Mutations of the p53 gene hallmark many human cancers. Several p53 mutant proteins acquire the capability to promote cancer progression and metastasis, a phenomenon defined as Gain of Oncogenic Function (GOF). The downstream targets by which GOF p53 mutants perturb cellular programs relevant to oncogenesis are only partially known. We have previously demonstrated that SLC25A1 (CIC) promotes tumorigenesis, while its inhibition blunts tumor growth. We now report that CIC is a direct transcriptional target of several p53 mutants. We identify a novel interaction between mutant p53 (mutp53) and the transcription factor FOXO-1 which is responsible for regulation of CIC expression levels. Tumor cells harboring mutp53 display higher CIC levels relative to p53 null or wild-type tumors, and inhibition of CIC activity blunts mutp53-driven tumor growth, partially overcoming GOF activity. CIC inhibition also enhances the chemotherapeutic potential of platinum-based agents. Finally, we found that elevated CIC levels predict poor survival outcome in tumors hallmarked by high frequency of p53 mutations. Our results identify CIC as a novel target of mutp53 and imply that the employment of CIC inhibitors may improve survival rates and reduce chemo-resistance in tumors harboring these types of mutations, which are among the most intractable forms of cancers.

In response to diverse genotoxic stimuli p53 is activated as transcription factor to exert its tumor-suppressor function. P53 activation requires protein stabilization, nuclear localization and posttranslational modifications in key residues that may influence p53 selection of target genes. Among them, serine 46 (Ser46) phosphorylation is considered specific for apoptotic activation. Hyperglicaemia, the high blood glucose condition, may negatively affect tumor response to therapies through several mechanisms, conferring resistance to drug-induced cell death. However, whether high glucose might modify p53Ser46 phosphorylation has never been addressed.

Understanding the mechanisms of autophagy induction and its role during chemotherapeutic treatments is of fundamental importance in order to manipulate it to improve the outcome of chemotherapy. In particular whether the bortezomib-induced autophagy plays a pro-survival or pro-death role is still controversial. In this study we investigated if bortezomib induced endoplasmic reticulum (ER) stress and activated autophagy in Primary Effusion Lymphoma (PEL) cells and how they influenced cell survival. We found that bortezomib induced up-regulation of the pro-survival and pro-death ER stress molecules BIP and CHOP and activated c-Jun NH2-terminal kinase (JNK), resulting in Bcl-2 phosphorylation and induction of autophagy. JNK and autophagy activation played a pro-survival role in this setting, thus their inhibition increased the bortezomib cytotoxic effect and PARP cleavage in PEL cells. Based on our results we suggest that the combination of bortezomib with JNK or autophagy inhibitors could be exploited to improve the outcome of therapy of this aggressive B cell lymphoma.

As an important cellular stress sensor phosphoprotein p53 can trigger cell cycle arrest and apoptosis and regulate autophagy. The p53 activity mainly depends on its transactivating function, however, how p53 can select one or another biological outcome is still a matter of profound studies. Our previous findings indicate that switching cancer cells in high glucose (HG) impairs p53 apoptotic function and the transcription of target gene PUMA.

Activation of p53-mediated gene transcription is a critical cellular response to DNA damage and involves a phosphorylation-acetylation cascade of p53. The discovery of differences in the response to different agents raises the question whether some of the p53 oncosuppressor functions might be exerted by different posttranslational modifications. Stress-induced homeodomain-interacting protein kinase-2 (HIPK2) phosphorylates p53 at serine-46 (Ser46) for p53 apoptotic activity; p53 acetylation at different C-terminus lysines including p300-mediated lysine-382 (Lys382) is also required for full activation of p53 transcriptional activity. The purpose of the current study was to evaluate the interplay among HIPK2, p300, and p53 in p53 acetylation and apoptotic transcriptional activity in response to drug by using siRNA interference, p300 overexpression or deacetylase inhibitors, in cancer cells.

Apigenin is a flavonoid widely distributed in plant kingdom that exerts cytotoxic effects against a variety of solid and haematological cancers. In this study, we investigated the effect of apigenin against primary effusion lymphoma (PEL), a KSHV-associated B cell lymphoma characterized by a very aggressive behavior, displaying constitutive activation of STAT3 as well as of other oncogenic pathways and harboring wtp53.

