Arginine and Arginases Modulate Metabolism, Tumor Microenvironment and Prostate Cancer Progression

Abstract

Arginine availability and activation of arginine-related pathways at cancer sites have profound effects on the tumor microenvironment, far beyond their well-known role in the hepatic urea cycle. Arginine metabolism impacts not only malignant cells but also the surrounding immune cells behavior, modulating growth, survival, and immunosurveillance mechanisms, either through an arginase-mediated effect on polyamines and proline synthesis, or by the arginine/nitric oxide pathway in tumor cells, antitumor T-cells, myeloid-derived suppressor cells, and macrophages. This review presents evidence concerning the impact of arginine metabolism and arginase activity in the prostate cancer microenvironment, highlighting the recent advances in immunotherapy, which might be relevant for prostate cancer. Even though further research is required, arginine deprivation may represent a novel antimetabolite strategy for the treatment of arginine-dependent prostate cancer.

Keywords: arginase; arginine; metabolism; nitric oxide; prostate cancer; tumor microenvironment.

Figure 1

Major cellular arginine metabolic routes and regulatory mechanisms. The aminoacid arginine is transported to the cell by CAT transporters or synthesized from citrulline. Intracellular arginine can either be metabolized by arginase I or II (ARGI or ARGII) to ornithine and urea (excreted out of the cell and of the body) or to NO and citrulline through iNOS catalysis, depending on iNOS/ARG ratio. ARGI expression and activity are regulated positively by anti-inflammatory and negatively via pro-inflammatory cytokines and NOHA, an intermediate in NO formation. In turn, iNOS activity is negatively regulated by agmatine, a decarboxylated product of arginine, and by anti-inflammatory cytokines, besides the positive pro-inflammatory and hypoxic stimulus. Preferential production of ornithine through ARG catalysis can either result in activation of the polyamines pathway (putrescine, spermidine, and spermine) or in ornithine shuttling to the mitochondria (where it might follow the pathway towards proline formation or be transformed in citrulline and shuttled back to the cytoplasm). Arginine antiported to the mitochondria will increase the ornithine pool after ARGII catalysis. When iNOS metabolizes arginine, the resulting products are citrulline and nitric oxide using BH4 as a co-factor. While NO might be transformed to peroxynitrite that may nytrosylate/nitrosate/nitrate key proteins to become pro-tumoral, citrulline formation will drive the ARG-NO recycling. Citrulline from UREA or ARG-NO cycles is converted to arginine-succinate (by ASS), which will result in arginine and fumarate (catalyzed by ASL). This last step represents a link with the Krebs cycle. Solid black lines with arrows indicate a positive effect, whereas solid lines with blunted ends specify an inhibitory effect. Dashed line indicates ornithine-citrulline antiport through mitochondrial membrane, whereas dotted line represents transportation of arginine into the mitochondria or transportation of P-5-C between cytoplasm and mitochondria. ASL, argininosuccinate lyase; ASS, argininosuccinate synthase; CPS-I, carbamoyl phosphate synthetase I; OAT, ornithine aminotransferase; ARG, arginase (type I and type II); ARG-NO cycle, arginine-nitric oxide cycle where iNOS is primarily involved in arginine metabolism; ADC, arginine decarboxylase; ODC, ornithine decarboxylase; OTC, ornithine transcarbamylase; iNOS, inducible nitric oxide synthase; L-HydroxyArg, L-hydroxyarginine; NO, nitric oxide; P-5-C, pyrroline-5-carboxylate; P5CR, pyrroline-5-carboxylate reductase; CAT1-3, cationic amino acid transporters (CAT1 is found in epithelial cells and CAT2 in macrophages); CCL2, Chemokine (C-C motif) ligand 2 or monocyte-chemoattractant protein 1; HIF1α, hypoxia-inducible factor 1 α; NFκB, nuclear factor κB; ORNT1, mitochondrial ornithine: citrulline antiporter; BH₄, tetrahydrobiopterin; DCAM, decarboxylated 5-adenonosylmethionine; Glu, glutamate; MTA, methylthioadenosine; NOHA, N-hydroxy-L-arginine; α-KG, α-ketoglutamate; p53, tumor protein 53; Ras, Ras oncogene; COX-2, cyclooxygenase 2; PTEN, phosphatase and tensin homolog; UREA cycle, urea cycle where ARGI is primarily involved in arginine metabolism.

