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Article

Alpha-Ketoglutarate Regulates Tnfrsf12a/Fn14 Expression via Histone Modification and Prevents Cancer-Induced Cachexia

by
Bryan I. Ruiz
,
Xazmin H. Lowman
,
Ying Yang
,
Qi Fan
,
Tianhong Wang
,
Hongmei Wu
,
Eric A. Hanse
and
Mei Kong
*
Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of California, Irvine, CA 92697, USA
*
Author to whom correspondence should be addressed.
Genes 2023, 14(9), 1818; https://doi.org/10.3390/genes14091818
Submission received: 30 August 2023 / Revised: 12 September 2023 / Accepted: 15 September 2023 / Published: 19 September 2023
(This article belongs to the Special Issue Signaling and Gene Regulation in Metabolism)
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Abstract

:
Previous studies have shown that inhibition of TNF family member FN14 (gene: TNFRSF12A) in colon tumors decreases inflammatory cytokine expression and mitigates cancer-induced cachexia. However, the molecular mechanisms underlying the regulation of FN14 expression remain unclear. Tumor microenvironments are often devoid of nutrients and oxygen, yet how the cachexic response relates to the tumor microenvironment and, importantly, nutrient stress is unknown. Here, we looked at the connections between metabolic stress and FN14 expression. We found that TNFRSF12A expression was transcriptionally induced during glutamine deprivation in cancer cell lines. We also show that the downstream glutaminolysis metabolite, alpha-ketoglutarate (aKG), is sufficient to rescue glutamine-deprivation-promoted TNFRSF12A induction. As aKG is a co-factor for histone de-methylase, we looked at histone methylation and found that histone H3K4me3 at the Tnfrsf12a promoter is increased under glutamine-deprived conditions and rescued via DM-aKG supplementation. Finally, expression of Tnfrsf12a and cachexia-induced weight loss can be inhibited in vivo by DM-aKG in a mouse cancer cachexia model. These findings highlight a connection between metabolic stress and cancer cachexia development.

1. Introduction

FN14 is a type 1 transmembrane protein with the gene name tumor necrosis factor receptor superfamily member 12A (TNFRSF12A). Previous studies have found the transcription of FN14 in a variety of tissues, such as the liver, heart, lung, and skeletal muscle [1,2,3,4]. FN14 is also found to be upregulated across a variety of high-grade solid tumors, including glioma, pancreatic, breast, non-small-cell lung cancer, and colorectal cancer (CRC) [5,6,7,8,9]. Importantly, it has been previously shown that specific inhibition of FN14 in CRC is able to decrease inflammatory cytokine expression as well as cancer-induced cachexia [10].
Cancer cachexia is defined as a multifactorial syndrome characterized by the progressive loss of skeletal muscle mass that cannot be entirely reversed with nutritional support [11]. As cachexia progresses, patient organs display a net negative energy and protein balance driven by a variable combination of reduced food intake and abnormal metabolism induced by the tumor. On the other hand, tumor cells often reprogram their metabolism to meet the increased requirement of rapid proliferation. For example, in colorectal cancer (CRC), it has been indicated that glutamine (Gln) is scavenged from muscle tissue to support cancer cell metabolism with increased expression of glutaminase [12,13]. Cancer cells use Gln in order to sustain proliferation and cell growth specifically by using Gln as a carbon source to drive the TCA during Warburg metabolism [14]. Moreover, Gln is used by proliferating cells to generate nucleotides, and glutathione is used to modulate ROS [15,16]. However, as a result of unchecked biosynthesis and outgrowth of vascular supply lines, the tumor is eventually unable to sustain intratumoral concentrations of Gln, leading tumor cells into a glutamine-deprived environment [17,18,19]. This phenomenon occurs across multiple cachexic cancer types as several studies show that solid tumors, such as colorectal cancer, melanoma, and pancreatic cancer, reside in glutamine-deprived tumor microenvironments (TME) [18,20,21]. However, how this tumor metabolic environment affects cancer-induced cachexia is largely unknown.
Alpha-ketoglutarate (aKG), an intermediate metabolite found two steps downstream from Gln during the process of glutaminolysis, has been shown to control gene expression by regulating the epigenetic architecture in normal and cancer cells [22,23]. Specifically, depletion of aKG from the TME has been shown to increase global histone methylation as a result of reduced activity of Jumonji histone demethylases (JHDMs), which use aKG as a cofactor [23]. In turn, this fosters hypermethylation of H3K4me3, a marker for active transcription [23,24,25]. This hypermethylated phenotype promotes cancer dedifferentiation via Wnt signaling, increases cancer cell survival, and upregulates systemic inflammatory pathways including VEGF, IL-1, and IL-8 within tumors [23]. Notably, this is reversible through supplementation of Gln or aKG in mice, and results in an increase in mice survival and a decrease in tumor burden [23,24]. Additionally, the supplementation of aKG and Gln have both been used in the treatment of cachexia-associated phenotypes. For example, aKG has been seen to reduce skeletal muscle protein degradation within the context of Duchenne muscular dystrophy, and Gln supplementation has been shown to both reduce loss of body mass as well as increase muscle protein synthesis in cancer cachexia [26,27,28]. Despite the efficacy of aKG and Gln supplementation in the prevention of cachexia within mice and rats, the mechanisms underlying the prevention of cancer-associated cachexia are less understood.
In this study, we show that FN14 expression is significantly upregulated in response to Gln deprivation across a variety of cancer cell lines. This effect is rescued by dimethyl alpha-ketoglutarate (DM-aKG) supplementation. These results provide support for therapeutic strategies that sustain Gln concentrations within the tumor microenvironment as a means of regulating FN14 expression and, subsequently, cachexia.

