Livia Manzella

The Insulin/IGF System in Colorectal Cancer Development and Resistance to Therapy

The insulin/insulin-like growth factor (IGF) system is a major determinant in the pathogenesis and progression of colorectal cancer (CRC). Indeed, several components of this signaling network, including insulin, IGF-1, IGF-2, the IGF-binding proteins, the insulin receptor (IR), the IGF-1 receptor (IGF-1R), and IR substrate proteins 1 and 2 contribute to the transformation of normal colon epithelial cells. Moreover, the insulin/IGF system is also implicated in the development of resistance to both chemotherapeutic drugs and epidermal growth factor receptor targeted agents. The identification of hybrid receptors comprising both the IR and IGF-1R adds further complexity to this signaling network. Thus, a comprehensive understanding of the biological functions performed by each component of the insulin/IGF system is required to design successful drugs for the treatment of CRC patients.

Suppression of Survivin Induced by a BCR-ABL/JAK2/STAT3 Pathway Sensitizes Imatinib-Resistant CML Cells to Different Cytotoxic Drugs

The BCR-ABL oncoprotein of chronic myelogenous leukemia (CML) displays exclusive cytoplasmic localization and constitutive tyrosine kinase activity leading to the activation of different pathways that favor cell proliferation and survival. BCR-ABL induces survivin expression at both the mRNA and protein level, thus inhibiting the apoptotic machinery of CML cells and contributing to the expansion of the leukemic clone. We report that, in human CML cell lines, BCR-ABL–mediated upregulation of survivin involves the JAK2/STAT3 pathway since silencing of either protein caused a consistent reduction in survivin expression. Cell lines unresponsive to imatinib mesylate (IM) because of BCR-ABL gene amplification were not resensitized to the drug after survivin downregulation. However, cells insensitive to IM because of point mutations in the BCR-ABL kinase domain were highly responsive to hydroxyurea (HU) after survivin silencing. To address the possible clinical applications of our results, we used shepherdin, a cell-permeable peptidomimetic compound that downregulates survivin expression by preventing its interaction with Hsp90. Incubation with shepherdin of immortalized cell lines both sensitive and resistant to IM enhanced cell death induced by HU and doxorubicin. Similarly, the combination of shepherdin with first- and second-generation tyrosine kinase inhibitors reduced the colony-forming potential of human progenitors derived from both patients with IM-sensitive and IM-resistant CML. These results suggest that strategies aimed at reducing survivin levels may represent a potential therapeutic option for patients with CML unresponsive to IM. Mol Cancer Ther; 12(6); 1085–98. ©2013 AACR.

Role of interferon regulatory factor 1 in monocyte/macrophage differentiation

Interferon regulatory factor‐1 (IRF‐1) has been recognized as an important tumor suppressor and growth regulatory transcription factor, which is also involved in cell differentiation. In this study we investigated the role of IRF‐1 in phorbol 12‐myristate 13‐acetate (PMA)‐induced monocyte/macrophage differentiation of human monoblastic U937 cells. For this purpose U937 cells were stably transfected with a vector overexpressing IRF‐1 antisense mRNA (U937 IRF‐1A cells) and with the SV‐40 empty vector (U937‐SV40 e.v. cells). We report here that U937 and U937‐SV40 e.v. cells differentiated into macrophage‐like cells upon PMA stimulation and showed IRF‐1 up‐regulation. On the contrary, U937 IRF‐1A cells stimulated with PMA kept an undifferentiated phenotype and proliferated actively. A direct correlation between induction of IRF‐1 and up‐regulation of IRF‐1 gene targets such as ornithine decarboxylase (ODC) and WAF‐1/CIP‐1 was also observed in U937 cells. On the other hand U937 IRF‐1A cells down‐regulated ODC and did not express WAF‐1. Results show that IRF‐1 plays a pivotal role in PMA‐induced monocyte/macrophage differentiation.

IRF5 promotes the proliferation of human thyroid cancer cells.

