www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 37 Research Paper XB130 is overexpressed in prostate cancer and involved in cell growth and invasion Bin Chen1,2,*, Mengying Liao3,*, Qiang Wei4,*, Feiye Liu5, Qinsong Zeng6, Wei Wang6, Jun Liu6, Jianing Hou7, Xinpei Yu8,9, Jian Liu9 1Department of Science and Training, General Hospital of Guangzhou Military Command of People’s Liberation Army, Guangzhou, Guangdong, China 2Guangzhou Huabo Biopharmaceutical Research Institute, Guangzhou, Guangdong, China 3Department Of Pathology, Peking University Shenzhen Hospital, Shenzhen, China 4Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China 5Cancer Center, Traditional Chinese Medicine-Integrated Hospital of Southern Medical University, Guangzhou, Guangdong, China 6Department of Urology, General Hospital of Guangzhou Military Command of People’s Liberation Army, Guangzhou, Guangdong, China 7Sun Yat-Sen University, Guangzhou, China 8Guangdong Provincial Key Laboratory of Geriatric Infection and Organ Function Support and Guangzhou Key Laboratory of Geriatric Infection and Organ Function Support, Guangzhou, Guangdong, China 9Center for Geriatrics, General Hospital of Guangzhou Military Command of People’s Liberation Army, Guangzhou, Guangdong, China *These authors contributed equally to this work Correspondence to: Xinpei Yu, email: [email protected] Jian Liu, email: [email protected] Keywords: XB130, adaptor protein, proliferation, invasion, Akt Received: January 27, 2016 Accepted: June 29, 2016 Published: August 05, 2016 ABSTRACT XB130 is a cytosolic adaptor protein involved in various physiological processes and oncogenesis of certain malignancies, but its role in the development of prostate cancer remains unclear. In current study, we examined XB130 expression in prostate cancer tissues and found that XB130 expression was remarkably increased in prostate cancer tissues and significantly correlated with increased prostate specific antigen (PSA), free PSA (f-PSA), prostatic acid phosphatase (PAP) and T classification. Patients with highly expressed XB130 had significantly decreased survival, which suggested XB130 as a possible prognostic indicator for prostate cancer. In vitro experiments showed that reduced XB130 expression restrained tumor growth both in vitro and in vivo. Furthermore, XB130 knockdown hindered transition of G1 to S phase in prostate cancer cell line DU145 and LNCap, which might contribute to the inhibition of cellular proliferation. Results from transwell assay demonstrated that downregulation of XB130 may attenuate invasion and metastasis of prostate cancer. Semiquantitative analysis of Western blot suggested that decreased XB130 expression was accompanied by diminished Akt signaling and EMT process. Thus, above observations suggest that XB130 may be a novel molecular marker and potent therapeutic target for prostate cancer. INTRODUCTION cancer with a low specificity of 18.67%–30.08% as single use in for biopsy [3]. To clarify the molecular mechanism Prostate cancer is the most common and second involved in prostate cancer is of great importance for lethal cancer in western male population [1, 2]. PSA is finding a potent and more effective diagnostic marker and the major significant marker for the diagnosis of prostate improving the treatment of prostate cancer. www.impactjournals.com/oncotarget 59377 Oncotarget Adaptor protein has a unique modular structure XB130 knockdown suppresses growth of prostate without enzymatic activity, which can be regulated by cancer in vitro multiple signaling. It consists of actin filament associated protein (AFAP), Src interacting/Signal integrating protein XB130 mRNA (Figure 2A) and protein (Figure 2B) (Sin) and Crk-associated substrate (CAS). Multiple studies levels were detected in prostate cancer cell line 22RV1, have proved this functions in mitosis, differentiation, LNCap, DU145, PC3, which showed that XB130 inflammation, cell survival, movement, and adhesion expressed in four prostate cancer cell lines at different through binding SH3 and SH2 domains and activating c-Src levels. DU145 and LNCap expressed more XB130 in four [4–6]. AFAP, as a small adaptor protein, is involved in cell prostate cancer cell lines than 22RV1 and PC3, thus were signal transduction, assembling of cell cytoskeleton and chosen for further knockdown studies. other cell functions, which can directly induce activation of To explore the effect of XB130 on proliferation c-Src by mechanical stretch. XB130, also named AFAP1L-2, of prostate cancer, we established two cell lines with is a 130kDa adaptor protein with 818 amino acids located downregulated XB130 expression and named as DU145/ on chromosome 10q25.3 [7]. As the substrate and regulator XB130- and