2. Materials and Methods

2.1. Cell Culture and Media

SW620, CT26, and PANC-1 cell lines were purchased from ATCC. Mouse-derived C26 cells were graciously donated by Dr. Andrea Bonnetto of Indiana State University. Gln-deprived and Gln-supplemented media were made with DMEM devoid of Gln and sodium-pyruvate (Corning, New York, NY, USA) supplemented with L-Glutamine (Omega Scientific, Tarzana, CA, USA), 10% dialyzed fetal bovine serum (Omega Scientific, Tarzana, CA, USA), and penicillin/streptomycin (Genesee Scientific, Morrisville, NC, USA). Cells were cultured at 37 °C with 5% CO2. Within Dulbecco’s MEM (DMEM) media, Dimethyl alpha-ketoglutarate (Tokyo Chemical Industry, Tokyo, Japan), Succinate (Sigma Aldrich, St. Lous, MO, USA), N-acetylcysteine (Sigma Aldrich, St. Lous, MO, USA), dialyzed fetal bovine serum (Omega Scientific, Tarzana, CA, USA), and D-Glucose (Sigma Aldrich, St. Lous, MO, USA) were supplemented as indicated. For hypoxia culture, cells were seeded in DMEM with 10% dialyzed FBS and 0.2 mM or 4 mM glutamine under 1% O2 using a BioSpherix Xvivo system (BioSpherix, Parish, NY, USA). The cells were maintained under hypoxic conditions for 24 h until collection. For amino acid deprivation experiments, DMEM with Low Glucose, w/o Amino Acids, Pyruvic Acid (Powder) (US Biological, Salem, MA, USA) was used. Nonessential amino acids (Sigma Aldrich, St. Lous, MO, USA), branched-chain amino acids (Sigma Aldrich, St. Lous, MO, USA), and essential Amino acids (Sigma Aldrich, St. Lous, MO, USA) were added according to the DMEM formulations found in the Table 1.

2.2. RNA Extraction and Quantitative Real-Time PCR

Total RNA was extracted and purified using Trizol according to the manufacturer’s instructions (Life Technologies, Carlsbad, CA, USA). Complementary DNA (cDNA) was made using 1 μg of RNA and the qScript cDNA Synthesis Kit (Quanta BioSciences, Beverly, MA, USA). Quantitative real-time PCR (qPCR) was performed using the SYBR Green PCR Master Mix (Quanta BioSciences, Beverly, MA, USA) on a Bio-Rad real-time PCR machine. Relative gene expression was calculated using the 2–∆∆Ct method and normalized to 18S or Actin RNA. The primers used in the study are shown in Table 2.

2.3. Flow Cytometry

Cells were washed once with cold PBS and trypsinized (Gibco, Grand Island, NY, USA). Cells measuring 2 × 106 were resuspended in fluorescence-activated cell sorting (FACS) staining buffer (FSB) (2% fetal bovine serum and 0.01% NaN3) for 20 min at 4 °C with 0.5 μg antibodies or isotype-matched IgG. Cells were then washed twice with FSB and fixed with FACS fixative (1% formaldehyde in PBS). Antibodies used were FN14 (CD266, eBioscience, Santa Clara, CA, USA) and Mouse IgG2b kappa Isotype Control (eBMG2b), PE, (eBioscience, Santa Clara, CA, USA). A total of 100,000 events were collected per sample. Flow analysis was conducted using a BD Fortessa X20 (BD Biosciences, San Jose, CA, USA).

2.4. Histone Extraction and Western Blot

Cells were washed twice with PBS and lysed with hypotonic lysis buffer with protease inhibitors (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, and Protease inhibitor (Roche, Mannheim, GER) on ice for 1 h. The lysate was then rotated overnight at 4 °C in a final concentration of 0.2 N H2SO4. The samples were then centrifuged, supernatants were collected, and histones were precipitated for 1 h on ice using 25% (w/v) TCA. The pellet was then washed three times with ice-cold acetone and dissolved in water. Membranes were blocked for 1 h in 5% non-fat milk in PBS with 0.05% Tween-20 (Sigma Aldrich) and incubated with antibodies overnight. Membranes (Bio-Rad) were visualized using horseradish-peroxidase-conjugated secondary antibodies (1:2000, Bio-Rad, Hercules, CA, USA) and Western Lightning Plus-ECL (PerkinElmer, Waltham, MA, USA). The immunoblots were normalized against Histone H3 using ImageJ. Antibodies used for the study include H3K4me3 (EMD Millipore, Burlington, MA, USA) at 1:1000 and H3 (Cell Signaling, Danvers, MA, USA) at 1:12,000.

2.5. Chromatin Immunoprecipitation Assay

The chromatin immunoprecipitation assay (ChIP) was performed using the ChIP assay kit (EMD Millipore, Burlington, MA, USA) according to the manufacturer’s guidelines. The cells were cultured in Gln-supplemented (4 mM) or Gln-deprived media with or without DM-aKG (4 mM) for 24 h. The cells were then crosslinked with 1% formaldehyde for 10 min at 37 °C. Cells were then washed twice with PBS containing protease inhibitors (Roche, Mannheim, GER) and lysed with SDS lysis buffer. The cell lysates were then sonicated to yield 200–1000 bp DNA fragments. The cell lysates were then centrifuged and supernatants were diluted with immunoprecipitation buffer with protease inhibitors (Roche, Mannheim, GER). The dilutant was then immunoprecipitated with 1 μg of H3K4me3 antibody (EMD Millipore, Burlington, MA, USA) or IgG overnight at 4 °C with rotation. Chromatin DNA was obtained according to the manufacturer’s specifications. The DNA was then analyzed via qPCR with the primers shown in Table 3.

