Prolyl endopeptidase inhibitor Y-29794 blocks the IRS1-AKT-mTORC1 pathway and inhibits survival and in vivo tumor growth of triple-negative breast cancer.

Prolyl endopeptidase (PREP), also known as prolyl oligopeptidase (POP), is an enzyme that cleaves short peptides (<30 amino acids in length) on the C-terminal side of proline. PREP is highly expressed in multiple carcinomas and is a potential target for cancer therapy. A potent inhibitor of PREP, Y-29794, causes long-lasting inhibition of PREP in mouse tissues. However, there are no reports on Y-29794 effects on cancer cell and tumor proliferation. Using cell line models of aggressive triple-negative breast cancer (TNBC), we show here that Y-29794 inhibited proliferation and induced death in multiple TNBC cell lines. Cell death induced by Y-29794 coincided with inhibition of the IRS1-AKT-mTORC1 survival signaling pathway, although stable depletion of PREP alone was not sufficient to reduce IRS1-AKT-mTORC1 signaling or induce death. These results suggest that Y-29794 elicits its cancer cell killing effect by targeting other mechanisms in addition to PREP. Importantly, Y-29794 inhibited tumor growth when tested in xenograft models of TNBC in mice. Induction of cell death in culture and inhibition of xenograft tumor growth support the potential utility of Y-29794 or its derivatives as a treatment option for TNBC tumors.

Critical roles for nitric oxide and ERK in the completion of prosurvival autophagy in 4OHTAM-treated estrogen receptor-positive breast cancer cells.

Autophagy is a mechanism of tamoxifen (TAM) resistance in ER-positive (ER+) breast cancer cells. In this study, we showed in ER+ MCF7 cells that 4-hydroxytamoxifen (4OHTAM) induced cellular nitric oxide (NO) that negatively regulates cellular superoxide (O2-) and cytotoxicity. 4OHTAM stimulated LC3 lipidation and formation of monodansylcadaverine (MDC)-labeled autophagic vesicles dependent on O2-. Depletion of NO increased O2- and LC3 lipidation, yet reduced formation of MDC-labeled autophagic vesicles. Instead, NO-depleted cells formed remarkably large vacuoles with rims decorated by LC3. The vacuoles were not labeled by MDC or the acidic lysosome-specific fluorescence dye acridine orange (AO). The vacuoles were increased by the late stage autophagy inhibitor chloroquine, which also increased LC3 lipidation. These results suggest NO is required for proper autophagic vesicle formation or maturation at a step after LC3 lipidation. In addition, 4OHTAM induced O2--dependent activation of ERK, inhibition of which destabilized lysosomes/autolysosomes upon 4OHTAM treatment and together with depletion of NO led to necrotic cell death. These results suggest an essential role for endogenous NO and ERK activation in the completion of pro-survival autophagy.

The prolyl peptidases PRCP/PREP regulate IRS-1 stability critical for rapamycin-induced feedback activation of PI3K and AKT.

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT)/mammalian target of rapamycin (mTOR) pathway conveys signals from receptor tyrosine kinases (RTKs) to regulate cell metabolism, proliferation, survival, and motility. Previously we found that prolylcarboxypeptidase (PRCP) regulate proliferation and survival in breast cancer cells. In this study, we found that PRCP and the related family member prolylendopeptidase (PREP) are essential for proliferation and survival of pancreatic cancer cells. Depletion/inhibition of PRCP and PREP-induced serine phosphorylation and degradation of IRS-1, leading to inactivation of the cellular PI3K and AKT. Notably, depletion/inhibition of PRCP/PREP destabilized IRS-1 in the cells treated with rapamycin, blocking the feedback activation PI3K/AKT. Consequently, inhibition of PRCP/PREP enhanced rapamycin-induced cytotoxicity. Thus, we have identified PRCP and PREP as a stabilizer of IRS-1 which is critical for PI3K/AKT/mTOR signaling in pancreatic cancer cells.

