Collagen I-induced VCAN/ERK signaling and PARP1/ZEB1-mediated metastasis facilitate OSBPL2 defect to promote colorectal cancer progression

Lin, Kang; Zhao, Yun; Tang, Yuqi; Chen, Ying; Lin, Moubin; He, Luwei

doi:10.1038/s41419-024-06468-1

Collagen I-induced VCAN/ERK signaling and PARP1/ZEB1-mediated metastasis facilitate OSBPL2 defect to promote colorectal cancer progression

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Published: 24 January 2024

Volume 15, article number 85, (2024)
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Cell Death & Disease

Collagen I-induced VCAN/ERK signaling and PARP1/ZEB1-mediated metastasis facilitate OSBPL2 defect to promote colorectal cancer progression

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Kang Lin^1,2,
Yun Zhao ORCID: orcid.org/0000-0002-7807-6094^3,4,5,
Yuqi Tang^1,2,
Ying Chen^1,2,
Moubin Lin ORCID: orcid.org/0000-0002-0686-688X^1,2,6 &
…
Luwei He ORCID: orcid.org/0000-0001-6564-848X^1,2

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Abstract

The global burden of colorectal cancer (CRC) has rapidly increased in recent years. Dysregulated cholesterol homeostasis facilitated by extracellular matrix (ECM) remodeling transforms the tumor microenvironment. Collagen I, a major with ECM component is highly expressed in colorectal tumors with infiltrative growth. Although oxysterol binding protein (OSBP)-related proteins accommodate tumorigenesis, OSBPL2, which is usually involved in deafness, is not associated with CRC progression. Therefore, we aimed to investigate the pathological function of OSBPL2 and identify the molecular link between ECM-Collagen I and OSBPL2 in CRC to facilitate the development of new treatments for CRC. OSBPL2 predicted a favorable prognosis in stage IV CRC and substantially repressed Collagen I-induced focal adhesion, migration, and invasion. The reduction of OSBPL2 activated ERK signaling through the VCAN/AREG/EREG axis during CRC growth, while relying on PARP1 via ZEB1 in CRC metastasis. OSBPL2 defect supported colorectal tumor growth and metastasis, which were suppressed by the ERK and PARP1 inhibitors SCH772984 and AG14361, respectively. Overall, our findings revealed that the Collagen I-induced loss of OSBPL2 aggravates CRC progression through VCAN-mediated ERK signaling and the PARP1/ZEB1 axis. This demonstrates that SCH772984 and AG14361 are reciprocally connective therapies for OSBPL2^Low CRC, which could contribute to further development of targeted CRC treatment.

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Introduction

Colorectal cancer (CRC) is a prevalent malignancy worldwide, with a high incidence and mortality [1]. Population aging has increased the CRC burden in China and the United States of America. This warrants further development of strategies for cancer prevention and treatment [Full size image

OSBPL2 modifies ERK signaling through the VCAN/AREG/EREG axis in Collagen I-induced cell growth but not in EMT

Transcriptome sequencing was performed to investigate the molecular mechanisms underlying OSBPL2 expression in CRC. RNA-seq analysis showed 564 and 668 genes whose expression was upregulated and downregulated, respectively, in OSBPL2 KD HCT116 cells compared to shcontrol cells (Fig. 5A). Proteoglycans in cancer and cell adhesion molecules were the two most enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (Fig. 5B). The two most differentially expressed genes were amphiregulin (AREG) and Versican (VCAN) (Table S1). AREG, a member of the epidermal growth factor (EGF) family, regulates mitogen-activated protein kinase (MAPK) signaling. The expression of epiregulin (EREG), another EGF, was upregulated by OSBPL2 knockdown.

