Dynamics of chromatin position in the interphase nucleus volume

In this article, we attempt to analyze the relationship between the processes which occur in the nucleus and the dynamics of chromatin, as well as to classify the changes in the position of chromatin in the cell nucleus during the lifetime of the cell. The proposed concept integrates possible types of chromatin movement within the nucleus.     

Advances in the computational and molecular understanding of the prostate cancer cell nucleus

Nuclear alterations are a hallmark of many types of cancers, including prostate cancer (PCa). Recent evidence shows that subvisual changes, ones that may not be visually perceptible to a pathologist, to the nucleus and its ultrastructural components can precede visual histopathological recognition of cancer. Alterations to nuclear features, such as nuclear size and shape, texture, and spatial architecture, reflect the complex molecular‐level changes that occur during oncogenesis. Quantitative nuclear morphometry, a field that uses computational approaches to identify and quantify malignancy‐induced nuclear changes, can enable a detailed and objective analysis of the PCa cell nucleus. Recent advances in machine learning–based approaches can now automatically mine data related to these changes to aid in the diagnosis, decision making, and prediction of PCa prognoses. In this review, we use PCa as a case study to connect the molecular‐level mechanisms that underlie these nuclear changes to the machine learning computational approaches, bridging the gap between the clinical and computational understanding of PCa. First, we will discuss recent developments to our understanding of the molecular events that drive nuclear alterations in the context of PCa: the role of the nuclear matrix and lamina in size and shape changes, the role of 3‐dimensional chromatin organization and epigenetic modifications in textural changes, and the role of the tumor microenvironment in altering nuclear spatial topology. We will then discuss the advances in the applications of machine learning algorithms to automatically segment nuclei in prostate histopathological images, extract nuclear features to aid in diagnostic decision making, and predict potential outcomes, such as biochemical recurrence and survival. Finally, we will discuss the challenges and opportunities associated with translation of the quantitative nuclear morphometry methodology into the clinical space. Ultimately, accurate identification and quantification of nuclear alterations can contribute to the field of nucleomics and has applications for computationally driven precision oncologic patient care.     

Nuclear matrix proteins in well and poorly differentiated human breast cancer cell lines

The nuclear matrix, besides providing the structural support of the nucleus, is involved in various cellular functions of the nucleus. Nuclear matrix proteins (NMPs), which are both tissue- and cell type–specific, are altered with transformation and state of differentiation. Furthermore, NMPs have been identified as informative markers of disease states. Here, the NMP profiles from human breast cancer cell lines and breast tumours were analyzed using two-dimension gel electrophoresis. We identified NMPs that are associated with well and poorly differentiated human breast cancer cells in vitro and in vivo. Five NMPs (NMBC 1–5) were found to be exclusive for well-differentiated human breast cancer cells, while one NMP (NMBC-6) was found to be present only in poorly differentiated human breast cancer cells. The identification of these proteins suggests the potential use of nuclear matrix proteins as prognostic indicators. J. Cell. Biochem. 66:9–15, 1997. © 1997 Wiley-Liss, Inc.     

Prelamin A, Zmpste24, misshapen cell nuclei, and progeria--new evidence suggesting that protein farnesylation could be important for disease pathogenesis

Prelamin A undergoes multistep processing to yield lamin A, a structural protein of the nuclear lamina. Prelamin A terminates with a CAAX motif, which triggers farnesylation of a C-terminal cysteine (the C of the CAAX motif), endoproteolytic release of the last three amino acids (the AAX), and methylation of the newly exposed farnesylcysteine residue. In addition, prelamin A is cleaved a second time, releasing 15 more residues from the C terminus (including the farnesylcysteine methyl ester), generating mature lamin A. This second cleavage step is carried out by an endoplasmic reticulum membrane protease, ZMPSTE24. Interest in the posttranslational processing of prelamin A has increased with the recognition that certain progeroid syndromes can be caused by mutations that lead to an accumulation of farnesyl-prelamin A. Recently, we showed that a key cellular phenotype of these progeroid disorders, misshapen cell nuclei, can be ameliorated by inhibitors of protein farnesylation, suggesting a potential strategy for treating these diseases. In this article, we review the posttranslational processing of prelamin A, describe several mouse models for progeroid syndromes, explain the mutations underlying several human progeroid syndromes, and summarize recent data showing that misshapen nuclei can be ameliorated by treating cells with protein farnesyltransferase inhibitors.     

