FAM64A Promotes Prostate Cancer Growth In Vivo and In Vitro

Background Prostate cancer (PCa) is the most common type of human cancer in males. However, the mechanisms underlying PCa tumorigenesis remained unclear. Methods The present study evaluated the expression levels of FAM64A in PCa by using 5 public datasets, including GSE8511, GSE45016, GSE55945, GSE38241 and GSE17951. Then, in vivo and in vitro assays were conducted to detect the biological functions of FAM64A in PCa. Microarray and bioinformatic analysis were carried out to detect the downstream targets and pathways regulated by FAM64A. Results In this study, we for rst time demonstrate FAM64A as a biomarker for PCa. FAM64A was found to be overexpressed in PCa compared to normal samples. Higher FAM64A expression were found in Gleason score (GS) ≥ 8 PCa compared to GS < 8 PCa samples, in N1 staging compared to N1 staging PCa samples, and T3/4 staging compared to T1 staging PCa. Moreover, higher FAM64A expression was correlated to shorter survival time in PCa. Knockdown of FAM64A signicantly suppressed PCa cell proliferation and colony formation, however, induced PCa apoptosis in vivo and in vitro. Bioinformatics analysis combined with microarray analysis revealed FAM64A played crucial roles in regulating multiple cancer related pathways, including cell-matrix adhesion and cAMP signaling pathway. Conclusions These results showed FAM64A could serve as a novel biomarker for PCa and will be helpful to understand the underlying FAM64A -related molecular mechanisms in the progression of PCa.


Introduction
As one of the most common types of human cancers in males worldwide, prostate cancer (PCa) had been a substantial economic and medical burden of both governments and households [1,2]. In the past decades, researchers made great efforts to identify key regulators of PCa progression. AR signaling [3], Wnt signaling [4] and MAPK signaling [5,6] were reported to be the most important pathways in PCa. Recently, emerging studies indicated non-coding RNAs, including miRNAs [7], lncRNAs [8,9] and circRNAs [10], were also participated in the regulation of PCa related pathways. For instance, miR-183 promoted PSA synthesis in PCa [11], PCAT19 induced PCa growth and metastasis by interacting with hRNPAB to promote cell cycle regulators expression [12]. However, the mechanisms regulating PCa tumorigenesis and progression remained largely unclear. Therefore, identifying the drivers involved in PCa progression is critical to advance our understanding of PCa and to explore novel therapeutic targets for this disease.
FAM64A was identifed as a CALM/AF10 interacting partner and a mitotic regulator which was involved in regulating metaphase-to-anaphase transition in cell cycle progression [13]. The abnormal cell cycle progression was considered as an important hallmark of cancer cells [14], suggested FAM64A may play an important role in human cancers. Consistent with this hypothesis, emerging studies showed FAM64A was overexpressed in leukemia [15], lymphoma [16] and breast cancer [17]. FAM64A was rst found to play its roles in leukemogenesis by interacting with CALM/AF10 or antagonizing the transactivation activity of CALM/AF10 [15]. In cervical carcinoma HeLa cells FAM64A was involved in regulate metaphase-to-anaphase transition but had no effect on cell proliferation [18]. Moreover, high expression of FAM64A was correlated to shorter survival time in triple-negative breast cancer, whose knockdown signi cantly suppressed cell growth in in basal-like breast cancer cells [17]. However, the roles of FAM64A in the progression of prostate cancer remained to be further investigated.
Here, we compared the expression of FAM64A in normal prostate and PCa samples using public datasets. Using PCa cell lines DU145 and PC-3, we examined the effects of FAM64A on PCa growth in vivo and in vitro. We thought this study could provide evidences to validate FAM64A as a novel biomarker for PCa.

Cell culture.
DU145 and PC-3 were obtained from Chinese Academy of Sciences Cell Bank. DU145 and PC-3 cells were cultured in RPMI-1640 medium (HyClone Laboratories; USA) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scienti c, Inc. USA ). DU145 and PC-3 cells were cultured at 37 °C in a humidi ed incubator with 5% CO2.

Cell cytometry and image analysis
Celigo® Image Cytometer (Nexcelom, Lawrence, MA, USA) was used to evaluate the number of cells by scanning green uorescence daily for 5 days at room temperature according to our previous reports [19].

In vivo tumor metastasis assays
shFAM64A -transfected or ctrol PC-3 cells were suspended in phosphate-buffered saline (PBS) and injected subcutaneously into the backs of six-week-old male BALB/c nude mice (Shanghai SLAC Laboratory Animals Co., Ltd. Shanghai, China). Tumor growth was measured using calipers over the course of 49 days. Tumor volume was calculated according to the formula: volume = 0.5 × length × width2. The mice were euthanized with CO2 and sacri ced on day 49. Tumor weight was measured and compared between two groups. All in vivo studies protocols were approved by the Shanghai Medical Experimental Animal Care Commission (Approval ID: ShCI-14-008).