Methylation of death-associated protein kinase is associated with cetuximab and erlotinib resistance.

Anti-EGFR therapy is among the most promising molecular targeted therapies against cancer developed in the past decade. However, drug resistance eventually arises in most, if not all, treated patients. Emerging evidence has linked epigenetic changes, such as DNA methylation at CpG islands, to the development of resistance to multiple anticancer drugs. In addition, genes that are differentially methylated have increasingly been appreciated as a source of clinically relevant biomarker candidates. To identify genes that are specifically methylated during the evolution of resistance to anti-EGFR therapeutic agents, we performed a methylation-specific array containing a panel of 56 genes that are commonly known to be regulated through promoter methylation in two parental non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) cell lines and their resistant derivatives to either erlotinib or cetuximab. We found that death-associated protein kinase (DAPK) was hypermethylated in drug-resistant derivatives generated from both parental cell lines. Restoration of DAPK into the resistant NSCLC cells by stable transfection re-sensitized the cells to both erlotinib and cetuximab. Conversely, siRNA-mediated knockdown of DAPK induced resistance in the parental sensitive cells. These results demonstrate that DAPK plays important roles in both cetuximab and erlotinib resistance, and that gene silencing through promoter methylation is one of the key mechanisms of developed resistance to anti-EGFR therapeutic agents. In conclusion, DAPK could be a novel target to overcome resistance to anti-EGFR agents to improve the therapeutic benefit, and further evaluation of DAPK methylation as a potential biomarker of drug response is needed.

DNA methylation as clinically useful biomarkers-light at the end of the tunnel.

A recent expansion of our knowledge about epigenetic changes strongly suggests that epigenetic rather than genetic features better reflect disease development, and consequently, can become more conclusive biomarkers for the detection and diagnosis of different diseases. In this paper we will concentrate on the current advances in DNA methylation studies that demonstrate a direct link between abnormal DNA methylation and a disease. This link can be used to develop diagnostic biomarkers that will precisely identify a particular disease. It also appears that disease-specific DNA methylation patterns undergo unique changes in response to treatment with a particular drug, thus raising the possibility of DNA methylation-based biomarkers for the monitoring of treatment efficacy, for prediction of response to treatment, and for the prognosis of outcome. While biomarkers for oncology are the most obvious applications, other fields of medicine are likely to benefit as well. This potential is demonstrated by DNA methylation-based biomarkers for neurological and psychiatric diseases. A special requirement for a biomarker is the possibility of longitudinal testing. In this regard cell-free circulating DNA from blood is especially interesting because it carries methylation markers specific for a particular disease. Although only a few DNA methylation-based biomarkers have attained clinical relevance, the ongoing efforts to decipher disease-specific methylation patterns are likely to produce additional biomarkers for detection, diagnosis, and monitoring of different diseases in the near future.

The MethDet: a technology for biomarker development.

Early detection and diagnosis of a disease in its presymptomatic form has to rely on biomarkers, and multiple laboratories are involved in their development and validation. In this article, we describe our work on a platform technology for a genome-wide analysis of DNA methylation while still using a small amount of sample - a biopsy, a section from a formalin-fixed paraffin-embedded tissue or a small volume (0.4 ml) of plasma from blood. This technology (methylation detection or MethDet) allows genome-wide association studies similar to the analysis of single-nucleotide polymorphisms. Instead of mostly static genetic differences, the MethDet technology tests disease-dependent changes of epigenetic makeup, which is closely related to the gene expression pattern of a disease. The MethDet assay has the capacity to utilize highly fragmented DNA (e.g., cell-free circulating DNA from plasma) to identify disease-specific changes, effects of treatment or changes in the disease activity.

Methylation patterns in cell-free plasma DNA reflect removal of the primary tumor and drug treatment of breast cancer patients.

