Development of Diagnostic SCAR Markers for Genomic DNA Amplifications in Breast Carcinoma by DNA Cloning of High-GC RAMP-PCR Fragments

Cancer is genetically heterogeneous regarding to molecular genetic characteristics and pathogenic pathways. A wide spectrum of biomarkers, including DNA markers, is used in determining genomic instability, molecular subtype determination and disease prognosis, and estimating sensitivity to different drugs in clinical practice. In a previous study, we developed highly effective DNA markers using improved random amplified polymorphic DNA (RAPD) with high-GC primers, which is a valuable approach for the genetic authentication of medicinal plants. In this study, we applied this effective DNA marker technique to generate genetic fingerprints that detect genomic alterations in human breast cancer tissues and then developed sequence-characterized amplified region (SCAR) markers. Three SCAR markers (BC10-1, BC13-4 and BC31-2) had high levels of genomic DNA amplification in breast cancer. The PHKG2 and RNF40 genes are either overlapping or close to the sequences of SCAR marker BC13-4, while SCAR marker BC10-1 is in the intron and overlap the DPEP1 gene, suggesting that alterations in the expression of these genes could contribute to cancer progression. Screening of breast cancer cell lines showed that the mRNA expression levels for the PHKG2 and DPEP1 were lower in non-tumorigenic mammary epithelial cell MCF10A, but elevated in other cell lines. The DPEP1 mRNA level in invasive ductal carcinoma specimens was significantly higher than that of the adjacent normal tissues in women. Taken together, high-GC RAMP-PCR provides greater efficacy in measuring genomic DNA amplifications, deletion or copy number variations. Furthermore, SCAR markers BC10-1 and BC13-4 might be useful diagnostic markers for breast cancer carcinomas

Effects of a particular heptapeptide on the IFN-α-sensitive CML cells

Abstract: Using the phage display biopanning technique, we have previously identified a heptapeptide KLWVIPQ which specifically bind to the surface of the IFN-α sensitive but not the IFN-α-resistant CML cells. The effects of this heptapeptide to the IFN-α-sensitive CML cells were investigated in the present study. IFN-α-sensitive KT-1/A3 and IFN-α-resistant KT-1/A3R CML cells were transfected by pEGFP KLWVIPQ expression vector and/or induced by IFN-α. WST-1 cell proliferation assay, flow cytometry and western blotting were performed to determine the effects of this heptapeptide and/or IFN-α on CML cells. The viability of the KT-1/A3 cells w as inhibited and apoptosis was induced by either expression of the heptapeptide KLWVIPQ or IFN-α treatment with concurrent up-regulation of P53 and down-regulation of P210bcr/abl. However, these effects were not observed in the IFN-α-resistant KT-1/A3R cells. These results suggest that the heptapeptide KLWVIPQ shares a similar mechanism w ith IFN-α in the regulat ion of CML cell growth and apoptosis, implying that the heptapeptide KLWVIPQ could be a novel target to go further into mechanisms of IFN-α sensitivity and/or resistance in CML

PIWI-interacting RNAs: Mitochondria-based biogenesis and functions in cancer

PIWI-interacting RNA (piRNAs), once thought to be mainly functioning in germlines, are now known to play an essential role in somatic and cancerous tissues. Ping-pong cycle initiation and mitochondria-based phased production constitute the core of the piRNA biogenesis and these two processes are well conserved in mammals, including humans. By being involved in DNA methylation, histone marker deposition, mRNA degradation, and protein modification, piRNAs also contribute to carcinogenesis partly due to oncogenic stress-induced piRNA dysregulation. Also, piRNAs play important roles in cancer stemness, drug resistance, and tumor immunology. Results from liquid biopsy analysis of piRNA can be used in both cancer diagnoses and cancer prognoses. A combination of targeting piRNA with other therapeutic strategies could be groundbreaking cancer treatment

