Adriano Angelucci

Inhibition of Protein Kinase c-Src Reduces the Incidence of Breast Cancer Metastases and Increases Survival in Mice: Implications for Therapy

c-Src is a proto-oncogene, belonging to the nonreceptor protein kinases family, which plays a prominent role in carcinogenesis. In this study, we tested the hypothesis that c-Src could promote breast cancer metastasis acting on several cell types and that pharmacological disruption of its kinase activity could be beneficial for the treatment of metastases. Female BALB/c-nu/nu mice were subjected to intracardiac injection of the human breast cancer cells MDA-MB-231 (MDA-231), which induced prominent bone and visceral metastases. These were pharmacologically reduced by treatment with the c-Src inhibitor [7-{4-[2-(2-methoxy-ethylamino-ethoxy]-phenyl}-5-(3-methoxy-phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine] CGP76030 (100 mg/kg/day p.o.), resulting in decreased morbidity and lethality. Metastases were more severe in mice injected with MDA-231 cells stably transfected with wild-type c-Src (MDA-231-SrcWT), whereas transfection in injected cells of a c-Src kinase-dead dominant-negative construct (MDA-231-SrcDN) resulted in reduced morbidity, lethality, and incidence of metastases similar to the mice treated with the inhibitor. An analogous beneficial effect of c-Src inhibition was observed in subcutaneous and intratibial implanted tumors. In vitro, c-Src suppression reduced MDA-231 cell aggressiveness. It also impaired osteoclast bone resorption both directly and by reducing expression by osteoblasts of the osteoclastogenic cytokines interleukin-1β and interleukin-6, whereas parathyroid hormone-related peptide was not implicated. c-Src was also modestly but consistently involved in the enhancement of endothelial cell proliferation in vitro and angiogenesis in vivo. In conclusion, we propose that c-Src disruption affects the metastatic process and thus is a therapeutic target for the treatment of breast cancer.

Osteoblast conditioned media contain TGF-β1 and modulate the migration of prostate tumor cells and their interactions with extracellular matrix components

Prostate cancers (PRCAs) frequently metastasize to bone. We show here that this process is facilitated by osteoblast‐mediated tumor cell recruitment. Transforming growth factor‐β1 (TGF‐β1) is produced by osteoblasts in a latent form and is activated by proteases in a cell‐dependent manner. This cytokine exhibits pleiotropic effects on cell‐extracellular matrix (ECM) interactions and may influence tumor cell invasion and metastasis. Our purpose was to identify the potential molecular mechanisms involved in osteoblast‐mediated cell recruitment and to characterize the effect of TGF‐β1 on adhesion, motility and invasiveness of a human prostate cancer cell line with high bone metastatic potential (PC3 cell line) in vitro. Conditioned media from osteoblast cultures (OB CM) enhanced PC3 cell chemotaxis and invasion of reconstituted basement membrane. These effects were blocked by a neutralyzing TGF‐β1 polyclonal antibody but not by elution of the OB CM in agarose‐heparin columns, suggesting that TGF‐β1, but not EGF‐like proteins, contribute to PC3 cell recruitment. In addition, TGF‐β1 directly induced chemotaxis and invasion of PC3 cells in a dose‐dependent manner. The TGF‐β1‐mediated invasion and motility were accompanied by increased PC3 cell adhesion, spreading and α2β1 and α3β1 integrin expression. These events are involved in the cell adhesion to several components of basement membrane and ECM and in the selective invasion of metastatic tumor cells. Our results suggest that TGF‐β1 can influence cellular recognition of ECM components by prostatic cancer cells and can modulate cell adhesion and invasion leading to increased invasive potential. Given the widespread tissue distribution of TGF‐β1, and the high levels present in the bone, this cytokine may be an important autocrine‐paracrine modulator of the bone invasive phenotype in vivo. Int. J. Cancer 81:395–403, 1999.