Abnormal DNA methylation is a feature of most types of cancer, which is reflected in cell-free circulating DNA in plasma. It is, however, unknown whether surgical removal of the tumor and subsequent therapy induces changes in plasma DNA methylation, which can be used to monitor treatment. In this pilot study, methylation in cell-free plasma DNA of 20 breast cancer patients was determined by the previously developed MethDet-56 technique. Samples at three time points were analyzed-before surgery (baseline), after surgery (to evaluate the effects of resection) and after surgery on tamoxifen therapy (to determine the effects of treatment). Methylation patterns of healthy controls were used as a reference for all comparisons. Seven promoters were differentially methylated (p < 0.05) in at least one comparison; three changed after surgery; another one changed after beginning of tamoxifen treatment; and four were differentially methylated in baseline versus combined treatment samples. Increased methylation of PR PROX, MDGI, PAX 5 and RARß2 at baseline (presurgery) diminished toward the healthy controls with the lowest methylation in the combined treatment group. Surgery alone decreased methylation in PAX 5 and RARß2, whereas tamoxifen treatment changed methylation only in the B promoter of ESR1. Methylation patterns in cell-free plasma DNA change after surgery and tamoxifen treatment, most significantly-after combined treatment. The baseline (presurgery) patterns become similar to those of healthy controls, suggesting that methylation patterns in cell-free plasma DNA may be used to monitor treatment.

Prolylcarboxypeptidase regulates proliferation, autophagy, and resistance to 4-hydroxytamoxifen-induced cytotoxicity in estrogen receptor-positive breast cancer cells.

Endocrine therapy with tamoxifen (TAM) significantly improves outcomes for patients with estrogen receptor-positive breast cancer. However, intrinsic (de novo) or acquired resistance to TAM occurs in a significant proportion of treated patients. To identify genes involved in resistance to TAM, we introduced full-length cDNA expression library into estrogen receptor-positive MCF7 cells and exposed them to a cytotoxic dose of 4-hydroxytamoxifen (4OHTAM). Four different library inserts were isolated from surviving clones. Re-introduction of the genes individually into naive MCF7 cells made them resistant to 4OHTAM. Cells overexpressing these genes had an increase in acidic autophagic vacuoles induced by 4OHTAM, suggesting their role in autophagy. One of them, prolylcarboxypeptidase (PRCP), was investigated further. Overexpression of PRCP increased cell proliferation, boosted several established markers of autophagy, including expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA in an ER-α dependent manner, and increased resistance to 4OHTAM. Conversely, knockdown of endogenous PRCP in MCF7 cells increased cell sensitivity to 4OHTAM and at the same time decreased cell proliferation and expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA. Inhibition of enzymatic activity of PRCP enhanced 4OHTAM-induced cytotoxicity in MCF7 cells. Cells with acquired resistance to 4OHTAM exhibited increased PRCP activity, although inhibition of PRCP prevented development of 4OHTAM resistance in parental MCF7 cells and restored response to 4OHTAM in MCF7 cells with acquired resistance to 4OHTAM. Thus, we have for the first time identified PRCP as a resistance factor for 4OHTAM resistance in estrogen receptor-positive breast cancer cells.

DNA methylation as a universal biomarker.

Cell-free circulating DNA carries not only tumor-specific changes in its sequence but also distinctive epigenetic marks, namely DNA methylation, in certain GC-rich fragments. These fragments are usually located within the promoters and first exons of many genes, comprising CpG islands. Analysis of DNA methylation using cell-free circulating DNA can facilitate development of very accurate biomarkers for detection, diagnosis, prediction of response to therapy and prognosis of outcomes. Recent data suggest that benign and inflammatory diseases have very specific methylation patterns within cell-free circulating DNA, which are different from the pattern of a malignant tumor of the same organ. In addition, specific methylation patterns have been detected for cancers of different organs, so a differential diagnosis of site-specific cancer appears feasible. Currently, cancer-related applications dominate the field, although methylation-based biomarkers may also be possible for other diseases, including neurodegenerative and psychiatric disorders.

Methylation profile of circulating plasma DNA in patients with pancreatic cancer.

