RUVBL1 ubiquitination by DTL promotes RUVBL1/2-ß-catenin-mediated transcriptional regulation of NHEJ pathway and enhances radiation resistance in breast cancer.

Radiotherapy effectiveness in breast cancer is limited by radioresistance. Nevertheless, the mechanisms behind radioresistance are not yet fully understood. RUVBL1 and RUVBL2, referred to as RUVBL1/2, are crucial AAA+ ATPases that act as co-chaperones and are connected to cancer. Our research revealed that RUVBL1, also known as pontin/TIP49, is excessively expressed in MMTV-PyMT mouse models undergoing radiotherapy, which is considered a murine spontaneous breast-tumor model. Our findings suggest that RUVBL1 enhances DNA damage repair and radioresistance in breast cancer cells both in vitro and in vivo. Mechanistically, we discovered that DTL, also known as CDT2 or DCAF2, which is a substrate adapter protein of CRL4, promotes the ubiquitination of RUVBL1 and facilitates its binding to RUVBL2 and transcription cofactor ß-catenin. This interaction, in turn, attenuates its binding to acetyltransferase Tat-interacting protein 60 (TIP60), a comodulator of nuclear receptors. Subsequently, ubiquitinated RUVBL1 promotes the transcriptional regulation of RUVBL1/2-ß-catenin on genes associated with the non-homologous end-joining (NHEJ) repair pathway. This process also attenuates TIP60-mediated H4K16 acetylation and the homologous recombination (HR) repair process. Expanding upon the prior study's discoveries, we exhibited that the ubiquitination of RUVBL1 by DTL advances the interosculation of RUVBL1/2-ß-catenin. And, it then regulates the transcription of NHEJ repair pathway protein. Resulting in an elevated resistance of breast cancer cells to radiation therapy. From the aforementioned, it is evident that targeting DTL-RUVBL1/2-ß-catenin provides a potential radiosensitization approach when treating breast cancer.

DDX18 drives tumor immune escape through transcription-activated STAT1 expression in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) resists to current treatments due to its inherent tumor heterogeneity, therapy-resistant cancer stem/initiating cells survival, and immune evasion in the immunosuppressive tumor microenvironment (TME). Here, the results show that clinical PDAC and adjacent tissues undergo distinct chromatin remodeling. Multiple omics analysis revealed DEAD-box RNA helicase 18 (DDX18), a carcinogenic gene with similar H3K4me3 profile, is up-regulated and correlates with poor survival in PDAC patients. We validated that DDX18 deposits on the STAT1 promoter region and counteracts H3K27me3 deposition on the STAT1 promoter sequence by modulating the formation of the PRC2 complex to up-regulate the expression of STAT1, which results in the up-regulation of PD-L1 expression, T lymphocyte accumulation and overactivation in the highly desmoplastic and immunosuppressive pancreatic TME. DDX18-STAT1 axis inhibition also affects stemness of cancer cells, epithelial-mesenchymal transition (EMT) and disrupts the immunosuppressive TME simultaneously, producing sustained remissions of aggressive PDAC by synergizing with anti-PD-L1 therapy. Combining DDX18 inhibition with anti-PD-L1 immunochemotherapy to treat PDAC patients will pave a new way for clinical treatment of patients with PDAC. This study found that clinical PDAC and adjacent pancreatic tissues undergo distinct chromatin remodeling featured by the upregulation of DEAD-box RNA helicase 18 (DDX18). We further validated that DDX18 deposits on the STAT1 promoter region and counteracts H3K27me3 deposition on the STAT1 promoter by modulating the formation of the PRC2 complex to up-regulate the expression of STAT1. DDX18-STAT1 axis enhances the stemness of cancer cells, the upregulation of PD-L1 expression, T lymphocyte accumulation and overactivation in the highly desmoplastic and immunosuppressive pancreatic TME.

Protein modifications throughout the lung cancer proteome unravel the cancer-specific regulation of glycolysis.

