Douglas I. Lin

p63 Is a Prostate Basal Cell Marker and Is Required for Prostate Development

The p53 homologue p63 encodes for different isotypes able to either transactivate p53 reporter genes (TAp63) or act as p53-dominant-negatives (DeltaNp63). p63 is expressed in the basal cells of many epithelial organs and its germline inactivation in the mouse results in agenesis of organs such as skin appendages and the breast. Here, we show that prostate basal cells, but not secretory or neuroendocrine cells, express p63. In addition, prostate basal cells in culture predominantly express the DeltaNp63alpha isotype. In contrast, p63 protein is not detected in human prostate adenocarcinomas. Finally, and most importantly, p63(-/-) mice do not develop the prostate. These results indicate that p63 is required for prostate development and support the hypothesis that basal cells represent and/or include prostate stem cells. Furthermore, our results show that p63 immunohistochemistry may be a valuable tool in the differential diagnosis of benign versus malignant prostatic lesions.

Mutations in Fbx4 Inhibit Dimerization of the SCFFbx4 Ligase and Contribute to Cyclin D1 Overexpression in Human Cancer

SCF(Fbx4) was recently identified as the E3 ligase for cyclin D1. We now describe cell-cycle-dependent phosphorylation and dimerization of Fbx4 that is regulated by GSK3beta and is defective in human cancer. We present data demonstrating that a pathway involving Ras-Akt-GSK3beta controls the temporal phosphorylation and dimerization of the SCF(Fbx4) E3 ligase. Inhibition of Fbx4 activity results in accumulation of nuclear cyclin D1 and oncogenic transformation. The importance of this regulatory pathway for normal cell growth is emphasized by the prevalence of mutations in Fbx4 in human cancer that impair dimerization. Collectively, these data reveal that inactivation of the cyclin D1 E3 ligase likely contributes to cyclin D1 overexpression in a significant fraction of human cancer.

Androgen-Dependent Regulation of Her-2/neu in Prostate Cancer Cells

The mechanisms underlying the progression of prostate cancer to a state of resistance to hormone ablation remain poorly understood. Here, we have investigated the relationship between androgen receptor (AR) and Her-2/neu in prostate cancer cells. Overexpression of Her-2/neu (c-ErbB2) activates the AR pathway and confers a survival and growth advantage to prostate cancer cells in an androgen-deficient milieu. In vitro, the absence of androgens or AR blockade induced Her-2/neu protein expression and phosphorylation. In contrast, upon readministration of androgens, Her-2/neu mRNA, protein, and phosphorylation levels decreased linearly with increasing concentrations of dihydrotestosterone as LNCaP cells reentered the cell cycle. In vivo, induction of Her-2/neu by castration in orthotopically injected LNCaP cells resulted in a progressive increase in prostate-specific antigen secretion into the mouse serum, indicating that Her-2/neu-mediated, AR-dependent transcription occurs following castration and results in tumor cell growth. Finally, selection of LNCaP cells stably transfected with short hairpin RNA specific for AR resulted in Her-2/neu overexpression. Similarly, knockdown of Her-2/neu led to induction of AR. However, when Her-2/neu and AR were simultaneously targeted, we observed cell death, whereas surviving cells retained low level expression of Her-2/neu. Thus, induction and activation of Her-2/neu occurs in an androgen-depleted environment or as a result of AR inactivation, promoting ablation-resistant survival of prostate cancer cells. These data provide the biochemical rationale to target Her-2/neu in hormone-refractory prostate cancer.

Disruption of cyclin D1 nuclear export and proteolysis accelerates mammary carcinogenesis

Cyclin D1 levels are maintained at steady state by phosphorylation-dependent nuclear export and polyubiquitination by SCFFBX4-αB crystallin. Inhibition of cyclin D1 proteolysis has been implicated as a causative factor leading to its overexpression in breast and esophageal carcinomas; however, the contribution of stable cyclin D1 to the genesis of such carcinomas has not been evaluated. We therefore generated transgenic mice wherein expression of either wild-type or a stable cyclin D1 allele (D1T286A) is regulated by MMTV-LTR. MMTV-D1T286A mice developed mammary adenocarcinomas at an increased rate relative to MMTV-D1 mice. Similar to human cancers that overexpress cyclin D1, D1T286A tumors were estrogen receptor-positive and exhibited estrogen-dependent growth. Collectively, these results suggest that temporal control of cyclin D1 subcellular localization and proteolysis is critical for maintenance of homeostasis within the mammary epithelium.

