Population-enriched innate immune variants may identify candidate gene targets at the intersection of cancer and cardio-metabolic disease.

Both cancer and cardio-metabolic disease disparities exist among specific populations in the US. For example, African Americans experience the highest rates of breast and prostate cancer mortality and the highest incidence of obesity. Native and Hispanic Americans experience the highest rates of liver cancer mortality. At the same time, Pacific Islanders have the highest death rate attributed to type 2 diabetes (T2D), and Asian Americans experience the highest incidence of non-alcoholic fatty liver disease (NAFLD) and cancers induced by infectious agents. Notably, the pathologic progression of both cancer and cardio-metabolic diseases involves innate immunity and mechanisms of inflammation. Innate immunity in individuals is established through genetic inheritance and external stimuli to respond to environmental threats and stresses such as pathogen exposure. Further, individual genomes contain characteristic genetic markers associated with one or more geographic ancestries (ethnic groups), including protective innate immune genetic programming optimized for survival in their corresponding ancestral environment(s). This perspective explores evidence related to our working hypothesis that genetic variations in innate immune genes, particularly those that are commonly found but unevenly distributed between populations, are associated with disparities between populations in both cancer and cardio-metabolic diseases. Identifying conventional and unconventional innate immune genes that fit this profile may provide critical insights into the underlying mechanisms that connect these two families of complex diseases and offer novel targets for precision-based treatment of cancer and/or cardio-metabolic disease.

Novel miRNA-31 and miRNA-200a-Mediated Regulation of Retinoblastoma Proliferation.

Retinoblastoma is the most common intraocular tumor in children. Current management includes broad-based treatments such as chemotherapy, enucleation, laser therapy, or cryotherapy. However, therapies that target specific pathways important for retinoblastoma progression could provide valuable alternatives for treatment. MicroRNAs are short, noncoding RNA transcripts that can regulate the expression of target genes, and their aberrant expression often facilitates disease. The identification of post-transcriptional events that occur after the initiating genetic lesions could further define the rapidly aggressive growth displayed by retinoblastoma tumors. In this study, we used two phenotypically different retinoblastoma cell lines to elucidate the roles of miRNA-31 and miRNA-200a in tumor proliferation. Our approach confirmed that miRNAs-31 and -200a expression is significantly reduced in human retinoblastomas. Moreover, overexpression of these two miRNAs restricts the expansion of a highly proliferative cell line (Y79), but does not restrict the growth rate of a less aggressive cell line (Weri1). Gene expression profiling of miRNA-31 and/or miRNA-200a-overexpressing cells identified differentially expressed mRNAs associated with the divergent response of the two cell lines. This work has the potential to enhance the development of targeted therapeutic approaches for retinoblastoma and improve the efficacy of treatment.

MicroRNAs-449a and -449b exhibit tumor suppressive effects in retinoblastoma.

Retinoblastoma is the most common pediatric cancer of the eye. Currently, the chemotherapeutic treatments for retinoblastoma are broad-based drugs such as vincristine, carboplatin, or etoposide. However, therapies targeted directly to aberrant signaling pathways may provide more effective therapy for this disease. The purpose of our study is to illustrate the relationship between the expressions of miRs-449a and -449b to retinoblastoma proliferation and apoptosis. We are the first to confirm an inhibitory effect of miR-449a and -449b in retinoblastoma by demonstrating significantly impaired proliferation and increased apoptosis of tumor cells when these miRNAs are overexpressed. This study suggests that these miRNAs could serve as viable therapeutic targets for retinoblastoma treatment.

Differentially expressed miRNAs in retinoblastoma.

MicroRNAs (miRNAs) are short non-coding RNA transcripts that have the ability to regulate the expression of target genes, and have been shown to influence the development of various tumors. The purpose of our study is to identify aberrantly expressed miRNAs in retinoblastoma for the discovery of potential therapeutic targets for this disease, and to gain a greater understanding of the mechanisms driving retinoblastoma progression. We report 41 differentially expressed miRNAs (p<0.05) in 12 retinoblastomas as compared to three normal human retinae. Of these miRNAs, many are newly identified as being differentially expressed in retinoblastoma. Further, we report the validations of five of the most downregulated miRNAs in primary human retinoblastomas (p<0.05), human retinoblastoma cell lines, and mouse retinoblastoma cell lines. This serves as the largest and most comprehensive retinoblastoma miRNA analysis to date with corresponding clinical and pathological characteristics. This is an essential step in the discovery of miRNAs associated with retinoblastoma progression, and in the identification of potential therapeutic targets for this disease.

