Claudia A. Benavente

A novel retinoblastoma therapy from genomic and epigenetic analyses

Retinoblastoma is an aggressive childhood cancer of the developing retina that is initiated by the biallelic loss of RB1. Tumours progress very quickly following RB1 inactivation but the underlying mechanism is not known. Here we show that the retinoblastoma genome is stable, but that multiple cancer pathways can be epigenetically deregulated. To identify the mutations that cooperate with RB1 loss, we performed whole-genome sequencing of retinoblastomas. The overall mutational rate was very low; RB1 was the only known cancer gene mutated. We then evaluated the role of RB1 in genome stability and considered non-genetic mechanisms of cancer pathway deregulation. For example, the proto-oncogene SYK is upregulated in retinoblastoma and is required for tumour cell survival. Targeting SYK with a small-molecule inhibitor induced retinoblastoma tumour cell death in vitro and in vivo. Thus, retinoblastomas may develop quickly as a result of the epigenetic deregulation of key cancer pathways as a direct or indirect result of RB1 loss.

Targeting the DNA Repair Pathway in Ewing Sarcoma

Ewing sarcoma (EWS) is a tumor of the bone and soft tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using three different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice.

Genetics and epigenetics of human retinoblastoma.

Retinoblastoma is a pediatric tumor of the developing retina from which the genetic basis for cancer development was first described. Inactivation of both copies of the RB1 gene is the predominant initiating genetic lesion in retinoblastoma and is rate limiting for tumorigenesis. Recent whole-genome sequencing of retinoblastoma uncovered a tumor that had no coding-region mutations or focal chromosomal lesions other than in the RB1 gene, shifting the paradigm in the field. The retinoblastoma genome can be very stable; therefore, epigenetic deregulation of tumor-promoting pathways is required for tumorigenesis. This review highlights the genetic and epigenetic changes in retinoblastoma that have been reported, with special emphasis on recent whole-genome sequencing and epigenetic analyses that have identified novel candidate genes as potential therapeutic targets.

Subcellular distribution and mitogenic effect of basic fibroblast growth factor in mesenchymal uncommitted stem cells.

Uncommitted mesenchymal stem cells (MSC), upon commitment and differentiation give rise to several mature mesenchymal lineages. Although the involvement of specific growth factors, including FGF2, in the development of committed MSC is known, the effect of FGF2 on uncommitted progenitors remains unclear. We have analyzed on a comparative basis, the subcellular distribution and mitogenic effect of FGF2 in committed and uncommitted MSC prepared from human bone marrow. Indirect immunofluorescence studies showed strong nuclear FGF2 staining in both progenitors; however, cytoplasmic staining was only detected in committed cells. Western blot analysis revealed the presence of 22.5 and 21-22 kDa forms of FGF2 in the nucleus of both progenitors; however, their relative content was higher in uncommitted than in committed cells. Exogenous FGF2 stimulated proliferation and sustained quiescence in committed and uncommitted cells, respectively. These results show that both type of progenitors, apart from morphological and proliferative differences, display specific patterns of response to FGF2.

Effects of Niacin Restriction on Sirtuin and PARP Responses to Photodamage in Human Skin

Sirtuins (SIRTs) and poly(ADP-ribose) polymerases (PARPs), NAD+-dependent enzymes, link cellular energy status with responses to environmental stresses. Skin is frequently exposed to the DNA damaging effects of UV irradiation, a known etiology in skin cancer. Thus, understanding the defense mechanisms in response to UV, including the role of SIRTs and PARPs, may be important in developing skin cancer prevention strategies. Here, we report expression of the seven SIRT family members in human skin. SIRTs gene expressions are progressively upregulated in A431 epidermoid carcinoma cells (SIRTs1 and 3), actinic keratoses (SIRTs 2, 3, 5, 6, and 7) and squamous cell carcinoma (SIRTs 1–7). Photodamage induces dynamic changes in SIRT expression with upregulation of both SIRT1 and SIRT4 mRNAs. Specific losses of SIRT proteins occur early after photodamage followed by accumulation later, especially for SIRT4. Niacin restriction, which decreases NAD+, the sirtuin substrate, results in an increase in acetylated proteins, upregulation of SIRTs 2 and 4, increased inherent DNA damage, alterations in SIRT responses to photodamage, abrogation of PARP activation following photodamage, and increased sensitivity to photodamage that is completely reversed by repleting niacin. These data support the hypothesis that SIRTs and PARPs play important roles in resistance to photodamage and identify specific SIRTs that respond to photodamage and may be targets for skin cancer prevention.

NAD in skin: therapeutic approaches for niacin.

The maintenance and regulation of cellular NAD(P)(H) content and its influence on cell function involves many metabolic pathways, some of which remain poorly understood. Niacin deficiency in humans, which leads to low NAD status, causes sun sensitivity in skin, indicative of deficiencies in responding to UV damage. Animal models of niacin deficiency demonstrate genomic instability and increased cancer development in sensitive tissues including skin. Cell culture models of niacin deficiency have allowed the identification of NAD-dependent signaling events critical in early skin carcinogenesis. Niacin restriction in immortalized keratinocytes leads to an increased expression and activity of NADPH oxidase resulting in an accumulation of ROS, providing a potential survival mechanism as has been shown to occur in cancer cells. Niacin deficient keratinocytes are more sensitive to photodamage, as both poly(ADP-ribose) polymerases and Sirtuins are inhibited by the unavailability of their substrate, NAD+, leading to unrepaired DNA damage upon photodamage and a subsequent increase in cell death. Furthermore, the identification of the nicotinic acid receptor in human skin keratinocytes provides a further link to niacins role as a potential skin cancer prevention agent and suggests the nicotinic acid receptor as a potential target for skin cancer prevention agents. The new roles for niacin as a modulator of differentiation and photo-immune suppression and niacin status as a critical resistance factor for UV damaged skin cells are reviewed here.

