Several Faces of Glutaminase Regulation in Cells

Metabolic reprogramming for cell growth and proliferation is one of the hallmarks of cancer (1). Among other roles, glutaminase (GLS) is responsible for fueling the tricarboxylic acid (TCA) cycle with glutamine-derived α-ketoglutarate, which supports cellular energy production and biosynthetic demands and thus promotes tumor growth and progression in several types of cancer (2). The anaplerotic reaction of replenishing glutamine carbons into the TCA cycle is essential for some tumors, where the rate of glucose uptake dramatically increases, resulting in lactate production, a phenomenon called the Warburg effect. Because GLS is an important enzyme for keeping up with the cancer cell's high rate of proliferation, it is not surprising that cells have developed many ways to regulate GLS function.

Indeed, GLS has been shown to be regulated transcriptionally, posttranscriptionally, and posttranslationally through various mechanisms including noncoding RNAs (ncRNA), such as miRNAs, and long noncoding RNAs (lncRNA; ref. 3). miRNAs are involved in the regulation of several metabolic enzymes. C-Myc is derived from the proto-oncogene and transcription factor MYC, which is known to be amplified and overexpressed in many types of cancer. In 2009, Gao and colleagues reported that the c-Myc transcriptionally represses miR-23a and miR-23b, resulting in a greater expression of their target protein, GLS (3). This was the first GLS regulatory mechanism reported to be involved with c-Myc.

In this issue of Cancer Research (4), Deng and colleagues elegantly describe a new axis of GLS regulation involving the lncRNA GLS-AS, an antisense RNA priming intron 17 of the GLS gene and c-Myc. These findings extend our knowledge on how Myc regulates GLS beyond the already described miRNA23a/b. Importantly, this report brings the Myc/GLS axis to the pancreatic cancer oncogenic scenario, because glutamine metabolism in this type of cancer has been thought to be controlled only by Kras. They discovered that GLS-AS binding to GLS pre-mRNA decreased mRNA stability and led to decreased protein levels. Moreover, they showed that, in pancreatic cancer, energy stress, mainly caused by glucose and glutamine deprivation, drives Myc expression, which downregulates GLS-AS expression, leading to increased levels of GLS. The GLS-AS/GLS pathway, in turn, mediates a reciprocal feedback to regulate Myc because the GLS levels control c-Myc (4). Whether GLS regulates c-Myc through its glutaminase activity or through a protein–protein-binding regulatory network is yet to be discovered.

The hypovascular nature of pancreatic cancer typically drives energy stress (mainly by glucose and glutamine deprivation), which often leads to therapy resistance and has been correlated with poor patient survival. Learning about resistance mechanisms may help identify new ways to effectively treat disease. This newly described Myc/GLS-AS/GLS regulatory axis highlights the importance of the interplay between c-Myc and GLS in a particularly crucial scenario such as nutrient stress and confirms GLS as a critical tumor-promoting gene.

This is not the first time that a lncRNA has been shown to regulate GLS in a posttranscriptional manner. Colon cancer transcript 2 (CCAT2), a lncRNA that is commonly overexpressed in colorectal cancer, regulates the alternative splicing of GLS variants, favoring glutaminase C (GAC) expression over kidney-type glutaminase (KGA; ref. 5). Specifically, CCAT2 regulates cancer metabolism in an allele-specific manner through differential binding to the cleavage factor I (CFI) complex, which is composed of the CFIm25 and CFIm68 subunits (5). The G allele is associated with an increased predisposition to colorectal cancer compared with the T allele and has a higher affinity for the CFIm25 subunit, leading to the selection of the poly(A) site within intron 14 of the GLS precursor mRNA (pre-mRNA) and resulting in preferential splicing of the GAC isoform (5). GAC overexpression augments glutamine metabolism, thus promoting colorectal cancer cell proliferation and metastasis (5).

In addition, Masamha and colleagues described a more complex GLS regulation mechanism involving CFIm25 and ncRNA (6). Depletion of the alternative polyadenylation (APA) regulator CFIm25 was shown to cause a shift toward the usage of a proximal poly(A) site within the KGA 3′ untranslated region (UTR), which altered splicing to favor the exclusion of the GAC 3′ UTR. Surprisingly, while miR-23a/b was capable of downregulating the shortened KGA 3′ UTR, it only had a minor impact on the full-length KGA 3′ UTR (which contains a previously described destabilizing AU-rich element site), suggesting that an additional potent negative posttranscriptional regulator of GLS expression exists beyond the previously described miRNA targeting site (3). Altogether, these results illustrate a complex interplay of alternative splicing, APA, mRNA decay, interfering RNA, and now, pre-mRNA destabilization by AS RNA binding within a single-gene unit. These processes describe the layered regulation that GLS evolved to meet the changing cellular environment. Intriguingly, GLS's paralog, GLS2, encoded by a second gene, is controlled by the lncRNA urothelial carcinoma–associated 1 (UCA1). UCA1 shows a sponge effect on miR-16, which binds to the GLS2 3′ UTR and regulates the GLS2 expression level, leading to a decrease in reactive oxygen species (ROS) formation in bladder cancer cells (7). Because GLS is under such an intricate net of RNA controlling, we have only started learning which RNA regulatory elements govern GLS2.

