RNAi screens identify HES4 as a regulator of redox balance supporting pyrimidine synthesis and tumor growth.

NADH/NAD+ redox balance is pivotal for cellular metabolism. Systematic identification of NAD(H) redox regulators, although currently lacking, would help uncover unknown effectors critically implicated in the coordination of growth metabolism. In this study, we performed a genome-scale RNA interference (RNAi) screen to globally survey the genes involved in redox modulation and identified the HES family bHLH transcription factor HES4 as a negative regulator of NADH/NAD+ ratio. Functionally, HES4 is shown to be crucial for maintaining mitochondrial electron transport chain (ETC) activity and pyrimidine synthesis. More specifically, HES4 directly represses transcription of SLC44A2 and SDS, thereby inhibiting mitochondrial choline oxidation and cytosolic serine deamination, respectively, which, in turn, ensures coenzyme Q reduction capacity for DHODH-mediated UMP synthesis and serine-derived dTMP production. Accordingly, inhibition of choline oxidation preserves mitochondrial serine catabolism and ETC-coupled redox balance. Furthermore, HES4 protein stability is enhanced under EGFR activation, and increased HES4 levels facilitate EGFR-driven tumor growth and predict poor prognosis of lung adenocarcinoma. These findings illustrate an unidentified mechanism, underlying pyrimidine biosynthesis in the intersection between serine and choline catabolism, and underscore the physiological importance of HES4 in tumor metabolism.

IGF1R-phosphorylated PYCR1 facilitates ELK4 transcriptional activity and sustains tumor growth under hypoxia.

The proline synthesis is importantly involved in tumor growth under hypoxia, while the underlying mechanism remains to be further investigated. Here we show that pyrroline-5-carpoxylate reductase-1 (PYCR1), displaying a constant nuclear localization, is phosphorylated by nuclear IGF1R at Tyrosine 135 under hypoxia; this phosphorylation promotes the binding of PYCR1 to ELK4 and thus PYCR1 recruitment to ELK4-targeted genes promoter. Under hypoxia, ELK4-binding ability and enzymatic activity of PYCR1 are both required for ELK4-Sirt7-mediated transcriptional repression and cell growth maintenance, in which PYCR1-catalyzed NAD+ production stimulates the deacetylation activity of Sirt7 on H3K18ac that restrains genes transcription. Functionally, PYCR1 Tyr-135 phosphorylation exerts supportive effect on tumor growth under hypoxia, and the level of PYCR1 Tyr-135 phosphorylation is associated with malignancy of colorectal cancer (CRC). These data uncover the relationship between the compartmentally metabolic activity of PYCR1 and genes transcription regulation, and highlight the oncogenic role of PYCR1 during CRC development.

The negative effect of G1958A polymorphism on MTHFD1 protein stability and HCC growth.

PURPOSE: Methylenetetrahydrofolate dehydrogenase (MTHFD1), a key enzyme on the folate pathway, has been implicated in the tumor development of distinct types of cancers. The single nucleotide polymorphism (SNP) of 1958G > A mutation in the coding region of MTHFD1 (arginine 653 is mutated into glutamine) has been detected in a significant proportion of clinical samples of hepatocellular carcinoma (HCC). METHODS : Hepatoma cell lines, 97H and Hep3B were used. The expression of MTHFD1 and SNP mutation protein was determined by immunoblotting analysis. The protein ubiquitination of MTHFD1 was detected by immunoprecipitation analysis. The post-translational modification sites and interacting proteins of MTHFD1 in the presence of G1958A SNP were identified by mass spectrometry. Metabolic flux analysis was used to detect the synthesis of relevant metabolites sourced from serine isotope. RESULTS: The present study showed G1958A SNP of MTHFD1, encoding MTHFD1 R653Q, was associated with the attenuated protein stability caused by ubiquitination-mediated protein degradation. Mechanistically, MTHFD1 R653Q displayed an enhanced binding to the E3 ligase TRIM21, which was responsible for the augmented ubiquitination, and MTHFD1 K504 was identified to be the primary ubiquitination site. The subsequent metabolite analysis revealed MTHFD1 R653Q resulted in the repressed flux of serine-derived methyl group into metabolite precursors for purine synthesis, and the compromised purine synthesis was demonstrated to be responsible for the impeded growth capability in MTHFD1 R653Q-expressing cells. Moreover, the suppressive effect of MTHFD1 R653Q expression in tumorigenesis was verified by xenograft analysis, and the relationship between MTHFD1 G1958A SNP and its protein levels was revealed in clinical human liver cancer specimens. CONCLUSION: Our results uncovered an unidentified mechanism underlying of the impact of G1958A SNP on MTHFD1 protein stability and tumor metabolism in HCC. which provides a molecular basis for the according clinical management when considering MTHFD1 as a therapeutic target.

