Targeting Spindle-Associated Protein HAUS6 Prevents Cell Growth of Colorectal Cancer Cells by Activating the p53/p21 Pathway

Background: HAUS6 participates in microtubule-dependent microtubule amplication, but its role in malignancies including colorectal cancer (CRC) has not been explored. We therefore assessed the potential oncogenic activities of HAUS6 in CRC. Methods: We investigated HAUS6 expression and its prognostic value in CRC by microarray, analysis of public datasets, quantitative reverse transcription - polymerase chain reaction (qRT-PCR) and immunohistochemistry. Biological functions of HAUS6 were investigated using loss-of-function and gain-of-function assays in vivo and/or in vitro. Effectors downstream of HAUS6 were identied using cDNA microarray, bioinformatics analysis, qRT-PCR and western-blotting in CRC cells. The putative mechanism by which HAUS6 knockdown elevated p53 and p21 expression were assessed by rescue experiment. The combination effects of HAUS6 knockdown and 5-uorouracil (5-FU) treatment in cultured CRC cells were further evaluated. Results: HAUS6 mRNA and protein expression is higher in CRC tissues, and high HAUS6 expression is correlated with shorter overall survival in CRC patients. HAUS6 knockdown in CRC cell lines suppressed cell growth in vitro and in vivo by inhibiting cell viability, survival and arresting cell cycle progression at G0/G1, while HAUS6 over-expression increased cell viability. We showed that these effects are dependent on activation of the p53/p21 signalling pathway by reducing p53 and p21 degradation. Moreover, HAUS6 knockdown enhanced the effects of 5-FU treatment in CRC cells by increasing activation of the p53/p21 pathway. Conclusions: Our study highlights a potential oncogenic role for HAUS6 in CRC. Targeting HAUS6 may be a promising novel prognostic marker and chemotherapeutic target for treating CRC patients.


Background
Colorectal cancer (CRC) is one of the leading causes of mortality worldwide [1]. Progression from microscopic pre-neoplastic lesions to larger adenomas involves a cascade of mechanisms including tumor suppressor gene mutation and bypass of cell cycle checkpoints [2]. Unfortunately, current treatment strategies have low success rates due to our poor understanding of the molecular mechanisms behind CRC progression. Hence, there is an urgent demand to understand the fundamental mechanisms underlying CRC development and to nd novel molecular targets [3].
Follow-up analysis of gene expression pro le microarrays from our previous study (GEO Submission: GSE113513) [4] showed that levels of mRNA encoding HAUS augmin like complex subunit 6 (HAUS6) are higher in CRC tissues than in adjacent noncancerous colorectal tissues. Our preliminary experiments further suggested that HAUS6 knockdown inhibits growth of CRC cells in culture (data not shown). These results suggest that HAUS6 has oncogenic potential in CRC.
HAUS6 is one of the 8 subunits of the augmin complex [5,6], which is required for microtubule (MT)dependent MT ampli cation during cell division independently of the centrosomes and chromatin [7][8][9][10]. HAUS6 promotes MT-dependent MT ampli cation by interacting with and recruiting neural precursor cellexpressed developmentally down-regulated protein 1 (NEDD1), a subunit of the NEDD1-γ-tubulin ring complex. Depletion of HAUS6 reduces MT density in the bipolar spindle [6]. Dissociation of the augmin complex or the presence of HAUS6 and/or γ-tubulin reduces MT formation and MT bundling, thereby affecting spindle assembly during mitosis and MT organization in interphase [11,12]. MTs, spindle poles and chromosomes form the main elements of the spindle architecture [13,14], which plays an essential role in organizing bipolar spindles and chromosomal segregation during mitosis [15]. Failure of these processes results in mitotic arrest and apoptosis and contributes to chromosome mis-segregation and aneuploidy, a hallmark of cancer [16]. However, the role of HAUS6 in cancer has never been evaluated to our knowledge. In the present study, we investigated the effects of HAUS6 expression on patient prognosis and on viability and proliferation of CRC cell lines in vitro and in vivo, as well as its potential underlying mechanisms.

