CD151 promotes Colorectal Cancer progression by a crosstalk involving CEACAM6, LGR5 and Wnt signaling via TGFβ1

CD151 impacts various signaling pathways in different cancers, and promotes colorectal cancer (CRC) cell malignancy by yet undefined mechanisms. This study aimed to comprehensively assess CD151's function in CRC. CD151 levels were significantly higher in CRC tissues and cells compared with controls in the tissue microarray. Cell viability, migration and invasion were suppressed by CD151 downregulation in CRC cells. Consistently, mouse xenografts were inhibited by CD151 silencing. RNA-seq revealed that multiple genes were significantly altered by CD151 knockdown in cultured CRC cells and xenografts. Particularly, transforming growth factor β1 (TGFβ1), carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) alongside CD151 were downregulated both in vitro and in vivo. Co-immunoprecipitation and mass spectrometry results were validated by qRT-PCR and immunoblot. Moreover, pull-down assay and immunofluorescence confirmed the associations of TGFβ1, CEACAM6 and LGR5 with CD151. This study demonstrated CEACAM6, LGR5 and Wnt pathway suppression by CD151 silencing might occur through TGFβ1 regulation, offering a comprehensive view of CD151's roles in colorectal carcinogenesis. Our findings provide an insight into the CD151-involved signaling network in CRC oncogenesis, which could be utilized to design novel targeted therapies against CD151-based signaling in treatment for CRC.


Introduction
Colorectal cancer (CRC) is one of the most malignant cancers around the world, causing death mostly by locoregional relapse and metastasis. CRC represents a growing health concern worldwide, especially in developing countries as the "Western" lifestyle is increasingly adopted [1]. Surgery and chemotherapy are commonly administered to CRC patients [2]. Currently, designing effective targeted therapies for cancer remains an important clinical challenge.

Ivyspring
International Publisher CD151 was reported to be associated with cancer cell's aggressive cell proliferation and invasiveness in various malignancies such as lung, colon, breast, liver and prostate cancers, through interactions with laminin-binding α3β1, α6β1, and α6β4 integrins [3][4][5]. CD151 expression is considered a poor prognostic factor in solid tumors such as colon cancer, breast cancer, gastric cancer, and esophageal squamous cell carcinoma. Indeed, a number of studies have shown that significant overexpression of CD151 is related to tumor cell motility, adhesion, proliferation and metastasis [6][7][8]. In addition, high expression of CD151 in all stages and subtypes of epithelial ovarian cancer was demonstrated by RNA-seq analysis [9]. However, several reports have indicated that CD151 expression depends on the stage of tumor progression [10][11][12]. In colorectal cancer patients, CD151 expression is high in early disease stage, but decreases with increasing invasion and metastasis caused by hypoxia [13,14].
Carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6 overexpression was reported in colonic hyperplastic polyps, adenomas, and adenocarcinomas, and was shown to have strong associations with colorectal cancer and increased invasiveness in liver CRC metastasis [10]. CEACAM6 is also regulated by human epidermal growth factor receptor 2 (HER2) through the TGFβ pathway [11]. Meanwhile, leucine rich repeat-containing G protein-coupled receptor 5 (LGR5), a colorectal cancer stem cell marker, is correlated with TGFβ and Wnt signaling, which could increase cell proliferation, survival, and metastasis in colon cancer [12,21,22].
Because complex genetic and non-genetic factors are involved in tumorigenesis, manipulating CD151 expression may be fundamental to understanding its key role in CRC tumorigenesis. Therefore, the present study is aimed to assess the functional role of CD151 in CRC and to explore the mechanism underlying its effects. Our findings provide an insight into the CD151-involved signaling networkin oncogenesis of CRC, which could be utilized to design novel targeted therapies against CD151-based signaling during the treatment for CRC.

Collection of carcinoma tissue samples
Colorectal cancer tissue and blood samples were collected from patients enrolled in the Second Department of General Surgery, Hebei Medical University Fourth Hospital (China). All tissue samples were immediately processed after surgical removal. Diagnosis and grading were histologically confirmed by two experienced pathologists. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This study was approved by the Ethical Committee of Hebei Medical University Fourth Hospital and Shanghai Pudong Hospital. Written consents were obtained from all patients.

