Clinicopathological and prognostic value of transforming acidic coiled-coil-containing protein 3 (TACC3) expression in soft tissue sarcomas

Transforming acidic coiled-coil-containing protein 3 (TACC3), a microtubule regulator, is associated with various cancers. However, the relationship between TACC3 and soft tissue sarcomas (STS) remains unclear. We investigated the expression of TACC3 in 136 STS patient samples using immunohistochemical (IHC) staining, and the statistical associations between TACC3 expression and clinicopathological characteristics were evaluated. Additionally, the expression levels of the tumor suppressor p53 and of the cell proliferation marker Ki-67 were also assessed by IHC. High TACC3 expression was detected in 94/136 of STS cases (69.1%), and significantly correlated with higher grade according to the French Fédération Nationale des Centres de Lutte Contre le Cancer system (P<0.0001), poorer tumor differentiation (P<0.0001), increased mitotic counts (P<0.0001), advanced stage per American Joint Committee on Cancer guidelines (P<0.0001), higher p53 expression (P = 0.0487), higher Ki-67 expression (P<0.0001), and undergoing postoperative therapy (P = 0.0001). Disease-free survival (DFS) and overall survival (OS) of patients with high TACC3 expression were significantly shorter (P<0.0001 and P<0.0001, respectively). On multivariate analyses, high TACC3 expression was an independent negative prognostic factor for both DFS and OS (hazard ratio [HR]: 3.074; P = 0.0235 and HR: 8.521; P = 0.0415, respectively). Our results suggest that TACC3 is an independent prognostic factor and may be a novel therapeutic target for the treatment of STS.


Introduction
Soft tissue sarcomas (STS) are rare, and comprise a heterogeneous group of mesenchymal tumors. Although they account for less than 1% of all human malignancies [1], they exhibit very aggressive behavior; approximately half of the patients with STS experience tumor a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 recurrence despite undergoing multimodal therapy, and more than one-third of patients die of their diseases [2]. Complete resection is the first-line treatment, and there are few confirmed treatment options available other than conventional chemotherapy [3,4]. Thus, the diagnosis and treatment of STS remain challenging for both physicians and patients.
Transforming acidic coiled-coil-containing protein 3 (TACC3) is a member of the TACC family, which is located in the 4p16.3 region of chromosome [5]. TACC3, an Aurora A kinase target, is essential for microtubule growth and stability when localizing to the centrosome during mitosis [6]. Microtubule fusion is necessary for the formation of the mitotic spindles, that segregate chromosomes. Abnormality of microtubules/centrosomes causes mitotic spindle defects and correlates with tumorigenesis and tumor progression; therefore, the involvement of TACC3 in various cancers has been reported [7]. Additionally, TACC3 depletion has been shown to induce p53-mediated apoptosis [8]. Despite the recent development of chemotherapeutic drugs that target mitotic proteins such as Aurora kinase and Polo-like kinase, their effects remain ambiguous [9]. Accordingly, TACC3 itself is being examined as a potentially novel therapeutic target.
In this study, we investigated TACC3 expression in STS using immunohistochemical (IHC) staining, and analyzed the association between TACC3 expression and clinicopathological characteristics as well as patient survival.

Patients and characteristics
We investigated 136 STS patients who underwent resection at Kurume University between 1990 and 2016, and retrieved formalin-fixed, paraffin-embedded (FFPE) specimens. The antibodies used for diagnoses were CAM5.2 (BD Biosciences, San Jose, CA, USA); CD3, MyoD1, Bcl-6, and CD34 (Leica Ltd., New Castle, UK); Bcl-2 and p16 (Ventana Medical Systems, Inc., Tucson, AZ, USA); MDM2 (Calbiochem, San Diego, CA, USA); CDK4 (Invitrogen, San Francisco, CA, USA); and vimentin, SMA, desmin, myogenin, S100, KP-1, AE1/AE3, EMA, CD20, and CD99 (Dako, Tokyo, Japan). Samples were reviewed and classified by 2 experienced pathologists (O.K. and M.H.) according to the 2013 World Health Organization (WHO) classification [1]. Patients with microscopic positive surgical margins, those with recurrence or metastasis at the initial visit, or those who received neoadjuvant therapies were excluded from the study. All patients underwent postoperative follow-up at least every other year after surgery. Clinical data were collected from patients' medical records. Our study was approved by the Research Ethics Committee of Kurume University, and written informed consent was obtained according to the Declaration of Helsinki.
All STS samples were cut into maximum cross sections and stained with hematoxylin and eosin for pathomorphological investigation. We selected sites that were most representative of the sample's pathological features, as determined by the 2 pathologists, and constructed tissue microarrays from 3-mm cores with a minimum of 2 cores per patient. If tissues were available, 3 cores per patient were used to compensate for STS tissue heterogeneity.
Determination of cut-off values. The cut-off value for TACC3 expression was determined by using receiver operating characteristic (ROC) curves and calculating the Youden index (Fig 1). FNCLCC grades were used as dichotomous variables (either 1 or 2/3), while the TACC3-positive rate was a continuous variable. The optimal cut-off value was calculated as 11%. Therefore, TACC3-positive rates of more than 11% were defined as high expression. Based on previous studies, high expression rates of p53 and Ki-67 were defined as greater than 10% positive staining [19,21,22].