2.6. Mouse Intestinal Crypt Isolation and Organoid Culture

Intestinal crypts from 6–8-week-old ApcMin/+ mice and wild-type mice (Jackson Laboratory, Bar Harbor, ME, USA) were isolated from tumor-free small intestinal tissues based on a previous study [29]. Crypts were cultured under IntestiCult™ organoid media supplemented with EGF, Noggin, and R-Spondin (Stemcell), and Primocin mixed 1:1 with Growth-Factor-Reduced Matrigel (Corning). An amount of 50 μL of the mixture was plated in a prewarmed 48-well plate and incubated for 10 min at 37 °C. For Gln deprivation experiments, Gln-free DMEM/F-12 (Gibco) was supplemented with L-Glutamine (Corning) or Dimethyl alpha-ketoglutarate (Tokyo Chemical Industry, Tokyo, Japan) as indicated.

2.7. Animal Models and Xenograft

All studies utilizing animals were performed according to the protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, Irvine, in compliance with ethical regulations. CD2F1 (Charles River) male mice aged 10-weeks-old were used for xenograft studies. For the xenograft, mice were injected subcutaneously with 0.5 × 106 C26 cells in 200 μL DMEM (Corning) devoid of FBS or antibiotics. Mice were placed randomly into two groups one-week post engraftment. Mouse weight, tumor length, and width were measured every 2–4 days using calipers; tumor volume was then calculated using the formula ½ (length × width2). Mice were euthanized upon 15% body mass loss, signs of ulceration/bleeding, or signs of lethargy. DM-aKG at 600 mg/kg or PBS was delivered intraperitoneally every 2–4 days beginning when tumor volume was at a minimum of 100 mm3. Estimated sample sizes for each animal study were based on preliminary data or prior experience with these models, anticipating group variance.

2.8. Statistics

Unpaired t-test and ordinary one-way ANOVA with post hoc Tukey’s test or Sidak’s correction were used to assess the statistical significance of mean differences and calculated using GraphPad Prism (v9) software. To determine the p values of DM-aKG versus PBS interpretational injection, ordinary one-way ANOVA with post hoc Tukey’s test was applied. For all statistics: ns represents p > 0.05, * represents p ≤ 0.05, ** represents p ≤ 0.01, *** represents p ≤ 0.001, and **** represents p ≤ 0.0001.

3. Results

3.1. Glutamine Deprivation Induces TNFRSF12A Expression

TNFRSF12A or FN14 is thought to be sufficient for cachexia and a link has been made between TNFRSF12A and CRC [8,10]. Additionally, it has been shown that tumor cells can adapt to Gln deprivation by altering gene expression [18,23,24]. To examine the effects of glutamine deprivation on TNFRSF12A CRC cell lines, human SW620 and mouse CT26 were Gln-deprived and the expression of TNFRSF12A was assayed via qPCR. We found that glutamine deprivation induces TNFRSF12A expression in both cell lines (Figure 1A). To confirm this induction, we further examined Tnfrsf12a expression in ApcMin/+ organoids under low Gln conditions, a system that highly resembles the in vivo profile of CRC. We found that glutamine deprivation significantly induces Tnfrsf12a expression in organoids (Figure 1B). Previous studies in glioma have shown that FN14 displays transient upregulation under serum-deprived conditions and TWEAK co-stimulation but decreases within 8 h [9]. We thus wanted to determine if Gln deprivation was able to sustain TNFRSF12A expression during Gln deprivation as CRC are chronically in a state of low Gln concentrations. As such, SW260 cells were cultured under low Gln conditions for 24 and 48 h. We found that TNFRSF12A expressions increased 5.6-fold over 24 h and further increased to 17.49-fold over 48 h (Figure 1C). These results show that Gln deprivation induces TNFRSF12A transcription, and this effect is sustained over time. Next, we asked whether induced TNFRSF12A expression results in increased FN14 protein expression. Using flow-cytometry-based analysis, we confirmed that the FN14 protein expression is also induced upon glutamine deprivation (Figure 1D).

3.2. TNFRSF12A Transcriptional Induction Is Specific to Glutamine Deprivation

As FN14 is over-expressed in a variety of tumors, such as CRC and pancreatic cancer, and nutrient deprivation is a common characteristic of the tumor microenvironment [8,21,30], we further asked if FN14 induction is specific to glutamine deprivation using different cancer lines under a few different metabolic stress conditions. Pancreatic cancer PANC-1, a cachexia-inducing cell line [31], and CRC SW620 cells were deprived of either Gln, glucose, or serum for 24 h, and the expression of TNFRSF12A mRNA was measured via qPCR. We found that only glutamine deprivation significantly induced TNFRSF12A expression in both cell lines (Figure 2A,B). FN14 expression was previously found to be upregulated in contexts of hypoxia [32]. Similarly, we found that hypoxia significantly induced TNFRSF12A mRNA expression. However, Gln deprivation induced TNFRSF12A expression and was found to be significantly higher than the hypoxia condition (Figure 2C). In addition, hypoxia cannot further promote Gln-deprivation-induced TNFRSF12A expression (Figure 2C). We also found TNFRSF12A mRNA expression is specific to Gln starvation as removal of non-essential amino acids, essential amino acids, and branch chain amino acids from media are unable to induce TNFRSF12A transcription (Figure 2D). Taken together, these results show Gln deprivation has a more pronounced effect in inducing TNFRSF12A expression compared with other metabolic stresses.

