermore, the EdU assay also revealed that DNA replication activity in QGP-1 cells could be reduced in ALKBH5-deficient cells and enhanced in ALKBH5-overexpression cells (Fig. 2G-J).Additionally, the migration and invasion ability of QGP-1 cells were also impaired by ALKBH5 depletion and ALKBH5 overexpression (Fig. 2K-N).These results suggested that ALKBH5 serves as an oncogene driving pNENs development.
Moreover, the stable transfected cell lines of ALKBH5 knockdown and overexpression in PNET were also constructed which showed significantly down-regulated and up-regulated ALKBH5 mRNA and protein levels (Additional file 1: Figure S1B, C).The CCK8 assay and colon formation assay both showed that ALKBH5 knockdown and over-expression in PNET caused significant inhibition and enhancement of cell viability and colony forming ability (Additional file 1: Figure S1D-G).The result of the EdU assay also showed that ALKBH5 knockdown and overexpression had the opposite effects on the DNA replication activity in PNET cells (Additional file 1: Figure S1H-K).Finally, the transwell assay also revealed that ALKBH5 knockdown inhibited the migration and invasion ability of PNET cells shown by the amount of the stained cells at the bottom of the chamber while ALKBH5 overexpression showed the opposite results (Additional file 1: Figure S1L-O).To explore the function of ALKBH5 in vivo, we construct the tumor xenograft models using QGP-1 cells with ALKBH5 knockdown.The results revealed that ALKBH5 knockdown inhibited tumor growth and weight (Fig. 1O-Q).The above results indicate that ALKBH5 plays a crucial role in tumor development.

ALKBH5 regulates fatty metabolism pathways
To determine potential ALKBH5-regulated signaling pathways, we initially performed RNA-seq between ALKBH5 depletion cells and control cells.The signal about tumor growth, including cell growth and death, cell division, angiogenesis, and G2/M transition of the mitotic cell cycle were significantly enriched, and correspond to the previous results of cell phenotype.The classical MAPK signaling pathway and PI3K-Akt-mTOR signaling pathway were expectedly enriched.Interestingly, two lipid metabolism-related pathways (lipid metabolism and response to fatty acid) were also identified by KEGG analysis and GO enrichment respectively (Fig. 3A).As mentioned before, dysregulated lipid metabolism plays an indispensable role in many cancers.However, the role of lipid metabolism in pNENs influenced by ALKBH5 has not been explored.Therefore, we next focused on the specific mechanism of ALKBH5 on lipid metabolism in pNENs.We firstly observed the biosynthesis of unsaturated fatty acid, fatty acid elongation, and glycerolipid metabolism pathways were significantly downregulated in ALKBH5-knockdown cells revealed by GSEA Fig. 4 FABP5 is a functionally important target gene of ALKBH5.A Differentially expressed genes between ALKBH5 knockdown and control groups in QGP-1 cells as determined by RNA-sequencing.B Peak profiles in m6A modification after ALKBH5 knockdown in QGP-1 cells as shown by MeRIP-sequencing.C The m6A consensus motif is present in QGP-1 cells.D Distribution of m6A peaks across the length of mRNAs in QGP-1 cells with or without ALKBH5 knockdown.E The volcano plot showed the distribution of genes both differential (up or down) methylation level and differential (up or down) gene expression level in ALKBH5 knockdown and control groups.F Venn diagram showed the down-regulated genes, genes with elevated m6A methylation levels, and lipid-associated genes after ALKBH5 knockdown.G, H The validation of four candidate genes was verified by qRT-PCR in QGP-1 cells.I The protein expression of FABP5 in cells with ALKBH5 knockdown.J The relative abundance of m6A sites along FABP5 mRNA in QGP-1 cells with or without ALKBH5 knockdown, as detected by MeRIP-seq.*p < 0.01, **p < 0.01, ***p < 0.001 (See figure on next page.)analysis (Fig. 3B).Moreover, we also detected the difference in the related index of lipid metabolism.The results revealed that ALKBH5 over-expression increased the total lipid droplet levels (Fig. 3C, D), suggesting increased lipid storage.ALKBH5 over-expression also decreased the content of free fatty acid which showed the activity of lipolysis may be enhanced by ALKBH5 (Fig. 3E).The amounts of total cholesterol and triglycerides also increased in cells with ALKBH5 over-expression (Fig. 3F,  G).In addition, abnormal lipid metabolism could be induced by the enhancement of lipid biosynthesis, decreased lipid catabolism, and increased fatty acid uptake.Hence, we also analyzed the expression of key molecules in QGP-1 cells with ALKBH5 knockdown and overexpression involved in lipid metabolism (Fig. 3H, I), including fatty acid synthesis (ACSL1, ACSL3, FABP5, SCD1, ACACA, MLYCD), fatty acid uptake (CD36), cholesterol biosynthesis (SRBF2, HMGCR ).The mRNA expression results revealed that the majority of molecules related to fatty acid synthesis, especially for FABP5, and fatty acid uptake were promoted by ALKBH5.However, the molecules related to cholesterol biosynthesis seem to be weakly influenced by ALKBH5.Taken together, the above results all showed that ALKBH5 may play a crucial role in regulating lipid metabolism for the development of pNENs.

