Breast cancer osteomimicry and its role in bone specific metastasis; an integrative,systematic review of preclinical evidence

Metastasis accounts for most of the deaths from breast cancer and the preference of invasive breast cancer metastasising to bone has been widely reported. However, the biological basis of breast cancer osteotropism is not fully understood. This paper provides, for the first time, an integrative, systematic review of evidence of molecular factors that have functional roles in the homing of metastatic breast cancer to the bone.Pubmed, Web of Science and EBSCOhost were searched using keywords and synonyms for molecular, metastasis, breast cancer and bone to identify articles published between January 2004 and August 2016. 4491 potentially relevant citations were retrieved. 63 articles met the inclusion criteria, which were primary studies reporting evidence of molecular factors that have functional roles in predisposing breast cancer bone metastasis in vivo. 12 of those 63 articles that additionally met quality criteria were included in the review. Extracted data were tabulated and key findings that indicated biological mechanisms involved in breast cancer metastasis to bone were synthesised.15 proteins expressed by breast cancer cells were identified as factors that mediate breast cancer bone metastasis: ICAM-1, cadherin-11, osteoactivin, bone sialoprotein, CCN3, IL-11, CCL2, CITED2, CXCR4, CTGF, OPN, CX₃CR1, TWIST1, adrenomedullin and Enpp1. Upregulation or overexpression of one or more of them by breast cancer cells resulted in increased breast cancer metastasis to bone in vivo, except for CCL2 where bone-metastatic cells showed a reduced expression of this factor. All factors identified, here expressed by breast cancer cells, are proteins that are normally expressed in the bone microenvironment and linked to physiologic bone functions. All have a functional role in one of more of the following: cell proliferation and differentiation, bone mineralization and remodelling, cell adhesion and/or chemokine signalling. Six of them (cadherin-11, ICAM-1, OPN, CX₃CR1, CCN3 and osteoactivin) have a reported function in cell adhesion and another eight (CCN3, osteoactivin, Enpp1, IL-11, CTGF, TWIST1, adrenomedullin and CITED2) are reported to be involved in cell proliferation and differentiation.This review collates and synthesises published evidence to increase our understanding of the biology of breast cancer osteomimicry in the development of bone metastasis. Findings of this review suggest that changes in expression of proteins in breast cancer cells that confer osteomimicry facilitate homing to bone to enable the development of bone metastasis.     

Tumor-Stromal Interactions in Bone Metastasis

The metastasis of tumor cells to distant organs is the primary cause of cancer-related mortality in most cancers. The interaction of tumor cells with local stroma at the metastatic site plays a critical role in metastatic dissemination and the establishment of metastases. These tumor-stromal interactions regulate several important steps including degradation of extracellular matrix, release of sequestered growth factors, and expression of chemokines, cytokines, and receptors on tumor cells and the interacting stromal cells. Breast, prostate, and lung cancers preferentially metastasize to bone. Tumor cell interactions with the bone microenvironment initiate a series of complex cellular interactions that promotes establishment of osteoclastic and/or osteoblastic metastasis. Understanding the interactions between tumor cells and the stroma is important to identify molecular targets to develop novel therapies aimed at reducing metastasis formation. In this article, we review the important mechanisms of tumor-stromal interaction in the development of bone metastasis.     

Loss of the Vitamin D Receptor in Human Breast Cancer Cells Promotes Epithelial to Mesenchymal Cell Transition and Skeletal Colonization

Expression of the vitamin D receptor (VDR) is thought to be associated with neoplastic progression. However, the role of the VDR in breast cancer metastasis to bone and the molecular mechanisms underlying this process are unknown. Employing a rodent model (female Balb/c nu/nu mice) of systemic metastasis, we here demonstrate that knockdown of the VDR strongly increases the metastatic potential of MDA-MB-231 human breast cancer cells to bone, resulting in significantly greater skeletal tumor burden. Ablation of VDR expression promotes cancer cell mobility (migration) and invasiveness, thereby facilitating skeletal colonization. Mechanistically, these changes in tumor cell behavior are attributable to shifts in the expression of proteins involved in cell adhesion, proliferation, and cytoskeletal organization, patterns characteristic for epithelial-to-mesenchymal cell transition (EMT). In keeping with these experimental findings, analyses of human breast cancer specimens corroborated the association between VDR expression, EMT-typical changes in protein expression patterns, and clinical prognosis. Loss of the VDR in human breast cancer cells marks a critical point in oncogenesis by inducing EMT, promoting the dissemination of cancer cells, and facilitating the formation of tumor colonies in bone. © 2019 American Society for Bone and Mineral Research.     