LNCap/XB130-, while control cell lines of signal transduction mediated by tyrosine kinases, XB130 were DU145/mock and LNCap/mock (Figure 2C). CCK8 phosphorylates tyrosine through SH2 and SH3 domains at assay (Figure 2D) showed that proliferation of DU145/ the N-terminal of Src [8]. The role of XB130 varies in cell XB130- was significantly slower than DU145/mock cells proliferation, survival, movement and invasion in different (F = 34.988, p = 0.02). Similar disparity was seen in malignancies [9–11]. In thyroid carcinoma, aberrant XB130 LNCap/XB130- cells (Figure 2D, F = 43.644, P = 0.000). expression induces cell death and enhances apoptosis [12]. Colony formation assay (Figure 3A and 3B) indicated that, Interestingly, high XB130 protein level improves survival knockdown of XB130 in DU145 (F = 95.663, p = 0.000) and sensitivity to 5-Fluorouracil in gastric cancer patients and LNCap (F = 92.789, p = 0.000) could suppress the [13] while correlates with higher pathological grade and formation of colony of both groups comparing to DU145/ poorer prognosis of pancreatic ductal carcinoma [14]. Thus mock and LNCap/mock. Downregulation of XB130 the association between XB130 and prostate cancer are resulted in repression of colony formation by nearly worthy to be explored. Our previous findings demonstrated 84% and 89% in DU145 and LNCap, respectively. that low to moderate XB130 expression were seen in normal, Thus, decreased XB130 led to inhibitive proliferation of hyperplastic and peritumoral prostate tissues, while elevated prostate cancer cell lines, which supported our previous XB130 expression was displayed in prostate cancer tissues conclusion that strong XB130 expression enhanced the (Data not shown), but whether XB130 can affect cellular growth of prostate cancer. In flow cytometry analysis growth and invasion in prostate cancer is unclear. Thus in (Figure 3C and 3D), DU145/XB130- (23.05% vs 26.92%, present study, we further investigated the linkage between p = 0.04) and LNCap/XB130- (18.86% vs 24.12%, XB130 and prognosis of prostate cancer and effects of p = 0.03) displayed strikingly shorter S phase than control XB130 on proliferation, invasion and metastasis of prostate cells. Although not statistically significant, G1 phase cancer cells. prolongation were shown in DU145/XB130- (66.16% vs 56.43%, p = 0.2) and LNCap/XB130- cells (70.45% vs RESULTS 65.43%, p = 0.3), which suggested that decreasing XB130 might subdue the transition of G1 to S phase in prostate XB130 is a novel prognostic indicator for cancer, which might be a cause for the restrain of cellular prostate cancer proliferation. We first examined XB130 expression in 210 cases XB130 knockdown inhibits tumor growth in vivo of prostate cancer with pathological diagnosis. Consistent with our previous findings (data not shown), XB130 We also investigated the effect of XB130 on the expression was remarkably increased in prostate cancer tumor growth using xenograft model in nude mice. Six days tissues as compared with hyperplastic and normal tissues after subcutaneous injection of 107 cells on the back of nude (Figure 1A). As Table 1 indicated, XB130 was positively mice, we started to assess the tumor growth by measuring expressed in 85.6% specimens and significantly correlated the tumor size when apparent tumors were seen on the back with increased prostate specific antigen (PSA) (p = 0.006), of mice. The tumor formation in the XB130 knockdown free PSA (f-PSA) (p = 0.036), prostatic acid phosphatase groups was significantly slower than in control groups. The (PAP) (p = 0.02) and T classification (p = 0.025). Kaplan– tumors of knockdown groups were strikingly smaller when Meier analysis (Figure 1B) revealed that patients with compared with control groups at day 18 and the difference highly expressed XB130 have significantly decreased became significant at day 21 after injection. As indicated in survival rate than those with lower XB130 expression Figure 4A and 4B, remarkable suppression of tumor size (p = 0.0121), which indicated that XB130 expression was and volume was seen in xenografts from DU145/XB130- inversely correlated with survival in prostate cancer. (p = 0.03) and LNCap/XB130- (p = 0.02) when compared www.impactjournals.com/oncotarget 59378 Oncotarget Table 1: Relation between XB130 expression and clinicopathologic variables inprostate cancer patients variables low expression high expression total t/χ2 P gleason 6.24 ± 1.84 7.04 ± 1.51 –3.295 0.001 PSA 43.08 ± 68.68 79.39 ± 143.15 0.006 f-PSA 8.71 ± 13.70 15.20 ± 22.80 0.036 T classification 1 11 (17.2%) 8 (5.5%) 19 (9.1%) 9.356 0.025 2 37 (57.8%) 102 (70.3%) 139 (66.5%) . 3 8 (12.5%) 11 (7.6%) 19 (9.1%) . 4 8 (12.5%) 24 (16.6%) 32 (15.3%) . PAP 1 6 (9.7%) 4 (2.7%) 10 (4.8%) 7.866 0.02