Andrew H. Fischer

Nuclear structure in cancer cells

Nuclear architecture — the spatial arrangement of chromosomes and other nuclear components — provides a framework for organizing and regulating the diverse functional processes within the nucleus. There are characteristic differences in the nuclear architectures of cancer cells, compared with normal cells, and some anticancer treatments restore normal nuclear structure and function. Advances in understanding nuclear structure have revealed insights into the process of malignant transformation and provide a basis for the development of new diagnostic tools and therapeutics.

Molecular profile and clinical-pathologic features of the follicular variant of papillary thyroid carcinoma. An unusually high prevalence of ras mutations.

The follicular variant (FV) of papillary thyroid carcinoma is characterized by a follicular growth pattern and cytologic features of papillary carcinoma. ret/PTC rearrangements are common in classic papillary thyroid carcinoma (PTC) and PAX8-PPAR gamma and ras mutations in follicular thyroid carcinoma. Their prevalence in FV has not been established. We studied these genetic alterations and clinical-pathologic features in 30 FV cases and compared those with 46 non-FV papillary carcinomas. FV cases revealed 1 ret/PTC rearrangement (3%) and 13 ras mutations (43%). Non-FV cases harbored 13 ret/PTC (28%) (P = .006) and no ras mutations (P = .0002). No PAX8-PPAR gamma was found in either group. FV cases demonstrated a significantly higher prevalence of tumor encapsulation, angiovascular invasion, and poorly differentiated areas and a lower rate of lymph node metastases. These data indicate that the FV of papillary carcinoma has a distinct set of molecular alterations and is characterized by a high frequency of ras point mutations.

Catastrophic Nuclear Envelope Collapse in Cancer Cell Micronuclei

During mitotic exit, missegregated chromosomes can recruit their own nuclear envelope (NE) to form micronuclei (MN). MN have reduced functioning compared to primary nuclei in the same cell, although the two compartments appear to be structurally comparable. Here we show that over 60% of MN undergo an irreversible loss of compartmentalization during interphase due to NE collapse. This disruption of the MN, which is induced by defects in nuclear lamina assembly, drastically reduces nuclear functions and can trigger massive DNA damage. MN disruption is associated with chromatin compaction and invasion of endoplasmic reticulum (ER) tubules into the chromatin. We identified disrupted MN in both major subtypes of human non-small-cell lung cancer, suggesting that disrupted MN could be a useful objective biomarker for genomic instability in solid tumors. Our study shows that NE collapse is a key event underlying MN dysfunction and establishes a link between aberrant NE organization and aneuploidy.

Papillary Thyroid Carcinoma Oncogene (RET/PTC) Alters the Nuclear Envelope and Chromatin Structure

Current evidence suggests the papillary thyroid carcinoma oncogene (RET/PTC) generates papillary thyroid carcinomas in one genetic step. We tested a resulting prediction that RET/PTC expression in thyroid epithelium should be sufficient to cause the changes in nuclear morphology diagnostic of this tumor. Primary cultures of human thyroid epithelial cells were infected with a RET/PTC retroviral construct. Morphological scoring by two independent cytopathologists shows RET/PTC expression by immunohistochemistry to be highly associated (p << 0.0001) with an irregular nuclear contour and a euchromatic appearance compared with non-expressing cells in the same cultures. The altered nuclear morphology is not due to gene transfer or transformation per se as primary thyroid cell cultures infected with a retroviral H-RAS construct differ from RET/PTC-infected cells by showing round nuclear envelopes and coarser chromatin, as determined by the independent scoring of two cytopathologists (p << 0.0001). In addition, RET/ PTC-transfected cells appear to disperse, whereas RAS-transfected cells grow as discrete colonies. The results provide additional support for the hypothesis that RET/PTC is sufficient to cause papillary thyroid carcinomas. A signaling pathway downstream of RET/ PTC leads to restructuring of the nuclear envelope and chromatin, and the signal does not depend entirely, if at all, on a RAS pathway.