骨桥蛋白在不同转移潜能肝癌细胞系及肝癌组织中表达意义

目的 研究不同转移潜能人肝癌细胞系及其肝癌组织中骨桥蛋白(OPN)表达情况,分析OPN表达与肝癌细胞侵袭转移潜能、肝癌术后复发转移的关系.方法 应用细胞免疫组化、半定量RT-PCR、Western Blot和ELISA检测不同转移潜能人肝癌细胞系SMMC-7721、MHCC97-L和HCCLM6 OPN表达情况.应用组织芯片、免疫组化检测200例肝癌组织中OPN表达.结果 OPN表达水平随着肝癌细胞侵袭转移潜能增加逐渐升高.肝癌组织中OPN表达与病人性别、年龄、血清AFP水平、肿瘤大小、有无包膜、细胞分化以及肝门淋巴结转移无明显相关性.而与肝癌TNM分期、门静脉癌栓和术后复发转移发生有明显相关(P<0.05).术后有复发转移病人的肝癌组织中66%表达OPN,而术后无复发转移病人的肝癌组织中仅37%表达OPN,两者比较差异具有显著性(P<0.05).结论 OPN表达与肝癌细胞细胞系侵袭转移潜能密切相关.肝癌组织中OPN表达可能是预测肝癌术后是否发生复发转移指标.     

Multifaceted Roles of Integrins in Breast Cancer Metastasis

Malignant breast cancer can be a debilitating disease due to metastasis to tissues such as brain or bone. The metastatic process involves the invasion of tumor cells into the adjacent tissue, followed by systemic dissemination and colonization of secondary organs. These processes require interactions between tumor cells and a changing microenvironment, which drive cell proliferation, migration, invasion and colonization, as well as promoting cell survival. The integrin family of cell adhesion receptors has been shown to play a critical role in all of these processes, consistent with their extracellular matrix binding properties. Experiments in cultured epithelial cells and in vivo models have demonstrated that integrins can promote various stages of metastasis by modulating the effects of growth factor receptors, extracellular proteases and chemotactic molecules. Integrins may therefore play a pivotal role in multiple mechanisms of metastasis. As a result, they represent promising targets for effective treatment of metastatic breast cancer.     

Kallikrein 4 is a potential mediator of cellular interactions between cancer cells and osteoblasts in metastatic prostate cancer

BACKGROUND: Prostate cancer (PCa) and bone cell interactions are critical in the metastatic phase. Kallikrein 4 (KLK4/hK4) is expressed in both PCa and mineralized tissues. We determined if KLK4/hK4 expression was associated with, and influenced by, the bone environment of metastatic PCa. METHODS: Immunohistochemistry, in vitro co-culture, cell migration, and attachment assays. RESULTS: hK4 was localized to tumor cells and osteoblasts in bone metastases. KLK4/hK4 increased in LNCaP and PC3 cells co-cultured with SaOs2 cells; SaOs2 KLK4/hK4 was unchanged. Co-culture did not affect cell proliferation but altered alkaline phosphatase activity/mRNA levels in SaOs2 cells. KLK4-transfected PC3 cells had increased migration towards SaOs2 conditioned medium and greater attachment to the bone-matrix proteins, collagens I and IV. CONCLUSIONS: hK4 expression and interaction with both tumor cells and osteoblasts suggests a role for hK4 in PCa bone metastasis. Whether this observation is unique to bone metastasis or reflects a role for hK4 in PCa metastasis generally is yet to be established.     

