Systemic inflammation promotes lung metastasis via E-selectin upregulation in mouse breast cancer model

Systemic inflammation might modulate the microenvironment in the lungs and promotes metastasis. E-selectin, an inflammation inducible endothelial cell adhesion molecule, has been reported to play an important role in homing metastatic cancer cells. To study the effects of E-selectin expression induced by systemic inflammation on breast cancer metastasis, we first treated BALB/c mice with lipopolysaccharide (LPS) to induce systemic inflammation. Pulmonary tissues were analyzed by wet/dry ratio, hematoxylin and eosin (H&E) staining and immunohistochemistry. Then 4T1 cells were injected via tail vein. Lung surface metastasis was counted and detected by histological analysis. LPS-induced E-selectin expression and tumor cells adhesion were assessed by western blotting and immunofluorescence. The circulating levels of proinflammatory cytokines in sera were evaluated by ELISA. Our results showed that a significant increase in breast cancer metastasis to lungs was observed in LPS-treated mice vs. the PBS-treated mice, accompanying with an increased E-selectin expression in pulmonary tissue of LPS-treated mice. In vitro studies showed a significant elevation of E-selectin production in MPVECs which enhanced the adhesion activity of 4T1 cells. Treatment with anti-E-selectin antibody significantly reduced the development of metastasis in vivo, and significantly reduced the adhesion of 4T1 cells to MPVECs in vitro. Our results suggest that systemic inflammation may increase the expression of E-selectin which mediated the lung metastasis of breast cancer in mouse model.     

Newly characterized motile sperm domain-containing protein 2 promotes human breast cancer metastasis

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among women worldwide. Breast cancer metastasis results in poor prognosis and increased mortality, but the mechanisms of breast cancer metastasis are yet to be fully resolved. Identifying distinctive proteins that regulate metastasis might be targeted to improve therapy in breast cancer. We previously described MOSPD2 as a surface membrane protein that regulates monocyte migration in vitro. In this study, we demonstrate for the first time that MOSPD2 has a major role in breast cancer cell migration and metastasis. MOSPD2 expression was highly elevated in invasive and metastatic breast cancer while it was absent or residual in normal tissue and in primary in situ tumors. In vitro experiments showed that silencing MOSPD2 in different breast cancer cell lines significantly inhibited cancer cell chemotaxis migration. Mechanistically, we found that silencing MOSPD2 profoundly abated phosphorylation events that are involved in breast tumor cell chemotaxis. In vivo, MOSPD2-silenced breast cancer cells exhibited marked impaired metastasis to the lungs. These results indicate that MOSPD2 plays a key role in the migration and metastasis of breast cancer cells and may be used to prevent the spreading of breast cancer cells and to mediate their death.     

CIP4 promotes metastasis in triple-negative breast cancer and is associated with poor patient prognosis

Signaling via epidermal growth factor receptor (EGFR) and Src kinase pathways promote triple-negative breast cancer (TNBC) cell invasion and tumor metastasis. Here, we address the role of Cdc42-interacting protein-4 (CIP4) in TNBC metastasis in vivo, and profile CIP4 expression in human breast cancer patients. In human TNBC cells, CIP4 knock-down (KD) led to less sustained activation of Erk kinase and impaired cell motility compared to control cells. This correlated with significant defects in 3D invasion of surrounding extracellular matrix by CIP4 KD TNBC cells when grown as spheroid colonies. In mammary orthotopic xenograft assays using both human TNBC cells (MDA-MB-231, HCC 1806) and rat MTLn3 cells, CIP4 silencing had no overt effect on tumor growth, but significantly reduced the incidence of lung metastases in each tumor model. In human invasive breast cancers, high CIP4 levels was significantly associated with high tumor stage, TNBC and HER2 subtypes, and risk of progression to metastatic disease. Together, these results implicate CIP4 in promoting metastasis in TNBCs.     