Statistical analysis
Clinicopathological features and TACC3 expression were analyzed using the χ 2 or Fisher's exact test. Disease-free survival (DFS) was defined as the duration between the day resection was performed and the day recurrence or metastasis was detected; overall survival (OS) was defined as the duration between the day resection was performed and the day of last follow-up or death. Both DFS and OS were evaluated using the Kaplan-Meier method and log-rank test. The impact of possible risk factors affecting DFS and OS were assessed by univariate and multivariate Cox regression analyses. A P-value less than 0.05 was considered statistically significant. The JMP, version 12 software (SAS Institute, Tokyo, Japan) was used for statistical analyses in this study.

Association of TACC3 expression with clinicopathological features
The statistical associations of TACC3 expression with specific clinicopathological features are shown in Table 3. Higher TACC3 expression was significantly associated with higher FNCLCC grade (P<0.0001), poorer tumor differentiation (P<0.0001), increased mitotic counts (P<0.0001),

Effect of TACC3 expression on DFS and OS
DFS and OS curves of patients with STS are shown in Fig 3. DFS in patients with high TACC3 expression was significantly shorter than in those with low TACC3 expression (P<0.0001) (Fig 3A), as was OS (P<0.0001) (Fig 3B).

Risk factors affecting DFS and OS
The results of univariate and multivariate analyses of potential risk factors that influence DFS and OS are shown in Tables 4 and 5

