The cross-talk between myeloid and mesenchymal stem cells of human bone marrow represents a biomarker of aging that regulates immune response and bone reabsorption

One of the mechanisms that characterizes the aging process of different organs is the accumulation of fat. Different authors have demonstrated that adipose tissue replaces the loss of other cell types, deriving from mesenchymal cells. During aging, there is substitution or trans-differentiation of mesenchymal cells with other cells having the same embryological origin. Newly formed adipocytes were also observed in the trabecular matrix of elderly people's bones, associated with myeloid cells. In this study, we have investigated the relationship between immature myeloid-derived suppressor cells (I-MDSCs) and mesenchymal stem cells (MSCs) in bone marrow (BM) samples harvested from 57 patients subjected to different orthopedic surgeries. Patients aged from 18 to 92 years were considered in order to compare the cellular composition of bone marrow of young and elderly people, considered a biomarker of immunity, inflammation, and bone preservation. The I-MDSC percentage was stable during aging, but in elderly people, it was possible to observe a strong basal immunosuppression of autologous and heterologous T cells' proliferation. We hypothesized that this pattern observed in elders depends on the progressive accumulation in the BM of activating stimuli, including cell-cell contact, or the production of different cytokines and proteins that induce the differentiation of bone marrow mesenchymal stem cells in adipocytes. The collected data provided underline the importance of specific biomarkers of aging that promote a reduction in immune response and incremented inflammatory pathways, leading to bone reabsorption in elderly people

Prostate Specific Membrane Antigen associates with Filamin A, beta1 integrin, pp130CAS and pSrc regulating beta1 integrin activation and survival of prostate cancer cells.

Il tumore della prostata \ue8 la seconda causa di morte tumore-correlata nella popolazione maschile dei paesi sviluppati. Evidenze precliniche hanno rivelato che virtualmente tutti i campioni di cancro avanzato della prostata presentano espressione elevata di Prostate Specific Membrane Antigen (PSMA), una proteina trans-membrana con funzione di folato-idrolasi/carbossipeptidasi. Nel 60% questa si associa con l\u2019attivazione basale delle vie di regolazione della sopravvivenza e con l\u2019aumentata espressione di p130CAS, il pi\uf9 importante adattatore nel complesso di segnalazione reclutato da beta1 integrina (beta1). L\u2019elevata espressione di PSMA nel tumore prostatico pi\uf9 aggressivo suggerisce che PSMA abbia un ruolo importante nella crescita e nella progressione del tumore stesso. Abbiamo gi\ue0 riportato che l\u2019aggregazione di PSMA, mediata da anticorpi specifici (cross-linking) induce l\u2019attivazione delle vie di segnalazione RAS-RAC-MAPK, la traslocazione di NF-kB, l\u2019espressione di IL-6 e la proliferazione in cellule LNCaP, un modello cellulare ampiamente utilizzato di tumore prostatico avanzato. In questo lavoro abbiamo studiato alcuni dei meccanismi molecolari necessari per l\u2019attivit\ue0 di segnalazione mediata da PSMA. PSMA ha un dominio intracellulare corto, privo di siti di autofosforilazione o di legame per chinasi o adattatori ed \ue8 quindi incapace di trasdurre segnali. Ha tuttavia un sito di legame per Filamin A (FLNa) attivo nella regolazione dell\u2019internalizzazione della molecola. E\u2019 noto che FLNa si associa anche con beta1, regolandone il traffico intracellulare e abbiamo ipotizzato quindi che FLNa potesse mediare una cooperazione tra PSMA e beta1 nell\u2019assemblaggio di una piattaforma di trasduzione. Questa ipotesi \ue8 stata verificata nelle seguenti linee cellulari: LNCaP, derivata da metastasi linfonodale ed esprimente PSMA, PC3-PSMA, originata da metastasi ossee, e dalla linea MCF7-PSMA, derivata da carcinoma mammario. PC3-PSMA e MCF7-PSMA sono state transfettate stabilmente con il gene umano PSMA. Inizialmente abbiamo dimostrato che il cross-linking di PSMA induce l\u2019attivazione concomitante delle vie di segnalazione AKT/mTOR/BAD e p38 e ERK1/2 MAPKs nelle tre linee cellulari, indipendentemente dalla loro differente origine. Il ruolo relativo delle singole vie nella sopravvivenza cellulare \ue8 stato valutato in LNCaP, mediante l\u2019inibizione farmacologica di PI3K, il principale attivatore di AKT (Wortmannina) o di p38 ed ERK1/2 (miscela di SB202190 e di PD98059 inibitore di MEK1, la chinasi attivante ERK). L\u2019apoptosi, sia reversibile che irreversibile, \ue8 stata quantificata rispettivamente mediante analisi citofluorimetrica