Neutrophil Gelatinase-Associated Lipocalin (NGAL) Predicts Response to Neoadjuvant Chemotherapy and Clinical Outcome in Primary Human Breast Cancer

In our previous work we showed that NGAL, a protein involved in the regulation of proliferation and differentiation, is overexpressed in human breast cancer (BC) and predicts poor prognosis. In neoadjuvant chemotherapy (NACT) pathological complete response (pCR) is a predictor for outcome. The aim of this study was to evaluate NGAL as a predictor of response to NACT and to validate NGAL as a prognostic factor for clinical outcome in patients with primary BC. Immunohistochemistry was performed on tissue microarrays from 652 core biopsies from BC patients, who underwent NACT in the GeparTrio trial. NGAL expression and intensity was evaluated separately. NGAL was detected in 42.2% of the breast carcinomas in the cytoplasm. NGAL expression correlated with negative hormone receptor (HR) status, but not with other baseline parameters. NGAL expression did not correlate with pCR in the full population, however, NGAL expression and staining intensity were significantly associated with higher pCR rates in patients with positive HR status. In addition, strong NGAL expression correlated with higher pCR rates in node negative patients, patients with histological grade 1 or 2 tumors and a tumor size <40 mm. In univariate survival analysis, positive NGAL expression and strong staining intensity correlated with decreased disease-free survival (DFS) in the entire cohort and different subgroups, including HR positive patients. Similar correlations were found for intense staining and decreased overall survival (OS). In multivariate analysis, NGAL expression remained an independent prognostic factor for DFS. The results show that in low-risk subgroups, NGAL was found to be a predictive marker for pCR after NACT. Furthermore, NGAL could be validated as an independent prognostic factor for decreased DFS in primary human BC.     

The low molecular weight (LMW) isoforms of cyclin E deregulate the cell cycle of mammary epithelial cells

Cyclin E in complex with CDK2 is a positive regulator of the G1 to S phase transition of the cell cycle and is responsible for cells passing the restriction point, committing the cell to another round of cell division. Cyclin E is overexpressed and proteolytically cleaved into low molecular weight (LMW) isoforms in breast cancer cell lines and tumor tissues compared to normal cells and tissues. These alterations in cyclin E are linked to poor prognosis in breast cancer patients. In order to evaluate the biological effects of the LMW cyclin E, immortalized mammary epithelial cells, 76NE6, were stably transfected with each of the three cyclin E constructs. Our results reveal that the LMW forms of cyclin E (T1 and T2) are biologically functional, as their overexpression in the immortalized cells increases the ability of these cells to enter S and G2/M phase by 2 fold over full length or vector-alone transfected cells, concomitant with an increased rate of cell proliferation. In addition, these LMW isoforms are biochemically hyperactive, shown by their ability to phosphorylate substrates such as histone H1 4 fold more in cells transfected with T1 or T2 versus cells transfected with the full length form. These results suggest that overexpression of the LMW forms of cyclin E is mitogenic, stimulating the cells to progress through the cell cycle much more efficiently than the full length cyclin E.     

Semi-high throughput method of measuring proteasome inhibition <Emphasis Type="Italic">in vitro</Emphasis> and in cultured cells

The ubiquitin proteasome–proteolytic pathway has emerged as one of the most significant pathways in modulating protein homeostasis under both normal and disease states. The use of proteasome inhibitors (PI) has played a pivotal role in understanding protein turn over. The main objective of this work was to develop a comprehensive, fast, and reliable, yet simple in vitro assay that would allow for the identification and characterization of a wide range of PIs. The assays consist of a 96-well plate high throughput (HTP) method to assess proteasome activity in Hs578T breast cancer cell extracts, purified 20S proteasome, using a fluorogenic substrate, Suc-leu-leu-val-tyr-7-AMC, specific to the chymotrypsin-like enzymatic activity of the proteasome. We showed that the chymotrypsin-like activity of the proteasome was inhibited in the two in vitro systems, albeit to different degrees. The assay system also includes two cell-based assays consisting of a vector expressing a fusion protein of green fluorescent protein (gfp) and Mouse Ornithine Decarboxylase (MODC) in Zs578T (parental Hs578T carrying the vector that expresses the fusion protein). In the cell-based assay analyses (qualitatively by microscopy and quantitatively by flow cytometry), treatment of Zs578T with PIs prevented the degradation of MODC, accumulated gfp, indicative of increased proteasome inhibition. Because no single assay represents a definitive proof of proteasome inhibitory activity, combined, these assays should serve as a comprehensive benchmark for the identification and partial characterization of novel inhibitors. In summary, the four-step assay protocol can easily be adapted into a high throughput format to rapidly screen unknown inhibitors.     

