Research ArticleExperimental Studies
Open Access

Increased Expression of Serca2b in the Adipose Tissue of a Cancer Cachexia Model

SATOKA KASAI, SHO SATO, KENTO NAMIKI, RINKA OBATA and KAZUMI YOSHIZAWA
In Vivo January 2026, 40 (1) 108-122; DOI: https://doi.org/10.21873/invivo.14177

Abstract

Background/Aim: Cancer cachexia is a complication that emerges in approximately 50-80% of patients with advanced cancer, characterized by symptoms such as lipoatrophy, skeletal muscle loss, metabolic abnormalities, and anorexia. While UCP1, a mitochondrial uncoupling protein, is implicated in lipolysis associated with cancer cachexia, the involvement of other thermogenic proteins remains unclear. In this exploratory study, we examined the expression of thermogenic genes in a mouse model of cancer cachexia.

Materials and Methods: Tumor-bearing mice were generated by injecting Colon-26 cells (C26) into the right flank of male BALB/c mice. The body weight and temperature, tumor volume, and food intake of these mice were recorded three times a week. After 46 days of C26 administration, the adipose tissue, muscle, tumor, and blood were isolated from the mice and analyzed for thermogenic gene expression and biochemical parameters.

Results: Quantitative reverse transcription PCR analysis revealed increased expression of Serca2b, a gene associated with Ucp1 independent thermogenesis, in adipose tissue of C26-bearing mice. A positive correlation between Serca2b and Ucp1 mRNA levels was observed. In addition, Serca2b expression was not responsive to norepinephrine in differentiated 3T3-L1 adipocytes.

Conclusion: Although the functional relevance of Serca2b up-regulation remains to be elucidated, these findings suggest a potential role for SERCA2b in adipose tissue remodeling during cachexia. This preliminary observation may serve as a foundation for future studies investigating calcium cycling and non-canonical thermogenesis in the pathophysiology of cancer cachexia.

Keywords:

Introduction

Cancer cachexia is defined as “a multifactorial syndrome characterized by the ongoing loss of skeletal muscle mass, with or without loss of fat mass, which cannot be fully reversed by conventional nutritional support and leads to progressive functional impairment” (1). The main symptoms of cancer cachexia are loss of skeletal muscle and fat weight, decreased appetite, and metabolic abnormalities (1). Despite its critical importance in cancer treatment, effective pharmacologic interventions to treat cancer cachexia are lacking largely due to insufficient social recognition and incomplete understanding at both basic and clinical research levels.

Adipose tissue is important for both nutrient supply and thermoregulation. White adipose tissue (WAT) stores large amounts of nutrients, such as lipids, in monocular white adipocytes and releases them as free fatty acids during starvation. Brown adipose tissue (BAT) expresses uncoupling protein-1 (UCP1), a mitochondrial protein responsible for non-shivering thermogenesis (2). Beige adipocytes, inducible within the WAT, also express UCP1 under sympathetic stimulation (3). Elevated UCP1 levels in patients with cachexia and mouse models suggest that enhanced thermogenesis is related to fat loss (2, 4).

Recent studies have proposed an alternative thermogenic pathway independent of UCP1. This pathway involves ATP-dependent Ca2+ cycling mediated by sarco/endoplasmic reticulum Ca2+-ATPase2b (SERCA2b), an endoplasmic reticulum membrane calcium pump, as well as ryanodine receptor 2 (RYR2) and inositol 1,4,5-trisphosphate receptor 1 (ITPR1) (5). C4orf3, also known as another regulin (ALN), has recently been identified as a negative regulatory factor that functions as a “molecular resistor” of SERCA2b-mediated Ca2+ transport (6). While SERCA2b has been implicated in thermogenic regulation, its expression profile and potential role in cancer cachexia remain largely unexplored.

In this exploratory study, we assessed the mRNA expression of thermogenesis-related genes in adipose tissues of C26 tumor-bearing mice with cachexia symptoms. Our primary aim was to identify potential alterations in Serca2b expression, with the goal of generating hypotheses for future mechanistic studies.

Materials and Methods

Animals. Eight-week-old male BALB/c mice, weighing approximately 23 g each, were obtained from Sankyo Labo Service Corporation, Inc. (Ibaraki, Japan). Mice were maintained in a controlled environment at the Institute for Experimental Animals, Tokyo University of Science, with a temperature of 23±1°C and a 12-hour light/dark cycle (lights on at 8:00 a.m.), using conventional mouse cages (136×208×115 mm). The cages had sawdust bedding, and the mice had access to food and water ad libitum. The mice were single-housed for 46 days starting from the day of cancer cell injection to individually monitor their food intake.

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Tokyo University of Science (approval number: Y22029), and were carried out in compliance with the “Fundamental Guidelines for Proper Conduct of Animal Experiments” established by the Ministry of Education, Culture, Sports, Science and Technology of Japan. Tumor-bearing mice were monitored daily, and humane endpoints were defined prior to the start of the experiment. Although tumor sizes in some mice slightly exceeded commonly referenced thresholds (e.g., University of Pennsylvania guidelines), this was approved by our IACUC.

Animal welfare was ensured by monitoring the health and behavior of the mice at least once daily, including assessments of food and water consumption, posture, mobility, coat condition, and signs of distress or pain. Once an animal met the predefined criteria, euthanasia was promptly performed using an overdose of isoflurane, a method widely recognized for its minimal invasiveness and ethical acceptability. Humane endpoints were established for this study, including situations in which mice showed significant distress, such as impaired ability to eat or drink due to pain or stress, or a reduction in estimated body weight after subtracting the calculated tumor mass (tumor-excluded body weight) exceeding 25%. In total, 11 mice were used (control group, n=5; C26 tumor-bearing group, n=6). One mouse reached the humane endpoint criterion on day 46 due to a >25% reduction in estimated tumor-excluded body weight, and was promptly euthanized. All other animals were humanely euthanized at the experimental endpoint, and no unexpected deaths occurred.