Kinase-Dependent and -Independent Roles of EphA2 in the Regulation of Prostate Cancer Invasion and Metastasis

Ligand-activated Eph tyrosine kinases regulate cellular repulsion, morphology, adhesion, and motility. EphA2 kinase is frequently up-regulated in several different types of cancers, including prostate, breast, colon, and lung carcinomas, as well as in melanoma. The existing data do not clarify whether EphA2 receptor phosphorylation or its simple overexpression, which likely leads to Eph kinase-independent responses, plays a role in the progression of malignant prostate cancer. In this study, we address the role of EphA2 tyrosine phosphorylation in prostate carcinoma cell adhesion, motility, invasion, and formation of metastases. Tumor cells expressing kinase-deficient EphA2 mutants, as well as an EphA2 variant lacking the cytoplasmic domain, are defective in ephrinA1-mediated cell rounding, retraction fiber formation, de-adhesion from the extracellular matrix, RhoA and Rac1 GTPase regulation, three-dimensional matrix invasion, and in vivo metastasis, suggesting a key role for EphA2 kinase activity. Nevertheless, EphA2 regulation of cell motility and invasion, as well as the formation of bone and visceral tumor colonies, reveals a component of both EphA2 kinase-dependent and -independent features. These results uncover a differential requirement for EphA2 kinase activity in the regulation of prostate carcinoma metastasis outcome, suggesting that although the kinase activity of EphA2 is required for the regulation of cell adhesion and cytoskeletal rearrangement, some distinct kinase-dependent and -independent pathways likely cooperate to drive cancer cell migration, invasion, and metastasis outcome.

Vesicle-associated urokinase plasminogen activator promotes invasion in prostate cancer cell lines.

The ability of a cell to modify the extracellular matrix is important in several pathophysiological alterations including tumorigenesis. Cell transformation is accompanied by changes in the surrounding stroma as a result of the action of specific proteases such as the urokinase plasminogen activator (uPA), which has been associated with invasive potential in many tumor types. In this study, we analyzed the release of vesicle-associated uPA by the aggressive prostatic carcinoma cell line PC3 and the implications of this release for the invasive behaviour of prostatic tumor cells. Zymography and Western blot analysis revealed the presence of vesicle-associated uPA in the high-molecular weight form. Vesicles adhered to and degraded both collagen IV and reconstituted basal membrane (Matrigel), and plasminogen enhanced the degradation in a dose-dependent manner. Addition of membrane vesicles shed by PC3 cells to cultures of the poorly invasive prostate cancer cell line LnCaP enhanced the adhesive and invasive capabilities of the latter, suggesting a mechanism involving substrate recognition and degradation. Together, these findings indicate that membrane vesicles can promote tumor invasion and point to the important role of vesicle-associated uPA in the extracellular compartment.

Osteoblast‐derived TGF‐β1 modulates matrix degrading protease expression and activity in prostate cancer cells

Tumor progression and metastasis may result in part from the selection of cell clones competent for survival, invasion and growth at secondary sites and characterized by loss of growth inhibitory responses, acquisition of increased adhesiveness and enhanced motility and protease expression. Transforming growth factor‐β1 (TGF‐β1) is produced by osteoblasts (OB) in a latent form and is activated by proteases in a cell‐dependent manner. We show here that OB conditioned medium (OB CM) modulates Matrigel invasion of a bone metastatic prostate cancer cell line (PC3) and that this effect is blocked by antibody against TGF‐β1 and by uPA/plasmin inhibitors, suggesting that TGF‐β1 can modulate OB‐mediated cell recruitment and that PC3 cells can activate TGF‐β1. TGF‐β1 induces uPA and PAI‐1 secretion and promotes binding of uPA at the external plasma membrane with increased membrane‐associated plasmin activity. Matrix metalloprotease‐9 (MMP‐9) is induced both in the medium and in the membrane associated form. Moreover, the balance between proteolytic activity and inhibition is crucial in the metastatic event. Indeed, the increment of PAI‐1 could have an important regulatory role on the extracellular proteolysis and might explain the decrease of net PA and gelatinolytic activities measured in the medium. In addition, PAI‐1 plays a regulative role localizing matrix degradation in some specific sites, such as areas of cell‐to‐cell or cell‐to‐ECM contacts. In conclusion, TGF‐β1 enhances PC3 Matrigel invasion by a uPA/plasmin‐dependent mechanism, also involving the MMP‐9, and thus may play a central role in malignant prostate tumor progression as a result of stimulating bone matrix invasion. Int. J. Cancer 85:407–415, 2000. ©2000 Wiley‐Liss, Inc.

Epidermal growth factor modulates prostate cancer cell invasiveness regulating urokinase-type plasminogen activator activity. EGF-receptor inhibition may prevent tumor cell dissemination.

