Chin-Lee Wu

A family of human cdc2-related protein kinases.

The p34cdc2 protein kinase is known to regulate important transitions in the eukaryotic cell cycle. We have identified 10 human protein kinases based on their structural relation to p34cdc2. Seven of these kinases are novel and the products of five share greater than 50% amino acid sequence identity with p34cdc2. The seven novel genes are broadly expressed in human cell lines and tissues with each displaying some cell type or tissue specificity. The cdk3 gene, like cdc2 and cdk2, can complement cdc28 mutants of Saccharomyces cerevisiae, suggesting that all three of these protein kinases can play roles in the regulation of the mammalian cell cycle. The identification of a large family of cdc2‐related kinases opens the possibility of combinatorial regulation of the cell cycle together with the emerging large family of cyclins.

Isolation and Characterization of Circulating Tumor Cells from Patients with Localized and Metastatic Prostate Cancer

Automated imaging of prostate-specific cancer cells from the blood provides a measure of circulating tumor cell half-life after tumor resection. Circling Cancers Out Oftentimes a patient and his or her clinician learn together at the flip of a radiological imaging scan that a solid tumor, previously removed, has returned either at the original site or at new locations to which it has spread. A failure of cancer treatment—but could it have been predicted? Whether it is freely floating tumor DNA or tumor cells, the circulation of cancer-derived material in the blood holds great promise for the early detection and prevention of cancer metastases. The accurate identification, enumeration, and molecular classification of blood-borne cells—although postulated more than 140 years ago—remain the greatest challenge. Now, in a small cohort of individuals with and without prostate cancer, Stott and colleagues have used a silicon microfluidic cell-capture technology that, when coupled to an automated imaging system, enables the detection and enumeration of prostate cancer cells fished out from the blood. These cells express a surface protein that uniquely identifies epithelial cells in the circulation. Once efficiently captured by antibody to this protein, the cells are counterstained with antibodies that are prostate-specific and that indicate cell proliferation, suggesting that these circulating cells are ready to repopulate distant metastatic sites. In their study, tumor cells obtained from the blood of cancer patients were monitored before and after surgery; some circulating cells persisted months after surgery while others rapidly declined shortly thereafter. Whether the persistence or disappearance of lurking cancer cells reflects an intrinsic capacity for reseeding remains to be established, but the system used in the study offers great potential for oncologists to detect cancer-related changes earlier and to monitor responses to drug treatments. In the hope of ultimately applying personalized treatments to cancer patients, based on “real-time” monitoring of the tumor cell genetic makeup, this approach to identifying these circulating cells early on moves us beyond the finality of the films currently offered to patients in the clinic. Rare circulating tumor cells (CTCs) are present in the blood of patients with metastatic epithelial cancers but have been difficult to measure routinely. We report a quantitative automated imaging system for analysis of prostate CTCs, taking advantage of prostate-specific antigen (PSA), a unique prostate tumor–associated marker. The specificity of PSA staining enabled optimization of criteria for baseline image intensity, morphometric measurements, and integration of multiple signals in a three-dimensional microfluidic device. In a pilot analysis, we detected CTCs in prostate cancer patients with localized disease, before surgical tumor removal in 8 of 19 (42%) patients (range, 38 to 222 CTCs per milliliter). For 6 of the 8 patients with preoperative CTCs, a precipitous postoperative decline (<24 hours) suggests a short half-life for CTCs in the blood circulation. Other patients had persistent CTCs for up to 3 months after prostate removal, suggesting early but transient disseminated tumor deposits. In patients with metastatic prostate cancer, CTCs were detected in 23 of 36 (64%) cases (range, 14 to 5000 CTCs per milliliter). In previously untreated patients followed longitudinally, the numbers of CTCs declined after the initiation of effective therapy. The prostate cancer–specific TMPRSS2-ERG fusion was detectable in RNA extracted from CTCs from 9 of 20 (45%) patients with metastatic disease, and dual staining of captured CTCs for PSA and the cell division marker Ki67 indicated a broad range for the proportion of proliferating cells among CTCs. This method for analysis of CTCs will facilitate the application of noninvasive tumor sampling to direct targeted therapies in advanced prostate cancer and warrants the initiation of long-term clinical studies to test the importance of CTCs in invasive localized disease.

Cables Links Cdk5 and c-Abl and Facilitates Cdk5 Tyrosine Phosphorylation, Kinase Upregulation, and Neurite Outgrowth

Cyclin-dependent kinase 5 (Cdk5) is a small serine/threonine kinase that plays a pivotal role during development of the CNS. Cables, a novel protein, interacts with Cdk5 in brain lysates. Cables also binds to and is a substrate of the c-Abl tyrosine kinase. Active c-Abl kinase leads to Cdk5 tyrosine phosphorylation, and this phosphorylation is enhanced by Cables. Phosphorylation of Cdk5 by c-Abl occurs on tyrosine 15 (Y15), which is stimulatory for p35/Cdk5 kinase activity. Expression of antisense Cables in primary cortical neurons inhibited neurite outgrowth. Furthermore, expression of active Abl resulted in lengthening of neurites. The data provide evidence for a Cables-mediated interplay between the Cdk5 and c-Abl signaling pathways in the developing nervous system.

RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance.

Circulating signals of drug resistance Cancer drugs often lose their effectiveness because tumors acquire genetic changes that confer drug resistance. Ideally, patients would be switched to a different drug before tumor growth resumes, but this requires early knowledge of how resistance arose. Miyamoto et al. have developed a non-invasive method to spot resistance by sequencing RNA transcripts in single circulating tumor cells (CTCs) (see the Perspective by Nanus and Giannakakou). For example, in prostate cancer patients, drug resistance was triggered by activation of the Wnt signaling pathway. But CTCs are rare and fragile, and the technology needs further development before it is used in clinical practice. Science, this issue p. 1351; see also p. 1283 Analysis of circulating tumor cells from prostate cancer patients reveals a mechanism that contributes to treatment failure. [Also see Perspective by Nanus and Giannakakou] Prostate cancer is initially responsive to androgen deprivation, but the effectiveness of androgen receptor (AR) inhibitors in recurrent disease is variable. Biopsy of bone metastases is challenging; hence, sampling circulating tumor cells (CTCs) may reveal drug-resistance mechanisms. We established single-cell RNA-sequencing (RNA-Seq) profiles of 77 intact CTCs isolated from 13 patients (mean six CTCs per patient), by using microfluidic enrichment. Single CTCs from each individual display considerable heterogeneity, including expression of AR gene mutations and splicing variants. Retrospective analysis of CTCs from patients progressing under treatment with an AR inhibitor, compared with untreated cases, indicates activation of noncanonical Wnt signaling (P = 0.0064). Ectopic expression of Wnt5a in prostate cancer cells attenuates the antiproliferative effect of AR inhibition, whereas its suppression in drug-resistant cells restores partial sensitivity, a correlation also evident in an established mouse model. Thus, single-cell analysis of prostate CTCs reveals heterogeneity in signaling pathways that could contribute to treatment failure.

In vivo association of E2F and DP family proteins.

The mammalian transcription factor E2F plays an important role in regulating the expression of genes that are required for passage through the cell cycle. This transcriptional activity is inhibited by association with the retinoblastoma tumor suppressor protein (pRB) or its relatives p107 and p103. The first cDNA from the E2F family to be cloned was designated E2F-1, and multiple E2F family members have now been identified. They bind to DNA as heterodimers, interacting with proteins known as DP. Here we demonstrate that DP is also a family of polypeptides with at least two members (hDP-1 and hDP-2). Both hDP-1 and hDP-2 bind to all E2F family members in vivo, and each complex is capable of activating transcription. However, the various E2F/DP complexes display strong differences in the ability to bind to either pRB or p107 in vivo, and the specificity of pRB or p107 binding is mediated by the E2F subunit.

Analysis of RNA-binding protein IMP3 to predict metastasis and prognosis of renal-cell carcinoma: a retrospective study

BACKGROUND Distant metastasis is the main cause of death from renal-cell carcinoma, and the metastatic potential of tumours is often unpredictable. We aimed to investigate whether IMP3, an oncofetal RNA-binding protein, can be used as a biomarker to predict metastasis and prognosis of renal-cell carcinoma. METHODS We studied 501 primary and metastatic renal-cell tumours. 371 patients with localised primary tumours were further investigated by use of survival analysis. We assessed IMP3 expression in tumour tissues by immunohistochemistry, and IMP3 mRNA and protein expression in selected tissues by quantitative real-time PCR and western blot analysis. FINDINGS Compared with non-metastatic renal-cell tumours, IMP3 expression was greatly increased not only in metastatic tumours but also in a subset of primary tumours that were likely to subsequently develop metastases. Patients with primary localised tumours that did not express IMP3 had a longer metastasis-free survival and overall survival than did those with tumours expressing IMP3 (p<0.0001). Patients with IMP3-positive localised tumours had a much lower 5-year metastasis-free survival than did those with IMP3-negative tumours (for stage I tumours, 44% vs 98%, hazard ratio 17.18 [95% CI 7.82-37.78]; stage II, 41% vs 94%, 10.14 [3.46-29.68]; stage III, 16% vs 62%, 4.04 [2.23-7.31]). IMP3 expression was also associated with reduced 5-year overall survival (stage I, 32% vs 89%, 6.44 [3.63-11.42]; stage II, 41% vs 88%, 6.93 [2.63-18.27]; stage III, 14% vs 58%, 3.46 [1.98-6.05]). Multivariable analysis of IMP3 status (positive vs negative) in primary tumours showed hazard ratios of 5.84 (95% CI 3.60-9.49) for metastasis-free survival and 4.01 (2.66-6.05) for overall survival (both p<0.0001), which were much higher than hazard ratios associated with other independent risk factors. INTERPRETATION IMP3 is an independent prognostic marker that can be used at initial diagnosis of renal-cell carcinoma to identify patients who have a high potential to develop metastasis and who might benefit from early systemic treatment.

