FLIP: A Targetable Mediator of Resistance to Radiation in Non-Small Cell Lung Cancer

Resistance to radiotherapy due to insufficient cancer cell death is a significant cause of treatment failure in non-small cell lung cancer (NSCLC). The endogenous caspase-8 inhibitor, FLIP, is a critical regulator of cell death that is frequently overexpressed in NSCLC and is an established inhibitor of apoptotic cell death induced via the extrinsic death receptor pathway. Apoptosis induced by ionizing radiation (IR) has been considered to be mediated predominantly via the intrinsic apoptotic pathway; however, we found that IR-induced apoptosis was significantly attenuated in NSCLC cells when caspase-8 was depleted using RNA interference (RNAi), suggesting involvement of the extrinsic apoptosis pathway. Moreover, overexpression of wild-type FLIP, but not a mutant form that cannot bind the critical death receptor adaptor protein FADD, also attenuated IR-induced apoptosis, confirming the importance of the extrinsic apoptotic pathway as a determinant of response to IR in NSCLC. Importantly, when FLIP protein levels were down-regulated by RNAi, IRinduced cell death was significantly enhanced. The clinically relevant histone deacetylase (HDAC) inhibitors vorinostat and entinostat were subsequently found to sensitize a subset of NSCLC cell lines to IR in a manner that was dependent on their ability to suppress FLIP expression and promote activation of caspase-8. Entinostat also enhanced the anti-tumor activity of IR in vivo. Therefore, FLIP down-regulation induced by HDAC inhibitors is a potential clinical strategy to radio-sensitize NSCLC and thereby improve response to radiotherapy. Overall, this study provides the first evidence that pharmacological inhibition of FLIP may improve response of NCSLC to IR

The BRCA1-binding protein BRAP2 can act as a cytoplasmic retention factor for nuclear and nuclear envelope-localizing testicular proteins

Regulation of nuclear protein import is central to many cellular processes such as development, with a key mechanism being factors that retain cargoes in the cytoplasm that normally localize in the nucleus. The breast cancer antigen BRCA1-binding protein BRAP2 has been reported as a novel negative regulator of nuclear import of various nuclear localization signal (NLS)-containing viral and cellular proteins, but although implicated in differentiation pathways and highly expressed in tissues including testis, the gamut of targets for BRAP2 action in a developmental context is unknown. As a first step towards defining the BRAP2 interactome, we performed a yeast-2-hybrid screen to identify binding partners of BRAP2 in human testis. Here we report characterization for the first time of three of these: the high mobility group (HMG)-box-domain-containing chromatin component HMG20A, nuclear mitotic apparatus protein NuMA1 and synaptic nuclear envelope protein SYNE2. Co-immunoprecipitation experiments indicate association of BRAP2 with HMG20A, NuMA1, and SYNE2 in testis, underlining the physiological relevance of the interactions, with immunohistochemistry showing that where BRAP2 is co-expressed with HMG20A and NuMA1, both are present in the cytoplasm, in contrast to their nuclear localization in other testicular cell types. Importantly, quantitative confocal microscopic analysis of cultured cells indicates that ectopic expression of BRAP2 inhibits nuclear localization of HMG20A and NuMA1, and prevents nuclear envelope accumulation of SYNE2, the first report of BRAP2 altering localization of a non-nuclear protein. These results imply for the first time that BRAP2 may have an important role in modulating subcellular localization during testicular development

Identification of sites of STAT3 action in the female reproductive tract through conditional gene deletion

