Ryo Kanai,Takeshi Uehara,Takahiro Yoshizawa,Masato Kamakura,Tomoyuki Nakajima,Yasuhiro Kinugawa,Mai Iwaya,Shiho Asaka,Masato Kitazawa,Tadanobu Nagaya,Hiroyoshi Ota
ARL4C is associated with epithelial-to-mesenchymal transition in colorectal cancer
ARL4C,Colorectal cancer,Tumor budding,RNA in situ hybridization,Colorectal adenocarcinoma
Background ADP-ribosylation factor-like protein 4 C (ARL4C) is a member of the ARF small GTP-binding protein subfamily. The ARL4C gene is highly expressed in colorectal cancer (CRC). ARL4C protein promotes cell motility, invasion, and proliferation. Methods We investigated the characteristics of ARL4C by comparing its expression at the invasion front and relationships with clinicopathological data using RNAscope, a highly sensitive RNA in situ method. Results In all cases, ARL4C expression was observed in cancer stromal cells and cancer cells. ARL4C expression in cancer cells was localized at the invasion front. In cancer stromal cells, ARL4C expression was significantly stronger in cases with high-grade tumor budding than in cases with low-grade tumor budding (P = 0.0002). Additionally, ARL4C expression was significantly increased in patients with high histological grade compared with those with low histological grade (P = 0.0227). Furthermore, ARL4C expression was significantly stronger in lesions with the epithelial-to-mesenchymal transition (EMT) phenotype compared with the non-EMT phenotype (P = 0.0289). In CRC cells, ARL4C expression was significantly stronger in cells that had the EMT phenotype compared with those with a non-EMT phenotype (P = 0.0366). ARL4C expression was significantly higher in cancer stromal cells than in CRC cells (P < 0.0001). Conclusion Our analysis reinforces the possibility that ARL4C expression worsens the prognosis of patients with CRC. Further elucidation of the function of ARL4C is desired.
ARL4C is associated with epithelial-to-mesenchymal transition in colorectal cancer

ADP-ribosylation factor-like protein 4 C (ARL4C) is a member of the ARF small GTP-binding protein subfamily. The ARL4C gene is highly expressed in colorectal cancer (CRC). ARL4C protein promotes cell motility, invasion, and proliferation.

We investigated the characteristics of ARL4C by comparing its expression at the invasion front and relationships with clinicopathological data using RNAscope, a highly sensitive RNA in situ method.

In all cases, ARL4C expression was observed in cancer stromal cells and cancer cells. ARL4C expression in cancer cells was localized at the invasion front. In cancer stromal cells, ARL4C expression was significantly stronger in cases with high-grade tumor budding than in cases with low-grade tumor budding (P = 0.0002). Additionally, ARL4C expression was significantly increased in patients with high histological grade compared with those with low histological grade (P = 0.0227). Furthermore, ARL4C expression was significantly stronger in lesions with the epithelial-to-mesenchymal transition (EMT) phenotype compared with the non-EMT phenotype (P = 0.0289). In CRC cells, ARL4C expression was significantly stronger in cells that had the EMT phenotype compared with those with a non-EMT phenotype (P = 0.0366). ARL4C expression was significantly higher in cancer stromal cells than in CRC cells (P < 0.0001).

Our analysis reinforces the possibility that ARL4C expression worsens the prognosis of patients with CRC. Further elucidation of the function of ARL4C is desired.