SUMOylation of YTHDF2 promotes mRNA degradation and cancer progression by increasing its binding affinity with m6A-modified mRNAs.

N 6-Methyladenosine (m6A) is the most abundant modification within diverse RNAs including mRNAs and lncRNAs and is regulated by a reversible process with important biological functions. Human YTH domain family 2 (YTHDF2) selectively recognized m6A-RNAs to regulate degradation. However, the possible regulation of YTHDF2 by protein post-translational modification remains unknown. Here, we show that YTHDF2 is SUMOylated in vivo and in vitro at the major site of K571, which can be induced by hypoxia while reduced by oxidative stress and SUMOylation inhibitors. SUMOylation of YTHDF2 has little impact on its ubiquitination and localization, but significantly increases its binding affinity of m6A-modified mRNAs and subsequently results in deregulated gene expressions which accounts for cancer progression. Moreover, Disease-free survival analysis of patients with lung adenocarcinoma derived from TCGA dataset reveals that higher expression of YTHDF2 together with higher expression of SUMO1 predicts poor prognosis. Our works uncover a new regulatory mechanism for YTHDF2 recognition of m6A-RNAs and highlight the importance of YTHDF2 SUMOylation in post-transcriptional gene expression regulation and cancer progression.

Recent advances in research on aspartate ß-hydroxylase (ASPH) in pancreatic cancer: A brief update.

Pancreatic cancer (PC) is a highly aggressive tumor, often difficult to diagnose and treat. Aspartate ß-hydroxylase (ASPH) is a type II transmembrane protein and the member of α-ketoglutarate-dependent dioxygenase family, found to be overexpressed in different cancer types, including PC. ASPH appears to be involved in the regulation of proliferation, invasion and metastasis of PC cells through multiple signaling pathways, suggesting its role as a tumor biomarker and therapeutic target. In this review, we briefly summarize the possible mechanisms of action of ASPH in PC and recent progress in the therapeutic approaches targeting ASPH.

SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function.

The methyltransferase like 3 (METTL3) is a key component of the large N6-adenosine-methyltransferase complex in mammalian responsible for N6-methyladenosine (m6A) modification in diverse RNAs including mRNA, tRNA, rRNA, small nuclear RNA, microRNA precursor and long non-coding RNA. However, the characteristics of METTL3 in activation and post-translational modification (PTM) is seldom understood. Here we find that METTL3 is modified by SUMO1 mainly at lysine residues K177, K211, K212 and K215, which can be reduced by an SUMO1-specific protease SENP1. SUMOylation of METTL3 does not alter its stability, localization and interaction with METTL14 and WTAP, but significantly represses its m6A methytransferase activity resulting in the decrease of m6A levels in mRNAs. Consistently with this, the abundance of m6A in mRNAs is increased with re-expression of the mutant METTL3-4KR compared to that of wild-type METTL3 in human non-small cell lung carcinoma (NSCLC) cell line H1299-shMETTL3, in which endogenous METTL3 was knockdown. The alternation of m6A in mRNAs and subsequently change of gene expression profiles, which are mediated by SUMOylation of METTL3, may directly influence the soft-agar colony formation and xenografted tumor growth of H1299 cells. Our results uncover an important mechanism for SUMOylation of METTL3 regulating its m6A RNA methyltransferase activity.

