Research ArticleExperimental Studies
Open Access

Contrasting Role of COMMD5 in Renal Cell Carcinoma: Tumor Suppression and Metastatic Enhancement

HIROYUKI MATSUDA, JIN IKEDA, MAIKO OGASAWARA-NOSOKO, MORITO ENDO, PAVEL HAMET and JOHANNE TREMBLAY
Anticancer Research April 2025, 45 (4) 1373-1385; DOI: https://doi.org/10.21873/anticanres.17523

Abstract

Background/Aim: Despite diagnostic and therapeutic advances, renal cell carcinoma (RCC) metastasis remains difficult to eliminate. Copper metabolism MURR1 domain-containing 5 (COMMD5), a protein involved in tubular epithelial integrity, is implicated in RCC tumorigenesis. This study explored its role in RCC metastasis.

Materials and Methods: Proliferation, adhesion, and survival under hypoxia in RCC cells over-expressing COMMD5 were evaluated. A murine model was used to evaluate metastatic potential in vivo. Clinical data were analyzed for correlations between COMMD5 expression and patient outcomes.

Results: COMMD5 over-expression facilitated cancer cell differentiation and suppressed proliferation but enhanced adhesion and survival under hypoxia. In vivo, it reduced metastatic lung nodule growth but increased the number of metastatic lung nodules. Clinically, high COMMD5 expression in normal kidneys correlated with smaller RCC size, while high COMMD5 expression in tumors was linked to poor metastasis-free survival.

Conclusion: COMMD5 plays dual roles in RCC progression, as a tumor suppressor and a metastasis enhancer.

Keywords:

Introduction

The global incidence of kidney cancer in 2020 was estimated at 431,288 newly diagnosed cases and 179,368 related deaths, according to the GLOBOCAN database, highlighting its significant health burden (1). Renal cell carcinoma (RCC) is the most prevalent malignant type of kidney cancer, accounting for nearly 90% of all kidney malignancies worldwide (2). RCC often progresses silently, with approximately 20 to 30% of patients presenting with metastatic disease at the time of the initial diagnosis. The lungs are the most common site of metastasis in RCC, followed by bone, accounting for 20-35% of all metastases. Other common sites include the lymph nodes, liver, adrenal glands, and brain. Despite advances in diagnostic imaging optimization and systemic therapies, the prognosis for metastatic RCC (mRCC) remains poor (3). The 5-year overall survival rate for patients with mRCC is approximately 20%, compared to over 90% for patients with localized disease (4, 5). Thus, continued research into effective treatment strategies to improve the prognosis of patients with advanced/metastatic RCC remains crucial.

The hypertension-related, calcium-regulated gene (HCaRG), also known as copper metabolism MURR1 domain-containing 5 (COMMD5), is characterized by a conserved COMM domain at the carboxy-terminal end and is highly expressed in the kidneys (6). We previously demonstrated that COMMD5 over-expression in murine proximal tubules (PTs) accelerated tubular repair in postischemic kidneys by facilitating redifferentiation of injured PT cells through the induction of p21Cip1/WAF1 through a p53-independent pathway, resulting in faster recovery of the renal function and improved murine survival (7). In addition, COMMD5 protects PTs from cisplatin-induced nephrotoxicity by maintaining tubular epithelial integrity and autophagy flux, thereby reducing mitochondrial dysfunction and apoptosis (8). Morphological assessments have shown that COMMD5 over-expression in cultured human embryonic kidney cells is associated with epithelial differentiation, including a cobblestone-like epithelial shape and a low proliferative state (9).