Materials And Methods
Bioinformatic analysis HAUS6 expression in CRC and noncancerous colorectal tissues was compared using the Oncomine database (www.oncomine.org), as described previously [4]. Correlation of HAUS6 expression with CRC patient survival was performed using the R2 Bioinformatic Platform (http://r2.amc.nl), as described previously [4]. The threshold of signi cance was set at p < 0.05.

Patients and specimens
Both CRC and matched adjacent non-cancerous tissues were collected from 36 CRC patients admitted to the First People's Hospital A liated with Fujian University of Traditional Chinese Medicine (FJTCM).
Clinicopathologic characteristics of patients are summarized in Supplementary Table S2. No patients received radio-or chemotherapy prior to surgery. Tissues were snap-frozen and stored in liquid nitrogen for quantitative RT-PCR or processed for immunohistochemistry. Samples were stained with hematoxylin and eosin, then examined by experienced pathologists, who assigned pathologic tumor-node-metastasis (p-TNM) grades according to International Union Against Cancer guidelines.
In addition, cDNA from 80 primary CRC and 15 adjacent noncancerous colorectal tissues were obtained from the cDNA-HColA095Su01 tissue cDNA array (Shanghai Outdo Biotech, Supplementary Quantitative RT-PCR Cells and tissue samples were lysed with RNAiso Plus reagent and total RNA was extracted according to the manufacturer's instructions. A total of 1 µg RNA was reverse-transcribed into cDNA using PrimeScript RT reagent kit and RT-qPCR was performed using SYBR Premix Ex Taq and the ABI 7500 Fast Real-Time PCR System (Applied Biosystems) according to manufacturer protocols. Primer sequences against HAUS6, TP53, CDKN1A and GAPDH are shown in Supplementary Table S5. GAPDH was used as an internal control.

Immunohistochemical staining
Tissues were processed using routine immunohistochemistry methods as in our previous study [4]. Tissue specimens collected in our laboratory were xed, sectioned and stained with primary antibodies against HAUS6 and Ki-67 then secondary biotinylated antibody (see supplementary Table S6), followed by incubation with horseradish peroxidase-conjugated streptavidin. Background staining was assessed by omitting the primary antibody. Five randomly selected elds were examined using a Leica microscope at 40 × magni cation.
For tissue arrays, slides were incubated with primary antibody against HAUS6 (dilution 1:500, Genetex) as described [4,17]. Images were captured using a Nano Zoomer 2.0 HT slide scanner and processed using Nano Zoomer Digital Pathology View 1.6 software. Staining intensity and the percentage of positively stained cells were determined as described [4,18]. Protein expression was calculated by multiplying the intensity and percentage scores together, giving expression scores ranging from 0 to 12.

Cell culture
Human CRC cell lines HCT116 and RKO were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Wild-type HCT116 cells (HCT116/p53 +/+ ) and p53 knockout HCT116 cells (HCT116/p53 −/− ) were a gift from Dr. Yao Lin (Fujian Normal University, Fujian, China) and originally obtained from Dr Bert Vogelstein (Johns Hopkins University, Baltimore, MD). All cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum, as well as 100 units/mL penicillin and 100 µg/mL streptomycin at 37 ℃ in a humidi ed atmosphere of 5% CO 2 . Cells were veri ed using short tandem repeat genotyping and examined for mycoplasma contamination using RT-PCR. 5-FU (Sigma-Aldrich) was dissolved in DMSO and used to treat control and HAUS6 knockdown cells; DMSO was used as a vehicle control.