Discussion
Our study revealed that TACC3 was highly expressed in more than two-thirds of STS patients, and that greater expression was associated with higher FNCLCC grade, poorer tumor differentiation, increased mitotic counts, advanced AJCC stage, higher p53 expression, higher Ki-67 expression, and undergoing postoperative therapy. The DFS and OS rates in STS patients with high TACC3 expression were significantly shorter. Furthermore, on multivariate analyses, high TACC3 expression was an independent negative prognostic factor for both DFS and OS in STS patients. Higher TACC3 expression was associated with poorer tumor differentiation of FNCLCC in STS. The relationship between higher TACC3 expression and poorer tumor differentiation was demonstrated in lung cancer [11], hepatocellular carcinoma [14], and cholangiocarcinoma [18]. In addition, TACC3 expression was suggested to be accelerated during the transition of breast cancer from ductal carcinoma in situ to invasive ductal carcinoma in microarray analysis [24]. Even though simple comparison is difficult due to the heterogeneity of STS, it is possible that, in STS, TACC3 is involved in the tumor differentiation as with other malignant tumors.  Higher TACC3 expression was associated with increased mitotic counts, and TACC3 was detected in almost all mitotic tumor cells by IHC (S1 Fig). These results are consistent with the known mechanism of TACC3 as a protein that is expressed specifically in the mitotic phase [5,7].
However, TACC3 was also expressed in many non-mitotic tumor cells on IHC. In normal human tissues, TACC3 expression is observed in the testis, spleen, thymus, and peripheral blood leukocytes, all of which are highly proliferative tissues [25]. Additionally, TACC3 expression is reportedly correlated with proliferation in hepatocellular carcinoma cells [14]. In  our study, greater expression of TACC3 was associated with that of Ki-67. Ki-67 is a nuclear antigen that is expressed throughout the cell cycle except during G0 phase, and is reflective of the cell's proliferative potential [26]. Therefore, our results suggest that TACC3 expression is a hallmark of STS cell proliferation. Higher TACC3 expression was associated with increased p53 expression; patients with high p53 expression tended to have poor prognoses, which is consistent with previous findings [19,21]. Generally, missense p53 gene mutations result in the accumulation of p53 proteins in the nuclei. On the other hand, p53 mutation is not always correlated with IHC findings, because mutant protein staining differs by both site and extent [27]. However, it was previously reported that p53 mutation correlates with nuclear immunoreaction of p53 protein in STS [21]; therefore, high TACC3 expression may indeed correlate with p53 mutation in STS.
High TACC3 expression was an independent predictor of poor prognosis in terms of both DFS and OS; this may be attributed to at least 3 possible explanations. First, overexpression of TACC3 promotes the PI3K/AKT and ERK signaling pathways that induce epithelial-mesenchymal transition, which enhances the migratory ability of tumor cells and increases their invasive capacity [28]. Second, TACC3 expression was associated with the expression of stem cell transcription factors, including Bmi-1, c-Myc, and Nanog, and the upregulation of TACC3 promoted tumor stem cell-like capabilities in hepatocellular carcinoma [13]; cancer stem cells can maintain themselves by self-replication to create additional daughter cells in their milieus [29]. Third, high levels of TACC3 reduce the efficiency of DNA repair, causing genomic instability and tumorigenesis [30]. However, p53-mediated apoptosis may not function since it is induced by the lack of TACC3 [8]. Taken together, high TACC3 expression may increase the aggressiveness of tumor cells by increasing their invasive capacities, stem cell-like properties, and antiapoptotic activities.
The relationships between high TACC3 expression and poor prognoses have been reported in various cancers to date [10][11][12][13][14][15][16][17][18]; however, cut-off values for high TACC3 on IHC have not The prognostic value of TACC3 in STS been clarified. In non-small cell carcinoma, the cut-off value of TACC3 was defined as 10% [10]. In a study of esophageal squamous cell carcinomas, however, the cut-off value for high TACC3 expression was determined by the immunoreactivity score, product of the staining intensity, and percentage of positive cells [12]; moreover, the rationale behind the selected cutoff value was not stated. In our case, we determined the cut-off value via ROC curve analysis and the Youden index using the FNCLCC grade, established grade of malignancy, and indicators of STS prognosis [20] as dichotomous variables, as well as the TACC3 positivity rate as a continuous variable. Hence, our derived cut-off value was not arbitrary, and should therefore be considered sound [31]. The depletion of TACC3 has been shown to suppress tumor growth without damaging normal tissues [32], cause premature senescence of tumor cells [33], and increase the susceptibility of tumor cells to paclitaxel (which disrupts the microtubule network) [34]. Furthermore, TACC3 inhibitors such as spindlactone (SPL) and SNIPER(TACC3) have been developed [35,36]. SPL interferes with TACC3 function by blocking the TACC3-TOGp complex and causing spindle defects in tumor cells, whereas SNIPER(TACC3) reduces TACC3 protein levels in tumor cells by inducing its polyubiquitylation and proteasomal degradation. These developments improve the prospects of TACC3 as a novel therapeutic target.
Our study has some limitations. First, the number of patients and histological types were relatively small. There are numerous histological types of STS, and further studies with larger numbers of patients that encompass these histological types are required to clarify common progression mechanisms. At the same time, STS are rare, and many studies included even fewer patients than ours, even though our results were ultimately consistent with theirs [19,21]. Second, TACC3 expression was evaluated only by IHC; additional investigations of genetic defects and transcriptional systems ought to reveal other biological roles for TACC3. Third, postoperative therapy was associated with high TACC3 expression and also correlated with shorter DFS and OS. These results suggest that surgeons might administer adjuvant treatment to more aggressive or higher-grade tumors. Our study was based on a retrospective analysis, which may be susceptible to selection bias. Therefore, prospective studies are required to validate our results.
In conclusion, we demonstrated that high TACC3 expression was correlated with aggressive clinicopathological features and unfavorable prognoses in STS patients. To our knowledge, ours is the first study of its kind to investigate TACC3 in STS. Our results expose the potential of TACC3 not only as a reliable prognostic biomarker but also as a novel and promising therapeutic target for the treatment of STS.