3.3. Dimethyl Alpha-Ketoglutarate Supplementation Is Sufficient to Inhibit TNFRSF12A Expression In Vitro

Once transported into the cell, Gln is broken down quickly by glutaminolysis into Glutamate (Glu) and then aKG which is not only a critical TCA component but can also serve as a co-factor for histone modification enzymes (Figure 3A). Specifically, aKG serves as a cofactor for the function of Jumonji histone demethylases and TET (ten–eleven translocation) methylcytosine dioxygenases [33,34]. Additionally, Gln can produce glutathione (GSH), which can be used to control redox conditions [35,36] (Figure 3A).
To ask if depleted Gln/Glutaminolysis and TCA byproducts are key effectors of Tnfrsf12a expression, CRC cells were deprived of Gln but supplemented with either DM-aKG, succinate (SUC), or antioxidant N-acetyl-L-cysteine (NAC). Interestingly, we found that a cell-permeable DM-aKG was able to rescue Gln-deprivation-induced Tnfrsf12a transcription, whereas supplementation with either SUC or NAC was unable to rescue Tnfrsf12a mRNA upregulation (Figure 3B). Next, to test if DM-aKG can reverse Gln-deprivation-induced Tnfrsf12a transcription, we cultured C26 or SW620 cells in Gln-free media for 16 h to induce Tnfrsf12a expression followed by the addback of glutamine, DM-aKG, or SUC. We found that addback with DM-aKG significantly suppressed Tnfrsf12a expression (Figure 3C,D). In addition, we found that DM-aKG was able to suppress FN14 protein expression in Gln-deprived culture (Figure 3E). Additionally, to test if Tnfrsf12a upregulation is relevant to CRC, we examined Tnfrsf12a expression in WT or ApcMin/+ mutant intestinal organoids and found that Tnfrsf12a expression is significantly higher in ApcMin/+ organoids. Additionally, supplementation of DM-aKG was able to decrease the expression of Tnfrsf12a in ApcMin/+ organoids (Figure 3F). Taken together, our findings show the expression of Tnfrsf12a can be suppressed by DM-aKG.

3.4. Dimethyl Alpha-Ketoglutarate Regulates Tnfrsf12a Expression via Histone Modification of H3K4me3

Previous data show that during Gln deprivation CRC undergoes global histone hypermethylation of H3K4me3 due in part to the loss of aKG and ability to activate histone demethylases [23,24]. This, in turn, results in increased oncogene expression. Upon treatment with either Gln or aKG, H3K4me3 methylation can be suppressed and leads to decreased expression of many oncogenes [23,24]. To test if Tnfrsf12a is regulated by histone demethylase activity, the methylation of H3K4me3 was probed in C26 cells upon Gln starvation and/or DM-aKG supplementation. As previously shown, H3K4me3 was found to increase during Gln starvation which is rescued by DM-aKG supplementation (Figure 4A). To test if Tnfrsf12a is regulated by histone demethylase activity, C26 cells were treated with adenosine dialdehyde (Adox), a global methyltransferase inhibitor [37,38]. We found that under Gln-deprived conditions, Adox was able to decrease the expression of Tnfrsf12a, indicating that transcriptional induction of Tnfrsf12a is in part through the regulation of methyltransferase activity (Figure 4B,C). Since the JARID1 family of JHDMs regulates the methylation of H3K4me3, we next tested to see if the JARID1 family serves to regulate the expression of Tnfrsf12a. PBIT is a more selective inhibitor than Adox that targets only members of the demethylases belonging to the JARID1 (Jumonji) family [39]. To verify if the expression of Tnfrsf12a is regulated by JARID1 family demethylases at H3K4, C26 cells were deprived of Gln for 16 h; afterward, DM-aKG or DM-aKG with 5 µM PBIT were added, and cells were collected after 8 h. We found that addback of DM-aKG with 5 µM PBIT resulted in a partial but significant rescue of Tnfrsf12a transcription (Figure 4D). To assess whether the Tnfrsf12a promoter region was regulated by H3K4me3, we performed chromatin immunoprecipitation (ChIP) of H3K4me3 and assessed for the Tnfrsf12a promoter region via qPCR. ChIP-qPCR of the Tnfrsf12a promoter region revealed that glutamine deprivation increases the extent of H3K4me3 at the Tnfrsf12a promoter and this effect can be mitigated via DM-aKG supplementation (Figure 4E). Together, our results show that H3K4me3 methylation increases at the Tnfrsf12a promoter under glutamine-deprived conditions and this effect is partly facilitated by JARID1 methyltransferase activity.

3.5. Dimethyl Alpha-Ketoglutarate Supplementation Is Sufficient to Rescue Cachexia and Tnfrsf12a Transcription In Vivo

Previous data show that inhibition of tumor-derived Fn14 is sufficient to perturb cachexia [10]. We then wanted to examine whether DM-aKG delivery via intraperitoneal injection was capable of blocking tumor-induced cachexia in vivo. C26 cells were xenografted into CD2F1 mice, an established CRC cachexia mouse model. We found that intraperitoneal (IP) injection of DM-aKG was able to decrease cachexia compared with PBS, respectively amounting to −5.48% versus −13.19% body mass loss over the course of 14 days without influencing tumor volume during that same duration (Figure 5A–C). However, if treatment was continued past 14 days, DM-aKG IP is capable of decreasing tumor volume growth as previously described [23]. Concurrently, DM-aKG treatment was sufficient to inhibit Tnfrsf12a transcription in the primary tumor (Figure 5D). Our findings establish a novel relationship between Gln deprivation that leads to histone methylation increases, and the induction of the cachexia-promoting factor FN14. As modeled in Figure 5E, the Gln-deficient condition promotes global H3K4me3 histone methylation due to decreased JHDM activity, which sustains the H3K4me3 methylation and upregulates Tnfrsf12a and FN14. Further, FN14 expression has been shown to promote a cachectic, wasting phenotype. We found this process can be rescued in vivo by supplementing DM-aKG, which mitigates cachexia-induced weight loss.

