Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer.

The Hippo pathway effectors Yes-associated protein 1 (YAP) and its homolog TAZ are transcriptional coactivators that control gene expression by binding to TEA domain (TEAD) family transcription factors. The YAP/TAZ-TEAD complex is a key regulator of cancer-specific transcriptional programs, which promote tumor progression in diverse types of cancer, including breast cancer. Despite intensive efforts, the YAP/TAZ-TEAD complex in cancer has remained largely undruggable due to an incomplete mechanistic understanding. Here, we report that nuclear phosphoinositides function as cofactors that mediate the binding of YAP/TAZ to TEADs. The enzymatic products of phosphoinositide kinases PIPKIα and IPMK, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (P(I3,4,5)P3), bridge the binding of YAP/TAZ to TEAD. Inhibiting these kinases or the association of YAP/TAZ with PI(4,5)P2 and PI(3,4,5)P3 attenuates YAP/TAZ interaction with the TEADs, the expression of YAP/TAZ target genes, and breast cancer cell motility. Although we could not conclusively exclude the possibility that other enzymatic products of IPMK such as inositol phosphates play a role in the mechanism, our results point to a previously unrecognized role of nuclear phosphoinositide signaling in control of YAP/TAZ activity and implicate this pathway as a potential therapeutic target in YAP/TAZ-driven breast cancer.

Elinzanetant: a phase III therapy for postmenopausal patients with vasomotor symptoms.

INTRODUCTION: Menopausal vasomotor symptoms (VMS) are experienced by most women and are often debilitating and can last for years. While hormone replacement therapy is effective, it carries risks that have impacted its wider use, and it can be contraindicated. There is a large unmet need for a safe, effective non-hormonal therapy. AREAS COVERED: The importance of the neurokinin (NK) system in the hypothalamic regulation of the vasomotor center has become clear. NK antagonists, previously developed for other indications, have therefore been investigated for the treatment of VMS. Elinzanetant is a potent antagonist at both NK1 (endogenous ligand Substance P) and NK3 (neurokinin B) receptors, whereas other related drugs in development are selective NK3 antagonists. Elinzanetant has been investigated in 2 Phase II trials for menopausal VMS, demonstrating rapid onset and dose-dependant efficacy for the relief of VMS and improvement in quality of life for up to 12 weeks. Phase III trials are underway in women both with physiological menopause and after treatment for breast cancer. EXPERT OPINION: Elinzanetant is a very promising non-hormonal approach to a highly prevalent symptom constellation, with rapid onset and high efficacy. Wider indications are being explored in current Phase III trials.

Anti-Mullerian hormone attenuates both cyclophosphamide-induced damage and PI3K signalling activation, while rapamycin attenuates only PI3K signalling activation, in human ovarian cortex in vitro.

