The glycolytic phenotype of the Warburg effect is associated with acidification of the tumor microenvironment. In this review, we describe how acidification of the tumor microenvironment may increase the invasive and degradative phenotype of cancer cells. As a template of an extracellular acidic microenvironment that is linked to proteolysis, we use the resorptive pit formed between osteoclasts and bone. We describe similar changes that have been observed in cancer cells in response to an acidic microenvironment and that are associated with proteolysis and invasive and metastatic phenotypes. This includes consideration of changes observed in the intracellular trafficking of vesicles, i.e., lysosomes and exosomes, and in specialized regions of the membrane, i.e., invadopodia and caveolae. Cancer-associated cells are known to affect what is generally referred to as tumor proteolysis but little direct evidence for this being regulated by acidosis; we describe potential links that should be verified.

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Accumulating evidence indicates that the endometrial extracellular matrix (ECM) modulates trophoblast adhesion during mouse blastocyst implantation. In previous studies of adhesion-competent mouse blastocysts, we have demonstrated that integrin-mediated fibronectin (FN)-binding activity on the apical surface of trophoblast cells is initially low, but becomes strengthened after embryos are exposed to FN. In the present study, we have examined whether the ligand-induced upregulation of trophoblast adhesion to FN is mediated by integrin signaling. The strengthening of adhesion to FN required integrin ligation, which rapidly elevated cytoplasmic-free Ca(2+). Chelation of intracellular Ca(2+) using BAPTA-AM, or inhibition of the Ca(2+)-dependent proteins, protein kinase C or calmodulin, significantly attenuated the effect of FN on binding activity. Furthermore, direct elevation of cytoplasmic Ca(2+) levels with ionomycin upregulated FN-binding activity, demonstrating that Ca(2+) signaling is required and sufficient for strong adhesion to FN. Ca(2+) signaling may induce protein trafficking, a known requirement for ligand-induced upregulation of FN-binding activity. Indeed, intracellular vesicles accumulated in adhesion-competent blastocysts, but were absent after exposure to either FN or ionomycin. These findings suggest that, during implantation, contact between peri-implantation blastocysts and FN elevates intracellular Ca(2+), which strengthens trophoblast adhesion to ECM through protein redistribution.

Integrin signaling modulates trophoblast adhesion to extracellular matrices during blastocyst implantation. Fibronectin (FN)-binding activity on the apical surface of trophoblast cells is strengthened after elevation of intracellular Ca(2+) downstream of integrin ligation by FN. We report here that phosphoinositide-specific phospholipase C (PLC) mediates Ca(2+) signaling in response to FN. Pharmacological agents used to antagonize PLC (U73122) or the inositol phosphate receptor (Xestospongin C) inhibited FN-induced elevation of intracellular Ca(2+) and prevented the upregulation of FN-binding activity. In contrast, inhibitors of Ca(2+) influx through either voltage-gated or non-voltage-gated Ca(2+) channels were without effect. Inhibition of protein tyrosine kinase activity by genistein, but not G-protein inhibition by suramin, blocked FN-induced intracellular Ca(2+) signaling and upregulation of adhesion, consistent with involvement of PLC-gamma. Confocal immunofluorescence imaging of peri-implantation blastocysts demonstrated that PLC-gamma2, but not PLC-gamma1 nor PLC-beta1, accumulated near the outer surface of the embryo. Phosphotyrosine site-directed antibodies revealed phosphorylation of PLC-gamma2, but not PLC-gamma1, upon integrin ligation by FN. These data suggest that integrin-mediated activation of PLC-gamma to initiate phosphoinositide signaling and intracellular Ca(2+) mobilization is required for blastocyst adhesion to FN. Signaling cascades regulating PLC-gamma could, therefore, control a critical feature of trophoblast differentiation during peri-implantation development.

Trophoblast cells of the peri-implantation blastocyst differentiate from a polarized epithelium, the trophectoderm, into invasive cells having an apical surface occupied by integrins that mediate adhesion to the extracellular matrix. Blastocyst differentiation was assessed during serum-free culture using a fibronectin binding assay with intact mouse blastocysts. Fibronectin binding activity became elevated during a 24-h "window" after approximately 72 h of culture. Blastocyst differentiation was unaffected by transcriptional inhibition with alpha-amanitin, however, exposure of cavitating morulae to the drug significantly delayed the onset of maximal fibronectin-binding activity. Inhibition of de novo protein synthesis with cycloheximide delayed development only when added during the first 24 h of blastocyst culture, indicating that proteins required for adhesion to fibronectin were synthesized at least 24 h before blastocyst differentiation was completed. Since blastocyst differentiation did not appear to be regulated temporally by gene expression, the possible role of protein trafficking was investigated using the inhibitor, brefeldin A. Brefeldin A caused a reversible, dose-dependent decrease in fibronectin-binding activity when added to the culture medium between 48 and 72 h of culture. During the period of brefeldin A sensitivity, alpha 5 beta 1 integrin, a major fibronectin receptor, translocated to the apical surface of trophoblast cells, as determined by immunohistochemistry and confocal microscopy. Mouse blastocysts expressed other integrins that recognize the central cell-binding domain of fibronectin, including the alpha v integrins and alpha llb beta 3, but not alpha4 which recognizes the lllCS site. Trafficking of alpha 5 beta 1, and possibly other integrins, to the apical surface of trophoblast cells appears to be a critical step in the differentiation of the mouse blastocyst to an invasive phenotype.