Involvement of non‑triple helical type VI collagen α1 chain, NTH α1(VI), in the proliferation of cancer cells
- Authors:
- Published online on: August 13, 2020 https://doi.org/10.3892/or.2020.7734
- Pages: 2297-2305
Abstract
Introduction
Type VI collagen secreted from host cells has been shown to influence the proliferation of cancer cells (1–3). High collagen type VI alpha 1 chain (COL6A1) mRNA expression was observed in astrocytoma using reverse transcription quantitative PCR (RT-qPCR) analysis (4). Overexpression of COL6A1 mRNA and the COL6A1 polypeptide was detected in non-small cell lung cancer using RT-qPCR and immunohistochemical (IHC) analyses (5). IHC analysis also indicated that COL6A1 expression is higher in castration-resistant prostate carcinoma than in adjacent normal tissue (6). In addition, gene expression profile analysis showed that COL6A1 mRNA expression is a predictive marker of poor prognosis in clear cell renal cell carcinoma (7). Proteomic analysis revealed that COL6A1 expression is associated with poor prognosis in patients with glioblastoma (8). Similarly, IHC analysis indicated that COL6A1 expression predicts the prognosis of patients with cervical cancer (9). Thus, previous reports suggest that type VI collagen, which contains the COL6A1 polypeptide as one of the three α chains, is important for cancer cell proliferation.
Zhu et al indicated that COL6A1 knockdown (KD) inhibits the proliferation of prostate cancer LNCaP cells. COL6A1 promotes proliferation through the JAK-STAT pathway (6). Owusu-Ansah et al also reported that COL6A1 is involved in the migration and invasion of highly metastatic pancreatic cancer BxPC-M8 cells (10). Recently, we reported the existence of a non-triple helical polypeptide encoded by COL6A1, NTH α1(VI), in the conditioned media of cancer cell lines (11). NTH α1(VI), rather than the COL6A1 polypeptide of type VI collagen, may be directly involved in cancer cell proliferation, migration, and invasion.
Extracellular matrix (ECM)-derived peptides, known as matrikines or matricryptins (hereafter, matricryptins), are defined as ‘enzymatic fragments of ECM containing exposed matricryptic sites’, according to Davis et al (12). Arresten, canstatin, and tumstatin are matricryptins derived from the NC1 domains of type IV collagen α1, α2, and α3 chains, respectively; they all possess anti-angiogenic and antitumor activities (13,14). It has been reported that endotrophin, the C5 domain derived from COL6A3, promotes malignant tumor progression (15). As described above, tumors express COL6A1 (5,6), whose expression is associated with the prognosis of patients with certain types of cancer (8,9). Additionally, Willumsen et al observed COL6A1- and COL6A3-derived peptides in some tumors (16); as indicated above, NTH α1(VI) is thought to be involved in cancer cell proliferation, migration, and invasion. Therefore, we evaluated whether NTH α1(VI) and/or derived peptide(s) are involved in cancer cell proliferation using highly metastatic human pancreatic cancer S2-VP10 cells, given that S2-VP10 cells constitutively express many matrix metalloproteases (17).
Materials and methods
Antibodies and type VI collagen
A mouse monoclonal antibody (#141) that recognizes COL4A1, NTH α1(IV), COL6A1, and NTH α1(VI) was prepared in our laboratory (Nippon Kayaku, Tokyo, Japan) and characterized as previously described (11,18,19). Rabbit anti-COL6A1 polyclonal antibody (cat. no. NBP1-59126) was purchased from Novus Biologicals. Rabbit anti-COL6A1 (N-term) polyclonal antibody (cat. no. AP6587a) and mouse anti-COL6A2 monoclonal antibody (cat. no. AT1585a) were purchased from Abgent Inc. Mouse anti-HSP90 monoclonal antibody (cat. no. 610418) was purchased from BD Biosciences. Horseradish peroxidase-labeled sheep anti-mouse (cat. no. NA931-1ML) and donkey anti-rabbit (cat. no. NA934-1ML) IgG antibodies were purchased from GE Healthcare. Human type VI collagen was purchased from BD Biosciences.
Small interfering RNAs (siRNAs)
Control and COL6A1 siRNAs were purchased from Thermo Fisher Scientific, Inc. The sequences of all siRNAs except for the control siRNA are shown in Table I.