Deficiency in surface expression of E-selectin ligand promotes lung colonization in a mouse model of breast cancer

Expression of sialyl Lewis(x) (sLe(x)) and sLe(a) on tumor cells is thought to facilitate metastasis by promoting cell adhesion to selectins on vascular endothelial cells. Experiments supporting this concept usually bypass the early steps of the metastatic process by employing tumor cells that are injected directly into the blood. We investigated the relative role of sLe(x) oligosaccharide in the dissemination of breast carcinoma, employing a spontaneous murine metastasis model. An sLe(x) deficient subpopulation of the 4T1 mammary carcinoma cell line was produced by negative selection using the sLe(x)-reactive KM93 MAb. This subpopulation was negative for E-selectin binding but retained P-selectin binding. Both sLe(x)-negative and -positive cells grew at the same rate; however, sLe(x)-negative cells spread more efficiently on plates and had greater motility in wound-scratch assays. Mice inoculated in the mammary fat pad with sLe(x)-negative and -positive variants produced lung metastases. However, the number of lung metastases was significantly increased in the group inoculated with the sLe(x)-negative variant (p = 0.0031), indicating that negative selection for the sLe(x) epitope resulted in enrichment for a subpopulation of cells with a high metastatic phenotype. Cell variants demonstrated significant differences in cellular morphology and pattern of tumor growth in primary and secondary tumor sites. These results strongly suggest that loss of sLe(x) may facilitate the metastatic process by contributing to escape from the primary tumor mass.     

The Role of chemokine receptor CXCR4 in breast cancer metastasis

Breast cancer is one of the leading causes of cancer related deaths worldwide. Breast cancer-related mortality is associated with the development of metastatic potential of primary tumor lesions. The chemokine receptor CXCR4 has been found to be a prognostic marker in various types of cancer, including breast cancer. Recent advances in the field of cancer biology has pointed to the critical role that CXCR4 receptor and its ligand CXCL12 play in the metastasis of various types of cancer, including breast cancer. Breast tumors preferentially metastasize to the lung, bones and lymph nodes, all of which represent organs that secrete high levels of CXCL12. CXCL12 acts as a chemoattractant that drives CXCR4-positive primary tumor cells towards secondary metastatic sites leading to the onset of metastatic lesions. Since its discovery in 2001, the CXCR4 field has progressed at a very fast rate and further studies have pointed to the role of CXCR4 in dissemination of tumor cells from primary sites, transendothelial migration of tumor cells as well as the trafficking and homing of cancer stem cells. This review summarizes the information that has been obtained over the years regarding the role of CXCL12-CXCR4 signaling in breast cancer, discusses its potential application to the development of new therapeutic tools for breast cancer control, and elucidates the potential therapeutic challenges which lie ahead and the future directions that this field can take for the improvement of prognosis in breast cancer patients.     

Ipriflavone inhibits osteolytic bone metastasis of human breast cancer cells in a nude mouse model

Osteolytic bone metastasis is a frequent problem in the treatment of cancer. Ipriflavone, a synthetic isoflavone that inhibits osteoclastic bone resorption, has been used for the treatment of osteoporosis in some countries. Some other isoflavones also exhibit an antitumor effect in vitro and in vivo. Here, we studied the effects of ipriflavone on osteolytic bone metastasis of MDA-231 human breast cancer cells injected intracardially into athymic nude mice (ICR-nu/nu). Daily oral administration of ipriflavone at 12 mg/mouse significantly inhibited the development of new osteolytic bone metastases (p < 0.05) and the progression of established osteolytic lesions (p = 0.01), prolonging the life of tumor-bearing mice (p = 0.01 vs. control). In addition, ipriflavone reduced the number of osteoclasts at the bone-cancer interface with no severe adverse effects on the host. In vitro, ipriflavone inhibited the proliferation and DNA synthesis of MDA-231 cells and blocked the ligand-induced phosphorylation of Tyr(845) of the EGFR. Ipriflavone did not promote apoptosis of MDA-231 cells. Our results show that ipriflavone not only directly inhibits the growth of cancer cells but also reduces osteoclasts to prevent the soft tissue tumor burden and osteolytic bone metastases. These findings raise the possibility that ipriflavone may be of use as a therapeutic agent against osteolytic bone metastasis.     

