CMTM6 promotes hepatocellular carcinoma invasion and metastasis and tumor-associated neutrophil immunoinfiltration through the Wnt/β-catenin pathway

doi:10.21203/rs.3.rs-4719551/v1

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CMTM6 promotes hepatocellular carcinoma invasion and metastasis and tumor-associated neutrophil immunoinfiltration through the Wnt/β-catenin pathway

https://doi.org/10.21203/rs.3.rs-4719551/v1

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Background

CMTM6 is closely associated with the onset and progression of different types of tumors. However, the exact mechanism by which CMTM6 operates in hepatocellular carcinoma remains unclear, necessitating further investigation.

Methods

The expression levels of CMTM6 in liver cancer tissues and cells were analyzed using immunohistochemistry and qRT-PCR. The study also investigated the relationship between CMTM6 expression in liver cancer tissues and clinical pathological characteristics and patient prognosis. Additionally, the proliferation and apoptosis of liver cancer cells with silenced or overexpressed CMTM6 were assessed, along with the measurement of β-catenin and Wnt1 protein expression levels. In vivo research was conducted using a mouse subcutaneous transplantation model. Furthermore, GSEA and KEGG enrichment analysis were performed to explore the regulatory mechanism of CMTM6. The study also examined the expression level of CD66b in tumor tissue using immunohistochemistry and analyzed the immune infiltration of CMTM6 and tumor-associated neutrophils (TANs).

Result

Increased expression levels of CMTM6 in liver cancer tissues and cells have been found to be associated with a poor prognosis for patients. Furthermore, overexpression of CMTM6 in liver cancer cells has been shown to promote cell proliferation and inhibit apoptosis. Mechanistically, the expression level of CMTM6 in liver cancer tissues has been observed to be positively correlated with the expression of β-catenin. Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis have demonstrated that CMTM6 is significantly enriched in the Wnt/β-catenin pathway and is involved in its regulation. Additionally, CMTM6 has been found to be associated with immune infiltration of tumor-associated neutrophils (TANs) in liver cancer tissues.

Conclusion

CMTM6 plays a crucial role in the development and progression of liver cancer by regulating the Wnt/β-catenin pathway through β-catenin. Additionally, CMTM6 has the ability to promote immune infiltration of tumor-associated neutrophils (TANs) in liver cancer tissues. Consequently, CMTM6 holds potential as both an early diagnostic marker and a novel therapeutic target for patients with liver cancer.

Hepatocellular carcinoma

CMTM6

Wnt/β-catenin signaling pathway

Tumor-associated neutrophils

Multiplication

Primary liver cancer, also known as hepato cellular carcinoma (HCC), is a prevalent tumor worldwide, with approximately 800,000 new cases reported annually [1]. HCC accounts for over 90% of primary liver cancer cases and its incidence continues to rise, making it the second most common malignant tumor in China, following lung cancer [2]. The onset of liver cancer is often subtle, with a long incubation period and no distinct symptoms in the early stages. However, once symptoms appear, usually in the middle or late stages, the mortality rate is alarmingly high [3–4]. Despite the advancements in new drugs like lenvatinib and immune checkpoint inhibitors, the 5-year survival rate for HCC patients remains low [5–6]. Consequently, there is an urgent need to investigate the regulatory mechanisms of HCC invasion and metastasis, as well as identify new diagnostic biomarkers and potential therapeutic targets.

Chemokine-like Factorlike MARVEL transmembrane domain-containing family member 6 (CMTM6) is a member of the chemotaxin-like factor superfamily. It is localized in human chromosome 3q23[7]. CMTM6 is a type III transmembrane protein consisting of 183 amino acids, primarily composed of a MARVEL domain and intracellular segments at both ends [8]. In vivo, CMTM6 can promote tumor occurrence and progression by affecting the activation of signaling pathways, and it may also be involved in the development of tumor drug resistance [9].

β-catenin, a subunit of the cadherin complex, plays a crucial role in intracellular signal transduction within the Wnt/β-catenin pathway [10]. It has been observed that β-catenin can facilitate the immune evasion of liver cancer cells and contribute to the development and progression of liver cancer through various mechanisms [11]. Moreover, an analysis of second-generation gene expression sequencing results indicated a positive correlation between the Wnt/β-catenin pathway and CMTM6 in triple-negative breast cancer [12]. In head and neck squamous cell cells, the expression of CMTM6 is suppressed by short hairpin RNA, resulting in decreased β-catenin protein expression [13]. These findings suggest that CMTM6 may be involved in the regulation of the Wnt/β-catenin pathway. However, the precise regulatory mechanism remains unclear and has not yet been validated in the context of liver cancer.

