Luca Sorvillo

Protein kinase A as a biological target in cancer therapy

Background: cAMP is a second messenger that plays a role in intracellular signal transduction of various stimuli. a major function of cAMP in eukaryotes is activation of cAMP-dependent protein kinase (PKA). PKA is the best understood member of the serine-threonine protein kinase superfamily, and is involved in the control of a variety of cellular processes. since it has been implicated in the initiation and progression of many tumors, PKA has been suggested as a novel molecular target for cancer therapy. Objective/methods: here, after describing some features of cAMP/PKA signaling that are relevant to cancer biology, we review targeting of PKA in cancer therapy, also discussing PKA as a biomarker for cancer detection and monitoring of therapy. Results/conclusions: PKA is an increasingly relevant biological target in the therapy and management of cancer.

Leptin enhances growth inhibition by cAMP elevating agents through apoptosis of MDA-MB-231 breast cancer cells.

Elevation of cAMP inhibits the proliferation and expression of transformed phenotype in several cell types, including breast cancer cells. Leptin has been shown to act as a mitogen/survival factor in many types of cancer cells. In the present work, we have studied the impact of cAMP elevation on leptin-induced proliferation of breast cancer cells. Here we report that treatment of estrogen receptor negative human breast cancer cell line MDA-MB-231 with leptin or cAMP elevating agents has positive and negative effects on cell proliferation, respectively. Surprisingly, we find that leptin strongly potentiates the anti-proliferative action of cAMP elevating agents, by concurring to cell cycle arrest at G1 phase and inducing apoptosis. Pretreatment with the PKA inhibitor KT-5720 completely prevented the anti-proliferative effects induced by the combination between leptin and cAMP elevating agents. The above anti-proliferative effects were paralleled by the decrease of cyclin D1 and A and by the increase of inhibitor p27kip1 cell cycle regulating protein levels. In these conditions we found also a strong decrease of anti-apopotic Bcl2 protein levels. Altogether, our data extend the evidence of adenylate cyclase/cAMP/PKA as a growth suppressor system and of leptin as a growth promoting factor in breast cancer cells. Remarkably, our results suggest that when cAMP levels are increased, leptin drives cells towards apoptosis, and that targeting both cAMP levels and leptin signalling might represent a simple novel way for therapeutic intervention in breast cancer.

Inorganic phosphate enhances sensitivity of human osteosarcoma U2OS cells to doxorubicin via a p53-dependent pathway.

Osteosarcoma is the most common malignant primary bone tumor in children and adolescents. The clinical outcome for osteosarcoma remains discouraging despite aggressive surgery and intensive radiotherapy and chemotherapy regimens. Thus, novel therapeutic approaches are needed. Previously, we have shown that inorganic phosphate (Pi) inhibits proliferation and aggressiveness of human osteosarcoma U2OS cells identifying adenylate cyclase, beta3 integrin, Rap1, ERK1/2 as proteins whose expression and function are relevantly affected in response to Pi. In this study, we investigated whether Pi could affect chemosensitivity of osteosarcoma cells and the underlying molecular mechanisms. Here, we report that Pi inhibits proliferation of p53‐wild type U2OS cells (and not of p53‐null Saos and p53‐mutant MG63 cells) by slowing‐down cell cycle progression, without apoptosis occurrence. Interestingly, we found that Pi strongly enhances doxorubicin‐induced cytotoxicity in U2OS, and not in Saos and MG63 cells, by apoptosis induction, as revealed by a marked increase of sub‐G1 population, Bcl‐2 downregulation, caspase‐3 activation, and PARP cleavage. Remarkably, Pi/doxorubicin combination‐induced cytotoxicity was accompanied by an increase of p53 protein levels and of p53 target genes mdm2, p21 and Bax, and was significantly reduced by the p53 inhibitor pifithrine‐alpha. Moreover, the doxorubicin‐induced cytotoxicity was associated with ERK1/2 pathway inhibition in response to Pi. Altogether, our data enforce the evidence of Pi as a novel signaling molecule capable of inhibiting ERK pathway and inducing sensitization to doxorubicin of osteosarcoma cells by p53‐dependent apoptosis, implying that targeting Pi levels might represent a rational strategy for improving osteosarcoma therapy. J. Cell. Physiol. 228: 198–206, 2013.

