Cell-Impermeable Inhibitors Confirm That Intracellular Human Transglutaminase 2 Is Responsible for the Transglutaminase-Associated Cancer Phenotype.

Transglutaminase 2 (TG2) is a multifunctional enzyme primarily responsible for crosslinking proteins. Ubiquitously expressed in humans, TG2 can act either as a transamidase by crosslinking two substrates through formation of an Nε(ɣ-glutaminyl)lysine bond or as an intracellular G-protein. These discrete roles are tightly regulated by both allosteric and environmental stimuli and are associated with dramatic changes in the conformation of the enzyme. The pleiotropic nature of TG2 and multi-faceted activities have resulted in TG2 being implicated in numerous disease pathologies including celiac disease, fibrosis, and cancer. Targeted TG2 therapies have not been selective for subcellular localization, such that currently no tools exist to selectively target extracellular over intracellular TG2. Herein, we have designed novel TG2-selective inhibitors that are not only highly potent and irreversible, but also cell impermeable, targeting only extracellular TG2. We have also further derivatized the scaffold to develop probes that are intrinsically fluorescent or bear an alkyne handle, which target both intra- and extracellular TG2, in order to facilitate cellular labelling and pull-down assays. The fluorescent probes were internalized and imaged in cellulo, and provide the first implicit experimental evidence that by comparison with their cell-impermeable analogues, it is specifically intracellular TG2, and presumably its G-protein activity, that contributes to transglutaminase-associated cancer progression.

Metabolic characterisation of transglutaminase 2 inhibitor effects in breast cancer cell lines.

Transglutaminase 2 (TG2), which mediates post-translational modifications of multiple intracellular enzymes, is involved in the pathogenesis and progression of cancer. We used 1 H-NMR metabolomics to study the effects of AA9, a novel TG2 inhibitor, on two breast cancer cell lines with distinct phenotypes, MCF-7 and MDA-MB-231. AA9 can promote apoptosis in both cell lines, but it is particularly effective in MD-MB-231, inhibiting transamidation reactions and decreasing cell migration and invasiveness. This metabolomics study provides evidence of a major effect of AA9 on MDA-MB-231 cells, impacting glutamate and aspartate metabolism, rather than on MCF-7 cells, characterised by choline and O-phosphocholine decrease. Interestingly, AA9 treatment induces myo-inositol alteration in both cell lines, indicating action on phosphatidylinositol metabolism, likely modulated by the G protein activity of TG2 on phospholipase C. Considering the metabolic deregulations that characterise various breast cancer subtypes, the existence of a metabolic pathway affected by AA9 further points to TG2 as a promising hot spot. The metabolomics approach provides a powerful tool to monitor the effectiveness of inhibitors and better understand the role of TG2 in cancer.

The Effect of Different Storage Conditions on Phytochemical Composition, Shelf-Life, and Bioactive Compounds of Voghiera Garlic PDO.

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.

Mir-29b in Breast Cancer: A Promising Target for Therapeutic Approaches.

The miR-29 family comprises miR-29a, miR-29b, and miR-29c, and these molecules play crucial and partially overlapped functions in solid tumors, in which the different isoforms are variously de-regulated and mainly correlated with tumor suppression. miR-29b is the most expressed family member in cancer, in which it is involved in regulating gene expression at both transcriptional and post-transcriptional levels. This review focuses on the role of miR-29b in breast cancer, in which it plays a controversial role as tumor suppressor or onco-miRNA. Here we have highlighted the dual effect of miR-29b on breast tumor features, which depend on the prevailing function of this miRNA, on the mature miR-29b evaluated, and on the breast tumor characteristics. Remarkably, the analyzed miR-29b form emerged as a crucial element in the results obtained by various research groups, as the most abundant miR-29b-3p and the less expressed miR-29b1-5p seem to play distinct roles in breast tumors with different phenotypes. Of particular interest are the data showing that miR-29b1-5p counteracts cell proliferation and migration and reduces stemness in breast tumor cells with a triple negative phenotype. Even if further studies are required to define exactly the role of each miR-29b, our review highlights its possible implication in phenotype-specific management of breast tumors.

Vav1 Selectively Down-Regulates Akt2 through miR-29b in Certain Breast Tumors with Triple Negative Phenotype.

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.

A Multidisciplinary Approach Establishes a Link between Transglutaminase 2 and the Kv10.1 Voltage-Dependent K+ Channel in Breast Cancer.

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.

UC.183, UC.110, and UC.84 Ultra-Conserved RNAs Are Mutually Exclusive with miR-221 and Are Engaged in the Cell Cycle Circuitry in Breast Cancer Cell Lines.