Glycolytic reprogramming is a typical feature of cancer. However, the cancer-specific modulation of glycolytic enzymes requires systematic elucidation. Here, we report a range of dysregulated modifications in association with a family of enzymes specifically related to the glycolysis pathway by systematic identification of delta masses at the proteomic scale in human non-small-cell lung cancer. The most significant modification is the delta mass of 79.967 Da at serine 58 (Ser58) of triosephosphate isomerase (TPI), which is confirmed to be phosphorylation. Blocking TPI Ser58 phosphorylation dramatically inhibits glycolysis, cancer growth, and metastasis. The protein kinase PRKACA directly phosphorylates TPI Ser58, thereby enhancing TPI enzymatic activity and glycolysis. The upregulation of TPI Ser58 phosphorylation is detected in various human tumor specimens and correlates with poor survival. Therefore, our study identifies a number of cancer-specific protein modifications spanned on glycolytic enzymes and unravels the significance of TPI Ser58 phosphorylation in glycolysis and lung cancer development.

Prognostic significance of miR-203 and ZEB1 expression in early-stage hepatocellular carcinoma.

Background: Approximately one-quarter of patients with early-stage hepatocellular carcinoma (HCC) suffer from tumor recurrence within the first year after hepatectomy. Identification of patients at high risk of recurrence and new therapeutic approaches are crucial to improve clinical outcome. This study aimed to assess the prognostic significance of miR-203 and Zinc finger E-box binding homeobox 1 (ZEB1) in early-stage HCC and explore the association between the expression of ZEB1 and miR-203 in HCC. Methods: Tissue microarray-based immunohistochemistry (IHC) and in situ hybridization (ISH) were performed to investigate ZEB1 and miR-203 expression in 73 patients with early-stage HCC and their correlation with clinicopathological features and prognosis of patients were analyzed. The prognostic value of the two factors was also measured by public KM plotter database. Quantitative reverse transcription PCR (qRT-PCR) assays were conducted to study the relationship between miR-203 and ZEB1. Transwell assays, Cell Counting Kit-8 (CCK-8) assays were performed to detect the roles of miR-203 in migration, invasion and proliferation of HCC cells. Results: We found low expression of miR-203 was associated significantly with tumor recurrence (P<0.001) and poor survival (P=0.020) of patients with early-stage HCC. Multivariate analysis revealed that low miR-203 expression was a poor prognostic factor for both overall survival (OS) (P=0.036) and recurrence free survival (RFS) (P=0.017). ZEB1 did not show any prognostic significance in our cohort. Correlation analysis indicated that there was no significant correlation between miR-203 and ZEB1 on both mRNA and protein levels. Furthermore, functional studies indicated that miR-203 repressed migration, invasion and proliferation of HCC cells in vitro. Conclusion: Our study suggested that miR-203 could be a novel predictor in early-stage HCC and might also be a potential molecular target for HCC therapy.

CT45A1 promotes the metastasis of osteosarcoma cells in vitro and in vivo through ß-catenin.

Increased expression of cancer/testis antigens (CTAs) is reported in various tumors. However, the unique role of CTAs in tumor genesis has not yet been verified. Here, we first report the functional role of CT45A1 in the carcinogenesis of osteosarcoma. RNA sequencing and immunohistochemistry confirmed that elevated expression of CT45A1 was detected in osteosarcoma, especially in metastatic tissues of osteosarcoma. Furthermore, osteosarcoma patients with poorer prognosis showed high expression of CT45A1. In cell tests, CT45A1 overexpression was shown to strengthen the proliferation, migration, and invasion abilities of osteosarcoma cells, while silencing CT45A1 markedly elicited the opposite effects in these tests by disrupting the activation of ß-catenin. In summary, we identify a novel role of CT45A1 in osteosarcoma. Furthermore, our results suggested that CT45A1 may contribute to the development of osteosarcoma and could be a possible therapeutic target for osteosarcoma patients.

