What happens if one day your cells start growing at a faster pace? You might tire faster on your morning run, notice a drop in the numbers on the weight scale, or even observe a bump that was not there before—a tumor. These common symptoms of cancer indicate the transformation of normal cells to cancerous cells, but how does this process occur? At UC San Diego’s Moores Cancer Center, Gutkind and Chen Labs study the regulation of mitosis in cancer cells, furthering our understanding of how normal cells turn cancerous.
In a normal cell, mitosis is the process of cell division in which a cell replicates its genetic information and splits into two identical daughter cells, which allows for organismal growth. Chemical signals from other cells and the environment initiate the process of cell division, however, mistakes can occur. For example, key proteins involved in regulatory processes of mitosis can accumulate mutations, increasing the risk of uncontrollable cell growth. Mutations are any change to an organism’s DNA sequence during DNA replication and cell division, occurring spontaneously or from exposure to carcinogens and other environmental factors. Over time, mutations accumulated in expressed regions of a gene can change protein function, which may be associated with disease traits, such as sickle-cell anemia or cystic fibrosis.
For a normal cell to undergo oncogenesis, or transform into a cancer cell, mutations must accumulate in two types of genes: tumor suppressor genes and proto-oncogenes. Tumor suppressors are responsible for slowing down the progression of the cell cycle. In a normal cell, they act like brake pedals on a car, coding for proteins that signal the cell to stop dividing and undergo cell death if something is wrong. Mutations that damage this regulatory mechanism may inactivate functions or reduce expression of tumor-suppressor genes. However, mutations in only tumor-suppressor genes are insufficient for cells to turn cancerous; mutations must also occur in proto-oncogenes. Normally, proto-oncogenes code for proteins responsible for cell proliferation but are inactivated in the cell once the processes they regulate are completed. With the accumulation of mutations, proto-oncogenes act like a gas pedal constantly being pressed, permanently activating the proteins and causing the cells to incessantly divide. With mutated tumor suppressor genes and proto-oncogenes, a normal cell experiences uncontrolled proliferation in a localized region of the body and eventually spread of invasive growth, or metastasis, becoming a cancer cell.
GPCRs: External Stimuli and the Kinase Cascade
Mutations in other regulatory cell signaling pathways of mitosis can similarly turn normal cells into cancerous cells. The Gutkind Lab at UC San Diego’s Moores Cancer Center studies G-protein coupled receptors (GPCRs), which are a varied family of proteins at the center of signal transduction pathways, linking a multitude of external stimuli to corresponding cellular activity. The GPCR can receive external chemical signals at its complementary binding site, like a key in a lock. Once a chemical signal binds at the binding site, the GPCR interacts with a G-protein (G(q)) to initiate a phosphorylation cascade inside the cell, like the clicking gears in an opening lock, activating proteins called activator kinases that lead to mitosis. Curiously, research shows a high occurrence of oncogenic mutations in GPCR and G-protein associated genes in most tumor types (O’Hayre). Because the GPCR signaling pathway is essential for normal mitosis, genetic mutations producing dysfunctional proteins in the pathway can lead to abnormal cell growth, resulting in cancer.
At the Gutkind Lab, researchers focus on mutations in G(q) and its role in the progression of uveal melanoma, a type of cancer in the eye. The lab previously concluded that gain-of-function mutations in the GNAQ, the gene coding for a subunit in G(q), lead to excessive cell proliferation (Gutkind). However, the question remains: which activator kinase is responsible?
The researchers aimed to determine which kinase was most essential to mitotic activity in cell lines as well as mouse models. The Gutkind Lab first conducted a genomic screen across all kinase-coding genes in the cell using CRISPR-Cas9 technology to individually inactivate each gene to monitor the result of mitosis without its corresponding kinase. This approach can identify a synthetic lethal, where the presence of an oncogenic mutations in two genes, often a proto-oncogene and a tumor suppressor gene, can turn the cell cancerous, but just one mutation will not. Synthetic lethality forms the basis of novel cancer therapy research, as any drug inhibiting the protein product of just one mutated gene will kill only cancerous cells and not healthy ones, and furthers research into targeting specific oncogenic mutations. The Gutkind Lab discovered that when a gene coding for focal adhesion kinase (FAK) was knocked out and the FAK protein was absent in the GNAQ-mutated pathway, cell proliferation halted despite the oncogenic mutation. FAK is a key player in regulating cell proliferation and migration or movement activity. The downstream effect of FAK showed its role in activating an oncogenic driver gene called YAP1 as well as inhibiting the HIPPO pathway that represses YAP1. Thus, by knocking out the FAK gene in a cell with a GNAQ mutation, the lab identified the FAK gene as a possible therapeutic target for treatment of uveal melanoma.
