Background
Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females worldwide [
1]. Despite the improvement in early clinical detection and therapy strategies of breast cancer, the prognosis of breast cancer patients still remains unsatisfying [
2]. Therefore, it is necessary to understand the mechanisms that underlie breast cancer progression and thus identify new precise therapeutic strategies.
Collagen triple helix repeat containing-1 (CTHRC1) is a secreted 28-kDa protein, which was firstly identified overexpressed in the adventitia and neointima of injured rat arteries, could inhibit collagen expression and increase cell migration [
3‐
5]. CTHRC1 was then found to be ubiquitously expressed in numerous cell types such as fibroblasts and smooth muscle cells, and aberrantly up-regulated in several malignant tumors, including melanoma, and cancers of gastrointestinal tract, liver, lung, ovary and so on [
6‐
8]. Yamamoto et al. [
9] have documented that cell surface anchored CTHRC1 can stabilize the physical interaction between Wnt ligands and Frizzled receptors, and selectively activate the planar cell polarity pathway of Wnt signaling to regulate cell motility and taxis. In our previous studies, we have found that CTHRC1 promotes non-small cell lung cancer (NSCLC) cell aggressiveness by targeting Wnt/β-catenin pathway, and may serve as a novel biomarker for NSCLC patients [
7]. Currently, there is little information about the role of CTHRC1 in breast cancer progression. Kharaishvili et al. [
10] performed immunohistochemical study of CTHRC1 in breast cancer with parallel analysis of periostin and versican and found that combined evaluation of stromal expression of CTHRC1 and periostin could serve as a potential marker for breast cancer bone metastasis prediction. Later Kim et al. [
11] found CTHRC1 over-expression was significantly associated with clinicopathological factors of poor prognosis in invasive ductal carcinoma. However, the functional role of CTHRC1 and its mechanism in breast cancer still remains unclear.
In the present study, we demonstrated that CTHRC1, negatively regulated by miR-30c, could promote breast cancer cell proliferation, invasion and migration and suppress cell apoptosis. In addition, our cohort study and further meta-analysis corroborated high expression of CTHRC1 in primary tumors of breast cancer patients is associated with poor clinical outcome.
Methods
Patients and tissue samples
Primary invasive ductal carcinomas of breast were obtained from 121 female patients at the Department of Breast and Thyroid Surgery, the First Affiliated Hospital, Sun Yat-sen University, from January 2007 to December 2012. Pathological diagnosis, as well as ER, PR and Her2 status, was verified by two different pathologists. Patients with invasive carcinomas, other than DCIS, underwent six cycles of postoperative adjuvant chemotherapy with FAC regimen (5-fluorouracil 500 mg/m
2, doxorubicin 50 mg/m
2, and cyclophosphamide 500 mg/m
2). Subsequently, patients with ER(+) tumors underwent endocrine therapy according to NCCN guideline. No distal metastasis was identified in the patients upon diagnosis. In addition, fresh samples of normal breast tissue, benign breast tumor tissues and invasive ductal carcinoma tissues were collected from patients who had undergone mastectomy or lumpectomy for benign or malignant breast diseases. All samples were snap-frozen for mRNA assessment and were collected with informed written consent from patients. The complete clinical and pathological features of these patients were collected and stored in our database by a researcher fellow. The study protocol followed the Ethical Guidelines of the 1975 Declaration of Helsinki, revised in 2000. All related procedures were performed with the approval of the Internal Review and the Ethics Boards of the First Affiliated Hospital, Sun Yat-sen University. All research protocols strictly complied with REMARK guidelines for reporting prognostic biomarkers in cancer [
12].
Immunohistochemistry
Archived paraffin-embedded tumor tissues collected from 121 consecutive patients with breast cancer treated in our hospital between 2007 and 2012 were used for tissue microarray construction and immunohistochemistry (IHC). The IHC was performed using the polymer HRP detection system (Zhongshan Goldenbridge Biotechnology, Beijing, China) and antibodies were listed in the Additional file
1: Table S3. CTHRC1 expression level was scored semi-quantitatively using the IRS (immunoreactive score) = SI (staining intensity) × PP (percentage of positive cells) as described [
13,
14]. Briefly, SI was determined as 0, negative; 1, weak; 2, moderate and 3, strong. PP was defined as 0, <1%; 1, 1–10%; 2, 11–50%; 3, 51–80% and 4, >80% positive cells. Five visual fields from different areas of each tumor were used for the IRS evaluation. IRS ≤ 4 was defined as low CTHRC1 expression and IRS > 4 were defined as high CTHRC1 expression.