4. Discussion

Mechanisms describing the regulation of FN14 include transcription factor ThPOK, cytokines, DNA methylation, and miRNAs, as well as physical cellular stress [5,40,41,42,43,44]. Our investigations across a variety of cancerous cell lines and ApcMin/+ organoids have shown that metabolic stress in the form of Gln deprivation is a major contributor to the regulation of Tnfrsf12a. We show that the upregulation of Tnfrsf12a is independent of the other major metabolic stressors known to affect cancer metabolism such as glucose and serum deprivation (Figure 2A,B). We also found that other amino acids could not rescue what glutamine deprivation achieves on its own (Figure 1A,B and Figure 2D). Interestingly, ROS activation of FN14 has been shown and Tnfrsf12a contains conserved hypoxia-inducible factor (HIF)-1α binding sites in retinal neovascularization models [32]. However, we found that supplementing with one dose of NAC was unable to consistently prevent Tnfrsf12a induction under glutamine deprivation (Figure 3A). Future experiments need to be performed with different doses and time periods to achieve a conclusion. Instead, our results suggest that Gln deprivation at the site of the tumor decreases the concentration of aKG, which stimulates Tnfrsf12a transcription as a form of metabolic adaptation that calls to distant sites for nutrients in the form of cachexia. Although TNFRSF12A expression increased in human tumors [5,6,7,8,9], especially those with high cachexia incidence, a correlation between TNFRSF12A levels with weight loss in patients has not been established due to lacking information concerning the presence of cachexia in current patient databases. It is interesting to note that the TCGA database analysis promisingly revealed positive correlations between many inflammatory cytokines implicated in cancer cachexia and Fn14 transcripts in patients [10].
We have previously shown that histone hypermethylation is induced in CRC tumors as a consequence of Gln deprivation [23]. Specifically, deficiency of Gln from the tumor microenvironment was shown to result in the methylation of H3K4, a marker for active transcription [24]. We also found that the mechanism of transcriptional induction of Tnfrsf12a is due in part to the decreased activity of JDHMs brought on by Gln deprivation. Further, the induction of Tnfrsf12a could only be rescued by the introduction of cell-permeable DM-aKG or Gln; the former serves as a cofactor for JDHMs, whereas the latter can be metabolized into aKG. Using the JARID1-specific inhibitor PBIT, we specifically pinpointed the JARID1 histone demethylases as regulators capable of decreasing the expression of Tnfrsf12a. These results were further corroborated by ChIP-qPCR of the H3K4me3, where in Gln-deprived conditions it showed increased H4K4 methylation at the Tnfrsf12a promoter, which was then decreased by DM-aKG supplementation. Interestingly, our study is not the first to discern a mechanism for the activation of FN14 regulated by epigenetic mechanisms in chronic disease states capable of inducing cachexia. Tajrishi et al. found that muscular denervation diminishes overall genomic DNA methylation and causes hypomethylation of TNFRSF12A at its CpG-rich promoter region via methyltransferase DMNT3A, thus facilitating its activation [4]. This finding is particularly interesting as previous data have shown that DNA CpG islands correlate closely with H3K4me3 methylation [45,46,47]. Additionally, the ability of FN14 to be induced under various contexts of global methylation may also account for its overexpression within the context of gliomas which are characteristically Gln-rich and Gln-addicted [9,48,49].
Furthermore, we found that the rescue of Tnfrsf12a transcription via DM-aKG was possible both in vitro as well as in vivo. In that, IP injection of DM-aKG was able to decrease the extent of body mass loss within mice, indicating that DM-aKG treatment may serve as an important avenue in the treatment of cachexia. Previous preclinical studies have already touched on the importance of aKG treatment in preventing skeletal muscle protein degradation [26,50,51]. However, sustaining subject weight using aKG in preclinical studies has proven difficult as studies using Ornithine alpha-ketoglutarate in the context of cancer-induced cachexia were unable to prevent body mass loss [50]. Our positive results may be the result of using cell-permeable DM-aKG, which may make delivery into the tumor and peripheral tissues more efficient. In summary, our findings establish a novel connection between Gln deprivation and the induction of the cachexia-inducing factor FN14. Our results show that under Gln-deprived conditions, global histone methylation of H3K4me3 increases, resulting in an upregulation of Tnfrsf12a and cachexia. This effect can be rescued both in vivo and in vitro through the introduction of DM-aKG, resulting in mitigation of cachexia-induced weight loss and FN14 expression. Ultimately, our results provide evidence for further exploration of aKG as a therapeutic strategy for cancer cachexia.

Author Contributions

B.I.R. and M.K. conceived and designed the project and wrote the manuscript. B.I.R., X.H.L., Q.F., T.W. and H.W. performed in vitro experiments. Y.Y. performed mouse experiments. The analyses were performed by B.I.R., M.K. supervised the project. E.A.H. contributed to manuscript preparation. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. All authors have read and agreed to the published version of the manuscript.

