Alternative splicing of ALCAM enables tunable regulation of cell-cell adhesion through differential proteolysis

Abstract

While many adhesion receptors are known to influence tumor progression, the mechanisms by which they dynamically regulate cell-cell adhesion remain elusive. We previously identified Activated Leukocyte Cell Adhesion Molecule (ALCAM) as a clinically relevant driver of metastasis and hypothesized that a tunable mechanism of ectodomain shedding regulates its contribution to dissemination. To test this hypothesis, we examined an under-explored ALCAM splice variant (ALCAM-Iso2) and demonstrated that loss of the membrane-proximal region of ALCAM (exon 13) increased metastasis four-fold. Mechanistic studies identified a novel MMP14-dependent membrane distal cleavage site in ALCAM-Iso2, which mediated a ten-fold increase in shedding, thereby decreasing cellular cohesion. Importantly, the loss of cohesion is not limited to the cell capable of shedding because the released extracellular domain diminished cohesion of non-shedding cells through disruption of ALCAM-ALCAM interactions. ALCAM-Iso2-dominated expression in bladder cancer tissue, compared to normal bladder, further emphasizes that ALCAM alternative splicing may contribute to clinical disease progression. The requirement for both the loss of exon 13 and the gain of metalloprotease activity suggests that ALCAM shedding and concomitant regulation of tumor cell adhesion is a locally tunable process.

Prognostic relevance of the Golgi mannosidase MAN1A1 in ovarian cancer: impact of N-glycosylation on tumour cell aggregation

“…Here, disruption of ALCAM-ALCAM interactions by blocking with a soluble recombinant ALCAM-Fc has been shown to reduce aggregation in different cell lines. 29 In our study, inhibition of α-mannosidases with kifunensine led to a dose-dependent mass shift of ALCAM in OvCa cells and an accumulation of this protein in the cytoplasm thereby potentially affecting its adhesive properties. The subcellular localisation of ALCAM has been previously described to impact the survival of OvCa patients.…”

Comparative proteomics analysis indicates that palmatine contributes to transepithelial migration by regulating cellular adhesion

“…Additionally, some other cellular adhesion molecules beyond those in AJs and TJs were upregulated in LPS-stimulated cells after their treatment with palmatine, including CD99, NGL1, SDC, CSPG, CNTNAP2 and ALCAM ( Figure 6), and the cell adhesion pathway was also activated ( Figure 5). These molecules often play a role in cell adhesion, migration and proliferation, which is especially true for CD99 and ALCAM, which are involved in leukocyte migration and adhesion (Pasello et al 2018), cell-cell interactions among the epithelia, and TEndoM (Hebron et al 2018), respectively. These results demonstrated that palmatine may increase cell adhesion and promote leukocyte migration during the inflammatory response in cells.…”

Negatively charged amino acids in the stalk region of membrane proteins reduce ectodomain shedding

“…Hebron et al (22) also previously reported the processing of ALCAM variant proteins. They showed that short ALCAM is more susceptible to processing than long ALCAM, as we show herein.…”

“…Because we could not detect this smaller fragment in the culture medium of our exon 13-knockout cells, which express mainly short ALCAM, we consider this discrepancy to be a result of differences in experimental conditions. We collected the culture media incubated with LPS-stimulated macrophage cells for 60 min, whereas Hebron et al (22) collected the culture media incubated with nonstimulated cancer cells for 24 h. Because ADAM17 is a stimulus-dependent enzyme (2, 18), ADAM17mediated shedding would be more prominent in our study. On the other hand, because the cancer cell line expressed high levels of MMP14 (23,24), MMP14-mediated processing might be emphasized in their study.…”