Autophagy is the principal catabolic response to nutrient starvation and is necessary to clear dysfunctional or damaged organelles, but excessive autophagy can be cytotoxic or cytostatic and contributes to cell death. Depending on the abundance of enzymes involved in molecule biosynthesis, cells can be dependent on uptake of exogenous nutrients to provide these molecules. Argininosuccinate synthetase 1 (ASS1) is a key enzyme in arginine biosynthesis, and its abundance is reduced in many solid tumors, making them sensitive to external arginine depletion. We demonstrated that prolonged arginine starvation by exposure to ADI-PEG20 (pegylated arginine deiminase) induced autophagy-dependent death of ASS1-deficient breast cancer cells, because these cells are arginine auxotrophs (dependent on uptake of extracellular arginine). Indeed, these breast cancer cells died in culture when exposed to ADI-PEG20 or cultured in the absence of arginine. Arginine starvation induced mitochondrial oxidative stress, which impaired mitochondrial bioenergetics and integrity. Furthermore, arginine starvation killed breast cancer cells in vivo and in vitro only if they were autophagy-competent. Thus, a key mechanism underlying the lethality induced by prolonged arginine starvation was the cytotoxic autophagy that occurred in response to mitochondrial damage. Last, ASS1 was either low in abundance or absent in more than 60% of 149 random breast cancer bio-samples, suggesting that patients with such tumors could be candidates for arginine starvation therapy.

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 ¼ 3.53, 95% CI: 1.37-9.70) and validation set (OR ¼ 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 g-H2AX in HCT-116 and SW480 cells post g-irradiation, which supports our finding that CRC patients with HMAG2-positive staining in primary tumors had augmented the efficacy of adjuvant radiotherapy (HR ¼ 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.

NCT02442414;Pre-results.

Farnesoid X receptor (FXR) (nuclear receptor subfamily 1, group H, member 4) is a member of nuclear hormone receptor superfamily, which plays essential roles in metabolism of bile acids, lipid, and glucose. We previously showed spontaneously hepatocarcinogenesis in aged FXR(-/-) mice, but its relevance to human hepatocellular carcinoma (HCC) is unclear. Here, we report a systematical analysis of hepatocarcinogenesis in FXR(-/-) mice and FXR expression in human liver cancer. In this study, liver tissues obtained from FXR(-/-) and wild-type mice at different ages were compared by microarray gene profiling, histological staining, chemical analysis, and quantitative real-time PCR. Primary hepatic stellate cells and primary hepatocytes isolated from FXR(-/-) and wild-type mice were also analyzed and compared. The results showed that the altered genes in FXR(-/-) livers were mainly related to metabolism, inflammation, and fibrosis, which suggest that hepatocarcinogenesis in FXR(-/-) mice recapitulated the progression of human liver cancer. Indeed, FXR expression in human HCC was down-regulated compared with normal liver tissues. Furthermore, the proinflammatory cytokines, which were up-regulated in human HCC microenvironment, decreased FXR expression by inhibiting the transactivity of hepatic nuclear factor 1α on FXR gene promoter. Our study thereby demonstrates that the down-regulation of FXR has an important role in human hepatocarcinogenesis and FXR(-/-) mice provide a unique animal model for HCC study.

Human mitochondrial pyrroline-5-carboxylate reductase (PYCR) is a house-keeping enzyme that catalyzes the reduction of Δ1-pyrroline-5-carboxylate to proline. This enzymatic cycle plays pivotal roles in amino acid metabolism, intracellular redox potential and mitochondrial integrity. Here, we hypothesize that PYCR1 might be a novel prognostic biomarker and therapeutic target for breast cancer. In this study, breast cancer tissue samples were obtained from Zhejiang University (ZJU set). Immunohistochemistry analysis was performed to detect the protein level of PYCR1, and Kaplan-Meier and Cox proportional analyses were employed in this outcome study. The prognostic significance and performance of PYCR1 mRNA were validated on 13 worldwide independent microarray data sets, composed of 2500 assessable breast cancer cases. Our findings revealed that both PYCR1 mRNA and protein expression were significantly associated with tumor size, grade and invasive molecular subtypes of breast cancers. Independent and pooled analyses verified that higher PYCR1 mRNA levels were significantly associated with poor survival of breast cancer patients, regardless of estrogen receptor (ER) status. For in vitro studies, inhibition of PYCR1 by small-hairpin RNA significantly reduced the growth and invasion capabilities of the cells, while enhancing the cytotoxicity of doxorubicin in breast cancer cell lines MCF-7 (ER positive) and MDA-MB-231 (ER negative). Further population study also validated that chemotherapy significantly improved survival in early-stage breast cancer patients with low PYCR1 expression levels. Therefore, PYCR1 might serve as a prognostic biomaker for either ER-positive or ER-negative breast cancer subtypes and can also be a potential target for breast cancer therapy.

Background: In addition to its essential role in ribonucleotide reduction, ribonucleotide reductase (RNR) small subunit, RRM2, has been known to play a critical role in determining tumor malignancy. Overexpression of RRM2 significantly enhances the invasive and metastatic potential of tumor. Angiogenesis is critical to tumor malignancy; it plays an essential role in tumor growth and metastasis. It is important to investigate whether the angiogenic potential of tumor is affected by RRM2.

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.

In this study, we describe the growth arrest DNA damage-inducible gene 45beta (GADD45beta), whose expression was significantly down-regulated in the hepatocellular carcinoma (HCC) microarray study and confirmed by Northern blot analysis. The results suggested that expression of GADD45beta was decreased in human liver cancer cell lines HepG2 and Hep3B, but not in normal human embryonic liver cell line CL-48 or normal liver tissue. Histochemistry study and real-time PCR further confirmed that GADD45beta staining in HCC was significantly decreased when compared to surrounding non-neoplastic liver tissue. In further studies of multiple human cancer tissues, GADD45beta strongly stained tissues such as colon cancer, breast cancer, prostate cancer, squamous cell cancer, lymphoma, and leiomyosarcoma, suggesting that the decreased expression of GADD45beta is specific to HCC. Eighty-five cases of primary HCC were further examined by immunohistochemistry and statistical analyses demonstrated that HCC scored lower than matched non-neoplastic liver tissues consistently and significantly. No staining occurred in 12.94% of HCC cases (score = 0, n = 11); 42.35% had weak staining (score = 1, n = 36); 27.06% had moderate staining (score = 2, n = 23); and 17.65% had staining as strong as normal tissue (score = 3, n = 15). Overall, surrounding non-neoplastic liver tissue was highly positive for GADD45beta compared to adjacent neoplastic liver tissues (P < 0.01). We further observed that down-regulation of GADD45beta expression was strongly correlated with differentiation (P < 0.01) and high nuclear grade (P < 0.01). Moreover, we found that expression of GADD45beta was inversely correlated to the presence of mutant p53 in HCC tissue (P < 0.05). Thus, the results of our study suggest that GADD45beta, which is down-regulated in most cases of HCC, remains an ideal candidate for development as a molecular marker in the diagnosis of HCC and as a potential therapeutic target.