Tissue microarray (TMA) construction and immunohistochemistry (IHC)
A total of 80 clinical specimens were collected in the Second Department of General Surgery, Hebei Medical University Forth Hospital in China. TMA construction has been described in detail as reported [23]. Briefly, after paraffin-embedding, 2-mm diameter tissue cylinders were punched from representative tissue areas. For IHC, 4-μm sections were deparaffinized in xylene and successively rehydrated with 85% and 75% ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide, antigen retrieval was performed by microwaving, and nonspecific binding was blocked via incubation with 10% normal rabbit serum (since primary antibodies were originated from the goat) or 3% bovine serum albumin (BSA; Solarbio, Beijing, China) at room temperature for 30 min. TMA slides were incubated with anti-CD151 antibody (1:50; Antibody Revolution, Boston, MA, USA) overnight at 4 °C. Diaminobenzidine (DAB; Wuhan Servicebio Technology Co., Ltd., Wuhan, China) reagent was used to detect positive cells with brown-yellow nuclei.

DNA transfection
The lentiviral vector pLV[Exp]-Puro-U6> hCD151[shRNA]-CMV>Luciferase with sh-CD151 (CD151 knockdown) and HT29-NC or HCT116-NC (lentiviral vector alone) as a negative control were synthesized by Fitgene Biotech (Guangzhou, China). HT29 and HCT116 cells were seeded in 24-well plates at 50% confluence on the day before transfection. For viral infection, titrated viral stocks were suitably diluted in blank medium to obtain the expected multiplicity of infection (MOI), and added to HT29 or HCT116 cell monolayers in wells containing polybrene at 5 μg/ml. The medium with the lentivirus was replaced with complete medium after 6 h. CD151 silencing in HT29 or HCT116 cells was screened by puromycin (2 μg/ml) treatment after 48 h. In addition, qRT-PCR was carried out to evaluate CD151 expression in cells.

Wound-healing assay
HT29-NC, HCT116-NC, sh-CD151 HT29 and sh-CD151 HCT116 cells (1.0×10 5 ) were seeded in 24-well cell culture plates, respectively, and grown overnight to a confluent monolayer. A pipette tip (10 µL) was used to generate a scratch on the cell culture, followed by two washes with phosphate-buffered saline (PBS). After culture for 48 h in McCoy's 5A medium supplemented with 2% serum, the migration of cells was evaluated by comparing the area difference between 0 and 48 h via a Motic digital medical image analysis system (Leica DM2500, Heidelberg, Germany).
After 48 h, the cells that migrated through the membrane were stained with 0.5% methylrosaniline chloride solution and counted in 5 random highpower fields. The data were represented as percent of the average number of cells per field under a light microscope.

Xenograft colon tumor mouse models
Male BALB/C nude mice (6-8 week old; 20-25g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China) and housed in a laminar flow cabinet under specific pathogen-free conditions. The animal experiments were approved by the Institutional Animal Care and Use Committee of Hebei Medical University (approval ID: SYXK2018-005).
Logarithmically growing cells were harvested after trypsinization, washed twice with McCoy's 5A medium and resuspended in sterile PBS. Cell suspensions (0.2 mL) adjusted to 2×10 6 /ml were injected subcutaneously into the flank right foreleg of each nude mouse. BALB/c nude mice were randomly divided into four groups, including the HT29-NC (n=6), HT29 sh-CD151 (n=6), HCT116-NC (n=7) and HCT116 sh-CD151 (n=7) groups.Meanwhile, mice were treated with firefly luciferin (150 mg/kg, Sciencelight; Shanghai, China) by intraperitoneal injection for bioluminescence image (BLI) analysis. Then, the nude mice were anesthetized with isoflurane 10 min post luciferin injection. BLIs were collected on an IVIS Spectrum CT in vivo Imaging System (PerkinElmer Inc., Waltham, MA, USA). Mouse body weights and tumor volumes were recorded every two days, with daily observations. The tumor volumes were calculated as V = (a×b 2 )/2 (cm 3 ), where a and b are the tumor's long and short diameters, respectively, to construct growth curves. The tumor tissues were excised and weighed after 14 days. Each tumor sample was immediately frozen in liquid nitrogen and stored at -80 °C.