BACKGROUND AND OBJECTIVES: Detection of pancreatic cancer by blood-based test may improve outcomes. We sought to establish the feasibility of a blood-based detection of pancreatic cancer through multiplexed array-mediated analysis of DNA methylation. METHODS: Methylation was assessed in each plasma sample using a panel of 56 frequently methylated genes. Methylation profiles in patients with ductal cell adenocarcinoma of the pancreas (n = 30) and healthy gender and age-matched controls (n = 30) were compared. Methylation was determined as described previously; a composite biomarker was developed for classification of cancer and normal samples. Sensitivity and specificity of the biomarker were estimated using 25 rounds of fivefold cross-validation. RESULTS: Five promoters were consistently selected for the classifier during cross-validation and comprised the final composite biomarker Five-fold cross-validation results indicate 76% sensitivity and 59% specificity of the biomarker, which included promoters of CCND2, SOCS1, THBS1, PLAU, and VHL. CONCLUSION: Differential methylation profiling of plasma DNA can detect ductal adenocarcinoma of the pancreas with significant accuracy and should be explored further. While additional improvement of biomarkers is necessary, the blood-based biomarker may be already useful as a first-line detection tool.

Array-based multiplex analysis of DNA methylation in breast cancer tissues.

Abnormal DNA methylation is well established for cancer cells, but a methylation-based diagnostic test is yet to be developed. One of the problems is insufficient accuracy of cancer detection in heterogeneous clinical specimens when only a single gene is analyzed. A new technique was developed to produce a multigene methylation signature in each sample, and its potential for selection of informative genes was tested using DNA from formalin-fixed, paraffin-embedded breast cancer tissues. Fifty-six promoters were analyzed in each of 138 clinical specimens by a microarray-based modification of the previously developed technique. Specific methylation signatures were identified for atypical ductal hyperplasia, ductal carcinoma in situ, and invasive ductal carcinoma. Informative promoters selected by Fisher's exact test were used for composite biomarker design using naïve Bayes algorithm. All informative promoters were unmethylated in disease compared with normal tissue. Cross-validation showed 72.4% sensitivity and 74.7% specificity for detection of ductal carcinoma in situ and invasive ductal carcinoma, and 87.5% sensitivity and 95% specificity for detection of atypical ductal hyperplasia. These results indicate that informative cancer-specific methylation signatures can be detected in heterogeneous tissue specimens, suggesting that a diagnostic assay can then be developed.

Biomarkers for early detection of breast cancer: what, when, and where?

Early detection of breast cancer reduces the suffering and cost to society associated with the disease. A sensitive assay to identify biomarkers that can accurately diagnose the onset of breast cancer using non-invasively collected clinical specimens is ideal for early detection. The earlier and more accurate the diagnostic biomarker can predict disease onset, the more valuable it becomes. Here, a brief review of existing and emerging approaches for breast cancer biomarker identification and analysis is presented. Those biomarkers found in biological fluids, blood in particular, apparently hold the best promise for fast development of screening assays. Autoantibodies and abnormal tumor-specific DNA methylation found in cell-free plasma DNA may provide the best opportunity for constructing multiplexed and highly redundant tests, which will be sufficiently specific and sensitive for early detection of breast cancer. It is expected that technologies developed for breast cancer detection will be useful for other types of cancer.

DNA methylation biomarkers of cancer: moving toward clinical application.

While different markers for cancer diagnosis have been known for at least a decade, the systematic search for biomarkers emerged only several years ago. In this article, I will concentrate on DNA methylation as a dynamic and robust platform for the development of cancer-specific biomarkers. Simultaneous analysis of a growing number of independent methylation events can create increasingly more precise and individualized diagnostics. The differential detection of methylated and unmethylated DNA can be accomplished through either chemical modification or digestion with methylation-sensitive restriction enzyme(s). The benefits and potential pitfalls of both these approaches for clinical sample analysis will be addressed.