A novel domain of caveolin‐2 that controls nuclear targeting: regulation of insulin‐specific ERK activation and nuclear translocation by caveolin‐2

Herein, we report that insulin‐activated extracellular signal‐regulated kinase (ERK) is translocated to the nuclear envelope by caveolin‐2 (cav‐2) and associates with lamin A/C in the inner nuclear membrane in response to insulin. We identified that the Ser¹⁵⁴–Val¹⁵⁵–Ser¹⁵⁶ domain on the C‐terminal of cav‐2 is essential for insulin‐induced phosphorylation and nuclear targeting of ERK and cav‐2. In human embryonic kidney 293T cells, ERK was not activated and translocated to the nucleus by insulin in comparison to insulin‐like growth factor‐1 (IGF‐1). However, insulin‐stimulated activation of ERK was induced by exogenous addition of cav‐2. The activated ERK associated and translocated with the cav‐2 to the nucleus. In turn, cav‐2 promoted phospho‐ERK interaction with lamin A/C in the inner nuclear membrane. In contrast, ERK, but not cav‐2, was phosphorylated and translocated to the nucleus by IGF‐1. The nuclear targeted phospho‐ERK failed to localize in the nuclear envelope in response to IGF‐1. Together, our data demonstrate that translocation of phospho‐ERK to the nuclear envelope is mediated by Ser¹⁵⁴–Val¹⁵⁵–Ser¹⁵⁶ domain of cav‐2 and this event is an insulin‐specific action.     

miR‐515‐5p controls cancer cell migration through MARK4 regulation

Here, we show that miR‐515‐5p inhibits cancer cell migration and metastasis. RNA‐seq analyses of both oestrogen receptor receptor‐positive and receptor‐negative breast cancer cells overexpressing miR‐515‐5p reveal down‐regulation of NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNAs. We demonstrate that miR‐515‐5p inhibits MARK4 directly 3′ UTR interaction and that MARK4 knock‐down mimics the effect of miR‐515‐5p on breast and lung cancer cell migration. MARK4 overexpression rescues the inhibitory effects of miR‐515‐5p, suggesting miR‐515‐5p mediates this process through MARK4 down‐regulation. Furthermore, miR‐515‐5p expression is reduced in metastases compared to primary tumours derived from both in vivo xenografts and samples from patients with breast cancer. Conversely, miR‐515‐5p overexpression prevents tumour cell dissemination in a mouse metastatic model. Moreover, high miR‐515‐5p and low MARK4 expression correlate with increased breast and lung cancer patients' survival, respectively. Taken together, these data demonstrate the importance of miR‐515‐5p/MARK4 regulation in cell migration and metastasis across two common cancers.     

Identification of PSF as a protein kinase Calpha-binding protein in the cell nucleus

Protein kinase C (PKC) isoforms are present in the cell nucleus in diverse cell lines and tissues. Since little is known about proteins interacting with PKC inside the cell nucleus, we used Neuro-2a neuroblastoma cells, in which PKCalpha is present in the nucleus, to screen for nuclear binding partners for PKC. Applying overlay assays, we detected several nuclear proteins which bind to PKCalpha. Specificity of binding was shown by its dependence on PKC activation by phorbol ester, calcium, and phosphatidylserine. The PKC-binding proteins were partially purified and analyzed by microsequencing and mass spectrometry. Four proteins could be identified: PTB-associated splicing factor (PSF), p68 RNA helicase, and the heterogeneous nuclear ribonucleoprotein (hnRNP) proteins A3 and L. In the case of PSF, binding to PKC could also be demonstrated in a GST-pull-down assay using GST-PKCalpha, expressed in insect cells. Phosphorylation experiments revealed that PSF is a weak in vitro substrate for PKCalpha.     

Promotion of human adipose‐derived stem cell proliferation mediated by exogenous nucleosides

Adult stem cells are becoming the best option for regenerative medicine because they have low tumourigenic potential and permit autologous transplantation, even without in vitro culture. Our objectives were to evaluate the effects of exogenous nucleosides on the proliferation of hASCs (human adipose‐derived stem cells), with or without co‐treatment with 5‐aza (5‐azacytidine), and to analyse the expression of lamin A/C during cardiomyocyte differentiation of these cells. We isolated hASCs from human lipoaspirates that were positive for mesenchymal stem cell markers. We found that 5‐aza induces a dose‐dependent inhibition of hASC proliferation [IC50 (inhibitory concentration 50): 5.37 μM], whereas exogenous nucleosides significantly promote the proliferation of hASCs and partially revert the antiproliferative effect of the drug. Multipotentiality of isolated hASCs was confirmed by adipogenic, osteogenic and cardiomyogenic induction. 5‐Aza‐induced cells expressed cardiac troponins I and T and myosin light chain 2, myocardial markers that were directly correlated with lamin A/C expression. Our results support the importance of the nucleoside supplementation of media to improve conditions for the expansion and maintenance of hASCs in culture. In addition, the quantification of lamin A/C expression appears to be a good marker for the characterization of cardiomyocyte differentiation of stem cells that has rarely been used.     

Pleomorphism of the nuclear envelope in breast cancer: a new approach to an old problem

In routine practice, nuclear pleomorphism of tumours is assessed by haematoxylin staining of the membrane-bound heterochromatin. However, decoration of the nuclear envelope (NE) through the immunofluorescence staining of NE proteins such as lamin B and emerin can provide a more objective appreciation of the nuclear shape. In breast cancer, nuclear pleomorphism is one of the least reproducible parameters to score histological grade, thus we sought to use NE proteins to improve the reproducibility of nuclear grading. First, immuno-fluorescence staining of NE as well as confocal microscopy and three-dimensional reconstruction of nuclei in cultured cells showed a smooth and uniform NE of normal breast epithelium in contrast to an irregular foldings of the membrane and the presence of deep invaginations leading to the formation of an intranuclear scaffold of NE-bound tubules in breast cancer cells. Following the above methods and criteria, we recorded the degree of NE pleomorphism (NEP) in a series of 273 invasive breast cancers tested by immunofluorescence. A uniform nuclear shape with few irregularities (low NEP) was observed in 135 cases or, alternatively, marked folds of the NE and an intranuclear tubular scaffold (high NEP cases) were observed in 138 cases. The latter features were significantly correlated (P-value <0.002) with lymph node metastases in 54 histological grade 1 and in 173 cancers with low mitotic count. Decoration of the NE might thus be regarded as a novel diagnostic parameter to define the grade of malignancy, which parallels and enhances that provided by routine histological procedures.     

Increasing the length of progerin's isoprenyl anchor does not worsen bone disease or survival in mice with Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the synthesis of a truncated prelamin A, commonly called progerin, that contains a carboxyl-terminal farnesyl lipid anchor. The farnesyl lipid anchor helps to target progerin to membrane surfaces at the nuclear rim, where it disrupts the integrity of the nuclear lamina and causes misshapen nuclei. Several lines of evidence have suggested that progerin's farnesyl lipid anchor is crucial for the emergence of disease phenotypes. Because a geranylgeranyl lipid is approximately 45-fold more potent than a farnesyl lipid in anchoring proteins to lipid membranes, we hypothesized that a geranylgeranylated version of progerin might be more potent in eliciting disease phenotypes. To test this hypothesis, we used gene targeting to create mice expressing geranylgeranylated progerin (Lmna(ggHG/+)). We then compared Lmna(ggHG/+) mice, side-by-side, with otherwise identical mice expressing farnesylated progerin (Lmna(HG/+)). Geranylgeranylation of progerin in Lmna(ggHG/+) cells and farnesylation of progerin in Lmna(HG/+) cells was confirmed by metabolic labeling. Contrary to our expectations, Lmna(ggHG/+) mice survived longer than Lmna(HG/+) mice. The Lmna(ggHG/+) mice also exhibited milder bone disease. The steady-state levels of progerin, relative to lamin C, were lower in Lmna(ggHG/+) mice than in Lmna(HG/+) mice, providing a potential explanation for the milder disease in Lmna(ggHG/+) mice.     

