Liver-Targeting Class I Selective Histone Deacetylase Inhibitors Potently Suppress Hepatocellular Tumor Growth as Standalone Agents.

Dysfunctions in epigenetic regulation play critical roles in tumor development and progression. Histone deacetylases (HDACs) and histone acetyl transferase (HAT) are functionally opposing epigenetic regulators, which control the expression status of tumor suppressor genes. Upregulation of HDAC activities, which results in silencing of tumor suppressor genes and uncontrolled proliferation, predominates in malignant tumors. Inhibition of the deacetylase activity of HDACs is a clinically validated cancer therapy strategy. However, current HDAC inhibitors (HDACi) have elicited limited therapeutic benefit against solid tumors. Here, we disclosed a class of HDACi that are selective for sub-class I HDACs and preferentially accumulate within the normal liver tissue and orthotopically implanted liver tumors. We observed that these compounds possess exquisite on-target effects evidenced by their induction of dose-dependent histone H4 hyperacetylation without perturbation of tubulin acetylation status and G0/G1 cell cycle arrest. Representative compounds 2 and 3a are relatively non-toxic to mice and robustly suppressed tumor growths in an orthotopic model of HCC as standalone agents. Collectively, our results suggest that these compounds may have therapeutic advantage against HCC relative to the current systemic HDACi. This prospect merits further comprehensive preclinical investigations.

Discovery and mechanisms of host defense to oncogenesis: targeting the ß-defensin-1 peptide as a natural tumor inhibitor.

Human beta-defensin-1 (hBD-1) is one of a number of small cationic host-defense peptides. Besides its well-known broad-spectrum antimicrobial function, hBD-1 has recently been identified as a chromosome 8p tumor-suppressor gene. The role of hBD-1 in modulating the host immune response to oncogenesis, associated with cell signaling and potential therapeutic applications, has become increasingly appreciated over time. In this study, multiple approaches were used to illustrate hBD-1 anti-tumor activities. Results demonstrate that hBD-1 peptide alters human epidermal growth factor receptor 2 (HER2) signal transduction and represses retroviral-mediated transgene expression in cancer cells. Loss of orthologous murine defense-1 (mBD1) in mice enhances nickel sulfate-induced leiomyosarcoma and causes mouse kidney cells to exhibit increased susceptibility to HPV-16 E6/7-induced neoplastic transformation. Furthermore, for the first time, a novel function of the urine-derived hBD-1 peptide was discovered to suppress bladder cancer growth and this may lead to future applications in the treatment of malignancy.

A mitochondrial DNA mutation influences the apoptotic effect of statins on prostate cancer.

BACKGROUND: Statins, 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are currently the most widely used cholesterol-lowering drugs. Previous epidemiological studies have suggested that there may be be an association between statin use and decreased risk of prostate cancer progression. Both inherited and somatic mutations of the mitochondrial genome are linked to prostate cancer. The purpose of this study was to determine if mitochondrial DNA (mtDNA) background and hence mitochondrial biochemistry can modulate the efficiency of statin as an anti-prostate cancer agent. METHODS: Cytoplasmic hybrid (cybrid) cell lines were constructed that contained a prostate cancer nucleus and either wild type or mutant mtDNA derived from a prostate cancer patient with the cytochrome oxidase subunit 1 gene mutation T6124C (Met74Thr). Multiple clones for each genotype were tested. After treating both wild type and mutant cells with increasing concentrations of simvastatin for 72 hr, cell proliferation and apoptosis were analyzed. RESULTS: Simvastatin inhibited both wild type and mutant cell proliferation. However, cells with the T6124C mtDNA mutation were more resistant to drug treatment than the wild type cells. In addition, analysis of caspase 3 assays and multiple proteins involved in cellular apoptosis demonstrated that mutant cells were more resistant to simvastatin treatment-induced apoptosis than wild type control cells. CONCLUSIONS: Simvastatin treatment induced apoptosis in human cybrid prostate cancer cells. The response to drug treatments was different depending on mitochondrial genotype. Therefore, the degree to which statins may affect prostate cancer progression may vary based on an individual's mtDNA background.

An inherited heteroplasmic mutation in mitochondrial gene COI in a patient with prostate cancer alters reactive oxygen, reactive nitrogen and proliferation.

Mitochondrial DNA (mtDNA) mutations have been found in many cancers but the physiological derangements caused by such mutations have remained elusive. Prostate cancer is associated with both inherited and somatic mutations in the cytochrome c oxidase (COI) gene. We present a prostate cancer patient-derived rare heteroplasmic mutation of this gene, part of mitochondrial respiratory complex IV. Functional studies indicate that this mutation leads to the simultaneous decrease in cytochrome oxidation, increase in reactive oxygen, and increased reactive nitrogen. These data suggest that mitochondrial DNA mutations resulting in increased reactive oxygen and reactive nitrogen generation may be involved in prostate cancer biology.

Mitochondrial DNA mutation stimulates prostate cancer growth in bone stromal environment.