**Fig. 5: OSBPL2 modifies ERK signaling through VCAN/AREG/EREG axis in Collagen I-induced cell growth not in EMT.**

VCAN is a cell adhesion molecule in the ECM that coordinates with EGF to modify MAPK signaling in OSBPL2 KD CRC cells (Fig. 5C). In the TCGA database, the expression of VCAN and COL1A1 was elevated in CRC. In addition, higher COL1A1 levels were associated with poor prognosis in stage II CRC (Fig. 5D–G). Although VCAN was not a prognostic marker in CRC, it positively correlated with COL1A1 (Fig. 5H). Additionally, VCAN correlated positively with AREG and EREG in stage I, and AREG correlated positively with EREG in all stages (Fig. 5I–K). Thus, the expression of AREG, EREG, and VCAN and the activation of MAPK signaling were examined. AREG, EREG, and VCAN levels were enhanced by OSBPL2 knockdown (Fig. 5L). Additionally, OSBPL2 deficiency stimulated ERK phosphorylation and VCAN accumulation following incubation with Collagen I (Fig. 5M). VACN knockdown by effective siRNA-2, 3 eliminated the phosphorylation of ERK and increased AREG levels in OSBPL2 KD CRC cells (Figs. 5N–O and S5A). Furthermore, the ERK inhibitor SCH772984 impaired Collagen I-induced colony formation, but not invasion. This was followed by the loss of OSBPL2 (Figs. 5P, Q and S5B, C). These findings demonstrate that a decrease in OSBPL2 provokes VCAN/AREG/AREG-mediated ERK signaling during CRC cell growth.

OSBPL2 protects against colorectal tumor growth by blocking ERK phosphorylation

We used a xenograft tumor model to assess the function of OSBPL2 in colorectal tumor growth in vivo. As shown in Fig. 6A–C, OSBPL2 deficiency significantly expedited the growth of tumors derived from CRC cells, except for SW620 cells. The average weights and volumes of xenograft tumors in mice injected with shcontrol HCT116, HT29, and LoVo cells reached 0.19 g, 0.18 g, and 0.16 g and 149.53 mm³, 187.18 mm³, and 199.83 mm³, respectively. The average weight and tumor volume of mice injected with OSBPL2 KD CRC cells increased by 169.41%, 100.48%, and 129.24% and 294.50%, 151.24%, and 202.70%, respectively. In contrast, OSBPL2 overexpression reduced the weights and volumes of HCT116 and HT29 cell-derived xenograft tumors by 43.18%, 20.62% and 53.58%, 37.69%, respectively (Fig. 6D–I). Consistent with these observations, Ki67 expression considerably increased after the downregulation of OSBPL2, whereas the upregulation of OSBPL2 markedly decreased Ki67 expression (Fig. 6J–L). The expression levels of VCAN, AREG, and EREG were evaluated in tumors derived from OSBPL2 KD CRC cells (Fig. 6M). OSBPL2 was negatively correlated with VCAN, AREG, and EREG, whereas there was a positive correlation between VCAN, AREG, and EREG in colorectal tumors (Fig. 6N–S). Similarly, ERK phosphorylation was elevated in xenograft tumors derived from OSBPL2 KD CRC cells (Fig. 6T). These results demonstrate that OSBPL2 defect facilitates colorectal growth by activating ERK signaling.

**Fig. 6: OSBPL2 protects against colorectal tumor growth *via* blocking ERK phosphorylation.**

The function of OSBPL2 on metastasis is mediated by PARP1 through ZEB1

A co-immunoprecipitation (co-IP) assay and mass spectrometry were used to identify the proteins interacting with OSBPL2. An interacting protein band appeared between 100 and 150 kDa (Fig. 7A), and PARP1 was identified based on the highest score (Fig. 7B). The interaction between OSBPL2 and PARP1 was verified using a co-IP assay (Figs. 7C and S6A). PARP1 plays a multifaceted role in DNA repair, chromatin remodeling, and PARylation (poly [ADP] ribosylation) of various EMT-related proteins such as E-cadherin, Snail1, ZEB1, Twist1, p65, and SMADs [41,42,43]. We constructed a PARP1 expression vector and siRNA and applied the PARP1 specific inhibitor AG14361 to validate the relationship between OSBPL2 and PARP1. The IC50 concentration of AG14361 was 13.33 μM, which suppressed cell proliferation without inducing cell death (Fig. S6B).