dell\u2019esposizione di Annexin V o mediante ELISA, misurando il rilascio citosolico dei complessi DNA-istoni. La pi\uf9 significativa e sinergica induzione di apoptosi \ue8 stata osservata quando entrambe le vie di segnalazione venivano inibite simultaneamente. Dato che il cross-linking di PSMA regola l\u2019attivazione delle due vie di segnalazione anti-apoptotiche, abbiamo valutato se il cross-linking di PSMA potesse \u201csalvare\u201d le cellule LNCaP dall\u2019apoptosi indotta dalla deprivazione di siero. I risultati hanno chiaramente indicato che effettivamente PSMA esercita questa attivit\ue0. Inoltre, l\u2019attivit\ue0 anti-apoptotica mediata da PSMA viene abrogata quando \ue8 l\u2019attivazione di ERK1/2 o AKT, dimostrando cos\uec che l\u2019attivit\ue0 di PSMA dipende dalla sua abilit\ue0 di attivare queste chinasi. Tra le MAPKs, ERK1/2 sembra prevalere su p38. Come gi\ue0 menzionato, il dominio citoplasmatico di PSMA \ue8, da un punto di vista strutturale, incapace reclutare complessi di trasduzione. Di conseguenza, abbiamo considerato FLNa, beta1 Src e p130CAS come potenziali partners molecolari. L\u2019analisi al microscopio confocale ha indicato che PSMA e beta1 solo strettamente localizzati, in membrana, nella maggior parte delle cellule LNCaP. L\u2019analisi degli immunoprecipitati ottenuti da lisati cellulari di LNCaP, successivamente al cross-linking di PSMA, ha mostrato che beta1 e FLNa venivano co-immunoprecipitate insieme con p130CAS (pp130CAS) e Src (pSrc) in forma fosforilata. Parimenti, PSMA, FLNa, pp130CAS e pSrc venivano co-immunoprecipitate con beta1. Da notare che il cross-linking di PSMA \ue8 rapidamente seguito dalla fosforilazione di Src e p130CAS. Studiando la possibile localizzazione delle varie molecole nei \u201clipid rafts\u201d di membrana di cellule non trattate, abbiamo localizzato PSMA nel compartimento resistente al detergente Lubrol WX (DRMs) insieme con FLNa, beta1, Src e p130CAS fosforilate, ma non con Src e p130CAS non fosforilate, suggerendo cos\uec che le DRMs potrebbero rappresentare il compartimento privilegiato per la formazione dei complessi molecolari. Il primo e forse pi\uf9 importante effetto della formazione del complesso di segnalazione di PSMA \ue8 l\u2019attivazione di beta1. Come dimostrato mediante citofluorimetria, la bassa espressione dell\u2019epitopo di attivazione di beta1, HUTS-21, nelle tre linee cellulari aumentava drammaticamente dopo 5 minuti dal cross-linking di PSMA, raggiungeva il picco dopo 10 minuti e declinava dopo 20 minuti, mentre l\u2019espressione totale di beta1 rimaneva stabile, indicando cos\uec che PSMA e beta1 intrattengono una relazione funzionalenelle cellule di carcinoma prostatico. E\u2019 stato quindi valutato il ruolo funzionale dei differenti partners nell\u2019attivazione di AKT ed ERK1/2 indotta da cross-linking di PSMA mediante silenziamento genico (siRNA) o inibizione dell\u2019attivit\ue0 chinasica indotta da inibitori farmacologici. La cinetica di attivazione di AKT ed ERK1/2 \ue8 apparsa significativamente ridotta in cellule LNCaP silenziate per FLNa, per beta1 o per p130CAS, se confrontata con cellule con fenotipo normale (wt), dimostrando cos\uec che tutte e tre le molecole sono necessarie per l\u2019attivit\ue0 di segnalazione di PSMA. Un ruolo rilevante viene svolto anche da pSrc, la cui inibizione farmacologica (inibitori SU6656 o PP1), ha ridotto notevolmente la fosforilazione di ERK e di AKT, indotta dall\u2019attivazione di PSMA. Gli effetti del silenziamento di FLNa o di beta1 o di p130CAS sull\u2019attivit\ue0 anti-apoptotica di PSMA sono stati quindi valutati utilizzando colture 3D in Matrigel. Le cellule LNCaP formano colonie in Matrigel, al 4% di Fetal Bovine Serum (FBS). Il numero di colonie viene decisamente aumentato in seguito all\u2019attivazione di PSMA, dimezzato dalla deprivazione di siero, ma completamente ripristinato dopo attivazione di PSMA. Le colonie in assenza di siero hanno mostrato il fenotipo \u201cshrink\u201d delle cellule apoptotiche. Il silenziamento di FLNa non ha influito sul numero di colonie al 4% di FBS, n\ue9 ha aggravato gli effetti della deprivazione di siero, se confrontate con le cellule wt. Tuttavia, ha abrogato la capacit\ue0 di PSMA di promuovere la formazione di colonie cos\uec come di impedire l\u2019apoptosi nelle due condizioni. Il silenziamento di beta1 o p130CAS ha cambiato drammaticamente la crescita di LNCaP nelle culture 3D. Il numero di colonie al 4% di FBS e allo 0% di FBS \ue8 crollato fino a un decimo di quello generato dalle wt e il cross-linking di PSMA ha fallito la sua attivit\ue0 in entrambe le condizioni. Collettivamente, i nostri risultati dimostrano per la prima volta che l\u2019espressione