Cell lines and cell culture
In this study, human breast cancer cell lines, SK-BR-3, MDA-MB-231, MDA-MB-468, MCF-7, and BT549 as well as MCF-10A breast epithelial line were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells’ characters were as described by Kao et al. [
15]. These cells were cultured according to the recommended protocols.
RNA extraction and real-time quantitative polymerase chain reaction (qRT-PCR)
TRIzol
® Reagent (Life Technologies, Carlsbad, CA) was used to isolate total RNA from frozen patient samples and cell lines according to the manufacturer’s protocol. cDNA was synthesized using the universal cDNA synthesis kit (Toyobo, tokyo, JP). The RNA was then reverse-transcribed to obtain cDNA by the universal cDNA synthesis kit (Toyobo, tokyo, JP) at 37 °C for 50 min. The cDNA was subjected to quantitative real-time PCR (qRT-PCR) using the SYBR Green PCR Kit (Roche Life Sciences, Switzerland) and the assay was performed on an PRISM 7300 Sequence Detection System (Applied Biosystems, CA). 18SrRNA was used as an internal control for CTHRC1 and U6 for microRNAs. The relative levels of expression were quantified and analyzed. The experiments were done in triplicate. The primers were all synthesized and bought from Sangon Biotech (Shanghai, China). The detailed procedure was as Gong et al. described [
16]. The primer sequences are listed in the Additional file
1: Table S2.
Western blot
Total proteins were extracted with RIPA lysis buffer and separated by SDS-PAGE and then transferred to the PVDF membrane (Roche Life Sciences, Switzerland). The membrane were blocked with 5% skimmed milk and incubated with the appropriate antibody. The antigen-antibody complex on the membrane was detected with enhanced chemiluminescence regents (Thermo Scientific, Waltham, MA). The antibodies are listed in the Additional file
1: Table S3.
Immunofluorescence for cells
Cells grow at glass coverslips, and then were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) with 0.2% Triton. Cells were then blocked for an hour with 1% bovine serum albumin (BSA) followed by incubation with primary antibody overnight at 4 °C. Cells were washed and incubated with appropriate secondary antibody and DAPI. The slides were imaged using an inverted fluorescence microscope DMI4000B (Leica, Wetzlar, Germany). The antibodies and reagents are listed in the Additional file
1: Tables S3 and S4.
Transfection
In this study, BT549 and HEK293T cells were transfected with miR-30c mimics, negative control, miR-30c inhibitor and inhibitor negative control. CTHRC1 expression vector without 3′-UTR was constructed by inserting its CDS sequence into the psiCHECK2 vector (Promega, USA). qRT-PCR analysis were performed to confirm the transfection efficiency (Additional file
2: Figure S3A). For siRNA and stable plasmid DNA transfection, cells were transfected into BT549 cells with specific siRNA duplexes targeting CTHRC1 or pcDNA3 vector expressing CTHRC1 construct using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. The overexpression level of CTHRC1 and silencing efficiency of each siRNA was assessed by qRT-PCR. Gain of CTHRC1 resulted in significant increase in its expression (Additional file
2: Figure S3B). CTHRC1-siRNA-2 caused the lowest level of CTHRC1 and then was selected for further study (Additional file
2: Figure S3C). The overexpression and knock-down level of CTHRC1 was further validated by western blot (Additional file
2: Figure S3D).
The above used sequences are listed in the Additional file
1: Table S5.
In vitro cell behavior assays
To assess the cell viability, the Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, USA) was used according to the manufacturer’s instructions. After transfection for 24 h, cells were seeded in 96-well plates. The cell viability was detected by adding WST-8 at a final concentration of 10%, and the absorbance of these samples were measured at 450 nm on a Microplate reader (Molecular Device, SpectraMax M5e, USA) every 24 h for 4 days. The data derived from triplicate samples are presented as mean ± SD.
For colony formation assay, cells were seeded into 6-well plates at a density of 500 cells/well and cultured for 2 weeks at 37 °C. The numbers of colonies per dish were counted after staining with crystal violet (Beyotime Institute of Biotechnology). Only positive colonies (diameter > 40 um) in the dishes were counted and compared.
Cell cycle analysis was conducted by flow cytometry (Beckman-Coulter, Fullerton, CA, USA) using a Propidium Iodide (PI) cell cycle detection kit (Beyotime Institute of Biotechnology, Beijing, China). Cell apoptosis analysis was conducted by flow cytometry (Beckman-Coulter, Fullerton, CA, USA) using Annexin V-FITC/PI Apoptosis Detection Kit (Keygen Biotechnology, Nanjing, China).
