Uncontrolled cell proliferation is the hallmark of cancer, and tumor cells have typically acquired damage to genes that directly regulate their cell cycles. Genetic alterations affecting p16(INK4a) and cyclin D1, proteins that govern phosphorylation of the retinoblastoma protein (RB) and control exit from the G1 phase of the cell cycle, are so frequent in human cancers that inactivation of this pathway may well be necessary for tumor development. Like the tumor suppressor protein p53, components of this "RB pathway," although not essential for the cell cycle per se, may participate in checkpoint functions that regulate homeostatic tissue renewal throughout life.

http://science.sciencemag.org/content/sci/274/5293/1672.full.pdf

Cancer Cell Cycles

Patterns of DNA methylation and chromatin structure are profoundly altered in neoplasia and include genome-wide losses of, and regional gains in, DNA methylation. The recent explosion in our knowledge of how chromatin organization modulates gene transcription has further highlighted the importance of epigenetic mechanisms in the initiation and progression of human cancer. These epigenetic changes -- in particular, aberrant promoter hypermethylation that is associated with inappropriate gene silencing -- affect virtually every step in tumour progression. In this review, we discuss these epigenetic events and the molecular alterations that might cause them and/or underlie altered gene expression in cancer.

The fundamental role of epigenetic events in cancer

Induction of apoptotic program in cell-free extracts : requirement for datp and cytochrome c

In a cell-free apoptosis system, mitochondria spontaneously released cytochrome c, which activated DEVD-specific caspases, leading to fodrin cleavage and apoptotic nuclear morphology. Bcl-2 acted in situ on mitochondria to prevent the release of cytochrome c and thus caspase activation. During apoptosis in intact cells, cytochrome c translocation was similarly blocked by Bcl-2 but not by a caspase inhibitor, zVAD-fmk. In vitro, exogenous cytochrome c bypassed the inhibitory effect of Bcl-2. Cytochrome c release was unaccompanied by changes in mitochondrial membrane potential. Thus, Bcl-2 acts to inhibit cytochrome c translocation, thereby blocking caspase activation and the apoptotic process.

https://science.sciencemag.org/content/sci/275/5303/1132.full.pdf

The Release of Cytochrome c from Mitochondria: A Primary Site for Bcl-2 Regulation of Apoptosis

Apoptosis is a major form of cell death, characterized initially by a series of stereotypic morphological changes. In the nematode Caenorhabditis elegans, the gene ced-3 encodes a protein required for developmental cell death. Since the recognition that CED-3 has sequence identity with the mammalian cysteine protease interleukin-1 beta-converting enzyme (ICE), a family of at least 10 related cysteine proteases has been identified. These proteins are characterized by almost absolute specificity for aspartic acid in the P1 position. All the caspases (ICE-like proteases) contain a conserved QACXG (where X is R, Q or G) pentapeptide active-site motif. Capases are synthesized as inactive proenzymes comprising an N-terminal peptide (prodomain) together with one large and one small subunit. The crystal structures of both caspase-1 and caspase-3 show that the active enzyme is a heterotetramer, containing two small and two large subunits. Activation of caspases during apoptosis results in the cleavage of critical cellular substrates, including poly(ADP-ribose) polymerase and lamins, so precipitating the dramatic morphological changes of apoptosis. Apoptosis induced by CD95 (Fas/APO-1) and tumour necrosis factor activates caspase-8 (MACH/FLICE/Mch5), which contains an N-terminus with FADD (Fas-associating protein with death domain)-like death effector domains, so providing a direct link between cell death receptors and the caspases. The importance of caspase prodomains in the regulation of apoptosis is further highlighted by the recognition of adapter molecules, such as RAIDD [receptor-interacting protein (RIP)-associated ICH-1/CED-3-homologous protein with a death domain]/CRADD (caspase and RIP adapter with death domain), which binds to the prodomain of caspase-2 and recruits it to the signalling complex. Cells undergoing apoptosis following triggering of death receptors execute the death programme by activating a hierarchy of caspases, with caspase-8 and possibly caspase-10 being at or near the apex of this apoptotic cascade.