COMMD5 levels were lower in cancer cell lines derived from glioblastoma, a partially differentiated RCC, and a moderately differentiated hepatocellular tumor than those in their corresponding normal cell lines (6). In addition, in patients with larger clear-cell RCC (ccRCC), COMMD5 levels are frequently suppressed not only in ccRCC specimens but also in non-tumorous PTs adjacent to tumors relative to adjacent kidney tissues in patients with smaller ccRCCs (10, 11). Based on our previous findings (10, 11), COMMD5 is not only localized intracellularly in cultured RCC cells but also secreted from PT cells, and it reduces malignant behaviors such as rapid proliferation, invasion and sphere formation. In a subcutaneous tumor model, mice injected with Renca cells, which were isolated from the murine renal cortical adenocarcinoma, demonstrated that the intracellular over-expression of COMMD5 suppressed in vivo tumor formation (10). However, its impact on the distant metastasis of RCC remains unknown. Thus, the purpose of this study was to investigate the impact of COMMD5 in RCC cells on the growth of metastatic lung tumors.

Materials and Methods

Stable transfection and cell culture. Renca cells were obtained from the American Type Culture Collection (Rockville, MD, USA). Renca cells were transfected with a control plasmid (pcDNA/Neo1; Thermo Fisher Scientific, Waltham, MA, USA) or a plasmid encoding rat COMMD5 using Attractene transfection reagent (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol (10). Transfected cells were first selected using medium containing G418 (Merck Millipore, Billerica, MA, USA). Single clones were isolated, and the cells were subsequently cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Sigma–Aldrich, St. Louis, MO, USA).

Western blotting. Cultured cells transfected with control plasmid (Neo-Renca) or COMMD5 expression plasmid (COMMD5-Renca) were lysed in a modified radioimmuno-precipitation assay protein extraction buffer (150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, 50 mM Tris-HCl, pH 7.4, 1 mM Na3VO4, 1 mM NaF, and 1 mM phenylmethylsulfonyl fluoride) supplemented with protease inhibitors (Roche Diagnostics, Risch-Rotkreuz, Switzerland), frozen/thawed, triturated, and centrifuged as previously described (7). Total proteins were mixed with sample buffer containing 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol and then heated to 95°C for 5 min. Equal amounts of each sample were loaded onto 8-15% polyacrylamide gels and transblotted to polyvinylidene difluoride membranes (GE Healthcare, Uppsala, Sweden). After blocking, the membranes were incubated overnight with primary antibodies against α-smooth muscle actin (SMA) (Abcam, Cambridge, MA, USA), COMMD5 (Proteintech Group, Chicago, IL, USA), E-cadherin (BD Biosciences, Franklin Lakes, NJ, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). This was followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) for 60 min. Immunocomplexes on the membranes were visualized using enhanced chemiluminescence (PerkinElmer, Shelton, CT, USA). The expression levels were normalized to the expression of GAPDH in each experiment.

Immunostaining. Neo- and COMMD5-Renca cells were grown on sterile cover slips under the abovementioned conditions (10). For E-cadherin staining, the cells were fixed and permeabilized in cold methanol. After blocking, the cells were incubated with an anti-E-cadherin antibody for 2 h at room temperature. For α-SMA staining, the cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton-X-100 in phosphate buffered saline (PBS). After blocking, the cells were incubated with an anti-α-SMA antibody overnight at 4°C. After incubation with secondary antibodies (Thermo Fisher Scientific), the samples were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and visualized using fluorescence microscopy (Olympus IX73; Olympus, Tokyo, Japan).

Cell proliferation and viability assays. Both stably transfected cells were seeded onto 96-well microliter plates under normoxic conditions. For hypoxic conditions, the cells were cultured in a humidified atmosphere with 5% CO2 and less than 1% O2. After 24 h of serum starvation, cell proliferation and viability assays were performed with the cell proliferation reagent WST-1 (Sigma–Aldrich) according to the manufacturer’s recommendations. WST-1 was added to the culture medium at each time point, and the cells were incubated at 37°C for 30 min. The absorption of WST-1 was measured at 450 nm using a Victor3 1420 Multilabel Counter (PerkinElmer). Cell viability is presented as the percentage of viable cells relative to that of controls under normoxic conditions.