The objective of this study was to examine the biological role and mechanism of CMTM6 in hepatocellular carcinoma (HCC). The findings demonstrated that CMTM6 expression was higher in HCC tissue samples compared to adjacent normal tissues. Additionally, CMTM6 was found to enhance the proliferation of HCC cells, suppress cell apoptosis, and facilitate the infiltration of tumor-associated neutrophils. The mechanism underlying these effects involves the regulation of Wnt/β-catenin signaling through β-catenin, which promotes the progression of HCC cells.

Pan-cancer analysis and immunocorrelation analysis were performed for CMTM6 in TIMER database

The TIMER (Tumor Immune Estimation Resource) database is a data analysis platform that utilizes the TCGA (https://cistrome.shinyapps.io/timer/) as its foundation. The Diff Exp module of TIMER was used to analyze the expression of CMTM6 in various cancers. Furthermore, the Gene module was employed to examine the correlation between CMTM6 and immune cell infiltration in liver cancer. The specific conditions for this analysis were as follows: (1) Gene Symbol: CMTM6; (2) Cancer Types: LIHC (Liver Hepatocellular Carcinoma); (3) Immune Infiltrates: B Cell, CD8 + T Cell, CD4 + Cell, Macrophage, Neutrophil, Dendritic Cell.

The expression and survival of CMTM6 in liver cancer tissues were analyzed by TCGA database

The Dynamic Analysis of gene expression profile data (gepia, http:∥ Gepia.cancer-pku.cn) database is a web analysis tool that utilizes TCGA and GTEx data. It provides various functions such as differential expression analysis, profile mapping, patient survival analysis, and related gene analysis. In this study, GEPIA was employed to analyze the expression difference between liver cancer tissues and normal tissues. The gene of interest was CMTM6, and the dataset selected was LIHC (Liver Hepatocellular Carcinoma). The default values were kept for other screening boxes. Additionally, an analysis of the gene sets enrichment for CMTM6 was conducted using the GSEA website (http://software.broadinstitute.org/gsea/index.jsp) to identify potential signaling pathways regulated by CMTM6.

Patient data and specimens

A total of 60 patients diagnosed with hepatocellular carcinoma and undergoing surgical treatment for the first time were selected as the study subjects. The patients were treated at the Department of Hepatobiliary and Pancreatic Surgery of the Affiliated Cancer Hospital of Xinjiang Medical University between January 2014 and December 2015. Tissue samples were collected during the operation and stored in a -80 degree biological freezer. All 60 patients strictly met the exclusion and inclusion criteria for liver cancer. The study protocol was approved by the Ethics Committee of the Affiliated Cancer Hospital of Xinjiang Medical University (Ethics approval number: k-2020023).

Immunohistochemistry

The SP method was used for immunohistochemical detection. Tissue samples from liver cancer patients were fixed in 10.0% formaldehyde for 18-24h, dehydrated, embedded in paraffin, and then cut into 4-micron-thick slices. The slices were placed in an oven at 65℃ for 2h. After dehydration, dewaxing, and trimming for antigen retrieval, each slice was treated with 50µl of 3%H2O2 and incubated at 37°C for 25min. The slices were then washed 3 times with PBS and incubated with a 1:500 dilution of rabbit anti-human CMTM6 monoclonal antibody for 5min each time. The slices were incubated overnight in the refrigerator at 4°C. After rewarming at room temperature for 1/3h, the slices were washed 3 times with PBS for 5min each time. The primary antibody was replaced with PBS buffer solution as a negative control, and then the secondary antibody was added and incubated at 37℃ for 30min. The slides were sealed with neutral gum before drying with xylene. The results were observed under a light microscope and the images were analyzed. The positive control sections provided by the ordering reagent company were used for the positive control, while the negative control was replaced by PBS for the primary antibody. The total score was calculated by dividing the sum of the staining intensity score and the positive cell proportion score. A score of ≤ 4 was classified as the negative group, and a score of > 4 was classified as the positive group. Two senior pathologists assessed the final results using the double-blind principle.

Cell culture and RNA extraction

Hepatocellular carcinoma cell lines (HepG2, HuH-7, MHCC97H) and normal liver cell lines (Lo2) were obtained from Suzhou Beina Chuanglian Biotechnology Co., LTD. For RNA extraction, liver cancer cell lines and normal liver cells were mixed with 1 ml of Trizol solution and incubated at room temperature for 15 minutes. Then, 200 µl of trichloromethane was added and mixed well, followed by another incubation at room temperature for 5 minutes. The mixture was then centrifuged at 12000 rpm at 4℃ for 15 minutes using a centrifuge with a radius of 10 cm. The supernatant was carefully transferred to another centrifuge tube, and an equal volume of isopropyl alcohol was added and mixed thoroughly. The sample was then placed at -20℃ for 5 minutes and centrifuged again at 4℃ for 12000 rpm for 15 minutes (centrifuge radius 10 cm). The supernatant was discarded, and the pellet was washed with 75% ethanol by centrifuging at 4℃ for 12000 rpm for 15 minutes (centrifuge radius 10 cm). After discarding the supernatant, the pellet was dissolved and precipitated with RNAse-free water. The RNA concentration was determined using a nucleic acid protein quantifier, and the RNA integrity was assessed by agarose gel electrophoresis.

