THE PI3K-AKT-MTOR SIGNAL TRANSDUCTION PATHWAY AND PI3K/MTOR INHIBITION IN CANINE OSTEOSARCOMA AND LYMPHOMA

Abstract

The research discussed herein presents an important opportunity to better understand and advance the treatment of canine osteosarcoma and lymphoid tumors, which carry a poor prognosis and serve as a valuable resource for the advancement of comparative oncology in the canine and human patient. Canine hematopoietic tumors are among the most common tumors in dogs and advancements in treatment are necessary to improve outcomes. Lymphoma represents one of the most common tumor types, accounting for 7-24% of all canine neoplasms and 80% of canine hematopoietic tumors, with many subtypes and heterogeneous biological behavior. Canine diffuse large B-cell lymphoma, which comprises 50% of all lymphoma cases, is also a valuable spontaneous animal model for non-Hodgkin lymphoma in humans. Canine osteosarcoma presents a significant clinical challenge in veterinary oncology. Due to the similarities in aggressive biologic behavior, mutation status, and gene expression profiles, the canine patient also provides a spontaneous animal model for osteosarcoma in humans. Advancements in the treatment of osteosarcoma and lymphoma have been slow to progress in recent years. In multiple tumor types of both species, including osteosarcoma and lymphoid neoplasms, there is similar dysregulation of signal transduction through phosphatidylinositol 3-kinase (PI3K), AKT serine/threonine kinase (AKT), and mechanistic target of rapamycin (mTOR), collectively known as the PI3K-AKT-mTOR pathway, which contributes to disease progression and poorer outcomes. In human medicine, there have been many attempts to induce cessation of signal transduction with halting progress due to the complexity of signal transduction feedback and resistance to broad or single-point pathway inhibition. Through these efforts we have come to better comprehend the potential benefits of targeted and multi-nodal PI3K-AKT-mTOR pathway inhibition as a component of cancer treatment. As is often the case in the veterinary field, the research in cellular signaling cascades in health and cancer development is not as completely documented for canine neoplasms as in human tumors. Presented in Chapter 1 is a review of the current knowledge of PI3K-AKT-mTOR signal transduction activity in health and disease, with a comparative focus on human and canine osteosarcoma and lymphoid tumors. The current body of knowledge regarding the role of PI3K-AKT-mTOR pathway in tumorigenesis serves as a basis for our investigation of the role of signal transduction in canine osteosarcoma and lymphoma and the efficacy of multi-nodal pathway inhibition with a dual PI3K/mTOR inhibitor, VDC597. In order to better understand the role of PI3K-AKT-mTOR signaling in these canine tumors, further investigation into pathway activity and the efficacy of dual-inhibition is warranted. We hypothesized that, in comportment with existing literature in canine and human tumors, there would be increased PI3K-AKT-mTOR activity in the canine osteosarcoma and lymphoid neoplastic samples and cells we examined. We further hypothesized that inhibition of signal transduction with VDC597 would result in antineoplastic effects that would provide a potentially beneficial additional component of chemotherapeutic protocols in the treatment of canine osteosarcoma and lymphoid tumors. Chapters 2 and 3 initially report the in vitro expression and activation of the pathway in canine osteosarcoma cells and lymphoid tumor cells, respectively. Subsequently, PI3K/mTOR dual-inhibition with VDC597 was examined for antineoplastic activity, as a single-agent treatment and in conjunction with currently used chemotherapy drugs for these tumor cell types. Growth inhibition and cell death assays were used to evaluate the efficacy of VDC597 for reducing cell survival and induction of apoptosis in these tumors. Western blots and immunohistochemistry were used to study the reduction in pathway activation. Scratch assays and Boyden chamber assays were used to investigate the inhibition of chemotactic and non-chemotactic migration and invasion in osteosarcoma cells. ELISA assays were used to examine the anti-angiogenic effects of the compound on vascular endothelial growth factor production by lymphoma, leukemia, and osteosarcoma cells. In all these tumor types, the results of this study demonstrate a dose-dependent: reduced signal transduction; increased cell death; reduced cell proliferation, migration, invasion, and vascular endothelial growth factor production in vitro. These effects were additive to mildly synergistic when VDC597 was combined with the chemotherapy drugs tested. Chapter 2 also presents our evaluation of the efficacy of oral administration of VDC597 for osteosarcoma treatment in a xenograft mouse model. Our results demonstrate reduced tumor growth and increased survival time in mice treated with VDC597 alone and in combination with carboplatin. We also used immunohistochemistry to evaluate pathway activation, and localization of a downstream target—the forkhead box O1 transcription factor (FOXO1)—in tumor samples from these mice. There was marked reduction in phosphorylation of pathway components and increased intranuclear FOXO1 immunolocalization in mice treated with VDC597, indicating a correlation between reduced tumor growth and inhibition of signal transduction. Based on these findings, we sought to use FOXO1 immunohistochemistry as a proxy for phosphoprotein detection in decalcified formalin fixed paraffin embedded osteosarcoma samples from canine patients, but found that, unlike the xenograft tumors which were treated with a PI3K/mTOR inhibitor, a correlation between tumor biologic behavior and FOXO1 immunoreactivity was not present in the patient derived tumor sections. Chapter 3 also presents findings from immunohistochemical evaluation of formalin-fixed paraffin embedded lymph node samples from patients diagnosed with B-cell lymphoma, in which the expression of phosphorylated components of the PI3K-AKT-mTOR pathway was examined for correlation to outcome data and prognostic indicators. We noted marked variation in the degree of phosphorylation in lymphoma samples between different patients. Major histocompatibility complex class II expression and post-treatment relapse status were correlated to phosphorylated AKT and phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 expression, respectively. There was no correlation, however, between phosphoprotein immunoreactivity and clinical outcome. Collectively, the results from our in vitro and in vivo xenograft experiments indicate there is a good basis for the use of dual-inhibition with VDC597 in the treatment of canine osteosarcoma and lymphoid tumors. Our immunohistochemical examination of osteosarcoma expression of FOXO1 and lymphoma expression of phospho-proteins within the signaling cascade indicate there is need for further investigation to better understand the variability and complexity of signal transduction in the canine patient to help better guide treatment regimens that involve such targeted inhibition. Through future studies to better characterize and correlate pathway expression by these tumors in the canine patient, we can better tailor treatment protocols. Future clinical trials will better demonstrate the potential efficacy of incorporation of VDC597 into cancer treatment in the canine patient and serve as a useful model for comparative oncology and similar inhibition in human tumors.