RNA sequencing
To obtain clear insights into the mechanism of CD151 in colon cancer in cultured cells and xenografts, RNA sequencing (RNA-seq) was performed to determine its impact on the transcriptomic profile (n=3). Total RNA was isolated with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) from triplicate samples of HT29-NC and HT29 sh-CD151 cells, as well as mouse xenograft HT29-NC and HT29 sh-CD151 groups. RNA concentrations were between 23.8 and 41 ng/μl. RIN values were ~9.4 for RNA integrity as assessed on an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). Paired-end libraries were synthesized with VAHTS Stranded mRNA-seq Library Prep Kit for Illumina® (Vazyme Biotech, Nanjing, China). Illumina sequencing libraries were constructed according to a modified strand-specific RNA-seq protocol [24]. Library construction and sequencing were performed by Shanghai Biotechnology Corporation (China). Significantly expressed genes (SEGs) were selected with False Discovery Rate (FDR) < 0.05 and a fold-change ≥2. Enrichment analysis of differentially expressed genes (DEGs) was conducted to identify enriched gene ontology (GO) terms (http://www.geneontology.org/), as well as signal transduction and biochemical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [25].

Co-immunoprecipitation (CoIP)
To determine proteins with potential interactions with CD151, CoIP was performed to assess whole cell extracts (WCEs) from HT29 cells. HT29 cells were lysed in lysis buffer (25 mM Tris, 150 mM NaCl, 1mM EDTA, 1% NP-40, 5% glycerol; pH 7.4). Total CD151 protein concentrations were measured by the BCA assay. CoIP was carried out with CoIP Kit (Pierce Co-immunoprecipitation kit; ThermoFisher Scientific; Waltham, MA, USA) according to the manufacturer's instructions. Briefly, HT29 cells were washed with PBS, lysed with cold immunoprecipitation lysate buffer, and centrifuged. Next, the cleared cell lysates were immunoprecipitated with anti-CD151 antibody or control normal mouse IgG (SC-2025) plus protein A Sepharose overnight at 4 °C. CD151 and its interacting proteins were precipitated with anti-CD151 antibody. SDS-polyacrylamide gel electrophoresis (PAGE) was performed, and the target proteins were extracted from the bands. The samples were analyzed by Mass Spectrometry, and CD151 interacting proteins were identified in a protein database.

Mass Spectrometry
Selected protein bands were in-gel digested by trypsin as previously described [26,27]. The extracted peptides were dissolved in 10 μl 0.1% formic acid. The peptide samples were loaded onto a C18 reverse phase column (100 μm 150 mm, ThermoFisher Scientific) coupled online to a QE-XF mass spectrometer (ThermoFisher Scientific) at a flow rate of 350 nl/min. Peptides were eluted with a gradient from 100% of A (0.1% formic acid) to 45% of B (0.1% formic acid and 95% acetonitrile) for 60 min.

Real time fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA from the abovementioned cells and tissues were isolated with ISOGEN reagent (Nippon Gene Co. Ltd., Kokyo, Japan) according to the manufacturer's instructions. RNA concentration and purity were assessed by spectrophotometry at 260 nm. A total of 1 µg of total RNA was converted into cDNA with Revert Aid first strand cDNA synthesis kit (ThermoFisher Scientific). The primers used are shown in Table 1. Real time fluorescence qRT-PCR was performed on a Real-Time PCR system (BIOER Co. Ltd., Kokyo, Japan) in reaction mixtures (20 μl) containing cDNA, primer pairs, and platinum SYBR Green QpcrSuperMix-UDG (Invitrogen) [28]. Table 1. Primers used in this study

Statistical analysis
Data are mean ± standard deviation (SD) from the indicated number of independently performed experiments. Statistical significance (P<0.05) was assessed by one-way analysis of variance (ANOVA) followed by the S-N-K test. Log-rank (Mantel-Cox) survival analysis was performed to assess the association of patient prognosis with CD151 expression on basis of the TCGA database.

