Elevated PAF1-RAD52 Axis Confers Chemoresistance to Human Cancers

Cisplatin- and gemcitabine-based chemotherapeutics represent a mainstay of cancer therapy for most solid tumors; however, resistance limits their curative potential. Here, we identify RNA polymerase II-associated factor 1 (PAF1) as a common driver of cisplatin and gemcitabine resistance in human cancers (ovarian, lung, and pancreas). Mechanistically, cisplatin- and gemcitabine-resistant cells show enhanced DNA repair, which is inhibited by PAF1 silencing. We demonstrate an increased interaction of PAF1 with RAD52 in resistant cells. Targeting the PAF1 and RAD52 axis combined with cisplatin or gemcitabine strongly diminishes the survival potential of resistant cells. Overall, this study shows clinical evidence that the expression of PAF1 contributes to chemotherapy resistance and worse clinical outcome for lethal cancers

Selective killing of homologous recombination-deficient cancer cell lines by inhibitors of the RPA:RAD52 protein-protein interaction.

Synthetic lethality is a successful strategy employed to develop selective chemotherapeutics against cancer cells. Inactivation of RAD52 is synthetically lethal to homologous recombination (HR) deficient cancer cell lines. Replication protein A (RPA) recruits RAD52 to repair sites, and the formation of this protein-protein complex is critical for RAD52 activity. To discover small molecules that inhibit the RPA:RAD52 protein-protein interaction (PPI), we screened chemical libraries with our newly developed Fluorescence-based protein-protein Interaction Assay (FluorIA). Eleven compounds were identified, including FDA-approved drugs (quinacrine, mitoxantrone, and doxorubicin). The FluorIA was used to rank the compounds by their ability to inhibit the RPA:RAD52 PPI and showed mitoxantrone and doxorubicin to be the most effective. Initial studies using the three FDA-approved drugs showed selective killing of BRCA1-mutated breast cancer cells (HCC1937), BRCA2-mutated ovarian cancer cells (PE01), and BRCA1-mutated ovarian cancer cells (UWB1.289). It was noteworthy that selective killing was seen in cells known to be resistant to PARP inhibitors (HCC1937 and UWB1 SYr13). A cell-based double-strand break (DSB) repair assay indicated that mitoxantrone significantly suppressed RAD52-dependent single-strand annealing (SSA) and mitoxantrone treatment disrupted the RPA:RAD52 PPI in cells. Furthermore, mitoxantrone reduced radiation-induced foci-formation of RAD52 with no significant activity against RAD51 foci formation. The results indicate that the RPA:RAD52 PPI could be a therapeutic target for HR-deficient cancers. These data also suggest that RAD52 is one of the targets of mitoxantrone and related compounds

Strategy for the accurate preoperative evaluation of the number of metastatic axillary lymph nodes in breast cancer

Summary: Background: After the ACOSOG Z0011 trial, it became important to evaluate the number of metastatic axillary lymph nodes (LNs) preoperatively. The purpose of this paper is to confirm whether the number of metastases can be accurately diagnosed by preoperative computed tomography (CT), ultrasound sonography (US), and US-guided fine-needle aspiration cytology (FNAC). Methods: We retrospectively analyzed the axillary LNs finding of preoperative CT/US of 470 breast cancer patients. Metastasis was suspected based on the following findings: LNs with a long-axis diameter of ≥10 mm or a short-axis diameter of ≥5 mm on CT, and LNs with the absence of a fatty hilum, focal cortical thickness or a cortical thickness ≥2 mm on US. We also examined the results of FNAC making a rapid bedside diagnosis (bedside-FNAC) of 162 LNs that were suspected to metastatic based on the US findings. Results: On CT, all cases with ≥3 LNs with a long-axis diameter of ≥10 mm and a short-axis diameter of ≥5 mm had metastasis. However, there was no relationship between the number of detected LNs and the number of metastases. On US, 75.7% of LNs with the absence of a fatty hilum and all LNs with cortical thickness ≥6 mm had metastasis. The accuracy of bedside-FNAC for suspicious LNs was 100%. Conclusions: Although we can pick up LNs that are likely to have metastasis on CT/US, it was impossible to accurately predict the number of metastases on CT/US. However, bedside-FNAC of suspicious LNs could accurately predict the number of metastases. Keywords: Bedside fine-needle aspiration cytology, Computed tomography, Lymph node metastasis, Rapid diagnosis, Ultrasound sonograph

Hematologic improvements in a myelodysplastic syndromes with myelofibrosis (MDS-F) patient treated with azacitidine

Myelodysplastic syndromes with myelofibrosis (MDS-F) is a poor prognostic hematopoietic disorder. Azacitidine was shown to prolong survival of high-risk MDS patients. However, the effects of azacitidine on MDS-F have yet to be elucidated. Azacitidine was administered to a 74-year-old man with MDS-F at a dose of 75 mg/m2/daily subcutaneously for 7 days every 28 days. Hematologic improvements were observed according to the International Working Group 2006 criteria after 8 cycles of the azacitidine treatment, and complete remission was achieved after 14 cycles. The grade of myelofibrosis was also improved. The therapeutic activity of azacitidine was confirmed in our MDS-F patient