Colorectal cancer (CRC) is one of the leading causes of death worldwide. As Western diet and lifestyle habits are increasingly adopted globally, CRC rates are rapidly increasing. In 2018, 1.8 million people were newly diagnosed with CRC, and approximately 880,000 people died from CRC [1]. CRC ranks third among all cancers in terms of incidence and second in terms of mortality. CRC is predominantly an adenocarcinoma [2]. The adenoma-carcinoma sequence is particularly well known in CRC carcinogenesis [3]. Furthermore, chromosomal instability, microsatellite instability (MSI), and the CpG island methylator phenotype have been found to be associated with carcinogenesis [4–6]. In recent years, omics-based analyses including those of the genome, epigenome, transcriptome, and metabolome have reconfirmed and integrated concepts related to carcinogenesis, which have progressed our understanding of carcinogenic mechanisms. Elucidation of the etiology of invasion and metastasis in CRC is progressing. In the RAS/RAF/MAPK pathway involving KRAS and BRAF, RAS activation promotes cell survival, tumor invasion, and metastasis [7]. KRAS mutation and aberrant TP53 expression regulate VEGF and VEGFR activity and promote cancer growth and migration [7]. However, much is still unknown about cancer invasion and metastasis. ADP-ribosylation factor-like protein 4 C (ARL4C) is a member of the ARF small GTP-binding protein subfamily [8]. Simultaneous activation of the Wnt/β-catenin and EGF/RAS pathways results in ARL4C gene expression, leading to epithelial cell morphological changes and tubular structure formation [9]. The ARL4C gene is highly expressed in CRC, lung adenocarcinoma, lung squamous cell carcinoma, and tongue squamous cell carcinoma [10] [11]. ARL4C also promotes cell motility, invasion, and proliferation [11]. Hu et al. reported that ARL4C causes peritoneal dissemination in gastric cancer [12]. ARL4C is a potential therapeutic target because tumor growth is suppressed by siRNA against ARL4C [10]. Several other studies have explored ARL4C as a potential therapeutic target [13–15]. It has been reported that the appearance of the invasion front affects the prognosis of CRC [16, 17]. In this study, we investigated the characteristics of ARL4C by comparing its expression in the invasion front and relationships with clinicopathological data in human CRC. We utilized an RNAscope kit from Advanced Cell Diagnostics (Hayward, CA, USA) to analyze ARL4C mRNA expression. This in situ hybridization technique is highly sensitive with minimal background noise, and utilizes a unique double “Z-shaped” probe that targets RNA sequences spanning approximately 18–25 bases. Upon hybridization, the probe binds to amplifier probes that recognize the chromogenic label. The RNAscope method is well-suited for semi-quantitative analysis and enables precise expression level analysis.

In total, 92 cases of CRC that were treated at Shinshu University (Matsumoto, Japan) between 2018 and 2020 were selected for this study. Among them, 14 cases were excluded because of insufficient samples available for evaluation, and 12 cases of mucinous adenocarcinoma were also excluded. Finally, 66 cases of CRC with invasion were examined.

