Search

Bone is the primarily preferred site for breast and prostate cancer to metastasize. Bone metastases are responsible for most deaths related to breast and prostate cancer. The bone’s particular microenvironment makes it conducive for the growth of cancer cells. Studies on bone metastasis have focused on the interaction between cancer cells and the bone microenvironment. Osteocytes, the most common cell type of bone tissue, have received little attention in bone metastasis, although they are master signal sensors, integrators, and skeleton transducers. They play an important role in regulating bone mass by acting on both osteoblasts and osteoclasts, through the release of proteins such as sclerostin, Dickkopf-1 (DKK-1), and fibroblast growth factor 23 (FGF23). Osteocytes have been extensively re-evaluated, in light of their multiple functions: with different experimental approaches, it has been shown that, indeed, osteocytes are actively involved in the colonization of bone tissue by cancer cells. The present review focuses on recent research on the role that osteocytes play in bone metastasis of breast and prostate cancers. Moreover, the studies here summarized open up perspectives for new therapeutic approaches focused on modulating the activity of osteocytes to improve the condition of the bone metastatic patients. A better understanding of the complex interactions between cancer cells and bone-resident cells is indispensable for identifying potential therapeutic targets to stop tumor progression and prevent bone metastases.

Bone metastases represent the main problem related to the progression of breast cancer, as they are the main cause of death for these patients. Unfortunately, to date, bone metastases are incurable and represent the main challenge for the researcher. Chemokines and cytokines affect different stages of the metastatic process, and in bone metastases, interleukin (IL) -6, IL-8, IL-1β, and IL-11 participate in the interaction between cancer cells and bone cells. This review focuses on IL-11, a pleiotropic cytokine that, in addition to its well-known effects on several tissues, also mediates certain signals in cancer cells. In particular, as IL-11 works on bone remodeling, it plays a relevant role in the osteolytic vicious cycle of bone resorption and tumour growth, which characterizes bone metastasis. IL-11 appears as a candidate for anti-metastatic therapy. Even if different therapeutic approaches have considered IL-11 and the downstream-activated gp130 signaling pathways activated downstream of gp130, further studies are needed to decipher the contribution of the different cytokines and their mechanisms of action in breast cancer progression to define therapeutic strategies.

The highlight of the molecular basis and therapeutic targets of the bone-metastatic process requires the identification of biomarkers of metastasis colonization. Here, we studied miR-34a-5p expression, and Met-receptor expression and localization in bone metastases from ductal breast carcinomas, and in ductal carcinomas without history of metastasis (20 cases). miR-34a-5p was elevated in non-metastatic breast carcinoma, intermediate in the adjacent tissue and practically absent in bone metastases, opposite to pair-matched carcinoma. Met-receptor biomarker was highly expressed and inversely correlated with miR-34a-5p using the same set of bone-metastasis tissues. The miR-34a-5p silencing might depend on aberrant-epigenetic mechanisms of plastic-bone metastases, since in 1833 cells under methyltransferase blockade miR-34a-5p augmented. In fact, 1833 cells showed very low endogenous miR-34a-5p, in respect to parental MDA-MB231 breast carcinoma cells, and the restoration of miR-34a-5p with the mimic reduced Met and invasiveness. Notably, hepatocyte growth factor (HGF)-dependent Met stabilization was observed in bone-metastatic 1833 cells, consistent with Met co-distribution with the ligand HGF at plasma membrane and at nuclear levels in bone metastases. Met-protein level was higher in non-metastatic (low grade) than in metastatic (high grade) breast carcinomas, notwithstanding miR-34a-5p-elevated expression in both the specimens. Thus, mostly in non-metastatic carcinomas the elevated miR-34a-5p unaffected Met, important for invasive/mesenchymal phenotype, while possibly targeting some stemness biomarkers related to metastatic phenotype. In personalized therapies against bone metastasis, we suggest miR-34a-5p as a suitable target of epigenetic reprogramming leading to the accumulation of miR-34a-5p and the down-regulation of Met-tyrosine kinase, a key player of the bone-metastatic process.

Hepatocyte growth factor (HGF) and transforming growth factor &beta, 1 (TGF&beta, 1) are biological stimuli of the micro-environment which affect bone metastasis phenotype through transcription factors, but their influence on the growth is scarcely known. In a xenograft model prepared with 1833 bone metastatic cells, derived from breast carcinoma cells, we evaluated mice survival and Twist and Snail expression and localization after competitive inhibition of HGF with NK4, or after blockade of TGF&beta, 1-type I receptor (RI) with SB431542: in the latter condition HGF was also measured. To explain the in vivo data, in 1833 cells treated with SB431542 plus TGF&beta, 1 we measured HGF formation and the transduction pathway involved. Altogether, HGF seemed relevant for bone-metastatic growth, being hampered by NK4 treatment, which decreased Twist more than Snail in the metastasis bulk. TGF&beta, 1-RI blockade enhanced HGF in metastasis and adjacent bone marrow, while reducing prevalently Snail expression at the front and bulk of bone metastasis. The HGF accumulation in 1833 cells depended on an auxiliary signaling pathway, triggered by TGF&beta, 1 under SB431542, which interfered in the transcription of HGF activator inhibitor type 1 (HAI-1) downstream of TGF&beta, activated kinase 1 (TAK1): HGF stimulated Twist transactivation. In conclusion, the impairment of initial outgrowth with NK4 seemed therapeutically promising more than SB431542 chemotherapy, a functional correlation between Twist and Snail in bone metastasis seemed to be influenced by the biological stimuli of the micro-environment, and the targeting of these phenotype biomarkers might inhibit metastasis plasticity and colonization, even if it would be necessary to consider the changes of HGF levels in bone metastases undergoing TGF&beta, 1-RI blockade.