Changes in retinoblastoma cell adhesion associated with optic nerve invasion.

In the 1970s, several human retinoblastoma cell lines were developed from cultures of primary tumors. As the human retinoblastoma cell lines were established in culture, growth properties and changes in cell adhesion were described. Those changes correlated with the ability of the human retinoblastoma cell lines to invade the optic nerve and metastasize in orthotopic xenograft studies. However, the mechanisms that underlie these changes were not determined. We used the recently developed knockout mouse models of retinoblastoma to begin to characterize the molecular, cellular, and genetic changes associated with retinoblastoma tumor progression and optic nerve invasion. Here we report the isolation and characterization of the first mouse retinoblastoma cell lines with targeted deletions of the Rb family. Our detailed analysis of these cells as they were propagated in culture from the primary tumor shows that changes in cadherin-mediated cell adhesion are associated with retinoblastoma invasion of the optic nerve prior to metastasis. In addition, the same changes in cadherin-mediated cell adhesion correlate with the invasive properties of the human retinoblastoma cell lines isolated decades ago, providing a molecular mechanism for these earlier observations. Most importantly, our studies are in agreement with genetic studies on human retinoblastomas, suggesting that changes in this pathway are involved in tumor progression.

AAV-mediated local delivery of interferon-beta for the treatment of retinoblastoma in preclinical models.

Interferon-beta (IFN-beta) has been found to have anti-tumor properties against a variety of malignancies through different mechanisms. However, clinical trials involving systemic administration of IFN-beta have been hampered by secondary toxicity and the short half-life of IFN-beta in the circulation. In order to circumvent these limitations, we have developed an adeno-associated viral (AAV) vector gene-therapy approach to deliver IFN-beta to tumors. In this study, we tested the efficacy of AAV-mediated local delivery of IFN-beta for the treatment of retinoblastoma in preclinical models. Retinoblastoma is an ideal candidate for gene-therapy-based anti-cancer treatment because target cell transduction and, therefore, IFN-beta delivery can be contained within the ocular environment, thereby minimizing systemic toxicity. We report here that retinoblastoma cell lines exhibit pleiotropic responses to IFN-beta consistent with previous studies on a variety of tumor cell lines. Intravitreal injection of AAV-IFN-beta resulted in efficient retinal infection and sustained IFN-beta production in the eye with minimal systemic exposure. Vector spread outside of the eye was not detected. Using our orthotopic xenograft model of retinoblastoma, we found that intravitreal injection of AAV-IFN-beta had a potent anti-tumor effect in vivo. These data suggest that AAV-mediated delivery of IFN-beta may provide a complementary approach to systemic chemotherapy which is the standard of care for retinoblastoma around the world.

Targeting MDM2 and MDMX in retinoblastoma.

Retinoblastoma is the third most common form of cancer in infants, and metastatic retinoblastoma is lethal in approximately 90% of cases. Early detection and aggressive therapy has resulted in a 95% probability of survival for retinoblastoma patients in the United States. However, the United States only represents 3-4% of the retinoblastoma cases worldwide. The majority of children diagnosed with retinoblastoma each year live in developing countries where the probability of survival is closer to 50%. This difference in survival rates reflects poor early detection rates and limited resources for the aggressive therapy necessary to treat retinoblastoma and manage the side effects associated with broad-spectrum systemic chemotherapy in young children. In order to have the most significant impact on retinoblastoma treatment in the United States and worldwide, current efforts have focused on local delivery of targeted chemotherapy. In this review, we summarize recent data showing that the p53 pathway is inactivated in 75% of retinoblastoma patients due to extra copies of the MDM2 and MDMX genes. A small molecule inhibitor of MDM2 called nutlin-3 can induce p53-mediated cell death in retinoblastoma cells. Subconjunctival delivery of nutlin-3 in preclinical models of retinoblastoma confirmed the efficacy of this approach in vivo. The advantage of local application of targeted chemotherapeutic agents such as nutlin-3 is that greater intraocular drug concentrations can be achieved without the side effects associated with systemic broad-spectrum chemotherapy. We propose that subconjunctival administration of targeted chemotherapy may be the best treatment option for children with retinoblastoma in the United States and throughout the developing world because it provides greater tumor response without the costs and complications associated with current treatment protocols.