SUMOylation Modification
Genes involved in other regulatory processes beyond cell signaling can also contribute to oncogenesis. The Chen Lab, also located at UC San Diego’s Moores Cancer Center, studies SUMOylation, a process involving small ubiquitin-like modifier (SUMO) proteins. Ubiquitin and SUMO proteins are small proteins involved in post-translational modifications and the regulation of cell proliferation. In normal cells, SUMOylation is blocked when the cell senses something wrong with the DNA replication phase of mitosis, preventing cells from dividing and triggering cell death during anaphase. However, during oncogenic pathways, SUMO proteins contain a gain-of-function mutation and SUMOylation is overactivated. Because oncogenesis amplifies cell cycle progression, SUMOylation happens more frequently and acts as a key driver of cell division in cancer cells.
To observe SUMOylation in cancer cells, graduate student researchers in the Chen Lab use immunohistochemistry, a powerful technique that takes advantage of antibody-antigen interactions to detect specific antigens in cells and tissues. Cancer cells of almost all cancer types, from blood cancer to solid tumors, have been studied to examine SUMOylation in oncogenic pathways and the cancer’s response to therapies. Using this technique, they look for SUMO proteins and the E1 and E2 enzymes, which are SUMO-activating enzymes found in all cells. According to Dr. Chen, E1 and E2 enzymes are “expressed at much higher levels in cancer cells than in normal cells.” In cancer patients, higher expression of these factors correlates with lower survival probability due to rapid oncogenesis driven by overactivation of SUMOylation.
Today, the Chen Lab focuses on developing inhibitors for SUMO proteins and the E1/E2 enzymes with hopes of decreasing SUMOylation in cancer cells and increasing long-term survival probability among patients. Through crystallography, the technique of determining the arrangement of atoms in a crystal structure, researchers recently determined the crystal structure of a complex between the E1 enzyme and a recently discovered inhibitor (C0H000). The SUMO E1 enzyme in its active conformation has specific residues essential for adenylation, a biological process of attaching an adenosine monophosphate (AMP) to the protein’s side-chain, and a second catalytic cysteine half-domain (SCCH). In this active conformation, the E1 enzyme binds to SUMO proteins in its active site and adenylation happens properly. Upon binding, the C0H000 inhibitor initiates a cascade of structural changes, including a 180-degree rotation of the SCCH and disordering adenylation sites, that lock the enzyme in an inactive conformation. The inhibitor prevents SUMO proteins from binding to the E1 since adenylation can no longer occur. With these exciting discoveries, there remains hope for the development of more inhibitors for SUMO proteins to be incorporated into cancer therapies.
What Comes Next?
The research conducted at the Gutkind Lab and the Chen Lab explores the regulation of mitosis when oncogenesis occurs, and contributes to identifying inhibitor drugs through clinical trials. The Gutkind Lab studies synthetic lethal in-cell signaling pathways to target oncogenes with greater specificity for a personalized approach to therapeutics. Moving forward, the lab will continue contributing to clinical trials with different drug combinations targeting the synthetic lethality of uveal melanoma, and also focus on the role of GPCRs in metastasis or cell migration. In the Chen Lab, researchers observed that with certain SUMO protein inhibitors, not only was SUMOylation blocked, but an antitumor response was also activated. The lab hopes to develop more SUMOylation inhibitor drugs and push them into clinical trials, to both prevent cancer cell proliferation and kill existing cancer cells in the body. As cancer research progressively focuses on the regulation of mitosis, cancer treatments may become more effective and act as a step toward personalized medicine.
Works Cited
Gutkind, Silvio. “A Platform of Synthetic Lethal Gene Interaction Networks Reveals That the GNAQ Uveal Melanoma Oncogene Controls the Hippo Pathway through FAK.” CellPress, Elsevier, 14 Feb. 2019, www.cell.com/cancer-cell/fulltext/S1535-6108(19)30043-1#secsectitle0015.
O’Hayre, M., Vázquez-Prado, J., Kufareva, I. et al. The emerging mutational landscape of G proteins and G-protein-coupled receptors in cancer. Nat Rev Cancer 13, 412–424 (2013). https://doi.org/10.1038/nrc3521.
Lv, Zongyang, et al. “Molecular Mechanism of a Covalent Allosteric Inhibitor of Sumo E1
Activating Enzyme.” Nature News, Nature Publishing Group, 4 Dec. 2018, www.nature.com/articles/s41467-018-07015-1.