Enhancing Activity of the Anaphase Promoting Complex to Reverse Multiple Drug Resistant Behaviour in Breast Cancer

Abstract

The development of multiple drug resistant (MDR) behaviour in cancer poses a significant challenge to effective treatment, yet this is not a rare event and occurs in up to 20% of breast, lung, colon, and hematological malignancies. MDR is defined as the presence of clinical resistance to cancer therapy including resistance to medical therapies not previously used. MDR can be innate, or acquired over the course of treatment. The underlying causes of MDR are clearly multifactorial, and there are several common themes that promote the behaviour: genomic instability, impaired stress response pathways, and dysregulated mitosis all promote MDR behavior. These cellular mechanisms exist as an interrelated network of pathways, as disruption of one often impacts another, such as the loss of mitotic regulation promoting genomic instability. This accumulation of mutations may then impair the activity of various stress responses pathways, thereby reducing the apoptotic response to chemotherapy and promoting MDR behaviour. The Anaphase Promoting Complex (APC) is an evolutionarily conserved, multi-subunit E3 ubiquitin ligase enzyme that targets selected proteins for proteasomal degradation during mitosis and G1. Its functions include activation of stress response pathways and regulated progression of mitosis, which protects the cell from genomic instability. Previously, a correlation had been established between dysfunction of the APC and cancer progression, primarily indicated by the accumulation of many APC substrates noted in multiple cancers. Recently, this relationship was directly implicated in the progression of cancer into an MDR state in canine lymphomas where relapse correlated with impaired APC function and remission restored APC activity. Therefore, we hypothesize that enhancing APC activity will subsequently restore chemosensitivity in MDR cancer. In yeast, novel peptides were identified that bound and activated the Saccharomyces cerevisiae (S. cerevisiae) APC. When stably expressed in the innately MDR breast cancer cell line MDA-MB-231, two of these peptides significantly enhanced APC activity. Significantly, peptide expression restored cytotoxic sensitivity to doxorubicin in this MDR cell population in accordance with relative APC activity, with greater APC activation producing greater chemosensitivity. An investigation of FOXO3A-dependent stress response pathways (suggested by previous studies) demonstrating a functional interaction between APC activity and yeast FOXO orthologs, to induce stress responses. We revealed FOXO3A signaling activity was increased upon APC-activating peptide expression, an elevation in FOXO3A activity, and therefore predicted a concurrent increase in stress-related apoptosis. As anticipated, peptide expression increased apoptosis with or without chemotherapy exposure. Peptide-dependent APC activity unexpectedly augmented mitotic dysregulation observed as an increased number of mitotic catastrophes, including chromosomal mis-segregation and micronuclei formation. The consequences of mitotic catastrophes are elevated genomic instability and DNA damage, which were found to be elevated. While this behaviour is typically considered to be tumorigenic, this phenomenon may enhance the cytotoxic effect of chemotherapy by inducing cell death via priming the cells with elevated genomic damage. Our results indicate that activation of the APC serves to enhance chemosensitivity in MDR cancer; however, the complicated mechanisms related to APC activity warrants further investigation into its relationship with MDR behaviour.