TaqMan probes
Collagen type IV alpha 1 (COL4A1), COL6A1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) TaqMan probes were purchased from Thermo Fisher Scientific, Inc.
Cell lines and cell cultures
The human breast cancer cell line, MDA-MB-436 (20), was purchased from the American Type Culture Collection and cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum (FBS; Tissue Culture Biologicals). The human pancreatic cancer cell line, S2-VP10 (17), was purchased from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Miyagi, Japan), and cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS. Both cell lines were cultured at 37°C in a 5% CO2 atmosphere. Notably, we confirmed that both cell lines were mycoplasma-negative.
Transfection of siRNAs and cell proliferation assays
Cells (1×104/well) were plated into 12-well plates and cultured at 37°C in a 5% CO2 atmosphere. After overnight incubation, the cells were transfected with the siRNAs indicated in the figure legends using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Briefly, 100 µl of OptiMEM (Thermo Fisher Scientific, Inc.) containing 10 pmol of siRNA was mixed with 100 µl of OptiMEM containing 2.0 µl of Lipofectamine RNAiMAX. The mixture was incubated at room temperature for 20 min. After incubation, the mixture was added to the cells, which were maintained at 37°C in a 5% CO2 atmosphere. Three hours later, the culture medium was replaced with fresh medium. After siRNA transfection, the indicated amount of type VI collagen, 1 ml of conditioned medium, or 1 ml of DMEM supplemented with 10% FBS was added to the cells. The cells were then cultured for 72 h at 37°C in a 5% CO2 atmosphere and subsequently subjected to a cell proliferation assay and cell lysate preparation. In some experiments, the siRNA-treated conditioned medium was collected at the end of the culture period. The conditioned medium was fractionated with Amicon Ultra-4 or Ultra-15 Centrifugal Filter Units (molecular weight cutoff, 10 kDa; Merck KGaA), as indicated in the section ‘Conditioned medium for the cell proliferation assay’; the rescue activities of the fractions were assessed in cell proliferation assays.
To evaluate cell proliferation, we calculated proportional proliferation (% of control) assessed by methylene blue staining as described in the section ‘Methylene blue staining.’
Methylene blue staining
We assessed cell proliferation by methylene blue staining as previously described with slight modifications (21,22). Briefly, the medium was removed from the wells and the plate was allowed to dry at room temperature. Five hundred microliters of methanol was added to each well, and the plate was kept at room temperature for 2 min to fix the cells. Next, methanol was removed from the wells and the plate was allowed to dry at room temperature. Thereafter, 500 µl of 0.05% methylene blue was added to each well, and the plate was incubated at room temperature for 30 min, after which the methylene blue solution was removed. The plate was washed three times with distilled water; 1 ml of 3% HCl was added to each well to dissolve the methylene blue stain, and the absorbance at 660 nm was measured using a SpectraMAX M3 plate reader (Molecular Devices, LLC) with SoftMAX Pro Software version 6.4.2 (Molecular Devices, LLC). All cell proliferation assays were performed in triplicate and each siRNA, drug treatment, and rescue experiment was repeated at least three times. Data are expressed as the mean ± SD.
Conditioned medium for the cell proliferation assay
A total of 2×105 S2-VP10 cells were plated per well in a 6-well plate and cultured for two days at 37°C in a 5% CO2 atmosphere. The conditioned medium obtained after incubation was used to evaluate the ability to rescue the repressed proliferation of KD cells. In some experiments, the conditioned media were fractionated in size using Amicon Ultra-4 or Ultra-15 Centrifugal Filter Units according to the manufacturer's instructions. The conditioned medium was centrifuged at 5,000 × g for 25 min at 4°C in the filter units, followed by filtration through a 0.45-µm polyvinylidene difluoride membrane filter (Merck KGaA); fractions containing molecules larger than 10 kDa (>10 kDa fraction) or smaller than 10 kDa (<10 kDa fraction) were collected separately. Nine volumes of DMEM were added to one volume of the >10 kDa fraction, whereas one volume of FBS was added to nine volumes of the <10 kDa fraction. Each fraction was used in the cell proliferation assay.
Concentration of <10 kDa fraction by acetone precipitation
The proteins were concentrated by acetone precipitation. Briefly, nine volumes of ice-cold acetone were added to one volume of the <10 kDa fraction and the mixture was incubated at −20°C for more than 2 h. This mixture was centrifuged at 15,000 × g for 10 min at 4°C, and the pellet was subjected to SDS-PAGE.
