Remco A. Spanjaard

Short-Chain Fatty Acid Inhibitors of Histone Deacetylases: Promising Anticancer Therapeutics?

Cancer is a disease in which cellular growth regulatory networks are disrupted. Lesions in well-characterized oncogenes and tumor suppressors often contribute to the dysregulation, but recent work has also uncovered the fundamental importance of enzymes that modulate the acetylation status of chromatin to the initiation or progression of cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are known to be involved in physiological cellular processes, such as transcription, cell cycle progression, gene silencing, differentiation, DNA replication, and genotoxic responses, but they are also increasingly being implicated in tumorigenesis. Butyrate is a short-chain fatty acid (SCFA) that acts as a HDAC inhibitor and is being clinically evaluated as an anti-neoplastic therapeutic, primarily because of its ability to impose cell cycle arrest, differentiation, and/or apoptosis in many tumor cell types, and its favorable safety profile in humans. Additionally, HDAC inhibitors could be used in combination with certain established antitumor therapeutics, such as those that target transcription, to augment clinical efficacy, and/or reduce toxicity. The molecular pathways of butyrate and related next-generation synthetic SCFAs in mediating these effects have not been fully elucidated, but HDAC inhibition is associated with regulation of critical cell cycle regulators, such as cyclin D, p21(CIP1/WAF1), and p27(KIP1). It is anticipated that a better understanding of this critical intersection between SCFAs, HDACs, and cell cycle control will lead to the design of novel treatment strategies for neoplasias. This review will summarize some of the recent research in these arenas of HDAC-directed cancer therapy and discuss the potential application of these agents in synergy with current chemotherapeutics.

Pancreatic cancer cells can evade immune surveillance via nonfunctional Fas (APO-1/CD95) receptors and aberrant expression of functional Fas ligand.

BACKGROUNDnThe Fas (APO-1/CD95) receptor/Fas ligand (FasR/FasL) system plays a key role in immune surveillance. We investigated the possibility of a tumor escape mechanism involving the FasR/FasL system in pancreatic cancer cells.nnnMETHODSnFourteen pancreatic cancer cell lines and 3 pancreatic cancer surgical specimens were studied for their expression of FasR and FasL by flow cytometry, immunoblotting, and immunohistochemistry, FasR function was tested with an anti-FasR antibody. FasL function was assessed by coculture assays using pancreatic cancer cells and FasR-sensitive Jurkat T-cells.nnnRESULTSnFasR was expressed in normal pancreas, in 14 of 14 pancreatic cancer cell lines, and in 3 of 3 surgical specimens. However, only 1 of 14 cancer cell lines expressed functional FasR when grown in monolayer, although 3 additional cell lines displayed functional FasR when cultured in suspension. Normal pancreas did not express FasL, whereas 14 of 14 cancer cell lines and 3 of 3 surgical specimens expressed FasL. FasL expressed by pancreatic cancer cells mediated killing of Jurkat T-cells in coculture assays (P < .005).nnnCONCLUSIONSnThese data suggest that pancreatic cancer cells have 2 potential mechanisms of evading Fas-mediated immune surveillance. A nonfunctional FasR renders them resistant to Fas-mediated apoptosis. The aberrant expression of functional FasL allows them to counterattack activated Fas-sensitive T-cells. Alone or in unison, these tumor escape mechanisms may contribute to the malignant and often rapid course of pancreatic cancer disease.

Effects of retinoic acid and sodium butyrate on gene expression, histone acetylation and inhibition of proliferation of melanoma cells

Retinoic acid (RA) induces growth-arrest of many tumor cell lines but it is an ineffective therapeutic against melanoma. We investigated whether the histone deacetylase (HDAC)-inhibitor sodium butyrate (BUT) can restore or potentiate the RA-response of RA-resistant human A375, and RA-responsive S91 murine melanoma cells. BUT induced expression of RARbeta and p21(waf1/cip1) mRNA in A375 cells but in S91 cells only p21(waf1/cip1) was induced. RA and BUT synergistically activated transcription of an RA-dependent reporter gene in S91, but not A375 cells. BUT increased histone H4-acetylation in both cell types. RA potentiated BUT-mediated inhibition of S91 cell proliferation, whereas A375 cells remained largely resistant to both compounds. HDAC-inhibitors may enhance the activity of RA on RA-responsive melanoma cells.