FABP5 has been identified as the target gene regulated by ALKBH5
To further insight into the mechanism of ALKBH5 regulating lipid metabolism in gene expression, both transcriptome and epitranscriptome sequencing in the QGP-1 cells inhibiting ALKBH5 and the vector group were performed.For the transcriptome sequencing data, a total of 537 genes was identified with significant differences and P-value was less than 0.05, including 433 up-regulated and 103 down-regulated genes (Fig. 4A).For the meRIP-seq date, we identified 16,950 unique m6A peaks in control group, 20,395 unique m6A peaks in sh-ALKBH5 group and 24,632 shared m6A peaks in both groups (Fig. 4B).Furthermore, the m6A modification peaks were mainly enriched in the intron region of the genes in both groups, and the UGGAC was the most common consensus motif in pNENs with ALKBH5 knockdown (Fig. 4C).ALKBH5 knockdown increased m6A enrichment primary in the 3ʹUTR and 5ʹUTR region (Fig. 4D).Next, we combined Methylated RNA immunoprecipitation (MeRIP) with an m6A-specific antibody followed by RNA sequencing (MeRIP-seq) and RNA sequencing to accurately identify the downstream targets of ALKBH5 (Fig. 4E).According to the overlapping part of RNA-seq and MERIP-seq between control and ALKBH5 knockdown, we found 201 genes with significant differences.Among this, we also eliminated 113 genes with down-regulated m6a levels which were inconsistent with high global m6A levels regulated by ALKBH5 knockdown.Moreover, the remaining four genes involved in lipid metabolism were singled out, including CHAT, FABP5, STARD9, and BPIFB2 (Fig. 4F).We next verified the mRNA levels of these candidate genes.The results showed FABP5 was the gene most significantly changed examined by qRT-PCR in ALKBH5 knockdown and overexpression cells (Fig. 4G, H).We also verified the protein expression of FABP5 in ALKBH5 knockdown cell lines (Fig. 4I).Furthermore, we predicted the m6A modification sites using IGV analysis.The results indicated that the peak in cells with ALKBH5 knockdown mainly localized in the 5ʹ untranslated region (UTR) (Fig. 4J), which is in line with previous studies suggesting that m6A modification in the 5ʹ UTR is closely associated with energy metabolism.The above results all showed FABP5 seems the target gene regulated by ALKBH5 in the development of cancer lipid metabolism.

