Partial Sperm beta1 Integrin Subunit Deletion Proves Its Involvement in Mouse Gamete Adhesion/Fusion

We have previously shown, using antibodies, that the sperm alpha6beta1 integrin is involved in mouse gamete fusion in vitro. Here we report the conditional knockdown of the sperm Itgb1 gene. It induced a drastic failure of sperm fusogenic ability with sperm accumulation in the perivitelline space of in vitro inseminated oocytes deleted or not for the Itgb1 gene. These data demonstrate that sperm, but not oocyte, beta1 integrin subunit is involved in gamete adhesion/fusion. Curiously, knockdown males were fertile in vivo probably because of the incomplete Cre-mediated deletion of the sperm Itgb1 floxed gene. Indeed, this was shown by Western blot analysis and confirmed by both the viability and litter size of pups obtained by mating partially sperm Itgb1 deleted males with females producing completely deleted Itgb1 oocytes. Because of the total peri-implantation lethality of Itgb1 deletion in mice, we assume that sperm that escaped the Itgb1 excision seemed to be preferentially used to fertilize in vivo. Here, we showed for the first time that the deletion, even partial, of the sperm Itgb1 gene makes the sperm unable to normally fertilize oocytes. However, to elucidate the question of the essentiality of its role during fertilization, further investigations using a mouse expressing a recombinase more effective in male germ cells are necessary.

Calpain-2 regulates hypoxia/HIF-induced amoeboid reprogramming and metastasis

SummaryHypoxia, through hypoxia inducible factor (HIF), drives cancer cell invasion and metastatic progression in various cancer types, leading to poor prognosis. In epithelial cancer, hypoxia further induces the transition to amoeboid cancer cell dissemination, yet the molecular mechanisms, relevance for metastasis, and effective interventions to combat hypoxia-induced amoeboid reprogramming remain unclear. Here, we identify calpain-2 as key regulator and anti-metastasis target of hypoxia-induced transition from collective to amoeboid dissemination of breast and head and neck (HN) carcinoma cells. Hypoxia-induced amoeboid dissemination occurred through low ECM-adhesive, bleb-based amoeboid movement, which effectively invaded into 3D collagen with low-oxidative and -glycolytic energy metabolism, revealing an microenvironmentally-induced, energy-conserving dissemination route in epithelial cancers. Hypoxia-induced calpain-2 mediated amoeboid conversion by de-activating beta1 integrins, through enzymatic cleavage of the focal adhesion adaptor protein talin-1. Consequently, targeted downregulation of calpain-2 or pharmacological intervention restored talin-1 integrity, beta1 integrin engagement and reverted blebbing-amoeboid to elongated phenotypes under hypoxia. Calpain-2 activity was required for hypoxia-induced blebbing-amoeboid conversion in the orthotopic mouse dermis, and upregulated in invasive HN tumor xenografts in vivo, and attenuation of calpain activity prevented hypoxia-induced metastasis to the lungs. This identifies the calpain-2/talin-1/beta1 integrin axis as mechanosignaling program and promising intervention target of plasticity of cancer cell invasion and metastasis formation in epithelial cancers under hypoxia.

TMIC-07. SYMBIOTIC MACROPHAGE-GLIOMA CELL INTERACTIONS REVEAL SYNTHETIC LETHALITY IN PTEN NULL GLIOMA

Heterotypic interactions across diverse cell types of the tumor microenvironment (TME) can enable tumor progression and offer points for potential therapeutic intervention. Employing Glioblastoma Multiforme (GBM) as a model system, we sought to (i) understand the nature of specific oncogenic signals in glioma cells that affects immune cells, and (ii) identify immune cell factors which may support glioma growth and survival. Combined profiling and functional studies of glioma cells established that PTEN deficiency, a signature GBM alteration, specifically tracks with an immune profile dominated by tumor-associated macrophages (TAMs, a key type of immune cells in GBM which can constitute up to half of the tumor mass) via upregulation of lysyl oxidase (LOX). Mechanistically, the PTEN-SRC/AKT axis regulates YAP1, which directly upregulates LOX expression and secretion in glioma cells via binding to the LOX promoter. The secreted LOX from glioma cells functions as a potent macrophage chemoattractant to promote macrophage recruitment via activation of the beta1 integrin-PYK2 pathway in macrophages. Consequently, these infiltrating macrophages secrete SPP1 (secreted phosphoprotein 1) which sustains glioma cell survival and stimulates angiogenesis. In human and mouse PTEN-null GBM models, but not PTEN-intact GBM models, LOX inhibition markedly inhibits tumor growth and extends the survival of tumor-bearing mice by decreasing macrophage infiltration, impairing SPP1 expression and attenuating angiogenesis. In silico analysis of human GBM shows that increased YAP1-LOX and b1 integrin-SPP1 signaling positively correlates with higher macrophage density and lower overall survival. Together, the symbiotic glioma (PTEN-YAP1-LOX) and macrophage (beta1 integrin-PYK2-SPP1) interactions provide novel therapeutic targets for this intractable disease.