Fluorescence quantitative PCR detection

Total RNA was extracted from HepG2, HuH-7, MHCC97, and Lo2 cellulite strains using the TRIzol method. cDNA was obtained through reverse transcription assay box. The reaction system consisted of SYBR Premix Ex Taq (6.0 µl), primer mixture (5 µmol/L) (0.3 µl), template (reverse transcription product) (0.6 µl), and RNase-Free Water (5.1 µl), making a total of 12 µl. The forward primer for CMTM6 was 5'-AGTGCCTTTCTTCTGAGTCTCCTT-3', and the reverse primer was 5'-CTTCAGCCCTAGTGGTATTTTCAG-3', resulting in an amplified fragment size of 205 bp. The forward primer for the internal reference was 5'-CACCATCTTCCAGGAGCGAG-3', and the reverse primer was 5'-AAATGAGCCCCAGCCTTC-3, resulting in an amplified fragment size of 230 bp. Two-step QRT-PCR was performed on the machine using the following reaction conditions: denaturation at 98 ℃ for 10 s, annealing at 62 ℃ for 20 s, extension at 72 ℃ for 30 s, for a total of 35 cycles. The extension step was carried out at 72 ℃ for 7 min and then cooled to 4 ℃. The experiment was repeated three times, and the relative expression of CMTM6 mRNA was calculated using the 2^−△△Ct method.

Construction of CMTM6 silent/overexpressed lentivirus stable strains

Hepatocellular carcinoma cells in a healthy growth state with a confluent rate of 90% were obtained and digested using pancreatic enzymes. The cells were then prepared into a single-cell suspension with a concentration of 3×104 cells/ml using complete media. The suspension was inoculated into 96-well plates and cultured at 37℃ and 5% CO2 for 24 hours to promote adherent culture. After 5 days, each well was treated with CCK8 Solution, which accounted for 10% of the total volume of the medium. The plates were then incubated further, and the OD value at 450nm was measured using an enzyme-labeled instrument after 1 hour. Transfection experiments were conducted in four groups: the blank group, no-load group, CMTM6 overexpression group, and CMTM6 silence group. Lentivirus-infected cells were screened and amplified after 96 hours of cell passage. CMTM6-shRNA1 was obtained from Shanghai Jierui Biotechnology Co., LTD. (Shanghai, China).

Determine the best shRNA filter

After infecting cells with three target SHRnas and negative control shRNA viruses, the expression level of CMTM6 was verified using qRT-PCR. The results indicated that the expression level of CMTM6 was significantly reduced when the three targets were knocked out, particularly with CMTM6 sh1. This knockout effect was further utilized in subsequent WB and other experiments.

The expression of β-catenin protein was verified by WB

Cells from the blank group, no-load group, and CMTM6 overexpression group, which exhibited good growth status and stable passage, were extracted using a conventional method. The protein concentration was determined using the BCA method. The protein samples were then separated using SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The membrane was incubated at 37°C for 2 hours and then overnight with the primary antibody. Subsequently, the membrane was washed three times with TBST and incubated with the secondary antibody at room temperature for 2 hours. After three additional washes, the expression of β-catenin protein was analyzed using the BIO-RAD imaging system. Images were captured and the strip gray values were quantified using Image J software for statistical analysis.

The CCK8 experiment

Hepatocellular carcinoma cells with a confluent rate of 80% were selected and prepared as a single cell suspension. They were then inoculated into a 96-well plate (100µl/well). After 24 hours, the old medium was discarded and cell intervention was carried out. Each group was repeated 3–5 times. The cell medium was absorbed and 10% CCK-8 solution was added. The plate was then incubated at 37℃ in an incubator. After the preset intervention time, the OD value was detected by an enzyme-labeler at a wavelength of 450 nm, 1–4 hours later.

Flow identification experiment

For the detection of apoptosis, liver cancer cells from each group were digested and collected. The cells were then washed three times with cold PBS. Next, the cells were suspended in 400µL of Annexin V binding solution, followed by the addition of 5µL of Annexin V-FITC staining solution. After incubating in the dark for 15 minutes, 10µL of PI staining solution was added and incubated for an additional 5 minutes. The proportion of apoptotic hepatoma cells in each group was determined using flow cytometry after passing through a 200 mesh filter.