Retinoic acid receptor-independent mechanism of apoptosis of melanoma cells by the retinoid CD437 (AHPN)

Retinoic acid (RA) induces differentiation of S91 melanoma cells through activation of RA receptor (RAR)γ without affecting cell viability. The novel RARγ-agonist CD437 (AHPN), however, also induces concomitant apoptosis through an unknown mechanism which was investigated here. By utilizing DNA microarray analysis, five apoptosis-associated, CD437-induced transcripts (CITs) were identified. Interestingly, all CITs are also regulated by p53 in a DNA damage response, and consistent with this interpretation, CD437 was found to cause DNA adduct-formation. However, p53 is not required for CD437-dependent regulation of CITs. Among this set of genes, induction of p21WAF1/CIP1 is likely to be responsible for early S-phase growth-arrest of CD437-treated cells, whereas ei24 is a critical mediator of CD437-induced apoptosis in S91 cells. These data suggest an RAR-independent mechanism in which CD437 causes DNA adduct-formation, resulting in induction of a p53-independent DNA damage response, and subsequent growth-arrest and apoptosis. CD437-mediated DNA adduct-formation may also explain its apoptotic effects in other cell types.

The apoptotic action of the retinoid CD437/AHPN: Diverse effects, common basis

Retinoids, such as all-TRANS-retinoic acid (RA), have found applications in several different types of (cancer) therapies. The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437 or AHPN), an RA receptor (RAR)gamma agonist, not only induces RARgamma-dependent differentiation, but in contrast to RA, it also induces RARgamma-independent apoptosis in many tumor cells. This observation makes this and similar new retinoids very interesting from a clinical perspective. Several genes have been associated with CD437/AHPN-mediated apoptosis, but the multiple activities of this compound and the apparent cell-type-specific responses to treatment have made it difficult to discern a common biochemical basis for the mechanism of its apoptotic action. In this brief review, we present a model which links all CD437/AHPN-associated apoptotic effects. CD437/AHPN rapidly induces DNA adduct formation through an as-yet unknown reaction which is independent of cell type. This is followed by a cell-type-specific, largely p53-independent DNA damage response which can result in engagement of multiple cell death pathways and activation of caspases as a common endpoint. At the same time, the RARgamma-dependent pathway leads to regulation of differentiation-associated, cell-type-specific genes. CD437/AHPN, with its simultaneous differentiation and apoptosis-inducing activities, is a good prototype for new drugs which may be clinically more efficacious than those with a single activity.

Telomerase inhibitors and 'T-oligo' as cancer therapeutics: contrasting molecular mechanisms of cytotoxicity.

Telomeres, the specialized structures that comprise the ends of chromosomes, form a closed structure, or t-loop, that is important in preventing genomic instability. Forced modulation of this structure, via overexpression of a dominant-negative form of telomere repeat binding factor 2, a protein critical for maintaining t-loop structure, for example, can result in the activation of DNA-damage responses, and ultimately cellular senescence or apoptosis. This response is also seen in normal somatic cells, where telomeres steadily decrease in length as cellular proliferation occurs owing to inefficient replication of terminal telomeric DNA. When telomere length becomes critically short, t-loop structure is compromised, and the cell undergoes senescence. Telomerase, the enzyme responsible for telomere length maintenance, is overexpressed in a majority of cancers. Its lack of expression in most normal somatic cells makes it an attractive target in designing cancer therapeutics. Compounds currently under development that seek to inhibit hTERT, the reverse transcriptase component of telomerase, include nucleoside analogs and the small molecule BIBR1532. Compounds inhibiting the RNA component of telomerase, hTERC, include peptide nucleic acids, 2–5A antisense oligonucleotides, and N3′–P5′ thio-phosphoramidates. Recently, an oligonucleotide sharing sequence homology with terminal telomeric DNA, termed ‘T-oligo’, has shown cytotoxic effects in multiple cancers in culture and animal models. Independent of telomerase function, T-oligo is thought to mimic the DNA-damage response a cell normally experiences when the telomere t-loop structure becomes dysfunctional. In this review, the molecular mechanisms attributed to telomerase inhibitors and T-oligo, as well as their potential as cancer therapeutics, are discussed.