Cell invasion and adhesion assays. The effect of COMMD5 on tumor cell invasiveness was assessed by measuring cell invasion with a CytoSelect™ 96-well cell invasion assay kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, a suspension of 2×106 Neo- and COMMD5-Renca cells in 100 μl of serum-free RPMI medium was placed on top of the basement membrane matrix inside the upper chamber and allowed to invade toward 10% fetal bovine serum through the matrix for 24 h and adhere to the bottom surface of the membrane of the insert. Non-invading cells were removed from the upper surface of the membrane, and invading cells on the bottom surface of the invasion membrane were lysed and quantified using CyQuant® GR Fluorescent Dye in a microplate reader at a wavelength of 480 nm.

Both Renca cells (5×106 cells/ml) were labelled with 5 μM calcein AM (Vybrant™ Cell Adhesion Assay Kit; Thermo Fisher Scientific) in serum-free RPMI for 30 min at 37°C and then were resuspended and added to 96-well microculture plates without coating. After being cultured at 37°C for 3 h, the cells were allowed to adhere, and then washed three times with serum-free RPMI. The plates were scanned to assay the number of adhered cells. The percentage of cell adhesion was calculated as the ratio of fluorescence between the washed and unwashed wells after subtraction of the background fluorescence from both values.

Animal experiment protocols. All procedures in this project conformed to the guidelines of the Canadian Council on Animal Care and were approved by the Animal Care Committee of the Research Centre, Centre Hospitalier de l’Université de Montréal (CRCHUM). Eight-week-old BALB/c male mice were purchased from Charles River Laboratories International, Inc. (Montreal, QC, Canada), and the animals were randomly assigned to two different groups (n=6 per group). For experimental metastasis assays, Neo- and COMMD5-Renca cells were resuspended in 100 μl of 0.1% bovine serum albumin in PBS and were injected intravenously into the lateral tail vein of anaesthetized BALB/c mice. After endovascular transplantation of Neo- and COMMD5-Renca cells, all mice were returned to their cages and allowed ad libitum access to food and water. At 2 weeks after cell transplantation, the mice were euthanized by carbon dioxide inhalation and their lungs were harvested for histological analyses.

Histological analysis. Lungs were fixed in Tissuefix (Biopharm Inc., Hatfield, AR, USA), embedded in paraffin, sectioned into 3-μm slices, and stained with hematoxylin and eosin at the Institute for Research in Immunology and Cancer (IRIC) in Montreal, Canada. The number of metastatic lung nodules on the maximum cut surface of the lungs was determined microscopically. The diameters (μm) of 100 randomly selected nodules in each experimental group were measured using Adobe Photoshop CS2 (Adobe Systems Corporation, San Jose, CA, USA).

Lung sections were incubated overnight with an anti-CD34 antibody (Abcam), as previously described (10), followed by visualization using a Cell & Tissue Staining Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s recommendations. Finally, the sections were counterstained with Mayer’s hematoxylin.

Patients and samples. This study was approved by the Examination Committee for Clinical Research of Nihon University Itabashi Hospital (RK-131213-9). Patients with kidney cancer who underwent surgery at Nihon University Itabashi Hospital (Tokyo, Japan) between 1997 and 2006 were enrolled. All patients provided their written informed consent prior to surgery. The samples were obtained from the archives of the Department of Pathology of Nihon University School of Medicine. Clinical characteristics, such as tumor diameter, histological RCC subtypes, and outcome, were retrieved from electronic records. The tumor size was based on the longest diameter of the pathological specimen. Kaplan–Meier curves were generated for time to metastasis-free survival, defined as the interval between primary resection and death from disease, disease metastasis and recurrence or the last follow-up date, and were compared using the log-rank test.

For immunohistochemical staining of COMMD5, sections from the RCC and renal cortex specimens were incubated overnight with an anti-COMMD5 antibody as previously described (10, 11), followed by visualization using a Cell & Tissue Staining Kit according to the manufacturer’s recommendations. All slides were reviewed by a pathologist blinded to the experimental groups. The COMMD5 staining index for RCCs and PTs was calculated as the product of the staining intensity and the percentage of positive area (range=1-4). COMMD5 expression levels were classified into low (indexes 1 and 2) and high (3 and 4) expression groups.

