Targeting BTK through microRNA in chronic lymphocytic leukemia.

Bruton's tyrosine kinase (BTK) is a critical mediator of survival in B-cell neoplasms. Although BTK inhibitors have transformed therapy in chronic lymphocytic leukemia (CLL), patients with high-risk genetics are at risk for relapse and have a poor prognosis. Identification of novel therapeutic strategies for this group of patients is an urgent unmet clinical need, and therapies that target BTK via alternative mechanisms may fill this niche. Herein, we identify a set of microRNAs (miRs) that target BTK in primary CLL cells and show that the histone deacetylase (HDAC) repressor complex is recruited to these miR promoters to silence their expression. Targeting the HDACs by using either RNA interference against HDAC1 in CLL or a small molecule inhibitor (HDACi) in CLL and mantle cell lymphoma restored the expression of the BTK-targeting miRs with loss of BTK protein and downstream signaling and consequent cell death. We have also made the novel and clinically relevant discovery that inhibition of HDAC induces the BTK-targeting miRs in ibrutinib-sensitive and resistant CLL to effectively reduce both wild-type and C481S-mutant BTK. This finding identifies a novel strategy that may be promising as a therapeutic modality to eliminate the C481S-mutant BTK clone that drives resistance to ibrutinib and provides the rationale for a combination strategy that includes ibrutinib to dually target BTK to suppress its prosurvival signaling.

Tcl1 protein functions as an inhibitor of de novo DNA methylation in B-cell chronic lymphocytic leukemia (CLL).

B-cell chronic lymphocytic leukemia (CLL) is the most common human leukemia. Deregulation of the T-cell leukemia/lymphoma 1 oncogene (TCL1) in mouse B cells causes a CD5(+) leukemia similar to aggressive human CLL. To examine the mechanisms by which Tcl1 protein exerts its oncogenic activity in B cells, we performed proteomics experiments to identify its interacting partners. We found that Tcl1 physically interacts with de novo DNA methylthansferases Dnmt3A and Dnmt3B. We further investigated the effects of Tcl1 up-regulation on the enzymatic activity of Dnmt3A and found that Tcl1 overexpression drastically inhibits Dnmt3A function. In addition, B cells from TCL1 transgenic mice showed a significant decrease in DNA methylation compared with WT controls. Similarly, CLL samples with high Tcl1 expression showed a decrease in DNA methylation compared with CLL samples with low Tcl1 expression. Given the previous reports of inactivating mutations of DNMT3A in acute myelogenous leukemia and myelodysplastic syndrome, our results suggest that inhibition of de novo DNA methylation may be a common oncogenic mechanism in leukemogenesis.

NOTCH1 mutations in CLL associated with trisomy 12.

Two recent studies reported whole-genome sequencing of chronic lymphocytic leukemia (CLL) samples and found repeated mutations in the XPO1 and NOTCH1 genes. XPO1 was found mutated in 2.4% of cases, while NOTCH1 was found mutated in 12.2% or 15.1% of CLL samples. Here we report the results of sequencing of XPO1 and NOTCH1 in 186 CLL cases. Our results confirmed frequency of XPO1 mutations. However, we found only 5 NOTCH1 mutations in 127 IGVH unmutated/ZAP70(+) CLL samples (4%), and one mutation was found in IGVH mutated/ZAP70(-) CLL for a total percentage of 1.5%. Because 4 of 6 mutated samples also showed trisomy 12, we sequenced NOTCH1 in an additional 77 cases with trisomy 12 CLLs, including 47 IGVH unmutated/ZAP70(+) cases. Importantly, we found 41.9% NOTCH1 mutation frequency in aggressive trisomy 12 CLL cases. Our data suggest that activation of NOTCH1 plays a critical role in IGVH unmutated/ZAP70(+) trisomy 12 CLL.

Tcl1 interacts with Atm and enhances NF-κB activation in hematologic malignancies.