胫骨内接种建立乳腺癌骨转移大鼠模型的特点

目的研究胫骨内接种MRMT-1细胞制作的乳腺癌骨转移大鼠模型在行为学、影像学、核医学、病理学和分子生物学等方面的特点。方法使用雌性SD大鼠,随机分为假手术组和模型组,使用胫骨内注射法制成乳腺癌骨转移模型。造模后第19天时进行疼痛测定;第21天取材,测定肿瘤体积,通过影像技术评估骨质缺损程度,核医学测定骨矿物质含量(BMC)和骨密度(BMD),HE染色观察形态,抗酒石酸酸性磷酸酶(TRAP)染色并计数破骨细胞,免疫组化法测定增殖细胞核抗原(PCNA)、护骨素(OPG)和核因子kB受体活化因子配体(RANKL),荧光实时定量RT-PCR测定甲状旁腺激素相关蛋白(PTHrP)。结果模型组在造模后第19天已出现机械痛觉超敏、机械痛觉过敏和热痛觉过敏(P0.01)。第21天取材后胫骨影像评分升高(P0.01),BMD下降(P0.05);肉眼观察肿瘤生长明显(P0.01),镜下可见溶骨病变为主的混合性骨质破坏;破骨细胞数量和活性增加(P0.01),PTHrP、OPG水平与OPG/RANKL比值均下降(P0.05、P0.01),而RANKL无明显变化。结论乳腺癌骨转移大鼠模型具有疼痛和骨质破坏的表现,但未表现出PTHrP和RANKL升高,其损伤途径是通过抑制OPG破坏了OPG-RANKL-RANK系统的平衡,引起破骨细胞过度激活,造成骨吸收作用亢进。     

TGFBI modulates tumour hypoxia and promotes breast cancer metastasis

Breast cancer metastasis is a complex process that depends not only on intrinsic characteristics of metastatic stem cells, but also on the particular microenvironment that supports their growth and modulates the plasticity of the system. In search for microenvironmental factors supporting cancer stem cell (CSC) growth and tumour progression to metastasis, we here investigated the role of the matricellular protein transforming growth factor beta induced (TGFBI) in breast cancer. We crossed the MMTV‐PyMT model of mammary gland tumorigenesis with a Tgfbi ^Δ/Δ mouse and studied the CSC content of the tumours. We performed RNAseq on wt and ko tumours, and analysed the tumour vasculature and the immune compartment by IHC and FACS. The source of TGFBI expression was determined by qPCR and by bone marrow transplantation experiments. Finally, we performed in silico analyses using the METABRIC cohort to assess the potential prognostic value of TGFBI. We observed that deletion of Tgfbi led to a dramatic decrease in CSC content and lung metastasis. Our results show that lack of TGFBI resulted in tumour vessel normalisation, with improved vessel perfusion and decreased hypoxia, a major factor controlling CSCs and metastasis. Furthermore, human data mining in a cohort of breast cancer patients showed that higher expression of TGFBI correlates with poor prognosis and is associated with the more aggressive subtypes of breast cancer. Overall, these data reveal a novel biological mechanism controlling metastasis that could potentially be exploited to improve the efficacy and delivery of chemotherapeutic agents in breast cancer.

Abbreviations

CSC

cancer stem cell

ECM

extracellular matrix

EMT

epithelial‐to‐mesenchymal transition

FACS

fluorescence‐activated cell sorting

TGFBI

transforming growth factor beta induced

     

Effect of zoledronic acid on the doxycycline-induced decrease in tumour burden in a bone metastasis model of human breast cancer

Bone is one of the most frequent sites for metastasis in breast cancer patients often resulting in significant clinical morbidity and mortality. Bisphosphonates are currently the standard of care for breast cancer patients with bone metastasis. We have shown previously that doxycycline, a member of the tetracycline family of antibiotics, reduces total tumour burden in an experimental bone metastasis mouse model of human breast cancer. In this study, we combined doxycycline treatment together with zoledronic acid, the most potent bisphosphonate. Drug administration started 3 days before the injection of the MDA-MB-231 cells. When mice were administered zoledronic acid alone, the total tumour burden decreased by 43% compared to placebo treatment. Administration of a combination of zoledronic acid and doxycycline resulted in a 74% decrease in total tumour burden compared to untreated mice. In doxycycline- and zoledronate-treated mice bone formation was significantly enhanced as determined by increased numbers of osteoblasts, osteoid surface and volume, whereas a decrease in bone resorption was also observed. Doxycycline greatly reduced tumour burden and could also compensate for the increased bone resorption. The addition of zoledronate to the regimen further decreased tumour burden, caused an extensive decrease in bone-associated soft tissue tumour burden (93%), and sustained the bone volume, which could result in a smaller fracture risk. Treatment with zoledronic acid in combination with doxycycline may be very beneficial for breast cancer patients at risk for osteolytic bone metastasis.     

