Solute carrier family 35 member D3 promotes colorectal cancer progression through AMPK signaling pathway

This article is aim to investigate the functional role and potential regulatory mechanisms of solute carrier family 35 member D3 (SLC35D3) in colorectal cancer. Beyond analyzing databases, western blot was used to detect and verify the expression of SLC35D3. Cell proliferation and invasion or migration ability were detected by CCK‐8, EdU, and Transwell assays. Animal experiments were conducted to verify if the biological function of SLC35D3 in vivo is consistent with that in vitro. Beyond SLC family pathway enrichment analysis, the relationship between SLC35D3 and metabolic pathway AMPK was explored using co‐immunoprecipitation and western blot. SLC35D3 is highly expressed in colorectal cancer (CRC) tissue. The overexpression or down regulation of SLC35D3 has been shown to promote or inhibit the biological functions of colorectal cancer, and similar experimental results can be verified in vivo experiments. Based on the high correlation between the SLC family and metabolic pathways, we chose the metabolic‐related AMPK pathway as the subject of our research. Co‐immunoprecipitation and protein analysis demonstrated that SLC35D3 may bind to AMPK molecules and regulate the AMPK pathway of colorectal cancer cells. Based on the above facts, SLC35D3 promotes colorectal cancer progression through regulating AMPK signaling pathway.


| INTRODUCTION
Colorectal cancer is a highly lethal cancer, ranking third in malignant tumors in China. 1,2 By 2022, there is a significant increase in colorectal cancer incidence and poor prognosis. 3 To identify more effective treatment strategies for colorectal cancer, it is essential to further investigate its pathogenesis.
Chemotherapy resistance in tumor cells may result from the decreased activity of drug transport, which may be due to the low expression of solute carrier transporters. 4 For instance, the down regulation of SLC4A4, which is responsible for bicarbonate transport, has been linked to poor prognosis of colorectal cancer. 4 Conversely, some solute carrier proteins have been shown to inhibit colorectal cancer growth through related pathways. 5 SLC35 is a family of nucleotide sugar transporters that play a crucial role in connecting the synthesis of activated sugars in the nucleus or cytosol. 6 Among these transporters, SLC35C1, which is responsible for guanosine diphosphate fucose (GDP-fucose) transportation, has been shown to restrict the development of colorectal cancer. 7 In contrast, SLC35A2, a transporter associated with glycosylation, has been found to promote the invasion, adhesion, metastasis, and membrane glycan profile in hepatocellular carcinoma (HCC) cells. 8 These findings indicate that differentially expressed solute carrier (SLC) proteins may play a pivotal role in the occurrence and development of colorectal cancer.
SLC35D3 is a protein that exhibits differential expression between colorectal cancer. Previous studies have suggested that SLC35D3 plays an important regulatory role in the nervous system, including inducing autophagy to ensure neuronal activity. 9 Furthermore, its mutations have been shown to be associated with metabolic syndrome in the nervous system. 10  We conducted plasmid transfections into CRC cells using jet-PRIME (Polyplus Transfection, France) following the protocol. SW480 and DLD1 cells were infected with SLC35D3-KO and HDR plasmids (Santa Cruz, USA) and selected with puromycin (Thermo Fisher Scientific, USA) for 2 weeks, while HCT116 and SW480 cells were infected with lentiviral particles for overexpression. Control lentivirus provided by the same company was used to infect cells in the control experimental group. The efficiency of lentiviral infection was assessed using western blot, and all cells were cultured in medium containing 4 μg/mL puromycin.

| Protein and western blot analysis
All cell samples will be treated with RIPA lysis buffer (Invitrogen, Thermo, USA) at a temperature of 4 C for 30 min, following centrifugation from the cultivation system at a speed of 300g. To ensure protein activity, the cleavage system will be

| Co-immunoprecipitation
Over 3 Â 10 7 HCT116 cells overexpressing SLC35D3 were collected by centrifugation at 300g. The extracted protein was divided into for parts and each sample was added to a low adsorption centrifuge tube.
Three different protein G beads, bound with the antibodies of IgG      which showed that SLC35D3 expression is significantly upregulated only in colorectal cancer ( Figure 1A) and in pheochromocytoma and paraganglioma ( Figure 1C). The same results can be found in the intestinal cell line. Validate by western blotting, we found that the expression of SLC35D3 was significantly higher in colorectal cancer cell lines than in HcoPEiC ( Figure 1B). Among these six cell lines, the expression level of SLC35D3 in SW480 was significantly higher than that in other colorectal cancer cells. Subsequently, DLD1 cells with relatively higher expression and HCT116 cells with relatively lower expression were selected for the next experiment. All the above results suggest that SLC35D3 may play a facilitating role in tumor development.

