The putative tumour suppressor protein Latexin is secreted by prostate luminal cells and is downregulated in malignancy.

Loss of latexin (LXN) expression negatively correlates with the prognosis of several human cancers. Despite association with numerous processes including haematopoietic stem cell (HSC) fate, inflammation and tumour suppression, a clearly defined biological role for LXN is still lacking. Therefore, we sought to understand LXN expression and function in the normal and malignant prostate to assess its potential as a therapeutic target. Our data demonstrate that LXN is highly expressed in normal prostate luminal cells but downregulated in high Gleason grade cancers. LXN protein is both cytosolic and secreted by prostate cells and expression is directly and potently upregulated by all-trans retinoic acid (atRA). Whilst overexpression of LXN in prostate epithelial basal cells did not affect cell fate, LXN overexpression in the luminal cancer line LNCaP reduced plating efficiency. Transcriptome analysis revealed that LXN overexpression had no direct effects on gene expression but had significant indirect effects on important genes involved in both retinoid metabolism and IFN-associated inflammatory responses. These data highlight a potential role for LXN in retinoid signaling and inflammatory pathways. Investigating the effects of LXN on immune cell function in the tumour microenvironment (TME) may reveal how observed intratumoural loss of LXN affects the prognosis of many adenocarcinomas.

ZNF521 Has an Inhibitory Effect on the Adipogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are multipotent progenitors present in the bone marrow stroma and in subcutaneous abdominal fat, an abundant and easily accessible source of MSCs with the ability to differentiate along multiple lineage pathways. The stem cell-associated transcription co-factor Zinc Finger Protein 521 (ZNF521/zfp521) has been implicated in the control of the homeostasis of hematopoietic, neural and osteo-adipogenic progenitors. Here we document through the analysis of a panel of human adipose-derived stem cells (hADSCs), that ZNF521 strongly inhibits the generation of mature adipocytes. Enforced overexpression of ZNF521 in these cells resulted in a significant delay and reduction in adipocyte differentiation upon exposure to inducers of adipogenesis. Of particular relevance, ZNF521 was able to inhibit the expression of ZNF423, recently identified as an essential commitment factor necessary for the generation of pre-adipocytes. Conversely, silencing of ZNF521 was found to significantly enhance the adipogenic differentiation of hADSCs. Inhibition of adipogenesis by ZNF521 was at least in part due to inhibition of EBF1. Taken together, these results confirm a role for ZNF521 as a key negative regulator of adipocyte differentiation of hADSCs.

Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells.

Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery.Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes.

Inhibition of the glucocorticoid receptor results in an enhanced miR-99a/100-mediated radiation response in stem-like cells from human prostate cancers.

Radiation therapy is a major primary treatment option for both localized early stage prostate cancer, and for advanced, regionally un-resectable, cancer. However, around 30% of patients still experience biochemical recurrence after radiation therapy within 10 years. Thus, identification of better biomarkers and new targets are urgently required to improve current therapeutic strategies. The miR-99 family has been shown to play an important role in the regulation of the DNA damage response, via targeting of the SWI/SNF chromatin remodeling factors, SMARCA5 and SMARCD1 in cell line models. In the present study, we have demonstrated that low expression of miR-99a and miR-100 is present in cell populations which are relatively radiation insensitive, for example in prostate cancer stem cells and in castration-resistant prostate cancer. Additionally, treatment of cells with the synthetic glucocorticoid, Dexamethasone resulted in decreased miR-99a and 100 expression, suggesting a new mechanism of miR-99a and 100 regulation in androgen-independent prostate cells. Strikingly, treatment of prostate cells with the glucocorticoid receptor inhibitor, Mifepristone was found to sensitize prostate cells to radiation by increasing the levels of miR-99a and miR-100. These results qualify the miR99 family as markers of radiation sensitivity and as potential therapeutic targets to improve efficiency of radiotherapy.

ZNF423 and ZNF521: EBF1 Antagonists of Potential Relevance in B-Lymphoid Malignancies.

