Elevated Levels of CTRP1 in Obesity Contribute to Tumor Progression in a p53-Dependent Manner

Simple Summary Obesity is regarded as a risk factor for various cancers. However, the molecular mechanisms linking obesity with cancer remain primarily uncharacterized. In this study, we demonstrate that CTRP1, an adiponectin paralogue, promotes tumor growth in a p53-dependent manner. Obese mice on a high-fat diet showed a higher level of CTRP1 protein in serum. It is also known that CTRP1 treatment contributes to tumor growth and cell migration. These results indicate that an elevated level of CTRP1 in obesity promotes tumor progression. Abstract Mounting evidence supports the relationship between obesity and cancer. However, the molecular mechanisms linking obesity with cancer remain largely uninvestigated. In this study, we demonstrate that the expression of C1q/TNF-related protein 1 (CTRP1), an adiponectin paralogue, contributes to tumor growth by regulating the tumor suppressor p53. In our study, obese mice on a high-fat diet showed higher serum CTRP1 levels. Through in vitro experiments, we showed that the secreted form of CTRP1 in the culture medium decreased p53 expression and p53-dependent transcription in the cells. Moreover, CTRP1 treatment enhanced colony formation and cell migration. These results collectively suggest that elevated levels of CTRP1 in obesity significantly contribute to tumor progression.


Introduction
Obesity is regarded as a risk factor for various cancers, and cancer incidence increases in the prevalence of risk factors such as obesity [1][2][3]. Recent reports show how obesity is associated with an increased incidence of at least 13 different cancers, including the following: endometrial, esophageal, renal, and pancreatic adenocarcinomas, as well as hepatocellular carcinoma, gastric cardia cancer, meningioma, multiple myeloma, and colorectal, postmenopausal breast, ovarian, gallbladder, and thyroid cancers [4]. Some of the probable biological mechanisms linking obesity and cancer include (1) insulin resistance and abnormal IGF-1 signaling, (2) sex hormone signaling, (3) subclinical chronic low-grade inflammation, and (4) alterations in the levels of adipocyte-derived factors among others [5][6][7][8].
p53 is an important tumor suppressor gene that regulates apoptosis, cell cycle, and cellular senescence [9]. While p53 is mutated in up to 50% of human cancers, wildtype p53 is functionally inactivated in various cancers by several mechanisms, such as promoter methylation and ubiquitin-mediated degradation [10]. Growth promoting signals decrease the level of p53 to inactivate its tumor suppressor function [11]. MDM2, a typical p53 ubiquitin ligase, gets activated by Akt-mediated phosphorylation and leads to p53 degradation [12,13].
HCT116 human colorectal cancer cells, MCF7 human breast cancer cells, A549 human lung cancer cells, H1299 human lung cancer cells, and HEK 293T human embryonic kidney cells were maintained in DMEM (Welgene, Seoul, Korea) containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and an antibiotic-antimycotic solution (Welgene). Cell proliferation was measured by the MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] assay. HCT116, MCF7, and A549 cells were seeded in 24-well culture plates (2 × 10 5 cells/well). After 24 h of plating, MTT solution was added at a final concentration of 1 mg/mL, and the mixture was incubated for 3 h. MTT was purchased from USB Corporation (Cleveland, OH, USA). A clonogenic assay was performed using crystal violet. HCT116, MCF7, and A549 cells were seeded in 6-well culture plates (10 3 cells/well). After 10 days of plating, the cells were fixed with 100% methanol and incubated for 10 min at -20 • C. Cells were then stained with crystal violet solution (20% methanol and 0.25% crystal violet) for 30 min. Crystal violet was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell conditioned media was collected from control lentivirus-and CTRP1 lentivirus-infected A549 cells. These cells were incubated for 48 h in serum-free media before collecting the conditioned media. The secreted proteincontaining supernatant media was collected and filtered through a 0.45 µm filter. After filtration, this media was added to the cells.

Virus Production and Transduction
Lentivirus was prepared by co-transfection of the lentiviral transfer vector with the psPAX2 envelope and pMD2.G packaging plasmids into HEK 293T cells using the calcium phosphate transfection method (2M CaCl 2 , 2X HEPES buffered saline (pH 7.2)) [25]. The media was changed 12 h after transfection. The virus-containing supernatant media was collected 48 h and 72 h after transfection and filtered through a 0.45 µm filter. After filtration, the media was concentrated using a Lenti-X concentrator (Clontech, Mountain View, CA, USA). Cells were infected in media containing 8 µg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA).