The hyperactivation of nuclear factor erythroid 2 p45-related factor 2 (NRF2), frequently found in many tumor types, can be responsible for cancer resistance to therapies and poor patient prognosis. Curcumin has been shown to activate NRF2 that has cytotprotective or protumorigenic roles according to tumor stage. The present study aimed at investigating whether the zinc-curcumin Zn(II)-curc compound, which we previously showed to display anticancer effects through multiple mechanisms, could induce NRF2 activation and to explore the underlying molecular mechanisms. Biochemical studies showed that Zn(II)-curc treatment increased the NRF2 protein levels along with its targets, heme oxygenase-1 (HO-1) and p62/SQSTM1, while markedly reduced the levels of Keap1 (Kelch-like ECH-associated protein 1), the NRF2 inhibitor, in the cancer cell lines analyzed. The silencing of either NRF2 or p62/SQSTM1 with specific siRNA demonstrated the crosstalk between the two molecules and that the knockdown of either molecule increased the cancer cell sensitivity to Zn(II)-curc-induced cell death. This suggests that the crosstalk between p62/SQSTM1 and NRF2 could be therapeutically exploited to increase cancer patient response to therapies.

Organometallic ruthenium (Ru)(II)-cymene complexes display promising pharmacological properties and might represent alternative therapeutic agents in medical applications. Polyphenols, such as curcumin and curcuminoids, display beneficial properties in medicine, including chemoprevention. Here we analyzed the anticancer effect of a cationic Ruthenium (Ru)(II)-cymene Bisdemethoxycurcumin (Ru-bdcurc) complex. The experimental data show that Ru-bdcurc induced cell death of colon cancer cells in vitro. In response to treatment, cancer cells activated the endoplasmic reticulum (ER)-resident chaperone GRP78/BiP and NRF2, the master regulators of the unfolded protein response (UPR) and the antioxidant response, respectively. Pharmacologic targeting of either NRF2 or BiP potentiated the cytotoxic effect of Ru-bdcurc. We also found that NRF2 and UPR pathways were interconnected as the inhibition of NRF2 reduced BiP protein levels. Mechanistically, the increased Ru-bdcurc-induced cell death, following NRF2 or BiP inhibition, correlated with the upregulation of the UPR apoptotic marker CHOP and with increased H2AX phosphorylation, a marker of DNA damage. The findings reveal that BiP and NRF2 interconnection was a key regulator of colon cancer cells resistance to Ru-bdcurc cytotoxic effect. Targeting that interconnection overcame the protective mechanism and enhanced the antitumor effect of the Ru-bdcurc compound.

Mutations of the p53 oncosuppressor gene are amongst the most frequent aberration seen in human cancer. Some mutant (mt) p53 proteins are prone to loss of Zn(II) ion that is bound to the wild-type (wt) core, promoting protein aggregation and therefore unfolding. Misfolded p53 protein conformation impairs wtp53-DNA binding and transactivation activities, favouring tumor growth and resistance to antitumor therapies. Screening studies, devoted to identify small molecules that reactivate mtp53, represent therefore an attractive anti-cancer therapeutic strategy. Here we tested a novel fluorescent curcumin-based Zn(II)-complex (Zn-curc) to evaluate its effect on mtp53 reactivation in cancer cells.

The Tyr705 STAT3 constitutive activation, besides promoting PEL cell survival, contributes to the maintenance of viral latency. We found indeed that its de-phosphorylation by AG490 induced KSHV lytic cycle. Moreover, Tyr705 STAT3 de-phosphorylation, mediated by the activation of tyrosine phosphatases, together with the increase of Ser727 STAT3 phosphorylation contributed to KSHV lytic cycle induction by TPA. We then observed that p53-p21 axis, essential for the induction of KSHV replication, was activated by the inhibition of Tyr705 and by the increase of Ser727 STAT3 phosphorylation. As a possible link between STAT3, p53-p21 and KSHV lytic cycle, we found that TPA and AG490 reduced the expression of KAP-1, promoting p53 stability, p21 transcription and KSHV lytic cycle activation in PEL cells.