Preparation of cell lysates
The cells were washed three times with phosphate-buffered saline (PBS, pH 7.4) and harvested. Lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, and 10 mM phenylmethylsulfonyl fluoride] was added to the cells, which were then incubated on ice for 30 min. After cell lysis, the mixture was centrifuged at 20,000 × g for 15 min at 4°C, and the supernatants were used as cell lysates. Protein concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Ten micrograms of cell lysate were subjected to western blotting.
Western blotting analysis
Western blotting was performed as previously described (11,18). Briefly, an equal amount of 2X concentrated sample buffer containing 2-mercaptoethanol (2-ME) [125 mM Tris-HCl (pH 6.8), 20% glycerol, 10% 2-ME, and 4% SDS] was added to each cell lysate or supernatant and heated at 95°C for 5 min. Under non-reducing conditions, a 2X concentrated sample buffer without 2-ME was used. The mixture was loaded onto a 7.5% SDS-PAGE gel, 5–20% SuperSep Ace precast gel (Wako Pure Chemical Industries), or 15–20% SuperSep Ace precast gel (Wako Pure Chemical Industries) and electrophoresed. Precision Plus Protein Dual Color Standards (Bio-Rad Laboratories, Inc.) were used as molecular weight markers. The gel was transferred to a polyvinylidene difluoride membrane (PerkinElmer), which was blocked with Tris-buffered saline-Tween 20 [TBS-T; 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] containing 5% skim milk (BD Biosciences). The membrane was incubated with predetermined antibodies and washed with TBS-T. All primary antibodies, except for the anti-COL6A1 (N-term) and anti-COL6A2 antibodies, were diluted to 1 µg/ml with TBS-T containing 5% skim milk prior to use. The anti-COL6A1 (N-term) and anti-COL6A2 antibodies were diluted to 1 µg/ml with Can Get Signal® (Toyobo). Bound antibodies were detected with horseradish peroxidase-labeled anti-mouse or anti-rabbit IgG secondary antibodies diluted to 0.25 µg/ml in TBS-T containing 5% skim milk. The washed membrane was developed with ECL Western Blotting Detection Reagent (GE Healthcare) and visualized on Hyperfilm ECL (GE Healthcare) according to the manufacturer's instructions. To reuse the membrane for some experiments, it was incubated at 55°C for 30 min in stripping buffer [62.5 mM Tris-HCl (pH 6.5), 100 mM 2-ME, and 2% SDS] and reprobed with another antibody as previously described (23).
Coomassie Brilliant Blue (CBB) staining
CBB staining was performed using a Rapid Stain CBB Kit (Nacalai Tesque) according to the manufacturer's instructions.
RNA extraction and cDNA synthesis
RNA was extracted and purified using an RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions.
cDNA was synthesized using purified RNA and SuperScript III (Thermo Fisher Scientific, Inc.) as follows. Briefly, 500 ng of RNA was mixed with 1 µl of 50 µM oligo d(T)20 (Thermo Fisher Scientific, Inc.) and 1 µl of 10 mM dNTP Mix (Thermo Fisher Scientific, Inc.). The mixture was adjusted to 13 µl with sterile distilled water, followed by heating at 65°C for 5 min. After heat denaturation, the mixture was incubated at 4°C for 1 min. Next, 4 µl of 5X First-Strand Buffer (Thermo Fisher Scientific, Inc.), 1 µl of 0.1 M DTT (Thermo Fisher Scientific, Inc.), 1 µl of RNaseOUT (Thermo Fisher Scientific, Inc.), and 1 µl of SuperScript III were added to the mixture. For cDNA synthesis, the mixture was incubated at 50°C for 30 min, followed by denaturation at 75°C for 15 min. The cDNA was used as a template in RT-qPCR.
