In this study, we show that the spindle-associated protein HAUS6 has an oncogenic role in CRC. HAUS6 mRNA and protein expression were both increased in CRC tissues compared to adjacent noncancerous colorectal tissues, and higher HAUS6 expression was correlated with shorter overall survival in CRC patients. HAUS6 knockdown suppressed tumor growth by inhibiting cell viability and survival as well as by arresting cells in G0/G1. HAUS6 knockdown exerted these effects by reducing p53 degradation and activating the p53/p21 signaling pathway.
Analysis from our own cDNA array [4] found that HAUS6 mRNA expression was increased in CRC tissues compared to adjacent noncancerous colorectal tissues. This was consistent with our analysis of multiple datasets from the Oncomine database, quantitative RT-PCR analysis of a cDNA tissue array, and immunohistochemical analysis of a tissue microarray. These results suggest that increased HAUS6 expression may be common in CRC and may play an essential role in CRC development. Moreover, survival analysis revealed a correlation between high HAUS6 mRNA and protein expression and shorter overall survival in CRC patients. This demonstrates that HAUS6 may be useful as a biomarker for CRC prognosis. However, HAUS6 expression should first be further investigated in other types of malignancies.
Cancer cells are characterized by an uncontrolled increase in cell proliferation [19, 20], which requires mitotic spindle formation. Nucleation of MTs is the initial step of mitotic spindle formation [15, 16], and loss-of-function in MT-dependent MT amplification results in mitotic arrest [21, 22]. Therefore, inhibition of mitotic spindle formation by targeting microtubule nucleation factors may be a promising anti-cancer strategy [23–25]. Many microtubule inhibitors, e.g. taxans, vinca alkaloids and paclitaxel, induce mitotic arrest by interfering with microtubule dynamics and have been used to treat cancer [26–28]. However, due to side effects in normal cells and acquired resistance in cancer cells, it is important to further explore the underlying mechanism of mitosis and find new targets for anticancer treatment. In the present study, we show that knockdown of the spindle assembly factor HAUS6 suppressed tumor growth in vivo and in vitro by inhibiting cell viability, survival and cell cycle progression. In contrast, HAUS6 over-expression obviously increased cell viability of HCT116 cells in vitro. We further show that HAUS6 knockdown enhanced the ability of 5-FU to decrease cell viability and survival and to arrest cells in G0/G1 and G2/M phases. These results suggest that HAUS6 may be a promising new target for anticancer treatments.
Microarray analysis of HAUS6 knockdown cells revealed 103 up-regulated genes and 171 down-regulated genes. Many of them, including CDKN1A [29], CyclinD1 [30], ROCK2 [31] and HMGA2 [32], have already been implicated in proliferation and cell cycle progression. One notable DEG was CDKN1A (p21), which arrests cell cycle progression at G1/S phase by binding to and inhibiting cyclin-dependent kinases [33]. We found that p21 protein levels were increased in HAUS6 knockdown cells, and that CDKN1A knockdown reversed the effects of HAUS6 knockdown on cell viability, cell survival and cell cycle arrest. Moreover, HAUS6 expression was inversely correlated with CDKN1A expression. These results indicate that CDKN1A may mediate the oncogenic effects of HAUS6. However, the regulatory effects of HAUS6 on p21 need to be further explored.
KEGG pathway enrichment analysis of DEGs in HAUS6 knockdown cells showed that the p53 pathway was one of the most enriched. The p53 protein upregulates p21 in response to stress stimuli by binding to two highly conserved p53 response elements in the p21 promoter [34, 35]. As a tumor suppressor, p53 is expressed at low levels under normal conditions due to MDM2, which mediates nuclear export of p53 and targets p53 for ubiquitination and degradation. Under stress conditions, p53 rapidly accumulates and activates p21 [33, 35, 36]. In our experiments, HAUS6 knockdown increased TP53 mRNA and protein expression. Knockout of p53 also abrogated the effects of HAUS6 knockdown on cell viability, survival and cell cycle arrest in HCT116 cells. Therefore, the suppression of tumor growth caused by HAUS6 knockdown depends at least partly on activation of p53. We further found that HAUS6 knockdown reduced the degradation of p53 and p21, suggesting a mechanism by which HAUS6 regulates p53.
Taken together, these results show that HAUS6 knockdown suppresses CRC tumor growth by increasing the stability of p53. Moreover, combining HAUS6 knockdown with 5-FU treatment led to even greater down-regulation of HAUS6 and up-regulation of p21 and p53 than 5-FU alone, suggesting a common therapeutic mechanism. The effect of HAUS6 on tumorigenesis and p53 expression is similar to that of Plk1, which recruits HAUS6 to spindle MTs and NEDD1 to the spindle and centrosomes during mitosis [6]. HAUS6 may therefore be a promising novel target for anticancer treatments. Further study should be done to investigate the mechanism by which HAUS6 knockdown activates the p53 pathway.