SUMO1 modification of KHSRP regulates tumorigenesis by preventing the TL-G-Rich miRNA biogenesis.

BACKGROUND: MicroRNAs (miRNAs) are important regulators involved in diverse physiological and pathological processes including cancer. SUMO (small ubiquitin-like modifier) is a reversible protein modifier. We recently found that SUMOylation of TARBP2 and DGCR8 is involved in the regulation of the miRNA pathway. KHSRP is a single stranded nucleic acid binding protein with roles in transcription and mRNA decay, and it is also a component of the Drosha-DGCR8 complex promoting the miRNA biogenesis. METHODS: The in vivo SUMOylation assay using the Ni2+-NTA affinity pulldown or immunoprecipitation (IP) and the in vitro E.coli-based SUMOylation assay were used to analyze SUMOylation of KHSRP. Nuclear/Cytosol fractionation assay and immunofluorescent staining were used to observe the localization of KHSRP. High-throughput miRNA sequencing, quantantive RT-PCR and RNA immunoprecipitation assay (RIP) were employed to determine the effects of KHSRP SUMO1 modification on the miRNA biogenesis. The soft-agar colony formation, migration, vasculogenic mimicry (VM) and three-dimensional (3D) cell culture assays were performed to detect the phenotypes of tumor cells in vitro, and the xenograft tumor model in mice was conducted to verify that SUMO1 modification of KHSRP regulated tumorigenesis in vivo. RESULTS: KHSRP is modified by SUMO1 at the major site K87, and this modification can be increased upon the microenvironmental hypoxia while reduced by the treatment with growth factors. SUMO1 modification of KHSRP inhibits its interaction with the pri-miRNA/Drosha-DGCR8 complex and probably increases its translocation from the nucleus to the cytoplasm. Consequently, SUMO1 modification of KHSRP impairs the processing step of pre-miRNAs from pri-miRNAs which especially harbor short G-rich stretches in their terminal loops (TL), resulting in the downregulation of a subset of TL-G-Rich miRNAs such as let-7 family and consequential tumorigenesis. CONCLUSIONS: Our data demonstrate how the miRNA biogenesis pathway is connected to tumorigenesis and cancer progression through the reversible SUMO1 modification of KHSRP.

miR186 suppresses prostate cancer progression by targeting Twist1.

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in north American men, and most its related deaths are due to advanced and metastatic PCa. However, the molecular mechanisms underlying PCa progression are still unclear. Here we use a pair of prostate cell lines P69/M12, which have the same genetic background and the highly metastatic cell line M12 is a subline derived from P69, to identify the pathogenesis of PCa. We find that a key miRNA--miR186 is significantly reduced in M12 compared to that in P69. Further, we validate that miR186 is also downregulated in human PCa specimens, most significantly in the metastatic patient specimens. The low miR186 expression is correlated with poor patient survival. Through knockdown or overexpression of miR186 in PCa cell lines, we discover that miR186 strongly inhibits cell motility, invasive, soft-agar colony formation, 3D culture growth and vasculogenic mimicry (VM) formation capacity, as well as the epithelial-to-mesenchymal transition (EMT) process by downregulation of its target Twist1. Moreover, the inverse relationship between the expression levels of miR186 and Twist1 is confirmed in vivo tumor metastasis experiment and clinical specimens. Taken together, our findings demonstrate an important role of miR186/Twist1 axis in the regulation of PCa progression, suggesting a potential application of miR186/Twist1 in PCa treatment.

A pilot randomized clinical study of the additive treatment effect of photodynamic therapy in breast cancer patients with chest wall recurrence.