Using an AMACR (P504S)/34βE12/p63 Cocktail for the Detection of Small Focal Prostate Carcinoma in Needle Biopsy Specimens

We assessed the usefulness of immunohistochemical analysis with a 3-antibody cocktail (alpha-methylacyl coenzyme A racemase [AMACR, or P504S], 34betaE12, p63) and a double-chromogen reaction for detection of limited prostate cancer in 138 needle biopsy specimens, including 82 with small foci of prostatic adenocarcinoma and 56 benign prostates. When carcinoma was present, red cytoplasmic granular staining (AMACR) in the malignant glands and cells and dark brown nuclear (p63) and cytoplasmic (34betaE12) staining in basal cells of adjacent nonmalignant glands were found. Of 82 cases of small foci of prostatic adenocarcinoma, 78 (95%) expressed AMACR; all malignant glands were negative for basal cell staining. All benign glands adjacent to malignant glands were recognized easily by basal cell marker positivity and little or no AMACR expression. No benign glands were simultaneously positive for AMACR and negative for basal cell markers (specificity, 100%). There were no differences in intensity and numbers of positive glands with double-chromogen staining compared with using 1-color staining. Our results indicate that immunohistochemistry with a 3-antibody cocktail and double chromogen is a simple and easy assay that can be used as a routine test, which overcomes the problems of studying small lesions in prostate needle biopsies with multiple immunohistochemical stains.

Targeting BRAFV600E with PLX4720 Displays Potent Antimigratory and Anti-invasive Activity in Preclinical Models of Human Thyroid Cancer

The role of the B-RafV600E mutation in aggressive thyroid cancers is examined.

The Ultrastructure of MCF-10A Acini

MCF‐10A human mammary epithelial cells cultured inside reconstituted basement membrane form acini that resemble the acinar structures of mammary lobules. This three‐dimensional culture system has been used for identifying and characterizing the signal transduction pathways controlling cell proliferation and death, and for studying their disregulation in malignant progression. We have compared the ultrastructure of MCF‐10A acini, MCF‐10A cells grown in monolayer, and the acinar structures of human breast lobules. The tissue architecture of MCF‐10A acini was formed by hemidesmosomes connected to a basement membrane and by abundant desmosomes between acinar cells. Intermediate filaments that joined into large and abundant filament bundles connected hemidesmosomes and desmosomes to sites at the nuclear surface. Fewer and thinner bundles of filaments were observed in monolayer MCF‐10A cells and even fewer in breast tissue. Tight junctions were observed between cells in breast tissue but missing in MCF‐10A acini. The cytoplasm of MCF‐10A acinar cells had a polar organization similar to that observed in breast tissue, with centrosomes and the Golgi apparatus on the apical side of the nucleus. MCF‐10A acinar nuclei had an irregular, frequently invaginated surface and had a single nucleolus. The distribution of heterochromatin was similar to that in the epithelial cells of breast tissue. The nuclei of monolayer MCF‐10A cells had multiple nucleoli, a more regular profile, and less heterochromatin. Electron microscopy has the resolution required to survey features of MCF‐10A cell and acinus architecture that may change with manipulations designed to induce malignant phenotypes. J. Cell. Physiol.

Molecular aspects of diagnostic nucleolar and nuclear envelope changes in prostate cancer

Prostate cancer is still diagnosed by pathologists based on subjective assessment of altered cell and tissue structure. The cellular‐level structural changes diagnostic of some forms of cancer are known to be induced by cancer genes, but the relation between specific cellular‐level structural features and cancer genes has not been explored in the prostate. Two important cell structural changes in prostate cancer—nucleolar enlargement and nuclear envelope (NE) irregularity—are discussed from the perspective that they should also relate to the function of the genes active in prostate cancer. Enlargement of the nucleolus is the key diagnostic feature of high‐grade prostatic intraepithelial neoplasia (PIN), an early stage that appears to be the precursor to the majority of invasive prostate cancers. Nucleolar enlargement classically is associated with increased ribosome production, and production of new ribosomes appears essential for cell‐cycle progression. Several cancer genes implicated in PIN are known (in other cell types) to augment ribosome production, including c‐Myc, p27, retinoblastoma, p53, and growth factors that impact on ERK signaling. However, critical review of the available information suggests that increased ribosome production per se may be insufficient to explain nucleolar enlargement in PIN, and other newer functions of nucleoli may therefore need to be invoked. NE irregularity develops later in the clonal evolution of some prostate cancers, and it has adverse prognostic significance. Nuclear irregularity has recently been shown to develop dynamically during interphase following oncogene expression, without a requirement for post‐mitotic NE reassembly. NE irregularity characteristic of some aggressive prostate cancers could reflect cytoskeletal forces exerted on the NE during active cell locomotion. NE irregularity could also promote chromosomal instability because it leads to chromosomal asymmetry in metaphase. Finally, NE irregularity could impact replication competence, transcriptional programming and nuclear pore function.