Molecular insights into prostate cancer progression: the missing link of tumor microenvironment

PURPOSE: Tumor cell genotype and phenotype have been considered the only determinants supporting cancer growth and metastasis. This review focuses on the published literature that suggests that tumor-microenvironment interaction has a decisive role in controlling local cancer growth, invasion and distant metastasis. As this review shows, genetic alterations in prostate cancer cells alone are not enough to confer metastatic status without a supporting tumor microenvironment. Effective therapeutic targeting requires a deeper understanding of the interplay between tumor and stroma. Approaches co-targeting tumor and stroma already show promise over the conventional targeting of tumor cells alone in preventing prostate cancer progression and eradicating preexisting or newly developed prostate cancers in bone and visceral organs. MATERIALS AND METHODS: A literature survey using the MEDLINE database was performed in basic and clinical publications relevant to tumor-host microenvironment interaction. Information pertinent to the biology and therapy of prostate cancer local growth and distant metastases was specifically emphasized. RESULTS: Tumor associated stroma actively fuel the progression of prostate cancer from localized growth to the invasion of surrounding tissues, and the development of distant bone and visceral organ metastasis. In concert with this progression tumor cells recovered from metastatic sites could represent a subpopulation of preexisting tumor cells or could be a newly acquired variant subsequent to tumor-stromal interaction. Experimental data from our laboratory and others suggest that permanent genetic and phenotypic changes occur in prostate cancer cells after 3-dimensional co-culture in vitro or when co-inoculated and grown with inductive stromal cells in vivo. These results support the idea that newly acquired variants are the dominant mechanism of prostate cancer progression. Intercellular communication between prostate cancer cells and organ specific stroma, including prostate and marrow stroma, could involve diffusible soluble and solid matrix molecules as mediators, leading to the development of metastasis. This presents a new opportunity for therapeutic targeting for the treatment of benign and malignant growth of the prostate glands. This review summarizes specific research implicating tumor-microenvironment interaction as the molecular basis of cancer progression, providing a rationale for targeting tumor and the tumor associated microenvironment in the management of androgen independent and bone metastatic prostate cancer progression in patients. CONCLUSIONS: Cancer is not a single cell disease. Aberrant cancer cells and their interactive microenvironment are needed for prostate cancer to progress to androgen independence and distant metastasis. It is highly plausible that newly evolved prostate cancer cell clones dominate cancer metastasis after cell-cell and cell-matrix interaction with the host microenvironment, rather than the selection or expansion of a preexisting prostate cancer cell clone(s). Based on this premise potential molecular targets in the microenvironment are especially emphasized. Further elucidation of the molecular mechanisms underlying tumor-stromal interaction may yield improved medical treatments for prostate cancer growth and metastasis.     

肿瘤干细胞表型CD44和CD24在乳腺癌骨转移模型骨髓中的表达及意义

目的 探讨人乳腺癌细胞转移到人骨的乳腺癌骨转移小鼠模型中骨髓肿瘤干细胞表型CD44和CD24的表达及其意义.方法 50只SCID小鼠随机分成实验组和对照组,其中实验组鼠背部植入人骨后随机均分3亚组:A组(MDA-MB-231干细胞亚群1×105个/只)、B组(同A组细胞1×106个/只)和C组(MDA-MB-231亲代细胞1×106个/只);对照组设为D组(阳性对照,未植入人骨,同C组细胞直接注射)、E组(阴性对照,植入人骨,生理盐水注射).各组8周后取人骨、鼠骨等行常规HE染色及CK、CD44、CD24、CXCR4、OPN免疫组织化学标记.Real-time PCR检测CD44、CXCR4、OPN的mRNA水平.结果 B组骨转移率最高(77.8%,P＜0.05).B组中人骨转移灶CD44、CXCR4、OPN抗原表达高于C、D组骨中的表达(均有P＜0.05);CD24抗原则在A、B组人骨转移灶中低表达与C、D组骨中的高表达无统计学意义(P＞0.05).B组CD44mRNA表达水平是C组的15.2倍、D组的21.1倍;B组CXCR4mRNA表达水平是C组8.4倍、D组28.4倍;B组OPN-mRNA表达水平是C组4.8倍、D组11.6倍;而B组CD24的mRNA表达显著低于C、D组(均为30%).结论 利用MDA-MB-231肿瘤干细胞亚群(CD44+/CD24-)可制备高转移率的"人源性"乳腺癌骨转移模型,其机制可能与CD44高表达有关.骨转移相关基因CXCR4、OPN转录上调可能参与其过程.     