Urokinase-type plasminogen activator receptor (uPAR) and Epidermal Growth Factor Receptor (EGFR) are ubiquitous receptors involved in the control of a variety of cellular processes frequently found altered in cancer cells. The EGFR has been recently described to play a transduction role of uPAR stimuli, mediating uPA-induced proliferation in highly malignant cells that overexpress uPAR. We compared the uPA production, the presence of uPAR, AR, EGFR and Her2 with the chemotaxis and the Matrigel invasion in ten human PCa cell lines and observed that: (1) the levels of Her2, but not of EGFR, as well as the uPA secretion, cell motility and Matrigel invasion were statistically higher in AR negative than in AR positive PCa cells; (2) the uPA secretion and uPA Rexpression were positively related to Matrigel invasion; (3) the EGF was able to stimulate chemotaxis and Matrigel invasion in a dose-dependent manner; (4) the EGF-induced cell migration was statistically higher inAR negative than in AR positive cells with a similar increase with respect to basal value (about 2.6 fold); (5) the Matrigel invasion was statistically higher in AR negative than in AR positive PCa cells also if the increment of Matrigel invasion after EGF treatment was statistically higher in AR positive respect to AR negative cells; (6) the EGF induced uPA secretion and its membrane uptake through the increment of uPAR; and (7) these effects were blocked by EGFR/Her2 tyrosine kinase inhibitors with IC(50) lower than those needed to inhibit cell proliferation and required PI3K/Akt, MAPK and PI-PLC activities as verified by inhibition experiments. These enzymatic activities were regulated in different manners in PTEN positive and negative cells. In fact, the inhibition of PI3K blocked the EGF-induced invasiveness in PTEN positive cells but not in PTEN negative cells, in which PI3K activity was not influenced by EGFR/Her2 activation, whereas the inhibition of MAPK was able to block the invasive phenomena in both cell types. Taken together, our data suggest that the blockade of EGFR could attenuate the invasive potential of PCa cells. In addition, considering that the EGFR expression is related to higher Gleason grade of PCa and that EGFR levels are increased after anti androgenic therapy, this therapeutic approach could slow down the metastasis formation which represents the most dramatic event of PCa progression.

Antiproliferative and proapoptotic activities of new pyrazolo[3,4-d]pyrimidine derivative Src kinase inhibitors in human osteosarcoma cells

Osteosarcoma is the most frequent primitive malignant tumor of the skeletal system, char acterized by an extremely aggressive clinical course that still lacks an effective treatment. Src kinase seems to be involved in the osteosarcoma malignant phenotype. We show that the treatment of human osteosarcoma cell lines with a new pyrazolo[3,4‐d]pyrimidine derivative Src inhibitor, namely SI‐83, impaired cell viability, with a half‐maximal inhibitory concentration of 12 μ.M in nonstarved cells and a kinetic different from that known for the Src inhibitor PP2. Analysis by terminal deoxynucleotidyl transferase‐mediated nick end labeling, Hoechst, and flow cytometric assay showed that SI‐83 induced apoptosis in SaOS‐2 cells. Moreover, SI‐83, by inhibiting Src phosphorylation, decreased in vivo osteosarcoma tumor mass in a mouse model. Finally, SI‐83 showed selectivity for osteosarcoma, since it had a far lower effect in primary human osteoblasts. These results show that human osteosarcoma had Src‐ dependent proliferation and that modulation of Src activity may be a therapeutic target of this new com pound with low toxicity for nonneoplastic cells—Spreafico, A., Schenone, S., Serchi, T., Orlandini, M., Angelucci, A., Magrini, D., Bernardini, G., Collodel, G., Di Stefano, A., Tintori, C., Bologna, M., Manetti, F., Botta, M., Santucci, A. Antiproliferative and proapo‐ ptotic activities of new pyrazolo[3,4‐d]pyrimidine deriv ative Src kinase inhibitors in human osteosarcoma cells. FASEBJ. 22, 1560–1571 (2008)

Bone-targeted doxorubicin-loaded nanoparticles as a tool for the treatment of skeletal metastases.

