Jacqueline A. Lees

Immunohistochemical Evidence of Loss of PTEN Expression in Primary Ductal Adenocarcinomas of the Breast

Germline mutations in PTEN, encoding a dual-specificity phosphatase on 10q23.3, cause Cowden syndrome (CS), which is characterized by a high risk of breast and thyroid cancers. Loss of heterozygosity of 10q22-24 markers and somatic PTEN mutations have been found to a greater or lesser extent in a variety of sporadic component and noncomponent cancers of CS. Among several series of sporadic breast carcinomas, the frequency of loss of flanking markers around PTEN is approximately 30 to 40%, and the somatic intragenic PTEN mutation frequency is <5%. In this study, we analyzed PTEN expression in 33 sporadic primary breast carcinoma samples using immunohistochemistry and correlated this to structural studies at the molecular level. Normal mammary tissue had a distinctive pattern of expression: myoepithelial cells uniformly showed strong PTEN expression. The PTEN protein level in mammary epithelial cells was variable. Ductal hyperplasia with and without atypia exhibited higher PTEN protein levels than normal mammary epithelial cells. Among the 33 carcinoma samples, 5 (15%) were immunohistochemically PTEN-negative; 6 (18%) had reduced staining, and the rest were PTEN-positive. In the PTEN-positive tumors as well as in normal epithelium, the protein was localized in the cytoplasm and in the nucleus (or nuclear membrane). Among the immunostain negative group, all had hemizygous PTEN deletion but no structural alteration of the remaining allele. Thus, in these cases, an epigenetic phenomenon such as hypermethylation, -ecreased protein synthesis or increased protein degradation may be involved. In the cases with reduced staining, 5 of 6 had hemizygous PTEN deletion and 1 did not have any structural abnormality. Finally, clinicopathological features were analyzed against PTEN protein expression. Three of the 5 PTEN immunostain-negative carcinomas were also both estrogen and progesterone receptor-negative, whereas only 5 of 22 of the PTEN-positive group were double receptor-negative. The significance of this last observation requires further study.

E2F-4 Switches from p130 to p107 and pRB in Response to Cell Cycle Reentry

The E2F transcription factor couples the coordinate expression of cell cycle proteins to their appropriate transition points. Its activity is controlled by the cell cycle regulators pRB, p107, and p130. These bind to E2F at defined but distinct stages of the cell cycle. Using specific antisera, we have identified the DP and E2F components of each of these species. Although present at very different levels, DP-1 and DP-2 are evenly distributed among each of these complexes. In contrast, the individual E2Fs have distinctly different binding profiles. Consistent with previous studies, E2F-1, E2F-2, and E2F-3 bind specifically to the retinoblastoma protein. In each case, their expression and DNA binding activity are restricted to post-G1/S fractions. Surprisingly, E2F-1 and E2F-3 make unequal contributions to the pRB-associated and free E2F activity, suggesting that these proteins perform different cell cycle functions. Most significantly, this study showed E2F-4 accounts for the vast majority of the endogenous E2F activity. In arrested cells, E2F-4 is sequestered by the p130 protein. However, as the cells pass the G1-to-S transition, the levels of pRB and p107 increase and E2F-4 now associates with both of these regulators. Despite this, a considerable amount of E2F-4 exists as free E2F. In G1 cells, this accounts for almost all of the free activity. Once the cells enter S phase, free E2F is composed of an equal mixture of E2F-4 and E2F-1.

Differential Nuclear and Cytoplasmic Expression of PTEN in Normal Thyroid Tissue, and Benign and Malignant Epithelial Thyroid Tumors

Germline mutations in PTEN (MMAC1/TEP1) are found in patients with Cowden syndrome, a familial cancer syndrome which is characterized by a high risk of breast and thyroid neoplasia. Although somatic intragenic PTEN mutations have rarely been found in benign and malignant sporadic thyroid tumors, loss of heterozygosity (LOH) has been reported in up to one fourth of follicular thyroid adenomas (FAs) and carcinomas. In this study, we examined PTEN expression in 139 sporadic nonmedullary thyroid tumors (55 FA, 27 follicular thyroid carcinomas, 35 papillary thyroid carcinomas, and 22 undifferentiated thyroid carcinomas) using immunohistochemistry and correlated this to the results of LOH studies. Normal follicular thyroid cells showed a strong to moderate nuclear or nuclear membrane signal although the cytoplasmic staining was less strong. In FAs the neoplastic nuclei had less intense PTEN staining, although the cytoplasmic PTEN-staining intensity did not differ significantly from that observed in normal follicular cells. In thyroid carcinomas as a group, nuclear PTEN immunostaining was mostly weak in comparison with normal thyroid follicular cells and FAs. The cytoplasmic staining was more intense than the nuclear staining in 35 to 49% of carcinomas, depending on the histological type. Among 81 informative tumors assessed for LOH, there seemed to be an associative trend between decreased nuclear and cytoplasmic staining and 10q23 LOH (P = 0.003, P = 0.008, respectively). These data support a role for PTEN in the pathogenesis of follicular thyroid tumors.

