Kinetochore-associated Complex Research Articles

Knockdown of SKA1 gene inhibits cell proliferation and metastasis in human adenoid cystic carcinoma

The spindle and kinetochore-associated complex subunit 1(SKA1) is a newly discovered gene, which has been associated with mitosis and tumorigenesis. However, its role insalivary adenoid cystic carcinoma (SACC) is still unknown, and the invasive and metastatic mechanism in SACC is still unclear. To explore the molecular mechanism of SKA1 in the process of malignant proliferation and metastasis in adenoid cystic cancer (ACC) cells, we employed lentivirus-mediated short hairpin RNA to knockdown SKA1 in SACC-83 cells. The results demonstrated that the lentivirus-mediated shRNA-targeting SKA1 lead to a significant down-regulation of SKA1 expression. Knockdown of SKA1 inhibited cell proliferation, cell invasion, migration and the cell cycle arrest. Furthermore, knockdown of SKA1 reduced the Ndc80, CDK4, Cyclin D1, Cyclin E1, Cyclin B1 and matrix metalloproteinase-9 (MMP-9) protein expression, but increased the p27 protein expression. These findings indicated that SKA1 might be a promising target for cancer gene therapy in human ACC.

Abstract 1922: miR-301 expression is deregulated in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma of myogenic derivation. Conversely to normal myoblasts, RMS cells are unable to differentiate, thus they proliferate indefinitely. MicroRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression by affecting both the stability and translation of mRNAs. They are fundamental regulators of myogenesis and their deregulation has been involved in the pathogenesis of RMS. We evaluate here the expression and regulation of miR-301, which has been related to tumorigenesis in other types of cancers. We report here that the expression of miR-301 is upregulated in primary samples of both the alveolar RMS, which bears the fusion protein PAX3-FOXO1 (P3F) (ARMS), and the fusion-negative embryonal RMS (ERMS) compared to normal muscle tissues. Similarly, it appears higher in RMS cell lines versus normal skeletal myoblasts. Using an inhibitor of miR-301 (CONTRAmiR) we demonstrate that this miRNA favors RMS cell mobility and down-regulates p21Cip1, PTEN and DICER1 levels. However, the inhibition of miR-301 with the CONTRAmiR does not reduce tumor cell proliferation suggesting that the regulation of cell cycle is not under the control of this miRNA in RMS. Interestingly, the expression of SKA2 (Spindle And Kinetochore Associated Complex Subunit 2), whose gene hosts miR-301, is also upregulated in our setting. Experiments are ongoing to determine whether miR-301 and SKA2 are associated with RMS pathogenesis and could be targets for an anti-RMS therapy. This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC). Citation Format: Rossella Rota, Alberto Gualtieri, Cristina Cossetti, Silvia Pomella, Laura Adesso, Franco Locatelli. miR-301 expression is deregulated in rhabdomyosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1922.

SKA1 regulates the metastasis and cisplatin resistance of non-small cell lung cancer.

Currently, chemotherapy with platinum-based drugs including cisplatin is the most effective therapy for the treatment of non-small cell lung carcinoma (NSCLC). However, the efficacy of chemotherapy is limited due to commonly developed drug resistance. Spindle and kinetochore-associated complex subunit 1 (SKA1) is part of a complex essential for stabilizing the attachment of spindle microtubules to kinetochores and for maintaining the metaphase plate during mitosis. In the present study, we aimed to investigate the role of SKA1 in the process of metastasis and drug resistance of NSCLC. We completed a series of experiments to investigate the function of SKA1 in NSCLC metastasis and drug resistance including qRT-PCR, immunohistochemistry and western blotting, as well as MTT, BrdU, wounded healing, Transwell and gelatin zymography assays. We demonstrated that the expression levels of SKA1 were elevated in NSCLC and were correlated with cancer progression and malignancy. We also reported that SKA1 positively regulated the proliferation and metastatic ability of NSCLC cells. In addition, we determined that SKA1 contributed to cisplatin resistance in NSCLC cells by protecting these cells from cisplatin-induced cell apoptosis. SKA1 also appeared to regulate the ERK1/2 and the Akt-mediated signaling pathways in NSCLC cells. SKA1 is required for metastasis and cisplatin resistance of non-small cell lung cancer.