乳腺癌骨转移机制研究进展

乳腺癌是一种容易发生骨转移的女性常见恶性肿瘤。乳腺癌细胞的特异性、骨微环境及两者间相互作用是形成骨转移的共同因素。乳腺癌细胞表达的趋化因子受体、整合素、溶骨因子和成骨因子等使肿瘤细胞易于扩散到骨,而骨微环境可以为肿瘤细胞的生长提供丰富的生长因子和细胞因子。一旦乳腺癌细胞侵入骨质,肿瘤细胞分泌的因子就会作用于骨的外在结构和内在结构（如造血干细胞、T细胞、血小板、内皮细胞等）,使骨质破坏且分泌相关因子反作用于癌细胞,从而引起转移的级联反应和恶性循环形成。     

CXCR4 peptide antagonist inhibits primary breast tumor growth,metastasis and enhances the efficacy of anti‐VEGF treatment or docetaxel in a transgenic mouse model

CXCR4 is a chemokine receptor implicated in the homing of cancer cells to target metastatic organs, which overexpress its ligand, stromal cell‐derived factor (SDF)‐1. To determine the efficacy of targeting CXCR4 on primary tumor growth and metastasis, we used a peptide inhibitor of CXCR4, CTCE‐9908, that was administered in a clinically relevant approach using a transgenic breast cancer mouse model. We first performed a dosing experiment of CTCE‐9908 in the PyMT mouse model, testing 25, 50 and 100 mg/kg versus the scrambled peptide in groups of 8–16 mice. We then combined CTCE‐9908 with docetaxel or DC101 (an anti‐VEGFR2 monoclonal antibody). We found that increasing doses of CTCE‐9908 alone slowed the rate of tumor growth, with a 45% inhibition of primary tumor growth at 3.5 weeks of treatment with 50 mg/kg of CTCE‐9908 (p = 0.005). Expression levels of VEGF were also found to be reduced by 42% with CTCE‐9908 (p = 0.01). In combination with docetaxel, CTCE‐9908 administration decreased tumor volume by 38% (p = 0.02), an effect that was greater than that observed with docetaxel alone. In combination with DC101, CTCE‐9908 also demonstrated an enhanced effect compared to DC101 alone, with a 37% decrease in primary tumor volume (p = 0.01) and a 75% reduction in distant metastasis (p = 0.009). In combination with docetaxel or an anti‐angiogenic agent, the anti‐tumor and anti‐metastatic effects of CTCE‐9908 were markedly enhanced, suggesting potentially new effective combinatorial therapeutic strategies in the treatment of breast cancer, which include targeting the SDF‐1/CXCR4 ligand/receptor pair.     

FGF13 promotes metastasis of triple-negative breast cancer

Triple-negative breast cancer (TNBC) represents 10–20% of all human ductal adenocarcinomas and has a poor prognosis relative to other subtypes, due to the high propensity to develop distant metastases. Hence, new molecular targets for therapeutic intervention are needed for TNBC. We recently conducted a rigorous phenotypic and genomic characterization of four isogenic populations of MDA-MB-231 human triple-negative breast cancer cells that possess a range of intrinsic spontaneous metastatic capacities in vivo, ranging from nonmetastatic (MDA-MB-231_ATCC) to highly metastatic to lung, liver, spleen and spine (MDA-MB-231_HM). Gene expression profiling of primary tumours by RNA-Seq identified the fibroblast growth factor homologous factor, FGF13, as highly upregulated in aggressively metastatic MDA-MB-231_HM tumours. Clinically, higher FGF13 mRNA expression was associated with significantly worse relapse free survival in both luminal A and basal-like human breast cancers but was not associated with other clinical variables and was not upregulated in primary tumours relative to normal mammary gland. Stable FGF13 depletion restricted in vitro colony forming ability in MDA-MB-231_HM TNBC cells but not in oestrogen receptor (ER)-positive MCF-7 or MDA-MB-361 cells. However, despite augmenting MDA-MB-231_HM cell migration and invasion in vitro, FGF13 suppression almost completely blocked the spontaneous metastasis of MDA-MB-231_HM orthotopic xenografts to both lung and liver while having negligible impact on primary tumour growth. Together, these data indicate that FGF13 may represent a therapeutic target for blocking metastatic outgrowth of certain TNBCs. Further evaluation of the roles of individual FGF13 protein isoforms in progression of the different subtypes of breast cancer is warranted.     

