Jeroen T. Buijs

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.

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.

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.

TGF-β and BMP7 interactions in tumour progression and bone metastasis

The skeleton is the second most frequent site of metastasis. However, only a restricted number of solid cancers, especially those of the breast and prostate, are responsible for the majority of the bone metastases. Metastatic bone disease is a major cause of morbidity, characterised by severe pain and high incidence of skeletal and haematopoietic complications (fractures, spinal cord compression and bone marrow aplasia) requiring hospitalisation. Despite the frequency of skeletal metastases, the molecular mechanisms for their propensity to colonise bone are poorly understood and treatment options are often unsatisfactory. TGF-β and the signalling pathway it controls appears to play major roles in the pathogenesis of many carcinomas, both in their early stages, when TGF-β acts to arrest growth of many cell types, and later in cancer progression when it contributes, paradoxically, to the phenotype of tumour invasiveness. Here we discuss some novel insights of the TGF-β superfamily—including BMPs and their antagonists—in the formation of bone metastasis. Increasing evidence suggests that the TGF-β superfamily is involved in bone homing, tumour dormancy, and development of micrometastases into overt bone metastases. The established role of TGF-β/BMPs and their antagonists in epithelial plasticity during embryonic development closely resembles neoplastic processes at the primary site as well as in (bone) metastasis. For instance, the tumour-stroma interactions occurring in the tissue of cancer origin, including epithelium-to-mesenchyme transition (EMT), bear similarities with the role of bone matrix-derived TGF-β in skeletal metastasis formation.

The BMP2/7 heterodimer inhibits the human breast cancer stem cell subpopulation and bone metastases formation.

Accumulating evidence suggests that a subpopulation of breast cancer cells, referred to as cancer stem cells (CSCs), have the ability to propagate a tumor and potentially seed new metastases. Furthermore, stimulation of an epithelial-to-mesenchymal transition by factors like transforming growth factor-β (TGFβ) is accompanied with the generation of breast CSCs. Previous observations indicated that bone morphogenetic protein-7 (BMP7) antagonizes the protumorigenic and prometastatic actions of TGFβ, but whether BMP7 action is mechanistically linked to breast CSCs has remained elusive. Here, we have studied the effects of BMP7, BMP2 and a BMP2/7 heterodimer on the formation of human breast CSCs (ALDHhi/CD44hi/CD24−/low) and bone metastases formation in a preclinical model of intra-cardiac injection of MDA-MB-231 cells in athymic nude (Balb/c nu/nu) mice. The BMP2/7 heterodimer was the most efficient stimulator of BMP signaling and very effectively reduced TGFβ-driven Smad signaling and cancer cell invasiveness. The tested BMPs—particularly the heterodimeric BMP2/7—strongly reduced the size of the ALDHhi/CD44hi/CD24−/low CSC subpopulation. In keeping with these in vitro observations, pretreatment of cancer cells with BMPs for 72 h prior to systemic inoculation of the cancer cells inhibited the formation of bone metastases. Collectively, our data support the notion that breast CSCs are involved in bone metastasis formation and describe heterodimeric BMP2/7 as a powerful TGFβ antagonist with anti-metastatic potency.

Brown adipose tissue takes up plasma triglycerides mostly after lipolysis

Brown adipose tissue (BAT) produces heat by burning TGs that are stored within intracellular lipid droplets and need to be replenished by the uptake of TG-derived FA from plasma. It is currently unclear whether BAT takes up FA via uptake of TG-rich lipoproteins (TRLs), after lipolysis-mediated liberation of FA, or via a combination of both. Therefore, we generated glycerol tri[3H]oleate and [14C]cholesteryl oleate double-labeled TRL-mimicking particles with an average diameter of 45, 80, and 150 nm (representing small VLDL to chylomicrons) and injected these intravenously into male C57Bl/6J mice. At room temperature (21°C), the uptake of 3H-activity by BAT, expressed per gram of tissue, was much higher than the uptake of 14C-activity, irrespective of particle size, indicating lipolysis-mediated uptake of TG-derived FA rather than whole particle uptake. Cold exposure (7°C) increased the uptake of FA derived from the differently sized particles by BAT, while retaining the selectivity for uptake of FA over cholesteryl ester (CE). At thermoneutrality (28°C), total FA uptake by BAT was attenuated, but the specificity of uptake of FA over CE was again largely retained. Altogether, we conclude that, in our model, BAT takes up plasma TG preferentially by means of lipolysis-mediated uptake of FA.

The aldehyde dehydrogenase enzyme 7A1 is functionally involved in prostate cancer bone metastasis

High aldehyde dehydrogenase (ALDH) activity can be used to identify tumor-initiating and metastasis-initiating cells in various human carcinomas, including prostate cancer. To date, the functional importance of ALDH enzymes in prostate carcinogenesis, progression and metastasis has remained elusive. Previously we identified strong expression of ALDH7A1 in human prostate cancer cell lines, primary tumors and matched bone metastases. In this study, we evaluated whether ALDH7A1 is required for the acquisition of a metastatic stem/progenitor cell phenotype in human prostate cancer. Knockdown of ALDH7A1 expression resulted in a decrease of the α2hi/αvhi/CD44+ stem/progenitor cell subpopulation in the human prostate cancer cell line PC-3M-Pro4. In addition, ALDH7A1 knockdown significantly inhibited the clonogenic and migratory ability of human prostate cancer cells in vitro. Furthermore, a number of genes/factors involved in migration, invasion and metastasis were affected including transcription factors (snail, snail2, and twist) and osteopontin, an ECM molecule involved in metastasis. Knockdown of ALDH7A1 resulted in decreased intra-bone growth and inhibited experimentally induced (bone) metastasis, while intra-prostatic growth was not affected. In line with these observations, evidence is presented that TGF-β, a key player in cancer invasiveness and bone metastasis, strongly induced ALDH activity while BMP7 (an antagonist of TGF-β signaling) down-regulated ALDH activity. Our findings show, for the first time, that the ALDH7A1 enzyme is functionally involved in the formation of bone metastases and that the effect appeared dependent on the microenvironment, i.e., bone versus prostate.

Integrin αv expression is required for the acquisition of a metastatic stem/progenitor cell phenotype in human prostate cancer.

Integrins participate in multiple cellular processes, including cell adhesion, migration, proliferation, survival, and the activation of growth factor receptors. Recent studies have shown that expression of αv integrins is elevated in the prostate cancer stem/progenitor cell subpopulation compared with more differentiated, committed precursors. Here, we examine the functional role of αv integrin receptor expression in the acquisition of a metastatic stem/progenitor phenotype in human prostate cancer. Stable knockdown of αv integrins expression in PC-3M-Pro4 prostate cancer cells coincided with a significant decrease of prostate cancer stem/progenitor cell characteristics (α2 integrin, CD44, and ALDH(hi)) and decreased expression of invasion-associated genes Snail, Snail2, and Twist. Consistent with these observations, αv-knockdown strongly inhibited the clonogenic and migratory potentials of human prostate cancer cells in vitro and significantly decreased tumorigenicity and metastatic ability in preclinical models of orthotopic growth and bone metastasis. Our data indicate that integrin αv expression is functionally involved in the maintenance of a highly migratory, mesenchymal cellular phenotype as well as the acquisition of a stem/progenitor phenotype in human prostate cancer cells with metastasis-initiating capacity.