A tissue microarray (TMA) was constructed as previously reported [18]. Briefly, the TMA was constructed from specimens fixed in 4% formaldehyde and embedded in paraffin. We collected clinicopathological data from the patients’ medical records. Two pathologists (T.U. and M.I.) re-evaluated the histological features of all specimens. One 3-mm core centered on the invasion front was created from a sample from each patient. The 3-mm core was sufficient to assess the pathological condition of the surroundings. The captured area was carefully selected for all hematoxylin–eosin staining specimens of pre-prepared excision material. Tumor budding (TB) was carefully investigated before the region used in the TMA was chosen. TB was graded as Bd1 (0–4 buds), Bd2 (5–9 buds), and Bd3 (≥ 10 buds) [19]. Furthermore, TB grades were categorized into low-grade (Bd1) and high-grade (Bd2 and Bd3). Immunohistochemistry was performed for E-cadherin (clone 36; dilution 1:2000; BD Biosciences, Franklin Lakes, NJ, USA) and vimentin (clone V9; dilution 1:50; Leica, Wetzlar, Germany). For antigen retrieval, sections were microwaved in 0.45% Tris/5 mM EDTA for 30 min. Detection of the primary antibodies was performed using an Envision detection system (Agilent Technologies, Santa Clara, CA, USA) in accordance with the manufacturer’s recommendations. In accordance with a previous report [18], membranous E-cadherin expression was divided into grades 0 to 3. Scores 0 and 1 were classified as E-cadherin negative, and scores 2 and 3 were classified as E-cadherin positive. For vimentin, clear positive staining in the cytoplasm of tumor cells was regarded as positive expression. Epithelial-mesenchymal transition (EMT) phenotypes were divided into (1) non-EMT type, which was defined as E-cadherin positive and vimentin negative, (2) incomplete EMT type, which was defined as E-cadherin negative and vimentin negative or E-cadherin positive and vimentin positive, and (3) complete EMT type, which was defined as E-cadherin negative and vimentin positive, in accordance with a report by Aruga et al. [20]. The incomplete and complete EMT types were analyzed together as the EMT phenotype group and the non-EMT type was analyzed as the non-EMT phenotype group. ARL4CRNA in situ hybridization. The detection of ARL4C mRNA was performed using an RNAscope kit (Advanced Cell Diagnostics, Hayward, CA, USA) in accordance with the manufacturer’s instructions [21]. The standard positive control (Mm-PPIB, ACD-313,902) and negative control (DapB, ACD-310,043) probes were used to ensure interpretable results. Brown punctate dots in the nucleus and/or cytoplasm indicated positive staining. ARL4C expression was quantified under a 20× objective lens (Olympus BX51, Tokyo, Japan) and was scored according to a five-grade scoring system. Furthermore, ARL4C mRNA expression was categorized into low expression (grades 0, 1+, and 2+) and high expression (grades 3 + and 4+). We analyzed the relationship between ARL4C expression and the clinicopathological data and prognosis of patients with CRC, with a focus on overall survival (OS).

To investigate the relationship between ARL4C expression and infiltrating immune cells in CRC, we analyzed 497 cases and 17,501 genes from the CRC dataset in the Pan-Cancer Atlas of The Cancer Genome Atlas (TCGA). We excluded pTis and pT1, POLE-mutated, and MSI-H colorectal adenocarcinoma, as well as cases and genes with missing information. We used the CIBERSORT algorithm to evaluate the expression levels of 22 types of immune cells in the TCGA databases.

Fisher’s exact test or the Wilcoxon rank-sum test were used to assess between-group differences. A P-value < 0.05 was considered significant. Spearman’s rank correlation coefficient analysis was used to assess correlations. The OS rates of CRC patients were calculated using the Kaplan–Meier method, and differences were compared using the log-rank test. All statistical analyses were performed using JMP Statistics software version 13 (JMP, Tokyo, Japan).

In all cases, ARL4C expression was observed in cancer stromal cells and cancer cells (Fig. 1). In cancer cells, ARL4C expression was localized at the invasion front. ARL4C expression was diffuse-positive in 17 cases and localized to the invasion front including TB in 49 cases. In cancer stromal cells, ARL4C expression were identified near cancer cells and ARL4C expression varied from diffuse to scattered patterns.

Clinicopathological data are shown in Table 1. In cancer stromal cells, ARL4C expression was significantly stronger in cases with high-grade TB than in cases with low-grade TB (P = 0.0002) (Fig. 2). Additionally, ARL4C expression was significantly stronger in cases with a high histological grade than in those with a low histological grade (P = 0.0227). Furthermore, ARL4C expression was significantly stronger in cases with the EMT phenotype compared with those with the non-EMT phenotype (P = 0.0289). In cancer cells, ARL4C expression was significantly stronger in cells with the EMT phenotype compared with those with the non-EMT phenotype (P = 0.0366). In cancer stromal cells, there was weak positive correlation between ARL4C expression and TB grades (r = 0.3526, P = 0.0037). However, in cancer cells, ARL4C expression was not correlated with TB grades (r = 0.1730, P = 0.1647).

ARL4C expression was significantly higher in cancer stromal cells than in cancer cells (P < 0.0001) (Fig. 3).