Expression and immunogenicity of oncofetal antigen-immature laminin receptor in human renal cell carcinoma

PURPOSE: The 32 to 44 kDa. oncofetal antigen-immature laminin receptor (OFA-iLR) is a multifunctional protein expressed by various tumors, including breast, lung, ovary and prostate carcinoma as well as lymphoma. OFA-iLR has been implicated in tumor invasiveness, metastasis and growth. Interferon-gamma producing effector T cells and interleukin (IL)-10 producing suppressor T cells specific for OFA-iLR have been described. MATERIALS AND METHODS: The 43515 IgG2a anti-OFA-iLR monoclonal antibody was used to detect OFA-iLR expression in human renal cell carcinoma tissue by flow cytometry and immunoblotting. Spontaneous or therapy induced immune responses against OFA-iLR were determined in patients with metastatic renal cell carcinoma. Proliferative and cytokine (interferon-gamma and IL-10) responses of peripheral blood mononuclear cells from patients with renal cell carcinoma against recombinant OFA-iLR were assessed. RESULTS: Using flow cytometry OFA-iLR was detected in all 13 tumors tested. Immunoblotting revealed differences in OFA-iLR expression in renal cell carcinoma and normal kidney tissue. OFA-iLR specific proliferative and cytokine responses of mononuclear cells were detected in all 6 patients tested. Importantly evidence was also obtained that treating metastatic renal cell carcinoma with tumor lysate pulsed dendritic cells would enhance OFA-iLR specific immunity. CONCLUSIONS: This study demonstrates that OFA-iLR is an immunogenic tumor associated antigen in human renal cell carcinoma. OFA-iLR specific effector T cells producing interferon-gamma may have a role in the control of tumor growth, whereas suppressor T cells producing IL-10 may promote tumor tolerance and, thus, tumor progression.     

Osteopontin is required for unloading-induced osteoclast recruitment and modulation of RANKL expression during tooth drift-associated bone remodeling,but not for super-eruption

Unloading of teeth results in extensive alveolar bone remodeling, causing teeth to move in both vertical (“super-eruption”) and horizontal direction (“drift”). In order to decipher the molecular mechanisms of unloading-induced bone remodeling during tooth movement, we focused on the role of osteopontin (OPN) in the un-opposed molar model, comparing wild-type (WT) and OPN-null mice. Our data indicated that OPN was not required for the continuous eruption of un-opposed teeth while OPN was necessary for the drift of teeth. OPN expression and osteoclast counts were greatly increased on alveolar bone surfaces facing the direction of the drift in WT mice, while osteoclast counts were diminished in OPN?/? mice. RANKL expression in the distal periodontal ligament of WT molars increased significantly by day 6 following unloading, while overall levels of RANKL expression were decreased in both WT and OPN-null mice. In vitro treatment of MC3T3 cells, WT BMCs and OPN?/? BMCs with recombinant OPN resulted in significantly increased RANKL expression in all three cell types. The PI3K and MEK/ERK pathway inhibitors Ly294002 and U0126 reduced RANKL expression levels in vitro. Treatment of BMCs and MC3T3 with OPN also resulted in increased ERK phosphorylation and reduced OPG levels. Together, our studies suggest that increased OPN expression during unloading-induced drifting of teeth enhances localized RANKL expression and osteoclast activity on drift-direction alveolar bone surfaces via extracellular matrix signaling pathways.     

