Introduction

Reprogramming energy metabolism is a hallmark of cancer, tumor cells undergo metabolic reprogramming to meet the demand of continuous cell proliferation (Hanahan and Weinberg 2011). Many metabolic pathways are changed in tumor cells, for example, aerobic glycolysis is enhanced, oxidative phosphorylation is reduced, and biosynthesis is increased. Altered metabolic pathways link macromolecules biosynthesis, redox homeostasis, and epigenetic modifications to tumor progression, metastasis and drug resistance (DeBerardinis and Chandel 2016). As the basic components of protein, amino acids are involved in a variety of important biological processes, such as metabolism, growth, and immunity (He et al. 2011). The uptake and secretion of multiple amino acids in tumors are quite different from those in normal tissues (Dunphy et al. 2018). Targeting amino acid metabolism for cancer treatment has always been the focus of attention.

Proline is the only imino acid that has an α-amino group in the pyrrolidine ring. Proline has its own metabolic pathway, and the metabolic pathway requires many enzymes to catalyze reactions. Delta-1-pyrroline-5-carboxylate synthase (P5CS) is a key enzyme in the biosynthesis of proline, which catalyzes the reduction of glutamic acid to Delta-1-pyrroline-5-carboxylic acid (P5C). Next, P5C is reduced to proline under the action of pyrroline-5-carboxylic acid reductase (PYCR) (Adams 1970). An important biological function of proline synthesis is to produce collagen, which is the main component of extracellular matrix (ECM). Prolyl 4-hydroxylases (P4Hs) are the enzymes that exist in the endoplasmic reticulum (ER) and play a central role in the biosynthesis of collagen (Myllyharju 2008). On the other hand, collagen can provide an additional source of proline for tumor cells under metabolic stress (Olivares et al. 2017). Proline dehydrogenase/proline oxidase (PRODH/POX) is the first step to catalyze the degradation of proline. It catalyzes the oxidation of L-proline, leading to the release of electrons, which participates in the electron transport chain (ETC) to generate reactive oxygen species (ROS) or ATP (Polyak et al. 1997; Donald et al. 2001). Although the proline metabolism regulation system still needs to be elucidated, more and more evidences show that proline metabolism can induce tumorigenesis and metastasis. In this review, we summarized the various proline metabolism-related enzymes mentioned above and their roles in cancer.

Proline metabolism

Proline metabolic pathway includes proline synthesis and catabolism. Proline is synthesized from glutamate or ornithine. First, the immediate precursor glutamate-γ-semialdehyde (GSAL) can be synthesized by P5CS in the mitochondria or ornithine aminotransferase (OAT) in the cytosol. Then, P5C is automatically transformed from GSAL. Finally, P5C is converted to proline by PYCR (Hu et al. 2008). In some cases, proline is hydroxylated by P4Hs, and used for the biosynthesis of collagen (Myllyharju 2003). In proline catabolism, proline is converted into P5C catalyzed by PRODH/POX. P5C can be re-metabolized to glutamate by pyrroline-5-carboxylate dehydrogenase (P5CDH) or to ornithine by OAT (Phang and Liu 2012) (Fig. 1).

Fig. 1
figure 1

Proline metabolic pathway

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Collagen and cancer

Collagen is the most abundant and widely distributed protein in animals, which accounts for about 25–30% of the total protein. In addition to being the main structural protein of ECM, collagen is also involved in many important physiological functions, such as cell adhesion and migration, cell signal transduction, and growth and development of tissues and organs (Ricard-Blum 2011; Xu et al. 2019). Abnormal collagen synthesis can lead to tumor invasion and metastasis.

Collagen has a triple helix structure, and the α chain constituting the triple helix has a repeating Gly-X-Y triplet, in which proline is usually located at the X position and 4-hydroxyproline (4Hyp) is located at the Y position. 4Hyp is necessary for the stability of the triple helix and the formation of collagen (Myllyharju and Kivirikko 2004). P4Hs hydroxylates the Y-site proline on the α chain Gly-X-Y repeat sequence of the triple helix region of collagen to 4Hyp. P4Hs is an α2β2 tetramer composed of two α subunits and two β subunits, and belongs to α ketoglutarate-dependent dioxygenase. The α subunit of P4Hs (P4HA) is the main catalytic subunit, including three subtypes (P4HA1, P4HA2, and P4HA3). The β subunit is a member of the protein disulfide isomerase family, which has only one subtype (P4HB) (Annunen et al. 1997). Recent studies have shown that P4Hs are closely related to cancer progression. The functions of P4Hs in cancer are summarized in Table1.