All surgical procedures were carried out under isoflurane anesthesia, with continuous measures taken to reduce pain and discomfort. Personnel involved in animal care were properly trained under the oversight of the IACUC of Tokyo University of Science. All procedures were conducted in accordance with accepted ethical and technical standards. Anesthesia was induced and maintained with isoflurane vapor delivered in oxygen at a nominal concentration of 2-3% (v/v). Isoflurane was applied onto a cotton pad placed in a small induction chamber, and mice inhaled the vapor until the desired anesthetic depth was reached. For euthanasia, animals were deeply anesthetized with isoflurane vapor at a nominal concentration of 4-5% (v/v in oxygen) until respiratory arrest was apparent. Death was verified by the absence of spontaneous respiration and heartbeat for >1 min, as confirmed by trained personnel. Blood samples were collected under deep anesthesia immediately before euthanasia from the abdominal aorta.

This study used only male mice to reduce variability related to hormonal fluctuations, and to maintain consistency with prior studies using the same cancer cachexia model. The potential influence of sex differences on the results should be considered in future investigations.

Cell culture. Colon-26 (C26) cells (RCB2657) and 3T3-L1 adipocytes (JCRB9014) were obtained from the RIKEN BioResource Research Center (RIKEN BRC, Ibaraki, Japan) and the Japan Collection of Research Bioresources Cell Bank (JCRB Cell Bank, Osaka, Japan), respectively. RIKEN BRC confirmed the species identity of C26 cells using polymerase chain reaction (PCR), enzyme digestion, and simple sequence length polymorphism (SSLP) analysis, and mycoplasma testing prior to distribution. Similarly, JCRB Cell Bank verified the species identity of 3T3-L1 cells by enzyme activity analysis and mycoplasma screening. In our laboratory, the cells were used within early passages after thawing. Although additional mycoplasma testing was not conducted post-acquisition, all cultures were maintained under sterile conditions and were carefully monitored for signs of contamination throughout the experiments.

C26 cells were grown in Roswell Park Memorial Institute-1640 (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S). 3T3-L1 adipocytes were grown in Dulbecco’s modified Eagle’s medium (DMEM; Fujifilm Wako Pure Chemical Corp.) containing 10% FBS and 1% P/S. All cells were cultured in a humidified atmosphere with 5% CO2 at 37°C. For adipocyte differentiation of 3T3-L1, cells were treated with DMEM containing 10% FBS, 0.5 mM isobutylmethylxantine, 1 μM dexamethasone, 5 μg/ml insulin, and 1 μM rosiglitazone. After 48 h, the cells were switched to DMEM containing 10% FBS, 5 μg/ml insulin, and 1 μM rosiglitazone for five days, during which the medium was replaced every one or two days. Next, the cells were treated with 100 ng/ml recombinant parathyroid hormone-related protein (PTHrP; Fujifilm Wako Pure Chemical Corp.) for 2 h, 100 nM norepinephrine (NE) (Sigma-Aldrich, St. Louis, MO, USA) for 4 h, 1% plasma for 24 h, and 3T3-L1 adipocyte-conditioned medium or C26 cell-conditioned medium for 24 h. To stimulate thermogenic gene expression, the cells were incubated with 10 μM forskolin for 4 h.

Cancer cachexia model. The mice were randomly divided into two groups with comparable average body weights. On day zero of the study, C26 cells (1×106), suspended in 200 μl phosphate-buffered saline (PBS), were subcutaneously implanted into the right flank of mice in the C26 tumor-bearing group (C26). An equal volume of PBS without tumor cells was injected into mice in the control group. Body weight, body temperature, tumor volume, and food intake of the mice were recorded every one or two days, beginning on day zero and ending on day 46, which was the last day of the experiment. To minimize stress on the animals, we measured body temperature using a non-invasive infrared thermometer. Body temperature was calculated as the average of three measurements taken over the abdomen of the mice using a non-contact infrared thermometer (FS-700, HuBDIC Co., Ltd., Tokyo, Japan). Surface body temperature has been reported to correlate with rectal temperature, and although infrared thermometry exhibits slightly greater variability than rectal probes, the difference is minimal (7). The shortest (x) and longest (y) diameters of the tumors were measured using digital calipers. The tumor volume (V) was calculated using the following formula: V=x×x×y×0.5. On day 46, all the mice were anesthetized with isoflurane inhalation. Blood was collected from the abdominal aorta into tubes containing EDTA-2K and centrifuged (2,000×g, 4°C, 15 min) to obtain the plasma. Tissues, including tumors, inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), BAT, and gastrocnemius muscle (GAS), were dissected and weighed. The tissues on the tumor side were fixed in 10% formaldehyde, while tissues on the opposite side of the tumor were soaked in RNAlater stabilization solution (Thermo Fisher Scientific, Waltham, MA, USA) until RNA extraction.

Grip strength test. The mice muscle strength was measured using an automated grip strength meter (GPM-100; Melquest, Toyama, Japan). The mice were allowed to grip the meter with their paws and then gently pulled backward in a horizontal plane from the tail base. The test was repeated three times per mouse, and the maximum strength exerted by the paws of each mouse was recorded.

Immunohistochemistry and morphological analysis. Mouse iWAT, eWAT, and BAT were embedded in paraffin and cut into 5-μm sections, and GAS was cut into 4-μm sections. The sections were stained with hematoxylin and eosin (HE). Images were captured using a bright-field microscope (BZ-X800; Keyence, Osaka, Japan). The adipocyte sizes of iWAT and eWAT were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA) with the Adiposoft plugin (8). Muscle fiber areas in the GAS were analyzed using StarDist (an ImageJ/Fiji Plugin) for detection (9) and MorphoLibJ (an ImageJ/Fiji Plugin) for size-based filtering (10).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Total RNA was isolated from mouse iWAT, eWAT, BAT, GAS, and 3T3-L1 adipocytes using the RNeasy Lipid Tissue Mini Kit (Qiagen, Venlo, the Netherlands), according to the manufacturer’s recommendations. RNA concentrations were quantified using Nanodrop LITE (Thermo Fisher Scientific). Complementary DNA was synthesized from total RNA using ReverTra Ace qPCR RT master mix (Toyobo, Osaka, Japan), following the manufacturer’s protocols. Gene expression analysis was performed using the Thunderbird SYBR qPCR mix (Toyobo) in the CFX Connect real-time PCR system (Bio-Rad, Hercules, CA, USA). Relative gene expression levels were calculated using standard curve analysis, in which PCR efficiency was calculated from the slope of the standard curve generated from serial dilutions of pooled cDNA samples. Gene expression was normalized to Actb (β-actin) as a reference gene. This method allows accurate quantification independent of amplification efficiency variation (11). For the comparison of gene expression levels among tissues, the 2−ΔΔCq method was applied (12). The primers used are listed in Table I.