**Fig. 7: The function of OSBPL2 on metastasis is dependent on PARP1 through ZEB1.**

AG14361 blocked the capabilities of migration and invasion induced by OSBPL2 in HCT116 cells (Fig. 7D–I). CRC cells were more sensitive to the PARP1 inhibitor, whereas the level of OSBPL2 was low. Hence, OSBPL2 competes with this inhibitor by trap** PARP1 during CRC metastasis. Knockdown of PARP1 by the two siRNAs markedly inhibited the metastatic function of OSBPL2 (Fig. 7J–M). However, the PARP1 overexpression did not impede the metastatic function of OSBPL2 (Fig. S6C–F). Otherwise, PARP1 had no impact on CRC cell adhesion to Collagen I or on the activation of ERK signaling (Fig. S6G–I).

Moreover, AG14361 blocked the upregulation of ZEB1 earlier and the downregulation of E-cadherin expression late (Fig. 7N). It substantially blocked invadopodia formation induced by OSBPL2 knockdown, which may be related to the phosphorylation of cortactin (Figs. 7O and S6J). These data indicate that PARP1 mediates OSBPL2 function in EMT through ZEB1.

OSBPL2 protects against colorectal tumor metastasis following tail vein and intrasplenic injection

To evaluate the function of OSBPL2 in CRC metastasis in vivo, we used tail vein and intrasplenic injection models. The tail vein injection model showed that the lack of OSBPL2 resulted in more metastatic nodules in the lungs, small intestines, and kidneys (Figs. 8A–D and S7A–F). Although there were no differences in the number of metastatic nodules in the lymph nodes, they were larger in the OSBPL2 KD group (Figs. 8C and S7B). Likewise, colorectal length was slightly shorter in the OSBPL2 KD group (Fig. S7F). The histological morphologies of the metastatic nodules are shown in Fig. 8E–G. Overexpression of OSBPL2 restrained tumor metastasis to the lymph nodes and lungs (Figs. 8H–K and S7G–L).

**Fig. 8: OSBPL2 protects against colorectal tumor metastasis *via* tail vein injection and intrasplenic injection.**

The intrasplenic injection model showed that OSBPL2 depletion increased the liver metastasis burden (Fig. 8L, S8A–S8C). As shown in Figs. 8M, and S8D, E, mice injected with OSBPL2 KD HT29 cells had more metastatic nodules in the small intestines and larger metastatic nodules in the colorectum. In contrast, OSBPL2 overexpression eliminated liver metastasis tumors (Figs. 8N and S8F–I). These results indicate that OSBPL2 protects against CRC metastasis.

SCH772984 and AG14361 relieve colorectal tumor progression induced by OSBPL2 defect

As ERK signaling and PARP1 play important roles OSBPL2 function, SCH772984 and AG14361 were used to treat CRC characterized by OSBPL2 deficiency. The effect of SCH772984 on CRC growth was evaluated using a xenograft tumor model (Fig. 9A). The ERK inhibitor SCH772984 significantly alleviated colorectal tumor growth induced by OSBPL2 knockdown, with reduced average tumor weights and volumes by 41.19% and 39.24%, respectively (Fig. 9B–D). SCH772984 treatment markedly suppressed the activation of ERK signaling in tumors derived from OSBPL2 KD CRC cells (Fig. 9E). Furthermore, SCH772984 substantially improved the poor survival induced by OSBPL2 knockdown (Fig. 9F). The effect of AG14361, a PARP1 inhibitor, on CRC metastasis was examined using a liver metastasis model (Fig. 9G). AG14361 markedly reduced the number of liver metastatic nodules induced by the loss of OSBPL2 (Fig. 9H). These data suggest that SCH772984 and AG14361 suppressed CRC progression in vivo.

**Fig. 9: SCH772984 and AG14362 inhibits OSBPL2 defect induced CRC progression.**

Discussion

ORPs modulate oncogenic signaling as oncogenes or tumor suppressors in testicular, hematologic, pancreatic, lung, and liver cancers [44,45,46,47]. Here, we show that OSBPL2 (ORP2) predicts a good prognosis for metastatic CRC. OSBPL2 defect promoted Collagen I-induced CRC cell growth, focal adhesion, migration, and invasion but impeded CRC cell proliferation without ECM. OSBPL2 regulated ERK signaling via the VCAN/AREG/EREG axis in CRC cell growth and modified EMT through PARP1. Most importantly, OSBPL2 inhibited colorectal tumor growth and distant metastasis, which were blocked by SCH772984 and AG14361 (Fig. 10).