/pdf/caspases-the-executioners-of-apoptosis-2w1nis6q3n.pdf

Caspases: the executioners of apoptosis

DNA methylation is a central epigenetic process involved in ­establishing normal cellular gene expression patterns and genome homeostasis. Aberrations in DNA methylation, leading to abnormal gene expression patterns, have now been linked to many human diseases, and are a nearly universal feature of human cancers. Because these DNA methylation changes can be stably transmitted during clonal outgrowth of cancer cells, they can carry the same importance as mutations in the initiation and progression of human cancers. Such somatic DNA methylation changes often occur earlier and more frequently than genome mutations during ­carcinogenesis, and have therefore provided a wealth of targets for translational opportunities in cancer biomarkers for diagnosis and risk stratification. Additionally, since these DNA methylation changes are epigenetic processes that are enzymatically mediated and do not alter the underlying DNA sequence, they can potentially be reversed by pharmacological inhibition of the epigenetic machinery, providing opportunities for cancer therapy. Therefore, understanding the genome-wide patterns of DNA methylation in normal and cancer cells has become of primary interest in cancer research. In this chapter, we will first provide an overview of DNA methylation as an epigenetic process in normal physiology and in carcinogenesis. Then we will describe some of the current and up-coming technologies used in analyzing DNA methylation patterns at a genome-wide level, and consider the strengths and limitations of each of these approaches.

Genome-Wide DNA Methylation Analysis in Cancer Research

3149 Intake of vegetables, particularly cruciferous vegetables, reduces prostate cancer risks.1 Sulforaphane, an isothiocyanate present in cruciferous vegetables, may protect against cancer development by inducing the expression of carcinogen-detoxification enzymes, such as glutathione S- transferases (GSTs).2,3 Upon consumption of glucosinolate compounds in cruciferous vegetables, isothiocyanates can be released during chewing via exposure of the glucosinolates to the plant enzyme myrosinase (thioglucoside glucohydrolase). In order to test whether sulforaphane and other isothiocyanates in broccoli sprouts can elevate the expression of chemoprotective enzymes in prostate tissues, both purified sulforaphane and a hot water extract of broccoli sprouts (broccoli sprout tea) were administered to adult male Fisher 344 rats, prone to develop prostate cancer when exposed to heterocyclic amine carcinogens,4 by gastric gavage. To monitor carcinogen-detoxification enzyme expression, RNA- and protein-containing extracts were prepared from liver, intestine, spleen and prostate tissues recovered 48 hours after treatment. GST activity was assessed using a CDNB assay.5 The expression of mRNAs encoding other chemoprotective enzymes was evaluated via hybridization to Affymetrix expression arrays. The results were that while both sulforaphane and broccoli sprout tea triggered increases in GST activity in the liver and the intestine, in a dose dependent manor, only the broccoli sprout tea caused increased GST activity in the prostate. mRNA expression profiling confirmed an induction of transcripts encoding chemoprotective enzymes in the different tissues. These findings suggest that consumption of cruciferous vegetables, containing glucosinolates, may effectively augment carcinogen defenses both in major metabolic organs, such as the liver and the intestine, as well as in cancer-prone organs, such as the prostate. References: 1Cohen JH et al. J. Natl. Cancer Inst. 92:61-68 (2000). 2Fahey JW et al. Proc. Natl. Acad. Sci., 94:10367-10372 (1997). 3Talalay P et al. Adv Enzyme Regul., 43:121-134 (2003). Shirai T et al. Cancer Res. 57:195-198 (1997). 5Habig WH et al. J. Biol. Chem. 249:7130-7139 (1974).

Hot water extracts from broccoli sprouts trigger induction of carcinogen-detoxification enzymes in prostate tissues.