The Hippo pathway is involved in human tumorigenesis and tissue repair. Here, we investigated the Hippo coactivator Yes-associated protein 1 (YAP1) and the kinase large tumor suppressor 1/2 (LATS1/2) in tumors of the parathyroid glands, which are almost invariably associated with primary hyperparathyroidism. Compared with normal parathyroid glands, parathyroid adenomas (PAds) and carcinomas show variably but reduced nuclear YAP1 expression. The kinase LATS1/2, which phosphorylates YAP1 thus promoting its degradation, was also variably reduced in PAds. Further, YAP1 silencing reduces the expression of the key parathyroid oncosuppressor multiple endocrine neoplasia type 1(MEN1), while MEN1 silencing increases YAP1 expression. Treatment of patient-derived PAds-primary cell cultures and Human embryonic kidney 293A (HEK293A) cells expressing the calcium-sensing receptor (CASR) with the CASR agonist R568 induces YAP1 nuclear accumulation. This effect was prevented by the incubation of the cells with RhoA/Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitors Y27632 and H1152. Lastly, CASR activation increased the expression of the YAP1 gene targets CYR61, CTGF, and WNT5A, and this effect was blunted by YAP1 silencing. Concluding, here we provide preliminary evidence of the involvement of the Hippo pathway in human tumor parathyroid cells and of the existence of a CASR-ROCK-YAP1 axis. We propose a tumor suppressor role for YAP1 and LATS1/2 in parathyroid tumors.

Bone is the most common site for breast cancer spread. In the pro-metastatic cell line 1833, derived from MDA-MB-231 breast adenocarcinoma cells, both hypoxia and hepatocyte growth factor (HGF) influence the effect of miR-125b on ETS proto-oncogene 1 transcription factor (ETS1). The effect of hypoxia inducible factor 1 alpha subunit (HIF1A), known to promote metastatic spread by upregulating prostaglandin endoperoxide synthase 2 (PTGS2), may be dampened by miR-125b targeting PTGS2. Here, we investigated whether miR-125b plays a role in breast cancer metastasis by measuring its activity in response to the chemotherapeutic agent NS-398 in a xenograft model. NS-398 is typically used in the clinic to target PTGS2. We also aimed to describe the molecular mechanisms in vitro, since the enhancement of epithelial properties may favor the efficacy of therapies. We report that in the xenograft model, miR-125b reduced metastasis to the bone. We also report suppression of PTGS2 enhanced survival by decreasing HIF1A in cells within the bone marrow. In 1833 cells transfected with a miR-125b mimic we observed several phenotypic changes including enhancement of the epithelial marker E-cadherin, a reduction of mesenchymal-associated genes and a reduction of WNT-associated stem cell signaling. Our findings suggest that in vivo, key players of the bone microenvironment promoting breast cancer spread are regulated by miR-125b. In future, biological molecules imitating miR-125b may enhance the sensitivity of chemotherapeutic agents used to counteract bone metastases.

We examined the influence of microenvironment stimuli on molecular events relevant to the biological functions of 1833-bone metastatic clone and the parental MDA-MB231 cells. (i) In both the cell lines, hepatocyte growth factor (HGF) and the osteoblasts’ biological products down regulated nuclear Ets-1-protein level in concomitance with endogenous miR-125b accumulation. In contrast, under hypoxia nuclear Ets-1 was unchanged, notwithstanding the miR-125b increase. (ii) Also, the 1833-cell invasiveness and the expression of Endothelin-1, the target gene of Ets-1/HIF-1, showed opposite patterns under HGF and hypoxia. We clarified the molecular mechanism(s) reproducing the high miR-125b levels with the mimic in 1833 cells. Under hypoxia, the miR-125b mimic maintained a basal level and functional Ets-1 protein, as testified by the elevated cell invasiveness. However, under HGF ectopic miR-125b downregulated Ets-1 protein and cell motility, likely involving an Ets-1-dominant negative form sensible to serum conditions; Ets-1-activity inhibition by HGF implicated HIF-1α accumulation, which drugged Ets-1 in the complex bound to the Endothelin-1 promoter. Altogether, 1833-cell exposure to HGF would decrease Endothelin-1 transactivation and protein expression, with the possible impairment of Endothelin-1-dependent induction of E-cadherin, and the reversion towards an invasive phenotype: this was favoured by Ets-1 overexpression, which inhibited HIF-1α expression and HIF-1 activity. (iii) In MDA-MB231 cells, HGF strongly and rapidly decreased Ets-1, hampering invasiveness and reducing Ets-1-binding to Endothelin-1 promoter; HIF-1α did not form a complex with Ets-1 and Endothelin-1-luciferase activity was unchanged. Overall, depending on the microenvironment conditions and endogenous miR-125b levels, bone-metastatic cells might switch from Ets-1-dependent motility towards colonization/growth, regulated by the balance between Ets-1 and HIF-1.