Reverse transcription quantitative PCR
The indicated mRNA expression was assessed by RT-qPCR using Rotor Gene Q (Qiagen) according to the manufacturer's instructions. Briefly, a reaction mixture containing 10 µl of 2X TaqMan Gene Expression Master Mix (Thermo Fisher Scientific, Inc.), 1 µl TaqMan probe (Thermo Fisher Scientific, Inc.), 1 µl cDNA, and 8 µl dH2O was prepared and subjected to qPCR. Cycling conditions were as follows: Pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 sec; annealing and extension at 60°C for 1 min; number of cycles, 40. After the reaction, the relative ΔCq of the target gene (ΔΔCq) was calculated using GAPDH mRNA as an internal standard (24). The expression level of COL6A2 mRNA was compared to that of COL6A1 mRNA. All RT-qPCR experiments were performed in duplicate, and each RT-qPCR experiment was repeated at least three times. Data are expressed as the mean ± SD.
Statistical analysis
All statistical analyses were carried out with Exsus version 8.1.0 software (CAC Croit Corp.), and were based on at least three independent experiments; the data are expressed as the mean ± SD. Dunnett's multiple comparison test was used to determine statistical differences among the four groups. Student's t-test was used to determine statistically significant differences between two groups. P<0.05 was considered as statistically significant.
Results
Expression of NTH α1(VI) by S2-VP10 cells: COL6A1 silencing reduces cell proliferation and NTH α1(VI) expression
S2-VP10 cells expressed the NTH α1(VI)/COL6A1 chain (Fig. 1A), whereas only faint expression of the COL6A2 polypeptide was found (Fig. 1B). Notably, a smear band was observed under non-reducing conditions (Fig. 1A). It is known that COL6A1 contains cysteine residues (1). Therefore, the smear band may have resulted from differences in intramolecular disulfide bonds. In addition, the COL6A2 mRNA expression level was 22.3±0.7% of the COL6A1 mRNA expression level (Fig. 1C). We found that MDA-MB-436 cells expressed COL6A1 and COL6A2 during our preliminary experiments (data not shown). Thus, we compared the COL6A1 mRNA/COL6A2 mRNA ratio between S2-VP10 and MDA-MB-436 cells. The results revealed that the COL6A2 mRNA expression level was comparable to that of COL6A1 mRNA expression (90.5±13.6% of the COL6A1 mRNA expression level) in the MDA-MB-436 cells (Fig. 1D), indicating that NTH α1(VI) is a major form of the COL6A1 gene product in S2-VP10 cells, as type VI collagen is composed of α1, α2, and α3 chains (1,25), requiring equal amounts of each chain.
COL6A1 KD repressed COL6A1 expression (Fig. 2A). Next, we confirmed that COL6A1 KD inhibited S2-VP10 cell proliferation as previously reported (6). Representative proliferation curves of S2-VP10 cells transfected with or without control siRNA are shown in Fig. 2B, indicating that the transfection of control siRNA does not affect the proliferation of S2-VP10 cells. Moreover, Fig. 2C indicates that COL6A1 KD inhibited the proliferation of S2-VP10 cells.
Requirement for fragmentation of NTH α1(VI) for enhanced proliferation
Repressed proliferation of S2-VP10 cells by COL6A1 KD suggests that COL6A1 gene products including type VI collagen, NTH α1(VI), and/or NTH α1(VI)-derived peptides are involved in cell proliferation. The rescue activity was evaluated by adding type VI collagen or the conditioned medium of S2-VP10 cells to COL6A1-silenced S2-VP10 cells; type VI collagen did not show rescue activity (Fig. 3A), whereas the conditioned medium showed rescue activity compared to DMEM supplemented with 10% FBS (Fig. 3B and C), indicating that NTH α1(VI) and/or derived peptides are responsible for the rescue activity. To test whether NTH α1(VI) or its fragments hold this activity, the conditioned medium was fractionated by size; rescue activity with a strength similar to that of non-fractionated conditioned medium was found in the <10 kDa fraction, whereas the >10 kDa fraction containing NTH α1(VI) showed no rescue activity (Fig. 3C-E), indicating that enhanced proliferation requires NTH α1(VI) fragmentation. To confirm that the active components in the <10 kDa fraction were derived from NTH α1(VI), we assessed the rescue activity of the <10 kDa fraction of COL6A1 KD cells. We observed that the <10 kDa fraction of control siRNA-treated cells was able to rescue cell proliferation (Fig. 4A and B), whereas the <10 kDa fraction of COL6A1 KD cells appeared to exhibit lower rescue activity (Fig. 4C). The slight rescue activity of the <10 kDa fraction of COL6A1 KD cells likely resulted from the small number of active fragments that may have been produced from preexisting NTH α1(VI) prior to KD treatment. Notably, we also confirmed that NTH α1(VI) with a size of 140 kDa was present in the >10 kDa fraction (Fig. 4D) and that the <10 kDa fraction contained only peptides less than 10 kDa in size (Fig. 4E). Therefore, fragmentation of NTH α1(VI) into fragments of less than 10 kDa was responsible for the enhanced proliferation of cancer cells.