STUDY QUESTION: What are the effects of cyclophosphamide exposure on the human ovary and can anti-Mullerian hormone (AMH) and rapamycin protect against these? SUMMARY ANSWER: Exposure to cyclophosphamide compromises the health of primordial and transitional follicles in the human ovarian cortex and upregulates PI3K signalling, indicating both direct damage and increased follicular activation; AMH attenuates both of these chemotherapy-induced effects, while rapamycin attenuates only PI3K signalling upregulation. WHAT IS KNOWN ALREADY: Studies primarily in rodents demonstrate that cyclophosphamide causes direct damage to primordial follicles or that the primordial follicle pool is depleted primarily through excessive initiation of follicle growth. This increased follicular activation is mediated via upregulated PI3K signalling and/or reduced local levels of AMH production due to lost growing follicles. Furthermore, while rodent data show promise regarding the potential benefits of inhibitors/protectants alongside chemotherapy treatment to preserve female fertility, there is no information about the potential for this in humans. STUDY DESIGN, SIZE, DURATION: Fresh ovarian cortical biopsies were obtained from 17 healthy women aged 21-41 years (mean ± SD: 31.8 ± 4.9 years) at elective caesarean section. Biopsies were cut into small fragments and cultured for 24 h with either vehicle alone (DMSO), the active cyclophosphamide metabolite 4-hydroperoxycyclophosphamide (4-HC) alone, 4-HC + rapamycin or 4-HC+AMH. Two doses of 4-HC were investigated, 0.2 and 2 µM in separate experiments, using biopsies from seven women (aged 27-41) and six women (aged 21-34), respectively. Biopsies from four women (aged 28-38) were used to investigate the effect of rapamycin or AMH only. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological analysis of ovarian tissue was undertaken for follicle staging and health assessment. Western blotting and immunostaining were used to assess activation of PI3K signalling by measuring phosphorylation of AKT and phosphorylated FOXO3A staining intensity, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to either dose of 4-HC caused an increase in the proportion of unhealthy primordial (P < 0.0001, both doses) and transitional follicles (P < 0.01 for low dose and P < 0.01 for high dose) compared to vehicle. AMH significantly reduced follicle damage by approximately half in both of the investigated doses of 4-HC (P < 0.0001), while rapamycin had no protective effect on the health of the follicles. Culture with AMH or rapamycin alone had no effect on follicle health. Activation of PI3K signalling following 4-HC exposure was demonstrated by both Western blotting data showing that 4-HC increased in AKT phosphorylation and immunostaining showing increased phosphorylated FOXO3A staining of non-growing oocytes. Treatment with rapamycin reduced the activation of PI3K signalling in experiments with low doses of 4-HC while culture with AMH reduced PI3K activation (both AKT phosphorylation and phosphorylated FOXO3A staining intensity) across both doses investigated. LIMITATIONS, REASONS FOR CAUTION: These in vitro studies may not replicate in vivo exposures. Furthermore, longer experiment durations are needed to determine whether the effects observed translate into irreparable deficits of ovarian follicles. WIDER IMPLICATIONS OF THE FINDINGS: These data provide a solid foundation on which to explore the efficacy of AMH in protecting non-growing ovarian follicles from gonadotoxic chemotherapies. Future work will require consideration of the sustained effects of chemotherapy treatment and potential protectants to ensure these agents do not impair the developmental competence of oocytes or lead to the survival of oocytes with accumulated DNA damage, which could have adverse consequences for potential offspring. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from TENOVUS Scotland, the Academy of Medical Sciences (to R.R.), the Medical Research Council (G1100357 to R.A.A., MR/N022556/1 to the MRC Centre for Reproductive Health), and Merck Serono UK (to R.A.A.). R.R., H.L.S., N.S., and E.E.T. declare no conflicts of interest. R.A.A. reports grants and personal fees from Roche Diagnostics and Ferring Pharmaceuticals, and personal fees from IBSA and Merck outside the submitted work. TRIAL REGISTRATION NUMBER: N/A.

Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins.

Reactive oxygen species (ROS) are generated by aerobic metabolism, and their deleterious effects are buffered by the cellular antioxidant response, which prevents oxidative stress. The nuclear factor erythroid 2-related factor 2 (NRF2) is a master transcriptional regulator of the antioxidant response. Basal levels of NRF2 are kept low by ubiquitin-dependent degradation of NRF2 by E3 ligases, including the Kelch-like ECH-associated protein 1 (KEAP1). Here, we show that the stability and function of NRF2 is regulated by the type I phosphatidylinositol phosphate kinase g (PIPKIg), which binds NRF2 and transfers its product phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) to NRF2. PtdIns(4,5)P 2 binding recruits the small heat shock protein HSP27 to the complex. Silencing PIPKIg or HSP27 destabilizes NRF2, reduces expression of its target gene HO-1, and sensitizes cells to oxidative stress. These data demonstrate an unexpected role of phosphoinositides and HSP27 in regulating NRF2 and point to PIPKIg and HSP27 as drug targets to destabilize NRF2 in cancer. In brief: Phosphoinositides are coupled to NRF2 by PIPKIγ, and HSP27 is recruited and stabilizes NRF2, promoting stress-resistance.

An interactive resource of molecular signalling in the developing human haematopoietic stem cell niche.