Metformin, the most used drug for the treatment of diabetes type 2 patients, has been shown to have anti-cancer properties. In this study, we found that metformin induced apoptosis in Primary Effusion Lymphoma (PEL) cells, an aggressive B cell lymphoma associated with KSHV against which the conventional therapies usually fail. The cytotoxic effect of metformin correlated with intracellular reactive oxygen species reduction, activation of AMPK, the inhibition of pro-survival pathways such as mTOR and STAT3 and the down-regulation of v-FLIP, a latent viral antigen that also plays a pivotal role in PEL cell survival. Interestingly, we found that metformin could be used to potentiate the bortezomib-mediated cytotoxicity against PEL cells and to inhibit the activation of KSHV lytic cycle, a side effect of this treatment that resulted in a block of autophagy in these cells. Mechanistically, metformin altered UPR activated by bortezomib, leading to a reduced expression of BiP, up-regulation of CHOP and down-regulation of Bcl-2. In summary, this study suggests that metformin could represent a promising strategy for the treatment of PEL alone or in combination with bortezomib. In the latter case, besides exerting a stronger cytotoxic effect, it might be used to restrain bortezomib-induced viral replication that is involved in the maintenance and progression of KSHV-associated malignancies.

Homeodomain-interacting protein kinase 2 (HIPK2) is a multifunctional protein that exploits its kinase activity to modulate key molecular pathways in cancer to restrain tumor growth and induce response to therapies. For instance, HIPK2 knockdown induces upregulation of oncogenic hypoxia-inducible factor-1 (HIF-1) activity leading to a constitutive hypoxic and angiogenic phenotype with increased tumor growth in vivo. HIPK2 inhibition, therefore, releases pathways leading to production of pro-inflammatory molecules such as vascular endothelial growth factor (VEGF) or prostaglandin E2 (PGE(2)). Tumor-produced inflammatory mediators other than promote tumour growth and vascular development may permit evasion of anti-tumour immune responses. Thus, dendritic cells (DCs) dysfunction induced by tumor-produced molecules, may allow tumor cells to escape immunosurveillance. Here we evaluated the molecular mechanism of PGE(2) production after HIPK2 depletion and how to modulate it.

Homeodomain-interacting protein kinase-2 (HIPK2), a transcriptional co-repressor with apoptotic function, can affect hypoxia-inducible factor 1 (HIF-1) transcriptional activity, through downmodulation of its HIF-1alpha subunit, in normoxic condition. Under hypoxia, a condition often found in solid tumors, HIF-1alpha is activated to induce target genes involved in chemoresistance, inhibition of apoptosis and tumor progression. Here, we investigated whether the HIPK2 overexpression could downregulate HIF-1alpha expression and activity in tumor cells treated with hypoxia-mimicking condition, and evaluated whether HIPK2-dependent downregulation of HIF-1alpha could sensitize chemoresistant tumor cells to adriamycin (ADR)-induced apoptosis.

Homeodomain-interacting protein kinase-2 (HIPK2) plays an essential role in restraining tumor progression as it may regulate, by itself or within multiprotein complexes, many proteins (mainly transcription factors) involved in cell growth and apoptosis. This study takes advantage of the recent finding that HIPK2 may repress the beta-catenin transcription activity. Thus, we investigated whether HIPK2 overexpression may down-regulate vascular endothelial growth factor (VEGF) levels (a beta-catenin target gene) and the role of beta-catenin in this regulation, in order to consider HIPK2 as a tool for novel anti-tumoral therapeutical approaches.

The oncogenic gammaherpesvirus Epstein-Barr virus (EBV) immortalizes in vitro B lymphocytes into lymphoblastoid cell lines (LCLs), a model that gives the opportunity to explore the molecular mechanisms driving viral tumorigenesis. In this study, we addressed the potential of quercetin, a widely distributed flavonoid displaying antioxidant, anti-inflammatory, and anti-cancer properties, in preventing EBV-driven B cell immortalization. The results obtained indicated that quercetin inhibited thectivation of signal transducer and activator of transcription 3 (STAT3) induced by EBV infection and reduced molecules such as interleukin-6 (IL-6) and reactive oxidative species (ROS) known to be essential for the immortalization process. Moreover, we found that quercetin promoted autophagy and counteracted the accumulation of sequestosome1/p62 (SQSTM1/p62), ultimately leading to the prevention of B cell immortalization. These findings suggest that quercetin may have the potential to be used to counteract EBV-driven lymphomagenesis, especially if its stability is improved.