SKP2 promotes breast cancer tumorigenesis and radiation tolerance through PDCD4 ubiquitination.

BACKGROUND: S-phase kinase-associated protein 2 (SKP2) is an oncogene and cell cycle regulator that specifically recognizes phosphorylated cell cycle regulator proteins and mediates their ubiquitination. Programmed cell death protein 4 (PDCD4) is a tumor suppressor gene that plays a role in cell apoptosis and DNA-damage response via interacting with eukaryotic initiation factor-4A (eIF4A) and P53. Previous research showed SKP2 may interact with PDCD4, however the relationship between SKP2 and PDCD4 is unclear. METHODS: To validate the interaction between SKP2 and PDCD4, mass spectrometric analysis and reciprocal co-immunoprecipitation (Co-IP) experiments were performed. SKP2 stably overexpressed or knockdown breast cancer cell lines were established and western blot was used to detect proteins changes before and after radiation. In vitro and in vivo experiments were performed to verify whether SKP2 inhibits cell apoptosis and promotes DNA-damage response via PDCD4 suppression. SMIP004 was used to test the effect of radiotherapy combined with SKP2 inhibitor. RESULTS: We found that SKP2 remarkably promoted PDCD4 phosphorylation, ubiquitination and degradation. SKP2 promoted cell proliferation, inhibited cell apoptosis and enhanced the response to DNA-damage via PDCD4 suppression in breast cancer. SKP2 and PDCD4 showed negative correlation in human breast cancer tissues. Radiotherapy combine with SKP2 inhibitor SMIP004 showed significant inhibitory effects on breast cancer cells in vitro and in vivo. CONCLUSIONS: We identify PDCD4 as an important ubiquitination substrate of SKP2. SKP2 promotes breast cancer tumorigenesis and radiation tolerance via PDCD4 degradation. Radiotherapy combine with SKP2-targeted adjuvant therapy may improve breast cancer patient survival in clinical medicine.

TCEA1 regulates the proliferative potential of mouse myeloid cells.

Leukemia is a malignance with complex pathogenesis and poor prognosis. Discovery of noval regulators amenable to leukemia could be of value to gain insight into the pathogenesis, diagnosis and prognosis of leukemia. Here, we conducted a large-scale shRNA library screening for functional regulators in the development of myeloid cells in primary cells. We identified eighteen candidate regulators in the primary screening. Those genes cover a wide range of cellular functions, including gene expression regulation, intracellular signaling transduction, nucleotide excision repair, cell cycle control and transcription regulation. In both primary screening and validation, shRNAs targeting Tcea1, encoding the transcription elongation factor A (SII) 1, exhibited the greatest influence on the proliferative potential of cells. Knocking down the expression of Tcea1 in the 32Dcl3 myeloid cell line led to enhanced proliferation of myeloid cells and blockage of myeloid differentiation induced by G-CSF. In addition, silence of Tcea1 inhibited apoptosis of myeloid cells. Thus, Tcea1 was identified as a gene which can influence the proliferative potential, survival and differentiation of myeloid cells. These findings have implications for how transcriptional elongation influences myeloid cell development and leukemic transformation.

miR-1204 targets VDR to promotes epithelial-mesenchymal transition and metastasis in breast cancer.

Plasmacytoma variant translocation 1 (PVT1) is an lncRNA that plays vital roles in breast cancer (BC) pathogenesis. Increasing evidence suggests that miRNAs that reside in the PVT1 locus are the main driver of the oncogenic roles of PVT1 in cancer. However, the oncogenic role and underlying mechanism of miR-1204, located in the PVT1 locus, in human cancer is still unclear. In this study, we discovered that increased expression of miR-1204 is associated with poor prognosis in BC. Moreover, miR-1204 promotes proliferation, epithelial-mesenchymal transition and invasion of BC cells both in vitro and in vivo. Mechanistic investigations demonstrated that VDR is a novel target gene of miR-1204. Interference of VDR restored miR-1204-mediated BC cell proliferation, tumorigenesis, and metastasis. Collectively, our results demonstrated that the miR-1204-VDR pathway exerts oncogenic effects in BC with potential therapeutic applications in blocking BC development and progression.

