Yanke Chen and Jing Luan contributed equally to this work.
Bone destruction and pain caused by cancer is one of the most devastating complications of cancer patients with bone metastases, and it seriously affects the quality of patients’ life. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell adhesion molecule with increased expression in a variety of tumors. This study focused to clarify the specific function of EMMPRIN in bone metastasis of breast cancer.
Adenovirus with shRNA-EMMPRIN was transfected into MRMT-1 rat breast carcinoma cells, and the MRMT-1 cells with different expression levels of EMMPRIN were implanted into the bone marrow cavity of rat tibia. Next, the effect of down-regulation of EMMPRIN was evaluated as follows: bone damage was detected by X-ray radiological and tartrate-resistant acid phosphatase staining; the tumor burden was evaluated by hematoxylin and eosin staining; the test of pain-related behaviors was assessed used the bilateral paw withdrawal mechanical threshold; and the levels of secretory factors in tumor conditioned medium were determined by using enzyme-linked immunosorbent assay.
We found that down-regulation of EMMPRIN in tumor cells can simultaneously reduce tumor burden, relieve cancer-induced bone destruction and pain.
EMMPRIN is expected to be a therapeutic target for relieving bone metastasis of breast cancer and alleviating cancer-induced bone destruction and pain. The method of targeting EMMPRIN may be a promising strategy for the treatment of cancer in the future.
Osseous metastasis is a disease in which some malignant tumors originating from other tissues are transferred to bone tissue by blood. Bone damage and pain are the main manifestations in osseous metastasis. Bone tissue is one of the most common distant metastases in patients with breast cancer and other cancers including prostate, lung, kidney, thyroid, and sarcoma, etc. [
Matrix metalloproteinases (MMPs) are a family of more than 20 proteases participate in a variety of physiological and pathological events. MMPs play important roles in tumor progression. Studies have shown that MMPs promote metastasis of malignant tumors [
In this study, we used an animal model in which MRMT-1 rat breast cancer cells were inoculated into the tibia bone marrow cavity of female rats to study the effects of EMMPRIN on bone destruction and pain. We found that down-regulation of EMMPRIN expression in breast cancer cells could simultaneously reduce tumor burden, bone destruction as well as pain caused by tumors. Our results suggest that EMMPRIN had multiple roles in tumor progression, and it might be developed into a potential new target to control pain and bone destruction.
MRMT-1 rat breast carcinoma cells were purchased from the Jiniou Biological Technology Company (China, Guangzhou). Cells were cultured in medium containing RPMI 1640 (ThermoFisher, Waltham, MA) with 10% fetal bovine serum (Gibco), at 37°C under a mixture of 95% air and 5% CO2.
OX47 expression in MRMT-1 cells was knocked down by lentiviral transduction. The construction of shRNA-EMMPRIN and the control of lentiviral vectors have been characterized in our previous study [
Female Sprague-Dawley rats weighing 180–200 g were provided by the Animal Center of School of Medicine, Xi’an Jiaotong University, and randomly divided into two groups, which were inoculated with control vector MRMT-1 cells or OX47 knockdown MRMT-1 cells.
Prior to implantation, these resuspended cells were counted and diluted to achieve a final concentration of 2×107 cells/mL. The surgery was performed as previously described and modified [
All behavioral tests were performed using the double-blind method. Rats were placed in cages individually, the bilateral paw withdrawal mechanical threshold was assessed by the application of an electronic von Freydevice (2290 Electrovonfrey, IITC, Woodland Hills, CA) as previously described [
On the 7th, 14th, 21st, and 28th day after tumor cell inoculation, the tumor-bearing rats were anesthetized. Then, bone damage was detected by X-ray using the E-COM digital radiographer system (Guangdong E-COM Technology Co., Ltd., Guangdong, China).
The tumor-bearing rats were deeply anesthetized by intra-peritoneal injection of overdose sodium pentobarbital (60 mg/kg). 0.01 M cold phosphate buffer saline (PBS; pH 7.4) followed by a fix solution which contained 4% paraformaldehyde in 0.01 M PBS was perfused into the rat heart. Then, the bilateral tibial bones and L4 spinal cord were taken and fixed with paraformaldehyde overnight at 4°C. Decalcification was performed with 4% EDTA for 1 week at room temperature. Frozen sections of the tibia were prepared by conventional methods and stained with hematoxylin and eosin (H&E).
