Patient-derived ovarian cancer organoids capture the genomic profiles of primary tumours applicable for drug sensitivity and resistance testing

The use of primary patient-derived organoids for drug sensitivity and resistance testing could play an important role in precision cancer medicine. We developed expandable ovarian cancer organoids in<3 weeks; these organoids captured the characteristics of histological cancer subtypes and replicated the mutational landscape of the primary tumours. Seven pairs of organoids (3 high-grade serous, 1 clear cell, 3 endometrioid) and original tumours shared 59.5% (36.1-73.1%) of the variants identified. Copy number variations were also similar among organoids and primary tumours. The organoid that harboured the BRCA1 pathogenic variant (p.L63*) showed a higher sensitivity to PARP inhibitor, olaparib, as well as to platinum drugs compared to the other organoids, whereas an organoid derived from clear cell ovarian cancer was resistant to conventional drugs for ovarian cancer, namely platinum drugs, paclitaxel, and olaparib. The overall success rate of primary organoid culture, including those of various histological subtypes, was 80% (28/35). Our data show that patient-derived organoids are suitable physiological ex vivo cancer models that can be used to screen effective personalised ovarian cancer drugs

Identification of a novel uterine leiomyoma GWAS locus in a Japanese population

Uterine leiomyoma is one of the most common gynaecologic benign tumours, but its genetic basis remains largely unknown. Six previous GWAS identified 33 genetic factors in total. Here, we performed a two-staged GWAS using 13,746 cases and 70,316 controls from the Japanese population, followed by a replication analysis using 3,483 cases and 4,795 controls. The analysis identified 9 significant loci, including a novel locus on 12q23.2 (rs17033114, P = 6.12 × 10−25 with an OR of 1.177 (1.141-1.213), LINC00485). Subgroup analysis indicated that 5 loci (3q26.2, 5p15.33, 10q24.33, 11p15.5, 13q14.11) exhibited a statistically significant effect among multiple leiomyomas, and 2 loci (3q26.2, 10q24.33) exhibited a significant effect among submucous leiomyomas. Pleiotropic analysis indicated that all 9 loci were associated with at least one proliferative disease, suggesting the role of these loci in the common neoplastic pathway. Furthermore, the risk T allele of rs2251795 (3q26.2) was associated with longer telomere length in both normal and tumour tissues. Our findings elucidated the significance of genetic factors in the pathogenesis of leiomyoma

Characteristics and clinicopathological features of patients with ovarian metastasis of endometrial cancer: a retrospective study

There are no criteria for patient selection for ovarian-preserving surgery for endometrial cancer (EC). In this study, intraoperative findings of ovarian swelling (OvS) and the clinicopathological features of patients with EC with or without ovarian metastasis were analysed to identify risk factors for ovarian metastasis. Patients who underwent surgery for EC between 2012 and 2019 at our hospital were enrolled. In univariate analysis, all features were significantly higher in metastasis(+) cases. In multivariate analysis, lymphatic space invasion (LSI), cervical stromal involvement (CSI), peritoneal dissemination, and OvS were significant risk factors. In univariate analysis in stage I and II cases classified without adnexal pathological factors, type 2 histologic type, LSI, CSI, and OvS were significantly higher in metastasis(+) cases. LSI, CSI, and OvS were significant risk factors in multivariate analysis. Patients with type 1 histologic type EC without myometrial invasion ≥1/2, CSI and extrauterine lesions are appropriate for ovarian preservation. IMPACT STATEMENT What is already known on this subject? The number of premenopausal patients with endometrial cancer (EC) is increasing. Bilateral oophorectomy for EC results in surgical primary ovarian insufficiency, and thus, surgery with ovarian preservation has been examined. However, there are few reports on risk factors for ovarian metastasis of EC and no established criteria for patient background or pathological factors to determine suitability for ovarian preservation surgery. What do the results of this study add? In univariate analysis, all pathological findings suggestive of disease progression were more frequent in cases with ovarian metastases. In multivariate analysis, lymphatic space invasion (LSI), cervical stromal involvement (CSI), peritoneal dissemination, and ovarian swelling (OvS) were identified as significant risk factors for ovarian metastasis. In an analysis of stage I and II cases classified without adnexal pathological factors, type 2 histologic type, LSI, CSI, and OvS were significantly more common in cases with ovarian metastasis, and LSI, CSI, and OvS emerged as significant risk factors for ovarian metastasis in multivariate analysis. What are the implications of these findings for clinical practice and/or further research? Patients with type 1 histologic type EC without depth of myometrial invasion ≥1/2, CSI, or extrauterine lesions may be appropriate cases for ovarian preservation

The Anaphase-Promoting Complex/Cyclosome Activator Cdh1 Modulates Rho GTPase by Targeting p190 RhoGAP for Degradation▿ †

