Lijun Xue

Overexpression of HMGA2 Promotes Metastasis and Impacts Survival of Colorectal Cancers

Purpose: This study aims to address the hypothesis that the high-mobility group A2 (HMGA2), an oncofetal protein, relates to survivability and serves as a prognostic biomarker for colorectal cancer (CRC). Experimental Design: This is a retroprospective multiple center study. The HMGA2 expression level was determined by performing immunohistochemistry on surgical tissue samples of 89 CRCs from a training set and 191 CRCs from a validation set. The Kaplan–Meier analysis and COX proportional hazard model were employed to analyze the survivability. Results: Multivariate logistic analysis indicated that the expression of HMGA2 significantly correlates with distant metastasis in training set (odds ratio, OR = 3.53, 95% CI: 1.37–9.70) and validation set (OR = 6.38, 95% CI: 1.47–43.95). Survival analysis revealed that the overexpression of HMGA2 is significantly associated with poor survival of CRC patients (P < 0.05). The adjusted HRs for overall survival were 2.38 (95% CI: 1.30–4.34) and 2.14 (95% CI: 1.21–3.79) in training and validation sets, respectively. Further investigation revealed that HMGA2 delays the clearance of γ-H2AX in HCT-116 and SW480 cells post γ-irradiation, which supports our finding that CRC patients with HMAG2-positive staining in primary tumors had augmented the efficacy of adjuvant radiotherapy (HR = 0.18, 95% CI: 0.04–0.63). Conclusion: Overexpression of HMGA2 is associated with metastasis and unequivocally occurred in parallel with reduced survival rates of patients with CRC. Therefore, HMGA2 may potentially serve as a biomarker for predicting aggressive CRC with poor survivability and as an indicator for better response of radiotherapy. Clin Cancer Res; 17(8); 2570–80. ©2011 AACR.

Metastasis-Suppressing Potential of Ribonucleotide Reductase Small Subunit p53R2 in Human Cancer Cells

Purpose: Previous gene transfection studies have shown that the accumulation of human ribonucleotide reductase small subunit M2 (hRRM2) enhances cellular transformation, tumorigenesis, and malignancy potential. The latest identified small subunit p53R2 has 80% homology to hRRM2. Here, we investigate the role of p53R2 in cancer invasion and metastasis. Experimental Design: The immunohistochemistry was conducted on a tissue array including 49 primary and 59 metastatic colon adenocarcinoma samples to determine the relationship between p53R2 expression and metastasis. A Matrigel invasive chamber was used to sort the highly invasive cells and to evaluate the invasion potential of p53R2. Results: Univariate and multivariate analyses revealed that p53R2 is negatively related to the metastasis of colon adenocarcinoma samples (odds ratio, 0.23; P < 0.05). The decrease of p53R2 is associated with cell invasion potential, which was observed in both p53 wild-type (KB) and mutant (PC-3 and Mia PaCa-2) cell lines. An increase in p53R2 expression by gene transfection significantly reduced the cellular invasion potential to 54% and 30% in KB and PC-3 cells, respectively, whereas inhibition of p53R2 by short interfering RNA resulted in a 3-fold increase in cell migration. Conclusions: Opposite regulation of hRRM2 and p53R2 in invasion potential might play a critical role in determining the invasion and metastasis phenotype in cancer cells. The expression level of ribonucleotide reductase small subunits may serve as a biomarker to predict the malignancy potential of human cancers in the future.

Ribonucleotide Reductase Small Subunit M2B Prognoses Better Survival in Colorectal Cancer

Ribonucleotide reductase subunit RRM2B (p53R2) has been reported to suppress invasion and metastasis in colorectal cancer (CRC). Here, we report that high levels of RRM2B expression are correlated with markedly better survival in CRC patients. In a fluorescence-labeled orthotopic mouse xenograft model, we confirmed that overexpression of RRM2B in nonmetastatic CRC cells prevented lung and/or liver metastasis, relative to control cells that did metastasize. Clinical outcome studies were conducted on a training set with 103 CRCs and a validation set with 220 CRCs. All participants underwent surgery with periodic follow-up to determine survivability. A newly developed specific RRM2B antibody was employed to carry out immunohistochemistry for determining RRM2B expression levels on tissue arrays. In the training set, the Kaplan-Meier and multivariate Cox analysis revealed that RRM2B is associated with better survival of CRCs, especially in stage IV patients (HR = 0.40; 95% CI = 0.18-0.86, P = 0.016). In the validation set, RRM2B was negatively related to tumor invasion (OR = 0.45, 95% CI = 0.19-0.99, P = 0.040) and lymph node involvement (OR = 0.48, 95% CI = 0.25-0.92, P = 0.026). Furthermore, elevated expression of RRM2B was associated with better prognosis in this set as determined by multivariate analyses (HR = 0.48, 95% CI = 0.26-0.91, P = 0.030). Further investigations revealed that RRM2B was correlated with better survival of CRCs with advanced stage III and IV tumors rather than earlier stage I and II tumors. Taken together, our findings establish that RRM2B suppresses invasiveness of cancer cells and that its expression is associated with a better survival prognosis for CRC patients.