The T-cell leukemia/lymphoma 1 (TCL1) oncogene is a target of chromosomal translocations and inversions at 14q31.2, and its rearrangement in T cells causes T-cell prolymphocytic leukemias. TCL1 dysregulation in B cells is responsible for the development of an aggressive form of chronic lymphocytic leukemia (CLL), the most common human leukemia. We have investigated the mechanisms underlying the oncogenic functions of Tcl1 protein using a mass spectrometry approach and have identified Atm (ataxia-telangiectasia mutated) as a candidate Tcl1-interacting protein. The Tcl1-Atm complex formation was validated by coimmunoprecipitation experiments. Importantly, we show that the association of Atm with Tcl1 leads to enhanced IκBα phosphorylation and ubiquitination and subsequent activation of the NF-κB pathway. Our findings reveal functional cross-talk between Atm and Tcl1 and provide evidence for a novel pathway that could be targeted in leukemias and lymphomas.

miR-29ab1 deficiency identifies a negative feedback loop controlling Th1 bias that is dysregulated in multiple sclerosis.

Th cell programming and function is tightly regulated by complex biological networks to prevent excessive inflammatory responses and autoimmune disease. The importance of microRNAs (miRNAs) in this process is highlighted by the preferential Th1 polarization of Dicer-deficient T cells that lack miRNAs. Using genetic knockouts, we demonstrate that loss of endogenous miR-29, derived from the miR-29ab1 genomic cluster, results in unrestrained T-bet expression and IFN-γ production. miR-29b regulates T-bet and IFN-γ via a direct interaction with the 3' untranslated regions, and IFN-γ itself enhances miR-29b expression, establishing a novel regulatory feedback loop. miR-29b is increased in memory CD4(+) T cells from multiple sclerosis (MS) patients, which may reflect chronic Th1 inflammation. However, miR-29b levels decrease significantly upon T cell activation in MS patients, suggesting that this feedback loop is dysregulated in MS patients and may contribute to chronic inflammation. miR-29 thus serves as a novel regulator of Th1 differentiation, adding to the understanding of T cell-intrinsic regulatory mechanisms that maintain a balance between protective immunity and autoimmunity.

Reprogramming of miRNA networks in cancer and leukemia.

We studied miRNA profiles in 4419 human samples (3312 neoplastic, 1107 nonmalignant), corresponding to 50 normal tissues and 51 cancer types. The complexity of our database enabled us to perform a detailed analysis of microRNA (miRNA) activities. We inferred genetic networks from miRNA expression in normal tissues and cancer. We also built, for the first time, specialized miRNA networks for solid tumors and leukemias. Nonmalignant tissues and cancer networks displayed a change in hubs, the most connected miRNAs. hsa-miR-103/106 were downgraded in cancer, whereas hsa-miR-30 became most prominent. Cancer networks appeared as built from disjointed subnetworks, as opposed to normal tissues. A comparison of these nets allowed us to identify key miRNA cliques in cancer. We also investigated miRNA copy number alterations in 744 cancer samples, at a resolution of 150 kb. Members of miRNA families should be similarly deleted or amplified, since they repress the same cellular targets and are thus expected to have similar impacts on oncogenesis. We correctly identified hsa-miR-17/92 family as amplified and the hsa-miR-143/145 cluster as deleted. Other miRNAs, such as hsa-miR-30 and hsa-miR-204, were found to be physically altered at the DNA copy number level as well. By combining differential expression, genetic networks, and DNA copy number alterations, we confirmed, or discovered, miRNAs with comprehensive roles in cancer. Finally, we experimentally validated the miRNA network with acute lymphocytic leukemia originated in Mir155 transgenic mice. Most of miRNAs deregulated in these transgenic mice were located close to hsa-miR-155 in the cancer network.

Modulation of mismatch repair and genomic stability by miR-155.

Inactivation of mismatch repair (MMR) is the cause of the common cancer predisposition disorder Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer (HNPCC), as well as 10-40% of sporadic colorectal, endometrial, ovarian, gastric, and urothelial cancers. Elevated mutation rates (mutator phenotype), including simple repeat instability [microsatellite instability (MSI)] are a signature of MMR defects. MicroRNAs (miRs) have been implicated in the control of critical cellular pathways involved in development and cancer. Here we show that overexpression of miR-155 significantly down-regulates the core MMR proteins, hMSH2, hMSH6, and hMLH1, inducing a mutator phenotype and MSI. An inverse correlation between the expression of miR-155 and the expression of MLH1 or MSH2 proteins was found in human colorectal cancer. Finally, a number of MSI tumors with unknown cause of MMR inactivation displayed miR-155 overexpression. These data provide support for miR-155 modulation of MMR as a mechanism of cancer pathogenesis.