View this table:
Table I.

Primers used for real-time qPCR analysis.

Enzyme-linked immunosorbent assay. The PTHrP concentration in the plasma was determined using the PTHrP (1-34) enzyme-linked immunosorbent assay (BMA Biomedicals, Augst, Switzerland), according to the manufacturer’s instructions.

Immunofluorescence staining. Immunofluorescence staining was performed on deparaffinized adipose tissues. To expose the target protein, antigen retrieval was performed by heating the tissue in a microwave oven for 5 min three times in a 10 mM sodium citrate buffer (pH 6.2). Following antigen retrieval, the tissue was blocked in 5% goat serum at approximately 25°C for 60 min. The tissues were then probed at approximately 25°C for 1 h in a humidified chamber at 1:50 dilution with a mouse monoclonal antibody that recognizes SERCA2 ATPase [Anti SERCA2 (F-1), Santa Cruz Biotechnology, Dallas, TX, USA]. Subsequently, the tissues were washed and probed at approximately 25°C for 1 h at a 1:100 dilution with a CoreLite488-conjugated goat anti-mouse IgG (H+L) (Thermo Fisher Scientific). They were then mounted with VECTASHIELD® Vibrance™ Antifade Mounting Medium (Vector Laboratories, Newark, CA, USA). Integrated density and the number of DAPI-positive cells were measured using ImageJ (National Institutes of Health).

Statistical analyses. The results are expressed as the mean±standard error of the mean. Control and C26 tumor-bearing mice were compared using a two-tailed Student’s t-test. A two-way analysis of variance (ANOVA) was performed to examine the interaction between the effects of C26 administration and the number of days after administration, followed by Bonferroni post hoc test for multiple comparisons. Pearson’s correlation analysis was performed to detect potential associations between control and C26 tumor-bearing mice in terms of body weight with tumor, calculated body weight without tumor, body temperature, accumulated food intake, tumor volume, tissue weight, grip strength, and the expression of thermogenic and thermogenesis-related genes using GraphPad Prism 9 (GraphPad Software, Inc., San Diego, CA, USA). Statistical significance was defined as p<0.05.

Results

Colon-26 tumor-bearing mice showed symptoms of cancer cachexia 46 days after Colon-26 administration. C26 tumor-bearing mice showed a significant increase in body weight (including tumor weight) after 31 days of C26 administration (Figure 1A; p<0.01 on day 31, p<0.001 on day 33, and p<0.0001 on days 35-46). However, after 40 days of C26 administration, they exhibited a significant decrease in the calculated body weight (excluding tumor weight) (Figure 1B; p<0.01 on days 40 and 45, p<0.001 on day 42, and p<0.0001 on day 46). This indicated that the overall increase in weight observed in C26 tumor-bearing mice could be attributed to an increase in tumor weight. Furthermore, the accumulated food intake across all time points after C26 administration was comparable in C26 tumor-bearing and control mice (Figure 1C). Although anorexia, a symptom of cachexia, was not observed in the C26 tumor-bearing mice, the decrease in calculated body weight (excluding tumor weight) observed in this experimental model can be attributed to metabolic abnormalities rather than to decreased food intake. The C26 cells implanted under the skin on the right side of the torso formed a tumor of measurable size after three or five days of treatment. When removed 46 days after treatment, part of the tumor had broken through the epidermis and was necrotic (Figure 1D). The body temperature of C26 tumor-bearing mice tended to decline compared with control mice on days 42 and 45 post-treatment (Figure 1E; p=0.0593 on day 42, p=0.0986 on day 45; two-way ANOVA post hoc Bonferroni’s test). This indicated a decrease in body temperature, one of the symptoms of cancer cachexia.

Figure 1.
Figure 1.
Figure 1.

Colon-26 tumor-bearing mice exhibit cancer cachexia-like symptoms. (A) Changes in body weight, including tumor weight, in control and C26 tumor-bearing mice. A two-way analysis of variance (ANOVA) showed a significant interaction between the effects of the C26 administration and the number of days after administration (F(20, 189)=8.130, p<0.0001). **p<0.01, control mice vs. C26 tumor-bearing mice on day 31, ***p<0.001, on day 33, ****p<0.0001, on days 35-46 (Bonferroni’s test). (B) Changes in body weight, excluding tumor weight, in control and C26 tumor-bearing mice. A two-way ANOVA reveals a significant interaction between the effects of the C26 administration and the number of days after administration (F(20, 189)=2.989, p<0.0001). **p<0.01, control mice vs. C26 tumor-bearing mice on days 40 and 45, ***p<0.001 on day 42, ****p<0.0001 on day 46 (Bonferroni’s test). (C) Accumulated food intake in control and C26 tumor-bearing mice. No difference is noted between the control and C26 tumor-bearing mice. (D) A photograph of the tumors collected 46 days after C26 administration. (E) Body temperature in control and C26 tumor-bearing mice. A two-way ANOVA revealed a significant effect of C26 administration (F(20, 189)=10.63, p<0.01). There are no statistically significant differences between the control and C26 tumor-bearing mice in post hoc Bonferroni’s test. Data are expressed as mean±SEM (n=5-6). C26: colon-26. (F) Significant decreases in the tissue weights of C26 tumor-bearing mice (iWAT, t=5.498, **p<0.001; eWAT, t=5.512, **p<0.001; BAT, t=2.263, *p<0.05; GAS, t=2.690, *p<0.05; Student’s t-test). Data are expressed as mean±SEM (n=5-6). (G) Hematoxylin and eosin (H&E)-stained images of iWAT, eWAT, BAT, and GAS. Representative sections stained with H&E. Scale bar, 100 μm. (H, I) Significant reductions in the adipocyte areas of iWAT (H) and eWAT (I) in C26 tumor-bearing mice (iWAT, t=4.843, **p<0.01; eWAT, t=5.065, **p<0.01; Student’s t-test). Data are expressed as mean±SEM (n=4). (J) Muscle fiber areas of GAS in C26 tumor-bearing mice. Data quantification was performed using Fiji. Data are expressed as mean±SEM (n=4). (K) Significant decrease in the grip strength of C26 tumor-bearing mice after 46 days of C26 administration (t=3.908, **p<0.01; Student’s t-test). Data are expressed as mean±SEM (n=5-6). C26: Colon-26; iWAT: inguinal white adipose tissue; eWAT: epididymal white adipose tissue; BAT: brown adipose tissue; GAS: gastrocnemius muscle.