Expression of SKA1 and MMP–9 in primary salivary adenoid cystic carcinoma: Correlation with tumor progression and patient prognosis

ABSTRACTConclusion The spindle and kinetochore-associated complex sub unit 1(SKA1) and matrix metalloproteinase-9 (MMP-9) are highly expressed in salivary adenoid cystic carcinoma (SACC), and SKA1 may be the novel, promising prognostic factor for clinical outcomes in head and neck adenoid cystic carcinoma patients. Objectives This study aimed to investigate the expression of SKA1 and MMP-9 in SACC tissues and the clinical significance. Methods SKA1 and MMP-9 expression in samples from 42 cases of SACC and 20 subjects with the normal tissue adjacent to carcinoma were detected by immunohistochemical analysis. Results The positive rate of SKA1 and MMP-9 staining was 78.6% and 66.7% in SACC, respectively, significantly higher than in normal salivary gland tissues (p < 0.001). Chi-square test showed that there was no significant correlation between SKA1 expression and MMP-9 expression in SACC tissues. However, SKA1 and MMP-9 expression was positively associated with advanced stage and solid histological pattern of SACC (p < 0.05). In addition, SKA1 and MMP-9 expression was positively associated with recurrence and perineural invasion, and survival time, respectively.

Effects of short interfering RNA-mediated gene silencing of SKA1 on proliferation of hepatocellular carcinoma cells

Background. Hepatocellular carcinoma (HCC) is one of the common causes of cancer resulting in death in China. Here, we found that spindle- and kinetochore-associated complex subunit 1 (SKA1) is a critical factor that plays an essential role in the regulation of HCC cell proliferation and apoptosis. Methods. Using immunohistochemistry in 38 HCC tissues, we showed that the expression of SKA1 was upregulated in HCC tissues compared with the normal tissues. Then, we investigated the effects of SKA1-targeted small interfering RNA (siRNA) on the HCC cell proliferation and apoptosis as of HCC cells. SKA1-targeted siRNA was delivered to HCC SMMC-7721 and Bel-7404 cells to evaluate its antiproliferation effects using lentivirus. Results. The lentivirus-mediated siRNA-targeting SKA1 treatment leads to a significant (p < 0.01) downregulation of SKA1 expression at mitochondrial RNA (mRNA) level. Knockdown of SKA1 inhibited HCC cell proliferation by inducing cell cycle arrest in the G0/G1 phase. Furthermore, lentivirus-mediated siRNA efficiently inhibited cell proliferation and colony formation while promoting the apoptosis. Conclusions. Of note, our data suggest that SKA1 might play an important role in the proliferation of HCC cells, and the absence of this gene in HCC may open promising therapeutic approaches for HCC.

Genetic Analysis of the Kinetochore DASH Complex Reveals an Antagonistic Relationship with the Ras/Protein Kinase A Pathway and a Novel Subunit Required for Ask1 Association

DASH is a microtubule- and kinetochore-associated complex required for proper chromosome segregation and bipolar attachment of sister chromatids on the mitotic spindle. We have undertaken a genetic and biochemical analysis of the DASH complex and uncovered a strong genetic interaction of DASH with the Ras/protein kinase A (PKA) pathway. Overexpression of PDE2 or deletion of RAS2 rescued the temperature sensitivity of ask1-3 mutants. Ras2 negatively regulates DASH through the PKA pathway. Constitutive PKA activity caused by mutation of the negative regulator BCY1 is toxic to DASH mutants such as ask1 and dam1. In addition, we have discovered two novel subunits of DASH, Hsk2 and Hsk3 (helper of Ask1), which are microproteins of fewer than 75 amino acids, as dosage suppressors of ask1 mutants. These are essential genes that colocalize with DASH components on spindles and kinetochores and are present in the DASH complex. Mutants in hsk3 arrest cells in mitosis with short spindles and broken spindle structures characteristic of other DASH mutants. Hsk3 is critical for the integrity of the DASH complex because in hsk3 mutants the association of Dam1, Duo1, Spc34, and Spc19 with Ask1 is greatly diminished. We propose that Hsk3 acts to incorporate Ask1 into the DASH complex.