The presence of hypoxic areas is common in all breast lesions but no data clearly correlate low oxygenation with the acquisition of malignant features by non-invasive cells, particularly by cells from ductal carcinoma in situ (DCIS), the most frequently diagnosed tumor in women.

Cells in non-invasive breast lesions are widely believed to possess molecular alterations that render them either susceptible or refractory to the acquisition of invasive capability. One such alteration could be the ectopic expression of the β2 isoform of phosphoinositide-dependent phospholipase C (PLC-β2), known to counteract the effects of hypoxia in low-invasive breast tumor-derived cells. Here, we studied the correlation between PLC-β2 levels and the propensity of non-invasive breast tumor cells to acquire malignant features. Using archival FFPE samples and DCIS-derived cells, we demonstrate that PLC-β2 is up-regulated in DCIS and that its forced down-modulation induces an epithelial-to-mesenchymal shift, expression of the cancer stem cell marker CD133, and the acquisition of invasive properties. The ectopic expression of PLC-β2 in non-transformed and DCIS-derived cells is, to some extent, dependent on the de-regulation of miR-146a, a tumor suppressor miRNA in invasive breast cancer. Interestingly, an inverse relationship between the two molecules, indicative of a role of miR-146a in targeting PLC-β2, was not detected in primary DCIS from patients who developed a second invasive breast neoplasia. This suggests that alterations of the PLC-β2/miR-146a relationship in DCIS may constitute a molecular risk factor for the appearance of new breast lesions. Since neither traditional classification systems nor molecular characterizations are able to predict the malignant potential of DCIS, as is possible for invasive ductal carcinoma (IDC), we propose that the assessment of the PLC-β2/miR-146a levels at diagnosis could be beneficial for identifying whether DCIS patients may have either a low or high propensity for invasive recurrence.

Vav1 is one of the signalling proteins normally restricted to hematopoietic cells that results ectopically expressed in solid tumors, including breast cancer. By immunohistochemical analysis on TMAs containing invasive breast tumor from patients without lymph node involvement, we have found that Vav1 is expressed in almost all investigated cancers and shows a peculiar localization inside the nucleus of tumor cells. High amounts of nuclear Vav1 are positively correlated with low incidence of relapse, regardless phenotype and molecular subtype of breast neoplasia. In particular, Kaplan-Meier plots showed an elevated risk of distant metastasis in patients with low Vav1 expression compared with patients with high Vav1 expression in their tumors. Experiments performed with breast tumor-derived cells indicated that Vav1 negatively modulates their invasiveness in vitro and their metastatic efficiency in vivo, possibly by affecting the expression of genes involved in invasion and/or metastasis of breast tumors. Since the high heterogeneity of breast tumors makes difficult to predict the evolution of early breast neoplasias, the evaluation of nuclear Vav1 levels may help in the characterization and management of early breast cancer patients. In particular, Vav1 may serve as a prognostic biomarker and a target for new therapies aimed to prevent breast cancer progression.

Osteosarcoma (OS) is a rare, insidious tumor of mesenchymal origin that most often affects children, adolescents, and young adults. While the primary tumor can be controlled with chemotherapy and surgery, it is the lung metastases that are eventually fatal. Multiple studies into the initial drivers of OS development have been undertaken, but few of these have examined innate immune/inflammatory signaling. A central figure in inflammatory signaling is the innate immune/stress-activated kinase double-stranded RNA-dependent protein kinase (PKR). To characterize the role of PKR in OS, U2OS, and SaOS-2 osteosarcoma cell lines were stably transfected with wild-type or dominant-negative (DN) PKR. Overexpression of PKR enhanced colony formation in soft agar (U2OS and SaOS-2), enhanced cellular migration (U2OS), and invasive migration (SaOS-2). In contrast, overexpression of DN-PKR inhibited attachment-independent growth, migration and/or invasion. These data demonstrate a role for inflammatory signaling in OS formation and migration/invasion and suggest the status of PKR expression/activation may have prognostic value.