UCbase & miRfunc: a database of ultraconserved sequences and microRNA function.

Four hundred and eighty-one ultraconserved sequences (UCRs) longer than 200 bases were discovered in the genomes of human, mouse and rat. These are DNA sequences showing 100% identity among the three species. UCRs are frequently located at genomic regions involved in cancer, differentially expressed in human leukemias and carcinomas and in some instances regulated by microRNAs (miRNAs). Here we present UCbase & miRfunc, the first database which provides ultraconserved sequences data and shows miRNA function. Also, it links UCRs and miRNAs with the related human disorders and genomic properties. The current release contains over 2000 sequences from three species (human, mouse and rat). As a web application, UCbase & miRfunc is platform independent and it is accessible at http://microrna.osu.edu/.UCbase4.

Association of a microRNA/TP53 feedback circuitry with pathogenesis and outcome of B-cell chronic lymphocytic leukemia.

CONTEXT: Chromosomal abnormalities (namely 13q, 17p, and 11q deletions) have prognostic implications and are recurrent in chronic lymphocytic leukemia (CLL), suggesting that they are involved in a common pathogenetic pathway; however, the molecular mechanism through which chromosomal abnormalities affect the pathogenesis and outcome of CLL is unknown. OBJECTIVE: To determine whether the microRNA miR-15a/miR-16-1 cluster (located at 13q), tumor protein p53 (TP53, located at 17p), and miR-34b/miR-34c cluster (located at 11q) are linked in a molecular pathway that explains the pathogenetic and prognostic implications (indolent vs aggressive form) of recurrent 13q, 17p, and 11q deletions in CLL. DESIGN, SETTING, AND PATIENTS: CLL Research Consortium institutions provided blood samples from untreated patients (n = 206) diagnosed with B-cell CLL between January 2000 and April 2008. All samples were evaluated for the occurrence of cytogenetic abnormalities as well as the expression levels of the miR-15a/miR-16-1 cluster, miR-34b/miR-34c cluster, TP53, and zeta-chain (TCR)-associated protein kinase 70 kDa (ZAP70), a surrogate prognostic marker of CLL. The functional relationship between these genes was studied using in vitro gain- and loss-of-function experiments in cell lines and primary samples and was validated in a separate cohort of primary CLL samples. MAIN OUTCOME MEASURES: Cytogenetic abnormalities; expression levels of the miR-15a/miR-16-1 cluster, miR-34 family, TP53 gene, downstream effectors cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) and B-cell CLL/lymphoma 2 binding component 3 (BBC3), and ZAP70 gene; genetic interactions detected by chromatin immunoprecipitation. RESULTS: In CLLs with 13q deletions the miR-15a/miR-16-1 cluster directly targeted TP53 (mean luciferase activity for miR-15a vs scrambled control, 0.68 relative light units (RLU) [95% confidence interval {CI}, 0.63-0.73]; P = .02; mean for miR-16 vs scrambled control, 0.62 RLU [95% CI, 0.59-0.65]; P = .02) and its downstream effectors. In leukemic cell lines and primary CLL cells, TP53 stimulated the transcription of miR-15/miR-16-1 as well as miR-34b/miR-34c clusters, and the miR-34b/miR-34c cluster directly targeted the ZAP70 kinase (mean luciferase activity for miR-34a vs scrambled control, 0.33 RLU [95% CI, 0.30-0.36]; P = .02; mean for miR-34b vs scrambled control, 0.31 RLU [95% CI, 0.30-0.32]; P = .01; and mean for miR-34c vs scrambled control, 0.35 RLU [95% CI, 0.33-0.37]; P = .02). CONCLUSIONS: A microRNA/TP53 feedback circuitry is associated with CLL pathogenesis and outcome. This mechanism provides a novel pathogenetic model for the association of 13q deletions with the indolent form of CLL that involves microRNAs, TP53, and ZAP70.

