The Role of the Ubiquitination–Proteasome Pathway in Breast Cancer: Use of Mouse Models for Analyzing Ubiquitination Processes

Turnover of several regulatory proteins results from targeted destruction via ubiquitination and subsequent degradation through the proteosome. The timely and irreversible degradation of critical regulators is essential for normal cellular function. The precise biochemical mechanisms that are involved in protein turnover by ubiquitin-mediated degradation have been elucidated using in vitro assays and cell culture systems. However, pathways that lead to ubiquitination of critical regulatory proteins in vivo are more complex, and have both temporal and tissue-specific differences. In vivo models will allow identification of substrates and enzymes of the ubiquitin–proteosome pathway that play important roles in selected tissues and diseases. In addition, assessment of the therapeutic efficacy of drugs designed to inhibit or enhance protein turnover by ubiquitination requires in vivo models. In the present review we describe selected examples of transgenic and knockout models of proteins that are known either to be regulated by ubiquitin-mediated degradation or to have a catalytic function in this process, and to play an important role in breast cancer. We outline the functions of these proteins in vivo and focus on knowledge gained in the comparison of in vivo behavior predicted from cell-free in vitro data or from experiments conducted in cell culture systems

O-GlcNAc transferase – an auxiliary factor or a full-blown oncogene?

The beta-linked N-acetyl-D-glucosamine (GlcNAc) is a posttranslational modification of serine and threonine residues catalyzed by the enzyme O-GlcNAc transferase (OGT). Increased OGT expression is a feature of most human cancers and inhibition of OGT decreases cancer cell proliferation. Antiproliferative effects are attributed to posttranslational modifications of known regulators of cancer cell proliferation, such as MYC, FOXM1, and EZH2. In general, OGT amplifies cell-specific phenotype, for example, OGT overexpression enhances reprogramming efficiency of mouse embryonic fibroblasts into stem cells. Genome-wide screens suggest that certain cancers are particularly dependent on OGT, and understanding these addictions is important when considering OGT as a target for cancer therapy. The O-GlcNAc modification is involved in most cellular processes, which raises concerns of ontarget undesirable effects of OGT-targeting therapy. Yet, emerging evidence suggest that, much like proteasome inhibitors, specific compounds targeting OGT elicit selective antiproliferative effects in cancer cells, and can prime malignant cells to other treatments. It is, therefore, essential to gain mechanistic insights on substrate specificity for OGT, develop reagents to more specifically enrich for O-GlcNAc-modified proteins, identify O-GlcNAc "readers," and develop OGT" small-molecule inhibitors. Here, we review the relevance of OGT in cancer progression and the potential targeting of this metabolic enzyme as a putative oncogene.Peer reviewe

De novo fatty acid synthesis at the mitotic exit is required to complete cellular division

Although the regulation of the cell cycle has been extensively studied, much less is known about its coordination with the cellular metabolism. Using mass spectrometry we found that lysophospholipid levels decreased drastically from G2/M to G1 phase, while de novo phosphatidylcholine synthesis, the main phospholipid in mammalian cells, increased, suggesting that enhanced membrane production was concomitant to a decrease in its turnover. In addition, fatty acid synthesis and incorporation into membranes was increased upon cell division. The rate-limiting reaction for de novo fatty acid synthesis is catalyzed by acetyl-CoA carboxylase. As expected, its inhibiting phosphorylation decreased prior to cytokinesis initiation. Importantly, the inhibition of fatty acid synthesis arrested the cells at G2/M despite the presence of abundant fatty acids in the media. Our results suggest that de novo lipogenesis is essential for cell cycle completion. This "lipogenic checkpoint" at G2/M may be therapeutically exploited for hyperproliferative diseases such as cancer.Instituto de Investigaciones Bioquímicas de La Plat

A Synthetic Form of Frizzled 8-Associated Antiproliferative Factor Enhances p53 Stability through USP2a and MDM2