Triple negative breast cancer (TNBC) represents the most aggressive breast tumor, showing a high intrinsic variability in terms of both histopathological features and response to therapies. Blocking the Akt signaling pathway is a well-studied approach in the treatment of aggressive breast tumors. The high homology among the Akt isoforms and their distinct, and possibly opposite, oncogenic functions made it difficult to develop effective drugs. Here we investigated the role of Vav1 as a potential down-regulator of individual Akt isozymes. We revealed that the over-expression of Vav1 in triple negative MDA-MB-231 cells reduced only the Akt2 isoform, acting at the post-transcriptional level through the up-modulation of miR-29b. The Vav1/miR-29b dependent decrease in Akt2 was correlated with a reduced lung colonization of circulating MDA-MB-231 cells. In cell lines established from PDX, the Vav1 induced down-modulation of Akt2 is strongly dependent on miR-29b and occurs only in some TNBC tumors. These findings may contribute to better classify breast tumors having the triple negative phenotype, and suggest that the activation of the Vav1/miR-29b axis, precisely regulating the amount of an Akt isozyme crucial for tumor dissemination, could have great potential for driving more accurate therapies to TNBCs, often not eligible or resistant to treatments.

The phosphoinositide-dependent signal transduction pathway has been implicated in the control of a variety of biologic processes, such as the regulation of cellular metabolism and homeostasis, cell proliferation and differentiation, and apoptosis. One of the key players in the regulation of inositol lipid signaling is the phospholipase Cβ1 (PI-PLCβ1), that hydrolyzes phosphatidylinositol 4,5-bisphosphate [PtIns(4,5)P2], giving rise to the second messengers inositol triphosphate and diacylglicerol. PI-PLCβ1 has been associated with the regulation of several cellular functions, some of which have not yet been fully understood. In particular, it has been reported that PI-PLCβ1 protects murine fibroblasts from oxidative stress-induced cell death. The mediators of oxidative stress, reactive oxygen species (ROS), have been shown to regulate major epigenetic processes, causing the silencing of tumor suppressors and enhancing the proliferation of leukemic cells under oxidative stress. Investigation of the interplay between ROS, PI-PLCβ1, and their signaling mediators in leukemia might therefore reveal innovative targets of pharmacological therapy in the treatment for leukemia. In this work, we demonstrate that in pro-B-lymphoblastic cells (Ba/F3), treated with H2O2, PI-PLCβ1b conferred resistance to cell death, promoting cell cycle progression and cell proliferation and influencing the expression of cyclin A and E. Interestingly, we found that, expression of PI-PLCβ1b affects the activity of caspase-3, caspase-7, and of several protein kinases induced by oxidative stress. In particular, PI-PLCβ1b expression completely abolished the phosphorylation of Erk1/2 MAP kinases, down-regulated phosphatase and tensin homolog (PTEN), and up-regulated the phosphorylation of Akt, thereby sustaining cellular proliferation.

Beyond its possible correlation with stemness of tumor cells, CD133/prominin1 is considered an important marker in breast cancer, since it correlates with tumor size, metastasis and clinical stage of triple-negative breast cancers (TNBC), to date the highest risk breast neoplasia.

Chemotherapy-induced differentiation of immature myeloid progenitors, such as acute myeloid leukemia (AML) cells or myeloid-derived suppressor cells (MDSCs), has remained a challenge for the clinicians. Testing our imidazo[1,2-b]pyrazole-7-carboxamide derivative on HL-60 cells, we obtained ERK phosphorylation as an early survival response to treatment followed by the increase of the percentage of the Bcl-xlbright and pAktbright cells. Following the induction of Vav1 and the AP-1 complex, a driver of cellular differentiation, FOS, JUN, JUNB, and JUND were elevated on a concentration and time-dependent manner. As a proof of granulocytic differentiation, the cells remained non-adherent, the expression of CD33 decreased; the granularity, CD11b expression, and MPO activity of HL-60 cells increased upon treatment. Finally, viability of HL-60 cells was hampered shown by the depolarization of mitochondria, activation of caspase-3, cleavage of Z-DEVD-aLUC, appearance of the sub-G1 population, and the leakage of the lactate-dehydrogenase into the supernatant. We confirmed the differentiating effect of our drug candidate on human patient-derived AML cells shown by the increase of CD11b and decrease of CD33+, CD7+, CD206+, and CD38bright cells followed apoptosis (IC50: 80 nM) after treatment ex vivo. Our compound reduced both CD11b+/Ly6C+ and CD11b+/Ly6G+ splenic MDSCs from the murine 4T1 breast cancer model ex vivo.