The development of the B-lymphoid cell lineage is tightly controlled by the concerted action of a network of transcriptional and epigenetic regulators. EBF1, a central component of this network, is essential for B-lymphoid specification and commitment as well as for the maintenance of the B-cell identity. Genetic alterations causing loss of function of these B-lymphopoiesis regulators have been implicated in the pathogenesis of B-lymphoid malignancies, with particular regard to B-cell acute lymphoblastic leukaemias (B-ALLs), where their presence is frequently detected. The activity of the B-cell regulatory network may also be disrupted by the aberrant expression of inhibitory molecules. In particular, two multi-zinc finger transcription cofactors named ZNF423 and ZNF521 have been characterised as potent inhibitors of EBF1 and are emerging as potentially relevant contributors to the development of B-cell leukaemias. Here we will briefly review the current knowledge of these factors and discuss the importance of their functional cross talk with EBF1 in the development of B-cell malignancies.

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells.

Cellular and molecular background underlying the diversity in therapeutic responses between primary tumours and metastases.

Metastasis, also called secondary neoplastic disease, is a tumour newly formed in a site different from that of origin, as a consequence of cancer progression and dissemination largely through blood and lymphatic vessels. The ability to form metastases is the main property that distinguishes malignant from benign tumours. Treatments for metastatic cancer are similar in practice to those for primary tumours, but such treatments are mostly palliative; indeed, almost all deaths caused by solid tumours occur in the metastatic phase. Increasing evidence supports the concept that therapies for primary tumours are inadequate to treat metastasis and can even promote formation of metastases, while exerting local growth control. Furthermore, recurrent tumours, which are denoted by increased aggressiveness and therapy resistance in comparison with the primary tumour, have an increased metastatic potential. Genetic modifications occurring during tumour progression lead to substantial differences between the primary and metastatic tumours. This emphasises the importance of designing novel therapies for metastasis. In the last decade, a number of studies have contributed to the understanding of the genetic rearrangements underlying the conversion of cancer cells into the metastasis founder cells. The present article aims at reviewing recent advances in metastasis research and attempts to discuss the reasons for which the therapeutic strategies against primary tumours may not satisfactorily address their metastatic counterparts.

Synergy between enzastaurin doxorubicin in inducing melanoma apoptosis.

Melanoma is resistant to most standard chemotherapeutics. We analysed the combined effect of doxorubicin and enzastaurin on cell death of four melanoma cell lines, namely G361, SK-MEL3, A375 and SAN. Enzastaurin IC50 was calculated by measure of growth inhibition with MTS assay and corresponded to 2 µM; the half maximal cytotoxicity of doxorubicin was obtained at 3 µM dose. Evaluation of combination index showed synergism (CI > 1) or additive effect (CI = 1) with all melanoma cell lines, with enzastaurin doses ≥0.6 µM and doxorubicin doses ≥1 µM. Combination of the two drugs resulted in increase in caspase 3 and 8 activation, in comparison with activation by single agents. Caspase 8 activation was impaired by TNFR-1 blocking. Our results show doxorubicin-stimulated production of TNFα, whereas enzastaurin-stimulated TNFR-1 expression on plasma membrane. The effect on TNFR-1 appeared to be mediated by PKCζ inhibition. Taken together, our findings suggest that enzastaurin increases doxorubicin-induced apoptosis of melanoma by a mechanism involving, at least in part, activation of the TNF-α signal.

Atorvastatin sensitises vascular smooth muscle cells, but not endothelial cells, to TNF-α-induced cell death.