Inhibition of breast cancer growth via miR-7 suppressing ALDH1A3 activity concomitant with decreasing breast cancer stem cell subpopulation

Breast cancer patients with high expression of aldehyde dehydrogenases (ALDHs) cell population have higher tolerability to chemotherapy since the cells posses a characteristic of breast cancer stem cells (BCSCs) that are resistant to conventional chemotherapy. In this study, we found that the ALDH-positive cells were higher in CD44⁺CD24⁻ and CD44⁺CD24⁻ESA⁺BCSCs than that in both BT549 and MDA-MB-231 cell lines but microRNA-7 (miR-7) level was lower in CD44⁺CD24⁻ and CD44⁺CD24⁻ESA⁺BCSCs than that in MDA-MB-231 cells. Moreover, miR-7 overexpression in MDA-MB-231 cells decreased ALDH1A3 activity by miR-7 directly binding to the 3′-untranslated region of ALDH1A3; while the ALDH1A3 expression was downregulated in MDA-MB-231 cells, the expressions of CD44 and Epithelium Specific Antigen (ESA) were reduced along with decreasing the BCSC subpopulation. Significantly, enforced expression of miR-7 in CD44⁺CD24⁻ESA⁺BCSC markedly inhibited the BCSC-driven xenograft growth in mice by decreasing an expression of ALDH1A3. Collectively, the findings demonstrate the miR-7 inhibits breast cancer growth via suppressing ALDH1A3 activity concomitant with decreasing BCSC subpopulation. This approach may be considered for an investigation on clinical treatment of breast cancers.     

The role of LMNA in adipose: a novel mouse model of lipodystrophy based on the Dunnigan-type familial partial lipodystrophy mutation

We investigated the role of LMNA in adipose tissue by developing a novel mouse model of lipodystrophy. Transgenic mice were generated that express the LMNA mutation that causes familial partial lipodystrophy of the Dunnigan type (FPLD2). The phenotype observed in FPLD-transgenic mice resembles many of the features of human FPLD2, including lack of fat accumulation, insulin resistance, and enlarged, fatty liver. Similar to the human disease, FPLD-transgenic mice appear to develop normally, but after several weeks they are unable to accumulate fat to the same extent as their wild-type littermates. One poorly understood aspect of lipodystrophies is the mechanism of fat loss. To this end, we have examined the effects of the FPLD2 mutation on fat cell function. Contrary to the current literature, which suggests FPLD2 results in a loss of fat, we found that the key mechanism contributing to the lack of fat accumulation involves not a loss, but an apparent inability of the adipose tissue to renew itself. Specifically, preadipocytes are unable to differentiate into mature and fully functional adipocytes. These findings provide insights not only for the treatment of lipodystrophies, but also for the study of adipogenesis, obesity, and insulin resistance.     

Compartmentalization of regulatory proteins in the cell nucleus

The cell nucleus is increasingly recognized as a spatially organized structure. In this review, the nature and controversies associated with nuclear compartmentalization are discussed. The relationship between nuclear structure and organization of proteins involved in the regulation of RNA polymerase II-transcribed genes is then discussed. Finally, very recent data on the mobility of these proteins within the cell nucleus is considered and their implications for regulation through compartmentalization of proteins and genomic DNA are discussed.     

Targeting the cell cycle in breast cancer: towards the next phase

Deregulation of the cell cycle is a hallmark of cancer that enables limitless cell division. To support this malignant phenotype, cells acquire molecular alterations that abrogate or bypass control mechanisms in signaling pathways and cellular checkpoints that normally function to prevent genomic instability and uncontrolled cell proliferation. Consequently, therapeutic targeting of the cell cycle has long been viewed as a promising anti-cancer strategy. Until recently, attempts to target the cell cycle for cancer therapy using selective inhibitors have proven unsuccessful due to intolerable toxicities and a lack of target specificity. However, improvements in our understanding of malignant cell-specific vulnerabilities has revealed a therapeutic window for preferential targeting of the cell cycle in cancer cells, and has led to the development of agents now in the clinic. In this review, we discuss the latest generation of cell cycle targeting anti-cancer agents for breast cancer, including approved CDK4/6 inhibitors, and investigational TTK and PLK4 inhibitors that are currently in clinical trials. In recognition of the emerging population of ER+ breast cancers with acquired resistance to CDK4/6 inhibitors we suggest new therapeutic avenues to treat these patients. We also offer our perspective on the direction of future research to address the problem of drug resistance, and discuss the mechanistic insights required for the successful implementation of these strategies.