Table 1 Biological implications of P4Hs on hallmarks of cancer
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P4HA1 is expressed in most tissues and cell types. The high expression of P4HA1 increases the production of collagen, thereby promotes tumor progression. Under hypoxia, hypoxia-inducible factor-1α (HIF-1α) activates P4HA1 to increase collagen deposition in breast cancer cells, thereby promoting metastasis (Gilkes et al. 2013). Moreover, in triple-negative breast cancer (TNBC), P4HA1 enhances HIF-1α stability to increase cancer cells stemness, accompanied by increased glycolysis and decreased oxidative phosphorylation (Xiong et al. 2018). Similarly, P4HA1 contributes to malignance of pancreatic cancer by regulating HIF-1α (Cao et al. 2019). Pyruvate can activate collagen hydroxylation to accelerate ECM remodeling by increasing the activity of P4HA1, and ultimately promote metastatic growth in breast cancer cells-driven lung metastatic niche (Elia et al. 2019).

P4HA2 has been identified to be associated with tumor progression and poor prognosis. In breast cancer, P4HA2 promotes tumor growth and invasiveness by enhancing collagen deposition (Xiong et al. 2014). In cervical cancer, P4HA2 promotes glycolysis through upregulating PGK1 and LDHA, leading to increased proliferation and migration (Li et al. 2019).

P4HB is overexpressed in bladder cancer, colon cancer and hepatocellular carcinoma (HCC). In bladder cancer, inhibition of P4HB decreases cell proliferation, promotes cell apoptosis, and sensitizes cancer cells to gemcitabine by activating endoplasmic reticulum stress (ERS) and apoptotic pathway (Wang et al. 2020). P4HB knockdown induces apoptosis through promoting ROS accumulation and inactivating the signal transducer and activator of transcription 3 (STAT3) signaling pathway in colon cancer (Zhou et al. 2019). Overexpressed P4HB promotes HCC progression by inducing epithelial to mesenchymal transition (EMT) and downregulating glucose-regulated protein 78 (GRP78) (Xia et al. 2017). Furthermore, P4HB is upregulated in chemo-resistant liver cancer sub-line HepG2/Adriamycin (ADR). P4HB knockdown decreases chemoresistance, migration and invasion abilities by inhibiting EMT, which is regulated by Snail and β-catenin pathway (Ma et al. 2020).

In addition, Collagen prolyl hydroxylation catalyzed by P4Hs and DNA/histones demethylation catalyzed by ten–eleven translocation (TET) DNA demethylases/Jumonji (JMJ) domain–containing histone demethylases share the same cofactor, l-ascorbic acid (vitamin C), they can integrate to control cell plasticity and promote cancer progression (D'Aniello et al. 2017). l-Proline can induce embryonic-stem-cell-to-mesenchymal-like transition (esMT), accompanied by the increase of H3K9 and H3K36 methylation levels. Otherwise, l-proline withdrawal or vitamin C addition reverses esMT by reducing H3K9 and H3K36 methylation levels (Comes et al. 2013). Collagen hydroxylation mediated by P4HA2 is increased in l-proline-induced esMT. Increasing collagen hydroxylation upregulates global DNA/histone methylation through reducing the activity of TET/JMJ demethylases, and regulates cell state transition (D'Aniello et al. 2019).

Proline synthesis and cancer

Enzymes involved in proline synthesis pathway can promote tumor progression (Table 2). Focusing on using metabolic profiling to determine the key role of proline and hydroxyproline metabolism in the hypoxic response of HCC (Tang et al. 2018), we found that hydroxyproline is accumulated in HCC tissues, and is correlated with the malignancy of HCC. In hypoxic tumor microenvironment, hypoxia activates proline synthesis by upregulating the expression of ALDH18A1, and leads to the accumulation of hydroxyproline by attenuating PRODH2 activity. Hydroxyproline enhances the stability of HIF-1α by inhibiting the hydroxylation of HIF-1α, thereby promoting HCC cell survival and sorafenib resistance. Moreover, inhibition of ALDH18A1 increases the sensitivity of lipogenesis inhibitor, and synergistically inhibits tumor growth in vivo (Liu et al. 2020a). In melanoma, proline synthesis is upregulated, and inhibition of ALDH18A1 impairs protein synthesis to decrease tumor development by activating general control nonderepressible 2 (GCN2) pathway (Kardos et al. 2015).