Colon cancer represents one of the most common and aggressive cancers in its advanced state. Among the most innovative anti-cancer approaches, the manipulation of UPR is a promising one, effective also against cancers carrying dysfunctional p53. Interestingly, it is emerging that UPR cross-talks with DDR and that targeting the interplay between these two adaptive responses may be exploited to overcome the resistance to the single DDR- and UPR-targeting treatments. Previous studies have highlighted the role of IRE1 alpha and PERK UPR sensors on DDR, while the impact of ATF6 on this process remains under-investigated. This study shows for the first time that ATF6 sustains the expression level of BRCA-1 and protects colon cancer cells from the cytotoxic effect of ER stressors DPE and Thapsigargin. At molecular level, ATF6 activates mTOR to sustain the expression of HSP90, of which BRCA-1 is a client protein. Therefore, pharmacological or genetic inhibition of ATF6 promoted BRCA-1 degradation and increased DNA damage and cell death, particularly in combination with Adriamycin. All together this study suggests that targeting ATF6 may not only potentiate the cytotoxic effect of drugs triggering ER stress but may render colon cancer cells more sensitive to Adriamycin and possibly to other DNA damaging agents used to treat colon cancer.

Homeodomain interacting protein kinase-2 (HIPK2) is an evolutionary conserved kinase that modulates several key molecular pathways to restrain tumor growth and induce p53-depending apoptotic cell-death in response to anticancer therapies. HIPK2 silencing in cancer cells leads to chemoresistance and cancer progression, in part due to p53 inhibition. Recently, hyperglycemia has been shown to reduce p53 phosphorylation at serine 46 (Ser46), the target residue of HIPK2, thus impairing p53 apoptotic function. Here we asked whether hyperglycemia could, upstream of p53, target HIPK2. We focused on the effect of high glucose (HG) on HIPK2 protein stability and the underlying mechanisms. We found that HG reduced HIPK2 protein levels, therefore impairing HIPK2-induced p53 apoptotic activity. HG-triggered HIPK2 protein downregulation was rescued by both proteasome inhibitor MG132 and by protein phosphatase inhibitors Calyculin A (CL-A) and Okadaic Acid (OA). Looking for the phosphatase involved, we found that protein phosphatase 2A (PP2A) induced HIPK2 degradation, as evidenced by directly activating PP2A with FTY720 or by silencing PP2A with siRNA in HG condition. The effect of PP2A on HIPK2 protein degradation could be in part due to hypoxia-inducible factor-1 (HIF-1) activity which has been previously shown to induce HIPK2 proteasomal degradation through several ubiquitin ligases. Validation analysed performed with HIF-1α dominant negative or with silencing of Siah2 ubiquitin ligase clearly showed rescue of HG-induced HIPK2 degradation. These findings demonstrate how hyperglycemia, through a complex protein cascade, induced HIPK2 downregulation and consequently impaired p53 apoptotic activity, revealing a novel link between diabetes/obesity and tumor resistance to therapies.

Kaposi's Sarcoma Herpesvirus (KSHV) is a gammaherpesvirus strongly linked to human cancer. The virus is also able to induce immune suppression, effect that contributes to onset/progression of the viral-associated malignancies. As KSHV may infect macrophages and these cells abundantly infiltrate Kaposi's sarcoma lesions, in this study we investigated whether KSHV-infection could affect macrophage polarisation to promote tumorigenesis.

Obesity is the result of white adipose tissue accumulation where excess of food energy is stored to form triglycerides. De novo lipogenesis (DNL) is the continuous process of new fat production and is driven by the transcription factor ChREBP. During adipogenesis, white adipocytes change their morphology and the entire cell volume is occupied by one large lipid droplet. Recent studies have implicated an essential role of autophagy in adipogenic differentiation, cytoplasmic remodelling and mitochondria reorganization. The phenolic monoterpenoid carvacrol (2-methyl-5-[1-methylethyl]phenol), produced by numerous aromatic plants, has been shown to reduce lipid accumulation in murine 3T3-L1 cells during adipogenic differentiation by modulating genes associated with adipogenesis and inflammation. Therefore, the aim of this study was to evaluate whether carvacrol could affect autophagy and ChREBP expression during adipogenic differentiation.

Activation of wild-type p53 in response to genotoxic stress occurs through different mechanisms including protein conformation, posttranslational modifications, and nuclear localization, leading to DNA binding to sequence-specific promoters. Zinc ion plays a crucial role in stabilizing p53/DNA binding to induce canonical target genes. Mutant p53 proteins undergo protein misfolding that can be counteracted by zinc. However, whether zinc supplementation might have a beneficial antitumor effect in wild-type p53-carrying cells in combination with drugs, has not been addressed so far.