Aberrant DNA methylation of gene promoters can be involved in silencing tumor suppressor genes in cancer. The protein ubiquitin-like containing PHD and ring finger domains 1 (UHRF1) is an essential component in the cellular machinery for DNA methylation maintenance and can couple DNA methylation and other epigenetic histone modifications. UHRF1 is thought to bind newly synthesized hemimethylated DNA and recruit DNA methyltransferases to copy the methylation pattern to the daughter DNA strand. Interfering with the ability of UHRF1 to bind methylated DNA can lead to the re-expression of epigenetically silenced genes including tumor suppressors, which positions UHRF1 as a target for anticancer drug development. We have created a high-throughput, time-resolved fluorescence resonance energy transfer (TR-FRET)-based assay to screen for inhibitors capable of disrupting the interaction between the UHRF1 SRA domain and hemi-methylated DNA. The assay measures the degree of TR-FRET between a 5′-fluorescein (FAM) labeled hairpin oligonucleotide and a terbium-conjugated antibody bound to the 6xHis-tag of the UHRF1_SRA domain polypeptide. In 384-well plate format, the assay achieved a Z9 factor of 0.74. We successfully screened the Library of Pharmacologically Active Compounds (LOPAC) for inhibitors of UHRF1_SRA binding to hemimethlylated DNA, and validated 6 hit compounds in a dose-response series. These include: aurintricarboxylic acid, NF449, 6-hydroxy-DL-DOPA, NF023, PPNDS and mitoxantrone. Additionally, we observed a decrease in methylation one week after treatment of DU145 cells with mitoxantrone at a concentration near its IC50 from the TR-FRET binding assay. This suggests that mitoxantrone, a known DNA topoisomerase II poison and general DNA intercalator, may interfere with methylation maintenance. In summary, we have established a novel method to identify small molecule inhibitors of the UHRF1-methylated DNA interface, and have shown that one hit compound, mitoxantrone, can perturb methylation maintenance near its IC50 for inhibition of SRA domain binding to methylated DNA. Citation Format: David A. Walker, Nicolas Wyhs, Hugh Giovinazzo, Srinivasan Yegnasubramanian, William G. Nelson. Development of a high-throughput screening assay to identify UHRF1 inhibitors via time-resolved fluorescence resonance energy transfer (TR-FRET). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5390. doi:10.1158/1538-7445.AM2014-5390

Abstract 5390: Development of a high-throughput screening assay to identify UHRF1 inhibitors via time-resolved fluorescence resonance energy transfer (TR-FRET)

Among men worldwide, prostate cancer has become the second most frequently diagnosed cancer and the sixth leading cause of cancer death, accounting for 240,890 new diagnoses and 33,720 deaths in the United States alone in 2011 (1, 2). Fortunately, with wide use of prostate cancer screening, early detection, and definitive local therapy, prostate cancer mortality rates have been falling in the United States—as much as 39% or more since 1990 (2). Nonetheless, randomized clinical trials of serum prostate-specific antigen (PSA) testing for prostate cancer screening have suggested that these gains have come at a significant cost: by one estimate, to save a single life from prostate cancer death, 1,410 men need to be screened, and 48 cases of prostate cancer need to be diagnosed and treated (3). On October 11, 2011, the US Preventive Services Task Force issued a draft report on PSA screening, assigning the practice a grade D recommendation and weighing in “against the service” because of “moderate or high certainty that the service has no net benefit or that the harms outweigh the benefits.” The firestorm of controversy triggered by this report highlights (i) the need for individualized approaches to prostate cancer screening at a population scale, so that screening services can be targeted in such a way as to minimize overdiagnosis, and (ii) the need to personalize treatment decisions, so that options like the use of active surveillance in lieu of definitive local therapy can be rationally selected in such a way as to reduce overtreatment (4, 5). In PNAS, Markert et al. (6) attempt to address these needs through the discovery of new molecular biomarkers for aggressive prostate cancer.

https://www.pnas.org/content/pnas/108/52/20861.full.pdf

Profiling prostate cancer

At the dawn of the new millennium, prostate cancer has become a major scourge
for men as they age in the developed world. In the United States, autopsy series
have revealed small prostate cancers in as many as 29% of men between age 30
and 40, and 64% of men between age 60 and 70 (1). With the current widespread
use of serum prostate-specific antigen (PSA) testing and digital rectal examination
in the United States for prostate cancer screening, the lifetime risk of a prostate cancer diagnosis has risen to about one in six, whereas the lifetime risk of death from
prostate cancer is on the order of 1 in 30 (2). Fortunately, since about 1994 or so,
with increased diagnosis and treatment of prostate cancer, age-adjusted prostate
cancer death rates have fallen steadily (2). Whether this trend reflects a benefit
of prostate cancer screening and/or early prostate cancer treatment on prostate
cancer mortality has not been fully resolved (3,4). Nonetheless, while some men
with prostate cancer do not appear to be at high risk for symptomatic or life-threatening prostate cancer progression, others continue to face the threat of death from
the disease.

William G. Nelson

Papers

Genome-Wide DNA Methylation Analysis in Cancer Research

Hot water extracts from broccoli sprouts trigger induction of carcinogen-detoxification enzymes in prostate tissues.

Abstract 5390: Development of a high-throughput screening assay to identify UHRF1 inhibitors via time-resolved fluorescence resonance energy transfer (TR-FRET)

Profiling prostate cancer

The Molecular Pathogenesis and Pathophysiology of Prostate Cancer