乳腺癌骨转移分子机制的研究进展

乳腺癌是女性最易患的恶性肿瘤,且发病率呈逐年上升趋势.大约有70%的患者在晚期发生骨转移,乳腺癌骨转移不仅可导致患者遭受贫血、骨折、截瘫、高血钙、疼痛和恶病质等痛苦,也是导致死亡率上升的重要原因.乳腺癌骨转移可分为若干个步骤,此篇综述介绍癌细胞的骨定向迁移及乳腺癌细胞和骨微环境的交互作用中涉及的重要因子.     

Modulatory effects of heparin and short-length oligosaccharides of heparin on the metastasis and growth of LMD MDA-MB 231 breast cancer cells in vivo

Expression of the chemokine receptor CXCR4 allows breast cancer cells to migrate towards specific metastatic target sites which constitutively express CXCL12. In this study, we determined whether this interaction could be disrupted using short-chain length heparin oligosaccharides. Radioligand competition binding assays were performed using a range of heparin oligosaccharides to compete with polymeric heparin or heparan sulphate binding to I(125) CXCL12. Heparin dodecasaccharides were found to be the minimal chain length required to efficiently bind CXCL12 (71% inhibition; P<0.001). These oligosaccharides also significantly inhibited CXCL12-induced migration of CXCR4-expressing LMD MDA-MB 231 breast cancer cells. In addition, heparin dodecasaccharides were found to have less anticoagulant activity than either a smaller quantity of polymeric heparin or a similar amount of the low molecular weight heparin pharmaceutical product, Tinzaparin. When given subcutaneously in a SCID mouse model of human breast cancer, heparin dodecasaccharides had no effect on the number of lung metastases, but did however inhibit (P<0.05) tumour growth (lesion area) compared to control groups. In contrast, polymeric heparin significantly inhibited both the number (P<0.001) and area of metastases, suggesting a differing mechanism for the action of polymeric and heparin-derived oligosaccharides in the inhibition of tumour growth and metastases.     

HER-2/neu genotype of breast cancer may change in bone metastasis

The genotype of breast cancer (BRC) is considered to be relatively stable during tumor progression, accordingly, determination of the estrogen receptor and HER-2/neu status is currently based on the primary tumor. However, recent data suggest that the gene expression profile of the metastatic lesion can be different compared to that of the primary BRC. Accordingly, it is possible that the HER-2/neu status is different in the metastatic lesion and the primary BRC. Since the bone is the most frequent metastatic site during the progression of BRC, we have analyzed the HER-2/neu status of 48 bone metastatic BRC cases by immunohistochemistry and fluorescent in situ hybridization, and it was possible to compare it to the primary site in 23 cases. The frequency of HER-2/neu amplification of BRC in the primary tumors was found to be 17.4% compared to 10.5% in bone metastases. Half of BRC cases with HER-2/neu amplification lost this genotype in bone metastases (4/23 versus 2/23, respectively) and even in the 2 cases where HER-2/neu amplification was retained in the metastases, the copy number was found to be decreased compared to the primary tumor. Based on our data and previous reports in the literature, we suggest to perform HER-2/neu testing both on primary tumor and samples obtained from BRC metastases, at least in case of primary tumors with HER-2/neu amplification, before introduction of HER-2/neu-targeting therapy. (Pathology Oncology Research Vol 12, No 3, 149–152)     

Pre-osteoblastic MC3T3-E1 cells promote breast cancer growth in bone in a murine xenograft model

The bones are the most common sites of breast cancer metastasis. Upon arrival within the bone microenvironment, breast cancer cells coordinate the activities of stromal cells, resulting in an increase in osteoclast activity and bone matrix degradation. In late stages of bone metastasis, breast cancer cells induce apoptosis in osteoblasts, which further exacerbates bone loss. However, in early stages, breast cancer cells induce osteoblasts to secrete inflammatory cytokines purported to drive tumor progressio...