Let-7a mimic transfection reduces chemotherapy-induced damage in a mouse ovarian transplantation model.

Pharmacological approaches offer a non-invasive and promising option for fertility preservation in young female cancer patients undergoing gonadotoxic therapy. The GnRH-agonists are the only clinically available drugs in this indication, but their use and mechanisms of protection are still controversial. Recently, we have investigated new targeted drugs based on microRNA (miRNA) replacement therapy, and have identified the let-7a miRNA as candidate for fertility preservation strategies. Here, the effect of let-7a replacement during chemotherapy exposure on follicular growth and oocyte maturation capacity was investigated using a mouse ovarian-kidney transplantation model. Newborn mouse ovaries were cultured under different conditions; control, chemotherapy exposure (4-hydroperoxycyclophosphamide, 4-HC), and co-treatment with 4-HC and let-7a mimic transfection (4-HC + let-7a). The ovaries were then transplanted under the kidney capsule of recipient mice and follicular growth, survival, and oocyte in vitro maturation were assessed after 3 weeks. The results showed that the follicular pool was highest in the control group but higher in the 4-HC + let-7a group than the 4-HC group. DNA-damage/apoptosis ratios were higher in all 4-HC-exposed groups compared to control but were reduced in the 4-HC + let-7a group. In addition, the post-transplantation oocyte in vitro maturation rate was higher in the 4-HC + let-7a group compared to the 4-HC group, suggesting better oocyte quality. These results provide new information regarding the beneficial effects of let-7a replacement against chemotherapy-induced ovarian damage and open new perspectives for future in vivo applications.

Letrozole-associated controlled ovarian hyperstimulation in breast cancer patients versus conventional controlled ovarian hyperstimulation in infertile patients: assessment of oocyte quality related biomarkers.

BACKGROUND: Fertility preservation (FP) protocols in case of breast cancer (BC) include mature oocyte cryopreservation following letrozole associated controlled ovarian hyperstimulation (Let-COH). To date, the impact of Let-COH on the follicular microenvironment has been poorly investigated, although a high androgen/estrogen ratio was previously associated with low oocyte quality. METHODS: In this prospective study, follicular fluid (FF) steroid levels (estradiol, testosterone, progesterone) and cumulus cell (CC) gene expression related to oocyte quality (HAS2, PTGS2, GREM1) were compared between 23 BC patients undergoing Let-COH for FP and 24 infertile patients undergoing conventional COH without letrozole. All patients underwent an antagonist COH cycle, and ovulation was triggered with hCG or GnRHa in both groups. RESULTS: FF estradiol levels were significantly lower while testosterone levels were significantly higher in the study group compared to controls irrespective of the trigger method. However, estradiol levels increased significantly with GnRHa triggering compared to hCG in the study group (median = 194.5 (95.4-438) vs 64.4 (43.8-152.4) ng/ml, respectively, p < 0.001), but not in the control group (median = 335.5 (177.5-466.7) vs 354 (179-511) ng/ml, respectively). After hCG trigger, Cumulus cell (CC) gene expression was lower in the study group compared to the control group, and difference was significant for PTGS2. Conversely, CC gene expression of PTGS2 and GREM1 was significantly higher in the study group compared to controls when ovulation was triggered with GnRHa. CONCLUSIONS: Let-COH triggered with hCG may negatively impact oocyte quality. However, ovulation triggering with GnRHa may improve the oocyte microenvironment and cumulus cell genes expression in Let-COH, suggesting a positive impact on oocyte quality in breast cancer patients. TRIAL REGISTRATION: Clinicaltrials.gov - NCT02661932 , registered 25 January 2016, retrospectively registered.

Folliculogenesis Is Not Fully Inhibited during GnRH Analogues Treatment in Mice Challenging Their Efficiency to Preserve the Ovarian Reserve during Chemotherapy in This Model.