The tissue weights of iWAT, eWAT, BAT, and GAS of C26 tumor-bearing mice significantly decreased (Figure 1F; iWAT, t=5.498, p<0.001; eWAT, t=5.512, p<0.001; BAT, t=2.263, p<0.05; and GAS, t=2.690, p<0.05). These reductions in tissue weight may be responsible for the decrease in body weight (excluding tumor weight) in C26 tumor-bearing mice. Histological analyses of adipose tissues revealed reduced adipocyte size and a multi-ocular lipid droplet phenotype in iWAT, eWAT, and BAT of C26 tumor-bearing mice (Figure 1G-I; iWAT, t=4.843, p<0.01; eWAT, t=5.065, p<0.01), which indicates atrophy of the adipose tissue attributed to cancer cachexia. In C26 tumor-bearing mice, BAT showed a decrease in the adipocyte area on HE-stained images but could not be quantified (Figure 1G). Histological and imaging analyses of the GAS revealed no muscle fiber atrophy (Figure 1G and J). No change in muscle fiber area was noted on histological images; however, the tissue weight (Figure 1F; GAS, t=2.690, p<0.05) and grip strength (Figure 1K; t=3.908, p<0.01) significantly decreased. Additionally, the expression of muscle degradation-related genes (Muscle atrophy F-box protein-1 and muscle specific-RING finger protein; Figure S1A, B) showed a tendency to increase, suggesting a functional decline and progressive degradation of muscle protein. However, this effect was not reflected in the muscle fiber cross-sectional area. Therefore, we concluded that C26 tumor-bearing mice also exhibited cancer cachexia symptoms in the muscle.

Expression of Serca2b mRNA increased in the adipose tissue of Colon-26 tumor-bearing mice. We analyzed the expression of Serca2b, a Ucp1-independent thermogenic gene, based on the assumption that a decrease in body temperature induces the expression of thermogenic genes. Serca2b mRNA expression in the iWAT, eWAT, and BAT of C26 tumor-bearing mice was significantly increased or tended to increase compared with that in control mice (Figure 2A; iWAT, t=2.685, p<0.05; eWAT, t=2.256, p=0.051; and BAT, t=2.500, p<0.05; Student’s t-test). None of the adipose tissues in C26 tumor-bearing mice exhibited a significant increase in Ucp1 mRNA expression (Figure 2B, left panel). To address baseline differences among tissues, we plotted relative Serca2b and Ucp1 expression across these tissues (Serca2b, Figure S2A; Ucp1, Figure 2B, right panel). The levels of Ucp1 expression were markedly higher in BAT than in iWAT and eWAT. We also plotted average amplification curves to visualize baseline differences across tissues (Figure S2B-D). As presented in Figure S2B, the amplification curve for Ucp1 in BAT reached the threshold cycles earlier than in iWAT and eWAT, reflecting its substantially high endogenous expression. In contrast, eWAT displayed considerably late and weak amplification, supporting the observation that fold changes in this tissue may appear large despite notably low absolute expression levels. Moreover, the differences in Serca2b expression among tissues were minimal (Figure S2A, C).

Figure 2.
Figure 2.
Figure 2.

Increased expression of Sarco/endoplasmic reticulum Ca2+-ATPase2b mRNA in adipose tissues of Colon-26 tumor-bearing mice. (A) Expression of Serca2b mRNA in C26 tumor-bearing mice. The expression is significantly increased or shows an increasing trend (iWAT, t=2.685, *p<0.05; eWAT, t=2.256, p=0.051; BAT, t=2.500, *p<0.05, Student’s t-test). Data are expressed as mean±SEM (n=5-6). (B) Expression of Ucp1 mRNA in iWAT, eWAT, and BAT of C26 tumor-bearing mice (left panel). No significant increases are observed. Right panel is relative Ucp1 expression across tissues. Data are expressed as mean±SEM (n=4-6). (C) Concentration of PTHrP in the plasma of C26 tumor-bearing mice shows an increasing trend compared to that of control mice (t=1.825, p=0.1013, Student’s t-test). Data are expressed as mean±SEM (n=5-6). (D) Effect of NE treatment (100 nM) for 2 h on the expression of Ucp1 mRNA in differentiated 3T3-L1 cells (F(2,9)=58.05, p<0.0001, one-way ANOVA; ****p<0.0001, post hoc Bonferroni’s test). A significant increase is observed. Data are expressed as mean±SEM (n=4). (E, F) Effect of C26 tumor-bearing mice plasma treatment on the expression of Serca2b and Ucp1 mRNA in differentiated 3T3-L1 cells. No significant effect is observed. Data are expressed as mean±SEM (n=4-6). (G, H) Effect of treatment with conditioned medium obtained from 2D cultures of C26 on the expression of Serca2b and Ucp1 mRNA in differentiated 3T3-L1 cells. Data are expressed as mean±SEM (n=4). C26: Colon-26; Ucp1: Uncoupling protein-1; Serca2b: Sarco/endoplasmic reticulum Ca2+-ATPase2b; iWAT: inguinal white adipose tissue; eWAT: epididymal white adipose tissue; BAT: brown adipose tissue; PTHrP: parathyroid hormone-related protein; NE: norepinephrine; CM: conditioned medium.