Western blot analysis
In accordance with standard Western-blot protocols, proteins were separated in 10% SDS-PAGE and transferred to PVDF membrane though Bio-Rad systems (Bio-Rad, Hercules CA, USA). Rabbit anti-FAM64A, and mouse anti-GAPDH antibodies were used in this study. The Quantity One software package (Bio-Rad, USA) was used for the quantitation of signal intensities.

Statistical analysis
Each experiment was performed for three times. student' T-test was used to calculate statistical signi cance between two groups. For more groups, one-way ANOVA followed by Newman-Keuls posthoc test was used. P-values < 0.05 was determined as statistical signi cance. Statistical analysis was performed using the SPSS software package, version 15.0 (SPSS, Inc., Chicago, IL, USA).

Results
3.1 FAM64A was up-regulated in prostate cancer.
The expression of FAM64A in normal and PCa samples remained unknown. Here, we screened GEO datasets and downloaded 5 public datasets to investigate FAM64A levels between PCa and normal samples, including GSE8511, GSE45016, GSE55945, GSE38241 and GSE17951. As shown in Fig. 1, we observed FAM64A was signi cantly overexpressed in PCa compared to normal samples by analyzing these GEO datasets. Furthermore, we validate FAM64A expression pro le in TCGA dataset, which was based on RNA-sequence method. We found FAM64A expression in PCa samples were higher than that in normal tissues by analyzing TCGA datasets (Fig. 1F).
3.2 Higher FAM64A expression was associated with shorter overall survival time in prostate cancer Furthermore, we evaluated the correlation between FAM64A levels and clinical varies in PCa. We found FAM64A was up-regulated in Gleason score (GS) ≥ 8 PCa and T3 and T4 stage PCa compared to GS < 7 PCa and T2 stage PCa ( Fig. 2A-B). Furthermore, our analysis showed FAM64A was overexpressed in N1 staging PCa samples in comparison with N0 staging PCa tissues (Fig. 2C). We also analyzed whether the dysregulation of FAM64A was correlated to overall survival time in prostate cancer using Kaplan-Meier method (Fig. 2D). These results suggested the dysregulation of FAM64A could serve as a novel biomarker for PCa.

Knockdown of FAM64A suppresses cell proliferation in prostate cancer
Our above analyses suggested that FAM64A may serve as an oncogene in PCa. Aiming to explore the functions of FAM64A in PCa, we knockdown its expression in PCa cells using a speci c shRNA against FAM64A. After transfecting PC-3 and DU145 with shFAM64A, we observed the expression of FAM64A was remarkably decreased compared to control groups. Meanwhile, western blot assay showed FAM64A protein level was reduced after transfection in DU145 cells (Fig. 3A-D).
Then, we measured the effect of FAM64A on PCa growth using Celigo cell counting assay for ve days. Compared with the control group, knockdown of FAM64A signi cantly reduced cell proliferation in PC-3 and DU145 by 3.76-and 6.41-fold, respectively (Fig. 3E-F). Next, a CCK-8 assay was conducted.
Consistent with above nding, we found knockdown of FAM64A suppressed PC-3 and DU145 cell proliferation rate (Fig. 4A-B).

Silencing of FAM64A inhibits prostate cancer cell colony formation
Furthermore, the cell colony formation assay was conducted to determine the roles of FAM64A in regulating PCa growth. As shown in Fig. 4, FAM64A knockdown remarkably inhibited the ability of PC-3 and DU145 cells to form colonies. The cell colonies in FAM64A knockdown group were signi cantly decreased by 75 percent and 85 percent, respectively.

Knockdown of FAM64A induced cell apoptosis in prostate cancer.
Deregulation in apoptotic pathways had been considered as a hallmark of human cancers. In present study, we accessed cell apoptosis in FAM64A knockdown group and control group by staining V-APC, which was analyzed by ow cytometry. Three days' post transfection, the number of PC-3 and DU145 apoptotic cells in FAM64A knockdown group were increased by 50% and 60% compared to control group, respectively. Taken together, these results showed FAM64A acted as an oncogene by inducing cell proliferation and reducing cell apoptosis.

