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The mitotic kinesin superfamily protein KIF14 is essential for cytokinesis and chromosome segregation and increased KIF14 expression is related to a variety of human cancers. In this study, we investigate KIF14 expression in association with clinical variables and the role of KIF14 during tumorigenesis. We found that KIF14 is overexpressed in most primary hepatocellular carcinoma (HCC) tissues compared with the adjacent normal liver tissues and KIF14 overexpression is associated with tumor grade (P = 0.002), stage (P = 0.013) and poor survival (P < 0.001). Downregulation of KIF14 decreased the capacity of proliferation both in vitro and in vivo. Furthermore, suppression of KIF14 not only decreases cancer cell migration but also induces apoptosis of cells with inactivation of the phosphatidylinositol 3-kinase-Akt signaling pathway. Therefore, our current study indicates that KIF14 promotes HCC carcinogenesis and may serve as a potential therapeutic target for human HCC.
Hepatocellular carcinoma (HCC), one of the most frequent visceral neoplasms worldwide, is a common fatal malignant tumor that is characterized by a high incidence recurrence, development of resistance to chemotherapy, and low sensitivity to radiation therapy. Improved surgical techniques, new chemotherapeutic agents, altered modes of chemotherapy delivery and emerging molecular targets have improved life expectancy; however, the incomplete understanding of its carcinogenic mechanisms leads to difficulties in selecting targeted treatment and contributes to a low survival rate for patients with HCC.
Microtubule-associated motor proteins have important roles in numerous cellular processes including intracellular transportation and cell division, with more than 45 members having been identified in mammalian cells. There is an indication that the abnormal expression and function of kinesins plays a key role in the development or progression of many kinds of human cancers. Better understanding of kinesin protein functions may translate into development of molecular-targeted therapy for various human cancers.[4, 5] Human KIF14 was first identified as a 6586 base-pair kinesin family 10 cDNA clone. Like all kinesins, KIF14 is a microtubule-dependent molecular motor, containing a kinesin motor domain and a forkhead-associated domain. KIF14 also contains a C-terminal citron kinase binding region and an N-terminal protein regulating cytokinesis binding region.[8, 9]
KIF14 has been predicted to be a possible oncogene in the 1q region, and was found to overexpress in four breast cancer cell lines compared with the normal breast tissues. Furthermore, the KIF14 mRNA was also more over expressed in retinoblastoma than in retina and showed that patients with an older age at diagnosis express higher levels of KIF14 mRNA, probably leading to chromosomal/genetic instability during tumorigenesis. KIF14 is regulated during the cell cycle, and is correlated with mitotic progression. Furthermore, KIF14, along with the microtubule-bundling protein PRC1 and citron kinase, with which it interacts, plays an important role in the cytokinesis, and depletion of KIF14 in HeLa cells results in the generation of binucleate cells, polyploidy and apoptosis. In addition, the transient KIF14 knockdown by siRNA in H1299 and HeLa cells significantly decreased the capacity of proliferation and colony formation, suggesting that KIF14 may have an important oncogenic role in cancer cells. Furthermore, KIF14 is also overexpressed in breast cancer and medulloblastoma cell lines, and some lung primary tumors, compared to appropriate normal tissues.[10, 13] In addition, KIF14 mRNA expression is a prognostic marker in breast, lung and ovarian cancer and potential therapeutic target for ovarian cancer.[7, 12, 14]
In the present study, we show that 23 of 30 primary HCC tissues display 1.5- to 46-fold increased KIF14 mRNA expression over matched normal samples. The KIF14 mRNA expression is associated with tumor grade and stage. Additionally, the patients with increased KIF14 levels show decreased disease free survival (DFS). The suppression of KIF14 inhibits proliferation and tumor formation both in vivo and in vitro. Furthermore, the downregulated expression KIF14 inhibits the migration and induces apoptosis via inactivation of Akt kinase in HCC cells.
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- Materials and Methods
- Disclosure Statement
In the present study, we investigated the involvement of the kinesin motor protein, KIF14, in human HCC. Our results indicate that KIF14 is overexpressed in human HCC and such overexpression is correlated significantly with tumors in advanced grade, metastasis and decreased DFS rate, suggesting that KIF14 plays an important role in human HCC carcinogenesis. Consistent with these findings, we show that ablation of KIF14 expression in human HCC cells inhibits cell proliferation in vitro and tumor formation in vivo. Furthermore, the suppression of KIF14 decreases the cell migration and promotes apoptosis via inactivation of Akt kinase.