Evidence for a dualistic model of high-grade serous carcinoma: BRCA mutation status, histology, and tubal intraepithelial carcinoma.

Most early adnexal carcinomas detected in asymptomatic women with germline BRCA mutations (BRCA+) present as serous tubal intraepithelial carcinomas (STIC). However, STICs are found in only ∼40% of symptomatic high-grade serous carcinomas (HGSCs) and less frequently in pseudoendometrioid variants of HGSC. Consecutive cases of untreated HGSC from BRCA+ and BRCA− women with detailed fallopian tube examination (SEE-FIM protocol) were compared. STIC status (+/−) was determined, and tumors were classified morphologically as SET (“SET”, >50% solid, pseudoendometrioid, or transitional) or classic predominate (“Classic”). SET tumors trended toward a higher frequency in BRCA+ versus BRCA− women (50% vs. 28%, P=0.11), had a significantly younger mean age than those with classic HGSC in BRCA− women (mean 56.2 vs. 64.8 y, P=0.04), and displayed a better clinical outcome in both groups combined (P=0.024). STIC was significantly more frequent in tumors from the BRCA− cohort (66% vs. 31%, P=0.017) and specifically the BRCA− tumors with classic morphology (83%) versus those with SET morphology (22%, P=0.003). Overall, several covariables—histology, BRCA status, age, coexisting STIC, and response to therapy—define 2 categories of HGSC with differences in precursor (STIC) frequency, morphology, and outcome. We introduce a dualistic HGSC model that could shed light on the differences in frequency of STIC between symptomatic and asymptomatic women with HGSC. This model emphasizes the need for further study of HGSC precursors to determine their relevance to the prevention of this lethal malignancy.

Phosphorylation of MCM3 on Ser-112 regulates its incorporation into the MCM2–7 complex

During late M and early G1, MCM2–7 assembles and is loaded onto chromatin in the final step of prereplicative complex (pre-RC) formation. However, the regulation of MCM assembly remains poorly understood. Cyclin-dependent kinase (CDK)-dependent phosphorylation contributes to DNA replication by initially activating pre-RCs and subsequently inhibiting refiring of origins during S and M phases, thus limiting DNA replication to a single round. Although the precise roles of specific MCM phosphorylation events are poorly characterized, we now demonstrate that CDK1 phosphorylates MCM3 at Ser-112, Ser-611, and Thr-719. In vivo, CDK1-dependent phosphorylation of Ser-112 triggers the assembly of MCM3 with the remaining MCM subunits and subsequent chromatin loading of MCMs. Strikingly, loss of MCM3 triggers the destabilization of other MCM proteins, suggesting that phosphorylation-dependent assembly is essential for stable accumulation of MCM proteins. These data reveal that CDK-dependent MCM3 phosphorylation contributes to the regulated formation of the MCM2–7 complex.

High grade serous ovarian carcinomas originate in the fallopian tube

High-grade serous ovarian carcinoma (HGSOC) is the most frequent type of ovarian cancer and has a poor outcome. It has been proposed that fallopian tube cancers may be precursors of HGSOC but evolutionary evidence for this hypothesis has been limited. Here, we perform whole-exome sequence and copy number analyses of laser capture microdissected fallopian tube lesions (p53 signatures, serous tubal intraepithelial carcinomas (STICs), and fallopian tube carcinomas), ovarian cancers, and metastases from nine patients. The majority of tumor-specific alterations in ovarian cancers were present in STICs, including those affecting TP53, BRCA1, BRCA2 or PTEN. Evolutionary analyses reveal that p53 signatures and STICs are precursors of ovarian carcinoma and identify a window of 7 years between development of a STIC and initiation of ovarian carcinoma, with metastases following rapidly thereafter. Our results provide insights into the etiology of ovarian cancer and have implications for prevention, early detection and therapeutic intervention of this disease.It has previously been proposed that high-grade serous ovarian carcinoma (HGSOC) may originate from the fallopian tube. Here, the authors analyze genetic aberrances in fallopian tube lesions, ovarian cancers, and metastases from HGSOC patients and establish the evolutionary origins of HGSOC in the fallopian tube.

