Malignant breast cancer cells that have entered the blood circulation from primary mammary fat pad tumors or are grown in end-over-end suspension culture assemble a characteristic, multi-globular polymeric fibronectin (polyFn) coat on their surfaces. Surface polyFn is critical for pulmonary metastasis, presumably by facilitating lung vascular arrest via endothelial dipeptidylpeptidase IV (CD26). Here, we show that cell-surface polyFn assembly is initiated by the state of suspension, is dependent upon the synthesis and secretion of cellular Fn, and is augmented in a dose-and time-dependent manner by plasma Fn. PolyFn assembly is regulated by protein kinase C⑀ (PKC⑀), which translocates rapidly and in increasing amounts from the cytosol to the plasma membrane and is phosphorylated. PolyFn assembly is impeded by select inhibitors of this kinase, i.e. bisindolylmaleimide I, Ro-32-0432, Gö6983, and Rottlerin, by the phorbol 12-myristate 13-acetate-mediated and time-dependent loss of PKC⑀ protein and decreased plasma membrane translocation, and more specifically, by stable transfection of lung-metastatic MTF7L breast cancer cells with small interfering RNA-PKC⑀ and dominant-negative PKC⑀ constructs (e.g. RD-PKC⑀). The inability to assemble a cell surface-associated polyFn coat by knockdown of endogenous Fn or PKC⑀ impedes cancer cells from metastasis to the lungs. The present studies identify a novel regulatory mechanism for polyFn assembly on blood-borne breast cancer cells and depict its effect on pulmonary metastasis.
Fibronectin (Fn) plays an important part in the branching morphogenesis of salivary gland, lung, and kidney. Here, we examine the effect of the conditional knockout of Fn in the mammary epithelium [FnMEp−/−] on postnatal mammary gland development, using Cre-loxP mediated gene knockout technology. Our data show that Fn deletion causes a moderate retardation in outgrowth and branching of the ductal tree in 5-week old mice. These defects are partially compensated in virgin 16-week old mice. However, mammary glands consisting of Fn-deficient epithelial cells fail to undergo normal lobuloalveolar differentiation during pregnancy. The severity of lobuloalveolar impairment ranged from lobular hypoplasia to aplasia in some cases and was associated with the amount of Fn protein recovered from these glands. Decreased rates of mammary epithelial cell proliferation accounted for delayed ductal outgrowth in virgin and lack of alveologenesis in pregnant FnMEp−/− mice. Concomitant decreased expression of integrin β1 (Itgb1) and lack of autophosphorylation of focal adhesion kinase (Fak) suggest that this pathology might, at least in part, be mediated by disruption of the Fn/Itgb1/Fak signaling pathway.
BackgroundHec1 (NDC80) is an integral part of the kinetochore and is overexpressed in a variety of human cancers, making it an attractive molecular target for the design of novel anticancer therapeutics. A highly potent first-in-class compound targeting Hec1, TAI-1, was identified and is characterized in this study to determine its potential as an anticancer agent for clinical utility.MethodsThe in vitro potency, cancer cell specificity, synergy activity, and markers for response of TAI-1 were evaluated with cell lines. Mechanism of action was confirmed with western blotting and immunofluorescent staining. The in vivo potency of TAI-1 was evaluated in three xenograft models in mice. Preliminary toxicity was evaluated in mice. Specificity to the target was tested with a kinase panel. Cardiac safety was evaluated with hERG assay. Clinical correlation was performed with human gene database.ResultsTAI-1 showed strong potency across a broad spectrum of tumor cells. TAI-1 disrupted Hec1-Nek2 protein interaction, led to Nek2 degradation, induced significant chromosomal misalignment in metaphase, and induced apoptotic cell death. TAI-1 was effective orally in in vivo animal models of triple negative breast cancer, colon cancer and liver cancer. Preliminary toxicity shows no effect on the body weights, organ weights, and blood indices at efficacious doses. TAI-1 shows high specificity to cancer cells and to target and had no effect on the cardiac channel hERG. TAI-1 is synergistic with doxorubicin, topotecan and paclitaxel in leukemia, breast and liver cancer cells. Sensitivity to TAI-1 was associated with the status of RB and P53 gene. Knockdown of RB and P53 in cancer cells increased sensitivity to TAI-1. Hec1-overexpressing molecular subtypes of human lung cancer were identified.ConclusionsThe excellent potency, safety and synergistic profiles of this potent first-in-class Hec1-targeted small molecule TAI-1 show its potential for clinically utility in anti-cancer treatment regimens.
A series of 4-aryl-N-arylcarbonyl-2-aminothiazoles of scaffold 4 was designed and synthesized as Hec1/Nek2 inhibitors. Structural optimization of 4 led to compound 32 bearing C-4' 4-methoxyphenoxy and 4-(o-fluoropyridyl)carbonyl groups that showed low nanomolar in vitro antiproliferative activity (IC50: 16.3-42.7 nM), high intravenous AUC (64.9 μM·h, 2.0 mg/kg) in SD rats, and significant in vivo antitumor activity (T/C = 32%, 20 mg/kg, IV) in mice bearing human MDA-MB-231 xenografts. Cell responses resulting from Hec1/Nek2 inhibition were observed in cells treated with 32, including a reduced level of Hec1 coimmunoprecipitated with Nek2, degradation of Nek2, mitotic abnormalities, and apoptosis. Compound 32 showed selectivity toward cancer cells over normal phenotype cells and was inactive in a [(3)H]astemizole competitive binding assay for hERG liability screening. Therefore, 32 is as a good lead toward the discovery of a preclinical candidate targeting Hec1/Nek2 interaction.
Current cytotoxic chemotherapy produces clinical benefit in patients with breast cancer but the survival impact is modest. To explore novel cytotoxic agents for the treatment of advanced disease, we have characterized a new and pharmacokinetically improved Hec1-targeted compound, TAI-95. Nine of 11 breast cancer cell lines tested were sensitive to nanomolar levels of TAI-95 (GI 50 ¼ 14.29-73.65 nmol/L), and more importantly, TAI-95 was active on a number of cell lines that were resistant (GI 50 > 10 mmol/L) to other established cytotoxic agents. TAI-95 demonstrates strong inhibition of in vivo tumor growth of breast cancer model when administered orally, without inducing weight loss or other obvious toxicity. Mechanistically, TAI-95 acts by disrupting the interaction between Hec1 and Nek2, leading to apoptotic cell death in breast cancer cells. Furthermore, TAI-95 is active on multidrug-resistant (MDR) cell lines and led to downregulation of the expression of P-glycoprotein (Pgp), an MDR gene. In addition, TAI-95 increased the potency of cytotoxic Pgp substrates, including doxorubicin and topotecan. Certain clinical subtypes of breast cancer more likely to respond to Hec1-targeted therapy were identified and these subtypes are the ones associated with poor prognosis. This study highlights the potential of the novel anticancer compound TAI-95 in difficult-to-treat breast cancers.
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