KIF14 is essential for cytokinesis and chromosome segregation. Several reports have been showed that the increased KIF14 expression is related to a variety of different cancers tumorigenesis, including HCC.[7, 11-14, 19-21] In this study, although we observed that KIF14 is upregulation in most primary HCC tissues compared to the paired normal tissues, KIF14 was overexpressed in 5 of 30 non-tumor tissues. These conflicting data suggested that KIF14 may have a different role in HCC carcinogenesis. In fact, previous studies indicate that increased KIF14 expression seems to be associated with more advanced, aggressive tumors and its expression increases pronouncedly with grade, fraction of tumor-positive nodes removed and percent invasive cells in patients with breast cancer. Because of the limited number of samples, Kim et al. did not observe any significant association between KIF14 expression levels and invasiveness, tumor grade, size and survival However, we observed an association between expression of KIF14 level and grade, stage or survival in HCC. Moreover, KIF14 has been reported to be an independent prognostic marker and potential therapeutic target in different cancers.[7, 12, 21] Further large-scale studies will be required to validate this possibility in HCC.
We next determined the potential role of KIF14 in tumor formation and progression. Our results indicate that suppression of KIF14 expression by RNAi in HCC cells inhibits cell proliferation in vitro and tumor formation in vivo. We show here that downregulation of KIF14 expression by siRNA moderately decreases proliferation but strongly decreases the ability of HepG2 and SMMC-7721 cells to form colonies. These in vitro results suggest that downregulation of KIF14 is not only cytotoxic, but also affects the capability of independent growth of cancer cells. This makes KIF14 a potential target for cancer therapy. The work of Carleton et al. showed that knockdown of KIF14 by siRNA in HeLa cells induced binucleation and apoptosis. Although, in our work, stable depletion of HepG2 cells induced significant apoptosis, stable deletion of KIF14 by shRNA did not induce binucleation in HepG2 cells (data not shown). This discrepancy could be explained in that the type (stable versus transient) and level of KIF14 knockdown may produce different cellular phenotypes, which could point to unique functions for KIF14 in different cell lines. Furthermore, our stable cones may be sensitive to multinucleation and induce apoptosis before any such phenotype can be characterized. Furthermore, our findings indicate that suppression of KIF14 not only inhibits cell division, but also decreases cell migration.
Previous studies demonstrated that KIF14 interacts with citron kinase, localizing this Rho effector kinase to the central spindle and citron kinase was a prognostic marker for DFS and the KIF14 was negatively related to citron kinase expression in lung cancer. These findings suggest that the expressions of KIF14 and citron kinase may be coordinately controlled to deregulate the central spindle and final phase of cytokinesis in HCC cells. It will be required for evaluating if KIF14 expression is predictive for response to treatment with spindle poisons such as Vinca Alkaloids and taxanes in future studies.
Apoptosis is a process of programmed cell death. Although normal cells use apoptosis to prevent abnormal cell proliferation, cancer cells often overcome the process and migrate to new sites for their abnormal growth. Therefore, the increased migration ability and resistance of apoptosis take an advantage for metastasis. Our results indicated that suppression of KIF14 in HCC cells decreases the growth and induces apoptosis. Although the exact mechanism by which KIF14 is directly involved in the regulation of apoptosis remains to be clarified, our results show that the cell proliferation/survival of phosphatidylinositol 3-kinase-Akt signaling pathway might be inhibited in depleted KIF14 expression cells, which is similar to the role of KIF18A during breast carcinogenesis. Further study is required to determine how KIF14 activates the phosphatidylinositol 3-kinase-Akt signaling pathway.
Although the exact function of KIF14 remains unclear, its role in aligning chromosomes at metaphase suggests that it could play an important role in the formation of aneuploidy, and its overexpression may function to accelerate the cell cycle during cancer progression. Whatever its mode of action, its specific overexpression in HCC and association with grade and survival, and inhibition of cell proliferation in vitro and tumor formation in vivo by suppression of KIF14 expression in HCC cells demonstrate its importance as a putative oncogene, therapeutic target and prognostic indicator.