Nuclear Structure, Organization, and Oncogenesis
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- Rynearson, A.L. & Sussman, C.R. J Gastrointest Canc (2011) 42: 112. doi:10.1007/s12029-011-9253-5
The genetic code has received a great amount of attention from investigators, and the media since its discovery, and then again with the sequencing of the human genome in 2000. A decade later, investigators are beginning to look beyond the raw sequence to other mechanisms that affect gene expression. The main function of the nucleus is to maintain the genome and regulate gene expression. Changes in the expression of genes can drastically change the properties of the cell therefore giving the nucleus a role as the cell's “command post.” In the past few years, one of the most notable discoveries in the study of the nucleus is that this organelle is not homogeneous. It is also not randomly organized; everything within the nucleus has a specific location with a specific function. Chromosome location within the nucleus relative to its center is directly related to transcription level. Additionally, there are specific regions of the nucleus where content and function differ. The various structures of the nucleus such as the membranes and matrix that supply support to the well protected chromatin offer ever increasing layers of complexity to the nucleus. Here, we focus on the nuclear matrix and its possible effects on signaling and cellular transformation leading to cancer.
The primary function of the nucleus is to maintain transcription and to transport RNA into the cytoplasm so that it may be translated into proteins to meet the needs of the cell. These tasks must be completed in the ever-changing microenvironment within which the cells, and therefore, their nuclei reside. Here, we focus on regulation and cancerous changes within the nucleus and their dramatic effects on gene expression. In particular, we examine regulation of chromosomal location within the nucleus, and its powerful effects on oncogenesis. Changes in location and binding of the DNA to different areas within the nucleus change the internal microenvironment which can change gene–gene interactions and therefore have dynamic changes in the transcription and translation of RNA resulting in alternate regulation of proteins. When control over these processes is altered and mistakes are made, the negative changes within the nuclear microenvironment have a great impact on the cell and can lead to chronic disease and cancer. By studying the process and knowing how the cell and nucleus function to regulate the genes within the nucleus we can gain insight into possible mechanisms of control with the idea of harnessing the power of the nucleus and directing it to aid in the treatment and therapy of chronic diseases, including cancer.
The nuclear envelope also functions in chromatin organization, regulation of gene expression, as well as nuclear positioning and migration. The outer nuclear membrane is continuous with the endoplasmic reticulum where the inner nuclear envelope joins the outer envelope at the nuclear pore complexes . The outer and inner nuclear membranes have proteins that are specific to each membrane. The outer nuclear membrane shares associated proteins with the endoplasmic reticulum while the inner membrane contains a unique set of proteins that allow it to maintain contact with the nuclear matrix.
The nuclear matrix is composed of lamins and other proteins that provide a cytoskeleton like structure just inside the nuclear envelope (Fig. 1) . This matrix exists to provide structure and support to the nucleus itself as well as an anchoring place for DNA, nuclear pores, and other nuclear proteins. Embedded within the nuclear matrix is the intranuclear space containing the genome. The genome is organized into regions called chromosome territories. Within territories, there are regions of high and low transcriptional activity called euchromatin and heterochromatin, respectively, with euchromatin towards the middle and heterochromatin towards the periphery [3, 4].
Chromosome Territories and Structure
During mitosis, the genome is highly condensed and organized into chromosomes, the physical units that separate during mitotic division. Following mitosis, chromosomes uncoil and are organized into chromosome territories . Chromosome territories that are located within close proximity to each other are considered to be in the same chromosome neighborhood. Organization and interaction of chromosome territories within chromosome neighborhoods have many effects on DNA expression.
A chromosome within one territory is more likely to interact with those chromosomes in nearby territories. For example, a gene loop may extend out of its chromosome territory to interact with a gene of another chromosome territory within its chromosome neighborhood. Interaction between territories may cause coordinated gene transcription or translocation. In fact, the frequency of chromosomal rearrangements is related to the proximity of chromosomes within the interphase nucleus .
Another important determinant of genetic activity and regulation is the location of genes within the chromosome territory [6, 7]. Genes that are more transcriptionally active are located centrally within the chromosome territory, while those that are less active are located more peripherally [3, 4].
Positioning of chromatin within chromosome territories is directed partially by base-pair sequence [5, 7]. Regions of the chromosome containing GC-rich genes tend to be located centrally, while regions containing AT-rich genes tend to be located peripherally. This is not a strict rule however; as AT-rich regions can also be found centrally and GC-rich regions can also be found peripherally. The non-random location of chromosomes and regions within them are conserved in mice, chicken and plants, and apply across species. Even foreign chromosomes segregate to their appropriate nuclear location, as demonstrated for human chromosomes in mouse nuclei . It is difficult to believe that this conservation would occur if nuclear architecture were not functionally significant.
