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Autophagy is a catabolic pathway involved both in tissue homeostasis and in cellular response to stress. The precise role of autophagy in cancer is still undefined and seems to depend on the tumor stage, appearing tumor-suppressive in physiological conditions and helpful to tumor progression in the established tumor. Here we analyzed by immunohistochemistry Beclin-1, p62, and LC3B, autophagic markers, in human specimens of normal breast, bone metastasis together with pair-matched invasive breast carcinoma of no special type (IBC-NST) as well as non-metastatic breast carcinoma, to disclose the possibility that they could be early prognostic indicators of the evolution of the disease toward the worst outcome. Different regions of metastatic carcinomas, i.e., areas adjacent to the tumor without signs of neoplastic growth, dysplastic lesions, and areas with invasive growth were considered. The pattern of autophagic parameters showed differences among the stages of breast carcinoma progression with a trend that indicated the activation of autophagic process in normal breast (Beclin-1 more elevated than p62), a pattern that was maintained in non-metastatic carcinoma. As the neoplasia proceeds with malignancy, the modification of the pattern of expression of autophagic markers (low ratio between Beclin-1 and p62) in areas of invasive growth of carcinomas suggested inhibition of the process. Of note, the parameters showed a different pattern in bone metastasis with respect to bone metastatic (bm)-IBC-NST, suggesting the reactivation of the autophagic process in the new growth site, helpful to the colonization. The course of autophagy markers during tumor progression could have a prognostic value towards bone metastasis and reveal different roles of the process in different phases of neoplastic growth. The understanding of the role of autophagy in bone metastasis could disclose new therapeutic targets to improve the conditions of patients., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest, (Copyright © 2024 Elsevier GmbH. All rights reserved.)

In a precision medicine perspective, among the biomarkers potentially useful for early diagnosis of cancers, as well as to define their prognosis and eventually to identify novel and more effective therapeutic targets, there are the long non-coding RNAs (lncRNAs). The term lncRNA identifies a class of non-coding RNA molecules involved in the regulation of gene expression that intervene at the transcriptional, post-transcriptional, and epigenetic level. Metastasis is a natural evolution of some malignant tumours, frequently encountered in patients with advanced cancers. Onset and development of metastasis represents a detrimental event that worsen the patient's prognosis by profoundly influencing the quality of life and is responsible for the ominous progression of the disease. Due to the peculiar environment and the biomechanical properties, bone is a preferential site for the secondary growth of breast, prostate and lung cancers. Unfortunately, only palliative and pain therapies are currently available for patients with bone metastases, while no effective and definitive treatments are available. The understanding of pathophysiological basis of bone metastasis formation and progression, as well as the improvement in the clinical management of the patient, are central but challenging topics in basic research and clinical practice. The identification of new molecular species that may have a role as early hallmarks of the metastatic process could open the door to the definition of new, and more effective, therapeutic and diagnostic approaches. Non-coding RNAs species and, particularly, lncRNAs are promising compounds in this setting, and their study may bring to the identification of relevant processes. In this review, we highlight the role of lncRNAs as emerging molecules in mediating the formation and development of bone metastases, as possible biomarkers for cancer diagnosis and prognosis, and as therapeutic targets to counteract cancer spread., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Maroni, Gomarasca and Lombardi.)

Bone metastasis is a serious and often lethal complication of particularly frequent carcinomas, such as breast and prostate cancers, which not only reduces survival but also worsens the patients' quality of life. Therefore, it is important to find new and/or additional therapeutic possibilities that can counteract the colonization of bone tissue. High adherence to the Mediterranean diet (MD) is effective in the prevention of cancer and improves cancer patients' health, thus, here, we considered its impact on bone metastasis. We highlighted some molecular events relevant for the development of a metastatic phenotype in cancer cells and the alterations of physiological bone remodeling, which occur during skeleton colonization. We then considered those natural compounds present in MD foods with a recognized role to inhibit or reverse the metastatic process both in in vivo and in vitro systems, and we reported the identified mechanisms of action. The knowledge of this bioactivity by the dietary components of the MD, together with its wide access to all people, could help not only to maintain healthy status but also to improve the quality of life of patients with bone metastases.