Given that SERCA2b is a calcium channel present on the endoplasmic reticulum membrane, we analyzed the expression of genes related to intracellular calcium homeostasis regulation. These genes included Ryr2; Itpr1; Voltage-dependent anion channel 1 (Vdac1); Cytochrome c oxidase subunit 8b (Cox8b), which encodes the last enzyme in the mitochondrial electron transport chain that drives oxidative phosphorylation; and C4orf3, which encodes a modulator of Ca2+ cycling thermogenesis (6). We used Cox8b to evaluate the mitochondrial electron transfer system in C26 tumor-bearing mice. Our results revealed significantly increased expression of Vdac1 mRNA in eWAT (Figure S3; t=3.058, p<0.05; Student’s t-test), whereas no significant differences were observed for the other genes examined.

We analyzed the induction of Serca2b mRNA expression in C26 tumor-bearing mice. PTHrP causes hypercalcemia in patients with cancer cachexia (13, 14), inducing Ucp1 mRNA expression in primary white adipose cells (15). Plasma PTHrP levels in C26 tumor-bearing mice showed an increasing tendency compared with those in control mice (Figure 2C; t=1.825, p=0.1013; Student’s t-test). Therefore, we added PTHrP or NE to adipocyte-like differentiated 3T3-L1 cells and examined their effect on the induction of Serca2b mRNA expression in adipocytes. Upon treatment of differentiated 3T3-L1 cells with PTHrP and NE for 4 and 2 h, respectively, Ucp1 mRNA expression remained unchanged in response to PTHrP, but significantly increased following NE induction (Figure 2D, right panel; p<0.0001; two-way ANOVA post hoc Bonferroni test). In contrast, Serca2b mRNA did not increase in 3T3-L1 adipocytes following PTHrP or NE stimulation (Figure 2D, left panel). In addition, no changes in Ucp1 or Serca2b mRNA expression were observed in differentiated 3T3-L1 cells to which plasma from C26 tumor-bearing mice was added (Figure 2E and F). Furthermore, 3T3-L1 cells did not show any changes in the expression of either Ucp1 or Serca2b mRNA when treated with C26-conditioned medium for 24 h (Figure 2G and H). This suggested that the conditioned medium obtained from 2D cultures of C26 cells does not contain substances that induce Ucp1 and Serca2b mRNA expression. Under the experimental conditions used in this study, no substance in the conditioned medium or plasma was found to increase Ucp1 or Serca2b mRNA expression in 3T3-L1 cells.

Expression of Serca2b mRNA positively correlated with the expression of Ucp1 mRNA. Pearson’s correlation coefficient was used to examine the correlation between Serca2b mRNA expression and cachexia symptoms in mice after 46 days of C26 administration. The heatmaps of Pearson’s correlation coefficient and p-value are shown in Figure 3A and B, respectively. Serca2b and Ucp1 mRNA expressions were positively correlated in iWAT, eWAT, and BAT (Figure 3A-E; iWAT, r=0.9063, p=0.0128; eWAT, r=0.970, p=0.0013; BAT, r=0.9218, p=0.0089). These results suggest that the expression of Serca2b mRNA in adipose tissue is not complementary to that of Ucp1 mRNA.

Figure 3.
Figure 3.

Expression of Sarco/endoplasmic reticulum Ca2+-ATPase2b mRNA positively correlated with uncoupling protein-1 mRNA expression on day 46. (A) A correlation matrix heatmap showing the values of Pearson’s correlation coefficient in double gradient color, the positive values in red, and the negative values in blue, ranging from −1 to 1. (B) Heatmap showing the p-value of Pearson’s correlation. Stronger correlations are represented by dark colors, and weaker correlations are represented by bright colors. (C-E) Scatter plots presenting a positive correlation between the ratio of Serca2b mRNA and Ucp1 mRNA expression in inguinal white adipose tissue (iWAT) (C), epididymal white adipose tissue (eWAT) (D), and brown adipose tissue (BAT) (E) of C26 tumor-bearing mice, respectively. (F, G) Scatter plots showing a positive correlation between the ratio of Itpr1 mRNA and Ryr2 mRNA expression (F) and C4orf3 mRNA expression (G) in eWAT of C26 tumor-bearing mice, respectively. Pearson’s correlation analysis and linear regression were carried out with a 95% confidence interval, and the resulting regression line is displayed on the graph.

We further examined the correlation between Serca2b and other Ca2+-cycling-related molecules, including Ryr2 and Itpr1. In eWAT, Ryr2 and Itpr1 expression exhibited a significant positive correlation (Figure 3F, r=0.9770, p=0.0221). In addition, C4orf3 was positively correlated with Itpr1 expression in eWAT (Figure 3G, r=0.9308, p=0.0070). However, we observed no significant correlation between C4orf3 and Serca2b itself.

Moreover, the body temperature positively correlated with GAS weight and grip strength (Figure S4A, B; GAS, r=0.8917, p=0.0169, strength, r=0.8297, p=0.0411), while Ucp1 expression in BAT correlated negatively with GAS weight (Figure S4C, r=−0.9353, p=0.0061). These findings suggest that muscle strength and weight play important roles in maintaining body temperature, and that lowering body temperature induces an increase in Ucp1 mRNA expression in BAT. GAS weight also negatively correlated with Serca2b and Ucp1 mRNA expression in BAT (Figure S4D, E; Serca2b, r=−0.8929, p=0.0166, Ucp1, r=−0.9665, p=0.0017), indicating that the correlation between skeletal muscle, body temperature, and thermogenesis was extremely strong in BAT. Additionally, food intake positively correlated with body weights (Figure S4F, r=0.8780, p=0.0214) and negatively correlated with plasma PTHrP concentration (Figure S4G, r=−0.8243, p=0.0436).