Elevated expression of UBE2T exhibits oncogenic properties in human prostate cancer.

Increased expression of ubiquitin-conjugating enzyme E2T (UBE2T) is reported in human prostate cancer. However, whether UBE2T plays any functional role in prostate cancer development remains unknown. We here report the first functional characterization of UBE2T in prostate carcinogenesis. Prostate cancer tissue array analysis confirmed upregulation of UBE2T in prostate cancer, especially these with distant metastasis. Moreover, higher level of UBE2T expression is associated with poorer prognosis of prostate cancer patients. Ectopic expression of UBE2T significantly promotes prostate cancer cell proliferation, motility and invasion, while UBE2T depletion by shRNA significantly inhibits these abilities of prostate cancer cells. Xenograft mouse model studies showed that overexpression of UBE2T promotes whereas UBE2T depletion inhibits tumor formation and metastasis significantly. Collectively, we identify critical roles of UBE2T in prostate cancer development and progression. These findings may serve as a framework for future investigations designed to more comprehensive determination of UBE2T as a potential therapeutic target.

FBXW7 negatively regulates ENO1 expression and function in colorectal cancer.

FBXW7 (F-box and WD40 domain protein 7) is a tumor suppressor frequently inactivated in human cancers. The precise molecular mechanisms by which FBXW7 exerts antitumor activity remain under intensive investigation and are thought to relate in part to FBXW7-mediated destruction of key cancer-relevant proteins. Enolase 1 (ENO1) possesses oncogenic activity and is often overexpressed in various human cancers, besides its critical role in glycolysis. However, the detailed regulatory mechanisms of ENO1 expression remain unclear. Here we show that the elevated expression of ENO1 was identified in FBXW7-depletion HCT116 cells through two-dimensional protein electrophoresis and mass spectrometry assays (2DE-MS). Subsequent western blotting and immunohistochemical assays confirmed that ENO1 expression reversely correlates with FBXW7 expression in several cells and colon cancer tissues. Furthermore, we show that FBXW7 physically binds to ENO1 and targets ENO1 for ubiquitin-mediated degradation. Functionally, we found that FBXW7 suppresses the ENO1-induced gene expression, lactate production, cell proliferation and migration. These findings suggest that ENO1 is a novel substrate of FBXW7, and its activity can be negatively regulated by FBXW7 at the posttranslational level. Our work provides a novel molecular insight into FBXW7-directed tumor suppression through regulation of ENO1.

CUL4A overexpression enhances lung tumor growth and sensitizes lung cancer cells to erlotinib via transcriptional regulation of EGFR.

BACKGROUND: CUL4A has been proposed as oncogene in several types of human cancer, but its clinical significance and functional role in human non-small cell lung cancer (NSCLC) remain unclear. METHODS: Expression level of CUL4A was examined by RT-PCR and Western blot. Forced expression of CUL4A was mediated by retroviruses, and CUL4A silencing by shRNAs expressing lentiviruses. Growth capacity of lung cancer cells was measured by MTT in vitro and tumorigenesis in vivo, respectively. RESULTS: We found that CUL4A was highly expressed in human lung cancer tissues and lung cancer cell lines, and this elevated expression positively correlated with disease progression and prognosis. Overexpression of CUL4A in human lung cancer cell lines increased cell proliferation, inhibited apoptosis, and subsequently conferred resistance to chemotherapy. On other hand, silencing CUL4A expression in NSCLC cells reduced proliferation, promoted apoptosis and resulted in tumor growth inhibition in cancer xenograft model. Mechanistically, we revealed CUL4A regulated EGFR transcriptional expression and activation, and subsequently activated AKT. Targeted inhibition of EGFR activity blocked these CUL4A induced oncogenic activities. CONCLUSIONS: Our results highlight the significance of CUL4A in NSCLC and suggest that CUL4A could be a promising therapy target and a potential biomarker for prognosis and EGFR target therapy in NSCLC patients.