Breast cancer is the most common type of cancer in women, and the occurrence of metastasis drastically worsens the prognosis and reduces overall survival. Understanding the biological mechanisms that regulate the transformation of malignant cells, the consequent metastatic transformation, and the immune surveillance in the tumor progression would contribute to the development of more effective and targeted treatments. In this context, microRNAs (miRNAs) have proven to be key regulators of the tumor-immune cells crosstalk for the hijack of the immunosurveillance to promote tumor cells immune escape and cancer progression, as well as modulators of the metastasis formation process, ranging from the preparation of the metastatic site to the transformation into the migrating phenotype of tumor cells. In particular, their deregulated expression has been linked to the aberrant expression of oncogenes and tumor suppressor genes to promote tumorigenesis. This review aims at summarizing the role and functions of miRNAs involved in antitumor immune response and in the metastasis formation process in breast cancer. Additionally, miRNAs are promising targets for gene therapy as their modulation has the potential to support or inhibit specific mechanisms to negatively affect tumorigenesis. With this perspective, the most recent strategies developed for miRNA-based therapeutics are illustrated.

The most serious aspect of neoplastic disease is the spread of cancer cells to secondary sites. Skeletal metastases can escape detection long after treatment of the primary tumour and follow-up. Bone tissue is a breeding ground for many types of cancer cells, especially those derived from the breast, prostate, and lung. Despite advances in diagnosis and therapeutic strategies, bone metastases still have a profound impact on quality of life and survival and are often responsible for the fatal outcome of the disease. Bone and the bone marrow environment contain a wide variety of cells. No longer considered a passive filler, bone marrow adipocytes have emerged as critical contributors to cancer progression. Released by adipocytes, adipokines are soluble factors with hormone-like functions and are currently believed to affect tumour development. Src-associated in mitosis of 68 kDa (Sam68), originally discovered as a protein physically associated with and phosphorylated by c-Src during mitosis, is now recognised as an important RNA-binding protein linked to tumour onset and progression of disease. Sam68 also regulates splicing events and recent evidence reports that dysregulation of these events is a key step in neoplastic transformation and tumour progression. The present review reports recent findings on adipokines and Sam68 and their role in breast cancer progression and metastasis.

Hepatocyte growth factor (HGF) and transforming growth factor β1 (TGFβ1) 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 TGFβ1-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 TGFβ1 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. TGFβ1-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 TGFβ1 under SB431542, which interfered in the transcription of HGF activator inhibitor type 1 (HAI-1) downstream of TGFβ-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 TGFβ1-RI blockade.

Bone is the primary site where some cancers develop secondary growth, particularly those derived from breast and prostate tissue. The spread of metastasis to distant sites relies on complex mechanisms by which only cells endowed with certain characteristics are able to reach secondary growth sites. Platelets play a pivotal role in tumour growth, by conferring resistance to shear stress to the circulating tumour cells and protection against natural killer cell attack. Mature polyploid megakaryocytes (MKs) reside in close proximity to the vascular sinusoids of bone marrow, where their primary function is to produce platelets. Emerging evidence has demonstrated that MKs are essential for skeletal homeostasis, due to the expression and production of the bone-related proteins osteocalcin, osteonectin, bone morphogenetic protein, osteopontin, bone sialoprotein, and osteoprotegerin. Debate surrounds the role that MKs play in the development of bone metastasis, which is the topic of this mini-review.

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., Competing Interests: The authors declare no conflict of interest.

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., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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.

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., Competing Interests: The authors declare no conflict of interest.

