Characterization of The Expression of The Heavy Chain of 4F2 Antigen As a Therapeutic Target in Prostate Cancer

The 4F2 cell-surface antigen heavy chain (4F2hc) forms a heterodimeric complex with L-type amino acid transporter 1 (LAT1) and transports large neutral essential amino acids. However, in contrast to the traditional role of LAT1 in various cancers, the role of 4F2hc has largely remained unknown. The role of 4F2hc in prostate cancer was studied. Treatment of C4-2 cells with si4F2hc was found suppress cellular growth and migratory and invasive abilities, with this effect occurring through the cell cycle, with a signicant decrease in S phase and a signicant increase in G0/G1 phase, suggesting cell cycle arrest. In addition, it was proven by RNA seq that the key to 4F2hc’s impact on cancer is SKP2. The expression of 4F2hc and LAT1 in prostate cancer cells suggests the importance of 4F2hc. Furthermore, si4F2hc, through the downstream target SKP2, upregulates the protein expression of cyclin-dependent kinase inhibitors (P21cip1, P27kip1). Multivariate analysis showed that high 4F2hc expression was an independent prognostic factor for progression-free survival (HR 11.54, p=0.0357). High 4F2hc was related to the clinical tumour stage (p=0.0255) and Gleason score (p=0.0035). Collectively, 4F2hc contributed signicantly to prostate cancer (PC) progression. 4F2hc may be a novel marker and therapeutic target in PC.


Introduction
Prostate cancer (PC) is the most commonly diagnosed malignancy and one of the most frequently diagnosed cancers in men 1,2 . Despite progress in the treatment of localized PC, management of locally advanced and metastatic disease is still a critical unmet need 3,4 . Androgen deprivation therapy (ADT) is the standard treatment for advanced PC 2 . However, the clinical bene t of ADT is only temporary, and it has always evolved into castration-resistant prostate cancer (CRPC) with different treatments 5,6 . A new generation of androgen receptor (AR)-targeting agents has been developed, such as the androgen biosynthesis inhibitor abiraterone and the AR inhibitor enzalutamide, and they have been shown to improve overall survival (OS) 6,7 . However, treatment resistance remains a signi cant challenge for CRPC. Some reports showed major improvement with abiraterone and enzalutamide treatment, whereas there was no decrease in serum prostate-speci c antigen (PSA) levels 8,9 . Thus, a novel no-AR therapeutic approach and biomarker candidates for CRPC remain a signi cant issue.
Cancer cells require substantial amounts of nutrients to grow and multiply, especially cancer cells that have lost normal reproductive function. Amino acids are essential nutrients that are transported to the cell by a selective transporter on the plasma membrane [10][11][12] . Amino acid transporters are required for tumour growth and proliferation. Several lines of evidence have shown that nutrient transporters are upregulated in cancer cells while supporting large-scale growth and reproduction 13 .
L-type amino acid transporter 1 (LAT1, SLC7A5), a systemic L amino acid transporter, requires a covalent association with 4F2hc for its functional expression in the plasma membrane 14 . It has been shown that LAT1 is abundantly expressed in various types of cancers, including non-small cell lung cancer, breast cancer, biliary tract cancer, pancreatic cancer, and prostate cancer [15][16][17][18][19] . Besides LAT1, several studies have shown an association between higher 4F2hc expression levels and worse prognosis in various types of cancer [20][21][22] . In addition, we recently identi ed 4F2hc as one of the main target genes for AR-V7.
Upregulation of 4F2hc through AR-V7 contributes to the progression of CRPC. 23 Based on the above-described ndings, it may be reasonable to expect that 4F2hc is a promising prognostic biomarker, as well as a molecular target, in PC. In this study, the oncogenic function of 4F2hc in PC and its relationship with the clinical outcome of PC patients was studied.