Introduction. Despite the increasing of number of patients with Sickle Cell Disease (SCD) in Italy, due to multi-ethnic migratory phenomena, a large percentage of Caucasian sickle population is already present in Italy mainly with b-thal/HbS genotype. Red cell transfusion is one effective treatment for both acute and chronic complications of SCD, while hydroxycarbamide (HC) is used to reduce the frequency of painful vaso-occlusive crises (VOCs) and decrease the need for blood transfusion. Through the National Comprehensive Reference Centers for SCD, the Italian Society of Thalassemia and Hemoglobinopathies (SITE), in collaboration with the Society Italian Transfusion Medicine and Immunohematology (SIMTI) and the Italian Association of Hematology and Pediatric Oncology (AIEOP) conducted a national survey to collect information on different therapeutic approaches used for SCD patients. Aim. To assess therapeutic approaches used a large Italian cohort of patients with SCD, accounting for age, genotype and ethnicity. Patients and Methods. Observational Longitudinal Systemic Multicentre Study (https://clinicaltrials.gov/ct2/show/NCT03397017). Data were collected from 2015 to 2018 through a standard web-based application (www.SITE-italia.org) encrypted by the Central Server. All the SCD patients, treated or not treated, were included in order to identify the overall number and all gave written informed consent. The study was approved by Ethics Committee of Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico of Milan, Italy. Results. Thirty-four centers were involved from 14 Italian regions and 1,579 patients were enrolled (802 male and 777 female; median age 23 years - IQR, 25th-75th 10-41 yrs). Genotype, age and ethnicity distribution are shown in Table 1A. As expected, the median age of non-Caucasian patients, mainly HbSS, is significantly lower than Caucasian ones (p Out of 1,579, 365 SCD patients (23%) did not receive any therapy. Acute transfusion regimen (ATR), Chronic transfusion regimen (CTR) and HC were given in monotherapy, respectively in 160, 226 and 197 patients, or in succession/combination in 631 (Table 1B), distributed throughout genotypes. The main reasons for ATR were acute anemia (384 events) and VOCs (352), followed by acute chest syndrome (ACS; 170), surgery (82), pregnancy (64), splenic sequestration (26), stroke (9); multi-organ failure (MOFs 6) and priapism (5). For CRT, it was acute anemia (306 events) and prevention of VOCs (371), ACS (107), primary stroke prevention (78) and secondary prevention stroke (55), pain HC-resistent (39) and leg ulcers (12). For 275 patients out of 631 it was possible to follow the timing of therapy switching (Table 1C). Of 275 patients, 67.6% switched from ATR/CRT to HC, 2.9% from HC to CRT and 6.5% stopped every therapy. Out of 275 patients, 104 were treated with overlapping therapeutic regimen. Discussion. The significant difference of age in Caucasian and non-Caucasian patients is probably due to the efficacy of the national prevention program of hemoglobinopathies, because the non-Caucasian patients are prevalently born out of Italy. The transfusional approach is similar in HbSS and b°-thal/HbS and b+-thal/HbS patients regarding both ATR and CTR. HbSC genotype needed less therapies(p Summary/Conclusion. The transfusional approach is similar in HbSS, b°-thal/HbS and b+-thal/HbS patients with similar indications, prevalently VOCs and anemia. The significant higher age in Caucasian cohort and the consequent long term follow up could be the cause of variable therapeutic approach observed, however Hydroxycarbamide seemed to be the therapy more frequently used and finally suggested to manage chronic manifestations. Figure. Figure. Disclosures Origa: Apopharma: Honoraria; Novartis: Honoraria; Bluebird Bio: Consultancy; Cerus Corporation: Research Funding. Forni:Apopharma: Other: DSM Board; Celgene: Research Funding; Novartis: Other: travel expenses, Research Funding; Shire: Research Funding; Roche: Consultancy.

The present study deals with the molecular mechanisms involved in the regulation of E-cadherin expression under hypoxia, because the adjustment of the amount of E-cadherin due to physical stimuli of the microenvironment might influence the colonization of metastasis to skeleton. We analyzed the effect of 1% oxygen tension, that is similar to that encountered in the bone marrow by metastatic cells spreading from breast carcinoma. The purpose was to evaluate the hypoxiaorchestrated control of E-cadherin transactivation via hypoxia inducible factor-1 (HIF-1) and peroxisome proliferator activated receptor-gamma (PPAR gamma), and the involvement of Hippo pathway members, as regulators of transcription factors. To give a translational significance to the study, we took into consideration human pair-matched ductal breast carcinoma and bone metastasis: E-cadherin and Wwox were expressed in bone metastasis but not in breast carcinoma, while HIF-1 alpha and TAZ seemed localized principally in nuclei of metastasis and were found in all cell compartments of breast carcinoma. A close examination of the regulatory mechanisms underlying E-cadherin expression in bone metastasis was done in 1833 clone derived from MDA-MB231 cells. Hypoxia induced E-cadherin only in 1833 clone, but not in parental cells, through HIF-1 and PPAR gamma activities, while Wwox decreased. Since Wwox was highly expressed in bone metastasis, the effect of ectopic Wwox was evaluated, and we showed E-cadherin transactivation and enhanced invasiveness in WWOX transfected 1833 cells. Also, hypoxia was additive with ectopic Wwox remarkably enhancing HIF-1 alpha nuclear shuttle and accumulation due to the lengthening of the half-life of HIF-1 alpha protein; under this experimental condition HIF-1 alpha appeared as a slower migrated band compared with control, in agreement with the phosphoiylation state. The in vitro data strongly supported the almost exclusive presence of HIF-1 alpha in nuclei of human-bone metastasis. Thus, we identified Wwox as a novel molecule in the HIF-1 alpha-HDM2 regulatory loop, necessary for the dynamic regulation of the HIF-1 alpha amount, and we suggested that the reduction of endogenous Wwox free pool under hypoxia might also be due to the interaction with HDM2, sequestering the E3 ubiquitin ligase. We highlighted the importance of nuclear HIF1 alpha in the biology of metastasis for the mesenchymal-epithelial transition: this phenotype was regulated by Wwox plus hypoxia through E-cadherin target gene, playing a pivotal role in bone metastasis colonization. (C) 2014 Elsevier Inc. All rights reserved.