MicroRNA Profiling of Salivary Duct Carcinoma Versus Her2/Neu Overexpressing Breast Carcinoma Identify miR-10a as a Putative Breast Related Oncogene.

Salivary duct carcinomas (SDC) and Her2/Neu3-overexpressing invasive breast carcinomas (HNPIBC/IBC) are histologically indistinguishable. We investigated whether common histopathologic and immunophenotypic features of SDC and IBC are mirrored by a similar microRNA (miRNA) profile. MiRNA profiling of 5 SDCs, 6 IBCs Her2/Neu3+, and 5 high-grade ductal breast carcinoma in situ (DCIS) was performed by NanoString platform. Selected miRNAs and HOXA1 gene were validated by RT-PCR. We observed similar miRNA expression profiles between IBC and SDC with the exception of 2 miRNAs, miR-10a and miR-142-3p, which were higher in IBC tumors. DCIS tumors displayed increased expression of miR-10a, miR-99a, miR-331-3p and miR-335, and decreased expression of miR-15a, miR-16 and miR-19b compared to SDC. The normal salivary gland and breast tissues also showed similar expression profiles. Interestingly, miR-10a was selectively increased in both IBC and normal breast tissue compared to SDC and normal salivary gland tissue. Moreover, our NanoString and RT-PCR data confirmed that miR-10a was upregulated in IBC and DCIS compared to SDC. Finally, we show downregulation of HOXA1, a miR-10 target, in IBC tumors compared to normal breast tissue. Taken together, our data demonstrates that, based on miRNA profiling, SDC is closely related to HNPIBC. Our results also suggest that miR-10a is differentially expressed in IBC compared to SDC and may have potential utility as a diagnostic biomarker in synchronous or metachronous malignant epithelial malignancies involving both organs. In addition, miR-10a could be playing an important role as a mammary-specific oncogene, involved in breast cancer initiation (DCIS) and progression (IBC), through mechanisms that include modulation of HOXA1 gene expression.

Circulating Micrornas Predict Survival of Patients with Tumors of Glial Origin.

The World Health Organization has recently introduced molecular prognostic-diagnostic biomarkers in the classification of Central Nervous System (CNS) tumors. In order to characterize subclasses of tumors that cannot find a precise location in the current classification, and, or cannot be tested because of scant material, it is important to find new molecular biomarkers in tissue and, or biological fluid samples. In this study, we identified serum microRNAs that could serve as biomarkers for the diagnosis and prognosis of patients with tumors of glial origin. We retrospectively analyzed microRNA expression in the serum extracellular vesicles of patients with tumors of glial origin. Extracellular vesicles RNA was analyzed by Nanostring. qRT-PCR confirmed 6 overexpressed microRNAs: hsa-miR-4443, hsa-miR-422a, hsa-miR-494-3p, hsa-miR-502-5p, hsa-miR-520f-3p, and hsa-miR-549a. Hsa-miR-4443 was the only microRNA that showed significant differences in most comparisons. In situ hybridization (ISH), confirmed that our signature was mostly expressed in cancer cells. Importantly, hsa-miR-549a and hsa-miR-502-5p expression predicted prognosis in patients with tumors of glial origin. Although more studies are needed, we demonstrated that serum vesicles microRNA profiles are promising diagnostic and prognostic molecular biomarkers that will find an actual application in the clinical practice of CNS tumors.

Role of complex cyclin d1/cdk4 in somatostatin subtype 2 receptor-mediated inhibition of cell proliferation of a medullary thyroid carcinoma cell line in vitro.