Western Blotting
Cells were harvested and resuspended in cell lysis buffer (150 mM NaCl, 50 mM HEPES (pH 7.5), 1% NP40) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). Whole-cell lysates were resolved by SDS-PAGE and transferred to PVDF membranes (GE Healthcare, Uppsala, Sweden) via Western blotting. Proteins were detected with a 1:1000 or 1:5000 dilution of the primary antibody using a chemiluminescence system (Dogen, Seoul, Korea). Images were acquired using the LAS4000 system (GE Healthcare, Uppsala, Sweden). The CTRP1 antibody was purchased from Invitrogen (Carlsbad, CA, USA), p21 antibody from Cell Signaling Technology (Danvers, MA, USA), and p53 antibody from Santa Cruz Biotechnology (Dallas, TX, USA). FR180204, ERK inhibitor, was purchased from Calbiochem (San Diego, CA, USA).

Immunofluorescence and Confocal Microscopy
Cells were grown on sterilized glass coverslips. The cells were then fixed with 4% paraformaldehyde. For immunostaining, cells were blocked with 3% BSA in PBS, stained with a 1:1000 dilution of the primary antibody in PBS, and stained with 1:1000 Alexa 488conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) or Alexa 568-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA). Images were captured using a Carl Zeiss LSM710 confocal microscope (Carl Zeiss, ObERKochen, Germany). The Image J software was used to analyze cell images (https://imagej.nih.gov/ij, accessed on 2 June 2021).

Animal and High-Fat Diet-Induced Obesity Mouse Model
Male C57BL/6 mice (6-week-old) were obtained from DBL (Seoul, Korea) and fed a lean control diet (Envigo, Indianapolis, IN, USA) or a high-fat diet (Research Diets, New Brunswick, NJ, USA) for 10 weeks. After 10 weeks, the control and high-fat diet mice were sacrificed, and blood was collected from the mice after anesthesia by puncturing the heart. Blood serum was isolated via centrifugation at 1500× g for 15 min following 30 min of clotting at room temperature.

Statistical Analysis
Statistical significance of differences, during analysis of Western blotting, quantitative RT-PCR, and MTT assay results, was evaluated via a two-tailed t-test using Excel software (Microsoft, Seattle, WA, USA). In most experiments, statistical significance was set at p < 0.05.