Epigenetic mechanisms influence molecular patterns important for the bone-metastatic process, and here we highlight the role of WW-domain containing oxidoreductase (Wwox). The tumor-suppressor Wwox lacks in almost all cancer types; the variable expression in osteosarcomas is related to lung-metastasis formation, and exogenous Wwox destabilizes HIF-1α (subunit of Hypoxia inducible Factor-1, HIF-1) affecting aerobic glycolysis. Our recent studies show critical functions of Wwox present in 1833-osteotropic clone, in the corresponding xenograft model, and in human bone metastasis from breast carcinoma. In hypoxic-bone metastatic cells, Wwox enhances HIF-1α stabilization, phosphorylation, and nuclear translocation. Consistently, in bone-metastasis specimens Wwox localizes in cytosolic/perinuclear area, while TAZ (transcriptional co-activator with PDZ-binding motif) and HIF-1α co-localize in nuclei, playing specific regulatory mechanisms: TAZ is a co-factor of HIF-1, and Wwox regulates HIF-1 activity by controlling HIF-1α. In vitro, DNA methylation affects Wwox-protein synthesis; hypoxia decreases Wwox-protein level; hepatocyte growth factor (HGF) phosphorylates Wwox driving its nuclear shuttle, and counteracting a Twist program important for the epithelial phenotype and metastasis colonization. In agreement, in 1833-xenograft mice under DNA-methyltransferase blockade with decitabine, Wwox increases in nuclei/cytosol counteracting bone metastasis with prolongation of the survival. However, Wwox seems relevant for the autophagic process which sustains metastasis, enhancing more Beclin-1 than p62 protein levels, and p62 accumulates under decitabine consistent with adaptability of metastasis to therapy. In conclusion, Wwox methylation as a bone-metastasis therapeutic target would depend on autophagy conditions, and epigenetic mechanisms regulating Wwox may influence the phenotype of bone metastasis.

We investigated the involvement of Hippo-related pathways in bone metastasis from breast cancer, by evaluating E-cadherin expression downstream of WWdomain-containing oxidoreductase (Wwox) and transcriptional co-activator with PDZ-binding motif (TAZ). These nuclear effectors functioned in a context-specific fashion on transcriptome, depending on breast-cancer aggressiveness and methylation state. Wwox and E-cadherin were found in human specimens of bone metastasis but not in primary-ductal breast carcinoma, while TAZ showed a characteristic localisation in metastasis nuclei. Wwox and E-cadherin were higher in 1833-metastatic clone with bone avidity than in parental-MDA-MB231 cells, while only metastatic cells presented TAZ. In 1833 cells, a complex interplay of transcriptional signalling controlled E-cadherin transactivation. Wwox and TAZ activated Hypoxia inducible factor-1 (HIF-1) binding to E-cadherin promoter, while Peroxisome proliferator-activated receptor γ (PPARγ) intervened in E-cadherin transactivation favouring and preventing Wwox and TAZ functions, respectively. Methylation impinged on Hippo-related pathways through Wwox and TAZ, modifying metastatic phenotype. The protract exposure to 5-azacytidine (Aza), by affecting methylation state modified the shape of 1833 cells, becoming mesenchymal as that of MDA-MB231 cells and reduced spontaneous-Matrigel invasion. The underlying-molecular mechanisms were diminutions of E-cadherin, Wwox, matrix metalloproteases 2 and 9, HIF-1- and PPARγ-activities, inversely correlated to Snail and nuclear-TAZ accumulations. Exogenous WWOX restored 1833-Aza invasion. Thus, 1833-Aza cells permitted to study the role played by methylation in metastasis plasticity, being E-cadherin loss part of an entire-gene reprogramming. Of note, bone-metastasis formation in 1833-Aza xenograft was partially impaired, prolonging mice survival. In conclusion, the methylation-heritable changes seemed important for cancer progression to establish bone metastasis engraftment/growth, by affecting steps requiring homotipic and/or heterotypic-adhesive properties and matrix degradation.