Somatostatin (SRIH) inhibits cell proliferation by interacting with five distinct SRIH receptor subtypes (SSTRs) activating several pathways in many tissues. We previously demonstrated that SRIH, by activating Src homology-2-containing protein, inhibits cell proliferation of the human medullary thyroid carcinoma cell line, TT, which expresses all SSTRs. However, the effects of SRIH on cell cycle proteins have not been investigated so far. We therefore evaluated the effects of SRIH and a selective SSTR2 agonist on cell cycle protein expression, mainly focusing on cyclin D1 and its associated kinases. Our data show that SRIH and the selective SSTR2 agonist, BIM-23120, reduce cell proliferation and DNA synthesis as well as induce a delay of the cell cycle in G(2)/M phase. Moreover, treatment with both SRIH and BIM-23120 decreases cyclin D1 levels, with a parallel increase in phosphocyclin D1 levels, suggesting protein degradation. Moreover, our data show an increase in glycogen synthase kinase-3beta activity, which triggers phosphorylation-dependent cyclin D1 degradation. Indeed, we observed a reduction in cyclin D1 protein half-life under treatment with SRIH or the SSTR2 selective agonist. A reduction in cdk4 protein levels is also observed with a parallel reduction in Rb phosphorylation levels at Ser-780. Our data indicate that the subtype 2 receptor-mediated antiproliferative effect of SRIH on TT cell proliferation may be exerted through a decrease in cyclin D1 levels.

Selective activation of somatostatin receptor subtypes differentially modulates secretion and viability in human medullary thyroid carcinoma primary cultures: potential clinical perspectives.

CONTEXT: Medullary thyroid carcinoma (MTC) is a rare tumor originating from thyroid parafollicular C cells. We previously demonstrated that somatostatin (SRIH) reduces cell growth in the human MTC cell line, TT, which expresses all SRIH receptor (SSTR) subtypes and responds differently to selective SSTR agonists. OBJECTIVE: To clarify the possible effects of SRIH analogs on hormone secretion and proliferation in MTC primary cultures, we evaluated SSTR expression and assessed the in vitro effects on calcitonin (CT) and chromogranin A secretion as well as cell viability of SRIH analogs interacting with SSTR1, SSTR2, and SSTR5. DESIGN: Thirty-five patients affected by MTC were recruited from 2003 to 2005. After total thyroidectomy, the samples were examined for CT, chromogranin A, and SSTR expression by RT-PCR. Primary cultures were developed and tested with SRIH analogs interacting with SSTR1, SSTR2, and SSTR5. RESULTS: We selected 18 MTC tumor samples, expressing SSTR1, SSTR2, and SSTR5. Two different groups were identified according to CT secretion inhibition by the clinically available SRIH analog, lanreotide. In the responder group, CT secretion was reduced by compounds interacting with SSTR1, SSTR2, and SSTR5, whereas cell viability was not affected. On the other hand, in the nonresponder group, CT secretion was reduced by the SSTR1 selective agonist, whereas cell viability was inhibited by SSTR2 selective agonists. CONCLUSIONS: Our data suggest that SRIH analogs might be useful in medical therapy of MTC because they could have antiproliferative effects despite the lack of antisecretory activity and vice versa.

SRC homology-2-containing protein tyrosine phosphatase-1 restrains cell proliferation in human medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) is a rare tumor originating from thyroid parafollicular C cells, where, in the inherited form, constitutive activation of the RET protooncogene is responsible for unrestrained cell proliferation. We previously demonstrated that somatostatin (SRIF) reduces cell growth in the human MTC cell line TT, which expresses all SRIF receptor (SSTR) subtypes and responds differently to selective SSTR agonists. The antiproliferative mechanism of SRIF and its analogs in MTC is still unclear. Src homology-2-containing protein tyrosine phosphatase-1 (SHP-1), a cytoplasmic protein tyrosine phosphatase (PTP), is activated by somatotropin release-inhibiting factor and reduces mutated RET autophosphorylation in a heterologous system. In this study, we explore the role of PTP activation, in particular of SHP-1, in TT cells, where RET is constitutively activated. In TT cells, SRIF stimulated the PTP activity of SHP-1, which was associated with proliferation inhibition and with reduction in the MAPK pathway activation. Blockade of PTP activity with sodium orthovanadate induced cell proliferation and MAPK phosphorylation and blunted the inhibitory effects of SRIF. Moreover, SHP-1 associates with SSTR2 depending on its activation. By using a MAPK kinase inhibitor, we demonstrated that TT cell growth depends on MAPK pathway activation. Furthermore, in TT cells overexpressing SHP-1, cell proliferation and MAPK signaling were strongly down-regulated, whereas in TT cells transfected with a dominant negative form of SHP-1, cell proliferation and MAPK signaling were markedly induced. Our data demonstrate that SRIF inhibitory effects on TT cell proliferation are mediated, at least in part, by SHP-1, which acts through a MAPK-dependent mechanism.

