LPA signaling is regulated through the primary cilium: a novel target in glioblastoma.

The primary cilium is a ubiquitous organelle presented on most human cells. It is a crucial signaling hub for multiple pathways including growth factor and G-protein coupled receptors. Loss of primary cilia, observed in various cancers, has been shown to affect cell proliferation. Primary cilia formation is drastically decreased in glioblastoma (GBM), however, the role of cilia in normal astrocyte or glioblastoma proliferation has not been explored. Here, we report that loss of primary cilia in human astrocytes stimulates growth rate in a lysophosphatidic acid (LPA)-dependent manner. We show that lysophosphatidic acid receptor 1 (LPAR1) is accumulated in primary cilia. LPAR1 signaling through Gα12/Gαq was previously reported to be responsible for cancer cell proliferation. We found that in ciliated cells, Gα12 and Gαq are excluded from the cilium, creating a barrier against unlimited proliferation, one of the hallmarks of cancer. Upon loss of primary cilia, LPAR1 redistributes to the plasma membrane with a concomitant increase in LPAR1 association with Gα12 and Gαq. Inhibition of LPA signaling with the small molecule compound Ki16425 in deciliated highly proliferative astrocytes or glioblastoma patient-derived cells/xenografts drastically suppresses their growth both in vitro and in vivo. Moreover, Ki16425 brain delivery via PEG-PLGA nanoparticles inhibited tumor progression in an intracranial glioblastoma PDX model. Overall, our findings establish a novel mechanism by which primary cilium restricts proliferation and indicate that loss of primary cilia is sufficient to increase mitogenic signaling, and is important for the maintenance of a highly proliferative phenotype. Clinical application of LPA inhibitors may prove beneficial to restrict glioblastoma growth and ensure local control of disease.

Dual Targeting of Mesenchymal and Amoeboid Motility Hinders Metastatic Behavior.

Commonly upregulated in human cancers, the scaffolding protein NEDD9/HEF1 is a known regulator of mesenchymal migration and cancer cell plasticity. However, the functional role of NEDD9 as a regulator of different migration/invasion modes in the context of breast cancer metastasis is currently unknown. Here, it is reported that NEDD9 is necessary for both mesenchymal and amoeboid individual cell migration/invasion in triple-negative breast cancer (TNBC). NEDD9 deficiency results in acquisition of the amoeboid morphology, but severely limits all types of cell motility. Mechanistically, NEDD9 promotes mesenchymal migration via VAV2-dependent Rac1 activation, and depletion of VAV2 impairs the ability of NEDD9 to activate Rac1. In addition, NEDD9 supports a mesenchymal phenotype through stimulating polymerization of actin via promoting CTTN phosphorylation in an AURKA-dependent manner. Interestingly, an increase in RhoA activity in NEDD9-depleted cells does not facilitate a switch to functional amoeboid motility, indicating a role of NEDD9 in the regulation of downstream RhoA signaling effectors. Simultaneous depletion of NEDD9 or inhibition of AURKA in combination with inhibition of the amoeboid driver ROCK results in an additional decrease in cancer cell migration/invasion. Finally, we confirmed that a dual targeting strategy is a viable and efficient therapeutic approach to hinder the metastasis of breast cancer in xenograft models, showcasing the important need for further clinical evaluation of this regimen to impede the spread of disease and improve patient survival.Implications: This study provides new insight into the therapeutic benefit of combining NEDD9 depletion with ROCK inhibition to reduce tumor cell dissemination and discovers a new regulatory role of NEDD9 in the modulation of VAV2-dependent activation of Rac1 and actin polymerization. Mol Cancer Res; 15(6); 670-82. ©2017 AACR.

Combination of Eribulin and Aurora A Inhibitor MLN8237 Prevents Metastatic Colonization and Induces Cytotoxic Autophagy in Breast Cancer.