Cdh1 is an activator of the anaphase-promoting complex/cyclosome and contributes to mitotic exit and G1 maintenance by targeting cell cycle proteins for degradation. However, Cdh1 is expressed and active in postmitotic or quiescent cells, suggesting that it has functions other than cell cycle control. Here, we found that homozygous Cdh1 gene-trapped (Cdh1GT/GT) mouse embryonic fibroblasts (MEFs) and Cdh1-depleted HeLa cells reduced stress fiber formation significantly. The GTP-bound active Rho protein was apparently decreased in the Cdh1-depleted cells. The p190 protein, a major GTPase-activating protein for Rho, accumulated both in Cdh1GT/GT MEFs and in Cdh1-knockdown HeLa cells. Cdh1 formed a physical complex with p190 and stimulated the efficient ubiquitination of p190, both in in vitro and in vivo. The motility of Cdh1-depleted HeLa cells was impaired; however, codepletion of p190 rescued the migration activity of these cells. Moreover, Cdh1GT/GT embryos exhibited phenotypes similar to those observed for Rho-associated kinase I and II knockout mice: eyelid closure delay and disruptive architecture with frequent thrombus formation in the placental labyrinth layer, respectively. Furthermore, the p190 protein accumulated in the Cdh1GT/GT embryonic tissues. Our data revealed a novel function for Cdh1 as a regulator of Rho and provided insights into the role of Cdh1 in cell cytoskeleton organization and cell motility

LATS1 phosphorylates the conserved N-terminal T7 residue of CDC26 <i>in vitro</i>.

<p>(A, C) Phosphorylation of CDC26 by recombinant LATS1. GST alone and GST-tagged wild-type (WT) and T7A-mutated CDC26 were incubated with the indicated amount (200ng/μl) of GST-LATS1 at 30°C for 30 min (A) or 100ng GST-LATS1 at the indicated time (C). The samples were then subjected to SDS-PAGE followed by autoradiography and Coomassie Blue staining. To adjust for differences in the reaction volume in (A), distilled water was added to the reactions in lanes 1, 5, and 9. (B) Quantitative analysis of the autoradiography data shown in (A). The intensity of each band was quantified and normalized to the intensity of the corresponding band on the Coomassie Blue-stained gel. Data are shown as the relative intensities compared with that in the reaction containing 1 μl (200 ng) of WT CDC26 and represent the mean ± standard deviation of three independent experiments. (D) GST-tagged wild-type (WT) and T7A-mutated CDC26-N (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118662#pone.0118662.g001" target="_blank">Fig. 1B</a>) were incubated with the indicated amount (200ng/μl) of GST-LATS1 at 30°C for 30 min. The samples were then subjected to SDS-PAGE followed by autoradiography and Coomassie Blue staining. Open arrowhead indicates autophophorylated GST-LATS1.</p

Phosphorylation of CDC26 affects APC/C assembly.

<p>(A) The effect of the T7D phosphor-mimic mutation of CDC26 on its ability to bind to APC/C. HeLa cells stably expressing FLAG alone (FLAG-mock) or FLAG-tagged wild-type, T7A-mutated, or T7D-mutated CDC26 mock were treated with a siRNA targeting the 3’UTR of endogenous CDC26. APC/C was immunoprecipitated using an anti-APC4 antibody and the immunoprecipitates, and 7% of the input fractions were analyzed by immunoblotting with the indicated antibodies. The input fractions were detected after long exposures of the blots (long exp). (B) The effect of nocodazole (noc)-induced phosphorylation of the T7 residue of CDC26 on the interaction of CDC26 with APC/C. HeLa cells were treated with or without nocodazole for 16 h and then APC/C was immunoprecipitated using anti-APC4 antibodies. The immunoprecipitates and 20% of the input fractions were analyzed by immunoblotting with the indicated antibodies. CDC27 in the input fractions were detected after long exposures of the blots (long exp). (C) Gel filtration chromatography analyses of HeLa cells stably expressing FLAG-tagged wild-type, T7A-mutated, or T7D-mutated CDC26. The cells were treated with a siRNA targeting the 3’UTR of endogenous CDC26. The lysates were fractioned on a column, and the fractions were analyzed by immunoblotting with the indicated antibodies. Vo, void volume.</p

LATS1-mediated phosphorylation of CDC26 modulates the ubiquitination of PLK1 <i>in vivo</i>.

<p>(A) The effects of wild-type (WT) and kinase-dead (KD) LATS1 on the ubiquitination of PLK1 <i>in vivo</i>. HEK293T cells were co-transfected with FLAG-tagged PLK1, Myc-tagged ubiquitin and a control vector (mock) or WT or KD LATS1, and then PLK1 was immunoprecipitated using an anti-FLAG antibody. The immunoprecipitates and input fractions were analyzed by immunoblotting with antibodies against c-Myc or FLAG. (B) The effect of a phosphor-mimic mutant of CDC26 (T7D) on the ubiquitination of PLK1 <i>in vivo</i>. HeLa cells were treated with a siRNA targeting the 3’UTR of endogenous CDC26 and then induced to express exogenous wild-type, T7A-mutated, or T7D-mutated CDC26 by adding doxycycline to the culture medium. The cells were then co-transfected with FLAG-tagged PLK1 and Myc-tagged ubiquitin and treated with the proteasome inhibitor MG132 (10 μM) for 12 h prior to cell collection. PLK1 was immunoprecipitated using an anti-FLAG antibody, and the immunoprecipitates and input fractions were analyzed by immunoblotting with antibodies against c-Myc or FLAG.</p