Funding

This material is based upon work supported by the NIH-R01CA244360 to M.K. and National Science Foundation Graduate Research Fellowship under Grant No. 1324585 to B.I.R.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Irvine.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available from the corresponding authors and the first author upon request.

Acknowledgments

We thank members of the Kong laboratory for helpful comments on the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Glutamine deprivation induces TNFRSF12A expression. (A) qPCR analysis of TNFRSF12A in human colorectal cancer cells SW620 and mouse CT26 cells cultured in glutamine (Gln)-deprived medium (0 mM) or 2 mM Gln for 24 h. (B) qPCR analysis of Tnfrsf12a in mouse ApcMin/+ organoids cultured in low-Gln medium (0.2 mM) or 3 mM Gln for 72 h. (C) qPCR analysis of Tnfrsf12a in SW620 cultured in low-Gln medium (0.2 mM) or 4 mM Gln for 24 h. (D) Mean fluorescence intensity (MFI) of anti-FN14 and representative histogram of FN14 protein expression levels in C26 cells cultured in Gln-deprived medium (0 mM) or 4 mM Gln for 24 h (n = 2). Data represent the mean ± SD (n = 3) unless specified. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.
Figure 1. Glutamine deprivation induces TNFRSF12A expression. (A) qPCR analysis of TNFRSF12A in human colorectal cancer cells SW620 and mouse CT26 cells cultured in glutamine (Gln)-deprived medium (0 mM) or 2 mM Gln for 24 h. (B) qPCR analysis of Tnfrsf12a in mouse ApcMin/+ organoids cultured in low-Gln medium (0.2 mM) or 3 mM Gln for 72 h. (C) qPCR analysis of Tnfrsf12a in SW620 cultured in low-Gln medium (0.2 mM) or 4 mM Gln for 24 h. (D) Mean fluorescence intensity (MFI) of anti-FN14 and representative histogram of FN14 protein expression levels in C26 cells cultured in Gln-deprived medium (0 mM) or 4 mM Gln for 24 h (n = 2). Data represent the mean ± SD (n = 3) unless specified. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.
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Figure 2. TNFRSF12A transcriptional induction is specific to glutamine deprivation. (A) qPCR analysis of TNFRSF12A in PANC1 cells cultured in 0 mM glutamine medium (Gln) (−) or 4 mM Gln (+) with either 4.5 g/L glucose (+), no glucose (−), 10% serum (+), or 1% serum (−). (B) qPCR analysis of TNFRSF12A in SW620 cells cultured in 0 mM Gln (−) or 4 mM Gln (+) with either 1.0 g/L glucose (+), no glucose (−), 10% serum (+), or 1% serum (−). (C) qPCR analysis of TNFRSF12A in SW620 cultured in 0.2 mM Gln medium (−) or 4 mM Gln (+) under 1% O2 (−) or 20% O2 (+) for 24 h. (D) qPCR analysis of TNFRSF12A in SW620 cultured in 0 mM Gln medium (−) or 4 mM Gln or media with (+) or without (−) nonessential amino acids (NEAAs), branched-chain amino acids (BCAAs), or essential amino acids (EAAs). Data represent the mean ± SD (n = 3). ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
Figure 2. TNFRSF12A transcriptional induction is specific to glutamine deprivation. (A) qPCR analysis of TNFRSF12A in PANC1 cells cultured in 0 mM glutamine medium (Gln) (−) or 4 mM Gln (+) with either 4.5 g/L glucose (+), no glucose (−), 10% serum (+), or 1% serum (−). (B) qPCR analysis of TNFRSF12A in SW620 cells cultured in 0 mM Gln (−) or 4 mM Gln (+) with either 1.0 g/L glucose (+), no glucose (−), 10% serum (+), or 1% serum (−). (C) qPCR analysis of TNFRSF12A in SW620 cultured in 0.2 mM Gln medium (−) or 4 mM Gln (+) under 1% O2 (−) or 20% O2 (+) for 24 h. (D) qPCR analysis of TNFRSF12A in SW620 cultured in 0 mM Gln medium (−) or 4 mM Gln or media with (+) or without (−) nonessential amino acids (NEAAs), branched-chain amino acids (BCAAs), or essential amino acids (EAAs). Data represent the mean ± SD (n = 3). ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