**Fig. 10: Schematic diagram of OSBPL2 function on CRC.**

We discovered that OSBPL2 alleviated Collagen I-induced focal adhesion by impairing FAK autophosphorylation at Y397, which binds to SRC family kinases. FAK regulates the expression of the cell cycle protein cyclin D or apoptosis through SRC/ERK, JNK, or PI3K/AKT signaling, thereby affecting cell survival and tumor growth [48]. FAK is highly expressed in metastatic CRC and is modulated by lipids [49, 50]. Although OSBPL2 accelerated CRC cell proliferation without the ECM, the reduction of focal adhesion and FAK signaling led to a decrease in stable OSBPL2 OE cell-derived colorectal tumor growth in the Collagen I-rich TME. Additionally, focal adhesion remodeling of the actin cytoskeleton is linked to anoikis and invasion [51]. Hence, OSBPL2 attenuated Collagen I-induced focal adhesion, thereby reducing invadopodia formation. Targeting both OSBPL2 and FAK may be an effective combination therapy for CRC. However, further investigation is required to validate this hypothesis [52].

In the present study, the VCAN/AREG/EREG axis synergistically regulated ERK signaling induced by decreased OSBPL2 expression. The expressions of AREG and EREG are higher in left-sided CRC than in right-sided CRC and is more sensitive to anti-EGFR therapy [53, 54]. In this study, we only used the ERK inhibitor SCH772984 to suppress the growth of tumors derived from OSBPL2^Low CRC cells. However, combined inhibition of the ECM genes, VCAN, and ERK signaling may be more effective for preventing OSBPL2 loss induced colorectal tumor growth.

Finally, we elucidated the correlation between OSBPL2 and PARP1 expression in CRC metastases. PARP1 is an ADP-ribosyltransferase enzyme that binds to single- and double-stranded DNA breaks during the DNA damage response [55]. We found that PARP1 is necessary for the metastatic function of OSBPL2. The expression and mutations of PARP1 must be examined before improving OSBPL2 level for CRC therapy, except in the early stages of CRC, in which PARP1 is overexpressed [56]. PARP1 expression is visualized using radiolabeled PARP1 inhibitors in clinical settings [57]. The mechanisms of action of its inhibitors, such as nicotinamide adenine dinucleotide (NAD) analogs, involve synthetic lethality and PARP1 trap**. However, dual-target inhibitors of PARP1 have been developed to overcome tumor toxicity and resistance [58]. As shown in Figs. 7 and 9, OSBPL2 deficiency can ameliorate the sensitivity of CRC to PARP1 inhibitors, and the PARP1 inhibitor AG14361 could eliminate metastasis induced by OSBPL2 defect.

This study had some limitations. As other ECM molecules, such as fibronectin and laminin, may coordinate with Versican to allow OSBPL2-regulated ERK signaling during CRC cell growth. Decrease of OSBPL2 stimulated growth of CRC cells, slightly induced VCAN accumulation and activated ERK signaling (Fig. S5D–F), the molecular mechanism needs to be further explored. Correspondingly, it is noteworthy how OSBPL2 regulates ECM degradation by matrix metalloproteinase and plasminogen activator systems.

Furthermore, based on IHC image of OSBPL2 in CRC, OSBPL2 was transported extracellularly. The future research direction of OSBPL2 as a secretory protein may be associated with exosomes acting on colorectal tumor microenvironment. And the diagnosis and drugs of OSBPL2 in CRC will be urgently developed.

Briefly, this study illustrates that OSBPL2 defect collaborates with ECM Collagen I to promote colorectal tumor growth and metastasis through two independent pathways and provides the basis for appropriate therapy using ERK and PARP1 inhibitors to treat OSBPL2^Low CRC.

Materials and methods

Reagents and equipment

The details of reagents and equipment used in this study were shown in Table S3.