Bone metastatic cells release bone microenvironment proteins, such as the matricellular protein SPARC (secreted protein acidic and rich in cysteine), and share a cell signaling typical of the bone metabolism controlled by Runx2. The megakaryocytes in the bone marrow engrafted by the metastases seem to be one of the principal microenvironment sources of the biological stimuli, implicated in the formation of an osteoblastic niche, and affecting metastasis phenotype and colonization. Educated platelets in the circulation might derive from megakaryocytes in bone metastasis. The evaluation of predictive markers in the circulating platelets might be useful for the stratification of patients for therapeutic purposes. The hypoxic environment in bone metastasis is one of the key regulators of the network of the biological soluble and structural components of the matrix. In bone metastatic cells under hypoxia, similar patterns of Runx2 and SPARC are observed, both showing downregulation. Conversely, hypoxia induces Endothelin 1, which upregulates SPARC, and these biological stimuli may be considered prognostic markers of bone metastasis in breast carcinoma patients.

Autophagy favours metastatic growth through fuelling energy and nutrients and resistance to anoikis, typical of disseminated-tumour cells. The autophagic process, mediated by a unique organelle, the autophagosome, which fuses with lysosomes, is divided into three steps. Several stages, especially early omegasome formation and isolation-membrane initiation, remain controversial; molecular mechanisms involve the small-GTPase Rab5a, which regulates vesicle traffic for autophagosome formation. We examined Rab5a involvement in the function of key members of ubiquitin-conjugation systems, Atg7 and LC3-lipidated, interacting with the scaffold-protein p62. Immunohistochemistry of Rab5a was performed in human specimens of bone metastasis and pair-matched breast carcinoma; the autophagic-molecular mechanisms affected by Rab5a were evaluated in human 1833 bone metastatic cells, derived from breast-carcinoma MDA-MB231 cells. To clarify the role of Rab5a, 1833 cells were transfected transiently with Rab5a-dominant negative, and/or stably with the short-hairpin RNA Atg7, were exposed to two inhibitors of autolysosome function, and LC3II and p62 expression was measured. We showed basal autophagy in bone-metastatic cells and the pivotal role of Rab5a together with Beclin 1 between the early stages, elongation of isolation membrane/closed autophagosome mediated by Atg7, and the late-degradative stages. This regulatory network might occur in bone-metastasis and in high-grade dysplastic lesions, preceding invasive-breast carcinoma and conferring phenotypic characteristics for dissemination.

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In order to become established in the skeleton, metastatic cells disseminating from the breast carcinoma need to acquire organ-specific traits. There are no effective predictors for who will develop bone metastasis to guide long-term predictive therapy. Our purpose was to individuate events critical for bone colonization to make a molecular classification of breast carcinoma useful for bone-metastasis outcome. In dysplasia adjacent to carcinoma and in pair-matched specimens of bone metastasis we examined SPARC expression and localization as well as Endothelin 1/ETAR signals by immunohistochemistry, and the evaluation of plasma levels of SPARC by ELISA was also performed. In patients with breast carcinoma metastasizing to bone, SPARC and Endothelin 1/ETAR axis were highly expressed from dysplasia until bone metastasis, but the SPARC plasma level was as low as that of normal women, in contrast to patients that never develop bone metastasis, suggesting that circulating SPARC was counter adhesive. Altogether, the early identification of SPARC/Endothelin 1/ETAR in dysplastic lesions would be important to devise therapies preventing metastasis engraftment, since often carcinoma cells spread to distant organs at the time or even before patients present with cancer.