OBJECTIVE: Stimuli activating vascular smooth muscle cell death can constrain the neointimal response to arterial damage and prevent vascular thickening. Conversely, endothelial cell death increases endothelial dysfunction and thrombosis risk. We investigated the combined effect of atorvastatin and TNF-α on vascular cell death. METHODS AND RESULTS: Cell death was investigated in cultures of human aortic smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs). Atorvastatin downregulated NF-κB and enhanced JNK activity and cell death in VSMC cultured with TNF- α. In the absence of TNF-α, percentages (mean and StDev) of annexin V positive cells were 17.4 ± 6.6%, 19.3 ± 5.9%, 22.9 ± 9.4% and 35.0 ± 20.0 % with 0, 1, 3 and 10 µM atorvastatin, respectively. The cytotoxic effect of statin was significant at the highest dose of 10 µM (p=0.001). In the presence of TNF-α, percentages of annexin V positive cells were 27.1 ± 10.6%, 34.2 ± 8.5%, 37.4 ± 14.6, and 54.1 ± 20.0% with 0, 1, 3 and 10 µM atorvastatin, respectively. The cytotoxic effect of statin was significant at each dose used (p≤0.02), in the presence of TNF-α. The cell death sensitising effect of atorvastatin was apparently mediated by down modulation of PKCß activity, because it was reproduced by the specific PKCß inhibitor LY317615 and prevented by the PKC activator phorbol-12-myristate-13-acetate (PMA). This effect was cell context dependent because it was not observed in HUVECs. PKCß was found to be constitutively active in VSMCs but not in HUVECs, thereby explaining the differential effect among the two cell types. Measurement of phosphoPKCß protein levels in arterial specimens confirmed increased activation of this kinase in the smooth muscle layer, in comparison with endothelium. We show that PKCß provides survival signals to vascular smooth muscle cells and not the endothelium. CONCLUSION: Our study suggests that atorvastatin enhances TNF-α-induced cell death in vascular smooth muscle- but not endothelial - cells; by a cell-context-dependent mechanism, involving PKCß inhibition.

Differential effect of atorvastatin and tacrolimus on proliferation of vascular smooth muscle and endothelial cells.

Although considered promising for use in drug-eluting stents (DES), tacrolimus failed clinically. Tacrolimus inhibits growth factor production but can also act as a growth factor on vascular smooth muscle cells (VSMC). This unexpected proliferative stimulus could reverse the beneficial effects of the drug on restenosis. We hypothesized that tacrolimus' association with statins, which lower cholesterol and impair cell proliferation, could restore tacrolimus' beneficial effect by abrogating the aberrant proliferative stimulus. Additionally, since maintenance of endothelial function represents a challenge for new-generation DES, we investigated the combined effect of tacrolimus and atorvastatin on endothelial cells. Human VSMC and umbilical vein endothelial cells (HUVEC) were incubated with 100 nM tacrolimus and increasing doses of atorvastatin (0-3.0 µM). Atorvastatin plus tacrolimus dose-dependently inhibited VSMC proliferation. The percentage of cells incorporating 5-bromo-2'-deoxyuridine (BrdU) in their DNA was 49 ± 14% under basal conditions, 62 ± 15% (P = 0.01) with tacrolimus, 40 ± 22% with 3 µM atorvastatin, and 30 ± 7% (P < 0.05) with 3 µM atorvastatin plus tacrolimus. Atorvastatin downregulated ß-catenin, Erk1 and Erk2, and cyclin B in tacrolimus-stimulated VSMC. In contrast, atorvastatin plus tacrolimus did not affect proliferation of endothelial cells. The percentage of HUVEC incorporating BrdU in their DNA was 47 ± 8% under basal conditions, 58 ± 6% (P = 0.01) with tacrolimus, 45 ± 4% with 3 µM atorvastatin, and 49 ± 1% with 3 µM atorvastatin plus tacrolimus. Both agents stimulated endoglin production by HUVEC. Taken together, these results suggest that, when combined with tacrolimus, atorvastatin exerts a dose-dependent antiproliferative effect on VSMC. In contrast, atorvastatin acts in concert with tacrolimus in HUVEC to stimulate production of endoglin, a factor that has an important role in endothelial repair. Our study supports the conclusion that prevention of postcoronary in-stent restenosis and late thrombosis may benefit of concomitant association of tacrolimus and high doses of atorvastatin.