Inactivation of the p53 pathway in retinoblastoma.

Most human tumours have genetic mutations in their Rb and p53 pathways, but retinoblastoma is thought to be an exception. Studies suggest that retinoblastomas, which initiate with mutations in the gene retinoblastoma 1 (RB1), bypass the p53 pathway because they arise from intrinsically death-resistant cells during retinal development. In contrast to this prevailing theory, here we show that the tumour surveillance pathway mediated by Arf, MDM2, MDMX and p53 is activated after loss of RB1 during retinogenesis. RB1-deficient retinoblasts undergo p53-mediated apoptosis and exit the cell cycle. Subsequently, amplification of the MDMX gene and increased expression of MDMX protein are strongly selected for during tumour progression as a mechanism to suppress the p53 response in RB1-deficient retinal cells. Our data provide evidence that the p53 pathway is inactivated in retinoblastoma and that this cancer does not originate from intrinsically death-resistant cells as previously thought. In addition, they support the idea that MDMX is a specific chemotherapeutic target for treating retinoblastoma.

Carcinoembryonic antigen-related cell adhesion molecule 1a-4L suppression of rat hepatocellular carcinomas.

Carcinoembryonic antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) is a member of the CEA family of immunoglobulin-like adhesion molecules with two major splice variants, CEACAM1(a)-4L and CEACAM1(b)-4S, differing in the length of their COOH-terminal cytoplasmic tail. Both forms are down-regulated in prostate and liver carcinomas relative to normal tissues. We have previously shown in a nude mouse xenograft model that restoration of CEACAM1(a)-4L expression in human prostate carcinoma cells (PC-3) suppresses tumorigenicity, an effect observed with carcinomas from several other tissues but never established for hepatocellular carcinomas. In this report, we have examined the effect of CEACAM1(a)-4L on tumorigenicity of 1682A, a rat hepatocellular carcinoma that grows on the omentum when injected into the peritoneal cavity. Results show that restoration of CEACAM1(a)-4L expression at levels 13- and 0.45-fold compared with negative controls or normal hepatocytes, respectively, completely suppressed the formation of 1682A tumor nodules on the omentum at 3 weeks after injection. In contrast, 1682A cells infected with CEACAM1(b)-4S or an empty retroviral vector formed multiple clusters of tumor nodules. Although tumor nodules of 1682A cells positive and negative for CEACAM1(a)-4L did not display significant differences in histologic organization, aggregates formed in vitro by 1682A-L were smaller in size and displayed enlarged intercellular spaces relative to their 1682A-V counterparts. Restoration of CEACAM1(a)-4L expression did not elevate levels of apoptosis but seemed to cause an increase in the length of G1. This is the first demonstration of CEACAM1(a)-4L-induced tumor suppression in liver carcinomas using a quantifiable i.p. syngeneic transplantation model.

Topotecan combination chemotherapy in two new rodent models of retinoblastoma.

Chemotherapy combined with laser therapy and cryotherapy has improved the ocular salvage rate for children with bilateral retinoblastoma. However, children with late-stage disease often experience recurrence shortly after treatment. To improve the vision salvage rate in advanced bilateral retinoblastoma, we have developed and characterized two new rodent models of retinoblastoma for screening chemotherapeutic drug combinations. The first model is an orthotopic xenograft model in which green fluorescent protein- or luciferase-labeled human retinoblastoma cells are injected into the eyes of newborn rats. The second model uses a replication-incompetent retrovirus (LIA-E(E1A)) encoding the E1A oncogene. Clonal, focal tumors arise from mouse retinal progenitor cells when LIA-E(E1A) is injected into the eyes of newborn p53-/- mice. Using these two models combined with pharmacokinetic studies and cell culture experiments, we have tested the efficacy of topotecan combined with carboplatin and of topotecan combined with vincristine for the treatment of retinoblastoma. The combination of topotecan and carboplatin most effectively halted retinoblastoma progression in our rodent models and was superior to the current triple drug therapy using vincristine, carboplatin, and etoposide. Vincristine had the lowest LC50 in culture but did not reduce tumor growth in our preclinical retinoblastoma models. Taken together, these data suggest that topotecan may be a suitable replacement for etoposide in combination chemotherapy for the treatment of retinoblastoma.