For the transwell invasion assay, 1 × 105 cells in serum-free medium were seeded into the upper chamber of 8-μm transwell inserts with a matrigel coated membrane (BD Biosciences, Franklin Lakes, NJ), while medium containing 10% bovine serum albumin was in the lower chamber. After several hours of incubation at 37 °C, gel and cells in the upper chamber were removed carefully and cells adhering to the underside of the membrane were stained with 0.1% crystal violet (Beyotime Institute of Biotechnology, Shanghai, China) and 20% methanol. The number of cells was counted under an inverted microscope (Nikon, Chiyoda-Ku, JP).
For the transwell migration assay, 5 × 104 cells in serum-free medium were seeded into the upper chamber of 8-μm transwell inserts (BD Biosciences, Franklin Lakes, NJ). And medium containing 10% bovine serum albumin was in the lower chamber. After several hours of incubation at 37 °C, cells in the upper chamber were removed carefully and cells adhering to the underside of the membrane were stained with 0.1% crystal violet (Beyotime Institute of Biotechnology, Shanghai, China) and 20% methanol. The number of cells was counted under an inverted microscope (Nikon, Chiyoda-Ku, JP).
All of the above experiments were performed in triplicate. The detailed procedure was as previously described [
7,
8].
Statistical analysis
All data were analyzed using the statistical software SPSS 17 for Windows (SPSS Inc.). The correlation between CTHRC1 expression and clinicopathological parameters was analyzed by Spearman rank-correlation analysis. Survival curves were constructed by Kaplan-Meier method and the difference of these groups were evaluated by using the log-rank test. The Cox proportional hazard regression model was used to identify factors that were independently associated with overall survival and recurrence-free survival. Only factor which was P < 0.05 in univariate analysis could be analyzed in multivariate Analysis. Continuous data in this study were presented as mean ± standard deviation (SD) from at least three independent experiments. The differences between groups were analyzed by Student’s t test when only two groups were compared or by one-way analysis of variance (ANOVA) when more than two groups were compared. Categorical data were analyzed with χ2 test or Fisher’s exact test. A two-tailed P < 0.05 was considered statistically significant. Meta-analyses were performed using RevMan 5.3 statistical software. We used Q-test and I
2
test to examine heterogeneity between studies. We used hazard ratio (HR) to evaluate the relationship of CTHRC1 expression with overall survival (OS) and recurrence-free survival (RFS) in breast cancer. To test publication bias, we utilized RevMan 5.3 software to construct a funnel plot. P < 0.05 was considered to indicate a significant difference.
Discussion
In breast cancer, around 15% of patients develop disseminated metastasis before or after diagnosis, and distant metastasis is responsible for approximately 90% of breast cancer-associated mortality [
25]. Activating invasion and metastasis is an important hallmark of cancer [
26]. The multistep process of invasion and metastasis has been schematized as a sequence of discrete steps, often termed the invasion-metastasis cascade [
27]. Research into the capability for invasion and metastasis has accelerated dramatically over the past decade and resulted in the development of hallmark-targeting therapies [
28]. However, a targeted therapeutic agent inhibiting one key pathway in a tumor may not completely shut off a hallmark capability, allowing some cancer cells to survive with residual function until they or their progeny eventually adapt to the selective pressure imposed by the therapy being applied [
26,
28]. Therefore, identifying more key regulators in cancer metastasis and targeting all of these supporting pathways therapeutically become increasingly important. In this study, we found CTHRC1 promoted cell proliferation, invasion and migration and suppressed cell apoptosis in breast cancer, which might be by activating GSK-3β/β-catenin signaling and inhibiting Bax/Caspase-9/Caspase-3 signaling respectively; and these biological functions of CTHRC1 could be directly negatively regulated by miR-30c. In addition, our cohort study and further meta-analysis validated high expression of CTHRC1 was associated with aggressive clinicopathological features and poor clinical outcome of breast cancer patients. Yet there still exist some potential problems to be further discussed and explored.
