Altruistic Donation of Surplus Embryos to Known and Unknown Recipients, The Dutch Approach.

BACKGROUND: Previous studies have shown that embryo donation can be a successful treatment for infertile couples,
however the willingness of Dutch couples to donate or accept embryos was unknown. The aim of this article is to
describe the protocol and results for altruistic embryo donation of the only embryo bank in the Netherlands.
Materials and Methods: This is a descriptive study. Since 2011, donated cryo-embryos from couples that have undergone
in vitro-fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatments, are being stored in our embryo
bank. The majority of the donated embryos were frozen on day 3 or 4 by slow freezing techniques. We perform a
thorough medical and psychological screening of donor couples and recipients, according to the protocol drawn up in
close collaboration with the Dutch Ministry of Health.
Results: Up to June 2021, 54 women have received embryos from our embryo bank, all single embryo transfers.
While the clinical pregnancy rate in 'unknown' embryo donations was relatively high (25.3%), the live birth rate
shows limited success (12.6%), partly due to high pregnancy loss through miscarriage. In known donation procedures,
the recipients tend to undergo more procedures, depending on the number of donated cryo-embryos. Twentyeight
women received embryos from known donors, with a clinical pregnancy rate per embryo transfer of 24%, and
live birth rate of 14%. In total, 82 recipients were granted donated cryo-embryos, twenty had an ongoing pregnancy
(24.4%), nineteen of whom have given birth to a healthy child (23%).
Conclusion: Altruistic embryo donation of embryos appears to be satisfying for the donors, as they are not obliged to destroy
their embryos, but instead help others build a family. Although success rates are still limited, partly due to the relatively high
miscarriage rates and inferior freezing techniques, to this date nineteen out of 82 recipients have given birth to a healthy child.

Mammary gland-specific ablation of focal adhesion kinase reduces the incidence of p53-mediated mammary tumour formation.

BACKGROUND: Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneous mammary tumour development caused by loss of tumour suppressor genes such as p53 is lacking. Alterations in the tumour suppressor gene p53 have been implicated in over 50% of human breast cancers. Given that elevated FAK expression highly correlates with p53 mutation status in human breast cancer, we set out to investigate the importance of FAK in p53-mediated spontaneous mammary tumour development. METHODS: To directly assess the role of FAK, we generated mice with conditional inactivation of FAK and p53. We generated female p53(lox/lox)/FAK(+/+)/WapCre, p53(lox/lox)/FAK(flox/+)/WapCre and p53(lox/lox)/FAK(flox/-)/WapCre mice, and mice with WapCre-mediated conditional expression of p53(R270H), the mouse equivalent of human p53(R273H) hot spot mutation, together with conditional deletion of FAK, P53(R270H/+)/FAK(lox/+)/WapCre and p53(R270H/+)/FAK(flox/-)/WapCre mice. All mice were subjected to one pregnancy to induce WapCre-mediated deletion of p53 or expression of p53 R270H, and Fak genes flanked by two loxP sites, and subsequently followed the development of mammary tumours. RESULTS: Using this approach, we show that FAK is important for p53-induced mammary tumour development. In addition, mice with the mammary gland-specific conditional expression of p53 point mutation R270H, the mouse equivalent to human R273H, in combination with conditional deletion of Fak showed reduced incidence of p53(R270H)-induced mammary tumours. In both models these effects of FAK were related to reduced proliferation in preneoplastic lesions in the mammary gland ductal structures. CONCLUSIONS: Mammary gland-specific ablation of FAK hampers p53-regulated spontaneous mammary tumour formation. Focal adhesion kinase deletion reduced proliferative capacity of p53 null and p53(R270H) mammary epithelial cells but did not lead to increased apoptosis in vivo. Our data identify FAK as an important regulator in mammary epithelial cell proliferation in p53-mediated and p53(R270H)-induced mammary tumour development.

Annexin A2 depletion delays EGFR endocytic trafficking via cofilin activation and enhances EGFR signaling and metastasis formation.

Enhanced epidermal growth factor receptor (EGFR) activity has been strongly linked to breast cancer progression and mediators of EGFR endocytosis may well be involved. We developed a semi-automated high-content fluorescence microscopy-based EGFR endocytosis screen to identify proteins that mediate EGFR endocytosis in human HBL100 breast cancer cells. Knockdown of 172 individual endocytosis and actin-regulatory genes with small interfering RNAs led to the identification of 14 genes of which the contribution to EGFR endocytosis in breast cancer is until now poorly defined, including DNAJC6, GDI2, FGD6, HAX1, NECAP2 and AnxA2. We show that depletion of the actin and endocytosis regulatory protein annexin A2 (AnxA2) in a panel of four triple negative breast cancer (TNBC) cell lines affected EGFR endocytosis. Depletion of AnxA2 in the aggressive and highly metastatic MDA-MB-231 TNBC cell line resulted in the inhibition of EGFR transport beyond the early endosomes. This inhibition coincided with enhanced epidermal growth factor (EGF)-induced cell migration and downstream signaling via c-Jun N-terminal kinase (JNK) and Akt. Moreover, AnxA2 knockdown increased lung metastasis formation in mice. The effect of AnxA2 knockdown on EGFR endocytosis in MDA-MB-231 was related to dephosphorylation/activation of the actin-severing protein cofilin, as re-expression of an inactive S3E-cofilin mutant, but not an active S3A-cofilin mutant, re-established EGFR endocytosis to control levels. Together, our data provide evidence for AnxA2 as a mediator of EGFR endocytosis and signaling in breast cancer via regulation of cofilin activation.

Human death effector domain-associated factor interacts with the viral apoptosis agonist Apoptin and exerts tumor-preferential cell killing.

Apoptin, a protein from chicken anemia virus without an apparent cellular homologue, can induce apoptosis in mammalian cells. Its cytotoxicity is limited to transformed or tumor cells, making Apoptin a highly interesting candidate for cancer therapy. To elucidate Apoptin's mechanism of action, we have searched for binding partners in the human proteome. Here, we report that Apoptin interacts with DEDAF, a protein previously found to associate with death effector domain (DED)-containing pro-apoptotic proteins, and to be involved in regulation of transcription. Like Apoptin, after transient overexpression, DEDAF induced apoptosis in various human tumor cell lines, but not in primary fibroblasts or mesenchymal cells. DEDAF-induced cell death was inhibited by the caspase inhibitor p35. Together with the reported association of DEDAF with a DED-containing DNA-binding protein in the nucleus and the transcription regulatory activity, our findings may provide a clue for the mechanism of Apoptin's actions in mammalian cells.