In the human genome, there are about 600 ultra-conserved regions (UCRs), long DNA sequences extremely conserved in vertebrates. We performed a large-scale study to quantify transcribed UCR (T-UCR) and miRNA levels in over 6000 cancer and normal tissue samples to find possible correlation between these kinds of regulatory molecules. Our analysis evidenced several non-coding RNAs showing negative co-regulation with miRNAs; among them, we focused on miR-221 to investigate any relationship with its pivotal role in the cell cycle. We have chosen breast cancer as model, using two cell lines with different phenotypes to carry out in vitro treatments with siRNAs against T-UCRs. Our results demonstrate that the expression of uc.183, uc.110, and uc.84 T-UCRs is mutually exclusive with miR-221 and is engaged in the regulation of CDKN1B expression. In addition, tests with a set of anticancer drugs, including BYL719, AZD5363, AZD8055, AZD7762, and XL765, revealed the modulation of specific T-UCRs without alteration of miR-221 levels.

The Motility and Mesenchymal Features of Breast Cancer Cells Correlate with the Levels and Intracellular Localization of Transglutaminase Type 2.

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.

The Molecular Networks of microRNAs and Their Targets in the Drug Resistance of Colon Carcinoma.

Drug resistance is one of the major forces driving a poor prognosis during the treatment and progression of human colon carcinomas. The molecular mechanisms that regulate the diverse processes underlying drug resistance are still under debate. MicroRNAs (miRNAs) are a subgroup of non-coding RNAs increasingly found to be associated with the regulation of tumorigenesis and drug resistance. We performed a systematic review of the articles concerning miRNAs and drug resistance in human colon cancer published from 2013 onwards in journals with an impact factor of 5 or higher. First, we built a network with the most studied miRNAs and targets (as nodes) while the drug resistance/s are indicated by the connections (edges); then, we discussed the most relevant miRNA/targets interactions regulated by drugs according to the network topology and statistics. Finally, we considered the drugs as nodes in the network, to allow an alternative point of view that could flow through the treatment options and the associated molecular pathways. A small number of microRNAs and proteins appeared as critically involved in the most common drugs used for the treatment of patients with colon cancer. In particular, the family of miR-200, miR34a, miR-155 and miR-17 appear as the most relevant microRNAs. Thus, regulating these miRNAs could be useful for interfering with some drug resistance mechanisms in colorectal carcinoma.

Ethanol-based garlic extract prevents malignant evolution of non-invasive breast tumor cells induced by moderate hypoxia.

BACKGROUND: In breast cancer, low oxygen availability is associated with a more aggressive phenotype and with malignant evolution of non-invasive cells. Natural compounds have long attracted attention in cancer treatment, and in recent years garlic (Allium sativum) organosulfur derivatives have been shown to negatively affect growth and invasion of tumor cells. METHODS: Homemade ethanol-based garlic extract (GE) was administered to MCF7 and MCF10DCIS breast tumor cell lines grown under moderate hypoxia. Cell cycle, epithelial-to-mesenchymal transition and cancer stem cell markers were evaluated. RESULTS: We revealed that, in the non-invasive MCF10DCIS cells but not in the post-EMT MCF7 cells, low oxygen availability induced the decrease of E-cadherin and the increase of vimentin and motility, that were prevented by GE administration. In both cell lines, treatment with GE counteracted the up-modulation of CD133 positive cells induced by hypoxia. CONCLUSIONS: Overall, our data firstly revealed anti-cancer properties of garlic in non-invasive breast cancer cells. In particular, they demonstrated a protective role of this natural product against the hypoxia-induced increase of molecules that play crucial roles in tumor evolution, suggesting that garlic derivatives can be considered in new approaches for preventing progression of breast tumors from non-invasive to infiltrating lesions.

Targeting the Vav1/miR­29b axis as a potential approach for treating selected molecular subtypes of triple­negative breast cancer.

MicroRNA (miR)­29b has been reported to play a controversial role in breast cancer, particularly triple­negative breast cancer (TNBC). Based on our previous data revealing that the PU.1­mediated expression of miR­29b in cells from acute myeloid leukemia is sustained by Vav1, the potential role of this multidomain protein in modulating miR­29b 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 patient­derived xenograft­derived TNBC cells were subjected to Vav1 modulation and reverse transcription quantitative PCR of miR­29b levels. The recruitment of CCAAT enhancer binding protein α (CEBPα) to miR­29b promoters was investigated by quantitative chromatin immunoprecipitation assays. It was found that Vav1 was essential for the recovery of mature miR­29b 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 miR­29b1/a cluster mediated by CEBPα. The present results suggest that Vav1 is a crucial modulator of miR­29b 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/miR­29b axis, as there is a lack of molecular­based treatments for TNBC.

Vav1 Sustains the In Vitro Differentiation of Normal and Tumor Precursors to Insulin Producing Cells Induced by all-Trans Retinoic Acid (ATRA).