Cyclooxygenase-2 inhibitors reverse chemoresistance phenotype in medullary thyroid carcinoma by a permeability glycoprotein-mediated mechanism.

OBJECTIVE: Medullary thyroid carcinoma (MTC) is a highly chemoresistant malignant neoplasia deriving from parafollicular C cells. Chemotherapy failure has been ascribed, at least in part, to the overexpression by MTC of the multidrug resistance 1 (MDR1) gene, encoding a transmembrane glycoprotein [permeability glycoprotein (P-gp)] that antagonizes intracellular accumulation of cytotoxic agents. P-gp expression and function in a rat model have been demonstrated to depend on cyclooxygenase (COX)-2 isoform levels, which are found elevated in many human cancers. The aim of our study was to investigate the role of the COX-2 pathway in modulating chemoresistance. DESIGN AND RESULTS: We investigated P-gp and COX-2 expression and then evaluated the sensitizing effects of COX-2 inhibitors on the cytotoxic effects of doxorubicin in the presence or in the absence of prostaglandin E2 in primary cultures and in a human MTC cell line, TT. Moreover, P-gp function has been studied. Our data show that TT cells express both MDR1 and COX-2 and that rofecoxib, a selective COX-2 inhibitor, sensitizes TT cells to the cytotoxic effects of doxorubicin, reducing P-gp expression and function. CONCLUSIONS: Our data suggest that these effects are mediated by a mechanism not involving the generation of prostaglandin E2, possibly implicating the synthesis of other COX-2 products.

Utilization of luminescent technology to develop a kinase assay: Cdk4 as a model system.

Protocols to assess kinase activity generally include radioactive methods, fluorescent polarization technology and the use of specific antibodies. Here, a simple, effective, non radioactive method to measure kinase activity of immunoprecipitated proteins is described. Cdk4, a cell cycle dependent enzyme, was immunoprecipitated from whole cell extracts and used in kinase reactions. This system has been developed taking advantage of the kinase-Glo reagent (Promega), based on ATP depletion technology, but with a wider range of applications. The original aim of the commercial kit is the evaluation of kinase activity of highly purified enzymes, while this system enabled the evaluation of native kinases, retrieved by immunoprecipitation. This method was highly homogeneous and did not require any kind of separation or purification as well. Moreover, it was suitable for basic research and may be useful for low-medium throughput pharmaceutical screening of chemical libraries.

A differentially expressed set of microRNAs in cerebro-spinal fluid (CSF) can diagnose CNS malignancies.

Central Nervous System malignancies often require stereotactic biopsy or biopsy for differential diagnosis, and for tumor staging and grading. Furthermore, stereotactic biopsy can be non-diagnostic or underestimate grading. Hence, there is a compelling need of new diagnostic biomarkers to avoid such invasive procedures. Several biological markers have been proposed, but they can only identify specific prognostic subtype of Central Nervous System tumors, and none of them has found a standardized clinical application.The aim of the study was to identify a Cerebro-Spinal Fluid microRNA signature that could differentiate among Central Nervous System malignancies.CSF total RNA of 34 neoplastic and of 14 non-diseased patients was processed by NanoString. Comparison among groups (Normal, Benign, Glioblastoma, Medulloblastoma, Metastasis and Lymphoma) lead to the identification of a microRNA profile that was further confirmed by RT-PCR and in situ hybridization.Hsa-miR-451, -711, 935, -223 and -125b were significantly differentially expressed among the above mentioned groups, allowing us to draw an hypothetical diagnostic chart for Central Nervous System malignancies.This is the first study to employ the NanoString technique for Cerebro-Spinal Fluid microRNA profiling. In this article, we demonstrated that Cerebro-Spinal Fluid microRNA profiling mirrors Central Nervous System physiologic or pathologic conditions. Although more cases need to be tested, we identified a diagnostic Cerebro-Spinal Fluid microRNA signature with good perspectives for future diagnostic clinical applications.