Ribonucleotide Reductase Small Subunit p53R2 Facilitates p21 Induction of G1 Arrest under UV Irradiation

p53R2, which is one of the two known ribonucleotide reductase small subunits (the other being M2), is suggested to play an important role in supplying deoxynucleotide triphosphates (dNTP) for DNA repair during the G(1) or G(2) phase of the cell cycle. The ability of p53R2 to supply dNTPs for repairing DNA damages requires the presence of a functional p53 tumor suppressor. Here, we report in vivo physical interaction and colocalization of p53R2 and p21 before DNA damage. Mammalian two-hybrid assay further indicates that the amino acids 1 to 113 of p53R2 are critical for interacting with the NH(2)-terminal region (amino acids 1-93) of p21. The binding between p21 and p53R2 decreases inside the nucleus in response to UV, the time point of which corresponds to the increased binding of p21 with cyclin-dependent kinase-2 (Cdk2), and the decreased Cdk2 activity in the nucleus at G(1). Interestingly, p53R2 dissociates from p21 but facilitates the accumulation of p21 in the nucleus in response to UV. On the other hand, the ribonucleotide reductase activity increases at the corresponding time in response to UV. These data suggest a new function of p53R2 of cooperating with p21 during DNA repair at G(1) arrest.

Structurally Dependent Redox Property of Ribonucleotide Reductase Subunit p53R2

p53R2 is a newly identified small subunit of ribonucleotide reductase (RR) and plays a key role in supplying precursors for DNA repair in a p53-dependent manner. Currently, we are studying the redox property, structure, and function of p53R2. In cell-free systems, p53R2 did not oxidize a reactive oxygen species (ROS) indicator carboxy-H2DCFDA, but another class I RR small subunit, hRRM2, did. Further studies showed that purified recombinant p53R2 protein has catalase activity, which breaks down H2O2. Overexpression of p53R2 reduced intracellular ROS and protected the mitochondrial membrane potential against oxidative stress, whereas overexpression of hRRM2 did not and resulted in a collapse of mitochondrial membrane potential. In a site-directed mutagenesis study, antioxidant activity was abrogated in p53R2 mutants Y331F, Y285F, Y49F, and Y241H, but not Y164F or Y164C. The fluorescence intensity in mutants oxidizing carboxy-H2DCFDA, in order from highest to lowest, was Y331F > Y285F > Y49F > Y241H > wild-type p53R2. This indicates that Y331, Y285, Y49, and Y241 in p53R2 are critical residues involved in scavenging ROS. Of interest, the ability to oxidize carboxy-H2DCFDA indicated by fluorescence intensity was negatively correlated with RR activity from wild-type p53R2, mutants Y331F, Y285F, and Y49F. Our findings suggest that p53R2 may play a key role in defending oxidative stress by scavenging ROS, and this antioxidant property is also important for its fundamental enzymatic activity.

Ribonucleotide reductase small subunit M2 serves as a prognostic biomarker and predicts poor survival of colorectal cancers

The overexpression of RRM2 [RR (ribonucleotide reductase) small subunit M2] dramatically enhances the ability of the cancer cell to proliferate and to invade. To investigate further the relevance of RRM2 and CRCs (colorectal cancers), we correlated the expression of RRM2 with the clinical outcome of CRCs. A retrospective outcome study was conducted on CRCs collected from the COH [(City of Hope) National Medical Center, 217 cases] and ZJU (Zhejiang University, 220 cases). IHC (immunohistochemistry) was employed to determine the protein expression level of RRM2, and quantitative real-time PCR was employed to validate. Multivariate logistic analysis indicated that the adjusted ORs (odds ratios) of RRM2-high for distant metastases were 2.06 [95% CI (confidence interval), 1.01–4.30] and 5.89 (95% CI, 1.51–39.13) in the COH and ZJU sets respectively. The Kaplan–Meier analysis displayed that high expression of RRM2 had a negative impact on the OS (overall survival) and PFS (progress-free survival) of CRC in both sets significantly. The multivariate Cox analysis further demonstrated that HRs (hazard ratios) of RRM2-high for OS were 1.88 (95% CI, 1.03–3.36) and 2.06 (95% CI, 1.10–4.00) in the COH and ZJU sets respectively. Stratification analysis demonstrated that the HR of RRM2 dramatically increased to 12.22 (95% CI, 1.62–258.31) in the MMR (mismatch repair) gene-deficient subgroup in the COH set. Meanwhile, a real-time study demonstrated that down-regulation of RRM2 by siRNA (small interfering RNA) could significantly and specifically reduce the cell growth and adhesion ability in HT-29 and HCT-8 cells. Therefore RRM2 is an independent prognostic factor and predicts poor survival of CRCs. It is also a potential predictor for identifying good responders to chemotherapy for CRCs.

