Gabri van der Pluijm

The Tumor Suppressor Smad4 Is Required for Transforming Growth Factor β–Induced Epithelial to Mesenchymal Transition and Bone Metastasis of Breast Cancer Cells

Transforming growth factor beta (TGF-beta) can act as suppressor and promoter of cancer progression. Intracellular Smad proteins (i.e., receptor regulated Smads and common mediator Smad4) play a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-beta, but their function in TGF-beta-induced invasion and metastasis is unclear. Here, we have investigated the role of Smad4 in a cellular and mouse model for TGF-beta-induced breast cancer progression. Consistent with its tumor suppressor function, specific silencing of Smad4 in NMuMG mammary gland epithelial cells using small hairpin RNA (shRNA)-expressing RNAi vectors strongly mitigated TGF-beta-induced growth inhibition and apoptosis. Smad4 knockdown also potently inhibited TGF-beta-induced epithelial to mesenchymal transition of NMuMG cells as measured by morphologic transformation from epithelial to fibroblast-like cells, formation of stress fibers, inhibition of E-cadherin expression, and gain of expression of various mesenchymal markers. Furthermore, we show that knockdown of Smad4 in MDA-MB-231 breast cancer cells strongly inhibited the frequency of bone metastasis in nude mice by 75% and significantly increased metastasis-free survival. Communication of MDA-MB-231 cells with the bone microenvironment, which is needed for optimal tumor cell growth and metastasis, may be affected in Smad4 knockdown cells as TGF-beta-induced expression of interleukin 11 was attenuated on Smad4 knockdown. Taken together, our results show that Smad4 plays an important role in both tumor suppression and progression of breast cancer cells.

High Aldehyde Dehydrogenase Activity Identifies Tumor-Initiating and Metastasis-Initiating Cells in Human Prostate Cancer

Metastatic progression of advanced prostate cancer is a major clinical problem. Identifying the cell(s) of origin in prostate cancer and its distant metastases may permit the development of more effective treatment and preventive therapies. In this study, aldehyde dehydrogenase (ALDH) activity was used as a basis to isolate and compare subpopulations of primary human prostate cancer cells and cell lines. ALDH-high prostate cancer cells displayed strongly elevated clonogenicity and migratory behavior in vitro. More strikingly, ALDH-high cells readily formed distant metastases with strongly enhanced tumor progression at both orthotopic and metastatic sites in preclinical models. Several ALDH isoforms were expressed in human prostate cancer cells and clinical specimens of primary prostate tumors with matched bone metastases. Our findings suggest that ALDH-based viable cell sorting can be used to identify and characterize tumor-initiating and, more importantly perhaps, metastasis-initiating cells in human prostate cancer.

The role of geranylgeranylation in bone resorption and its suppression by bisphosphonates in fetal bone explants in vitro : A clue to the mechanism of action of nitrogen-containing bisphosphonates

Bisphosphonates, synthetic compounds used in the treatment of skeletal disorders, suppress osteoclast‐mediated bone resorption by a yet unidentified mechanism. Previous studies showed that some bisphosphonates can inhibit enzymes of the mevalonate pathway, and nitrogen‐containing bisphosphonates inhibit protein prenylation in mouse macrophages. In the present study, we examined the involvement of the mevalonate pathway in basal and bisphosphonate‐inhibited osteoclastic resorption in fetal mouse long bone explants, an experimental model representative of the in vivo action of bisphosphonates. Mevastatin inhibited bone resorption at concentrations similar to those of the potent bisphosphonate ibandronate. This effect could be totally reversed by the addition of mevalnate and geranylgeraniol but not farnesol. The first two intermediates but not the latter could also stimulate basal bone resorption. The inhibitory effect of ibandronate on bone resorption could be totally reversed by the addition of geranylgeraniol and to a small extent only by mevalonate and farnesol, indicating that the bisphosphonate acts at a level of the mevalonate pathway different from that of mevastatin. Histologic sections of ibandronate‐treated bone explants showed further rescue of functioning osteoclasts during concomitant treatment with geranylgeraniol. Finally, the reversibility of bisphosphonate inhibited osteoclastic resorption by geranylgeraniol was also demonstrated for the potent nitrogen‐containing bisphosphonates alendronate, olpadronate, and risedronate but not for the non–nitrogen‐containing bisphosphonates clodronate and etidronate. These studies demonstrate that protein geranylgeranylation but not farnesylation is important for osteoclast‐mediated bone resorption and that nitrogen‐containing bisphosphonates exert their antiresorptive action probably by affecting enzymes of the mevalonate pathway involved in the generation of geranylgeranyl pyrophosphate.

Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease.

The development of novel anti-cancer strategies requires more sensitive and less invasive methods to detect and monitor in vivo minimal residual disease in cancer models. Bone marrow metastases are indirectly detected by radiography as osteolytic and/or osteosclerotic lesions. Marrow micrometastases elude radiographic detection and, therefore, more sensitive methods are needed for their direct identification. Injection of cancer cells into the left cardiac ventricle of mice closely mimics micrometastatic spread. When luciferase-transfected cells are used, whole-body bioluminescent reporter imaging can detect microscopic bone marrow metastases of approximately 0.5 mm(3) volume, a size below the limit in which tumors need to induce angiogenesis for further growth. This sensitivity translates into early detection of intramedullary tumor growth, preceding the appearance of a radiologically evident osteolysis by approximately 2 weeks. Bioluminescent reporter imaging also enables continuous monitoring in the same animal of growth kinetics for each metastatic site and guides end-point analyses specifically to the bones affected by metastatic growth. This model will accelerate the understanding of the molecular events in metastasis and the evaluation of novel therapies aiming at repressing initial stages of metastatic growth.

Bone morphogenetic protein 7 in the development and treatment of bone metastases from breast cancer.

Bone morphogenetic protein 7 (BMP7) counteracts the physiological epithelial-to-mesenchymal transition (EMT), a process that is indicative of epithelial plasticity. Because EMT is involved in cancer, we investigated whether BMP7 plays a role in breast cancer growth and metastasis. In this study, we show that decreased BMP7 expression in primary breast cancer is significantly associated with the formation of clinically overt bone metastases in patients with > or = 10 years of follow-up. In line with these clinical observations, BMP7 expression is inversely related to tumorigenicity and invasive behavior of human breast cancer cell lines. Moreover, BMP7 decreased the expression of vimentin, a mesenchymal marker associated with invasiveness and poor prognosis, in human MDA-MB-231 (MDA-231)-B/Luc(+) breast cancer cells under basal and transforming growth factor-beta (TGF-beta)-stimulated conditions. In addition, exogenous addition of BMP7 to TGF-beta-stimulated MDA-231 cells inhibited Smad-mediated TGF-beta signaling. Furthermore, in a well-established bone metastasis model using whole-body bioluminescent reporter imaging, stable overexpression of BMP7 in MDA-231 cells inhibited de novo formation and progression of osteolytic bone metastases and, hence, their metastatic capability. In line with these observations, daily i.v. administration of BMP7 (100 mug/kg/d) significantly inhibited orthotopic and intrabone growth of MDA-231-B/Luc(+) cells in nude mice. Our data suggest that decreased BMP7 expression during carcinogenesis in the human breast contributes to the acquisition of a bone metastatic phenotype. Because exogenous BMP7 can still counteract the breast cancer growth at the primary site and in bone, BMP7 may represent a novel therapeutic molecule for repression of local and bone metastatic growth of breast cancer.

Epithelial plasticity, cancer stem cells and bone metastasis formation.