Frizzled 8-associated Antiproliferative Factor (APF) is a sialoglycopeptide urinary biomarker of interstitial cystitis/painful bladder syndrome (IC/PBS), a chronic condition of unknown etiology with variable symptoms that generally include pelvic and/or perineal pain, urinary frequency, and urgency. We previously reported that native human APF suppresses the proliferation of normal bladder epithelial cells through a mechanism that involves increased levels of p53. The goal of this study was to delineate the regulatory mechanism whereby p53 expression is regulated by APF. Two APF-responsive cell lines (T24 bladder carcinoma cells and the immortalized human bladder epithelial cell line, TRT-HU1) were treated with asialo-APF (as-APF), a chemically synthesized form of APF. Biochemical analysis revealed that as-APF increased p53 levels in two ways: by decreasing ubiquitin specific protease 2a (USP2a) expression leading to enhanced ubiquitination of murine double minute 2 E3 ubiquitin ligase (MDM2), and by suppressing association of p53 with MDM2, thus impairing p53 ubiquitination. Biological responses to as-APF were suppressed by increased expression of wild type, but not mutant USP2a, which enhanced cell growth via upregulation of a cell cycle mediator, cyclin D1, at both transcription and protein levels. Consistent with this, gene silencing of USP2a with siRNA arrested cell proliferation. Our findings suggest that APF upregulates cellular p53 levels via functional attenuation of the USP2a-MDM2 pathway, resulting in p53 accumulation and growth arrest. These data also imply that targeting USP2a, MDM2, p53 and/or complex formation by these molecules may be relevant in the development of novel therapeutic approaches to IC/PBS

O-GlcNAc transferase couples MRE11 to transcriptionally active chromatin to suppress DNA damage

Background: Transcription, metabolism and DNA damage response are tightly regulated to preserve the genomic integrity, and O-GlcNAc transferase (OGT) is positioned to connect the three. Prostate cancer is the most common cancer in men, and androgen-ablation therapy halts disease progression. However, a significant number of prostate cancer patients develop resistance against anti-androgens, and this incurable disease is termed castration-resistant prostate cancer (CRPC). We have shown that combined inhibition of OGT and the transcription elongation kinase CDK9 induce CRPC-selective anti-proliferative effects. Here, we explain the functional basis for these combinatorial effects. Methods: We used comprehensive mass spectrometry profiling of short-term CDK9 inhibitor effects on O-GlcNAcylated proteins in an isogenic cell line system that models transition from PC to CRPC. In addition, we used both ChIP-seq and RNA-seq profiling, and pulldown experiments in multiple CRPC models. Finally, we validated our findings in prostate cancer patient samples. Results: Inhibition of CDK9 results in an OGT-dependent remodeling of the proteome in prostate cancer cells. More specifically, the activity of the DNA damage repair protein MRE11 is regulated in response to CDK9 inhibition in an OGT-dependent manner. MRE11 is enriched at the O-GlcNAc-marked loci. CDK9 inhibition does not decrease the expression of mRNAs whose genes are bound by both O-GlcNAc and MRE11. Combined inhibition of CDK9 and OGT or MRE11 further decreases RNA polymerase II activity, induces DNA damage signaling, and blocks the survival of prostate cancer cells. These effects are seen in CRPC cells but not in normal prostate cells. Mechanistically, OGT activity is required for MRE11 chromatin-loading in cells treated with CDK9 inhibitor. Finally, we show that MRE11 and O-GlcNAc are enriched at the prostate cancer-specific small nucleotide polymorphic sites, and the loss of MRE11 activity results in a hyper-mutator phenotype in patient tumors. Conclusions: Both OGT and MRE11 are essential for the repair of CDK9 inhibitor-induced DNA damage. Our study raises the possibility of targeting CDK9 to elicit DNA damage in CRPC setting as an adjuvant to other treatments.Peer reviewe

De novo fatty acid synthesis at the mitotic exit is required to complete cellular division

Although the regulation of the cell cycle has been extensively studied, much less is known about its coordination with the cellular metabolism. Using mass spectrometry we found that lysophospholipid levels decreased drastically from G2/M to G1 phase, while de novo phosphatidylcholine synthesis, the main phospholipid in mammalian cells, increased, suggesting that enhanced membrane production was concomitant to a decrease in its turnover. In addition, fatty acid synthesis and incorporation into membranes was increased upon cell division. The rate-limiting reaction for de novo fatty acid synthesis is catalyzed by acetyl-CoA carboxylase. As expected, its inhibiting phosphorylation decreased prior to cytokinesis initiation. Importantly, the inhibition of fatty acid synthesis arrested the cells at G2/M despite the presence of abundant fatty acids in the media. Our results suggest that de novo lipogenesis is essential for cell cycle completion. This "lipogenic checkpoint" at G2/M may be therapeutically exploited for hyperproliferative diseases such as cancer.Instituto de Investigaciones Bioquímicas de La Plat