Increased Serca2b expression in BAT of C26 tumor-bearing mice. To investigate the expression of Serca2b protein in adipose tissue, fluorescent immunohistochemistry was performed (Figure 4). No significant variation in the integrated density of Serca2b per cell in the iWAT and eWAT of the C26-treated group was noted, despite the advanced multilocularization (Figure 4A and B). Conversely, in the BAT of the C26-treated group, integrated density of Serca2b per cell was significantly increased (Figure 4A and C; t=2.480, p<0.05; Student’s t-test).

Figure 4.
Figure 4.
Figure 4.

Integrated density of Sarco/endoplasmic reticulum Ca2+-ATPase2b per cell in brown adipose tissue (BAT) significantly increased on day 46. (A) Fluorescent immunohistochemistry image of Sarco/endoplasmic reticulum Ca2+-ATPase2b (Serca2b) in inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), and BAT in Colon-26 (C26)-tumor bearing mice. Scale bar, 200 μm. (B, C) Quantification of fluorescent immunohistochemistry image was performed using ImageJ. A significant increase is observed in BAT (C) of C26-rumor bearing mice (p<0.05, t=2.480, Student’s t-test), but no change in iWAT (B). The severe atrophy in the eWAT of C26-tumor bearing mice did not to obtain sufficient images for use in image analysis. Data are expressed as mean±SEM (n=4).

Discussion

In this study, we observed increased expression of Serca2b mRNA in the WAT and BAT, as well as elevated protein levels of Serca2b in BAT of C26 tumor-bearing mice exhibiting symptoms of cancer cachexia. These mice exhibited symptoms that included reduced body weight (excluding tumor mass), muscle weakness, and adipose atrophy. SERCA2b is a predominant isoform of the sarco/endoplasmic reticulum Ca2+-ATPase in adipocytes (5, 16), classically associated with Ca2+ homeostasis (17). Recent studies suggest that it may contribute to heat generation through ATP-dependent calcium cycling, independent of UCP1 activity (5).

Although the functional importance of Serca2b up-regulation in cachexia remains to be determined, it is noteworthy that its expression increased with a slight decrease in body temperature (Figure 1D) and tended toward hypercalcemia in cancer cachexia (18). These findings suggest that Serca2b may be involved in compensatory thermogenic responses through calcium cycling, potentially acting as an alternative mechanism to UCP1-mediated thermogenesis.

In contrast to previous studies reporting increased UCP1 expression in cachexia (19, 20), we did not observe a substantial change in Ucp1 mRNA levels in our model, even at advanced stages of weight loss. This discrepancy suggests that UCP1 expression may vary depending on disease progression or experimental conditions. Notably, the observed up-regulation of Serca2b at later stages (around day 46) may reflect the activation of alternative thermogenic pathways, independent of Ucp1.

In vitro experiments revealed that norepinephrine stimulation induced Ucp1 expression in 3T3-L1 adipocytes, but did not affect Serca2b levels. This supports the idea that Serca2b is not regulated through canonical β3-adrenergic signaling. Additionally, although plasma PTHrP levels showed a non-significant trend toward elevation in C26 tumor-bearing mice, Ucp1 expression remained unaltered by PTHrP treatment in 3T3-L1 cells, in contrast to previous findings (15). Such differences may be attributable to cell type or differentiation status.

Serca2b and Ucp1 mRNA levels were positively correlated across individual mice, despite apparently distinct regulatory mechanisms. This observation may suggest a shared physiological context that enhances the expression of both genes, such as systemic energy stress or compensatory thermogenic demand. Moreover, skeletal muscle loss may affect thermoregulatory homeostasis, potentially enhancing BAT activity in response to body temperature reduction (Figure S4A-C), as supported by prior studies (2, 21).

Notably, we observed significant correlations between Ryr2, Itpr1, and C4orf3 expression in eWAT, whereas Serca2b did not exhibit such associations. eWAT inherently exhibits considerably low levels of Ucp1 expression, suggesting that this depot may rely more heavily on alternative Ca2+-cycling pathways for thermogenesis and metabolic regulation than other fat depots. Therefore, the strong correlations between Ryr2, Itpr1, and C4orf3 support the notion that under cachectic conditions, coordinated regulation of genes involved in Ca2+ dynamics may represent a compensatory mechanism in this Ucp1-deficient depot. Given that C4orf3 functions as a molecular resistor for SERCA2b-mediated Ca2+ transport, its specific association with Itpr1 rather than Serca2b may further indicate a distinct role in IP3R-dependent Ca2+ signaling in eWAT.

Lastly, we observed a negative correlation between plasma PTHrP levels and food intake (Figure S3G), which may reflect the anorexigenic effects of tumor-derived factors as previously proposed (22). However, the mechanisms underlying these associations remain speculative and warrant further investigation.

Taken together, our results provide preliminary evidence that Serca2b expression is altered in adipose tissues during cancer cachexia. This observation suggests that SERCA2b may represent an adaptive response to metabolic stress in cachectic states and highlights a potential thermogenic mechanism that operates independently of UCP1. While functional validation is beyond the scope of this study, these findings support the hypothesis that calcium cycling and UCP1-independent thermogenesis may be involved in the pathophysiology of cachexia. Identifying such alternative thermogenic pathways is crucial for deepening our understanding of adipose tissue remodeling in cancer cachexia and may serve as a foundation for the development of novel therapeutic strategies targeting metabolic dysfunction in this syndrome.

Recently, the approval of anamorelin as a therapeutic agent for cancer cachexia has marked a new era in cachexia research (23). Moreover, increasing evidence has linked cancer to alterations in the SERCA system, as exemplified by reports showing enhanced SERCA ATPase activity in the skeletal muscle of tumor-bearing mice (24). In this context, the present study provides a foundation for future investigations aimed at elucidating the role of SERCA2b in adipose tissue during cancer cachexia.

