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.

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.

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.

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An increase in fibroblast growth factor-1 (FGF-1) is established as part of the cause of several important cancers including breast cancer, but the mechanisms by which it induces malignant behavior are not known. We now report that the protein 80K-H, a substrate for PKC, appears to be part of this mechanism and that it is increased in breast cancer and localizes to the nucleus as part of the mechanism. Our conclusion is based on an examination of a total of 58 biopsy specimens from human breast cancer patients for the presence of relationships between the 80K-H protein and the following: fibroblast growth factor receptor-1 (FGFR-1), tumor grade, microvessel counts (MVC), estrogen receptor (ER) and progesterone receptor (PgR) status. Based on histological grading and immunohistochemical (IHC) assays, we found strong direct relationships between 80K-H and FGFR-1 (r = 0.49, p = 0.003) and tumor grade (r = 0.42, p = 0.006). A trend for a direct relationship was observed with PgR (r=0.27, p=0.087). Notably, 80K-H immunostaining was largely limited to the epithelial cells of the mammary ducts. Subsequently, we studied the effects of FGF-1 on 80K-H in cultured human mammary carcinoma epithelial cells in order to establish a more direct relationship between these two molecules. We observed that FGF-1 treatment of MCF-7 cells stimulated translocation of 80K-H protein to the cell nucleus, as demonstrated by subcellular fractionation studies. Maximal intranuclear 80K-H was observed approximately 30 minutes following FGF-1 treatment. In addition, FGF-1 treatment of MCF-7 cells increased growth and invasion of MCF-7 cells, as demonstrated by cell proliferation and a modified Boyden chamber assay, respectively. Further support for 80K-H nuclearization was provided by the immunostaining of human breast cancer specimens and computer-assisted identification of a putative nuclear localization signal (NLS) near the amino terminus of 80K-H protein structure. These data support the existence of a previously unrecognized FGF-1/80K-H nuclear pathway in progression of human breast cancer and suggest that 80K-H may be useful for the assessment of breast tumor progression.