MicroRNA-145 Impairs Classical Non-Homologous End-Joining in Response to Ionizing Radiation-Induced DNA Double-Strand Breaks via Targeting DNA-PKcs.

DNA double-strand breaks (DSBs) are one of the most lethal types of DNA damage due to the fact that unrepaired or mis-repaired DSBs lead to genomic instability or chromosomal aberrations, thereby causing cell death or tumorigenesis. The classical non-homologous end-joining pathway (c-NHEJ) is the major repair mechanism for rejoining DSBs, and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a critical factor in this pathway; however, regulation of DNA-PKcs expression remains unknown. In this study, we demonstrate that miR-145 directly suppresses DNA-PKcs by binding to the 3'-UTR and inhibiting translation, thereby causing an accumulation of DNA damage, impairing c-NHEJ, and rendering cells hypersensitive to ionizing radiation (IR). Of note, miR-145-mediated suppression of DNA damage repair and enhanced IR sensitivity were both reversed by either inhibiting miR-145 or overexpressing DNA-PKcs. In addition, we show that the levels of Akt1 phosphorylation in cancer cells are correlated with miR-145 suppression and DNA-PKcs upregulation. Furthermore, the overexpression of miR-145 in Akt1-suppressed cells inhibited c-NHEJ by downregulating DNA-PKcs. These results reveal a novel miRNA-mediated regulation of DNA repair and identify miR-145 as an important regulator of c-NHEJ.

53BP1-ACLY-SLBP-coordinated activation of replication-dependent histone biogenesis maintains genomic integrity.

p53-binding protein 1 (53BP1) regulates the DNA double-strand break (DSB) repair pathway and maintains genomic integrity. Here we found that 53BP1 functions as a molecular scaffold for the nucleoside diphosphate kinase-mediated phosphorylation of ATP-citrate lyase (ACLY) which enhances the ACLY activity. This functional association is critical for promoting global histone acetylation and subsequent transcriptome-wide alterations in gene expression. Specifically, expression of a replication-dependent histone biogenesis factor, stem-loop binding protein (SLBP), is dependent upon 53BP1-ACLY-controlled acetylation at the SLBP promoter. This chain of regulation events carried out by 53BP1, ACLY, and SLBP is crucial for both quantitative and qualitative histone biogenesis as well as for the preservation of genomic integrity. Collectively, our findings reveal a previously unknown role for 53BP1 in coordinating replication-dependent histone biogenesis and highlight a DNA repair-independent function in the maintenance of genomic stability through a regulatory network that includes ACLY and SLBP.

Correction: miR146a-mediated targeting of FANCM during inflammation compromises genome integrity.

[This corrects the article DOI: 10.18632/oncotarget.10275.].

ABHD12 Knockdown Suppresses Breast Cancer Cell Proliferation, Migration and Invasion.

BACKGROUND/AIM: Alpha/beta-hydrolase domain containing 12 (ABHD12) is a serine hydrolase that regulates immunological and neurological mechanisms. This study aimed to elucidate the oncogenic effect of ABHD12 on human breast cancer. MATERIALS AND METHODS: ABHD12 expression was confirmed in breast cancer tissues and breast cancer cell lines by immunohistochemistry and quantitative RT-PCR. To determine the role of ABHD12, ABHD12 siRNA-suppressed breast cancer cells (MCF7 and MDA-MB-231 cells) were investigated for cell proliferation, migration, and invasion capabilities using MTT assays, EdU assays, colony formation assays, and Boyden chamber assays. RESULTS: Immunohistochemical staining showed a higher ABHD12 expression in breast cancer tissues than in normal tissues. Additionally, ABHD12 knockdown was found to inhibit cell growth, proliferation, migration, and invasion in breast cancer cells. CONCLUSION: ABHD12 plays a crucial role in cell proliferation, migration, and invasion of breast cancer cells.

Oncogenic Ras downregulates mdr1b expression through generation of reactive oxygen species.

In the present study, we investigated the effect of oncogenic H-Ras on rat mdr1b expression in NIH3T3 cells. The constitutive expression of H-RasV12 was found to downregulate the mdr1b promoter activity and mdr1b mRNA expression. The doxorubicin-induced mdr1b promoter activity of the H-RasV12 expressing NIH3T3 cells was markedly lower than that of control NIH3T3 cells. Additionally, there is a positive correlation between the level of H-RasV12 expression and a sensitivity to doxorubicin toxicity. To examine the detailed mechanism of H-RasV12-mediated down-regulation of mdr1b expression, antioxidant N-acetylcysteine (NAC) and NADPH oxidase inhibitor diphenylene iodonium (DPI) were used. Pretreating cells with either NAC or DPI significantly enhanced the oncogenic H-Ras-mediated down-regulation of mdr1b expression and markedly prevented doxorubicin-induced cell death. Moreover, NAC and DPI treatment led to a decrease in ERK activity, and the ERK inhibitors PD98059 or U0126 enhanced the mdr1b-Luc activity of H-RasV12-NIH3T3 and reduced doxorubicin-induced apoptosis. These data suggest that RasV12 expression could downregulate mdr1b expression through intracellular reactive oxygen species (ROS) production, and ERK activation induced by ROS, is at least in part, contributed to the downregulation of mdr1b expression.

c-Fos-dependent miR-22 targets MDC1 and regulates DNA repair in terminally differentiated cells.