Table 2 Functions of enzymes involved in proline synthesis in cancer
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PYCR has three subtypes, PYCR1, PYCR2 and PYCRL. PYCR1-mediated proline synthesis is necessary for HCC cells proliferation and tumor growth (Ding et al. 2020). PYCR1 promotes cell proliferation and inhibits cell apoptosis via activating c-Jun N-terminal kinase/insulin receptor substrate 1 (JNK/IRS1) pathway in HCC (Zhuang et al. 2019). PYCR1 is accumulated in prostate cancer, highly expressed PYCR1 promotes cell proliferation. However, inhibition of PYCR1 induces cell cycle arrest and cell apoptosis (Zeng et al. 2017). In non-small cell lung cancer (NSCLC), elevated PYCR1 expression enhances cell proliferation and represses cells apoptosis through regulating p38 signaling pathway or cyclin D1, Bcl-2 and Bcl-xl expression (Wang et al. 2019; Cai et al. 2018). Moreover, PYCR1 also accelerates migration and invasion of NSCLC by inducing EMT (Sang et al. 2019). 2500 assessable breast cancer cases confirmed that PYCR1 closely related with tumor size, grades and molecular subtypes. In breast cancer cells, inhibition of PYCR1 reduces the abilities of cancer cells growth and invasion by inhibiting ERK pathway and MMP9 activity (Ding et al. 2017). In addition, PYCR1 can promote proliferation and invasion of bladder cancer cells by the increase of Akt/Wnt/β-catenin signaling (Du et al. 2021). PYCR2 silence decreases cells proliferation by activating (AMP-activated protein kinase) AMPK/mTOR pathway induced autophagy in melanoma (Ou et al. 2016). Together, PYCR1 induces cancer progression by promoting cell proliferation and inhibiting cell apoptosis or enhancing migration and invasion abilities. These findings suggest that PYCR1 may be a novel prognostic marker and a potential therapeutic target for cancer.

OAT is another enzyme that catalyzes proline synthesis. High expression of OAT promotes tumor growth of HCC (Zigmond et al. 2015). In NSCLC, OAT promotes proliferation, invasion and migration by upregulating miR-21 (Liu et al. 2019).

In addition, latest studies show that mitochondrial NADP(H) is important for the synthesis of proline. NADK2 is responsible for catalyzing NAD(H) to the generation of NADP(H). NADP(H) depletion through inhibiting NADK2 causes proline auxotrophy, finally inhibiting cell proliferation (Tran et al. 2021; Zhu et al. 2021).

Proline catabolism and cancer

PRODH/POX is a mitochondrial enzyme which catalyzes the transformation of proline to P5C (Johnson and Strecker 1962). PRODH/POX functions as a tumor suppressor to induce apoptosis or cell cycle arrest. POX can catalyze ROS generation to cause cell apoptosis (Donald et al. 2001). Nuclear factor of activated T cells (NFAT) signaling is activated and MEK/ERK pathway is inhibited in POX-induced apoptosis (Liu et al. 2006). Furthermore, POX is reduced in colorectal cancer. Overexpression of POX inhibits cancer cells proliferation and reduces tumor growth, and ultimately induces cell apoptosis. The cyclooxygenase-2 (COX-2) pathway, the epidermal growth factor receptor (EGFR) pathway and the Wnt/β-catenin pathway participate in POX-induced apoptosis (Liu et al. 2008). In addition, POX can induce G2 cell cycle arrest to reduce tumor formation by impairing HIF signaling (Liu et al. 2009).

PRODH also functions as an oncogene to promote cancer cells survival, proliferation, and metastasis. Under conditions of nutrient stress, proline can be obtained from the breakdown of collagen, proline is further degraded by PRODH to generate glutamate and α-ketoglutarate (α-KG), subsequently enter the TCA cycle to produce ATP to sustain cell survival (Pandhare et al. 2009). Moreover, proline derived from collagen promotes cell survival and proliferation of pancreatic ductal adenocarcinoma (PDAC) under conditions of limited nutrition, and PRODH-mediated proline metabolism is essential for the tumor growth (Olivares et al. 2017). Under H2O2 stress, PRODH protects prostate carcinoma cells against hydrogen peroxide-induced cell death by activating Akt and Forkhead box O 3a (FoxO3a) (Natarajan et al. 2012). Under the hypoxic tumor microenvironment, high expression of PRODH/POX induces protective autophagy by generating ROS (Liu et al. 2012a; Liu and Phang 2012). In NSCLC, PRODH promotes tumorigenesis by inducing EMT and activating IKKα (Liu et al. 2020b). In breast cancer, PRODH-mediated proline catabolism supports 3D growth, and promotes the formation of lung metastasis (Elia et al. 2017). Together, the role of PRODH in cancer may depend on cell types or tumor microenvironment.