Statistical analysis. Data are presented as the mean±standard error of the mean (SEM). Comparisons of data between groups were performed using an analysis of variance followed by a t-test. Pearson’s chi-square test was used for analyzing COMMD5 expression and distant metastasis in patients with RCC. Kaplan–Meier analysis was used for survival outcomes. p-Values of <0.05 were considered statistically significant. All analyses were performed using JMP (ver. 14, SAS Institute Inc., Cary, NC, USA).

Results

COMMD5 facilitates the differentiation of mouse renal adenocarcinoma cells. To test whether COMMD5 can reduce malignant behaviors, such as rapid proliferation, invasion and adhesion, in mRCC, we evaluated the impact of its over-expression in Renca cells. The cells were stably transfected with a control plasmid (Neo-Renca) or an expression plasmid containing rat COMMD5 cDNA (COMMD5-Renca) as previously described (12). Consistent with the results of our previous study (10), the total COMMD5 protein expression, including that of exogenous rat COMMD5 and endogenous murine COMMD5, was clearly higher in stably transfected COMMD5-Renca cells than in Neo-Renca cells, which expressed only endogenous murine COMMD5 (Figure 1A). Cellular morphological changes, such as epithelial–mesenchymal transition (EMT), have been implicated in cancer progression and metastasis (13). In comparison to COMMD5-Renca cells, Neo-Renca control cells exhibited a more spindle-shaped morphology, whereas the over-expression of COMMD5 induced a cobblestone-like epithelial morphology in COMMD5-Renca cells (Figure 1B). The hallmark of EMT is the down-regulation of E-cadherin (a marker of epithelial integrity) and the up-regulation of α-SMA (a mesenchymal marker) (14, 15). As expected, E-cadherin was only detected in COMMD5-Renca cells, whereas α-SMA was highly expressed in Neo-Renca cells (Figure 1A and B).

Figure 1.
Figure 1.

Copper metabolism MURR1 domain-containing 5 (COMMD5) facilitates differentiation of the mouse renal adenocarcinoma cells. (A) Western blotting of COMMD5 and differentiation markers in Renca clones. Total (exogenous and endogenous) COMMD5 protein levels, revealed by western blotting, are higher in COMMD5-Renca cells than in Neo-control cells. E-cadherin is only detected in COMMD5-Renca cells while α-smooth muscle actin (SMA) expression is lower in COMMD5-Renca cells than in Neo-Renca cells. (B) Photographs and immunostaining of differentiation markers illustrate the difference in cell morphology. COMMD5 promotes the differentiation of Renca cells. Scale bars, 50 μm.

COMMD5 inhibits tumor cell proliferation but improved adhesion and viability. To investigate the effect of COMMD5 on tumor cell growth, we initially performed a cell proliferation assay using Neo- and COMMD5-Renca cells. The results showed that COMMD5 over-expression significantly inhibited the proliferation of Renca cells after 24 h compared to Neo-Renca cells (Figure 2A), consistent with our previous findings in other cell types (7, 11). To assess the impact of COMMD5 on tumor cell invasiveness, suspensions of Neo- and COMMD5-Renca cells were deposited on top of a basement membrane matrix. After 24 h, the cells that had invaded through the matrix were lysed and quantified, revealing no significant effect of COMMD5 over-expression on tumor cell invasion in this model (Figure 2B). Since tumor cells must adhere to the lung endothelium before extravasating into the tissue to establish lung metastases, we also assessed the effect of COMMD5 over-expression on Renca cell adhesion (Figure 2C). COMMD5 over-expression increased the percentage of adherent Renca cells after 3 h relative to Neo-Renca cells.

Figure 2.
Figure 2.