CUL4A induces epithelial-mesenchymal transition and promotes cancer metastasis by regulating ZEB1 expression.

The ubiquitin ligase CUL4A has been implicated in tumorigenesis, but its contributions to progression and metastasis have not been evaluated. Here, we show that CUL4A is elevated in breast cancer as well as in ovarian, gastric, and colorectal tumors in which its expression level correlates positively with distant metastasis. CUL4A overexpression in normal or malignant human mammary epithelial cells increased their neoplastic properties in vitro and in vivo, markedly increasing epithelial-mesenchymal transition (EMT) and the metastatic capacity of malignant cells. In contrast, silencing CUL4A in aggressive breast cancer cells inhibited these processes. Mechanistically, we found that CUL4A modulated histone H3K4me3 at the promoter of the EMT regulatory gene ZEB1 in a manner associated with its transcription. ZEB1 silencing blocked CUL4A-driven proliferation, EMT, tumorigenesis, and metastasis. Furthermore, in human breast cancers, ZEB1 expression correlated positively with CUL4A expression and distant metastasis. Taken together, our findings reveal a pivotal role of CUL4A in regulating the metastatic behavior of breast cancer cells.

Involvement of CUL4A in regulation of multidrug resistance to P-gp substrate drugs in breast cancer cells.

CUL4A encodes a core component of a cullin-based E3 ubiquitin ligase complex that regulates many critical processes such as cell cycle progression, DNA replication, DNA repair and chromatin remodeling by targeting a variety of proteins for ubiquitination and degradation. In the research described in this report we aimed to clarify whether CUL4A participates in multiple drug resistance (MDR) in breast cancer cells. We first transfected vectors carrying CUL4A and specific shCUL4A into breast cancer cells and corresponding Adr cells respectively. Using reverse transcription polymerase chain reactions and western blots, we found that overexpression of CUL4A in MCF7 and MDA-MB-468 cells up-regulated MDR1/P-gp expression on both the transcription and protein levels, which conferred multidrug resistance to P-gp substrate drugs, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. On the other hand, silencing CUL4A in MCF7/Adr and MDA-MB-468/Adr cells led to the opposite effect. Moreover, ERK1/2 in CUL4A-overexpressing cells was highly activated and after treatment with PD98059, an ERK1/2-specific inhibitor, CUL4A-induced expression of MDR1/P-gp was decreased significantly. Lastly, immunohistochemistry in breast cancer tissues showed that P-gp expression had a positive correlation with the expression of CUL4A and ERK1/2. Thus, these results implied that CUL4A and ERK1/2 participated in multi-drug resistance in breast cancer through regulation of MDR1/P-gp expression.

C2ORF40 suppresses breast cancer cell proliferation and invasion through modulating expression of M phase cell cycle genes.

Recently, it has been suggested that C2ORF40 is a candidate tumor suppressor gene in breast cancer. However, the mechanism for reduced expression of C2ORF40 and its functional role in breast cancers remain unclear. Here we show that C2ORF40 is frequently silenced in human primary breast cancers and cell lines through promoter hypermethylation. C2ORF40 mRNA level is significantly associated with patient disease-free survival and distant cancer metastasis. Overexpression of C2ORF4 0 inhibits breast cancer cell proliferation, migration and invasion. By contrast, silencing C2ORF40 expression promotes these biological phenotypes. Bioinformatics and FACS analysis reveal C2ORF40 functions at G2/M phase by downregulation of mitotic genes expression, including UBE2C. Our results suggest that C2ORF40 acts as a tumor suppressor gene in breast cancer pathogenesis and progression and is a candidate prognostic marker for this disease.