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Figure 3. Dimethyl alpha-ketoglutarate supplementation is sufficient to inhibit TNFRSF12A expression in vitro. (A) Schematic diagram of glutamine metabolism created with BioRender. (B) qPCR analysis of Tnfrsf12a in C26 cells cultured in conditions as indicated: 0 mM Gln (−), 4 mM Gln (+), 4 mM Dimethyl alpha-ketoglutarate (DM-aKG) (+), 5 mM N-acetylcysteine (NAC) (+), or 5 mM Succinate (SUC) (+). (C) qPCR analysis of Tnfrsf12a in C26 cells cultured in 0 mM Gln for 16 h. An amount of 2 mM Gln (+) or 2 mM DM-aKG (+) were then added back into Gln-deprived media for 8 h. (D) qPCR analysis of TNFRSF12A in SW620 cells cultured in 0 mM Gln for 16 h. An amount of 2 mM Gln (+), 4 mM Dm-aKG (+), or 5 mM SUC (+) were then added back into Gln-deprived media for 8 h. (E) Mean fluorescence intensity (MFI) of anti-FN14 and representative histogram of FN14 protein expression levels in C26 cells cultured in 0 mM Gln, 4 mM Gln, or 4 mM DM-aKG for 24 h. (F) qPCR analysis of Tnfrsf12a in wild type (−) or ApcMin/+ (+) organoids cultured in low-Gln medium (0.2 mM) (−) or 3 mM Gln (+) for 72 h. For all experiments, data represent the mean ± SD (n = 3). ns p > 0.05, * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001.
Figure 3. Dimethyl alpha-ketoglutarate supplementation is sufficient to inhibit TNFRSF12A expression in vitro. (A) Schematic diagram of glutamine metabolism created with BioRender. (B) qPCR analysis of Tnfrsf12a in C26 cells cultured in conditions as indicated: 0 mM Gln (−), 4 mM Gln (+), 4 mM Dimethyl alpha-ketoglutarate (DM-aKG) (+), 5 mM N-acetylcysteine (NAC) (+), or 5 mM Succinate (SUC) (+). (C) qPCR analysis of Tnfrsf12a in C26 cells cultured in 0 mM Gln for 16 h. An amount of 2 mM Gln (+) or 2 mM DM-aKG (+) were then added back into Gln-deprived media for 8 h. (D) qPCR analysis of TNFRSF12A in SW620 cells cultured in 0 mM Gln for 16 h. An amount of 2 mM Gln (+), 4 mM Dm-aKG (+), or 5 mM SUC (+) were then added back into Gln-deprived media for 8 h. (E) Mean fluorescence intensity (MFI) of anti-FN14 and representative histogram of FN14 protein expression levels in C26 cells cultured in 0 mM Gln, 4 mM Gln, or 4 mM DM-aKG for 24 h. (F) qPCR analysis of Tnfrsf12a in wild type (−) or ApcMin/+ (+) organoids cultured in low-Gln medium (0.2 mM) (−) or 3 mM Gln (+) for 72 h. For all experiments, data represent the mean ± SD (n = 3). ns p > 0.05, * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001.
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Figure 4. Tnfrsf12a expression is regulated by histone modification of H3K4me3. (A) Western blot of H3K4me3 in C26 cells cultured in 0.1 mM glutamine (Gln) (−), 2 mM Gln (+), or (+) 4 mM dimethyl alpha-ketoglutarate (DM-aKG) (+) annotated with band density relative to H3. (B) Western blot of H3K4me3 in C26 cells cultured in 0.1 mM Gln (−) and 2 mM Gln (+), with (+) or without (−) 50 µM Adox annotated with a band density relative to H3. (C) qPCR analysis of Tnfrsf12a in C26 cells cultured in 0 mM Gln (−) and 4 mM Gln (+), with (+) or without (−) 50 µM Adox. (D) qPCR analysis of Tnfrsf12a in C26 cells cultured in 0 mM Gln medium for 16 h. An amount of 4 mM Gln (+), 4 mM Dm-aKG (+), or 5 µM PBIT (+) were then added back into Gln-deprived media for 8 h. (E) ChIP analysis of H3K4me3 levels on the promoter region of Tnfrsf12a in C26 cells cultured in 0 mM Gln (−), 4 mM Gln (+), and 4 mM DM-aKG (+). Data represent the mean ± SD (n = 3). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
Figure 4. Tnfrsf12a expression is regulated by histone modification of H3K4me3. (A) Western blot of H3K4me3 in C26 cells cultured in 0.1 mM glutamine (Gln) (−), 2 mM Gln (+), or (+) 4 mM dimethyl alpha-ketoglutarate (DM-aKG) (+) annotated with band density relative to H3. (B) Western blot of H3K4me3 in C26 cells cultured in 0.1 mM Gln (−) and 2 mM Gln (+), with (+) or without (−) 50 µM Adox annotated with a band density relative to H3. (C) qPCR analysis of Tnfrsf12a in C26 cells cultured in 0 mM Gln (−) and 4 mM Gln (+), with (+) or without (−) 50 µM Adox. (D) qPCR analysis of Tnfrsf12a in C26 cells cultured in 0 mM Gln medium for 16 h. An amount of 4 mM Gln (+), 4 mM Dm-aKG (+), or 5 µM PBIT (+) were then added back into Gln-deprived media for 8 h. (E) ChIP analysis of H3K4me3 levels on the promoter region of Tnfrsf12a in C26 cells cultured in 0 mM Gln (−), 4 mM Gln (+), and 4 mM DM-aKG (+). Data represent the mean ± SD (n = 3). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