PC tissue specimens
A total of 82 clinical PC tissue samples were obtained from patients who underwent radical prostatectomy at Chiba University Hospital between 2006 and 2014. The present study was conducted in accordance with ethical standards that promote and ensure respect and integrity for all human subjects and the Declaration of Helsinki. The study was approved by the Review Board (approval number 408), and the patients signed a written, informed consent form.
Cell culture and transfection DU145 and C4-2 cell lines were obtained from the RIKEN Cell Bank (Tsukuba, Japan). PC cell lines were supplemented with 10% foetal bovine serum (FBS) in RPMI 1640 culture medium and maintained in a humidi ed atmosphere incubator (95% air, 5% CO 2 , 37 °C). Lipofectamine RNAiMAX reagent (Invitrogen) was used, and siRNA was transfected with PC cells. Detailed experimental methods have been previously reported. 35 mRNA expression evaluation Total RNA was isolated using the RNeasy Mini Kit. cDNA was synthesized with the ImProm-II™ Reverse (Promega, Madison, WI, USA). mRNA expression was carried out as described previously 35 with ABI 7300 (Applied Biosystems, Foster, CA, USA) and SYBR Green PCR Master Mix (QPS-201, Toyobo, Japan). GAPDH (internal control) and PCR primers used in this study are listed in Table S1.

Western blot analysis
Protein expression levels were measured using GAPDH as the control. Protein samples (24 mg) were subjected to SDS-PAGE and transferred to Hybond-C membranes (GE Healthcare, Chicago, IL, USA). The membranes were then blocked (5% skim milk, 30 min, 37 °C). The primary antibody was incubated overnight at 4 °C. Detailed experimental methods have been previously reported. 35 Immunohistochemistry (IHC) Immunohistochemistry procedures were performed according to a previously described method. 35 The slides were treated with endogenous peroxidase (30% hydrogen peroxide solution, 100% methanol, 10 min), and then incubated with anti-4F2hc and anti-SKP2 (4 °C overnight). Finally, the slides were lightly counterstained with haematoxylin, dehydrated with ethanol, cleared with xylene, and mounted.
IHC scores were as follows: 3 intense staining; 2 moderate; 1 very weak; and 0 no staining. IHC scores were calculated as follows: IHC score = 3× (mean percentage of intensely stained cells in the eld) +2× (mean percentage of moderately stained cells in the eld) +1× (mean percentage of weakly stained cells in the eld). Two independent investigators blinded to patient clinical status scored each specimen.
Growth assay, cell migration, and invasion assay Detailed experimental methods have been previously reported. 35 Following the manufacturers' instructions, the migration assay was performed using the Cell Counting Kit-8 (343-07623, Dojindo, Japan) and the Falcon Permeable Support Plate (353097, Corning, NY, USA), and the invasion assay used the Matrigel invasion chamber (354480, Corning).

Statistical analysis
The Kaplan-Meier method and univariate and multivariate Cox proportional models were used for statistical analyses. Mathematical calculations were performed using JMP Pro 15 (SAS Institute, Cary, NC, USA).