Recent findings suggest that the inhibition of Aurora A (AURKA) kinase may offer a novel treatment strategy against metastatic cancers. In the current study, we determined the effects of AURKA inhibition by the small molecule inhibitor MLN8237 both as a monotherapy and in combination with the microtubule-targeting drug eribulin on different stages of metastasis in triple-negative breast cancer (TNBC) and defined the potential mechanism of its action. MLN8237 as a single agent and in combination with eribulin affected multiple steps in the metastatic process, including migration, attachment, and proliferation in distant organs, resulting in suppression of metastatic colonization and recurrence of cancer. Eribulin application induces accumulation of active AURKA in TNBC cells, providing foundation for the combination therapy. Mechanistically, AURKA inhibition induces cytotoxic autophagy via activation of the LC3B/p62 axis and inhibition of pAKT, leading to eradication of metastases, but has no effect on growth of mammary tumor. Combination of MLN8237 with eribulin leads to a synergistic increase in apoptosis in mammary tumors, as well as cytotoxic autophagy in metastases. These preclinical data provide a new understanding of the mechanisms by which MLN8237 mediates its antimetastatic effects and advocates for its combination with eribulin in future clinical trials for metastatic breast cancer and early-stage solid tumors. Mol Cancer Ther; 15(8); 1809-22. ©2016 AACR.

Prometastatic NEDD9 Regulates Individual Cell Migration via Caveolin-1-Dependent Trafficking of Integrins.

UNLABELLED: The dissemination of tumor cells relies on efficient cell adhesion and migration, which in turn depends upon endocytic trafficking of integrins. In the current work, it was found that depletion of the prometastatic protein, NEDD9, in breast cancer cells results in a significant decrease in individual cell migration due to impaired trafficking of ligand-bound integrins. NEDD9 deficiency does not affect the expression or internalization of integrins but heightens caveolae-dependent trafficking of ligand-bound integrins to early endosomes. Increase in mobility of ligand-bound integrins is concomitant with an increase in tyrosine phosphorylation of caveolin-1 (CAV1) and volume of CAV1-vesicles. NEDD9 directly binds to CAV1 and colocalizes within CAV1 vesicles. In the absence of NEDD9, the trafficking of ligand-bound integrins from early to late endosomes is impaired, resulting in a significant decrease in degradation of ligand-integrin complexes and an increase in recycling of ligand-bound integrins from early endosomes back to the plasma membrane without ligand disengagement, thus leading to low adhesion and migration. Reexpression of NEDD9 or decrease in the amount of active, tyrosine 14 phosphorylated (Tyr14) CAV1 in NEDD9-depleted cells rescues the integrin trafficking deficiency and restores cellular adhesion and migration capacity. Collectively, these findings indicate that NEDD9 orchestrates trafficking of ligand-bound integrins through the attenuation of CAV1 activity. IMPLICATIONS: This study provides valuable new insight into the potential therapeutic benefit of NEDD9 depletion to reduce dissemination of tumor cells and discovers a new regulatory role of NEDD9 in promoting migration through modulation of CAV1-dependent trafficking of integrins.

NEDD9 depletion leads to MMP14 inactivation by TIMP2 and prevents invasion and metastasis.

UNLABELLED: The scaffolding protein NEDD9 is an established prometastatic marker in several cancers. Nevertheless, the molecular mechanisms of NEDD9-driven metastasis in cancers remain ill-defined. Here, using a comprehensive breast cancer tissue microarray, it was shown that increased levels of NEDD9 protein significantly correlated with the transition from carcinoma in situ to invasive carcinoma. Similarly, it was shown that NEDD9 overexpression is a hallmark of highly invasive breast cancer cells. Moreover, NEDD9 expression is crucial for the protease-dependent mesenchymal invasion of cancer cells at the primary site but not at the metastatic site. Depletion of NEDD9 is sufficient to suppress invasion of tumor cells in vitro and in vivo, leading to decreased circulating tumor cells and lung metastases in xenograft models. Mechanistically, NEDD9 localized to invasive pseudopods and was required for local matrix degradation. Depletion of NEDD9 impaired invasion of cancer cells through inactivation of membrane-bound matrix metalloproteinase MMP14 by excess TIMP2 on the cell surface. Inactivation of MMP14 is accompanied by reduced collagenolytic activity of soluble metalloproteinases MMP2 and MMP9. Reexpression of NEDD9 is sufficient to restore the activity of MMP14 and the invasive properties of breast cancer cells in vitro and in vivo. Collectively, these findings uncover critical steps in NEDD9-dependent invasion of breast cancer cells. IMPLICATIONS: This study provides a mechanistic basis for potential therapeutic interventions to prevent metastasis.