ISG15 inhibits IFN- a -Resistant liver cancer cell growth

Hepatocellular carcinoma (HCC) is one of the most prevalent tumors worldwide. Interferon-a (IFN-a) has been widely used in the treatment of HCC, but patients eventually develop resistance. ISG15 ubiquitin-like modifier (ISG15) is a ubiquitin-like protein transcriptionally regulated by IFN-a which shows antivirus and antitumor activities. However, the exact role of ISG15 is unknown. In the present study, we showed that IFN-a significantly induced ISG15 expression but failed to induce HepG2 cell apoptosis, whereas transient overexpression of ISG15 dramatically increased HepG2 cell apoptosis. ISG15 overexpression increased overall protein ubiquitination, which was not observed in cells with IFN-a-induced ISG15 expression, suggesting that IFN-a treatment not only induced the expression of ISG15 but also inhibited ISG15-mediated ubiquitination. The tumor suppressor p53 and p21 proteins are the key regulators of cell survival and death in response to stress signals such as DNA damage. We showed that p53 or p21 is only up regulated in HepG2 cells ectopically expressing ISG15, but not in the presence of IFN-a-induced ISG15. Our results suggest that ISG15 overexpression could be developed into a powerful gene-therapeutic tool for treating IFN-a-resistant HCC. © 2013 Xin-xing Wan et al

Resveratrol Enhances Polyubiquitination-Mediated ARV-7 Degradation in Prostate Cancer Cells

Although androgen deprivation therapy (ADT) serves as the primary treatment option for localized or metastatic prostate cancer, most cases eventually develop into castration-resistant prostate cancer (CRPC). However, androgen receptor (AR) continues to be functional in CRPC through various mechanisms, including the development of AR splicing variants, especially ARV7. Since it lacks the ligand binding domain but retains the intact DNA binding domain, ARV7 is constitutively active, which makes ARV7-positive prostate cancer responsive to neither abiraterone nor enzalutamide. In this study, we explored the effect of resveratrol on ARV7 transcriptional activity and the potential for development of resveratrol as a treatment for ARV7-positive prostate cancer. First, we ectopically expressed ARV7 in PC3 cells, an AR-negative prostate cancer cell line, and demonstrated that resveratrol is capable of inhibiting ARV7 transcriptional activity by downregulating ARV7 protein levels. Of note, resveratrol does not affect the mRNA levels of ARV7 nor its nuclear translocation. Next, we demonstrated that resveratrol is capable of downregulating the levels of the endogenously expressed ARV7 as well as AR target gene mRNAs in 22RV1 prostate cancer cells. Mechanistically, resveratrol downregulates ARV7 by enhancing ARV7 polyubiquitination and subsequent proteasome-mediated degradation. These findings suggest that resveratrol could be a potential treatment for ARV7-positive CPRC

Genetic Identification and Molecular Modeling Characterization Reveal a Novel PROM1 Mutation in Stargardt4-like Macular Dystrophy

Stargardt disease-4 (STGD4) is an autosomal dominant complex, genetically heterogeneous macular degeneration/dystrophy (MD) disorder. In this paper, we used targeted next generation sequencing and multiple molecular dynamics analyses to identify and characterize a disease-causing genetic variant in four generations of a Chinese family with STGD4-like MD. We found a novel heterozygous missense mutation, c.734T\u3eC (p.L245P) in the PROM1 gene. Structurally, this mutation most likely impairs PROM1 protein stability, flexibility, and amino acid interaction network after changing the amino acid residue Leucine into Proline in the basic helix-loop-helix leucine zipper domain. Molecular dynamic simulation and principal component analysis provide compelling evidence that this PROM1 mutation contributes to disease causativeness or susceptibility variants in patients with STGD4-like MD. Thus, this finding defines new approaches in genetic characterization, accurate diagnosis, and prevention of STGD4-like MD

Tripartite motif containing 28 (TRIM28) promotes breast cancer metastasis by stabilizing TWIST1 protein.

TRIM28 regulates its target genes at both transcriptional and posttranscriptional levels. Here we report that a TRIM28-TWIST1-EMT axis exists in breast cancer cells and TRIM28 promotes breast cancer metastasis by stabilizing TWIST1 and subsequently enhancing EMT. We find that TRIM28 is highly expressed in both cancer cell lines and advanced breast cancer tissues, and the levels of TRIM28 and TWIST1 are positively correlated with the aggressiveness of breast carcinomas. Overexpression and depletion of TRIM28 up- and down-regulates the protein, but not the mRNA levels of TWIST1, respectively, suggesting that TRIM28 upregulates TWIST1 post-transcriptionally. Overexpression of TRIM28 in breast cancer cell line promotes cell migration and invasion. Knockdown of TRIM28 reduces the protein level of TWIST1 with concurrent upregulation of E-cadherin and downregulation of N-cadherin and consequently inhibits cell migration and invasion. Furthermore, Immunoprecipitation and GST pull-down assays demonstrated that TRIM28 interacts with TWIST1 directly and this interaction is presumed to protect TWIST1 from degradation. Our study revealed a novel mechanism in breast cancer cells that TRIM28 enhances metastasis by stabilizing TWIST1, suggesting that targeting TRIM28 could be an efficacious strategy in breast cancer treatment