Study limitations. First, the tumor burden in some mice exceeded the commonly recommended thresholds for experimental cancer models. Although this was reviewed and approved by our IACUC, large tumor size may influence systemic metabolism and the progression of cachexia, and thus the results should be interpreted with caution. Future studies will employ alternative tumor models or earlier time points to minimize tumor burden and to confirm whether the observed changes in Serca2b expression occur independently of tumor size. Second, while Serca2b protein expression was evaluated using immunohistochemistry, this method has limited quantitative reliability. To confirm protein-level changes, future analyses using more quantitative approaches such as western blotting or ELISA will be required. Third, the number of animals per group was relatively small (5-6), which may reduce the statistical power and limit the generalizability of the findings. Although we observed consistent trends across individual animals, future studies using larger cohorts will be required to confirm the robustness and reproducibility of our findings.

Acknowledgements

The Authors would like to thank Editage (www.editage.jp) for editing this manuscript.

Footnotes

  • Authors’ Contributions

    Satoka Kasai: Conceptualization, Methodology, Validations, Formal analysis, Investigation, Writing-Original Draft, Visualization, Supervision, Project administration, Funding acquisition; Sho Sato, Kento Namiki, and Rinka Obata: Methdology, Validations, Formal analysis, Investigation; Kazumi Yoshizawa: Resources, Supervision.

  • Supplementary Material

    Available at: https://doi.org/10.6084/m9.figshare.30444413

  • Conflicts of Interest

    The Authors declare that they have no competing interest in relation to this study.

  • Funding

    This work was supported by JSPS KAKENHI Grant No. JP23737769.

  • Artificial Intelligence (AI) Disclosure

    During the preparation of this manuscript, a large language model (ChatGPT-4o, OpenAI) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning–based image enhancement tools.

  • Received September 26, 2025.
  • Revision received October 15, 2025.
  • Accepted October 16, 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).