ALKBH5 over-expression up-regulated FABP5 mRNA levels in an m6A-IGF2BP2-dependent manner
Next, to further demonstrated whether ALKBH5 regulates FABP5 in an m6A-dependant manner, we first performed the immunofluorescence to examine the expression of the m6A levels in QGP-1 cells with ALKBH5 knockdown (Fig. 5A).Next, we also examined the global m6A levels in ALKBH5 knockdown and over-expression cell lines.The results showed that the m6A levels were also changed in corresponding cell lines (Fig. 5B, C).We also treated QGP-1cells with DAA, an RNA methylation inhibitor, to evaluate the expression of FABP5 in ALKBH5 knockdown cell lines.The result also showed the amount of m6A and FABP5 was (See figure on next page.)Fig. 5 ALKBH5 stabilizes FABP5 mRNA in an m6A-dependent manner.A The expression of m6A in QGP-1 cells with ALKBH5 knockdown was detected by immunofluorescent imaging.B, C The global m6A levels in ALKBH5 knockdown and overexpression cell lines.D The relative expression of FABP5 mRNA in QGP-1 cells treated with DAA.E RIP-qRT-PCR revealing binding enrichment of m6A to FABP5 mRNA in QGP-1 cells with or without ALKBH5 knockdown.F The relative FABP5 mRNA expression in cells with IGF2BP2 knockdown.G The protein expression of FABP5 in QGP-1 cells with IGF2BP2 knockdown was verified by western blots.H RIP-qRT-PCR revealing binding enrichment of IGF2BP2 to FABP5 in QGP-1 cells.I Individual GSEA plots of regulation of mRNA metabolic process and regulation of mRNA stability pathway in RNA-seq data from QGP-1 cells with ALKBH5 knockdown.J FABP5 mRNA half-life (t1/2) was tested at the indicated time points by qRT-PCR in QGP-1 cells with ALKBH5 knockdown.K The knockdown rate of IGF2BP2 was verified by western blots.IG2BP2 inhibition inhibits the growth and motility of pNENs.The CCK-8 (L), colony formation (M, N), and EdU (O, P) assays were applied to evaluate the proliferation ability of QGP-1 cells with the knockdown of IGF2BP2.Q, R Transwell assays of QGP-1 cells with IGF2BP2 knockdown were applied to measure their migration and invasion abilities, magnification: × 100.*p < 0.01, **p < 0.01, ***p < 0.001 negatively correlated (Fig. 5D).In addition, we conducted RIP to investigate the close interaction between m6A and FABP5.It revealed that the successful combination between m6a and FABP5 and the less combination in ALKBH5 over-expression cells (Fig. 5E).Next, we also examined the specific readers when FABP5 was regulated by ALKBH5.IGF2BP2 was predicated to bind and recognized these m6A modifications by the software prediction and verified by qRT-PCR and western blots (Fig. 5F,  G).RIP results also indicated that IGF2BP2 directly binds FABP5 (Fig. 5H).The above results all revealed that IGF2BP2 interacted with FABP5 through m6A modification.Moreover, it has been demonstrated ALKBH5 plays an indispensable role in mRNA stability [36].From the sequencing of me-RIP, we found the m6A modification of FABP5 was located in 5ʹUTR which may participate in mRNA stability.GSEA results based on RNA-seq data showed that the RNA metabolic process and regulation of mRNA stability signaling pathway was inhibited in the ALKBH5 knockdown group compared to the control group (Fig. 5I).By treating cells with ActD to test this hypothesis, which inhibits nascent mRNA transcription, we detected the remaining intracellular mRNA in QGP-1cells with or without ALKBH5 knockdown.We found that the expression of remaining mRNA levels of FABP5 deceased much faster compared with control cells, suggesting that ALKBH5 sustained the mRNA stability of FABP5 (Fig. 5J).To further explore the role of IGF2BP2 functions in pNENs, we also knockdown the expression of IGF2BP2 in QGP-1 cells (Fig. 5K) and performed a series of phenotypic experiments in IGF2BP2 deficient cells which revealed the carcinogenic effect of ALKBH5 played in pNENs (Fig. 5L-R).

FABP5 restores the malignant effects of ALKBH5 in pNENs in vitro and in vivo
To further examine the role of FABP5 regulated by ALKBH5 in cell lines, including the phenotype of proliferation and lipid metabolism, a series of rescue experiments in QGP-1 cells were performed in ALKBH5 over-expression cell lines with or without FABP5 knockdown.First, we constructed the stable ALKBH5 over-expression with FABP5 knockdown cell lines using lentivirus verified by western blots (Fig. 6A, B).ALKBH5 over-expression promoted cell proliferation and increased the colony formation of QGP-1 cells, which could be rescued by stable knockdown of FABP5 (Fig. 6C-E).Rescue with knockdown of FABP5 also restored the increased DNA replication capacity caused by ALKBH5 over-expression in QGP-1 cells (Fig. 6F, G).Moreover, FABP5 inhibition could also partly rescue the ability of migration and invasion in ALKBH5 over-expression cell lines (Fig. 6H,  I).Next, some changes in lipid metabolism were also detected in ALKBH5 over-expression QGP-1cells with or without FABP5 knockdown.The results showed that cells over-expressed ALKBH5 with more accumulation of lipid droplets, less free fatty acids, and higher amounts of cholesterol and triglyceride, which could be rescued by FABP5 knockdown (Fig. 6J-N).Moreover, in line with in vitro results, ALKBH5 over-expression led to the higher rate of tumor formation in subcutaneous xenograft models of QGP-1 cells, proved by the higher tumor weight and volume, which could also be rescued by FABP5 knockdown (Fig. 6O-Q).Moreover, the level of ALKBH5 and Ki-67 was also enhanced by ALKBH5 over-expression and reduced by FABP5 knockdown again (Fig. 6R).The above results indicated that ALKBH5-medicated FABP5 plays a crucial role in lipid metabolism and tumor development.

FABP5 promotes the proliferation, migration, invasion, lipid metabolism of pNENs
To further evaluate the biological function of FABP5, we initially examined the expression of FABP5 in pNENs tissues and pNENs cell lines verified by immunohistochemical staining and immunofluorescence (Fig. 7A, Additional file 1: Figure S2A).Next, we knocked down and over-expressed FABP5 by lentivirus in pNENs.The protein levels of FABP5 in QGP-1 cells and PNET were detected by western blotting respectively.(Fig. 7B, C, Additional file 1: Figure S2B-C).Subsequently, CCK8 assay and colon formation were performed to evaluate the proliferation rates of pNENs cells.Silencing FABP5 inhibited the proliferation of pNENs cells, whereas FABP5 over-expression increased pNENs cell proliferation (Fig. 7D-G, Additional file 1: Figure S2D-G).EdU (See figure on next page.)Fig. 7 The altered expression of FABP5 promotes proliferation, migration, and lipid metabolism of QGP-1 cells.A Representative images of FABP5 immunohistochemistry staining in pNENs samples were shown.B, C The knockdown and overexpression rate of FABP5 was detected by western blotting.D-J FABP5 knockdown inhibited the growth of pNENs, indicated by the results of CCK-8 (D, E), colony formation (F, G), and EdU (H-K) assays in QGP-1 cells with FABP5 knockdown and overexpression, Edu magnification: ×200.L-O The transwell analysis was conducted to examine the effect of FABP5 knockdown on the migration and invasion capabilities of QGP-1 cells, magnification: ×100.P LDs (lipid droplets) were detected using Nile red in QGP-1 cells with FABP5 knockdown and overexpression, along with the results of relative fluorescence intensity, magnification: ×200.Q-S The relative amounts of free fatty acids, triglycerides, and cholesterol were measured in QGP-1 cells with FABP5 knockdown.*p < 0.01, **p < 0.01, ***p < 0.001, ****p < 0.0001 assay also showed that the DNA replication capacity of pNENs cells was hampered by FABP5 knockdown.Conversely, ectopic FABP5 enhanced DNA replication activity in pNENs cells (Fig. 7H-K, Additional file 1: Figure S2H-K).In addition, we also evaluated the function of FABP5 in migration and invasion by transwell assay.The results revealed that silencing FABP5 resulted in a lower migration and invasion capacity evidenced by less stained pNENs cells at the bottom of the chamber.Conversely, over-expression of FABP5 enhanced the ability of migration and invasion in pNENs cells (Fig. 7L-O, Additional file 1: Figure S2L-O).To further prove the role of FABP5 in lipid metabolism in pNENs.The Nile red staining showed more lipid droplet accumulation in FABP5 over-expression cell lines (Fig. 7P, Additional file 1: Figure S2P).Similarly, FABP5 knockdown could increase the amounts of free fatty acids in QGP-1 cells and PNET cells (Fig. 7Q, Additional file 1: Figure S2Q).Triglyceride and total cholesterol levels were decreased in FABP5-knockdown cells (Fig. 7R-S, Additional file 1: Figure S2R-S).The above results revealed FABP5 may play an indispensable role in lipid metabolism.