The prostate cancer bone marrow niche: more than just ‘fertile soil'

The hematopoietic stem cell (HSC) niche in the bone marrow has been studied extensively over the past few decades, yet the bone marrow microenvironment that supports the growth of metastatic prostate cancer (PCa) has only been recently considered to be a specialized ‘niche'' as well. New evidence supports the fact that disseminated tumor cells (DTCs) of PCa actually target the HSC niche, displace the occupant HSCs and take up residence in the pre-existing niche space. This review describes some of the evidence and mechanisms by which DTCs act as molecular parasites of the HSC niche. Furthermore, the interactions between DTCs, HSCs and the niche may provide new targets for niche-directed therapy, as well as insight into the perplexing clinical manifestations of metastatic PCa disease.     

骨桥蛋白和MMP-10在膀胱尿路上皮癌中的表达及临床意义

目的检测膀胱尿路上皮癌中骨桥蛋白（OPN）和金属基质蛋白酶10（MMP-10）的表达,探索二者与膀胱癌的病理分级和临床分期及临床预后的关系,比较二者之间的相关性。方法应用免疫组化方法检测60例膀胱移行细胞癌和20例正常膀胱壁标本中的OPN和MMP-10的表达,并分析二者表达与肿瘤分期、分级、复发、淋巴结转移之间的关系。结果20例正常膀胱组织中,17例（85.0%）OPN表达为阴性,3例（15.0%）为弱阳性,而MMP-10未见表达;OPN在膀胱癌组织中的阳性率为73.3％（44/60）,MMP-10在膀胱癌组织中的阳性表达率为63.3％（38/60）,OPN和MMP-10在膀胱尿路上皮癌组织和正常膀胱组织表达中有明显差异（P＜0.05）。膀胱尿路上皮癌组织中,OPN及MMP-10的表达与组织病理学分级、临床分期、复发及转移呈正相关,膀胱尿路上皮癌组织中,二者之间的表达亦呈正相关关系。结论OPN及MMP-10表达强度同肿瘤分级、分期、复发、转移有关,可能作为临床评估膀胱尿路上皮癌进展及肿瘤预后的指标及在膀胱癌的进展中起协同作用。     

食管鳞癌G3BP和骨桥蛋白的表达及其对患者预后的影响

目的研究食管鳞癌组织中GTP酶激活蛋白SH3功能区结合蛋白(G3BP)和骨桥(OPN)蛋白的表达及其与食管鳞癌生物学行为之间的关系。方法采用免疫组织化学方法检测80例食管鳞癌组织中G3BP和OPN蛋白的表达情况,探讨它们与食管鳞癌的肿瘤大小、组织分化程度、TNM分期、淋巴结转移和预后的关系。结果(1)G3BP蛋白在食管鳞癌组织中的阳性表达率为71.3%,它在淋巴结转移组的阳性表达率高于无淋巴结转移组(Z=-2.283,P=0.022);而与肿瘤大小、组织分化程度、TNM分期无关(P>0.05);G3BP蛋白阳性表达组患者的术后生存时间较阴性表达组短,差异有统计学意义(P=0.000)。(2)OPN蛋白在食管鳞癌组织中的阳性表达率为100%,OPN蛋白的表达水平与组织分化程度(X~2=10.766,P=0.005)及淋巴结转移(Z=-2.289,P=0.022)有关,而与肿瘤大小和TNM分期无关(P>0.05);OPN蛋白的表达水平越高,患者术后生存时间越短(P=0.000)。(3)G3BP蛋白和OPN蛋白在食管鳞癌组织中的表达之间存在正相关性(r(?)=0.376,P=0.001)。结论食管鳞癌组织G3BP和OPN蛋白的表达与淋巴结转移情况及患者的预后有密切关系。     