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 ß-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 ß-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 ß-cells. Graphical abstract.

Vav1 Down-Modulates Akt2 Expression in Cells from Pancreatic Ductal Adenocarcinoma: Nuclear Vav1 as a Potential Regulator of Akt Related Malignancy in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive tumor malignancy worldwide, mainly due to uncontrolled metastasis. Among the numerous molecules deregulated in PDAC, different members of the Akt pathways are of great importance because they are involved in tumor cell proliferation, migration, and invasion. We have recently demonstrated that Vav1, ectopically expressed in solid tumors, is capable of down-modulating expression and/or activation of specific Akt isoforms in breast cancer cells. By using pancreatic cell lines expressing different basal levels of Vav1, we demonstrated here that Vav1 down-regulates the expression of Akt2, known to correlate with tumor metastases and resistance to therapy. In particular, while the silencing of Vav1 is sufficient to induce Akt2, its up-modulation reduces Akt2 levels only when Vav1 accumulates inside the nucleus of PDAC cells. Moreover, in PDAC tissues, we revealed that high nuclear levels of Vav1 correlate with low Akt2 expression. Although we cannot demonstrate the mechanisms involved, our results provide new insights into the role of Vav1 in PDAC and, as targeting specific members of the Akt family is a promising therapeutic chance in solid tumors, they suggest that Vav1, by down-modulating Akt2, has potential as a molecular target in PDAC.

Pulsed Electromagnetic Fields Modulate miRNAs During Osteogenic Differentiation of Bone Mesenchymal Stem Cells: a Possible Role in the Osteogenic-angiogenic Coupling.

Despite the high intrinsic ability of bone tissue to regenerate, bone healing fails in some pathological conditions and especially in the presence of large defects. Due to the strong relationship between bone development and vascularization during in vivo bone formation and repair, strategies promoting the osteogenic-angiogenic coupling are crucial for regenerative medicine. Increasing evidence shows that miRNAs play important roles in controlling osteogenesis and bone vascularization and are important tool in medical research although their clinical use still needs to optimize miRNA stability and delivery. Pulsed electromagnetic fields (PEMFs) have been successfully used to enhance bone repair and their clinical activity has been associated to their ability to promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs). In this study we investigated the potential ability of PEMF exposure to modulate selected miRNAs involved in the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs). We show that, during in vitro hBMSC differentiation, PEMFs up-modulate the expression of miR-26a and miR-29b, which favor osteogenic differentiation, and decrease miR-125b which acts as an inhibitor miRNA. As PEMFs promote the expression and release of miRNAs also involved in angiogenesis, we conclude that PEMFs may represent a noninvasive and safe strategy to modulate miRNAs with relevant roles in bone repair and with the potential to regulate the osteogenic-angiogenic coupling.

Imidazo[1,2-b]pyrazole-7-Carboxamide Derivative Induces Differentiation-Coupled Apoptosis of Immature Myeloid Cells Such as Acute Myeloid Leukemia and Myeloid-Derived Suppressor Cells.

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.

Integrative proteomic and functional analyses provide novel insights into the action of the repurposed drug candidate nitroxoline in AsPC-1 cells.

We recently identified nitroxoline as a repurposed drug candidate in pancreatic cancer (PC) showing a dose-dependent antiproliferative activity in different PC cell lines. This antibiotic is effective in several in vitro and animal cancer models. To date, the mechanisms of nitroxoline anticancer action are largely unknown. Using shotgun proteomics we identified 363 proteins affected by nitroxoline treatment in AsPC-1 pancreatic cancer cells, including 81 consistently deregulated at both 24- and 48-hour treatment. These proteins previously unknown to be affected by nitroxoline were mostly downregulated and interconnected in a single highly-enriched network of protein-protein interactions. Integrative proteomic and functional analyses revealed nitroxoline-induced downregulation of Na/K-ATPase pump and ß-catenin, which associated with drastic impairment in cell growth, migration, invasion, increased ROS production and induction of DNA damage response. Remarkably, nitroxoline induced a previously unknown deregulation of molecules with a critical role in cell bioenergetics, which resulted in mitochondrial depolarization. Our study also suggests that deregulation of cytosolic iron homeostasis and of co-translational targeting to membrane contribute to nitroxoline anticancer action. This study broadens our understanding of the mechanisms of nitroxoline action, showing that the drug modulates multiple proteins crucial in cancer biology and previously unknown to be affected by nitroxoline.

CD133 in Breast Cancer Cells: More than a Stem Cell Marker.