Bone metastases contribute to morbidity in patients with common cancers, and conventional therapy provides only palliation and can induce systemic side effects. The development of nanostructured delivery systems that combine carriers with bone-targeting molecules can potentially overcome the drawbacks presented by conventional approaches. We have recently developed biodegradable, biocompatible nanoparticles (NP) made of a conjugate between poly (D,L-lactide-co-glycolic) acid and alendronate, suitable for systemic administration, and directly targeting the site of tumor-induced osteolysis. Here, we loaded NP with doxorubicin (DXR), and analyzed the in vitro and in vivo activity of the drug encapsulated in the carrier system. After confirming the intracellular uptake of DXR-loaded NP, we evaluated the anti-tumor effects in a panel of human cell lines, representative for primary or metastatic bone tumors, and in an orthotopic mouse model of breast cancer bone metastases. In vitro, both free DXR and DXR-loaded NP, (58-580 ng/mL) determined a significant dose-dependent growth inhibition of all cell lines. Similarly, both DXR-loaded NP and free DXR reduced the incidence of metastases in mice. Unloaded NP were ineffective, although both DXR-loaded and unloaded NP significantly reduced the osteoclast number at the tumor site (P = 0.014, P = 0.040, respectively), possibly as a consequence of alendronate activity. In summary, NP may act effectively as a delivery system of anticancer drugs to the bone, and deserve further evaluation for the treatment of bone tumors.

Receptor Activator of NF-κB Ligand Enhances Breast Cancer–Induced Osteolytic Lesions through Upregulation of Extracellular Matrix Metalloproteinase Inducer/CD147

Breast cancer shows a strong predilection to metastasize to bone. Cell surface glycoprotein extracellular matrix metalloproteinase inducer (EMMPRIN)/CD147 induces metalloproteinases (MMP) and vascular endothelial growth factor (VEGF), which may support osteoclastic activity and increased incidence of breast cancer bone metastases. In support of this hypothesis, we observed that MDA-MB-231 human breast tumor cells engineered to overexpress EMMPRIN strongly induced osteolytic lesions in immunodeficient mice, which was blunted by in vivo treatment with an EMMPRIN blocking antibody. Similarly, these cells exhibited increased expression of MMP-9 and VEGF relative to control cells. Treatment of MDA-MB-231 cells with the osteoclastogenic cytokine receptor activator of NF-kappaB ligand (RANKL) upregulated EMMPRIN expression with a parallel increase of MMP-9 and VEGF. Conditioned medium from osteoblasts similarly increased EMMPRIN, MMP-9, and VEGF expression in cells. Osteoblast treatment with the RANKL decoy receptor osteoprotegerin abolished this effect. EMMPRIN overexpression stimulated MDA-MB-231 cell invasion but not proliferation. Conversely, small interfering RNA-mediated knockdown of EMMPRIN downregulated MMP-9 and VEGF basal expression and RANKL-stimulated expression, and reduced cell invasion. Our results argue that EMMPRIN drives breast cancer-induced osteolytic lesions and that activation of the RANKL pathway increases EMMPRIN in osteotropic tumor cells, in turn enhancing tumor-induced bone resorption.

EphA2 induces metastatic growth regulating amoeboid motility and clonogenic potential in prostate carcinoma cells.

EphA2 kinase regulates cell shape, adhesion, and motility and is frequently overexpressed in several cancers, including melanoma, prostate, breast, and colon cancers and lung carcinoma. Although a function in both tumor onset and metastasis has been proposed, the role played by EphA2 in tumor progression is still debated. In melanoma, EphA2 has been reported to affect cell migration and invasiveness allowing cells to move by a proteolysis-independent strategy, commonly referred as amoeboid motility. With the aim to understand the role of EphA2 in prostate cancer metastatic spreading, we stably silenced EphA2 expression in a model of aggressive metastatic prostate carcinoma. Our results show that EphA2 drives the metastatic program of prostate carcinoma, although its involvement greatly differs among metastatic steps. Indeed, EphA2 expression (i) greatly affects prostate carcinoma cell motility style, guiding an amoeboid movement based on Rho-mediated cell rounding and independent from metalloprotases, (ii) is ineffective on transendothelial migration, adhesion onto extracellular matrix proteins, and on resistance to anoikis, (iii) regulates clonogenic potential of prostate carcinoma, thereby increasing anchorage-independent growth and self-renewal, prostasphere formation, tumor onset, dissemination to bone, and growth of metastatic colonies. Our finding indicate that EphA2-overexpressing prostate carcinoma cells gain an invasive benefit from their amoeboid motility style to escape from primary tumors and then, enhancing their clonogenic potential successfully target bone and grow metastases, thereby acknowledging EphA2 as a target for antimetastatic therapy of aggressive prostate cancers. Mol Cancer Res; 9(2); 149–60. ©2011 AACR.