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The genome‑wide copy number analysis of circulating tumor cells (CTCs) provides a promising prognostic biomarker for survival in breast cancer liver metastasis (BCLM) patients. The present study aimed to confirm the prognostic value of the presence of CTCs in BCLM patients. We previously developed an assay for the genome‑wide pattern differences in copy number variations (CNVs) as an adjunct test for the routine imaging and histopathologic diagnosis methods to distinguish newly diagnosed liver metastases and recurrent liver metastases. Forty‑three breast cancer patients were selected for this study in which 23 newly diagnosed and 20 recurrent liver metastases were diagnosed by histopathology and 18F‑FDG PET/CT imaging. CTCs were counted from all patients using the CellSearch system and were confirmed by cytomorphology and three‑color immunocytochemistry. Genomic DNA of single CTCs was amplified using multiple annealing and looping based amplification cycles (MALBAC). Then, we compared the CTC numbers of newly diagnosed and recurrent BCLM patients using Illumina platforms. A high CTC frequency (>15 CTCs/7.5 ml blood) was found to be correlated with disease severity and metastatic progression, which suggests the value for CTCs in the diagnosis of BCLM in comparison with pathohistology and PET/CT imaging (P>0.05). Moreover, CTCs isolated from BCLM patients remained an independent prognostic detection factor associated with overall survival (P=0.0041). Comparison between newly diagnosed and recurrent liver metastases revealed different frequencies of CNVs (P>0.05). Notably, the CNV pattern of isolated CTCs of recurrent BCLM patients was similar to recurrent liver metastases (nearly 82% of the gain/loss regions). Functional enrichment analysis identified 25 genes as a CNV signature of BCLM. Among them, were defensin and β‑defensin genes, which are significantly associated with anti‑angiogenesis and immunomodulation signaling pathways. High CTC frequencies are effective in the evaluation and differentiation between newly diagnosed liver metastases from recurrent liver metastases. Future clinical studies will be necessary to fully determine the prognostic potential of CTC cluster signatures in patients with BCLM.

BackgroundKinesin superfamily of proteins (KIFs) has been broadly reported to play an indispensable role in the biological process. Recently, emerging evidence reveals its oncogenic role in various cancers. However, the prognostic, oncological, and immunological values of KIFs have not been comprehensively explored in pancreatic ductal adenocarcinoma (PDAC) patients. We aimed to illustrate the relationship between KIFs and pancreatic ductal adenocarcinoma by using bioinformatical analysis.MethodsWe use GEPIA, Oncomine datasets, cBioPortal, LOGpc, TIMER, and STRING bioinformatics tools and web servers to investigate the aberrant expression, prognostic values, and oncogenic role of KIFs. The two-gene prognostic model and the correlation between KIFs and KRAS and TP53 mutation were performed using an R-based computational framework.ResultsOur results demonstrated that KIFC1/2C/4A/11/14/15/18A/18B/20B/23 (we name it prognosis-related KIFs) were upregulated and associated with unfavorable clinical outcome in pancreatic cancer patients. KIF21B overexpression is associated with better clinical outcome. The KIFC1/2C/4A/11/14/15/18A/18B/20B/23 profiles were significantly increased compared to grade 1 and grade 2/3. Besides, KIFC1/2C/4A/11/14/15/18A/18B/20B/23 was significantly associated with the mutation status of KRAS and TP53.Notably, most prognosis-related KIFs have strong correlations with tumor growth and myeloid-derived suppressor cells infiltration (MDSCs). A prognostic signature based on KIF20B and KIF21B showed a reliable predictive performance. Receiver operating characteristic (ROC) curve was employed to assess the predictive power of two-gene signature. Consequently, the gene set enrichment analysis (GSEA) showed that KIF20B and KIF21B’s overexpression was associated with the immunological and oncogenic pathway activation in pancreatic cancer. Finally, real-time quantitative PCR (RT-qPCR) was utilized to investigate the expression pattern of KIF20B and KIF21B in pancreatic cancer cell lines and normal pancreatic cell.ConclusionsKnowledge of the expression level of the KIFs may provide novel therapeutic molecular targets and potential prognostic biomarkers to pancreatic cancer patients.