CD151 is upregulated in CRC tissue samples and associated with reduced patient survival
To investigate whether CD151 protein expression is altered in CRC, a TMA slide was generated with CRC and para-carcinoma tissue samples from 80 patients. IHC was performed to evaluate CD151 expression and analyze its associations with clinicopathologic features. As shows in Figure 1A, epithelial structure in CRC was irregular, with significant variations in cell nucleus size and shape. The CD151 protein was highly expressed in CRC tissues. There was a negative correlation between CD151 expression levels and the survival rate of CRC patients. Further analysis showed a statistically significant association of CD151 expression with survival in all validation data sets (P=0.006, Chi-square=11.77). High CD151 expression (n=539) resulted in lower overall survival compared with the low CD151 expression group (n=59) in CRC patients ( Figure 1B). CD151 expression was significantly increased in 5 colon cancer tissue samples compared with paired non-cancerous samples as assessed by Western blot (P<0.05, Figure  1C and D). In agreement, CD151 mRNA levels were increased in colon cancer tissue specimens compared with paired non-cancerous ones as determined by qRT-PCR (n=32, P<0.05, Figure 1E). The clinicopathological characteristics and CD151 expression statuses assessed by IHC in 80 CRC patients are summarized in Table 3. Two sided Fisher's exact analysis indicated a significant correlation between CD151 expression and distant metastasis (pM0 vs pM1; P=0.001). No significant associations between CD151 expression and other clinicopathologic variables were found.

CD151 inhibits proliferation, migration and invasion of colon cancer cells
As shows in Figure 2A and B, CD151 was knocked down in HT29 and HCT116 cells, as examined by Western blot and qRT-PCR. To verify whether CD151 is required for colon cancer progression, we investigated cell proliferation, migration and invasion in sh-CD151 HT29 and HCT116 cells. As shown in Figure 2C and D, the sh-CD151 groups in both cell lines showed decreased cell viability at 24-72 h (all P<0.05). In wound healing assay, the cells migrated significantly less after CD151 silencing ( Figure 3A, B and E; P<0.05). In agreement, the transwell assay indicated that cell migration and invasion were inhibited upon CD151 silencing ( Figure  3C, D and F), in both HT29 and HCT116 cells ( Figure  3A-F).

Figure 3. Knockdown of CD151 inhibits migration and invasion of HT29 and HCT116 cells. A, B and E.
Migrative activities were suppressed in sh-CD151 HT29 and HCT116 cells as determined by the wound healing assay. C, D and F. Cell invasion abilities were inhibited in the transwell assay for both sh-CD151 HT29 and sh-CD151 HCT116 cells. Data are represented as mean ± SD derived from at least three independent experiments. * P < 0.05, **P < 0.01 compared with the negative control (HT29-NC or HCT116-NC) groups. Scale bars, 50 µm for A, B; 20 µm for C, D.

CD151 knockdown suppresses colon cancer growth in vivo
To further investigate the role of CD151 in tumorigenesis, we evaluated the effect of CD151 knockdown on HT29 and HCT116 cell-related nude mouse xenografts. As shown in Figure 4A, C-E, tumor growth was significantly slower in the sh-CD151 groups compared with the control groups, with reduced tumor volumes and weights. The control group of HCT116 cells showed time-dependently increased luminescence, which was markedly reduced by CD151 silence. As shown in Figure 4B, luminescence decreased in the HCT116 sh-CD151 group indicating that tumor progression was repressed by reducing CD151 expression.

CD151 knockdown alters multiple cancerassociated biological processes in colon cancer
To investigate the molecular mechanisms by which CD151 knockdown regulates tumorigenesis, the transcriptomic profiles of HT29 cells and derived colon xenografts in nude mice were examined by RNA-seq. In this study, 724 and 586 significantly differentially expressed genes (SDEGs) were found in cell and xenograft groups, respectively, which included 243 SDEGs found in both cell and xenograft groups. A total of 279 exclusive and 114 common SDEGs were upregulated, while 202 exclusive and 129 common SDEGs were downregulated in HT29 cells treated with sh-CD151 versus control cells. Meanwhile, 117 exclusive and 314 common SDEGs were upregulated, while 226 exclusive and 329 common SDEGs were downregulated in colon xenografts from cells treated with sh-CD151 versus controls. GO and KEGG pathway analysis of DEGs in HT29 cells and xenografts revealed that CD151 was involved in multiple cancer signaling pathways, including the TGFβ, Wnt, Notch, Hedgehog and vascular endothelial growth factor (VEGF) signaling pathways, and regulated signal transduction effectors such as LGR5, bone morphogenetic protein 4 (BMP4) and TGFβ receptor type 1 (TGFβR1) (Figure 5).