Copper metabolism MURR1 domain-containing 5 (COMMD5) over-expression inhibits Renca cell proliferation but enhances adhesion and viability. (A) Tumor cell growth curves of Renca clones. COMMD5-Renca cells proliferate less than Neo-controls. (B) Cells were plated onto a basement membrane matrix and allowed to invade. After 24 h, cells that had invaded through the matrix were quantified, showing that COMMD5 over-expression does not affect Renca cell invasion. (C) Among suspended Neo- and COMMD5-Renca cells, those that adhered to an uncoated plate after 3 h of culture were quantified. The percentage of adherent cells was calculated as the ratio of fluorescence between washed and unwashed wells. COMMD5 over-expression increases the number of adherent Renca cells relative to Neo-controls. (D) Cell viability in Neo- and COMMD5-Renca cells under hypoxic conditions. COMMD5 over-expression improves tumor cell viability after 48 h under hypoxic conditions. Values indicate the mean±standard error of the mean (n=8). Significantly different at: **p<0.01. NS: Not significant.

To some extent, hypoxic conditions eliminate tumor cells that are more sensitive to hypoxia. Conversely, tumor cells that survive hypoxia exposure often acquire EMT phenotypes, leading to increased cell motility and metastasis (16). Therefore, we examined the adaptation of Renca clones to hypoxic conditions for survival (Figure 2D). After the initial hypoxic shock, 70-80% of Neo- and COMMD5-Renca cells were eliminated within 24 h, with no significant difference observed between the two clones. However, COMMD5 over-expression significantly (p<0.01) increased cell viability after 48 h under hypoxic conditions.

Effects of COMMD5 on experimental lung metastasis. We next assessed the effects of COMMD5 on metastatic tumor growth in vivo in an experimental lung metastatic model. COMMD5-Renca cells or Neo-Renca cells were administered by intravenous tail vein injection to six syngeneic BALB/c mice per group. The number and size of the metastatic lung nodules were measured 14 days after injection. The number of metastatic lung nodules in the mice injected with COMMD5-Renca cells was 2.6-fold greater than that in mice injected with Neo-Renca cells (Figure 3A and B). However, the mean size of the metastatic lung nodules in the mice injected with COMMD5-Renca cells was significantly smaller (p< 0.01) than that in mice injected with Neo-Renca cells (Figure 3C). The total lung weight in mice injected with COMMD5-Renca cells was greater than that in mice injected with Neo-Renca cells (Figure 3D). To confirm the antiangiogenic effect of COMMD5 on experimental lung metastasis, we performed immunohistochemical staining of tumor microvessels with an anti-CD34 antibody (Figure 4) (17). In comparison to mice injected with Neo-Renca cells, the metastatic lung nodules in mice injected with COMMD5-Renca cells had fewer CD34-positive microvessels and endothelial cells. Overall, our results demonstrated that COMMD5 enhanced the adherence of Renca cells to the lung endothelium, whereas mRCC cell proliferation was suppressed by the over-expression of COMMD5.

Figure 3.
Figure 3.

Copper metabolism MURR1 domain-containing 5 (COMMD5) increases the number of metastatic lung nodules in an experimental mouse model of lung metastasis. (A) Representative images of hematoxylin and eosin staining show smaller and more numerous metastatic lung nodules in mice injected intravenously with COMMD5-Renca cells relative to mice injected with Neo-Renca cells. Scale bars, 1 mm. (B) Metastatic lung nodules were counted on the maximum cut surface of the lungs. The number of lung metastatic nodules in the mice injected with COMMD5-Renca cells is 2.6-fold greater than that in the mice injected with Neo-Renca cells. (C) The diameters of 100 randomly selected nodules in each experimental group were measured. The mean size of the metastatic lung nodules is smaller in the mice injected with COMMD5-Renca cells than in the mice injected with Neo-Renca cells. (D) The mean lung weight of mice at 14 days after the implantation of COMMD5-Renca cells is greater than that of the mice injected with Neo-Renca cells. Values indicate the mean±standard error of the mean (n=6). Significantly different at: *p<0.05; **p<0.01.

Figure 4.
Figure 4.