The model of subcutaneous tumor transplantation in nude mice was established

A subcutaneous transplanted tumor model was constructed using 6-week-old female BALB/c nude mice with the same body weight. The mice used in the study were obtained from Hangzhou Ziyuan Experimental Animal Technology Co., LTD. Hepatocellular carcinoma cells, either silenced or overexpressed with CMTM6, were used in the experiment. The cells in the logarithmic growth stage were digested, centrifuged, and re-suspended in PBS. The cell concentration was adjusted to 5×107/ml and injected into the groin fat pad of each nude mouse. A total of 1 point was injected into each mouse, with 0.2ml injected into each transplant site. Tumor volume was measured every 3 days starting from day 3 using the formula: tumor volume = 0.5 × long diameter × short diameter^{^2}. After 15 days of tumor formation, the mice were euthanized, and the tumor body was removed. The weight and volume of the tumor were measured. A portion of the tumor body was cut and paraffin embedded for immunohistochemical detection and weighing comparison.

Statistical analysis

Statistical processing was performed using SPSS 22.0 software. A T-test was used to compare between two groups, while one-way ANOVA and corresponding multiple comparisons were used for multiple groups. Counting data were measured using the chi-square test and expressed as n (%). The differences between groups were analyzed using the Kaplan-Meier method and log-rank method. Cox univariate analysis was conducted to analyze the expression of CMTM6 and its clinicopathological features and prognosis. A significance level of P < 0.05 was considered statistically significant.

CMTM6 is differentially expressed in a variety of tumors and leads to poor prognosis in patients with liver cancer

Pan-cancer analysis of CMTM6 was conducted using the TIMER database. The study found that in bladder urothelial carcinoma (BLCA), breast cancer (BRCA), colon cancer (COAD), esophageal cancer (ESCA), renal papillary cell carcinoma (KIRP), hepatocellular liver cancer (LIHC), lung squamous carcinoma (LUSC), prostate cancer (PRAD), gastric adenocarcinoma (STAD), and thyroid cancer, the expression level of CMTM6 in tumor tissues was higher than that in adjacent normal tissues (THCA). Conversely, in chromophobe cell carcinoma of the kidney (KICH), the expression level of CMTM6 was lower in tumor tissues compared to adjacent normal tissues. However, no significant difference in CMTM6 expression level was observed between tumor tissues and adjacent normal tissues in cervical squamous cell carcinoma (CESC), bile duct carcinoma (CHOL), head and neck squamous cell carcinoma (HNSC), renal clear cell carcinoma (KIRC), lung adenocarcinoma (LUAD), pancreatic cancer (PAAD), rectal adenocarcinoma (READ), and endometrial carcinoma (UCEC), as shown in Fig. 1A. Verification through the GEPIA database revealed that the expression level of CMTM6 in liver cancer tissues was significantly higher than that in adjacent normal tissues (P < 0.001), as shown in Fig. 1B. Furthermore, prognosis analysis of liver cancer patients indicated that increased expression of CMTM6 in liver cancer tissues was associated with poor prognosis, as shown in Fig. 1C.

CMTM6 expression is upregulated in liver cancer and leads to poor prognosis

Immunohistochemical analysis of tumor tissues and adjacent normal tissues from 60 liver cancer patients revealed that CMTM6 was expressed in the cell membrane and cytoplasm (Fig. 1D). The positive expression rate of CMTM6 was approximately 61.7% in tumor tissues and 25.0% in adjacent normal tissues (Fig. 1E). Statistical analysis demonstrated a significantly higher expression level of CMTM6 in cancer tissues compared to adjacent tissues (χ² = 5.288, P = 0.021) (Fig. 1F).

Correlation analysis between the expression level of CMTM6 and clinicopathological features in liver cancer tumor tissues showed a significant association with the preoperative AFP level (χ² = 6.287, P = 0.041) and the degree of tumor differentiation (χ² = 7.855, P = 0.042) (Table 1). Logistic regression analysis revealed that the preoperative AFP level and tumor differentiation degree were risk factors for CMTM6 expression and positively correlated with its expression level (Table 2). Follow-up analysis of 60 patients indicated that up-regulation of CMTM6 expression was associated with a decrease in relapse-free survival time (P = 0.024) (Fig. 1G). Consequently, CMTM6 expression is upregulated in liver cancer tissues and is indicative of poor prognosis in patients.