Tartrate-resistant acid phosphatase (TRAP), a marker of activate osteoclasts, is currently the most widely used index to evaluate ability of bone resorption. Frozen sections of the tibia were stained using the TRAP kit (Genmed Scientific, Arlington, MA) according to the manufacturer’s protocol. The numbers of TRAP-positive multinucleated osteoclasts were counted in four random regions of each section.
In the study, immunohistochemical analyses of c-Fos and glial-fibrillary acidic protein (GFAP) on spinal cord were performed to examine neurochemical alterations in tumor-bearing rats. Frozen spinal cord tissues were cut into 10 μm on a freezing microtome and the gelatin-coated slides were collected and processed. For c-Fos and GFAP immunohistochemistry, the detailed methods of immunostaining have been characterized in our previous studies [
The levels of MMP2 (Catalog #F16170, Westang, Shanghai, China), MMP9 (Catalog#orb381134, Biorbyt, Wuhan, China), osteocrin (Catalog#E00357, Wksubio, Shanghai, China), osteoactivin (Catalog#E00355, Wksubio), BMPs (Catalog#FK-L1802, Fanke, Shanghai, China) in tumor conditioned medium (TCM) were determined by using enzyme-linked immunosorbent assay (ELISA) assay kits. Experiments were carried out according to the manufacturer’s instructions. The absorbance value was acquired using a VersaMax Tunable MicroPlate Reader (Molecular Devices, Sunnyvale, CA) at 490 nm.
All data were expressed as means±standard error of mean. Student’s t test for random measures was performed followed by
To construct the OX47 knocked down MRMT-1 cell line and its control cell line, a lentivirus containing the target sequence of OX47 or scrambled sequence was transduced into MRMT-1 cells. The down-regulation of OX47 was confirmed by qRT-PCR (
MRMT-1 cells were implanted into the medullary cavity of rat tibia. The tumor burden level in the femur was quantified by H&E staining. As the tumor cells proliferated rapidly, after 28 days of injection, the bone marrow cavity of the control group was filled with a large number of tumor masses. Compared with the shRNA-control group, OX47 low-expression MRMT-1 cells grew slowly and the tumor burden area of rat was significantly reduced (
In order to observe the changes in bone structure after tumor cell inoculation, the bone structure examination was performed on the rats by radiological examination system. On the 7th day after the injection of tumor cells, we did not observe any osteopenia and bone destruction. However, on the 14th day after the injection, some loss of medullary bone and obvious cortical bone erosion were observed. As the deterioration progressed, on the 21st day after the injection, a full-thickness cortical bone loss and displaced fracture were observed in the control group. The representative examples are shown in
Our previous study reported that EMMPRIN was highly expressed in cancer cells and promoted tumor growth and metastasis by regulating the secretion of soluble factors, including MMPs, osteocrin, osteactivin, and BMPs [
We used von Frey hair esthesiometer to assess the dynamic change of mechanical allodynia of rats a few days after MRMT-1 cell implantation. As can be seen from
The reduction in the paw withdrawal threshold to von Frey hair stimulation occurred not only on the surgical side hind paw (
In addition, we performed immunohistochemical analysis of c-Fos (an immediate-early gene for assessing sensory neuron activity) and GFAP (a glial marker). We found that compared with sham-operated rats, the number of c-Fos–positive neurons and GFAP-labeled glial cells in the ipsilateral dorsal horn of the rats increased significantly after inoculation of cancer cells. What’s more, knockdown of OX47 in the cancer cells significantly reduced staining of c-Fos and GFAP in the corresponding segments of the ipsilateral spinal cord (
Studies have shown that EMMPRIN is highly expressed in a variety of tumors. It plays important roles in promoting tumor growth, angiogenesis, invasion, and metastasis [
EMMPRIN affects adjacent local microenvironment by promoting the secretion of matrix metalloproteins (MMPs) [
In our study, inhibition of EMMPRIN alleviated cancer pain. The relevant mechanism maybe it simultaneously inhibited cancer growth and bone destruction. In addition, we observed that inhibition of EMMPRIN also significantly reduced the number of c-Fos–positive neurons and GFAP-positive glial cells in the corresponding segments of the ipsilateral spinal cord. Previous studies have revealed a stereotypic set of neurochemical changes occured in the spinal cord in animals with cancer-induced pain, these areas receive sensory input from the tumor-bearing bone, and lead neurons activation and glial cells proliferation [
In summary, EMMPRIN plays significant multi-effect roles in the process of breast cancer cells metastasis to bone. Inhibition of EMMPRIN showed multifaceted inhibitory effects on cancer progression, including reducing tumor burden and bone destruction, as well as reducing pain. This information will help to develop new therapeutic strategies to synergistically improve the survival and quality of life for patients with cancer metastasized to bone.
