Claudio Festuccia

Bombesin Stimulates Growth of Human Prostatic Cancer Cells In Vitro

Cell proliferation of the human prostatic carcinoma cell line PC3 and of the epithelial cell strain PMU 23 derived from a primary culture of a stage III prostatic carcinoma was enhanced dose dependently by adding 0.1 nM to 10.0 nM bombesin (BMBS) to the culture medium. The growth stimulation was specifically inhibited by antibodies versus Gastrin Releasing Peptide (GRP) crossreacting with BMBS. Presence of BMBS‐positive neuroendocrine cells in human prostate and measurable amounts of BMBS‐like peptides in prostatic fluid were reported previously. In a binding assay using 125I‐GRP, it was possible to demonstrate the presence of saturable specific receptors on PC3 cells, numerically comparable with those measured on small cell lung cancer cell lines. By immunofluorescence, however, no BMBS immunoreactivity on PC3 cells could be demonstrated. These observations suggest that BMBS plays a role in prostatic epithelium growth and that prostatic carcinoma may have an autocrine or paracrine proliferation stimulus within the gland microenvironment.

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.

Biological rationale for the use of DNA methyltransferase inhibitors as new strategy for modulation of tumor response to chemotherapy and radiation

Epigenetic modifications play a key role in the patho-physiology of many tumors and the current use of agents targeting epigenetic changes has become a topic of intense interest in cancer research. DNA methyltransferase (DNMT) inhibitors represent a promising class of epigenetic modulators. Research performed yielded promising anti-tumorigenic activity for these agents in vitro and in vivo against a variety of hematologic and solid tumors. These epigenetic modulators cause cell cycle and growth arrest, differentiation and apoptosis. Rationale for combining these agents with cytotoxic therapy or radiation is straightforward since the use of DNMT inhibitor offers greatly improved access for cytotoxic agents or radiation for targeting DNA-protein complex. The positive results obtained with these combined approaches in preclinical cancer models demonstrate the potential impact DNMT inhibitors may have in treatments of different cancer types. Therefore, as the emerging interest in use of DNMT inhibitors as a potential chemo- or radiation sensitizers is constantly increasing, further clinical investigations are inevitable in order to finalize and confirm the consistency of current observations.The present article will provide a brief review of the biological significance and rationale for the clinical potential of DNMT inhibitors in combination with other chemotherapeutics or ionizing radiation. The molecular basis and mechanisms of action for these combined treatments will be discussed herein.

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.

Nucleo-cytoplasmic transport as a therapeutic target of cancer

Shuttling of specific proteins out of the nucleus is essential for the regulation of the cell cycle and proliferation of both normal and malignant tissues. Dysregulation of this fundamental process may affect many other important cellular processes such as tumor growth, inflammatory response, cell cycle, and apoptosis. It is known that XPO1 (Exportin-1/Chromosome Region Maintenance 1/CRM1) is the main mediator of nuclear export in many cell types. Nuclear proteins exported to the cytoplasm by XPO1 include the drug targets topoisomerase IIα (topo IIα) and BCR-ABL and tumor suppressor proteins such as Rb, APC, p53, p21, and p27. XPO1 can mediate cell proliferation through several pathways: (i) the sub-cellular localization of NES-containing oncogenes and tumor suppressor proteins, (ii) the control of the mitotic apparatus and chromosome segregation, and (iii) the maintenance of nuclear and chromosomal structures. The XPO1 protein is elevated in ovarian carcinoma, glioma, osteosarcoma, pancreatic and cervical cancer. There is a growing body of research indicating that XPO1 may have an important role as a prognostic marker in solid tumors. Because of this, nuclear export inhibition through XPO1 is a potential target for therapeutic intervention in many cancers. The best understood XPO1 inhibitors are the small molecule nuclear export inhibitors (NEIs; Leptomycin B and derivatives, ratjadones, PKF050-638, valtrate, ACA, CBS9106, selinexor/KPT-330, and verdinexor/KPT-335). Selinexor and verdinexor are orally bioavailable, highly potent, small molecules that are classified as Selective Inhibitors of Nuclear Export (SINE). KPT-330 is the only NEI currently in Phase I/II human clinical trials in hematological and solid cancers. Of all the potential targets in nuclear cytoplasmic transport, the nuclear export receptor XPO1 remains the best understood and most advanced therapeutic target for the treatment of cancer.