All-trans retinoic acid (ATRA) promotes the development and the function of insulin producing cells and induces partial differentiation of pancreatic tumor cells. A number of evidences clearly indicate that the ATRA mediated signaling may have a substantial role in therapeutic approaches based on restoration of functional β-cells. Among the proteins up-regulated by ATRA, Vav1 is involved in maturation and function of haematopoietic cells and is essential for retinoids induced differentiation of tumor promyelocytes. The presence of Vav1 in solid tissues, including pancreas, is considered ectopic and no role in the differentiation of human epithelial cells has so far been described. We demonstrated here that Vav1 sustains the maturation to β-cells of the normal precursors human Biliary Tree Stem/progenitor Cells (hBTSCs) induced by a differentiation medium containing ATRA and that, in the mature normal pancreas, insulin-producing cells express variable levels of Vav1. Using pancreatic ductal adenocarcinoma (PDAC)-derived cells, we also revealed that the ATRA induced up-modulation of Vav1 is essential for the retinoid-induced trans-differentiation of neoplastic cells into insulin producing cells. The results of this study identify Vav1 as crucial molecule in ATRA induced maturation of insulin producing cells and suggest this protein as a marker for new strategies ended to restore functional β-cells. Graphical abstract.

We have investigated motility in breast cancer cell lines in association with the expression of Transglutaminase type 2 (TG2) as well as upon the administration of Doxorubicin (Dox), an active cytotoxic agent that is employed in chemotherapy. The exposure of MCF-7 cells to the drug increased TG2 levels, triggering epithelial-mesenchymal transition (EMT), thereby supporting cell motility. The effects of Dox on the movement of MCF-7 cells were counteracted by treatment with NC9, a TG2 inhibitor, which induced morphological changes and also reduced the migration of MDA-MB-231 cells exhibiting high levels of TG2. The physical association of TG2 with the cytoskeletal component vimentin appeared pivotal both in drug-treated MCF-7 and in MDA-MB-231 cells and seemed to be independent of the catalytic activity of TG2. NC9 altered the subcellular distribution of TG2 and, consequently, the co-localization of TG2 with vimentin. Furthermore, NC9 induced a nuclear accumulation of TG2 as a prelude to TG2-dependent gene expression modifications. Since enzyme activity can affect both motility and nuclear functions, targeting of this protein could represent a method to improve therapeutic interventions in breast tumors, particularly those to control progression and to limit drug resistance.

Voghiera garlic is an Italian white garlic variety which obtained in 2010 the Protected Designation of Origin. It is widely used for culinary purposes or as an ingredient for supplement production due to its phytochemical compositions. The storage conditions seem to be crucial to retain the high quality of garlic bulbs and their by-products, taking into account the high importance of organosulfur and phenolic compounds for the bioactive potency of garlic and its shelf-life. This study aims to examine the effect of storage on the phytochemical composition, biological effects, and shelf-life of Voghiera garlic PDO. In detail, we considered (i) -4 °C (industrial storage) for 3, 6, and 9 months; (ii) +4 °C for 3 months (home conservation), and (iii) -4 °C for 3 months, plus +4 °C for another 3 months. We focused our attention on the organosulfur compounds, total condensed tannins, flavonoids, phenolic compounds, and related antioxidant activity changes during the storage period. To evaluate the bioactive effects, the Voghiera garlic extracts at different storage conditions were administered to a breast cancer cell line, while antioxidant and anti-inflammatory activity was detected using macrophage RAW 264.7 cells. We observed a decrease in sulfur compounds after 6 months which correlated to a decrease in bioactive effects, while the number of antioxidant compounds was stable during the storage period, showing the good effect of refrigerated temperature in maintaining garlic bulb shelf-life.

Since the multifunctionality of transglutaminase 2 (TG2) includes extra- and intracellular functions, we investigated the effects of intracellular administration of TG2 inhibitors in three breast cancer cell lines, MDA-MB-231, MDA-MB-436 and MDA-MB-468, which are representative of different triple-negative phenotypes, using a patch-clamp technique. The first cell line has a highly voltage-dependent a membrane current, which is low in the second and almost absent in the third one. While applying a voltage protocol to responsive single cells, injection of TG2 inhibitors triggered a significant decrease of the current in MDA-MB-231 that we attributed to voltage-dependent K+ channels using the specific inhibitors 4-aminopyridine and astemizole. Since the Kv10.1 channel plays a dominant role as a marker of cell migration and survival in breast cancer, we investigated its relationship with TG2 by immunoprecipitation. Our data reveal their physical interaction affects membrane currents in MDA-MB-231 but not in the less sensitive MDA-MB-436 cells. We further correlated the efficacy of TG2 inhibition with metabolic changes in the supernatants of treated cells, resulting in increased concentration of methyl- and dimethylamines, representing possible response markers. In conclusion, our findings highlight the interference of TG2 inhibitors with the Kv10.1 channel as a potential therapeutic tool depending on the specific features of cancer cells.