Proteins identified by CoIP-MS and validation of LGR5, TGFβ1, and CEACAM6 as binding partners of CD151
CoIP coupled with MS was performed to further investigate the proteins interacting with CD151. A total of 2268 and 2014 proteins were identified in the experimental and control groups, respectively. Of these, 472 proteins had fold changes greater than two in the experimental group compared with the control group. GO and KEGG pathway analysis indicated that they were involved in multiple signaling pathways, including TGFβ1 signaling, the VEGF/Wnt/Jak-STAT signaling pathway, as well as pathways in the cell cycle, cancer and colorectal cancer (Figure 6A-C). GO enrichment analysis showed that specific proteins, including TGFβ1, adenomatous polyposis coli (APC), CTNB1, low-density lipoprotein receptor-related protein (LRP) 5 (LRP5), LRP6, JAK1, ST14, and TP53BP1, were hypothetical CD151-interacting proteins in colon cancer. The CoIP-MS results were consistent with RNA-seq data. To further verify LGR5, CEACAM6, and TGFβ1 as potential proteins interacting with CD151, a pull-down assay was performed, confirming that CD151 could interact with LGR5, CEACAM6 and TGFβ1 in vitro ( Figure  6D-F). Moreover, immunofluorescence indicated that LGR5 co-localized with CD151 in HCT116 cells. As confirmed in Figure  7, red fluorescence (LGR5) and green fluorescence (CD151) were overlapped on HT29 cells (Figure 7).    LGR5, CEACAM6 and Wnt pathways are suppressed by shCD151 in HT29 and HCT116 cells in vitro and in vivo Western blot and qRT-PCR demonstrated significantly decreased expression levels of LGR5, CEACAM6, β-catenin, Wnt3a and Wnt5a in cultured HT29 and HCT116 cells after CD151 silencing at the protein and mRNA levels, respectively (Figure 8). Similar findings were obtained in mouse xenografts (Figure 9).

TGFβ1 activates LGR5, CEACAM6 and Wnt signaling after CD151 silencing
It was previously reported that TGFβ1 upregulation after CD151 silencing could revive malignancy, suggesting that CD151 may promote renal cell carcinoma partially by regulating TGFβ1 amounts and inducing TGFβ1/Smad signaling [16]. Therefore, to further investigate the effects of TGFβ1 on LGR5, CEACAM6 and Wnt signaling effectors with CD151 knockdown in this study, sh-CD151 HT29 and HCT116 cells were treated with TGFβ1 (0.05, 0.1 and 0.5 ng/ml) for 48 h. Compared with the sh-CD151 groups, LGR5, CEACAM6, β-catenin, Wnt3a and Wnt5a protein and mRNA levels were starkly increased in the sh-CD151+TGFβ1 groups; Meanwhile, TGFβ1 additive had no impacts on CD151 in the aforementioned groups (Figure 10).   LGR5, CEACAM6, Wnt3a and Wnt5a were remarkably upregulated at both protein and mRNA levels after treatment with TGFβ1 at 0.05, 0.10 and 0.50 ng/ml, respectively, on sh-CD151 transfected HT29 cells (sh-CD151+TGFβ1 group versus sh-CD151 group). C and D. Western blot and qRT-PCR showed TGFβ1, LGR5, CEACAM6, Wnt3a and Wnt5a were remarkably upregulated at both protein and mRNA levels after treatment with TGFβ1 at 0.05, 0.10 and 0.50 ng/ml, respectively, on sh-CD151 transfected HCT116 cells (sh-CD151+TGFβ1 group versus sh-CD151 group). TGFβ1 additive had no impacts on CD151 in the aforementioned groups.