The emergence of definitive human haematopoietic stem cells (HSCs) from Carnegie Stage (CS) 14 to CS17 in the aorta-gonad-mesonephros (AGM) region is a tightly regulated process. Previously, we conducted spatial transcriptomic analysis of the human AGM region at the end of this period (CS16/CS17) and identified secreted factors involved in HSC development. Here, we extend our analysis to investigate the progression of dorso-ventral polarised signalling around the dorsal aorta over the entire period of HSC emergence. Our results reveal a dramatic increase in ventral signalling complexity from the CS13-CS14 transition, coinciding with the first appearance of definitive HSCs. We further observe stage-specific changes in signalling up to CS17, which may underpin the step-wise maturation of HSCs described in the mouse model. The data-rich resource is also presented in an online interface enabling in silico analysis of molecular interactions between spatially defined domains of the AGM region. This resource will be of particular interest for researchers studying mechanisms underlying human HSC development as well as those developing in vitro methods for the generation of clinically relevant HSCs from pluripotent stem cells.

Reproduction in life and death: should cancer patients with a poor prognosis be offered fertility preservation interventions?

In the context of a cancer diagnosis, fertility preservation interventions are used to mitigate the potential impact of gonadotoxic cancer treatment upon fertility. They provide patients with cancer the option to freeze their reproductive material to have their own biological child following treatment. The evidence suggests some clinicians are less likely to have fertility preservation discussions with patients who have an aggressive or metastatic cancer which has a poor prognosis. Although this is contrary to current policy recommendations, there is a lack of guidance relating to offering fertility preservation in the context of a poor prognosis to support clinicians. Controversy surrounds posthumous reproduction, and whether the wishes of the cancer patient, when living and deceased should take precedence over others' wellbeing. We consider the question of whether cancer patients with a poor prognosis should be offered FP from an ethics perspective. We structure the paper around key arguments to which multiple ethical principles might pertain, first establishing a central argument in favour of offering fertility preservation based on respect for autonomy, before exploring counterarguments. We conclude by proposing that a defeasible assumption should be adopted in favour of offering fertility preservation to all cancer patients who might benefit from it. It is important to recognise that patients could benefit from fertility preservation in many ways, and these are not limited to having a parenting experience. The burden of proof rests on the clinician in collaboration with their multi-disciplinary team, to show that there are good grounds for withholding the offer.

Regulation of Cell Adhesion and Migration via Microtubule Cytoskeleton Organization, Cell Polarity, and Phosphoinositide Signaling.

The capacity for cancer cells to metastasize to distant organs depends on their ability to execute the carefully choreographed processes of cell adhesion and migration. As most human cancers are of epithelial origin (carcinoma), the transcriptional downregulation of adherent/tight junction proteins (e.g., E-cadherin, Claudin and Occludin) with the concomitant gain of adhesive and migratory phenotypes has been extensively studied. Most research and reviews on cell adhesion and migration focus on the actin cytoskeleton and its reorganization. However, metastasizing cancer cells undergo the extensive reorganization of their cytoskeletal system, specifically in originating/nucleation sites of microtubules and their orientation (e.g., from non-centrosomal to centrosomal microtubule organizing centers). The precise mechanisms by which the spatial and temporal reorganization of microtubules are linked functionally with the acquisition of an adhesive and migratory phenotype as epithelial cells reversibly transition into mesenchymal cells during metastasis remains poorly understood. In this Special Issue of "Molecular Mechanisms Underlying Cell Adhesion and Migration", we highlight cell adhesion and migration from the perspectives of microtubule cytoskeletal reorganization, cell polarity and phosphoinositide signaling.

Measuring ovarian toxicity in clinical trials: an American Society of Clinical Oncology research statement.

Anticancer agents can impair ovarian function, resulting in premature menopause and associated long-term health effects. Ovarian toxicity is not usually adequately assessed in trials of anticancer agents, leaving an important information gap for patients facing therapy choices. This American Society of Clinical Oncology (ASCO) statement provides information about the incorporation of ovarian toxicity measures in trial design. ASCO recommends: (1) measurement of ovarian toxicity in relevant clinical trials of anticancer agents that enrol post-pubertal, pre-menopausal patients; (2) collection of ovarian function measures at baseline and at 12-24 months after anticancer agent cessation, as a minimum, and later in line with the trial schedule; and (3) assessment of both clinical measures and biomarkers of ovarian function. ASCO recognises that routine measurement of ovarian toxicity and function in cancer clinical trials will add additional complexity and burden to trial resources but asserts that this issue is of such importance to patients that it cannot continue to be overlooked.

Regulation of Phosphoinositide Signaling by Scaffolds at Cytoplasmic Membranes.