The Level of CTRP1 in Serum Is Elevated in Obesity
We used a high-fat diet-induced obese mouse model to identify the relationship between serum CTRP1 levels and obesity. The high-fat diet resulted in higher weight gain in mice when compared with the ones fed with a normal diet ( Figure 1C). We analyzed serum CTRP1 expression in mice using Western blotting, which showed significantly higher CTRP1 levels in the serum of the obese mice ( Figure 1A,B). To assess CTRP1 expression in obese animals, we used the GEO profiles database in NCBI (https://www.ncbi.nlm.nih. gov/geoprofiles/, accessed on 2 June 2021). In rats, CTRP1 mRNA levels were elevated in diet-induced obese mice epididymal fat ( Figure 1D) [26]. In humans, CTRP1 mRNA levels were found to be higher in abdominal adipocytes of obese people than in abdominal adipocytes of normal people, irrespective of their sex ( Figure 1E) [27].
We used a high-fat diet-induced obese mouse model to identify the relationship between serum CTRP1 levels and obesity. The high-fat diet resulted in higher weight gain in mice when compared with the ones fed with a normal diet ( Figure 1C). We analyzed serum CTRP1 expression in mice using Western blotting, which showed significantly higher CTRP1 levels in the serum of the obese mice ( Figure 1A,B). To assess CTRP1 expression in obese animals, we used the GEO profiles database in NCBI (https://www.ncbi.nlm.nih.gov/geoprofiles/, accessed on 2 June 2021). In rats, CTRP1 mRNA levels were elevated in diet-induced obese mice epididymal fat ( Figure 1D) [26]. In humans, CTRP1 mRNA levels were found to be higher in abdominal adipocytes of obese people than in abdominal adipocytes of normal people, irrespective of their sex ( Figure 1E) [27]. The protein level of CTRP1 in serum is increased in high-fat diet mice. Mice were fed with a control diet or a high-fat diet for 10 weeks. After feeding, an equal amount of mouse blood serum was subject to Western blotting with anti-CTRP1 antibody (upper panel). Albumin was visualized by Ponceau S staining (bottom panel). Images of the uncropped western blots can be found in Figure S1. (B) The level of CTRP1 protein was quantified and depicted in the graph. The graph shows the average and standard error. Control vs highfat diet rat, *** p < 0.005 (n = 6). (C) The weights of the mice are shown in the graph. (D) GEO data analysis shows an elevated level of CTRP1 mRNA in obese rats. GEO data were used for analysis. The protein level of CTRP1 in serum is increased in high-fat diet mice. Mice were fed with a control diet or a high-fat diet for 10 weeks. After feeding, an equal amount of mouse blood serum was subject to Western blotting with anti-CTRP1 antibody (upper panel). Albumin was visualized by Ponceau S staining (bottom panel). Images of the uncropped western blots can be found in Figure S1. (B) The level of CTRP1 protein was quantified and depicted in the graph. The graph shows the average and standard error. Control vs high-fat diet rat, *** p < 0.005 (n = 6). (C) The weights of the mice are shown in the graph. (D) GEO data analysis shows an elevated level of CTRP1 mRNA in obese rats. GEO data were used for analysis. (E) GEO data shows that the level of CTRP1 mRNA is elevated in obese humans (abdominal subcutaneous adipocytes), * p < 0.05.
We further tested this hypothesis by overexpressing CTRP1 in Caco2 cells, a p53 null cell line. Western blotting showed that the p53-dependent transcription of p21 did not decrease in this case ( Figure 3A). Next, we examined the mRNA expression levels of p53 Control vs CTRP1 overexpression, * p < 0.05, ** p < 0.01. Images of the uncropped western blots can be found in Figure S2. (C) CTRP1 decreases p53. MCF7 cells, A549 cells, and HCT116(+/+) cells were infected with either control lentivirus or lentivirus encoding CTRP1, and the cells were fixed and immunostained with anti-p53 antibody (green) and anti-CTRP1 antibody (red). Scale bar, 10 µm. (D) CTRP1 overexpression decreased the mRNA level of p53 and p21. The level of p53 and p21 mRNA was examined by quantitative RT-PCR in control and CTRP1 overexpressed cells. * p < 0.05, *** p < 0.005. (E) The expression levels of GADD45α, NOXA, PIG3, and SESN2 mRNAs were measured using quantitative RT-PCR * p < 0.05, ** p < 0.01, *** p < 0.005.
Next, we examined whether CTRP1 expression decreased p53 protein levels and p53-dependent transcription. Western blot analysis showed that the expression levels of p53 protein and p21 protein, a well-known transcriptional target of p53, were significantly reduced in CTRP1 overexpressed cancer cell lines MCF7, A549, and HCT116 ( Figure 2B). Confocal microscopy confirmed that the overexpression of CTRP1 downregulated p53 protein expression ( Figure 2C). Our investigation also revealed that CTRP1 overexpression inhibited the expression of p53 and p21 at the transcript level. (Figure 2D). We also examined the mRNA expression levels of p53-dependent genes NOXA, GADD45α, PIG3, and SESTRIN2, which exhibited significant downregulation ( Figure 2E). These results indicated that CTRP1 overexpression resulted in the decreased expression of p53 and p53-dependent genes.
We further tested this hypothesis by overexpressing CTRP1 in Caco2 cells, a p53 null cell line. Western blotting showed that the p53-dependent transcription of p21 did not decrease in this case ( Figure 3A). Next, we examined the mRNA expression levels of p53 and p53 target genes by quantitative RT-PCR, and the levels of p53, p21, NOXA, GADD45, PIG3, and SESTRIN2 did not show any significant decrease ( Figure 3B,C). These results indicated that CTRP1 decreased transcription of p53-dependent genes in the presence of p53.
Cancers 2021, 13, x FOR PEER REVIEW 7 of 14 and p53 target genes by quantitative RT-PCR, and the levels of p53, p21, NOXA, GADD45, PIG3, and SESTRIN2 did not show any significant decrease ( Figure 3B,C). These results indicated that CTRP1 decreased transcription of p53-dependent genes in the presence of p53.