Approximately 7% of cancers arising in children and 1% of those arising in adults are soft tissue sarcomas (STS). Of these malignancies, rhabdomyosarcoma (RMS) is the most common. RMS survival rates using current therapeutic protocols have remained largely unchanged in the past decade. Thus, it is imperative that the main molecular drivers in RMS tumorigenesis are defined so that more precise, effective, and less toxic therapies can be designed. Curcumin, a common herbal supplement derived from plants of the Curcuma longa species, has an exceptionally low dietary biotoxicity profile and has demonstrated anti-tumorigenic benefits in vitro. In this study, the anti-tumorigenic activity of curcumin was assessed in rhabdomyosarcoma cell lines and used to identify the major pathways responsible for curcumin's anti-tumorigenic effects. Curcumin treatment resulted in cell cycle arrest, inhibited cell migration and colony forming potential, and induced apoptotic cell death. Proteome profiler array analysis demonstrated that curcumin treatment primarily influenced flux through the AKT-mammalian target of rapamycin (mTOR), signal transducer and activator of transcription (STAT), AMP-dependent kinase (AMPK), and p53 associated pathways in a rhabdomyosarcoma subtype-specific manner. Thus, the strategic, combinational therapeutic targeting of these pathways may present the best option to treat this group of tumors.

MicroRNA (miR)‑29b has been reported to play a controversial role in breast cancer, particularly triple‑negative breast cancer (TNBC). Based on our previous data revealing that the PU.1‑mediated expression of miR‑29b in cells from acute myeloid leukemia is sustained by Vav1, the potential role of this multidomain protein in modulating miR‑29b levels in breast tumor cells, in which Vav1 is ecstopically expressed and shows a nuclear accumulation, was investigated. Breast cancer cell lines with various phenotypes and patient‑derived xenograft‑derived TNBC cells were subjected to Vav1 modulation and reverse transcription quantitative PCR of miR‑29b levels. The recruitment of CCAAT enhancer binding protein α (CEBPα) to miR‑29b promoters was investigated by quantitative chromatin immunoprecipitation assays. It was found that Vav1 was essential for the recovery of mature miR‑29b in breast cancer cell lines, and that it promoted the expression of the miRNA in TNBC cells of the mesenchymal molecular subtype by sustaining the transcription of the miR‑29b1/a cluster mediated by CEBPα. The present results suggest that Vav1 is a crucial modulator of miR‑29b expression in breast tumor cells, and this finding may help identify strategies that may be useful in the management of TNBC by targeting the Vav1/miR‑29b axis, as there is a lack of molecular‑based treatments for TNBC.

It has been recently demonstrated that high pre-treatment levels of miR-29b positively correlated with the response of patients with acute myeloid leukaemia (AML) to hypomethylating agents. Upmodulation of miR-29b by restoring its transcriptional machinery appears indeed a tool to improve therapeutic response in AML. In cells from acute promyelocytic leukaemia (APL), miR-29b is regulated by PU.1, in turn upmodulated by agonists currently used to treat APL. We explored here the ability of PU.1 to also regulate miR-29b in non-APL cells, in order to identify agonists that, upmodulating PU.1 may be beneficial in hypomethylating agents-based therapies. We found that PU.1 may regulate miR-29b in the non-APL Kasumi-1 cells, showing the t(8;21) chromosomal rearrangement, which is prevalent in AML and correlated with a relatively low survival. We demonstrated that the PU.1-mediated contribution of the 2 miR-29b precursors is cell-related and almost completely dependent on adequate levels of Vav1. Nuclear PU.1/Vav1 association accompanies the transcription of miR-29b but, at variance with the APL-derived NB4 cells, in which the protein is required for the association of PU.1 with both miRNA promoters, Vav1 is part of molecular complexes to the PU.1 consensus site in Kasumi-1. Our results add new information on the transcriptional machinery that regulates miR-29b expression in AML-derived cells and may help in identifying drugs useful in upmodulation of this miRNA in pre-treatment of patients with non-APL leukaemia who can take advantage from hypomethylating agent-based therapies.