The Nuclear Matrix
A rich network of intranuclear proteins, called the nuclear matrix, is instrumental in allowing maintenance and regulation of chromosome structure [6, 8]. The nuclear matrix was first defined as the non-chromatin structures of the nucleus that remained after the removal of chromatin. The nuclear matrix is also referred to as the nucleoskeleton or scaffold, and contains the nucleoli, nuclear envelope, and nuclear membranes. The nuclear matrix composition in human cells has been proven to be cell type specific. Additionally, it varies between normal cells and tumors and among tumor types .
There are four major subtypes of lamins associated with the nuclear matrix, Lamin A, B1, B2, and C [11, 12]. Lamin A tends to be absent in undifferentiated cells, whereas all four subtypes, as well as some minor subtypes, are generally expressed in adult somatic cells. Three genes encode for the lamins that make up the nuclear matrix. LMNA, LMNB1, and LMNB2 located on chromosome 1, 5, and 19, respectively. Alternate splicing and posttranslational modifications are responsible for alternative subtypes that are cell specific.
The building block of the nuclear matrix is dimers of these subtypes of lamins . The lamins not only interact with themselves to form dimers, they interact with transcription factors, chromatin, DNA, and other proteins of the inner nuclear membrane including lamin B receptor, MAN1, emerin, lamina-associated polypeptide 1 (lap) LAP2B, small nesprin1 isoforms, and SUNs. SUNs interact with large nesprin 2 isoforms which are large proteins in the outer nuclear membrane and interact with actin, linking the nuclear lamina to the cytoskeleton.
During mitosis and nuclear disassembly, lamins become phosphorylated, causing depolymerization and dissociation of lamin fibers . This dissociation allows for the separation of the chromosomes and completion of mitosis. As soon as the chromosomes separate the lamins begin to reform around the chromosomes as they begin to de-condense, the matrix attachment regions reform and the nuclear scaffold is restored. This restoration allows for continued control of the nuclear contents.
Nuclear Changes Associated with Oncogenesis
Gross Anatomical Nuclear Changes in Cancer
Changes in cellular and nuclear appearance with disease were noted as early as 1887 by Boveri [13, 14]. Changes include obvious differences in the size and shape of cancer cells and their nuclei . Multiple nuclear changes can be characteristics of grade and stage. The mechanisms for change and their functional consequences are not well understood.
In small cell carcinoma the malleable nuclei appear to have frequent crush artifacts. Thyroid, and types of ovarian cancer show long clefts and grooves in the nucleus . Adenocarcinomas, and other cancers show poly-lobulations, indentations, and folds in the nucleus all suggesting an increase in the deregulation, and possible loss of control mechanisms. Nuclear size and number increase in cancer cells compared to normal cells.
Numerous changes in nucleoli, sites of ribosomal RNA synthesis and processing, have been noted in cancer . In many cancers, enlarged nucleoli with marked cell to cell variation in the numbers and sizes of the nucleoli are seen. In some precancerous lesions of the cervix and small cell anaplastic lung carcinoma, inconspicuous nucleoli are noted. In Hodgkins disease and large cell lung carcinoma, there is an increase in size and number of nucleoli. Interestingly, the increase in number of nucleoli is not associated with increased growth of the tumor cells and therefore must be associated with some other function of the tumor cells.
Another alteration in the cancer cell nucleoli is the formation of the perinuclear compartment . This compartment forms a tight meshwork with the nucleolar surface and is only present in cancer cells. In breast cancer, the perinuclear compartment appears to be associated with progression of disease. Although it is speculated that the perinuclear compartment may have a role in processing specific RNAs associated with RNA polymerase III, further studies need to be done to confirm its true role in cancer cells.
Yet another abnormality of the cancerous nuclei is the promyelocytic leukemia body (PML body) located in the nucleus . It is known to contain tumor suppressor genes in normal cells. Tumor suppressor p53 is thought to undergo posttranslational modifications within this doughnut shaped sub-region of the nucleus. In cancer cells the PML body is altered and/or mislocalized. Disruption of such a major compartment of tumor suppressors has potentially huge effects on cellular function. However, it is currently not known whether this is a cause or effect of tumorigenesis.
Nuclear Structural Changes in Cancer
The diseases resulting from its alteration evidence the importance of the nuclear lamina to maintaining cellular homeostasis. Mutations in lamin are linked with colorectal cancer as well as over 20 other diseases, including cardiomyopathy, Emery Dreifuss muscular dystrophy, Charcot–Marie–Tooth disorder type 2B1, and adult onset autosomal dominant leukodystrophy [12, 17].
