Clinicopathological Significance of Loss of ARID1A Immunoreactivity in Ovarian Clear Cell Carcinoma

Recent genome-wide analysis has demonstrated that somatic mutations in ARID1A (BAF250) are the most common molecular genetic changes in ovarian clear cell carcinoma (OCCC). ARID1A mutations, which occur in approximately half of OCCC cases, lead to deletion of the encoded protein and inactivation of the putative tumor suppressor. In this study, we determined the significance of loss of ARID1A immunoreactivity with respect to several clinicopathological features in a total of 149 OCCCs. First, we demonstrated that ARID1A immunohistochemistry showed concordance with the mutational status in 91% of cases with 100% sensitivity and 66% specificity. Specifically, among 12 OCCC cases for which ARIDA mutational status was known, ARIDIA immunoreactivity was undetectable in all 9 cases harboring ARID1A mutations and was undetectable in one of 3 cases with wild-type ARID1A. With respect to the entire cohort, ARID1A immunoreactivity was undetectable in 88 (59%) of 149 OCCCs. There was no significant difference between ARID1A negative and positive cases in terms of histopathologic features, age, clinical stage, or overall survival. In conclusion, this study provides further evidence that mutations in ARID1A resulted in loss of ARID1A protein expression in OCCC, although no significant differences between ARID1A positive and negative cases were observed with respect to any clinicopathological features examined

Loss of the SxxSS Motif in a Human T-Cell Factor-4 Isoform Confers Hypoxia Resistance to Liver Cancer: An Oncogenic Switch in Wnt Signaling

PURPOSE: Aberrantly activated Wnt/β-catenin signaling is important in hepatocellular carcinoma (HCC) development. Downstream gene expressions involving the Wnt/β-catenin cascade occur through T-cell factor (TCF) proteins. Here, we show the oncogenic potential of human TCF-4 isoforms based on the expression of a single conserved SxxSS motif. METHODS: We investigated the TCF-4J and K isoform pair characterized by the presence (K) or absence (J) of the SxxSS motif. The mRNA expression profiles were examined in 47 pairs of human HCCs and adjacent non-cancerous liver tissues by RT-PCR. Proliferation, sphere assays and immunoblot analysis were performed under normoxia and hypoxia conditions. The ability of HCC cells overexpressing TCF-4J (J cells) and K (K cells) to grow as solid tumors in nude mice was explored. RESULTS: TCF-4J expression was significantly upregulated in HCC tumors compared to corresponding peritumor and normal liver and was preferentially expressed in poorly differentiated HCCs. In contrast, TCF-4K was downregulated in those same HCC tumors. TCF-4J-overexpressing HCC cells (J cells) revealed a survival advantage under hypoxic conditions, high proliferation rate and formation of aggregates/spheres compared to overexpression of TCF-4K (K cells). The hypoxic J cells had high expression levels of HIF-2α and EGFR as possible mechanisms to promote tumorigenesis. Increased stability of HIF-2α under hypoxia in J cells was associated with a decreased level of von Hippel-Lindau (VHL) protein, a known E3 ligase for HIF-αs. In a xenograft model, the J cells rapidly developed tumors compared to K cells. Tumor tissues derived from J cells exhibited high expression levels of HIF-2α and EGFR compared to the slow developing and small K cell derived tumors. CONCLUSIONS: Our results suggest that the specific TCF-4J isoform, which lacks a regulatory SxxSS motif, has robust tumor-initiating potential under hypoxic conditions

FXYD3 functionally demarcates an ancestral breast cancer stem cell subpopulation with features of drug-tolerant persisters

乳がんの再発を起こす原因細胞を解明. 京都大学プレスリリース. 2023-11-16.The heterogeneity of cancer stem cells (CSCs) within tumors presents a challenge in therapeutic targeting. To decipher the cellular plasticity that fuels phenotypic heterogeneity, we undertook single-cell transcriptomics analysis in triple-negative breast cancer (TNBC) to identify subpopulations in CSCs. We found a subpopulation of CSCs with ancestral features that is marked by FXYD domain–containing ion transport regulator 3 (FXYD3), a component of the Na⁺/K⁺ pump. Accordingly, FXYD3⁺ CSCs evolve and proliferate, while displaying traits of alveolar progenitors that are normally induced during pregnancy. Clinically, FXYD3⁺ CSCs were persistent during neoadjuvant chemotherapy, hence linking them to drug-tolerant persisters (DTPs) and identifying them as crucial therapeutic targets. Importantly, FXYD3⁺ CSCs were sensitive to senolytic Na⁺/K⁺ pump inhibitors, such as cardiac glycosides. Together, our data indicate that FXYD3⁺ CSCs with ancestral features are drivers of plasticity and chemoresistance in TNBC. Targeting the Na⁺/K⁺ pump could be an effective strategy to eliminate CSCs with ancestral and DTP features that could improve TNBC prognosis

Inhibitory Effects of the New Bombesin Receptor Antagonist RC-3095 on the Luteinizing Hormone Release in Rats