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Figure 5. Dimethyl alpha-ketoglutarate supplementation is sufficient to rescue Tnfrsf12a transcription and cachexia in vivo. (A) CD2F1 mice with subcutaneous injection of C26 cells. Dimethyl alpha-ketoglutarate (DM-aKG) 600 mg/kg or PBS was delivered intraperitoneally every 2–4 days (arrows). Weight was measured every 2–4 days (non-xenograft (NX) PBS, n = 4; C26 PBS IP and C26 DM-aKG IP, n = 5). (B) Tumor volume was measured every 2–4 days (NX PBS, n = 4; C26 PBS IP and C26 DM-aKG IP n = 5). (C) Final weight change from initial weight of CD2F1 mice with NX or C26 cells intraperitoneally injected with PBS or DM-aKG. (D) qPCR analysis of the DM-aKG- (C26 DM-aKG IP, n = 3) or PBS- (C26 PBS IP, n = 4) treated tumors. (E) Schematic of the proposed mechanism created with BioRender. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.
Figure 5. Dimethyl alpha-ketoglutarate supplementation is sufficient to rescue Tnfrsf12a transcription and cachexia in vivo. (A) CD2F1 mice with subcutaneous injection of C26 cells. Dimethyl alpha-ketoglutarate (DM-aKG) 600 mg/kg or PBS was delivered intraperitoneally every 2–4 days (arrows). Weight was measured every 2–4 days (non-xenograft (NX) PBS, n = 4; C26 PBS IP and C26 DM-aKG IP, n = 5). (B) Tumor volume was measured every 2–4 days (NX PBS, n = 4; C26 PBS IP and C26 DM-aKG IP n = 5). (C) Final weight change from initial weight of CD2F1 mice with NX or C26 cells intraperitoneally injected with PBS or DM-aKG. (D) qPCR analysis of the DM-aKG- (C26 DM-aKG IP, n = 3) or PBS- (C26 PBS IP, n = 4) treated tumors. (E) Schematic of the proposed mechanism created with BioRender. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.
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Table 1. Amino Acid deprivation working concentrations.
Table 1. Amino Acid deprivation working concentrations.
Nonessential
Amino Acids (NEAAs)		Essential
Amino Acids (EAAs)		Branched-Chain
Amino Acids (BCAAs)
MetaboliteConc. (mM)MetaboliteConc. (mM)MetaboliteConc. (mM)
Glycine0.4L-Lysine·HCl0.8L-Isoleucine0.8
L-Arginine·HCl0.4L-Methionine0.2L-Leucine0.8
L-Cystine·2HCl0.2L-Phenylalanine0.4L-Valine0.8
L-Serine0.4L-Histadine·HCl0.2
L-Tyrosine
disodium salt dihydrate		0.4L-Threonine0.8
L-Tryptophan0.08
Media Metabolite Supplementations:
NEAA-deprived mediaEAA only
EAA-deprived mediaNEAA only
BCAA-deprived mediaEAA + NEAA only
Table 2. Primers used in the study for qPCR.
Table 2. Primers used in the study for qPCR.
TitleSequenceFigure
m-Tnfrsf12a (FWD)5′-GTGTTGGGATTCGGCTTGGT-3′Figures 1A,B, 3B,C, 4C,D and 5D
m-Tnfrsf12a (REV)5′-GGCAGAAGTCGCTGTGTGGT-3′Figures 1A,B, 3B,C, 4C,D and 5D
m-Tnfrsf12a (FWD)5′-GCAGATCCTCGTGTTGGGAT-3′Figure 3F
m-Tnfrsf12a (REV)5′-GGACAAGAAGCGCAGTCCAT-3′Figure 3F
h-TNFRSF12A (FWD)5′-CTGGCTCCAGAACAGAAAGG-3′Figure 1A
h-TNFRSF12A (REV)5′-GGGCCTAGTGTCAAGTCTGC-3′Figure 1A
h-TNFRSF12A (FWD)5′-TTT GGT CTG GAG ACG ATG CC-3′Figures 1C, 2A–D and 3D
h-TNFRSF12A (REV)5′-TGAATGATGAGTGGG CGA GC-3′Figures 1C, 2A–D and 3D
m-18s (FWD)5′-CGCTTCCTTACCTGGTTGAT-3′Figures 1A,B, 3B,C,F, 4C,D and 5D
m-18s (REV)5′-GAGCGACCAAAGGAACCATA-3′Figures 1A,B, 3B,C,F, 4C,D and 5D
h-18S (FWD)5′-ACCCGTTGAACCCCATTCGTG-3′Figures 1C, 2A–C and 3D
h-18S (REV)5′-GCCTCACTAAACCATCCAATC GG-3′Figures 1C, 2A–C and 3D
h-ACTB (FWD)5′-CACCAACTGGGAGGACAT-3′Figure 1A
h-ACTB (REV)5′-GCACAGCCTGGATAGCAAC-3′Figure 1A
h-ACTB (FWD)5′-CACCAACTGGGACGACAT -3′Figure 2D
h-ACTB (REV)5′-GCACAGCCTGGATAGCAAC -3′Figure 2D
m-Tnfrsf12a (FWD)5′-GTGTTGGGATTCGGCTTGGT-3′Figures 1A,B, 3B,C, 4C,D and 5D
Table 3. Primers used in the study for ChIP qPCR.
Table 3. Primers used in the study for ChIP qPCR.
TitleSequenceFigure
m-Tnfrsf12a Promoter (FWD)5′-TTTCCTTCGCTCCACATCGT-3′Figure 4E
m-Tnfrsf12a Promoter (REV)5′-ATTCAACACAGTCCCGCCAA-3′Figure 4E
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MDPI and ACS Style

Ruiz, B.I.; Lowman, X.H.; Yang, Y.; Fan, Q.; Wang, T.; Wu, H.; Hanse, E.A.; Kong, M. Alpha-Ketoglutarate Regulates Tnfrsf12a/Fn14 Expression via Histone Modification and Prevents Cancer-Induced Cachexia. Genes 2023, 14, 1818. https://doi.org/10.3390/genes14091818

AMA Style

Ruiz BI, Lowman XH, Yang Y, Fan Q, Wang T, Wu H, Hanse EA, Kong M. Alpha-Ketoglutarate Regulates Tnfrsf12a/Fn14 Expression via Histone Modification and Prevents Cancer-Induced Cachexia. Genes. 2023; 14(9):1818. https://doi.org/10.3390/genes14091818

Chicago/Turabian Style

Ruiz, Bryan I., Xazmin H. Lowman, Ying Yang, Qi Fan, Tianhong Wang, Hongmei Wu, Eric A. Hanse, and Mei Kong. 2023. "Alpha-Ketoglutarate Regulates Tnfrsf12a/Fn14 Expression via Histone Modification and Prevents Cancer-Induced Cachexia" Genes 14, no. 9: 1818. https://doi.org/10.3390/genes14091818

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