Oncogenic regulators and substrates of the anaphase promoting complex/cyclosome are frequently overexpressed in malignant tumors - PubMed (original) (raw)

Oncogenic regulators and substrates of the anaphase promoting complex/cyclosome are frequently overexpressed in malignant tumors

Norman L Lehman et al. Am J Pathol. 2007 May.

Abstract

The fidelity of cell division is dependent on the accumulation and ordered destruction of critical protein regulators. By triggering the appropriately timed, ubiquitin-dependent proteolysis of the mitotic regulatory proteins securin, cyclin B, aurora A kinase, and polo-like kinase 1, the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase plays an essential role in maintaining genomic stability. Misexpression of these APC/C substrates, individually, has been implicated in genomic instability and cancer. However, no comprehensive survey of the extent of their misregulation in tumors has been performed. Here, we analyzed more than 1600 benign and malignant tumors by immunohistochemical staining of tissue microarrays and found frequent overexpression of securin, polo-like kinase 1, aurora A, and Skp2 in malignant tumors. Positive and negative APC/C regulators, Cdh1 and Emi1, respectively, were also more strongly expressed in malignant versus benign tumors. Clustering and statistical analysis supports the finding that malignant tumors generally show broad misregulation of mitotic APC/C substrates not seen in benign tumors, suggesting that a "mitotic profile" in tumors may result from misregulation of the APC/C destruction pathway. This profile of misregulated mitotic APC/C substrates and regulators in malignant tumors suggests that analysis of this pathway may be diagnostically useful and represent a potentially important therapeutic target.

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Figures

Figure 1

Figure 1

Model for pRb- and APC/C-dependent control of S phase and early mitosis. G1 proliferation control genes upstream of Emi1 (shown in blue) regulate the E2F-dependent expression of Emi1 and certain APC/C substrates (cyclin A, Plk1, and securin). Accumulation of Emi1 stabilizes APC/C substrates vital for progression through S phase (blue) and mitosis (pink). When overexpressed Emi1, or the mitotic control APC/C substrates Skp2, Plk1, securin, or aurora A can induce mitotic catastrophe. In the absence of p53, these genomically unstable cells survive and lead to tumor formation.

Figure 2

Figure 2

Validation of anti-Emi1 immunohistochemical staining. A: An Emi1-immunopositive ovarian clear cell carcinoma was stained with anti-Emi1 antibody showing characteristic cytoplasmic Emi1 immunoreactivity (top) or antibody preincubated with recombinant antigen eliminating Emi1-specific immunostaining (bottom). B: HCT116 cells transfected with control siRNAs specific for green fluorescent protein (top) or siRNAs specific for Emi1 (bottom) were fixed and immunostained for Emi1. Cells transfected with Emi1 siRNA express considerably less Emi1 as indicated by the decrease in red AEC chromagen labeling of the cells and elimination of the Emi1 band by Western blot (right).

Figure 3

Figure 3

Analysis of Emi1 protein expression in human tumors. Emi1 is highly expressed in retinoblastoma (eye whole mount and retinoblastoma rosette) and other major tumor types (TMA cores). The viable peripheral tumor tissue within the eye whole mount is Emi1 immunopositive, whereas the necrotic center is nonreactive. Normal lymph nodes and breast tissue exhibit less intense Emi1 staining than lymphomas and breast ductal cancer, respectively. Benign fibrocystic breast tissue and endometrial stroma show very little Emi1 staining in the ductal carcinoma in situ and endometrial cancer cores, respectively. Positive immunoreactivity is indicated by the brown diaminobenzidine chromagen.

Figure 4

Figure 4

Summary of TMA analysis of Emi1 and APC/C substrate protein expression in human tumors. TMAs representing different classes of tumors, grouped according to developmental tissue origin, were analyzed by immunohistochemical staining. Numbers in boxes indicate the percentage of positive tumors (specific numbers of immunopositive and total tumors surveyed are summarized in Supplemental Table 2, see http://ajp.amjpathol.org). Green indicates a low percentage of immunopositive tumors (<33% of cases); dark red, an intermediate percentage (33 to 66%); and bright red, a high percentage of Emi1-positive tumors (>66%). Benign tumors are highlighted by the blue boxes and less aggressive malignant tumors by yellow boxes. World Health Organization grades are indicated for astrocytomas and follicular lymphomas. Histopathological grades are also listed for colon adenocarcinomas. Adenoca, adenocarcinoma; Ca, carcinoma; CIS, carcinoma in situ; DFSP, dermatofibrosarcoma protuberans; esoph., esophageal; GI, gastrointestinal; HN, head and neck; MPNST, malignant peripheral nerve sheath tumor; PNET, primitive neuroectodermal tumor.

Figure 5

Figure 5

Hierarchical clustering analysis of Emi1, proliferation control, and mitotic control proteins in human tumors. For each tumor type, immunopositivity for Emi1, proliferation control (cyclins D1, D2, E, and A; β-catenin; p27Kip1; Bcl2; and Ki67), and mitotic control/APC/C (aurora A, Plk1, securin, Cdh1, and Skp2) substrates were identified. Dendrograms for neural tumor (A) and lymphoma (B) TMAs are shown. Green rectangles indicate a tumor showing no immunopositivity, dark red indicates moderate positivity (3 to 29% of tumor cells positive), and bright red indicates high positivity (>30% tumor cells positive). Gray rectangles indicate unscorable TMA cores. The dendograms on the top horizontal axes show clusters of proliferation control proteins and APC/C substrate proteins. Oncogenic APC/C substrates securin, aurora A, Plk1, and Skp2 (red) form a cluster with Emi1 distinct from broader proliferation markers such as Ki67, cyclin A, and Bcl-2 in malignant neural tumors and lymphomas. The pink rectangles on the right vertical axes represent Emi1-positive tumors, which are largely malignant and positive for both proliferation and APC/C clusters. The yellow rectangles indicate mostly malignant tumors that are largely proliferation and APC/C cluster positive but Emi1 negative. The blue rectangles represent mostly benign or low-grade tumors that are predominantly proliferation cluster positive and APC/C cluster negative. ALCL, anaplastic large cell lymphoma; DLBCL, diffuse large B-cell lymphoma; FL1–3, follicular lymphoma grades 1 through 3; NKC, natural killer cell (lymphoma); NLP, nodular lymphocyte predominant Hodgkin’s lymphoma; CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; PTLD, posttransplant lymphoproliferative disorder.

Figure 6

Figure 6

Hierarchical clustering analysis of Emi1, proliferation control, and mitotic control proteins in human connective tissue tumors. Data are represented as in Figure 4. DFSP, dermatofibrosarcoma protuberans; DSRCT, desmoplastic small round cell tumor; GIST, gastrointestinal stromal cell tumor; MFH, malignant fibrous histiocytoma; MPNST, malignant peripheral nerve sheath tumor; NOS, type not otherwise specified; PNET, primitive neuroectodermal tumor; SFT, solitary fibrous tumor.

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