Fused in Sarcoma (FUS) Expression May Predict Survival Outcomes of Patients With Advanced Squamous Cell Cervical Carcinoma

Because of its role in preserving DNA/RNA stability, there is an increasing number of studies addressing the relationship between Fused in Sarcoma (FUS) expression and cancer. However, the disparity in FUS/FUS oncogenic vs. tumor-suppressive roles may be attributed to the complex molecular pathways associated with FUS regulation in different cancer types, and its role in cervical carcinogenesis remains largely unexplored. Methods: We determined FUS protein expression in specimens of 61 patients with advanced cervical cancer. Long-term (> 10 years) clinical follow-up data for these patients were available, and we determined disease-free, cancer-related and overall survival as related to FUS expression. Results: There were no signicant associations between FUS expression and patients’ age, tumor grade, and acute/late toxicity events related to treatment (either radiation alone or chemoradiation). However, multivariate Cox regression analysis for disease-free survival (recurrence), overall survival (death) and cancer-related survival showed that patients with high average FUS expression fared signicantly better than their counterparts with low average FUS expression, both in terms of disease-free survival (HR = 0.31; 95%CI 0.12 to 0.77; p = 0.01) and cancer-related survival (HR = 0.41; 95%CI 0.17 to 0.98; p = 0.04). Conclusions: Our study shows that high FUS protein expression in advanced cervical cancer specimens is a potent harbinger of better prognosis, and can as such be used in clinical practice to help characterize patients and, possibly, plan treatment and follow-up strategies.


Introduction
RNA metabolism and DNA repair are regulated by several genes (1,2). In this context, the Fused in Sarcoma (FUS) oncogene, located on chromosome 16 and initially idendi ed in human liposarcoma, has been described to encode a 526 amino acid DNA/RNA binding protein which seems to be involved in gene transcription, RNA transport and translation (3). Of special interest in oncology, it seems that FUS helps safeguard genomic stability; during DNA damage repair, FUS is one of the proteins rstly recruited to the DNA damage site (4,5).
Knockdown of FUS during cell growth leads to defects in DNA damage recovery (6). Loss of FUS in the nucleus affects transcription, alternative splicing and also DNA repair (7).
Because of its role in preserving DNA stability, there is an increasing number of studies addressing the relationship between FUS expression and cancer, such as in cervical, brain (glioma), liposarcoma and lung cancers (8-11). Con icting results, however, have been reported, depending on the particular cancer type studied and whether FUS-related mRNA, protein expression, or genomic stability are being evaluated. For instance, C-terminal mutations in FUS have been shown to explain 5-10% of cases of familial amyotrophic sclerosis (12), but FUS mutations or SNPs were not found in 96 cases of liposarcoma (10), leading the authors of that study to assume that mutations in FUS may not play a role in sarcomagenesis. Later, one study described that elevated FUS expression was negatively associated with prognosis of patients with non-small cell lung cancer (11), and one experimental study revealed that the knockdown of FUS inhibited the viability, migration and tube formation of glioma cells (9). Further, studies on prostate and cervical cancer have suggested potential associations between FUS/FUS. Haile et al (2011) found that FUS is a co-activator of androgen receptor in prostate cancer (13), and Chen et al (2019) reported that circRNA_0000285 promotes cervical cancer by up-regulating FUS in human tissues (8).
We developed a long-term randomized clinical trial to explore the e cacy of chemoradiation versus radiation alone for the treatment of advanced (stage IIIB-FIGO 2009) (14) cervical cancer. Follow-up results covering ten years have now been published (15,16). We took advantage of the vast clinical follow-up data and the availability of tumor specimens from patients included in our randomized trial to further explore the association between FUS expression and cervical cancer prognosis.

Selection of cases
For this study, we selected patients who had participated in a randomized clinical trial aimed at comparing radiotherapy alone (RT) vs. radiotherapy + cisplatin (CRT) for the treatment of advanced cervical cancer (Concomitant cisplatin plus radiotherapy and high-dose-rate brachytherapy versus radiotherapy alone for stage IIIB squamous cell cervical cancer: a randomized controlled trial) (15). This study was For this present study, only patients with available para n-embedded cervical specimens were included. Trial and patient details were described elsewhere (15). Of the 147 patients originally included in the trial, only 61 had cervical material available for analysis (31 in the CRT group, 30 in RT group). Ethics approval for the present analyses was obtained from regulatory authorities in Brazil (Research Ethics Committee approval CAAE# 55014816.5.0000.5404, 11 December 2017). All methods were carried out in accordance with guidelines and regulations from the hospital institutional review board and Ethics Comittee. Follow-up assessments were performed every 4 months in the rst 2 years following the treatment, every 6 months in the third year and therefore once a year.
Immunohistochemistry assay for FUS detection 5mm thick sections of the available samples were stained with hematoxylin and eosin (HE) to ascertain presence of cervical cancer in the For statistical reasons, the percentage of positive cells was further categorized into < 50%, 50-75% and > 90%. Finally, we multiplied the percentage of stained cells by staining intensity, in three distinct elds, and calculated the average of that product (hereinafter referred to as average of three elds).

Statistical analysis
In order to determine the best threshold for the average of three elds to diagnose recurrence, we produced a Receiver Operating Characteristic (ROC), with the resulting optimal cutoff point being 0.233. This cutoff point was used in subsequent analysis to separate patients into two groups, according to the average of three elds (Low if ≤ 0.233 and High if > 0.233). In Tables 1 and 2, we compared the frequencies of key patients' and tumor characteristics as related to either staining intensity, percentage of stained cells and average expression of 3 elds using Chi-squares (or Fisher's exact test where appropriate). In Table 3, we used Cox Proportional Hazard models to evaluate the disease-free, overall survival and cancer-related survival of the patients, as related to FUS expression (average of 3 elds), age, tumor grade and trial allocation group. All calculations were performed using the R Environment for Statistical Computing (18), assuming p < 0.05 (95% con dence intervals) as signi cant. Table 1 shows FUS protein expression characteristics (staining intensity, percentage of stained cells and the average expression of 3 elds) as related to patient age, tumor grade, treatment allocation (either CRT or RT), acute and late toxicity events. There were no signi cant associations between any of FUS expression characteristics and the variables analysed.

Results
In

Discussion
After examining our long-term prospective data, we were able to infer that patients with advanced cervical cancer with high FUS expression in their pathological samples had signi cantly better disease-free and cancer-related survival probabilities compared to those without a high FUS expression level. Other factors associated with survival were age < 50 years and being treated with radiotherapy alone, but the association between FUS expression and survival persisted even after adjustments for age and the treatment allocation. We also found that FUS expression must be determined at several hotspot expression points, since only when examining the average expression of 3 elds we obtained a signi cant association with survival.
Some previous studies focused on the laboratorial aspects of FUS interaction with cancer (19)(20)(21), and less than a handful examined the clinical implications of FUS expression. In this regard, our study is a major stride towards understanding whether FUS protein expression may be used as a tool to prognosticate cervical cancer, since we used a large clinical database on prospectively followed cervical cancer patients to evaluate whether the molecule bore a relationship with survival.
It is important to highlight that our results somewhat contradict previous laboratory ndings on how FUS operates in cervical cancer.
Recently, a Chinese study showed that the expression level of FUS was positively correlated with the expression of circRNA_0000285 and, subsequently, that the knockdown of circRNA_0000285 signi cantly inhibited the formation and metastasis of cervical cancer in nude mice. We must emphasize, however, that those authors did not test for a direct relationship between FUS expression at the protein level and cervical cancer proliferation and metastasis (8). On the other hand, in an attempt at summarizing data about FUS gene expression in association with prostate cancer, Ghanbarpanah and cols. (22) reported that FUS may prevent the growth of prostate cancer cells by down-regulating proliferator factors such as Cdk6 and cyclin D1, and up-regulating Cdk and p27. Further, an immuno-histochemical analysis showed that FUS expression had an inverse relationship with the degree of prostate cancer, which in turn suggests that patients whose tumors have a high FUS expression may experience less bone pain and theoretically enjoy a longer survival. These ndings are in alignment with those from a study on cell cultures published in 2010 (23), in which the authors posited that FUS has some features of a putative tumor suppressor: FUS overexpression promoted growth inhibition and apoptosis of prostate cancer cells, whereas its knockdown led to prostate cancer cell proliferation.
Two studies on hepatocarcinoma have also suggested a positive association between FUS expression and better prognosis. Ma and colleagues (24) examined the association between miR-378 expression and liver cancer cell migration using real-time quantitative PCR, and found that miR-378 overexpression enhanced cell proliferation, migration and liver cell invasion by down-regulating Fus expression.
Further, Bao and colleagues (25) examined the effects FUS has on hepatocellular carcinoma progression in HuH7 and MHCC97 cells, and found that overexpression of FUS decreased cell viability, migration, invasion and stemness, in addition to activating the Hippo pathway, which in turn is an important signaling pathway that regulates organ growth and tissue size (26). All those phenomena associated with FUS overexpression resulted in signi cant inhibition of hepatocellular carcinoma progression.
The small sample size is a weakness of this study because it prevented the analysis of more variables, such as other prognostic factors. However, this study showed a clear statistical association between FUS expression and the prognosis of cervical cancer, which strongly suggests the existence of a relevant biological event. This notwithstanding, further studies with large patient cohorts are need to con rm the results of this work.
In synthesis, our study suggests an important association between FUS protein expression and cervical cancer prognosis. As mentioned above, the disparity in FUS/FUS oncogenic vs. tumor-suppressive roles may be attributed to the complex molecular pathways associated with FUS regulation in different cancer types. In the context of cervical cancer, owing to our prospective dataset, this study suggest that FUS acts as a tumor suppressor. Low cost approaches such as immunohistochemistry can be used in clinical practice to determine FUS protein expression in cervical cancer specimens, which as demonstrated here may be a predictor of longer survival in advanced cases. Con rmatory data from larger studies is pending. Author's disclosures of Potencial Con icts of Interest

Declarations
The authors indicated no potencial con icts of interest.