Background: Increasing evidence suggests that deubiquitinase USP7 participates in tumor progression by various mechanisms and serves as a potential therapeutic target. However, its functional role in melanoma remains elusive and needs to be investigated. Methods: The down-regulation of USP7 in A375 human melanoma cells was determined by high resolution liquid chromatography mass spectrometry (LC-MS/MS). The effects of USP7 expression on proliferation and apoptosis of melanoma cells were examined by western blot, Immunohistochemical and flow cytometry. Further study knock-down of USP7 in A375 cell lines, especially knockout USP7 using CRISPR-Cas9, verified USP7 regulate cell proliferation in vivo and in vitro .Western blot and qRT-PCR, Immunofluorescence were performed to investigate the mechanisms by which USP7 mediated melanoma growth through regulating the PI3K/Akt/FOXO pathways activity Results: Proteomic and western blotting analysis show that inhibition of USP7 increases expression of PRKAB1, CASP7, and PPP2R3A, attenuates expression of ATP6V0C and PEX11B. Furthermore, ATP6V0C and PEX11B are proposed to be the substrate for USP7 function. Conclusions: Our findings demonstrate that PI3K/Akt/FOXO and AMPK signaling pathways can be regulated by USP7 during melanoma progression and provide USP7 as an attractive anticancer target for melanoma.

BACKGROUND Accumulating data point to intermediate-conductance calcium-activated potassium channel (IKCa1) as a key player in controlling cell cycle progression and proliferation of human cancer cells. However, the role that IKCa1 plays in the growth of human cervical cancer cells is largely unexplored. MATERIAL AND METHODS In this study, Western blot analysis, immunohistochemical staining, and RT-PCR were first used for IKCa1protein and gene expression assays in cervical cancer tissues and HeLa cells. Then, IKCa1 channel blocker and siRNA were employed to inhibit the functionality of IKCa1 and downregulate gene expression in HeLa cells, respectively. After these treatments, we examined the level of cell proliferation by MTT method and measured IKCa1 currents by conventional whole-cell patch clamp technique. Cell apoptosis was assessed using the Annexin V-FITC/Propidium Iodide (PI) double-staining apoptosis detection kit. RESULTS We demonstrated that IKCa1 mRNA and protein are preferentially expressed in cervical cancer tissues and HeLa cells. We also showed that the IKCa1 channel blocker, clotrimazole, and IKCa1 channel siRNA can be used to suppress cervical cancer cell proliferation and decrease IKCa1 channel current. IKCa1 downregulation by specific siRNAs induced a significant increase in the proportion of apoptotic cells in HeLa cells. CONCLUSIONS IKCa1 is overexpressed in cervical cancer tissues, and IKCa1 upregulation in cervical cancer cell linea enhances cell proliferation, partly by reducing the proportion of apoptotic cells.

Diffuse large B-cell lymphoma (DLBCL) is a clinically aggressive B-cell non-Hodgkin’s lymphoma (NHL) with high treatment difficulty and high relapse rate. The bromodomain and extra-terminal (BET) proteins play significant roles in supporting the transcription of known DLBCL oncogene MYC, which provides a way for the development of targeted therapeutic agents to address this kind of malignant tumor. Here, we reported a novel benzoxazinone derivative YLT-LL-11 as potential BRD4 inhibitor and further investigated the biological activities against DLBCL. The results suggested that YLT-LL-11 inhibited cell growth against a panel of human hematopoietic malignancies cell lines in a dose- and time-dependent manner. In addition, flow cytometry and Western blotting assays showed that YLT-LL-11 inhibited the proliferation of a DLBCL cell line OCI-LY10 via inducing G0/G1 cell cycle arrest with regulation of the cyclin-dependent kinases (CDKs) expression. Furthermore, YLT-LL-11 facilitated OCI-LY10 cell apoptosis by up-regulation of pro-apoptotic protein BAX and down-regulation of anti-apoptotic protein Bcl-2. Taken together, these results revealed that BRD4 inhibitor YLT-LL-11 can down-regulate growth-associated transcription factors MYC in DLBCL thus resulted in cell growth inhibition and apoptosis.

Direct treatment of ER (+) breast cancer with Formestane diminishes the tumor within weeks. This is unlikely due to lack of estrogens alone. We proposed that it is the negative influence of androgens on the growth of ER(+) breast cancer. We investigated the influence of Formestane and Exemestane and of their major androgenic metabolites 4-hydroxytestosterone and 17-hydroexemestane on the proliferation of MCF-7 cells and ZR-75-1 cells. Inhibitory effects could be prevented by antiandrogens and siRNA. Activation of the AR in MCF-7 and U2-OS cells was tested by reporter gene assays. In vivo androgenicity was evaluated using the Hershberger assay. Influence on the cell cycle was demonstrated by flow-cytometry. Influence of androgens on the activity of CCND1 was demonstrated by Chip-qPCR. Antitumor activity was determined by topical treatment of DMBA tumors. We found that breast cancer cells can metabolize Formestane and Exemestane to androgenic compounds which inhibit proliferation. This can be explained by hindering the accessibility of CCND1 by histone modification. Androgenic metabolites can abolish the growth of DMBA-tumors and prevent the appearance of new tumors. The lack of cross-resistance between steroidal and nonsteroidal aromatase inhibitors is due to inhibitory effects of androgenic steroidal metabolites on the production of cyclin D1. These sterols not only inhibit proliferation of cancer cells but can also stop the growth of DMBA cancers upon direct absorption into the tumor. The quick and considerable effect on ER(+) tumors may open a new avenue for neodjuvant treatment.