CMTM6 promotes the proliferation of hepatoma cells

Normal liver cells (Lo2) and liver cancer cells (HepG2, HuH-7, MHCC97H) were cultured to investigate the expression levels of CMTM6. qRT-PCR was used to detect CMTM6 levels in both normal liver cells and liver cancer cells (Fig. 2E). The expression level of CMTM6 was significantly higher in hepatocellular carcinoma cells (HepG2, HuH-7, and MHCC97H) (3.321 ± 0.815, 3.151 ± 0.543, and 3.161 ± 0.492) compared to normal liver cells (Lo2, 31.011 ± 0.169), and this difference was statistically significant (P < 0.05). Refer to Fig. 2A and 2B for detailed information. Further qRT-PCR analysis revealed that the expression level of CMTM6 was highest in HepG2 cells among the three HCC cells, thus making it the focus of subsequent studies. Following infection of cells with 3 target shrnas and negative control shRNA viruses, the expression level of CMTM6 was verified through qRT-PCR. The results demonstrated that the expression level of CMTM6 in the Blank group and shRNA-NC group (1.014 ± 0.183, 1.002 ± 0.161) was significantly higher than that in the CMTM6SH1 group, CMTM6SH2 group, and CMTM6 group. The expression of CMTM6 in the sh3 group was 0.411 ± 0.240, 0.616 ± 0.152, and 0.437 ± 0.161, respectively, and these differences were statistically significant (P < 0.05), indicating a successful knockout effect (Figs. 2C and 2D). The knockout effect of the CMTM6 sh1 group was the most pronounced, and this target was selected for WB and other experiments.

CCK8 was used to measure cell proliferation in each group (Fig. 3C). The survival rates of the Blank group, hRNA-NC group, CMTM6-shRNA group, CMTM6-OE group, and Vector group were 100.000 ± 7.205%, 98.196 ± 5.422%, 44.041 ± 5.845%, 154.514 ± 3.087%, and 95.150 ± 6.4%, respectively (Fig. 3A). Upon analysis, it was observed that compared to the Blank group and the shRNA-NC group, the cell survival rate significantly decreased after CMTM6 knockout, while the cell survival rate significantly increased in the CMTM6 overexpression group, with statistical significance (P < 0.05) (Fig. 3B). In conclusion, CMTM6 has a biological function in promoting the proliferation of hepatoma cells.

The subcutaneous transplantation tumor model of CMTM6-silenced/overexpressed mouse liver cancer was constructed and divided into three groups: the model group, CMTM6-OE group, and CMTM6-shRNA group (Fig. 4A). Daily recording of subcutaneous tumor size showed that the growth rate of subcutaneously transplanted tumors in the CMTM6-OE group was significantly higher than that in the CMTM6-shRNA group (Fig. 4C). After sacrificing the mice, the subcutaneously transplanted tumors were dissected for detection. The tumor weight in the CMTM6-OE group was significantly higher than that in the CMTM6-shRNA group, with statistical significance (P < 0.05) (Fig. 4B). In summary, our findings suggest that CMTM6 plays a role in promoting the occurrence and development of liver cancer cells.

CMTM6 inhibits apoptosis of hepatoma cells

The number of apoptotic cells in each group was determined using flow cytometry (Fig. 3F). The apoptosis rates for the Blank group, CMTM6-shRNA group, CMTM6-OE group, and Vector group were 4.450 ± 1.527%, 4.960 ± 1.123%, 22.290 ± 2.187%, and 3.670 ± 0.141%, respectively (Fig. 3D). The analysis revealed that the apoptosis of cells significantly increased after CMTM6 knockout compared to the blank group and the negative control group. Conversely, the apoptosis of cells in the CMTM6 overexpression group significantly decreased (P < 0.05) (Fig. 3E). In conclusion, CMTM6 has a biological function of inhibiting the apoptosis of hepatoma cells.

There is a correlation between the expression of CMTM6 and β-catenin in liver cancer

Studies have found that Wnt1 and β-catenin play key roles in the Wnt/β-catenin pathway and influence signal transmission within the pathway [14]. To further investigate the regulatory mechanism of CMTM6 in liver cancer, immunohistochemical analysis was conducted on cancer tissues and adjacent tissues from 60 liver cancer patients. The results revealed that Wnt1 was expressed in the nucleus (Fig. 5A), but there was no significant difference in its expression level between cancer tissues and adjacent control tissues (χ² = 0.287, P = 0.592, Fig. 5B, 5C). Moreover, there was no significant correlation between Wnt1 expression and relapse-free survival (Fig. 5I). On the other hand, β-catenin was expressed in both the cytoplasm and nucleus (Fig. 5E), and its expression level in cancer tissues was significantly higher than that in adjacent tissues (χ² = 2.732, P = 0.009, Fig. 5F, 5G). The up-regulation of β-catenin expression was associated with a decrease in recurrence-free survival time for patients (Fig. 5J). Correlation analysis indicated a positive correlation between the expression levels of CMTM6 and β-catenin in tumor tissues (χ² = 4.671, P = 0.031, Fig. 5H), but no significant correlation was observed with Wnt1 (χ² = 0.006, P = 0.936, Fig. 5D). In conclusion, there may be a regulatory relationship between CMTM6 and β-catenin in liver cancer tissues.

