RRM2 is well-known as small subunit of RNR, a rate-limiting enzyme for dNDP synthesis required for DNA replication [1–3]. High RNR enzymatic activity is associated with tumor progression and resistance to various cellular stressors such as chemotherapeutic agents and ionizing radiation [6–8]. Increased RRM2 and RNR enzymatic activity were reported to present in highly metastatic tumor cells and tissues [9–11]. Therefore, RRM2 is also an important therapeutic target for DNA replication-dependent diseases such as cancer. Recently, many studies have demonstrated that RRM2 plays additional roles in determining the malignant potential of tumor cells. For example, elevated expression of RRM2 has been found to increase the drug-resistant properties of cancer cells and significantly enhance the invasive potential of many human cancer cells [3, 9, 10, 15], whereas knockdown of RRM2 expression results in the reversal of drug-resistance and suppressed tumor growth, and decreased metastasis potential [14, 16, 25]. Studies have identified that overexpression of both RRM1 and RRM2 leads increased RR activities and expended dNTP pools [[9, 13, 15], and ]. Interestingly, two recent studies have the studies found that overexpression of RRM1 suppresses invasion and metastasis formation of tumors through induction of PTEN pathway [9–11]. These data suggest two subunits of RNR play opposing roles in tumor progression and malignancy; beside they cooperate in dNTP production.
Tumor angiogenesis is necessary for the growth and metastasis of many tumors. Potential of angiogenic switch is a critical character of tumor malignancy [17–19]. Tumor angiogenesis and metastatic formation are intrinsically connected [20, 27]. Accumulating evidences support RRM2 possesses oncogene-like properties; it plays a potential role in tumor malignancy and metastasis. In this study, we therefore focused on the effect of RRM2 on tumor angiogenesis. We investigated the effect of overexpression of RRM2 on the production of angiogenesis regulatory factors in human cancer cells. Our study demonstrated that overexpression of RRM2 decreased the production of TSP-1 and increased that of VEGF in KB cells. Because hypoxia is a critical factor regulating VEGF expression through stabilizing its transcriptional factor HIF-1 protein and it is also an essential factor for tumor development . We also examined the effect of RRM2 on VEGF expression under hypoxia condition. The data demonstrated that under hypoxia condition overexpression of RRM2 further increased VEGF protein in KB cells. To further confirm the regulation, we measured TSP-1 and VEGF expression in KB and LNCaP cells after RRM2 knockdown by its specific siRNA. Contrast to overexpression of RRM2, we found knockdown of RRM2 expression caused an increased TSP-1 and decreased VEGF expression in both KB and LNCaP cells. Considering the potential therapeutic application of RNA interference for RRM2 such as siRNA, antisense oligo-nucleotide [16, 25], our above finding is potentially important, and it also indicates that the therapy based on RNA interference for RRM2 may potentially inhibit tumor angiogenesis beside its known inhibitory effects [16, 25]. The changes in the expression of angiogenic factors: TSP-1 and VEGF induced by RRM2 in tumor cells did result in increased angiogenic activity in vitro, as shown by the increased chemotactic activity for endothelial cells. Moreover, tumors generated in vivo by RRM2-overexpressing cells showed a higher vascular density compared to controls. We supposed that higher vascular density might partially contribute to growth advantage of RRM2-overexpressing cancer cells. Consistent with what we expected, the increased vascularization caused by overexpression of RRM2 did confer a growth advantage to RRM2-overexpressing cancer cells.
VEGF is significantly associated with poor outcome in human various malignancies [29, 30]. The increased production of VEGF is significant for various cancers development. Exposure of tumor cells to hypoxia is a common finding in solid tumors. Hypoxia induces a myriad of adaptive changes within tumor cells, which result in increased anaerobic glycolysis, new blood vessel formation and genetic instability . Consistently, a previous study by Graff et al  also found that elevated RNR caused by overexpression of RRM2 could overrule long-lasting arrest of DNA-synthesis after severe hypoxia insult, and cancer cells with elevated RRM2 were more resistant to hypoxia. TSP-1 is an important inhibitor of angiogenesis, and its suppression is crucial for the angiogenic switch in many tumor models [[32, 33], and ]. Additionally, TSP-1 is an inhibitor of invasion; it inhibits the activity of matrix metalloproteinase-9 (MMP9), which causes release of vascular endothelial growth factor sequestered in the extracellular matrix, thereby increasing invasion potential . Coincidentally, a recent study of Duxbury et al  also found that overexpression of RRM2 increased pancreatic adenocarcinoma cellular invasiveness and MMP-9 expression.
The earlier study by Wright et al [12, 13] reported that mouse RRM2 synergize with a variety of oncogenes including h-ras, h-rac, v-src, a-raf, c-myc, etc in cellular transformation and tumorigenic potential to normal fibroblast cells through a variety of signal pathways. For example, cooperation with Ras, overexpression of mouse RNR small subunit R2 significantly increases membrane-associated Raf-1 protein and MAPK-2 activity, finally further activates MAPK pathway . Oncogenes not only promote aberrant cellular mitogenesis, but also have an important impact on tumor formation and growth through an indirect mechanism, namely, by driving tumor angiogenesis [20–22]. Oncogenes such as v-myc, c-jun, v-src h-ras etc down-regulate the TSP-1 expression [20–22]. Additionally, some of them – for example, h-ras and c-myc – also significantly up-regulate VEGF expression . Contrast to oncogenes, tumor suppressor genes such as p53, Rb and PTEN etc stimulate TSP-1 expression [20–22]. Interestingly, our previous study found that in cancer cells, even without any stress RRM2 and p53R2 are bound to wild-type p53 protein. In response to UV irradiation, RRM2 and p53R2 dissociate themselves from p53 and form RNR with RRM1 . It is interesting to further examine whether elevated RRM2 has any impact on p53 function through the protein-protein interaction. Coincidently, we found that knockdown of RRM2 leads a significantly elevated p53 protein in both KB and LNCaP (unpublished data); we are focusing on the correlations among these proteins. Since p53 acts as a negative controller of angiogenesis, and the loss of functional p53 results in high VEGF and low TSP-1 production, with consequent increase in angiogenic activity . The mechanisms through which RRM2 regulate TSP-1 and VEGF expression requires further investigation.