di PSMA conferisce un notevole vantaggio alle cellule di carcinoma prostatico. Il reclutamento di un complesso di segnalazione che include FLNa, beta1, pp130CAS e pSrc, promuove la crescita delle cellule tumorale e contrasta gli stimoli apoptotici. Per quanto riguarda l\u2019attivazione di beta1 nelle cellule silenziate, abbiamo osservato dei risultati decisamente inattesi. L\u2019attivazione di PSMA \ue8 stata seguita da una forte attivazione di beta1. Tuttavia, questa attivazione non \ue8 mai risultata tempo\u2013dipendente n\ue9 seguita dall\u2019attivazione di ERK o AKT. Ulteriori indagini sono necessarie a riguardo.Prostate cancer (PCa) is the second cause of cancer-related death in males of developed countries. Pre-clinical evidence has revealed that virtually all specimens of advanced PCa express great amounts of Prostate Specific Membrane Antigen (PSMA), a transmembrane folate-hydrolase/ carboxypeptidase, associated in 60% of the samples with activation of survival pathways, with an increase of p130CAS, the major scaffolding protein of beta1 integrin signaling complex. Up-regulated PSMA expression in aggressive prostate tumors provides a selective advantage for tumor cells and a role for PSMA in cancer cell growth and progression is therefore envisaged. We have previously reported that PSMA clustering induces RAS-RAC-MAPK pathway activation, NF-kB translocation, IL-6 gene expression and proliferation in LNCaP cells, a widely used cellular model of advanced PCa cells. We here investigated some of the mechanisms required for signaling activity of PSMA. PSMA has a short intracellular cytodomain, devoid of phopshorylation or docking sites for kinase and adaptor and therefore unable to signal directly. However, it exposes a functional recognition site for Filamin A (FLNa). Recent evidence indicates that FLNa associates also with beta1 integrin (beta1) regulating its intracellular trafficking. This led us to make the hypothesis that FLNa could mediate a cooperation between PSMA and beta1 in the assembly of a signaling platform which may mediate, among others, the activation of anti apoptotic pathways. Our cellular system was constituted by the LNCaP cell line, derived from a lymphnodal metastasis and expressing PSMA endogenously, the PC3-PSMA cell line, originated from bone metastasis of PCa and the MCF7-PSMA cell line, derived from breast carcinoma. PC3-PSMA and MCF7-PSMA were stably transfected with the human PSMA gene. In the first part of the work we have investigated the signaling pathways engaged by PSMA activation (PSMA cross-linking), demonstrating that PSMA induced the parallel activation of both AKT/mTOR/BAD and p38 and ERK1/2 MAPKs pathways in the cell lines, irrespectively of their different origin. The relative role of the two pathways in the basal cell survival was evaluated next in LNCaP by using different doses of specific inhibitors of Wortmannin (inhibitor of PI3K, the main activator of AKT) or a mix of SB202190 (inhibitor of p38) and PD 98059 (inhibitor of MEK1, the ERK-activating kinase). Reversible and irreversible apoptosis was quantified respectively by FACS analysis of Annexin V exposure or ELISA measuring the cytosolic release of DNA\u2013hyston complexes. The most significant and synergic induction of apoptosis was observed when both pathways were inhibited simultaneously with a mix of all drugs used at maximal doses. As PSMA cross-linking regulates the activation of the two anti apoptotic pathways we next assayed if PSMA cross-linking could rescue LNCaP from apoptosis induced by serum withdrawal. Results clearly indicated that this was the case. Moreover, PSMA-induced rescue could be abrogated, by inhibiting MAPK p38 or ERK1/2 or PI3K pathways, thus demonstrating the PSMA activity depended on its ability to activate AKT, p38 and ERK1/2. Among MAPKs, ERK1/2 seemed to prevail on p38. As previously mentioned, PSMA cytodomain is by structurally unable to start the assembly of a signaling complex. We therefore considered FLNa, beta1, Src and p130CAS as potential molecular partners. Confocal analysis indicated that PSMA and beta1 were located in close proximity on the membrane of the majority of LNCaP cells. Immunoblotting of immunoprecipitates prepared from cell lysate of LNCaP cells after PSMA activation, showed that beta1 and FLNa could be pulled down from PSMA IPs, together with phopsphorylated p130CAS and Src. Conversely, PSMA, FLNa, pp130CAS and pSrc could be pulled down from beta1 IPs. Noteworthy, PSMA activation was rapidly followed by Src and p130CAS phosphorylation. In investigating possible location of the various molecules in the lipid rafts of untreated cells we found PSMA in Lubrol WX-detergent resistant membranes (DRMs) with FLNa, beta1, phosphorylated Src and p130CAS, but not with not phosphorylated Src and p130CAS, thus suggesting that DRMs could be a privileged membrane location for the complex formation. The first and perhaps most important effect of this complex assembly was the beta1 activation. As assessed by cytofluorometry, the very low expression of HUTS-21 activation epitope on beta1, in all the three PSMA-positive cell lines analysed, raised dramatically 5 min after PSMA cross-linking, peaked at 10 min and declined at 20 min whereas the total expression of beta1 remained stable throughout the experimental time, thus indicating that PSMA and beta1 entertained a very remarkable relationship in PCa cells. The role of the different partners of the PSMA-signaling complex, in the anti-apoptotic activity, was determined by siRNA silencing or chemical blockage. The phosphorylation of AKT and ERK1/2, induced by PSMA cross-linking, was greatly and significantly reduced at all points of the time courses in FLNa or beta1 or p130CAS silenced LNCaP if compared to the wt cells, thus demonstrating that both molecules were required for PSMA signaling activity. Src activity was as well needed, as specific Src inhibition, induced by SU6656 or PP1 drug, inhibited ERK phosphorylation and AKT phosphorylation, induced by PSMA activation. The effects of FLNa or beta1 or p130CAS silencing on PSMA-mediated rescue from apoptosis, were assayed by using 3D cultures. LNCaP cells do formed colonies in Matrigel at 4% FBS. The number of colonies was strongly increased upon PSMA activation, halved by serum deprivation but was fully restored if PSMA was activated. Colonies in starved samples showed the \u201cshrinking\u201d phenotype of apoptotic cells. FLNa silencing did not affect the ability of LNCaP cells to form colonies at 4% FBS, nor aggravated the effects of starvation if compared to wt cells. However, it abrogated the promoting/rescuing effect of PSMA cross-linking in the two conditions. Beta1 or p130CAS silencing changed dramatically the LNCaP growth in 3D culture. The number of colonies at 4% FBS and at 0% FBS dropped down up to one tenth of that of wt cells and PSMA cross-linking lacked activity in both conditions. Very unexpected results were instead observed regarding the beta1 activation in LNCaP, PC3-PSMA and MCF7-PSMA cells silenced for FLNa or p130CAS. PSMA activation was followed by a strong activation of beta1. However, this activation was not anymore time-dependent, nor followed by activation of ERK or AKT. More investigation is needed on this matter. In all, our results demonstrate for the first time that PSMA expression gives a remarkable advantage to PCa cells by recruiting a signaling complex, including FLNa, beta1, p130CAS and Src, and thereby supporting the activation of antiapototic and pro-proliferative signaling pathways

Prostate-specific membrane antigen (PSMA) assembles a macromolecular complex regulating growth and survival of prostate cancer cells "in vitro" and correlating with progression "in vivo"

The expression of Prostate Specific-Membrane Antigen (PSMA) increases in high-grade prostate carcinoma envisaging a role in growth and progression. We show here that clustering PSMA at LNCaP or PC3-PSMA cell membrane activates AKT and MAPK pathways thus promoting proliferation and survival. PSMA activity was dependent on the assembly of a macromolecular complex including filamin A, beta1 integrin, p130CAS, c-Src and EGFR. Within this complex beta1 integrin became activated thereby inducing a c-Src-dependent EGFR phosphorylation at Y1086 and Y1173 EGF-independent residues. Silencing or blocking experiments with drugs demonstrated that all the complex components were required for full PSMA-dependent promotion of cell growth and/or survival in 3D culture, but that p130CAS and EGFR exerted a major role. All PSMA complex components were found assembled in multiple samples of two high-grade prostate carcinomas and associated with EGFR phosphorylation at Y1086. The expression of p130CAS and pEGFRY1086 was thus analysed by tissue micro array in 16 castration-resistant prostate carcinomas selected from 309 carcinomas and stratified from GS 3+4 to GS 5+5. Patients with Gleason Score 645 resulted negative whereas those with GS 655 expressed p130CAS and pEGFRY1086 in 75% and 60% of the cases, respectively.Collectively, our results demonstrate for the first time that PSMA recruits a functionally active complex which is present in high-grade patients. In addition, two components of this complex, p130CAS and the novel pEGFRY1086, correlate with progression in castration-resistant patients and could be therefore useful in therapeutic or surveillance strategies of these patients