Expression of a novel oncofetal mRNA-binding protein IMP3 in endometrial carcinomas: diagnostic significance and clinicopathologic correlations.

Insulin-like growth factor-II mRNA-binding protein 3 (IMP3) is a newly identified oncofetal mRNA-binding protein that is involved in embryogenesis and carcinogenesis of some malignant neoplasms. To investigate the diagnostic and clinicopathologic significance of this protein in endometrial carcinomas, we evaluated immunohistochemical expression of IMP3 in the two most common forms of endometrial malignancies, endometrioid adenocarcinoma and serous carcinoma. We selected 167 endometrial adenocarcinoma cases including 122 cases of endometrioid adenocarcinoma and 45 cases of serous carcinoma. Twenty samples of benign endometrium obtained from 20 patients with nonmalignant uterine lesions were used as controls. Positive immunohistochemical stain for IMP3 was identified in all serous carcinoma cases, among which, 39 (86%) and 3 (7%) cases showed IMP3 immunoreactivity in >50%, and 21–50, or 6–20% of tumor cells, respectively. Immunohistochemical reaction intensity for IMP3 was identified to be strong in 38 (84%) and intermediate in 7 (16%) cases of serous carcinoma. Fifty-four (44%) cases of endometrioid adenocarcinoma were negative for IMP3. Thirty (25%), 20 (16%), 10 (8%), and 8 (7%) cases of endometrioid adenocarcinoma demonstrated positive immunoreactivity for IMP3 in 1–5, 6–20, 21–50, and >50% of the tumor cells. Strong IMP3-staining intensity was noted in 34 (28%), intermediate in 26 (21%), and weak in 8 (7%) cases of endometrioid adenocarcinoma. All 20 control cases were negative for IMP3. To compare p53 with IMP3 expressions, we found that 35 (78%) of the serous carcinoma cases showed strong p53 immunohistochemical activity in >50% of the tumor cell nuclei. In contrast, 11 of 112 (10%) endometrioid adenocarcinoma cases demonstrated strong p53 positivity in >50% of the tumor cell nuclei. In conclusion, our findings demonstrate significant expression of IMP3 in serous carcinoma as compared to endometrioid adenocarcinoma (P<0.0001). Expression of IMP3 and p53 may be helpful biomarkers in the distinction of endometrial serous carcinoma from endometrioid adenocarcinoma. In addition, expression of IMP3 in endometrioid adenocarcinoma correlates with higher nuclear and architecture grades of the tumor (P=0.0000 and P=0.0002, respectively).

Nuclear envelope irregularity is induced by RET/PTC during interphase.

Nuclear envelope (NE) irregularity is an important diagnostic feature of cancer, and its molecular basis is not understood. One possible cause is abnormal postmitotic NE re-assembly, such that a rounded contour is never achieved before the next mitosis. Alternatively, dynamic forces could deform the NE during interphase following an otherwise normal postmitotic NE re-assembly. To distinguish these possibilities, normal human thyroid epithelial cells were microinjected with the papillary thyroid carcinoma oncogene (RET/PTC1 short isoform, known to induce NE irregularity), an attenuated version of RET/PTC1 lacking the leucine zipper dimerization domain (RET/PTC1 Deltazip), H (V-12) RAS, and labeled dextran. Cells were fixed at 6 or 18 to 24 hours, stained for lamins and the products of microinjected plasmids, and scored blindly using previously defined criteria for NE irregularity. 6.5% of non-injected thyrocytes showed NE irregularity. Neither dextran nor RAS microinjections increased NE irregularity. In contrast, RET/PTC1 microinjection induced NE irregularity in 27% of cells at 6 hours and 37% of cells at 18 to 24 hours. RET/PTC1 Deltazip induced significantly less irregularity. Since irregularity develops quickly, and since no mitoses and only rare possible postmitotic cells were scored, postmitotic NE re-assembly does not appear necessary for RET/PTC signaling to induce an irregular NE contour.