Acquisition of an invasive phenotype of cancer cells in primary tumors is an absolute requirement for bone metastasis. The majority of bone metastases is derived from epithelial cancers, particularly those of the breast and prostate. Accumulating evidence suggest that transformed epithelial cells can activate embryonic programs of epithelial plasticity and switch from a sessile, epithelial phenotype to a motile, mesenchymal phenotype also referred to as epithelial-to-mesenchymal transition (EMT). Induction of EMT can, therefore, lead to invasion of surrounding stroma, intravasation, dissemination and colonization of distant sites. In bone/bone marrow disseminated tumor cells can partially regain their original epithelial characteristics via a mesenchymal-to-epithelial transition (MET) as glandular structures in bone metastasis are frequently observed. To date, the importance of epithelial plasticity in cancer cells disseminated to the bone/bone marrow microenvironment has remained largely elusive. Interestingly, a number of growth factors that play a prominent role in EMT induction in the primary tumor have been identified as important stimulators of skeletal metastasis formation. Recent studies have demonstrated that EMT may render cancer cells with properties of stem cells, which in turn can lead to escape from immune surveillance, increased resistance to apoptosis, diminished senescence and, last-but-not least, therapy resistance. This review will discuss current concepts regarding the role of epithelial plasticity in the multistep processes of bone metastasis, the issue of minimal residual disease, cancer stem cells and the importance of EMT in the development of novel targeted drug therapy.

The TGF-β/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system

Transforming growth factor-β (TGF-β) has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stages. In contrast to the mechanisms by which TGF-β induces growth arrest, the pathways that mediate tumor invasion are not well understood. Here, we describe a TGF-β-dependent invasion assay system consisting of spheroids of MCF10A1 normal breast epithelial cells (M1) and RAS-transformed (pre-)malignant derivatives (M2 and M4) embedded in collagen gels. Both basal and TGF-β-induced invasion of these cell lines was found to correlate with their tumorigenic potential; M4 showing the most aggressive behavior and M1 showing the least. Basal invasion was strongly inhibited by the TGF-β receptor kinase inhibitor SB-431542, indicating the involvement of autocrine TGF-β or TGF-β-like activity. TGF-β-induced invasion in premalignant M2 and highly malignant M4 cells was also inhibited upon specific knockdown of Smad3 or Smad4. Interestingly, both a broad spectrum matrix metalloproteinase (MMP) inhibitor and a selective MMP2 and MMP9 inhibitor mitigated TGF-β-induced invasion of M4 cells, while leaving basal invasion intact. In line with this, TGF-β was found to strongly induce MMP2 and MMP9 expression in a Smad3- and Smad4-dependent manner. This collagen-embedded spheroid system therefore offers a valuable screening model for TGF-β/Smad- and MMP2- and MMP9-dependent breast cancer invasion.

Monitoring Metastatic Behavior of Human Tumor Cells in Mice with Species-Specific Polymerase Chain Reaction: Elevated Expression of Angiogenesis and Bone Resorption Stimulators by Breast Cancer in Bone Metastases

Tumor‐stroma interactions are of primary importance in determining the pathogenesis of metastasis. Here, we describe the application of sensitive competitive polymerase chain reaction (PCR) techniques for detection and quantitation of human breast cancer cells (MDA‐MB‐231) in an in vivo mouse model of experimental metastasis. Human‐specific oligonucleotide primers in competitive PCR reactions were used to quantify the amount of MDA‐MB‐231 cells per tissue per organ. Using this species‐specific (semi)quantitative PCR approach, gene expression patterns of (human) tumor cells or (mouse) stromal cells in metastatic lesions in the skeleton or soft tissues were investigated and compared. In all metastatic lesions, MDA‐MB‐231 cells express angiogenic factors (vascular endothelial growth factors [VEGFs]; VEGF‐A, ‐B, and ‐C) and bone‐acting cytokines (parathyroid hormone‐related protein [PTHrP] and macrophage colony‐stimulating factor [M‐CSF]). In these metastases, PECAM‐1‐positive blood vessels and stromal cells of mouse origin are detected. The latter express angiogenic factors and markers of sprouting vessels (VEGF receptors flt‐1/flk‐1/flk‐4 and CD31/PECAM‐1). Strikingly, steady‐state messenger RNA (mRNA) levels of VEGF‐A and ‐B and the major bone resorption stimulators PTHrP and M‐CSF by tumor cells were elevated significantly in bone versus soft tissues (p ≤ 0.05, p ≤ 0.0001, p ≤ 0.001, and p ≤ 0.05, respectively), indicating tissue‐specific expression of these tumor progression factors. In conclusion, MDA‐MB‐231 breast cancer cells express a variety of factors in vivo that have been implicated in metastatic bone disease and that correlate with poor survival of patients with breast cancer. We hypothesize that the observed up‐regulated expression of angiogenic and bone‐resorbing factors by the breast cancer cells in the skeleton underlie the clinically observed osteotropism of breast cancer cells and pathogenesis of osteolytic bone metastases. The application of the species‐specific competitive PCR‐based assay in vivo can provide new information concerning the involvement of gene families in tumor progression and metastatic disease and greatly facilitates the study of tumor‐stroma interactions in cancer invasion and metastasis.