Cytoplasmic phosphoinositides (PI) are critical regulators of the membrane-cytosol interface that control a myriad of cellular functions despite their low abundance among phospholipids. The metabolic cycle that generates different PI species is crucial to their regulatory role, controlling membrane dynamics, vesicular trafficking, signal transduction, and other key cellular events. The synthesis of phosphatidylinositol (3,4,5)-triphosphate (PI3,4,5P3) in the cytoplamic PI3K/Akt pathway is central to the life and death of a cell. This review will focus on the emerging evidence that scaffold proteins regulate the PI3K/Akt pathway in distinct membrane structures in response to diverse stimuli, challenging the belief that the plasma membrane is the predominant site for PI3k/Akt signaling. In addition, we will discuss how PIs regulate the recruitment of specific scaffolding complexes to membrane structures to coordinate vesicle formation, fusion, and reformation during autophagy as well as a novel lysosome repair pathway.

Ovarian stimulation and oocyte cryopreservation in females and transgender males aged 18 years or less: a systematic review.

Background: Fertility preservation is an important healthcare focus in the paediatric and adolescent population when gonadotoxic treatments are required. Ovarian stimulation (OS) resulting in oocyte cryopreservation is a well-established fertility preservation option in the adult population. It's utility, however, is little known in young patients. The purpose of this review was to synthesise the available literature on OS in patients ≤18 years old, to identify gaps in current research and provide suggestions for future research directions. Methods: Using PRISMA guidelines, a systematic review of the literature was performed for all relevant full-text articles published in English in Medline, Embase, the Cochrane Library and Google Scholar databases. The search strategy used a combination of subject headings and generic terms related to the study topic and population. Two reviewers independently screened studies for eligibility, extracted data and assessed the risk of bias. Characteristics of the studies, objectives and key findings were extracted and summarised in a narrative synthesis. Results: Database search and manual review identified 922 studies, 899 were eliminated based on defined exclusion criteria. Twenty-three studies were included and comprised 468 participants aged ≤18 years who underwent OS (median 15.2, range 7-18 years old). Only three patients were premenarchal, and four patients were on treatment to suppress puberty. Patients had OS for a broad range of indications including oncology treatment, transgender care and Turner syndrome. A total of 488 cycles of OS were completed, with all but 18 of these cycles (96.3%) successfully resulting in cryopreserved mature oocytes (median 10 oocytes, range 0-35). Fifty-three cycles (9.8%) were cancelled. Complications were rare (<1%). One pregnancy was reported from a female who had OS aged 17 years old. Conclusion: This systematic review demonstrates that OS and oocyte cryopreservation is achievable in young females however there are only a few cases in the literature describing OS in premenarcheal children or those who have suppressed puberty. There is little proof that OS can lead to pregnancy in adolescents, and no proof that this can be achieved in premenarchal girls. Therefore it should be regarded as an innovative procedure for adolescents and experimental for premenarcheal girls. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=265705, identifier CRD42021265705.

Fertility assessment and treatment in adolescent and young adult cancer survivors.

In the survivorship setting, adolescent and young adult (AYA) cancer survivors frequently demonstrate little knowledge of infertility risk, are unclear regarding their fertility status, and may under- or overestimate their treatment-related risk for infertility. In female AYA survivors, ovarian function usually parallels fertility, and can be assessed with serum hormone levels and ultrasonography. Posttreatment fertility preservation may be appropriate for survivors at risk for primary ovarian insufficiency. In male AYA survivors, fertility and gonadal function are not always equally affected, and can be assessed with a semen analysis and serum hormones, respectively. As reproductive health issues are commonly cited as an important concern by survivors of AYA cancer, multidisciplinary care teams including oncology, endocrinology, psychology, and reproductive medicine are advocated, with the aim of optimal provision of fertility advice and care for AYA cancer survivors.

Lipid transfer proteins initiate nuclear phosphoinositide signaling.