E2Fs Regulate Adipocyte Differentiation

When preadipocytes reenter the cell cycle, PPAR gamma expression is induced, coincident with an increase in DNA synthesis, suggesting the involvement of the E2F family of cell cycle regulators. We show here that E2F1 induces PPAR gamma transcription during clonal expansion, whereas E2F4 represses PPARg amma expression during terminal adipocyte differentiation. Using a combination of in vivo experiments with knockout and chimeric animals and in vitro experiments, we demonstrate that the absence of E2F1 impairs, whereas depletion of E2F4 stimulates, adipogenesis. E2Fs hence represent the link between proliferative signaling pathways, triggering clonal expansion, and terminal adipocyte differentiation through regulation of PPAR gamma expression. This underscores the complex role of the E2F protein family in the control of both cell proliferation and differentiation.

Rb regulates fate choice and lineage commitment in vivo

Mutation of the retinoblastoma gene (RB1) tumour suppressor occurs in one-third of all human tumours and is particularly associated with retinoblastoma and osteosarcoma. Numerous functions have been ascribed to the product of the human RB1 gene, the retinoblastoma protein (pRb). The best known is pRb’s ability to promote cell-cycle exit through inhibition of the E2F transcription factors and the transcriptional repression of genes encoding cell-cycle regulators. In addition, pRb has been shown in vitro to regulate several transcription factors that are master differentiation inducers. Depending on the differentiation factor and cellular context, pRb can either suppress or promote their transcriptional activity. For example, pRb binds to Runx2 and potentiates its ability to promote osteogenic differentiation in vitro. In contrast, pRb acts with E2F to suppress peroxisome proliferator-activated receptor γ subunit (PPAR-γ), the master activator of adipogenesis. Because osteoblasts and adipocytes can both arise from mesenchymal stem cells, these observations suggest that pRb might play a role in the choice between these two fates. However, so far, there is no evidence for this in vivo. Here we use mouse models to address this hypothesis in mesenchymal tissue development and tumorigenesis. Our data show that Rb status plays a key role in establishing fate choice between bone and brown adipose tissue in vivo.

Mutation and Expression Analyses Reveal Differential Subcellular Compartmentalization of PTEN in Endocrine Pancreatic Tumors Compared to Normal Islet Cells

The pathogenesis of sporadic endocrine pancreatic tumors (EPTs) is still primarily unknown. Comparative genomic hybridization studies revealed loss of 10q in a significant number (nine of 31) of EPTs. The tumor suppressor gene PTEN lies on 10q23, and so, is a candidate to play some role in EPT pathogenesis. Germline PTEN mutations are found in Cowden and Bannayan-Riley-Ruvalcaba syndromes, whereas somatic mutations and deletions are found in a variety of sporadic cancers. The mutation and expression status of PTEN in EPTs has not yet been examined. Mutation analysis of the entire coding region of PTEN including splice sites was performed in 33 tumors, revealing one tumor with somatic L182F (exon 6). Loss of heterozygosity of the 10q23 region was detected in eight of 15 informative malignant (53%) and in none of seven benign EPTs. PTEN expression was assessed in 24 available EPTs by immunohistochemistry using a monoclonal anti-PTEN antibody. Of these 24, 23 tumors showed strong immunoreactivity for PTEN. Only the EPTs with PTEN mutation lacked PTEN protein expression. Although normal islet cells always exhibited predominantly nuclear PTEN immunostaining, 19 of 23 EPTs had a predominantly cytoplasmic PTEN expression pattern. Exocrine pancreatic tissue was PTEN-negative throughout. PTEN mutation is a rare event in malignant EPTs and PTEN protein is expressed in most (23 of 24) EPTs. Thus, intragenic mutation or another means of physical loss of PTEN is rarely involved in the pathogenesis of EPTs. Instead, either an impaired transport system of PTEN to the nucleus or some other means of differential compartmentalization could account for impaired PTEN function. Loss of heterozygosity of the 10q23 region is a frequent event in malignant EPTs and might suggest several hypotheses: a different tumor suppressor gene in the vicinity of PTEN might be principally involved in EPT formation; alternatively, 10q loss, including PTEN, seems to be associated with malignant transformation, but the first step toward neoplasia might involve altered subcellular localization of PTEN.