Terminally differentiated cells have a reduced capacity to repair double-stranded breaks (DSB) in DNA, however, the underlying molecular mechanism remains unclear. Here, we show that miR-22 is upregulated during postmitotic differentiation of human breast MCF-7 cells, hematopoietic HL60 and K562 cells. Increased expression of miR-22 in differentiated cells was associated with decreased expression of MDC1, a protein that plays a key role in the response to DSBs. This downregulation of MDC1 was accompanied by reduced DSB repair, impaired recruitment of the protein to the site of DNA damage following IR. Conversely, inhibiting miR-22 enhanced MDC1 protein levels, recovered MDC1 foci, fully rescued DSB repair in terminally differentiated cells. Moreover, MDC1 levels, IR-induced MDC1 foci, and the efficiency of DSB repair were fully rescued by siRNA-mediated knockdown of c-Fos in differentiated cells. These findings indicate that the c-Fos/miR-22/MDC1 axis plays a relevant role in DNA repair in terminally differentiated cells, which may facilitate our understanding of molecular mechanism underlying the downregulating DNA repair in differentiated cells.

miR146a-mediated targeting of FANCM during inflammation compromises genome integrity.

Inflammation is a potent inducer of tumorigenesis. Increased DNA damage or loss of genome integrity is thought to be one of the mechanisms linking inflammation and cancer development. It has been suggested that NF-κB-induced microRNA-146 (miR146a) may be a mediator of the inflammatory response. Based on our initial observation that miR146a overexpression strongly increases DNA damage, we investigated its potential role as a modulator of DNA repair. Here, we demonstrate that FANCM, a component in the Fanconi Anemia pathway, is a novel target of miR146a. miR146a suppressed FANCM expression by directly binding to the 3' untranslated region of the gene. miR146a-induced downregulation of FANCM was associated with inhibition of FANCD2 monoubiquitination, reduced DNA homologous recombination repair and checkpoint response, failed recovery from replication stress, and increased cellular sensitivity to cisplatin. These phenotypes were recapitulated when miR146a expression was induced by overexpressing the NF-κB subunit p65/RelA or Helicobacter pylori infection in a human gastric cell line; the phenotypes were effectively reversed with an anti-miR146a antagomir. These results suggest that undesired inflammation events caused by a pathogen or over-induction of miR146a can impair genome integrity via suppression of FANCM.

mRNA 3'-UTR shortening is a molecular signature of mTORC1 activation.

Mammalian target of rapamycin (mTOR) enhances translation from a subset of messenger RNAs containing distinct 5'-untranslated region (UTR) sequence features. Here we identify 3'-UTR shortening of mRNAs as an additional molecular signature of mTOR activation and show that 3'-UTR shortening enhances the translation of specific mRNAs. Using genetic or chemical modulations of mTOR activity in cells or mouse tissues, we show that cellular mTOR activity is crucial for 3'-UTR shortening. Although long 3'-UTR-containing transcripts minimally contribute to translation, 3-'UTR-shortened transcripts efficiently form polysomes in the mTOR-activated cells, leading to increased protein production. Strikingly, selected E2 and E3 components of ubiquitin ligase complexes are enriched by this mechanism, resulting in elevated levels of protein ubiquitination on mTOR activation. Together, these findings identify a previously uncharacterized role for mTOR in the selective regulation of protein synthesis by modulating 3'-UTR length of mRNAs.

MicroRNA-22 Suppresses DNA Repair and Promotes Genomic Instability through Targeting of MDC1.

MDC1 is critical component of the DNA damage response (DDR) machinery and orchestrates the ensuring assembly of the DDR protein at the DNA damage sites, and therefore loss of MDC1 results in genomic instability and tumorigenicity. However, the molecular mechanisms controlling MDC1 expression are currently unknown. Here, we show that miR-22 inhibits MDC1 translation via direct binding to its 3' untranslated region, leading to impaired DNA damage repair and genomic instability. We demonstrated that activated Akt1 and senescence hinder DDR function of MDC1 by upregulating endogenous miR-22. After overexpression of constitutively active Akt1, homologous recombination was inhibited by miR-22-mediated MDC1 repression. In addition, during replicative senescence and stress-induced premature senescence, MDC1 was downregulated by upregulating miR-22 and thereby accumulating DNA damage. Our results demonstrate a central role of miR-22 in the physiologic regulation of MDC1-dependent DDR and suggest a molecular mechanism for how aberrant Akt1 activation and senescence lead to increased genomic instability, fostering an environment that promotes tumorigenesis.