Background: 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., Methods: 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., Results: 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., Conclusions: 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 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 hypoxia-orchestrated control of E-cadherin transactivation via hypoxia inducible factor-1 (HIF-1) and peroxisome proliferator activated receptor-γ (PPARγ), 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-1α 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 PPARγ 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-1α nuclear shuttle and accumulation due to the lengthening of the half-life of HIF-1α protein; under this experimental condition HIF-1α appeared as a slower migrated band compared with control, in agreement with the phosphorylation state. The in vitro data strongly supported the almost exclusive presence of HIF-1α in nuclei of human-bone metastasis. Thus, we identified Wwox as a novel molecule in the HIF-1α-HDM2 regulatory loop, necessary for the dynamic regulation of the HIF-1α 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 HIF-1α 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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Here we show that the fate of osteolytic bone metastasis depends on the balance among autophagy, anoikis resistance and ossification, and that the hepatocyte growth factor (HGF) signaling pathway seems to have an important role in orchestrating bone colonization. These findings are consistent with the pathophysiology of bone metastasis that is influenced by the cross-talk of supportive and neoplastic cells through molecular signaling networks. We adopted the strategy to target metastasis and stroma with the use of adenovirally expressed NK4 (AdNK4) and Dasatinib to block HGF/Met axis and Src activity. In human bone metastatic 1833 cells, HGF conferred anoikis resistance via Akt and Src activities and HIF-1α induction, leading to Bim isoforms degradation. When Src and Met activities were inhibited with Dasatinib, the Bim isoforms accumulated conferring anoikis sensitivity. The proviability effect of HGF, under low-nutrient stress condition, was related to a faster autophagy deactivation with respect to HGF plus Dasatinib. In the 1833 xenograft model, AdNK4 switched metastasis vasculature to blood lacunae, increasing HIF-1α in metastasis. The combination of AdNK4 plus Dasatinib gave the most relevant results for mice survival, and the following molecular and cellular changes were found to be responsible. In bone metastasis, we observed a hypoxic condition - marked by HIF-1α - and an autophagy failure - marked by p62 without Beclin-1. Then, osteolytic bone metastases were largely prevented, because of autophagy failure in metastasis and ossification in bone marrow, with osteocalcin deposition. The abnormal repair process was triggered by the dysfunctional autophagy/anoikis interplay. In conclusion, the concomitant blockade of HGF/Met axis and Src activity seemed to induce HIF-1α in metastasis, whereas the bone marrow hypoxic response was reduced. As a consequence, anoikis resistance might be hampered favoring, instead, autophagy failure and neoformation of woven bone trabeculae. Mice survival was, therefore, prolonged by overcoming an escape strategy adopted by metastatic cells by disruption of tumor-stroma coevolution, showing the importance of autophagy inhibition for the therapy of bone metastasis.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Metastatic cells switch between different modes of migration through supramolecular plasticity mechanism(s) still largely unknown. The aim of the present paper was to clarify some molecular aspects of the epigenetic control of migration of 1833-bone metastatic cells compared to MDA-MB231-parental mammary carcinoma cells. Active c-Src overexpression enhanced 1833-cell spontaneous migration and CXCR4-mediated chemoinvasion toward CXCL12 ligand. Only in metastatic cells, in fact, c-Src seemed to stabilize nuclear CXCR4-protein receptor possibly due to tyrosine phosphorylation, by impairing protein-degradative smear and causing instead an electrophoretic-mobility shift; the cytosolic steady-state level of CXCR4 was enhanced, and the protein appeared also phosphorylated. These findings suggested the triggering of unique signaling pathways in metastasis for homing of breast-cancer cells to congenial environment of specific organs. Microenvironmental stimuli activating c-Src might influence Ets1 binding to CXCR4 promoter and consequent transactivation, as well as CXCR4 post-translational regulatory mechanisms such as phosphorylation. Enhancement of Ets1 activity and CXCR4 induction by c-Src overexpression were prevented by histone deacetylase (HDAC) blockade. In contrast, HDAC inhibition with trichostatin A increased cytosolic phosphorylated CXCR4 expression in MDA-MB231 cells, but Ets1 involvement was practically unneeded. c-Src might be suggested as a bio-marker predicting metastasis sensitivity patterns to HDAC inhibitors. Rationally designed and individualized therapy may become possible as more is learned about the target molecules of HDAC's inhibitory agents and their roles, as undertaken for CXCR4 that is likely to be crucial for homing, angiogenesis and survival in a c-Src-dependent manner in bone-metastatic mammary cells., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The aim of this article is to identify nuclear co-localization of COX-2 and HIF-1α in human-bone metastasis of breast cancer, index of transcriptionally activated cells and functional for gene expression. In particular, we verified whether hypoxia exerted a direct role on metastasis-gene expression or through COX-2 signaling, due to the relevance for clinical implications to individuate molecular targets for diagnosis and therapy. The experiments were performed in vitro with two metastatic clones, 1833 and MDA-231BO, and the parental MDA-MB231 cells, in vivo (1833-xenograft model), and in human-bone metastasis specimens. In 1833 cells in vitro, COX-2 signaling pathway was critical for nuclear HIF-1α-protein expression/translocation, mechanisms determining HIF-1 activity and gene expression. The data were corroborated by immunohistochemistry in human-bone metastasis specimens. COX-2 and HIF-1α showed wide co-localization in the nucleus, indicative of COX-2-nuclear import in transcriptionally activated metastatic cells and consistent with COX-2-HIF-1α functional interaction. A network of microenvironmental signals controlled COX-2 induction and HIF-1 activation downstream. In fact, hypoxia through HGF and TGF-β1 autoregulatory loops triggered a specific array of transcription factors responsible for COX-2 transactivation. The novelty was that HGF and TGF-β1 biological signals were produced by hypoxic metastatic cells and, therefore, the microenvironment seemed to be modified by metastatic-cell engraftment in the bone. In agreement, HIF-1α expression in bone marrow supportive cells occurred in metastasis-bearing animals. Altogether, the data supported the pre-metastatic-niche theory. Our observations might be useful to design therapies against bone metastasis, by affecting the phenotype changes of metastatic cells occurring at the secondary growth site through COX-2-HIF-1 interaction.