Figure 2

Schematic representation of arginine metabolism and the interplay between cellular players in the prostate tumor microenvironment. The activities of ARGs and iNOS are illustrated, together with arginine-activated downstream pathways in cellular components of the tumor microenvironment (androgen-responsive prostate cancer cell, macrophages, antitumor T-cell, MSCs). The most relevant pathophysiological implications of arginine metabolism are reduced cancer immunosurveillance and a stimulatory action in prostate malignant cells towards cancer progression. Solid black lines with arrows indicate the main enzymatic activity or movement of molecules, whereas dashed lines indicate alternative metabolic pathways or suppression of the movement of molecules. Solid blue lines with arrows designate a stimulatory effect in enzyme activity, while solid red lines with blunt ends specify inhibition of enzyme activity or transporter activity. While pro-tumoral M2 macrophages present increased activity of ARGs with subsequent proline and polyamines production that result in collagen deposition and higher cell proliferation, the antitumoral M1 macrophages (commonly found out of the tumor microenvironment) have an overactive iNOS pathway with resulting pro-inflammatory stimulus (NRF2L2 and NFKB1 overexpression), reduced cell growth and production of reactive oxygen (hydrogen peroxide) and nitrogen species (peroxynitrites) that ultimately induce cytotoxicity and tissue injury. The iNOS and ARGs enzymes are tightly regulated by cytokine and metabolic circuits, although these enzymes also directly activate biochemical circuits that negatively regulate each other. As a resulting effect of arginine metabolism and capture in M2 macrophages, several pro-tumoral growth factors are produced and secreted to the tumor microenvironment (VEGF, EGF, TGFβ), while reducing the extracellular pool of free arginine that will contribute to their immune suppression and tissue repair phenotype. By producing IL-4, IL-10, and TGF-β1 anti-inflammatory cytokines, tumor cells also contribute not only to macrophages differentiation towards M2 but also to the regulation of arginine metabolism in macrophages (activating ARGs and downregulating iNOS). Together with tumor cells and MDSCs, macrophages also contribute to increased levels of urea as a result of higher ARGs activity in these cells, which will impair mTOR signaling and T-cell receptor CD3 ζ-chain subunit mRNA translation, thus resulting in hindered antitumor T-cell activation. The hypoxic tumoral microenvironment increases HIF1A expression and protein production by tumor cells, which signals downstream to increase arginine-iNOS pathway activation that results in increased production of nitric oxide, and reactive nitrogen and oxygen species. While NO can impact antitumor T-cell activation through nitrosylation of target proteins and suppression of the IL-2/IL-2R pathway, peroxynitrites may hamper CCL2 binding and prevent T-cell chemoattraction towards tumors, and reactive oxygen species can influence negatively antitumor T-cell activation. Moreover, the particularity of prostate tumor cells’ dependence on androgens implies its influence in ARG1 and ARG2 expression, which will potentiate the urea cycle towards polyamines and proline production with resulting increases in cell proliferation and collagen synthesis. In addition, the overexpression of ARG1 and ARG2 will lead to ARGs exportation out of the cell, where they might sequester extracellular arginine, further increasing immunosuppression. MDSCs also metabolize arginine either through the ARG-NO cycle or the UREA cycle (Figure 1). MDSCs in the tumor microenvironment might secrete NO, reactive oxygen species, and ARGs receptors out of the cell, which will contribute to the suppression of IL-2/IL-2R pathway of T-cells, decreased activation of antitumor T-cells and sequestering of extracellular arginine, respectively, ultimately leading to reduced cancer immunosurveillance. 5-DHT, 5alpha-dihydrotestosterone; AR, androgen receptor; ARG1, gene coding for the arginase type I; ARG2, gene coding for the arginase type II; CAT, cationic amino acid transporter; CCL2, chemokine-CC motif-ligand 2; CCR2, chemokine-CC motif-receptor 2; EGF, epidermal growth factor; eIF2α, eukaryotic initiation factor 2α; GCN2, general control of nutrition; HIF-1α, hypoxia inducible factor 1 alpha; HIF1A, gene coding for the hypoxia inducible factor 1 alpha; IFNγ, interferon γ; IL-1, interleukine 1; IL-10, interleukine 10; IL-12, interleukine 12; IL-13, interleukine 13; IL2, gene coding for the interleukine 2; IL-2, interleukine 2; IL-2R, interleukine 2 receptor; IL-4, interleukine 4; IL-8, interleukine 8; iNOS, inducible nitric oxide synthase; MDSC, myeloid-derived suppressor cells; mTOR, mammalian target of rapamycin; NFE2L2, gene coding for the nuclear factor erythroid 2-like 2 (Nrf2); NFKB1, gene coding for the nuclear factor kappa-b subunit 1; NO, nitric oxide; OAT, ornithine aminotransferase; ARG, arginase (type I and type II); ODC, ornithine decarboxylase; RNOS, reactive nitrogen species; ROS, reactive oxygen species; TCR CD3 ζ-chain, CD3 ζ-chain in T cell receptor; TCR, T-cell receptor; TGFβ, transforming growth factor beta; TNFα, tumoral necrosis factor α; VEGF, vascular endothelial growth factor.

Figure 3

Arginine metabolism according to prostate cancer hormonal status informs plausible therapies. During early AD PCa development, androgens are available and contribute to increased immunosuppression and proliferative stimulus through ARGII mediation. In this setting, increased ASS activity confers resistance to ADI administration, and the most benefit is likely to derive from immunotherapy. Conversely, emergence of the androgen-independent phase of disease, after acquired resistance to ADT therapy, is associated with tumor cell survival in very low androgen levels with subsequent low ASS and ARGII expression and normal immune activity (conferring higher response rate to ADI treatment). ADT, androgen deprivation therapy; ADI, arginine deiminase; ARGII, arginase II; ASS, argininosuccinate synthase.