This study demonstrates for the first time that binding of calcium-activated calmodulin to a minimal interaction site within the disordered N-terminal domain is required for the essential Aurora-A activity in mitosis and in regulation of ciliary disassembly.

NEDD9 is an established marker of invasive and metastatic cancers. NEDD9 downregulation has been shown to dramatically reduce cell invasion and metastasis in multiple tumors. The mechanisms by which NEDD9 regulates invasion are largely unknown. In the current study, we have found that NEDD9 is required for MMP14 enzymatic recovery/recycling through the late endosomes to enable disengagement of tissue inhibitor of matrix metalloproteinase 2 (TIMP2) and tumor invasion. Depletion of NEDD9 decreases targeting of the MMP14/TIMP2 complex to late endosomes and increases trafficking of MMP14 from early/sorting endosomes back to the surface in a small GTPase Arf6-dependent manner. NEDD9 directly binds to Arf6-GAP, ARAP3, and Arf6 effector GGA3 thereby facilitating the Arf6 inactivation required for MMP14/TIMP2 targeting to late endosomes. Re-expression of NEDD9 or a decrease in Arf6 activity is sufficient to restore MMP14 activity and the invasive properties of tumor cells. Importantly, NEDD9 inhibition by Vivo-Morpholinos, an antisense therapy, decreases primary tumor growth and metastasis in xenograft models of breast cancer. Collectively, our findings uncover a novel mechanism to control tumor cells dissemination through NEDD9/Arf6-dependent regulation of MMP14/TIMP2 trafficking, and validates NEDD9 as a clinically relevant therapeutic target to treat metastatic cancer.

The scaffolding protein NEDD9 is an established pro-metastatic marker in several cancers. Nevertheless, the molecular mechanisms of NEDD9 driven metastasis in cancers remain ill defined. Here, using a comprehensive breast cancer (BCa) tissue microarray, it was show 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 BCa 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 (CTCs) 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. Re-expression of NEDD9 is sufficient to restore the activity of MMP14 and the invasive properties of BCa cells in vitro and in vivo. Collectively, these findings uncover critical steps in NEDD9-dependent invasion of BCa cells. Implications This study provides a mechanistic basis for potential therapeutic interventions to prevent metastasis.

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.

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. Additionally, 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 in order to impede the spread of disease and improve patient survival.

Tumor cell heterogeneity poses a major hurdle in the treatment of cancer. Mammary cancer stem-like cells (MaCSCs), or tumor-initiating cells, are highly tumorigenic sub-populations that have the potential to self-renew and to differentiate. These cells are clinically important, as they display therapeutic resistance and may contribute to treatment failure and recurrence, but the signaling axes relevant to the tumorigenic phenotype are poorly defined. The zinc-finger transcription factor Kruppel-like factor 4 (KLF4) is a pluripotency mediator that is enriched in MaCSCs. KLF4 promotes RAS-extracellular signal-regulated kinase pathway activity and tumor cell survival in triple-negative breast cancer (TNBC) cells. In this study, we found that both KLF4 and a downstream effector, microRNA-206 (miR-206), are selectively enriched in the MaCSC fractions of cultured human TNBC cell lines, as well as in the aldehyde dehydrogenase-high MaCSC sub-population of cells derived from xenografted human mammary carcinomas. The suppression of endogenous KLF4 or miR-206 activities abrogated cell survival and in vivo tumor initiation, despite having only subtle effects on MaCSC abundance. Using a combinatorial approach that included in silico as well as loss- and gain-of-function in vitro assays, we identified miR-206-mediated repression of the pro-apoptotic molecules programmed cell death 4 (PDCD4) and connexin 43 (CX43/GJA1). Depletion of either of these two miR-206-regulated transcripts promoted resistance to anoikis, a prominent feature of CSCs, but did not consistently alter MaCSC abundance. Consistent with increased levels of miR-206 in MaCSCs, the expression of both PDCD4 and CX43 was suppressed in these cells relative to control cells. These results identify miR-206 as an effector of KLF4-mediated prosurvival signaling in MaCSCs through repression of PDCD4 and CX43. Consequently, our study suggests that a pluripotency factor exerts prosurvival signaling in MaCSCs, and that antagonism of KLF4-miR-206 signaling may selectively target the MaCSC niche in TNBC.

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 induced 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. This preclinical data provides a new understanding of the mechanisms by which MLN8237 mediates its anti-metastatic effects and advocates for its combination with eribulin in future clinical trials for metastatic breast cancer and early stage solid tumors.

Photoreceptor cells are specialized neurons with a sensory cilium carrying an elaborate membrane structure, the outer segment (OS). Inherited mutations in genes involved in ciliogenesis frequently result in OS malformation and blindness. ADP-ribosylation factor-like 2 (ARL2) has recently been implicated in OS formation through its association with Binder of ARL2 (BART or ARL2BP), a protein linked to inherited blinding disease. To test the role of ARL2 in vision we created a transgenic mouse model expressing a tagged-dominant active form of human ARL2 (ARL2-Q70L) under a rod-specific promoter. Transgenic ARL2-Q70L animals exhibit reduced photoreceptor cell function as early as post-natal day 16 and progressive rod degeneration. We attribute loss of photoreceptor function to the defective OS morphogenesis in the ARL2-Q70L transgenic model. ARL2-Q70L expression results in shortened inner and outer segments, shortened and mislocalized axonemes and cytoplasmic accumulation of rhodopsin. In conclusion, we show that ARL2-Q70L is crucial for photoreceptor neuron sensory cilium development. Future research will expand upon our hypothesis that ARL2-Q70L mutant interferes with microtubule maintenance and tubulin regulation resulting in impaired growth of the axoneme and elaboration of the photoreceptor outer segment.