P504S/α-methylacyl-CoA racemase: A useful marker for diagnosis of small foci of prostatic carcinoma on needle biopsy

Establishing a definitive diagnosis of malignancy in prostate needle biopsies with very small foci of adenocarcinoma is a major diagnostic challenge for surgical pathologists. A positive diagnostic marker specific for prostatic adenocarcinoma may enhance our ability to detect limited prostate cancer and reduce errors in diagnosis. P504S, also known as &agr;-methylacyl-CoA racemase, recently identified by cDNA subtraction and microarray technology, might serve as such a specific marker because it has been demonstrated to be highly expressed in prostatic adenocarcinoma, but not in benign prostatic glands. However, whether small foci of carcinoma can be reliably detected by this marker is a crucial question for its clinical application. The aim of this study was to assess the utility of P504S immunohistochemistry in detecting small amounts of prostate cancer in prostate needle biopsies. A total of 142 prostate needle biopsies, including 73 cases with a small focus of prostatic adenocarcinoma (≤1 mm) and 69 benign prostates, were examined by using immunohistochemistry for P504S and high molecular weight cytokeratin (34&bgr;E12). P504S immunoreactivity was found in 69 of 73 cases (94.5%) of carcinoma but not in any benign prostates (0 of 69) or benign glands adjacent to malignant glands. The 34&bgr;E12 immunostaining confirmed the absence of basal cells in the focus of carcinoma in all 73 cases. The high specificity and sensitivity of P504S in the detection of minimal prostatic adenocarcinoma indicated its potential diagnostic value in clinical practice. Using a combination of P504S and 34&bgr;E12 can help the diagnosis of limited prostatic adenocarcinoma on needle biopsy.

Metabolic Characterization of Human Prostate Cancer with Tissue Magnetic Resonance Spectroscopy

Diagnostic advancements for prostate cancer have so greatly increased early detections that hope abounds for improved patient outcomes. However, histopathology, which guides treatment, often subcategorizes aggressiveness insufficiently among moderately differentiated Gleason score (6 and 7) tumors (>70% of new cases). Here, we test the diagnostic capability of prostate metabolite profiles measured with intact tissue magnetic resonance spectroscopy and the sensitivity of local prostate metabolites in predicting prostate cancer status. Prostate tissue samples (n = 199) obtained from 82 prostate cancer patients after prostatectomy were analyzed with high-resolution magic angle spinning proton magnetic resonance spectroscopy, and afterwards with quantitative pathology. Metabolite profiles obtained from principal component analysis of magnetic resonance spectroscopy were correlated with pathologic quantitative findings by using linear regression analysis and evaluated against patient pathologic statuses by using ANOVA. Paired t tests show that tissue metabolite profiles can differentiate malignant from benign samples obtained from the same patient (P < 0.005) and correlate with patient serum prostate-specific antigen levels (P < 0.006). Furthermore, metabolite profiles obtained from histologically benign tissue samples of Gleason score 6 and 7 prostates can delineate a subset of less aggressive tumors (P < 0.008) and predict tumor perineural invasion within the subset (P < 0.03). These results indicate that magnetic resonance spectroscopy metabolite profiles of biopsy tissues may help direct treatment plans by assessing prostate cancer pathologic stage and aggressiveness, which at present can be histopathologically determined only after prostatectomy.

Expression of dominant-negative mutant DP-1 blocks cell cycle progression in G1.

Unregulated expression of the transcription factor E2F promotes the G1-to-S phase transition in cultured mammalian cells. However, there has been no direct evidence for an E2F requirement in this process. To demonstrate that E2F is obligatory for cell cycle progression, we attempted to inactivate E2F by overexpressing dominant-negative forms of one of its heterodimeric partners, DP-1. We dissected the functional domains of DP-1 and separated the region that facilitate heterodimer DNA binding from the E2F dimerization domain. Various DP-1 mutants were introduced into cells via transfection, and the cell cycle profile of the transfected cells was analyzed by flow cytometry. Expression of wild-type DP-1 or DP-1 mutants that bind to both DNA and E2F drove cells into S phase. In contrast, DP-1 mutants that retained E2F binding but lost DNA binding arrested cells in the G1 phase of the cell cycle. The DP-1 mutants that were unable to bind DNA resulted in transcriptionally inactive E2F complexes, suggesting that the G1 arrest is caused by formation of defective E2F heterodimers. Furthermore, the G1 arrest instigated by these DP-1 mutants could be rescued by coexpression of wild-type E2F or DP protein. These experiments define functional domains of DP and demonstrate a requirement for active E2F complexes in cell cycle progression.

Non-destructive quantitation of spermine in human prostate tissue samples using HRMAS 1H NMR spectroscopy at 9.4 T

We present the results of a study of human prostate specimens evaluated by high resolution magic angle spinning 1H nuclear magnetic resonance (NMR) spectroscopy at 400 MHz (9.4 T) and by quantitative histopathology. We demonstrate that NMR and pathology data can be obtained from the same intact specimens, and report for the first time a linear correlation between the NMR measured concentration of spermine, a proposed endogenous inhibitor to prostate cancer growth, and the volume percentage of normal prostatic epithelial cells as quantified by histopathology. Our results show that NMR may serve as a critical tool for the investigation of the inhibitory mechanism of spermine in human subjects.