Interference with the Microenvironmental Support Impairs the De novo Formation of Bone Metastases In vivo

Interference with the microenvironmental growth support is an attractive therapeutic strategy for repressing metastatic tumor growth. Bone is a highly dynamic tissue that is continuously remodeled by bone resorption and subsequent bone formation. Growth factors supporting bone metastatic growth are released especially during bone resorption. Differently from most other tissues, drugs that can limit local turnover, such as bisphosphonates, are available for bone. In the present study, we tested the hypothesis that inhibition of bone turnover can affect development and growth progression of experimental bone metastasis. Whole-body bioluminescent reporter imaging was used for the detection, monitoring, and quantification in vivo of the growth progression of bone metastases induced by intracardiac or intraosseous injection of luciferase-transfected breast cancer cells (MDA-231-B/luc+) to nude mice. Suppression of bone turnover by bisphosphonates, before bone colonization by cancer cells, inhibited by a great extent the number of developing bone metastasis. Tumor growth in the few, but still developing, bone metastases was affected only transiently. Reduction of bone turnover had no effect on growth progression of bone metastases, which were already established when bisphosphonate treatment was initiated, despite a substantial reduction in osteolysis. Therefore, cancer cells metastatic to bone, after an initial growth phase that depends on the interaction with the local stroma, become independent of microenvironmental growth factor support and progress autonomously. Inhibition of bone turnover may represent a useful adjuvant therapy especially for cancer patients at risk to develop bone metastasis.

Role and Localization of Urokinase Receptor in the Formation of New Microvascular Structures in Fibrin Matrices

Fibrin or a fibrinous exudate can facilitate angiogenesis in many pathological conditions. In vitro, the outgrowth of capillary-like structures in fibrin can be mimicked by exposing human microvascular endothelial cells (hMVECs) to an angiogenic growth factor and tumor necrosis factor (TNF)-alpha. Urokinase-type plasminogen activator (u-PA) and plasmin activities are required for this angiogenic process. This study focuses on the role and localization of the u-PA receptor (u-PAR) in newly formed microvascular structures. The u-PAR-blocking monoclonal antibody (MAb) H-2 completely inhibited the formation of capillary-like tubular structures induced by exposure of hMVECs to basic fibroblast growth factor and TNF-alpha. This was accompanied by a several-fold increase in u-PA accumulation in the conditioned medium. The effect of MAb H-2 was not caused by blocking cellular activation by u-PA/u-PAR interaction, as the amino-terminal fragment (ATF) of u-PA, which also activates u-PAR, prevented tube formation. In addition, the inhibition by MAb H-2 was not due to an effect of the antibody on u-PAR-vitronectin binding. These data show that inhibition of tube formation can be caused not only by inhibition of u-PA or plasmin activities but also by unavailability of the u-PAR for cell-bound proteolysis. Immunohistochemical analysis showed that in in vitro angiogenesis u-PAR and u-PA were localized on the invading, tube-forming hMVECs and not on the endothelial cells that are located on top of the fibrin matrix. u-PAR and u-PA were also prominently expressed on endothelial cells of neovessels present in an atherosclerotic plaque. These data may give more insight into the role of u-PAR in repair-associated angiogenesis.