Knockdown of FAM64A suppressed prostate cancer growth in vivo
The effects of FAM64A knockdown on tumor growth were analyzed in vivo using nude mouse models.
The luciferase-expressing DU145 cells were constructed to race tumors in live animals. By using an IVIS system, we found luciferase signaling in FAM64A knockdown group was decreased compared to control group. In vivo tumor growth curve showed that knockdown of FAM64A signi cantly suppressed PCa growth in nude mouse. The mice were euthanized with CO2 and sacri ced on day 34. Tumor weight was measured and the results showed the tumor weight in FAM64A knockdown group was less than that in negative control group (Fig. 6).

Identi cation of FAM64A downstream targets in PCa using microarray analysis
To understand the mechanisms of FAM64A in regulating PCa progression, we used a gene expression microarray to identify FAM64A targeting genes in DU145 cells. A total of 259 genes (including 75 upregulated and 184 down-regulated genes) were identi ed as downstream targets of FAM64A in PCa.
DAVID system was used to predict the potential functions of FAM64A in PCa. GO analysis showed FAM64A was involved in regulating cell-matrix adhesion, chondrocyte proliferation, cell division, type I interferon signaling pathway, extracellular matrix organization, apoptotic process, cellular response to insulin stimulus, autophagy, fatty acid metabolic process, and regulation of translation. KEGG analysis showed FAM64A was associated with the regulation of Pertussis, Complement and coagulation cascades, Hematopoietic cell lineage, Arginine biosynthesis, and cAMP signaling pathway (Fig. 7).

Discussion
PCa is the most common type of human cancer in males. However, the mechanisms underlying PCa tumorigenesis remained unclear. In this study, we for rst time demonstrate FAM64A as a biomarker for PCa. FAM64A was found to be overexpressed and correlated to shorter survival time in PCa. Knockdown of FAM64A signi cantly suppressed PCa cell proliferation and colony formation, however, induced PCa apoptosis in vivo and in vitro. Bioinformatics analysis combined with microarray analysis revealed FAM64A played crucial roles in regulating multiple cancer related pathways, including cell-matrix adhesion and cAMP signaling pathway.
Previous studies indicated FAM64A was involved in regulating cancer progression. FAM64A was a CALM/AF10 co-factor, which regulated cell cycle processes in cells. FAM64A was up-regulated in a few types of cancers, such as leukemia and breast cancer. For instance, FAM64A overexpression was correlated to shorter survival time in triple-negative breast cancer. In this study, we found FAM64A was signi cantly overexpressed in PCa compared to normal samples by analyzing GEO datasets and TCGA database. Moreover, we found PCa was up-regulated in advanced N staging and T staging PCa samples.
Higher FAM64A was associated with shorter disease-free survival time in PCa. These results suggested FAM64A could act as a biomarker for PCa prognosis.
The functions of FAM64A in PCa remained unknown. Loss of function assay showed knockdown of FAM64A suppresses cell proliferation and colony formation in PCa. However, silencing of FAM64A induced cell apoptosis in PCa. Of note, nude mouse models were used to evaluate the effects of FAM64A on PCa growth in vivo. The results showed knockdown of FAM64A suppressed prostate cancer growth in vivo. Furthermore, bioinformatics analysis and microarray analysis were conducted to explore the mechanisms of FAM64A in regulating PCa progression. Bioinformatics analysis showed FAM64A was involved in regulating cell-matrix adhesion, chondrocyte proliferation, cell division, type I interferon signaling pathway, extracellular matrix organization, apoptotic process, cellular response to insulin stimulus, autophagy, fatty acid metabolic process and cAMP signaling pathway.
By constructing FAM64A related PPI network, we identi ed MAPK8, PRKACA, MAPK9, MX1, APP, GBP1, GNAI1, ITGA2, and ITGA6 as key mediators of FAM64A regulating PCa progression. MAPK8 and MAPK9 were reported to regulate autophagy in non-small cell lung cancer [20]. PRKACA restored anti-apoptotic signaling in breast cancer [21]. ITGA2 was found to be dysregulated in various cancers, including Gastric Cancer [22], pancreatic cancer [23], breast cancer [24] and prostate cancer [25]. Knockdown of ITGA2 promotes Gastric Cancer apoptosis and suppressed cell migration [22]. In prostate cancer samples, the methylation levels in the ITGA2 promotes were signi cantly decreased compared to normal tissues. ITGA6 was a hypoxia-responsive gene, which enhanced cancer stem cell activity and metastatic phenotypes in breast cancers [26]. These reports together with our nding showed FAM64A could serve as a novel biomarker for PCa and will be helpful to understand the underlying FAM64A -related molecular mechanisms in the progression of PCa.

Availability of data and materials
All relevant data that support the ndings of this study are available from the corresponding author upon reasonable request.

Ethics approval
All animals were housed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC 2016-0298).

Consent for publication
Not applicable.