PURPOSE: This study investigated the additive effect of photodynamic therapy (PDT) plus traditional radiotherapy (RT) for patients with breast cancer and chest wall recurrence. METHODS: A total of 40 patients with recurrent breast cancer were prospectively randomized to receive RT alone (group A, n=20) or PDT and RT in combination (group B, n=20). Traditional RT at a dose of 50 Gy was delivered in 25 fractions with or without exposure to 5-aminolevulinic acid and red light as PDT. RESULTS: The response rates were not statistically different between the groups, but more patients achieved a complete response (CR) in group B (50%) than in group A (20%). The median time to CR in group B was significantly shorter than that in group A (109.6 days vs. 175.2 days, p=0.001). Adverse event profiles were not different between the groups. CONCLUSION: An additive antitumor effect is demonstrated with additional PDT to RT. This combination therapy might reduce the duration of exposure to RT, but further investigation is warranted.

Clinical pathological characteristics and prognostic analysis of diabetic women with luminal subtype breast cancer.

This study selected luminal-type breast cancer patients as the study subjects. The patients were divided into groups according to the presence of diabetes and the types of medication used, and the patients' clinicopathological characteristics and prognostic indicators were explored. A total of 5,785 patients with luminal-type breast cancer admitted to Tianjin Medical University Cancer Institute and Hospital between January 2002 and December 2006 were selected as the study subjects. The subjects included 680 breast cancer patients with diabetes and 5,105 breast cancer patients without diabetes. The patients were divided into Luminal A, Luminal B (high ki67), and Luminal B (her-2/neu+) subtypes. Each subtype was further divided into a metformin group, a non-metformin group, and a nondiabetic group. The research indicators included breast cancer mortality, age, body mass index (BMI), amenorrhea, the presence of cardiovascular and cerebrovascular disease, pathological stage, pathological type, lymph node involvement, vessel carcinoma embolus, and the chemotherapy and endocrine regimen. A Kaplan-Meier analysis was conducted to analyze the differences in breast cancer mortality rates among the groups. The Cox proportional hazard model was adopted to detect independent factors related to prognosis. Kaplan-Meier univariate analysis showed that for the Luminal A, Luminal B (high ki67), and Luminal B (her-2/neu+) subtypes, the cancer-specific mortality rates differed significantly among the metformin, non-metformin, and nondiabetic groups. The 5-year survival rates were 94%, 82%, and 91% (P = 0.002); 93.5%, 81%, and 89% (P < 0.001); and 84%, 77%, and 83% (P = 0.035) for the subtypes within each group, respectively. Cox regression multivariate analysis showed that compared with the metformin group, all three subtypes of the, the non-metformin group showed poorer prognosis (hazard ratio [HR], 3.579; 95% confidence interval [CI], 1.506-8.506 [P = 0.004]; HR, 3.232; 95% CI, 1.839-5.678 [P < 0.001]; HR, 2.034; 95% CI,1.019-4.059 [P = 0.044] for Luminal A, Luminal B (high ki67), and Luminal B (her-2/neu+, respectively). Compared with the metformin group, the diabetic group showed poorer prognosis only for the Luminal B (high ki67) subtype (HR, 1.762; 95% CI, 1.033-3.005 [P = 0.038]). In addition, for the Luminal A, Luminal B (high ki67), and Luminal B (her-2/neu+) subgroups, there was a higher proportion of elderly patients (P < 0.001) and postmenopausal patients (P < 0.001) in the metformin and non-metformin groups than in the nondiabetic group. Moreover, the probability of having cardiovascular and cerebrovascular disease was also higher (P < 0.001) in the metformin and non-metformin groups. For the Luminal B (high ki67) and Luminal B (her-2/neu +) subgroups, there was a higher proportion of obese patients in the metformin and non-metformin groups (P < 0.001). In terms of clinical characteristics, for the Luminal B (high ki67) subtype, the proportion of patients with invasive ductal carcinoma was lower in the non-metformin group than in the other two groups (P = 0.001). In both the metformin and non-metformin groups, the proportion of T3/4 patients was higher (P < 0.001), the proportion of patients with lymph node metastasis was higher (P = 0.001), and the proportion of patients with vessel carcinoma embolus was higher (P = 0.001) compared with the nondiabetic group. In conclusion, compared with the metformin group, the non-metformin group had a poorer prognosis for all subtypes of luminal breast cancer. In the diabetic group, only patients with the Luminal B (high ki67) subtype exhibited a poorer prognosis. Therefore, different diabetes medication may have a different impact on the prognosis of different subtypes of luminal breast cancer.