Conditioned Media-Derived CTRP1 Decreased p53 and p53-Dependent Transcription
Next, we demonstrated that the expression of CTRP1 affects the expression of p53 and p53-dependent transcription (Figure 2). Since CTRP1 is a secreted glycoprotein, we examined whether the secreted form of CTRP1 affects p53 and p53-dependent transcription. To determine the function of secreted CTRP1 protein, we collected conditioned media from A549 cells expressing CTRP1 protein and incubated MCF7, A549, and HCT116 cells with the conditioned media ( Figure 4A). Incubation with the conditioned media resulted in the downregulation of p53 and p21 protein levels in MCF7, A549, and HCT116 cells, indicating that the secreted form of CTRP1 decreased p53 function ( Figure 4B,C). A similar decrease in the mRNA levels of p53 and p21 was seen in the cells incubated with conditioned media ( Figure 4D). We also found that the transcription of p53-dependent genes was downregulated by treating the cells with conditioned media-derived CTRP1 ( Figure 4E). Finally, we used confocal microscopy to confirm the downregulation of p53

Conditioned Media-Derived CTRP1 Decreased p53 and p53-Dependent Transcription
Next, we demonstrated that the expression of CTRP1 affects the expression of p53 and p53-dependent transcription (Figure 2). Since CTRP1 is a secreted glycoprotein, we examined whether the secreted form of CTRP1 affects p53 and p53-dependent transcription.
To determine the function of secreted CTRP1 protein, we collected conditioned media from A549 cells expressing CTRP1 protein and incubated MCF7, A549, and HCT116 cells with the conditioned media ( Figure 4A). Incubation with the conditioned media resulted in the downregulation of p53 and p21 protein levels in MCF7, A549, and HCT116 cells, indicating that the secreted form of CTRP1 decreased p53 function ( Figure 4B,C). A similar decrease in the mRNA levels of p53 and p21 was seen in the cells incubated with conditioned media ( Figure 4D). We also found that the transcription of p53-dependent genes was downregulated by treating the cells with conditioned media-derived CTRP1 ( Figure 4E). Finally, we used confocal microscopy to confirm the downregulation of p53 by conditioned medium-derived CTRP1 ( Figure 4F). These results collectively indicate that secreted CTRP1 affects p53 expression and p53-dependent transcription. by conditioned medium-derived CTRP1 ( Figure 4F). These results collectively indicate that secreted CTRP1 affects p53 expression and p53-dependent transcription.

CTRP1 Enhances Cell Proliferation and Migration
The crucial tumor-suppressive role of p53 led us to hypothesize that CTRP1 overexpression might result in increased tumor progression by inhibiting p53. We tested this hypothesis using a clonogenic assay. Lentivirus-mediated CTRP1 overexpression increased colony formation in MCF7, A549, and HCT116 cells ( Figure 5A). Next, we incubated the cancer cells with conditioned media containing CTRP1 and found that the secreted form of CTRP1 also significantly increased colony formation ( Figure 5B). However, incubation of the p53-null Caco2 cells with conditioned medium did not increase colony formation, indicating that CTRP1 increases colony formation in a p53-dependent manner ( Figure 5C). These results indicated that CTRP1 positively regulates cell proliferation, thereby having a potential tumorigenic role. Since p53 also regulates the cell cycle, we examined whether CTRP1 contributes to cell cycle progression by regulating p53. When the cancer cells were incubated with CTRP1 conditioned medium, the percentage of S and G2/M phase cells was higher in MCF-7, A549, and HCT116 cells ( Figure 5D). These data indicate that CTRP1 conditioned medium activates cell proliferation and cell cycle progression.