Regulation of proline metabolic enzymes

POX encoded by PIG6 is originally found to be transcriptionally activated by p53 in p53-dependent apoptosis (Donald et al. 2001). PRODH is regulated positively by peroxisome proliferator-activated receptor (PPARγ). Specifically, the PPARγ ligand troglitazone can induce apoptosis by enhancing the binding of PPARγ in the POX promoter and increasing POX expression (Pandhare et al. 2006). On the other hand, oxidized low-density lipoproteins (oxLDLs) can upregulate POX expression to initiate pro-survival autophagy through activating PPARγ (Zabirnyk et al. 2010). The activation of AMPK can induce POX expression to promote cancer cell survival under glucose deprivation and hypoxia conditions (Pandhare et al. 2009; Liu et al. 2012a).

In addition, there is a negative correlation between miR-23b* and POX protein expression in renal cancer. Overexpression of miR-23b* decreases POX expression to promote cell proliferation (Liu et al. 2010). In prostate cancer cells and Burkitt lymphoma cells, c-MYC inhibits the expression of PRODH to promote cancer cells proliferation and survival mainly by the increase of miR-23b* (Liu et al. 2012b). The detailed PRODH network is shown in Fig. 2.

Fig. 2
figure 2

PRODH functions as tumor suppressor or tumor survival factor. PRODH is regulated positively () or negatively () (indicated in red). PRODH activates () or inhibits () many signaling pathways and biological process (indicated in green)

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PYCR1 can be regulated in many ways (Fig. 3). At transcriptional level, c-MYC upregulates the expression of PYCR1 to increase proline synthesis from glutamine, which is critical for tumor cell growth (Liu et al. 2012b, 2015; Li et al. 2021). Besides, the transcription factor myeloid zinc finger 1(MZF1) enhances ALDH18A1 and PYCR1 expression, thereby promoting the synthesis of proline and invasion ability of neuroblastoma cells (Fang et al. 2019).

Fig. 3
figure 3

Regulation of PYCR1 in cancer. PYCR1 is upregulated by c-MYC and MZF1. High expressed PYCR1 promotes proline synthesis. PYCR1 is deacetylated by SIRT3. Deacetylated PYCR1 enhances PYCR1 enzymatic activity to increase proline synthesis. kindlin-2, DJ-1, ORAOV1, and KSHV K1 bind to PYCR1 to increase PYCR1-mediated proline synthesis

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At the post-translational level, PYCR1 is acetylated by CBP and deacetylated by SIRT3. The acetylation of PYCR1 inhibits tumor cell growth. Oppositely, the deacetylation of PYCR1 enhances PYCR1 enzymatic activity, increasing proline synthesis promotes tumor cell growth (Chen et al. 2019).

In terms of protein–protein interaction, cell mechano-environment such as ECM stiffening significantly promotes kindlin-2 translocation to the mitochondria from the cytosol, and enhances the interaction between kindlin-2 and PYCR1. The interaction elevates PYCR1 level, consequently increases proline synthesis and lung adenocarcinoma cell proliferation. On the contrary, knockdown of kindlin-2 results in tumor growth inhibition and mortality rate reduction by downregulating PYCR1 (Guo et al. 2019). Moreover, DJ-1, oral cancer overexpressed 1 (ORAOV1), and Kaposi’s sarcoma-associated herpesvirus (KSHV) K1 can bind to PYCR1 and increase PYCR1-mediated proline synthesis, which is critical for anti-oxidative stress protection or tumorigenesis (Yasuda et al. 2013; Togashi et al. 2014; Choi et al. 2020). In addition, PINCH-1 promotes the interaction between kindlin-2 and PYCR1 to increase proline synthesis, thereby promoting tumor growth (Guo et al. 2020).

Conclusion

Various studies have provided an increasingly clear understanding of the different and important functions of proline-related metabolic enzymes in cancer. There is increasing evidence that proline metabolism is related to cancer progression. The changes in proline metabolism of cancer cells are related to the proliferation, metastasis and drug resistance of cancer cells. The hypoxic tumor microenvironment can upregulate the expression of ALDH18A1 to promote proline synthesis, resulting in the accumulation of hydroxyproline, thereby promoting the survival of HCC and the resistance of sorafenib. PYCR1 induces cancer progression by promoting cell proliferation and inhibiting cell apoptosis or enhancing the abilities of migration and invasion. The function of PRODH to promote tumor cell apoptosis or tumor cell survival and metastasis depends on the influence of different cell types and tumor microenvironment. These findings indicate that proline metabolic enzymes participate in a variety of tumor disease processes. A further understanding of the pathway of proline metabolism will provide new strategies and ideas for cancer diagnosis and treatment.