| SLC35D3 knockout inhibits colorectal cancer cell proliferation, invasion, and migration in vitro
Previous experiments demonstrate that SLC35D3 could be a relevant factor in predicting survival differences in lymph node metastases 11 without direct evidence on the function of SLC35D3 in colorectal cancer. To verify the specific functions of SLC35D3, SW480, and DLD1 were transfected to keep stable SLC35D3 knockout, which has been confirmed by western blot (Figure 2A). The result of CCK-8 showed that after knockout of SLC35D3, the proliferation of CRC cells was significantly reduced ( Figure 2B). The same conclusion can be obtained through EdU proliferation experiments in SW480 ( Figure 2C). Meanwhile, we also detected the invasion and migration effects of SW480 and DLD1 cells through corresponding transwell experiment. It was confirmed that knockout of SLC35D3 has a consistent inhibitory effect on cell invasion and migration ( Figure 2D). All above indicate that SLC35D3 plays a role as an oncogene in the genesis and metastasis of CRC cells.

| SLC35D3 overexpression boosts colorectal cancer cell proliferation, invasion, and migration in vitro
To further define the biological function of SLC35D3 in the progression of CRC, the matched plasmid/lentivirus was used to establish stable overexpression SLC35D3 cells of SW480 and HCT116. The efficiency has been verified by western blot assays ( Figure 3A).
Results consistent with the SLC35D3 knockout cells were obtained in SLC35D3 overexpression cell lines, that is the upregulated SLC35D3 enhanced the progression activity of CRC cells, including proliferation, invasion, and migration ( Figure 3B-D). Therefore, these results demonstrated that SLC35D3 promoted CRC development.

| SLC35D3 knockout inhibits colorectal cancer tumor growth and metastasis in vivo
In order to further explore whether the expression of SLC35D3 can similarly affect CRC tumor progression in vivo, we used stably SLC35D3 knockout SW480 and wild type cells to establish xenotransplantation models and via tail vein injection to construct metastasis models. Consistent with the experimental results in vitro, compared with the control group, the knockout group had a significant reduction in subcutaneous tumors ( Figure 4A-C,E). However, there was no significant difference in body weight between the two groups ( Figure 4D). Similarly, the number of mouse lung metastasis models constructed with knockout SLC35D3 cells was significantly reduced ( Figure 4F,G). The tumor tissues of mice were stained with HE and ki-67, lung tissues stained with HE ( Figure 4H) to test the proliferation ability. The SLC35D3 gene knockout group had fewer solid tissues and lower proliferative activity. It can be seen that SLC35D3 also promotes the growth and metastasis of CRC cells in vivo.

| SLC35D3 promotes colorectal cancer progression via AMPK signaling pathway
It has been shown in previous study that the triggering of AMPK pathway can downregulate the ERK axis to inhibit the development of tumors. 14 Analysis of the database indicates that the SLC family is closely related to the metabolic pathway ( Figure 5A). As a pathway F I G U R E 5 The relevance between SLC35D3 and AMPK signaling pathway. (A) Functional and pathway enrichment analyses of SLCs in the TCGA database. (B) Co-immunoprecipitation assays were performed to confirmed the binding relationship between SLC35D3 and AMPK. (C) Western blot was used to detected the phosphorylation level of AMPK and ERK between SLC35D3-WT and SLC35D3-KO SW480 cells, and finally perform relative analysis of the AMPK protein bands. Protein analysis were based on three independent replicates. *p < .05 and ***p < .001. closely related to metabolism, AMPK pathway played an important role in the process of colorectal cancer. It has been demonstrated that AMPK-α is downregulated in some tumor tissues. 15 Additionally, the application of AMPK activator metformin has been shown to inhibit abnormal lesion formation and reduce the incidence of tumors. 16,17 The above evidence indicates that AMPK may play an anti-tumor role in cancer. In order to confirm whether SLC35D3 interacts with the classical metabolic pathway AMPK, we performed co-immunoprecipitation experiments to validate the direct or indirect protein binding between SLC35D3 and AMPK molecules ( Figure 5B). In stable SLC35D3 over-expression cells of SW480, the results of western blotting showed that the phosphorylation level of AMPK was reduced while the subsequent phosphorylated expression of ERK was significantly increased.

| DISCUSSION
With the continuous updating of measurement, the detection rate of colorectal cancer continues to increase. 18 As the second most deadly cancer, the incidence and fatalities related to CRC are growing both in income and developing countries. 19 Colorectal cancer has more than 50% probability of distant metastasis. Although surgical treatment has always been the first-line treatment for colorectal cancer, we still need to further explore the mechanism of colorectal cancer to find a better treatment plan.
SLC family transports the substrates to another side of biological membranes through an electrochemical potential difference or an ion gradient generated by primary active transporters. 20 SLC35D3 is a member of solute carrier 35, the nucleotide sugar transporter, while some member of it have been proved to play a role in the occurrence and development of colorectal cancer. Although SLC35D3 was only found to play a role in blood system diseases before, some studies showed that SLC35D3 is related to the methylation-trend in the colorectal cancer. 12 Protein glycosylation achieves diversity and functional regulation of proteins by selectively modifying proteins and connecting different lengths and numbers of sugar residues. 21 While the substrates of SLC35 family members currently under study are all nucleotide-linked products, a significant number of family members have not had their substrates and functions fully explored. For proteins such as SLC35A4, A5, and F2, which may not directly affect cell survival or proliferation by transporting substrates, they may play a regulatory role in cells by coupling with other members of the same family with transport activity. [22][23][24] Among them, SLC35F2 has been shown to act as an initiator in pancreatic and prostate cancers, exerting important regulatory effects. 25