The effects of bombesin receptor antagonist RC-3095 and gastrin-releasing peptide [GRP (14–27)] on basal luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels, as well as on the release of LH and FSH in response to LH-releasing hormone, were studied in normal and castrated male rats. We also examined the release of LH and FSH after intracerebroventricular injection of RC-3095 under different types of anesthesia (urethane, sodium pentobarbital, Metofane). Following injection of 10 µg RC-3095 into the lateral brain ventricle, serum LH levels were rapidly and significantly (p < 0.001) lowered. This effect lasted for at least 60 min and was not affected by the type of anesthetic used. Serum FSH levels were not affected by intracerebroventricular administration of RC-3095 or GRP (14–27), indicating different central control mechanisms between LH and FSH release. The suppressive effect of RC-3095 on LH release could be completely prevented by prior intracerebroventricular administration of GRP (14–27) at a dose of 1 µg, but intravenous administration of either peptide RC-3095 or GRP (14–27) did not change the basal levels of LH, FSH, and testosterone. The pituitary LH response to LH-releasing hormone was also not modified by intravenous administration of RC-3095. These results indicate that bombesin-like peptides might be involved in control of LH release from the pituitary by an action on the CNS which is mediated by specific bombesin receptors

Effect of Luteinizing Hormone-Releasing Hormone Analogs on the Rat Ovarian Follicle Development

Our objective was to study the direct action of luteinizing hormone-releasing hormone (LHRH) agonist buserelin and LHRH antagonist Cetrorelix (SB-75) on cell proliferation and differentiation in the rat ovarian follicle. Preovulato-ry follicles were isolated from PMSG-primed immature rats and incubated in the presence or absence of hCG (10 IU/ml), buserelin (10-9-10-6M) or Cetrorelix (10-9-10-6M) for 12 h in vitro. Buserelin induced meiotic maturation of the follicle-enclosed oocytes dose-dependently. The percentage of oocytes with germinal vesicle breakdown at 10-6M buserelin (73.3%) did not differ from that of hCG-treated control (73.3%). Buserelin also significantly stimulated prostaglandin E2 and progesterone production by follicles, but not estradiol production. Granulosa cells were obtained from the preovulatory follicles and cultured for 5 days. Epidermal growth factor (EGF) stimulated granulosa cell growth at concentrations of 1, 10 and 100 ng/ml. In contrast, both buserelin and Cetrorelix inhibited granulosa cell growth dose-dependently in the range of 10-10-10-5 M, with Cetrorelix inducing a greater growth inhibition than buserelin. Electrophoretic analysis of genomic DNA extracted from granulosa cells treated with 10-6M concentration of either LHRH analog revealed a definitive ladder pattern of oligonucleosomal length DNA fragments characteristic of apoptosis. Western blotting detected that EGF-induced tyrosine phosphorylation was not affected by either analog. These results demonstrate that LHRH agonist and antagonist inhibit directly proliferation of granulosa cells through apoptosis, without interference with EGF receptor phosphorylation, whereas LHRH agonist stimulates cell differentiation in the preovulatory follicle

Recovery of Pituitary‐Gonadal Function in Male Rats after Long‐Term Suppression Induced by a Single Injection of Microcapsules of LH‐RH Antagonist Cetrorelix (SB‐75)

The clinical utility of luteinizing hormone‐releasing hormone (LH‐RH) analogs can be greatly enhanced by a sustained delivery system, which could maintain elevated peptide levels in the blood for prolonged periods of time, up to several weeks. Recently, we developed long‐acting microcapsules and microgranules of the LH‐RH antagonist SB‐75. In this study, we examined the suppressive effects of a single injection of microcapsules of antagonist SB‐75 on gonadotropin and testosterone secretion, as well as on fertility, in male rats and the reversibility of those effects. Serum SB‐75 levels were measured by RIA. A dose of 20 mg of microcapsules/rat containing 3.58 mg of antagonist in poly(D,L‐lactide‐co‐glycolide), administered intramuscularly produced SB‐75 levels higher than 20 ng/ml for approximately 24 days, and a significant elevation was maintained until day 90. Serum testosterone was decreased to castration values for 164 days and LH levels were suppressed below the detection limit of the RIA for a period of 102 days. Serum FSH was suppressed by more than 90%, as compared to control animals, for a period of 58 days and remained significantly decreased until day 164 after the injection. This treatment also caused a significant decrease in the weights of the testes, seminal vesicles, and ventral prostate 30 days after peptide administration. The histology of the testes from the treated rats showed that spermatogenesis was totally depressed. No mature elongated or round spermatids were found in the seminiferous tubules, spermatocytes being the most advanced germ cell form in 99.5% of the testicular tubules. Ten months after injection, a complete recovery in organ weights, hormonal levels, and fertility was observed. Histological studies revealed a complete recovery of spermatogenesis, with 100% of seminiferous tubules containing mature elongated spermatids. All treated rats proved to be able to impregnate normal female rats. The offspring were normal, with no evidence of genetic abnormalities. The overall results demonstrate the efficacy of SB‐75 microcapsules in suppressing the pituitary‐gonadal axis for a prolonged period of time and show that the long‐term suppression of gonadal function induced by chronic treatment with antagonist SB‐75 is completely reversible