BACKGROUND AND OBJECTIVES: Mitochondrial DNA (mtDNA) mutations, inherited and somatically acquired, are common in clinical prostate cancer. We have developed model systems designed to study specific mtDNA mutations in controlled experiments. Because prostate cancer frequently metastasizes to bone we tested the hypothesis that mtDNA mutations enhance prostate cancer growth and survival in the bone microenvironment. METHODS: The pathogenic nucleotide position (np) 8993 mDNA mutation was introduced into PC3 prostate cancer cells by cybrid formation. Wild-type and mutant cybrids were grown as nude mouse subcutaneous xenografts with or without bone stromal cell co-inoculation. Cybrids were also grown in the intratibial space. Tumor growth was assayed by direct tumor measurement and luciferase chemiluminescence. Gene expression was assayed using cDNA microarrays confirmed by real time PCR, western blot analysis and immunohistochemistry. RESULTS: Cybrids with the 8,993 mtDNA mutation grew faster than wild-type cybrids. Further growth acceleration was demonstrated in the bone microenvironment. A 37 gene molecular signature characterized the growth advantage conferred by the mtDNA mutation and bone microenvironment. Two genes of known importance in clinical prostate cancer, FGF1 and FAK, were found to be substantially upregulated only when both mtDNA mutation and bone stromal cell were present. CONCLUSIONS: The ATP6 np 8,993 mtDNA mutation confers a growth advantage to human prostate cancer that is most fully manifest in the bone microenvironment. The identification of specific molecular alterations associated with mtDNA mutation and growth in bone may allow new understanding of prostate cancer bone metastasis.

Human beta-defensin-1, a potential chromosome 8p tumor suppressor: control of transcription and induction of apoptosis in renal cell carcinoma.

Human beta-defensin-1 (hBD-1) is a candidate tumor suppressor gene located on chromosome 8p23. Previously, we showed that cancer-specific loss of hBD-1 was found in 90% of renal clear cell carcinomas and in 82% of prostate cancers. To investigate the possible mechanisms of decreased gene expression and determine the function of hBD-1 protein in urological cancers, we sequenced hBD-1 gene coding regions in prostatic and renal cancer samples. We then analyzed the frequency distribution of promoter polymorphisms and determined the effect of these base changes on transcriptional activity of the hBD-1 promoter. A polymorphism at -688 bases upstream of the ATG start codon affects hBD-1 promoter activity, leading to a rate of reporter gene transcription that is 40% to 50% lower than the wild-type sequence when tested in either DU145 or TSU-Pr1 cell lines. In addition, a polymorphism at -44 bases was shown to enhance transcription up to 2.3 times more than the wild-type sequence in the same cell lines. In addition, three novel hBD-1 promoter mutations were found in renal and prostate cancer clinical samples. An iso-5-aza-2'-deoxycytidine treatment was effective in transcription up-regulation in DU145, suggesting a possible upstream methylation-dependent effect. Synthetic hBD-1 peptide inhibited bladder cancer cell TSU-Pr1 proliferation. Overexpression of the hBD-1 gene in renal cancer cells SW156 resulted in caspase-3-mediated apoptosis. These data support the hypothesis that hBD-1 is a potential tumor suppressor gene for urological cancers. Promoter point mutations may be responsible for cancer-specific loss of hDB-1 expression.

Automated sequencing of complete mitochondrial genomes from laser-capture microdissected samples.

Mitochondrial DNA mutations have been related to both aging and a variety of diseases such as cancer. Due to the relatively small size of the genome (16 kb) and with the use of automated DNA sequencing, the entire genome can be sequenced from clinical specimens in days. We present a reliable approach to complete mitochondrial genome sequencing from laser-capture microdissected human clinical cancer specimens that overcome the inherent limitations of relatively small tissue samples and partial DNA degradation, which are unavoidable when laser-capture microdissection is used to attain pure populations of cells from heterogeneous tissues obtained from surgical procedures. The acquisition of sufficient template combined with a standard set of 18 pairs of PCR primers allows for the efficient amplification of the genome. Subsequent single-stranded amplification is performed using 36 sequencing primers, and samples are run on an ABI PRISM 3100 Genetic Analyzer. The use of this procedure should allow even investigators with little experience sequencing from clinical specimens success in complete mitochondrial genome sequencing.

Cancer-specific loss of beta-defensin 1 in renal and prostatic carcinomas.

In a previous large-scale gene expression profiling study of renal epithelial neoplasms, human beta-defensin-1 (DEFB1) was found to be significantly down-regulated in conventional clear cell (renal) carcinoma. We have now completed an expanded expression analysis of this gene. We performed immunohistochemical analysis for the DEFB1 protein in clinical specimens of both renal cell carcinoma and prostate cancer. In a subset of prostate cancers, we performed laser capture microdissection and RT-PCR to correlate mRNA levels with protein levels. Overall, 82% of prostate cancers exhibit either complete loss of protein expression or only minimal expression, whereas the adjacent benign epithelium retained expression in all cases. Similarly, 90% of renal cell carcinomas show cancer-specific loss of DEFB1 protein. In the prostate cancer subset analysis, mRNA levels correlate with protein levels. We have thus demonstrated the cancer-specific down-regulation of DEFB1 in a large sample of prostatic and renal carcinomas and validated one of the key findings of previous cancer gene profiling studies of prostatic and renal neoplasia.