Copper metabolism MURR1 domain-containing 5 (COMMD5) inhibits tumor angiogenesis in metastatic lung nodules. Representative images of immunostaining with an anti-CD34 antibody. COMMD5 over-expression decreases CD34-positive microvessels and endothelial cells in the experimental lung metastasis model on day 14. Scale bars, 100 μm.

Expression of COMMD5 in RCC is associated with distant metastasis. In our related study, COMMD5 was found to be frequently under-expressed, not only in ccRCC specimens but also in PTs adjacent to large ccRCCs in patients with worse clinical outcomes (10). In this study, human RCC samples from 130 patients (mean age: 60.8±0.10 years, SEM; 70.8% male, 29.2% female) were immunohistochemically stained with an anti-COMMD5 antibody (Figure 5A). The histological subtypes of the samples were as follows: clear cell type (n=116), granular cell type (n=4), spindle cell type(n=1), pleomorphic cell type (n=1), papillary cell type (n=1), and mixed cell type (n=7). Samples were obtained along with adjacent non-neoplastic kidney tissue. Patients were initially divided into two groups based on the intensity of COMMD5 staining in PTs adjacent to RCCs (Figure 5A, panels a-d). The mean tumor diameter was significantly smaller (p< 0.05) when the COMMD5 expression level was high in adjacent PTs than when the COMMD5 expression level was low (Figure 5B). In a cohort of 74 patients with low COMMD5 expression in PTs, 26 developed metastases in distant organs, including the lungs, bones, brain, and liver. In contrast, among 56 patients with high COMMD5 expression in PTs, only nine were diagnosed with distant metastasis within ten years after surgery (Table I). In addition, a Kaplan–Meier plot demonstrated that high COMMD5 levels in PTs were significantly (p=0.0254) associated with improved metastasis-free survival (Figure 5C). These findings suggest that high COMMD5 expression in normal PTs may act as a protective factor against tumor growth and metastasis in RCCs (11).

Figure 5.
Figure 5.
Figure 5.

High levels of Copper metabolism MURR1 domain-containing 5 (COMMD5) expression in renal cell carcinomas (RCCs) are associated with distant metastasis. (A) Representative immunohistochemical staining of COMMD5 was performed on sections from patients with RCC, including cases with and without metastasis, as well as non-neoplastic kidney tissue specimens adjacent to the tumors. The immunostaining of the clear cell-type is shown in panels a-d. Strong COMMD5 intensity is primarily observed in normal kidney tissue specimens located adjacent to smaller RCCs (panels in a and b) and in some RCCs with distant metastasis (panels in b and d). Immunostaining of the granular cell type is shown in panels e and f. COMMD5 staining intensity is reduced in 70% of RCCs and 57% of renal proximal tubules (PTs) from patients with larger tumors. Scale bars, 100 μm. (B) High COMMD5 levels in PTs are associated with smaller RCC tumor sizes. Patients were classified into high- and low-COMMD5-level groups based on COMMD5 expression in PTs adjacent to tumors. (C) Five-year metastasis-free survival curves of patients with RCC show that high COMMD5 levels in normal PTs predict a better prognosis. (D) Patients were classified into high- and low-COMMD5-level groups based on COMMD5 expression in RCC. No significant differences in the tumor size are observed between these groups. (E) The five-year metastasis-free survival curves of patients with RCC show that high COMMD5 levels in RCCs are associated with a poor prognosis. Values indicate the mean±standard error of the mean. Significantly different at: *p<0.05. NS: Not significant.

View this table:
Table I.

Pearson chi-square test to evaluate the association between copper metabolism MURR1 domain-containing 5 (COMMD5) and distant metastasis of renal cell carcinoma (RCC).