Activation of cyclin-dependent kinase 2 by full length and low molecular weight forms of cyclin E in breast cancer cells

Cyclin E, a positive regulator of the cell cycle, controls the transition of cells from G(1) to S phase. Deregulation of the G(1)-S checkpoint contributes to uncontrolled cell division, a hallmark of cancer. We have reported previously that cyclin E is overexpressed in breast cancer and such overexpression is usually accompanied by the appearance of low molecular weight isoforms of cyclin E protein, which are not present in normal cells. Furthermore, we have shown that the expression of cyclin E low molecular weight isoforms can be used as a reliable prognostic marker for breast cancer to predict patient outcome. In this study we examined the role of cyclin E in directly activating cyclin-dependent kinase (CDK) 2. For this purpose, a series of N-terminal deleted forms of cyclin E corresponding to the low molecular weight forms detected only in cancer cells were translated in vitro and mixed with cell extracts. These tumor-specific N-terminal deleted forms of cyclin E are able to activate CDK2. Addition of cyclin E into both normal and tumor cell extracts was shown to increase the levels of CDK2 activity, along with an increase in the amount of phosphorylated CDK2. The increase in CDK2 activity was because of cyclin E binding to endogenous CDK2 in complex with endogenous cyclin E, cyclin A, or unbound CDK2. The increase in CDK2 phosphorylation was through a pathway involving cyclin-activating kinase, but addition of cyclin E to an extract containing unphosphorylated CDK2 can still lead to increase in CDK2 activity. Our data suggest that the ability of high levels of full-length and low molecular weight forms of cyclin E to activate CDK2 may be one mechanism that leads to the constitutive activation of cyclin E.CDK2 complexes leading to G(1)/S deregulation and tumor progression.     

SNARE bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of membrane fusion

Sec1/Munc18 (SM) proteins activate intracellular membrane fusion through binding to cognate SNAP receptor (SNARE) complexes. The synaptic target membrane SNARE syntaxin 1 contains a highly conserved H_abc domain, which connects an N-peptide motif to the SNARE core domain and is thought to participate in the binding of Munc18-1 (the neuronal SM protein) to the SNARE complex. Unexpectedly, we found that mutation or complete removal of the H_abc domain had no effect on Munc18-1 stimulation of fusion. The central cavity region of Munc18-1 is required to stimulate fusion but not through its binding to the syntaxin H_abc domain. SNAP-25, another synaptic SNARE subunit, contains a flexible linker and exhibits an atypical conjoined Q_bc configuration. We found that neither the linker nor the Q_bc configuration is necessary for Munc18-1 promotion of fusion. As a result, Munc18-1 activates a SNARE complex with the typical configuration, in which each of the SNARE core domains is individually rooted in the membrane bilayer. Thus, the SNARE four-helix bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of fusion.     

Low molecular weight cyclin E is associated with p27-resistant,high-grade,high-stage and invasive bladder cancer

Expression of low molecular weight (LMW) isoforms of cyclin E is a strong predictor of poor outcome in patients with breast cancer. The purpose of this study was to examine the expression of full-length and LMW cyclin E in bladder cancer cell lines and patient tumors. We used western blotting, immunoprecipitation and kinase assays to examine the expression and activity of key cell cycle-regulatory proteins in various human bladder cell lines, both tumorigenic and non-tumorigenic. We also analyzed cyclin E expression, kinase activity and immune complex binding partners in 43 tissue samples from grade 2 and 3 transitional cell carcinomas. Cyclin E was overexpressed and LMW isoforms were present only in bladder cancer cells. Overexpression of LMW isoforms of cyclin E and increased cyclin E kinase activity were both significantly associated with tumorigenicity of the bladder cell lines (p = 0.005 and 0.022, respectively). Binding of the cyclin-dependent kinase inhibitors p21 and p27 to LMW cyclin E did not inhibit the kinase activity of cyclin E and cyclin-dependent kinase 2 in primary tumor samples overexpressing LMW cyclin E. Full-length and LMW cyclin E were significantly overexpressed in grade 3 tumors compared with grade 2 tumors (p = 0.004). Finally, LMW cyclin E levels were significantly associated with a non-papillary growth pattern (p = 0.031) and invasiveness (p = 0.021) of the bladder tumors and poor overall survival (p = 0.06). These results suggest that LMW cyclin E can be used as a new prognostic marker for bladder cancer.Key words: cyclin E, p27, Cdk2 kinase, bladder cancer, cell cycle     

LMW-E/CDK2 deregulates acinar morphogenesis, induces tumorigenesis, and associates with the activated b-Raf-ERK1/2-mTOR pathway in breast cancer patients