The membrane-localized phosphatidylinositol (PI) 3-kinase (PI3K)/Akt pathway regulates cell growth and is aberrantly activated in cancer. Recent studies reveal a distinct nuclear PI3K/Akt pathway involving PI phosphate (PIP) kinases that bind the tumor suppressor protein p53 (wild-type and mutant) to generate nuclear p53-polyphosphoinositide (PIP n ) complexes that activate Akt. In the membrane pathway, PI transfer proteins (PITPs) transport PI, the precursor of PIP n s, to endomembranes to enable PIP n synthesis. In contrast, nuclear PIP n signaling relies on poorly characterized non-membranous PIP n pools. Here we show that PITPs accumulate in the non-membranous nucleoplasm in response to stress and are necessary to generate nuclear PIP n pools. Class I PITPα/ß bind p53 to form p53-PIP n complexes that activate nuclear Akt in response to stress, which inhibits apoptosis. These findings demonstrate an unexpected function for PITPα/ß in nuclear PIP n signaling by generating membrane-free, protein-linked PIP n pools that are modified by PIP kinases/phosphatases to regulate protein function. In brief: Phosphatidylinositol transfer proteins initiate the nuclear protein-associated PIP n network in membrane-free regions.

Genes in loci genetically associated with polycystic ovary syndrome are dynamically expressed in human fetal gonadal, metabolic and brain tissues.

Background: Polycystic ovary syndrome (PCOS) is a heterogeneous disorder, affecting around 10% of women of reproductive age, with infertility, depression or anxiety, obesity, insulin resistance and type 2 diabetes as risk factors. The cause of PCOS is not known but there is a predisposition to developing PCOS in adult life that arises during fetal or perinatal life. PCOS also has a genetic predisposition and a number of genetic loci associated with PCOS have been identified. These loci contain 25 candidate genes which are currently being studied to define the syndrome. Although the name PCOS suggests a syndrome of the ovary, PCOS has also been associated with the central nervous system and other organ systems in the body due to the wide variety of symptoms it presents. Methods: Here, we examined the expression patterns of PCOS candidate genes in gonadal (ovary and testis), metabolic (heart, liver and kidney) and brain (brain and cerebellum) tissues during the first half of human fetal development and postnatally until adulthood using public RNA sequencing data. This study is an initial step for more comprehensive and translational studies to define PCOS. Results: We found that the genes were dynamically expressed in the fetal tissues studied. Some genes were significantly expressed in gonadal tissues, whilst others were expressed in metabolic or brain tissues at different time points prenatally and/or postnatally. HMGA2, FBN3 and TOX3 were highly expressed during the early stages of fetal development in all tissues but least during adulthood. Interestingly, correlation between expression of HMGA2/YAP1 and RAD50/YAP1 were significant in at least 5 of the 7 fetal tissues studied. Notably, DENND1A, THADA, MAPRE1, RAB5B, ARL14EP, KRR1, NEIL2 and RAD50 were dynamically expressed in all postnatal tissues studied. Conclusions: These findings suggest that these genes have tissue- or development-specific roles in multiple organs, possibly resulting in the various symptoms associated with PCOS. Thus the fetal origin of a predisposition to PCOS in adulthood could arise via the effects of PCOS candidate genes in the development of multiple organs.

Long-term follow-up to assess criteria for ovarian tissue cryopreservation for fertility preservation in young women and girls with cancer.