CMTM6 promotes the proliferation and development of liver cancer cells through the Wnt/β-catenin pathway

β-catenin is a core protein in the Wnt/β-catenin signaling pathway [14]. To further investigate the relationship between CMTM6 and β-catenin, enrichment analysis of CMTM6 was conducted using GSEA and KEGG. The results suggested that CMTM6 may have a regulatory relationship with Wnt/β-catenin (Fig. 6A, 6B). The expression levels of Wnt1 in the Blank group, shRNA-NC group, CMTM6-shRNA group, Vector group, and CMTM6-OE group were 1.055 ± 0.339, 1.019 ± 0.095, 0.614 ± 0.066, 1.101 ± 0.441, and 1.7, respectively, as detected by qRT-PCR (88 ± 0.330). Similarly, the expression levels of β-catenin were 1.079 ± 0.212, 1.028 ± 0.079, 0.506 ± 0.079, 1.180 ± 0.360, and 1.967 ± 0.303, respectively (Fig. 6C). Statistical analysis revealed that compared to the blank group and the negative control group, the CMTM6 knockout group showed significantly reduced expression levels of CMTM6, Wnt1, and β-catenin, while the CMTM6 overexpression group exhibited significantly increased expression levels of these proteins, with statistical significance (P < 0.05) (Fig. 6E, 6F, 6G). Western blot detection also demonstrated that the expression levels of CMTM6, Wnt1, and β-catenin proteins were significantly higher in the CMTM6 overexpression group compared to the blank group and the negative control group (Fig. 6D). In conclusion, our findings suggest that CMTM6 may play a role in promoting the proliferation and development of liver cancer by regulating the Wnt/β-catenin pathway through β-catenin.

The expression level of CMTM6 in hepatocellular carcinoma is correlated with immune infiltration of tumor-associated neutrophils

To further explore the mechanism of CMTM6 in promoting liver cancer, we analyzed the relationship between CMTM6 expression and immune cell infiltration (B cells, CD4⁺ T cells, CD8⁺ T cells, DC, neutrophils, and macrophages) using the TIMER database. Our analysis revealed that the expression level of CMTM6 was positively correlated with neutrophil immunoinfiltration (r = 0.383, p = 1.58e − 13), B lymphocytes (r = 0.218, p = 4.67e − 5), and CD8⁺ T cells (r = 0.149, p = 5.81e − 3). Furthermore, CD4⁺ T cells (r = 0.386, p = 1.09e − 13), macrophages (r = 0.367, p = 2.50e − 12), and dendritic cells (r = 0.268, p = 5.30e − 07) also showed a positive correlation with immunoinfiltration, as depicted in Fig. 7A.

To validate these findings, we performed immunohistochemical detection of the neutrophil marker CD66b in 60 liver cancer tissues. The results showed that CD66b was expressed in the cell membrane and cytoplasm (Fig. 7B), and its expression was up-regulated in cancer tissues (Fig. 7C). Moreover, CD66b expression was found to be positively correlated with CMTM6 expression (χ² = 24.825, P = 0.001, Fig. 7D). However, there was no correlation between high expression of CD66b and relapse-free survival (P = 0.856), as shown in Fig. 7E. In conclusion, our findings suggest that CMTM6 plays a role in promoting tumor-associated neutrophil immune infiltration in hepatocellular carcinoma.

With the study of the chemotin-like factor superfamily (CMTM), it has been discovered that the members of this superfamily encode product proteins that are structurally similar to chemokines. These proteins have the ability to activate and chemotactically attract a large number of immune cells, while also influencing the proliferation and invasion of tumor cells. The CMTM family is also widely present in other systems of the body, playing a significant role [15–16]. Research has revealed that CMTM1 is an effective indicator of chemotherapy resistance and poor prognosis in NSCLC patients [17]. CMTM2, to some extent, affects HIV-l transcription, as well as sperm and testosterone production [18]. CMTM3 is an independent prognostic indicator of gastric cancer. CMTM4 regulates the cell cycle and influences cell growth [19]. CMTM5 is an independent prognostic indicator of oral squamous cell carcinoma [20]. As a member of the CMTM family, CMTM6 is a widely expressed protein involved in epigenetic regulation, embryo development, and tumorigenesis [21]. Several experiments have confirmed that CMTM6, as a cancer cell, has a regulatory relationship with the surface expression of PD-L1 cells in both mouse and human bone marrow cells [22]. CMTM6 co-locates with PD-L1 in the plasma membrane and recirculates endosomes, preventing PD-L1 from being degraded in lysosomes [23]. Additionally, knockout of CMTM6 reduces PD-L1 expression but does not affect the expression of MHC class I molecules on the cell surface. This significantly weakens the inhibition of tumor-specific T cell activity [24]. These findings establish CMTM6 as a major regulator of PD-L1, a key immune checkpoint, and a potential therapeutic target for tumor cell immune evasion [25]. Therefore, building upon previous research, this project delves into an in-depth study on the biological function and mechanism of CMTM6 in liver cancer from a fresh perspective.