BACKGROUND Accumulating data point to intermediate-conductance calcium-activated potassium channel (IKCa1) as a key player in controlling cell cycle progression and proliferation of human cancer cells. However, the role that IKCa1 plays in the growth of human cervical cancer cells is largely unexplored. MATERIAL AND METHODS In this study, Western blot analysis, immunohistochemical staining, and RT-PCR were first used for IKCa1protein and gene expression assays in cervical cancer tissues and HeLa cells. Then, IKCa1 channel blocker and siRNA were employed to inhibit the functionality of IKCa1 and downregulate gene expression in HeLa cells, respectively. After these treatments, we examined the level of cell proliferation by MTT method and measured IKCa1 currents by conventional whole-cell patch clamp technique. Cell apoptosis was assessed using the Annexin V-FITC/Propidium Iodide (PI) double-staining apoptosis detection kit. RESULTS We demonstrated that IKCa1 mRNA and protein are preferentially expressed in cervical cancer tissues and HeLa cells. We also showed that the IKCa1 channel blocker, clotrimazole, and IKCa1 channel siRNA can be used to suppress cervical cancer cell proliferation and decrease IKCa1 channel current. IKCa1 downregulation by specific siRNAs induced a significant increase in the proportion of apoptotic cells in HeLa cells. CONCLUSIONS IKCa1 is overexpressed in cervical cancer tissues, and IKCa1 upregulation in cervical cancer cell linea enhances cell proliferation, partly by reducing the proportion of apoptotic cells.

CULLIN 4 (CUL4)-DAMAGED DNA binding protein 1 (DDB1)-based ubiquitin E3 ligase modulates diverse cellular processes including repair of damaged genomic DNA. In this study, an uncharacterized gene termed as DDB1-Interacting protein 2 (DDI2) was identified in yeast two-hybrid screening with bait gene DDB1. The co-immunoprecipitation (co-IP) assays further demonstrated that DDI2 is associated with tomato DDB1-CUL4 complex in vivo. It appears that DDI2 encodes an ortholog of proliferating cell nuclear antigen (PCNA). Confocal microscope observation indicated that DDI2-GFP fusion protein was localized in nuclei. The expression of DDI2 gene is constitutive but substantially enhanced by UV-C irradiation. The transgenic tomato plants with overexpression or knockdown of DDI2 gene displayed the increased or decreased tolerance, respectively, to UV-C stress and chemical mutagen cisplatin. The quantitative analysis of UV-induced DNA lesions indicated that the dark repair of DNA damage was accelerated in DDI2 overexpression lines but delayed in knockdown lines. Conclusively, tomato DDI2 gene is required for UV-induced DNA damage repair and plant tolerance to UV stress. In addition, fruits of DDI2 transgenic plants are indistinguishable from that of wild type, regarding fresh weight and nutrient quality. Therefore, overexpression of DDI2 offers a suitable strategy for genetic manipulation of enhancing plant tolerance to UV stress.

Epigenetic modification generally refers to phenotypic changes by a mechanism other than changes in DNA sequence and plays a significant role in developmental processes. In this study, we found that overexpression of one alternatively spliced tomato DDB1 transcript, DDB1(F) that is prevalently present in all tested tissues, resulted in reduction of organ size. Transgenic plants constitutively expressing the DDB1(F) from a strong cauliflower mosaic virus (CaMV) 35S promoter displayed moderately reduced size in vegetative organs (leaves and stems) and radically decreased size in reproductive organs (flowers, seeds and fruits), in which several genes encoding negative regulators for cell division were upregulated. Significantly, reduction of organ size conferred by overexpression of DDB1(F) transgene appears not to segregate in the subsequent generations, suggesting the phenotypic alternations are manipulated in an epigenetic manner and can be transmitted over generations. This notion was further substantiated by analysis of DNA methylation level at the SlWEE1 gene (encoding a negative regulator of cell division), revealing a correlation between less methylation in the promoter region and elevated expression level of this gene. Thus, our results suggest DDB1 plays an important role in regulation of the epigenetic state of genes involved in organogenesis, despite the underlying mechanism remains to be elucidated.