Evidence for differential effects of selective somatostatin receptor subtype agonists on alpha-subunit and chromogranin a secretion and on cell viability in human nonfunctioning pituitary adenomas in vitro.

Somatostatin (SRIF) analogs interacting with SRIF receptor (SSTR) subtypes SSTR2 and SSTR5 reduce hormone secretion of pituitary adenomas, but their antiproliferative effects are still controversial. We investigated the in vitro effects of SRIF and SSTR-selective agonists interacting with SSTR1 (BIM-23926), SSTR2 (BIM-23120), SSTR5 (BIM-23206), or both SSTR2 and SSTR5 (BIM-23244) on alpha-subunit and chromogranin A secretion and on cell viability of 12 nonfunctioning pituitary adenomas (NFA) expressing SSTR1, SSTR2, and SSTR5, as assessed by RT-PCR. Treatment with SRIF or BIM-23206 did not modify alpha-subunit and chromogranin A secretion, which was significantly inhibited by BIM-23926, BIM-23120, and BIM-23244. SRIF and BIM-23120 did not influence cell viability, which was significantly promoted by BIM-23206 and BIM-23244 and reduced by treatment with BIM-23926. These results demonstrate that, in the selected NFA, the SSTR1-selective agonist inhibits secretory activity and cell viability, the SSTR2-selective agonist inhibits secretion but not cell viability, and the SSTR5-selective agonist does not influence secretion but promotes cell viability. These data can explain the lack of inhibitory effects of currently used SRIF analogs and suggest that drugs acting potently and preferentially on SSTR1 might be useful for medical treatment of NFA.

MicroRNAs as main players in the pathogenesis of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. The accumulation of mature CD5(+) B-lymphocytes in bone marrow, peripheral blood, and lymphoid organs due to decreased apoptosis is a characteristic of this malignancy. MicroRNAs (miRNAs) are small noncoding RNAs able to regulate the expression of many target genes, including the main apoptosis regulators BCL2 and MCL1. miRNAs play key roles in the pathogenesis of CLL, including specific miRNAs located at the 13q14 chromosomal region that is often deleted or mutated in patients with CLL. In this paper, we review new investigations that underscore the significance of miRNAs for CLL pathogenesis.

MicroRNA profiles discriminate among colon cancer metastasis.

MicroRNAs are being exploited for diagnosis, prognosis and monitoring of cancer and other diseases. Their high tissue specificity and critical role in oncogenesis provide new biomarkers for the diagnosis and classification of cancer as well as predicting patients' outcomes. MicroRNAs signatures have been identified for many human tumors, including colorectal cancer (CRC). In most cases, metastatic disease is difficult to predict and to prevent with adequate therapies. The aim of our study was to identify a microRNA signature for metastatic CRC that could predict and differentiate metastatic target organ localization. Normal and cancer tissues of three different groups of CRC patients were analyzed. RNA microarray and TaqMan Array analysis were performed on 66 Italian patients with or without lymph nodes and/or liver recurrences. Data obtained with the two assays were analyzed separately and then intersected to identify a primary CRC metastatic signature. Five differentially expressed microRNAs (hsa-miR-21, -103, -93, -31 and -566) were validated by qRT-PCR on a second group of 16 American metastatic patients. In situ hybridization was performed on the 16 American patients as well as on three distinct commercial tissues microarray (TMA) containing normal adjacent colon, the primary adenocarcinoma, normal and metastatic lymph nodes and liver. Hsa-miRNA-21, -93, and -103 upregulation together with hsa-miR-566 downregulation defined the CRC metastatic signature, while in situ hybridization data identified a lymphonodal invasion profile. We provided the first microRNAs signature that could discriminate between colorectal recurrences to lymph nodes and liver and between colorectal liver metastasis and primary hepatic tumor.