Analysis of 4F2hc expression and 4F2hc knockdown in PC cell lines
In PC cell lines, the highest 4F2hc protein expression was observed in DU145 cells, followed by PC-3 and C4-2 cells ( Fig. 1A; p=0.0061, p=0.0804, p=0.0026, and p=0.0200; respectively). The expression level of 4F2hc was studied by IHC. Positive immunostaining for 4F2hc was detected in the cell membrane and cytoplasm by IHC of PC specimens. A strong 4F2hc immune response was detected in the cell membrane of cancer lesions, whereas normal adjacent tumour (NAT) mainly showed weak immunostaining. Regarding the IHC scores of 4F2hc staining for NAT and PC lesions, NAT ranged from 0 to 150 (median=20), whereas PC lesions ranged from 0 to 300 (median=90). The IHC score was signi cantly higher for PC lesion 4F2hc staining than for NAT ( Fig. 1B; p=0.0001). Compared with LAT1 expression, signi cantly higher 4F2hc expression was observed in C4-2 and DU145 cells ( Fig. 1C; p=0.0014 and p=0.0020; respectively). According to the basal expression level in Figure 1A, C4-2 and DU145 cells were used for subsequent functional analysis. The expression levels of si4F2hc (si4F2hc-1 and si4F2hc-2) were signi cantly decreased in C4-2 and DU145 cells compared with those in the Negative Control (Nega) ( Fig. 1D and E; D: p=0.0026 and p=0.0043; E: p=0.0034 and p=0.0017; respectively. The functional role of 4F2hc in PC Si4F2hc suppresses cell proliferation, migration, and invasion Levels of 4F2hc mRNA were signi cantly suppressed following transfection of C4-2 and DU145 cells with si4F2hc (si4F2hc-1 and si4F2hc-2) compared with mock-transfected cells or those transfected with Nega. The si4F2hc (si4F2hc-1 and si4F2hc-2)-transfected cells showed signi cant growth reduction compared to the Nega ( Fig. 2A and B; A: p=0.0131 and p=0.0012; B: p=0.0177 and p=0.0033; respectively). Furthermore, si4F2hc-transfected cells also demonstrated signi cant decreases in migration (C: p=0.0075 and p=0.0003; D: p=0.0011 and p=0.0050; respectively) and invasion (E: p=0.0027 and p=0.0015; F: p=0.0079 and p=0.0073; respectively) activities compared with Nega ( Fig. 2C to F).

Analyse downstream genes by RNA-seq
To further elucidate the mechanism underlying inhibition by si4F2hc, the gene target and downstream signals of si4F2hc were investigated. A comparative RNA-seq analysis was performed using si4F2hc (si4F2hc-1 and si4F2hc-2) and Nega. Figure 3A shows a heat map of changes in the RNA-seq analysis under the in uence of si4F2hc. In order to make the heat map more concrete, the most down-regulated gene was selected, and Metascape gene analysis was used (http://www.metascape.org). These commonly downregulated genes were markedly associated with the cell cycle (-log10=43.84), DNA replication (-log10=26.75), and cell division (-log10=23.88) (Fig. 3B). Con rmation of target genes downregulated at the top of the cell cycle was performed by real-time PCR analysis. Finally, SKP2 was identi ed as a target gene of 4F2hc (Fig. 3C). To study the functional role of SKP2, the growth of siSKP2transfected cells was monitored for ve days. The increase in SiSKP2 (siSKP2-1 and siSKP2-2)transfected cells was signi cantly reduced compared to Nega ( Fig. 3D; p=0.0002 and p=0.0005).
In order to study the associations among 4Fhc, SKP2, and LAT1 expressions, the effects of si4F2hc on SKP2 and 4F2hc expressions and the effects of siSKP2 on 4F2hc and LAT1 expressions were studied.