Recent studies using digital image analysis and quantitative nuclear morphometry show that nuclear changes correlate with cancer prognosis. Debes et al. studied prostate cancer cells transfected with p300 and induced nuclear alterations such as diameter, perimeter, and absorbance which also correlate with aggressive features of prostate cancer . P300 has the function of inducing histone acetylation through its histone acetyltransferase activity which changes the chromatin structure. The authors suggest that the changes induced by the transcriptional co-activator p300 may be mediated by lamin A/C. These changes indicate clinical importance as one could imagine them as possible targets for therapy.
Proteins that attach the genome to the nuclear matrix have been shown to be altered in some types of cancer, p114 for example is found in breast cancer, but not in normal breast tissue . Other matrix attachment proteins such as SATB1 have been targets for possible treatment. SATB1 null mice have shown that localization of some genes that normally bind to SATB1 have been altered in location and could mimic the deregulation associated with disease.
Changes in nuclear shape described above in cancerous cells are often associated with heteromeric changes as well as altered gene expression. It is possible that the degradation of the normal nuclear matrix including the lamina allows the heterochromatic regions of the genome to move away from the periphery, which alters the organization and the transcriptional regulation of the nucleus causing cell transformation. Nuclear shape irregularities combined with loss of heterochromatin are two key features of papillary thyroid carcinoma . Cremer et al. tested the positioning of chromosome territories in normal and cancer cell lines and showed that in seven of eight cell lines a partial disturbance in the radial positioning of the chromosome territory was observed. It is unknown whether the positioning causes the changes in transcription and therefore the transformation, or if the transformation causes changes in the organization of the nucleus and therefore changes the location of the DNA.
As described above, evidence suggests the possibility of a chromosome location code within the nucleus. Recently, a comparison was done of chromosome territory organization in cancerous and non-cancerous lung fibroblast cells . The chromosome territory organization of six chromosome pairs was analyzed by a novel computational geometric approach. The chromosomes examined have a preferred three-dimensional arrangement distinct for each cell type that is conserved from one cell to the next. By contrast, when compared to cancer cells of the same cell type, the profile was strikingly different. Thus, cells with different functions or cancer cells have different preferential location of their chromosomes and therefore altered chromosome neighborhoods resulting in altered gene regulation, silencing, and transcription. This idea fits with the dogma of altered gene expression in cancer cells, and may explain a mechanism for increased number of translocations as the chromosome neighborhoods are physically moved from their preferred neighborhoods to their malignant neighborhoods. The result of this transformation would be possible altered gene expression, and loss of cell cycle control, which is also consistent with the malignant phenotype.
The Future of the Nucleus as a Therapeutic Target in Pancreatic Cancer
Nuclear changes are integral to the development and progression of pancreatic cancer. The Pancreas Intraepithelial Neoplasm (PanIN) grading guidelines of 1999, used by pathologists to access the grade of duct lesions in the pancreas, state that the nucleus undergoes visual changes as the grade increases . In fact, the transition of PanIN-1B to PanIN-2 requires changes in nuclear shape. Furthermore, a PanIN-2 lesion may include some loss of polarity, nuclear crowding, enlarged nuclei, pseudo-stratification, and hyperchromatism where a PanIN-3 lesion is characterized by a loss of nuclear polarity, nuclear irregularities, and prominent (macro) nucleoli .
Further evidence implicating nuclear changes in pancreatic cancer is that several proteins that regulate nuclear shape and are involved in cancer of other tissue types (as described above) are also expressed in the pancreas . It is not unreasonable to speculate that P53, Lamins, p300, p114, and SATB1 expression in the pancreas could be altered similarly to contribute to the transformation of normal pancreatic cells. In fact, roles for P53 and Myc specifically have been demonstrated in pancreatic cancer [23, 24], and may also be differentially regulated by alterations in SATB1 and PML bodies.
In summary, it is difficult to imagine that nuclear changes do not contribute to the transformation from one cell type into another in pancreatic cancer, and many other cancers. We suggest that the internal nuclear architecture must transform to accommodate the drastic differences in gene transcription and gene/gene interactions that occur in pancreatic cancer. Therapeutic targeting of these nuclear changes represents a novel and promising approach to treating this devastating disease which has not seen improvement in therapies for the past 35 years.
We thank Dr. Martin E. Fernandez-Zapico for helpful comments. CRS received support from AHA (SDG 0630137 N), ACS Young Investigator Award, and Mayo Clinic Cancer Center.