Expression Of Nuclear Matrix Proteins Research Articles

Abstract A033: Identification of cancer-specific alterations in expression of nuclear matrix proteins through proteomic and RNAsequencing analyses of ductal carcinoma in situ

Abstract Background and Objectives: In 2021 breast cancer (BC) was the most common cancer affecting women and the second deadliest cancer in the U.S. Ductal Carcinoma In Situ (DCIS) is the earliest identifiable pre-invasive breast cancer lesion. Approximately 20-25% of diagnosed breast cancer cases are ductal carcinoma in situ (DCIS). Estimates show that 14 to 50% of DCIS cases progress to invasive breast cancer. We used a novel tissue-engineering system established in the Latimer laboratory to generate model systems representative of different molecular and histological subtypes of all stages of BC, including DCIS. Using a mass spectrometry (MS)-based relative quantitative methodology, we analyzed one novel DCIS cell line, the matching isogenic contralateral cell line and the non-tumor adjacent cell line, as well as two non-diseased breast reduction mammoplasty cell lines and the established stage IV BC cell lines MCF7 and MDA MB231. RNA sequencing analysis included additional cell lines from stages I, II and III. In order to limit the number of proteins for proteomic assessment, nuclear matrix proteins (NMPs) were targeted for analysis. NMPs have previously been linked with cancers of the prostate, colon, and breast. Maldi-TOF/TOF-MS analysis identified 270 NMPs with high confidence. Our objective was to identify NMPs with specifically altered expression in DCIS and later stages of BC compared to non-diseased breast and contralateral breast. Such markers may help to accurately identify invasive DCIS and improve early detection of BC. Results: We identified two nuclear matrix proteins that are significantly downregulated in DCIS and all other stages of BC in our collection. This downregulation was also apparent through mRNA expression analysis. These proteins are upregulated in non-diseased breast reduction tissue and contra-lateral DCIS derived from the same patient. We also identified four NMPs that were significantly upregulated in DCIS and all other BC stages compared to non-diseased breast reduction tissue and contralateral DCIS. Gene expression analyses showed that these four genes were upregulated with BC progression. Finally, we identified nine NMPS with significant differences in expression between DCIS and all stages of BC versus non-diseased breast reduction tissue and contralateral DCIS where gene expression did not follow the same trend as proteomic analysis. Conclusion: Our findings suggest that the changes that occur in the nuclear matrix of DCIS vs. normal breast epithelium could provide a basis for the development of prognostic biomarkers for DCIS and allow for targeted prevention of progression to invasive disease. This work was supported by grant W81XWH-0701-0660 from the DOD CDMRP BC program. Citation Format: Ali Almutairy, Abdullah Alhamed, Stephen G. Grant, Miranda Sarachine Falso, Billy W. Day, Jean J. Latimer. Identification of cancer-specific alterations in expression of nuclear matrix proteins through proteomic and RNAsequencing analyses of ductal carcinoma in situ [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A033.

Temporal Changes in Nucleus Morphology, Lamin A/C and Histone Methylation During Nanotopography-Induced Neuronal Differentiation of Stem Cells.

Stem cell differentiation can be regulated by biophysical cues such as nanotopography. It involves sensing and integration of these biophysical cues into their transcriptome with a mechanism that is yet to be discovered. In addition to the cytoskeletal and focal adhesion remodeling, nanotopography has also been shown to modulate nucleus morphology. Here, we studied the effect of nanotopography on the temporal changes in nuclei of human embryonic stem cells (hESCs) and human mesenchymal stem cells (hMSCs). Using a high throughput Multi-architecture (MARC) chip analysis, the circularity of the stem cell nuclei changed significantly on different patterns. Human ESCs and MSCs showed different temporal changes in nucleus morphology, lamin A/C expression and histone methylation during topography-induced neuronal differentiation. In hESCs, the expression of nuclear matrix protein, lamin A/C during neuronal differentiation of hESCs on PDMS samples were weakly detected in the first 7 days of differentiation. The histone 3 trimethylation on Lysine 9 (H3K9me3) decreased after differentiation initiated and showed temporal changes in their expression and organization during neuronal differentiation. In hMSCs, the expression of lamin A/C was significantly increased after the first 24 h of cell culture. The quantitative analysis of histone methylation also showed a significant increase in hMSCs histone methylation on 250 nm anisotropic nanogratings within the first 24 h of seeding. This reiterates the importance of cell-substrate sensing within the first 24 h for adult stem cells. The lamin A/C expression and histone methylation shows a correlation of epigenetic changes in early events of differentiation, giving an insight on how extracellular nanotopographical cues are transduced into nuclear biochemical signals. Collectively, these results provide more understanding into the nuclear regulation of the mechanotransduction of nanotopographical cues in stem cell differentiation.

Expression of nuclear matrix proteins binding matrix attachment regions in prostate cancer. PARP-1: New player in tumor progression.