Initially correlated with hematopoietic precursors, the surface expression of CD133 was also found in epithelial and nonepithelial cells from adult tissues in which it has been associated with a number of biological events. CD133 is expressed in solid tumors as well, including breast cancer, in which most of the studies have been focused on its use as a surface marker for the detection of cells with stem-like properties (i.e., cancer stem cells (CSCs)). Differently with other solid tumors, very limited and in part controversial are the information about the significance of CD133 in breast cancer, the most common malignancy among women in industrialized countries. In this review, we summarize the latest findings about the implication of CD133 in breast tumors, highlighting its role in tumor cells with a triple negative phenotype in which it directly regulates the expression of proteins involved in metastasis and drug resistance. We provide updates about the prognostic role of CD133, underlining its value as an indicator of increased malignancy of both noninvasive and invasive breast tumor cells. The molecular mechanisms at the basis of the regulation of CD133 levels in breast tumors have also been reviewed, highlighting experimental strategies capable to restrain its level that could be taken into account to reduce malignancy and/or to prevent the progression of breast tumors.

Clusterin enhances AKT2-mediated motility of normal and cancer prostate cells through a PTEN and PHLPP1 circuit.

Clusterin (CLU) is a chaperone-like protein with multiple functions. sCLU is frequently upregulated in prostate tumor cells after chemo- or radiotherapy and after surgical or pharmacological castration. Moreover, CLU has been documented to modulate the cellular homolog of murine thymoma virus akt8 oncogene (AKT) activity. Here, we investigated how CLU overexpression influences phosphatidylinositol 3'-kinase (PI3K)/AKT signaling in human normal and cancer epithelial prostate cells. Human prostate cells stably transfected with CLU were broadly profiled by reverse phase protein array (RPPA), with particular emphasis on the PI3K/AKT pathway. The effect of CLU overexpression on normal and cancer cell motility was also tested. Our results clearly indicate that CLU overexpression enhances phosphorylation of AKT restricted to isoform 2. Mechanistically, this can be explained by the finding that the phosphatase PH domain leucine-rich repeat-containing protein phosphatase 1 (PHLPP1), known to dephosphorylate AKT2 at S474, is markedly downregulated by CLU, whereas miR-190, a negative regulator of PHLPP1, is upregulated. Moreover, we found that phosphatase and tensin homolog (PTEN) was heavily phosphorylated at the inhibitory site S380, contributing to the hyperactivation of AKT signaling. By keeping AKT2 phosphorylation high, CLU dramatically enhances the migratory behavior of prostate epithelial cell lines with different migratory and invasive phenotypes, namely prostate normal epithelial 1A (PNT1A) and prostatic carcinoma 3 (PC3) cells. Altogether, our results unravel for the first time a circuit by which CLU can switch a low migration phenotype toward a high migration phenotype, through miR-190-dependent downmodulation of PHLPP1 expression and, in turn, stabilization of AKT2 phosphorylation.

Ectopic expression of PLC-ß2 in non-invasive breast tumor cells plays a protective role against malignant progression and is correlated with the deregulation of miR-146a.

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 ß2 isoform of phosphoinositide-dependent phospholipase C (PLC-ß2), known to counteract the effects of hypoxia in low-invasive breast tumor-derived cells. Here, we studied the correlation between PLC-ß2 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-ß2 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-ß2 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-ß2, was not detected in primary DCIS from patients who developed a second invasive breast neoplasia. This suggests that alterations of the PLC-ß2/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-ß2/miR-146a levels at diagnosis could be beneficial for identifying whether DCIS patients may have either a low or high propensity for invasive recurrence.

Protective role of all-trans retinoic acid (ATRA) against hypoxia-induced malignant potential of non-invasive breast tumor derived cells.

BACKGROUND: 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. METHODS: By using a DCIS-derived cell line, we evaluated the effects of low oxygen availability on malignant features of non-invasive breast tumor cells and the possible role of all-trans retinoic acid (ATRA), a well-known anti-leukemic drug, in counteracting the effects of hypoxia. The involvement of the ß2 isoform of PI-PLC (PLC-ß2), an ATRA target in myeloid leukemia cells, was also investigated by specific modulation of the protein expression. RESULTS: We demonstrated that moderate hypoxia is sufficient to induce, in DCIS-derived cells, motility, epithelial-to-mesenchymal transition (EMT) and expression of the stem cell marker CD133, indicative of their increased malignant potential. Administration of ATRA supports the epithelial-like phenotype of DCIS-derived cells cultured under hypoxia and keeps down the number of CD133 positive cells, abrogating almost completely the effects of poor oxygenation. We also found that the mechanisms triggered by ATRA in non-invasive breast tumor cells cultured under hypoxia is in part mediated by PLC-ß2, responsible to counteract the effects of low oxygen availability on CD133 levels. CONCLUSIONS: Overall, we assigned to hypoxia a role in increasing the malignant potential of DCIS-derived cells and we identified in ATRA, currently used in treatment of acute promyelocytic leukemia (APL), an agonist potentially useful in preventing malignant progression of non-invasive breast lesions showing hypoxic areas.