The αvß6 integrin is transferred intercellularly via exosomes.

Exosomes, cell-derived vesicles of endosomal origin, are continuously released in the extracellular environment and play a key role in intercellular crosstalk. In this study, we have investigated whether transfer of integrins through exosomes between prostate cancer (PrCa) cells occurs and whether transferred integrins promote cell adhesion and migration. Among others, we have focused on the αvß6 integrin, which is not detectable in normal human prostate but is highly expressed in human primary PrCa as well as murine PrCa in Pten(pc-/-) mice. After confirming the fidelity of the exosome preparations by electron microscopy, density gradient, and immunoblotting, we determined that the αvß6 integrin is actively packaged into exosomes isolated from PC3 and RWPE PrCa cell lines. We also demonstrate that αvß6 is efficiently transferred via exosomes from a donor cell to an αvß6-negative recipient cell and localizes to the cell surface. De novo αvß6 expression in an αvß6-negative recipient cell is not a result of a change in mRNA levels but is a consequence of exosome-mediated transfer of this integrin between different PrCa cells. Recipient cells incubated with exosomes containing αvß6 migrate on an αvß6 specific substrate, latency-associated peptide-TGFß, to a greater extent than cells treated with exosomes in which αvß6 is stably or transiently down-regulated by shRNA or siRNA, respectively. Overall, this study shows that exosomes from PrCa cells may contribute to a horizontal propagation of integrin-associated phenotypes, which would promote cell migration, and consequently, metastasis in a paracrine fashion.

Somatic mutation of GRIN2A in malignant melanoma results in loss of tumor suppressor activity via aberrant NMDAR complex formation.

The ionotropic glutamate receptors (N-methyl-D-aspartate receptors (NMDARs)) are composed of large complexes of multi-protein subunits creating ion channels in the cell plasma membranes that allow for influx or efflux of mono- or divalent cations (e.g., Ca(2+)) important for synaptic transmissions, cellular migration, and survival. Recently, we discovered the high prevalence of somatic mutations within one of the ionotropic glutamate receptors, GRIN2A, in malignant melanoma. Functional characterization of a subset of GRIN2A mutants demonstrated a loss of NMDAR complex formation between GRIN1 and GRIN2A, increased anchorage-independent growth in soft agar, and increased migration. Somatic mutation of GRIN2A results in a dominant negative effect inhibiting the tumor-suppressive phenotype of wild-type (WT) GRIN2A in melanoma. Depletion of endogenous GRIN2A in melanoma cells expressing WT GRIN2A resulted in increased proliferation compared with control. In contrast, short-hairpin RNA depletion of GRIN2A in mutant cell lines slightly reduced proliferation. Our data show that somatic mutation of GRIN2A results in increased survival, and we demonstrate the functional importance of GRIN2A mutations in melanoma and the significance that ionotropic glutamate receptor signaling has in malignant melanoma.

Integrin αvß6 promotes an osteolytic program in cancer cells by upregulating MMP2.

The molecular circuitries controlling osseous prostate metastasis are known to depend on the activity of multiple pathways, including integrin signaling. Here, we demonstrate that the αvß6 integrin is upregulated in human prostate cancer bone metastasis. In prostate cancer cells, this integrin is a functionally active receptor for fibronectin and latency-associated peptide-TGF-ß1; it mediates attachment and migration upon ligand binding and is localized in focal contacts. Given the propensity of prostate cancer cells to form bone metastatic lesions, we investigated whether the αvß6 integrin promotes this type of metastasis. We show for the first time that αvß6 selectively induces matrix metalloproteinase 2 (MMP2) in vitro in multiple prostate cancer cells and promotes osteolysis in vivo in an immunodeficient mouse model of bone metastasis through upregulation of MMP2, but not MMP9. The effect of αvß6 on MMP2 expression and activity is independent of androgen receptor in the analyzed prostate cancer cells. Increased levels of parathyroid hormone-related protein (PTHrP), known to induce osteoclastogenesis, were also observed in αvß6-expressing cells. However, by using MMP2 short hairpin RNA, we demonstrate that the αvß6 effect on bone loss is due to upregulation of soluble MMP2 by the cancer cells, not due to changes in tumor growth rate. Another related αv-containing integrin, αvß5, fails to show similar responses, underscoring the significance of αvß6 activity. Overall, these mechanistic studies establish that expression of a single integrin, αvß6, contributes to the cancer cell-mediated program of osteolysis by inducing matrix degradation through MMP2. Our results open new prospects for molecular therapy for metastatic bone disease.

Premature senescence and increased TGFß signaling in the absence of Tgif1.

Transforming growth factor ß (TGFß) signaling regulates cell cycle progression in several cell types, primarily by inducing a G1 cell cycle arrest. Tgif1 is a transcriptional corepressor that limits TGFß responsive gene expression. Here we demonstrate that primary mouse embryo fibroblasts (MEFs) lacking Tgif1 proliferate slowly, accumulate increased levels of DNA damage, and senesce prematurely. We also provide evidence that the effects of loss of Tgif1 on proliferation and senescence are not limited to primary cells. The increased DNA damage in Tgif1 null MEFs can be partially reversed by culturing cells at physiological oxygen levels, and growth in normoxic conditions also partially rescues the proliferation defect, suggesting that in the absence of Tgif1 primary MEFs are less able to cope with elevated levels of oxidative stress. Additionally, we show that Tgif1 null MEFs are more sensitive to TGFß-mediated growth inhibition, and that treatment with a TGFß receptor kinase inhibitor increases proliferation of Tgif1 null MEFs. Conversely, persistent treatment of wild type cells with low levels of TGFß slows proliferation and induces senescence, suggesting that TGFß signaling also contributes to cellular senescence. We suggest that in the absence of Tgif1, a persistent increase in TGFß responsive transcription and a reduced ability to deal with hyperoxic stress result in premature senescence in primary MEFs.

Inhibition of CtBP1 activity by Akt-mediated phosphorylation.

Pc2 (Cbx4) is a member of the chromobox family of polycomb proteins, and is a SUMO E3 ligase for the transcriptional corepressor CtBP1. Here, we show that both CtBP1 and Pc2 are phosphorylated by the kinase Akt1, which is activated by growth factor signaling via the PI3-kinase pathway. In the presence of Pc2, phosphorylation of CtBP1 is increased, and this requires interaction of both CtBP1 and Akt1 with Pc2. Pc2 promotes CtBP1 phosphorylation by recruiting Akt1 and, in part, by preventing de-phosphorylation of activated Akt1. Alteration of the Akt-phosphorylated residue in CtBP1 to a phosphomimetic results in decreased CtBP1 dimerization, but does not prevent interaction with other transcriptional regulators. The phosphomimetic mutant of CtBP1 is expressed at a lower level than the wild type protein, resulting in decreased transcriptional repression. We show that this CtBP1 mutant is targeted for poly-ubiquitylation and is less stable than the wild type protein. Co-expression of Pc2 and Akt1 results in both phosphorylation and ubiquitylation of CtBP1, thereby targeting CtBP1 for degradation. This work suggests that Pc2 might coordinate multiple enzymatic activities to regulate CtBP1 function.