Previous studies have shown that the expression level of CMTM6 is increased in liver cancer tissues, and it induces the EMT process of liver cancer cells, thereby promoting the proliferation, migration, and invasion of liver cancer [32]. In this project, we analyzed CMTM6 using the TIMER database and TCGA database, and found that CMTM6 is differentially expressed in various tumors, including hepatocellular carcinoma. Increased expression of CMTM6 is associated with a poor prognosis in liver cancer patients. To validate the biological information analysis, we conducted immunohistochemistry and qRT-PCR analyses on liver cancer tissues and cells. Consistent with previous studies, we found that CMTM6 expression is upregulated in liver cancer, leading to a poor prognosis for patients. To elucidate the biological function of CMTM6 in liver cancer, we established a stable transfected HCC cell line with CMTM6 silencing/overexpression. In vivo and in vitro experiments using CCK8 assay, flow cytometry assay, and nude mouse tumor formation assay revealed that upregulation of CMTM6 expression promotes cell proliferation and inhibits cell apoptosis. This provides evidence that CMTM6 can enhance the biological functions involved in tumor development.

The Wnt signaling pathway is a highly conserved pathway in biological evolution and is closely associated with tumor development [26]. Currently, there are 19 known Wnt proteins, which can be classified into two types based on their dependence on the β-catenin protein for activation: the classical Wnt/β-catenin signaling pathway and the non-classical pathways (JNK pathway and Wnt/Ca2 + pathway) [27]. Among these, the Wnt/β-catenin signaling pathway is better understood due to its earlier discovery. Studies have reported that the Wnt/β-catenin signaling pathway is abnormally activated in approximately 66% of liver cancer patients, leading to enhanced tumor cell proliferation, metastasis, drug resistance, and formation of dry (sic) [28]. The activation of the Wnt/β-catenin signaling pathway is closely associated with metabolic reprogramming in tumor cells [29]. In this project, we observed a significant up-regulation of β-catenin expression in liver cancer tissues, which is consistent with previous literature on the abnormal activation of the Wnt/β-catenin signaling pathway. Furthermore, we explored the correlation between the expression levels of CMTM6 and β-catenin in this project.

In order to elucidate the mechanism by which CMTM6 promotes HCC progression, GSEA and KEGG enrichment analysis were conducted, revealing potential regulatory effects of CMTM6 on the Wnt/β-catenin pathway. Notably, the expression levels of key proteins in the Wnt/β-catenin pathway, namely Wnt1 and β-catenin, were altered upon silencing/overexpression of CMTM6 in hepatoma cells. This provides evidence that CMTM6 regulates the Wnt/β-catenin pathway through β-catenin to facilitate liver cancer cell proliferation. Previous studies have also indicated a potential association between high CMTM6 expression and increased infiltration of TANs in various tumors, including liver cancer. However, the specific molecular mechanism underlying this regulation remains unclear [30]. TANs play a crucial role in immune and inflammatory responses within the tumor microenvironment (TME), contributing to tumorigenesis and progression [31]. These cells exhibit plasticity and can be polarized into either the N1 phenotype by type I interferon or the N2 phenotype by transforming growth factor-β (TGF-β) [32]. The N1 phenotype of TANs exerts anti-tumor effects by enhancing cytotoxicity, reducing immunosuppression through the production of reactive oxygen species (ROS), tumor necrosis factor-α, and intercellular adhesion molecule-1, and downregulating arginase expression [33]. Further investigation of the biological function of CMTM6 revealed a correlation between its expression level in liver cancer tissues and the polarization of the N2 subtype (CD66b) of tumor-associated neutrophils. This finding suggests that CMTM6 may influence the tumor immune microenvironment and mediate immune evasion of tumor cells, thereby providing a potential avenue for future research.

In conclusion, this study discovered that the increased expression of CMTM6 in liver cancer tissues is associated with a poor prognosis in patients. It can also serve as an independent risk factor for liver cancer prognosis. CMTM6 plays a role in regulating the Wnt/β-catenin pathway and promoting the proliferation of hepatocellular carcinoma cells by influencing the expression of β-catenin. Furthermore, the study also revealed that CMTM6 can impact the immune infiltration of tumor-associated neutrophils in tumor tissues, indicating a significant relationship between CMTM6 and the tumor immune microenvironment. These findings suggest that CMTM6 could potentially be used as a biomarker and a novel therapeutic target for liver cancer, providing valuable insights for future research

Ethical Approval

The study was approved by the Independent Ethics Committee of Tumor Hospital Affiliated to Xinjiang Medical University in 2023. The ethics lot number: G-2023032.

Funding

This work was supported by Science and Technology Department of Xinjiang Uygur Autonomous Region, Key Laboratory Open Project (2023D04030).