Wnt/β-catenin signaling plays a critical role in a variety of biological processes, including cell proliferation, migration, polarity establishment and stem cell self-renewal [
24,
29]. Deregulation of Wnt/β-catenin signaling is associated with development of various cancers, including breast cancer [
30,
31]. Wnt ligands initiate this signaling by interacting with cell surface receptors Frizzled (Fz) and low-density lipoprotein receptor-related protein 6 (LRP6). Subsequent recruitment of the downstream signal mediators Dvl to Fz and Axin to LRP6 results in disassembly of the β-catenin destruction complex consisting of Axin, adenomatous polyposis coli (APC), GSK-3β and casein kinase 1 (CK1), then leading to the nuclear translocation of β-catenin. Binding of β-catenin with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) activates the transcription of Wnt target genes, such as c-Myc, Cyclin D1 and MMP7, ultimately initiating cell proliferation, invasion and migration [
32,
33]. In this study we presented evidence that CTHRC1 could promote GSK-3β phosphorylation at Ser9 and thus GSK3β-mediated phosphorylation of β-catenin at Ser-33, Ser-37, and Thr-41 [
34] was inhibited. Therefore, ubiquitin-dependent degradation of β-catenin was prevented, after which β-catenin accumulated in cytoplasm and then translocated into nuclear, thereby activating this signaling for cell proliferation and metastasis. However, how CTHRC1 promoted phosphorylation of GSK-3β remains to be further studied. Furthermore, it has been widely recognized that the activation of Wnt/β-catenin signaling can induce epithelial-mesenchymal transition (EMT). EMT is a vital initiator of and a contributor to tumor metastasis, during which, polarized epithelial cells lost their cell-cell junctions and converted into individual, non-polarized, motile and invasive mesenchymal cells [
35]. Accumulating researches have focused on the role of EMT in breast cancer progression, but still remain inconclusive [
36‐
38]. In this study, we also found it seemed that interfering the expression of CTHRC1 might change the morphology of BT549 cells when performing cell immunofluorescence, indicating CTHRC1 might also regulate EMT of breast cancer cells. Thus in future study, we will also focus on whether CTHRC1 regulates breast cancer progression by targeting EMT.
In addition, we found CTHRC1 could suppress breast cancer cell apoptosis. Apoptosis is a form of programmed cell death that occurs in a physiological setting and is indispensable for the preservation of tissue homeostasis and embryonic development [
26,
39]. Importantly, apoptosis down-regulation is a major cause of tumorigenesis, cancer progression and chemo-resistance in various cancers, including breast cancer [
40,
41]. The apoptotic machinery is composed of upstream regulators and downstream effectors, and controlled by counterbalancing pro- and antiapoptotic members of the Bcl-2 family of regulatory proteins [
42]. The archetype, Bcl-2, along with its closest relatives (Bcl-xL, etc.) are inhibitors of apoptosis, acting in large part by binding to and thereby suppressing two proapoptotic triggering proteins (Bax and Bak) [
22]. When relieved of inhibition by antiapoptotic relatives, Bax and Bak disrupt the integrity of the outer mitochondrial membrane, causing the release of proapoptotic signaling proteins, key of which is cytochrome C. Then a cascade of caspases (Caspase-9, Caspase-3) is activated, that act via their proteolytic activities to induce the multiple cellular changes associated with the apoptotic program [
23,
43]. In this study, we identified CTHRC1 could down-regulate Bax and thus inhibit the activation of Caspase-9 and Caspase-3. However, exactly how CTHRC1 inactivates the Bax/Caspase-9/Caspase-3 pathway needs to be further explored.
Last but not least, in this study we identified CTHRC1 as an independent prognostic factor for breast cancer patients, suggesting it might be used for molecular classification of breast cancer. Precision medicine is about matching the right drugs to the right patients [
44]. We supposed those breast cancer patients with CTHRC1 high-expression could be characterized into poor prognosis group, whose therapeutic effect of traditional strategies was not so good, and might be treated with strategies designed to up-regulate miR-30c. The potential of miRNAs in regulating different cellular pathways and as mediators of interactions between cells make them ideal drug targets. MicroRNA-based strategies for anti-cancer therapy are becoming increasingly possible, and promising results have been achieved with the use of miRNA antagonists and miRNA mimics in preclinical studies. Moreover, due to advances in the understanding of specific miRNA modulatory role in immune system and tumor-mediated immunity, researchers are also developing novel miRNA-based interventions for cancer immunotherapy [
45]. However, successful delivery directed to specific tissue and cell, represents a big challenge for in vivo miRNA-mediated anti-cancer therapy.
Acknowledgements
We thank Prof. Zun-fu Ke (Department of Pathology, the First Affiliated Hospital of Sun Yat-sen University) and his colleagues for the help of pathological diagnoses and guidance. We also thank Prof. Xuan-xian Peng (Center for Proteomics and Metabolomics, State Key Laboratory of Biocontrol, School of Life Sciences, MOE Key Lab Aquat Food Safety, School of Life Sciences, Sun Yat-sen University) for the help of guidance in mechanism study.