QM-FISH analysis of the genes involved in the G1/S checkpoint signaling pathway in triple-negative breast cancer.

This study was conducted to analyze copy number alterations (CNAs) of the genes involved in the G1/S checkpoint signaling pathway of triple-negative breast cancer (TNBC) and to evaluate their clinical value in the prognosis of TNBC. Quantitative multi-gene fluorescence in situ hybridization was used to study CNAs of the genes involved in the G1/S checkpoint signaling pathway, including cyclin d1 (CCND1), c-Myc, p21, cell-cycle-checkpoint kinase 2 gene, p16, retinoblastoma (Rb1), murine double minute 2 (Mdm2) and p53, in 60 TNBC samples and 60 non-TNBC samples. In comparison with the non-TNBC samples, CNAs of the genes involved in the G1/S checkpoint signaling pathway were more frequently observed in the TNBC samples (p = 0.000). Out of a total of eight genes, six (CCND1, c-Myc, p16, Rb1, Mdm2, and p53) exhibited significantly different CNAs between the TNBC group and the non-TNBC group. Univariate survival analysis revealed that the gene amplification of c-Myc (p = 0.008), Mdm2 (p = 0.020) and the gene deletion of p21 (p = 0.004), p16 (p = 0.015), and Rb1 (p = 0.028) were the independent predictive factor of 5-year OS for patients with TNBC. Cox multivariate analysis revealed that the gene amplification of c-Myc (p = 0.026) and the gene deletion of p21 (p = 0.019) and p16 (p = 0.034) were independent prognostic factors affecting the 5-year OS for TNBC. CNAs of the genes involved in the G1/S checkpoint signaling pathway presented a higher rate of incidence in TNBC than in non-TNBC, which could indicate one of the molecular mechanisms for the specific biological characteristics of TNBC. The genes c-Myc, p21, and p16 were correlated with the prognosis of TNBC and therefore may have potential clinical application values in the prognostic prediction of TNBC.

Everolimus in combination with letrozole inhibit human breast cancer MCF-7/Aro stem cells via PI3K/mTOR pathway: an experimental study.

This study evaluated the effects of an mTOR inhibitor everolimus alone or in combination with letrozole on MCF-7/Aro (MCF-7 cells stably transfected with CYP19) in vitro and in vivo. In vitro studies, full-length CYP19 (aromatase) was cloned in a plasmid transfer vector pH ß-Aro and then transfected into MCF-7 stem cells which were ESA(+)CD44(+)CD24(-/low) sorted by flow cytometry. MTT assays were used to quantify the inhibitory effect of the drugs on MCF-7/Aro stem cells (SCs) and non-stem cells (NSCs). Apoptosis and the cell cycle distributions of stem cells were examined by flow cytometry. The tumorigenicity of stem cells after treatment was investigated by soft agar colony formation assays. In vivo studies, the BALB/c mice were injected with MCF-7/Aro SCs, and the different treatments were administered. After necropsy, the expression of KI67, CD31, AKT1, phospho-AKT (Thr308), and mTOR was analyzed by immunohistochemistry. In vitro, compared with MCF-7/Aro NSCs, there were greater resistance to the standard treatment doses of letrozole and everolimus in MCF-7/Aro SCs (17- and 15-fold, respectively). Treatment with everolimus or letrozole resulted in growth inhibition of SCs in a dose-dependent manner. Compared with single-agent therapy, the combination of everolimus with letrozole was more effective in the inhibition of cell growth (P < 0.001) and tumorigenicity (P < 0.01). In addition, an increase in G1 cell cycle arrest and increases in early apoptosis were observed in the combination treatment group compared with either single-agent group. In vivo, the xenograft tumor sizes were significantly decreased in everolimus alone group compared to control group, and everolimus plus letrozole therapy was much more effective compared with either single agent alone (P < 0.01). Compared with everolimus alone, the combination of everolimus and letrozole reduced the expression of KI67, mTOR, and phospho-AKT (Thr308; P < 0.01). Everolimus has effective inhibition on aromatase-overexpressing stem cell in vitro and in vivo. The combination everolimus and letrozole could be more effective than either drug alone.