DNA methylation is an important component of the epigenetic machinery that regulates the malignancy of Ewing sarcoma (EWS), the second most common primary bone tumor in children and adolescents. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming and the DNMT1 enzyme has been demonstrated to have an important role in both maintaining the epigenome and controlling cell cycle. Here, we showed that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 induces a specific depletion of DNMT1 and affects EWS tumor proliferation through a mechanism that is independent on DNA methylation. Depletion of DNMT1 causes perturbation of the cell cycle, with an accumulation of cells in the G1 phase, and DNA damage, as revealed by the induction of γH2AX foci. These effects elicited activation of p53-dependent signaling and apoptosis in p53wt cells, while in p53 mutated cells, persistent micronuclei and increased DNA instability was observed. Treatment with MC3343 potentiates the efficacy of DNA damaging agents such as doxorubicin and PARP-inhibitors (PARPi). This effect correlates with increased DNA damage and synergistic tumor cytotoxicity, supporting the use of the DNMTi MC3343 as an adjuvant agent in treating EWS.

To explore the potential of a chip-based miniaturized capillary gel electrophoresis device in a quantitative evaluation of the human tear protein profile and to validate the method.

A substantial number of ductal carcinoma in situ (DCIS) detected by mammography never progress to invasive ductal carcinoma (IDC) and current approaches fail to identify low-risk patients not at need of adjuvant therapies. We aimed to identify the key miRNAs protecting DCIS from malignant evolution, that may constitute markers for non-invasive lesions. We studied 100 archived DCIS samples, including pure DCIS, DCIS with adjacent IDC and pure DCIS from patients with subsequent IDC in contralateral breast or no recurrence. A DCIS derived cell line was used for molecular and cellular studies. A genome wide study revealed that pure DCIS has higher miR-126 and miR-218 expression than DCIS with adjacent IDC lesions or than IDC. The down-regulation of miR-126 and miR-218 promoted invasiveness in vitro and, in patients with pure DCIS, was associated with later onset of IDC. Survival studies of independent cohorts indicated that both miRNAs play a protective role in IDC. The clinical findings are in agreement with the miRNAs' roles in cell adhesion, differentiation and proliferation. We propose that miR-126 and miR-218 have a protective role in DCIS and represent novel biomarkers for the risk assessment in women with early detection of breast cancer.

The malignant potential of triple negative breast cancer (TNBC) is also dependent on a sub-population of cells with a stem-like phenotype. Among the cancer stem cell markers, CD133 and EpCAM strongly correlate with breast tumor aggressiveness, suggesting that simultaneous targeting of the two surface antigens may be beneficial in treatment of TNBC. Since in TNBC-derived cells we demonstrated that PLC-β2 induces the conversion of CD133high to CD133low cells, here we explored its possible role in down-modulating the expression of both CD133 and EpCAM and, ultimately, in reducing the number of TNBC cells with a stem-like phenotype.

Targeting different members of the Akt pathways is a promising therapeutic chance in solid tumors including breast cancer. The variable expression levels of Akt isoforms with opposite effects on tumor growth and metastasis, however, make it difficult to select the inhibitors to be used for specific breast tumor subtypes. Using in vitro and in vivo models, we demonstrated here that Vav1, ectopically expressed in invasive breast tumors derived cells, downmodulates Akt acting at expression and/or activation levels depending on tumor subtype. The decreased p-Akt1 (Ser473) levels are a common effect of Vav1 upmodulation, suggesting that, in breast tumor-derived cells and independently of their phenotype, Vav1 interferes with signaling pathways ended to specifically recruit Akt1. Only in ER-negative cell lines, the silencing of Vav1 induced the expression but not the activation of Akt2. A retrospective analysis of early invasive breast tumors allowed to establish the prognostic significance of the p-Akt/Vav1 relationship. In particular, low Vav1 levels negatively influence the follow-up of patients with low p-Akt in their primary tumors and subjected to adjuvant chemotherapy. As the use of specific or pan Akt inhibitors may not be sufficient or may even be detrimental, increasing the levels of Vav1 could be a new approach to improve breast cancer outcomes. This might be particularly relevant for tumors with a triple-negative phenotype, for which target-based therapies are not currently available.