Animal experiments were approved by the Animal Use Subcommittee of the Animal Protection Committee of Xi’an Jiao tong University, and all procedures were conducted under the guidance of the Animal Protection and Use Institutions Committee.
Conceived and designed the analysis: Chen Y, Jiang T, Gou X.
Collected the data: Chen Y, Jiang T, Cai D, Sun C, Wang X, Zhao X, Gou X.
Contributed data or analysis tools: Chen Y, Jiang T, Cai D, Sun C, Wang X, Zhao X, Gou X.
Performed the analysis: Chen Y, Luan J, Jiang T, Cai D, Sun C, Wang X, Zhao X, Gou X.
Wrote the paper: Luan J.
Conflicts of interest relevant to this article was not reported.
This work was supported by the National Natural Science Foundation of China (81101744 to Yanke Chen), Projects of International Cooperation and Exchanges Natural Science Foundation of ShaanXi Province of China (2017KW-059 to Yanke Chen) and the Scientific Research and Sharing Platform Construction Project of Shaanxi Province (2018PT-09 to Yanke Chen). The data that support the findings of this study are available from the corresponding author upon reasonable request.
Down-regulation of extracellular matrix metalloproteinase inducer (OX47) inhibited growth of MRMT-1 rat breast carcinoma cells
Down-regulation of extracellular matrix metalloproteinase inducer (OX47) alleviated cancer-induced bone destruction. Representative radiographs images (A) and quantification (B) of bone destruction in control-shRNA group and OX47-shRNA group on the 14th and 21st day after MRMT-1 cells injection, shRNA control vs. shRNA OX47; **p < 0.01. (C, D) Representative tartrate-resistant acid phosphatase staining photomicrographs (C) and quantification (D) of osteoclasts in control-shRNA group (n=6) and OX47-shRNA group (n=6) on the 21st day after MRMT-1 cells injection, shRNA control vs. shRNA OX47; **p < 0.01. (E) Knockdown of OX47 increased the pH value of tumor conditioned medium (TCM) which collected from MRMT-1 cells incubating for 12–72 hours, shRNA control vs. shRNA OX47; *p < 0.05. (F) Enzyme-linked immunosorbent assay quantitative results of solvable factors in TCM, shRNA control vs. shRNA OX47; *p < 0.05, **p < 0.01.
Down-regulation of extracellular matrix metalloproteinase inducer (OX47) relieved cancer-induced pain. Ipsilateral (A) and contralateral (B) hind paw withdrawal responses to von Frey hair stimulation in MRMT-1 cells (OX47 is knocked down or not) injected rats, shRNA control vs. shRNA OX47; *p < 0.05, **p < 0.01; shRNA control vs. Sham; #p < 0.01. PWMT, paw withdrawal mechanical threshold.
Immunohistochemical analysis of c-Fos (an immediate-early gene for assessing sensory neuron activity) and glial fibrillary acidic protein (GFAP; a glial marker). (A, B) Representative photomicrographs of c-Fos and GFAP immunostaining in the dorsal horn within spinal L4 segments of sham rats and tumor-bearing rats (A). GFAP and c-Fos positive cells were counted by examining six different visual fields under a microscope at 200× magnification in a blinded manner. Multiple comparisons were performed with one-way ANOVA, ****p < 0.0001 (B).