Lentiviral transduction
Lentiviruses encoding shRNA against HAUS6 or CDKN1A or encoding control shRNA (Supplementary  Table S7 for sequences) and enhanced green uorescent protein (EGFP) under control of the CMV promoter were obtained from Shanghai GeneChem (Shanghai, China). CRC cells were transduced with lentivirus at a multiplicity of infection (MOI) of 10 for 72 h before analysis. To examine effects of HAUS6 over-expression on cell proliferation, HCT116 cells were transduced for 72 h with a lentiviral vector (MOI = 10) encoding full-length human HAUS6 (2,946 nt, GenBank accession NM_017645; Shanghai GeneChem). Transductants were selected for 2 weeks using 1 µg/mL puromycin.

Cell viability analysis
Cell viability was measured every day for 5 days by CCK-8 assay. Brie y, transduced cells were reseeded into 96-well plates at a density of 2000 cells/well with 100 µL medium/well. CCK-8 dye was added to each well and cells were incubated for 2 h at 37 ˚C. Cell viability was measured at 450 nm.

Cell survival analysis
Transduced cells were reseeded into 12-well or 6 well plates at a density of 500 or 1000 cells/well and incubated for 10-12 days at 37 °C under 5% CO2 in a humidi ed incubator. Colonies were xed with 4% paraformaldehyde for 20 min and stained with 0.1% of crystal violet for 20 min. Colonies were photographed and the number of colonies counted. Data were normalized relative to control cells.

In vivo experiments
Male pathogen-free athymic nude mice (6-8 weeks old) were purchased from Shanghai Laboratory Animal Co., Ltd. (Shanghai, China) and maintained in a speci c pathogen-free (SPF) facility.
Tranduced CRC cells (1 × 10 6 ) in 100 µL RPMI-1640 medium containing 50% matrigel were subcutaneously injected into the ank of nude mice (n = 8). Tumor growth was monitored from day 7 after injection on every second day for 19 days. Tumor volume was calculated as (1/2) (length × width 2 ), where length and width refer to the longest longitudinal and transverse diameters.
Tumor growth was also measured in terms of GFP intensity using an IVIS Spectrum whole live-animal imaging system (PerkinElmer). Mice were anesthetized with iso urane and sacri ced by neck dislocation on day 26 after injection, and tumors were dissected and weighed. Collected tissues were processed for immunohistochemistry.

Microarray analysis
HCT116 cells were transduced with lentivirus encoding shRNA speci c for HAUS6 or control shRNA for 72 h. Brie y, total RNA was extracted with Trizol reagent and analyzed by CapitalBio using the human GeneChip Primeview array (Affymetrix) according to the manufacturer's instructions. Microarray images were scanned using GeneChip Scanner 3000 and analyzed using GeneChip GCOS 1.4 software (Affymetrix). Genes with a p-value < 0.05 and fold change > 2 were de ned as differentially expressed. The raw microarray data can be found in the NCBI Gene Expression Omnibus (GEO Submission: GSE140326). Enriched pathways in DEGs were identi ed using KEGG pathway enrichment analysis.
Cells were lysed with RIPA lysis buffer and the protein concentration was determined using BCA assay.
The proteins (50 µg) were then separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked with BSA and incubated overnight at 4 °C with primary antibody against HAUS6, p53, or p21 (Supplementary Table 6). The membranes were then washed three times with TBST, and incubated at room temperature for 1 h with horseradish peroxidase-conjugated goat anti-rabbit. The blots were visualized using enhanced chemiluminescence according to the manufacturer's protocols.

Statistical analysis
Statistical analysis was performed using SPSS 20.0 (IBM, Chicago, IL, USA). Data are presented as mean ± SD. The median HAUS6 expression in CRC tissues was used as the cutoff to divide tumors into "low expression" or "high expression". The relationship between HAUS6 expression and overall survival was analyzed using Kaplan-Meier curves and the log-rank test. Differences between two groups were assessed using the independent or paired Student's t test, while differences among three or more groups were assessed using one-way ANOVA. Survival differences were assessed using the log-rank test. The correlation between HAUS6 and CDKN1A expression was analyzed using Pearson's correlation. A p-value of < 0.05 was considered statistically signi cant.