References

  1. ↵
    1. Fearon K,
    2. Strasser F,
    3. Anker SD,
    4. Bosaeus I,
    5. Bruera E,
    6. Fainsinger RL,
    7. Jatoi A,
    8. Loprinzi C,
    9. Mac-Donald N,
    10. Mantovani G,
    11. Davis M,
    12. Muscaritoli M,
    13. Ottery F,
    14. Radbruch L,
    15. Ravasco P,
    16. Walsh D,
    17. Wilcock A,
    18. Kaasa S,
    19. Baracos VE
    : Definition and classification of cancer cachexia: an international consensus. Lancet Oncol 12(5): 489-495, 2011. DOI: 10.1016/S1470-2045(10)70218-7
    OpenUrlCrossRefPubMed
  2. ↵
    1. Cannon B,
    2. Nedergaard J
    : Brown adipose tissue: function and physiological significance. Physiol Rev 84(1): 277-359, 2004. DOI: 10.1152/physrev.00015.2003
    OpenUrlCrossRefPubMed
  3. ↵
    1. Bartelt A,
    2. Heeren J
    : Adipose tissue browning and metabolic health. Nat Rev Endocrinol 10(1): 24-36, 2014. DOI: 10.1038/nrendo.2013.204
    OpenUrlCrossRefPubMed
  4. ↵
    1. Tsoli M,
    2. Moore M,
    3. Burg D,
    4. Painter A,
    5. Taylor R,
    6. Lockie SH,
    7. Turner N,
    8. Warren A,
    9. Cooney G,
    10. Oldfield B,
    11. Clarke S,
    12. Robertson G
    : Activation of thermogenesis in brown adipose tissue and dysregulated lipid metabolism associated with cancer cachexia in mice. Cancer Res 72(17): 4372-4382, 2012. DOI: 10.1158/0008-5472.CAN-11-3536
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Ikeda K,
    2. Kang Q,
    3. Yoneshiro T,
    4. Camporez JP,
    5. Maki H,
    6. Homma M,
    7. Shinoda K,
    8. Chen Y,
    9. Lu X,
    10. Maretich P,
    11. Tajima K,
    12. Ajuwon KM,
    13. Soga T,
    14. Kajimura S
    : UCP1-independent signaling involving SER-CA2b-mediated calcium cycling regulates beige fat thermogenesis and systemic glucose homeostasis. Nat Med 23(12): 1454-1465, 2017. DOI: 10.1038/nm.4429
    OpenUrlCrossRefPubMed
  6. ↵
    1. Auger C,
    2. Li M,
    3. Fujimoto M,
    4. Ikeda K,
    5. Yook JS,
    6. O’Leary TR,
    7. Caycedo MPH,
    8. Xiaohan C,
    9. Oikawa S,
    10. Verkerke ARP,
    11. Shinoda K,
    12. Griffin PR,
    13. Inaba K,
    14. Stimson RH,
    15. Kajimura S
    : Identification of a molecular resistor that controls UCP1-independent Ca(2+) cycling thermogenesis in adipose tissue. Cell Metab 37(6): 1311-1325.e9, 2025. DOI: 10.1016/j.cmet.2025.03.009
    OpenUrlCrossRefPubMed
  7. ↵
    1. Kawakami Y,
    2. Sielski R,
    3. Kawakami T
    : Mouse body temperature measurement using infrared thermometer during passive systemic anaphylaxis and food allergy evaluation. J Vis Exp (139): 58391, 2018. DOI: 10.3791/58391
    OpenUrlCrossRef
  8. ↵
    1. Galarraga M,
    2. Campión J,
    3. Muñoz-Barrutia A,
    4. Boqué N,
    5. Moreno H,
    6. Martínez JA,
    7. Milagro F,
    8. Ortiz-de-Solórzano C
    : Adiposoft: automated software for the analysis of white adipose tissue cellularity in histological sections. J Lipid Res 53(12): 2791-2796, 2012. DOI: 10.1194/jlr.D023788
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Schmidt U,
    2. Weigert M,
    3. Broaddus C,
    4. Myers G
    : Cell detection with star-convex polygons. In: Medical Image Computing and Computer-Assisted Intervention – MICCAI 2018, Granada, Spain, 2018. Cham, Switzerland, Springer International Publishing pp 265-273, 2018. DOI: 10.1007/978-3-030-00934-2_30
    OpenUrlCrossRef
  10. ↵
    1. Legland D,
    2. Arganda-Carreras I,
    3. Andrey P
    : MorphoLibJ: integrated library and plugins for mathematical morphology with ImageJ. Bioinformatics 32(22): 3532-3534, 2016. DOI: 10.1093/bioinformatics/btw413
    OpenUrlCrossRefPubMed
  11. ↵
    1. Pfaffl MW
    : A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9): e45, 2001. DOI: 10.1093/nar/29.9.e45
    OpenUrlCrossRefPubMed
  12. ↵
    1. Livak KJ,
    2. Schmittgen TD
    : Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25(4): 402-408, 2001. DOI: 10.1006/meth.2001.1262
    OpenUrlCrossRefPubMed
  13. ↵
    1. Burtis WJ,
    2. Brady TG,
    3. Orloff JJ,
    4. Ersbak JB,
    5. Warrell RP Jr.,
    6. Olson BR,
    7. Wu TL,
    8. Mitnick ME,
    9. Broadus AE,
    10. Stewart AF
    : Immunochemical characterization of circulating parathyroid hormone–related protein in patients with humoral hypercalcemia of cancer. N Engl J Med 322(16): 1106-1112, 1990. DOI: 10.1056/NEJM199004193221603
    OpenUrlCrossRefPubMed
  14. ↵
    1. Abou-Samra AB,
    2. Jüppner H,
    3. Force T,
    4. Freeman MW,
    5. Kong XF,
    6. Schipani E,
    7. Urena P,
    8. Richards J,
    9. Bonventre JV,
    10. Potts JT Jr.
    : Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium. Proc Natl Acad Sci U S A 89(7): 2732-2736, 1992. DOI: 10.1073/pnas.89.7.2732
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Kir S,
    2. White JP,
    3. Kleiner S,
    4. Kazak L,
    5. Cohen P,
    6. Baracos VE,
    7. Spiegelman BM
    : Tumour-derived PTH-related protein triggers adipose tissue browning and cancer cachexia. Nature 513(7516): 100-104, 2014. DOI: 10.1038/nature13528
    OpenUrlCrossRefPubMed
  16. ↵
    1. Ikeda K,
    2. Yamada T
    : UCP1 dependent and independent thermogenesis in brown and beige adipocytes. Front Endocrinol (Lausanne) 11: 498, 2020. DOI: 10.3389/fendo.2020.00498
    OpenUrlCrossRefPubMed
  17. ↵
    1. Chemaly ER,
    2. Troncone L,
    3. Lebeche D
    : SERCA control of cell death and survival. Cell Calcium 69: 46-61, 2018. DOI: 10.1016/j.ceca.2017.07.001
    OpenUrlCrossRefPubMed
  18. ↵
    1. Fearon K,
    2. Arends J,
    3. Baracos V
    : Understanding the mechanisms and treatment options in cancer cachexia. Nat Rev Clin Oncol 10(2): 90-99, 2013. DOI: 10.1038/nrclinonc.2012.209
    OpenUrlCrossRefPubMed
  19. ↵
    1. Petruzzelli M,
    2. Schweiger M,
    3. Schreiber R,
    4. Campos-Olivas R,
    5. Tsoli M,
    6. Allen J,
    7. Swarbrick M,
    8. Rose-John S,
    9. Rincon M,
    10. Robertson G,
    11. Zechner R,
    12. Wagner EF
    : A switch from white to brown fat increases energy expenditure in cancer-associated cachexia. Cell Metab 20(3): 433-447, 2014. DOI: 10.1016/j.cmet.2014.06.011
    OpenUrlCrossRefPubMed
  20. ↵
    1. Han J,
    2. Meng Q,
    3. Shen L,
    4. Wu G
    : Interleukin-6 induces fat loss in cancer cachexia by promoting white adipose tissue lipolysis and browning. Lipids Health Dis 17(1): 14, 2018. DOI: 10.1186/s12944-018-0657-0
    OpenUrlCrossRefPubMed
  21. ↵
    1. Fukagawa NK,
    2. Kenney WL,
    3. Buskirk ER
    : Functional consequences of sarcopenia: Effects on thermoregulation. J Gerontol A Biol Sci Med Sci 50A(Special): 78-85, 1995. DOI: 10.1093/gerona/50a.special_issue.78
    OpenUrlCrossRefPubMed
  22. ↵
    1. Hashimoto H,
    2. Azuma Y,
    3. Kawasaki M,
    4. Fujihara H,
    5. Onuma E,
    6. Yamada-Okabe H,
    7. Takuwa Y,
    8. Ogata E,
    9. Ueta Y
    : Parathyroid hormone-related protein induces cachectic syndromes without directly modulating the expression of hypothalamic feeding-regulating peptides. Clin Cancer Res 13(1): 292-298, 2007. DOI: 10.1158/1078-0432.CCR-06-1487
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Watanabe H,
    2. Oshima T
    : The latest treatments for cancer cachexia: an overview. Anticancer Res 43(2): 511-521, 2023. DOI: 10.21873/anticanres.16188
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Irazoki A,
    2. Frank E,
    3. Pham TCP,
    4. Braun JL,
    5. Ehrlich AM,
    6. Haid M,
    7. Riols F,
    8. Hansen CHF,
    9. Jørgensen AR,
    10. Andersen NR,
    11. Hidalgo-Corbacho L,
    12. Meneses-Valdes R,
    13. Ali MS,
    14. Raun SH,
    15. Modvig JL,
    16. Gallero S,
    17. Larsen S,
    18. Gerhart-Hines Z,
    19. Jensen TE,
    20. Rohm M,
    21. Treebak JT,
    22. Fajardo VA,
    23. Sylow L
    : Housing temperature impacts the systemic and tissue-specific molecular responses to cancer in mice. J Cachexia Sarcopenia Muscle 16(2): e13781, 2025. DOI: 10.1002/jcsm.13781
    OpenUrlCrossRef
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
Increased Expression of Serca2b in the Adipose Tissue of a Cancer Cachexia Model
SATOKA KASAI, SHO SATO, KENTO NAMIKI, RINKA OBATA, KAZUMI YOSHIZAWA
In Vivo Jan 2026, 40 (1) 108-122; DOI: 10.21873/invivo.14177
Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords