Jerusa Araújo Quintão Arantes Faria

SET domain-containing Protein 4 (SETD4) is a Newly Identified Cytosolic and Nuclear Lysine Methyltransferase involved in Breast Cancer Cell Proliferation.

Cancer is comprised of a multitude of epigenetic abnormalities, including the global loss and regional gain of DNA methylation as well as alterations in histone methylation. Here, we characterize a new methyltransferase, SET domain-containing protein 4 (SETD4), which is involved in breast carcinogenesis. Quantitative real-time PCR (qPCR) showed elevated expression levels of SETD4 in several breast cancer cell lines. SETD4 overexpression was confirmed by western blot analysis suggesting a correlation between high expression of SETD4 and a lack of the estrogen receptor (ER) in breast cancer. In addition, cell fractionation studies and confocal immunofluorescence revealed the nuclear and non-nuclear localization of this new protein. SETD4 knockdown in breast cancer cell lines significantly suppressed their proliferation and delayed the G1/S cell cycle transition without affecting apoptosis. Furthermore, western blot analysis showed that knockdown of SETD4 decreased cyclin D1 expression, revealing the involvement of SETD4 in cell cycle regulation. These data imply that SETD4 plays a crucial role in breast carcinogenesis and could be a novel molecular target for the development of new strategies for the diagnosis and treatment of breast cancer.

Inner nuclear membrane localization of epidermal growth factor receptor (EGFR) in spontaneous canine model of invasive micropapillary carcinoma of the mammary gland.

The epidermal growth factor receptor (EGFR) has been described in the nucleus of primary tumors. Accumulation of EGFR at the nucleus is linked to DNA synthesis and cell proliferation, but the pathological significance of nuclear EGFR is not completely understood. The aim of this study was to investigate the nuclear localization of EGFR in invasive micropapillary carcinoma (IMPC) that is an aggressive neoplasm of canine mammary gland. Confocal immunofluorescence of formalin and paraffin-embedded tissue was used to access the subcellular localization of EGFR. Our results demonstrated that EGFR co-localizes with the inner nuclear envelope marker, Lamin B1 in IMPC. Furthermore, EGFR was not localized within the nucleus or at the inner nuclear envelope membrane in mammary carcinoma in mixed tumor (CMT) that is associated with a better prognosis than other malignant histological types. This finding could be useful as a predictive biomarker of therapeutic response for IMPC.

Genomic and phenotypic profiles of two Brazilian breast cancer cell lines derived from primary human tumors

Breast cancer is the most common type of cancer among women worldwide. Research using breast cancer cell lines derived from primary tumors may provide valuable additional knowledge regarding this type of cancer. Therefore, the aim of this study was to investigate the phenotypic profiles of MACL-1 and MGSO-3, the only Brazilian breast cancer cell lines available for comparative studies. We evaluated the presence of hormone receptors, proliferation, differentiation and stem cell markers, using immunohistochemical staining of the primary tumor, cultured cells and xenografts implanted in immunodeficient mice. We also investigated the ability of the cell lines to form colonies and copy number alterations by array comparative genomic hybridization. Histopathological analysis showed that the invasive primary tumor from which the MACL-1 cell line was derived, was a luminal A subtype carcinoma, while the ductal carcinoma in situ (DCIS) that gave rise to the MGSO-3 cell line was a HER2 subtype tumor, both showing different proliferation levels. The cell lines and the tumor xenografts in mice preserved their high proliferative potential, but did not maintain the expression of the other markers assessed. This shift in expression may be due to the selection of an ‘establishment’ phenotype in vitro. Whole-genome DNA evaluation showed a large amount of copy number alterations (CNAs) in the two cell lines. These findings render MACL-1 and MGSO-3 the first characterized Brazilian breast cancer cell lines to be potentially used for comparative research.

Cytoplasmic-targeted parvalbumin blocks the proliferation of multipotent mesenchymal stromal cells in prophase.

IntroductionMultipotent mesenchymal stromal cells (MSCs) have gained considerable interest because of their potential use in the treatment of a variety of diseases and injuries. Although remarkable advancements have been made in clinical studies, substantial concerns still regard the safety of MSCs. Some evidence suggests that MSCs can spontaneously generate a population of cells with tumorigenic potential. Thus, studying the molecular mechanisms that control the proliferation of MSCs may be a necessary step toward the development of strategies for safe clinical practice. Ca2+ is a second messenger that mediates a wide range of cellular responses, including the regulation of cell proliferation, but little is known about its function in MSCs. The aim of this study was to investigate the effects of targeted Ca2+ buffering on MSCs proliferation in vitro.MethodsHere, we used an adenoviral (Ad) vector encoding the Ca2+ chelator protein parvalbumin (PV) fused to a nuclear exclusion signal (NES) and the Discosoma red fluorescent protein (DsRed) to investigate the function of cytoplasmic Ca2+ signals on MSC proliferation. Confocal microscopy was used to demonstrate that PV-NES-DsRed was expressed in the cytoplasm. Ca2+ signaling was monitored by using Fluo-4-AM. Fluorescence-activated cell sorting (FACS) analysis of cells that were stained with propidium iodide was used as a quantitative measure of cell death. The mitotic index was assessed by immunofluorescence, and the expression of cyclins was examined with Western blot.ResultsOur results show that the Ad-PV-NES-DsRed fusion protein decreased serum-induced Ca2+ signaling and blocked the proliferation of rat adipose-derived MSCs (AT-MSCs) in prophase. FACS analysis revealed that Ad-PV-NES-DsRed did not induce cell death in AT-MSCs. Furthermore, Western blot analysis demonstrated that Ad-PV-NES-DsRed reduced extracellular signal-regulated kinase (Erk1/2) phosphorylation and cyclin B1 expression. Buffering cytosolic Ca2+ did not alter the expression of cyclins A/D1/D2/D3/E and E2.ConclusionsOur results show that cytoplasmic Ca2+ signals are important for AT-MSCs progression beyond prophase because of their effects on Erk phosphorylation and cyclin B1 expression.

Mesoporous silica SBA-16/hydroxyapatite-based composite for ciprofloxacin delivery to bacterial bone infection

The development of systems that can prevent infections and also ensure bone integration as well as regeneration have been of great interest for pharmaceutical technology. In this study, we show the synthesis of surface-functionalized mesoporous silica (SBA-16) and silica composed of calcium phosphate (SBA-16/HA) particles in order to be applied as efficient drug delivery carriers. The particles were synthesized, functionalized with 3-aminopropyltriethoxysilane (APTES) by post-synthesis grafting and loaded with the osteomyelitis antibiotic agent ciprofloxacin. Moreover, the diethylenetriaminepentacetic acid (DTPA) was anchored in silica-APTES to allow measurements of biological process at molecular and cellular levels. Particles were physicochemically characterized by small angle X-ray scattering (SAXS), elemental analysis (CHN), thermogravimetric analysis (TGA), N2 adsorption and zeta potential analysis. Functionalized silica particles were radiolabeled with technetium-99m showing high radiochemical yields and high radiolabeled stability. In vivo experiments results showed higher bone uptake of the SBA-16/HAAPTES than SBA-16APTES. In addition, bactericidal efficacy of these particles was tested against microorganisms present in bone infection, and our composites had bactericidal efficiency comparable to free-ciprofloxacin. In summary, taking into account the great potential of these silica mesoporous and nanocomposite structures to carry molecules, besides their bactericidal efficacy, these materials are promising candidates for bone infection treatment.Graphical abstract

Phospholipase C delta 4 (PLCδ4) is a nuclear protein involved in cell proliferation and senescence in mesenchymal stromal stem cells

Ca2+ is an important second messenger, and it is involved in many cellular processes such as cell death and proliferation. The rise in intracellular Ca2+ levels can be due to the generation of inositol 1,4,5-trisphosphate (InsP3), which is a product of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by phospholipases C (PLCs), that leads to Ca2+ release from endoplasmic reticulum by InsP3 receptors (InsP3R). Ca2+ signaling patterns can vary in different regions of the cell and increases in nuclear Ca2+ levels have specific biological effects that differ from those of Ca2+ increase in the cytoplasm. There are PLCs in the cytoplasm and nucleus, but little is known about the functions of nuclear PLCs. This work aimed to characterize phenotypically the human PLCδ4 (hPLCδ4) in mesenchymal stem cells. This nuclear isoform of PLC is present in different cell types and has a possible role in proliferative processes. In this work, hPLCδ4 was found to be mainly nuclear in human adipose-derived mesenchymal stem cells (hASC). PLCδ4 knockdown demonstrated that it is essential for hASC proliferation, without inducing cell death. An increase of cells in G1, and a reduction of cells on interphase and G2/M in knockdown cells were seen. Furthermore, PLCδ4 knockdown increased the percentage of senescent cells, p16INK4A+ and p21Cip1 mRNAs expression, which could explain the impaired cell proliferation. The results show that hPLCδ4 is in involved in cellular proliferation and senescence in hASC.

Translocation of Epidermal Growth Factor (EGF) to the nucleus has distinct kinetics between adipose tissue-derived mesenchymal stem cells and a mesenchymal cancer cell lineage

Nuclear Epidermal Growth Factor Receptor (EGFR) has been associated with worse prognosis and treatment resistance for several cancer types. After Epidermal Growth Factor (EGF) binding, the ligand-receptor complex can translocate to the nucleus where it functions in oncological processes. By three-dimensional quantification analysis of super-resolution microscopy images, we verified the translocation kinetics of fluorescent conjugated EGF to the nucleus in two mesenchymal cell types: human adipose tissue-derived stem cells (hASC) and SK-HEP-1 tumor cells. The number of EGF clusters in the nucleus does not change after 10 min of stimulation with EGF in both cells. The total volume occupied by EGF clusters in the nucleus of hASC also does not change after 10 min of stimulation with EGF. However, the total volume of EGF clusters increases only after 20 min in SK-HEP-1 cells nuclei. In these cells the nuclear volume occupied by EGF is 3.2 times higher than in hASC after 20 min of stimulation, indicating that translocation kinetics of EGF differs between these two cell types. After stimulation, EGF clusters assemble in larger clusters in the cell nucleus in both cell types, which suggests specific sub-nuclear localizations of the receptor. Super-resolution microscopy images show that EGF clusters are widespread in the nucleoplasm, and can be localized in nuclear envelope invaginations, and in the nucleoli. The quantitative study of EGF-EGFR complex translocation to the nucleus may help to unravel its roles in health and pathological conditions, such as cancer.