CTRP1 Enhances Cell Proliferation and Migration
The crucial tumor-suppressive role of p53 led us to hypothesize that CTRP1 overexpression might result in increased tumor progression by inhibiting p53. We tested this hypothesis using a clonogenic assay. Lentivirus-mediated CTRP1 overexpression increased colony formation in MCF7, A549, and HCT116 cells ( Figure 5A). Next, we incubated the cancer cells with conditioned media containing CTRP1 and found that the secreted form of CTRP1 also significantly increased colony formation ( Figure 5B). However, incubation of the p53-null Caco2 cells with conditioned medium did not increase colony formation, indicating that CTRP1 increases colony formation in a p53-dependent manner ( Figure 5C). These results indicated that CTRP1 positively regulates cell proliferation, thereby having a potential tumorigenic role. Since p53 also regulates the cell cycle, we examined whether CTRP1 contributes to cell cycle progression by regulating p53. When the cancer cells were incubated with CTRP1 conditioned medium, the percentage of S and G2/M phase cells was higher in MCF-7, A549, and HCT116 cells ( Figure 5D). These data indicate that CTRP1 conditioned medium activates cell proliferation and cell cycle progression.  Because p53 is an essential regulator of cell migration, we next examined the effect of CTRP1 on this cellular phenotype. The wound-healing assay showed that cell migration with CTRP1 conditioned medium was significantly faster than that of the control ( Figure 6A,B). These results indicated that CTRP1 is also involved in cell migration.
days. After incubation, cell proliferation was measured by crystal violet assay. * p < 0.05, ** p < 0.01. (B) CTRP1 CM increase cell proliferation. HCT116, MCF7, and A549 cells were seeded at a density of 10 3 cells per well and incubated for 10 days with either control CM or CTRP1 CM. After 10 days of incubation, cells were measured by crystal violet assay. *** p < 0.005. (C) CTRP1 CM did not increase the cell proliferation in p53 null cells (Caco2). (D) CTRP1 CM enhances cell cycle progression. Control CM and CTRP1 CM were analyzed by flow cytometry, and the cell cycle population (%) were calculated and shown in the graph * p < 0.05, ** p < 0.01, *** p < 0.005.
Because p53 is an essential regulator of cell migration, we next examined the effect of CTRP1 on this cellular phenotype. The wound-healing assay showed that cell migration with CTRP1 conditioned medium was significantly faster than that of the control ( Figure  6A,B). These results indicated that CTRP1 is also involved in cell migration.

CTRP1 Induces Cell Proliferation through the Activation of the ERK Signaling Pathway
Finally, we examined the expression of various cell signaling proteins to determine the mechanism of regulation of p53 and p53-dependent transcription by CTRP1. Our investigations revealed that CTRP1 conditioned medium increased the levels of phospho-ERK protein in MCF7, A549, and HCT116 cells ( Figure 7A). Since the activation of ERK signaling contributes to the downregulation of p53, we examined whether the inhibition of ERK signaling prevents p53 downregulation. We used FR180204 to inhibit Erk signaling and FR180204 treatment did not induce dramatic cell death at 10 µ M. We found that ERK inhibitor treatment blocks CTRP1-mediated decrease in p53 and p21 protein ( Figure  7B) and mRNA expression ( Figure 7C). These results collectively indicate that ERK signaling is essential for CTRP1-mediated transcription of p53 and p53 target genes.

CTRP1 Induces Cell Proliferation through the Activation of the ERK Signaling Pathway
Finally, we examined the expression of various cell signaling proteins to determine the mechanism of regulation of p53 and p53-dependent transcription by CTRP1. Our investigations revealed that CTRP1 conditioned medium increased the levels of phospho-ERK protein in MCF7, A549, and HCT116 cells ( Figure 7A). Since the activation of ERK signaling contributes to the downregulation of p53, we examined whether the inhibition of ERK signaling prevents p53 downregulation. We used FR180204 to inhibit Erk signaling and FR180204 treatment did not induce dramatic cell death at 10 µM. We found that ERK inhibitor treatment blocks CTRP1-mediated decrease in p53 and p21 protein ( Figure 7B) and mRNA expression ( Figure 7C). These results collectively indicate that ERK signaling is essential for CTRP1-mediated transcription of p53 and p53 target genes. The level of p-ERK protein was quantified with an anti-p-ERK antibody and depicted in the graph (* p < 0.05, ** p < 0.01). Images of the uncropped western blots can be found in Figure S6. (B) ERK inhibitor treatment rescues p53 and p21 levels via inhibition of the ERK pathway. Cells were treated with either control or ERK inhibitor (FR180204, 10 μM) for 24 h. After treatment, cells were harvested and probed with the indicated antibodies. Images of the uncropped western blots can be found in Figure S7. (C) The level of p53 and p21 protein was quantified and depicted in the graph (* p < 0.05, ** p < 0.01, *** p < 0.005).