Clinicopathological and prognostic characteristics of triple- negative breast cancer (TNBC) in Chinese patients: a retrospective study.

AIMS: To determine the clinical, pathological and prognostic features associated with triple-negative breast cancer (TNBC). METHODS: Clinical and histologic data of 21,749 breast cancer patients who were treated at Tianjin Medical University Cancer Institute and Hospital between July 2002 and December 2011 were collected. Patients were divided into two groups: those with TNBC and those with other types of breast cancer. Patients and tumor characteristics were compared between the two groups using the Chi-square test. The prognostic results of 9,823 patients in the study population were also analyzed to determine long-term survival rates in the two groups of breast cancer patients. RESULTS: Among the breast cancer patients treated in our hospital between 2003 and 2011, 10.4%-13.5% of them had triple-negative breast cancers. Data analyses revealed significant differences in disease onset age, family history of breast cancer, tumor size, tumor histologic grade, lymph note positivity and metastatic status between TNBC and non-TNBC patients. There were also significant differences in 5-year, 7-year and 9-year disease-free and 7-year and 9-year overall survival probability between the groups. CONCLUSIONS: TNBC are associated with younger disease onset age, larger tumor size, higher rate of axillary lymph node positivity, and higher tumor histologic grade. TNBC is also related to family history of breast cancer, increased metastatic risk and poor prognosis.

Clinical pathological characteristics and prognostic analysis of 1,013 breast cancer patients with diabetes.

The purpose of this study was to investigate the clinical, pathological, and prognostic characteristics of breast cancer patients with diabetes. In total, the study included 1,013 breast cancer patients with diabetes and 4,621 breast cancer patients without diabetes. Patients with diabetes were further divided into the metformin- and nonmetformin-treated subgroups. The percentage of elderly patients (P < 0.001), obese patients (P < 0.001), and menopausal patients (P < 0.001) as well as the percentage of patients with cardio-cerebrovascular complications (P < 0.001), negative PR (P < 0.001) expression, or low Ki67 expression (P = 0.001) tended to be higher in the diabetic group. In addition, these patients had later pathological stages (P < 0.001), more lymph node metastasis (P = 0.014), and a lower percentage of them were on the anthracycline-based chemotherapy regimen (P = 0.003). The diabetic group was further divided into the metformin and nonmetformin-treated subgroups. The pairwise comparison between the metformin-treated subgroup and the control group did not show a significant difference in the pathological stages (P = 0.0079). However, the HER-2 positive rate tended to be lower in the metformin-treated subgroup than in the nonmetformin-treated subgroup (P = 0.002). No significant difference was found in the lymph node status between the nonmetformin-treated subgroup and the control group (P = 0.057), while the nonmetformin-treated subgroup was associated with higher HER-2 positive expression (P = 0.002). The median follow-up time for this study was 68 months (10-120 months). In Kaplan-Meier analysis, The diabetic group predicted worse survival compared with the control group (P < 0.001) with 5-year survival rates of 79 and 82 %, respectively. The breast cancer mortality rates in the metformin-treated subgroup, the nonmetformin-treated subgroup, and the control group were significantly different (long-rank test, P < 0.001), and the 5-year survival rates were 88, 73, and 82 %, respectively. As shown in the multivariate survival analysis using Cox's regression model, compared with the control group, the metformin-treated subgroup was associated with lower mortality risk (HR 0.762; 95 % CI 0.6-0.968; P = 0.026), whereas the nonmetformin-treated subgroup was associated with higher mortality risk (HR 1.708; 95 % CI 1.461-1.997; P < 0.001). In conclusion, the diabetic group is associated with poor prognosis. Compared with the control group, the metformin-treated subgroup is associated with better clinical outcomes, while nonmetformin-treated subgroup with poorer prognosis. The selection of different antidiabetic drugs may impact the prognosis of breast cancer patients with diabetes.