Discussion
We showed that peptides of less than 10 kDa, possibly derived from NTH α1(VI) fragmentation, are involved in the proliferation of S2-VP10 cells. Higher expression of collagen type VI alpha 1 chain (COL6A1) is observed in some tumors (4–6), and its expression is associated with poor prognosis (7–9). Zhu et al also indicated that COL6A1 overexpression promotes the proliferation of prostate cancer LNCaP cells, and that COL6A1 expression is higher in patients with castration-resistant prostate cancer (6). In addition, secretion of the COL6A1 fragment is upregulated in some types of cancers, including pancreatic cancer (16). This suggests that the peptide fragments described herein are involved in cell proliferation within tumors. Recently, Chen et al reported that COL6A1 KD suppresses the proliferation and migration of human aortic vascular smooth muscle cells (26), suggesting that the peptides are also involved in vascular development.
Collagen-derived matricryptins (endostatin, arresten, canstatin, and tumstatin) have anti-angiogenic and antitumor activities (13,14). Hamano et al indicated that tumstatin is generated by MMP-9 proteolysis in vivo (27). It has also been reported that S2-VP10 cells constitutively express many matrix metalloproteases (MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, and MMP-14) and show a highly metastatic phenotype (17). In addition, Willumsen et al indicated that the serum level of MMP-generated COL6A1 fragments was upregulated in some types of cancers (16). Similarly, the peptide fragments in the present study may have been generated by one or more of these proteases.
Our results suggest that the growth-promoting mechanism of the peptides is not shared with type VI collagen. As previously indicated, arresten, canstatin, and tumstatin show anti-angiogenic and antitumor activities; these matricryptin sequences are located in non-collagenous NC1 domains. The biological activities of these matricryptins have also been reported to be mediated through integrins (13,14). In addition, endotrophin, the C5 Kunitz-like domain of COL6A3, promotes malignant tumor progression via transforming growth factor β signaling (15). This indicates that non-collagenous regions in collagens have biological functions and that these activities are mediated via specific receptors. COL6A1 has three von Willebrand factor A (vWA) modules, one in its N-terminus and two in its C-terminus (1,28,29). Whittaker and Hynes suggested that the vWA modules in collagen proteins, including COL6A1, are involved in protein-protein interactions with other matrix proteins and possibly with cells (30), suggesting that vWA modules are important for peptide activity.
We showed that the mRNA and protein expression of COL6A2 was low in S2-VP10 cells. This suggests that the chain stoichiometry between COL6A1 and COL6A2 is different. Merl-Pham et al also reported that the chain stoichiometry abnormality between COL6A1 and COL6A2 was observed in the extracellular matrix of human lung fibroblasts; they concluded that one of the causes of the stoichiometry abnormality is the existence of NTH α1(VI) (31). This strongly suggests that the origin of the peptides is NTH α1(VI); however, the possibility that the origin of the peptides is a different type VI collagen is not completely excluded. Thus, it is essential to identify the peptide sequences. We are currently investigating this fraction by liquid chromatography-tandem mass spectrometry analysis; the sequence may also provide information about peptide generation as MMPs and other proteases contain consensus sequences for peptide cleavage.
In conclusion, we demonstrated i) that peptides of less than 10 kDa are involved in the proliferation of pancreatic cancer S2-VP10 cells and ii) the peptides are derived from NTH α1(VI) fragmentation.
Acknowledgements
The authors would like to thank Takashi Aijima, Aya Kikitsu, and Naoko Takahara for their experimental support.
Funding
This work was supported in part by a grant from the Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sport, Science, and Technology, Japan, 2014–2018 (S1411005 to YI).
Availability of data and materials
The datasets and materials used during the present study are available from the corresponding author upon reasonable request.
Author's contributions
TS designed, conducted and performed the experiments, and wrote the manuscript. KT, KS, HS, TH, YI and MM designed, reviewed and edited the manuscript. MM designed, conducted and supervised this project. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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