Inorganic phosphate inhibits growth of human osteosarcoma U2OS cells via adenylate cyclase/cAMP pathway

In order to elucidate how phosphate regulates cellular functions, we investigated the effects of inorganic phosphate (Pi) on adenylate cyclase (AC)/cyclic AMP (cAMP) axis. Here we describe that Pi treatment of human osteosarcoma U2OS cells results in a decrease of both intracellular cAMP levels and AC activity, and in a cell growth inhibition. The phosphate‐triggered effects observed in U2OS cells are not a widespread phenomenon regarding all cell lines, since other cell lines screened respond differently to parallel Pi treatments. In U2OS cell line, the AC activity/cAMP downregulation is accompanied by significant variations in the levels of some membrane proteins belonging to the AC system. Remarkably, the above effects are blunted by pharmacological inhibition of sodium‐dependent phosphate transport. Moreover, 8‐Br‐cAMP and other cAMP‐elevating agents, such as IBMX and forskolin, interestingly, prevent the cell growth inhibition in response to phosphate. Our results enforce the increasing evidences of phosphate as a signaling molecule, identifying in U2OS cell line the AC/cAMP axis, as a novel‐signaling pathway modulated by phosphate to ultimately affect cell growth. J. Cell. Biochem.

cAMP Elevation Down-Regulates β3 Integrin and Focal Adhesion Kinase and Inhibits Leptin-Induced Migration of MDA-MB-231 Breast Cancer Cells.

Abstract Breast cancer is one of the most common malignancies and a major cause of cancer death among women worldwide. The high mortality rate associated with breast cancer is mainly due to a propensity of the tumor to metastasize, even if small or undetectable. Given the relevant role of leptin in breast cancer growth and metastasis, novel strategies to counteract biological effects of this obesity-linked cytokine are warranted. Recently, we demonstrated that in MDA-MB-231 breast cancer cells, intracellular cAMP elevation completely abrogates both ERK1/2 and STAT3 phosphorylation in response to leptin. Very surprisingly, this provided evidence that when cAMP levels are increased, leptin drives cells towards apoptosis associated with a marked decrease of Bcl2 protein levels and accompanied by down-regulation of protein kinase A (PKA). The aim of the current study was to investigate the role of cAMP in leptin-associated motility of breast cancer cells. Here we show that cAMP elevation completely prevents leptin-induced migration of MDA-MB-231 breast cancer cells. Interestingly, the inhibition by cAMP-elevating agents of leptin-mediated cell migration is accompanied by a strong decrease of β3 integrin subunit and focal adhesion kinase (FAK) protein levels. Analysis of the underlying cAMP-dependent molecular mechanisms revealed that PKA blockers partly counteract the inhibition of leptin-induced migration and completely prevent the antiproliferative action by cAMP elevation. Moreover, a cAMP analogue that specifically activates Epac and not PKA has an inhibitory effect on leptin-induced cell migration as well. The present study confirms initial evidence for the efficacy of cAMP elevation against oncogenic effects of leptin, identifies β3 integrin subunit and FAK as proteins strongly down-regulated by cAMP elevation, and suggests that both cAMP/PKA- and cAMP/Epac-dependent pathways are involved in inhibition of leptin-induced migration of MDA-MB-231 breast cancer cells. The potential clinical significance and therapeutic applications of our data are discussed.

Inorganic Phosphate Prevents Erk1/2 and Stat3 Activation and Improves Sensitivity to Doxorubicin of MDA-MB-231 Breast Cancer Cells.