To further explore the potential effects of COMMD5 in RCCs on distant metastasis, we divided patients into two groups based on the intensity of COMMD5 staining in their tumors. Intracellular over-expression of COMMD5 in Renca cells inhibited proliferation (Figure 2A) and in vivo tumor formation (10); however, no significant differences in tumor size were observed based on the COMMD5 expression levels (Figure 5D). Contrary to our expectations, among a cohort of 39 patients with high COMMD5 expression in RCCs, 25 developed metastases in distant organs. In contrast, only 10 of 91 patients with low COMMD5 expression were diagnosed with distant metastases within ten years after surgery (Table I). Moreover, the Kaplan–Meier analysis demonstrated that high COMMD5 levels in RCCs were significantly (p<0.0001) associated with poor metastasis-free survival (Figure 5E). These findings suggest that high COMMD5 expression in RCC may be a risk factor for tumor metastasis.

Discussion

Cancer metastasis remains a significant challenge in oncology, accounting for the vast majority of cancer-related deaths (18). While localized RCC can be effectively managed with surgical interventions, and treatments such as cytokine-based therapy and checkpoint inhibitor-based immunotherapy have been introduced for mRCC, patients with mRCC still have a poor prognosis and a high mortality rate (19-21). Thus, there is a need for improved treatment strategies, including the use of biomarkers for treatment selection and early detection methods. In this study, we demonstrated that COMMD5 exerts context-dependent effects in RCC. In non-neoplastic PTs, high COMMD5 expression was associated with smaller tumor size, a lower incidence of distant metastasis, and better metastasis-free survival. In contrast, elevated COMMD5 expression in RCCs was correlated with a higher incidence of distant metastasis and poorer metastasis-free survival. However, COMMD5 over-expression in Renca cells was able to inhibit cell proliferation in vitro and suppress in vivo tumor growth of metastatic lung nodules in a syngeneic murine model. These findings highlight the paradoxical role for COMMD5 in RCC progression.

We initially reported that COMMD5 was highly expressed in the kidneys of hypertensive rats and stimulated the repair of ischemic-injured PTs (6, 7). Furthermore, COMMD5 alleviated drug-induced oxidative stress and toxicity in PTs by maintaining the epithelial cell phenotype (8). In addition, COMMD5 secreted from PT cells was shown to reduce malignant behaviors such as proliferation, invasion and sphere formation in kidney cancer cell lines (11). Based on these findings, we hypothesize that the absence of COMMD5 in PTs may lead to uncontrolled de-differentiation of the epithelium, increasing the risk of kidney injury progression and oncogenesis of RCC (22). Consistent with our previous studies, COMMD5 over-expression in Renca cells induced an epithelial-like morphology, as evidenced by increased E-cadherin expression and reduced α-SMA levels (Figure 1) (9, 10). These morphological changes suggest that COMMD5 promotes mesenchymal-epithelial transition, a process traditionally associated with less aggressive features, such as reduced proliferation (Figure 2) and invasion (11).

These phenotypic changes induced by COMMD5 enhanced cell adhesion and adaptability to hypoxic conditions (Figure 2). These alterations in cell behavior are noteworthy because the metastatic cascade depends not only on proliferation but also on the ability of tumor cells to adhere, survive in circulation, and colonize distant organs. Findings from murine models further complexify the role of COMMD5 in tumor biology. We previously demonstrated that COMMD5 over-expression in Renca cells inhibited the tumorigenesis and angiogenesis of RCC in a syngeneic subcutaneous tumor transplantation model in mice (10). In an experimental metastasis model, mice intravenously injected with COMMD5-Renca cells developed 2.6 times more metastatic lung nodules than controls did, although the individual nodules were significantly smaller (p<0.01) (Figure 3). Furthermore, reduced microvessel density was observed in COMMD5-positive metastatic lung nodules (Figure 4). These findings suggest that the antiproliferative effects of COMMD5 may limit the enlargement of the primary tumor and each metastatic deposit, whereas the enhanced viability and adhesion properties of circulating tumor cells induced by COMMD5 facilitate their initial seeding within the lung vasculature.