The prognostic value of ARL4C expression in CRC was analyzed by the Kaplan–Meier method and log-rank test. The median OS for the study patients was 24 (range; 17–34) months. A significant difference in OS was not found between CRC patients in the ARL4C-high expression group [median OS: 23 (range; 17.5–33.5) months] and ARL4C-low expression group [median OS: 32 (range; 12.75–45.5) months] (log-rank test, P = 0.6921).

Our analysis revealed that ARL4C had weak but significant positive correlations with M2 macrophages (r = 0.413, P < 0.001) and M1 macrophages (r = 0.342, P < 0.001). However, there was no significant correlation between ARL4C expression and the other 20 types of immune cells evaluated by CIBERSORT. These findings suggest that ARL4C may play a role in the polarization of macrophages in CRC.

Although some prognostic involvement of ARL4C expression in colorectal carcinoma has been suggested, our analysis reinforces the possibility that ARL4C expression worsens the prognosis in CRC. Moreover, ARL4C expression has been identified in cancer cells in other reports, but in our study, ARL4C expression was identified in cancer cells as well as cancer stromal cells. Furthermore, ARL4C expression in the cancer stromal cells was stronger, suggesting that ARL4C expression in cancer stromal cells may have various effects on the tumor microenvironment. Moreover, ARL4C expression in cancer stromal cells was associated with poorly differentiated adenocarcinoma components and higher TB grade. Both are known to be factors that worsen a prognosis, suggesting ARL4C expression in cancer stromal cells is of great prognostic significance. ARL4C was highly expressed in cancer cells and cancer stromal cells with the EMT phenotype. This is consistent with previous reports of other cancers [12]. ARL4C expression appeared to be strongest at the invasion front. Because TB grade has a strong influence on prognosis, the association between ARL4C expression and TB grade may have a strong influence on cancer metastasis. Further analysis of ARL4C in cancer cells of TB is necessary. ARL4C expression in cancer stromal cells has not been reported by immunostaining, so ARL4C expression may be an RNA in situ phenomenon only. However, previous ARL4C immunostaining suggests ARL4C positivity in cancer stromal cells [22]. Recently, the pathogenesis of cancer-associated fibroblasts (CAFs) has been elucidated. TGF-β1 is an important factor in CAFs. Xie et al. reported that ARL4C was highly correlated with TGF-β1 signaling [23]. Silencing ARL4C inhibited SMAD phosphorylation, which is a downstream factor of TGF-β1 signaling. Conversely, co-expression of ARL4C and TGF-β1 worsened the prognosis of gastric cancer patients. ADP-ribosylation factor 6 (ARF6) is downstream of ARL4C and has been reported to play an important role in promoting gastric cancer EMT [24, 25]. Therefore, ARL4C may promote EMT by activating ARF6. A similar phenomenon may also occur in CRC. Harada et al. found strong ARL4C expression in invasive pseudopods in pancreatic cancer [26]. Invasive pseudopods are cells closely related to EMT [27], and are usually identified in TB regions [28]. Therefore, it has been speculated that ARL4C expression is increased in the TB region and is involved in EMT. Our results support these conclusions. A Gene Set Enrichment Analysis (GSEA) study showed that ARL4C expression was positively correlated with an EMT gene set [12]. In the same paper, Hu et al. concluded that ARL4C expression was a poor prognostic factor. Therefore, a detailed analysis of ARL4C and prognosis is desired in the future. Silencing ARL4C markedly inhibited gastric cancer cell proliferation and metastasis [23]. It is strange that our study did not show a significant difference in stage, which is closely related to prognosis. The reason may be related to the small number of cases. The analysis of markers that reinforce ARL4C, such as ARF6, may also be necessary. Our study had some limitations. This study had a relatively small sample size, which may have led to unreliable estimates. Expression analysis using cultured cells is desirable to clarify the causal relationship between ARL4C and prognosis.

Our analysis supports the possibility that ARL4C expression worsens prognosis. Further elucidation of the function of ARL4C is desired.