Due to its expression profile, triple-negative breast cancer (TNBC) is refractory to the most effective targeted therapies available for breast cancer treatment. Thus, cytotoxic chemotherapy represents the mainstay of treatment for early and metastatic TNBC. Therefore, it would be greatly beneficial to develop therapeutic approaches that cause TNBC cells to increase their sensitivity to cytotoxic drugs. Inorganic phosphate (Pi) is emerging as an important signaling molecule in many cell types. Interestingly, it has been shown that Pi greatly enhances the sensitivity of human osteosarcoma cell line (U2OS) to doxorubicin. We investigated the effects of Pi on the sensitivity of TNBC cells to doxorubicin and the underlying molecular mechanisms, carrying out flow cytometry-based assays of cell-cycle progression and cell death, MTT assays, direct cell number counting and immunoblotting experiments. We report that Pi inhibits the proliferation of triple-negative MDA-MB-231 breast cancer cells mainly by slowing down cell cycle progression. Interestingly, we found that Pi strongly increases doxorubicin-induced cytotoxicity in MDA-MB-231 cells by apoptosis induction, as revealed by a marked increase of sub-G1 population, Bcl-2 downregulation, caspase-3 activation and PARP cleavage. Remarkably, Pi/doxorubicin combination-induced cytotoxicity was dynamically accompanied by profound changes in Erk1/2 and Stat3 protein and phosphorylation levels. Altogether, our data enforce the evidence of Pi acting as a signaling molecule in MDA-MB-231 cells, capable of inhibiting Erk and Stat3 pathways and inducing sensitization to doxorubicin of TNBC cells, and suggest that targeting Pi levels at local sites might represent the rationale for developing effective and inexpensive strategies for improving triple-negative breast cancer therapy.

Integrating leptin and cAMP signalling pathways in triple-negative breast cancer cells.

Breast cancer is a major cause of cancer death in women in the world. Triple-negative breast cancers, which accounts for 10-20% of all mammary tumours, are characterised by an aggressive phenotype, are often found in younger women and have been associated with poor prognosis. Obesity increases the risk for triple-negative breast cancer occurrence. Because triple-negative breast cancer patients are unresponsive to current targeted therapies and other treatment options are only partially effective, new pharmacological approaches are warranted. The obesity-linked adipokine, leptin, is a well known mitogen/survival factor in breast cancer cells and several studies have addressed the role of leptin in breast cancer pathogenesis and progression. Surprisingly, recent in vitro studies have shown that leptin enhances the anti-proliferative effects of cAMP elevation in triple-negative breast cancer cells by apoptosis induction. In the current review, we discuss on the role of cAMP as a growth suppressor and of leptin as a growth promoting factor in breast cancer cells and we will focus on the molecular pathways involved in the antiproliferative interaction between leptin and cAMP elevation. The rationale for the possible development of a simple, cheap and innovative approach for therapeutic intervention in triple-negative breast cancer, based on the use of cAMP elevating drugs at lower and tolerable doses, will be also discussed.

Synergistic cytotoxic effects of inorganic phosphate and chemotherapeutic drugs on human osteosarcoma cells

Novel therapeutic approaches are required for the treatment of osteosarcoma. Combination chemotherapy is receiving increased attention in order to identify compounds that may increase the therapeutic index of clinical anticancer drugs. In this regard, naturally occurring molecules with antitumor activity and with limited toxicity to normal tissues have been suggested as possible candidates for investigation of their synergistic efficacy in combination with antineoplastic drugs. Inorganic phosphate (Pi) is an essential nutrient for living organisms. Relevantly, Pi has emerged as an important signaling molecule capable of modulating multiple cellular functions by altering signal transduction pathways, gene expression and protein abundance in many cell types. Previously, we showed that Pi inhibits proliferation and aggressiveness of U2OS human osteosarcoma cells and that Pi is capable of inducing sensitization of osteosarcoma cells to doxorubicin in a p53-dependent manner. In this study, we extended the role of Pi in the chemosensitivity of osteosarcoma cells to other anticancer drugs. Specifically, we report and compare the antiproliferative effects of a combination between Pi and doxorubicin, Taxol® and 5-fluorouracil (5-FU) treatments. We found that Pi increases the antiproliferative response to both Taxol and doxorubicin to a similar extent. On the other hand, Pi did not potentiate the anticancer effects induced by 5-FU. These effects were paralleled by apoptosis induction and were cell cycle-dependent. The clinical significance of our data and their potential therapeutic applications for improving osteosarcoma treatment are discussed.