Data availability

This manuscript has not been previously published and is not under consideration by another journal. The authors declare that they have no competing interests. The data generated or analyzed during this study are available from the corresponding author upon reasonable request.

Author Contributions

Panpan Kong was responsible for experimental design and article writing. Huan Yang and Huifang Liu were responsible for the analysis and processing of experimental data. Xiaogang Dong and Yimu were responsible for collecting clinical data and conducting bioinformatics analysis. Dong Yan was responsible for revising, polishing, and providing financial support for the article.

Informed Consent Statement

Informed consent was obtained from all individual participants who were included in the study. The data supporting the conclusions of this article are included within the article. For the present study, the raw datasets used and analysis are available from the corresponding author upon reasonable request.

Acknowledgments

Acknowledgements: The author would like to express gratitude to all those who have contributed to the research and writing of this paper. Special thanks are extended to Professor Dong Yan, the corresponding author, for providing financial support and assistance in designing the article.

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TABLE 1 | Correlation between the expression of CMTM6, β-catenin and clinicopathological features in hepatocellular carcinoma

Characteristics	CMTM6 Expression				β-catenin Expression
	Low	High	χ²	P	Low	High	χ²	P
Gebder
Male	16	30	1.051	0.356	18	28	1.418	0.356
Female	7	7			8	6
Age
＜60	12	22	0.307	0.603	13	21	0.830	0.435
≥60	11	15			13	13
Tumor size(cm)
＜5	10	19	0.352	0.603	10	19	1.791	0.230
≥5	13	18			16	15
Tumor capsule
Yes	10	19	0.352	0.603	10	19	1.791	0.202
N0	13	18			16	15
Number of tumors
Single	19	27	0.736	0.534	21	25	0.432	0.555
Multiple	4	10			5	9
Vascular invasion
Yes	6	11	0.093	0.779	5	12	1.872	0.249
NO	17	26			21	22
Portal vein tumor thrombus
Yes	15	15	4.678	0.412	16	14	2.783	0.048
NO	8	22			10	20
Cirrhosis
Yes	11	20	0.220	0.791	10	21	4.13	0.045
NO	12	17			16	13
AFP（ug/L）
＜13.4	13	14	6.287	0.041	6	21	1.852	0.396
13.4≤AFP＜400	5	16			6	15
≥400	5	7			1	11
Hepatitis
Yes	15	25	0.035	0.851	4	36	9.624	0.002
NO	8	12			9	11
Differentiation degree
Low	10	7	7.855	0.042	5	12	1.075	0.584
Medium	11	24			6	29
High	2	6			2	6
AJCC stage
I	11	13	4.678	0.051	14	10	4.480	0.047
II	3	13			4	12
III	9	11			8	12

TABLE 2 | Logistic regression analysis of the relationship between clinicopathological characteristics and the expression of CMTM6 in hepatocellular carcinoma

Characteristics	β	SE	OR	*95%CI*	*P-value*
Differentiation degree （Low vs Medium）	2.632	1.094	15.84	1.606~17.121	0.021
Differentiation degree （Medium vs High）	2.253	1.321	11.21	1.231~92.715	0.038
AFP （＜13.4 vs 3.4≤AFP＜400）	3.112	1.423	12.34	1.332~93.134	0.042
AFP （3.4≤AFP＜400 vs ≥400）	2.895	1.456	12.12	2.021~92.134	0.045

No competing interests reported.

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CMTM6 promotes hepatocellular carcinoma invasion and metastasis and tumor-associated neutrophil immunoinfiltration through the Wnt/β-catenin pathway

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Abstract

Background

Methods

Result

Conclusion

Figures

Background

Materials and methods

Pan-cancer analysis and immunocorrelation analysis were performed for CMTM6 in TIMER database

The expression and survival of CMTM6 in liver cancer tissues were analyzed by TCGA database

Patient data and specimens

Immunohistochemistry

Cell culture and RNA extraction

Fluorescence quantitative PCR detection

Construction of CMTM6 silent/overexpressed lentivirus stable strains

Determine the best shRNA filter

The expression of β-catenin protein was verified by WB

The CCK8 experiment

Flow identification experiment

The model of subcutaneous tumor transplantation in nude mice was established

Statistical analysis

Results

CMTM6 is differentially expressed in a variety of tumors and leads to poor prognosis in patients with liver cancer

CMTM6 expression is upregulated in liver cancer and leads to poor prognosis

CMTM6 promotes the proliferation of hepatoma cells

CMTM6 inhibits apoptosis of hepatoma cells

There is a correlation between the expression of CMTM6 and β-catenin in liver cancer

CMTM6 promotes the proliferation and development of liver cancer cells through the Wnt/β-catenin pathway

The expression level of CMTM6 in hepatocellular carcinoma is correlated with immune infiltration of tumor-associated neutrophils

Discussion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

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