The de-ubiquitinating enzyme Unp interacts with the retinoblastoma protein.

The ubiquitin pathway is involved in the proteolytic turnover of many short-lived cellular regulatory proteins. Since selective degradation of substrates of this system requires the covalent attachment of a polyubiquitin chain to the substrates, degradation could be counteracted by de-ubiquitinating enzymes (or isopeptidases) which selectively remove the polyubiquitin chain. Unp is a human isopeptidase with still poorly understood biological functions. Here, we show that cellular Unp specifically interacts with the retinoblastoma gene product (pRb).

SCF Fbx4/alphaB-crystallin cyclin D1 ubiquitin ligase: a license to destroy.

Cyclin D1 is an allosteric regulator for cyclin-dependent kinases 4 and 6 (CDK4/6). The cyclin D/CDK4 kinase promotes G1/S transition through the posttranslational modification and the subsequent inactivation of the retinoblastoma (Rb) protein and related family members (p107 and p130). Accumulation of cyclin D1 is tightly regulated through various mechanisms including transcription, protein localization and ubiquitin-dependent proteolysis. While regulators of cyclin D1 gene expression have been under considerable scrutiny, the identity of the protein complex that targets cyclin D1 protein for degradation, the putative E3 ubiquitin ligase, has remained obscure. In a recent report, Lin et al [1] describe the identification and characterization of a novel SCF, wherein FBX4 and αB-crystallin serve as specificity factors that direct ubiquitination of phosphorylated cyclin D1. As cyclin D1 overexpression in human cancer has been postulated to occur through the loss of degradation machinery, the identification of the SCFFbx4/αB-crystallin ligase will allow new experimental approaches that address mechanisms of cyclin D1 overexpression in human cancer.

Characterization of Clinical Cases of Collecting Duct Carcinoma of the Kidney Assessed by Comprehensive Genomic Profiling

BACKGROUND Collecting duct carcinoma (CDC) is a rare type of renal cell carcinoma (RCC) originating from the renal medulla. Clinical outcomes are poor, and there are no consensus guidelines to guide therapy. OBJECTIVE To determine genomic alterations (GAs) in a series of patients with locally advanced or metastatic CDC for whom genomic profiling was performed during the course of clinical care. DESIGN, SETTING, AND PARTICIPANTS Formalin-fixed, paraffin-embedded blocks or slides were obtained for 17 patients with CDC. DNA was extracted and comprehensive genomic profiling was performed in a laboratory certified under the Clinical Laboratory Improvement Amendments. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Bayesian algorithms and local alignment algorithms were used to detect substitutions and insertions/deletions, respectively. A comparison to normal control samples was used to detect copy number alterations. Clinically relevant GAs (CRGAs) were defined as those linked to approved or investigational targeted therapies. RESULTS AND LIMITATIONS The median age in the cohort was 53 yr (range 26-73), and 14 primary tumors and three metastatic sites assessed. A total of 36 GAs were detected in this series of patients, with an average of 2.1 GAs per case. The most common GAs were in NF2 (5/17, 29%), SETD2 (4/17, 24%), SMARCB1 (3/17, 18%), and CDKN2A (2/17, 12%). Of nine cases assessed for FH GAs, two patients had FH homozygous loss. A limitation is that targeted interrogation of genes known to be implicated in other cancers was performed, so mutations outside of these cannot be excluded. CONCLUSIONS Recurrent CRGAs were detected in this series of CDC cases and suggest a possible benefit from targeted therapy. In particular, mTOR inhibitors may be of interest in patients with NF2 alterations. Alterations in FH and SMARCB1 also occurred in a mutually exclusive manner to NF2 alterations. PATIENT SUMMARY This report provides important genomic insights into collecting duct carcinoma, a rare type of renal cell carcinoma with a very aggressive course. These insights could further rationalize the use of targeted therapies for rare tumors according to the individual genomic alterations harbored.