Inorganic Phosphate as a Novel Signaling Molecule with Antiproliferative Action in MDA-MB-231 Breast Cancer Cells

Abstract Inorganic phosphate (Pi) is an essential nutrient for living organisms. It plays a key role in diverse physiological functions, including osteoblast differentiation and skeletal mineralization. Relevantly, Pi is emerging as an important signaling molecule capable of modulating multiple cellular functions by altering signal transduction pathways, gene expression, and protein abundance in many cell types. To our knowledge, the consequences of elevated Pi on behavior of breast cancer cells have been poorly addressed. In this study we investigate the effects of Pi on proliferation of MDA-MB-231 breast cancer cells. We report that Pi inhibits proliferation of MDA-MB-231 cells by slowing cell cycle progression, without apoptosis occurrence. We found that Pi causes cells to accumulate in G1 phase in a time-dependent manner. Accordingly, G1 accumulation was associated with a decrease of cyclin A and cyclin E and an increase of cell cycle inhibitors p21 and p27 protein levels, respectively. Moreover, the Pi-induced antiproliferative effect was dynamically accompanied by profound changes in ERK1/2 and STAT3 protein and phosphorylation levels in response to Pi. Altogether, our data represent the first evidence of Pi acting as a novel signaling molecule in MDA-MB-231 breast cancer cells, capable of eliciting a strong antiproliferative action and suggest that targeting Pi levels at local sites might represent the rationale for developing novel strategies for therapeutic intervention in triple-negative breast cancer.

Inorganic phosphate as a signaling molecule: a potential strategy in osteosarcoma treatment.

Inorganic phosphate (Pi) is an essential nutrient to living organisms. It plays a key role in diverse biological processes, including osteoblast differentiation and skeletal mineralization. Maintenance of proper Pi homeostasis is a critical event, as any deviation from that state can lead to several acute and chronic disease states and influence the ageing process and lifespan. Serum Pi level is maintained within a narrow range through a complex interplay between intestinal absorption, exchange with intracellular and bone storage pools, renal tubular reabsorption and depends mainly on the activity of Na/Pi cotransporters. Pi is abundant in the diet and intestinal absorption of Pi is efficient and minimally regulated. The kidney is a major regulator of Pi homeostasis and can increase or decrease its Pi reabsorptive capacity to accommodate Pi need. Relevantly, Pi is emerging as an important signalling molecule capable of modulating multiple cellular functions by altering signal transduction pathways, gene expression and protein abundance in many cell types. However, little is known about the initial events involving the detection of changes in serum or local Pi concentrations and the subsequent downstream regulation cascade. Previously, we provided evidence that Pi inhibits proliferation and aggressiveness of human osteosarcoma U2OS cells identifying adenylate cyclase, beta3 integrin, Rap1, ERK1/2 as proteins whose expression and function are relevantly affected in response to Pi. More recently, we demonstrated that Pi is capable also of inducing sensitization of osteosarcoma cells to doxorubicin in a p53-dependent manner and through a mechanism involving ERK1/2 down-regulation. This review summarizes the current knowledge regarding inorganic phosphate as a novel specific signaling molecule in bone and other cell types in mammals and discuss how targeting Pi levels at local sites might represent a potential strategy for improving osteosarcoma therapy.