The STAT3 transcription factor is a pleiotropic transducer of signalling by hormones, growth factors and cytokines that has been identified in the female reproductive tract from oocytes and granulosa cells of the ovary to uterine epithelial and stromal cells. In the present study we used transgenic models to investigate the importance of STAT3 for reproductive performance in these different tissues. The Cre-LoxP system was used to delete STAT3 in oocytes by crossing Stat3^fl/fl with Zp3-cre+ mice, or in ovarian granulosa cells and uterine stroma by crossing with Amhr2-Cre+ mice. Surprisingly, deletion of STAT3 in oocytes had no effect on fertility indicating that the abundance of STAT3 protein in maturing oocytes and fertilized zygotes is not essential to these developmental stages. In Stat3^fl/fl;Amhr2-cre+ females impaired fertility was observed through significantly fewer litters and smaller litter size. Ovulation rate, oocyte fertilization and development to blastocyst were unaffected in this line; however, poor recombination efficiency in granulosa cells had yielded no net change in STAT3 protein abundance. In contrast, uteri from these mice showed STAT3 protein depletion selectively from the stomal compartment. A significant reduction in number of viable fetuses on gestational day 18, increased fetal resorptions and disrupted placental morphology were evident causes of the reduced fertility. In conclusion, this study defines an important role for STAT3 in uterine stromal cells during embryo implantation and the development of a functional placenta

Reduced fetal viability and placental morphology in <i>Stat3^fl/fl;Amhr2-cre⁺</i> mice.

<p>Mid-saggital sections of placental tissue recovered on day 17.5 pc from <i>Stat3^fl/fl; Amhr2-cre⁺</i> females <b>(A, C)</b> and <i>Stat3^fl/+;Amhr2-cre⁺</i> females <b>(B, D)</b> stained with hematoxylin and eosin. A moderately thicker junctional zone (Jz) compared with control genotypes was observed in <i>Stat3^fl/fl;Amhr2-cre⁺</i> mice, while the labyrinthine (La) zone appears comparable in thickness. Representative images are shown. Representative examples of 7-9 mice per genotype. Bars in A&B are 2 mm; bars in C and d are 400 µm</p

STAT3 deficiency in uterine stroma and aberrant uterine morphology in <i>Stat3^fl/fl; Amhr2-cre⁺</i> mice.

<p>Transverse sections of uteri immunostained for STAT3 demonstrate the lack of STAT3 in <b>(A, B)</b><i>Stat3^fl/fl;Amhr2-cre⁺</i> uterine stromal cells, while luminal and glandular epithelial cells had equivalent STAT3 in all genotypes. <b>(C, D)</b><i>Stat3^fl/fl;Amhr2-cre^-</i>, <b>(E, F)</b><i>Stat3^fl/+;Amhr2-cre⁺.</i> Representative examples of 5 mice for each genotype are shown. Scale bars in A, C and E 500 µm, in B, D, and E 100 µm.</p

The effect of maternal STAT3 deficiency on fetal and placental parameters at day 18 of pregnancy.

<p>Values are mean ± SD. Data were compared by independent samples t-test. Values with different superscript characters were significantly different P<0.05.</p

Subfertility in <i>Stat3^fl/fl;Amhr2-cre⁺</i> mice.

<p>Reproductive ability of the <i>Stat3;Amhr2Cre</i> line was tracked over a 30 week breeding period. (<b>A</b>) Cumulative number of pups born (characters indicate statistical significance, <i>P</i><0.05, one way ANOVA and Tukey's <i>post hoc</i> test). (<b>B</b>) Average number of litters per 30 day period. (<b>C</b>) Average number of pups per litter (asterix indicates statistical significance from other groups,<i>P</i><0.05, one way ANOVA and Tukey's <i>post hoc</i> test) (n = 5 breeder pairs per genotype). (<b>D</b>) Ovulation rates were assessed in both hormonally stimulated (eCG+hCG) and naturally mated (<b>E</b>) mice (n≥8 mice per group). (<b>F</b>) Rate of embryo development was assessed in knock out and wild type groups following in vitro fertilization (n≥6 mice per group). (<b>G</b>) Recombination of the <i>Stat3</i> gene in COCs was confirmed by PCR with the amplification of a 310 bp truncated product in heterozygous (<i>Stat3^fl/+;Amhr2-cre⁺</i>) and knock out (<i>Stat3^fl/fl;Amhr2-cre⁺</i>) mice. (<b>H</b>) Abundance of STAT3 protein was analysed by Western blot of GC collected from all 4 genotypes of the <i>STAT3;Amhr2Cre</i> line. Pooled GC samples from 3 mice shown, equivalent sample loading (10 µg protein extract per lane) was confirmed by subsequent β-actin analysis of the same blot. (<b>I</b>) STAT3 immuno staining (brown) in ovaries from mice after completion of the 30 week breeding period, bar  = 500 µm.</p