Discussion
The present study comprehensively assessed the role of CD151 in CRC, and demonstrated that CD151 could promote malignancy via multiple pathways, both in cultured cells and in xenograft mouse models.
It has been reported that overexpression of CD151 enhances tumor invasion and metastasis, and reduces overall survival in various cancers [29,30]. In this study, CD151 was upregulated in CRC tissue samples, and a negative correlation was found between CD151 expression and the survival rate of CRC patients, corroborating the above conclusion. In addition, CD151 silencing was shown to suppress cell migration and invasion in cultured CRC cells in vitro, and reduce tumor growth in mouse xenograft models. RNA-seq further demonstrated that CD151 modulated several molecular processes responsible for CRC progression, including TGFβR1 binding, cell motility, activation of calcium channel inhibitors, biological response to stress, immune system activation, signal transducer activities, and et al.
Impacts of CD151 silencing were observed in both cultured cells and mouse xenografts. Among the SDEGs common in both cells and xenograft groups, 114 were upregulated and 129 were downregulated, among which TGFβ1, CEACAM6 and LGR5 were found to be downregulated along with CD151. These findings suggest that TGFβ1, CEACAM6 and LGR5 might be correlated with CD151 in CRC. These data were consistent with previous studies reporting that CD151 promotes cell migration, invasion and metastasis via TGFβ1/Smad signaling [19,31]. Here, high TGFβ1 expression was responsible for the aggressiveness of CRC both in vitro and in vivo. Among the well-known TGFβ family members (TGFβ1, TGFβ2, and TGFβ3), CRC patients are more likely to show recurrence bearing high levels of TGFβ1, which regulates a number of biological processes such as CRC cell proliferation, differentiation, apoptosis, extracellular matrix adhesion and immune response [16,26,27,32].
This study updated our knowledge on CD151 function in cancer progression. Notably, LGR5 co-downregulation with CD151 was identified in xenograft models by RNA-seq, and then validated by Western blot and qRT-PCR in cultured CRC cells and xenograft models. CoIP followed by MS was utilized to confirm that TGFβ1, LRP5, LRP6, CEACAM6 and LGR5 may act as specific CD151 binding partners in vivo. Previous reports revealed that LRP5/6 could function as co-receptors in the Wnt signaling pathway by binding to LGR5 [33]. Here we provided the first direct evidence showing that the TGFβ1-CEACAM6-LGR5 complex interacts with CD151 via RNA-seq, Co-IP-MS, pull-down assay and immunofluorescence. These results suggest that TGFβ1, CEACAM6, and LGR5 act as CD151 binding proteins in CRC. Consistently, we demonstrated that CD151 knockdown in CRC could downregulate TGFβ1, CEACAM6 and LGR5 expression levels. On the other hand, sh-CD151 transfected cells treated with TGFβ1 additive could exhibit restored expression of CEACAM6, LGR5, Wnt3a, Wnt5a and β-catenin. These findings demonstrated that suppression of CEACAM6, LGR5 and Wnt signaling by CD151 silencing may revive via TGFβ1 upregulation, confirming that CD151 promotes CRC progression by a crosstalk involving CEACAM6, LGR5 and Wnt signaling via TGFβ1. Consistently, previous studies reported that regulatory T-cells (Tregs) promote LGR5 upregulation in gastric cancer cells through TGFβ1 signaling, with a likely involvement of Wnt signaling; Meanwhile, high LGR5 levels upregulated by TGFβ1 was thought to deteriorate gastric cancer patient prognosis [34]. However, whether Tregs also affect CD151 expression levels deserves further attention.
Although the current study provided novel insights into the mechanism by which CD151 induces malignancy, further investigation is required to explore the exact mechanism underlying the interaction and regulation between CD151 and the aforementioned proteins. Additional molecules screened in RNA-seq and CoIP also need to be further assessed, and future efforts will be directed towards that end.

Conclusion
Taken together, this study demonstrated that CD151 plays a pivotal role in CRC progression, during which process CD151 functions through TGFβ1 to regulate CEACAM6, LGR5 and Wnt signaling, thus offering a comprehensive view of CD151's roles in colorectal carcinogenesis. Our findings provide an insight into the CD151-involved signaling network in oncogenesis of CRC, which could be utilized to design novel targeted therapies against CD151-based signaling during the treatment for CRC. Chinese Medicine (grant No. XCXZZSS2020002) to H.B.W.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.