Our translational research deals with the influence of microenvironment on the phenotype and colonization of bone metastases from breast carcinoma, and on pre-metastatic niche formation. The aim of the present study was to clarify the origin of hepatocyte growth factor (HGF), ligand of Met receptor, the control of the axis HGF/Met by DNA methylation, and its importance for the nexus supportive cells-metastatic cells and for metastasis outgrowth. In bone metastasis of the 1833-xenograft model, DNA methyltransferase blockade using the chemotherapic drug 5-aza-2'-deoxycytidine (decitabine) strongly reduced the expression of HGF/Met receptor axis and of E-cadherin, with decrease of metastasis wideness and osteolysis, prolonging mice survival. Thus, DNA methylation events acted as commanders of breast carcinoma cells metastatizing to bone influencing the epithelial phenotype. HGF emerged as a bone-marrow stimulus, and the exosomes seemed to furnish HGF to metastatic cells. In fact, decitabine treatment similarly affected some markers of these microvesicles and HGF, indicating that its supply to recipient cells was prevented. Notably, in bone metastasis the hypomethylation of HGF, Met and E-cadherin promoters did not appear responsible for their elevated expression, but we suggest the involvement of hypermethylated regulators and of Wwox oncosuppressor, the latter being affected by decitabine. Wwox expression increased under decitabine strongly localizing in nuclei of bone metastases. We hypothesize a role of Wwox in Met activity since in vitro Wwox overexpression downregulated the level of nuclear-Met protein fragment and Met stability, also under long exposure of 1833 cells to decitabine. HGF enhanced phosphoMet and the activity in nuclei, an effect partially prevented by decitabine. Altogether, the data indicated the importance to target the tumor microenvironment by blocking epigenetic mechanisms, which control critical events for colonization such as HGF/Met axis and Wwox, as therapy of bone metastasis.

We have previously shown that cancer cells can protect themselves from apoptosis induced by type 1 interferons (IFNs) through a ras-->MAPK-mediated pathway. In addition, since IFN-mediated signalling components STATs are controlled by PPAR gamma we studied the pharmacological interaction between recombinant IFN-beta and the PPAR-gamma agonist troglitazone (TGZ). This combination induced a synergistic effect on the growth inhibition of BxPC-3, a pancreatic cancer cell line, through the counteraction of the IFN-beta-induced activation of STAT-3, MAPK and AKT and the increase in the binding of both STAT-1 related complexes and PPAR-gamma with specific DNA responsive elements. The synergism on cell growth inhibition correlated with a cell cycle arrest in G0/G1 phase, secondary to a long-lasting increase of both p21 and p27 expressions. Blockade of MAPK activation and the effect on p21 and p27 expressions, induced by IFN-beta and TGZ combination, were due to the decreased activation of STAT-3 secondary to TGZ. alone also increased p21 and p27 expression through STAT-1 phosphorylation and this effect was attenuated by the concomitant activation of IFNbeta-induced STAT-3-activation. The combination induced also an increase in autophagy and a decrease in anti-autophagic bcl-2/beclin-1 complex formation. This effect was mediated by the inactivation of the AKT-->mTOR-dependent pathway. To the best of our knowledge this is the first evidence that PPAR-gamma activation can counteract STAT-3-dependent escape pathways to IFN-beta-induced growth inhibition through cell cycle perturbation and increased autophagic death in pancreatic cancer cells. (C) 2011 Elsevier Inc. All rights reserved.

Here, we show that NF-κB-HIF-1 interaction contributed to breast cancer metastatic capacity by means of an incomplete epithelial/mesenchymal transition and influencing migration, as shown in 1833 (human) and 4T1 (mouse) metastatic cells after different stimuli. The 1833 and the transforming growth factor-β1–exposed 4T1 cells showed both epithelial (E-cadherins) and mesenchymal (N-cadherins and vimentin) markers, and common mechanisms contributed to the retention of certain epithelial characteristics and the control of migration. The complex NF-κB-HIF-1 reciprocal regulation and the enhanced c-Jun expression played a functional role in exacerbating the invasiveness of 1833 cells after p50/p65 transfection and of 4T1 cells exposed to transforming growth factor-β1. Twist expression seemed to exert a permissive role also regulating epithelial/mesenchymal transition markers. After c-Src wild-type (Srcwt) transfection, c-Src-signal transducer overexpression in 1833 cells increased HIF-1 transactivating activity and invasiveness, and changed E-cadherin/N-cadherin ratio versus mesenchymal phenotype. The transcription factor pattern and the motile phenotype of metastatic 1833 cells were influenced by p65-lysine acetylation and HDAC-dependent epigenetic mechanisms, which positively regulated basal NF-κB and HIF-1 activities. However, HDAC3 acted as a corepressor of NF-κB activity in parental MDA-MB231 cells, thus explaining many differences from the derived 1833 clone, including reduced HIF-1α and c-Jun expression. Invasiveness was differently affected by HDAC knockdown in 1833 and MDA-MB231 cells. We suggest that acetylation/deacetylation are critical in establishing the bone-metastatic gene signature of 1833 cells by regulating the activity of NF-κB and HIF-1, and further clarify the epigenetic control of transcription factor network in the motile phenotype of 1833 cells. (Mol Cancer Res 2009;7(8):1328–41)