Cell culture

We established cell culture systems for five cell lines using DMEM or RPMI 1640 media supplemented with 10% FBS and 1% P/S. Specifically, HEK293T cells and CRC cell lines HCT116 and SW620 were cultured in DMEM, while the other two CRC cell lines, HT29 and LoVo, were cultured in RPMI 1640 medium. All cells are cultured in a humidified incubator at a constant temperature of 37 °C with 5% CO₂. All cells were sourced from the Shanghai Life Academy of Sciences cell library (Shanghai, China).

Tissue microarrays

From January 2015 to December 2018, we collected 627 tissue samples (adjacent normal tissues, n = 312; CRC tissues, n = 315), from Yangpu Hospital, Tongji University (Shanghai, China) with the consent of surgical patients. CRC patients at stages I to IV provided all samples used in this study, which were reviewed by experienced pathologists prior to being utilized. A TMA was created using formalin-fixed and paraffin-embedded specimens. The utilization of all tissue samples has obtained approval from the Hospital Ethics Committee. We downloaded pan-cancer samples containing mRNA sequencing data of 19,131 samples from the UCSC database (https://xenabrowser.net/). We conducted a log₂(x + 0.001) transformation on the OSBPL2 mRNA expression levels of 34 cancer samples and generated a bar chart illustrating the expression of OSBPL2 in pan-cancer.

Immunohistochemistry (IHC) and H&E staining

The tissue slides featuring paraffin sections were subjected to deparaffinization with xylene and ethanol. The activity of endogenous peroxidase was blocked by subjecting the sample to a 15-min treatment with 3% H₂O₂ at room temperature. To perform antigen retrieval on tissue slides, they were heated in a citrate buffer for 20 min, followed by blocking with a DPBS solution containing 10% FBS at 37 °C for 15 min. Subsequently, OSBPL2 or Ki67 was added to the tissue slides and incubated overnight at 4°C. The staining was performed with the DAB substrate, followed by incubating the slides with a biotin-conjugated secondary antibody using a general SP kit. We designed a two-dimensional scoring system to assess the immunoreactivity level of the prepared slides. The scoring ranges from 0 to 3 for staining intensity and from 0 to 4 for the proportion of positive cells. During the process of H&E staining of tissue slides, the first step involves fixing the slides. Subsequently, the slides are stained by adding Harris-modified hematoxylin solution and 1% aqueous eosin Y solution, and the staining process is continued for 1–2 min until completion. Dehydration was carried out by immersing the slides in ascending alcohol solutions and xylene. The data obtained from the experiment were analyzed using the Image J software.

OSBPL2 knockdown and overexpression

The pCDH-PURO vector was used to clone full-length OSBPL2. Subsequently, the construction of short hairpin RNA (shRNA) for OSBPL2 was achieved using the pLKO.1-PURO vector, with use of the following target sequences:

shOSBPL2-1: 5′-GGATTACTTTGAGCGGAATTT-3′;

shOSBPL2-2: 5′-GGGAGAAACGTATGAATTAAT-3′;

shOSBPL2-3: 5′-GAAGATTTAGGATTCAGATTT-3′;

scramble control: 5′-CCTAAGGTTAAGTCGCCCTCG-3′.

Lipofectamine 3000 was utilized to facilitate co-transfection of packaging plasmids (psPAX2/pMD2.G) and viral vectors (pCDH and pLKO.1) in HEK293T cells. Following a 48-h incubation period, the cell medium was passed through a 0.45 μm filter. Following a 48-h viral medium infection, cells were subjected to selection using puromycin to establish stable cell lines.

RNA isolation and real-time PCR

We extracted total RNA from the target cells using Trizol reagent, following the standard protocol. After determining the RNA concentration, the total RNA samples were reverse transcribed using RT master mix. Real-time PCR was performed using SYBR Green. All aforementioned steps were conducted in compliance with the provided protocols. Data were evaluated using the 2^−ΔΔCt method, with real-time PCR primer details available in Supplementary Table S2.