ALKBH5 causes the accumulation of lipids in pNENs through PI3K/Akt/mTOR axis
Here, we focus on the PI3K/Akt/mTOR signaling pathway through the results of KEEG.To investigate the role of ALKBH5 in regulating the PI3K/Akt signaling pathway in pNENs.We examined the expression of key markers of PI3K/Akt/mTOR signaling pathway, the results showed that the expression of PI3K, P-Akt, and P-mTOR were all up-regulated in cells with ALKBH5 knockdown, whereas the expression of these markers showed the opposite behaviors in cells with ALKBH5 over-expression (Fig. 8A-C).Moreover, to prove the role of FABP5 regulated by ALKBH5 for the PI3K/Akt/mTOR signaling pathway.We also examine these markers in cells with ALKBH5 over-expression and ALKBH5 over-expression with FABP5 knockdown.It indicated that ALKBH5 overexpression could activate the PI3K/Akt/mTOR signaling pathway.However, it was also been partly rescued by the inhibition of FABP5 in ALKBH5 over-expression cells (Fig. 8D).The above results all showed the PI3K/Akt/ mTOR signaling pathway was regulated by ALKBH5 and regulated by FABP5.We next examine whether the ability of lipid formation induced by ALKBH5 could be compromised by Rapamycin, a kind of mTOR inhibitor, the results revealed that this inhibition abrogated the lipid formation ability of QGP-1 cells enhanced by ALKBH5.(Fig. 8E-I).

Discussion
ALKBH5, an important component in maintaining the dynamic balance of m6A modification, has become increasingly recognized as a widely up-regulated biomarker in the development of different cancers, including head and neck squamous cell carcinoma [37], colorectal cancer [38], epithelial ovarian cancer [39], pancreatic neuroendocrine neoplasms [40], and is correlated with poor clinical prognosis in patients.However, recent studies also reported the inhibitory role of ALKBH5 in many cancers, such as gastric cancer [41], and osteosarcoma [42].Whether ALKBH5 played a role in cancer promoting or cancer inhibiting may depend on the different target genes regulated by ALKBH5.Alternatively, ALKBH5 is also regulated by different gens in different tumors.The processing of ALKBH5 functions may involve in the different reading proteins.In addition, the controversial role of ALKBH5 in different cancers might be the existence of genetic and epigenetic heterogeneities among the cancer cell lines and primary tumor specimens utilized by different research groups.Moreover, the regions of the same or different mRNA transcripts bind by ALKBH5 is inconsistent and lead to different fates of the target transcripts.Such as, ALKBH5 promotes the stability of DDX58 mRNA through HNRNPC-mediated RNA stability enhancement due to increased m6A abundance on the 3′-region of DDX58 mRNA [37].ALKBH5-driven 5ʹ UTR m6A demethylation fine-tunes SF3B1 translation to impacts genome stability and leukemia progression [43].Consequently, additional comprehensive investigations are essential to elucidate and resolve these discrepancies.Although there have been reports indicating the upregulation of ALKBH5 in pNENs [40], the precise role and underlying mechanisms of ALKBH5 in pNENs remain elusive.Our study also indicated that the expression of ALKBH5 in pNENs was significantly increased and that m6A modification mediated by ALKBH5 promoted the survival, proliferation, migration, invasion, and lipid metabolism of pNENs cells.