As many chemotherapy regimens induce follicular depletion, fertility preservation became a major concern in young cancer patients. By maintaining follicles at the resting stage, gonadotropin-releasing hormone analogues (GnRHa) were proposed as an ovarian-protective option during chemotherapy. However, their efficacy and mechanisms of action remain to be elucidated. Mice were dosed with cyclophosphamide (Cy, 100-500 mg/kg i.p) to quantify follicular depletion and evaluate apoptosis at different times. We observed a dose-dependent depletion of the follicular reserve within 24 hours after Cy injection with a mean follicular loss of 45% at the dose of 200mg/kg. Apoptosis occurs in the granulosa cells of growing follicles within 12 hours after Cy treatment, while no apoptosis was detected in resting follicles suggesting that chemotherapy acutely affects both resting and growing follicles through different mechanisms. We further tested the ability of both GnRH agonist and antagonist to inhibit oestrus cycles, follicular growth and FSH secretion in mice and to protect ovarian reserve against chemotherapy. Although GnRHa were efficient to disrupt oestrus cycles, they failed to inhibit follicular development, irrespective of the doses and injection sites (sc or im). Around 20% of healthy growing follicles were still observed during GnRHa treatment and serum FSH levels were not reduced either by antagonist or agonist. GnRHa had no effect on Cy-induced follicular damages. Thus, we showed that GnRHa were not as efficient at inhibiting the pituitary-gonadal axis in mice as in human. Furthermore, the acute depletion of primordial follicles observed after chemotherapy does not support the hypothesis that the ovary may be protected by gonadotropin suppression.

Evaluation of quantitative polymerase chain reaction markers for the detection of breast cancer cells in ovarian tissue stored for fertility preservation.

OBJECTIVE: To develop molecular tools increasing the sensitivity of breast cancer micrometastases detection within ovarian tissue cryopreserved for fertility preservation. DESIGN: Expression of breast markers was evaluated by quantitative polymerase chain reaction in ovarian tissue from patients with benign or cancerous diseases. Suspected tissues were long-term xenografted into mice. SETTING: Academic research institute. PATIENT(S): Patients undergoing a fertility preservation procedure. INTERVENTION(S): Ovarian tissue was processed for RNA extraction and quantitative polymerase chain reaction analysis. Cryopreserved ovarian cortex from patients with breast cancer or benign disease was grafted for 6 months into severe combined immunodeficiency mice. MAIN OUTCOMES MEASURE(S): Predictive values of mammaglobin 1 (MGB-1), gross cystic disease fluid protein-15 (GCDFP-15), small breast epithelial mucine (SBEM), and mammaglobin 2 (MGB-2) to detect breast cancer cells in ovarian tissue, and the potential development of cancerous disease after xenograft of ovarian cortex from breast cancer patients. RESULT(S): MGB-1 and GCDFP-15 presented the highest predictive values to detect breast cancer micrometastases in the ovarian cortex, with an efficiency reaching 100% and 77%, respectively. The MGB-2 assay resulted in a high false-positive rate (47%) in the ovarian cortex but could be used to detect breast cancer cells in ovarian medulla. MGB-1 was detected in three of five ovarian cortex samples from early-stage breast cancer patients but not in the ovarian tissue from advanced breast cancer patients (none of 10). None of the mice grafted with ovarian tissue expressing these markers developed cancerous disease. CONCLUSION(S): MGB-1, GCDFP-15, and MGB-2 can serve as molecular markers for the detection of breast cancer micrometastases within the ovarian tissue of breast cancer patients. However, the clinical relevance of such a highly sensitive assay must be further investigated.

SOX9 regulates ERBB signalling in pancreatic cancer development.

OBJECTIVE: The transcription factor SOX9 was recently shown to stimulate ductal gene expression in pancreatic acinar-to-ductal metaplasia and to accelerate development of premalignant lesions preceding pancreatic ductal adenocarcinoma (PDAC). Here, we investigate how SOX9 operates in pancreatic tumourigenesis. DESIGN: We analysed genomic and transcriptomic data from surgically resected PDAC and extended the expression analysis to xenografts from PDAC samples and to PDAC cell lines. SOX9 expression was manipulated in human cell lines and mouse models developing PDAC. RESULTS: We found genetic aberrations in the SOX9 gene in about 15% of patient tumours. Most PDAC samples strongly express SOX9 protein, and SOX9 levels are higher in classical PDAC. This tumour subtype is associated with better patient outcome, and cell lines of this subtype respond to therapy targeting epidermal growth factor receptor (EGFR/ERBB1) signalling, a pathway essential for pancreatic tumourigenesis. In human PDAC, high expression of SOX9 correlates with expression of genes belonging to the ERBB pathway. In particular, ERBB2 expression in PDAC cell lines is stimulated by SOX9. Inactivating Sox9 expression in mice confirmed its role in PDAC initiation; it demonstrated that Sox9 stimulates expression of several members of the ERBB pathway and is required for ERBB signalling activity. CONCLUSIONS: By integrating data from patient samples and mouse models, we found that SOX9 regulates the ERBB pathway throughout pancreatic tumourigenesis. Our work opens perspectives for therapy targeting tumourigenic mechanisms.

