Overview

It is critical to discover the mechanisms of normal cell cycle regulation if we are to fully understand what goes awry in cancer cells. The research in the Calvi lab focuses on defining the mechanisms of normal cell division, cell cycle checkpoints, and DNA replication. An important goal is to define the many variations on the canonical cell cycle and checkpoint themes during development. Another important goal is to define how errors in these processes contribute to genome instability and cancer. To achieve these goals, we use the powerful tools available in the fruit fly Drosophila melanogaster. We are also collaborating to translate our findings to human cells. Our research is uncovering new mechanisms that regulate the cell cycle and protect genome integrity.

Cell division cycles and checkpoints are altered during both development and cancer. We have focused on a cell cycle variation that results in polyploid cells: the endocycle (Figure 1). Endocycling cells grow and periodically duplicate their DNA without dividing (G/S cycle), and ultimately become large, polyploid cells. Endocycles are a normal growth program that occurs during the development of a wide array of organisms including humans (Figure 1). Emerging evidence indicates that cells also switch to endocycles in response to conditional stimuli during wound healing, tissue regeneration, and cancer (Figure 2). We refer to these as induced endocycling cells (iECs). Despite the ubiquity and importance of endocycles, much remains unknown about their regulation.

We are currently using genetic, molecular, cell, and genomic approaches to define how polyploid cell cycles are regulated. Recent findings have revealed a transcriptome network that is remodeled when cells switch from mitotic division to polyploid cell cycles (Figure 3). This transcriptome remodeling occurs in developmental endocycling cells (devECS) and induced endocycling cells (iECS) that switch to endocycles in response to conditional signals. An important remaining question is how this remodeling contributes to polyploid cycles during tissue regeneration and cancer.

Evidence suggests that cancer cells can switch to an endocycle, polyploidize, and then return to an error-prone mitosis that contributes to aneuploidy (Figure 4). Together with the Walczak lab, we are modeling in flies and human cells how a resumption of mitotic division in polyploid cells contributes to genome instability, drug resistance, and cancer (Figure 5).

DNA damage activates checkpoints that arrest the cell cycle and mobilize proteins to fix the damage. Excessive DNA damage can trigger a programmed cell death known as apoptosis. The inability to properly execute apoptosis is a known cause of cancer predisposition in humans.

We discovered that the apoptotic response to DNA damage is repressed in endocycling cells (Figure 6). We showed that the mechanism of this repression is chromatin-based silencing of the expression of genes regulated by the fly ortholog of the p53 tumor suppressor (Figure 7). We are currently investigating how this chromatin silencing is linked to cell cycle regulation, and how it prevents transcriptional activation by p53.

Genomic DNA must be fully and accurately replicated and then segregated into two daughter cells when cells divide. DNA replication that is uncoordinated from cell division causes “replication stress” and DNA damage, which is sensed and corrected by cell cycle checkpoints (Figure 8). Defects in both DNA replication and cell cycle checkpoints lead to high rates of DNA mutation (genome instability) and cause cancer. We are using novel approaches in Drosophila to discover molecular mechanisms that regulate DNA replication and how replication errors result in genome instability.

It is known that problems with DNA replication cause genome instability and heritable developmental syndromes in humans. Yet, it is still not fully understood how DNA replication is regulated and executed during multicellular development. Filling this knowledge gap is important for developing new diagnostics and therapies.

We have identified a gene in Drosophila that is required for DNA replication—humpty dumpty (hd). Hd protein sequence and DNA replication function are conserved in eukaryotes. Recent evidence suggests that it acts at replication forks, but the range and specificity of its molecular functions remain ill-defined. We have shown that mutations in hd causes genome instability and impairs Drosophila development (Figure 9).

It is now known that mutations in the human ortholog of hd is the genetic cause of heritable developmental malformations in children. We are using Drosophila to further define hd molecular function, and to model how the mutant alleles from children impair this function.