E2F activity is regulated by cell cycle-dependent changes in subcellular localization.

E2F directs the cell cycle-dependent expression of genes that induce or regulate the cell division process. In mammalian cells, this transcriptional activity arises from the combined properties of multiple E2F-DP heterodimers. In this study, we show that the transcriptional potential of individual E2F species is dependent upon their nuclear localization. This is a constitutive property of E2F-1, -2, and -3, whereas the nuclear localization of E2F-4 is dependent upon its association with other nuclear factors. We previously showed that E2F-4 accounts for the majority of endogenous E2F species. We now show that the subcellular localization of E2F-4 is regulated in a cell cycle-dependent manner that results in the differential compartmentalization of the various E2F complexes. Consequently, in cycling cells, the majority of the p107-E2F, p130-E2F, and free E2F complexes remain in the cytoplasm. In contrast, almost all of the nuclear E2F activity is generated by pRB-E2F. This complex is present at high levels during G1 but disappears once the cells have passed the restriction point. Surprisingly, dissociation of this complex causes little increase in the levels of nuclear free E2F activity. This observation suggests that the repressive properties of the pRB-E2F complex play a critical role in establishing the temporal regulation of E2F-responsive genes. How the differential subcellular localization of pRB, p107, and p130 contributes to their different biological properties is also discussed.

E2F4 Is Essential for Normal Erythrocyte Maturation and Neonatal Viability

The retinoblastoma protein (pRB) plays a key role in the control of normal development and proliferation through the regulation of the E2F transcription factors. We generated a mutant mouse model to assess the in vivo role of the predominant E2F family member, E2F4. Remarkably, loss of E2F4 had no detectable effect on either cell cycle arrest or proliferation. However, E2F4 was essential for normal development. E2f4-/- mice died of an increased susceptibility to opportunistic infections that appeared to result from craniofacial defects. They also displayed a variety of erythroid abnormalities that arose from a cell autonomous defect in late stage maturation. This suggests that E2F4 makes a major contribution to the control of erythrocyte development by the pRB tumor suppressor.

Metastatic osteosarcoma induced by inactivation of Rb and p53 in the osteoblast lineage.

Mutation of the RB-1 and p53 tumor suppressors is associated with the development of human osteosarcoma. With the goal of generating a mouse model of this disease, we used conditional and transgenic mouse strains to inactivate Rb and/or p53 specifically in osteoblast precursors. The resulting Rb;p53 double mutant (DKO) animals are viable but develop early onset osteosarcomas with complete penetrance. These tumors display many of the characteristics of human osteosarcomas, including being highly metastatic. We established cell lines from the DKO osteosarcomas to further investigate their properties. These immortalized cell lines are highly proliferative and they retain their tumorigenic potential, as judged by their ability to form metastatic tumors in immunocompromised mice. Moreover, they can be induced to differentiate and, depending on the inductive signal, will adopt either the osteogenic or adipogenic fate. Consistent with this multipotency, a significant portion of these tumor cells express Sca-1, a marker that is typically associated with stem cells/uncommitted progenitors. By assaying sorted cells in transplant assays, we demonstrate that the tumorigenicity of the osteosarcoma cell lines correlates with the presence of the Sca-1 marker. Finally, we show that loss of Rb and p53 in Sca-1-positive mesenchymal stem/progenitor cells is sufficient to yield transformed cells that can initiate osteosarcoma formation in vivo.

Bmi1 is critical for lung tumorigenesis and bronchioalveolar stem cell expansion

Understanding the pathways that control epithelial carcinogenesis is vital to the development of effective treatments. The Polycomb group family member Bmi1 is overexpressed in numerous epithelial tumors, but its role in their development has not been established. We now show a key role for Bmi1 in lung adenocarcinoma. Whereas lung development occurs normally in Bmi1-deficient mice, loss of Bmi1 decreases the number and progression of lung tumors at a very early point in an oncogenic K-ras-initiated mouse model of lung cancer. This correlates with a defect in the ability of Bmi1-deficient putative bronchiolalveolar stem cells (BASCs) to proliferate in response to the oncogenic stimulus. Notably, in the absence of oncogenic K-ras, Bmi1-deficient BASCs show impaired proliferation and self-renewal capacity in culture and after lung injury in vivo. Abrogated lung cancer development and BASC self-renewal occur partially in a p19ARF-dependent manner. Our data suggest that Bmi1 deficiency suppresses tumor development by limiting the expansion potential of BASCs, the apparent lung cancer cells of origin. Because Bmi1 is elevated in additional tumor types, this suggests that Bmi1 plays a key role in regulating proliferation of both stem cells and tumor cells in diverse adult epithelial tissues.