Xanthohumol regulates miR-4749-5p-inhibited RFC2 signaling in enhancing temozolomide cytotoxicity to glioblastoma.

AIMS: Xanthohumol (XN), a natural prenylated flavonoid isolated from Humulus lupulus L. (hops), possess the therapeutic effects in glioblastoma multiforme (GBM), which is a grade IV aggressive glioma in adults. However, low bioavailability and extractive yield limit the clinical applications of XN. To comprehensively investigate XN-mediated gene networks in inducing cell death is helpful for drug development and cancer research. Therefore, we aim to identify the detailed molecular mechanisms of XN's effects on exhibiting cytotoxicity for GBM therapy. METHODS AND KEY FINDINGS: XN significantly induced GBM cell death and enhanced temozolomide (TMZ) cytotoxicity, a first-line therapeutic drug of GBM. XN-mediated transcriptome profiles and canonical pathways were identified. DNA repair signaling, a well-established mechanism against TMZ cytotoxicity, was significantly correlated with XN-downregulated genes. Replication factor C subunit 2 (RFC2), a DNA repair-related gene, was obviously downregulated in XN-treated cells. Higher RFC2 levels which occupied poor patient survival were also observed in high grade GBM patients and tumors. Inhibition of RFC2 reduced cell viability, induced cell apoptosis, and enhanced both XN and TMZ cytotoxicity. By intersecting array data, bioinformatic prediction, and in vitro experiments, microRNA (miR)-4749-5p, a XN-upregulated microRNA, was identified to target to RFC2 3'UTR and inhibited RFC2 expression. A negative correlation existed between miR-4749-5p and RFC2 in GBM patients. Overexpression of miR-4749-5p significantly promoted XN- and TMZ-mediated cytotoxicity, and reduced RFC2 levels. SIGNIFICANCE: Consequently, we suggest that miR-4749-5p targeting RFC2 signaling participates in XN-enhanced TMZ cytotoxicity of GBM. Our findings provide new potential therapeutic directions for future GBM therapy.

Genome-wide RNAi Screening Identifies RFC4 as a Factor That Mediates Radioresistance in Colorectal Cancer by Facilitating Nonhomologous End Joining Repair.

PURPOSE: Neoadjuvant chemoradiotherapy (neoCRT) is a standard treatment for locally advanced rectal cancer (LARC); however, resistance to chemoradiotherapy is one of the main obstacles to improving treatment outcomes. The goal of this study was to identify factors involved in the radioresistance of colorectal cancer and to clarify the underlying mechanisms. EXPERIMENTAL DESIGN: A genome-wide RNAi screen was used to search for candidate radioresistance genes. After RFC4 knockdown or overexpression, colorectal cancer cells exposed to X-rays both in vitro and in a mouse model were assayed for DNA damage, cytotoxicity, and apoptosis. Moreover, the regulatory effects and mechanisms of RFC4 in DNA repair were investigated in vitro. Finally, the relationships between RFC4 expression and clinical parameters and outcomes were investigated in 145 patients with LARC receiving neoCRT. RESULTS: RFC4, NCAPH, SYNE3, LDLRAD2, NHP2, and FICD were identified as potential candidate radioresistance genes. RFC4 protected colorectal cancer cells from X-ray-induced DNA damage and apoptosis in vitro and in vivo. Mechanistically, RFC4 promoted nonhomologous end joining (NHEJ)-mediated DNA repair by interacting with Ku70/Ku80 but did not affect homologous recombination-mediated repair. Higher RFC4 expression in cancer tissue was associated with weaker tumor regression and poorer prognosis in patients with LARC treated with neoCRT, which likely resulted from the effect of RFC4 on radioresistance, not chemoresistance. CONCLUSIONS: RFC4 was identified as a radioresistance factor that promotes NHEJ-mediated DNA repair in colorectal cancer cells. In addition, the expression level of RFC4 predicted radiotherapy responsiveness and the outcome of neoadjuvant radiotherapy in patients with LARC.

Kaposi's sarcoma-associated herpesvirus LANA recruits the DNA polymerase clamp loader to mediate efficient replication and virus persistence.

Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects tumor cells and persists as a multiple-copy, extrachromosomal, circular episome. To persist, the viral genome must replicate with each cell cycle. The KSHV latency-associated nuclear antigen (LANA) mediates viral DNA replication and persistence, but little is known regarding the underlying mechanisms. We find that LANA recruits replication factor C (RFC), the DNA polymerase clamp [proliferating cell nuclear antigen (PCNA)] loader, to drive DNA replication efficiently. Mutated LANA lacking RFC interaction was deficient for LANA-mediated DNA replication and episome persistence. RFC depletion had a negative impact on LANA's ability to replicate and maintain viral DNA in cells containing artificial KSHV episomes or in infected cells, leading to loss of virus. LANA substantially increased PCNA loading onto DNA in vitro and recruited RFC and PCNA to KSHV DNA in cells. These findings suggest that PCNA loading is a rate-limiting step in DNA replication that is incompatible with viral survival. LANA enhancement of PCNA loading permits efficient virus replication and persistence, revealing a previously unidentified mechanism for KSHV latency.

Phosphoinositide 3-kinase beta controls replication factor C assembly and function.

Genomic integrity is preserved by the action of protein complexes that control DNA homeostasis. These include the sliding clamps, trimeric protein rings that are arranged around DNA by clamp loaders. Replication factor C (RFC) is the clamp loader for proliferating cell nuclear antigen, which acts on DNA replication. Other processes that require mobile contact of proteins with DNA use alternative RFC complexes that exchange RFC1 for CTF18 or RAD17. Phosphoinositide 3-kinases (PI3K) are lipid kinases that generate 3-poly-phosphorylated-phosphoinositides at the plasma membrane following receptor stimulation. The two ubiquitous isoforms, PI3Kalpha and PI3Kbeta, have been extensively studied due to their involvement in cancer and nuclear PI3Kbeta has been found to regulate DNA replication and repair, processes controlled by molecular clamps. We studied here whether PI3Kbeta directly controls the process of molecular clamps loading. We show that PI3Kbeta associated with RFC1 and RFC1-like subunits. Only when in complex with PI3Kbeta, RFC1 bound to Ran GTPase and localized to the nucleus, suggesting that PI3Kbeta regulates RFC1 nuclear import. PI3Kbeta controlled not only RFC1- and RFC-RAD17 complexes, but also RFC-CTF18, in turn affecting CTF18-mediated chromatid cohesion. PI3Kbeta thus has a general function in genomic stability by controlling the localization and function of RFC complexes.

Replication factor C recruits DNA polymerase delta to sites of nucleotide excision repair but is not required for PCNA recruitment.

Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3' termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5' phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA.

c-jun/AP-1 activation does not affect the antiproliferative activity of phenethyl isothiocyanate, a cruciferous vegetable-derived cancer chemopreventive agent.

Cruciferous vegetable-derived isothiocyanates (ITCs) display potent cancer chemopreventive activity, but also markedly stimulate oncogenic activator protein 1 (AP-1). AP-1 is well known to promote cell survival and proliferation. We examined the impact of AP-1 activation on antiproliferative activity of ITCs, using bladder cancer cells and phenethyl isothiocyanate (PEITC) as models. AP-1 transactivation induced by PEITC was almost completely suppressed by a dominant-negative c-jun (TAM67). However, suppression of AP-1 transactivation did not affect PEITC-induced apoptosis or cell-cycle arrest. Moreover, we previously showed that in response to ITC treatment c-jun was predominantly stimulated among AP-1 family members largely by c-jun N-terminal kinase (JNK) [Food Chem Toxicol 2005; 43: 1373-1380], but neither JNK inhibition nor forced expression of c-jun altered the antiproliferative activity of PEITC. In addition, cyclin D1, which is considered as an AP-1 target gene and promotes cell proliferation, was markedly elevated in PEITC-treated cells. Unexpectedly, neither TAM67 or JNK inhibition, nor forced c-jun expression had a significant impact on cyclin D1 induction by PEITC, indicating that c-jun/AP-1 does not play an important role in cyclin D1 induction by PEITC. In conclusion, despite the known role of c-jun/AP-1 as a stimulator of cell growth and proliferation, our data show that its activation does not diminish the antiproliferative activity of PEITC and is not responsible for cyclin D1 induction by PEITC.