Western blot

For total protein extraction, the target cells were first washed with PBS, followed by cell lysis using RIPA lysis buffer containing protease and phosphatase inhibitors. After BCA quantification, the samples were mixed with loading buffer and boiled for 10 min to obtain total protein samples. Protein separation was carried out using SDS-PAGE, and the total proteins were subsequently transferred onto PVDF membranes. After blocking with 5% non-fat milk for 1 h, the membranes were incubated overnight at 4 °C with primary antibodies. The antibodies used in this study are as follows: OSBPL2, DDDDK-Tag, E-Cadherin, PARP1, Vimentin, FAK, Phospho-FAK-Y397, Phospho-FAK-Y576/577, Phospho-FAK-Y925, Versican, ERK1/2, Phospho-ERK1/2 (Thr202/Tyr204), ZEB1, β-actin and β-tubulin. Visualization of the proteins was achieved through ECL methodology following incubation with secondary antibodies targeting Goat anti-Mouse IgG and Goat anti-Rabbit IgG. The protein levels of OSBPL2 and β-actin were quantified using Image J software.

Cell proliferation detection

In this study, we used a CCK8 kit to determine the viability of target cells. An appropriate amount of CCK8 reagent was added to the 96-well plate to be tested. The plate was then incubated in a cell culture incubator at 37 °C for 2 h. After incubation, we measured the absorbance at 450 nm using a microplate reader to assess cellular viability. Additionally, we performed colony formation assays to assess cell proliferation capability. We seeded 200 CRC cells into each individual well of a 6-well plate, cultured them for approximately 10 days, and subsequently harvested and fixed the cells for staining. For the cloning formation experiment that required Collagen I (40 μg/ml) or fibronectin (40 μg/ml) stimulation, Collagen I or fibronectin was added to DPBS and thoroughly mixed on ice. Subsequently, the mixture was added to the plate, sealed with parafilm, and then incubated overnight at 4 °C. After removing excess liquid, cells were seeded as described previously. For the cloning formation experiment involving laminin stimulation (1–2 μg/cm²), laminin was added to DPBS and thoroughly mixed on ice. The resulting mixture was then applied to the plate, sealed with parafilm, and incubated for 2 h at 37 °C. After being washed three times with DPBS, the plate was ready for cell seeding. When visible cell colonies were observed, the cells were harvested. Cell fixation was performed using 4% paraformaldehyde at 37 °C for 15 min. After being washed three times with PBS, the cells were stained with a crystal violet staining solution. For cells subjected to flow cytometry analysis, after harvesting, they are first fixed with paraformaldehyde, and then permeabilized using 0.2% Triton X-100. Subsequently, the prepared Ki67 primary antibody is added to the cells and incubated on ice for 1 h. Cell proliferation analysis was carried out with the aid of BD Accuri™ C6 flow cytometer. Analysis of the results was performed with BD Accuri™ C6 software.

Cell adhesion assay

The CRC cells were subjected to a process of washing and starving for 8 h. Following the aforementioned procedures, place the pre-coated 96-well plate with collagen I at room temperature for 1 h. After aspirating the liquid from the wells, add serum-free culture medium (0.1% BSA) to the plate. Then, seed the cells (5 × 10⁴) into the plate and incubate for 2 h. Cells were incubated for another 4 h in complete medium. Finally, a 2-h incubation with CCK8 reagent was conducted before detection.

Migration and invasion quantification

For the detection of cell migration and invasion capabilities, we used Transwell chambers. The target cells were digested from the dishes, centrifuged, and washed with serum-free culture medium. Subsequently, the cells were resuspended in an appropriate volume of serum-free culture medium and then seeded into the upper chamber. For the wells of the 24-well plate, medium containing 10% FBS was added beforehand. The chamber was coated with matrigel for invasion assay. After a 48-h incubation, stain the fixed CRC cells with crystal violet, followed by their detection and quantification through utilization of an inverted optical microscope and ImageJ software, respectively.

Immunofluorescence staining

Fix the CRC cells or tissues on the slides with 4% paraformaldehyde for 15 min, followed by washing with PBS three times, and then permeabilize using 0.2% Triton X-100. Incubate the prepared slides with primary antibodies against Paxillin, Cortactin, E-cadherin, TRITC Phalloidin and DDDDK-Tag. The secondary antibodya were FITC Goat Anti-Mouse IgG, FITC Goat Anti-Rabbit IgG, Cy3 Goat Anti-Rabbit IgG and Cy3 Goat Anti-Mouse IgG. Olympus BX53 microscope was employed for fluorescence confocal image capturing. Later, Image J software was used for analyzing the average fluorescence intensity.

