DNA modifications such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are epigenetic marks known to affect global gene expression in mammals. Given their prevalence in the human genome, close correlation with gene expression and high chemical stability, these DNA epigenetic marks could serve as ideal biomarkers for cancer diagnosis. Taking advantage of a highly sensitive and selective chemical labeling technology, we report here the genome-wide profiling of 5hmC in circulating cell-free DNA (cfDNA) and in genomic DNA (gDNA) of paired tumor and adjacent tissues collected from a cohort of 260 patients recently diagnosed with colorectal, gastric, pancreatic, liver or thyroid cancer and normal tissues from 90 healthy individuals. 5hmC was mainly distributed in transcriptionally active regions coincident with open chromatin and permissive histone modifications. Robust cancer-associated 5hmC signatures were identified in cfDNA that were characteristic for specific cancer types. 5hmC-based biomarkers of circulating cfDNA were highly predictive of colorectal and gastric cancers and were superior to conventional biomarkers and comparable to 5hmC biomarkers from tissue biopsies. Thus, this new strategy could lead to the development of effective, minimally invasive methods for diagnosis and prognosis of cancer from the analyses of blood samples.

Posttranslational neddylation of cullins in the Cullin-Ring E3 ligase (CRL) complexes is needed for proteolytic degradation of CRL substrates, whose accumulation induces cell-cycle arrest, apoptosis, and senescence. The Nedd8-activating enzyme (NAE) is critical for neddylation of CRL complexes and their growth-promoting function. Recently, the anticancer small molecule MLN4924 currently in phase I trials was determined to be an inhibitor of NAE that blocks cullin neddylation and inactivates CRL, triggering an accumulation of CRL substrates that trigger cell-cycle arrest, apoptosis, and senescence in cancer cells. Here, we report that MLN4924 also triggers autophagy in response to CRL inactivation and that this effect is important for the ability of MLN4924 to suppress the outgrowth of liver cancer cells in vitro and in vivo. MLN4924-induced autophagy was attributed partially to inhibition of mTOR activity, due to accumulation of the mTOR inhibitory protein Deptor, as well as to induction of reactive oxygen species stress. Inhibiting autophagy enhanced MLN4924-induced apoptosis, suggesting that autophagy is a survival signal triggered in response to CRL inactivation. In a xenograft model of human liver cancer, MLN4924 was well-tolerated and displayed a significant antitumor effect characterized by CRL inactivation and induction of autophagy and apoptosis in liver cancer cells. Together, our findings support the clinical investigation of MLN4924 for liver cancer treatment and provide a preclinical proof-of-concept for combination therapy with an autophagy inhibitor to enhance therapeutic efficacy. Cancer Res; 72(13); 3360-71. Ó2012 AACR.

Our goal was to study the prevalence of systemic sclerosis (SSc) subtypes, autoantibody profile, and pulmonary fibrosis in a large group of Han Chinese. Chinese SSc patients (n=419) were recruited from a multicenter study including hospitals and outpatient clinics in China. All patients met the American College of Rheumatology classification criteria for SSc. Anti-topoisomerase (ATA), anti-centromere (ACA), anti- RNA polymerase III (anti-RNAP3), and anti-U1- ribonucleoprotein (anti-U1RNP) were detected utilizing commercially available kits. The clinical and autoantibody information in Chinese patients was compared to that in the US Caucasian patients (n=834), recruited from the Genetics versus Environment in Scleroderma Outcome Study and Scleroderma Family Registry. Chi-square test was utilized for the abovementioned comparisons. Chinese patients showed 40.3 % limited (lcSSc) and 59.7 % diffuse (dcSSc) forms of SSc. ATA was found in 59.9 %, ACA in 13.4 %, anti-RNAP3 in 1.3 %, and anti-U1RNP in 18 % of Chinese SSc patients. Compared to US patients (65.1 % lcSSc, 34.9 % dcSSc, ATA in 18.7 %, ACA in 32.4 %, anti-RNAP3 in 17.4 %, and anti-U1RNP in 2.8 %), Chinese SSc patients are significantly higher in dcSSc and the frequencies of ATA and anti-U1RNP, but lower in ACA and anti-RNAP3. In addition, pulmonary fibrosis was observed in 78 % Chinese SSc patients and was strongly associated with the presence of ATA. The present study represents the first report of SSc features in a large group of Chinese patients. Clinical subtypes and the frequencies of SSc-related autoantibodies in Chinese SSc patients are significantly different from those in SSc patients of the US Caucasian descent.

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