Finding New Treatments for Rhabdomyosarcoma
Mary Baylies, PhD
Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma, representing 3 to 5 percent of all childhood cancers. RMS is classified into two subtypes, alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS).
ARMS is notoriously aggressive and associated with metastasis. The outcome for patients with metastatic or recurrent disease remains dismal with an estimated five-year survival of less than 30 percent. Approximately 14 percent of children with RMS have metastatic disease at the time of initial diagnosis. These data emphasize a critical need to identify novel therapeutic agents that limit RMS metastasis and improve survival rates, which is the key goal of this proposal.
With between 250 and 350 new cases diagnosed each year, RMS is a rare cancer lacking major investments in treatment development. Diseases with comparatively low numbers of newly diagnosed cases can profit from drug-repurposing screens of compounds approved by the US Food and Drug Administration. Drugs identified in these screens can be prioritized for clinical trials
We have adopted a relatively high-throughput protocol for drug screens in the Drosophila system and found three compounds that reduce ARMS metastasis. We hypothesize that compounds from an FDA-approved library will selectively inhibit ARMS development or metastasis in this efficient and novel in vivo model. Positives recovered from this screen will be tested for efficacy against human RMS cell lines.
Contralateral Breast Cancers: Independent Cancers or Metastases?
Colin Begg, PhD
Women diagnosed with breast cancer frequently develop cancer in the opposite breast, either simultaneously or subsequently. These cancers have traditionally been classified as new, independent primaries. However it is possible that a proportion of the tumors represent metastases from the original primary to the contralateral breast.
Indeed, using modern molecular pathologic technology investigators have demonstrated in other cancer sites, notably lung cancer, that many tumors classified as independent are in fact metastases. A resolution of this issue is important in breast cancer to allow individualization of the extent of surgery on the contralateral breast and axilla based on a diagnosis of metastatic disease versus new primary cancer, and to direct the choice and duration of systemic therapy in patients diagnosed with contralateral breast cancer (CBC).
Additionally, evidence of good survival outcomes after resection of limited metastatic sites such as the contralateral breast and axilla, followed by aggressive systemic therapy, would have significant implications for the management of some cancers. This includes patients presenting with stage IV breast cancer and an intact primary tumor, or those who develop metastases to isolated, surgically respectable sites in this era of improved targeted therapies.
Although a number of studies have examined the clonal relatedness of CBCs, these investigations have been based exclusively on small sample sizes and employed technology with limited resolution and diagnostic accuracy. We have the opportunity to provide resolution to the issue by assembling tumor specimens from both breasts for a large number of CBC cases from our tumor archives and other available sources, and using up-to- date sequencing technology and specialized statistical methods to classify the tumor pairs as either independent or of clonal origin.
ARMS is associated with chromosomal translocations generating the fusion oncoprotein PAX3/7- FOXO1. This fusion protein acts as an aberrant transcription factor, altering protein networks in muscle cells and ultimately causing tumor formation and metastasis. Targets of PAX7-FOXO1 are therefore candidates for the development of new drugs and could serve as additional biomarkers for the disease.
Our preliminary data has identified one such target. To identify additional target genes, we will carry out a limited RNAi screen in Drosophila based on comparative transcriptome analysis between fly and human. Top candidates from the fly screen will be validated in human tumor cell lines.
If successful, these investigations will identify compounds for clinical trials and novel drug targets or protein networks for ARMS treatment.
Neutrophil Mediated Antimetastatic Therapy
Robert Benezra, PhD
Cancer-related mortality is usually associated with metastasis, the spreading of cancer from a primary tumor to distant organs. Previous studies suggest that looking at the events that determine the site of future metastasis have shown that the primary tumor can modify distant organs and prepare them for the arrival of tumor cells.
In addition, our preliminary data suggests that the primary tumor can induce an antimetastatic immune response and delay the formation of distant metastases. This response is mediated by neutrophils — a subset of white blood cells that play a role in inflammation and immune responses to infections.
Neutrophils are mobilized by the primary tumor and accumulate in the pre-metastatic lungs prior to the arrival of tumor cells. Tumor-entrained neutrophils (TENs) but not naïve neutrophils can induce tumor cell death in vitro by initiating physical contact with the tumor cells and generating H2O2. In vivo, depletion of neutrophils results in enhanced metastatic progression while transfusion of tumor-entrained neutrophils can dramatically reduce the development of lung metastases.