Elastase-mediated cleavage of cyclin E generates low molecular weight cyclin E (LMW-E) isoforms exhibiting enhanced CDK2-associated kinase activity and resistance to inhibition by CDK inhibitors p21 and p27. Approximately 27% of breast cancers express high LMW-E protein levels, which significantly correlates with poor survival. The objective of this study was to identify the signaling pathway(s) deregulated by LMW-E expression in breast cancer patients and to identify pharmaceutical agents to effectively target this pathway. Ectopic LMW-E expression in nontumorigenic human mammary epithelial cells (hMECs) was sufficient to generate xenografts with greater tumorigenic potential than full-length cyclin E, and the tumorigenicity was augmented by in vivo passaging. However, cyclin E mutants unable to interact with CDK2 protected hMECs from tumor development. When hMECs were cultured on Matrigel, LMW-E mediated aberrant acinar morphogenesis, including enlargement of acinar structures and formation of multi-acinar complexes, as denoted by reduced BIM and elevated Ki67 expression. Similarly, inducible expression of LMW-E in transgenic mice generated hyper-proliferative terminal end buds resulting in enhanced mammary tumor development. Reverse-phase protein array assay of 276 breast tumor patient samples and cells cultured on monolayer and in three-dimensional Matrigel demonstrated that, in terms of protein expression profile, hMECs cultured in Matrigel more closely resembled patient tissues than did cells cultured on monolayer. Additionally, the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E-expressing patient samples, and activation of this pathway was associated with poor disease-specific survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (a pan kinase inhibitor targeting b-Raf) effectively prevented aberrant acinar formation in LMW-E-expressing cells by inducing G1/S cell cycle arrest. LMW-E requires CDK2-associated kinase activity to induce mammary tumor formation by disrupting acinar development. The b-Raf-ERK1/2-mTOR signaling pathway is aberrantly activated in breast cancer and can be suppressed by combination treatment with roscovitine plus either rapamycin or sorafenib.     

DEAR1 Is a Dominant Regulator of Acinar Morphogenesis and an Independent Predictor of Local Recurrence-Free Survival in Early-Onset Breast Cancer

Background

Breast cancer in young women tends to have a natural history of aggressive disease for which rates of recurrence are higher than in breast cancers detected later in life. Little is known about the genetic pathways that underlie early-onset breast cancer. Here we report the discovery of DEAR1 (ductal epithelium–associated RING Chromosome 1), a novel gene encoding a member of the TRIM (tripartite motif) subfamily of RING finger proteins, and provide evidence for its role as a dominant regulator of acinar morphogenesis in the mammary gland and as an independent predictor of local recurrence-free survival in early-onset breast cancer.

Methods and Findings

Suppression subtractive hybridization identified DEAR1 as a novel gene mapping to a region of high-frequency loss of heterozygosity (LOH) in a number of histologically diverse human cancers within Chromosome 1p35.1. In the breast epithelium, DEAR1 expression is limited to the ductal and glandular epithelium and is down-regulated in transition to ductal carcinoma in situ (DCIS), an early histologic stage in breast tumorigenesis. DEAR1 missense mutations and homozygous deletion (HD) were discovered in breast cancer cell lines and tumor samples. Introduction of the DEAR1 wild type and not the missense mutant alleles to complement a mutation in a breast cancer cell line, derived from a 36-year-old female with invasive breast cancer, initiated acinar morphogenesis in three-dimensional (3D) basement membrane culture and restored tissue architecture reminiscent of normal acinar structures in the mammary gland in vivo. Stable knockdown of DEAR1 in immortalized human mammary epithelial cells (HMECs) recapitulated the growth in 3D culture of breast cancer cell lines containing mutated DEAR1, in that shDEAR1 clones demonstrated disruption of tissue architecture, loss of apical basal polarity, diffuse apoptosis, and failure of lumen formation. Furthermore, immunohistochemical staining of a tissue microarray from a cohort of 123 young female breast cancer patients with a 20-year follow-up indicated that in early-onset breast cancer, DEAR1 expression serves as an independent predictor of local recurrence-free survival and correlates significantly with strong family history of breast cancer and the triple-negative phenotype (ER⁻, PR⁻, HER-2⁻) of breast cancers with poor prognosis.

Conclusions

Our data provide compelling evidence for the genetic alteration and loss of expression of DEAR1 in breast cancer, for the functional role of DEAR1 in the dominant regulation of acinar morphogenesis in 3D culture, and for the potential utility of an immunohistochemical assay for DEAR1 expression as an independent prognostic marker for stratification of early-onset disease.     

Deregulation of cyclin E meets dysfunction in p53: closing the escape hatch on breast cancer

In this review, we focus on pathways intersecting through p53 and cyclin E, highlighting how oncogenic effects of cyclin E deregulation, especially overexpression of shortened or low molecular weight (LMW) forms of cyclin E protein, are amplified by loss of regulatory control through p53 to promote tumor development. Expression of cyclin E protein promotes progression into S-phase, an activity opposed by p53-regulated activation of checkpoint controls or apoptosis. Loss of p53 function is an escape hatch by which tumor cells, initiated by a number of means including cyclin E deregulation, can avoid cell cycle arrest or cell death and progress through further stages of unchecked deregulation and growth. To determine how this escape hatch is opened and, ultimately, how to close it, we must understand the networks of normal signaling and processing in a cell and where they intersect.