Study approval
Collection of the clinic samples and related experiments were approved by the Ethics Committee of FJTCM. Written, informed consent was obtained from all patients or their families. All animal maintenance and procedures were performed in strict accordance with the "Guide for the Care and Use of Laboratory Animals" and the "Principles for the Utilization and Care of Vertebrate Animals" and approved by the Animal Committee of FJTCM.

HAUS6 is highly expressed in CRC tissues
We rst compared HAUS6 expression in cDNA samples from 14 matched pairs of primary CRC tumors and non-cancerous tissues from a cDNA array (GEO Submission: GSE113513). HAUS6 expression was higher in CRC tumors than non-cancerous tissues (Fig. 1A). Analysis of publicly available tumor expression data from the Oncomine database (http://www.oncomine.com/) also showed higher HAUS6 mRNA levels in CRC tissues than non-cancerous tissues (Supplementary Table S1). Quantitative RT-PCR analysis of HAUS6 expression in clinical samples of 36 CRC tissues and adjacent non-cancerous colorectal tissues from our laboratory and a cDNA tissue array containing 15 pairs of CRC primary tumors and surrounding non-cancerous tissues also showed higher HAUS6 mRNA expression in CRC tissues ( Fig. 1B-C). Immunohistochemistry of 10 pairs of CRC samples randomly selected from the above mentioned 36 pairs, as well as analysis of a tissue microarray of 260 pairs of CRC samples (Shanghai Outdo Biotech), con rmed that HAUS6 protein levels were higher in CRC primary tumor tissues (Fig. 1D HAUS6 expression is associated with poor patient prognosis Kaplan-Meier analysis of 80 patients whose primary tumor tissues were sampled on the cDNA array mentioned above did not reveal any difference in patient survival between tumors with low and high HAUS6 mRNA expression (data not shown). In contrast, analysis of data from 194 CRC patients (high expression: n = 79; low expression: n = 115) from the public microarray dataset using the R2 bioinformatic platform showed that high HAUS6 mRNA expression was associated with shorter overall survival ( Fig. 2A). Similarly, tumors with high HAUS6 protein levels were associated with shorter overall survival in 280 CRC patients (