STUDY QUESTION: Do the Edinburgh Selection Criteria correctly identify female cancer patients under the age of 18 who are at risk of premature ovarian insufficiency (POI) as candidates for ovarian tissue cryopreservation (OTC)? SUMMARY ANSWER: Patient assessment using these criteria accurately identifies those at risk of POI, who can be offered OTC and future transplantation as a means of fertility preservation. WHAT IS KNOWN ALREADY: Treatment for childhood cancer can have adverse consequences on future fertility; at the time of diagnosis, fertility risk assessment should be undertaken in order to identify patients to whom fertility preservation should be offered. The Edinburgh selection criteria, based on planned cancer treatment and patient health status, are utilized to identify those at high risk and therefore eligible for OTC. However, this procedure is not without risk and there are few data on the efficacy of the procedure in prepubertal patients. As such, long-term follow-up of reproductive outcomes is necessary, to ensure that OTC is being offered appropriately. STUDY DESIGN, SIZE, DURATION: Cohort study encompassing all females diagnosed with cancer under the age of 18 in South East Scotland, from 1 January 1996 to 30 April 2020. Patients were followed up for reproductive outcomes to assess for diagnosis of POI. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 638 eligible patients were identified; patients under the age of 12 or deceased before the age of 12 were excluded from the study, leaving a study population of 431 patients. Electronic records were reviewed for reproductive function, assessed by current menstruation, pregnancy (in the absence of POI diagnosis), reproductive hormone measurements, pubertal progression, or diagnosis of POI. Patients on hormonal contraception (other than for treatment of POI or panhypopituitarism with no history of gonadatoxic treatment) were excluded from analysis (n = 9). Analysis on remaining 422 patients was carried out using the Kaplan-Meier methods, with POI as the defined event, and Cox proportional hazards model. MAIN RESULTS AND THE ROLE OF CHANCE: In the study population of 431 patients, median ages at diagnosis and analysis were 9.8 and 22.2 years, respectively. Reproductive outcomes were unavailable in 142 patients; the assumption was made that these patients did not have POI, but a subanalysis excluding these patients was also performed. Of the 422 patients aged >12 at analysis and not taking hormonal contraception, OTC was offered to 37 patients and successfully performed in 25 patients. Of the 37 patients offered OTC (one at time of relapse), nine (24.3%) developed POI. Of the 386 not offered OTC, 11 (2.9%) developed POI. The probability of developing POI was significantly higher in those offered OTC (hazard ratio [HR] 8.7 [95% CI 3.6-21]; P < 0.0001), even when those patients with unknown outcomes were excluded from the analysis (HR 8.1 [95% CI 3.4-20]; P < 0.001). All patients offered OTC who developed POI did so after treatment for primary disease; in those not offered OTC, five patients (45.5%) developed POI after treatment for disease relapse. LIMITATIONS, REASONS FOR CAUTION: A significant number of patients had unknown reproductive outcomes; many of these patients were engaged in ongoing follow-up but did not have documented reproductive assessment. This may have introduced bias to the analysis and highlights the need for reproductive follow-up as part of routine cancer aftercare. In addition, the relatively young age of the patient population and short duration of follow-up in some cases demonstrates the need for ongoing follow-up of this cohort. WIDER IMPLICATIONS OF THE FINDINGS: The prevalence of POI after childhood cancer is low, but the Edinburgh selection criteria remain a robust tool for selecting those at high risk at the time of diagnosis, to offer OTC appropriately. However, disease relapse necessitating more intensive treatments remains a challenge. This study additionally highlights the importance of routine assessment and documentation of reproductive status in haematology/oncology follow-up. STUDY FUNDING/COMPETING INTEREST(S): K.D. is supported by a CRUK grant (C157/A25193). This work was undertaken in part in the MRC Centre for Reproductive Health, (supported by MRC grant MR/N022556/1). R.A.A. has received consulting fees from Ferring and Roche Diagnostics; payment from Merck and IBSA for educational events; and laboratory materials from Roche Diagnostics. The other authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Granulocyte-colony stimulating factor does not prevent in vitro cisplatin-induced germ cell reduction in immature human and mouse testis.

BACKGROUND: Currently there are no established fertility preservation options for pre-pubertal boys facing cancer treatment. Granulocyte-colony stimulating factor (G-CSF) treatment has been proposed to be chemoprotective against spermatogonial cell loss in an alkylating chemotherapy model of busulfan treated adult mice. Having previously shown that exposure to the alkylating-like chemotherapy cisplatin resulted in a reduction in germ cell numbers in immature human testicular tissues, we here investigate whether G-CSF would prevent cisplatin-induced germ cell loss in immature human and mouse (fetal and pre-pubertal) testicular tissues. METHODS: Organotypic in vitro culture systems were utilised to determine the effects of clinically-relevant concentrations of G-CSF in cisplatin-exposed immature testicular tissues. Human fetal (n = 14 fetuses) and mouse pre-pubertal (n = 4 litters) testicular tissue pieces were cultured and exposed to cisplatin or vehicle control for 24 hrs and analysed at 72 and 240 hrs post-exposure. Combined G-CSF and cisplatin exposure groups explored varying concentrations and duration of G-CSF supplementation to the culture medium (including groups receiving G-CSF before, during and after cisplatin exposure). In addition, effects of G-CSF supplementation alone were investigated. Survival of total germ cell and sub-populations were identified by expression of AP2γ and MAGE-A4 for human gonocytes and (pre)spermatogonia, respectively, and MVH and PLZF, for mouse germ cells and putative spermatogonial stem cells (SSCs) respectively, were quantified. RESULTS: Exposure to cisplatin resulted in a reduced germ cell number in human fetal and mouse pre-pubertal testicular tissues at 240 hrs post-exposure. Germ cell number was not preserved by combined exposure with G-CSF using any of the exposure regimens (prior to, during or after cisplatin exposure). Continuous supplementation with G-CSF alone for 14 days did not change the germ cell composition in either human or mouse immature testicular tissues. CONCLUSIONS: This study demonstrates that exposure to G-CSF does not prevent cisplatin-induced germ cell loss in immature human and mouse testicular tissues in an in vitro system.