VCAN and PARP1 RNA interference

Human PARP1 small interfering RNAs (siRNAs) were procured from TSINGKE (Bei**g, China). The manufacturer’s protocol was followed for siRNA transfection, utilizing Lipofectamine RNAiMAX. Provided below are the siRNA target sequences:

siVCAN-1: 5′-GATTGGTCAGGACTACAAA-3′;

siVCAN-2: 5′-CACCTGTTATCCUACTGAA-3′;

siVCAN-3: 5′-GGCTGTTATGGAGATAAGA-3′;

siPARP1-1: 5′-GGTTGACAGAGATTCTGAA-3′;

siPARP1-2: 5′-GAAAGTGTGTTCAACTAAT-3′.

Co-IP

Using the method mentioned in the Western blot section, the CRC cells were lysed, and the total protein extracted was used for Co-IP assays. IgG and protein A/G agarose beads were used to pre-clear the protein. The pre-clearing process was conducted for 1 h at 4 °C. Following the pre-clearing process, fresh protein A/G magnetic beads and a specific antibody (or IgG) were added to the lysates. Finally, incubate the mixture overnight at 4 °C. Sequential washing with PBS was done three times on the beads the following day. Elution of the washed beads was carried out using protein sample loading buffer, followed by SDS-PAGE.

Coomassie Brilliant Blue-staining of protein gels

The gels were treated with Coomassie Brilliant Blue on a rocking platform for 30 min and destained using a solution of Milli-Q water.

Animal experiments and treatments

We purchased six-week-old male nude mice (BALB/cA-nu/nu) from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. These mice were housed in appropriate living conditions, with a 12-h light–dark cycle. We provided the mice with adequate food and sterile drinking water, and we regularly changed them. Additionally, we cleaned the cages and replaced the bedding on a regular basis. Animals are randomized based on the body weight. To initiate the subcutaneous tumor model, 65 mice were injected with stable CRC cell lines (1 × 10⁶–3 × 10⁶ cells) subcutaneously in the flank. Tumor volume (V) was assessed one week after injection by measuring with digital calipers at intervals of 3–4 days, with this formula: V (mm³) = 0.5 × length × width². Tumors were harvested and weighed before their tumor volume reached 1000 mm³. For SCH772984 treatment, CRC cells were injected following the aforementioned protocol. When tumor volumes reached 30 mm³, these mice (n = 20) were intraperitoneal injected with DMSO or SCH772984 group (5 mg/kg) daily for 5 days. The subsequent measurements of tumor volume and tumor harvesting were executed as previously delineated. The endpoint for survival was a tumor exceeding 500 mm³.

For the tumor metastasis model, 2 × 10⁶ CRC cells were evenly mixed, then injected into the tail vein of mice (n = 23). The weights of the mice were measured, and their conditions was observed every 3-4 days. These mice were euthanized 35 ± 2 days after surgery. For the hepatic metastasis model, a solution of 2–4 × 10⁶ HCT116 or HT29 cells was injected into the splenic parenchyma after opening the upper abdominal wall. We observed the condition of mice (n = 23) and measured their body weight every 3–4 days. These mice were euthanized 42 ± 2 days after surgery. For AG14361 treatment, 5 days after the intrasplenic injection, the mice (n = 20) were intraperitoneal injected with DMSO or AG14361 group (5 mg/kg) daily for 5 days. The use of animals for this study adhered to the guidelines set by the Animal Center of Tongji University.

Statistical analysis

For statistical analyses in the study, GraphPad Prism was utilized. The data presented in this study is primarily shown as mean ± SEM (standard error of the mean). For immunohistochemistry results, we display the data as mean ± SD (standard deviation). For TCGA dataset, expression between tumor and normal samples were evaluated via employment of R software (version 3.6.4), with significant analysis executed by means of non-paired Wilcoxon Rank Sum and Signed Rank Tests. For comparisons between two groups, we employed paired or unpaired Student’s t-tests. For multiple group comparisons, multiple t-tests were performed, with one t-test per row. The overall survival was generated using the Kaplan-Meier method and analyzed with the log-rank test. In this study, each experiment was followed by at least 3 replicates, P < 0.05 indicates a statistically significant difference between the data.