BackgroundInterferon Regulatory Factor 5 is a transcription factor that regulates the expression of genes involved in the response to viral infection and in the stimulation of the immune system. Moreover, multiple studies have demonstrated that it negatively regulates cell growth and oncogenesis, favoring cell differentiation and apoptosis.Thyroid carcinoma represents 98% of all thyroid malignancies and has shown a steady increase in incidence in both the USA and western European countries.FindingsWe investigated the expression, localization and function of IRF5 in thyroid cancer cells and found that it is highly expressed in both primary and immortalized thyroid carcinomas but not in normal thyrocytes. IRF5 levels were variably modulated by Interferon alpha but IRF5 only localized in the cytoplasmic compartment, thus failing to induce p21 expression as previously reported in different cell models. Furthermore, ectopic IRF5 increased both the proliferation rate and the clonogenic potential of malignant thyroid cells, protecting them from the cytotoxic effects of DNA-damaging agents. These results were directly attributable to IRF5, as demonstrated by the reduction in colony-forming ability of thyroid cancer cells after IRF5 silencing. An IRF5-dependent induction of endogenous B-Raf observed in all thyroid cancer cells might contribute to these unexpected effects.ConclusionsThese findings suggest that, in thyroid malignancies, IRF5 displays tumor-promoting rather than tumor-suppressor activities.

BCR-ABL residues interacting with ponatinib are critical to preserve the tumorigenic potential of the oncoprotein

Patients with chronic myeloid leukemia in whom tyrosine kinase inhibitors (TKIs) fail often present mutations in the BCR‐ABL catalytic domain. We noticed a lack of substitutions involving 4 amino acids (E286, M318, I360, and D381) that form hydrogen bonds with ponatinib. We therefore introduced mutations in each of these residues, either preserving or altering their physicochemical properties. We found that E286, M318, I360, and D381 are dispensable for ABL and BCR‐ABL protein stability but are critical for preserving catalytic activity. Indeed, only a “conservative” I360T substitution retained kinase proficiency and transforming potential. Molecular dynamics simulations of BCR‐ABLI360T revealed differences in both helix αC dynamics and protein‐correlated motions, consistent with a modified ATP‐binding pocket. Nevertheless, this mutant remained sensitive to ponatinib, imatinib, and dasatinib. These results suggest that changes in the 4 BCR‐ABL residues described here would be selected against by a lack of kinase activity or by maintained responsiveness to TKIs. Notably, amino acids equivalent to those identified in BCR‐ABL are conserved in 51% of human tyrosine kinases. Hence, these residues may represent an appealing target for the design of pharmacological compounds that would inhibit additional oncogenic tyrosine kinases while avoiding the emergence of resistance due to point mutations.—Buffa, P., Romano, C., Pandini, A., Massimino, M., Tirrò, E., Di Raimondo, F., Manzella, L., Fraternali, F., Vigneri, P. G. BCR‐ABL residues interacting with ponatinib are critical to preserve the tumorigenic potential of the oncoprotein. FASEB J. 28, 1221–1236 (2014). www.fasebj.org

Possible role of the transcription factor interferon regulatory factor 1 (IRF-1) in the regulation of ornithine decarboxylase (ODC) gene expression during IFNγ macrophage activation

In this report we discuss the role of interferon regulatory factor 1 (IRF‐1) in the regulation of ornithine decarboxylase (ODC) transcription during IFNγ human macrophage activation. We show that a binding sequence for the transcription factor IRF‐1 is contained in the first intron of the human ODC gene (from nt +2711 to nt +2722) we demonstrate that the level of expression of IRF‐1 increases in human macrophages and in the human promonocytic cell line, U937, previously differentiated in monocytes/macrophages by phorbol myristate acetate (PMA), after 2 h of IFNγ stimulation. We also show that the hamster tk‐ts13 cell line, stably transfected with the IRF‐1 cDNA, over‐expresses ODC. In addition, a specific complex was detected, by gel‐shift assay after incubating a 20 bp double‐stranded oligomer containing the binding sequence for IRF‐1 with nuclear proteins extracted from human macrophages and from (PMA‐differentiated) U937 cells stimulated with IFNγ for 2 h.