Lipid accumulation is a driving force in tumor development, as it provides tumor cells with both energy and the building blocks of phospholipids for construction of the cell membranes.Increasing evidence underscores the importance of lipid metabolism in both the initiation and progression of tumorigenesis.Consequently, targeting the process of lipid metabolism for cancer is an optimal strategy for anti-cancer treatment.Current studies have shown that abnormal lipid metabolism may be associated with different types of cancer pathogenesis.Such as, altered lipid metabolism was shown to involve in the progression of glioblastoma [44].Yin Yang 1(YY1) facilitated hepatocellular carcinoma cell lipid metabolism and tumor progression [45].Moreover, lipid metabolism also plays an indispensable role in drug resistance [46].For example, Stearoyl-CoA desaturase (SCD1) accelerated lipid droplet formation to alleviate chemotherapyinduced ER stress and increase drug resistance in gastric cancer [47].Interestingly, the altered lipid metabolism was also observed in the tumor micro-environment.Depletion of fatty acid transporter FATP2 in melanoma cells in an aged micro-environment inhibited lipid accumulation and disrupted their mitochondrial metabolism.In addition, the increased level of cholesterols regulated by cholesterol acyltransferase 1 (ACAT1) could enhance CD8 + T cell proliferation and support anti-tumor immunotherapy [48].Interestingly, consistent with the dual role of ALKBH5 played in tumors, FABP5 seems also function contradictory effect in different cancers.In colorectal cancer, FABP5 overexpression exerted an inhibitory influence on cancer progression by reducing lipid accumulation [49].On the contrary, FABP5 also play a carcinogenesis role by promoting lipid metabolism in osteosarcoma [50].This contradictory role may attribute to up-down regulatory mechanisms are different in different cancers, and are also influenced by the interaction of cancer with other organs in the body, thereby warranting further exploration.In the current study, we have uncovered the upregulated ALKBH5 activated FABP5 to promote the lipid metabolism of cancer.
Several studies have demonstrated that the intricate interplay between m6A modification and metabolic reprogramming furnished tumor cells with remarkable adaptability to evolving environmental conditions during tumorigenesis.For the connection between m6A modification and glycolysis, m6A-dependent glycolysis enhances the proliferation of colorectal cancer [51,52].Elevated METTL3 expression promoted tumor angiogenesis and glycolysis in gastric cancer.In addition, the FTO/ m6A/PFKP/LDHB axis is targeted by R-2-hydroxyglutarate, resulting in the suppression of aerobic glycolysis in leukemia.For m6A modification and lipid metabolism, ACSL4 mediates the function of METTL5 on fatty acid metabolism and HCC progression.The aberrant m6A modification promotes lipogenesis and contributes to the progression of hepatocellular carcinoma [53].In addition, the malignant progression of bladder cancer is promoted by m6A-induced lncDBET through FABP5-mediated lipid metabolism [31].In our study, we demonstrated m6A modified FABP5 plays a crucial role in the progression of pNENs through the altered lipid metabolism.Similarity, a similar study in colorectal cancer has also demonstrated the positive connection between ALKBH5 and FABP5 [49].Nevertheless, the role of other key enzymes involved in m6A functions in the lipid metabolism of other metabolic reprogramming in pNENs are still little known.Moreover, the other epigenetics which not restrict the level of RNA, such as DNA methylation, ubiquitination, phosphorylation, and so on, is still little known in the progression of pNENs, especially for metabolic reprogramming.
MTOR signaling pathway, a central signaling pathway controlling tumor metabolism, is one of the signaling pathways that has a fundamental role in the regulation of PI3K/Akt and mTOR signaling pathway function in gastrointestinal cancer [54].In colorectal cancer, m6A methylated EphA2 promotes vasculogenic mimicry via PI3K/Akt signaling [55].Furthermore, METTL3 promotes the proliferation of retinoblastoma cells by activating PI3K-Akt-mTOR signaling pathways [56].Here, we focus on the PI3K/Akt/mTOR signaling pathway through the results of KEEG.The results of western blots have also demonstrated that ALKBH5 medicated FABP5 is include in the involvement of PI3K/Akt/mTOR signaling.Above all, everolimus, a pharmacological mTOR inhibitor widely used for advanced pNENs patient treatment, the lower expression of ALKBH5 may further synergize with everolimus to suppress mTOR activation and inhibit cancer cell growth.