In normal tissues, transforming growth factor-β1 contributes to wound healing and immunosuppression (23). However, in cancer cells, it promotes EMT and tumor microenvironment formation, facilitating metastasis and immune evasion. Similarly, the dual role of COMMD5, which plays a protective role in normal PT cells while being implicated in metastasis of RCC cells, highlights the critical importance of the cellular context. In normal tissues, COMMD5 appears to maintain tubular epithelial integrity and facilitate redifferentiation for repair after injury (7, 8). Conversely, in cancer cells, it may enhance the viability of circulating tumor cells and adhesive interactions, promoting colonization in distant organs. These findings are also supported by clinical investigations, suggesting that the expression of COMMD5 could serve as a valuable prognostic biomarker. While high COMMD5 levels in normal PTs are associated with favorable outcomes, elevated COMMD5 expression in RCC cells may predict a greater risk of metastatic dissemination (Figure 5). Therefore, careful assessment of the COMMD5 expression in both RCC and surrounding PTs might be necessary to fully evaluate its prognostic potential. In fact, chromosomal abnormalities and genetic alterations leading to the amplification of COMMD5 and other COMMD proteins have also been observed in other types of cancer, suggesting a role in tumor progression (24, 25). Additional experiments involving larger and more diverse patient populations are necessary to confirm or clarify the prognostic significance of COMMD5 in relation to RCC progression.

In conclusion, our data reveals a seemingly contradictory role of COMMD5 in RCC. While high COMMD5 expression in normal PTs appears to protect against primary tumor growth, the over-expression of COMMD5 in RCC promotes lung metastasis despite inhibiting proliferation and facilitating EMT in tumor cells. This versatility aligns with recent evidence showing that certain tumor suppressors can play a context-dependent role, limiting tumor growth during early progression but inadvertently promoting metastasis later by enabling tumor cells to survive in hostile microenvironments and adhere to distant organs (26-28). Thus, these observations emphasize the need for a nuanced approach when considering COMMD5 as a potential therapeutic target for RCC. We have reported that mechanistically, COMMD5 binds to endosomal Rab5 and facilitates the transport of the epidermal growth factor receptor to sorting endosomes by organizing actin and microtubule networks (29). Consequently, COMMD5 promotes the dephosphorylation of ErbB-2, thereby inhibiting the phosphatidylinositol-3-kinase-Akt pathway, which promotes tumor growth (10). Future studies should extend these findings to additional cancer cell lines and animal models that more accurately recapitulate human biology and disease (30, 31). Future research is also essential to elucidate the molecular mechanisms underlying the dual functions of COMMD5 in RCC occurrence and progression, as well as to validate its clinical utility. Determining whether modulation of COMMD5 activity could improve patient outcomes will be a crucial step in advancing RCC treatment.

Acknowledgements

The Authors are grateful to Prof. Satoru Takahashi and Dr. Kenya Yamaguchi at the Department of Urology, Nihon University School of Medicine for sample collection, and to Suzanne Cossette for her expert technical assistance and advice. This work was supported in part by the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research [18K08226 to H.M.].

Footnotes

  • Authors’ Contributions

    Conception and design: Hiroyuki Matsuda, Jin Ikeda, and Johanne Tremblay; Experiments, Data analysis and Interpretation: Hiroyuki Matsuda, Jin Ikeda, Maiko Ogasawara-Nosoko, and Morito Endo; Resources: Pavel Hamet and Johanne Tremblay; Article Writing: All Authors; Final approval of article: All Authors.

  • Conflicts of Interest

    The Authors declare no conflicts of interest in association with the present study.

  • Received February 21, 2025.
  • Revision received March 10, 2025.
  • Accepted March 11, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Contrasting Role of COMMD5 in Renal Cell Carcinoma: Tumor Suppression and Metastatic Enhancement
HIROYUKI MATSUDA, JIN IKEDA, MAIKO OGASAWARA-NOSOKO, MORITO ENDO, PAVEL HAMET, JOHANNE TREMBLAY
Anticancer Research Apr 2025, 45 (4) 1373-1385; DOI: 10.21873/anticanres.17523
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