Pyrrolidinedithiocarbamate Induces Apoptosis in Human Acute Myelogenous Leukemic Cells Affecting NF-κB Activity

Pyrrolidindithiocarbamate (PDTC), is a metal chelator widely used to study the activation of redox sensitive transcription factors. Recently it has been demonstrated that it manifests pro-oxidant properties. The nuclear factor-Kappa B (NF-κB) transcription factor can both promote cell survival and induce apoptosis depending on cell type and context in response to genotoxic stress. In our previous study we reported that in acute myelogenous leukemia CD34+ cells PDTC stimulates apoptosis, whereas in CD34+ cells of healthy volunteers PDTC was ineffective. This cytotoxicity was dependent on the generation of superoxide anion and oxidized glutathione. In this article we have shown that the pro-oxidant effect of PDTC in AML cells induces NF-κB activity. These findings imply a role for NF-κB in the survival of normal cells with respect to leukemic cells, suggesting that NF-κB activity and function differs according to tumor cell phenotype.

Biomarkers and prognostic factors for malignant pleural mesothelioma

The increasing incidence and the dismal prognosis of malignant pleural mesothelioma calls for the identification of biomarkers that will allow a timely diagnosis; display prognostic value; and, predict the response to pharmacological agents employed for the treatment of the disease. Biomarkers associated with early diagnosis currently include mesothelin in combination with miRNA miR-103a-3p. As for prognostic biomarkers, the Cancer and Leukemia Group B (CALGB) and the European Organization for Research and Treatment of Cancer (EORTC) scores take into account different hematological and clinical parameters that distinguish patients with good prognosis from those with inferior outcomes. Fluorodeoxyglucose-PET, microarray expression data, neutrophil-to-lymphocyte ratios, c-MET expression, Ki-67 ratios and fibulin-3 levels have also been associated with disease outcome. Finally, thymidylate synthase protein cutoffs may predict mesothelioma response to the association of pemetrexed with a platinum derivative.

IRF5 is a target of BCR-ABL kinase activity and reduces CML cell proliferation

Interferon regulatory factor 5 (IRF5) modulates the expression of genes controlling cell growth and apoptosis. Previous findings have suggested a lack of IRF5 transcripts in both acute and chronic leukemias. However, to date, IRF5 expression and function have not been investigated in chronic myeloid leukemia (CML). We report that IRF5 is expressed in CML cells, where it interacts with the BCR-ABL kinase that modulates its expression and induces its tyrosine phosphorylation. Tyrosine-phosphorylated IRF5 displayed reduced transcriptional activity that was partially restored by imatinib mesylate (IM). Interestingly, a mutant devoid of a BCR-ABL consensus site (IRF5(Y104F)) still presented significant tyrosine phosphorylation. This finding suggests that the oncoprotein phosphorylates additional tyrosine residues or induces downstream signaling pathways leading to further IRF5 phosphorylation. We also found that ectopic expression of IRF5 decreases the proliferation of CML cell lines by slowing their S-G2 transition, increasing the inhibition of BCR-ABL signaling and enhancing the lethality effect observed after treatment with IM, α-2-interferon and a DNA-damaging agent. Furthermore, IRF5 overexpression successfully reduced the clonogenic ability of CML CD34-positive progenitors before and after exposure to the above-indicated cytotoxic stimuli. Our data identify IRF5 as a downstream target of the BCR-ABL kinase, suggesting that its biological inactivation contributes to leukemic transformation.

New Insights in Thyroid Cancer and p53 Family Proteins

Thyroid cancers are common endocrine malignancies that comprise tumors with different clinical and histological features. Indeed, papillary and follicular thyroid cancers are slow-growing, well-differentiated tumors, whereas anaplastic thyroid cancers are undifferentiated neoplasias that behave much more aggressively. Well-differentiated thyroid carcinomas are efficiently cured by surgery and radioiodine, unlike undifferentiated tumors that fail to uptake radioactive iodine and are usually resistant to chemotherapy. Therefore, novel and more effective therapies for these aggressive neoplasias are urgently needed. Whereas most genetic events underlying the pathogenesis of well-differentiated thyroid cancers have been identified, the molecular mechanisms that generate undifferentiated thyroid carcinomas are still unclear. To date, one of the best-characterized genetic alterations leading to the development of poorly differentiated thyroid tumors is the loss of the p53 tumor suppressor gene. In addition, the existence of a complex network among p53 family members (p63 and p73) and their interactions with other factors that promote thyroid cancer progression has been well documented. In this review, we provide an update on the current knowledge of the role of p53 family proteins in thyroid cancer and their possible use as a therapeutic target for the treatment of the most aggressive variants of this disease.