Prostate cancer (PCa) displays infrequent point mutations, whereas genomic rearrangements are highly prevalent. In eukaryotes, the genome is compartmentalized into chromatin loop domains by the attachment to the nuclear matrix (NM), and it has been demonstrated that several recombination hot spots are situated at the base of loops. Here, we have characterized the binding between NM proteins and matrix attachment regions (MARs) in PCa. Nontumor and 44 PCa tissues were analyzed. More aggressive tumors were characterized by an increase in the complexity of the NM protein patterns that was synchronous with a decrease in the number of proteins binding the MAR sequences. PARP-1 was the protein that showed the most evident changes. The expression of the PARP-1 associated with NM increased and it was dependent on tumor aggressiveness. Immunohistochemical analysis showed that the protein was significantly overexpressed in tumor cells. To explore the role of PARP-1 in PCa progression, PCa cells were treated with the PARP inhibitor, ABT-888. In androgen-independent PC3 cells, PARP inhibition significantly decreased cell viability, migration, invasion, chromatin loop dimensions and histone acetylation. Collectively, our study provides evidence that MAR-binding proteins are involved in the development and progression of PCa. PARP could play a key role in the compartmentalization of chromatin and in the development of the more aggressive phenotype. Thus, PARP can no longer be viewed only as an enzyme involved in DNA repair, but that its role in chromatin modulation could provide the basis for a new therapeutic approach to the treatment of PCa.

Aberrant expression of nuclear matrix proteins during HMBA-induced differentiation of gastric cancer cells

To investigate the aberrant expression of nuclear matrix proteins in human gastric cancer cells before and after hexamethylene bisacetamide (HMBA) treatment. Proteomics analysis of differential nuclear matrix proteins was performed by two dimensional electrophoresis polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The expression levels of three nuclear matrix proteins were further confirmed by Western blotting and their locations in nuclear matrix filament were observed by quantum dots-based immunofluorescence. Proteomics analysis showed that 43 protein spots were significantly changed due to HMBA treatment. Fifteen proteins were identified in the HMBA-induced differentiation of gastric tumor cells. Eight proteins spots were down-regulated while seven were up-regulated. Among these proteins, prohibitin, nucleophosmin and hnRNP A2/B1 were significantly decreased in HMBA-treated human gastric cancer cells, and their locations in nuclear matrix were altered by HMBA. Our results proved the alteration of specific nuclear matrix proteins during the differentiation of human gastric cancer cells. And the aberrant expressions of nuclear matrix proteins were of significance in revealing the regulatory mechanism of tumor cell proliferation and differentiation. The aberrant expressions and intracellular redistributions of nuclear matrix proteins before and after HMBA treatment indicated that nuclear matrix proteins play a pivotal role in the differentiation of gastric cancer cells.

Differential expression of nuclear matrix proteins during the differentiation of human neuroblastoma SK‐N‐SH cells induced by retinoic acid

To investigate the alteration of nuclear matrix proteins (NMPs) during the differentiation of neuroblastoma SK-N-SH cells induced by retinoic acid (RA), differentiation markers were detected by immunocytochemistry and NMPs were selectively extracted and subjected to two-dimensional gel electrophoresis analysis. Immunocytochemical observation demonstrated that the expression of neuronal markers was up-regulated in SK-N-SH cells following RA treatment. Meanwhile, 52 NMPs (41 of which were identified) changed significantly during SK-N-SH differentiation; four of these NMPs were further confirmed by immunoblotting. This study suggests that the differentiation of neuroblastoma cells was accompanied by the altered expression of neuronal markers and NMPs. The presence of some differentially expressed NMPs was related to the proliferation and differentiation of neuroblastomas. Our results may help to reveal the relationship between NMPs and neuroblastoma carcinogenesis and reversion, as well as elucidate the regulatory principals driving neural cell proliferation and differentiation.

Alteration of nuclear matrix-intermediate filament system and differential expression of nuclear matrix proteins during human hepatocarcinoma cell differentiation

To investigate the association between the configurational and compositional changes of nuclear matrix and the differentiation of carcinoma cells. Cells cultured with or without 5 x 10(-3) mmol/L of hexamethylene bisacetamide (HMBA) on Nickel grids were treated by selective extraction and prepared for whole mount observation under electron microscopy. The samples were examined under transmission electron microscope. Nuclear matrix proteins were selectively extracted and subjected to subcellular proteomics study. The protein expression patterns were analyzed by PDQuest software. Spots of differentially expressed nuclear matrix proteins were excised and subjected to in situ digestion with trypsin. The peptides were analyzed by matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Data were submitted for database searching using Mascot tool (www.matrixscience.com). The nuclear matrix (NM) and intermediate filament (IF) in SMMC-7721 hepatocarcinoma cells were found relatively sparse and arranged irregularly. The nuclear lamina was non-uniform, and two kinds of filaments were not tightly connected. After induction for differentiation by HMBA, the NM-IF filaments were concentrated and distributed uniformly. The heterogeneous population of filaments, including highly branched ultrathin filaments could also be seen in the regular meshwork. The connection between the two kinds of filaments and the relatively thin, condensed and sharply demarcated lamina composed of intermediate-sized filaments was relatively fastened. Meanwhile, 21 NM proteins changed remarkably during SMMC-7721 cell differentiation. Four proteins, i.e. mutant Pyst1, hypothetical protein, nucleophosmin 1, and LBP were downregulated, whereas four other proteins, eIF6, p44 subunit, beta-tubulin, and SIN3B were upregulated with the last one, SR2/ASF found only in the differentiated SMMC-7721 cells. The induced differentiation of SMMC-7721 cells by HMBA is accompanied by the configurational changes of nuclear matrix-intermediate filament (NM-IF) system and the compositional changes of nuclear matrix protein expression. These changes may be important morphological or functional indications of the cancer cell reversion.

Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells.

The events of cell death and the expression of nuclear matrix protein (NMP) have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide. By means of TUNEL assay, the nuclei displayed a characteristic morphology change, and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h. Nucleosomal DNA fragmentation was observed after treatment for 4 h. The morphological change of HL-60 cells, thus, occurred earlier than the appearance of DNA ladder. Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis. Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells. Western blotting was then performed on three nuclear matrix acssociated proteins, PML, HSC70 and NuMA. The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment, while NuMA, a nuclear mitotic apparatus protein, was down regulated. These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

The expression of the nuclear matrix proteins NuMA, topoisomerase II-α, and -β in bone and osseous cell culture: regulation by parathyroid hormone

Bone cells undergo changes in cell structure during phenotypic development. Parathyroid hormone (PTH) induces a change in osteoblast shape, a determinant of collagen expression. We hypothesize that alterations in bone cell shape reflect and direct gene expression as governed, in part, by nuclear organization. In this study, we determined whether the expression of nuclear matrix proteins that mediate nuclear architecture, NuMA, topoisomerase II (topo II)-α, and -β, were altered during osteoblast development and response to PTH in vivo. NuMA forms an interphase nuclear scaffold in some cells, the absence of which may accommodate alterations in nuclear organization necessary for specific functions. Topo II enzymes are expressed in bone cells; the α-isoform is specific to proliferating cells. We used immunohistochemistry and flow cytometry to determine whether NuMA is expressed in the primary spongiosa of the rat metaphyseal femur and whether expression of NuMA, topo II-α, and II-β changes during osteoblast development or with PTH treatment. NuMA and topo II-β were expressed in marrow cells, osteoblasts, osteocytes, and chondrocytes. These proteins were not detected in osteoclasts in vivo, but were observed in cultured cells. Bone marrow cells expressed topo II-α. All three proteins were expressed in cultures of rat osteoblast-like UMR-106 cells. PTH treatment downregulated the number of topo II-α-immunopositive cells, correlated with a decrease in S-phase cells, in both bone tissue and cell culture. We conclude that, in vivo, nuclear matrix composition is altered during bone cell development and that anabolic doses of PTH attenuate the proliferative capacity of osteogenic cells, in part, by targeting topo II-α expression.

Expression of nuclear matrix proteins in rat liver tissue

We have studied the synthesis of nuclear matrix proteins as it occurs in the rat liver. To investigate their kinetics in tissue, nuclear matrix proteins were prepared from liver of rats injected with radioactive methionine. Synthesis of lamins was not observed in quiescent hepatocytes although they were the principal proteins of this subcellular fraction, suggesting that lamins are very stable in the liver. When hepatocytes were stimulated to divide by partial hepatectomy, only synthesis of lamin B was initiated. Many proteins not visible on Coomassie blue-stained gels were detectable by autoradiography. In the nuclear matrix extracts of quiescent hepatocytes, one of the most prominently labeled ones was a protein of 70 kDa. After hepatectomy, an additional protein of 62 kDa was detectable. These proteins were visible 1 h after the injection of radioactivity, but were no longer observed in nuclear matrices prepared 24 h after injection. These experiments indicate that in addition to lamins, two nuclear matrix proteins are present in the rat liver that were not detected previously, perhaps because of their rapid turnover.

Cell cycle-dependent expression of nuclear matrix proteins of ehrlich ascites cells studied by in vitro translation

A combination of methods was used to study the cell cycle-dependent expression of nuclear matrix proteins of Ehrlich ascites cells: 1. 1. Separation of asynchronous cells growing in vivo into fractions of G1-, S- and G2-phase cells by centrifugal elutriation with less than 10% cross-contamination. 2. 2. Isolation of poly(A +) RNA populations from total cytoplasmic RNA by affinity chromatography on messenger affinity paper (mAP). 3. 3. In vitro translation of poly(A +) RNA from asynchronous and phase synchronous cells. 4. 4. Immunoprecipitation of in vitro synthesized nuclear matrix proteins by a monoclonal antibody with anti-lamin specificity (PKB8) and by a polyspecific anti-nuclear matrix serum (AMS5) followed by analysis of immunoprecipitated materials on SDS-polyacrylamide gels. The results indicate that mRNAs for nuclear matrix-associated proteins including the lamins B and C are either exclusively or at least predominantly present in the cytoplasm of cells in S phase suggesting a high rate of in vivo synthesis of these proteins during S phase. This is consistent with an anticipated biological function of the nuclear matrix which is considered to organize parental and newly synthesized DNA in higher order structures.