In summary, the current research explored the biological role and mechanism of ALKBH5 on pNENs lipid metabolism and malignment behavior.We demonstrated that m6A modification medicated by ALKBH5 activates the mTOR signaling pathway to promote the lipid metabolism of pNENs through regulating the expression of FABP5, thus promoting the malignant progression of pNENs.

Fig. 1
Fig. 1 Increased expression of ALKBH5 is observed in pNENs.A Representative images of ALKBH5 immunohistochemistry staining in pNENs samples were shown.B The protein expression of ALKBH5 from four pairs of pNENs samples showed by western blots (T: tumor, N: normal).C, D The mRNA and protein expression of ALKBH5 in normal pancreatic cell lines compared to pNENs.E The expression of ALKBH5 was detected by immunofluorescent imaging.F The amount of m6A in normal pancreatic cell lines HPNE and pNENs cell lines PENT and QGP-1.**p < 0.01, ***p < 0.001

Fig. 2
Fig.2ALKBH5 knockdown inhibits proliferation, migration, and invasion of QGP-1 cells in vitro and in vivo.A, B The mRNA and protein expression of ALKBH5 in QGP-1 cells were assessed by qRT-PCR and western blotting (Scr represents scramble which means control group with disrupted rna sequence; Oc represents overexpression control).C, D CCK-8 proliferation assays were carried out in QGP-1 cells with ALKBH5 knockdown and overexpression.E, F Colony formation assays were conducted in QGP-1 cells with ALKBH5 knockdown and overexpression.Column diagrams showed the relative colony numbers of each group.G-J EdU assays were carried out to evaluate the proliferation of cells with ALKBH5 knockdown and overexpression and a positive rate of EdU was calculated, magnification: ×200.K-N Representative images of transwell assays to evaluate the migration and invasion capacity of QGP-1 cells with ALKBH5 knockdown and overexpression, magnification: ×100.Quantification data showed the relative transwell numbers of cells which passed through the chamber membrane.O-Q Representative images of tumors and comparison of the tumor volume and weight between ALKBH5-deficient groups and scramble groups in QGP-1 cells.*p < 0.01, **p < 0.01, ***p < 0.001 (See figure on next page.)

Fig. 6
Fig. 6 Knockdown of FABP5 reverses the proliferation, migration, and invasion of cells with ALKBH5 over-expression.A qRT-PCR and B Western blotting were performed to examine FABP5 expression in different transfected cell groups.C-I Rescue experiments by CCK-8 (C), colony formation (D, E), EdU assays (F, G), and Transwell assays (H, I) were conducted to evaluate the effect of FABP5 interference on the growth and motility of QGP-1 cells with over-expressed ALKBH5.J-N Rescue experiments were performed in QGP-1 cells with over-expressed ALKBH5 to evaluate the effect of FABP5 interference on the amounts of LDs (lipid droplets) (J, K), FFA (free fatty acids) (L), total cholesterol level (M), and triglyceride (N).O-R Representative subcutaneous xenograft tumor image from the indicated groups (O).Tumors were removed 4 weeks after Subcutaneous implantation, followed by volume calculation and weight measurement (P, Q).Representative IHC staining images of ALKBH5, FABP5, and Ki67 (R).*p < 0.01, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 8
Fig. 8 ALKBH5 regulates lipid metabolism of pNENs by modulating PI3K/Akt/mTOR signaling pathway.A KEGG enrichment barplot based on ALKBH5 knockdown and scramble RNA-seq data.B, C Expression levels of PI3K/Akt/mTOR signaling pathway related proteins as determined by western blotting after ALKBH5 knockdown and over-expression in pNENs cells.D Expression levels of PI3K/Akt/mTOR signaling pathway related proteins as determined by western blotting in ALKBH5 over-expression cells with or without FABP5 knockdown.E, F LDs (lipid droplets) were detected using Nile red in QGP-1 cells from the indicated groups, along with the results of relative fluorescence intensity, magnification: ×200.G-I The cellular content of free fatty acids (FFA), triglycerides, and total cholesterol were detected in QGP-1 cells from the indicated groups.*p < 0.01, **p < 0.01, ***p < 0.001 (See figure on next page.)