Discussion
In this study, we highlight the oncogenic role of CTRP1, a member of the adipokine paralogue family, and demonstrate how it activates cell proliferation by inhibiting p53 and p53-dependent transcription. CTRP1 was initially identified to be produced in adipose tissues, and we showed that the level of CTRP1 in mouse serum is elevated in highfat diet-fed obese mice than in mice fed with a normal diet. Incubation of cancer cells with the secreted form of CTRP1 downregulated p53 and p53-dependent transcription. We also demonstrated that the secreted form of CTRP1 activates colony formation and cell cycle progression. Since the serum level of CTRP1 protein is elevated in obese mice, these results suggest that obesity-induced CTRP1 expression contributes to cancer progression. The level of p-ERK protein was quantified with an anti-p-ERK antibody and depicted in the graph (* p < 0.05, ** p < 0.01). Images of the uncropped western blots can be found in Figure S6. (B) ERK inhibitor treatment rescues p53 and p21 levels via inhibition of the ERK pathway. Cells were treated with either control or ERK inhibitor (FR180204, 10 µM) for 24 h. After treatment, cells were harvested and probed with the indicated antibodies. Images of the uncropped western blots can be found in Figure S7. (C) The level of p53 and p21 protein was quantified and depicted in the graph (* p < 0.05, ** p < 0.01, *** p < 0.005).

Discussion
In this study, we highlight the oncogenic role of CTRP1, a member of the adipokine paralogue family, and demonstrate how it activates cell proliferation by inhibiting p53 and p53-dependent transcription. CTRP1 was initially identified to be produced in adipose tissues, and we showed that the level of CTRP1 in mouse serum is elevated in high-fat diet-fed obese mice than in mice fed with a normal diet. Incubation of cancer cells with the secreted form of CTRP1 downregulated p53 and p53-dependent transcription. We also demonstrated that the secreted form of CTRP1 activates colony formation and cell cycle progression. Since the serum level of CTRP1 protein is elevated in obese mice, these results suggest that obesity-induced CTRP1 expression contributes to cancer progression.
Many reports support the hypothesis that obesity is positively correlated to tumor progression [1]. Adipose tissue secretes various adipokines, and their association with cancer is diverse. While adiponectin is inversely correlated to the progression of breast, colorectal, and endometrial cancers, leptin is associated with an increased risk of endometrial and renal cancers [28]. Here, we demonstrate that CTRP1, a protein secreted from adipose tissue, can activate tumor cell growth. While CTRP1 belongs to the adiponectin family, our data shows that the function of CTRP1 is different from that of the other adiponectins. In this study, we demonstrate that CTRP1 has the potential to activate tumor cell growth and migration.
p53 is the most important tumor suppressor, and we demonstrate that CTRP1 decreases p53 expression and p53-dependent transcription. We show how CTRP1 downregulates p53 in MCF-7, A549, and HCT116 cells, which express wild-type p53. However, such oncogenic effects of CTRP1 are not seen in a p53-null cell line (Figures 3 and 5). These results indicate that the tumor-promoting effect of CTRP1 is wild-type p53-dependent. p53 mRNA expression was downregulated by CTRP1 overexpression and by the secreted form of CTRP1. The level of p53 protein is usually regulated by ubiquitin-mediated degradation. However, CTRP1 treatment downregulates p53 mRNA and protein levels (Figures 2 and 4). These results suggest that CTRP1 regulates p53 at the transcriptional level, while treatment with an ERK inhibitor treatment increased p53 mRNA expression by abrogating the effects of CTRP1 (Figure 7). These results collectively support that ERK signaling downregulates p53 expression at the transcriptional level. Our novel finding suggests that secreted CTRP1 protein regulates p53 transcription by modulating the ERK signaling pathway. Further mechanistic study will be required to elucidate the function of CTRP1 protein.
Even though CTRP1 expression or secreted CTRP1 activates tumor cell proliferation in a p53-dependent manner, the role of CTRP1 extends far beyond the modulation of tumor progression by cancer cells. CTRP1 can act as a growth factor to promote cancer cell growth, similar to other growth factors. It is worth examining whether CTRP1 can induce tumor initiation, although our hypothesis suggests that it does not. Further research with CTRP1 knockout or transgenic mice will elucidate the long-term effects of CTRP1 on cancer initiation.
The findings from our study suggest that since elevated levels of CTRP1 contribute to tumor cell growth and migration, this adipocyte-secreted protein might be a potential link between obesity and carcinogenesis. Uncovering the molecular mechanisms of CTRP1 may provide a greater understanding of how obesity increases the risk of carcinogenesis. Further in-depth clinical studies are required to prove the correlation between serum CTRP1 levels and the degree of cancer progression.

Conclusions
Here, we demonstrate that obese mice on a high-fat diet showed higher serum CTRP1 levels, and the secreted form of CTRP1 in culture medium decreased p53 expression and p53-dependent transcription in cells. CTRP1 treatment enhanced colony formation and cell migration. These results indicate that elevated levels of CTRP1 in obesity contribute to tumor progression.