Impact of Anti-HER2 Therapy Alone and With Weekly Paclitaxel on the Ovarian Reserve of Young Women With HER2-Positive Breast Cancer.

BACKGROUND: The potential gonadotoxicity of anti-HER2 agents remains largely unknown, and limited, conflicting evidence exists for taxanes. Antimüllerian hormone (AMH) is an established biomarker of ovarian reserve that may aid in quantifying anticancer treatment-induced gonadotoxicity. PATIENTS AND METHODS: The present biomarker analysis of the randomized phase III neoadjuvant NeoALTTO trial included premenopausal women aged ≤45 years at diagnosis of HER2-positive early breast cancer with available frozen serum samples at baseline (ie, before anticancer treatments), at week 2 (ie, the "biological window" of anti-HER2 therapy alone), and/or at the time of surgery (ie, after completing paclitaxel + anti-HER2 therapy, before starting adjuvant chemotherapy). RESULTS: The present analysis included 130 patients with a median age of 38 years (interquartile ratio [IQR], age 33-42 years). AMH values at the 3 time points differed significantly (P<.001). At baseline, median AMH levels were 1.29 ng/mL (IQR, 0.56-2.62 ng/mL). At week 2, a small but significant reduction in AMH levels was observed (median, 1.10 ng/mL; IQR, 0.45-2.09 ng/mL; P<.001). At surgery, a larger significant decline in AMH levels was observed (median, 0.01 ng/mL; IQR, 0.01-0.03 ng/mL; P<.001). Although the type of anti-HER2 treatment (trastuzumab and/or lapatinib) did not seem to impact the results, age and pretreatment ovarian reserve had a major influence on treatment-induced gonadotoxicity risk. CONCLUSIONS: This NeoALTTO biomarker analysis showed that anti-HER2 therapies alone had limited gonadotoxicity but that the addition of weekly paclitaxel resulted in marked AMH decline with possible negative implications for subsequent ovarian function and fertility.

Potential ovarian toxicity and infertility risk following targeted anti-cancer therapies.

Unlike traditional chemotherapy agents which are generally cytotoxic to all cells, targeted anti-cancer therapies are designed to specifically target proliferation mechanisms in cancer cells but spare normal cells, resulting in high potency and reduced toxicity. There has therefore been a rapid increase in their development and use in clinical settings, including in curative-intent treatment regimens. However, the targets of some of these drugs including kinases, epigenetic regulatory proteins, DNA damage repair enzymes and proteasomes, have fundamental roles in governing normal ovarian physiology. Inhibiting their action could have significant consequences for ovarian function, with potentially long-lasting adverse effects which persist after cessation of treatment, but there is limited evidence of their effects on reproductive function. In this review, we will use literature that examines these pathways to infer the potential toxicity of targeted anti-cancer drugs on the ovary. Lay summary: Compared to traditional chemotherapy agents, anti-cancer therapies are thought to be highly effective at targeting cancer cells but sparing normal cells, resulting in reduced drug side effects. However, many of processes within the cells that these drugs affect are also important for the ovary to work normally, so suppressing them in this way could have long-lasting implications for female fertility. This review examines the potential toxicity of anti-cancer therapies on the ovary.

Cancer, Fertility and Me: Developing and Testing a Novel Fertility Preservation Patient Decision Aid to Support Women at Risk of Losing Their Fertility Because of Cancer Treatment.