Much effort has been devoted to determining how metastatic cells and microenvironment reciprocally interact. However, the role of biological stimuli of microenvironment in controlling molecular events in bone metastasis from breast carcinoma for mesenchymal-epithelial transition (MET) is largely unknown. The purpose of the present paper was to clarify (1) the influence of hepatocyte-growth factor (HGF) and transforming growth factorβ1 (TGFβ1) on the phenotype of bone-metastatic 1833 and parental MDA-MB231 cells; (2) the hierarchic response of Twist and Snail controlled by Wwox co-factor, that might be critical for the control of 1833-adhesive properties via E-cadherin. We studied under HGF and TGFβ1 the gene profiles—responsible for epithelial-mesenchymal transition (EMT), versus the revertant MET phenotype—making the correspondence with 1833 morphology and the relation to HGF-dependent control of TGFβ1 signalling. In particular, the activation of Twist program and the underlying molecular mechanisms were investigated, considering the role of endogenous and exogenous Wwox with siRNAWWOX and the expression vector transfection, to clarify whether Twist affected E-cadherin transactivation through a network of transcription factors and regulators. HGF and TGFβ1 oppositely affected the expression of Wwox in 1833 cells. Under HGF, endogenous Wwox decreased concomitant with Twist access to nuclei and its phosphorylation via PI3K/Akt pathway. Twist activated by HGF did not influence the gene profile through an E-box mechanism, but participated in the interplay of PPARγ/Ets1/NF-kB-transcription factors, triggering E-cadherin transactivation. Altogether, HGF conferred MET phenotype to 1833 cells, even if this was transient since followed by TGFβ1-signalling activation. TGFβ1 induced Snail in both the cell lines, with E-cadherin down-regulation only in 1833 cells because in MDA-MB231 cells E-cadherin was practically absent. Exogenous Wwox activated metastatic HIF-1, with Twist as co-factor. HGF and TGFβ1 of bone-metastasis microenvironment acted co-ordinately, influencing non redundant pathways regulated by Twist program or Snail-transcription factor, with reversible MET switch. This process implicated different roles for Wwox in the various steps of the metastatic process including colonization, with microenvironmental/exogenous Wwox that activated HIF-1, important for E-cadherin expression. Interfering with the Twist program by targeting the pre-metastatic niche stimuli could be an effective anti-bone metastasis therapy.

The pathogenesis of bone metastasis is unclear, and much focus in metastatic biology and therapy relays on epigenetic alterations. Since DNA-methyltransferase blockade with 5-aza-2′-deoxycytidine (dAza) counteracts tumour growth, here we utilized dAza to clarify whether molecular events undergoing epigenetic control were critical for bone metastatization. In particular, we investigated the patterns of secreted-protein acidic and rich in cysteine (SPARC) and of Endothelin 1, affected by DNA methyltransferases in tumours, with the hypothesis that in bone metastasis a coordinate function of SPARC and Endothelin 1, if any occurs, was orchestrated by DNA methylation. To this purpose, we prepared a xenograft model with the clone 1833, derived from human-MDA-MB231 cells, and dAza administration slowed-down metastasis outgrowth. This seemed consequent to the reductions of SPARC and Endothelin 1 at invasive front and in the bone marrow, mostly due to loss of Twist. In the metastasis bulk Snail, partly reduced by dAza, might sustain Endothelin 1-SPARC cooperativity. Both SPARC and Endothelin 1 underwent post-translational control by miRNAs, a molecular mechanism that might explain the in vivo data. Ectopic miR29a reduced SPARC expression also under long-term dAza exposure, while Endothelin 1 down-regulation occurred in the presence of endogenous-miR98 expression. Notably, dAza effects differed depending on in vivo and in vitro conditions. In 1833 cells exposed to 30-days dAza, SPARC-protein level was practically unaffected, while Endothelin 1 induction depended on the 3′-UTR functionality. The blockade of methyltransferases leading to SPARC reduction in vivo, might represent a promising strategy to hamper early steps of the metastatic process affecting the osteogenic niche.

As experimental evidence suggests that leptin may have direct effects on peripheral tissues, we investigated some of the transductional molecules induced by leptin in C2C12 cells. In immunoprecipitation experiments using anti-p85 antibodies (a regulatory subunit of phosphatidylinositol-3-kinase; PI3K), we observed a significant increase in PI3K activity. Immunoblot analyses showed that Akt, GSK3, ERK1, ERK2, and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation significantly increased after leptin treatment. Protein kinase C (PKC)-zeta was also activated by leptin, as documented by an immunocomplex kinase assay and immunoblotting experiments. The treatment of C2C12 cells with Wortmannin before leptin administration inhibited induction of the phosphorylation of ERKs (extracellular signal-regulated kinases) but not that of p38 MAPK, whereas pre-treatment with a PKC-zeta inhibitor partially decreased ERK phosphorylation. Taken together, our in vitro results further support the hypothesis that leptin acts acutely on skeletal muscle tissue through some of the components of insulin signalling, including PKC-zeta.

Experimental evidence suggests that leptin operates on the tissues, including skeletal muscle, also by modulating gene expression. Using electrophoretic mobility shift assays, we have shown that physiological doses of leptin promptly increase the binding of C2C12 cell nuclear extracts to peroxisome proliferator-activated receptor (PPAR) response elements in oligonucleotide probes and that all three PPAR isoforms participate in DNA-binding complexes. We pre-treated C2C12 cells with AACOCF{sub 3}, a specific inhibitor of cytosolic phospholipase A{sub 2} (cPLA{sub 2}), an enzyme that supplies ligands to PPARs, and found that it abrogates leptin-induced PPAR DNA-binding activity. Leptin treatment significantly increased cPLA{sub 2} activity, evaluated as the release of [{sup 3}H]arachidonic acid from pre-labelled C2C12 cells, as well as phosphorylation. Further, using MEK1 inhibitor PD-98059 we showed that leptin activates cPLA{sub 2} through ERK induction. These results support a direct effect of leptin on skeletal muscle cells, and suggest that the hormone may modulate muscle transcription also by precocious activation of PPARs through ERK-cPLA{sub 2} pathway.