骨桥蛋白在大肠癌和大肠癌肝转移灶中的表达及其临床意义

目的：检测骨桥蛋白（osteopontin OPN)mRNA在大肠癌及大肠癌肝转移灶中的表达,并探讨其在大肠癌发生、发展及肝转移中的临床意义。方法：提取44例大肠癌及癌旁正常组织,20例大肠癌肝转移灶及3例正常肝组织RNA,采用RT－PCR半定量法检测其OPN mRNA的表达。并用原位杂交行组织定位检测。结果：44例大肠癌组织OPN表达高于癌旁正常组织（t=4.89,P=0.000)。44例大肠癌组织OPN mRNA表达率为18．2％,而在20例肝转移灶中表达率达80．0％（χ^2=22.42,P=0.000）。原位杂交表明,OPN mRNA定位于大肠癌细胞浆。结论：OPN mRNA在大肠癌组织中表达高于正常组织,而在大肠癌肝转移灶中呈更高表达,提示OPN可能是大肠癌肝转移的预测指标之一。     

Smad4在人前列腺癌细胞系LNCaP和ARCaP中的差异表达及意义

目的:探讨TGF-β/Smads信号转导的核心Smad4在前列腺癌转移潜能获得中的作用及可能的机制。方法:用Millicell体外侵袭试验观察LNCaP和ARCaP系IF-11、IA-8细胞株转移潜能的差异情况,分别应用Western印迹法和逆转录PCR鉴定这3种细胞株中Smad4蛋白及mRNA的差异表达。结果:ARCaP系IF-11、IA-8细胞株的体外侵袭潜能明显高于LNCaP细胞(P<0.01);但Smad4蛋白在低转移潜能的LNCaP细胞中高表达,而在高转移潜能的IF-11、IA-8中表达缺失(P<0.01),Smad4 mRNA表达结果与蛋白一致。结论:Smad4在转移潜能不同的前列腺癌细胞系间存在差异表达,Smad4表达缺失可能是晚期前列腺癌发生侵袭转移的机制之一。     

TGF-beta promotes the establishment of renal cell carcinoma bone metastasis.

Bone metastases develop in approximately 30% of patients with RCC, and the mechanisms responsible for this phenomenon are unknown. We found that TGF-beta1 stimulation of RCC bone metastasis cells promotes tumor growth and bone destruction possibly by stimulating paracrine interactions between tumor cells and the bone. INTRODUCTION: Bone metastasis is a frequent complication and causes marked morbidity in patients with renal cell carcinoma (RCC). Surprisingly, the specific mechanisms of RCC interaction with bone have been scarcely studied despite the inability to prevent or effectively treat bone metastasis. Bone is a reservoir for various growth factors including the pleiotropic cytokine TGF-beta1. TGF-beta1 has been shown to have tumor-supportive effects on advanced cancers and evidence suggests its involvement in promoting the development of breast cancer bone metastasis. Here, we studied the potential role of TGF-beta1 in the growth of RCC bone metastasis (RBM). MATERIALS AND METHODS: To inhibit TGF-beta1 signaling, RBM cells stably expressing a dominant-negative (DN) TGF-betaRII cDNA were generated. The in vivo effect of TGF-beta1 on RBM tumor growth and osteolysis was determined by histological and radiographic analysis, respectively, of athymic nude mice after intratibial injection of parental, empty vector, or DN RBM cells. The in vitro effect of TGF-beta1 on RBM cell growth was determined after TGF-beta1 treatment by MTT assay. RESULTS: TGF-beta1 and the TGF-beta receptors I and II (TGF-betaRI/II) were consistently expressed in both RBM tissues and cell lines. Inhibition of TGF-beta1 signaling in RBM cells significantly reduced tumor establishment and osteolysis observed in vivo after injection into the murine tibia, although no effect on tumor establishment was observed after injection of RBM cells subcutaneously or into the renal subcapsule. Treatment of five RBM cell lines with TGF-beta1 in vitro either had no effect (2/5) or resulted in a significant inhibition (3/5) of cell growth, suggesting that TGF-beta1 may promote RBM tumor growth indirectly in vivo. CONCLUSIONS: TGF-beta1 stimulation of RBM cells plays a role in promoting tumor growth and subsequent osteolysis in vivo, likely through the initiation of tumor-promoting paracrine interactions between tumor cells and the bone microenvironment. These data suggest that inhibition of TGF-beta1 signaling may be useful in the treatment of RBM.