Data availability

The RNA-seq data generated during the current study are available in NCBI Sequence Read Archive (SRA), BioProject ID: PRJNA1003685, https://www.ncbi.nlm.nih.gov/sra/PRJNA1003685. All data analyzed in this study are available from the corresponding author on reasonable request.

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Acknowledgements

We are grateful to clinical research and translational medicine center of Yangpu Hospital for providing CRC samples.

Funding

This study was funded by the National Natural Science Foundation of China (32200623, 32370755, 32130025 and 32293232), the National Key Research and Development Program of China (2020YFA0509000), Project supported by Shanghai Municipal Science and Technology Major Project.

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Center for Clinical Research and Translational Medicine, Yangpu Hospital, School of Medicine, Tongji University, Shanghai, China
Kang Lin, Yuqi Tang, Ying Chen, Moubin Lin & Luwei He
Institute of Gastrointestinal Surgery and Translational Medicine, School of Medicine, Tongji University, Shanghai, China
Kang Lin, Yuqi Tang, Ying Chen, Moubin Lin & Luwei He
State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, China
Yun Zhao
School of Life Science and Technology, ShanghaiTech University, Shanghai, China
Yun Zhao
Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China
Yun Zhao
Department of General Surgery, Yangpu Hospital, School of Medicine, Tongji University, Shanghai, China
Moubin Lin

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Kang Lin
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Yun Zhao
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Yuqi Tang
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Ying Chen
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Moubin Lin
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Luwei He
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KL carried out the experiments and analyzed data. YQT and YC offered help for KL. YZ discussed the manuscript. MBL supervised the study. LWH designed research concept, supervised the study and wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Moubin Lin or Luwei He.

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The authors declare no competing interests.

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This study was approved by the Ethics Committee at the Yangpu Hospital, Tongji University (Shanghai, China). All participants signed the informed consent form. The animal experiments were executed in line with the documents of the Animal Center of Tongji University (Shanghai, China).

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Lin, K., Zhao, Y., Tang, Y. et al. Collagen I-induced VCAN/ERK signaling and PARP1/ZEB1-mediated metastasis facilitate OSBPL2 defect to promote colorectal cancer progression. Cell Death Dis 15, 85 (2024). https://doi.org/10.1038/s41419-024-06468-1

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Received: 15 August 2023
Revised: 10 January 2024
Accepted: 11 January 2024
Published: 24 January 2024
DOI: https://doi.org/10.1038/s41419-024-06468-1
Springer Nature Limited

Collagen I-induced VCAN/ERK signaling and PARP1/ZEB1-mediated metastasis facilitate OSBPL2 defect to promote colorectal cancer progression

Abstract

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Introduction

OSBPL2 modifies ERK signaling through the VCAN/AREG/EREG axis in Collagen I-induced cell growth but not in EMT

OSBPL2 protects against colorectal tumor growth by blocking ERK phosphorylation

The function of OSBPL2 on metastasis is mediated by PARP1 through ZEB1

OSBPL2 protects against colorectal tumor metastasis following tail vein and intrasplenic injection

SCH772984 and AG14361 relieve colorectal tumor progression induced by OSBPL2 defect

Discussion

Materials and methods

Reagents and equipment

Cell culture

Tissue microarrays

Immunohistochemistry (IHC) and H&E staining

OSBPL2 knockdown and overexpression

RNA isolation and real-time PCR

Western blot

Cell proliferation detection

Cell adhesion assay

Migration and invasion quantification

Immunofluorescence staining

VCAN and PARP1 RNA interference

Co-IP

Coomassie Brilliant Blue-staining of protein gels

Animal experiments and treatments

Statistical analysis

Data availability

References

Acknowledgements

Funding

Author information

Authors and Affiliations

Contributions

Corresponding authors

Ethics declarations

Competing interests

Ethics approval and consent to participate

Additional information

Supplementary information

Supplementary file

Original Data File

Reproducibility Checklist

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