HAUS6 knockdown inhibits CRC cell growth and proliferation in vitro
We next assessed the effects of HAUS6 knockdown on growth and proliferation of CRC cell lines.
Lentiviral transduction with vectors expressing short hairpin (sh)RNAs against HAUS6 down-regulated HAUS6, as expected ( Fig. 3A-B; Supplementary Fig. S1A-B). CRC cells transduced with HAUS6 shRNAs had lower cell viability than those transduced with control RNAs (Fig. 3C; Supplementary Fig. S1C). In contrast, lentiviral transduction with vectors over-expressing HAUS6 up-regulated HAUS6 protein expression, and increased the cell viability of HCT116 cells based on the CCK8 assay ( Fig. 3A-C, right   panel). Moreover, HAUS6 knockdown also decreased the number of colonies formed by CRC cells (Fig. 3D). Cell cycle analysis revealed that HAUS6 knockdown increased the percentage of cells in G0/G1 and decreased the percentage of cells in S phase (Fig. 3E). However, HAUS6 knockdown did not affect cell apoptosis in HCT116 cells ( Supplementary Fig. S2).
HAUS6 knockdown suppresses CRC cell growth and proliferation in vivo.
We next used a xenograft mouse model to assess the effects of HAUS6 knockdown on CRC cell growth and proliferation in vivo. HAUS6 knockdown strongly reduced tumor volume (Fig. 4A) and decreased uorescence of tumor cells (Fig. 4B) in nude mice injected with GFP-expressing CRC cells. Final tumor weights were also lower in mice injected with HAUS6 knockdown cells than in those injected with control cells (Fig. 4C). Immunohistochemistry of the excised tumors showed that Ki-67 protein expression was decreased in HAUS6 knockdown cells (Fig. 4D), suggesting that HAUS6 knockdown suppresses CRC tumor growth by inhibiting cell proliferation.
Microarray analysis of HAUS6 knockdown HCT116 cells revealed a total of 274 differentially expressed genes (DEGs) in CRC (Fig. 5A-B; GEO Submission: GSE140326). CDKN1A (encoding p21 protein) was upregulated in HAUS6 knockdown cells (Fig. 5C), and this was con rmed in both HCT116 and RKO cells by quantitative RT-PCR (Fig. 5D) and western blotting (Fig. 5E). Transduction with shRNAs speci c for HAUS6 and CDKN1A reversed the up-regulation of p21 observed in HAUS6 knockdown cells (Fig. 5F). HAUS6 knockdown decreased cell viability and colony formation, whereas CDKN1A knockdown had the opposite effect and attenuated the effects of HAUS6 knockdown (Fig. 5G-H). HAUS6 knockdown increased the percentage of cells in G0/G1 and reduced the percentage in S phase, which were reversed by CDKN1A knockdown (Fig. 5I). Analysis of microarray expression data (GEO Submission: GSE113513) showed that CDKN1A was down-regulated in CRC tissues (Fig. 5J) and its expression correlated negatively with HAUS6 expression (Fig. 5K).
HAUS6 knockdown suppresses cell proliferation by activating the p53 pathway.
KEGG pathway enrichment analysis of DEGs in HAUS6 knockdown cells identi ed the p53 pathway as the one of the most enriched signalling pathways (Fig. 6A). Given the key regulatory effects of p53 on cell proliferation and CDKN1A transcription, we next explored the effect of HAUS6 on the p53 pathway. HAUS6 knockdown increased TP53 expression in HCT116 and RKO cells at the levels of mRNA ( Fig. 6B-C) and protein (Fig. 6D). The inhibitory effects of HAUS6 knockdown on cell viability were attenuated by knockout of p53 ( Fig. 6E-F) or treatment with the p53 inhibitor PFT-α (Fig. 6G). Similarly, p53 knockout blocked the inhibitory effects of HAUS6 knockdown on cell survival (Fig. 6H) and cell cycle progression (Fig. 6I-J). To determine the molecular mechanism by which HAUS6 knockdown regulates p53, we evaluated p53 protein stability in RKO cells expressing wild-type p53. Protein levels of p53 and its downstream p21 decreased over time in sh-Ctrl cells, but remained stable after HAUS6 knockdown (Fig. 6K).
We further assessed the effect of HAUS6 on the response of CRC cells to 5-uorouracil (5-FU) treatment. 5-FU treatment down-regulated HAUS6 and up-regulated p53 and p21 protein expression. These effects were further enhanced by HAUS6 knockdown (Fig. 7A). Moreover, HAUS6 knockdown reduced viability of HCT116 cells, regardless of whether they were treated with 5-FU (Fig. 7B). Combining HAUS6 knockdown with 5-FU led to greater suppression of HCT116 colony formation (Fig. 7C) than 5-FU alone, as well as higher percentages of cells in G0/G1 and G2/M and lower percentage in S phase (Fig. 7D-E).

Discussion
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 rst 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 ampli cation 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][24][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][27][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 nd 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.
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.

Conclusions
In summary, we show that HAUS6 upregulation is associated with shorter survival of CRC patients and increased cell viability in cultured CRC cells, while HAUS6 knockdown inhibits tumor growth and enhances 5-FU treatment by activating the p53/p21 pathway. HAUS6 may be a useful prognostic marker and chemotherapeutic target in CRC.

Declarations
Ethics approval and consent to participate Collection of the clinic samples and related experiments were approved by the Ethics Committee of FJTCM. Written, informed consent was obtained from all patients or their families. All animal maintenance and procedures were performed in strict accordance with the "Guide for the Care and Use of Laboratory Animals" and the "Principles for the Utilization and Care of Vertebrate Animals" and approved by the Animal Committee of FJTCM.

Consent for publication
All authors have agreed to publish this manuscript.

Competing interests
The authors have declared that no competing interest exists.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.