BRCA1-associated growth arrest is RB-dependent

BRCA1 is a susceptibility gene for breast and ovarian cancer with growth-inhibitory activity for which the mechanism of action remains unclear. When introduced into cells, BRCA1 inhibits growth of some but not all cell lines. In an attempt to uncover the mechanism of growth suppression by BRCA1, we examined a panel of cell lines for their ability to reduce colony outgrowth in response to BRCA1 overexpression. Of all variables tested, only those cells with wild-type pRb were sensitive to BRCA1-induced growth suppression. In cells with an intact rb gene, inactivation of pRb by HPV E7 abrogates the growth arrest imposed by BRCA1. In accordance with these observations, we found that BRCA1 could not suppress BrdUrd uptake in primary fibroblasts from rb−/− mice and exhibited an intermediate ability to inhibit DNA synthesis in rb+/− as compared with rb+/+ cells. We further found that the BRCA1 protein complexes with the hypophosphorylated form of pRb. This binding is localized to amino acids 304–394 of BRCA1 protein and requires the ABC domain of pRb. In-frame deletion of BRCA1 fragment involved in interaction with pRb completely abolished the growth-suppressive property of BRCA1. Although it has been reported that BRCA1 interacts with p53, we find the p53 status did not affect the ability of BRCA1 to suppress colony formation. Our data suggest that the growth suppressor function of BRCA1 depends, at least in part, on Rb

Prognostic impact of peripheral blood Wilms’ tumour 1 mRNA expression levels in response to azacytidine in MDS: A single-centre analysis

To determine the impact of peripheral blood (PB) Wilms’ tumour 1 (WT-1) mRNA levels in patients with primary myelodysplastic syndromes (MDS), we analysed the relationships between several clinical variables at the time of diagnosis and the haematological response of patients treated with azacytidine. We observed overall responses in 20 (63%) patients; there were no significant differences in clinical variables, including bone marrow blast counts, IPSS scores and IPSS-R risk scores, between responders and non-responders. The responders’ PB WT-1 mRNA levels were significantly lower than those of non-responders (P = 0.03). PB WT-1 mRNA expression could be a marker for predicting the response to azacytidine in patients with de novo MDS

Determination of cell fate in skeletal muscle following BMP gene transfer by in vivo electroporation

We previously developed a novel method for gene transfer, which combined a non-viral gene expression vector with transcutaneous in vivo electroporation. We applied this method to transfer the bone morphogenetic protein (BMP) gene and induce ectopic bone formation in rat skeletal muscles. At present, it remains unclear which types of cells can differentiate into osteogenic cells after BMP gene transfer by in vivo electroporation. Two types of stem cells in skeletal muscle can differentiate into osteogenic cells: muscle-derived stem cells, and bone marrow-derived stem cells in the blood. In the present study, we transferred the BMP gene into rat skeletal muscles. We then stained tissues for several muscle-derived stem cell markers (e.g., Pax7, M-cadherin), muscle regeneration-related markers (e.g., Myod1, myogenin), and an inflammatory cell marker (CD68) to follow cell differentiation over time. Our results indicate that, in the absence of BMP, the cell population undergoes muscle regeneration, whereas in its presence, it can differentiate into osteogenic cells. Commitment towards either muscle regeneration or induction of ectopic bone formation appears to occur five to seven days after BMP gene transfer.</p

Assessment of ATP8B1 Deficiency in Pediatric Patients With Cholestasis Using Peripheral Blood Monocyte-Derived Macrophages

Progressive familial intrahepatic cholestasis type 1 (PFIC1), a rare inherited recessive disease resulting from a genetic deficiency in ATP8B1, progresses to liver failure. Because of the difficulty of discriminating PFIC1 from other subtypes of PFIC based on its clinical and histological features and genome sequencing, an alternative method for diagnosing PFIC1 is desirable. Herein, we analyzed human peripheral blood monocyte-derived macrophages (HMDM) and found predominant expression of ATP8B1 in interleukin-10 (IL-10)-induced M2c, a subset of alternatively activated macrophages. SiRNA-mediated depletion of ATP8B1 in IL-10-treated HMDM markedly suppressed the expression of M2c-related surface markers and increased the side scatter (SSC) of M2c, likely via impairment of the IL-10/STAT3 signal transduction pathway. These phenotypic features were confirmed in IL-10-treated HMDM from four PFIC1 patients with disease-causing mutations in both alleles, but not in those from four patients with other subtypes of PFIC. This method identified three PFIC1 patients in a group of PFIC patients undiagnosed by genome sequencing, an identical diagnostic outcome to that achieved by analysis of liver specimens and in vitro mutagenesis studies. In conclusion, ATP8B1 deficiency caused incomplete polarization of HMDM into M2c. Phenotypic analysis of M2c helps to identify PFIC1 patients with no apparent disease-causing mutations in ATP8B1