Other lncRNAs have been shown to regulate cellular energetics and biosynthetic pathways in cancer. For example, lncRNA NBR2 activates the 5′ AMP-activated protein kinase (AMPK) pathway under conditions of glucose deprivation in various types of cancer cells. LncRNA UCA1 promotes glycolysis by activating the mTOR-STAT3/miR143-HK2 cascade and reduces ROS production, promoting mitochondrial glutaminolysis in bladder cancer cells. LncRNA CRNDE increases glucose transporter-4 (GLUT4) transcription and contributes to glucose intake, promoting the aerobic glycolysis of cancer cells. LncRNA ANRIL is upregulated in nasopharyngeal carcinoma, promoting glucose transporter-1 (GLUT1) and lactate dehydrogenase overexpression, thereby increasing glucose uptake for glycolysis and promoting cancer (all studies reviewed in ref. 8). These are only a few examples of lncRNAs that have been found to regulate the key enzymes that integrate malignant cell transformation and metabolism reprogramming toward tumor progression. The intricate network of ncRNAs acting on metabolism players has yet to be completely defined.

GLS is not only regulated by the c-Myc/ncRNAs axes. The transcription factor c-Jun, derived from the proto-oncogene JUN and activated downstream of oncogenic Rho-GTPase signaling, binds to the GLS promoter region, leading to elevated GLS gene expression and glutaminase activity in breast cancer (reviewed in ref. 9). Wang and colleagues reported that GLS isoforms are activated by oncogenic Rho-GTPases in a manner dependent on NFκB in B-cell lymphoma and breast cancer (reviewed in ref. 9). Their findings suggest that NFκB does not elevate basal glutaminase activity in cancer cells by directly affecting the expression of the enzyme itself, but by influencing the expression of a protein kinase or a regulatory protein that promotes its phosphorylation posttranslationally. It was also found that the Ser314 phosphorylation site on GAC is regulated by the NFκB/PKCε axis and that high levels of GAC phosphorylation correlate with poor survival rates in patients with lung cancer (10).

The results presented by Deng and colleagues (4), along with other studies, have revealed that GLS is tightly regulated through a series of fine-tuned mechanisms orchestrated by several regulatory proteins and RNAs, many of which are oncogenic. This intricate network of regulation denotes the importance of GLS for maintaining and/or shifting cellular metabolism. The interplay between GLS and Myc, an oncogene of overspread importance in cancer, strengthens its tumor-promoting role via metabolic reprogramming. However, although Myc has found different ways to control GLS, it is clearly not the only cancer-promoting agent that acts on this protein, because GLS is regulated through multiple mechanisms, some of which may yet to be discovered. Indeed, Deng and colleagues found a positive correlation between GLS and MYC (with a negative correlation found between GLS and GLS-AS mRNA levels) in a relatively small cohort; this is in contrast to their findings in two other larger publicly available datasets (4). In this sense, it is important to state that a positive correlation between Myc and GLS is likely genetically/epigenetically background-specific and also dependent on the tumor microenvironment.

The c-Myc/GLS-AS/GLS is a branch of a complex regulatory network where all the players are yet to be identified. Therefore, besides detecting the driving oncogenes affecting a given tumor, it is also necessary to learn the landscape of ncRNAs and search for markers that could better predict glutamine dependence, implying a glutaminase inhibition sensitivity. Moreover, glutaminase inhibition by ncRNA knockdown, small molecules, or, as suggested by Deng and colleagues, the use of GLS-AS has consistently been revealed to only decrease primary tumor volume and the number of metastatic foci. However, none of the approaches, which have been tested on several distinct mouse models and different tumor types, have led to tumor elimination. This necessitates the search for other targets that can be used in association with glutaminase inhibition to generate efficient synthetic lethality, which can be particularly interesting in situations of nutrient stress.

In summary, GLS is a tumor-promoting metabolic enzyme under the control of several signaling pathways and regulatory RNAs that fine-tune its protein and activity levels. Although accumulated evidence argues in favor of GLS being a key enzyme behind tumor growth and malignancy, it is becoming clear that targeting this enzyme for treating cancer has its caveats, and a greater understanding of context-dependent glutamine addiction is mandatory.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.