Experimental evidence suggests that leptin may exert direct effects on peripheral tissues. In this study we investigated some transductional molecules in skeletal muscle, after intraperitoneal leptin injection in wild-type and ob/ob mice. By immunoprecipitation and immunoblotting with anti-phosphotyrosine antibodies, we observed a modified pattern of phosphotyrosine proteins. We then identified an increase in JAK2, IRS1 and IRS2 tyrosine-phosphorylation and in their association with p85, a subunit of PI3K. The increase in PI3K activity in immunoprecipitated p85 did not reach statistical significance, however, both Akt and GSK3 resulted significantly hyper-phosphorylated. Bad, an Akt substrate involved in cell survival, appeared modified in its phosphorylation. ERK1, ERK2 and p38 MAP kinase phosphorylation significantly increased, even if the latter only in wild-type animals. Finally, by EMSA experiments, we documented that leptin increased the DNA binding capacity of Stat3 homodimers and AP-1. Thus, leptin appears to activate, within minutes, some insulin signalling molecules. Stat3 and AP-1 activation by gene expression remodelling could subsequently trigger more leptin-specific effects. Further, leptin might play a still underestimated role in cell survival.

Our translational research deals with the influence of microenvironment on the phenotype and colonization of bone metastases from breast carcinoma, and on pre-metastatic niche formation. The aim of the present study was to clarify the origin of hepatocyte growth factor (HGF), ligand of Met receptor, the control of the axis HGF/Met by DNA methylation, and its importance for the nexus supportive cells-metastatic cells and for metastasis outgrowth. In bone metastasis of the 1833-xenograft model, DNA methyltransferase blockade using the chemotherapic drug 5-aza-2′-deoxycytidine (decitabine) strongly reduced the expression of HGF/Met receptor axis and of E-cadherin, with decrease of metastasis wideness and osteolysis, prolonging mice survival. Thus, DNA methylation events acted as commanders of breast carcinoma cells metastatizing to bone influencing the epithelial phenotype. HGF emerged as a bone-marrow stimulus, and the exosomes seemed to furnish HGF to metastatic cells. In fact, decitabine treatment similarly affected some markers of these microvesicles and HGF, indicating that its supply to recipient cells was prevented. Notably, in bone metastasis the hypomethylation of HGF, Met and E-cadherin promoters did not appear responsible for their elevated expression, but we suggest the involvement of hypermethylated regulators and of Wwox oncosuppressor, the latter being affected by decitabine. Wwox expression increased under decitabine strongly localizing in nuclei of bone metastases. We hypothesize a role of Wwox in Met activity since in vitro Wwox overexpression downregulated the level of nuclear-Met protein fragment and Met stability, also under long exposure of 1833 cells to decitabine. HGF enhanced phosphoMet and the activity in nuclei, an effect partially prevented by decitabine. Altogether, the data indicated the importance to target the tumor microenvironment by blocking epigenetic mechanisms, which control critical events for colonization such as HGF/Met axis and Wwox, as therapy of bone metastasis.

The present study was undertaken to clarify the function(s) of Endothelin-1 and its receptors ETAR and ETBR in osteolytic-bone metastasis from breast cancer, and their regulation by hepatocyte and transforming growth factors (HGF, TGF-β) and hypoxia. The aim was to evaluate the adaptability of bone metastasis to microenvironmental stimuli through Endothelin-1-mediated epithelial-mesenchymal transition (EMT), or the reverse process MET, and through osteomimicry possible key features for bone colonization. We compared low (MCF-7) and high (MDA-MB231) invasive-breast carcinoma cells, and 1833-bone metastatic clone, with human pair-matched primary breast-carcinomas and bone metastases. Parental MDA-MB231 and the derived 1833-clone responded oppositely to the stimuli. In 1833 cells, TGF-β and hypoxia increased Endothelin-1 release, altogether reducing invasiveness important for engraftment, while Endothelin-1 enhanced MDA-MB231 cell invasiveness. The Endothelin-1-autocrine loop contributed to the cooperation of intracellular-signaling pathways and extracellular stimuli triggering MET in 1833 cells, and EMT in MDA-MB231 cells. Only in 1833 cells, HGF negatively influenced transactivation and release of Endothelin-1, suggesting a temporal sequence of these stimuli with an initial role of HGF-triggered Wnt/β-catenin pathway in metastatization. Then, Endothelin-1/ETAR conferred MET and osteomimetic phenotypes, with Runt-related transcription factor 2 activation and metalloproteinase 9 expression, contributing to colonization and osteolysis. Findings with human pair-matched primary ductal carcinomas and bone metastases gave a translational significance to the molecular study. Endothelin-1, ETAR and ETBR correlated with the acquisition of malignant potential, because of high expression already in the in situ carcinoma. These molecular markers might be used as predictive index of aggressive behavior and invasive/metastatic phenotype.