Nicotinic Acid Receptor Abnormalities in Human Skin Cancer: Implications for a Role in Epidermal Differentiation

Background Chronic UV skin exposure leads to epidermal differentiation defects in humans that can be largely restored by pharmacological doses of nicotinic acid. Nicotinic acid has been identified as a ligand for the human G-protein-coupled receptors GPR109A and GPR109B that signal through Gi-mediated inhibition of adenylyl cyclase. We have examined the expression, cellular distribution, and functionality of GPR109A/B in human skin and skin derived epidermal cells. Results Nicotinic acid increases epidermal differentiation in photodamaged human skin as judged by the terminal differentiation markers caspase 14 and filaggrin. Both GPR109A and GPR109B genes are transcribed in human skin and in epidermal keratinocytes, but expression in dermal fibroblasts is below limits of detection. Receptor transcripts are greatly over-expressed in squamous cell cancers. Receptor protein in normal skin is prominent from the basal through granular layers of the epidermis, with cellular localization more dispersive in the basal layer but predominantly localized at the plasma membrane in more differentiated epidermal layers. In normal human primary and immortalized keratinocytes, nicotinic acid receptors show plasma membrane localization and functional Gi-mediated signaling. In contrast, in a squamous cell carcinoma derived cell line, receptor protein shows a more diffuse cellular localization and the receptors are nearly non-functional. Conclusions The results of these studies justify future genetic and pharmacological intervention studies to define possible specific role(s) of nicotinic acid receptors in human skin homeostasis.

Brg1 coordinates multiple processes during retinogenesis and is a tumor suppressor in retinoblastoma.

Retinal development requires precise temporal and spatial coordination of cell cycle exit, cell fate specification, cell migration and differentiation. When this process is disrupted, retinoblastoma, a developmental tumor of the retina, can form. Epigenetic modulators are central to precisely coordinating developmental events, and many epigenetic processes have been implicated in cancer. Studying epigenetic mechanisms in development is challenging because they often regulate multiple cellular processes; therefore, elucidating the primary molecular mechanisms involved can be difficult. Here we explore the role of Brg1 (Smarca4) in retinal development and retinoblastoma in mice using molecular and cellular approaches. Brg1 was found to regulate retinal size by controlling cell cycle length, cell cycle exit and cell survival during development. Brg1 was not required for cell fate specification but was required for photoreceptor differentiation and cell adhesion/polarity programs that contribute to proper retinal lamination during development. The combination of defective cell differentiation and lamination led to retinal degeneration in Brg1-deficient retinae. Despite the hypocellularity, premature cell cycle exit, increased cell death and extended cell cycle length, retinal progenitor cells persisted in Brg1-deficient retinae, making them more susceptible to retinoblastoma. ChIP-Seq analysis suggests that Brg1 might regulate gene expression through multiple mechanisms. Summary: The SWI/SNF protein Brg1 controls cell cycle length, cell cycle exit and cell survival, and is required for cell differentiation and retinal lamination, in the developing mouse retina.

The cyclin-dependent kinase inhibitor flavopiridol (alvocidib) inhibits metastasis of human osteosarcoma cells

Osteosarcoma is the most common primary malignant neoplasm of bone and typically occurs in children and young adults. As a highly metastatic malignancy, 15–20% of osteosarcoma patients are diagnosed after the tumor has already metastasized (typically to the lungs), which translates to 5-year survival rates of <40%. Here, we tested the effect of the cyclin-dependent kinase (CDK) inhibitor flavopiridol (alvocidib) in U2OS, SaOS-2, SJSA-1, and 143B osteosarcoma tumor cells in vitro and in vivo. Our results show that flavopiridol can drastically decrease survival in these osteosarcoma cell lines at nanomolar concentrations and induce mitotic catastrophe in p53-null osteosarcomas. We also performed transcriptome analysis (RNA-seq) of flavopiridol-treated osteosarcoma cells, which revealed significant changes in genes coding for proteins involved in cell-cell and cell-matrix adhesions, including cadherin 3 (CDH3) and 4 (CDH4). These transcriptional changes translated to a striking reduction in the ability of osteosarcoma cells to migrate and invade in vitro. Further, in vivo assessment of the effects of flavopiridol on osteosarcoma metastasis resulted in a significant reduction in the number of lung metastases in mice treated with flavopiridol at concentrations that are physiologically tolerable. This study suggests that flavopiridol, likely in combination with other cytotoxic chemotherapeutic agents, may be a promising drug for the treatment of osteosarcoma.

Genetically Engineered Mouse and Orthotopic Human Tumor Xenograft Models of Retinoblastoma

Retinoblastoma is a rare pediatric cancer of the developing retina that initiates with biallelic inactivation of the RB1 gene. Murine models of retinoblastoma provide excellent tools for preclinical studies as well as for the study of the biological processes that drive tumorigenesis following Rb loss. In this chapter, we describe several genetically engineered mouse and orthotopic human xenograft models of retinoblastoma and discuss the advantages and disadvantages of these models.