We have identified the tumor-secreted factors that stimulate the antimetastatic neutrophil response and shown that while these factors may contribute to primary tumor progression, they concomitantly stimulate an anti-metastatic response at the metastatic site. To gain additional insight into the antimetastatic role of neutrophils and explore their therapeutic potential, we will further characterize tumor-entrained neutrophils and test their antimetastatic potential in a variety of preclinical settings.
Real-Time In Vivo Marker Detection for Rapid Measurement of Metastasis Onset
Daniel A. Heller, PhD
The study of metastatic cancer presents several challenges that may be addressed using improved sensor technologies. These challenges include detecting metastasis at early stages and discerning indolent versus aggressive tumors. Both tasks require simple, reliable, inexpensive, and non-invasive methods for detection.
We will perform real-time and multiplexed in vivo quantification of metastatic cancer markers. The elevated serum levels of several antigenic biomarkers have been shown to mark the onset of metastasis. Using biofunctionalized single-walled carbon nanotubes (SWCNTs), we will develop unique sensors to detect the concentration of these biomarkers in real-time, with high sensitivity and selectivity, and from within the organism.
Developing Novel Strategies to Target Tumor Cell-Macrophage Interactions in Brain Metastasis
Johanna A. Joyce, PhD
Metastasis to distant sites, including the brain, remains the single most lethal aspect of breast cancer. Despite important advances in the treatment of localized breast cancer, few therapies succeed at combating disseminated disease.
Indeed, the critical need for improved therapy for women with metastatic breast cancer is underscored by the fact that only 5 to 10 percent of these patients survive five or more years after their diagnosis. This is a particularly dire problem for breast cancer patients whose disease has spread to the brain, as there are currently no effective therapies for treating brain metastasis.
In addition, as the incidence of brain metastasis often exceeds 30 percent in both HER2-positive and triple-negative breast cancer subtypes, a significant number of breast cancer patients are affected by this disease annually in the United States. Clearly, novel insights are required to improve treatment of breast to brain metastases.
One new paradigm comes from the observation that noncancerous stromal cells in the tumor microenvironment can make critical contributions to promote malignancy. While the tumor microenvironment has emerged as an important regulator of cancer progression in other organ sites, and hence is considered a potential therapeutic target, our knowledge of the brain tumor microenvironment remains very limited.
We will address this knowledge gap and specifically focus our investigation on tumor-associated macrophages and microglia in the brain. We have interesting preliminary data supporting the tumor-promoting functions of these cell types.
We hypothesize that macrophages in the brain microenvironment significantly contribute to promoting the extravasation, seeding and outgrowth of metastatic breast cancer cells in this secondary site. Targeting these cells, or the tumor-promoting factors they produce, might lead to novel mechanistic insights into brain metastasis.
We will use animal models to evaluate different small molecule inhibitors targeted against these factors in preclinical trials. This project has the potential to rapidly translate new findings into innovative strategies to successfully treat metastatic breast cancer.
Metastasis in the Zebrafish System
Richard White, MD, PhD
Metastasis exerts its physiologic effects via an interaction between the tumor cells and the host cells within individual organs. Because of this, an understanding of the factors that promote metastasis will require increasingly sophisticated in vivo models that recapitulate each step of metastasis.
In recent years, the zebrafish has emerged as an important tool in cancer research because of its capacity for high-resolution in vivo imaging coupled with rapid and large-scale genetic manipulation. We have developed a zebrafish model of melanoma that closely resembles the human disease at histological and gene expression levels.
We will use the zebrafish as a platform to discover new genes and pathways associated with metastasis. We will first develop quantitative assays for melanoma metastasis using a transparent adult zebrafish strain known as casper. With these tools, we will then characterize intratumoral and metastatic heterogeneity using the Brainbow fate mapping system. Tumor heterogeneity is an essential factor underlying treatment resistance and failure during disseminated disease.
In addition, we will investigate whether stress-induced mutation via upregulation of dinB/polK mechanistically underlies the generation of heterogeneity during tumor progression. This would have broad implications across different cancer types, since mechanisms generating heterogeneity would be a rational therapeutic target in metastatic disease.
Our discoveries in the zebrafish will be complemented by investigation of parallel mechanisms in human tissues, leveraging the strengths of each system to investigate fundamental dynamics of metastatic progression.