Here, we report a complex regulation of endothelin-1 (ET-1) axis driven by epigenetic reactions in 1833-bone metastatic cells, emphasizing the importance in skeletal metastasis from breast carcinoma. Inhibitors of histone deacetylases, trichostatin A (TSA), and of DNA methylases, 5'-Azacytidine (Aza), caused, respectively, reduction and increase in 1833 cell invasiveness, without affecting the basal migration of parental MDA-MB231 cells. Of note, in the two cell lines exposed to Aza the blockade of the ET-1 receptor ETAR with BQ-123 oppositely changed invasive properties. Even if in MDA-MB231 cells the ET-1 axis was scarcely influenced by epigenetic reactions, ETAR remarkably decreased after Aza. In contrast, in 1833 cells Aza exposure enhanced ET-1 coupled to ETAR wild type, being also ETAR truncated form increased, and invasiveness was stimulated. Under demethylation, the increase in ET-1 steady state protein level in 1833 clone seemed regulated at transcriptional level principally via Ets1 transcription factor. In fact, actinomycin D almost completely prevented ET-1 mRNA induction due to Aza. Only in 1833 cells, TSA exposure inactivated ET-1 axis, with reduction of the expression of ET-1 and ETAR mutated form, in agreement with Matrigel invasion decrease. This treatment favoured the ET-1 repressional control, taking place at the level of mRNA stability due to the 3'-untranslated region in the ET-1 gene, and also decreased transcription via NF-kB. Environmental conditions that alter the balance between epigenetic reactions might, therefore, affect metastasis migratory mode influencing ET-1 axis.

We have investigated the mechanisms of signal transduction in the response of liver to heat shock in vivo. By immunoblot experiments we have shown that heat shock decreases the electrophoretic mobility of the 40 and 43 kDa mitogen activated protein kinases (MAPKs) and we have found a significant increase of MAPK activity measured as phosphotransferase capacity of both cytosolic extracts and MAPK immunoprecipitates. To elucidate the signalling pathway which accounts for MAPK activation, we focused our attention on its upstream factors, Raf and Res. We have shown that, heat shock activates Raf-1 kinase and causes an increase in phosphotyrosine content of the 52 kDa She protein accompanied by an increment in the amount of coimmunoprecipitated Grb2. These findings provide the first evidence that the Ras-Raf-MAPK pathway is activated in liver during heat shock in vivo.

The human adipose-tissue derived stem/stromal cells (hASCs) are an interesting source for bone-tissue engineering applications. Our aim was to clarify in hASCs the role of acetylation in the control of Runt-related transcription factor 2 (Runx2) and Peroxisome proliferator activated receptor (PPAR) γ. These key osteogenic and adipogenic transcription factors are oppositely involved in osteo-differentiation. The hASCs, committed or not towards bone lineage with osteoinductive medium, were exposed to HDACs chemical blockade with Trichostatin A (TSA) or were genetically silenced for HDACs. Alkaline phosphatase (ALP) and collagen/calcium deposition, considered as early and late osteogenic markers, were evaluated concomitantly as index of osteo-differentiation. TSA pretreatment, useful experimental protocol to analyse pan-HDAC-chemical inhibition, and switch to osteogenic medium induced early-osteoblast maturation gene Runx2, while transiently decreased PPARγ and scarcely affected late-differentiation markers. Time-dependent effects were observed after knocking-down of HDAC1 and 3: Runx2 and ALP underwent early activation, followed by late-osteogenic markers increase and by PPARγ/ALP activity diminutions mostly after HDAC3 silencing. HDAC1 and 3 genetic blockade increased and decreased Runx2 and PPARγ target genes, respectively. Noteworthy, HDACs knocking-down favoured the commitment effect of osteogenic medium. Our results reveal a role for HDACs in orchestrating osteo-differentiation of hASCs at transcriptional level, and might provide new insights into the modulation of hASCs-based regenerative therapy.

The hypoxic microenvironment of bone marrow favours the bone metastasis process. Hypoxia inducible factor (HIF)-1α is hallmark for hypoxia, correlating with poor prognosis and radio/chemotherapy resistance of primary-breast carcinoma. For bone metastasis, the molecular mechanisms involved in HIF-1α expression and HIF-1 (α/β heterodimer)-transcription factor activity are scarcely known. We studied the role played by HIF-1 in the cross-talk between neoplastic and supportive-microenvironmental cells. Also, WWdomain-containing oxidoreductase (Wwox) and transcriptional co-activator with PDZ-binding motif (TAZ) were taken into consideration evaluating whether these Hippo-pathway effectors affect bone-metastatic phenotype through HIF-1 activity. Considering bone-metastasis specimens, nuclear HIF-1α-TAZ co-localisation occurred in neoplastic and supportive cells, such as fibroblasts and endotheliocytes. Based on these data, the functional importance was verified using 1833-bone metastatic clone under hypoxia: nuclear HIF-1α and TAZ expression increased and co-immunoprecipitated, activating HIF-1-DNA binding and transactivation. In contrast, Wwox localised at perinuclear level in neoplastic cells of bone metastasis, being almost absent in supportive cells, and Wwox-protein expression diminished in hypoxic-1833 cells. Thus, TAZ regulation of HIF-1 activity might be important for bone-secondary growth, participating in metastasis-stroma cross-talk. Further, TAZ and HIF-1α-protein levels seemed correlated. In fact, blocking cyclooxygenase-2 with NS398 in hypoxic-1833 cells, not only HIF-1α decreased but also molecular-mechanism(s) upstream of the Hippo pathway were triggered: LATS-dependent TAZ phosphorylation seemed responsible for TAZ nucleus/cytoplasm translocation and degradation. In the 1833-xenograft model, NS398 largely prevented the outgrowth of bone-metastatic cells, probably related to remarkable-extracellular matrix assembly. We gained clinical insight into HIF-1α and TAZ as candidate biomarkers for bone avidity, relevant for early-therapeutic intervention against bone metastasis.