Repressive Effects of Resveratrol on Androgen Receptor Transcriptional Activity

The chemopreventive effects of resveratrol (RSV) on prostate cancer have been well established; the androgen receptor (AR) plays pivotal roles in prostatic tumorigenesis. However, the exact underlying molecular mechanisms about the effects of RSV on AR have not been fully elucidated. A model system is needed to determine whether and how RSV represses AR transcriptional activity.The AR cDNA was first cloned into the retroviral vector pOZ-N and then integrated into the genome of AR-negative HeLa cells to generate the AR(+) cells. The constitutively expressed AR was characterized by monitoring hormone-stimulated nuclear translocation, DNA binding, and transcriptional activation, with the AR(-) cells serving as controls. AR(+) cells were treated with RSV, and both AR protein levels and AR transcriptional activity were measured simultaneously. Chromatin immunoprecipitation (ChIP) assays were used to detect the effects of RSV on the recruitment of AR to its cognate element (ARE).AR in the AR (+) stable cell line functions in a manner similar to that of endogenously expressed AR. Using this model system we clearly demonstrated that RSV represses AR transcriptional activity independently of any effects on AR protein levels. However, neither the hormone-mediated nucleus translocation nor the AR/ARE interaction was affected by RSV treatment.We demonstrated unambiguously that RSV regulates AR target gene expression, at least in part, by repressing AR transcriptional activity. Repressive effects of RSV on AR activity result from mechanisms other than the affects of AR nuclear translocation or DNA binding

Integrated Optimization for Biofuel Management Associated with a Biomass-Penetrated Heating System under Multiple and Compound Uncertainties

The biofuel management of a biofuel-penetrated district heating system is complicated due to its association with multiple and polymorphic uncertainties. To handle uncertainties and system dynamic complexities, an inexact two-stage compound-stochastic mixed-integer programming technique is proposed, innovatively based on the integration of different uncertain optimization approaches. The proposed technique can not only address the inexact recourse problems sourced from multiple and compound uncertainties existing in the pre-regulated biofuel supply–demand match mode, but can also quantitatively analyze the conflicts between the economic target that minimizes the system cost and the risk preference that maximizes the heating service satisfaction. The developed model is applied to a real-world biofuel management case study of a district heating system to obtain the optimal biofuel management schemes subject to supply–demand, policy requirement constraints, and the financial minimization objective. The results indicate that biofuel allocation and expansion schemes are sensitive to the multiple and compound uncertainty inputs, and the corresponding biofuel-deficit change trends of three heat sources are obviously distinct with the system’s condition, varying due to the complicated interactions of the system’s components. Beyond that, a potential trade-off relationship between the heating cost and the constraint-violation risk can be obtained by observing system responses with thermalization coefficient varying

Integrated Optimization for Biofuel Management Associated with a Biomass-Penetrated Heating System under Multiple and Compound Uncertainties

The biofuel management of a biofuel-penetrated district heating system is complicated due to its association with multiple and polymorphic uncertainties. To handle uncertainties and system dynamic complexities, an inexact two-stage compound-stochastic mixed-integer programming technique is proposed, innovatively based on the integration of different uncertain optimization approaches. The proposed technique can not only address the inexact recourse problems sourced from multiple and compound uncertainties existing in the pre-regulated biofuel supply–demand match mode, but can also quantitatively analyze the conflicts between the economic target that minimizes the system cost and the risk preference that maximizes the heating service satisfaction. The developed model is applied to a real-world biofuel management case study of a district heating system to obtain the optimal biofuel management schemes subject to supply–demand, policy requirement constraints, and the financial minimization objective. The results indicate that biofuel allocation and expansion schemes are sensitive to the multiple and compound uncertainty inputs, and the corresponding biofuel-deficit change trends of three heat sources are obviously distinct with the system’s condition, varying due to the complicated interactions of the system’s components. Beyond that, a potential trade-off relationship between the heating cost and the constraint-violation risk can be obtained by observing system responses with thermalization coefficient varying