Loss of putative tumor suppressor EI24/PIG8 confers resistance to etoposide.

Expression of p53‐target gene EI24/PIG8 is lost in invasive breast cancers, suggesting that EI24/PIG8 is a tumor suppressor that prevents tumor spreading, and partially mediates p53‐attributed tumor suppressor activity. EI24/PIG8 also has pro‐apoptotic activity indicating that loss of EI24/PIG8 may modulate sensitivity to chemotherapy. Here it is demonstrated that suppression of EI24/PIG8 in fibroblasts and breast cancer cells significantly inhibits the apoptotic response to etoposide treatment. These findings suggest that loss of EI24/PIG8 contributes significantly to resistance of cells to chemotherapeutic agents that function through p53, and identify the EI24/PIG8 status as a potentially new prognostic marker of chemotherapy responsiveness.

Tumor necrosis factor receptor superfamily member TROY is a novel melanoma biomarker and potential therapeutic target

Incidence of melanoma continues to rise, and a better understanding of its genetics will be critical to improve diagnosis and develop new treatments. Here, we search for novel melanoma‐specific genes that may serve as biomarkers and therapeutic targets by using an in vitro genetic screen. One identified cDNA encoded TROY, a member of the tumor necrosis factor receptor superfamily (TNFRSF). TROY is widely expressed during embryogenesis, but in adults expression is restricted to hair follicles and brain. However, TROY had never been associated with melanoma, and it was selected for further study. First we show that expression in melanoma is specific by semiquantitative RT‐PCR analysis of a large panel of established tumor cell lines. Next, specificity of expression was evaluated by immunohistochemistry analysis of primary cell cultures and patient tissues. TROY is expressed in 2/2 primary melanoma cells and 45/45 melanoma tissue samples (p < 0.0001). With the exception of sebaceous glands, TROY is not expressed in normal skin biopsies (p < 0.0001) or primary skin cell cultures that contain keratinocytes and epidermal melanocytes, nor is it expressed in other skin tumor cells (p < 0.0001). Finally, we show that TROY regulates melanoma growth, because replication of melanoma cells with reduced TROY levels through treatment with short‐interfering RNA was significantly decreased relative to control cells (p < 0.004). In summary, TROY is the first TNFRSF member that is a biomarker for melanoma. TROY also presents a potentially novel cell surface signaling target for inhibitors, cell and/or antibody‐based immunotherapies.

Identification of LTR‐specific small non‐coding RNA in FeLV infected cells

The U3‐LTR region of leukemia viruses transactivates cancer‐related signaling pathways through the production of a non‐coding RNA transcript although the role of this transcript in virus infection remains unknown. In this study we demonstrate for the first time that an long terminal repeat (LTR)‐specific small non‐coding RNA is produced from a feline leukemia virus (FeLV)‐infected feline cell line. RNA cloning identified this as a 104 base transcript that originates from the U3‐LTR region. We also demonstrate that in in vitro assays this LTR‐RNA transcript activates NFκB signaling. Taken together, our findings suggest a possible role for this LTR transcript in FeLV pathogenesis.

Differential Effects of Short-Chain Fatty Acids on Head and Neck Squamous Carcinoma Cells

Objectives/Hypothesis Head and neck squamous cell carcinoma (HNSCC) is a major cause of mortality. Despite advances in therapeutic modalities, recurrences and second primaries are commonly observed. Biological agents that can suppress growth of tumors that are otherwise difficult to treat are greatly needed. The present study examined the effects of short‐chain fatty acids on HNSCC cell lines.