RRM2B Suppresses Activation of the Oxidative Stress Pathway and is Up-regulated by P53 During Senescence

RRM2B is the DNA damage-inducible small subunit of ribonucleotide reductase, the rate-limiting enzyme in de novo deoxyribonucleoside triphosphate synthesis. Although RRM2B is implicated in DNA repair and the maintenance of mitochondrial DNA content, the regulation and function of RRM2B in senescence have not been previously established. Here, we show that RRM2B is highly induced in a p53-dependent manner during senescence in primary human fibroblast IMR90 cells and is expressed at higher levels in senescent precancerous human prostatic intraepithelial neoplasm lesions compared to adjacent normal prostate glands. Paradoxically, silencing RRM2B expression leads to an increase in the level of reactive oxygen species, mitochondrial membrane depolarization, and premature senescence in a p38MAPK- and p53-dependent manner in young fibroblasts. Consistently, induction of senescence is accelerated in Rrm2b deficient mouse embryo fibroblasts. Our data demonstrate that RRM2B is induced by stress signals prior to the onset of senescence and prevents premature oxidative stress-induced senescence.

Redox Property of Ribonucleotide ReductaseSmall Subunit M2 and p53R2

Human ribonucleotide reductase (RR) small subunits, M2 and P53R2, play key roles in forming RR holoenzyme and supplying nucleotide precursors for DNA replication and repair. Currently, we are studying the redox property, structure, and function of hRRM2 and p53R2. In the cell-free system, p53R2 did not oxidize a reactive oxygen species (ROS) indicator Carboxy-H(2)DCFDA, but hRRM2 did. Further studies demonstrated that purified recombinant p53R2 protein has the catalase activity to scavenge H(2)O(2). Over-expression of p53R2 reduced intracellular ROS and protected the mitochondrial membrane potential against oxidative stress, whereas over-expression of hRRM2 did not result in the collapse of mitochondrial membrane potential. Our findings suggest that p53R2 may play a key role in defending oxidative stress by scavenging ROS, and this antioxidant property is also important for its enzymatic activity.

Prognostic and therapeutic significance of ribonucleotide reductase small subunit M2 in estrogen-negative breast cancers

BackgroundRibonucleotide reductase (RR) is an essential enzyme involved in DNA synthesis. We hypothesized that RR subunit M2 (RRM2) might be a novel prognostic and predictive biomarker for estrogen receptor (ER)-negative breast cancers.MethodsIndividual and pooled survival analyses were conducted on six independent large-scale breast cancer microarray data sets; and findings were validated on a human breast tissue set (ZJU set).ResultsGene set enrichment analysis revealed that RRM2-high breast cancers were significantly enriched for expression of gene sets that increased in proliferation, invasiveness, undifferentiation, embryonic stem/progenitor-like phenotypes, and poor patient survival (p < 0.01). Independent and pooled analyses verified that increased RRM2 mRNA levels were associated with poor patient outcome in a dose-dependent manner. The prognostic power of RRM2 mRNA was comparable to multiple gene signatures, and it was superior to TNM stage. In ER-negative breast cancers, RRM2 showed more prognostic power than that in ER-positive breast cancers. Further analysis indicated that RRM2 was a more accurate prognostic biomarker for ER-negative breast cancers than the pathoclinical indicators and uPA. A new RR inhibitor, COH29, could significantly enhance the chemosensitivity to doxorubicin in ER-negative MDA-MB-231 cells, but not in ER-positive MCF-7 cells.ConclusionRRM2 appears to be a promising prognostic biomarker and therapeutic target for ER-negative breast cancer patients.

Abstract 4183: Ribonucleotide reductase small subunit M2B associates with better survival of colorectal cancers through inhibiting tumor invasion and metastasis

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Previous studies have demonstrated that RRM2B suppresses the invasion and metastasis of cancer cells. Here, we examined the hypothesis that RRM2B may be related to better survivability and serve as a prognostic biomarker for colorectal cancer (CRC). The RRM2B-overexpressing transfectant HCT-116/RRM2B or the corresponding vector control transfectant was implanted into NSG mouse to generate orthotopic xenograft mouse models. The lung and liver metastasis occurred in 60% of the HCT-116/vector mice (3 of 5 cases) but in none of the HCT-116/RRM2B mice (0 of 6 cases). The outcome studies were conducted on a training set with 103 CRC cases and were validated on a set with 220 consecutive CRC cases. All participants underwent surgery and periodically followed-up to determine survivability. Immunohistochemistry (IHC) was conducted on tissue arrays to determine the expression level of RRM2B. In the training set, the Kaplan-Meier and multivariate COX analysis revealed that the RRM2B is associated with better overall survival (OS) of CRC patients, especially in stage IV CRCs (Hazard ratio, HR=0.40; 95% CI 0.18-0.86). This result has been validated as HRs of RRM2B were 1.02 (95% CI 0.31-4.58) and 0.53(95% CI 0.29-1.01) for stage 0-II and stage III-IV, respectively. Multivariate analysis indicated that the adjusted HR of RRM2B was 0.48 (95% CI 0.26-0.91). Further analysis suggested that RRM2B significantly reduces the relative risk of death due to colon cancer (HR = 0.38; 95% CI 0.16-0.87) but not rectal cancer. Therefore, we conclude that RRM2B plays a critical role in suppressing invasiveness ability of cancer cells and it associates with a better prognosis for CRC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4183. doi:10.1158/1538-7445.AM2011-4183