To clarify the reciprocal interaction between human-breast cancer metastatic cells and bone microenvironment, we studied the influence of HGF/Met system on a proposed-prognostic marker of aggressiveness, the beta-catenin/Wnt pathway. For in vitro and in vivo experiments we used 1833-bone metastatic clone, derived from human-MDA-MB231 cells. In osteolytic bone metastases and in metastatic cells, Met was expressed in nuclei and at plasma membrane, and abnormally co-localised at nuclear level with beta-catenin and the tyrosine phosphorylated c-Src kinase. Thus, in 1833 cells nuclear-Met COOH-terminal fragment and beta-catenin-TCF were constitutively activated, possibly by receptor and non-receptor tyrosine kinases. The activity of the gene reporter TOPFLASH (containing multiple TCF/LEF-consensus sites) was measured, as index of beta-catenin functionality. In 1833 cells, human and mouse HGF increased Met and beta-catenin tyrosine phosphorylation and expression in nuclear and perinuclear compartments, beta-catenin nuclear translocation via Kank and TOPFLASH transactivation. Human HGF was autocrine/intracrine in bone metastasis, and mouse HGF originating from the adjacent host-bone marrow, was found inside the metastatic nuclei. Parental MDA-MB231 cell nuclei did not show functional beta-catenin, for TCF-transactivating activity, and the regulation by HGF. Our study highlighted the importance of the metastasis-stroma interaction in human-breast cancer metastatisation and first identified the HGF/nuclear Met/phospho-c-Src/beta-catenin-TCF/Wnt pathway as a potential-therapeutic target to delay establishment/progression of bone metastases by affecting the aggressive phenotype., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Here, we show that NF-kappaB-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-beta1-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-kappaB-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-beta1. 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-kappaB and HIF-1 activities. However, HDAC3 acted as a corepressor of NF-kappaB activity in parental MDA-MB231 cells, thus explaining many differences from the derived 1833 clone, including reduced HIF-1alpha 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-kappaB and HIF-1, and further clarify the epigenetic control of transcription factor network in the motile phenotype of 1833 cells.