Let-7b and miR-495 stimulate differentiation and prevent metaplasia of pancreatic acinar cells by repressing HNF6.

BACKGROUND & AIMS: Diseases of the exocrine pancreas are often associated with perturbed differentiation of acinar cells. MicroRNAs (miRNAs) regulate pancreas development, yet little is known about their contribution to acinar cell differentiation. We aimed to identify miRNAs that promote and control the maintenance of acinar differentiation. METHODS: We studied mice with pancreas- or acinar-specific inactivation of Dicer (Foxa3-Cre/Dicer(loxP/-) mice), combined (or not) with inactivation of hepatocyte nuclear factor (HNF) 6 (Foxa3-Cre/Dicer(loxP/-)/Hnf6-/- mice). The role of specific miRNAs in acinar differentiation was investigated by transfecting cultured cells with miRNA mimics or inhibitors. Pancreatitis-induced metaplasia was investigated in mice after administration of cerulein. RESULTS: Inhibition of miRNA synthesis in acini by inactivation of Dicer and pancreatitis-induced metaplasia were associated with repression of acinar differentiation and with induction of HNF6 and hepatic genes. The phenotype of Dicer-deficient acini depends on the induction of HNF6; overexpression of this factor in developing acinar cells is sufficient to repress acinar differentiation and to induce hepatic genes. Let-7b and miR-495 repress HNF6 and are expressed in developing acini. Their expression is inhibited in Dicer-deficient acini, as well as in pancreatitis-induced metaplasia. In addition, inhibiting let-7b and miR-495 in acinar cells results in similar effects to those found in Dicer-deficient acini and metaplastic cells, namely induction of HNF6 and hepatic genes and repression of acinar differentiation. CONCLUSIONS: Let-7b, miR-495, and their targets constitute a gene network that is required to establish and maintain pancreatic acinar cell differentiation. Additional studies of this network will increase our understanding of pancreatic diseases.

Role of the ductal transcription factors HNF6 and Sox9 in pancreatic acinar-to-ductal metaplasia.

OBJECTIVE: Growing evidence suggests that a phenotypic switch converting pancreatic acinar cells to duct-like cells can lead to pancreatic intraepithelial neoplasia and eventually to invasive pancreatic ductal adenocarcinoma. Histologically, the onset of this switch is characterised by the co-expression of acinar and ductal markers in acini, a lesion called acinar-to-ductal metaplasia (ADM). The transcriptional regulators required to initiate ADM are unknown, but need to be identified to characterise the regulatory networks that drive ADM. In this study, the role of the ductal transcription factors hepatocyte nuclear factor 6 (HNF6, also known as Onecut1) and SRY-related HMG box factor 9 (Sox9) in ADM was investigated. DESIGN: Expression of HNF6 and Sox9 was measured by immunostaining in normal and diseased human pancreas. The function of the factors was tested in cultured cells and in mouse models of ADM by a combination of gain and loss of function experiments. RESULTS: Expression of HNF6 and Sox9 was ectopically induced in acinar cells in human ADM as well as in mouse models of ADM. HNF6 and, to a lesser extent, Sox9 were required for repression of acinar genes, for modulation of ADM-associated changes in cell polarity and for activation of ductal genes in metaplastic acinar cells. CONCLUSIONS: HNF6 and Sox9 are new biomarkers of ADM and constitute candidate targets for preventive treatment in cases when ADM may lead to cancer. This work also shows that ectopic activation of transcription factors may underlie metaplastic processes occurring in other organs.