Regulation of SKP2 and the cell cycle pathway by 4F2hc
Given the key function of SKP2 in the cell cycle and its molecular interplay with P21cip1 and P27cip1, a potential role of SKP2 in cell cycle progression was investigated. Treatment of C4-2 cells with si4F2hc-1 led to a signi cant decrease in the S phase and a substantial increase in the G0/G1 phase, suggesting cell cycle arrest (Fig. 4A to C). The percentage of si4F2hc-transfected cells was higher in the G0/G1 phase than in Nega-transfected cells (Fig. 4D). Next, whether 4F2hc regulation is associated with the expression of cyclin SKP2 in these cells and the regulation of the expression level or activation state of downstream signals were investigated. Western blot analysis showed signi cant downregulation of SKP2 in a state in which 4F2hc was inhibited, inhibition of phosphorylation of MAPK and AKT, and upregulation of protein expression by cyclin-dependent kinase inhibitors (P21cip1, P27kip1) (Fig. 3E).
4F2hc and LAT1 expression in PC tissue and association with clinical variables The clinical signi cance of 4F2hc was further investigated, along with examination of 4F2hc expression in PC specimens, by IHC. Positive immunostaining for 4F2hc was detected in the cell membrane and cytoplasm. Intense immunostaining for 4F2hc was detected in cancerous lesions, whereas nonmalignant lesions showed negative or weak immunostaining. Positive immunostaining for SKP2 was detected in the nucleus, mainly on the super cial central compartment of the tumour rather than in the tumour margins. Fig. 5 is a representative IHC result for 4F2hc and SKP2 staining. Sections were stained with haematoxylin and eosin ( Next, the prognostic clinical signi cance of 4F2hc and LAT1 expression was evaluated statistically. The patients' characteristics are listed in Table 1. Specimens were divided into two groups based on median 4F2hc and LAT1 IHC scores. The results showed that the high-4F2hc expression group showed signi cantly shorter progression-free survival (PFS) than the no-low 4F2hc expression group (Fig. 6A; p=0.0034). Although the high LAT1 expression group tended to show shorter survival, LAT1 expression was not related to PFS (Fig. S3A; p=0.1085). When combining 4F2hc and LAT1 expression, the high 4F2hc/high LAT1 expression group showed the worst PFS (p=0.0071), followed by the high 4F2hc/low LAT1 or low 4F2hc/high LAT1 expression group (others) (p=0.2297). In contrast, the low 4F2hc/low LAT1 expression group showed good PFS (Fig. S3B).
Next, associations between clinicopathological characteristics and 4F2hc protein expression were investigated. On multivariate analysis, high 4F2hc expression (HR 11.54, p=0.0357) and high clinical tumour stage (CT stage) (HR 4.22, p=0.0280) were also identi ed as independent prognostic factors for PFS ( Table 2). The relationships between 4F2hc expression and various clinical factors were determined.
High 4F2hc expression was associated with high age (p=0.0162), high cT stage (p=0.0255), and high Gleason score (GS) (p=0.0035) in PC patients (Table 3). However, high LAT1 expression was only associated with high GS (p=0.0399) in PC patients (Table S2).

4F2hc and SKP2 expression in PC tissue and associations with clinical variables
The prognostic clinical signi cance of 4F2hc and SKP2 expressions was evaluated statistically. The high SKP2 expression group also showed a signi cantly shorter PFS ( Fig. 6B; p=0.0040). When expressed in conjunction with 4F2hc and SKP2, the high 4F2hc/SKP2 high expression group showed the worst PFS (p=0.0017), followed by the high 4F2hc/low SKP2 or low 4F2hc/high SKP2 expression group (others) (p=0.0623). In contrast, the low 4F2hc/low LAT1 expression group showed good PFS (Fig. 6C). The relationship between SKP2 expression and various clinical factors was determined. High SKP2 expression was associated with high cT stage (p=0.0277), high GS (p=0.0138), high PSA (p=0.0458), and high PSAD (p=0.0360) in PC patients (Table 4).