Janus kinase JAK1 maintains the ovarian reserve of primordial follicles in the mouse ovary

STUDY QUESTION Is the Janus kinase and signal transducer and activator of transcription (JAK-STAT) signalling pathway involved in ovarian follicle development and primordial follicle activation? SUMMARY ANSWER JAK1 is a key factor involved in the regulation of primordial follicle activation and maintenance of the ovarian reserve. WHAT IS KNOWN ALREADY A series of integrated, intrinsic signalling pathways (including PI3K/AKT, mTOR and KITL) are responsible for regulating the ovarian reserve of non-growing primordial follicles and ultimately female fertility. The JAK-STAT signal transduction pathway is highly conserved with established roles in cell division and differentiation. Key pathway members (specifically JAK1, STAT3 and SOCS4) have been previously implicated in early follicle development. STUDY DESIGN, SIZE, DURATION A laboratory animal study was undertaken using the C57Bl/6 inbred mouse strain as a model for human ovarian follicle development. To determine which Jak genes were most abundantly expressed during primordial follicle activation, mRNA expression was analysed across a developmental time-course, with ovaries collected from female mice at post-natal days 1 (PND1), 4 (PND4), 8 (PND8), as well as at 6 weeks (6WK) and 7 months (7MTH) (n ≥ 4). Functional analysis of JAK1 was performed on PND2 mouse ovaries subjected to in vitro explant culture treated with 12.5 μM Ruxolitinib (JAK inhibitor) or vehicle control (DMSO) for 48 h prior to histological assessment (n ≥ 4). PARTICIPANTS/MATERIALS, SETTING, METHODS The expression and localization of the JAK family during ovarian follicle development in the C57Bl/6 inbred mouse strain were evaluated using quantitative PCR, immunoblotting and immunolocalisation. Functional studies were undertaken using the JAK inhibitor Ruxolitinib to investigate the underpinning cellular mechanisms via biochemical in vitro inhibition and histological assessment of intact neonate ovaries. All experiments were replicated at least three times using tissue from different mice unless otherwise stated. MAIN RESULTS AND THE ROLE OF CHANCE Jak1 is the predominant Jak mRNA expressed in the C57Bl/6 mouse ovary across all developmental time-points assessed (P ≤ 0.05). Forty-eight hour inhibition of JAK1 with Ruxolitinib of PND2 ovaries in vitro demonstrated concomitant acceleration of primordial follicle activation and apoptosis (P ≤ 0.001) and upregulation of downstream JAK-STAT pathway members STAT3 and suppressors of cytokine signalling 4 (SOCS4). LARGE-SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Results are shown in one species, the C57Bl/6 mouse strain as an established model of human ovary development. Ruxolitinib also inhibits JAK2, with decreased efficacy. However, Jak2 mRNA had limited expression in the mouse ovary, particularly at the neonatal stages of follicle development, thus any effect of Ruxolitinib on primordial follicle activation was unlikely to be mediated via this isoform. WIDER IMPLICATIONS OF THE FINDINGS This study supports a key role for JAK1 in the maintenance and activation of primordial follicles, with potential for targeting the JAK-STAT pathway as a method of regulating the ovarian reserve and female fertility. STUDY FUNDING AND COMPETING INTEREST(S) This project has been funded by the Australian National Health and Medical Research Council (G1600095) and The Hunter Medical Research Institute Bob and Terry Kennedy Children's Research Project Grant in Pregnancy & Reproduction (G1501433). All authors declare no conflict of interest