Background: Women with a new cancer diagnosis face complex decisions about interventions aiming to preserve their fertility. Decision aids are more effective in supporting decision making than traditional information provision. We describe the development and field testing of a novel patient decision aid designed to support women to make fertility preservation treatment decisions around cancer diagnosis. Methods: A prospective, mixed-method, three stage study involving: 1) co-development of the resource in collaboration with a multi-disciplinary group of key stakeholders including oncology and fertility healthcare professionals and patient partners (n=24), 2) alpha testing with a group of cancer patients who had faced a fertility preservation treatment decision in the past (n=11), and oncology and fertility healthcare professionals and stakeholders (n=14) and, 3) beta testing with women in routine care who had received a recent diagnosis of cancer and were facing a fertility preservation treatment decision (n=41) and their oncology and fertility healthcare professionals (n=3). Ten service users recruited from a closed Breast Cancer Now Facebook group and the support group Cancer and Fertility UK also provided feedback on CFM via an online survey. Results: A 60-page paper prototype of the Cancer, Fertility and Me patient decision aid was initially developed. Alpha testing of the resource found that overall, it was acceptable to cancer patients, healthcare professionals and key stakeholders and it was considered a useful resource to support fertility preservation treatment decision-making. However, the healthcare professionals felt that the length of the patient decision aid, and elements of its content may be a barrier to its use. Subsequently, the prototype was reduced to 40 pages. During beta testing of the shortened version in routine care, women who received the resource described its positive impact on their ability to make fertility preservation decisions and support them at a stressful time. However, practical difficulties emerged which impacted upon its wider dissemination in clinical practice and limited some elements of the evaluation planned. Discussion: Women receiving the decision aid within the cancer treatment pathway found it helped them engage with decisions about fertility preservation, and make better informed, values-based care plans with oncology and fertility teams. More work is needed to address access and implementation of this resource as part of routine oncology care pathways.

A p53-phosphoinositide signalosome regulates nuclear AKT activation.

The tumour suppressor p53 and PI3K-AKT pathways have fundamental roles in the regulation of cell growth and apoptosis, and are frequently mutated in cancer. Here, we show that genotoxic stress induces nuclear AKT activation through a p53-dependent mechanism that is distinct from the canonical membrane-localized PI3K-AKT pathway. Following genotoxic stress, a nuclear PI3K binds p53 in the non-membranous nucleoplasm to generate a complex of p53 and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), which recruits AKT, PDK1 and mTORC2 to activate AKT and phosphorylate FOXO proteins, thereby inhibiting DNA damage-induced apoptosis. Wild-type p53 activates nuclear AKT in an on/off fashion following stress, whereas mutant p53 dose-dependently stimulates high basal AKT activity. The p53-PtdIns(3,4,5)P3 complex is dephosphorylated to p53-phosphatidylinositol 4,5-bisphosphate by PTEN to inhibit AKT activation. The nuclear p53-phosphoinositide signalosome is distinct from the canonical membrane-localized pathway and insensitive to PI3K inhibitors currently in the clinic, which underscores its therapeutic relevance.

Survival after breast cancer in women with a subsequent live birth: Influence of age at diagnosis and interval to subsequent pregnancy.

BACKGROUND: There remains a considerable concern among both patients and oncologists that having a live birth (LB) after breast cancer might adversely impact survival. METHODS: analysis of survival in a national cohort of women with breast cancer diagnosed at age 20-39 years between 1981 and 2017 (n = 5181), and subsequent LB using Scottish Cancer Registry and national maternity records. Cases had at least one subsequent LB, each was matched with up to six unexposed cases without subsequent LB, accounting for guaranteed time bias. RESULTS: In 290 women with a LB after diagnosis, overall survival was increased compared to those who did not have a subsequent LB, HR 0.65 (95%CI 0.50-0.85). Women with subsequent LB who had not had a pregnancy before breast cancer showed increased survival (HR 0.56, 0.38-0.82). There was a progressively greater interaction of subsequent LB with survival with younger age, thus for women aged 20-25 years, HR 0.30 (0.12-0.74) vs. those aged 36-39, HR 0.89 (0.42-1.87). In women with LB within five years of diagnosis, survival was also increased (HR 0.66; 0.49-0.89). Survival following LB was similar to unexposed women by ER status (both positive and negative) and in those known to have been exposed to chemotherapy. CONCLUSIONS: This analysis provides further evidence that for the growing number of women who wish to have children after breast cancer, LB does not have a negative impact on overall survival. This finding was confirmed within subgroups, including the youngest women and those not previously pregnant.