Vitamin D Protects Human Endothelial Cells from H2O2 Oxidant Injury Through the Mek/Erk-Sirt1 Axis Activation

Endothelium homeostasis alterations govern the pathogenesis of cardiovascular diseases. Several studies show that vitamins anti-oxidant proprieties rescue the endothelial functions adversely affected by oxidative stress in several diseases. We investigated the vitamin D anti-oxidant potential in human endothelial cells exposed to H2O2 oxidative stress. Vitamin D protected endothelial cells against H2O2 oxidative stress counteracting the superoxide anion generation, the apoptosis and blocking the extrinsic caspase cascade by positively controlling phospho-active ERKs level. MEKs/ERKs inhibitor U0126 reverted the vitamin D anti-oxidant effects. Characterizing the vitamin D downstream effector, we found that vitamin D up-regulated SirT-1 and reverted the SirT-1 down-regulation induced by H2O2. ERKs activation by vitamin D strictly correlated with SirT-1 protein accumulation since both MEKs/ERKs inhibition and ERK1/2 silencing decreased SIRT-1. SirT-1 inhibition by Sirtinol reverted the vitamin D anti-oxidant effects. Thus, vitamin D significantly reduced the endothelial malfunction and damage caused by oxidative stress, through the activation of MEKs/ERKs/SirT-1 axis.

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.

In vitro regulation of pericellular proteolysis in prostatic tumor cells treated with bombesin

Bombesin is a potent inducer of signal trasduction pathways involved in the proliferation and invasion of androgen‐insensitive prostatic tumor cells. This study examines the bombesin‐mediated modulation of pericellular proteolysis, monitoring cell capability to migrate and invade basement membranes, using a chemo‐invasion assay and analyzing protease production. The results suggest that bombesin could modulate the invasive potential of prostatic cell lines regulating secretion and cell‐surface uptake of uPA and MMP‐9 activation. In fact, in PC3 and DU145 cells but not in LNCaP cells, urokinase‐type plasminogen activator (uPA) and plasminogen activator inhibitor‐1 (PAI‐1) are induced by bombesin treatment. Bombesin also stimulates cell proliferation and this effect can be inhibited blocking uPA by antibodies and/or uPA inhibitor p‐aminobenzamidine. Moreover, HMW‐uPA induces cell proliferation in LNCaP cells, which do not produce uPA in the basal conditions, while PC3 and DU145 cell growth is supported by autocrine production of uPA. The increment of uPA activity on the external plasma membrane causes an increased pericellular plasmin activation. This effect is inhibited by antibodies against uPA and by p‐aminobenzamidine. Similarly to EGF, bombesin stimulates secretion and activation of MMP‐9 and TIMP‐1 production. MMP‐9 activation can be also obtained by HMW‐uPA treatment, suggesting that plasma‐membrane‐bound uPA can start a proteolytic cascade involving MMP‐9. Therefore, in in vitro assays, bombesin is able to modulate pericellular proteolysis and cell proliferation, differently distributing and activating proteolytic activities. This effect can be related to the “non‐random” degradation of the extracellular matrix in which membrane uPA‐uPAreceptor complexes could start bombesin‐induced directional protein degradation during metastatic spread. Int. J. Cancer 75:418–431, 1998.

Plasminogen activator system modulates invasivecapacity and proliferation in prostatic tumor cells

The malignant phenotype of prostatic tumor cells correlates with the expression of both uPA and itscell-membrane receptor (uPAR); however, there is little information concerning the role of cell-bound uPAin matrix degradation and invasion. Our results suggest that cell-associated uPA plays a key role in regulat-ingthe amount of plasmin present at the surface of prostatic carcinoma (PRCA) cells and show that differ-entialproduction of uPA corresponds with the capacity to bind and activate plasminogen. In addition, weprovide direct evidence that both uPA secretion and the presence of uPA-uPAR complexes characterize theinvasive phenotype of PRCA cells and suggest the existence of several pathways by which tumor cells acquireplasmin activity. LNCaP cells (which do not produce uPA but express uPAR) may activate plasmin throughexogenous uPA. In vivo, the source of uPA may be infiltrating macrophages and/or fibroblasts as observedin several other systems. PAI-1 accumulation in the conditioned medium (CM) limits plasmin action to thepericellular microenvironment. Our results indicate that MMP-9 and MMP-2 are also activated by plasmingenerated by cell-bound but not by soluble, extracellular uPA. Plasmin activation and triggering of the pro-teolyticcascade involved in Matrigel invasion is blocked by antibodies against uPA (especially by anti- A-chainof uPA which interacts with uPAR) and by PA inhibitors such as p-aminobenzamidine which mayregulate levels of cell-bound uPA. uPA may also regulate growth in PRCA cells. Indeed, antibodies againstuPA A-chain (and also p-aminobenzamidine treatment) interfere with the ATF domain and inhibit cell growthin uPA-producing PC3 and DU145 prostate cancer cell lines, whereas exogenous uPA (HMW-uPA with ATF)induces growth of LNCaP prostate tumor cell line. These data support the hypothesis that in prostatic can-cerpatients at risk of progression, uPA/plasmin blockade may be of therapeutic value by blocking both growthof the primary tumor and dissemination of metastatic cells. ©Kluwer Academic Publishers