p63 Promotes Cell Survival through Fatty Acid Synthase

There is increasing evidence that p63, and specifically ΔNp63, plays a central role in both development and tumorigenesis by promoting epithelial cell survival. However, few studies have addressed the molecular mechanisms through which such important function is exerted. Fatty acid synthase (FASN), a key enzyme that synthesizes long-chain fatty acids and is involved in both embryogenesis and cancer, has been recently proposed as a direct target of p53 family members, including p63 and p73. Here we show that knockdown of either total or ΔN-specific p63 isoforms in squamous cell carcinoma (SCC9) or immortalized prostate epithelial (iPrEC) cells caused a decrease in cell viability by inducing apoptosis without affecting the cell cycle. p63 silencing significantly reduced both the expression and the activity of FASN. Importantly, stable overexpression of either FASN or myristoylated AKT (myr-AKT) was able to partially rescue cells from cell death induced by p63 silencing. FASN induced AKT phosphorylation and a significant reduction in cell viability was observed when FASN-overexpressing SCC9 cells were treated with an AKT inhibitor after p63 knockdown, indicating that AKT plays a major role in FASN-mediated survival. Activated AKT did not cause any alteration in the FASN protein levels but induced its activity, suggesting that the rescue from apoptosis documented in the p63-silenced cells expressing myr-AKT cells may be partially mediated by FASN. Finally, we demonstrated that p63 and FASN expression are positively associated in clinical squamous cell carcinoma samples as well as in the developing prostate. Taken together, our findings demonstrate that FASN is a functionally relevant target of p63 and is required for mediating its pro-survival effects

AKT1[low] quiescent cancer cells persist after neoadjuvant chemotherapy in triple negative breast cancer

Background: Absence of pathologic complete response (pCR) to neoadjuvant chemotherapy (NACT) correlates with poor long-term survival in patients with triple negative breast cancer (TNBC). These incomplete treatment responses are likely determined by mechanisms that enable cancer cells to resist being killed. However, the detailed characterization of a drug-resistant cancer cell state in residual TNBC tissue after NACT has remained elusive. AKT1(low) quiescent cancer cells (QCCs) are a quiescent, epigenetically plastic, and chemotherapy-resistant subpopulation initially identified in experimental cancer models. Here, we asked whether QCCs exist in primary tumors from patients with TNBC and persist after treatment with NACT. Methods: We obtained pre-treatment biopsy, post-treatment mastectomy, and metastatic specimens from a retrospective cohort of TNBC patients treated with NACT at Massachusetts General Hospital (n = 25). Using quantitative automated immunofluorescence microscopy, QCCs were identified as AKT(low)/H3K9me2(low)/HES1(high) cancer cells using prespecified immunofluorescence intensity thresholds. QCCs were represented in 2D and 3D digital tumor maps and QCC percentage (QCC-P) and QCC cluster index (QCC-CI) were determined for each sample. Results: We showed that QCCs exist as non-random and heterogeneously distributed clusters within primary breast tumors. In addition, these QCC clusters persist after treatment with multi-agent, multi-cycle, neoadjuvant chemotherapy in both residual primary tumors and nodal and distant metastases in patients with triple negative breast cancer. Conclusions: These first-in-human data potentially qualify AKT1(low) quiescent cancer cells as a non-genetic cell state that persists after neoadjuvant chemotherapy in triple negative breast cancer patients and warrants further study

Literature Lab: a method of automated literature interrogation to infer biology from microarray analysis

<p>Abstract</p> <p>Background</p> <p>The biomedical literature is a rich source of associative information but too vast for complete manual review. We have developed an automated method of literature interrogation called "Literature Lab" that identifies and ranks associations existing in the literature between gene sets, such as those derived from microarray experiments, and curated sets of key terms (i.e. pathway names, medical subject heading (MeSH) terms, etc).</p> <p>Results</p> <p>Literature Lab was developed using differentially expressed gene sets from three previously published cancer experiments and tested on a fourth, novel gene set. When applied to the genesets from the published data including an <it>in vitro </it>experiment, an <it>in vivo </it>mouse experiment, and an experiment with human tumor samples, Literature Lab correctly identified known biological processes occurring within each experiment. When applied to a novel set of genes differentially expressed between locally invasive and metastatic prostate cancer, Literature Lab identified a strong association between the pathway term "FOSB" and genes with increased expression in metastatic prostate cancer. Immunohistochemistry subsequently confirmed increased nuclear FOSB staining in metastatic compared to locally invasive prostate cancers.</p> <p>Conclusion</p> <p>This work demonstrates that Literature Lab can discover key biological processes by identifying meritorious associations between experimentally derived gene sets and key terms within the biomedical literature.</p