Discussion
The present study demonstrated several novel ndings. 4F2hc expression seems to be one of the promising therapeutic targets in PC. Of the major clinical factors, including LAT1 expression, 4F2hc expression was the most signi cant prognostic factor in PC patients. The inhibition of 4F2hc function prevents the progression of several PC cell types. Furthermore, SKP2 was newly identi ed as a novel target of 4F2hc. Current evidence indicated the contribution of the cell cycle pathway as a central downstream mechanism of 4F2hc 22,24 . 4F2hc expression is increased in various human neoplasms, such as gastric cancer, pulmonary pleomorphic carcinoma, and neuroendocrine carcinoma [20][21][22] . Moreover, it has been reported that increased 4F2hc expression is signi cantly associated with shorter survival outcomes, cell proliferation, and metastasis 25 . 4F2hc binds with LAT1 on the membranous surface of cancer cells 26 . After the discovery of LAT1, it was also shown that the other ve members of the solute carrier family 7 bind to 4F2hc as a light chain to form a different heterodimeric amino acid transporter. Heterodimerization is essential for its functional expression 27 . Interestingly, relevant research shows that ATF4 regulates 4F2hc, LAT1, and ASCT1 gene, with low expression in healthy prostate tissue and early prostate cancer. However, they are signi cantly increased in metastatic prostate cancer, indicating the essential nutrients required for transporters to promote metastatic prostate cancer 28 .
In previous studies, the oncogenic function of 4F2hc was demonstrated. Several groups reported that 4F2hc inhibits the proliferation of cancer cells by inhibiting the cell cycle. The FACS assay was performed in C4-2 cells. Si4F2hc effectively inhibited the S phase, with a signi cant increase in the G0/G1 period, suggesting cell cycle arrest. These data are consistent with two previous studies reporting the effect of 4F2hc on human osteosarcoma and thymic epithelial tumors 24,29 . 4F2hc has been shown to affect cancer cell proliferation through the AKT, MAPK, and cell cycle related P21 and P27 signal pathways.
SKP2 is related to a cell cycle signal pathway. It is worth noting that overexpression of SKP2 is associated with the promotion and aggravation of many tumors 30,31 . The cell cycle signal pathway is activated in PC. Therefore, inhibition of SKP2 promotes the deterioration of cancer by targeting promotion of p21 and p27 and by cell senescence 32 . Reports show that overexpression of SkP2 coupled with underexpression of p27 is characteristic of CRPC 33 . In line with this evidence, the present data implicated the participation of SKP2 through alteration of the S phase of the cell cycle in the 4F2hc-related pathway. Previous studies demonstrated that mucin 1, mucin 16, and mucin 5B were the downstream genes of 4F2hc in gastric carcinoma cells 34 , although the effect of 4F2hc on transcription remains mostly unknown. In the present study, a comprehensive whole transcriptome shotgun sequencing analysis was conducted using the C4-2 PC cell line and Metascape gene analysis. The data showed that downregulated genes were markedly associated with the cell cycle, DNA replication, and cell division, and identi ed SKP-2 as a speci c target gene among the downregulated genes in C4-2 cells.
Nevertheless, there are several limitations in this study. First, the data were obtained from a single institution, and the number of patients and the follow-up periods were limited. A prospective, multiinstitutional study would be ideal to objectively assess the prognostic signi cance of 4F2hc. Second, the mechanism of how 4F2hc regulates SKP2 remained to be identi ed. We are currently performing a chip assay to verify any direct association between two genes. Third, although the major binding partner of 4F2hc may be LAT1, the functional relationships with other transporters need to be determined.
Collectively, the present results suggest that, in line with its high expression frequency, 4F2hc may be a promising prognostic marker for PC patients. Thus, it is possible that high levels of 4F2hc expression, together with a high level of LAT1 expression, in surgical specimens may indicate the need to follow-up the patient carefully with frequent imaging in order to take precautions against recurrence.

Figure 2
Functional signi cance of 4F2hc in DU145 and C4-2 cells Si4F2hc (si4F2hc-1 and si4F2hc-2) inhibits C4-2 and DU145 cell proliferation (A and B), migration (C and D), and invasion (E and F). Nega indicates negative siRNA control. Data represent three independent experiments with similar results. P-values were calculated by the Mann-Whitney U-test. *p<0.05, **p<0.01. Figure 3 4F2hc RNA-sequencing analysis RNA-seq analysis was performed with si4F2hc (si4F2hc-1 and si4F2hc-2) and compared with a control group (A). The most down-regulated gene is selected, and Metascape gene analysis is used (B). Si4F2hc concentration-dependent effect on candidate genes was assessed by real-time PCR (C). SiSKP2 (siSKP2-1 and siSKP2-2) inhibits C4-2 cell proliferation (D). Data represent three independent experiments with similar results. P-values were calculated by the Mann-Whitney Utest. ***p<0.001. Figure 4 4F2hc signalling pathway Cell cycle analysis was performed with si4F2hc (si4F2hc-1 and si4F2hc-2) and compared with a control group (A to D). After 72 h or 96 h, si4F2hc (si4F2hc-1 and si4F2hc-2) downregulation of SKP2, phosphorylation of AKT and MAPK, and increased expression of p21 and p27 (E). Data represent three independent experiments with similar results.  Progression-free survival of PC patients categorized by 4F2hc and SKP2 expression Prognostic signi cance of 4F2hc expression for progression-free survival (PFS) (A), and prognostic signi cance of